Stimulated Aldosterone Release Research Articles

Abstract 067: Suppression Of The Renin Angiotensin Aldosterone System In Human Hypertensive Pregnancy

In healthy pregnancy, elevated renin activity stimulates aldosterone release via angiotensin II (Ang II) signaling. Despite the known importance in blood volume expansion, mechanisms of aldosterone secretion during pregnancy are not well understood. In vitro / in vivo evidence that aldosterone concentrations are disproportionally elevated compared to renin activity suggest altered sensitivity to Ang II or nonangiotensin stimulation during pregnancy. We used a novel LC-MS/MS-based methodology to quantify serum biomarkers of the renin-angiotensin-aldosterone system (RAAS) in order to evaluate renin-aldosterone relationships in healthy and hypertensive human pregnancy. We recruited 73 pregnant individuals with no known hypertension and followed them prospectively for the development of gestational hypertension or pre-eclampsia outcomes (HYP, n=27), no adverse outcomes (No AO, n=27), or adverse outcomes that were non-hypertensive (AO, Non-HYP, n=20). Concentrations of Ang peptides I and II and aldosterone were quantified from first and third trimester serum. Blood pressure was elevated starting in the first trimester in women who developed HYP outcomes (P<0.01). Renin activity (calculated by [Ang I + Ang II]) and aldosterone concentrations significantly increased from first to third trimester in No AO and AO Non-HYP (renin activity, 30% and 37% increase; aldosterone, 230% and 238% increase, respectively, P<0.001). This was in contrast to HYP pregnancies, where renin activity and aldosterone increased by 19% and 70%, respectively, and were not significant(P=0.54). Further, third trimester aldosterone was significantly lower, median (interquartile range) = 492 (260 - 827) pmol/L compared to No AO = 828 (467-1778) and AO Non-HYP = 1046 (527 - 1368) pmol/L (P<0.01). There was a significant increase in the aldosterone to Ang II ratio over gestation in No AO and AO Non-HYP pregnancies(P<0.01), but not HYP. Taken together, we propose that activity of the RAAS is suppressed in pregnancies that develop hypertension, likely a compensatory response to already-elevated BP. Further, non-hypertensive pregnancy is characterized by a preferential increase in aldosterone, where mechanisms may be impaired in pregnancies that develop hypertension.

The microRNA cluster miR-17~92 is induced by aldosterone and estrogen and inhibits aldosterone signaling in the kidney distal nephron

Background: Hypertension affects more than one billion people worldwide. A key regulator of blood pressure homeostasis is aldosterone, the final signaling hormone of the renin-angiotensin-aldosterone-signaling (RAAS) system. Aldosterone increases sodium (Na+) transport in the kidney distal nephron to regulate blood volume. We previously demonstrated that aldosterone alters the expression of microRNAs (miRs) in collecting duct epithelial cells to modulate the Na+ transport response to aldosterone. In this study a novel miR cluster, miR-17~92, is investigated for its ability to alter aldosterone signaling in the kidney in a sex-specific manner. Methods: The cortical collecting duct (CCD), mCCD-cl1, cell line were cultured on permeable filter supports to measure Na+ transport by short-circuit current recordings in Ussing chambers. In vivo regulation of miRs by aldosterone was confirmed using freshly isolated CCD cells from male and female mice placed on low Na+ diets. Dual-luciferase assays validated miR targeting of the serum and glucocorticoid kinase (SGK1). CCD cells were treated with estrogen to determine the impact on aldosterone stimulation and Na+ transport. RT-qPCR quantified miR and mRNA expression and western blot analysis quantified protein expression of the RAAS signaling cascade in mCCD cells after aldosterone and estrogen stimulation. Results: The miR-17~92 cluster expression is increased after aldosterone and estrogen stimulation in cultured mCCD cells. A sex-specific upregulation of the miR-17~92 cluster was observed in isolated CCD cells from female mice placed on a low-Na+ diet to stimulate aldosterone release. This increase was not observed in male mice. Members of the miR cluster, miRs-19a&b, are predicted to bind to the 3’-untranslated region of SGK1 and this was confirmed using luciferase assays. Overexpression of miR-19 in mCCD cells using miR mimics significantly inhibited aldosterone stimulation of Na+ transport via the epithelial sodium channel (ENaC). Estrogen pretreatment of mCCD blunted a subsequent aldosterone stimulation. This blunting of aldosterone signaling was due to a decrease in SGK1 mRNA and protein levels, for both estrogen stimulated and miR-19 mimic overexpressing cells. Conclusion: The miR-17~92 cluster is regulated by aldosterone and estrogen. Mir-19 targets SGK1 and reduces its expression. This, in turn, reduces the aldosterone-induced ENaC transport. The regulation of miR-17~92 is sex-specific and estrogen responsive, and targets SGK1 to downregulate sodium transport in the CCD. This could contribute to sex differences in sodium homeostasis. NIH 5T32DK061296-20 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Severe Case of Hypokalemic Metabolic Alkalosis: A Quiz

