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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 M 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-M
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).
[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)
![]() |
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
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).
|
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.
|
|
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).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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+ + ClDoes
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.
![]() |
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alvarez, J.,
M. Montero,
and
J. Garcia-Sancho.
Cytochrome P-450 may link intracellular Ca2+ stores with plasma membrane Ca2+ influx.
Biochem. J.
274:
193-197,
1991[Medline].
2.
Bender, A. S.,
J. T. Neary,
J. Blicharska,
L.-O. B. Norenberg,
and
M. D. Norenberg.
Role of calmodulin and protein kinase C in astrocytic cell volume regulation.
J. Neurochem.
58:
1874-1882,
1992[Medline].
3.
Bender, A. S.,
and
M. D. Norenberg.
Calcium dependence of hypoosmotically induced potassium release in cultured astrocytes.
J. Neurosci.
14:
4237-4243,
1994[Abstract].
4.
Berridge, M. J.
Inositol triphosphate and calcium signalling.
Nature
361:
315-325,
1993[Medline].
5.
Bourke, R. S.,
H. K. Kimelberg,
C. R. West,
and
A. H. Bremer.
The effect of HCO3 on the swelling and ion uptake of monkey cerebellar cortex under conditions of raised extracellular potassium.
J. Neurochem.
25:
323-328,
1975[Medline].
6.
Bourke, R. S.,
and
D. B. Tawer.
Fluid compartmentation and electrolytes of cat cerebral cortex in vitro. I. Swelling and solute distribution in mature cerebral cortex.
J. Neurochem.
13:
1071-1097,
1966[Medline].
7.
Delouze, C.,
A. Duvoid,
and
N. Hussy.
Properties and glial origin of osmotic-dependent release of taurine from the rat supraoptic nucleus.
J. Physiol. (Lond.)
507:
463-471,
1998
8.
Frangakis, M. V.,
and
H. K. Kimelberg.
Dissociation of neonatal rat brain by dispase for preparation of primary astrocyte cultures.
Neurochem. Res.
9:
1689-1698,
1984[Medline].
9.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985[Abstract].
10.
Hallopainen, I.,
P. Kontro,
and
S. S. Oja.
Release of preloaded taurine and hypotaurine from astrocytes in primary culture: stimulation by Ca2+-free media.
Neurochem. Res.
10:
123-131,
1985[Medline].
11.
Hansen, A. J.
Effect of anoxia on ion distribution in the brain.
Physiol. Rev.
65:
101-148,
1985
12.
Hoffmann, E. K.,
and
I. H. Lambert.
Amino acid transport and cell volume regulation in Ehrlich ascites tumor cells.
J. Physiol. (Lond.)
338:
613-625,
1983[Abstract].
13.
Hussy, N.,
C. Deleuze,
A. Pantaloni,
M. G. Desarmenien,
and
F. Moos.
Agonist action of taurine on glycine receptors in rat supraoptic magnocellular neurons: possible role in osmoregulation.
J. Physiol. (Lond.)
502:
609-621,
1997[Abstract].
14.
Huxtable, R. J.
Physiological actions of taurine.
Physiol. Rev.
72:
101-163,
1992
15.
Jalonen, T.,
V. Varga,
K. Harticainen,
R. Janaky,
and
S. S. Oja.
Anion conductance blocked by divalent cations in cultured rat astrocytes.
Ann. NY Acad. Sci.
633:
583-585,
1991[Medline].
16.
Kimelberg, H. K.
Current concepts of brain edema. Review of laboratory investigations.
J. Neurosurg.
83:
1051-1059,
1995[Medline].
17.
Kimelberg, H., K.,
D. J. Bonville,
and
S. Goderie.
Use of 51Cr cell labelling to distinguish between release of radiolabelled amino acids from primary astrocyte cultures being due to efflux or cell damage.
Brain Res.
622:
237-242,
1993[Medline].
18.
Kimelberg, H., K.,
Z. Cai,
P. Rastogi,
C. J. Charniga,
S. Goderie,
V. Dave,
and
T. O. Jalonen.
Transmitter-induced calcium responses differ in astrocytes acutely isolated from rat brain and in culture.
J. Neurochem.
68:
1088-1098,
1997[Medline].
19.
Kimelberg, H., K.,
S. K. Goderie,
S. Higman,
S. Pang,
and
R. A. Waniewski.
Swelling-induced release of glutamate, aspartate, and taurine from astrocyte cultures.
J. Neurosci.
10:
1583-1591,
1990[Abstract].
20.
Kimelberg, H. K.,
E. Rutledge,
S. Goderie,
and
C. Charniga.
Astrocytic swelling due to hypotonic or high K+ medium causes inhibition of glutamate and aspartate uptake and increases their release.
J. Cereb. Blood Flow Metab.
15:
409-416,
1995[Medline].
