Lactate exposure shapes the metabolic and transcriptomic profile of CD8+ T cells.

Introduction: CD8+ T cells infiltrate virtually every tissue to find and destroy infected or mutated cells. They often traverse varying oxygen levels and nutrient-deprived microenvironments. High glycolytic activity in local tissues can result in significant exposure of cytotoxic T cells to the lactate metabolite. Lactate has been known to act as an immunosuppressor, at least in part due to its association with tissue acidosis. Methods: To dissect the role of the lactate anion, independently of pH, we performed phenotypical and metabolic assays, high-throughput RNA sequencing, and mass spectrometry, on primary cultures of murine or human CD8+ T cells exposed to high doses of pH-neutral sodium lactate. Results: The lactate anion is well tolerated by CD8+ T cells in pH neutral conditions. We describe how lactate is taken up by activated CD8+ T cells and can displace glucose as a carbon source. Activation in the presence of sodium lactate significantly alters the CD8+ T cell transcriptome, including the expression key effector differentiation markers such as granzyme B and interferon-gamma. Discussion: Our studies reveal novel metabolic features of lactate utilization by activated CD8+ T cells, and highlight the importance of lactate in shaping the differentiation and activity of cytotoxic T cells.

Homology- and cross-resistance of Lactobacillus plantarum to acid and osmotic stress and the influence of induction conditions on its proliferation by RNA-Seq.

In this study, homology- and cross-resistance of Lactobacillus plantarum L1 and Lactobacillus plantarum L2 to acid and osmotic stress were investigated. Meanwhile, its proliferation mechanism was demonstrated by transcriptomic analysis using RNA sequencing. We found that the homologous-resistance and cross-resistance of L. plantarum L1 and L. plantarum L2 increased after acid and osmotic induction treatment by lactic acid and sodium lactate solution in advance, and the survival rate of live bacteria was improved. In addition, the count of viable bacteria of L. plantarum L2 significantly increased cultivated at a pH 5.0 with a 15% sodium lactate sublethal treatment, compared with the control group. Further study revealed that genes related to membrane transport, amino acid metabolism, nucleotide metabolism, and cell growth were significantly upregulated. These findings will contribute to promote high-density cell culture of starter cultures production in the fermented food industry.

Time to fatigue on lactate threshold and supplementation with sodium bicarbonate in middle-distance college athletes / Tiempo hasta la fatiga sobre el umbral láctico y suplementación con bicarbonato de sodio en corredores de medio fondo universitarios

Introduction: There have been many researches that have attempted to improve sports performance based on supplementation with different buffer substances. Within this group of substances, sodium bicarbonate supplementation has been widely used in both cyclic and acyclic sports. In the case of cyclic sports, it has been tried to improve the performance of pedestrian races at different percentages of peak oxygen uptake. However, the results on intensity over anaerobic threshold have been contradictory. Objective: To determine the performance variation based on execution in a endurance test after sodium bicarbonate administration. The second aim to evaluate the levels of blood lactate in the finish of endurance test. Material and method: Five college athletes of middle-endurance and endurance race runners were subjects in the study. The variables measured were: effort maximum time (in seconds) measured through a endurance test, and maximum lactate post effort. All athletes were administered sodium bicarbonate (0.3 g·Kg-1 body mass) or a placebo (0.045 g·Kg-1 body mass) an hour before each endurance test. Student’s t-test (lactate) and Wilcoxon (time) was used for the statistical analysis. The size of the effect was calculated with Cohen’s d-test. Results: The effort maximum time showed a significant increase (P < 0.042; size of the effect = 0.852) as well as lactate concentrations post effort (P < 0.022; size of the effect = 1.987). Conclusions: The results of the study showed that the supplementation whit sodium bicarbonate generates an increase in the performance and lactate concentrations post effort, when the race speed surpass by seven percent the anaerobic threshold speed

