Zone-specific regulation of two messenger RNAs for P450c11 in the adrenals of pregnant and nonpregnant rats.

Adrenal mitochondria possess two steroidogenic cytochrome P450s. P450c11 converts deoxycorticosterone to corticosterone and aldosterone, and P450scc converts cholesterol to pregnenolone. These P450s receive electrons from NADPH via adrenodoxin reductase and adrenodoxin. A single bovine P450c11 protein has 11-hydroxylase, 18-hydroxylase, and 18-oxidase activities, but this series of enzymatic steps may be mediated by more than one enzyme in rats. Enzymatic assays of purified rat mitochondrial proteins have suggested that one enzyme found in all zones of the adrenal cortex has both 11- and 18-hydroxylase activities, whereas another enzyme, found exclusively in the zona glomerulosa, catalyzes 18-hydroxylation and 18-oxidation of corticosterone. We studied the number and zonal distribution of P450c11 mRNA species in the rat adrenal and how these mRNAs are regulated in the adrenals of normal and pregnant rats. Rats synthesize two similar, but distinct, P450c11 mRNAs. One, P450c11A, is found in both the zona glomerulosa and fasciculata/reticularis, whereas the second, P450c11B, is found only in the zona glomerulosa. The abundance of neither P450c11A mRNA nor P450c11B mRNA is affected by a high-salt diet. However, when rats receive a low-salt diet, P450c11A mRNA decreases and P450c11B mRNA increases. Dexamethasone decreases the amount of P450c11A mRNA without affecting P450c11B mRNA. The combination of a high-salt diet and dexamethasone decreases the amount of both mRNAs further to almost undetectable amounts. Rats given a low-salt diet and dexamethasone have a dramatic increase in the abundance of P450c11B mRNA. Thus both forms of P450c11 mRNA are regulated independently in the rat adrenal cortex. In situ hybridization studies show that only the P450c11 found in the zona glomerulosa is regulated by salt treatment in vivo, whereas glucocorticoid treatment in vivo regulates P450c11 in all zones. In the adrenals of pregnant rats, P450c11B is regulated in a similar fashion to its regulation in the nonpregnant rat adrenal, despite major differences in sodium retention and intravascular volume in pregnant and nonpregnant rats. In the pregnant rat, a low-salt diet increases the abundance of P450c11B to a greater degree than in the nonpregnant rat. By contrast, dexamethasone does not diminish the abundance of P450c11A mRNA in the pregnant rat but reduces it to an almost undetectable amount in the nonpregnant rat. Thus, the regulation of glucocorticoid and mineralocorticoid production in the pregnant and nonpregnant rat occurs by different mechanisms.     

Molecular mechanism of cytochrome P-450-dependent aldosterone biosynthesis in the adrenal cortex.

In the adrenal cortex, the potent mineralocorticoid, aldosterone, is produced in the zoba glomerulosa but not in the zona fasciculata/reticularis. In rodents and humans, two distinct species of P-450(C18) (aldosterone synthase) and P-450(11beta) (11beta-hydroxylase) are expressed in the adrenal cortex. The selective expression of cytochrome P-450 species in different zones contributes to zone specificity of aldosterone synthesis. In the cow and pig, only one molecular species of P-450(11beta) having both 11beta-hydroxylase and aldosterone synthase activity is expressed throughout the adrenal cortex. P-450(11beta) in the zona fasciculata/reticularis catalyzes the formation of corticosterone but not that of aldosterone from 11-deoxycorticosterone; the same enzyme in the zona glomerulosa produces aldosterone from the same substrate, indicating that a local factor in mitochondria is likely to be involved in the selective suppression of the aldosterone synthetic activity of P-450(11beta) in the zona fasciculata/reticularis. The zone specificity of aldosterone synthesis catalyzed by P-450(11beta) in the bovine adrenal cortex appears to be due to differences in interactions between P-450(11beta) and P-450(SCC) in mitochondria in different cortical zones. Thus, two modes exist for aldosterone biosynthesis in mammals: rodent-human and bovine-porcine modes.     

Zonal distribution and regulation of adrenal renin in a transgenic model of hypertension in the rat.

