Sensitivity of orexin-A binding to phospholipase C inhibitors, neuropeptide Y, and secretin.
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.
Kane
J K
JK
Department of Pharmacology, University of Tennessee College of Medicine, Memphis 38163, USA.
Tanaka
H
H
Parker
S L
SL
Yanagisawa
M
M
Li
M D
MD
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000819:agonists / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D047908:Intracellular Signaling Peptides and Proteins; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D009478:Neuropeptide Y / Q000378:metabolism*; D009479:Neuropeptides / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D064446:Orexin Receptors; D000068797:Orexins; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D016548:Prosencephalon / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012633:Secretin / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; C000596229:HCRT protein, human; D047908:Intracellular Signaling Peptides and Proteins; D009478:Neuropeptide Y; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D012633:Secretin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010741:Phospholipases A; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C105598:phosphatidylcholine-specific phospholipase C; D020962:Heterotrimeric GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D008274:Magnesium; D002118:Calcium