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PubMed1993

Cytoprotection by inhibition of chloride channels: the mechanism of action of glycine and strychnine.

Previous studies have demonstrated that strychnine mimics the cytoprotective effects of glycine (1) and that strychnine binds specifically to renal proximal tubules (RPT) at cytoprotective concentrations (2). The goal of this study was to determine a mechanism by which strychnine and glycine are cytoprotective. Antimycin A (0.1 microM) caused chloride influx subsequent to mitochondrial inhibition and prior to the release of lactate dehydrogenase (LDH) activity (a marker of cell death/lysis). The addition of strychnine or glycine prevented the chloride influx and LDH release. The chloride channel inhibitors ethacrynic acid, furosemide, anthracene-9-carboxylic acid, DIDS, and SITS decreased LDH release in RPT exposed to antimycin A with a rank order of potency of DIDS > ethacrynic acid = furosemide = anthracene-9-carboxylic acid > SITS. These data, in conjunction with the preceeding paper, indicate a critical role for chloride influx in cell death/lysis; support the existence of a novel strychnine binding site on the plasma membrane of RPT that is coupled to a chloride channel; and suggest that glycine and strychnine are cytoprotective through their inhibition of chloride influx.

Animals↗
PubMed1982

Effect of choleretics on canalicular transport of protoporphyrin in the rat liver.

The major route of protoporphyrin elimination is biliary secretion. To clarify the nature of the secretory process, maximal canalicular secretion of protoporphyrin was determined under basal conditions and after treatment with various choleretics. The maximal secretion of protoporphyrin under basal conditions was 0.07 +/- 0.01 micrograms.min-1.100 g body wt-1. Infusion of physiological amounts of sodium taurocholate increased protoporphyrin secretion 13-fold (0.90 +/- 0.02), primarily by increasing the biliary protoporphyrin concentration. Biliary protoporphyrin secretion tended to plateau in spite of a continued rise in both biliary bile acid secretion and concentration. Infusion of sodium dehydrocholate increased protoporphyrin secretion, but to only 35% of that achieved by sodium taurocholate. Ethacrynic acid and phenobarbital increased bile flow over controls but failed to enhance protoporphyrin transport. Thus, canalicular secretion of protoporphyrin was maximally enhanced by micelle-forming bile acids and unaffected by nonbile acid choleretics. The observed limitation of protoporphyrin secretion may be related to achievement of a canalicular transport maximum or to a toxic effect of protoporphyrin on the transport process.

Animals↗
PubMed1996

Identification of efflux systems for large anions and anionic conjugates as the mediators of methotrexate efflux in L1210 Cells.

Two ATP-dependent efflux systems for methotrexate have been identified in inside-out vesicles from an L1210 mouse cell variant with a defective influx carrier for methotrexate. Transport at 40 muM [3H]methotrexate was separated by inhibitors into two components comprising 62 and 38% of total transport activity. The predominant route was inhibited by low concentrations of indoprofen (Ki=2.5 muM, 4-biphenylacetic acid (Ki=5.3 muM), and flurbiprofen (Ki=5.2 muM, whereas the second component showed a high sensitivity to the glutathione conjugates of bromosulfophthalein (Ki=0.08 muM), ethacrynic acid (Ki=0.52 muM, and 1-chloro-2,4-dinitrobenzene (Ki=0.77 muM). Bilirubin ditaurate was a potent inhibitor of both transport components (Ki=1.5 and 0.17 muM, respectively). Separation of transport activities without interference from the other route was achieved by adding an excess (100 muM) of either the glutathione conjugate of ethacrynic acid or biphenylacetic acid. Double-reciprocal plots of transport at various substrate concentrations gave Km values of 170 and 250 muM for methotrexate transport via the anion-sensitive and conjugate-sensitive routes, respectively. A comparison of inhibitor specificities indicated that the anion-sensitive transport activity in vesicles represents efflux system II for methotrexate in intact cells and is the same system identified previously in vesicles as an anion/anion conjugate pump. The conjugate-sensitive activity corresponds to efflux system I for methotrexate in intact cells and is the same system identified in vesicles as the high-affinity glutathione conjugate pump.

Adenosine Triphosphate↗
PubMed1996

Dexamethasone inhibits trabecular cell retraction.

