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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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Effect of successive exposures of rabbit red cells (not density separated) to 2 mM NEM at 0°C. Cells were washed in and suspended at 5% hematocrit in HEPES-buffered physiological saline. Suspensions were chilled to <2°C and NEM was added from a freshly prepared 1 M stock solution in dimethylformamide to a final concentration of 2 mM. After 15 min on ice, cells were washed in cold HPS, and the treatment with NEM was repeated once or twice as indicated. Cells were finally washed twice with HPS, suspended in isosmotic flux medium, and incubated 20 min at 37°C before adding 86Rb+; influx was measured for 20 min at 37°C. Data from two different cell preparations are shown (mean ± range).
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Figure 6: Effect of successive exposures of rabbit red cells (not density separated) to 2 mM NEM at 0°C. Cells were washed in and suspended at 5% hematocrit in HEPES-buffered physiological saline. Suspensions were chilled to <2°C and NEM was added from a freshly prepared 1 M stock solution in dimethylformamide to a final concentration of 2 mM. After 15 min on ice, cells were washed in cold HPS, and the treatment with NEM was repeated once or twice as indicated. Cells were finally washed twice with HPS, suspended in isosmotic flux medium, and incubated 20 min at 37°C before adding 86Rb+; influx was measured for 20 min at 37°C. Data from two different cell preparations are shown (mean ± range).

Mentions: For experiments involving transport activation by swelling, low pH, or Mg2+ depletion, cells were separated on Percoll-Renograffin as previously described (Al-Rohil and Jennings 1989) to select the least dense one third of cells. The lower-density fractions are enriched in younger cells, which have a higher volume-dependent KCC activity (Brugnara and Tosteson 1987; Canessa et al. 1987; Al-Rohil and Jennings 1989). The NEM activation experiments in Fig. 6Fig. 7Fig. 8Fig. 9 used unseparated red cells; comparable experiments with density-separated cells gave indistinguishable results. If blood had been stored more than a few hours, cells were washed three times and incubated 60–90 min at 37°C in HEPES-buffered physiological saline (HPS: 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na-phosphate, 10 mM HEPES, pH 7.4) plus 10 mM glucose to try to establish a reproducible steady state.


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

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

Effect of successive exposures of rabbit red cells (not density separated) to 2 mM NEM at 0°C. Cells were washed in and suspended at 5% hematocrit in HEPES-buffered physiological saline. Suspensions were chilled to <2°C and NEM was added from a freshly prepared 1 M stock solution in dimethylformamide to a final concentration of 2 mM. After 15 min on ice, cells were washed in cold HPS, and the treatment with NEM was repeated once or twice as indicated. Cells were finally washed twice with HPS, suspended in isosmotic flux medium, and incubated 20 min at 37°C before adding 86Rb+; influx was measured for 20 min at 37°C. Data from two different cell preparations are shown (mean ± range).
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Related In: Results  -  Collection

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Figure 6: Effect of successive exposures of rabbit red cells (not density separated) to 2 mM NEM at 0°C. Cells were washed in and suspended at 5% hematocrit in HEPES-buffered physiological saline. Suspensions were chilled to <2°C and NEM was added from a freshly prepared 1 M stock solution in dimethylformamide to a final concentration of 2 mM. After 15 min on ice, cells were washed in cold HPS, and the treatment with NEM was repeated once or twice as indicated. Cells were finally washed twice with HPS, suspended in isosmotic flux medium, and incubated 20 min at 37°C before adding 86Rb+; influx was measured for 20 min at 37°C. Data from two different cell preparations are shown (mean ± range).
Mentions: For experiments involving transport activation by swelling, low pH, or Mg2+ depletion, cells were separated on Percoll-Renograffin as previously described (Al-Rohil and Jennings 1989) to select the least dense one third of cells. The lower-density fractions are enriched in younger cells, which have a higher volume-dependent KCC activity (Brugnara and Tosteson 1987; Canessa et al. 1987; Al-Rohil and Jennings 1989). The NEM activation experiments in Fig. 6Fig. 7Fig. 8Fig. 9 used unseparated red cells; comparable experiments with density-separated cells gave indistinguishable results. If blood had been stored more than a few hours, cells were washed three times and incubated 60–90 min at 37°C in HEPES-buffered physiological saline (HPS: 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na-phosphate, 10 mM HEPES, pH 7.4) plus 10 mM glucose to try to establish a reproducible steady state.

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