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![Concentration response of effects of PGE 2 on cAMP levels and 300 M UTP-promoted AA release of MDCK-D 1 cells. Cells were assayed for [ 3 H]AA or in parallel for cAMP content as described under "Experimental Procedures" and in the legend to Fig. 1. q, AA; E, cAMP.](profile/Michael-Samardzija/publication/13109911/figure/fig2/AS:601690709438471@1520465652516/Concentration-response-of-effects-of-PGE-2-on-cAMP-levels-and-300-M-UTP-promoted-AA.png)
Concentration response of effects of PGE 2 on cAMP levels and 300 M UTP-promoted AA release of MDCK-D 1 cells. Cells were assayed for [ 3 H]AA or in parallel for cAMP content as described under "Experimental Procedures" and in the legend to Fig. 1. q, AA; E, cAMP.
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In Madin-Darby canine kidney D1cells extracellular nucleotides activate P2Y receptors that couple to several signal transduction pathways, including stimulation of multiple phospholipases and adenylyl
cyclase. For one class of P2Y receptors, P2Y2 receptors, this stimulation of adenylyl cyclase and increase in cAMP occurs via the conversion of phosp...
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Extracellular nucleotides transmit signals into the cells through the P2 family of cell surface receptors. These receptors
are amply expressed in human blood vessels and participate in vascular tone control; however, their signaling mechanisms remain
unknown. Here we show that in smooth muscle cells of isolated human chorionic arteries, the activat...
Citations
... The pathways involved in the crosstalk between P2Y receptors and PGE 2 on macrophages have been established using biochemical (inhibitors and activators of signal transduction pathways), pharmacological (mainly through the use of agonists and antagonists of the EP and P2Y receptors) and genetic (cells lacking P2X 7 receptor or COX-2; expressing a COX-2 transgene or expressing different constructs of the proteins that participate in the signal-transduction pathways) approaches [24, 71,88,115,131,132]. Based on the data from these different strategies it was concluded that PKD1 phosphorylation at S916 is a necessary condition to suppress PGE 2 -dependent UTP-mediated Ca 2+ -mobilization. ...
The role of extracellular nucleotides as modulators of inflammation and cell stress is well established. One of the main actions of these molecules is mediated by the activation of purinergic receptors (P2) of the plasma membrane. P2 receptors can be classified according to two different structural families: P2X ionotropic ion channel receptors, and P2Y metabotropic G protein-coupled receptors. During inflammation, damaged cells release nucleotides and purinergic signaling occurs along the temporal pattern of the synthesis of pro-inflammatory and pro-resolving mediators by myeloid and lymphoid cells. In macrophages under pro-inflammatory conditions, the expression and activity of cyclooxygenase 2 significantly increases and enhances the circulating levels of prostaglandin E2 (PGE2), which exerts its effects both through specific plasma membrane receptors (EP1-EP4) and by activation of intracellular targets. Here we review the mechanisms involved in the crosstalk between PGE2 and P2Y receptors on macrophages, which is dependent on several isoforms of protein kinase C and protein kinase D1. Due to this crosstalk, a P2Y-dependent increase in calcium is blunted by PGE2 whereas, under these conditions, macrophages exhibit reduced migratory capacity along with enhanced phagocytosis, which contributes to the modulation of the inflammatory response and tissue repair.
... P2Y 2 R antagonists may in fact be useful for a range of excessive inflammatory reactions, including atherosclerosis. This is because the P2Y 2 R was found to activate cytosolic phospholipase A 2 , which in turn promotes the release of arachidonic acid and the subsequent synthesis of prostaglandins and leukotrienes (Seye et al., 2002;Welch, Carlson, Shi, Myatt, & Kishore, 2003;Xing, Post, Ostrom, Samardzija, & Insel, 1999;Xu et al., 2002;Xu et al., 2003). The potential of P2Y 2 R blockade in the treatment of atherosclerosis is further emphasized by the following observations: the receptor appears to mediate the uptake of lowdensity lipoprotein in vascular smooth muscle cells and contribute to vascular inflammation by increasing vascular cell adhesion molecule-1 expression. ...
