Aldosterone secretion in patients with septic shock: a prospective study.

OBJECTIVE: To assess serum levels of the main factors that regulate the activation of the zona glomerulosa and aldosterone production in patients with septic shock, as well as their response to a high-dose (250 µg) adrenocorticotropic hormone (ACTH) stimulation test. SUBJECTS AND METHODS: In 27 patients with septic shock, baseline levels of aldosterone, cortisol, ACTH, renin, sodium, potassium, and lactate were measured, followed by a cortrosyn test. RESULTS: Renin correlated with baseline aldosterone and its variation after cortrosyn stimulation. Baseline cortisol and its variation did not correlate with ACTH. Only three patients had concomitant dysfunction of aldosterone and cortisol secretion. CONCLUSIONS: Activation of the zona glomerulosa and zona fasciculata are independent. Aldosterone secretion is dependent on the integrity of the renin-angiotensin-aldosterone system, whereas cortisol secretion does not appear to depend predominantly on the hypothalamic-pituitary-adrenal axis. These results suggest that activation of the adrenal gland in critically ill patients occurs by multiple mechanisms.

Mechanism of angiotensin II-induced phospholipase D activation in bovine adrenal glomerulosa cells.

Based on previous data demonstrating activation of phospholipase D (PLD) in response to angiotensin II (AngII), we have hypothesized a role for PLD in mediating aldosterone secretion from bovine adrenal glomerulosa cells. In this study we demonstrate that a PLD-generated signal(s) is required for the AngII-elicited secretory response, since interfering with lipid second messenger formation using a primary alcohol inhibited AngII-induced aldosterone secretion, but not that elicited by incubation with a hydrophilic cholesterol analog, 22(R)-hydroxycholesterol, which bypasses signaling pathways. Three mechanisms for hormonal activation of PLD have been described in other systems: direct receptor coupling, activation through protein kinase C (PKC) and a combination of these two mechanisms. Our results indicate that the PKC activator, phorbol 12-myristic 13-acetate (PMA), is able to activate PLD, and that receptor engagement is apparently not necessary for PLD activation in response to this agent. Maximal doses of AngII and PMA produced no additive effect on PLD activation, suggesting that these two agents function through a common PKC pathway. This interpretation was confirmed by the ability of a PKC inhibitor, Gö 6976, to inhibit partially AngII-induced PLD activation. Finally, treatment with the calcium ionophores A23187 or ionomycin or the calcium channel agonist BAY K8644 had no effect on PLD activity. Likewise, inhibiting calcium influx with high-dose nitrendipine affected neither basal PLD activity nor that stimulated by AngII. Thus, our results suggest a role for PKC, independent of calcium influx, in mediating AngII-induced PLD activation in glomerulosa cells.

Inhibitory effect of evodiamine on aldosterone release by Zona glomerulosa cells in male rats.

Evodiamine is a bioactive alkaloid extracted from a Chinese herb named Wu-Chu-Yu, which possesses thermoregulatory, analgesic, and cardiovascular effects. Some studies have demonstrated that evodiamine reduces blood pressure through acting on endothelium and smooth muscle cells to produce a vasodilatory effect, but whether it affects aldosterone secretion is unclear. The purpose of this study was to examine the effect of evodiamine on aldosterone release in adrenal zona glomerulosa (ZG) cells. ZG cells were isolated from the adrenal glands of adult male rats and incubated with angiotensin II (Ang II, 1x10(-7) M) and 3H-pregnenolone in the presence or absence of evodiamine (1x10(-6)-1x10(-3) M) at 37 degrees C for one hour. The concentration of aldosterone in the media was measured by a radioimmunoassay. The level of radioactivity incorporated into aldosterone and its precursors after incubation of ZG cells with 3H-pregnenolone was analyzed by thin-layer chromatography. The results demonstrated that evodiamine decreased the basal level of and Ang II-induced release level of aldosterone in rat ZG cells. Administration of evodiamine also decreased the level of radioactivity incorporated into 3H-corticosterone and 3H-aldosterone following incubation of ZG cells with 3H-pregnenolone. This suggest that evodiamine affects aldosterone release in rat adrenal glomerulosa cells by acting on Ang II-associated pathway and reducing the activity of 11 beta-hydroxylase (an enzyme which coverts deoxycorticosterone to corticosterone) during the steroidogenesis of aldosterone.

