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PubMed2005

A new class of glutathione S-transferase from the hepatopancreas of the red sea bream Pagrus major.

To elucidate drug deposition and metabolism in cultured marine fishes, in a previous study we isolated and purified the GSTs (glutathione S-transferases) from the hepatopancreas of the red sea bream Pagrus major that contained 25 and 28 kDa GST subunits. The 25 kDa GST subunits encoded by two genes (GSTA1 and GSTA2) have been identified as Alpha-class GSTs. In the present study, we performed the molecular cloning and characterization of the GSTR1 gene encoding the 28 kDa GST subunit from the Pa. major hepatopancreas. The nucleotide sequence of GSTR1 was composed of an ORF (open reading frame) of 675 bp encoding a protein of 225 residues with a predicted molecular mass of 25.925 Da. A search of the BLAST protein database revealed that the deduced amino acid sequence of GSTR1 was structurally similar to that of GSTs derived from other fishes such as largemouth bass (Micropterus salmoides) and plaice (Pleuronectes platessa). The genomic DNA containing the GSTR1 gene was found to consist of six exons and five introns quite distinct from mammalian Theta-class GSTs. We have purified and characterized the recombinant GSTR1 enzyme (pmGSTR1-1) which showed activity only towards 1-chloro-2,4-dinitrobenzene, although it had no detectable activity towards cumene hydroperoxide, 1,2-dichloro-4-nitrobenzene, ethacrynic acid, 4-hydroxynonenal and p-nitrobenzyl chloride. Moreover, pmGSTR1-1 revealed remarkable heat instability (melting temperature Tm=30.3+/-0.11 degrees C). Collectively, our results indicated that the characteristic GST genes including GSTR1 have been conserved and functional in fishes. Therefore we designate them 'Rho-class', a new class of GSTs.

Amino Acid Sequence↗
PubMed2002

Influence of temperature, pH, oxygenation, water-type and substrate on biomarker responses in the freshwater clam Corbicula fluminea (Müller).

We investigated the influence of abiotic factors on responses of components of detoxification metabolism of phases I and II, indicators related to oxidative stress and propionylcholinesterase, proposed as biomarkers of pollution in Corbicula fluminea. Combined effects of temperature (10 and 20 degrees C), water-type (water purified by reverse-osmosis, lake water from the collection site and tap water) and habitat substrate (presence and absence of sand) were assessed in a five-day experiment. Additionally, clams were exposed to hypoxia or submitted to acidic, neutral or alkaline conditions at 20 degrees C, in lake water, without sand, for five days. Responses of biomarkers were generally higher when clams were placed in tap water, except for level of peroxidised lipids (PL). Catalase (CAT) and NADH-cytochrome c reductase (NADH-red.) activities increased whereas propionylcholinesterase (PChE) activity decreased in absence of sand. Decreasing temperature resulted in depressing PChE and NADPH-cytochrome c reductase (NADPH-red.) activities. Hypoxia induced an increase of CAT activity and glutathione S-transferase activity towards ethacrynic acid and a decrease of PL level. CAT and NADH-red. activities as well as PL level were reduced when clams were exposed to acidic and/or alkaline conditions. These results indicate that effects of abiotic factors should be taken into account in environmental studies.

Analysis of Variance↗
PubMed2001

Heterologous expression and characterization of alternatively spliced glutathione S-transferases from a single Anopheles gene.

