Regulation of OX sub(1) orexin/hypocretin receptor-coupling to phospholipase C by Ca super(2+) influx

• Published in: British journal of pharmacology Vol. 150; no. 1; pp. 97 - 104
• Main Authors: Johansson, L, Ekholm, ME, Kukkonen, J P
• Format: Journal Article
• Language: English
• Published: 01.01.2007
• ISSN: 0007-1188
• DOI: 10.1038/sj.bjp.0706959

Background and purpose: Orexin (OX) receptors induce Ca super(2+) elevations via both receptor-operated Ca super(2+) channels (ROCs) and the "conventional" phospholipase C (PLC)-Ca super(2+) release-store-operated Ca super(2+) channel (SOC) pathways. In this study we assessed the ability of these different Ca super(2+) influx pathways to amplify OX sub(1) receptor signalling to PLC in response to stimulation with the physiological ligand orexin-A. Experimental approach: PLC activity was assessed in CHO cells stably expressing human OX sub(1) receptors. Key results: Inhibition of total Ca super(2+) influx by reduction of the extracellular [Ca super(2+)] to 1 mu M effectively inhibited the receptor-stimulated PLC activity at low orexin-A concentrations (by 93% at 1 nM), and this effect was gradually reduced by higher orexin-A concentrations. A similar but weaker inhibitory effect (84% at 1 nM) was obtained on depolarization to similar to 0mV, which disrupts most of the driving force for Ca super(2+) entry. The inhibitor of the OX sub(1) receptor-activated ROCs, tetraethylammonium chloride (TEA), was somewhat less effective than the reduction in extracellular [Ca super(2+)] at inhibiting PLC activation, probably because it only partially blocks ROCs. The partial inhibitor of both ROCs and SOCs, Mg super(2+), and the SOC inhibitors, dextromethorphan, SKF-96365 (1-[ beta -(3-(4-methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole HCI) and 2-APB (2-aminoethoxydiphenyl borate), inhibited PLC activity at low concentrations of orexin-A, but were not as effective as TEA. Conclusions and implications: Both ROCs and SOCs markedly amplify the OX sub(1) receptor-induced PLC response, but ROCs are more central for this response. These data indicate the crucial role of ROCs in orexin receptor signalling.