Regulation of the adrenal renin angiotensin system in cultured bovine zona glomerulosa cells: effect of catecholamines.

The renin-angiotensin system consists of two main enzymes, renin and angiotensin-converting enzyme, which lead to the formation of angiotensin-II. Angiotensin-II is a potent vasoconstrictor and stimulates the production of aldosterone. In this study we examined the effect of ACTH, potassium, (Bu)2cAMP (dbcAMP), and catecholamines on the adrenal renin-angiotensin system. To study the production of renin and aldosterone in vitro, we developed a monolayer culture of bovine zona glomerulosa cells in serum-free medium. Collagenase-dispersed zona glomerulosa cells were incubated in Pasadena Foundation for Medical Research-4 medium containing 10% fetal calf serum for 72 h, and the medium was replaced with serum-free medium for the next 24 h of the experimental period. The cells during this 24 h were exposed to various doses of ACTH, potassium, dbcAMP, and sympathomimetic agents. ACTH and dbcAMP stimulated aldosterone secretion, and this secretion was associated with an increase in renin activity in cells and medium. Aldosterone was also stimulated by high doses of potassium, and potassium had a stimulatory effect on the secretion of renin in medium. Catecholamines had a weak stimulating effect on aldosterone secretion and were potent stimulators of adrenal renin activity in cells and medium. Dopamine had no significant effect on basal aldosterone secretion or renin activity in cells and medium. In conclusion, these data indicate that adrenal renin is synthesized in bovine zona glomerulosa cells in vitro, and that ACTH and dbcAMP stimulate adrenal renin and aldosterone production. Furthermore, adrenal renin, like renal renin, may be under the control of the sympathetic nervous system.     

Role of the adrenal renin-angiotensin system on adrenocorticotropic hormone- and potassium-stimulated aldosterone production by rat adrenal glomerulosa cells in monolayer culture

The rat zona glomerulosa has a renin-angiotensin system that appears to function as an autocrine or paracrine system in the regulation of aldosterone production. To further investigate dynamic changes of production of renin and aldosterone in vitro we developed a primary monolayer culture of rat adrenal glomerulosa cells in serum-free medium. Collagenase-dispersed glomerulosa cells were incubated in PFMR-4 medium containing 10% fetal calf serum for 48 hours; the medium was then replaced with serum-free PFMR-4 medium. The cell viability and the aldosterone secretion were stable over the additional 48 hours in the serum-free control medium. After incubation for 24 hours in the serum-free medium, the cells were exposed to high K+ or adrenocorticotropic hormone (ACTH) for another 24 hours. ACTH stimulated aldosterone secretion, and this increased secretion was associated with an increase in renin activity (cell active renin, from 15.56 +/- 0.71 to 45.75 +/- 5.69; cell inactive renin, from 0.67 +/- 0.54 to 8.75 +/- 3.40; medium inactive renin, from 5.58 +/- 1.16 to 106.20 +/- 14.01 pg angiotensin I (Ang I)/micrograms protein/3 hr). Aldosterone was also stimulated by high K+. This increase was also associated with an increase in active renin in the cells (from 15.08 +/- 1.80 to 23.26 +/- 2.15 pg Ang I/micrograms protein/3 hr) and an increase in inactive renin in the medium (from 10.87 +/- 1.62 to 21.37 +/- 3.20 pg Ang I/micrograms protein/3 hr). Addition of the angiotensin converting enzyme inhibitor lisinopril attenuated both ACTH- and high K(+)-stimulated aldosterone secretion significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of nitric oxide on aldosterone synthesis and nitric oxide synthase activity in glomerulosa cells from bovine adrenal gland

