Didier Communi

Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase.

Clones encoding a new human P2Y receptor, provisionally called P2Y11, have been isolated from human placenta complementary DNA and genomic DNA libraries. The 1113-base pair open reading frame is interrupted by one intron. The P2Y11 receptor is characterized by considerably larger second and third extracellular loops than the subtypes described so far. The deduced amino acid sequence exhibits 33% amino acid identity with the P2Y1 receptor, its closest homolog. Northern blot analysis detected human P2Y11 receptor messenger RNA in spleen and HL-60 cells. The level of P2Y11 transcripts was strongly increased in these cells after granulocyte differentiation induced by retinoic acid or dimethyl sulfoxide. The new receptor was stably expressed in 1321N1 astrocytoma and CHO-K1 cells, where it couples to the stimulation of both the phosphoinositide and adenylyl cyclase pathways, a unique feature among the P2Y family. The rank order of agonists potency was: ATP > 2-methylthio-ATP >>> ADP, whereas UTP and UDP were inactive, indicating that it behaves as a selective purinoceptor.

Pharmacological characterization of the human P2Y11 receptor

The human P2Y11 receptor is coupled to both the phosphoinositide and the cyclic AMP pathways. A pharmacological characterization of the recombinant human P2Y11 receptor has been conducted following stable expression in two different cell lines: the 1321N1 astrocytoma cells for inositol trisphosphate measurements and the CHO‐K1 cells for cyclic AMP assays. The rank order of potency of a series of nucleotides was almost identical for the two pathways: ATPγS∼BzATP>dATP>ATP>ADPβS>2MeSATP. ADPβS, AMPαS and A3P5PS behaved as partial agonists of the human P2Y11 receptor. At high concentrations, these three nucleotides were able to partially inhibit the ATP response. Suramin was a more potent antagonist than reactive blue 2, whereas pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid was completely inactive. The P2Y11 receptor proved to be sensitive to suramin in a competitive way with an apparent Ki value of 0.82±0.07 μM. The ATP derivative AR‐C67085 (2‐propylthio‐β, γ‐dichloromethylene‐D‐ATP), a potent inhibitor of ADP‐induced platelet aggregation, was the most potent agonist of the P2Y11 receptor, among the various nucleotides tested. The pharmacological profile of the recombinant human P2Y11 receptor is closely similar to that of the cyclic AMP‐coupled P2 receptor recently described in HL‐60 cells, suggesting that it is the same receptor.

Advances in signalling by extracellular nucleotides. the role and transduction mechanisms of P2Y receptors.

Nucleotides are ubiquitous intercellular messengers whose actions are mediated by specific receptors. Since the first clonings in 1993, it is known that nucleotide receptors belong to two families: the ionotropic P2X receptors and the metabotropic P2Y receptors. Five human P2Y receptor subtypes have been cloned so far and a sixth one must still be isolated. In this review we will show that they differ by their preference for adenine versus uracil nucleotides and triphospho versus diphospho nucleotides, as well as by their transduction mechanisms and cell expression.

Receptors responsive to extracellular pyrimidine nucleotides.

Extracellular adenine nucleotides have long been known to mediate pharmacological responses via several subtypes of P2 purinoceptors. More recently, however, similar responses to uracil nucleotides have been demonstrated. Here, Didier Communi and Jean-Marie Boeynaems discuss the evidence to suggest that distinct pyrimidinoceptors do indeed exist, although they do not appear to constitute a separate receptor family of their own.

