A2B adenosine and P2Y2 receptors stimulate mitogen-activated protein kinase in human embryonic kidney-293 cells. cross-talk between cyclic AMP and protein kinase c pathways.
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Mitogen-activated protein kinase (MAPK) cascades underlie long-term mitogenic, morphogenic, and secretory activities of purinergic receptors. In HEK-293 cells, N-ethylcarboxamidoadenosine (NECA) activates endogenous A2BARs that signal through Gs and Gq/11. UTP activates P2Y2 receptors and signals only through Gq/11. The MAPK isoforms, extracellular-signal regulated kinase 1/2 (ERK), are activated by NECA and UTP. H-89 blocks ERK activation by forskolin, but weakly affects the response to NECA or UTP. ERK activation by NECA or UTP is unaffected by a tyrosine kinase inhibitor (genistein), attenuated by a phospholipase C inhibitor (U73122), and is abolished by a MEK inhibitor (PD098059) or dominant negative Ras. Inhibition of protein kinase C (PKC) by GF 109203X failed to block ERK activation by NECA or UTP, however, another PKC inhibitor, Ro 31-8220, which unlike GF 109203X, can block the zeta-isoform, and prevents UTP- but not NECA-induced ERK activation. In the presence of forskolin, Ro 31-8220 loses its ability to block UTP-stimulated ERK activation. PKA has opposing effects on B-Raf and c-Raf-1, both of which are found in HEK-293 cells. The data are explained by a model in which ERK activity is modulated by differential effects of PKC zeta and PKA on Raf isoforms. (+info)
Inhibition of phospholipase A2-mediated arachidonic acid release by cyclic AMP defines a negative feedback loop for P2Y receptor activation in Madin-Darby canine kidney D1 cells.
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In Madin-Darby canine kidney D1 cells 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 phospholipase A2 (PLA2)-generated arachidonic acid (AA) to prostaglandins (e.g. PGE2). These prostaglandins then stimulate adenylyl cyclase activity, presumably via activation of prostanoid receptors. In the current study we show that agents that increase cellular cAMP levels (including PGE2, forskolin, and the beta-adrenergic agonist isoproterenol) can inhibit P2Y receptor-promoted AA release. The protein kinase A (PKA) inhibitor H89 blocks this effect, suggesting that this feedback inhibition occurs via activation of PKA. Studies with PGE2 indicate that inhibition of AA release is attributable to inhibition of mitogen-activated protein kinase activity and in turn of P2Y receptor stimulated PLA2 activity. Although cAMP/PKA-mediated inhibition occurs for P2Y receptor-promoted AA release, we did not find such inhibition for epinephrine (alpha1-adrenergic) or bradykinin-mediated AA release. Taken together, these results indicate that negative feedback regulation via cAMP/PKA-mediated inhibition of mitogen-activated protein kinase occurs for some, but not all, classes of receptors that promote PLA2 activation and AA release. We speculate that receptor-selective feedback inhibition occurs because PLA2 activation by different receptors in Madin-Darby canine kidney D1 cells involves the utilization of different signaling components that are differentially sensitive to increases in cAMP or, alternatively, because of compartmentation of signaling components. (+info)
Desensitization of P2Y2 receptor-activated transepithelial anion secretion.
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Desensitization of P2Y2 receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epithelial diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y2 receptors, freshly excised or cultured murine gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure short-circuit current (Isc), an index of electrogenic anion secretion. Luminal treatment with nucleotide receptor agonists increased the Isc with a potency profile of ATP = UTP > 2-methylthioATP >> alpha,beta-methylene-ATP. RT-PCR revealed the expression of P2Y2 receptor mRNA in the MGEP cells. The desensitization of anion secretion required a 10-min preincubation with the P2Y2 receptor agonist UTP and increased in a concentration-dependent manner (IC50 approximately 10(-6) M). Approximately 40% of the anion secretory response was unaffected by maximal desensitizing concentrations of UTP. Recovery from UTP-induced desensitization was rapid (<10 min) at preincubation concentrations less than the EC50 (1.9 x 10(-6) M) but required progressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+ mobilization desensitized with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ ionophore ionomycin was unaffected by preincubation with a desensitizing concentration of UTP. It was concluded that 1) desensitization of transepithelial anion secretion stimulated by the P2Y2 receptor agonist UTP is time and concentration dependent; 2) recovery from desensitization is prolonged (>90 min) at UTP concentrations >10(-5) M; and 3) UTP-induced desensitization occurs before the operation of the anion secretory mechanism. (+info)
P2u receptor-mediated release of endothelium-derived relaxing factor/nitric oxide and endothelium-derived hyperpolarizing factor from cerebrovascular endothelium in rats.
