The Orexin OX sub(1) Receptor Activates a Novel Ca super(2+) Influx Pathway Necessary for Coupling to Phospholipase C

Ca super(2+) elevations in Chinese hamster ovary cells stably expressing OX sub(1) receptors were measured using fluorescent Ca super(2+) indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca super(2+) elevations with an EC sub(50) around 1 nM. When the extracellular [Ca super(2+)] was reduced to a submicromolar concentration, the EC sub(50) was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mM external Ca super(2+) was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca super(2+). The shift in the potency was not caused by depletion of intracellular Ca super(2+) but by a requirement of extracellular Ca super(2+) for production of inositol 1,4,5-trisphosphate. Fura-2 experiments with the "Mn super(2+)-quench technique" indicated a direct activation of a cation influx pathway by OX sub(1) receptor independent of Ca super(2+) release or pool depletion. Furthermore, depolarization of the cells to +60 mV, which almost nullifies the driving force for Ca super(2+) entry, abolished the Ca super(2+) response to low concentrations of orexin-A. The results thus suggest that OX sub(1) receptor activation leads to two responses, (i) a Ca super(2+) 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.