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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.

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.

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.

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.

A number of human diseases are linked to local reduced oxygen availability. Hypoxemia, the condition in which oxygen partial pressure in blood falls below 40 mmHg, generates a distress which leads the cells in the vascular wall to activate a genetic program inducing a homeostatic response. The effectiveness of this response is conditioned by the degree and duration of the hypoxic stress and depends on the equilibrium among several factors which are worked out mainly in the vascular endothelial cell layer. Among them are vasoconstrictors such as angiotensin II, endothelins, prostaglandins and thromboxans, and vasodilators such as nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor. A present challenge of the research is understanding the physiological and pathophysiological relevance of the growing body of data collected, disclosing the potential therapeutical application of the basic knowledge in this field.

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.

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.

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.

Recent investigations have indicated the involvement of proteasome in programmed cell death. The present studies show that although peptide aldehyde inhibitors of proteasome are by themselves weak inducers of apoptosis, they inhibit the apoptotic effect of the anticancer drug etoposide in rat thymocytes. Acetyl-Leu-Leu-norvalinal (LLnV-al) and other related peptide aldehydes inhibited the increase in caspase activity and DNA fragmentation that followed treatment with etoposide and their effect was related to their potency as proteasome inhibitors. To inhibit etoposide-induced apoptosis, LLnV-al must be present within 3 h of treatment with etoposide, in the same way as the inhibitor of protein synthesis cycloheximide must be. Etoposide caused a rapid accumulation of p53 protein that was not inhibited by LLnV-al, which was also a strong inducer of p53. Peptide aldehydes were also weak activators of caspase activity, suggesting that the same mechanism, i.e. the blocking of proteasome function, both triggers apoptosis and inhibits the effect of etoposide. These results are consistent with a model in which proteasome is selectively involved in the pathway used by etoposide to induce cell suicide.

Nitric oxide synthases (NOS) are different widely expressed enzymes which produce the molecular messenger nitric oxide. The neuronal isoform of NOS (nNOS) is involved in several processes of the cell metabolism, most of which are, at present, not fully understood (neurotransmission, smooth muscle motility, myoblast and myocyte biology and others). In skeletal muscle nNOS is present mainly at the plasmalemma, where it is attached to the dystrophin-related proteins; in fact, in pathologies involving dystrophin, nNOS is altered as well. We report that in aged rats the nNOS amount in skeletal muscle increases both in the soluble and microsomal fractions and that an additional intracytoplasmic localisation appears., (Copyright 1998 Academic Press.)

The AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases that are believed to protect cells against environmental and nutritional stress. In the present study the hypothesis of a protective role for AMPK against thymocyte apoptosis has been tested. It is shown that AMPK is expressed in rat thymocytes that contain the transcript for the a1 isoform of the AMPK catalytic subunit and can be activated by treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a well-established activator of AMPK. AICAR is not toxic and prevents glucocorticoid-induced apoptosis in the same concentration range used to activate AMPK. At concentrations higher than 1 mM, AICAR fully restores cell viability and inhibits DNA laddering in dexamethasone-treated thymocytes. Furthermore, AICAR blocks the dexamethasone-induced activation of caspase 3-like enzymes, which are believed to play a pivotal role in apoptotic cell death. Activation of AMPK by oligomycin, which depletes thymocytes of ATP, is also correlated to inhibition of caspase 3-like activity in dexamethasone-treated cells. However, AICAR and oligomycin do not exert any protective action when apoptosis is induced by staurosporine. These results indicate that AICAR is a powerful inhibitor of glucocorticoid-induced apoptosis and suggest that AMPK activation may interfere with a step in the apoptotic cascade triggered by dexamethasone.

