GRK2 expression and catalytic activity are essential for vasoconstrictor/ERK-stimulated arterial smooth muscle proliferation.

Prolonged vasoconstrictor signalling found in hypertension, increases arterial contraction, and alters vessel architecture by stimulating arterial smooth muscle cell (ASMC) growth, underpinning the development of re-stenosis lesions and vascular remodelling. Vasoconstrictors interact with their cognate G protein coupled receptors activating a variety of signalling pathways to promote smooth muscle proliferation. Here, angiotensin II (AngII) and endothelin 1 (ET1), but not UTP stimulates ASMC proliferation. Moreover, siRNA-mediated depletion of endogenous GRK2 expression, or GRK2 inhibitors, compound 101 or paroxetine, prevented AngII and ET1-promoted ASMC growth. Depletion of GRK2 expression or inhibition of GRK2 activity ablated the prolonged phase of AngII and ET-stimulated ERK signalling, while enhancing and prolonging UTP-stimulated ERK signalling. Increased GRK2 expression enhanced and prolonged AngII and ET1-stimulated ERK signalling, but suppressed UTP-stimulated ERK signalling. In ASMC prepared from 6-week-old WKY and SHR, AngII and ET1-stimulated proliferation rates were similar, however, in cultures prepared from 12-week-old rats AngII and ET1-stimulated growth was enhanced in SHR-derived ASMC, which was reversed following depletion of GRK2 expression. Furthermore, in ASMC cultures isolated from 6-week-old WKY and SHR rats, AngII and ET1-stimulated ERK signals were similar, while in cultures from 12-week-old rats ERK signals were both enhanced and prolonged in SHR-derived ASMC, and were reversed to those seen in age-matched WKY-derived ASMC following pre-treatment of SHR-derived ASMC with compound 101. These data indicate that the presence of GRK2 and its catalytic activity are essential to enable pro-proliferative vasoconstrictors to promote growth via recruitment and activation of the ERK signalling pathway in ASMC.

[Effect of multi-glycosides of Tripterygium wilfordii on renal injury in diabetic kidney disease rats through NLRP3/caspase-1/GSDMD pyroptosis pathway].

This study investigated the effect of multi-glycosides of Tripterygium wilfordii(GTW) on renal injury in diabetic kidney disease(DKD) rats through Nod-like receptor protein 3(NLRP3)/cysteine-aspartic acid protease-1(caspase-1)/gsdermin D(GSDMD) pyroptosis pathway and the mechanism. To be specific, a total of 40 male SD rats were randomized into the normal group(n=8) and modeling group(n=34). In the modeling group, a high-sugar and high-fat diet and one-time intraperitoneal injection of streptozotocin(STZ) were used to induce DKD in rats. After successful modeling, they were randomly classified into model group, valsartan(Diovan) group, and GTW group. Normal group and model group were given normal saline, and the valsartan group and GTW group received(ig) valsartan and GTW, respectively, for 6 weeks. Blood urea nitrogen(BUN), serum creatinine(Scr), alanine ami-notransferase(ALT), albumin(ALB), and 24 hours urinary total protein(24 h-UTP) were determined by biochemical tests. The pathological changes of renal tissue were observed based on hematoxylin and eosin(HE) staining. Serum levels of interleukin-1ß(IL-1ß) and interleukin-18(IL-18) were detected by enzyme-linked immunosorbent assay(ELISA). Western blot was used to detect the expression of pyroptosis pathway-related proteins in renal tissue, and RT-PCR to determine the expression of pyroptosis pathway-related genes in renal tissue. Compared with the normal group, the model group showed high levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1ß and IL-18(P<0.01), low level of ALB(P<0.01), severe pathological damage to kidney, and high protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01). Compared with the model group, valsartan group and GTW group had low levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1ß and IL-18(P<0.01), high level of ALB(P<0.01), alleviation of the pathological damage to the kidney, and low protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01 or P<0.05). GTW may inhibit pyroptosis by decreasing the expression of NLRP3/caspase-1/GSDMD in renal tissue, thereby relieving the inflammatory response of DKD rats and the pathological injury of kidney.

Effects of uridine and nucleotides on hemostasis parameters.

