J Physiol Volume 507, Number 1, 147-162, February 15, 1998
The Journal of Physiology (1998), 507.1, pp. 147-162
© Copyright 1998 The Physiological Society
Intracellular Ca2+ regulation by the leech giant glial cell
Wolfgang Nett and Joachim W. Deitmer
Abteilung für Allgemeine Zoologie, FB Biologie, Universität Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany
Received 30 July 1997; accepted after revision 10 October 1997.
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ABSTRACT |
- We have measured the intracellular Ca2+ concentration, [Ca2+]i, and the intracellular Na+ concentration, [Na+]i, with the fluorescent dyes fura-2 (for Ca2+) and SBFI (for Na+) in situ in giant glial cells of the central nervous system of the leech Hirudo medicinalis.
- The basal [Ca2+]i was 79 ± 35 nM (n = 27) in cells voltage clamped at -70 to -80 mV, and 75 ± 29 nM (mean ± s.d., n = 82) in unclamped cells at a mean membrane potential of -67 ± 6 mV.
- Removal of external Na+ evoked a small reversible [Ca2+]i increase of 29 ± 21 nM (n = 27) in cells voltage clamped at -70 to -80 mV, and of 35 ± 18 nM (n = 37) in unclamped cells. This [Ca2+]i increase, and the time constant of the subsequent [Ca2+]i recovery after Na+ re-addition, did not change significantly with the holding potential between -110 and -60 mV.
- The basal [Na+]i was 5·6 ± 1·3 mM (n = 18). Increasing [Na+]i by inhibiting the Na+-K+ pump with 100 µM ouabain had no effect on the [Ca2+]i rise upon removal of external Na+.
- The time course of recovery from a [Ca2+]i load mediated by voltage-dependent Ca2+ influx during depolarization in high K+ was unaffected by the removal of external Na+.
- Cyclopiazonic acid (10 µM), an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a transient increase in [Ca2+]i of 28 ± 11 nM (n = 5), and significantly slowed the recovery from imposed [Ca2+]i loads.
- Iontophoretic injection of orthovanadate, an inhibitor of P-type ATPases including the plasma membrane Ca2+-ATPase, caused a persistent increase in the basal [Ca2+]i of 163 ± 101 nM (n = 5) in standard saline, and of 427 ± 338 nM in Na+-free saline (n = 5). Vanadate injection significantly slowed the recovery from [Ca2+]i loads. Removal of external Na+ during vanadate injection induced an additional, reversible [Ca2+]i increase of 254 ± 64 nM (n = 3).
- The results suggest that the low basal [Ca2+]i in these glial cells is predominantly maintained by a Ca2+-ATPase in the plasma membrane. This ATPase is also the main Ca2+ extruder after an intracellular Ca2+ load, while intracellular stores appear to contribute little to this recovery. A Na+-Ca2+ exchanger seems to play a minor role in the maintenance of basal [Ca2+]i in these cells, but becomes prominent when the plasma membrane Ca2+-ATPase is blocked.
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INTRODUCTION |
Like most other cells, glial cells maintain their intracellular free Ca2+ concentration ([Ca2+]i) at low levels of between 50 and 100 nM, against a large electrochemical gradient. Studies have shown that glial cells respond with transient changes in [Ca2+]i to a variety of external stimuli (Verkhratsky & Kettenmann, 1996). Glial cells possess voltage-gated Ca2+ channels (MacVicar, 1984), and ligand-operated ion channels, e.g. ionotropic glutamate receptors of the AMPA/kainate type, which have been shown to be Ca2+ permeable in different types of glial cells (Burnashev et al. 1992; Müller, Möller, Berger, Schnitzer & Kettenmann, 1992; Munsch, Nett & Deitmer, 1994). Stimulation of neurones can also induce an increase in glial [Ca2+]i, as has been shown for astrocytes in organotypically cultured slices of the rat hippocampus (Dani, Chernjavsky & Smith, 1992), perisynaptic Schwann cells of the frog neuromuscular junction (Robitaille, 1995) and the giant glial cell in the central nervous system of the leech (Rose, Lohr & Deitmer, 1995). The amplitude and time course of these changes in [Ca2+]i, however, are not only determined by the amount of Ca2+ influx, but also depend on the capacity of intracellular Ca2+-buffering systems, on sequestration of Ca2+ into organelles and on Ca2+-extrusion mechanisms. Since intracellular sequestration and buffering of Ca2+ is limited, all Ca2+ that enters the cell has to be extruded from the cell via the plasma membrane.
Two main mechanisms for Ca2+ extrusion from cells have been described: a Ca2+-transporting ATPase and a Na+- Ca2+ exchanger. In cultured astrocytes of the rat, a Na+-Ca2+ exchanger has been reported (Goldmann, Yarowsky, Juhaszova, Krueger & Blaustein, 1994; Takuma, Matsuda, Hashimoto, Asano & Baba, 1994), and some evidence exists for the presence of a plasma membrane Ca2+-ATPase in mouse glial cell cultures of the oligodendrocyte lineage (Kirischuk, Möller, Voitenko, Kettenmann & Verkhratsky, 1995). However, in rat brain slices, mRNA for the plasma membrane Ca2+-ATPase was only found in neurones, not in glial cells (Carafoli & Stauffer, 1994).
We have now investigated the contribution of the different Ca2+ extrusion mechanisms to the regulation of [Ca2+]i in a single, identified glial cell in situ, by employing microfluorometric measurements of intracellular Ca2+ and Na+ combined with electrophysiological recordings. We present evidence for Na+-dependent Ca2+ extrusion at low basal Ca
levels, and at high [Ca2+]i of several hundred nanomoles per litre when the Ca2+-ATPases were inhibited by orthovanadate injection. Our results suggest that the Ca2+-ATPases in the plasma membrane are the main regulatory mechanism of a low basal [Ca2+]i in these cells. Some of these results have been presented in abstract form (Nett & Deitmer, 1997).