Three cDNA sequences of glutathione S-transferase (GST), adgst1-2, adgst1-3 and adgst1-4, which are alternatively spliced products of the adgst1AS1 gene, were obtained from fourth instar larvae of Anopheles dirus mosquito by reverse transcriptase PCR reactions. The nucleotide sequences of these three cDNAs share >67% identity and the translated amino acid sequences share 61-64% identity. A comparison of the An. dirus to the An. gambiae enzymes shows that adGST1-2 versus agGST1-4, adGST1-3 versus agGST1-5 and adGST1-4 versus agGST1-3 have 85, 92 and 85% amino acid sequence identity, respectively, which confirms that orthologous isoenzymes occur across anopheline species. These three proteins were expressed at high levels, approximately 15-20 mg from 200 ml of E. coli culture. The recombinant enzymes were purified by affinity chromatography on an S-hexylglutathione agarose column. The subunit sizes of adGST1-2, adGST1-3 and adGST1-4 are 24.3, 23.9 and 25.1 kDa. The recombinant enzymes have high activities with 1-chloro-2,4-dinitrobenzene (CDNB), detectable activity with 1,2-dichloro-4-nitrobenzene but markedly low activity with ethacrynic acid and p-nitrophenethyl bromide. adGST1-3 was shown to be the most active enzyme from the kinetic studies. Permethrin inhibition of CDNB activity, at varying concentrations of CDNB, was significantly different, being uncompetitive for adGST1-2, noncompetitive for adGST1-3 and competitive for adGST1-4. In contrast, permethrin inhibition with varying glutathione concentrations was noncompetitive for all three GSTs. Despite the enzymes being splicing products of the same gene and sharing identical sequence in the N-terminal 45 amino acids, these GSTs show distinct substrate specificities, kinetic properties and inhibition properties modulated by the differences in the C-terminus.

Alternative Splicing↗
PubMedDate not supplied

Cloning, expression and characterization of an insect class I glutathione S-transferase from Anopheles dirus species B.

Insect class I glutathione S-transferases (GSTs) were expressed from cDNA obtained from larvae of the Thai malaria vector. Anopheles dirus in a PCR RACE (rapid amplification of cDNA ends) reaction using a primer to the conserved N-terminal region of An. gambiae class I GSTs and a synthetic oligo d(T)-adaptor primer. Seven different plasmids, resulting from sub-cloning of an original single 0.7 Kb PCR band, were picked at random and sequenced. Four of these were clearly GSTs on the basis of putative amino acid sequence conservation. All the sequences had a conserved N-terminal region, but were highly divergent at the C-terminus. The variability in the PCR products suggests that there is a high level of GST class I isoenzyme variability in larval An. dirus. One of the subclones from the PCR reaction contained a full coding region of the cDNA for GST. This had a putative amino acid sequence which was 76 and 91% identity to the An. gambiae GST class I, agGST 1-5 and agGST 1-6 respectively, but only 48% identity to agGST 1-2. The catalytically active enzyme, expressed in Escherichia coli, was strongly immuno-cross reactive with antisera raised against the two An. gambiae class I GSTs. The expressed enzyme was purified to homogeneity from an E. coli cell lysate by S-hexylglutathione agarose affinity chromatography. The enzyme had a high specific activity with CDNB, and also used DCNB and ethacrynic acid as substrates. In addition, it had peroxidase and DDTase activity and its activity with CDNB, was strongly inhibited by a range of organophosphorus and pyrethroid insecticides. This is consistent with the predicted role of this GST class in insecticide resistance.

Amino Acid Sequence↗
PubMed1980

Studies on the mechanism and characteristics of action of a uricosuric diuretic, indacrinone (MK-196).

1 The renal action of indacrinone (MK-196), a phenoxyacetic acid derivative with diuretic and uricosuric properties, has been studied in fifteen male subjects. 2 Increasing single doses of up to 60 mg of oral indacrinone produced a linear increase in urinary volume and excretion of Na+ and Cl-, whilst the responses of urinary K+, Ca2+, Mg2+ and uric acid excretion, rose to a plateau at the 40 mg dose. 3 Indacrinone evoked a rapid diuretic response which reached a maximum of 2-4 h and was largely complete at 8-12 h after administration. 4 During maximal hydration, indacrinone produced a substantial fall in fractional free water clearance (CH2O), from 8.89% to 5.83% of the filtered load of water, associated with an increase in osmolal clearance, from 1.38% to 5.78% of the filtered load of solute. The reduction in CH2O was of the same order as that produced by a dose of ethacrynic acid with comparable saluretic activity and significantly greater than that produced by an equi-saluretic dose of hydrochlorothiazide. These findings imply an action of indacrinone upon solute transport in the diluting segments of the distal tubule. 5 At the time of maximal indacrinone-induced saluresis, which amounted to an increase from 0.48% to 4.61% of the filtered load of NaCl, fractional urate clearance increased from 5.16% to 12.24% of the filtered load of uric acid. 6 Indacrinone is a long acting diuretic, sharing some properties in common with both loop diuretics and benzothiadiazines. The results are discussed in relation to structure-activity amongst derivatives of phenoxyacetic acid.

