Is a Ca2+ -ATPase involved in Ca2+ regulation during capacitation and the acrosome reaction of guinea-pig spermatozoa?

The Ca2+-ATPase antagonists quercetin and ethacrynic acid accelerated the onset of the acrosome reaction in guinea-pig spermatozoa incubated in the continuous presence of Ca2+, whereas furosemide had no effect, and sodium orthovanadate only affected sperm motility. When spermatozoa were preincubated in a 'Ca2+-free' medium, quercetin and ethacrynic acid shortened capacitation time: spermatozoa incubated for 1 h in 100-200 microM-ethacrynic acid showed 60-80% acrosome reactions when Ca2+ was added. Such spermatozoa were able to fertilize zona-free hamster eggs. Our results therefore point to the possible involvement of a Ca2+-ATPase in the regulation of intracellular Ca2+ in spermatozoa. Cysteine and dithiothreitol, both disulphide reducing agents, prevented the effects of quercetin and ethacrynic acid, suggesting that sulphydryl groups may be important for the expression of Ca2+-ATPase activity. Lysophosphatidylserine (LS) also prevented the stimulatory effect of ethacrynic acid, an effect similar to that shown by LS on lysophosphatidylcholine (LC). It is argued that both LS and LC could exert their action through an effect on the Ca2+-ATPase.     

The Relationship between Cell Membrane Potassium Ion Transport and Glycolysis : The effect of ethacrynic acid

Cell membrane transport of K⁺ stimulates the rate of glycolysis in Ehrlich ascites tumor cells. A study of the characteristics of this relationship indicates that the stimulation occurs under anaerobic as well as under aerobic conditions. The data suggest that glycolysis is stimulated by a K⁺ transport mechanism that is coupled to Na⁺ transport because the effect is blunted or abolished when the principal intracellular ion is lithium or choline. This stimulus to glycolysis is blocked by ouabain and ethacrynic acid, agents that have been shown to inhibit monovalent cation transport in erythrocytes. In contrast to the action of ouabain, glycolysis is inhibited by ethacrynic acid in Ehrlich ascites tumor cells in the absence of cell membrane K⁺ transport. In studies with ghost-free hemolysates of human erythrocytes and with cytosol prepared from Ehrlich ascites tumor cells, ethacrynic acid significantly blocks lactate formation from fructose diphosphate demonstrating the direct inhibitory effect of this agent on one or more enzymes of the Embden-Meyerhof pathway. Since ethacrynic acid has no influence on lactate formation in intact erythrocytes utilizing an endogenous substrate, the presumptive site of inhibition is proximal to the 3-phosphoglycerate level.     

Effects of ethacrynic acid and furosemide on phosphory-lation reactions of kidney mitochondria. Inhibition of the adenine nucleotide translocase

Previous reports that ethacrynic acid and furosemide diminish mitochondrial P : O ratios and reduce (Na⁺ + K⁺)-ATPase activity suggested that these diuretics may inhibit mitochondrial phosphorylation reactions. This possibility was initially studied by determining the effects of ethacrynic acid and furosemide on [³²P]ATP exchange activity of rat kidney mitochondria. Concentrations of both drugs at 10⁻⁴ M or greater, significantly inhibited [³²P]ATP exchange. To investigate the mechanism of this inhibition, the effects of ethacrynic acid and furosemide on the ATPase activity of intact mitochondria and sonicated submitochondrial particles were determined. Both diuretics inhibited ATPase activity of intact mitochondria at 10⁻⁴ M. In contrast, ATPase of submitochondrial particles was significantly less susceptible to inhibition by the diuretics. These results suggested that ethacrynic acid and furosemide inhibit adenine nucleotide transport across the mitochondrial membrane. This was directly tested by determining the effects of the diuretics on the mitochondrial adenine nucleotide translocase. At 5 · 10⁻⁴ M, both ethacrynic acid and furosemide significantly inhibited adenine nucleotide transport. These findings suggest that ethacrynic acid and furosemide may diminish renal tubular solute reabsorption by direct inhibition of adenine nucleotide transport across the mitochondrial inner membrane.     

Effects of ethacrynic acid and furosemide on phosphorylation reactions of kidney mitochondria. Inhibition of the adenine nucleotide translocase.

