Guinea Pig Adrenal Cortex Research Articles

Downregulation of CYP2D16 by ACTH in the Guinea Pig Adrenal Cortex: Time Course, Reversibility, and Mechanism of Action

Studies were done to investigate the actions of ACTH on the expression of CYP2D16 in the guinea pig adrenal cortex. Guinea pigs were treated with ACTH for 1, 3, or 7 days. In addition, some animals received ACTH for 7 days and were then untreated for an additional 3 or 7 days to test for reversibility of ACTH actions. ACTH treatment caused a time-dependent decrease in the rates of adrenal microsomal bufuralol metabolism, a CYP2D-catalyzed reaction; hepatic bufuralol metabolism was unaffected by ACTH. Adrenal enzyme activity was significantly reduced by ACTH within 1 day and decreased by 80% after 7 days. Western blotting and in situ hybridization analyses revealed corresponding declines in adrenal CYP2D16 protein and mRNA concentrations. Nuclear runoff assays indicated that ACTH treatment inhibited CYP2D16 expression at the transcriptional level. Adrenal 17α-hydroxylase activities were increased by ACTH treatment, but CYP17 protein concentrations were not affected. Following cessation of ACTH administration, the rates of adrenal bufuralol metabolism and CYP2D16 protein and mRNA concentrations returned to control levels within 7 days. The results demonstrate that ACTH has a relatively rapid and reversible effect to inhibit adrenal CYP2D16 transcription, thereby decreasing adrenal xenobiotic metabolism. Thus, the actions of ACTH on CYP2D16 expression are opposite to those on other adrenal P-450 isozymes, indicating unique regulatory mechanisms.

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.

Bimodal Expression of CYP2D16 in the Guinea Pig Adrenal Cortex

Prior studies revealed an apparent bimodal distribution of adrenal xenobiotic-metabolizing activities in outbred guinea pigs. High activities were characteristic of most animals but a minority had very low rates of metabolism. The present studies were done to determine the basis for this distribution. Among the enzymatic activities abundant in guinea pig adrenal microsomes is bufuralol 1′-hydroxylation, a marker for CYP2D isozymes. Western blot analyses revealed that adrenal expression of CYP2D16, the guinea pig CYP2D analogue, was highly correlated with xenobiotic-metabolizing activities. Of 12 animals studied, 4 had low levels of adrenal CYP2D16 expression and correspondingly low bufuralol 1′-hydroxylase and benzphetamine N-demethylase activities. In contrast, adrenal expression of steroidogenic P450 isozymes (CYP17 and CYP21) and the corresponding steroid hydroxylase activities were similar in all animals. There was also no evidence of polymorphism in hepatic xenobiotic metabolism. The results suggest that the bimodal expression of adrenal CYP2D16 is at least partly responsible for the variability in adrenal xenobiotic metabolism, and that this pattern of expression is not applicable to other adrenal P450 isozymes or to hepatic CYP2D16. Further studies are needed to determine if genetic polymorphism is responsible for the mode of adrenal CYP2D16 expression.

Effects of ACTH administration on zonation of the guinea pig adrenal cortex

Experiments were done to determine the actions of ACTH on the morphologic and functional characteristics of the zona fasciculata (ZF) and zona reticularis (ZR) in the guinea pig adrenal cortex. In control guinea pigs, a number of morphologic differences distinguished the ZF from the ZR, including the presence of far more lipid in the ZF than in the ZR. Treatment with ACTH decreased the lipid droplet content of the ZF cells, equalizing the amount of lipid in the two zones. Other morphologic differences between the ZF and ZR were also diminished by ACTH treatment. Immunohistochemical analyses indicated that CYP17 protein was found in both the ZF and ZR in control animals, but with greater immunostaining intensity in the ZF. The enzyme protein distribution corresponded with higher 17 α-hydroxylase activity in the ZF than in the ZR. After ACTH treatment, the intensity of staining and enzyme activities in the two zones were similar, attributable largely to increases in the ZR. In situ hybridization and immunohistochemistry showed that in control animals CYPD216 was highly expressed in the ZR but not in the ZF. ACTH treatment dramatically reduced the intensity of CYP2D16 mRNA and protein staining in the ZR. Bufuralol 1′-hydroxylase activity, a marker for CYP2D subfamily members, was also decreased significantly in the ZR by ACTH treatment. The data indicate that administration of ACTH to guinea pigs has opposite effects on the expression of CYP17 and CYP2D16 in the ZR, and diminishes or eliminates some of the structural and functional differences between the ZF and ZR. The results suggest a role for ACTH in establishing and maintaining adrenocortical zonation.