P2Y receptors (P2YRs) are a family of G protein-coupled receptors activated by extracellular nucleotides. Physiological P2YR agonists include purine and pyrimidine nucleoside di- and triphosphates, such as ATP, ADP, UTP, UDP, nucleotide sugars, and dinucleotides. Eight subtypes exist, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14, which represent current or potential future drug targets. Here we provide a comprehensive overview of ligands for the subgroup of the P2YR family that is activated by uracil nucleotides: P2Y2 (UTP, also ATP and dinucleotides), P2Y4 (UTP), P2Y6 (UDP), and P2Y14 (UDP, UDP-glucose, UDP-galactose). The physiological agonists are metabolically unstable due to their fast hydrolysis by ectonucleotidases. A number of agonists with increased potency, subtype-selectivity and/or enzymatic stability have been developed in recent years. Useful P2Y2R agonists include MRS2698 (6-01, highly selective) and PSB-1114 (6-05, increased metabolic stability). A potent and selective P2Y2R antagonist is AR-C118925 (10-01). For studies of the P2Y4R, MRS4062 (3-15) may be used as a selective agonist, while PSB-16133 (10-06) represents a selective antagonist. Several potent P2Y6R agonists have been developed including 5-methoxyuridine 5'-O-((Rp)α-boranodiphosphate) (6-12), PSB-0474 (3-11), and MRS2693 (3-26). The isocyanate MRS2578 (10-08) is used as a selective P2Y6R antagonist, although its reactivity and low water-solubility are limiting. With MRS2905 (6-08), a potent and metabolically stable P2Y14R agonist is available, while PPTN (10-14) represents a potent and selective P2Y14R antagonist. The radioligand [3H]UDP can be used to label P2Y14Rs. In addition, several fluorescent probes have been developed. Uracil nucleotide-activated P2YRs show great potential as drug targets, especially in inflammation, cancer, cardiovascular and neurodegenerative diseases.
... The P2Y nucleotide receptors are a family of G protein-coupled receptors that consists of eight mammalian subtypes. These are the P2Y 1 -like receptors P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 , as well as the P2Y 12 -like subtypes P2Y 12 , P2Y 13 , and P2Y 14 [1]. The numbers missing in this sequence correspond to nonmammalian orthologs or to receptors that have structural similarities but do not respond to nucleotides. ...
... Blocking this receptor, on the other hand, could be useful for treating atherosclerosis and excessive inflammation, since the P2Y 2 receptor activates cytosolic phospholipase A 2 , which in turn promotes the release of arachidonic acid and subsequent synthesis of prostaglandins and leukotrienes [11][12][13][14][15]. Furthermore, P2Y 2 receptor antagonists may be useful as novel cancer therapeutics, as the P2Y 2 receptor was shown to promote tumor metastasis via opening of the endothelial barrier [16]. ...
... The Xfect™ Transfection Reagent kit was purchased from Clontech (Saint-Germain-en-Laye, France). (11) 5-Bromo-2,4-dichloropyrimidine (12) [33,34] A mixture of 5-bromouracil (11,7.64 g, 40 mmol), N,N-dimethylaniline (7.3 g, 60 mmol) and freshly distilled phosphorus oxychloride (24 ml) was stirred at 120-130°C for 80 min. After cooling to 50-60°C, the resulting black solution was poured into icewater. ...
The Gq protein-coupled, ATP- and UTP-activated P2Y2 receptor is a potential drug target for a range of different disorders, including tumor metastasis, inflammation, atherosclerosis, kidney disorders, and osteoporosis, but pharmacological studies are impeded by the limited availability of suitable antagonists. One of the most potent and selective antagonists is the thiouracil derivative AR-C118925. However, this compound was until recently not commercially available and little is known about its properties. We therefore developed an improved procedure for the synthesis of AR-C118925 and two derivatives to allow up-scaling and assessed their potency in calcium mobilization assays on the human and rat P2Y2 receptors recombinantly expressed in 1321N1 astrocytoma cells. The compound was further evaluated for inhibition of P2Y2 receptor-induced β-arrestin translocation. AR-C118925 behaved as a competitive antagonist with pA2 values of 37.2 nM (calcium assay) and 51.3 nM (β-arrestin assay). Selectivity was assessed vs. related receptors including P2X, P2Y, and adenosine receptor subtypes, as well as ectonucleotidases. AR-C118925 showed at least 50-fold selectivity against the other investigated targets, except for the P2X1 and P2X3 receptors which were blocked by AR-C118925 at concentrations of about 1 μM. AR-C118925 is soluble in buffer at pH 7.4 (124 μM) and was found to be metabolically highly stable in human and mouse liver microsomes. In Caco2 cell experiments, the compound displayed moderate permeability indicating that it may show limited peroral bioavailability. AR-C118925 appears to be a useful pharmacological tool for in vitro and in vivo studies.
