The role of extracellular conditions during CaCo-2 cells growth: a preliminary study for numerical model validation.

OBJECTIVES: One important limitation in cell therapy protocols, and regenerative medicine (an innovative and promising strategy for different pathologies treatment), is the lack of knowledge about cells engraftment, proliferation and differentiation. In order to allow an efficient and successful cell transplant, it is necessary to predict the logistics, economic and timing issues during cellular injection. It has been reported that several parameters, such as cells number, temperature and extracellular pH (pH0) value can influence metabolic pathways and cellular growth. Numerical analysis and model can help to reduce and understand the effects of the above environmental conditions on cell survival. The aim of this paper is to develop the first step of cells transplantation in order to identify "in vitro", which parameters can be useful to develop and validate a numerical model, able to evaluate "in vivo" cells engraftment and proliferation. MATERIAL AND METHODS: We studied the variation of extracellular parameters--such as medium volume, buffer system, nutrient concentrations and temperature on human colon carcinoma cells (CaCo-2) "in vitro culture"--pursuing the goal of understanding in deeper details cellular processes such as growth, metabolic activity, survival and pH0. RESULTS: Results showed that CaCo-2 cells growth and mortality increase after two days in culture when cells were suspended in 3.5 ml volume to respect of 10 ml volume. Different temperature values influenced CaCo-2 cells growth and metabolic activity showing a direct relationship with the volume of the medium. CONCLUSIONS: Our results describe as CaCo-2 cell growth, metabolic activity, mortality and extracellular pH were influenced by extracellular parameters, enabling us to develop and validate a numerical model to be use to predict cells engraftment and proliferation.

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.

Cardiac resynchronization therapy: could a numerical simulator be a useful tool in order to predict the response of the biventricular pacemaker synchronization?

BACKGROUND AND OBJECTIVES: Cardiac resynchronization therapy (CRT) can be considered as an established therapy for patients with moderate or severe heart failure (HF), depressed systolic function and a wide QRS complex. Biventricular stimulation through the CRT is applied at patients with an intra and/or inter-ventricular conduction delay. The goal of this technique is to resynchronize contraction between and within ventricles. A numerical model of the cardiovascular system, together with the numerical model of the biventricular pacemaker (BPM), can be an useful tool to study the better synchronization of the BPM in order to reduce the inter-ventricular and/or intra-ventricular conduction delay. SUBJECTS AND METHODS: Within a group of patients which were representative of the most common disease etiologies of heart failure, seven patients, affected by dilated cardiomyopathy undergoing CRT with BPM, were studied and simulated using the numerical model of the cardiovascular system CARDIOSIM. The patients were submitted to echocardiographic evaluation (with pulsate Doppler and tissue Doppler imaging) and electrocardiography evaluation in order to evaluate intra-ventricular and/or inter-ventricular dyssynchrony. These evaluations were made three times: the first one before BPM implantation, the second and the third one respectively within seven days and six months after BPM implantation. Also haemodynamic parameters were measured. Using the software simulator, the pathological conditions before CRT, within seven days and within six months since CRT were reproduced for each patients in order to evaluate the following haemodynamic parameters: the end-systolic and end-diastolic left ventricular volume, the systolic pulmonary arterial pressure, the systolic, diastolic and mean aortic blood pressure and the ejection fraction. Also the trend of the left ventricular elastance was studied for each patient in order to evaluate the benefits produced by the CRT. RESULTS: The results obtained by means the numerical simulator were in good agreement with clinical data measured on the patients. For each patient also the evolution of the left ventricular elastance was in accordance with the literature data. CONCLUSION: The cardiovascular numerical model seems to be a useful tool to study the synchronization of the BPM in order to reduce the inter-ventricular and/or intra-ventricular conduction delay and to reproduce the condition of a patient.

Mesenchymal stem cell interaction with a non-woven hyaluronan-based scaffold suitable for tissue repair.

The fabrication of biodegradable 3-D scaffolds enriched with multipotent stem cells seems to be a promising strategy for the repair of irreversibly injured tissues. The fine mechanisms of the interaction of rat mesenchymal stem cells (rMSCs) with a hyaluronan-based scaffold, i.e. HYAFF(R)11, were investigated to evaluate the potential clinical application of this kind of engineered construct. rMSCs were seeded (2 x 10(6) cells cm(-2)) on the scaffold, cultured up to 21 days and analysed using appropriate techniques. Light (LM), scanning (SEM) and transmission (TEM) electron microscopy of untreated scaffold samples showed that scaffolds have a highly porous structure and are composed of 15-microm-thick microfibres having a rough surface. As detected by trypan blue stain, cell adhesion was high at day 1. rMSCs were viable up to 14 days as shown by CFDA assay and proliferated steadily on the scaffold as revealed by MTT assay. LM showed rMSCs in the innermost portions of the scaffold at day 3. SEM revealed a subconfluent cell monolayer covering 40 +/- 10% of the scaffold surface at day 21. TEM of early culture showed rMSCs wrapping individual fibres with regularly spaced focal contacts, whereas confocal microscopy showed polarized expression of CD44 hyaluronan receptor; TEM of 14-day cultures evidenced fibronexus formation. Immunohistochemistry of 21-day cultures showed that fibronectin was the main matrix protein secreted in the extracellular space; decorin and versican were seen in the cell cytoplasm only and type IV collagen was minimally expressed. The expression of CD90, a marker of mesenchymal stemness, was found unaffected at the end of cell culture. Our results show that HYAFF(R)11 scaffolds support the adhesion, migration and proliferation of rMSCs, as well as the synthesis and delivery of extracellular matrix components under static culture conditions without any chemical induction. The high retention rate and viability of the seeded cells as well as their fine modality of interaction with the substrate suggest that such scaffolds could be potentially useful when wide tissue defects are to be repaired as in the case of cartilage repair, wound healing and large vessel replacement.

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.

Characterization of highly purified ornithine decarboxylase from rat heart.

A highly purified preparation of heart ornithine decarboxylase was obtained from isoproterenol-treated rats. The molecular and catalytic properties of the cardiac enzyme were investigated. The isoelectric point of the enzyme appeared to be 4.9, and the molecular weight was estimated to be 54000 by SDS-polyacrylamide gel electrophoresis. Under nondenaturing conditions, the molecular weight of the partially purified enzyme was 10000-110000 as determined by gel filtration, whereas a significantly lower (Mr approx. 70000) value was obtained for purified ornithine decarboxylase. Both Km for the substrate and Vmax were affected by the dithiothreitol concentration in the assay mixture. In particular, the Km for ornithine was found to be about 0.09 mM in the presence of 2.9 mM dithiothreitol and appeared to decrease at lower dithiothreitol concentrations. The Km for pyridoxal phosphate was about 0.09 microM; putrescine and lysine inhibited the enzyme competitively, with Ki values of 1.3 and 11.7 mM, respectively. The existence of two different forms of ornithine decarboxylase in cardiac tissue was indicated by DEAE-cellulose chromatography.

Incorporation of [14C]hypoxanthine into cardiac adenine nucleotides: effect of aging and post-ischemic reperfusion.

In order to investigate whether the 'hypoxanthine salvage' pathway of the cardiac muscle is modified with age, we aerobically perfused isolated hearts of 4-month- and 22-month-old male Wistar rats for 20 min with 0.18 microM [14C]hypoxanthine. A second group of hearts was subjected to a 30-min ischemic perfusion (95% reduction of the coronary flow), followed by 20 min of reperfusion. In this last 20 min, the perfusate contained the same concentration of [14C]hypoxanthine used under the aerobic condition. After 20 min of aerobic perfusion the myocardial levels of ATP were significantly lower (15%) in aged than young rat hearts, whilst no age-related differences were observed at the end of the reperfusion. In the young rats the incorporation of the isotope into ATP, ADP, and AMP was significantly higher (192%, 226%, and 300%, respectively), after 20 min of reperfusion with respect to the aerobic values. On the contrary, in the aged hearts, no significant change in the rate of [14C]-incorporation into ATP was observed during reperfusion, despite an increase of the [14C]-incorporation into ADP and AMP. Moreover, the content of each labeled adenine nucleotide was significantly higher in aged than young hearts at the end of the aerobic period, whereas the incorporation of the labeled hypoxanthine was not affected by age after 20 min of reperfusion. The release of uric acid into coronary effluents was greater (50%) in aged than young rats during the reperfusion period, but no age-dependent differences in the isotope incorporation into uric acid were observed. These data indicate that in the aged rat heart, perfused under aerobic conditions, there is an increased incorporation of hypoxanthine into ATP, although it does not further increase during postischemic reperfusion.

Modulation of ornithine decarboxylase activity and ornithine decarboxylase-antizyme complex in rat heart by hormone and putrescine treatment.

