[Endocochlear potential of the inner ear and its modification as affected by dihydrostreptomycin and ethacrynic acid].
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Adenosine Triphosphate↗
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Reversible conjugation of ethacrynic acid with glutathione and human glutathione S-transferase P1-1.
The reversibility of the conjugation reaction of the diuretic drug ethacrynic acid (EA), an alpha,beta-unsaturated ketone, with glutathione and glutathione S-transferase P1-1 (GST P1-1) has been studied. When the glutathione conjugate of EA was incubated with a 5-fold molar excess of N-acetyl-L-cysteine or GST P1-1, a time-dependent transfer of EA to N-acetyl-L-cysteine or GST P1-1 was observed. With increasing pH, the pseudo first order rate constants of transfer of EA to N-acetyl-L-cysteine increased from 0.010 h-1 (pH 6.4) to 0.040 h-1 (pH 7.4) and 0.076 h-1 (pH 8.4). From the fact that preincubation of GST P1-1 with 1-chloro-2,4-dinitrobenzene reduced the incorporation of [14C]EA from 0.94 +/- 0.21 (SD) to 0.16 +/- 0.02 mol EA/mol subunit and from automated Edman degradation of the major radioactive peptide isolated after pepsin digestion of the [14C]EA-labeled enzyme, it was concluded that the reaction of EA takes place with cysteine 47 of GST P1-1. When GST P1-1 was inactivated with a 5-fold molar excess of EA, adding an excess of glutathione resulted in full restoration of the catalytic activity in about 120 h. These findings may have several implications. Under normal physiological conditions the inhibition of GST P1-1 by covalent binding of EA would be reversed by glutathione, leaving reversible inhibition by the glutathione conjugate of EA and by EA itself as the main mechanism of inhibition; however, when glutathione levels are low the covalent inhibition might be predominant, resulting in a completely different time course for the inhibition.
Ethacrynic Acid↗
Characteristics of ethacrynic acid highly sensitive Mg2+-ATPase in microsomal fractions of the rat brain: functional molecular size, inhibition by SITS and stimulation by Cl-.
Studies were performed to characterize ethacrynic acid (EA) highly sensitive Mg2+-ATPase isolated from microsomal fractions of the rat brain. The functional molecular sizes of the EA highly sensitive and EA less sensitive Mg2+-ATPases, estimated by a radiation inactivation method, were 480 and 80 kDa, respectively. An anion transport inhibitor, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) inhibited the EA highly sensitive Mg2+-ATPase activity. The type of inhibition was uncompetitive with respect to ATP, and the inhibition was suppressed by anions such as Cl-, Br- and I-. Chloride ions stimulated enzyme activity with an increase in Vmax, but not in Km, for ATP. Anions tested also increased the enzyme activity in the following order of decreasing potency: Cl- greater than Br- greater than CH3COO- = I- greater than SO4(2-) = HCO3- greater than SO3(2-). These results suggest that EA highly sensitive Mg2+-ATPase is a relatively large molecule with anion-sensitive sites that affect the ATP hydrolyzing activity and the SITS binding capacity through anions, with Cl- being the most potent.
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
The nature of the ototoxic actions of ethacrynic acid upon the mammalian endolymph system. I. Functional aspects.
The endolymphatic changes produced by an intravenous injection of 60 mg kg-1 ethacrynic acid were followed for up to 120 min using conventional and ion-sensitive (Na+, K+ and pH) microelectrodes in the rat. They were found to be caused by three distinct effects upon the endolymph system. Initially, the drug completely inhibited the strial potential-producing and cation-transporting processes. Recovery began quickly and was rapid at first. Then its rate declined considerably, probably due to diminution in strial energy production of delayed onset and prolonged duration. Coincident with these actions upon active transport, there was a decrease in the overall cation permeability of the endolymph system. This followed a different time course and affected K+ much more than Na+. The findings also provided further information about the mechanisms responsible for the normal endolymphatic composition. Qualitatively similar results were obtained in a subsidiary study on guinea pigs.
Animals↗
Effect of ethacrynic acid, a glutathione-S-transferase inhibitor, on nitroglycerin-mediated cGMP elevation and vasorelaxation of rabbit aortic strips.
