Effects of inhibitors of Na+,K+-ATPase on the membrane potentials and neurotransmitter efflux in rat brain slices.

The potassium potential EK, of rat brain slices was estimated by determining the uptake of 86Rb+. The ERb was the same for slices prepared from five rostral brain regions, the average value being 66.4 mV. The ERb values in the presence of 20 microM ouabain were only slightly lower than the resting values; increasing concentrations of ouabain above 20 microM resulted in a graded depolarization in all five brain regions. High concentrations (1 mM) of two other inhibitors of Na+,K+-ATPase, dihydro-ouabain and strophanthidin, produced no more depolarization than did 20 microM ouabain. Competitive binding studies indicated that the differential effects were due to the relative binding to brain slices. Erythrosin B, an inhibitor of Na+,K+-ATPase, had no measurable effect on ERb. Intermediate concentrations of the Na+/H+ ionophore monensin slightly hyperpolarized striatal slices, whereas the same monensin concentrations plus 20 microM ouabain, 1 mM strophanthidin or 70 microM erythrosin B resulted in marked depolarization. Measurement of the membrane potential via uptake of methyltriphenylphosphonium cation indicated that ERb was indeed a valid estimation of the membrane potential. EK was measured directly by monitoring 42K+ uptake in striatal slices and was found to be essentially identical to ERb. Uptake of 22Na+ was consistent with the values for ERb or EK. Several conditions that resulted in little or no measurable depolarization of striatal slices did induce efflux of exogenously loaded GABA and dopamine; these conditions included 20 microM ouabain, 1 mM dihydro-ouabain or strophanthidin, and 70 microM erythrosin B. Neurotransmitter efflux in the absence of general cell depolarization was not accompanied by altered rates of respiration or decreased ATP levels.

Effect of pyrithiamine treatment on potassium ion fluxes in rat cortical slices.

The effect of thiamine deficiency on energy-requiring processes in brain tissue was studied by comparing cortical slices prepared from control and pyrithiamine-treated rats. Veratridine was used to stimulate energy metabolism by opening voltage-sensitive sodium channels resulting in enhanced Na+/K+ pumping; subsequent tetrodotoxin addition closed the sodium channels. Pyrithiamine-treated slices showed both lower basal and veratridine-stimulated respiration rates compared to control slices. K+ was released from the tissue upon addition of veratridine and was taken up again upon addition of tetrodotoxin. The movement of K+ was monitored directly with a K+-sensitive electrode as well as by measuring the rubidium diffusion potential. There was no difference between control and pyrithiamine-treated slices in K+ fluxes in response to veratridine and tetrodotoxin. The extent of reuptake of K+ upon tetrodotoxin addition was inversely related to the extracellular Ca2+ concentration and to the incubation temperature. Veratridine resulted in a marked decrease in tissue levels of ATP and creatine phosphate; these levels remained quite low upon tetrodotoxin addition. Despite the different respiration rates, control and pyrithiamine-treated slices showed the same ATP and creatine phosphate levels in response to veratridine and tetrodotoxin.

The Na+,K+-ATPase: a plausible trigger for voltage-independent release of cytoplasmic neurotransmitters.

A comparison was made between the releasability of eight neurotransmitters from eight regions of mouse brain in response to either 60 mM-K+ or 20 microM-ouabain, a specific inhibitor of the Na+,K+-ATPase. With few exceptions, all transmitters were released by either or both agents from each brain region examined. Potassium was superior in releasing the biogenic amines and acetylcholine, while the putative amino acid transmitters were generally releasable by both agents. Measurements of tissue depolarization using [3H]-tetraphenylphosphonium uptake indicated that 60 mM-K+ is capable of depolarizing brain tissue above the threshold necessary for initiating an action potential, but 20 microM-ouabain is not. The pattern of release by ouabain coupled with its failure to depolarize brain tissue at 20 microM suggests that inhibition of the Na+,K+-ATPase is capable of releasing cytoplasmic neurotransmitters in a voltage-independent manner.

Thiamine deficiency and glyoxylic acid.

The effect of thiamine deficiency on glyoxylic acid metabolism in mice and rats was investigated to determine whether the vitamin deficiency results in gross effects on glyoxylate levels via an alteration in the activity of alpha-ketoglutarate:glyoxylate carboligase. Thiamine-deprived or pyrithiamine-treated mice did not show a decreased oxidation of [1-14C]glyoxylate to respiratory CO2; there was some decrease in the conversion of [2-14C]glyoxylate into CO2 by pyrithiamine-treated mice, but not by thiamine-deprived animals. Dietary thiamine deprivation caused a decrease in carboligase levels in liver but no effect on levels in three brain regions. Pyrithiamine treatment had no significant effect on liver carboligase activities, but did decrease the levels in cerebrum, cerebellum and brainstem. Thiamine-deprived and pyrithiamine-treated mice showed decreased urinary glycolic acid excretion Glyoxylic acid excretion by thiamine-deprived rats was monitored in order to re-examine a previous report by another laboratory that glyoxyluria occurs under these conditions. Trace amounts of glyoxylate could be detected in the urine of rats fed thiamine-deficient diet for 3-5 weeks, but urinary glyoxylate was not detectable at later stages of thiamine deprivation. These results do not support a significant role for alpha-ketoglutarate:carboligase activity in the primary etiology of thiamine deficiency syndromes.

Cellular localization of alpha-ketoglutarate: glyoxylate carboligase in rat tissues.

alpha-Ketoglutarate: glyoxylate carboligase activity has been reported by other laboratories to be present in mitochondria and in the cytosol of mammalian tissues; the mitochondrial activity is associated with the alpha-ketoglutarate decarboxylase moiety of the alpha-ketoglutarate dehydrogenase complex. The cellular distribution of the carboligase has been re-examined here using marker enzymes of known localization in order to monitor the composition of subcellular fractions prepared by differential centrifugation. Carboligase activity paralleled the activity of the mitochondrial matrix enzyme citrate synthase in subcellular fractions prepared from rat liver, heart and brain as well as from rabbit liver. Whole rat liver mitochondria upon lysis released both carboligase and citrate synthase. The activity patterns of several other extramitochondrial marker enzymes differed significantly from that of carboligase in rat liver. In addition, the distribution pattern of carboligase was similar to that of alpha-ketoglutarate decarboxylase and of alpha-ketoglutarate dehydrogenase complex. The data indicate that alpha-ketoglutarate: glyoxylate carboligase activity is located exclusively within the mitochondria of the rat and rabbit tissues investigated. There is no evidence for a cytosolic form of the enzyme. Thus the report from other laboratory that the molecular etiology of the human genetic disorder hyperoxaluria type I is a deficiency of cytosolic carboligase must be questioned.