Abstract
In this study, we have compared the abilities of orexin-A and orexin-B and variants of orexin-A to activate different Ca2+responses (influx and release) in human OX1 and OX2 receptor- expressing Chinese hamster ovary cells. Responses mediated by activation of both receptor subtypes with either orexin-A or -B were primarily dependent on extracellular Ca2+, suggesting similar activation of Ca2+influx as we have previously shown for orexin-A and OX1receptors. Amino acid-wise truncation of orexin-A reduced its ability to activate OX1 and OX2 receptors, but the response mediated by the OX2 receptor was more resistant to truncation than the response mediated by the OX1 receptor. We also performed a sequential replacement of amino acids 14 to 26 with alanine in the truncated orexin-A variant orexin-A14–33. Replacement of the same amino acids produced a fall in the potency for each receptor subtype, but the reduction was less prominent for the OX2 receptor. The most marked reduction was produced by the replacement of Leu20, Asp25, and His26 with alanine. Interestingly, extracellular Ca2+ dependence of responses to some of the mutated peptides was different from those of orexin-A and -B. The mutagenesis also suggests that although the determinants required from orexin-A for binding to and activation of the receptor are highly conserved between the orexin receptor subtypes, the OX2receptor requires fewer determinants. This might in part explain why orexin-B has the affinity and potency equal to orexin-A for this subtype, although it has 10- to 100-fold lower affinity and potency for the OX1 receptor.
Footnotes
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This study was supported by European Union contracts ERBBIO4CT960699 and QLG3-CT-2002-00826, by the Swedish Medical Research Council, the Cancer Research Fund of Sweden, the Lars Hierta Foundation, the Göran Gustafsson Foundation, the Novo Nordisk Foundation, the Academy of Finland, and the Sigrid Jusélius Foundation.
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DOI: 10.1124/jpet.102.048025
- Abbreviations:
- [Ca2+]e
- extracellular free calcium concentration
- [Ca2+]i
- intracellular free calcium concentration
- CHO
- Chinese hamster ovary
- CNS
- central nervous system
- Δ[Ca2+]i
- change in [Ca2+]i([Ca2+]i/stimulated − [Ca2+]i/basal)
- EC50
- concentration producing half-maximal response
- EGTA
- ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid
- IP3
- inositol 1,4,5-trisphosphate
- N
- the number of batches of cells for the measurements
- pEC50
- −log(EC50)
- probenecid
- p-(dipropylsulfamoyl)benzoic acid
- TBM
- TES-buffered medium
- TES
- 2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino) ethanesulfonic acid
- Received December 13, 2002.
- Accepted January 14, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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