Correspondence to: Michael L. Jennings, Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 W. Markham St., Mail Slot 505, Little Rock, AR 72205. Fax:501-686-8167 E-mail:jenningsmichaell{at}exchange.uams.edu.
Released online: 15 November 1999
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Abstract |
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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 Mg2+) 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 Mg2+-depleted rabbit red cells is not affected by cell volume. KCC activation kinetics were examined in cells pretreated with NEM at 0°C, washed, and then incubated at higher temperatures. The forward rate constant for activation has a very high temperature dependence (Ea ~ 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kinetics were examined by swelling cells at 37°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 Mg2+, acid pH, and NEM.
Key Words: osmoregulation, red blood cell, phosphatase, kinase, kinetics
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INTRODUCTION |
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A fundamental property of cells is the capacity to regulate cell volume. The water permeability of most cell membranes is sufficiently high that cell volume is determined by the total amount of intracellular solute. Regulation of intracellular solute takes place by way of negative feedback mechanisms in which the synthesis, degradation, influx, or efflux of various solutes is increased or decreased to correct perturbations in cell volume. Mechanisms for increasing total cell solute include regulated osmolyte synthesis (
The vertebrate red blood cell is a simple system in which to investigate mechanisms of cell volume regulation. The mechanisms of volume regulation in red cells are limited to post-translational events, and some aspects of osmoregulation (e.g., gene regulation; see
To understand the mechanisms by which cell volume affects transport, it will be important to identify, in kinetic and biochemical terms, the sequence of events associated with activation and inactivation of transport. An early attempt to analyze the kinetics of activation and inactivation of KCC was based on a simple two-state model (
The two-state model for KCC regulation is undoubtedly an oversimplification. Even if the transporter does exist in only two main functional states, the rate constants for activation and/or inactivation themselves are very likely regulated by forward and reverse rate processes (e.g.,
The purpose of the present work is to obtain quantitative estimates of the rate constants for the rate-limiting activation and inactivation events that regulate KCC. Rabbit red cells were chosen as an experimental system because they exhibit a relatively large, volume-dependent KCC flux (
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MATERIALS AND METHODS |
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Materials
Blood was obtained from healthy New Zealand white rabbits by either venipuncture or cardiac puncture, the latter in animals that were being killed for the purposes of obtaining other tissues for study in other laboratories. All animal procedures were in compliance with American Physiological Society guidelines. Some of the experiments were carried out using rabbit blood purchased from Pel-Freez; results obtained with blood from Pel-Freez were indistinguishable from those with blood from laboratory animals. Most experiments were performed using blood that had been stored <3 d at 4°C. Okadaic acid and ionophore A23187 were purchased from Calbiochem Corp. 86Rb+ was purchased as RbCl from DuPont NEN. All salts and buffers were purchased from Sigma Chemical Co. or Fisher Chemicals.
Cell Preparation
For experiments involving transport activation by swelling, low pH, or Mg2+ depletion, cells were separated on Percoll-Renograffin as previously described (
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NEM Treatment
Cells were washed in HPS, suspended at 5% hematocrit in HPS, and chilled until the temperature of the suspension was <2°C. NEM was then added from a freshly prepared 1-M stock solution in dimethylformamide to a final concentration of 2 mM. Control suspension received dimethylformamide at the same final concentration (0.2%). The suspensions were incubated 15 min on ice, washed once, and resuspended in HPS on ice. For some aliquots of cells, the NEM treatment was repeated once or twice more. After all NEM treatments were complete, the cells were washed twice in ice-cold HPS before further incubations. In some experiments, 0.1% ß-mercaptoethanol was included to remove all traces of remaining NEM; results were identical whether or not ß-mercaptoethanol was included in the wash medium.
