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PubMedDate not supplied

Activity of glutathione-dependent enzymes in long term diabetes. II. Glutathione contents and activity of glutathione-dependent enzymes: S-transferase and peroxidase in the kidney cytosol of alloxan induced diabetic rats.

In the kidney of diabetic rats the elevated GSH concentration was accompanied with the 25% increase of cytosolic Se-dependent glutathione peroxidase (Se-GSHPx) activity. The activity of cytosol glutathione S-transferase (GST) was decreased to 55% of the control with p-nitrobenzyl chloride, and was unchanged with 1-chloro-2,4-dinitrobenzene, and ethacrynic acid. The activity of GST with cumene hydroperoxide (a good substrate for determination the activity of non Se-GSHPx) in diabetic animals stay unchanged too. Insulin treatment of diabetic rats restores the normal glycemia and the body weight, strongly reduces glucosuria and reverses the morphological changes observed in the kidney. The activity of GST and cytosol Se-GSHPx, as well as GSH content, returned to a normal values after insulin treatment, while the activity of non Se-GSHPx was reduced of about 50% in relation to the control values.

Alloxan↗
PubMed1998

Role of glutathione S-transferases in the resistance of human colon cancer cell lines to doxorubicin.

The anthracycline doxorubicin has little activity against colorectal cancers. It is hypothesized that this is attributable to a multifactorial resistance mechanism in which the glutathione S-transferases (GST) may play a role. We studied the relationship between GST expression and doxorubicin resistance in four human colon adenocarcinoma cell lines (HT-29, LoVo, SW620, and Caco-2), with the goal of modulating GST activity to overcome resistance. Caco-2 cells were the most resistant to doxorubicin, showing an IC50 value approximately 80- to 90-fold higher than HT-29 or LoVo and 600-fold higher than SW620. Total GST catalytic activity was significantly higher in Caco-2 cells compared with the other lines. All four cell lines expressed GST-pi at the catalytic activity, protein, and mRNA levels; however, no significant differences were observed among the cell lines. GST-mu expression was not detectable at the protein and mRNA levels, and the four cell lines displayed very low catalytic activity toward a GST-mu-selective substrate. Caco-2 cells showed a unique, highly expressed GST-alpha-immunoreactive band that was not detected in the other lines; however, the glutathione peroxidase activity of Caco-2 cells was the lowest among the four cell lines. Neither ethacrynic acid nor glutathione analogues that function as GST class-selective inhibitors were able to potentiate the cytotoxic effects of doxorubicin in these colon cancer cell lines, as demonstrated in both microplate colorimetric and clonogenic assays. The multidrug resistance-associated protein and P-glycoprotein were either not detectable or expressed at such low levels that they are not likely to contribute to the differences in doxorubicin sensitivity observed among these cell lines.

Antibiotics, Antineoplastic↗
PubMed1998

Role of ions and ion channels in the regulation of Atlantic croaker sperm motility.

The effects of the osmolarity and ionic composition of the external media and ion-channel blockers on the induction of sperm motility in the marine teleost, Atlantic croaker (Micropogonias undulatus) were investigated. An in vitro sperm motility assay was developed to determine treatment effects on the percentage of motile sperm, sperm speed, and turning rate, using a computer-aided motion-analysis system. Maximum activation of sperm motility occurred in saline with an osmolarity of 680 mOs/kg. Potassium caused a decrease in the percentage of motile sperm, but only at high nonphysiological concentrations in the presence of high amounts of calcium. Calcium caused an increase in sperm velocity and turning rate. Percent motility was inhibited by the potassium-channel blockers, 4-aminopyridine and veratrine, the calcium-channel blockers, verapamil, diltiazem, and nifedipine; the sodium-channel blocker, amiloride, and the chloride-channel blocker, ethacrynic acid. In addition, the calcium-channel blockers caused a decrease in sperm velocity and turning rate. These results provide evidence for the role of potassium, calcium, and possibly sodium, and chloride ions in Atlantic croaker sperm motility. In addition, this study demonstrates that membrane-bound ion channels are involved in the motility of sperm from a marine teleost.

Animals↗
PubMed1998

Inhibition of apoptosis is the cause of resistance to doxorubicin in human breast adenocarcinoma cells.

