Dispersed Zona Glomerulosa Cells Research Articles

G Protein Receptors 7 and 8 Are Expressed in Human Adrenocortical Cells, and Their Endogenous Ligands Neuropeptides B and W Enhance Cortisol Secretion by Activating Adenylate Cyclase- and Phospholipase C-Dependent Signaling Cascades

Neuropeptides B and W (NPB and NPW) are regulatory peptides that act via two subtypes of G protein-coupled receptors, named GPR7 and GPR8. RT-PCR demonstrated the expression of these receptors in both zona glomerulosa and zona fasciculata-reticularis (ZF/R) cells of the human adrenal cortex. NPB and NPW did not affect aldosterone secretion from dispersed zona glomerulosa cells but enhanced cortisol production from ZF/R cells, NPB being more effective than NPW. NPB evoked sizable cAMP and inositol triphosphate responses from ZF/R cells, which were abrogated by the adenylate cyclase inhibitor SQ-22536 and the phospholipase C inhibitor U-73122, respectively. Cortisol response to NPB was lowered by either SQ-22536 and the protein kinase (PK) A inhibitor H-89 or U-73122 and the PKC inhibitor calphostin-C and abolished by the simultaneous exposure to H-89 and calphostin-C. NPW elicited only a rise in cAMP production from dispersed ZF/R cells, and its cortisol response was suppressed by both SQ-22536 and H-89. PreproNPB and preproNPW mRNAs were detected in human adrenal cortexes. We conclude that: 1) NPB and NPW exert a secretagogue action on human ZF/R cells, probably acting in an autocrine-paracrine manner; and 2) the effect of NPB is mediated by both the adenylate cyclase/PKA and the phospholipase C/PKC cascades, whereas that of NPW involves only the activation of the former signaling pathway.

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.

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.

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.

Pituitary adenylate cyclase-activating polypeptide and PACAP receptor expression and function in the rat adrenal gland

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a basic 38-amino acid peptide, which acts through three main G protein-coupled VIP/PACAP receptor subtypes, called PAC1, VPAC1 and VPAC2. We have investigated the expression and function of PACAP and its receptors in the rat adrenal gland. Reverse transcription (RT)-polymerase chain reaction (PCR) and radioimmune assay (RIA) allowed the detection of PACAP expression as mRNA and protein exclusively in adrenal medulla (AM). RT-PCR and quantitative autoradiography, using [(125)I]PACAP and selective VIP/PACAP receptor ligands, demonstrated the expression of PAC1 only in AM, and VPAC1 and VPAC2 in both AM and zona glomerulosa (ZG), PACAP receptor expression being absent in zona fasciculata/reticularis (ZF/R). PACAP38 concentration-dependently increased aldosterone secretion from dispersed ZG cells and catecholamine secretion from AM tissue, the maximal effective concentration being 10(-7) M. ZF/R cells did not display any secretory response to PACAP38. Aldosterone response of ZG cells to 10(-7) M PACAP38 was unaffected by the PAC1-antagonist (A) PACAP(6-38), and significantly decreased by the VPAC1-A [Ac-His(1),D-Phe(2),Lys(15),Arg(16)]VIP(3-7) GRF(8-27)-NH(2). Catecholamine response of AM tissue to PACAP38 was reduced, but not abolished, by both PAC1-A and VPAC1-A. The VPAC2 agonist (ago) Ro25-1553 elicited sizeable secretory responses from both ZG cells and AM tissue. PACAP38 (10(-7) M) evoked a marked rise in cyclic-AMP (cAMP) and inositol-1,4,5-triphosphate (IP3) production by ZG cells and AM tissue. cAMP response of ZG cells was lowered by VPAC1-A, and that of AM tissue by both PAC1-A and VPAC1-A. IP3 response of ZG cells and AM tissue was unaffected by PAC1-A and decreased by VPAC1-A. VPAC2-ago did not affect cAMP release, but raised IP3 production by both ZG cells and AM tissue. Aldosterone response of ZG cells and catecholamine response of AM tissue to PACAP38 (10(-7) M) were reduced by the adenylate cyclase (AC) and phospholipase-C (PLC) inhibitors (I) SQ-22536 and U-73122, as well as by the protein kinase (PK)A-I H-89 and PKC-I calphostin-C. Conversely, the secretory responses of both ZG and AM preparations to VPAC2-ago were annulled by PLC-I, lowered by PKC-I, and unaffected by either AC-I or PKA-I. Collectively, our findings allow us to conclude that in the rat adrenals: i) PACAP biosynthesis exclusively occurs in the AM; ii) ZG cells are provided with functional VPAC1 and VPAC2 receptors, whose activation by PACAP evokes a moderate aldosterone response; iii) AM cells possess all the subtypes of VIP/PACAP receptors, whose activation by PACAP elicits a marked catecholamine response; and iv) PAC1 receptors are coupled to the AC-dependent cascade, VPAC1 receptors to both the AC- and PLC-dependent cascades, and VPAC2 receptors exclusively to the PLC-dependent cascade.