Localization of CYP2D16 in the guinea pig adrenal cortex by immunohistochemistry and in situ hybridization

Recent reports indicate that the cytochrome P450 isozyme, CYP2D16, is expressed at high levels in the inner regions of the guinea pig adrenal cortex and may contribute to xenobiotic and/or steroid metabolism in the gland. In the present studies, immunohistochemical and in situ hybridization techniques were employed to definitively establish the localization of CYP2D16 within the adrenal cortex. In male guinea pigs of various ages, CYP2D16 protein and mRNA were highly localized to the zona reticularis (ZR); none was detectable in the zona fasciculata (ZF), zona glomerulosa (ZG) or the medulla. In contrast, the steroidogenic P450 isozyme, CYP17, was distributed throughout the ZF and ZR. From the earliest stages of development of the ZR, CYP2D16 staining was intense. As guinea pigs aged, the ZR progressively enlarged and comprised a proportionately greater amount of the cortex. At all ages, CYP2D16 was uniformly distributed throughout only the ZR. Coinciding with the age-related growth of the ZR and increase in adrenal CYP2D16 content was an increase in adrenal xenobiotic-metabolizing activity. The results establish that CYP2D16 has an intraadrenal localization that is unique among P450 isozymes, suggesting novel regulatory mechanisms and indicating that CYP2D16 may serve as a specific marker for ZR cells. The increase in CYP2D16 expression with age probably accounts for increasing levels of xenobiotic metabolism and may also contribute to an increase in intraadrenal cortisol degradation in older animals.

Effects of prolactin on DNA synthesis, cholesterol stores, and steroidogenesis in the adrenal cortex of dexamethasone-treated guinea pigs

The effect of prolactin on the guinea pig adrenal cortex is studied in a model of dexamethasone-induced atrophy. Prolactin elevates the content of free and total cholesterol per tissue weight but does not affect the specific activity of hydrocortisone synthesized in the presence of labeled cholesterol. It is concluded that prolactin has no direct effect on hormonopoiesis but activates proliferative processes in the adrenal cortex.

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.

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

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

Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs.

Prior in vitro investigations demonstrated that the P450 suicide substrate, 1-aminobenzotriazole (ABT), was a potent inhibitor of xenobiotic metabolism but had no effect on steroidogenic enzymes in the guinea pig adrenal cortex. Studies were done to determine if ABT administration of guinea pigs in vivo also selectively inhibited adrenal xenobiotic metabolism. At single doses of 25 or 50 mg/kg, ABT effected rapid decreases in spectrally detectable adrenal P450 concentrations. The higher dose caused approx. 75% decreases in microsomal and mitochondrial P450 levels within 2 h. The decreases in P450 were sustained for 24 h but concentrations returned to control levels within 72 h. Accompanying the ABT-induced decreases in adrenal P450 content were proportionately similar decreases in P450-mediated xenobiotic and steroid metabolism. Microsomal benzo(a)pyrene hydroxylase, benzphetamine N-demethylase, 17 alpha-hydroxylase and 21-hydroxylase activities were decreased to 20-25% of control values by the higher dose of ABT. Mitochondrial 11 beta-hydroxylase and cholesterol sidechain cleavage activities were similarly diminished by ABT treatment. Adrenal 3 beta-hydroxysteroid dehydrogenase activity, by contrast, was not affected by ABT, indicating specificity for P450-catalyzed reactions. The results demonstrate that ABT in vivo is a non-selective inhibitor of adrenal steroid- and xenobiotic-metabolizing P450 isozymes. The absence of ABT effects on steroid metabolism in vitro suggests that an extra-adrenal metabolite may mediate the in vivo inhibition of steroidogenesis.

