Orexin-A-induced Ca2+ entry -: Evidence for involvement of TRPC channels and protein kinase C regulation

The orexins are peptide transmitters/hormones, which exert stimulatory actions in many types of cells via the G-protein-coupled OX1 and OX2 receptors. Our previous results have suggested that low (subnanomolar) concentrations of orexin-A activate Ca2+ entry, whereas higher concentrations activate phospholipase C, Ca2+ release, and capacitative Ca2+ entry. As shown here, the Ca2+ response to subnanomolar orexin-A concentrations was blocked by activation of protein kinase C by using different approaches (12-O-tetradecanoylphorbol acetate, dioctanoylglycerol, and diacylglycerol kinase inhibition) and protein phosphatase inhibition by calyculin A. The Ca2+ response to subnanomolar orexin-A concentrations was also blocked by Mg2+, dextromethorphan, and tetraethylammonium. These treatments neither affected the response to high concentrations of orexin-A nor the thapsigargin-stimulated capacitative entry. The capacitative entry was instead strongly suppressed by SKF96365. An inward membrane current activated by subnanomolar concentrations of orexin-A and the currents activated upon transient expression of trpc3 channels were also sensitive to Mg2+, dextromethorphan, and tetraethylammonium. Responses to subnanomolar concentrations of orexin-A (Ca2+ elevation, inward current, and membrane depolarization) were voltage-dependent with a loss of the response around -15 mV. By using reverse transcription-PCR, mRNA for the trpc1-4 channel isoforms were detected in the CHO-hOX1-C1 cells. The expression of truncated TRPC channel isoforms, in particular trpc1 and trpc3, reduced the response to subnanomolar concentrations of orexin-A but did not affect the response to higher concentrations of orexin-A. The results suggest that activation of the OX1 receptor leads to opening of a Ca2+-permeable channel, involving trpc1 and -3, which is controlled by protein kinase C.