Ghrelin, a 28 amino acid peptide secreted mainly from stomach endocrine cells, is the endogenous ligand of human GHS-R1a (Kojima et al., 1999) and known for its regulator effects on growth hormone secretion and appetite. However, ghrelin receptors have also been identified in human cardiovascular tissue and ghrelin is a potent vasodilator both in vitro and in vitro(Katugampola et al., 2001; Wiley & Davenport, 2002). These cardiovascular effects and the presence of ghrelin mRNA in a wide range of peripheral tissues suggests the existence of a site of ghrelin synthesis in cardiovascular tissues. Our aim was to determine the cellular distribution of ghrelin and GHS-R1a in human tissue using immunocytochemistry and fluorescent double staining.

Human tissue was obtained with ethical approval. Sections (30µm) of fresh frozen ventricle (n=7 patients) atrium (n=5), coronary artery (n=12), radial artery (n=6), left internal mammary artery (n=15), saphenous vein (n=14), kidney (n=3) and lung (n=4) were stained using rabbit-anti-ghrelin (RaG) or rabbit-anti-GHS-R1a (RaGR) primary antisera in a peroxidase-anti-peroxidase protocol. Adjacent sections were double labelled using RaG antisera and mouse-anti-van Willebrand Factor (vWF) monoclonal antibody (vWFma) or RaGR antisera and vWFma or mouse-anti-smooth muscle a-actin monoclonal antibody, detected by fluorescence labelled secondary antibodies. Sections were viewed using a standard light or confocal laser scanning microscope.

Ghrelin-like immunoreactivity (ghrelin-LI) was detected in endothelial cells of all vessels examined, confirmed by the co-localisation with the endothelial marker vWF (Fig.1). GHS-R1a-like immunoreactivity (GHSR-LI) was present in blood vessels and double staining with the endothelial marker vWF or the smooth muscle marker a-actin revealed that GHSR-LI localised to endothelial cells and vascular smooth muscle cells. GHSR-LI was also present in atrial and ventricular cardiomyocytes.

Fig.1:Photomicro-graphs show ghrelin-like immunoreactivity in endothelial cells of small coronary arteries (a) and saphenous vein (b). Scalebar=50µm

We report for the first time ghrelin-LI in vascu-lar endothelial cells of all tissues examined. This suggests that ghrelin is secreted from endothelial cells and may elicit cardiovascular effects acting on GHSR-LI, the receptor that we detected on cardiomyocytes and vascular smooth muscle cells. Furthermore, the presence of GHSR-LI on endothelial cells makes us hypothesise a negative feedback loop regulating endothelial ghrelin synthesis. Ghrelin may therefore represent a novel paracrine vascular mediator. In obesity, circulating ghrelin levels decrease (Shiiya et al., 2002). Obesity is also associated with atherosclerosis and endothelial dysfunction. We therefore speculate, that endothelial dysfunction in obese patients may result in impaired endothelial ghrelin synthesis. The resulting loss of ghrelin-mediated vasodilation may contribute to the development of hypertension in obese individuals.

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