Influx Measurements
Cells were suspended at 2% hematocrit in "standard flux medium" (155 mM NaCl, 5 mM KCl, 10 mM HEPES hemisodium, pH 7.5 at 25°C, 10-4 M ouabain). Depending on the design of the experiment, 86Rb+ (0.51 µCi/ml) was present at the outset or was added after incubating the cells in flux medium for various intervals (see figure legends). At timed intervals after exposure of cells to 86Rb+, the intracellular radioactivity was determined as described previously (
Activation by Low pH
The time course of activation of KCC by low pH was determined by adding MOPS from a 1-M stock solution to a final concentration of 1518 mM. Within a short time after extracellular acidification, the intracellular pH reaches Donnan equilibrium with the extracellular pH (e.g.,
Determination of Intracellular Mg2+
Intracellular Mg2+ was estimated colorimetrically in cells that were prepared as in the flux experiments. Cells were incubated at 2% hematocrit at 25°C in 160 mM NaCl, 10 mM HEPES, pH 7.45, 1 mM EDTA. Two aliquots (1 ml) were removed before addition of ionophore A23187 and mixed with 10 ml of cold 160 mM KCl/10 mM HEPES, centrifuged 2 min at 4,000 rpm, and the supernatants were removed. A23187 (1020 µM final) was then added, and further aliquots were removed, centrifuged, and supernatants removed. The cell pellets were lysed in 0.5 ml water, heated for 2 min in boiling water, and allowed to cool. The tubes were then centrifuged, and 0.1 ml of the supernatant was mixed with 0.9 ml of Mg2+ color reagent containing calmagite (1-[1-hydroxy-4-methyl-2-phenylazo]-2-naphthol-4-sulfonic acid; Sigma Diagnostics). Absorbances were compared with those of standards, and the results were expressed as micromoles Mg2+ per milliliter cells.
Calculation of Activation and Inactivation Delay Times
In all these experiments, the main measured parameter is the lag time for the transition from one steady state to another. The rate of this transition (inverse lag time) was calculated as follows. The time course of accumulation of intracellular 86Rb+ [*K+] after a step change in conditions at t = 0 is given by the following expression (
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(1) |
where J0 is the initial flux (micromoles per milliliter cells minute-1), J1 is the flux in the new steady state, is the lag time for establishing the new steady state, and [*K+]int = 0 is the amount of intracellular tracer at the time of the step change in conditions. In most experiments, the change in conditions is at the time of first exposure of cells to 86Rb+; in these cases, [*K+]int = 0 represents the small amount of tracer that is not removed by the washing procedure. For the activation experiments in this paper, J0 and [*K+]int = 0 were determined directly in a parallel suspension in isosmotic medium at physiological pH.
The influx data were fit (Sigma Plot; Jandel Scientific) to Equation 1, with two adjustable parameters, and J1, for most experiments. In the NEM activation experiments, the steady state flux J1 was determined independently in a parallel suspension that had been incubated 1520 min at 37°C to allow KCC to activate. In these experiments, the only adjustable parameter in the curve fits was the lag time
, which can be estimated accurately when all the other parameters are determined independently. In the inactivation experiments (Figure 10 Figure 11 Figure 12), the initial flux was estimated in swollen cells, and the lag time was determined in a two-parameter fit (
and J1). The estimate of
is reasonably accurate in an inactivation experiment (despite the two-parameter fit) because the final steady state flux is small.
