Adrenal Zona Glomerulosa Cells Research Articles

Angiotensin II-Mediated Protein Kinase D Activation Stimulates Aldosterone and Cortisol Secretion in H295R Human Adrenocortical Cells

Protein kinases are important mediators in intracellular signaling. Angiotensin II is the most important modulator of adrenal zona glomerulosa cell physiology. Angiotensin II regulates steroidogenesis and proliferation among many other metabolic processes. H295R human adrenal cells are a widely used experimental model to study adrenal cell physiology and metabolism. We screened for protein kinase expression levels using the Kinetwork system in H295R cells after 3 h angiotensin II treatment. Protein kinase D (PKD) was the protein kinase that suffers the most dramatic changes. PKD is a member of a new class of serine/threonine protein kinases that is activated by phosphorylation. Our studies indicated that angiotensin II time- and dose-dependently increased PKD phosphorylation, which occurred within 2 min of angiotensin II treatment and at concentrations as low as 1 nm. PKD phosphorylation was also dose-dependently increased by the PKC activator phorbol 12-myristate 13-acetate. Angiotensin II-mediated PKD phosphorylation was blocked by several PKC inhibitors. Furthermore, PKCepsilon translocation inhibitor peptide decreased angiotensin II-mediated PKD phosphorylation, and PKCepsilon down-regulation by RNA interference also decreased PKD phosphorylation mediated by angiotensin II. Cotransfection of constitutively active PKD mutant constructs up-regulated aldosterone synthase and 11beta-hydroxylase expression in reporter assays. Constitutively active PKD mutants increased aldosterone and cortisol secretion under angiotensin II stimulatory conditions. This study reveals that PKD is an intracellular signaling mediator of angiotensin II regulation of steroidogenesis in human adrenal cells. These data provide new insights into the molecular mechanisms involved in angiotensin II-induced physiological and pathophysiological events in adrenal cells.

Probenecid inhibits adrenal zona glomerulosa cell aldosterone release

This study examines the effect of the organic anion transporter inhibitor, probenecid, on aldosterone release and Fluo-3 acetoxymethyl ester dye sequestration from cultured bovine adrenal zona glomerulosa (ZG) cells. ZG cells were isolated and maintained in primary culture in Hams F12 medium supplemented with antioxidants, fetal bovine serum and antibiotics prior to their use in aldosterone or Fluo-3 experiments. Aldosterone release was quantified by enzyme-linked immunosorbant assay. Probenecid inhibited angiotensin II-stimulated aldosterone release in a concentration dependent manner and aldosterone release was completely inhibited with 10 mM probenecid. Potassium, adrenocorticotropic hormone, and 8-bromoadenosine 3′,5′-cyclic monophosphate-stimulated aldosterone release was similarly inhibited by probenecid treatment. The inhibitory effects of probenecid were also observed in ZG cells stimulated with exogenous 25-hydroxycholesterol and progesterone. Fluo-3 fluorescence was increased by probenecid treatment. Treatment of ZG cells with 10 mM probenecid during the Fluo-3 dye loading process caused a dramatic increase in visible fluorescence and more than doubled the average pixel intensity in fluorescing angiotensin II-stimulated ZG cells as compared to control. We conclude that probenecid inhibits transport of Fluo-3 dye by organic anion transporters in the cell membrane of the cultured ZG cell. We further conclude that probenecid interferes with either the enzymes involved in the process of aldosterone synthesis or a transporter required for aldosterone synthesis or release from the ZG cell. This work was supported by grants from the University of Wisconsin-Green Bay.