The effects of ethacrynic acid (ECA), an inhibitor of glutathione-S-transferase, on both the pharmacologic and biochemical responses of aortic tissue to nitroglycerin (GTN) were evaluated. Using the rabbit aortic strip model, relaxation responses to 0.6 microM GTN were measured with and without ECA (0.2 mM) pretreatment. These same strips were frozen, and the concentrations of cGMP in the strips were measured using a 3H-labeled radioimmunoassay. Both the relaxation response and the increase in cGMP upon GTN treatment were reduced significantly by pretreatment of the strips with ECA. A correlation was observed between the decreases in the pharmacodynamic and biochemical responses upon ECA pretreatment. cGMP levels in strips treated with sodium nitroprusside, which generates nitric oxide by mechanisms distinct from that for organic nitrates, were not decreased by ECA pretreatment. These observations suggest that the mechanism of GTN action involves a glutathione-S-transferase-mediated metabolic step for GTN and that the isozyme(s) involved in this activation process may be inhibited by ECA.
Animals↗
Ethacrynic acid rapidly and selectively abolishes blood flow in vessels supplying the lateral wall of the cochlea.
The mechanisms underlying the ototoxicity of ethacrynic acid (EA) are not fully understood. Previous studies have focused on morphologic and enzymatic changes in the stria vascularis. The current experiment shows that one of the earliest effects of EA is ischemia, resulting from impaired blood flow in vessels supplying the lateral wall of the cochlea. Inner ear microcirculation, endocochlear potentials, compound action potentials (CAP), cochlear microphonics (CM) and summating potentials (SP) were monitored over time in chinchillas following a single injection of EA (40 mg/kg i.v.). At all times after EA injection, blood vessels supplying the spiral lamina, modiolus, and vestibular end organs appeared normal. In contrast, lateral wall (spiral ligament and stria vascularis) vessels were poorly stained with eosin 2 min after EA injection, and devoid of red blood cells at 30 min post EA. Decline, but not recovery, of CAP, CM and SP followed the microcirculation changes in the lateral wall. Reperfusion was delayed in stria vascularis arterioles relative to other lateral wall vessels. The ischemia-reperfusion caused by EA would be expected to generate large quantities of free radicals, which may trigger or contribute to the cellular, enzymatic, and functional pathologies that have been described in detail previously.
Action Potentials↗
Late dosing with ethacrynic acid can reduce gentamicin concentration in perilymph and protect cochlear hair cells.
A key factor in the well-known interaction between ethacrynic acid (EA) and aminoglycoside antibiotics (AABs) is disruption of the blood-labyrinth barrier (BLB), leading to rapid entry of EA and AABs into the cochlear fluids. The idea that the blood-labyrinthine fluid concentration gradient might be utilized in a protective manner was tested in the current experiment. We hypothesized that administering EA when gentamicin (GM) levels are higher in the cochlea than in the blood might actually reduce cochlear damage by permitting efflux of GM from the cochlear fluids into the bloodstream, down a concentration gradient and across a temporarily disrupted BLB. Guinea pigs received 1, 11, 14 or 20 injections of GM (125 mg/kg i.m.). Approximately half of the animals also received a single injection of EA (40 mg/kg i.v.) either concurrently or 12-18 h after the last GM injection. Concurrent injection of EA significantly increased GM concentration in serum and perilymph at all time points sampled (2.5, 5-8, and 12 h post injection). Compared to animals that received GM only, animals that received a delayed injection of EA had a significantly lower GM concentration in perilymph, lower thresholds of the compound action potential, and less outer hair cell loss. Collectively, the evidence suggests that EA can reduce GM ototoxicity if it is administered 12-18 h after GM, but the mechanism remains to be elucidated. The results may have implications for the clinical management of aminoglycoside ototoxicity in humans, as well as for understanding the mechanisms underlying AAB/EA interactions.
Action Potentials↗
The phase 2 enzyme inducers ethacrynic acid, DL-sulforaphane, and oltipraz inhibit lipopolysaccharide-induced high-mobility group box 1 secretion by RAW 264.7 cells.
