Sodium pump and Na+/H+ antiport restoration in erythrocytes from cancer patients in remission.

We previously showed that in erythrocytes from cancer patients, the sodium pump is decreased and the optimal intracellular pH for Na+/H+ antiport activity is shifted toward an acidic value. We now have studied these sodium transporters in erythrocytes from patients in remission. Moreover, we intended to explain why the transporters were impaired in erythrocytes, which have no apparent bearing on cancer tissues. The sodium pump was studied through a microcalorimetric method, and the Na+/H+ antiport by a titrimetric method. In patients in remission the sodium pump activity returned to normal: 15.10 +/- 6.00 vs. 14.12 +/- 5.28 mW/l cells for remission and control, respectively. The optimal intracellular pH for Na+/H+ antiport activity was identical in remission and control: 6.09 +/- 0.23 vs. 6.10 +/- 0.10. Restoration of sodium pump activity and optimal intracellular pH for Na+/H+ antiport activity in erythrocytes were thus linked to remission. Moreover, we showed that the impairments of the sodium transporters were due to the presence of plasma-borne factors, the existence of which explained why the sodium transporters were impaired in erythrocytes.

A non ouabain-like inhibitor of the sodium pump in uremic plasma ultrafiltrates and urine from healthy subjects.

A non ouabain-like inhibitor of the sodium pump was separated from uremic plasma ultrafiltrates and normal urine. Under the same chromatographic conditions (C18 column and a gradient of acetonitrile as eluant), ouabain was eluted in a fraction different from the inhibitor. Affinity chromatography based on the formation of a complex between Na,K-ATPase and the inhibitor achieved the differentiation ouabain. Without magnesium and sodium phosphate, ouabain could not bind to enzyme whereas the inhibitor did. A study of Na,K-ATPase enzyme kinetics showed the inhibitor was not competitive for K+, which further differentiates it from ouabain. It was uncompetitive for ATP and seemed competitive for Na+. These results indicate that the inhibitor acts inside the cell, unlike ouabain, and thus its action mechanism appears to be original.

Structure-property relationships of trimetazidine derivatives and model compounds as potential antioxidants.

Twenty-five compounds (trimetazidine derivatives and other compounds, mostly having a free phenolic group) were examined for their radical scavenging and antioxidant properties. Their reaction with DPPH (2,2-diphenyl-1-picrylhydrazyl) as a measure of radical scavenging capacity was assessed by two parameters, namely EC50 (the concentration of antioxidant decreasing DPPH by 50%), and log Z, a kinetic parameter proposed here and derived from initial second-order rate constants and antioxidant/DPPH ratios. Antioxidant activities were determined by the inhibition of lipid peroxidation and albumin oxidation. The most active compounds were derivatives having a trolox or hydroquinone moiety. Physicochemical and structural properties were determined by molecular modeling as lipophilicity (virtual log P calculations) and H-Surf (solvent-accessible surface of hydroxyl hydrogen) and by quantum mechanical calculations (deltaH(ox) = oxidation enthalpy; deltaH(abs) = enthalpy of hydrogen abstraction). QSAR models were derived to identify molecular mechanisms responsible for the reactivity toward the DPPH radical and for the inhibition of lipid peroxidation. A useful prediction of antioxidant capacity could be achieved from calculated molecular properties and the kinetic parameter developed here.

Evidence for the existence of [3H]-trimetazidine binding sites involved in the regulation of the mitochondrial permeability transition pore.

