Nitric oxide synthesis is impaired in glutathione-depleted human umbilical vein endothelial cells

Ghigo, Dario; Alessio, P.; Foco, A; Bussolino, Federico; Costamagna, Costanzo; Heller, Regine; Garbarino, Giovanni; Pescarmona, Gianpiero; Bosìa, Amalia

doi:10.1152/ajpcell.1993.265.3.c728

Cited by 94 publications

(47 citation statements)

References 20 publications

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“…Determination of Endothelial Cell GSH Content-The 5,5Ј-dithiobis(2-nitrobenzoic acid) colorimetric method was used to measure glutathione (19), the predominant soluble thiol in EC (20). In brief, cells were cultured in 60-mm dishes, and post-confluent monolayers (Ϸ1 ϫ 10 6 cells/dish) were preincubated in culture medium at 37°C for 16 h in the presence of increasing concentrations of BSO or for 1 h in the presence of increasing concentrations of NAC.…”

Section: Methodsmentioning

confidence: 99%

“…3), suggesting that intracellular ROS generated by flow may mediate BMK1 activation in BAEC. To test this hypothesis, we utilized BSO (a selective inhibitor of ␥-glutamylcysteine synthetase) and NAC (a well known precursor of glutathione synthesis) to change the levels of GSH, the intracellular reduced form of glutathione, which is the most important cellular non-protein-reducing thiol antioxidant (20). Treatment of cells with varying concentrations of NAC (3-30 mM) did not attenuate flow-induced BMK1 activation (Fig.…”

Section: Shear Stress Stimulates Bmk1 In Baec: Time and Forcementioning

confidence: 99%

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Fluid Shear Stress Stimulates Big Mitogen-activated Protein Kinase 1 (BMK1) Activity in Endothelial Cells

Yan

Takahashi

Okuda

et al. 1999

Journal of Biological Chemistry

166

130

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Mitogen-activated protein (MAP) kinases including ERK1/2 and JNK play an important role in shear stressmediated gene expression in endothelial cells (EC).A new MAP kinase termed big MAP kinase 1 (BMK1/ERK5) has been shown to phosphorylate and activate the transcription factor MEF2C, which is highly expressed in EC. To determine the effects of shear stress on BMK1, bovine aortic EC were exposed to steady laminar flow (shear stress ‫؍‬ 12 dynes/cm 2 ). Flow activated BMK1 within 10 min with peak activation at 60 min (7.1 ؎ 0.6-fold) in a force-dependent manner. Flow was the most powerful activator of BMK1, significantly greater than H 2 O 2 or sorbitol. An important role for non-Src tyrosine kinases in flow-mediated BMK1 activation was demonstrated by inhibition with herbimycin A, but not with the Src inhibitor PP1 or overexpression of kinaseinactive c-Src. BMK1 activation was calcium-dependent as shown by inhibition with 1,2-bis(2-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid/acetoxymethyl ester or thapsigargin. As shown by specific inhibitors or activators, flow-mediated BMK1 activation was not regulated by the following: intracellular redox state; intracellular NO; protein kinase A, C, or G; calcium/ calmodulin-dependent kinase; phosphatidylinositol 3-kinase; or arachidonic acid metabolism. In summary, flow potently stimulates BMK1 in EC by a mechanism dependent on a tyrosine kinase(s) and calcium mobilization, but not on c-Src, redox state, or NO production.Fluid shear stress of blood, which modulates vessel structure and function, is one of the most important hemodynamic forces recognized and transduced by endothelial cells. Shear stress is also important in the pathogenesis of atherosclerosis because atherosclerotic plaques occur preferentially at arterial locations that experience turbulent flow or low shear stress. Increases in shear stress stimulate rapid secretion of vasoactive molecules, including nitric oxide, prostacyclin, and endothelin.Changes in shear stress also cause long-term alterations in vessel structure and function by regulating protein and gene expression. For example, shear stress stimulates expression of platelet-derived growth factor A-and B-chains, tissue plasminogen activator, endothelial nitric-oxide synthase, and endothelin (1, 2).The mechanisms by which endothelial cells sense mechanical stimuli and convert them to biochemical signals are not well characterized. Much experimental evidence indicates that the cellular response to shear stress is similar to the response to G protein-coupled receptors and growth factor receptors, which involves activation of a complex array of phosphorylation cascades. The mitogen-activated protein (MAP) 1 kinases respond to diverse stimuli, including physical stress, oxidative stress, and UV light, and play pivotal roles in a variety of cell functions. Thus, MAP kinases are excellent candidates to mediate mechanotransduction in endothelial cells.MAP kinases are serine/threonine protein kinases. Four subfamilies of MAP kinases have been identified, i...

