The orexin OX1 receptor regulates Ca2+ entry via diacylglycerol-activated channels in differentiated neuroblastoma cells
2006 (English)In: Journal of Neuroscience, ISSN 0270-6474, E-ISSN 1529-2401, Vol. 26, no 42, 10658-10666 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2006. Vol. 26, no 42, 10658-10666 p.
baculovirus, calcium, differentiation, neuroblastoma, orexin, TRP channel
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:uu:diva-153664DOI: 10.1523/JNEUROSCI.2609-06.2006ISI: 000241727300009PubMedID: 17050705OAI: oai:DiVA.org:uu-153664DiVA: diva2:417415