UTP binding and phosphoinositidase C activation in ampulla from frog semicircular canal.
Pyrimidine nucleotide-sensitive phosphoinositidase C activity (PLC), previously identified in frog semicircular canal ampulla, was pharmacologically characterized. Binding of [(3)H]UTP and abilities of unlabeled nucleotide analogs to inhibit binding and to stimulate PLC in myo-[(3)H]inositol-loaded ampullas were determined. Specific [(3)H]UTP binding was competitively inhibited by UTP [apparent dissociation binding constant = 0.8 microM; Hill coefficient = 0.7]. Scatchard analysis revealed a minor class of high-affinity binding sites [45 fmol UTP bound/microgram protein; dissociation constant (K(D1)) = 0.4 microM] and a major class of moderate-affinity binding sites (365 fmol UTP bound/microgram protein; K(D2) = 10 microM). The stereospecificity pattern for UTP analog recognition was UMP > UDP >/= ADP = UTP = dTTP > adenosine 5'-O-(3-thiotriphosphate) = ATP = CTP = 2'-and 3'-O-4-(benzoylbenzoyl)-ATP (Bz-ATP) >/= AMP >/= 2-methylthio-ATP = alpha,beta-methylene-ATP > uridine = diadenosine tetraphosphate (Ap(4)A); cAMP and adenosine were inactive. Antagonist recognition pattern was DIDS = pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) = reactive blue 2 > suramin. The rank order of potencies for agonist-induced PLC activation was UDP >/= UTP >/= Ap(4)A >/= UMP = Bz-ATP; uridine was inactive. UTP-stimulated PLC activity was inhibited by DIDS = reactive blue 2 = PPADS > suramin. These results suggest that the population of [(3)H]UTP-labeled binding sites is heterogeneous, with a low number of high-affinity UTP receptors whose function(s) need to be determined and a large number of moderate-affinity receptors triggering PLC activation.
Teixeira
M
M
Institut National de la Santé et de la Recherche Médicale, Unité 426, Faculté de Médecine Xavier Bichat, 75870 Paris Cedex 18, France.
Ferrary
E
E
Butlen
D
D
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005260:Female; D007700:Kinetics; D008297:Male; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D011869:Radioligand Assay; D011895:Rana ridibunda; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D012665:Semicircular Canals / Q000737:chemistry / Q000201:enzymology*; D014316:Tritium; D014544:Uridine Triphosphate / Q000378:metabolism* / Q000494:pharmacology*
D010975:Platelet Aggregation Inhibitors; D018048:Receptors, Purinergic P2; D014316:Tritium; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D011732:Pyridoxal Phosphate; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D014544:Uridine Triphosphate