Expression of P2Y receptors in cell lines derived from the human lung

Northern blotting experiments have been performed with RNA extracted from several cell lines derived from the human lung in order to detect P2Y1, P2Y2, P2Y4 and P2Y6 mRNA. We have investigated the 1HAEo− and 16HBE14o− epithelial cell lines derived from the airway epithelium, the A549 cell line displaying properties of type II alveolar epithelial cells, the CALU‐3 serous cells, the 6CFSMEo− submucosal cells and the HASMSC1 airway smooth muscle cells. We have also evaluated one pancreatic epithelial cell line called CFPAC‐1. These experiments revealed that P2Y2 and P2Y6 mRNA are co‐expressed in the 1HAEo−, 16HBE14o− and A549 epithelial cell lines. The CFPAC‐1 pancreatic cell line was strongly positive for the P2Y2 receptor. No signal was obtained for the P2Y1 and P2Y4 receptors. We have then performed RT–PCR experiments with specific oligonucleotides of these last two P2Y receptors with the RNA used for the Northern blotting experiments. P2Y4 mRNA was detected in five cell lines: 1HAEo−, 16HBE14o−, 6CFSMEo−, HASMSC1 and CFPAC‐1. P2Y1 mRNA was only detected in the CALU‐3 cell line. Inositol trisphosphates assays have identified a response typical of the P2Y2 receptor in the 1HAEo− and the 16HBE14o− airway epithelial cell lines which co‐express P2Y2 and P2Y6 mRNA. By contrast, the 6CFSMEo− submucosal cells expressed a UTP‐specific response which displayed pharmacological characteristics compatible with the human P2Y4 receptor: in particular, there was no response to UDP or ATP and the UTP effect was totally inhibited by pertussis toxin.

Involvement of multiple P2Y receptors and signaling pathways in the action of adenine nucleotides diphosphates on human monocyte‐derived dendritic cells

Adenosine 5′‐triphosphate (ATP), which is released from necrotic cells, induces a semimaturation state of dendritic cells (DC), characterized by the up‐regulation of costimulatory molecules and the inhibition of proinflammatory cytokines. This action is mediated by cyclic adenosine monophosphate (cAMP) and involves the P2Y11 receptor. As DC express the ecto‐enzyme CD39, which converts ATP into adenosine 5′‐diphosphate (ADP), the effects of adenine nucleotides diphosphates on molecular signaling [intracellular calcium ([Ca2+]i), cAMP, extracellular signal‐regulated kinase 1 (ERK1)], costimulatory molecule expression (CD83), and cytokine production [interleukin (IL)‐12, tumor necrosis factor α (TNF‐α), IL‐10] were investigated in human monocyte‐derived DC. ADP, 2‐methylthio‐ADP, and ADPβS had no effect on cAMP, increased [Ca2+]i, and stimulated the phosphorylation of ERK1. The effect on ERK1 was inhibited by AR‐C69931MX, a P2Y12 and P2Y13 antagonist. On the contrary the effect on [Ca2+]i was neither inhibited by AR‐C69931MX or by the P2Y1 antagonist MRS‐2179. Both effects were inhibited by pertussis toxin. ADPβS alone was less potent for up‐regulation of CD83 than ATPγS and did not increase the CD83 expression by DC stimulated with lipopolysaccharide (LPS). Similar to ATPγS, ADPβS inhibited the release of IL‐12p40, IL‐12p70, and TNF‐α stimulated by LPS (1–100 ng/ml). The inhibitory effect of ADPβS on IL‐12 release was neither reversed by AR‐C69931MX or by MRS‐2179. The two nucleotides had opposite effects on IL‐10 production: inhibition by ADPβS and potentiation by ATPγS. In conclusion, ATP can modulate the function of DC, directly via a cAMP increase mediated by the P2Y11 receptor and indirectly via its degradation into ADP, which acts via Gi‐coupled receptors coupled to ERK activation and calcium mobilization. These distinct mechanisms converge on the inhibition of inflammatory cytokine production, particularly IL‐12, but have a differential effect on IL‐10.

Involvement of multiple receptors in the actions of extracellular ATP: the example of vascular endothelial cells.

