University of Southern California Dissertations and Theses

On the mechanism of ACTH stimulation of adrenal steroidogenesis

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On the mechanism of ACTH stimulation of adrenal steroidogenesis

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Content ON THE MECHANISM OF ACTH STIMULATION OF ADRENAL STEROIDOGENESIS by Julia Bell A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biochemistry) September 1973 UN IVERSITY O F S O U T H E R N C A LIFO R N IA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES, CALIFORNIA S 0 0 0 7 This dissertation, written by ................. under the direction of h.§.T... Dissertation Com mittee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillm ent of requirements of the degree of D O C T O R O F P H IL O S O P H Y V Dean Da,* ( Z c u ^ u i C ^ y < }75 DISSERTATION COMMITTEE TABLE OF CONTENTS Page ACKNOWLEDGMENTS iv LIST OF TABLES V LIST OF FIGURES vi INTRODUCTION 1 LITERATURE REVIEW 5 Cholesterol Cholesterol Utilization Corticosteroid Biosynthesis Steroid Hydroxylases Absorption Properties of Cytochrome P450 Stimulation of Corticosteroidogenesis by ACTH STATEMENT OF PROBLEM 45 MATERIALS AND METHODS 48 Measurement of Steady-State Concentrations of Adrenal Pyridine and Adenine Nucleo tides, and the Substrates of Glycolysis and the Krebs Cycle Cyclic AMP Measurements Corticosterone Measurements Preparation of Mitochondria Preparation of Microsomes Preparation of Cholesterol-Lecithin Emulsions Absorption Spectra Incubation Conditions of Hydroxylation Studies Measurement of Steroid Incubation Products P450 Assay ii MATERIALS AND METHODS (Continued) Conditions for Determining the In Vivo Effects of ACTH and Cycloheximide on tKe Levels of Endogenous Cholesterol and DOC-Bound P450 (Table VI) Sources of Important Reagents RESULTS Control of NADPH Production Control of Glycolysis ACTH and the Steroid Hydroxylases DISCUSSION Intramitochondrial NADPH Production ACTH-Induced Cyclic AMP Accumulation and Activation of Glycolysis Spin State Changes Induced in Cytochrome P450 Substrate Metabolism by Rat Adrenal Mito chondria ACTH Activation of Cholesterol Transport to Cytochrome P450 The Simpson, et al Hypothesis for ACTH Activation of Adrenal Steroidogenesis Cholesterol Transport Process SUMMARY AND CONCLUSIONS REFERENCES CITED ACKNOWLEDGMENTS I will always be indebted to m y husband, Richard, and to my s is te r, Sandra, for th eir love, understanding, and encouragement which enabled me to complete this work. I wish to thank Dr. Boyd Harding, my advisor, for securing financial aid to support m y graduate studies, and m y other committee members, Dr. Visser and Dr. Sadee, for their helpful criticisms of the manuscript. M y special thanks to radio station K BCA for making the long hours tolerable by providing 24-hour jazz music. LIST OF TABLES Table I. II. III. IV. V. VI. Page Effects of ACTH on Levels of Krebs Cycle 61 Intermediates Pyridine and Adenine Nucleotides Some Conditions Affecting Oxidized-Re- 64 duced Pyridine Nucleotide Ratios Distribution of Pyridine Nucleotides in 66 Rat Adrenal Bovine Adrenal Cortex Microsomal P450 104 Effects of Inhibitors and ACTH on Sub- 119 strate Metabolism by Rat Adrenal Mito chondria In Vivo Effects of ACTH and Cycloheximide 124 on Levels of Endogenous Cholesterol and DOC-Bound P450 v LIST OF FIGURES Figure 1. 2 . 3. 4. 5. 6 . 7. 8 . 9. 10. 11. Mechanism of Cholesterol Side-Chain Cleavage (SCC) by Adrenal Cortex Synthesis of Adrenal Corticoids from Pregnenolone Summary of Changes in Glycolytic Inter mediates Two Minutes After ACTH Kinetics of Changes in Glycolytic Inter mediates Following 100 milliunits (mu) ACTH The Effect of Cycloheximide on the ACTH- Induced Crossovers The Effect of 100 milliunits (mu) ACTH on Cyclic AMP Levels The Effect of 100 milliunits (mu) ACTH on Rat Plasma Corticosterone Levels Titration of Deoxycorticosterone (DOC) on Rat Adrenal Mitochondria 20a-hydroxycholesterol-Induced Difference Spectra of Rat Adrenal Mitochondria and Bovine Adrenocortical Mitochondria and Submitochondrial Particles The Effect of Electron Donors on Rat Adrenal Mitochondria and Bovine Adreno cortical Mitochondria and Submitochon drial Particles The Effect of Pregnenolone on Rat Adrenal Mitochondria and Bovine Adrenocortical Mitochondria Page 3 4 68 71 73 75 77 81 84 86 89 vi Figure 12. Titration of Deoxycorticosterone (DOC) and Cholesterol on Rat Adrenal Mito chondria 13. Rate of Binding of Cholesterol to Rat Adrenal Side-Chain Cleavage (SCC) P450 14. 20a-hydroxycholesterol-Induced Difference Spectra of Rat Adrenal Mitochondria 15. Pregnenolone-Induced Difference Spectra of Rat Adrenal Mitochondria 16. The Effect of Electron Donors on Bovine Adrenocortical Mitochondria 17. The Effect of Cholesterol on Bovine Adreno cortical Submitochondrial Particles 18. The Effect of Isocitrate on Rat Adrenal Mitochondria 19. The Effect of 17a-hydroxyprogesterone on Bovine Adrenocortical Microsomes 20. The Effect of Pregnenolone on Bovine Adrenocortical Microsomes 21. The Effect of Various Agents on the Rate of Cholesterol Side-Chain Cleavage (SCC) 22. The Effect of Various Agents on the Rate of Deoxycorticosterone (DOC) llg-hydroxy- lation 23. Cholesterol Saturation Curve 24. 20a-hydroxycholesterol Saturation Curve 25. Deoxycorticosterone (DOC) Saturation Curve Page 91 93 95 97 99 100 102 105 107 109 112 113 114 115 vli Figure 26. 27. Cholesterol Side-Chain Cleavage (SCC) Progress Curves 20a-hydroxycholesterol Side-Chain Cleavage (SCC) and Deoxycorticosterone (DOC) 113-hydroxylation Progress Curves Page 116 117 viii CHAPTER I INTRODUCTION The endocrine or hormonal system is important in mammalian homeostasis since along with the nervous system it comprises the major control system by which all bodily functions are regulated. The adrenal glands, which lie at the superior poles of the kidneys, are a part of the endocrine system and are each composed of a medulla and a cortex. As far as is known, no direct functional relation ship exists between the medulla and the cortex. The adrenal medulla secretes the hormones, epinephrine and norepinephrine and is functionally related to the sympathetic nervous system while the adrenal cortex secretes mineralocorticoids, glucocorticoids, and small amounts of the androgenic hormones. The mineralocorticoids are so named because they are especially affect the electrolytes of the extracellular fluids--sodium, potassium, and chloride, in particular. The glucocorticoids have gained this name because they exhibit an important effect in increasing blood glucose concentrations. The glucocorticoids also produce altera tions in protein metabolism and in the tissue inflammatory 2 response. The androgens, such as dehydroisoandrosterone, appear to have no essential function in the adult. Although a number of different steroid hormones have been isolated from the adrenal cortex, only three are of major importance to the endocrine functions of the normal adult vertebrate organism--the mineralocorticoid, aldosterone, and the glucocorticoids, cortisol (the major excretory product in primates and fishes) and corticos terone (the predominant product in rodents). These steroid hormones are produced from cholesterol, which is either extracted from the plasma or synthesized from acetyl CoA (1,2). Pregnenolone, the major intermediate in the pathway of hormone synthesis, is derived from cholesterol in the mitochondrion. The metabolic pathways after pregnenolone include 33-ol dehydrogenation, A^-isomerization, and 21-hydroxylation (which occur in adrenocortical microsomes) and 113- and 18- hydroxylation (which occur in adrenocorti cal mitochondria). These metabolic pathways are shown in Figures 1 and 2. Glucocorticoid secretion by the adrenal cortex is under hormonal control by adrenocorticotropin hormone (ACTH). The tropic effects of this hormone on adrenal steroidogenesis will be the major consideration of this thesis. 3 ploim o .*■' choU iltrol (m ilochondnol) chcJattorol ‘ O H ! X-1 ^ HO-Y1 '-< i 2 0 a hydroiycliolattorol j A ] CIPNKOJ* J—J I 2 0 o , 2 2 dihydrcxycholtifm il i ?h 3 * r ' [ i p n h . o 2] pragnanolona + * bocoproic o ld th y d a Fig. 1.— Mechanism of cholesterol side-chain cleavage by adrenal cortex. 4 pra grunolona I I i h 2- c p « o A. "MICROSOMES" ,3£cJZ d a h rd ro g an o tai IT f 3 *"(17 M r a i r l m ) 17 hydfoiyprogw ffona tio m aro ia l C H j c*o aS6 ( 2 1 h y d ro x y fat^ pfOQ«»HfOO« o # I (IS hydroiylota) I (1 1 / hydroijrloia) { C H j O H O OH B. MITOCHONDRIA ?£r to rtk o u I C H 2 0 H O (11/K y d fo ijrlo ta ) t*0 '"“ as6 0— — DOC |d*o«ycert>ee»foo4 Q tk o tta w w a J. Q O s (IS hydroiylosa) Hr C l * 18 hydtoiycpflicotlw ont v j / ^ (dahydroganota) A ld o tttro n a Fig. 2.— Synthesis of adrenal corticoids from pregnenolone: major pathways. Literature Review Cholesterol Although it has been known for some time that cholesterol could serve as a precursor of adrenal hormones (3), Hechter, et al.'s study (4) showing that cortisol had a higher specific radioactivity than adrenal cholesterol 14 following acetate-C incorporation into the perfused bovine adrenal, raised the question of whether cholesterol was an obligatory intermediate. In response to Hechter's study, several investiga tors (5-7) suggested the existence of pathways other than those involving cholesterol, but Werbin and Chaikoff's (1) demonstration that the specific radioactivities of urine cortisol and adrenal cholesterol were equal after long-term feeding of cholesterol-4-C1^ to guinea pigs, reestablished cholesterol's role as an obligatory intermediate in the conversion of acetate to adrenocortical steroids. The Werbin and Chaikoff study was later confirmed by Krum, e_t al. (2), using dogs as the experimental animal. The disparity in labeling by acetate reported by Hechter, et al., and others (8,9) was explained by Ichii and Kobayashi (10) . These investigators showed that after 14 incubation of rat adrenals with acetate-l-C , the cholesterol radioactivity was higher in the mitochondria than in the mitochondrial supernatant, and that it was further increased after ACTH. Since the specific activity of corticosterone was still higher than that of mitochon drial cholesterol, it appeared that only a fraction of mitochondrial cholesterol was directly accessible for corticoid synthesis. The existence of a small, rapidly replenished pool of cholesterol which serves as the direct precursor of steroid hormones had previously been proposed by Hayano, et al.(11), and similar observations have been made following the incubation of rat corpora lutea with 14 acetate-l-C and luteinizing hormone (12) . While it is now generally accepted that corticos teroids are synthesized from a restricted pool of free cholesterol in the adrenal, the origin of this cholesterol, i.e., whether it originates from the plasma or is liberated from the hydrolysis of cytoplasmic cholesterol esters, its subcellular location in the adrenal, and how it is effected by ACTH, are still being investigated and will be discussed in a later section. Cholesterol Utilization The metabolic pathway from cholesterol to pregnenolone is shown in Figure 1. This step is of particular importance in corticosteroid biosynthesis because both ACTH and cyclic 3'5’-AMP have been shown to exert their principal effects here (13,14). The initial products of cholesterol side-chain cleavage (SCC) are pregnenolone and isocaproaldehyde, which is subsequently oxidized to isocaproic acid (15). Although the scission of the cholesterol side-chain is thought by some investi gators to involve hydroxylation at C-20 and C-22, the exact nature and sequence of formation of intermediates has yet to be established. 20a-hydroxycholesterol was first implicated as an intermediate by the in vivo trapping experiments of Solomon (16) . More direct confirmation was provided by Shimizu, et al. (17), who showed that 20a-hydroxycholes- terol could serve as a precursor of pregnenolone. An intermediate formed during the conversion of 20a-hydroxy- cholesterol to pregnenolone was later identified as 20a,22R-dihydroxycholesterol (18,19). Like 20a-hydroxy- cholesterol, 20a,22R-dihydroxycholesterol and 22R-hydroxy- cholesterol can also serve as precursors of pregnenolone (18,20). Attempts to isolate these intermediates have provided conflicting results. The isolation of 20a-hydroxycholesterol by Ichii, el: al. (21), could not be duplicated by Koritz, et al. (22). Using tritiated internal standards, crystalline 22R-hydroxycholesterol and 20a,22R-dihydroxycholesterol have been isolated by Dixon, et al. (23), who propose that these sterols are the naturally occurring constituents of adrenal tissue. A reasonable explanation of these conflicting results has been given by Simpson and Boyd (24). Based on the 8 competitive inhibition of cholesterol SCC by 20a-hydroxy- cholesterol, these authors have proposed that the 20a and 22R-hydroxylations may occur at the same enzyme site or the reaction may be concerted, 20a-hydroxycholesterol never being formed. If the hydroxylations at C-20 and C-22 are concerted, the roles of 20a-hydroxycholesterol, 22R- hydroxycholesterol, and 20,22R-dihydroxycholesterol as physiologic substrates becomes questionable. Corticosteroid Biosynthesis Once synthesized, pregnenolone is transformed to progesterone by two distinct enzymes, an NAD+-dependent 38-hydroxysteroid dehydrogenase and a 3-oxosteroidA^-A^- isomerase. Activity of these enzymes was first found in the microsomal fraction of ox adrenal homogenates (25), and until recently, this was thought to be their sole subcellular location. The presence of 38-hydroxysteroid dehydrogenase activity in the mitochondrial fraction of a rat adrenal homogenate was first reported by McCune, et^ ad. (26) . This finding was confirmed by Basch, £t al. (27), who showed that 31 per cent of the dehydrogenase activity was associated with the mitochondrial fraction of rat adrenal homogenates compared to 45 per cent in the microsomal fraction. Analyses of the two fractions revealed no significant differences in the apparent Km for 9 pregnenolone, dehydroepiandrosterone or NAD, suggesting that a common enzyme was involved. A similar bimodal dis tribution of both the dehydrogenase and isomerase enzyme activities has also been found in bovine adrenal cortex (28). These findings are important since the conversion of pregnenolone to progesterone in the mitochondria would eliminate the apparent necessity of pregnenolone transport out of the mitochondrion, a process thought by some investigators (29), to be rate-limiting in corticosteroid- ogenesis, and therefore to be facilitated by ACTH. The major pathways for the synthesis of gluco corticoids through progesterone are shown in Figure 2. This route was established by Hechter and Pincus (30) in some of the earliest work done on steroid biosynthesis. Products isolated in the effluent of bovine adrenal glands perfused with various steroids indicated a definite order of hydroxylations. Progesterone was converted to both 17-hydroxyprogesterone and 21-hydroxyprogesterone (11- deoxycorticosterone). 17-hydroxyprogesterone would undergo 21-hydroxylation, but 11-deoxycorticosterone was not readily 17-hydroxylated. The last step was llg-hydroxylation. Although more refined techniques have shown that this order is not absolute, that llg-hydroxy- progesterone, for instance, will undergo 21-hydroxylation to corticosterone (31,32), the low rates of formation of the alternative intermediates indicate that they play a minor role under normal conditions. 10 Steroid Hydroxylases As shown in Figures 1 and 2, most of the reactions in steroid biosynthesis are hydroxylations. These reac tions are a subgroup of biological oxidations first dis covered by Mason (33) in which atmospheric oxygen is introduced into the substrate instead of serving as the terminal acceptor of electrons transferred to it from substrate through carrier systems and cytochrome oxidase. The requirements for 110-hydroxylation have been determined. Hayano and Dorfman (34) demonstrated the requirement for an intermediate of the tricarboxylic acid cycle such as fumarate, succinate or malate. Grant (35) found that NADPH would serve as the electron donor and that electron donation by other substances occurred through NADPH formation. Molecular oxygen, which is incorporated 18 into the hydroxyl function, has been shown by O2 studies to be the other major requirement for the reaction (36). Studies with tritiated steroids (37) indicate that the reaction is a direct replacement of a hydrogen atom with a hydroxyl, the hydroxyl having the same steric position. The chemical identity of the "activated" oxygen species which binds to reduced P450 and is subsequently incorporated into the steroid nucleus is still uncertain. Perhydroxyl or hydroxy radicals (38), superoxide ions 11 (39,40), "oxenoid" species (41) and steroidal alkoxy radicals (42) have all been proposed as initial inter mediates in the cytochrome P450-catalyzed hydroxylation mechanism. In addition, hydroperoxide intermediates have been proposed in cholesterol SCC (43-46) and steroid 113-hydroxylation (47). The complete stoichiometry of all of the adrenal steroid hydroxylation reactions has not been determined but they are assumed to be similar to the 21-hydroxylation in adrenal microsomes (48) which is shown below: SH + NADPH + H+ + 02 = SOH + NADP+ + H20 In 1957, Ryan and Engel (49) demonstrated that the C21-hydroxylation of 17-hydroxyprogesterone by bovine adrenocortical microsomes was inhibited by carbon monoxide. Although their evidence was incomplete, they also believed that the inhibition was reversed by light. However, no carbon monoxide (CO)-combining pigment was detected spectrophotometrically in these preparations. The subsequent discovery (50,51) of a CO-combining pigment with a 450nm absorption maximum in liver microsomes led Estabrook, ejt al. (48) , to the identification of the CO- binding chromophore in bovine adrenal cortex microsomes. The identity of the chromophore as the 21-hydroxylase was based on photochemical action spectra in which the CO inhibition of 21-hydroxylation was reversed at greatest 12 efficiency with 450nm light. A subsequent characterization of the CO-combining pigment in liver microsomes by Omura and Sato (52) revealed that it was a hemoprotein with atypical b-type cytochrome properties, which were attributed by these investigators to the tight association of the protein with the microsomal membrane. Omura and Sato named the pigment cytochrome P450. Cytochrome P450 was discovered in bovine and rat adrenal cortex mitochondria by Harding, et al. (53). Light reversal studies have since proven that P450 catalyzes llg-hydroxylation of deoxycorticosterone (DOC) and llg- deoxycortisol (54), side-chain cleavage of cholesterol (24), and 18-hydroxylation of corticosterone (55). In addition to membrane-bound cytochrome P450, two soluble protein components are necessary for llg-hydroxy- lation (56-58) and for cholesterol side-chain cleavage (SCC) (59). One of these components is a non-heme iron containing protein, adrenodoxin (58) and the other is a flavoprotein, adrenodoxin reductase. Adrenodoxin and adrenodoxin reductase oxidize NADPH and reduce P450. The reaction sequence is thought to proceed from NADPH, through the flavoprotein to adrenodoxin and finally to P450. Adrenodoxin contains two gram atoms of iron and two moles of labile sulfur per mole of the protein and has a molecular weight of 12,500 with a single polypeptide chain 13 consisting of 114 amino acid residues (60). A recent report by Huang and Kimura (61) indicates that adrenodoxin accepts one reducing equivalent and has a midpoint potential of -0.27 volts at pH 7.0. The reduction of adrenodoxin and of similar electron transport iron-sulfur proteins causes a bleaching of the optical absorption spectra of these proteins con comitant with the appearance, at liquid nitrogen tempera tures, of a characteristic electron spin resonance (ESR) signal at or close to g = 1.94 (62-64). Both iron and sulfur contribute to this signal (65-67) which presumably reflects the valence, spin state, and environment of the iron in the protein. In spite of similar oxidation-reduction potentials, iron-sulfur chromophores, and amino acid sequences, adrenodoxin and another 2Fe - 2S protein, putidaredoxin, are nonexchangeable in the steroid and camphor hydroxyla- tions which they catalyze, indicating a possible specificity of interaction of the iron-sulfur proteins with the reductases and cytochrome P450. The molecular and catalytic properties of adreno doxin reductase, partially purified from bovine adrenal glands, have recently been studied (68). The flavoprotein contains one mole of FAD per mole of enzyme, two per cent (w/w) hexose, and has a molecular weight of approximately 54,000. The oxidation-reduction potential for the coupled 14 oxidized reductase-fully reduced reductase is -0.274 volts at pH 7.0. Adrenodoxin reductase was found to accept two electrons from NADPH. Since adrenodoxin is a one electron acceptor, it has been postulated that the flavoprotein may shuttle between the fully reduced form and the half reduced form as does NADPH-cytochrome C reductase (69) . An alternative possibility is that two moles of oxidized adrenodoxin accept two equivalents of electrons from the fully reduced reductase. The detailed oxidation-reduction mechanism of adrenodoxin reductase awaits further investigation. Like adrenodoxin, adrenodoxin reductase is very similar to, but non-exchangeable with, the flavoprotein from Pseudomonas putida, putidaredoxin reductase. The 1:1 molar complex produced between adrenodoxin and the reductase (70) makes it likely that the non-exchangeability in the catalytic activities among these components may arise from the specificity of the binding between the flavin and iron-sulfur components. The electron-transport sequence involved in P450- mediated hydroxylations in adrenocortical microsomes and mitochondria have several marked differences. The primary difference seems to be the complete lack of involvement of adrenodoxin or an immunochemically similar iron-sulfur protein in microsomal C-21 hydroxylation (71). The flavoproteins involved in these two electron transport 15 systems are also quite different. While the microsomal flavoprotein can apparently directly reduce cytochrome C and is assumed to be identical with NADPH-cytochrome C reductase (72,73), the mitochondrial flavoprotein cannot (74,75). In addition, the flavoprotein of adrenocortical microsomes exhibits partial immunochemical identity with the hepatic microsomal flavoprotein, while there is no immunochemical similarity between the mitochondrial flavoprotein (adrenodoxin reductase) and the flavoprotein isolated from hepatic microsomes (76). Whether there is direct electron transfer between the NADPH-specific flavoprotein and cytochrome P450 in adrenocortical microsomal hydroxylations, or whether an unknown component, immunochemically dissimilar to adrenodoxin is involved, is still under investigation. Whereas NADPH generated extramitochondrially or in the cytoplasm can be utilized for the C-21 and C-17 hydroxylations of progesterone in adrenal cortex micro somes, adrenocortical mitochondrial hydroxylations (choles terol SCC, C-ll, and C-18) are dependent on NADPH generated intramitochondrially. Extramitochondrial NADPH is impermeable to the mitochondrial membrane unless it is disrupted with high concentrations of calcium or ultrasound (77). Intramitochondrial NADPH is produced by the action of mitochondrial dehydrogenases (78,79) which transfer 16 hydrogen and electrons from Krebs Cycle intermediates in part via an energy controlled pyridine nucleotide trans- hydrogenase (80-82), and in part by the direct reduction of NADP by the malic enzyme in bovine adrenocortical mitochondria (77-83) and by a NADP-specific isocitrate dehydrogenase in rat adrenal mitochondria (84). The relative contribution of these various pathways to the production of intramitochondrial NADPH in vivo is largely a matter of conjecture since none of the studies cited have reproduced the cellular environment of the mito chondria or examined substrate flux in the intact cell. Absorption Properties of Cytochrome P450 All of the mammalian P450 enzymes thus far studied are closely associated with either mitochondrial or microsomal membranes. Since most attempts to solubilize these P450 enzymes result in their degradation to an enzymatically inactive form called P420 (52) , investigators have been dependent for the most part on spectrophotometric techniques as a means of studying their catalytic properties. Three areas of the visible region are of interest in studying hemoproteins. The largest absorption is called the Soret band and is located in the 390-420nm region. Two smaller absorptions, located in the 500-600nm range, are called the a and 8 bands. The porphyrin of the heme are 17 primarily responsible for these three absorptions (85). In addition to the a, 8, and soret bands, there may also occur two charge transfer bands, one at 600-650 nm and the other at 450-500nm. These charge transfer bands are so called because they arise in response to photon excitation in which an electron is transferred from a porphyrin nitrogen to the metal cation (86). Difference spectroscopy has been the major method used in studying changes in the above-mentioned absorption bands in turbid solutions of particulate cytochrome P450 preparations. This technique was also successfully used by Chance and Williams (87) to study the oxidation and reduction of the respiratory cytochromes. In this method the turbid preparation is in both the sample and reference cuvettes with a large end-on photomultiplier tube next to the cuvettes. Such an arrangement tends to cancel out effects of turbidity and of settling, and also permits greater efficiency in collecting the scattered light. The disadvantage of this method is that only changes in absorptions can be studied. The relationship of these changes to the absolute spectra must be inferred. In difference spectroscopy, absorptions represent differences between the spectra of chromophores in the sample and reference cuvettes. Peaks represent the appearance of a new pigment or an increase in the extinc tion of an existing pigment in the sample cuvette. 