| The physiological significance of ATP as extracellular signalling molecules is well established. ATP is released in a regulated manner from many types of cells,and released nucleotides act as the transmitters from the nerve terminal. Because of the species difference and the diversity of receptors, the physiological functions of extracellular nucleotides were less well understood. Like ATP, as a signal transmitter, UTP regulates physiological and pathological processes of the body. P2 receptor which is a category of P receptor.,Its endogenous ligands are ATP and UTP, and their hydrolysis products—ADP and UDP. P2 receptor is divided into ligand-gated ion channel receptor-P2X . and G-protein-coupled receptor-P2Y. These two types of receptors have many subtypes.In contrast to the compelling evidence for the extracellur signaling role of ATP,the hypothesis that UTP may also fufill an autocrine/paracrine role has only recently gained experimental support. The research of UTP become a new hot spot. Under normal conditions, cells continue to release UTP in order to maintain the normal physiological function. In some pathological cases, cells also can release UTP. For example, after myocardial infarction, the concentration of UTP increased in the coronary sinus blood. The source of UTP may be myocardial cells and vascular endothelial cells. In other studies ,they found that myocardial tolerance to hypoxia / reoxygenation injury enhanced when pretreatment with UTP .In vivo experiments also showed that UTP can improve cardiac function and morphological index after myocardial infarction in rats. However the mechanism of UTP preconditioning is not clear, moreover, adenosine which is the analogues of uridine can mediate the effects of myocardial ischemic preconditioning. This study was designed to discover the receptor which specifically mediate the preconditioning protective effect of UTP as well as to clarify its possible mechanism.This study was divided into two partsPart 1.Indirect in vivo experiment: isolated heart perfusion experiment after administration of drugs in vivo.Aim: To explore the antagonistic effect of uridine triphosphate (UTP) on myocardial ischemia/reperfusion injury(I/RI) in rats.Methods: Twenty four SD rats were randomly divided into 4 groups: control group (9 g/L normal saline, i.v.), UTP group(4.4μg/kg i.v.), UTP and suramin(SRM, antagonist of P2Y receptor) group (4.4μg/kg i.v.+30μg/kg i.v.) and SRM group(30μg/kg i.v.). 24 h after drug injection, hearts were isolated from the rats and perfused with K-H solution by Langendorff system. Hearts were subjected to 25 min ischemia followed by 40 min reperfusion. The hemodynamics indexs were measured and ECG was recorded. The coronary effluent was collected for measuring lactate dehydrogenas(LDH) level. Ultrastructure of myocardial was observed by transmission electron microscope. The remaining myocardial tissue was fixed with 4% paraformaldehyde and embedded for slice, samples of myocardial tissue were prepared for immunofluorescence staining to confirme the existence of P2Y2 receptors and P2Y4 receptors in rats'hearts .Results: After 25 min ischemia, the UTP group had significantly better recovery percentage of left ventricular function (P<0.01) and lower LDH level(P<0.01) compared with control group,the arhythmia frequency was also lower(P<0.01) than control group. The myocardial ultrastructure of UTP group was generally normal. The cardio-protective effect of UTP were eliminated by SRM. Myocardial biopsy and immunofluorescence staining showed that the distribution of P2Y2 receptor and P2Y4 receptors in mature rat heart.Conclusion: UTP pretreatment can protect rat's hearts from I/RI, which is cancelled by the P2Y receptors'antagonist suramin, indicating that the myocardial protective effect of UTP is mediated by P2Y receptors. As the affinity of UTP to the receptors of P2Y2 and P2Y4 is high and these two receptors are widely distributed in the mature myocardium, then the myocardial protective effect of UTP may be mediated through these two subtypes of P2Y receptor.Part2. Myocardial cells experimentsAim and Methods: 1.ATP on hypoxia / reoxygenation injury.Aim: To investigate whether ATP preconditioning can protect myocardial cells from hypoxia / reoxygenation injury like UTP.Methods: Experimental groups:①normal control group;②positive control group which is hypoxia / reoxygenation group (hypoxia 12h, reoxygenation 4h);③low dose of ATP pretreatment group (pretreatment of 3μM ATP 24h prior hypoxia / reoxygenation);④middle dose of ATP treatment group (ATP15μM);⑤high dose of ATP treatment group (ATP 50μM).2. Effect of UTP on apoptosis of myocardial cell after hypoxia / reoxygenation injury.Aim: To investigate the inhinbition effect of UTP to cardiomyocyte apoptosis which induced by hypoxia / reoxygenation injury.Methods: Experimental groups:①normal control group;②positive control group which is hypoxia / reoxygenation group (hypoxia 12h, reoxygenation 4h)③UTP preconditioning group (pretreatment of 50μM UTP 24h prior hypoxia / reoxygenation);④UTP and SRM handling group (SRM 300μM, 15min later treatment with 50μMUTP). Collection of hypoxia / reoxygenation treated myocardial cells were detected by flow cytometry.3. The effect of Ca2+ in UTP preconditioning before myocardial hypoxia / reoxygenation injuryAim: To investigate the role of extracellular Ca2 + in P2Y receptor-mediated cardio-protective effect of UTP preconditioning.Methods: Experimental groups:①normal control group;②positive control group which is hypoxia / reoxygenation group (hypoxia 12h, reoxygenation 4h);③The Ca2 + containing medium UTP preconditioning group (24h before hypoxia / reoxygenation treatment with 50μM UTP );④T he Ca2 +-free medium UTP preconditioning group (Before hypoxia / reoxygenation treatment , the original medium was replaced by Ca2 +-free medium and treatment with 50μMUTP, then replaced with Ca2 + containing medium). The LDH released from the myocardial cells after hypoxia / reoxygenation treatment was analyzed using Automatic biochemical analyzer.Results:1. The release of LDH from the myocardial cells which were pretreated with low dose and mid dose of ATP after hypoxia / reoxygenation showed no significant difference compared with the control group,.The release of LDH in the high dose group increased compared with the control group.2. The percentage of early apoptosis in UTP pretreatment group was markedly decreased, while SRM combined with UTP pretreatment can increase the percentage of early apoptosis in myocardial cells.3. In compared with the control group, the release of LDH after hypoxia / reoxygenation in the Ca2+-free medium and medium containing Ca2+ reduced significantly. However, difference was found little between these two groups.Conclusions:1. ATP preconditioning can not produce protective effect on myocardial cells. As the affinity of rat's P2Y4 receptor to UTP is the same with ATP, but ATP could not active P2Y2 receptor fully, so we may conclude that myocardial protection of UTP is mainly mediated by the receptor of P2Y2 rather than P2Y4 receptor.2. Pretreatment of UTP can inhibit apoptosis of myocardial cells,this may be the reasen of UTP induced myocardial-protection. 3. When UTP acting on myocardial cells in the Ca2+-free medium, the ability of anti-hypoxia / reoxygenation injury did not compromised. It may elucidate that the mechanism of myocardial-protection is not related with the extracellular Ca2+.
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