P2Y receptors are metabotropic G-protein-coupled receptors, which are involved in many important biologic functions in the central nervous system including retina. Subtypes of P2Y receptors in retinal tissue vary according to the species and the cell types. We examined the molecular and pharmacologic profiles of P2Y purinoceptors in retinoblastoma cell, which has not been identified yet. To achieve this goal, we used Ca2+ imaging technique and western blot analysis in WERI-Rb-1 cell, a human retinoblastoma cell line. ATP (10 microM) elicited strong but transient [Ca2+]i increase in a concentration-dependent manner from more than 80% of the WERI-Rb-1 cells (n=46). Orders of potency of P2Y agonists in evoking [Ca2+]i transients were 2MeS-ATP>ATP>>UTP=alphabeta-MeATP, which was compatible with the subclass of P2Y1 receptor. The [Ca2+]i transients evoked by applications of 2MeS-ATP and/or ATP were also profoundly suppressed in the presence of P2Y1 selective blocker (MRS 2179; 30 microM). P2Y1 receptor expression in WERI-Rb-1 cells was also identified by using western blot. Taken together, P2Y1 receptor is mainly expressed in a retinoblastoma cell, which elicits Ca2+ release from internal Ca2+ storage sites via the phospholipase C-mediated pathway. P2Y1 receptor activation in retinoblastoma cell could be a useful model to investigate the role of purinergic [Ca2+]i signaling in neural tissue as well as to find a novel therapeutic target to this lethal cancer.
Adenosine Triphosphate ; Blotting, Western ; Calcium ; Cell Line ; Central Nervous System ; Humans ; Phospholipases ; Receptors, G-Protein-Coupled ; Receptors, Purinergic P2Y ; Receptors, Purinergic P2Y1 ; Retina ; Retinaldehyde ; Retinoblastoma

BACKGROUND: Dual therapy with aspirin and clopidogrel has emerged as the gold standard therapy for patients treated with drug-eluting stents (DES). However, there is variability in patients' responses to this antiplatelet therapy, and some patients continue to show ischemic recurrences after therapy. The purpose of the study was to compare the simultaneously obtained results of various platelet-function tests for assessing the prevalence of antiplatelet resistance in coronary artery disease patients undergoing DES therapy. METHODS: A total of 66 patients were administered a loading dose of aspirin, clopidogrel, and cilostazol at least 12 hr before stenting. The results of VerifyNow (Accumetrics, USA), multiplate analyzer (Dynabyte Medical, Germany), and vasodilator-stimulated phosphoprotein/P2Y12 (Biocytex, France) assays were compared with those of light transmission aggregometry (LTA) analysis. RESULTS: The P2Y12 reaction units and P2Y12% inhibition values obtained using the VerifyNow assay showed strong correlation (r) with the results of the LTA analysis. All tests results showed low concordance in defining the antiplatelet resistance in patients, and the degrees of agreement were as follows: 0 for aspirin reaction units; 0.25, P2Y12% inhibition; 0, aspirin-sensitive patients' identification test; 0.21, ADPtest; and 0.14, platelet reactivity index, expressed as the kappa statistics. The prevalence of aspirin and clopidogrel resistances in patients resulted in remarkable variations, from 0% to 22.7% and from 9.1% to 48.5%, respectively. CONCLUSIONS: The clinical usefulness of the different assays for the correct classification of patients in terms of antiplatelet resistance remains unclear. Further studies are required to determine the best method for correlating the occurrences of adverse ischemic events.
Aged ; Aspirin/*administration & dosage ; Coronary Artery Disease/*drug therapy ; Drug Resistance ; Drug Therapy, Combination ; Drug-Eluting Stents ; Female ; Humans ; Male ; Middle Aged ; Platelet Aggregation/drug effects ; Platelet Aggregation Inhibitors/*administration & dosage ; Platelet Function Tests ; Purinergic P2Y Receptor Antagonists/administration & dosage ; Receptors, Purinergic P2Y12/metabolism ; Tetrazoles/administration & dosage ; Ticlopidine/administration & dosage/*analogs & derivatives

