Reactivity of mitochondrial sulfhydryl groups toward dithionitrobenzoic acid and bromobimanes under oligomycin-inhibited and uncoupling conditions

Thiol reactivity was determined in rat heart mitochondria using chromophores of differing polarities: monobromobimane (MB), dithionitrobenzoate (NbS2), and bromobimane-q (MQ). The purpose of this study is to correlate reaction rates of protein thiols in the mitochondrial membrane with the oligomycin-inhibited and uncoupled states: In all cases investigated the reactivity of -SH groups toward MB decreases under the above conditions. In parallel with an increase of their uncoupling activities the uncouplers reduce the reaction rate of thiol groups toward NbS2 and, progressively, toward MQ, indicating differences in sensitivity of thiol groups to uncouplers depending on the polarity of the environment. The pattern of -SH reactivity under inhibition by oligomycin resembles that of carbonylcyanide-p-trifluoromethoxyphenylhydrazone. Functional changes of the mitochondrial membrane probably correlate with reactivity/polarity changes of membrane -SH groups. Masking of membrane thiol groups thus is not specific for uncouplers but is also observed under inhibition with oligomycin.     

Mitochondrial sulfhydryl groups under oligomycin-inhibited, aging, and uncoupling conditions: beneficial influence of cardioprotective drugs

Uncoupling, oligomycin-inhibited, and aging/swelling conditions comprise three models for mitochondrial dysfunction. In these models, the effects of cardioprotective agents on rat heart mitochondrial membrane -SH reactivity have been studied. For -SH detection two different chromophores were used: dithionitrobenzoate (NbS2) and monobromobimane (MB). The objective of this study is to reveal the influence of three cardioprotective substances against the loss of membrane -SH reactivity: (i) The thiol reagent 2-mercaptopropionylglycine (MPG) prevents the decrease of thiols caused by carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), aging, and oligomycin measured with MB and NbS2, and the diminution by oleate detected with MB. The small amount of MPG (6 nmol/mg protein), necessary for the protection, agrees with oligomycin sensitivity of the -SH groups concerned. (ii) The active metabolite of molsidomine, 3-morpholinosydnonimine (SIN-1), protects against the decrease of thiols by FCCP, oleate, and aging monitored with MB. In the case of oligomycin -SH groups accessible to NbS2 are protected. (iii) Another antianginal drug, isosorbidedinitrate (ISDN) does not protect membrane thiol groups. In contrast to SIN-1, ISDN probably requires enzymatic activation. It is suggested that MPG as well as SIN-1 may help to restitute the original -SH status of the mitochondrial membrane.     

Different effects of endotoxic shock on the respiratory function of liver and heart mitochondria in rats

This study was designed to clarify whether mitochondrial function/dysfunction and reactive oxygen species (ROS) production have a temporal relationship with organ failure during endotoxic shock. Adult male Sprague-Dawley rats were divided into three groups receiving 1) isotonic saline (control group, n = 16); 2) 8 mg/kg lipopolysaccharide (LPS; n = 8); or 3) 20 mg/kg LPS (n = 8) intraperitoneally under short anesthesia with 3.5% of isoflurane. After 16 h, animals were killed to analyze plasma, rat liver mitochondria (RLM), and rat heart mitochondria (RHM). In accordance with plasma analysis, LPS-treated rats were divided into "responders" and "nonresponders" with high and low levels of alanine aminotransferase and creatine, respectively. RHM from responders had significantly lower respiratory activity in state 3, suggesting a decreased rate of ATP synthesis. In contrast, RLM from responders had significantly higher respiratory activity in state 3 than both nonresponders and the control group. This increase was accompanied by a decrease in phosphate-to-oxygen ratio values, which was not observed in RHM. ROS generation determined with a spin probe, 1-hydroxy-3-carboxypyrrolidine, neither revealed a difference in RHM between LPS and control groups nor between responders and nonresponders. In contrast, RLM isolated from responders showed a marked increase in ROS production compared with both the control group and nonresponders. Our data demonstrate that 1) RHM and RLM respond to endotoxic shock in a different manner, decreasing and increasing respiratory activity, respectively, and 2) there is a temporal relationship between ROS production in RLM (but not in RHM) and tissue damage in rats subjected to LPS shock.     