This study investigated the effects of two NO-releasing agents, diethylenetriamine-NO (deta-NO) and sodium nitroprusside (SNP), on basal, ACTH-, and angiotensin II (AngII)-stimulated aldosterone production in glomerulosa cells from bovine adrenal gland. NO donors inhibited basal and ACTH- or AngII-stimulated aldosterone synthesis in a concentration-dependent manner. Deta-NO and SNP also provoked a concentration-dependent stimulation of cGMP production. However, cGMP was not responsible for the inhibition of aldosterone secretion, because a cGMP analog did not reproduce the inhibitory effect. Moreover, soluble guanylyl cyclase or protein kinase G inhibitors did not revert the inhibitory effect of NO on aldosterone production. NO donors did not modify ACTH-stimulated cAMP production or AngII-stimulated PLC activity stimulation, but inhibited 22[R] hydroxycholesterol- or pregnenolone-stimulated aldosteronogenesis. NO can be synthesized in bovine glomerulosa cells because nitrite production was determined and characterization of NOS activity was also performed. Nitrite accumulation was not modified in the presence of ACTH, AngII, or other factors used to induce iNOS. NOS activity that showed a Michaelis-Menten kinetic was NADPH- and calcium-dependent and was inhibited by two competitive inhibitors, L-NAME and L-NMMA. These results show that NO inhibits aldosterone production in glomerulosa cells acting on P450scc and other P450-dependent steroidogenic enzymes, and these cells display NOS activity suggesting that NO can be produced by constitutive NOS isozymes.     

Tumor necrosis factor and interleukin-1 are potent inhibitors of angiotensin-II-induced aldosterone synthesis

Cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), mediate many inflammatory and cellular responses. However, the effects of TNF and IL-1 on basal and angiotensin-II (AII)-stimulated aldosterone synthesis are not known. We studied the effect of recombinant and purified TNF and IL-1 on basal as well as AII-, ACTH-, and K+-induced aldosterone synthesis in isolated rat adrenal glomerulosa cells. Since we have previously shown that AII action is mediated by activation of the 12-lipoxygenase (12LO) pathway of arachidonic acid, we also evaluated the effects of these cytokines on the 12LO product 12-hydroxyeicosatetraenoic acid (12HETE) using a validated RIA technique. TNF at 2.5 and 5.0 ng/ml produced a dose-dependent inhibition of AII-induced aldosterone synthesis [AII, 39.0 +/- 3.3 ng/10(6) cells.h; AII plus TNF (5.0 ng/ml), 14.3 +/- 1.6; P less than 0.001 vs. AII; AII plus TNF (2.5 ng/ml), 24.7 +/- 3.2; P less than 0.01 vs. AII]. Similarly, TNF at 5.0 ng/ml also attenuated the stimulatory effect of ACTH (10(-9) M). However, K+-induced aldosterone synthesis was not altered. TNF also did not alter basal aldosterone levels. AII, as previously shown, stimulates 12HETE synthesis (basal, 608 +/- 114 pg/10(5) cells.h; versus AII, 1268 +/- 197; P less than 0.02). TNF at concentrations of 1.0-5.0 ng/ml produced a dose-dependent inhibition of AII stimulatory action on 12HETE synthesis [AII plus TNF (1.0 ng/ml), 650 +/- 26 pg, P less than 0.03 vs. AII; AII plus TNF (5.0 ng/ml), 390 +/- 46; P less than 0.01 vs. AII plus TNF (1.0 ng/ml)]. In addition, 12HETE at 10(-8) M completely restored the effects of AII during blockage by TNF. Purified human IL-1 (75% beta, 25% alpha) as well as recombinant human IL-1 beta at concentrations as low as 50 pg/ml inhibited AII-induced aldosterone synthesis. IL-1 beta did not alter ACTH- or K+-induced aldosterone synthesis and, in fact, had a tendency to potentiate ACTH effects. These results suggest that the cytokines TNF and IL-1 are potent inhibitors, particularly of AII action in the adrenal glomerulosa cell. Therefore, local or systemically produced TNF or IL-1 may be important negative modulators of aldosterone synthesis.     

Adrenal renin: A possible local hormonal regulator of aldosterone production

Summary The complete renin-angiotensin system is present in the adrenal cortex; prorenin, renin, angiotensinogen, angiotensin I and II, and converting enzyme. Most of the renin found is probably synthesized there since the renin concentration increases after nephrectomy, and the mRNA for renin is present. The renin-angiotensin system has the highest activity in the zona glomerulosa cells, the site of aldosterone formation. A low-sodium dist or a high-potassium diet, or nephrectomy markedly inereases the adrenal renin concentration in the zona glomerulosa cells without any effect on the fasciculata-medullary cells. There is a close correlation between adrenal renin and aldosterone production. The adrenal renin angiotensin system may be a local regulator of aldosterone production.     

Role of angiotensin II receptor subtypes on the regulation of aldosterone secretion in the adrenal glomerulosa zone in the rat.

