• cytotoxic swelling;
  • volume regulation;
  • excitoxicity;
  • protease-activated receptors


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Thrombin levels increase in brain during ischemia and hemorrhagic episodes, and may contribute to excitotoxic neural damage. This study examined the effect of thrombin on glutamate efflux from rat cortical cultured astrocytes using 3H-d-aspartate as radiotracer. The glutamate efflux was initiated by addition of 100 mM K+ plus 1 mM ouabain (K/O) to replicate extracellular and intracellular ionic changes that occur during cerebral ischemia. Upon exposure to K/O, astrocytes swelled slowly and progressively with no evidence of volume regulation. The K/O-induced swelling was inhibited by 65% with bumetanide and 25% with BaCl2, suggesting contribution of Na+/K+/Cl co-transporter and Kir channels. K/O-elicited 3H-d-aspartate that consisted of two phases. The first transient component of the release corresponded to 13.5% of total 3H-d-aspartate loaded. It was markedly reduced (61%) by the glutamate transporter blocker DL-threo-b-Benzyloxyaspartic acid and weakly inhibited (21%) by the volume-sensitive anion channel blocker 4-[(2-Butyl-6,7dichloro-2-cyclopentyl-2,3-dihidro-1oxo-1H-inden-5-yl)oxy] butanoic acid (DCPIB). During the second sustained phase of release, cells lost 45% of loaded of 3H-d-aspartate via a mechanism that was insensitive to DL-threo-b-Benzyloxyaspartic acid but nearly completely suppressed by DCPIB. Thrombin (5 U/mL) had only marginal effects on the first phase but strongly potentiated (more than two-fold) 3H-d-aspartate efflux in the second phase. The effect of thrombin effect was proportional to cell swelling and completely suppressed by DCPIB. Overall our data showed that under K/O swelling conditions, thrombin potently enhance glutamate release via volume-sensitive anion channel. Similar mechanisms may contribute to brain damage in neural pathologies which are associated with cell swelling, glutamate efflux and increased thrombin levels.

Abbreviations used:

100 mM K+ plus 1 mM ouabain


4-[(2-Butyl-6,7dichloro-2-cyclopentyl-2,3-dihidro-1oxo-1H-inden-5-yl)oxy] butanoic acid


DL-threo-b-Benzyloxyaspartic acid


N+/K+/Cl co-transporter


inwardly rectifying K+ channel


proteinase activated receptor


4,4′-diisothiocyanostilbene-2,2′-disulfonic acid


5-nitro-2-(3-phenylpropylamino)benzoic acid.

Swelling of brain cells, predominantly astrocytes, occurs either by a decrease in external osmolarity, or under isosmotic conditions by redistribution of ions and organic osmolytes which accumulate into the cells, generating the driving force for water influx. Isosmotic swelling occurs in brain associated with pathologies such as epilepsies, ischemia, hepatic encephalopathy and cranial trauma (Mongin and Kimelberg 2004; Pasantes-Morales and Franco 2005). Astrocytes are the brain cells which predominantly swell under these conditions, as consequence of their crucial role of clearance from the extracellular space, of potential injuring molecules such as K+, ammonium, or lactate, thus maintaining an optimal environment for neuronal function (Leis et al. 2005; Norenberg et al. 2005; Syková and Nicholson 2008). Mechanisms of uptake and/or metabolism operate specifically in astrocytes to accomplish this homeostatic function (Chen and Swanson 2003). However, during the progress of pathologies, the clearance capacities of astrocytes may be exceeded or forced to operate at maximal rate, a situation in which astrocytes not only fail to restore homeostasis, but may trigger responses that exacerbate and spread the original damage (Mongin and Kimelberg 2004; Pasantes-Morales and Franco 2005). Swelling is an early expression of this exceeded buffering capacity of astrocytes. Astrocyte swelling occurs in ischemia due to K+ and Cl accumulation followed by osmotically-driven water. The enhanced extracellular K+ levels, which may reach concentrations of up to 80 mM, generate an ionic imbalance harmful for neuronal excitability (Walz 2000; Rossi et al. 2007; Doyle et al. 2008). Also involved in astrocytic K+ clearance is the Na+/K+ ATPase (Leis et al. 2005). If, as in ischemia, the ATPase activity is reduced or impaired, the dissipation of Na+ and K+ transmembrane gradients may further contribute to swelling and to disturb in addition, the normal operation of transporters which use the driving force of these gradients for the uptake of a variety of molecules, including the highly neurotoxic excitatory amino acid glutamate (Camacho and Massieu 2006; Doyle et al. 2008; Malarkey and Parpura 2008). This situation contributes to neuronal death by excitotoxicity, particularly at the perifocal areas of global ischemia (Won et al. 2002; Rossi et al. 2007). Under these conditions, any additional factor enhancing glutamate efflux from brain cells will aggravate the excitotoxic damage. Thrombin may be one of such factors.

