Immunocytochemical localization of the urotensin-II receptor, UT, to rat and human tissues: relevance to function.

We have examined whether differential expression of UT receptors in cardiovascular tissues from rats and humans may account for the diverse vascular actions reported for urotensin-II. We found UT immunoreactivity ubiquitously expressed in arterial and venous smooth muscle and cardiomyocytes in both species, however, compared to human, levels of UT immunoreactivity in rat vascular endothelial cells was below the level for detection. In rat skeletal muscle cells UT receptor localized to the sarcolemma, a pattern comparable to that for isoforms of nitric oxide synthase suggesting that urotensin-II mediated hindquarter vasodilatation may involve release of nitric oxide from skeletal muscle fibers.

Apelin reduces myocardial reperfusion injury independently of PI3K/Akt and P70S6 kinase.

Apelin, the endogenous ligand of the G protein-coupled APJ receptor, is a peptide mediator with emerging regulatory actions in the heart. The aim of the present studies was to explore potential roles of the apelin/APJ system in myocardial ischaemia/reperfusion injury. To determine the cardiac expression of apelin/APJ and potential regulation by acute ischaemic insult, Langendorff perfused rat hearts were subjected to regional ischaemia (left coronary artery occlusion, 35 min) or ischaemia followed by reperfusion (30 min). Apelin and APJ mRNA expression were then determined in ventricular myocardium by rt-PCR. Unlike APJ mRNA expression, which remained unchanged, apelin mRNA was upregulated 2.4 fold in ventricular myocardium from isolated rat hearts undergoing ischaemia alone, but returned back to control levels after 30 min reperfusion. We then proceeded to test the hypothesis that treatment with exogenous apelin is protective against ischaemia/reperfusion injury. Perfused hearts were subjected to 35 min left main coronary artery occlusion and 120 min reperfusion, after which infarct size was determined by tetrazolium staining. Exogenous Pyr(1)-apelin-13 (10(-8 )M) was perfused either from 5 min prior to 15 min after coronary occlusion, or from 5 min prior to 15 min after reperfusion. Whilst ineffective when used during ischaemia alone, apelin administered during reperfusion significantly reduced infarct size (47.6+/-2.6% of ischaemic risk zone compared to 62.6+/-2.8% in control, n=10 each, p<0.05) in hearts subject to temporary coronary occlusion followed by reperfusion. This protective effect was not abolished by co-administration of the PI3K inhibitor wortmannin (10(-7 )M, infarct size 49.8+/-4.1%, n=4) or the P70S6 kinase inhibitor rapamycin (10(-9 )M, 41.8+/-8.8%, n=4). In conclusion these results suggest that apelin may be a new and potentially important cardioprotective autacoid, upregulated rapidly after myocardial ischaemia and acting through an unknown pathway.

Functional and immunocytochemical evidence for a role of ghrelin and des-octanoyl ghrelin in the regulation of vascular tone in man.

OBJECTIVE: Ghrelin is the recently identified endogenous ligand for the ghrelin receptor GHS-R1a and known to regulate growth hormone secretion and appetite. Ghrelin also is a potent vasodilator and improves cardiac performance after systemic administration. Generally, the octanoyl modification on Ser3 has been considered essential for biological activity. Recently however, cardiovascular actions of des-octanoyl ghrelin have been reported in rodents. Our aim was to investigate if ghrelin and ghrelin receptor protein are expressed within the human vasculature, to determine if des-octanoyl ghrelin, like ghrelin, is a vasodilator in human artery and to test the acute effect of ghrelin peptides on cardiac contractility. METHODS: Distribution of ghrelin and ghrelin receptor was determined using standard immunocytochemistry and confocal microscopy. Ghrelin peptides were tested for vasodilator actions in human isolated arteries and their effect on cardiac contractility was investigated in human isolated paced atria. RESULTS: Immunoreactive ghrelin was detected in endothelial cells of human arteries and veins where it localized to intracellular vesicles but not to the Weibel-Palade bodies of the regulated pathway, suggesting constitutive ghrelin production. Specific antisera detected ghrelin receptor on vascular smooth muscle cells and cardiomyocytes. Ghrelin (pD2=8.60+/-0.1, Emax=55.8+/-8.9, mean+/-standard error of the mean) and des-octanoyl ghrelin (pD2=8.8+/-0.2, Emax=54.7+/-5.3) showed comparable (P>0.05) endothelium independent vasodilator potency and efficacy in reversing endothelin-1 induced constriction in human artery. Neither ghrelin nor des-octanoyl ghrelin had effects on contractile force in paced atria. CONCLUSIONS: We show widespread expression of ghrelin and its cognate receptor in the human cardiovascular system with functional evidence suggesting a role for both ghrelin and the more abundant endogenous form des-octanoyl ghrelin in the paracrine regulation of vascular tone in man.

