Effects of cholecalciferol on the translocation of calcium by non-everted chick ileum in vitro.
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An apparatus is described that allows perfusion of a non-everted segment of intestine in vitro and the study of the accumulation of substances within the mucosal cells. The translocation of Ca(2+) by rachitic-chick ileum and the effect of pretreatment with cholecalciferol was investigated, with the following conclusions. (1) Entry of Ca(2+) across the microvilli into mucosal cells is by diffusion; it does not require metabolic energy or the presence of any other inorganic ions. (2) Pretreatment of the chick with cholecalciferol causes increased permeability of the microvillus to Ca(2+) in both directions (lumen to cell, cell to lumen). The increased transport brought about by cholecalciferol in vivo can be partially mimicked by sodium dodecyl sulphate added in vitro. (3) The sign and the magnitude of the electrical potential difference prevailing across the ileum does not influence Ca(2+) transport. (4) Exit of Ca(2+) from the mucosal cell is temperature-sensitive, requires metabolic energy and Na(+). (5) Pretreatment with cholecalciferol caused increased movement of Ca(2+) out of the cell across the basement membranes. This effect of cholecalciferol given in vivo could be markedly increased by the presence of dicyclohexylcarbodi-imide in the perfusion fluid. These observations suggested that cholecalciferol increased Ca(2+) entry (and exit) at the mucosal surface and also caused Ca(2+) to be more available to the pump at the serosal surface. (+info)
Non-sensory cells in the deafened organ of Corti: approaches for repair.
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After moderate cochlear trauma, hair cells degenerate and their places are taken by phalangeal scars formed by differentiated supporting cells. A short time after trauma, these supporting cells can respond to an induced expression of genes which signal hair cell differentiation during normal development and transdifferentiate into new hair cells. However, these non-sensory cells often lose their differentiated features after severe insults or prolonged hearing loss and become a simple, flat epithelium. The flat organ of Corti can serve as a substrate for gene- and stem cell-based therapies. Despite its prevalence, the flat epithelium is not well characterized. Recent data show that cells of the flat epithelium can divide and maintain the structural confluence of the membranous labyrinth. The mitotic potential of these cells should facilitate production of cells for therapies based on recapitulation of development or insertion of stem cells. (+info)
Mode of stimulation by adenosine 3':5'-cyclic monophosphate of the sodium efflux in barnacle muscle fibres.
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1. Giant fibres of the barnacle Balanus nubilus have been used as a preparation for studying the mode of action of cAMP on sodium transport. 2. It is shown that a concentration of cAMP as low as 10(-6)M, when micro-injected, causes a sharp rise in the radio-Na efflux. Ouabain fails to reverse the cAMP effect. 3. The magnitude of the response of the Na efflux to cAMP is markedly reduced by pre-injecting 100 or 500 mM-EGTA solutions or by omitting Ca2+ from the bathing medium. Both together fail to bring about a greater reduction in the response. 4. The response to cAMP is greatly reduced by pre-injecting the protein inhibitor of Walsh and practically abolished by pre-injecting 500 mM-EGTA and soaking in Ca-free artificial sea water, ASW. 5. The Ca2+-independent component of the Na efflux which is also stimulated by cAMP is shown to involve Na for H exchange. The magnitude of this exchange is governed by external pH. 6. The Na efflux into Ca2+-free, Li+-ASW is shown to be markedly stimulated by injecting cAMP, an effect which is enhanced by reducing external pH. 7. The Na efflux at 0 degrees C is stimulated by injecting cAMP. This is shown to be related to activation of the protein kinase by cAMP and to depend on the presence of external Ca2+. 8 (i) Ethacrynic acid when injected reduces the ouabain-insensitive Na efflux into HEPES-Ca2+-free ASW at pH 6-3. These same fibres show a marked response to cAMP. (II) The ouabain-insensitive Na efflux into HCO3-, Ca2+-free ASW from fibres pre-treated with ethacrynic acid fails to respond to external acidification. This is interpreted as indicating that ethacrynic acid inactivates the CO2-sensitive adenyl cyclase system. These same fibres when injected with cAMP show a marked response. (iii) Stimulation of the ouabain-insensitive Na efflux into HCO-3, Ca2+-free ASW by external acidification is reversed by injecting ethacrynic acid. These fibres when injected with cAMP show a reduced response. 9. It is concluded that: (i) stimulation of the Na efflux by injected cAMP is mainly due to activation of cAMP-dependent protein kinase; (ii) the underlying exchange mechanism consists of Na:Ca and Na:H exchange. Interaction of Ca2+ with a phosphorylated membrane, thereby modifying permeability remains as a real possibility; (iii) the site of action of CO2 and ethacrynic acid is the adenyl cyclase system. 10. The implications of activation of the adenyl cyclase system by CO2 and Na:H exchange are briefly touched upon. (+info)
Ethacrynic acid-sensitive and ATP-dependent Cl- transport in the rat kidney.
