Acute Aldosterone Research Articles

Chronic aldosterone administration alters aquaporin expression and localization in the murine kidney

The steroid hormone aldosterone is an important regulator of fluid balance and electrolyte homeostasis. While acute aldosterone signaling is canonically associated with fluid retention due to its role in promoting sodium reabsorption through ENaC in the distal nephron, chronic aldosterone exposure has been shown to exacerbate urine production in a rat model of diabetes insipidus through its actions on aquaporin 2 (AQP2) localization and expression. In our studies, we aimed to characterize not only the effect of chronic aldosterone administration on AQP2 expression and localization, but also its effect on the basolateral water channels, AQP3 and AQP4. Given the large urinary output we observed in our animals following chronic aldosterone treatments, we hypothesized all three of these channels would show reduced expression.Aldosterone was administered to male C57Bl/6 mice at a dose of 200 μg/kg/day for 28 days via subcutaneous minipumps. Aldosterone treatment caused a significant diuresis as compared to control animals by day 15 (Ctl 4.8 ± 1.6 ml; Aldo 8.4 ± 2.4 ml), and this difference persisted for the remainder of the experiment. Mice given aldosterone also consumed more water as compared to their control counterparts (Ctl 4.0 ± 1.2 ml; Aldo 9.6 ± 2.3 ml). Urine osmolality was measured by freeze-point depression, with a significant decrease in urinary concentration being noted during the daytime by day 4 (Ctl 1219.8 ± 473.4 mOsm/kg; Aldo 638.8 ± 143.8 mOsm/kg) and during the night by day 17 (Ctl 905.3 ± 175.1 mOsm/kg; Aldo 522.2 ± 127.3 mOsm/kg). At the end of the 28 days of aldosterone administration, mice receiving aldosterone had a higher whole blood [Na + ] (Ctl 146 ± 1.9mmol/l; Aldo 152 ± 1.3mmol/l) and lower whole blood [K+] (Ctl 4.8 ± 0.56mmol/l; Aldo 3.0 ± 0.34mmol/l) as measured by iSTAT cartridge with terminal cardiac blood draw.Kidneys were examined by Western blot and immunofluorescent staining for expression and localization of the three AQPs. AQP2 expression decreased after both 14 and 28 days of aldosterone administration, with line scan analysis showing a decreased signal present at the apical membrane. AQP3 expression was decreased in the basolateral membranes of principal cells within the cortex of the kidney but increased in cells within the inner medulla after 28 days of aldosterone. AQP4 levels were significantly increased in AQP2-positive cells of the inner stripe of the outer medulla and the inner medulla, suggesting that this channel may be upregulated to compensate for changes in the other two aquaporins. Potassium supplementation to separate the effects of aldosterone administration from hypokalemia did not rescue the expression of these channels.Overall, our studies reveal that chronic aldosterone administration in healthy mice leads to polyuria and polydipsia, with decreased apical expression of AQP2 and variable changes in AQP3 and AQP4 dependent on location within in the kidney. This study was funded by NIH grants R01 DK130901 (to SS), R01 HL147818 and R01 DK129285 (to TRK), and P30 DK079307 (the Pittsburgh Center for Kidney Research). SMM was supported by T32 DK061296, T32 DK007052, and a grant from Relypsa. 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.

Intrinsic Adrenal TWIK-Related Acid-Sensitive TASK Channel Dysfunction Produces Spontaneous Calcium Oscillations Sufficient to Drive AngII (Angiotensin II)-Unresponsive Hyperaldosteronism.

