Purinergic P2Y Receptors Research Articles

Extracellular adenosine 5ʹ-diphosphate promotes MCP-1/CCL2 expression via the P2Y13 purinergic receptor/ERK signaling axis in temporomandibular joint-derived mouse fibroblast-like synoviocytes

BackgroundTemporomandibular joint osteoarthritis (TMJ-OA) causes cartilage degeneration, bone cavitation, and fibrosis of the TMJ. However, the mechanisms underlying the fibroblast-like synoviocyte (FLS)-mediated inflammatory activity in TMJ-OA remain unclear.Methods and resultsReverse transcription-quantitative polymerase chain reaction analysis revealed that the P2Y1, P2Y12, and P2Y13 purinergic receptor agonist adenosine 5ʹ-diphosphate (ADP) significantly induces monocyte chemotactic protein 1 (MCP-1)/ C–C motif chemokine ligand 2 (CCL2) expression in the FLS1 synovial cell line. In contrast, the uracil nucleotide UTP, which is a P2Y2 and P2Y4 agonist, has no significant effect on MCP-1/CCL2 production in FLS1 cells. In addition, the P2Y13 antagonist MRS 2211 considerably decreases the expression of ADP-induced MCP-1/CCL2, whereas ADP stimulation enhances extracellular signal-regulated kinase (ERK) phosphorylation. Moreover, it was found that the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor U0126 reduces ADP-induced MCP-1/CCL2 expression.ConclusionADP enhances MCP-1/CCL2 expression in TMJ FLSs via P2Y13 receptors in an MEK/ERK-dependent manner, thus resulting in inflammatory cell infiltration in the TMJ. Collectively, the findings of this study contribute to a partial clarification of the signaling pathway underlying the development of inflammation in TMJ-OA and can help identify potential therapeutic targets for suppressing ADP-mediated purinergic signaling in this disease.

Altered expression of microglial markers of phagocytosis in schizophrenia

BackgroundCognitive disturbances in schizophrenia have been linked to a lower density of dendritic spines on pyramidal neurons in the prefrontal cortex (PFC). Complement component C4, which has previously been found at higher levels in schizophrenia, marks synapses for phagocytosis by microglia. Thus, elevated consumption of dendritic spines by microglia mediated through excessive complement activity may play a role in lower spine density in schizophrenia. However, it is unclear if microglia themselves have the molecular capacity for enhanced phagocytosis of spines in schizophrenia. MethodsTranscript levels for complement components and microglia-specific phagocytic markers were quantified using quantitative PCR in the PFC of 62 matched pairs of schizophrenia and unaffected comparison subjects and in antipsychotic-exposed monkeys. ResultsRelative to comparison subjects, schizophrenia subjects had higher mRNA levels for C4 (+154 %); C1q (+69 %), which initiates the classical complement pathway that includes C4; and for microglia-specific markers that enable phagocytic activity including TAM receptor tyrosine kinases Axl (+27 %) and MerTK (+27 %) and lysosome-associated glycoprotein CD68 (+27 %) (all p ≤ .042). Transcript levels for microglial phagocytic markers were correlated with C4 mRNA levels in schizophrenia subjects (all r ≥ 0.31, p ≤ .015). We also found further evidence consistent with microglial activation in schizophrenia, including higher mRNA levels for THIK1 (TWIK-related halothane-inhibited potassium channel: +30 %) and lower mRNA levels for the purinergic receptor P2Y12 (−27 %) (all p ≤ .016). Transcript levels were unchanged in antipsychotic-exposed monkeys. ConclusionsThese results are consistent with the presence of increased complement activity and an elevated molecular capacity of microglia for phagocytosis in the same schizophrenia subjects.

Progressive Transcriptional Changes in the Amygdala Implicate Neuroinflammation in the Effects of Repetitive Low-Level Blast Exposure in Male Rats.