Glucocorticosteroids such as dexamethasone (Dex) are known to cause an increased resistance to aqueous outflow in the intact and cultured eye. We investigated whether Dex treatment of cultured endothelial or trabecular meshwork (TM) cells might interfere with the cell separations and retraction induced by the facility-enhancing agents ethacrynic acid (ECA), cytochalasin B and the calcium chelator EGTA. Our hypothesis was that Dex-induced changes in the response of our model cells in vitro might serve as a paradigm for those produced in the cells of the outflow pathway, perhaps through influencing the changing dimensions of the pathway for aqueous humor through the juxtacanalicular tissue and/or inner wall of Schlemm's canal. We treated calf pulmonary artery endothelial (CPAE) and human and porcine TM cells with Dex (1-100 microns, 1-9 days), and then assessed monolayer and cytoskeletal integrity by immunofluorescence microscopy for tubulin and direct fluorescence staining for F-actin after exposure to the agents named above. We found that Dex-pretreated CPAE and TM cells gradually (over 5-7 days) became refractory to the effects of both ECA and EGTA, but not to cytochalasin B. Despite the preservation of general cell shape and attachment after ECA in Dex-treated cells, microtubule disruption still took place as in controls. Dex-treated cells also demonstrated a reorganization of filamentous actin staining after ECA and EGTA. Combination experiments of ECA and EGTA in Dex-treated cells suggested that the Dex effects were due to a greater strength of cell-to-cell and cell-to-substrate attachment, possibly due to interference with the normal cellular signaling required for coordinated cellular retraction and junctional disruption.

Actins↗
PubMed2006

Oxidative stress and Mrp2 internalization.

Oxidative stress in the liver is sometimes accompanied by cholestasis. We have described the internalization of multidrug resistance-associated protein 2/ATP-binding cassette transporter family 2 (Mrp2/Abcc2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress in rat liver. However, the signaling pathway and regulatory molecules have not been investigated. In the present study, we investigated the mechanism of EA-induced Mrp2 internalization using isolated rat hepatocyte couplets (IRCHs). The Mrp2 index, defined as the ratio of Mrp2-positive canalicular membrane staining in IRCHs per number of cell nuclei, was significantly reduced by treatment with EA. This reduction was abolished by a nonspecific protein kinase C (PKC) inhibitor Gö6850, a Ca(2+) chelator, EGTA, but not by a protein kinase A (PKA)-selective inhibitor, a Ca(2+)-dependent conventional PKC (cPKC) inhibitor Gö6976, or a protein kinase G (PKG) inhibitor (1 microM). Moreover, an increase in the intracellular Ca(2+) level and NO release into medium were observed shortly after the EA treatment. Both of these increases, as well as Mrp2 internalization, were completely blocked by EGTA. In conclusion, EA produced a reduction in GSH, Ca(2+) elevation, NO production, and nPKC activation in a sequential manner, finally leading to Mrp2 internalization.

ATP-Binding Cassette Transporters↗
PubMed1975

ATPase activity of kidney mitochondria stimulated by sodium.

ATPase activity of intact mitochondria may be induced by the presence of sodium. Mitochondria obtained from kidney cortex or medulla demonstrate a higher sodium-stimulated ATPase than mitochondria prepared from liver. This difference is not due to a greater capacity of kidney mitochondria for ATP hydrolysis. Sodium-stimulated ATPase activity of mitochondria is not further enhanced by magnesium or potassium, nor is it inhibited by ouabain. Furthermore, the ATPase of submitochondrial particles is not enhanced by the presence of sodium. These results indicate that the ATPase activity of mitochondria which is stimulated by sodium is unrelated to the microsomal Na-K-ATPase. mitochondrial sodium-stimulated ATPase is affected by pH, anionic composition, furosemide, and ethacrynic acid. The demonstration that mitochondrial ATP hydrolysis can be activated by sodium suggests that intracellular sodium may directly influence intracellular energy metabolism.

Adenosine Triphosphatases↗
PubMed1994

Differential combined effect of cadmium and nickel on hepatic and renal glutathione S-transferases of the guinea pig.

