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Nitric oxide donors regulate nitric oxide synthase in bovine pulmonary artery endothelium
Author(s) -
Chen JianXiong,
Berry Leonard C.,
Tanner Miles,
Chang Mike,
Myers R. Paul,
Meyrick Barbara
Publication year - 2001
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/1097-4652(200101)186:1<116::aid-jcp1005>3.0.co;2-x
Subject(s) - snap , nitric oxide , nitric oxide synthase , sodium nitroprusside , endothelial nos , chemistry , enos , nitric oxide synthase type iii , medicine , endothelium , endothelial stem cell , endocrinology , microbiology and biotechnology , andrology , biology , biochemistry , in vitro , computer graphics (images) , computer science
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 μM 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 Ca 2+ 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 μM); but A23187 (3 μM)‐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 ± 2.5 fmol cGMP/μg 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 Ca 2+ , 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. J. Cell. Physiol. 186:116–123, 2001. © 2001 Wiley‐Liss, Inc.