Stimulatory effect of the substance P antagonist spantide-II on aldosterone secretion of dispersed rat zona glomerulosa cells.

Substance P (SP) did not affect either basal or agonist-stimulated aldosterone production by dispersed rat zona glomerulosa (ZG) cells. In contrast, the SP-receptor antagonist spantide-II (SPA), at 10(-8)/10(-6) M concentrations, markedly raised basal and 10(-9) M ACTH, but not 10(-9) M angiotensin II-stimulated aldosterone secretion. The secretagogue effect of 10(-6) M SPA was annulled by SP (10(-6) M) and the protein kinase (PK)-C inhibitor Ro31-8220 (10(-6) M), but was unaffected by the PKA inhibitor H-89 (10(-5) M). In light of these findings the following conclusions can be drawn: (i) SP does not exert a physiologically relevant direct modulatory action on aldosterone secretion of rat ZG cells; (ii) a receptor-independent inhibitory interaction is likely to occur between SP and SPA molecules; and (iii) SPA activates, through a receptor-independent mechanism, phosphoinositide signaling pathway in rat ZG cells.

Possible role for mitochondrial calcium in angiotensin II- and potassium-stimulated steroidogenesis in bovine adrenal glomerulosa cells.

In adrenal zona glomerulosa cells, the action of angiotensin II (Ang II) and of potassium (K+) on aldosterone synthesis is mediated by the Ca2+ messenger system. The major part of the steroidogenic pathway takes place inside the mitochondria, and Ca2+ must enter the mitochondrial matrix to stimulate the steroidogenic cascade. To examine how changes in the cytosolic free calcium concentration ([Ca2+]c) induced by Ang II and K+ are relayed into the mitochondrial matrix, we transfected bovine adrenal zona glomerulosa cells in primary culture with a chimeric complementary DNA encoding for the signal presequence targeting human cytochrome c oxidase subunit VIII to the matrix, linked to a complementary DNA coding for the Ca2+-sensitive photoprotein aequorin. Resting mitochondrial free calcium concentration ([Ca2+]m) amounted to 0.41 +/- 0.18 microM (n = 40). Ang II induced a concentration-dependent (EC50 = 11.3 +/- 6.0 nM), biphasic rise of [Ca2+]m. After a large transient initial peak (5.13 +/- 0.89 microM, n = 28), [Ca2+]m decreased to a plateau that remained higher than basal [Ca2+]m for several minutes in the presence of the hormone. By contrast, studies in cells transfected with cytosolic aequorin indicated that the rise of [Ca2+]c triggered by Ang II was confined to 1.34 +/- 0.26 microM (n = 17). In Ca2+-free medium, a reduced peak [Ca2+]m response to Ang II occurred without a secondary plateau. On readdition of extracellular Ca2+, in the presence of the hormone, the resulting Ca2+ influx was accompanied by small rise of [Ca2+]m. The mitochondrial uncoupler, carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone, prevented the Ang II-induced [Ca2+]m rise but not the [Ca2+]c response, thus demonstrating the mitochondrial location of transfected aequorin. In contrast to Ang II, K+ (13 mM) induced a sustained [Ca2+]c response, which was relayed without amplification into the mitochondrial matrix as a plateau of[Ca2+]m. This plateau of[Ca2+]m was suppressed by the addition of the dihydropyridine, nifedipine (200 nM). The inhibitor of the mitochondrial Na+/Ca2+ exchanger, CGP37157, reduced significantly the rate of decrease of [Ca2+]m following the peak induced by Ang II. In cells whose [Ca2+]c was clamped at various levels (0.05-0.860 microM) with ionomycin, a concentration-dependent stimulation of pregnenolone output was induced by Ca2+. Under these conditions, the output of pregnenolone--the early product of steroidogenesis--was markedly potentiated by CGP37157. These results suggest the existence of microdomains of high [Ca2+]c elicited by Ang II in the proximity of mitochondria. Moreover, our observations are consistent with a mitochondrial site of action for calcium in the activation of the steroidogenic cascade.