When male guinea pigs were given a single dose of Cd (2.0 mg Cd2+/kg, ip) 72 hr prior to sacrifice, the hepatic reduced glutathione (GSH) level did not change although glutathione S-transferase (GST) activities toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EAA), and 1,2-epoxy-3-(p-nitrophenoxy) propane (ENPP) increased significantly as compared to controls. Cd did not change the renal GSH level and GST activities toward CDNB and EAA. However, significant increase was observed in the GST activity for DCNB whereas GST activity for ENPP was significantly inhibited by Cd. When the animals were given a single dose of Ni (14.8 mg Ni2+/kg, sc) 16 hr prior to sacrifice, significant increases were observed in hepatic GSH level and GST activities toward CDNB, DCNB, EAA and ENPP. Ni, however, depressed the renal GSH level and GST activities toward CDNB, DCNB and ENPP significantly. The renal GST activity toward EAA remained unaltered. For the combined treatment, guinea pigs received the single dose of Ni 56 hr after the single dose of Cd and then they were killed 16 hr later. In these animals, no significant alteration was observed in the hepatic GSH level. The augmentation of elevation was observed in hepatic GST activities toward CDNB and DCNB. Combined metal treatment did not potentiate the elevation of hepatic GST activities toward EAA and ENPP to any greater degree. The depression of renal GSH level was significantly ameliorated by the combined treatment. Combination treatment potentiated the depression of renal GST activity for ENPP but not for CDNB.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Ototoxicity or teratogenicity. An analysis of drug-induced effects on the early development of the mammalian otocyst.

The 13th gestational day inner ear (otocyst stage) is particularly vulnerable to toxic influence from the environment during its further development. Ethacrynic acid in a concentration of 1 and 10 micrograms/ml caused complete arrest in development, and this was not overcome when withdrawing the drug after in vitro exposure to ethacrynic acid during 3 days. Gentamicin exposure at 1 and 10 micrograms/ml caused dysmorphogenesis but did not inhibit cytodifferentiation which, however, often was poor. Higher concentrations of gentamicin (100 micrograms/ml) had a more generalized toxic effect not only on epithelial but also on mesenchymal tissue.

Abnormalities, Drug-Induced↗
PubMed2000

Compensation of the siphoning effect in nonaqueous capillary electrophoresis by vial lifting.

Increasing the sample load in nonaqueous capillary electrophoresis through the use of wide-bore capillaries is a good way to scale up analytical separations to semipreparative level. However, obtaining high efficiency requires the use of special instrumentation to eliminate siphoning. When wide-bore capillaries are employed, relatively large solvent volumes are transported from inlet to outlet vial, and due to the difference in liquid levels a siphoning flow from outlet to inlet is established. Siphoning induces a deviation from the plug-like flow profile and adversely affects the separation efficiency. In this study the use of wide-bore capillaries in nonaqueous capillary electrophoresis was examined with compensation for siphoning by lifting of the inlet vial. The inlet vial is raised at a speed appropriate for maintaining equal levels of liquid in the inlet and outlet vials. The optimal lift rate was determined empirically from a series of runs in which the lift rate was varied. As well, a simple theoretical model was devised for the calculation of lift rates. The model was successfully applied for the 200 microm and 320 microm ID capillaries but for the 530 microm ID capillary the predicted optimal lift rate was too low. Evidently this was because the theory was unable to account for the effect of siphoning on the migration times. Three model compounds, bumetanide, furosemide and ethacrynic acid, were separated using an acetonitrile-ethanol mixture (50:50, v/v) with potassium acetate (1 mM) or ammonium acetate (5 mM) as electrolyte. Good separation of bumetadine and ethacrynic acid was obtained even with a 530 microm ID capillary when the lift rate was carefully optimized. Without elimination of siphoning the peaks would not have been detectable. The viscosities and electrical conductivities of the electrolyte solution measured at different temperatures showed that viscosity as well as conductivity decreased with increasing temperature. The temperature dependence of the conductivity was used to estimate the temperature inside the CE capillary.

Electric Conductivity↗
PubMed1996

Drug effects on triiodothyronine uptake by rat anterior pituitary cells in vitro.

