Volume-dependent taurine release from cultured astrocytes requires permissive [Ca2+]i and calmodulin

Alexander A. Mongin1,2, Zhaohui Cai2,3, and Harold K. Kimelberg1,2,3

1 Division of Neurosurgery, 3 Department of Pharmacology and Neuroscience, and 2 Neuroscience and Neuropharmacology Research Group, Albany Medical College, Albany, New York 12208


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell swelling results in regulatory activation of multiple conductive anion pathways permeable toward a broad spectrum of intracellular organic osmolytes. Here, we explore the involvement of extracellular and intracellular Ca2+ in volume-dependent [3H]taurine efflux from primary cultured astrocytes and compare the Ca2+ sensitivity of this efflux in slow (high K+ medium induced) and fast (hyposmotic medium induced) cell swelling. Neither Ca2+-free medium nor Ca2+-channel blockers prevented the volume-dependent [3H]taurine release. In contrast, loading cells with the membrane-permeable Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suppressed [3H]taurine efflux by 65-70% and 25-30% under high-K+ and hyposmotic conditions, respectively. Fura 2 measurements confirmed that BAPTA-AM, but not Ca2+-free media, significantly reduced resting intracellular Ca2+ concentration ([Ca2+]i). The calmodulin antagonists trifluoperazine and fluphenazine reversibly and irreversibly, respectively, inhibited the high-K+-induced [3H]taurine release, consistent with their known actions on calmodulin. In hyposmotic conditions, the effects were less pronounced. These data suggest that volume-dependent taurine release requires minimal basal [Ca2+]i and involves calmodulin-dependent step(s). Quantitative differences in Ca2+/calmodulin sensitivity of high-K+-induced and hyposmotic medium-induced taurine efflux are due to both the effects of the inhibitors on high-K+-induced cell swelling and their effects on transport systems and/or signaling mechanisms determining taurine efflux.

cell swelling; brain edema; anion channels; calcium dependence; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; trifluoperazine; intracellular calcium concentration


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AS IN MOST CELL TYPES, cultured astrocytes exposed to hyposmotic medium undergo swelling and then regulate their volume back toward normal due to activation of K+ and Cl- efflux through volume-sensitive ion channels (3, 39). Some free amino acids, especially taurine, may also participate in cell volume regulation in astrocytes (19, 38, 40), as in other cell types (12). Taurine-deficient astrocytes have higher volumes than control cells and show incomplete volume restoration in hyposmotic medium (30). In neural cells in vivo and in vitro, taurine efflux is more sensitive to volume changes compared with other organic anions and K+ (37, 38, 57). These findings support the view that taurine may play a role as a major cell osmoregulatory molecule in the brain.

Taurine and other amino acids may have a special importance in volume regulation when cell swelling is promoted by increases in extracellular K+, since net efflux of K+ and Cl- is likely to be suppressed under this condition. The high-K+-induced release of taurine and other amino acids from cultured astrocytes is mediated primarily by volume-sensitive anion channels (20, 41, 45). The molecular nature of these channels is currently being investigated (for review, see Refs. 35 and 51). There is very limited work on the intracellular mechanisms linking the change in cell volume with the activation of transport pathways. In the case of hyposmotic swelling, Ca2+ has been considered as a candidate for a volume signaling molecule in astrocytes (2, 32), as in many other cell types (26). The Ca2+ dependence of high-K+-induced taurine release in astrocytes is controversial. Both dependence (43) and independence (24, 41) have been reported. In comparisons of hyposmotic and high-K+-induced astrocytic swelling and volume regulation, it should be noted that, depending on magnitude and rate of volume changes, different regulatory transport pathways and signaling mechanisms may be expected (50). An additional important point is that treatments that inhibit high-K+-induced amino acid release may affect entry of KCl to cause cell swelling in addition to, or instead of, inhibition of volume-dependent efflux, which will also cause inhibition of amino acid release. For hyposmotic-induced swelling, inhibition of the initial volume changes occurring from water influx is deemed much less likely. To help resolve some of these problems, we studied in the present work how treatments that differentially affect intracellular Ca2+ concentration ([Ca2+]i) alter both high-K+ medium- and hyposmotic medium-induced cell volume alterations and associated [3H]taurine release in cultured astrocytes.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
RESULTS
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Materials. [3H]taurine (specific activity 228 mCi/mg) and Na251CrO4 (specific activity 571 mCi/mg) were obtained from Amersham (Arlington Heights, IL) and DuPont NEN Research Products (Boston, MA), respectively. Dispase (neutral protease dispase grade II) was purchased from Boehringer Mannheim (Indianapolis, IN). All cell culture reagents were from GIBCO (Grand Island, NY). Fura 2-AM was purchased from Molecular Probes (Eugene, OR). Flurphenazine-N-2-chloroethane dihydrochloride was from Calbiochem (San Diego, CA). Trifluoperazine, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, and other chemicals, unless otherwise specified, were from Sigma Chemical (St. Louis, MI).

Cell cultures. Primary astrocyte cultures were prepared from the cerebral cortex of newborn Sprague-Dawley rats, as described by Frangakis and Kimelberg (8). The dissociated cells were seeded on poly-D-lysine-coated 18 × 18-mm coverslips (Bellco Biotechnology, Vineland, NJ), and cultures were used after 3-4 wk when the cells reached confluence. Immunocytochemistry showed that >95% of the cells stained positively for the astrocytic marker glial fibrillary acid protein. Cell viability was routinely monitored by trypan blue exclusion and was always >95%.