In our previous paper we have described the isolation and characterization of a doxorubicin (DOX) resistant subline of breast adenocarcinoma SC6 cells. These cells were obtained after the treatment with low, clinically relevant doses of doxorubicin. They became cross-resistant to different wide used cytostatics. The expression of several genes involved in mitotic signal transduction, as well as cathepsins D and L, was similar in both parental and doxorubicin treated cells. The aim of this study was to examine the molecular mechanisms involved in resistance of these cells to doxorubicin. Activity of plasma membrane Pgp was examined in parental and resistant cells due to rhodamine-accumulation assay. The involvement of glutathione (GSH) and glutathione S-transferase (GST) in resistance to doxorubicin was determined in MTT modified assay due to the addition of specific inhibitors: buthionine sulfoximine (for GSH) or ethacrynic acid (for GST). The kinetic of apoptosis was followed after the treatment with DOX in control and SC6 cells by fluorescent microscope. The occurrence of apoptosis was confirmed by analysing DNA fragmentation in agarose gel. Our results indicate that P-glycoprotein, glutathione or glutathione transferases were not involved in resistance of SC6 cells to doxorubicin. However, the apoptosis was inhibited in doxorubicin-resistant cells. Therefore, even low doses of doxorubicin can induce the resistance to this drug due to inhibition of apoptosis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Regulation of human dihydrodiol dehydrogenase by Michael acceptor xenobiotics.

A human oxidoreductase (H-37) that is overexpressed in ethacrynic acid-resistant HT29 colon cells (Ciaccio, P. J., Stuart, J.E., and Tew, K.D. (1993) Mol. Pharmacol. 43, 845-853) has been identified as a dihydrodiol dehydrogenase. Translated protein from a dihydrodiol dehydrogenase cDNA isolated from a library prepared from ethacrynic acid-resistant HT29 cell poly(A+) RNA was recognized by anti-H-37 IgG and was identical in molecular weight with H-37. The isolated cDNA was identical in both nucleotide and amino acid sequences with the recently cloned liver dihydrodiol dehydrogenase (Stolz, A., Hammond, L., Lou, H., Takikawa, H., Ronk, M., and Shively, J.E. (1993) J. Biol. Chem. 268, 10448-10457). Using this cDNA as probe, we have examined its induction by Michael acceptors. The steady state dihydrodiol dehydrogenase mRNA level in the ethacrynic acid-resistant line was increased 30-fold relative to that of wild-type cells. Twenty-four hour treatment of wild-type cells with ethacrynic acid or dimethyl maleate increased mRNA 10-fold and 5-fold, respectively. These changes are accompanied by both increased protein expression and increased NADP-dependent 1-acenaphthenol oxidative activity in cell cytosol. In gel shift assays, compared to wild type controls, increased binding of NAD(P)H quinone oxidoreductase human antioxidant response element (hARE) DNA to redox labile protein complexes present in treated and resistant cell nuclear extract was observed. Ethacrynic acid induced CAT activity 2-fold in Hepa1 cells stably transfected with NAD(P)H quinone oxidoreductase hARE-tk-CAT chimeric gene construct. Thus, dihydrodiol dehydrogenase protein is inducible by de novo synthesis from mRNA by structurally related monofunctional inducer Michael acceptors. Altered in vitro binding of nuclear protein to the hARE is indirect evidence for the involvement of an element similar to hARE in the regulation of dihydrodiol dehydrogenase by these agents.

Alcohol Oxidoreductases↗
PubMed1981

Sodium-chloride transport in the thick ascending limb of Henle's loop. Oxygen consumption studies in isolated cells.

Isolated cells were prepared from the medullary thick ascending limb of Henle's loop (TALH) and the response of oxygen consumption was correlated with the active chloride transport system found in these cells. Oxygen consumption was 31.6 microliters O2/mg protein . h and inhibited 50% by the absence of either sodium or chloride in the incubation medium. The absence of both sodium and chloride produced no further inhibition of oxygen consumption. Ouabain (10(-4) M) inhibited oxygen consumption by 50% and the inhibitory effect depended on the presence of both sodium and chloride in the incubation medium. Further, furosemide inhibited oxygen consumption by a maximum of 50% at 10(-3) M and also had no inhibitory effect if either sodium or chloride were absent. Furosemide had no effect on the Na, K-ATPase activity or ATP levels of the TALH cells. Thus, the data suggest or ATP levels of the TALH cells. Thus, the data suggest that 50% of the oxygen consumption of the TALH cells is related to the movement of sodium and chloride into the cell and that the ions may be transported in a coupled manner. In addition the effect of various diuretics on oxygen consumption in the isolated TALH cells was tested. The diuretics could be grouped in three categories: (1) highly effective in inhibiting chloride-dependent oxygen consumption with an apparent inhibitory constant (Ki) of around 10(-6) M, including the diuretics furosemide, bumetanide, ethacrynic acid-cysteine and piretanide, (2) diuretics which were less effective in inhibiting oxygen consumption with an apparent Ki of around 10(-4) M, HOE 740 and ethacrynic acid, and (3) diuretics which were ineffective in inhibiting chloride-dependent oxygen consumption, amiloride and hydrochlorothiazide.