18 Alternatively, a peak can represent a decrease in extinction of a pigment in the reference cuvette. A trough is representative of an extinction decrease in the sample or an extinction increase in the reference. A peak associated with a trough can represent a shift of the maximum of a peak in either the sample or reference cuvette. Substrate interaction with cytochrome P450 was first reported by Narashimhulu, et_ al. (88) . These investigators found that successive additions of 17a- hydroxyprogesterone (a substrate for the microsomal 21-hydroxylase) to adrenal cortex microsomes produced a difference spectrum which was characterized by the instant and synchronous formation of a 420nm trough and an associated 390nm peak. The amount of 17a-hydroxypro- gesterone required to produce a half maximal difference spectrum was 2pM. Other substrates for the 21-hydroxylase produced identical spectral changes when added to the microsomal preparation. The ability of NADPH to abolish the steroid-induced spectral changes and of CO to inhibit this NADPH-supported disappearance, suggested to these investigators that the spectral changes were in some way related to steroid hydroxylation. If dithionite or NADPH in the absence of O 2 were added, the substrate difference spectrum was abolished. The spectrum could be regenerated by the re-addition of 02- 19 Based on these findings, Narasimhulu, el: al. concluded that steroids converted the reduced form of a pigment into a form which was readily oxidized. They therefore attrib uted the substrate-induced difference spectrum to an oxidized minus reduced spectrum of the pigment. It was later found that similar difference spectra were induced in adrenal cortex mitochondria by 11-deoxy- cortisol and deoxycorticosterone, substrates of 118- hydroxylation (89), and in the P450-catalyzed drug oxidation system of liver microsomes by substrates such as hexobarbital, phenobarbital, and amobarbital (90). The concentrations required for the difference spectra in liver microsomes were in the mM range (compared to PM range in adrenocortical mitochondria and microsomes) and were in close agreement with the Michaelis constants (Km) for these substrates. An additional type of spectral change was induced in liver microsomes by the substrate aniline. This difference spectrum had a 430nm peak and a 390nm trough. Again, the Ks and Km were in close agreement, 0.8mM and 0.7mM, respectively. Remmer, £t al.(90) , reasoned that the substrate effect in liver microsomes was due to an interaction with P450 since pretreatment of animals with drugs to induce synthesis of P450 also altered the substrate difference spectra. More convincing evidence for P450 participation as the chromophore group in the substrate-induced 20 difference spectra was obtained by Imai and Sato (91,92). These investigators found that the magnitude of the sub strate spectral change was proportional to P450 but not to cytochrome b,- concentration in a variety of liver microso mal preparations. Partially purified cytochrome bg, the only other heme protein in microsomes, showed no spectral interaction with drugs. The fact that ethyl isocyanide, a chemical which binds to cytochrome P450 competed with aniline and aminopyrine for spectral interaction and that the loss of substrate spectrum paralleled the conversion of P450 to the enzymatically inactive species, P420, were further proof that P450 was the pigment involved. Difference spectra produced in bovine adrenal cortex mitochondria were studied in more detail by Oldham, et al. (93). They found that all 113-hydroxylase sub strates induced troughs at 530 and 575nm in addition to the 420nm trough and 385nm peak previously reported, and that these spectral effects were intensified following the addition of malate to the sample and reference cuvettes. Non-substrates such as corticosterone, progesterone, 17a-hydroxyprogesterone, and cortisol did not interact spectrally when added to the mitochondrial preparations. An additional difference spectrum, produced by adding malate to the sample cuvette of a bovine adrenal mitochondrial preparation pre-treated with succinate, antimycin a and KCN (to cancel out the absorption 21 contributed by cytochrome b of the respiratory chain), was also reported. This spectrum with peaks at 420, 530, and 570 nm and a trough at 385nm was the reciprocal of the steroid-induced difference spectrum, and was thought by Oldham, et al., to represent the reduced minus oxidized difference spectrum of cytochrome P450. The ability of graded additions of CO to shift the 420nm peak induced by malate to 450nm proved that cytochrome P450 was the hemoprotein involved in this difference spectrum. Based on these data, Oldham, et^ al., concluded, as Narasimhulu, et al., had earlier, that substrates interacted with reduced cytochrome P450 to shift it to a more oxidized state. These conclusions were contradicted by Cammer and Estabrook (94) and Whysner and Harding (95) who demon strated by two different methods that substrate interaction 3+ occurred with P450 in the Fe oxidation state. Pauling (96) established that oxidized, Fe^+ hemoproteins can be divided into two main classes. The members of one class have spin moments approximately that of five unpaired electrons, spin 5/2 and magnetic moment 5.92 (Bohr magneton), whereas the members of the other class have magnetic moments approaching that of one unpaired electron, spin 1/2 and magnetic moment 1.73 B. Complexes in the first group are referred to as high spin complexes and those in the second group as low spin complexes. 22 In addition to magnetic susceptibility measure ments, unpaired electrons can also be detected by electron spin resonance (ESR) spectroscopy. High and low spin 3+ oxidized Fe hemoproteins have characteristic ESR spectra. The high spin (S = 5/2) hemoproteins have ESR signals at about g = 6.0 and 2.0, whereas the low spin (S = 1/2) hemoproteins have signals centered around g = 2 with three anisotropic components. Alkaline methemoglobin, for example, has ESR signals at g = 2.61, 2.19, and 1.82 (97). Hashimoto, et al. (98), were the first to demon strate the low spin P450 spectrum when they found ESR signals at g = 2.41, 2.25, and 1.91 in rabbit liver microsomes. Since the spectrum was shown not to be due to cytochrome b^, the other hemoprotein present in these particles, it was named Fe . Subsequent studies (99-101) indicated that the Fe spectrum was the ESR spectrum of A low spin liver microsomal P450. Low spin ESR signals associated with P450 in adrenal cortex mitochondria and microsomes were found by Cammer, £t aJ. (102) . In the adrenal mitochondria the signals were at g = 2.42 and 2.26; the third anisotropic component at g = 1.91 was obscured by the adrenodoxin signal at 1.94. In 1961, Brill and Williams (103) established that in addition to magnetic susceptibility and ESR measure ments, the spin state of the ferric iron of hemoproteins 23 could be determined by analyzing the absorption spectra of ferric porphyrin complexes. A change from low spin to high spin being characterized by the appearance of two new charge transfer absorption bands, one at about 600-650nm and a second at 450-500nm, a decrease in the ot and 3 absorption bands, and a shift in the soret absorption maximum to shorter wavelengths. A spin state change in the opposite direction, i.e., from high spin to low spin would produce opposite changes in the absorption spectrum. After analyzing the substrate-induced changes in the visible absorption of cytochrome P450 according to the Brill and Williams criteria, Whysner, el; al. (104) and Mitani and Horie (105) suggested that substrates changed the spin state of the ferric iron of P450 from low spin to high spin. Whysner, e£ al. (106), showed that two 113-hydroxylase substrates, 11-deoxycortisol and deoxy corticosterone (DOC) interacted with fully oxidized P450 in bovine adrenocortical submitochondrial particles to produce difference spectra characterized by peaks at 385, 500, and 645nm and troughs at 420, 535, and 570nm (Type I difference spectra). 20a-hydroxycholesterol, a substrate for the 22R-hydroxylase, produced opposite spectral effects (Type II difference spectrum). The changes produced by 11-deoxycortisol and DOC reflected a shift of the soret absorption to shorter wavelength (420nm to 385nm), a decrease in the a and 3 absorptions (535 and 570nm), and 24 an increase in the charge transfer absorption bands (500 to 645nm). According to the criteria of Brill and Williams, these changes indicated a low spin (S = 1/2) to high spin (S = 5/2) transition of the Fe^+ of P450. The opposite spectral changes produced by 20a-hydroxy- cholesterol, i.e., peaks at 420, 535, and 570nm and troughs at 385, 500, and 645nm (Type II difference spectrum), were indicative of a high spin to low spin shift in the ferric iron of P450. The ability of 11-deoxycortisol and DOC to decrease, and of 20a-hydroxycholesterol to increase the g = 2.42, 2.26, and 1.91 low spin signals of P450 were indirect confirmation of these spin state changes. A new low field ESR signal at g = 7.9, attributable to high spin P450 was also reported by Whysner, et al. (106) . Although atypical because of its position and low intensity, several lines of evidence, including a DOC- induced increase and a 20a-hydroxycholesterol-induced decrease in the height of the 7.9 signal, indicates that it represents high spin P450. Mitani and Horie (105) have described a similar low field signal. Absolute spectra and magnetic susceptibility measurements on a soluble P450 enzyme isolated from Pseudomonas putida in the presence and absence of substrate have since provided direct confirmation of these substrate- induced spin state changes (107). The possible importance of these substrate-induced 25 spin state changes to the reaction mechanism of P450 was first suggested by the work of Gigon, £t al. (108). These authors demonstrated that substances which cause the low to high spin change or which induce Type I spectra in liver microsomes enhance the rate of reduction of P450 and substances which effect the high spin to low spin spectral change (Type II difference spectrum) decrease its reduction rate. Similar studies have also been reported using bovine adrenocortical mitochondria (109,110). These reports and spectrophotometric studies which reveal that the substrate- induced difference spectrum persists under steady-state hydroxylation conditions (88,93) suggest that the slow step in the P450 reaction sequence is the reduction of sub strate-bound oxidized P450. The near identity of the concentrations of 11-deoxy cortisol and 20a-hydroxycholesterol required to produce half-maximal changes in the visible and ESR absorption and in the rates of llg- and 22R-hydroxylation reported by Whysner, et_ al. is consistent with the hypothesis that the substrate-induced low to high spin shifts in the Fe^+ of cytochrome P450 regulates the rate of hydroxylation by increasing the rate-limiting reduction of P450. However, their inability to demonstrate a cholesterol-induced low to high spin shift in their submitochondrial particles, and the fact that 20a-hydroxycholesterol, an hydroxy- latable substrate, produced high to low spin shifts in the 3+ Fe of P450, are inconsistent with this hypothesis. In addition, the ability of malate, a non-substrate, to in duce a type II difference spectrum or a high to low spin shift in bovine adrenocorticol mitochondria (93) appears also to be incompatible with such an hypothesis. Reasonable explanations for some of these incon sistencies were given by Whysner, et^ al. The inability of cholesterol to produce a type I difference spectrum (420nm troughs and 390nm peaks) or a low to high spin change was thought to reflect the high concentration of endogenous cholesterol-bound P450 present in their sub-mitochondrial particles. Mitani and Horie (113) has previously demonstrated that the substrate-induced difference spectrum of cholesterol is observed only when mitochondrial preparations are depleted of endogenous cholesterol. The 20a-hydroxycholesterol-induced type II spectrum (420nm peak and 390nm trough) or high to low spin change was thought to arise from the competitive displacement of cholesterol from its P450 enzyme, leaving the enzyme in the non-steroid bound or low spin state. Stimulation of Corticosteroidogenesis by ACTH After ACTH administration to hypophysectomized rats, corticosterone synthesis increases within 3 minutes, reaches a maximum in ten to fifteen minutes, and maintains this level of secretion for a period of time directly 27 related to the amount of ACTH injected (112). Although a number of possibilities have been proposed, the exact mechanism of this ACTH-mediated increase in cortico- steroidogenesis is still under investigation. A. NADPH Production The initial report of Sweat and Lipscomb in 1955 (113), and of many others since, that NADPH is required for steroid hydroxylation prompted a number of investi gations directed toward understanding the control of steroid biosynthesis in terms of the control of NADPH generation. One of the earliest and most accepted of these studies was that of Haynes and Berthet (114). These investigators demonstrated an ACTH-stimulated increase in phosphorylase activity in bovine adrenal slices. When a NADPH-generating system or glycogen plus a preparation of liver phosphory lase was substituted for ACTH in the incubation medium, corticosteroid production was stimulated. Activation of phosphorylase therefore appeared to be an initial response of the adrenal gland in the sequence of metabolic con versions of glycogen to glucose-6-phosphate oxidation. Haynes (115) later demonstrated that ACTH induced the accumulation of cyclic AMP in the adrenal cortex. Finally, Haynes, Koritz, and Peron (116) reported that cyclic AMP could induce corticoid production in bisected 28 rat adrenal glands and that the response elicited was equal to or greater than that produced by ACTH. Since a cyclic AMP-induced activation of liver and heart phosphorylase had been demonstrated earlier (117), Haynes and Berthet concluded that ACTH, via cyclic AMP, activated phos phorylase which in turn caused the production of glucose- 6-phosphate. The aerobic metabolism of G-6-P via the hexose monophosphate shunt resulting, finally, in the pro duction of NADPH which stimulated an increase in cortico steroid production. Aside from the ACTH-induced increase in cyclic AMP production, a number of findings since the formulation of this hypothesis suggest that it is incorrect. The studies of Koritz and Peron (118), Peron and Koritz (119), and Peron (120), which suggest that ACTH may increase the biosynthesis of adrenal corticoids as a result of mech anisms other that phosphorylase activation, the inability of Kobayashi, et al. (121) to demonstrate an ACTH-mediated stimulation of phosphorylase activity in rat adrenal glands incubated iji vitro, the report by Koritz and Peron (124) that cyclic AMP did not increase phosphorylase activity of fresh or frozen adrenal homogenates although increased steroid output was obtained, and the in vivo study by Yago et^ al. , (123) in which phosphorylase activity was actually decreased in adrenal glands of animals after ACTH injection, are all difficult to reconcile with the 29 proposal that activation of phosphorylase is obligatory in the sequence preceeding the proposed rate-limiting step (NADPH production). The limitation of adrenal corticosteroidogenesis by the rate of NADPH production has also been advocated by McKerns. He proposes that ACTH acts primarily by acti vating glucose-6-phosphate dehydrogenase (124). McKerns suggests that ACTH may bind to glucose-6-phosphate de hydrogenase either by hydrogen bonding or by electro static attraction to induce a conformational change in its structure. The new enzyme conformation would then increase the efficiency of hydrogen transfer from glucose- 6-phosphate to NADP (125). The inability of several investigators to demonstrate an increase in glucose-6- phosphate dehydrogenase activity after ACTH stimulation in vivo (126,127) or in vitro (114,128), makes the signi ficance of McKerns' data questionable. In addition to the criticisms cited above, a number of other findings suggest that the hypothesis that cyto plasmic NADPH is rate-limiting in corticosteroidogenesis may be incorrect. Harding and Nelson (129) showed that the pyridine nucleotide concentrations of adrenocortical cells in the resting rat were very high and that although adrenal steroid secretion fell precipitously within thirty minutes of hypophysectomy, no change in adrenal NADPH or NADP was observed for seventy-two hours. The activities 30 of several NADPH-generating enzymes were also not signi ficantly diminished within this period (130). In addition, Peron and McCarthy (131) found the same patterns of pyridine nucleotide content in cell fractions of normal animals, hypophysectomized animals, and animals treated with ACTH. Their data indicate no significant loss or accumulation of cytoplasmic or intramitochondrial NADPH, and also suggest that coenzyme concentration is not crit ical to corticosteroid hydroxylations. The high rate of steroid hydroxylation supported by NADPH generated intramitochondrially (77), and the in ability of extramitochondrial NADPH to permeate the mito chondria where the rate-limiting hydroxylations are postulated to occur (13), also argue against an ACTH- mediated increase in cytoplasmic NADPH as a mechanism for controlling the production of corticoids in vivo. B. Cholesterol Side-Chain Cleavage In a study in which bovine adrenal glands were per fused with radioactive cholesterol,progesterone,and acetate Stone and Hechter (13) found that ACTH increased the con version of cholesterol-^C into products (corticosterone and hydrocortisone) by 1800% compared to 17% and 140% for 14 progesterone and acetate- C, respectively. Although these results can also be explained by differences in the endo genous pool sizes of the three substrates, it was con cluded that ACTH stimulated the conversion of cholesterol 31 but not of acetate or progesterone, to corticosteroids. On the basis of an earlier kinetic study (132), the site of ACTH stimulation was further restricted to between cholesterol and pregnenolone. The Stone and Hechter hypothesis is generally accept ed, but the specific mechanism by which ACTH stimulates the rate-limiting conversion of cholesterol to pregnenolone is still very much in question. C. End-Product Inhibition by Pregnenolone A qualitative relationship was observed by Hirschfield and Koritz (133) between substances like calcium, fatty acids, and sodium lauryl sulfate, which cause swelling of adrenal mitochondria and which stimulate pregnenolone synthesis in this organelle. Both the swelling phenomenon and the stimulation of pregnenolone synthesis were in hibited by ATP. Since the relationship was not quantita tive, e.g., oleate produced a good stimulation of preg nenolone synthesis but caused little mitochondrial swell ing, it was concluded that mitochondrial swelling by it self is not the critical factor in the stimulation of pregnenolone synthesis, but rather, a modification of the mitochondrial membrane, which may have swelling as one of its consequences, was considered of prime importance. In a later study(134) the administration of ACTH to both normal and hypophysectomized rats resulted in a 30% increase in mitochondrial pregnenolone synthesis. This increase in pregnenolone synthesis could be eliminated by the addition of ATP to the incubation medium. Based on their previous observation that ATP inhibits adrenal mitochondrial swelling and the associated increase in pregnenolone formation, it was suggested that ACTH, via cyclic AMP, may control steroidogenesis by affecting the permeability of the mitochondrial membrane. The ACTH-induced change in the permeability properties of the mitochondrial membrane was considered to affect pregnenolone synthesis in two possible ways. The entry of exogenous NADPH, normally impermeable to the mito chondrial membrane could be facilitated, or alternatively, the transport of pregnenolone, an inhibitor of cholesterol SCC (135) out of the mitochondria could be increased. The first alternative, or an increase in pregnenolone syn thesis due to the increase of available NADPH was ruled out by several experimental observations, and it was con cluded that the ACTH stimulation of pregnenolone synthesis was due to the increased rate of removal of this steroid from the mitochondria. Although Koritz and Kumar (136) have recently reported additional findings in support of this hypothesis, the biomodal distribution of 3$-hydroxysteroid dehydrogenase and 3-oxosteroid A1 *-A5 isomerase (the enzymes which convert pregnenolone to progesterone) in adrenocortical mito chondria and microsomes (25,26) would appear to eliminate 33 the necessity of pregnenolone egress out of the mito chondria and to make its role as a physiological in hibitor of cholesterol SCC questionable. While the Koritz hypothesis that ACTH controls steroidogenesis by determining the rate of efflux of mito chondrial pregnenolone appears not be feasible, the ACTH- stimulated increase in pregnenolone synthesis from endo genous adrenal cholesterol is an important observation and will be discussed more fully in another section of this thesis. D. Protein Synthesis Ferguson's report (137) that puromycin (138) pre vented the steroidogenic response of rat adrenal quarters to ACTH prompted the current hypothesis that protein synthesis may be an essential component of ACTH respon siveness. In this study puromycin was also shown to in hibit steroidogenesis activated by cyclic AMP, but had no effect on the increase in steroidogenesis produced by NADPH. This latter observation was taken as evidence that puromycin was not directly inhibiting the catalytic function of enzymes concerned in steroidogenesis. The basal production of steroids occurring in the absence of ACTH or cyclic AMP was also not affected by puromycin. In an attempt to establish a causal relationship between protein synthesis and ACTH responsiveness, Ferguson (139) later showed that 1) both in vitro processes were 34 inhibited by identical concentrations of puromycin, 2) structural analogues of puromycin, known to have no effect on protein synthesis, also had no inhibitory effect on ACTH responsiveness, and 3) both the puromycin-induced inhibition of amino acid incorporation into adrenal protein and the puromycin-inhibited responsiveness to ACTH were re versible— both processes following the same time course when the antibiotic was removed from the medium. Chloramphenicol (140) and cycloheximide (141) , two antibiotics structurally unrelated to puromycin were also shown to inhibit amino acid incorporation into adrenal protein and the ACTH-induced increase in corticoid pro duction (142) . In answer to the argument that the rapid response of steroidogenesis to the administration of ACTH and the rapid decline in steroid hormone secretion which follows its removal from the circulation could not be explained by a process which depended on the synthesis of new pro tein, Garren et al., (143) investigated this phenomenon in vivo by measuring corticosterone secretion directly from the adrenal vein of rats. It was shown that the administration of either puromycin or cycloheximide in concentrations (30 and 10 milligrams, respectively) that blocked adrenal protein synthesis markedly inhibited the increased rate of corticosterone secretion which immedi ately follows intravenous injection of ACTH (112). 