BACKGROUND AND OBJECTIVES: Extracellular purines and pyrimidines regulate various physiological responses via cell surface receptors known as purinoreceptors, and may exert autocrine or paracrine effects on ion transport, fluid transport, ciliary beat frequency and mucin secretion. This study aims to investigate the expression patterns of such purinoreceptors found in normal human nasal epithelial (NHNE) cells. MATERIALS AND METHOD: In RT-PCR, the mRNAs for several P2X (P2X3, P2X4, P2X7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12) receptors were identified in NHNE cells. Functional localizations of P2 receptors were investigated by measuring [Ca2+]i increases in a membrane-specific manner using a double-perfusion chamber. Absence of the responses of -Me ATP and 2MeS-ATP excluded functionally active P2X3, P2X4, and P2Y1 receptors as far as [Ca2+]i increase was concerned. RESULTS: Applications with ATP and UTP revealed that luminal membranes of NHNE cells express P2Y2 and P2Y6 receptors and basolateral membranes P2Y2 receptors. Expressions of P2Y2 and P2Y6 receptors in NHNE cells were further verified by the immunoblotting using specific antibodies. In addition, the results with BzATP indicated that the P2Y11 receptor may be present on the luminal side. CONCLUSION: The NHNE cells express functionally active P2Y2, P2Y6 and P2Y11 receptors in a membrane-specific pattern, which may play an important role in the control of mucin and fluid secretion in NHNE cells.
Adenosine Triphosphate ; Antibodies ; Calcium ; Epithelial Cells* ; Humans* ; Immunoblotting ; Ion Transport ; Membranes ; Mucins ; Nasal Mucosa ; Phenobarbital ; Purines ; Pyrimidines ; Receptors, Cell Surface ; Receptors, Purinergic P2Y1 ; Receptors, Purinergic P2Y2 ; Receptors, Purinergic* ; RNA, Messenger ; Uridine Triphosphate

BACKGROUND AND OBJECTIVES: Nucleotide binding to purinergic P2Y receptors contributes to the regulation of fluid and ion transport in the middle ear epithelial cells. Here, we investigated the regulatory mechanism of the P2Y2 receptor agonist, uridine-5'-triphosphate (UTP), on Cl- transport in cultured normal human middle ear epithelial (NHMEE) cells. MATERIALS AND METHOD: Electrophysiological measurements were performed in monolayers of cultured NHMEE cells. Short circuit currents (Isc) were measured from the cells mounted in Ussing chambers under various conditions. RESULTS: Apical addition of UTP in presence of amiloride evoked a transient rise and a sustained response in Isc due to Cl- efflux. Application of different Cl- channel blockers to the apical side of the cells significantly decreased UTP-induced Isc. Niflumic acid (NFA), a known blocker of Ca(2+)-activated chloride channels (CACC), and CFTRinh172, a selective inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR), partially inhibited the UTP-induced Cl- secretion, respectively. CONCLUSION: Cl- transport across the airway epithelia plays a predominant role in regulating airway hydration. In this study, UTP is shown to increase both CACC and CFTR-dependent Cl- secretion in NHMEE cells, suggesting their role in fluid and ion transport in the middle ear epithelium.
Amiloride ; Chloride Channels ; Cystic Fibrosis ; Cystic Fibrosis Transmembrane Conductance Regulator ; Ear, Middle ; Epithelial Cells ; Epithelium ; Humans ; Ion Channels ; Ion Transport ; Niflumic Acid ; Receptors, Purinergic P2Y ; Uridine Triphosphate

BACKGROUND/AIMS: Newer P2Y12 inhibitors, such as prasugrel and ticagrelor, have greater antiplatelet efficacy but may increase the risk of bleeding. In this study, we compared the pharmacodynamic efficacy of prasugrel and ticagrelor in East Asian patients with acute coronary syndrome (ACS). METHODS: We selected 83 ACS patients undergoing percutaneous coronary intervention who were discharged with 90 mg ticagrelor twice daily (n = 24), 10 mg prasugrel daily (n = 39) or 5 mg prasugrel daily (n = 20). After 2 to 4 weeks, on-treatment platelet reactivity (OPR) was assessed in terms of P2Y12 reaction units (PRUs) using the VerifyNow P2Y12 assay (Accumetrics). We compared East Asian (85 < PRU < or = 275) and Caucasian (85 < PRU < or = 208) criteria for assessing the therapeutic window of OPR. RESULTS: OPR was lowest in the ticagrelor group, followed by the 10 mg prasugrel and 5 mg prasugrel groups (49.1 ± 29.9 vs. 83.7 ± 57.1 vs. 168.5 ± 60.8, respectively; p < 0.001). The 5 mg prasugrel group had the highest proportion of patients with OPR values within the therapeutic window, followed by the 10 mg prasugrel and ticagrelor groups (90.0% vs. 46.2% vs. 12.5%, respectively; p < 0.001 for East Asian criteria; 60.0% vs. 43.6% vs. 12.5%, respectively; p < 0.001 for Caucasian criteria). CONCLUSIONS: Short-term administration of 5 mg prasugrel facilitated maintenance within the therapeutic window of OPR compared with the 10 mg prasugrel and ticagrelor groups. Thus, 5 mg prasugrel daily may be the optimal antiplatelet regimen for stabilized East Asian ACS patients.
Acute Coronary Syndrome/blood/diagnosis/ethnology/*therapy ; Adenosine/administration & dosage/adverse effects/*analogs & derivatives/pharmacology ; Aged ; *Asian Continental Ancestry Group ; Blood Platelets/*drug effects/metabolism ; Drug Administration Schedule ; Drug Monitoring/methods ; European Continental Ancestry Group ; Female ; Hemorrhage/chemically induced ; Humans ; Male ; Middle Aged ; *Percutaneous Coronary Intervention/adverse effects ; Pilot Projects ; Platelet Aggregation Inhibitors/administration & dosage/adverse effects ; Platelet Function Tests ; Prasugrel Hydrochloride/administration & dosage/adverse effects/*pharmacology ; Purinergic P2Y Receptor Antagonists/administration & dosage/adverse effects/*pharmacology ; Receptors, Purinergic P2Y12/blood/*drug effects ; Republic of Korea ; Retrospective Studies ; Risk Factors ; Time Factors ; Treatment Outcome

