Sungsam Cho

Melatonin induces γ-glutamylcysteine synthetase mediated by activator protein-1 in human vascular endothelial cells

Abstract In the present study, we show that melatonin induces the expression of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme of glutathione (GSH) synthesis, in ECV304 human vascular endothelial cells. One micromolar melatonin induced the expression of γ-GCS mRNA followed by an increase in the concentration of GSH with a peak at 24 h. An electrophoretic mobility shift assay showed that melatonin stimulates the DNA-binding activity of activator protein-1 (AP-1) as well as retinoid Z receptor/retinoid receptor-related orphan receptor α (RZR/RORα). ECV304 cells transiently transfected with a plasmid containing the γ-GCS promoter-luciferase construct showed increased luciferase activity when treated with melatonin. The melatonin-dependent luciferase activity was found in the γ-GCS promoter containing AP-1 site. The luciferase activity mediated by AP-1 was repressed in the promoter containing RZR/RORα site. In addition, cell cycle analysis showed that melatonin increases the number of cells in the G0/G1 phase; however, treatment of the cells with buthionine sulfoximine, a specific inhibitor of γ-GCS, abolished the effect of melatonin on the cell cycle, suggesting induction of cell arrest by melatonin requires GSH. As conclusion, induction of GSH synthesis by melatonin protects cells against oxidative stress and regulates cell proliferation.

Overexpression of glutathione S-transferase π enhances the adduct formation of cisplatin with glutathione in human cancer cells

In this paper, we provide direct evidence that glutathione S-transferase pi (GSTpi) detoxifies cisplatin (CDDP). We used human colonic cancer HCT8 cells sensitive and resistant to CDDP, the level of cisplatin-glutathione adduct (DDP-GSH) being higher in the resistant cells. There was an overexpression of GSTpi mRNA in these CDDP-resistant cells. Incubation of the cells with CDDP resulted in the formation of DDP-GSH dependent on the CDDP concentration and the incubation time. The formation of DDP-GSH was abolished when the cells were pre-treated with ethacrynic acid or ketoprofen, inhibitors of GSTpi. Purified GSTpi also catalyzed the formation of DDP-GSH in vitro, with an apparent Km of 0.23 mM for CDDP and an apparent Vmax of 4.9 nmol/min/mg protein. The increase in DDP-GSH produced by GSTpi was linear with incubation time up to 3 h and optimal of pH 7.4. A GSTpi transfectant cell line was constructed in HCT8 cells using a pcDNA3.1 (-)/Myc-His B with an expression vector containing cDNA for GSTpi. Transfection of GSTpi cDNA into HCT8 cells resulted in an increase in the expression of GSTpi by 1.4-fold in parallel with an augmentation of the formation of DDP-GSH. These results suggest that GSTpi plays a role in the formation of DDP-GSH and the acquisition of resistance to CDDP in cancer cells.

Effects of sevoflurane with and without nitrous oxide on human cerebral circulation : Transcranial Doppler study

Background This study was designed to evaluate the effects of sevoflurane with and without nitrous oxide on human middle cerebral artery (MCA) flow velocity, cerebrovascular carbon dioxide reactivity, and autoregulation compared with the awake state using transcranial Doppler ultrasonography. Methods In 14 patients, the time‐mean middle cerebral artery flow velocity (Vmca) was measured when the end‐tidal carbon dioxide level was approximately 30, 40, and 50 mmHg under the following conditions: (1) awake; (2) with 2% (1.2 MAC) sevoflurane; and (3) with 1.2 MAC sevoflurane‐60% nitrous oxide. In six other patients, the cerebrovascular autoregulation during anesthesia was determined using intravenous phenylephrine to increase blood pressure. Results Sevoflurane (1.2 MAC) significantly decreased Vmca compared with the awake value at each level of end‐tidal carbon dioxide, whereas 1.2 MAC sevoflurane‐60% nitrous oxide did not exert significant influence. The Vmca in normocapnic patients decreased from 69 cm/s to 55 cm/s with 1.2 MAC sevoflurane and then increased to 70 cm/s when nitrous oxide was added. Sevoflurane (1.2 MAC) with and without 60% nitrous oxide had a negligible effect on cerebrovascular carbon dioxide reactivity. A phenylephrine‐induced increase of mean arterial pressure did not influence Vmca during anesthesia. Conclusions Sevoflurane (1.2 MAC) reduced Vmca compared with the awake condition, whereas the addition of nitrous oxide caused Vmca to increase toward the values obtained in the awake condition. The cerebrovascular carbon dioxide reactivity and autoregulation were well maintained during 1.2 MAC sevoflurane with and without 60% nitrous oxide.

Regulation of γ-glutamycysteine synthetase expression in response to oxidative stress

Glutathione (GSH) is synthesized by the activity of two ATP-requiring GSH synthesizing enzymes. Gammaglutamylcysteine synthetase (γ-GCS) is the rate limiting enzyme for the GSH synthesis. γ-GCS is a heterodimer of heavy, catalytic subunit and light, regulatory subunit and responsive to many stresses, such as heat shock, oxidative stress or cytokines. To know the regulation of the expression of γ-GCS gene, in the present study, we show evidences that γ-GCS heavy subunit is upregulated by oxidative stress by ionizing radiation and TNF-α mediated by nuclear factor-κB (NF-κB), and impairment of the expression of γ-GCS by TNF-α in diabetic condition. Furthermore we describe the importance of GSH in the regulation of NF-κB subunits.