In nonthyroidal illness, numerous drugs such as glucocorticoids, dopamine, fenclofenac, furosemide and diphenylhydantoin may modify the close inverse-feedback relationship between circulating thyroid hormones and TSH. Such effects could involve altered hypothalamic TRH secretion, a direct effect on TSH production by the thyrotroph, alterations in circulating free thyroid hormone concentrations, or changes in thyroid hormone uptake by the thyrotroph. We therefore examined the effect of nonsteroidal antiinflammatory drugs (NSAID), diuretics, the synthetic flavonoid EMD 21388, and diphenylhydantoin, on [125I]T3 cellular uptake in rat pituitary primary cell cultures. Uptake of [125I]T3 (cell-associated counts of washed cells) was measured at 15 min after the addition of 50 pmol/L [125I]T3 in protein-free medium (37 degrees C, pH 7.4). Uptake of [125I]T3 by pituitary cells was 6.0 +/- 1.7% of total counts (mean +/- SD, n = 18). Unlabeled T3 (10 mumols/L) displaced 92% of total uptake. The IC50 of unlabeled T3 for the displacement of [125I]T3 was 1.2 mumols/L. T4 and rT3 were approximately 10% as effective as T3 itself in inhibiting [125I]T3 uptake, while triac did not affect cellular [125I]T3 uptake. Inhibition of [125I]T3 uptake at drug concentrations of 100 mumols/L was seen with the diuretics, furosemide (9%), bumetanide (14%), piretanide (12%) and ethacrynic acid (76%), the NSAID, meclofenamic acid (35%) and fenclofenac (52%), EMD 21388 (49%), and the anticonvulsant, diphenylhydantoin (23%). Aspirin, up to 500 mumols/L, had no effect on [125I]T3 uptake. Our results indicate that ethacrynic acid, meclofenamic acid, fenclofenac, EMD 21388 and diphenylhydantoin affect plasma membrane T3 uptake in the pituitary. This potential influence on TSH release will be contrary to the previously-demonstrated direct inhibitory effect of these drugs on TSH release.

Animals↗
PubMed2000

The influence of coordinate overexpression of glutathione phase II detoxification gene products on drug resistance.

Glutathione (GSH), glutathione S-transferases (GSTs), and the multidrug resistance-associated protein 1 (MRP1) have been independently studied for their contributions to drug resistance. Single cDNA transfection experiments have provided inconsistent and disparate conclusions with respect to the importance of GSH and GST in conferring a resistant phenotype. Because these three proteins can act as a concerted coordinated pathway, we reasoned that equivalent increases may be required for enhanced resistance to be expressed. We have assembled these proteins together, or in various combinations, to determine whether they show cooperativity in determining drug response. Increased expression through single cDNA transfection of GSTpi, gamma-glutamylcysteine synthetase (gamma-GCS) (regulatory plus catalytic subunits), or MRP1 enhanced resistance to a number of anticancer drugs. Cotransfection of GSTpi and GCS, gave higher resistance to doxorubicin, etoposide, and vincristine than with either alone. Resistance toward chlorambucil and ethacrynic acid was similar in cells overexpressing either component or overexpressing GST alone. Coexpression of GSTpi with MRP1 conferred significant resistance above that seen for MRP1 alone to chlorambucil, etoposide, ethacrynic acid, and vincristine. The combination of GCS and MRP1 did not afford additional resistance above MRP1 alone. When all three were transfected, significantly higher levels of resistance were found for doxorubicin and etoposide. These results support the concept that coordinate enhancement of focal thiol elements of detoxification pathways provides a more efficient protective phenotype than do single components alone.

3T3 Cells↗
PubMed1996

Busulfan-glutathione conjugation catalyzed by human liver cytosolic glutathione S-transferases.

We have examined the catalytic activity of glutathione S-transferases (GST) in the conjugation of busulfan with glutathione (GSH) in human liver cytosol, purified human liver GST, and cDNA-expressed GST-alpha 1-1. Human liver microsomes and cytosol were incubated with 40 microM busulfan and 1 mM GSH. Cytosol catalyzed the formation of the GSH-busulfan tetrahydrothiophenium ion (THT+) in a concentration-dependent manner, whereas microsomes lacked activity. The total and spontaneous rates of THT+ formation increased with pH (pH range, 6.50-7.75), with the maximum difference at pH 7.4. Due to the limited aqueous solubility of busulfan, a K(m) for busulfan was not determined. The intrinsic clearance (Vmax/K(m)) of busulfan conjugation was 0.167 microliter/min/mg with 50-1200 microM busulfan and 1 mM GSH. GSH Vmax and K(m) for busulfan conjugation were 30.6 pmol/min/mg and 312 microM, respectively. Ethacrynic acid (0.03-15 microM) inhibited cytosolic busulfan-conjugating activity with 40 microM busulfan and 1 mM GSH. Enzyme-mediated THT+ formation was decreased 97% by 15 microM ethacrynic acid with no effect on the spontaneous reaction. In incubations with affinity-purified liver GST and GST-alpha 1-1, the intrinsic clearance for busulfan conjugation was 0.87 and 2.92 microliters/min/mg, respectively. Busulfan is a GST substrate with a high K(m) relative to concentrations achieved clinically (1-8 microM).

Antineoplastic Agents, Alkylating↗
PubMed1988

Differential induction of rat hepatic glutathione S-transferase isoenzymes by hexachlorobenzene and benzyl isothiocyanate. Comparison with induction by phenobarbital and 3-methylcholanthrene.

