Department of Biology, Syracuse University, Syracuse, New York 13244-1220
Submitted 30 December 2003 ; accepted in final form 18 March 2004
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ABSTRACT |
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erythrocytes; cell volume regulation; amiloride; kinetics of sodium ion influx
NHE-1, like other isoforms, is stimulated by shrinkage and by acidification of the cells and also by growth factors. Intracellular acidification activates NHE-1 through an allosteric site, where H+ binding causes an alkaline shift in the set point for activation of NHE by H+ (3). Growth factors and osmotic shrinkage also shift the set point for activation of the exchanger by intracellular H+ (9, 19, 24). The molecular identity of this allosteric H+ binding site is not known, but structure and function studies have provided clues about its location in the NHE molecule and also of the location of the shrinkage-sensitive domains (4, 33). Activation of NHE-1 by growth factors involves phosphorylation at multiple sites on the transporter (28). However, increased phosphorylation of NHE-1 does not accompany activation by cell shrinkage (10).
We report here studies on the kinetics of activation of NHE-1 in dog red blood cells by external Na+. Mature dog red blood cells are unusual in lacking Na+-K+ pumps and having intracellular Na+ and K+ near their plasma concentrations (25). This property is shared with cat red blood cells and may be characteristic of red blood cells of carnivores in general. At physiological external Na+ concentration ([Na+]o), there is little Na+ gradient, and at low [Na+]o, there is a large, outwardly directed Na+ gradient.
At low [Na+]o in isotonic media, there was activation of NHE by increasing [Na+]o. At [Na+]o >40 mM in isotonic media, there was a progressive decline in NHE as though Na+ inhibited it. In osmotically shrunken cells (isotonic media + 120 mM sucrose), NHE was greatly stimulated, >10-fold at 150 mM [Na+]o. In these shrunken cells, there was no inhibition of NHE at high [Na+]o, and Na+ influx through NHE was a hyperbolic function of [Na+]o, typical of activated NHE in other cells (16, 18, 21, 34) and consistent with interaction of Na+ with a single external substrate site per transporter. K1/2, the Na+ concentration at half-maximal activation of NHE, was 58.5 mM in shrunken dog red blood cells. The results suggest that there are external sites on dog red cells where Na+ inhibits NHE, and shrinkage activates NHE in part by reducing this inhibition by [Na+]o. The K1/2 for Na+ binding to these inhibitory sites in isotonic media was 82.3 mM.
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MATERIALS AND METHODS |
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Na+ influxes. Unidirectional influxes of Na+ were measured by using 22Na as a tracer (PerkinElmer, Boston, MA). Cells were washed in media appropriate for the experiments; the washing required 5 min. The fluxes were measured over 10 min; all measurements were made in triplicate. NHE was taken as the amiloride-inhibitable Na+ influx (1 mM amiloride). Two amiloride derivatives, 5-(N,N-hexamethylene)-amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA), specific inhibitors of NHE (17), were used to confirm that the amiloride-inhibitable Na+ influx is NHE. N-methyl-D-glucamine (NMDG+) was used as the substitute cation for Na+. The methods for calculating the influxes were slight modifications of earlier methods (27). Fluxes are expressed as millimoles per liter cells per hour when performed in isotonic media. Fluxes measured in shrunken cells were corrected to the original, physiological cell volume by using the hemoglobin concentrations of the flux samples. These fluxes are expressed as millimoles per original liter cells per hour.
It is important that the influx of Na+ be a linear function of time when the measurements of fluxes are made. Figure 1 shows the time course of amiloride-inhibitable Na+ influxes during 8 min in cells in 150 mM Na+, both isotonic and hypertonic. The data were well fit by straight lines. One other experiment of similar design gave the same results.
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Intracellular pH. The method for measuring intracellular pH was a modification of earlier methods (1, 6). After the desired treatments, 0.5-ml cell samples were packed by centrifugation. The cells were suspended in 15 volumes of unbuffered isotonic medium and packed again. The packed cells were lysed by three cycles of freezing in dry ice/methanol and thawing in a room temperature water bath. The lysates were diluted with 1 ml of deionized water. The pH of each lysate was determined in triplicate by using a combination electrode.
