Effect of 5-week moderate intensity endurance training on the oxidative stress, muscle specific uncoupling protein (UCP3) and superoxide dismutase (SOD2) contents in vastus lateralis of young, healthy men.

In the present study fifteen male subjects (age: 22.7 ± 0.5 years; BMI: 23.5 ± 0.6 kg x m⁻²; VO2(max) 46.0 ± 1.0 mL x kg⁻¹ x min⁻¹) performed 5 week moderate intensity endurance training. The training resulted in a significant increase in maximal oxygen uptake (VO2(max)) (P=0.048) and power output reached at VO2(max) (P=0.0001). No effect of training on the uncoupling protein 3 (UCP3) content in the vastus lateralis was found (P>0.05). The improvement of physical capacity was accompanied by no changes in cytochrome-c and cytochrome-c oxidase contents in the vastus lateralis (P>0.05). However, the training resulted in an increase (P=0.02) in mitochondrial manganese superoxide dismutase (SOD2) content in this muscle. Moreover, a significant decrease (P=0.028) in plasma basal isoprostanes concentration [F2isoprostanes](pl) accompanied by a clear tendency to lower (P=0.08) gluthatione disulfide concentration [GSSG](pl) and tendency to higher (P=0.08) total antioxidant capacity (TAC) was observed after the training. We have concluded that as little as 5 weeks of moderate intensity endurance training is potent to improve physical capacity and antioxidant protection in humans. Surprisingly, these effects occur before any measurable changes in UCP3 protein content. We postulate that the training-induced improvement in the antioxidant protection at the muscle level is due to an increase in SOD2 content and that therefore, the role of UCP3 in the enhancement of physical capacity and antioxidant protection, at least in the early stage of training, is rather questionable.

Transcriptional upregulation of the human MRP2 gene expression by serine/threonine protein kinase inhibitors.

Transcriptional regulation by cellular signalling pathways of multidrug resistance proteins that pump anticancer drugs out of cells is one of key issues in the development of the multidrug resistance phenotype. In our study, we have used the reporter gene approach as well as determination of mRNA levels in two cancer cell lines of human origin, MCF-7 and A549, to study the regulation of multidrug resistance proteins 2 and 3 (MRP2 AND MRP3) by serine/threonine protein kinases. Since a prototypic PKC inducer, PMA, caused a marked upregulation of transcription from both human MRP2 and MRP3 promoters, a role for PKC isoforms in positive control of expression of these proteins could be postulated. Interestingly, broad-spectrum serine-threonine protein kinase inhibitors which also inhibit PKC, staurosporine and H-7, stimulated expression from the MRP2 promoter instead of inhibiting it. This effect was not seen for MRP3. MRP2 induction by staurosporine and H-7 was shown to have phenotypic consequences in whole cells, rendering them more resistant to etoposide and increasing their ability to export calcein through the plasma membrane. These results point to the involvement of serine/threonine protein kinases in negative regulation of the human MRP2 gene and to the necessity of testing novel anti-cancer drugs acting as protein kinase inhibitors with regard to their potential ability to induce multidrug resistance.

Structure-activity relationship studies of protective function of nitroxides in Fenton system.

The aim of this study was to evaluate the effect of piperidine nitroxides and their amine precursors on deoxyribose oxidation in the Fenton system. Protecting activity of nitroxides was found to be concentration-dependent and strongly influenced by ring substituents, while secondary amines did not provide any protection. The reported results suggest a mechanism of nitroxide action through iron oxidation rather than through direct scavenging of hydroxyl radicals. Moreover, presented data point to the danger of interference of nitroxides during the TBARS assay procedure.

Phosphorylation of Smad7 at Ser-249 does not interfere with its inhibitory role in transforming growth factor-beta-dependent signaling but affects Smad7-dependent transcriptional activation.

Smad proteins are major components in the intracellular signaling pathway of transforming growth factor-beta (TGF-beta), and phosphorylation is an important mechanism in regulation of their functions. Smad7 was identified as a potent inhibitor of TGF-beta-dependent signaling. We have identified serine 249 in Smad7 as a major phosphorylation site, the phosphorylation of which was not affected by TGF-beta1. Abrogation of the phosphorylation by substitution of Ser-249 with alanine or aspartic acid residues did not affect the ability of Smad7 to inhibit TGF-beta1 and BMP7 signaling. No differences were found in the stability or in the intracellular distribution of Smad7 mutants compared with the wild-type molecule. However, Smad7 fused to the DNA-binding domain of GAL4 induced transcription from a reporter with mutated TATA minimal promoter in a Ser-249-dependent manner. Moreover, a reporter with the SV40 minimal promoter was inhibited by GAL4-Smad7, and this effect was also dependent on Ser-249 phosphorylation. The amplitude of effects on transcriptional regulation was dependent on cell type. Our results suggest that phosphorylation of Smad7, unlike phosphorylation of the receptor-regulated Smads, does not regulate TGF-beta signaling but rather affects TGF-beta-independent effects of Smad7 on transcriptional regulation.

Analysis of the properties of the N-terminal nucleotide-binding domain of human P-glycoprotein.

Human P-glycoprotein, the MDR1 gene product, requires both Mg(2+)-ATP binding and hydrolysis to function as a drug transporter; however, the mechanism(s) defining these events is not understood. In the present study, we explored the nature of Mg(2+)-ATP binding in the N-terminal nucleotide-binding domain of human P-glycoprotein and identified the minimal functional unit required for specific ATP binding. Recombinant proteins encompassing amino acids within the region beginning at 348 and ending at 707 were expressed in Escherichia coli, purified from inclusion bodies under denaturing conditions, and renatured by rapid dilution. The ability of ATP to interact with these proteins was examined by use of the photoactive ATP analogue [alpha-(32)P]-8-azido-ATP. Photoaffinity labeling of recombinant proteins identified the region between amino acids 375 and 635 as the region necessary to obtain specific ATP-binding properties. Specific protein labeling was saturable, enhanced by Mg(2+), and inhibited by ATP. Recombinant proteins confined within the region beginning at amino acid 392 and ending at amino acid 590 demonstrated nonspecific [alpha-(32)P]-8-azido-ATP labeling. Nonspecific labeling was not enhanced by Mg(2+) and was inhibited only by high concentrations of ATP. Using a D555N mutated protein, we found that the conserved aspartate residue in the Walker B motif plays a role in magnesium-enhanced ATP-binding. Taken together, these data define the region of the N-terminal nucleotide-binding domain of P-glycoprotein that is required for specific ATP binding and suggest that magnesium may play a role in stabilizing the ATP-binding site.

Transport of organic anions by multidrug resistance-associated protein in the erythrocyte.

The active transport of oxidized glutathione and glutathione S-conjugates has been demonstrated for the first time in erythrocytes and this cell remained the main subject of research on the "glutathione S-conjugate pump" for years. Further studies identifled the "glutathione S-conjugate pump" as multidrug resistance-associated protein (MRP). Even though cells overexpressing MRP and isolated MRP provide useful information on MRP structure and function, the erythrocyte remains an interesting model cell for studies of MRP1 in its natural environment, including the substrate specificity and ATPase activity of the protein.

Is the glutathione S-conjugate pump a flippase?

A hypothesis of the flippase nature of the glutathione S-conjugate transport is presented. Experimental premises for this hypothesis include interaction of glutathione S-conjugates with the membrane, as demonstrated by their effects on membrane fluidity, quenching of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene fluorescence and induction of echinocytosis by 2,4-dinitrophenyl-S-glutathione (DNP-SG). This hypothesis can rationalize, i. a., observations of the enhancement of DNP-SG transport by butanol and stimulation of erythrocyte membrane Mg(2+)-ATPase activity by albumin-coupled DNP-SG.

An electron spin resonance assay of glutathione S-conjugate transport.

A method for studying export of glutathione S-conjugates from cells is proposed based on the use of the spin label tempo-maleimide. This compound is conjugated intracellularly with glutathione and the concentration of the exported conjugate is measured in cell supernatants in an electron spin resonance spectrometer after reoxidation with ferricyanide. This method allows for measurements of micromolar concentrations of the conjugate and requires low amounts of cells.

Identification of the multidrug-resistance protein (MRP) as the glutathione-S-conjugate export pump of erythrocytes.

The identification of the multidrug resistance protein (MRP) as a conjugate export pump in several cell types suggested its involvement in the long-known glutathione-S-conjugate transport across erythrocyte membranes. We investigated the ATP-dependent transport of glutathione S-conjugates in human erythrocyte and erythroleukemia cell membrane vesicles using the endogenous conjugate leukotriene C4 (LTC4), known to be a high-affinity substrate for MRP, in addition to S-(2,4-dinitrophenyl)glutathione. The kinetic parameters, including the Km value for LTC4 of 118 +/- 5 nM and the inhibition constants for transport of both substrates for the quinoline-based inhibitor MK 571, were similar to those obtained for transport mediated by recombinant MRP. Direct photoaffinity labeling of human erythrocyte membranes with [3H]LTC4 revealed a major binding protein of about 190 kDa which was immunoprecipitated by an anti-MRP serum. The radiolabeling of this protein was specifically suppressed by the transport inhibitor MK 571. Several additional anti-MRP sera detected the protein of about 190 kDa in human erythrocyte and erythroleukemia cell membranes. These data identify for the first time the glutathione-S-conjugate transporting protein in erythrocyte membranes.

Peroxides inhibit the glutathione S-conjugate pump.

Tert-Butyl hydroperoxide (100-300 microM) was found to inhibit the active efflux of dinitrophenyl-S-glutathione (DNP-SG) from human erythrocytes. From among amino acid peroxides generated by irradiation of amino acid (proline, valine and leucine) solutions, valine hydroperoxide (150 microM) had a similar effect on the DNP-SG transport. As the transport of glutathione S-conjugates is an important step of cellular detoxication, these results indicate that oxidative stress may impair cellular resistance to chemical stress of other kinds. t-Butyl and amino acid peroxides upon interaction with hemoglobin and whole erythrocytes produce free radicals which may be responsible for the damage to the glutathione S-conjugate pump of the erythrocyte membrane.

Transport of bimane-S-glutathione in human erythrocytes.

Export of glutathione S-conjugate of bimane (BSG) was studied in human erythrocytes. Characteristics of the BSG transport is similar to that of dinitrophenyl-S-glutathione (DNP-SG). BSG transport has two kinetic components, one of high affinity and low capacity (Km = 7.4 +/- 0.2 mumol/ml cells, Vm = 2.7 +/- 0.1 nmol/min per ml RBC) and another of low affinity and high capacity (Km = 242 +/- 8 mumol/ml cells, Vm = 9.6 +/- 1.6 nmol/min per ml RBC). BSG export is inhibited by vanadate (Ki = 65 +/- 6 microM) and fluoride (Ki = 11.4 +/- 0.8 mM). Activation energy of the transport is 67 +/- 7 kJ/mol. BSG transport is independent of membrane potential; its rate increases with pH in the pH range of 6-8, in line with the assumption that the anionic conjugate is cotransported with proton. BSG import to erythrocyte membrane inside-out vesicles is stimulated by ATP. Fluorimetric measurements of BSG export require low amounts of cells and may also be useful for other cell types as an alternative to studies of glutatione S-conjugate transport using radioactive substrates.

Effect of inhibitors on the transport of dinitrophenyl-S-glutathione in human erythrocytes.

Effect of inhibitors on and pH dependence of the export of dinitrophenyl-S-glutathione (DNP-SG), a glutathione S-conjugate formed upon in vivo conjugation of 1-chloro-2,4-dinitrobenzene to glutathione, was studied in intact human erythrocytes. The transport was inhibited by orthovanadate (IC50 = 80 microM) and fluoride (IC50 = 9 nM). Erythrocyte anion exchange (Band 3) protein inhibitors DIDS and SITS did not exert any significant effect on the transport. The transport rate increased with increasing extracellular pH in the range of 6.0-8.0. The Arrhenius activation energy of the process was 72.4 +/- 1.3 kJ/mol. Membrane potential, extracellular sodium/potassium concentration ratio and extracellular osmolality in the range 300-600 mOsm did not influence the export rate.