Several purinergic receptors have been identified on platelets which are involved in hemostatic and thrombotic processes. The aim of the present study was to investigate the effects of uridine and its nucleotides on platelet aggregation and hemostasis in platelet-rich plasma (PRP) and whole blood. The effects of uridine, UMP, UDP, and UTP at different final concentrations (1 to 1000 µM) on platelet aggregation were studied using an aggregometer. In PRP samples, platelet aggregation was induced by ADP, collagen and epinephrine 3 min after addition of uridine, UMP, UDP, UTP and saline (as a control). All thromboelastogram experiments were performed at 1000 µM final concentrations of uridine and its nucleotides in whole blood. UDP and UTP were also tested in thromboelastogram with PRP. Our results showed that UDP, and especially UTP, inhibited ADP- and collagen-induced aggregation in a concentration-dependent manner. In whole blood thromboelastogram experiments, UDP stimulated clot formation while UTP suppressed clot formation. When thromboelastogram experiments were repeated with PRP, UTP's inhibitory effect on platelets was confirmed, while UDP's stimulated clot forming effect disappeared. Collectively, our data showed that UTP inhibited platelet aggregation in a concentration-dependent manner and suppressed clot formation. On the other hand, UDP exhibited distinct effects on whole blood or PRP in thromboelastogram. These data suggest that the difference on effects of UTP and UDP might have arisen from the different receptors that they stimulate and warrant further investigation with regard to their in vivo actions on platelet aggregation and hemostasis.

Effect of multi-glycosides of Tripterygium wilfordii on renal injury in diabetic kidney disease rats through NLRP3/caspase-1/GSDMD pyroptosis pathway / 中国中药杂志

This study investigated the effect of multi-glycosides of Tripterygium wilfordii(GTW) on renal injury in diabetic kidney disease(DKD) rats through Nod-like receptor protein 3(NLRP3)/cysteine-aspartic acid protease-1(caspase-1)/gsdermin D(GSDMD) pyroptosis pathway and the mechanism. To be specific, a total of 40 male SD rats were randomized into the normal group(n=8) and modeling group(n=34). In the modeling group, a high-sugar and high-fat diet and one-time intraperitoneal injection of streptozotocin(STZ) were used to induce DKD in rats. After successful modeling, they were randomly classified into model group, valsartan(Diovan) group, and GTW group. Normal group and model group were given normal saline, and the valsartan group and GTW group received(ig) valsartan and GTW, respectively, for 6 weeks. Blood urea nitrogen(BUN), serum creatinine(Scr), alanine ami-notransferase(ALT), albumin(ALB), and 24 hours urinary total protein(24 h-UTP) were determined by biochemical tests. The pathological changes of renal tissue were observed based on hematoxylin and eosin(HE) staining. Serum levels of interleukin-1β(IL-1β) and interleukin-18(IL-18) were detected by enzyme-linked immunosorbent assay(ELISA). Western blot was used to detect the expression of pyroptosis pathway-related proteins in renal tissue, and RT-PCR to determine the expression of pyroptosis pathway-related genes in renal tissue. Compared with the normal group, the model group showed high levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1β and IL-18(P<0.01), low level of ALB(P<0.01), severe pathological damage to kidney, and high protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01). Compared with the model group, valsartan group and GTW group had low levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1β and IL-18(P<0.01), high level of ALB(P<0.01), alleviation of the pathological damage to the kidney, and low protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01 or P<0.05). GTW may inhibit pyroptosis by decreasing the expression of NLRP3/caspase-1/GSDMD in renal tissue, thereby relieving the inflammatory response of DKD rats and the pathological injury of kidney.

Indoxyl sulfate decreases uridine adenosine tetraphosphate-induced contraction in rat renal artery.

The protein-bound uremic toxin indoxyl sulfate has negative effects on a variety of physiological activities including vascular function. Uridine adenosine tetraphosphate (Up4A), a new dinucleotide molecule affects vascular function including induction of vasocontraction, and aberrant responsiveness to Up4A is evident in arteries from disorders such as hypertension and diabetes. The link between indoxyl sulfate and the Up4A-mediated response is, however, unknown. We used Wistar rat's renal arteries to see if indoxyl sulfate will affect Up4A-mediated vascular contraction. In renal arteries of indoxyl sulfate, the contractile response generated by Up4A was dramatically reduced compared to the non-treated control group. Indoxyl sulfate increased endothelin-1-induced contraction but had no effect on phenylephrine, thromboxane analog, or isotonic K+-induced renal arterial contractions. UTP, ATP, UDP, and ADP-produced contractions were reduced by indoxyl sulfate. CH223191, an aryl hydrocarbon receptor (AhR) antagonist, did not reverse Up4A, and UTP contraction decreases caused by indoxyl sulfate. The ectonucleotidase inhibitor ARL67156 prevents indoxyl sulfate from reducing Up4A- and UTP-mediated contractions. In conclusion, we discovered for the first time that indoxyl sulfate inhibits Up4A-mediated contraction in the renal artery, possibly through activating ectonucleotidase but not AhR. Indoxyl sulfate is thought to play a function in the pathophysiology of purinergic signaling.

