Cholecystokinin and adrenal-cortex secretion.

Cholecystokinin, or CCK, is a 33-amino acid peptide, originally considered a gut hormone, that acts via two subtypes of receptors, named CCK1-R and CCK2-R. CCK, along with its receptors, has been subsequently localized in the central nervous system, where it exerts, among other fuctions, antiorexinogenic actions. In this survey, we describe findings indicating that CCK, similar to other peptides modulating food intake (e.g., neuropeptide Y, leptin, and orexins), is also able to regulate the function of the hypothalamo-pituitary-adrenal axis, acting on both its central and peripheral branches. CCK stimulates aldosterone secretion via specific receptors (CCK1-Rs and CCK2-Rs in rats, and CCK2-Rs in humans) located in zona glomerulosa cells and coupled to the adenylate cyclase-dependent signaling cascade; and enhances glucocorticoid secretion from zona fasciculata-reticularis cells via an indirect mechanism mainly involving the CCK2-R-mediated stimulation of corticotropin-releasing hormone-dependent ACTH release.

Adrenomedullin (AM) and AM receptor type 2 expression is up-regulated in prostate carcinomas (PC), and AM stimulates in vitro growth of a PC-derived cell line by enhancing proliferation and decreasing apoptosis rates.

Adrenomedullin (AM) is a hypotensive peptide, that acts via the calcitonin receptor-like receptor (CRLR), whose interaction with the subtypes 2 and 3 of a family of receptor activity-modifying proteins (RAMP) gives rise to two distinct AM receptors, named AM1 and AM2 receptors. AM derives from the post-translational proteolytic cleavage of pro(p)AM, the last step of which involves the conversion of the inactive AM to active AM by the peptidyl-glycine alpha-amidating monooxigenase (PAM). Compelling evidence suggests that AM, in addition to exerting its well-known regulatory action on blood pressure and water and electrolyte balance, also possesses a growth promoting effect in several normal and neoplastic tissues, including human prostate. Conventional reverse transcription (RT)-polymerase chain reaction (PCR) demonstrated the expression of pAM, PAM, CRLR and RAMP(1-3) mRNAs in both prostate hyperplasias (PH) and carcinomas (PC), and semiquantitative PCR showed that pAM, PAM and RAMP3 mRNA expression was higher in PCs than PHs. Radioimmunoassay measured higher concentrations of immunoreactive AM in PCs than PHs. The expression of pAM, CRLR and RAMP1,2 mRNAs was also detected in the PC-derived cell lines PC-3 and DU-145, RAMP3 expression being restricted to the latter line. AM did not affect the growth rate (duplication time) of PC-3 cells, but it did significantly increase that of DU-145 cells. The growth promoting effect of AM was found to ensue from both the rise in the proliferation rate and the lowering in the apoptosis rate of DU-145 cells. These effects of AM were counteracted by the AM receptor antagonists CGRP(8-37) and AM(22-52), the former antagonist, which is more selective for AM2 than AM1 receptors, being more effective than the latter one. Both antagonists were per se able to induce a slow, but significant decrease in the basal growth rate of DU-145 cells by inhibiting proliferation and enhancing apoptosis, again CGRP(8-37) being more effective than AM(22-52). Taken together, our findings allow us to suggest that: i) endogenous AM system plays an important autocrine-paracrine growth promoting action in the human prostate, being possibly involved in the development of the malignant phenotype of epithelial cells; and ii) the tumor promoting effect of AM in the human prostate is mainly mediated by the AM2 receptor (CRLR/RAMP3) subtype.

Ghrelin inhibits in vitro angiogenic activity of rat brain microvascular endothelial cells.