Diuresis↗
PubMed1999

Gene structure, expression and chromosomal localization of murine theta class glutathione transferase mGSTT1-1.

We have isolated and characterized a cDNA and partial gene encoding a murine subfamily 1 Theta class glutathione transferase (GST). The cDNA derived from mouse GSTT1 has an open reading frame of 720 bp encoding a peptide of 240 amino acids with a calculated molecular mass of 27356 Da. The encoded protein shares only 51% deduced amino acid sequence identity with mouse GSTT2, but greater than 80% deduced amino acid sequence identity with rat GSTT1 and human GSTT1. Mouse GSTT1-1 was expressed in Escherichia coli as an N-terminal 6x histidine-tagged protein and purified using immobilized-metal affinity chromatography on nickel-agarose. The yield of the purified recombinant protein from E. coli cultures was approx. 14 mg/l. Recombinant mouse GSTT1-1 was catalytically active towards 1, 2-epoxy-3-(p-nitrophenoxy)propane, 4-nitrobenzyl chloride and dichloromethane. Low activity towards 1-menaphthyl sulphate and 1-chloro-2,4-dinitrobenzene was detected, whereas mouse GSTT1-1 was inactive towards ethacrynic acid. Recombinant mouse GSTT1-1 exhibited glutathione peroxidase activity towards cumene hydroperoxide and t-butyl hydroperoxide, but was inactive towards a range of secondary lipid-peroxidation products, such as the trans-alk-2-enals and trans,trans-alka-2,4-dienals. Mouse GSTT1 mRNA is most abundant in mouse liver and kidney, with some expression in intestinal mucosa. Mouse GSTT1 mRNA is induced in liver by phenobarbital, but not by butylated hydroxyanisole, beta-napthoflavone or isosafrole. The structure of mouse GSTT1 is conserved with that of the subfamily 2 Theta class GST genes mouse GSTT2 and rat GSTT2, comprising five exons interrupted by four introns. The mouse GSTT1 gene was found, by in situ hybridization, to be clustered with mouse GSTT2 on chromosome 10 at bands B5-C1. This region is syntenic with the location of the human Theta class GSTs clustered on chromosome 22q11.2. Similarity searches of a mouse-expressed sequence tag database suggest that there may be two additional members of the Theta class that share 70% and 88% protein sequence identity with mouse GSTT1, but less than 55% sequence identity with mouse GSTT2.

Amino Acid Sequence↗
PubMed1980

Renal effects of indanone in dogs and rats: modification by a cyclooxygenase inhibition.

The effects of indomethacin, a PG synthesis inhibitor, on the renal hemodynamic and diuretic responses to indanone were evaluated in 10 rats and 12 dogs. In anaesthetized rats, 50 mg/kg of indanone, an indanyloxyacetic acid diuretic, increased water excretion 20--30 times, sodium excretion 80--100 times, and potassium excretion 4 times, without marked changes in GFR and BP. Indomethacin pretreatment, 2 mg/kg, did not significantly reduce these responses. In vehicle pretreated dogs, urine flow, and sodium and potassium excretions increased 9,7 and 2 fold, respectively, after 4 mg/kg indanone. These responses were significantly diminished in dogs pretreated with 4 mg/kg indomethacin. Mean RBF was reduced 17% (P less than 0.02) after 10 min of indanone infusion (0.2 mg/kg/min). Indomethacin failed to alter this effect, suggesting the action of indanone differs from that of furosemide and ethacrynic acid, agents know to cause PG mediated renal vasodilation. The cyclooxygenase inhibitor did inhibit the diuretic response to indanone in these dogs. The dissimilar renal excretory response in the two species may reflect a more effective blockade by indomethacin of the tubular secretion of indanone in dogs than in rats. Alternatively, PGs may have different renal effects in the two species, or cyclooxygenase was inhibited to a greater extent in dogs than in rats.

Animals↗
PubMed1998

Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA.

The canalicular (apical) membrane of the hepatocyte contains an ATP-dependent transport system for organic anions, known as the multispecific organic anion transporter (cMOAT). The deduced amino acid sequence of cMOAT is 49% identical to that of the human multidrug resistance- associated protein (MRP) MRP1, and cMOAT and MRP1 are members of the same sub-family of adenine nucleotide binding cassette transporters. In contrast to MRP1, cMOAT was predominantly found intracellularly in nonpolarized cells, suggesting that cMOAT requires a polarized cell for plasma membrane routing. Therefore, we expressed cMOAT cDNA in polarized kidney epithelial MDCK cell lines. When these cells are grown in a monolayer, cMOAT localizes to the apical plasma membrane. We demonstrate that cMOAT causes transport of the organic anions S-(2,4-dinitrophenyl)-glutathione, the glutathione conjugate of ethacrynic acid, and S-(PGA1)-glutathione, a substrate not shown to be transported by organic anion transporters previously. Transport is inhibited only inefficiently by compounds known to block MRP1. We also show that cMOAT causes transport of the anticancer drug vinblastine to the apical side of a cell monolayer. We conclude that cMOAT is a 5'-adenosine triphosphate binding cassette transporter that potentially might be involved in drug resistance in mammalian cells.

ATP-Binding Cassette Transporters↗
PubMed2005

Sulfur amino acid restriction induces the pi class of glutathione S-transferase expression in primary rat hepatocytes.

The regulation of genes by amino acids is attracting increasing attention. In the present study, we investigated the restriction of expression of the pi class of glutathione S-transferase (GST Yp) by sulfur amino acids. Hepatocytes isolated from male Sprague-Dawley rats were cultured with L-15-based medium containing low (LSAA; 0.1 mmol/L L-methionine and 0.1 mmol/L L-cysteine) or high (HSAA; 0.5 mmol/L L-methionine and 0.2 mmol/L L-cysteine) amounts of sulfur amino acids for up to 6 d. Cellular protein contents did not differ between LSAA- and HSAA-treated cells over the entire period. In contrast, glutathione concentrations were suppressed by the LSAA medium and on d 6 were only 20% of those of HSAA-treated cells (P < 0.05). As shown by immunoblot analysis, GST Yp protein levels were greater in LSAA-treated cells than in HSAA-treated cells (P < 0.05). The induction of GST Yp by L-methionine and L-cysteine restriction was not affected by insulin and dexamethasone, but the latter suppressed GST Yp expression (P < 0.05). LSAA increased GST Yp mRNA levels and GST activity toward ethacrynic acid (P < 0.05). GST Yp induction occurred only in cells with a limited supply of L-methionine; restriction of L-isoleucine, L-leucine, L-lysine, and L-phenylalanine had no significant effect. In contrast with the induction of GST Yp, the expression of the GST isoforms Ya and Yb was not changed by amino acid restriction. In conclusion, hepatic GST Yp gene expression is upregulated by a limited availability of sulfur amino acids.

Amino Acids, Sulfur↗
PubMed2005

Two glutathione S-transferase isoenzymes purified from Bulinus truncatus (Gastropoda: Planorbidae).

We purified and characterized two major glutathione S-transferase isoenzymes (GST2 and GST3) from snail Bulinus truncatus (Mollusca, Gastropoda, Planorbidae) tissue. The Km with respect to 1-chloro-2, 4-dinitrobenzene (CDNB) for both isoenzymes was increased as the pH decreased. Km of both isoenzymes with respect to glutathione (GSH) doubled when the pH was increased from 6.0 to 6.5. Acid inactivated GST2 and GST3 and the two enzymes were almost inactive at pH 3.5. However, they retain the full activity for at least 20 h when incubated at pH between 6.0 and 9.0. The optimum temperature was 45 degrees C for GST2 and 50 degrees C for GST3. The half lifetime at 50 degrees C was 70 min and 45 min for GST2 and GST3 isoenzymes, respectively. Addition of 5 mM GSH to the incubation buffer increased the half life of both isoenzymes more than fourfold. The activation energy for catalyzing the conjugation of CDNB was 1.826 and 3.435 kcal/mol for GST2 and GST3, respectively. I50 values for Cibacron blue, bromosulphophthalein, indocyanine green, hematin and ethacrynic acid were 0.76 microM, 47.9 microM, 7.59 microM, 0.03 microM and 0.79 microM for GST2, and 0.479 microM, 79.4 microM, 89.1 microM, 32.4 microM and 1.15 microM for GST3, respectively. Cibacron blue and indocyanine green were non-competitive inhibitors, while hematin was a mixed inhibitor. Bromosulphophthalein was found to be a competitive inhibitor for GST2 and a mixed inhibitor for GST3.

Animals↗
PubMed2005

Molecular cloning and characterization of alpha-class glutathione S-transferase genes from the hepatopancreas of red sea bream, Pagrus major.

Two distinct cDNAs corresponding to GSTA1 and GSTA2 genes encoding glutathione S-transferases (GSTs) from the hepatopancreas of red sea bream, Pagrus major were cloned and sequenced. A comparison of the nucleotide sequences of GSTA1 and GSTA2 revealed 98% identity and their derived amino acid sequences had 96% similarity. Both genes could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. Genomic DNA cloning showed that both GSTA1 and GSTA2 genes consisted of six exons and five introns. In a comparison of genomic DNAs, the structures of GSTA1 and GSTA2 differed. In addition, Southern-blot analysis indicated that at least two kinds of alpha-class GSTs existed in the P. major genome. In order to biochemically characterize the recombinant enzymes (pmGSTA1-1 and pmGSTA2-2), both clones were highly expressed in Escherichia coli. The purified pmGSTA1-1 and pmGSTA2-2 exhibited glutathione conjugating activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide, while neither pmGSTs show detectable activity toward 1,2-dichloro-4-nitrobenzene, ethacrynic acid, 4-hydroxynonenal, or p-nitrobenzyl chloride. Despite their high level of amino acid sequence identity, the pmGSTs had quite different enzyme-kinetic parameters.

Amino Acid Sequence↗
PubMed1991

Role of membrane sulfhydryl groups in stimulation of renin secretion by sulfhydryl reagents.

The present study was designed to address the reactivity and accessibility of the particular class of sulfhydryl groups involved in the regulatory process of renin secretion. Both mercurial (such as P-chloromercuriphenyl sulfonate [PCMPS] and non-mercurial sulfhydryl reagents (for example, 6,6-dithiodinicotinic acid [DTDN]), which very slowly penetrate the cell membrane of intact cells, stimulated renin secretion. The membrane permeant sulfhydryl reagent N-ethylmaleimide had no effect on renin secretion but its membrane impermeant derivative, stilbene maleimide, strongly stimulated secretion. Furthermore, disulfide reducing agents such as dithiothreitol (DTT) had no effect on renin secretion at low concentrations, but strongly inhibited it at high concentrations. Several reagents which are known to primarily deplete cellular reduced glutathione were without effect on renin secretion. The stimulation of renin secretion by PCMPS was rapid in onset, and prevented and reversed by DTT and L-cysteine. Furthermore, the maximal stimulatory effect of PCMPS was not additive to that by diuretics with sulfhydryl reactivity (such as, ethacrynic acid and mersalyl). The stimulatory effect of PCMPS was not affected by diuretics which lack sulfhydryl reactivity (such as, bumetanide and furosemide). These results suggest that sulfhydryl reagents of both with and without diuretic activity stimulate renin secretion by reacting with specific class of sulfhydryl groups which are readily accessible from the extracellular compartment. In addition, these results provide further support the possibility that a sulfhydryl-disulfide interchange in the membrane may play a regulatory role in the renin secretory process.

4-Chloromercuribenzenesulfonate↗
PubMed1993

Affinity labeling of glutathione S-transferase, isozyme 4-4, by 4-(fluorosulfonyl)benzoic acid reveals Tyr115 to be an important determinant of xenobiotic substrate specificity.

Incubation of 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, with the 4-4 isozyme of rat liver glutathione S-transferase at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. The rate of inactivation exhibits a nonlinear dependence on 4-FSB concentration from 0.50 to 7.85 mM, with kmax = 0.082 min-1 and a KI of 1.95 mM. Nearly 1 mol of reagent/mol of enzyme subunit is incorporated when the enzyme is maximally inactivated. Protection against incorporation and inactivation is provided by bromosulfophthalein, a competitive inhibitor with respect to the hydrophobic substrate, 1-chloro-2,4-dinitrobenzene (CDNB), suggesting that the reaction occurs in the binding site of the xenobiotic substrate. Fractionation by high-performance liquid chromatography of a tryptic digest of inactivated enzyme yields a single, modified, 14-residue peptide containing Tyr115 as the altered amino acid. Modified and control enzymes have comparable affinities for glutathione, as indicated by fluorescence titration. In contrast, as distinguished from the control enzyme, modified enzyme does not adsorb to a column of an agarose-linked Cibacron Blue derivative, indicating that it has lost its ability to bind a hydrophobic substrate analogue. These results are supported by kinetic characteristics of modified and control enzymes: upon modification of the enzyme with 4-FSB, the apparent Km for glutathione is unchanged, while the apparent Km for CDNB increases dramatically from 193 to 1690 microM. When the reaction of 4-FSB with enzyme is monitored, the final percent residual activity is found to be dependent on the substrate used in the assay: 11% for CDNB, 20% for ethacrynic acid, 2.5% for trans-stilbene oxide, and 2% for trans-4-phenyl-3-butene-2-one. Analysis of the kinetics of modified enzyme suggests that Tyr115 of glutathione S-transferase, isozyme 4-4, contributes to xenobiotic substrate binding and, when certain types of substrates are employed, is involved in catalysis.

Affinity Labels↗
PubMed1992

Cloning and heterologous expression of cDNA encoding class alpha rat glutathione transferase 8-8, an enzyme with high catalytic activity towards genotoxic alpha,beta-unsaturated carbonyl compounds.

A cDNA clone, lambda GTRA8, encoding rat glutathione transferase subunit 8 has been isolated from a lambda gt10 rat hepatoma cDNA library. The previously known amino acid sequence of the enzyme was used to design primers for a polymerase chain reaction that yielded a 0.3 kb DNA fragment from the hepatoma library. The 0.3 kb fragment was used as a probe for screening and a 0.9 kb cDNA clone containing a complete open reading frame was obtained. After DNA sequencing and subcloning into an expression vector, the enzyme was expressed in Escherichia coli and purified. Specific activities and kcat./Km values were determined for a number of substrates, including alpha,beta-unsaturated carbonyl compounds. The highest activity was obtained with 4-hydroxyalkenals and with acrolein, genotoxic products of lipid peroxidation. In addition, the rat class Alpha glutathione transferase 8-8 displays high catalytic activity in the reaction between glutathione and the diuretic drug ethacrynic acid, a compound normally considered as a substrate characteristic for class Pi glutathione transferases.

Amino Acid Sequence↗
PubMed1996

A Cl- pump in rat brain neurons.