Mechanisms of histamine-induced relaxation in bovine small adrenal cortical arteries

Adrenal steroidogenesis is closely correlated with increases in adrenal blood flow. Many reports have studied the regulation of adrenal blood flow in vivo and in perfused glands, but until recently few studies have been conducted on isolated adrenal arteries. The present study examined vasomotor responses of isolated bovine small adrenal cortical arteries to histamine, an endogenous vasoactive compound, and its mechanism of action. In U-46619-precontracted arteries, histamine (10(-9)-5 x 10(-6) M) elicited concentration-dependent relaxations. The relaxations were blocked by the H(1) receptor antagonists diphenhydramine (10 microM) or mepyramine (1 microM) (maximal relaxations of 18 +/- 6 and 22 +/- 6%, respectively, vs. 55 +/- 5% of control) but only partially inhibited by the H(2) receptor antagonist cimetidine (10 microM) and the H(3) receptor antagonist thioperamide (1 microM). Histamine-induced relaxations were also blocked by the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NA, 30 microM; maximal relaxation of 13 +/- 7%) and eliminated by endothelial removal or L-NA combined with the cyclooxgenase inhibitor indomethacin (10 microM). In the presence of adrenal zona glomerulosa (ZG) cells, histamine did not induce further relaxations compared with histamine alone. Histamine (10(-7)-10(-5) M) concentration-dependently increased aldosterone production by adrenal ZG cells. Compound 48/80 (10 microg/ml), a mast cell degranulator, induced significant relaxations (93 +/- 0.6%), which were blocked by L-NA plus indomethacin or endothelium removal, partially inhibited by the combination of the H(1), H(2), and H(3) receptor antagonists, but not affected by the mast cell stabilizer sodium cromoglycate (1 mM). These results demonstrate that histamine causes direct relaxation of small adrenal cortical arteries, which is largely mediated by endothelial NO and prostaglandins via H(1) receptors. The potential role of histamine in linking adrenal vascular events and steroid secretion requires further investigation.

Human renal mesangial cells produce aldosterone in response to low-density lipoprotein (LDL)

Systemic aldosterone plays an important role in the development of the microvascular disease and glomerular damage of the kidney in patients with diabetes mellitus and hyperlipidemia. Here, we investigated the possibility of local production of aldosterone in the kidney, using human primary glomerular mesangial cells. These cells produced both pregnenolone and aldosterone measured by specific radioimmunoassay and/or gas chromatography/mass spectrometry (GC/MS) methods. The production of both steroids was significantly stimulated by treatment with LDL, while angiotensin II had a synergistic effect. Adrenocorticotropic hormone (ACTH) and (Bu)2cAMP, on the other hand, failed to stimulate aldosterone production by these cells, suggesting that the local production of this steroid by mesangial cells is regulated differently from that of adrenal zona glomerulosa cells. Mesangial cells expressed the mRNA of the LDL receptor and steroidogenic enzymes, such as P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), 21-hydroxylase and CYP11B2. Mesangial cells also expressed mRNA of the mineralocorticoid receptor (MR), and LDL stimulated its abundance by three-fold, while spironolactone, a completive antagonist of aldosterone, completely abolished this LDL effect. Since MR is a known mineralocorticoid-responsive gene as well as an intracellular receptor molecule for this steroid, these results suggest that locally produced aldosterone is biologically active, stimulating the transcription rates of the mineralocorticoid-responsive genes by activating the MR in mesangial cells. These pieces of evidence indicate that human mesangial cells are an aldosterone-producing tissue in which LDL plays a major regulatory role. Therefore, human renal mesangial endocrine system may contribute to local aldosterone concentrations and effects in the renal glomerulus independently of the systemic renin–angiotensin–aldosterone system and may participate in the development and progression of glomerular damage in several pathologic conditions.

Inhibition of agonist-stimulated aldosterone production from adrenal zona glomerulosa cells by ANP is mediated by GC-A

Atrial natriuretic peptide (ANP) is a potent inhibitor of agonist-induced aldosterone synthesis in zona glomerulosa (ZG) cells of the adrenal gland. Cellular ANP effects are mediated by two receptors: GC-A, a membrane guanylyl cyclase, and GC-C that lacks guanylyl cyclase activity. To identify which one of these two receptors is involved in inhibitory pathway of aldosterone production we developed a new model of isolation and primary culture of mouse ZG cells. Aldosterone concentration in the blood was not different in wild type and GC-A knockout mice. ANP (10 nM) significantly inhibited basal, forskolin, and Angiotensin II-stimulated aldosterone production from wild type mice, while in GC-A knockout mice ANP had no inhibitory effect on aldosterone production. Inhibition of forskolin or ACTH stimulated aldosterone production by ANP was connected with increased concentration of cGMP, stimulation of cGMP-stimulated phosphodiesterase (PDE2), and decrease of intracellular cAMP content. IBMX and the PDE2-selective inhibitor MEP-1 completely prevented ANP inhibitory effect on cAMP-stimulated aldosterone production. In contrast, ANP-dependent inhibition of Ang II-stimulated aldosterone production was not connected with PDE2 or PKG activity indicating that other cGMP-dependent mediator(s)/mechanism(s) are involved in regulation of Ang II-stimulated aldosterone production from ZG cells by cGMP.