Previous reports that ethacrynic acid and furosemide diminish mitochondrial P : O ratios and reduce (Na+ + K+)-ATPase activity suggested that these diuretics may inhibit mitochondrial phosphorylation reactions. This possibility was initially studied by determining the effects of ethacrynic acid and furosemide on [32P]ATP exchange activity of rat kidney mitochondria. Concentrations of both drugs at 10(-4) M or greater, significantly inhibited [32P]ATP exchange. To investigate the mechanism of this inhibition, the effects of ethacrynic acid and furosemide on the ATPase activity of intract mitochondria and sonicated submitochondrial particles were determined. Both diuretics inhibited ATPase activity of intact mitochondria at 10(-4) M. In contrast, ATPase of submitochondrial particles was significantly less susceptible to inhibition by the diuretics. These results suggested that ethacrynic acid anf furosemide inhibit adenine nucleotide transport across the mitochondrial membrane. This was directly tested by determining the effects of the diretics on the mitochondrial adenine nucleotide translocase. At 5-10(-4) M, both ethacrynic acid and furosemide significantly inhibited adenine nucleotide transport. These findings suggest that ethacrynic acid and furosemide may diminish renal tubular solute reabsorption by direct inhibition of adenine nucleotide transport across the mitochondrial inner membrane.     

Selective inhibition of sodium-linked and sodium-independent bicarbonate/chloride antiport in Vero cells

The effects of compounds previously described to inhibit anion transport were tested for their ability to inhibit anion antiport in Vero cells as measured by uptake of 36Cl- by chloride self-exchange and as bicarbonate-linked uptake of 22Na+. While 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited both processes, ethacrynic acid and probenecid selectively inhibited the uptake of 36Cl-. Low concentrations of pyridoxal phosphate and picrylsulfonic acid selectively inhibited the bicarbonate linked uptake of 22Na+, while higher concentrations of these compounds also inhibited the uptake of 36Cl-. Measurements of the internal pH indicated that ethacrynic acid inhibits Na+-independent HCO-3/Cl- exchange, while it has no measurable effect on Na+-linked bicarbonate-dependent regulation of the internal pH. Conversely, picrylsulfonic acid selectively inhibits the latter process. The results indicate that anion antiport in Vero cells occurs by two independent processes.     

Ethacrynic acid analogues lacking the α,β-unsaturated carbonyl unit—Potential anti-metastatic drugs

A series of ethacrynic acid analogues, lacking the α,β-unsaturated carbonyl unit, was synthesized and subsequently evaluated for their ability to inhibit the migration of human breast cancer cells, MCF-7/AZ. Several of the analogues were already active in the low micromolar range, whereas ethacrynic acid itself shows no potential to inhibit the migration of these cancer cells. Preliminary studies show that the presence of one or more methoxy groups at the phenyl ring of ethacrynic acid is important in order for the ethacrynic acid analogues to demonstrate an inhibitory effect on the migration.     

Stimulation of renal tubular transport by ethacrynic acid in rats of different ages.

The transport system for organic acids in the kidney is not fully developed in the neonatal period. The effect of repeated administrations of ethacrynic acid on the renal excretion of p-aminohippurate (PAH) was studied in rats of different ages. Pretreatment with ethacrynic acid was followed by an increase in the renal excretion of PAH in 33-, 55-, 105- and 240-day-old rats but not in newborn rats. In 55-day-old rats the increase in renal excretion of PAH after pretreatment with ethacrynic acid was not associated with any consistent change of the glomerular filtration rate. It is concluded from these results that the stimulation of transport processes in the kidney by ethacrynic acid and some other drugs is linked with their affinity to tissue proteins.     

Potential difference responses to nutrient K+, Na+ and Cl- changes with varying nutrient HCO3- in resting frog stomach

The effects of changes in nutrient concentrations of K+, Na+ and Cl- on the transmucosal potential difference (PD) and resistance were compared for 25 and 5 mM nutrient HCO3- in resting fundus. With 25 mM HCO3-, increase of K+ from 4 to 40 mM, decrease of Na+ from 100 to 10 mM and decrease of Cl- from 81 to 8.1 mM gave, 10 min after the change, delta PD values of -23.2, -15.1 and -21.3 mV, respectively. With 5 mM HCO3-, the same changes in nutrient ion concentration gave delta PD values of -11.9, -9.4 and -10.0 mV, respectively. From these results, in going from 25 to 5 mM HCO3-, it follows that the resistances of the ionic pathways for K+, Na+ and Cl- increased. The anomalous PD response following the increase in nutrient K+ from 4 to 40 mM with 5 mM nutrient HCO3- gave further evidence that the resistance of the simple K+ conductance pathway increased prior to the increase to 40 mM K+. The fact that 2 mM Ba2+ in the 25 mM HCO3- nutrient gave a smaller increase in resistance, compared to the decrease in nutrient HCO3- from 25 to 5 mM, supported the inference that resistances of ion pathways other than that of the K+ pathway increased.     