Introducción: Han sido muchas las investigaciones que han intentado mejorar el rendimiento deportivo en base a la suplementación con diferentes sustancias buffer. Dentro de este grupo de sustancias, la suplementación con bicarbonato de sodio ha sido ampliamente utilizada tanto en deportes cíclicos como acíclicos. En el caso de deportes cíclicos, se ha experimentado mejorar el rendimiento de carreras pedestres a diferentes porcentajes del consumo máximo de oxigeno. Sin embargo, los resultados a intensidades por sobre el umbral láctico han sido contradictorios. Objetivo: Determinar la variación del rendimiento en base al tiempo de ejecución y producción de lactato post esfuerzo en una prueba de resistencia a una intensidad por sobre el UL posterior a la suplementación con bicarbonato de sodio. Material y métodos: Cinco atletas de medio fondo y fondo universitarios fueron parte del estudio. Las variables medidas fueron: tiempo máximo de esfuerzo (segundos) evaluado a través de una prueba de tiempo límite y lactato máximo post esfuerzo. Una hora antes de cada prueba de tiempo límite los atletas fueron suplementados con bicarbonato de sodio (0,3 g·Kg-1 masa corporal) o un placebo (0,045 g·Kg-1 masa corporal). Para el análisis estadístico se utilizó la prueba t de Student (lactato) y Wilcoxon (tiempo). El tamaño del efecto fue calculado con la prueba d de Cohen. Resultados: El tiempo máximo de esfuerzo tuvo incrementos significativos (p < 0,042; tamaño del efecto = 0,852), al igual que las concentraciones de lactato post esfuerzo (p < 0,022; tamaño del efecto = 1,987). Conclusión: Los resultados del estudio mostraron que la suplementación con bicarbonato de sodio genera un aumento en el rendimiento y en las concentraciones de lactato post esfuerzo, cuando la velocidad de desplazamiento sobrepasa un siete por ciento la velocidad de umbral láctico

Dynamics of organohalide-respiring bacteria and their genes following in-situ chemical oxidation of chlorinated ethenes and biostimulation.

Application of Fenton's reagent and enhanced reductive dechlorination are currently the most common remediation strategies resulting in removal of chlorinated ethenes. In this study, the influence of such techniques on organohalide-respiring bacteria was assessed at a site contaminated by chlorinated ethenes using a wide spectrum of molecular genetic markers, including 16S rRNA gene of the organohalide-respiring bacteria Dehaloccocoides spp., Desulfitobacterium and Dehalobacter; reductive dehalogenase genes (vcrA, bvcA) responsible for dechlorination of vinyl chloride and sulphate-reducing and denitrifying bacteria. In-situ application of hydrogen peroxide to induce a Fenton-like reaction caused an instantaneous decline in all markers below detection limit. Two weeks after application, the bvcA gene and Desulfitobacterium relative abundance increased to levels significantly higher than those prior to application. No significant decrease in the concentration of a range of chlorinated ethenes was observed due to the low hydrogen peroxide dose used. A clear increase in marker levels was also observed following in-situ application of sodium lactate, which resulted in a seven-fold increase in Desulfitobacterium and a three-fold increase in Dehaloccocoides spp. after 70 days. An increase in the vcrA gene corresponded with increase in Dehaloccocoides spp. Analysis of selected markers clearly revealed a positive response of organohalide-respiring bacteria to biostimulation and unexpectedly fast recovery after the Fenton-like reaction.

Nutritional Strategies to Modulate Intracellular and Extracellular Buffering Capacity During High-Intensity Exercise.