The hypertensive transgenic rat [TGR (mRen-2)27] is a genetic model of hypertension in which transfection of the Ren-2 mouse renin gene into rats results in severe hypertension. These transgenic rats express a high level of renin in the adrenal gland, and the hypertension is ameliorated by treatment with angiotensin-converting enzyme inhibitors. In this study we investigated the distribution of adrenal renin in the TGR rat and examined the regulation of adrenal renin in a monolayer culture of adrenal cells. High concentrations of active renin and prorenin were found in the adrenal capsular (glomerulosa) and decapsular (fasciculata-medullary) portions of the TGR adrenal. This is in contrast with the Sprague-Dawley (S-D) rat, in which adrenal renin is found mostly in the active form and located primarily in the glomerulosa cells. The zonal distribution of aldosterone was also different in the TGR, with substantial amounts of aldosterone in the zona fasciculata as well as in the glomerulosa, while in the S-D rat, aldosterone is limited to the zona glomerulosa. In the primary monolayer culture of glomerulosa cells, TGR cells had significantly higher levels of active renin and prorenin and showed an increased response to ACTH and high potassium in the medium. Renin activity in the medium was predominantly in the form of prorenin and significantly higher than that in the S-D rat. Cultured fasciculata cells from TGR also produce renin that is stimulated by ACTH, but not by a high potassium concentration. Renin activity in the adrenal homogenate, medium, and plasma from TGR rats was completely inhibited by the renin inhibitor (CP 71362; 1 microM), but only slightly inhibited (12.3 +/- 3%) by a monoclonal antibody that inhibits renin activity from S-D rat tissues by 79.2 +/- 2.5%, suggesting that renin in the plasma and adrenal glands from TGR appears to derive primarily from mouse renin. In conclusion, the TGR (mRen-2)27 rats have higher than normal levels of adrenal renin, and the cultured cells show an exaggerated renin response to ACTH and potassium. The distribution of the renin enzyme in the adrenal zones of the TGR is similar to the distribution of mouse adrenal renin.     

The 21-hydroxylase activity in the glomerulosa and fasciculata of the adrenal cortex in congenital adrenal hyperplasia

In the two clinical syndromes of congenital adrenal hyperplasia due to a 21-hydroxylation defect of adrenal steroidogenesis, the simple virilizing and the salt-wasting forms, the 21-hydroxylase activity was studied considering the zona fasciculata and the zona glomerulosa of the adrenal cortex as two separate glands under different regulation. To test this hypothesis, we stimulated adrenal steroidogenesis by ACTH infusion or dietary sodium restriction in eight patients with congenital adrenal hyperplasia (four patients with the simple virilizing form and four with the salt-wasting form of congenital adrenal hyperplasia) and in six normal children. Both the 17-hydroxy and 17-deoxy pathways of adrenocortical steroid biosynthesis were examined by measuring serum concentrations of 17-hydroxyprogesterone, cortisol, progesterone, deoxycorticosterone, corticosterone, and aldosterone and the excretion of free deoxycorticosterone, 18-hydroxydeoxycorticosterone, corticosterone, 18-hydroxycorticosterone, cortisol, and aldosterone. We considered the steroids 18-hydroxycorticosterone and aldosterone to be primarily of zona glomerulosa origin. These studies indicated that the zona fasciculata of both the salt-wasting and the simple virilizing forms is defective in 21-hydroxylation of 17-hydroxy and 17-deoxy steroids. The zona glomerulosa demonstrated deficient 21-hydroxylation only in the salt-wasting form, whereas in the simple virilizing form, the glomerulosa was spared this defect.     

Developmental changes in steroidogenic enzymes in human postnatal adrenal cortex: immunohistochemical studies

Adrenarche is considered to occur as a result of intra-adrenal changes in steroidogenic enzymes involved in C19 steroid production. The present study was conducted because developmental changes in steroidogenic enzymes have not been examined well in human postnatal adrenal. Twenty-four specimens of nonpathological human adrenals from 7 months to 62 years retrieved from autopsy files. Immunohistochemistry for P450 side-chain cleavage (P450scc), 17alpha hydroxylase (P450c17), dehydroepiandrosterone sulfotransferase (DHEA-ST), P450 oxidoreductase, cytochrome b5, and 3beta-hydroxysteroid dehydrogenase (3betaHSD) was per-formed in these specimens, and the immuno-intensity was evaluated using CAS 200 computed image analysis system. Immunoreactivity of P450scc was marked in the zona glomerulosa, fasciculata and reticularis in the adrenal glands of all the cases examined. P450c17 and DHEA-ST immunoreactivity was weak in the zona fasciculata and reticularis in the adrenals of age 7 months to 5 years, but thereafter became prominent in the zona reticularis. Immunoreactivity of P450 oxidoreductase and cytochrome b5, components of the electron transfer system hypothesized to regulate the 17-20 lyase activity of P450c17, was weak in all three zones of adrenal cortex from 7 months to 5 years, and became more marked in the zona reticularis after age 5 years. 3betaHSD immunoreactivity was marked in all three zones of the adrenal cortex from 7 months to 8 years but thereafter decreased in the zona reticularis. These data suggest that the human adrenal zona reticularis markedly begins to develop morphologically and functionally at around 5 years of age. The increased level of P450c17, DHEA-ST, P450 oxidoreductase, and cytochrome b5, and the decreased level of 3betaHSD in the reticularis is likely to contribute to increased C19 steroid production during adrenarche.     

