Relations Between Intracellular Ions and Energy Metabolism Under Acidotic Conditions: A Study with Nigericin in Synaptosomes, Neurons, and C6 Glioma Cells

Abstract: Effects of nigericin were investigated in rat brain synaptosomes, cultured neurons, and C6 glioma cells to characterize the relations among ATP synthesis, [Na⁺]_i., [K⁺]_i, and [Ca²⁺]_i, and pH under conditions when [H⁺]_i is substantially increased and transmembrane electrical potential is decreased. Intracellular acidification and loss of K⁺ were accompanied by enhanced oxygen consumption and lactate production and a decrease in cellular energy level. Changes in the last three parameters were attenuated by addition of 1 mM ouabain. In synaptosomes treated with nigericin, neither respiration nor glycolysis was affected by 0.3 μM tetrodotoxin, whereas 1 mM amiloride reduced lactate production by 20% but did not influence respiration. In C6 cells, amiloride decreased the nigericin-stimulated rate of lactate generation by about 50%. The enhancement by nigericin of synaptosomal oxygen uptake and glycolytic rate decreased with time. However, there was only a small reduction in respiration and none in glycolysis in C6 cells. Measurements with ion-selective microelectrodes in neurons and C6 cells showed that nigericin also caused a rise in [Ca²⁺], and [Na⁺]., The increase in [Na⁺], in C6 cells was partially reversed by 1 mM amiloride. It is concluded that nigericin-induced loss of K⁺ and subsequent depolarization lead to an increase in Na⁺ influx and stimulation of the Na⁺/K⁺ pump with a consequent rise in energy utilization; that acidosis inhibits mitochondrial ATP production; that a rise in [H⁺] does not decrease glycolytic rate when the energy state (a fall in [ATP] and rises in [ADP] and [AMP]) is simultaneously reduced; that a fall in [K⁺], depresses both oxidative phosphorylation and glycolysis; and that the nigericin-induced alterations in ion levels and activities of energy-producing pathways can explain some of the deleterious effects of ischemia and hypoxia.     

Effects of High-Potassium-Induced Depolarization on Amino Acid Chemistry of the Dorsal Cochlear Nucleus in Rat Brain Slices

High K⁺ was used to depolarize glia and neurons in order to study the effects on amino acid release from and concentrations within the dorsal cochlear nucleus (DCN) of brain slices. The release of glutamate, -aminobutyrate (GABA) and glycine increased significantly during exposure to 50 mM K⁺, while glutamine and serine release decreased significantly during and/or after exposure, respectively. After 10 min of exposure to 50 mM K⁺, glutamine concentrations increased in all three layers of DCN slices, to more than 5 times the values in unexposed slices. In the presence of a glutamate uptake blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), glutamine concentrations in all layers did not increase as much during 50 mM K⁺. Similar but smaller changes occurred for serine. Mean ATP concentrations were lower in 50 mM K⁺-exposed slices compared to control. The results suggest that depolarization, such as during increased neural activity, can greatly affect amino acid metabolism in the cochlear nucleus.     

Mg2+-dependent (Na+ + K+)- and Na+-ATPases in the kidneys of the gilthead bream (Sparus auratus L.)

• 1.1. The (Na⁺ + K⁺)- and Na⁺-ATPases, both present in kidney microsomes of Sparus auratus L., have different activities and optimal assay conditions as, in the first of the two stocks of fish used (A), the spec. act. of the former is 51.7 μmol P_i mg prot⁻¹ hr⁻¹ at pH 7.5, 100 mM Na⁺, 10 mM K⁺, 17.5 mM Mg²⁺, 7.5 mM ATP and that of the latter is 6.5 μmol P_i mg prot⁻¹ hr⁻¹ at pH 6.5, 40 mM Na⁺, 4.0 mM Mg²⁺, 2.5 mM ATP.
• 2.2. Ouabain and vanadate specifically inhibit the (Na⁺ + K⁺)-ATPase but not the Na⁺-ATPase that is preferentially inhibited by ethacrynic acid.
• 3.3. While the (Na⁺ + K⁺)-ATPase is strictly specific for ATP and Na⁺, Na⁺-ATPase can be activated by various monovalent cations and, apart from ATP, hydrolyses CTP, though less efficiently.
• 4.4. The second stock B, subjected to higher salinity than A, shows an acidic shifted Na⁺-ATPase optimal pH, opposed to the stability of that of the (Na⁺ + K⁺)-ATPase, a decreased (Na⁺ + K⁺)-ATPase and a strikingly depressed Na⁺-ATPase.
• 5.5. The results are compared with literature data and discussed on the basis of the presumptive different roles as well as functional prevalence in various salinities of the two ATPases.