Intramuscular acidosis is a contributing factor to fatigue during high-intensity exercise. Many nutritional strategies aiming to increase intra- and extracellular buffering capacity have been investigated. Among these, supplementation of beta-alanine (~3-6.4 g/day for 4 weeks or longer), the rate-limiting factor to the intramuscular synthesis of carnosine (i.e. an intracellular buffer), has been shown to result in positive effects on exercise performance in which acidosis is a contributing factor to fatigue. Furthermore, sodium bicarbonate, sodium citrate and sodium/calcium lactate supplementation have been employed in an attempt to increase the extracellular buffering capacity. Although all attempts have increased blood bicarbonate concentrations, evidence indicates that sodium bicarbonate (0.3 g/kg body mass) is the most effective in improving high-intensity exercise performance. The evidence supporting the ergogenic effects of sodium citrate and lactate remain weak. These nutritional strategies are not without side effects, as gastrointestinal distress is often associated with the effective doses of sodium bicarbonate, sodium citrate and calcium lactate. Similarly, paresthesia (i.e. tingling sensation of the skin) is currently the only known side effect associated with beta-alanine supplementation, and it is caused by the acute elevation in plasma beta-alanine concentration after a single dose of beta-alanine. Finally, the co-supplementation of beta-alanine and sodium bicarbonate may result in additive ergogenic gains during high-intensity exercise, although studies are required to investigate this combination in a wide range of sports.

Comparative Proteomic Insights into the Lactate Responses of Halophilic Salinicoccus roseus W12.

Extremophiles use adaptive mechanisms to survive in extreme environments, which is of great importance for several biotechnological applications. A halophilic strain, Salinicoccus roseus W12, was isolated from salt lake in Inner Mongolia, China in this study. The ability of the strain to survive under high sodium conditions (including 20% sodium lactate or 25% sodium chloride, [w/v]) made it an ideal host to screen for key factors related to sodium lactate resistance. The proteomic responses to lactate were studied using W12 cells cultivated with or without lactate stress. A total of 1,656 protein spots in sodium lactate-treated culture and 1,843 spots in NaCl-treated culture were detected by 2-dimensional gel electrophoresis, and 32 of 120 significantly altered protein spots (fold change > 2, p < 0.05) were identified by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. Among 21 successfully identified spots, 19 proteins were upregulated and 2 were downregulated. The identified proteins are mainly involved in metabolism, cellular processes and signaling, and information storage and processing. Transcription studies confirmed that most of the encoding genes were upregulated after the cells were exposed to lactate in 10 min. Cross-protecting and energy metabolism-related proteins played an important role in lactate tolerance for S. roseus W12.

Comparative transcriptome analysis reveals different molecular mechanisms of Bacillus coagulans 2-6 response to sodium lactate and calcium lactate during lactic acid production.

Lactate production is enhanced by adding calcium carbonate or sodium hydroxide during fermentation. However, Bacillus coagulans 2-6 can produce more than 180 g/L L-lactic acid when calcium lactate is accumulated, but less than 120 g/L L-lactic acid when sodium lactate is formed. The molecular mechanisms by which B. coagulans responds to calcium lactate and sodium lactate remain unclear. In this study, comparative transcriptomic methods based on high-throughput RNA sequencing were applied to study gene expression changes in B. coagulans 2-6 cultured in non-stress, sodium lactate stress and calcium lactate stress conditions. Gene expression profiling identified 712 and 1213 significantly regulated genes in response to calcium lactate stress and sodium lactate stress, respectively. Gene ontology assignments of the differentially expressed genes were performed. KEGG pathway enrichment analysis revealed that 'ATP-binding cassette transporters' were significantly affected by calcium lactate stress, and 'amino sugar and nucleotide sugar metabolism' was significantly affected by sodium lactate stress. It was also found that lactate fermentation was less affected by calcium lactate stress than by sodium lactate stress. Sodium lactate stress had negative effect on the expression of 'glycolysis/gluconeogenesis' genes but positive effect on the expression of 'citrate cycle (TCA cycle)' genes. However, calcium lactate stress had positive influence on the expression of 'glycolysis/gluconeogenesis' genes and had minor influence on 'citrate cycle (TCA cycle)' genes. Thus, our findings offer new insights into the responses of B. coagulans to different lactate stresses. Notably, our RNA-seq dataset constitute a robust database for investigating the functions of genes induced by lactate stress in the future and identify potential targets for genetic engineering to further improve L-lactic acid production by B. coagulans.

