RESUMEN
Thrombin effect increasing swelling-induced glutamate efflux was examined in cultured cortical astrocytes, cerebellar granule neurons (CGN), hippocampal and cortical neurons. Hypotonic glutamate efflux (monitored by D-[(3)H]aspartate) from cortical astrocytes was increased by thrombin (5 U/mL) to reach 16% of the cell pool in 5 min. Thrombin had lower effects in CGN, and marginal effects in hippocampal and cortical neurons. These differences were related to the magnitude of thrombin-evoked cytosolic calcium rise. The protease-activated receptor 1 is expressed in astrocytes and neurons. In astrocytes exposed to chemical ischemia (sodium iodoacetate plus sodium azide) D-[(3)H]aspartate release showed a first phase (20-40 min) of initial low efflux which progressively increases; and a second phase (40-60 min) of larger efflux coincident with cell swelling. Efflux at the first phase was 52% inhibited by the glutamate transporter blocker DL-threo-ß-benzyloxyaspartic-acid (TBOA) and 11% by the volume-sensitive pathway blocker phloretin. At the second phase, efflux was reduced 52 and 38% respectively, by these blockers. In CGN D-[(3)H]aspartate efflux increased sharply and then decreased. This efflux was 32% reduced by calcium omission, 46% by TBOA and 32% by 4-[(2-butyl-6,7dichloro-2-cyclopentyl-2,3-dihydro-1oxo-1H-inden-5-yl)oxy] butanoic-acid. Thrombin enhanced this release by 32%. Ischemic treatment increased astrocyte mortality from 4% in controls to 39 and 61% in ischemia and ischemia plus thrombin, respectively. Cell death was prevented by phloretin. CGN viability was unaffected by the treatment. These results suggest that coincidence of swelling and thrombin during ischemia elevates extracellular glutamate prominently from astrocyte efflux, which may endanger neurons in vivo.
Asunto(s)
Ácido Aspártico/metabolismo , Astrocitos/metabolismo , Hiponatremia/metabolismo , Neuronas/metabolismo , Trombina/toxicidad , Tritio/metabolismo , Animales , Astrocitos/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Neuronas/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Gyroxin, a thrombin-like enzyme isolated from Crotalus durissus terrificus venom and capable of converting fibrinogen into fibrin, presents coagulant and neurotoxic activities. The aim of the present study was to evaluate such coagulant and toxic properties. Gyroxin was isolated using only two chromatographic steps - namely gel filtration (Sephadex G-75) and affinity (Benzamidine Sepharose 6B) - resulting in a sample of high purity, as evaluated by RP-HPLC C2/C18 and electrophoretic analysis that showed a molecular mass of 30 kDa. Gyroxin hydrolyzed specific chromogenic substrates, which caused it to be classified as a serine proteinase and thrombin-like enzyme. It was stable from pH 5.5 to 8.5 and inhibited by Mn²+, Cu²+, PMSF and benzamidine. Human plasma coagulation was more efficient at pH 6.0. An in vivo toxicity test showed that only behavioral alterations occurred, with no barrel rotation. Gyroxin was not able to block neuromuscular contraction in vitro, which suggests that its action, at the studied concentrations, has no effect on the peripheral nervous system.