     

Inhibition of Na+, K+-ATPase activity by δ-aminolevulinic acid

-Aminolaevulinic acid (ALA) has been shown to be toxic to cultured neurons and glia at concentrations as low as 10 M. In an attempt to elucidate the mechanism of toxicity, the effects of ALA on membrane ATPase activity were investigated. Exposure of neuron cultures to 1 mM ALA for 7 days caused a substantial decrease in both Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities. At lower concentrations, ALA affected only the Na⁺, K⁺-component. ALA appeared to act directly, inhibiting Na⁺, K⁺-ATPase activity in rat brain cortex membrane preparations at 10 M Although this effect was slight, it may well represent the mechanism of action of ALA, since ouabain, a potent inhibitor of Na⁺, K⁺-ATPase activity, proved to be more toxic to cultured neurons than ALA. Furthermore, cardiac glycoside overdosage causes neurological disturbances which are very similar to those observed in the acute attack of porphyria.     

Effects of aldosterone on Na transport in the toad bladder I. Glycolysis and lactate production under aerobic conditions

Previous studies indicated that aldosterone enhances active Na⁺ transport, glycolysis, lactate production and respiration of the toad bladder. Evidence was also presented that the changes in glycolysis and lactate production were secondary to the changes in active Na⁺ transport. Further analysis of the relationships between metabolism and Na⁺ transport was undertaken with the aid of two inhibitors of pyruvate metabolism, oxythiamine and phenylpyruvate. These inhibitors prevented the aldosterone-induced increase in oxidation of [6-¹⁴C]glucose but had little effect on the increase in lactate production. In contrast, the effect on Na⁺ transport (i.e., I_sc) was completely inhibited by oxythiamine plus phenylpyruvate with glucose as substrate. The effect on Na⁺ transport, however, was obtained wth the by-pass substrates, oxaloacetate plus ß-hydroxybutyrate, in the presence of these inhibitors. These results implied that steroidal enhancement of lactate production and Na⁺ transport were independent effects. To evaluate whether an increase in Na⁺ transport, per se would augment lactate production, the responses were evaluated under conditions of an imposed Na⁺ gradient (mucosal Na⁺ = 5 mM; serosal Na⁺ = 110 mM). Addition of NaCl to the mucosal media evoked the same increase in I_sc as the addition of aldosterone; both additions increased I_sc more than two-fold. Aldosterone reduced lactate production under these conditions while the re-addition of NaCl had no effect on lactate formation. These results are consistent with an action of aldosterone on pathways involved in oxidative energy metabolism, and suggest that the activation of glycolysis may be a function of the net balance between energy production and utilization.     

Studies on the relationship between glycolysis and (Na++K+)-ATPase in cultured cells

In several tissues a coupling between glycolysis and (Na⁺+K⁺)-ATPase has been observed. We report here studies on the coupling of glycolysis and (Na⁺+K⁺)-ATPase in Rous-transformed hamster cells and Ehrlich ascites tumor cells. The rate of (Na⁺+K⁺)-ATPase was estimated by the initial rate of ouabain-sensitive K⁺ influx after K⁺ reintroduction to K⁺-depleted cells. Experiments were performed with cells producing ATP via oxidative phosphorylation alone (i.e., lactate sole substrate), glycolysis alone (i.e., glucose as substrate in the absence of oxygen or with antimycin A), or glycolysis and oxidative phosphorylation (i.e., glucose as substrate in the presence of oxygen). The cells produced ATP at approximately the same rate under all of these conditions, but the initial rate of K⁺-influx was approx. 2-fold higher when AtP was produced from glycolysis. Changes in cell Na⁺ due to other transport processes related to glycolysis, such as Na⁺-H⁺ exchange, Na⁺-glucose cotransport, and K⁺-H⁺ exchange were ruled out as mediators of this effect on (Na⁺+K⁺)-ATPase. These data suggest that glycolysis is more effective than oxidative phosphorylation in providing ATP to (Na⁺+K⁺)-ATPase to these cultured cells.     

