Difluoromethylornithine inhibits hypertrophic, pro-fibrotic and pro-apoptotic actions of aldosterone in cardiac cells.

Recent studies have shown that aldosterone may play a critical role in the transition to heart failure and that heart is a direct target of the action of aldosterone, which can provoke hypertrophy and apoptosis of isolated cardiomyocytes and also increase the expression of genes that favor tissue fibrosis. Early work from this and other laboratories has established a link between the aliphatic polyamines and cardiac hypertrophy, while more recently an involvement of polyamines even in cell death and survival has emerged. In the present study we have treated cardiac cells, i.e. rat H9c2 cardiomyoblasts and neonatal cardiomyocytes, with (D, L)-2-(difluoromethyl)ornithine, a specific inhibitor of polyamine biosynthesis, to investigate the effects of polyamines in relation to the hypertrophic, pro-fibrotic and pro-apoptotic actions of aldosterone. The results indicate that inhibition of polyamine biosynthesis may prevent or attenuate the adverse actions of aldosterone, by modulating the expression of genes related to cardiac hypertrophy and fibrosis, as well as the levels of proteins and the activities of enzymes that control apoptosis.

Polyamine biosynthesis as a target to inhibit apoptosis of non-tumoral cells.

Growing evidence suggests a role for polyamines in apoptosis, although the relationship appears to be complex. alpha-Difluoromethylornithine (DFMO), a largely used ornithine decarboxylase inhibitor, is cytostatic, hardly cytotoxic and may even increase the resistance of tumour cells to some apoptotic stimuli. This may represent a problem in cancer therapy, where the killing of tumoral cells would be a desired effect, but could be an advantage in other pathological contexts related to an excess of apoptosis, such as cardiovascular diseases, stem cell transplantation, arthritis and infections. In different cellular models, polyamine depletion following treatment with polyamine biosynthesis inhibitors appears to inhibit mitochondrial and death receptor pathways of apoptosis by affecting key proteins. These studies indicate that inhibition of polyamine biosynthesis may prevent or reduce the apoptotic response triggered by a variety of stimuli in non-tumoral cells, such as cardiac cells, stem cells, chondrocytes, macrophages and intestinal epithelial cells.

Signal transduction pathways linking polyamines to apoptosis.

Polyamines are important multifunctional cellular components and are classically considered as mediators of cell growth and division. Recently polyamines have been also implicated in cell death. Now it appears that polyamines are bivalent regulators of cellular functions, promoting proliferation or cell death depending on the cell type and on environmental signals. This review draws a picture about the role of polyamines in signalling pathways related to apoptotic cell death and the proposed molecular targets of these polycations at the level of the apoptotic cascade. Solid evidence indicates that polyamines may affect the mitochondrial and postmitochondrial phases of apoptosis, by modulating cytochrome c release from mitochondria and activation of caspases. Recently, polyamines have been also implicated in the regulation of the premitochondrial phase of apoptosis, during which upstream apoptotic signal transduction pathways are activated. The studies reviewed here suggest that polyamines may participate in loops involving interaction with signal transduction pathways and activation/expression of proteins that may control cell death or cell growth.

Polyamine depletion inhibits etoposide-induced NF-kappaB activation in transformed mouse fibroblasts.

In a previous research, we have shown that adequate levels of polyamines are required in transformed mouse fibroblasts for the correlated activations of MAPK subtypes (ERK and JNK) and caspases induced by etoposide and leading to apoptosis. We report now that the treatment of fibroblasts with etoposide also elicited a progressive and sustained increase of NF-kappaB activation. The DNA binding activity of p65 NF-kappaB subunit was increased up to approximately 4-fold and was accompanied by enhancement of p65 phosphorylation. A two days pre-treatment of fibroblasts with alpha-difluoromethylornithine (DFMO), which caused polyamine depletion, provoked a slight activating effect when given alone, but markedly inhibited the etoposide-induced increases in p65 DNA binding and phosphorylation. The NF-kappaB inhibiting effect of DFMO was prevented by the addition of exogenous putrescine, which restored the intracellular content of polyamines. Selective inhibitors of the etoposide-stimulated MAPK subtypes also reduced NF-kappaB activation. Moreover, pharmacological NF-kappaB inhibition reduced the increase in caspase activity and cell death elicited by etoposide, suggesting that NF-kappaB is involved in signaling to apoptosis. The results of the present study, together with our previous findings, suggest that polyamines play a permissive role in the pathways triggered by etoposide and leading to cell death of fibroblasts, by supporting the activation of MAPKs, NF-kappaB and caspases.