Ion channel mutations in calcium regulating genes strongly associate with AngII (angiotensin II)-independent aldosterone production. Here, we used an established mouse model of in vivo aldosterone autonomy, Cyp11b2-driven deletion of TWIK-related acid-sensitive potassium channels (TASK-1 and TASK-3, termed zona glomerulosa [zG]-TASK-loss-of-function), and selective pharmacological TASK channel inhibition to determine whether channel dysfunction in native, electrically excitable zG cell rosette-assemblies: (1) produces spontaneous calcium oscillatory activity and (2) is sufficient to drive substantial aldosterone autonomy. We imaged calcium activity in adrenal slices expressing a zG-specific calcium reporter (GCaMP3), an in vitro experimental approach that preserves the native rosette assembly and removes potentially confounding extra-adrenal contributions. In parallel experiments, we measured acute aldosterone production from adrenal slice cultures. Absent from untreated WT slices, we find that either adrenal-specific genetic deletion or acute pharmacological TASK channel inhibition produces spontaneous oscillatory bursting behavior and steroidogenic activity (2.4-fold) that are robust, sustained, and equivalent to activities evoked by 3 nM AngII in WT slices. Moreover, spontaneous activity in zG-TASK-loss-of-function slices and inhibitor-evoked activity in WT slices are unresponsive to AngII regulation over a wide range of concentrations (50 pM to 3 µM). We provide proof of principle that spontaneous activity of zG cells within classic rosette assemblies evoked solely by a change in an intrinsic, dominant resting-state conductance can be a significant source of AngII-independent aldosterone production from native tissue.

Aldosterone Increases Vascular Permeability in Rat Skin.

The aim of this study was to evaluate the effect of acute aldosterone (ALDO) administration on the vascular permeability of skin. ALDO was injected intradermally into rats, and vascular permeability was measured. Eplerenone (EPL), a selective mineralocorticoid receptor (MR) antagonist, was used. Skin biopsies were carried out for immunohistochemical (IHC) staining, and polymerase chain reactions were performed to analyze the expression of MR, 11β-hydroxysteroid dehydrogenase type 2, von Willebrand factor (vWF), vascular endothelial growth factor (VEGF), and zonula occludens 1. Our study showed the presence of MR in the rat skin vasculature for the first time. It was found that ALDO injection resulted in a more than 30% increase in vascular permeability and enhanced the endothelial exocytosis of vWF. The effect of ALDO diminished after EPL administration. An accumulation of vWF and a reduction in VEGF IHC staining were observed following chronic EPL administration. No effect of ALDO or EPL on the mRNA expression of the studied genes or skin structure was observed. The results suggest that ALDO increases vascular permeability in the skin via an MR-dependent mechanism. This effect of ALDO on skin microcirculation may have important therapeutic implications for diseases characterized by increased levels of ALDO and coexisting skin microangiopathy.

Acute Aldosterone Administration Increases Vascular Resistance in Patients with Type 2 Diabetes

IntroductionIndividuals with type 2 diabetes (T2D) have an increased risk of developing macrovascular dysfunctions such as arteriosclerosis and reduced blood flow, which is not improved by blood‐glucose management. Chronic elevations in plasma aldosterone levels could contribute to endothelial dysfunction. A shift in the balance between constrictor and dilator responses to aldosterone could be involved in the detrimental actions of aldosterone.HypothesisWe tested the hypothesis that T2D is associated with a shift from a vasodilator to vasoconstrictor response to aldosterone.MethodsIn 6 subjects with T2D and 11 control subjects, we measured leg hemodynamics during 5 min of incremental doses of infused aldosterone (0.2 and 5 ng/min/L leg volume) in the femoral artery. Leg blood flow (LBF; ultrasound Doppler) and mean arterial blood pressure (MAP) was measured. Femoral arterial and venous blood samples were also collected. Data was analyzed with a one‐way repeated measures ANOVA (within groups) and two‐way ANOVA (between groups).ResultsIn the subjects with T2D, LBF and leg vascular conductance (LVC) decreased during aldosterone infusion at the high dose (P<0.05), whereas there was no change in LBF and LVC in the control group. Aldosterone infusion did not change MAP and there was no difference between groups. Venous aldosterone levels increased during infusion of aldosterone in both groups (P<0.05) and no differences were observed between groups.ConclusionThese preliminary results suggest that T2D is associated with a vasoconstrictor response to physiological levels of infused aldosterone.Support or Funding InformationIndependent Research Fund Denmark and The Region of Southern DenmarkThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Potential of ACTH in the Genesis of Primary Aldosteronism