Salty taste deficits in CALHM1 knockout mice.

Genetic ablation of calcium homeostasis modulator 1 (CALHM1), which releases adenosine triphosphate from Type 2 taste cells, severely compromises the behavioral and electrophysiological responses to tastes detected by G protein-coupled receptors, such as sweet and bitter. However, the contribution of CALHM1 to salty taste perception is less clear. Here, we evaluated several salty taste-related phenotypes of CALHM1 knockout (KO) mice and their wild-type (WT) controls: 1) In a conditioned aversion test, CALHM1 WT and KO mice had similar NaCl avoidance thresholds. 2) In two-bottle choice tests, CALHM1 WT mice showed the classic inverted U-shaped NaCl concentration-preference function but CALHM1 KO mice had a blunted peak response. 3) In brief-access tests, CALHM1 KO mice showed less avoidance than did WT mice of high concentrations of NaCl, KCl, NH(4)Cl, and sodium lactate (NaLac). Amiloride further ameliorated the NaCl avoidance of CALHM1 KO mice, so that lick rates to a mixture of 1000 mM NaCl + 10 µM amiloride were statistically indistinguishable from those to water. 4) Relative to WT mice, CALHM1 KO mice had reduced chorda tympani nerve activity elicited by oral application of NaCl, NaLac, and sucrose but normal responses to HCl and NH(4)Cl. Chorda tympani responses to NaCl and NaLac were amiloride sensitive in WT but not KO mice. These results reinforce others demonstrating that multiple transduction pathways make complex, concentration-dependent contributions to salty taste perception. One of these pathways depends on CALHM1 to detect hypertonic NaCl in the mouth and signal the aversive taste of concentrated salt.

A high-sensitive and non-radioisotopic fluorescence dye method for evaluating bacterial adhesion to denture materials.

Oral bacteria adhered to dental material surfaces are known to cause various oral diseases. This study aimed to develop a highsensitive and non-radioisotopic fluorescence dye method for quantification of oral bacteria (Streptococcus, Actinomyces and Veillonella) adhered to denture material surfaces. The amount of adhered bacteria was estimated from the fluorescence intensity derived from resazurin, which is reduced by bacterial metabolic reactions. The addition of bacterial metabolic substrates (glucose for Streptococcus and Actinomyces and sodium lactate for Veillonella) to the reaction mixture increased the fluorescence intensity by 2.3-110 times, subsequently improved the sensitivity. Furthermore, an experimental device having silicon wells containing test material was carefully designed for accurate quantification of bacteria adhered to test materials. The improved resazurin method using a new experimental device successfully enabled the quantification of bacterial adhesion to polymethyl methacrylate and other three conventional denture materials.

Exogenous L-lactate clearance in adult horses.

OBJECTIVE: To determine endogenous production of L-lactate and the clearance of exogenous sodium L-lactate (ExLC) in healthy adult horses. DESIGN: A sodium L-lactate solution (1 mmol/kg body weight qs to 500 mL final volume in 0.9% NaCl) was adminstered IV over 15 minutes. Blood samples for L-lactate concentration [LAC] measurement were collected immediately prior to infusion, at 5, 10, and 15 minutes during infusion and at 1 minute intervals for 15 minutes, at 30, 45, 60, 120, and 180 minutes postinfusion. Disposition modeling and pharmacokinetic analysis was performed using proprietary software. SETTING: University Teaching Hospital. ANIMALS: Six clinically healthy adult horses. MEASUREMENTS AND MAIN RESULTS: Median (range) baseline [LAC] was 0.43 (0.20-0.72) mmol/L for samples obtained every 3 hours over the 24 hours prior to ExLC and demonstrated variability primarily associated with horse. Median [LAC] immediately prior to ExLC was 0.43 (0.35-0.52) mmol/L. A 2-compartment model was used to specify the pharmacokinetic parameters. Median (range) ExLC was 1.05 (0.073-1.75) L·h(-1) ·kg(-1) and t(1/2) ß was 29.54 (20.8-38.6) min. Median lactate production based on basal [LAC] immediately prior to ExLC was was 0.49 (0.31-0.93) mmol·h(-1) ·kg(-1) . CONCLUSIONS: ExLC in healthy adult horses is greater than that of hyperlactemic human patients but similar to normolactemic-sick human patients examined using the same model, supporting development of species, and disease specific ExLC parameters.