A Severe Case of Hypokalemic Metabolic Alkalosis: A Quiz

S-12-7: THE RENIN ANGIOTENSIN SYSTEM IN BASIC AND CLINICAL RESEARCH

The renin-angiotensin-system (RAS) is one of the oldest known hormonal systems preserved in multiple species over millions of years. A key enzyme of the system, renin, was discovered in 1898, and ever since a huge body of research has continuously revealed physiological and pathophysiological features of the RAS. Classically, these include, via the so-called angiotensin AT1 receptor, powerful vasoconstriction, regulation of the body fluid balance by inducing thirst, stimulation of aldosterone release from the adrenal cortex, renal salt retention, stimulation of the sympathetic nervous system and, more recently discovered, pro-fibrotic and pro-inflammatory effects. Pathophysiological actions of the RAS, with its extension to those of aldosterone (RAAS), include the induction of arterial hypertension, cardiac and vascular hypertrophy, lung and renal fibrosis, vascular atherosclerosis, perivascular fibrosis or chronic inflammation and others. Attempts to inhibit the RAS in hypertension and other cardiovascular diseases first led to the clinical introduction of ACE-inhibitors some forty years ago, followed by selective antagonists of the AT1 receptor, the sartans. After a multitude of evidence-generating trials and global success in clinical practice, both approaches are now firmly established in the therapy of hypertension as well as cardio-metabolic and renal diseases. More recently, research has focused on the so-called protective arm of the RAS, mediated by stimulation of the angiotensin AT2 receptor by or by the Mas receptor stimulated by the angiotensin 1–7 peptide. Activation of these systems has exhibited beneficial effects in numerous preclinical studies via opposing AT1-mediated actions and beyond. Other degradation peptides of the angiotensin precursor, angiotensinogen, may also exhibit protective effects via these receptors. Agonists for the AT2 receptor as well as for the ACE2-Mas system have been developed and are currently under clinical evaluation. It remains to be seen whether the pharmacological stimulation of the protective arm of the RAS will become an effective clinical tool in various cardiovascular and metabolic indications. Theoretically, it would make sense not only to block the harmful RAS but, at the same time, also stimulate the beneficial actions of the system.

PGE2 stimulates aldosterone release via cAMP/sPRR/β-catenin pathway in mouse inner medullary collecting duct cells

Increasing evidence showed the generation of aldosterone in the collecting duct (CD), a major site for the synthesis and action of prostaglandin E2 (PGE2). Both aldosterone and PGE2 are stimulated by Na+ depletion or K+ loading or angiotensin II. However, what happens first and the correlation between PGE2 and aldosterone in the CD remains obscure. PGE2 stimulates aldosterone release in the adrenal zona glomerulosa cells via E-prostanoid (EP) receptors by activating the cAMP/cAMP-responsive element-binding pathway. Here, we further tested the hypothesis that the synthesis of aldosterone is stimulated by PGE2 in CD cells and the underlying mechanism. In primary mouse inner medullary CD cells, PGE2 incubation significantly stimulated aldosterone release in parallel with an increase of CYP11B2 (cytochrome P-450, family 11, subfamily B, polypeptide 2), (pro)renin receptor (PRR), and prorenin protein expression and upregulation of medium soluble PRR (sPRR), prorenin/renin, and cAMP levels, both of which were attenuated by the EP1 antagonist SC-19220 and the protein kinase C (PKC) inhibitor calphostin C. PGE2-induced release of aldosterone, prorenin/renin, and sPRR was also abolished by the protein kinase A inhibitor H89. Both a PRR antagonist PRO20 and β-catenin inhibitor ICG001 blunted PGE2 or sPRR-induced aldosterone and prorenin/renin secretion. Exogenous sPRR protein incubation not only stimulated the release of aldosterone and prorenin/renin but also reversed PF429242 (an inhibitor of subtilisin kexin isozyme-1, which can block endogenous sPRR generation)-attenuated aldosterone and prorenin/renin secretion. Therefore, we conclude that PGE2 increases CD aldosterone synthesis through the EP1 receptor via the PKC/cAMP/sPRR/β-catenin pathway.