Ubc9 and Protein Inhibitor of Activated STAT 1 Activate Chicken Ovalbumin Upstream Promoter-Transcription Factor I-mediated Human CYP11B2 Gene Transcription

Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the -129/-114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.

Measurement of steroid synthesis in zona glomerulosa cells by liquid chromatography–electrospray ionization–mass spectrometry: inhibition by nitric oxide

A liquid chromatography–electrospray ionization–mass spectrometry method was developed to simultaneously determine the concentrations of aldosterone, corticosterone, cortisol, deoxycorticosterone, pregnenolone, and progesterone in bovine adrenal zona glomerulosa (ZG) cells. Steroids were extracted by liquid–liquid extraction, separated on a reverse-phase C 18 column, ionized by electrospray, and detected by single-quadrupole mass spectrometry in a positive ion mode. All steroids formed sodium adducts at high abundance. Factors affecting the formation and signal of sodium adducts were investigated. The limits of detection ( S/ N = 3) using selected ion monitoring are 2 pg for these steroids and 10 pg for pregnenolone. DETA NONOate, a nitric oxide donor, inhibited the basal, angiotensin-II-stimulated, and 25-hydroxycholesterol-stimulated syntheses of these steroids in ZG cells in a concentration-dependent manner. The technique demonstrates the ability to determine the individual steroid in each enzymatic step of aldosterone synthesis and the activity of steroidogenic enzymes in adrenal ZG cells.

Role of voltage-gated calcium channels in potassium-stimulated aldosterone secretion from rat adrenal zona glomerulosa cells

The mineralocorticoid aldosterone plays an important role in the regulation of plasma electrolyte homeostasis. Exposure of acutely isolated rat adrenal zona glomerulosa cells to elevated K + activates voltage-gated calcium channels and initiates a calcium-dependent increase in aldosterone synthesis. We developed a novel 96-well format aldosterone secretion assay to rapidly evaluate the effect of known T- and L-type calcium channel antagonists on K +-stimulated aldosterone secretion and better define the role of voltage-gated calcium channels in this process. Reported T-type antagonists, mibefradil and Ni 2+, and selected L-type antagonist dihydropyridines, inhibited K +-stimulated aldosterone synthesis. Dihydropyridine-mediated inhibition occurred at concentrations which had no effect on rat α1H T-type Ca 2+ currents. In contrast, below 10 μM, the L-type antagonists verapamil and diltiazem showed only minimal inhibitory effects. To examine the selectivity of the calcium channel antagonist-mediated inhibition, we established an aldosterone secretion assay in which 8Br-cAMP stimulates aldosterone secretion independent of extracellular calcium. Mibefradil remained inhibitory in this assay, while the dihydropyridines had only limited effects. Taken together, these data demonstrate a role for the L-type calcium channel in K +-stimulated aldosterone secretion. Further, they confirm the need for selective T-type calcium channel antagonists to better address the role of T-type channels in K +-stimulated aldosterone secretion.

Ghrelin enhances the growth of cultured human adrenal zona glomerulosa cells by exerting MAPK-mediated proliferogenic and antiapoptotic effects

