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

This study examined the notion that exogenous generation of nitric oxide (N O) modulates NOS gene expression and activity. Bovine pulmonary artery endo thelial cells (BPAEC) were treated with the NO donors, 1 mM SNAP (S-nitroso -N-acetylpenicillamine), 0.5 mM SNP (sodirrm nitroprusside) or 0.2 muM NONO ate (spermine NONOate) in medium 199 containing 2% FBS. Controls included u ntreated cells and cells exposed to 1 mM NAP (N-acetyl-D-penicillamine). NO S activity was assessed using a fibroblast-reporter cell assay; intracellul ar Ca2+ concentrations were assessed by Fura-2 microfluorometry; and NO rel ease was measured by chemiluminescence. Coristitutive endothelial (e) and i nducible (i) NOS gene and protein expression were examined by northern and western blot analysis, respectively. Two hours exposure to either SNAP or N ONOate caused a significant elevation in NO release from the endothelial ce lls (SNAP = 51.4 +/- 5.9; NONOate = 23.8 +/- 4.2; control = 1 4.5 +/- 2.8 m uM); but A23187 (3 muM)-slimulated 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 +/- 2.5 fmol cGMP/mug per 10(6 ) cells). Exposure to SNAP and SNP, but not NONOate, for 1 h caused an incr ease 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 e xpression. NAP had little effect on either eNOS gene expression, activity o r 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 express ion and activity are regulated by a positive-feedback regulatory action of exogenous NO. i. Cell. Physiol. 186:126-123, 2001. (C) 2001 Wiley-Liss, Inc .