Relationship between mitochondrial lipid peroxidation and alpha-tocopherol levels in the guinea-pig adrenal cortex.

Lipid peroxidation in mitochondria from the functionally distinct inner (zona reticularis) and outer (zona fasciculata + zona glomerulosa) zones of the guinea-pig adrenal cortex was investigated. Ferrous ion (Fe2+)-induced lipid peroxidation was far greater in inner than outer zone mitochondria. Ascorbic acid similarly initiated lipid peroxidation to a greater extent in inner zone mitochondrial preparations. Differences in the unsaturated fatty acid content of inner and outer zone mitochondria could not account for the regional differences in lipid peroxidation. Total fatty acid concentrations were greater in the outer than in the inner zone, and the relative amounts of each fatty acid were similar in the two zones. However, mitochondrial concentrations of alpha-tocopherol, an antioxidant known to inhibit lipid peroxidation, were approx. 5-times greater in the outer than inner zone. The results demonstrate that there are regional differences in mitochondrial lipid peroxidation in the adrenal cortex which may be attributable to differences in alpha-tocopherol content. Thus, alpha-tocopherol may serve to protect outer zone mitochondrial enzymes from the consequences of lipid peroxidation and thereby contribute to some of the functional differences between the zones of the adrenal cortex.

Regulation of adrenal and hepatic alpha-tocopherol content by androgens and estrogens.

Previous studies demonstrated that alpha-tocopherol concentrations were far greater in adrenal glands and in livers from female rats than in those from males. Studies were done to investigate the role of androgens and estrogens in the regulation of adrenal and hepatic alpha-tocopherol content. In males and females, adrenal concentrations of alpha-tocopherol were approx. 10-fold greater than those in liver and the highest concentrations of alpha-tocopherol were in the crude mitochondrial fractions in both organs. Castration of female rats decreased alpha-tocopherol concentrations in adrenals and in livers. Proportionately similar declines occurred in both organs and in all subcellular fractions. The effects of castration were prevented by estradiol replacement at the time of surgery. Gonadectomy in male rats had effects opposite to those in females, increasing adrenal and hepatic alpha-tocopherol concentrations. Testosterone administration to castrated males prevented the increases in adrenal and hepatic alpha-tocopherol content. Neither castration nor gonadal hormone replacement in either sex had any effect on plasma alpha-tocopherol levels or on cytosolic ascorbic acid concentrations in adrenals or livers. The results indicate a role for estrogens and androgens in the regulation of adrenal and hepatic concentrations of alpha-tocopherol. The opposing effects of androgens and estrogens fully account for the sex differences in tissue alpha-tocopherol levels in rats.

Lipid peroxidation in guinea pig lung microsomes.

The effects of substances known to influence lipid peroxidation were studied in guinea pig lung microsomes by measuring the formation of malonaldehyde in vitro. Incubation of lung microsomes at 37 degrees C results in lipid peroxidation which appears to be an enzymatic process but is not dependent upon iron. Lipid peroxidation can be initiated non-enzymatically in lung microsomes by Fe2+, but ascorbate and Fe3+ have very little effect on malonaldehyde formation. The effects of NADPH on lipid peroxidation are dependent upon the concentration of Fe2+ in the incubation medium. At concentrations of Fe2+ between 0.05 mM and 1 mM, addition of NADPH causes an increase in lipid peroxidation over that produced by Fe2+ alone. This stimulation by NADPH is an enzymatic process and phosphate is required for the maximal effect. Addition of NADPH to lung microsomes in the presence of Fe3+ does not increase malonaldehyde formation over that produced by Fe3+ alone, suggesting that NADPH does not influence lipid peroxidation by maintaining iron in the reduced form. At concentrations of Fe2+ greater than 1 mM, NADPH inhibits Fe2+-induced lipid peroxidation in normal microsomes and in microsomes in which enzymes have been inactivated with heat. This latter result suggests that the inhibition by NADPH is at least partially non-enzymatic. The result suggests that the inhibition by NADPH is at least partially non-enzymatic. The results of all of these experiments are discussed and compared with those obtained during lipid peroxidation in liver microsomes. We conclude that the processes involved in pulmonary microsomal lipid peroxidation differ significantly from those in hepatic microsomes.