Isotope efflux measurements. Astrocytes grown on coverslips were incubated in an incubator in 5% CO2-95% air at 37°C overnight in 2.5 ml of MEM containing 10% heat-inactivated horse serum plus 8 µCi/ml [3H]taurine (final concentration of 270 nM). In some experiments, 16 µCi/ml Na251CrO4 (final concentration of 230-270 nM) was also added to the incubation medium. The appearance of 51Cr in the perfusate during release experiments can be used to monitor whether an increase in [3H]taurine release is due to cell detachment or lysis (17).

Before the start of efflux measurements, which were done at 35°C, the cells were preincubated for 10 min in HEPES-buffered solution at 35°C to remove extracellular isotope and serum-containing medium and adapt the cells to experimental conditions. The basal medium for efflux measurements consisted of (in mM) 122 NaCl, 3.3 KCl, 0.4 MgSO4, 1.3 CaCl2, 1.2 KH2PO4, 10 D-glucose, and 25 HEPES. NaOH (10 N) was used to adjust the pH to 7.4. After the washing procedure, the coverslips were inserted into a Lucite perfusion chamber with a depression precisely cut in the bottom to accommodate the 18 × 18 × 0.19-mm (width × height × thickness) glass coverslip. The chamber has a teflon screw top and when screwed down leaves a space above the cells of around 100 µm in height. The cells were perfused at a flow rate of 1.0 ml/min in an incubator set at 34.5-35°C with the basal HEPES-buffered medium (for composition, see above) or with high-K+ medium in which NaCl was replaced with an equimolar amount of KCl to give a final extracellular K+ concentration ([K+]o) of 100 mM. The volume of the chamber above the coverslip is ~32 µl and allows a complete change of the perfusion buffer in <2 min, as directly determined by removal of a trypan blue-containing solution. The osmolarities of all buffers were measured by a freezing point osmometer (Advanced Instruments, Needham Heights, MA) and were in the range of 285-290 mosM. Fractions (1 min) were collected. At the end of experiment, the cells were removed from the coverslips with a solution containing 1% SDS plus 4 mM EDTA. When 51Cr was present, the samples were first counted in a Clini-C LKB 1272 counter (Pharmacia, Gaitherburg, MD). Ecoscint (5 ml, National Diagnostics, Atlanta, GA) was then added, and each fraction was counted for 3H in a Packard Tri-Carb 1900TR liquid scintillation analyzer (Packard Instrument, Meriden, CT) set at 2.0-18.6 keV to exclude 51Cr radioactivity. Percent fractional release for each time point was calculated by dividing radioactivity released in each 1-min interval by the radioactivity left in the cells (the sum of all the radioactive counts in the remaining fractions up to the beginning of the fraction being measured, plus the radioactivity left in the cell digest) and multiplying by 100.

For measurement of hyposmotic-induced [3H]taurine release, an identical experimental design was used. In hyposmotic medium, the concentration of NaCl was reduced by 50 mM (final osmolarity was 190 mosM).

Cell volume measurements. Astrocytic cell volume was measured at 35°C using an electric impedance method (33), with minor modifications. The glass coverslips (no. 1.5; 0.19 ± 0.01 mm thickness) plus cells were perfused at a constant rate of ~0.4-0.5 ml/min in the same Lucite chamber as used for the efflux experiments. Silver wire electrodes were inserted on both sides of the channel. These were soldered to an insulated copper wire and connected in series through a 1 MOmega resistor to a lock-in amplifier (model SR510; Stanford Research Systems, Sunnyvale, CA) that supplied a 500-Hz, 5-V signal to the system. At this frequency, current does not pass through the cells. As the volume of the monolayer increases, the volume of the solution above the cells available for current flow decreases proportionally, resulting in an increase in the measured resistance. Because current is constant due to a 1-MOmega series resistor, changes in voltage are directly proportional to changes in resistance of the solution above the monolayer and therefore directly related to the volume of solution above the cells. Because the resistivities of basal and high-K+ media were different, the resistivity of all experimental solutions was first measured in the chamber containing a blank coverslip, and all results were normalized in terms of medium resistivity. When different drugs were added to solutions, any effect on resistance was also checked but usually no effect was observed. To measure hyposmotic medium-induced volume regulation, 50 mM NaCl in isotonic medium was replaced with 100 mM D-mannitol to assure that Na+ concentration and medium resistivity would not differ between isotonic and hypotonic media (33).

Calibrations and calculations, which were done in previous studies (33), showed that a 1% change in resistance of the volume measurement chamber approximately correspond to a 20-25% change in total volume of the cell monolayer. Because we did not perform calibrations for the experiments presented in this study, all data are given as a normalized resistance signal.

[Ca2+]i microspectrofluorometry. Free [Ca2+]i measurements were performed with a monochromator-based Deltascan spectrofluorometric system (model RF D-4010; PTI, South Brunswick, NJ) with dual excitation at 350- and 380-nm wavelengths and a bandpass of 2 nm. Fluorescent emission was measured at 510 nm using a Nikon Diaphot microscope to which a photomultiplier was attached. Primary cultures on coverslips were loaded with 4 µM fura 2-AM in basal HEPES-buffered medium for 30 min at 37°C. The coverslips were rinsed several times with the same solution without fura 2-AM and allowed to equilibrate at 37°C. The coverslip with loaded cells was then placed in a PDMI-2 open perfusion chamber with a TC-202 bipolar temperature controller (Medical Systems, Greenville, NY) and maintained at 37°C.

