The G-protein coupled receptor KISS1, the molecular switch in puberty, has a novel localisation in the cardiovascular system

Helen Kirby, Rhoda Kuc, Anthony Davenport
University of Cambridge, Cambridge, United Kingdom

Kisspeptin (KP)-54, the endogenous ligand of the GPCR KISS1 (GPR54) (Ohtaki et al. 2001), is known to have roles in metastasis suppression and placentation (Lee et al. 1996), processes involving angiogenesis. Recently it has also been found to act as a molecular switch for puberty (Seminara et al., 2003). We have previously described a novel distribution of KISS1 to endothelial cells and smooth muscle cells of human umbilical vein and the atherosclerosis-prone coronary artery and aorta. We have also reported constrictor function of KP-54, and its cleavage products KP-10 and KP-13, in human umbilical vein and coronary artery (Mead et al., 2007). The rat is an invaluable model species for the study of disease. However, to date, no investigations have been made into the cardiovascular actions of kisspeptins in this species so we aimed to determine the cellular distribution of the receptor protein and pharmacologically characterise the receptor in rat cardiovascular tissues.

Receptor protein was detected by Western blotting on heart homogenates (n=3) from male Sprague-Dawley rats (350-450g) euthanised by CO2 inhalation, using SDS-PAGE and a semi-dry blotting method, before probing with rabbit anti-KISS1 and detection via the ECl method. For localisation by immunocytochemistry, 10μm cryostat-cut heart and aorta sections were incubated with rabbit anti-KISS1 antibody and detection via a peroxidase/antiperoxidase method. For cellular colocalisation, sections of heart were incubated with smooth muscle α-actin (SMαA) or von Willebrand factor (vWF) and rabbit anti-KISS1. For receptor autoradiography, sections were incubated with 0.2nM [125I]KP-13 for 2 hours at 22°C. Sections were washed, air dried and apposed to radiation-sensitive film for 7 days before determination of specific binding (amol/mm2). For saturation binding assays, sections were incubated with increasing concentrations (4pM–2nM) of [125I]KP-13 and non-specific binding determined by inclusion of 1μM KP-10.

Western blot analysis of a novel anti-KISS1 (rat) antibody revealed a single band of 43kDa in all 3 samples, corresponding to the KISS1 protein. Immunocytochemistry and autoradiography showed widespread distribution of KISS1 throughout the rat heart, with high expression in the aorta, intramyocardial blood vessels and cardiomyocytes (aorta: 22.3±2.5 amol/mm2, n=3; myocardium: 29.1±10.1 amol/mm2, n=5). Dual-labelling revealed colocalisation with both a smooth muscle and an endothelial cell marker. [125I]KP-13 bound with high affinity, KD=0.44±0.14nM and Bmax=11.1±2.2 fmol/mg (n=6) and was specific and saturable.

In conclusion, we have detected KISS1 throughout rat heart and, for the first time, have shown it to be localised to both vascular endothelial cells and smooth muscle cells. We have shown the presence of high affinity binding sites for [125I]KP-13 with a subnanomolar KD and a receptor density comparable to other vasoactive peptides such as endothelin and ghrelin. Our data suggest that KISS1 has a distribution in rat similar to that in human and hence may have a role as a vascular transmitter in this species.

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Seminara, S.B., et al. (2003). N Engl J Med, 349, 1614-1627