Bernard Robaye

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.

The P2Y11 Receptor Mediates the ATP-Induced Maturation of Human Monocyte-Derived Dendritic Cells

Recently, it has been shown that ATP and TNF-α synergize in the activation and maturation of human dendritic cells (DC); the effect of ATP was reproduced by hydrolysis-resistant derivatives of ATP and was blocked by suramin, suggesting the involvement of a P2 receptor, but the particular subtype involved was not identified. In this report we confirm that ATP and various derivatives synergize with TNF-α and LPS to induce the maturation of human monocyte-derived DC, as revealed by up-regulation of the CD83 marker and the secretion of IL-12. The rank order of potency of various analogs (AR-C67085 > adenosine 5′-O-(3-thiotriphosphate) = 2′- and 3′-O-(4-benzoyl-benzoyl) ATP > ATP > 2-methylthio-ATP) was close to that of the recombinant human P2Y11 receptor. Furthermore, these compounds activated cAMP production in DC, in a xanthine-insensitive way, consistent with the involvement of the P2Y11 receptor, which among P2Y subtypes has the unique feature of being dually coupled to phospholipase C and adenylyl cyclase activation. The involvement of the P2Y11/cAMP/protein kinase A signaling pathway in the nucleotide-induced maturation of DC is supported by the inhibitory effect of H89, a protein kinase A inhibitor. Taken together, our results demonstrate that ATP activates DC through stimulation of the P2Y11 receptor and subsequent increase in intracellular cAMP.

Autocrine Purinergic Receptor Signaling Is Essential for Macrophage Chemotaxis

Amplification of outside-in chemotactic signaling by inside-out purinergic signaling drives macrophage migration. Self-Help Migration Immune cells such as neutrophils and macrophages migrate to sites of infection or inflammation by following gradients of chemoattractants. These include chemokines, which can be released by other cells at the target site; components of the complement system, such as C5a; and bacterial products, such as the formylated peptide, fMLP. While they navigate along the chemoattractant gradient toward their destination, cells are also exposed to other signals, some of which may compete with the chemoattractant that the cells were already following. Another level of complexity in the regulation of cell migration came from the discovery that migrating neutrophils release adenosine triphosphate (ATP), which then functions in an autocrine fashion through the purinergic receptor P2Y2 to enhance migration; cells deficient in P2Y2 have impaired gradient sensing. Kronlage et al. provide evidence that autocrine ATP signaling is also required for the migration of macrophages in vitro and in an in vivo model. The authors found that more than one type of ATP receptor type as well as metabolites of ATP contributed to the migratory responses of macrophages; furthermore, ATP was not released through pannexin-1 proteins, as has been suggested for neutrophils. Together, these data suggest that autocrine purinergic receptor signaling may play a general role in regulating the chemotactic responses of immune cells. Chemotaxis, the movement of cells along chemical gradients, is critical for the recruitment of immune cells to sites of inflammation; however, how cells navigate in chemotactic gradients is poorly understood. Here, we show that macrophages navigate in a gradient of the chemoattractant C5a through the release of adenosine triphosphate (ATP) and autocrine “purinergic feedback loops” that involve receptors for ATP (P2Y2), adenosine diphosphate (ADP) (P2Y12), and adenosine (A2a, A2b, and A3). Whereas macrophages from mice deficient in pannexin-1 (which is part of a putative ATP release pathway), P2Y2, or P2Y12 exhibited efficient chemotactic navigation, chemotaxis was blocked by apyrase, which degrades ATP and ADP, and by the inhibition of multiple purinergic receptors. Furthermore, apyrase impaired the recruitment of monocytes in a mouse model of C5a-induced peritonitis. In addition, we found that stimulation of P2Y2, P2Y12, or adenosine receptors induced the formation of lamellipodial membrane protrusions, causing cell spreading. We propose a model in which autocrine purinergic receptor signaling amplifies and translates chemotactic cues into directional motility.

The purinergic receptor P2Y2 receptor mediates chemotaxis of dendritic cells and eosinophils in allergic lung inflammation.

To cite this article: Müller T, Robaye B, Vieira RP, Ferrari D, Grimm M, Jakob T, Martin SF, Di Virgilio F, Boeynaems J‐M, Virchow JC, Idzko M. The purinergic receptor P2Y2 receptor mediates chemotaxis of dendritic cells and eosinophils in allergic lung inflammation. Allergy 2010; 65: 1545–1553.

Knockout Mice Reveal a Role for P2Y6 Receptor in Macrophages, Endothelial Cells, and Vascular Smooth Muscle Cells

P2Y receptors are G-protein-coupled receptors activated by extracellular nucleotides. The P2Y6 receptor is selectively activated by UDP, and its transcript has been detected in numerous organs, including the spleen, thymus, intestine, blood leukocytes, and aorta. To investigate the biological functions of this receptor, we generated P2Y6-null mice by gene targeting. The P2Y6 knockout (KO) mice are viable and are not distinguishable from the wild-type (WT) mice in terms of growth or fertility. In thioglycollate-elicited macrophages, the production of inositol phosphate in response to UDP stimulation was lost, indicating that P2Y6 is the unique UDP-responsive receptor expressed by mouse macrophages. Furthermore, the amount of interleukin-6 and macrophage-inflammatory protein-2, but not tumor necrosis factor-α, released in response to lipopolysaccharide stimulation was significantly enhanced in the presence of UDP, and this effect was lost in the P2Y6 KO macrophages. The endothelium-dependent relaxation of the aorta by UDP was abolished in KO P2Y6 mice. The contractile effect of UDP on the aorta, observed when endothelial nitric-oxide synthase is blocked, was also abolished in P2Y6-null mice. In conclusion, we generated P2Y6-deficient mice and have shown that these mice have a defective response to UDP in macrophages, endothelial cells, and vascular smooth muscle cells. These observations might be relevant to several physiopathological conditions such as atherosclerosis or hypertension.

Extracellular adenine nucleotides modulate cytokine production by human monocyte‐derived dendritic cells: dual effect on IL‐12 and stimulation of IL‐10

To clarify the functional consequences of adenine nucleotides action on human monocyte‐derived dendritic cells (DC), we have systematically compared the effects of adenosine 5′‐O‐(3‐thiotriphosphate) (ATPγS), an ATP analog active on the P2Y11 receptor, on the responses to three DC stimuli, TNF‐α, LPS, sCD40L, tested at various concentrations, using two different IL‐12 assays. We observed that ATPγS potentiated the IL‐12p40 release induced by TNF‐α, but also by lipopolysaccharides (LPS) and soluble CD40 ligand (sCD40L). This potentiation was observed as long as the IL‐12p40 concentration under agonist stimulation remained below a threshold value close to 10 ng/ml; inhibition was observed above this value. The combinations ATPγS‐TNF‐α and ATPγS‐sCD40L were unable to induce detectable bioactive IL‐12p70 production and at concentrations of LPS that induced a significant stimulation of IL‐12p70, the effect of ATPγS was purely inhibitory. Our results also show that ATPγS synergized with LPS and sCD40L, but not TNF‐α, to stimulate IL‐10 production. In conclusion, we have clarified the discrepancies in the literature concerningthe action of adenine nucleotides on DC and our study supports the concept that, like prostaglandin E2 and other agents increasing cyclic AMP, they favor either a Th2 response or tolerance.

NTPDase1 (CD39) controls nucleotide-dependent vasoconstriction in mouse

AIMS Extracellular nucleotides are vasoactive molecules. The concentrations of these molecules are regulated by ectonucleotidases. In this study, we investigated the role of the blood vessel ectonucleotidase NTPDase1, in the vasoconstrictor effect of nucleotides using Entpd1(-/-) mice. METHODS AND RESULTS Immunofluorescence, enzyme histochemistry, and HPLC analysis were used to evaluate both NTPDase expression and activity in arteries and isolated vascular smooth muscle cells (VSMCs). Vascular reactivity was evaluated in vitro and mean arterial blood pressure was recorded in anesthetized mice after nucleotide i.v. infusion. Expression of nucleotide receptors in VSMCs was determined by RT-PCR. Entpd1(-/-) mice displayed a dramatic deficit of nucleotidase activity in blood vessel wall in situ and in VSMCs in comparison to control mice. In aortic rings from Entpd1(-/-) mice, UDP and UTP induced a potent and long-lasting constriction contrasting with the weak response obtained in wild-type rings. This constriction occurred through activation of P2Y(6) receptor and was independent of other uracil nucleotide-responding receptors (P2Y(2) and P2Y(4)). UDP infusion in vivo increased blood pressure and this effect was potentiated in Entpd1(-/-) mice. In addition, pressurized mesenteric arteries from Entpd1(-/-) mice displayed an enhanced myogenic response, consistent with higher local concentrations of endogenously released nucleotides. This effect was inhibited by the P2 receptor antagonist RB-2. CONCLUSION NTPDase1 is the major enzyme regulating nucleotide metabolism at the surface of VSMCs and thus contributes to the local regulation of vascular tone by nucleotides.

