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At least 1,063 records · Page 59Linked to original sources
PubMed2006

Fast gas chromatographic/mass spectrometric determination of diuretics and masking agents in human urine: Development and validation of a productive screening protocol for antidoping analysis.

An analytical procedure was developed for the fast screening of 16 diuretics (acetazolamide, althiazide, amiloride, bendroflumethiazide, bumetanide, canrenoic acid, chlorthalidone, chlorthiazide, clopamide, ethacrynic acid, furosemide, hydrochlorthiazide, hydroflumethiazide, indapamide, triamterene, trichlormethiazide) and a masking agent (probenecid) in human urine. The whole method involves three analytical steps, including (1) liquid/liquid extraction of the analytes from the matrix, (2) their reaction with methyl iodide at 70 degrees C for 2 h to form methyl derivatives, (3) analysis of the resulting mixture by fast gas chromatography/electron impact mass spectrometry (fast GC/EI-MS). The analytical method was validated by determining selectivity, linearity, accuracy, intra and inter assay precision, extraction efficiencies and signal to noise ratio (S/N) at the lowest calibration level (LCL) for all candidate analytes. The analytical performances of three extraction procedures and five combination of derivatization parameters were compared in order to probe the conditions for speeding up the sample preparation step. Limits of detection (LOD) were evaluated in both EI-MS and ECNI-MS (electron capture negative ionization mass spectrometry) modes, indicating better sensitivity for most of the analytes using the latter ionization technique. The use of short columns and high carrier gas velocity in fast GC/MS produced efficient separation of the analytes in less than 4 min, resulting in a drastic reduction of the analysis time, while a resolution comparable to that obtained from classic GC conditions is maintained. Fast quadrupole MS electronics allows high scan rates and effective data acquisition both in scan and selected ion monitoring modes.

Diuretics↗
PubMed1977

Effects of replacing medium sodium by choline, caesium, or rubidium, on water and ion contents of renal cortical slices.

1. Renal cortical slices from rat, rabbit, and guinea-pig were incubated in media in which choline, caesium or rubidium replaced sodium.2. Slices of rabbit and guinea-pig renal cortex incubated in oxygenated choline Ringer decreased in volume initially and did not swell over 3 hr at 25 degrees C. There was a steady loss of potassium. Inhibition of metabolism (N(2) + 1 mM iodoacetamide) caused some swelling. Ouabain, 10 mM, in choline Ringer affected neither loss of potassium nor tissue water content.3. Slices of rat renal cortex similarly incubated in choline Ringer swelled over 3 hr at 25 degrees C whether or not metabolism was inhibited; ouabain (15 mM) affected neither tissue potassium loss nor tissue water content.4. Incubation in choline Ringer containing either 0.2 mMp-chloromercuribenzoic acid, or 1 mM ethacrynic acid increased the tissue water content of guinea-pig renal cortical slices.5. Depletion of cellular potassium (by preliminary incubation in oxygenated potassium-free sodium Ringer with 10 mM ouabain at 30 degrees C) resulted in increased tissue water content when rabbit renal cortical slices were subsequently incubated in oxygenated choline Ringer at 25 degrees C for 3 hr.6. There was no evidence of energy-dependent extrusion of water or ions from either equilibrated rat or rabbit renal cortical slices leached at 0.5 degrees C and then reincubated at 25 degrees C in choline Ringer.7. Rat and guinea-pig renal cortical slices leached at 0.5 degrees C and reincubated at 25 degrees C swelled in rubidium Ringer and in caesium Ringer. There was no evidence of energy-dependent water or ion extrusion when metabolism was restored after leaching in either of these media. Metabolizing rat slices but not guinea-pig slices swelled faster than slices whose metabolism was inhibited.8. These results lend no support to the mechano-chemical hypothesis which ascribes cellular volume regulation to a contractile mechanism squeezing isotonic extracellular fluid from the cells. Instead it is suggested that cellular water content in these experiments reflects the balance between the rate of loss of potassium (and chloride) from the cells and the rate of uptake of extracellular cation (and chloride) into the cells - these rates reflecting both the electrochemical potential gradients of the ions and membrane permeability to them. The implications in relation to the hypothesis of ouabain-insensitive cellular volume regulation are discussed.