Developmental expression of genes for the stereoidogenic enzymes P450scc (20,22-desmolase), P450c17 (17 alpha-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase) in the human fetus

Fetal adrenal steroidogenesis is required for the production of placental estrogen, and fetal testicular steroidogenesis is required for the development of male external genitalia. We studied the ontogeny and tissue specificity of expression of the genes for three steroidogenic enzymes: P450scc (the cholesterol side-chain cleavage enzyme), P450c17 (17 alpha-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase) in the human fetus. RNA from fetal tissues was probed with homologous human P450scc, P450c17, and P450c21 cDNAs cloned in our laboratory. At 20-21 weeks gestation, P450scc mRNA was most abundant in the adrenal, followed by testis, placenta, and ovary. P450c17 mRNA was also most abundant in the adrenal, followed by testis and ovary, but was undetectable in the placenta. P450c21 mRNA was detected only in the adrenal. None of these mRNAs was detected in kidney, liver, spleen, intestine, or muscle. Twenty-two fetal testis samples (13-25.8 weeks gestation) were studied. P450scc and P450c17 mRNAs were most abundant at 14-16 weeks and diminished to 35 and 19% of their peak values, respectively, by 20-25.8 weeks. Ovarian P450scc and P450c17 mRNAs were present, respectively, in only 6.2% and 1.8% of the maximum amount in the testis and did not vary detectably from 14.9 to 21.5 weeks gestation. The testicular and ovarian steroidogenic enzyme mRNA data correlate well with previously reported changes in gonadal steroidogenesis with gestational age. The presence of P450scc mRNA, but not P450c17 mRNA, in the placenta indicates that the placenta is able to initiate the synthesis of some steroid hormones, but is not able to synthesize estrogen de novo. Since P450c21 was found only in the adrenal, the extraadrenal 21-hydroxylation of progesterone to deoxycorticosterone, a common event in the fetus, is probably mediated by an enzyme(s) other than P450c21.     

The effect of hypoxia from birth on the regulation of aldosterone in the 7-day-old rat: plasma hormones, steroidogenesis in vitro, and steroidogenic enzyme messenger ribonucleic acid.

Adaptation to hypoxia in the neonate requires an appropriate adrenocortical response. The purpose of this study was to examine the adaptation of the aldosterone pathway in rat pups exposed to hypoxia in vivo from birth to 7 days of age. Neonatal rats (with their lactating dams) were exposed to normoxia (21% O2) or hypoxia (12% O2) continuously for 7 days from birth. Trunk blood was collected, and entire adrenal glands were processed from 7-day-old rats to study the activity of the steroidogenic pathway in dispersed cells and isolated mitochondria, for measurement of expression of the steroidogenic enzyme messenger RNAs (mRNAs) by RT-competitive PCR and in situ hybridization histochemistry, for measurement of zona glomerulosa width by immunohistofluorescent staining for P450c11AS protein, and for measurement of mitochondrial number and distribution by transmission electron microscopy. Exposure to hypoxia for 7 days from birth resulted in a marked increase in plasma ACTH, corticosterone, and aldosterone with no change in PRA. Aldosteronogenesis and P450c11AS activity were both augmented in dispersed cells; this effect was lost in isolated mitochondria (from entire adrenal glands) using a permeable substrate for P450c11AS. There was no significant effect of hypoxia on expression of the steroidogenic enzyme mRNAs measured by RT-competitive PCR or in situ hybridization histochemistry. Finally, hypoxia had no effect on mitochondrial number or stereology as assessed by transmission electron microscopy or on zona glomerulosa width as assessed by staining for P450c11AS protein. We conclude that, as opposed to that in adults, hypoxia in the neonate results in an augmentation of aldosteronogenesis. This effect is not accounted for by a change in steroidogenic enzyme mRNA expression, zona glomerulosa width (i.e. hyperplasia), or mitochondrial number or distribution. This functional augmentation of aldosteronogenesis may be due to a change in mitochondrial permeability to steroid substrates and/or the effect of cytosolic factors that control mitochondrial steroidogenesis.     

