Claudio M. Caldarera

Role of oxygen in the cellular damage induced by re-oxygenation of hypoxic heart

Abstract The role of oxygen in the induction of cellular damages during re-oxygenation of hypoxic hearts was studied in isolated Langendorff perfused rat hearts. An increase in the period of hypoxia of up to 80 min resulted in a decrease in both superoxide dismutase and glutathione peroxidase activity. This decrease continued when the hearts perfused in hypoxia for periods of 40 and 60 min were re-oxygenated for an additional 20 min. Under these conditions, re-oxygenation caused an increase in lipid peroxidation which was evaluated as malondialdehyde formation. In comparison to aerobic hearts, the cellular contents of acid soluble thiol groups, insoluble thiol groups and reduced glutathione were all decreased by hypoxia, while oxidized glutathione remained unchanged. The re-admission of oxygen to hypoxic hearts produced a further reduction in thiol groups and reduced glutathione. In contrast to well-oxygenated rat hearts, perfusion with substrate-free hypoxic medium resulted in a marked release of reduced glutathione into coronary effluents, which also continued after re-oxygenation. In hypoxia the release of oxidized glutathione began after 30 min of perfusion and slightly increased during the next 60 min of hypoxia. Re-oxygenation after 60 min of hypoxia did not enhance hypoxic oxidized glutathione release. These results suggest that re-oxygenation is able to induce lipid peroxidative damage in cardiac rat tissue when hypoxic substrate-free perfusions had previously reduced the cellular defensive mechanisms capable of neutralizing the toxic reactions mediated by oxygen metabolites.

Oxygen-mediated myocardial damage during ischameia and reperfusion: Role of the cellular defences against oxygen toxicity

The possibility that myocardial ischaemia alters the defence mechanisms against oxygen toxicity has been investigated. Ischaemia was induced in isolated, perfused rabbit hearts by reducing coronary flow from 25 ml/min to 1 ml/min for 90 min. Two different degrees of ischaemic damage have been achieved using either spontaneously beating or electrically stimulated hearts. The effects of post-ischaemic reperfusion were also followed for 30 min. Tissue activity of superoxide dismutase (SOD), glutathione peroxidase and reductase (GPD and GRD) have been determined together with tissue content of reduced and oxidized glutathione (GSH and GSSG) and of protein SH groups. The changes in myocardial ATP and CP content and release of CPK and of GSH and GSSG were also determined. Systolic and diastolic pressures were continuously monitored. In the spontaneously beating hearts ischaemia induced a reduction of tissue GSH and protein SH groups. On reperfusion there was a recovery of mechanical function, a transient release of GSH into the coronary effluent and an increase of tissue GSH. In the paced hearts, ischaemia resulted in 50% reduction of mitochondrial SOD activity together with a reduction of tissue GSH and protein SH groups. Reperfusion induced a massive release of CPK and of GSH and GSSG, a further reduction of tissue GSH concomitant with an increase of GSSG and no recovery of mechanical function. GPD and GRD activity were not affected by ischaemia and reperfusion. These data indicate that severe ischaemia induces a reduction of the protective mechanisms against oxygen toxicity.

Induction of nitric oxide synthase mRNA expression. Suppression by exogenous nitric oxide.

The reactive nitrogen species, nitric oxide (NO), plays an important role in the pathogenesis of neurodegenerative diseases. The suppression of NO production may be fundamental for survival of neurons. Here, we report that pretreatment of human ramified microglial cells with nearly physiological levels of exogenous NO prevents lipopolysaccharide (LPS)/tumor necrosis factor α (TNFα)-inducible NO synthesis, because by affecting NF-κB activation it inhibits inducible Ca-independent NO synthase isoform (iNOS) mRNA expression. Using reverse transcriptase polymerase chain reaction, we have found that both NO donor sodium nitroprusside (SNP) and authentic NO solution are able to inhibit LPS/TNFα-inducible iNOS gene expression; this effect was reversed by reduced hemoglobin, a trapping agent for NO. The early presence of SNP during LPS/TNFα induction is essential for inhibition of iNOS mRNA expression. Furthermore, SNP is capable of inhibiting LPS/TNFα-inducible nitrite release, as determined by Griess reaction. Finally, using electrophoretic mobility shift assay, we have shown that SNP inhibits LPS/TNFα-elicited NF-κB activation. This suggests that inhibition of iNOS gene expression by exogenous NO may be ascribed to a decreased NF-κB availability.

