UTP binding and phosphoinositidase C activation in ampulla from frog semicircular canal
| Autor: | Daniel Butlen, Marie Teixeira, Evelyne Ferrary |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Male
P2Y receptor Physiology Uridine Triphosphate 4 4'-Diisothiocyanostilbene-2 2'-Disulfonic Acid Tritium Binding Competitive Radioligand Assay chemistry.chemical_compound Physiology (medical) medicine Animals heterocyclic compounds Inner ear Ampulla Rana ridibunda Uridine triphosphate Semicircular canal Phosphoric Diester Hydrolases Receptors Purinergic P2 Chemistry Semicircular Canals Cell biology Enzyme Activation Kinetics medicine.anatomical_structure Biochemistry Pyridoxal Phosphate GRENOUILLE Platelet aggregation inhibitor Female Platelet Aggregation Inhibitors UTP binding |
| Zdroj: | American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 279:R803-R812 |
| ISSN: | 1522-1490 0363-6119 |
| DOI: | 10.1152/ajpregu.2000.279.3.r803 |
| Popis: | Pyrimidine nucleotide-sensitive phosphoinositidase C activity (PLC), previously identified in frog semicircular canal ampulla, was pharmacologically characterized. Binding of [3H]UTP and abilities of unlabeled nucleotide analogs to inhibit binding and to stimulate PLC in myo-[3H]inositol-loaded ampullas were determined. Specific [3H]UTP binding was competitively inhibited by UTP [apparent dissociation binding constant = 0.8 μM; Hill coefficient = 0.7]. Scatchard analysis revealed a minor class of high-affinity binding sites [45 fmol UTP bound/μg protein; dissociation constant ( KD1) = 0.4 μM] and a major class of moderate-affinity binding sites (365 fmol UTP bound/μg protein; KD2= 10 μM). 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 = α,β-methylene-ATP > uridine = diadenosine tetraphosphate (Ap4A); 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 ≥ Ap4A ≥ 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 [3H]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. |
| Databáze: | OpenAIRE |
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