Chronic mental health problems are common among military veterans who sustained blast-related traumatic brain injuries. The reasons for this association remain unexplained. Male rats exposed to repetitive low-level blast overpressure (BOP) exposures exhibit chronic cognitive and post-traumatic stress disorder (PTSD)-related traits that develop in a delayed fashion. We examined blast-induced alterations on the transcriptome in four brain areas (anterior cortex, hippocampus, amygdala, and cerebellum) across the time frame over which the PTSD-related behavioral phenotype develops. When analyzed at 6 weeks or 12 months after blast exposure, relatively few differentially expressed genes (DEGs) were found. However, longitudinal analysis of amygdala, hippocampus, and anterior cortex between 6 weeks and 12 months revealed blast-specific DEG patterns. Six DEGs (hyaluronan and proteoglycan link protein 1 [Hapln1], glutamate metabotropic receptor 2 [Grm2], purinergic receptor P2y12 [P2ry12], C-C chemokine receptor type 5 [Ccr5], phenazine biosynthesis-like protein domain containing 1 [Pbld1], and cadherin related 23 [Cdh23]) were found altered in all three brain regions in blast-exposed animals. Pathway enrichment analysis using all DEGs or those uniquely changed revealed different transcription patterns in blast versus sham. In particular, the amygdala in blast-exposed animals had a unique set of enriched pathways related to stress responses, oxidative phosphorylation, and mitochondrial dysfunction. Upstream analysis implicated tumor necrosis factor (TNF)α signaling in blast-related effects in amygdala and anterior cortex. Eukaryotic initiating factor eIF4E (EIF4e), an upstream regulator of P2ry12 and Ccr5, was predicted to be activated in the amygdala. Quantitative polymerase chain reaction (qPCR) validated longitudinal changes in two TNFα regulated genes (cathepsin B [Ctsb], Hapln1), P2ry12, and Grm2. These studies have implications for understanding how blast injury damages the brain and implicates inflammation as a potential therapeutic target.

P2Y1 receptor in the colonic submucosa of rats and its association with opioid‑induced constipation.

The aim of the present study was to explore the expression changes of P2Y purinergic receptor 1 (P2Y1) in the distal colonic submucosa of opioid-induced constipation (OIC) rats and its association with the occurrence of OIC, an OIC rat model was generated by intraperitoneal injection of loperamide hydrochloride, a selective agonist of µ-opioid receptors (MORs). At 7 days post-treatment, the model was assessed by analyzing stool scores and calculating the gastrointestinal (GI) transit ratio of rats. The distribution of P2Y1-expressing neurons in the colonic submucosal plexus was demonstrated by immunofluorescence (IF). Western blotting was performed to evaluate the expression changes of MOR, P2Y1 and ATP synthase subunit β (ATPB) proteins in the colonic submucosa, while reverse transcription-quantitative PCR (RT-qPCR) analysis was performed to determine the relative mRNA expression of MOR and P2Y1. After 7 days, the feces of OIC rats exhibited an appearance of sausage-shaped pieces and both the stool weight and GI transit ratio of OIC rats were significantly decreased. IF revealed co-expression of P2Y1 and calbindin and MOR and ATPB in the nerve cells of the distal colonic submucosal plexus. Moreover, RT-qPCR analysis showed that the MOR mRNA levels were significantly increased in the distal colonic submucosa of OIC rats, while mRNA levels of P2Y1 were decreased. WB showed that in the distal colonic submucosa of OIC rats, MOR protein expression was increased, whereas that of P2Y1 was significantly decreased. GI transit ratio analysis suggested that the P2Y agonist ATP significantly relieved constipation symptoms in rats, while the P2Y inhibitor MRS2179 aggravated these symptoms. Finally, P2Y1 expression change was shown to be associated with the occurrence of OIC, while expression of MOR and P2Y1 was associated with OIC development in rats.

Endothelial Extracellular Vesicles Regulate Platelet Purinergic Signaling in Sickle Cell Disease

Introduction. Acute chest syndrome (ACS) is a type of acute lung injury and the leading cause of mortality among sickle cell disease (SCD) patients. ACS is often preceded by thrombocytopenia and involves massive thrombosis across pulmonary artery branches. Although, released during hemolysis, adenosine diphosphate (ADP) is known to activate platelets by stimulating their P2Y1 and P2Y12 purinergic receptors, antagonists of P2Y12 have not shown any benefit in ACS therapy, justifying the need for better understanding of purinergic signaling in SCD. CD39 maintains ADP homeostasis by degrading excessive ADP. Though CD39 inhibits ADP-dependent platelet activation, its role in the pathophysiology of SCD is still unidentified. Methods. To evaluate SCD platelet response to ADP in vivo we used a state-of-the-art intravital lung microscopy and a novel in vivo model of ADP-triggered thrombocytopenia in transgenic humanized SCD mice. Additionally, both mouse and human SCD platelet ADP-dependent aggregation was examined using in vitro turbidimetric aggregation assay. Hemin, a major host-derived damage associated molecular pattern associated with SCD, was incubated with Human Lung Micro-Vascular Endothelial Cells (HMVEC-L). Extracellular vesicles (EVs) were obtained from mouse and human plasma samples, and cell culture medium samples using size exclusion chromatography. Obtained EVs were subjected to nanoparticle tracking analysis. EVs CD39 levels and activity were determined using ELISA, western blot and malachite green phosphate assays, respectively. Results. Intravital lung microscopy and in vivo thrombocytopenia studies revealed that intravascular administration of ADP triggered acute pulmonary thrombosis in control but not in SCD mice. In vitro aggregation study confirmed our in vivo findings and demonstrated impaired SCD mouse and human platelet response to ADP, which was, further, significantly augmented by a specific CD39 inhibitor. Hemin triggered shedding of CD39-bearing EVs by HMVEC-L. Indeed, we discovered that isolated from mouse and human plasma endothelial cell-derived SCD EVs expressed higher CD39 levels and activity in comparison to control EVs. Conclusions. Our findings suggest that CD39-bearing EVs prevent ADP-mediated platelet aggregation and pulmonary thrombosis in SCD. Current study explains why P2Y12 blockers are not effective in SCD therapy.