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Ouabain- and furosemide-insensitive and ATP-dependent Cl- uptake was demonstrated in rat renal membrane vesicles. Such a Cl- uptake activity was prominent in cortical plasma membrane fractions with high activities of Cl(-)-ATPase and Na+, K(+)-ATPase. The membrane vesicles accumulated Cl- in an osmotically reactive manner with the following sequence of nucleotide specificity: ATP greater than ITP greater than UTP greater than GTP greater than CTP., beta, gamma-Methylene ATP, ADP and AMP had no effect. ATP-dependent Cl- uptake was markedly inhibited by a Cl(-)-ATPase inhibitor, ethacrynic acid (0.3 mM), but not affected by an H(+)-ATPase inhibitor, N,N'-dicyclohexylcarbodiimide (0.1 mM). These findings suggest that an ethacrynic acid-sensitive and ATP-driven (+info)
Characterization of the binding of 8-anilinonaphthalene sulfonate to rat class Mu GST M1-1.
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Phase I study of thiotepa in combination with the glutathione transferase inhibitor ethacrynic acid.
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The glutathione transferases comprise a family of isoenzymes, one or more of which are involved in the conjugation of alkylating agents to glutathione (GSH). Increased GSH transferase activity has been shown to underlie acquired resistance to several alkylating agents. Ethacrynic acid inhibits the isoenzymes of GSH transferase with 50% inhibitory concentration values ranging from 0.3 to 6.0 microM and has been shown to restore sensitivity to alkylating agents in drug-resistant animal tumor models. We entered 27 previously treated patients with advanced cancer on a study of ethacrynic acid (25 to 75 mg/m2 p.o. every 6 h for 3 doses) and thiotepa (30 to 55 mg/m2 i.v. 1 h after the second dose of ethacrynic acid). The major toxicity of ethacrynic acid was diuresis, which was observed at every dose level; in addition, severe metabolic abnormalities occurred at 75 mg/m2. At 50 mg/m2, the diuretic effects were manageable. Myelosuppression was the most important effect of the combination. Two of seven courses of ethacrynic acid, 50 mg/m2, and thiotepa, 55 mg/m2, were associated with grade 3 or 4 neutropenia and/or thrombocytopenia. Nausea/vomiting greater than or equal to grade 2 was observed in 16% of courses. GSH transferase activity was assayed spectrophotometrically in the peripheral mononuclear cells of all patients. At each dose level, activity decreased following ethacrynic acid administration, with recovery by 6 h. Administration of ethacrynic acid, 50 mg/m2, resulted in a mean nadir of transferase activity of 37% of control. The pharmacokinetics of thiotepa and its principal metabolite TEPA were studied in 23 patients. The plasma disappearance of thiotepa fit a two-compartment open model with a terminal half-life of approximately 2 h. Plasma TEPA levels peaked at a mean of 2.16 h following thiotepa administration. The harmonic mean terminal half-life of TEPA was 10.4 h, and the TEPA area under the curve (AUC) did not increase with increasing thiotepa dose. The AUC of thiotepa was approximately twice, and the clearance about one-half, of the values obtained in a previous study of single agent thiotepa. The AUC of TEPA was lower than that previously observed. The data suggest that ethacrynic acid inhibits enzymes involved in the metabolic disposition of thiotepa, including its oxidative desulfuration to TEPA. The severity of the platelet toxicity was correlated with the AUC of thiotepa, but not with that of TEPA. This combination of thiotepa and ethacrynic acid will be tested further in Phase II trials. (+info)
Glutathione participates in the regulation of mitophagy in yeast.
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