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), two subtypes of which have been identified and named GHS-R1a and GHS-R1b. Evidence has been provided that ghrelin and its receptors are expressed in the adrenal gland, and we have investigated the possible role of the ghrelin system in the functional regulation of the human adrenal cortex. Reverse transcription–polymerase chain reaction detected the expression of both subtypes of GHS-Rs exclusively in the zona glomerulosa (ZG). Ghrelin did not significantly affect either basal or agonist-stimulated aldosterone secretion from cultured ZG cells. In contrast, ghrelin raised proliferative activity and decreased apoptotic deletion rate of ZG cells, the maximal effective concentration being 10 −8 M. The growth effects of 10 −8 M ghrelin on cultured ZG cells were not affected by either the protein kinase (PK)A and PKC antagonists H-89 and calphostin-C or the mitogen-activated PK (MAPK) p38 antagonist SB-293580, but were abolished by both the tyrosine kinase (TK) and MAPK p42/p44 antagonists tyrphostin-23 (10 −5 M) and PD-98059 (10 −4 M), respectively. Ghrelin (10 −8 M) enhanced TK and MAPK p42/p44 activities of ZG cells. Preincubation with 10 −5 M tyrphostin-23 blocked the ghrelin-induced stimulation of both TK and MAPK p42/p44, while preincubation with 10 −4 M PD-98059 only annulled MAPK p42/p44 stimulation. Collectively, our findings allow us to conclude that ghrelin, acting via GHS-Rs exclusively located in the ZG, enhances the growth of human adrenal cortex, through a mechanism involving the activation of the TK-dependent MAPK p42/p44 cascade.

Reciprocal influences between the signalling pathways regulating proliferation and steroidogenesis in adrenal glomerulosa cells.

The main regulators of aldosterone secretion in adrenal gland zona glomerulosa (ZG) cells are the hormones angiotensin II (Ang II) and adrenocorticotrophin (ACTH) and small increases in the extracellular potassium (K(+)) concentration. The action of these agonists is mediated by different signalling systems - ACTH is mediated by cAMP and activation of protein kinase A while Ang II and K(+) activate two protein kinases, Ca(2+)-calmodulin-dependent protein kinase (CamK) and diacylglycerol-dependent protein kinase (PKC). Ang II, besides being one of the main agonists for the secretion of aldosterone, also stimulates proliferation of ZG cells, a process mediated by mitogen-activated protein kinases (MAPKs). Recent studies aimed at elucidating the molecular mechanisms underlying cell proliferation have shown that calcineurin is the principal regulator of MAPKs activity. The purpose of this review is to discuss experimental evidence of possible reciprocal influences between the signalling pathways regulating proliferation and steroidogenesis in ZG cells.

Do human vascular endothelial cells produce aldosterone?

Extraadrenal production of aldosterone has been reported in several tissues, including vascular endothelial cells. The implications of local production of aldosterone in certain nonepithelial target tissues in normal and pathological physiology could be very important and merits further investigation. Human vascular endothelial cells have been reported to synthesize aldosterone under the regulation of angiotensin II. However, discrepancies are noted upon close scrutiny, the most important of which are the relative large efficiency of deoxycorticosterone conversion to aldosterone and the rate of aldosterone production in comparison to the adrenal zona glomerulosa cells. We investigated the production of aldosterone in three different human vascular endothelial cell lines, two from human umbilical veins, one from human pulmonary artery endothelial cells using a very sensitive ELISA method. Cells were incubated with the secretagogues angiotensin II, ACTH, and K(+), at various physiological concentrations with and without 1 microm deoxycorticosterone as additional substrate. In addition, RT-PCR was used to detect expression of the mRNA for the aldosterone synthase gene using a protocol developed by us that detects very low expression in subregions of the human brain. Our results failed to demonstrate mRNA for the aldosterone synthase gene or aldosterone biosynthesis in human endothelial cells.

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.

CGMP-dependent Protein Kinase Type II Regulates Basal Level of Aldosterone Production by Zona Glomerulosa Cells without Increasing Expression of the Steroidogenic Acute Regulatory Protein Gene

