The P2Y(11) receptor is hypothesized to link to both G(s) and G(q), although this proposition is based on expression and separate assays of G(s) and G(q) function in different cell types [J Biol Chem 1997;272:31969-31973]. We have cloned and characterized a canine P2Y(11)-like (cP2Y(11)) receptor from cultured Madin Darby canine kidney (MDCK-D1) cells. When cP2Y(11) receptors are expressed in canine thymocyte (CF2Th) cells that normally lack functional purinergic responses, ADP beta S stimulates phosphatidylinositol (PI) hydrolysis, Ca(2+) mobilization, and cAMP accumulation. Pharmacologic analysis indicates that the stimulation of cAMP production is direct and not a result of eicosanoid synthesis, activation of PKC, or elevation of cell Ca(2+). The rank order of potency for stimulation of PI hydrolysis by cP2Y(11) receptors (adenosine 5'-(2-O-thio) diphosphate = 2-methylthio-ADP >/= 2-methylthio-ATP >> ADP > ATP) differs from that of hP2Y(11) receptors. Microscopic examination of MDCK-D1 cells expressing carboxyl-terminal green fluorescent protein (GFP)-tagged cP2Y(11) (cP2Y(11)-GFP) receptors indicates primarily basolateral (BL) targeting. BL addition of 200 microM ADP beta S to confluent monolayers of MDCK-D1 cells produces an increase in short circuit current (I(sc)) (11.6 +/- 1.6 microA/cm(2)) whereas apical addition of agonist has no effect, confirming targeting of functional endogenous P2Y(11) receptors to the BL surface. In contrast, when either cP2Y(11) or cP2Y(11)-GFP is overexpressed in MDCK-D1 cells, the sensitivity of I(sc) to BL agonist increases by nearly 2 orders of magnitude, as if receptor density normally limited agonist potency; moreover, apical addition of ADP beta S now produces an increase in I(sc) but with low potency. The data support the BL localization of cP2Y(11) receptors and receptor coupling to changes in I(sc) in MDCK-D1 cells except in cases in which receptors are overexpressed; receptor overexpression leads to altered sensitivities and sites of coupling to physiologic responses.

Cells commonly co-express multiple receptor subtypes that recognize the same physiological agonist, but it is difficult to define which among such receptor subtypes mediates a particular response. This can be a particularly vexing problem if subtype-selective agonists and antagonists are not available. One such example is P2Y receptors, which respond to ATP and other nucleotides, are expressed in a variety of tissues and cell types, and for which few subtype-selective antagonists exist (2-4). Our laboratory has undertaken a series of studies related to signal transduction by P2Y receptors in the renal epithelial cell line, MDCK 1 -D 1 (reviewed in Refs. 5 and 6). P2Y receptors in MDCK-D 1 cells modulate membrane potential and short circuit current, and the receptors regulate phospholipases, intracellular [Ca 2ϩ ], prostaglandin E 2 (PGE 2 ) formation, and cAMP accumulation (1,(7)(8)(9)(10)(11)(12)(13)(14).Cyclooxygenase (COX) inhibitors, such as indomethacin (indo), have proven useful for studying P2Y receptors in MDCK-D 1 cells (1, 12). Agonist-stimulated cAMP accumulation in MDCK-D 1 cells occurs via both indo-sensitive and -insensitive pathways. Response to the P2Y 2 agonist UTP is entirely indo-sensitive, whereas response to ATP is partially sensitive and to 2-methylthio-ATP (MT-ATP) is insensitive (1). These findings suggest that UTP, and ATP in part, stimulate P2Y 2 receptors to cause COX-mediated (perhaps by both COX1 and COX2, see Ref. 15) formation of arachidonic acid metabolites (e.g. PGE 2 ), which activate EP receptors to stimulate cAMP formation, while ATP and MT-ATP can also enhance cAMP formation via an indo-insensitive P2Y receptor pathway (1).The present studies were designed to characterize more fully the nature of the latter pathway. Our working hypothesis, based in part on initial results obtained with MDCK-D 1 cells (12), was that the indo-resistant response might represent a P2Y 1 receptor effect. The current data show results not consistent with this hypothesis but instead suggest a key role for another receptor, the P2Y 11 receptor, in indo-resistant cAMP formation. The findings directly document a role for P2Y 11 receptors in stimulation of adenylyl cyclase activity and in potentially contributing to autocrine-paracrine regulation by nucleotides. EXPERIMENTAL PROCEDURESCell Culture-MDCK-D 1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% mixed serum (85% horse serum, 15% fetal bovine serum). Cells were used in assays at 60 -80% confluency. GFP-tagged cP2Y 11 receptor-overexpressing MDCK-D 1 cells were cultured from the stable cell line prepared by Zambon et al. (14).Measurement of cAMP Accumulation in Normal and GFP-tagged cP2Y 11 Intact Cells-Prior to the treatment of the cells, the growth medium was removed, and cells were equilibrated for 30 min at 37°C in serum-free 20 mM HEPES-buffered Dulbecco's modified Eagle's medium (DMEH, pH 7.4). Subsequently, cells were incubated in fresh DMEH and various agents as shown in the figures. Incubations with th...

