Purine and pyrimidine nucleotide-sensitive phospholipase A2 in ampulla from frog semicircular canal

Abstract

This study was attempted to characterize pharmacologically the P2Y receptors triggering phospholipase A2 (PLA2) activation in ampulla from frog semicircular canal. A microassay was developed to screen the abilities of UTP analogs to stimulate [3H]arachidonic acid release by labeled ampullas. At 26°C UTP induced a dose-dependent and saturable increase of PLA2 activity (apparent activation constant 1.3 ± 0.4 μM, Hill coefficient 0.9 ± 0.2, maximal stimulating factor 2.0 ± 0.1). The rank order of potency of agonists for PLA2 activation was UTP ≥ UDP > adenosine 5′- O-(2-thiodiphosphate) = adenosine 5′- O-(3-thiotriphosphate) ≥ ATP = 2-methylthio-ATP ≥ ADP = diadenosine tetraphosphate ≥ α,β-methylene-ATP = CTP > 2′ and 3′- O-(4-benzoylbenzoyl)-ATP ≥ AMP = UMP >> uridine and adenosine. UTP- and 2-methylthio-ATP-induced PLA2 activations were inhibited by U-73122, GF-109203X, and methyl arachidonyl fluorophosphate. Basal activity was stimulated by phorbol ester and epinephrine and reduced by vasotocin, isoproterenol, prostaglandin E2, cAMP, and forskolin. H-89 restored the cAMP- and forskolin-inhibited PLA2 activities. Results indicate that P2Y receptor-mediated PLA2 stimulation requires phopholipase C and protein kinase C activations and basal activity is inhibited by agonist-stimulated cAMP-dependent mechanisms.