The role of AII receptors subtypes, AT1 and AT2, in the regulation of aldosterone secretion was studied in adrenal glomerulosa cells and membranes from rats on normal and low sodium intake, using AII receptor subtype-specific antagonists. In adrenal glomerulosa cells, more than 90% of the receptors were AT1 and there was a good correlation between the potencies of the antagonists to inhibit ligand binding, and AII-stimulated aldosterone production and inositol phosphate formation. The inhibition of basal and ACTH-stimulated cAMP by AII was also abolished by the AT1, but not the AT2, antagonist. Sodium restriction for 6 days increased both receptor subtypes in the same proportion, but only the AT1 antagonist inhibited AII-stimulated aldosterone production. The data demonstrate that AT1 receptor mediates the regulatory actions of AII in the adrenal zona glomerulosa.     

Effects of dibutyryl adenosine 3',5'-monophosphate, angiotensin II, and atrial natriuretic factor on phosphorylation of a 17,600-dalton protein in adrenal glomerulosa cells

Rat adrenal glomerulosa cells were incubated with [32P]phosphate and (Bu)2AMP (dbcAMP), angiotensin II, and atrial natriuretic factor (ANF). Incorporation of [32P]phosphate into cellular proteins was analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. dbAMP stimulated phosphorylation of a 17.6K protein, while angiotensin II did not stimulate it. ANF did not affect the protein phosphorylation, whether the cells were in the basal state or stimulated by dbcAMP or angiotensin II. On the other hand, ANF markedly inhibited angiotensin II-stimulated aldosterone production, but only slightly inhibited dbcAMP-stimulated aldosterone. These results suggest that in rat adrenal glomerulosa cells phosphorylation of the 17.6K protein may have a relationship with the stimulatory effect of cAMP on aldosterone production; however, neither angiotensin II nor ANF affected the phosphorylation of this protein, and phosphorylation of the 17.6K protein is not an obligatory step in the regulation of aldosterone production.     

Calcitonin gene-related peptide is an inhibitor of aldosterone secretion

This study examined the effects of calcitonin gene-related peptide (CGRP) on aldosterone secretion both in vivo and in vitro. Intravenous administration of CGRP (0.01 micrograms/kg) in 6 conscious dogs produced a significant decrease in plasma aldosterone concentration from 68 +/- 12 pg/ml to 28 +/- 11 pg/ml (p less than 0.05) despite a mild but significant elevation of plasma renin activity. In an in vitro study using isolated rabbit adrenal glomerulosa cells CGRP reduced the basal aldosterone secretion in a dose-related manner and furthermore 10(-9) M CGRP inhibited the aldosterone secretion stimulated by 10(-8) M angiotensin II. From these results it is suggested that CGRP has an inhibitory effect on aldosterone secretion.     

Ovine fetal adrenal synthesis of cortisol: regulation by adrenocorticotropin, angiotensin II and transforming growth factor-beta.

An increase in cortisol production by the fetal adrenal cortex is an important prepartum event. The increase in ovine fetal adrenal synthesis of cortisol appears to rely in part on the ACTH induction of 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha) which occurs before parturition. In the present study we examined the effect of ACTH treatment on cortisol production and P-450(17) alpha expression using primary cultures of ovine fetal adrenal cells. In addition, we examined the effects of angiotensin II (A-II) and transforming growth factor-beta (TGF beta) on ACTH-treated cells. We have demonstrated previously that these factors modulate P-450(17 alpha) levels in adult ovine and bovine adrenal cells. Fetal ovine adrenal cells were isolated from 126- to 130-day fetuses (term = 144 +/- 3 days) and placed in monolayer cell culture. After 1 day in culture the cells were treated with ACTH (10 nM) with or without A-II (0.1-100 nM) or TGF beta (1-100 pM). Medium content of cortisol was low under basal conditions, whereas ACTH-stimulated cortisol production by 10- to 100-fold. A-II and TGF beta inhibited ACTH-induced cortisol production by 70-90%. In addition, 3 days of treatment with ACTH caused a greater than 10-fold induction of P-45017 alpha enzyme activity in fetal adrenal cells. A-II and TGF beta inhibited the ability of ACTH to induce P-45017 alpha activity by at least 75%. Using an antibody to P-45017 and immunoblotting techniques the effects of ACTH, A-II, and TGF beta on enzyme activity were observed to correspond to cellular levels of P-45017 alpha protein. The inhibitory effects of TGF beta and A-II could not be overcome by the cAMP analog (Bu)2cAMP. Interestingly, the expression of the enzyme 3 beta-hydroxysteroid dehydrogenase was much less sensitive to inhibition by A-II or TGF beta. The ability of A-II and TGF beta to suppress P-45017 alpha expression could play a role in determining the pathway of steroidogenesis and specifically the amount of cortisol produced by fetal adrenocortical cells in vivo.     