1. Trimetazidine is an anti-ischaemic drug effective in different experimental models but its mechanism of action is not fully understood. Data indicate that mitochondria could be the main target of this drug. The aim of this work was to investigate the binding of [3H]-trimetazidine on a purified preparation of rat liver mitochondria. 2. [3H]-trimetazidine binds to two populations of mitochondrial binding sites with Kd values of 0.96 and 84 microM. The total concentration of binding sites is 113 pmol mg(-1) protein. Trimetazidine binding sites are differently distributed. The high-affinity ones are located on the outer membranes and represent only a small part (4%) of total binding sites, whereas the low-affinity ones are located on the inner membranes and are more abundant (96%) with a Bmax=108 pmol mg(-1) protein. 3. Drug displacement studies with pharmacological markers for different mitochondrial targets showed that [3H]-trimetazidine binding sites are different from previously described mitochondrial sites. 4. The possible involvement of [3H]-trimetazidine binding sites in the regulation of the mitochondrial permeability transition pore (MTP), a voltage-dependent channel sensitive to cyclosporin A, was investigated with mitochondrial swelling experiments. Trimetazidine inhibited the mitochondrial swelling induced by Ca2+ plus tert-butylhydroperoxide (t-BH). This effect was concentration-dependent with an IC50 value of 200 microM. 5. Assuming that trimetazidine effectiveness may be related to its structure as an amphiphilic cation, we compared it with other compounds exhibiting the same chemical characteristic both for their ability to inhibit MTP opening and to displace [3H]-trimetazidine bound to mitochondria. Selected compounds were drugs known to interact with various biological membranes. 6. A strong correlation between swelling inhibition potency and low-affinity [3H]-trimetazidine binding sites was observed: r=0.907 (n=24; P<0.001). 7. These data suggest that mitochondrial sites labelled with [3H]-trimetazidine may be involved in the MTP inhibiton.

Calcium channel blockers correct in vitro mitochondrial toxicity of cellulose acetate.

We studied the action of rinse solutions from cellulose acetate hemodialyzers on isolated mitochondria. We showed that concentrates from the rinses impaired the adenosine 5'-triphosphate (ATP) synthesis as reflected by the decrease in respiration during state 3 and in P/O ratio. This impairment results from a calcium release from mitochondria that is induced by rinse solution concentrates. The release, triggering the mitochondrial calcium carrier, would explain the decrease in ATP synthesis. Moreover, rinse solution concentrates hinder mitochondrial calcium storage. The rise in cytosolic calcium in hemodialyzed patients may be related, at least in part, to these findings, since a lack of ATP impairs the ATP-dependent cellular calcium-extrusion pumps. We also showed that calcium channel blockers, at therapeutically relevant doses, restore ATP synthesis and calcium storage in mitochondria impaired by rinse solution concentrates. Finally, these in vitro results were confirmed by experiments on cells in culture proving that Diltiazem counteracts the cytotoxicity of rinse solution concentrates. These findings are consistent with observations that these drugs suppress the increase in leukocyte cytosolic calcium in dialyzed patients. Moreover, this would help explain the efficiency of calcium channel blockers in cells without L-calcium channels.

Differential effects of zidovudine and zidovudine triphosphate on mitochondrial permeability transition and oxidative phosphorylation.

1. The effects of zidovudine (ZDV) and zidovudine triphosphate (ZDV-3P) on Ca2+-induced mitochondrial permeability transition (MPT), respiratory control ratio (RCR) and ATP synthesis have been investigated on isolated rat liver mitochondria. 2. ZDV slightly but significantly decreased RCR and ATP synthesis but was ineffective in inhibiting MPT. In contrast, ZDV-3P did not alter RCR and ATP synthesis but strongly inhibited MPT (IC50 = 3.0 +/- 0.9 microM). 3. The effect of ZDV-3P on mitochondrial swelling required a preincubation time. When incubated 10 min with mitochondria, ZDV-3P (8 microM) totally inhibited the rate of swelling. 4. ADP, ATP and atractyloside, which are agents known to interact with the mitochondrial adenine nucleotide carrier (ANC), antagonized the effect of ZDV-3P on mitochondrial swelling. Indeed, the IC50 value of ZDV-3P increased from 3.0 to 17.4, 93.6 and 66.5 microM, in the presence of 20 microM, ADP, ATP or atractyloside, respectively. 5. ZDV-3P did not displace [3H]-ATP from its mitochondrial binding site(s) whereas ADP and atractyloside did, suggesting that ZDV-3P and [3H]-ATP do not share the same binding sites. 6. ZDV-3P did not affect either mitochondrial respiration or ATP synthesis but inhibited Ca2+-dependent mitochondrial swelling. It was concluded that mitochondrial toxic effects observed during the chronic administration of ZDV cannot be related to its active metabolite (ZDV-3P).