Effects of lipid peroxidation on adrenal microsomal monooxygenases.

Incubation of guinea pig adrenal microsomes with 10(-6) M ferrous (Fe2+) ion and adrenal cytosol initiated high levels of lipid peroxidation as measured by the production of malonaldehyde. Cytosol or Fe2+ alone had little effect on microsomal malonaldehyde formation. When microsomes were incubated in the presence of Fe2+ and cytosol, malonaldehyde levels continued to increase for at least 60 min. Accompanying the lipid peroxidation was a decline in adrenal microsomal monooxygenase activities. The rates of metabolism of xenobiotics (benzphetamine demethylase, benzo[a]pyrene hydroxylase) as well as steroids (21-hydroxylation) decreased as malonaldehyde levels increased. In addition, cytochrome P-450 levels, NADPH- and NADH-cytochrome c reductase activities, and substrate interactions with cytochrome(s) P-450 decreased as lipid peroxidation progressed. Inhibition of lipid peroxidation by increasing microsomal protein concentrations during the incubation period prevented the changes in microsomal metabolism. Malonaldehyde had no direct effects on adrenal microsomal enzyme activities. The results indicate that lipid peroxidation may have significant effects on adrenocortical function, diminishing the capacity for both xenobiotic and steroid metabolism.

Mechanism of action of prolactin on adrenocortical steroid secretion in hypophysectomized female rats.

Studies were carried out to determine the actions of PRL on adrenocortical function in hypophysectomized female rats in the presence and absence of ACTH. PRL administration alone decreased 5 alpha-reductase activity but did not significantly affect the rates of corticosterone secretion or peripheral plasma corticosterone concentrations. The activities of several steroidogenic enzymes (cholesterol desmolase, 11 beta-hydroxylase, and 21-hydroxylase) were also unaffected by PRL. Adrenal steroidogenesis was increased by ACTH treatment, as expected, resulting in an increase in corticosterone secretion. However, since adrenal 5 alpha-reductase activity was higher in ACTH-treated hypophysectomized rats than in normal animals with intact pituitary glands, large amounts of 5 alpha-dihydrocorticosterone (DHB) and 3 beta, 5 alpha-tetrahydrocorticosterone (THB) were also secreted. PRL, when administered in combination with ACTH, potentiated the effecte levels. PRL did not affect cholesterol side chain cleavage, 11 beta-hydroxylation, or 21-hydroxylation in ACTH-treated rats. However, administration of PRL to ACTH-treated rats lowered adrenal 5 alpha-reductase activity, decreasing DHB and THB secretion. The decrease in DHB and THB secretion approximated the increase in corticosterone output. The results indicate that, in the presence of ACTH, PRL increases corticosterone secretion by decreasing intraadrenal degradation of corticosterone and not by enhancing steroidogenesis.

Is growth hormone the pituitary feminizing factor mediating the actions of estradiol on hepatic drug and steroid metabolism?

Previous studies have established that the effects of estradiol (E2) on hepatic steroid and drug metabolism are demonstrable only in the presence of the pituitary gland. Studies were carried out to test the hypothesis that GH is the pituitary feminizing factor mediating the actions of E2 on hepatic metabolism. E2 and GH administered to castrated male rats had similar effects on hepatic enzymes, decreasing the oxidataive metabolism of drugs [ethylmorphine demethylation, aniline, hydroxylation, and benzo(a)pyrene hydroxylation) and increasing steroid (corticosterone) delta 4-hydrogenase activity. None of these effects of E2 or GH could be demonstrated in hypophysectomized (hypox) rats. However, GH administration to T4- or ACTH-treated hypox rats resulted in some of the changes in drug and steroid metabolism seen in animals with intact pituitary glands. The actions of GH on hepatic microsomal enzymes were fully demonstrable in hypox rats receiving both T4 and ACTH. E2 had no effects in T4 plus ACTH-treated hypox rats. These and prior observations are consistent with the hypothesis that GH mediates the actions of E2 on hepatic microsomal drug- and steroid-metabolizing enzymes. The data also indicates that the cations of GH on hepatic metabolism are dependent upon the interactions with still other endocrine factors.

Differences in microsomal steroid metabolism between the inner and outer zones of the guinea pig adrenal cortex.

Previous investigations established that cells isolated from the outer zone (zona fasciculata + zona glomerulosa) of the guinea pig adrenal cortex produced far more cortisol and androstenedione than those from the inner zone (zona reticularis). Studies were done to determine whether differences in microsomal metabolism might contribute to the zonal differences in steroid secretion. Cytochromes P-450 and b5 concentrations were greater in inner zone microsomes as were the magnitudes of the type I difference spectra produced by progesterone and 17 alpha-hydroxyprogesterone. Basal NADPH-cytochrome P-450 reductase activity was greater in the outer zone, but steroid substrates (progesterone, 17 alpha-hydroxyprogesterone) increased reductase activity in the inner zone and decreased activity in the outer zone. 21-Hydroxylase activity was far greater in inner than outer zone microsomes, but 17 alpha-hydroxylase activity was greater in the outer zone. As a result, progesterone was converted primarily to 17 alpha-hydroxyprogesterone by outer zone microsomes, but 11-deoxycorticosterone was the major metabolite produced by inner zone preparations. In addition, with 17 alpha-hydroxyprogesterone as substrate, the major product produced by outer zone microsomes was androstenedione, indicating relatively high C17-20-lyase activity. Inner zone microsomes by contrast, converted 17 alpha-hydroxyprogesterone primarily to the 21-hydroxylated metabolite, 11-deoxycortisol, with little production of androstenedione. The rate of conversion of pregnenolone to progesterone was also greater with outer than inner zone microsomes. The results suggest that differences in the patterns of microsomal steroid metabolism contribute to the greater secretion of cortisol and androstenedione by adrenocortical outer zone cells than by inner zone cells.

Functional zonation of the guinea pig adrenal cortex: differences in mitochondrial steroid metabolism between the inner and outer zones.

Previous studies established that cells isolated from the chromatically distinct inner (primarily zona reticularis) and outer (zona fasciculata + zona glomerulosa) zones of the guinea pig adrenal cortex had vastly different steroidogenic capabilities; the outer zone produced far more cortisol than the inner zone. The mechanism(s) responsible for those differences were investigated by comparing mitochondrial steroid metabolism in the inner and outer zones. Cytochrome P-450 concentrations were similar in the two zones, but 11 beta-hydroxylase activity was approximately twice as great in the outer zone. More importantly, cholesterol sidechain cleavage, the rate-limiting step in steroidogenesis, was nearly 10 times greater in outer than inner zone mitochondria. Free cholesterol concentrations were also far higher in outer zone mitochondria. The results suggest that the relatively low level of steroid secretion by cells of the zona reticularis is attributable, at least in part, to deficiencies in mitochondrial cholesterol content and/or metabolism.

Changes in adrenal microsomal cytochrome(s) P-450 with aging in the guinea pig.

Studies were carried out to investigate the mechanism(s) responsible for the changes in adrenal microsomal mixed function oxidase activity which occur with aging (30-200 days) in guinea pigs. With aging, the rate os metabolism of xenobiotics [ethylmorphine and benzo(a)pyrene] by adrenal microsomes increased 3- to 5-fold. Steroid 17 alpha- and 21-hydroxylations, when expressed per mg protein, were similar in immature (30 days old) and mature (200 days old) animals. Adrenal microsomal NADPH- and NADH-cytochrome c reductase activities and cytochrome b5 concentrations increased wih aging, but cytochrome P-450 concentrations were not significantly different in young and old guinea pigs. Maximal type I difference spectra produced by steroids were the same in adrenal microsomes from 30- and 200-day-old guinea pigs, but the ethylmorphine-induced spectrum was far greater in the older animals. Progesterone enhanced NADPH-cytochrome P-450 reductase activity to about the same extent in adrenal microsomes from 30- and 200-day-old guinea pigs. Ethylmorphine had no effect on the rate of reduction of cytochrome P-450 in adrenals from young animals but produced a 4-fold increase in activity in adrenals from older animals. The results demonstrate selective changes in adrenal xenobiotic metabolism with aging and suggest that changes in the composition and/or reactivity of adrenal cytochromes P-450 are responsible for the effects of aging.