The renin-angiotensin-aldosterone system plays a pivotal role in the regulation of salt and water homeostasis. Here, we demonstrate the expression and functional role of cGMP-dependent protein kinases (PKGs) in rat adrenal cortex. Expression of PKG II is restricted to adrenal zona glomerulosa (ZG) cells, whereas PKG I is localized to the adrenal capsule and blood vessels. Activation of the aldosterone system by a low sodium diet up-regulated the expression of PKG II, however, it did not change PKG I expression in adrenal cortex. Both, activation of PKG II in isolated ZG cell and adenoviral gene transfer of wild type PKG II into ZG cells enhanced aldosterone production. In contrast, inhibition of PKG II as well as infection with a PKG II catalytically inactive mutant had an inhibitory effect on aldosterone production. Steroidogenic acute regulatory (StAR) protein that regulates the rate-limiting step in steroidogenesis is a new substrate for PKG II and can be phosphorylated by PKG II in vitro at serine 55/56 and serine 99. Stimulation of aldosterone production by PKG II in contrast to stimulation by PKA did not activate StAR gene expression in ZG cells. The results presented indicate that PKG II activity in ZG cells is important for maintaining basal aldosterone production.

Endogenous adrenomedullin system regulates the growth of rat adrenocortical cells cultured in vitro

The expression of adrenomedullin (AM) system (AM and its receptors), as mRNA and protein, has been detected in the mammalian adrenal zona glomerulosa (ZG) cells. Evidence has been also provided that exogenous AM is able to enhance in vivo and in vitro the proliferative activity of ZG cells. However, the possibility that endogenous AM system may act as a physiological ZG growth regulator has not yet been demonstrated. Hence, we investigated whether the prolonged (48–72 h) suppression of AM gene transcription by a specific antisense oligonucleotide or the long-lasting (24–96 h) blockade of AM receptors by the selective antagonist AM 22–52 are able to affect the growth of rat ZG cells cultured in vitro. Freshly dispersed cells were incubated for 3 h with an AM antisense or a scrambled oligonucleotide, then they were cultured for 48 or 72 h, and proAM mRNA expression and AM content was checked by reverse transcription-polymerase chain reaction and radioimmune assay, respectively. Other ZG cells were cultured in the presence of AM and/or AM 22–52. Growth assay showed that AM (10 −8 M) decreased and AM 22–52 (10 −6 M) increased the duplication time of cultured cells. AM (10 −8 M) raised proliferation index and decreased apoptotic index of cultured cells, and AM 22–52 reversed these effects. AM 22–52 (from 10 −7 to 10 −6 M) and pAM gene suppression by the antisense oligonucleotide significantly lowered proliferation index and increased apoptotic index of cultured cells, both these effects being abrogated by AM (10 −8 M). It is concluded that endogenous AM system plays a relevant role in the autocrine–paracrine regulation of cultured rat ZG-cell growth.

Role of the endogenous adrenomedullin system in regulating the secretion and growth of rat adrenal cortex.

The expression of components of the adrenomedullin (AM) system (AM and its receptors) has been detected in mammalian adrenal zona glomerulosa (ZG) cells, and evidence has been provided that AM is able to inhibit agonist-stimulated aldosterone secretion from and to enhance the proliferative activity of ZG cells. However, there has been no evidence that the endogenous AM system acts as a physiological regulator of ZG function. Hence, we investigated whether the suppression of AM gene transcription by a specific antisense oligodeoxynucleotide (ODN) is able to alter the secretion and growth of rat ZG cells cultured in vitro. ZG cell cultures were examined 0, 2, 4, 6 and 8 days after treatment with scrambled sense (S)-ODN (control cultures) and AM antisense (A)-ODN. Control cultures, as well as freshly dispersed ZG cells and ODN-untreated cultures, expressed AM as mRNA and protein. A-ODN treatment suppressed AM expression within 4 days and the suppression lasted until day 6. Confluent control cultures displayed basal and angiotensin-II (Ang-II), K(+)- and adrenocorticotropic hormone (ACTH)-stimulated aldosterone secretions similar to those of ODN-untreated cultures. A-ODN treatment magnified the aldosterone response to Ang-II and K+ at days 4 and 6 (but not at day 8), without affecting the basal or ACTH-stimulated secretion. As compared to ODN-untreated and control cultures, non-confluent A-ODN-treated ones showed a 40% elongation in the duplication time, a significant decrease in the proliferation index, and a marked rise in apoptotic index from day 4 to day 8. In conclusion, our study validates the use of A-ODN to block the endogenous AM system, showing that suppression of AM-synthesis requires at least 2 days to become appreciable and persists for at least 6 days. Moreover, it provides the first evidence that endogenous AM plays a physiological role in cultured rat ZG cells, by exerting a buffering action on their acute secretory response to Ang-II and K+ and by maintaining normal basal proliferative and apoptotic activities.

