ATP Activates cAMP Production via Multiple Purinergic Receptors in MDCK-D1 Epithelial Cells
BLOCKADE OF AN AUTOCRINE/PARACRINE PATHWAY TO DEFINE RECEPTOR PREFERENCE OF AN AGONIST*
- Steven R. Post,
- L. Christian Rump‡,
- Alex Zambon,
- Richard J. Hughes,
- Mihaela D. Buda,
- J. Paul Jacobson,
- Cecilia C. Kao and
- Paul A. Insel§
- From the Department of Pharmacology-0636, University of California, San Diego, La Jolla, California 92093-0636
Abstract
Extracellular nucleotides regulate function in many cell types via activation of multiple P2-purinergic receptor subtypes. However, it has been difficult to define which individual subtypes mediate responses to the physiological agonist ATP. We report a novel means to determine this by exploiting the differential activation of an autocrine/paracrine signaling pathway. We used Madin-Darby canine kidney epithelial cells (MDCK-D1) and assessed the regulation of cAMP formation by nucleotides. We found that ATP, 2-methylthio-ATP (MT-ATP) and UTP increase cAMP production. The cyclooxygenase inhibitor indomethacin completely inhibited UTP-stimulated, did not inhibit MT-ATP-stimulated, and only partially blocked ATP-stimulated cAMP formation. In parallel studies, ATP and UTP but not MT-ATP stimulated prostaglandin production. By pretreating cells with indomethacin to eliminate the P2Y2/prostaglandin component of cAMP formation, we could assess the indomethacin-insensitive P2 receptor component. Under these conditions, ATP displayed a ten-fold lower potency for stimulation of cAMP formation compared with untreated cells. These data indicate that ATP preferentially activates P2Y2 relative to other P2 receptors in MDCK-D1 cells (P2Y1 and P2Y11, as shown by reverse transcriptase polymerase chain reaction) and that P2Y2 receptor activation is the principal means by which ATP increases cAMP formation in these cells. Blockade of autocrine/paracrine signaling can aid in dissecting the contribution of multiple receptor subtypes activated by an agonist.
Footnotes
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↵* This work was supported by National Institutes of Health Grants (GM 40781, GM 31987, and HL 53773), a grant from the California affiliate of the American Lung Association (to S. R. P.), and a grant (Forschungsstipendium) from the Deutsche Forschungsgemeinschaft, Bonn, Germany (to L. C. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Present address: Innere Medizin IV, Universitaetsklinik Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany.
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↵§ To whom correspondence should be addressed. Tel.: 619-534-2295; Fax: 619-822-1007; E-mail: pinsel{at}ucsd.edu.
- Abbreviations:
- MDCK-D1
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clonal Madin-Darby canine kidney cells
- PGE2
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prostaglandin E2
- MT-ATP
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2-methylthioadenosine 5′-triphosphate
- RT
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reverse transcriptase
- PCR
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polymerase chain reaction.
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- Received December 24, 1997.
- Revision received May 21, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