Dopaminergic modulation of aldosterone secretion in the rat

The dopamine antagonist metoclopramide (MCP) has been shown to acutely stimulate aldosterone secretion in vivo. To determine whether a dopaminergic mechanism is involved in the regulation of aldosterone secretion, we examined the effect of minipump infusion of MCP (iv) and/or angiotensin II (AII;sc) upon plasma aldosterone, adrenal capsular AII receptors, and 18-hydroxylase activity in rats maintained on high sodium intake. During normal sodium intake, plasma aldosterone was elevated from 8.3 +/- 1.3 to 35.4 +/- 3.2 ng/dl after 2-day infusion of a nonnatriuretic dose of AII (25 ng/min) and to 15.0 +/- 1.8 ng/dl after the infusion of 1.2 micrograms/min MCP. AII receptors were unchanged by MCP infusion, and rose from 1014 +/- 98 to 1638 +/- 98 fmol/mg after AII infusion. During high sodium intake, the infusion of either AII or MCP alone produced no change in plasma aldosterone or AII receptors. However, after simultaneous infusion of AII and MCP, plasma aldosterone rose from 4.5 +/- 1.2 to 32.5 +/- 2.7 ng/dl, AII receptors increased from 969 +/- 35 to 1607 +/- 280 fmol/mg, and 18-hydroxylase activity, measured as the conversion of corticosterone to aldosterone by isolated mitochondria, rose from 29.5 +/- 1.67 to 40.6 +/- 2.9 pmol/mg . min. These adrenal responses induced by the combined treatment with AII and MCP were similar to the effects of AII infusion during normal sodium intake, indicating that MCP exerts a permissive action upon the trophic effects of AII on the adrenal cell during high sodium intake. These actions of MCP were completely abolished by the simultaneous infusion of dopamine (2 micrograms/min), suggesting that the effects of MCP on adrenal function are due to its dopaminergic antagonist properties. In collagenase-dispersed adrenal glomerulosa cells, only supraphysiological concentrations of dopamine in the incubation medium (10-100 microns) inhibited basal, AII-stimulated, and ACTH-stimulated aldosterone production, and these inhibitory effects were not reversed by high concentrations of MCP. Also, MCP itself inhibited both basal and stimulated aldosterone production. These results suggest that the stimulatory actions of MCP in vivo are exerted through liberation of other local regulators, rather than directly upon the adrenal glomerulosa cell. These findings have defined a mechanism by which the primary regulatory action of AII upon aldosterone secretion can be modulated during high sodium intake by dopaminergic inhibition of adrenal glomerulosa function.     

Serotonin increases interleukin-6 release and decreases tumor necrosis factor release from rat adrenal zona glomerulosa cells in vitro

Interleukin-6 (IL-6) and tumor necrosis factor (TNF) are secreted by rat adrenal zona glomerulosa cells. Serotonin increases the release of aldosterone, corti-costerone, and cortisol from the adrenal cortex. Therefore, the effects of serotonin on IL-6 and TNF release from rat adrenal zona glomerulosa cells were investigated. Cultures of rat adrenal zona glomerulosa cells were enzymatically prepared and cultured for 4–6 d. The cells were then exposed to serum-free RPMl-1640 medium containing vehicle (RPMl medium alone), serotonin, and/or endotoxin, interleukin-1β, or adrenocorticotrophic hormone (ACTH). Following a 5-h incubation, medium was removed from the cells, and IL-6 and TNF content of this medium determined with bioassays. Serotonin (1–1000 nM) increased basal IL-6 release from zona glomerulosa cells, but inhibited basal TNF release from these cells. Endotoxin and interleukin-1β (IL-1β) increased IL-6 and TNF release from zona glomerulosa cells. Serotonin potentiated IL-6 release stimulated by endotoxin and IL-1β, but inhibited TNF release stimulated by these agents. Serotonin potentiated ACTH-stimulated IL-6 release. Serotonin had no effect on IL-6 release from rat anterior pituitary cells. Because IL-6, TNF, and serotonin modify the release of aldosterone and glucocorticoids from adrenal cells, the stimulatory effects of serotonin on aldosterone and glucocorticoid release may be mediated in part by the effects of serotonin on IL-6 and TNF release from adrenal cells.     

