Thrombin potentiates d-aspartate efflux from cultured astrocytes under conditions of K+ homeostasis disruption
Article first published online: 3 OCT 2009
© 2009 The Authors. Journal Compilation © 2009 International Society for Neurochemistry
Journal of Neurochemistry
Volume 111, Issue 6, pages 1398–1408, December 2009
How to Cite
Vázquez-Juárez, E., Hernández-Benítez, R., López-Domínguez, A. and Pasantes-Morales, H. (2009), Thrombin potentiates d-aspartate efflux from cultured astrocytes under conditions of K+ homeostasis disruption. Journal of Neurochemistry, 111: 1398–1408. doi: 10.1111/j.1471-4159.2009.06418.x
- Issue published online: 20 NOV 2009
- Article first published online: 3 OCT 2009
- Received June 12, 2009; revised manuscript received August 26, 2009; accepted September 14, 2009.
- cytotoxic swelling;
- volume regulation;
- protease-activated receptors
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.