Effects of tocopherol depletion on the regional differences in adrenal microsomal lipid peroxidation and steroid metabolism.

Studies were done to assess the contribution of alpha-tocopherol to the regional differences in microsomal lipid peroxidation (LP) and steroid metabolism in the guinea pig adrenal cortex. In normal guinea pigs, ferrous ion (Fe2+)- and ascorbic acid-induced LP are far greater in microsomal preparations from the inner adrenal zone (zona reticularis) than in those from the outer zones (zona fasciculata plus zona glomerulosa). The amounts of unsaturated fatty acids, substrates for LP, are similar in the two zones, but alpha-tocopherol concentrations are 4-5 times greater in outer than inner zone microsomes. Tocopherol depletion by dietary deprivation had little effect on LP in vitro in inner zone microsomes, but substantially increased LP in outer zone preparations. As a result, tocopherol deficiency eliminated the zonal differences in microsomal LP. Unsaturated FFA concentrations were lower in tocopherol-deficient microsomal preparations than in those from tocopherol-sufficient animals, suggesting peroxidative losses in vivo. Tocopherol deficiency decreased steroid C-17,20 lyase activity in outer zone microsomes, but had no effect on activity in inner zone preparations, eliminating the normal zonal difference in activity (outer greater than inner). The results indicate that alpha-tocopherol is a major determinant of adrenal LP and is responsible for the regional differences in microsomal LP in guinea pig adrenal cortex; the effects of ascorbic acid on LP in each zone are also affected by alpha-tocopherol. alpha-Tocopherol may influence the functional zonation of the adrenal cortex by selectively protecting outer zone steroidogenic enzymes from oxidative degradation.

Site of action of growth hormone on adrenocortical steroidogenesis in rats.

Studies were carried out to define the mechanism of action of growth hormone on adrenocortical steroidogenesis in hypophysectomized female rats. ACTH administration for 7 days increased corticosterone secretion in vivo and corticosterone production by adrenal tissue in vitro. Adrenal mitochondrial and microsomal cytochrome P-450 concentrations as well as the activities of cytochrome P-450-dependent enzymes (cholesterol sidechain cleavage, 11beta-hydroxylase, 21-hydroxylase) were also increased by ACTH. Administration of bovine growth hormone alone to hypophysectomized rats had no effect on any of the parameters evaluated. However, when given in combination with ACTH, growth hormone synergistically enhanced the effects of ACTH on cholesterol sidechain cleavage activity and corticosterone secretion. The magnitude of the pregnenolone-induced difference spectrum in adrenal mitochondria, indicative of cholesterol binding to cytochrome P-450, was also increased by growth hormone, but neither cytochrome P-450 content nor the activities of other steroidogenic enzymes were affected. The results indicate that growth hormone interacts with ACTH to promote corticosterone secretion by increasing the association of cholesterol with adrenal mitochondrial cytochrome P-450, thereby increasing the activity of cholesterol sidechain cleavage, the rate-limiting step in steroidogenesis.

Role of adrenal 5alpha-reductase activity in determining the responsiveness of hypophysectomized rats to ACTH.

Studies were conducted to examine the contribution of adrenal 5alpha-reductase to the phenomenon of diminished adrenal "responsiveness" to ACTH after hypophysectomy in rats. Rats hypophysectomized for 1 week secreted small amounts of corticosterone (B), 5alpha-dihydrocorticosterone (DHB) and 3beta,5alpha-tetrahydrocorticosterone (R) acutely after ACTH. Adrenal reductase activity in vitro at that time was high. After replacement with ACTH for 24 h, B,DBH, and R secretion increased slightly. Reductase activity remained high. Treatment with ACTH for 2 days further stimulated secretion of DHB and R but not B. Reductase activity was unaffected. Enzyme activity declined at 3 days concomitant with a proportionately greater increase in B than DHB and R secretion. Only after 7 days of ACTH did B secretion exceed DHB and R output. At that time, reductase activity was still lower. The results establish the functional significance of 5alpha-reductase activity as a regulatory site for the action of ACTH in determining the composition of adrenocortical secretory products in hypophysectomized rats.