In vitro mitochondrial test to assess haemodialyser biocompatibility.

BACKGROUND: This paper describes an in vitro mitochondrial test to assess the biocompatibility of haemodialysers. METHODS: We tested on isolated liver mitochondria the effect of solutions obtained by an aqueous rinse of different haemodialysers (cuprophane, cellulose acetate, Hemophan, polyacrylonitrile, polymethylmethacrylate, polysulphone, polyamide). Moreover, to determine the penetration into the cell and the cytotoxicity of these solutions from haemodialysers, we examined the effect of rinse solutions on HT29-D4 cells. RESULTS: Our results showed that rinse solutions from haemodialysers decrease the mitochondrial ATP synthesis. Cuprophane has the most marked effect, and the synthetic membranes exhibited only mild effects. Rinse solutions penetrated the cell and were cytotoxic by acting on mitochondria in the cell. In this respect, cellulosic membranes were the most toxic. CONCLUSION: Taken together our findings lead to a classification of haemodialyser membranes which is identical to one based on criteria such as activation of complement (cuprophane > other cellulosics > synthetics). Moreover isolated mitochondria make it possible to differentiate among the synthetic membranes. Isolated mitochondria thus appear to be a good in vitro test to assess the biocompatibility of haemodialysers.

[Clinical pilot study on repeated use of heparin, vancomycin, colimycine locked flush solution for central intravenous implanted catheter in oncology]. / Etude clinique pilote de l'utilisation repétée dune solution héparine, vancomycine, colimycine en verrouillage systématique des chambres implantables avec cathéter centeral en cancérologie.

With an anti-infectious and an antithrombotic prophylaxis aims, a locked flush solution including heparin and vancomycin, was used systematically for implantable venous access system for each patient, from january to april 1995. Since the 6th of april 1995, in order to widen the antibiotic spectrum on Gram negative bacteriae, we added colimycin at the flush solution. In 1995, 342 hospitalised patients held this type of venous access and received chemotherapy and/or radiochemotherapy for cancer. Two thousand six hundred thirty three manipulations were done, 575 with the first flush solution, 2058 with the second. During the year, 15 implantable access system (4.4%) were considered as infected, only 3 (0.9%) were removed, in the first period. THe infectious rate seemed to be stable, but the bacterial assessment to be modified between the two periods. The Gram negative bacterial infections seemed to decrease with colimycin addition (33% versus 50%). These results must be confirmed by a long term and/or randomized study.

Sodium pump and Na+/H+ activities in uremic erythrocytes. A microcalorimetric and pH-metric study.

The sodium pump and Na+/H+ antiport activities in red blood cells from uremic hemodialyzed patients were measured concomitantly. The patients selected (n = 35) were normotensive and free of intercurrent illness known to affect Na transport. The Na pump activity of intact red blood cells in suspension in their own plasma was measured by flow microcalorimetry. The Na+/H+ antiport activity of the erythrocytes from the same patients was determined by a titrimetric technique. The mean global value of the sodium pump was lower in uremics than in controls (13.3 +/- 0.6 vs. 11.3 +/- 0.8 mW/l cells, P < 0.05). The Na+/H+ antiport maximal activity was decreased in uremics (2.9 +/- 0.3 vs. 4.6 +/- 0.5 mmol H+/l cells/h, P < 0.05). Our results thus confirm that uremia per se can affect sodium transport. Moreover it has been shown that a decrease in Na+/H+ antiport activity is able to counteract an impairment of sodium pump. The decrease found in this study could thus explain, at least in part, the absence of hypertension in the patients studied despite their decreased sodium pump activity.

Comparison of the effects of cyclosporine A and trimetazidine on Ca(2+)-dependent mitochondrial swelling.