35 It was further reasoned by these investigators that the hypothetical protein would have to demonstrate an extremely rapid turnover in order to regulate the rapid onset and decline in steroidogenesis which follows ad ministration and removal of ACTH. Contraiwise, if a long- lived protein was synthesized in response to ACTH, then blocking the synthesis of the protein after ACTH had already stimulated steroidogenesis would have no effect on the continued secretion of corticosterone. This hypothesis was tested by the injecting cycloheximide into hypo- physectomized rats after maximum rates of steroidogenesis were induced by the administration of 50 milliunits of ACTH. The results showed a rapid fall-off in the rate of steroidogenesis with a rapid return to baseline despite the fact that the ACTH administered was sufficient to maintain maximum levels of steroid hormone for more than 60 minutes. These data were interpreted to mean that ACTH stimu lates steroidogenesis by initiating the synthesis of a protein with a rapid turnover rate, the level of which determines the rate of steroidogenesis. ACTH was there fore thought to be required for its continued synthesis and presence. The protein appeared to be synthesized from a stable messenger RNA since the injection of actino- mycin D into hypophysectomized rats in amounts that in hibited RNA synthesis for as long as 8 hours had no effect 36 on the stimulation of steroidogenesis by ACTH. Similar in vitro results with actinomycin D had been reported earlier (144). Several observations indicate that the site of cyclo heximide inhibition of steroidogenesis is between choles terol and pregnenolone. Inhibition at a step prior to the formation of cholesterol seems unlikely since in jection of cycloheximide into hypophysectomized rats, in amounts that blocked both adrenal protein synthesis and ACTH-stimulated steroidogenesis did not interfere with the incorporation of H-acetate into adrenal cholesterol (145). The normal production of corticosterone from pregnenolone, progesterone, and deoxycorticosterone despite the presence of inhibitory concentrations of cycloheximide in the media suggest that the pathway after pregnenolone is not dependent on protein synthesis (145). These findings, plus the demonstration that previous ad ministration of cycloheximide to rats prevents the ACTH- induced depletion of adrenal cholesterol (148) are in ferential proof that cycloheximide prevents ACTH action by blocking the conversion of cholesterol to pregnenolone, the currently accepted rate-limiting step in the pathway of steroid hormone production. Based on the observation that cycloheximide (at a concentration which inhibits ACTH-stimulated steroido genesis) does not interfere with the ACTH-activated 37 hydrolysis of cholesterol esters located in the soluble cytoplasm of the adrenal (146), Garren (14 7) concludes that the hypothetical protein synthesized by ACTH via cyclic AMP facilitates the interaction of extramitochondrial cholesterol with the intramitochondrial cholesterol SCC enzyme system by some mechanism yet to be determined. In spite of the fact that 1) the hypothetical labile protein synthesized in response to ACTH has not been found, 2) there is as yet no evidence which indicates that ACTH increases the incorporation of amino acids into adrenal protein in a time sequence compatible with the rapid onset of ACTH-stimulated steroidogenesis, and 3) the antibiotics used in these studies have been shown to produce many other effects in addition to inhibiting protein synthesis (148-152), the hypothesis that protein synthesis is intimately connected with the ACTH-induced increase in corticosteroidogenesis is difficult to refute and is still very much accepted. E. Cyclic AMP The cyclic nucleotide 315’-AMP has been strongly im plicated as the intracellular intermediary in the readout of a number of peptide hormones and biogenic amines, including: glucagon, ACTH, vasopressin, thyroid-stimulating hormone, luteinizing hormone, parathormone, catechol amines, and serotonin (153-156). In characteristic target tissues and cells, these signals activate a membrane- 38 associated enzyme, adenyl cyclase, that converts ATP to 3'5*-AMP. The generated cyclic nucleotide in turn initi ates a sequential set of reactions (157) which differ in various specialized cell types in each case leading to a specific cellular response characteristic of hormonal stimulation. It was first shown by Haynes (115) that the addition of ACTH to slices of bovine adrenal cortex caused an in crease in the content of 3'5*-AMP. A later demonstration that cyclic AMP stimulated steroidogenesis in adrenocortical tissue (116) led to the proposal that cyclic AMP acts as the intracellular mediator of ACTH to stimulate cortico steroidogenesis . The relationship between ACTH-mediated adrenal cyclic AMP accumulation and increased rates of steroidogenesis was studied more extensively by Grahame-Smith et al. (158). These investigators found a six-fold increase in cyclic AMP one minute after the addition of ACTH to bisected rat adrenal glands. The rise in cyclic concentration clearly preceeded the increased rate of corticosterone biosynthesis which did not rise perceptibly until two minutes after ACTH. The similar time course required for different doses of ACTH to increase both adrenal cyclic accumulation and corticosterone synthsis in vitro, as well as the equipo- tency displayediby certain ACTH analogues on these two 39 processes, were also supportive of the idea that the ACTH- induced increases in corticoid biosynthesis are mediated through cyclic AMP. Since cycloheximide inhibited the ACTH-induced stimulation of corticosterone production but failed to alter the rise in cyclic in their studies, Grahame-Smith et al., concluded that cyclic AMP accumulation in response to ACTH is not dependent on protein synthesis and must occur prior to the synthesis of the labile protein postulated by Garren £t al., (143) to be involved in the steroido genic effect of ACTH. The increased adrenal cyclic concentration results from an increase in synthesis of the nucleotide brought about by an ACTH-induced stimulation of adrenal adenyl cyclase rather than from a decrease in its destruction by phosphodiesterase (158,159). In contrast to the enzyme in rat adipose tissue, adrenal adenyl cyclase shows high specificity for ACTH; other peptide hormones such as insulin, glucagon, and thyrotropin are not effective (149). The intracellular location of adrenal adenyl cyclase is a matter of controversy. Hechter et al., (160) reported a high adenyl cyclase activity in adrenal cortex mitochon dria. In confirmation of these data, Schimmer (161) also found the adenyl cyclase activity of adrenocortical tumor cells to be enriched in the 11,000 x g or mitochondrial 40 fraction. Another group of workers (162) prepared a fraction from bovine adrenal cortex enriched in plasma membranes which binds ACTH, and fragments and analogs of the hormone and which also contains an ACTH-sensitive adenyl cyclase. Although radioactive ACTH did bind to mitochondrial fractions in their study, this was thought to be due to nonspecific binding or to the contamination of these fractions with plasma membranes, since the adenyl cyclase activity was highest in the lightest fractions Cplasma membranes) and was virtually absent from fractions that had the greatest concentration of mitochondria. The subcellular distribution of adenyl cyclase in the adrenal cortex was investigated in greater detail by Satre, et al (163). These investigators found that all crude particulate fractions separated from a bovine adrenal cortex homogenate contained significant adenyl cyclase activity, with the main portion of the total activity recoverable in the crude mitochondrial fraction. However, upon further purification of the mitochondrial microsomal fractions (purification was judged by the increase in specific activity of marker enzymes), the adenyl cyclase activity was nearly ten times lower in the mitochondria than in the microsomes. These results strongly argue against a mitochondrial localization of adenyl cyclase in the adrenal cortex. Prior to activating adenyl cyclase, ACTH is first 41 bound to receptor molecules which are presumably located on the plasma membrane (164,165). In contrast to other lipolytic hormones such as adrenaline and glucagon, trace amounts of calcium are required for the ACTH stimulation of adenyl cyclase (166). By studying separately hormone binding and hormone-induced adenyl cyclase activation in subcellular membrane particles isolated from adrenal tumors, Lefkowitz, £t al., (167) established that calcium is re quired at a step or steps between these two processes. While the binding of ACTH to its adrenal receptor was in tact in the absence of calcium, the ACTH activation of adenyl cyclase was totally inhibited. Once cyclic AMP has been synthesized via the ACTH- induced activation of adenyl cyclase, several different mechanisms have been proposed to account for the increase in adrenal corticosteroids which results. Roberts £t al^., (168) postulate that cyclic AMP en hances steroidogenesis by stimulating directly an early rate-limiting step in cholesterol utilization in adreno cortical mitochondria. This hypothesis is based on experiments in which 50 pM cyclic AMP stimulated the utilization of exogenous cholesterol (20-100 yM) by NADPH permeable, cholesterol-depleted "leaky" mitochondria pre pared by repeated washing in 0.25 M sucrose - 0.8 mM Tris- HC1 (pH 7.1) followed by incubation at 37°C in a buffer composed of 24 mM NaHCO^ and 130 mM KC1. One millimolar 42 (1 mM) cyclic AMP also stimulated cholesterol utilization in coupled mitochondria (using succinate as electron donor). The primary effect of cyclic AMP in both "leaky" and coupled mitochondria was to increase the rate of formation of pregnenolone from the exogenous cholesterol. The conversion of pregnenolone to progesterone was not activated. Neither 5'-AMP, 5'-ADP, or ATP were effective in increasing the utilization of cholesterol by the mito chondrial preparations. These results have not been con firmed by other investigators. Akhtar ejt al., (169) also propose that cyclic AMP may increase steroidogenesis by acting at the mitochondrial level. These investigators observed that cyclic AMP causes an inhibition of the rotenone insensitive oxidation of NADH in isolated bovine adrenocortical mitochondria. Consistent with this observation, they propose that the in creased cellular level of NADH (resulting from the cyclic AMP-induced inhibition of NADH oxidation) is used to gen erate intramitochondrial NADPH (via the concerted actions of the mitochondrial NAD-linked malate dehydrogenase and the NADP-linked malic enzyme) which in turn mediates an increase in corticoid production by providing reducing equivalents to the hydroxylating pathway. The most accepted of the mechanisms proposed for cyclic AMP activation of adrenal steroidogenesis is 43 the one advocated by Gill and Garren. In a series of re ports (170-173), these investigators described the isola tion, from bovine adrenal cortex, of a cyclic AMP-dependent protein phosphokinase which is located in both the cytosol and endoplasmic reticulum. A specific receptor for cyclic AMP was identified in the cytosol of the adrenal cell (171) and was later demonstrated to be associated with a protein kinase in a regulatory complex (172). The cyclic AMP receptor inhibits the activity of the protein phos phokinase when complexed to the enzyme; cyclic AMP activ ates the enzyme by binding to the receptor, causing it to dissociate from the enzyme moiety. The receptor and kinase also exist in a regulatory complex in the adrenal endoplasmic reticulum (173). Ex tensively purified adrenal cortical ribosomes, without associated protein kinase activity, were prepared as substrate for the cyclic AMP-dependent protein kinase partially purified from the adrenal cortex cytosol. The phosphorylation of ribosomes was shown to be dependent upon both cyclic AMP and protein kinase. Phosphorylation of the ribosomes without the addition of the protein kinase was minimal and was not stimulated by cyclic AMP. In another set of experiments it was shown that the substrate phosphorylated by the protein kinase is a pro tein associated with the 80S ribosome. The serine and thereonine residues of the protein were phosphorylated 44 as has been observed with other substrates of this enzymic : reaction (174). Although the function of the ribosome- associated protein was not defined by thees studies, the removal from the ribosomes of the substrate of the cyclic AMP-dependent protein kinase reaction by concentrated KC1 was thought to indicate that the ribosome-associated pro tein is relatively specific for the enzymic reaction. This demonstration that the adrenal cell contains a cyclic AMP-dependent protein kinase that phosphorylates protein tightly associated with ribosomes supports the eart ier suggestion by this group of workers (143), that ACTH, acting through cyclic AMP, regulates adrenal function by modulating protein synthesis at the level of translation of mRNA. CHAPTER II STATEMENT OF PROBLEM The experiments of Stone and Hechter (13) which indicate that the conversion of cholesterol to pregnenolone (cholesterol SCC) is rate-limiting in adrenal cortico- steroidogenesis has prompted a great deal of experiment ation directed toward understanding at which point this reaction is hormonally stimulated. Although a number of attractive hypothesis have been proposed, investigators in this area are still in pursuit of more definitive answers. The discovery of cytochrome P450, and the subsequent elucidation of the electron transport pathway involved in adrenal steroidogenesis, has provided new insight into the mechanism of steroid bydroxylation reactions in general and consequently of the cholesterol SCC enzyme system. In adrenal mitochondrial steroid hydroxylations, of which cholesterol SCC is an example, electrons from intra- mitochondrially generated NADPH are transferred to sub strate-bound P450 which subsequently binds oxygen,activates it, and incorporates it into the steroid nucleus. The reduction of substrate-baund P450 appears to be rate- limiting in this reaction sequence (108,110,88,93). Based 45 46 on these facts, it seems logical to speculate that the rate of adrenal corticosteroidogenesis, and therefore of cholesterol SCC, may be limited in vivo by the rate of generation of intramitochondrial NADPH, by the formation of cholesterol-bound P450, or by both of these processes. If ACTH activates steroidogenesis by increasing the rate of generation of intramitochondrial NADPH, this might be detected by measuring the steady-state concentrations of the Kreb cycle substrates and the oxidized and reduced pyridine nucleotides in adrenal glands removed before and after the administration of ACTH to hypophysectomized animals. Since one of the pathways of intramitochondrial NADPH production is via an energy-dependent transhydro- genase reaction, ACTH stimulation may also be expected to affect the entire energy metabolism of the adrenal cell. This perturbation could possibly be detected by examining the concentrations of glycolytic substrates before and after ACTH stimulation. To determine whether ACTH stimulates the transport of cholesterol to the SCC P450 enzyme requires a technique which specifically detects cholesterol-bound P450. For this reason, chemical procedures and gas chromatography which measure total mitochondrial cholesterol concen trations are not usable. The type II difference spectrum produced by the interaction of 20a-hydroxycholesterol with cholesterol-bound P450 (112) seemed to provide a suitable 4 7 I } technique. To determine whether ACTH activates the cholesterol SCC enzyme system per se, entailed measuring the rates of cholesterol SCC in mitochondria isolated from hypophy- sectomized rats and from hypophysectomized rats treated with ACTH. To do this, a cholesterol solution soluble in aqueous solution and capable of binding to cytochrome P450 had to be prepared. Some of these results have been published (177-179). CHAPTER III MATERIALS AND METHODS Measurement of Steady-State Concentrations of Adrenal Pyridine and Adenine Nucleotides, and the Substrates of Glycolysis and the Krebs Cycle A. Preparation of Perchloric Acid Adrenal Extracts Female Albino rats weighing approximately 150-200 grams were hypophysectomized by the transaural approach. Four hours post-hypophysectomy, one adrenal gland (which served as a control) was collected under ether anesthesia and immediately immersed into liquid nitrogen. Following the injection of ACTH for a prescribed time period, the remaining adrenal gland was collected into liquid nitrogen All subsequent manipulations were done essentially accord ing to the procedures described by Williamson (178). The frozen control glands from 25-30 rats were pooled and powdered in a mortar and pestle immersed in liquid nitrogen. The frozen powder was deproteinized by homo genization in 5% cold perchloric acid, with care taken to avoid thawing of the powder during acidification. Aliquots of the homogenate were removed for protein determinations before centrifugihg at 15,000 x g for 15 minutes. The same procedure was followed for the ACTH 48 49 stimulated glands. Measured volumes of the supernatants were neutralized to pH 6.0 with 3N J^CO^, and the pre- cipated potassium perchlorate was removed by centrifugation in the cold. The extracts were kept frozen between analyses, and the least stable intermediates, such as pyruvate, phos- phoenolpyruvate, oxalacetate, and NADP, were analyzed in fresh extracts. Analysis of extracts for hexose mono- and diphosphates, dihydroxyacetone phosphate and ATP on successive days showed that these intermediates were stable to degradation at least over a 4-day period. B. Preparation of Alkaline Adrenal Extracts Aliquots of the frozen adrenal gland powders obtained as described above were mixed with 1 ml of 1.5 N ethanolic KOH (equal volumes of water and ethanol) while still cold, and heated with mixing for 60 seconds at 55°C. The clear digest was cooled, and 1 ml of cold 0.5 M triethanolamine hydrochloride, pH 6.5, was added slowly with mixing. The extracts were carefully neutralized to pH 8.0 with 2 N HC1 during vigorous mixing and centrifuged at. 20,000 x g for 20 minutes in the cold. These extracts were used for the assay of NADH and NADPH. C. Analytical Methods The metabolic intermediates were all measured enzy matically by coupling the reactions to appropriate enzymes involving the oxidation or reduction of di- or triphos- 50 phopyridine nucleotides. An Eppendorf fluorometer, equipped with a primary filter permitting the transmission of only the 366 nm mercury vapor line as the source of activating light, was used for all of the measurements. A Varian G-14 recorder, with a 1-mAMP input for full scale deflection, was used to record the fluorescence changes. The pyridine and adenine nucleotides and the glyco lytic and Krebs cycle substrates in the adrenal extracts were analyzed by modifications of methods described in the literature (179-182). Citrate was determined with aconitase (180) , which was purified up to the first ammonium sulfate fractionation step by the method of Morrison (183). A 0.1 M triethanol- amine buffer, pH 7.4, was used for the assay of NADP, NADH, NADPH, glucose-6-phosphate, fructose-6-phosphate, fructose- 1, 6-diphosphate, and triose-phosphate. NADH and NADPH were measured in the same cuvette, which contained 0.1 mM pyruvate, 0.1 mM a-ketoglutarate, and 0.1 mM ammonium chloride as substrates, by the addition of lactic dehydro genase followed by glutamic dehydrogenase when the reaction utilizing NADH had reached completion. A buffer contain ing 50 mM triethanolamine buffer, 10 mM MgC^, and 5 mM EDTA (pH 7.0), was used for the determination of ADP and AMP. ATP in the adrenal extract was determined with hexokinase and glucose-6-phosphate dehydrogenase (180). An Alkaline buffer of 0.2 M glycine and 0.4 M hydrazine 51 hydrate, pH 9.5, was used for the assay of NAD and malate. 