Etude des mécanismes d'action de l'ATP extracellulaire sur l'endothélium vasculaire

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Transduction mechanisms of p2 purinergic receptors: Role of phospholipase c and calcium

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Evidence that ATP, ADP and AMP are not ligands of the striatal adenosine A2A receptors.

It has been claimed recently that, in several cell types, ATP can induce a stimulation of cAMP production which is sensitive to methylxanthine inhibition and is not mediated by the ATP degradation product, adenosine. One explanation for these results would be direct activation of adenosine A2 receptors by ATP itself. We have therefore investigated whether adenine nucleotides are ligands of adenosine A2A receptors from bovine striatum. We show here that ATP, ADP, AMP and their phosphorothioates analogues (ATP gamma S, ADP beta S and AMP alpha S), at a 100 microM concentration, produced a 83-91% inhibition of the binding of [3H]CGS21680, an adenosine A2A receptor agonist, to striatum membranes. However, this action was inhibited by adenosine deaminase or by adenosine 5'-O-(alpha, beta-methylene)diphosphate (APCP), an inhibitor of 5'-nucleotidase-mediated AMP degradation. The effects of adenosine deaminase and APCP were dependent on their concentration. These results indicate that ATP, ADP and even AMP can exert an effect on the adenosine A2A receptors only through their breakdown into adenosine by ectonucleotidases.In VitroJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Evidence that a form of ATP uncomplexed with divalent cations is the ligand of P2y and nucleotide/P2u receptors on aortic endothelial cells

1. The response of bovine aortic endothelial cells to adenosine 5'-triphosphate (ATP) is mediated by both P2y and nucleotide/P2u receptors. In order to determine which form of the nucleotide is the true ligand of these receptors, we have investigated the effects of divalent cations on ATP-, uridine 5'-triphosphate (UTP)- and 2 methylthioadenosine 5'-triphosphate (2MeSATP)-induced inositol phosphate accumulation in these cells. 2. Omisson of Mg2+ from a calcium-free incubation buffer caused a shift to the left of the ATP concentration-action curve. 3. In the presence of EDTA (1 mM), the basal level of inositol trisphosphate (InsP3) was markedly increased and the absolute maximal response to ATP was decreased; however, the response to low concentrations of ATP was enhanced. 4. When the results were plotted in terms of calculated ATP4- concentrations, the concentration-response curves obtained in the presence of 1.25 mM Mg2+ lay closer to the respective curves obtained when Mg2+ was omitted from the medium or when Mg2+ was omitted and EDTA (1 mM) was added. The curves became almost superimposable when the baseline value was subtracted. 5. A similar shift to the left of the concentrations-action curves was also observed with both UTP and 2MeSATP. 6. Our data provide evidence that a form of ATP uncomplexed with divalent cation is the preferential agonist of both the nucleotide/P2u and the P2y receptors expressed on bovine aortic endothelial cells.Journal ArticleResearch Support, Non-U.S. Gov'tFLWINinfo:eu-repo/semantics/publishe

Pharmacological characterization of the human P2Y4 receptor

info:eu-repo/semantics/publishedPurines '96 Molecular, Pharmacological and Therapeutic Advances, Milan (Italy) - July 6-9, 199

Heterogeneity of ATP receptors in aortic endothelial cells. Involvement of P2y and P2u receptors in inositol phosphate response.

Extracellular ATP plays an important role in the regulation of prostacyclin and nitric oxide release from vascular endothelial cells. These cellular responses to ATP are generally attributed to the stimulation of the P2y subtype of P2 purinergic receptors. However, it has recently been suggested that two types of ATP receptors might coexist on endothelial cells. To evaluate this hypothesis, we examined the effects of P2y receptor agonists 2-methylthioadenosine 5'-triphosphate (2MeSATP) and 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) and of UTP on the accumulation of inositol phosphates in bovine aortic endothelial cells. BzATP, 2MeSATP, and UTP produced a smaller maximal effect than ATP. The effects of 2MeSATP and UTP were additive, whereas the effects of ATP and either UTP or 2MeSATP were not. Prior exposure to UTP reduced the subsequent response to UTP to 12% of the control response, whereas the response to 2MeSATP was decreased to 61%. Reciprocally, preincubation with 2MeSATP reduced the subsequent response to 2MeSATP to 23% of the control response, whereas the response to UTP was reduced to 73%. Pertussis toxin pretreatment decreased the response to both ATP and UTP (65% and 70% inhibition, respectively), whereas the response to 2MeSATP was not modified. Our data support the hypothesis that two classes of receptors recognizing ATP are expressed on bovine aortic endothelial cells.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Cloning and functional expression of a human uridine nucleotide receptor.

