| 10860858 |
Kane JK, Tanaka H, Parker SL, Yanagisawa M, Li MD: Sensitivity of orexin-A binding to phospholipase C inhibitors, neuropeptide Y, and secretin. Biochem Biophys Res Commun. 2000 Jun 16;272(3):959-65.
The binding of [(125) I] orexin-A (Ox-A) to particulates from Chinese hamster ovary (CHO) cells expressing the cloned orexin-A receptor, or from rat forebrain areas, was sensitive to blockers of phosphatidylinositol-specific phospholipase C (PtdIns-PLC) U-73122 and ET-18-OCH (3), little affected by phospholipase A (2) inhibitor quinacrine, and not sensitive to D609, a xanthate inhibitor of phosphatidylcholine-selective PLC. Interaction of the receptor with a PtdIns-PLC was further indicated by a large sensitivity of the binding to Ca (2+). Up to 50% of the binding was sensitive to the G-protein nucleotide site agonist GTP-gamma-S. Ligand attachment to the orexin-A receptor thus depends on an association with both PtdIns-PLC and G-protein alpha-subunits. In all paradigms examined, the binding of [(125) I] orexin-A was competed by human/rat neuropeptide Y (hNPY) and porcine secretin with a potency similar to orexin-A (IC (50) range 30-100 nM). The rank order of potency for NPY-related peptides was hNPY > porcine peptide YY (pPYY) > (Leu (31), Pro (34)) human PYY > human PYY (3-36) > hNPY free acid > human pancreatic polypeptide. Among secretin-related peptides, the rank order of potency was porcine secretin > or = orexin-A > human pituitary adenylate cyclase-activating peptide > orexin-B > porcine vasoactive intestinal peptide. Among opioid peptides, rat beta-endorphin and camel delta-endorphin were much less active than NPY and secretin, and two enkephalins were inactive at 1 microM. In view of high abundance of NPY in forebrain, the above cross-reactivity could indicate a significant contribution of NPY to signaling via orexin-A receptors. |
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