Thiamine in Excitable Tissues: Reflections on a Non-Cofactor Role

in Metabolic Brain Disease (1994), 9(3), 183-209

Mechanism of Thiamine Transport in Neuroblastoma Cells. Inhibition of a High Affinity Carrier by Sodium Channel Activators and Dependence of Thiamine Uptake on Membrane Potential and Intracellular Atp

in Journal of Biological Chemistry (1994), 269(20), 14379-14385

Nerve cells are particularly sensitive to thiamine deficiency. We studied thiamine transport in mouse neuroblastoma (Neuro 2a) cells. At low external concentration, [14C]thiamine was taken up through a ... [more ▼]

Nerve cells are particularly sensitive to thiamine deficiency. We studied thiamine transport in mouse neuroblastoma (Neuro 2a) cells. At low external concentration, [14C]thiamine was taken up through a saturable high affinity mechanism (Km = 35 nM). This was blocked by low concentrations of the Na+ channel activators veratridine (IC50 = 7 +/- 4 microM) and batrachotoxin (IC50 = 0.9 microM). These effects were not antagonized by tetrodotoxin and were also observed in cell lines devoid of Na+ channels, suggesting that these channels are not involved in the mechanism of inhibition. At high extracellular concentrations, thiamine uptake proceeds essentially via a low affinity carrier (Km = 0.8 mM), insensitive to veratridine but blocked by divalent cations. In both cases, the uptake was independent on external sodium, partially inhibited (10-35%) by depolarization and sensitive to metabolic inhibitors. A linear relationship between the rate of thiamine transport and intracellular ATP concentration was found. When cells grown in a medium of low thiamine concentration (6 nM) were exposed to 100 nM extracellular thiamine, a 3-fold increase in intracellular thiamine diphosphate was observed after 2 h while the concomitant increase in intracellular free thiamine was barely significant. These data suggest a secondary active transport of thiamine, the main driving force being thiamine phosphorylation rather than the sodium gradient. [less ▲]

Subcellular Localization and Compartmentation of Thiamine Derivatives in Rat Brain

in Biochimica et Biophysica Acta (1994), 1222(1), 1-6

The subcellular distribution of thiamine derivatives in rat brain was studied. Thiamine diphosphate content was highest in the mitochondrial and synaptosomal fractions, and lowest in microsomal, myelin ... [more ▼]

The subcellular distribution of thiamine derivatives in rat brain was studied. Thiamine diphosphate content was highest in the mitochondrial and synaptosomal fractions, and lowest in microsomal, myelin and cytosolic fractions. Only 3-5% of total thiamine diphosphate was bound to transketolase, a cytosolic enzyme. Thiamine triphosphate was barely detectable in the microsomal and cytosolic fraction, but synaptosomes were slightly enriched in this compound compared to the crude homogenate. Both myelin and mitochondrial fractions contained significant amounts of thiamine triphosphate. In order to estimate the relative turnover rates of these compounds, the animals received an intraperitoneal injection of either [14C]thiamine or [14C]sulbutiamine (isobutyrylthiamine disulfide) 1 h before decapitation. The specific radioactivities of thiamine compounds found in the brain decreased in the order: thiamine > thiamine triphosphate > thiamine monophosphate > thiamine diphosphate. Incorporation of radioactivity into thiamine triphosphate was more marked with [14C]sulbutiamine than with [14C]thiamine. The highest specific radioactivity of thiamine diphosphate was found in the cytosolic fraction of the brain, though this pool represents less than 10% of total thiamine diphosphate. Cytosolic thiamine diphosphate had a twice higher specific radioactivity when [14C]sulbutiamine was used as precursor compared with thiamine though no significant differences were found in the other cellular compartments. Our results suggest the existence of two thiamine diphosphate pools: the bound cofactor pool is essentially mitochondrial and has a low turnover; a much smaller cytosolic pool (6-7% of total TDP) of high turnover is the likely precursor of thiamine triphosphate. [less ▲]

Thiamine Triphosphate Activates an Anion Channel of Large Unit Conductance in Neuroblastoma Cells

in Journal of Membrane Biology (1993), 136(3), 281-8

In neuroblastoma cells, the intracellular thiamine triphosphate (TTP) concentration was found to be about 0.5 microM, which is several times above the amount of cultured neurons or glial cells. In inside ... [more ▼]