Does the function of adenine nucleotide translocase in fatty acid uncoupling depend on the type of mitochondria?

The stimulation of respiration by long-chain fatty acids and FCCP was studied with oligomycin-inhibited mitochondria from rat liver, heart and kidney tissue. By addition of equal amounts of palmitate and oleate, mitochondrial respiration was increased in the order RLM less than RKM less than RHM. Using the classical protonophore FCCP, this difference could not be observed. Inhibition of oleate-stimulated respiration by carboxyatractyloside decreased in the order RHM greater than RKM greater than RLM. As CAT sensitivity of oleate-stimulated respiration and the mitochondrial ANT content were found to be correlated, it is suggested that the weak CAT sensitivity of oleate-stimulated respiration of RLM [(1989) Biochim. Biophys. Acta 977, 266-272] is due to the low content of ANT.     

Reactivity of the sulphydryl groups of the mitochondrial phosphate carrier

The rate of reaction of - SH groups of the mitochondrial phosphate carrier with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) and N-ethylmaleimide (MalNEt) was followed by measuring the inhibition of phosphate transport. The changes in the rate of reaction caused by alterations of the ionic composition of the matrix were compared with changes of the total intramitochondrial phosphate content, the intramitochondrial K+ content and the value of intramitochondrial pH. The ionic composition was manipulated by addition of valinomycin to non-respiring or to respiring mitochondria and by addition of inorganic phosphate to respiring and non-respiring mitochondria. From all these variables it was the changes of the intramitochondrial pH which correlated with the - SH group reactivity. Internal acidification decreased and internal alkalinization increased the rate of reaction of mitochondrial phosphate carrier with both Nbs2 and MalNEt. Nbs2 did not penetrate the inner mitochondrial membrane as assayed by determination of the acid-soluble thiol content of the matrix. From this fact it follows that the Nbs2-reactive SH groups of the carrier were accessible from the outer surface of the inner membrane in our experiments. It is concluded that intramitochondrial pH modifies the reactivity of the externally oriented - SH groups indirectly. A hypothesis is presented according to which protonation and deprotonation of the carrier molecule on the inner side could induce a conformational change of the whole protein altering also the microenvironment of the - SH groups near the opposite surface.     

Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2′-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiols

1. The u.v.-spectral characteristics of 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs(2)), 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py), 4,4'-dipyridyl disulphide (4-Py-S-S-4-Py), 5-mercapto-2-nitrobenzoic acid (Nbs), 2-thiopyridone (Py-2-SH) and 4-thiopyridone (Py-4-SH) were determined over a wide range of pH and used to calculate their acid dissociation constants. 2. The reactions of l-cysteine, 2-mercaptoethanol and papain with the above-mentioned disulphides were investigated spectrophotometrically in the pH range 2.5-8.5. 3. Under the conditions of concentration used in this study the reactions of both low-molecular-weight thiols with all three disulphides resulted in the stoicheiometric release of the thiol or thione fragments Nbs, Py-2-SH and Py-4-SH at all pH values. The rates of these reactions are considerably faster at pH8 than at pH4, which suggests that the predominant reaction pathway in approximately neutral media is nucleophilic attack of the thiolate ion on the unprotonated disulphide. 4. The reaction of papain with Nbs(2) is markedly reversible in the acid region, and the pH-dependence of the equilibrium constant for this system in the pH range 5-8 at 25 degrees C and I=0.1 is described by: [Formula: see text] 5. Papain reacts with both 2-Py-S-S-2-Py and 4-Py-S-S-4-Py in the pH range 2.5-8.5 to provide release of the thione fragments, stoicheiometric with the thiol content of the enzyme. 6. Whereas the ratios of the second-order rate constant for the reaction at pH4 to that at pH8 for the cysteine-2-Py-S-S-2-Py reaction (k(pH4)/k(pH8)=0.015) and for the papain-4-Py-S-S-4-Py reaction (k(pH4)/k(pH8)=0.06) are less than 1, that for the papain-2-Py-S-S-2-Py reaction is greater than 1 (k(pH4)/k(pH8)=15). 7. This high reactivity of papain has been shown to involve reaction of the thiol group of cysteine-25, the enzyme's only cysteine residue, which is part of its catalytic site. 8. That this rapid and stoicheiometric reaction of the thiol group of native papain is not shown either by low-molecular-weight thiols or by the thiol group of papain after its active conformation has been destroyed by acid or heat denaturation, strongly commends 2-Py-S-S-2-Py as one of the most useful papain active-site titrants discovered to date. This reagent has been shown to allow accurate titration of papain active sites in the presence of up to 10-fold molar excess of l-cysteine and up to 100-fold molar excess of 2-mercaptoethanol.     