3-phosphoglycerate was measured by means of reactions coupled to glyceraldehyde-3-phosphate dehydrogenase and 2- phosphoglycerate by reactions coupled to lactic dehydro genase. NAD and NADP were made up in water and added directly to the cuvettes immediately prior to the assay. NADH, made up in 0.1 M triethanolamine buffer, pH 8.0, was used similarly. Substrates were stored frozen for short periods as stock solutions and were added directly to the cuvette. The recovery of known amounts of ATP, glucose- 6-phosphate, fructose-1,6-diphosphate, NAD, and NADPH added to extracts ranged from 94 to 100%. Cyclic AMP Measurements One adrenal gland (which served as the control) was collected from 4 hour hypophysectomized female albino rats (200 gm body weight) under ether anesthesia and immediately immersed into liquid nitrogen. Following the infusion of 100 milliunits ACTH for a prescribed time period the other adrenal was collected into liquid nitro gen. The cyclic AMP content of each control and stimu lated gland was measured using the radioisotopic displace ment method (184). Corticosterone Measurements The effect of 100 milliunits ACTH on corticosterone production by rat adrenals in vivo was studied using the 52 method of Lipscomp and Nelson (112) with certain modifi cations. Female rats weighing approximately 200 gm were hypophysectomized by the transaural approach. Four hours post-hypophysectomy heparin was injected into the right femoral vein of the anesthesized rats and a needle was threaded through the left adrenal vein almost to its entrance. Baseline blood samples were collected from the adrenal venous flow at 1 minute intervals for 3 minutes. ACTH was then injected into the left femoral vein and 1 minute blood samples were collected for 10 minutes following ACTH injection. Corticosterone in the plasma samples were extracted with chloroform and measured according to the method of Silber e_t £il. (185) . Preparation of Mitochondria A. Bovine Adrenocortical Mitochondria Bovine adrenal glands were collected from steers atthe Pride Meat Packing Company of Vernon, California, within 30 minutes of their death, and stored in crushed ice. Follow ing transport of the glands to the laboratory, all sub sequent manipulations were carried out at 4-6°C. The capsule and medulla were removed from the defatted glands by dissection, and the remaining cortical tissue was minc ed and placed in ten volumes of 0.25 M sucrose - 1% albumin (pH 7.2). The cortical tissue was then brought into solu tion using a minimum of passes in a loose-fitting, re ground tenbrock homogenizer, followed by an additional pass 53 in a standard tenbrock homogenizer. The resulting homo- genate was centrifuged at 900 x g for 10 minutes to remove the whole cells and connective tissue. Mitochondria were isolated from the supernatant by centrifugation at 8,700 x g for 10 minutes. The mitochondria were washed by re- suspending the 8,700 x g pellet in 3 volumes of sucrose- albumin and resedimenting at 8,700 x g. The washed mito chondrial pellet was resuspended in 0.25 M sucrose-1% albumin. B. Bovine Adrenocortical Submitochondrial Particles Submitochondrial particles were prepared according to the procedure of Kielly and Bronk (186). Mitochondrial pellets, prepared as described above, were resuspended in 0.03 M phosphate, pH 7.2, and subjected to sonic oscilla tion for 40 seconds using the probe of a Branson Model S-75 sonifier operating at 20Kc/min. Particles from the sonified preparation were collected by sedimenting at 105,000 x g for 30 minutes, washed once, and resedimented at the same forces. The final sediment was resuspended in 0.25 M sucrose-1% albumin. C. Rat Adrenal Mitochondria Adrenal glands were removed from 10-30 ether-anesthe- sized, laporotomized 200 gm female rats and placed immedi ately into ice-cold sucrose (0.25 M)-albumin (1%). All subsequent procedures were done at 4-6°C.The pooled adrenal glands were defatted, placed in 10 volumes of sucrose- 54 i albumin, and brought into solution with minimum passes in a glass homogenizer. Once washed mitochondria were obtained as described for the preparation of bovine adrenocortical mitochondria. Preparation of Microsomes Bovine adrenocortical and rat adrenal microsomes were prepared by sedimenting the supernatants obtained from the respective mitochondrial pellets at 105,000 x g for 60 minutes. The resulting sediments were washed once by re- suspending in sucrose-albumin and resedimenting at the same force for 60 minutes. Preparation of Cholesterol-Lecithin Emulsions Cholesterol and 20a-hydroxycholesterol-lecithin emul sions were prepared essentially according to the procedure of Hoyes and Saunders (187). One half milliliter (0.5 ml) of a 10% egg"lecithin"hexane solution was placed in a 10 ml beaker and brought to dryness under a stream of dry nitrogen. A 1% solution was prepared by dissolving the dried egg lecithin in 5 mis of water. The 1% egg lecithin solution was then added to 30 mg of cholesterol dissolved in a minimum of petroleum ether and magnetically stirred. Ether was removed from the lecithin-cholesterol mixture by bubbling with dry nitrogen. The ether-free mixture was then brought into solution by sonicating with a Branson sonifier for 10 minutes at 30 second intervals. Excess lecithin was removed from the 10 min sonicated solution by 55 centrifuging at 8,700 x g for 10 minutes in the cold. The resulting supernatant was stored at 5°C for approximately one month. 20a-hydroxycholesterol-egg lecithin emulsions were similarly prepared. Cholesterol and 20a-hydroxy- cholesterol concentrations in these emulsions were deter mined by the micromethod of Glick £t al., (188). Absorption Spectra Difference absorption spectroscopy was measured with a Cary Model 14 equipped with a scattered transmission accessory. Baseline values were obtained with sample and reference cuvettes containing suspensions oftfre particulate P450 preparations in phosphate buffer (16 mM, pH 7.2) containing MgC^ (6 mM) and KC1 (96 mM) . The baseline was corrected at each 50 nm using the multipot adjustment. The reagent was then added to the sample cuvette and the spectrum recorded. All spectra were obtained at room temperature using a 0 - 0.1 absorbancy slide wire. Wave length calibrations showed an error or less than 0.1 nm. All spectra were obtained with 1 cm pathlength cuvettes. Due to a residual 420 nm peak which occurred in the adjust ed baseline, all difference spectra have been plotted with the baseline absorption substracted. Incubation Conditions of Hydroxyl at ion' Studies Incubations were done at 37°C in Erlenmeyer flasks which were shaken continuously in a Dubonoff shaker. Each flask contained mitochondria, phosphate buffer (16 mM, pH 56 7.2), MgC^ (6 mM), KC1 (96 mM), actimycin A (4 yg), and NADP (0.45 mM) . Mitochondria were pre-incubated with the steroid substrates for 10 minutes and the reactions were started with 50 mM isocitrate. Aliquots were taken from 15 to 60 seconds for measurement of products. For 118- hydroxylation, DOC concentration ranged from 1 to 80 yM, containing 0.062 yCi of DOC-^C. For 22R-hydroxylation, 20a-hydroxycholesterol concentration ranged from 1 to 72 yM, containing 1.25 yCi of 20a-hydroxycholesterol-7 H. The reaction medium contained in addition, rat adrenal microsomes equivalent to 0.017 mgN, NAD (5 mM), and NADPH (10 mM). For cholesterol SCC, cholesterol concentration ranged from 7 to 623 yM containing 0.25 yCi of cholesterol- 14 4 C. The reaction medium contained in addition, rat adrenal microsomes equivalent to 0.017 mgN, NAD (5 mM), and NADPH (10 mM). Measurement of Steroid Incubation Products Aliquots from the 118-hydroxylase assays were ex tracted with chloroform, and paper chromatographed in a benzene-formamide system. For the 22R-hydroxylase assay, aliquots extracted with ethyl acetate and with chloro form were chromatographed in a formamide-heptane system. Aliquots from the cholesterol SCC assay were extracted with ethyl acetate and with chloroform and chromatographed in a cyclohexane-propylene glycol system. Radioactive peaks were determined quantitatively on a Tracerlab 4ir 57 radiochromatogram strip counter. P450 Assay The mitochondrial and microsomal preparations were diluted in phosphate buffer (16 mM, pH 7.7) containing MgC^ (6 mM) and KC1 (96 mM) to a volume of 6.0 mis. 2.5 mis were placed in the sample and reference cuvettes and a baseline recorded. A few crystals of ^25^0^ were then added to the sample and reference cuvettes and the sample cuvette was subsequently gassed with carbon monoxide for 30 seconds. The absorption at 450 nm was measured, and the concentration of P450 was calculated using the molar -1 -1 extinction of 91 cm mM (52). Conditions For Determining the In. Vivo Effects of ACTH and Cycloheximide on The Levels of Endogenous Cholesterol and DOC-Bound P450 (Table VI) All adrenal glands were removed using ether anesthesia. Rats were hypophysectomized for 44 hours and ACTH stimula tion was achieved by infusing (I.V.) 100 munits of ACTH (in 0.10 ml NaCl) for 10 minutes. Anoxia was maintained by clamping the blood supply to the adrenal gland for 3 minu tes. When used, 10 mg cycloheximide (in 0.10 mg ethanol) was injected I.P.for 20 minutes before removal of adrenals. In the experiments using hypophysectomized rats,one adrenal was removed as a control prior to ACTH stimulation. SOURCES OF IMPORTANT REAGENTS Calbiochem: Nicotinamide-adenine dinucleotide, 58 Ikapharm, Ltd: Sigma Chemical Company: New England Nuclear: Matheson Scientific: Gilmore Liquid Air Co: Airco: reduced (NADH), oxidized (NAD ), nicotinamide adenine dinucleo tide phosphate, reduced (NADPH), oxidized (NADP), adenosine-5'- triphosphate (ATP), adenosine-5' -diphosphate (ADP), adenosine-3* -5’-monophosphate (cyclic AMP), malate, isocitrate, a-keto- glutarate, glutathione, glut athione reductase, glucose-6- phosphate, glucose-6-phosphate dehydrogenase, glucose-l-phos- phate, fructose-6-phosphate, fructose-1,6-diphosphate, di- hydroxyacetone phosphate, glycer aldehyde phosphate, 3-phospho- glyceric acid, 2-phosphoglyceric acid, phosphoenolpyruvate, pyruvate, citrate, cholesterol. 20a-hydroxycholesterol Deoxycorticosterone, 17a-hydroxy progesterone, 17a-hydroxy- pregnenolone, progesterone, pregnenolone, egg lecithin, sodium succinate, adreno- corticotrophin hormone (ACTH), cycloheximide, aminoglutethimide antimycin A. 14 Deoxycorticosterone-4- C, 20a- hydroxycholesterol-7a- H, chol esterol^-1^. Carbon Monoxide Liquid nitrogen, isopentane Dry nitrogen gas Matheson, Coleman § Bell:Chloroform, ethyl acetate, methanol, formamide, benzene, heptane, sucrose, potassium chloride, hydrazine sulfate, sodium hydroxide, potassium carbonate. J.T. Baker Laboratory Chemicals: Magnesium chloride, sodium dithionite. United States International Chemicals: Ethanol CHAPTER IV RESULTS Control of NADPH Production Mitochondrial steroid hydroxylations are supported in vitro by NADPH generated directly by NADP-linked mito chondrial dehydrogenases and/or indirectly by an energy- dependent transhydrogenase which converts NAD to NADPH. To determine whether ACTH stimulates the activities of the dehydrogenases and transhydrogenase prior to increasing the rate of steroidogenesis, the steady-state concen trations of Krebs cycle substrates and pyridine nucleotides were measured before and after ACTH administration to hypo physectomized rats. Adrenal glands were removed from ether-anesthesized animals and plunged immediately into liquid nitrogen. Glands removed prior to the administra tion of ACTH were used as controls. Pyridine and adenine nucleotides and Krebs cycle substrates were measured in perchloric acid extracts of the frozen glands according to the procedures described under Materials and Methods. As shown in Table I, no significant changes in either the Krebs cycle substrates or pyridine nucleotides were detected two minutes after ACTH stimulation. Measurements at earlier time periods were essentially the same as those shown in Table I. 60 61 TABLE I EFFECTS OF ACTH ON LEVELS OF KREBS CYCLE INTERMEDIATES PYRIDINE AND ADENINE NUCLEOTIDES Kreb cycle substrates, pyridine and adenine nucleo tides were measured according to procedures described in Methods. Values shown are means + standard error of the mean. Rat Adrenal Control ACTH, 2 min. Component m^moles/gm protein Citrate 2,472 + 456 2,114 + 468 Isocitrate 298 + 41 303 + 36 a-Ketoglutarate 149 + 13 160 + 14 Malate 1,226 + 147 1,092 + 187 Oxaloacetate 31 + 3 40 + 5 NAD 2,400 + 178 2,625 + 354 NADH 237 + 40 238 + 22 NADP 397 + 53 453 + 58 NADPH 403 + 50 468 + 50 ATP 15,483 + 513 11,833 + 379 ADP 1,760 + 215 1,438 + 231 AMP 521 + 120 94-7 + 320 62 Several experiments were done to determine whether changes in the pyridine nucleotides were occurring, but were being masked by a shift to a new steady state redox level during the collection procedure. The results of these experiments are summarized in Table II. Normal rats were used in all experiments. Altered collection condi tions are indicated by A, B, C, and D. The controls in the first three experiments (A,B, and C) consisted of collect ing one of the glands of each of the experimental rats into liquid nitrogen as rapidly as possible as was the usual procedure. The control value for D is an average of the controls in A, B, and C. A. To test the effectiveness of liquid nitrogen as a coolant, glands were collected into isopentane cooled to -140°C. Isopentane has been reported to be a more efficient coolant because of its ability to transfer heat rapidly Cl893• A layer of gas is formed with liquid nitrogen and consequently the heat transfer is retarded (190) . As judged by the ratio of oxidized to reduced NAD and NADP, there was no significant difference between the two cool ants in these studies. B. Glands were collected and placed on a watch glass for two minutes before being put into liquid nitrogen. The NAD/NADH ratio dropped 72% and the NADP/NADPH ratio showed a 37% decrease compared to immediate immersion of the glands in liquid nitrogen. 63 C. Rats were sacrificed by diaphragm cutting before collecting the glands into liquid nitrogen. This pro cedure resulted in a 74% decrease in the NAD/NADH ratio and a 40% decrease in the NADP/NADPH ratio:. D. Glands were collected into liquid nitrogen after rats were allowed to breathe carbon monoxide instead of the usual ether anesthesia. Under these conditions the ratio of NAD/NADH dropped 85% and the NADP/NADPH ratio, 80%. These results indicate that the lack of change in the ratio of oxidized to reduced pyridine nucleotides after ACTH infusion was probably not due to an inadequate collection procedure since significant shifts toward reduction were observed when the method of collection was substantially altered. Changes in the pyridine nucleotide redox state could also be masked by reciprocal changes occurring simultaneously in the cytoplasm and mitochondria (oxidized pyridine nucleotides in the cytoplasm becoming reduced due to the activation of a process like glycolysis and reduced mitochondrial pyridine nucleotides becoming oxidized due to increased steroidogenesis). To test this hypothesis, the distribution of NAD and NADP in the whole homogenate and mitochondria were determined. A homogenate consisting of 60 adrenal glands in 30 mis of 0.25 M sucrose was made. After the 900 x g sediment 64 TABLE II SOME CONDITIONS AFFECTING OXIDIZED-REDUCED PYRIDINE NUCLEOTIDE RATIOS CONDITION NAD NADH NADP nmoles/gm Protein NADPH NAD NADH NADP NADPH A. Isopentane 3,080 165 330 417 18.65 0.79 Control 2,320 139 304 388 16.70 0.78 B. Rm. Temp. (2 min.) 1,780 626 222 478 2.84 0.46 Control 2,750 268 295 396 10.25 0.74 C. Death 2,000 469 183 523 4.26 0.35 Control 3,510 207 291 495 16.95 0.59 D. Carbon Monoxide 1,625 748 102 724 2.18 0.14 Control 2,855 201 297 426 14.60 0.70 65 was discarded a mitochondrial fraction was obtained by resuspending the 25,000 x g pellet in sucrose. As shown in Table III, the mitochondria contained approximately one third of the total NAD and NADP when compared to the whole homogenate. These results therefore indicate that it is possible for the oxidized-reduced pyridine nucleotide ratio to remain constant following ACTH stimulation if most of the mitochondrial pyridine nucleotides become oxidized at the same time that 301 of the cytoplasmic pyridine nucleotides become reduced. The lack of change in the Kreb cycle substrates fol lowing ACTH stimulation suggests that none of the enzymes associated with these intermediates are rate-limiting prior to the onset of corticoid production. The constancy of the pyridine nucleotide values after ACTH is not as easily interpreted since the distribution of these cofactors in the cytoplasm and mitochondria make it impossible to rule out an ACTH-induced stimulation of the energy-depen dent transhydrogenase in the time period preceeding the increase in corticosterone synthesis. The suggested decrease in the ATP/ADP-AMP ratio shown in Table I would be compatible with such a stimulation. ’ Control of Glycolysis Alterations in the rate of generation of mitochondrial reducing equivalents could of course be regulated by the rate of flow of intermediates to the mitochondria and 66 TABLE III DISTRIBUTION OF PYRIDINE NUCLEOTIDES IN RAT ADRENAL FRACTION NAD NADH NADP (nmoles) NADPH TOTAL Whole Homog. 869 57.9 275 219 1420 Mitochondria 309 ---- 176 --- 485 67 consequently by the rate of glucose transport into the adrenal cell. An insulin-like action of ACTH on the adrenal is suggested by the data reported by Schonbaum et al., (191) which showed that optimum ACTH stimulation of their adrenal slices depended on the presence of glucose in the incubation medium, and by Hechter and Lester's (192) demonstration of an ACTH-induced increase in the D-xylose, mannitol, and aminoisobutyric acid spaces in adrenal slices. Such an explanation for the mechanism of action of ACTH on the adrenal metabolism would explain the alterations in steroid biosynthesis along with changes in the entire metabolism of the adrenal cell. Since the decreased ATP/ADP*AMP ratio following ACTH shown in Table I could mediate and activation of glycoly sis by increasing the rate of phosphofructokinase (193), the effect of ACTH on the concentrations of glycolytic intermediates was studied in hypophysectomized rats. The experimental procedure is described under METHODS. Figure 3 shows a summary of several such experiments. The concentration of each intermediate in nmoles per gram protein with the standard error of its mean is shown for the control glands along the bottom of the figure. The per cent change in the concentration of these intermediates two minutes after ACTH stimulation is plotted on the or dinate with the standard error of the mean indicated by brackets around each point. Statistically significant 68 <0.0005 <00005 <0475 300 [< 5ooq5 1 <00005 I <000.25 I <0 . 10 <0.05 <0.05 200 PERCENT OF CONTROL 100 G IP MuMotot^mP. W ±3 3PG A 278±4I 0«P FDP G A P PEP 5 «± 37 21+3 38+2 40±5 i Fig. 3.— Suiranary of changes in glycolytic inter mediates two minutes after ACTH. Values of glycolytic intermediates are expressed as a percentage of the control values obtained before 100 mu ACTH infusion. The concentration of each intermediate in nmoles per gram protein with its standard error of the mean is shown for the control glands along the bottom of the figure. The control and stimulated values for each experiment were obtained from pooled adrenals from at least 20 rats. 69 changes (p< 0.01) are shown along the bottom of the figure. A decrease in the hexose phosphates, glucose-l-phos- phate (G1P), glucose-6-phosphate (G6P), and fructose-6- phosphate (F6P), with a simultaneous increase in fructose- 1, 6-diphosphate (FDP) and dihydroxyacetone phosphate (DHAP), are clearly shown. This accumulation and depletion of intermediates represents a crossover (194) in substrate concentration between F6P and FDP and suggest an activation of phosphofructokinase (PFK). As shown, the concentration of glyceraldehyde phosphate (GAP) returns to the control level in the stimulated glands. This is followed by a significant increase in 3-phosphoglyceric acid (3PGA), phosphoenolpyruvate (PEP) and pyruvate. The fall in GAP relative to DHAP, along with the increase in the remain ing three carbon acids of the glycolytic system describes another crossover in substrate concentrations between GAP and 1,3-diphosphoglyceric acid (1,3 DPGA), and suggests an activation of glyceraldehyde phosphate dehydrogenase (GAPDH). Changes in lactate concentration were not used to measure flux in these experiments because of the possible contribution of pyruvate to the Krebs cycle. A. Kinetics of Glycolytic Intermediate Changes Following ACTH To detect the earliest effect of ACTH on the glycolytic intermediates, groups of 30 hypophysectomized rats were 70 infused with 100 milliunits of ACTH for 10, 20, 30, 120, and 600 seconds. Figure 4 shows the results of these studies. The dotted line marked 60 seconds sodium chloride, and the light dashed line marked 10 seconds, both roughly- paralleling the 100% line, show that neither sodium chloride infusion for 1 minute nor ACTH stimulation for 10 seconds, produces a change in the intermediates. Twenty- seconds after ACTH infusion, shown by the heavy unbroken line, the beginning of a crossover at PFK is seen. This crossover becomes progressively more pronounced at 30 seconds and 120 seconds as shown by the light dashed line and the heavy dashed line, respectively. Beginning in the two minute stimulated study, the additional crossover at GAPDH appears. At ten minutes after ACTH, when the rate of steroidogenesis has plateaued the crossovers are still evident as shown by the dotted line marked 600 seconds. B. Effect of Cycloheximide on the ACTH-Induced Crossovers Since several studies have shown that new protein synthesis may be essential for the increase in steroid biosynthesis induced by ACTH, it was of interest to examine the effect of cycloheximide on the ACTH-induced crossovers. Hypophysectomized rats were divided into two groups of twenty-five. One group was stimulated with 100 milli units of ACTH for 2 minutes as in previous experiments, while the second group received 10 mg of cycloheximide 20 71 PERCENT OF CONTROL 4 0 0 3 0 0 200 100 G1P F6P DAP 3PGA PY R. G6P FDP GAP PEP Fig. 4.— Kinetics of changes in glycolytic inter mediates following 100 mu ACTH. Values of glycolytic intermediates are expressed as a percentage of the control values obtained before ACTH infusion at the indicated time periods. The control and stimulated values of each experi ment were obtained from pooled adrenals from at least 20 rats. 72 minutes before the ACTH was administered. Figure 5 shows the results of this experiment. The heavy line again shows the effect of ACTH stimulation for 2 minutes on the concentrations of glycolytic intermediates, with crossovers at PFK and GAPDH. As shown by the broken line, infusion of cycloheximide prior to ACTH did not alter the apparent activation of these glycolytic enzymes. Blood samples collected from cycloheximide-treated animals showed that the ACTH-induced increase in plasma corticosterone was inhibited at this concentration of the antibiotic in agree ment with previous reports (143). C. Relationship of Cyclic AMP to the ACTH-induced Cross overs Although an increase in cyclic AMP prior to 1 minute after ACTH has not been demonstrated, the reported activ ation of PFK in liver flukes by cyclic AMP (193), and the well-documented increase in adrenal cyclic AMP following ACTH stimulation, clearly raised the possibility that the relationship of cyclic AMP changes to PFK activation were causally related. Figure 6 shows a study of the cyclic AMP response to ACTH infusion. Cyclic AMP concentrations were measured using the enzymatic radioisotope displacement method (184). Each point represents the assay of one adrenal gland, indicated in nanomoles on the ordinate. The open circles show the control levels, the filled circles, the stimulated 73 PERCENT OF CONTROL 600 400 CYCLOHEXIMIDE (20min) ♦ACTH (2min.) 200 G1P F6P 3PGA DAP PYR. G6P FDP GAP PEP Fig. 5.— Effect of cycloheximide on the ACTH-induced crossovers. Changes in the glycolytic intermediates were measured before and after the infusion of 100 mu ACTH for 2 minutes. The dashed line shows the effect of 10 mg cycloheximide administered 20 minutes prior to ACTH. 74 levels. The horizontal bars at each time period represent the means of the stimulated values. A significant and almost linear increase in cyclic AMP is shown beginning at 10 seconds after ACTH infusion, the earliest time period examined and continuing through the three minute period. This is the earliest reported increase in adrenal cyclic AMP, and clearly preceeds the activation of PFK. The activation of adrenal PFK is compatible with the activation in other tissues where PFK has been found to be a rate-limiting enzyme for glycolysis (193). This enzyme is usually under allosteric control by a number of effect ors including AMP, inorganic phosphate, ATP, citrate, and in some instances, cyclic AMP (193). The significant rise in cyclic AMP at 10 seconds after ACTH and its sustained elevation through 180 seconds are compatible with this compound functioning as a mediator of the activation of PFK as has been observed in other tissues. Failure of the citrate levels to change at any time after ACTH infusion would seem to indicate that this substrate is not involved in the changes in adrenal PFK measured here. Unlike PFK, GAPDH has a high maximal capacity in most tissues, and for this reason is not normally thought to be rate-limiting. A regulatory role has been observed, how ever, in aerobic-anaerobic transitions in brain and heart 75 20 40 60 80 10 0 1 2 0 140 60 180 TIME (SECONDS) AFTER ACTH Fig. 6.