Effect of natrium ionophore on aldosterone production in the rat adrenal gland

The effect of the natrium ionophore, monensin, on aldosterone production was studied using rat adrenal collagenase-dispersed capsular cells. Monensin inhibited aldosterone production in a dose-dependent fashion (10(-9)-10(-6)M). Although monensin inhibited both ACTH- and angiotensin II (AT-II)-stimulated aldosterone production, its inhibitory effect on ACTH-stimulated aldosterone production was greater than that on AT-II-stimulated aldosterone production. The inhibitory effect of monensin on aldosterone production was not accompanied by significant changes in cAMP production. Furthermore, (Bu)2cAMP-stimulated aldosterone production was inhibited by monensin. These results suggest that the inhibitory effect of monensin on aldosterone production is due to an increase in intracellular sodium ion concentration, and that monensin acts at step(s) distal to the generation of cAMP. The differences in the inhibitory effects of monensin on the steroidogenic action of AT-II and that of ACTH may be related to the presence of different steroidogenic mechanisms, including calcium ion dependency.     

Control of aldosterone production by angiotensin II is mediated by two guanine nucleotide regulatory proteins

The involvement of guanine nucleotide regulatory proteins in the steroidogenic response of the adrenal glomerulosa to angiotensin II (AII) was investigated by analyzing the effects of Bordetella pertussis toxin (PT) on several aspects of AII action. These included receptor binding, stimulation of aldosterone production and GTPase activity, inhibition of cAMP production, and attenuation of the aldosterone response at high angiotensin concentrations. Pretreatment of glomerulosa cells with PT abolished the inhibitory effects of both AII and somatostatin (SRIF) on ACTH-stimulated cAMP production. Under the same incubation conditions, the stimulation of aldosterone secretion by submaximal and maximal steroidogenic concentrations of AII was completely unaffected by the toxin. However, the attenuation of steroid responses seen with supramaximal concentrations of AII was abolished. In addition, the ability of SRIF to inhibit AII-stimulated steroid production was markedly reduced by PT treatment. The binding of [125I]AII to high affinity sites in intact cells and particulate fractions, and modulation of the binding by guanine nucleotides, were unaffected by toxin pretreatment, even under conditions where a 40-41K protein was completely ADP ribosylated. In contrast, the toxin substantially diminished the binding of [125I]Tyr0-SRIF to SRIF receptors in glomerulosa cells (by 50% after 5 h and by 90% after 20 h). These results indicate that Ni or a similar protein probably mediates the inhibition of cAMP formation by AII and the attenuation of the steroid response by high concentrations of AII as well as the inhibitory actions of SRIF in the adrenal glomerulosa cell. Furthermore, the lack of effect of PT on AII binding and stimulation of GTPase activity suggests the existence of an additional pertussis-insensitive guanine nucleotide-regulatory protein that is activated by lower concentrations of AII and mediates the stimulation of aldosterone production.     

Effect of adrenocorticotrophin on cortisol and androstenedione secretion from dispersed cells of guinea-pig adrenal zonae fasciculata and reticularis

We have studied cortisol and androstenedione secretion by dispersed cells of the outer zona fasciculata (ZF) plus zona glomerulosa, and the inner zona reticularis (ZR) plus medulla of the guinea-pig adrenal. The ZF and ZR were microdissected apart, the cells dispersed and incubated (200 000 cells/ml) for 90 min in the presence of adrenocorticotrophin (ACTH; 500 ng/l), dibutyryl cyclic AMP (dbcAMP; 1 mmol/l), pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol and 21-deoxycortisol. The steroid concentrations were 5-25 mumol/l. Cortisol secretion was assayed by radioimmunoassay. There was no detectable cortisol secretion (less than 50 nmol/l) from the ZR in the controls (no additive) or after dbcAMP stimulation. Adrenocorticotrophin-stimulated cortisol secretion was also low (range less than 50-340 nmol/l). In contrast the ZF secreted 177-379 (control), 828-2052 (dbcAMP) and 2863-9735 (ACTH) nmol cortisol/l. There was no detectable (i.e. less than 2 nmol/l) cAMP production by ZR or ZF either basally (no ACTH) or after ACTH stimulation (500 ng/l). Challenge of the ZR cells with each cortisol precursor steroid (5 mumol/l) increased (P less than 0.05) cortisol secretion over that seen with the corresponding basal and ACTH-stimulated controls. Thus pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol and 21-deoxycortisol (converted directly to cortisol by 21-hydroxylase) gave rise to (mean +/- S.D., n = 4) 406 +/- 86, 680 +/- 180, 1307 +/- 111, 1141 +/- 234 and 3160 +/- 419 nmol cortisol/l respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma levels of prorenin and Renin in blacks and whites: their relative abundance and associations with plasma aldosterone concentration

