Basal Aldosterone Secretion Research Articles

PS-BPR02-7: SACUBITRIL VALSARTAN FOR REFRACTORY HYPERTENSION CAUSED BY PRIMARY ALDOSTERONISM: A CASE SERIES

Introduction: Primary aldosteronism (PA) is the disease characterized by inappropriately elevated autonomous adrenal aldosterone secretion, leading to the development of refractory hypertension. Patients with PA show higher nighttime blood pressure (BP) and lower nocturnal BP decline than those with essential hypertension. Angiotensin receptor neprilysin inhibitors (ARNIs) are a new class of cardiovascular agents characterized by their dual action on the renin angiotensin system and the natriuretic peptide system. Atrial natriuretic peptide has been shown to inhibit basal aldosterone secretion from adrenal cells in vitro. Case Presentation: A man in his 50 s was referred to our hospital due to vision loss and diagnosed with hypertensive retinopathy. His serum potassium level was below 3.5 mEq/L. A captopril test confirmed the diagnosis of PA, though a CT image revealed no adrenal adenoma. According to his discretion, medical treatment including a mineralocorticoid receptor antagonist, a calcium channel blocker, an angiotensin receptor blocker (ARB), an angiotensin converting enzyme inhibitor (ACEI), an alpha blocker and a beta blocker, were commenced. However, the result of ambulatory bloop pressure monitoring (ABPM) revealed his mean BP of 143/91 mmHg and his mean nighttime BP of 148/89 mmHg, indicating riser pattern. Switching from an ACEI, imidapril (10 mg per day), and an ARB, azilsartan (40 mg per day), to sacubitril/valsartan (200 mg per day) decreased his mean BP to 138/89 mmHg. Furthermore, his mean nighttime BP decreased to 122/79 mmHg, indicating that his nocturnal hypertension was improved by sacubitril/valsartan. Similar favorable effects of Sacubitril/valsartan on BP were found in three other patients with refractory hypertension caused by PA. Discussion: ARNIs are an appropriate treatment option for refractory hypertension caused by PA.

The Endocrine‐Disrupting Chemical Di‐(2‐ethylhexyl) phthalate (DEHP) Increases Corticosteroid Secretion in Human Adrenocortical Cells

Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine-disrupting chemical that is widely used as a plasticizer in the production of polyvinyl chloride containing plastics. DEHP is a component of many common household items (toys, packaging, furniture, etc.) and medical devices, resulting in significant and chronic human exposure. Human exposure to DEHP has been linked to androgen disruption and developmental toxicity, including cryptorchidism, decreased anogenital distance, and decreased testosterone levels. Other adrenal corticosteroids are also susceptible to the endocrine-disrupting effects of DEHP. One study has demonstrated that fetal exposure to DEHP results in altered adrenal aldosterone production in adult rats. Due to these potential effects of DEHP on adrenal cortex steroidogenesis, the focus of our studies is to characterize the effect of DEHP on aldosterone secretion in a human adrenocortical cell line. HAC15 cells were exposed to vehicle or various concentrations of DEHP (10 nM-30 μM). After 72 h, cell viability, aldosterone secretion, and gene expression of enzymes and cofactors involved in aldosterone synthesis was examined. No effects on cell viability were observed with the concentrations studied. Basal aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 75.6±3.5; 10 μM DEHP, 7735.2±151.3; n=8). Ang II-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 4278.9±110.2; 10 μM DEHP, 18651.7±572.2; n=8). ACTH-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 124.0±3.7; 10 μM DEHP, 23455.8±272.7; n=8). Gene expression of most enzymes and cofactors involved in aldosterone synthesis were increased by 10 μM DEHP. Aldosterone synthase (CYP11B2) had the greatest induction at 175.2±2.0 fold. These data support the evidence that DEHP disrupts the regulation of aldosterone secretion and provides further evidence that DEHP is an endocrine disruptor.

L- and T-type calcium channels control aldosterone production from human adrenals.