Control of survival of proliferating L1210 cells by soluble guanylate cyclase and p44/42 mitogen-activated protein kinase modulators.

Intracellular signaling pathways involved in the survival of proliferating L1210 leukemia cells were investigated by using specific modulators. Among the various inhibitors tested, only 1H-[1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase (sGC) inhibitor, was found to induce a marked increase in caspase activity, which was associated with a loss of cell viability and a reduction in cGMP content. ODQ also provoked the processing of caspases-3 and -9, release of cytochrome c and, as early events, reduction of Bcl-2 content and dephosphorylation of Bad at Ser 112. Furthermore, YC-1, an sGC activator, and 8-Br-cGMP, a cell-permeant analogue of cGMP, exerted some protection against various apoptotic stimuli, such as serum deprivation or spermine accumulation. Although PD98059 (2'-amino-3'-methoxyflavone), an inhibitor of the p44/42 mitogen-activated protein kinase (MAPK) pathway, did not increase basal caspase activity, and ODQ did not affect p44/42 MAPK phosphorylation significantly, phorbol myristate acetate stimulated p44/42 MAPK and reduced caspase activation induced by ODQ, serum deprivation, and spermine in a p44/42-dependent manner. SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)1H-imidazole), a p38 MAPK inhibitor, also partially protected against ODQ-induced apoptosis by increasing p44/42 MAPK phosphorylation. In conclusion, these results suggest that sGC may be relevant both for survival of L1210 cells under basal growing conditions and for protection against various apoptotic stimuli. p44/42 MAPK activation may also confer some protection from apoptosis, but apparently through a pathway largely independent of cGMP.

Effect of polyamine depletion on caspase activation: a study with spermine synthase-deficient cells.

Activation of the caspase proteases represents a central point in apoptosis. The requirement for spermine for the processes leading to caspase activation has been studied in transformed embryonic fibroblasts obtained from gyro (Gy) mutant male mice. These cells lack spermine synthase activity and thus provide a valuable model to study the role of spermine in cell processes. Gy fibroblasts do not contain spermine and have a higher spermidine content. However, when compared with fibroblasts obtained from normal male littermates (N cells), Gy fibroblasts were observed to grow normally. The lack of spermine did not affect the expression of Bcl-2, and caspases 3 and 9 were activated by etoposide in both N and Gy cells, indicating that spermine is dispensable for caspase activation. Spermine deficiency did not significantly influence caspase activity in cells treated with etoposide, cycloheximide or staurosporine, but sensitized the cells to UV irradiation, which triggered significantly higher caspase activity in Gy cells compared with N cells. alpha-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis that is able to deplete cells of putrescine and spermidine, but usually does not influence spermine content, was able to produce a more complete polyamine depletion in Gy cells. This depletion, which included spermine deficiency, dramatically increased caspase activation and cell death in Gy fibroblasts exposed to UV irradiation. On the other hand, in either N or Gy cells, DFMO treatment did not influence caspase activity triggered by staurosporine, but inhibited it when the inducers were cycloheximide or etoposide. In Gy cells depleted of polyamines by DFMO, polyamine replenishment with either spermidine or spermine was sufficient to restore caspase activity induced by etoposide, indicating that, in this model, polyamines have an interchangeable role in supporting caspase activation. Therefore, spermine is not required for such activation, and the effect and specificity of polyamine depletion on caspase activity may be very different, depending on the role of polyamines in the specific death pathways engaged by different stimuli. Some inducers of apoptosis, for example etoposide, absolutely require polyamines for caspase activation, yet the lack of polyamines, particularly spermine, strongly increases caspase activation when induced by UV irradiation.

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 (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.

Signaling pathways leading to the induction of ornithine decarboxylase: opposite effects of p44/42 mitogen-activated protein kinase (MAPK) and p38 MAPK inhibitors.