Differential effects of adrenocorticotropin in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex.

Studies were performed to compare the effects of ACTH treatment in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex. In untreated animals, CYP2D16 protein and messenger RNA (mRNA) expression as well as xenobiotic-metabolizing activities (bufuralol 1'-hydroxylase, benzphetamine N-demethylase, and benzo(a)pyrene hydroxylase) were far greater in the inner (zona reticularis) than the outer (zona fasciculata plus zona glomerulosa) zones of the cortex. ACTH treatment for 3 or 7 days significantly decreased the rates of xenobiotic metabolism in both the inner and outer adrenal zones. Western and Northern blot analyses revealed that adrenal CYP2D16 protein and mRNA concentrations were significantly decreased by ACTH. In contrast to its inhibitory effects on CYP2D16, ACTH treatment increased steroid 17 alpha-hydroxylase activity in the adrenal inner zone, but did not affect outer zone activity. Microsomal CYP17 protein concentrations were not affected by ACTH despite increases in CYP17 mRNA levels in both zones. The results indicate that ACTH causes down-regulation of adrenal CYP2D16, probably at the transcriptional level. Thus, modulation of CYP2D16 by ACTH is opposite that for the steroidogenic P450 isozymes, suggesting unique regulatory mechanisms. In addition, the data suggest that posttranscriptional mechanisms contribute to ACTH regulation of 17 alpha-hydroxylase activity in the guinea pig adrenal cortex.

Zonal differences in adrenocortical lipid peroxidation: role of alpha-tocopherol.

Studies were done to evaluate the relationship between alpha-tocopherol (alpha-T) concentrations and lipid peroxidation (LP) in vitro in microsomal preparations from the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Microsomes were incubated with ferrous ion (Fe2+) to promote free radical production, and alpha-T levels and LP were monitored after various incubation times. alpha-T concentrations were far lower in inner than outer zone preparations and were rapidly depleted from inner zone microsomes by incubation with Fe2+. Coinciding with alpha-T depletion was a large and rapid increase in LP. With outer zone microsomes, alpha-T depletion required more than 30 min, and very little LP was demonstrable during this period. However, once alpha-T depletion occurred, LP was rapidly initiated and reached levels similar to those obtained with inner zone preparations. Inhibition of LP by MnCl2 prevented the Fe(2+)-induced declines in alpha-T in both zones. The results demonstrate the importance of alpha-T as a modulator of adrenal LP and indicate that the zonal differences in LP are largely attributable to the differences in alpha-T concentrations.

Effects of adrenocorticotropic hormone and dexamethasone on adrenal and hepatic alpha-tocopherol concentrations.

Studies were done to determine the effects of ACTH treatment on adrenal alpha-tocopherol (alpha-T) concentrations in female rats. Administration of dexamethasone (DEX) to inhibit endogenous ACTH secretion increased whole adrenal alpha-T levels as well as the fractional amount in adrenal cytosol. Adrenal ascorbic acid (AA) concentrations were unaffected by DEX. DEX treatment also had no effect on hepatic AA content but decreased alpha-T concentrations in the liver. The subcellular distribution of alpha-T in the liver was not altered by DEX. Administration of ACTH to DEX-treated animals decreased adrenal alpha-T content and restored the pattern of subcellular distribution to that seen in controls. ACTH had no effect on hepatic alpha-T concentrations or subcellular distribution. ACTH treatment also had no effect on AA concentrations in adrenals or livers. The results demonstrate that ACTH has a role in the regulation of adrenal alpha-T but the mechanism(s) involved remain to be determined. The data also indicate that glucocorticoids such as DEX directly influence hepatic alpha-T levels independent of their effects on ACTH secretion.

