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Extracellular nucleotides are responsible for pleiotropic effects in the vasculature. Uracyl nucleotides are vasoactive and trophic agents and promote inflammation. The participation of specific P2 receptors in these effects remains undefined and their potential contribution in arterial hypertension is unknown. Objective To evaluate the contribution of the UDP receptor P2Y6 in hypertension in mouse. Methods Arterial contraction was evaluated using a wire myograph. Blood pressure was measured following nucleotides iv infusion and experimental hypertension was induced either by Angiotensine-II (Ang-II 1mg/kg/j) or DOCA-salt (1%) in uni-nephrectomized mice. Histological approaches, immunofluorescence and RTqPCR were used to evaluate the nature of vascular remodeling. Results P2Y6 displayed the highest arterial expression level among other P2Y receptors. Contraction of conductance (thoracic aorta) and resistance (mesenteric) arteries was abrogated in P2ry6-/- mice in response to UDP and UTP while other vasoconstrictor induced normal responses. P2Y6 receptor triggered a moderated intracellular calcium increase while RhoA (calcium facilitating pathway) activation was abrogated in P2ry6-/- mice. Both genetic deletion and pharmacological blockade of P2Y6 receptor abolished Ang-II-induced blood pressure increase (40 mmHg in wild type mice). By contrast, hypertensive response in DOCA-salt was equivalent in both genotypes. Following Ang-II treatment, P2ry6-/- mice developed a reduced arterial hypertrophic remodeling and fibrosis but equivalent immune cell recruitment/infiltration compared to wild type. These changes were corroborated to reduced mRNA expressions of TGFβ and NADPH oxidase subunits. Conclusions Vascular P2Y6 receptor contributes to exaggerated vascular tone, hypertrophy and fibrosis in the context of Ang-II-dependent hypertension. Its absence or pharmacological blockade limits vascular damages and prevents blood pressure increase associated to hypertension. [ABSTRACT FROM AUTHOR]

Background & Aims: During progression of liver disease, inflammation affects survival of hepatocytes. Endogenous release of adenosine triphosphate (ATP) in the liver activates purinergic P2 receptors (P2R), which regulate inflammatory responses, but little is known about the roles of these processes in the development of acute hepatitis. Methods: We induced acute hepatitis in C57BL/6 mice by intravenous injection of concanavalin A and then analyzed liver concentrations of ATP and expression of P2R. We assessed P2Y 2 R −/− mice and C57BL/6 wild-type mice injected with suramin, a pharmacologic inhibitor of P2YR. Toxic liver failure was induced in mice by intraperitoneal injection of acetaminophen. Hepatocyte-specific functions of P2R signaling were analyzed in primary mouse hepatocytes. Results: Induction of acute hepatitis in wild-type C57BL/6 mice released large amounts of ATP from livers and induced expression of P2Y2R. Liver damage and necrosis were greatly reduced in P2Y 2 R −/− mice and C57BL/6 mice given injections of suramin. Acetaminophen-induced liver damage was reduced in P2Y 2 R −/− mice. Analysis of liver-infiltrating immune cells during acute hepatitis revealed that expression of P2Y2R in bone marrow−derived cells was required for liver infiltration by neutrophils and subsequent liver damage. Hepatic expression of P2Y2R interfered with expression of genes that regulate cell survival, and promoted tumor necrosis factor−α-mediated cell death, in a cell-autonomous manner. Conclusions: Extracellular ATP and P2Y2R have cell-type specific, but synergistic functions during liver damage that regulate cellular immune responses and promote hepatocyte death. Reagents designed to target P2Y2R might be developed to treat inflammatory liver disease. [ABSTRACT FROM AUTHOR]

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques. [ABSTRACT FROM AUTHOR]