Besides the role of thrombin in blood coagulation, this molecule exerts a variety of effects on brain cells, which depending on thrombin concentration may be either cytoprotective or cytotoxic (Wang and Reiser 2003). Thrombin is present in brain in low concentrations, which dramatically increase in ischemia as well as in other hemorrhagic or traumatic episodes (Xi et al. 2003; De Castro Ribeiro et al. 2006; Hua et al. 2007). Thrombin effects occur through PAR-1, PAR-3 and PAR-4 receptors, activated by a proteolytic cleavage mechanism via G protein-coupled signaling pathways (Coughlin 2000). The PAR receptors are present in astrocytes (Junge et al. 2004). The link of thrombin with glutamate efflux here investigated is based on recent reports showing that ligand activation of G protein-coupled receptors, including PAR receptors, potentiates the swelling-evoked efflux of amino acids such as taurine and glutamate, which in a variety of cells are acting as osmolytes and in brain may have the dual role of osmolytes and neurotransmitters (Fisher et al. 2008; Vázquez-Juárez et al. 2008).

In a previous study, we showed a marked effect of thrombin increasing hyposmotic-swelling induced glutamate efflux from cultured astrocytes (Ramos-Mandujano et al. 2007). The purpose of the present study is to investigate whether thrombin potentiates glutamate efflux evoked by isosmotic swelling under conditions disturbing the astrocytic capacity for K+ clearance, i.e. high extracellular K+ levels and ATPase blockade by ouabain (Leis et al. 2005). If this occurs, thrombin may exacerbate neurotoxicity and brain damage in pathologies concurrent with a disturbed K+ homeostasis. Due to the time required for the experiments, the non-metabolizable analogue of glutamate, d-aspartate, was used in this study as tracer for glutamate.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References


Basal medium Eagle, fetal bovine serum and fura-2AM were purchased from Invitrogen. Ouabain octahydrate, l-glutamine (non-animal source), bumetanide, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and the synthetic peptide SFLLRN were from Sigma–Aldrich Chemical (St Louis, MO, USA). FSLLRN, TFLLR and AYPGKF were from Bachem Americas (Torrance, CA, USA). Pen Strep (penicillin streptomycin) was from GIBCO, Invitrogen’s brand, Invitrogen (Carlsbad, CA, USA). DCPIB (4-[(2-Butyl-6,7dichloro-2-cyclopentyl-2,3-dihidro-1oxo-1H-inden-5-yl)oxy] butanoic acid) and DL-TBOA were from TOCRIS Bioscience (Ellisville, MO, USA), and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and PPACK from Calbiochem (San Diego, CA, USA). Thrombin was from Vital Products (bovine plasma origin, specific activity > 2000 U/mg protein). Radiactive molecules [2,3-3H]-Taurine and d-[2,3-3H]-aspartic acid were from American Radiolabeled Chemicals Inc. (St Louis, MO, USA) and Amersham Biosciences (Buckinghamshire, UK) respectively. Salts for preparation of medium solutions were from J.T. Baker (NaCl, KCl, KH2PO4, MgSO4, CaCl2, and BaCl2), Sigma Chemicals (d-(+)-Glucose) and Roche (Indianapolis, IN, USA) (Hepes).

Cell cultures

Cortical astrocyte cultures were obtained from Wistar 1 day-old rat pups. Dissociated cell suspensions were plated at 3 × 106 cells in 35 mm Petri dishes, with basal medium Eagle, 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin. For cell volume measurements astrocytes were grown on coverslips set over 60 mm Petri dishes with 1.5 × 106 cells. Cultures were incubated at 37°C in humidified 5% CO2/95% air atmosphere, until confluence.


Isosmotic control medium contained (in mM) 135 NaCl, 5 KCl, 1.7 KH2PO4, 1.17 MgSO4, 1 CaCl2, 5 glucose, 10 HEPES (300 mOsm, with pH 7.4). Isosmotic K/O solutions were prepared replacing 100 mM NaCl with 100 mM KCl, plus 1 mM ouabain. Hyposmotic solution (210 mOsm) was prepared correspondingly reducing NaCl. Osmolarity was verified by a freezing point osmometer from Precision Systems Inc. (Natick, MA, USA).

