Jerzy W. Lazarewicz

N‐Methylaspartate‐Evoked Liberation of Taurine and Phosphoethanolamine In Vivo: Site of Release

Abstract: The effect of N‐methyl‐d,l‐aspartic acid (NMA) on extracellular amino acids was studied in the rabbit hippocampus with the brain dialysis technique. Administration of 0.5 or 5 mM NMA caused a concentration‐dependent liberation of taurine and phosphoethanolamine (PEA). Taurine increased by 1,200% and PEA by 2,400% during perfusion with 5 mM NMA whereas most other amino acids rose by 20–100%. The effect of NMA appeared to be receptor‐mediated, as coperfusion with D‐2‐amino‐5‐phosphonovaleric acid curtailed the NMA response by some 90%. The NMA‐stimulated release of taurine and PEA was suppressed when Ca2+ was omitted and further inhibited when Co2+ was included in the perfusion medium. The effect of NMA was mimicked by the endogenous NMA agonist quinolinic acid and the partial NMA agonist d,l‐cis‐2,3‐piperidine dicarboxylic acid. Although the NMA‐evoked release of taurine and PEA was Ca2+‐dependent in vivo, NMA had no effect on Ca2+ accumulation in hippocampal synaptosomes. The previously reported NMA‐induced activation of dendritic Ca2+ spikes and the lack of effect on synaptosomal Ca2+ uptake suggest that taurine and PEA are released from sites other than nerve terminals, possibly from dendrosomatic sites. This notion was strengthened by the absence of an effect of NMA on the efflux of radiolabelled taurine from hippocampal synaptosomes. In contrast, high K+ stimulated synaptosomal uptake of Ca2+ and release of taurine.

Homocysteine-induced acute excitotoxicity in cerebellar granule cells in vitro is accompanied by PP2A-mediated dephosphorylation of tau.

Our results demonstrate that acute exposure of primary rat cerebellar granule cell cultures to homocysteine at millimolar concentrations induces a glutamate receptor-mediated decrease in tau protein phosphorylation, which is accompanied by excitotoxic neuronal damage. This contrasts with the previously reported hyperphosphorylation of tau in homocysteine-treated neurons, and with the assumption that hyperhomocysteinemia is one of the risk factors in Alzheimer disease, in which abnormal hyperphosphorylation of tau protein leads to neurofibrillary degeneration. The mechanisms of homocysteine neurotoxicity have been explained mainly either by disturbances in methylation processes, that may also lead to the accumulation of phosphorylated tau due to reduced activity of tau-selective protein phosphatase 2A, or by excitotoxicity. Since the relationships between homocysteine excitotoxicity and tau phosphorylation are unclear, the aim of this study was to characterize these processes in neurons acutely treated with homocysteine at neurotoxic concentrations, and to link them to the activities of glutamate receptors and protein phosphatase 2A. Within 24h following a 30 min exposure of neuronal cultures to 20mM d,l-homocysteine, significant neurotoxicity was induced. This could be reduced by treatment with an uncompetitive NMDA receptor antagonist, MK-801 (0.5 microM), or by mGlu1 and mGlu5 receptor antagonists, LY367385 and MPEP, respectively (both at 25 microM). Western blot analysis showed a rapid decrease in immunostaining of phospho-tau, 2h after incubation of cell cultures with 15 mM D,L-homocysteine, which persisted for 6h after the insult. Application of MK-801, LY367385 or okadaic acid (100 nM), an inhibitor of protein phosphatases 1 and 2A, significantly prevented dephosphorylation of tau, implying a role for the activation of glutamate receptors and protein phosphatase 2A. The phosphorylation of tau may be increased or reduced by treatment with homocysteine, and the nature of the cellular response to this sulfur-containing amino acid depends on the neuronal phenotype.

