title: Cholecystokinin activates orexin/hypocretin neurons through the cholecystokinin A receptor.
abstract: Orexin A and B are neuropeptides implicated in the regulation of sleep/wakefulness and energy homeostasis. The regulatory mechanism of the activity of orexin neurons is not precisely understood. Using transgenic mice in which orexin neurons specifically express yellow cameleon 2.1, we screened for factors that affect the activity of orexin neurons (a total of 21 peptides and six other factors were examined) and found that a sulfated octapeptide form of cholecystokinin (CCK-8S), neurotensin, oxytocin, and vasopressin activate orexin neurons. The mechanisms that underlie CCK-8S-induced activation of orexin neurons were studied by both calcium imaging and slice patch-clamp recording. CCK-8S induced inward current in the orexin neurons. The CCKA receptor antagonist lorglumide inhibited CCK-8S-induced activation of orexin neurons, whereas the CCKB receptor agonists CCK-4 (a tetrapeptide form of cholecystokinin) and nonsulfated CCK-8 had little effect. The CCK-8S-induced increase in intracellular calcium concentration was eliminated by removing extracellular calcium but not by an addition of thapsigargin. Nifedipine, omega-conotoxin, omega-agatoxin, 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride, and SNX-482 had little effect, but La3+, Gd3+, and 2-aminoethoxydiphenylborate inhibited CCK-8S-induced calcium influx. Additionally, the CCK-8S-induced inward current was dramatically enhanced in the calcium-free solution and was inhibited by the cation channel blocker SKF96365, suggesting an involvement of extracellular calcium-sensitive cation channels. CCK-8S did not induce an increase in intracellular calcium concentration when membrane potential was clamped at -60 mV, suggesting that the calcium increase is induced by depolarization. The evidence presented here expands our understanding of the regulation of orexin neurons and the physiological role of CCK in the CNS.
authors.affiliation: Department of Molecular Pharmacology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Natsuko
authors.initials: N
authors.lastname: Tsujino
authors.forename: Akihiro
authors.initials: A
authors.lastname: Yamanaka
authors.forename: Kanako
authors.initials: K
authors.lastname: Ichiki
authors.forename: Yo
authors.initials: Y
authors.lastname: Muraki
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Kilduff
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Yagami
authors.forename: Satoru
authors.initials: S
authors.lastname: Takahashi
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Goto
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sakurai
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002766:Cholecystokinin / Q000502:physiology*; D004553:Electric Conductivity; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects; D045604:Extracellular Fluid / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009433:Neural Inhibition / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D009479:Neuropeptides / Q000378:metabolism*; D064446:Orexin Receptors; D000068797:Orexins; D018408:Patch-Clamp Techniques; D043886:Receptor, Cholecystokinin A / Q000378:metabolism / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012844:Sincalide / Q000031:analogs & derivatives / Q000494:pharmacology; D009435:Synaptic Transmission / Q000187:drug effects
chemical_list: C090019:8-sulfocholecystokinin octapeptide; C000596229:HCRT protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D043886:Receptor, Cholecystokinin A; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C492603:Ycam-2 fluorescent protein; C507867:yellow cameleon 2.1; D002766:Cholecystokinin; D012844:Sincalide; D002118:Calcium