Synthesis and anticancer activity of new coumarin-3-carboxylic acid derivatives as potential lactatetransportinhibitors.

As an oncometabolite, lactate plays a very important role in tumor proliferation, metastasis, angiogenesis, immune escape and other tumor biological functions. Pharmacological inhibition oflactate transport has been viewed as a promising therapeutic strategy to target a range of human cancers. In this study, a series of new coumarin-3-carboxylic acid derivatives 5a-t and 9a-b were synthesized and evaluated as lactate transport inhibitors. Their cytotoxic activity has been tested against three cell lines high-expressing and low-expressing monocarboxylate transporter 1 (MCT1) which acts as the main carrier for lactate. Compound 5c-e, 5g-i and 5m-o showed significant cytotoxicity and good selectivity against Hela and HCT116 cell lines with high MCT1 expression. Notably, coumarin-3-hydrazide 5o, the lead molecule with the most potent cytotoxic activity, exhibitedsignificant anti-proliferationandapoptosisinductioneffects. Further studies revealed that compound 5o decreased the expression level of target MCT1, and suppressed the energetic metabolism of Hela and HCT116 cells byremarkably reducing glucoseconsumptionandlactate production. Additionally, compound 5o induced intracellular lactate accumulation and inhibited lactate uptake, which implied that it blocked lactate transport via MCT1. These results indicate a good start point for the development of lactate transport inhibitors as new anticancer agents.

Danshensu attenuates scopolamine and amyloid-ß-induced cognitive impairments through the activation of PKA-CREB signaling in mice.

Alzheimer's disease (AD) is an important chronic neurodegenerative disorder and is mainly associated with cognitive dysfunction. At present, bioactive compounds from traditional medicinal plants have received much attention for the enhancement of cognitive function. Danshensu, a phenolic acid isolated from herbal medicines, has various pharmacological activities in the central nervous system, including anxiolytic-like and neuroprotective properties. The present study aimed to investigate the ameliorating effects of danshensu on scopolamine- and amyloid-ß (Aß) protein-induced cognitive impairments in mice. Danshensu (3 and 10 mg/kg, p.o.) effectively ameliorated scopolamine-induced cognitive dysfunction in mice, as measured in passive avoidance and Y-maze tasks. In a mechanistic study, danshensu inhibited monoamine oxidase A (MAO-A) activity but not MAO-B. Additionally, danshensu treatment increased the dopamine level and the phosphorylation levels of protein kinase A (PKA) and cAMP response element binding protein (CREB), in the cortex of the brain. Furthermore, the ameliorating effect of danshensu against scopolamine-induced cognitive impairment was fully blocked by H89, a PKA inhibitor. Finally, danshensu also ameliorated Aß-induced cognitive impairments in an animal model of AD. The results revealed that danshensu treatment significantly improved scopolamine and Aß-induced cognitive impairments in mice by facilitation of dopamine signaling cascade such as PKA and CREB due to MAO-A inhibition. Thus, danshensu could be used as a promising therapeutic agent for preventing and treating AD.

Short communication: 3-phenyllactic acid production in milk by Pediococcus pentosaceus SK25 during laboratory fermentation process.

3-Phenyllactic acid (PLA) is a broad-spectrum antimicrobial compound, produced by a wide range of lactic acid bacteria. A novel lactic acid bacteria strain with high PLA-producing ability, Pediococcus pentosaceus SK25, was isolated from traditional Chinese pickles. When grown in de Man, Rogosa, Sharpe broth at 30°C for 36h, this strain produced 135.6mg/L of PLA. Using this strain as starter for milk fermentation, 47.2mg/L of PLA was produced after fermentation for 12h. The PLA production was significantly improved by phenylalanine supplement, but was completely inhibited by tyrosine supplement.

Anxiolytic-like effect of danshensu [(3-(3,4-dihydroxyphenyl)-lactic acid)] in mice.

