The orexin OX1 receptor activates a novel Ca2+ influx pathway necessary for coupling to phospholipase C

Ca2+ elevations in Chinese hamster ovary cells stably expressing OX, receptors were measured using fluorescent Ca2+ indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca2+ elevations with an EC50 around 1 nM. When the extracellular [Ca2+] was reduced to a submicromolar concentration, the EC50 was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mM external Ca2+ was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca2+. The shift in the potency was not caused by depletion of intracellular Ca2+ but by a requirement of extracellular Ca2+ for production of inositol 1,4,5-trisphosphate. Fura-a experiments with the Mn2+-quench technique indicated a direct activation of a cation influx pathway by OX, receptor independent of Ca2+ release or pool depletion. Furthermore, depolarization of the cells to +60 mV, which almost nullifies the driving force for Ca2+ entry, abolished the Ca2+ response to low concentrations of orexin-A. The results thus suggest that OX, receptor activation leads to two responses, (i) a Ca2+ influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.