Aldosterone, which plays a key role in the regulation of blood pressure, is produced by zona glomerulosa (ZG) cells of the adrenal cortex. Exaggerated overproduction of aldosterone from ZG cells causes primary hyperaldosteronism. In ZG cells, calcium entry through voltage-gated calcium channels plays a central role in the regulation of aldosterone secretion. Previous studies in animal adrenals and human adrenal adrenocortical cell lines suggest that the T-type but not the L-type calcium channel activity drives aldosterone production. However, recent clinical studies show that somatic mutations in L-type calcium channels are the second most prevalent cause of aldosterone-producing adenoma. Our objective was to define the roles of T and L-type calcium channels in regulating aldosterone secretion from human adrenals. We find that human adrenal ZG cells mainly express T-type CaV3.2/3.3 and L-type CaV1.2/1.3 calcium channels. TTA-P2, a specific inhibitor of T-type calcium channel subtypes, reduced basal aldosterone secretion from acutely prepared slices of human adrenals. Surprisingly, nifedipine, the prototypic inhibitor of L-type calcium channels, also decreased basal aldosterone secretion, suggesting that L-type calcium channels are active under basal conditions. In addition, TTA-P2 or nifedipine also inhibited aldosterone secretion stimulated by angiotensin II- or elevations in extracellular K+. Remarkably, blockade of either L- or T-type calcium channels inhibits basal and stimulated aldosterone production to a similar extent. Low concentrations of TTA-P2 and nifedipine showed additive inhibitory effect on aldosterone secretion. We conclude that T- and L-type calcium channels play equally important roles in controlling aldosterone production from human adrenals.

MON-035 Aldosterone Secretion in a Human Adrenal Cell Line Is Enhanced by the Endocrine-Disrupting Chemical Di-(2-Ethylhexyl) Phthalate (DEHP)

Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine-disrupting chemical that is widely used as a plasticizer in the production of polyvinyl chloride containing plastics. DEHP is a component of many common household items (toys, packaging, furniture, etc.) and medical devices, resulting in significant and chronic human exposure. Human exposure to DEHP has been linked to androgen disruption and developmental toxicity, including cryptorchidism, decreased anogenital distance, and decreased testosterone levels. Other adrenal corticosteroids are also susceptible to the endocrine-disrupting effects of DEHP. One study has demonstrated that fetal exposure to DEHP results in altered adrenal aldosterone production in adult rats. Due to these potential effects of DEHP on adrenal cortex steroidogenesis, the focus of our studies is to characterize the effect of DEHP on aldosterone secretion in a human adrenocortical cell line. HAC15 cells were exposed to vehicle or various concentrations of DEHP (10 nM-30 μM). After 72 h, cell viability, aldosterone secretion, and gene expression of enzymes and cofactors involved in aldosterone synthesis was examined. No effects on cell viability were observed with the concentrations studied. Basal aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 75.6±3.5; 10 μM DEHP, 7735.2±151.3; n=8). Ang II-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 4278.9±110.2; 10 μM DEHP, 18651.7±572.2; n=8). ACTH-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 124.0±3.7; 10 μM DEHP, 23455.8±272.7; n=8). Gene expression of most enzymes and cofactors involved in aldosterone synthesis were increased by 10 μM DEHP. Aldosterone synthase (CYP11B2) had the greatest induction at 175.2±2.0 fold. These data support the evidence that DEHP disrupts the regulation of aldosterone secretion and provides further evidence that DEHP is an endocrine disruptor.

Nicotinamide phosphoribosyltransferase and the hypothalamic-pituitary-adrenal axis of the rat.