Male Wistar rats were treated with hexachlorobenzene, benzyl isothiocyanate, phenobarbital or 3-methylcholanthrene. Hepatic cytosolic glutathione S-transferase (GST) activity was determined with the substrates 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, ethacrynic acid and trans-4-phenyl-3-buten-2-one. Cytosolic glutathione peroxidase activity was measured with cumene hydroperoxide. GST activity toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene and ethacrynic acid was enhanced by all compounds, hexachlorobenzene and 3-methylcholanthrene causing the largest and the smallest increase respectively. Trans-4-phenyl-3-buten-2-one-conjugating activity exhibited only small changes, while peroxidase activity with cumeme hydroperoxide was not changed by any of the inducing agents. GST isoenzymes were purified on S-hexylglutathione Sepharose 6B and separated by means of FPLC-chromatofocusing, to evaluate effects on the GST isoenzyme pattern. Hexachlorobenzene and phenobarbital both caused an increase in the relative amounts of subunits 1 and 3 when compared with subunits 2 and 4 respectively. For 3-methylcholanthrene only induction of subunit 1 was observed, possibly due to the relatively low induction levels of total GST activity. In benzyl isothiocyanate-treated animals, an induction of subunit 3 was found as well as an increase in the relative amount of subunit 2. Thus, benzyl isothiocyanate behaves differently from hexachlorobenzene, phenobarbital and 3-methylcholanthrene as an inducing agent of rat hepatic glutathione S-transferases.

Animals↗
PubMed1997

Role of O6-methylguanine-DNA methyltransferase, glutathione transferase M3-3 and glutathione in resistance to carmustine in a human non-small cell lung cancer cell line.

O6-methylguanine-DNA methyltransferase (MGMT), glutathione transferase (GST) M3-3 and glutathione (GSH) have all been implicated in the resistance of cells to the cytostatic drug carmustine. U1810, a human non-small cell lung cancer cell line, expresses all of these putative resistance factors. The U1810 cells show a 4.4-fold lower sensitivity to carmustine compared with the U1690 cell line, a human small cell lung cancer cell line lacking detectable levels of both MGMT and GST M3-3. We investigated the effect of the MGMT inhibitor O6-benzylguanine, the GST inhibitor ethacrynic acid and the GSH synthesis inhibitor D,L-buthionine-S,R-sulfoximine (BSO) on the cytotoxicity of carmustine to U1810 cells. No potentiation to carmustine was observed after treatment with ethacrynic acid, while a 2-fold potentiation was found after exposure to O6-benzylguanine. Depletion of GSH with BSO showed a similar sensitising effect as that obtained with O6-benzylguanine. Thus, MGMT and GSH are the predominant resistance factors to carmustine in the U1810 cell line, whereas it is unclear whether GST M3-3 plays any role.

Antineoplastic Agents, Alkylating↗
PubMed1998

Antioxidant and detoxifying enzymes in the liver and kidney of pheasants after intoxication by herbicides MCPA and ANITEN I.

The activity of antioxidant and detoxifying enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase, glutathione-S-transferase (GST), the contents of thiobarbituric acid reactive substances, and the superoxide dismutase and glutathione-S-transferase isoenzyme patterns, were determined in the liver and kidney of pheasants after acute intoxication by herbicides MCPA and ANITEN I. In the liver, the activity of antioxidant enzymes was significantly decreased in the group given ANITEN I. New superoxide dismutase isoforms (pI 6.30, 6.85, 7.00) and higher intensity of isoform with pI 6.60 were observed after isoelectrofocusing in all experimental groups. In the kidney, the activity of superoxide dismutase was significantly decreased, and a higher intensity of superoxide dismutase isoforms (pI 6.00 and 6.60) was observed in all experimental groups. The contents of thiobarbituric acid reactive substances were significantly increased in the group with ANITEN I. The glutathione-S-transferase isoenzyme pattern was studied by using subunit-specific substrates and by Western blotting. The activity of glutathione-S-transferase with ethacrynic acid and cross-reactivity with rat subunit 7 was lower in all experimental groups in the kidney and liver, except in the liver of the group given a higher dose of ANITEN I. In this group, we have found a 2.10-fold higher activity to ethacrynic acid and a strong induction of subunit 7.

2-Methyl-4-chlorophenoxyacetic Acid↗
PubMed1992

Glutathione S-transferase pi in an arsenic-resistant Chinese hamster ovary cell line.