Animals↗
PubMed1991

Extracellular MgATP activates the Cl-/HCO3- exchanger in single rat cardiac cells.

1. The effect of extracellular MgATP on cytosolic pH (pHi) was investigated in single rat cardiac cells loaded with the pH-sensitive probe Snarf-1. 2. Basal pHi in HEPES-buffered solution (containing 4.4 mM-NaHCO3) was 7.08. MgATP induced a transient acidification followed by an alkalinization. The latter is prevented by ethylisopropylamiloride (EIPA) and has been attributed to the activation of the Na+/H+ antiport. The MgATP-induced acidification reached a maximal value of 0.42 +/- 0.03 pH units (U pH). It was concentration dependent with a K0.5 of 2.6 microM-MgATP. This acidification was also observed with the same magnitude in the presence of the more physiological Krebs-bicarbonate buffer but was greatly reduced in nominally HCO3-free HEPES. 3. The MgATP-induced acidification was prevented by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), probenecid and ethacrynic acid but not by bumetanide. It was dependent upon the external chloride concentration. The K0.5[Cl-] was 9 mM and the maximal acidification required 60 mM-Cl-. 4. MgATP accelerated the recovery from an alkalinization triggered by a pulse of NH4Cl. The nucleotide also facilitated the efflux of HCO3- when the cell was switched from a Krebs-bicarbonate buffer gassed with 5% CO2 to an HEPES buffer. 5. The acidification was only evoked by MgATP and its poorly hydrolysable analogues but not by the other nucleotides (ADP, GTP (guanosine triphosphate), CTP (cytidine triphosphate) UTP (urodine triphosphate), ITP (inositol triphosphate) nor by adenosine. It required the presence of Mg2+ ions. 6. These results provide evidence that MgATP activates the Cl-/HCO3- exchanger and that this activation accounts for the acidification. Such an activation could not be related to the P1- or the P2-purinergic receptors since it requires triphosphate adenylic compounds and Mg2+ ions. This leads us to suggest the existence of a putative P3-type of purinergic receptor.

Adenosine Triphosphate↗
PubMed1993

Na(+)-dependent sulfate transport in opossum kidney cells is DIDS sensitive.

Sulfate transport was examined in OK/E cells, a clonal subline of opossum kidney cells that express several differentiated functions of the proximal tubule. Extracellular Na+ stimulated [35S]sulfate uptake five- to sixfold. Hill analysis demonstrating the dependence of sulfate uptake on Na+ concentration yielded a Hill coefficient of 1.5 and a Michaelis constant (KNa+) of 23 mM. Na(+)-dependent sulfate uptake was increased by lowering the pH from 7.4 to 6.4, decreased by raising the pH to 8.4 and inhibited by a 10-fold molar excess of SO3(2-), S2O3(2-) and CrO4(2-), but not by phosphate. The Na(+)-mediated component of sulfate uptake was saturable and kinetic parameters were estimated [Michaelis constant (Km) = 2.4 +/- 0.2 mM and maximum velocity (Vmax) = 125 +/- 15 pmol.mg protein-1.min-1]. Omitting extracellular Cl- resulted in a significant increase in the affinity of the carrier for sulfate (Km = 0.5 mm), without changing Vmax, consistent with competitive inhibition by Cl-. Na(+)-dependent sulfate uptake in OK/E cells was also inhibited by HCO3- [half-maximal inhibitory concentration (IC50) = 7 mM], 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, IC50 = 0.9 microM), 0.5 mM picrylsulfonic acid and 0.1 mM ethacrynic acid, but not by 1 mM amiloride. Na(+)-dependent, DIDS-sensitive sulfate uptake was also expressed in the parental OK cell line and was not influenced by serum or 3,3',5-triiodo-L-thyronine. We conclude that Na(+)-dependent sulfate uptake in OK/E cells observes many of the features of Na(+)-sulfate cotransport in the renal brush-border membrane and provides a useful model to investigate the regulation of renal sulfate transport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Effects of ouabain, amiloride, monensin, and other agents on ovine parotid secretion.