Aldosterone is a homeostatic hormone, rising in volume depletion, sodium deficiency, and potassium loading, in response to angiotensin11 and elevation of plasma potassium. Pathophysiologically, in primary aldosteronism (PA) aldosterone levels are inappropriate for the patient’s sodium and potassium status, and thus outside the normal feedback loop. ACTH is equivalent with A11 and [K+] in elevating aldosterone: its effects differ from those of the other secretagogues in four ways. First, it is not sustained; second, it raises aldosterone and cortisol secretion with equal potency; third, it is outside the normal feedback loops, reflecting the epithelial action of aldosterone; and finally its possible role in driving inappropriate aldosterone secretion (aka PA) is not widely recognized. Thirty years ago, it was shown that on a fixed sodium intake of 175 meq/day 36 of 100 unselected hypertensives, in whom PA has been excluded on contemporary criteria, had 24 h urinary aldosterone levels above the upper limit of normotensive controls. More recently, the dexamethasone enhanced fludrocortisone suppression test (FDST) showed 29% of unselected hypertensives to have plasma aldosterone concentrations above the upper limit of normotensive controls. In subjects negative for PA on the FDST, 27% were extremely hyper-responsive to ultra-low dose ACTH infusion; the remaining 73% showed minimal aldosterone elevation, as did normotensive controls: all three groups had negligible cortisol responses. On treadmill testing, no differences were found between groups in (minimally altered) ACTH and cortisol levels: hyper-responders to ultra-low ACTH, however, showed a major elevation in PAC. The implications of these studies, when validated, are substantial for PA, in that approximately half of hypertensive patients appear to show inappropriate aldosterone levels for their sodium status. The physiological role(s) of ACTH as an acute aldosterone secretagogue, and the mechanisms whereby its continuous secretion is curtailed, remain to be established.

Aldosterone Acutely Enhances Coronary Artery Contractile Responsiveness without Inducing Endothelial Dysfunction

Inappropriate activation of the renin‐angiotensin‐aldosterone system (RAAS) contributes to increased contractility of the vasculature in many disease states. Activation of vascular mineralocorticoid receptors (MR) has recently been implicated as an important regulator of vascular function. In vascular smooth muscle, genomic mechanisms of MR activation have been implicated in vascular contractility, however, little is known regarding non‐genomic MR signaling as a modulator of vascular contractility. Based on available evidence, we hypothesized that acute aldosterone incubation of murine coronary arteries ex vivo would enhance coronary contractility via non‐genomic MR activation in smooth muscle cells (SMC). Wire myography experiments were conducted on isolated coronary arteries from C57BL/6J mice. Contractile responses to KCl (10mM–45mM) were determined by repeated measures design before and following one hour aldosterone treatment (10nM or 100nM). KCl induced dose‐dependent coronary vasoconstriction that was enhanced by aldosterone incubation in a dose dependent fashion. Vasoconstriction to 20mM KCl was doubled following incubation with 10nM aldosterone and increased >3‐fold following incubation with 100nM aldosterone. Pretreatment with the selective MR antagonist eplerenone (10μM) did not significantly reduce enhanced KCl vasoconstriction in response to 100nM aldosterone. Furthermore, endothelium‐dependent coronary vasodilation to acetylcholine was unchanged following one hour aldosterone incubation regardless of dose. In conclusion, acute aldosterone incubation enhances coronary vasoconstrictor responses to KCl that, at least in response to high dose aldosterone (100nM), is not dependent on MR activation. Importantly, this effect appears to be smooth muscle‐specific and not secondary to aldosterone‐induced endothelial dysfunction as acute aldosterone incubation did not impair endothelium‐dependent coronary vasodilation. These results highlight important vascular cell‐specific effects of aldosterone that may underlie enhanced coronary contractility in disease states associated with inappropriate RAAS activation.Support or Funding InformationFunded by VA CDA‐2 IK2 BX002030 and the American Physiological Society.

