Adrenalectomy Blocks the Compensatory Increases in UT-A1 and AQP2 in Diabetic Rat Kidney

In normal rats we showed that glucocorticoids participate in the downregulation of UT-A1 protein abundance in the inner medullary tip and in lowering of basal and vasopressin-stimulated facilitated urea permeability in terminal IMCDs. To examine the relevance of this response to a rat model of human disease, we studied rats with uncontrolled diabetes mellitus (DM) induced by streptozotocin (STZ), since these rats have increased corticosterone production and urea excretion. We found that at 3 days of DM, UT-A1 protein abundance is downregulated in the inner medullary tip compared to pair-fed control rats, while DM for more than 7 days caused an increase in UT-A1. To test whether adrenal steroids could be a mechanism contributing to the latter increase, we studied adrenalectomized rats (ADX), ADX rats given STZ to induce diabetes (ADX + STZ), and ADX + STZ rats receiving exogenous aldosterone or dexamethasone. In contrast to control rats, UT-A1 protein abundance was not increased by prolonged DM in the ADX rats. Aquaporin 2 (AQP2) was not increased in the inner medullas of 10-day DM rats either. However, UT-A1 protein abundance was significantly reduced in the inner medullary tips from both diabetic aldosterone-treated (40 ± 2%) and dexamethasone-treated (43 ± 2%) ADX rats compared to diabetic ADX rats without steroid replacement. AQP2 was unaffected by steroid hormone treatments. Thus, both mineralocorticoids and glucocorticoids downregulate UT-A1 protein abundance in rats with uncontrolled diabetes mellitus for 10 days. These results suggest that: 1) the increase in UT-A1 observed in DM is dependent upon having adrenal steroids present; and 2) adrenal steroids are not sufficient to enable the compensatory rise in UT-A1 to a steroid-deficient diabetic animal.     

The relationship between adrenal vascular events and steroid secretion: the role of mast cells and endothelin.

The actions of ACTH on the adrenal cortex are known to be 2-fold. In addition to increased steroidogenesis, ACTH also causes marked vasodilation, reflected by an increased rate of blood flow through the gland. Our studies, using the in situ isolated perfused rat adrenal preparation, have shown that zona fasciculata function and corticosterone secretion are closely related to vascular events, with an increase in perfusion medium flow rate causing an increase in corticosterone secretion, in the absence of any known stimulant. These observations give rise to two important questions: how does ACTH stimulate blood flow; and how does increased blood (or perfusion medium) flow stimulate steroidogenesis? Addressing the first question, we have recently identified mast cells in the adrenal capsule, and shown that Compound 48/80, a mast cell degranulator, mimics the actions of ACTH on adrenal blood flow and corticosterone secretion. We have also demonstrated an inhibition of the adrenal vascular response to ACTH in the presence of disodium cromoglycate, which prevents mast cell degranulation. We conclude, therefore, that ACTH stimulates adrenal blood flow by its actions on mast cells in the adrenal capsule. Addressing the second question, we looked at the role of endothelin in the rat adrenal cortex. Endothelin 1, 2 and 3 caused significant stimulation of steroid secretion by collagenase dispersed cells from both the zona glomerulosa and the zona fasciculata. A sensitive response was seen, with significant stimulation at an endothelin concentration of 10(-13) mol/l or lower. Endothelin secretion by the in situ isolated perfused rat adrenal gland was measured using the Amersham assay kit. Administration of ACTH (300 fmol) caused an increase in the rate of immunoreactive endothelin secretion, from an average of 28.7 +/- 2.6 to 52.6 +/- 6 fmol/10 min (P less than 0.01, n = 5). An increase in immunoreactive endothelin secretion was also seen in response to histamine, an adrenal vasodilator, which stimulates corticosterone secretion in the intact gland, but has no effect on collagenase-dispersed cells. From these data we conclude that endothelin may mediate the effects of vasodilation on corticosterone secretion, and this mechanism may explain some of the differences in response characteristics between the intact gland and dispersed cells.     

Molecular and binding characteristics of IP3 receptors in bovine spermatozoa

We have shown the presence of inositol 1,4,5-triphosphate (IP3) receptors in bovine spermatozoa. These receptors are mainly localized and functionally associated with the acrosome region. Molecular characterization of these bovine IP3 receptors has shown that the functional size of the IP3 binding domain is a protein of 66+/-2 kDa, in agreement with the size of both bovine adrenal cortex and bovine adrenal medullar chromaffin cells IP3 receptors. In contrast, bovine cerebellum IP3 receptor displays molecular weight of 220+/-5 kDa, a value in agreement with data in the literature. Bovine IP3 receptors have a one-affinity state characterized by a low affinity (Kd 750 nM) and a relatively high density (7.5 pmol/mg protein). They are functional and release internal calcium upon the binding of the second messenger. Moreover, the finding that the specific A1 adenosine receptor agonist R-PIA elicits almost the same effect as IP3 might be of some help in understanding the physiological role of these inhibitory adenosine receptors in mammalian spermatozoa.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

Further evidence that the mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are involved in the acute regulation of steroidogenesis.

In previous studies we and others have described several mitochondrial proteins which are synthesized in response to acute hormone stimulation in several steroidogenic tissues. In both MA-10 mouse Leydig tumor cells and primary cultures of rat adrenal cortex cells, these proteins consist of a family of 37 kilodalton (kDa) and 32 kDa precursor forms and fully processed forms which are 30 kDa in molecular weight. The nature of the appearance of these proteins and their subcellular localization to the mitochondria, the site of the rate limiting step in steroidogenesis, has led to the speculation that they may be involved in the acute regulation of steroidogenesis. In the present study we have taken advantage of another steroidogenic cell, the R2C rat Leydig tumor cell, to perform studies which further indicate that these mitochondrial proteins are involved in the regulation of steroidogenesis. Unlike the MA-10 cell which requires hormone stimulation for steroid production, the R2C cell is a constitutive progesterone producer whose steroid production cannot be further increased with hormone stimulation. We have shown that the R2C cell line is less sensitive to the inhibition of steroid production by the metal chelator orthophenanthroline (OP) than is the MA-10 cell. We have demonstrated that progesterone production and the 30 kDa mitochondrial proteins remain present in the R2C cells at a concentration of OP which completely inhibits progesterone production and totally eliminates the 30 kDa proteins in MA-10 cells. As further evidence for the role of these proteins in steroidogenic regulation, we have isolated several revertants of the R2C parent (P) cell line which have lost the ability to synthesize progesterone constitutively, but which can be stimulated to synthesize this steroid by trophic hormone and cAMP analog. In these revertants, designated (R), the normally constitutively present 30 kDa proteins are greatly decreased compared to controls, but reappear in large amounts following hormone stimulation. Taken together, these data provide further evidence that the 30 kDa mitochondrial proteins are involved in the acute regulation of steroidogenesis in Leydig cells.     

Corticotropin increases protein tyrosine phosphatase activity by a cAMP-dependent mechanism in rat adrenal gland.

Corticotropin signal transduction pathway involves serine/threonine protein phosphorylation. Recent reports suggest that protein tyrosine dephosphorylation may also be an integral component of that pathway. The present study was performed to investigate the role played by protein tyrosine phosphatases (PTPs) on acute response to corticotropin and the hypothetical regulation of PTPs by this hormone. We have used two powerful cell permeant PTP inhibitors, phenylarsine oxide (PAO) and pervanadate (PV), in order to examine the relevance of PTP activity on hormone-stimulated and 8-bromo-adenosine 3',5'-phosphate (8Br-cAMP is a permeant analogue of adenosine 3',5'-phosphate)-stimulated steroidogenesis in adrenal zona fasciculata (ZF) cells. In both cases, PAO and PV inhibited the steroid production in a dose-dependent fashion, and had no effect on steroidogenesis supported by a permeant analogue of cholesterol. The effect of hormonal stimulation on PTP activity was analyzed in rat adrenal ZF. In vivo corticotropin treatment reduced phosphotyrosine content in endogenous proteins and produced a transient increase of PTP activity in the cytosolic fraction, reaching a maximum (twofold) after 15 min. Incubation of adrenal ZF with 8Br-cAMP also produced PTP activation, suggesting that it can be mediated by cAMP-dependent protein kinase (PKA)-dependent phosphorylation. Detection of PTP activity in an in-gel assay showed three corticotropin-stimulated soluble PTPs with molecular masses of 115, 80 and 50 kDa. In summary, we report for the first time a hormone-dependent PTP activation in a steroidogenic tissue and provide evidence that PTP activity plays an important role in corticotropin signal pathway, acting downstream of PKA activation and upstream of cholesterol transport across the mitochondrial membrane.     

New signal transduction mechanisms of atrial natriuretic factor: inhibition of phosphorylation of protein kinase C and A 240 kDa protein in adrenal cortical plasma membrane by cGMP dependent and independent mechanisms

The effects of synthetic atrial natriuretic factor (ANF) on the state of protein phosphorylation in plasma membranes of bovine adrenal cortex have been studied in vitro. ANF (1x10(-8)M - 1x10(-7)M) specifically inhibited the phosphorylation of two distinct proteins of 78 kDa and 240 kDa. Immunoblotting with specific antiserum to protein kinase C produced evidence that 78 kDa protein is most likely the protein kinase C whose phosphorylation is inhibited by both ANF and cGMP. However, cGMP did not affect the phosphorylation of 240 kDa protein, indicating a new cGMP-independent mechanism of ANF action in the adrenal, which is compatible with the lack of action of cGMP and its analogs in ANF-induced inhibition of aldosterone secretion from adrenal cortex. The inhibition of phosphorylation of putative protein kinase C by ANF or cGMP indicates a hitherto unknown signal transduction mechanism of ANF.     

Regional distribution of microsomal drug and steroid metabolism in the guinea pig adrenal cortex

The regional distribution of steroid and drug metabolism was studied in intact cells and microsomal fractions obtained from the chromatically distinct inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Cells isolated from the outer cortical zone produced far more cortisol than cells from the inner zone and cortisol production was stimulated by adrenocorticotropic hormone only in cells from the outer zone. Among the factors which may contribute to the greater cortisol production by the outer zone are a higher rate of 17 alpha-hydroxylation and ratio of 17 alpha- to 21-hydroxylase activities in that zone, both of which favor cortisol synthesis. In contrast, steroid 21-hydroxylase activity was far greater than 17 alpha-hydroxylase activity in microsomes obtained from the inner zone of the adrenal cortex. Microsomal metabolism of various xenobiotics such as benzo(a)pyrene and ethylmorphine proceeded far more rapidly in the inner than outer cortical zone. The zonal differences in metabolism appeared to result in part from differences in the ability of xenobiotics to interact with microsomal cytochromes P-450 in the two zones. The results indicate that the inner zone has a minor role in cortisol production by the adrenal cortex, but its involvement in the production of other steroids cannot be excluded. In contrast, the inner zone appears to have the major role in the metabolism of at least some xenobiotics which may account for its greater vulnerability to the toxic effects of chemicals requiring metabolic activation.     

Purification and characterization of 3β-hydroxysteroid-dehydrogenase/isomerase from bovine adrenal cortex

The formation of 4-ene-3-ketosteroids from 3β-hydroxy-5-ene precursors is an obligatory step in the biosynthesis of hormonal steroids such as glucocorticoids, mineralocorticoids, estrogens and androgens. In the adrenal cortex, pregnenolone, 17-hydroxy-pregnenolone and dehydroisoandrosterone are converted to progesterone, 17-hydroxy-progesterone and androstenedione, respectively, by the enzymatic system 3β-hydroxy-5-ene steroid dehydrogenase and 3-keto-5-ene steroid isomerase (3β-HSD/I).

The present work reports a two step purification procedure which yields an homogenous preparation of 3β-HSD/I from bovine adrenal cortex. It uses solubilization of the microsomal proteins followed by two chromatographic steps, i.e. DEAE-cellulose and heparine-sepharose columns. The enzyme was obtained as an homogeneous protein exhibiting an apparent molecular size of 45 kDa upon SDS-gel electrophoresis and of 81 kDa upon gel filtration. The purified enzyme exhibits both the 5-ene-3β-ol steroid dehydrogenase and isomerase activities in contrast to previous work using a more complex procedure which yielded a final preparation having lost its isomerase activity [Hiwatashi et al., Biochem. J. 98 (1985) 1519–1525]. N-terminal aminoacid (29 residues) sequence of the purified protein was determined and was found identical to that predicted from the nucleic acid sequence of the recently identified enzyme cDNA [Zhas et al. FEBS Lett. 259 (1989) 153–157].     

