Abstract
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.
Footnotes
-
Send reprint requests to: Dr. Paul A. Insel, Department of Pharmacology, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0636. E-mail:pinsel{at}ucsd.edu
-
↵1 The work has been supported by National Institutes of Health Grant GM 31987.
-
↵2 The recipient of a Fulbright Fellowship.
- Abbreviations:
- PPADS
- pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate
- DMEM
- Dulbecco's modified Eagle's medium
- MDCK-D1
- clonal variant of Madin-Darby canine kidney
- PLA
- phospholipase A
- cPLA2
- cytosolic PLA2
- AA
- arachidonic acid and arachidonic acid metabolite
- 2MeSATP
- 2-methylthioadenosine triphosphate
- PMA
- phorbol-12-myristate-13-acetate
- ADPβS
- adenosine-5′-O-(2-thio)diphosphate
- Received April 19, 1999.
- Accepted September 17, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|