Bacterial polyphosphates induce CXCL4 and synergize with complement anaphylatoxin C5a in lung injury.

Polyphosphates are linear polymers of inorganic phosphates that exist in all living cells and serve pleiotropic functions. Bacteria produce long-chain polyphosphates, which can interfere with host defense to infection. In contrast, short-chain polyphosphates are released from platelet dense granules and bind to the chemokine CXCL4. Here, we report that long-chain polyphosphates induced the release of CXCL4 from mouse bone marrow-derived macrophages and peritoneal macrophages in a dose-/time-dependent fashion resulting from an induction of CXCL4 mRNA. This polyphosphate effect was lost after pre-incubation with recombinant exopolyphosphatase (PPX) Fc fusion protein, demonstrating the potency of long chains over monophosphates and ambient cations. In detail, polyphosphate chains >70 inorganic phosphate residues were required to reliably induce CXCL4. Polyphosphates acted independently of the purinergic P2Y1 receptor and the MyD88/TRIF adaptors of Toll-like receptors. On the other hand, polyphosphates augmented LPS/MyD88-induced CXCL4 release, which was explained by intracellular signaling convergence on PI3K/Akt. Polyphosphates induced Akt phosphorylation at threonine-308. Pharmacologic blockade of PI3K (wortmannin, LY294002) antagonized polyphosphate-induced CXCL4 release from macrophages. Intratracheal polyphosphate administration to C57BL/6J mice caused histologic signs of lung injury, disruption of the endothelial-epithelial barrier, influx of Ly6G+ polymorphonuclear neutrophils, depletion of CD11c+SiglecF+ alveolar macrophages, and release of CXCL4. Long-chain polyphosphates synergized with the complement anaphylatoxin, C5a, which was partly explained by upregulation of C5aR1 on myeloid cells. C5aR1-/- mice were protected from polyphosphate-induced lung injury. C5a generation occurred in the lungs and bronchoalveolar lavage fluid (BALF) of polyphosphate-treated C57BL/6J mice. In conclusion, we demonstrate that polyphosphates govern immunomodulation in macrophages and promote acute lung injury.

Antimalarial and Plasmodium falciparum serpentine receptor 12 targeting effect of FDA approved purinergic receptor antagonist

Intraerythrocytic stages of Plasmodium falciparum responsible for all clinical manifestations of malaria are regulated by array of signalling cascades that represent attractive targets for antimalarial therapy. G-protein coupled receptors (GPCRs) are druggable targets in the treatment of various pathological conditions, however, there is limited understanding about the role of GPCRs in malaria pathogenesis. In Plasmodium, serpentine receptors (PfSR1, PfSR10, PfSR12 and PfSR25) with GPCR-like membrane topology have been reported with the finite knowledge about their potential as antimalarial targets. We analyzed the localization of these receptors in malaria parasite by immunofluorescence assays. All four receptors were expressed in blood stages with PfSR12 expressing more in late intraerythrocytic stages. Further, we evaluated the druggability of PfSR12 using FDA-approved P2Y purinergic receptor antagonist, Prasugrel and its active metabolite R138727, which is proposed to be specific towards PfSR12. Interestingly, biophysical analysis indicated strong binding between PfSR12 and R138727 as compared to the prodrug Prasugrel. This binding interaction was further confirmed by thermal shift assay. Treatment of parasite with Prasugrel and R138727 resulted in growth inhibition of P. falciparum indicating an important role of purinergic signalling and PfSR12 in parasite survival. Next, progression studies indicated the inhibitory effect of Prasugrel begins in late erythrocyte stages corroborating with PfSR12 expression at these stages. Furthermore, Prasugrel also blocked in vivo growth of malaria parasite in a mouse experimental model. This study indicates the presence of P2Y type of purinergic signalling in growth and development of malaria parasite and suggests PfSR12, putative purinergic receptor druggability through Prasugrel. Communicated by Ramaswamy H. Sarma

ATP Promotes Oral Squamous Cell Carcinoma Cell Invasion and Migration by Activating the PI3K/AKT Pathway via the P2Y2-Src-EGFR Axis.