Secretion by the parotid gland of Na-replete and -depleted sheep was investigated by examining the effects of modifiers of ionic transfer on salivary composition and flow rate. These agents were infused into the arterial blood supply of the vascularly isolated gland in anesthetized sheep. Ouabain inhibited Na+-K+ exchange in the ducts caused by Na depletion and restored the [Na+], [K+], and osmolality to close to those of Na-replete saliva. Ouabain also inhibited Cl- -HCO3- exchange in the ducts in Na repletion and depletion. Amiloride partially inhibited Na+-K+ exchange in Na depletion without affecting Cl- -HCO3- exchange. Monensin potentiated Na+-K+ exchange in Na repletion and depletion. Amiloride and monensin gained access to the saliva, but furosemide and ethacrynic acid were almost totally excluded, and, up to 10(-3) M in blood, they did not affect salivary composition or flow rate. Methazolamide gained free access to saliva but was without effect. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid at 10(-3) M slightly increased salivary [Na+] and [HPO4(2-)]. The results indicate potent effects of ouabain on basolateral Na+-K+ pumps and of amiloride and monensin on transcellular delivery of Na+ to these pumps, but ouabain had no effect on salivary flow rate until O2 consumption approached zero and secretion failed. The findings do not support a proposal that the salivary secretion depends on a Cl- -dependent furosemide-sensitive system energized by Na+-K+-ATPase-dependent Na pumps.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2006

Aryloxoalcanoic compounds induce resistance to antibiotic therapy in urinary tract infection caused by Escherichia coli.

Clofibric acid (CL) is a compound used to control hypertriglyceridemia, and ethacrynic acid (ET) is administered to enhance diuresis. These compounds are structurally analogous to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), as they have a chlorinated phenoxy moiety. As these agents are mainly excreted by the renal route, they could potentially coexist with Escherichia coli in the urinary tract of infected patients. Induction of the in vitro resistance of E. coli to hydrophilic antibiotics was determined by increasing the values of the minimum inhibitory concentration (2-40-fold). These results correlated with drastically inhibited expression of the hydrophilic bacterial channel OmpF. In vivo assays were performed in ascending urinary tract infection in female BALB/c mice. Treatment with the hydrophilic antibiotic cephalexin 25 mg kg(-1) day(-1) by the oral route diminished renal infection. The CFU mean values in the kidneys were between 75% and 89% lower than those in animals without treatment. Simultaneous exposure to CL (at a therapeutic dose, 28.6 mg kg(-1) day(-1)) did not change the effect of the treatment. In contrast, ET at 2.9 mg kg(-1) day(-1) or 2,4-D at 70 mg kg(-1) day(-1) inhibited the antibiotic therapeutic effect. Moreover, 2,4-D dramatically increased bacterial infection after 9 days of exposure.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1988

Possible involvement of glutathione S-transferases in the cell growth of C6 astroglioma cells.

The changes of glutathione S-transferase activity were investigated using rat brain astroglioma C6 cells that were synchronized at different phases of the cell cycle. The enzyme showed two significant activity peaks at G2 and G1 phases. Furthermore, when C6 glioma cells were exposed to a culture medium supplemented with specific glutathione S-transferase inhibitors, ethacrynic acid and caffeic acid, cell growth was remarkably suppressed. These results suggest that glutathione S-transferases may be closely related to the mechanism of cell proliferation.

Animals↗
PubMed1994

Poly[(amino acid ester)phosphazenes] as substrates for the controlled release of small molecules.