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BACKGROUND AND PURPOSE: Stimulation of P2u purinoceptors by UTP on endothelium dilates the rat middle cerebral artery (MCA) through the release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) and an unknown relaxing factor. The purpose of this study was to determine whether this unknown relaxing factor is endothelium-derived hyperpolarizing factor (EDHF). METHODS: Rat MCAs were isolated, cannulated, pressurized, and luminally perfused. UTP was added to the luminal perfusate to elicit dilations. RESULTS: Resting outside diameter of the MCAs in one study was 209+/-7 micrometer (n=10). The MCAs showed concentration-dependent dilations with UTP administration. Inhibition of NO synthase with NG-nitro-L-arginine methyl ester (L-NAME) (1 micromol/L to 1 mmol/L) did not diminish the maximum response to UTP but did shift the concentration-response curve to the right. Scavenging NO with hemoglobin (1 or 10 micromol/L) or inhibition of guanylate cyclase with ODQ (1 or 10 micromol/L) had effects on the UTP-mediated dilations similar to those of L-NAME. In the presence of L-NAME, dilations induced by 10 micromol/L UTP were accompanied by 13+/-2 mV (P<0.009) hyperpolarization of the vascular smooth muscle membrane potential (-28+/-2 to -41+/-1 mV). Iberiotoxin (100 nmol/L), blocker of the large-conductance calcium-activated K channels, sometimes blocked the dilation, but its effects were variable. Charybdotoxin (100 nmol/L), also a blocker of the large-conductance calcium-activated K channels, abolished the L-NAME-insensitive component of the dilation to UTP. CONCLUSIONS: Stimulation of P2u purinoceptors on the endothelium of the rat MCA released EDHF, in addition to EDRF/NO, and dilated the rat MCA by opening an atypical calcium-activated K channel. (+info)
Signaling in human osteoblasts by extracellular nucleotides. Their weak induction of the c-fos proto-oncogene via Ca2+ mobilization is strongly potentiated by a parathyroid hormone/cAMP-dependent protein kinase pathway independently of mitogen-activated protein kinase.
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Extracellular nucleotides acting through specific P2 receptors activate intracellular signaling cascades. Consistent with the expression of G protein-coupled P2Y receptors in skeletal tissue, the human osteosarcoma cell line SaOS-2 and primary osteoblasts express P2Y1 and P2Y2 receptors, respectively. Their activation by nucleotide agonists (ADP and ATP for P2Y1; ATP and UTP for P2Y2) elevates [Ca2+]i and moderately induces expression of the c-fos proto-oncogene. A synergistic effect on c-fos induction is observed by combining ATP and parathyroid hormone, a key bone cell regulator. Parathyroid hormone elevates intracellular cAMP levels and correspondingly activates a stably integrated reporter gene driven by the Ca2+/cAMP-responsive element of the human c-fos promoter. Nucleotides have little effect on either cAMP levels or this reporter, instead activating luciferase controlled by the full c-fos promoter. This induction is reproduced by a stably integrated serum response element reporter independently of mitogen-activated protein kinase activation and ternary complex factor phosphorylation. This novel example of synergy between the cAMP-dependent protein kinase/CaCRE signaling module and a non-mitogen-activated protein kinase/ternary complex factor pathway that targets the serum response element shows that extracellular ATP, via P2Y receptors, can potentiate strong responses to ubiquitous growth and differentiative factors. (+info)
Human P2Y2 receptor polymorphism: identification and pharmacological characterization of two allelic variants.