Adenosine Triphosphate↗
PubMed1977

Factors affecting the distribution of chloride ions in rat renal outer medulla.

1. The intracellular Cl- concentration, [Cl-]1, of rat renal outer medullary slices has been studied in iso-osmolal media containing 42, 93, 144, 189 or 225 mM chloride, [Cl-]0. Equilibrium values for [Cl-]1 were attained within 25-50 min from the start of incubation such that [Cl-]1/[Cl-]0 = 0-46. This ratio was independent of [Cl-]0 within the range studied. Intracellular Na+ was unaffected, and only minor variations of cell volume were observed (calculated from slice weight changes and [14C]carboxyl inulin spaces. 2. When [Cl-]0 = 189 mM, [Cl-]1 remained constant at 87 mM for up to 50 min, indicating that these figures may represnet the interstitial [Cl-] and mean intracellular [Cl-] respectively in outer medulla. 3. Omission of bicarbonate from medium containing 189 mM-Cl- caused an increase in [Cl-]1/[Cl-]0 to 0-58, which was not significantly affected by anoxia or by the presence of arsenite (5 X 10(-3) M) or 2,4-dinitrophenol (10(-3) M). Significant further increases were observed in the presence of iodoacetic acid (5 X 10(-3) M) (0-70), acetazolamide (10(-3) M or 5 M 10(-3) M) (0-71) and iodacetic acid plus 2,4-dinitrophenol (0-85). The addition of the diuretic agents ouabain, ethacrynic acid-cysteine and frusemide (all 10(-3) M) to 189 mM-Cl- media containing bicarbonate reduced [Cl-]1/[Cl-]0 to 0-36, 0-37 and 0-42 respectively. 4. The mean 36Cl- space of outer medulla after 50 min incubation in 189 mM Cl- medium was 49-7 +/- 2-1 micronl./100 mg wet wt. The volume of distribution was not significantly affected by ouabain, ethacrynic acid-cysteine or frusemide (10(-3) mM). 5. Net efflux of 36Cl- from slices loaded with isotope into 'cold' 189 mM Cl medium showed three components with rate constants of 69 X 10(-3), 18 X 10(-3) and 9-9 X 10(-5) sec-1 respectively. Efflux was not affected by ouabain, ethacrynic acid-cysteine or frusemide (10(-3) mM). 6. The main conclusions drawn from this study are: (i) the interstitial fluid Cl- concentration of normally hydrated rat outer medulla is approx. 189 mM; (ii) the [Cl-]1/[Cl-]0 ratio 0-46 may represent a chiefly passive distribution maintained by the opposing gradient of a second anion, probably bicarbonate; (iii) the energy required to maintain unequal distribution of Cl- in the absence of external bicarbonate is derived chiefly from glycolysis, with a small aerobic component; (iv) there may be two intracellular chloride pools whose net rates of Cl- exchange differ by a factor of approx. 180.

Animals↗
PubMed1985

Influence of muzolimine and other diuretics on human red cell Na+, K+-cotransport.

It is known that the diuretic activity of so-called loop diuretics is characterized by the ability of such compounds to inhibit the Na+, K+-cotransport system of the cell membrane. A good correlation has been demonstrated of the inhibitory activity of several furosemide- and ethacrynic acid-related compounds with their natriuretic activity in man. It was the aim of the study to examine, wether muzolimine has Na+, K+-cotransport inhibitory activity, and if so, if this occurs in a dose range which might be relevant for its natriuretic activity. For reasons of comparison, several other diuretics with high ceiling activity were studied. Cotransport inhibitory activities were compared with natriuretic activities in rats. Na+, K+-cotransport was measured by determination of furosemide-sensitive, ouabain-insensitive Na+-extrusion from Na+-loaded human red blood cells. Whereas the natriuretic activity of muzolimine is equal to that of furosemide in rats and superior in man, its cotransport inhibitory activity is merely 1/13 of that of furosemide. Additionally, only 50% inhibition is reached. The inhibitory activity of muzolimine, in contrast to ethacrynic acid, is not enhanced in the presence of equimolar cysteine. Muzolimine therefore appears to act via other mechanisms than furosemide-like diuretics. However, it cannot be excluded that muzolimine might act by an active metabolite.

Adult↗
PubMed1983

Effect of osmotic shocks on rabbit kidney cortex slices.

