Under physiological conditions, adenosine triphosphate (ATP) is present at low levels in the extracellular milieu, being massively released by stressed or dying cells. Once outside the cells, ATP and related nucleotides/nucleoside generated by ectonucleotidases mediate a high evolutionary conserved signaling system: the purinergic signaling, which is involved in a variety of pathological conditions, including inflammatory diseases. Extracellular ATP has been considered an endogenous adjuvant that can initiate inflammation by acting as a danger signal through the activation of purinergic type 2 receptors—P2 receptors (P2Y G-protein coupled receptors and P2X ligand-gated ion channels). Among the P2 receptors, the P2X7 receptor is the most extensively studied from an immunological perspective, being involved in both innate and adaptive immune responses. P2X7 receptor activation induces large-scale ATP release via its intrinsic ability to form a membrane pore or in association with pannexin hemichannels, boosting purinergic signaling. ATP acting via P2X7 receptor is the second signal to the inflammasome activation, inducing both maturation and release of pro-inflammatory cytokines, such as IL-1β and IL-18, and the production of reactive nitrogen and oxygen species. Furthermore, the P2X7 receptor is involved in caspases activation, as well as in apoptosis induction. During adaptive immune response, P2X7 receptor modulates the balance between the generation of T helper type 17 (Th17) and T regulatory (Treg) lymphocytes. Therefore, this receptor is involved in several inflammatory pathological conditions. In infectious diseases and cancer, P2X7 receptor can have different and contrasting effects, being an angel or a demon depending on its level of activation, cell studied, type of pathogen, and severity of infection. In neuroinflammatory and neurodegenerative diseases, P2X7 upregulation and function appears to contribute to disease progression. In this review, we deeply discuss P2X7 receptor dual function and its pharmacological modulation in the context of different pathologies, and we also highlight the P2X7 receptor as a potential target to treat inflammatory related diseases.
Background Decreased endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production are critical contributors to endothelial dysfunction and vascular complications observed in many diseases, including diabetes mellitus. Extracellular nucleotides activate eNOS and increase NO generation, however the mechanism of this observation is not fully clarified. Methods and Results To elucidate the signaling pathway(s) leading to nucleotide-mediated eNOS phosphorylation at Ser-1177, human umbilical vein endothelial cells (EC) were treated with several nucleotides including, ATP, UTP, and ADP in the presence or absence of selective inhibitors. These experiments identified P2Y1, P2Y2 and possibly P2Y4 as the purinergic receptors involved in eNOS phosphorylation, and demonstrated that this process was adenosine-independent. Nucleotide-induced eNOS phosphorylation and activity were inhibited by BAPTA-AM (an intracellular free calcium chelator), rottlerin (a protein kinase C (PKC) delta inhibitor) and PKC delta siRNA. In contrast, blockade of AMP-activated protein kinase (AMPK), calcium/calmodulin-dependent kinase (CaMK) II, CaMK kinase (CaMKK), serine/threonine protein kinase B (Akt), protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK) and p38 mitogen-activated protein kinase (MAPK) did not affect nucleotide-mediated eNOS phosphorylation. Conclusions The present study indicates that extracellular nucleotide-mediated eNOS phosphorylation is calcium and PKC delta dependent. This newly identified signaling pathway opens new therapeutic avenues for the treatment of endothelial dysfunction.
Abstract-AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis and is activated in response to cellular stress, including hypoxia/ischemia and hyperglycemia. The stress events are accompanied by rapid release of extracellular nucleotides from damaged tissues or activated endothelial cells (EC) and platelets. We demonstrate that extracellular nucleotides (ATP, ADP, and UTP, but not UDP) and adenosine independently induce phosphorylation and activation of AMPK in human umbilical vein EC (HUVEC) by the mechanism that is not linked to changes in AMP:ATP ratio. HUVEC express NTPDases, as well as 5Ј-nucleotidase; hence, nucleotides can be metabolized to adenosine. However, inhibition of 5Ј-nucleotidase had no effect on ATP/ADP/UTP-induced phosphorylation of AMPK, indicating that AMPK activation occurred as a direct response to nucleotides. Nucleotide-evoked phosphorylation of AMPK in HUVEC was mediated by P2Y1, P2Y2, and/or P2Y4 receptors, whereas P2Y6, P2Y11, and P2X receptors were not involved. The nucleotide-induced phosphorylation of AMPK was affected by changes in the concentration of intracellular Ca 2ϩ and by Ca 2ϩ /calmodulin-dependent kinase kinase (CaMKK), although most likely it was not dependent on LKB1 kinase. Adenosine-induced phosphorylation of AMPK was not mediated by P1 receptors but required adenosine uptake by equilibrative nucleoside transporters followed by its (intracellular) metabolism to AMP. Moreover, adenosine effect was Ca 2ϩ and CaMKK independent, although probably associated with upstream LKB1. We hypothesize that P2 receptors and adenosine transporters could be novel targets for the pharmacological regulation of AMPK activity and its downstream effects on EC function. is an evolutionarily conserved enzyme that acts as an ultrasensitive energy charge sensor and regulates cell energy metabolism. 1 AMPK is a heterotrimeric Ser/Thr kinase consisting of a catalytic ␣ subunit and regulatory  and ␥ subunits. AMPK is activated in response to ATP depletion associated with an increase in the intracellular AMP:ATP ratio. This ATP consumption can originate from pathological cellular stress, such as heat shock, hypoxia, or ischemia, and from physiological exerciseinduced skeletal muscle contraction. 2 AMPK can also be phosphorylated and activated by the mechanism independent of changes in AMP:ATP levels. 3 AMPK is activated allosterically by AMP (up to 5-fold) and by phosphorylation of Thr172 on the ␣ subunit (50-to 100-fold), which is catalyzed by upstream kinases, including LKB1 and CaMKK. 4,5 Activated AMPK turns on catabolic pathways that generate ATP and turns off pathways that consume ATP by phosphorylation of multiple targets. These include glycogen synthase, acetylcoenzyme A carboxylase (ACC), and hydroxymethylglutarylcoenzyme A (HMG-CoA) reductase. Because AMPK is central in controlling the metabolism of glucose and fatty acids, its role in obesity and type 2 diabetes is of major importance. AMPK is expressed in skeletal muscle, brain, liver, and pancre...
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