Sexual Dimorphic Regulation of MicroRNAs Alters Sodium Transport in the Kidney Distal Nephron

BackgroundHypertension affects more than one billion people worldwide. A key regulator of blood pressure homeostasis is aldosterone, the final signaling hormone of the renin‐angiotensin‐aldosterone‐signaling (RAAS) system. Aldosterone increases sodium (Na+) transport in the kidney distal nephron to regulate blood volume. Premenopausal women are less likely to develop hypertension than age‐matched men, due to both lower aldosterone levels and estrogen signaling. We previously demonstrated that aldosterone alters the expression of microRNAs (miRs) in collecting duct epithelial cells to modulate the Na+ transport response to aldosterone. However, the sex‐specific regulation of miRs and role of estrogen to alter miR expression in the kidney distal nephron has not been explored. This study investigated the hypothesis that estrogen alters aldosterone signaling in the distal nephron by regulating the expression of miRs in collecting duct epithelia.MethodsPrimary cortical collecting duct (CCD) cells derived from male and female mice as well as the mCCD‐cl1 cell line were cultured on permeable filter supports to measure Na+ transport by short‐circuit current recordings in Ussing chambers. Cells were incubated with estrogen for time and dose responses to determine the impact on aldosterone stimulation and Na+ transport. RT‐qPCR quantified miR and mRNA expression and western blot analysis quantified protein expression after aldosterone and estrogen stimulation. In vivo regulation of miRs by aldosterone was confirmed using freshly isolated CCD cells from male and female mice placed on low Na+ diets.ResultsA sex‐specific upregulation of the miR‐17~92 cluster was observed in female mice placed on a low‐Na+ diet to stimulate aldosterone release. MiRs‐19a&b were also upregulated in mCCD cells stimulated with estrogen. Estrogen pretreatment blunted aldosterone stimulation, by targeting the serum and glucocorticoid induced kinase (SGK1) mRNA. Luciferase assays demonstrated that miRs‐19a&b bind to the 3’‐UTR of SGK1. Overexpression of miR‐19 in mCCD cells using miR mimics significantly inhibited aldosterone stimulation of Na+ transport. Conversely, aldosterone stimulation was greater in mCCD cells transfected with a miR‐19 inhibitor (antagomir).ConclusionThe miR‐17~92 cluster is regulated by aldosterone and estrogen. Mir‐19 targets SGK1 and may account for estrogen’s inhibition of aldosterone signaling. This miR cluster may be responsible, in part, for the observed sex‐specific differences in aldosterone signaling. Ongoing studies aim to determine if altering the expression of miR‐17~92 disrupts RAAS signaling in the kidney in vivo.

Novel high molecular weight albumin-conjugated angiotensin II activates β-arrestin and G-protein pathways.