— The effect of 100 mu ACTH on cyclic AMP levels. Each point represents the assay of one adrenal gland (approximately 25 mg) indicated in nmoles on the ordinate. The open circles show the control levels, and the filled circles the stimulated levels. The horizontal bars at each time period represent the means of the stimulated values. 76 tissues (195). Presumably, the NADH:NAD ratio mediates this control by regulating the rate of this oxidative reaction. Although the data in Table I does not show evidence for such a shift in the NADHrNAD ratio, it is possible that this change is occurring, but is being masked by reciprocal changes occurring simultaneously in the cyto plasm and mitochondrial compartments as discussed earlier. As the data in Figure 7 indicates, plasma cortico sterone levels did not significantly rise until approxi mately 3 minutes after the infusion of 100 milliunits ACTH. The increased cyclic AMP concentrations which occurred at 10 seconds, and the activation of PFK which occurred at 20 seconds after ACTH, clearly preceeded the increased rate of corticosterone synthesis and the three events therefore appear to be causally related. Since the rate of steroid biosynthesis has been shown to increase in response to ACTH concentrations as low as 0.03 milliunits (196), the concentrations of glycolytic intermediates were measured before and after the adminis tration of 0.5, 1.0, and 2.0 milliunits of ACTH. At these lower hormone concentrations there was no significant difference between the control and stimulated glycolytic substrate concentrations, indicating that none of the enzymes of glycolysis were activated. Although cyclic AMP concentrations were not measured at these lower doses of ACTH, studies by other investigators (197) show that 77 150 i ~c 100 i i i I - o - 8 . 0 4 0 TIME (MIN) 0 -4.0 Fig. 7.— Effect of 100 mu ACTH on rat plasma cortico sterone levels. Plasma corticosterone was measured accord ing to the procedures described under Methods before and after the infusion of 100 mu ACTH. The various symbols represent samples taken from individual rats. 78 there is also a lack of correlation between the amount of ACTH required to increase adrenal cyclic AMP accumulation and the amount required to increase the rate of corticoid synthesis. These data suggest that ACTH may activate steroido genesis in at least two different ways. At low levels of the hormone, some rate-limiting aspect of the steroid bio synthetic pathway may be directly stimulated, whereas at high concentrations of ACTH, when the adrenal is under prolonged stimulus, the energy demands of hydroxylation might initiate other processes such as the cyclic AMP- induced activation of glycolysis. Such an activation of glycolysis would provide reducing equivalents to the hydroxylation pathway as a secondary response to the hormonal stimulus. ACTH and the Steroid Hydroxylases The 420 nm troughs and the 390 nm peaks (type I diff erence spectra) induced by steroid substrates, and the 420 nm peaks and 390 nm troughs (type II difference spectra) induced, by certain inhibitors in the visible absorption of adrenal cytochrome P450 have been used as one of the primary means of studying the catalytic properties of these membrane-bound enzymes. It has been shown (105,106) that the substrate-in duced type I spectra represent a change in the spin state of the ferric iron of P450 from the low spin state (S=l/2) 79 to the high spin state (S=5/2), and the inhibitor-induced changes in the visible absorption (type II spectra) are indicative .of a change in the spin state in the reverse direction, or from high spin to low spin. Substances which induce type I difference spectra or which change the spin state of P450 from low spin to high spin increase its rate of reduction, and substances which change the enzyme's spin state from high spin to low spin decrease its rate of reduction (108-110). Since P450 reduction appears to be the rate-limiting step in the hydroxylation of steroids (88,93), these spin state changes become important regulators of the enzymatic activity of the enzyme and of steroidogenesis in general. Although the use of spectrophotometric techniques have proven invaluable in studying the catalytic properties of P450, the cholesterol SCC enzyme system, which is believed to be rate-limiting in the production of cortico steroids, and therefore to be controlled by ACTH, has not been adequately studied. The limited solubility of chol esterol in aqueous solution, and the fact that most choles terol SCC preparations already contain high levels of cholesterol when isolated, have complicated most attempts to study this enzyme system. In addition to these difficulties, the formation (in adrenal mitochondrial P45Q preparations) of two. distinct difference spectra (type I and type II) by 20a- 80 hydroxycholesterol (198) and of type II difference spectra by malate (93) and pregnenolone (199) have been difficult to interpret and have raised doubts as to the specificity of the substrate and inhibitor-induced spectral changes. A. Mitochondrial P450 Difference Spectra Since very few of the adrenal P450 spectral studies reported in the literature have been performed on rat mitochondria, the binding of several steroids was studied in this tissue. Figure 8 shows the difference spectra induced by titrating incremental concentrations of deoxycortico sterone (DOC) into a preparation of rat mitochondria. The 420 nm troughs and the associated 390 nm peaks (type I difference spectra) reflect the transformation of cyto chrome P450 from an inactive 420 nm form (low spin P450) to an active 390 nm state (high spin P450) which permits enhanced electron flow to the cytochrome (200). A linearized plot of these titration data from which the DOC spectral dissociation constant, (203), was calculated is also shown in Fig. 8. The Kg of 0.25 pM DOC suggests that the 118-hydroxylase has a high affinity for DOC, and is considerably lower than the Kg of 7.0 pM previously reported for titrations of DOC with bovine adrenocortical submitochondrial particles (106). Since Ks. values calculated in this manner are dependent on P450 concentration (201) and are therefore not true equilibrium 81 O — 0 . 0 1 ai 0.005 M in -1 -0 .0 0 5 -Q01 4 0 0 45 0 WAVELENGTH (nm) 5 0 0 5.0 2 . 0 Fig. 8.— Titration of DOC on rat adrenal mitochondria. Mitochondria equivalent to 1 pM P450 were suspended in phosphate buffer (19 mM, pH 7.2) containing MgCl2 (6 mM) and KCl (96 mM). The solid lines (____) show the effect of 0.33, 0.66, and 1.6 pM DOC (dissolved in ethanol) added to the sample cuvette. Equal volumes of ethanol were added to the reference cuvette. The dashed line (-----) shows the spectrum obtained 3 minutes after the addition of antimycin a (4 pg) and isocitrate (47 mM) to the sample and reference cuvettes of the maximally titrated DOC curve. Also shown is the linearized plot of these saturation data from which a DOC Ks of 0.25 pM was calculated. 82 constants, the discrepancy may be explained by the diff erent concentrations of P450 used in the two studies. The dotted line in Figure 8 shows the dissipation of the maximally titrated spectrum 3 minutes after adding antimycin A and isocitrate to the sample and reference cuvettes. The disappearance of the type I spectrum in the presence of NADPH generation and oxygen indicates that high spin P450 activated by DOC for the subsequent redox reactions has returned to the inactive 420 nm form following the apparent hydroxylation of DOC to cortico sterone. This is an important demonstration since Narasimhulu (202) has shown that non-hydroxylatable steroids are also capable of inducing type I difference spectra and therefore of activating P450 for reduction. The type I spectra formed by these non-substrates can be distinguished only by their persistence under conditions supporting hydroxylation (NADPH generation and oxygen). 20a-hydroxycholesterol, a substrate of 22R-hydroxy- lation, is also an inhibitor of cholesterol SCC (24). This steroid can form two different spectral species in bovine adrenocortical mitochondria (198). These diff erential effects of 20a-hydroxycholesterol on bovine mitochondria have been explained by Harding,eit al^ (110). Using bovine adrenocortical submitochondrial particles, depleted of endogenous cholesterol by acetone extraction, these investigators showed that the type II spectrum formed 83 by 20a-hydroxycholesterol resulted from the interaction of this steroid with cholesterol-bound high spin P450. When added to the acetone-extracted particles in the absence of exogenous cholesterol, 20a-hydroxycholesterol formed a type I difference spectrum. Figure 9 shows the spectra resulting from the addition of 20a-hydroxycholesterol to bovine adrenocortical sub- mitochondrial particles (curve A), bovine adrenocortical mitochondria (curve B), and rat adrenal mitochondria (curve C). Although these three preparations contain identical concentrations of P450, the magnitude and kind of spectrum induced by 20a-hydroxycholesterol is different. 20a- hydroxycholesterol produced a type II spectrum in the bovine preparations (curves A and B), and a shifted type I spectrum (trough at 430 nm, peak at 410 nm) when added to the rat preparation. Based on Harding, et al's data which showed that the 2Oa-hydroxycholesterol-induced type II spectrum results from the combination of this steroid with cholesterol-bound P450, this study indicates that rat adrenal mitochondria contain less endogenous cholesterol- bound P450 than do bovine adrenocortical mitochondria. Spectral studies on mitochondria prepared from whole bovine adrenal glands indicated that this difference is not due to the presence of the medulla in the rat adrenal mito chondrial preparations. 84 0.04 I E o CO z UJ Q _l < p - 0 02 Q . O 3 5 0 4 0 0 5 0 0 4 5 0 WAVELENGTH (nm) Fig. 9.— 20a-hydroxycholesterol-induced difference spectra of rat adrenal mitochondria and bovine adreno cortical mitochondria and submitochondrial particles. Mitochondria and submitochondrial particles equivalent to 1 |iM P450 were suspended in phosphate buffer as described in Fig. 8. Sample cuvettes contained 10 |aM 20a-hydroxy- cholesterol (dissolved in ethanol) in Curve C (rat mito chondria) , 17 |jM 2Oa-hydroxycholesterol in Curve B (bovine mitochondria), and 40 |iM 20a-hydroxycholesterol in Curve A (bovine submitochondrial particles). Equal volumes of ethanol were added to the reference cuvettes. 85 The increased type II spectrum produced by 20a- hydroxycholesterol in the sonicated bovine adrenal mito chondrial preparation (submitochondrial particles, curve A) is supportive of the idea that a restricted pool of mitochondrial cholesterol exists (10), and that this cholesterol becomes bound to cytochrome P450 when the mitochondria are sonically disrupted. The different concentrations of 20a-hydroxycholesterol required to produce the maximal spectral change in Figure 9 is consistent with the idea that a variable concentration of endogenous cholesterol-bound high spin P459 is contain ed in the three preparations. Malate (93) and pregnenolone (199) also produce type II spectra in bovine adrenocortical mitochondria. Although no proof is available, it has been suggested that the malate-induced type II spectrum results from the meta bolism of endogenous cholesterol in these mitochondria (203). The origin of the pregnenolone-induced type II spectrum has not been explained. In view of the different results obtained when 20a-hydroxycholesterol was added to rat and bovine adrenal mitochondria in the previous experi ment, the interaction of electron donors and pregnenolone with these preparations was studied. The effect of electron donors on rat adrenal mito chondria, and on bovine adrenocortical mitochondria and submitochondrial particles are compared in Figure 10. 86 0 0 4 I E U i os a z C O z U J a - i 8 E o 4 0 0 4 5 0 5 0 0 WAVELENGTH (nm) Fig. 10.— Effect of electron donors on rat adrenal mitochondria and bovine adrenocortical mitochondria and submitochondrial particles. Mitochondria and submito chondrial particles plus supernatant equivalent to 1 pM P450 were suspended in phosphate buffer. Sample and reference cuvettes in Curves A and B contained 4 pg anti- mycin a and 47 mM succinate. Sample cuvettes contained 47 mM isocitrate in Curve A, 20 mM malate in Curve B, and 0.3 mM NADPH in Curve C. 87 Isocitrate and malate were used as the NADPH-generating substrates for the rat and bovine adrenal mitochondria, respectively. NADPH, which can permeate the sonically disrupted mitochondrial membrane, was employed as electron donor for the submitochondrial particles. To cancel out the absorption of reduced cytochrome b, antimycin A and succinate were added to the sample and reference cuvettes in curves A and B. In addition, an aliquot of the super natant fraction (obtained from sonicating the bovine adrenal mitochondria) containing the additional electron transport factors, adrenodoxin and adrenodoxin reductase, was added to the submitochondrial particles. As shown in Figure 10, the maximum type II spectrum produced by electron donor in bovine adrenal mitochondria (curve B) is larger than the spectrum produced in rat mitochondria (curve A). Sonication of the bovine adrenal mitochondria increased th.e electron donor-induced type II spectrum (curve C). The pattern of spectral changes produced in this study is consistent with the results of the previous experiment and seem to support the hypothesis that the electron donor-induced type II spectrum results from the hydroxylation of endogenous cholesterol-bound P450 in the sample cuvette, returning the enzyme to the cholesterol-free, 420 nm or low spin form. The difference spectra produced by the addition of pregnenolone to rat mitochondria (curve A) and bovine 88 mitochondria (curve B) are shown in Figure 11. Again, the magnitude of the type II spectrum induced by preg nenolone in bovine adrenocortical mitochondria is larger than the corresponding spectrum produced in rat mito chondria. As discussed earlier, the high concentration of endogenous cholesterol-bound P450 contained in most adrenal mitochondrial preparations and the poor solubility of cholesterol in aqueous solution have made the cholesterol SCC enzyme system extremely difficult to study. Since the spectral studies presented above seemed to indicate that rat adrenal mitochondria contain low concentrations of endogenous cholesterol-bound P450, considerable time was spent preparing a soluble cholesterol solution with which, the side-chain cleavage enzyme system in these mitochondria could be studied. Soluble cholesterol-lecithin emulsions had previously been prepared In our laboratory (199). Although these emulsions could induce high spin P450 or type I spectra in a partially purified P450 preparation (204) and in acetone-extracted submitochondrial particles (110), no difference spectra were produced when the cholesterol emulsions were added to intact rat or bovine adrenal mitochondria. After numerous alterations in the procedure used to prepare these emulsions, and in the type of lecit hin used, it was discovered that storage at 5°C for 89 500 450 400 350 WAVELENGTH (nm) Fig. 11.— Effect of pregnenolone on rat adrenal mito chondria and bovine adrenocortical mitochondria. Mito chondria equivalent to 1 nM P450 were suspended in phos phate buffer. The sample cuvettes contained 11.7 joM pregnenolone (dissolved in ethanol) in Curve A and 94 nM pregnenolone in Curve B. Equal volumes of ethanol were added to the reference cuvettes. 90 approximately one month was necessary before the chol esterol -egg lecithin emulsions and were able to bind cyto chrome P450. The titration of DOC (solid lines) and cholesterol (dashed lines) into a preparation of rat adrenal mito chondria is shown in Figure 12. The 420 nm troughs and 390 nm peaks induced by cholesterol indicates that this substrate, like DOC, transforms inactive 420 nm or low spin P4S0 to the active 390 nm or high spin state, and represents the first demonstration of cholesterol-induced spin state changes in intact adrenal mitochondria. The substantial reduction of the maximally induced type I spectrum three minutes after the addition of isocitrate and antimycin A to the sample and reference cuvettes (dotted line) indicates a return of the cholesterol and DOC-activated high spin P450 to the inactive low spin state following the apparent hydroxylation of the steroids. Although both DOC and cholesterol produce identical type I difference spectra, the concentration of steroid required to produce the maximum trough in each case is very different (1.6 yM DOC and 104 yM cholesterol). The average cholesterol Ks value (36 yM per yM P450) calcu lated from a number of titration studies is approximately 100-fold higher than the average DOC Kg (.25 yM/yM P450) and suggests that the cholesterol P450 enzyme has a much lower affinity for its substrate. 91 0 .0 2 0.0 oz -0.02 SOS' 450 (nm) 400 WAVELENGTH (nm) 3 5 0 5 0 0* 60 30 Fig. 12.— Titration of DOC and cholesterol on rat mitochondria. Mitochondria equivalent to 1.2 jaM P450 were suspended in phosphate buffer. The solid lines ( -----) show the effect of successive DOC additions (0.33, 0.66, and 1.6 pM) to the sample cuvette (equal volumes of ethanol added to the reference cuvette). The dashed lines (-----) represent cholesterol additions (20.8, 62.4, and 104 pM) to the sample cuvette of the maximally titrated DOC curve (equal volumes of lecithin were added to the reference cuvette). The dotted line (....) shows the spectrum produced by adding isocitrate (47 mM), antimycin a (4 pg), and NADP (0.4 mM) to the sample and reference cuvettes of the maximally titrated DOC and cholesterol curve. A linear plot of cholesterol titration in rat mito chondria is shown at the bottom. Open circles (o o) show the linear plot of cholesterol titrated in the absence of DOC and the crosses (x x) show the same titration done on mitochondria presaturated with 2.9 pM DOC. Mitochondria equivalent to 0.8 pM P450 suspended in phosphate buffer were used in both studies. The apparent Ks in the presence and absence of DOC is 33 pM cholesterol. 92 The additive nature of the DOC and cholesterol-induced spectral changes, and the identical cholesterol Kg values calculated in the presence and absence of DOC, support previous conclusions (106,198) that either two separate P450 enzymes are involved in 118-hydroxylation and chol esterol SCC or DOC and cholesterol are independently bound on one P450 enzyme. The difference in the maximum 420 nm trough con sistently induced by saturating concentrations of chol esterol (0.0203) and DOC (0.0114) is probably not due to a higher extinction coefficient of cholesterol-bound P450, but rather to the higher, concentration of P450 available for binding the exogenous cholesterol (200). The lower concentration of free P450 apparently available for binding by exogenous DOC may be due to a larger con centration of endogenous DOC-bound P450 contained in these mitochondria. The rate of binding of cholesterol to rat adrenal mitochondrial P450 is shown in Figure 13. Unlike the 118-hydroxylase substrates, DOC and 11-deoxycortisol which bind almost instaneously to. cytochrome P450 (93), the 420 nm trough induced by cholesterol does not reach a maximum until approximately 10 minutes after the sterol addition. The binding rate is not dependent on the con centration of cholesterol or P45Q and permeation of the mitochondrial membrane also appears not to be a factor 93 0020 0015- § 3 < 0005 GO 120 TIME (MIN) Fig. 13.— Rate of binding of cholesterol to rat adrenal SCC P450. Rat mitochondria equivalent to 0.8 P450 were diluted in phosphate buffer. 32 |aM cholesterol was added to the sample cuvette (lecithin to the re ference) , and the absorbancy changes at 420 nm were recorded at the indicated time periods. 94 since the binding rate is significantly decreased in ace tone-extracted submitochondrial particles (205) and in a soluble SCC P450 preparation (206) . B. Dependence of Type II Difference Spectra on Steroid- Bound P450 With a cholesterol solution capable of binding to mitochondrial P450, it became possible to investigate more directly the origin of the type II spectra induced by 20a-hydroxycholesterol, pregnenolone, and electron donors. The effect of 20a-hydroxycholesterol on rat adrenal mitochondria is shown in curve A, Figure 14. Also shown are the effects of 20a-hydroxycholesterol on rat mito chondria presaturated with exogenous cholesterol (curve B), and DOC (curve C). Curve D shows the spectrum obtained immediately after the addition of isocitrate and antimycin A to the sample and reference cuvettes of curve B. These data show that 20a-hydroxycholesterol can pro duce two different spectral species when added to rat mitochondria, a type I spectrum or high spin P450 in the absence of exogenous cholesterol (curve A) and a type II spectrum or low spin P450 when the mitochondria have been presaturated with cholesterol (curve B). Presaturation of the mitochondria with DOC has no effect on the 20a- hydroxycholesterol spectrum as seen by comparing curves A and C. When isocitrate and antimycin A are added to the sample 95 k o 1 1 1 Z U J a . u z > - t 2 UJ a u o -002 500 400 450 WAVELENGTH (nm) I I Fig. 14.— 20a-hydroxycholesterol-induced difference spectra of rat adrenal mitochondria. Mitochondria equi valent to 1 pM P450 were suspended in phosphate buffer. Sample cuvettes contained 10 |jM 20a-hydroxycholesterol in Curves A and C, and 25 |aM 20a-hydroxycholesterol in Curve B. Sample and reference cuvettes contained 46 |aM choles terol in Curve B and 6 DOC in Curve C. Curve D shows the spectrum obtained 3 minutes after the addition of antimycin a (4 (jg) and isocitrate (47 mM) to the sample and reference cuvette of Curve B. 96 and reference cuvettes of curve B, the type II spectrum is immediately displaced by a type I spectrum (curve D) which, disappears with time. This result is consistent with the explanation that electron donor removes (by SCC) the endogenous and exogenous cholesterol in the sample cuvette, curve B, returning cytochrome P450 to the unbound or low spin form. 20a-hydroxycholesterol (a hydroxylatable subs trate) then combines with the low spin P450 released by SCC, activates it to the high spin state, and is itself subsequently hydroxylated as indicated by the disappear ance of the type I spectrum. Figure 15 shows the addition of pregnenolone to rat mitochondria (curve A) and rat mitochondria presaturated with cholesterol (curve B) and DOC (curve D). The result of adding isocitrate and antimycin A to the sample and reference cuvettes of curve B is shown in curve E. The increased magnitude of the type II spectrum obtained when pregnenolone is added to mitochondria presaturated with cholesterol (compare curves A and B) but not DOC (compare curves. A and C) suggests that like 20a-hydroxycholesterol, pregnenolone also combines with cholesterol-bound P450 to change it from the high spin state to the low spin state, The addition of electron donor to the sample and re ference cuvettes of curve B rapidly dissipates the type II spectrum presumably' by hydroxylating th.e endogenous and exogenous cholesterol. Unlike 20a-hydroxycholesterol, 97 /\ I 002 £ £ UJ IE U z V-B > t CO z UJ a _I < u & o -002 5 0 0 4 0 0 4 5 0 W/WE LENGTH ( r a n ) Fig. 15.