Evidence for an autocrine-paracrine role of adrenomedullin in the cultured rat adrenal zona glomerulosa cells

Rat adrenomedullin (ADM) is a 50-amino acid hypotensive and vasodilating peptide, which derives from the posttranslational proteolytic cleavage of pro(p)ADM. ADM acts via at least two subtypes of receptors, named L1-receptor (L1-R) and calcitonin receptor-like receptor (CRLR). CRLR functions as a calcitonin gene-related peptide or a selective ADM receptor depending on the expression of the subtype 1 or the subtypes 2 and 3 of a family of receptor-activity-modifying proteins (RAMPs). Adrenal zona glomerulosa (ZG) is one of the main target tissues of ADM, which has been shown to exert a potent inhibitory effect on aldosterone secretion acting through ADM[22-52]-sensitive receptors. Reverse transcription (RT)-polymerase chain reaction (PCR) consistently allowed the detection of pADM mRNA in the ZG, but not zona fasciculata-reticularis (ZF/R) cells of the rat adrenal cortex. Immunocytochemistry and radioimmune assay showed a weak but sizeable expression of ADM protein in the ZG, but not inner adrenocortical layers. ZG cells expressed peptidyl-glycine alpha-amidating monooxigenase, the enzyme converting immature ADM to the mature peptide, thereby suggesting their potential ability to produce active ADM. RT-PCR demonstrated the presence in ZG, but not ZF/R cells, of the specific mRNAs of L1-R, CRLR and RAMPs (especially RAMP2). ZG cells were cultured in vitro for 24 or 48 h in the presence of ADM (10(-8) M) and/or its receptor antagonist ADM[22-52] (10(-6) M). ADM increased proliferation index and lowered apoptotic index of cultured cells, and the effects were annulled by ADM[22-52]. ADM[22-52] alone was ineffective in 24 h cultures, but moderately decreased proliferation index and raised apoptotic index in 48 h cultures. In conclusion, our study provides evidence that i) rat ZG cells express ADM and ADM receptor of L1 and CRLR/RAMP2 subtypes, which both are sensitive to ADM[22-52]; and ii) endogenous ADM system modulates in an autocrine/paracrine manner ZG growth, by stimulating cell proliferation and reducing cell apoptotic deletion.

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...

Stimulation of recombinant Ca(v)3.2, T-type, Ca(2+) channel currents by CaMKIIgamma(C).

Molecular cloning of low-voltage activated (LVA) T-type calcium channels has enabled the study of their regulation in heterologous expression systems. Here we investigate the regulation of Ca(v)3.2 alpha(1)-subunits (alpha1H) by calcium- and/or calmodulin-dependent protein kinase II (CaMKII). 293 cells stably expressing alpha1H were transiently transfected with CaMKIIgamma(C). Using the whole-cell recording configuration, we observed that elevation of pipette free Ca(2+) (1 microM) in the presence of CaM (2 microM) increases T-type channel activity selectively at negative potentials, evoking an 11 mV hyperpolarizing shift in the half-maximal potential (V(1/2)) for activation. The V(1/2) of channel inactivation is not altered by Ca(2+)/CaM. These effects reproduced modulation observed in adrenal zona glomerulosa cells. The potentiation by Ca(2+)/CaM was dependent on the co-expression of CaMKIIgamma(C) and required Ca(2+)/CaM-dependent kinase activity. Peptide (AIP) and lipophilic (KN-62) protein kinase inhibitors prevented the Ca(2+)/CaM-induced changes in channel gating without altering basal Ca(v)3.2 channel activity (27 nM free Ca(2+)) as did replacing pipette ATP with adenylyl imidodiphosphate (AMP-PNP), a non-hydrolysable analogue. CaMKII-dependent potentiation of channel opening resulted in significant increases in apparent steady-state open probability (P(o)) and sustained channel current at negative voltages. Under identical conditions, CaMKII activation did not regulate the activity of Ca(v)3.1 channels, the first cloned member (alpha1G) of the T-type Ca(2+) channel family. Our results provide the first evidence for the differential regulation of two members of the Ca(v)3 family by protein kinase activation and the first report reconstituting CaMKII-dependent regulation of any cloned Ca(2+) channel.