Oral cancer is one of the most common malignancies of the head and neck, and approximately 90% of oral cancers are oral squamous cell carcinomas (OSCCs). The purinergic P2Y2 receptor is upregulated in breast cancer, pancreatic cancer, colorectal cancer, and liver cancer, but its role in OSCC is still unclear. Here, we examined the effects of P2Y2 on the invasion and migration of oral cancer cells (SCC15 and CAL27). The BALB/c mouse model was used to observe the involvement of P2Y2 with tumors in vivo. P2Y2, Src, and EGFR are highly expressed in OSCC tissues and cell lines. Stimulation with ATP significantly enhanced cell invasion and migration in oral cancer cells, and enhanced the activity of Src and EGFR protein kinases, which is mediated by the PI3K/AKT signaling pathway. P2Y2 knockdown attenuated the above ATP-driven events in vitro and in vivo. The PI3K/AKT signaling pathway was blocked by Src or EGFR inhibitor. Extracellular ATP activates the PI3K/AKT pathway through the P2Y2-Src-EGFR axis to promote OSCC invasion and migration, and thus, P2Y2 may be a potential novel target for antimetastasis therapy.

The blockage of downstream P2Y2 receptor signaling inhibits the prostate cancer cell adhesion to endothelial cells

AimsProstate cancer is the second most frequently malignancy in men worldwide. Most deaths are caused by metastasis, and tumor cell dissemination involves the interaction with endothelial cells. However, the endothelial cell signaling involved in such interaction is not entirely understood. The tumor microenvironment contains extracellular ATP, an endogenous agonist of the purinergic P2Y2 receptor (P2Y2R). P2Y2R signaling changes endothelial cell phenotype, which may be relevant to cancer pathophysiology. Therefore, we hypothesized that P2Y2R activation could favor the metastatic prostate cancer cells adhesion to endothelial cells. Main methodsFor adhesion assays, confluent endothelial cells EA.hy926 were treated with P2Y2R agonists before adding and imaging stained DU-145 cells. Alternatively, fluorescent probes and antibodies were used to determine intracellular endothelial Ca2+, nitric oxide (NO), and flow cytometry assays. Key findingsEndothelial P2Y2R activation with ATP, UTP, or the selective agonist 2-thio-UTP increased DU-145 cell adhesion to EA.hy926 cells. This effect required endothelial cell Ca2+ mobilization and relied on the endothelial expression of VCAM-1 and ICAM-1. Conversely, inhibiting this proadhesive endothelial phenotype could impair DU-145 cell adhesion. To evaluate this, we chose atorvastatin based on its notable improvement of endothelial cell dysfunction. Atorvastatin blocked UTP-induced DU-145 cell adhesion to endothelial cell monolayer in a NO-dependent manner, unveiling a P2Y2R and NO signaling crosstalk. SignificanceEndothelial P2Y2R signaling contributes to the adhesion of metastatic prostate cancer cells suggesting that the downstream signaling blockade by statins could be a putative mechanism to reduce prostate cancer metastasis.

Early Dry Eye Disease Onset in a NOD.H-2h4 Mouse Model of Sjögren's Syndrome.

PurposeTo develop a mouse model of human dry eye disease (DED) for investigation of sex differences in autoimmune-associated dry eye pathology.MethodsOcular surface disease was assessed by quantifying corneal epithelial damage with lissamine green stain in the NOD.H-2h4,IFNγ−/−,CD28−/− (NOD.H-2h4 DKO) mouse model of Sjögren's syndrome (SS). Lacrimal gland function was assessed by tear volume quantification with phenol red thread and lacrimal gland inflammation (i.e., dacryoadenitis) was assessed by quantification of immune cell foci, flow cytometric analysis of immune cell composition, and expression of proinflammatory markers.ResultsThe NOD.H-2h4 DKO mouse model of SS exhibits greater age-dependent increases in corneal damage than in NOD.H-2h4 parental mice and demonstrates an earlier disease onset in females compared to males. The severity of ocular surface disease correlates with loss of goblet cell density, increased conjunctivitis, and dacryoadenitis that is more pronounced in NOD.H-2h4 DKO than NOD.H-2h4 mice. B cells dominate lacrimal infiltrates in 16-week-old NOD.H-2h4 and NOD.H-2h4 DKO mice, but T helper cells and macrophages are also present. Lacrimal gland expression of proinflammatory genes, including the P2X7 and P2Y2 purinergic receptors, is greater in NOD.H-2h4 DKO than NOD.H-2h4 mice and correlates with dacryoadenitis.ConclusionsOur results demonstrate for the first time that autoimmune dry eye disease occurs in both sexes of NOD.H-2h4 DKO and NOD.H-2h4 mice, with earlier onset in female NOD.H-2h4 DKO mice when compared to males of the same strain. This study demonstrates that both NOD.H-2h4 and NOD.H-2h4 DKO mice are novel models that closely resemble SS-related and sex-dependent DED.

