Ambient pressure upregulates nitric oxide synthase in a phosphorylated-extracellular regulated kinase- and protein kinase C-dependent manner.

Purpose Using endothelial cell/smooth muscle cell (SMC) cocultures, we have demonstrated that pressurized endothelial cell coculture inhibits SMC proliferation and promotes apoptosis, and that this effect is transferable through pressurized endothelial medium. We now hypothesized that endothelial nitric oxide synthase (eNOS) plays a significant role in mediating these pressure-induced effects. Methods Conditioned media from endothelial cells and SMCs exposed to ambient and increased pressure were transferred to recipient SMCs. We counted cells after 5 days of incubation with these media and evaluated eNOS and inducible NOS (iNOS) levels by Western blot. Results Conditioned media from pressurized endothelial cells significantly decreased recipient SMC counts. This effect was sustained when N -nitro-L-arginine-methyl ester (L-NAME) was added to recipient cells but abolished when L-NAME was added to donor cells. SMCs were then exposed to control and pressurized conditions in monoculture or in coculture with endothelial cells. Pressure and coculture caused similar increase in iNOS levels but had no additive effect in combination. Finally, endothelial cells were exposed to control and pressurized environments. Pressure caused a 24% ± 1.6% increase in eNOS protein ( P = .04, n=12). This effect was sustained when cells were treated with L-NAME (32% ± 1.6% increase, P = .02) but abolished when endothelial cells were treated with calphostin C or PD98059 to block protein kinase C (PKC) or extracellular regulated kinase (ERK). Pressure also increased endothelial phosphorylated ERK (p-ERK) by 1.8-fold to 2.6-fold compared with control conditions after exposure of 2, 4, and 6 hours ( P = .02, n=4). This increase was sustained after pretreatment with calphostin C. Conclusion Pressure modulates endothelial cell effects on SMC growth by increasing eNOS in an ERK-dependent and PKC-dependent manner.