Vol. 292, Issue 1, 346-350, January 2000

Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), a Putative P2Y₁ Receptor Antagonist, Blocks Signaling at a Site Distal to the Receptor in Madin-Darby Canine Kidney-D₁ Cells¹

Darakhshanda Shehnaz², Brian Torres, Maria A. Balboa and Paul A. Insel

Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California

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 P2Y₁ receptor-selective blocker in Madin-Darby canine kidney (MDCK-D₁) cells. We found that the key action of PPADS in MDCK-D₁ cells was blockade of signaling at a postreceptor site. PPADS blocked UTP (P2Y₂)-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 P2Y₁ and P2Y₁₁ 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 Ca²⁺ transients or PKC activation. Inhibition of melittin-stimulated AA release by PPADS suggested that the target of PPADS action may either be a phospholipase A₂ (PLA₂) or a site distal to PLA₂, but PPADS did not inhibit Ca²⁺-dependent PLA₂ activity in MDCK-D₁ 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.