Ghrelin, a 28-amino acid peptide originally isolated from rat stomach, is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R). Evidence has been provided that ghrelin and GHS-Rs are highly expressed in the cardiovascular system, including endothelial cells (ECs), of which they regulate the growth in vitro. It, therefore, seemed worthwhile to investigate the effect of ghrelin on in vitro angiogenesis, using cultures of rat ECs derived from brain microvessels (neuromicrovascular ECs, NECs). ECs, when cultured on a supportive matrix, form a network of tubule-like structures, and such process is enhanced by the classic angiogenic factors, including fibroblast growth factor-2 (FGF-2). After seeding on Matrigel-coated wells, NECs formed within 18 h a meshwork of capillary-like structures; vinblastine (2 x 10(-12) M) disrupted the meshwork, while FGF-2 (50 ng/ml) increased its density. Ghrelin (10(-8) M) exerted a vinblastine-like effect and counteracted the stimulatory action of FGF-2. Computerized image-analysis confirmed these observations. FGF-2 enhanced the proliferation rate and lowered the apoptotic rate of NECs cultured on plastic wells, and ghrelin exerted opposite effects and completely reversed the proliferogenic and antiapoptotic actions of FGF-2. In contrast to vinblastine, ghrelin did not increase lactate dehydrogenase release from cultured NECs, thereby ruling out the possibility that its effects may ensue from an aspecific cytotoxic action. FGF-2 enhanced tyrosine kinase (TK) and mitogen activated protein kinase (MAPK) p42/p44 activities of NECs. Ghrelin significantly decreased TK and MAPK p42/p44 activities and effectively counteracted the effect of FGF-2. Taken together, the present findings indicate that ghrelin exerts a marked in vitro antiangiogenic action, and that the mechanism underlying this effect involves the inhibition of TK/MAPK-dependent cascades.

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.

Up-regulation of the adrenomedullin system mediates hypotension and hypoaldosteronism induced by simulated microgravity.

We recently demonstrated that prolonged simulated microgravity (SMG) induced hypotension and hypoaldosteronism in rats, and gathered preliminary evidence for an involvement of circulating adrenomedullin (AM). Thus, we aimed to investigate whether short-term SMG elicits the same effects, and whether up-regulation of adrenal AM system plays a relevant role. Rats were exposed for 8 days to SMG in the form of hindlimb unweighting, and then, along with control animals, were given an intraperitoneal injection of AM22-52 and/or angiotensin-II (Ang-II) (100 nmoles/kg) or the saline vehicle. Systolic blood pressure (SBP) was measured by tail-cuff sphygmomanometry. The adrenal expression of AM was assayed by semiquantitative RT-PCR. The plasma concentrations of aldosterone (PAC) and AM, and adrenal AM content were measured by RIA. Short-term SMG induced significant decreases in SBP and PAC. Conversely, both the plasma and adrenal levels of AM, and adrenal AM mRNA were enhanced in SMG-exposed animals. The SMG-induced hypotension and hypoaldosteronism were reversed by AM22-52, an AM-receptor antagonist, thereby demonstrating a causal link between these effects and the up-regulation of AM system. SMG hampered SBP and PAC responses to Ang-II; the co-administration of AM22-52 restored these responses. These findings accord well with the known ability of AM to counteract the effects of Ang-II on both blood vessels and adrenocortical cells. Taken together, our findings allow us to conclude that up-regulation of the adrenal AM system i) occurs early and takes part in the adaptative changes occurring during SMG conditions; and ii) may account for both hypotension and hypoaldosteronism on returning to the normogravitational environment.

Zidovudine-induced alterations in the heart and vascular smooth muscle of the rat.

OBJECTIVE: We investigated the effects of zidovudine (AZT) on cardiac and vascular smooth muscle function and morphology in rats. METHODS: Four adult male Wistar-Kyoto rats received AZT in drinking water for 240 days; four rats served as controls. Echocardiographic examination and systolic blood pressure (SBP) measurement were performed. At the end of treatment the rats were sacrificed, their hearts were weighed and vascular smooth muscle contractile and relaxing properties were evaluated in vitro on endothelium-intact aortic rings. Morphological studies were performed on cardiac and aortic myocytes by light and electron microscopy. RESULTS: AZT-treated rats (AZT-Rs) showed higher SBP, greater heart weight and, as revealed by echocardiography, greater interventricular septum thickness. Electron microscopy revealed mitochondrial swelling in myocardiocytes in AZT-Rs. Reduced response to contractile stimuli and enhanced relaxation in response to charbacol were observed in the aortic rings of AZT-Rs. The aortic myocytes of AZT-Rs contained apparently unaffected ultrastructural features, but light microscopy suggested their marked enlargement. CONCLUSIONS: AZT treatment for 240 days in rats induces a modest increase in SBP, hypertrophy of the interventricular septum and modified vascular smooth muscle responsiveness. The role of mitochondria in these AZT-induced cardiovascular alterations remains to be established.