Rabbit kidney cortex slices behave an osmometers when withstanding hyperosmotic or hyposmotic shocks of amplitude up to pi 1/pi 2 = 1.25. For hyposmotic shocks of amplitude larger than or equal to pi 1/pi 2 = 1.50, the maximum swelling achieved is less than what can be expected on the basis of the van't Hoff relation, thereby indicating that a volume regulation process is taking place. Volume regulation in kidney slices can be dissociated into two distinct phases. The first one, of swelling limitation, is very rapid and keeps maximum cell volume at values lower than expected when the tissue is considered as an osmometer. This phase is followed by a slow volume readjustment process during which volume progressively decreases towards control values. The major intracellular osmotic effector loss during both swelling limitation and volume readjustment is Na+. The overall volume regulation process is insensitive to furosemide, vanadate, and bumetanide. Swelling limitation is blocked by addition of ouabain. Contrary to what has been believed previously, there is, however, no need to implicate control of the activity of a ouabain-sensitive, Na+/K+ pump in the Na-dependent volume regulation mechanism.

Amino Acids↗
PubMed1986

Intracellular sodium, potassium and magnesium concentration, ouabain-sensitive 86rubidium-uptake and sodium-efflux and Na+, K+-cotransport activity in erythrocytes of normal male subjects studied on two occasions.

The red cell Na+,K+-ATPase pump activity estimated by the ouabain-sensitive 86Rb-uptake or Na+-efflux, the Na+,K+-cotransport activity measured either by the furosemide-sensitive K+- or Na+- efflux or by the ethacrynic acid-sensitive 86Rb-uptake as well as the intraerythrocyte concentration of sodium, potassium and magnesium were studied in 29 normal male subjects with one to three weeks interval between the first and second blood sampling. Both the red cell sodium and potassium concentration, the erythrocyte ouabain-sensitive 86Rb-uptake and Na+-efflux, the furosemide-sensitive Na+- and K+-efflux and the ethacrynic acid-sensitive 86Rb-uptake are stable over time in the same individual. The furosemide-sensitive Na+-and K+-efflux is significantly related to the ethacrynic acid-sensitive 86Rb-uptake. The intraerythrocyte Na+ concentration was negatively related to the ouabain-sensitive 86Rb-uptake, but not to the ouabain-sensitive Na+-efflux; it was, however, negatively related to the rate constant for the ouabain-sensitive Na+-efflux.

Adult↗
PubMed1968

Red-cell transport defect in patients with cystic fibrosis and in their parents.

The ouabain-sensitive and the ethacrynic acid-sensitive sodium efflux from erythrocytes of patients with cystic fibrosis are both decreased. Furthermore, the ouabain-sensitive adenosine triphosphatase activity is diminished in the red blood cell ghosts of these patients. Perhaps of greater significance is the fact that ethacrynic acid-sensitive sodium efflux is clearly diminished in the erythrocytes of the asymptomatic parents of these sick children. This defect in sodium transport may be valuable for detecting the heterozygous carrier state.

Adenosine Triphosphatases↗
PubMed1979

The influence of chloride ions on renal outer medullary cell volume.

1. Slices of rat renal outer medulla have been incubated in media made hyperosmotic (540 and 1055 m-osmole/kg H2O) by the addition of urea, and containing variable concentrations of Cl (90, 144, 189 and 215 mM) and constant concentrations of Na (180 mM) and K (5.9 mM). A small number of incubations have been conducted in the presence of 100 mM-Na. 2. Changes in cell volume during incubation have been calculated on the basis of initial and final slice weight and inulin space. 3. The capacity of cells to shrink in response to extracellular osmotic stress was related principally to the external Cl concentration rather than to osmolality, increases in concentration being associated with enhanced shrinkage. Shrinkage was accompanied by net loss of cellular Cl. The ratio between intra- and extracellular Cl concentration (ca. 0.41) remained constant in all media. 4. In media containing low Cl concentration (90 mM), reduction of media Na concentration to 100 mM enhanced shrinkage. This effect was not observed when medium Cl concentration was greater than 90 mM. 5. Ethacrynic acid-cysteine (1 mM) significantly impaired the shrinkage response to extracellular osmotic stress, and caused cell swelling in media of relatively low Cl concentration and osmolality. It did not abolish the dependency of cell volume upon Cl concentration. There was marked reduction in the net amount of Cl lost from cells. 6. Ethacrynic acid-cysteine caused an increase in cellular Na content only in media containing 540 m-osmole/kg H2O and Cl concentrations less than 215 mM. 7. Ouabain (1 mM) inhibited cell shrinkage to a lesser extent than ethacrynic acid-cysteine in all media except that causing the greatest shrinkage under control conditions (215 mM-Cl/1055 m-osmole/kg H2O). It is suggested that a ouabain-sensitive process may play an increasingly important role in Cl-related cell shrinkage as this becomes more pronounced. 8. The findings are consistent with the view that Cl ions influence cell volume both through their effective external osmotic pressure and by means of Cl-specific process; the latter is associated with net loss of cellular Cl. A dependence of this loss upon Na/k exchange-linked metabolism is inferred, but the present findings do not permit the active or passive nature of the extrusion to be defined.

Animals↗