The Interplay of Endothelial P2Y Receptors in Cardiovascular Health: From Vascular Physiology to Pathology.

The endothelium plays a key role in blood vessel health. At the interface of the blood, it releases several mediators that regulate local processes that protect against the development of cardiovascular disease. In this interplay, there is increasing evidence for a role of extracellular nucleotides and endothelial purinergic P2Y receptors (P2Y-R) in vascular protection. Recent advances have revealed that endothelial P2Y1-R and P2Y2-R mediate nitric oxide-dependent vasorelaxation as well as endothelial cell proliferation and migration, which are processes involved in the regeneration of damaged endothelium. However, endothelial P2Y2-R, and possibly P2Y1-R, have also been reported to promote vascular inflammation and atheroma development in mouse models, with endothelial P2Y2-R also being described as promoting vascular remodeling and neointimal hyperplasia. Interestingly, at the interface with lipid metabolism, P2Y12-R has been found to trigger HDL transcytosis through endothelial cells, a process known to be protective against lipid deposition in the vascular wall. Better characterization of the role of purinergic P2Y-R and downstream signaling pathways in determination of the endothelial cell phenotype in healthy and pathological environments has clinical potential for the prevention and treatment of cardiovascular diseases.

Extracellular Nucleotides and Histamine Suppress TLR3- and RIG-I-Mediated Release of Antiviral IFNs from Human Airway Epithelial Cells.

Respiratory viruses stimulate the release of antiviral IFNs from the airway epithelium. Previous studies have shown that asthmatic patients show diminished release of type I and type III IFNs from bronchial epithelia. However, the mechanism of this suppression is not understood. In this study, we report that extracellular nucleotides and histamine, which are elevated in asthmatic airways, strongly inhibit release of type I and type III IFNs from human bronchial airway epithelial cells (AECs). Specifically, ATP, UTP, and histamine all inhibited the release of type I and type III IFNs from AECs induced by activation of TLR3, retinoic acid-inducible gene I (RIG-I), or cyclic GMP-AMP synthase-STING. This inhibition was at least partly mediated by Gq signaling through purinergic P2Y2 and H1 receptors, but it did not involve store-operated calcium entry. Pharmacological blockade of protein kinase C partially reversed inhibition of IFN production. Conversely, direct activation of protein kinase C with phorbol esters strongly inhibited TLR3- and RIG-I-mediated IFN production. Inhibition of type I and type III IFNs by ATP, UTP, histamine, and the proteinase-activated receptor 2 (PAR2) receptor agonist SLIGKV also occurred in differentiated AECs grown at an air-liquid interface, indicating that the suppression is conserved following mucociliary differentiation. Importantly, histamine and, more strikingly, ATP inhibited type I IFN release from human airway cells infected with live influenza A virus or rhinovirus 1B. These results reveal an important role for extracellular nucleotides and histamine in attenuating the induction of type I and III IFNs from AECs and help explain the molecular basis of the suppression of IFN responses in asthmatic patients.