In neuroblastoma cells, the intracellular thiamine triphosphate (TTP) concentration was found to be about 0.5 microM, which is several times above the amount of cultured neurons or glial cells. In inside-out patches, addition of TTP (1 or 10 microM) to the bath activated an anion channel of large unit conductance (350-400 pS) in symmetrical 150 mM NaCl solution. The activation occurred after a delay of about 4 min and was not reversed when TTP was washed out. A possible explanation is that the channel has been irreversibly phosphorylated by TTP. The channel open probability (Po) shows a bell-shaped behavior as a function of pipette potential (Vp). Po is maximal for -25 mV < Vp < 10 mV and steeply decreases outside this potential range. From reversal potentials, permeability ratios of PCl/PNa = 20 and PCl/Pgluconate = 3 were estimated. ATP (5 mM) at the cytoplasmic side of the channel decreased the mean single channel conductance by about 50%, but thiamine derivatives did not affect unit conductance; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM) increased the flickering of the channel between the open and closed state, finally leading to its closure. Addition of oxythiamine (1 mM), a thiamine antimetabolite, to the pipette filling solution potentiates the time-dependent inactivation of the channel at Vp = -20 mV but had the opposite effect at +30 mV. This finding corresponds to a shift of Po towards more negative resting membrane potentials. These observations agree with our previous results showing a modulation of chloride permeability by thiamine derivatives in membrane vesicles from rat brain. [less ▲]

Application of High-Performance Liquid Chromatography to the Study of Thiamine Metabolism and in Particular Thiamine Triphosphatase

in Journal of Chromatography. A (1991), 566(2), 397-408

Thiamine triphosphate can be found in most tissues at very low levels, but its role is unknown. Organs and muscles that generate electrical impulses are particularly rich in this compound. This paper ... [more ▼]

Thiamine triphosphate can be found in most tissues at very low levels, but its role is unknown. Organs and muscles that generate electrical impulses are particularly rich in this compound. This paper describes a thiamine triphosphatase from the electrical organ of Electrophorus electricus. The activity of this enzyme, as measured by a high-performance liquid chromatographic method, is closely anion-regulated. Furthermore, thiamine triphosphate increases chloride uptake in membrane vesicles prepared from rat brain. Our results suggest that this compound could play an important role in the regulation of chloride permeability. [less ▲]

Determination of Thiamin and Its Phosphate Esters in Cultured Neurons and Astrocytes Using an Ion-Pair Reversed-Phase High-Performance Liquid Chromatographic Method

in Analytical Biochemistry (1991), 198(1), 52-59

A sensitive method, based on fluorescence detection, for the determination of thiamin derivatives after precolumn derivatization is described. The separation is achieved on a PRP-1 column using ion-pair ... [more ▼]

A sensitive method, based on fluorescence detection, for the determination of thiamin derivatives after precolumn derivatization is described. The separation is achieved on a PRP-1 column using ion-pair reversed-phase HPLC. This method is especially well adapted to the detection of thiamin triphosphate in complex mixtures such as tissue extracts. The detection limit for TTP is 50 fmol. The contents of thiamin derivatives were determined in primary cultures of rat cerebellar granule neurons and cerebral astrocytes. The amount of TTP is about five times higher in neurons than in astrocytes. Thus in rat brain TTP seems to be essentially associated with neurons and the intracellular concentration is estimated to be about 0.2 microM. Our results suggest the existence, in nerve cells, of specific regulatory mechanisms not related to the blood-brain barrier and responsible for the maintenance of thiamin homeostasis in brain. [less ▲]

Regulation of Ion Uptake in Membrane Vesicles from Rat Brain by Thiamine Compounds

in Biochemical and Biophysical Research Communications (1990), 171(3), 1137-1144

We examined the effects of thiamine derivatives on ion uptake in rat brain membrane vesicles. Thiamine triphosphate (1 mM) and pyrithiamine (0.1 mM) increase chloride uptake. Preincubation of crude ... [more ▼]

We examined the effects of thiamine derivatives on ion uptake in rat brain membrane vesicles. Thiamine triphosphate (1 mM) and pyrithiamine (0.1 mM) increase chloride uptake. Preincubation of crude homogenate with thiamine or pyrithiamine increases chloride uptake while oxythiamine has the reverse effect. Thiamine and oxythiamine also affect 22Na+ and 86Rb+ uptake in the same way as for 36Cl- but to a lesser extent. Thiamine-dependent 36Cl- uptake is activated by sodium bicarbonate (10 mM) and partially inhibited by bumetanide (0.1 mM) and 2,4-dinitrophenol (0.1 mM). Preincubation with thiamine increases the thiamine triphosphate content of the vesicles. The hypothesis that TTP is the activator of a particular chloride uptake mechanism is discussed. [less ▲]

