Intracellular Lactate Concentrations Research Articles

Simulating NIRS and MRS measurements during cerebral hypoxia-ischaemia in piglets using a computational model.

We present a group analysis of the changes in cerebral haemodynamics, and the oxidation state of cytochrome-c-oxidase measured using broadband near-infrared spectroscopy (NIRS) and intracellular pH measured by phosphorous (31P) magnetic resonance spectroscopy (MRS) during and after cerebral hypoxia-ischaemia (HI) in 15 piglets. We use a previously published computational model of cerebral metabolism in the piglet [1] to integrate these measurements and simulate HI. We successfully simulate changes in cellular metabolism including shifts in intracellular pH observed in the piglet brain during HI. In this process, we optimise physiological parameters in the model identified through sensitivity analysis (such as the rate of glucose metabolism and intracellular lactate concentration), to fit simulated and measured data. The model fits the data reasonably and suggests a 20 % drop in glucose consumption, a ~65 % increase in lactate concentration and ~35 % drop in the cerebral metabolic rate of oxygen (CMRO2) during HI.

Catabolite Control Protein E (CcpE) Is a LysR-type Transcriptional Regulator of Tricarboxylic Acid Cycle Activity in Staphylococcus aureus

The tricarboxylic acid cycle (TCA cycle) is a central metabolic pathway that provides energy, reducing potential, and biosynthetic intermediates. In Staphylococcus aureus, TCA cycle activity is controlled by several regulators (e.g. CcpA, CodY, and RpiRc) in response to the availability of sugars, amino acids, and environmental stress. Developing a bioinformatic search for additional carbon catabolite-responsive regulators in S. aureus, we identified a LysR-type regulator, catabolite control protein E (CcpE), with homology to the Bacillus subtilis CcpC regulator. Inactivation of ccpE in S. aureus strain Newman revealed that CcpE is a positive transcriptional effector of the first two enzymes of the TCA cycle, aconitase (citB) and to a lesser extent citrate synthase (citZ). Consistent with the transcriptional data, aconitase activity dramatically decreased in the ccpE mutant relative to the wild-type strain. The effect of ccpE inactivation on citB transcription and the lesser effect on citZ transcription were also reflected in electrophoretic mobility shift assays where CcpE bound to the citB promoter but not the citZ promoter. Metabolomic studies showed that inactivation of ccpE resulted in increased intracellular concentrations of acetate, citrate, lactate, and alanine, consistent with a redirection of carbon away from the TCA cycle. Taken together, our data suggest that CcpE is a major direct positive regulator of the TCA cycle gene citB.

Downregulation of CD147 expression by RNA interference inhibits HT29 cell proliferation, invasion and tumorigenicity in vitro and in vivo

We investigated the effect of CD147 silencing on HT29 cell proliferation and invasion. We constructed a novel short hairpin RNA (shRNA) expression vector pYr-mir30-shRNA. The plasmid was transferred to HT29 cells. The expression of CD147, MCT1 (lactate transporters monocarboxylate transporter 1) and MCT4 (lactate transporters monocarboxylate transporter 4) were monitored by quantitative PCR and western blotting, respectively. The MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) activities were determined by gelatin zymography assay, while the intracellular lactate concentration was determined by the lactic acid assay kit. WST-8 assay was used to determine the HT29 cell proliferation and the chemosensitivity. Invasion assay was used to determine the invasion of HT29 cells. In addition, we established a colorectal cancer model, and detected CD147 expression in vivo. The results showed that the expression of CD147 and MCT1 was significantly reduced at both mRNA and protein levels, and also the activity of MMP-2 and MMP-9 was reduced. The proliferation and invasion were decreased, but chemosensitivity to cisplatin was increased. Invivo, the CD147 expression was also significantly decreased, and reduced the tumor growth after CD147 gene silencing. The results demonstrated that silencing of CD147 expression inhibited the proliferation and invasion, suggesting CD147 silencing might be an adjuvant gene therapy strategy to chemotherapy.