Protective role of glutathione synthesis in response to oxidized low density lipoprotein in human vascular endothelial cells

Impairment of endothelial cells by oxidized low density lipoprotein (OxLDL) is believed to be the first step in atherogenesis. It is also believed that oxidative stress/antioxidant imbalance is involved in the cell damage by OxLDL. However, little is known about the interaction between OxLDL and antioxidants. In this study, we show that treatment of human vascular endothelial cells with OxLDL caused a gradual increase of glutathione (gamma-glutamylcysteinyl glycine, GSH) levels in 24 h. OxLDL increased the intracellular levels of reactive oxygen species (ROS) and stimulated the expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme for the GSH synthesis, the mitogen-activated protein kinase (MAPK) activity, and the AP-1-DNA binding activity. The luciferase activity of gamma-GCS promoter containing AP-1 site was activated by OxLDL. Collectively, OxLDL induces gamma-GCS expression mediated by AP-1 resulting in an increase of GSH levels. The MAPK activity stimulated by ROS may be involved in the activation of AP-1. The increase in GSH by OxLDL may afford cellular protection against OxLDL-induced oxidative stress.

The effects of propofol with and without ketamine on human cerebral blood flow velocity and CO2 response

UNLABELLED The combination of propofol and ketamine has been used for total IV anesthesia. This study was designed to clarify the effects of propofol-ketamine anesthesia on cerebral circulation by using transcranial Doppler ultrasonography. In Study 1, we examined the time course of time-mean middle cerebral artery blood flow velocity (Vmca) after ketamine (n = 10) or saline (n = 6) administration during propofol anesthesia. In Study 2, CO(2) responses were measured under the following conditions: awake (Group C, n = 7), propofol anesthesia (Group D, n = 7), and propofol-ketamine anesthesia (Group E, n = 8). Ketamine administration during propofol anesthesia administration did not affect Vmca, mean arterial pressure, or heart rate. Vmca under normocapnia in Groups D and E were 36 +/- 3 and 37 +/- 3 cm/s (mean +/- SE), respectively. The values were significantly lower than that of Group C (70 +/- 3 cm/s). The CO(2) response slopes of Groups D and E were significantly lower than that of Group C, although there was no significant difference between Groups D and E. These results suggest that ketamine does not influence Vmca or the cerebrovascular CO(2) response during propofol anesthesia administration, although the sample size in each group was small. IMPLICATIONS Our study suggests that ketamine does not influence middle cerebral artery blood flow velocity or the cerebrovascular CO(2) response assessed by transcranial Doppler ultrasonography during propofol anesthesia administration in patients without neurological complications.

Direct protective effects of dexmedetomidine against myocardial ischemia-reperfusion injury in anesthetized pigs.

ABSTRACT Systemic administration of &agr;2-adrenergic agonists has been shown to protect ischemic myocardium, but the direct effects on ischemia-reperfused myocardium have not yet been clarified. This study was carried out to determine the effects of intracoronary dexmedetomidine (DEX) on the myocardial ischemia-reperfusion injury in anesthetized pigs. In open-chest pigs, the left anterior descending coronary artery was perfused through an extracorporeal circuit from the carotid artery. They received intracoronary infusion of DEX at a rate of 1 ng · mL−1 (group LD, n = 9), 10 ng · mL−1 (group MD, n = 9), or 100 ng · mL−1 (group HD, n = 9) of coronary blood flow or vehicle (group C, n = 12) for 30 min before ischemia. Myocardial stunning was produced by 12-min ischemia of the perfused area of left anterior descending coronary artery and 90-min reperfusion. The effect on reperfusion-induced arrhythmias was evaluated using the incidence of ventricular tachycardia or fibrillation after reperfusion. Regional myocardial contractility was evaluated with segment shortening (%SS). Dexmedetomidine significantly reduced the incidence of reperfusion-induced ventricular arrhythmias. Dexmedetomidine significantly improved the recovery of percentage segment shortening at 90 min after reperfusion (32.6% ± 3.1% in group C, 58.2% ± 2.1% in group LD, 61.1% ± 1.8% in group MD, and 72.0% ± 2.0% in group HD). Dexmedetomidine suppressed the increase in plasma norepinephrine concentration after reperfusion. The results indicate that DEX would exert the protective effect against ischemia-reperfusion injury by the direct action on the myocardium, which is not mediated through the central nervous system. ABBREVIATIONS VT—ventricular tachycardia VF—ventricular fibrillation NE—norepinephrine DEX—dexmedetomidine PaCO2—arterial carbon dioxide tension PaO2—arterial oxygen tension LAD—left anterior descending coronary artery CBF—coronary blood flow LV—left ventricular LVP—left ventricular pressure LVdP/dtmax—peak rate of increase in left ventricular pressure %SS—percentage of segment shortening RMBF—regional myocardial blood flow endo/epi—subendocardial to subepicardial

The effects of dexmedetomidine on left ventricular function during hypoxia and reoxygenation in isolated rat hearts.