Effects of neuromedin-K on the rat hypothalamo-pituitary-adrenal axis.

The effects of neuromedin-K (NMK) on the rat hypothalamo-pituitary-adrenal (HPA) axis were studied both in vivo and in vitro. A subcutaneous injection of 1 nmol/100 g NMK evoked a rise in plasma ACTH level at 30 min, increased plasma corticosterone (B) concentration (PBC) at 60 and 120 min, and did not alter plasma aldosterone (ALDO) concentration (PAC). The administration of 3 nmol/100 g NMK induced a rise in plasma ACTH level at 120 min and a drop of PBC at 30 min; it increased PBC and PAC at 60 and 120 min. NMK did not affect basal B secretion of dispersed zona fasciculata/reticularis (ZF/R) cells, but markedly enhanced basal ALDO production by dispersed zona glomerulosa (ZG) cells (minimal and maximal effective concentrations were 10(-9) M and 10(-7) M). Video-imaging analysis showed that NMK (10(-8) M) increased intracellular Ca2+ concentration in dispersed ZG cells, but not in ZF/R ones. These findings indicate that NMK exerts a complex modulatory action on the rat HPA axis: low doses of NMK appear to evoke a transient stimulation of ACTH release, while high doses seem to exert a short-term inhibition of glucocorticoid synthesis followed by the compensatory hypersecretion of ACTH; moreover, elevated doses of NMK also exert a strong ALDO secretagogue action by acting directly on the ZG cells.

Blocking T-type calcium channels with tetrandrine inhibits steroidogenesis in bovine adrenal glomerulosa cells.

Tetrandrine, an alkaloid extracted from a Chinese medicinal herb traditionally used in hypertension treatment, inhibited aldosterone production induced in bovine adrenal glomerulosa cells by either potassium ion, angiotensin II, or ACTH in a concentration-dependent manner (IC50 = 10 microM). The inhibition of the response to potassium by tetrandrine had a pattern very similar to that of nickel, a blocker of T-type calcium channels. In addition, tetrandrine prevented calcium influx induced by potassium or angiotensin II without affecting the calcium release phase stimulated by the hormone. The effect of tetrandrine on voltage-activated barium currents was investigated using the whole cell configuration of the patch clamp technique. T-type currents were isolated by recording the slowly deactivating currents elicited during repolarization of the cell to -65 mV after various depolarizing pulses. These currents were blocked by micromolar concentrations of the drug. The voltage sensitivity of channel activation was not affected by tetrandrine; nevertheless, the drug significantly slowed the deactivation of the current. The action of tetrandrine did not require the activation of the channel. Tetrandrine also affected L-type currents, as assessed after inactivating T channels for 100 msec, but at higher concentrations of the drug. Thus, tetrandrine affects with a similar potency aldosterone production, calcium influx, and T-type calcium channel activity. This finding strongly suggests a role for these channels in calcium signaling and control of steroidogenesis in adrenal glomerulosa cells.

Regulation of renin gene expression in rat adrenal zona glomerulosa cells.

