Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), a putative P2Y(1) receptor antagonist, blocks signaling at a site distal to the receptor in Madin-Darby canine kidney-D(1) cells.
Substantial evidence documents the potential importance of P2Y receptor subtypes in the regulation of cellular responses, but few selective antagonists exist for these receptors. In the current study, we assessed the use of pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate (PPADS) as a putative P2Y(1) receptor-selective blocker in Madin-Darby canine kidney (MDCK-D(1)) cells. We found that the key action of PPADS in MDCK-D(1) cells was blockade of signaling at a postreceptor site. PPADS blocked UTP (P2Y(2))-stimulated accumulation of cAMP [which is dependent on arachidonic acid (AA) metabolism by cyclooxygenase] but not that by 2-methyl thio-adenosine triphosphate (2MeSATP; which is independent of cyclooxygenase and has been attributed to P2Y(1) and P2Y(11) receptors). By contrast, PPADS inhibited AA release mediated by both 2MeSATP and UTP. PPADS displayed uncompetitive antagonism in blockade of AA release in response to 2MeSATP. PPADS also inhibited AA release stimulated by various nucleotides, phenylephrine, and bradykinin, implying that the effect does not involve the inhibition of a specific receptor. Because PPADS also inhibited ionomycin-, thapsigargin-, and phorbol-12-myristate-13-acetate-promoted AA release, it appears to act at a site distal to an increase in intracellular Ca(2+) transients or PKC activation. Inhibition of melittin-stimulated AA release by PPADS suggested that the target of PPADS action may either be a phospholipase A(2) (PLA(2)) or a site distal to PLA(2), but PPADS did not inhibit Ca(2+)-dependent PLA(2) activity in MDCK-D(1) cell homogenate. The data indicate that PPADS blocks AA release in response to multiple compounds and suggest caution in the use of this compound for distinguishing P2Y receptor subtypes.
Shehnaz
D
D
Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA 92093-0636, USA.
Torres
B
B
Balboa
M A
MA
Insel
P A
PA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D020555:GTP-Binding Protein Regulators / Q000037:antagonists & inhibitors*; D007668:Kidney / Q000187:drug effects; D008555:Melitten / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D058472:Receptors, Purinergic P2Y1; D014544:Uridine Triphosphate / Q000494:pharmacology
D020555:GTP-Binding Protein Regulators; D010975:Platelet Aggregation Inhibitors; D058919:Purinergic P2 Receptor Antagonists; D058472:Receptors, Purinergic P2Y1; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D008555:Melitten; D016718:Arachidonic Acid; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011493:Protein Kinase C; D010741:Phospholipases A; D002118:Calcium; D014544:Uridine Triphosphate