Zonal expression of endothelial nitric oxide synthase in sheep and rhesus adrenal cortex.

There is mounting evidence that nitric oxide (NO) may inhibit adrenal steroidogenesis by binding to the heme group of P450 enzymes, particularly the rate-limiting steps cholesterol side-change cleavage P450, aldosterone synthase P450, and 17 alpha-hydroxylase/C(17/20)-lyase P450. Using immunohistochemistry, nitrotyrosine was detectable throughout the ovine adrenal cortex, and endothelial NO synthase (eNOS) was further identified in zona glomerulosa (ZG) and at a higher level throughout the zona fasciculata, increasing toward the medulla. Caveolin-1, 90-kDa heat shock protein, ERK-1/2, and Akt, all known and proposed regulators of eNOS activity, were detected throughout the ovine adrenal cortex. Western immunoblotting confirmed the identity of these proteins as well as the absence of neuronal NOS, inducible NOS, caveolin-2, and caveolin-3. Through dual immunostaining we further identified for the first time a zona intermedia without strong staining for 17 alpha-hydroxylase/C(17/20)-lyase P450 or angiotensin II type 1 receptor, but positive for eNOS. Rhesus adrenals also stained positively for eNOS, but staining was seen only in the ZG and zona reticularis. We conclude that eNOS may play a role in controlling zone-specific aldosterone synthase vs. 11 beta-hydroxylase activities of the single CYP11B gene in sheep. In the rhesus monkey, NO may modulate ZG aldosterone synthase, but it is not needed for control of the distinct 11 beta-hydroxylase in the zona fasciculata. In the zona reticularis, however, eNOS may control C(19) steroid production at the level of 17 alpha-hydroxylase vs. 17,20-lyase activity otherwise unopposed by 3beta-hydroxysteroid dehydrogenase.     

Immunohistochemistry of cytochrome P-450 21-hydroxylase: microscopic examination of the enzyme in the bovine adrenal cortex and kidney

Cytochrome P-450 specific for steroid 21-hydroxylase (P-450C21) localized in bovine adrenal cortex and kidney was immunocytochemically observed by the peroxidase-antiperoxidase method using a specific antibody. P-450C21 was present in all three zones of the adrenal cortex. Immunoreactivity for P-450C21 was intense in the zona glomerulosa and inner reticularis and faint in the area between the zona glomerulosa and outer fasciculata, probably representing the zona intermedia. The positive stain was only observed in parenchymal cells. The immunoreactivity varied within each zone, especially in the zona reticularis. In the kidney, immunoreactivity for P-450C21 was exclusively localized in the distal and cortical and medullary collecting tubules. This corresponds to the site of mineralocorticoid action in the kidney. No immunoreactivity was observed in the liver and aorta.     

Lipoxygenase products as common intermediates in cyclic AMP-dependent and -independent adrenal steroidogenesis in rats

Aldosterone secretion from adrenal glomerulosa cells can be stimulated by angiotensin II (AII), extracellular potassium and ACTH. Mitochondria from these cells respond to intracellular factors generated by AII (cyclic AMP (cAMP)-independent steroidogenesis) and ACTH (cAMP-dependent steroidogenesis), suggesting that the two-signal-transduction mechanisms are linked by a common intermediate. We have evaluated this hypothesis by stimulating mitochondria from the unstimulated zona glomerulosa with a subcellular post-mitochondrial fraction (PMF) obtained from the zona glomerulosa after stimulation with AII or from the fasciculata gland after stimulation with ACTH; the subcellular fractions were also tested on mitochondria from fasciculata cells. PMFs obtained after incubation of adrenal zona glomerulosa with or without AII (0.1 microM) or ACTH (0.1 nM) were able to increase net progesterone synthesis 4.5-fold in mitochondria isolated from unstimulated rat zona glomerulosa. AII-pretreated PMFs from the zona glomerulosa also stimulated steroidogenesis by mitochondria from zona fasciculata cells. Separate experiments showed that inhibitors of arachidonic acid release and metabolism (bromophenacyl bromide, nordihydroguaiaretic acid, caffeic acid or esculetin) blocked corticosterone production in fasciculata cells stimulated with ACTH, suggesting that arachidonic acid could be the common intermediate in the actions of AII and ACTH on steroid synthesis. Evidence to support this concept was obtained from experiments in which the formation of an activated PMF by treatment of zona fasciculata with ACTH was blocked by the presence of the same inhibitors. Moreover, the inhibitory effects of these substances on PMF activation by ACTH were overcome by exogenous arachidonic acid and, in addition, arachidonic acid release was stimulated by ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen-dependence of ACTH-stimulated aldosterone and corticosterone synthesis in the rat adrenal cortex: developmental aspects

