Polyamines, NO and cGMP mediate stimulation of DNA synthesis by tumor necrosis factor and lipopolysaccharide in chick embryo cardiomyocytes

Objective: We have recently shown that tumor necrosis factor-alpha (TNF alp ha) and lipopolysaccharide (LPS) stimulate DNA synthesis in chick embryo ca rdiomyocytes (CMs). The aim of the present research was to investigate the pathways involved in this mitogenic response. Methods: CMs were isolated fr om 10-day-old chick embryos and grown to confluence. After 20 h of serum st arvation the cells were treated with TNF alpha and LPS, and/or specific ago nists and antagonists to manipulate the levels of polyamines, NO, cGMP and their biosynthetic enzymes ornithine decarboxylase (ODC), nitric oxide synt hase (NOS) and soluble guanylate cyclase (sGC). ODC, NOS, sGC activities an d cGMP contents were determined by radiochemical procedures. DNA synthesis was determined by incorporation of [H-3]-thymidine. Results: Treatment of C Ms with TNF alpha: and LPS increased cell number and [3H]-thymidine incorpo ration. Addition of TNF alpha and LPS provoked an induction of ODC, with co nsequent polyamine accumulation, and a more delayed enhancement of NOS acti vity, which appeared to be independent of the activation of the ODC-polyami ne system. TNF alpha and LPS treatment also enhanced cGMP level in CMs and both polyamine and NO biosyntheses appeared to be required. Experiments wit h specific inhibitors of ODC and NOS, as well as with inhibitors of sGC and cGMP-dependent protein kinase (PKG), showed that polyamine-, NO- and cGMP- dependent pathways are required for the mitogenic action of TNF alpha and L PS. Moreover, addition of exogenous polyamines to untreated cells raised th e cGMP level in a NO-dependent fashion, and enhanced [3H]-thymidine incorpo ration. The latter: effect was inhibited by sGC or PKG inhibitors. Treatmen t of quiescent cells with NO donors, 8-bromo-cGMP or YC-1, an sGC activator , also promoted DNA synthesis. Furthermore, putrescine and NO donor can add itively activate sGC in cell-free extracts. Conclusion: TNF alpha, and LPS stimulate DNA synthesis in chick embryo CMs and this effect is mediated by polyamines, NO and intracellular cGMP. (C) 2001 Elsevier Science BN. All ri ghts reserved.