Ornithine decarboxylase was present in a cryptic, complexed form in an amount approximately equivalent to that of free ornithine decarboxylase activity in adult rat heart. Addition of isoproterenol (10 mg/kg) caused a notable rise in ornithine decarboxylase activity and a simultaneous decrease in the amount of the complexed enzyme. During the period of ornithine decarboxylase decay, when cardiac putrescine content had reached high values, the level of the complex increased above that of the control. Administration of putrescine (1.5 mmol/kg, twice) or dexamethasone (4 mg/kg) produced a decrease of heart ornithine decarboxylase activity, while it did not remarkably affect the level of complexed ornithine decarboxylase, therefore raising significantly the ratio of bound to total ornithine decarboxylase. Putrescine also elicited the appearance of free antizyme, concomitantly with the disappearance of free ornithine decarboxylase activity after 3-4 h of treatment. These results indicate that a significant amount of ornithine decarboxylase occurs in an inactive form in the heart under physiological conditions and that its absolute and relative levels may vary following stimuli which affect heart ornithine decarboxylase activity.

Effect of oxygen radicals and hyperoxia on rat heart ornithine decarboxylase activity.

Rat heart ornithine decarboxylate activity from isoproterenol-treated rats was inactivated in vitro by reactive species of oxygen generated by the reaction xanthine/xanthine oxidase. Reduced glutathione, dithiothreitol and superoxide dismutase has a protective effect in homogenates and in partially purified ornithine decarboxylase exposed to the xanthine/xanthine oxidase reaction, while diethyldithiocarbamate, which is an inhibitor of superoxide dismutase, potentiated the damage induced by O2- on enzyme activity. Dithiothreitol at concentrations above 1.25 mM had an inhibitory effect upon supernatant ornithine decarboxylase activity, while at 2.5 mM it was most effective in the recovery of ornithine decarboxylase activity, after the purification of the enzyme by the ammonium sulphate precipitation procedure. The ornithine decarboxylase inactivated by the xanthine/xanthine oxidase reaction showed a higher value of Km and a reduction of Vmax with respect to control activity. The exposure of rats to 100% oxygen for 3 h reduced significantly the isoproterenol-induced heart ornithine decarboxylase activity. The injection with diethyldithiocarbamate 1 h before hyperoxic exposure further reduced heart ornithine decarboxylase activity.

Effects of dexamethasone on spermidine N1-acetyltransferase and ornithine activities in rat spleen.

Treatment of rats with the glucocorticoid dexamethasone causes an increase in the activity of cytosolic spermidine N1-acetyltransferase both in the spleen and thymus, but not, however, in liver, kidney or lung. The induced spermidine N1-acetyltransferase activity in the spleen catalyses acetylation of spermidine as well as spermine and sym-norspermidine, but not of diamines and histones. The enzyme induction depends on the dose of dexamethasone, and is suppressed by cycloheximide, which suggests that de novo protein synthesis is required for the action of this glucocorticoid. N1-acetylspermidine accumulates in the spleen after dexamethasone treatment, while spermidine progressively decreases and is partly converted into putrescine, the content of which transiently increases. In accordance with previous reports, dexamethasone was found to cause a rapid and large fall in the activity of spleen ornithine decarboxylase which was effected via the appearance of an inhibitor of the enzyme. Glucocorticoids exert large catabolic effects on lymphoid tissues, and further selectively affect the activities of spermidine N1-acetyltransferase and ornithine decarboxylase in the thymus and spleen. These latter selective responses may represent an important early event in lymphoid tissue response to glucocorticoid hormones.

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.

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.

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.

Green tea modulation of inducible nitric oxide synthase in hypoxic/reoxygenated cardiomyocytes.

Hypoxia/reoxygenation (H/R) is one of the causes of the increased expression of inducible nitric oxide synthase (iNOS) in cardiomyocytes. Since an aberrant NOS induction has detrimental consequences, we evaluated the effect of a green tea extract (GTE) on the NOS induction and activity in H/R-cardiomyocytes to define a nutritional strategy. Cultured rat cardiomyocytes were exposed to H/R in the presence of two concentrations of a green tea extract (GTE), which is reported to inhibit NOS expression and activity in different cells. In cultured cardiomyocytes two NOS isoforms were constitutively expressed, but only iNOS was induced by H/R. GTE supplementation at the lowest concentration, comparable to that in human plasma after dietary consumption, was ineffective, while the highest, comparable to that achievable by dietary supplements, counteracted the effect of H/R on iNOS induction and activity. It is necessary to verify in humans the relationship between the modulation of NO production and green tea dietary consumption.