Glycyrrhetinic acid as inhibitor or amplifier of permeability transition in rat heart mitochondria

Glycyrrhetinic acid (GE), a hydrolysis product of glycyrrhizic acid, one of the main constituents of licorice root, is able, depending on its concentration, to prevent or to induce the mitochondrial permeability transition (MPT) (a phenomenon related to oxidative stress) in rat heart mitochondria (RHM). In RHM, below a threshold concentration of 7.5 microM, GE prevents oxidative stress and MPT induced by supraphysiological Ca2+ concentrations. Above this concentration, GE induces oxidative stress by interacting with a Fe-S centre of Complex I, thus producing ROS, and amplifies the opening of the transition pore, once again induced by Ca2+. GE also inhibits Ca2+ transport in RHM, thereby preventing the oxidative stress induced by the cation. However, the reduced amount of Ca2+ transported in the matrix is sufficient to predispose adenine nucleotide translocase for pore opening. Comparisons between observed results and the effects of GE in rat liver mitochondria (RLM), in which the drug induces only MPT without exhibiting any protective effect, confirm that it interacts in a different way with RHM, suggesting tissue specificity for its action. The concentration dependence of the opposite effects of GE, in RHM but not RLM, is most probably due to the existence of a different, more complex, pathway by means of which GE reaches its target. It follows that high GE concentrations are necessary to stimulate the oxidative stress capable of inducing MPT, because of the above effect, which prevents the interaction of low concentrations of GE with the Fe-S centre. The reported results also explain the mechanism of apoptosis induction by GE in cardiomyocytes.     

Glycyrrhetinic acid as inhibitor or amplifier of permeability transition in rat heart mitochondria

Glycyrrhetinic acid (GE), a hydrolysis product of glycyrrhizic acid, one of the main constituents of licorice root, is able, depending on its concentration, to prevent or to induce the mitochondrial permeability transition (MPT) (a phenomenon related to oxidative stress) in rat heart mitochondria (RHM). In RHM, below a threshold concentration of 7.5 μM, GE prevents oxidative stress and MPT induced by supraphysiological Ca²⁺ concentrations. Above this concentration, GE induces oxidative stress by interacting with a Fe-S centre of Complex I, thus producing ROS, and amplifies the opening of the transition pore, once again induced by Ca²⁺. GE also inhibits Ca²⁺ transport in RHM, thereby preventing the oxidative stress induced by the cation. However, the reduced amount of Ca²⁺ transported in the matrix is sufficient to predispose adenine nucleotide translocase for pore opening. Comparisons between observed results and the effects of GE in rat liver mitochondria (RLM), in which the drug induces only MPT without exhibiting any protective effect, confirm that it interacts in a different way with RHM, suggesting tissue specificity for its action. The concentration dependence of the opposite effects of GE, in RHM but not RLM, is most probably due to the existence of a different, more complex, pathway by means of which GE reaches its target. It follows that high GE concentrations are necessary to stimulate the oxidative stress capable of inducing MPT, because of the above effect, which prevents the interaction of low concentrations of GE with the Fe-S centre. The reported results also explain the mechanism of apoptosis induction by GE in cardiomyocytes.     