Cl(-)-stimulated and ethacrynic acid-sensitive ATPase (Cl(-)-ATPase) of plasma membrane origin in the rat brain is a candidate for an active outwardly directed Cl- translocating system. Biochemistry of Cl(-)-ATPase and ATP-dependent Cl- transport (Km values for ATP and Cl-, nucleotide specificity, pH dependency, and sensitivity to ethacrynic acid) suggested that Cl(-)-ATPase is an ATP-driven Cl- pump. Activity of the reconstituted Cl(-)-ATPase/pump increased in the presence of phosphatidylinositol-4-monophosphate, and this pump activity further increased at an inside-positive membrane potential or in the presence of a protonophore, suggesting that the Cl(-)-ATPase/pump is an electrogenic Cl- transporter, probably regulated by phosphoinositide turnover in vivo. In cultured hippocampal pyramidal cell-like neurons from embryonic rat brain, ethacrynic acid and ATP-consuming treatment increased, but furosemide, an inhibitor of Na+/K+/Cl- cotransporter, decreased, [Cl-]i when monitored using Cl(-)-sensitive fluorescent probes. The stationary levels of [Cl-]i were lower and the effects of ethacrynic acid were more prominent in perikarya than in dendrites, while the effects of furosemide were more obvious in dendrites than in perikarya. The lower perikaryonic [Cl-]i and the marked effects of ethacrynic acid were observed in the later stage rather than in the early stage of culture. Thus, region-specific localization and developmental changes in the activities of Cl- transporters probably result in uneven and age-dependent distribution of Cl- in the neurons.

Adenosine Triphosphatases↗
PubMed1981

[Demonstration and properties of a Cl-/HCO3--ATPase in the avian salt gland (author's transl)].

An anion-stimulated, Mg2+-dependent, ouabain-insensitive ATPase is present in salt gland homogenates of domestic ducks (Anas platyrhynchos). The enzyme is unspecifically stimulated by various inorganic and organic anions including amino and sulfonic acids which are often used as buffer components (e. g. histidine, Bicin, PIPES, MES and HEPES). Therefore, the demonstration of ATPase stimulation by chloride strongly depends on the type and concentration of the buffer used and may also largely interfere with the stimulation caused by other anions present in the incubation medium. Of the inorganic anions tested chloride and bicarbonate appear to be the favorite physiological activators, but the possible role of carbonic acids in the stimulation of the anion-dependent ATPase should not be neglected. Km values are approximately 5.8 mM for Cl- and approximately 8.7 mM for HCO-3-activation. Maximal ATPase stimulation is obtained at 25 mM Cl- and approximately 30 mM HCO-3, respectively. The simultaneous presence of bicarbonate decreases chloride affinity and Vmax, and shifts the chloride optimum to lower concentrations. ATP is the most preferred substrate. Maximal activation by Cl- and HCO-3 occurs at ATP concentrations between 0.5 and 1 mM. ATP affinity increases in the presence of Cl- and HCO-3, respectively. Both chloride and bicarbonate require a Mg2+ to ATP ratio of approximately 0.5 and a pH value of 8.0 to 8.5 for optimal stimulation. Stimulation by Cl- and HCO-3 is inhibited by thiocyanate, cyanate and by the diuretic drugs furosemide, ethacrynic acid and mersalyl. Incubation media adapted for the simultaneous demonstration of both chloride and bicarbonate activation contained 10 to 20 mM histidine-Tris buffer at pH 8.0 to 8.5, 150 mM sucrose, 0.2 mM ouabain, 0.5 mM magnesium acetate, 1 mM ATP, (pH adjusted to 8.0-8.5 with Tris or NaOH), with and without 25 mM sodium chloride or 25 mM sodium bicarbonate.

Adenosine Triphosphatases↗
PubMed1987

Inhibition of the Cl-/NaCO3- anion exchanger by xipamide in human red blood cells.

