Nitric oxide effects on cell growth: GMP-dependent stimulation of the AP-1 transcription complex and cyclic GMP-independent slowing of cell cycling.

1. The role of nitric oxide (NO) in the control of cell growth is controversial since both stimulation and (more often) inhibition have been demonstrated in various cell types. In order to reinvestigate the problem and identify the sites of NO action, we have employed murine NIH-3T3 fibroblasts overexpressing epidermal growth factor (EGF) receptors. 2. The effects of four structurally-unrelated NO donors: S-nitroso-N-acetyl penicillamine, S-nitroso-L-glutathione, 3-morpholinosydnonimine and isosorbide dinitrate (0.01-3 mM) on EGF (10 nM)-stimulated cell growth were estimated by both thymidine incorporation and the colorimetric MTT assay, while those of a messenger generated in response to NO, cyclic GMP, were revealed by the use of 8-Br cyclic GMP (0.01-3 mM) as well as of blockers of guanylyl cyclase and cyclic GMP-dependent kinase I. 3. Studies were focused on: (i) multiple signalling events, including receptor-induced tyrosine phosphorylations, phosphorylation of mitogen-activated protein kinase, activation of the AP-1 transcription complex and deoxyribonucleotide synthesis; (ii) the progression through the cell cycle, dissected out by the use of staurosporine (1 nM), lovastatin (10 microM), mimosine (200 microM), hydroxyurea (1 mM) and nocodazole (1.5 microM). 4. NO was found to have no effects on the phosphorylation events of the growth factor cascade. In contrast, later processes were modified by the messenger but with opposite effects. 5. A cyclic GMP-dependent stimulation of growth was shown to be sustained in part by the activation of the AP-1 transcription complex, while a predominant, cyclic GMP-independent inhibition was found to be mediated by both the negative regulation of ribonucleotide reductase and the marked slowing down of the cell cycle occurring at early and late G1 and during the S phase. 6. Although multiple and apparently conflicting, the effects of NO here described could work coordinately in a general programme of cell growth regulation. In particular, the cyclic GMP-dependent actions might function as rapid modulatory events, while the effects on cell cycle might operate collectively as a multi-switch process whenever growth inhibition is required.