Carla Pignatti

Antiproliferative effect of sildenafil on human pulmonary artery smooth muscle cells

AbstractPulmonary arterial hypertension (PAH) is characterized by vasoconstriction and by obstructive changes of the pulmonary vasculature including smooth muscle cell proliferation which leads to medial hypertrophy and subsequent luminal narrowing. Sildenafil, an orally active inhibitor of cGMP phosphodiesterase–type–5, exerts pulmonary vasodilator activity in PAH patients. We evaluated the effects of sildenafil on growth of cultured human pulmonary artery smooth muscle cells (PASMC). The results indicate that sildenafil reduced DNA synthesis stimulated by PDGF and dose dependently inhibited PASMC proliferation. These effects were paralleled by a progressive increase in cGMP content, followed by an accumulation of cAMP. The treatment with 8–bromo–cGMP or dibutyryl–cAMP mimicked all the effects of sildenafil. On the other hand, treatment of PASMC with inhibitors of cGMP–dependent protein kinase (PKG) or cAMP–dependent protein kinase (PKA) reversed the antiproliferative effect of sildenafil. In addition, sildenafil inhibited the phosphorylation of ERK, a converging point for several pathways leading to cell proliferation. This effect was partially reduced by PKG inhibition and completely abolished by PKA inhibition.We conclude that sildenafil exerts an antiproliferative effect on human PASMC that is mediated by an interaction between the cGMP–PKG and the cAMP–PKA activated pathways, leading to inhibition of PDGF–mediated activation of the ERK.

Spermine causes caspase activation in leukaemia cells

Exposure of several leukaemia cell types to the polyamine spermine triggered caspase activation. In HL60 cells, the onset of caspase activity correlated with the accumulation of spermine, and was accompanied by the processing of the caspase‐3 precursor and the digestion of the substrate proteins PARP and gelsolin. Spermine also induced the accumulation of cytochrome c in the cytosol. Caspase activation triggered by spermine was not blocked by antioxidants or inhibition of polyamine oxidase. The deregulation of polyamine uptake strongly sensitised the cells to spermine‐induced caspase activation. These data show that an excessive intracellular level of spermine triggers caspase activation that is not mediated by oxidative mechanisms, and suggest a model where elevated free cytosolic polyamines may act as transducers of a death message.

Signal transduction pathways linking polyamines to apoptosis

Summary.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.

Polyamines directly induce release of cytochrome c from heart mitochondria

Cytochrome c release from mitochondria to the cytosol represents a critical step in apoptosis, correlated to the activation of the caspase cascade. In this report, we show that addition of micromolar concentrations of polyamines to isolated rat heart mitochondria induces the release of cytochrome c. Spermine, which is effective at concentrations of 10-100 microM, is more potent than spermidine, whereas putrescine has no effect up to 1 mM. The release of cytochrome c caused by spermine is a rapid, saturable and selective process that is independent of mitochondria damage. Spermine, unlike polylysine, is able to release a discrete amount of cytochrome c from intact, functional mitochondria. The cytochrome c-releasing power of spermine is not affected by cyclosporin A, differently from the effect of permeability transition inducers. In a cardiac cell-free model of apoptosis, the latent caspase activity of cytosolic extracts from cardiomyocytes could be activated by cytochrome c released from spermine-treated heart mitochondria. These data indicate a novel mechanism of cytochrome c release from the mitochondrion, and suggest that prolonged and sustained elevation of polyamines, characteristic of some pathologies such as heart hypertrophy, could be involved in the development of apoptosis.

Caspase activation in etoposide-treated fibroblasts is correlated to ERK phosphorylation and both events are blocked by polyamine depletion.

Activation of the extracellular signal‐regulated kinases (ERKs) 1 and 2 is correlated to cell survival, but in some cases ERKs can act in signal transduction pathways leading to apoptosis. Treatment of mouse fibroblasts with 20 μM etoposide elicited a sustained phosphorylation of ERK 1/2, that increased until 24 h from the treatment in parallel with caspase activity. The inhibitor of ERK activation PD98059 abolished caspase activation, but caspase inhibition did not reduce ERK 1/2 phosphorylation, suggesting that ERK activation is placed upstream of caspases. Both ERK and caspase activation were blocked in cells depleted of polyamines by the ornithine decarboxylase inhibitor α‐difluoromethylornithine (DFMO). In etoposide‐treated cells, DFMO also abolished phosphorylation of c‐Jun NH2‐terminal kinases triggered by the drug. Polyamine replenishment with exogenous putrescine restored the ability of the cells to undergo caspase activation and ERK 1/2 phosphorylation in response to etoposide. Ornithine decarboxylase activity decreased after etoposide, indicating that DFMO exerts its effect by depleting cellular polyamines before induction of apoptosis. These results reveal a role for polyamines in the transduction of the death signal triggered by etoposide.

SPERMINE TRIGGERS THE ACTIVATION OF CASPASE-3 IN A CELL-FREE MODEL OF APOPTOSIS

Polyamines are ubiquitous organic cations required for cell proliferation. However, some evidence suggested that their excessive accumulation can induce apoptosis. We show here that, in a post‐nuclear extract from U937 cells, the addition of spermine triggers the death program, represented by cytochrome c exit from mitochondria, the dATP‐dependent processing of pro‐caspase‐3 and the onset of caspase activity. Spermine is more effective than spermidine, whereas putrescine has no effect. Polyamine acetylation abolishes their pro‐apoptotic power. These data demonstrate a direct mechanism responsible for polyamine toxicity and also suggest that an excessive elevation of free polyamines could be involved in the transduction of a death signal.

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.

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.

Reduced mechanical activity of perfused rat heart following morphine or enkephalin peptides administration

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.

NF-κB and ERK cooperate to stimulate DNA synthesis by inducing ornithine decarboxylase and nitric oxide synthase in cardiomyocytes treated with TNF and LPS

We previously reported that tumor necrosis factor‐α (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‐κB (NF‐κB) in the induction of nitric oxide synthase (NOS) and ornithine decarboxylase (ODC), the key enzyme in polyamine biosynthesis. In addition NF‐κB 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‐κB activation. In conclusion, these results indicate that NF‐κB, 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.