Treatment of serum-starved, human ECV304 cells with histamine or ATP elicited a transient induction of ornithine decarboxylase (ODC), a key enzyme in polyamine synthesis, to an extent similar to that provoked by phorbol myristate acetate or serum re-addition. All these agents also provoked an increase in active phosphorylated p44/42 mitogen-activated protein kinase (MAPK) and p38 MAPK. The involvement of p44/42 MAPK and p38 MAPK in the induction of ODC was investigated by using selective inhibitors. U0126 and PD98059, two specific p44/42 MAPK kinase inhibitors, prevented the induction of ODC elicited by any stimulus employed, whereas SB203580 and SB202190, which are widely used as p38 MAPK inhibitors, enhanced ODC induction in a way that appeared dependent on p44/42 MAPK activation. By using inhibitors of other key signaling proteins that may lead to activation of p44/42 MAPK, we provide evidence that protein kinase C, but not phosphoinositide 3-kinase, is involved in histamine-stimulated ODC induction. These results show that the p44/42 MAPK pathway, but not p38 MAPK, is essential for ODC induction stimulated either by agonists of G-protein-coupled receptors, phorbol esters, or serum, and suggest that the inhibition of ODC induction may be an important event in the antiproliferative response to p44/42 MAPK pathway inhibitors.

p44/42 mitogen-activated protein kinase is involved in the expression of ornithine decarboxylase in leukaemia L1210 cells.

The involvement of p44/42 mitogen-activated protein kinase (MAPK) in the induction of ornithine decarboxylase (ODC) was investigated by using PD98059, a specific MAPK-kinase (MEK1/2) inhibitor, and other signal-transduction inhibitors. In d,l-alpha-difluoromethylornithine (DFMO)-resistant L1210 cells stimulated to grow from quiescence, treatment with PD98059 inhibited p44/42 MAPK phosphorylation and the induction of ODC activity and protein. A marked reduction of the accumulation of mature ODC mRNA and its intron-containing precursor was observed, whereas ODC turnover was hardly affected. PD98059 also reduced the content of antizyme, but not that of antizyme mRNA. U0126, a novel and more potent inhibitor of MEK1/2, provoked a dose-dependent inhibition of ODC induction at lower concentrations with respect to PD98059. Other effective inhibitors of ODC induction proved to be genistein, manumycin A, herbimycin A, LY294002, wortmannin and KT5823, suggesting the involvement of other key proteins of signal-transduction pathways, i.e. Ras, Src, phosphatidylinositol 3-kinase and cGMP-dependent protein kinase, which may have a positive impact on MAPK. Cells kept in a DFMO-free medium, and thus containing high levels of putrescine and spermidine, showed enhanced MAPK phosphorylation and lower sensitivity to PD98059, compared with cells maintained in the presence of DFMO. In conclusion, these results indicate that the activation of p44/42 MAPK may favour the expression of ODC, and that polyamines, in turn, may affect the phosphorylation state of MAPK.

Nitric oxide can function as either a killer molecule or an antiapoptotic effector in cardiomyocytes.

Caspase enzymes are a family of cysteine proteases that play a central role in apoptosis. Recently, it has been demonstrated that caspases can be S-nitrosylated and inhibited by nitric oxide (NO). The present report shows that in chick embryo heart cells (CEHC), NO donor molecules such as S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione, spermine-NO or sodium nitroprusside inhibit caspase activity in both basal and staurosporine-treated cells. However, the inhibitory effect of NO donors on caspase activity is accompanied by a parallel cytotoxic effect, that precludes NO to exert its antiapoptotic capability. N-Acetylcysteine (NAC) at a concentration of 10 mM blocks depletion of cellular glutathione and cell death in SNAP-treated CEHC, but it poorly affects the ability of SNAP to inhibit caspase activity. Consequently, in the presence of NAC, SNAP attenuates not only caspase activity but also cell death of staurosporine-treated CEHC. These data show that changes in the redox environment may inhibit NO-mediated toxicity, without affecting the antiapoptotic capability of NO, mediated by inhibition of caspase enzymes. NO may thus be transformed from a killer molecule into an antiapoptotic agent.

Nitric oxide mediates either proliferation or cell death in cardiomyocytes. Involvement of polyamines.

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.

Modulation of the induction of ornithine decarboxylase by some opioid receptor agonists in immune cells and cardiomyocytes.

