G-Protein-Coupled Receptor KISS1; novel vasoconstrictor action in human coronary artery and umbilical vein

Emma J. Mead, Rhoda E. Kuc, Janet J. Maguire & Anthony P. Davenport. Clinical Pharmacology Unit, University of Cambridge, Level 6, Centre for Clinical Investigation, Box 110 Addenbrooke’s Hospital, Cambridge, CB2 2QQ, UK.

The kisspeptins, KP-54, KP-14, KP-13 and KP-10, are cleavage products of the KiSS-1 gene, which have recently been paired with the orphan G-protein coupled receptor KISS1 (GPR54)(Ohtaki et al., 2001). Previously the KPs have been described as potent inhibitors of metastasis and unexpectedly, as a molecular switch for puberty(Harms et al., 2003; Seminara et al., 2003). We have recently discovered that in addition to these actions, KPs function as equipotent and equiefficacious vasoconstrictors of rat aorta in vitro (Mead et al., 2005). Interestingly, we have also shown that KISS1 mRNA has a remarkably discrete localisation to the smooth muscle of large diameter blood vessels originating from the aorta-gonad-mesonephros; aorta, coronary artery (CA) and umbilical vein (UV). Furthermore we identified localisation of KISS1 and KPs to vascular endothelial cells. We aimed to characterise expression of KISS1 in the smooth muscle of human aorta, and to detect if KPs are circulating in human plasma using our novel radioligand [125I]KP-13, and finally to test if KP-54 functions as a constrictor of human CA and UV.

Saturation binding curves were constructed using a 2 hour incubation with increasing concentrations (7.8pM-4nM) of [125I]KP-13 on sections of human aorta. Non-specific binding was determined by addition of an excess (1µM) of KP-13(Davenport et al., 2005). Binding data was analysed using iterative, non-linear, curve fitting programmes, all values are expressed as mean±s.e.mean. Radioimmunoassay was carried out in plasma from n=4 individuals, using [125I]KP-13, and anti-KP-10-NH2 (human) serum with standard charcoal detection. Isolated rings of human CA or UV were bathed in oxygenated Krebs’ solution and isometric force measurements taken. Optimum resting tension was determined by responses to 100mM KCl at incrementally increasing levels of basal tension. Cumulative concentration-response curves were constructed to KP-54 (1x10-12-3x10-7M) and terminated by addition of 100mM KCl to determine maximum contractile response. Responses are expressed as % post KCl and data were fitted to a four parameter logistic equation.

Table 1. Constrictor activity of KP-54 in human CA and UV. n= number of tissues in which responses were recorded.

Saturable, high affinity binding, in the sub-nanomolar range (KD 0.37±0.17 nM, Bmax; 6.18±0.58 fmol/mg), of [125I]KP-13 was detected in smooth muscle of human aorta and KPs were detected circulating at 0.7fmol/ml. KP-54 functioned as a vasoconstrictor in both CA and UV, as we predicted from expression of KISS1 on the smooth muscle of these vessels. We have developed a novel radioligand for the quantification of KISS1 in human tissue and have detected low levels of KPs circulating in plasma. Importantly, we have identified a previously undescribed role of KP-54 as a constrictor of human CA and UV.


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