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

The binding of [¹²⁵I] 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-OCH₃, little affected by phospholipase A₂ 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 Ca²⁺. Up to 50% of the binding was sensitive to the G-protein nucleotide site agonist GTP-γ-S. Ligand attachment to the orexin-A receptor thus depends on an association with both PtdIns-PLC and G-protein α-subunits. In all paradigms examined, the binding of [¹²⁵I]orexin-A was competed by human/rat neuropeptide Y (hNPY) and porcine secretin with a potency similar to orexin-A (IC₅₀ range 30-100 nM). The rank order of potency for NPY-related peptides was hNPY > porcine peptide YY (pPYY) > (Leu³¹, Pro³⁴) human PYY > human PYY( 3-36) > hNPY free acid > human pancreatic polypeptide. Among secretin-related peptides, the rank order of potency was porcine secretin > orexin-A > human pituitary adenylate cyclase-activating peptide > orexin-B > porcine vasoactive intestinal peptide. Among opioid peptides, rat β-endorphin and camel δ-endorphin were much less active than NPY and secretin, and two enkephalins were inactive at 1 μM. 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. (C) 2000 Academic Press.