Mechanisms of cytokine induced NO-mediated cardiac fibroblast apoptosis

This study examined the role of nitric oxide (NO) in cytokine-induced apoptosis in adult cardiac fibroblasts (CFbs). In cultured adult rat CFbs, IL-1β (5 ng/ml), but not interferon-γ (10 ng/ml) or tumor necrosis factor-α (10 ng/ml), induced inducible NO synthase (iNOS) expression and NO production that was associated with an increase in caspase-3 activity and apoptotic cell death. Apoptotic frequency was reduced by the iNOS inhibitor S-methylisothiourea (3 × 10^-5 M). Apoptosis in response to IL-1β was attenuated by the caspase-3 inhibitor [Z-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DVED-FMK)] but not by inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazolo[4,3-alquinoxalin-1-one (ODQ). IL-1β-induced CFb apoptosis was associated with an increase in p53 and Bax protein expression with no changes in Bcl-2 or Bcl-X_L. Nuclear condensation and fragmentation occurred when isolated nuclei were exposed to an NO donor {Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate) 10^-5 M} an effect that was not blocked by the peroxynitrite scavenger Mn(III)tetrakis(4-benzoic acid) porphyrin chloride. Moreover, Mn(III)tetrakis(4-benzoic acid) porphyrin chloride attenuated but did not eliminate IL-1β-induced CFb apoptosis, indicating that the proapoptotic effect of NO can occur independently of its conversion to peroxynitrite. Our results demonstrate that IL-1β-induced iNOS expression can trigger NO-dependent apoptosis in adult CFbs, which appears to result from DNA damage and may be mediated by a p53-dependent apoptotic pathway.