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B Sarkadi

Publications and source records attributed to B Sarkadi.

107 records · Page 6Linked to original sources
PubMed1978

Lithium transport pathways in human red blood cells.

In human red cells, Li is extruded against its own concentration gradient if the external medium contains Na as a dominant cation. This uphill net Li extrusion occurs in the presence of external Na but not K, Rb, Cs, choline, Mg, or Ca, is ouabain-insensitive, inhibited by phloretin, and does not require the presence of cellular ATP. Li influx into human red cells has a ouabain-sensitive and a ouabain-insensitive but phloretin-sensitive component. Ouabain-sensitive Li influx is competitively inhibited by external K and Na and probably involves the site on which the Na-K pump normally transports K into red cells. Ouabain does not inhibit Li efflux from red cells containing Li concentrations below 10 mM in the presence of high internal Na or K, whereas a ouabain-sensitive Li efflux can be measured in cells loaded to contain 140 mM Li in the presence of little or no internal Na or K. Ouabain-insensitive Li efflux is stimulated by external Na and not by K, Rb, Cs, choline, Mg, or Ca ions. Na-dependent Li efflux does not require the presence of cellular ATP and is inhibited by phloretin, furosemide, quinine, and quinidine. Experiments carried out in cells loaded in the presence of nystatin to contain either only K or only Na show that the ouabain-insensitive, phloretin-inhibited Li movements into or out of human red cells are stimulated by Na on the trans side and inhibited by Na on the cis side of the red cell membrane. The characteristics of the Na-dependent unidirectional Li fluxes and uphill Li extrusion are similar, suggesting that they are mediated by the same Na-Li countertransport system.

Adenosine Triphosphate↗
PubMed1978

Kinetics and stoichiometry of Na-dependent Li transport in human red blood cells.

This paper describes the kinetics and stoichiometry of a tightly coupled Na-Li exchange transport system in human red cells. The system is inhibited by phloretin and furosemide but not by ouabain. Li influx by this system increases and saturates with increasing concentrations of external Li and internal Na and is inhibited competitively by external Na. Comparable functions relate Li efflux and Na efflux to internal and external Li and Na concentrations. Analysis of these relations yields the following values for the ion concentrations required to half-maximally activate the transport system: internal Na and Li 9.0 and 0.5 mM, respectively, external Na and Li 25 and 1.5 mM, respectively. The system performs a 1:1 exchange of Na and Li moving in opposite directions across the red cell membrane. We found no evidence for a simultaneous transport of more than one Na and Li by the system. The maximum transport rate of Na-dependent Li transport varied between 0.1 and 0.37 mmol/(liter of cells X h) in the red cells of the five normal male subjects studied. No significant variations between individual subjects were observed for bicarbonate-stimulated Li transport and for the residual Li fluxes which occur in the absence of bicarbonate and in the presence of ouabain plus phloretin.

Biological Transport, Active↗
PubMed1977

Transport parameters and stoichiometry of active calcium ion extrusion in intact human red cells.

Ca2+-transport and its energy consumption were studied in intact human red cells loaded with Ca2+ by the aid of the ionophore A23187. After the complete elimination of the ionophore the passive Ca2+-permeability of the membrane returned to its normal low value, except when the intracellular Ca2+-concentration was higher than 3 mM or the ATP level fell below 100 muM. Within these limits the rate of Ca2+-extrusion was independent of the cellular ATP content but was greatly enhanced by increasing [Ca2+]i and reached a plateau at about 1 mM intracellular Ca2+-concentration. The maximum rate of Ca2+-efflux was about 85 mumol/l of cells per min at 37 degrees C, pH 7.4. The activation energy of active Ca2+-extrusion was found to be 15 200 cal/mol, and the optimum pH in the suspension was 7.7. Ca2+-efflux was not connected with the counter-transport of cations. The Ca2+-pump was not affected by ouabain or oligomycin and only partial inhibition could be achieved by the SH-reagents: ethacrynic acid, N-ethylmaleimide and p-chloromercuribenzoate or with propranolol and ruthenium red. An 80 to 95% inhibition of the active Ca2+-extrusion was brought about by 50-250 muM lanthanum, which in the above concentrations caused no aggregation or haemolysis. The inhibition of the Ca2+-pump by lanthanum was found to be reversible, the site of inhibition being at the external surface of the cell membrane. To examine the energy consumption of the Ca2+-extrusion, ATPase activity was assessed by measuring inorganic phosphate liberation in Ca2+-loaded red cells the metabolism of which was inhibited by iodoacetamide + Na+-tetrathionate. Ca2+-activated ATPase activity connected with the Ca2+-pump was distinguished from other Ca2+-ATPases by using the non-penetrating inhibitor, lanthanum. The molar ratio of Ca2+-transported per ATP split was found to be 2 : 1.

Adenine Nucleotides↗
PubMed1977

Kinetic studies on the calcium-dependent potassium transport in human red blood cells.

The kinetics of the Ca-dependent K transport across the human red cell membrane were studied under experimental conditions allowing the separation of this step from the previous membrane events. Net potassium and tracer potassium fluxes were measured and a self-consistent model was constructed for calculating the ionic permeability values. All the experimental results could be interpreted by means of a nonsaturable neutral K selective channel. In net K efflux measurements the accompanying C1 movement was found to be rate-limiting, and the charge-carrying C1 permeability of the red cell membrane could be calculated.

Biological Transport↗
PubMed1976

The use of ionophores of rapid loading of human red cells with radioactive cations for cation-pump studies.

Techniques are described for the rapid loading of intact human red cells with radioactive isotopes of alkali cations or Ca2+ by using ionophorous compounds (nigericin, gramicidin D and A 23187). Loading was rapid and efficient if the membrane potential of the cells was rendered more negative inside. After cation loading the ionophores could be bound to albumin and removed by repeated washings. The ATP and 2,3-DPG contents of the cells were practically unaltered by this treatment. Passive membrane permeability to Na+ and Ca2+ returned to normal. Loaded erythrocytes pumped out Na+ in a ouabain-sensitive and Ca2+ in a lanthanum-sensitive way. Ca2+ -loaded red cells were microspherocytes and exhibited a rapid K+ -efflux. Parallel with the extrusion of Ca2+ cells regained their biconcave shape and normal passive permeability to K+.

Adenosine Triphosphate↗
PubMedDate not supplied

Functional studies on the MRP1 multidrug transporter: characterization of ABC-signature mutant variants.

BACKGROUND: MRP1 is a key multidrug resistance ATP-binding Cassette (ABC) transporter in tumor cells. A functionally important signature motif is conserved within all ABC domains. Our current studies aimed to elucidate the role of these motifs in the cooperation of MRP1 ABC domains. MATERIALS AND METHODS: We designed human MRP1 mutants based on a bacterial ABC structure. Conserved leucines (Leu) were replaced by arginines (Arg), while glycines (Gly) were substituted for aspartic acids (Asp). The activity of these mutants was assayed by measuring ATPase activity and vesicular transport. ATP-binding and transition-state formation were studied by a photoreactive ATP analog. RESULTS: The Leu to Arg mutants retained both ATPase and transport activity, while the Gly to Asp mutants were inactive in all functional assays, while showing normal ATP-binding. CONCLUSION: Our results reinforce the notion that a single mutation in one of the ABC-signature regions affects the function of the whole protein. The relative role of the conservative leucines and glycines in MRP1 indicates a similar three-dimensional structure within the catalytic center of various ABC proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Parallel functional and immunological detection of human multidrug resistance proteins, P-glycoprotein and MRP1.

The proper assessment of the expression and drug extrusion activity of multidrug resistance proteins in various tumor cells is a challenging clinical laboratory problem. Recently, we have introduced a fluorescent dye (calcein) accumulation assay for the estimation of the functional expression of both P-glycoprotein (MDR1) and the multidrug resistance-associated protein (MRP1). Since both MDR1 and MRP1 decrease the intracellular accumulation of the fluorescent free calcein, by applying appropriate inhibitors of MDR1 and MRP1, the transport activity of these proteins could be quantitatively and selectively estimated in fluorometry or flow-cytometry assays. In the present work single-cell fluorescence digital imaging has been applied to characterize the kinetics and inhibitor-sensitivity of calcein accumulation in a mixture of HL60 MRP1 and NIH 3T3 MDR1 cells. Subsequent immunofluorescence labeling was performed by the anti-MDR1 monoclonal antibody (mAb) UIC2 in the same cell population. We report that the double labeling approach, based on the single cell calcein accumulation assay and an immunofluorescence detection, provides good sensitivity and selectivity for the simultaneous functional and immunological detection of cellular MDR1 and MRP1.

3T3 Cells↗