Regulation of OX1 orexin/hypocretin receptor-coupling to phospholipase C by Ca2+ influx

Background and purpose: Orexin (OX) receptors induce Ca2+ elevations via both receptor-operated Ca2+ channels (ROCs) and the conventional phospholipase C (PLC)-Ca2+ release-store-operated Ca2+ channel (SOC) pathways. In this study we assessed the ability of these different Ca2+ influx pathways to amplify OX, 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 OX1 receptors. Key results: Inhibition of total Ca2+ influx by reduction of the extracellular [Ca2+] 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 0 mV, which disrupts most of the driving force for Ca2+ entry. The inhibitor of the OX, receptor-activated ROCs, tetraethylammonium chloride (TEA), was somewhat less effective than the reduction in extracellular [Ca2+] at inhibiting PLC activation, probably because it only partially blocks ROCs. The partial inhibitor of both ROCs and SOCs, Mg2+, and the SOC inhibitors, dextromethorphan, SKF-96365 (1-[beta-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole HCl) 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 OX1 receptor-induced PLC response, but ROCS are more central for this response. These data indicate the crucial role of ROCs in orexin receptor signalling.