Mechanisms transducing the aldosterone secretagogue signal of endothelins in the human adrenal cortex

Evidence has been provided that the 21-amino acid hypertensive peptide endothelin (ET)-1 exerts a potent secretagogue effect on human adrenocortical zona glomerulosa (ZG), acting through two receptor subtypes, called ETA and ETB, the signaling mechanism(s) of which has (have) not yet been investigated. Collagenase dispersed human ZG cells were obtained from normal adrenals of patients undergoing nephrectomy/adrenalectomy for renal cancer. The selective ETA- and ETB-receptor activation was obtained by exposing dispersed cells to ET-1 plus the ETB-receptor antagonist BQ-788 and to the ETB-receptor agonist BQ-3020, respectively. The phospholipase (PL) C inhibitor U-73122 abolished ETA receptor-mediated secretory response, but only partially prevented the ETB receptor-mediated one. The phosphatidylinositol 3-kinase inhibitor wortmannin, the calmodulin inhibitor W-7 and the protein kinase (PK) C inhibitor calphostin-C significantly blunted the secretory responses ensuing from the activation of both receptor subtypes. When added together, calphostin-C and wortmannin or W-7 abolished ETA-mediated secretory response, but only decreased ETB-mediated one. The ETB receptor-, but not the ETA receptor-mediated aldosterone response was partially reversed by the cyclooxygenase (COX) inhibitor indomethacin, which when added together with U-73122 abolished it. ETA-receptor activation raised inositol triphosphate (IP3) production from dispersed ZG cells, while ETB-receptor stimulation enhanced both IP3 and prostaglandin-E2 production. Collectively, our findings indicate that ETs stimulate aldosterone secretion from human ZG cells, acting through ETA receptors exclusively coupled to PLC/PKC-dependent pathway and ETB receptors coupled to both PLC/PKC- and COX-dependent cascades.

Cholecystokinin Stimulates Aldosterone Secretion from Dispersed Rat Zona Glomerulosa Cells, Acting Through Cholecystokinin Receptors 1 and 2 Coupled with the Adenylate Cyclase-Dependent Cascade

Cholecystokinin is a regulatory peptide, that acts through two subtypes of receptors, 1 and 2. RT-PCR demonstrated the expression of both cholecystokinin receptors 1 and 2 genes in the zona glomerulosa, but not the zona fasciculata-reticularis, of rat adrenals. Autoradiography demonstrated the presence of abundant [(125)I]cholecystokinin-binding sites in the zona glomerulosa, but not the zona fasciculata-reticularis, which were displaced by both cholecystokinin receptor 1- and 2-selective antagonists (cholecystokinin 1-A and 2-A). Cholecystokinin increased basal aldosterone secretion from dispersed zona glomerulosa cells without affecting corticosterone secretion from zona fasciculata-reticularis cells. The aldosterone response to cholecystokinin was blunted by cholecystokinin 1-A and 2-A, which when added together abolished it. ACTH-stimulated aldosterone production was not affected by cholecystokinin; in contrast, cholecystokinin potentiated aldosterone response to both angiotensin II and K(+). Cholecystokinin enhanced cAMP, but not IP(3), release by dispersed zona glomerulosa cells. The aldosterone response to cholecystokinin was abolished by the adenylate cyclase inhibitor SQ-22536 and the PKA inhibitor H-89, but not by either the PLC inhibitor U-73122 or the PKC inhibitor calphostin C. In conclusion, our study provides evidence that cholecystokinin, acting through cholecystokinin receptors 1 and 2 coupled with the adenylate cyclase/PKA cascade, exerts a sizeable secretagogue action on rat zona glomerulosa cells.

