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[3H]taurine and D-[3H]aspartate release from astrocyte cultures are differently regulated by tyrosine kinases

Alexander A. Mongin1,2, Jyoti M. Reddi1, Carol Charniga1,2, and Harold K. Kimelberg1,2,3

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


    ABSTRACT
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Volume-dependent anion channels permeable for Cl- and amino acids are thought to play an important role in the homeostasis of cell volume. Astrocytes are the main cell type in the mammalian brain showing volume perturbations under physiological and pathophysiological conditions. We investigated the involvement of tyrosine phosphorylation in hyposmotic medium-induced [3H]taurine and D-[3H]aspartate release from primary astrocyte cultures. The tyrosine kinase inhibitors tyrphostin 23 and tyrphostin A51 partially suppressed the volume-dependent release of [3H]taurine in a dose-dependent manner with half-maximal effects at ~40 and 1 µM, respectively. In contrast, the release of D-[3H]aspartate was not significantly affected by these agents in the same concentration range. The inactive analog tyrphostin 1 had no significant effect on the release of both amino acids. The data obtained suggest the existence of at least two volume-dependent anion channels permeable to amino acids in astrocyte cultures. One of these channels is permeable to taurine and is under the control of tyrosine kinase(s). The other is permeable to both taurine and aspartate, but its volume-dependent regulation does not require tyrosine phosphorylation.

regulatory volume decrease; swelling-activated anion channels; excitatory amino acids; tyrosine phosphorylation; tyrphostins


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MOST VERTEBRATE CELLS studied so far release free inorganic and organic osmolytes when swollen by exposure to hypotonic media (reviewed in Refs. 5 and 13). This enables the cells to return their volumes to normal through a process termed regulatory volume decrease (RVD). The volume-dependent release of Cl- and organic osmolytes, including amino acids and polyols, appears to be mediated by volume-sensitive anion channels, the molecular natures of which are presently being extensively investigated (12, 17, 27). Currently, major attention is focused on a volume-sensitive Cl-/organic osmolyte channel characterized by outward rectification, cytosolic ATP dependency, and intermediate unitary conductance (20-80 pS) that is ubiquitously present in animal cells (17, 27). It has been termed volume-sensitive organic osmolyte anion channel (VSOAC) (27) or volume expansion-sensing outwardly rectifying (VSOR) anion channel (17). It is now generally believed that this channel is responsible for the volume-dependent release of structurally dissimilar organic osmolytes.

Although electrophysiological studies of the VSOAC/VSOR channel show that its biophysical properties (voltage dependence, outward rectification, single-channel conductance, ion selectivity) are relatively conserved through most cell types, there is no consensus on the intracellular mechanisms by which cell volume regulates the channel. There is substantial evidence that the volume-dependent activation of the channel requires nonhydrolytic ATP binding (reviewed in Refs. 17 and 27) and may be modulated in some cell types by protein kinase C- or protein kinase A-related intracellular protein phosphorylation (2, 7, 20). However, several recent studies showed that tyrosine protein phosphorylation is an essential step for the volume regulation of anion currents with properties corresponding to those of the VSOAC/VSOR channel. Activation of whole cell anion current or 125I- efflux in hyposmotic media may be completely prevented by protein tyrosine kinase (PTK) inhibitors or strongly suppressed by nonhydrolyzable analogs of ATP (26, 28, 29, 31, 32). In the present report, we show that the involvement of tyrosine kinases in the volume-dependent activation of amino acid fluxes in primary astrocyte cultures varies with the amino acid being measured.


    MATERIALS AND METHODS
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Materials. Tyrphostin 1 and tyrphostin 23 were obtained from Sigma Chemical (St. Louis, MO). Tyrphostin A51 and epidermal growth factor (EGF) from murine submaxillary glands were from Calbiochem (San Diego, CA). [3H]taurine (sp act 30.3 Ci/mmol) and D-[3H]aspartate (sp act 10.5 Ci/mmol) were from NEN Life Sciences Products (Boston, MA). All culture reagents were from GIBCO (Grand Island, NY). Other chemicals were obtained from Sigma Chemical.

Cell cultures. Primary astrocyte cultures were prepared from the cerebral cortices of newborn Sprague-Dawley rats, as described previously (3). They were used after ~3-4 wk, when the cells reached a confluent monolayer. Immunocytochemistry showed that >95% of the cells stained positively for the astrocytic marker glial fibrillary acid protein.

Efflux measurements. Amino acid efflux measurements were performed with [3H]taurine and D-[3H]aspartate, as described previously (16, 22). Briefly, astrocytes grown on glass coverslips (18 × 18 mm; no. 11/2; Bellco Biotechnology, Vineland, NJ) were preloaded overnight with [3H]taurine (8 µCi/ml, 264 nM) or D-[3H]aspartate (8 µCi/ml, 762 nM). The coverslips were placed in a Lucite perfusion chamber (total volume ~100 µl) and were superfused at 1 ml/min with isosmotic or hyposmotic medium. Isosmotic medium consisted of (in mM) 122 NaCl, 3.3 KCl, 0.4 MgSO4, 1.3 CaCl2, 1.2 KH2PO4, 10 D-glucose, and 25 HEPES. The pH was adjusted to 7.4 by the addition of NaOH. Hyposmotic medium contained (in mM) 72 NaCl, 3.3 KCl, 0.4 MgSO4, 1.3 CaCl2, 1.2 KH2PO4, 10 D-glucose, and 25 HEPES (pH 7.4). The osmolarities of isotonic and hypotonic media were 285 and 190 mosM, respectively, as verified with a freezing-point osmometer (Advanced Instruments, Needham Heights, MA). The 1-min fractions were collected and subjected to liquid scintillation counting. The rate of amino acid efflux was calculated as the fractional release (percentage of isotope remaining in the cells at the beginning of each 1-min interval) by using a custom-prepared computer program (22).


    RESULTS AND DISCUSSION
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It has been established that hyposmotic cell swelling triggers the activation of PTKs and downstream signaling cascades in several cell types (28, 29, 31), including C6 glioma cells (25) and cultured primary astrocytes (23). This activation appears to play an important role in the volume-dependent regulation of anion fluxes because inhibitors of PTKs and other members of the same signaling cascade, e.g., ERK1 and ERK2 kinases, suppress hyposmotic medium-activated whole cell anion currents and 125I- efflux in various cells (26, 29, 31, 32), including primary astrocyte cultures (1). We evaluated whether such inhibition is characteristic of swelling-induced taurine and D-aspartate release in primary astrocytes, which has been shown to be mediated in these cells by an anion channel(s) (9, 11, 18, 19) showing a pharmacological sensitivity close to that of the VSOAC/VSOR channel (22).

In our experiments the tyrosine kinase inhibitors tyrphostin 23 and tyrphostin A51 inhibited the volume-sensitive release of [3H]taurine in primary astrocyte cultures in a dose-dependent manner (Fig. 1, A and B). Because there was a variation between the maximal rates of [3H]taurine release under control hyposmotic conditions in different culture preparations, in the range of 11-23%, all data were normalized to controls performed with the same cell preparation on the same day, which were very constant. Significant inhibition by tyrphostin A51 at a concentration as low as 100 nM was observed. The maximal inhibition did not exceed 50-55% and was saturated at concentrations of 50-100 µM, with the half-maximal effect at 1.2 ± 1.3 µM (Fig. 1B). This value is very close to the IC50 for the inhibition of EGF receptor kinase activity (IC50 = 800 nM) (15). In the concentration range of 0.32-10 µM, the inhibitory potency of tyrphostin 23 was lower than that of tyrphostin A51, a finding that is consistent with the lower potency of tyrphostin 23 for inhibiting PTKs (IC50 for EGF receptor kinase = 35 µM) (4, 15). However, unlike that of tyrphostin A51, the tyrphostin 23 effect did not saturate at intermediate levels of inhibition up to a concentration of 320 µM, and computer fitting gave a maximal inhibition of ~90% and an IC50 of 41.0 ± 39.8 µM (Fig. 1B). We think this difference in the action of these two tyrphostins might be due to nonspecific inhibition of the anion channel by high concentrations of tyrphostin 23. Indeed, tyrphostin 1, an analog of tyrphostins with minimal potency for inhibiting tyrosine kinase activity and a structure similar to that of tyrphostin 23 (4), had no effect on volume-dependent [3H]taurine release at low concentrations, whereas at 320 µM it suppressed the release by 15-20% (Fig. 1B).