Nicotinamide phosphoribosyltransferase (Nampt), also termed visfatin, catalyses the rate‑limiting step in the nicotinamide adenine dinucleotide (NAD) salvage pathway. In addition to its intracellular function (iNampt), extracellular Nampt (eNampt) also affects numerous intracellular signalling pathways. The current study investigated the role of Nampt in the regulation of the hypothalamic‑pituitary‑adrenal (HPA) axis in rats. At 1h after intraperitoneal administration of eNampt (4µg/100g) in adult male rats, serum adrenocorticotropic hormone(ACTH) and aldosterone levels remained unchanged, while corticosterone levels were notably elevated compared with the control group, as determined by ELISA. The results of reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) demonstrated that, in the hypothalami of eNampt‑treated rats, the mRNA expression levels of Fos proto‑oncogene, which is also termed c‑Fos, were not significantly different compared with the control group; however, the mRNA expression levels of proopiomelanocortin (POMC) were markedly increased in the pituitary gland of eNampt‑treated rats compared with the control group. Furthermore, in hypothalamic explants, ELISA results demonstrated that the addition of the eNampt protein exhibited no effect on corticotropin‑releasing hormone (CRH) release into the incubation medium and prevented potassium ion‑induced CRH release. Additionally, the eNampt‑induced increase in ACTH output by pituitary gland explants was not statistically significant, compared with the control group. However, RT‑qPCR indicated that exposure of pituitary gland explants to eNampt and CRH increased the levels of POMC mRNA expression; the effect of eNampt, but not CRH, was inhibited by FK866, which is a specific Nampt inhibitor. In primary rat adrenocortical cell cultures, eNampt exhibited no effect on basal aldosterone or corticosterone secretion, while increases in aldosterone and corticosterone levels in response to ACTH were retained. To assess the potential role of iNampt in the regulation of adrenal steroidogenesis, experiments involving a specific Nampt inhibitor, FK866, were performed. Exposure of cultured cells to FK866 notably lowered basal aldosterone and corticosterone output compared with the control group, and completely eliminated the response of cultured cells to ACTH. The results of the present study indicated that the injected eNampt may have increased the corticosterone serum levels by acting at the pituitary level. In addition, iNampt may exert a tonic stimulating effect on the secretion of aldosterone and corticosterone from rat adrenocortical cells, as normal iNampt levels were required to retain the response of cultured rat adrenocortical cells to ACTH. Thus, these data suggest an important physiological role of both iNampt and eNampt in the regulation of the HPA axis activity in the rat.

Abstract 054: Visinin Like Protein 1 Regulation of Aldosterone Biosynthesis

Visinin-Like Protein-1(Vsnl1) is a member of the EF-hand calcium sensor proteins. Its mRNA was found to be upregulated in aldosterone-producing adenomas and to be expressed in the zona glomerulosa (ZG) of the rat and human adrenal. We studied the expression pattern of Vsnl1 and its co-localization with the CYP11B2 enzyme and ZG marker Dlk1 (ZOG) using double and triple immunofluorescence in adrenal glands of rats on normal, high and low sodium diets and the effect of Vsnl1 on calcium mobilization and aldosterone biosynthesis in the human adrenocarcinoma cell line, the HAC15 cell. In rat adrenals, Vsnl1 was expressed only in ZG, in the same cells as Dlk1, in both functional and undifferentiated ZG cells. CYP11B2 co-localized with Vsnl1 in nearly all ZG cells of rats on a low sodium diet. Co-expression of CYP11B2 with Vsnl1 in adrenals of rats on normal and high sodium diets was significantly less. HAC15 were transduced with a lentivirus carrying Vsnl1 or an empty virus. Three days later cells were incubated with vehicle, A-II 10 nM, Forskolin 10μM, or potassium 16 mM. Aldosterone and cortisol were measured after 24 hr. Basal secretion of aldosterone was not increased; cortisol was. Aldosterone and cortisol secretion were greater after A-II, forskolin or potassium stimulation in cells overexpressing Vsnl1. Intracellular calcium measured by Fluo-4 AM dye and proliferation were increased by Vsnl1 overexpression. HAC15 cells were transduced with 2 different shRNA for Vsnl1. Aldosterone synthesis was reduced in HAC15 cells transduced with the shRNA for Vsnl1 stimulated with A-II. Vsnl1 has a myristoylation consensus sequence at the N-terminal domain. Site directed mutagenesis of Vsnl1 at the 2 position (G2A) resulted in a significant decrease in the aldosterone response to A-II. In summary, the calcium sensor protein Vsnl1 is ZG specific and detected in both steroidogenically active and inactive cells, as well as undifferentiated cells in the subcapsular area of the rat adrenal. Vsnl1 expression and activity in HAC15 cell correlate positively with calcium mobilization, proliferation and aldosterone synthesis.

NEFM (Neurofilament Medium) Polypeptide, a Marker for Zona Glomerulosa Cells in Human Adrenal, Inhibits D1R (Dopamine D1 Receptor)-Mediated Secretion of Aldosterone.