Methylglyoxal impairs ATP- and UTP-induced relaxation in the rat carotid arteries.

Although methylglyoxal (MGO), a highly reactive dicarbonyl compound, influences the functioning of the vasculature, modulating its effects on vascular reactivity to various substances remains unclear, especially purinoceptor ligands. Therefore, we sought to investigate the direct effects of MGO on relaxation induced by adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) in isolated rat carotid arteries. When carotid arteries were exposed to MGO (420 µM for 1 h), relaxation induced by acetylcholine or sodium nitroprusside was not affected by MGO. However, ATP- and UTP-induced relaxation was impaired by MGO compared with the control. In both ATP- and UTP-induced relaxation, endothelial denudation, incubation with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine or the selective P2Y purinoceptor 2 (P2Y2) receptor antagonist AR-C118925XX reduced relaxation in both the control and MGO groups, while the differences between the control and MGO groups were eliminated. The cyclooxygenase (COX) inhibitor indomethacin inhibited the differences in ATP/UTP-mediated relaxations between the control and MGO groups. Moreover, N-acetyl-L-cysteine (NAC), an antioxidant, could augment carotid arterial relaxation induced by ATP/UTP in the presence of MGO. MGO increased arachidonic acid-induced contraction, which was suppressed by NAC. Following both ATP/UTP stimulation, MGO increased the release of prostanoids. These results suggest that MGO impaired ATP- and UTP-induced relaxation in carotid arteries, which was caused by suppressed P2Y2 receptor-mediated signaling and reductions in endothelial NO. Moreover, MGO partially contributed to COX-derived vasoconstrictor prostanoids through increased oxidative stress.

Characterisation of P2Y receptor subtypes mediating vasodilation and vasoconstriction of rat pulmonary artery using selective antagonists.

Pulmonary vascular tone is modulated by nucleotides, but which P2 receptors mediate these actions is largely unclear. The aim of this study, therefore, was to use subtype-selective antagonists to determine the roles of individual P2Y receptor subtypes in nucleotide-evoked pulmonary vasodilation and vasoconstriction. Isometric tension was recorded from rat intrapulmonary artery rings (i.d. 200-500 µm) mounted on a wire myograph. Nucleotides evoked concentration- and endothelium-dependent vasodilation of precontracted tissues, but the concentration-response curves were shallow and did not reach a plateau. The selective P2Y2 antagonist, AR-C118925XX, inhibited uridine 5'-triphosphate (UTP)- but not adenosine 5'-triphosphate (ATP)-evoked relaxation, whereas the P2Y6 receptor antagonist, MRS2578, had no effect on UTP but inhibited relaxation elicited by uridine 5'-diphosphate (UDP). ATP-evoked relaxations were unaffected by the P2Y1 receptor antagonist, MRS2179, which substantially inhibited responses to adenosine 5'-diphosphate (ADP), and by the P2Y12/13 receptor antagonist, cangrelor, which potentiated responses to ADP. Both agonists were unaffected by CGS1593, an adenosine receptor antagonist. Finally, AR-C118925XX had no effect on vasoconstriction elicited by UTP or ATP at resting tone, although P2Y2 receptor mRNA was extracted from endothelium-denuded tissues using reverse transcription polymerase chain reaction with specific oligonucleotide primers. In conclusion, UTP elicits pulmonary vasodilation via P2Y2 receptors, whereas UDP acts at P2Y6 and ADP at P2Y1 receptors, respectively. How ATP induces vasodilation is unclear, but it does not involve P2Y1, P2Y2, P2Y12, P2Y13, or adenosine receptors. UTP- and ATP-evoked vasoconstriction was not mediated by P2Y2 receptors. Thus, this study advances our understanding of how nucleotides modulate pulmonary vascular tone.

