Regulation of OX 1 orexin/hypocretin receptor‐coupling to phospholipase C by Ca 2+ influx

Background and purpose: Orexin (OX) receptors induce Ca 2+ elevations via both receptor‐operated Ca 2+ channels (ROCs) and the “conventional” phospholipase C (PLC)–Ca 2+ release–store‐operated Ca 2+ channel (SOC) pathways. In this study we assessed the ability of these different Ca 2+ influx pathways to amplify OX 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 1 receptors. Key results: Inhibition of total Ca 2+ influx by reduction of the extracellular [Ca 2+ ] to 1 μM 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 ∼0 mV, which disrupts most of the driving force for Ca 2+ entry. The inhibitor of the OX 1 receptor‐activated ROCs, tetraethylammonium chloride (TEA), was somewhat less effective than the reduction in extracellular [Ca 2+ ] at inhibiting PLC activation, probably because it only partially blocks ROCs. The partial inhibitor of both ROCs and SOCs, Mg 2+ , and the SOC inhibitors, dextromethorphan, SKF‐96365 (1‐[β‐(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 OX 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. British Journal of Pharmacology (2007) 150 , 97–104. doi: 10.1038/sj.bjp.0706959