... Because our data indicated that augmentation in perfusion from US cavitation is mediated, in part, through the endothelial P2Y 2 receptor, we investigated the signaling pathways that are thought to mediate the vasoactive effects of P 2 Y receptor activation, including calcium-dependent phosphorylation of eNOS and phospholipase-A 2mediated production of vasoactive arachidonic acid metabolites. 16,[28][29][30] Skeletal muscle from wild-type mice exposed to ultrasound-induced MB cavitation demonstrated significant increases in phosphorylated eNOS by enzyme-linked immunosorbent assay (Figure 2c). Because eNOS phosphorylation can occur by alternative non-P2Y-receptor-mediated processes, including K ATP and A 2B receptor activation, H 2 O 2 , and other endothelialderived hyperpolarizing factors, 21,22,31 we also evaluated phospho-eNOS in P2Y 2 -/mice with and without inhibition of adenosine receptors. ...
... Irrespective of which receptor pathway was stimulated, the downstream events involve both activation of eNOS and production of prostanoids such as prostacyclin presumably downstream from calcium-dependent PLA 2 activity. 16,21,30 The mechanistic insights from this study are important for several reasons. They are important for understanding how to optimize cavitation for therapeutic benefit beyond the preliminary observations that we have performed in mice with limb ischemia and patients with SCD. ...
Background:
- Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signalling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation.
Methods:
-Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for ten minutes after intravenous injection of 2×10(8) lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signalling pathways were assessed by studying interventions that either (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or KATP channels; or (3) inhibited downstream signalling pathways involving endothelial nitric oxide synthase (eNOS) or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease (SCD).
Results:
-Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hrs in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with SCD. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced a nearly 40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or through adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of eNOS abolished the effects of therapeutic ultrasound, indicating downstream signalling through both NO and prostaglandins.
Conclusions:
-Therapeutic ultrasound using microbubble cavitation to increase muscle perfusion relies on shear-dependent increases in ATP which can act through a diverse portfolio of purinergic signalling pathways. These events can reverse hindlimb ischemia in mice for >24 hours, and increase muscle blood flow in patients with sickle cell disease.Clinical Trial Registration-NCT01566890 https://clinicaltrials.gov.
... Phospholipase-A-mediated arachidonic acid (AA) and TXB 2 formation will be inhibited by an elevation of cAMP. Consequently, cardioprotection of sildenafil is due to NOS enhancement, cGMP elevation and therefore cGMP-dependent protein kinase (PKG) activation through K + /ATP channel stimulation [30]. ...
PDE5 inhibitors (PDE5inhs) have proven to be of great impact in the treatment of numerous human extra-sexual diseases and their chronic use may induce endothelial rehabilitation. This study aimed to assess the effects of PDE5inhs at chronic administration to explore the possible endothelial cyto-protective and thrombo-resistance effects.
One hundred New Zealand white male rabbits were divided into four groups. The first group (control, C) received 1 ml saline/kg, the second group (S) received 10 mg/kg sildenafil, the third group (V) received 2 mg/kg vardenafil, and the fourth group (T) received 2 mg/kg tadalafil in saline I.P. three times weekly for 4 weeks. Blood samples were collected and plasma was isolated for determination of 2,3-dinor-6-keto-prostaglandin F-1α (PGF1α), 11-dehydro-TXB2 (TXB2), fibrinogen, calcium levels, prothrombin (PT), and thrombin times (TT).