Amplified Ca2+ dynamics and accelerated cell proliferation in breast cancer tissue during purinergic stimulation.

Intracellular Ca2+ dynamics shape malignant behaviors of cancer cells. Whereas previous studies focused on cultured cancer cells, we here used breast organoids and colonic crypts freshly isolated from human and murine surgical biopsies. We performed fluorescence microscopy to evaluate intracellular Ca2+ concentrations in breast and colon cancer tissue with preferential focus on intracellular Ca2+ release in response to purinergic and cholinergic stimuli. Inhibition of the sarco-/endoplasmic reticulum Ca2+ ATPase with cyclopiazonic acid elicited larger Ca2+ responses in breast cancer tissue, but not in colon cancer tissue, relative to respective normal tissue. The resting intracellular Ca2+ concentration was elevated, and ATP, UTP and acetylcholine induced strongly augmented intracellular Ca2+ responses in breast cancer tissue compared with normal breast tissue. In contrast, resting intracellular Ca2+ levels and acetylcholine-induced increases in intracellular Ca2+ concentrations were unaffected and ATP- and UTP-induced Ca2+ responses were smaller in colon cancer tissue compared with normal colon tissue. In accordance with the amplified Ca2+ responses, ATP and UTP substantially increased proliferative activity-evaluated by bromodeoxyuridine incorporation-in breast cancer tissue, whereas the effect was minimal in normal breast tissue. ATP caused cell death-identified with ethidium homodimer-1 staining-in breast cancer tissue only at concentrations above the expected pathophysiological range. We conclude that intracellular Ca2+ responses are amplified in breast cancer tissue, but not in colon cancer tissue, and that nucleotide signaling stimulates breast cancer cell proliferation within the extracellular concentration range typical for solid cancer tissue.

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.

The P2Y2 Receptor C-Terminal Tail Modulates but Is Dispensable for ß-Arrestin Recruitment.

The P2Y2 receptor (P2Y2R) is a G protein-coupled receptor that is activated by extracellular ATP and UTP, to a similar extent. This allows it to play roles in the cell's response to the (increased) release of these nucleotides, e.g., in response to stress situations, including mechanical stress and oxygen deprivation. However, despite its involvement in important (patho)physiological processes, the intracellular signaling induced by the P2Y2R remains incompletely described. Therefore, this study implemented a NanoBiT® functional complementation assay to shed more light on the recruitment of ß-arrestins (ßarr1 and ßarr2) upon receptor activation. More specifically, upon determination of the optimal configuration in this assay system, the effect of different (receptor) residues/regions on ßarr recruitment to the receptor in response to ATP or UTP was estimated. To this end, the linker was shortened, the C-terminal tail was truncated, and phosphorylatable residues in the third intracellular loop of the receptor were mutated, in either singly or multiply adapted constructs. The results showed that none of the introduced adaptations entirely abolished the recruitment of either ßarr, although EC50 values differed and time-luminescence profiles appeared to be qualitatively altered. The results hint at the C-terminal tail modulating the interaction with ßarr, while not being indispensable.

Structural basis for the allosteric inhibition of UMP kinase from Gram-positive bacteria, a promising antibacterial target.

Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5'-monophosphate (UMP) kinase is a promising target for novel antibiotic discovery as it is essential for bacterial survival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regulation with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.

Converging purinergic and immune signaling pathways drive IL-6 secretion by Fragile X cortical astrocytes via STAT3.

The symptoms of Fragile X syndrome (FXS) are driven in part by abnormal glial-mediated function. FXS astrocytes release elevated levels of immune-related factors interleukin-6 (IL-6) and tenascin C (TNC), and also demonstrate increased purinergic signaling, a pathway linked to signaling factor release. Here, in cortical astrocytes from the Fmr1 knockout (KO) FXS mouse model, purinergic agonism enhanced TNC secretion and STAT3 phosphorylation, two processes linked to elevated IL-6 secretion in FXS, while STAT3 knockdown and TLR4 antagonism normalized Fmr1 KO IL-6 release. We therefore suggest that purinergic signaling and immune regulatory pathways converge to drive FXS cortical pro-inflammatory responses.

UTP is a regulator of in vitro and in vivo angiogenic properties of cardiac adipose-derived stem cells.