AIMS: Danshensu [3-(3,4-dihydroxyphenyl)-lactic acid], a phenylpropanoid compound isolated from Prunella vulgaris var. lilacina, is a well-known antioxidant. Although its antioxidant activity and cardioprotective effect have been reported, the pharmacological properties of danshensu in the central nervous system remain unclear. We investigated whether danshensu exerts anxiolytic-like activity in mice. MAIN METHODS: We conducted monoamine oxidase A (MAO-A) inhibition assay on danshensu in vitro, and behavioral tests including the elevated plus-maze test (EPM), the hole-board test, the rotarod test and the open field test were employed. KEY FINDINGS: We found that danshensu significantly inhibited the activity of MAO-A in vitro. The administration of danshensu (3 or 10mg/kg) produced a significant anxiolytic-like effect in the EPM and hole-board test. In addition, no changes in the spontaneous locomotor activity and no myorelaxant effects were observed compared to the control group; these effects were confirmed with the open field test and the rotarod test. Moreover, the anxiolytic-like properties of danshensu were antagonized by a dopamine D1 receptor antagonist (SCH 23390) but not by a 5-HT1A receptor antagonist (WAY 100635) or an α1-adrenergic receptor antagonist (prazosin). SIGNIFICANCE: These results indicate that danshensu exerts its anxiolytic-like properties, in part, through dopaminergic neurotransmitter signaling.

Chronic treatment with tandospirone, a 5-HT(1A) receptor partial agonist, suppresses footshock stress-induced lactate production in the prefrontal cortex of rats.

Serotonin 1A receptor (5-HT1A-R) agonists have been demonstrated to elicit antidepressant and anxiolytic effects. Lactate has been considered to play a major role in energy metabolism in the brain. Specifically, extracellular lactate concentrations (eLAC) have been suggested to reflect neural activity. Mild physical (e.g., handling) and non-physical (e.g., psychological) stressors have been shown to increase eLAC in several brain regions, including the medial prefrontal cortex (mPFC) and basolateral amygdala (BLA). Using in vivo microdialysis technique, we measured eLAC in the mPFC and BLA of rats under electric footshock stress to clarify the effect of repeated injection procedure (saline, once daily for 14 days) as a stressor on brain energy metabolism. Then, we examined the effect of chronic treatment with tandospirone, a 5-HT1A-R partial agonist, on eLAC during footshock stress in the mPFC. Footshock stress led to an increase in eLAC both in the mPFC and BLA in rats without injections. Repeated saline injection increased basal eLAC in the BLA, while footshock-induced lactate increment was reduced. In the mPFC, repeated saline injection did not affect basal eLAC and footshock-induced eLAC increments. Chronic treatment with tandospirone, at 0.2 and 1.0 mg/kg/day, but not 2.0 mg/kg/day, attenuated footshock stress-induced eLAC elevation in the mPFC. These observations suggest that eLAC in the BLA is sensitive to repeated exposure to physical stress. Data also indicate chronic treatment with tandospirone diminishes acute energy demands during neural activation in the mPFC. The implications of the present findings in relation to clinical efficacy of 5-HT1A agonists are discussed.

Low dose of dichloroacetate infusion reduces blood lactate after submaximal exercise in horses / Baixa dose de infusão de dicloroacetato reduz o lactato sanguíneo após exercício submáximo em cavalos

The acute administration of an indirect activator of the enzyme pyruvate dehydrogenase (PDH) in human athletes causes a reduction in blood lactate level during and after exercise. A single IV dose (2.5m.kg-1) of dichloroacetate (DCA) was administered before a submaximal incremental exercise test (IET) with five velocity steps, from 5.0 m.s-1 for 1 min to 6.0, 6.5, 7.0 and 7.5m.s-1 every 30s in four untrained mares. The blood collections were done in the period after exercise, at times 1, 3, 5, 10, 15 and 20 min. Blood lactate and glucose (mM) were determined electro-enzymatically utilizing a YSI 2300 automated analyzer. There was a 15.3% decrease in mean total blood lactate determined from the values obtained at all assessment times in both trials after the exercise. There was a decrease in blood lactate 1, 3, 5, 10, 15 and 20 min after exercise for the mares that received prior DCA treatment, with respective mean values of 6.31±0.90 vs 5.81±0.50, 6.45±1.19 vs 5.58±1.06, 6.07±1.56 vs 5.26±1.12, 4.88±1.61 vs 3.95±1.00, 3.66±1.41 vs 2.86±0.75 and 2.75±0.51 vs 2.04±0.30. There was no difference in glucose concentrations. By means of linear regression analysis, V140, V160, V180 and V200 were determined (velocity at which the rate heart is 140, 160, 180, and 200 beats/minute, respectively). The velocities related to heart rate did not differ, indicating that there was no ergogenic effect, but prior administration of a relatively low dose of DCA in mares reduced lactatemia after an IET.

