Abstract
The orexins are peptide transmitters/hormones, which exert stimulatory actions in many types of cells via the G-protein-coupled OX(1) and OX(2) receptors. Our previous results have suggested that low (subnanomolar) concentrations of orexin-A activate Ca(2+) entry, whereas higher concentrations activate phospholipase C, Ca(2+) release, and capacitative Ca(2+) entry. As shown here, the Ca(2+) 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 Ca(2+) response to subnanomolar orexin-A concentrations was also blocked by Mg(2+), 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 Mg(2+), dextromethorphan, and tetraethylammonium. Responses to subnanomolar concentrations of orexin-A (Ca(2+) 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 OX(1) receptor leads to opening of a Ca(2+)-permeable channel, involving trpc1 and -3, which is controlled by protein kinase C.
Highlights
The orexins are peptide transmitters/hormones, which exert stimulatory actions in many types of cells via the G-protein-coupled OX1 and OX2 receptors
Effect of Mg2ϩ and Ion Channel Blockers on Orexin-A Evoked Ca2ϩ Elevation in Cell Suspensions—To distinguish the orexinactivated Ca2ϩ influx pathway from intracellular release and capacitative Ca2ϩ entry, we tested the effect of different inhibitors of cation channels on the response to low and high concentrations of orexin-A (Ox-A) and capacitative entry activated by thapsigargin in CHO-hOX1-C1
Application of 5 mM Mg2ϩ was less effective and blocked the trpc3 current by 49.6 Ϯ 2.0% (n ϭ 6). Orexins and their receptor can be found scattered in many brain areas at low density, but recently they have been detected outside the central nervous system, in organs involved in feeding and energy metabolism
Summary
The orexins are peptide transmitters/hormones, which exert stimulatory actions in many types of cells via the G-protein-coupled OX1 and OX2 receptors. The Ca2؉ response to subnanomolar orexin-A concentrations was blocked by Mg2؉, dextromethorphan, and tetraethylammonium These treatments neither affected the response to high concentrations of orexin-A nor the thapsigargin-stimulated capacitative entry. Activation of a novel Ca2ϩ influx pathway was suggested The existence of such a pathway is indirectly suggested by the dependence of the Ca2ϩ response on a negative internal membrane potential and the activation of a robust influx of Mn2ϩ ions in the absence of store discharge [9]. The identity of this Ca2ϩ influx pathway and the mechanisms involved in its activation remain unresolved. These include storeoperated Ca2ϩ channels and second messenger-operated channels as well as Ca2ϩ-activated Ca2ϩ channels
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
