Biomedical subjects

Lingyun Wu

Publications and source records attributed to Lingyun Wu.

At least 19 recordsLinked to original sources

PubMed2006

Effects of hydrogen sulfide on homocysteine-induced oxidative stress in vascular smooth muscle cells.

Hydrogen sulfide (H(2)S) is an important gasotransmitter that generated in mammalian cells from l-cysteine metabolism. Little is known about its protective role in oxidative stress. In the present study, we investigated whether H(2)S could affect homocysteine (HCY)-induced cytotoxicity and oxidative stress in vascular smooth muscle cells. Cultured A-10 cells were exposed to HCY treatment in the presence or absence of NaHS (donor of H(2)S). HCY induced cytotoxicity, increased levels of H(2)O(2), ONOO(-), and O2- in a time- and concentration-dependent manner. Low levels of NaHS (30 or 50microM) protected A-10 cells from cytotoxicity, decreased the production of H(2)O(2), ONOO(-), and O2- in the presence of HCY. Furthermore, NaHS enhanced inhibitory effects of NAC, GSH, DPI, SOD, L-NAME, or vitamin C on oxidized DCF or O2- formation induced by HCY. In conclusion, our findings provide the first evidence that low levels of H(2)S decrease reactive oxygen species and improve cell viability and by doing so limit cellular damage induced by HCY.

Acetylcysteine↗

PubMed2006

Sustained normalization of high blood pressure in spontaneously hypertensive rats by implanted hemin pump.

Treatment of established hypertension, especially for prolonged control of this pathogenic process, represents a great challenge. To upregulate the expression of heme oxygenase (HO) to lower blood pressure (BP) of spontaneously hypertensive rats (SHRs), we administered hemin to 12-week-old adult SHRs through subcutaneously implanted osmotic minipumps for 3 consecutive weeks (the hemin protocol). Systolic BP of SHRs was normalized 123+/-2 mm Hg (n=20; P<0.001) and this normalization maintained for 9 months after the removal of hemin pumps. At the end of the hemin protocol, HO-1 expression, HO activity, soluble guanylyl cyclase expression, and cGMP content were all increased, but phosphodiesterase-5 expression was downregulated in the mesenteric arteries. The hemin protocol also reversed SHR-featured arterial eutrophic inward remodeling and decreased expression levels of vascular endothelial growth factor. These changes lasted 9 months after the hemin protocol. Our study, thus, formulates a novel hemin protocol that will not only normalize BP in SHRs with established hypertension but, more importantly, will also provide long-lasting antihypertension protection. Sustained upregulation of HO-1-linked signaling pathways and reversal of vascular remodeling in peripheral blood vessels mediate likely the antihypertensive effect of the hemin protocol.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2006

Fructose-induced peroxynitrite production is mediated by methylglyoxal in vascular smooth muscle cells.

Methylglyoxal (MG), a highly reactive molecule, has been implicated in the development of insulin resistance. We investigated whether fructose, a precursor of MG, induced ONOO(-) generation and whether this process was mediated via endogenously increased MG formation. Fructose significantly increased MG generation in vascular smooth muscle cells (VSMCs) in a concentration and time dependent manner. The intracellular production of MG was increased by 153+/-23% or 259+/-28% after cells were treated 6 h with fructose (15 mM or 30 mM), compared with production from untreated cells (p<0.01, n=4 for each group). A significant increase in the production of ONOO(-), NO, and O(2)(*-), was found in the cells treated with fructose (15 mM) or MG (10 microM). Fructose- or MG-induced ONOO(-) generation was significantly inhibited by MG scavengers, including reduced glutathione or N-acetyl-l-cysteine, and by O(2)(*-) or NO inhibitors, such as diphenylene iodonium, superoxide dismutase or N-nitro-l-arginine methyl ester. Moreover, an enhanced iNOS expression was also observed in the cells treated directly with MG which was significantly inhibited when co-application with N-acetyl-l-cysteine. Our results demonstrated that fructose is capable of inducing a significant increase in ONOO(-) production, which is mediated by an enhanced formation of endogenous MG in VSMCs.

Acetylcysteine↗

PubMed2006

Structural and functional changes in human insulin induced by methylglyoxal.