Simulated microgravity impairs aldosterone secretion in rats: possible involvement of adrenomedullin.

The prolonged exposure to microgravity (MG) or simulated MG (SMG) has been reported to cause hypotension, mainly due to reduced vascular contractility, and dysregulation of fluid and electrolyte balance. However, the mechanism(s) involved in these MG- or SMG-induced effects is not yet completely elucidated. Hence, we investigated in the rat the effect of prolonged (15 day) SMG, in the form of hindlimb unweighting, on the renin-angiotensin-aldosterone system (RAAS), as well as on atrial natriuretic peptide (ANP) and adrenomedullin (ADM), two hypotensive peptides that play a major role in the regulation of RAAS activity by inhibiting adrenal aldosterone secretion. SMG caused a mild hypotension in rats, associated with the blockade of body weight gain. Plasma aldosterone concentration and basal and agonist-stimulated in vitro aldosterone secretion from adrenal slices were decreased, and plasma renin activity was moderately increased. Neither Na(+) and K(+) serum concentrations nor ACTH and corticosterone blood levels were significantly affected. Plasma ANP concentration did not display significant alterations, while ADM blood concentration underwent a marked rise. The administration of the ADM-receptor antagonist ADM-(22-52) during the last 3 days of hindlimb unweighting reversed the SMG-induced hypotension and hypoaldosteronism. Collectively, these findings allow us to suggest that prolonged SMG impairs RAAS activity in rats, through a mechanism probably involving upregulation of the ADM system. Both hypoaldosteronism and increased ADM secretion may contribute to the development of hypotension during prolonged exposure to SMG.

Dual ACE and NEP inhibitor MDL-100,240 prevents and regresses severe angiotensin II-dependent hypertension partially through bradykinin type 2 receptor.

OBJECTIVE: To investigate the effects of the dual angiotensin-converting enzyme (ACE) + neutral endopeptidase (NEP) inhibitor, MDL-100,240 (MDL), on hypertension and cardiovascular damage in male heterozygous transgenic Ren2 rats. METHODS: Blood-pressure-matched 5-week-old transgenic rats were allocated to receive a placebo, MDL (40 mg/kg body weight) or ramipril (5 mg/kg body weight) for 8 weeks. During the last 4 weeks, the bradykinin B2 receptor antagonist, icatibant (0.5 mg/kg body weight), was also administered subcutaneously via osmotic minipumps to 50% of the transgenic rats receiving MDL or ramipril. We measured blood pressure, heart weight, structural changes in the aorta and small resistance mesenteric arteries, and the plasma concentrations of adrenomedullin, aldosterone, atrial natriuretic peptide and cGMP. To verify if MDL could regress long-standing hypertension and full-blown cardiovascular damage, 3-month-old transgenic rats received MDL subcutaneously (3 and 10 mg/kg body weight, osmotic minipumps) for 4 weeks. RESULTS: Compared with placebo, MDL decreased blood pressure (P < 0.001) and prevented left ventricular hypertrophy (P < 0.001), being as effective as ramipril. Hypertrophy and dilatation of the aorta and hypertrophy of the resistance arterioles were all prevented by MDL. Plasma aldosterone was decreased by MDL (P < 0.001), but not by ramipril. Icatibant blunted the decrease in blood pressure (P < 0.001), decreased cGMP concentrations and blunted the decrease in cross-sectional area of the resistance arteries in MDL-treated, but not in ramipril-treated, transgenic rats. In 3-month-old transgenic rats, MDL normalized blood pressure, regressed left ventricular hypertrophy and decreased adrenomedullin concentrations. CONCLUSIONS: The dual ACE+NEP inhibitor MDL prevented and regressed severe hypertension and cardiovascular damage, even in this model of severe angiotensin II-dependent hypertension with pronounced cardiovascular damage. Enhancement of the effects of bradykinin has a role in such favourable outcomes.

11beta-Hydroxysteroid dehydrogenase types 1 and 2 are up- and downregulated in cortisol-secreting adrenal adenomas.