Inhibition of chloride permeability and sodium transport in frog skin by mercusal and ethacrynic acid

Potential difference across the frog skin is increased 1-2 min after addition of 0.063-1.0 mg/ml ethacrynic acid or 0.2-1.0 mg/ml mercusal to outside Ringer solution. Within this time the short-circuit current remains unchanged or increased. Potential difference and short-circuit current are diminished after the addition of ethacrynic acid or mercusal to inside solution. This effect is similar to that of ouabain. These findings suggest that ethacrynic acid and mercusal inhibit chloride channel in the apical cell membrane, and inhibit sodium transport in the basolateral membrane.     

Changes in mitochondrial shape and distribution induced by ethacrynic acid and the transient formation of a mitochondrial reticulum

We have examined the effect of ethacrynic acid on mitochondrial morphology and distribution as well as on cellular toxicity in cultured human fibroblasts, African Green Monkey B-SC-1 kidney cells, and Chinese hamster ovary cells. Treatment of the above cells with 66 μM ethacrynic acid causes no reduction in cell viability after 2 h but is cytotoxic upon prolonged (6–7 days) exposure. Ethacrynic acid treatment for up to 2 h is found to cause novel shape changes and redistribution of mitochondria, as assessed by immunofluorescence and electron microscopy. Early effects include the transient formation of a mitochondrial reticulum involving the majority of mitochondria, and these reticula are aligned along microtubules. At later times within 2 h, mitochondrial distributions become disoriented (show no association with microtubules), and an aggregation and final positioning of mitochondria around the nucleus is observed. Whole mount electron microscopy shows that mitochondria in treated cells increase in length and form junctions, indicating reticula result from mitochondrial fusion. Electron microscopy of sections through ethacrynic acid induced reticula demonstrates structural continuity in mitochondria at branch points and the presence of regular cristae. Staining of endoplasmic reticulum and mitochondria in intact cells with the cyanine dye 3,3′-dihexyloxacarbocyanine iodide provides evidence of concurrent aggregation of endoplasmic reticulum. Rhodamine 123 staining of living cells followed by immunofluorescent labeling of mitochondria in the same cells indicates that all mitochondria retain a transmembrane potential during the druginduced shape changes and redistributions. The described effects of ethacrynic acid on mitochondrial morphology as well as on cellular toxicity are completely prevented by 0.5 mM dithiothreitol, indicating that ethacrynic acid is acting as a sulfhydryl reagent to produce the observed effects. The above observations also indicate that ethacrynic acid effects on mitochondrial morphology are an early event in the drug-induced cytotoxicity. The generation of varied mitochondrial morphologies by fusion and fission of mitochondria and its modulation by agents such as ethacrynic acid are discussed. © 1994 wiley-Liss, Inc.     

Effects of ethacrynic acid on ion transport and energy metabolism in slices of avian salt gland and of mammalian liver and kidney cortex

Summary Ethacrynic acid greatly inhibited net transport of ions and aerobic, energyconserving metabolism in slices of avian salt gland, rat liver, and rat and guinea-pig kidney cortex. The effects of increasing concentrations of ethacrynic acid on the transport of Na⁺, K⁺ and Cl^– ran closely parallel to its effects on tissue ATP levels and respiration. The concentration needed for maximal inhibition of transport reduced ATP levels by 80–90%. Respiration was reduced by 80–90% in salt gland and kidney cortex, and by a maximum of 30% in liver slices. The effects of low concentrations of ethacrynic acid required time to become fully manifest in some tissues, and the development of transport inhibition followed a similar course to decline of respiration and ATP levels. Ca²⁺ extrusion by liver cells was inhibited by ethacrynic acid. The concentration dependence of the inhibition was similar to that shown by the other transport systems inhibited. There was no distinction evident between the sensitivity of Na⁺ extrusion and of K⁺ accumulation to the diuretic. Lactate production increased as respiration decreased in the presence of increasing concentrations of ethacrynic acid. We conclude that ethacrynic acid acted primarily as an inhibitor of mitochondrial respiration and ATP synthesis in the tissue slices, and that inhibition of ion transport was a nonspecific consequence of the failure of the energy supply.     