Three different poly[(amino acid ester)phosphazenes] have been examined in order to investigate their possible use as drug delivery vehicles. The three polymers are poly[di(ethyl glycinato)phosphazene], poly[di(ethyl alanato)phosphazene] and poly[di(benzyl alanato)phosphazene]. These macromolecules either share the same amino acid residue or the same ester group, and this facilitated comparisons of the hydrolytic decomposition and the small molecule release profiles of the polymers. The polymers were synthesized by treatment of poly(dichlorophosphazene) with an excess of the appropriate amino acid ester. Tetrahydrofuran solutions of each polymer were then thoroughly mixed with ethacrynic acid, a diuretic, or Biebrich Scarlet, an azo dye. Films cast from these solutions were immersed in aqueous media (pH 7) at 25 degrees C and at 37 degrees C for approximately 1400 h. During these experiments, the release of the small molecules was monitored by UV/visible spectroscopy. The molecular weight decline and the mass loss of the polyphosphazene films were measured.

Amino Acids↗
PubMed1969

Sodium transport in turtle erythrocytes. Apparent stimulation of exchange diffusion by anaerobiosis.

Studies were performed on Na and K transport by red blood cells of the freshwater turtle under anaerobic and aerobic conditions. Although it had previously been assumed that cation transport in turtle red blood cells was dependent on respiration, the present data show greater Na efflux rates in N(2) than in O(2). However, ouabain inhibited Na transport by the same amount quantitatively in O(2) and N(2) gas phases. Thus there was no difference in ouabain-sensitive or "pump" Na transport rates. Na influx rates were higher in nitrogen than in air and potassium influx rates were not significantly different under aerobic and anaerobic conditions. Moreover in the absence of sodium in the bathing medium no difference between air and nitrogen could be discovered. Finally with ethacrynic acid plus ouabain there was an additional decrease in Na efflux but there was a persisting difference between air and nitrogen. These studies do not rule out the existence of a ouabain-insensitive ethacrynic acid-inhibitable flux; however, they suggest that at least part of the activation of Na efflux observed in N(2) was due to increased exchange diffusion.

Adenosine Triphosphate↗
PubMed1991

Combined in vitro modulation of adriamycin resistance.

In a P-glycoprotein-negative cell line, GLC4-Adr90, a 75-fold acquired Adriamycin (Adr) resistance coincided with a reduced cellular Adr level, an increased detoxifying capacity (glutathione (GSH) and glutathione S-transferase (GST) elevated), and a reduced topoisomerase-II (topo-II) activity compared with the parent cell line GLC4. The effect on Adr resistance of buthionine sulfoximine (BSO, GSH synthesis inhibitor), was studied alone or in combination with verapamil (drug-efflux inhibitor), docosahexaenoic acid (membrane lipid domain affector), ethacrynic acid (GST inhibitor), aphidicolin (DNA-polymerase-alpha inhibitor) or novobiocin (NOV, topo-II inhibitor). Cytotoxicity was tested using a microculture tetrazolium assay. In GLC4-Adr90, BSO and NOV increased Adr-induced cytotoxicity 12.9-fold and 1.8-fold respectively. The combination of BSO plus NOV showed an additive effect, decreasing the Adr resistance factor from 75 to 2.7. Combination of modulators of Adr resistance directed at different resistance mechanisms appears promising in vitro.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Optimization of bivalent glutathione S-transferase inhibitors by combinatorial linker design.

Dimeric glutathione S-transferases (GSTs) are pharmacological targets for several diseases, including cancer. Isoform specificity has been difficult to achieve due to their overlapping substrate selectivity. Here we demonstrate the utility of bivalent GST inhibitors and their optimization via combinatorial linker design. A combinatorial library with dipeptide linkers emanating symmetrically from a central scaffold (bis-3,5-aminomethyl benzoic acid, AMAB) to connect two ethacrynic acid moieties was prepared and decoded via iterative deconvolution, against the isoforms GSTA1-1 and GSTP1-1. The library yielded high affinity GSTA1-1 selective inhibitors (70-120-fold selectivity) and with stoichiometry of one inhibitor: one GSTA1-1 dimer. Saturation Transfer Difference (STD) NMR with one of these inhibitors, with linker structure (Asp-Gly-AMAB-Gly-Asp) and K(D) = 42 nM for GSTA1-1, demonstrates that the Asp-Gly linker interacts tightly with GSTA1-1, but not P1-1. H/D exchange mass spectrometry was used to map the protein binding site and indicates that peptides within the intersubunit cleft and in the substrate binding site are protected by inhibitor from solvent exchange. A model is proposed for the binding orientation of the inhibitor, which is consistent with electrostatic complementarity between the protein cleft and inhibitor linker as the source of isoform selectivity and high affinity. The results demonstrate the utility of combinatorial, or "irrational", linker design for optimizing bivalent inhibitors.

