Nitric oxide induces oxidative stress and mediates cytotoxicity to human cavernosal cells in culture

Nitric oxide (NO) is a product of nitric oxide synthase (NOS) activity and is recognized as the main mediator of penile erection by induction of caver nosal smooth muscle relaxation. Although excessive NO can be generated via inducible NOS activation under certain inflammatory and noninflammatory con ditions, for example, in response to TGF-beta and gamma -IFN (the proinflam matory cytokines), the effect of excessive NO produced as reactive nitrogen radical (NO.-) in the corpora cavernosa is not known. The present study wa s designed to evaluate whether the effect of NO.- on human cavernosal cells in primary culture is via oxidative stress. Cell growth was monitored by D NA synthesis, and mitochondrial function was evaluated by adenosine triphos phate (ATP) production. Primary culture was initiated with explants from hu man corpora cavernosa, and the monolayer cavernosal cells (passage 2-3) wer e plated on 12-well tissue culture plates. At 70%-80% confluency, the cells were incubated with varying concentrations of sodium nitroprusside (SNP) f or 16 hours. The cell growth (DNA synthesis) was monitored by measuring PH] thymidine incorporation, ATP levels (nanomoles per 10(4) cells) were measu red by chemiluminescence assay using a luminometer, the total oxidative str ess was monitored by measuring the levels of 8-iso PGF(2 alpha) (picograms per milliliter) by using an enzyme-linked immunosorbent assay kit, and NO p roduction was monitored by accumulation of nitrite levels (micrometer per 1 04 cells). Human cavernosal smooth muscle cells (HCSMC) exposed to SNP (0 t o 0.8 mM) exhibited a dose-dependent (two- to fivefold) decrease in DNA and ATP synthesis, accompanied by a two- to threefold increase in the levels o f 8-iso PGF(2 alpha) and about an eightfold increase in nitrite accumulatio n. These findings suggest that the NO released by SNP (>0.8 mM) exhibited a significant cytotoxicity to HCSMC, mediated by increased oxidative stress to these cells.