Immunocytochemical localization of the urotensin-II receptor, UT, to rat and human tissues: Relevance to function
Introduction
Urotensin-II (U-II) is a 12 amino acid peptide that was originally discovered in the urophysis of the teleost fish, Gillichthys mirabilis [35]. A gene encoding a human form of U-II was subsequently cloned [9] and the G protein-coupled receptor UT, a human homologue of the rat orphan receptor GPR14/SENR [31], [41], identified as the endogenous receptor for the human undecapeptide U-II (hU-II) [2]. In fish, U-II has a variety of endocrine functions and has been demonstrated to constrict fish smooth muscle [4], [35]. The fish peptide, containing the characteristic cyclic hexapeptide that is essential for the biological activity of U-II from all species [28], also shows vasoconstrictor properties in the blood vessels from mammals [16], [20], suggesting an evolutionary conserved role for this receptor system in the regulation of cardiovascular function. This is supported by the discovery of genes for UT and U-II in a number of mammalian and non-mammalian species including the flounder [26], frog [9], mouse [8], pig [33] and cat [3].
Messenger RNA (mRNA) encoding both UT [2], [11], [25] and U-II [2], [8], [12] is abundantly expressed in the cardiovascular tissue of rats and humans, with the highest levels of UT mRNA expression observed, unexpectedly, in skeletal muscle [11]. Accordingly, UT receptors have been identified, using radioligand binding, in human myocardium, blood vessels and skeletal muscle [29] as well as in rat blood vessels [20], [29]. U-II has been reported to be the most potent vasoconstrictor of mammalian blood vessels discovered [2] and also elicits endothelium-dependent vasodilatation. Localization of the peptide to vascular endothelial cells of healthy [2], [30] and diseased [12], [30] human blood vessels using immunocytochemistry suggest that U-II acts as an autocrine/paracrine mediator at least in the vasculature. Whether U-II induces vasoconstriction or vasodilatation appears to depend on the species (for example, U-II contracts human arteries and veins in vitro [29] but is arterioselective in monkey [2]) and the vascular bed investigated and on the presence or absence of a functional endothelium, for review see refs. [10], [28]. In the conscious rat, in vivo, the effect of infused U-II is an increase in heart rate, blood pressure and vascular conductance in the hindquarters, effects attenuated by the nitric oxide synthase (NOS) inhibitor L-NAME and the non-selective cyclo-oxygenase inhibitor indomethacin [15].
To examine whether differential expression of UT receptors in cardiovascular tissues from rats and humans may account for the diversity of vascular actions observed for U-II we have investigated the distribution of UT in a representative panel of rat tissues using standard immunocytochemistry. In skeletal muscle, additional experiments were performed to localize NOS isoforms and the endothelial cell marker von Willebrand factor for comparison. We employed fluorescent double labeling confocal microscopy to identify the specific cell types expressing UT in rat compared to human tissue and in human umbilical vein endothelial cells (HUVECs) to establish morphological evidence for the involvement of either the synthetic or secretory pathways in the production and release of endothelial U-II.
Section snippets
Materials
Rabbit anti-UT receptor (human) antibody was purchased from Lifespan Biosciences (Seattle, WA, USA). UT receptor blocking peptide was from MBL International Corporation (Woburn, MA, USA). Human urotensin-II and rabbit anti-human urotensin-II antibody were from Peptide Institute Inc. (Osaka, Japan). Mouse anti-NOS I (brain/neuronal NOS), anti-NOS III (endothelial NOS) and anti-NOS II (inducible NOS) monoclonal antibodies were from Affiniti Research Products Ltd. (Exeter, UK). Mouse anti-human
Immunocytochemistry
In rat heart, UT-LI was localized to cardiomyocytes of the left and right atrium and ventricle (Fig. 2A). UT-LI was also detected in hindquarter skeletal muscle and in smooth muscle cells forming the medial layer of blood vessels from the rat heart, hindquarters (Fig. 2B), kidney (Fig. 2C) and mesentery (Fig. 2D and H). UT-LI was absent or below the level of detection in endothelial cells lining these blood vessels (Fig. 2H). In rat skeletal muscle UT-LI appeared to localize to regions of the
Discussion
U-II has been shown, in vitro and in vivo, to contract [5], [23], [27], [28], [29], [30], [34] and relax [39] human arteries and veins and to elicit effects on human heart [36] actions presumed to be due to stimulation of UT receptors. We have now substantiated this hypothesis by demonstrating the presence of UT-LI in human cardiovascular tissues, confirming the ubiquitous localization of UT receptor protein to vascular smooth muscle and endothelial cells of human large and small diameter
Acknowledgments
Supported by grants from the British Heart Foundation, Cambridge European and Isaac Newton Trusts. We thank the Consultant and theatre staff of Papworth Hospital for collection of human tissues. HUVECs were a kind gift from Dr. Jun Wang (Department of Medicine, University of Cambridge).
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