A administração aguda de um ativador indireto da enzima piruvato desidrogenase (PD) em atletas da espécie humana provoca redução na concentração de lactato sanguíneo durante e após exercício. Uma dose única, intravenosa de 2.5m.kg-1 de dicloroacetato (DCA) foi administrada antes de um exercício teste incremental submáximo (ETI) com cinco etapas de velocidade sendo 5,0 ms-1 por 1 minuto e 6,0, 6,5, 7,0, e 7,5 ms-1 a cada 30 segundos em quatro éguas destreinadas. As coletas de sangue foram realizadas no período após o exercício, nos momentos de 1, 3, 5, 10, 15 e 20 min. Lactato e glicose (mM) foram determinados electro-enzimaticamente utilizando um analisador automático (YSI 2300). Houve redução de 15,3% no lactato sanguíneo total médio que foi determinado a partir dos valores obtidos em todos os momentos de avaliação em ambos os testes, após o exercício. Houve diminuição na lactatemia 1, 3, 5, 10, 15 e 20 minutos após exercício para as éguas que receberam infusão de DCA, com os respectivos valores médios de 6,31±0,90 versus 5,81±0,50, 6,45±1,19 versus 5,58±1,06, 6,07±1,56 versus 5,26±1,12, 4,88±1,61 versus 3,95±1,00, 3,66±1,41 versus 2,86±0,75 e 2,75±0,51 versus 2,04±0,30. Não houve diferença nas concentrações de glicose. Por meio de análise de regressão linear, V140, V160, V180 e V200 foram determinados (velocidades em que as taxas cardíacas alcançam 140, 160, 180 e 200 bpm, respectivamente). As velocidades relacionadas com a frequência cardíaca não diferiram, indicando que não houve efeito ergogênico, mas a administração prévia de uma dose relativamente baixa de DCA em éguas reduziu a lactatemia após um ETI.

Elevated blood lactate is not a primary cause of anorexia in tumor-bearing rats.

Tumor-bearing (TB) rats exhibit elevated concentrations of lactate in blood contiguous with the development of anorexia. Continuous intravenous infusion of lactate into non-TB rats reduced food intake at plasma concentrations lower than those observed in anorectic TB rats. Levels of neuropeptide Y (NPY) were elevated in the ventromedial (VMH) and dorsomedial hypothalamic regions of lactate-infused rats. The addition of the enhancer of pyruvate dehydrogenase activity, dichloroacetate (DCA), to the drinking water of TB rats (0.1-0.4%) normalized blood lactate concentration but had no significant effect on anorexia. However, the elevated concentration of NPY in the VMH of anorectic TB rats was also normalized by the DCA treatment. No alterations in regional hypothalamic levels of corticotropin-releasing factor were observed within any treatment conditions. These results suggest that, although hyperlactatemia may be involved in maintaining elevated NPY concentrations in anorectic TB rats, it does not appear to be a significant factor in the etiology of experimental cancer anorexia.

Acidogenicity of buccal plaque after a single rinse with amine fluoride - stannous fluoride mouthrinse solution.

Caries and gingivitis prevention may benefit from chemotherapeutic plaque control, therefore we compared in a cross-over study with 5 subjects the anti-acidogenic effects of a single use of AmF-SnF2 mouthrinse solutions (Meridol with and without 5% alcohol) with baseline and with the effects of a placebo and a chlorhexidine mouthrinse (CHX). Buccal plaque was collected 0.5, 3 and 8 h after the subjects used one of the mouthrinses, each time before and after a rinse with 10% sucrose to induce lactic acid production. Samples were analysed for acid anions by capillary electrophoresis and for protein. At 0.5 h after the use of AmF-SnF2 or CHX, the concentration of acetate in resting plaque was 70% lower than at baseline or after using the placebo. Average post-sucrose acetate and lactate concentrations in the placebo group were 30-80% higher than at baseline; up to 3 h this difference was significant. 8 h after using AmF-SnF2 or CHX, the post-sucrose acetate and lactate concentrations were still 30-50% lower than after the placebo, and up to 40% lower than at baseline. To conclude, AmF-SnF2 in both Meridol formulations and CHX were shown to have a similar potency to inhibit acid production after a single rinse.