Each [Ca2+]i determination was done on a single cell. To change medium in the chamber, basal, high-K+, or drug-containing HEPES-buffered medium was completely changed three times using a Pasteur pipette at the beginning of each 20-min experimental period and then carefully replaced with the same medium at 5-min intervals to mimic perfusion conditions.

Ca2+ calibration was performed for fura 2 in selected cells by equilibrating [Ca2+]i at varying extracellular Ca2+ concentration ([Ca2+]i) values in the presence of 5 µM ionomycin. [Ca2+]i was calculated from the formula of Grynkiewicz et al. (9)
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>d</SUB>&bgr;(R − R<SUB>min</SUB>) / (R<SUB>max</SUB> − R)
Rmax was obtained in a solution containing 4 mM CaCl2 and ionomycin. Rmin was obtained in a solution without Ca2+ and containing 5 mM EGTA, also with 5 µM ionomycin; beta  was the ratio of fluorescence values excited at 380 nm without and with Ca2+. The dissociation constant (Kd) was assumed to be the same as in vitro, under likely similar conditions, namely 224 nM (9).

Statistical analysis. Data are presented as means ± SE of three to eight experiments performed on four different astrocyte preparations. The difference between control and inhibitor-treated groups was analyzed by one-way classification ANOVA followed by comparison using Student's t-test. For multiple comparisons, Bonferonni's correction was applied.


    RESULTS
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Influence of high-K+ medium on cell volume, [3H]taurine release, and [Ca2+]i in primary astrocytic cultures. Exposure of primary astrocytes to 100 mM KCl medium induced a progressively increasing cell swelling (Fig. 1A). When high-K+ medium was replaced with basal medium, cell volume was restored to near initial levels within 5-7 min (Fig. 1A). The high-K+-induced cell swelling was accompanied by stimulation of [3H]taurine release (Fig. 1B). Successive exposures to high K+ medium led to an increase of labeled amino acid release, but each peak was somewhat smaller than the previous one. This made it problematic to quantitatively compare the actions of different drugs on taurine efflux in one experiment. Therefore, we measured both the average maximal fractional release during a 20-min exposure to high [K+]o, as observed at minute 20, and the sum of fractional releases (peak area) over a 20-min exposure to high [K+]o of the first peak of K+-induced amino acid release, which was quite constant regarding both these parameters, under different conditions. Under control conditions, the mean maximal rate of release was 3.79 ± 0.21%/min (n = 8, mean ± SE) and the average area of the peak over the entire 20-min exposure was 29.34 ± 0.53% (n = 8, mean ± SE). The second and subsequent responses were more variable with regard to amplitude and area. Unlike [3H]taurine, 51Cr efflux, used for measuring cell loss or lysis (17), was very low (0.03-0.08%/min) and rarely varied during the whole experimental period in basal and high-K+ conditions (Fig. 1B).


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Fig. 1.   Effect of high-K+ medium on cell volume (A, D, and G), [3H]taurine release (B, E, and H), and intracellular Ca2+ concentration ([Ca2+]i) (C, F, and I) in primary cortical astrocyte cultures under different conditions. A: high-K+-induced astrocytic swelling. Data are means ± SE of 3 experiments. B: [3H]taurine and 51Cr release from astrocytes exposed to medium with KCl concentration isosmotically elevated to 100 mM by replacing NaCl as shown by bars. Results are representative of 8 experiments in 4 different culture preparations. C: [Ca2+]i response in a single cell during a 20-min exposure to medium containing 100 mM KCl. This represents the behavior seen in 2 of 9 cells. See text for further details. D: effect of Ca2+-free medium on high-K+-induced astrocytic swelling. Data are means ± SE of 3 experiments. E: effect of Ca2+-free medium on [3H]taurine and 51Cr release. Results are representative of 4 experiments. F: effect of extracellular Ca2+ deprivation on [Ca2+]i under basal and high K+ conditions. Results are representative of 6 experiments. Arrows indicate 3 complete changes of medium in superfusion chamber at the start of Ca2+ removal. G: effect of loading with 10 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM on astrocytic cell volume under basal and high K+ conditions. Data are means ± SE of 4 experiments. H: influence of loading with 10 µM BAPTA-AM on high-K+-induced [3H]taurine release compared with control performed on the same culture preparation. Results are representative of 5 experiments. I: influence of BAPTA loading on [Ca2+]i levels registered in a single cell. Results are representative of 3 experiments.

We also monitored the influence of high K+ medium on [Ca2+]i under the same conditions and within the time intervals used for isotope efflux studies. Resting [Ca2+]i was calculated to be ~110-130 nM. The single cell responses to high [K+]o were very variable. Two of nine cells showed very sharp increases in [Ca2+]i, up to 600-700 nM with fast normalization within 1-2 min to basal levels; this was followed by a small progressive [Ca2+]i elevation (Fig. 1C). In three other cells, high K+ induced a smaller [Ca2+]i rise (50-150 nM) with slower normalization (within 3-5 min) and no subsequent secondary increase during the 20-min exposure to high K+. This type of [Ca2+]i signal was similar to the second exposure to high K+ shown in Fig 1F. Finally, four cells demonstrated no response to high K+ or even a decrease of [Ca2+]i (data not shown). It should be stressed that astrocytes not responding to high K+ showed a typical [Ca2+]i peak when exposed to 20 µM ATP, as normally seen in cultured astrocytes (18).

