Cultured Zona Glomerulosa Cells Research Articles

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.

Ghrelin and growth hormone secretagogue receptor are expressed in the rat adrenal cortex: evidence that ghrelin stimulates the growth, but not the secretory activity of adrenal cells

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), which has been originally isolated from rat stomach. Evidence has been previously provided that adrenal gland possesses abundant ghrelin-displaceable GHS-Rs, but nothing is known about the possible role of ghrelin in the regulation of adrenocortical function. Reverse transcription-polymerase chain reaction demonstrated the expression of ghrelin and GHS-R in the rat adrenal cortex, and high adrenal concentrations of immunoreactive ghrelin were detected by radioimmune assay (RIA). Autoradiography localized abundant [ 125I]ghrelin binding sites in the adrenal zona glomerulosa (ZG) and outer zona fasciculata (ZF). Ghrelin (from 10 −10 to 10 −8 M) did not affect either basal steroid hormone (pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, 18-hydroxycorticosterone and aldosterone) secretion from dispersed ZG and zona fasciculata/reticularis (ZF/R) cells (as evaluated by quantitative high pressure liquid chromatography), or basal and agonist-stimulated aldosterone and corticosterone production from cultured ZG and ZF/R cells, respectively (as measured by RIA). Ghrelin (10 −8 and 10 −6 M) raised basal, but not agonist-stimulated, proliferation rate of cultured ZG cells (percent of cells able to incorporate 5-bromo-2′-deoxyuridine), without affecting apoptotic deletion rate (percent of cells able to incorporate biotinylated nucleosides into apoptotic DNA fragments). The tyrosine kinase (TK) inhibitor tyrphostin-23 and the p42/p44 mitogen-activated protein kinase (MAPK) inhibitor PD-98059 abolished the proliferogenic effect of 10 −8 M ghrelin, while the protein kinase A and C inhibitors H-89 and calphostin-C were ineffective. Ghrelin (10 −8 M) stimulated TK and MAPK activity of dispersed ZG cells, and the effect was abolished by preincubation with tyrphostin-23 and PD-98059, respectively. Tyrphostin-23 annulled ghrelin-induced activation of MAPK activity. Taken together, the present findings indicate that (i) ghrelin and GHS-R are both expressed in the rat adrenal cortex, ghrelin binding sites being very abundant in the ZG; (ii) ghrelin does not affect the secretory activity of rat adrenocortical cells, but significantly enhances the proliferation rate of cultured ZG cells, without affecting apoptotic deletion rate; and (iii) the ZG proliferogenic action of ghrelin involves the TK-dependent activation of the p42/p44 MAPK cascade.

Expression and function of vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, and their receptors in the human adrenal gland.

VIP and pituitary adenylate cyclase-activating polypeptide (PACAP) are two regulatory peptides that possess remarkable amino acid sequence homology and act through common receptors, named PAC(1), VPAC(1), and VPAC(2). PAC(1) receptor is selective for PACAP, whereas VPAC(1) and VPAC(2) receptors bind both VIP and PACAP. We have investigated the expression and function of VIP, PACAP, and their receptors in the zona glomerulosa (ZG), zonae fasciculata and reticularis, and adrenal medulla (AM) of the human adrenal cortex. RT-PCR and RIA detected VIP and PACAP expression exclusively in AM cells. RT-PCR demonstrated the presence of PAC(1) mRNA only in AM and of VPAC(1) and VPAC(2) mRNAs in both ZG and AM cells. VIP and PACAP concentration-dependently increased aldosterone and catecholamine secretion from cultured ZG and AM cells. The catecholamine response to both peptides was higher than the aldosterone response, and the secretagogue action of PACAP was more intense than that of VIP. The aldosterone response of cultured ZG cells to VIP or PACAP was unaffected by the PAC(1) receptor antagonist PACAP-(6-38) (PAC(1)-A), but was significantly decreased by the VPAC(1) receptor antagonist [Ac-His(1),D-Phe(2),Lys(15),Arg(16)]VIP-(3-7),GH-releasing factor-(8-27)-NH(2) (VPAC(1)-A). The catecholamine response of cultured AM cells to VIP was lowered by VPAC(1)-A and unaffected by PAC(1)-A; conversely, the catecholamine response to PACAP was reduced by both PAC(1)-A and VPAC(1)-A. Simultaneous exposure to both antagonists did not abolish the catecholamine response to PACAP. Collectively, our findings allow us to conclude that in human adrenals 1) VIP and PACAP biosynthesis exclusively occurs in AM cells; 2) ZG cells are provided with functional VPAC(1) and VPAC(2) receptors, whose activation by VIP or PACAP elicits a moderate aldosterone response; 3) AM cells possess PAC(1), VPAC(1), and VPAC(2) receptors, whose activation evokes a marked catecholamine response; and 4) the catecholamine response to PACAP is more intense than that to VIP, because it is mediated by all subtypes of VIP/PACAP receptors.