BACKGROUND: 11beta-Hydroxysteroid dehydrogenase types 1 and 2 (11betaHSD1 and 11betaHSD2) are two isoenzymes that convert inactive glucocorticoids (e.g., cortisone) to their active forms (e.g., cortisol) and vice versa. Abundant evidence indicates that 11betaHSD2 is expressed as mRNA and protein in both adrenal cortex and adrenal tumors. In contrast, 11betaHSD1 has been investigated to a much lesser degree. We therefore studied and compared the expression and activity of the two isoenzymes in the human adrenal cortex (HAC) and cortisol-secreting adenomas (CSAs). METHODS: Six HAC and six CSA specimens were studied. 11betaHSD1 and 11betaHSD2 gene expression was studied by conventional and semiquantitative reverse transcription-polymerase chain reaction. 11betaHSD1 and 11betaHSD2 activity was assayed by measuring the capacity of both microsomal fraction and tissue fragments to convert [3H]cortisone to [3H]cortisol and vice versa. Steroid hormones were separated and purified by high-performance liquid chromatography, and cortisol concentration was measured by radioimmunoassay. RESULTS: Semiquantitative reverse transcription-polymerase chain reaction and enzymatic assay demonstrated higher 11betaHSD1 expression and activity and lower 11betaHSD2 expression and activity in CSAs than in HACs. CSA slices secreted larger amounts of cortisol than did HAC specimens, and the cholesterol side-chain-cleaving enzyme inhibitor aminoglutethimide, by blocking the early step of steroid synthesis, reduced cortisol secretion by approximately 70%. Aminoglutethimide decreased [3H]cortisol production from [3H]cortisone and increased [3H]cortisone production from [3H]cortisol in both tissues, thereby annulling differences in 11betaHSD1 and 11betaHSD2 activity between HACs and CSAs. CONCLUSION: Collectively, our findings indicate that 1) both 11betaHSD isoenzymes are expressed as mRNA and proteins in the HAC and CSA, with 11betaHSD1 upregulated and 1betaHSD2 downregulated in CSAs; and 2) 11betaHSD1 and 11betaHSD2 activity is positively and negatively correlated with the intracellular concentration of steroid hormones. Hence, 11betaHSD isoenzymes could act as amplifiers of the secretagogue effect of agonists and could contribute to the elevated hormonal secretion of CSAs.

Aortic smooth muscle cell phenotypic modulation and fibrillar collagen deposition in angiotensin II-dependent hypertension.

BACKGROUND: We investigated the effect of nifedipine, AT-1 and ET-1 receptor blockade on arterial smooth muscle cell phenotypes and collagen deposition in TGRen2 transgenic rat (TGR). METHODS: Four-week-old TGR were blood pressure (BP)-matched and allocated to receive a placebo (n=8), the calcium antagonist nifedipine (n=6), the AT-1 specific receptor antagonist irbesartan (n=6), the ET(A)/ET(B) antagonist bosentan (n=6) or the ET(A)-selective antagonist BMS-182874 (n=5). Sprague-Dawley normotensive rats served as controls (n=6). After 4 weeks of treatment animals were euthanized and the left ventricle (LV) and the structural changes in intracardiac arterioles and aorta were assessed histomorphometrically. Smooth muscle cell phenotypes and fibrillar collagen content of the aortic wall were evaluated by immunostaining, using differentiation markers-specific antibodies and Syrius red staining, respectively. The changes in ET(A) and ET(B) receptor density were also assessed with quantitative autoradiography. RESULTS: Compared to placebo, only irbesartan lowered BP (P<0.001) and prevented LV and small resistance artery hypertrophy. The aorta of placebo-treated TGR showed an increase in foetal-type smooth muscle cell content and fibrillar collagen staining, compared to controls. These changes were blunted by irbesartan, which increased ET(A) receptors in the arterial wall, enhanced by BMS-182874 and unaffected by bosentan. Nifedipine also blunted both the VSMC and collagen changes despite having no effect on BP and ET(A) receptors. CONCLUSIONS: In TGRen2, vascular hypertrophy entails both smooth muscle cell phenotypic modulation and collagen deposition. These alterations do not follow closely the BP changes and seem to imply the dihydropyridine-sensitive calcium channels.

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.