title: Orexin A-induced extracellular calcium influx in prefrontal cortex neurons involves L-type calcium channels.
abstract: Orexins, novel excitatory neuropeptides from the lateral hypothalamus, have been strongly implicated in the regulation of sleep and wakefulness. In this study, we explored the effects and mechanisms of orexin A on intracellular free Ca2+ concentration ([Ca2+]i) of freshly dissociated neurons from layers V and VI in prefrontal cortex (PFC). Changes in [Ca2+]i were measured with fluo-4/AM using confocal laser scanning microscopy. The results revealed that application of orexin A (0.1-1 microM) induced increase of [Ca2+]i in a dose-dependent manner. This elevation of [Ca2+]i was completely blocked by pretreatment with selective orexin receptor 1 antagonist SB 334867. While depletion of intracellular Ca2+ stores by the endoplasmic reticulum inhibitor thapsigargin (2 pM), [Ca2+]i in PFC neurons showed no increase in response to orexin A. Under extracellular Ca2+-free condition, orexin A failed to induce any changes of Ca2+ fluorescence intensity in these acutely dissociated cells. Our data further demonstrated that the orexin A-induced increase of [Ca2+]i was completely abolished by the inhibition of intracellular protein kinase C or phospholipase C activities using specific inhibitors, BIS II (1 microM) and D609 (10 microM), respectively. Selective blockade of L-type Ca2+ channels by nifedipine (5 microM) significantly suppressed the elevation of [Ca2+]i induced by orexin A. Therefore, these findings suggest that exposure to orexin A could induce increase of [Ca2+]i in neurons from deep layers of PFC, which depends on extracellular Ca2+ influx via L-type Ca2+ channels through activation of intracellular PLC-PKC signaling pathway by binding orexin receptor 1.
authors.affiliation: Department of Physiology, Third Military Medical University, 30 Gao tanyan Street, Chongqing 400038, China.
authors.forename: J X
authors.initials: JX
authors.lastname: Xia
authors.forename: S Y
authors.initials: SY
authors.lastname: Fan
authors.forename: J
authors.initials: J
authors.lastname: Yan
authors.forename: F
authors.initials: F
authors.lastname: Chen
authors.forename: Y
authors.initials: Y
authors.lastname: Li
authors.forename: Z P
authors.initials: ZP
authors.lastname: Yu
authors.forename: Z A
authors.initials: ZA
authors.lastname: Hu
mesh_terms: D000814:Aniline Compounds; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology*; D018613:Microscopy, Confocal; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D009479:Neuropeptides / Q000494:pharmacology*; D009543:Nifedipine / Q000494:pharmacology; D000068797:Orexins; D017397:Prefrontal Cortex / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D019284:Thapsigargin / Q000494:pharmacology; D014966:Xanthenes
chemical_list: D000814:Aniline Compounds; D020746:Calcium Channels, L-Type; C409648:Fluo 4; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D000068797:Orexins; D014966:Xanthenes; D019284:Thapsigargin; D009543:Nifedipine; D002118:Calcium