The involvement of phosphatidylinositol 3-kinase (PI3K) in the induction of ornithine decarboxylase (ODC) was investigated by using specific PI3K inhibitors. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, treatment with LY294002 inhibited cell growth and provoked a complete block of the induction of ODC activity (IC50 approximately 2 microM) and ODC protein. Some reduction in the accumulation of ODC mRNA was also observed, whereas ODC turnover was not affected significantly. Wortmannin, another specific inhibitor of PI3K, structurally unrelated to LY294002, also inhibited ODC induction with an IC50 of about 10 nM. These results indicate that PI3K activity is required for the induction of ODC, possibly affecting both ODC mRNA level and translation. Since p70 S6 kinase (p70S6K) is considered an important mediator of PI3K action in several experimental systems, the effect of rapamycin, which can lead to selective inhibition of p70S6K, was also investigated. Rapamycin inhibited p70S6K activity and produced ODC inhibiting effects similar to those elicited by LY294002. However, LY294002 and wortmannin at concentrations which inhibited almost completely PI3K activity did not decrease p70S6K activity, suggesting that p70S6K does not mediate the PI3K effects on ODC, but may lie on a separate pathway in this experimental model.

In quiescent thymocytes, mitochondrial de-energization was not correlated to apoptotic death. In fact, thymocytes treated with oligomycin, a highly specific inhibitor of ATP synthase, alone or with atractyloside to block ATP translocation from the cytoplasm, were alive, even if their mitochondria were depolarized, as revealed by flow cytometry after Rhodamine 123 staining. Furthermore, oligomycin was a powerful inhibitor of apoptosis induced in rat thymocytes by dexamethasone and, to a lesser extent, by the calcium ionophore A23187 and etoposide, but was without effect when apoptosis was induced by staurosporine, and increased cell death in mitogen-treated thymocytes. The inhibition of apoptosis was confirmed by morphological criteria, inhibition of inter-nucleosomal DNA fragmentation and inhibition of the loss of membrane integrity. The anti-apoptotic effect of oligomycin in cells treated with A23187 or etoposide was correlated to the inhibition of protein synthesis, while inhibition of apoptosis induced by dexamethasone, already evident at an oligomycin concentration of 10 ng/ml, was instead strictly correlated to the effect exerted on the cellular ATP level. Thymocyte apoptosis triggered by dexamethasone was blocked or delayed by inhibitors of respiratory-chain uncouplers, inhibitors of ATP synthase and antioxidants: a lasting protection from dexamethasone-induced apoptosis was always correlated to a drastic and rapid reduction in ATP level (31-35% of control), while a delay in the death process was characterized by a moderate decrease in ATP (73-82% of control). Oligomycin inhibited the specific binding of radioactive corticosteroid to thymocyte nuclei, confirming the inhibitory effect of ATP depletion on glucocorticoid binding and suggesting that ATP depletion is a common mediator of the anti-apoptotic action of different effectors in glucocorticoid-induced apoptosis. In conclusion, the reported data indicate that ATP may act as a cellular modulator of some forms of apoptosis, depending on the death trigger, and that in quiescent cells the de-energization of mitochondria is not necessarily linked to apoptosis.

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.

Acetylcholine-induced, endothelium-dependent relaxation of norepinephrine-precontracted aortic strips, was severely impaired after exposure to a hypoxanthine/xanthine oxidase reaction generating oxygen radicals. This effect was more evident in aortic strips of aging rats (24 months old) in comparison to young rats (3 months old). The addition of authentic .NO (1 microM) completely relaxed aortic strips exposed to oxidative stress both in young and aging rats. In vitro EPR measurements showed that the .NO signal was reduced by enzymatic O2.- generating reaction. The activity of a partial purified preparation of constitutive NO synthase from rat cerebellum was significantly decreased after exposure to exogenous oxygen radicals. Pretreatment of aortic strips with 100 microM alpha-tocopherol-phosphate, produced a significant improvement of acetylcholine-dependent relaxation in the aortic strips exposed to oxidative stress, particularly in the aged vessel. The content of malondialdehyde in aortic tissue did not change after oxidative stress or alpha-tocopherol pretreatment. Alpha-tocopherol was unable to recover the NO synthase activity depressed in vitro by hypoxanthine/xanthine oxidase reaction. This study confirms that an oxidative stress impairs the endothelium-mediated vasodilation. Alpha-tocopherol pretreatment protects the vessel against this damage. The mechanism of action of alpha-tocopherol is unknown, but seems unrelated to an antioxidant activity.

