Purine and pyrimidine nucleotide-sensitive phospholipase A(2) in ampulla from frog semicircular canal

This study was attempted to characterize pharmacologically the P2Y receptors triggering phospholipase A(2) (PLA(2)) activation in ampulla from frog semicircular canal. A microassay was developed to screen the abilities of UTP analogs to stimulate [(3)H]arachidonic acid release by labeled ampullas. At 26 degrees C UTP induced a dose-dependent and saturable increase of PLA(2) activity (apparent activation constant 1.3 +/- 0.4 microM, Hill coefficient 0.9 +/- 0.2, maximal stimulating factor 2.0 +/- 0.1). The rank order of potency of agonists for PLA(2) activation was UTP > or = UDP > adenosine 5'-O-(2-thiodiphosphate) = adenosine 5'-O-(3-thiotriphosphate) > or = ATP = 2-methylthio-ATP > or = ADP = diadenosine tetraphosphate > or = alpha,beta-methylene-ATP = CTP > 2' and 3'-O-(4-benzoylbenzoyl)-ATP > or = AMP = UMP >> uridine and adenosine. UTP- and 2-methylthio-ATP-induced PLA(2) 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 E(2), cAMP, and forskolin. H-89 restored the cAMP- and forskolin-inhibited PLA(2) activities. Results indicate that P2Y receptor-mediated PLA(2) stimulation requires phopholipase C and protein kinase C activations and basal activity is inhibited by agonist-stimulated cAMP-dependent mechanisms.