Vascular smooth muscle cell activation depends on NOS-1-derived NO and consequent peroxynitrite generation

Background

Low levels of nitric oxide (NO) play a key role in vascular tonus maintenance. Previous results from our laboratory show that hypotension and mortality during sepsis are prevented by the early administration of NOS-1 inhibitors. The aim of this study was thus to investigate the role of NOS-1 and NOS-3-derived NO and of other reactive oxygen species (ROS) in smooth muscle cell activation.

Materials and methods

Smooth muscle cell line of rat aorta (A7r5) was used. Control cells and NOS-1 or NOS-3 silenced cells (siNOS-1 and siNOS-3, respectively) were stimulated with LPS 1 µg/ml and IFN 200 U/ml (LPS/IFN). NO and ROS production was assessed with fluorescent probes. NOS content was evaluated by western blot and NOS-2 activity was indirectly measured by Griess reaction. Further, control cells were treated for 30 minutes with a NO scavenger (c-PTIO), a NOS inhibitor (7-NI) or a NADPH oxidase inhibitor (DPI) before stimulation. Immunofluorescence was used to evaluate protein nitration and NF-κB nuclear translocation. To confirm the role of peroxynitrite in cell activation, control cells were stimulated with a sub-effective amount of LPS/IFN together with a NO donor and a superoxide anion generator and treated with a NOS-2 inhibitor 4 hours after stimulation. Griess reaction was performed 48 hours after. Statistical comparisons were performed by two-way ANOVA followed by the Bonferroni test.

Results

A7r5 control cells stimulated with LPS/IFN presented a rapid increase in intracellular NO and ROS content. These increases were prevented by c-PTIO, 7-NI and DPI, as well as in siNOS-1 and siNOS-3 cells. NOS-2 was only expressed after cell stimulation. Control cells incubated with c-PTIO or 7-NI and stimulated with LPS/IFN presented a diminished NOS-2 expression and activity. Only in siNOS-1 cells was NOS-2 expression and activity also reduced. Nuclear translocation of NF-κB and positive nitrotyrosine reaction were reduced in c-PTIO or 7-NI treated groups. Sub-effective concentrations of LPS/IFN did not induce significant nitrite production. However, when sub-effective LPS/IFN was associated with the production of low concentrations of peroxynitrite, nitrite accumulation was as high as in cells stimulated with activating concentrations of LPS/IFN.

Conclusions

We show for the first time the importance of NOS-1-derived NO and peroxynitrite for smooth muscle cell activation. Cell stimulation with LPS/IFN causes an early NOS-1-derived NO pulse and a ROS pulse that forms peroxynitrite. The interplay between these species seems to be key events for NF-κB nuclear translocation and NOS-2 expression.