PDE5inhs significantly increase PGF1α, calcium levels, PT and TT (p < 0.001) when compared with baseline data or with the saline group at the end of treatment. In contrast, PDE5inhs significantly decrease TXB2 and fibrinogen levels (p < 0.001) when compared either with their baseline data or with the saline group at the end of treatment. The tadalafil group showed a lower increase in PGF1α (p < 0.001), lower decrease in TXB2 (p < 0.001), and higher increase in calcium levels (p < 0.01, p < 0.05), lower increase in PT and TT levels (p < 0.001) when compared with sildenafil or vardenafil.
The prolonged use of PDE5inhs has time-dependent mild to moderate endothelial cyto-protective, thrombo-resistance anti-inflammatory and anti-nociception effects via activation of endothelial NOS (eNOS), increase of PGI2 synthesis and decrease of fibrinogen with significant increase in PT and TT.
... Antagonism between PKA and ERK signaling has been reported [44]. P2Y signaling is diminished by PKA [45], whereas P2X signaling is augmented by PKA [46]. Moreover, P2Y receptors inhibit adenylyl cyclase and PKA activation [47]. ...
Extracellular ATP is elevated by transient ischemia and is a potent signaling molecule in the central nervous system. ATP promotes neuron survival from serum starvation by activating P2Y purinergic receptors. ATP also activates IL-6 production and phosphorylation of Stat3 that promotes neuron survival. The transcription cofactor LMO4 is a positive mediator of IL-6/Stat3 signaling. Here, we found that LMO4 and the pro-survival factor cIAP2 (cellular inhibitor of apoptosis protein 2) are rapidly upregulated in neurons exposed to elevated extracellular ATP. Blocking LMO4 upregulation using siRNA in F11 cells blunted cIAP2 upregulation and abolished the early protective effect of ATP. Similar results were obtained using primary cortical neurons from LMO4 null mice, suggesting that LMO4 is required for ATP to protect neurons from hypoxia-induced apoptosis. Whereas increased Stat3 phosphorylation occurs after LMO4 and cIAP2 induction, the rapid upregulated phosphorylation of ERK and CREB may account for increased LMO4 and cIAP2 by ATP. ATP signaling through ERK and CREB activated LMO4 promoters and ERK activation increased LMO4 protein stability in F11 cells. Taken together, our studies reveal that LMO4 is a rapidly induced downstream effector of ATP signaling that promotes neuron survival from hypoxia.
... The RGD sequence in the P2Y 2 receptor also has been shown to play an integrin-independent role in targeting of the receptor to the apical membrane of Madin-Darby canine kidney cells (Qi et al., 2005). P2Y 2 receptor activation increases the synthesis and/or release of arachidonic acid (AA), prostaglandins and nitric oxide (NO) (Lustig et al., 1992; Pearson et al., 1992a,b; Xing et al., 1999; Xu et al., 2002b Xu et al., , 2003 Welch et al., 2003 ). In primary murine astrocytes, P2Y 2 receptors mediate the activation of calcium-dependent and calcium-independent PKCs and ERK1/2 that can activate cytosolic phospholipase A 2 , leading to production of AA (Gendron et al., 2003; Xu et al., 2003), the precursor of eicosanoids, prostaglandins, and leukotrienes (Balsinde et al., 2002). ...
There have been many advances in our knowledge about different aspects of P2Y receptor signaling since the last review published by our International Union of Pharmacology subcommittee. More receptor subtypes have been cloned and characterized and most orphan receptors de-orphanized, so that it is now possible to provide a basis for a future subdivision of P2Y receptor subtypes. More is known about the functional elements of the P2Y receptor molecules and the signaling pathways involved, including interactions with ion channels. There have been substantial developments in the design of selective agonists and antagonists to some of the P2Y receptor subtypes. There are new findings about the mechanisms underlying nucleotide release and ectoenzymatic nucleotide breakdown. Interactions between P2Y receptors and receptors to other signaling molecules have been explored as well as P2Y-mediated control of gene transcription. The distribution and roles of P2Y receptor subtypes in many different cell types are better understood and P2Y receptor-related compounds are being explored for therapeutic purposes. These and other advances are discussed in the present review.