Abstract 4074: The effects of the HIF pathway inhibitor NSC-134754 on glucose metabolism

Abstract The master regulator in hypoxic cell survival and adaptation is the hypoxia-inducible factor-1 (HIF-1) pathway. Altered HIF-1 regulated gene expression drives a tumour towards a more malignant phenotype. HIF-1 is thus a therapeutic target, and small molecule inhibitors such as NSC-134754 are being investigated. Glucose transporter 1 (GLUT-1) is an important downstream target of the HIF-1 pathway, with fundamental roles in intracellular glucose flux and availability. Given the intrinsic role of GLUT-1 in glucose metabolism, the ability of NSC-134754 to evoke a unique metabolic response in vitro and in vivo was investigated. Human PC3LN5 prostate tumour cells were treated with 7.5µM NSC-134754 and incubated in a humidified hypoxic workstation (1.0% O2) for 24h. Western blot analysis demonstrated a reduction in GLUT-1 expression in treated cells compared to controls. GLUT-1 immunocytofluorescence confirmed control cells exhibited extensive membranous and diffuse cytoplasmic expression compared to treated cells. Culture media and cell extracts were collected and metabolites analysed using 1H magnetic resonance spectroscopy (MRS). A significant decrease in glucose consumption (p<0.01) and lactate production (p<0.05) in NSC-134754 treated cells compared to control was determined. No significant differences in intracellular lactate concentration were found. However, treatment significantly increased intracellular glucose by >3 fold compared to control cells (p<0.01). Glutamine/glutamate metabolism was also significantly increased in NSC-134754 treated cells (p<0.05). Mice bearing established (∼1cm diameter) orthotopic PC3LN5 prostate tumours were treated with either 100mg/kg i.p. NSC-134754 (n=4) or vehicle alone (n=5) for 24h. Subsequent semi-quantitative immunohistochemical analyses revealed a significant 28 ± 2% decrease in GLUT-1 membrane expression in the treated tumours compared to controls (p<0.01). These data suggest that NSC-134754 has a profound effect on glucose metabolism in vitro and in vivo. Treatment of PC3LN5 cells for 24h in hypoxia significantly reduced the expression and distribution of GLUT-1, decreased glucose uptake and reduced lactate production. A reduction in glucose transport and lactate production have been shown to be indicators of response to therapy. The increased uptake and metabolism of glutamine/glutamate may reflect compensatory mechanisms for reduced glycolysis in order to sustain cellular bioenergetics. Interestingly, we have also shown that NSC-134754 increased intracellular glucose accumulation. Many glycolytic enzymes are known gene targets of HIF, thus a reduction in their availability (and hence glycolysis) could lead to glucose trapping. To our knowledge, this is a novel finding of a small molecule inhibitor of the HIF pathway. Increased intracellular glucose with decreased glucose uptake has been reported with other therapeutics in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4074. doi:10.1158/1538-7445.AM2011-4074

Microfluidic cell arrays for metabolic monitoring of stimulated cardiomyocytes

An array of PDMS microchambers was aligned to an array of sensor electrodes and stimulating microelectrodes, which was used for the electrochemical monitoring of the metabolic activity of single isolated adult ventricular myocytes inside the chamber array, stimulated within a transient electric field. The effect of the accumulation of metabolic byproducts in the limited extracellular volume of the picolitre chambers was demonstrated by measuring single muscle cell contraction optically, while concomitant changes in intracellular calcium transients and pH were recorded independently using fluorescent indicator dyes. Both the amplitude of the cell shortening and the magnitude of the intracellular calcium transients decreased over time and both nearly ceased after 20 min of continuous stimulation in the limited extracellullar volume. The intracellular pH decreased gradually during 20 min of continuous stimulation after which a dramatic pH drop was observed, indicating the breakdown of the intracellular buffering capacity. After continuous stimulation, intracellular lactate was released into the microchamber through cell electroporation and was detected electrochemically at a lactate microbiosensor, within the chamber. A mitochondrial uncoupler was used to mimic ischaemia and thus to enhance the cellular content of lactate. Under these circumstances, intracellular lactate concentrations were found to have risen to approximately 15 mM. This array system has the potential of simultaneous electrochemical and optical monitoring of extracellular and intracellular metabolites from single beating heart cells at a controlled metabolic state.

