Thrombin potentiates D-aspartate efflux from cultured astrocytes under conditions of K+ homeostasis disruption.
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-6,7-dichloro-2-cyclopentyl-2,3-di-hydro-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.
División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México DF, México.
Erika
E
Vázquez-Juárez
Reyna
R
Hernández-Benítez
Alejandra
A
López-Domínguez
Herminia
H
Pasantes-Morales
D000818:Animals; D000831:Animals, Newborn; D001224:Aspartic Acid / Q000494:pharmacology; D001253:Astrocytes / Q000187:drug effects*; D001464:Barium / Q000494:pharmacology; D002034:Bumetanide / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D003517:Cyclopentanes / Q000494:pharmacology; D026603:D-Aspartic Acid / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006490:Hemostatics / Q000494:pharmacology*; D006706:Homeostasis / Q000187:drug effects*; D007189:Indans / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D013654:Taurine / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors; D014316:Tritium / Q000378:metabolism
C443139:4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-one-5-yl)oxybutyric acid; D003517:Cyclopentanes; D004791:Enzyme Inhibitors; D006490:Hemostatics; D007189:Indans; D009842:Oligopeptides; D049994:Sodium Potassium Chloride Symporter Inhibitors; C120673:benzyloxyaspartate; D002034:Bumetanide; D014316:Tritium; D013654:Taurine; D001464:Barium; D001224:Aspartic Acid; D026603:D-Aspartic Acid; D010042:Ouabain; D013917:Thrombin; D011188:Potassium; D002118:Calcium