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Section: Methodsmentioning

confidence: 99%

Section: Shear Stress Stimulates Bmk1 In Baec: Time and Forcementioning

confidence: 99%

Fluid Shear Stress Stimulates Big Mitogen-activated Protein Kinase 1 (BMK1) Activity in Endothelial Cells

Yan

Takahashi

Okuda

et al. 1999

Journal of Biological Chemistry

166

130

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“…Since NO formation is catalysed by NOS, which induces the conversion of l-arginine to l-citrulline and NO with a 1: 1 stoichiometry [26], we measured l-3 H-citrulline synthesis from l-3 H-arginine to evaluate NO production in VSMC. The method used here to measure NO in VSMC is a modification of a technique described previously [27] and has been used by our research group in human umbilical vein endothelial cells [24]. Briefly, cultured hVSMC, put in 35-mm dishes, were washed once with HEPES buffer (145 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l MgSO 4 , 10 mmol/l HEPES sodium salt, 10 mmol/l glucose and 1 mmol/l CaCl 2 , pH 7.4) and incubated in 1 ml of the same buffer at 37°C for 20 min.…”

Section: Methodsmentioning

confidence: 99%

“…The present study has been carried out to clarify, in hVSMC, the following points: i) the kinetics of the insulin-induced increase of cGMP and cAMP concentrations; ii) the ability of NO to enhance both cGMP and cAMP, evaluated by studying the effects of NO donors; iii) the NO involvement in the short-term influence exerted by insulin on cGMP and cAMP; iv) the ability of insulin to increase NO production in a few minutes; v) the presence of Ca 2+ -dependent NOS activity for which we measured NO production and cyclic nucleotide concentrations after hVSMC incubation with the Ca 2+ ionophore ionomycin, a powerful shortterm stimulus for the NO synthesis mediated by Ca 2+ -dependent NOS activity [24]; and, vi) the expression of mRNA for the endothelial isoform of cNOS (ecNOS), using the northern blot hybridization technique.…”

Section: : 831±839]mentioning

confidence: 99%

Human vascular smooth muscle cells express a constitutive nitric oxide synthase that insulin rapidly activates, thus increasing guanosine 3′ : 5′-cyclic monophosphate and adenosine 3′ : 5′-cyclic monophosphate concentrations

et al. 1999

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Aims/hypothesis. Insulin incubation of human vascular smooth muscle cells (hVSMC) for 120 min increases both guanosine 3¢ : 5¢-cyclic monophosphate (cGMP) and adenosine 3¢ : 5¢-cyclic monophosphate (cAMP) and these effects are blocked by inhibiting nitric oxide synthase (NOS). These data suggest that insulin activates a constitutive Ca 2+ -dependent NOS (cNOS), not described at yet in hVSMC. To test this hypothesis, we evaluated in hVSMC: i) the kinetics of the insulin-induced enhancement of the two cyclic nucleotides; ii) the ability of nitric oxide (NO) to increase both cyclic nucleotides; iii) NO involvement in the short-term influence of insulin on both cyclic nucleotides; iv) the ability of insulin to increase NO production in a few minutes; v) the presence of a cNOS activity; vi) the expression of mRNA for cNOS. Methods. In hVSMC incubated with insulin, NO donors and the Ca 2+ ionophore ionomycin, we measured cAMP and cGMP (RIA); in hVSMC incubated with insulin and ionomycin we measured NO, evaluated as l-( 3 H)-citrulline production from l-( 3 H)-arginine; by northern blot hybridization, we measured the expression of cNOS mRNA. Results. i) By incubating hVSMC with 2 nmol/l insulin for 0±240 min, we observed an increase of both cGMP and cAMP (ANOVA: p = 0.0001). Cyclic GMP rose from 0.74 ± 0.01 to 2.62 ± 0.10 pmol/10 6 cells at 30 min (p = 0.0001); cAMP rose from 0.9 ± 0.09 to 11.65 ± 0.74 pmol/10 6 cells at 15 min (p = 0.0001). ii) Sodium nitroprusside (100 mmol/l) and glyceryltrinitrate (100 mmol/l) increased both cGMP and cAMP (p = 0.0001). iii) The effects of insulin on cyclic nucleotides were blocked by NOS inhibition. iv) An increase of NO was observed by incubating hVSMC for 5 min with 2 nmol/l insulin (p = 0.0001). v) Ionomycin (1 mmol/l) enhanced NO production (p = 0.0001) and increased both cyclic nucleotides (p = 0.0001). vi) hVSMC expressed mRNA of cNOS. Conclusion/interpretation. Human VSMC express cNOS, which is rapidly activated by insulin with a consequent increase of both cGMP and cAMP, suggesting that insulin-induced vasodilation in vivo is not entirely endothelium-mediated. [Diabetologia (1999) 42: 831±839]