Strain Differences in Adrenal Microsomal Steroid Metabolism in Guinea Pigs

We recently reported that CYP2D16, a xenobiotic-metabolizing P450 isozyme, was expressed at higher levels in adrenal microsomes from inbred Strain 13 guinea pigs than in those from outbred English Short Hair (ESH) animals. Studies were done to determine if there also were strain differences in adrenal microsomal steroid metabolism. In both inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zone preparations of the adrenal cortex, 21-hydroxylase activities were greater in microsomes from ESH than from Strain 13 guinea pigs. By contrast, 17-hydroxylase activities were similar in the two strains. In both strains, 21-hydroxylase activities were greater in inner than outer zone microsomes, but the opposite was found for 17-hydroxylase activities (outer>inner). Northern and Western analyses revealed higher levels of CYP21 mRNA and protein in adrenals from ESH than Strain 13 guinea pigs, but there were no strain differences in CYP17 mRNA or protein concentrations. Despite the zonal differences in adrenal 17-hydroxylase and 21-hydroxylase activities, CYP17 and CYP21 mRNA and protein levels were similar in the inner and outer zones within each strain of guinea pig. The results demonstrate strain differences in microsomal steroid metabolism that are explained by differences in CYP21 expression. By contrast, the zonal differences in steroid hydroxylase activities may be attributable to post-translational mechanisms.     

Participation of cyclic nucleotides, protein kinase A and C in realization of n-acylethanolamine action in human adrenocortical cells

The messenger mechanisms mediating N-acylethanolamines (NAE) regulatory signals in the adrenal cortex were studied. An analysis of the mechanisms of realization of NAE effects in the post-operation human adrenal cortex was carried out in vitro. Influence of NAE mix on cAMP and cGMP level, protein kinase A and C activity in sub-cellular fraction of adrenocorticocytes and homogenates of conditionally normal adrenal cortex tissues was investigated. It was shown, that N-acylethanolamines treatment resulted in a decrease of cAMP level in adrenocortical cells. cGMP level is not changed in these conditions. The rise of protein kinase C activity was obtained in the membrane fraction after N-acylethanolamines in vitro treatment (3.3 microg/ml). Activity of cAMP-dependent protein kinase A significantly decreased in cytosol fraction of adrenocorticocytes. It was concluded, that steroid genesis activation is determined by protein kinase C activation, inhibition is determined by cAMP-dependent messenger system.     

The Involvement of Nek2 and Notch in the Proliferation of Rat Adrenal Cortex Triggered by POMC-Derived Peptides

The adrenal gland is a dynamic organ that undergoes constant cell turnover. This allows for rapid organ remodeling in response to the physiological demands of the HPA axis, which is controlled by proopiomelanocortin (POMC)-derived peptides, such as adrenocorticotropic hormone (ACTH) and N-Terminal peptides (N-POMC). In the rat adrenal cortex, POMC-derived peptides trigger a mitogenic effect, and this process increases cyclins D and E, while inhibiting p27Kip1. The goal of the present study was to further explore the mitogenic effect of ACTH and synthetic N-POMC_1–28 peptides by investigating the differences in the expression of key genes involved in the cell cycle of the rat adrenal cortex, following inhibition of the HPA axis. Moreover, we evaluated the differences between the inner and outer fractions of the adrenal cortex (ZF-fraction and ZG-fraction) in terms of their response patterns to different stimuli. In the current study, the inhibition of the HPA axis repressed the expression of Ccnb2, Camk2a, and Nek2 genes throughout the adrenal cortex, while treatments with POMC-derived peptides stimulated Nek2, gene and protein expression, and Notch2 gene expression. Furthermore, Notch1 protein expression was restricted to the subcapsular region of the cortex, an area of the adrenal cortex that is well-known for proliferation. We also showed that different regions of the adrenal cortex respond to HPA-axis inhibition and to induction with POMC-derived peptides at different times. These results suggest that cells in the ZG and ZF fractions could be at different phases of the cell cycle. Our results contribute to the understanding of the mechanisms involved in cell cycle regulation in adrenocortical cells triggered by N-POMC peptides and ACTH, and highlight the involvement of genes such as Nek2 and Notch.     

