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‐6,7dichloro‐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.     

Modulation of pro-survival and death-associated pathways under retinal ischemia/reperfusion: effects of NMDA receptor blockade

Loss of retinal ganglion cells occurs in a variety of pathological conditions, including central retinal artery occlusion, diabetes and glaucoma. Using an experimental model of retinal ischemia induced by transiently raise the intraocular pressure (IOP), In this study, we report the original observation that ischemic retinal ganglion cells death is associated with the transient deactivation of the pro-survival kinase Akt and activation of GSK-3beta followed, during reperfusion, by a longer lasting, PI3K-dependent, activation of Akt and phosphorylation of GSK-3beta. Under these experimental conditions, retinal ischemia induced the expression of Bad, a pro-apoptotic protein, member of the Bcl-2 family. The detrimental effects yielded by the ischemic stimulus were minimized by intravitreal administration of the NMDA receptor antagonist, MK801, that reduced the expression of Bad and significantly increased Akt phosphorylation. In conclusion, our present results contribute to unravel the mechanisms underlying retinal damage by high IOP-induced transient ischemia in rat. In addition, these data implicate the pro-survival PI3K/Akt pathway and the observed reduced expression of Bad in the neuroprotection afforded by MK801.     

The mechanism of 1,2,3,4-tetrahydroisoquinolines neuroprotection: the importance of free radicals scavenging properties and inhibition of glutamate-induced excitotoxicity

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), unlike several other tetrahydroisoquinolines, displays neuroprotective properties. To elucidate this action we compared the effects of 1MeTIQ with 1,2,3,4-tetrahydroisoquinoline (TIQ), a compound sharing many activities with 1MeTIQ (among them reducing free radicals formed during dopamine catabolism), but offering no clear neuroprotection. We found that the compounds similarly inhibit free-radical generation in an abiotic system, as well as indices of neurotoxicity (caspase-3 activity and lactate dehydrogenase release) induced by glutamate in mouse embryonic primary cell cultures (a preparation resistant to NMDA toxicity). However, in granular cell cultures obtained from 7-day-old rats, 1MeTIQ prevented the glutamate-induced cell death and 45Ca2+ influx, whereas TIQ did not. This suggested a specific action of 1MeTIQ on NMDA receptors, which was confirmed by the inhibition of [3H]MK-801 binding by 1MeTIQ. Finally, we demonstrated in an in vivo microdialysis experiment that 1MeTIQ prevents kainate-induced release of excitatory amino acids from the rat frontal cortex. Our results indicate that 1MeTIQ, in contrast to TIQ, offers a unique and complex mechanism of neuroprotection in which antagonism to the glutamatergic system may play a very important role. The results suggest the potential of 1MeTIQ as a therapeutic agent in various neurodegenarative illnesses of the central nervous system.     

Cofilin activation mediates Bax translocation to mitochondria during excitotoxic neuronal death

During excitotoxic neuronal death, Bax translocates to the mitochondria where it plays an important role by contributing to the release of proapoptotic factors. However, how Bax translocates to the mitochondria during excitotoxicity remains poorly understood. Herein, our data suggest the presence of a novel signalling mechanism by which NMDA receptor stimulation promotes Bax translocation. This signalling pathway is triggered by dephosphorylation of cofilin. Once dephosphorylated, cofilin might interact physically with Bax acting as a carrier for it, translocating it to the mitochondria, where it contributes to mitochondrial membrane despolarization, permeabilization and to the release of apoptotic factors, thus leading to neuronal death. Lack-of-function studies indicate that only the Slingshot family of phosphatases, more specifically the enzyme Slingshot 1L phosphatase, but not cronophin participates in the cofilin activation process during excitotoxicity. Indeed, cofilin-mediated Bax translocation seems to be a key event in excitotoxic neuronal death as knock down of either cofilin or Slingshot 1L phosphatase has a marked neuroprotective effect on NMDA-mediated neuronal death. This novel biochemical pathway may therefore be a good target to develop future therapeutic molecules for neurodegenerative diseases.