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RESULTS |
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Single Exponential Time Course for Approach to the Steady State
The purpose of these experiments is to obtain quantitative estimates of the rate constants for the rate-limiting activation and inactivation events in the regulation of rabbit red cell KCC. Experimentally, under a wide variety of conditions, the time course of activation by cell swelling or NEM is not distinguishable from a single exponential in red cells from rabbit (
Activation by Low pH
It is known that low intracellular pH (e.g., pH 6.9) activates KCC in human and LK sheep red cells (
Figure 3 shows the time course of acid activation at 25°C. For comparison, the flux was also activated by hypotonic cell swelling in the same preparation of cells. The flux activates much more slowly at 25° than at 37°C for both modes of activation. The lag time at 25°C is 6080 min for activation by swelling and low pH. This lag time is in the same range as that measured previously (average 55 min) for swelling-activated KCC at 25°C (
Activation by Low Mg2+
In human and LK sheep red cells, depletion of Mg2+ at normal cell volume activates KCC (
Volume Dependence of Transport in Low Mg2+ Cells
Figure 5 shows that, in cells that have been depleted of Mg2+, there is only a very minor effect of cell volume on the KCC flux. A slight volume dependence of the flux in Mg2+-depleted cells is observed even in NO3- medium. Varying the osmolality from 410 to 185 mosmol/kg (2.45.4 [osmol/kg]-1) caused the Cl--dependent flux to change by <20%. The same results were observed in two other experiments. In one earlier experiment, there appeared to be a decrease in the flux in very hypertonic solutions (>400 mosmol/kg), but in the range between 200 and 400 mosmol/kg there is essentially no effect of volume on KCC in Mg2+-depleted rabbit red cells. In contrast, KCC is still volume sensitive in LK sheep red cells, though less so than in cells with normal Mg2+ (
Measurement of One-Way Activation after NEM Pretreatment
The above experiments (Figure 1 Figure 2 Figure 3 Figure 4) indicate that the time course of activation of KCC is similar after step changes in cell volume, pH, or Mg2+, suggesting that the same event is rate limiting for all three modes of activation, and it is of interest to try to measure the rate constant for this event. The measured lag time for transport activation in general depends not only on the rate-limiting activation event, but also on the rate constant for inactivation. The most direct way to estimate the rate constant for activation is to devise conditions in which the rate of inactivation is negligible. Under these conditions, the measured rate of activation is very nearly equal to the rate constant for the rate-limiting step in the activation process.
One approach to measuring activation kinetics under conditions of maximal activation would be to measure transport at very high cell volume. However, extreme cell swelling may cause prelytic leaks or other abnormalities. Instead of trying to activate transport maximally by cell swelling, we used NEM, which has long been known to activate KCC (see
To characterize the kinetics of activation by NEM more precisely, cells were incubated with 2 mM NEM for 15 min at 0°C, and residual NEM was removed by washing at 0°C. Transport was then activated by incubating for 1520 min at 37°C. Figure 6 shows that activation by NEM is nearly maximal after two NEM treatments (2 mM; 15 min) at 0°C. Further treatments with NEM at low temperature do not cause significant further activation or inhibition. Therefore, as originally shown by
The fluxes shown in Figure 6 were measured by adding 86Rb+ after incubating NEM-pretreated cells for 1520 min at 37°C. If pretreated cells were exposed to 86Rb+ without preincubation at 37°C, the influx was initially small but increased with a single exponential time course to the same steady state level as preincubated cells (Figure 7). The lag time for KCC activation at 37°C was estimated in five separate preparations of cells that had been treated twice with 2 mM NEM on ice. The lag time was 9.2 ± 1.4 (SD) min. The lag time was also measured in five preparations of cells that had been treated once with 2 mM NEM. The lag time in this case was slightly shorter (7.6 ± 0.6 min), as expected if the flux is not quite maximally activated and the reverse rate constant is not completely inhibited. From these experiments, we conclude that the rate constant for the rate-limiting forward step in transport activation in NEM-treated cells is ~0.11/min at 37°C.
Activation Rate Constant Is Very Dependent on Temperature
The temperature dependence of the activation rate was measured by treating with NEM at 0°C, and then measuring the time course of 86Rb+ influx at 25°C. The flux in the fully activated state at 25°C was measured by pretreating cells with NEM at 0°C, incubating 1520 min at 37°C to activate >90% of the transporters (Figure 6), and then shifting the temperature back to 25°C for the flux measurement. Figure 8 shows that the lag time for activation at 25°C is much longer than at 37°C. In four experiments (with either one or two pretreatments with NEM), the activation lag time was 75 ± 13 (SD) min, which is a factor of about eight longer than at 37°C. The apparent activation energy of the rate-limiting activation process is ~32 kCal/mol in this temperature range.
Lack of Effect of Cell Volume on the Activation Rate Constant
Figure 9 shows that the activation rate constant is not detectably dependent on cell volume in NEM-pretreated cells. Cells were preincubated with NEM at 0°C, and the activation rate was measured as in Figure 7 in isotonic and hypotonic media. The activation rate in swollen cells is indistinguishable from that in cells of normal volume. In agreement with earlier data obtained under different conditions of NEM treatment (
Kinetics of KCC Inactivation
The above experiments indicate that the rate-limiting kinetic step in transport activation is not dependent on cell volume, in agreement with our previous proposal that the main volume-dependent process is the inactivation step (
The results of the experiment in Figure 10 plus two additional experiments are summarized in Figure 11. A single experiment with fluxes at 37° instead of 25°C is shown in Figure 12. Again, the rate of inactivation continues to increase as cell volume decreases, in a volume range where the steady state KCC flux is very small. Therefore, the rate of inactivation does not reach a limiting value as cell volume decreases. This finding is evidence that the major volume-dependent step in transport regulation is the rate-limiting inactivation event (see below).