Changes in mitochondrial and microsomal lipid peroxidation and fatty acid profiles in adrenal glands, testes, and livers from alpha-tocopherol-deficient rats.

Studies were done to evaluate the effects of alpha-tocopherol deficiency in rats on the fatty acid composition and sensitivity to lipid peroxidation (LP) of mitochondria and microsomes from adrenal glands, testes, and livers. In control (alpha-tocopherol-sufficient) animals, adrenal concentrations of alpha-tocopherol were approximately 10 times greater than those in livers and testes. Dietary deficiency of alpha-tocopherol for 8 weeks decreased adrenal and hepatic concentrations by 80-90% and testicular concentrations by approximately 60-70%. Incubation of testicular or hepatic mitochondria and microsomes from control rats with FeSO(4) (1.0 mM) caused a time-dependent stimulation of LP as indicated by the formation of thiobarbituric acid reactive substances (TBARS); the rate of TBARS production increased in preparations from alpha-tocopherol-deficient animals. TBARS formation was not demonstrable in adrenal mitochondria or microsomes from alpha-tocopherol sufficient rats, but reached high levels in alpha-tocopherol-deficient preparations. The fatty acid composition of mitochondria and microsomes was tissue-dependent. In particular, arachidonic acid comprised approximately 40% of the total fatty acids in adrenal membranes, but only 20-25% in testes and livers. alpha-Tocopherol deficiency increased oleic acid concentrations in adrenal and hepatic mitochondria and microsomes but not in testes. In all three tissues, linoleic acid concentrations decreased by approximately 50%, but arachidonic acid levels were unaffected by alpha-tocopherol deficiency. The results indicate a close relationship between tissue sensitivity to LP in vitro and alpha-tocopherol concentrations. Nonetheless, any oxidative stress in vivo caused by alpha-tocopherol deficiency seems to spare arachidonic acid in mitochondria and microsomes but decreases linoleic acid concentrations. It is possible that because of the important physiological functions of arachidonic acid, metabolic adaptations serve to maintain membrane content during periods of oxidative stress.

Species differences in adrenal lipid peroxidation: role of alpha-tocopherol.

Previous reports have noted high levels of lipid peroxidation (LP) in vitro in a variety of adrenocortical preparations. However, we have observed that susceptibility to adrenal LP seems to vary considerably from species to species. The current study was done to confirm these apparent species differences in adrenal LP in vitro and to determine if they were attributable to differences in alpha-tocopherol content. Incubation of mitochondrial or microsomal preparations from guinea pig or rabbit adrenal glands with ferrous ion (Fe2+) caused a time-dependent increase in the formation of thiobarbituric acid reactive substances (TBARS) accompanied by depletion of alpha-tocopherol. By contrast, incubation of adrenal mitochondria or microsomes from rats or monkeys with Fe2+ had little or no detectable effect on TBARS and basal adrenal alpha-tocopherol levels were five to ten-fold greater than those in guinea pigs or rabbits. In addition, there was little change in alpha-tocopherol concentrations during incubation of rat or monkey adrenal tissue. Dietary alpha-tocopherol deficiency in rats reduced adrenal alpha-tocopherol to concentrations approximating those in guinea pigs. Incubation with Fe2+ induced high levels of TBARS in adrenal mitochondria and microsomes from the alpha-tocopherol deficient rats. Conversely, dietary alpha-tocopherol supplementation in rabbits increased adrenal alpha-tocopherol levels and prevented Fe2+ induced TBARS formation in mitochondria and microsomes. The results indicate that there are large species differences in adrenal susceptibility to LP in vitro and that these differences are at least partly attributable to species differences in adrenal alpha-tocopherol concentrations.

Changes in adrenocortical function in male and female guinea-pigs during maturation.