Effects of Ca2+-free medium and Ca2+ channel blockers on high-K+-induced cell volume changes, [3H]taurine release, and [Ca2+]i. When we exposed cells to high-K+, Ca2+-free medium containing 50 µM EGTA, K+-evoked cell swelling and [3H]taurine release were shown to be not statistically different from control, high-K+ conditions (summarized in Table 1); this was consistent with previous observations (24, 41). However, because a complete change of medium in our superfusion chamber could be achieved only within 2 min, it left a possibility for activation of Ca2+-dependent intracellular events within this time frame. Therefore, we preincubated cells for 15 min in Ca2+-free conditions before exposing them to elevated [K+]o. Superfusion of cells with Ca2+-free medium containing 50 µM EGTA led to modest cell swelling under basal (low [K+]o) conditions (Fig. 1D, first response). The subsequent high-K+-induced cell swelling in extracellular Ca2+-free medium was somewhat slower but not significantly different compared with control high-K+-induced cell swelling (Fig. 1D).

                              
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Table 1.   Effects of Ca2+-free medium containing 50 µM EGTA, agents affecting intracellular Ca2+, and calmodulin inhibitors on high-[K+]o-induced [3H]taurine release from primary cultured cortical astrocytes

If the cells were exposed to Ca2+-free medium 15 min before and during the first exposure to high [K+]o, the stimulated specific [3H]taurine release, determined as the difference between total [3H]taurine efflux and 51Cr releases both being expressed in the same way as fractional isotope release (see MATERIALS AND METHODS), was inhibited by 20% compared with control high-[K+]o-induced responses (Table 1). Also, the release was inhibited compared with the second exposure to high [K+]o in the same cell preparation (Fig. 1E). As shown in Fig. 1E, exposure to Ca2+-free medium increased cell loss (or lysis) under basal conditions and in high-[K+]o medium, as evaluated by 51Cr efflux. If Ca2+-free media were applied after the first or second exposure to high [K+]o, the increases in cell loss/lysis were much more pronounced (data not shown), probably mirroring lower stability of cell membranes or altered cell adhesion properties.

The replacement of basal medium with Ca2+-free buffer containing 50 µM EGTA (assuming that in nominally Ca2+-free medium Ca2+ concentration is <= 1-2 µM, the calculated free [Ca2+]o is <= 15 nM) decreased [Ca2+]i to a new equilibrium level, which was 10-25 nM less compared with that in basal Ca2+-containing conditions (Fig. 1F). There was no further decrease in [Ca2+]i during a 20-min exposure to Ca2+-free EGTA-containing medium in either basal or high-[K+]o conditions. In two of six measured cells, [Ca2+]o deprivation induced an increase of [Ca2+]i within the first several seconds (Fig. 1F). This first response may be attributed to Ca2+ release from intracellular stores due to extracellular Ca2+ signal or volume changes (61). After cells were returned to Ca2+-containing medium, [Ca2+]i elevated to the starting level and the cells were subsequently able to respond to high K+ (Fig. 1F).

Although we did not find long-lasting alterations of [Ca2+]i after exposure to high-K+ medium in most of the measured cells (see above), we could not exclude that Ca2+ entry into astrocytes during depolarization with high [K+]o (22, 23) contributed to high-[K+]o-induced [3H]taurine release in a subpopulation of the cells and that we are observing only the average effects. The Ca2+-free medium did not seem to be a very good control because of nonspecific alterations in membrane permeability and cell volume increases. Therefore, we checked the effects of several Ca2+ channel blockers on high-[K+]o-induced [3H]taurine release. Concentrations of 500 µM Co2+, 100 µM Cd2+, and 50 µM verapamil have been found to completely inhibit voltage-dependent as well as volume-dependent Ca2+ channels in Madin-Darby canine kidney cells (26) and presynaptic nerve terminals (27). In our experiments, 50 µM verapamil and 500 µM Co2+ produced no significant inhibition of high-[K+]o-induced [3H]taurine release (Table 1). In contrast, 100 µM Cd2+ strongly inhibited the taurine efflux evoked by elevated [K+]o (Table 1).

Effect of intracellular Ca2+ chelators on astrocytic cell volume, [3H]taurine release, and [Ca2+]i. To further evaluate a possible involvement of [Ca2+]i in high-[K+]o-induced amino acid release, we loaded astrocytes with the acetoxymethyl ester of the Ca2+ chelator BAPTA, which is known to permeate cells in vitro (54) and to decrease depolarization- and ligand-induced [Ca2+]i elevation by complexing with Ca2+ (55). To avoid any of the nonspecific alterations observed in Ca2+-free medium, all BAPTA experiments were done in the presence of 1 mM CaCl2. As shown in Fig. 1I, loading with 10 µM BAPTA-AM, even in Ca2+-containing medium, led to a significant decrease in [Ca2+]i, down to levels of 40-50 nM. After washout of external BAPTA-AM, [Ca2+]i only slightly increased. In two of three BAPTA-loaded cells, we observed slight increases in [Ca2+]i following 100 mM [K+]o challenge; the most pronounced response is presented in Fig. 1I.

Volume measurements showed that BAPTA-AM pretreatment failed to affect high-[K+]o-induced astrocytic swelling (Fig. 1G). However, under basal conditions, loading with BAPTA-AM induced a fast and drastic volume increase followed by volume regulation with a time course similar to that previously observed in hyposmotic medium (33, 39, 40).

The 30-min superfusion with 10 µM BAPTA-AM before application of high-[K+]o medium inhibited K+-evoked [3H]taurine release by 60-70% (Fig. 1H, Table 1). Loading with 3 µM BAPTA-AM (see Fig. 3B) or with 4 µM fura 2-AM (Fig. 2, Table 1) produced no significant inhibition of the high-[K+]o-induced [3H]taurine release. Under basal conditions, an initial transient [3H]taurine release was observed in some 10 µM BAPTA-AM-treated (Fig. 3A) and 4 µM fura 2-AM-treated (Fig. 2) cells.


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Fig. 2.   Influence of loading with 4 µM fura 2-AM (20 min) on basal and high-K+-induced [3H]taurine release from primary cultured astrocytes compared with control performed on the same day using the same culture preparation. Results are representative of 3 experiments.



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Fig. 3.   Effect of loading with BAPTA-AM on hyposmotic medium-induced cell volume changes and taurine release in primary astrocytes. A: effect of superfusion with 3 and 10 µM BAPTA-AM (30 min) on the swelling-induced [3H]taurine release compared with control performed on the same day using the same culture preparation. Each curve represents 3-5 experiments. B: comparison of sensitivity of hyposmotic medium-induced (open bars) and high-K+-induced (hatched bars) astrocytic [3H]taurine release to loading with BAPTA-AM. Maximal rates of fractional release under hyposmotic conditions or during 20-min exposure to high K+ medium were normalized to respective controls. Data are means ± SE of 3-6 experiments. * P < 0.05, hyposmotic vs. high-K+ conditions. C: effect of loading with 10 µM BAPTA-AM on cell volume under basal and hyposmotic conditions. Data are means ± SE of 3 experiments.

Comparison of the sensitivity of high-[K+]o- and hyposmotic medium-induced [3H]taurine release to changes in [Ca2+]i. Volume-dependent taurine release under hyposmotic conditions was less sensitive to chelation of intracellular Ca2+ compared with the release induced by elevated [K+]o. As seen in Fig. 3, A and B, a 30-min preexposure to 3 µM BAPTA-AM was ineffective in preventing hyposmotic medium-induced [3H]taurine release; 10 µM BAPTA-AM produced a moderate 25-30% inhibition of the release vs. 75% inhibition of the release under hyposmotic and high-[K+]o conditions, respectively.

Cell volume measurements performed under similar conditions showed decreased hyposmotic swelling of the BAPTA-treated cells, whereas the rate of regulatory volume decrease (RVD) was not significantly changed (Fig. 3C, exposure to hyposmotic medium). Once again, under basal conditions, exposure of cells to 10 µM BAPTA-AM led to drastic transient increases in cell volume followed by an isosmotic volume restoration (Fig. 3C and Fig. 1G, exposure to BAPTA-AM).

Influence of calmodulin antagonists on high-[K+]o- and hyposmotic medium-induced [3H]taurine release. Because volume regulation in hyposmotic medium and the associated activation of K+ and anion fluxes are calmodulin-dependent processes in many cells (for review, see Ref. 26), we checked the effect of calmodulin inhibitors on [3H]taurine efflux under conditions of raised external K+. A reversible calmodulin inhibitor trifluoperazine (25) reversibly reduced high-[K+]o-induced [3H]taurine release (Fig. 4A and Table 1). The inhibition was concentration dependent, with an IC50 of ~10 µM (Fig. 4B). Long-term treatment, i.e., more than 30 min, with 25 µM trifluoperazine led to a progressive increase of cell loss evaluated as 51Cr release and [3H]taurine efflux, which was presumably due to cytotoxic effects of this drug (data not shown). At concentrations higher than 25 µM, increases of both basal and high-K+-induced amino acid release (Fig. 4B) and cell loss measured with 51Cr (data not shown) were observed even after short-term exposures (<= 20 min) to trifluoperazine. The irreversible calmodulin inhibitor fluphenazine (1) produced potent (over 80%) inhibition of swelling-induced [3H]taurine efflux at a concentration of 25 µM (Fig. 4C, Table 1). As expected, its effect was completely irreversible (Fig. 4C).


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Fig. 4.   Effect of calmodulin inhibitors on volume-dependent [3H]taurine release. A: effect of 25 µM trifluoperazine on high-K+-induced taurine release compared with control performed on the same culture preparation. Results are representative of 5 experiments. B: dose-response curve for the trifluoperazine effect on the maximal rate of fractional [3H]taurine release during a 20-min exposure to 100 mM KCl + trifluoperazine. Calmodulin inhibitor was added 20 min before and during exposure to high K+. Each point is mean ± SE of 3-8 experiments in 4 different culture preparations. C: effect of 25 µM fluphenazine-N-2-chloroethane dihydrochloride on high-K+-induced taurine release compared with control release. Results are representative of 3 experiments. D: effect of 10 and 25 µM trifluoperazine on hyposmotic medium-induced [3H]taurine release compared with control performed on the same culture preparation on the same day. Osmolarity of hypotonic medium was 190 mosM. E: comparison of dose-response curves for the trifluoperazine effects on taurine release under hyposmotic (, ) and high-K+ (black-triangle, triangle ) conditions. Maximal fractional %releases (solid symbols) or areas of the peak (open symbols) were normalized to the control values. Each point represents mean ± SE of 3-6 experiments. * P < 0.05, hyposmotic vs. high-K+ conditions. F: effect of 25 µM trifluoperazine on high-K+-induced astrocytic swelling. Data are means ± SE of 3 (control) or 5 (trifluoperazine) experiments.

Under high-[K+]o conditions, the effect of calmodulin antagonists on taurine release may be due to specific inhibition of calmodulin-dependent volume signal transduction, direct interaction of the drugs with a volume-dependent anion channel, or inhibition of KCl-induced cell swelling. To help distinguish these possibilities, we studied the influence of trifluoperazine on [3H]taurine release under hyposmotic conditions. In this case, anticalmodulin agents appeared not to affect cell swelling due to water influx following the imposed osmotic gradient (2) but did block swelling-induced K+ release (3, 58) and cell volume regulation (2, 58). The maximal fractional efflux rate of hyposmotic medium-induced [3H]taurine release varied between different culture preparations. Therefore, we compared the influence of trifluoperazine on control values of efflux measured in the same cell preparation on the same day. Astrocytes were pretreated with different concentrations of trifluoperazine for 15-20 min and then subjected to an osmotic gradient of 100 mosM. Both maximal fractional release and area of peak were compared with control values. Trifluoperazine was a less effective blocker of volume-dependent amino acid release in hyposmotic medium compared with high-[K+]o conditions (Fig. 4, D and E). Also, in contrast to the identical effects of trifluoperazine on maximal rate and area of high-[K+]o-induced taurine release, we found that the area of the hyposmotic release is less sensitive to trifluoperazine pretreatment than the maximal fractional release: 15% vs. 40% inhibition for area and fractional release, respectively (Fig. 4E). This discrepancy may be due to confounding effects of calmodulin antagonists on RVD (see discussion).

To evaluate the possibility of whether the trifluoperazine effects were due to decreased cell swelling, we studied its influence on high-[K+]o-induced cell volume increase. Trifluoperazine at 25 µM inhibited K+-evoked astrocytic swelling (Fig. 4F). Unlike BAPTA-AM, it did not affect cell volume under basal isosmotic conditions.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is well established that brain tissue swells in isosmotic high K+ medium (5, 6), which may be used as a model for cytotoxic (cellular) edema (16). In pathophysiological conditions, such as ischemia, anoxia, or head injury, external K+ can rise to 80 mM (11, 48). The main cell type contributing to brain cellular edema seems to be astroglial cells, which in vitro accumulate K+ both by Donnan uptake of KCl and through a diuretic-sensitive carrier mechanism (16, 59, 60). In brain tissue as well as in cell cultures, K+-evoked astrocytic swelling is slow and is not followed by cell volume regulation, presumably due to the inability of the cells to lose KCl in the face of high extracellular K+ (33, 44). Several studies have shown mobilization of taurine under conditions that likely increase cell volume in brain (47, 57). Swelling-induced release of taurine in pathophysiological conditions in vivo may be beneficial due to partial compensation for cell volume increases or diminution of the level of neuronal excitability, since taurine is an inhibitory neuromodulator (14). In the supraoptic nucleus, volume-dependent taurine efflux from glial cells was found to inhibit neuronal activity and was suggested to contribute to a neuroendocrine loop of body fluid homeostasis through the modulation of vasopressin release (7, 13).

External Ca2+ deprivation does not prevent high-K+-induced taurine efflux but does affect glial cell volume and taurine release under basal conditions. In general, our findings confirm previous observations that high-K+-induced release of [3H]taurine (24, 41) and D-[3H]aspartate (45) is independent of extracellular Ca2+. We have not found any significant effect of Ca2+-free medium on K+-induced [3H]taurine release when extracellular Ca2+ was omitted during exposure to high [K+]o and observed only 15-20% inhibition of response when Ca2+ was omitted 15 min before and during [K+]o elevation. The latter effect may be due to small inhibition of cell swelling, which was below the limit of statistical significance (see Fig. 1D). However, as seen from our data, complications of experiments in which Ca2+-free medium is used include nonspecific alterations of plasma membrane permeability, cell lysis, or cell detachment as well as [Ca2+]o-dependent cell volume changes. Such possibilities are not usually checked. We observed loss of radioactivity from astrocytes preloaded with 51Cr in EGTA-containing Ca2+-free medium as well as in nominally Ca2+-free medium. These effects increased with time of superfusion, and, after 30-40 min, the nonspecific [Ca2+]o-dependent [3H]taurine release exceeded values of the specific release by severalfold (not shown). Interestingly, exposure to high-[K+]o medium decreased 51Cr efflux evoked by Ca2+ deprivation, possibly by making the membranes more stable by diminishing the transmembrane electric potential, as seen in phospholipid bilayers (52). The specific [3H]taurine release (corrected for cell loss/lysis) was still significantly stimulated in basal Ca2+-free conditions. Measurements of cell volume revealed that deprivation of extracellular Ca2+ induced fast and relatively long-lasting cell swelling, which also contributed to the [Ca2+]o-dependent taurine release. Cell swelling under Ca2+-free conditions has previously been shown in a suspension of cultured astrocytes by Olson et al. (36) using a Coulter counter for cell volume monitoring. Nonspecific alterations of membrane permeability, cell loss/lysis, and/or volume changes induced by Ca2+-free medium may also explain some data in previous studies. A pronounced increase of basal taurine release in Ca2+-free EGTA-containing medium has been shown in brain slices (34), synaptosomes (46), and cultured astrocytes (10, 43).