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DISCUSSION |
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The experiments described above have provided the most quantitative information to date on the rate-limiting events in activation and inactivation of a volume-regulatory transporter. The rates were measured under conditions where, in the final steady state, KCC is either fully activated by NEM (Figure 6 Figure 7 Figure 8 Figure 9) or nearly fully inactivated by shrinkage (Figure 10 Figure 11 Figure 12). Kinetic analysis is much simpler under these conditions than it is when an intermediate (and unknown) percentage of transporters are in the activated state. The data will be discussed below in terms of specific current models for activation and inactivation of KCC. However, it is useful first to summarize the main experimental findings without reference to particular models. (a) Activation of KCC by sudden acidification, depletion of Mg2+, cell swelling, or NEM takes place with a time course that is consistent with a single rate-limiting activation event. (b) In contrast to LK sheep red cells (
Interdependence of Volume and pH Signals
The acidification experiments in Figure 1 Figure 2 Figure 3 show that activation of KCC by acidification has a similar time course to activation by hypotonic swelling. It should be pointed out that, in these experiments, there was slight cell swelling in addition to the acidification. Acidification of red cells is accompanied by a net influx of Cl- (e.g.,
A complete study of the relationship between acid activation and swelling activation in rabbit red cells was not performed because it is already clear from the work of
Three-State Models of Transport Regulation
Earlier data on activation and inactivation rates (
In rabbit red cells there is very little effect of cell volume on KCC in Mg2+-depleted cells (Figure 5). Instead, the cells behave similarly to NEM-treated cells; KCC is activated and is not strongly affected by volume. The influx was measured at an extracellular K+ concentration of 5 mM, which is well below the apparent Michaelis constant for extracellular K+ (
The three-state model of
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(2) |
where KBC is the equilibrium constant k23/k32 for the rapid second step.
In rabbit red cells, -1 is smaller in swollen cells than in cells of normal size (
The inactivation experiments in Figure 10 Figure 11 Figure 12 provide a way to address the question of whether the slow or the rapid step is the major volume-dependent step. Suppose, for example, that the rapid (B to C) step were the only volume-dependent step. If so, then KBC must decrease as cell volume decreases. As KBC becomes small, the steady state number of C states ([C]ss) decreases in proportion to KBC, but the relaxation rate -1 should reach a limiting value (Equation 3 and Equation 4):
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(3) |
and
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(4) |
In other words, if the major volume-dependent process is the rapid B to C transition, then the rate of inactivation should become independent of cell volume in the limit of low cell volume. The data in Figure 10 Figure 11 Figure 12 show that, experimentally, the rate of inactivation continues to increase as cell volume decreases in a range of cell volumes where nearly all the transporters are inactivated in the steady state. This indicates that the rate constant k21 for the rate-limiting inactivation event is strongly dependent on cell volume. These data are the best evidence to date that the rate-limiting inactivation event is also the main volume-dependent event. It is significant that the KCC inactivation rate is strongly dependent on cell volume at physiological cell volumes. This indicates that, even though most of the transporters are inactivated under physiological conditions, modulation of KCC by small changes in cell volume is a real physiological mechanism for maintaining normal cell volume over the long life of the cell.