OBJECTIVE: To investigate the clinical features and prognostic factors of acute lymphoblastic leukemia (ALL) children with P2RY8-CRLF2 gene rearrangement. METHODS: A total of 108 children with B-cell ALL (B-ALL) were diagnosed and systematically treated according to Chinese Children's Leukemia Group (CCLG) -ALL 2008 in our hospital from January 2016 to December 2016. The 108 patients were divided into two groups according to the result of mutiplex polymerase chain reaction: group with P2RY8-CRLF2 gene rearrangement and group without P2RY8-CRLF2 gene rearrangement. The ALL children with P2RY8-CRLF2 gene rearrangement were all treated by CCLG-ALL 2008 high-risk group (HR) regimens, and the ALL children in group without P2RY8-CRLF2 gene rearrangement received different intensity chemotherapy according to clinical risk classification. RESULTS: Five (4 male and 1 female) out of 108 patients with B-ALL had P2RY8-CRLF2 gene rearrangement. In the 5 B-ALL patients with P2RY8-CRLF2 gene rearrangement, the median age of the was 4 (2-6) years old and the median WBC count was 26.2 (2.46-525.1)×10 CONCLUSION The early treatment response and prognosis of ALL children with P2RY8-CRLF2 gene rearrangement are worse, and more effective protocol is needed for this subtype patients.
Child ; Child, Preschool ; Disease-Free Survival ; Female ; Gene Rearrangement ; Humans ; Male ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; Prognosis ; Receptors, Cytokine/genetics* ; Receptors, Purinergic P2Y/genetics*

The regulated transport of salt and water is essential to the integrated function of many organ systems, including the respiratory, reproductive, and digestive tracts. Airway epithelial fluid secretion is a passive process that is driven by osmotic forces, which are generated by ion transport. The main determinant of a luminally-directed osmotic gradient is the mucosal transport of chloride ions (Cl(-)) into the lumen. As with many epithelial cells, a number of classic signal transduction cascades are involved in the regulation of ion transport. There are two well-known intracellular signaling systems: an increase in intracellular Ca(2+) concentration ([Ca(2+)]i) and an increase in the rate of synthesis of cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP). Therefore, Cl(-) secretion is primarily activated via the opening of apical Ca(2+)- or cAMP-dependent Cl(-) channels at the apical membrane. The opening of basolateral Ca(2+)- or cAMP-activated K(+) channels, which hyperpolarizes the cell to maintain the driving force for Cl(-) exit through apical Cl(-) channels that are constitutively open, is also important in regulating transepithelial ion transport. P2Y receptors are expressed in the apical and/or basolateral membranes of virtually all polarized epithelia to control the transport of fluid and electrolytes. Human airway epithelial cells express multiple nucleotide receptors. Extracellular nucleotides, such as UTP and ATP, are calcium-mobilizing secretagogues. They are released into the extracellular space from airway epithelial cells and act on the same cell in an autocrine fashion to stimulate transepithelial ion transport. In addition, recent data support the role of P2Y receptors in releasing inflammatory cytokines in the bronchial epithelium and other immune cells.
Biological Transport ; Cell Membrane ; physiology ; Chloride Channels ; physiology ; Cyclic AMP ; physiology ; Cytokines ; immunology ; Epithelial Cells ; physiology ; Epithelium ; immunology ; physiology ; Humans ; Ion Transport ; Receptors, Purinergic P2Y ; immunology ; physiology ; Signal Transduction