... Signal transduction mediated by the P2Y 2 receptor subtype has been extensively investigated (Fig. 2). P2Y 2 receptors due to their Gα q/11 -dependent coupling to PLCβ increase the IP 3 -mediated release of Ca 2+ from intracellular stores and the DAG-induced activation of PKC that, in turn, increases the synthesis and/or release of AA, prostaglandins, and nitric oxide (NO) [78, 100, 101, 138,146147148. In primary murine astrocytes, P2Y 2 receptors mediate the activation of both calcium-dependent and calcium-independent PKCs and ERK1/2, leading to the activation of cytosolic phospholipase A 2 and the production of AA [148] , the precursor of eicosanoids, prostaglandins , and leukotrienes [7]. ...
P2 receptors for extracellular nucleotides are divided into two categories: the ion channel receptors (P2X) and the G-protein-coupled receptors (P2Y). For the P2X receptors, signal transduction appears to be relatively simple. Upon activation by extracellular ATP, a channel comprised of P2X receptor subunits opens and allows cations to move across the plasma membrane, resulting in changes in the electrical potential of the cell that, in turn, propagates a signal. This regulated flux of ions across the plasma membrane has important signaling functions, especially in impulse propagation in the nervous system and in muscle contractility. In addition, P2X receptor activation causes the accumulation of calcium ions in the cytoplasm, which is responsible for activating numerous signaling molecules. For the P2Y receptors, signal transduction is more complex. Intracellular signaling cascades are the main routes of communication between G-protein-coupled receptors and regulatory targets within the cell. These signaling cascades operate mainly by the sequential activation or deactivation of heterotrimeric and monomeric G proteins, phospholipases, protein kinases, adenylyl and guanylyl cyclases, and phosphodiesterases that regulate many cellular processes, including proliferation, differentiation, apoptosis, metabolism, secretion, and cell migration. In addition, there are numerous ion channels, cell adhesion molecules and receptor tyrosine kinases that are modulated by P2Y receptors and operate to transmit an extracellular signal to an intracellular response. These intracellular signaling pathways and their regulation by P2 receptors are discussed in this review.
... Cancer Cells-Exogenous ATP (17,26,27) as well as mechanical (28) and hypotonic stress (29) have been previously shown to activate arachidonic acid metabolism and prostaglandin generation. It is, however, presently not known whether prostaglandins are involved in hypotonic stress-induced ATP release and whether prostaglandin synthesis requires the presence of ATP released upon hypotonic stress of cells. ...
ATP is released in many cell types upon mechanical strain, the physiological function of extracellular ATP is largely unknown, however. Here we report that ATP released upon hypotonic stress stimulated prostate cancer cell proliferation, activated purinergic receptors, increased intracellular [Ca(2+)](i), and initiated downstream signaling cascades that involved MAPKs ERK1/2 and p38 as well as phosphatidylinositol 3-kinase (PI3K). MAPK activation, the calcium response as well as induction of cell proliferation upon hypotonic stress were inhibited by preincubation with the ATP scavenger apyrase, indicating that hypotonic stress-induced signaling pathways are elicited by released ATP. Hypotonic stress increased prostaglandin E(2) (PGE(2)) synthesis. Consequently, ATP release was inhibited by antagonists of PI3K (LY294002 and wortmannin), phospholipase A(2) (methyl arachidonyl fluorophosphonate (MAFP)), cyclooxygenase-2 (COX-2) (indomethacin, etodolac, NS398) and 5,8,11,14-eicosatetraynoic acid (ETYA), which are involved in arachidonic acid metabolism. Furthermore, ATP release was abolished in the presence of the adenylate cyclase (AC) inhibitor MDL-12,330A, indicating regulation of ATP-release by cAMP. The hypotonic stress-induced ATP release was significantly blunted when the ATP-mediated signal transduction cascade was inhibited on different levels, i.e. purinergic receptors were blocked by suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), the Ca(2+) response was inhibited upon chelation of intracellular Ca(2+) by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ERK1,2 as well as p38 were inhibited by UO126 and SB203580, respectively. In summary our data demonstrate that hypotonic stress initiates a feed forward cycle of ATP release and purinergic receptor signaling resulting in proliferation of prostate cancer cells.