Madin-Darby canine kidney (MDCK)-D1 cells, a canine renal epithelial cell line, co-express at least three different P2Y receptor subtypes: P2Y(1), P2Y(2), and P2Y(11) (24). Stimulation of P2Y receptors in these cells results in the release of arachidonic acid (AA) and metabolites and the elevation of intracellular cAMP. To define in more precise terms the signaling contributed by the MDCK-D1 P2Y(2) (cP2Y(2)) receptor, we have cloned and heterologously expressed it in CF2Th (canine thymocyte) cells, a P2Y(2)-null cell. Analysis by RT-PCR indicated that canine P2Y(2) receptors are expressed in skeletal muscle, spleen, kidney, lung, and liver. When expressed in CF2Th cells, cP2Y(2) receptors promoted phospholipase C-mediated phosphatidylinositol (PI) hydrolysis [uridine 5'-triphosphate > or = ATP > adenosine 5'-diphosphate > 2MT-ATP] and mobilization of intracellular Ca(2+). In contrast to their actions in MDCK-D1 cells, cP2Y(2) receptors did not stimulate formation of cAMP or AA release when expressed in CF2Th cells. The data indicate that cell setting plays an essential role in the ability of P2Y receptors to regulate AA release and cAMP formation. In particular, renal epithelial cells preferentially express components critical for cP2Y(2)-induced cAMP formation, including the expression of enzymes involved in the generation and metabolism of AA and receptors that respond to PGE(2).

1. Madin-Darby canine kidney (MDCK) cells, a well- differentiated renal epithelial cell line derived from distal tubule/collecting duct, respond to extracellular nucleotides by altering ion flux and the production of arachidonic acid-derived products, in particular prostaglandin E2 (PGE2). Our work has defined the receptors and signalling events involved in such responses. 2. We have found evidence for expression of at least three P2Y receptor subtypes (P2Y1, P2Y2 and P2Y11) in MDCK-D1 cells, a subclone from parental MDCK. 3. These receptors appear to couple to increases in calcium and protein kinase C activity, probably via a Gq/G11-mediated activation of phospholipase C. 4. In addition, P2Y receptor activation can promote a prominent increase in cAMP. This includes both a P2Y2 receptor-mediated cyclo-oxygenase (COX)-dependent component and another COX-independent component mediated by other P2Y receptors. 5. We have documented that changing media in which cells are grown releases ATP and, in turn, activates P2Y receptors. Such release of ATP contributes in a major way to basal cAMP levels in these cells. 6. The data indicate that MDCK cells are a useful model to define the regulation of epithelial cells by extracellular nucleotides. Of particular note, spontaneous or stretch-induced release of ATP and subsequent activation of one or more P2Y receptors contributes to establishing the basal activity of signalling pathways.