Stimulation of endogenous nitric oxide production is involved in the inhibitory effect of adrenomedullin on aldosterone secretion in the rat

Adrenomedullin (AM) (10 −8 M) partially suppressed aldosterone response of dispersed rat zona glomerulosa (ZG) cells to 10 mM K +, and the nitric oxide (NO) synthase inhibitors L-NAME (10 −3 M) and 1400W (10 −4 M) effectively counteracted this effect of AM. The NO donor L-Arginine (L-Arg) (10 −5 M) decreased both basal and K +-stimulated aldosterone secretion. The guanylate-cyclase inhibitor Ly-83583, at a concentration (10 −4 M) abolishing either the guanylate-cyclase activator guanylin- or L-Arg-induced cGMP release from dispersed ZG cells, did not affect the aldosterone antisecretagogue action of AM and L-Arg. AM (10 −8 M) evoked a moderate increase in cGMP release by dispersed ZG cells, and the effect was blocked by both 10 −4 M Ly-83583 and 10 −3 M L-NAME. Collectively, these findings allow us (1) to confirm that NO inhibits aldosterone secretion through a cGMP-independent mechanism; and (2) to suggest that stimulation of endogenous NO synthesis plays a role in the mechanisms underlying the inhibitory effect of AM on K +-stimulated aldosterone secretion from rat ZG cells.

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.

Comparison of the signaling mechanisms involved in the ETB receptor-mediated secretagogue action of endothelin-1 on dispersed zona glomerulosa cells and capsule-zona glomerulosa preparations of the rat adrenal gland.

Endothelin-1 (ET-1) is a hypertensive peptide, which is expressed in the rat adrenal gland, where it stimulates aldosterone secretion from zona glomerulosa (ZG) by activating the ETb receptor subtype. A higher effectiveness of ET-1 has been frequently observed when the integrity of adrenal tissue is preserved. Hence, we compared the aldosterone secretagogue action of ET-1 on dispersed rat ZG cells and capsule-ZG strips. ET-1 concentration-dependently raised aldosterone output by both preparations with similar potency. However, the efficacy of the maximal effective concentration of ET-1 (10-8 M) was about 2.7-fold higher in capsule-ZG strips. The ETb-receptor antagonist BQ-788 (10-7 M) abolished aldosterone response to 10-8 M ET-1 in both ZG preparations, while the ETa receptor antagonist BQ-123 was ineffective. The aldosterone secretagogue action of 10-8 M ET-1 on dispersed ZG cells was concentration-dependently suppressed by the protein kinase (PK) inhibitor calphostin-C. Conversely, both calphostin-C and the nitric oxide (NO) synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) evoked a concentration-dependent partial reversal of the aldosterone response to 10-8 M ET-1 of capsule-ZG strips. The NO donor L-arginine enhanced basal aldosterone yield of capsular strips, but not dispersed ZG cells. The PKA, cyclooxygenase and lipoxygenase inhibitors H-89, indomethacin and phenidone, as well as the beta-adrenoceptor antagonist l-alprenolol, were ineffective. Collectively, these findings allow us to conclude that in the rat i) the ETb receptor-mediated PKC activation is the main signaling mechanism involved in the direct stimulatory effect of ET-1 on ZG cells; and ii) the higher responsiveness of capsular strips to ET-1 may be accounted for by the ETb receptor-mediated release by stromal elements of NO, which in turn increases aldosterone secretion from ZG cells in a paracrine manner.