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“…Since NO synthesis is catalyzed by NOS, which induces the conversion of L-arginine to L-citrulline and NO with a 1:1 stoichiometry (46,50), the presence of NOS activity in cultured CSMC was checked as L- [ 3 H]-citrulline production from L-[ 3 H]-arginine. Briefly, cultured CSMC in 35 mm dishes were washed once with Hepes buffer (145 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l MgSO 4 , 10 mmol/l Hepes sodium salt, 10 mmol/l glucose and 1 mmol/l CaCl 2 , pH 7.4) and incubated in 1 ml of the same buffer at 37 8C for 20 min.…”

Section: Evaluation Of Nos Activitymentioning

confidence: 99%

Insulin influences the nitric oxide cyclic nucleotide pathway in cultured human smooth muscle cells from corpus cavernosum by rapidly activating a constitutive nitric oxide synthase

Anfossi

Massucco

Mattiello

et al. 2002

European Journal of Endocrinology

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Aims: We have evaluated, in cultured human cavernosal smooth muscle cells, the expression and activity of calcium-dependent constitutive nitric oxide synthase (cNOS) and the ability of insulin to induce nitric oxide (NO) production and to increase intracellular cyclic nucleotides guanosine 3 0 ,5 0 -cyclic monophosphate (cGMP) and adenosine 3 0 ,5 0 -cyclic monophosphate (cAMP). Methods: cNOS mRNA was detected by RT-PCR amplification, cNOS protein by immunofluorescence, cNOS activity as L-[3 H]-arginine and cyclic nucleotides by radioimmunoassay. Results: cNOS mRNA and cNOS protein were found in cultured cells; cNOS activity was increased by 5-min exposure to 1 mmol/l calcium ionophore ionomycin (from 0:1094^0:0229 to 0:26850 :0560 pmol=min per mg cell protein, P ¼ 0:011) and to 2 nmol/l insulin (from 0:1214^0:0149 to 0:2045^0:0290 pmol=min per mg cell protein, P ¼ 0:041). Insulin increased both cGMP and cAMP in a dose-and time-dependent manner (i.e. with 2 nmol/l insulin, cGMP rose from 2:710 :10 to 6:80^0:40 pmol=10 6 cells at 30 min, P ¼ 0:0001; cAMP from 1:26^0:06 to 3:020 :30 pmol=10 6 cells at 60 min, P ¼ 0:0001). NOS inhibitor N G -monomethyl-L-arginine and phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 blunted these effects of insulin. The action of insulin on cyclic nucleotides persisted in the presence of phosphodiesterase inhibition, guanylate cyclase activation by NO donors and adenylate cyclase activation by Iloprost or forskolin. Conclusion: Human cavernosal smooth muscle cells, by expressing cNOS activity, are a source of NO and not only its target; in these cells, insulin rapidly activates cNOS through a PI 3-kinase pathway, with a consequent increase of both cyclic nucleotides, thus directly influencing the mechanisms involved in penile vascular tone and interplaying with classical haemodynamic mediators.

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Nitric oxide synthesis is impaired in glutathione-depleted human umbilical vein endothelial cells

Cited by 94 publications

References 20 publications

Fluid Shear Stress Stimulates Big Mitogen-activated Protein Kinase 1 (BMK1) Activity in Endothelial Cells

Fluid Shear Stress Stimulates Big Mitogen-activated Protein Kinase 1 (BMK1) Activity in Endothelial Cells

Human vascular smooth muscle cells express a constitutive nitric oxide synthase that insulin rapidly activates, thus increasing guanosine 3′ : 5′-cyclic monophosphate and adenosine 3′ : 5′-cyclic monophosphate concentrations

Insulin influences the nitric oxide cyclic nucleotide pathway in cultured human smooth muscle cells from corpus cavernosum by rapidly activating a constitutive nitric oxide synthase

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