Chelerythrine rapidly induces apoptosis through generation of reactive oxygen species in cardiac myocytes

The role of protein kinase C (PKC) inhibition in cardiac myocyte apoptosis has not been well understood. We investigated the mechanism, by which chelerythrine, a commonly used PKC inhibitor, induces potent myocyte death. Chelerythrine (6-30 microm) rapidly induced pyknosis, shrinkage and subsequent cell death in cardiac myocytes. Chelerythrine-induced myocyte death was accompanied by nuclear fragmentation and activation of caspase-3 and -9, while it was prevented by XIAP, suggesting that the cell death is due to apoptosis. Higher concentrations of chelerythrine caused necrotic cell death where neither cell shrinkage nor caspase activation was observed. Intravenous injection of chelerythrine (5 mg/kg) also increased apoptosis in adult rat hearts in vivo. Downregulation of the phorbol 12-myristate 13-acetate (PMA)-sensitive PKC failed to affect chelerythrine-induced apoptosis, while anti-oxidants, including N-acetyl-L-cysteine (NAC) and glutathione, inhibited it, suggesting that generation of reactive oxygen species (ROS) rather than inhibition of PMA-sensitive PKC mediates chelerythrine-induced cardiac myocyte apoptosis. Chelerythrine caused cytochrome c release from mitochondria, which was significantly inhibited in the presence of NAC, suggesting that ROS mediates chelerythrine-induced cytochrome c release. Partial inhibition of cytochrome c release by Bcl-X(L) significantly reduced chelerythrine-induced apoptosis. These results suggest that chelerythrine rapidly induces cardiac myocyte apoptosis and that production of ROS, possibly H(2)O(2), and subsequent cytochrome c release from mitochondria play an important role in mediating chelerythrine-induced rapid cardiac myocyte apoptosis.     

Aldosterone stimulates matrix metalloproteinases and reactive oxygen species in adult rat ventricular cardiomyocytes

Matrix metalloproteinases (MMPs), aldosterone, and reactive oxygen species (ROS) are implicated in myocardial remodeling. Although ROS, cytokines, and neurohormones regulate MMP in cardiac fibroblasts, it is unknown whether aldosterone regulates MMP in cardiomyocytes. Therefore, we tested the hypothesis that aldosterone regulates MMP in cultured adult rat ventricular myocytes (ARVMs). ARVMs were treated with aldosterone for 24 hours, and MMP-2 and MMP-9 activities were measured by zymography. Aldosterone (50 nmol/L) increased MMP-2 (43+/-5%) and MMP-9 (55+/-15%; P<0.001 for both) activities. Pretreatment with spironolactone (100 nmol/L) abolished the aldosterone-induced increase in MMP activities. Aldosterone (50 nmol/L; 30 minutes) increased mitogen/extracellular signal-regulated kinase (MEK) (31+/-3%) and extracellular signal-regulated kinase 1/2 (ERK1/2; 41+/-7%; P<0.001 for both) phosphorylation. U0126 (10 micromol/L), an MEK1/2 inhibitor, abolished the aldosterone-induced increase in MMP activities. Aldosterone increased intracellular ROS as assessed by dichlorofluorescein diacetate (27+/-4%; P<0.05). This increase was inhibited by apocynin, an NADPH oxidase inhibitor. Apocynin likewise inhibited aldosterone-induced ERK1/2 phosphorylation and the increase in MMP activities. Furthermore, the antioxidants MnTMPyP and N-acetylcysteine inhibited the aldosterone-induced increase in ERK1/2 phosphorylation and MMP activities, respectively. Protein kinase C (PKC) is implicated in the nongenomic effects of aldosterone. To test the role of PKC, ARVMs were pretreated with chelerythrine, a PKC inhibitor. Chelerythrine prevented the aldosterone-induced increase in ERK1/2 phosphorylation and MMP activities. Thus, aldosterone induces MMP activity in ARVM via activation of the mineralocorticoid receptor, PKC, and ROS-dependent activation of the MEK/ERK pathway. NADPH oxidase is a likely source of ROS in this system.     