Lack of Effect of Cell Volume on the Rate-limiting Forward Activation Step
In the context of the three-state model, NEM could activate transport by increasing k12, decreasing k21, or increasing KBC. NEM probably does not have a large stimulatory effect on k12, because the rate of activation in NEM-pretreated cells is relatively slow. Accordingly, NEM must cause a large decrease in k21 and/or increase in KBC. In either case, the measured rate of activation -1 is approximately equal to the forward activation rate constant k12 under conditions of maximum NEM activation, because the second term in Equation 2 is very small when activation is maximal. Experimentally, cell swelling has no detectable effect on the activation step k12 (Figure 9). This is consistent with our earlier data (
Large Temperature Dependence of the Rate-limiting Activation Event
Although volume has very little effect on the activation rate constant, temperature has a very large effect. Earlier data on the effect of temperature on rabbit red cell KCC (
The high temperature dependence of k12 is in agreement with the recent work of
Biochemical Nature of the Rate-limiting Activation Process
Inhibitors of serine-threonine protein phosphatases prevent activation of KCC (
It is of interest to compare the temperature dependence of the activation rate constant k12 with those for known protein phosphatases.
If k12 does represent a protein dephosphorylation event, then k21 most likely represents phosphorylation mediated by a serine-threonine protein kinase, as suggested previously (
Predicting Steady State Fluxes from Activation and Inactivation Rate Constants
It is of interest to ask whether the measured rate constants for activation and inactivation are consistent with the observed effects of volume on the steady state fluxes. In the two-state model, the steady-state flux is related to the inverse lag time as shown in Equation 5 (
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(5) |
Therefore, if the only volume-dependent parameter is k21, then the flux should be directly proportional to the lag time. In Figure 11 and Figure 12, both the flux and the lag time are strongly dependent on cell volume, but the flux varies somewhat more rapidly than does the lag time. Therefore, the volume dependence of the inactivation rate constant k21, by itself, does not appear to be sufficient to account for the entire volume dependence of the flux. As discussed above, we have no direct evidence for volume dependence of the activation rate constant k12, although the estimates of k12 were necessarily made in NEM-treated cells, and it is possible that, without NEM treatment, k12 is volume dependent. Another possibility is that a rapid step in the activation is volume dependent, as found in LK sheep red cells by
Comparison of Rabbit and LK Sheep Red Cells
Although there are differences between the current results and those obtained in LK sheep red cells (
Variations on the Three-State Model
The three-state model of
Many variations on simple cascade-type models are possible, including those in which a tyrosine kinase modulates the activity of a serine/threonine phosphatase (
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Acknowledgements |
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The authors are grateful to Mark Adame, Anjali Shinde, and Daniel Brown for assistance with some of the flux experiments described here. Anil Namboodiripad assisted with the Mg2+ determinations.
This work was supported by National Institutes of Health research grant R01 GM 26861-20.
Submitted: 2 August 1999
Revised: 7 October 1999
Accepted: 8 October 1999
HPS, HEPES-buffered physiological saline; KCC, K+/Cl- cotransport; NEM, N-ethylmaleimide; PP1, protein phosphatase 1
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Appendix |
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Relaxation Rate for Three-State Models
A general three-state model for regulation of KCC is shown in Scheme 3 (
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(A1) |
and
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(A2) |
where [A]ss, [B]ss, and [C]ss are the steady state fractions of the three forms of the transporter. It is assumed that there are only three states; therefore,
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(A3) |
and
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(A4) |
The above equations can be solved for [C]ss:
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(A5) |
It is assumed that the B to C transition is much faster than the A to B transition (
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(A6) |
The relaxation rate for the whole system is then derived as follows. Define Y as the difference between the concentration of A at time t and the concentration of A in the final steady state:
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(A7) |
From conservation of mass, it must be true that:
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(A8) |
Equation A6 and Equation A8 can be combined to produce:
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(A9) |
The first order differential equation for the rate of change of Y is:
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(A10) |
Substituting for [A] (Equation A7) and [B] (Equation A9) into Equation A10 gives:
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(A11) |
The terms containing [A]ss and [B]ss sum to zero (Equation A1), leaving
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(A12) |
Therefore, the relaxation rate (inverse lag time), -1, is the following:
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(A13) |
Equation A5 and Equation A13 can be rewritten in terms of KBC (k23/k32), the equilibrium constant for the rapid (B to C) step:
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(A14) |
and
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(A15) |
The relaxation rate -1 for a three-state model (with a rapid second step), then, is similar to that for a two-state model, except that the term in k21 is divided by (1 + KBC).
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