In order to isolate new subtypes of P2 purinoceptors, sets of degenerate oligonucleotide primers were synthesized on the basis of the best conserved segments in the published sequences of the chick brain P2Y/P2Y1 and murine neuroblastoma P2U/P2Y2 receptors. Their use in polymerase chain reaction (PCR) experiments on human genomic DNA amplified, among other things, a 712-base pair sequence, that was used as a probe to screen a human genomic DNA library. Several clones corresponding to a single locus were isolated, and the sequence analysis revealed an intronless 1095-base pair open reading frame. The deduced amino acid sequence is consistent with a G protein-coupled receptor and exhibits 51% identity with the human P2Y2 receptor and 35% with the chick P2Y1 receptor. A close comparison with the human P2Y2 sequence reveals the conservation of histidine 262, arginine 265, lysine 289, and arginine 292, which were reported to be involved in nucleotide binding (Erb, L. Garrad, R. Wang, Y. Quinn, T. Turner, J. T. and Weisman, G. A. (1995) J. Biol. Chem. 270, 4185-4188). Northern blot analysis detected a 1.8-kilobase messenger RNA in human placenta. The coding sequence was inserted in the pcDNA3 vector in order to transfect 1321N1 human astrocytoma cells. In cells stably expressing the receptor, UTP and UDP stimulated the formation of inositol phosphates with equivalent potency and maximal effect, ATP behaved as a partial agonist, and ADP was almost inactive. We have thus cloned a new member of the G protein-coupled P2 purinergic receptor family, which functionally behaves as a pyrimidinergic receptor.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Pharmacological characterization of the human P2Y4 receptor.

The P2Y4 receptor is a new member of the P2Y family which functionally behaves as a pyrimidinergic receptor. The pharmacological properties of the human P2Y4 receptor have been characterized following its stable expression in 1321N1 astrocytoma cells. UTP induced a biphasic accumulation of inositol trisphosphates, with an early peak at 30 s followed by a smaller but more sustained accumulation. ATP was a pure antagonist at early times and later behaved as a partial agonist. At 20 min, the rank order of potency of various nucleotides was the following: UTP > UDP = deoxy UTP > 5-bromo-UTP > ITP > ATP. Diadenosine polyphosphates also stimulated the production of inositol trisphosphates (after 20 min), more potently than ATP, but their maximal effect represented only 20-25% of that of UTP. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid inhibited strongly the UTP response, whereas suramin was inactive and reactive blue 2 had an intermediate effect. Pertussis toxin inhibited the response to UTP at early times (62 +/- 5% inhibition at 30 s), but its effect was no longer observed at 5 or 20 min. It is speculated that the P2Y4 receptor can exist in two distinct activation states differing in terms of time-course, specificity for uridine nucleotides and G-protein coupling.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Coexpression of P2Y and P2U receptors on aortic endothelial cells. Comparison of cell localization and signaling pathways.

Depending on the vascular bed considered, the actions of ATP on the endothelium are mediated by either P2Y or P2U receptors. The two types of receptors seem to coexist on bovine aortic endothelial cells, where they are both coupled to phospholipase C. In this study, we have investigated whether they are truly coexpressed on the same cells and whether their signaling pathways diverge beyond phospholipase C activation. Measurements of [Ca2+]i in single cells showed that almost all bovine aortic endothelial cells are responsive to both 2-methylthio-ATP (2MeSATP), an agonist of P2Y receptors, and UTP, an agonist of P2U receptors. UTP stimulated the release of prostacyclin from freshly isolated bovine aortic endothelial cells, even when they were exposed to cycloheximide at the time of their collection: this indicates that P2U receptors must already be expressed on endothelial cells in situ and do not appear during cell culture. The time course of inositol phosphate (InsP) accumulation and the relative proportion of Ins(1,4,5)P3, Ins(1,3,4,5)P4, and Ins(1,3,4)P3 were similar in cells stimulated by 2MeSATP or UTP. UTP and 2MeSATP both stimulated the hydrolysis of phosphatidylcholine by phospholipase D, as reflected by the release of [3H]choline from prelabeled cells. The responses to both agents were blocked after downregulation of protein kinase C, resulting from a prolonged exposure to phorbol 12-myristate 13-acetate: this blockade occurred at a step distal to phospholipase C activation. A single difference between the two pathways has been identified: the effect of 2MeSATP on InsP3 was significantly more inhibited after a short exposure to phorbol 12-myristate 13-acetate than that of UTP.(ABSTRACT TRUNCATED AT 250 WORDS)Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Cloning of human pyrimidinergic receptors

F. I.2,3000FLWINinfo:eu-repo/semantics/publishe