Effect of potassium and ammonium ions upon glycolysis catalyzed by an extract of rat brain

The stimulatory effect of ammonium and potassium ions upon glycolysis, as catalyzed by an extract prepared from an acetone powder of rat brain, has been investigated. The effect is most pronounced when small amounts of adenosine triphosphate (ATP) are employed and it becomes progressively less apparent as the ATP concentration is increased.Unlike the extracts and homogenates obtained from fresh brain tissue (1,2), glycolysis by the acetone extracts is not inhibited significantly by sodium ion. This is apparently due to the low apyrase content of these extracts. In addition the experiments indicate that the NH₄⁺ and K⁺ have direct stimulatory effects which are not due to antagonism between Na⁺ and NH₄⁺ or K⁺. This suggestion gains credence by the observation that the same concentration of NH₄⁺ is required for stimulation whether or not the concentration of Na⁺ is reduced through substitution of trishydroxymethylaminomethane buffer for the NaHCO₃ buffer. Apparently NH₄⁺ and K⁺ serve to maintain ATP in these systems by initiating or accelerating phosphorylation reactions. In so doing, they prevent the formation of adenylic acid which is fairly rapidly dephosphorylated in those systems by a 5-nucleotidase.     

The glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Transport mechanism, regulation by ATP and characterization of the glutamine/glutamate antiport

The glutamine/amino acid transporter solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes has been previously identified as the ASCT2 transporter. The reconstituted transporter catalyses an antiport reaction in which external glutamine and Na⁺ are cotransported in exchange with internal glutamine (or other amino acids). The glutamine-Na⁺ cotransport occurred with a 1:1 stoichiometry. The concentration of Na⁺ did not influence the Km for glutamine and vice versa. Experimental data obtained by a bi-substrate analysis of the glutamine-Na⁺ cotransport, together with previous report on the glutamine_ex/glutamine_in pseudo bi-reactant analysis, indicated that the transporter catalyses a three-substrate transport reaction with a random simultaneous mechanism. The presence of ATP in the internal compartment of the proteoliposomes led to an increase of the Vmax of the transport and to a decrease of the Km of the transporter for external Na⁺. The reconstituted glutamine/amino acid transporter was inhibited by glutamate; the inhibition was more pronounced at acidic pH. A kinetic analysis revealed that the inhibition was competitive with respect to glutamine. Glutamate was also transported in exchange with glutamine. The external Km of the transporter for glutamate (13.3 mM) was slightly higher than the internal one (8.3 mM). At acidic pH the external but not the internal Km decreased. According with the Km values, glutamate should be transported preferentially from inside to outside in exchange for external glutamine and Na⁺.     

Salinity-dependence of the properties of gill (Na++K+-ATPase in rainbow trout Oncorhynchus mykiss

• 1.1. The expected higher gill (Na⁺+K⁺)-ATPase activity in rainbow trout adapted to brackish water (BW) with respect to fresh water (FW) is accompanied by some changes in the enzyme kinetics while the enzyme sensitivity to ouabain is unaffected
• 2.2. Maximal activation is attained under the optimal conditions of 4 mM ATP, 7.5 mM Mg²⁺, 50 mM Na⁺, 2.5 mM K⁺, pH 7.0 in FW, and 3 mM ATP, 10 mM Mg²⁺, 100 mM Na⁺, 10 mM K⁺, pH 7.5 in BW.
• 3.3. The change of the enzyme activation kinetics by Mg²⁺, ATP, Na⁺ and K⁺ from simple saturation in FW to cooperativity in BW and other habitat-dependent variations including the pH alkaline shift in BW are hypothetically related to an adaptive significance to the different environmental salinity.
• 4.4. Gill total lipids and phospholipids are 30% lower in BW than in FW while their ratio is constant; some differences in gill total lipid fatty acid composition between FW and BW do not significantly affect the unsaturation parameters.