Binding Sites↗
PubMed1981

Urinary excretion of bile acids in cholestasis: evidence for renal tubular secretion in man.

1. The apparent renal clearance of intravenously injected [14C]glycocholate and [3H]chenodeoxycholate-3-sulphate was estimated in 22 patients with cholestasis. The degree of protein binding of the isotopes in serum from these patients was determined. The effects of pharmacological agents, changes in urine flow rate and pH on renal clearance was studied. 2. The mean renal clearance of [14C]glycocholate was 1 . 7 +/- 0 . 4 ml/min (mean +/- SEM), and that of [3H]chenodeoxycholate-3-sulphate was 6 . 4 +/- 0 . 9 ml/min. [14C]Glycocholate was 80 . 1% protein bound and [3H]chenodeoxycholate-3-sulphate 96 . 5% protein bound. 3. Comparisons of the observed clearance rates with those calculated on the basis of glomerular filtration of the unbound fraction suggest that whereas [14C]glycocholate is predominantly reabsorbed by the renal tubules, [3H]chenodeoxycholate-3-sulphate appears in the urine mainly as the result of tubular secretion. 4. Probenecid, ethacrynic acid, frusemide and bendrofluazide decreased the clearance of both bile acids, implying competition for secretion via the proximal tubular organic acid secretory pathway between these compounds and bile acids. 5. Passive non-ionic diffusion does not seem to be an important mechanism in the renal excretion of bile acids as changes in urine flow rate and pH did not influence bile acid clearance. 6. A greater affinity of the proximal tubular organic acid secretory pathway for sulphated than for non-sulphated bile acids may explain the higher observed renal clearance rate of sulphated bile acids.

Aged↗
PubMed1990

Enzymatic formation of prostaglandin F2 alpha in human brain.

Prostaglandin (PG)E2 9-ketoreductase, which catalyzes the conversion of PGE2 to PGF2 alpha, was purified from human brain to apparent homogeneity. The molecular weight, isoelectric point, optimum pH, Km value for PGE2, and turnover number were 34,000, 8.2, 6.5-7.5, 1.0 mM, and 7.6 min-1, respectively. Among PGs tested, the enzyme also catalyzed the reduction of other PGs such as PGA2, PGE1, and 13,14-dihydro-15-keto PGF2 alpha, but not that of PGD2, 11 beta-PGE2, PGH2, PGJ2, or delta 12-PGJ2. The reaction product formed from PGE2 was identified as PGF2 alpha by TLC combined with HPLC. This enzyme, as is the case for carbonyl reductase, was NADPH-dependent, preferred carbonyl compounds such as 9,10-phenanthrenequinone and menadione as substrates, and was sensitive to indomethacin, ethacrynic acid, and Cibacron blue 3G-A. The reduction of PGE2 was competitively inhibited by 9,10-phenanthrenequinone, which is a good substrate of this enzyme, indicating that the enzyme catalyzed the reduction of both substrates at the same active site. These results suggest that PGE2 9-ketoreductase, which belongs to the family of carbonyl reductases, contributes to the enzymatic formation of PGF2 alpha in human brain.

Amino Acids↗
PubMed1988

Enhancement of eicosanoid synthesis in mouse peritoneal macrophages by the organic mercury compound thimerosal.