CD147 silencing inhibits lactate transport and reduces malignant potential of pancreatic cancer cells in in-vivo and in-vitro models

Background: CD147 (basigin, EMMPRIN) is a multifunctional, highly conserved glycoprotein enriched in pancreatic ductal adenocarcinomas (PDACs) which is associated with poor prognosis in many malignancies. The role of CD147 in pancreatic cancer, however, remains elusive. Methods and Results: Silencing of CD147 by RNA interference (RNAi) reduced the proliferation rate of MiaPaCa2 and Panc1 cells. CD147 is required for the function and expression of the monocarboxylate transporters MCT1 and MCT4 that are expressed in human PDAC cells as demonstrated by real-time reverse transcription-PCR (RT-PCR) as well as immunohistology. MCT1 and MCT4 are the natural transporters of lactate, and MiaPaCa2 cells exhibited a high rate of lactate production, which is characteristic for the Warburg effect, an early hallmark of cancer that confers a significant growth advantage. Further induction of lactate production by sodium azide in MiaPaCa2 cells increased MCT1 as well as MCT4 expression. CD147 silencing inhibited the expression and function of MCT1 and MCT4 and resulted in an increased intracellular lactate concentration. Addition of exogenous lactate inhibited cancer cell growth in a dose-dependent fashion. In vivo, knock-down of CD147 in MiaPaCa2 cells by inducible short hairpin RNA (shRNA)-mediated CD147 silencing reduced invasiveness through the chorioallantoic membrane of chick embryos (CAM assay) and inhibited tumourigenicity in a xenograft model in nude mice. Conclusion : The function of CD147 as an ancillary protein that is required to sustain the expression and function of MCT1 and MCT4 is involved in the association of CD147 expression with the malignant potential of pancreatic cancer cells exhibiting the Warburg effect.

CD147 silencing inhibits lactate transport and reduces malignant potential of pancreatic cancer cells in in vivo and in vitro models

Background:CD147 (basigin, EMMPRIN) is a multifunctional, highly conserved glycoprotein enriched in pancreatic ductal adenocarcinomas (PDACs) which is associated with poor prognosis in many malignancies. The role of CD147 in pancreatic...

Increased cerebral lactate during hypoxia may be neuroprotective in newborn piglets with intrauterine growth restriction

Intrauterine growth restriction (IUGR) can increase susceptibility to perinatal hypoxic brain injury for reasons that are unknown. Previous studies of the neonatal IUGR brain have suggested that the cerebral mitochondrial capacity is reduced but the glycolytic capacity increased relative to normal weight (NW) neonates. In view of these two factors, we hypothesized that the generation of brain lactate during a mild hypoxic insult would be greater in neonatal IUGR piglets compared to NW piglets. Brain lactate/N-acetylaspartate (NAA) ratios and apparent diffusion coefficients (ADCs) were determined by proton magnetic resonance spectroscopy and imaging of the brain before, during and after hypoxia in seven neonatal piglets with asymmetric IUGR and six NW piglets. During hypoxia, IUGR piglets had significantly higher brain lactate/NAA ratios than NW piglets (P=0.046). The lactate response in the IUGR piglets correlated inversely with apoptosis in the thalamus and frontal cortex of the brain measured 4 h post hypoxia (Pearson's r=0.86, P<0.05). Apoptosis in IUGR piglets with high brain lactate was similar to that in the NW piglets whereas IUGR piglets with low brain lactate had significantly higher apoptosis than NW piglets (P=0.019). ADCs in the high lactate IUGR piglets were significantly lower during hypoxia than in all the other piglets. This signifies increased diffusion of water into brain cells during hypoxia, possibly in response to increased intracellular osmolality caused by high intracellular lactate concentrations. These findings support previous studies showing increased susceptibility to hypoxic brain injury in IUGR neonates but suggest that increased glycolysis during hypoxia confers neuroprotection in some IUGR piglets.

