Evidence for binding and function of Neuromedin U-25 in the human cardiovascular system

John D. Mitchell, 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.

Neuromedin U (NMU) has been paired with the previously designated orphan G-protein coupled receptors NMU1 and NMU2 (Howard et al., 2000). In rats, NMU elicited a pressor response (Minamino et al., 1985) with reduced mesenteric blood flow reported in conscious animals in vivo (Gardiner et al., 1990), the latter suggesting local vasoconstriction. We have recently demonstrated potent vasoconstrictor responses to human neuromedin U-25 (NMU-25) in human coronary and radial artery (Maguire & Davenport, 2003). To further identify the role of NMU receptors in the human cardiovascular system we have extended the in vitro studies to human vein and characterised binding of [125I]-NMU-25 in human heart.

For saturation binding assays, 15µm sections of left ventricle from patients with dilated cardiomyopathy were pre-incubated for 5 min in 20mM Tris-HCl buffer (5mM MgCl 2, 2mM EGTA and 0.1mg/ml bacitracin, pH7.4) and subsequently for 60 min with increasing concentrations of [125I]-NMU-25. Non-specific binding was defined using 1µM NMU-25. Sections were washed in Tris-HCl buffer (50mM, pH7.4) at 4oC for 10 min and counted. Data were analysed using the KELL suite of programs (Biosoft, Cambridge, UK). For receptor autoradiography, heart sections were incubated for 60 min with 0.1nM [125I]-NMU-25. Non-specific binding was defined as above. Sections were washed, air-dried and apposed to radiation-sensitive film for 3 days. For in vitro studies, rings (4mm) of endothelium-denuded human saphenous vein (SV) were mounted in 5ml organ baths containing oxygenated Krebs’ solution (37 oC) for isometric force measurements. Cumulative concentration-response curves were constructed to NMU-25 (1x10-10 to 3x10-7 M) and terminated by addition of 100mM KCl to determine maximum contractile response. NMU-25 responses were expressed as % KCl response and data fitted to a four parameter logistic equation (FigP, Biosoft, Cambridge, UK) to obtain values of potency (pD2) and efficacy (Emax). All data are expressed as mean±s.e.m. and n values are the number of patients from whom tissue was obtained.

In human left ventricle, [125I]-NMU-25 bound with high affinity, KD = 58 ± 20 pM, and Bmax = 4.44 ± 0.71 fmol/mg protein (n=8) that was specific and saturable. A single site was preferred to a two-site model. Receptor autoradiography indicated that in heart binding was to cardiomyocytes. NMU-25 contracted human SV with pD 2 = 8.76 ± 0.34, equivalent to its potent effect on human artery, and Emax = 39.6 ± 6.2 %KCl (n=9).

n conclusion, we have determined binding characteristics for [125I]-NMU-25 in human heart. The KD value we obtained is comparable to that reported for binding of [125I]-NMU-25 to cells expressing either NMU1 or NMU2 (Brighton et al., 2004). The receptor subtypes expressed in human heart remain to be identified. Functionally, we have identified NMU-25 as a potent constrictor of human large arteries and veins.

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This work is funded by a studentship awarded by the British Heart Foundation.