Internucleosomal DNA fragmentation and cell death induced by dexamethasone in rat thymocytes were inhibited when cells were cultured in 95% N2/5% CO2 atmosphere, in which oxygen was rapidly reduced to under 0.5%. DNA fragmentation was delayed by a less severe hypoxia in 5% oxygen whilst in cell cultured in high oxygen atmosphere (95% O2) cell death was increased. On the other hand, prolonged oxygen deprivation caused an increase of spontaneous apoptotic cell death. Hypoxia also inhibited DNA fragmentation induced by calcium ionophore A23187, but not by topoisomerase inhibitor camptothecin. These data support the hypothesis of the involvement of oxygen reactive species in calcium-mediated apoptosis and suggest a complex role of oxygen in the modulation of programmed cell death.

Pathological conditions that cause oxidative stress can affect DNA integrity. The aim of this research was to study the protective effect of spermine against DNA damage induced by an oxygen-radical generating system. Deoxyguanosine and DNA were separately dissolved in phosphate buffer and incubated for 1 h at 40 degrees C in the presence of 50 mM H2O2/10 mM ascorbic acid. Single nucleosides and their products of oxidation were then obtained by enzymatic digestion of DNA. The compounds were separated by micellar electrokinetic capillary chromatography (MECC) with SDS-modified mobile phase and detected at 254 nm. Two major products of DNA oxidation have been identified as derivatives of deoxyguanosine with electrophoretic properties different from 8-hydroxy-2'-deoxyguanosine. When the oxidation of DNA was carried out in the presence of 0.1 mM spermine, the formation of the two by-products of deoxyguanosine was markedly reduced. On the contrary, spermine did not prevent the oxidation of deoxyguanosine alone, suggesting that the polyamine should be bound to the DNA strands to exert its antioxidative effect.

This study investigated the biosynthesis of ubiquinone in isolated and perfused hearts of young and aged rats exposed to ischemia and reperfusion. A first group of hearts was used to determine the changes in coenzyme Q9 (CoQ9) and coenzyme Q10 (CoQ10) concentrations at mitochondrial and microsomal level after 30 min of ischemia (98% reduction of the preischemic flow) and 60 min of reperfusion. A second group was utilized to evaluate the rate of CoQ9 and CoQ10 biosynthesis in the membranes by dissolving two ubiquinone precursors, p-OH-[U-14C]benzoate and mevalonolactone, in the perfusion buffer. The hearts were aerobically perfused for 60 min in the presence of the precursors either immediately after the equilibration period or following 30 min ischemia. The young rat hearts showed a 30% reduction in the mitochondrial levels of CoQ9 after ischemia and reperfusion with respect to the preischemic values (P < 0.05 and P < 0.01, respectively). On the contrary, the mitochondrial CoQ9 content was not modified under these conditions in the aged hearts. At the end of reperfusion, the biosynthesis of mitochondrial CoQ9 and CoQ10 was higher in the young rats (P < 0.05), and lower in the aged rats (P < 0.05), with respect to the aerobic perfusion. In both young and aged rats minor changes in CoQ9 concentrations and biosynthesis were observed at microsomal level. These results indicate that myocardial reperfusion decreases the mitochondrial content of ubiquinone and stimulates CoQ9 biosynthesis in young rats but not in aged rats.

The GSH level in myocardial tissue represents an important defense mechanism against oxygen toxicity. Since the ischemia-induced depletion of GSH might favour the cytotoxicity of oxygen-derived free radicals produced during reperfusion, we assessed the effects of the GSH donor, glutathione monoethylester, in anaesthetized pigs subjected to 90 minutes of coronary occlusion followed by 30 minutes reperfusion. The drug was infused intracoronarily at a dose of 1 mg/ml (0.5 ml/min) throughout the experimental period. After coronary occlusion and reperfusion, we found a decrease in GSH, ADP, ATP and phosphocreatine levels in reperfused compared with non-ischemic tissue. Less evident were the differences in mitochondrial function, there being only a reduction in the reperfused tissue of the respiratory control index and state 3 respiration values when pyruvate was used as substrate. The infusion with glutathione monoethylester decreased the depletion of tissue GSH and improved the GSH/GSSG ratio, particularly in the non-ischemic tissue. Moreover, the drug decreased the mitochondrial dysfunction at the level of pyruvate utilization and partially prevented the fall in ATP in the reperfused tissue. This study confirms a possible protective effect of glutathione monoethylester in the prevention of reperfusion-induced myocardial damage.