Effect of pH and KCl on aggregation state and sulphydryl groups reactivity of rat skeletal muscle AMP deaminase

The effects of pH and KCl on sedimentation properties and SH groups reactivity of rat skeletal muscle AMP deaminase have been investigated. The values obtained for apparent molecular weight are consistent with an association of AMP deaminase subunits in response to increasing KCl concentration. Increasing pH value from 6.0 to 8.0 causes a reduction in the apparent molecular weight of the enzyme at high KCl concentration, which can be interpreted as due to a deprotonation-induced isomerization process. Removal of Zn2+ from AMP deaminase has effect similar to alkalinization in modifying the sedimentation properties of the enzyme. In the native enzyme at high K+ concentration about 7, 9 and 12 SH groups can be titrated with Nbs2, approximately 1, 2 and 4 SH groups reacting as fast sets, at pH 6.0, 7.0 and 8.0, respectively. Substitution of the 12 SH groups reactive with Nbs2 at pH 8.0 has no effect on the pH-dependent allosteric behaviour of the enzyme. Removal of K+ causes considerable changes in the reactivity of AMP deaminase towards Nbs2, unmasking a class of additional SH groups, so that the total number of titratable SH groups approaches that of 30 determined in denaturing conditions. In the enzyme previously treated with N-ethylmaleimide to alkylate the fast reacting class of SH groups, the class of additional SH groups are substituted by Nbs2 at basic pH, but not at acidic pH, with a concomitant reduction of the enzyme activity.     

Hepatic mitochondrial inner membrane properties and carnitine palmitoyltransferase A and B. Effect of diabetes and starvation.

Intact mitochondria and inverted submitochondrial vesicles were prepared from the liver of fed, starved (48 h) and streptozotocin-diabetic rats in order to characterize carnitine palmitoyltransferase kinetics and malonyl-CoA sensitivity in situ. In intact mitochondria, both starved and diabetic rats exhibited increased Vmax., increased Km for palmitoyl-CoA, and decreased sensitivity to malonyl-CoA inhibition. Inverted submitochondrial vesicles also showed increased Vmax. with starvation and diabetes, with no change in Km for either palmitoyl-CoA or carnitine. Inverted vesicles were uniformly less sensitive to malonyl-CoA regardless of treatment, and diabetes resulted in a further decrease in sensitivity. In part, differences in the response of carnitine palmitoyltransferase to starvation and diabetes may reside in differences in the membrane environment, as observed with Arrhenius plots, and the relation of enzyme activity and membrane fluidity. In all cases, whether rats were fed, starved or diabetic, and whether intact or inverted vesicles were examined, increasing membrane fluidity was associated with increasing activity. Malonyl-CoA was found to produce a decrease in intact mitochondrial membrane fluidity in the fed state, particularly at pH 7.0 or less. No effect was observed in intact mitochondria from starved or diabetic rats, or in inverted vesicles from any of the treatment groups. Through its effect on membrane fluidity, malonyl-CoA could regulate carnitine palmitoyltransferase activity on both surfaces of the inner membrane through an interaction with only the outer surface.     

Control of the NADPH supply and GSH recycling for oxidative stress management in hepatoma and liver mitochondria

To unveil what controls mitochondrial ROS detoxification, the NADPH supply and GSH/GSSG recycling for oxidative stress management were analyzed in cancer and non-cancer mitochondria. Therefore, proteomic and kinetomic analyses were carried out of the mitochondrial (i) NADPH producing and (ii) GSH/GSSG recycling enzymes associated to oxidative stress management. The protein contents of the eight enzymes analyzed were similar or even higher in AS-30D rat hepatoma mitochondria (HepM) than in rat liver (RLM) and rat heart (RHM) mitochondria, suggesting that the NADPH/GSH/ROS pathway was fully functional in cancer mitochondria.The Vmax values of IDH-2 were much greater than those of GDH, TH and ME, suggesting that IDH-2 is the predominant NADPH producer in the three mitochondrial types; in fact, the GDH reverse reaction was favored. The Vmax values of GR and GPx were lower in HepM than in RLM, suggesting that the oxidative stress management is compromised in cancer mitochondria. The Km values of IDH-2, GR and GPx were all similar among the different mitochondrial types.Kinetic modeling revealed that the oxidative stress management was mainly controlled by GR, GPx and IDH. Modeling and experimentation also revealed that, due to their higher IDH-2 activity and lower GPx activity presumably by acetylation, HepM (i) showed higher steady-state NADPH levels; (ii) required greater peroxide concentrations to achieve reliable steady-state fluxes and metabolite concentration; and (iii) endured higher peroxide concentrations without collapsing their GSH/GSSG ratios. Then, to specifically prompt lower GSH/GSSG ratios under oxidative stress thus compromising cancer mitochondria functioning, GPx should be re-activated.     

