The protective action of tetrodotoxin and (+/-)-kavain on anaerobic glycolysis, ATP content and intracellular Na+ and Ca2+ of anoxic brain vesicles

Because recent reports point to Na+ channel blockers as protective agents directed against anoxia-induced neuronal damage including protection of anaerobic glycolysis, the influences of tetrodotoxin (TTX) and (+/-)-kavain on anoxic rat brain vesicles were investigated with respect to lactate synthesis, vesicular ATP content and cytosolic free Na+ and Ca2+ ([Na+](i), [Ca2+](i)), both of the latter determined fluorometrically employing SBFI and FURA-2, respectively. After anoxia, basal lactate production was increased from 2.9 to 9.8 nmol lactate/min/mg protein. Although lactate synthesis seemed to be stable for at least 45 min of anoxia, as deduced from the linearity of lactate production, the ATP content declined continuously with a half life (tau(1/2)) of 14.5 min, indicating that anaerobic glycolysis was insufficient to cover the energy demand of anoxic vesicles. Correspondingly, [Na+](i) and [Ca2+](i) increased persistently after anoxia by 22.1 mmol/l Na+ and 274.9 nmol/l Ca2+, determined 6.3 min after onset. An additional stimulation of vesicles with veratridine accelerated the drop of ATP (tau(1/2) = 5.1 min) and provoked a massive Na+ overload, which levelled off to 119 mmol/l Na+ within a few minutes. Concomitantly, [Ca2+](i) increased linearly with a rate of 355 nmol Ca2+/l/min. Despite the massive perturbation of ion homeostasis, lactate production was unaffected during the first 8 min of veratridine stimulation. However, complete inhibition of lactate synthesis took place 30 min after veratridine was added. The Na+ channel blockers TTX and (+/-)-kavain, if applied before anoxia, preserved vesicular ATP content, diminished anoxia-induced increases in [Na+](i) and [Ca2+](i) and prevented both the veratridine-induced increases of [Na+](i) and [Ca2+](i) and the inhibition of lactate production. The data indicate a considerable Na+ influx via voltage-dependent Na+ channels during anoxia, which speeds up the decline in ATP and provokes an increase in [Ca2+](i). A massive Na+ and Ca2+ overload induced by veratridine failed to influence lactate synthesis directly, but initiated its inhibition. (C) 1997 Elsevier Science Ltd.