High level production of 5-aminolevulinic acid by Propionibacterium acidipropionici grown in a low-cost medium.

5-Aminolevulinic acid (ALA) is an intermediate in the biosynthesis of tetrapyrroles. Its current production is expensive. We have developed a low-cost medium for Propionibacterium acidipropionici to produce extracellular ALA. When grown at 35 °C on a medium containing 3 % (w/v) food-grade sodium lactate supplemented with 18 g glycine/l, 4.05 g succinate/l, 1.8 g glucose/l, pH 7, it produced ALA up to 7.7 g/l over 6 days. Plant-growth promoting activity assays showed that the ALA was biologically active.

Renal lactate elimination is maintained during moderate exercise in humans.

Reduced hepatic lactate elimination initiates blood lactate accumulation during incremental exercise. In this study, we wished to determine whether renal lactate elimination contributes to the initiation of blood lactate accumulation. The renal arterial-to-venous (a-v) lactate difference was determined in nine men during sodium lactate infusion to enhance the evaluation (0.5 mol x L(-1) at 16 ± 1 mL x min(-1); mean ± s) both at rest and during cycling exercise (heart rate 139 ± 5 beats x min(-1)). The renal release of erythropoietin was used to detect kidney tissue ischaemia. At rest, the a-v O(2) (CaO(2)-CvO(2)) and lactate concentration differences were 0.8 ± 0.2 and 0.02 ± 0.02 mmol x L(-1), respectively. During exercise, arterial lactate and CaO(2)-CvO(2) increased to 7.1 ± 1.1 and 2.6 ± 0.8 mmol x L(-1), respectively (P < 0.05), indicating a -70% reduction of renal blood flow with no significant change in the renal venous erythropoietin concentration (0.8 ± 1.4 U x L(-1)). The a-v lactate concentration difference increased to 0.5 ± 0.8 mmol x L(-1), indicating similar lactate elimination as at rest. In conclusion, a -70% reduction in renal blood flow does not provoke critical renal ischaemia, and renal lactate elimination is maintained. Thus, kidney lactate elimination is unlikely to contribute to the initial blood lactate accumulation during progressive exercise.

Intracellular Ca2+ modulation during short exposure to ischemia-mimetic factors in isolated rat ventricular myocytes.

We investigated the effects of different ischemia-mimetic factors on intracellular Ca2+ concentration ([Ca2+]i). Ventricular myocytes were isolated from adult Wistar rats, and [Ca2+]i was measured using fluorescent indicator fluo-4 AM by confocal microscopy. Intracellular pH was measured using c5-(and-6)-carboxy SNARF-1 AM, a dual emission pH-sensitive ionophore. Myocytes were exposed to hypoxia, extracellular acidosis (pH(o) 6.8), Na-lactate (10 mM), or to combination of those factors for 25 min. Monitoring of [Ca2+]i using fluo-4 AM fluorescent indicator revealed that [Ca2+]i accumulation increased immediately after exposing the cells to Na-lactate and extracellular acidosis, but not during cell exposure to moderate ischemia. Increase in [Ca2+]i during Na-lactate exposure decreased to control levels at the end of exposure period at extracellular pH 7.4, but not at pH 6.8. When combined, Na-lactate and acidosis had an additive effect on [Ca2+]i increase. After removal of solutions, [Ca2+]i continued to rise only when acidosis, hypoxia, and Na-lactate were applied together. Analysis of intracellular pH revealed that treatment of cells by Na-lactate and acidosis caused intracellular acidification, while short ischemia did not significantly change intracellular pH. Our experiments suggest that increase in [Ca2+]i during short hypoxia does not occur if pH(i) does not fall, while extracellular acidosis is required for sustained rise in [Ca2+]i induced by Na-lactate. Comparing to the effect of Na-lactate, extracellular acidosis induced slower [Ca2+]i elevation, accompanied with slower decrease in intracellular pH. These multiple effects of hypoxia, extracellular acidosis, and Na-lactate are likely to cause [Ca2+]i accumulation after the hypoxic stress.