The control of ACTH-stimulated steroidogenesis under decreasing levels of O(2) is not fully understood. The purpose of this study was to examine the effects of decreased O(2) in vitro on rat adrenocortical steroid synthesis at different stages of development. Of interest was the evaluation of the effect of low O(2) on steroidogenesis during the stress hyporesponsive period of the neonate. Rats were killed at 7, 14, or 42 days of age, adrenals collected and capsules (zona glomerulosa, ZG) separated from subcapsules (zona fasciculata/reticularis, ZFR). Cells were dispersed and placed into glass vials each gassed with a different level of O(2) (21, 5, 2, 1, or 0% O(2)). The entire steroidogenic pathway was analyzed by measuring ACTH-stimulated cAMP, corticosterone and aldosterone production during a 2 h incubation. In addition, the early (P450 scc) and late (P450c11 beta and P450 aldo) pathway activities were examined in the presence of cyanoketone. The PO(2) for half-maximal activity (P(50)) for aldosterone synthesis in ZG cells from 7- and 42-day-old rats was approximately 28 mmHg and 7 mmHg respectively, indicating that cells from older rats were more resistant to inhibition by low O(2). The P(50) for cAMP production from the ZG was approximately 14 mmHg for both age groups. The P(50) for corticosterone synthesis was approximately 28 mmHg and <7 mmHg in ZFR cells from 7- and 42-day-old cells respectively. The only enzyme activities affected by low O(2) (<35 mmHg) were P450 aldo and P450 scc. Moderate decreases in O(2) (from approximately 150 mmHg) decreased aldosteronogenesis, possibly due to observed decreases in cAMP generation, but not due to decreases in steroidogenic enzyme activity (7-day-old). Severe decreases in O(2) presumably inhibited P450 aldo through a direct effect on enzyme activity (both ages). P450 scc activity (including cholesterol transport) also seems to be decreased by very low O(2) (7-day-old). These findings illustrate a novel developmental alteration in O(2)-regulated steroid production, and may have implications for neonatal health and disease.     

Hormonal regulation of messenger ribonucleic acids for P450scc (cholesterol side-chain cleavage enzyme) and P450c17 (17 alpha-hydroxylase/17,20-lyase) in cultured human fetal adrenal cells

ACTH has acute and long term effects on adrenal steroidogenesis by week 14 of fetal life. We used human fetal adrenal cells to investigate the long term effect of physiological doses of ACTH on mRNAs for P450scc (the cholesterol side-chain cleavage enzyme) and P450c17 (17 alpha-hydroxylase/17,20-lyase). Monolayer cultures of 18- to 24-week gestation fetal zone adrenal cells were maintained in the presence and absence of 10(-9) or 10(-8) M ACTH for up to 12 days. As assessed by RNA dot blots probed with cloned homologous human cDNAs, ACTH increased P450scc and P450c17 mRNAs 4- and 9-fold, respectively, over control values on day 7 of culture. ACTH-mediated stimulation was slightly less on day 12 of culture. The ACTH-mediated accumulation of those mRNAs were time dependent. When cells were exposed to a single 10(-8)-M dose of ACTH, the amount of P450scc and P450c17 mRNA was increased by 24 h, reaching a maximum at 48 h and diminishing by 72 h. When cells were maintained in 10(-8) M ACTH continuously, mRNA for both enzymes accumulated in a similar pattern, reaching a peak at 48 h but remaining at nearly maximal values thereafter, up to 96 h. Dibutyryl cAMP (10(-3) M) mimicked these stimulatory actions of ACTH, although its effect was greater at 24 h and more stable up to 96 h. Angiotensin II (1-100 ng/mL) and hCG (1-100 ng/mL) had no effect on accumulation of P450scc and P450c17 mRNAs. The production of both dehydroepiandrosterone sulfate and cortisol also was stimulated by ACTH, suggesting that the increased mRNAs were translated into active enzymes. These results indicate that ACTH induces human fetal adrenal cells to accumulate mRNAs for both P450scc and P450c17; this effect of ACTH is probably mediated by cAMP. Chronic 96-h stimulation of human fetal adrenal cells did not diminish their responsiveness to ACTH. Together with our earlier studies of the human fetal adrenal, these data indicate that fetal adrenal tissue does not exhibit the desensitization to trophic hormone stimulation characteristic of adult tissue.     