The ability of cardiac adipose-derived stem cells (cADSC) to differentiate into multiple cell types has opened new perspectives in cardiac cell-based regenerative therapies. P2Y nucleotide receptors have already been described as regulators of adipogenic differentiation of cADSC and bone marrow-derived stem cells. In this study, we defined UTP as a regulator of cADSC endothelial differentiation. A daily UTP stimulation of cADSC during endothelial predifferentiation increased their capacity to form an endothelial network in matrigel. Additionally, pro-angiogenic UTP target genes such as epiregulin and hyaluronan synthase-1 were identified in predifferentiated cADSC by RNA sequencing experiments. Their regulation by UTP was confirmed by qPCR and ELISA experiments. We then evaluated the capacity of UTP-treated predifferentiated cADSC to increase post-ischemic revascularization in mice subjected to left anterior descending artery ligation. Predifferentiated cADSC treated or not with UTP were injected in the periphery of the infarcted zone, 3 days after ligation. We observed a significant increase of capillary density 14 and 30 days after UTP-treated predifferentiated cADSC injection, correlated with a reduction of cardiac fibrosis. This revascularization increase was not observed after injection of UTP-treated cADSC deficient for UTP and ATP nucleotide receptor P2Y2. The present study highlights the P2Y2 receptor as a regulator of cADSC endothelial differentiation and as a potential target for the therapeutic use of cADSC in post-ischemic heart revascularization.

Nucleotides-Induced Changes in the Mechanical Properties of Living Endothelial Cells and Astrocytes, Analyzed by Atomic Force Microscopy.

Endothelial cells and astrocytes preferentially express metabotropic P2Y nucleotide receptors, which are involved in the maintenance of vascular and neural function. Among these, P2Y1 and P2Y2 receptors appear as main actors, since their stimulation induces intracellular calcium mobilization and activates signaling cascades linked to cytoskeletal reorganization. In the present work, we have analyzed, by means of atomic force microscopy (AFM) in force spectroscopy mode, the mechanical response of human umbilical vein endothelial cells (HUVEC) and astrocytes upon 2MeSADP and UTP stimulation. This approach allows for simultaneous measurement of variations in factors such as Young's modulus, maximum adhesion force and rupture event formation, which reflect the potential changes in both the stiffness and adhesiveness of the plasma membrane. The largest effect was observed in both endothelial cells and astrocytes after P2Y2 receptor stimulation with UTP. Such exposure to UTP doubled the Young's modulus and reduced both the adhesion force and the number of rupture events. In astrocytes, 2MeSADP stimulation also had a remarkable effect on AFM parameters. Additional studies performed with the selective P2Y1 and P2Y13 receptor antagonists revealed that the 2MeSADP-induced mechanical changes were mediated by the P2Y13 receptor, although they were negatively modulated by P2Y1 receptor stimulation. Hence, our results demonstrate that AFM can be a very useful tool to evaluate functional native nucleotide receptors in living cells.

CaMK II/Ca2+ dependent endoplasmic reticulum stress mediates apoptosis of hepatic stellate cells stimulated by transforming growth factor beta 1.

Previous studies by our group have demonstrated that the calcium imbalance in rat hepatic stellate cells (HSCs) can induce endoplasmic reticulum stress (ERS) and promote cell apoptosis. KN-62, an inhibitor of Calmodulin kinase II (CaMK II), can decrease the expression of CaMK II that plays a major role in regulating the steady state of intracellular Ca2+. Uridine triphosphate (UTP) plays a biological role in increasing indirectly the level of intracellular Ca2+. In the experiment, we demonstrate that KN-62 and UTP can inhibit the proliferation and promote the apoptosis in HSCs, increase the level of intracellular Ca2+ and the expression of ERS protein GRP78, and increase the apoptosis protein Caspase-12 and Bax expression, while decrease the expression of Bcl-2 protein. Our findings indicate that the CaMK II/Ca2+ signaling pathway regulates the ERS apoptosis pathway and induces HSC apoptosis.

P2Y2 receptor activation promotes esophageal cancer cells proliferation via ERK1/2 pathway.