Cell volume measurements

Volume measurements were performed by estimating the changes in relative cell volume with a large-angle light-scattering system (McManus et al. 1993;Pedersen et al. 2002). Astrocytes cultured on coverslips were placed at 50o angle relative to the excitation light in a cuvette filled with isosmotic K/O. To test the effect of hyposmolarity the cuvette was filled with isosmotic medium during 2 min and then distilled water was added to attain the osmolarity required. Cells were excited at 585 nm with an argon arc lamp (emission was detected at 585 nm). Data are expressed as the inverse of the emission signal as light intensity inversely correlates with cell volume, according to the equation lo/lt (where lo = the emission signal average when basal signal has been reached just before the stimulus; lt = emission signal at time t). It should be noticed that absolute volume values cannot be obtained with this method, but that it is useful mainly to comparatively evaluate changes in cell volume evoked by different conditions or in the presence of inhibitors. This has to be considered for all mentions to cell volume made through the manuscript.

Release experiments

Astrocytes preloaded 1 h with 3H-d-aspartate or 3H-taurine (0.3 μCi/mL) were washed and superfused at 1 mL/min with isosmotic control medium up to reach a stable efflux baseline. Then, medium was replaced by isosmotic K/O solution superfused continued during 40 min. To test the effect of blockers DL-threo-b-Benzyloxyaspartic acid (TBOA) and DCPIB, cells were 30 min pre-incubated with the inhibitor or the corresponding vehicle. Thrombin (5 U/mL) was added as indicated in the figures. At the end of the experiment, radioactivity in samples (collected during 45 min) and that remaining in cells was measured in a liquid scintillation counter. Results are expressed as radioactivity released per minute as percentage of the total radioactivity incorporated during loading.

Ca2+ measurements

To estimate changes in [Ca2+]i astrocytes cultured on rectangular coverslips in plastic dishes (60 mm), were incubated with fura-2/AM (2 μM) for 40 min. The coverslips were then gently washed in control medium to remove the extracellular dye and were placed at a 50° angle relative to the excitation light path in a cuvette filled with control medium or with K/O solution in a Fluoromax-3, Horiba luminescence spectrometer. Excitation wavelength was alternated between 340 and 380 nm and fluorescence intensity was monitored at 510 nm. The values obtained through this procedure were used to calculate the ratio of fluorescence intensity (fluorescence at 340 nm/fluorescence at 380 nm).

Data analysis

Statistical differences between experimental groups were determined by Student′s t-test and analysis of variance (anova) followed by Tukey test, statistically significant differences were considered at *p < 0.05. All data are given as mean ± SEM.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

High extracellular K+ concentration and ouabain (K/O) increased astrocyte volume under isosmotic conditions

Cultured astrocytes exposed to 100 mM KCl and 1 mM ouabain (isosmotic solution made by equiosmolar reduction of NaCl) (K/O) showed a continuous increase in cell volume. Swelling started immediately after the treatment and progressively increased to reach a maximum after 27 min. No significant further swelling was observed up to 50 min. Maximal K/O-induced astrocyte swelling (evaluated with the limitations inherent to the light-scattering method) was 16% in average over cell volume in controls (Fig. 1a). K/O-elicited swelling was markedly reduced (66%) when astrocytes were treated with 10 μM bumetanide, a known blocker of the Na+/K+/Cl co-transporter (NKCC1). Swelling was also decreased (27%) by 200 μM BaCl2, (barium) which at this concentration acts as blocker of inwardly rectifying K+ channels. Treatment with the two blockers simultaneously, abolished astrocyte swelling (Fig. 1a). Astrocytes exposed to 30% hyposmotic solution show immediate swelling, with maximal peak of 22% over the basal value, attained almost immediately after the stimulus. Thereafter, a progressive reduction in cell volume was observed (Fig. 1b). This result, included in the present study with comparative purposes, confirms other reports showing the typical regulatory volume decrease in cultured astrocytes (Pasantes-Morales et al. 1994; Olson et al. 1995; Cardin et al. 1999). Thrombin added to the control medium elicited a marginal increase of 2.1 ± 0.84% (n = 6) of astrocyte volume.


