4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and Brain-Ischemia

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Astrocytes; Brain; Brain Edema; Brain Injuries; Brain Ischemia; Calcium; Cell Size; Cells, Cultured; Excitatory Amino Acids; Humans; Hypotonic Solutions; Ion Channels; Ion Transport; Nerve Tissue Proteins; Neurons; Potassium; Reperfusion Injury; Taurine

GABA is known to be the inhibitory neurotransmitter in the majority of brain stem nuclei. The release of GABA has been extensively studied both in vivo and in vitro in higher brain areas, whereas the mechanisms of release in the brain stem have not been systemically characterized. The properties of preloaded [3H]GABA were now investigated in mouse brain stem slices, using a superfusion system. The basal release was enhanced by K+ stimulation (50 mM K+) and under various cell-damaging conditions (ischemia, hypoglycemia, the presence of free radicals and metabolic poisons). No K+-stimulated release was discernible in the absence of Ca2+, indicating that the release was at least partly Ca2+-dependent. Moreover, the release was increased when Na+ or Cl- was omitted from the superfusion medium. GABA and beta-alanine stimulated the release, confirming the involvement of the reversed function of GABA transporters. Incubation of the slices with the anion channel inhibitors diisothiocyanostilbene and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate and with the Cl- uptake inhibitor 9-anthracenecarboxylic acid also reduced GABA release, demonstrating that a part of it comprises leakage through anion channels. All these mechanisms were involved in the ischemia-induced GABA release, which was over 4-fold greater than the release in normoxia. Contrary to the other brain areas, GABA release in the brain stem was not affected by ionotropic glutamate receptors but may be modulated by metabotropic receptors. This ischemia-induced GABA release might constitute an important mechanism against excitotoxicity, protecting the brain stem under cell-damaging conditions.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Anthracenes; Brain Ischemia; Brain Stem; Female; gamma-Aminobutyric Acid; Glutamic Acid; Ion Channels; Male; Mice

The release of preloaded [3H]glycine from hippocampal slices from 7-day-old and 3-month-old (adult) mice was studied in different cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and the presence of free radicals and metabolic poisons, using a superfusion system. Glycine release was greatly enhanced in all the above conditions in both age groups, with the exception of hypoxia in developing mice. This coincides with the increased susceptibility to seizures and excitotoxicity during postnatal development. The ischemia-induced release of glycine was Ca2+-independent at both ages. The release was potentiated by exogenously applied glycine but not in Na+-free conditions, indicating the involvement of Na+-dependent transporters operating outwards. The Cl- channel blockers 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate and diisothiocyanostilbene-2,2'-disulphonate generally reduced the ischemia-induced release, suggesting that this occurs through anion channels in both developing and adult mice. Furthermore, in the adult hippocampus riluzole and amiloride inhibited the release, indicating that Na+ channels also contribute to the ischemia-evoked release. Since glycine is an essential factor in glutamate-induced Ca2+ channel opening at the N-methyl-D-aspartate receptor, the elevated levels of glycine, together with the increased release of excitatory amino acids, must obviously collaborate in the development of ischemic neuronal damage.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Aging; Animals; Animals, Newborn; Brain Ischemia; Female; Glycine; Hippocampus; Hypoglycemia; Hypoxia; In Vitro Techniques; Ion Channels; Male; Mice; Mice, Inbred Strains; Oxidative Stress

Neuronal and glial cell swelling occurs rapidly in ischemia as part of the cytotoxic response. Astrocytic swelling is known to result in large extracellular increases in certain amino acids, including glutamate, aspartate and taurine, as part of the regulatory volume decrease (RVD) response inherent to these and other cells. RVD in astrocytic cultures is inhibited by anion channel blockers. In this study, we compared the effects of three anion channel blockers on the ischemia/reperfusion-evoked release of amino acids from the in vivo rat cerebral cortex. Twenty minutes of four vessel cerebral ischemia caused significant increases in cortical superfusate levels of aspartate, glutamate, GABA, taurine and phosphoethanolamine. During reperfusion there were delayed increases in the level of glycine, alanine and serine. Glutamine levels were not affected. Cl- channel blockers, 4-acetamido-4'-isothiocyanostrilbene-2,2'-disulfonic acid (SITS, 2 mM), 5-nitro-2-(3-phenyl-propylamino)benzoic acid (NPPB, 350 microM) and dipyridamole (200 microM) depressed basal releases of glutamate and taurine and the ischemia/reperfusion-evoked releases of aspartate, glutamate, taurine and phosphoethanolamine. The results suggest that diffusion of amino acids through an anion channel may be partially responsible for the elevated extracellular levels of excitotoxic and other amino acids that occur during cerebral ischemia/reperfusion.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amino Acids; Animals; Aspartic Acid; Brain Ischemia; Cerebral Cortex; Glutamic Acid; Ion Channels; Male; Nitrobenzoates; Rats; Rats, Sprague-Dawley

We have examined the extracellular pH (pHe) during spreading depression and complete cerebral ischemia in rat parietal cortex utilizing double-barrelled H+ liquid ion exchanger microelectrodes. The baseline pHe of the parietal cortex was 7.33 at a mean arterial PCO2 of 38 mm Hg. Following spreading depression and cerebral ischemia, highly reproducible triphasic changes in pHe occurred, which were intimately related to the negative deflection in tissue potential (Ve). The changes in pHe for spreading depression (n = 23) were a small initial acidic shift, beginning before the rapid change in Ve, followed by a rapid transient alkaline shift of 0.16 pH units, the onset of which coincided with the negative deflection in Ve. A prolonged acidic shift of 0.42 pH units then occurred. The maximal decrease in pHe was to 6.97 and the mean duration of the triphasic pHe change was 7.8 min. The lactate concentration in brain cortex increased from baseline 1.2 mM to 7.0 mM (n = 6) during the maximal acidic change in spreading depression. In addition, lactate levels correlated well with resolution of the pHe changes during spreading depression. The triphasic pHe changes following complete cerebral ischemia were an initial acidic shift of 0.43 pH units which developed over 2 min, then an alkaline shift of 0.10 pH units coincident with the negative deflection in Ve, and a final acidic shift of 0.26 pH units. The terminal pHe was 6.75. Superfusion of the cortex with inhibitors of carbonic anhydrase (acetazolamide), Na+/H+ counter transport (amiloride), and Cl-/HCO-3 countertransport (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) altered the triphasic pHe changes in a similar fashion for both spreading depression and cerebral ischemia, providing insights into the pHe regulatory mechanisms in mammalian brain.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acetazolamide; Acid-Base Equilibrium; Amiloride; Animals; Brain; Brain Ischemia; Extracellular Space; Homeostasis; Hydrogen-Ion Concentration; Lactates; Male; Microelectrodes; Parietal Lobe; Rats; Rats, Inbred Strains; Time Factors