The Ca2+ channel blockers verapamil (50 µM) and Co2+ (500 µM), known to suppress voltage- and volume-dependent Ca2+ channels (26, 27), did not significantly affect high-[K+]o-evoked [3H]taurine release, supporting its independence of [Ca2+]o. Inhibition by 100 µM Cd2+ may be due to direct inhibition of a swelling-activated anion channel, since blockade of astrocytic anion channels by millimolar concentrations of Cd2+ and Zn2+ has been demonstrated using the excised patch-clamp technique (15). Also, in human astrocytoma cells, the voltage-dependent anion currents were suppressed approximately twofold by concentrations of Cd2+ and Zn2+ as low as 25 and 100 µM, respectively (56).

Evidence for the requirement of a minimal basal [Ca2+]i for K+-evoked stimulation of taurine efflux. Only ~50% of cultured cells in our preparations showed immediate K+-evoked elevations of [Ca2+]i. The absence of a depolarization-induced [Ca2+]i signal in approximately half of the cells may be because not all the astrocytes in our cultures express a sufficient density of voltage-dependent Ca2+ channels. In earlier work, cultured mouse astrocytes expressing L-type voltage-dependent Ca2+ channels demonstrated quite different responses to high [K+]o, ranging from elevation to a significant decrease depending on the age of the culture (23). In our experiments, observations showed that [Ca2+]i increased only during the first 2-5 min and then returned to near basal levels. A subsequent secondary long-lasting [Ca2+]i rise was rarely seen. The specificity of this secondary response may be placed in doubt, and it may be ascribed to unstable Ca2+ levels over long time intervals employed in our experiments. Therefore, the gradual increase of amino acid efflux always observed during the whole period of exposure to high [K+]o is not associated with a general measured rise in [Ca2+]i.

In contrast to Ca2+-free medium, loading of astrocytes with BAPTA-AM potently inhibited the high-[K+]o-induced [3H]taurine release even in Ca2+-containing medium. As verified by measurements of fura 2 fluorescence, exposure to BAPTA-AM decreased [Ca2+]i to 45-60 nM, which is close to the minimal [Ca2+]i of 50 nM required for activation of volume-dependent anion channels in vascular endothelial cells (53). However, in different cell types, the dependence on [Ca2+]i may vary. In Ehrlich ascites tumor cells, volume-activated anion currents were observed at [Ca2+]i buffered to 25 nM (42).

A possible problem with [Ca2+]i measurements is that the average fura 2 signal does not accurately reflect local Ca2+ concentration, which may by of primary influence. If we compare Fig. 1G with Fig. 1I, it may be seen that loading with BAPTA-AM led to fast cell swelling within 2 min, whereas the average Ca2+ concentration dropped below a presumed "permissive" level within 4-6 min. Why didn't we observe any immediate volume-dependent taurine release? The explanation may be that local [Ca2+]i near the membrane is decreased faster than the average measurable [Ca2+]i. In Ca2+-free medium (Fig. 1, D and E) or when lower Ca2+ chelator concentrations were applied (Fig. 2), isosmotic cell swelling did induce taurine release. An additional issue has been recently raised by Speake et al. (49) who showed that the lack of [Ca2+]i elevation in lacrimal acinar cells loaded with fura 2 and exposed to hyposmotic medium is artifactual due to buffering of cytosolic Ca2+ by fura 2. RVD in these cells was also suppressed when they were loaded with fura 2. We did not find any significant effect of loading with fura 2 on high-K+-induced taurine release and cell volume changes in our preparation. Therefore, even if fura 2 increased intracellular Ca2+ buffering capacity and interfered with high-[K+]o-induced internal Ca2+ rise, which we cannot completely exclude, it was not significant to affect volume-dependent processes induced under high-[K+]o conditions.

Involvement of calmodulin in high-[K+]o-induced cell swelling and taurine release from cultured astrocytes. The main intracellular pathway for Ca2+-dependent regulation of cellular processes is activation of calmodulin and subsequent stimulation of Ca2+/calmodulin-dependent enzymes such as protein kinases (4, 31). It is well established that calmodulin is involved in hyposmotic medium-induced RVD and electrolyte efflux in many cell types (21, 26), including cultured astrocytes (2, 3, 58). Because swelling under conditions of raised K+ differs from hyposmotic swelling with respect to both the rate and the magnitude of the volume changes, different ion transport and intracellular signaling mechanisms may be activated (50). To our knowledge, no data exist on the involvement of calmodulin in high-[K+]o-induced astrocytic cell volume increase and associated amino acid fluxes. We found that the reversible calmodulin antagonist trifluoperazine potently and reversibly inhibited high-[K+]o-evoked taurine release. The IC50 of ~10 µM for this inhibition was comparable to the Kd of 5.8 µM for binding of trifluoperazine to calmodulin in vitro (25). The irreversible calmodulin antagonist fluphenazine also produced irreversible effects in our system.