Figure 1.  Swelling of cortical astrocytes exposed to isosmotic K/O and effects of the NKCC cotransporter blocker bumetanide and the Kir channel blocker barium. (a) Representative traces of volume changes in cells exposed to KCl (100 mM), replacing equiosmolar NaCl, plus 1 mM ouabain (K/O), and the effect of bumetanide (10 μM) barium (200 μM) and both. Bumetanide (10 μM) was preincubated 30 min in control medium before treatment with K/O, and was present throughout the experiment. Barium (200 μM) was added at the time of exposure to K/O medium. Results in bars (means ± SE of 6–10 experiments) correspond to maximal swelling expressed as percentage over volume under control conditions. (b) Astrocyte swelling and volume regulation in 30% hyposmotic medium (H-30%). *Significantly different from control K/O p < 0.05.

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Treatment with K/O increased d-aspartate via two different routes

Astrocytes loaded with 3H-d-aspartate and superfused with control medium, released labeled d-aspartate at a rate of 0.5% per min. Treatment with K/O increased d-aspartate release with a biphasic pattern. In the first phase, a fast and large increase in d-aspartate was observed, reaching a peak release of 1.7%, 3 min after the stimulus (Fig. 2a). Then, efflux slowly decreased showing release fractions of 1.6–1.2% per min during about 8 min. Thereafter, a second phase of release was observed, in which d-aspartate efflux increased slowly and progressively, without showing any decline during the time of the experiment (40 min). (Fig. 2a). The amount of d-aspartate released during the first phase corresponds to 13.5% of loaded d-aspartate, and the amount released in the second phase is 31% resulting in a total release of 45% (Fig. 2a).


Figure 2. d-Aspartate efflux from cortical astrocytes evoked by K/O and effect of swelling and carrier blockers. Astrocytes preloaded with 3H-d-aspartate were superfused (1 mL/min) with control medium during 4 min, and then (arrow) with the same control medium (□) or the isosmotic K/O medium (100 mM KCl replacing the equivalent NaCl), and 1 mM ouabain (•). Points represent the radioactivity released at each fraction, expressed as percentage of total radioactivity accumulated during loading. (a) The time-course of 3H-D-aspartate showing two phases, a first phase (solid bottom line), activated and inactivated within 10 min after exposure to K/O, and a progressively increasing second phase of release (bottom dashed line) during the next 30 min. In bars is the amount of 3H-D-aspartate release at each phase, expressed as percentage of the total label accumulated in loading. (b) 3H-D-aspartate release in K/O during the first phase (10 min after K/O, solid bottom line) and effects of 5 μM TBOA and 10 μM DCPIB and both. (c) Efflux and effect of blockers TBOA, DCPIB during the second phase (min 15–40 after K/O). (□) Control; (•) K/O; (inline image) K/O plus DCPIB; (inline image) K/O plus TBOA. (d). Effect of cell swelling blockers bumetanide (10 μM) and barium (200 μM) on D-aspartate release in K/O during the second phase (30–40 min after K/O). Bars represent net efflux, i.e. release under the K/O condition minus release without K/O, in the presence of the blockers. Results are means ± SE of 6–8 experiments. *Significantly different from control K/O p < 0.05.

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The mechanism of d-aspartate efflux during the two identified phases was investigated using the Na+-dependent glutamate carrier blocker TBOA, and DCPIB, a specific and potent blocker of the volume-sensitive anion channel. Cells were incubated with the blockers during 20 min before treatment with K/O. Fig. 2b shows a marked inhibitory effect of TBOA on the first phase of d-aspartate efflux, which is reduced up to 61%. DCPIB showed also an inhibitory effect, decreasing d-aspartate efflux by 21%. Simultaneous treatment with the two blockers reduced the efflux up to 84% (Fig. 2b). An opposite inhibition pattern was found for the second release phase of d-aspartate, which was essentially insensitive to TBOA but markedly reduced by DCPIB (Fig. 2c). These results suggest that swelling is the stimulus to evoke the second phase of d-aspartate release. This was confirmed by the marked reduction of d-aspartate efflux when cell swelling was inhibited by bumetanide (59%), barium (19%) or bumetanide plus barium (81%) (Fig. 2d). Results on the time-course and mechanisms of d-aspartate efflux confirm those reported by Rutledge and Kimelberg (1996).

D-Aspartate efflux evoked by K/O is potentiated by thrombin

Thrombin added at the same time as K/O evoked a small but significant enhancement of D-aspartate efflux (Fig. 3a), but when added 15 min after exposure to K/O, once the first release phase has almost inactivated, a large potentiation of d-aspartate efflux was observed, increasing by 101% the K/O-evoked efflux (Fig. 3b). An even higher potentiation of d-aspartate efflux of about 117% was observed when thrombin was applied when the second phase has progressed, 30 min after the stimulus (Fig. 3c). Thrombin evoked release of d-aspartate was unaffected by TBOA and essentially abolished by DCPIB, NPPB or DIDS (Fig. 4a). These results suggest that the effect of thrombin occurs primarily on d-aspartate fluxes across the volume-activated anion channel. In further support to this notion, the thrombin potentiated d-aspartate efflux was essentially suppressed by preventing astrocyte swelling with bumetanide and barium (Fig. 4b).