Thiamine Triphosphatase in the Membranes of the Main Electric Organ of Electrophorus Electricus: Substrate-Enzyme Interactions

in Journal of Neurochemistry (1989), 53(3), 738-746

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate (TTP). Membrane fractions prepared from this tissue contain a thiamine triphosphatase that is strongly ... [more ▼]

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate (TTP). Membrane fractions prepared from this tissue contain a thiamine triphosphatase that is strongly activated by anions and irreversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion transport inhibitor. Kinetic parameters of the enzyme are markedly affected by the conditions of enzyme preparation: In crude membranes, the apparent Km is 1.8 mM and the pH optimum is 6.8, but trypsin treatment of these membranes or their purification on a sucrose gradient decreases both the apparent Km (to 0.2 mM) and the pH optimum (to 5.0). Anions such as NO3- (250 mM) have the opposite effect, i.e., even in purified membranes, the pH optimum is now 7.8 and the Km is 1.1 mM; at pH 7.8, NO3- increases the Vmax 24-fold. TTP protects against inhibition by DIDS, and the KD for TTP could be estimated to be 0.25 mM, a value close to the apparent Km measured in the same purified membrane preparation. Thiamine pyrophosphate (0.1 mM) did not protect against DIDS inhibition. At lower (10(-5)-10(-6) M) substrate concentrations, Lineweaver-Burk plots of thiamine triphosphatase activity markedly deviate from linearity, with the curve being concave downward. This suggests either anticooperative binding or the existence of binding sites with different affinities for TTP. The latter possibility is supported by binding data obtained using [gamma-32P]TTP. Our data suggest the existence of a high-affinity binding site (KD of approximately 0.5 microM) for the Mg-TTP complex.(ABSTRACT TRUNCATED AT 250 WORDS) [less ▲]

Phosphorylated Thiamine Derivatives and Cortical Activity in the Baboon Papio Papio: Effect of Intermittent Light Stimulation

in Journal of Neurochemistry (1989), 53(1), 80-87

The effect of intermittent light stimulation (ILS) on the distribution of thiamine derivatives in three brain areas (occipital, motor, and premotor) was compared in photosensitive and nonphotosensitive ... [more ▼]

The effect of intermittent light stimulation (ILS) on the distribution of thiamine derivatives in three brain areas (occipital, motor, and premotor) was compared in photosensitive and nonphotosensitive baboons. ILS induces paroxysmal discharges in the motor and premotor areas of photosensitive animals only. In baboons submitted to ILS, thiamine triphosphate (TTP) decreases in both photosensitive and nonphotosensitive animals; thiamine monophosphate (TMP) increases in photosensitive animals, which present ILS-induced paroxysmal discharges, whereas it is unaffected in nonphotosensitive animals. The variations are the most significant in the occipital (visual) cortex. A consumption of TTP may result from electrical activity induced by light stimulation in the occipital area. No correlation between ILS-induced paroxysmal activity and a decrease in TTP contents was found. However, photosensitive animals are affected differently from nonphotosensitive animals, as their content of TMP in the cerebral cortex increases on stimulation. However, as long as the exact role of thiamine compounds in relation to membrane excitability in the nervous system remains unknown, it is impossible to conclude whether the differences observed in the metabolism of thiamine compounds are the cause or the consequence of the photosensitivity in the baboon Papio papio. [less ▲]

Synthesis of [Gamma-32p]Thiamine Triphosphate

in Analytical Biochemistry (1988), 169(2), 274-278

We developed a novel chemical synthesis of thiamine triphosphate which allows us to incorporate 32P in the gamma position. The reaction is based on the condensation of [32P]orthophosphoric acid and ... [more ▼]

We developed a novel chemical synthesis of thiamine triphosphate which allows us to incorporate 32P in the gamma position. The reaction is based on the condensation of [32P]orthophosphoric acid and thiamine diphosphate in the presence of ethyl chloroformate. After purification by two ion-exchange purification steps, the thiamine derivative has a specific radioactivity of 10 Ci/mmol. The average final yield synthesis is about 10%. [less ▲]