Emerging roles of apelin in biology and medicine.

The family of apelin peptides is derived from a single gene and activates the 7-transmembrane G-protein-coupled receptor (GPCR) APJ. Apelins have been shown to be involved in the regulation of cardiovascular function and fluid homeostasis and interestingly represent substrates for ACE2, a carboxypeptidase recently described as a novel key enzyme in the renin-angiotensin-aldosterone system (RAS). APJ has further been reported to be a coreceptor for the infection of CD4-positive cells with HIV in the central nervous system (CNS). Apelin-36 and shorter C-terminal sequences have different potencies and efficacies in regulating these functions. Shorter sequences, especially (Pyr(1))apelin-13, are potent regulators of cardiovascular function, while longer peptides such as apelin-36 are more effective in inhibiting human immunodeficiency virus (HIV) infection by blocking the HIV coreceptor APJ. The pyroglutamate modification characteristic of the short apelin peptide (Pyr(1))apelin-13 indicates paramount biological importance of this peptide. The aim of this review is to compile conclusive evidence for the involvement of apelin/APJ in the regulation of cardiovascular function and HIV pathology, emphasizing the properties of this receptor system that may make it a successful future drug target.

Immunocytochemical localisation of the apelin receptor, APJ, to human cardiomyocytes, vascular smooth muscle and endothelial cells.

The novel G protein-coupled receptor APJ, recently paired with the proposed cognate peptide ligand apelin, mediates potent vasodilator and positive inotropic actions in rats. Radioligand binding showed apelin receptors in rat and human heart and human large conduit vessels. The specific cell types expressing the receptor, however, have not been determined. Apelin, the cognate receptor ligand, is present in endothelial cells. However, the exact pathway of endothelial apelin synthesis and secretion is not known. We therefore investigated the cellular distribution of APJ receptor-like immunoreactivity (APJ-LI) in a range of human tissues using immunocytochemistry and fluorescent double staining confocal microscopy. The same techniques were applied to determine the intracellular localisation of apelin-like immunoreactivity (apelin-LI) in cultured human umbilical vein endothelial cells (HUVECs). APJ-LI is present in endothelial cells, vascular smooth muscle cells and cardiomyocytes. Apelin-LI localises to secretory vesicles and the Golgi complex/endoplasmic reticulum of HUVECs. Apelin-LI does not co-localise with von Willebrand factor in Weibel-Palade bodies, suggesting synthesis of apelin via the constitutive pathway. The proximity of receptor and ligand in the human vasculature, together with evidence for local vascular apelin synthesis, suggests an important role for APJ/apelin as a paracrine cardiovascular regulator system.

Cellular distribution of immunoreactive urotensin-II in human tissues with evidence of increased expression in atherosclerosis and a greater constrictor response of small compared to large coronary arteries.