21.
Kirk, J.,
and
K. Kirk.
Inhibition of volume-activated I and taurine efflux from HeLa cells by P-glycoprotein blockers correlates with calmodulin inhibition.
J. Biol. Chem.
269:
29389-29394,
1994
22.
MacVicar, B. A.
Voltage-dependent Ca channels in glial cells.
Science
226:
1345-1347,
1984[Medline].
23.
MacVicar, B. A.,
D. Hochman,
M. J. Delay,
and
S. Weiss.
Modulation of intracellular Ca2+ in cultured astrocytes by influx through voltage-activated Ca2+ channels.
Glia
4:
448-455,
1991[Medline].
24.
Martin, D. L.,
V. Madelian,
B. Seligmann,
and
W. Shain.
The role of osmotic pressure and membrane potential in K+-stimulated taurine release from cultured astrocytes and LRM55 cells.
J. Neurosci.
10:
571-577,
1990[Abstract].
25.
Massom, L.,
H. Lee,
and
H. W. Jarrett.
Trifluoperazine binding to porcine brain calmodulin and skeletal muscle troponin C.
Biochemistry
29:
671-681,
1990[Medline].
26.
McCarty, N. A.,
and
R. G. O'Neil.
Calcium signaling in cell volume regulation.
Physiol. Rev.
72:
1037-1061,
1992
27.
Mongin, A. A.,
S. L. Aksentsev,
S. N. Orlov,
and
S. V. Konev.
Hypoosmotic shock activates Ca2+ channels in isolated nerve terminals.
Neurochem. Int.
31:
835-843,
1997[Medline].
28.
Mongin, A. A.,
Z. Cai,
and
H. K. Kimelberg.
Intracellular Ca2+ and calmodulin are involved in high K+-induced astrocytic swelling and amino acid release (Abstract).
Soc. Neurosci. Abstr.
24:
2013,
1998.
29.
Mongin, A. A.,
J. M. Reddi,
C. Charniga,
and
H. K. Kimelberg.
[3H]taurine and D-[3H]aspartate release from astrocyte cultures are differentially regulated by tyrosine kinases.
Am. J. Physiol.
276 (Cell Physiol. 45):
C1226-C1230,
1999
30.
Moran, J.,
T. E. Maar,
and
H. Pasantes-Morales.
Impaired cell volume regulation in taurine deficient cultured astrocytes.
Neurochem. Res.
19:
415-420,
1994[Medline].
31.
Niki, I.,
H. Yokokura,
T. Sudo,
M. Kato,
and
H. Hidaka.
Ca2+ signalling and intracellular Ca2+ binding proteins.
J. Biochem. (Tokyo)
120:
685-698,
1996[Abstract].
32.
O'Connor, E., R.,
and
H. K. Kimelberg.
Role of calcium in astrocyte volume regulation, ion and amino acid release.
J. Neurosci.
13:
2638-2650,
1993[Abstract].
33.
O'Connor, E., R.,
H. K. Kimelberg,
C. R. Keese,
and
I. Giaever.
Electrical impedance method for measuring volume changes in astrocytes.
Am. J. Physiol.
264 (Cell Physiol. 33):
C471-C478,
1993
34.
Oja, S. S.,
and
P. Kontro.
Cation effects on taurine release from brain slices: comparison to GABA.
J. Neurosci. Res.
17:
302-311,
1987[Medline].
35.
Okada, Y.
Volume expansion-sensing outward-rectifier Cl channel: fresh start to the molecular identity and volume sensor.
Am. J. Physiol.
273 (Cell Physiol. 42):
C755-C789,
1997
36.
Olson, J. E.,
D. Fleischhacker,
W. B. Murray,
and
D. Holtzman.
Control of astrocyte volume by intracellular and extracellular Ca2+.
Glia
3:
405-412,
1990[Medline].
37.
Olson, J. E.,
and
G. Z. Li.
Increased potassium, chloride and taurine conductance in astrocytes during hypoosmotic swelling.
Glia
20:
254-261,
1997[Medline].
38.
Pasantes-Morales, H.,
J. Moran,
and
A. Shousboe.
Volume-sensitive release of taurine from cultured astrocytes: properties and mechanism.
Glia
3:
427-432,
1990[Medline].
39.
Pasantes-Morales, H.,
R. A. Murray,
L. Lilja,
and
J. Moran.
Regulatory volume decrease in cultured astrocytes. I. Potassium-and chloride-activated permeability.
Am. J. Physiol.
266 (Cell Physiol. 35):
C165-C171,
1994
40.
Pasantes-Morales, H.,
R. A. Murray,
R. Sanchez-Olea,
and
J. Moran.
Regulatory volume decrease in cultured astrocytes. II. Permeability pathway to amino acids and polyols.
Am. J. Physiol.