Effect of age on phosphorylated compounds and mechanical activity of isolated rat heart: a 31P-NMR study.

OBJECTIVE: The aim was to test the hypothesis that aging may change the function and energetics of isolated hearts subjected to an increased work load induced by varying the calcium concentration ([Ca2+]) in the perfusion fluid from 0.5 to 1.0, 1.5, or 2.0 mM. METHODS: Female Wistar rats aged 4, 12-14, and 22-24 months were used. Their hearts were perfused through the aorta and changes in myocardial phosphorylated compound concentration were measured by means of 31P-nuclear magnetic resonance spectroscopy and biochemical analysis. Myocardial contractility was measured in situ in closed heart anaesthetised animals and was followed in vitro during perfusion. RESULTS: The contractile indices measured in situ revealed a decrease with aging of the left ventricular developed pressure and dP/dtmax, while heart rate did not show any significant difference. In all age groups the stepwise increase in [Ca2+] caused a graded increase in left ventricular pressure in the perfused hearts. In aged rats, the left ventricular pressure associated with the different concentrations of Ca2+ was significantly lower than in young rats. In all three groups the myocardial content of inorganic phosphorus (Pi) increased in response to a rise in [Ca2+] and left ventricular pressure. The ATP content of the hearts remained constant in all three groups at each value of [Ca2+] induced left ventricular pressure. However, both ATP and total adenine nucleotide contents of the hearts were lower in aged rats. When the alteration in Pi due to each increase in [Ca2+] was expressed in relation to the rise in left ventricular developed pressure, this relationship was not significantly different in the three groups of hearts. CONCLUSIONS: The reduced mechanical activity of aged rat hearts is not due to a diminished efficiency of the mechanisms transferring high energy phosphates.

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.

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.

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.

Age-dependent differences of ATP breakdown and ATP-catabolite release in ischemic and reperfused hearts.

The hearts of young (6 months) and aged (24 months) rats, paced at a frequency of 300 bpm, were perfused by the Langendorff technique and subjected to: 20 min of equilibration perfusion, 30 min of global ischemia (95% reduction of the coronary flow) and 20 min of reperfusion. The control group was equilibrated for 20 min and then aerobically perfused for 50 min. After 20 min of stabilization, ATP and ADP levels and the adenine nucleotide pool were significantly higher in young than aged hearts (15% increase), but no modifications were found between the two age groups after 50 min of aerobic perfusion. Even the energy charge did not change under aerobic conditions. At the end of the ischemic period the levels of ATP and ADP decreased to a similar extent in young and aged hearts. After 20 min of reperfusion the myocardial level of ATP remained lower in comparison to the preischemic and control values in both age groups. At the end of the reperfusion there was a decrease in energy charge and creatine phosphate levels in the aged group in respect to the young group. The concentrations of adenosine, hypoxanthine and xanthine in coronary effluents did not change during ischemia and reperfusion irrespective of the age of the animals. On the contrary, the release of uric acid during ischemia and reperfusion was greater in aged than young hearts (90% increase). Moreover, the level of inosine in perfusates during the ischemic period was significantly lower in the 24-month-old group (30% decrease). These results are in accordance with the increased purine nucleoside phosphorylase activity and the decreased hypoxanthine phosphorybosyl-transferase activity found in the myocardium of the aged vs. young rats at the end of the reperfusion period. These data indicate that in the aged rat hearts, when exposed to ischemic and reperfusion conditions, there is a modification of purine breakdown which leads to a greater production of uric acid in respect to that found in young hearts.

Mitochondrial production of oxygen free radicals in the heart muscle during the life span of the rat: peak at middle age.

Mitochondria extracted from Wistar rat hearts at 3, 14-18 and 24 months of age showed no change in state 3-mitochondrial respiration measured in the presence of glutamate or succinate. Again no changes were found in the SMP-O2- production at the level of the rotenone-inhibited region, whilst at the level of the antimycin-inhibited region there was a marked increase in O2- production in the group of 14-18-month-old rats. In the same age period, the production of mitochondrial H2O2 supported by glutamate or succinate and the level of GSSG increased in comparison to the young group, accompanied by a decrease in the GSH level. Mitochondrial TBARS levels did not change during a life span, while a progressive accumulation in the mitochondrial lipofuscin content with age was measured.