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We previously reported that tumor necrosis factor-alpha (TNF) and lipopolysaccharide (LPS) stimulate DNA synthesis in chick embryo cardiomyocytes (CM) via nitric oxide and polyamine biosynthesis. Here we show an involvement of nuclear factor-kappaB (NF-kappaB) in the induction of nitric oxide synthase (NOS) and ornithine decarboxylase (ODC), the key enzyme in polyamine biosynthesis. In addition NF-kappaB activation appears to favor survival of CM by reducing caspase activation. TNF and LPS also stimulate phosphorylation of extracellular signal-regulated kinase (ERK), which is required for the changes in ODC and caspase activity, but not for NOS induction or NF-kappaB activation. In conclusion, these results indicate that NF-kappaB, in cooperation with ERK, plays a pivotal role in the growth stimulating effects of TNF and LPS, leading to the induction of both ODC and NOS and to the reduction of caspase activity.

Objective: We have recently shown that tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS) stimulate DNA synthesis in chick embryo cardiomyocytes (CMs). The aim of the present research was to investigate the pathways involved in this mitogenic response., Methods: CMs were isolated from 10-day-old chick embryos and grown to confluence. After 20 h of serum starvation the cells were treated with TNFalpha and LPS, and/or specific agonists and antagonists to manipulate the levels of polyamines, NO, cGMP and their biosynthetic enzymes ornithine decarboxylase (ODC), nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC). ODC, NOS, sGC activities and cGMP contents were determined by radiochemical procedures. DNA synthesis was determined by incorporation of [3H]-thymidine., Results: Treatment of CMs with TNFalpha and LPS increased cell number and [3H]-thymidine incorporation. Addition of TNFalpha and LPS provoked an induction of ODC, with consequent polyamine accumulation, and a more delayed enhancement of NOS activity, which appeared to be independent of the activation of the ODC-polyamine system. TNFalpha and LPS treatment also enhanced cGMP level in CMs and both polyamine and NO biosyntheses appeared to be required. Experiments with 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 TNFalpha and LPS. Moreover, addition of exogenous polyamines to untreated cells raised the cGMP level in a NO-dependent fashion, and enhanced [3H]-thymidine incorporation. The latter effect was inhibited by sGC or PKG inhibitors. Treatment 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 additively activate sGC in cell-free extracts., Conclusion: TNFalpha and LPS stimulate DNA synthesis in chick embryo CMs and this effect is mediated by polyamines, NO and intracellular cGMP.

Nitric oxide (NO) is a molecule involved in several signal transduction pathways leading either to proliferation or to cell death. Induction of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, represents an early event preceding DNA synthesis. In some cell types increased ODC activity seems to be involved in cytotoxic response. We investigated the role of NO and ODC induction on the events linked to cell proliferation or to cell death in cultured chick embryo cardiomyocytes. Exposure of cardiomyocytes to tumor necrosis factor (TNF) and lipopolysaccharide (LPS) caused NO synthase (NOS) and ODC induction as well as increased incorporation of [3H]-thymidine. This last effect was blocked by a NOS inhibitor and was strongly reduced by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. Sodium nitroprusside (SNP), an exogenous NO donor, inhibited the increases of NOS and ODC activities and abolished the mitogenic effect of TNF and LPS. Moreover, SNP alone caused cell death in a dose dependent manner. The cytotoxicity of SNP was not affected by DFMO while it was prevented by antioxidants. The results suggest that different pathways would mediate the response of cardiomyocytes to NO: they can lead either to ODC induction and DNA synthesis when NO is formed through NOS induction or to growth inhibition and cell death, when NO is supplied as NO donor. Increased polyamine biosynthesis would mediate the proliferative response of NO, while the cytotoxicity of exogenous NO seems to involve some oxidative reactions and to depend on the balance between NO availability and cellular redox mechanisms.

Biochemical and structural changes occurring in the myocardium with aging are mainly resulting from the association of a general tissue atrophy with the hypertrophy of the remaining myocytes. Whilst hypertrophy seems to be a compensatory process to the loss of cardiomyocytes and to a mild systolic hypertensive condition that accompanies elderly people, atrophy should be the modification more closely related to aging 'per se.' In support to the free radical theory of aging, several signs of oxidative damage have been shown in the aged heart, such as lipofuscin accumulation, decreased phospholipid unsaturation index, greater formation of both hydrogen peroxide and 8-hydroxy-2'deoxyguanosine. As a compensatory reaction, the activities of the main oxygen-radical scavenger enzymes are stimulated in the mitochondria of aged rat heart. Endothelium-mediated vasoregulation is more susceptible to oxidative stress in aged with respect to young rats, suggesting that also the vasculature can be negatively influenced by the oxygen free radicals generated during aging. The possible primary role of oxygen free radicals in the development of myocardial atrophy is also discussed.

The activities of enzymes involved in adenine nucleotide metabolism and the concentration of their metabolic products were studied in the hearts of chickens from birth to advanced age. In particular, in order to investigate the main mechanisms which contribute to ensure availability of adenine nucleotides during ageing of the heart, IMP concentration and the activities of enzymes involved in its turnover were studied. In newborn animals, AMP degradation, though limited in amount, was found to lead to the final products of purine metabolism. In fact, the activity of hypoxanthine phosphoribosyl-transferase (HPRT)-the salvage enzyme of IMP-was not detected. On the contrary, in young chickens, the low concentration of final products of purine metabolism, together with a remarkable activity of HPRT and a high concentration of IMP, indicates that metabolic flux converges on the salvage pathway. In adult chickens, an increase of purine catabolism was observed. This, together with an optimal concentration of endogenous adenine nucleotides, is indicative of a particularly high AMP metabolism. Finally, in chickens of advanced age, a reduced purine catabolism appeared to take place, thus contributing to the maintenance of the adenine nucleotide pool. In ageing heart, a major role of IMP turnover probably consists in the preservation of adenine nucleotides and in the recovery of high-energy phosphates.

