Effects of magnesium on nitric oxide synthase activity in endothelial cells

Magnesium modulates endothelium-dependent vasodilation in intact blood vessels. Therefore, the effects of magnesium on nitric oxide (NO) release by isolated endothelial cells and nitric oxide synthase (NOS) activity in endothelial cell homogenates were studied. Unstimulated and bradykinin-stimulated NO release by porcine aortic endothelial cell (PAEC) monolayers were unaffected by 30 min of exposure to magnesium concentrations varying from 0.010 to 10.0 mM. In contrast, when A-23187-stimulated cells were exposed to 0.01, 3.16, and 10.0 mM MgCl2, NO release was decreased by 11.3 +/- 1.8, 11.7 +/- 3.0, and 20.3 +/- 7.2%, respectively, compared with cells exposed to 1.0 mM MgCl2 (P < 0.01). These data suggested that a change in the intracellular magnesium concentration had an effect on NO release, in contrast to a change in the extracellular concentration, which did not have an effect. To further assess this possibility, crude NOS extracts were prepared from PAEC and exposed to MgCl2. NOS activity was measured via the conversion of L-[3H]arginine to L-[3H]citrulline. Increasing the concentration of MgCl2 by 1.0, 3.16, and 10.0 mM caused a 16.0 +/- 6.8, 17.1 +/- 1.7, and 38.6 +/- 5.3% decrease in citrulline formation, respectively (P < 0.05), suggesting a direct inhibition of NOS by MgCl2. No significant difference in the degree of inhibition of NOS activity was found between MgSO4 and MgCl2, thus ruling out a nonspecific chloride effect. In addition, increasing the concentration of NaCl to 15 mM had no effect on NOS activity, ruling out a nonspecific osmotic effect [101.6 +/- 10.5% of control activity, P = not significant (NS)].(ABSTRACT TRUNCATED AT 250 WORDS)

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Increased expression of endothelial cell nitric oxide synthase (ecNOS) in afferent and glomerular endothelial cells is involved in glomerular hyperfiltration of diabetic nephropathy

Diabetologia ◽

10.1007/s001250051088 ◽

1998 ◽

Vol 41 (12) ◽

pp. 1426-1434 ◽

Cited By ~ 110

Author(s):

H. Sugimoto ◽

K. Shikata ◽

M. Matsuda ◽

M. Kushiro ◽

Y. Hayashi ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Diabetic Nephropathy ◽

Endothelial Cell ◽

Nitric Oxide Synthase ◽

Glomerular Hyperfiltration ◽

Glomerular Endothelial Cells ◽

Oxide Synthase

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Abstract 15396: Maintenance of Endothelial NO Release after Inhibition of NADPH Oxidase 4

Circulation ◽

10.1161/circ.130.suppl_2.15396 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Claudia Eickholt ◽