Our previous studies indicated that the amount of renin present in cultured adrenal zona glomerulosa cells increased after stimulation with adrenocorticotropic hormone or potassium. In the present study, we investigated the effects of adrenocorticotropic hormone or potassium on renin gene expression in cultured rat adrenal zona glomerulosa cells. The amount of rat renin messenger RNA (mRNA) was measured by complementary DNA synthesis and the competitive polymerase chain reaction method. The effects of adrenocorticotropic hormone or potassium on adrenal zona glomerulosa cell renin activity and renin mRNA content were compared with the activity and content of control cells. After 1 and 4 hours of stimulation by adrenocorticotropic hormone or potassium, total renin in the medium increased slightly; at the same time, the percent change in the amount of renin mRNA was 281% and 291%, respectively, in the adrenocorticotropic hormone-stimulated group and 218% and 348%, respectively, in the potassium-stimulated group. Twenty-four hours after adrenocorticotropic hormone or potassium stimulation, total renin in the medium increased significantly, by 689% and 220%, respectively; percent change in the renin mRNA content was 754% and 278%, respectively. These results demonstrate that adrenocorticotropic hormone and potassium increased the activity of adrenal renin through an increase in the level of renin mRNA.

Calcitonin gene-related peptide depresses the growth and secretory activity of rat adrenal zona glomerulosa.

The bolus ip. injection of rat calcitonin gene-related peptide (CGRP) (5 pm. kg-1) significantly lowered plasma aldosterone concentration (PAC) in rats, despite a mild rise in plasma renin activity. Natremia, kalaemia and the blood levels of ACTH or corticosterone were not affected. Similar results were obtained after prolonged (5 days) sc. infusion of rats with CGRP (1 pm. kg-1. h-1). Moreover, CGRP infusion caused a notable atrophy of the zona glomerulosa (ZG) and its parenchymal cells, as well as a clearcut reduction in the surge of PAC evoked by a bolus injection of a high dose of angiotensin-II (100 micrograms. kg-1). From these results it is suggested that CGRP exerts an inhibitory effect on the growth and secretory activity of ZG in rats.

In vivo regulation of gene expression of enzymes controlling aldosterone synthesis in rat adrenal.

We studied the effect of alterations in the intake of sodium and potassium as well as changes in circulating adrenocorticotropin (ACTH) on the expression of the two rate-limiting systems of aldosterone formation in the rat. Low sodium and high potassium intake promoted time-dependent increases in the zona glomerulosa cytochrome P450scc (P450scc) and cytochrome P450c11 (P450c11) protein and mRNA levels, but no changes were found in the zona fasciculata-reticularis. In addition, these responses were associated with markedly elevated transcriptional activities. To further define the contribution of P450c11 and P450c18 (aldosterone synthase) in response to these differing intakes, we evaluated their mRNA levels using gene-specific oligonucleotide probes. P450c18 mRNA was restricted to the zona glomerulosa, whereas P450c11 mRNA was detected in both zona glomerulosa and zona fasciculata-reticularis. Furthermore, only P450c18 mRNA was induced by both low sodium or high potassium intake, as P450c11 mRNA levels remained unchanged. Captopril, an inhibitor of angiotensin-I converting enzyme, abolished the enhancing effects of the low sodium regimen on P450scc and P450c18 mRNA levels. Captopril also suppressed the augmentation of P450c18 mRNA observed with potassium supplementation but had no effect on P450scc mRNA levels. When the hypocholesterolemic drug 4-aminopyrazolopyrimidine (4-APP) was administered to rats for 3 consecurive days, both the level of plasma ACTH and the adrenal content of mRNA encoding P450scc increased 24 h post final injection. The coadministration of dexamethasone with 4-APP prevented these increases. In contrast, the mRNA content of P450c11 remained at control levels. In conclusion, this work demonstrates that variations in the intake of sodium and potassium act on the expression of the CYP11B2 gene, but not on that of the CYP11B1 gene. Moreover angiotensin-II (A-II) is an important factor in this mechanism of action. Both ions also enhance the expression of the CYP11A1 gene. A-II appears to participate in the mechanism of action of the low sodium intake at this level. Another mechanism is postulated for the action of potassium supplementation since captopril did not prevent the increased expression of the CYP11A1 gene. In addition, the fact that 4-APP enhanced the mRNA level of P450scc but not that of P450c, also demonstrates different regulation of the P450s involved at the early and final steps of aldosteroone formation in the rat adrenal zona glomerulosa in vivo.