We detected urotensin-II-like immunoreactivity in the endothelium of normal human blood vessels from heart, kidney, placenta, adrenal, thyroid and umbilical cord. Immunoreactivity was also detected in endocardial endothelial and kidney epithelial cells. In atherosclerotic coronary artery, immunoreactivity localized to regions of macrophage infiltration. Urotensin-II constricted human atherosclerotic epicardial coronary arteries with pD2=10.58 +/- 0.46 (mean +/- S.E.M.) and Emax=11.4 +/- 4.2% KCl and small coronary arteries with pD2=9.25 +/- 0.38 and Emax=77 +/- 16% KCl. Small coronary arteries clearly exhibited a greater maximum response to urotensin-II than epicardial vessels. This enhanced responsiveness may be of importance in heart failure, where circulating concentrations of U-II are increased, or in atherosclerosis where focally up-regulated urotensin-II production may act down stream to produce significant vasospasm, compromising blood flow to the myocardium. We conclude that urotensin-II is a locally released vasoactive mediator that may be an important regulator of blood flow particularly to the myocardium and may have a specific role in human atherosclerosis.

Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells.

Apelin, the proposed endogenous peptide ligand of the novel G-protein-coupled receptor APJ, has been shown to possess potent vasodilator and positive inotropic effects in rats and humans in vivo. However, in humans, no endogenous source of apelin has been reported. Therefore, based on the presence of APJ and mRNA encoding apelin in human tissues, we investigated the expression of apelin in fresh-frozen human tissue from right atrium, left ventricle, lung, kidney, adrenal and large conduit vessels using immunocytochemistry. Apelin-like immunoreactivity (apelin-LI) was detected in vascular endothelial cells lining blood vessels in the human heart, kidney, adrenal gland and lung and in endothelial cells of large conduit vessels. Apelin-LI was also present in endocardial endothelial cells lining recesses of the right atrium. Apelin-LI was not present or below the level of detection in cardiomyocytes, Purkinje's cells, pulmonary or renal epithelial cells, secretory cells of the adrenal gland, vascular smooth muscle cells, adipocytes, nerves and connective tissue. The restricted presence of apelin-LI in endothelial cells suggests that endothelial apelin may play a role as a locally secreted cardiovascular mediator acting on APJ receptors present on the vascular smooth muscle and on cardiac myocytes to regulate vascular tone and cardiac contractility.

[Pyr1]apelin-13 identified as the predominant apelin isoform in the human heart: vasoactive mechanisms and inotropic action in disease.

Apelin receptors, present on vascular smooth muscle cells, endothelium, and cardiomyocytes, are activated by the family of apelin peptides to elicit cardiovascular effects in experimental animals, but functional activity in humans has not been studied in detail. We detected low levels of apelin immunoreactivity in plasma of volunteers consistent with an autocrine/paracrine action and detected apelin immunoreactivity in the supernatant from human cultured endothelial cells. We found that [Pyr(1)]apelin-13 was the predominant isoform in cardiac tissue from patients with coronary artery disease. We tested the hypothesis that apelins have vascular and cardiac actions in human tissues in vitro and compared responses to [Pyr(1)]apelin-13, apelin-13, and apelin-36. In endothelium-intact mammary artery, all 3 of the apelins induced concentration-dependent vasodilatation with comparable potency (EC(50): 0.6 to 1.6 nM; maximum response: 40% to 50%). Vasodilatation was abolished after endothelial removal or preincubation with indomethacin but was unaffected by preincubation with N(G)-nitro-L-arginine methyl ester, indicating involvement of prostanoids but not NO in dilatation by apelins in this patient group. Apelins were potent constrictors of endothelium-denuded saphenous vein (EC(50): 0.6 to 1.6 nM; maximum response: 17% to 26%) and mammary artery ([Pyr(1)]apelin-13; EC(50): 0.2 nM; maximum response: 29%). In paced atrial strips, all 3 of the peptides increased the force of contraction with subnanomolar potencies (EC(50): 40 to 125 pM). For the first time, we demonstrate that the 3 principal forms of apelin have comparable potency and efficacy in human cardiovascular tissues. Apelins are potent endothelium-dependent vasodilators acting via a prostanoid-dependent mechanism; however, removal of the endothelium revealed direct vasoconstrictor actions in both the artery and vein. Furthermore, in human cardiac tissue, the apelin peptides are among the most potent endogenous positive inotropic agents yet reported.