Cyclosporine A (CsA) is a known potent inhibitor of pro-oxidant-induced mitochondrial swelling. In the present study we show that CsA's effect is only transient when the liver mitochondrial swelling in induced by Ca2+ plus tert-butylhydroperoxide (t-BH). After an initial inhibition, swelling is worsened by CsA as evidenced by an extent of mitochondrial swelling that exceeds that of the control. Unlike CsA, trimetazidine (TMZ), an anti-ischemic drug decreases both the extent and the rate of the swelling with an IC50 value of 214 +/- 24 microM. Its inhibition effect on the initial swelling rate mimicks that of CsA but the mechanism may be independent. During long-term swelling. TMZ counteracts the worsening effect of CsA. The inhibition of swelling induced by TMZ is assessed by the fact that TMZ significantly increases the EC50 of Ca(2+)-induced mitochondrial swelling (46.6 +/- 6.0 to 85 +/- 10 microM, P < 0.01), without affecting its cooperativity. Apparently, TMZ seems to behave like trifluoperazine (TFP), a phospholipase A2 inhibitor that, under our experimental conditions, inhibits the mitochondrial swelling induced by Ca2+ and t-BH with an IC50 value of 25 +/- 10 microM. Both drugs are able to protect mitochondria from both phases (early and late) of the swelling, especially the late, which is enhanced in the presence of CsA. TFP and other phospholipase A2 inhibitors were able to displace [3H]TMZ from its mitochondrial binding sites whereas CsA was ineffective. We suggest that TMZ, like TFP, inhibits the CsA insensitive mechanism involved in the swelling process which is responsible for the worsening effect observed in the presence of CsA when the swelling is generated by Ca2+ and t-BH.

Potential interest of anti-ischemic agents for limiting cyclosporin A nephrotoxicity.

Chronic administration of cyclosporin A induces nephrotoxicity in humans. This is related to a cyclosporin A-induced constriction of afferent glomerular arterioles and mesangial cells, which leads to a decrease in filtration pressure and creatinine clearance. Afterwards, cellular lesions are observed involving mainly tubular atrophy and interstitial fibrosis, both of which are nonspecific. The initial mechanism of its toxicity is not clearly explained. The current pharmacological approach is symptomatic in order to counteract or minimize the consequences of a prime cause, which still remains to be defined. However, cyclosporin A has a deletereous effect on mitochondrial functions and mainly on ATP synthesis, which occurs when Ca2+ accumulates in matrix mitochondria. The effects of trimetazidine, an antischemic drug used in the treatment of angina pectoris, have been assessed. This drug is effective in experimental models of hypoxia induced by cyclosporin A: it restores ATP synthesis previously decreased by Ca2+ and cyclosporin A, and releases a part of Ca2+ excess accumulated by mitochondria at concentrations reached in humans at usual dosage regimens. At higher concentrations, it reverses the mitochondrial permeability transition previously generated (opened) by Ca2+ and a pro-oxidant such as terbutylperoxide (t-BH). It was also observed that trimetazidine does not modify the immunosuppressive effects of cyclosporin A in various models. These data suggest that nephrotoxicity of cyclosporin A is not irrevocably linked to its immunosuppressive effect but that it may be possible to counteract at least partly its nephrotoxic effects without altering its effectiveness in preventing graft rejection.

A controlled one-year trial of dextromethorphan in amyotrophic lateral sclerosis.

In a one-year parallel group double-blind placebo-controlled study of dextromethorphan (1.5 mg/kg) in amyotrophic lateral sclerosis, no significant differences were observed in the rate of progression (Norris scale) in comparing 24 patients randomly assigned to the dextromethorphan group and 25 patients randomly assigned to the placebo group. Of the 24 patients in the dextromethorphan group, 17 had limb onset and 7 had bulbar onset disease; average duration of disease was 12.5 +/- 6 months and sex ratio (M:F) was 1.4:1. Of the 25 patients in the placebo group, 18 had limb onset and 7 had bulbar onset disease; average duration of disease was 9.9 +/- 6 months and sex ratio (M:F) was 1.55:1. Dextromethorphan is a weak noncompetitive N-methyl-D-aspartate (NMDA) antagonist and higher doses or other potent NMDA receptor antagonists should be tested.

Trimetazidine reverses calcium accumulation and impairment of phosphorylation induced by cyclosporine A in isolated rat liver mitochondria.