Distribution, functional role, and signaling mechanism of adrenomedullin receptors in the rat adrenal gland

Adrenomedullin (ADM) is a hypotensive peptide, highly expressed in the mammalian adrenal medulla, which belongs to a peptide superfamily including calcitonin gene-related peptide (CGRP) and amylin. Quantitative autoradiography demonstrated the presence of abundant [ 125I]ADM binding sites in both zona glomerulosa (ZG) and adrenal medulla. ADM binding was selectively displaced by ADM(22–52), a putative ADM-receptor antagonist, and CGRP(8–37), a ligand that preferentially antagonizes the CGRP1-receptor subtype. ADM concentration-dependently inhibited K +-induced aldosterone secretion of dispersed rat ZG cells, without affecting basal hormone production. Both ADM(22–52) and CGRP(8–37) reversed the ADM effect in a concentration-dependent manner. ADM counteracted the aldosterone secretagogue action of the voltage-gated Ca 2+-channel activator BAYK-8644, and blocked K +- and BAYK-8644-evoked rise in the intracellular Ca 2+ concentration of dispersed ZG cells. ADM concentration-dependently raised basal catecholamine (epinephrine and norepinephrine) release by rat adrenomedullary fragments, and again the response was blocked by both ADM(22–52) and CGRP(8–37). ADM increased cyclic-AMP release by adrenal-medulla fragments, but not capsule-ZG preparations, and the catecholamine response to ADM was abolished by the PKA inhibitor H-89. Collectively, the present findings allow us to draw the following conclusions: (1) ADM modulates rat adrenal secretion, acting through ADM(22–52)-sensitive CGRP1 receptors, which are coupled with different signaling mechanisms in the cortex and medulla; (2) ADM selectively inhibits agonist-stimulated aldosterone secretion, through a mechanism probably involving the blockade of the Ca 2+ channel-mediated Ca 2+ influx; (3) ADM raises catecholamine secretion, through the activation of the adenylate cyclase/PKA signaling pathway.

Orexins stimulate corticosterone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade

Orexins-A and B are two novel hypothalamic peptides, which, like leptin and neuropeptide-Y (NPY), are involved in the central regulation of feeding. Since leptin and NPY were found to modulate adrenal function, we have examined whether orexins are able to directly affect rat adrenal steroid secretion. Both orexin-A and orexin-B raised basal corticosterone secretion of dispersed rat zona fasciculata–reticularis (ZF/R) cells, their maximal effective concentration being 10 −8 M. In contrast, orexins did not affect either maximally ACTH (10 −9 M)-stimulated corticosterone production by ZF/R cells or the basal and agonist-stimulated aldosterone secretion of dispersed zona glomerulosa cells. The ACTH-receptor antagonist corticotropin-inhibiting peptide (10 −6 M) annulled corticosterone response of ZF/R cells to ACTH (10 −9 M), but not to orexins (10 −8 M). Orexins (10 −8 M) enhanced cyclic-AMP release by ZF/R cells, and the selective inhibitor of protein-kinase A (PKA) H-89 (10 −5 M) abolished corticosterone responses to both ACTH (10 −9 M) and orexins (10 −8 M). A subcutaneous injection of both orexins (5 or 10 nmol/kg) evoked a clear-cut increase in the plasma concentration of corticosterone (but not aldosterone), the effect of orexin-A being significantly more intense than that of orexin-B. Collectively, these findings suggest that orexins exert a selective and direct glucocorticoid secretagogue action on the rat adrenals, acting through a receptor-mediated activation of the adenylate cyclase/PKA-dependent signaling pathway.

Gastric inhibitory polypeptide stimulates glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase-dependent signaling pathway