Mitochondrial NADP-dependent malic enzyme of cod heart. Rate of forward and reverse reaction

The mitochondrial NADP-dependent malic enzyme (EC 1.1.1.40) was purified about 300-fold from cod Gadus morhua heart to a specific activity of 48 units (mumol/min)/mg at 30 degrees C. The possibility of the reductive carboxylation of pyruvate to malate was studied by determination of the respective enzyme properties. The reverse reaction was found to proceed at about five times the velocity of the forward rate at a pH 6.5. The Km values determined at pH 7.0 for pyruvate, NADPH and bicarbonate in the carboxylation reaction were 4.1 mM, 15 microM and 13.5 mM, respectively. The Km values for malate, NADP and Mn2+ in the decarboxylation reaction were 0.1 mM, 25 microM and 5 microM, respectively. The enzyme showed substrate inhibition at high malate concentrations for the oxidative decarboxylation reaction at pH 7.0. Malate inhibition suggests a possible modulation of cod heart mitochondrial NADP-malic enzyme by its own substrate. High NADP-dependent malic enzyme activity found in mitochondria from cod heart supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate.     

Palladacycles catalyse the oxidation of critical thiols of the mitochondrial membrane proteins and lead to mitochondrial permeabilization and cytochrome c release associated with apoptosis

Permeabilization of the mitochondrial membrane has been extensively associated with necrotic and apoptotic cell death. Similarly to what had been previously observed for B16F10-Nex2 murine melanoma cells, PdC (palladacycle compounds) obtained from the reaction of dmpa (N,N-dimethyl-1-phenethylamine) with the dppe [1,2-ethanebis(diphenylphosphine)] were able to induce apoptosis in HTC (hepatoma, tissue culture) cells, presenting anticancer activity in vitro. To elucidate cell site-specific actions of dmpa:dppe that could respond to the induction of apoptosis in cancer cells in the present study, we investigated the effects of PdC on isolated RLM (rat liver mitochondria). Our results showed that these palladacycles are able to induce a Ca2+-independent mitochondrial swelling that was not inhibited by ADP, Mg2+ and antioxidants. However, the PdC-induced mitochondrial permeabilization was partially prevented by pre-incubation with CsA (cyclosporin A), NEM (N-ethylmaleimide) and bongkreic acid and totally prevented by DTT (dithiothreitol). A decrease in the content of reduced thiol groups of the mitochondrial membrane proteins was also observed, as well as the presence of membrane protein aggregates in SDS/PAGE without lipid and GSH oxidation. FTIR (Fourier-transform IR) analysis of PdC-treated RLM demonstrated the formation of disulfide bonds between critical thiols in mitochondrial membrane proteins. Associated with the mitochondrial permeabilization, PdC also induced the release of cytochrome c, which is sensitive to inhibition by DTT. Besides the contribution to clarify the pro-apoptotic mechanism of PdC, this study shows that the catalysis of specific protein thiol cross-linkage is enough to induce mitochondrial permeabilization and cytochrome c release.     

Cross-reconstitution of isolated F1-ATPase from potato tuber mitochondria with F1-depleted beef heart and yeast submitochondrial particles

Cross-reconstitution of isolated potato mitochondrial F1-ATPase with F1-depleted beef heart and yeast submitochondrial particles is reported. Potato F1 binds to the heterologous membrane and confers oligomycin sensitivity on the ATPase activity of the reconstituted system. Binding of F1 is promoted by the presence of Mg2+ with the maximal stimulatory effect at 20 mM. Mg2+ increase the sensitivity to oligomycin of the reconstituted system consisting of potato F1 and yeast membranes, however, they do not influence oligomycin sensitivity of potato F1 and beef heart membranes.     