Molecular Cloning and Sequencing of a Guinea Pig Cytochrome P4502D (CVP2D16): High Level Expression in Adrenal Microsomes

Studies were done to characterize a guinea pig adrenal microsomal P450 that had been linked with xenobiotic metabolism in the inner zone of the gland. N-terminal amino acid sequencing of the isolated protein revealed homology with members of the CYP2D subfamily. A human CYPD2D6 cDNA probe was used to screen a guinea pig adrenal cDNA library and a full-length clone was obtained having an open reading frame encoding a 500 amino acid protein. The sequence was found to be highly homologous with all members of the CYP2D subfamily and was designated CYP2D16. The N-terminal sequence of 38 amino acids obtained from the protein microsequencing was identical to that deduced from the nucleotide sequence of the cloned CYP2D16. Northern blot analysis confirmed that CYP2D16 is expressed at high levels in the inner zone of the guinea pig adrenal cortex. The results suggest that CYP2D 16 may account, at least in part, for the high rates of xenobiotic metabolism in the guinea pig adrenal.

Biochemical alterations in guinea pig adrenal cortex following administration of PD 132301-2, an inhibitor of acyl-CoA:cholesterol acyltransferase.

To assess whether previously reported ultrastructural alterations of adrenocortical mitochondria induced by the acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor PD 132301-2 are accompanied by functional deficits in tissue energy stores, phosphorylated adenine nucleotide levels in guinea pig adrenal cortex were quantitated. Adrenals of male guinea pigs were obtained at 1, 2, 6, or 24 hours after oral administration of 100 mg/kg PD 132301-2 or 0.5% methylcellulose vehicle. In treated animals, ATP levels and ATP/ADP ratios were decreased approximately 50% with concurrent increases in AMP. Calculated energy charge was also decreased; these decreases were maximal by 6 hours. Cholesterol esterification in adrenal cortex was inhibited, resulting in progressive accumulation of free cholesterol up to 3-fold over control by 24 hours, consistent with the ACAT inhibitory activity of the drug. These data suggest that PD 132301-2 distributes to the adrenal cortex where early alterations of tissue bioenergetics occur in a time frame consistent with ultrastructural alterations of mitochondria.

Zonal differences in adrenocortical lipid peroxidation: Role of α-tocopherol

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

Regulation of 21-hydroxylase activity by steroids.

In this study, we investigated the effect of steroids on guinea pig and bovine adrenal steroidogenesis, especially 21-hydroxylase activity. Analysis of guinea pig adrenal steroids indicated the presence of high concentrations of androstenedione in the guinea pig adrenal; furthermore, in vitro studies using guinea pig adrenal cortex cells in primary culture confirmed that androstenedione is one of the major C19 steroids produced and secreted. The direct action of steroids on steroid production by adrenal cells was investigated. Our data indicate that steroids themselves increase C19 steroid synthesis and inhibit glucocorticoid production by guinea pig adrenal cells without affecting gene expression for steroidogenic enzymes. Incubation of a series of C19 steroids, namely, androstenedione, with guinea pig adrenal cell cultures demonstrated that the decrease in 21-hydroxylase activity is largely independent of the androgenic activity of C19 steroids. RU38486, a synthetic C18 steroid possessing a 4-ene-3-ketosteroid with an aryl group at position 11 and a very low affinity for the androgen receptor, also irreversibly altered 21-hydroxylase activity. An effect of RU38486 on 21-hydroxylase activity was also demonstrated in bovine adrenal cells. Further studies with bovine adrenal cells showed that the decrease in 21-hydroxylase activity induced by RU38486 was accompanied by a small but significant inhibition of P450c21 protein levels at both basal and ACTH-stimulated levels. In summary, our data indicate that alteration of 21-hydroxylase activity by steroids is likely due to a direct action on P540c21 protein, and the levels of androstenedione in the adrenal are high enough to inhibit 21-hydroxylase activity.

