L-arginine inhibits apoptosis via a NO-dependent mechanism in Nb2 lymphomacells

Prolactin (PRL) inhibits apoptosis and stimulates proliferation of the PRL- dependent rat Nb2 lymphoma cell line by divergent signaling pathways. Nitri c oxide (NO) was recently identified as a downstream regulator of PRL actio n, and as an inhibitor of apoptosis in immune cells. In the present study, the role of NO in PRL-regulated Nb2 cell function was investigated. Nb2 cel ls expressed the endothelial nitric oxide synthase (eNOS) isoform, whereas neuronal NOS (nNOS) and inducible NOS (iNOS) mRNAs were undetectable. The e NOS mRNA was abundantly expressed in PRL-deprived, growth-arrested cells bu t decreased by at least 3-fold at 3-24 h following PRL treatment. Downregul ation of eNOS was not accompanied by a corresponding decrease in the eNOS p rotein, the level of which remained constant for at least 24 h after PRL tr eatment. PRL had no effect on the phosphorylation state or subcellular redi stribution of the eNOS enzyme, or on production of NO by Nb2 cells. However , increasing concentrations of L-arginine (NOS substrate) alone increased N O production in these cells and significantly enhanced PRL-stimulated cell proliferation. NO releasers (SNAP, DEA/NO, SIN-1) also significantly enhanc ed Nb2 cell proliferation in the presence of a submaximal dose of PRL (0.12 5 ng/ml). In the absence of PRL, the NO releasers alone promoted cell survi val and maintained a viable cell density significantly higher than that of untreated PRL-deprived cells. L-arginine or the NO releaser DEA/NO alone si gnificantly inhibited apoptosis in Nb2 cells deprived of PRL for 5 days. Ex pression of the anti-apoptotic gene bcl-2, which was stimulated within 1 h by PRL, was upregulated by L-arginine or DEA/NO alone at 2 h and 8 h, respe ctively. These findings suggest that NO produced by eNOS inhibits apoptosis and promotes the survival of growth-arrested Nb2 lymphoma cells via a prol actin-independent, Bcl-2-mediated pathway. J. Cell. Biochem. 77:624-634,200 0. (C) 2000 Wiley-Liss, Inc.