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.

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.

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.

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.

Proteomic technologies in the study of kinases: novel tools for the investigation of PKC in the heart.

Recent developments in the field of protein separation allows for the analysis of qualitative and quantitative global protein changes in a particular state of a biological system. Due to the enormous number of proteins potentially present in a cell, sub-fractionation and the enrichment of specific organelles are emerging as a necessary step to allow a more comprehensive representation of the protein content. The proteomic studies demonstrate that a key to understand the mechanisms underlying physiological or pathological phenotypes lies, at least in part, in post-translational modifications (PTMs), including phosphorylation of proteins. Rapid improvements in proteomic characterization of amino acid modifications are further expanding our comprehension of the importance of these mechanisms. The present review will provide an overview of technologies available for the study of a proteome, including tools to assess changes in protein quantity (abundance) as well as in quality (PTM forms). Examples of the recent application of these technologies and strategies in the field of kinase signalling will be provided with particular attention on the role of PKC in the heart. Studies of PKC-mediated phosphorylation of cytoskeletal, myofilament and mitochondrial proteins in the heart have provided great insight into the phenotypes of heart failure, hypertrophy and cardioprotection. Proteomics studies of the mitochondria have provided novel evidences for kinase signalling cascades localized to the mitochondria, some of which are known to involve various isoforms of PKC. Proteomics technologies allow for the identification of the different PTM forms of specific proteins and this information is likely to provide insight into the determinants of morphological as well as metabolic mal-adaptations, both in the heart and other tissues.

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.

Interleukin-6 gene polymorphism is an age-dependent risk factor for myocardial infarction in men.

Several studies show that inflammatory components may contribute to atherosclerosis and increase the risk for myocardial infarction (MI). Interleukin-6 (IL-6) is a key pro-inflammatory and immune-modulatory cytokine of relevance for cardiovascular diseases. In this case-control study, 200 patients with MI and 257 healthy controls were genotyped for the polymorphism present in -174 promoter region of the IL-6 gene. Plasma concentrations of IL-6 and C-reactive protein (CRP) in a group of patients and controls were measured. The -174 C allele was associated with an increased risk of developing MI (OR = 2.886, c.i. = 1.801-4.624, P = 0.0001) in older patients, while no association was found in younger ones. The IL-6 plasma levels were higher in patients with MI carrying the CC genotype than in GG patients (CC carriers, IL-6 = 2.97 pg mL(-1) vs. GG carriers = 1.81 pg mL(-1), P = 0.016). A positive correlation of IL-6 levels with those of CRP in serum from patients with MI was also found. Data from this study suggest that the C allele of the promoter polymorphism in the IL-6 gene is a risk factor for MI in the elderly, and the production of the IL-6 is differentially affected by different genotypes of the IL-6 -174 promoter polymorphism.

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.

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

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

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

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

Polyamines, NO and cGMP mediate stimulation of DNA synthesis by tumor necrosis factor and lipopolysaccharide in chick embryo cardiomyocytes.

OBJECTIVE: We have recently shown that tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS) stimulate DNA synthesis in chick embryo cardiomyocytes (CMs). The aim of the present research was to investigate the pathways involved in this mitogenic response. METHODS: CMs were isolated from 10-day-old chick embryos and grown to confluence. After 20 h of serum starvation the cells were treated with TNFalpha and LPS, and/or specific agonists and antagonists to manipulate the levels of polyamines, NO, cGMP and their biosynthetic enzymes ornithine decarboxylase (ODC), nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC). ODC, NOS, sGC activities and cGMP contents were determined by radiochemical procedures. DNA synthesis was determined by incorporation of [3H]-thymidine. RESULTS: Treatment of CMs with TNFalpha and LPS increased cell number and [3H]-thymidine incorporation. Addition of TNFalpha and LPS provoked an induction of ODC, with consequent polyamine accumulation, and a more delayed enhancement of NOS activity, which appeared to be independent of the activation of the ODC-polyamine system. TNFalpha and LPS treatment also enhanced cGMP level in CMs and both polyamine and NO biosyntheses appeared to be required. Experiments with specific inhibitors of ODC and NOS, as well as with inhibitors of sGC and cGMP-dependent protein kinase (PKG), showed that polyamine-, NO- and cGMP-dependent pathways are required for the mitogenic action of TNFalpha and LPS. Moreover, addition of exogenous polyamines to untreated cells raised the cGMP level in a NO-dependent fashion, and enhanced [3H]-thymidine incorporation. The latter effect was inhibited by sGC or PKG inhibitors. Treatment of quiescent cells with NO donors, 8-bromo-cGMP or YC-1, an sGC activator, also promoted DNA synthesis. Furthermore, putrescine and NO donor can additively activate sGC in cell-free extracts. CONCLUSION: TNFalpha and LPS stimulate DNA synthesis in chick embryo CMs and this effect is mediated by polyamines, NO and intracellular cGMP.

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.

Molecular mechanisms of response to low oxygen tension in the vascular wall.

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.

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.

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

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

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

Nitric oxide (NO) is a molecule involved in several signal transduction pathways leading either to proliferation or to cell death. Induction of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, represents an early event preceding DNA synthesis. In some cell types increased ODC activity seems to be involved in cytotoxic response. We investigated the role of NO and ODC induction on the events linked to cell proliferation or to cell death in cultured chick embryo cardiomyocytes. Exposure of cardiomyocytes to tumor necrosis factor (TNF) and lipopolysaccharide (LPS) caused NO synthase (NOS) and ODC induction as well as increased incorporation of [3H]-thymidine. This last effect was blocked by a NOS inhibitor and was strongly reduced by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. Sodium nitroprusside (SNP), an exogenous NO donor, inhibited the increases of NOS and ODC activities and abolished the mitogenic effect of TNF and LPS. Moreover, SNP alone caused cell death in a dose dependent manner. The cytotoxicity of SNP was not affected by DFMO while it was prevented by antioxidants. The results suggest that different pathways would mediate the response of cardiomyocytes to NO: they can lead either to ODC induction and DNA synthesis when NO is formed through NOS induction or to growth inhibition and cell death, when NO is supplied as NO donor. Increased polyamine biosynthesis would mediate the proliferative response of NO, while the cytotoxicity of exogenous NO seems to involve some oxidative reactions and to depend on the balance between NO availability and cellular redox mechanisms.

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.

Simultaneous detection of reduced and oxidized glutathione in tissues and mitochondria by capillary electrophoresis.

We have developed a rapid and precise method for glutathione quantitation by capillary electrophoresis, that allows a low amount of both redox forms to be measured. Small fragments of rat heart or liver tissues (20 mg wet weight) and the corresponding mitochondria (1 mg protein) were homogenized in 1% perchloric acid and the acid-soluble phase ultrafiltered by centrifugation with a microconcentrator (Mr cut-off 3000 Da). The analysis was performed at a constant temperature (28 degrees C) using a Beckman P/ACE System 2100, equipped with a UV absorbance detector set to 200 nm. The limit of quantitation in heart tissue was 1.8 microM for GSH and 1.2 microM for GSSG. Myocardial concentrations of GSH and GSSG were 8.1 +/- 2.6 and 0.45 +/- 0.15 (nmol/mg protein +/- S.D.), respectively. The ratio of GSH to GSSG was 17.8 +/- 1.3 for heart tissue, whereas it was much higher (>100) in the mitochondria. An oxidative stress decreased the myocardial tissue GSH/GSSG ratio, indicating that the CE analysis of both glutathione forms is also a useful method to study biological redox modification.