P2Y11 Receptors Activate Adenylyl Cyclase and Contribute to Nucleotide-promoted cAMP Formation in MDCK-D1Cells

Abstract

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...

Cross talk between P2 purinergic receptors modulates extracellular ATP-mediated interleukin-10 production in rat microglial cells

“…Indeed, exposure to these nucleotides increased the release of IL-10 (Figures 1 and 3), and IL-10 release mediated by ATP-γS (10 µM), BzATP (100 µM), and dATP (1 mM) was down-regulated by an adenylate cyclase inhibitor, SQ22536, and a PKA inhibitor, H-89 ( Figure 5B). These results are consistent with previous findings that P2Y 11 receptors activate adenylate cyclase and contribute to cAMP formation (Torres et al, 2002). Recent studies showed that ATP-γS enhanced LPS-induced IL-10 production in human monocyte-derived dendritic cells (Marteau et al, 2004), and that ATP-γS and BzATP inhibited the production of TNF-α, IL-8, and MIP-1β in human mast cells through a G s -coupled receptor (Feng et al, 2004).…”

Secretion of IL-2 and IFN-γ, But Not IL-4, by Antigen-Specific T Cells Requires Extracellular ATP

“…The authors thought that ATP␥S might be functioning as a P2 agonist, but it failed to induce calcium flux in CD4 ϩ T cells. Furthermore, the CD4 ϩ T cells did not express P2Y 11 receptors, which could account for the increase in cAMP because they are the only P2 receptors that are coupled to both adenylyl cyclase and phospholipase C pathways (38,40). Our data suggest that E-NTPDase might be the target for ATP␥S-induced inhibition of cytokine secretion by activated CD4 ϩ T cells in the study reported by Duhant et al (34).…”

Activity-Dependent Autocrine-Paracrine Activation of Neuronal P2Y Receptors

“…In hepatoma cells (Junankar et al, 2002), for example, released ATP is degraded to ADP to stimulate P2Y 1 receptors. In epithelial (Torres et al, 2002) and osteoblastic (You et al, 2002) cells, in contrast, released ATP itself activates P2Y 11 and P2Y 2 receptors, respectively. In PC12 cells, both types of autocrine-paracrine regulation occur under resting conditions and during depolarization and involve P2Y 12 and P2Y 2 receptors, respectively.…”

“…Therefore, neuronal ATP release is depolarization and Ca 2ϩ dependent (von Kugelgen et al, 1994). In non-neural tissues, such as liver, kidney, bones, and blood vessels, released nucleotides subserve autocrine-paracrine functions by activating certain P2Y receptors (Gerasimovskaya et al, 2002;Junankar et al, 2002;Schwiebert et al, 2002;Torres et al, 2002;You et al, 2002). In neurons, action potential-and Ca 2ϩ -dependent release of ATP contributes to synaptic transmission via P2X receptors (Robertson et al, 2001); however, much less is known about the activation of neuronal P2Y receptors by released nucleotides.…”