Adrenal cortisol secretion is greater in female than male guinea-pigs and declines with maturation in animals of both sexes. In an attempt to determine the intra-adrenal mechanisms responsible for age and sex influences on corticosteroid output, adrenocortical enzyme activities were compared in sexually immature (3 weeks) and mature (17 weeks) animals. Adrenal mitochondrial protein concentration decreased with maturation in male and female guinea-pigs. 11beta-Hydroxylase activity in adrenal mitochondria was also lower in mature than immature guinea-pigs but greater in males than females. Neither mitochondrial cytochrome P-450 concentration nor cholesterol side-chain cleavage activity varied with age or sex. Adrenal microsomal protein concentration and 21-hydroxylase activity were similar in male and female guinea-pigs of the same age but far greater in mature than immature animals. Microsomal cytochrome P-450 concentration was unaffected by age or sex. Adrenal delta4-steroid (cortisol) hydrogenase activity increased with maturation in both male and female guinea-pigs and was higher in males than females. These observations indicate that cortisol secretion, as modified by age and sex, correlates closely with adrenal steroid reductive but not oxidative metabolism, suggesting that changes in delta4-hydrogenase activity are responsible, at least in part, for the decline in adrenal secretion during maturation in guinea-pigs.

Suppression of lipid peroxidation in adrenal microsomes following ACTH administration to guinea pigs.

Previous studies demonstrated high levels of lipid peroxidation (LP) in the guinea pig adrenal cortex. The present studies were done to determine if adrenal LP activity was influenced by ACTH, the major hormonal regulator of the gland. Guinea pigs were treated with ACTH for 1, 3 or 7 days. In addition, some guinea pigs received ACTH for 7 days and were killed 3 or 7 days later. After treatment, adrenal microsomal fractions were prepared and incubated in vitro with 1 mM ferrous sulfate to initiate LP. ACTH treatment caused a progressive decrease in adrenal LP; activity was almost totally inhibited within 3 days. The inhibitory effects of ACTH on LP were dose-dependent. Following cessation of ACTH treatment, adrenal LP gradually returned toward control levels. Microsomal concentrations of linoleic acid, a major substrate for adrenal LP, were increased by ACTH administration and then also returned to control levels after cessation of treatment. There were no significant changes in adrenal alpha-tocopherol or beta-carotene concentrations resulting from ACTH treatment. The results indicate that ACTH has a role in the regulation of adrenal LP. The actions of ACTH cannot be attributed to an increase in adrenal content of the antioxidants, alpha-tocopherol and beta-carotene, or to a decrease in LP substrate. The actions of ACTH to inhibit LP may contribute to an increase in adrenal hormone production by protecting steroidogenic enzymes from peroxidative degradation.

Conversion of spironolactone to 7 alpha-thiomethylspironolactone by hepatic and renal microsomes.

Recent observations indicate that 7 alpha-thiomethylspironolactone is an important circulating metabolite of the mineralocorticoid antagonist spironolactone (SL). Studies were carried out to determine possible sites and pathways of 7 alpha-thiomethyl-SL formation and, in particular, to evaluate SL metabolism by guinea pig hepatic and renal microsomal preparations. In the absence of S-adenosylmethionine (SAM), liver and kidney microsomes rapidly converted SL to 7 alpha-thio-SL as the only metabolite. The rate of 7 alpha-thio-SL production was greater in liver than kidney. In the presence of SAM, 7 alpha-thio-SL was further converted to 7 alpha-thiomethyl-SL by liver and kidney microsomes. The rates of methylation with 7 alpha-thio-SL as substrate were three to four times greater for liver than for kidney, but the Km values were similar (approximately 30 microM) in the two issues. Maximal enzyme activity was obtained with SAM concentrations of 25-200 microM. NADPH had no effect on SL or 7 alpha-thio-SL metabolism by liver or kidney microsomes. To determine if a pathway involving the C-S lyase enzyme might contribute to circulating 7 alpha-thiomethyl-SL levels in vivo, guinea pigs were treated with SL or its dethioacetylated derivative, canrenone, and plasma metabolites were analyzed by HPLC. Both 7 alpha-thiomethyl-SL and canrenone were found to be circulating metabolites in SL-treated animals, but only canrenone was identified in the plasma of canrenone-treated guinea pigs. The results indicate that the liver and kidney are potential sites of 7 alpha-thiomethyl-SL production and that its formation probably does not involve the C-S lyase pathway.