Signaling pathways activated during acute aldosterone stimulation in kidney distal convoluted tubule cells

The kidney distal convoluted tubule (DCT) plays an important role in modulating body sodium balance and blood pressure. Long‐term effects of aldosterone to increase sodium reabsorption in the DCT by modulating the function of the NaCl cotransporter (NCC) and the epithelial sodium channel (ENaC) are well described. However, potential effects of aldosterone to acutely modulate NCC or ENaC function in the DCT and the signaling cascades responsible remain largely uncharacterized. Here, we used stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics to map global changes in protein phosphorylation in mouse kidney convoluted tubule cells (mpkDCT) following acute (30 min) treatment with aldosterone (1μM). A total of 8136 unique proteins, containing 14838 different phosphopeptides were identified. Of these, 148 phosphopeptides were significantly up regulated, and 96 phosphopeptides were significantly downregulated in abundance following acute aldosterone treatment. Motif analysis indicated that basophilic motifs were preferential targets for upregulated sites upon aldosterone stimulation, whereas proline‐directed motifs were prominent for downregulated sites. Analysis of motif prediction indicated that the phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K) and CAMKK dependent pathways may be involved in aldosterone‐induced signaling response by the DCT. Acute application of aldosterone did not significantly alter cAMP levels in mpkDCT cells. Ongoing analysis and pharmacological inhibition studies will help clarify and verify these systems biology findings and determine novel new acute aldosterone‐mediated signaling networks important for blood pressure regulation.

Activation of the Endogenous Renin-Angiotensin-Aldosterone System or Aldosterone Administration Increases Urinary Exosomal Sodium Channel Excretion.

Urinary exosomes secreted by multiple cell types in the kidney may participate in intercellular signaling and provide an enriched source of kidney-specific proteins for biomarker discovery. Factors that alter the exosomal protein content remain unknown. To determine whether endogenous and exogenous hormones modify urinary exosomal protein content, we analyzed samples from 14 mildly hypertensive patients in a crossover study during a high-sodium (HS, 160 mmol/d) diet and low-sodium (LS, 20 mmol/d) diet to activate the endogenous renin-angiotensin-aldosterone system. We further analyzed selected exosomal protein content in a separate cohort of healthy persons receiving intravenous aldosterone (0.7 μg/kg per hour for 10 hours) versus vehicle infusion. The LS diet increased plasma renin activity and aldosterone concentration, whereas aldosterone infusion increased only aldosterone concentration. Protein analysis of paired urine exosome samples by liquid chromatography-tandem mass spectrometry-based multidimensional protein identification technology detected 2775 unique proteins, of which 316 exhibited significantly altered abundance during LS diet. Sodium chloride cotransporter (NCC) and α- and γ-epithelial sodium channel (ENaC) subunits from the discovery set were verified using targeted multiple reaction monitoring mass spectrometry quantified with isotope-labeled peptide standards. Dietary sodium restriction or acute aldosterone infusion similarly increased urine exosomal γENaC[112-122] peptide concentrations nearly 20-fold, which correlated with plasma aldosterone concentration and urinary Na/K ratio. Urine exosomal NCC and αENaC concentrations were relatively unchanged during these interventions. We conclude that urinary exosome content is altered by renin-angiotensin-aldosterone system activation. Urinary measurement of exosomal γENaC[112-122] concentration may provide a useful biomarker of ENaC activation in future clinical studies.

PP.39.23

Objective: We showed previously that acute aldosterone (ALDO) infusion resulted in impairment of the fibrinolytic process and activation of coagulation cascade, consequently leading to augmentation of stasis-induced venous thrombosis in rats. This prothrombotic effect of ALDO was only partially mediated by the mineralocorticoid receptor (MR). Bearing in mind that ALDO potentiates the angiotensin II (Ang II) effects, in the present study we investigated the role of Ang II in the mechanism of ALDO prothrombotic action. Design and method: Male Wistar rats (300–350 g, n = 8–12) were used in this study. Aldosterone (ALDO, 30 μg/kg/h) or 0,4% ethanol (VEH; 2 ml/kg/h) was infused into the femoral vein 5 minutes before the induction of venous thrombosis and was continued for 1 h. Valsartan (VAL, 10 mg/kg), AT1 antagonist, was injected as a bolus into femoral vein 5 minutes before ALDO infusion. Venous thrombosis was performed by the vena cava ligation. The formed thrombus was removed and weighted after 24 h. Plasminogen activator inhibitor (PAI-I), tissue plasminogen activator (t-PA), thrombin activatable fibrinolysis inhibitor (TAFI) plasma levels were measured by ELISA. Results: We observed that one-hour infusion of ALDO resulted in a significant increase in venous thrombus weight (p < 0.01) and markedly elevated incidence of venous thrombosis, paralleled by significant increase in PAI-1 (p < 0.001) and TAFI (p < 0.001) as well as decrease in t-PA (p < 0.001) plasma levels in comparison to VEH. VAL administered together with ALDO reduced thrombus formation (p < 0.05) and diminished the incidences of thrombosis. We also observed that pretreatment with VAL reversed slightly the ALDO-induced decrease in t-PA level and reduced significantly TAFI (p < 0.05) and PAI-1 plasma levels (p < 0.05, vs ALDO). Conclusions: This study provides evidence that prothrombotic effect of ALDO is also mediated by the angiotensin II AT1 receptor. The mechanism of ALDO-induced AT1 activation involved impairment of the fibrinolytic process. The present study supplies our knowledge about the multiple mechanism of ALDO prothrombotic action, which is mediated via MR and AT1 receptors.