Endothelial Pannexin1‐purinergic signaling pulmonary hypertension

Endothelial cell (EC) Ca2+ signals dilate small pulmonary arteries (PAs) and maintain a low resting pulmonary arterial pressure (PAP). Impairment of endothelial Ca2+ signaling mechanisms is a crucial contributor to elevated PAP in pulmonary hypertension (PH). Studies in systemic arteries show that flow/shear stress activates endothelial Ca2+ signals to cause vasodilation. Although PAs are a “high‐flow” vascular bed, flow‐induced endothelial Ca2+ signaling mechanisms have not been investigated in PH. In this regard, we recently showed that Ca2+ influx through endothelial transient receptor potential vanilloid 4 (TRPV4EC) ion channels dilates PAs and lowers resting PAP (Daneva et al., PNAS, 2021). Moreover, flow/shear stress activates the efflux of adenosine triphosphate (ATP) through endothelial Pannexin 1 (Panx1EC) in PAs. Panx1EC‐effluxed ATP, in turn, stimulates endothelial purinergic P2Y2 receptor (P2Y2REC) to increase TRPV4EC channels activity (Daneva et al., eLife, 2021). I hypothesized that impaired Panx1EC—P2Y2REC—TRPV4EC signaling axis reduces flow‐induced dilation of PAs and elevates PAP in PH. Confocal Ca2+ imaging studies in fluo‐4‐loaded en face PAs (~ 50 mm) showed that the baseline and agonist (GSK1016790A or GSK101)‐induced activity of TRPV4EC sparklets (individual Ca2+ influx signals through TRPV4EC channels) is reduced in the mice exposed to Sugen 5416 + chronic hypoxia (Su+CH; 3 weeks; 10% O2) compared to the normoxic control mice, indicating a decrease in TRPV4EC channel activity in PH. The expression of TRPV4 channels at the mRNA level, however, was not altered in Su+CH mice. The activation of TRPV4EC sparklets by exogenous ATP (1 mM) was also reduced in PAs from Su+CH mice, suggesting an impairment of ATP‐P2Y2REC‐TRPV4EC channel signaling axis in PH. Accordingly, ATP‐induced, endothelium‐dependent dilation was absent in PAs from Su+CH mice. We previously showed that basal ATP efflux in PAs predominantly occurs through endothelial Panx1, and flow/shear stress increases ATP efflux entirely through the activation of endothelial Panx1. Bioluminescence measurements of extracellular ATP concentration showed lower baseline ATP levels in PAs from Su+CH mice than control mice, suggesting a decrease in ATP efflux in PH. Importantly, flow/shear stress (4 – 15 dynes/cm2) increased the efflux of ATP from normal mice, but not in PAs from Su+CH mice, indicating that flow/shear stress‐Panx1EC signaling is also impaired in PH. Together, these data support the idea that flow‐Panx1EC and P2Y2REC‐TRPV4EC signaling are impaired in PH. Targeting individual elements of this pathway may provide novel therapeutic options in PH and other pulmonary vascular disorders characterized by endothelial dysfunction. Future studies will assess the individual signaling linkages of the flow‐Panx1EC‐P2Y2REC‐TRPV4EC signaling axis in PH.

UDP/P2Y6 contributes to enhancing LPS-induced acute lung injury by regulating neutrophil migration

Neutrophils play a prominent role in the inflammatory response and are a critical factor in the pathogenesis of acute lung injury (ALI). Despite a deep understanding of neutrophil accumulation in the pulmonary microvasculature during the process of this disease, the regulatory mechanism of neutrophil recruitment remains unclear. This study aimed to explore the functions and signaling pathways of the purinergic receptor P2Y6 in mediating the innate immune response in ALI. P2Y6-deficient mice, bone marrow chimeras, and neutrophilic chimeras were created in this work to explore the function of P2Y6 in ALI. The results indicated that the extracellular nucleotide UDP was released as a dangerous signal and activated P2Y6 to promote the inflammatory response and pulmonary damage during the process of ALI. P2Y6 deficiency may mitigate deterioration of this disease, including reduced ALI-related inflammatory factor release and immune cell invasion. Bone marrow and neutrophil chimeras and adoptive transfer in mice showed that P2Y6 expression on neutrophils contributed to neutrophil infiltration into lung tissues induced by UDP. Further work indicated that P2Y6 was involved in the neutrophil migration capability through the ErK signaling pathway by mediating the deformation of F-actin filaments and pseudopodia formation during cell recruitment to pulmonary tissue. Here, we provide evidence for the mechanism by which the purinergic receptor P2Y6 contributes to ALI development by regulating neutrophil infiltration into lung tissues. These data indicated that P2Y6 might be a potential therapeutic target for the treatment of this acute severe disease.

MicroRNA 320a and Membrane Antigens as Tools to Evaluate the Pathophysiology of Platelets Stored in Blood Banks.

Our research group, through the analysis of miRNomes in platelet concentrates (PCs) stored in blood banks, identified and validated the miR-127 and miR-320a miRNAs as biomarkers of platelet storage lesions (PSLs) in PCs. In order to validate the miRNAs 127 and 320a methodologically, as PSL biomarkers in a large number of PC bags, we also evaluated important immunological markers involved in the platelet activation/aggregation process—the CD62P receptor (P-selectin), the surface glycoproteins (GP) IIb/IIIa, and the purinergic P2Y12 receptor—via flow cytometry. The miRNAs miR-127 and miR-320a were quantified by real-time quantitative PCR (RT-qPCR). To carry out this study, 500 collection tubes were used at the upper edge of the PC bags containing platelets. Each tube was divided into seven equal parts (totaling 3500 samples) for platelet analysis from 7 different storage days, where the 1st day represents the high-quality control, and the 7th day corresponds to the low-quality control of the platelets. After analyzing all parameters during storage days, it was concluded that the relative quantification of miR-320a below 0.50 and the CD62P receptor below 27.92% are reliable indicators of the absence of storage lesions in blood banks. We believe that the values found in the expression of the CD62P receptor legitimize the use of the miR-320a and miR-127 miRNAs to build a kit capable of accurately measuring whether the stored platelets are suitable for transfusion.