The diuretic ethacrynic acid (EA) has been shown to inhibit signaling by the proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB). Accordingly, we sought to determine whether this compound is capable of inhibiting the release of cytokines [interleukin (IL)-6 and IL-10] and NO from RAW 264.7 murine macrophage-like cells stimulated with lipopolysaccharide (LPS). Additionally, we sought to determine whether EA can inhibit secretion of high-mobility group box 1 (HMGB1), a nuclear protein that is secreted by immunostimulated macrophages and functions in the extracellular milieu as a proinflammatory mediator. In a concentration-dependent manner, EA inhibited secretion of IL-6, IL-10, nitric oxide, and HMGB1. As expected, EA inhibited NF-kappaB DNA binding in LPS-stimulated RAW 264.7 cells. Treating these cells with pyrrolidine dithiocarbamate, SN50 (amino acid sequence AAVALLPAVLLALLAPVQRKRQKLMP) or 5-(thien-3-yl)-3-aminothiophene-2-carboxamide (SC-514) also inhibited LPS-induced NF-kappaB DNA binding, but these compounds failed to inhibit LPS-induced HMGB1 secretion. These findings suggested that inhibition of HMGB1 secretion by EA might occur via a mechanism unrelated to the NF-kappaB signaling pathway. Because EA is an electrophilic compound that is known to be capable of inducing expression of so-called phase 2 proteins, we sought to determine whether two other phase 2 enzyme inducers, oltipraz and DL-sulforaphane, also are capable of inhibiting HMGB1 release from immunostimulated macrophages. Incubating RAW 264.7 cells with either oltipraz or DL-sulforaphane inhibited LPS-induced HMGB1 secretion. Moreover, both EA and DL-sulforaphane inhibited relocalization of nuclear HMGB1 into the cytoplasm of LPS-stimulated RAW 264.7 cells. These data suggest that phase 2 inducers may exert anti-inflammatory effects by inhibiting secretion of the cytokine-like nuclear protein HMGB1.
Active Transport, Cell Nucleus↗
Solubilization and separation of ethacrynic acid (EA) highly sensitive and EA less sensitive Mg2+-ATPases in the rat brain.
Rat brain microsomal Mg2+-ATPases with two distinct activities: ethacrynic acid (EA) highly sensitive and EA less sensitive Mg2+-ATPase activities were solubilized by the combined treatment with 10 mM 3-(3-chlolamidopropyl)-dimethylammonio-1-propane-sulfate (CHAPS) and 30 mM octyl-beta-D-glucoside. The solubilized enzymes had properties similar to those of the membrane-bound enzyme in microsomes with respect to the sensitivity to EA and Cl-, although the optimal pH and the affinity to ATP were slightly altered after the solubilization. Fast protein liquid chromatography of the solubilized enzymes on an anion-exchanger (Mono Q) column with a linear NaCl gradient (0-1.0 M) yielded separate peaks for EA highly sensitive and EA less sensitive Mg2+-ATPase activities at 0.1 and 0.35 M NaCl, respectively. Polyacrylamide gradient gel electrophoresis of the samples from the peak-fractions of EA highly sensitive and EA less sensitive Mg2+-ATPase activities yielded prominent bands at 600 and 70 kDa, respectively. These results indicate that EA highly sensitive Mg2+-ATPase is solubilized and separated from EA less sensitive Mg2+-ATPase as a large enzyme molecule with anion-sensitive sites.
Animals↗
Induction of mitochondrial fusion by cysteine-alkylators ethacrynic acid and N-ethylmaleimide.
Mitochondrial fusion and fission are important aspects of eukaryotic cell function that permit the adoption of varied mitochondrial morphologies depending upon cellular physiology. We previously observed that ethacrynic acid (EA) induced mitochondrial fusion in cultured BSC-1 and CHO/wt cells. However, the mechanism responsible for it was not clear since EA has a number of known cellular effects including glutathione (GSH) depletion and alkylation of cysteine residues. To gain insight, we have tested the effects of a variety of compounds on EA induced cellular toxicity and mitochondrial fusion. N-acetyl cysteine (NAC), a GSH precursor, was found to abrogate both the toxic and fusion-inductive effects, whereas diethylmaleate (dEM), a GSH depletor, potentiated both these effects in a dose-dependent manner. However, treatment with dEM alone, which depleted GSH to the same degree as EA, did not induce mitochondrial fusion. These results indicate that although detoxification of EA via formation of GSH conjugates is dependant upon GSH levels, the depletion of GSH by EA is not responsible for its effect on mitochondrial fusion. Dihydro-EA (DH-EA), a saturated EA analogue, lacked EA's toxicity and effect on fusion, indicating that the alpha,beta-unsaturated ketone is central to its observed effects. N-ethylmaleimide (NEM), another well-known cysteine-alkylator, also induced mitochondrial fusion at near toxic concentrations. These data suggests that cysteine-alkylation is the causative factor for fusion and toxicity. In live BSC-1 cells, EA induced fusion of mitochondria occurred very rapidly (<20 min), which suggests that it is inducing fusion by modifying certain critical cysteine residue(s) in proteins involved in the process.