Chemical codes of sensory neurons innervating the guinea-pig adrenal gland

Retrograde neuronal tracing in combination with double-labelling immunofluorescence was applied to distinguish the chemical coding of guinea-pig primary sensory neurons projecting to the adrenal medulla and cortex. Seven subpopulations of retrogradely traced neurons were identified in thoracic spinal ganglia T1-L1. Five subpopulations contained immunolabelling either for calcitonin gene-related peptide (CGRP) alone (I), or for CGRP, together with substance P (II), substance P/dynorphin (III), substance P/cholecystokinin (IV), and substance P/nitric oxide synthase (V), respectively. Two additional subpopulations of retrogradely traced neurons were distinct from these groups: neurofilament-immunoreactive neurons (VI), and cell bodies that were nonreactive to either of the antisera applied (VII). Nerve fibers in the adrenal medulla and cortex were equipped with the mediator combinations I, II, IV and VI. An additional meshwork of fibres solely labelled for nitric oxide synthase was visible in the medulla. Medullary as well as cortical fibres along endocrine tissue apparently lacked the chemical code V, while in the external cortex some fibre exhibited code III. Some intramedullary neuronal cell bodies revealed immunostaining for nitric oxide synthase, CGRP or substance P, providing an additional intrinsic adrenal innervation. Perikarya, immunolabelled for nitric oxide synthase, however, were too few to match with the large number of intramedullary nitric oxide synthase-immunoreactive fibres. A non-sensory participation is also supposed for the particularly dense intramedullary network of solely neurofilament-immunoreactive nerve fibres. The findings give evidence for a differential sensory innervation of the guinea-pig adrenal cortex and medulla. Specific sensory neuron subpopulations suggest that nervous control of adrenal functions is more complex than hitherto believed.

Adrenal activation of carbon tetrachloride: role of microsomal P450 isozymes

Previous investigations demonstrated that carbon tetrachloride (CCl 4) was activated by adrenal microsomes, resulting in various functional changes and ultimately in necrosis of the zona reticularis of the gland. Experiments were done to identify the adrenal P450 isozyme(s) involved in the bioactivation of CCl 4. Incubation of microsomes from the zona reticularis (ZR) of the guinea pig adrenal cortex with CCl 4 plus NADPH caused initiation of lipid peroxidation, covalent binding of CCl 4-derived radioactivity to protein, and degradation of cytochrome(s) P450. Preincubation of the microsomal preparations with inhibitory antibodies to P450 17α or P450 C12 decreased the corresponding enzyme activities (17α-hydroxylation and 21-hydroxylation), but did not affect the activation of CCl 4. 1-Aminobenzotriazole (ABT), a suicide inhibitor of some P450 isozymes, decreased the enzyme activities catalysed by an adrenal 52 000 Da (52 kDa) isozyme, but had no effect on the function of P450 17α or P450 C21. However, ABT completely inhibited the CCl 4-induced LP and covalent binding in adrenal microsomes. The results indicate that adrenal CCl 4 activation is catalysed by the 52 kDa P450 isozyme and not by the steroid hydroxylases. Localization of the 52 kDa isozyme to the ZR probably accounts for the selective necrosis of this region of the gland by CCl 4.

Estrogen and hydroxysteroid sulfotransferases in guinea pig adrenal cortex: cellular and subcellular distributions.