Coy Brunssen ◽

Heike Langbein ◽

Melanie Brux ◽

Anja Hofmann ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Shear Stress ◽

Nitric Oxide Synthase ◽

Nadph Oxidase ◽

Protein Expression ◽

Nadph Oxidase 4 ◽

No Release ◽

Mrna And Protein Expression ◽

Oxide Synthase

The NADPH oxidase isoform NOX4 mainly produces H 2 O 2 . Previously we could show, that NOX4 is the major endothelial NOX isoform and constitutively active. Regulation of NOX4 and formation of reactive oxygen species are involved in NO release. The transcription factor NRF2 is a key mediator of cellular adaptation to redox stress. Therefore, regulation of NOX4 on the transcriptional level by NRF2 might be directly linked to NO release and endothelial function. Besides endothelial nitric oxide synthase (eNOS), a role of neuronal nitric oxide synthase (nNOS) in endothelial NO and H 2 O 2 release has been proposed. In this study, endothelial cells (HUVEC) were constantly exposed to high laminar shear stress (24 h, 30 dyn/cm 2 ). Application of shear stress stimulated NO formation and induced elongation of endothelial cells in the direction of flow. Lentiviral overexpression of NOX4 strongly increased endothelial H 2 O 2 release, while downregulation using shNOX4 decreased H 2 O 2 release. Furthermore, application of shear stress caused downregulation of NOX4 as well as upregulation of eNOS and antioxidative response via NRF2 and its target genes NQO-1 and HMOX-1. H 2 O 2 can affect NO release by eNOS. We could show that downregulation of NOX4 leads to upregulation of eNOS mRNA and protein expression accompanied by attenuation of NRF2 pathway under flow conditions. Determination of NO release confirmed these results. This supports a compensatory mechanism maintaining a stable NO release after NOX4 inhibition in response to flow. Attenuation of NRF2 by shNRF2 inhibited shear stress-dependent induction of NRF2 mRNA and protein expression and its target genes. In addition, shNRF2 enhanced the shear stress-dependent downregulation of NOX4. Finally, we detected a stable mRNA and protein expression of nNOS in endothelial cells, which was unaffected by flow. Interestingly, downregulation of NOX4 resulted in induction of nNOS expression. In conclusion, our data suggest an important role of H 2 O 2 production by NADPH oxidase 4 in maintaining NO release and endothelial function in response to flow. Inactivation of NADPH oxidase 4 attenuates NRF2 pathway and increases neuronal nitric oxide synthase as compensatory mechanisms.

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Integrin-linked kinase regulates endothelial cell nitric oxide synthase expression in hepatic sinusoidal endothelial cells

Liver International ◽

10.1111/liv.12606 ◽

2014 ◽

Vol 35 (4) ◽

pp. 1213-1221 ◽

Cited By ~ 7

Author(s):

Mahnoush S. Shafiei ◽

Songling Lui ◽

Don C. Rockey

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Endothelial Cell ◽

Nitric Oxide Synthase ◽

Sinusoidal Endothelial Cells ◽

Integrin Linked Kinase ◽

Oxide Synthase ◽

Hepatic Sinusoidal Endothelial Cells

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Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress

AJP Cell Physiology ◽

10.1152/ajpcell.1995.269.6.c1371 ◽

1995 ◽

Vol 269 (6) ◽

pp. C1371-C1378 ◽

Cited By ~ 396

Author(s):

M. Uematsu ◽

Y. Ohara ◽

J. P. Navas ◽

K. Nishida ◽

T. J. Murphy ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Shear Stress ◽

Endothelial Cell ◽

Nitric Oxide Synthase ◽

Mrna Expression ◽

K Channel ◽

Shear Stresses ◽

Aortic Endothelial Cells ◽

Oxide Synthase

Shear stress enhances expression of Ca(2+)-calmodulin-sensitive endothelial cell nitric oxide synthase (ecNOS) mRNA and protein in bovine aortic endothelial cells (BAEC). The present studies were performed to investigate mechanisms responsible for regulation of ecNOS mRNA expression by shear stress and to determine if this induction of ecNOS mRNA is accompanied by an enhanced nitric oxide (NO) production. Shear stresses of 15 dyn/cm2 for 3-24 h resulted in a two- to threefold increase of ecNOS mRNA content quantified by Northern analysis in BAEC. Shear stresses (1.2-15 dyn/cm2) for 3 h resulted in an induction of ecNOS mRNA in a dose-dependent manner. In human aortic endothelial cells, shear stresses of 15 dyn/cm2 for 3 h also resulted in ecNOS mRNA induction. In BAEC, this induction in ecNOS mRNA was prevented by coincubation with actinomycin D (10 micrograms/ml). The K+ channel antagonist tetraethylammonium chloride (3 mM) prevented increase in ecNOS mRNA in response to shear stress. The ecNOS promotor contains putative binding domains for AP-1 complexes, potentially responsive to activation of protein kinase C (PKC). However, selective PKC inhibitor calphostin C (100 nM) did not inhibit ecNOS induction by shear stress. Finally, production of nitrogen oxides under both basal conditions and in response to the calcium ionophore A-23187 (1 microM) by BAEC exposed to shear stress was increased approximately twofold compared with cells not exposed to shear stress. These data suggest that ecNOS mRNA expression is regulated by K+ channel opening, but not by activation of PKC, and that shear not only enhances ecNOS mRNA expression but increases capacity of endothelial cells to release NO.

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