Gastric inhibitory polypeptide (GIP) is a 42-amino acid peptide, belonging to the VIP-secretin-glucagon superfamily, some members of this group are able to regulate adrenocortical function. GIP-receptor mRNA has been detected in the rat adrenal cortex, but investigations on the effect of GIP on steroid-hormone secretion in this species are lacking. Hence, we have investigated the distribution of GIP binding sites in the rat adrenal gland and the effect of their activation in vivo and in vitro. Autoradiography evidenced abundant [ 125I]GIP binding sites exclusively in the inner adrenocortical layers, and the computer-assisted densitometric analysis of autoradiograms demonstrated that binding was displaced by cold GIP, but not by either ACTH or the selective ACTH-receptor antagonist corticotropin-inhibiting peptide (CIP). The intraperitoneal (IP) injection of GIP dose-dependently raised corticosterone, but not aldosterone plasma concentration: the maximal effective dose (10 nmol/rat) elicited a twofold increase. GIP did not affect aldosterone and cyclic-AMP release by dispersed zona glomerulosa cells. In contrast, GIP enhanced basal corticosterone secretion and cyclic-AMP release by dispersed inner adrenocortical cells in a concentration-dependent manner, and the maximal effective concentration (10 −7 M) evoked 1.5- and 2.4-fold rises in corticosterone and cyclic-AMP production, respectively. GIP (10 −7 M) did not display any additive or potentiating effect on corticosterone and cyclic-AMP responses to submaximal or maximal effective concentrations of ACTH. The corticosterone secretagogue action of 10 −7 M GIP was abolished by the protein kinase A (PKA) inhibitor H-89 (10 −5 M), and unaffected by CIP (10 −6 M). Collectively, these findings indicate that GIP exerts a moderate but statistically significant stimulatory effect on basal glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase/PKA-dependent signaling pathway.

Mechanisms and receptor subtypes involved in the stimulatory action of endothelin-1 on rat adrenal zona glomerulosa.

Endothelin (ET)-1 is the prototype of a family of 21-amino acid residue hypertensive peptides, acting through two subtypes of receptors, named ETA and ETB. ETs and their receptors are expressed in the adrenal cortex and medulla, and ET-1 enhances both corticosteroid and catecholamine release. ET-1 concentration-dependently (from 10(-11) to 10(-8) M) increased aldosterone secretion of both dispersed rat zona glomerulosa (ZG) cells and adrenal slices containing a core of medullary chromaffin tissue, but the response of the latter preparations was significantly more intense than that of the formers. The stimulatory effect of 10(-8) M ET-1 on dispersed ZG cells was blocked by the ETB-receptor antagonist BQ-788 (10(-7) M), but not by the ETA-receptor antagonist BQ-123 (10(-7) M); conversely, both ET-receptors antagonists counteracted aldosterone response of adrenal slices to ET-1. The -adrenoceptor antagonist l-alprenolol (10(-6) M) did not affect aldosterone response of dispersed ZG cells to ET-1 (10(-8) M), but it significantly lowered that of adrenal slices. l-Alprenolol also counteracted the aldosterone response of adrenal slices to the pure activation of ETB or ETA receptors, as obtained by using the selective ETB-receptor agonist BQ-3020 (10(-8) M) or ET-1 (10(-8) M) plus BQ-788 (10(-7) M). ET-1 concentration-dependently (from 10(-9) to 10(-8)/10(-7) M) stimulated catecholamine release by adrenal slices, and the effect was counteracted by both BQ-123 and BQ-788 (10(-7) M). Collectively, our findings suggest that, when the integrity of adrenal tissue is preserved, a two-fold mechanism underlies the aldosterone secretagogue action of ET-1 in the rat: i) a direct mechanism mediated by ETB receptors located on ZG cells; and ii) an indirect mechanism involving the ETA and ETB receptor-mediated local release of catecholamines, which in turn stimulate ZG cells in a paracrine manner.

Distribution and functional significance of angiotensin-II AT1-AND AT2-Receptor subtypes in the rat adrenal gland

The distribution and the functional significance of angiotensin-II (ANG-II) receptor subtypes. AT1 and AT2, in the rat adrenal gland has been investigated in vitro Autoradiographic assessment of the selective displacement of [125I]ANG-II binding by selective ligands of the two receptor subtypes indicated that zona glomerulosa (ZG) was provided with both AT1 and AT2, and adrenal medulla (AM) almost exclusively with AT2 receptors ANG-II (10−9 M) evoked a marked rise in the secretion of aldosterone by dispersed ZG cells and catecholamines by AM fragments The selective AT1-receptor antagonist DuP753 blocked aldosterone response to ANG-II, while the selective AT2-receptor antagonist PD123319 was ineffective. Catecholamine response to ANG-II was inhibited by PD123319 and only moderately affected by high concentrations of DuP753. The selective AT2-receptor agonist CGP42112 did not change basal aldosterone release of ZG cells, but concentration-dependently enhanced basal catecholamine release by AM fragments. In light of these findings the conclusion is drawn that in the rat the aldosterone secretagogue effect of ANG-II is exclusively mediated by the AT1 receptors present in the ZG, while the catecholamine secretagogue action preminently involves the activation of AT2 receptor located on medullary chromaffin cells

