CD36 mediates the cardiovascular action of growth hormone-releasing peptides in the heart.

Growth hormone-releasing peptides (GHRPs) are known as potent growth hormone secretagogues whose actions are mediated by the ghrelin receptor, a G protein-coupled receptor cloned from pituitary libraries. Hexarelin, a hexapeptide of the GHRP family, has reported cardiovascular activity. To identify the molecular target mediating this activity, rat cardiac membranes were labeled with a radioactive photoactivatable derivative of hexarelin and purified using lectin affinity chromatography and preparative gel electrophoresis. A binding protein of M(r) 84 000 was identified. The N-terminal sequence determination of the deglycosylated protein was identical to rat CD36, a multifunctional glycoprotein, which was expressed in cardiomyocytes and microvascular endothelial cells. Activation of CD36 in perfused hearts by hexarelin was shown to elicit an increase in coronary perfusion pressure in a dose-dependent manner. This effect was lacking in hearts from CD36-null mice and hearts from spontaneous hypertensive rats genetically deficient in CD36. The coronary vasoconstrictive response correlated with expression of CD36 as assessed by immunoblotting and covalent binding with hexarelin. These data suggest that CD36 may mediate the coronary vasospasm seen in hypercholesterolemia and atherosclerosis.

Identification and characterization of a new growth hormone-releasing peptide receptor in the heart.

Hexarelin, a synthetic hexapeptide of the growth hormone-releasing peptide (GHRP) family with strong growth hormone (GH)-releasing activity, features protecting activity against postischemic ventricular dysfunction in hearts from GH-deficient and senescent rats. To document whether hexarelin action is mediated through specific cardiac receptors, perfusion of Langendorff rat hearts with hexarelin and binding studies were carried out. In the Langendorff rat heart system, hexarelin induced a dose-dependent increase in coronary perfusion pressure. Nifedipine, chelerythrine, and bisindolylmaleimide partially inhibited the vasoconstriction induced by hexarelin, suggesting that this effect was mediated at least in part by L-type Ca(2+) channels and protein kinase C. In contrast, diclofenac and 1-(7-carboxyheptyl)imidazole were without effect, suggesting that prostaglandins and thromboxanes were not involved in the coronary vasoconstriction induced by hexarelin. To characterize the hexarelin binding sites in the rat heart, [(125)I]Tyr-Bpa-Ala-hexarelin was used as photoactivatable radioligand in saturation and competitive binding studies. We specifically labeled a hexarelin receptor with an M(r) of 84 000 in rat cardiac membranes. Saturation binding curves revealed a single class of binding sites with a K(d) of 14.5 nmol/L and a density of 91 fmol/mg of protein. Competition binding studies gave an IC(50) of 2.9 micromol/L for hexarelin; MK-0677 and EP51389, both potent GH secretagogues, did not displace the binding of the photoactivatable derivative from rat cardiac membranes. Interestingly, both compounds were devoid of any vasoconstrictive activity. These results suggest the existence of a new class of hexarelin receptor in the heart, whose role in the regulation of the coronary vascular tone is yet to be determined.

Binding sites for growth hormone-releasing peptide.

Growth hormone-releasing peptides (GHRPs) are known to release growth hormone (GH) in vivo and in vitro by a direct action on receptors in anterior pituitary cells. Measurement of second messengers released following somatotroph stimulation suggests the existence of more than one GHRP receptor subtype in the hypothalamic-pituitary system. Furthermore, hexarelin, a hexapeptide of the GHRP family and a potent GH secretagogue, is reported to increase left ventricular ejection fraction, suggesting the expression of specific myocardial GHRP binding sites. In order to confirm such a hypothesis, a photoactivatable derivative of hexarelin, Tyr-p-benzoyl phenylalanine-Ala-hexarelin, was developed. A putative GHRP receptor with an apparent relative molecular mass of 57,000 was specifically labelled and characterized in human, bovine and porcine anterior pituitary membranes using this hexarelin derivative. The existence of myocardial binding sites was also demonstrated using the same approach. The differential binding affinity of GHRP analogues to cardiac tissue raises the possibility of the existence of distinct GHRP receptor subtypes in the pituitary and the cardiovascular system, for which physiological roles have yet to be determined.