Elevated methylglyoxal (MG) levels have been reported in insulin-resistance syndrome. The present study investigated whether MG, a highly reactive metabolite of glucose, induced structural and functional changes of insulin. Incubation of human insulin with MG in vitro yielded MG-insulin adducts, as evidenced by additional peaks observed on mass spectrometric (MS) analysis of the incubates. Tandem MS analysis of insulin B-chain adducts confirmed attachment of MG at an arginine residue. [3H]-2-deoxyglucose uptake by 3T3-L1 adipocytes was significantly and concentration-dependently decreased after the treatment with MG-insulin adducts, in comparison with the effect of native insulin at the same concentrations. A significant decrease of glucose uptake induced by MG-insulin adducts was also observed in L8 skeletal muscle cells. MG alone had no effect on glucose uptake or the transcriptional expression of insulin receptor. Unlike native insulin, MG-insulin adducts did not inhibit insulin release from pancreatic beta-cells. The degradation of MG-insulin through liver cells was also decreased. In conclusion, MG modifies insulin by attaching to internal arginine residue in beta-chain of insulin. The formation of this MG-insulin adduct decreases insulin-mediated glucose uptake, impairs autocrine control of insulin secretion, and decreases insulin clearance. These structural and functional abnormalities of insulin molecule may contribute to the pathogenesis of insulin resistance.

3T3 Cells↗

PubMed2006

Pro-apoptotic effect of endogenous H2S on human aorta smooth muscle cells.

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). The CSE/H2S system has been shown to play an important role in regulating cellular functions in different systems. In the present study, we overexpressed CSE in human aorta smooth muscle cells (HASMCs) using a recombinant defective adenovirus containing CSE gene (Ad-CSE). Infection of HASMCs with Ad-CSE resulted in a significant increase in the expression of CSE protein and H2S production. Ad-CSE transfection inhibited cell growth and stimulated apoptosis, as evidenced by cell viability assay, Hoechst 33258 staining, TUNEL, and caspase 3 activation. CSE-mediated apoptosis was associated with an increased ERK and p38 MAPK activation, up-regulation of p21(Cip/WAK-1), and down-regulation of cyclin D1 expression. After inhibiting endogenous background CSE gene expression, direct administration of H2S at 100 microM induced apoptosis of HASMCs. The other two endproducts of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, failed to do so. These results demonstrate that overexpression of CSE stimulates SMC apoptosis due to an increased endogenous production of H2S. Adenovirus-mediated transfer of CSE gene may provide a novel therapeutic approach in treating vascular diseases linked to abnormal cellular proliferation and vascular remodeling.

Aorta↗

PubMed2006

Modulation of cardiac and aortic peroxisome proliferator-activated receptor-gamma expression by oxidative stress in chronically glucose-fed rats.

BACKGROUND: The aim of the present study was to examine the chronic effects of alpha-lipoic acid on proliferator peroxisome activated receptors-gamma (PPAR-gamma) and PPAR-alpha expressions in cardiovascular tissues of chronically hypertensive insulinoresistant rats. We have also evaluated the chronic effects of high levels of insulin, glucose, or both on superoxide anion (O(2)(-)) production in aortic smooth muscle cells (SMCs) in the presence or in absence of pioglitazone, a PPAR-gamma agonist. METHODS: The PPAR-gamma and PPAR-alpha expressions were measured by Western blot. The oxidative stress was evaluated by measuring the O(2)(-) production using the lucigenin method. RESULTS: Increases in blood pressure, in aortic O(2)(-) production, in glucose or insulin levels, and in insulin resistance, as well as the decrease in PPAR-gamma protein levels in aorta and heart tissues were prevented or attenuated in glucose-treated rats fed with lipoic acid. Chronic treatment with pioglitazone prevented the marked increase in O(2)(-) production in cultured SMCs chronically treated with high insulin combined or not with high glucose levels. CONCLUSIONS: The combined therapy with the antioxidant alpha-lipoic acid restored PPAR-gamma levels in cardiovascular tissues and attenuated or prevented the development of insulin resistance and hypertension in chronically glucose-fed rats. Moreover, the finding that pioglitazone was also efficient in preventing the increase in oxidative stress in SMCs treated with high insulin combined with high glucose concentrations supports the hypothesis that the activation of PPAR-gamma activity can counteract the oxidative stress that seems to be implicated in the development of hypertension and insulin resistance.

Animals↗

PubMed2006

Altered expression of BK channel beta1 subunit in vascular tissues from spontaneously hypertensive rats.