Effects of Cd2+ on ATP-driven membrane potential in beef heart mitochondrial H+-ATPase: a study using the voltage-sensitive probe oxonol VI

Beef heart mitochondrial H+-ATPase (F1-F0) vesicles were prepared by lysolecithin extraction of ETPH. ATP-driven membrane potential was monitored indirectly by following absorbance changes of the potential-sensitive dye oxonol VI. The steady-state potential was discharged by oligomycin and/or Cd2+ (a dithiol reagent). At 13 degrees C, the agents appeared to act synergistically; at 24 degrees C the data were equivocal. When Cd2+ was added before energization, the membrane potential was markedly attenuated. Both effects of Cd2+ were inhibited by dithiothreitol. The activation energy for oligomycin-sensitive ATPase exhibited a discontinuity at 16 degrees C. However, the temperature dependence of the rate of potential discharge by oligomycin showed no such discontinuity. The results are discussed in terms of the involvement of thiol groups in proton translocation and the thermotropic behavior of the membrane vesicles.     

Respiration and mitochondrial ATPase in energized mitochondria during isoproterenol-induced cell injury of myocardium.

1. Respiration of mitochondria, membrane potential and mitochondrial ATPase under energized conditions were studied in rat myocardium during cell injury induced by treatment with isoproterenol. 2. Increase in the state 4 rate of respiration and ADP:O ratio, as well as decrease in the state 3 rate and Respiratory Control Ratio (RCR) were found. 3. The optimum pH for RCR and for maximum ATPase activity was shifted to lower values. 4. The state 3 respiration was more sensitive to oligomycin inhibition. 5. The mitochondria showed lower ability to generate membrane potential. 6. An increase in the K0.5 values for catalytic sites II and III of mitochondrial ATPase at pH 7.4 and 5.5 was found. 7. These results are consistent with alterations on the integrity of mitochondrial membrane, and corroborate with the hypothesis of changes on the mitochondrial ATPase during isoproterenol-induced cell injury of myocardium.     

Monovalent ion stimulated adenosine triphosphatase from oat roots

Monovalent ion stimulated ATPase activity from oat (Avena sativa) roots has been found to be associated with various membrane fractions (cell wall, mitochondrial and microsomal) of oat roots. The ATPase requires Mg²⁺ (or Mn⁺²) but is further stimulated by K⁺ and other monovalent ions. The monovalent ions are ineffective in the absence of the divalent activating cation. The ATPase has been described with respect to monovalent ion specificity, temperature, pH, substrate specificity, and Mg²⁺ and K⁺ concentrations. It was further shown that oligomycin inhibits a part of the total ATPase activity and on the basis of the oligomycin sensitivity it appears that at least 2 membrane associated ATPases are being measured. The mitochondrial fraction is most sensitive to oligomycin and the microsomal fraction is least sensitive to oligomycin. The oligomycin insensitive ATPase appears to be stimulated more by K⁺ than the oligomycin sensitive ATPase.     

Biogenesis of mitochondria 23. The biochemical and genetic characteristics of two different oligomycin resistant mutants ofSaccharomyces cerevisiae under the influence of cytoplasmic genetic modification

Two classes ofSaccharomyces cerevisiae mutants resistant to oligomycin, an inhibitor of mitochondrial membrane bound ATPase are described. Biochemical analysis shows thatin vitro the mitochondrial ATPase of both types of mutant are sensitive to oligomycin.In vivo sensitivity of the mutants to oligomycin can be demonstrated following anaerobic growth of the cells, which grossly alters the mitochondrial membrane and renders the ATPase of the mutants sensitive to oligomycin. It is concluded that the mutation to oligomycin resistance in both mutant types is phenotypically expressed as a change in the mitochondrial membrane. The intact mitochondrial membrane in the wild type cell is freely permeable to oligomycin, whereas the resistant mutant is impermeable to oligomycin; alteration of the mitochondrial membrane during isolation of the organelle or physiological modification of the membranes of the mitochondria by anaerobic growth renders the membranes permeable.These mitochondrial membrane mutants differ in their cross-reference patterns and their genetics. One is resistant to oligomycin only, and behaves like previously reported cytoplasmic mutants. The other shows cross-resistance to inhibitors of mitochondrial protein synthesis as well as to oligomycin; although the mutant appears to arise from a single step mutation its genetic properties are complex and show part-nuclear and part-cytoplasmic characteristics. The implications of the observations are discussed.     

