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

Biochem Biophys Res Commun. 2000 Jun 16;272(3):959-65. doi: 10.1006/bbrc.2000.2880.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding, Competitive
  • CHO Cells
  • Calcium / metabolism
  • Calcium / pharmacology
  • Carrier Proteins / antagonists & inhibitors*
  • Carrier Proteins / metabolism*
  • Cricetinae
  • Enzyme Inhibitors / metabolism*
  • Enzyme Inhibitors / pharmacology
  • Guanosine 5'-O-(3-Thiotriphosphate) / pharmacology
  • Heterotrimeric GTP-Binding Proteins / agonists
  • Heterotrimeric GTP-Binding Proteins / metabolism
  • Humans
  • Inhibitory Concentration 50
  • Intracellular Signaling Peptides and Proteins*
  • Magnesium / metabolism
  • Magnesium / pharmacology
  • Male
  • Neuropeptide Y / metabolism*
  • Neuropeptides / antagonists & inhibitors*
  • Neuropeptides / metabolism*
  • Orexin Receptors
  • Orexins
  • Phosphatidylinositol Diacylglycerol-Lyase
  • Phosphoinositide Phospholipase C
  • Phospholipases A / antagonists & inhibitors
  • Prosencephalon / drug effects
  • Prosencephalon / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Receptors, G-Protein-Coupled
  • Receptors, Neuropeptide / antagonists & inhibitors
  • Receptors, Neuropeptide / genetics
  • Receptors, Neuropeptide / metabolism
  • Secretin / metabolism*
  • Signal Transduction / drug effects
  • Type C Phospholipases / antagonists & inhibitors*
  • Type C Phospholipases / metabolism

Substances

  • Carrier Proteins
  • Enzyme Inhibitors
  • HCRT protein, human
  • Intracellular Signaling Peptides and Proteins
  • Neuropeptide Y
  • Neuropeptides
  • Orexin Receptors
  • Orexins
  • Receptors, G-Protein-Coupled
  • Receptors, Neuropeptide
  • Secretin
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Phospholipases A
  • Type C Phospholipases
  • Phosphoinositide Phospholipase C
  • phosphatidylcholine-specific phospholipase C
  • Heterotrimeric GTP-Binding Proteins
  • Phosphatidylinositol Diacylglycerol-Lyase
  • Magnesium
  • Calcium