The role of ATP and ADP as intercellular mediators is now well established. The presence of the nucleotides in extracellular fluids can result from several mechanisms: cell lysis, selective permeabilization of the plasma membrane and exocytosis of secretory vesicles, such as platelet dense bodies. Extracellular adenine nucleotides are rapidly degraded by ectonucleotidases expressed inter alia on the surface of endothelial cells. They act on cells via the family of P2 receptors which encompasses more than 5 subtypes, some of which have been cloned recently. The P2T, P2U and P2Y receptors belong to the superfamily of receptors coupled to G proteins, whereas the P2X receptor is a cation channel and the P2Z receptor a non-selective pore. ATP and ADP stimulate the endothelial production of prostacyclin (PGI2) and nitric oxide (NO), two vasodilators and inhibitors of platelet aggregation, via an increase in cytosolic Ca2+. This action of adenine nucleotides is believed to limit the extent of intravascular platelet aggregation and to help localize thrombus formation to areas of endothelial damage. The endothelial response to nucleotides is mediated by at least two distinct subtypes of P2 receptors, P2Y and P2U, both coupled to phospholipase C.

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)

Coexpression of Several Types of Metabotropic Nucleotide Receptors in Single Cerebellar Astrocytes

Abstract: We have examined the expression of mRNA for several P2Y nucleotide receptors by northern blot analysis in purified type 1 cerebellar astrocyte cultures. These results suggest that different P2Y subtypes could be responsible for ATP metabotropic calcium responses in single type 1 astrocytes. To identify these subtypes we have studied the pharmacological profile of ATP calcium responses using fura‐2 microfluorimetry. All tested astrocytes responded to ATP and UTP stimulations evoking similar calcium transients. Most astrocytes also responded to 2‐methylthioATP and ADP challenges. The agonist potency order was 2‐methylthioATP > ADP > ATP = UTP. Cross‐desensitization experiments carried out with ATP, UTP, and 2‐methylthioATP showed that 2‐methylthioATP and UTP interact with different receptors, P2Y1 and P2Y2 or P2Y4. In a subpopulation of type 1 astrocytes, ATP prestimulation did not block UTP responses, and UDP elicited clear intracellular Ca2+ concentration responses at very low concentrations. 2‐MethylthioATP and UTP calcium responses exhibited different sensitivity to pertussis toxin and different inhibition patterns in response to P2 antagonists. The P2Y1‐specific antagonist N6‐methyl‐2′‐deoxyadenosine 3′,5′‐bisphosphate (MRS 2179) specifically blocked the 2‐methylthio‐ATP responses. We can conclude that all single astrocytes coexpressed at least two types of P2Y metabotropic receptors: P2Y1 and either P2Y2 or P2Y4 receptors. Moreover, 30‐40% of astrocytes also coexpressed specific pyrimidine receptors of the P2Y6 subtype, highly selective for UDP coupled to pertussis‐toxin insensitive G protein.

Modulation of murine dendritic cell function by adenine nucleotides and adenosine: Involvement of the A2B receptor

Adenosine triphosphate has previously been shown to induce semi‐mature human monocyte‐derived dendritic cells (DC). These are characterized by the up‐regulation of co‐stimulatory molecules, the inhibition of IL‐12 and the up‐regulation of some genes involved in immune tolerance, such as thrombospondin‐1 and indoleamine 2,3‐dioxygenase. The actions of adenosine triphosphate are mediated by the P2Y11 receptor; since there is no functional P2Y11 gene in the murine genome, we investigated the action of adenine nucleotides on murine DC. Adenosine 5′‐(3‐thiotriphosphate) and adenosine inhibited the production of IL‐12p70 by bone marrow‐derived DC (BMDC). These inhibitions were relieved by 8‐p‐sulfophenyltheophylline, an adenosine receptor antagonist. The use of selective ligands and A  2B–/– BMDC indicated the involvement of the A2B receptor. A microarray experiment, confirmed by quantitative PCR, showed that, in presence of LPS, 5′‐(N‐ethylcarboxamido) adenosine (NECA, the most potent A2B receptor agonist) regulated the expression of several genes: arginase I and II, thrombospondin‐1 and vascular endothelial growth factor were up‐regulated whereas CCL2 and CCL12 were down‐regulated. We further showed that NECA, in combination with LPS, increased the arginase I enzymatic activity. In conclusion, the described actions of adenine nucleotides on BMDC are mediated by their degradation product, adenosine, acting on the A2B receptor, and will possibly lead to an impairment of Th1 response or tolerance.