Binding of neuromedin U to cell surface recombinat human receptors is essentially irreversible under phisiological confdintions Brighton P.J, Szekeres P.G* & Willars G.B. Department of Cell Physiology and Pharmacology, University of Leicester, and *GlaxoSmithKline, Harlow, UK.	Print Abstract Search PubMed for: Brighton PJ Szekeres PG Willars GB

The neuropeptide neuromedin U (NmU) mediates contraction of smooth muscle from a variety of tissues and may be an important regulator of blood flow, cancer, stress, anxiety, and obesity. Two G-protein coupled receptors for NmU (NmU-R1 and NmU-R2) have been cloned that have ~50% sequence homology and couple to phosphoinositide and Ca²⁺ signalling.

Addition of 10nM human NmU-25 (at 37°C) to fluo-3-loaded HEK-293 cells expressing recombinant human NmU-R1 or -R2 caused a robust (2-3 fold of basal), rapid (~5s) peak elevation of intracellular [Ca²⁺] ([C²⁺]_i) followed by a much lower (1.2-1.4 fold of basal) sustained phase. The pEC₅₀ values for the peaks were 9.41+0.09 and 9.37+0.06 for NmU-R1 and -R2 respectively; mean+sem, n=3. In re-challenge experiments, a second addition of 10nM NmU-25 following extensive washing with Kreb's buffer (pH 7.4) failed to elevate [Ca²⁺]_i. Although this suggested desensitisation, this was inconsistent with both a sustained [Ca²⁺]_i elevation and prolonged (60 min) NmU-mediated accumulation (~30 fold of basal) of [³H]-inositol phosphates in Li⁺-blocked, [³H]-inositol labelled cells. Using three approaches we examined whether the absence of a second NmU-mediated [Ca²⁺]_i response was a consequence of the inability to remove receptor-bound NmU.

Firstly, perfusion of buffer alone subsequent to challenge with 10nM NmU-25 failed to lower [Ca²⁺]_i. In contrast, during sustained [Ca²⁺]_i elevation caused by activation of endogenous G_q/11-coupled muscarinic M₃ receptors with 100µM carbachol, perfusion of buffer returned [Ca²⁺]_i to basal levels.

Secondly we used the phenomenon of crosstalk between G_q/11- and G_s-coupled receptors. Thus, activation of endogenous ß2-adrenoceptors with 10µM arterenol did not elevate [Ca²⁺]_i. However, addition of arterenol during a carbachol-mediated plateau phase, caused a robust, transient [Ca²⁺]_i response. This crosstalk was dependent on continued activation of G_q/11-coupled receptors as no Ca²⁺ response to arterenol was seen after removal of carbachol. Addition of arterenol following activation of either NmU receptor also caused robust (2 fold of basal) Ca²⁺ signals. Crosstalk was still evident following perfusion in an attempt to remove NmU.

Finally we used fluorescently tagged NmU-8 (Cy3b-NmU-8). Following addition of 10nM Cy3b-NmU-8 at 20°C, confocal imaging of cells expressing either receptor showed strong membrane fluorescence that was not seen after pre-addition of 1µM NmU-25 or in wild-type cells. This fluorescence was not diminished by perfusion of buffer (12min) but was removed by lowering buffer pH to 2.0. Following return to pH 7.4, addition of Cy3b-NmU-8 resulted in membrane fluorescence.

Thus, NmU receptors do not rapidly and fully desensitise. Furthermore, binding of NmU to cell-surface, recombinant human receptors is essentially irreversible. Indeed, incubation of cells with Cy3b-NmU-8 for 12 min at 20° (or 5min at 37°C) resulted in punctate intracellular fluorescence, suggesting internalization of the ligand with the receptor.

Acknowledgements: BBSRC and GSK for funding; J. Scott and M. Ruediger (GSK) for Cy3b-NmU-8.