Mitochondria extracted from the hearts of Wistar rats aged 6 and 24 months showed similar values for the respiratory control index (RCI), state 3 oxygen consumption (QO2) and ADP/O measured using glutamate or succinate as substrates; with the exception that the QO2 of the aged rats was lower than that of the young rats in the presence of glutamate. The consumption of O2 induced by 2-oxoglutarate and ADP was similar in both age groups. Concentrations of external free Ca2+ ranging from 0.2 to 0.8 microM produced an increase in O2 consumption and ATP formation in the young mitochondria, with a maximum effect at 0.2 microM external free Ca2+. Little or no change in O2 consumption and ATP formation was evident in aged mitochondria following incubations in concentrations of external free Ca2+ ranging from 0.2 to 0.8 microM. The continuous rate of formation of ATP, measured in the presence of 0.2 microM external free Ca2+ using a luminescence method, confirmed the previous results. This study indicates that the cardiac mitochondrial phosphorylating system of aged rats is poorly sensitive to variations in external free calcium.

In the myocardial cell, stimulation of the K opioid receptor is involved in the modulation of cytosolic calcium and pH homeostasis, as well as in the regulation of myofilament responsiveness to calcium. In the present study, we found that prodynorphin mRNA, which encodes for the synthesis of a common precursor of opioid peptides interacting with K sites, is synthesized both in atrial and ventricular tissue of the rat heart. In adult cultured rat ventricular cardiomyocytes, the level of prodynorphin mRNA did not differ from that detected in the original ventricular tissue. This finding indicates that the myocardial cell is a source for prodynorphin gene expression and has the potential for an intrinsic synthesis of dynorphin-related peptides.

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.

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.

Isolation of thymocytes from rat thymus resulted in the disappearance of the high activity of ornithine decarboxylase (ODC) that characterizes the thymus of young rats, together with the appearance of an antizyme-like ODC inhibiting activity, which showed a chromatographic profile that resembled that of dexamethasone-treated rat thymus. Omission of serum or addition of dexamethasone or spermidine did not affect appreciably the extent of the antizyme-like activity. On the other hand, a variety of hormonal effectors, i.e. insulin, glucagon, adrenalin and T3, as well as the phorbol ester, PMA or the mitogen, concanavalin A (Con A) induced ODC activity in cultured thymocytes together with the disappearance of the antizyme-like activity. A paradoxical, transient induction of ODC was caused by the transcriptional inhibitor, actinomycin D. Complexed ODC was detected in rat thymus, but not in thymocytes, either quiescent or stimulated by mitogens. These results indicate that thymic lymphocytes can express either ODC activity or its inhibitor depending on the hormonal and proliferative status of the cells.

The purpose of the present study was to evaluate the biosynthesis of coenzyme Q9 (CoQ9) in isolated and perfused young (6 months) and aged (24 months) rat hearts, either under aerobic perfusion condition or during postischemic reperfusion. The young and aged hearts have been divided into 2 groups: Group A, aerobic perfusion for 60 min with recirculating Krebs-Henseleit solution, containing 0.8 microM p-OH-[U-14C]benzoate plus 2.5 mM mevalonlactone; Group B, severe ischemic perfusion for 30 min, followed by 60 min of reperfusion under the same experimental condition of Group A. At the end of the reperfusion the mitochondrial content of CoQ9 was lower in young than aged rat hearts (p < 0.01). In Group A the incorporation of the labeled precursor into mitochondrial CoQ9 was greater in the hearts of aged than young rats (p < 0.01); on the contrary, in Group B this incorporation was significantly reduced in aged than in young rats (p < 0.05). Thus, it is possible that, in the aged rat heart, the higher activity of CoQ9 biosynthesis is related to an elevated turnover of the coenzyme due to the aging process; moreover, this activity is partially reduced by an ischemic-reperfusion stress.

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.

