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The objective of this study was to elucidate the mechanisms by which nebivolol, a cardio-selective beta-adrenergic receptor antagonist, inhibits rat aortic smooth muscle cell (RASMC) proliferation. Nebivolol was compared with DETA-NO and S-nitroso-N-acetylpenicillamine (SNAP), two nitric oxide (NO) donor agents, and alpha-difluoromethylornithine (DFMO), a known inhibitor of ornithine decarboxylase (ODC). All four test agents inhibited RASMC proliferation in a concentration-dependent manner, with nebivolol being the most potent (IC(50) = 4.5 microM), whereas atenolol, another relatively selective beta(1)-blocker, was inactive. DFMO, nebivolol, and DETA-NO interfered with cell proliferation in a cell-density-dependent manner, the lower the cell density the greater the inhibition of cell proliferation. The cytostatic effects of nebivolol and DETA-NO were completely independent of cyclic GMP, as neither ODQ (cytosolic guanylyl cyclase inhibitor) nor zaprinast (cyclic GMP phosphodiesterase inhibitor) affected the antiproliferative action of nebivolol or DETA-NO. The cytostatic effects of nebivolol, SNAP, and DFMO were largely prevented by the addition of excess putrescine, but not ornithine, to cell cultures. Moreover, nebivolol caused a marked reduction in the intracellular levels of putrescine, spermidine, and spermine. Like DFMO, nebivolol and DETA-NO interfered with the G(1)-phase to S-phase cell cycle transition in RASMC. These observations confirm previous findings that DFMO and NO interfere with RASMC proliferation by inhibiting ODC and polyamine production and provide evidence that nebivolol works by the same mechanism.

The objective of this study was to elucidate the mechanism by which cyclic AMP increases arginase activity in cultured human Caco-2 tumor cells. Caco-2 cells were incubated for 24 h in the presence of 8-bromo cyclic AMP or forskolin, and the cells were harvested, lysed, and assayed for total arginase activity. Both test agents increased arginase activity by twofold, and this was attributed to the induction of the arginase II isoform. Both arginase II mRNA and protein showed increased expression in response to 8-bromo cyclic AMP and forskolin, and these effects were inhibited by H-89 (protein kinase A inhibitor), enhanced by okadaic acid (phosphatase inhibitor), and enhanced by 1-methyl-3-isobutylxanthine (cyclic nucleotide phosphodiesterase inhibitor). Cyclic GMP did not appear to be involved in arginase II induction. These observations indicate that cyclic AMP stimulates arginase II gene expression by mechanisms involving activation of protein kinase A and consequent activation of appropriate transcription factors.

In an attempt to identify potent inhibitors of inducible (type II) NO synthase (iNOS) for use in cell culture systems, we found that two S-substituted isothioureas were very potent in cell culture but one such compound also interfered with the induction of NO synthase. S-Ethylisothiourea (EITU) and S-aminoethylisothiourea (AEITU) were found to be much more potent than NG-methylarginine, NG-nitroarginine methy lester, or aminoguanidine as inhibitors of NO production by cultured RAW 264.7 cell macrophages activated by lipopolysaccharide (LPS). The approximate EC50 values as inhibitors of NO production, assessed by 24-h accumulation in cell culture media, were 10 microM (EITU), 30 microM (AEITU), 300 microM (NG-methylarginine), and 1000 microM (aminoguanidine). EITU was found to inhibit NO production by activated macrophages without interfering with the induction of iNOS. More specifically, EITU failed to influence transcription of iNOS mRNA (Northern blot analysis), translation of iNOS protein (pulse experiments), or degradation of translated iNOS protein (pulse-chase experiments). In contrast, however, AEITU interfered markedly with the induction of iNOS by mechanisms attributed to inhibition of translation of iNOS mRNA into functional protein as well as acceleration of degradation of already translated iNOS protein. These observations indicate that AEITU should not be used in cell culture experiments where the intent is solely to assess the consequences of inhibition of iNOS catalytic activity.