BackgroundAll renin arises from prorenin. The proportion of renin relative to prorenin could influence overall renin-angiotensin-aldosterone activity. We sought to determine whether prorenin levels were related to extracellular volume, as reflected by the levels of plasma renin activity (PRA), and to aldosterone.MethodsWe analyzed plasma levels of prorenin, renin, and aldosterone, as well as their interactions, in 129 young blacks and whites.ResultsBlacks had lower plasma renin concentration (PRC) and PRA, but had prorenin levels similar to whites (69 pg/ml in blacks vs. 62 pg/ml in whites, P = 0.41). As a result, the renin-to-total renin ratio was significantly lower in blacks (11.5% in blacks as compared to 19.8% in whites; P = 0.0001). Because prorenin also resides in tissues including the adrenal where it can bind to a specific receptor to generate angiotensin II, we examined the relationship of prorenin levels to plasma aldosterone concentrations (PAC). While a positive association between PRC and PAC was found in both blacks and whites, PAC was positively related to prorenin in whites (P = 0.04) but negatively in blacks, an observation that we hypothesize was due to reduced prorenin-to-renin conversion in blacks.ConclusionsWe observed a disproportionately high level of prorenin in blacks. These high circulating prorenin levels however do not result in greater adrenal angiotensin II and aldosterone production in healthy young blacks.American Journal of Hypertension 2012; doi:10.1038/ajh.2012.83.     

The role of calcium in steroidogenesis in fetal zone cells of the human fetal adrenal gland

The human fetal adrenal gland is composed primarily of fetal zone (FZ) cells, which have a high rate of steroidogenesis. The purpose of this study was to examine the role of calcium in the regulation of steroidogenesis by FZ cells. Dispersed FZ cells were incubated in Krebs-Ringers medium at 37 C for 3 h in the presence of ACTH, (Bu)2cAMP, or forskolin in addition to various drugs. The medium contents of dehydroepiandrosterone sulfate (DS), cortisol (F), and cAMP were quantified by RIA. After the addition of ACTH (10(-10)-10(-5) M), DS and cAMP secretion increased. The addition of EGTA to the medium inhibited ACTH- and forskolin-stimulated DS, F, and cAMP secretion by 50% as well as (Bu)2cAMP-stimulated steroidogenesis. The addition of calcium (10(-5)-10(-2) M) had only a slight effect on the secretion of DS or F in the absence of ACTH or (Bu)2cAMP. In the presence of ACTH and (Bu)2cAMP, however, increasing amounts of calcium resulted in a 2- to 3-fold increase in the rates of DS and F secretion. The addition of either A23187, a calcium ionophore, or verapamil, a calcium channel blocker, inhibited ACTH-stimulated DS and F secretion by 90%. The rate of cAMP formation was greater after ACTH plus verapamil treatment than after ACTH treatment alone, whereas A23187 inhibited ACTH-stimulated cAMP secretion to basal levels. Both A23187 and verapamil inhibited ACTH- and cAMP-stimulated pregnenolone secretion. The metabolism of 22R-hydroxycholesterol to pregnenolone was inhibited by A23187 and verapamil. In conclusion, our results suggest that extracellular calcium is important for activation of the human adrenal FZ cell adenylate cyclase system, while intracellular calcium plays a multifaceted role in controlling steroid production.     

Isolation and structure of corticostatin peptides from rabbit fetal and adult lung.

A 34-amino acid peptide and three other structurally related peptides were isolated from rabbit fetal and adult lung. These cationic arginine- and cysteine-rich peptides inhibit corticotropin (ACTH)-stimulated rat adrenal cell corticosterone production. The peptide was called corticostatin (CSI). CSI was purified by reverse-phase HPLC and was shown to be homogenous from its amino acid analysis. Its sequence was determined on a gas-phase sequenator. The structure of CSI is Gly-Ile-Cys-Ala-Cys-Arg-Arg-Arg-Phe-Cys-Pro-Asn-Ser-Glu-Arg-Phe-Ser-Gly- Tyr-Cys - Arg-Val-Asn-Gly-Ala-Arg-Tyr-Val-Arg-Cys-Cys-Ser-Arg-Arg. CSI was found to markedly inhibit ACTH-stimulated corticosterone production by rat adrenal cells in vitro but did not affect basal levels. CSI did not affect the stimulation of aldosterone synthesis by angiotensin II in rat zona glomerulosa cells but it did suppress ACTH-stimulated aldosterone synthesis in whole adrenal cells, demonstrating that CSI is a specific inhibitor of ACTH-stimulated corticosteroid synthesis. The minimum effective concentration of CSI inhibiting ACTH-stimulated (33 pM) corticosterone production was 5 nM (20 ng/ml), the ED50 (50% effective dose) was 25 nM and steroidogenesis was completely inhibited at concentrations greater than 500 nM (2 micrograms/ml).     

Chlorbutol, a new inhibitor of aldosterone biosynthesis identified during examination of heparin effect on aldosterone production

This study was designed to examine the mechanism whereby routine heparin therapy inhibits adrenal aldosterone production. In bovine adrenal glomerulosa cell suspensions, pure heparin, in concentrations up to 500 U/ml, had no significant effect on basal or angiotensin II-stimulated aldosterone production. A therapeutic preparation of heparin for parenteral use containing the preservative chlorbutol (2.8 X 10(-2) M) inhibited aldosterone production [67 +/- 8.7% (+/- SE); P less than 0.005]. Chlorbutol alone, in a dose-dependent manner, inhibited basal aldosterone production from 1548 +/- 355 to 316 +/- 152 pg/ml (P less than 0.001) and inhibited angiotensin II-stimulated production from 4950 +/- 724 to 589 +/- 257 pg/ml (P less than 0.001). To elucidate the inhibitory mechanism of chlorbutol, we used trilostane, an inhibitor of the conversion of pregnenolone to progesterone, and aminoglutethimide, an inhibitor of the conversion of cholesterol to pregnenolone. Aldosterone production was completely suppressed by each inhibitor. Pregnenolone accumulation in trilostane-treated cells fell from 9.70 +/- 1.66 to 1.40 +/- 0.28 ng/ml (P less than 0.005) with the addition of chlorbutol. Aldosterone accumulation from corticosterone added to aminoglutethimide-treated cells fell from 715 +/- 96 to 348 +/- 59 pg/ml (P less than 0.02) in cells incubated with chlorbutol. Thus, chlorbutol is a potent inhibitor of aldosterone production, inhibiting both the early biosynthetic phase and, to a lesser extent, the late phase. Since chlorbutol is a widely used pharmaceutical preservative and has a slow metabolic clearance, these findings may be of toxicological significance and may account for the inhibition of aldosterone production previously attributed to heparin.     

Atrial natriuretic polypeptide inhibits cortisol secretion as well as aldosterone secretion in vitro from human adrenal tissue

The effect of alpha-human atrial natriuretic polypeptide (ANP) on adrenal steroidogenesis was studied in human adrenal tissues obtained surgically from four patients with Cushing's syndrome due to an adrenal adenoma and five patients with an aldosterone-producing adenoma (APA). ANP significantly inhibited basal and ACTH (3.4 X 10(-8) M)-stimulated cortisol and aldosterone secretion in both the adenomas and adjacent adrenocortical tissues from patients with Cushing's syndrome. ANP inhibited ACTH-stimulated, but not basal, secretion of cortisol and aldosterone in the adjacent tissues from patients with APA. In addition, ANP significantly inhibited both basal and ACTH-, angiotensin II (10(-6) M)-, and potassium chloride (10 mM)-stimulated secretion of aldosterone from the adenomas of patients with APA. ANP-induced changes in cortisol and aldosterone secretion were accompanied by a decrease in cAMP and an increase in cGMP secretion. These results suggest that ANP may be a possible regulator of cortisol as well as aldosterone secretion in humans, and these effects might be due to concomitant alteration in cyclic nucleotide metabolism.