... In ATCC–MDCK (Post et al. 1996Post et al. , 1998 Xing et al. 1999) and C7–MDCK cells (Orlov et al. 1999), ATP sharply augments cAMP production in a manner partially inhibited by indomethacin, suggesting an autocrine PLA 2 /AA/COX/PGE-mediated pathway of PKA activation. This hypothesis is consistent with data showing activation of PLA 2 (Post et al. 1996; Xing et al. 1999) and augmented production of AA (Firestein et al. 1996; Xing et al. 1997 Xing et al. , 1999) and PGE 2 (Post et al. 1998) in ATP-treated ATCC–MDCK cells. ...
... In ATCC–MDCK (Post et al. 1996Post et al. , 1998 Xing et al. 1999) and C7–MDCK cells (Orlov et al. 1999), ATP sharply augments cAMP production in a manner partially inhibited by indomethacin, suggesting an autocrine PLA 2 /AA/COX/PGE-mediated pathway of PKA activation. This hypothesis is consistent with data showing activation of PLA 2 (Post et al. 1996; Xing et al. 1999) and augmented production of AA (Firestein et al. 1996; Xing et al. 1997 Xing et al. , 1999) and PGE 2 (Post et al. 1998) in ATP-treated ATCC–MDCK cells. To examine the role of PLA 2 in P 2Y -induced Cl − secretion, we treated the cells with an inhibitor of this enzyme, AACOCF 3 , or with its inactive structural analogue AACOCH 3 . ...
... In ATCC–MDCK (Post et al. 1996Post et al. , 1998 Xing et al. 1999) and C7–MDCK cells (Orlov et al. 1999), ATP sharply augments cAMP production in a manner partially inhibited by indomethacin, suggesting an autocrine PLA 2 /AA/COX/PGE-mediated pathway of PKA activation. This hypothesis is consistent with data showing activation of PLA 2 (Post et al. 1996; Xing et al. 1999) and augmented production of AA (Firestein et al. 1996; Xing et al. 1997 Xing et al. , 1999) and PGE 2 (Post et al. 1998) in ATP-treated ATCC–MDCK cells. To examine the role of PLA 2 in P 2Y -induced Cl − secretion, we treated the cells with an inhibitor of this enzyme, AACOCF 3 , or with its inactive structural analogue AACOCH 3 . ...
This study examines the mechanism of P 2Y-induced Cl- secretion in monolayers of C7-Madin-Darby canine kidney (MDCK) cells triggered by basolateral application of ATP and measured as transcellular short current (I(SC)). Both ATP-induced arachidonic acid (AA) synthesis and I(SC) in ATP-treated cells were abolished by the phosholipase A2 (PLA2) inhibitor, AACOCF3. The cyclo-oxygenase inhibitor indomethacin decreased I(SC) and cAMP production in ATP-treated cells with an IC50 of approximately 0.3 microm. ATP led to rapid activation of cAMP-dependent protein kinase A (PKA), as estimated by phosphorylation of a vasodilator-stimulated phosphoprotein. PKA activity and I(SC) evoked by ATP, as well as by prostaglandin E1 (PGE1), were diminished in the presence of the PKA inhibitor H-89 or an adenovirus-mediated expression of PKA-inhibitor protein, PKI. In contrast, indomethacin completely blocked the increment of PKA and I(SC) triggered by ATP and AA, but did not affect PKA activation and I(SC) detected with PGE1. The kinetics of [Ca2+]i elevation in ATP- and thapsigargin-treated cells were similar and suppressed by the Ca(2+)i chelator BAPTA. Neither baseline nor maximal increment of ATP-induced I(SC) was affected by thapsigargin and BAPTA. Real-time PCR showed that C7 cells express more mRNA for P 2Y1 and P 2Y2 than for other P 2Y receptor subtypes. The rank order of potency (2MeSATP > ATP > ADP > UTP) indicates that P 2Y1 rather than P 2Y2 receptors contribute to PKA and I(SC) activation. Viewed collectively, these data show that Cl- secretion in C7-MDCK monolayers treated with basolateral ATP is triggered by P 2Y1 receptors and is mediated by subsequent [Ca2+]i-independent activation of PLA2 and PKA.