Figure 3.  Effect of thrombin on K/O-induced efflux of D-aspartate from cultured cortical astrocytes (a)3H-d-aspartate efflux during 10 min after K/O exposure (first phase) and the potentiation by 5 U/mL thrombin, added at the arrow. (b)3H-d-aspartate efflux during 30 min after K/O and the potentiation by thrombin added at the arrow. (c) K/O-evoked 3H-d-aspartate efflux during 40 min and the potentiation by thrombin added at the arrow. Points represent the radioactivity released at each fraction, expressed as percentage of total accumulated under the following conditions: (•) K/O; (bsl00072) K/O plus thrombin. Bars in b and c show 3H-d-aspartate released in 10 fractions (bottom line) in the presence or absence of thrombin and represent net efflux (K/O minus control release) under the indicated conditions, Points in the curves and results in bars, are means ± SE of six experiments. *Significantly different from controls in the absence of thrombin p < 0.05.

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Figure 4.  Effects of swelling-, carrier- and volume-sensitive pathway-blockers on K/O-evoked d-aspartate potentiated by thrombin. (a) Effects of TBOA, DCPIB, NPPB and DIDS on d-aspartate released during the last 10 min of perfusion with K/O plus thrombin (bottom line). (b) Effects of the K/O induced swelling blockers bumetanide and barium on the d-aspartate release as in (a). Astrocytes were pre-incubated with the blockers, 5 μM TBOA, 10 μM DCPIB, 50 μM NPPB or DIDS, 10 μM bumetanide, during 30 min, and then the experiment was carried out as in Fig. 2. Barium (200 μM) was added at the time of K/O treatment. Bars represent net efflux (K/O minus control release) under the indicated conditions, and are means ± SE of 4–6 experiments. *Significantly different from K/O plus thrombin condition p < 0.05.

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K/O evoked taurine efflux is potentiated by thrombin

Taurine efflux examined in the same conditions exhibited only a small increase as result of K/O exposure in the initial minutes, corresponding to the first phase of release (Fig. 5a). Thereafter, taurine efflux increased continuously during the next 30 min with no sign of inactivation. Taurine released was 8% and 35% in the first and second phase, respectively, resulting in a total release of 43% (Fig. 5a). Taurine efflux evoked by K/O was abolished by DCPIB (Fig. 5a). Thrombin increased taurine efflux, by a DCPIB-sensitive mechanism (Fig. 5b).


Figure 5.  Effect of K/O on 3H-taurine release, its potentiation by thrombin and the effect of DCPIB. (a) Astrocytes were preloaded with 3H-taurine and treated as in Fig. 2. (b) Time-course of K/O-evoked taurine efflux and the effect of thrombin (5 U/mL) added 30 min after K/O exposure (bottom line) and the effect of DCPIB (10 μM) on thrombin potentiation. DCPIB treatment as described in Fig. 2. Points represent the radioactivity released at each fraction, expressed as percentage of total 3H-taurine accumulated under the following conditions: (□) Control; (•) K/O; (inline image) ) K/O plus DCPIB; (bsl00083) K/O plus thrombin (bsl00001) K/O plus thrombin plus DCPIB. Bars in (a) represent net efflux (K/O minus control release) in the two phases of release. Bars in b show taurine released in 10 fractions (bottom line) in the presence or absence of thrombin. *Significantly different at p < 0.05.

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The mechanisms of thrombin effect increasing taurine and d-aspartate efflux

The mechanism mediating thrombin effects on K/O efflux of d-aspartate likely involves its action as a protease on a PAR receptor. This is supported by results in Fig. 6a, showing how thrombin pretreatment with the protease inhibitor PPACK, prevented the thrombin potentiation of K/O-evoked d-aspartate efflux. The PAR-1 isoform seems that predominantly involved in thrombin effect, since the PAR-1 agonists SFLLRN and TFLLR (5 μM), fully replicate the effect of thrombin on d-aspartate efflux, while the scrambled peptide, FSLLRN was without effect (Fig. 6b). The PAR-4 agonist AYPGKF (50 μM) was essentially ineffective (Fig. 6b).