Availability of the common precursor arachidonic acid represents the fundamental prerequisite of the cellular eicosanoid synthesis. The amount of free arachidonic acid is regulated not only by phospholipases, which liberate this polyunsaturated fatty acid from lipid pools, but also by the reacylating enzyme acylCoA:lysophosphatide acyltransferase. We have previously shown (Goppelt-Strübe, G., C.-F. Körner, G. Hausmann, D. Gemsa, and K. Resch. Control of Prostanoid Synthesis: Role of Reincorporation of Released Precursor Fatty Acids. Prostaglandins 32:373. 1986.) that the organic mercury compound thimerosal in murine peritoneal macrophages inhibits arachidonic acid reincorporation into cellular lipids, thereby leading to an enhanced prostanoid synthesis. In this report we show that the production of leukotriene C4 was also increased after the addition of thimerosal to mouse peritoneal macrophages in a time and dose dependent manner. Concomitantly, thimerosal led to a significant rise of the intracellular calcium concentration as measured by fura-2 fluorescence. Simultaneous addition of thimerosal and indomethacin or exogeneous arachidonic acid to the cells resulted in a synergistic enhancement of leukotriene C4 synthesis. On the other hand, another sulfhydryl group blocking agent, ethacrynic acid, was found to be ineffective in increasing leukotriene C4 levels even in combination with exogeneous arachidonic acid. Thimerosal therefore provides a helpful tool in studying the basic regulatory mechanisms of the cellular leukotriene synthesis.

Animals↗
PubMed1984

Renal venous and urinary PGE2 output during intrarenal arachidonic acid infusion in dogs.

Inferences about total renal (venous and urinary) PGE2 output from determinations of urinary excretion rates (U PGE2 V) cannot be made unless the distribution of PGE2 between renal venous plasma and urine is known. Therefore, in the present study on intact kidneys of anesthetized dogs both urinary excretion of PGE2 and the renal venous output (the product of plasma flow and venous concentration of PGE2) was determined during low and high rates of renal PGE2 synthesis. PGE2 was measured in urine and arterial and renal venous plasma by radioimmunoassay during the following conditions: (1) Hydropenia. In the control condition U PGE2 V averaged 0.041 +/- 0.012 pmol/g . min and varied between 4 and 70% of the total PGE2 output. With infusion of arachidonic acid (AA, 160 micrograms/kg . min) into the renal artery total PGE2 output increased from 0.18 +/- 0.03 to 3.23 +/- 0.51 pmol/g . min, whereas arterial concentrations of PGE2 were unchanged. The urinary fraction still varied between 6 and 46% of total renal PGE2 output. (2) High urine flows caused by mannitol, saline or saline and ethacrynic acid (ECA) infusion. These procedures did not stimulate total renal PGE2 output and the urinary fraction varied between 4 and 49%. ECA combined with saline infusion increased the urinary fraction significantly to 34.7 +/- 4.0%. AA increased the total PGE2 output as during hydropenia, but the urinary fraction fell to 13% in 13 dogs and was unchanged at about 8% in six dogs. On average the urinary fraction of total PGE2 output was significantly lower than in hydropenia. Thus, the urinary fraction of total renal PGE2 output is not constant, and urinary excretion of PGE2 does not give reliable information about renal synthetic rates of prostaglandins.

Animals↗
PubMed1994

Enzymatic characteristics of chimeric mYc/rYc1 glutathione S-transferases.