3,4,3',4'-Tetrachlorobiphenyl distribution and induced effects in the rat adrenal gland. Localization in the zona fasciculata.

The distribution of radiolabeled 3,4,3',4'-tetrachlorobiphenyl (TCB) and TCB-induced effects on serum and adrenal gland retinoid content, and adrenal gland morphology was studied by liquid scintillation counting, high performance liquid chromatography, light microscopic autoradiography, and transmission electron microscopy. Adult, female WAG/Rij rats received a single intraperitoneal injection of either vehicle (corn oil), 15 mg TCB/kg, or 200 mg TCB/kg body weight and were sacrificed (N = 3 per group) at 1, 3, 7, and 14 days after treatment. One rat of the high dose group that was sacrificed at each sampling time had received radiolabeled compound (containing 1.85 mCi of 3H-TCB). At day 1, the adrenal gland had the greatest concentration of radioactivity (dpm x 10(-6)/gm wet tissue) of any organ examined. There was a selective distribution of radiolabeled compound to the zona fasciculata accompanied by morphometric evidence of hypertrophy of the zona fasciculata. The vast majority of 3H-TCB present in the adrenal gland was parent compound at all time periods. Serum retinol content was significantly decreased in the high dose group by 61 and 54% at days 3 and 7, respectively. No significant decrease in adrenal gland retinoid content occurred at any time in this study, but in contrast, adrenal gland retinol and retinyl palmitate content was increased. Serum cortisol levels were transiently decreased in the high dose group. Ultrastructural alterations were only observed in cells of the zona fasciculata. Predominant changes included mitochondrial hypertrophy and concentric whorling lamellar arrays of the membranes of the outer mitochondrial compartment and mitochondrial cristae. The results of this study indicate that the rat adrenal gland is an early target organ after TCB intoxication, and that there is an early and selective distribution of TCB in the rat adrenal gland accompanied by morphologic alterations in the sites of compound localization. The results further suggest that the observed morphologic changes did not result from hypovitaminosis A.

Calcium stimulates steroidogenesis in permeabilized bovine adrenal cortical cells.

The effect of Ca2+ on steroid production was examined in electropermeabilized bovine adrenal zona glomerulosa and fasciculata cells. The cells were superfused with a medium mimicking cytosolic ionic content but deprived of Ca2+. The permeabilized glomerulosa cells produced aldosterone at a low basal rate. Upon addition of NADP+ to the medium, a transient and concentration-dependent (EC50 = 6 microM) peak of aldosterone production occurred. When the superfusion medium was supplemented with buffered Ca2+ at submicromolar concentrations, a concentration-dependent and sustained increase of aldosterone output was observed. The maximal response (2-3 times the basal secretion rate) was achieved with 1-2 microM ambient free Ca2+, and the EC50 for Ca2+ was 0.5 microM. The continuous presence of NADP+ was found to be necessary for a Ca2+ effect. The Ca2+-induced aldosterone response was entirely blocked by ruthenium red (1 microM), an inhibitor of mitochondrial Ca2+ uptake, and by W-7 (5 microM), a calmodulin inhibitor. Qualitatively and quantitatively similar results were obtained for corticosterone production in adrenal fasciculata cells. These results show that permeabilized adrenal cortical cells retain the ability to produce steroids. Moreover, Ca2+ influx into the mitochondria and Ca2+/calmodulin-dependent reactions appear to be critical steps in the activation of steroidogenesis. These studies provide a further direct link between cytosolic free calcium concentration and biological responses induced by steroidogenic, calcium-mobilizing stimulators in the adrenal cortex.