Animals↗
Cell membrane alterations in the stria vascularis of the guinea pig after ethacrynic acid treatment studied by freeze-fracture.
A freeze-fracture examination of the stria vascularis during the first 2 h after injection of ethacrynic acid was performed. This showed a re-distribution of the particles on the membrane fracture faces of both marginal and intermediate cells. As oedematous spaces developed, particle-poor, vesicle-like structures were found associated with both cell types. The tight junctions at the apices of the marginal cells and around basal cells were unaffected.
Animals↗
[Morphologic studies of the toxicity of ethacrynic acid in the spiral prominence].
Ultrastructural changes in the guinea pig spiral prominence were studied at various times after a single intravenous injection of ethacrynic acid (40 mg/kg body-weight). Initial swelling of endolymph-facing epithelial cells was followed by dilatation of the intercellular spaces and marked shrinkage of the stroma cells surrounding the spiral prominence vessel. While the changes in the stria vascularis and the spiral prominence progressed at about the same pace, the regression to normal ultrastructure set in earlier in the spiral prominence.
Animals↗
[Measurement of the concentrations of PGF2 alpha and PG12 in the cochlear lateral wall of guinea pigs following intravenous injection of ethacrynic acid].
The concentrations of prostaglandin F2 alpha(PGF2 alpha) and prostacyclin (PGI2) in the cochlear lateral wall of guinea pigs following intravenous injection of ethacrynic acid (EA) were measured with radioimmunoassay. After injection of EA (60mg/kg) 30, 60, 120 minutes, the concentrations of PGF2 alpha and PGI2 in the cochlear lateral wall of three experimental groups were not significantly different from control ones respectively. The results suggested that there was no remarkable relationship between PGF2 alpha PGI2 and the changes of the microcirculation of cochlear lateral wall caused by EA.
6-Ketoprostaglandin F1 alpha↗
Selective inhibition of leukotriene C4 synthesis and natural killer activity by ethacrynic acid.
We have investigated the role of arachidonic acid (AA) metabolism in natural killer (NK) cell activity. Human nonadherent (NA) peripheral blood lymphocytes were used as effector cells against 51Cr-labeled K562 target cells. Synthesis of leukotriene C4 (LTC4) is dependent on glutathione S-transferase (GST). We have chosen to study three putative GST inhibitors, namely, ethacrynic acid (ET), caffeic acid (CA), and ferulic acid (FA), with regard to NK activity and with regard to their effect on AA metabolism. The GST inhibitors inhibited NK lysis when added directly to the NK assay. The GST inhibitors inhibited LTC4 synthesis as induced by calcium ionophore A23187 in a dose-dependent manner similar to their inhibition of NK activity. However, only ET was selective, for it had little effect on LTB4, 5-hydroxyeicosatetraenoic acid, and prostaglandin E2 synthesis. LTC4 synthesis was associated with the NK-enriched fractions obtained from discontinuous Percoll gradients. NK-specific anti-Leu-11b antibody and C treatment could abrogate NK activity and LTC4 synthesis. ET was also inhibitory when NA cells were cultured at 37 degrees C for 18 hr. In this case, LTC4 could reverse the inhibitory effect of ET. Our data suggest that LTC4 plays an important role in NK activity.
Adult↗
Stimulation of renin secretion by ethacrynic acid is independent of Na(+)-K(+)-2Cl- cotransport.
This study investigated the cellular mechanism of stimulation of renin secretion by the loop diuretic ethacrynic acid (EA) in rabbit renal cortical slices. The diuretic rapidly stimulated renin secretion reversibly and in a concentration-dependent manner. The stimulation was independent of the presence of Na+, Cl-, Ca2+, or other loop diuretics (furosemide and bumetanide) in the incubation media, suggesting that the stimulation in vitro was not dependent on the inhibitory effect of the diuretic on Na(+)-K(+)-2Cl-cotransport. The findings do not support the macula densa hypothesis. The stimulation by the diuretic was prevented and reversed by thiols such as cysteine and dithiothreitol, which also prevented and reversed the stimulation of renin secretion by the nondiuretic sulfhydryl reagent P-chloromercuriphenyl-sulfonate (PCMPS). These results suggest that EA stimulates renin secretion in vitro via reversible chemical reactions with specific membrane sulfhydryl groups that may have no functional role in the Na(+)-K(+)-2Cl- cotransport.