The ability of natural and synthetic opioids to modulate the induction of ornithine decarboxylase (ODC) was investigated in immune cells and cardiomyocytes in culture. In particular, Leu-enkephalin, which shows preference for delta-receptors, enhanced ODC activity in both thymocytes and cardiomyocytes, whereas the effect of U-50488H, a synthetic kappa-selective agonist, was cell-specific. In thymocytes, U-50488H markedly inhibited the induction of the enzyme elicited by the mitogen concanavalin A (Con A) or by a combined treatment with PMA and A23187, and also reduced basal ODC activity. However the drug did not affect ODC induced by other stimuli. The inhibition of the induction of ODC activity was accompanied by a reduction of ODC mRNA level and an acceleration of ODC turnover. The action of U-50488H in thymocytes does not appear to be mediated by kappa or other classical opioid receptors lacking both stereospecificity and antagonist sensitivity, but may involve a pertussis toxin-sensitive G protein. Splenocytes also showed the ODC inhibiting effect of U-50488H, although they were less sensitive compared to thymocytes. In contrast, U-50488H enhanced ODC activity in cardiomyocytes and this effect was blocked by a specific kappa-antagonist. In conclusion, these results indicate that some opioid agonists can modulate ODC expression in non neural cells. In particular, kappa-opioid receptors may be involved in the U-50488H action in cardiomyocytes, and a distinct site, linked to inhibition of cell proliferation, may operate in immune cells.

Enzymes involved in guanine monophosphate metabolism of aging chicken heart.

The activities of enzymes involved in GMP metabolism were studied in the heart of aging chickens. In newborn (1-day-old) animals, GMP breakdown apparently leads to the final products of purine metabolism, as the activity of hypoxanthine-guanine phosphoribosyl-transferase (HGPRT), the salvage enzyme of GMP is not detectable. On the contrary, HGPRT shows maximal activity in young (20-day-old) chickens, when xanthine oxidase activity is very low, indicating that the metabolic flux converges on the salvage pathway. Again, maximal activity of the catabolic enzymes and a limited resort to the salvage pathway characterize GMP metabolism of adult (12-month-old) hearts. Finally, in aged (30-month-old) chickens, a reduced GMP catabolism and a greater utilization of the salvage pathway might contribute to the maintenance of the guanine nucleotide pool. In conclusion, the pattern of the activities of enzymes relating to GMP metabolism in the aging heart, compared to AMP metabolism, indicates a parallel temporal regulation of the purine pathways.

Beta 2-adrenergic receptor-stimulated increase in cAMP in rat heart cells is not coupled to changes in Ca2+ dynamics, contractility, or phospholamban phosphorylation.

Previous studies have shown that both beta 1- and beta 2-adrenergic receptors (AR) are present in rat ventricular myocytes, but stimulation of these receptor subtypes elicits qualitatively different cellular responses (Xiao, R.-P., and Lakatta, E. G. (1993) Circ. Res. 73, 286-300). In the present study, the biochemical mechanism underlying the distinct beta AR subtype actions have been investigated. Although both beta 1AR and beta 2AR stimulation increased total cellular cAMP in suspensions of rat ventricular myocytes to a similar extent, the maximum elevation of the membrane bound cAMP by beta 2AR stimulation was only half of that induced by beta 1AR stimulation, suggesting that stimulation the beta AR subtypes leads to different compartmentation of cAMP. The effects of beta 1AR stimulation on Ca2+ transient (indexed by the transient increase in indo-1 fluorescence ration after excitation) and contraction amplitude (measured via photodiode array) and their kinetics closely paralleled the increase in cAMP. In contrast, the increase in both membrane bound and total cAMP content after beta 2AR stimulation were completely dissociated from the effects of beta 2AR stimulation to increase the amplitudes of cytosolic Ca2+ transient and contraction. Furthermore, beta 2AR stimulation did not phosphorylate phospholamban to the same extent as did beta 1AR stimulation. This finding provides a mechanism for the failure of beta 2AR stimulation to accelerate the kinetics of the Ca2+i (cytosolic Ca2+) transient and contraction. These results indicate that the effects of beta 2AR stimulation on Ca2+i transient and contraction are uncoupled from the cAMP production and cAMP-dependent protein phosphorylation and indicate that, in addition to coupling to adenylate cyclase, beta 2AR stimulation also activates other signal transduction pathway(s) to produce changes in cytosolic Ca2+ and contraction.

Influence of Mg2+ on the in vitro responsiveness of adenylate cyclase from hearts of aging rats.