This report describes for the first time the cellular and subcellular localization of estrogen sulfotransferase (EST) as well as the subcellular localization of hydroxysteroid sulfotransferase (HST) in the mammalian adrenal cortex. A 34-kilodalton EST and two HSTs with 3 alpha- and 3 beta-hydroxysteroid substrate specificities (32 and 33 kilodaltons, respectively) were previously purified from guinea pig adrenal cortex and characterized. Western blots were used to establish that two antisera generated against EST were highly specific for EST, whereas three antisera generated against the HSTs were highly specific for the HSTs, but did not distinguish between the 3 alpha- and 3 beta HSTs. Light and electron microscopic immunoperoxidase labeling with these antisera revealed that the sulfotransferases were expressed only within the ACTH-responsive layers of the guinea pig adrenal cortex, with EST localized to zona fasciculata and zona reticularis cells, and the HSTs confined to the zona reticularis. No labeling was detected in the zona glomerulosa or adrenal medulla. EST was concentrated in cell nuclei; sparse labeling was distributed throughout the cytoplasm. HST labeling was intense in smooth endoplasmic reticulum of zona reticularis cells, but was absent from nuclei. Ovoid inclusions about 1-4 microns in diameter, with no limiting membrane, were observed in zona reticularis cells; these inclusions were strongly labeled for both EST and HSTs. No gender-specific differences in distribution or labeling intensity were apparent. The high concentration of EST immunoreactivity in nuclei suggests that EST may play a role in modulating the ability of active estrogens to regulate gene expression in ACTH-responsive cells. The distribution of HST labeling suggests that sulfonation of adrenocortical 3-hydroxysteroids takes place largely within smooth endoplasmic reticulum in the zona reticularis in adult guinea pigs.

Estrogen and hydroxysteroid sulfotransferases in guinea pig adrenal cortex: cellular and subcellular distributions

This report describes for the first time the cellular and subcellular localization of estrogen sulfotransferase (EST) as well as the subcellular localization of hydroxysteroid sulfotransferase (HST) in the mammalian adrenal cortex. A 34-kilodalton EST and two HSTs with 3 alpha- and 3 beta-hydroxysteroid substrate specificities (32 and 33 kilodaltons, respectively) were previously purified from guinea pig adrenal cortex and characterized. Western blots were used to establish that two antisera generated against EST were highly specific for EST, whereas three antisera generated against the HSTs were highly specific for the HSTs, but did not distinguish between the 3 alpha- and 3 beta HSTs. Light and electron microscopic immunoperoxidase labeling with these antisera revealed that the sulfotransferases were expressed only within the ACTH-responsive layers of the guinea pig adrenal cortex, with EST localized to zona fasciculata and zona reticularis cells, and the HSTs confined to the zona reticularis. No labeling was detected in the zona glomerulosa or adrenal medulla. EST was concentrated in cell nuclei; sparse labeling was distributed throughout the cytoplasm. HST labeling was intense in smooth endoplasmic reticulum of zona reticularis cells, but was absent from nuclei. Ovoid inclusions about 1-4 microns in diameter, with no limiting membrane, were observed in zona reticularis cells; these inclusions were strongly labeled for both EST and HSTs. No gender-specific differences in distribution or labeling intensity were apparent. The high concentration of EST immunoreactivity in nuclei suggests that EST may play a role in modulating the ability of active estrogens to regulate gene expression in ACTH-responsive cells. The distribution of HST labeling suggests that sulfonation of adrenocortical 3-hydroxysteroids takes place largely within smooth endoplasmic reticulum in the zona reticularis in adult guinea pigs.

Maturational changes in steroidogenesis in the inner and outer zones of the guinea pig adrenal cortex

