Accumulation of cytochrome P450 induced by proteasome inhibition during cardiac ischemia.

Increasing evidence shows that cytochrome P450 (CYP) contributes to cardiac reperfusion injury. However, there have been few reports about the roles of CYPs in cardiac ischemia. The aim of the present study was to investigate the CYP expression and activity during ischemia using an in vivo rat model of myocardial infarction. Cardiac ischemia was evoked by ligation of the left anterior descending coronary artery for 1 h. The protein levels of CYP 2C6, 2E1 and 2J3 increased in the ischemic region of the rat hearts, while the mRNA levels of CYPs were unchanged. CYP 2C6 activity was significantly elevated in the ischemic region, and the activities of 2E1 and 2J3 tended to increase during ischemia. The proteasome activity decreased and the expression of ubiquitinated proteins increased in the ischemic region. Remarkably, ubiquitinated CYP 2C6, 2E1 and 2J3 were detected in the ischemic area, suggesting that CYP proteins accumulate in the ischemic region as a result of the suppression of their degradation due to the reduction of proteasome activity. The amounts of reactive oxygen species and protein carbonyls increased, and proteasome subunits, Rpt3 and Rpt5, were carbonylated in the ischemic region of the hearts, indicating that the proteasome is oxidized during ischemia. Taken together, our findings indicate that CYPs, especially CYP 2C6, were accumulated by oxidative impairment of the proteasome in the ischemic region of rat hearts. Accumulated CYPs might be involved in myocardial infarction and dysfunction during reperfusion.

Effects of sulfaphenazole derivatives on cardiac ischemia-reperfusion injury: association of cytochrome P450 activity and infarct size.

Cardiac ischemia-reperfusion injury is evoked by reactive oxygen species (ROS). We previously reported that sulfaphenazole (SPZ) attenuated cardiac ROS levels and ischemia-reperfusion injury in rats. SPZ has distinct two actions: a) elimination of ROS and b) inhibition of cytochrome P450 (CYP) that is responsible for ROS production. The aim of this study is to determine which action contributes to the attenuation of cardiac ischemia-reperfusion injury using SPZ and its derivatives [acetyl-SPZ (Ac-SPZ) and dichloro-SPZ (2Cl-SPZ)]. Administration of 2Cl-SPZ or SPZ prior to ischemia significantly reduced myocardial infarct size, myocardial lipid peroxides, and ROS levels. In addition, they inhibited rat cardiac CYP activity. However, Ac-SPZ neither reduced infarct size nor inhibited cardiac CYP activity. The three compounds had similar effects on ROS scavenging activity in that they scarcely scavenged hydrogen peroxide and superoxide anions but reduced hydroxyl radicals with the same efficacy. The serum concentration of each compound was almost the same until 24 h after reperfusion. Collectively, our findings indicate that the suppressive effects of SPZ and 2Cl-SPZ on ischemia-reperfusion injury are associated with the reduction of ROS levels, which is primarily due to a decrease in ROS production via inhibition of cardiac CYP, not via ROS scavenging activity.

Suppression of myocardial ischemia-reperfusion injury by inhibitors of cytochrome P450 in rats.

The effects of inhibitors of cytochrome P450 on myocardial regional ischemia-reperfusion injury were examined in rats. Ischemia-reperfusion injury was evoked by ligation of the left anterior descending coronary artery for 1 h, followed by reperfusion for 24 h. Injuries were evident in causing infarction, decreases in left ventricular systolic pressure and left ventricle (dP/dt max)/P and an increase in left ventricular end-diastolic pressure. Increases in lipid peroxidation and reactive oxygen species levels in the ischemic region were observed. Intravenous injection of the potent cytochrome P450 inhibitor sulfaphenazole at 10 and 30 mg/kg at the time of reperfusion reduced infarct size by 41.7 and 73.2%, respectively; and improved cardiac function accompanied by the decrease in content of lipid peroxide and reactive oxygen species in the area at risk. Cardiac testosterone metabolism was inhibited by sulfaphenazole administration, indicating its inhibitory effects on cardiac cytochrome P450 activity. Another cytochrome P450 inhibitor, cimetidine, given intravenously, had similar effects to sulfaphenazole on reperfusion injury. Taken together, these results indicate that reactive oxygen species derived from cytochrome P450 play an important part in myocardial regional ischemia-reperfusion injury in vivo, and strongly support the hypothesis that cytochrome P450 inhibitors are promising therapeutic agents for cardiac ischemia-reperfusion injury.

Sustained contraction and endothelial dysfunction induced by reactive oxygen species in porcine coronary artery.

A combination of purine and xanthine oxidase (XOD) dose-dependently elicited sustained contraction of porcine coronary arterial rings and resulted in increased concentrations of superoxide anions and hydrogen peroxide. These contractile responses appeared, with a delay, after the application of purine and XOD, used as a reactive oxygen species (ROS)-generating system. Coronary arteries precontracted with prostaglandin F(2alpha) failed to relax in response to substance P after exposing the arterial preparation to this ROS-generating system. The contractile response of the coronary artery to the ROS-generating system was almost completely inhibited by catalase (130 U/ml), and was partially inhibited by superoxide dismutase (60 U/ml), or mannitol (30 mM). A voltage-dependent L-type Ca(2+) channel antagonist, nicardipine, had no effect on contraction. Dysfunction of endothelial cells was completely prevented by catalase, but not by superoxide dismutase or mannitol. These results suggest that superoxide anions, hydrogen peroxide and hydroxyl radicals might be involved in eliciting sustained, delayed-onset coronary artery contraction, which is not related to L-type Ca(2+) channels. They also suggest that hydrogen peroxide might play a major role in endothelial dysfunction of the porcine coronary artery.