Effects of chronic NaHCO3 ingestion during interval training on changes to muscle buffer capacity, metabolism, and short-term endurance performance

This study determined the effects of altering the H(+) concentration during interval training, by ingesting NaHCO(3) (Alk-T) or a placebo (Pla-T), on changes in muscle buffer capacity (beta m), endurance performance, and muscle metabolites. Pre- and posttraining peak O(2) uptake (V(O2 peak)), lactate threshold (LT), and time to fatigue at 100% pretraining V(O2 peak) intensity were assessed in 16 recreationally active women. Subjects were matched on the LT, were randomly placed into the Alk-T (n = 8) or Pla-T (n = 8) groups, and performed 8 wk (3 days/wk) of six to twelve 2-min cycle intervals at 140-170% of their LT, ingesting NaHCO(3) or a placebo before each training session (work matched between groups). Both groups had improvements in beta m (19 vs. 9%; P < 0.05) and V(O2 peak) (22 vs. 17%; P < 0.05) after the training period, with no differences between groups. There was a significant correlation between pretraining beta m and percent change in beta m (r = -0.70, P < 0.05). There were greater improvements in both the LT (26 vs. 15%; P = 0.05) and time to fatigue (164 vs. 123%; P = 0.05) after Alk-T, compared with Pla-T. There were no changes to pre- or postexercise ATP, phosphocreatine, creatine, and intracellular lactate concentrations, or pH(i) after training. Our findings suggest that training intensity, rather than the accumulation of H(+) during training, may be more important to improvements in beta m. The group ingesting NaHCO(3) before each training session had larger improvements in the LT and endurance performance, possibly because of a reduced metabolic acidosis during training and a greater improvement in muscle oxidative capacity.

Point:Counterpoint: Lactic acid accumulation is an advantage/disadvantage during muscle activity

Point:Counterpoint: Lactic acid accumulation is an advantage/disadvantage during muscle activity

Extracellular hyper‐osmolality increases resting skeletal muscle lactate in mammalian skeletal muscle

The current study investigated the affects of acute alterations in extracellular osmolality on resting in-vitro skeletal muscle. Isolated, whole muscle strips (Soleus and EDL) from 5 week old Long Evans rats were incubated for 60 min in an organ bath containing Sigma Medium-199 (resting tension (1g), bubbled with 95:5 O2:CO2, 30 ± 2°C, and pH 7.4). Three different osmotic states were used; an iso-osmotic (290±10 Osm) (CON), hyper-osmotic (400 ±10 Osm) (HYPR), and hypo-osmotic (190 ± 10 Osm) (HYPO). After incubation, the relative water content decreased with HYPR, and increased with HYPO in both muscle types. As a result of the HYPO type I fiber area was increased in the Soleus (HYPO = 4066.3 ± 634.4μm2, CON = 2990 ± 364.1 μm2, n=12, p<0.05), whereas only TYPE II fiber area was increased in the EDL (HYPO = 2419.3 ± 681.5 μm2, CON = 1729.1 ± 513.1μm2, n>11, p<0.001). HYPR resulted in increased muscle lactate concentrations (EDL, p<0.001; Soleus, p<0.001, n=51) in both muscle types, but no extracellular lactate content was detected. Glycogen content decreased in EDL as a result of HYPR (HYPR=79.3 ± 11.8 mmol/kg, ISO=94.3.3 ± 16.1, p<0.01), whereas glycogen content in the soleus increased in HYPO (HYPO= 92.5 ± 12.2 mmol/kg, ISO=76.6 ± 18.5 mmol/kg, p<0.05). ATP and PCr concentrations confirmed muscle viability. Therefore, acute exposure of skeletal muscle to extracellular osmolality increases intracellular concentrations of lactate. The specific mechanisms underlying these changes in lactate remain to be determined. Supported by NSERC, Canada.

Detection of intracellular lactate with localized diffusion { 1H– 13C}-spectroscopy in rat glioma in vivo

The aim of this study was to compare the diffusion characteristic of lactate and alanine in a brain tumor model to that of normal brain metabolites known to be mainly intracellular such as N-acetylaspartate or creatine. The diffusion of 13C-labeled metabolites was measured in vivo with localized NMR spectroscopy at 9.4 T (400 MHz) using a previously described localization and editing pulse sequence known as ACED-STEAM (‘adiabatic carbon editing and decoupling’). 13C-labeled glucose was administered and the apparent diffusion coefficients of the glycolytic products, { 1H– 13C}-lactate and { 1H– 13C}-alanine, were determined in rat intracerebral 9L glioma. To obtain insights into { 1H– 13C}-lactate compartmentation (intra- versus extracellular), the pulse sequence used very large diffusion weighting (50 ms/μm 2). Multi-exponential diffusion attenuation of the lactate metabolite signals was observed. The persistence of a lactate signal at very large diffusion weighting provided direct experimental evidence of significant intracellular lactate concentration. To investigate the spatial distribution of lactate and other metabolites, 1H spectroscopic images were also acquired. Lactate and choline-containing compounds were consistently elevated in tumor tissue, but not in necrotic regions and surrounding normal-appearing brain. Overall, these findings suggest that lactate is mainly associated with tumor tissue and that within the time-frame of these experiments at least some of the glycolytic product ([ 13C] lactate) originates from an intracellular compartment.

