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The Role of Akt in the Protective Effect of Morphine on Mitochondrial Damage and the Mechanism by Which Morphine Activates Akt

Author: XuZuoMan
Tutor: XuZheLong;TianZuo
School: North China Coal Medical
Course: Pathology and Pathophysiology
Keywords: Morphine PKB/Akt PTEN Ser/Thr phosphatases ROS Mitochondrial KATP channel
CLC: R541
Type: Master's thesis
Year: 2010
Downloads: 34
Quote: 0
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Abstract


Background:Although Akt has been demonstrated to play a role in the cardioprotective effect of morphine on myocardial ischemia/reperfusion (I/R) injury, little is known about the mechanism underlying morphine-induced Akt activation.Objective:This study aimed to test if morphine prevents oxidant-induced mitochondrial damage through activation of Akt and to determine the molecular mechanism by which morphine regulates Akt activity.Methods:Rat heart tissue-derived H9c2 cells were used.And cardiac H9c2 cells were exposed to H2O2 (600μM) for 20 min to induce mitochondrial oxidant damage.Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE) and the mPTP opening was evaluated with changes inΔΨm. Immunofluorescence assay was used to test Akt (Ser473) phosphorylation.Meanwhile phosphorylated Akt (Ser473), PTEN (Ser308/ Thr382/383) and total PTEN were determined with Western blot. PP2A activity was determined by using a Ser/Thr phosphatase assay kit.Intracellular ROS were determined by loading cells with H2DCFDA.Results:1 Cardiac H9c2 cells treated with 0.1μM morphine(83.8±2.6%) showed a significant increase in TMRE fluorescence intensity compared to the control group (47.1±4.1%), indicating that morphine can prevent oxidant-induced mitochondrial damage. This protective effect of morphine was reversed by both the PI3K inhibitor wortmannin (1μM) and the Akt inhibitor AKTI (10μM) (66.8±13.2%, 55.9±4.3%, respectively), indicating that Akt accounts for the protective action of morphine.2 Compared to the control group, cells treated with morphine showed a significant increase in phosphorylation of Akt (Ser473) (155.2±6.2%). This effect of morphine was abolished by wortmannin (1μM) (69.4±14.6%), indicating that Akt mediates the cardioprotective effect of morphine.3 Morphine (0.1μM) did not alter both the total and phosphorylated PTEN. Further experiments with cells treated with morphine did not result in the appearance of the higher-mobility form of PTEN (the oxidized form). All these results suggest that PTEN may not mediate the action of morphine on Akt.4 Morphine (70.9±3.9%) significantly inhibited PP2A activity. In support, the protein Ser/Thr phosphatase inhibitor okadaic acid (20 nM, OA) (70.3±20.9%) markedly increased Akt (Ser473) phosphorylation and OA (66.0±36.6%) mimicked the action of morphine by preventing oxidant-induced loss ofΔΨm. These results suggest that morphine may activate Akt by inhibiting Ser/Thr phosphatases.5 Morphine (162.0±7.4%) significantly increased DCF fluorescence compared to the control group (106.5±1.8%), indicating that morphine can produce ROS in cardiac cells. The effect of morphine was inhibited by MPG (300μM) (89.4±3.3%), suggesting that morphine may inhibit Ser/Thr phosphatases via ROS. The protective effect of morphine on mitochondrial oxidant damage was partially but significantly inhibited by MPG (67.3±6.1%) and morphine-induced Akt phosphorylation was inhibited by MPG (118.3±6.9%). Based on these observations, it is highly likely that morphine activates Akt by inactivating Ser/Thr phosphatases via ROS. by the mitochondrial KATP channel closer 5HD (500μM) (117.1±3.0%), suggesting that morphine generates ROS by opening mitochondrial KATP channels.Conclusions:In summary, we have demonstrated that morphine prevents oxidant-induced mitochondrial damage by positively regulating Akt activity. But the important regulator of the PI3K/Akt signaling PTEN appears not to be involved in the action of morphine. The regulation of Akt activity by morphine is attributed to inhibiting protein Ser/Thr phosphatases via ROS. Mitochondrial KATP channel opening is responsible for morphine-induced ROS generation. 6 This effect of morphine (0.1μM) (162.0±7.4%) on ROS was reversed

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CLC: > Medicine, health > Internal Medicine > Heart, blood vessels ( circulatory ) disease > Heart disease
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