Half-molar sodium-lactate solution has a beneficial effect in patients after coronary artery bypass grafting.

OBJECTIVE: To compare two solutions for fluid resuscitation in post-coronary artery bypass grafting (CABG) surgery patients: Ringer's lactate (RL) versus a new solution containing half-molar sodium-lactate (HL). DESIGN: Prospective randomized open label study. SETTING: The first 12 h post-CABG surgery in an intensive care unit (ICU). PATIENTS: There were 230 patients enrolled in the study: 208 were analyzed, with 109 from the HL group and 99 from the RL group. INTERVENTIONS: Patients received over the first 12 h post-CABG 10 ml kg BW(-1) HL solution in the HL group versus 30 ml kg BW(-1) of RL solution in the RL group. MEASUREMENTS AND RESULTS: Hemodynamic status, body fluid balance and inotrope utilization were compared in the two groups. Post-operative cardiac index increase was significantly higher in HL than in RL (P = 0.02), while mean arterial pressure and other hemodynamic parameters were comparable together with urinary output, indicating similar tissue perfusion in both the groups despite a much lower fluid infusion in the HL group. Therefore, a significant negative fluid balance was achieved in the HL but not in the RL group (-790 +/- 71 vs. +43 +/- 115 mL 12 h(-1), P < 0.0001 for HL and RL, respectively). None of the enrolled patients exhibited side effects related to the treatment. CONCLUSION: Half-molar lactate solution is effective for fluid resuscitation in post-CABG patients. Compared to Ringer's Lactate, its use results in a significantly higher cardiac index with less volume being infused, resulting in a very negative post-operative body fluid balance.

Co-metabolic degradation of bensulfuron-methyl in laboratory conditions.

The present study deals with the degradation of bensulfuron-methyl by microorganisms cultured with different sources of carbon, nitrogen and phosphorus. Addition of carbon source accelerated the degradation of bensulfuron-methyl under co-metabolism process. Sodium lactate was the best carbon source for the degradation of bensulfuron-methyl, compared to other carbon sources studied, and the degradation ratio of bensulfuron-methyl reached 79.5%, whereas only 34.6 and 29.7% were removed in the presence of glucose and sucrose, respectively. Supplement of nitrogen source also enhanced degradation of bensulfuron-methyl. However, no significant differences were observed in the loss of bensulfuron-methyl between organic nitrogen and inorganic source. Phosphate buffer was supplemented into the media to maintain neutral conditions for the advantage of the strain growth since increase in pH value was observed. An orthogonal array design was applied to arrange main factors singled out for investigating the influence of factor and interaction between them on the degradation of bensulfuron-methyl. Statistical analysis showed that the concentration of sodium lactate, bensulfuron-methyl and inoculum size were the main effects, and the interaction of sodium lactate and bensulfuron-methyl was of statistical significance.

A novel exclusion mechanism for carbon resource partitioning in Aggregatibacter actinomycetemcomitans.