Determination of intracellular pH and compartmentation using diffusion-weighted NMR spectroscopy with pH-sensitive indicators.

The intracellular pH (pHi) of a series of cancer cell lines was determined using the pH-sensitive indicators imidazole (Im) or histidine (His) and diffusion-weighted (DW) proton NMR spectroscopy. The DW method allows the observation at high magnetic field gradient values of only the slow-moving (intracellular) components, thus ensuring complete separation between intra- and extracellular components. Using the chemical shift difference (deltadelta) between the imidazole ring C2-H and C4(5)-H peaks, we were able to measure the pHi independently of chemical shift standardization. With His, the cell lines gave pHi values of approximately 6.5-7.0, whereas with Im, a second, more acidic compartment (pHi = 5.5-5.8) was also observed. An inverse correlation was also found between pHi and the intracellular lactate concentration. This method may be applicable to in vivo pH determinations.

Efficacy of Dichloroacetate as a Lactate‐Lowering Drug

Dichloroacetate (DCA) decreases blood, cerebral spinal fluid, and intracellular lactate concentrations by activating the mitochondrial pyruvate dehydrogenase enzyme complex. The authors reviewed the efficacy of this investigational drug in the treatment of acquired or congenital forms of lactic acidosis from data in 40 English-language publications. The hypolactatemic effect of DCA occurs over a broad range of pretreatment lactate concentrations and is directly related to the baseline lactate level. The maximum lactate-lowering effect of the drug is dependent on its dose but independent of time following its administration. Recent clinical studies of acquired lactic acidosis suggest that DCA could be rapidly effective in reducing this risk factor of mortality in patients with mild hyperlactatemeia.

Efficacy of Dichloroacetate as a Lactate-Lowering Drug

Dichloroacetate (DCA) decreases blood, cerebral spinal fluid, and intracellular lactate concentrations by activating the mitochondrial pyruvate dehydrogenase enzyme complex. The authors reviewed the efficacy of this investigational drug in the treatment of acquired or congenital forms of lactic acidosis from data in 40 English-language publications. The hypolactatemic effect of DCA occurs over a broad range of pretreatment lactate concentrations and is directly related to the baseline lactate level. The maximum lactate-lowering effect of the drug is dependent on its dose but independent of time following its administration. Recent clinical studies of acquired lactic acidosis suggest that DCA could be rapidly effective in reducing this risk factor of mortality in patients with mild hyperlactatemeia.

Adaptation and maladaptation of the heart in diabetes: Part II: potential mechanisms.

The prevailing concept of the heart’s response to changes in its environment is a complex network of inter-connecting signal transduction cascades.1 In such a scheme, the focus is on communication of various cell surface receptors, heterotrimeric G-proteins, protein kinases, and transcription factors.2–4⇓⇓ Diabetes is a disorder of metabolic dysregulation. At first glance it appears that metabolism and the metabolic consequences of diabetes do not fit into this signal-response coupling scheme. Two questions arise. First, is metabolism simply an “effect” rather than a “cause” of adaptation? Second, is metabolism only a by-product of signal transduction-induced adaptation, allowing equilibrium (and therefore maintenance of function) in the presence of the other adaptational responses? An alternative is to take a new, less restricted view of metabolism. Beyond its stereotypical function as a provider of ATP, alterations in metabolic flux within the cell create essential signals for the adaptation of the heart to situations such as diabetes. This concept is novel for the heart, but has already been considered in the liver. Like the phosphorylation events occurring in signal transduction cascades, changes in metabolic flux are extremely rapid. For example, translocation of GLUT4 to the cell surface in response to insulin occurs within a second.5 We have previously found that increases or decreases in workload also change metabolic fluxes in seconds.6,7⇓ Therefore, changes in metabolites are rapid enough to allow them to act as signaling molecules. Many of these acute changes in metabolic flux are brought about by the same signal transduction cascades believed to be involved in the adaptation of the heart to changes in its environment. Phosphatidylinositol 3-kinase, Ca2+, and protein kinase C, all of which play a role in cardiac adaptation, regulate metabolism in the heart.8,9⇓ Metabolic signals therefore provide a …