Inhibition of apoptosis in pulmonary endothelial cells by altered pH,mitochondrial function,and ATP supply

We investigated the effect of altered extracellular pH, mitochondrial function, and ATP content on development of apoptosis in human pulmonary artery endothelial cells after treatment with staurosporine (STS). STS produced a concentration- and time-dependent increase in caspase-3 activity in pH 7.4 medium that reached a peak at 6 h. The increase in caspase activity was associated with significant DNA fragmentation. Fluorescent imaging of treated monolayers in pH 7.4 medium with Hoechst-33342-propidium iodide demonstrated a large percentage of apoptotic cells ( approximately 40%) with no evidence of necrosis. Caspase activity, DNA fragmentation, and percentage of apoptotic cells were reduced after STS treatment in acidic media (pH 7.0 and 6.6). The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM inhibited STS-induced apoptosis, whereas the rise in intracellular Ca2+concentration in STS-treated cells in pH 7.4 medium was reduced in pH 7.0 medium. These results suggest that one mechanism for inhibitory effects of acidosis may be a pH-induced alteration in Ca2+ signaling. Treatment with STS in the presence of oligomycin (10 microM), an inhibitor of the mitochondrial F(0)F(1)-ATPase, in glucose-free media abolished caspase activation and DNA fragmentation in association with severe ATP depletion ( approximately 2% of control cells). Imaging demonstrated a change in the mode of cell death from apoptosis to necrosis under these conditions. This change was linked to the level of ATP depletion, because STS treatment in the absence of glucose or the presence of oligomycin in media with glucose still leads to apoptosis in the presence of only moderate ATP depletion. These results demonstrate that pH, mitochondrial function, and ATP supply are important variables that regulate STS-induced apoptosis in human pulmonary artery endothelial cells.     

Quantitative dehydrogenase histochemistry with exogenous electron carriers (PMS,MPMS, MB)

Summary The relative efficiencies of phenazine methosulfate (PMS), 1-methoxy-phenazine methosulfate (MPMS) and Meldola Blue (MB) as electron carriers were determined biochemically (non-enzymic NADH-tetrazolium salt-test) and by quantitative histochemistry (heart and kidney slices; succinate dehydrogenase, SDH; lactate dehydrogenase, LDH). MPMS developed the highest electron transfer velocity in biochemical assays. The reaction was independent of the pH value between 7.0–8.5. PMS and MB always showed a lower transfer ability in biochemical tests which was higher with iodonitrotetrazolium chloride (INT) than with nitro blue tetrazolium chloride (NBT). A distinct pH dependence was demonstrable with MB in this respect, preferentially using INT as tetrazolium salt.Quantitative histochemical results with electron carriers are often at variance with biochemical ones. MPMS leads to somewhat higher demonstrable activities only in the determination of the NAD-dependent LDH, whereas MB results in somewhat higher LDH activity than PMS (reaction medium with agarose). MB and PMS yielded almost equally high activities in the demonstration of the flavoprotein-dependent SDH using a reaction medium with agarose. With an aqueous reaction medium, PMS resulted in higer SDH activities than MB. MPMS always had the lowest efficiency in electron transfer ability using an aqueous or agarose containing reaction medium (SDH). With PVA in the reaction medium (SDH determination) PMS was clearly superior to MPMS. MB showed only a small transfer activity under these conditions because PVA seems to bind MB almost completely. It is concluded that in histochemistry an appropriate electron carrier and electron carrier concentration must be determined for different incubation conditions, tissues, tissue preparations and dehydrogenases studied. General statements about the efficiency or inefficiency of an electron carrier as a result of only one incubation condition does not seem to be justified.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)