Characterization of a mitochondrial matrix protease catalyzing the processing of adrenodoxin precursor

Adrenodoxin (Ad) is synthesized as a larger precursor (preAd) by cytoplasmic polysomes and then transported into mitochondria concomitant with its proteolytic processing to the mature form. The protease in bovine adrenal cortex mitochondria, which converts preAd to the mature form, is a metalloprotease in the matrix (Sagara, Y., Ito, A. & Omura, T. (1984) J. Biochem. 96, 1743-1752). In this study, the protease was purified about 100-fold from the matrix fraction of bovine adrenal cortex mitochondria. The partially purified protease converted not only preAd, but also the precursors of malate dehydrogenase (MDH) and 27 kDa protein (P-27) to the corresponding mature forms. However, it was inactive toward the precursors of P-450(SCC) and of P-450(11 beta). Since isolated rat liver mitochondria can import and process preAd as efficiently as bovine adrenal cortex mitochondria, we partially purified a preAd-processing protease from rat liver mitochondria and compared its properties with those of the bovine adrenal cortex enzyme. The properties of the rat liver protease were indistinguishable from those of the bovine adrenal cortex enzyme in molecular weight determined from Sephadex G-150 gel filtration, metal requirement and ability to process preMDH and preP-27. The rat liver enzyme was also inactive toward the precursors of P-450(SCC) and P-450(11 beta). These results indicate the presence in both adrenal cortex and liver mitochondria of the same type of processing protease, which processes preAd and also the precursors of some other mitochondrial proteins.     

The subcellular distribution of the nonspecific lipid transfer protein (sterol carrier protein 2) in rat liver and adrenal gland

The distribution of the nonspecific lipid transfer protein (i.e., sterol carrier protein 2) over the various subcellular fractions from rat liver and adrenal gland was determined by enzyme immunoassay and immunoblotting. This distribution is very different in each of these two tissues. In liver, 66% of the transfer protein is present in the membrane-free cytosol as compared to 19% in the adrenal gland. In the latter tissue, the transfer protein is mainly found in the lysosomal/peroxisomal and the microsomal fraction at a level of 1093 and 582 ng per mg total protein, respectively (i.e., 17% and 35% of the total), and to a lesser extent in the mitochondrial fraction (11% of the total). Of all the membrane fractions isolated, the microsomal fraction from the liver and the mitochondrial fraction from the adrenal gland have the lowest levels of the transfer protein (i.e., 168 ng and 126 ng per mg total protein, respectively). These low levels correlate poorly with the active role proposed for this transfer protein in the conversion of cholesterol into bile acids and steroid hormones in these fractions. Using immunoblotting, it was demonstrated that in addition to the transfer protein (14 kDa) a cross-reactive 58 kD protein was present in the supernatant and the membrane fractions of both tissues. Cytochemical visualization in adrenal tissue with specific antibodies against the nonspecific lipid transfer protein showed that immunoreactive protein(s) were present mainly in the peroxisome-like structures.     

Complex role of the mitochondrial targeting signal in the function of steroidogenic acute regulatory protein revealed by bacterial artificial chromosome transgenesis in vivo

The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green fluorescent protein to steroidogenic cells of the adrenal cortex and gonads. Thereafter, we targeted expression of either wild-type StAR (WT-StAR) or a mutated StAR protein lacking the mitochondrial targeting signal (N47-StAR). In the context of mice homozygous for a StAR knockout allele (StAR-/-), all StAR activity derived from the StAR transgenes, allowing us to examine the function of the proteins that they encode. The WT-StAR transgene consistently restored viability and steroidogenic function to StAR-/- mice. Although the N47-StAR protein was reportedly active in transfected COS cells and mitochondrial reconstitution experiments, the N47-StAR transgene rescued viability in only 40% of StAR-/- mice. Analysis of lipid deposits in the primary steroidogenic tissues revealed a hierarchy of StAR function provided by N47-StAR: florid lipid deposits were seen in the adrenal cortex and ovarian theca region, with milder deposits in the Leydig cells. Our results confirm the ability of StAR lacking its mitochondrial targeting signal to perform some essential functions in vivo but also demonstrate important functional defects that differ from in vitro studies obtained in nonsteroidogenic cells.     