To study the ability of a novel bovine serum albumin-angiotensin II (BSA-Ang II) conjugate to effect responses of the AT1 angiotensin II receptor subtype mediated by the G-protein-coupled and the beta-arrestin pathways. Angiotensin II (Ang II) was conjugated with bovine serum albumin and compared with Ang II for competition binding to AT1 receptors, to stimulate aldosterone release from adrenocortical cells, to promote beta-arrestin binding to AT1 receptors, to promote calcium mobilization, and stimulate drinking of water and saline by rats. The BSA-Ang II conjugate was less potent competing for AT1R binding, but was equally efficacious at stimulating aldosterone release from H295R adrenocortical cells. Both BSA-Ang II and Ang II stimulated calcium mobilization and beta-arrestin binding to AT1 receptors. BSA-Ang II and Ang II stimulated water appetite equivalently but BSA-Ang II stimulated saline appetite more than Ang II. Both BSA-Ang II and Ang II were considerably more potent at causing calcium mobilization than β-arrestin binding. Addition of a high molecular weight molecule to Ang II reduced its AT1 receptor binding affinity, but did not significantly alter stimulation of aldosterone release or water consumption. The BSA-Ang II conjugate caused a greater saline appetite than Ang II suggesting that it may be a more efficacious agonist of this beta-arrestin-mediated response than Ang II. The higher potency calcium signaling response suggests that the G-protein-coupled responses predominate at physiological concentrations of Ang II, while the beta-arrestin response requires pathophysiological or pharmacological concentrations of Ang II to occur.

Abstract 128: Leptin: A Regulator of Aldosterone Synthase Expression & Aldosterone Secretion in Visceral Adipocytes, in Mice

Obesity is associated with inappropriately high aldosterone levels, which contribute to the development of metabolic and cardiovascular disorders. The origin of these high aldosterone levels is incompletely understood. We recently demonstrated that the adipocyte-derived hormone leptin regulates aldosterone synthase (CYP11B2) expression and stimulates aldosterone release from adrenal zona glomerulosa cells. Recent studies demonstrate that adipocytes express CYP11B2 and secrete aldosterone. However, the mechanisms regulating aldosterone release from adipocytes remain unclear. Likewise, whether visceral (Visc) and subcutaneous (SubQ) adipose tissue contribute to a similar extent to aldosterone production is unknown. We tested the hypothesis that leptin increases adipocyte CYP11B2 expression and aldosterone production and investigated whether Visc and SubQ adipose tissues respond similarly to leptin. Immunostaining of mouse adipose tissue cross-sections and isolated mature adipocytes revealed that Visc and SubQ adipose tissue express leptin receptors. Treatment of mouse freshly isolated mature adipocytes, non-differenciated (stromal fraction) and differentiated adipocytes revealed that leptin dose-dependently increased CYP11B2 expression and aldosterone production in Visc adipose tissue only. Although leptin receptor and CYP11B2 levels were similar in SubQ and Visc adipocytes, SubQ adipocytes were unresponsive to leptin. The physiological relevance of these in vitro data was tested by measuring plasma aldosterone levels in mice deprived of adipose tissue (lipodystrophic mice) treated with leptin. Absence of adipose tissue in lipodystrophic mice blunted leptin-induced increases in aldosterone levels (WT-vehicle: 471±82 vs. WT-Leptin: 1699±396, p<0.05; KO-vehicle: 539±71 vs. KO+leptin: 787±156, NS). The human relevance of these data was determined by reporting that CYP11B2 expression gradually increased with body mass index in human mediastinal and omental fat depots. In summary these data strongly suggest that leptin regulates CYP11B2 levels and aldosterone release in visceral adipose tissue and that leptin-induced, adipocyte-derived aldosterone may contribute to obesity-associated hyperaldosteronism.

High potassium promotes mutual interaction between (pro)renin receptor and the local renin-angiotensin-aldosterone system in rat inner medullary collecting duct cells.

(Pro)renin receptor (PRR) is predominantly expressed in the collecting duct (CD) with unclear functional implication. It is not known whether CD PRR is regulated by high potassium (HK). Here, we aimed to investigate the effect of HK on PRR expression and its role in regulation of aldosterone synthesis and release in the CD. In primary rat inner medullary CD cells, HK augmented PRR expression and soluble PPR (sPRR) release in a time- and dose-dependent manner, which was attenuated by PRR small interfering RNA (siRNA), eplerenone, and losartan. HK upregulated aldosterone release in parallel with an increase of CYP11B2 (cytochrome P-450, family 11, subfamily B, polypeptide 2) protein expression and upregulation of medium renin activity, both of which were attenuated by a PRR antagonist PRO20, PRR siRNA, eplerenone, and losartan. Similarly, prorenin upregulated aldosterone release and CYP11B2 expression, both of which were attenuated by PRR siRNA. Interestingly, a recombinant sPRR (sPRR-His) also stimulated aldosterone release and CYP11B2 expression. Taken together, we conclude that HK enhances a local renin-angiotensin-aldosterone system (RAAS), leading to increased PRR expression, which in turn amplifies the response of the RAAS, ultimately contributing to heightened aldosterone release.