When applied to rat liver mitochondria in contact with Ca++, cyclosporine A (CsA) induced both an accumulation of this ion and a decrease in oxidative phosphorylation. Trimetazidine (TMZ) reversed both phenomena in a dose-dependent manner. These two effects were demonstrated in separate experiments. A decrease in oxidative phosphorylation was observed with succinate as substrate. V3 and P/O (ratio corresponds to the number of ADP molecules added in the medium per oxygen atom consumed during phosphorylation and represents the yield of ATP synthesis) were simultaneously decreased by CsA (1 microM) and restored by TMZ. Ca++ accumulation in mitochondria was observed when it was added to the mitochondrial suspension; its uptake was followed by a new equilibrium. CsA prolonged its duration, whereas TMZ reduced it in a dose-dependent manner. The same phenomenon was observed when ADP was used instead of CsA. Ca++ efflux from mitochondria could be induced by TMZ without the addition of CsA. It was immediate and always partial and followed by a reuptake process only observed at concentrations of TMZ of >1 microM. Compared with ruthenium red, which blocks Ca++ uniporter, TMZ seemed to act on Ca++ efflux mechanisms. Interestingly, low TMZ doses promote a Ca++ efflux process without activating reentry mechanism, which may explain the correction of deleterious effect of CsA on V3 and P/O. As nephrotoxicity observed in humans after CsA chronic administration is considered to be related, at least in part, to an alteration of Ca++ intracellular homeostasis, TMZ seems to be a candidate for alleviation of CsA nephrotoxic effects in humans.

Impairment of sodium pump and Na+/H+ antiport in erythrocytes isolated from cancer patients.

We sought to determine whether the impairment of sodium pump activity and Na+/H+ exchange reported in tumorigenic cells was specific to these cells or more general. Sodium pump activity and Na+/H+ exchange were measured in erythrocytes from 49 cancer patients and 51 healthy subjects. Cancer patients with a newly detected cancer or in relapse and without associated pathologies known to modify these sodium transporters were included in this study. Two sodium pump statuses reflecting its physiological modulation were evidenced for healthy subjects (10.3 +/- 0.2 and 19.4 +/- 0.8 mW/liter of cells). In cancer patients, only one basal status lower than those of controls was observed (8.3 +/- 0.5 mW/liter of cells; P < 0.001). Cooperativity of the Na+/H+ antiporter is the same in cancer patients and controls (2.58 +/- 0.27 versus 2.60 +/- 0.15). The intracellular pH (pHi) dependence curve of the antiporter was shifted toward more acidic values, and optimal pH1 was lower in cancer patients than in controls (5.80 +/- 0.03 versus 6.08 +/- 0.02; P < 0.0001). The mean maximal rate and the Km of H+ for the Na+/H+ antiporter were higher: 8.4 +/- 1.2 versus 4.6 +/- 0.4 mmol H+/liter of cells/h (P < 0.01) and 514 +/- 12 versus 322 +/- 16 nM (P < 0.05), respectively. Alterations of these Na+ transporters, therefore, were not restricted to cancerous cells. Among the alterations, the acidic shift in the pHi dependence of Na+/H+ exchange appears associated with cancer because this behavior has never been reported in other pathologies.

Lack of pharmacodynamic interaction between trimetazidine and cyclosporin A in human lymphoproliferative and mouse delayed hypersensitivity response models.

The hypothesis of an interaction between trimetazidine and the immunosuppressive effect of cyclosporin A was investigated in two models: a) ex vivo, the lymphoproliferative response of normal human lymphocytes to phytohemagglutinin and a murine monoclonal antibody against the CD3 T-lymphocyte membrane complex; b) in vivo, the delayed hypersensitivity response model in mouse. The uptake of methyl-3H-thymidine was measured in both models. For the lymphoproliferative response, statistical analysis showed that there was a significant inhibitory effect of cyclosporin A on cell proliferation (P < 0.001) and confidence intervals obtained by ANOVA showed the equivalence of the results when trimetazidine was combined with cyclosporin A (all CI95% < or = 10). In the delayed hypersensitivity model, cyclosporin A was also found to be very effective in inhibiting the immune response (P < 0.001), while trimetazidine did not interfere with cyclosporin A's effect. It was concluded that trimetazidine exerted neither an immunostimulatory nor an immunosuppressive effect in the two models, suggesting of the absence of interaction between trimetazidine and cyclosporin A's effectiveness when both drugs are given in combination.