Effect of sporulation and recovery medium on the heat resistance and amount of injury of spores from spoilage bacilli

AIMS: To assess the influence of sporulation media on heat resistance, and the use of stress recovery media to measure preservation injury of spores of five representative spoilage bacilli. METHODS AND RESULTS: Bacillus spores prepared on nutrient agar supplemented with Ca2+, Mg2+, Mn2+, Fe2+ and K+ were more heat-resistant than spores obtained from nutrient agar with Mn2+. This increased heat resistance correlated with a decrease in the protoplast water content as determined by buoyant density sedimentation. The degree of preservation injury severity could be assessed on media containing NaCl at moderate pH and organic acids at acid pH. Ca-DPA, K+ or proline were added to the recovery media to demonstrate that heat probably caused injury to both spore germination and the outgrowth system. SIGNIFICANCE AND IMPACT OF THE STUDY: The metal content of sporulation media can strongly effect the validity of preservation resistance studies. The distinctive recovery media developed here can be relevant for assessing and comparing new preservation technologies.     

Inhibitory effects of ethacrynic acid analogues lacking the α,β-unsaturated carbonyl unit and para-acylated phenols on human cancer cells

A series of ethacrynic acid analogues, lacking the α,β-unsaturated carbonyl unit, was synthesized and subsequently evaluated for their ability to inhibit the migration of human breast cancer cells, Hs578Ts(i)8 as well as of human prostate cancer cells, C4-2B. These cell lines provide a good model system to study migration and invasion, since they represent metastatic cancer. Our studies show that ethacrynic acid analogues with methyl substituents at the aromatic ring demonstrate no inhibitory effect on the migration of both cancer cell lines, whereas a precursor in the synthesis of these ethacrynic acid analogues (II-1, a para-acylated m-cresol) is an excellent inhibitor of the migration of both cancer cell lines.     

Interaction of ethacrynic acid and cysteine with renal medullary adenylate cyclase

Results of the present study indicate that (1) ethacrynic acid, dihydroethacrynic acid, and the cysteine adduct of ethacrynic acid inhibit plasma membrane-bound adenylate cyclase from canine renal medulla; (2) reaction with a sulfhydryl group is not essential for inhibition by ethacrynic acid and its derivatives, but may contribute quantitatively to the inhibition; and (3) cysteine enhances the activity of renal medullary adenylate cyclase in the presence of a vasopressin analog or sodium fluoride.Observations support the view that ethacrynic acid and its cysteine adduct interfere with the action of vasopressin on the distal nephron at the site of renal medullary adenylate cyclase.     

Possible mechanism of unconjugated bilirubin toxicity on renal tissue

1. The effects of unconjugated bilirubin on rat renal tissue metabolism and organic anion transport were investigated using cortical slices. 2. Unconjugated bilirubin in the medium decreased slice-to-medium ratio of p-aminohippurate, altered intracellular Na+ and K+, and decreased ATP content without modifications of (Na+-K+) ATPase. 3. The effects were similar to those of ethacrynic acid and cyanide but less marked. 4. The presence of probenecid blocked the effect of pigment on intracellular electrolytes. 5. The results suggest that pigment is taken up by renal tissue using the organic anion transport system, and within the cell inhibits ATP production.     

Effects of phenol compounds, glutathione analogues and a diuretic drug on glutathione S-transferase, glutathione reductase and glutathione peroxidase from canine erythrocytes.

1. Phenol compounds (ellagic acid, quercetin and purpurogallin), glutathione analogues (S-hexylglutathione and S-octylglutathione) and a diuretic drug (ethacrynic acid) were compared for their inhibitory effects on glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) in the canine erythrocytes. 2. All these compounds inhibited GST activity; quercetin was found to be the most potent inhibitor. 3. Ellagic acid, purpurogallin, quercetin and ethacrynic acid inhibited GR activity; S-hexylglutathione and S-octylglutathione had no effect on GR and GSH-Px activities. 4. Quercetin and purpurogallin inhibited GST non-competitively toward glutathione, whereas ellagic acid showed a competitive inhibition. Ellagic acid and purpurogallin inhibited GR non-competitively toward oxidized glutathione.     