266 (Cell Physiol. 35):
C172-C178,
1994
41.
Pasantes-Morales, H.,
and
A. Schousboe.
Release of taurine from astrocytes during potassium-evoked swelling.
Glia
2:
45-50,
1989[Medline].
42.
Pedersen, S. F.,
J. Prenen,
G. Droogmans,
E. K. Hoffmann,
and
B. Nilius.
Separate swelling- and Ca2+-activated anion currents in Ehrlich ascites tumor cells.
J. Membr. Biol.
163:
97-110,
1998[Medline].
43.
Philibert, R. A.,
K. L. Rogers,
A. J. Allen,
and
G. R. Dutton.
Dose-dependent K+-stimulated efflux of endogenous taurine from primary astrocyte cultures is Ca2+-dependent.
J. Neurochem.
51:
122-126,
1988[Medline].
44.
Rutledge, E. M.,
M. Ashner,
and
H. K. Kimelberg.
Pharmacological characterization of swelling-induced D-[3H]aspartate release from primary astrocytic cultures.
Am. J. Physiol.
274 (Cell Physiol. 43):
C1511-C1520,
1998
45.
Rutledge, E. M.,
and
H. K. Kimelberg.
Release of [3H]-D-aspartate from primary astrocyte cultures in response to raised external potassium.
J. Neurosci.
16:
7803-7811,
1996
46.
Sieghart, W.,
and
K. Heckl.
Potassium-evoked release of taurine from synaptosomal fractions of cerebellar cortex.
Brain Res.
116:
538-543,
1976[Medline].
47.
Solis, J. M.,
A. S. Herranz,
O. Herreras,
and
R. Martin del Rio.
Weak organic acids induce taurine release through an osmotic-sensitive process in in vivo rat hippocampus.
J. Neurosci. Res.
26:
159-167,
1990[Medline].
48.
Somjen, G., G.
Extracellular potassium in the mammalian central nervous system.
Annu. Rev. Physiol.
41:
159-177,
1979[Medline].
49.
Speake, T.,
I. J. Douglas,
and
P. D. Brown.
The role of calcium in the volume regulation of rat lacrimal acinar cells.
J. Membr. Biol.
164:
283-291,
1998[Medline].
50.
Strange, K.
Are all volume changes the same?
News Physiol. Sci.
9:
223-228,
1994.
51.
Strange, K.,
F. Emma,
and
P. S. Jackson.
Cellular and molecular physiology of volume-sensitive anion channels.
Am. J. Physiol.
270 (Cell Physiol. 39):
C711-C730,
1996
52.
Sugar, I. P.
The effects of external electric fields on the structure of lipid bilayers.
J. Physiol. Paris
77:
1035-1042,
1981[Medline].
53.
Szucs, G.,
S. Heinke,
G. Droogmans,
and
B. Nilius.
Activation of the volume-sensitive chloride current in vascular endothelial cells requires a permissive intracellular Ca2+ concentration.
Pflügers Arch.
431:
467-469,
1996[Medline].
54.
Tsien, R. Y.
A non-disruptive technique for loading calcium buffers and indicators into cells.
Nature
290:
527-528,
1981[Medline].
55.
Tymianski, M.,
M. C. Wallace,
I. Spigelman,
M. Uno,
P. L. Carlen,
C. H. Tator,
and
M. P. Charlton.
Cell-permeant Ca2+ chelators reduce early excitotoxic and ischemic neuronal injury in vitro and in vivo.
Neuron
11:
221-235,
1993[Medline].
56.
Ullrich, N.,
and
H. Sontheimer.
Biophysical and pharmacological characterization of chloride currents in human astrocytoma cells.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1511-C1521,
1996
57.
Verbalis, J. G.,
and
S. R. Gullans.
Hyponatremia causes large sustained reduction in brain content of multiple organic osmolytes in rats.
Brain Res.
567:
274-282,
1991[Medline].
58.
Vitarella, D.,
D. J. DiRisio,
H. K. Kimelberg,
and
M. Aschner.
Potassium and taurine release are highly correlated with regulatory volume decrease in neonatal primary rat asrocyte cultures.
J. Neurochem.
63:
1143-1149,
1994[Medline].
59.
Walz, W.
Swelling and potassium uptake in cultured astrocytes.
Can. J. Physiol. Pharmacol.
65:
1051-1057,
1987[Medline].
60.
Walz, W.,
and
E. C. Hicks.
Carrier-mediated KCl accumulation accompanied by water movement is involved in the control of physiological K+ levels by astrocytes.
Brain Res.
343:
44-51,
1985[Medline].
61.
Zanotti, S.,
and
A. Charles.
Extracellular calcium sensing by glial cells: low extracellular calcium induces intracellular calcium release and intercellular signaling.
J. Neurochem.
69:
594-602,
1997[Medline].