— Pregnenolone-induced difference spectra of rat adrenal mitochondria. Mitochondria equivalent to 1 pM P450 were suspended in phosphate buffer. The sample cuvettes contained 11.7 pM pregnenolone in Curves A and C, 71 pM pregnenolone in Curve B. Sample and reference cuvettes contained 46 |jM cholesterol in Curve B and 6 ( j i M DOC in Curve C. Curve E shows the spectrum obtained 3 minutes after the addition of antimycin a (4 jag) and iso citrate (47 mM) to the sample and reference cuvettes of Curve B. 98 pregnenolone does not combine with the low spin P450 re leased after SCC to form a transient type I spectrum, which is compatible with the fact that it is not a substrate for the mitochondrial hydroxylase. Several experiments were done to determine whether the malate-induced type II spectra produced in bovine adrenal mitochondria results from the metabolism of endo genous cholesterol in these preparations as previously suggested (203). Figure 16 shows that a number of Kreb cycle substrates capable of supporting steroid hydroxylation induce similar type II difference spectra in bovine adrenocortical mito chondria. This demonstration supports the idea that the malate-induced type II spectrum represents 420 nm low spin P450 released after the hydroxylation of cholesterol-bound high spin P450. The broken line in Figure 17 shows the addition of cholesterol to a preparation of bovine adrenocortical sub- mitochondrial particles. The failure of exogenous chol esterol to induce a type I spectrum in these particles suggests that the SCC P450 enzyme is already bound with endogenous cholesterol. It has previously been demon strated that the addition of NADPH to these sonicated mitochondria results in a large type II difference spectrum Ccurve C, Figure 8). If this type II difference spectrum represents low spin P450 released after cholesterol SCC, 99 004 g O z E CO z UJ o _l s f c o y - 002 500 400 450 WAVELENGTH (nm) Fig. 16.— Effect of electron donors on bovine adreno cortical mitochondria. Mitochondria equivalent to 1.7 (iM P450 were suspended in phosphate buffer. Sample and re ference cuvettes contained 6.6 \xg antimycin a and 16.6 mM succinate in all curves. Sample cuvettes contained 13 mM malate in Curve A, 16.6 mM a-ketoglutarate in Curve B, 16.6 mM isocitrate in Curve C, and 1.7 mM ATP in Curve D. 100 I E 0 . 0 2 H 2 S £ CO z U l o £ -002 500 450 400 WAVELENGTH (nm) Fig. 17.— Effect of cholesterol on bovine adreno cortical submitochondria1 particles. Submitochondrial particles plus supernatant were suspended in phosphate buffer. The dashed line (-----) shows the addition of 60 |oM cholesterol to the sample cuvette. The solid line shows the addition of 60 |iM cholesterol the sample cuvette of submitochondrial particles plus supernatant incubated for 10 minutes at room temperature with 0.3 mM NADPH, followed by the addition of 3.3 mM glutathione and gluta thione reductase. 101 it was reasoned tliat pre-incubation of the submitocliondriai particles with. NADPH followed by removal of the excess NADPH with glutathione and glutathione reductase, should result in a preparation free of endogenous cholesterol- bound P450, and capable of binding exogenous cholesterol. The large type I difference spectrum now induced by exogenous cholesterol in the NADPH-treated submitochondrial particles (solid line, Figure 17), suggests that this reasoning was correct. Figure 18 shows the effects of isocitrate added to rat mitochondria (curve A), rat mitochondria presaturated with cholesterol (curve B), with DOC (curve C), and with chol esterol plus aminoglutethimide (curve D). Also shown in the spectrum obtained by adding aminoglutethimide to the sample and reference cuvettes of curve A. The intensification of the isocitrate-induced diff erence spectrum in mitochondria presaturated with either exogenous cholesterol (curve B), or DOC (curve C) suggests that the type II spectrum induced by electron donors in adrenal mitochondrial preparations can result from the metabolism of endogenous cholesterol, DOC, or presumably other P450-bound steroid intermediates contained in these preparations. This hypothesis is further supported by the demonstration that aminoglutethimide, an inhibitor of cholesterol SCC (2 0 7 ), prevents the formation of the type II spectrum (compare curves A and E) . 102 I E o £ £ IaJ c e u z >- b ( n z i i i a s £ o 500 450 400 Wfl/ELENGTH (nm) Pig. 18.— The effect of isocitrate on rat adrenal mitochondria. Rat mitochondria equivalent to 1 pM P450 were diluted with phosphate buffer. Sample and reference cuvettes in all curves contained 4 pg antimycin a and 47 mM succinate. In addition, sample and reference cuvettes contained 46 pM cholesterol in Curve B, 2.9 |iM DOC in Curve C, 44 |jM aminoglutethimide in Curve E, and 46 pM cholesterol plus 44 pM aminoglutethimide in Curve D. Sample cuvettes in all curves contained 47 mM isocitrate. 103 C. Substrate Difference Spectra in Bovine Adrenocortical Microsomes Difference spectra produced by the interaction of steroid substrates with bovine adrenocortical microsomes were studied to determine whether these organelles, like adrenal mitochondria, also contained endogenous P450-bound steroid intermediates. The maximum 420 nm absorption changes produced by the addition of 17a-hydroxyprogesterone, pregnenolone, and 17a-hydroxypregnenolone to bovine adrenal cortex micro somes are shown in Table IV. The positive values indicate that all substrates produce type I difference or high spin P450 except pregnenolone, which, as indicated by the negative absorbancy difference, forms a small type II spectrum or low spin P450. The large type II spectrum induced by NADPH is similar to the malate-induced diff erence spectrum in bovine adrenal mitochondria and suggests that these microsomal preparations also contain endogenous P45Q-bound steroid intermediates. Partial removal of the bound intermediates by success ive washing of the microsomal preparation (three washes) increases the type I spectrum formed by each intermediate. This is illustrated in Tahle IV by the increased optical density values and in Figure 19 by the increased type I spectrum induced hy one of these substrates, 17a-hydroxy- progesterone ( c u r v e b compared to curve A). Successive TABLE IV 104 BOVINE ADRENAL CORTEX MICROSOMAL P450 Effects of steroids on bovine adrenocortical micro somes. Microsomes equivalent to 1 jaM P450 were suspended in phosphate buffer (16 mM, pH 7.2) containing MgCl2 (6 mM) and KCl (96 mM). Difference spectra were produced by titrating steroids into the sample cuvette until the maximum absorbancy change was reached. Washes were achieved by resuspending the particles in 0.25 M sucrose-1% albumin and sedimenting at 100,000 x g for 30 minutes. NADPH- induced difference spectra were obtained by adding 200 |jM NADPH to the sample cuvette and 150 nM NADH to both the sample and reference cuvettes. ^450nm ” A420nm SUBSTRATE (mm) ONE WASH THREE WASHES 0.65mM NADPH + THREE WASHES PROGESTERONE 12 .0117 .0190 .0226 17-OH PROGESTERONE 12 .0170 .0292 .0331 PREGNENOLONE 6.7 -.0013 .0036 .0101 17-OH PREGNENOLONE 15 .0113 .0204 .0287 NADPH 200 -.0317 — — 17-OH PROGESTERONE + 27 — — .0390 NADPH 200 —— .0072 105 i i I J 0 2 k < j z $ o z CO z U J a - I < u l - o . o 4 0 0 4 5 0 5 0 0 WAVELENGTH (nm) Fig. 19.— Effect of 17a-hydroxyprogesterone on bovine adrenocortical microsomes. Microsomes equivalent to 1 |jM P450 were suspended in phosphate buffer. Sample cuvettes contained 12.7 (iM 17a-hydroxyprogesterone in Curve A and 12.1 |oM 17a-hydroxyprogesterone in Curve B. The micro somes in Curve B were washed once by resuspending in sucrose-albumin (0.25-1%) and resedimenting at 100,000 x g for 30 minutes. Curve C shows the spectrum obtained 3 minutes after the addition of 200 nM NADPH to the sample and reference cuvettes of Curve B. 106 washing also changed the pregnenolone-induced spectrum from type II to type I as indicated by the positive value in Table IV and by the difference in curves B and D, Figure 20. As indicated in the last column in Table IV, depletion of endogenous substrates in these microsomal preparations by pre-incubation with NADPH in the presence of oxygen followed by washing out of the reduced cofactor, further increased the amount of high spin P450 formed by all of the steroid intermediates. The type I difference spectrum formed by 17a-hydroxy- progesterone was rapidly dissipated in the presence of NADPH as shown in Table IV and in curve C, Figure 19. Similar results were obtained when NADPH was added to the type I difference spectra formed by the other microsomal substrates. D. Substrafe Metabolism by Rat Adrenal Mitochondria All reported measurements of the in vitro rate of cholesterol SCC have indicated that this reaction is several times slower than the rate of llg-hydroxylation. Based on these measurements, it has been postulated that ACTH stimulates adrenal steroidogenesis by increasing the activity of the inherently low cholesterol SCC enzyme system (.17) . Two observations in the preceeding experiments sugg ested that the rate of cholesterol SCC, in the contrast 107 b o 002 1 ae . o z < / > z UJ o u I - a. o -Q02 400 500 450 WAVELENGTH (nm) Fig. 20.— Effect of pregnenolone on bovine adreno cortical microsomes. Microsomes equivalent to 1 | j iM P450 were suspended in phosphate buffer. Sample cuvettes con tained 13.4 (jtM pregnenolone in Curve A, 107 |aM pregnenolone in Curve B, 8.0 pM pregnenolone in curve C and 128 [oM pregnenolone in Curve D. The microsomes in Curve C and D were washed once by resuspending in 0.25 M sucrose-1% albumin and resedimenting at 100,000 x g for 30 minutes. 108 to previous reports, may be quite rapid the similar time course (4 minutes) required for the type II difference spectra obtained by adding electron donor to mitochondria presaturated with exogenous cholesterol and with exogenous DOC to reach a maximum (Figure 18, curves B and C), and the rapid disappearance (3 minutes) of the type I spectrum induced by high concentrations of exogenous cholesterol in the presence of electron donor (Figure 12). The low cholesterol SCC activity previously reported may have been due to high concentrations of endogenous cholesterol-bound P450 in the mitochondrial preparations, or to inadequate binding of the ethanolic solutions of cholesterol (used in most of these studies) to cytochrome P45Q.-For this reason, the relative rates of cholesterol* SCC, 20a-hydroxycholesterol SCC, and DOC llB-hydroxylation were re-investigated in rat adrenal mitochondria under conditions where the substrates for these enzyme systems were pre-bound to their P450 enzymes. An initial investigation of the cholesterol SCC re action indicated that the rate was significantly increased by a number of agents. The effects of these agents on the 15 second rate are shown in Figure 21. The control rate was obtained with rat adrenal mitochondria pre-incubated with 78 pM cholesterol for 10 minutes at 37°C followed by the addition of NADP and NAD. Side-chain cleavage was initiated in all cases with 50 mM isocitrate. 109 Fig. 21.— Effects of various agents on the rate of cholesterol SCC. Mitochondria equivalent to 0.15 mg N were preincubated with 78 piM cholesterol at 37°C for 10 minutes followed by the addition of 0.45 mM NADP and 5 mM NAD (Control). Side-chain cleavage was initiated in all curves by the addition of 50 mM isocitrate. Other addi tions: antimycin a (4 ng), NADPH (10 mM), NADPH generator (14 mM glucose-6-phosphate, 10 mM NADP, and 1 e.u. glucose- 6-phosphate dehydrogenase), microsomes (0.017 mg N), and CaCl2 (10 mM). 110 As shown by the second bar, the rate was significantly increased when the respiratory chain was inhibited by antimycin A. Although isocitrate provides NADPH for the hydroxylating pathway directly, via a NADP-linked dehydro genase, this experiment suggests that the respiratory chain is competing either for isocitrate (via NAD-linked isocitrate dehydrogenase) or for NADPH via NADPH cyto chrome c reductase. Bars 3 - 5 depict the stimulatory effect of NADPH on the rate of cholesterol SCC. NADPH apparently does not increase the rate by entering the mitochondria since there is a significant reduction of the rate in the absence of isocitrate (compare bars 4 and 9) and since the mitochondr ial membrane has to be disrupted (with 10 mM calcium) before NADPH will support the reaction in the absence of isocitrate (bar 8). Based on these results, it appears that NADPH reduces some extra-mitochondrial component which is either inhibitory to the SCC reaction in the oxi dized state, or is able to stimulate the rate in the re duced state. NADPH reduction of adrenodoxin reductase, which may have leaked out of the mitochondria during their isolation, would be compatible with these results provided the flavoprotein, following its reduction, is able to re enter the mitochondria and participate in the remaining redox reaction leading to hydroxylation. The ability of the microsomes to stimulatethe reaction Ill (bar 6) appears to be related to an indirect action such as increasing the rate of removal of pregnenolone (an inhibitor of cholesterol SCC) from the mitochondria, since the rate with microsomes alone (in the absence of mitochondria) was dramatically decreased (bar 10 compared to bar 6) . While the rate of 20a-hydroxycholesterol SCC was similarly stimulated by NADPH and by the addition of micro somes, the rate of 110-hydroxylation was slightly inhibited by these agents as shown in Figure 22. These results are consistent with the idea that a common P450 enzyme is in volved in cholesterol and 20a-hydroxycholesterol SCC, and that this enzyme is distinct from the 113-hydroxylase. Figures 23, 24, and 25 show saturation curves for rat mitochondrial SCC of cholesterol and 20a-hydroxychol- esterol, and for llg-hydroxylation of DOC. . Also shown are the linear plots of these saturation data from which the apparent Km and V for each reaction was calculated. max The calculated Km values are 90.0, 20.0 and 8.0 yM for cholesterol, 20a-hydroxycholesterol, and DOC, respectively. The Vmax for cholesterol SCC (1,000 nmoles products/min- mgN) and 20a-hydroxycholesterol SCC (910 nmoles products/ min/mgN) are approximately four times greater than the f°r 113-hydroxylation (250 nmoles/min/mgN). IUcLX Progress curves of the three reactions are shown in Figures 26 and 27. Whereas corticosterone is the only 112 30 20 8 S o • » 9 ’ S < < + + <M * + C M * X a . 3 z + * £ + # Fig. 22.— Effects of various agents on the rate of DOC llj3-hydroxylation. Mitochondria equivalent to 0.115 mg N were preincubated with 10 \xM DOC for 10 minutes at 37°C, followed by the addition of 0.45 mM NADP (Control). Other additions: antimycin a (4 ng), bovine serum albumin, BSA, (0.1%), NAD (5 mM), NADPH generator (14 mM glucose-6-phos- phate, 10 mM NADP, and 1 e.u. glucose-6-phosphate dehydro genase) microsomes (0.017 mg N). 113 800- ao Z s i 600- < k o. i n XIO' 4.0- Z 400- s c "^20 20 40 60 l/S X KJ3 200 200 400 600 liM CHOLESTEROL Fig. 23.— Cholesterol saturation curve. Mitochondria equivalent to 0.109 mg N were incubated with 15.6 to 623 pM cholesterol containing 0.25 (jiCi of cholesterol-4-14c. Other conditions and quantitation of steroids are described under Methods. 114 800 to 80' i / i 3 I C 200 400 6&> I/S X 10s -200 20 40 60 1M20H-0H CHOLESTEROL Fig. 24.— 20a-hydroxycholesterol saturation curve. Mitochondria equivalent to 0.080 mg N were incubated with 2.4 to 72 pM 20a-hydroxycholesterol containing 1.25 jiCi °f 20a-hydroxycholesterol-73H. Other conditions and quantita tion of steroids are described under Methods. 115 300 ec «/> 5200 ec 100 - M 0 40 liM DOC 0.20 S/yO.IO 20 40 Fig. 25.— DOC saturation curve. Mitochondria equi valent to 0.12 mg N were incubated with 2 to 50 pM DOC containing 0.062 pCi of DOC-.^C according to the pro cedure described under Methods. 116 300 - 200 100 P o la r -x -P re g ■•-Prog • P r o g • P r o g • P o la r 30 60 30 60 S E C O N D S z l c c 900 600 300 200 Sfc(5 550" 30 60 S E C O N D S Fig. 26.— Cholesterol side-chain cleavage progress curves. 26A. Mitochondria equivalent to 0.115 mg N were incubated with 78 ^M cholesterol under conditions described under Methods, except microsomes were omitted from the incubate. 26B. Same as 26A, except microsomes equivalent to 0.017 mg N were added. 26C. Same as 26B, except the cholesterol concentration was increased to 600 |iM. 26D. Same as 26B, except the reaction was sampled over a longer time period. 117 iso i £ ■Polar Prog z 100- m 90 - i 30 SECONDS 300 ■ S o o r o o z 200 9 z m i c 100 30 60 SECONDS ! i i Pig. 27.— 20a-hydroxycholesterol SCC and 110-hydroxy- lation progress curves. 27A. Mitochondria equivalent to 0.115 mg N were incubated with 24 |jM 20a-hydroxycholesterol as described under Methods, except microsomes were omitted from the incubate. 27B. Same as 27A, except microsomes equivalent to 0.017 mg N were added. 27C. Mitochondria equivalent to 0.12 mg N were incubated with 60 ^M DOC as described under Methods. 118 product resulting from the 118-h.ydroxylation of DOC (.Fig- ure 27, curve C) , the side-chain cleavage of cholesterol — and 20a-hydroxycholesterol results in the formation of sev eral products (Figure 26 and 2 7, curves A and B). The cholesterol and 20a-hydroxycholesterol curves both show a rapid fall-off in the reaction rates with time. The gradual increase in product formation up to 10 minutes after the initiation of cholesterol SCC (curve D, Figure26) indicates that the fall-off is not caused by an inactivated hydroxylase system. The rate fall-off also appears not to be due to end-product inhibition by pregnenolone since the addition of microsomes significantly decreased the concentration of pregnenolone formed in the incubates (curves B, Figures 26 and 27), but failed to correct the non-linear rates. E. Effects of Inhibitors and ACTH on Substrate Metabolism Table V shows the effects of 20a-hydroxycholesterol, pregnenolone, and aminoglutethimide on the side-chain cleavage of cholesterol and the effect of ACTH on the Km and V v values calculated from cholesterol and 20a- max hydroxycholesterol SCC and DOC llg-hydroxylation rates. 20a-hydroxycholesterol appears to competitively in hibit cholesterol SCC in this study since the cholesterol Km is increased almost eight times and the Vma:xis unchanged in the presence of 20 pM of this steroid. Although these data are in agreement with two previous reports (24,106), TABLE V 119 EFFECTS OF INHIBITORS AND ACTH ON SUBSTRATE METABOLISM BY RAT ADRENAL MITOCHONDRIA Effects of inhibitors and ACTH on substrate metabolism by rat adrenal mitochondria. Mitochondria isolated from the adrenals of normal rats, hypophysectomized (44 hours) rats, and hypophysectomized rats treated with ACTH (100 munits for 10 minutes), were incubated with cholesterol, 20a-hydroxycholesterol, and DOC as described under Methods. Km and Vmax values were calculated from linear plots of the saturation data as shown in Figures 23, 24, and 25. Normal ]% and Vmax values are means + standard error of the mean. SUBSTRATE CONDITION (pM) vmax nmoles products or corticosterone/min/ mgN Cholesterol Normal 129 + 11 1,131 + 125 + 20a-OH Choi. (20 pM) 800 1,250 + Pregnenolone (50 pM) 190 1,650 + Aminoglutethimide (25 pM) 143 334 Cholesterol Hypox'd 100 1,250 Cholesterol Hypox'd + ACTH 125 1,000 2 0 a-Hydroxycho1. Normal 35 + 5 938 + 52 2 0 a-Hydroxycho1. Hypox1d 22 936 DOC Normal 5 + 1 219 + 7.5 DOC Hypox'd 6 582 120 it would appear that the ability of 20a-hydroxycholesterol to inhibit cholesterol SCC in a competitive or non-competi tive manner would depend on the experimental design, since it has been shown (Figure 14) that this sterol can combine both free cytochrome P450 (E) as well as with cholesterol- bound P450 (ES). In the study presented in Table V, 20a- hydroxycholesterol and cholesterol were both added in the 10 minute pre-incubation period. Since 20a-hydroxy- cholesterol apparently has a greater affinity for free cytochrome P450 (Km = 20 yM) than does cholesterol (Km = 90 yM), it is preferentially bound to the enzyme and com petitive inhibition of cholesterol SCC results. If, how ever, 20a-hydroxycholesterol had been added after the 10 minute pre-incubation period, when the cholesterol-P450 enzyme-substrate had been maximally formed, non-competitive inhibition of cholesterol SCC should have resulted since 20a-hydroxycholesterol would be inhibiting the reduction of P450 by combining with the high spin ES complex in this case and not with the free enzyme. The lack of inhibition by 50 yM pregnenolone in the study shown in Table V is due, a p p a r e n t l y ,to-the metabolism of this steroid during the pre-incubation period by the 36-hydroxysteroid dehydrogenase and isomerase present in the mitochondria. In other studies 20 yM pregnenolone added after the pre-incubation period resulted in a 54% inhibition of cholesterol SCC. In the latter case, since 121 pregnenolone was added after the cholesterol-P450 complex was allowed to form, the inhibition is probably non competitive and results from a pregnenolone-induced con version of cholesterol-bound high spin P450 to the inactive non-reducible low spin state as demonstrated earlier (Fig ure 15) . Aminoglutethimide interacts with bovine and rat adrenal mitochondria to produce shifted (peaks at 420 nm and troughs at 392 nm) type II difference spectra (93). These type II spectra are apparently different than the ones produced by 20a-hydroxycholesterol and pregnenolone in adrenal mitochondria since the ESR signals of low spin ferric P450 are not just increased in the presence of aminoglutethimide, but are shifted to even lower fields (93). McIntosh and Salhnick (109) have shown that the -NJ^ group of aminoglutethimide is probably responsible for its inhibitory action and that this agent, as well as other nitrogen-containing compounds such as amphenone and mety- rapone, inhibit steroid hydroxylation by preventing the reduction of cytochrome P450. The results in Table V which show that 25 yM aminoglutethimide non-competitively inhibits cholesterol SCC (as judged by the constancy of the cholesterol Km and the almost four-fold decrease in the V of the reaction) are consistent with the McIntosh and max Salhanick data. ACTH appears to have no direct effect on the enzyme 122 system involved in the side-chain cleavage of cholesterol and 20a-hydroxycholesterol, since the Km and Vmax values obtained from the incubation of these substrates with mitochondria from normal rats and from long-term hypo physectomized rats were essentially the same. The signi ficance of the increased V value obtained by incubating nicix DOC with mitochondria isolated from hypophysectomized rats compared to mitochondria from normal rats is not clear although the data are reproducible. F. In Vivo Effects of ACTH and Cycloheximide on Endogenous Cholesterol' and DOC-Bound P450 The low level of endogenous cholesterol-bound highspin; P450 contained in these rat adrenal mitochondria, together with the high side-chain cleaving activity of cholesterol- bound P450, suggested that the in vivo production of corticosteroids may be stimulated by an ACTH-mediated transport of cholesterol to the SCC P450 enzyme. To test this hypothesis, the levels of endogenous cholesterol and DOC-bound P450 in mitochondria isolated from hypophy sectomized rats, and hypophysectomized rats treated with ACTH were compared. The levels of endogenous cholesterol-bound P450 in the various mitochondrial samples were compared by asse ssing the magnitude of the type I spectra induced by exogenous cholesterol and by the type II spectra induced by 20a-hydroxycholesterol and by pregnenolone. The 123 magnitude of the type I spectra induced by exogenous DOC was used to compare the various levels of endogenous DOC- bcund P450, and the magnitude of the isocitrate-induced type II spectra to compare the levels of both endogenous cholesterol and DOC-bound P450. The results of these experiments are summarized in Table VI. The primary effect of ACTH administered to hypo physectomized rats was to increase the concentration of endogenous DOC-bound P450 as judged by the 40% decrease in the type I spectrum obtained when DOC was added to the mitochondrial preparation (0.0068 compared to 0.0110). Endogenous cholesterol-bound P450 was also increased in these ACTH-treated samples, but to a lesser degree since: 1) the exogenous cholesterol-induced type I spectrum showed only a 25% reduction (0.0168 compared to 0.0225), 2) there was no change in the pregnenolone-induced type II spectrum as seen by comparing the treated and control values (-0.0089 and -0.0088), and 3) 20a-hydroxycholesterol continued to produce a type I spectrum. The increased isocitrate-induced type II spectrum (-0.0142 compared to -0.0044) reflects the higher levels of both cholesterol and DOC-bound