Aldosterone synthase inhibition: cardiorenal protection in animal disease models and translation of hormonal effects to human subjects.

BackgroundAldosterone synthase inhibition provides the potential to attenuate both the mineralocorticoid receptor-dependent and independent actions of aldosterone. In vitro studies with recombinant human enzymes showed LCI699 to be a potent, reversible, competitive inhibitor of aldosterone synthase (Ki = 1.4 ± 0.2 nmol/L in humans) with relative selectivity over 11β-hydroxylase.MethodsHormonal effects of orally administered LCI699 were examined in rat and monkey in vivo models of adrenocorticotropic hormone (ACTH) and angiotensin-II-stimulated aldosterone release, and were compared with the mineralocorticoid receptor antagonist eplerenone in a randomized, placebo-controlled study conducted in 99 healthy human subjects. The effects of LCI699 and eplerenone on cardiac and renal sequelae of aldosterone excess were investigated in a double-transgenic rat (dTG rat) model overexpressing human renin and angiotensinogen.ResultsRat and monkey in vivo models of stimulated aldosterone release predicted human dose– and exposure–response relationships, but overestimated the selectivity of LCI699 in humans. In the dTG rat model, LCI699 dose-dependently blocked increases in aldosterone, prevented development of cardiac and renal functional abnormalities independent of blood pressure changes, and prolonged survival. Eplerenone prolonged survival to a similar extent, but was less effective in preventing cardiac and renal damage. In healthy human subjects, LCI699 0.5 mg selectively reduced plasma and 24 h urinary aldosterone by 49 ± 3% and 39 ± 6% respectively (Day 1, mean ± SEM; P < 0.001 vs placebo), which was associated with natriuresis and an increase in plasma renin activity. Doses of LCI699 greater than 1 mg inhibited basal and ACTH-stimulated cortisol. Eplerenone 100 mg increased plasma and 24 h urinary aldosterone while stimulating natriuresis and increasing renin activity. In contrast to eplerenone, LCI699 increased the aldosterone precursor 11-deoxycorticosterone and urinary potassium excretion.ConclusionsThese results provide new insights into the cardiac and renal effects of inhibiting aldosterone synthase in experimental models and translation of the hormonal effects to humans. Selective inhibition of aldosterone synthase appears to be a promising approach to treat diseases associated with aldosterone excess.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0340-9) contains supplementary material, which is available to authorized users.

Resistant hypertension—complex mix of secondary causes and comorbidities

Resistant hypertension—complex mix of secondary causes and comorbidities

Angiotensin II Type 1 Receptor–associated Protein

See related article, pp 1203–1210 Most physiological actions of angiotensin (Ang) II are mediated by its binding to the Ang II type 1 receptors (AT1R). These include, but are not limited to, vasoconstriction, increased renal sodium and water reabsorption, as well as the stimulation of aldosterone release, thirst, vasopressin secretion, and cell growth. Not surprisingly, the biochemistry of the AT1R has been a subject of intense research for >30 years. These studies revealed that the cytoplasmic tail of the AT1R is an important site for several protein-to-protein interactions, phosphorylation, and thereby the regulation of receptor signaling, desensitization, and endocytosis.1 Accordingly, considerable efforts have been made to identify and to study molecules with affinity to this region; one of such molecules is the angiotensin receptor–associated protein (ATRAP). ATRAP is an 18-kDa protein that was first identified and cloned in 1999 using a yeast 2-hybrid assay.2 Successive in vitro studies showed that ATRAP lacks catalytic activity.3 Instead, ATRAP is a membrane protein with 3 predicted transmembrane domains and a cytoplasmic tail that is important for its interactions with the carboxyl terminal of the AT1R.3 ATRAP colocalizes with the AT1R mostly in intracellular compartments, it induces AT1R internalization and reduces its signaling.2,3 Thus, available data indicate that ATRAP is a negative modulator of AT1R activity. ATRAP is made by many organs, including the heart, testis, and adrenal glands. However, by far the kidneys are the site of highest expression.2 Studies using gene targeting technology have provided valuable insights into the …