Adrenomedullin and its receptors are expressed in the zona glomerulosa of human adrenal gland: evidence that ADM enhances proliferation and decreases apoptosis in cultured ZG cells

Adrenomedullin (ADM) is a hypotensive peptide, that derives from the proteolytic cleavage of pro(p)ADM and acts through two subtypes of receptors, called L1-receptor (L1-R) and calcitonin receptor-like receptor (CRLR). CRLR may function 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 proteins, named receptor-activity-modifying protein (RAMPs). Reverse transcription (RT)-polymerase chain reaction (PCR) consistently allowed the detection of pADM mRNA in zona glomerulosa (ZG), but not zona fasciculata-reticularis (ZF/R) cells of eight human adrenal cortexes. Despite the rather high level of pADM mRNA, immunocytochemistry and radioimmunoassay showed that the expression of ADM as protein in the ZG was weak. However, 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 RAMPs1-3. Semiquantitative PCR showed that L1-R expression was higher than that of CRLR, while the level of expression of the three RAMPs was nearly the same. ADM (10(-8) M) inhibited both angiotensin-II (10(-9) M)- and K(+) (10(-2) M)-stimulated aldosterone secretion from cultured ZG cells, without affecting basal production. ADM (10(-8) M) also increased proliferative activity and lowered apoptotic deletion of cultured cells. All the effect of ADM were reversed by the ADM-receptor antagonist ADM(22-52). In conclusion our study provides evidence that i) human ZG cells express ADM and ADM receptors of both L1 and CRLR/PAMP2,3 subtypes; and ii) through the activation of these receptors, ADM exerts an aldosterone antisecretagogue action and a growth promoting effect on cultured ZG cells, the latter an effect which includes both stimulation of proliferation and inhibition of apoptosis. Taken together, these findings make it likely that endogenous ADM system plays a potentially important role in the paracrine/ autocrine regulation of human adrenal cortex.

Endothelin-1[1–31], acting as an ETA-receptor selective agonist, stimulates proliferation of cultured rat zona glomerulosa cells

Endothelin-1 (ET-1)[1–31] is a novel hypertensive peptide that mimics many of the vascular effects of the classic 21 amino acid peptide ET-1[1–21]. However, at variance with ET-1[1–21] that enhances aldosterone secretion from cultured rat zona glomerulosa (ZG) cells by acting via ETB receptors, ET-1[1–31] did not elicit such effect. Both ET-1[1–21] and ET-1[1–31] raised the proliferation rate of cultured ZG cells, the maximal effective concentration being 10 −8 M. This effect was blocked by the ETA-receptor antagonist BQ-123 and unaffected by the ETB-receptor antagonist BQ-788. Quantitative autoradiography showed that ET-1[1–21] displaced both [ 125I]PD-151242 binding to ETA receptors and [ 125I]BQ-3020 binding to ETB receptors in both rat ZG and adrenal medulla, while ET-1[1–31] displaced only [ 125I]BQ-3020 binding. The tyrosine kinase (TK) inhibitor tyrphostin-23 and the p42/p44 mitogen-activated protein kinase (MAPK) inhibitor PD-98059 abolished the proliferogenic effect of ET-1[1–31], while the protein kinase-C (PKC) inhibitor calphostin-C significantly reduced it. ET-1[1–31] (10 −8 M) stimulated TK and MAPK activity of dispersed ZG cells, an effect that was blocked by BQ-123. The stimulatory action of ET-1[1–31] on TK activity was annulled by tyrphostin-23, while that on MAPK activity was reduced by calphostin-C and abolished by either tyrphostin-23 and PD-98059. These data suggest that ET-1[1–31] is a selective agonist of the ETA-receptor subtype, and enhances proliferation of cultured rat ZG cells through the PKC- and TK-dependent activation of p42/p44 MAPK cascade.