Metabolism of trinitrobenzene sulfonic acid by the rat colon produces reactive oxygen species.

Recent studies have shown that intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in ethanol or intramural injection of TNBS in saline produces an acute and possibly chronic colitis in rats. It has been assumed that interstitial TNBS initiates the inflammatory response via macrophage-mediated recognition and degradation of TNBS-modified mucosal cells and proteins. However, it is known that certain flavoproteins and/or reductants interact with compounds containing the nitro functional group to generate pro-inflammatory, nitrogen-centered free radicals and reactive oxygen metabolites. The objective of this study was to assess the ability of the rat colon, using either colon homogenates, isolated colonocytes, or intestinal interstitial fluid, to produce reactive oxygen species via enzymatic and/or nonenzymatic metabolism of TNBS. It was found that the addition of TNBS (1 mmol/L) to the 10,000 x g supernatant of rat colon homogenates increased the rate of superoxide production from normally undetectable levels to 2.6 +/- 0.23 nmol.min-1.mg protein-1. Addition of nicotinamide adenine dinucleotide, reduced form (NADH; 1 mmol/L) to colon homogenates containing TNBS significantly enhanced superoxide production to 10.4 +/- 0.9 nmol.min-1.mg-1. Similarly, addition of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH; 1 mmol/L) to colon extracts containing TNBS produced an even further increase in the rate of superoxide formation to 25.2 +/- 1.1 nmol.min-1.mg-1. Addition of NADH or NADPH to the colon homogenate in the absence of TNBS produced no detectable superoxide formation, suggesting that TNBS was required for the enhanced oxidative metabolism. In a separate series of experiments, it was found that isolated colonocytes produced small but significant amounts of superoxide (3.15 +/- 0.6 nmol/2 x 10(6) cells) that were significantly increased in the presence of ethanol to 6.55 +/- 1.14 nmol/2 x 10(6) cells. Using purified preparations of two flavoproteins found in the rat colon, it was shown that the addition of TNBS (1 mmol/L) to purified NADH dehydrogenase or glutathione reductase increased the rate of superoxide formation by these enzymes from normally undetectable levels to 1.6 nmol/min and 1.2 nmol/min, respectively. In addition, it was found that intestinal interstitial fluid (lymph) initiated redox cycling of TNBS such that 28.1 +/- 1.6 nmol of oxygen was consumed per minute per milliliter of lymph. This increase in oxygen consumption was inhibited by the addition of superoxide dismutase and catalase. One possible metabolite involved in both mucosal and lymph-mediated metabolism of TNBS is ascorbic acid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Motexafin gadolinium generates reactive oxygen species and induces apoptosis in sensitive and highly resistant multiple myeloma cells

Motexafin gadolinium (MGd), an expanded porphyrin, is a tumor-selective redox-mediator that reacts with many intracellular reducing metabolites. Because redox mechanisms mediate apoptosis in multiple myeloma, we hypothesized that disruption of redox balance by MGd would result in cellular cytotoxicity in myeloma. We examined the effects of MGd on cellular cytotoxicity, apoptosis, reactive oxygen species (ROS) production, and intracellular drug uptake in dexamethasone-sensitive (C2E3), dexamethasone-resistant (1-310 and 1-414) chemotherapy-sensitive (8226-RPMI) and highly chemotherapy-resistant (DOX-10V) myeloma cells. We found complete inhibition of proliferation and cytotoxicity in each sensitive and resistant cell line with 24-hour exposure to clinically relevant concentrations of 50 muM MGd and 50 to 100 microM ascorbate, which was required for the effect. The mechanism of cytotoxicity was related to induction of apoptosis as demonstrated by alteration in mitochondrial membrane potential and elevated annexin V expression. This was accompanied by depletion of intracellular glutathione and increased ROS production. Moreover, catalase substantially abrogated MGd-induced cell death. Using fluorescence microscopy and flow cytometry, we found intracellular uptake of MGd and intracellular ROS production. MGd also induced apoptosis in fresh malignant cells from patients with multiple myeloma. These studies provide a rationale for clinical investigation of this novel redox-mediating agent in patients with multiple myeloma and related disorders.     

Oxidized low density lipoprotein induces apoptosis via generation of reactive oxygen species in vascular smooth muscle cells

OBJECTIVE: Death of vascular smooth muscle cell (VSMC) induced by oxidized LDL (oxLDL) can occur by both necrosis and apoptosis which may contribute to plaque instability and rupture. Reactive oxygen species (ROS) induces apoptosis in VSMC and is involved in oxLDL action, we tested the hypothesis here that a coupling exists between ROS generation and apoptosis of oxLDL-treated VSMC. METHODS: Cultured VSMC from rat aorta were treated with oxLDL, apoptosis and necrosis were distinguished by using FITC-annexin V label and propidium iodide stain, analyzed by flow cytometry. ROS generation of VSMCs was detected by the fluorescence intensity of DCF. Apoptosis was also determined by cleavage of procaspase-3. RESULTS: OxLDL induced apoptosis (3 h) in a dose-dependent manner and reached maximum (with near-basal necrosis) at a concentration of 300 microg/ml. At this and lower (100 microg/ml) concentration, oxLDL, but not native LDL, stimulated ROS production rapidly (< or =5 min) and ROS level remained elevated for at least 45 min. Catalase and deferoxamine reduced both oxLDL-induced apoptosis and ROS generation. Superoxide dismutase and benzoic acid neither reduced the oxLDL-induced ROS generation nor inhibited apoptosis. Since oxLDL-induced ROS generation were inhibited by nordihydroguaiaretic acid and rotenone, lipoxygenase and mitochondrial pathways could be involved. In addition, catalase, deferoxamine, and N-acetylcysteine inhibited oxLDL-induced cleavage of procaspase-3 as well. CONCLUSIONS: ROS generation and apoptosis are tightly coupled in oxLDL-treated VSMCs. Antioxidants that reduced ROS level inhibited apoptosis, those that did not reduce ROS level were ineffective. Both mitochondrial and lipoxygenase activities may be involved.     

Lysine triggers apoptosis through a NADPH oxidase-dependent mechanism in human renal tubular cells

Progressive chronic kidney disease (CKD) is common in lysinuric protein intolerance (LPI), a primary inherited aminoaciduria characterized by massive Lysine excretion in urine. However, by which mechanisms Lysine may cause kidney damage to tubule cells is still not understood. This study determined whether Lysine overloading of human proximal tubular cells (HK-2) in culture enhances apoptotic cell loss and its associated mechanisms. Overloading HK-2 with Lysine levels reproducing those observed in urine of patients affected by LPI (10?mM) increased apoptosis (+30%; p?相似文献   

Long-lived Indy induces reduced mitochondrial reactive oxygen species production and oxidative damage

Decreased Indy activity extends lifespan in D. melanogaster without significant reduction in fecundity, metabolic rate, or locomotion. To understand the underlying mechanisms leading to lifespan extension in this mutant strain, we compared the genome-wide gene expression changes in the head and thorax of adult Indy mutant with control flies over the course of their lifespan. A signature enrichment analysis of metabolic and signaling pathways revealed that expression levels of genes in the oxidative phosphorylation pathway are significantly lower in Indy starting at day 20. We confirmed experimentally that complexes I and III of the electron transport chain have lower enzyme activity in Indy long-lived flies by Day 20 and predicted that reactive oxygen species (ROS) production in mitochondria could be reduced. Consistently, we found that both ROS production and protein damage are reduced in Indy with respect to control. However, we did not detect significant differences in total ATP, a phenotype that could be explained by our finding of a higher mitochondrial density in Indy mutants. Thus, one potential mechanism by which Indy mutants extend life span could be through an alteration in mitochondrial physiology leading to an increased efficiency in the ATP/ROS ratio.     

