From human to horse: binding and function of neuromedin U in the equine vasculature

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.

Acute laminitis, a vascular disease of the foot, affects over 8000 horses annually (Hinckley and Henderson, 1996), causing displacement of the pedal bone and considerable pain. Pathogenesis seems to involve an ischaemic-reperfusion injury with subsequent activation of matrix metalloproteinases and laminar separation (Bailey et al., 2004). Pharmacotherapy, in the form of vasodilators, has had variable clinical success, suggesting novel mediators involved in disease progression remain to be discovered. The aim of the current study is to translate orphan GPCR pharmacology in human vascular beds to the equine digital circulation. One potential candidate is neuromedin U (NMU); we have recently demonstrated potent and efficacious vasoconstrictor responses to NMU-25 in human coronary artery, radial artery (Maguire & Davenport, 2003) and saphenous vein (Mitchell et al., 2006).

Equine digital artery (EDA) and vein (EDV) were obtained from 6 horses slaughtered at a local abattoir. For saturation binding assays, sections of EDA and EDV were pre-incubated for 5 min in 20mM Tris-HCl buffer (5mM MgCl2, 2mM EGTA and 0.1mg/ml bacitracin, pH7.4) and subsequently incubated with increasing concentrations of [125I]-NMU-25 (2pM – 1nM) for 60 min. Non-specific binding was defined with 10μM NMU-25. Sections were washed in Tris-HCl buffer (50mM, pH7.4) at 4°C for 10 min, air-dried and apposed to radiation-sensitive film, together with 125I-microscale standards, for 5 days. Films were analysed by computer-assisted densitometry and data evaluated using the KELL suite of programmes (Biosoft, Cambridge, UK). For in vitro studies, isolated rings (4mm) of endothelium-denuded digital vessels were mounted in organ baths containing oxygenated Krebs’ solution for isometric force measurements. Cumulative concentration-response curves were constructed to NMU-25 (1x10-11 to 3x10-7 M) and terminated with 0.1M 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 for potency (pD2) and efficacy (Emax). All data are expressed as mean ± s.e.m. and n values are the number of horses from which tissue was obtained.

In EDA and EDV, [125I]-NMU-25 binding was specific, saturable and of high affinity (KD: 0.22 ± .0.06nM, 0.49 ± 0.18nM respectively), with receptor densities of 4.09 ± 0.20 and 4.89 ± 0.27 fmol/mg protein (n=3). Receptor autoradiography indicated that binding was to the medial smooth muscle layer of these vessels. Functionally, NMU-25 constricted EDV with equivalent potency to ET-1 (pD2: NMU-25 7.75 ± 0.28 (n=4); ET-1 7.67 ± 0.31 (n=5)), although it was significantly less effective at contraction (Emax: NMU-25 11.69 ± 0.43 %KCl; ET-1 66.49 ± 7.62 %KCl). No responses were obtained in EDA.

For the first time, we have identified binding and function for NMU in equine digital vessels. Future aims are to establish whether NMU is present as a local transmitter in the equine digital circulation and to verify the identity of the [125I]-NMU-25 binding site. In addition, it is yet to be determined whether receptor or peptide expression are altered in laminitis.

Bailey S. et al. (2004) Veterinary Journal, 167, 129-142
Hinckley K.A. & Henderson I.W. (1996) Proceedings of the 35 th Congress of BEVA
Maguire J.J. & Davenport A.P. (2003) Proceedings of the British Pharmacological Society at http://www.pa2online.org/Vol1Issue2abst028P.html
Mitchell J.D. et al. (2006) Proceedings of the British Pharmacological Society at http://www.pa2online.org/abstracts/Mitchell_Horsham.pdf

We would like to thank The RCVS Trust and British Heart Foundation.