Heterogeneity among aldosterone-producing adenomas (APAs) has been highlighted by the discovery of somatic mutations. KCNJ5 mutations predominate in large zona fasciculata (ZF)-like APAs; mutations in CACNA1D, ATP1A1, ATP2B3, and CTNNB1 are more likely to be found in small zona glomerulosa (ZG)-like APAs. Microarray comparison of KCNJ5 mutant versus wild-type APAs revealed significant differences in transcriptomes. NEFM, encoding a neurofilament subunit which is a D1R (dopamine D1 receptor)-interacting protein, was 4-fold upregulated in ZG-like versus ZF-like APAs and 14-fold more highly expressed in normal ZG versus ZF. Immunohistochemistry confirmed selective expression of NEFM (neurofilament medium) polypeptide in ZG and in ZG-like APAs. Silencing NEFM in adrenocortical H295R cells increased basal aldosterone secretion and cell proliferation; silencing also amplified aldosterone stimulation by the D1R agonist, fenoldopam, and inhibition by the D1R antagonist, SCH23390. NEFM coimmunoprecipitated with D1R, and its expression was stimulated by fenoldopam. Immunohistochemistry for D1R was mainly intracellular in ZG-like APAs but membranous in ZF-like APAs. Aldosterone secretion in response to fenoldopam in primary cells from ZF-like APAs was higher than in cells from ZG-like APAs. Transfection of mutant KCNJ5 caused a large reduction in NEFM expression in H295R cells. We conclude that NEFM is a negative regulator of aldosterone production and cell proliferation, in part by facilitating D1R internalization from the plasma membrane. Downregulation of NEFM in ZF-like APAs may contribute to a D1R/D2R imbalance underlying variable pharmacological responses to dopaminergic drugs among patients with APAs. Finally, taken together, our data point to the possibility that ZF-like APAs are in fact ZG in origin.

Differential effects of down-regulated steroidogenic factor-1 on basal and angiotensin II-induced aldosterone secretion.

Aldosterone synthase (CYP11B2) is responsible for the final step in aldosterone synthesis and is importantly regulated by angiotensin-II (Ang II) through diverse pathways. However, under pathological conditions, such as in hyperaldosteronism, the regulation becomes disordered. The transcription factor steroidogenic factor-1 (SF-1) is important in regulating the endocrine system and is overexpressed in aldosterone-producing adenoma (APA), a common cause of hyperaldosteronism. Overexpression of SF-1 has been extensively studied, but little in-depth information is available regarding the effects of inhibitory SF-1 on CYP11B2 and Ang II. In this paper, we have investigated the roles of down-regulated SF-1 in basal and Ang II-induced CYP11B2 expression using SF-1-specific short hairpin RNA. Inhibitory SF-1 was found to decrease the sensitivity of CYP11B2 and aldosterone to Ang II stimulation, whereas a down-regulation of SF-1 enhanced basal CYP11B2 expression and aldosterone production in H295R cells. Considering these differential effects of SF-1 on aldosterone production, these results might provide a new insight into the understanding of hyperaldosteronism.

Expression of the spexin gene in the rat adrenal gland and evidences suggesting that spexin inhibits adrenocortical cell proliferation

Spexin (SPX, also called NPQ) is a recently identified, highly conserved peptide which is processed and secreted. We analysed the SPX gene and its protein product in the rat adrenal gland to ascertain whether SPX is involved in the regulation of corticosteroid secretion of and growth of adrenocortical cells. In adult rat adrenal glands the highest levels of SPX mRNA were present in the glomerulosa (ZG) and fasciculate/reticularis (ZF/R) zones. High SPX gene expression levels were found in freshly isolated adult rat ZG and ZF/R cells. In cultured adrenocortical cells the levels of SPX mRNA were lower than in freshly isolated cells. SPX mRNA expression levels were found to be 2–3 times higher during days 90–540 of postnatal development than found during days 2–45. Prolonged ACTH administration lowered and dexamethasone increased adrenal SPX mRNA levels in vivo. Adrenal enucleation produced a significant linear increase in SPX mRNA levels, with the highest value occurring at day 8 after surgery, with control values taken on day 30 after enucleation. Immunohistochemistry revealed SPX-like immunoreactivity in the entire cortex of the adult male rat and in enucleation-induced regenerating cortex. A concentration of 10–6 M SPX peptide stimulated basal aldosterone secretion by freshly isolated ZG. In prolonged exposure of adrenocortical cell primary cultures to SPX (10–6 M) resulted in a small increase in corticosterone secretion and a notable decrease in BrdU incorporation. The results suggest the direct involvement of SPX in the regulation of adrenocortical cell proliferation; however, the mechanism of action remains unknown.

Influence of trifluoperazine on ACTH- or angiotensin-stimulated mineralocorticoid and glucocorticoid secretion in man.

During stimulation of adrenocortical secretion the calcium--calmodulin system is activated to a different extent, depending on the secretagogue substance. In the submitted paper the influence of therapeutic doses of the calmodulin inhibitor, trifluoperazine, on aldosterone and cortisol secretion stimulated by ACTH or by activation of endogenous angiotensin by furosemide was investigated in healthy subjects. Trifluoperazine already in amounts of 6 mg/day administered for one week inhibited the "basal" aldosterone secretion assessed in a vertical position (p less than 0.01) and ACTH stimulated secretion (during the 30th minute p less than 0.05). The basal aldosterone secretion assessed in a horizontal position was not affected by trifluoperazine, similarly as it did not affect the secretory response to endogenous angiotensin activated by furosemide, regardless whether a dose of 6 mg or 12 mg/day was used. ACTH stimulated cortisol blood levels were after trifluoperazine insignificantly but constantly lower throughout the test, while they were not altered by trifluoperazine in the furosemide test. The plasma calcium level was not significantly affected by trifluoperazine. It may be concluded that trifluoperazine alters ACTH stimulated mineralocorticoid secretion, while it does not influence angiotensin stimulated secretion. The revealed differences in adrenocortical response to trifluoperazine in vivo cannot be explained merely by a different sensitivity of the calcium-calmodulin system to stimulation by two different secretagogues, but by interaction of some regulatory mechanisms influenced by trifluoperazine with adrenocortical secretion.

Precerebellin-related genes and precerebellin 1 peptide in the adrenal gland of the rat: Expression pattern, localization, developmental regulation and effects on corticosteroidogenesis

Precerebellin (Cbln)-related peptides are known to modulate the secretory activity and growth of the adrenal gland. However, precise expression of the Cbln-related genes and Cbln1 peptide in the adrenal remains unclear. Therefore, we investigated, using RT-PCR, QPCR, Western blotting, immunohistochemistry and hormonal assays, their expression in the adrenals of adult rats and in the course of postnatal ontogenesis. Of the 4 known Cblns, Cbln(1-3) mRNAs were found in the adrenal gland of the adult male rats. Expression patterns of Cbln1 and 3 were similar to each other and different from that of Cbln2. Highest expression of the Cbln1 and 3 genes was observed in the zona glomerulosa (ZG), lower expression was noted in the fasciculata/reticularis and lowest expression was observed in the adrenal medulla. Expression of these genes was also present in freshly isolated rat adrenocortical cells. On the contrary, by means of classic RT-PCR, we demonstrated the presence of mRNAs of CBLN(1-4) in the human adrenal gland. In the rat, highest expression of the Cbln1 and 3 genes was found at postnatal day 2 and was somewhat lower at day 90. On the contrary, expression of the Cbln2 gene was low in adrenals of 2-day-old rats and notably higher at the remaining time points studied (up to day 360). Cerebellin (CER)-like immunoreactivity was observed in the membranes of the adrenal ZG cells, while in the medulla, immunoreactive substances were localized primarily in the cytoplasm of chromaffin cells. Cbln1-like immunoreactivity was present mainly in the cortex of the gland, and reaction products were noted both in the membranes and cytoplasm of adrenocortical cells. Semiquantitative evaluation of Cbln1 protein expression in compartments of the adrenal gland of the adult rat revealed a higher concentration of Cbln1 protein in the cortex than in the medulla of studied rats. We also found that both CER and desCER stimulated basal aldosterone secretion by freshly isolated ZG cells. Thus, the present study provided precise data on expression of the Cbln-related genes and Cbln1 peptide in the adrenal gland of the rat and on their developmental regulation. We also found that, contrary to the human adrenal, in the rat adrenal gland, the Cbln4 gene was not expressed at the mRNA level.