Effects of nonylphenol on aldosterone release from rat zona glomerulosa cells

Alkylphenol ethoxylate, which consists of approximately 80% nonylphenol ethoxylate (NPE), is a major nonionic surfactant. Nonylphenol (NP), the primary degradation product of NPE, has been reported to interfere with reproduction in fish, reptiles, and mammals by inducing cell death in the gonads and by affecting other reproductive parameters. However, the effects of NP on rat adrenal zona glomerulosa cells (ZG) and the underlying mechanisms remain unclear. In this study, we explored the effects of NP on aldosterone release. ZG cells were incubated with NP in the presence or absence of the secretagogues angiotensin II (ANG II), potassium, 8-Br-cAMP, 25-OH-cholesterol, corticosterone or cyclopiazonic acid (CPA). After performing radioimmunoassay (RIA) and Western blot analysis, we found that (1) NP stimulated aldosterone release in cells induced by ANG II, KCl, 8-Br-cAMP, 25-OH-cholesterol, corticosterone, and CPA; (2) NP triggered the release of higher amounts of pregnenolone in cells treated with vehicle and 25-OH-cholesterol+trilostane than in cells treated with other compounds; and (3) the stimulatory effect of NP seemed to be mediated through steroidogenic acute regulatory protein (StAR) and aldosterone synthase activity. These observations suggest that the effects of NP are mediated via increased free Ca2+ in the cytoplasm.

Expression of the epithelial Na + channel and other components of an aldosterone response pathway in human adrenocortical cells

We have unexpectedly found expression of the epithelial Na + channel (ENaC) in human adrenocortical cells and tested the hypothesis that these cells contain the components of an aldosterone response pathway. Tissue was obtained from patients undergoing adrenalectomy and mRNA and protein expression of recognised components of an aldosterone-response pathway were determined by RT-PCR and Western blotting. The effects of mineralocorticoid receptor agonists and antagonists, amiloride analogues, and extracellular Na + on basal and stimulated aldosterone release from immortalised (H295R) cells were determined by radioimmunoassay. Expression of mRNA for α-, β- and γ-subunits of ENaC, the mineralocorticoid receptor, Nedd4L, Sgk1 and 11β hydroxysteroid dehydrogenase type II was confirmed in human adrenal cortex. Using Western blotting α-, β- and γ-ENaC expression was demonstrated in adrenocortical cells. Measurements of 24 h aldosterone release from H295R cells showed stimulation by K + and angiotensin II, suppression by both Na + and high-concentration 5-( N-ethyl- N-isopropyl) amiloride (EIPA, blocker of Na +–H + exchange) and no change with benzamil (ENaC blocker). 22Na-uptake into H295R cells was inhibited by EIPA, but not by benzamil. Our experiments suggest that the components of an aldosterone response pathway are present in human adrenal cortex. Studies in H295R cells, however, suggest that ENaC is not an important mediator of 22Na-uptake or aldosterone production. Further studies are required to determine the importance of an adrenal aldosterone response pathway.

Severe loss‐of‐function mutations in the adrenocorticotropin receptor (ACTHR, MC2R) can be found in patients diagnosed with salt‐losing adrenal hypoplasia

ObjectiveFamilial glucocorticoid deficiency type I (FGD1) is a rare form of primary adrenal insufficiency resulting from recessive mutations in the ACTH receptor (MC2R, MC2R). Individuals with this condition typically present in infancy or childhood with signs and symptoms of cortisol insufficiency, but disturbances in the renin-angiotensin system, aldosterone synthesis or sodium homeostasis are not a well-documented association of FGD1. As ACTH stimulation has been shown to stimulate aldosterone release in normal controls, and other causes of hyponatraemia can occur in children with cortisol deficiency, we investigated whether MC2R changes might be identified in children with primary adrenal failure who were being treated for mineralocorticoid insufficiency.DesignMutational analysis of MC2R by direct sequencing.PatientsChildren (n = 22) who had been diagnosed with salt-losing forms of adrenal hypoplasia (19 isolated cases, 3 familial), and who were negative for mutations in DAX1 (NR0B1) and SF1 (NR5A1).ResultsMC2R mutations were found in three individuals or kindred (I: homozygous S74I; II: novel compound heterozygous R146H/560delT; III: novel homozygous 579-581delTGT). These changes represent severely disruptive loss-of-function mutations in this G-protein coupled receptor, including the first reported homozygous frameshift mutation. The apparent disturbances in sodium homeostasis were mild, manifest at times of stress (e.g. infection, salt-restriction, heat), and likely resolved with time.ConclusionsMC2R mutations should be considered in children who have primary adrenal failure with apparent mild disturbances in renin-sodium homeostasis. These children may have been misdiagnosed as having salt-losing adrenal hypoplasia. Making this diagnosis has important implications for treatment, counselling and long-term prognosis.