The influence of [Mg2+] on the basal or stimulated activity of adenylate cyclase from the hearts of young (1 month old) and aged (24 months old) rats has been investigated in vitro. The basal activity of cardiac adenylate cyclase, and its responsiveness to stimulatory or inhibitory effectors, declined with age. This is probably due to alterations at the catalytic moiety of the signal transduction system, such as an impairment in the affinity of the catalytic moiety for ATP and a lower capacity of the catalytic moiety to bind activated stimulatory (Gs) or inhibitory (Gi) guanine nucleotide binding proteins. Compared to the enzyme from the heart of aged rats, unstimulated adenylate cyclase from the heart of young rats was more sensitive to an increase in [Mg2+] in the incubation mixture, as shown by a greater increase in basal activity and in the affinity of the enzyme for ATP. An increase in [Mg2+] counteracted the inhibitory effect of spermine on adenylate cyclase more effectively in young rats than in aged rats. On the other hand, an increase in [Mg2+] facilitated the stimulation of adenylate cyclase by Gpp(NH)p, isoproterenol and forskolin more in aged rats than in young rats. GDP beta S prevented the positive effect of high [Mg2+] on the stimulation of adenylate cyclase by forskolin, suggesting that an increased [Mg2+] favors the activation of Gs or the formation of functional complexes between the catalytic moiety and Gs. We suggest that aging leads to a higher requirement for Mg2+ at the allosteric site on the catalytic moiety whose occupancy is essential for the full expression of stimulated activity.

[Variations in the functionality of cardiac adenyl cyclase as a function of age]. / Variazioni della funzionalità dell'adenilico ciclasi cardiaca in funzione dell'età.

The metabolic and functional activity of the heart closely depends on cAMP and therefore on the integrity of adenylate cyclase (AC) system. Alterations of this signal transduction system might be co-responsible for the impairment of cardiac performance observed with aging. Evidence is here provided that basal activity of cardiac membrane-bound (48,000 x g) AC significantly declines with the age of the rat (1, 12, 24 month-old). This is accompanied with the decrease of cAMP content, which leads to the fall of cAMP/cGMP molar ratio a possible final determinant of cardiac performance. Kinetic analyses indicate that aging is associated with a net increase of the Km of a cardiac AC, while the Vmax is unaffected. Besides, the response in vitro of AC from 24-month-old heart to the inhibitor spermine or a different stimulants, such as Gpp (NH) p, isoproterenol, PGE1 or forskolin, is significantly lower than that of AC from 1 month-old one. The suggestion is made that aging causes an impairment in the capability of the catalytic moiety of cardiac AC to make functional complexes with activated guanine nucleotide binding proteins.

Influence of docosahexaenoic acid on phosphatidylinositol metabolism in cultured cardiomyocytes.

Polyunsaturated fatty acids are involved at several steps in the turnover of phosphatidylinositol mediated by alpha 1-adrenoceptors. We have cultured neonatal rat cardiomyocytes in a medium containing docosahexaenoic acid (C22:6n3) and have investigated the effects produced by the change in the fatty acid composition of phosphatidylinositol on alpha 1-adrenoceptor-mediated phosphatidylinositol metabolism. The experimental cells, in which phosphatidylinositol was significantly enriched in docosahexaenoic acid and depleted in arachidonic acid, showed a reduced ability to incorporate labeled inositol into phospholipids in comparison with control cells, both in basal conditions and after stimulation by phenylephrine. This decreased incorporation led to a reduced availability of inositol phospholipids, substrates of phospholipase C, and to a reduced production of inositol phosphates under basal conditions.

Effect of bacterial toxins on spermine-induced inhibition of adenylate cyclase activity of cultured heart cells.

The exposure of quiescent cultures of cardiac cells to 1 microM spermine for 2 hours leads to an increase of the content of intracellular polyamines and to a 40% decrease of basal adenylate cyclase activity. The response of adenylate cyclase to stimulation by PGE1 is reduced by about 50% after spermine treatment. The effects of the amine on adenylate cyclase are completely prevented by pretreating the cells with pertussis toxin which blocks the activation of the inhibitory guanine binding protein (Gi). In vitro experiments with adenylate cyclase from cells pre-treated with pertussis toxin show that spermine fails to reduce basal enzyme activity and to counteract the stimulation by PGE1 or forskolin. Cholera toxin, which blocks the deactivation of the stimulatory protein (Gs), does not influence the effects of spermine either in vivo or in vitro. The results suggest that spermine acts through the activation of Gi. This hypothesis is supported by the fact that, in vitro, the inhibition of stimulated adenylate cyclase by the amine is synergistic with that of a stable analog of GDP, GDP beta S, which causes deactivation of Gs.

Study on the role of endogenous polyamines in glucagon, isoproterenol or serum-mediated induction of tyrosine aminotransferase in cultured heart cells.

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.