Both TASK-3 and TREK-1 two-pore loop K channels are expressed in H295R cells and modulate their membrane potential and aldosterone secretion

The rate of aldosterone synthesis by adrenal glomerulosa cells relies on the selective permeability of the glomerulosa cell to K(+) ions. In rodent and bovine adrenal glomerulosa cells, this background potassium current is provided by a two-pore loop potassium (K2P) channel: largely TASK-3 in the rat and TREK-1 in the cow. The nature of the K2P channel in the human adrenal cortex is not known, and we have addressed this issue here using the H295R human adrenal cell line. We show that these cells express mRNA and protein for both TASK-3 and TREK-1 K2P channels. Using a potentiometric dye (FMP), we also show that TASK-3 and TREK-1 channel modulators can affect the membrane potential of H295R cells. Transfecting H295R cells with TASK-3 or TREK-1 dominant-negative mutants (TASK-3 G95E or TREK-1 G144E) produced depolarization of H295R cells and altered K-stimulated aldosterone secretion. Finally, transfection of a constitutively active mutant of Galpha(q) into H295R cells (GTPase-deficient Galpha(q)-QL) depolarized them and increased basal aldosterone secretion. Taken together, our data support both TASK-3 and TREK-1 as being functionally operational in the H295R cell line. This suggests that human adrenal glomerulosa cells may utilize both of these K2P channels for their background potassium current.

Effects of neuropeptides B and W on the rat pituitary-adrenocortical axis: In vivo and in vitro studies

Neuropeptides (NP) B and W are hypothalamic peptides involved in the regulation of feeding and neuro-endocrine axes. Evidence has been provided that NPB and NPW act on both the central and the peripheral branches of the rat hypothalamic-pituitary-adrenocortical axis, and we carried out in vivo and in vitro studies to gain insight into this topic. Reverse transcription-polymerase chain reaction showed the expression of NPB, NPW and their receptors in both adrenal cortex (zonae glomerulosa and fasciculata-reticularis) and adrenal medulla, where immunocytochemistry also detected the presence of abundant NPB- and NPW-immunoreactivity. The acute subcutaneous administration of NPB (0.5 or 1.5 nmol/100 g) did not alter ACTH plasma concentration, while that of NPW (1.5 nmol/100 g) decreased it. Neither NPB nor NPW affected the blood level of aldosterone, while both peptides (0.5 nmol/100 g) raised that of corticosterone. NPB (10(-6) M) lowered ACTH-stimulated aldosterone secretion, and basal and ACTH-stimulated corticosterone production from adrenal quarters containing both cortical and medullary tissues. NPW (10(-6) M) enhanced basal aldosterone secretion from adrenal quarters, and the effect was suppressed by the beta-adrenoceptor antagonist l-alprenolol (10(-5) M). NPW did not affect corticosterone production. Collectively, our findings allow us to draw the following tentative conclusions: i) ACTH-independent extra-adrenal mechanism(s) are operative in vivo, by which NPB and NPW stimulate adrenal glucocorticoid, but not mineralocorticoid secretion; ii) in vitro the interaction of NPB with adrenal medulla activates unknown mechanism(s) hampering adrenocortical steroidogenic machinery; and iii) NPW stimulates in vitro aldosterone secretion by enhancing the release of medullary catecholamines, which in turn activate beta-adrenoceptors located on zona glomerulosa cells.

Evidence for a Role of Vasopressin in the Control of Aldosterone Secretion in Primary Aldosteronism:in Vitroandin VivoStudies

Arginine vasopressin (AVP) stimulates steroid secretion from the normal human adrenal gland and some cortisol-producing adrenocortical tumors or hyperplasia through activation of the V(1a) receptor. The objective of the study was to investigate in vitro and in vivo the possible involvement of AVP in the physiopathology of primary aldosteronism. The design of the study included immunohistochemical, pharmacological, and molecular studies on aldosterone-producing adenoma (APA), followed by a monocentric, crossover trial of the orally active V(1a) receptor antagonist, SR 49059, in a double blind, randomized, and placebo-controlled fashion. The study was conducted at a university hospital and research laboratory. The study population included eight untreated patients with primary aldosteronism, four with APA and four with idiopathic hyperaldosteronism. Aldosterone secretion of APA cells in vitro and plasma aldosterone, renin, and ACTH were measured. SR 49059 (200 mg once daily) or placebo was administered during two 1-wk treatment periods separated by a 2-wk washout. We observed the occurrence of AVP-containing cells in APA tissues. Administration of AVP to perifused APA cells induced an increase in aldosterone production, which was inhibited by a specific V(1a) antagonist. RT-PCR analysis showed the expression of V(1a) receptor mRNA in most APAs studied. In APA patients, SR 49059 did not induce any effect on basal aldosterone secretion but provoked a plasma aldosterone response to orthostatism (P < 0.03) and strengthened the positive correlation between plasma aldosterone and ACTH. The present study indicates that functional V(1a) receptors are present in APA and suggests that AVP may exert an autocrine/paracrine control of aldosterone secretion in APA tissues.