Protein kinase C and Src family kinases mediate angiotensin II-induced protein kinase D activation and acute aldosterone production

Recent evidence has shown a role for the serine/threonine protein kinase D (PKD) in the regulation of acute aldosterone secretion upon angiotensin II (AngII) stimulation. However, the mechanism by which AngII activates PKD remains unclear. In this study, using both pharmacological and molecular approaches, we demonstrate that AngII-induced PKD activation is mediated by protein kinase C (PKC) and Src family kinases in primary bovine adrenal glomerulosa cells and leads to increased aldosterone production. The pan PKC inhibitor Ro 31-8220 and the Src family kinase inhibitors PP2 and Src-1 inhibited both PKD activation and acute aldosterone production. Additionally, like the dominant-negative serine-738/742-to-alanine PKD mutant that cannot be phosphorylated by PKC, the dominant-negative tyrosine-463-to-phenylalanine PKD mutant, which is not phosphorylatable by the Src/Abl pathway, inhibited acute AngII-induced aldosterone production. Taken together, our results demonstrate that AngII activates PKD via a mechanism involving Src family kinases and PKC, to underlie increased aldosterone production.

Angiotensin II-Induced Protein Kinase D Activates the ATF/CREB Family of Transcription Factors and Promotes StAR mRNA Expression

Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

Acute aldosterone treatment enhances SPAK and NCC phosphorylation

Acute aldosterone treatment enhances SPAK and NCC phosphorylation

Study of the mechanisms of aldosterone prothrombotic effect in rats

We investigated the role of primary haemostasis, fibrinolysis, nitric oxide (NO) and oxidative stress as well as mineralocorticoid receptors (MR) in acute aldosterone prothrombotic action. Venous thrombosis was induced by stasis in Wistar rats. Aldosterone (ALDO; 10, 30, 100 µg/kg/h) was infused for 1 h. Eplerenone (EPL; 100 mg/kg, p.o.), a selective MR antagonist, was administered before ALDO infusion. Bleeding time (BT) and platelet adhesion to collagen were evaluated. The expression of nitric oxide synthase (NOS), NADPH oxidase, superoxide dismutase (SOD) and plasminogen activator inhibitor (PAI-1) was measured. NO, malonyl dialdehyde (MDA) and hydrogen peroxide (H(2)O(2)) plasma levels were assayed. Significant enhancement of venous thrombosis was observed after ALDO infusion. ALDO shortened BT and increased platelet adhesion. Marked increases were observed in PAI-1, NADPH oxidase and SOD mRNA levels. MDA and H(2)O(2) levels were augmented in ALDO-treated groups, and NOS expression and NO level were decreased. EPL reduced ALDO effects on thrombus formation, primary haemostasis, PAI-1 expression and MDA level. Short-term ALDO infusion enhances experimental venous thrombosis in the mechanism involving primary haemostasis, fibrinolysis, NO and oxidative stress-dependent pathways. The MR antagonist only partially diminished the ALDO effects, suggesting the involvement of additional mechanisms.