Percent cell water.
Cells, 0.4 ml, were packed in 1.5-ml tared microfuge tubes by centrifugation for 5 min in a Fisher Scientific microcentrifuge. The supernatant solutions were carefully aspirated from the tubes, along with the top
1 mm of cells. The tubes were weighed, giving wet weights. The tubes were dried to constant weight at 80°C and weighed again, giving dry weights. A correction was made for loss of
1.5 mg weight of the tubes during drying, determined by using two empty tubes in each experiment. All measurements were made in triplicate. Dry weights were corrected to dry volume by using a density of 1.17 g/ml for dry cell contents, mostly hemoglobin (15). This permitted expression of percent cell water (volume/volume).
Intracellular Na+ concentrations.
Cell samples of 0.04 ml were washed three times in isotonic NMDG Cl and lysed in 5 ml of deionized water. The samples were analyzed by using a PerkinElmer AAnalyst 100 atomic absorption spectrometer in the emission mode. Standards of 2080 µM Na+ were used.
Statistical analysis. Differences among more than two means were analyzed by using a one-factor ANOVA and the Fisher protected least significant difference test between pairs of means.
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RESULTS |
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Activation of NHE by acidification?
At low [Na+]o, NHE runs in reverse, owing to the high cell Na+ concentration ([Na+]c) and the outwardly directed Na+ gradient. This may cause acidification of the cells that would activate NHE. To test this, intracellular pHs were measured in cells in isotonic media over a range of [Na+]o values after incubation for 530 min with or without amiloride. There was no suggestion of a change in pH with either time or [Na+]o (Table 1); there was no hint of acidification as time proceeded. Protons taken up by reversed NHE at low [Na+]o are buffered, and the activation of NHE at low [Na+]o is not due to acidification of the cells. As will be shown below, the Na+ loss in 2 mM [Na+]o in 15 min is a little less than 10 mmol/l cells. A corresponding H+ influx would mean about two protons per hemoglobin molecule (5 mM hemoglobin in mammalian erythrocytes). Results with amiloride were indistinguishable from the controls.
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NHE is inhibited by external Na+. Figure 4 showed that there is no loss of Na+ in 15 min from cells in 40 mM [Na+]o. Therefore, as [Na+]o is raised from 40 mM and NHE is inhibited, [Na+]c remains constant (it did not decrease as [Na+]o was lowered). Therefore, the inhibition of exchange with the increase in [Na+]o is due to Na+ acting on the exchanger at external sites.
Na+ influx in cells in hypertonic media. Figure 6A shows unidirectional Na+ influxes in cells shrunken in hypertonic medium, 415 mosmol/kgH2O (isotonic medium and 120 mM sucrose) over a range of [Na+]o values, total influx (solid circles), and influx with amiloride (solid squares). The fluxes in isotonic media from Fig. 2 are included for comparison (open circles and squares). Osmotic shrinkage greatly stimulated total Na+ influx. Shrinkage was without effect on the amiloride-insensitive Na+ influx.
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Almost all of the increase in influx caused by shrinkage can be accounted for by relief of the inhibition of NHE by Na o+. The part of flux in hypertonic media that is not due to the relief of inhibition is the flux in isotonic medium. To obtain the Na+ flux that is due to relief of inhibition, the individual fluxes in isotonic medium were subtracted from the calculated fluxes in hypertonic media. The difference curve for relief of inhibition is given by the open symbols. The difference curve was fitted using the Hill equation, giving a good fit to the data. The K1/2 was 82.3 ± 2.2, and the nH was 1.43 ± 0.02. This K1/2 for inhibition (K1/2 i) was greater than the K1/2 for activation (K1/2 a), 58.5 mM, and Na+ inhibits at sites with a lower apparent affinity than that of the sites at which it activates. The nH was greater than unity, indicating more than one inhibitory site for Na+ on each transporter. The Jmax for activation by 120 mM sucrose was 161 ± 8 mmol·original liter cells1·h1. The Jmax for the difference curve was 146 ± 2 mmol·original liter cells1·h1. The Jmax for the difference curve was less than the Jmax for hyperosmotic activation because of the positive NHE in isotonic medium at 150 mM [Na+]o. If the NHE flux in isotonic medium had decreased to zero at 150 mM [Na+]o, then the two Jmax values would likely have been the same. The difference between the Jmax values suggests that NHE in isotonic medium does not go to zero as [Na+]o is raised above 150 mM.