Figure 6.  Effect of PPACK and PAR agonists on K/O-evoked D-aspartate release. (a) Thrombin pre-incubated 30 min with PPACK (1 μM) was applied to cells exposed to K/O during 30 min as in Fig. 4c. (b) Effect of the synthetic peptides, PAR-1 agonists, SFLLRN or TFLLR (5 μM), FSLLRN (scrambled peptide), the PAR-4 agonist, AYPGKF (50 μM), were added to K/O-treated cells replacing thrombin, 30 min after K/O as in Fig. 3c. Bars in (a) represent D-aspartate released in the last 10 fractions (bottom line) by K/O, K/O plus thrombin and K/O plus PPACK-treated thrombin. In (b) bars illustrate d-aspartate release in the presence of the peptides as in (a). Results are means ± SE of four experiments. *Significantly different from K/O plus thrombin, by p < 0.05.

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Thrombin interaction with PAR receptors is known to elicit a marked increase in cytosolic Ca2+ concentration ([Ca2+]i). This effect is consistently observed in a large variety of cells and was also found in the present study in cortical cultured astrocytes treated with K/O. The magnitude of thrombin effect increasing K/O-induced d-aspartate release was markedly different during the time of the experiment as above described. To exclude a variation in the extent of thrombin-evoked [Ca2+]i rise during the experiment as the reason for this difference, the change in [Ca2+]i was examined when thrombin was applied 2 min (Fig. 7a), 15 or 30 min after K/O stimulus (Fig. 7b and c). Figure 7b shows no difference in [Ca2+]i elevation at any of the times examined.


Figure 7.  Changes in [Ca2+]i elicited by thrombin in astrocytes at different times of exposure to K/O: (a). Thrombin added 2 min after K/O exposure (b). 15 min after K/O. (c) 30 min after K/O. Results are expressed as ratio of fluorescence intensity (340/380 nm) (d). Quantification of the peak of [Ca2+]i response for each condition (bars were obtained from the peak value minus the basal K/O value). [Ca2+]i was measured with fura-2AM (2 μM) as detailed in Methods. Means ± SE from 6 to 10 experiments.

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The present results showed swelling in cultured astrocytes under isosmotic conditions, evoked by treatment with high extracellular K+ concentrations and ouabain (K/O). The swelling time-course observed contrasts notably with that induced by 30% reduction in osmolarity in the same preparation (cultured astrocytes) (Pasantes-Morales et al. 1994; Olson et al. 1995; Cardin et al. 2003 and present results). Whereas maximal volume under the hyposmotic condition was attained almost immediately after the stimulus, it required about 30 min to be reached in K/O-treated cells. Another remarkable difference is that while hyposmotic swelling is followed by an active process of volume recovery, in the K/O-treated cells there is no evidence of volume regulation, though a plateau is reached at a certain time.

Astrocyte swelling elicited by K/O treatment is the consequence of K+ and Cl accumulation, followed by osmotically obligated water (Ransom et al. 1996; Walz 2000). A small proportion may come also from intracellular Na+ raised by Na+/K+ ATPase blockade, which is though, counteracted by the suppressed K+ accumulation via the ATPase. Under this condition, net K+ uptake is accomplished primarily by activation of the electroneutral co-transporter NKCC. NKCC1 is the isoform expressed in cultured astrocytes and there is evidence in support of the substantial contribution of this transporter to the uptake phase of K+ clearance by astrocytes (Walz 1987; Su et al. 2002a, b; Mongin 2007). The present results showing a marked reduction in K/O-induced swelling when NKCC1 is blocked by bumetanide are in line with these previous observations. The glial inwardly rectifying K+ channels of the Kir family channels are proposed as an additional pathway for K+ accumulation and K+ buffering. The Kir4.1 isoform is expressed in cultured and in situ astrocytes and constitute the major part of the astrocytic Kir conductance (Olsen and Sontheimer 2008; Benesova et al. 2009). We found that blockade of Kir channels with barium led to a mild decrease of astrocyte swelling, suggesting a modest contribution of this mechanism to K+ uptake in cultured cortical astrocytes.

The time-course of K/O-induced swelling in cortical cultured astrocytes showed no evidence of an efficient volume regulation, which contrasts with the fast volume recovery observed after hyposmotic swelling. This is a predictable result since the typical regulatory volume decrease observed under hyposmotic conditions is accomplished to a large extent, by K+ and Cl extrusion (Wehner 1998; Stutzin and Hoffmann 2006) which cannot occur in high extracellular K+ concentrations. The pool of organic osmolytes, including glutamate, taurine and myo-inositol (Rutledge and Kimelberg 1996; Cardin et al. 1999; Isaacks et al. 1999), is mobilized, attenuating the magnitude of swelling, and is presumably responsible for the observed swelling plateau, but appears insufficient to accomplish cell volume recovery when the K/O condition persists.

