The orexin OX1 receptor regulates Ca2+ entry via diacylglycerol-activated channels in differentiated neuroblastoma cells.
We studied the cellular response to orexin type 1 receptor (OX1R) stimulation in differentiated IMR-32 neuroblastoma cells. In vitro differentiation of IMR-32 cells with 5-bromo-2'-deoxyuridine leads to a neuronal phenotype with long neurite extensions and an upregulation of mainly N-type voltage-gated calcium channels. Transduction of differentiated IMR-32 cells with baculovirus harboring an OX1R-green fluorescent protein cDNA fusion construct resulted in appearance of fluorescence that was confined mainly to the plasma membrane in the cell body and to neurites. Application of orexin-A to fluorescent cells led to an increase in intracellular free Ca2+ concentration, [Ca2+]i. At low nanomolar concentrations of orexin-A, the response was reversibly attenuated by removal of extracellular Ca2+, by application of a high concentration (10 mM) of Mg2+, and by the pharmacological channel blocker dextromethorphan. A diacylglycerol, dioctanoylglycerol, but not thapsigargin or depolarization with potassium, mimicked the OX1R response with regard to Mg2+ sensitivity. A reverse transcription-PCR screening identified mRNAs for all transient receptor potential canonical (TRPC) channels, including TRPC3, TRPC6, and TRPC7, which are known to be activated by diacylglycerol. Expression of a dominant-negative TRPC6 channel subunit blunted the responses to both dioctanoylglycerol and OX1R stimulation. The results suggest that the OX1R activates a Ca2+ entry pathway that involves diacylglycerol-activated TRPC channels in neuronal cells.
Näsman
Johnny
J
A. I. Virtanen Institute for Molecular Sciences, Department of Neurobiology, University of Kuopio, FIN-70211 Kuopio, Finland. johnny.nasman@uku.fi
Bart
Genevieve
G
Larsson
Kim
K
Louhivuori
Lauri
L
Peltonen
Hanna
H
Akerman
Karl E O
KE
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D004075:Diglycerides / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007313:Insecta; D008274:Magnesium / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D064446:Orexin Receptors; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000502:physiology*
D015220:Calcium Channels; D004075:Diglycerides; D064446:Orexin Receptors; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; C044295:1,2-dioctanoylglycerol; D008274:Magnesium; D002118:Calcium