Single-nucleotide polymorphisms of the SLC17A9 and P2RY12 genes are significantly associated with phantom tooth pain.

Phantom tooth pain (PTP) is a rare and specific neuropathic pain that occurs after pulpectomy and tooth extraction, but its cause is not understood. We hypothesized that there is a genetic contribution to PTP. We focused on solute carrier family 17 member 9 (SLC17A9)/vesicular nucleotide transporter (VNUT) and purinergic receptor P2Y12 (P2RY12), both of which have been associated with neuropathic pain and pain transduction signaling in the trigeminal ganglion in rodents. We sought to corroborate these associations in humans. We investigated gene polymorphisms that contribute to PTP. We statistically examined the association between genetic polymorphisms and PTP vulnerability in 150 patients with orofacial pain, including PTP, and 500 healthy subjects. We found that the rs735055 polymorphism of the SLC17A9 gene and rs3732759 polymorphism of the P2RY12 gene were associated with the development of PTP. Carriers of the minor allele of rs735055 and individuals who were homozygous for the major allele of rs3732759 had a higher rate of PTP. Carriers of the minor allele of rs735055 reportedly had high SLC17A9 mRNA expression in the spinal cord, which may increase the storage and release of adenosine triphosphate. Individuals who were homozygous for the major allele of rs3732759 may have higher P2RY12 expression that is more active in microglia. Therefore, these carriers may be more susceptible to PTP. These results suggest that specific genetic polymorphisms of the SLC17A9 and P2RY12 genes are involved in PTP. This is the first report on genes that are associated with PTP in humans.

Rat Hepatocellular Primary Cells: A Cellular and Genetic Assessment of the Chitosan Nanoparticles-Induced Damage and Cytotoxicity.

Chitosan (CH) is a non-toxic vital polymer that is derived naturally from chitin. Due to its anti-bacterial and anti-fungal properties, it has attracted researchers' attention. The anti-bacterial activity of 1-3 CH is ideal in an acidic medium due to its weak solubility at pH levels higher than 6.5. The type of CH and the degree of its polymerization affect its anti-microbial activity, as well as some of its other chemical and physical properties. The present study was conducted to investigate the damage induced by chitosan nanoparticles (CHNPs) at various concentrations on the cultured rat hepatic cells. The CHNPs were synthesized by the ionotropic gelation of CH with sodium tripolyphosphate anions. Hepatic cells were cultured from tissues freshly isolated from the liver of normal laboratory rats. Cells were allowed to reach a confluence level before the treatment with CHNPs. In total, five different concentrations of CHNPs were used, and cell cytotoxicity was evaluated using the MTT assay. The genetic expression of P2Y1, P2Y2, and P2Y4 purinergic receptors was evaluated on the cellular level using Qualitative Reverse Transcription Polymerase Chain Reaction technique. The primary culture of rat hepatic cells was thoroughly exposed to a range of CHNPs. Under normal conditions, the cells showed normal cellular morphology with clearly defined borders and normal nuclear structure. Apoptotic cellular damage was observed in the cultured hepatic cells when exposed to CHNPs. Moreover, irregular cellular morphology and heavy pigmentation were noticed in the hepatic cells when exposed to a high concentration of CHNPs. Purinergic receptor gene expression indicated an inflammatory response by an increased gene fold change post-exposure to CHNPs. This study concludes that CHNPs have a strong cytotoxic effect on the cultured rat hepatic cells. Overall, CHNPs showed an inhibitory response to hepatic cells evoking a purine receptor-mediated inflammatory response.

Blockade of the P2Y2 Receptor Attenuates Alcoholic Liver Inflammation by Targeting the EGFR-ERK1/2 Signaling Pathway.