The curve for NHE in isotonic media fitted by using constants for activation and inhibition.
The following equation, with components of both activation and inhibition, was used to fit the data for NHE in isotonic media:
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DISCUSSION |
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The hyperbolic kinetics of NHE in 120 mM sucrose media are consistent with activation of NHE by Na+ at a single site on each exchanger. Hyperbolic kinetics for Na+ influx via NHE activated by acidification or shrinkage have been reported in several studies on the NHE-1, -2, and -3 isoforms (16, 21, 34, 36). The K1/2 a values (K1/2 for activation) ranged from 10 to 50 mM [Na+]o. The K1/2 a in dog red blood cells is at the high end of this range.
The Jmax measured in 120 mM sucrose media may not be the maximum velocity of the transporter. Measurements in more hypertonic media were attempted, but acceptable results could not be obtained, owing to the high rates of transport and distortion of the curve at the high [Na+]o values. The kinetic constants might be quantitatively different at higher Jmax values, but the general conclusions would be the same.
There are few results in the literature on the kinetics of Na+ influx through unstimulated NHE. In rat thymocytes, total Na+ influx was not much greater than the amiloride-insensitive flux, so the kinetics of unstimulated NHE could not be characterized (8). NHE was measured in unstimulated Chinese hamster lung fibroblasts at 130, 15, and 1 mM [Na+]o. There was measurable NHE at 1 mM [Na+]o, but not at the higher [Na+]o values (24). This effect at 1 mM Na+ was attributed to an intracellular acidification by 0.15 pH units in cells at low [Na+]o. Intracellular buffering to pH 7.35 abolished the stimulation of NHE at 1 mM [Na+]o. In the present work on dog red blood cells, it is clear that the stimulation of NHE at low [Na+]o is not due to acidification.
Extracellular Li+ interacts in a complex manner with NHE of brush-border membrane vesicles from rabbit renal cortex (11). Li+ inhibits at the extracellular surface by competing with Na+; Li+ has a lower K1/2 than Na+ (by 12-fold) and a lower Jmax (by
3-fold). In addition, Li+ interacts with a separate modifier site, where it inhibits NHE noncompetitively with a very high affinity (Ki
50 µM). Amiloride also binds to this site, resulting in noncompetitive inhibition of NHE. There was no evidence for Na+ binding to this modifier site (11), making it unlikely that this site is the same as the Na+ inhibitory site of the present study. K+ is a competitive, dead-end inhibitor of NHE-1 with a very low affinity (K1/2
180 mM) (21). There is an early report of slight inhibition of NHE by Cs+ (14) and another report of no effect of Cs+ on NHE (12). There is evidence for inhibition of NHE by extracellular Ca2+ (26, 32), although at which site(s) is not known.
When the intracellular allosteric H+ sites are unoccupied, the NHE flux is low. Acidification or shrinkage caused H+ binding at these sites and stimulation of NHE (3, 9, 19, 24). The newly described allosteric inhibitory Na+ sites also maintain NHE low in isotonic medium but when they are occupied by Na+. Cell shrinkage reduces Na+ affinity at these sites, and NHE is stimulated. Therefore, two allosteric sites participate in shrinkage activation of NHE. The H+ site leads to activation when it is occupied, and the Na+ site does so when it is not. The relationship between the allosteric H+ and Na+ sites remains to be elucidated.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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