Taurine and d-aspartate efflux elicited by K/O was comparatively examined in the present study, and marked differences were found in the release pattern between the two amino acids. In contrast to the fast and large release of d-aspartate observed immediately after K/O exposure, only a marginal increase in taurine efflux was observed. Differences in the carrier properties may contribute to the difference observed, since while glutamate transporter is Na+ and K+-dependent, taurine carrier is only Na+-dependent and consequently is less influenced by changes in external K+. Results showing that prevention of taurine efflux by DCPIB, a specific blocker of the volume-sensitive anion channel (Decher et al. 2001), points to this pathway as the main route for taurine translocation.

The release of d-aspartate from astrocytes was also increased by K/O treatment, as previously reported (Rutledge and Kimelberg 1996). The efflux time-course shows two different phases: an initial phase, of fast activation and inactivation, and a second phase, of delayed and progressive efflux, detectable as long as the K/O condition persists. The pharmacological profile of these two phases revealed two different mechanisms for release. The initial phase, markedly reduced (60%) by the carrier blocker TBOA, is then likely occurring via the transport reversal, a condition favored by the dissipation of ionic gradients and depolarization. Interestingly, a fraction of 21% of d-aspartate release in this first phase was reduced by DCPIB, the volume-sensitive pathway blocker, suggesting that even small changes in cell volume as occurring in the first minutes after treatment with K/O, enhance glutamate efflux via this pathway. The simultaneous presence of TBOA and DCPIB reduced 95% the d-aspartate efflux from this first fraction, excluding mechanisms other than swelling and carrier-mediated efflux as contributors to d-aspartate release. The second phase of d-aspartate efflux showed a markedly different time-course as compared with the initial phase, and a different pattern of sensitivity to TBOA and DCPIB. While the carrier blocker had no effect, d-aspartate efflux was abolished by DCPIB, NPPB or DIDS, a result that points to swelling as the main stimulus for this release. The swelling-dependent phase of d-aspartate efflux is also evident by the effect preventing this efflux when cell swelling is reduced by treatment with bumetanide and barium. A previous study has shown a strong inhibitory effect of bumetanide on d-aspartate release elicited by high K+ concentrations (Su et al. 2002a, b), a result confirmed in the present results. All these observations clearly establish that d-aspartate efflux is elicited by both, depolarization/dissipation of the ionic gradients and cell swelling, and proceeds via two different routes, as has been previously demonstrated by Rutledge and Kimelberg (1996). The same conclusion has been reached after substantial evidence regarding glutamate efflux in a variety of experimental models of ischemia, in vitro and in vivo (Nelson et al. 2003; Phillis and O’Regan 2003; Mongin and Kimelberg 2004; Swanson et al. 2004; Kimelberg 2005). This similarity is expected since the experimental paradigm of the present study replicates intracellular and extracellular ionic changes that occur during cerebral ischemia, and has been often considered as an ischemic-like model (Rutledge and Kimelberg 1996). In contrast to glutamate, taurine efflux, which is also reported to be released in ischemia models (Phillis and O’Regan 2003; Mongin and Kimelberg 2004), seems to respond largely to swelling.

The main interest of the present study was to investigate whether thrombin potentiates glutamate efflux under ischemic-like conditions, thus potentially aggravating the risk of excitotoxicity. It should be noticed that in all experiments, d-aspartate was used as tracer for glutamate. Glutamate participates in multiple reactions related to brain energetic demands, and excitability (Dienel and Hertz 2005; Rossi et al. 2007) and in astrocytes particularly, glutamate is actively metabolized via glutamine synthetase (Isaacks et al. 1999). Therefore, the amount of glutamate released by K/O and K/O plus thrombin may be lower than that of d-aspartate. If this is too low to promote excitotoxicity remains to be demonstrated.

We showed in a previous report a marked effect of thrombin increasing glutamate efflux from cultured astrocytes swollen by hyposmolarity (Ramos-Mandujano et al. 2007) and the present study demonstrates a similar effect of thrombin in a model of isosmotic swelling, in this case elicited by intracellular K+, Na+ and Cl intracellular accumulation. As above mentioned, in contrast to the immediate and fast increase in cell volume after a hyposmotic stimulus, swelling under the K/O condition has a temporal pattern allowing us to demonstrate that thrombin potentiation of glutamate efflux, d-aspartate in this case, occurs with a magnitude proportional to the degree of swelling. A small but significant effect of thrombin increasing d-aspartate release was observed within the first minutes after the stimulus, when only minute changes in cell volume occur. Later, the potentiation by thrombin is much higher, with a magnitude related to the extent of swelling. In full accordance with this conclusion, when swelling is prevented by bumetanide and barium, the thrombin-potentiated D-aspartate efflux was essentially suppressed.