The activity of adenylate deaminase, adenylate phosphatase and adenosine deaminase, as well as the endogenous content of adenine nucleotides, was examined in the heart of ageing chickens. In new-born (1-day-old) and young (20-day-old) chickens, AMP degradation in the heart seems to proceed preferentially through deamination, while in adult (1-year-old) through dephosphorylation. Compared with the adult heart, a 2-year-old one exhibits a decline of AMP catabolism. The total adenine nucleotide content and the concentration of ATP are higher in adult and aged chicken hearts, than in new-born and young ones. Adaptive mechanisms might occur in the heart of ageing chickens to ensure an adequate availability of adenine nucleotides.

The effect of polyamines on the cellular concentrations of cyclic AMP was studied. It was shown that 1 microM-spermine caused a decrease in cyclic AMP in chick-embryo heart cells, chick-embryo fibroblasts, neuroblastoma, glioma and neuroblastoma-glioma hybrid cells, grown in culture. A similar decrease was observed when polyamines were added to cells in the presence of a phosphodiesterase inhibitor or after stimulating the cells with various hormones. Noradrenaline was used in cultures of heart cells, prostaglandin E1 and adenosine for neuroblastoma and neuroblastoma-glioma hybrids, whereas isoproterenol was used for the stimulation of glioma cells. Polyamines at higher concentrations were either without effect or caused a slight increase in cyclic AMP. Spermidine (10 microM) also caused a decrease in cellular cyclic AMP, as did 0.1 microM-putrescine. It is suggested that the effect of polyamines on cellular cyclic AMP may be explained by the effect of these polycations on the activity of cellular phosphodiesterase.

Preincubation for 23 hr of confluent chick embryo heart cell cultures with increasing doses of a phospholipidic complex produces a progressive increase of [14C] leucine uptake and of its incorporation into proteins when compared to control cultures maintained with 0.5% foetal calf serum. Conversely, a longer time of exposure (47 and 71 hr) to the same doses of phospholipids exhibits a negative effect on both parameters. The changes in protein specific activity appear to be due to a real different capability of the cells in utilizing the amino acid taken up rather than to the changes in is endogenous availability.

The activity of aminoacid transport, as measured by alpha-aminoisobutyrate uptake, has been studied in confluent myocardial cell cultures exposed to different oxygen tensions. The results obtained indicate that the rate of cellular uptake and accumulation of the inert aminoacid increase with time as the fraction of oxygen is reduced. When alpha-aminoisobutyrate was added in presence of all other aminoacids of the medium, the effect of oxygen was also evident, suggesting a mechanism which overcomes the competitive action of the other aminoacids assigned to the same transport system of alpha-aminoisobutyrate (A system). The modulation of aminoacid transport activity may represent one of the possible mechanisms by which environmental oxygen affect the rate of cellular protein synthesis.

Cyclic AMP (cAMP) and cyclic GMP (cGMP) have been implicated as intracellular signals in the transition from a resting to a growing state. This suggestion comes from observations showing that the addition of growth promoting factors to quiescent cell cultures causes a rapid and transient decrease in cAMP and an increase in cGMP contents [9, 11] and that exogenous cAMP or cGMP congeners reduce or stimulate cell growth respectively [6, 13]. In view of this antagonistic effect elicited by the two nucleotides, it has been suggested that a fall in cAMP/cGMP ratio might be the triggering event for the initiation of cell proliferation [6]. Since polyamines correlate positively with active cell division [7], a possible involvement of these biogenic polycations in the regulation of cellular cyclic nucleotide contents is worthwhile investigating. Our previous reports have shown indeed that in different cultured cell types, spermine, spermidine and putrescine, at relatively low doses, are able to reduce cAMP content [3] by increasing cAMP-dependent phosphodiesterase activity (cAMP-PDE) [4] and to counteract the action of different cAMP-mediated effectors [3]. Besides endogenous polyamines seem to be involved in the cAMP-mediated induction of cAMP-PDE, as observed in heart cell cultures [4]. This report shows that the addition of each individual polyamine to confluent and serum-restricted heart cell cultures, while lowering cAMP content, induces an early and rapid increase of cGMP content by reducing the rate of its degradation.

In the isolated and perfused rat heart, the addition of morphine, methionine-enkephalin or leucine-enkephalin to the coronary perfusate, significantly reduces the mechanical activity by negatively affecting both the heart rate and the developed tension. These effects are dose dependent and maximally evident with leucine-enkephalin. Furthermore all the opioids strongly reduce the activity of isoproterenol-stimulated hearts. The suggestion is made that opioid peptides directly influence the cardiac mechanical activity possibly by interacting with membrane-receptor systems.

In confluent and serum-starved embryonic heart cell cultures, the addition of serum (10%), glucagon (GLU, 0.1 microM) or isoproterenol (ISO, 10 microM), causes the onset of ornithine decarboxylase (ODC) activity, with a maximum after 5-6 hr. This is paralleled by polyamine accumulation and by the induction of TAT, which, in the case of GLU and ISO, exhibits maximal activity at 4-3 hr respectively, followed by a net decline. Cyclic AMP (cAMP) also accumulates after exposure to GLU or ISO. However, under different conditions of ODC inhibition, serum fails to induce TAT, thus supporting a relevant role of cellular polyamines in serum action. Conversely, cAMP and TAT responses to GLU or ISO are markedly improved under prevention of polyamine accumulation, which also leads to a longer lasting TAT inducibility. The suggestion is made that polyamines are not required in the cAMP-dependent mechanism of TAT induction, but rather in the restoration of the basal activity of the enzyme.