The mechanism of action of classical loop diuretics of the 2- or 3-amino-5-sulfamoylbenzoic acid and (aryloxy)acetic acid families involves competition with chloride for a common site on the (Na+, K+, 2Cl-) co-transport system. However this is not the mechanism of action of some high-ceiling diuretics like muzolimine, MK 473, xipamide, indapamide and clopamide, which are not carboxylic acids. We evaluated three of these latter diuretics (xipamide, muzolimine and clopamide) for their inhibitory effects on five ion transport systems in human red blood cells: (i) Cl(-)-dependent (Na+, K+) co-transport, (ii) (NaCO3-/Cl-) anion exchanger, (iii) (Cl-, K+) co-transport, (iv) Na+, K+ pump and (v) Na+: Li+ counter-transport; and on one ion channel the Ca2+-dependent, K+ channel. All erythrocyte transport pathways were resistant to the three diuretics studied (IC50 of 10(-3) M or higher) with one remarkable exception, the (NaCO3-/Cl-) anion exchanger. This transport system was inhibited by xipamide (IC50 of 2.5 +/- 0.4 X 10(-5) M, mean +/- S.D. of five experiments) and less potently by muzolimine (IC50 of 1.1 +/- 0.3 X 10(-4) M, mean +/- S.D. of three experiments). Clopamide only inhibited the anion exchanger at high concentrations (IC50 of about 10(-3) M). Xipamide, the most potent diuretic in this test, was at least one order of magnitude more active than furosemide, ethacrynic acid, hydrochlorothiazide and amiloride. Inhibition of the anion carrier could be involved in the diuretic action (inhibition of CO2-stimulated NaCl absorption in the TAL) and/or in the antihypertensive action (inhibition of net NaCO3- influx and secondarily of Ca2+ influx through Na+: Ca2+ exchange in vascular smooth muscle cells of xipamide).

Bicarbonates↗
PubMed2002

A major human arsenic metabolite, dimethylarsinic acid, requires reduced glutathione to induce apoptosis.

Inorganic arsenicals are important environmental toxicants and carcinogens in humans. In mammals, including humans, inorganic arsenicals often undergo methylation, forming compounds such as dimethyarsinic acid (DMA). Recent evidence indicates DMA is a complete carcinogen in rodents while evidence for inorganic arsenicals as carcinogens in rodents remains equivocal. Thus, we studied the molecular mechanisms of in vitro cytolethality of DMA compared to that of the trivalent inorganic arsenical, sodium arsenite, using a rat liver epithelial cell line (TRL 1215). Arsenite was very cytotoxic in these cells (LC(50) = 35 microM after 48 h of exposure). With arsenite exposure, most dead cells showed histological and biochemical evidence of necrosis. Arsenite cytotoxicity increased markedly when cellular GSH was depleted with the glutathione synthase inhibitor, L-buthionine-[S,R]-sulfoximine (BSO). In contrast, DMA was nearly 3 orders of magnitude less cytotoxic (LC(50) = 1.5 mM) although evidence showed the predominating form of death was apoptosis. Surprisingly, GSH depletion actually decreased DMA-induced apoptosis. A glutathione scavenger, diethyl maleate (DEM), and a glutathione reductase inhibitor, carmustine, also prevented DMA-induced apoptosis. These data indicate that DMA requires intracellular GSH to induce apoptosis. Ethacrynic acid (EA), an inhibitor of glutathione S-transferase (GST) that catalyzes GSH-substrate conjugation, acivicin, an inhibitor of gamma-glutamyltranspeptidase (GGT) which catalyzes the initial breakdown of GSH-substrate conjugates, and aminooxyacetic acid (AOAA), an inhibitor of beta-lyase which catalyzes the final breakdown of GSH-substrate conjugates, all were effective in suppressing DMA-induced apoptosis. These findings indicate that DMA likely is conjugated in some form with GSH, and that it is this conjugate that induces apoptosis during subsequent metabolic reactions.

Animals↗