Effects of taurine on calcium binding and accumulation in rabbit hippocampal and cortical synaptosomes

The effect of taurine on Ca(2+) binding and uptake was studied with rabbit brain cortical and hippocampal synaptosomes. Taurine (25 mM) increased by 25% the high affinity (45)Ca(2+) binding in the cortical fraction and by 55% in hippocampal synaptosomes but had no effect on low affinity Ca(2+) binding. Taurine decreased significantly the fluorescence of the chlorotetracycline-hydrophobic Ca(2+) chelate probe in both synaptosomal fractions which suggests a shift of bound Ca(2+) from the hydrophobic to the hydrophilic part of the membranes. The uptake of (45)Ca(2+) by rabbit brain synaptosomes, when measured in control and 65 mK K(+)-containing media, was not influenced by taurine. However, taurine inhibited significantly the (45)Ca(2+) uptake in synaptosomes incubated in media containing moderately increased K(+) concentrations (14 and 20 mM K(+)). The effects of taurine are discussed in conjunction with its stabilizing effect on excitable membranes.

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.

Effects of kainic acid on brain calcium fluxes studied in vivo and in vitro

Abstract: The effect of in vivo administration of kainic acid into the rabbit hippocampus was studied with brain dialysis and subsequent determination of the Ca2+ concentration in the dialysate. When included in the perfusing medium, kainic acid as well as veratridine induced a decrease in extracellular Ca2+. The effect of kainic acid (but not of veratridine) was insensitive to tetrodotoxin. In vitro studies revealed no effect of kainic acid on 45Ca2+ uptake by isolated astrocytes, but showed an enhancement of synaptosomal 45Ca2+ accumulation. This was, however, only 25% of the stimulatory effect of high K+ depolarization. Glutamate activated synaptosomal Ca2+ uptake, whereas dihydrokainate had no effect. The up take evoked by kainate and glutamate was independent of the K+ level in the medium which indicates the involvement of other than voltage‐sensitive Ca2+ channels. The results confirm previous findings that kainic acid promotes the uptake of Ca2+ in brain cells. Kainate affects Ca2+ fluxes pre‐ and postsynaptically. Presynaptic Ca2+ influx may be mediated by chemically gated mechanisms.

Excitotoxic neuronal injury in acute homocysteine neurotoxicity: Role of calcium and mitochondrial alterations

In this study we tested if calcium imbalance and mitochondrial dysfunction, which have been implicated in the conventional mechanisms of excitotoxicity induced by glutamate (Glu), are also involved in homocysteine (Hcy) neurotoxicity. Primary cultures of rat cerebellar granule cells were incubated for 30 min in the presence of 25 mM D,L-Hcy or 1mM Glu. At these concentrations both amino acids induced comparable neurodegeneration and chromatin condensation, evaluated after 24 h using the propidium iodide and Hoechst 33258 staining. These effects were partially prevented by cyclosporin A (CsA), but not FK506. Hcy-induced release of [(3)H]inositol phosphates and increase in intracellular calcium level (evaluated with fluo-3 fluorescent probe) were weakly expressed. Hcy- and Glu-induced mitochondrial swelling was visualized under electron microscope, and the release of Cytochrome c was evaluated using immunocytochemical method and confocal microscopy. Comparing to Glu, the effects of Hcy were slightly less expressed and less sensitive to CsA, while FK506 did not modify mitochondrial alterations. These data indicate that mitochondrial alterations play a similar role in acute Hcy and Glu neurotoxicity, although the mechanisms triggering Glu- and Hcy-evoked mitochondrial dysfunction seem to differ, Hcy toxicity being less dependent on calcium.

Calcium fluxes in cultured and bulk isolated neuronal and glial cells.

Abstract— The influx and efflux of 45Ca has been studied in cultured human glioma and mouse neuroblastoma cells and in isolated fractions enriched in synaptosomes, neuronal and astrocytic perikarya from rabbit brain.

Dual effect of DL-homocysteine and S-adenosylhomocysteine on brain synthesis of the glutamate receptor antagonist, kynurenic acid.