Effects of neuropeptides B and W on the secretion and growth of rat adrenocortical cells

Neuropeptide-B (NPB) and neuropeptide-W (NPW) are recently discovered endogenous ligands of the GPR7- and GPR8-receptors (R), which in humans are expressed in the hypothalamus and probably involved in the regulation of energy homeostasis and neuroendocrine axes. GPR8-Rs are absent in rodents, where the GPR8-like-R has been described. Reverse transcription-polymerase chain reaction detected the expression of NPB, NPW, GPR7-R and GPR8-like-R mRNAs in rat adrenocortical cells (both freshly-dispersed and 4-day-cultured cells). NPB did not acutely (60-min exposure) alter basal aldosterone secretion from freshly dispersed zona glomerulosa cells, while NPW raised it. Both NPB and NPW enhanced ACTH-stimulated aldosterone secretion and did not affect either basal or ACTH-stimulated corticosterone production by dispersed zona fasciculata/reticularis (ZF/R) cells. The prolonged (4-day) exposure to NPW, but not NPB, raised corticosterone secretion from cultured ZF/R cells, and both neuropeptides increased the proliferation rate of cultured cells. Taken together, our findings indicate that NPB and NPW affect rat adrenocortical function, so they may be included in that large family of peptides involved in the autocrine-paracrine stimulation of secretion and growth of adrenal cortex.

Cholecystokinin (CCK) stimulates aldosterone secretion from human adrenocortical cells via CCK2 receptors coupled to the adenylate cyclase/protein kinase A signaling cascade.

Cholecystokinin (CCK) IS a regulatory peptide that acts via two receptor subtypes, CCK1-R and CCK2-R. RT-PCR demonstrated the expression of both CCK1-R and CCK2-R in the zona glomerulosa (ZG), but not zona fasciculata-reticularis cells of the human adrenal cortex. CCK and the CCK2-R agonist pentagastrin enhanced basal aldosterone secretion from ZG cells without affecting cortisol production from zona fasciculata-reticularis cells. The aldosterone response to CCK and pentagastrin was suppressed by a CCK2-R antagonist, but not by a CCK1-R antagonist. Pentagastrin evoked a sizeable cAMP, but not inositol triphosphate, response from ZG cells, whereas CCK plus CCK2-R antagonist was ineffective. The cAMP response to pentagastrin was abrogated by CCK2-R antagonist or the adenylate cyclase inhibitor SQ-22536, and the aldosterone response was abolished by both SQ-22536 and the protein kinase A inhibitor H-89. Both CCK and pentagastrin increased steroidogenic acute regulatory protein mRNA expression in ZG cells; the effect was abrogated by CCK2-R antagonist. We conclude that CCK exerts secretagogue action on human ZG cells, acting through CCK2-Rs coupled to the adenylate cyclase/protein kinase A signaling cascade, which, in turn, stimulates the expression of steroidogenic acute regulatory protein, the rate-limiting step of steroidogenesis.

Expression of the beacon gene in the rat adrenal gland: Direct inhibitory effect of beacon[47-73] on aldosterone secretion from dispersed adrenal zona glomerulosa cells

Beacon gene was recently identified in the rat hypothalamus, and there is evidence that beacon may be involved in the functional regulation of neuroendocrine axes. Reverse transcription-polymerase chain reaction and immunocytochemistry showed the expression of beacon mRNA and protein in the rat adrenal gland, especially in the cortex. Beacon[47-73], at a concentration over 10(-7) M decreased basal aldosterone secretion from dispersed rat zona glomerulosa (ZG) cells, without affecting the ACTH-stimulated one. Basal and agonist-stimulated corticosterone secretion from dispersed zona fasciculata-reticularis cells and catecholamine release from adrenomedullary slices were unaffected by beacon[47-73]. The suppressive effect of beacon[47-73] on aldosterone secretion from ZG cells was abolished by either H-89 or calphostin-C, which are inhibitors of protein kinase A and C signaling cascades. Taken together, these findings allow us to suggest that beacon can be included in the group of regulatory peptides involved in the fine tuning of ZG secretory activity.