A glutathione S-transferase (GST) was purified from an arsenic-resistant Chinese hamster ovary cell line, SA7. The SA7 GST was shown to catalyse the conjugation of glutathione and ethacrynic acid, a specific substrate for Pi class GST. Its N-terminal amino-acid sequence has 80% identical residues to that of rat GST P and human GST pi. Thus, the GST purified from SA7 cells belongs to the Pi family. Treatment with Cibacron Blue or ethacrynic acid, which are GST inhibitors, significantly decreased the resistance of SA7 cells to sodium arsenite. On the other hand, pretreatment of SA7N cells, a partial revertant of SA7 cells, with sublethal doses of sodium arsenite, cadmium acetate or zinc sulphate resulted in re-elevation of GST activities and the cells regained the arsenic resistance. The regained arsenic resistance was well correlated with the levels of GST pi which were induced dose-dependently by zinc sulphate. Heat-shock treatment (45 degrees C for 10 min) did not increase GST pi expression or arsenic resistance of SA7N cells. The results indicate that GST pi is possibly involved in the mechanism of arsenic detoxification.

Amino Acid Sequence↗
PubMed1982

Evidence for the nonenzymatic and irreversible binding of cytembena to rat liver microsomes in vitro.

Characteristics of the irreversible binding of cytembena (CYT) to rat liver microsomal proteins have been investigated in vitro. Binding of [14C]CYT to rat liver microsomal proteins remained unchanged in the presence of SKF-525A or after heat denaturation and was not dependent upon the presence of pyridine nucleotide (NADPH or NADH). Ethacrynic acid, cysteine, dithiothreitol, and lysine were found to block the irreversible binding of [14C]CYT to microsomal proteins in a dose-dependent manner. The rank order of effectiveness as inhibitors of CYT binding was ethacrynic acid greater than cysteine greater than dithiothreitol greater than lysine. In other studies, CYT was shown to preferentially form adducts with cysteine rather than lysine or glycine. Using structural analogs and metabolites of CYT, it was found that 4-methoxybenzoylacrylic acid and beta-benzoylacrylic acid were effective competitors of [14C]CYT binding to liver microsomal proteins. By contrast, 4-methoxybenzoylpropionic acid, 5-(4-methoxyphenyl)dihydro-2(3H)furanone and 4-hydroxy-4-(4-methoxyphenyl)butyric acid were ineffective as inhibitors of the irreversible binding of CYT. These data suggest that sulfhydryl groups are involved in the nonenzymatic binding of CYT and that the presence of a carbon-carbon double bond in CYT, in debrominated metabolites or structural analogs, is requisite for an interference of the binding of CYT to microsomal proteins.

Acrylates↗
PubMed1976

[New aspects on the mode of action of cardiac glycosides].

A dissociation of the therapeutic from the toxic effects of cardiac glycosides has repeatedly been described. Whereas it is generally accepted that the toxic effects of cardiac glycosides are based on an inhibition of the Na+-K+-ATPase, the mechanism of action of therapeutic concentrations of cardiac glycosides still remains uncertain. To test the hypothesis, that cardiac glycosides might be transported into a distinct compartment of the myocardium with the Na+-K-ATPase acting as a carrier, the interaction of some inhibitors of this enzyme (digitoxin, dihydroouabain, cassaine, N-ethylmaleimide, p-hydroxy-mercuribenzoate, ethacrynic acid, spironolactone) with ouabain was studied at different levels of cardiac glycoside actions: Myocardial function, cardiac uptake and subcellular distribution and binding to the Na+-K+-ATPase. The following results were obtained: All cardioactive drugs (ethacrynic acid and spironolactone showed no such effects) reduced dose-dependently the inotropic action of ouabain and in high concentrations increased its toxicity. The same drugs inhibited dose-dependently the cardiac uptake of ouabain without affecting the subcellular distribution pattern of ouabain. The binding of ouabain to the Na+-K+-ATPase was influenced in a similar way by these drugs, showing a competitive type of interaction with digitoxin, dihydroouabain and cassaine and a non-competitive mechanism with N-ethylmaleimide and p-hydroxymercuribenzoate. These results support the concept of a cardiac glycoside-ATPase interaction as a basis for the therapeutic action of these drugs. This may be explained either by a direct influence of cardiac glycosides on the ATPase activity and/or by a carrier mediated cardiac glycoside-transport into a distinct compartment of the myocardial cell.

Action Potentials↗