Stress-protective effect of the synthetic ACTH-like peptide leucocorticotropin

We found that the tritium-labeled synthetic ACTH-like octapeptide leucocorticotropin corresponding to the 81-88 sequence of the precursor of human interleukin-1alpha ([3H]GKVLKKRR) is bound by the ACTH receptor of rat adrenal cortex with a high affinity and specificity (Kd 2.2 +/- 0.1 nM). This peptide was shown to exert no effect on the adenylate cyclase activity of the membranes of rat adrenal cortex in the concentration range from 1 to 1000 nM. Leucocorticotropin administration three times at doses of 10-20 microg/animal did not change the level of hydroxycorticosteroids (11-HOCS) in the rat adrenal glands in the absence of temperature action. At the same time, the peptide abolishes (at a dose of 20 microg/animal, three times) or significantly decreases (at a dose of 10 microg/animal, three times) the dramatic increase in the 11-HOCS content in the adrenal glands occurring in the case of cold or heat shock. Thus, leucocorticotropin normalizes the 11-HOCS level in the rat adrenal cortex during stress. The stress-protective effect of the peptide is mediated through the ACTH receptor.     

High- and low-affinity binding sites for vasoactive intestinal peptide (VIP) in the rat kidney revealed by light microscopic autoradiography

The distribution of VIP binding sites in rat kidney and adrenal gland has been examined by light microscopic autoradiography. A fully characterized mono-iodinated molecular form of VIP (M-125-I-VIP) which maintains the biological activity of the native peptide, was used for this study. Two types of VIP binding sites, with high and low affinity, have been identified. High affinity sites are associated with (i) glomerular structures in the cortex, (ii) the inner stripe of the outer medulla, possibly corresponding to Henle's loops and distal tubules, (iii) radiated structures in the inner zone of the medulla, likely to represent labeling of collecting ducts and/or vascular bundles and (iv) the adrenal cortex. Autoradiographic grains associated with low affinity sites are present diffusely throughout the renal cortex, possibly corresponding to labeling of tubular and/or vascular structures, and throughout the adrenal gland. These observations further delineate a role of VIP in renal and neuroendocrine function.     

Biphasic regulation by N, 2′-O-dibutyryl adenosine 3′, 5′-cyclic monophosphate (dbcAMP) of steroid 21-hydroxylase activity in rat hepatocytes

Steroid 21-hydroxylase activity has been identified in many tissues, including liver. But it is possible that the enzyme found in the liver is different from adrenal 21-hydroxylase. In the adrenal cortex, steroid 21-hydroxylase activity is increased by corticotropin (ACTH); the effect of ACTH is mediated by cyclic AMP (cAMP), and presumably involves a cAMP-dependent protein kinase (PKA). It is not yet clear, however, how extra-adrenal steroid 21-hydroxylase activity is regulated. In the present study, we examined the effect of N⁶, 2′-O-dibutyryl adenosine 3′,5′-cyclic monophosphate (dbcAMP), forskolin, N-[2-(methylamino)ethyl]5-isoquinolinesulfonamide (H-8) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on steroid 21-hydroxylase activity in primary cultures of rat hepatocytes to determine the nature of regulation of extra-adrenal steroid 21-hydroxylase activity. Steroid 21-hydroxylase activity in hepatocytes incubated with 10⁻¹¹M dbcAMP for 24 h was 1.6 times higher than that in control hepatocytes untreated with dbcAMP. On the other hand, steroid 21-hydroxylase activity decreased by 20 and 50% when the cells were incubated with 10⁻⁵ and 10⁻³ M dbcAMP, respectively. The stimulatory effect of 10⁻¹¹ M dbcAMP was not blocked by 10⁻⁵ M H-8 (PKA inhibitor), but the inhibitory effect of 10⁻⁵ or 10⁻³ M cAMP was. TPA did not alter the activity of steroid 21-hydroxylase. These findings indicate that the steroid 21-hydroxylase in rat liver is regulated by mechanisms different from those in the adrenal glands.