The AT2 receptor-mediated stimulation of adrenal catecholamine release may potentiate the AT1 receptor-mediated aldosterone secretagogue action of angiotensin-II in rats

The role played by AT1 and AT2 receptors in the mediation of angiotensin-II (ANG-II) aldosterone secretagogue action has been investigated in vitro using different types of rat adrenal preparations. ANG-II enhanced aldosterone secretion of dispersed zona glomerulosa (ZG) cells in a concentration-dependent manner (EC50, 3 × 10−10 M), and its effect was annulled by the AT1-receptor antagonist DuP753 and unaffected by the AT2-receptor antagonist PD123319. ANG-II was significantly more effective in stimulating aldosterone secretion when capsule-ZG and adrenal slices containing medullary chromaffin cells were used (EC50, 1 × 10−12 M and 7 × 10−12 M, respectively); moreover, both DuP753 and PD 123319 caused partial reversals (intense and moderate, respectively) of the responses to ANG-II, and when added together annulled them. The β-adrenoceptor antagonist l-alprenolol did not affect aldosterone response to ANG-II of dispersed ZG cells, but exerted a PD123319-like effect on the responses of capsule-ZG and adrenal slices In light of these findings we conclude that, when the integrity of adrenal tissue is preserved, ANG-II stimulates aldosterone secretion by activating both AT1 and AT2 receptors, the major role being played by AT1 receptors located on ZG cells. The activation of AT2 receptors probably elicits the local release of catecholamines, which in turn enhance aldosterone secretion in a paracrine manner acting through the β-adrenoceptors with which ZG cells are provided.

Neuropeptide-Y family of peptides in the autocrine-paracrine regulation of adrenocortical function.

Neuropeptide-Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are a family of 36-amino-acid peptides, which are widely distributed in the body and act through several subtypes of G-protein-coupled Y receptors. The three members of the NPY-family peptides are contained in the adrenal gland, where they exert various autocrine/paracrine regulatory functions. Binding sites for NPY are present in adrenal medulla and zona glomerulosa (ZG), where also several NPY-ergic fibers end. Binding sites for PYY and PP are prevalently located in the inner adrenocortical zones and adrenal medulla. NPY and PYY inhibit aldosterone secretion of dispersed ZG cells, but this effect has probably to be considered nonspecific and toxic in nature. On the contrary, there are indications that NPY may indirectly stimulate ZG cells, by eliciting the release of catecholamines, which in turn enhance aldosterone secretion. Evidence is also available that NPY modulates the secretory response of ZG cells to their main agonists. PP is able to raise glucocorticoid secretion acting directly on the inner adrenocortical cells. The physiological relevance of these effects of NPY-family peptides remains to be addressed by future experimental studies employing more selective Y receptor agonists and antagonists. In contrast, indirect evidence is available that endogenous NPY-family peptides might play an important role in the modulation of adrenocortical secretory action under paraphysiological or pathological conditions such as like aging, hypoglycemic stress and pheochromocytomas.

Short circuit current in rat adrenal capsule/glomerulosa

Angiotensin II (AII) and potassium are the primary regulators of aldosterone secretion by adrenal zona glomerulosa cells. Electrophysiological studies using isolated adrenal tissues and dispersed zona glomerulosa cells show that stimulation by these secretagogues results in depolarisation of the plasma membrane and the opening of voltage-sensitive ion channels. The concept that these cells act together to create a polarity within the gland has not been examined.Whole adrenal capsule/glomerulosa preparations were studied in Ussings chambers. An increase in [KC1] to either side of the capsule resulted in a concentration-related change in short-circuit current (SCC). KC1 added externally caused an increase in SCC, indicating net inward movement of positive ions or net outward movement of negative ions. Internal KC1 had a smaller opposite effect. Use of the non-specific potassium channel blocker tetraethylammonium chloride (TEA) resulted in an increase in SCC regardless of which side the addition was made, although on occasions the responses to TEA and internal KC1 were unexpectedly reversed. The aldosterone antagonist spironolactone produced a variable change in SCC suggesting an autocrine/paracrine role for aldosterone on the adrenal cortex. No responses were observed with the addition of AII, ACTH or aldosterone, though these may be present in excess. The results suggest that ion gradients may be created by stimulation that conceivably have a role in cellular organisation.