Esophageal cancer is a prominent worldwide illness that is divided into two main subtypes: esophageal squamous cell carcinoma and esophageal adenocarcinoma. Mortality rates are alarming, and the understanding of the mechanisms involved in esophageal cancer development, becomes essential. Purinergic signaling is related to many diseases and among these various types of tumors. Here we studied the effects of the P2Y2 receptor activation in different types of esophageal cancer. Esophageal tissue samples of healthy controls were used for P2Y2R expression quantification. Two human esophageal cancer cell lines Kyse-450 (squamous cell carcinoma) and OE-33 (adenocarcinoma) were used to perform in vitro analysis of cell proliferation, migration, adhesion, and the signaling pathways involved in P2Y2R activation. Data showed that P2Y2R was expressed in biopsies of patients with ESCC and adenocarcinoma, as well as in the two human esophageal cancer cell lines studied. The RT-qPCR analysis demonstrated that OE-33 cells have higher P2RY2 expression than Kyse-450 squamous cell line. Results showed that P2Y2R activation, induced by ATP or UTP, promoted esophageal cancer cells proliferation and colony formation. P2Y2R blockage with the selective antagonist, AR-C 118925XX, led to decreased proliferation, colony formation and adhesion. Treatments with ATP or UTP activated ERK 1/2 pathway in ESCC and ECA cells. The P2Y2R antagonism did not alter the migration of esophageal cancer cells. Interestingly, the esophageal cancer cell lines presented a distinct profile of nucleotide hydrolysis activity. The modulation of P2Y2 receptors may be a promising target for esophageal cancer treatment.

P2Y2 Receptor Induces L. amazonensis Infection Control in a Mechanism Dependent on Caspase-1 Activation and IL-1ß Secretion.

Leishmaniasis is a neglected tropical disease caused by an intracellular parasite of the genus Leishmania. Damage-associated molecular patterns (DAMPs) such as UTP and ATP are released from infected cells and, once in the extracellular medium, activate P2 purinergic receptors. P2Y2 and P2X7 receptors cooperate to control Leishmania amazonensis infection. NLRP3 inflammasome activation and IL-1ß release resulting from P2X7 activation are important for outcomes of L. amazonensis infection. The cytokine IL-1ß is required for the control of intracellular parasites. In the present study, we investigated the involvement of the P2Y2 receptor in the activation of NLRP3 inflammasome elements (caspase-1 and 11) and IL-1ß secretion during L. amazonensis infection in peritoneal macrophages as well as in a murine model of cutaneous leishmaniasis. We found that 2-thio-UTP (a selective P2Y2 agonist) reduced parasite load in L. amazonensis-infected murine macrophages and in the footpads and lymph nodes of infected mice. The antiparasitic effects triggered by P2Y2 activation were not observed when cells were pretreated with a caspase-1 inhibitor (Z-YVAD-FMK) or in macrophages from caspase-1/11 knockout mice (CASP-1,11-/-). We also found that UTP treatment induced IL-1ß secretion in wild-type (WT) infected macrophages but not in cells from CASP-1,11-/- mice, suggesting that caspase-1 activation by UTP triggers IL-1ß secretion in L. amazonensis-infected macrophages. Infected cells pretreated with IL-1R antagonist did not show reduced parasitic load after UTP and ATP treatment. Our in vivo experiments also showed that intralesional UTP treatment reduced both parasite load (in the footpads and popliteal lymph nodes) and lesion size in wild-type (WT) and CASP-11-/- but not in CASP-1,11-/- mice. Taken together, our findings suggest that P2Y2R activation induces CASP-1 activation and IL-1ß secretion during L. amazonensis infection. IL-1ß/IL-1R signaling is crucial for P2Y2R-mediated protective immune response in an experimental model of cutaneous leishmaniasis.

Expanding Role of Dopaminergic Inhibition in Hypercapnic Responses of Cultured Rat Carotid Body Cells: Involvement of Type II Glial Cells.

