L-arginine inhibits apoptosis via a NO-dependent mechanism in Nb2 lymphoma cells.
Prolactin (PRL) inhibits apoptosis and stimulates proliferation of the PRL-dependent rat Nb2 lymphoma cell line by divergent signaling pathways. Nitric oxide (NO) was recently identified as a downstream regulator of PRL action, 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 cells expressed the endothelial nitric oxide synthase (eNOS) isoform, whereas neuronal NOS (nNOS) and inducible NOS (iNOS) mRNAs were undetectable. The eNOS mRNA was abundantly expressed in PRL-deprived, growth-arrested cells but decreased by at least 3-fold at 3-24 h following PRL treatment. Downregulation of eNOS was not accompanied by a corresponding decrease in the eNOS protein, the level of which remained constant for at least 24 h after PRL treatment. PRL had no effect on the phosphorylation state or subcellular redistribution of the eNOS enzyme, or on production of NO by Nb2 cells. However, increasing concentrations of L-arginine (NOS substrate) alone increased NO production in these cells and significantly enhanced PRL-stimulated cell proliferation. NO releasers (SNAP, DEA/NO, SIN-1) also significantly enhanced Nb2 cell proliferation in the presence of a submaximal dose of PRL (0.125 ng/ml). In the absence of PRL, the NO releasers alone promoted cell survival and maintained a viable cell density significantly higher than that of untreated PRL-deprived cells. L-arginine or the NO releaser DEA/NO alone significantly inhibited apoptosis in Nb2 cells deprived of PRL for 5 days. Expression 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, respectively. These findings suggest that NO produced by eNOS inhibits apoptosis and promotes the survival of growth-arrested Nb2 lymphoma cells via a prolactin-independent, Bcl-2-mediated pathway.
Dodd
F
F
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.
Limoges
M
M
Boudreau
R T
RT
Rowden
G
G
Murphy
P R
PR
Too
C K
CK
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001120:Arginine / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004727:Endothelium / Q000201:enzymology; D020287:In Situ Nick-End Labeling; D008070:Lipopolysaccharides / Q000494:pharmacology; D008223:Lymphoma / Q000378:metabolism*; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism* / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010766:Phosphorylation; D011233:Precipitin Tests; D011388:Prolactin / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D008070:Lipopolysaccharides; D020033:Protein Isoforms; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D009569:Nitric Oxide; D011388:Prolactin; D001120:Arginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat