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

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)

Download Full-text

Nitric Oxide Synthase Isoforms and Glomerular Hyperfiltration in Early Diabetic Nephropathy

Journal of the American Society of Nephrology ◽

10.1681/asn.v11171 ◽

2000 ◽

Vol 11 (1) ◽

pp. 71-79

Author(s):

ROLAND VEELKEN ◽

KARL F. HILGERS ◽

ANDREA HARTNER ◽

ALEXANDER HAAS ◽

KIRSTEN P. BÖHMER ◽

...

Keyword(s):

Nitric Oxide ◽

Diabetic Nephropathy ◽

Nitric Oxide Synthase ◽

Renal Plasma Flow ◽

Diabetic Rats ◽

Glomerular Hyperfiltration ◽

Molecular Evidence ◽

Inos Mrna ◽

Oxide Synthase ◽

And Control

Abstract. This study tested the hypothesis that nitric oxide (NO)-mediated renal vasodilation due to the activity of the inducible nitric oxide synthase (iNOS) contributes to glomerular hyperfiltration in diabetic rats. Two weeks after induction of diabetes mellitus by streptozotocin, mean arterial BP (MAP), GFR (inulin clearance), and renal plasma flow (RPF) (para-aminohippurate clearance) were measured in conscious instrumented rats. Diabetic rats had elevated GFR (3129 ± 309 μl/min versus 2297 ± 264 μl/min in untreated control rats, P < 0.05) and RPF (10526 ± 679 μl/min versus 8005 ± 534 μl/min), which was prevented by chronic insulin treatment. Intravenous administration of 0.1 and 1 mg of L-imino-ethyl-lysine (L-NIL), an inhibitor of iNOS, did not affect MAP, GFR, or RPF, either in diabetic or control rats. A higher L-NIL dose (10 mg) increased MAP and decreased RPF in diabetic rats significantly (n = 6, P < 0.05), but not in controls (n = 6). In addition, 0.1 mg of NG-nitro-L-arginine methyl ester (L-NAME), a nonselective blocker of NOS isoforms, decreased GFR (2389 ± 478 μl/min) and RPF (7691 ± 402 μl/min) in diabetic animals to control levels, while renal hemodynamics in normoglycemic rats were not altered. Higher L-NAME doses (1 and 10 mg) reduced GFR and RPF in diabetic and control rats to identical levels. In glomeruli isolated from diabetic and control rats, neither iNOS mRNA nor iNOS protein expression was detected. In contrast, increased protein levels of endothelial constitutive NOS (ecNOS) were found in glomeruli of diabetic rats compared with controls. By immunohistochemistry, ecNOS but not iNOS staining was observed in the endothelium of preglomerular vessels and in diabetic glomeruli. These results support the notion that increased NO availability due to greater abundance of ecNOS contributes to the pathogenesis of glomerular hyperfiltration in early experimental diabetic nephropathy. In contrast, we found no functional or molecular evidence for increased glomerular expression and activity of iNOS in diabetic rats.

Download Full-text

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

Download Full-text

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.

Download Full-text