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 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

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