Opioid receptors in rat cardiac sarcolemma: effect of phenylephrine and isoproterenol

The present study demonstrates the presence of opioid receptors in the rat cardiac sarcolemma isolated by the hypotonic LiBr-shock procedure. Opioid binding was measured by using [3H]U69 593, [3H](2-D-penicillamine,5-D-penicillamine)enkephalin ([3H]DPDPE) or [3H][D-Ala2,MePhe4,Gly-(ol)5]enkephalin ([3H]DAGO) as selective radioligands for K, delta and mu opioid receptors, respectively. Both the K- and delta-selective ligands exhibited highly specific (75-86%) binding, saturable at a concentration of about 20 nM. No specific binding for the selective agonist DAGO was observed. A marked increase in both [3H]U69 593 and [3H]DPDPE binding was observed after incubation of the sarcolemma with the alpha-adrenoceptor agonist phenylephrine or with the beta-adrenoceptor agonist isoproterenol. These stimulatory effects were associated with an increase in the Bmax values, a decrease in the Kd values, and were completely antagonized by the respective antagonists phentolamine and propranolol.

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.

Green tea protection of hypoxia/reoxygenation injury in cultured cardiac cells

Antioxidant-rich diets exert a protective effect in diseases involving oxidative damage. Among dietary components, green tea is an excellent source of antioxidants. In this study, cultured neonatal rat cardiomyocytes were used to clarify the protective effect of a green tea extract on cell damage and lipid peroxidation induced by different periods of hypoxia followed by reoxigenation. Cultures of neonatal rat cardiomyocytes were exposed to 2--8 hr hypoxia, eventually followed by reoxygenation, in the absence or presence of alpha-tocopherol or green tea. LDH release and the production of conjugated diene lipids were measured, and appeared linearly related to the duration of hypoxia. During hypoxia, both LDH release and conjugated diene production were reduced by alpha-tocopherol and, in a dose dependent manner, by green tea, the 50 &mgr;g/ml being the most effective dose. Reoxygenation caused no further increase in LDH leakage, while it caused a significant increase in conjugate dienes, which absolute value was lower in antioxidant supplemented cells. Anyway, the ratio between conjugated diene production after hypoxia and after reoxygenation was similar in all groups, indicating that the severity of free radical-induced reoxygenation injury is proportional to the severity of previous hypoxic injury. Since hypoxic damage is reduced by alpha-tocopherol and green tea, our data suggest that any nutritional intervention to attenuate reoxygenation injury must be directed toward the attenuation of the hypoxic injury. Therefore, recommendations about a high dietary intake of antioxidants may be useful not only in the prevention, but also in the reduction of cardiac injury following ischemia.

H9c2 cardiac myoblasts undergo apoptosis in a model of ischemia consisting of serum deprivation and hypoxia: inhibition by PMA

Cardiac myocytes undergo apoptosis under condition of ischemia. Little is known, however, about the molecular pathways that mediate this response. We show that serum deprivation and hypoxia, components of ischemia in vivo, resulted in apoptosis of rat ventricular myoblast cells H9c2. Hypoxia alone did not induce significant apoptosis for at least 48 h, but largely increased the proapoptotic action of serum deprivation. H9c2 cells apoptosis is evidenced by an increase in terminal (TdT)‐mediated dUTP nick end‐labeling‐positive nuclei and by activation of caspases 3, 6, 7 and 9, and loss of mitochondrial functions. In this model of simulated ischemia, represented by serum deprivation plus hypoxia, cardiomyoblasts apoptosis was associated with a p53‐independent Bax accumulation and with a down‐regulation of Bcl‐xL, whereas the levels of cIAP‐1, cIAP‐2 and X‐IAP proteins did not change. Phorbol‐12‐myristate‐13‐acetate significantly reduced the induction of apoptosis, inhibiting caspase 3 cleavage, Bax accumulation, Bcl‐xL down‐regulation as well as restoring cell viability.

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.

Mitochondrial function and superoxide generation from submitochondrial particles of aged rat hearts

A decrease in heart function with ageing might be related to an impairment of mitochondrial function, since these organelles produce the greatest fraction of ATP in the myocyte. Mitochondria extracted from Wistar rat hearts at 3, 14, 18 and 24 months of age were employed to evaluate the changes of the respiratory activity during lifetime. A slight decrease of the respiratory rate (QO2) was observed in the 14 month group with respect to the 3 month group when succinate was used as substrate, whereas the respiratory control index (RCI) in the presence of glutamate or succinate increased in the 24 month group. The latter result may be related to a condition of moderate hypertrophy that generally occurs in the ageing heart. Submitochondrial particles (SMP) were also prepared to study the superoxide radicals (O2-) production at the level of rotenone or antimycin-inhibited regions of the respiratory chain. A strong elevation in the O2- generation was observed in the antimycin-inhibited region at 14 months of age; on the contrary, the rate of O2- production remained unchanged in the 24 month group in comparison to the youngest group. These observations correlate well with the enhanced tissue level of oxidized glutathione that was observed at 14 and 18 months of age. The products of lipid peroxidation (TBARS) did not change in the rat heart at any of the ages measured, whereas the levels of fluorescent substances progressively increased beginning from 18 months of age, with a greater extent in the mitochondrial compartment. The present study suggests that age does not substantially affect mitochondrial respiration and energy output in the rat heart, while a greater production by cardiac mitochondria of superoxide anions in the adult rats (14 months) might accelerate the fluorescent pigment formation.