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.
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.
Department of Pharmacology-0636, University of California, San Diego, La Jolla, California 92093-0636, USA.
S R
SR
Post
L C
LC
Rump
A
A
Zambon
R J
RJ
Hughes
M D
MD
Buda
J P
JP
Jacobson
C C
CC
Kao
P A
PA
Insel
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D019898:Autocrine Communication / Q000187:drug effects; D000242:Cyclic AMP / Q000096:biosynthesis*; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004285:Dogs; D004847:Epithelial Cells / Q000187:drug effects*; D007213:Indomethacin / Q000494:pharmacology; D019899:Paracrine Communication / Q000187:drug effects; D011453:Prostaglandins / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000145:classification / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; D015398:Signal Transduction / Q000187:drug effects; D013498:Suramin / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology
D016861:Cyclooxygenase Inhibitors; C546382:P2RY1 protein, human; C109734:P2RY11 protein, human; C546385:P2RY2 protein, human; D011453:Prostaglandins; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; D013873:Thionucleotides; D016718:Arachidonic Acid; D013498:Suramin; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D014544:Uridine Triphosphate; D007213:Indomethacin; C008501:2-methylthio-ATP