GPR30, Mineralocorticoid Receptors, and the Rapid Vascular Effects of Aldosterone

See related article, pp 442–451 It is now generally accepted that in common with other steroid hormones aldosterone has rapid nongenomic effects, in addition to those mediated via DNA-directed, RNA-mediated protein synthesis. The currently accepted physiology of aldosterone was primarily charted by nephrologists, who understandably focused on the epithelial (and genomic) effects of aldosterone on urinary electrolytes, and the homeostatic changes in aldosterone secretion in response to sodium deficiency or potassium loading. The most acute physiological stimulus to aldosterone secretion, however, is assumption of the upright posture. Given this, it is not inappropriate to seek responses, similarly acute, to this rapid change in plasma aldosterone levels. In the paper by Gros et al,1 the authors conclusively show that aldosterone at low picomolar concentrations can act rapidly via both GPR30 (originally an “orphan” G protein–coupled receptor, subsequently an erstwhile membrane estrogen receptor) and classic mineralocorticoid receptors (MR) over a range of parameters. These include extracellular signal-regulated kinase (ERK) 1/2 activation and myosin light chain phosphorylation in rat aortic vascular smooth muscle cells (VSMC) in vitro; for ERK activation, aldosterone has equivalent action via both receptors at low picomolar concentrations. The effects on GPR30 appear mineralocorticoid specific: in rat aortic endothelial cells, with MR expression below detection levels by Western blotting, aldosterone increased ERK activation with an EC50 < 10 pM, an action abrogated by the GPR30 antagonist G15. In contrast, the effect of estradiol on inhibition of ERK activation was unaffected by G15 but blocked by the estrogen receptor α (ERα) antagonist ICI-182780. The first point to be made is that these studies firmly establish GPR30 as a bona fide receptor for aldosterone, given the low picomolar concentrations used (except for myosin light chain phosphorylation, where inexplicably 10 nmol/L aldosterone was used, and Figures 8 and 9 point in …

Angiotensin‐II induced protein kinase D activation via src kinase and protein kinase C mediates aldosterone secretion in bovine adrenal glomerulosa cells

ObjectiveTo study the mechanism of angiotensin II (AngII) induced protein kinase D (PKD) activation in modulating aldosterone secretion in bovine adrenal glomerulosa (BAG) cells.MethodsAdenoviral vectors were used to overexpress S738/742A PKD mutant which is not phosphorylatable by protein kinase C (PKC), Y463F PKD mutant, and Src wildtype as well as a dominant negative K296R/Y528F Src mutant. Supernatants were stored for aldosterone assay, and the cells processed for immunoblot analysis. Samples were immunoprecipitated with either PKD, or anti‐phosphotyrosine antibodies and reciprocally blotted. Blots were quantified and statistically significant differences were determined when p&lt;0.05.ResultsOverexpression of non‐phosphorylatable S738/742A and the Y463F PKD mutants downregulated PKD activation and decreased acute (30‐minute) aldosterone secretion. The dominant negative K296R/Y528F Src mutant, which blocks Src activation, attenuated PKD phosphorylation and aldosterone secretion. AngII induced an increase in tyrosine phosphorylation of PKD presumably at the tyrosine 463 residue.ConclusionAngiotensin II (AngII) activates protein kinase D in BAG cells via a mechanism involving Src kinase and PKC, to underlie increased aldosterone production.Funding SupportNational Institutes of Health/National Heart, Lung and Blood InstituteAmerican Heart Association

Rapid Aldosterone Signaling and Vascular Reactivity: Relax or Don't Do It

Rapid Aldosterone Signaling and Vascular Reactivity: Relax or Don't Do It

Effects of CP-532,623 and Torcetrapib, Cholesteryl Ester Transfer Protein Inhibitors, on Arterial Blood Pressure