Hypoxia resulting from apnea in patients with sleep apnea is an important factor in heart disease. We designed the present study to determine whether dexmedetomidine (DEX) has a direct protective effect against hypoxia-reoxygenation-induced left ventricular dysfunction without systemic hemodynamic and humoral effects. Isolated rat hearts were exposed to 60-min hypoxia followed by 30-min reoxygenation with 0, 10, or 100 nM DEX prehypoxia administration (n = 7 each group). In a second experiment (n = 7), 100 nM DEX was administered posthypoxia. In a third experiment (n = 7 each group), an &agr; 2 antagonist, yohimbine was given with and without 100 nM DEX prehypoxia administration. DEX prehypoxia, but not posthypoxia, administration significantly improved the recovery of left ventricular developed pressure after reoxygenation (0, 10, 100 nM DEX prehypoxia or 100 nM DEX posthypoxia values were 53 ± 6, 64 ± 9, 78 ± 13, or 62 ± 12 mm Hg [mean ± sd]) and reversed by yohimbine, 58 ± 8 mm Hg, respectively. We conclude that DEX exerts the direct protective effect on the left ventricular dysfunction caused by hypoxia-reoxygenation through mainly &agr; 2-adrenergic stimulation before and during the hypoxic period.

Cardioprotection induced by olprinone, a phosphodiesterase III inhibitor, involves phosphatidylinositol-3-OH kinase-Akt and a mitochondrial permeability transition pore during early reperfusion

PurposeIschemic preconditioning is mediated by the activation of phosphatidylinositol-3-OH kinase-Akt (PI3K-Akt) and by the inhibition of the opening of a mitochondrial permeability transition pore (mPTP) during early reperfusion. Preischemic administration of the phosphodiesterase type III inhibitor olprinone protects the myocardium against infarction, but its mechanism has not been fully clarified. We hypothesized that this olprinone-induced cardioprotective effect was mediated by the activation of PI3K-Akt and by the inhibition of mPTP during early reperfusion.MethodsPentobarbital-anesthetized rats (n = 42) subjected to 30-min coronary occlusion followed by 2-h reperfusion, received olprinone (20 µg·kg−1) or saline (control) in the preischemic phase in the presence or absence of the PI3K-Akt inhibitor wortmannin (0.6 mg·kg−1) or the mPTP opener atractyloside (5 mg·kg−1) before 5 min of reperfusion. The myocardial infarct size was expressed as a percentage of the area at risk. All values were expressed as means ± SD. Statistical comparisons within groups were made using repeated-measures analysis of variance (ANOVA), followed by a paired t-test, and comparisons among groups were analyzed using a two-way ANOVA, followed by the Tukey-Kramer test.ResultsMean arterial pressure and heart rate showed no significant differences within or among groups. The preischemic administration of olprinone significantly reduced the infarct size (12 ± 4%) as compared with that in the control group (43 ± 4%). Wortmannin or atractyloside abolished the protective effect of olprinone (42 ± 11% or 41 ± 10%).ConclusionThe olprinone-induced cardioprotective effect could be exerted via the activation of PI3K-Akt and the inhibition of mPTP during early reperfusion.

Nicardipine normalizes elevated levels of antioxidant activity in response to xanthine oxidase-induced oxidative stress in hypertensive rat heart

It has been reported that the production of oxygen radicals mediated by xanthine oxidase (XO) is stimulated in hypertensive cardiovascular endothelium, suggesting involvement of oxidative stress in pathogenesis of hypertension. In this study we estimated the effect of nicardipine, a calcium blocker, on the oxidative stress and antioxidant activities in left ventricles from spontaneously hypertensive rat (SHR) and stroke-prone SHR (SHRSP). The activity of XO increased 3.5-fold in SHR and 6.2-fold in SHRSP compared to that in normal controls (WKY). Interestingly, the levels of glutathione (GSH) and the activity of its synthesizing enzyme (gamma-glutamylcysteine synthetase, gamma-GCS) elevated concomitantly in SHR and SHRSP: the level of GSH increased 1.2-fold in SHR and 1.3-fold in SHRSP. The activity of gamma-GCS was elevated 1.5-fold in SHR and 2.4-fold in SHRSP, accompanying an increase in the expression of its mRNA. Treatment of these rats with nicardipine, for 4 weeks improved blood pressure, from 176 +/- 10 to 140 +/- 8 mmHg in SHR, and from 201 +/- 11 to 167 +/- 5 mmHg in SHRSP, respectively, and decreased wet weight of heart, levels of GSH, and the activities of XO and gamma-GCS. Nicardipine reduced the expression of gamma-GCS mRNA. Collectively, these results suggest that reactive oxygen species produced by XO in hypertensive rat heart cause induction of the expression of gamma-GCS and nicardipine plays a role in reducing the oxidative stress in hypertensive heart.