Comparative effect of pituitary adenylate cyclase-activating polypeptide on aldosterone secretion in normal bovine and human tumorous adrenal cells.

The purpose of this study was to investigate the mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in stimulating aldosterone production in two different models: bovine adrenal zona glomerulosa (ZG) cells in primary culture and the human adrenocortical carcinoma cell line H295R. PACAP binds to two major groups of receptors: type I, which prefers PACAP38 and PACAP27 over vasoactive intestinal peptide (VIP); and type II, which has approximately equal affinity for PACAP38, PACAP27, and VIP. The type I subclass comprises multiple splice variants that can be distinguished by their specificity to PACAP38 and PACAP27 in their activation of adenylate cyclase and phospholipase C. Type II PACAP/ VIP receptors couple only to AC. In bovine ZG cells, PACAP38 and PACAP27 stimulated aldosterone production in a dose-dependent manner, whereas VIP was ineffective. In H295R cells, PACAP38, PACAP27, and VIP dose-dependently stimulated aldosterone production with roughly the same ED50. In bovine ZG cells, PACAP38 and PACAP27 stimulated cAMP production with similar efficacy, whereas VIP had no effect. In H295R cells, all three peptides stimulated cAMP accumulation. PACAP38 and PACAP27 also activated PLC in bovine ZG cells as they induced an increase in Ins(1,4,5)Ps production. In H295R cells, neither of these peptides was able to stimulate IP turnover. These results indicate that PACAP stimulation of aldosterone production is mediated by the PVR1s or the PVR1hop splice variants of the type I PACAP-specific receptor subtype in bovine ZG cells, whereas only type II PACAP/VIP receptors seemed to occur in the human H295R cell line. In addition, PACAP-stimulated aldosterone production was inhibited by atrial natriuretic peptide in bovine and human adrenocortical cells, however not by the same mechanism. This further supports species-specific and/or cell type-specific signaling pathways for PACAP in the regulation of aldosterone production.

Pituitary adenylate-cyclase activating polypeptide (PACAP) evokes long-lasting secretion and de novo biosynthesis of bovine adrenal medullary neuropeptides.

Recently, the pituitary adenylate-cyclase activating polypeptide (PACAP) has emerged as a potential noncholinergic neuromodulator of adrenal medullary function. In support of this hypothesis, we documented PACAP's effects on the secretion and biosynthesis of neuropeptides by cultured bovine chromaffin cells. Data presented in this study indicate that PACAP is a potent and efficacious secretagogue of leucine-enkephalin which was coreleased with catecholamines with identical profiles. In comparison to nicotinic activation, however, rates of PACAP-induced secretion were substantially slower but persisted for several hours causing a prolonged increase in the tonic release of both transmitters and peptides. Interestingly, renewal of intracellular pools of neuropeptides was also stimulated by PACAP but not the vasoactive intestinal peptide (VIP). Indeed, the higher incorporation of [35S]-labeled amino acids into atrial and brain natriuretic peptides (ANP, BNP) provided strong evidence that PACAP directly activated de novo biosynthesis. Of particular importance was PACAP's net preferential stimulation of the biosynthesis of BNP, similar to the differential regulation by protein kinase A (PK-A) and protein kinase C (PK-C) activators we have previously the differential regulation by protein kinase A (PK-A) and protein kinase C (PK-C) activators we have previously reported. PACAP-induced secretion and biosynthesis appeared to be mediated by the PACAP-specific type I receptors known to activate adenylate cyclase and phospholipase C. We verified that PACAP did indeed stimulate the production of cyclic AMP and inositol phosphates in our cell system. These findings suggest that the dual signaling properties of type I receptors may be important for PACAP's differential effect on the biosynthesis of natriuretic peptides. We conclude that PACAP might assume important noncholinergic trans-synaptic regulation of the adrenal medulla by releasing and modifying intragranular catecholamine and neuropeptide contents.