雌激素与活性氧

大量证据表明活性氧(ROS)对心脑血管疾病的发生和发展有重要影响。它可以直接氧化膜脂质和DNA,造成细胞氧化损伤和异常;同时还作为信使参与细胞各种生理和病理活动,例如调节基因表达和信号转导。近几年,雌激素对心脑血管疾病的保护作用越来越受到人们的重视,它主要参与细胞内的氧化应激反应,但在某些方面也存在争议。     

Angiotensin II induces afterdepolarizations via reactive oxygen species and calmodulin kinase II signaling

Renin-angiotensin system inhibitors significantly reduce the incidence of arrhythmias. However, the underlying mechanism(s) is not well understood. We aim to test the hypothesis that angiotensin II (Ang II) induces early afterdepolarizations (EADs) and triggered activities (TAs) via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-ROS-calmodulin kinase II (CaMKII) pathway. ROS production was analyzed in the isolated rabbit myocytes loaded with ROS dye. Ang II (1-2 μM) increased ROS fluorescence in myocytes, which was abolished by Ang II type 1 receptor blocker losartan, NADPH oxidase inhibitor apocynin, and antioxidant MnTMPyP, respectively. Action potentials were recorded using the perforated patch-clamp technique. EADs emerged in 27 out of 41 (66%) cells at 15.8 ± 1.6 min after Ang II (1-2 μM) perfusion. Ang II-induced EADs were eliminated by losartan, apocynin, or trolox. The CaMKII inhibitor KN-93 (n = 6) and inhibitory peptide (AIP) (n = 4) also suppressed Ang II-induced EADs, whereas the inactive analogue KN-92 did not. Nifedipine, a blocker of L-type Ca current (I_Ca²⁺_,L), or ranolazine, an inhibitor of late Na current (I_Na⁺), abolished Ang II-induced EADs. The effects of Ang II on major membrane currents were evaluated using voltage clamp. While Ang II at same concentrations had no significant effect on total outward K⁺ current, it enhanced I_Ca.L and late I_Na, which were attenuated by losartan, apocynin, trolox, or KN-93. We conclude that Ang II induces EADs via intracellular ROS production through NADPH oxidase, activation of CaMKII, and enhancement of I_Ca,L and late I_Na. These results provide evidence supporting a link between renin-angiotensin system and cardiac arrhythmias.     

High glucose induces myocardial cell injury through increasing reactive oxygen species production

Objective: To study the injury effect and molecular mechanism of high glucose on myocardial cells. Methods: Myocardial cells H9 c2 were cultured and divided into the control group treated with DMEM containing 5.5 mmol/L glucose, the high glucose group treated with DMEM containing 35 mmol/L glucose, and the N-acetylcysteine(NAC) group pre-treated with 1000μmol/L NAC and treated with DMEM containing 1000 μmol/L NAC and 35 mmol/L glucose.The production of ROS and the expression of mitochondria pathway apoptosis molecules in cells as well as the contents of collagen and collagen metabolism molecules were measured.Results: After 8 h, 16 h and 24 h of treatment, ROS RFU as well as Bax, CytC, Caspase-3 and Caspase-9 protein expression in cells and Col-I, Col-Ⅲ, PINP and PⅢNP protein levels in culture medium of high glucose group were higher than those of control group, Bcl-2 protein expression were lower than those of control group, but CTX-Ⅰ protein levels in culture medium were not significantly different from those of control group; after 24 h of treatment, Bax, CytC,Caspase-3 and Caspase-9 protein expression in cells as well as Col-Ⅰ, Col-Ⅲ, PINP and PIIINP protein levels in culture medium of NAC group were lower than those of high glucose group whereas Bcl-2 protein expression was higher than that of high glucose group. Conclusions:High glucose can induce myocardial cell apoptosis, increase collagen synthesis and accelerate interstitial fibrosis by increasing the production of reactive oxygen species.