ILLUMINATE, the phase 3 morbidity and mortality trial of the cholesteryl ester transfer protein (CETP) inhibitor, torcetrapib, plus atorvastatin terminated in 2006. The underlying morbidity and mortality cause remains undetermined. In addition to lipoprotein changes, off-target increases in blood pressure (BP), sodium, bicarbonate, and aldosterone and potassium decreases were described. We report nonclinical and clinical studies using torcetrapib and a closely related CETP inhibitor, CP-532,623, to further characterize this pharmacology. Pressor effects of torcetrapib and CP-532,623 were observed in monkeys and human subjects. CETP inhibition and high-density lipoprotein cholesterol elevation were demonstrated. In humans, high- versus low-dose CP-532,623 produced significantly greater pressor effects despite similar maximal CETP inhibition. Inhibition of CETP was seen 48 hours post dose, whereas BP elevation dissipated by 24 hours, temporally dissociating CETP inhibition from BP changes. These data, and structural similarities between the compounds, support the conclusion that the BP effects are related to chemotype. We also observed an acute aldosterone increase without changes in renin in monkeys. Continuous BP measurements showed persistent elevations, whereas aldosterone changes were transient, suggesting that increases in BP were not directly the result of renin-angiotensin-aldosterone system activation and may, in part, be due to direct effects on blood vessels or other nongenomic effects.

C-Fos Mediates Angiotensin II-Induced Aldosterone Production and Protein Synthesis in Bovine Adrenal Glomerulosa Cells

Angiotensin II (AngII), potassium ion, and ACTH are the main factors controlling aldosterone biosynthesis in adrenal glomerulosa cells. AP-1 response elements for the immediate early gene products, c-Fos and c-Jun, have been identified, among others, in the promoter of the steroidogenic acute regulatory (StAR) protein gene, whose expression is acutely regulated by activators of aldosterone production. In bovine glomerulosa cells, AngII treatment led to a rapid and transient increase in c-fos mRNA expression, c-Fos protein expression, and c-Fos phosphorylation. Inhibition of the ERK1/2 MAPK pathway abolished the effect of AngII on c-fos mRNA, protein, and phosphorylation. EMSA and chromatin immunoprecipitation experiments demonstrated that c-Fos binds with c-Jun to the proximal StAR promoter and that AngII treatment increases the amount of c-Fos bound to the promoter. Overexpression of a dominant-negative form of c-Fos with adenoviral vectors inhibited StAR mRNA and StAR protein expression as well as aldosterone biosynthesis in response to AngII. The dominant-negative c-Fos also prevented the increase in protein synthesis induced by AngII in glomerulosa cells, as assessed by [(3)H]leucine incorporation. These results indicate that AngII rapidly induces c-Fos expression and posttranslational modifications. Furthermore, a heterodimeric c-Fos/c-Jun complex binds to the proximal StAR promoter in glomerulosa cells, thus activating StAR gene expression and acute aldosterone biosynthesis. Finally, c-Fos also contributes to other functional responses to the hormone, such as protein synthesis.

Acute aldosterone infusion enhances thrombosis development in normotensive rats

Acute aldosterone infusion enhances thrombosis development in normotensive rats

Rapid effects of aldosterone on clonal human vascular smooth muscle cells

It has been increasingly appreciated that aldosterone elicits acute vascular effects through nongenomic signaling pathways. Our previous studies demonstrated that aldosterone attenuated phenylephrine-mediated constriction in intact vessels [via phosphatidylinositol 3-kinase-dependent nitric oxide synthase activation] but enhanced vasoconstrictor responses in endothelium-denuded arteries. To determine the mechanism of this vasoconstrictor response, we assessed the effect of aldosterone on myosin light-chain phosphorylation and contraction in clonal adult human vascular smooth muscle cells. Acute aldosterone exposure mediated dose-dependent myosin light-chain phosphorylation, inhibited by spironolactone and phosphatidylinositol 3-kinase inhibition. These rapid effects of aldosterone were mimicked by estradiol and hydrocortisone and were also inhibitable by both spironolactone and eplerenone. In parallel to its effects on myosin light-chain phosphorylation, aldosterone mediated dose-dependent contraction responses that were inhibited by spironolactone. Comparable contractile responses were seen with both 17beta-estradiol and hydrocortisone. In total, these data are consistent with a mechanism of acute aldosterone-mediated contraction common to both glucocorticoids and estrogen. Steroid-mediated vasoconstriction may represent an important pathobiological mechanism of vascular disease, especially in the setting of preexisting endothelial dysfunction.