Is activation of volume-sensitive anion channel(s) or (K+ + Cl-) accumulation under high-K+ conditions the calmodulin-dependent step(s)? In the case of hyposmotic swelling, calmodulin antagonists did not affect astrocytic volume increase but did inhibit RVD (2, 58). The inhibitory effect of trifluoperazine on the early rates of [3H]taurine release in hyposmotic medium thus shows that the volume-dependent anion channel itself or volume signal transduction machinery is a likely target for calmodulin antagonists. However, if we calculated the effect of trifluoperazine on total area of the hyposmotic medium-induced [3H]taurine release, it seemed less inhibited compared with the maximal fractional percent release (Fig. 4E). This was in contrast to uniform effects of the drug on maximal release rate and area of high-[K+]o-induced release (Fig. 4E). We explain this paradox by the effect of trifluoperazine on volume regulation. Prolonged hyposmotic cell swelling in the presence of trifluoperazine (2, 58) masks the inhibitory effect of the drug on the anion channel. As shown in Fig. 4D, 25 µM trifluoperazine clearly inhibited the initial hyposmotic [3H]taurine release, whereas, at the 7th-10th min of exposure, we see a stimulation compared with control release. Thus the high sensitivity of the K+-evoked [3H]taurine release to calmodulin antagonists may involve two mechanisms, i.e., a direct effect on the anion channel or channel regulatory machinery and via suppression of high-[K+]o-induced cell swelling (Fig. 4F).

Does high-K+-induced and hyposmotic medium-induced taurine release from cultured astrocytes involve different transport and signaling mechanisms? Both hyposmotic- and high-[K+]o-induced [3H]taurine release in our experiments were sensitive to chelation of intracellular Ca2+ and calmodulin inhibitors, revealing a qualitative similarity of volume-dependent transport and signaling mechanisms under both conditions. However, a more detailed comparison of the BAPTA-AM and trifluoperazine effects showed some differences. Thus high-K+-induced [3H]taurine release was more sensitive to both [Ca2+]i chelation and calmodulin inhibition. A reasonable explanation for this is the inhibitory effects on (K+ + Cl-) accumulation and cell swelling. This is true in the case of calmodulin inhibitors but seems unlikely for the action of intracellular Ca2+ chelators. Alternatively, it could be that different types of anion channels contribute to hyposmotic medium- and high-[K+]o medium-induced [3H]taurine release. Our recent observation on two components of hyposmotically induced [3H]taurine efflux, sensitive and insensitive to inhibition of tyrosine kinases, supports the latter possibility (29). We have found that K+-evoked astrocytic D-[3H]aspartate release also requires permissive [Ca2+]i and is sensitive to BAPTA-AM loading, similar to high-[K+]o-induced taurine release (28). It has been shown that loading of brain cells in vivo with Ca2+ chelators strongly reduces ischemic brain injury, which was explained by prevention of excitotoxic neuronal death (55). Some component of protection by chelators may be due to inhibition of excitatory amino acid (EAA) release from astrocytes (16). It also seems that calmodulin inhibitors may be useful in the search for new pharmacological agents for treatment of ischemia and brain edema. Possible roles for their action include prevention of cytotoxic cell swelling and suppression of volume-dependent EAA release.

In conclusion, volume-dependent [3H]taurine release requires a permissive level of [Ca2+]i, in agreement with an earlier observed requirement of a volume-dependent anion channel for [Ca2+]i (53). However, high-[K+]o medium- and hyposmotic medium-induced amino acid release show quantitative differences in intracellular Ca2+ sensitivity. This may be due to multiple anion transport pathways activated under hyposmotic conditions, as was established in a recent study (29), some of which may be Ca2+ insensitive or possesses low Ca2+ sensitivity. In addition, high-[K+]o-induced astrocytic swelling is insensitive (or possesses low sensitivity) to alterations of extracellular and intracellular Ca2+ concentration. Calmodulin inhibitors suppress both the hyposmotic medium-induced and to a larger extent the high-[K+]o-evoked [3H]taurine release and also inhibit high-[K+]o-induced cell swelling. Therefore, the pronounced effects of calmodulin antagonists on the high-[K+]o-induced taurine release likely involve both a direct effect on the swelling-activated channel or channel regulatory machinery and an indirect action via inhibition of cell swelling.


    ACKNOWLEDGEMENTS

We are grateful to Carol Charniga for expert preparation of astrocytic cultures.


    FOOTNOTES

This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-35205 to H. K. Kimelberg and by International Research Fellowship Award F05 TW-05329 from the Fogarty International Center, National Institutes of Health, to A. A. Mongin.

Present address of A. A. Mongin: Institute of Photobiology, Belarussian Academy of Sciences, Minsk 220072, Belarus.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: H. K. Kimelberg, Div. of Neurosurgery, MC-60, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (Email: hkimelberg{at}ccgateway.amc.edu).

Received 17 March 1999; accepted in final form 25 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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