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

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

C-type natriuretic peptide as a possible local modulator of aldosterone secretion in bovine adrenal zona glomerulosa

Although atrial and brain natriuretic peptides are well known to be involved in the regulation of cardiovascular and endocrine functions as circulating hormones, the roles of the C-type natriuretic peptide (CNP) remain unknown. We examined the effects of CNP on the secretion of aldosterone and cyclic nucleotides from bovine adrenal zona glomerulosa cells in culture. CNP produced a dose-dependent increase in the basal secretion of cGMP, with an EC50 of 3.8 x 10(-10)M. CNP significantly inhibited the ACTH-induced increase in aldosterone and cAMP in a dose-related manner, with an IC50 of 3.6 x 10(-10)M. Although ACTH itself did not increase cGMP secretion, the addition of CNP elicited a significant increase in cGMP secretion. The effects of CNP on the basal secretion of cGMP and the ACTH-induced secretion of aldosterone were significantly reversed by a nonpeptide natriuretic peptide receptor antagonist, HS-142-1. CNP immunoreactivity was localized in the zona glomerulosa by immunohistochemical staining. In addition, expression of CNP messenger RNA and natriuretic peptide B receptor messenger RNA was demonstrated by RT-PCR in the zona glomerulosa tissue and cells in culture. These findings suggest that CNP is a local factor regulating ACTH-induced aldosterone secretion through a guanylyl cyclase-cGMP pathway.

C-type natriuretic peptide as a possible local modulator of aldosterone secretion in bovine adrenal zona glomerulosa.

Although atrial and brain natriuretic peptides are well known to be involved in the regulation of cardiovascular and endocrine functions as circulating hormones, the roles of the C-type natriuretic peptide (CNP) remain unknown. We examined the effects of CNP on the secretion of aldosterone and cyclic nucleotides from bovine adrenal zona glomerulosa cells in culture. CNP produced a dose-dependent increase in the basal secretion of cGMP, with an EC50 of 3.8 x 10(-10)M. CNP significantly inhibited the ACTH-induced increase in aldosterone and cAMP in a dose-related manner, with an IC50 of 3.6 x 10(-10)M. Although ACTH itself did not increase cGMP secretion, the addition of CNP elicited a significant increase in cGMP secretion. The effects of CNP on the basal secretion of cGMP and the ACTH-induced secretion of aldosterone were significantly reversed by a nonpeptide natriuretic peptide receptor antagonist, HS-142-1. CNP immunoreactivity was localized in the zona glomerulosa by immunohistochemical staining. In addition, expression of CNP messenger RNA and natriuretic peptide B receptor messenger RNA was demonstrated by RT-PCR in the zona glomerulosa tissue and cells in culture. These findings suggest that CNP is a local factor regulating ACTH-induced aldosterone secretion through a guanylyl cyclase-cGMP pathway.

Adrenal receptors for natriuretic peptides and inhibition of aldosterone secretion in calf zona glomerulosa cells in culture.

Atrial and brain natriuretic peptides specifically bind to primary cultures of calf adrenal glomerulosa cells. Binding of both natriuretic peptides to the same receptor has been proved by: a Dixon plot showing competitive effects for the binding of 125I-labeled brain natriuretic peptide in the presence of increasing concentrations of unlabeled atrial natriuretic peptide; a Scatchard plot showing a lower dissociation constant (Kd) for atrial natriuretic peptide than for brain natriuretic peptide binding, but the maximum binding (Bmax) values were the same; autoradiography of sodium dodecyl sulfate polyacrylamide gels after cross-linking of 125I-labeled atrial natriuretic peptide and 125I-labeled brain natriuretic peptide, showing the same molecular weights for both peptide receptors--a single 66-kD band in whole cells and a main band at 125 kD in membranes. C-Type atrial natriuretic peptide only slightly displaced atrial natriuretic peptide binding. Angiotensin II- and potassium-mediated stimulation of aldosterone production were inhibited strongly and to the same degree by atrial and brain natriuretic peptide but only slightly by C-type atrial natriuretic peptide. Stimulation of aldosterone production mediated by adrenocorticotropin was only partially inhibited by atrial and brain natriuretic peptide, while baseline aldosterone was not affected. These results suggest that atrial and brain natriuretic peptide bind to the same receptors and provoke the same effects on aldosterone production. The weak effects found with C-type atrial natriuretic peptide suggest that the primary culture of calf adrenal glomerulosa cells contain the guanylate cyclase A receptor.

Transforming growth factor-beta 1 inhibits aldosterone biosynthesis in cultured bovine zona glomerulosa cells.