P450. When hypophysectomized rats were stimulated with ACTH and subsequently the adrenal gland made anoxic, the amount of endogenous cholesterol-bound P450 was significantly in creased as indicated by the 60% decrease in the cholesterol TABLE VI In vivo effects of ACTH and cycloheximide on levels of endogenous cholesterol and DOC-bound P450. Cholesterol, DOC, 2Oa-hydroxycholesterol, and pregnenolone were each titrated into the sample cuvettes of mitochondrial suspensions containing 1 pM P450 in 16 mM phosphate buffer, pH 7.2 until the maximum absorbancy change was reached. The isocitrate-induced difference spectra were obtained by adding succinate (47 mM) and antimycin a (4 pg) to both the sample and reference cuvettes and 50 mM isocitrate to the sample cuvette only. Other conditions are described under Methods. Normal values shown are means + standard error of the mean. MAXIMUM P.P. A (450 - 420nm) pM P-450 Condition Cholesterol DOC Pregnenolone 20cOH Choi Isocitrate Normal .0203 +.0021 .0114 +.0008 (-).0066 + .0006 .0036 +.0008 (-).0078 + .0007 Hypox'd .0225 .0110 (-).0088 — (-).0044 Hypox'd, ACTH .0168 .0068 (-).0089 .0033 (-).0142 Hypox'd, Anoxia .0205 .0162 .0021 .0024 (-).0093 Hypox'd, ACTH, Anoxia .0086 .0110 (-).0055 (-).0140 (-).0292 Normal, Cycloheximide .0092 .0104 (-).0112 (-).0008 (-).0214 Normal, ACTH, Anoxia .0142 .0118 — (-).0076 (-).0156 125 induced type I spectrum (0*0086 compared to 0-0205), and also by the fact that 20 a-hydroxycholesterol and preg nenolone now induce type II spectra. The amount of endo genous DOC-bound P450 was also increased as shown by the 30% decrease in the DOC-induced type I spectrum (0.0110 compared to 0.0162). The large increase in the isocitrate- induced type II spectrum (-0.0292 compared to ^0.0093) is compatible with the increased levels of both cholesterol and DOC-bound P450. When mitochondria are isolated from normal rats treat ed with cycloheximide, the amount of endogenous cholesterol- bound P450 is increased as indicated by the 55% decrease in the type I spectrum produced by exogenous cholesterol CO.0092 compared to 0.0202), and also by the increased pregnenolone, 20 a-hydroxycholesterol, and isocitrate-in duced type II spectra. The amount of endogenous DOC- bound P450 was not increased since there was relatively no change in the intensity of the type I spectrum induced by exogenous DOC (0.0104 compared to 0.0114). Mitochondria isolated from normal rats treated with ACTH and anoxia gave results similar to the ones obtained with cyclo- heximide. The results of these experiments which show that the levels of endogenous DOC and cholesterol-bound P450 are increased in mitochondria isolated from hypophysectomized rats treated with ACTH indicate that ACTH activates 126 ' steroidogenesis by transporting cholesterol to the SCC P450 enzyme, and that under conditions supporting hydro- xylation (NADPH and oxygen), this cholesterol is rapidly hydroxylated. The rapid SCC activity is further evidenced by the fact that the amount of endogenous cholesterol- bound high spin P450 is significantly increased only when hydroxylation is inhibited by anaerobiosis, or if the animals are pretreated with cycloheximide. The cyclo heximide data are compatible with the explanation that this agent causes a build-up of endogenous cholesterol- bound P4S0 by inhibiting cholesterol SCC (143). CHAPTER V DISCUSSION The exact mechanism by which ACTH activates the side- chain cleavage of cholesterol, the proposed rate-limiting reaction in the biosynthesis of adrenal corticosteroids (13), is not understood. It has been proposed that ACTH facilitates the reaction by: 1) transporting cholesterol, via a labile protein, from the adrenal cytoplasm into the mitochondria (147), 2) transporting pregnenolone, an inhibitor of cholesterol SCC, out of the mitochondria (136), 3) increasing the inherently low activity of the cholesterol hydroxylase system, presumably by changing the Km or V x of the SCC reaction (17), and 4) providing NADPH to the system (169). The rate-limiting step in the overall steroid hydro- xylation mechanism is thought to be the reduction of subs trate-bound P450 by NADPH (200,208). Based on this hypothesis, experiments were done to determine whether cholesterol SCC is limited by the rate of generation of intramitochondrial NADPH, or whether the SCC enzyme system is activated by an ACTH-mediated transport of cholesterol to the SCC P450 enzyme. Ihtramitochondrial NADPH Production Failure of the steady-state concentrations of the 127 128 oxidized and reduced pyridine nucleotides and of the Krebs cycle substrates to change at any time after the adminis tration of ACTH to hypophysectomized rats suggested that none of the NADPH-producing mitochondrial dehydrogenases or the pyridine nucleotide transhydrogenase were rate- limiting, and therefore activated by ACTH, prior to the increase in plasma corticosterone. A subsequent study of the NAD and NADP distribution in the adrenal showed, how ever, that one third of the oxidized pyridine nucleotides were contained in the mitochondria compared to the whole homogenate. This indicated that the changes in the oxidized/reduced pyridine nucleotide ratio following ACTH stimulation could have been masked by reciprocal changes occurring simultaneously in the cytoplasm and mitochondria. For example, an ACTH-mediated reduction of most of the mitochondrial pyridine nucleotides, with a concomitant oxidation of 30% of the cytoplasmic cofactors would not be detected in measurements of the total adrenal pyridine nucleotides. Reciprocal changes, such as the one cited above, occurring prior to the stimulation of steroidogenesis, would of course, by compatible with the hypothesis that ACTH increases corticoid production by increasing the rate of generation of intramitochondrial NADPH. Since none of the activities of the NAD and NADP-linked dehydrogenases appeared to be rate-limiting (as judged by the lack of 129 change in the Kreb cycle substrates), the increase in intramitochondrial NADPH, if it occurs, would probably result from an ACTH-induced activation of the energy-linked pyridine transhydrogenase which converts NAD to NADPH. The production of NADPH via the energy-linked transhydro genase instead of by the NADP-linked isocitrate dehydro genase, would appear to be an unsuitable mechanism for the adrenal cell to adopt in response to stress, since the former pathway, but not the latter, would probably severe ly comprise the energy metabolism of the cell by competing for high energy intermediates and NADH from the pools available to oxidative phosphorylation (81). For this reason, it is concluded that the rate of generation of intramitochondrial NADPH is probably not rate-limiting in steroidogenesis. Data reported by several other in vestigators are consistent with this view. Harding and Nelson (129) reported no change in the rat adrenal NADPH/NADP ratio at a time when corticosteroid secretion had dropped almost to zero following hypophy- sectomy. These investigators also found that hypophy- sectomy did not significantly alter the activities of rat adrenal isocitrate dehydrogenase or the pyridine nucleo tide transhydrogenase (130). Greenberg and Glick (2Q9) reported that the administration of ACTH caused significant increases in oxidized pyridine nucleotides in all zones of the adrenal gland but no demonstrable effect on the reduced 130 pyridine nucleotide levels. Peron and McCarthy (131) found; the same patterns of pyridine nucleotide content in rat adrenal mitochondria isolated from normal, hypophysectomiz ed, and hypophysectomized animals treated with ACTH. Monitoring the adrenal gland fluorescence of hypophy sectomized rats, Chance, et al., (210) observed a rapid oxidation of pyridine nucleotide fluorescence immediately following the injection of one and ten milliunits of ACTH. ACTH-Induced Cyclic AMP Accumulation and Activation of Glycolysis In this study the concentration of adrenal cyclic AMP was increased ten seconds after the administration of 100 milliunits of ACTH to hypophysectomized rats. The ACTH- mediated increase in cyclic was followed by an activation of glycolysis which was indicated by an increase in the activities of phosphofructokinase at 20 seconds, and glyceraldehyde phosphate dehydrogenase at 2 minutes after ACTH. The increased concentration of cyclic AMP and the activation of glycolysis clearly preceeded the increased rate of corticosterone production, suggesting that the latter process may have been activated by a process which affected the entire energy metabolism of the adrenal cell. A later finding which showed that corticosterone production was increased at lower doses of ACTH while the activities of phosphofructokinase and glyceraldehyde phosphate were not, indicated that glycolysis activation 131 and steroidogenesis maybe associated or causally related only at high doses of ACTH. Beall and Sayers (19 7) have observed that low doses of ACTH (5-25 yunits) stimulated steroidogenesis in iso lated adrenal cells without causing detectable changes in the cyclic AMP concentration. Intermediate doses of ACTH (50-250 yunits) induced parallel increases in cyclic AMP and corticosterone accumulation, while large doses of ACTH (250-10,000 yunits) caused additional increases in the concentration of cyclic AMP without causing further increases in corticosterone production. In addition, the in vivo studies reported by Grahame-Smith et al., (158) showed that there is a 25-fold difference between the dose of ACTH required to acutely and maximally stimulate steroidogenesis, and the dose required to produce maximal accumulation of adrenal cyclic AMP. These observations suggest that the ACTH-induced accumulation of cyclic AMP measured in the present study may mediate the activation of glycolysis by acting as a positive effector of phos phof ructokinase, and that these events are not causally related to the increased rate of corticoid secretion produced by low doses of ACTH. Kowal (211), who reported an in vitro activation of glycolysis by ACTH, also con cludes that this activation is not directly related to the ACTH-induced increase in steroidogenesis. 132 ; Spin State Changes Induced In Cytochrome P450 . The variable formation of type I spectra by substrates capable of being hydroxylated (212,213), the formation of both type I and type II spectra by 20a-hydroxycholesterol (198), and the formation of type II spectra by malate (93) and pregnenolone (199) in adrenocortical preparations have been difficult to reconcile with the proposed substrate and inhibitor-induced control of P450 reduction and steroid hydroxylation, and have caused a number of investigators to question the specificity of the spectral changes and therefore their relevance to the P450 reaction mechanism. In the present study, it was discovered that rat adrenal mitochondria contain lower levels of endogenous cholesterol-bound high spin P450 than do bovine adreno cortical mitochondria and submitochondrial particles. Consistent with this observation, soluble cholesterol- lecithin emulsions added to these rat preparations pro duced large type I difference spectra which disappeared rapidly in the presence of electron donor and oxygen. Taking advantage of the fact that high spin SCC P450 could be induced in these rat adrenal mitochondria, explanations for the anomalous difference spectra produced in adrenal mitochondria by 20a-hydroxycholesterol, pregnenolone, and electron donors were sought. It was demonstrated that the type II difference spectra: induced by pregnenolone in bovine and rat adrenal 133 mitochondria are due to the binding of this steroid to endogenous cholesterol-bound high spin P450 to convert it to the inactive low spin state. Pregnenolone does not interact with DOC-bound high spin P450, nor, apparently, can it bind with cholesterol-free P450 since no spectral effects are observed with this steroid in the presence of low spin P450 released by the electron donor-induced metabolism of cholesterol (curve E, Figure 15), or when it (pregnenolone) is added to a cholesterol-depleted prepara tion of bovine adrenocortical submitochondrial particles (205) . The substantial inhibition of cholesterol SCC ob tained in this study when pregnenolone was added to mito chondria presaturated with exogenous cholesterol (to allow .maximum formation of high spin P450) , is consistent with the proposal that this steroid inhibits the reduction of cholesterol-activated high spin P450 by changing it to the inactive low spin state. The 20a-hydroxycholesterol-induced type II spectrum produced in bovine adrenocortical mitochondria was shown also to result from the conversion of cholesterol-bound high spin P450 to the inactive low spin state, thus con firming a previous report (110). This sterol, like pregnenolone, did not interact with DOC-bound high spin P450. In the presence of electron donor, the type II spectrum induced by 20a-hydroxycholesterol was rapidly 134 dissipated and replaced by a transient type I spectrum. These results are compatible with the explanation that 20a-hydroxycholesterol binds to the low spin SCC P450 (released by the electron donor-induced metabolism of cholesterol), activates it to the high spin state, and is itself hydroxylated (indicated by the disappearance of the type I spectrum with time). This explanation has been confirmed by recently reported results (214), and is consistent with the competitive inhibition of cholesterol SCC which was obtained in the present study when mito chondria were pre-incubated with cholesterol and 20a- hydroxycholesterol prior to the addition of electron donor. Type II difference spectra induced by the addition of electron donors to bovine and rat adrenal mitochondria were shown to represent low spin P450 released by the hydroxylation of endogenous cholesterol, DOC, or pre sumably other P450-bound steroid intermediates contained in these preparations. These data therefore provide an explanation for the significant rates of oxygen consump tion observed when malate is added to bovine adrenocortical mitochondria in the presence of various respiratory chain inhibitors (77) . A similar conclusion regarding the difference spectra induced by. electron donors in adrenal mitochondria has been reached by Simpson et\ hi, (215) . In contrast to the data obtained in this study, however, these investigators 135 attributed the type II spectra induced by. electron donor in their bovine adrenocortical mitochondria solely to low spin SCC P450 released by the metabolism of endogenous cholesterol. The fact that endogenous DOC (or some other steroid) -bound P450 was also present in their mito chondrial preparation is suggested by the type II spectrum they obtained when malate was added to mitochondria pre saturated with pregnenolone. This spectrum was thought by these investigators to represent a malate-reducible b-type cytochrome present in bovine adrenal cortex mito chondria along with SCC high spin P450. Evidence has also been presented which indicates that the type II spectrum induced by the addition of NADPH to bovine adrenal microsomes results from the metabolism of endogenous P450-bound steroid intermediates contained in these preparations as well. In analogy with the sit uation in adrenal mitochondria, these endogenous P450-bound intermediates were shown to greatly alter both the magni tude and kind of difference spectra induced by exogenous steroid substrates added to bovine adrenocortical micro- somes. In addition to providing support for the specificity of the substrate and inhibitor-induced spectral changes produced in P450 preparation by explaining a:number of anomalous spectral results previously reported, these experiments also suggested a qualitative method ofdetecting, 136 cholesterol-bound high spin P450 which was later used to determine the effects of ACTH on cholesterol transport to SCC P450. Substrate Metabolism by Rat Adrenal Mitochondria The fact that the rat mitochondria used in this study contained low levels of endogenous cholesterol-bound P450 and exogenous cholesterol induced high spin P450 in these preparations, allowed the rates of cholesterol SCC and 113- hydroxylation to be examined under conditions where the substrates for these systems were pre-bound to their P450 enzymes. Since it had been established that substrate binding to P450 increases its reducibility, this method of studying the hydroxylases was considered essential for assessing their true activity for determining the effects of ACTH on this activity. The results of these studies indicate that the maxi mum rate of cholesterol SCC is approximately four times greater than the maximum rate of DOC 113-hydroxylation. ACTH seems to have no direct effect on the side-chain cleaving activity of the cholesterol P450 enzyme since similar Km and V values were calculated from chol- max esterol incubations using mitochondria isolated from normal rats, hypophysectomized rats, and hypophysectomized rats, treated with ACTH. : These findings are substantially different from earlier studies which show that the rate of 1 1 3 -hydroxylation is several times faster than the rate of 137 cholesterol SCC. The slow side-chain cleaving rates prev iously reported can probably be attributed to the high levels of endogenous cholesterol-bound P450 contained in the mitochondrial preparations used, and/or to the in ability of ethanolic cholesterol solutions to induce high spin P450 in these preparations. Km values calculated from these in vitro cholesterol and DOC incubations (129 yM cholesterol and 6.0 yM DOC) suggest that the 113-hydroxylase has a much higher affinity for its substrate than does the SCC P450 enzyme. To determine whether this affinity difference might be related to solvent effects, Km values were calculated from in cubations using ethanolic solutions of 20a-hydroxychol- esterol and 20a-hydroxycholesterol-lecithin emulsions. Both incubations produced similar Km values. The low affinity of the SCC P450 enzyme for chol esterol could still, of course, be related to competition between P450 and the cholesterol-lecithin micelles, or to an altered cholesterol binding site resulting from the preparation of the mitochondria. However, the even lower affinity of the liver microsomal P450 enzyme for its substrates (mM range), and the varying abilities of several oxygenated cholesterol derivatives to bind to the SCC P450 enzyme (213).suggests that the affinity difference between this enzyme and the 113-hydroxylase may be related to the more hydrophobic structure of the cholesterol 138 molecule. The competitive inhibition of cholesterol SCC by 20a- hydroxycholesterol, and several similarities in the data obtained from the cholesterol and 20a-hydroxycholesterol incubations point to these two substrates being hydroxy- lated by the same enzyme system. The Vmax values calcu lated for both reactions are similar, both reactions are similarly affected by NADPH and microsomes, and progress curves indicate a rapid fall-off in the rates of both 20a-hydroxycholesterol and cholesterol SCC with time. The fall-off in the rates of these reactions appears not to be due to end-product inhibition by pregnenolone since the addition of microsomes significantly decreased the concentration of pregnenolone formed in the incubates, but failed to correct the non-linear rates. The addition of several agent, including cyclic AMP, albumin, and inc reased concentrations of isocitrate, NAD, and NADP, were all without effect on the kinetics of the reactions. Other experiments showed that the non-linear rates were not caused by aging of the mitochondria, and lecithin seemed not to be inhibitory since similar progress curves were obtained from incubations using ethanolic solutions of 20 a-hydroxycholesterol. Koritz and Kumar (136), and more recently Brownie et al., (216) have reported a similar fall-off in the rate of pregnenolone formation from endogenous cholesterol in rat 139 adrenal mitochondria. Brownie e_t al., have suggested that this fall-off is due to the slow rate of cholesterol binding to the SCC P450 enzyme. This explanation seems unlikely in the present study since the mitochondria were pre-incubated with exogenous cholesterol (until the maximum high spin P450 was formed) prior to the initiation of hydroxylation, and since similar kinetics were observed for the SCC of 20a-hydroxycholesterol even though this substrate appears to form high spin P450 as readily as DOC does (198). Since linear rates of cholesterol SCC are observed in a reconstituted system consisting of acetone-extracted submitochondrial particles, adrenodoxin, and adrenodoxin reductase (176), an alternative explana tion for these data is that either the non-heme iron pro tein or flavoprotein of the electron transport system may have become rate-limiting during the preparation of the mitochondria. The results of these hydroxylation studies have con tradicted two of the mechanisms previously proposed for the limitation of adrenal steroidogenesis. The rapid con version of pregnenolone to products in the presence of microsomes, and the inability of pregnenolone to inhibit cholesterol SCC except under conditions where the chol- esterol-P450 complex is allowed to build up (a situation that wouldn't exist in vivo in the presence of reducing equivalents),suggest that pregnenolone can pass unrestricted 140 through, the outer mitochondrial membrane and does not normally act as an end-product inhibitor of cholesterol metabolism. The activity of the cholesterol hydroxylase system appears not to be inherently low as a number of pre vious studies have indicated, and ACTH seems to have no effect on either the ICm or V of the SCC reaction. max ACTH Activation of Cholesterol Transport to Cytochrome P450 Since the rate of corticosteroidogenesis appeared not to be limited by the rate of generation of intra- mitochondrial NADPH, and since ACTH seemed to have no effect on the cholesterol hydroxylase system per se, experiments were done to determine whether ACTH activated steroidogenesis by transporting cholesterol to the SCC P450 enzyme. The results of these experiments which showed that ACTH stimulation of hypophysectomized rats increased the level of both endogenous cholesterol and DOC-bound P45Q in the mitochondria isolated from these animals, seems to indicate that cholesterol transport to the SCC P450 enzyme is stimulated by ACTH, and that the transported cholesterol is rapidly metabolized. More direct proof of this conclusion was obtained from experiments in which the transported cholesterol, resulting from ACTH stimu lation, was prevented' from leaving the SCC P450 enzyme by an anoxia-induced inhibition of steroid hydroxylation. 