L-[U-14C] lactate binding to a 43 kDa protein in plasma membranes of Candida utilis.

To identify the putative lactate transporter protein of Candida utilis, plasma membranes from cells grown either on lactic acid (presence of lactate proton symport) or glucose (absence of lactate proton symport) were incubated with L-[U-14C]lactic acid and the membrane proteins were then separated by SDS-PAGE. A well-defined peak of radioactivity occurred in the lane of the gel containing plasma membrane proteins from lactic-acid-grown cells but not from glucose-grown cells. Binding was inhibited by unlabelled pyruvate and lactate, whereas succinate and citrate were not inhibitory. The monocarboxylate transporter inhibitor of animal cells, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, competitively inhibited the lactate proton symport in the whole yeast and also inhibited lactate binding to proteins of isolated plasma membranes. The polypeptide pattern of plasma membranes from lactic-acid-grown cells revealed a 43 kDa polypeptide associated with the peak of labelled lactate. Altogether the results suggest that this polypeptide is either the lactate transporter or a component of it.

Amrinone prevents the inhibition of muscle pyruvate dehydrogenase complex activity during sepsis.

A decreased proportion of active pyruvate dehydrogenase complex (PDH) in skeletal muscle has been implicated as an important factor in elevating plasma lactate concentrations in hypermetabolic sepsis. The mediators of the septic process responsible for the inhibition of PDH complex in muscle are unknown. To assess the role of tumor necrosis factor in mediating the effects of sepsis, the effect of daily injections of amrinone (5 mg/kg/day), which inhibits the release of tumor necrosis factor during sepsis, on the proportion of PDH in the active form (PDHa) was investigated in a model of chronic hypermetabolic sepsis. In skeletal muscle from untreated septic rats, PDHa was decreased 50%. Treatment of septic rats with amrinone for 5 days prevented the sepsis-induced decrease in PDHa. Sepsis caused a 2.5-fold elevation in plasma lactate concentrations. The maintenance of the PDH complex activity at control values following injection of amrinone in septic rats was associated with reduced lactate concentrations in plasma. Thus, amrinone prevented the sepsis-induced abnormalities in skeletal muscle PDH activity and plasma lactate concentrations.

L(+)-lactate binding to a protein in rat skeletal muscle plasma membranes.

Biochemical studies were conducted to determine the location of a putative lactate transport protein in rat skeletal muscle plasma membranes (PM). PM (50-100 micrograms protein) were incubated with [U-14C] L(+)-lactate, in the presence or absence of unlabeled monocarboxylates or potential inhibitors, after which proteins were separated by SDS-PAGE. Gel slices (2 mm) were cut and analyzed for 14C. [U-14C] L(+)-lactate was bound to plasma membranes in the 30 to 40 kDa molecular mass range. Binding of [U-14C] L(+)-lactate was inhibited by N-ethylmaleimide, unlabeled L-lactate and pyruvate, and in a dose dependent manner by alpha-cyano-4-hydroxycinnamate (r = 0.995), but not by cytochalasin-B. The inhibition of [U-14C] L(+)-lactate binding was similar to the inhibition of lactate transport. Therefore the transport of L(+)-lactate across skeletal muscle plasma membranes involves a polypeptide of 30 to 40 kDa.

Evidence for a lactate-anion exchanger in the rat jejunal basolateral membrane.

