Purinergic receptors mediate endothelial dysfunction and participate in atherosclerosis

Atherosclerosis is the main pathological basis of cardiovascular disease and involves damage to vascular endothelial cells (ECs) that results in endothelial dysfunction (ED). The vascular endothelium is the key to maintaining blood vessel health and homeostasis. ED is a complex pathological process involving inflammation, shear stress, vascular tone, adhesion of leukocytes to ECs, and platelet aggregation. The activation of P2X4, P2X7, and P2Y2 receptors regulates vascular tone in response to shear stress, while activation of the A2A, P2X4, P2X7, P2Y1, P2Y2, P2Y6, and P2Y12 receptors promotes the secretion of inflammatory cytokines. Finally, P2X1, P2Y1, and P2Y12 receptor activation regulates platelet activity. These purinergic receptors mediate ED and participate in atherosclerosis. In short, P2X4, P2X7, P2Y1, and P2Y12 receptors are potential therapeutic targets for atherosclerosis.

Quantitative Super-Resolution Imaging for the Analysis of GPCR Oligomerization

G-protein coupled receptors (GPCRs) are known to form homo- and hetero- oligomers which are considered critical to modulate their function. However, studying the existence and functional implication of these complexes is not straightforward as controversial results are obtained depending on the method of analysis employed. Here, we use a quantitative single molecule super-resolution imaging technique named qPAINT to quantify complex formation within an example GPCR. qPAINT, based upon DNA-PAINT, takes advantage of the binding kinetics between fluorescently labelled DNA imager strands to complementary DNA docking strands coupled to protein targeting antibodies to quantify the protein copy number in nanoscale dimensions. We demonstrate qPAINT analysis via a novel pipeline to study the oligomerization of the purinergic receptor Y2 (P2Y2), a rhodopsin-like GPCR, highly expressed in the pancreatic cancer cell line AsPC-1, under control, agonistic and antagonistic conditions. Results reveal that whilst the density of P2Y2 receptors remained unchanged, antagonistic conditions displayed reduced percentage of oligomers, and smaller numbers of receptors in complexes. Yet, the oligomeric state of the receptors was not affected by agonist treatment, in line with previous reports. Understanding P2Y2 oligomerization under agonistic and antagonistic conditions will contribute to unravelling P2Y2 mechanistic action and therapeutic targeting.

Increased Purinergic Responses Dependent on P2Y2 Receptors in Hepatocytes from CCl4-Treated Fibrotic Mice

Inflammatory and wound healing responses take place during liver damage, primarily in the parenchymal tissue. It is known that cellular injury elicits an activation of the purinergic signaling, mainly by the P2X7 receptor; however, the role of P2Y receptors in the onset of liver pathology such as fibrosis has not been explored. Hence, we used mice treated with the hepatotoxin CCl4 to implement a reversible model of liver fibrosis to evaluate the expression and function of the P2Y2 receptor (P2Y2R). Fibrotic livers showed an enhanced expression of P2Y2R that eliminated its zonal distribution. Hepatocytes from CCl4-treated mice showed an exacerbated ERK-phosphorylated response to the P2Y2R-specific agonist, UTP. Cell proliferation was also enhanced in the fibrotic livers. Hepatic transcriptional analysis by microarrays, upon CCl4 administration, showed that P2Y2 activation regulated diverse pathways, revealing complex action mechanisms. In conclusion, our data indicate that P2Y2R activation is involved in the onset of the fibrotic damage associated with the reversible phase of the hepatic damage promoted by CCl4.

Extracellular ATP activates store-operated Ca2+ entry in white adipocytes: functional evidence for STIM1 and ORAI1

In the present study, we have applied ratiometric measurements of intracellular Ca2+ concentrations ([Ca2+]i) to show that extracellularly applied ATP (adenosine triphosphate) (100 µM) stimulates store-operated Ca2+ entry (SOCE) in 3T3-L1 adipocytes. ATP produced a rapid increase in [Ca2+]i consisting of an initial transient elevation followed by a sustained elevated phase that could be observed only in the presence of extracellular Ca2+. Gene expression data and [Ca2+]i recordings with uridine-5′-triphosphate or with the phospholipase C (PLC) inhibitor U73122 demonstrated the involvement of purinergic P2Y2 receptors and the PLC/inositol trisphosphate pathway. The [Ca2+]i elevation produced by reintroduction of a Ca2+-containing intracellular solution to adipocytes exposed to ATP in the absence of Ca2+ was diminished by known SOCE antagonists. The chief molecular components of SOCE, the stromal interaction molecule 1 (STIM1) and the calcium release-activated calcium channel protein 1 (ORAI1), were detected at the mRNA and protein level. Moreover, SOCE was largely diminished in cells where STIM1 and/or ORAI1 had been silenced by small interfering (si)RNA. We conclude that extracellular ATP activates SOCE in white adipocytes, an effect predominantly mediated by STIM1 and ORAI1.

mTOR and differential activation of mitochondria orchestrate neutrophil chemotaxis

Neutrophils use chemotaxis to locate invading bacteria. Adenosine triphosphate (ATP) release and autocrine purinergic signaling via P2Y2 receptors at the front and A2a receptors at the back of cells regulate chemotaxis. Here, we examined the intracellular mechanisms that control these opposing signaling mechanisms. We found that mitochondria deliver ATP that stimulates P2Y2 receptors in response to chemotactic cues, and that P2Y2 receptors promote mTOR signaling, which augments mitochondrial activity near the front of cells. Blocking mTOR signaling with rapamycin or PP242 or mitochondrial ATP production (e.g., with CCCP) reduced mitochondrial Ca2+ uptake and membrane potential, and impaired cellular ATP release and neutrophil chemotaxis. Autocrine stimulation of A2a receptors causes cyclic adenosine monophosphate accumulation at the back of cells, which inhibits mTOR signaling and mitochondrial activity, resulting in uropod retraction. We conclude that mitochondrial, purinergic, and mTOR signaling regulates neutrophil chemotaxis and may be a pharmacological target in inflammatory diseases.