Dopamine (DA) is a well-studied neurochemical in the mammalian carotid body (CB), a chemosensory organ involved in O2 and CO2/H+ homeostasis. DA released from receptor (type I) cells during chemostimulation is predominantly inhibitory, acting via pre- and post-synaptic dopamine D2 receptors (D2R) on type I cells and afferent (petrosal) terminals respectively. By contrast, co-released ATP is excitatory at postsynaptic P2X2/3R, though paracrine P2Y2R activation of neighboring glial-like type II cells may boost further ATP release. Here, we tested the hypothesis that DA may also inhibit type II cell function. When applied alone, DA (10 µM) had negligible effects on basal [Ca2+]i in isolated rat type II cells. However, DA strongly inhibited [Ca2+]i elevations (Δ[Ca2+]i) evoked by the P2Y2R agonist UTP (100 µM), an effect opposed by the D2/3R antagonist, sulpiride (1-10 µM). As expected, acute hypercapnia (10% CO2; pH 7.4), or high K+ (30 mM) caused Δ[Ca2+]i in type I cells. However, these stimuli sometimes triggered a secondary, delayed Δ[Ca2+]i in nearby type II cells, attributable to crosstalk involving ATP-P2Y2R interactions. Interestingly sulpiride, or DA store-depletion using reserpine, potentiated both the frequency and magnitude of the secondary Δ[Ca2+]i in type II cells. In functional CB-petrosal neuron cocultures, sulpiride potentiated hypercapnia-induced Δ[Ca2+]i in type I cells, type II cells, and petrosal neurons. Moreover, stimulation of type II cells with UTP could directly evoke Δ[Ca2+]i in nearby petrosal neurons. Thus, dopaminergic inhibition of purinergic signalling in type II cells may help control the integrated sensory output of the CB during hypercapnia.

Impaired UTP-induced relaxation in the carotid arteries of spontaneously hypertensive rats.

Uridine 5'-triphosphate (UTP) has an important role as an extracellular signaling molecule that regulates inflammation, angiogenesis, and vascular tone. While chronic hypertension has been shown to promote alterations in arterial vascular tone regulation, carotid artery responses to UTP under hypertensive conditions have remained unclear. The present study investigated carotid artery responses to UTP in spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY). Accordingly, our results found that although UTP promotes concentration-dependent relaxation in isolated carotid artery segments from both SHR and WKY after pretreatment with phenylephrine, SHR exhibited significantly lower arterial relaxation responses compared with WKY. Moreover, UTP-induced relaxation was substantially reduced by endothelial denudation and by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine in both SHR and WKY. The difference in UTP-induced relaxation between both groups was abolished by the selective P2Y2 receptor antagonist AR-C118925XX and the cyclooxygenase (COX) inhibitor indomethacin but not by the thromboxane-prostanoid receptor antagonist SQ29548. Furthermore, we detected the release of PGE2, PGF2α, and PGI2 in the carotid arteries of SHR and WKY, both at baseline and in response to UTP. UTP administration also increased TXA2 levels in WKY but not SHR. Overall, our results suggest that UTP-induced relaxation in carotid arteries is impaired in SHR perhaps due to impaired P2Y2 receptor signaling, reductions in endothelial NO, and increases in the levels of COX-derived vasoconstrictor prostanoids.

Calcium signaling in interdental cells during the critical developmental period of the mouse cochlea.

The tectorial membrane (TM), a complex acellular structure that covers part of the organ of Corti and excites outer hair cells, is required for normal hearing. It consists of collagen fibrils and various glycoproteins, which are synthesized in embryonic and postnatal development by different cochlear cell types including the interdental cells (IDCs). At its modiolar side, the TM is fixed to the apical surfaces of IDCs, which form the covering epithelium of the spiral limbus. We performed confocal membrane imaging and Ca2+ imaging in IDCs of the developing mouse cochlea from birth to postnatal day 18 (P18). Using the fluorescent membrane markers FM 4-64 and CellMask™ Deep Red on explanted whole-mount cochlear epithelium, we identified the morphology of IDCs at different z-levels of the spiral limbus. Ca2+ imaging of Fluo-8 AM-loaded cochlear epithelia revealed spontaneous intracellular Ca2+ transients in IDCs at P0/1, P4/5, and P18. Their relative frequency was lowest on P0/1, increased by a factor of 12.5 on P4/5 and decreased to twice the initial value on P18. At all three ages, stimulation of IDCs with the trinucleotides ATP and UTP at 1 and 10 µM elicited Ca2+ transients of varying amplitude and shape. Before the onset of hearing, IDCs responded with robust Ca2+ oscillations. At P18, after the onset of hearing, ATP stimulation either caused Ca2+ oscillations or an initial Ca2+ peak followed by a plateau while the UTP response was unchanged from that at pre-hearing stage. Parameters of spontaneous and nucleotide-evoked Ca2+ transients such as amplitude, decay time and duration were markedly reduced during cochlear development, whereas the kinetics of the Ca2+ rise did not show relevant changes. Whether low-frequency spontaneous Ca2+ transients are necessary for the formation and maintenance of the tectorial membrane e.g. by regulating gene transcription needs to be elucidated in further studies.