Characterization of recombinant human orexin receptor pharmacology in a Chinese hamster ovary cell-line using FLIPR.
The cellular mechanisms underlying the physiological effects of the orexins are poorly understood. Therefore, the pharmacology of the recombinant human orexin receptors was studied using FLIPR. Intracellular calcium ([Ca2+]i) was monitored in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX1) or orexin-2 (OX2) receptors using Fluo-3AM. Orexin-A and orexin-B increased [Ca2+]i in a concentration dependent manner in CHO-OX1 (pEC50=8.03+/-0.08 and 7. 30+/-0.08 respectively, n=5) and CHO-OX2 (pEC50=8.18+/-0.10 and 8. 43+/-0.09 respectively, n=5) cells. This response was typified as a rapid peak in [Ca2+]i (maximal at 6 - 8 s), followed by a gradually declining secondary phase. Thapsigargin (3 microM) or U73122 (3 microM) abolished the response. In calcium-free conditions the peak response was unaffected but the secondary phase was shortened, returning to basal values within 90 s. Calcium (1.5 mM) replacement restored the secondary phase. In conclusion, orexins cause a phospholipase C-mediated release of calcium from intracellular stores, with subsequent calcium influx.
Smart
D
D
Neuroscience Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK. Darren_2_Smart@sbphrd.com
Jerman
J C
JC
Brough
S J
SJ
Rushton
S L
SL
Murdock
P R
PR
Jewitt
F
F
Elshourbagy
N A
NA
Ellis
C E
CE
Middlemiss
D N
DN
Brown
F
F
D000814:Aniline Compounds; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D064446:Orexin Receptors; D000068797:Orexins; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014966:Xanthenes
D000814:Aniline Compounds; D002118:Calcium; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010739:Phospholipase D; D011493:Protein Kinase C; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D011994:Recombinant Proteins; D019284:Thapsigargin; D010738:Type C Phospholipases; D014966:Xanthenes; C000596229:HCRT protein, human; C059715:Fluo-3