Increased serum level of homocysteine, a sulfur‐containing amino acid, is considered a risk factor in vascular disorders and in dementias. The effect of homocysteine and metabolically related compounds on brain production of kynurenic acid (KYNA), an endogenous antagonist of glutamate ionotropic receptors, was studied. In rat cortical slices, DL‐homocysteine enhanced (0.1–0.5 mM) or inhibited (concentration inducing 50% inhibition [IC50] = 6.4 [5.5–7.5] mM) KYNA production. In vivo peripheral application of DL‐homocysteine (1.3 mmol/kg intraperitoneally) increased KYNA content (pmol/g tissue) from 8.47 ± 1.57 to 13.04 ± 2.86 (P < 0.01; 15 min) and 11.4 ± 1.72 (P < 0.01; 60 min) in cortex, and from 4.11 ± 1.54 to 10.02 ± 3.08 (P < 0.01; 15 min) in rat hippocampus. High concentrations of DL‐homocysteine (20 mM) applied via microdialysis probe decreased KYNA levels in rabbit hippocampus; this effect was antagonized partially by an antagonist of group I metabotropic glutamate receptors, LY367385. In vitro, S‐adenosylhomocysteine acted similar to but more potently than DL‐homocysteine, augmenting KYNA production at 0.03–0.08 mM and reducing it at ≥0.5 mM. The stimulatory effect of S‐adenosylhomocysteine was abolished in the presence of the L‐kynurenine uptake inhibitors L‐leucine and L‐phenyloalanine. Neither the N‐methyl‐D‐aspartate (NMDA) antagonist CGS 19755 nor L‐glycine influenced DL‐homocysteine‐ and S‐adenosylhomocysteine‐induced changes of KYNA synthesis in vitro. DL‐Homocysteine inhibited the activity of both KYNA biosynthetic enzymes, kynurenine aminotransferases (KATs) I and II, whereas S‐adenosylhomocysteine reduced only the activity of KAT II. L‐Methionine and L‐cysteine, thiol‐containing compounds metabolically related to homocysteine, acted only as weak inhibitors, reducing KYNA production in vitro and inhibiting the activity of KAT II (L‐cysteine) or KAT I (L‐methionine). The present data suggest that DL‐homocysteine biphasically modulates KYNA synthesis. This seems to result from conversion of compound to S‐adenosylhomocysteine, also acting dually on KYNA formation, and in part from the direct interaction of homocysteine with metabotropic glutamate receptors and KYNA biosynthetic enzymes. It seems probable that hyperhomocystemia‐associated brain dysfunction is mediated partially by changes in brain KYNA level.

Homocysteine-Evoked 45Ca Release in the Rabbit Hippocampus Is Mediated by Both NMDA and Group I Metabotropic Glutamate Receptors: In Vivo Microdialysis Study

This in vivo microdialysis study compared the effects of NMDA and d,l-homocysteine (Hcy) administered via dialysis medium on 45Ca efflux from prelabeled rabbit hippocampus. Application of these agonists evoked dose-dependent, and sensitive to MK-801, opposite effects: NMDA decreased the 45Ca radioactivity in the dialysate, whereas Hcy induced the release of 45Ca. The latter effect was potentiated by glycine, inhibited by the antagonist of group I metabotropic glutamate receptors (mGluR) LY367385, and mimicked by t-ADA, an agonist of these receptors. Electron microscopic examination of pyramidal neurones in the CA1 sector of the hippocampus in the vicinity of the microdialysis probe after NMDA application demonstrated swelling of mitochondria, which was prevented by cyclosporin A. This study shows, for the first time, Hcy-induced activation of both group I mGluR and NMDA receptors, which may play a role in acute Hcy neurotoxicity. We present new applications of brain microdialysis in studies on excitotoxicity and neuroprotection.

Extracellular calcium in the hippocampus of unanesthetized rabbits monitored with dialysis-perfusion

Extracellular levels of Ca2+ in the rabbit hippocampus were investigated in vivo with perfusion-dialysis. A thin semipermeable tubing was implanted and perfused at a constant rate with a Ca2+-free medium. Ca2+ levels in the dialysate were measured with an ion-specific electrode or as radioactivity after labeling of the endogenous pool with 45CaCl2. The system was characterized with model studies. The usefulness of the method is demonstrated for studies of, for example chemically evoked changes in Ca2+ concentrations extracellularly in CNS regions of non-anesthetized, freely moving rabbits. Moreover, measurements of Ca2+ changes are easily correlated with neurotransmitter levels in the same samples.