141 The rapid rate of cholesterol SCC suggested by these in vivo experiments are consistent with the results of the in vitro cholesterol incubations presented earlier and with the findings reported previously by Dexter, et al. (217) which showed that ACTH stimulated the accumulation of ad renal cholesterol, but that this accumulation could only be detected in the presence of aminoglutethimide, an in hibitor of cholesterol SCC. Cycloheximide administered to normal rats seemed also to inhibit cholesterol SCC since the level of cholesterol- bound P450 was increased in these mitochondrial samples as well. These cycloheximide results are in agreement with a number of previous reports which have indicated that this antibiotic inhibits steroidogenesis by blocking the conversion of cholesterol to pregnenolone (142-146). Although the mechanism of cycloheximide inhibition of cholesterol SCC has not been determined, Davis and Garren (145) have postulated that the antibiotic blocks the ACTH-induced synthesis of a labile protein which they postulate (147) facilitates the rate of conversion of cholesterol to pregnenolone by transporting free cyto plasmic cholesterol into the mitochondria. A study re ported by Dexter, et a1. (218) suggests that cycloheximide may inhibit steroidogenesis by interacting directly with the SCC P450 enzyme. These investigators showed that hypophysectomized rats treated with ACTH demonstrated a 142 greater in vivo uptake by adrenal tissue of H-cholesterol from plasma than did hypophysectomized control animals. In vivo administration of cycloheximide in amounts that blocked adrenal protein synthesis did not block the ACTH- 3 induced uptake of H-cholesterol hut instead increased 3 the adrenal H-cholesterol concentration approximately seven times. Similar results were obtained with amino glutethimide . Aminoglutethimide and other nitrogen-containing com pounds have been shown to inhibit the hydroxylation of steroids by decreasing the rate of reduction of cyto chrome P450 (109) . Although unlike aminoglutethimide, cycloheximide has not been shown to inhibit steroid hydroxylation in isolated mitochondria, the fact that this antibiotic is also nitrogen-containing leaves open the possibility that it too may interact directly with the SCC P450 enzyme to change it to the low spin state and thus decrease the rate at which it is reduced. The results of the present study which show that the level of endogenous cholesterol-bound P450 is increased in mitochondria iso lated from rats pre-treated with cycloheximide are consis tent with this postulated inhibition mechanism. Th.e anoxia-induced increase in the level of endogenous cholesterol-bound P450 in the mitochondria isolated from hypophysectomized and normal rats treated with ACTH suggest that the higher levels of endogenous cholesterol-bound 143 P450 contained in bovine adrenocortical mitochondria com pared to rat adrenal mitochondria may be due to the fact that the bovine mitochondria were prepared from anoxic glands collected from dead animals, whereas the rat mitochondria were prepared from more aerobic glands collected from live animals. The Simpson, et al. Hypothesis for ACTH Activation of. Adrenal Steroidogenesis While these studies were in progress, another group of workers (216,219) obtained data which led them to also conclude that ACTH activates cholesterol SCC by increas ing the transport or binding of cholesterol to the SCC P450 enzyme. In their experiments normal rats were subjected to ether anesthesia stress for 7-10 minutes as a means of raising their plasma ACTH levels (220). Control rats were either injected with cycloheximide or kept in a quiescent state. Adrenal glands were collected from the three groups of animals following decapitation. Using cyanoketone (an inhibitor of steroid 33-01 dehydrogenase) to prevent pregnenolone metabolism, the time-course of [4-^C] pregnenolone formation from [4-^C] cholesterol as a function of preincubation time was stu died. In one set of experiments mitochondria were pre incubated with [4-^C] cholesterol for 5 and 10 minutes and cholesterol SCC initiated by the addition of isocitrate. In the other experiment [4-^C] cholesterol and isocitrate 144 were added simultaneously. r 14 i In the experiment where L4- CJ cholesterol and isocitrate were added simultaneously, side-chain cleavage of the tracer substrate was linear up to 20 minutes of incubation. However, when the mitochondria were pre incubated with the radioactive cholesterol for 5 minutes prior to the addition of isocitrate, there was an initial rapid formation of [4-^C] pregnenolone lasting 5 minutes, after which the rate slowed down to that seen when pre incubation had been omitted. Preincubation with radio active cholesterol for 10 minutes prior to the addition of isocitrate, again increased the initial rapid phase of pregnenolone formation but after 5 minutes the rate slowed down to that seen in the absence of pre-incubation. In another study, pregnenolone synthesis or SCC from endogenous cholesterol was measured in mitochondria isolated- from ether-stressed, quiescent, and cycloheximide-treated rats. In all cases, cholesterol SCC followed a biphasic time-course with an initial rapid phase followed by a much slower phase. In the mitochondria from cycloheximide- treated and quiescent rats, the initial phase of preg nenolone formation was complete by the time the first sample was taken at 2 minutes. The amount of pregnenolone formed in the quiescent rat mitochondria was approximately double the amount formed in the mitochondria isolated from the cycloheximide- 145 treated rats. In the mitochondria isolated from the rats subjected to stress, over 3 times as much pregnenolone was formed in the initial phase compared to the amount formed in the cycloheximide-treated group. Pregnenolone syn thesis from rats treated with cycloheximide and subse quently subjected to. ether stress was not reported, but was said to be identical with the rates observed in mito chondria isolated from rats given cycloheximide alone. A study of cholesterol depletion in mitochondria from the three.rgroups of animals indicated that the initial levels of cholesterol were quite similar. The greatest drop in cholesterol content occurred in the mitochondria isolated from the stressed rats, which presumably account ed for the increased pregnenolone formation in these mitochondria. Difference spectra produced in the various mito chondrial samples showed that the type II spectra induced by pregnenolone and isocitrate in mitochondria from stressed rats were 2 - 3 times larger than the corres ponding spectra in mitochondria isolated from quiescent and cycloheximide-treated rats. In contrast, stress treatment had no effect on the reduced carbon monoxide difference spectrum or upon the type I difference spectrum induced by the 1 IB -hydroxylase substrate, DOC. In addit ion no difference was found in the rates of 11B -hydroxy lation of added DOC in mitochondria isolated from rats 146 treated with cycloheximide and from rats given the stand ard ether stress. The biphasic rate of pregnenolone formation from endogenous cholesterol in these experiments was thought by Simpson, et ad to indicate that only a fraction of the total cholesterol in rat adrenal mitochondria is available for side-chain cleavage, and that when this pool of available cholesterol is depleted, the rate of side-chain cleavage becomes dependent on the mobilization of chol esterol from other mitochondrial sites, which is the rate-limiting process. This hypothesis was thought to be supported by their [4-^C] cholesterol experiments. In the absence of pre incubation, the conversion of tracer [4-^C] cholesterol to [4-^C] pregnenolone was linear with time, unlike the biphasic time-course of pregnenolone from endogenous cholesterol. However, preincubation of the adrenal mitochondria with radioactive cholesterol prior to the addition of isocitrate resulted in a biphasic conversion of the tracer cholesterol, and the magnitude of the initial phase was dependent upon the duration of the preincubation. Since data from other of their experi ments had indicated that the biphasic rate of cholesterol SCC was not due to end-product inhibition by pregneno lone, or to the depletion of cholesterol, these investi gators suggested that these results can best be explained i f 147 by assuming that the rate of side-chain cleavage of added tracer cholesterol is limited by the rate at which it is transported or bound to a site from which it can readily be metabolized. In mitochondria isolated from rats whose blood ACTH levels had been raised by ether-stress, there was an in crease in the rate of pregnenolone formation from endo genous cholesterol. This effect was concluded to be mainly on the initial phase of cholesterol SCC since there was no appreciable increase in mitochondrial cholesterol levels. These results suggested to the authors that one action of ACTH is to affect the distribution of chol esterol within the mitochondria in such a way that more can react with SCC cytochrome P450. The increased type II difference spectra induced by pregnenolone and isocitrate in the mitochondria from stressed animals compared to cycloheximide-treated and quiescent animals were cited as proof of the ACTH-induced increase in the association of cholesterol with the SCC P450 enzyme. Based on these findings it was concluded that in the intact gland ACTH results in an increase in that fraction of the mitochondrial cholesterol which is in a reactive complex with cytochrome P450, perhaps via an increase in the transport or binding of cholesterol to the SCC P450 enzyme from other intramitochondrial binding sites. In vivo or in the isolated mitochondria this would mean 148 that more cholesterol is available for SCC. Since cyclo heximide blocked the effect of ether-stress, and there fore presumably of ACTH, within minutes after its adminis tration, it was suggested that some labile protein (143), synthesized in response to ACTH, may be involved in the transport or binding of cholesterol to SCC P450. Although similar conclusions were reached regarding the acute action of ACTH on steroidogenesis, the results of some of the experiments reported by Simpson,et al. differ substantially from those obtained in the present study. Firstly, although all adrenal glands used in the present study were collected from animals subjected to ether anesthesia and laporotomy, a stressful condition which has been shown to instantaneously raise plasma ACTH levels and keep them elevated for 60 minutes (220), the amount of endogenous cholesterol-bound P450 was not significantly increased in the mitochondria isolated from these glands. This is shown by the similar magnitude of the optical density values obtained from titration studies with mitochondria isolated from normal and hypophysecto mized rats in Table VI. This lack of accumulation of cholesterol-bound P450 in the mitochondria isolated from normal animals subjected to stress is compatible with a high rate of cholesterol SCC since under these circumstances, the cholesterol, once transported in the process stimulated by ACTH, would 149 be expected to disappear rapidly and not to accumulate on the SCC P450 enzyme. The increased levels of endogenous cholesterol-bound P450 found in the mitochondria isolated from rats sub jected to stress in the study reported by Simpson et^ al. appears not to be compatible with a rapid rate of chol esterol SCC since the transported cholesterol, instead of being* metabolized, remains bound to the SCC P450 enzyme. If the reduction of substrate-bound high spin P450 is the factor which limits the rate at which steroids are hydroxylated (208) substrate-activated high spin P450 would probably be expected to accumulate only under conditions where the supply of NADPH or oxygen are limited. Based on the results obtained in the present study which indicate that the rate of cholesterol SCC is ex tremely rapid and that-the rate of generation of intra- mitochondrial NADPH is not limiting in steroidogenesis, it is suggested that the accumulation of cholesterol-bound P450 in the Simpson et al. study is due to an anoxia- induced inhibition of cholesterol SCC since the adrenal •glands were collected after the animals had been sacrified by decapitation. Our experiments with cycloheximide have also yielded conflicting results. In the Simpson et aT. study prior administration of cycloheximide to animals subjected to stress seemed to prevent the ACTH-induced transport of 150 cholesterol to the SCC P450 enzyme. Based on these results they have suggested that a labile protein synthesized in response to ACTH is functional in the transport or binding of cholesterol to cytochrome P450. In the present study cholesterol transport appeared not to be inhibited by cycloheximide since the level of endogenous cholesterol-bound P450 was increased, not decreased, in the mitochondria isolated from animals pre treated with this antibiotic. These results have been explained by suggesting a direct action of cycloheximide on the SCC hydroxylase as opposed to an inhibition of ACTH-induced protein synthesis. The reasons for the discrepancies between the results obtained with cyclo heximide in this study and in the Simpson et_ al. study are unclear. Cholesterol Transport Process A knowledge of the immediate source of cholesterol used for the ACTH-induced synthesis of glucocorticoids would appear to be essential to an understanding of how cholesterol is transported to the SCC P450 enzyme and to an understanding of how this transport mechanism maybe facilitated by ACTH. It has been demonstrated that the pool of adrenal free cholesterol which apparently serves as the precursor of glucocorticoids produced in response to ACTH (10) can be derived both from the uptake of free plasma cholesterol (1) and from the hydrolysis of 151 cholesterol esters stored in the adrenal cytoplasm (221). Both the uptake of plasma cholesterol and the hydrolysis of cholesterol esters are stimulated by ACTH. The intraadrenal location of the free cholesterol pool is a matter of controversy. Some investigators claim that adrenocortical mitochondria contain large concen trations of cholesterol (222,10), while other reports indicate that the concentration is normally quite low (168). Studies by another group of investigators (223) 3 show that 70 to 80 percent of the H-cholesterol injected into rats becomes segregated into lipid droplets within the adrenal cortical cell. They suggest that cellular mechanisms probably exist which cause mobilization of the cholesterol from the lipid droplets to the mitochondria when steroidogenesis is stimulated by ACTH. The origin of the free cholesterol which contributes to the increased rate of corticosteroidogenesis in rat adrenal glands has been studied by Ichii et^ al_., (224) . 3 14 By injecting rats with H- and C-cholesterol for 3 days and 4 hours prior to their death, respectively, these investigators were able to label most of the adrenal free 14 cholesterol with C and the bulk of the ester chol- 3 esterol with H. The interconversion of ester and free cholesterol and corticosterone following vivo ACTH stimulation was determined by examining the ^H/^C ratios of free and ester cholesterol in the mitochondria and 152 post-mitochondrial fractions. and the ^H/^C ratio of plasma corticosterone obtained from control and ACTH treated animals. While the results of the Ichii £t al., study clearly indicated that the free cholesterol liberated via the ACTH- induced hydrolysis of adrenal cytoplasmic esters is ... .. utilized for the biosynthesis of corticosterone, the much 3 ,14 lower H/ C ratios of plasma corticosterone compared to mitochondrial free cholesterol following ACTH stimu lation, suggested to these authors that the cholesterol derived from plasma is readily utilized for corticoid synthesis before mixing with the endogenous cholesterol pool in the adrenal mitochondria. According to the Simpson e_t al. hypothesis, ACTH activates steroidogenesis by transporting cholesterol from restricted pools in the mitochondria to a smaller more reactive mitochondrial pool which is accessible to the SCC P450 enzyme. The hypothesis that only a fraction of the total mitochondrial cholesterol is reactive with or can readily bind to the SCC enzyme was used by these investigators to explain the biphasic kinetics of preg nenolone synthesis from endogenous cholesterol. Biphasic kinetics or a rapid fall-off in the rate of SCC from exogenous cholesterol were also obtained in the present study. Since the mitochondria in these experiments were presaturated with exogenous cholesterol prior to the 153 initiation of hydroxylation, and since similar biphasic kinetics were observed for the SCC of 20a-hydroxychol- esterol, a substrate which forms high spin P450 as readily as DOC does (198), it is suggested that the biphasic rate at which the side-chain is cleaved from endogenous and exogenous cholesterol is not due to the slow binding of cholesterol to the SCC P450 enzyme but may be caused by a limitation of adrenodoxin or adrenodoxin reductase result ing from the preparation of the mitochondria as discussed earlier. Therefore, while a restricted pool of cholesterol does seem to exist in adrenal mitochondria which can serve as the precursor for corticoid synthesis, it is postu lated that the immediate source of the cholesterol used for glucocorticoid synthesis during stress or following ACTH stimulation, is plasma cholesterol which is transported directly to the SCC P450 enzyme without mixing with the mitochondrial free cholesterol pool. Following the cholesterol-induced activation of low spin SCC P450 to the high spin state, and its subsequent reduction by adrenodoxin, it is envisioned that the remaining reactions, which lead ultimately to the production of corticosteroids, proceed without limitation. CHAPTER VI SUMMARY AND CONCLUSIONS Changes occurring in the whole adrenal and in adrenal mitochondria following the in vivo administration of ACTH to hypophysectomized rats have been used to determine in what manner or at which point, the conversion of chol esterol to pregnenolone (the rate-limiting reaction in the biosynthetic pathway leading to glucocorticoid production) may be hormonally stimulated. Based on previous findings which suggest that the reduction of substrate-bound high spin P450 may be the rate-limiting step in steroid hydroxylation, evidence of an ACTH-induced increase in the rate of generation of intramitochondrial NADPH and/or of an increase in the formation of cholesterol-bound high spin P450 was sought. None of the NAD or NADP-linked mitochondrial dehydro genases appeared to be rate-limiting prior to the increase in corticosteroid production since the steady-state con centrations of Krebs cycle substrates failed to change following the administration of ACTH to hypophysecto mized rats. Although activation of the energy-dependent transhydrogenase (which converts NAD to NADPH in adreno cortical mitochondria) following ACTH stimulation could not 154 155 be ruled out by these experiments, based on data presented previously by a number of other investigators (129-31,209, 210), and on the severe compromise such an activation would probably place on the energy metabolism of the ; . adrenal cell (81), it is concluded that the increase in corticosteroid synthesis resulting from ACTH stimulation is not caused by the increased production of NADPH via the energy-dependent transhydrogenase reaction. Difference spectra produced by the addition of steroid substrates and inhibitors to bovine adrenocortical mito chondria and microsomes have been extensively used to study some of the catalytic properties of the membrane- bound llg- and 21-hydroxylase enzyme systems. The low concentration of endogenous cholesterol-bound high spin P450 found to be contained in the rat mitochondria used in this study, and the subsequent preparation of a soluble cholesterol-lecithin emulsion capable of binding the SCC P450 enzyme in these mitochondria, allowed some of the catalytic properties of the cholesterol SCC enzyme to be similarly studied. It was demonstrated that bovine adrenocortical mito chondria and microsomes contain significant concentrations of endogenous P450-bound steroid intermediates which greatly influence both the kind (type I or type II) and magnitude of the difference spectra induced by exogenous steroid substrates and inhibitors and electron donors 156 added to these preparations. The type II difference spectra (420 nm troughs and 390 nm peaks) induced by 20a-hydroxycholesterol and pregnenolone in bovine adrenal mitochondria were shown to result from the interaction of these steroids with endogenous cholesterol-bound high spin P450 to convert it to a less active low spin state. 20a-hydroxycholesterol, but not pregnenolone, was shown to also interact with cholesterol-free low spin SCC P450 to activate it to the high spin state. The competitive inhibition of cholesterol SCC obtained when 20a-hydroxycholesterol and cholesterol were prein cubated with rat adrenal mitochondria prior to the initiation of hydroxylation, and the lack of inhibition of SCC obtained in similar studies with pregnenolone, sup port these spectral data. The type II difference spectra generated by the addition of malate to bovine adrenocortical mitochondria, of isocitrate to rat adrenal mitochondria, and of NADPH to bovine adrenocortical microsomes and submitochondrial particles, were shown to represent low spin P450 released by the metabolism of endogenous steroid substrates. A similar conclusion regarding the origin of these type II spectra has been reached by other investigators (215). In vitro incubations performed under conditions where the substrates for the hydroxylases were pre-bound to their P450 enzymes, indicate that the vmax of 157 cholesterol SCC (1,131 nmoles products/min/mgN) and 20a-hydroxycholesterol SCC (938 nmoles products/min/mgN) are approximately four times greater than the V of DOC nictx llg-hydroxylation (250 nmoles corticosterone/min/mgN). The similar V and Km values calculated from incubations UlclX using mitochondria isolated from longterm (44 hours) hypophysectomized rats and from hypophysectomized treated with ACTH, suggest that ACTH has no direct effect on the activity of the cholesterol SCC enzyme system. The levels of endogenous cholesterol and DOC-bound high spin P450 in mitochondria isolated from normal rats, hypophysectomized rats treated with ACTH and with ACTH followed by anoxia, were determined by titrating these preparations with 20a-hydroxycholesterol, pregnenolone, DOC, cholesterol, and isocitrate. The increased con centrations of endogenous cholesterol and DOC-bound high spin P450 in the mitochondria isolated from ACTH-.stimu- lated hypophysectomized rats compared to the hypophy sectomized controls, suggest that cholesterol transport to the SCC P450 enzyme is stimulated by ACTH and that the transported cholesterol is rapidly metabolized. This conclusion was further substantiated by the increased concentration of endogenous cholesterol-bound P450 in the mitochondria isolated from ACTH-stimulated hypophysectomized rats whose adrenals had been rendered anoxic following stimulation to inhibit steroid 158 hydroxylation. These findings support the earlier report by Dexter ejt al. , (218) which demonstrated that the ACTH- induced increase in adrenal free cholesterol could only be detected in the presence of aminoglutethimide, an inhibitor of cholesterol SCC. The concentration of endogenous cholesterol-bound high spin P450 was also increased in mitochondria isolated from normal rats treated with cycloheximide. 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T he m ajority of users indicate th a t the textual content is of greatest value, however, a som ewhat higher quality reproduction could be made from "p h o to g rap h s" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered a t additional charge by writing th e O rder Departm ent, giving the catalog num ber, title, author and specific pages you wish reproduced. 5. PLEASE NOTE: Some pages m ay have indistinct print. Filmed as received. Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 t I 74-1663 BELL, Julia, 1940- 0N THE MECHANISM OF ACTH STIMULATION OF f ADRENAL STEROIDOGENESIS. I I ' i University of Southern California, Ph.D., 1973 | Biochemistry University Microfilms, A X E R O X Company, Ann Arbor, Michigan THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.

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Creator Bell, Julia, 1940- (author)

Core Title On the mechanism of ACTH stimulation of adrenal steroidogenesis

Contributor Digitized by ProQuest (provenance)

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1973-09

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Advisor Harding, Boyd W. (committee chair), Sadee, Wolfgang (committee member), Visser, Donald W. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-713997

Unique identifier UC11364316

Identifier 7401663.pdf (filename),usctheses-c18-713997 (legacy record id)

Legacy Identifier 7401663.pdf

Dmrecord 713997

Document Type Dissertation

Rights Bell, Julia

Type texts

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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