title: Inducible nitric oxide synthase mRNA and activity in glomerular mesangial cells.
abstract: Previous studies have suggested that glomerular mesangial cells produce nitric oxide (NO), using measurements of the NO decomposition products, NO2- and NO3-. We have now directly measured NO in the headspace above rat mesangial cell cultures, using a chemiluminescence analyzer. In addition, we examined mesangial cell RNA for inducible NO synthase (iNOS). We found no detectable NO in the headspace or iNOS mRNA in unstimulated mesangial cells. However, after four hours of incubation with LPS (10 micrograms/ml), iNOS mRNA was apparent and after six hours, significant increases in NO were detected. Both of these parameters continued to increase for at least 24 hours. Significant increases in NO2-/NO3- in the media and cGMP in the mesangial cells were also detected after 24 hours of incubation with LPS. The induction of iNOS mRNA by LPS was markedly inhibited by actinomycin D and dexamethasone, as was the accumulation of NO2-/NO3- in the media. Cycloheximide significantly inhibited NO2-/NO3- in the media of LPS-treated cells, but had little effect on induction of iNOS mRNA by LPS. We conclude that rat mesangial cells possess an iNOS, with activity and regulation similar to that described in macrophages. Furthermore, we demonstrate the activity of this enzyme by direct measurement of NO and its decomposition products, NO2- and NO3-. We suggest that production of NO by glomerular mesangial cells could occur, even when macrophage infiltration is not present, and could, thereby, modulate glomerular and tubular functions within the kidney.
authors.affiliation: VA Medical Center, Minneapolis, Minnesota.
authors.forename: P J
authors.initials: PJ
authors.lastname: Shultz
authors.forename: S L
authors.initials: SL
authors.lastname: Archer
authors.forename: M E
authors.initials: ME
authors.lastname: Rosenberg
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D008070:Lipopolysaccharides; D012333:RNA, Messenger; D003609:Dactinomycin; D009569:Nitric Oxide; D003513:Cycloheximide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases