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

Abstract

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.

Cannabinoid Regulation of Nitric Oxide Synthase I (nNOS) in Neuronal Cells

“…In order to determine levels of gene expression of the three NOS isoforms, total RNA was isolated, and cDNA was amplified using previously published primers designed to detect expression of nNOS, eNOS, and iNOS genes (Dodd et al 2000 ). Analysis by PCR indicated the expression of nNOS, but not eNOS or iNOS (Fig.…”

“…Following RNA isolation and quantitation, 1 μg of RNA from each sample was reverse-transcribed according to the manufacturer’s specifications of the GeneAmp Gold RNA PCR Core kit. The resulting cDNA was amplified (35 cycles, AmpliTaqGold DNA polymerase and GeneAmp Gold kits) using primers described in previous studies to identify NOS isozymes (Dodd et al 2000 ) and 18S ribosomal RNA as the loading control (28 cycles; Goidin et al 2001 ). PCR products were detected from agarose gels (1.6%) after electrophoresis in TBE buffer containing 50 ng/ml ethidium bromide.…”

Prolactin activates mammalian target-of-rapamycin through phosphatidylinositol 3-kinase and stimulates phosphorylation of p70S6K and 4E-binding protein-1 in lymphoma cells

“…Total cell lysates from about 30 to 50!10 6 cells/treatment were prepared for immunoprecipitation in RIPA buffer containing protease inhibitors as previously described (Dodd et al 2000). Immunocomplexes or cell lysates were used for SDS-PAGE (4-20% gels) and followed by western analysis.…”

Decreased Mitochondrial Proton Leak and Reduced Expression of Uncoupling Protein 3 in Skeletal Muscle of Obese Diet-Resistant Women

“…Relative-quantitative RT-PCR was performed using the Quantum RNA 18S Internal Standards kit from Ambion (Austin, TX). This kit has been previously shown to accurately determine relative changes in gene expression between samples (18). Briefly, 2.5 g total RNA was used to synthesize first-strand cDNA using 10 mol/l random decamer primers (Ambion) and 200 units Moloney murine leukemia virus (MMLV) reverse transcriptase (Life Technologies, Burlington, Canada), with incubation at 42°C for 1 h. UCP3 primers (UCP3 forward 5Ј-CCTCGTTACCTTTCCACTGG-3Ј and UCP3 reverse 5Ј-GGCAGAGACAAAGTGGCAGG-3Ј) were designed from human UCP3 cDNA sequence information to amplify a 615-bp sequence common to both known splice variants of the UCP3 mRNA.…”