NITRIC-OXIDE INHIBITS LIPOPOLYSACCHARIDE-INDUCED APOPTOSIS IN PULMONARY-ARTERY ENDOTHELIAL-CELLS

Our group recently reported that cultured sheep pulmonary artery endot helial cells (SPAECs) became resistant to lipopolysaccharide (LPS)-ind uced apoptosis several days after constitutive synthesis of nitric oxi de (NO) after adenoviral (Ad) transfer of inducible NO synthase (iNOS) or exposure to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) (E . Tzeng, Y.-M. Kim, B. R. Pitt, A. Lizonova. I. Kovesdi, and T. R. Bil liar. Surgery 122: 255-263, 1997). In the present study, we confirmed this observation by establishing stable transfectants after retroviral gene transfer [replication-deficient retrovirus (DFG)] of human iNOS (DFG-iNOS) SPAECs and then used all three approaches (Ad, DFG, and SNA P) to determine underlying mechanisms of this phenomenon. Continuous e ndogenous production of NO in itself did not cause apoptosis as assess ed by phase-contrast microscopy, nuclear morphology, and internucleoso mal DNA fragmentation. Prolonged (72-96 h) synthesis of NO, however, a fter DFG- or replication-deficient adenovirus (Ad.CMV)-iNOS or SNAP (1 00 mu M, 96 h) inhibited LPS-induced apoptosis. The kinetics of such p rotection suggested that NO may be inducing other gene products. Ad-me diated transfer of manganese superoxide dismutase (MnSOD) decreased th e sensitivity of wild-type SPAECs to LPS-induced apoptosis. MnSOD, how ever, was not induced in an NG-monomethyl-L-arginine (L-NMMA)-sensitiv e time-dependent fashion after Ad.CMV-iNOS. Other inducible genes that may be affected by NO and that may protect against potential oxidant- mediated LPS-induced apoptosis including 70-kDa heat shock protein, he me oxygenase-l, metallothionein, and Bcl-2 also were not elevated in a n L-NMMA-sensitive, time-dependent fashion. Although the candidate gen e product underlying NO-induced protection remains unclear, we did not e that prolonged synthesis of NO inhibited LPS-induced activation of a n interleukin-1 beta-converting enzyme-like cysteine protease (cystein e protease protein-32-like) in a dithiothreitol-sensitive fashion, sug gesting that S-nitrosylation of an important downstream target of conv ergence of apoptotic signals may contribute to the sensitivity of SPAE Cs to LPS.