Correlation of blood pressure (BP) with expression levels of large-conductance, voltage- and Ca2+-activated K+ (BK) channel beta1 subunit in vascular tissues from spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and Sprague-Dawley rats (SD) at different ages was investigated. Systolic BP and BK beta1 expression in mesenteric arteries at either mRNA or protein levels were not different among 4-week-old SHR, WKY, and SD. With hypertension developed at 7 weeks and reached plateau at 12 weeks, expression levels of BK beta1 mRNA in mesenteric arteries and aortae from SHR during this period of time were significantly higher than in age-matched normotensive WKY. The BK beta1 protein expression was significantly higher in mesenteric arteries from 12-week-old but not 7-week-old SHR when compared with age-matched WKY and SD. The BK beta1 protein levels in aortae were not different among 7-week-old SHR, WKY, and SD but were significantly lower in 12-week-old WKY than in age-matched SHR and SD. Captopril treatment normalized BP of 12-week-old SHR. This treatment downregulated BK beta1 protein in mesenteric arteries but upregulated it in aortae. No significant difference in BK alpha subunit expression was detected in mesenteric arteries from three strains of rats as well as the captopril-treated SHR. It appears that expression patterns of BK beta1 in vascular tissues vary depending on tissue types, animal age, and animal strains. Expression of BK beta1 in mesenteric arteries is closely correlated with BP in SHR. Increased BK beta1 expression in mesenteric arteries may represent a compensatory reaction to limit the development of hypertension.

Animals↗

PubMed2006

Is methylglyoxal a causative factor for hypertension development?

Hypertension is a life-threatening disease that is associated with increased cardiovascular risks. Causes and mechanisms for hypertension development remain poorly understood. Methylglyoxal (MG), a highly reactive molecule, is a metabolite of sugar. Increased circulation and tissue levels of MG have been documented not only in diabetes but also in hypertension. Many recent studies also link MG-induced vascular damage to the pathogenic process of hypertension. As such, an etiological role of MG in hypertension development is proposed.

Animals↗

PubMed2005

Dietary approaches to positively influence fetal determinants of adult health.

An imbalance between production and scavenging of oxidants is a commonality found in factors that result in fetal determinants that negatively affect adult health. We reasoned that a dietary intervention that promotes oxidant scavenging through phase 2 protein induction would have positive effects on fetal programming of adult health. Previously, we demonstrated that a diet containing broccoli sprouts high in glucoraphanin (Grn), precursor of the phase 2 protein inducer sulforaphane, decreased oxidative stress and associated problems in male spontaneously hypertensive stroke-prone rats (SHRsp). We hypothesized that placing females on a Grn-containing (Grn+) diet would have similar positive effects and that the adult offspring of such females would have lower blood pressures and less tissue inflammation than offspring from mothers on control diet. We demonstrate that female SHRsp on a Grn+ diet had decreased oxidative stress and associated problems such as hypertension than females on control diet. The offspring of females on Grn+ diet also had lower blood pressures and less tissue inflammation in adulthood regardless of diet, with offspring placed on a Grn+ diet having the best health outcomes. We conclude that reducing oxidative stress in pregnant females has profound outcomes in the health of their adult offspring.

Aging↗

PubMed2005

Mild palladium-catalyzed selective monoarylation of nitriles.

Two new palladium-catalyzed procedures for the arylation of nitriles under less basic conditions than previously reported have been developed. The selective monoarylation of acetonitrile and primary nitriles has been achieved using alpha-silyl nitriles in the presence of ZnF2. This procedure is compatible with a variety of functional groups, including cyano, keto, nitro, and ester groups, on the aryl bromide. The arylation of secondary nitriles occurred in high yield by conducting reactions with zinc cyanoalkyl reagents. These reaction conditions tolerated base-sensitive functional groups, such as ketones and esters. The combination of these two methods, one with alpha-silyl nitriles and one with zinc cyanoalkyl reagents, provides a catalytic route to a variety of benzylic nitriles, which have not only biological significance but utility as synthetic intermediates. The utility of these new coupling reactions has been demonstrated by a synthesis of verapamil, a clinically used drug for the treatment of heart disease, by a three-step route from commercial materials that allows convenient variation of the aryl group.

Anti-Arrhythmia Agents↗

PubMed2005

Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms.