[Pharmacological modulation of mitochondrial oxidative phosphorylation: inhibition by cyclosporine A, restoration by trimetazidine]. / Modulation pharmacologique de la phosphorylation oxydative mitochondriale: la cyclosporine A l'inhibe, la trimétazidine la restaure.

When applied to a suspension of isolated mitochondria extracted from rat hepatocytes, cyclosporine A decreases ATP synthesis and induces Ca2+ accumulation. Both effects are considered as possible determinants, even partly, of renal toxicity observed with this drug. Trimetazidine antagonizes both effects at concentrations easily reached in man with therapeutic dosages. It is concluded that the association of both drugs may improve the renal tolerance of Cyclosporine A.

Opposite effects of urea on hemoglobin-oxygen affinity in anemia of chronic renal failure.

We studied the action of urea on the spin-spin relaxation rate of 2,3-diphosphoglycerate (2,3-DPG) phosphorus atoms in normal and uremic erythrocytes. At concentrations from 10 to 60 mM, urea increased the relaxation rates of 2,3-DPG P-3 phosphorus atoms. This evidenced a stronger binding of 2,3-DPG to hemoglobin (Hb), suggesting that the deoxyform of Hb was stabilized. This hypothesis was confirmed by measurements of the association constant of oxygen to hemoglobin (K) in normal erythrocytes in presence of urea concentrations in the range of those observed in uremic patients (30 mM). Indeed, the observed decrease in K suggests that the T structure of hemoglobin is stabilized. By contrast, with higher urea concentrations (120 mM), measurements of P50 showed an increase in the hemoglobin affinity for oxygen (decrease in P50). Moreover, the relaxation rates of 2,3-DPG P-3 phosphorus atoms were not modified, which is consistent with the simultaneous increase of K. This may be attributed to the formation of carbamylated hemoglobin in presence of urea. These results suggest two opposite effects of urea on Hb-O2 affinity: the first reinforces 2,3-DPG-Hb binding and leads to a decrease in O2 affinity; the second, mediated by carbamylation of Hb, hinders the binding of 2,3-DPG and increases the O2 affinity. These findings are consistent with the fact that, despite the presence of carbamylated hemoglobin, uremic patients do not present increased Hb-O2 affinity.

Is the beneficial effect of calcium channel blockers against cyclosporine A toxicity related to a restoration of ATP synthesis?

ATP synthesis inhibited by Cyclosporine A is restored by calcium channel blockers: nifedipine, verapamil, bepridil, diltiazem. ATP synthesis was estimated using liver mitochondria by measuring the rate of respiration during state 3 and a measure of the yield of ATP synthesis, the P/O ratio. The study of calcium fluxes through mitochondrial membrane indicates that calcium channel blockers counteract the mitochondrial calcium storage induced by cyclosporine A. If the restoration of ATP synthesis observed in vitro also occurred in vivo, the increase in ATP pool might contribute to a better functioning of the Ca2+ extrusion pumps of the cells, thereby maintaining the cytosolic calcium concentration (Cai), in the normal range. The nephrotoxicity of cyclosporine A appears to be due to a vasoconstrictive effect related to an increased Cai. This result may account for the reduction of clinical cyclosporine A toxicity by calcium channel blockers. Verapamil appears to be the most efficient in restoring ATP synthesis.

Modulation defect of sodium pump evidenced in diabetic patients by a microcalorimetric study.

Sodium pump activity of intact erythrocytes in their own plasma was measured by microcalorimetry in 41 healthy subjects and 35 insulin-dependent diabetic patients. Results show that modulation of the sodium pump is altered in diabetic patients. Addition of insulin increases functioning of the Na(+)-K+ pump in controls but has no effect in diabetic patients. These subjects show a slower response of the Na(+)-K+ pump to the inhibitory effect of ouabain. Cross-incubation experiments suggest that these findings may be explained by the existence of a plasmatic factor that impairs the modulation of the sodium pump in diabetic patients.