Transforming growth factors (TGF beta s) are emerging as possible autocrine regulators of steroidogenesis in a variety of steroid hormone-producing cells. Our laboratory has recently shown that TGF beta 1 is a potent inhibitor of basal and ACTH- and (Bu)2cAMP-stimulated aldosterone production. In this study, we investigated the effects of TGF beta 1 on potassium- and angiotensin-II (A-II)-stimulated aldosterone and the mechanisms by which TGF beta 1 inhibits aldosterone biosynthesis. Cultured zona glomerulosa cells were incubated in serum-free PFMR-4 medium in the presence and absence of TGF beta 1. To investigate the effects of TGF beta 1 on the early pathway of aldosterone biosynthesis, we studied the production of pregnenolone in the presence of the cyanoketone derivative WIN 19,578, which blocks the conversion of pregnenolone to progesterone. TGF beta 1 inhibited pregnenolone production from 133.9 +/- 30.1 to 68.7 +/- 25.4 ng/10(6) cells.h, and the ACTH-stimulated production of pregnenolone was inhibited from 764.6 +/- 127.7 to 141.0 +/- 2.2 ng/10(6) cells.h. In contrast, TGF beta 1 did not inhibit 25-hydroxycholesterol-stimulated pregnenolone production. To study the late pathway of aldosterone production, we added the steroid precursors deoxycorticosterone and corticosterone. TGF beta 1 significantly inhibited deoxycorticosterone- and corticosterone-stimulated aldosterone production by over 50%. TGF beta 1 inhibited the AII- and potassium-induced synthesis of aldosterone. These observations show that TGF beta 1 inhibits AII- and potassium-induced aldosterone synthesis and the early pathway of aldosterone biosynthesis by interfering with the transport of cholesterol across the mitochondrial membrane as well as inhibiting the late pathway of aldosterone biosynthesis.

The Product of the CYP11B2 Gene Is Required for Aldosterone Biosynthesis in the Human Adrenal Cortex

The steroid 11 beta-hydroxylase (P450c11) enzyme is responsible for the conversion of 11-deoxycortisol to cortisol in the zona fasciculata of the adrenal cortex. Animal studies have suggested that this enzyme or a closely related isozyme is also responsible for the successive 11 beta- and 18-hydroxylation and 18-oxidation of deoxycorticosterone required for aldosterone synthesis in the zona glomerulosa. There are two distinct 11 beta-hydroxylase genes in man, CYP11B1 and CYP11B2, which are predicted to encode proteins with 93% amino acid identity. We used a sensitive assay based on the polymerase chain reaction to analyze the expression of the CYP11B1 and B2 genes. Transcripts of CYP11B1 were detected at high levels in surgical specimens of normal adrenals and also in an aldosterone-secreting adrenal tumor. Transcripts of CYP11B2 were found at low levels in normal adrenals, but at a much higher level in the aldosterone-secreting tumor. CYP11B2 mRNA levels were increased in cultured zona glomerulosa cells by physiological levels of angiotensin-II. The entire coding regions of both CYP11B1 and B2 cDNAs were cloned from the tumor mRNA. Expression of these cDNAs in cultured COS-1 cells demonstrated that the CYP11B1 product could only 11 beta-hydroxylate 11-deoxycortisol or deoxycorticosterone, whereas the CYP11B2 product could also 18-hydroxylate cortisol or corticosterone. A small amount of aldosterone was synthesized from deoxycorticosterone only in cells expressing CYP11B2 cDNA. These data demonstrate that the product of CYP11B2 is required for the final steps in the synthesis of aldosterone.

Endothelin binding to cultured calf adrenal zona glomerulosa cells and stimulation of aldosterone secretion.

Endothelins are a group of potent vasoconstrictors whose structure was deduced from genomic DNA. ET-1 was first isolated from culture supernatants from porcine endothelial cells and ET-3 was identified from a rat DNA library. We report on the binding of 125I-ET-1 to zona glomerulosa cells in culture and on its ability to stimulate aldosterone secretion. Cultured calf adrenal zona glomerulosa cells have saturable, high affinity [Kd = 1.00 +/- 0.17 X 10(-10) M (SEM)] receptors which bind ET-1 in a temperature and time dependent manner. Binding was specific and angiotensin II, vasopressin, ANP, BNP, apamin, calcium channel agonists or antagonists did not interact with the receptor. ET-3 displaced 125I-ET-1 from the receptor with a relative potency of 0.39 +/- 0.1% (SEM) that of ET-1. ET-1 incubated with cultured glomerulosa cells stimulated aldosterone secretion in a dose dependent manner but it was less potent than angiotensin II. ET-3 had less than 1% the relative potency of ET-1 stimulating aldosterone secretion. This data suggest that ET-1 is an independent stimulator of aldosterone secretion and we are speculating that it might be important in those situations, like in malignant hypertension, where endothelial damage might result in increased ET-1 production.