Nitric oxide and cyclic guanosine monophosphate stimulate apoptosis via activation of the Fas-FasL pathway.

Abstract

Inappropriately exaggerated response of pulmonary vascular cells to inflammatory mediators may be one mechanism that leads to acute (or adult) respiratory distress syndrome. Nitric oxide (NO) is induced following such exaggerated responses and may have a variety of biological effects, including induction of apoptosis. The mechanism by which NO causes apoptosis is unknown; however, Fas (CD95) and Fas ligand (FasL) (CD95L) have been implicated. We hypothesized that NO-induced apoptosis in pulmonary vascular smooth muscle cells is mediated through a Fas-FasL pathway. Cultured human and rat pulmonary artery smooth muscle cells (PASMCs) were exposed to soluble FasL (0-5 ng/ml), the NO donor S(G)-nitroso-N-acetyl pencillamine (SNAP) (0-50 microg/ml), and/or anti-FasL (0-100 microg/ml) for 12 h. Apoptosis was measured using in situ DNA nick end labeling and flow cytometry. Changes in Fas and FasL protein levels were assessed via Western blot analysis. Messenger RNA (mRNA) abundance of apoptosis-related genes was determined using a ribonuclease protection assay. Rat PASMCs exposed to FasL show a dose-dependent increase in apoptosis. Human PASMCs are less responsive to FasL. Addition of anti-FasL to rat PASMCs treated with 10(-5) M SNAP decreases apoptosis levels compared to SNAP treated alone. FasL and Fas receptor proteins are increased in response to 10(-3) to 10(-4) M SNAP or 10(-6) M 8-bromo-cyclic guanosine monophosphate (cGMP). The mRNA abundance of Fas, FasL, and other apoptosis-related genes is increased in response to 10(-6) M 8-bromo-cGMP but not 8-bromo-cyclic adenosine monophosphate. Nitric oxide-induced apoptosis in rat and human PASMCs is mediated, at least in part, through the Fas-FasL pathway, with cGMP increasing the expression of Fas and FasL.