The effect of thrombin found in the present study, in agreement with that observed on hyposmotic glutamate efflux, involved a protease-activated receptor (PAR), mainly the PAR-1 isoform, which is present in astrocytes (Wang et al. 2002; Wang and Reiser 2003; Junge et al. 2004). Thrombin activation of PAR receptors elicits a signaling pathway resulting in [Ca2+]i increase in astrocytes confirming its effect in numerous cell types. Thrombin increased [Ca2+]i in astrocytes from two main sources, extracellular Ca2+ and Ca2+ from the endoplasmic reticulum stores (Ramos-Mandujano et al. 2007). As above mentioned, thrombin potentiation of d-aspartate and taurine efflux was higher when thrombin was applied after longer times after the K/O treatment. This pattern is not due to any difference in the extent of thrombin-elicited [Ca2+]i elevation, which was found to be the same all along the experiment. It seems, in contrast, related to the degree of cell swelling which is progressively increasing. Altogether, these results show that glutamate (d-aspartate) efflux can be enhanced in swollen cells under isosmotic conditions including those replicating ischemia, provided that a threshold swelling is attained.

The thrombin-elicited increase in glutamate efflux from astrocytes might contribute to ischemic-induced neuronal death by excitotoxicity (Feustel et al. 2004; Mongin 2007) particularly since brain thrombin levels notably increase in ischemia. Other observations relate thrombin with excitotoxicity, such as the ischemia-induced up-regulation of PAR receptors (Xi et al. 2003) for which thrombin is the main substrate, or reported thrombin action increasing the efficiency of the glutamate NMDA-type receptor, which may exacerbate glutamate potential damage (Gingrich et al. 2000; Lee et al. 2007; Sharp et al. 2008). Altogether, these observations point to a possible effect of thrombin aggravating the excitotoxic damage known to occur in ischemia. In support to this possibility is the resistance to ischemic damage observed in transgenic mice defective in PAR-1, and the increased neuronal survival by treatment with PAR-1 blockers argatroban and hirudin (Kawai et al. 1996; Striggow et al. 2000, 2001; Karabiyikoglu et al. 2004).

The effects of thrombin increasing d-aspartate efflux were found abolished by preventing swelling with bumetanide and barium. Also taurine and d-aspartate release were suppressed by DCPIB. Altogether, these results point to the swelling-activated permeability pathway as the site of thrombin influence. DCPIB is a specific and potent blocker of the volume-sensitive glutamate efflux from astrocytes as shown by previous results from us and others (Abdullaev et al. 2006; Ramos-Mandujano et al. 2007). DCPIB and other Cl channel blockers also inhibit the swelling-induced efflux of organic osmolytes (Abdullaev et al. 2006; Shennan 2008). There is still controversy on whether the swelling-sensitive Cl channel itself is the permeability pathway for the organic osmolytes, including glutamate, a controversy raised by consistent observation of an inhibitory effect of essentially all the volume-sensitive Cl channel blockers on the volume-sensitive efflux of organic osmolytes. If this means that the same pathways carries both Cl and organic osmolytes or that they are so closely interconnected that blockade of one, blocks also the other one, is still uncertain. In any event, there is evidence of a strong effect of DCPIB reducing swelling-induced glutamate efflux in astrocytes and more recently DCPIB was also shown to prevent glutamate efflux evoked by middle cerebral artery occlusion-induced ischemia in adult rat. In support to the critical role played by cell swelling as a route for glutamate efflux leading to excitotoxic damage in ischemia, DCPIB showed a significant reduction of the infarct volume in this in vivo ischemia model (Zhang et al. 2008).


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We deeply acknowledge the valuable technical assistance of Ms. Claudia Peña-Segura, and Dr. Gerardo Ramos-Mandujano. This work was supported in part by grants from IMPULSA-03 and DGAPA IN203410, UNAM and 46465 from CONACYT, México. This study is part of the requirements for the PhD degree in Biomedical Sciences of Erika Vázquez-Juárez at the Universidad Nacional Autónoma de México, with a CONACYT, Mexico fellowship.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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