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METHODS |
Preparation
The experiments were performed on isolated segmental ganglia of the leech Hirudo medicinalis. The giant glial cells of the neuropile were exposed in a modified version of the preparation of Munsch & Deitmer (1992). In brief, the ventral ganglion capsule was removed mechanically. The ganglia were then incubated for 30 min in 2 mg ml-1 collagenase-dispase (Boehringer-Mannheim), dissolved in a modified Leibowitz-15 (L-15) medium (see below) at room temperature (20-25°C). After enzyme treatment, the ganglia were thoroughly washed with enzyme-free medium. Neurones overlying the giant glial cells were removed by suction with a fire-polished glass micropipette (tip diameter, 50-100 µm). Ganglia were then transferred into standard leech saline (see below) for experimentation.
Solutions
Original L-15 medium (Gibco) was modified by dilution (1: 3) with a salt solution of the following composition (mM): CaCl2, 6·87; MgCl2, 2·51; KCl, 3·32; sodium malate, 20·1; sodium pyruvate, 12·5; Hepes, 15; glucose, 15; plus 0·3 % (v/v) gentamicin (stock solution, 10 mg ml-1), adjusted to pH 7·4 with NaOH.
The standard leech saline had the following composition (mM): NaCl, 85; KCl, 4; CaCl2, 2; MgCl2, 1; Hepes, 10; adjusted to pH 7·4 with NaOH. In Na+-free solutions, 85 mM N-methyl-D-glucamine (NMDG), Tris, choline or Li+ were used in place of Na+, and pH was adjusted to 7·4 with HCl or LiOH. In high-K+ solutions, 16 mM NaCl was replaced with KCl to a final K+ concentration of 20 mM. In Ca2+-free solutions, CaCl2 was replaced with MgCl2, and 0·5 mM EGTA was added. Cyclopiazonic acid (CPA), thapsigargin and methoxyverapamil (D600; all obtained from Sigma) were dissolved in DMSO and then added to the saline. The DMSO concentration of the experimental solutions was below 0·1 % in each experiment.
Solutions for calibration of the Ca2+-sensitive dye fura-2 were as follows. pCa (mM): NaCl, 85; KCl, 4; MgCl2, 3; EGTA, 0·5; Hepes, 10; adjusted to pH 7·4 with NaOH. pCa 7 (mM): KCl, 90; CaCl2, 5; EGTA, 10; Mops, 10; adjusted to pH 7·29 with KOH. pCa 2 (mM): KCl, 80; CaCl2, 10; MgCl2, 1; Hepes, 10; adjusted to pH 7·4 with NaOH. 2Mn (mM): KCl, 80; CaCl2, 2; MgCl2, 1; MnCl2, 2; Hepes, 10; adjusted to pH 7·4 with NaOH. Ionomycin (10 µM; Sigma) was added to the pCa7, pCa2 and 2Mn solutions for equilibration of extra- and intracellular Ca2+.
The calibration solutions for the Na+-sensitive dye SBFI had the following composition (mM): (NaCl + KCl), 100; Hepes, 10; CaCl2, 2; MgCl2, 1; adjusted to pH 7·4 with KOH. Ouabain (1 mM), gramicidin (3 µM) and monensin (10 µM; all obtained from Sigma) were added to block intracellular Na+ regulation and to permeabilize the cell membrane to Na+ ions. Different Na+ concentrations were prepared by exchanging NaCl with KCl.
Microelectrodes
In all experiments double-barrelled, theta-type electrodes were used. One barrel was filled with 3 M KCl for the recording of the membrane potential. The second barrel was filled with ion-sensitive dye (12 mM fura-2, 11 mM SBFI or 19 mM 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF); all obtained from Molecular Probes), dissolved in 100 mM KCl or in 200 mM potassium acetate. In two-electrode voltage-clamp experiments a second, single-barrelled microelectrode filled with 2 M potassium acetate, adjusted to pH 7 with HCl, was impaled into the cell for current injection. When vanadate was injected, the second microelectrode was filled with 50 mM sodium orthovanadate, adjusted to pH 9 with HCl. All electrodes were bevelled on a rotating disc.
Measurement of intracellular Ca2+, Na+ and pH
The intracellular free Ca2+ concentration, [Ca2+]i, was measured by determining the ratio of fura-2 fluorescence at 350 nm (F350) and 380 nm (F380) excitation. The fluorescence ratio F350/F380 of fura-2 was calibrated in situ according to Munsch & Deitmer (1995) using the equation described by Grynkiewicz, Poenie & Tsien (1985):
[Ca2+]i = KD [(R - Rmin)/(Rmax - R)] × Sf2/Sb2, (1)
where Rmin and Rmax are the fluorescence ratio values for Ca2+-free and Ca2+-saturating conditions, respectively, and Sf2/Sb2 the ratio of fluorescence values for Ca2+-free/Ca2+-bound dye measured at 380 nm excitation wavelength. KD is the apparent dissociation constant between Ca2+ and the dye. In calibration experiments, one channel of the theta-microelectrode was filled with a 0·5 M EGTA solution, adjusted to pH 7·3 with KOH, the second channel contained the dye solution. For the determination of Rmin and Sf2, EGTA was injected with a hyperpolarizing current pulse into the cell whilst perfusing the experimental chamber with pCa solution, Rmax and Sb2 were determined in the pCa2 calibration solution, the pCa7 calibration solution was used to estimate KD, and the fluorescence was quenched in the 2Mn calibration solution for the determination of the background fluorescence. In these calibration experiments, the following calibration parameters were obtained: Rmin = 0·53 ± 0·07, Rmax = 8·43 ± 1·6, Sf2/Sb2 = 6·88 ± 1·2 and KD = 204 ± 91 nM (mean ± S.D., n = 5).
Changes in [Na+]i were measured with sodium-binding benzofuran isophthalate (SBFI; Minta & Tsien, 1989), also excited at 350 and 380 nm. The SBFI ratio was calibrated in solutions with various Na+ concentrations ([Na+] + [K+] = 100 mM; Fig. 1A), containing 3 µM gramicidin, 10 µM monensin and 1 mM ouabain. In vivo excitation spectra in different Na+ concentrations are shown in the inset of Fig. 1A. The correlation between the fluorescence ratio and the Na+ concentration in the selected range of 0-100 mM Na+ appeared non-linear (Fig. 1B) and could be fitted to an equation similar to that used for the fura-2 calibration (Harootunian, Kao, Eckert & Tsien, 1989):
[Na+]i = K [(R - Rmin)/(Rmax - R)]. (2)
The expression KD × Sf2/Sb2 was substituted by a single parameter K, because saturating Na+ concentrations were not applied to determine Sb2. The following parameters were obtained for the SBFI calibration: Rmin = 1·01, Rmax = 3·87 and K = KD × Sf2/Sb2 = 41·48 mM. When the extracellular K+ concentration was changed during the calibration procedure from 95 to 80 mM at a constant Na+ concentration of 5 mM, an increase in the fluorescence ratio of 0·012 was measured, indicating an apparent decrease in [Na+]i of 0·22 mM. The pH sensitivity of SBFI is controversial. Whereas Harootunian et al. (1989) found no significant shift in the SBFI ratio during an acidification of 0·4 pH units, Rose & Ransom (1996) reported an apparent [Na+]i increase of 6·1 mM due to a similar acidification. When we changed the pH of the 10 mM Na+ calibration solution from 7·4 to 7·1, which is near the estimated intracellular pH of the giant glial cells (Nett & Deitmer, 1996a), a decrease in the SBFI ratio of 0·035 was observed, which indicated an apparent [Na+]i decrease of 0·77 mM.
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Figure 1. In situ calibration of SBFI
A, fluorescence ratio F350/F380 measured at different Na+ concentrations. The inset shows the corresponding excitation spectra of the dye-filled cell. a.u., arbitrary units. B, mean values (± S.D.) of the fluorescence ratio at different Na+ concentrations. The numbers above the symbols indicate the number of experiments. The continuous line was fitted through the data points as described in the text.
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BCECF was used to measure pHi, using the calibration procedure described by Nett & Deitmer (1996a). The calibration solutions of different pH values contained 80 mM K+ and 10 µM nigericin (Molecular Probes), a K+/H+ ionophore.
Experiments were performed using a Deltascan dual excitation spectrofluorometer (PTI, Wedel, Germany) in which shutters, monochromator settings and the data acquisition were controlled by software and interfaces from PTI. The dyes were excited with light from a 75 W xenon arc lamp, alternately at 350 and 380 nm for determination of [Ca2+]i and [Na+]i, and at 440 and 495 nm for measuring pHi, with a bandwidth of 4 nm through the epifluorescence port of a Zeiss Axioskop FS microscope with a × 40 water-immersion objective. Fluorescence intensity between 500 and 530 nm was measured for determining [Ca2+]i and [Na+]i, and above 520 nm for pHi, using a photon-counting photomultiplier tube (PTI). Measurements were limited to a field of view slightly larger than the cell body of the injected glial cell. Background correction was performed before the fluorescence ratio was calculated.
Statistics
All data are given as means ± standard deviation (S.D.). The statistical significance of differences between mean values was tested using Student's t test. Differences were indicated as statistically significant when the error probability was < 0·05.
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RESULTS |
Resting [Ca2+]i and [Na+]i and the effect of external Na+ removal
The basal [Ca2+]i of the giant glial cells, measured with fura-2, was 75 ± 29 nM (mean ± S.D., n = 82) at a mean membrane potential of -66 ± 6 mV (n = 82). The [Ca2+]i values ranged from 25 to 137 nM.
Substitution of NMDG for external Na+ induced a rise in [Ca2+]i of 35 ± 18 nM (n = 37; Fig. 2A). This [Ca2+]i increase was totally blocked, or even reversed to a small [Ca2+]i decrease, in Ca2+-free saline with 0·5 mM EGTA (Fig. 2B), indicating that the [Ca2+]i rise upon Na+ removal was due to Ca2+ influx. This [Ca2+]i increase, however, was not affected by 0·5 mM methoxyverapamil (D600; data not shown), which blocks voltage-dependent Ca2+ influx into the cells (Munsch & Deitmer, 1997).
In cells voltage clamped to membrane potentials between -70 and -80 mV, the basal [Ca2+]i was 79 ± 35 nM (n = 27), i.e. very similar to the [Ca2+]i in unclamped cells. The [Ca2+]i increase observed upon removal of external Na+ in these cells amounted to 29 ± 21 nM (n = 27; Fig. 2C). Thus, [Ca2+]i increased on average by 47 and 36 % in unclamped and voltage-clamped cells, respectively, following removal of external Na+. These [Ca2+]i changes were not significantly different (P = 0·2).
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Figure 2. The effects of external Na+ removal on [Ca2+]i and [Na+]i
A, records of [Ca2+]i (lower trace) and membrane potential (Em, upper trace) of a current-clamped giant glial cell during Na+ removal. B, [Ca2+]i recording during Na+ removal in Ca2+-free saline. C, [Ca2+]i recording in a voltage-clamped cell. Vh, holding potential. D, recording of [Na+]i during external Na+ removal.
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The mean resting [Na+]i of the giant glial cells, measured with the fluorescent dye SBFI, was 5·6 ± 1·3 mM (n = 18). It decreased to values of around 1 mM or below after Na+ was removed from the saline (Fig. 2D). This decrease in [Na+]i did not depend on external Ca2+, suggesting that Na+ does not primarily leave the cell via a Ca2+-dependent mechanism. The mean maximum rate of the [Na+]i decrease was 7·4 ± 2·5 mM min-1 (n = 8).
We also tested the effect of Na+ substitutes other than NMDG, in particular Tris, choline, and Li+, on the basal [Ca2+]i. The amplitude of the [Ca2+]i rise was similar with all of these Na+ substitutes (Fig. 3). The time course of the [Ca2+]i increase was similar when Na+ was replaced with either NMDG or Tris (Fig. 3A). Replacing Na+ with choline (Fig. 3A) or Li+ (Fig. 3B) induced a somewhat faster increase in [Ca2+]i. Since choline and Li+ also induced large inward currents (not shown), indicating some additional effect of these cations, Na+ was replaced with NMDG in most of the subsequent experiments.
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Figure 3. Effects of different Na+ substitutes
Records of [Ca2+]i in voltage-clamped giant glial cells during removal of external Na+. Na+ was replaced with either NMDG, Tris or choline at a Vh of -80 mV (A), and with Tris or Li+ at a Vh of -70 mV (B).
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Changes in holding potential
The [Ca2+]i increase in Na+-free saline suggested a possible role for Na+-Ca2+ exchange as a mechanism to regulate the basal [Ca2+]i. In most tissues in which Na+-Ca2+ exchange has been demonstrated, this exchange was shown to be electrogenic, in general with a stoichiometry of three Na+ ions for one Ca2+ ion (Eisner & Lederer, 1985; Rasgado-Flores & Blaustein, 1987). This electrogenicity renders the exchange sensitive to the membrane potential. We therefore studied the [Ca2+]i rise upon removal of external Na+, and the fall in [Ca2+]i upon re-addition of Na+ to the saline at different holding potentials (Vh) between -60 and -110 mV (Fig. 4). The range of holding potentials was not extended to more positive membrane potentials, to exclude possible interference from voltage-operated Ca2+ channels, which are activated at around -50 mV (Munsch & Deitmer, 1995).
In seven out of twenty experiments, a decrease in basal [Ca2+]i of 6-50 nM was observed when Vh was stepped to more negative potentials (by 10-40 mV), while in three cells, basal [Ca2+]i increased by 9-20 nM. In ten cells, an effect of changes in Vh on basal [Ca2+]i could not be observed (Fig. 4).
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Figure 4. Na+ removal at different holding potentials
[Ca2+]i changes (upper trace) induced by Na+ removal at different holding potentials (lower trace) between -65 and -85 mV.
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In Fig. 5A, the basal [Ca2+]i of twenty cells is plotted against the holding potential. To account for the different values of [Ca2+]i in different cells, the [Ca2+]i values were normalized to the [Ca2+]i at a holding potential of -80 mV for each individual cell. There was a weak but significant correlation between basal [Ca2+]i and Vh (r = 0·42, n = 47).
In contrast, the amplitude of the [Ca2+]i increase upon Na+ removal did not appear to be dependent on Vh between -60 and -110 mV (Figs 4 and 5B). The [Ca2+]i increase averaged from twenty-three voltage-clamped cells was 30 ± 21 nM, and could not be correlated with the actual holding potential (r = 0·05, n = 48; Fig. 5B).
For analysis of the time course of the [Ca2+]i recovery upon Na+ re-addition, the [Ca2+]i decay was fitted with a single exponential (Fig. 5C). There was no significant correlation (r = 0·17) between the time constant of [Ca2+]i recovery and the holding potential between -60 and -110 mV. The time constants for the decay in [Ca2+]i were pooled with a mean of 56 ± 37 s (n = 46, twenty-three experiments). In two out of twenty-three cells, the [Ca2+]i decay could not be fitted sufficiently by only one time constant and in addition showed a much larger time constant.
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Figure 5. Summary of Na+-dependent effects
Plots of the basal [Ca2+]i (A), the amplitude of [Ca2+]i increases induced by Na+ removal (B), and the time constant ( ) of the recovery of [Ca2+]i after Na+ re-addition (C) against the holding potential. The values of the basal [Ca2+]i were normalized to the [Ca2+]i at -80 mV holding potential. The correlation coefficients (r) of the linear regressions are indicated in the top left-hand corner of each graph.
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In summary, with the experimental protocols employed here, we could not detect any dependence of the Na+-dependent [Ca2+]i changes on the membrane potential between -60 and -110 mV in these glial cells, indicating that the mechanisms underlying the observed [Ca2+]i rise and subsequent recovery might not be electrogenic.
The effects of intracellular Na+ loading
Na+-Ca2+ exchange is believed to be very sensitive to the [Na+]i (Mullins, Tiffert, Vassort & Whittembury, 1983). We therefore studied the effect of Na+ loading of the cell on the [Ca2+]i rise, following removal of external Na+. The [Na+]i was increased by adding 100 µM ouabain to the saline to block the Na+-K+ pump (Fig. 6A). Within 5 min of ouabain treatment the [Na+]i increased from 5 to 15 mM; removal of external Na+ led to a fast [Na+]i decrease below 2 mM (Fig. 6A; n = 3). Removal of external Na+ evoked a rise in [Ca2+]i of 26 ± 8 nM after 5 min of ouabain treatment, compared with 28 ± 10 nM before ouabain application in the same experiments (n = 6). This indicates that elevating the [Na+]i seems to have no influence on the [Ca2+]i increase induced by Na+ removal (Fig. 6B, bottom trace).
Longer exposure to ouabain (> 10 min) caused a depolarization of the cell membrane beyond -50 mV (Fig. 6B, upper trace), presumably due to a decrease in the transmembrane K+ gradient. This depolarization, which continued for about 1 min after the removal of ouabain, was accompanied by a large [Ca2+]i increase. The ouabain-induced Ca2+ rise could be inhibited by application of 500 µM D600 (n = 3; data not shown), indicating Ca2+ influx through voltage-dependent Ca2+ channels rather than a contribution of a reversed Na+-Ca2+ exchanger.
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Figure 6. Inhibition of the Na+-K+-ATPase with ouabain
Recordings of [Na+]i (A), [Ca2+]i and Em (B) during removal of external Na+ in the presence and absence of 100 µM ouabain.
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Recovery from intracellular Ca2+ loads
In most cells in which Na+-Ca2+ exchange has been described, it has been suggested that this exchanger has a high capacity for Ca2+ transport, but a low affinity for intracellular Ca2+. This would indicate that Na+-Ca2+ exchange becomes prominent only at higher [Ca2+]i. Since the mean basal [Ca2+]i of the giant glial cells was below 100 nM, according to this hypothesis a Na+-Ca2+ exchanger would not be expected to contribute much to the maintenance of the low resting Ca2+ levels, but might be challenged during [Ca2+]i loads.
The giant glial cells can be Ca2+ loaded in high-K+ saline solutions (Munsch et al. 1994; Munsch & Deitmer, 1995). Raising the extracellular K+ concentration from 4 to 20 mM depolarized the cell membrane by 30 ± 7 mV to around -35 mV (n = 10), which induced a large Ca2+ influx through voltage-dependent Ca2+ channels; this increased the [Ca2+]i to, on average, 287 ± 80 nM (n = 14).
High-K+ saline solutions were repeatedly applied, and the recoveries of [Ca2+]i after restoring the normal external K+ concentration were compared in the presence and in the absence of external Na+ (Fig. 7A). The depolarization-induced Ca2+ transients of the experiment shown in Fig. 7A were normalized and superimposed, indicating a similar time course of the [Ca2+]i recoveries in Na+-free and in Na+-containing saline (Fig. 7B). The high-K+-induced [Ca2+]i rise sometimes appeared more rapid in Na+-free than in Na+-containing saline. This is presumably due to a faster and larger depolarization caused by an increase in the K+ permeability in Na+-free saline (Nett & Deitmer, 1996b) that leads to an earlier and larger activation of voltage-dependent Ca2+ channels. The [Ca2+]i recoveries were fitted by a double exponential decay, yielding two time constants, 1 and 2. The fast time constant, 1, was 12·5 ± 8·4 s in standard saline, and 13·7 ± 8·2 s in Na+-free saline (n = 20), and the slow time constant, 2, was 78 ± 43 s in standard saline, and 102 ± 56 s in Na+-free saline (n = 20). The mean values of 1 and 2 in the presence and in the absence of external Na+ were not significantly different, as calculated by Student's paired t test (P = 0·61 and 0·08, respectively), indicating that Na+-driven Ca2+ transport did not contribute to the Ca2+ extrusion during [Ca2+]i rises up to about 300 nM.
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Figure 7. High-K+-induced [Ca2+]i transients in the presence and absence of external Na+
A, membrane potential (Em) and intracellular Ca2+ ([Ca2+]i). B, normalized [Ca2+]i transients of the experiment shown in A, as indicated by the corresponding numbers (1-3).
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To assess whether Na+-Ca2+ exchange could potentially contribute to the recovery after an intracellular Ca2+ load, the Na+ and Ca2+ gradients and the reversal potential for the exchanger were determined for this type of experimental protocol (Fig. 8). Assuming a stoichiometry of the exchanger of three Na+ ions for one Ca2+ ion, the reversal potential is given by:
ENa-Ca = 3ENa - 2ECa, (3)
where ENa-Ca is the reversal potential of the transporter and ENa and ECa are the Nernst equilibrium potentials for Na+ and Ca2+, respectively (see also Blaustein, 1988). The direction of the net Ca2+ transport across the cell membrane by the assumed Na+-Ca2+ exchanger is determined by the difference between the membrane potential (Em) and ENa-Ca. When Em is more negative than ENa-Ca, net Ca2+ outward movement could be driven by Na+-Ca2+ exchange, whereas Ca2+ influx via the exchanger would occur when Em is more positive than ENa-Ca. We averaged the changes in [Ca2+]i, [Na+]i (Fig. 8A and B) and Em recorded during the experiments in high-K+ saline, and calculated the mean course of ECa, ENa and ENa-Ca (Fig. 8C).
The mean resting [Ca2+]i in this set of experiments was 85 nM and increased to 280 nM in high-K+ saline (Fig. 8A). [Na+]i decreased from 4·6 mM in standard saline to 2·9 mM during high-K+ application (Fig. 8B). The mean equilibrium potentials for Ca2+ and Na+ were calculated to be +128 and +76 mV, and changed in the high-K+ saline to +112 and +90 mV, respectively (Fig. 8C). ENa-Ca was calculated with these values for ECa and ENa; on average, ENa-Ca changed from -30 to +30 mV, whilst Em decreased from -65 to -35 mV in the high-K+ saline. At any time during the protocol, ENa-Ca was positive to Em. The difference between ENa-Ca and Em became even larger in high-K+ saline. Thus, at all phases of this experimental protocol there was an inwardly directed driving force for an assumed 3Na+-1Ca2+ exchange, which therefore would, in principle, be capable of contributing to the recovery from an imposed Ca2+ load.
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Figure 8. Calculation of the reversal potential of a Na+-Ca2+ exchanger
Averaged traces of [Ca2+]i (A) and [Na+]i (B) were used to calculate the equilibrium potentials for Ca2+ (ECa; C, top trace) and Na+ (ENa; C, second trace) in standard and high-K+ saline. The reversal potential of the Na+-Ca2+ exchanger (ENa-Ca) was then calculated according to eqn (3) (C, third trace). The bottom trace shows the averaged course of the membrane potential, Em.
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Inhibition of intracellular Ca2+-ATPases
One possible mechanism for short term [Ca2+]i regulation is the sequestration of Ca2+ into the endoplasmic reticulum. The Ca2+ uptake into these stores is achieved by Ca2+-ATPases in the organellar membranes. In most cells these Ca2+-ATPases can be selectively blocked by cyclopiazonic acid (CPA; Seidler, Jona, Vegh & Martonosi, 1989).
CPA (10 µM) induced a transient increase in the [Ca2+]i of 28 ± 11 nM (Fig. 9A, n = 5). In addition, CPA had a small, but significant effect on the time course of the recovery from [Ca2+]i loads (Fig. 9B and C). The time constants obtained from double exponential fits were 1 = 5·9 ± 2·5 s and 2 = 47 ± 22 s in standard saline, and 1 = 8·1 ± 2·1 s and 2 = 45 ± 20 s in the presence of CPA. The difference in the fast time constant, 1, in CPA-containing and in standard saline was significant (P < 0·05), suggesting that Ca2+ uptake into CPA-sensitive stores contributes to some extent to the recovery from [Ca2+]i loads.
In contrast, application of 0·1-1 µM thapsigargin, another inhibitor of intracellular Ca2+-ATPases, neither changed basal [Ca2+]i in the glial cells, nor the time course of the recovery following a [Ca2+]i load induced by the high-K+ saline (n = 5; data not shown).
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Figure 9. Effects of cyclopiazonic acid on the basal [Ca2+]i and on the recovery after [Ca2+]i loads
A, transient increase in [Ca2+]i during application of 10 µM cyclopiazonic acid (CPA). B, high-K+-induced [Ca2+]i transients in the presence and absence of CPA. C, normalized [Ca2+]i transients.
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Effects of vanadate injection on basal [Ca2+]i, [Na+]i and pHi
In many cells, including neurones and glial cells, Ca2+ efflux occurs via ATP-dependent pumps in the plasma membrane (Carafoli & Stauffer, 1994). Orthovanadate is known to block P-type ATPases, including Ca2+-ATPases and other ATPases such as the Na+-K+ pump (Bond & Hudgins, 1980).
Orthovanadate was iontophoretically injected into the giant glial cells with currents of -5 to -10 nA. This induced an increase in the [Ca2+]i of 163 ± 101 nM (n = 5, Fig. 10). Subsequent removal of external Na+ now evoked an additional, large [Ca2+]i increase of 254 ± 64 nM (n = 3). Restoring external Na+ induced a rapid [Ca2+]i fall by several hundred nanomolar, even during continuous vanadate injection (Fig. 10). This indicates that Na+-dependent [Ca2+]i regulation significantly contributes to the Ca2+ extrusion during inhibition of the plasma membrane Ca2+-ATPase. When the vanadate injection current was turned off, [Ca2+]i decreased further and levelled at a value below 200 nM (but still somewhat higher than the basal [Ca2+]i at the beginning of the experiment).
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Figure 10. Effect of vanadate injection and removal of external Na+ on [Ca2+]i
High-K+-induced [Ca2+]i transients and Em during iontophoretic injection of orthovanadate, in the presence and absence of external Na+.
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Figure 11. Effect of vanadate injection on pHi and [Na+]i
pHi changes (BCECF fluorescence ratio 440 nm/495 nm; A) and [Na+]i (B) during vanadate injection and subsequent removal of external Na+. NH4Cl (20 mM) was applied in A to demonstrate active pH regulation in the cell.
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The recovery of [Ca2+]i from additional Ca2+ loads imposed by a voltage-activated Ca2+ influx in high-K+ saline also appeared to be slowed during vanadate injection. This was analysed in another experimental protocol (see below; Fig. 12).
We also measured the pHi and the [Na+]i during vanadate injection to assess the effects of vanadate on pHi and [Na+]i, which may influence the [Ca2+]i measurements on their own. During 10 min of vanadate injection, the pHi changed by less than 0·1 pH unit (Fig. 11A). Only when the external Na+ was removed did the pHi change, falling at a rate of 0·02 pH units min-1 due to inhibition of Na+-dependent pHi regulation. For comparison, brief application of NH4Cl (20 mM) produced a large intracellular alkalinization and a subsequent acidification by several tenths of a pH unit, followed by pHi recovery. Thus, vanadate injection has only negligible effects on pHi in this glial cell (n = 4).
Due to inhibition of the Na+-K+ pump by vanadate, the [Na+]i increased (Fig. 11B). Following the removal of external Na+, [Na+]i decreased in spite of continuous vanadate injection, which was reversed upon restoring the external Na+ concentration.
The rise in [Na+]i during vanadate injection might force the Na+-Ca2+ exchanger into a reversed mode (see above). Therefore, in another type of experiment, external Na+ was removed before vanadate injection was started (Fig. 12A). In Na+-free saline, vanadate injection induced a [Ca2+]i increase of 427 ± 338 nM (n = 5). In all experiments Na+ re-addition induced a fast decrease in [Ca2+]i of several hundred nanomolar, even during continuous vanadate injection. This indicates that Na+-dependent [Ca2+]i regulation plays a prominent role in this glial cell during inhibition of the plasmalemmal Ca2+-ATPases (see above).
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Figure 12. Effect of vanadate injection on [Ca2+]i and on the recovery from [Ca2+]i loads in the absence of external Na+
A, records of membrane potential (upper trace) and of [Ca2+]i. B and C, normalized [Ca2+]i transients in Na+-containing (B) and Na+-free saline (C). The numbers (1-4) indicate the individual transients that were normalized to compare the [Ca2+]i recoveries.
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Effect of vanadate injection on the recovery from [Ca2+]i loads
[Ca2+]i loads were imposed by applying high-K+ saline (20 mM) to the glial cell. In order to prevent a rise in [Na+]i during vanadate injection (see above, and Fig. 11B), and hence secondary effects on intracellular Ca2+ regulation, vanadate injection was started in the absence of external Na+ (Fig. 12A). The Ca2+ loads imposed were normalized and then superimposed to compare the time course of the recovery (Fig. 12B and C). The [Ca2+]i recovery time constants were determined by a double exponential fit. In three out of five experiments of this kind, the recoveries in Na+-free saline could be fitted with a single exponential. Therefore only the fast time constants were compared. During vanadate injection, this time constant of [Ca2+]i recovery increased significantly from 8·6 ± 6·6 to 23·2 ± 19·8 s (n = 8, P < 0·03) in standard, Na+-containing saline, and from 11·3 ± 6·5 to 33·5 ± 22·5 s (n = 5, P < 0·05) in Na+-free saline. Thus, the Ca2+-ATPase of the plasma membrane appears to play a major role in the recovery from [Ca2+]i loads.
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DISCUSSION |
Measurement of [Ca2+]i and [Na+]i
Our measurements of basal [Ca2+]i are in reasonable agreement with previous [Ca2+]i measurements in the giant glial cells, although our mean value of 79 nM is in the upper range of previously reported values (32 ± 23 nM, Munsch & Deitmer, 1992; 76 ± 17·5 nM, Deitmer & Munsch, 1992).
The mean resting [Na+]i was 1·3 mM lower than the value previously reported for the giant glial cells with ion-sensitive microelectrode measurements (Deitmer, 1991). This might be due to a somewhat higher [Na+]i when the cells were impaled with double- or triple-barrelled microelectrodes, but it cannot be excluded that this discrepancy is due to some error sources of the SBFI calibration procedure. The calibration procedure assumes the equilibration of intra- to extracellular concentrations of the measured ion, due to the presence of ionophores. We have shown for the calibration of the pH-sensitive dye BCECF that this assumption may not necessarily apply for the giant glial cells of the leech (Nett & Deitmer, 1996a).
Na+-dependent regulation of [Ca2+]i
Recently, the presence of a Na+-Ca2+ exchanger in vertebrate glial cells has been reported (Goldmann et al. 1994; Takuma et al. 1994). To our knowledge, the present study is the first that gives evidence for the presence of a Na+-Ca2+ exchanger in invertebrate glial cells, by measuring membrane potential or membrane current simultaneously with the [Ca2+]i.
In the leech giant glial cells, removal of external Na+ induced an increase in [Ca2+]i of about 35 nM. This increase depended on the presence of external Ca2+, indicating a Ca2+ influx through the plasma membrane. It could not be prevented by voltage-clamping the cells or by blocking voltage-activated Ca2+ channels, suggesting that Ca2+ did not enter the cells through voltage-activated Ca2+ channels. This Ca
-dependent increase in [Ca2+]i, induced by removal of extracellular Na+, suggests the presence of a Na+-Ca2+ exchanger in these cells.
[Ca2+]i increases of similar amplitude upon Na+ removal were reported for cultured cortical rat astrocytes (Goldmann et al. 1994), where Na+-Ca2+ exchanger mRNA and protein were identified (Goldmann et al. 1994; Takuma et al. 1994). Small [Ca2+]i increases of about 30 nM upon Na+ removal were also found in rat hippocampal neurones (Mironov, 1995). In cultured mouse hippocampal neurones, removal of external Na+ slowed the recovery from imposed intracellular Ca2+ loads (Koch & Barish, 1994). In contrast, snail neurones do not appear to possess a Na+-Ca2+ exchange, because they did not respond to external Na+ removal with any change in the [Ca2+]i (Kennedy & Thomas, 1995), even when Ca2+-ATPases were blocked with vanadate.
However, attempts to challenge the Na+-Ca2+ exchange in the giant glial cells by increasing the [Na+]i using ouabain failed to accelerate or augment the increase in the [Ca2+]i induced by Na+ removal. An increase in [Ca2+]i during the application of ouabain could only be seen after the cells depolarized, presumably due to the breakdown of the transmembrane K+ gradient. This [Ca2+]i increase was therefore most probably due to a Ca2+ influx through voltage-activated Ca2+ channels.
During our experiments with ouabain, the [Na+]i reached a maximum level of about 15-20 mM during the first 5-10 min of application. In Bergmann glial cells it has been demonstrated that the Na+-Ca2+ exchanger contributes to the Ca2+ influx only when [Na+]i rises above 20-30 mM (Kirishuk, Kettenmann & Verkhratsky, 1997). We therefore cannot exclude the possibility that larger [Na+]i loads are needed to induce a [Ca2+]i rise by activating the reversed mode of the Na+-Ca2+ exchanger.
In our experiments we could not observe any effect of the membrane potential between -60 and -110 mV on the [Ca2+]i increase during Na+ removal, and on the [Ca2+]i recovery rate after Na+ re-addition. There was, however, a slightly positive correlation between Vh and the basal [Ca2+]i, which is in line with the existence of a Na+-Ca2+ exchanger contributing to the maintenance of a low resting [Ca2+]i. The effects of the membrane potential on the Na+-dependent [Ca2+]i rise might possibly be masked by opposite effects on some Ca2+ influx pathways. A more negative membrane potential increases the Ca2+ electrochemical driving force, and hence might raise a Ca2+ influx through Ca2+-permeable leak channels.
In our experiments using high-K+ saline solutions to induce Ca2+ loads, there was no significant contribution of a Na+-dependent mechanism to the recovery from intracellular Ca2+ loads in the leech glial cells, although the equilibrium of the Na+-Ca2+ exchange was calculated to be always more positive than Em. Even when a higher initial value for the basal [Na+]i was used, e.g. 6·9 mM, measured with ion-sensitive microelectrodes (Deitmer, 1991), the calculated ENa-Ca was always more positive than Em.
Thus, in spite of a clearly reproducible [Ca2+]i rise upon external Na+ removal, we could not demonstrate any electrogenicity or dependency on [Na+]i of this [Ca2+]i increase, or any effect of Na+ removal on the recovery from [Ca2+]i loads. Even if some inadequacy of voltage space clamp might have obscured the sensitivity of the Na+-dependent [Ca2+]i rise to membrane potential changes, a lack of sensitivity to the [Na+]i is hard to reconcile with the known properties of a Na+-Ca2+ exchanger. Therefore, the small [Ca2+]i rise upon Na+ removal might have been due to some other, as yet unknown, effect of Na+-free saline. An increased background Ca2+ permeability of the plasma membrane, e.g. induced by Na+ removal, might result in a rise of basal [Ca2+]i. We are not aware of a mechanism reported whereby external Na+ can affect the Ca2+ permeability of the plasma membrane, but this might be worth studying.
When Ca2+-ATPases were blocked with orthovanadate, however, the glial cells responded with a large and reversible [Ca2+]i increase of several hundred nanomolar to the removal of external Na+. This suggests the presence of a Na+-dependent component of the [Ca2+]i regulation under these conditions that might be masked by the action of the plasma membrane Ca2+-ATPase under more physiological conditions, i.e. without inhibition of the Ca2+-ATPases. In contrast, in snail neurones, which have been shown to lack any Na+-Ca2+ exchange, no effect of Na+ removal on [Ca2+]i was observed even during vanadate injection (Kennedy & Thomas, 1995).
In snail neurones and in many other neurones, the plasma membrane Ca2+-ATPase exchanges Ca2+ for H+ (Schwiening, Kennedy & Thomas, 1993). This could lead to an intracellular alkalinization during inhibition of Ca2+ extrusion via this mechanism. The leech giant glial cell, however, showed no significant pHi change upon inhibition of the Ca2+-ATPase with vanadate. This is in line with previous findings that leech giant glial cells, in contrast to snail neurones, regulate their pHi and [Ca2+]i independently (Deitmer, Schneider & Munsch, 1993).
Role of intracellular Ca2+ stores
Inhibition of the intracellular store ATPase with CPA induced a transient increase in [Ca2+]i. This finding is in line with many other studies in which [Ca2+]i increased when intracellular Ca2+ uptake into the endoplasmic reticulum was blocked. Surprisingly, thapsigargin, which is supposed to act irreversibly on the same Ca2+-ATPase (Lytton, Westlin & Hanley, 1991), induced no increase in [Ca2+]i in these glial cells, even at concentrations of up to 1 µM. A similar insensitivity to thapsigargin was reported for Ca2+ stores in bee photoreceptors (B. Walz, personal communication).
The [Ca2+]i increase induced by CPA was relatively small (< 30 nM), compared with the CPA-induced [Ca2+]i increases in mouse astrocytes, which were up to 400 nM (Golovina, Bambrick, Yarowsky, Krueger & Blaustein, 1996). This indicates the existence of Ca2+ stores with possibly a small capacity in the leech giant glial cells. Furthermore, the recovery of [Ca2+]i after an intracellular Ca2+ load was only weakly reduced during application of CPA.
Role of a plasma membrane Ca2+-ATPase
Orthovanadate is an inhibitor of P-type ATPases including the Na+-K+-ATPase, the Ca2+-ATPase of the endoplasmic reticulum and the plasma membrane Ca2+-ATPase. Injection of orthovanadate resulted in a marked increase in [Ca2+]i in the leech giant glial cells. As the experiments with CPA show, intracellular stores play only a minor role in the regulation of [Ca2+]i. An increase in [Na+]i and a possible subsequent reversal of a Na+-Ca2+ exchanger due to the inhibition of the Na+-K+-ATPase could be circumvented by previous removal of external Na+. Thus, the [Ca2+]i increase during orthovanadate injection was most probably due to the inhibition of the plasma membrane Ca2+-ATPases. Our experiments suggested a major role for this Ca2+-ATPase in the maintenance of the basal [Ca2+]i, and in the recovery from intracellular Ca2+ loads. This is somewhat in contrast to reports on cultured astrocytes, in which other mechanisms, e.g. the Na+-Ca2+ exchanger and Ca2+ uptake into the endoplasmic reticulum, contribute greatly to intracellular Ca2+ regulation (Goldmann et al. 1994; Golovina et al. 1996).
A central role for a plasma membrane Ca2+-ATPase has been reported for dorsal root ganglion neurones of the rat (Benham, Evans & McBain, 1992), which also possess a Na+-Ca2+ exchanger, and for snail neurones, in which the Ca2+-ATPase is the only Ca2+ extrusion mechanism (Kennedy & Thomas, 1995).
Since the basal [Ca2+]i was elevated during vanadate injection, there might be a varying influence of Ca2+ buffering on the time course of the Ca2+ recovery. Ca2+ buffer may saturate at the high [Ca2+]i observed, or diffuse relatively slowly, leading to a reduced recovery rate after a Ca2+ load. On the other hand, the rates of Ca2+ extrusion via the plasma membrane and of Ca2+ uptake into intracellular stores are expected to increase at high [Ca2+]i. The relatively fast [Ca2+]i recovery during orthovanadate injection in Na+-free saline might support the presence of additional Ca2+ regulation mechanisms at high [Ca2+]i, e.g. Ca2+ uptake into mitochondria. This has been shown to contribute to the Ca2+ recovery at high levels of [Ca2+]i in various cell types (Thayer & Miller, 1990; White & Reynolds, 1997), but it is as yet unknown whether mitochondrial Ca2+ sequestration contributes to the Ca2+ regulation in the leech glial cells.
At basal levels of [Ca2+]i and up to concentrations of some 100 nM, however, the plasma membrane Ca2+-ATPase appears to be the most important mechanism for intracellular Ca2+ regulation in these cells.
Conclusion
The leech giant glial cells possess different mechanisms leading to either an increase or decrease in [Ca2+]i (summarized in Fig. 13). Activation of voltage-activated Ca2+ channels (Munsch & Deitmer, 1995), ionotropic glutamate receptors of the AMPA/kainate type (Munsch et al. 1994; Munsch & Deitmer, 1997) or metabotropic glutamate receptors (Lohr & Deitmer, 1997) result in an increase in [Ca2+]i. The maintenance of the basal [Ca2+]i in these cells is, according to the present study, predominantly achieved by a plasma membrane Ca2+-ATPase. This mechanism is also the most prominent for the recovery from an intracellular Ca2+ load. Additionally, a Na+-Ca2+ exchanger can contribute to the maintenance of the [Ca2+]i, in particular, when the Ca2+-ATPases are blocked. Ca2+ uptake into the endoplasmic reticulum contributes relatively little to the recovery from Ca2+ loads, and appears not to be involved in the maintenance of the low basal [Ca2+]i.
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Figure 13. Schematic drawing of the mechanisms that influence the [Ca2+]i of the giant glial cell
Ca2+ influx can occur through voltage-activated Ca2+ channels (VACC) and through ionotropic glutamate receptors of the AMPA/kainate type (Glu-AMPA/KA-R). The activation of metabotropic glutamate receptors (mGlu-R) induces Ca2+ release from the endoplasmic reticulum (ER). The extrusion of Ca2+ from the cytoplasm is predominantly achieved by the action of a plasma membrane Ca2+-ATPase. Additionally, Ca2+ uptake into intracellular stores via a CPA-sensitive Ca2+-ATPase contributes to the recovery from Ca2+ loads. Na+-dependent mechanisms may contribute either to a Ca2+ increase or to a decrease, depending on the Na+ transmembrane electrochemical gradient, which is maintained by an ouabain-sensitive Na+-K+-ATPase.
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Acknowledgements
We thank F. W. Lischka and C. Lohr for their comments on an earlier version of the manuscript. We gratefully acknowledge financial support by the Deutsche Forschungsgemeinschaft (De 231/9-2,9-3). W. N. was a recipient of a scholarship of the Landesgraduiertenförderung of Rheinland-Pfalz.
Corresponding author
W. Nett: Abteilung für Allgemeine Zoologie, FB Biologie, Universität Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany.
Email: wnett{at}rhrk.uni-kl.de
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Introduction