Mice are resistant to aflatoxin carcinogenicity primarily due to expression of a glutathione S-transferase (mYc) with high catalytic activity toward aflatoxin B1-8,9-epoxide (AFBO). In contrast, rats are more sensitive to aflatoxin carcinogenicity due to the constitutive expression of a glutathione S-transferase with relatively low catalytic activity toward AFBO (rYc1). To identify the contribution of different regions of the mYc protein that confer high catalytic activity toward AFBO, six chimeric mYc/rYc1 GST enzymes were generated utilizing full and partial restriction enzyme digestions at two conserved StyI sites in the mYc and rYc1 complementary DNAs (between amino acid residues 56-57 and 142-143). Recombinant wild-type and chimeric glutathione S-transferases were bacterially expressed, affinity purified, and their catalytic activities measured toward AFBO, delta 5-androstene-3,17-dione, 1-chloro-2,4-dinitrobenzene, and ethacrynic acid. The set of chimeras displayed a wide range of catalytic activities toward the substrates assayed. The chimeras with the greatest activity toward AFBO were 1:56rat-57: 221mouse and 1:56mouse-57:142rat-143:221mouse, with AFBO conjugating activities 200 and 8 times greater than wild-type rYc1, respectively. These results demonstrate that the residues that confer high AFBO conjugation activity in mYc are located in the region spanning residues 57-221.

Aflatoxin B1↗
PubMed2006

Characterization of hepatic glutathione S-transferases in coho salmon (Oncorhynchus kisutch).

The glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes which protect against chemical injury. In contrast to mammals, GST expression in fish has not been extensively characterized, especially in the context of detoxifying waterborne pollutants. In the Northwestern United States, coho salmon (Oncorhynchus kisutch) are an important species of Pacific salmon with complex life histories that can include exposure to a variety of compounds including GST substrates. In the present study we characterized the expression of coho hepatic GST to better understand the ability of coho to detoxify chemicals of environmental relevance. Western blotting of coho hepatic GST revealed the presence of multiple GST-like proteins of approximately 24-26kDa. Reverse phase HPLC subunit analysis of GSH affinity-purified hepatic GST demonstrated six major and at least two minor potential GST isoforms which were characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI MS-MS) and Fourier transform-ion cyclotron resonance (FT-ICR) MS analyses. The major hepatic coho GST isoforms consisted of a pi and a rho-class GST, whereas GSTs representing the alpha and mu classes constituted minor isoforms. Catalytic studies demonstrated that coho cytosolic GSTs were active towards the prototypical GST substrate 1-chloro-2,4-dinitrobenzene, as well as towards ethacrynic acid and nitrobutyl chloride. However, there was no observable cytosolic GST activity towards the pesticides methyl parathion or atrazine, or products of oxidative stress, such as cumene hydroperoxide and 4-hydroxynonenal. Interestingly, coho hepatic cytosolic fractions had a limited ability to bind bilirubin, reflecting a potential role in the sequestering of metabolic by-products. In summary, coho salmon exhibit a complex hepatic GST isoform expression profile consisting of several GST classes, but may have a limited a capacity to conjugate substrates of toxicological significance such as pesticides and endogenous compounds associated with cellular oxidative stress.

Amino Acid Sequence↗
PubMed1982

Agents for the treatment of brain injury. 1. (Aryloxy)alkanoic acids.

Blunt and ischemic injuries of the brain have been shown to result in swelling that is predominantly limited to a single cell type, the astrocyte, within the complex cellular mosiac of cerebral gray matter. Evaluation of various diuretic (aryloxy)acetic acids in vitro using incubating cat brain slices and primary astrocyte cultures identified compounds with marked ability to inhibit brain tissue swelling. Some of the compounds significantly reduced the mortality and morbidity following acceleration/deceleration brain injury in anesthesized cats. A variety of (indanyloxy)alkanoic acids were synthesized which were analogous to the dually active (indanyloxy)acetic acids. Some of the 4-(indanyloxy)butanoic acids were found to be devoid of diuretic activity but to possess equal or greater activity than the dually active compounds in the in vitro and in vivo brain assays. Selected examples from both the (indanyloxy)acetic and 4-(indanyloxy)butanoic acid series showed marked chiral effects, with one enantiomer generally exhibiting a much greater activity than the other. A clinical study of severely head-injured patients treated with ethacrynic acid demonstrated a significantly improved outcome when compared to controls. These data suggest a clinical advantage for the nondiuretic (aryloxy)alkanoic acids which possess in vitro and in vivo activities in the cat brain assays that are comparable or superior to dually active compounds.

Animals↗