Effects of sodium-transport inhibitors on diuresis and mid-gut (Na+ + K+)-ATPase in the tsetse fly Glossina morsitans

The effects of four inhibitors of specific sodium-transport mechanisms on diuresis in the tsetse fly Glossina morsitans, have been determined. Ouabain (1.0, 0.1 mM) and ethacrynic acid (1.0, 0.2 mM) reduced the rate of water loss, whereas amiloride (1.0 mM) and furosemide (1.0 mM) did not. The effects of ouabain, ethacrynic acid and meal size upon the anterior mid-gut (Na⁺ + K⁺)-ATPase activity were also determined. For ouabain, the negative logarithm causing 50% inhibition of (Na⁺ + K⁺)-ATPase (pI₅₀) was 6.0, whilst ethacrynic acid together with meal size did not affect the activity of this enzyme. These results show that diuresis in this insect involves the active transport of sodium ions by both electrogenic and $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ exchange pumps.     

Na+, K+ and Cl- transport in isolated small intestinal cells from guinea pig. Evidences for the existence of a second Na+ pump

Isolated small intestinal epithelial cells, after incubation at 4 degrees C for 30 min, reach ion concentrations (36 mM K+, 113 mM Na+ and 110 mM Cl-) very similar to those of the incubation medium. Upon rewarming to 37 degrees C, cells are able to extrude Na+, Cl- and water and to gain K+. Na+ extrusion is performed by two active mechanisms. The first mechanism, transporting Na+ by exchanging it for K+, is inhibited by ouabain and is insensitive to ethacrynic acid. It is the classical Na+ pump. The second mechanism transports Na+ with Cl- and water, is insensitive to ouabain but is inhibited by ethacrynic acid. Both mechanisms are inhibited by dinitrophenol and anoxia. The second Na+ extruding mechanism could be the Na+/K+/2Cl- cotransport system. However, this possibility can be ruled out because the force driving cotransport would work inwards, and because Na+ extrusion with water loss continues after substitution of Cl- by NO3-. We propose that enterocytes have a second Na+ pump, similar to that proposed in proximal tubular cells.     

Cysteine enhances in vivo natriuretic potency of ethacrynic acid in the rat

Total renal blood flow, glomerular filtration rate, and renal excretory function were determined in anesthetized rats treated with intravenous infusion of ethacrynic acid, 0.36 mg.min-1.kg-1, alone or in combination with cysteine. Simultaneously, the corticomedullary electrolyte gradient was evaluated in vivo from measurement of tissue electrical admittance (reciprocal impedance). Renal hemodynamics was not altered by drug infusion. Sodium excretion increased 1.7-fold with ethacrynic acid alone and 5-fold after the addition of cysteine. Tissue electrolytes of inner medulla decreased much more in rats given ethacrynic acid plus cysteine. We conclude that the addition of cysteine to intravenous infusion of ethacrynic acid greatly enhances its in vivo natriuretic potency in the rat.     

Effect of single dose of diuretics on renal magnesium excretion in man, with special reference to their site of action.

The administration of a single dose of furosemide, ethacrynic acid and polythiazide to healthy individuals under conditions of maximum water diuresis produces a significant increase in renal magnesium excretion. Elevated Mg excretion displayed a direct correlation to renal sodium excretion after furosemide (r=0.689, p less than 0.001), ethacrynic acid (r=0.869, p less than 0.001) and polythiazide (r=0.586, p less than 0.01). The slopes of the various regression lines did not differe significantly from each other or from the slope of the regression line characterizing this correlation for mannitol (r= 0.603, p less than 0.01). A significant linear correlation was likewise found between the excretion of Mg and total osmotically active substances after furosemide (r=0.783, p less than 0.001), ethacrynic acid (r=0.88, p less than 0.001) and polythiazide (r=0.646, p less than 0.01). The regression lines of the given correlations did not differ significantlyfrom each other, but their slopes were significantly higher than that of the regression line for the correlation after mannitol (r=0.454, p less than 0.01). The findings indicate that tubular Mg transport is influenced both by a decrease in tubular Na resorption in the diluting segment (polythiazide) and by an effect on Na resorption in the parts of the nephron proximal to the diluting segment of the nephron (furosemide, ethacrynic acid).