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.
Gleitz
J
J
University Clinics Ulm, Institute of Naturheilkunde, Germany.
Tosch
C
C
Beile
A
A
Peters
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000693:Anaerobiosis; D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology*; D001572:Benzofurans; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002545:Brain Ischemia / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; D016257:Fura-2; D006019:Glycolysis / Q000187:drug effects*; D002534:Hypoxia, Brain / Q000378:metabolism*; D007700:Kinetics; D007773:Lactates / Q000378:metabolism; D008297:Male; D009243:NAD / Q000378:metabolism; D010101:Oxygen Consumption; D011753:Pyrones / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000378:metabolism*; D026941:Sodium Channel Blockers; D013779:Tetrodotoxin / Q000494:pharmacology*
D000927:Anticonvulsants; D001572:Benzofurans; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; D007773:Lactates; D011753:Pyrones; D026941:Sodium Channel Blockers; D009243:NAD; C061665:sodium-binding benzofuran isophthalate; D013779:Tetrodotoxin; D000255:Adenosine Triphosphate; D012964:Sodium; D002118:Calcium; D016257:Fura-2; C011889:kavain