Further investigations on the effects of neuropeptide Y on the secretion and growth of rat adrenal zona glomerulosa

NPY is a regulatory peptide, high levels of which are contained in adrenal glands of several mammals and which is co-released with catecholamines during various stressful conditions. The acute and chronic effects of NPY on adrenocortical secretion and growth were studied in the rat. NPY concentration-dependently increased aldosterone (ALDO), but not corticosterone (B) secretion of adrenal slices (maximal effective concentration was 10 −7 M). Two competitive inhibitors of NPY receptors, named PYX-1 and PYX-2, were found to dose-dependently inhibit ALDO response of adrenal preparations to 10 −7 M NPY; PYX-2 was more efficient than PYX-1, and at a concentration of 10 −5 M completely annulled the effect of 10 −7 M NPY. The acute bolus intraperitoneal (i.p.) injection of NPY (3 nmol/kg) raised plasma ALDO concentration (PAC), but not that of B (PBC); this effect of NPY was blocked by the simultaneous injection of PYX-2 (300 nmol/kg). The prolonged i.p. infusion with NPY (3 nmol/kg/h for 7 days) increased PAC (but not PBC) and induced a marked hypertrophy of the zona glomerulosa (ZG) and its parenchymal cells; dispersed ZG cells obtained from NPY-infused rats displayed a significantly enhanced basal and maximally agonist-stimulated ALDO production. The simultaneous infusion with PYX-2 (300 nmol/kg/h) completely annulled all these effects of NPY. The acute or chronic administration of PYX-2 alone did not evoke any apparent effect on the ZG secretion and growth. In light of these findings the following conclusions can be drawn: (i) NPY is able to stimulate not only the secretion, but also the growth of adrenal ZG in rats, via a receptor-mediated mechanism (since this effect is blocked by PYX-2); (ii) endogenous NPY does not play a prominent role in the physiological maintenance of secretion and growth of rat ZG (since PYX-2 alone is ineffective); (iii) NPY may play a crucial role in the fine tuning of the ZG functions in conditions requiring an increased release of mineralocorticoid hormones.

Effects of neuromedin-k on the rat hypothalamo pituitary-adrenal axis

The effects of neuromedin-K (NMK) on the rat hypothalamo-pituitary-adrenal (HPA) axis were studied both in vivo and in vitro. A subcutaneous injection of 1 nmol/100 g NMK evoked a rise in plasma ACTH level at 30 min, increased plasma corticosterone (B) concentration (PBC) at 60 and 120 min, and did not alter plasma aldosterone (ALDO) concentration (PAC). The administration of 3 nmol/100 g NMK induced a rise in plasma ACTH level at 120 min and a drop of PBC at 30 min; it increased PBC and PAC at 60 and 120 min. NMK did not affect basal B secretion of dispersed zona fasciculata/reticularis (ZF/R) cells, but markedly enhanced basal ALDO production by dispersed zona glomerulosa (ZG) cells (minimal and maximal effective concentrations were 10 −9 M and 10 −7 M). Video-imaging analysis showed that NMK (10 −8 M) increased intracellular Ca 2+ concentration in dispersed ZG cells, but not in ZF/R ones. These findings indicate that NMK exerts a complex modulatory action on the rat HPA axis: low doses of NMK appear to evoke a transient stimulation of ACTH release, while high doses seem to exert a short-term inhibition of glucocorticoid synthesis followed by the compensatory hypersecretion of ACTH; moreover, elevated doses of NMK also exert a strong ALDO secretagogue action by acting directly on the ZG cells.