PurposeIt is well known that inflammation plays a key role in complex pathological progressions of alcohol-associated liver disease (ALD). To date, effective therapy for ALD is lacking. P2Y2 receptor (P2Y2R), a G protein-coupled P2Y purinergic receptor, represents a novel pharmacological target in many inflammations.MethodsThe alcohol-associated liver injury and inflammation mouse model was established. The effect of P2Y2R on alcohol-induced liver injury and inflammation was evaluated using quantitative real-time PCR, Western blot and immunohistochemical assay. An alcohol-stimulated (100 mmol/L, for 24 h) AML-12 cell model was established. Different agonists, antagonists and P2Y2R siRNA were used to explore the possible mechanisms of P2Y2R.ResultsIn vivo, results showed that the hepatoprotective effect of P2Y2R blockade by significantly suppressed liver structural abnormalities and lipid infiltration, and decreased levels of ALT/AST and TNF-α/IL-1β in the high dosage group of suramin (20 mg/kg) compared to control diet (CD)-fed mice. At the same time, we found that alcohol feeding promoted the phosphorylation of EGFR and ERK1/2, both of which were effectively inhibited by suramin (20 mg/kg). In vitro, suramin or P2Y2R silencing effectively inhibited the phosphorylation of EGFR and ERK1/2, similar to the down-regulated effects of their corresponding inhibitors (EGFR inhibitor AG1478 and ERK1/2 inhibitor U0126) accompanied by reduced levels of TNF-α and IL-1β compared to alcohol-induced AML-12 cell. In addition, we found that silencing P2Y2R attenuated the apoptosis of hepatocyte.ConclusionOur findings suggest that P2Y2R regulates alcoholic liver inflammation by targeting the EGFR-ERK1/2 signaling pathway and plays an important role in hepatocyte apoptosis, which may provide new ideas for the development of methods to treat ALD.

Noradrenaline and ATP regulate adiponectin exocytosis in white adipocytes: Disturbed adrenergic and purinergic signalling in obese and insulin-resistant mice

White adipocyte adiponectin exocytosis is triggered by cAMP and a concomitant increase of cytosolic Ca2+ potentiates its release. White adipose tissue is richly innervated by sympathetic nerves co-releasing noradrenaline (NA) and ATP, which may act on receptors in the adipocyte plasma membrane to increase cAMP via adrenergic receptors and Ca2+ via purinergic receptors. Here we determine the importance of NA and ATP for the regulation of white adipocyte adiponectin exocytosis, at the cellular and molecular level, and we specifically detail the ATP signalling pathway. We demonstrate that tyrosine hydroxylase (enzyme involved in catecholamine synthesis) is dramatically reduced in inguinal white adipose tissue (IWAT) isolated from mice with diet-induced obesity; this is associated with diminished levels of NA in IWAT and with a reduced ratio of high-molecular-weight (HMW) to total adiponectin in serum. Adiponectin exocytosis (measured as an increase in plasma membrane capacitance and as secreted product) is triggered by NA or ATP alone in cultured and primary mouse IWAT adipocytes, and enhanced by a combination of the two secretagogues. The ATP-induced adiponectin exocytosis is largely Ca2+-dependent and activated via purinergic P2Y2 receptors (P2Y2Rs) and the Gq11/PLC pathway. Adiponectin release induced by the nucleotide is abrogated in adipocytes isolated from obese and insulin-resistant mice, and this is associated with ∼70% reduced abundance of P2Y2Rs. The NA-triggered adiponectin exocytosis is likewise abolished in “obese adipocytes”, concomitant with a 50% lower gene expression of beta 3 adrenergic receptors (β3ARs). An increase in intracellular Ca2+ is not required for the NA-stimulated adiponectin secretion. Collectively, our data suggest that sympathetic innervation is a principal regulator of adiponectin exocytosis and that disruptions of this control are associated with the obesity-associated reduction of circulating levels of HMW/total adiponectin.

P2Y1 purinergic receptor identified as a diabetes target in a small-molecule screen to reverse circadian β-cell failure.

The mammalian circadian clock drives daily oscillations in physiology and behavior through an autoregulatory transcription feedback loop present in central and peripheral cells. Ablation of the core clock within the endocrine pancreas of adult animals impairs the transcription and splicing of genes involved in hormone exocytosis and causes hypoinsulinemic diabetes. Here, we developed a genetically sensitized small-molecule screen to identify druggable proteins and mechanistic pathways involved in circadian β-cell failure. Our approach was to generate β-cells expressing a nanoluciferase reporter within the proinsulin polypeptide to screen 2640 pharmacologically active compounds and identify insulinotropic molecules that bypass the secretory defect in CRISPR-Cas9-targeted clock mutant β-cells. We validated hit compounds in primary mouse islets and identified known modulators of ligand-gated ion channels and G-protein-coupled receptors, including the antihelmintic ivermectin. Single-cell electrophysiology in circadian mutant mouse and human cadaveric islets revealed ivermectin as a glucose-dependent secretagogue. Genetic, genomic, and pharmacological analyses established the P2Y1 receptor as a clock-controlled mediator of the insulinotropic activity of ivermectin. These findings identify the P2Y1 purinergic receptor as a diabetes target based upon a genetically sensitized phenotypic screen.