Adrenomedullin stimulates DNA synthesis of rat adrenal zona glomerulosa cells through activation of the mitogen-activated protein kinase-dependent cascade.

Adrenal zona glomerulosa cells are provided with adrenomedullin receptors. Adrenomedullin has recently been found to enhance proliferation of cultured rat vascular smooth muscle cells and zona glomerulosa cells. To investigate whether adrenomedullin affects rat zona glomerulosa proliferative activity through the tyrosine kinase and extracellular signal regulated kinases (ERKs) pathways. Dispersed rat zona glomerulosa cells were cultured in vitro for 24 h and then exposed to adrenomedullin (10(-7) mol/l), alone or in the presence of tyrphostin-23 (10(-5) mol/l) or PD-98059 (10(-4) mol/l), for 24 or 48 h. To assess the rate of DNA synthesis, 5-bromo-2'-deoxyuridine (BrdU, 20 mg/ml) was also added to the medium and BrdU-positive cells were detected by immunocytochemistry. The expression of ERKs and the effect of adrenomedullin on ERKs phosphorylation and activity were assayed in dispersed zona glomerulosa cells. Adrenomedullin significantly increased the percentage of BrdU-positive (phase-S) zona glomerulosa cells; this effect was blocked by either the tyrosine kinase inhibitor, tyrphostin-23, or the mitogen-activated protein kinase kinase (MEK-1) inhibitor, PD-98059. Both zona glomerulosa and zona fasciculata/reticularis express ERK-1 (44 kDa) and ERK-2 (42 kDa) isoforms. However, adrenomedullin phosphorylated ERK-1 and ERK-2 only in the zona glomerulosa; this effect was blunted by the MEK-1 inhibitor, PD98059, and by the calcitonin gene-related peptide type 1 (CGRP-1) receptor antagonist, CGRP8-37, but not by the adrenomedullin C-terminal fragment, ADM22-52. Adrenomedullin stimulates the growth of rat zona glomerulosa cells through activation of CGRP-1 receptor, linked to the tyrosine kinase-MEK-1-ERKs signalling pathway. These results confirm the complex role played by this peptide in the regulation of zona glomerulosa cell physiology.

Control of CYP11B2 Gene Expression through Differential Regulation of Its Promoter by Atypical and Conventional Protein Kinase C Isoforms

We reported previously that the protein kinase C (PKC) inhibitor GF109203X stimulated the hamster CYP11B2 promoter activity in transfected NCI-H295 cells. PKCalpha, -epsilon, and -zeta were detected in hamster adrenal zona glomerulosa and NCI-H295 cells, and PKCtheta in NCI-H295 cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) inhibited basal and stimulated cytochrome P450 aldosterone synthase mRNA expression by angiotensin (AII), dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP), or KCl in NCI-H295 cells. Basal CYP11B2 promoter activity was inhibited in cells cotransfected with constitutively active (CA) PKCalpha, -epsilon, and -theta mutants, whereas it was increased with CA-PKCzeta. Dominant negative (DN) PKCalpha, -theta, -epsilon, and -zeta mutants stimulated the promoter activity. AII-, KCl-, and Bt2cAMP-stimulatory effects were abolished in cells cotransfected with CA-PKCalpha, -epsilon, or -theta. The effect of Bt2cAMP was abolished by CA-PKCzeta but AII and KCl were still able to enhance the promoter activity. DN-PKCalpha, -epsilon, -theta, or -zeta did not inhibit these effects. Gö6976 enhanced promoter activity, providing further evidence that PKCalpha was involved. Various CYP11B2 promoter constructs were used to identify the area associated with TPA and PKC inhibition. TPA and CA-PKCalpha, -epsilon, or -theta abolished the effects of AII, KCl, and Bt2cAMP on the activity of -102 and longer constructs. In summary, our findings suggest that the hamster CYP11B2 gene is under differential control by conventional (alpha) and atypical (zeta) PKC.