Effects of preproglucagon-derived peptides and exendins on steroid-hormone secretion from dispersed adrenocortical cells of normal and streptozotocin-induced diabetic rats

Many lines of evidence have shown that preproglucagon-derived peptides affect steroid secretion from dispersed adrenocortical cells, and that streptozotocin (STZ)-induced experimental diabetes alters adrenocortical-cell function. Hence, we compared the effects of glucagon, glucagon-like peptide (GLP)-1 and GLP-2 on basal and ACTH-stimulated secretion of dispersed adrenocortical cells from normal and STZ-induced diabetic rats. We also examined the effects of exendins (EX) 3 and 4, because EX4 is known to be a potent and long-lasting agonist of GLP-1 receptors. STZ-induced diabetes moderately enhances basal and ACTH-stimulated secretion from dispersed zona glomerulosa (ZG) cells, without significantly affecting corticosterone production from dispersed zona fasciculata-reticularis (ZF/R) cells. In normoglycemic rats, glucagon increased basal aldosterone and corticosterone secretion from ZG and ZF/R cells, GLP-2 raised both basal and ACTH-stimulated aldosterone secretion and ACTH-stimulated corticosterone output, and EX4 increased basal corticosterone secretion. In contrast, glucagon, GLP-2 and EX4 did not elicit secretory responses from adrenocortical cells of diabetic rats. GLP-1 and EX3 did not alter secretion of dispersed adrenocortical cells of either normal or STZ-treated rats. Taken together, our findings indicate that preproglucagon-derived peptides enhance steroid secretion from adrenocortical cells of normal, but not STZ-induced diabetic rats. It is suggested that the prolonged exposure to low concentrations of insulin causes unresponsiveness of adrenocortical cells to glucagon, GLP-2 and EX4, which may contribute to the hyporeninemic hypoaldosteronism and alterations in glucocorticoid metabolism occurring in experimental diabetes.

Effects of adrenomedullin and its fragment 22-52 on basal and ACTH-stimulated secretion of cultured rat adrenocortical cells

Adrenomedullin (ADM) and its receptors are expressed in the adrenal cortex, where ADM is currently known to inhibit agonist-stimulated aldosterone secretion from zona glomerulosa (ZG), without affecting either basal aldosterone release or the secretory activity of zona fasciculata-reticularis (ZF/R) cells. These functional findings have been obtained using freshly dispersed adrenocortical cells, but evidence has been provided that ADM is able to enhance basal aldosterone secretion from rat capsule-ZG preparations. Hence, we investigated the effect of ADM and ADM22-52, a putative antagonist of ADM receptors, on the secretory activity of rat adrenal cell cultured in vitro for 72 h. Cultures were exposed for 3 or 24 h to 10(-7) M ADM and/or ADM22-52, in the absence or the presence of 10(-8) M ACTH, and the concentration of aldosterone and corticosterone in the culture medium was measured by radioimmune assay. ADM and/or ADM22-52 raised basal aldosterone secretion at 3 h, but not 24 h exposure, and did not affect ACTH-stimulated aldosterone production. Corticosterone secretion was not changed at 3 h. In contrast, at 24 h exposure ADM22-52 alone or with ADM decreased basal corticosterone secretion; ADM evoked a small rise in ACTH-stimulated corticosterone production, and the effect was annulled by ADM22-52. These puzzling findings are interpreted in light of the fact that i) our cultures were actually a mixture of ZG, ZF/R and medullary chromaffin cells; ii) ADM stimulates adrenomedullary cells to release catecholamines, which are able to enhance aldosterone secretion from ZG cells; and iii) the prolonged exposure to ADM may modify, under in vitro culture conditions, ZF/R cells, switching their phenotype from an ADM-unresponsive to an ADM-responsive one. Our study casts doubts on the selectivity of ADM22-52 as ADM receptor antagonist, and stresses that great caution must be used in comparing adrenal-secretion findings obtained with different in vitro techniques.

Stimulatory Effects of Hyperprolactinemia on Aldosterone Secretion in Ovariectomized Rats

BackgroundTo evaluate the effects of hyperprolactinemia on aldosterone secretion and its mechanisms of action in ovariectomized (OVX) rats.MethodsHyperprolactinemia was induced by the transplantation of rat anterior pituitary (AP) glands under...