Extracellular carbonic anhydrase activity facilitates lactic acid transport in rat skeletal muscle fibres.

1. In skeletal muscle an extracellular sarcolemmal carbonic anhydrase (CA) has been demonstrated. We speculate that this CA accelerates the interstitial CO2/HCO3- buffer system so that H+ ions can be rapidly delivered or buffered in the interstitial fluid. Because > 80 % of the lactate which crosses the sarcolemmal membrane is transported by the H+-lactate cotransporter, we examined the contributions of extracellular and intracellular CA to lactic acid transport, using ion-selective microelectrodes for measurements of intracellular pH (pHi) and fibre surface pH (pHs) in rat extensor digitorum longus (EDL) and soleus fibres. 2. Muscle fibres were exposed to 20 mM sodium lactate in the absence and presence of the CA inhibitors benzolamide (BZ), acetazolamide (AZ), chlorzolamide (CZ) and ethoxzolamide (EZ). The initial slopes (dpHs/dt, dpHi/dt) and the amplitudes (DeltapHs, DeltapHi) of pH changes were quantified. From dpHi/dt, DeltapHi and the total buffer factor (BFtot) the lactate fluxes (mM min-1) and intracellular lactate concentrations ([lactate]i) were estimated. 3. BFtot was obtained as the sum of the non-HCO3- buffer factor (BFnon-HCO3) and the HCO3- buffer factor (BFHCO3). BFnon-HCO3 was 35 +/- 4 mM pH-1 for the EDL (n = 14) and 86 /- 16 mM pH-1 for the soleus (n = 14). 4. In soleus, 10 mM cinnamate inhibited lactate influx by 44 % and efflux by 30 %; in EDL, it inhibited lactate influx by 37 % and efflux by 20 %. Cinnamate decreased [lactate]i, in soleus by 36 % and in EDL by 45 %. In soleus, 1 mM DIDS reduced lactate influx by 18 % and efflux by 16 %. In EDL, DIDS lowered the influx by 27 % but had almost no effect on efflux. DIDS reduced [lactate]i by 20 % in soleus and by 26 % in EDL. 5. BZ (0.01 mM) and AZ (0.1 mM), which inhibit only the extracellular sarcolemmal CA, led to a significant increase in dpHs/dt and pHs by about 40 %-150 % in soleus and EDL. BZ and AZ inhibited the influx and efflux of lactate by 25 %-50 % and reduced [lactate]i by about 40 %. The membrane-permeable CA inhibitors CZ (0.5 mM) and EZ (0.1 mM), which inhibit the extracellular as well as the intracellular CAs, exerted no greater effects than the poorly permeable inhibitors BZ and AZ did. 6. In soleus, 10 mM cinnamate inhibited the lactate influx by 47 %. Addition of 0.01 mM BZ led to a further inhibition by only 10 %. BZ alone reduced the influx by 37 %. 7. BZ (0.01 mM) had no influence on the Km value of the lactate transport, but led to a decrease in maximal transport rate (Vmax). In EDL, BZ reduced Vmax by 50 % and in soleus by about 25 %. 8. We conclude that the extracellular sarcolemmal CA plays an important role in lactic acid transport, while internal CA has no effect, a difference most likely attributable to the high internal vs. low extracellular BF(non-HCO3). The fact that the effects of cinnamate and BZ are not additive indicates that the two inhibitors act at distinct sites on the same transport pathway for lactic acid.

A model of low-flow ischemia and reperfusion in single, beating adult cardiomyocytes.

