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Characterization of recombinant human orexin receptor pharmacology in a Chinese hamster ovary cell‐line using FLIPR
Author(s) -
Smart D,
Jerman J C,
Brough S J,
Rushton S L,
Murdock P R,
Jewitt F,
Elshourbagy N A,
Ellis C E,
Middlemiss D N,
Brown F
Publication year - 1999
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0702780
Subject(s) - chinese hamster ovary cell , phospholipase c , thapsigargin , calcium , calcium in biology , orexin , orexin receptor , receptor , endocrinology , medicine , biology , chemistry , pharmacology , microbiology and biotechnology , neuropeptide
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 ([Ca 2+ ] i ) was monitored in Chinese hamster ovary (CHO) cells stably expressing orexin‐1 (OX 1 ) or orexin‐2 (OX 2 ) receptors using Fluo‐3AM. Orexin‐A and orexin‐B increased [Ca 2+ ] i in a concentration dependent manner in CHO‐OX 1 (pEC 50 =8.03±0.08 and 7.30±0.08 respectively, n =5) and CHO‐OX 2 (pEC 50 =8.18±0.10 and 8.43±0.09 respectively, n =5) cells. This response was typified as a rapid peak in [Ca 2+ ] i (maximal at 6–8 s), followed by a gradually declining secondary phase. Thapsigargin (3 μ M ) or U73122 (3 μ M ) 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 m M ) replacement restored the secondary phase. In conclusion, orexins cause a phospholipase C‐mediated release of calcium from intracellular stores, with subsequent calcium influx. British Journal of Pharmacology (1999) 128 , 1–3; doi: 10.1038/sj.bjp.0702780