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Fig. 1.   Influence of protein tyrosine kinase (PTK) inhibitors on volume-dependent [3H]taurine release from primary astrocytic cultures. A: representative experiments showing effects of 32 µM tyrphostin 1 (T1), tyrphostin 23 (T23), and tyrphostin A51 (T51) on [3H]taurine release in 1 cell preparation. Cells were preincubated with tyrphostins under isosmotic (285 mosM) conditions for 15 min and then subjected to 190 mosM hyposmotic medium (HYPO) containing same concentration of drugs. B: dose-response curves for effects of tyrphostin 1, tyrphostin 23, and tyrphostin A51 on volume-dependent [3H]taurine release. Each point represents mean ± SE of 3-6 experiments involving 3 different culture preparations. Computer fitting and calculation of IC50 values were performed with Origin 4.1 (Microcal, Northampton, MA) by sigmoidal fitting. [PTK inhibitor], concentration of PTK inhibitor.

Our observations involving the inhibition of swelling-induced [3H]taurine efflux by tyrphostins are consistent with previous studies regarding the effects of PTK inhibitors on volume-dependent anion currents (26, 32) and 125I- efflux (29, 31). The VSOAC/VSOR channel is permeable to fairly large anions, including taurine and glutamate (reviewed in Refs. 17 and 27). In primary astrocyte cultures, the volume-dependent anion current is consistent with the properties of the VSOAC/VSOR channel and is completely suppressed by PTK inhibitors (1). This is slightly different from our data because we found that two tyrphostins acting as specific inhibitors of PTKs gave only partial inhibition of [3H]taurine release. The PTK inhibitors tyrphostin B46 and genistein were found to inhibit the swelling-activated efflux of [3H]taurine from calf pulmonary artery endothelial cells by 52.5 and 50.6% at 10 and 50 µM, respectively, (32) but it was not established if complete inhibition of amino acid release was possible.

Because the cell swelling-induced release of excitatory amino acids likely contributes to brain damage in head trauma and ischemia (8, 10), we further investigated the influence of tyrosine kinase inhibitors on the volume-dependent release of D-[3H]aspartate. As in the case of taurine, all the data were normalized to controls performed on the same culture preparations on the same day. It was found previously that the sensitivity of astrocytic volume-dependent D-[3H]aspartate release to a variety of anion channel inhibitors was very similar to that of the VSOAC/VSOR channel (22). Surprisingly, we found that swelling-induced D-[3H]aspartate efflux was not inhibited but rather was marginally activated after the treatment of the cells with 3.2-100 µM tyrphostin 23 or 10-100 µM tyrphostin A51 (Fig. 2, A and B), in contrast to volume-dependent [3H]taurine release. Once more, tyrphostin 1 had no significant effect up to a concentration of 100 µM (Fig. 2B). The reason for the stimulation of D-[3H]aspartate efflux in tyrphostin-treated astrocytes is presently unclear. The stimulation may be due to slightly larger cell swelling because in other cell types PTK inhibitors have been shown to block RVD (14). Our data suggest that in cultured astrocytes different volume-dependent anion channels mediate excitatory amino acid efflux and taurine release. Although the existence of several classes of swelling-activated Cl- channels is well established (reviewed in Refs. 12 and 27) and there is evidence for the coexpression of multiple swelling-activated Cl- channels in a single epithelial cell (34), the contribution of multiple channels to volume-dependent organic osmolyte release has not yet been to our knowledge demonstrated, except for one report discriminating volume-dependent sorbitol/betaine and myo-inositol/taurine transport pathways in renal inner medullary collecting duct cultured cells (21). It is noteworthy that, in agreement with our findings for primary astrocyte cultures, different intracellular signaling mechanisms were found to be involved in the regulation of separate swelling-activated transport systems in the renal epithelium (21).


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Fig. 2.   Influence of PTK inhibitors on volume-dependent D-[3H]aspartate release from primary astrocytic cultures. A: representative experiments showing effects of tyrphostin 1 and tyrphostin A51 on D-[3H]aspartate release in 1 cell preparation. B: dose-response curves for effects of tyrphostin 1, tyrphostin 23, and tyrphostin A51 on volume-dependent D-[3H]aspartate release. Data are means ± SE of 3-4 experiments involving 3 different culture preparations.

We speculate that the VSOAC/VSOR channel is the major channel determining volume-dependent whole cell conductance in primary astrocyte cultures and that its activation involves tyrosine kinase-related signaling pathways. Another channel, which is not affected by tyrosine kinases, is predominantly involved in excitatory amino acid efflux and, perhaps because of a lower Cl- permeability, does not contribute substantially to whole cell anion currents. This simple assumption may also explain the difference between complete inhibition of anion currents (1, 14, 26, 32) and partial inhibition of [3H]taurine release (this paper) by PTK inhibitors. Although the VSOAC/VSOR channel in C6 glioma cells was found to be permeable to taurine, as well as aspartate and glutamate (6), in primary cultured astro-cytes RVD was completely suppressed by the replacement of extracellular Cl- with taurine but was not affected by high extracellular concentrations of glutamate or aspartate, confirming the low permeability of volume-sensitive channels to the last two amino acids (19). Similarly, in hyposmotically swollen lymphocytes, very poor membrane permeability for glutamate was found (14).

The tyrphostins were primarily designed as inhibitors of receptor-linked PTKs such as EGF receptor kinase (4, 15). The presence of EGF receptors, as well as EGF-stimulated cell proliferation and EGF-related intracellular tyrosine phosphorylation, in astrocyte cultures is widely described (24, 30, 33). To evaluate the involvement of receptor PTKs in the volume-dependent activation of amino acid fluxes, we studied the effects of EGF on basal and swelling-induced amino acid release. Under basal conditions, EGF did not significantly affect either [3H]taurine or D-[3H]aspartate efflux (data not shown). A short-term pretreatment with EGF (3 min, which was comparable to the time of complete change of medium in the superfusion chamber) before the exposure of astrocytes to hyposmotic medium led to a decrease of the volume-dependent release of [3H]taurine and D-[3H]aspartate by 18.7 ± 4.5 (n = 6, P < 0.05) and 23.4 ± 7.2% (n = 8, P < 0.05), respectively (data not shown). After 15 min of preincubation with EGF, the volume-dependent [3H]taurine release was also decreased by 14.2 ± 5.4% (n = 8, P < 0.05), whereas no significant effect on D-[3H]aspartate efflux was found (data not shown). These results are in conflict with either 1) the expected activation of amino acid efflux under basal conditions if we assume that EGF receptor tyrosine kinase is a volume signal-transducing element or 2) the potentiation of volume-dependent efflux if we suppose that EGF receptor-related tyrosine phosphorylation is a factor modulating volume-dependent amino acid release. Therefore, the tyrphostin-sensitive PTK involved in the swelling-induced activation of taurine release in cultured astrocytes is not the EGF receptor kinase, and its molecular identity remains to be established. Other possible candidates, p125 focal adhesion kinase (28), p56lck src-product-like tyrosine kinase (14), and downstream ERK1 and ERK2 kinases (1, 23, 25, 29) have been proposed as signal transducers in other cells.

In conclusion, in primary astrocyte cultures hyposmotic medium-induced cell swelling leads to the activation of at least two anion channels permeable for amino acids with different mechanisms of volume signal transduction. One channel, which may correspond to the VSOAC/VSOR channel, is permeable to taurine (and Cl-) but not to excitatory amino acids (EAAs) and requires EGF receptor-independent, tyrphostin-sensitive tyrosine kinase activity for hyposmotic stimulation. The second channel is permeable to taurine and EAAs (and Cl-) and is not controlled by tyrosine kinases. An important implication of our study is the possibility of being able to distinguish between the volume-dependent Cl-/taurine channel and the volume-dependent channel for EAAs by using tyrphostins as a molecular tool. The former channel is required for cell volume regulation. Although the normal function of the latter channel is unclear, it can likely contribute to brain damage in the several pathologies in which astrocytic swelling occurs (10).


    ACKNOWLEDGEMENTS

A. A. Mongin and J. M. Reddi contributed equally to the experiments.


    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.

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, Division of Neurosurgery, MC-60, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: hkimelberg{at}ccgateway.amc.edu).

Received 21 December 1998; accepted in final form 9 February 1999.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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