BACKGROUND/AIMS: The mechanism by which lactate, absorbed from the intestinal lumen or generated within the epithelium, crosses the basolateral membrane of the enterocyte and enters the bloodstream has not previously been characterized in detail. METHODS: L-lactate uptake into and efflux from isolated jejunal basolateral membrane vesicles was investigated at room temperature using rapid filtration techniques. RESULTS: Furosemide sensitive uptake of L-lactate was unaffected by cis sodium or proton gradients but could be stimulated by a trans gradient of bicarbonate and chloride. Kinetic analysis showed uptake to consist of a saturable component with a Michaelis constant (Km) of 3.2 mmol/L and a maximum velocity (Vmax) of 67 pmol.mg protein-1 x s-1 and a nonsaturable alpha-4-hydroxy-cinnamic acid insensitive component. Pyruvate, butyrate, acetate, valerate, and propionate competitively inhibited lactate uptake into the vesicles. Efflux of lactate from preloaded vesicles was furosemide sensitive and accelerated by a trans bicarbonate gradient as well as by 10 mmol/L acetate, butyrate, and pyruvate. CONCLUSIONS: It is concluded that there is a short chain-fatty acid carrier system in the intestinal basolateral membrane, which operates as an anion exchanger.

Mechanisms of stimulus-evoked intracellular acidification in frog nerve fibres.

Measurements of cytoplasmic pH (pHi) in frog nerve fibers (sciatic nerve and its thin bundles) were performed by using fluorescein diacetate. Earlier it had been established that veratridine (VER) treatment of the nerve greatly enhances the stimulus-evoked intracellular acidification (SEIA) which becomes irreversible after blockade of the Na+/K+ pump with ouabain. Present experiments have shown that inhibition of lactic acid production by iodacetamide (5 mM) or blockade of Cl- influx by SITS do not prevent or attenuate the VER- and stimulus-evoked decrease in pHi. Blockade of Na+/H+ exchange by EIPA impedes pHi recovery following repetitive stimulation. Lowering of external pH (pHe) to 6.5 enhances, while elevation of pHe to 9.5 greatly diminishes SEIA, both in the presence or absence of VER. The hypothesis is put forward that SEIA results from excessive influx of H+ and Na+ into the fiber via activated Na+ channels: internal Na+ suppresses Na+/H+ exchange which potentiates the pHi decrease caused by H+ influx.

Iron- and lactic acid-induced lipid peroxidation in rat kidney homogenates and slices.

Lactic acid 4 mM acted as a lipoperoxidant by increasing production of thiobarbituric acid-reactive substances (TBARS) in rat kidney slices and homogenates. This effect occurred mainly when slices and homogenates were incubated in a pH 5.4 medium conductive to full expression of compound acidity. TBARS increase was only slight when incubation was performed in Krebs buffer, pH 7.4. Moreover, sodium lactate 4 mM increased TBARS production only when homogenates were incubated in the pH 5.4 medium. Deferoxamine (1 mM) inhibited the prooxidant effect of lactic acid 4 mM, and TBARS increase was correlated with iron release. The iron mobilized may come from reserves where it is weakly bound or from ferritin; and ascorbic acid, present in low quantities in kidney, might trigger the release of this product.

Inhibition by noradrenaline and adrenaline of the increase in glucose and lactate output and decrease in flow after sympathetic nerve stimulation in perfused rat liver: possible involvement of protein kinase C.

In perfused rat liver stimulation of the hepatic nerve plexuses increased via alpha 1-receptors glucose and lactate output decreased flow and caused an overflow of noradrenaline into the hepatic vein. Infusion of noradrenaline and adrenaline also elicited similar metabolic and hemodynamic alterations via alpha 1-receptors, whereas infusion of isoproterenol via beta 2-receptors enhanced glucose output and slightly reduced lactate release without affecting flow. The influence of circulating catecholamines on the nerve stimulation-dependent changes was investigated. Noradrenaline (100 nmol/L) or adrenaline (40 nmol/L) but not isoproterenol (1 mumol/L), which themselves caused about half-maximal alterations, strongly inhibited the nerve stimulation-induced increase in glucose and lactate output and decrease in flow but had no effect on noradrenaline overflow. The protein kinase C activator (4 beta)phorbol 12-myristate, 13-acetate (100 nmol/L) but not its analog (4 alpha)phorbol 12,13-didecanoate (100 nmol/L) strongly inhibited the metabolic and hemodynamic changes caused by nerve stimulation or noradrenaline infusion. The protein kinase C inhibitor H7 (20 mumol/L) partially prevented the inhibition of the nerve actions by noradrenaline. The results lead us to conclude that noradrenaline and adrenaline inhibited the metabolic and hemodynamic nerve actions by means of a mechanism involving protein kinase C rather than presynaptic alpha-receptors or beta-receptors. The catecholamines apparently increased via alpha 1-receptors inositol 1,4,5-trisphosphate, which in turn enhanced cytosolic Ca2+ and thus altered metabolism and in part hemodynamics, and diacylglycerol, which in turn activated protein kinase C and thus feedback inhibited the signal chain from alpha 1-receptors via G proteins to phospholipase C.

