Nitric oxide donors regulate nitric oxide synthase in bovine pulmonary artery endothelium

Abstract

This study examined the notion that exogenous generation of nitric oxide (NO) modulates NOS gene expression and activity. Bovine pulmonary artery endothelial cells (BPAEC) were treated with the NO donors, 1 mM SNAP (S-nitroso-N-acetylpenicillamine), 0.5 mM SNP (sodium nitroprusside) or 0.2 microM NONOate (spermine NONOate) in medium 199 containing 2% FBS. Controls included untreated cells and cells exposed to 1 mM NAP (N-acetyl-D-penicillamine). NOS activity was assessed using a fibroblast-reporter cell assay; intracellular Ca2+ concentrations were assessed by Fura-2 microfluorometry; and NO release was measured by chemiluminescence. Constitutive endothelial (e) and inducible (i) NOS gene and protein expression were examined by northern and western blot analysis, respectively. Two hours exposure to either SNAP or NONOate caused a significant elevation in NO release from the endothelial cells (SNAP = 51.4 +/- 5.9; NONOate = 23.8 +/- 4.2; control = 14.5 +/- 2.8 microM); but A23187 (3 microM)-stimulated NO release was attenuated when compared to controls. Treatment with either SNAP or NONOate for 2 h also resulted in a significant increase in NOS activity in endothelial homogenates (SNAP = 23.6 +/- 2.5; NONOate= 29.8 +/- 7.7; control = 14.5 +/- 2.5fmol cGMP/microg per 10(6) cells). Exposure to SNAP and SNP, but not NONOate, for 1 h caused an increase in intracellular calcium. Between 4 and 8 h, SNAP and NONOate caused a 2- to 3-fold increase in eNOS, but not iNOS, gene (P < 0.05) and protein expression. NAP had little effect on either eNOS gene expression, activity or NO production. Our data indicate that exogenous generation of NO leads to a biphasic response in BPAEC, an early increase in intracellular Ca2+, and increases in NOS activity and NO release followed by increased expression of the eNOS gene, but not the iNOS gene. We conclude that eNOS gene expression and activity are regulated by a positive-feedback regulatory action of exogenous NO.

Nitric Oxide Generation by NO Donors Is Enhanced Following Balloon Injury in the Porcine Coronary Artery

“…This is in agreement with previous studies of in vitro injury where some 60% of the response to the endotheliumdependent vasodilator calcimycin remained (Kennedy et al 2003). A recent study demonstrated that incubation of cultured endothelial cells with NO donors for 2 h can upregulate eNOS gene expression by the cells (Chen et al 2001). In the present study, an identical incubation time had no such effect on eNOS expression and caused no change in NF-κB, a transcription factor involved in upregulation of gene expression.…”

Hypoxia increases Hsp90 binding to eNOS via PI3K-Akt in porcine coronary artery endothelium

“…NOS activity was measured as an increase in cGMP using a fibroblast-reporter cell line (RFL-6) as previously described. 29 Briefly, RFL-6 cells in six-well plates (1 Â 10 6 cells/well) were incubated for 20 min at 371C in HEPES. A 500 ml aliquot of cell particulates from each of the samples (see above) was added to the RFL-6 cells and the mixture was incubated for a further 5 min.…”

Endothelial Nitric Oxide Synthase

“…Also, given the evidence indicating that NO may modulate expression of the varied NOS isoform mRNAs, 96,97 including eNOS itself, it remains possible that the varied in vitro and in vivo models may reflect differences in feedback regulatory mechanisms.…”