Isoflurane favorably modulates guanosine triphosphate cyclohydrolase-1 and endothelial nitric oxide synthase during myocardial ischemia and reperfusion injury in rats

Abstract

Background: The authors investigated the hypothesis that isoflurane modulates nitric oxide (NO) synthesis and protection against myocardial infarction through time-dependent changes in expression of key NO regulatory proteins, guanosine triphosphate cyclohydrolase (GTPCH)-1, the rate-limiting enzyme involved in the biosynthesis of tetrahydrobiopterin and endothelial nitric oxide synthase (eNOS).

Methods: Myocardial infarct size, NO production (ozone-mediated chemiluminescence), GTPCH-1, and eNOS expression (real-time reverse transcriptase polymerase chain reaction and western blotting) were measured in male Wistar rats with or without anesthetic preconditioning (APC; 1.0 minimum alveolar concentration isoflurane for 30 min) and in the presence or absence of an inhibitor of GTPCH-1, 2,4-diamino-6-hydroxypyrimidine.

Results: NO2 production (158 ± 16 and 150 ± 13 pmol/mg protein at baseline in control and APC groups, respectively) was significantly (P < 0.05) increased 1.5 ± 0.1 and 1.4 ± 0.1 fold by APC (n = 4) at 60 and 90 min of reperfusion, respectively, concomitantly, with increased expression of GTPCH-1 (1.3 ± 0.3 fold; n = 5) and eNOS (1.3 ± 0.2 fold; n = 5). In contrast, total NO (NO2 and NO3) was decreased after reperfusion in control experiments. Myocardial infarct size was decreased (43 ± 2% of the area at risk for infarction; n = 6) by APC compared with control experiments (57 ± 1%; n = 6). 2, 4-Diamino-6-hydroxypyrimidine decreased total NO production at baseline (221 ± 25 and 175 ± 31 pmol/mg protein at baseline in control and APC groups, respectively), abolished isoflurane-induced increases in NO at reperfusion, and prevented reductions of myocardial infarct size by APC (60 ± 2%; n = 6).

Conclusion: APC favorably modulated a NO biosynthetic pathway by up-regulating GTPCH-1 and eNOS, and this action contributed to protection of myocardium against ischemia and reperfusion injury.

Figure 1

Schematic diagram depicting the experimental protocols used to determine myocardial infarct size and modulation of nitric oxide in rats in vivo. APC, anesthetic preconditioning; DAHP, 2,4-diamino-6-hydroxypyrimidine; LV, left ventricle; MAC, minimum alveolar concentration; OCC, coronary artery occlusion.

Figure 2

Myocardial infarct size depicted as a percentage of the area at risk (% of AAR) for infarction in control (CON) rats and in rats subjected to anesthetic preconditioning (APC) in the absence or presence of 2,4-diamino-6-hydroxypyrimidine (DAHP). Data are expressed as mean ± SE. *P < 0.05 vs. control; †P < 0.05 vs. APC alone.

Figure 3

Time-dependent changes in NO⁻₂ production in control rats and in rats subjected to anesthetic preconditioning (APC) with or without 2,4-diamino-6-hydroxypyrimidine (DAHP), before (PreOcc) and during coronary artery occlusion (Occ), and after reperfusion (60 and 90 min). Data are expressed as mean ± SE. *P < 0.05 vs. PreOcc baseline; †P < 0.05 vs. control at the same time point; ‡P < 0.05 vs. respective control without DAHP.

Figure 4

Time-dependent changes in total NO (NO⁻₂ and NO⁻₃: NO_x) production in control rats and in rats subjected to anesthetic preconditioning (APC) with or without 2,4-diamino-6-hydroxypyrimidine (DAHP), before (PreOcc) and during coronary artery occlusion (Occ), and after reperfusion (60 and 90 min). Data are expressed as mean ± SE. *P < 0.05 vs. PreOcc baseline; †P < 0.05 vs. control at the same time point; ‡P < 0.05 vs. respective control without DAHP.

Figure 5

Time-dependent changes in guanosine triphosphate cyclohydrolase (GTPCH)-1 (panel A) and endothelial nitric oxide synthase (eNOS; panel B) gene expression in control (CON: panels A and B) rats and in rats subjected to anesthetic preconditioning (APC: panels C and D), before (PreOcc) and during coronary artery occlusion (Occ), and after reperfusion (30, 60 and 90 min). Data are expressed as mean ± SE. *P < 0.05 vs. PreOcc baseline; †P < 0.05 vs. control at the same time point.

Figure 6

Time-dependent changes in guanosine triphosphate cyclohydrolase (GTPCH)-1 (panel A) and endothelial nitric oxide synthase (eNOS; panel B) protein expression in control (CON) rats and in rats subjected to anesthetic preconditioning (APC), before (PreOcc) and during coronary artery occlusion (Occ), and after reperfusion (60 and 90 min). Representative western blots are shown above the summary data. Data are expressed as mean ± SE. *P < 0.05 vs. PreOcc baseline; †P < 0.05 vs. control at the same time point.