The present study was undertaken to comprehensively characterize low-flow ischemia and reperfusion in single adult cardiomyocytes and to determine whether it is important to control contractile activity. The ischemia-mimetic solution was hypoxic, acidic (pH 6.0), and deficient in glucose but contained elevated KCl. Cardiomyocytes were stimulated to contract throughout ischemia and during reperfusion with control perfusate. After the ischemia-reperfusion insult, cells exhibited poor recovery of active cell shortening, a decrease in passive cell length, increased frequency of necrosis, lower ATP content, and evidence of the generation of oxygen-derived free radicals within the cells. Intracellular lactate concentration increased, pH decreased, and Ca(2+) transients were depressed during the ischemic insult, but the latter two parameters recovered partially on reperfusion. Basal intracellular Ca(2+) concentration was elevated during ischemia and early into reperfusion. Recovery was attenuated in cells that were electrically stimulated to contract throughout ischemia. The duration of ischemia, stimulation frequency, and composition of the ischemia-mimetic solution were important variables. The inclusion of 10 mM lactate in the ischemia-mimetic solution significantly aggravated all the parameters examined above. Our data demonstrate that 1) an ischemia-mimetic solution administered to single, isolated adult cardiomyocytes can reproduce many of the responses observed in whole hearts, 2) caution should be used in adding lactate to an ischemic solution, and 3) it is important to stimulate contractile activity throughout ischemia to reproduce the effects of ischemia in whole hearts.

Effects of lactate on force production by mouse EDL muscle: implications for the development of fatigue

Numerous studies suggest that the accumulation of lactate during exercise contributes to the fatigue process. This notion is based on close negative correlations between force and intracellular muscle lactate concentrations during fatigue and recovery. In this investigation, we attempted to determine if lactate directly affects muscle force output. This was accomplished by incubating mouse extensor digitorum longus muscles in extracellular concentrations of 10, 20, 30 and 50 mM L-(+)-lactate at 21 and 37°C and monitoring force output. At 21°C, 30 and 50 mM, extracellular lactate significantly reduced tetanic force (Po, 250 ms, 100 Hz) to 95 and 93% of initial, respectively. In addition, the rate of force development (+dP/dt) was reduced to 93 and 89% of initial. At 37°C, the effects of extracellular lactate were augmented as Po was reduced to 73 and 62% of initial and +dP/dt was reduced to 55 and 44% of initial at 30 and 50 mM, respectively. We next sought to determine if the reduction in Po was due to altered sarcoplasmic reticulum (SR) function using a muscle homogenate fraction. The rate of AgNO3-induced SR Ca2+ release was depressed by 31% in the presence of 25 mM lactate. These results suggest that elevated lactate depresses force production by whole muscle and may play some role in the fatigue process. In addition, it appears that lactate depresses force production, in part, by inhibiting Ca2+ release from the SR.Key words: fatigue, skeletal muscle, sarcoplasmic reticulum, calcium.

Effects of lactate on force production by mouse EDL muscle: implications for the development of fatigue.

Numerous studies suggest that the accumulation of lactate during exercise contributes to the fatigue process. This notion is based on close negative correlations between force and intracellular muscle lactate concentrations during fatigue and recovery. In this investigation, we attempted to determine if lactate directly affects muscle force output. This was accomplished by incubating mouse extensor digitorum longus muscles in extracellular concentrations of 10, 20, 30 and 50 mM L-(+)-lactate at 21 and 37 degrees C and monitoring force output. At 21 degrees C, 30 and 50 mM, extracellular lactate significantly reduced tetanic force (Po 250 ms, 100 Hz) to 95 and 93% of initial, respectively. In addition, the rate of force development (+dP/dt) was reduced to 93 and 89% of initial. At 37 degrees C, the effects of extracellular lactate were augmented as Po was reduced to 73 and 62% of initial and +dP/dt was reduced to 55 and 44% of initial at 30 and 50 mM, respectively. We next sought to determine if the reduction in Po was due to altered sarcoplasmic reticulum (SR) function using a muscle homogenate fraction. The rate of AgNO3-induced SR Ca2+ release was depressed by 31% in the presence of 25 mM lactate. These results suggest that elevated lactate depresses force production by whole muscle and may play some role in the fatigue process. In addition, it appears that lactate depresses force production, in part, by inhibiting Ca2+ release from the SR.