4-Chloromercuribenzenesulfonate↗
Increased levels of glutathione S-transferase pi transcript as a mechanism of resistance to ethacrynic acid.
Subpopulations of HT 29 human colon carcinoma cells (HT/M and HT/S) were selected for resistance to the glutathione S-transferase (GST) inhibitor ethacrynic acid (EA). Both clones displayed a 2-fold resistance to the selection agent and required its constant presence for the maintenance of the resistant phenotype. Purification and characterization of GST isoforms showed similar profiles in the wild-type (WT) and EA-resistant clones, with microheterogeneous forms of the pi isoenzyme detected in each case. Metabolism of EA in vitro in the presence of GSH and the isolated GST from each cell line was characterized by a biphasic disappearance of the parent drug; the initial rate at which each of these enzymes metabolized EA was similar. These enzymes also displayed similar Km values for 1-chloro-2,4-dinitrobenzene. However, the amount of GST isolated per total cellular protein was 3.0-fold in HT/M and 1.6-fold in HT/S relative to WT in the continuous presence of EA. Under these conditions GST activity was increased by 2.3-fold in HT/M and 3.2-fold in HT/S as were GSH levels (2.7- and 4.1-fold for HT/M and HT/S respectively). When EA was removed, enzyme activity and GSH concentrations decreased to values similar to those of the WT. Slot-blot and Southern analyses of the DNA gave no evidence of GST-pi-gene amplification or rearrangement. However, RNA analyses by both slot-blot and Northern studies indicate a 2.5-3.5-fold elevation in the GST pi transcript in the EA-resistant population. Results from these studies indicate that: (1) maintenance of the EA-resistant phenotype requires constant presence of the agent; (2) the 2-fold resistance to EA can be quantitatively related to a 2-3-fold increase in GST activity and amount which appears to be the result of a 2.5-3.5-fold elevation in GST transcript; (3) EA, a Michael-reaction acceptor, can induce GST at the transcriptional level.
Cell Division↗
Ethacrynic acid: acute hemodynamic effects and influence on the in vivo and in vitro response to nitroglycerin in the dog.
We examined the hemodynamic effect of ethacrynic acid (EA), a diuretic, and sulfhydryl binding reagent in the dog to determine whether EA attenuated the response to nitroglycerin (GTN) in vivo or in vitro in rings of dorsal pedal artery (DPA). Six dogs (group A) received infusions of GTN (21 micrograms/min i.v.) before and after EA (0.75 mg/kg). EA produced a marked diuresis [289 ml +/- 41 (SD) urine during 60 min]; 10 min after EA and before substantial diuresis, there was a transient increase in heart rate (HR) from 117 beats/min +/- 29 to 143 beats/min +/- 28 and in mean arterial pressure (MAP) from 137 mm Hg +/- 18 to 144 mm Hg +/- 17 (p less than 0.005). Intravenous GTN resulted in a similar decline in systolic blood pressure before (14.7% +/- 6.4) and after (9.4% +/- 8.1) EA. We also studied two additional groups of dogs that received either EA (0.75 mg/kg) or saline, and urine output was replaced with saline. Similar results were obtained as with group A. In the in vitro studies, 75 rings of DPA from 14 dogs were pretreated with EA at low (1.6 X 10(-5) M), medium (8.3 X 10(-5) M), or high (1.7 X 10(-4) M) doses or EA solvent (control) for 30 min, and dose-response curves were performed for GTN (10(-9) to 10(-5) M). In control rings, the maximum relaxation achieved with GTN was 89% +/- 5.8 inhibition of phenylephrine-induced tone; with both the medium and high EA doses, the response to GTN was partially attenuated.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
[Functional morphology of stria vascularis after treatment with ethacrynic acid or atoxyl (author's transl)].
Surface alterations of the stria vascularis and Reissner's membrane were studied in guinea pigs following intravenous or intraperitoneal administration of ethacrynic acid or atoxyl. DC-potential was measured in the same animals during intoxication. In addition we studied changements in potassium concentration (perilymph, endolymph) of the atoxyl-treated animals.
Action Potentials↗