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. CONCLUSION: 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.
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. CONCLUSION: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.