Transient treatments with l-glutamate and threo-β-hydroxyaspartate induce swelling of rat cultured astrocytes

Abstract

We characterized swelling of rat cultured astrocytes induced by l-glutamate and its analogues. Among l-glutamate receptor agonists, l-glutamate, l-aspartate, l-cysteic acid, dl-homocysteic acid, quisqualate and (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) increased astrocytic intracellular volume (³H-OMG space), while kainate, and N-methyl-d-aspartate did not. Threo-β-hydroxyaspartate (TBHA), d-aspartate and l-trans-pyrrolidine-2,4-dicarboxylic acid, high-affinity substrates for Na⁺-dependent l-glutamate transporters, increased astrocytic ³H-OMG space. l-Glutamate (0.5 mM) increased astrocytic ³H-OMG space to 300% of control in 40–60 min. The increase in ³H-OMG space by 1 mM TBHA was comparable to the l-glutamate-induced one. After a 10 min-exposure to 0.5 mM l-glutamate, astrocytic ³H-OMG space was further increased to 200% even in the absence of l-glutamate. Astrocytes transiently exposed to l-glutamate did not increase their cell volume in K⁺-free medium and in the presence of 1 mM ouabain, a Na⁺-K⁺ATPase inhibitor. The increase after a transient exposure was also observed by a treatment of 1 mM TBHA, but not by 0.5 mM quisqualate. These results suggest that the volume increases after a transient treatment are mediated by activation of Na⁺-dependent l-glutamate transporter.

Introduction

Brain edema is a mortal pathological state generally observed on brain insults such as head trauma, ischemia and hypoxia. Pathogenesis of brain edema has been classified into two factors (Chan and Fishman, 1985). One pathogenesis is called vasogenic edema, which is caused by an increased permeability of brain capillary to blood macromolecules. The other one, cytotoxic edema is characterized by a marked swelling of brain cells. Astrocytic swelling is observed in many brain pathological states (Kimelberg and Ransom, 1986). On several brain insults, an excess amount of l-glutamate appears in extracellular fluid and the following overactivation of l-glutamate receptors results in degradation of neuronal cells (Jacobsson and Fowler, 1999, Tapia et al., 1999). The excess l-glutamate release is also involved in generation of brain edema. It has been shown that excitatory amino acid receptor antagonists attenuated brain edema following ischemia and other experimental brain insults (Katayama et al., 1992, Okiyama et al., 1995, Simon et al., 1986, Westergren and Johansson, 1993). Therefore, agents modulating excitability of l-glutamate are expected to have therapeutic values in the treatment against acute brain injuries (Vizi et al., 1996, Vizi et al., 1997, Yamashita et al., 1998).

The mechanisms underlying astrocytic swelling on brain injuries have been examined in cultured astrocytes. l-Glutamate and its analogues induce swelling of cultured astrocytes (Bender et al., 1998, Chan et al., 1990, Hansson, 1994, Hansson et al., 1994, Koyama et al., 1991b, Schneider et al., 1992). The l-glutamate-induced swelling of cultured astrocytes has different properties from those of hypotonic medium-induced swelling. Astrocytes increases their volume on a delayed onset in response to treatment with l-glutamate (Koyama et al., 1991a, Koyama et al., 1994). While astrocytes swollen by a hypotonic medium rapidly turn their cell volume to normal level by the “regulatory volume decrease mechanisms” (Bender et al., 1992, Kimelberg and Frangakis, 1985), astrocytes treated with l-glutamate keep the increased volume for a few hours even after removal of l-glutamate (Koyama et al., 1991a). Examinations on the mechanism of l-glutamate-induced swelling of cultured astrocytes suggest two different action sites of l-glutamate, i.e., metabotropic l-glutamate receptors and Na⁺-dependent transporters (Hansson, 1994, Bender et al., 1998). In this study, the l-glutamate-induced swelling of cultured astrocytes was further characterized by using its analogue compounds. We found that a transient treatment with high-concentrations of l-glutamate was sufficient to induce the delayed swelling of cultured astrocytes. And the effect of l-glutamate was mimicked by threo-β-hydroxyaspartate (TBHA), a selective substrate for Na⁺-dependent l-glutamate transporters.