Adenosine thiamine triphosphate (AThTP) inhibits poly(ADP-ribose) polymerase-1 (PARP-1) activity.

Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) has been demonstrated to result in various stress-related diseases, including diabetes mellitus. Deficiency of cellular nicotinamide adenine dinucleotide (NAD(+)) content, consumed by PARP-1 to add ADP-ribose moieties onto target proteins, contributes to pathophysiological conditions. Adenosine thiamine triphosphate (AThTP) exists in small amounts in mammals; however, the function(s) of this metabolite remains unresolved. The structure of AThTP resembles NAD(+). Recent experimental studies demonstrate beneficial impacts of high-dose thiamine treatment of diabetic complications. These findings have led us to hypothesize that AThTP may modulate the activity of PARP-1. We have chemically synthesized AThTP and evaluated the effect of AThTP on recombinant PARP-1 enzyme activity. AThTP inhibited the PARP-1 activity at 10 µM, and a structural model of the PARP-1-AThTP complex highlighted the AThTP binding site. The results provide new insights into the pharmacological importance of AThTP as an inhibitor of PARP-1.

Thiamine triphosphate activates an anion channel of large unit conductance in neuroblastoma cells.

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.

The effects of thiamin and its phosphate esters on dopamine release in the rat striatum.

The effect of thiamin and its phosphate esters on dopamine (DA) release was examined in the rat striatum using an in vivo microdialysis. Intrastriatal administration of thiamin triphosphate (TTP) or thiamin diphosphate (TDP) induced DA release, but thiamin monophosphate (TMP) or thiamin did not show any change. In the absence of Ca2+ in the perfusate, TTP did not increase the DA release. omega-Conotoxin did not decrease the TTP-dependent DA release. These findings suggest that, in contrast to TMP and thiamin, TTP and TDP may play a specific role in DA release from nerve terminals.

Thiamin triphosphate does not affect contraction of skinned fibers.

Effects of thiamin triphosphate (TTP) on contraction of chemically skinned fibers prepared from the extensor digitorum longus muscle of guinea pigs were investigated. The addition of TTP at 40 microM concentration affected neither Ca2+ uptake nor Ca(2+)-induced Ca2+ release by sarcoplasmic reticulum. Contraction of myofibril was not affected by TTP either.

Regulation of ion uptake in membrane vesicles from rat brain by thiamine compounds.

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.

Thiamine triphosphatase in the membranes of the main electric organ of Electrophorus electricus: substrate-enzyme interactions.

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)

[Effect of thiamine phosphates on the activity of regulatory enzymes of the pyruvate dehydrogenase complex]. / Vliianie tiaminfosfatov na aktivnost' reguliatornykh fermentov piruvatdegidrogenaznogo kompleksa.

The effect of thiamine triphosphate (ThTP) and thiamine diphosphate (ThDP) on the activity of rat liver pyruvate dehydrogenase complex regulatory enzymes (kinase and phosphatase) was studied in experiments with isolated enzyme preparations. It is shown that ThDP caused a pronounced activation of pyruvate dehydrogenase phosphatase (Ka is equal to 65.0 nM). ThTP inhibits phosphatase competitively against the substrate--the phosphorylated pyruvate dehydrogenase complex. The both thiamine phosphates inhibit the pyruvate dehydrogenase kinase activity almost similarly in concentrations exceeding 10 microM. The physiological significance of the antagonistic action of ThDP and ThTP on the pyruvate dehydrogenase phosphatase activity is discussed.

Thiamine phosphorylated derivatives and bioelectrogenesis.

Kinetic as well as thermodynamic considerations favour the idea that the change in sodium conductance explaining the action potential, must result from a bimolecular reaction system. The fact that thiamine phosphorylated derivatives are associated with the specific protein forming the sodium channel could well mean that these thiamine derivatives and more specifically thiamine triphosphate are directly involved in the conductance change.

Alterations in uptake and metabolism of aspartate and glutamate in brain of thiamine deficient animals.

The high affinity uptake systems of aspartate, glutamate, glycine and taurine were studied in synaptosomal preparations isolated from the cerebellum, medulla-pons and the telencephalon of rats made thiamine deficient (TD) by diet or pyrithiamine (PT). There was a significant enhancement in the uptake of asparate/glutamate (probably transported by the same carrier) by the synaptosomal preparations of the cerebellum only, in both groups of thiamine-deficient animals as compared to controls. This was due to an increase in the number of uptake sites and not to an alteration of the binding affinity. Aspartate levels decreased significantly in all three brain areas of PT-treated animals and this change was greatest in the medulla-pons and the cerebellum and least in the cortex. Glutamate and serine levels were significantly decreased only in the medulla-pons whilst the concentration of glutamine was significantly increased in the three brain regions studied. The changes in both uptake and levels of amino acids in TD rats were reversed by thiamine therapy. Though the uptake studies do not discriminate between an altered aspartergic or glutamergic system, the changes in the levels of aspartate in the cerebellum suggest that the aspartergic system is involved. Since earlier studies showed a selective impairment in the high affinity uptake of serotonin by cerebellar synaptosomes, thiamine deficiency could impair cerebellar function by inducing an imbalance in its neurotransmitter systems.