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1. In the process of cloning the human P2Y2 receptor in order to establish 1321N1 cell lines expressing this receptor, we detected a gene polymorphism characterized by an arginine 334 to cysteine 334 transition. 2. The frequency distribution of the polymorphism was studied in a European population. We observed that 66% of the tested persons are homozygotes R/R, 29% are heterozygotes R/C and 5% are homozygotes C/C. The frequency of the R allele was 0.8 versus 0.2 for the C allele. 3. We stably expressed each form of the human P2Y2 receptor into 1321N1 cells and isolated clones by limiting dilution. The effects of nucleotides and antagonists on inositol trisphosphate accumulation and cyclic AMP formation were compared between the two cell lines. 4. The time-courses of inositol trisphosphate accumulation as well as concentration-response curves characterizing the effects of UTP, ATP, AP4A and ATP gamma S were mostly similar, except for slight kinetic differences (slower time-course with the 334C form). 5. The sensitivity to pertussis toxin of inositol trisphosphates accumulation was critically dependent on the agonist concentration and stimulation duration, suggesting the involvement of a Gi.0 protein during the early stimulation by low nucleotide concentrations. No inhibition of cyclic AMP accumulation could be detected. These properties were observed with both polymorphic receptors. (+info)
Regulation of epidermal homeostasis through P2Y2 receptors.
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1. Previous studies have indicated a role for extracellular ATP in the regulation of epidermal homeostasis. Here we have investigated the expression of P2Y2 receptors by human keratinocytes, the cells which comprise the epidermis. 2. Reverse transcriptase-polymerase chain reaction (RT - PCR) revealed expression of mRNA for the G-protein-coupled, P2Y2 receptor in primary cultured human keratinocytes. 3. In situ hybridization studies of skin sections revealed that P2Y2 receptor transcripts were expressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y2 receptor transcripts to the basal layer of the epidermis, the site of cell proliferation. 4. Increases in intracellular free Ca2+ concentration ([Ca2+]i) in keratinocytes stimulated with ATP or UTP demonstrated the presence of functional P2Y receptors. 5. In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTP and ATPgammaS were found to stimulate the proliferation of keratinocytes. 6. Using a real-time firefly luciferase and luciferin assay we have shown that under static conditions cultured human keratinocytes release ATP. 7. These findings indicate that P2Y2 receptors play a major role in epidermal homeostasis, and may provide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis. (+info)
Nucleotide-regulated calcium signaling in lung fibroblasts and epithelial cells from normal and P2Y(2) receptor (-/-) mice.
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To test for the role of the P2Y(2) receptor (P2Y(2)-R) in the regulation of nucleotide-promoted Ca(2+) signaling in the lung, we generated P2Y(2)-R-deficient (P2Y(2)-R(-/-)) mice and measured intracellular Ca(2+)(i) responses (DeltaCa(2+)(i)) to nucleotides in cultured lung fibroblasts and nasal and tracheal epithelial cells from wild type and P2Y(2)-R(-/-) mice. In the wild type fibroblasts, the rank order of potencies for nucleotide-induced DeltaCa(2+)(i) was as follows: UTP >/= ATP >> ADP > UDP. The responses induced by these agonists were completely absent in the P2Y(2)-R(-/-) fibroblasts. Inositol phosphate responses paralleled those of DeltaCa(2+)(i) in both groups. ATP and UTP also induced Ca(2+)(i) responses in wild type airway epithelial cells. In the P2Y(2)-R(-/-) airway epithelial cells, UTP was ineffective. A small fraction (25%) of the ATP response persisted. Adenosine and alpha,beta-methylene ATP were ineffective, and ATP responses were not affected by adenosine deaminase or by removal of extracellular Ca(2+), indicating that neither P1 nor P2X receptors mediated this residual ATP response. In contrast, 2-methylthio-ADP promoted a substantial Ca(2+)(i) response in P2Y(2)-R(-/-) cells, which was inhibited by the P2Y(1) receptor antagonist adenosine 3'-5'-diphosphate. These studies demonstrate that P2Y(2)-R is the dominant purinoceptor in airway epithelial cells, which also express a P2Y(1) receptor, and that the P2Y(2)-R is the sole purinergic receptor subtype mediating nucleotide-induced inositol lipid hydrolysis and Ca(2+) mobilization in mouse lung fibroblasts. (+info)