H2S is an important gasotransmitter, generated in mammalian cells from L-cysteine metabolism. As it stimulates K(ATP) channels in vascular smooth muscle cells, H2S may also function as an endogenous opener of K(ATP) channels in INS-1E cells, an insulin-secreting cell line. In the present study, K(ATP) channel currents in INS-1E cells were recorded using the whole-cell and single-channel recording configurations of the patch-clamp technique. K(ATP) channels in INS-1E cells have a single-channel conductance of 78 pS. These channels were activated by diazoxide and inhibited by gliclazide. ATP (3 mm) in the pipette solution inhibited K(ATP) channels in INS-1E cells. Significant amount of H2S was produced from INS-1E cells in which the expression of cystathinonie gamma-lyase (CSE) was confirmed. After INS-1E cells were transfected with CSE-targeted short interfering RNA (CSE-siRNA) or treated with DL-propargylglycine (PPG; 1-5 mm) to inhibit CSE, endogenous production of H2S was abolished. Increase in extracellular glucose concentration significantly decreased endogenous production of H2S in INS-1E cells, and increased insulin secretion. After transfection of INS-1E cells with adenovirus containing the CSE gene (Ad-CSE) to overexpress CSE, high glucose-stimulated insulin secretion was virtually abolished. Basal K(ATP) channel currents were significantly reduced after incubating INS-1E cells with a high glucose concentration (16 mm) or lowering endogenous H2S level by CSE-siRNA transfection. Under these conditions, exogenously applied H2S significantly increased whole-cell K(ATP) channel currents at concentrations equal to or lower than 100 microm. H2S (100 microm) markedly increased open probability by more than 2-fold of single K(ATP) channels (inside-out recording) in native INS-1E cells (n = 4, P < 0.05). Single-channel conductance and ATP sensitivity of K(ATP) channels were not changed by H2S. In conclusion, endogenous H2S production from INS-1E cells varies with in vivo conditions, which significantly affects insulin secretion from INS-1E cells. H2S stimulates K(ATP) channels in INS-1E cells, independent of activation of cytosolic second messengers, which may underlie H2S-inhibited insulin secretion from these cells. Interaction among H2S, glucose and the K(ATP) channel may constitute an important and novel mechanism for the fine control of insulin secretion from pancreatic beta-cells.

Adenosine Triphosphate↗

PubMed2005

Direct stimulation of K(ATP) channels by exogenous and endogenous hydrogen sulfide in vascular smooth muscle cells.

ATP-sensitive K+ (K(ATP)) channels in vascular smooth muscle cells (VSMC) are important targets for endogenous metabolic regulation and exogenous drug therapy. H2S, as a novel gasotransmitter, has been shown to relax rat aortic tissues via opening of K(ATP) channels. However, interaction of H2S, exogenous-applied or endogenous-produced, with K(ATP) channels in resistance artery VSMC has not been delineated. In the present study, using the whole-cell and single-channel patch-clamp technique, we demonstrated that exogenous H2S activated K(ATP) channels and hyperpolarized cell membrane in rat mesenteric artery VSMC. H2S enhanced the amplitude of whole-cell K(ATP) currents with an EC50 value of 116 +/- 8.3 microM and increased the open probability of single K(ATP) channels. H2S hyperpolarized membrane potentials by -12 mV in nystatin-perforated VSMC. Furthermore, inhibition of endogenous H2S production with D,L-propargylglycine (PPG) reduced whole-cell K(ATP) currents. PPG alone had no effect on unitary K(ATP) channel currents in cell-free membrane patches. In addition, effects of H2S on K(ATP) channels and membrane potentials were independent of cGMP-mediated phosphorylation. This study demonstrated modulation of K(ATP) channel activity by exogenous and endogenous H2S in resistance artery VSMC, thus helping elucidate cardiovascular functions of this endogenous gas.

Animals↗

PubMed2005

The effect of hydroxylamine on KATP channels in vascular smooth muscle and underlying mechanisms.

Hydroxylamine (HA) is a putative intermediate in the conversion of l-arginine to nitric oxide (NO). HA was reported to cause the relaxation of precontracted aorta strips; however, the ionic mechanisms of HA-induced vasorelaxation were not yet known. In the present study, the whole-cell patch-clamp technique was used to examine the effects of HA on ATP-sensitive K+ (K(ATP)) currents and membrane potentials in vascular smooth muscle cells from rat mesenteric arteries and underlying mechanisms. It was found that bath-applied HA reversibly enhanced K(ATP) currents in a concentration-dependent fashion with an EC50 of 54 +/- 3.4 microM and hyperpolarized the cell membrane from -48 +/- 5.2 to -65 +/- 7.5 mV (n = 6, p < 0.01). The increase in K(ATP) currents induced by HA was suppressed by superoxide dismutase (-380 +/- 45 to -160 +/- 20 pA, n = 4, p < 0.01) and N-acetyl-L-cysteine (-385 +/- 55 to -150 +/- 16 pA, n = 5, p < 0.01), indicating the involvement of different free radicals, including superoxide anion. Hypoxanthine/xanthine oxidase increased not only basal K(ATP) currents, but also HA-enhanced K(ATP) currents (from -355 +/- 40 to -480 +/- 62 pA, n = 6, p < 0.05). Sodium nitroprusside, a spontaneous NO donor, and a membrane-permeable cGMP analog (8-bromo-cGMP) were without effects on HA-enhanced K(ATP) currents or basal K(ATP) currents. Our results indicate that HA augmented K(ATP) channel activity and hyperpolarized cell membrane, possibly via increased free radical generation.

Adenosine Triphosphate↗

PubMed2005

Methylglyoxal-induced nitric oxide and peroxynitrite production in vascular smooth muscle cells.

Methylglyoxal (MG) is a metabolite of glucose. Our previous study demonstrated an elevated MG level with an increased oxidative stress in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats. Whether MG causes the generation of nitric oxide (NO) and superoxide anion (O2*-), leading to peroxynitrite (ONOO-) formation in VSMCs, was investigated in the present study. Cultured rat thoracic aortic SMCs (A-10) were treated with MG or other different agents. Oxidized DCF, reflecting H2O2 and ONOO- production, was significantly increased in a concentration- and time-dependent manner after the treatment of SMCs with MG (3-300 microM) for 45 min-18 h (n = 12). MG-increased oxidized DCF was effectively blocked by reduced glutathione or N-acetyl-l-cysteine, as well as L-NAME (p < 0.05, n = 12). Both O2*- scavenger SOD and NAD(P)H oxidase inhibitor DPI significantly decreased MG-induced oxidized DCF formation. MG significantly and concentration-dependently increased NO and O2*- generation in A-10 cells, which was significantly inhibited by L-NAME and SOD or DPI, respectively. In conclusion, MG induces significant generation of NO and O2*- in rat VSMCs, which in turn causes ONOO- formation. An elevated MG level and the consequential ROS/RNS generation would alter cellular signaling pathways, contributing to the development of different insulin resistance states such as diabetes or hypertension.

Animals↗

PubMed2005

Vascular methylglyoxal metabolism and the development of hypertension.

OBJECTIVES: The pathogenic process of diabetes mellitus is associated with increased methylglyoxal (MG). MG causes non-enzymic glycation of proteins to form irreversible advanced glycation endproducts (AGEs). However, the correlation between MG and essential hypertension is unknown. The aim of the present study was to investigate whether MG, MG-induced AGEs, and oxidative stress were increased in the aorta of spontaneously hypertensive rats (SHR) and whether an increased formation of MG and related AGEs was correlated with the development of high blood pressure in these rats. METHODS: High-performance liquid chromatography (HPLC) was used to determine MG and reduced glutathione levels in plasma and aorta. MG-induced AGEs, N(epsilon)-carboxyethyl-lysine (CEL) and N(epsilon)-carboxymethyl-lysine (CML), in aorta were determined using immunohistochemistry. Hydrogen peroxide and superoxide levels in aorta and glutathione peroxidase and reductase activities were also determined. RESULTS: Aortic and plasma MG levels were significantly elevated in SHR, but not in Wistar-Kyoto (WKY) rats, at 8, 13 and 20 weeks of age, in parallel with blood pressure increase. Immunohistochemistry revealed more intense staining for CML and CEL in aorta from SHR than those of WKY rats from 8 weeks onwards. Most of the staining was localized to endothelial cells. Superoxide and hydrogen peroxide levels were significantly elevated in aorta of SHR at 13 weeks, whereas reduced glutathione levels, glutathione peroxidase and glutathione reductase activities were significantly decreased compared to WKY rats. CONCLUSIONS: Increased aortic MG, AGE formation and oxidative stress were associated with blood pressure increase in SHR, which may cause endothelial dysfunction and altered vascular reactivity.

Age Factors↗

PubMed2005

Carbon monoxide: endogenous production, physiological functions, and pharmacological applications.

