Expression of inducible nitric oxide synthase in cultured smooth muscle cells from rat mesenteric lymphatic vessels.
OBJECTIVE
The objective was to devise a method for establishing cultures of rat mesenteric lymphatic vessel smooth muscle cells (LSMC) and to investigate if inducible nitric oxide synthase (iNOS) expression could be activated in LSMC treated with bacterial lipopolysaccharide (LPS).


METHODS
LSMC were successfully grown from explanted rat lymphatic microvessels and maintained by subculture. Treatment of LSMC for 24 h with LPS (1-100 microg/mL) activated iNOS protein induction, associated with (1) assay of increased nitrite concentrations in the medium representing cellular nitric oxide synthesis, and (2) demonstration of iNOS in cell extracts by Western blotting.


RESULTS
The protein synthesis inhibitor cycloheximide (10 microM) blocked both LPS-induced nitrite formation and iNOS protein expression in LSMC. 1400 W (1 microM), a selective iNOS inhibitor, prevented LPS-induced nitrite formation but not iNOS expression. As well as induction of iNOS by LPS, "constitutive" iNOS was present in some cultures, producing nitrite in amounts that were also subsequently reduced after cell treatment with 1400 W.


CONCLUSIONS
Rat mesenteric LSMC produce nitrite and express iNOS in response to bacterial LPS. Cultured LSMC may provide a useful model for studying mechanisms of iNOS induction in relation to possible influences of iNOS upon lymphatic vessel function.
Robertson
Deborah A F
DA
Department of Pharmacology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK.
Hughes
Gwen A
GA
Lyles
Geoffrey A
GA
D019840:2-Propanol / Q000494:pharmacology; D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D003513:Cycloheximide / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007097:Imines / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000008:administration & dosage / Q000494:pharmacology; D042601:Lymphatic Vessels / Q000166:cytology* / Q000201:enzymology; D008297:Male; D008643:Mesentery / Q000033:anatomy & histology*; D009130:Muscle, Smooth / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000032:analysis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
D004791:Enzyme Inhibitors; D007097:Imines; D008070:Lipopolysaccharides; C496401:N-((3-(aminomethyl)phenyl)methyl)ethanimidamide; D009573:Nitrites; D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D019840:2-Propanol