K+ secretion activated by luminal P2Y2 and P2Y4 receptors in mouse colon.

Extracellular nucleotides are important regulators of epithelial ion transport, frequently exerting their action from the luminal side. Luminal P2Y receptors have previously been identified in rat distal colonic mucosa. Their activation by UTP and ATP stimulates K+ secretion. The aim of this study was to clarify which of the P2Y receptor subtypes are responsible for the stimulated K+ secretion. To this end P2Y2 and P2Y4 knock‐out mice were used to measure distal colonic ion transport in an Ussing chamber. In mouse (NMRI) distal colonic mucosa, luminal UTP and ATP with similar potency induced a rapid and transient increase of the transepithelial voltage (Vte) (UTP: from −0.81 ± 0.23 to 3.11 ± 0.61 mV, n= 24), an increase of equivalent short circuit current (Isc) by 166.9 ± 22.8 μA cm−2 and a decrease of transepithelial resistance (Rte) from 29.4 ± 2.4 to 23.5 ± 2.0 Ω cm2. This effect was completely inhibited by luminal Ba2+ (5 mm, n= 5) and iberiotoxin (240 nm, n= 6), indicating UTP/ATP‐stimulated K+ secretion. RT‐PCR analysis of isolated colonic crypts revealed P2Y2, P2Y4 and P2Y6 specific transcripts. The luminal UTP‐stimulated K+ secretion was still present in P2Y2 receptor knock‐out mice, but significantly reduced (ΔVte: 0.83 ± 0.26 mV) compared to wild‐type littermates (ΔVte: 2.08 ± 0.52 mV, n= 9). In P2Y4 receptor knock‐out mice the UTP‐induced K+ secretion was similarly reduced. Luminal UTP‐stimulated K+ secretion was completely absent in P2Y2/P2Y4 double receptor KO mice. Basolateral UTP showed no effect. In summary, these results indicate that both the P2Y2 and P2Y4 receptors are present in the luminal membrane of mouse distal colonic mucosa, and stimulation of these receptors leads to K+ secretion.

P2Y13 receptor is critical for reverse cholesterol transport.

A major atheroprotective functionality of high‐density lipoproteins (HDLs) is to promote “reverse cholesterol transport” (RCT). In this process, HDLs mediate the efflux and transport of cholesterol from peripheral cells and its subsequent transport to the liver for further metabolism and biliary excretion. We have previously demonstrated in cultured hepatocytes that P2Y13 (purinergic receptor P2Y, G protein–coupled, 13) activation is essential for HDL uptake but the potential of P2Y13 as a target to promote RCT has not been documented. Here, we show that P2Y13‐deficient mice exhibited a decrease in hepatic HDL cholesterol uptake, hepatic cholesterol content, and biliary cholesterol output, although their plasma HDL and other lipid levels were normal. These changes translated into a substantial decrease in the rate of macrophage‐to‐feces RCT. Therefore, hallmark features of RCT are impaired in P2Y13‐deficient mice. Furthermore, cangrelor, a partial agonist of P2Y13, stimulated hepatic HDL uptake and biliary lipid secretions in normal mice and in mice with a targeted deletion of scavenger receptor class B type I (SR‐BI) in liver (hypomSR‐BI–knockoutliver) but had no effect in P2Y13 knockout mice, which indicate that P2Y13‐mediated HDL uptake pathway is independent of SR‐BI–mediated HDL selective cholesteryl ester uptake. Conclusion: These results establish P2Y13 as an attractive novel target for modulating RCT and support the emerging view that steady‐state plasma HDL levels do not necessarily reflect the capacity of HDL to promote RCT. (HEPATOLOGY 2010)