The bacterium Aggregatibacter actinomycetemcomitans is a common commensal of the human oral cavity and the putative causative agent of the disease localized aggressive periodontitis. A. actinomycetemcomitans is a slow-growing bacterium that possesses limited metabolic machinery for carbon utilization. This likely impacts its ability to colonize the oral cavity, where growth and community composition is mediated by carbon availability. We present evidence that in the presence of the in vivo relevant carbon substrates glucose, fructose, and lactate A. actinomycetemcomitans preferentially metabolizes lactate. This preference for lactate exists despite the fact that A. actinomycetemcomitans grows faster and obtains higher cell yields during growth with carbohydrates. The preference for lactate is mediated by a novel exclusion mechanism in which metabolism of lactate inhibits carbohydrate uptake. Coculture studies reveal that A. actinomycetemcomitans utilizes lactate produced by the oral bacterium Streptococcus gordonii, suggesting the potential for cross-feeding in the oral cavity.

High power density from a miniature microbial fuel cell using Shewanella oneidensis DSP10.

A miniature microbial fuel cell (mini-MFC) is described that demonstrates high output power per device cross-section (2.0 cm2) and volume (1.2 cm3). Shewanella oneidensis DSP10 in growth medium with lactate and buffered ferricyanide solutions were used as the anolyte and catholyte, respectively. Maximum power densities of 24 and 10 mW/m2 were measured using the true surface areas of reticulated vitreous carbon (RVC) and graphite felt (GF) electrodes without the addition of exogenous mediators in the anolyte. Current densities at maximum power were measured as 44 and 20 mA/m2 for RVC and GF, while short circuit current densities reached 32 mA/m2 for GF anodes and 100 mA/m2 for RVC. When the power density for GF was calculated using the cross sectional area of the device or the volume of the anode chamber, we found values (3 W/m2, 500 W/m3) similar to the maxima reported in the literature. The addition of electron mediators resulted in current and power increases of 30-100%. These power densities were surprisingly high considering a pure S. oneidensis culture was used. We found that the short diffusion lengths and high surface-area-to-chamber volume ratio utilized in the mini-MFC enhanced power density when compared to output from similar macroscopic MFCs.

The influence of intracellular lactate and H+ on cell volume in amphibian skeletal muscle.

The combined effects of intracellular lactate and proton accumulation on cell volume, Vc, were investigated in resting Rana temporaria striated muscle fibres. Intracellular lactate and H+ concentrations were simultaneously increased by exposing resting muscle fibres to extracellular solutions that contained 20-80 mm sodium lactate. Cellular H+ and lactate entry was confirmed using pH-sensitive electrodes and 1H-NMR, respectively, and effects on Vc were measured using confocal microscope xz-scanning. Exposure to extracellular lactate up to 80 mm produced significant changes in pH and intracellular lactate (from a pH of 7.24 +/- 0.03, n = 8, and 4.65 +/- 1.07 mm, n = 6, respectively, in control fibres, to 6.59 +/- 0.03, n = 4, and 26.41 +/- 0.92 mm, n = 3, respectively) that were comparable to those observed following fatiguing stimulation (6.30-6.70 and 18.04 +/- 1.78 mm, n = 6, respectively). Yet, the increase in intracellular osmolarity expected from such an increase in intracellular lactate did not significantly alter Vc. Simulation of these experimental results, modified from the charge difference model of Fraser & Huang, demonstrated that such experimental manoeuvres produced changes in intracellular [H+] and [lactate] comparable to those observed during muscle fatigue, and accounted for this paradoxical conservation of Vc through balancing negative osmotic effects resulting from the net cation efflux that would follow a titration of intracellular membrane-impermeant anions by the intracellular accumulation of protons. It demonstrated that with established physiological values for intracellular buffering capacity and the permeability ratio of lactic acid and anionic lactate, P(LacH): P(Lac-), this would provide a mechanism that precisely balanced any effect on cell volume resulting from lactate accumulation during exercise.