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Volume-sensitive K(+)/Cl(-) cotransport in rabbit erythrocytes. Analysis of the rate-limiting activation and inactivation events.

Jennings ML - J. Gen. Physiol. (1999)

Bottom Line: The forward rate constant for activation has a very high temperature dependence (E(a) approximately 32 kCal/mol), but is not affected measurably by cell volume.The rate of transport inactivation increases steeply as cell volume decreases, even in a range of volumes where nearly all the transporters are inactive in the steady state.This finding indicates that the rate-limiting inactivation event is strongly affected by cell volume over the entire range of cell volumes studied, including normal cell volume.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA. jenningsmichaell@exchange.uams.edu

ABSTRACT
The kinetics of activation and inactivation of K(+)/Cl(-) cotransport (KCC) have been measured in rabbit red blood cells for the purpose of determining the individual rate constants for the rate-limiting activation and inactivation events. Four different interventions (cell swelling, N-ethylmaleimide [NEM], low intracellular pH, and low intracellular Mg(2+)) all activate KCC with a single exponential time course; the kinetics are consistent with the idea that there is a single rate-limiting event in the activation of transport by all four interventions. In contrast to LK sheep red cells, the KCC flux in Mg(2+)-depleted rabbit red cells is not affected by cell volume. KCC activation kinetics were examined in cells pretreated with NEM at 0 degrees C, washed, and then incubated at higher temperatures. The forward rate constant for activation has a very high temperature dependence (E(a) approximately 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kinetics were examined by swelling cells at 37 degrees C to activate KCC, and then resuspending at various osmolalities and temperatures to inactivate most of the transporters. The rate of transport inactivation increases steeply as cell volume decreases, even in a range of volumes where nearly all the transporters are inactive in the steady state. This finding indicates that the rate-limiting inactivation event is strongly affected by cell volume over the entire range of cell volumes studied, including normal cell volume. The rate-limiting inactivation event may be mediated by a protein kinase that is inhibited, either directly or indirectly, by cell swelling, low Mg(2+), acid pH, and NEM.

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(Top) Time course of total cellular Mg2+ in rabbit red cells incubated at 2% hematocrit in 155 mM NaCl, 5 mM KCl, 1 mM EDTA, 10 mM HEPES, pH 7.5, 25°C. At t = 0, ionophore A23187 was added to a final concentration of 10 μM, 0.2% ethanol. (Bottom) Time course of influx of 86Rb+ at 25°C in the following media (all containing 10−4 M ouabain, 10 mM HEPES, pH 7.5, 1 mM EDTA, 0.2% ethanol; mM): 155 NaCl, 5 KCl (•); 155 NaCl, 5 KCl, 10 μM A23187 (▴); 155 NaNO3, 5 KNO3, 10 μM A23187 (▪). The solid curve corresponds to , with a lag time of 81 min.
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Figure 4: (Top) Time course of total cellular Mg2+ in rabbit red cells incubated at 2% hematocrit in 155 mM NaCl, 5 mM KCl, 1 mM EDTA, 10 mM HEPES, pH 7.5, 25°C. At t = 0, ionophore A23187 was added to a final concentration of 10 μM, 0.2% ethanol. (Bottom) Time course of influx of 86Rb+ at 25°C in the following media (all containing 10−4 M ouabain, 10 mM HEPES, pH 7.5, 1 mM EDTA, 0.2% ethanol; mM): 155 NaCl, 5 KCl (•); 155 NaCl, 5 KCl, 10 μM A23187 (▴); 155 NaNO3, 5 KNO3, 10 μM A23187 (▪). The solid curve corresponds to , with a lag time of 81 min.

Mentions: In human and LK sheep red cells, depletion of Mg2+ at normal cell volume activates KCC (Brugnara and Tosteson 1987; Bergh et al. 1990; Delpire and Lauf 1991a; Dunham et al. 1993). The time course of activation after a step decrease in Mg2+ has not been reported. We found that Mg2+-depleted rabbit red cells do not tolerate incubation at 37°C for more than ∼30 min; accordingly, Mg2+ depletion experiments were performed at lower temperature, either 25° or 30°C. Fig. 4 (top) shows that it is possible to deplete rabbit red cells of Mg2+ in <5 min by addition of ionophore A23187 and EDTA, in agreement with the work of Flatman and Lew 1980 on human red cells. Sudden Mg2+ depletion causes activation of KCC with a time course (Fig. 4, bottom) similar to that observed after stimulation by cell swelling or low pH. The 86Rb+ flux stimulated by low Mg2+ is inhibited by replacement of Cl− with NO3− and is also inhibited by preincubation with okadaic acid (data not shown).


Volume-sensitive K(+)/Cl(-) cotransport in rabbit erythrocytes. Analysis of the rate-limiting activation and inactivation events.

Jennings ML - J. Gen. Physiol. (1999)

(Top) Time course of total cellular Mg2+ in rabbit red cells incubated at 2% hematocrit in 155 mM NaCl, 5 mM KCl, 1 mM EDTA, 10 mM HEPES, pH 7.5, 25°C. At t = 0, ionophore A23187 was added to a final concentration of 10 μM, 0.2% ethanol. (Bottom) Time course of influx of 86Rb+ at 25°C in the following media (all containing 10−4 M ouabain, 10 mM HEPES, pH 7.5, 1 mM EDTA, 0.2% ethanol; mM): 155 NaCl, 5 KCl (•); 155 NaCl, 5 KCl, 10 μM A23187 (▴); 155 NaNO3, 5 KNO3, 10 μM A23187 (▪). The solid curve corresponds to , with a lag time of 81 min.
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Related In: Results  -  Collection

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Figure 4: (Top) Time course of total cellular Mg2+ in rabbit red cells incubated at 2% hematocrit in 155 mM NaCl, 5 mM KCl, 1 mM EDTA, 10 mM HEPES, pH 7.5, 25°C. At t = 0, ionophore A23187 was added to a final concentration of 10 μM, 0.2% ethanol. (Bottom) Time course of influx of 86Rb+ at 25°C in the following media (all containing 10−4 M ouabain, 10 mM HEPES, pH 7.5, 1 mM EDTA, 0.2% ethanol; mM): 155 NaCl, 5 KCl (•); 155 NaCl, 5 KCl, 10 μM A23187 (▴); 155 NaNO3, 5 KNO3, 10 μM A23187 (▪). The solid curve corresponds to , with a lag time of 81 min.
Mentions: In human and LK sheep red cells, depletion of Mg2+ at normal cell volume activates KCC (Brugnara and Tosteson 1987; Bergh et al. 1990; Delpire and Lauf 1991a; Dunham et al. 1993). The time course of activation after a step decrease in Mg2+ has not been reported. We found that Mg2+-depleted rabbit red cells do not tolerate incubation at 37°C for more than ∼30 min; accordingly, Mg2+ depletion experiments were performed at lower temperature, either 25° or 30°C. Fig. 4 (top) shows that it is possible to deplete rabbit red cells of Mg2+ in <5 min by addition of ionophore A23187 and EDTA, in agreement with the work of Flatman and Lew 1980 on human red cells. Sudden Mg2+ depletion causes activation of KCC with a time course (Fig. 4, bottom) similar to that observed after stimulation by cell swelling or low pH. The 86Rb+ flux stimulated by low Mg2+ is inhibited by replacement of Cl− with NO3− and is also inhibited by preincubation with okadaic acid (data not shown).

Bottom Line: The forward rate constant for activation has a very high temperature dependence (E(a) approximately 32 kCal/mol), but is not affected measurably by cell volume.The rate of transport inactivation increases steeply as cell volume decreases, even in a range of volumes where nearly all the transporters are inactive in the steady state.This finding indicates that the rate-limiting inactivation event is strongly affected by cell volume over the entire range of cell volumes studied, including normal cell volume.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA. jenningsmichaell@exchange.uams.edu

ABSTRACT
The kinetics of activation and inactivation of K(+)/Cl(-) cotransport (KCC) have been measured in rabbit red blood cells for the purpose of determining the individual rate constants for the rate-limiting activation and inactivation events. Four different interventions (cell swelling, N-ethylmaleimide [NEM], low intracellular pH, and low intracellular Mg(2+)) all activate KCC with a single exponential time course; the kinetics are consistent with the idea that there is a single rate-limiting event in the activation of transport by all four interventions. In contrast to LK sheep red cells, the KCC flux in Mg(2+)-depleted rabbit red cells is not affected by cell volume. KCC activation kinetics were examined in cells pretreated with NEM at 0 degrees C, washed, and then incubated at higher temperatures. The forward rate constant for activation has a very high temperature dependence (E(a) approximately 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kinetics were examined by swelling cells at 37 degrees C to activate KCC, and then resuspending at various osmolalities and temperatures to inactivate most of the transporters. The rate of transport inactivation increases steeply as cell volume decreases, even in a range of volumes where nearly all the transporters are inactive in the steady state. This finding indicates that the rate-limiting inactivation event is strongly affected by cell volume over the entire range of cell volumes studied, including normal cell volume. The rate-limiting inactivation event may be mediated by a protein kinase that is inhibited, either directly or indirectly, by cell swelling, low Mg(2+), acid pH, and NEM.

Show MeSH
Related in: MedlinePlus