Endothelium-Derived Steroidogenic Factor Enhances Angiotensin II-Stimulated Aldosterone Release by Bovine Zona Glomerulosa Cells

Endothelium-derived steroidogenic factor (EDSF) is an endothelial peptide that stimulates aldosterone release from bovine adrenal zona glomerulosa (ZG) cells. The regulation of aldosterone release by combinations of EDSF and angiotensin II (AII) or EDSF and ACTH was investigated. Endothelial cells (ECs) and EC-conditioned media (ECCM) increased aldosterone release from ZG cells, an activity attributed to EDSF. AII (10−12 to 10−8m) and ACTH (10−12 to 10−9m) also stimulated the release of aldosterone from ZG cells. The stimulation by AII, but not ACTH, was greatly enhanced when ZG cells were coincubated with ECs. AII was metabolized by ECs to peptides identified by mass spectrometry as angiotensin (1-7) and angiotensin IV. There was very little metabolism of AII by ZG cells. Neither of these two AII metabolites altered aldosterone release from ZG cells, so they could not account for the enhanced response with ECs. AII-induced aldosterone release from ZG cells was enhanced by ECCM but not cell-free conditioned medium. This enhanced response was not due to increased EDSF release from ECs by AII. The synergistic effect of EDSF and AII was apparent when AII was added during or after the generation of ECCM and not observed when the AII component of the enhancement was blocked by the AII antagonist, losartan. These studies indicate that EDSF enhances the steroidogenic effect of AII. In the adrenal gland, ECs are in close anatomical relationship with ZG cells and may sensitize ZG cells to the steroidogenic action of AII by releasing EDSF.

Up-regulation of the Angiotensin II Type 1 Receptor by the MAS Proto-oncogene Is Due to Constitutive Activation of Gq/G11 by MAS

Coexpression of the MAS proto-oncogene with the angiotensin II type 1 (AT(1)) receptor in CHO-K1 cells has been reported to increase the number of [(3)H]angiotensin II-binding sites, although MAS does not bind [(3)H]angiotensin II. In HEK293 cells stably expressing AT(1) receptor-cyan fluorescent protein (CFP), MAS-yellow fluorescent protein (YFP) expression from an inducible locus caused strong up-regulation of AT(1) receptor-CFP amounts and [(3)H]angiotensin II binding levels. The time course of AT(1) receptor-CFP up-regulation was also markedly slower than that of induction of MAS expression. These effects were not mimicked by induced expression of I138D MAS-YFP, a mutant unable to cause constitutive loading of [(35)S]guanosine 5'-O-(thiotriphosphate) onto the phospholipase Cbeta-linked G protein Galpha(11). Protein kinase C (PKC) inhibitors and the selective Galpha(q)/Galpha(11) inhibitor YM-254890 fully blocked MAS-induced up-regulation of AT(1) receptor-CFP amounts, whereas the PKC activator phorbol 12-myristate 13-acetate produced strong up-regulation of AT(1) receptor-CFP without induction of MAS-YFP expression and in the presence of I138D MAS-YFP. The C-terminal tail of the AT(1) receptor is a known target for PKC-mediated phosphorylation. In cells stably expressing a C-terminally truncated version of the AT receptor, induction of MAS expression did not up-regulate the truncated construct levels. These data demonstrate that the ability of MAS to up-regulate AT(1) receptor levels reflects the constitutive capacity of MAS to activate Galpha(q)/Galpha(11) and hence stimulate PKC-dependent phosphorylation of the AT(1) receptor.