Studies were done to determine the effects of age on steroidogenesis in the inner (zona reticularis) and outer (zona fasciculta plus glomerulosa) zones of the guinea pig adrenal cortex. In 35-day-old animals, cortisol production by adrenal outer zone cells was approximately twice as great as that by inner zone cells. With aging, cortisol secretion by inner zone cells decreased to very low levels, but there was no detectable change in the capacity for cortisol production by the outer zone. However, the outer zone comprised a progressively decreasing fraction of the total adrenal mass in older animals. To determine the basis for the decline in cortisol production by inner zone cells with aging, the activities of several steroidogenic enzymes were determined. Microsomal 21-hydroxylase activity was greater in the inner than outer zone but was not significantly affected by age. By contrast, 17α-hydroxylase activity was greater in the outer zone at all ages, and decreased with aging in the inner but not the outer zone. Mitochondrial cholesterol sidechain cleavage and 11β-hydroxylase activities were also higher in the outer than inner zone and declined in the zone only in older animals. The decrease in inner zone cholesterol sidechain cleavage activity with aging was proportionately greater than the age-dependent changes in other enzyme activities. The results indicate that the effects of aging on steroidogenesis are both zone- and enzyme-specific. The overall decline in cortisol secretion by the guinea pig adrenal cortex with aging is attributable to both a decrease in cortisol production by the cells of the zone reticularis and a disproportionate increase in the mass of the gland comprised by this zone. The decrease in cortisol secretion correlates closely with a decline in cholesterol sidechain cleavage activity in the zona reticularis, and may be causally related.

Steroidogenesis in guinea pig adrenal cortex: Effects of ACTH on steroid secretion and steroidogenic enzyme activities and expression

In this study, we investigated guinea pig adrenal steroidogenesis, specially, C19 steroid production. Analysis of adrenal steroids by high performance liquid chromatography and gas chromatography indicated the presence of androstenedione and 11β-hydroxyandrostenedione. Adrenal androstenedione and 11β-hydroxyandrostenedione levels were stimulated by ACTH administration while only 11β-hydroxyandrostenedione was increased in plasma. In vitro studies using adrenal cortex cells in primary culture confirmed that 11β-hydroxyandrostenedione is the major C19 steroid secreted. The chronic treatment of guinea pig with ACTH stimulated all adrenal post-pregnenolone enzyme activities and decreased P 450c17 mRNA levels while P 450scc, 3β-hydroxysteroid dehydrogenase and P450c11 mRNAs remained unaffected. Treatment of adrenal cells in primary culture with ACTH for 72 h changed the distribution of steroids secreted and decreased 21-hydroxylase activity while 17α-hydroxylase and 17,20-lyase activities were increased favoring C19 steroid production. In ACTH-treated cells, the mRNA levels for P450c21 and P450c17 increased and reached a peak at 18 h. Our data indicate that treatment with ACTH stimulates adrenal steroidogenic capacity by increasing steroid secretion and causes transcriptional and post-transcriptional effects on steroidogenic enzymes gene expression. Finally, the direct action of steroids on steroid production by adrenal cells in primary culture was investigated. Our data indicate that steroids themselves increase C19 steroid synthesis and inhibit glucocorticoid production without affecting gene expression for steroidogenic enzymes.

Differential effects of adrenocorticotropic hormone on steroid hydroxylase activities in the inner and outer zones of the guinea pig adrenal cortex

We have studied the effects of ACTH treatment on steroid hydroxylase activities in the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Animals received 5 or 10 U of ACTH daily for 6 days and enzyme activities were then assessed in isolated microsomal or mitochondrial preparations. In control animals, microsomal cytoochrome P-450 concentrations were greater in the inner than outer zone, but mitochondrial P-450 levels were similar in the two zones. Microsomal 17α-hydroxylase and mitochondrial 11β-hydroxylase activities were greater in the outer than inner zone, but microsomal 21-hydroxylase activity was greater in the inner zone. ACTH treatment decreased cytochrome P-450 concentrations in inner but not outer zone microsomes; mitochondrial P-450 levels were unaffected in both zones. ACTH caused a dose-dependent increase in inner zone 17α-hydroxylase activity and decrease in 21-hydroxylase activity without affecting the activity of either enzyme in outer zone microsomes. ACTH also decreased 11β-hydroxylase activity in outer but not inner zone mitochondrial preparations. The net effect of ACTH treatment was to diminish the differences in steroid metabolism between the two zones. The results indicate that the effects of ACTH on steroid hydroxylase activities are both zone- and enzyme-dependent, suggesting the existence of multiple and independent regulatory mechanisms.