The H295R human adrenocortical cell line contains functional atrial natriuretic peptide receptors that inhibit aldosterone biosynthesis.

The inhibitory effect of atrial natriuretic peptide (ANP) on angiotensin II (AII)-stimulated aldosterone secretion has been previously studied in rat and bovine adrenal zona glomerulosa cells in primary culture. However the understanding of the mode of action of ANP at the molecular level has been hampered by limitations of those primary cell culture systems and by the lack of cell lines from human adrenal cortex. Here we demonstrate the presence of fully functional ANP receptors in the recently characterized AII-responsive adrenocortical carcinoma cell line H295R. Specific saturable binding of 125I-rANP to H295R cell membrane preparations revealed a single class of high affinity binding sites with a density of 20 fmol/mg of protein. The pharmacological profile of this ANP receptor was documented by competitive binding of 125I-rANP with naturally occurring natriuretic peptides. rANP was the most potent with a Kd of 42 pM. pBNP32 was less potent with a Kd of 174 pM. 125I-rANP binding was not competed by pCNP (NPRB-specific ligand) nor by C-ANF (NPRC-specific ligand). Photoaffinity labeling of membrane preparations with 125I-BPA-ANP revealed a single specific protein of molecular weight around 130 kDa. This protein was further identified by immunodetection with a specific antibody directed to the human ANP-specific receptor NPRA. Natriuretic peptides stimulated cGMP production by the receptor-coupled guanylate cyclase with the same specificity. Aldosterone production by AII-stimulated H295R cells was dose-dependently inhibited by rANP with an ED50 of 1.5 nM. In addition, we used this model to test two chimeric analogs of ANP and BNP. pBNP1 and pBNP3 were, respectively, 4- and 2-fold more potent than rANP in competing for 125I-rANP binding with Kd of 10 and 20 pM. pBNP1 was 24-fold more potent in inhibiting AII-stimulated aldosterone production with ED50 of 63 pM. pBNP1 is therefore the most potent natriuretic peptide analog tested. In summary, the human H295R cell line contains NPRA receptors positively coupled to the particulate guanylate cyclase and that antagonize angiotensin II stimulation of aldosterone secretion.

A role for protein tyrosine kinase in the steroidogenic pathway of angiotensin II in bovine zona glomerulosa cells.

Stimulation of aldosterone synthesis in bovine adrenal zona glomerulosa (ZGB) cells by angiotensin II (AngII) is believed to be mediated by the phospholipase C (PLC) pathway that results in the increase of cytosolic free calcium concentration and in the activation of protein kinase C (PKC). However, the cell proliferation and contraction associated with AngII action are known to be mediated in part by protein tyrosine kinases (PTK). To assess the potential role of PTK in the stimulatory effect of AngII on adrenal steroidogenesis, the actions of a series of PTK inhibitors on this metabolic pathway were examined in isolated ZGB cells. Tyrphostin 23 (TP23) caused a dose-dependent inhibition of AngII-stimulated aldosterone production with an IC50 of 15 microM and reached complete inhibition at 100 microM. Genistein (GS) was more potent with an IC50 of 35 nM and complete inhibition at 10 microM. The stimulation of aldosterone production by the calcium-mobilizing agent thapsigargin (Thaps) was also dose-dependently inhibited by TP and GS with the same potency. A specific PKC inhibitor, calphostin C (0.1 microM) caused only a 51.7% inhibition of AngII-stimulated aldosterone production. In the same way, a specific Ca2+/calmodulin-dependent protein kinase inhibitor, KN-62 (1 microM), reduced aldosterone production stimulated by AngII by 64%. As expected, thapsigargin-stimulated aldosterone biosynthesis was not affected by calphostin C, but was completely inhibited by KN-62. These results demonstrate for the first time that protein tyrosine kinase activity is part of the angiotensin II signalling pathway in bovine zona glomerulosa cells. The activation of this PTK occurs subsequently to the mobilization of intracellular calcium. This calcium-dependent protein tyrosine kinase pathway is essential for the steroidogenic response to AngII in bovine zona glomerulosa cells.