Immune cell function is modulated by changes in extracellular nucleotide levels. Here we used reverse transcription-PCR analyses, single cell Ca2+ imaging, and knock-out mice to define the receptors mediating nucleotide-induced Ca2+ signaling in resident peritoneal macrophages. In Ca2+ -free buffer, the potent (K0.5 <1 μM) stimulatory effect of UTP (or ATP) on endoplasmic reticulum (ER) Ca2+ release was abolished in cells isolated from P2Y2/P2Y4 double knock-out mice. More- over, P2Y40/-, but not P2Y2-/-, macrophages responded to UTP. In P2Y2-/- macrophages, we could elicit Ca2+ responses to ‘pure’ P2X receptor activation by applying ATP in buffer containing Ca2+. Purified UDP and ADP were ineffective agonists, although modest UDP-induced Ca2+ responses could be elicited in macrophages after ‘activation’ with lipopolysaccharide and interferon-γ. Notably, in Ca2+-free buffer, UTP-induced Ca2+ transients decayed within 1 mm, and there was no response to repeated agonist challenge. Measurements of ER [Ca2+] with mag-fluo-4 showed that ER Ca2+ stores were depleted under these conditions, When extracellular Ca2+ was available, ER Ca2+ stores refilled, but Ca2+ increased to only ∼40% of the initial value upon repeated UTP challenge. This apparent receptor desensitization persisted in GRK2+/- and GRK6-/- macrophages and after inhibition of candidate kinases protein kinase C and calmodulin-dependent kinase II. Initial challenge with UTP also reduced Ca2+ mobilization by complement component C5a (and vice versa). In conclusion, homologous receptor desensitization is not the major mechanism that rapidly dampens Ca2+ signaling mediated by P2Y2, the sole Gq-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y6 receptor expression) increases following macrophage activation. [ABSTRACT FROM AUTHOR]

The activation of Toll-like receptors (TLRs) by lipopolysaccharide or other ligands evokes a proinflammatory immune response, which is not only capable of clearing invading pathogens but can also inflict damage to host tissues. It is therefore important to prevent an overshoot of the TLR-induced response where necessary, and here we show that extracellular ATP is capable of doing this in human monocytes. Using reverse transcription-PCR, we showed that monocytes express P2Y1, P2Y2, P2Y4, P2Y11, and P2Y13 receptors, as well as several P2X receptors. To elucidate the function of these receptors, we first studied Ca2+ signaling in single cells. ATP or UTP induced a biphasic increase in cytosolic Ca2+, which corresponded to internal Ca2+ release followed by activation of store-operated Ca2+ entry. The evoked Ca2+ signals stimulated Ca2+-activated K+ channels, producing transient membrane hyperpolarization. In addition, ATP promoted cytoskeleton reorganization and cell migration; however, unlike chemoattractants, the migration was non-directional and further analysis showed that ATP did not activate Akt, essential for sensing gradients. When TLR2, TLR4, or TLR2/6 were stimulated with their respective ligands, ATPγS profoundly inhibited secretion of proinflammatory cytokines (tumor necrosis factor-α and monocyte chemoattractant protein-1) but increased the production of interleukin-10, an anti-inflammatory cytokine. In radioimmune assays, we found that ATP (or ATPγS) strongly increased cAMP levels, and, moreover, the TLR-response was inhibited by forskolin, whereas UTP neither increased cAMP nor inhibited the TLR-response. Thus, our data suggest that ATP promotes non-directional migration and, importantly, acts as a ‘host tissue damage’ signal via the Gs protein-coupled P2Y11 receptor and increased cAMP to negatively regulate TLR signaling. [ABSTRACT FROM AUTHOR]

The aim of the present study was to identify biochemical pathways driving the resistance of endothelial cells to apoptosis induced by tumour necrosis factor-α (TNF). (1) Although nuclear factor-kappa B (NF-κB) was activated by TNF, its inhibition by MG-132 failed to sensitize these cells. (2) The activation of protein kinase C (PKC) by phorbol ester completely abolished the TNF-induced cell death. (3) The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (Wo) triggered apoptosis and enhanced the TNF-induced cell death. (4) The MEK inhibitor PD98059 did not affect the TNF-induced apoptotic process. (5) The p38 is activated by TNF and its inhibition by SB203580 sensitized the cells to TNF. This is correlated with the inhibition of phosphorylation of heat-shock protein of 27 kDa (HSP27).These results indicate that TNF activates NF-κB, which does not drive any anti-apoptotic response, and p38, which plays an anti-apoptotic function probably through HSP27 phosphorylation. Moreover, PKC and PI3K are involved in the control of survival pathways. [Copyright &y& Elsevier]