Over the last decade, studies have unraveled many aspects of endogenous production and physiological functions of carbon monoxide (CO). The majority of endogenous CO is produced in a reaction catalyzed by the enzyme heme oxygenase (HO). Inducible HO (HO-1) and constitutive HO (HO-2) are mostly recognized for their roles in the oxidation of heme and production of CO and biliverdin, whereas the biological function of the third HO isoform, HO-3, is still unclear. The tissue type-specific distribution of these HO isoforms is largely linked to the specific biological actions of CO on different systems. CO functions as a signaling molecule in the neuronal system, involving the regulation of neurotransmitters and neuropeptide release, learning and memory, and odor response adaptation and many other neuronal activities. The vasorelaxant property and cardiac protection effect of CO have been documented. A plethora of studies have also shown the importance of the roles of CO in the immune, respiratory, reproductive, gastrointestinal, kidney, and liver systems. Our understanding of the cellular and molecular mechanisms that regulate the production and mediate the physiological actions of CO has greatly advanced. Many diseases, including neurodegenerations, hypertension, heart failure, and inflammation, have been linked to the abnormality in CO metabolism and function. Enhancement of endogenous CO production and direct delivery of exogenous CO have found their applications in many health research fields and clinical settings. Future studies will further clarify the gasotransmitter role of CO, provide insight into the pathogenic mechanisms of many CO abnormality-related diseases, and pave the way for innovative preventive and therapeutic strategies based on the physiologic effects of CO.

Animals↗

PubMed2005

The pro-oxidant role of methylglyoxal in mesenteric artery smooth muscle cells.

Methylglyoxal (MG), a highly reactive metabolite of glucose, causes non-enzymatic glycation of proteins to form irreversible advanced glycation endproducts (AGEs). The present study investigated whether methylglyoxal induced oxidative stress and activated nuclear factor kappa B (NF-kappaB) in freshly isolated and cultured smooth muscle cells (SMCs) from rat mesenteric artery. The treatment of cells with MG (50 or 100 micromol/L) induced a significant increase in AGE formation and oxidation of DCF. MG-enhanced generation of AGEs and the oxidation of DCF was markedly inhibited by antioxidant n-acetylcysteine (NAC, 600 micromol/L). MG at a concentration of 100 micromol/L increased the heme-oxygenase-1 expression in these cells. Moreover, MG activated NF-kappaB p65, indicated by an increased immuno cytochemistry stain for NF-kappaB p65 located in the nucleus after the treatment of mesenteric artery SMCs with MG. MG-induced activation of NF-kappaB p65 was inhibited by NAC. In summary, MG significantly increases oxidative stress and activates NF-kappaB p65 in mesenteric artery SMCs. The pro-oxidant role of methylglyoxal may contribute to various pathological changes of SMCs from resistance arteries.

Animals↗

PubMed2004

Cystathionine gamma-lyase overexpression inhibits cell proliferation via a H2S-dependent modulation of ERK1/2 phosphorylation and p21Cip/WAK-1.

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway. CSE uses L-cysteine as a substrate to produce hydrogen sulfide (H2S). The CSE/H2S system has been shown to play an important role in regulating cellular functions in different systems. In the present study, we used CSE stably overexpressed HEK-293 cells to explore the effect of the CSE/H2S system on cell growth and proliferation. The overexpression of CSE resulted in increases in CSE mRNA levels, CSE proteins, and intracellular H2S production rates, as well as the inhibition of cell proliferation and DNA synthesis. These effects were accompanied by a sustained ERK activation and up-regulation of the cyclin-dependent kinase inhibitor p21Cip/WAK-1. Blocking the action of ERK with U0126 inhibited the induction of p21Cip/WAK-1, suggesting that ERK activation functions upstream of p21Cip/WAK-1 activation to initiate the CSE overexpression-induced cell growth inhibition. The antiproliferative effect of CSE is likely mediated by endogenously produced H2S because the H2S scavenger methemoglobin (10 microm) significantly decreased the H2S production rate and reversed the antiproliferative effect afforded by CSE. Exogenous H2S (100 microm) also inhibited cell proliferation. However, the other CSE-catalyzed products, ammonium and pyruvate, failed to inhibit cell proliferation. Methemoglobin also abolished the inhibitory effect of exogenous H2S on cell proliferation. Moreover, exogenous H2S induced a sustained ERK and p21Cip/WAK-1 activation. These findings support the hypothesis that endogenously produced H2S may play a fundamental role in cell proliferation and survival.

Blotting, Western↗