1. The effect of dichlororibofuranosylbenzimidazole (DiCl-RB), an inhibitor of hnRNA synthesis and casein kinase-2 activity, on ornithine decarboxylase (ODC) was investigated in a difluoromethylornithine (DFMO) resistant, ODC overproducing cell line. 2. In cells growing in the absence of DFMO, DiCl-RB provoked a marked, but transient increase in ODC activity and immunoreactive ODC content. 3. The ODC response to DiCl-RB was prevented by cycloheximide and was not due to stabilization of the enzyme. 4. The dibromo derivative analogue (DiBr-RB) exerted similar effects on ODC, but was effective at lower concentrations. 5. The halogenated ribofuranosylbenzimidazoles were ineffective in cells growing in the presence of DFMO and containing higher levels of ODC protein.

Polyamines (putrescine, spermidine, and spermine) are widely distributed in animal and vegetal tissues, where their intracellular concentration strictly correlates with normal and pathological cell growth and protein synthesis. By means of a sensitive HPLC technique, the fasting plasma concentrations of polyamines were measured serially in 11 patients who underwent partial hepatic resection because of focal liver lesions. Samples were obtained before surgery and over the next 6 months, during hepatic regeneration. Liver volume was also measured by ultrasound on the basis of the 3 maximum diameters of the liver. From 2 to 4 weeks after surgery, plasma putrescine increased by a maximum of 78%, and spermidine by approximately 50%. No changes were observed in spermine levels. The spermidine/spermine ratio nearly doubled during liver regeneration. The volume of the liver decrease from 1505 [SD 236] ml to 743 [151] ml after resection, and returned to nearly normal values after 6 months (1231 [100] ml, p < 0.05 vs. basal values). The liver regeneration rate was highest 2-4 weeks after resection, and declined thereafter, when prevailing polyamine concentrations returned to normal. These data show that liver regeneration is accompanied by a significant increase in fasting putrescine and spermidine concentrations, which might be biochemical signals of active liver cell regeneration.

In order to investigate the energetic status of the aged heart during ischemia and reperfusion we perfused female Wistar rats 6, 12 and 24 month old. The hearts were subjected to 15 min of global total ischemia plus 30 min of reperfusion. NMR spectra were collected during the entire experimental period to have the in vivo monitoring of the changes in intracellular pH and intracellular ATP, PCr and Pi contents. In the first 8 min of ischemia the fall of pH was similar in the 3 groups of rats, while at the end of the ischemic period the young rat hearts showed an intracellular pH significantly lower than aged rat hearts. At the end of reperfusion, ATP and PCr contents appeared significantly higher in the adult and aged hearts as compared to the young. The Pi content, on the contrary, was significantly lower in aged than young rat hearts. We suggest that the hearts of adult and aged animals, at the end of reperfusion, showed larger energetic recovery, in our experimental conditions of brief ischemia, than young hearts.

Both contraction and relaxation times are prolonged in cardiac muscle of senescent animals. This is in part explained by an alteration of excitation-contraction coupling due to an increased duration of the action potential, reduced biosynthesis of the Ca(2+)-stimulated ATPase pump of sarcoplasmic reticulum, and prevalence of the V3 isoform of myosin with slow ATPase activity. The response to catecholamine decreases with aging because of a defective transmission of alpha and beta adrenergic stimulation mediated respectively by phosphoinositide hydrolysis and adenylate cyclase. Cardiac energetics is also impaired in the aged myocardium, since ATP and creatine phosphate levels are reduced by about 20%. This reduction seems in part the consequence of defective mitochondrial function, especially in fatty acid oxidation and ATP translocation to the cytoplasm. In this paper we have discussed the possibility that oxygen free radicals may be a cause of myocardial senescence, by damaging the nuclear and mitochondrial genomes as well as membranes and other cellular components.

The transcriptional inhibitors actinomycin D and dichlororibofuranosylbenzimidazole induced ornithine decarboxylase activity in isolated, quiescent thymocytes, which otherwise did not show detectable levels of the enzyme. This paradoxical induction was transient and dependent on the presence of serum and continuous protein synthesis. However, alpha-amanitin, another inhibitor of transcription, did not affect ornithine decarboxylase activity. Dichlororibofuranosylbenzimidazole and actinomycin D were unable to enhance the activity of spermidine acetyltransferase or S-adenosyl-methionine decarboxylase, which are other inducible and short-lived enzymes involved in the metabolism of polyamines.