Sensitivity of orexin-A binding to phospholipase C inhibitors, neuropeptide Y, and secretin

The binding of [I-125] orexin-A (Ox-A) to particulates from Chinese hamster ovary (CHO) cells expressing the cloned orexin-A receptor, or from rat for ebrain areas, was sensitive to blockers of phosphatidylinositol-specific ph ospholipase C (PtdIns-PLC) U-73122 and ET-18-OCH3, little affected by phosp holipase A(2) inhibitor quinacrine, and not sensitive to D609, a xanthate i nhibitor of phosphatidylcholine-selective PLC. Interaction of the receptor with a PtdIns-PLC was further indicated by a large sensitivity of the bindi ng to Ca2+. Up to 50% of the binding was sensitive to the G-protein nucleot ide site agonist GTP-gamma-S. Ligand attachment to the orexin-A receptor th us depends on an association with both PtdIns-PLC and G-protein alpha-subun its. In all paradigms examined, the binding of [I-125]orexin-A was competed by human/rat neuropeptide Y (hNPY) and porcine secretin with a potency sim ilar to orexin-A (IC50 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 se cretin greater than or equal to orexin-A > human pituitary adenylate cyclas e-activating peptide > orexin-B > porcine vasoactive intestinal peptide. Am ong opioid peptides, rat beta-endorphin and camel delta-endorphin were much less active than NPY and secretin, and two enkephalins were inactive at 1 mu M. In view of high abundance of NPY in forebrain, the above cross-reacti vity could indicate a significant contribution of NPY to signaling via orex in-A receptors. (C) 2000 Academic Press.