Inhibition of lactate-induced swelling by dichloroacetate in human astrocytoma cells.

High levels of tissue lactate exacerbate tissue damage that results from cerebral ischemia and reperfusion injury that follows. Post-ischemic treatment with dichloroacetate (DCA) facilitates a decrease in lactate in the central nervous system (CNS) of animals during reperfusion following experimental ischemia, thus it may help to ameliorate ischemic cell damage. It has been suggested that the lactate lowering effect is mediated through a stimulatory effect of DCA on pyruvate dehydrogenase (PDHC) activity. We have studied such a hypothesis in a human astrocytoma derived cell line, UC-11MG. Under conditions resembling those of the ischemic tissue (i.e. high lactate and low pH) these cells accumulate lactate, driven by the inwardly directed proton gradient, and swell as a consequence of the osmotic effect of intracellular lactate. We have demonstrated that DCA increases PDHC activity and also reduces lactate-induced swelling. However, we also found that these two effects could be uncoupled and that the ability of DCA to prevent swelling is still present in the absence of any stimulation of PDHC. We also demonstrated that DCA competitively inhibits the uptake of lactate (Ki = 1.9 mM) and increases the efflux of lactate in a trans-acting manner that suggests the presence of a lactate-DCA exchange. We present a mechanism by which reduction in the rate of lactate uptake could account for the observed inhibition of swelling. This effect of DCA on lactate transport indicates another possible mechanism of action for DCA in facilitating the decrease in lactate observed in vivo during reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the beta-adrenergic-response in cultured rat heart cells. II. Mammary-derived growth inhibitor (MDGI) blocks induction of beta-adrenergic supersensitivity. Dissociation from lipid-binding activity of MDGI.

'Mammary-derived growth inhibitor (MDGI)' is a 14.5 kDa polypeptide with growth-inhibitory activity for various mammary epithelial cells in vitro which is highly homologous to cardiac fatty acid-binding protein (H-FABP). Here we describe a new biological activity of MDGI: Inhibition of L(+)-lactate-, arachidonic acid- and 15-S-hydroxyeicosatetraenoic acid-induced supersensitivity of neonatal rat heart cells for beta-adrenergic stimulation, concerning particularly a small population of beta 2-receptors. Synthetic peptides corresponding to the MDGI-sequence, residue 121-131 mimic the effect of MDGI. Measurements of lipid-binding to MDGI and synthetic peptides excluded the binding of arachidonic acid, 15-S-hydroxyeicosatetraenoic acid or beta-adrenergic agonists to MDGI or the peptides as the mechanism for this effect. Also, no direct interference of MDGI and the synthetic peptides with the binding of the beta-adrenergic agent CGP 12177 to its receptor on A431 cells could be detected. We suggest that MDGI and the peptides act by interference with the function of the beta 2-adrenergic receptor and that this mechanism might also be relevant for the growth-inhibitory activity of MDGI. Furthermore, the data point to a possible function of H-FABP for the modulation of beta-adrenergic sensitivity of cardiac myocytes.

[Pathophysiological and clinical evaluation of the effectiveness of dalargin in the treatment of arterial occlusive diseases of the lower extremities]. / Patofiziologicheskaia i klinicheskaia otsenka éffektivnosti dalargina pri lechenii obliteriruiushchikh zabolevanii arterii nizhnikh konechnostei.

Eighty seven patients with Stages II-III vascular occlusions in the lower extremities were examined. The clinical and biochemical parameters of the course of the disease were found to improve in the patients who received in earlier periods dalargin supplemented to the combined therapy than in those from a matched group. There was a decrease in femoral venous blood lactate concentrations in the diseased extremity, blood cholesterol and parathyroid hormone levels. The mechanisms responsible for effects of dalargin on metabolic parameters and clinical indices of the patients' condition were also discussed. Whether it is possible to use dalargin in the combined therapy for extremity vascular occlusive diseases is considered.