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 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.
Larsson
Kim P
KP
A. I. Virtanen Institute for Molecular Sciences, Department of Neurobiology, Laboratory of Cell Biology, University of Kuopio, P. O. Box 1627, FIN-70211 Kuopio, Finland.
Peltonen
Hanna M
HM
Bart
Genevieve
G
Louhivuori
Lauri M
LM
Penttonen
Annika
A
Antikainen
Miia
M
Kukkonen
Jyrki P
JP
Akerman
Karl E O
KE
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D017931:DNA Primers; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology*; D007473:Ion Channels / Q000502:physiology*; D017136:Ion Transport; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000494:pharmacology*; D000068797:Orexins; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000378:metabolism*
D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; D007473:Ion Channels; D009479:Neuropeptides; D000068797:Orexins; D011493:Protein Kinase C; D008274:Magnesium; D002118:Calcium