Role of NOS-NO-NHE1 pathway in sevoflurane postconditioning-induced mitigation of myocardial ischemia/reperfusion injury in rats in vitro

Objective To evaluate the role of NOS-NO-NHE1 pathway in sevoflurane postconditioning-induced mitigation of myocardial ischemia/reperfusion (I/R) injury in rats in vitro.Methods Sprague-Dawley rats,weighing 270-350 g,were heparinized and anesthetized with intraperitoneal chloral hydrate.Their hearts were rapidly excised and perfused in a Langendorff apparatus with oxygenated (95％ O2-5％ CO2) K-H solution.After 15-20 min of stabilization,72 isolated rat hearts which were successfully perfused in a Langendorff apparatus were randomly divided into 6 groups (n =12 each) using a random number table:sham operation group (group S),sevoflurane group (group Sev),group I/R,sevoflurane postconditioning group (group SP),sevollurane postconditioning + L-NAME (the NOS inhibitor) group (group SPL),and L-NAME group (group L).The hearts were subjected to ischemia for 30 min followed by 120 min reperfusion in I/R,SP,SPL and L groups.In group S,the hearts were continuously perfused with K-H solution for 180 min.In group Sev,the hearts were perfused with K-H solution for 60 min,with K-H solution saturated with 2.5％ sevoflurane for 15 min,and then with K-H solution for 105 min.In group I/R,the hearts were perfused with K-H solution for 30 min,and perfusion was then suspended for 30 min followed by 120 min reperfusion.In SP group,the hearts were perfused with K-H solution saturated with 2.5 ％ sevollurane for 15 min starting from the beginning of reperfusion,and then with K-H solution for 105 min.In SPL group,the hearts were perfused with K-H solution saturated with 100 μmol/L L-NAME for 60 min starting from the beginining of reperfusion,2.5％ sevoflurane was simultaneously introduced into K-H solution and maintained for 15 min,and then the hearts were perfused with K-H solution for 60 min.In group L,the hearts were perfused with K-H solution saturated with 100 μmol/L L-NAME for 60 min starting from the beginning of reperfusion and then with K-H solution for 60 min.Myocardial specimens were obtained at the end of reperfusion for determination of NO and NAD+ contents and NOS activity (by spectrophotometry),expression of total NHE1 (t-NHE1),phosphor-NHE (p-NHE) (by Western blot),and NHE1 mRNA (by RT-PCR),and myocardial infarct size (by TTC) and for microscopic examination of myocardial ultrastructure.Results Compared with group S,the myocardial infarct size was significantly enlarged,the levels of NO,NOS and NAD + were decreased,and the expression of p-NHE and NHE1 mRNA was up-regulated in I/R,SP,SPL and L groups,and no significant changes were found in the indices mentioned above in group Sev.Compared with group I/R,the myocardial infarct size was significantly decreased,the levels of NO,NOS and NAD+ were increased,and the expression of p-NHE and NHE1 mRNA was down-regulated in group SP,and no significant changes were found in the indices mentioned above in SPL and L groups.Compared with group SP,the myocardial infarct size was significantly enlarged,the levels of NO,NOS and NAD+ were decreased,and the expression of p-NHE and NHE1 mRNA was up-regulated in SPL and L groups.The pathological changes of myocardium were significantly mitigated in SP group as compared with I/R and SPL groups.Conclusion Sevollurane postconditioning enhances myocardial NOS activity,promotes NO synthesis,and inhibits the function of NHE1,thus reducing myocardial I/R injury in rats. Key words: Nitric oxide synthase;  Nitric oxide;  Cation transport proteins;  Myocardial reperfusion injury ;  Anesthetics, inhalation