Role of prostaglandins in adrenal steroidogenesis

Using an in vitro rat glomerulosa and fasciculata cell preparation, we studied the role of porstaglandins (PGs) as potential mediators of angiotensin II-, ACTH-, and potassium-induced adrenal steroidogenesis. The glomerulosa cells primarily produced prostacyclin under basal and stimulated conditions, whereas the fasciculata cells primarily produce PGF2 alpha. Using 6-keto-PGF1 alpha (the stable hydrolysis product of prostacyclin) and PGF2 alpha as indices of PG production in glomerulosa and fasciculata cells, respectively, we noted no significant changes in PGs during angiotensin II-, ACTH-, or potassium-induced steroidogenesis. Pretreatment with indomethacin significantly (P less than 0.01) inhibited PG production, but did not alter adrenal steroidogenesis. Furthermore, the administration of exogenous PGs did not affect steroidogenesis. We conclude that PGs do not mediate steroidogenesis from rat glomerulosa or fasciculata cells.     

Expression of cytochrome P450 cholesterol side chain cleavage and 3beta-hydroxysteroid dehydrogenase during embryogenesis in chicken adrenal glands and gonads

Expression of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNAs was examined in chicken embryonic adrenal glands and gonads between days 4 and 12 of incubation. In situ hybridization analysis showed that 3beta-HSD mRNA appeared on day 5 of incubation in the adrenal glands and on day 6 in the gonads, while P450scc mRNA was expressed on day 7 in both the adrenal glands and the gonads. Cells expressing both enzyme mRNAs were distributed in the steroidogenic tissues of the adrenal glands and in the medullary cords of the gonads. From days 9 to 11 of incubation, P450scc mRNA expression was not found in the majority of both the adrenal glands and the gonads, but was detected again in both on day 12, although 3beta-HSD mRNA was constitutively expressed during this period. Changes in the expression pattern of P450scc mRNA are paralleled by changes in the plasma corticosterone level reported previously. Therefore, it is suggested that P450scc is essential to embryogenesis.     

The role of the adrenal gland in hypertensive transgenic rat TGR(mREN2)27.

The TGR(mREN2)27 is a new monogenetic rat model in hypertension research. As the mouse Ren-2d renin gene is integrated into their genome, they develop fulminant hypertension between 5 and 15 weeks of age, with blood pressure maxima of 300 mm Hg. Their plasma renin-angiotensin system (RAS) is suppressed, but the transgene is highly expressed in the adrenal gland, so we investigated its possible role in steroid metabolism and the pathogenesis of hypertension. During the phase of hypertension development (between 6-18 weeks), the urinary excretion of deoxycorticosterone (DOC), corticosterone (B), 18-hydroxycorticosterone, and aldosterone is 1.5- to 2.5-fold elevated compared with that in Sprague-Dawley (SD) rats (P less than 0.0005) despite the suppressed plasma RAS. Moreover, the adrenal gland in TGR(mREN2)27 shows an increased maximal response to ACTH stimulation in regard to urinary excretion of DOC (after ACTH, 244 +/- 42 ng/24 h in TGR; 62 +/- 10 ng/24 h in SD; P less than 0.0005) and B (after ACTH, 5144 +/- 346 ng/24 h in TGR; 2607 +/- 324 ng/24 h in SD; P less than 0.0005). Additionally, plasma prorenin in TGR was stimulated more than 10-fold, indicating transgene regulation by ACTH. Since spironolactone treatment did not lower the blood pressure in TGR, hypertension solely due to hypermineralocorticoism is unlikely. Our results indicate that the adrenal steroid metabolism is markedly stimulated in young TGR, and the absolute increase in urinary DOC and B after ACTH injections is enhanced, possibly due to a stimulated local intraadrenal RAS.