The contractile and diastolic function of smooth muscle cells (SMCs) is closely related to penile erection and erectile dysfunction (ED). In addition to nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S), sulfur dioxide (SO2), estrogen receptor (ER), P2Y receptor, perivascular tissue (PVT), and calcium activated potassium channel (Kca) are found to be involved in the relaxation of SMCs. This review updates the mechanisms of the relaxation of SMCs and its relationship with ED.
Carbon Monoxide ; physiology ; Erectile Dysfunction ; etiology ; physiopathology ; Humans ; Hydrogen Sulfide ; metabolism ; Male ; Muscle Contraction ; Muscle, Smooth ; Myocytes, Smooth Muscle ; physiology ; Nitric Oxide ; physiology ; Penile Erection ; physiology ; Potassium Channels, Calcium-Activated ; physiology ; Receptors, Estrogen ; physiology ; Receptors, Purinergic P2Y ; physiology ; Sulfur Dioxide ; metabolism

OBJECTIVEThe present study aimed to explore the role of P2Y(1) receptor in glial fibrillary acidic protein (GFAP) production and glial cell line-derived neurotrophic factor (GDNF) secretion of astrocytes under ischemic insult and the related signaling pathways.

METHODSUsing transient right middle cerebral artery occlusion (tMCAO) and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, enzyme linked immunosorbent assay (ELISA) were used to investigate location of P2Y(1) receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules.

RESULTSBlockage of P2Y(1) receptor with the selective antagonist N(6)-methyl-2'-deoxyadenosine 3',5'-bisphosphate diammonium (MRS2179) reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y(1) receptor caused elevation of phosphorylated Akt and cAMP response element binding protein (CREB), and reduction of phosphorylated Janus kinase2 (JAK2) and signal transducer and activator of transcription3 (STAT3, Ser727). After blockage of P2Y(1) receptor and deprivation of oxygen-glucose-serum, AG490 (inhibitor of JAK2) reduced phosphorylation of STAT3 (Ser727) as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase1/2 (MEK1/2) U0126, an important molecule of Ras/extracellular signal-regulated kinase (ERK) signaling pathway, decreased the phosphorylation of JAK2, STAT3 (Ser727), Akt and CREB.

CONCLUSIONThese results suggest that P2Y(1) receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.

Animals ; Astrocytes ; metabolism ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Glial Cell Line-Derived Neurotrophic Factor ; biosynthesis ; Glial Fibrillary Acidic Protein ; biosynthesis ; Infarction, Middle Cerebral Artery ; metabolism ; RNA, Messenger ; analysis ; Rats ; Receptors, Purinergic P2 ; metabolism ; Receptors, Purinergic P2Y1 ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; physiology

BACKGROUNDAspirin is widely used in the secondary prevention of coronary artery diseases, including myocardial infarction, stroke, and vascular related deaths. However, the antiplatelet effect of aspirin appears to be variable and aspirin resistance (AR) is currently still controversial for Chinese patients. The aim of this study was to describe the prevalence of AR, and identify possible risk factors associated with a lack of response to aspirin treatments in patients with unstable coronary artery disease.

METHODSPlatelet function tests with arachidonic acid (ARA) and urinary 11-dehydro-thromboxane B2 (11-DH-TXB2) concentrations were performed in 262 patients with unstable coronary artery disease who had not been taking aspirin before admission. ARA induced platelet aggregation and 11-DH-TXB2 were detected to evaluate the functional and biochemical responses to aspirin before and on days 1, 4, and 10 after aspirin administration. Six-month follow-up was completed in patients who developed AR to evaluate the effect of aspirin in a long-term treatment. GP1Bα (C1018T), Pl (A1/A2), P2Y1 (A1622G), TBXA2R (T924C) were also detected to evaluate the influence of genetic variant on aspirin responsiveness.

RESULTSA total of 8.8% of patients were indentified as AR at the first day after aspirin treatment. The level of urine 11-DH-TXB2 in the AR group was higher compared to non-AR group (P < 0.05). There was no relationship between ARA induced platelet aggregation and urinary 11-DH-TXB2 levels (r = 0.038, P = 0.412). The results of DNA sequencing showed that TBXA2R-924TT homozygotes had a significantly high rate of AR. Logistic regression demonstrated that diabetes was an independent risk factor of AR.

CONCLUSIONSIn the beginning period of administration, aspirin was not a sufficient factor that inhibits platelet aggregation. TBXA2R-924T allele was involved in AR. Diabetes was an independent risk factor of AR.

Aged ; Aged, 80 and over ; Arachidonic Acid ; pharmacology ; Aspirin ; therapeutic use ; Coronary Artery Disease ; drug therapy ; genetics ; Diabetes Mellitus, Type 2 ; Female ; Genotype ; Humans ; Male ; Membrane Glycoproteins ; genetics ; Middle Aged ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; therapeutic use ; Platelet Function Tests ; Platelet Glycoprotein GPIb-IX Complex ; Polymerase Chain Reaction ; Polymorphism, Genetic ; genetics ; Receptors, Purinergic P2Y1 ; genetics ; Receptors, Thromboxane A2, Prostaglandin H2 ; genetics ; Thromboxane B2 ; analogs & derivatives ; urine