P.1.c.028 The administration of the cannabinoid CB1 receptor antagonist AM-251 blocked the anxiolytic and sedative actions of benzodiazepines

We have unexpectedly found expression of the epithelial Na+ channel (ENaC) in human adrenocortical cells and tested the hypothesis that these cells contain the components of an aldosterone response pathway. Tissue was obtained from patients undergoing adrenalectomy and mRNA and protein expression of recognised components of an aldosterone-response pathway were determined by RT-PCR and Western blotting. The effects of mineralocorticoid receptor agonists and antagonists, amiloride analogues, and extracellular Na+ on basal and stimulated aldosterone release from immortalised (H295R) cells were determined by radioimmunoassay. Expression of mRNA for α-, β- and γ-subunits of ENaC, the mineralocorticoid receptor, Nedd4L, Sgk1 and 11β hydroxysteroid dehydrogenase type II was confirmed in human adrenal cortex. Using Western blotting α-, β- and γ-ENaC expression was demonstrated in adrenocortical cells. Measurements of 24 h aldosterone release from H295R cells showed stimulation by K+ and angiotensin II, suppression by both Na+ and high-concentration 5-(N-ethyl-N-isopropyl) amiloride (EIPA, blocker of Na+–H+ exchange) and no change with benzamil (ENaC blocker). 22Na-uptake into H295R cells was inhibited by EIPA, but not by benzamil. Our experiments suggest that the components of an aldosterone response pathway are present in human adrenal cortex. Studies in H295R cells, however, suggest that ENaC is not an important mediator of 22Na-uptake or aldosterone production. Further studies are required to determine the importance of an adrenal aldosterone response pathway.

Angiotensin receptors: distribution, signalling and function

Angiotensin II (Ang II) is a multi-functional hormone that plays a major role in regulating blood pressure and cardiovascular homoeostasis. The actions of Ang II are mediated by at least two receptor subtypes, designated AT(1) and AT(2). In addition, other angiotensin receptors have been identified which may recognize other angiotensin peptide fragments; however, until now only the AT(1) and AT(2) receptor have been cloned in animals or humans. Most of the well-described actions of Ang II, such as vasoconstriction, facilitation of sympathetic transmission, stimulation of aldosterone release and promotion of cellular growth are all mediated by the AT(1) receptor. Much less is known about the function of the AT(2) receptor, but recent studies suggest that it may play a role in mediating anti-proliferation, cellular differentiation, apoptosis and vasodilatation. In this review, we discuss recent advances in our understanding of Ang II receptors, in particular, their distribution, signalling and function.

Endothelial cell nitric oxide inhibits aldosterone synthesis in zona glomerulosa cells: modulation by oxygen.

The regulation of aldosterone synthesis by endogenous nitric oxide (NO) was examined in cultured cells of the adrenal cortex. Endothelial NO synthase (eNOS) was detected by Western blot in cultured adrenal endothelial cells (ECs) but not in zona glomerulosa (ZG) cells or adrenal fibroblasts. Neither inducible (iNOS) nor neuronal NOS (nNOS) isoforms were detected in the cells. Only ECs had NOS activity and converted [(3)H]L-arginine to [(3)H]L-citrulline. Angiotensin II (ANG II, 100 nM) increased EC production of nitrate/nitrite by 2.4-fold. Coincubation with ECs or treatment with DETA nonoate increased the fluorescence of ZG cells loaded with an NO-sensitive dye, diaminofluorescein 2 diacetate (DAF-2 DA). DETA nonoate inhibited ANG II (1 nM) and potassium (10 mM) -stimulated aldosterone release in a concentration-related manner. This inhibitory effect of NO was enhanced >10-fold by decreasing the oxygen concentration from 21 to 8%. Coincubation of EC and ZG cells in 8% oxygen inhibited ANG II-induced aldosterone release, and inhibition was reversed by blockade of NOS. These findings indicate that adrenal EC-derived NO inhibits aldosterone release by cultured ZG cells and that the sensitivity to NO inhibition is increased at low oxygen concentrations.