     

Kinetics of Na+, K+-ATPase Inhibition by a Rat Brain Endogenous Factor (II-E)

Previous work from this laboratory led to the isolation by gel filtration and anionic exchange HPLC of a rat brain fraction named II-E, which highly inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity. In this study we evaluated the kinetics of such inhibition and found that inhibitory potency was independent of Na⁺(1.56–200 mM), K⁺(1.25–40 mM), or ATP (1–8 mM) concentration. Hanes-Woolf plots indicated that II-E decreases Vmax but does not alter K_Mvalue, and suggested uncompetitive inhibition for Na⁺, K⁺or ATP. However, II-E became a stimulator at 0.5 mM ATP concentration. It is postulated that this brain factor may modulate ionic transport at synapses, thus participating in central neurotransmission.     

Effect of calcium on brain metabolism in vitro

In attempts to distinguish between direct and indirect effects of Ca on brain cell metabolism, respiration, glycolysis, ATP, phosphocreatine, incorporation of [¹⁴C] leucine into protein, and accumulation of⁴⁵Ca was determined in brain slices. Incubation was carried out in normal salt-balanced medium, in high-potassiumor ouabain-containing medium under aerobic and anaerobic conditions. Calcium ions inhibited slightly glycolysis and respiration in normal medium and activated amino acid incorporation into proteins. Levels of ATP and phosphocreatine remained normal. These effects were interpreted as due to a stabilization of plasma membranes by Ca ions to prevent their spontaneous depolarization. Incubation of slices in high-potassium and ouabain media in aerobic conditions in the presence of Ca resulted in activation of respiration and glycolysis, decrease of ATP and phosphocreatine levels, and inhibition of amino acid incorporation into proteins. The disturbances in energy metabolism, caused by the respiration-linked Ca uptake in brain mitochondria and concomitant inhibition of oxidative phosphorylation, may lead to the inhibition of amino acid incorporation into proteins. An increase in Ca levels in the cytoplasm may only be expected in anaerobic conditions during the incubation in high-potassium and ouabain media. This is manifested by a direct inhibition of glycolysis by Ca ions and a drastic decrease of ATP and phosphocreatine in slices. The results suggest that stimulation of aerobic glycolysis and inhibition of anaerobic glycolysis by Ca may explain the unknown mechanism of the so-called reversed Pasteur effect of brain slices incubated in high-potassium media.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na⁺ is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac muscle, the exact mechanisms of low Na⁺-induced increases in [Ca²⁺]_i are not completely defined. To gain information in this regard, we examined the effects of low Na⁺ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca²⁺]_i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na⁺-induced increase in [Ca²⁺]_i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na⁺ did not alter the ATP-induced increase in [Ca²⁺]_i in the cardiomyocytes. This increase in [Ca²⁺]_i due to low Na⁺ and elevated KCl was dependent on the extracellular concentration of Ca²⁺ (0.25–2.0 mM). The L-type Ca²⁺-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na⁺, KCl-induced increase in [Ca²⁺]_i without significantly affecting the response to low Na⁺ alone. The low Na⁺, high KCl-induced increase in [Ca²⁺]_i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na⁺–Ca²⁺ exchanger, both sarcolemmal Na⁺–K⁺ATPase, as well as the sarcoplasmic reticulum Ca²⁺-pump play prominent roles in the low Na⁺-induced increase in [Ca²⁺]_i. (Mol Cell Biochem 263: 151–162, 2004)     

Nitration as a Mechanism of Na+, K+-ATPase Modification During Hypoxia in the Cerebral Cortex of the Guinea Pig Fetus

Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide free radicals leading to peroxynitrite production. The present study tests the hypothesis that hypoxia results in NO-mediated modification of Na⁺, K⁺-ATPase in the fetal brain. Studies were conducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO₂ of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P₂ membrane fractions were prepared from normoxic and hypoxic fetuses and divided into untreated and treated groups. The membranes were treated with 0.5 mM peroxynitrite at pH 7.6. The Na⁺, K⁺-ATPase activity was determined at 37°C for five minutes in a medium containing 100 mM NaCl, 20 mM KCl, 6.0 mM MgCl₂, 50 mM Tris HCl buffer pH 7.4, 3.0 mM ATP with or without 10 mM ouabain. Ouabain sensitive activity was referred to as Na⁺, K⁺-ATPase activity. Following peroxynitrite exposure, the activity of Na⁺, K⁺-ATPase in guinea pig brain was reduced by 36% in normoxic membranes and further 29% in hypoxic membranes. Enzyme kinetics was determined at varying concentrations of ATP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alters the affinity of the active site of Na⁺, K⁺-ATPase for ATP and decreases the Vmax by 35% in hypoxic membranes. When compared to untreated normoxic membranes Vmax decreases by 35.6% in treated normoxic membranes and further to 52% in treated hypoxic membranes. The data show that peroxynitrite treatment induces modification of Na⁺, K⁺-ATPase. The results demonstrate that peroxynitrite decreased activity of Na⁺, K⁺-ATPase enzyme by altering the active sites as well as the microenvironment of the enzyme. We propose that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na⁺, K⁺-ATPase activity.     

Voltage-dependent inhibition of the sodium pump by external sodium: Species differences and possible role of the N-terminus of the α-subunit

Currents generated by the Na⁺/K⁺ ATPase were measured under voltage clamp in oocytes of Xenopus laevis. The dependence of pump current on external [Na⁺] was investigated for the endogenous Xenopus pump as well as for wild-type and mutated pumps of electroplax of Torpedo californica expressed in the oocytes. The mutants had -subunits truncated before position Lys²⁸ (K28) or Thr²⁹ (T29) of the N-terminus. The currents generated by all variants of pump molecules in the presence of 5 mM K⁺ show voltage-dependent inhibition by external [Na⁺]. The apparent K₁ values increase with membrane depolarisation, and the potential dependence can be described by the movement of effective charges in the electrical potential gradient across the membrane. Taking into account Na⁺-K⁺ competition for external binding to the E₂P form, apparent K₁ values and effective charges for the interaction of the Na⁺ ions with the E₂P form can be estimated. For the Xenopus pump the effective charge amounts to 1.1 of an elementary charge and the K₁ value at 0 mV to 44 mM. For the wild-type Torpedo pump, the analysis yields values of 0.73 of an elementary charge and 133 mM, respectively. Truncation at the N-terminus removing a lysinerich cluster of the a-subunit of the Torpedo pump leads to an increase of the effective charge and decrease of the K₁ value. For K28, values of 0.83 of an elementary charge and 117 mM are obtained, respectively. If LyS²⁸ is included in the truncation (·T29), the effective charge increases to 1.5 of an elementary charge and the apparent K₁ value is reduced to 107 mM. The K, values for pump inhibition by external Na⁺, calculated by taking into account Na⁺-K⁺ competition, are smaller than the K_/12 values determined in the presence of 5 mM [K⁺]. The difference is more pronounced for those pump variants that have higher K_m, values. The variations of the parameters describing inhibition by external [Na⁺] are qualitatively similar to those described for the stimulation of the pumps by external [K⁺] in the absence of extracellular [Na⁺]. The observations may be explained by an acess channel within the membrane dielectric that has to be passed by the external Na⁺ and K⁺ ions to reach or leave their binding sites. The potential-dependent access and/or the interaction with the binding sites shows species differences and is affected by cytoplasmic lysine residues in the N-terminus.     

Kinetics of Na+-, K+-ATPase Inhibition by an Endogenous Modulator (II-A)

We have previously reported the isolation by gel filtration and anionic exchange HPLC of two brain Na⁺, K⁺-ATPase inhibitors, II-A and II-E, and kinetics of enzyme interaction with the latter. In the present study we evaluated the kinetics of synaptosomal membrane Na⁺, K⁺-ATPase with II-A and found that inhibitory activity was independent of ATP (2–8 mM), Na⁺ (3.1–100 mM), or K⁺ (2.5–40 mM) concentration. Hanes-Woolf plots showed that II-A decreases V_max in all cases; K_M value decreased for ATP but remained unaltered for Na⁺ and K⁺, indicating respectively uncompetitive and noncompetitive interaction. However, II-A became a stimulator at 0.3 mM K⁺ concentration. It is postulated that brain endogenous factor II-A may behave as a sodium pump modulator at the synaptic region, an action which depends on K⁺ concentration.     

Brain plasmamembrane NA+:,K+-ATPase is inhibited by acetylated tubulin

Membranes from brain tissue contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. The hydrophobic behavior of this tubulin is due to the formation of a complex with the -subunit of Na⁺,K⁺-ATPase. In the present work we show that the interaction of tubulin with Na⁺K⁺-ATPase inhibits the enzyme activity. We found that the magnitude of the inhibition is correlated with: (1) concentration of the acetylated tubulin isoform present in the tubulin preparation used, and (2) amount of acetylated tubulin isoform isolated as a hydrophobic compound. In addition, some compounds involved in the catalytic action of Na⁺K⁺-ATPase were assayed to determine their effects on the inhibitory capability of tubulin on this enzyme. The inhibitory effect of tubulin was only slightly decreased by ATP at relatively low nucleotide concentration (0.06 mM). NaCl (1-160 mM) and KCl (0.2-10 mM) showed no effect whereas inorganic phosphate abolished the inhibitory effect of tubulin in a concentration-dependent manner.     

Sodium transport in red blood cells of lamprey LAmpetra fluviatilis

• 1.1. Unidirectional Na⁺ influx in lamprey red blood cells was determined using ²²Na as a tracer.
• 2.2. Total Na⁺ uptake and amiloride-inhibitable Na⁺ influx increased in a saturable fashion as a function of external Na⁺ concentration (Na_e).
• 3.3. At 141 mM Na_e, the average value of net Na⁺ influx was 13 ± 1.1 and the amiloride-sensitive Na⁺ influx was 5.3±1.1 mmol/l cells per hr (±SE).
• 4.4. The amiloride-sensitive component of Na⁺ influx was significantly activated by 10⁻⁵ M isoproterenol, by 2 × 10⁻⁵ M DNP, and by cell shrinkage.
• 5.5. Furosemide (1 mM) had no effect on the Na⁺ transport in red cells.
• 6.6. The residual amiloride-insensitive component of Na⁺ transport was a linear function of Na_e in the range of 5–141 mM. This transport seems to be accounted for by simple diffusion.

     

Role of MMP-2 in inhibiting Na+ dependent Ca2+ uptake by H2O2 in microsomes isolated from pulmonary smooth muscle

Treatment of microsomes (preferentially enriched with endoplasmic reticulum) isolated from bovine pulmonary artery smooth muscle tissue with H₂O₂ (1 mM) markedly stimulated matrix metalloproteinase activity and also inhibited Na⁺ dependent Ca²⁺ uptake. Electron micrograph revealed that H₂O₂ (1 mM) does not cause any damage to the microsomes. MMP-2 and TIMP-2 were determined to be the ambient protease and corresponding antiprotease of the microsomes. Pretreatment with vitamin E (1 mM) and TIMP-2 (50 g/ml) reversed the effect produced by H₂O₂ (1 mM) on Na⁺ dependent Ca²⁺ uptake in the microsomes. However, H₂O₂ (1 mM) caused changes in MMP-2 activity and Na⁺ dependent Ca²⁺ uptake were not reversed upon pretreatment of the microsomes with a low concentration of 5 g/ml of TIMP-2 which otherwise reversed MMP-2 (1 g/ml) mediated increase in ¹⁴C-gelatin degradation and inhibition of Na⁺ dependent Ca²⁺ uptake. Combined treatment of the microsomes with a low dose of MMP-2 (0.5 g/ml) and H₂O₂ (0.5 mM) inhibited Na⁺ dependent Ca²⁺ uptake in the microsomes compared to the respective low dose of either of them. Direct treatment of TIMP-2 (5 g/ml) with H₂O₂ (1 mM) abolished the inhibitory effect of the inhibitor on ¹⁴C-gelatinolytic activity elicited by 1 g/ml of MMP-2. Thus, one of the mechanisms by which H₂O₂ activates MMP-2 could be due to inactivation of TIMP-2 by the oxidant. The resulting activation of MMP-2 subsequently inhibits Na⁺ dependent Ca²⁺ uptake in the microsomes. (Mol Cell Biochem 270: 79–87, 2005)