Metabolic effects of induced alkalosis during progressive forearm exercise to fatigue.

Metabolic alkalosis induced by sodium bicarbonate (NaHCO(3)) ingestion has been shown to enhance performance during brief high-intensity exercise. The mechanisms associated with this increase in performance may include increased muscle phosphocreatine (PCr) breakdown, muscle glycogen utilization, and plasma lactate (Lac(-)(pl)) accumulation. Together, these changes would imply a shift toward a greater contribution of anaerobic energy production, but this statement has been subject to debate. In the present study, subjects (n = 6) performed a progressive wrist flexion exercise to volitional fatigue (0.5 Hz, 14-21 min) in a control condition (Con) and after an oral dose of NaHCO(3) (Alk: 0.3 g/kg; 1.5 h before testing) to evaluate muscle metabolism over a complete range of exercise intensities. Phosphorus-31 magnetic resonance spectroscopy was used to continuously monitor intracellular pH, [PCr], [P(i)], and [ATP] (brackets denote concentration). Blood samples drawn from a deep arm vein were analyzed with a blood gas-electrolyte analyzer to measure plasma pH, Pco(2), and [Lac(-)](pl), and plasma [HCO(3)(-)] was calculated from pH and Pco(2). NaHCO(3) ingestion resulted in an increased (P < 0.05) plasma pH and [HCO(3)(-)] throughout rest and exercise. Time to fatigue and peak power output were increased (P < 0.05) by approximately 12% in Alk. During exercise, a delayed (P < 0.05) onset of intracellular acidosis (1.17 +/- 0.26 vs. 1.28 +/- 0.22 W, Con vs. Alk) and a delayed (P < 0.05) onset of rapid increases in the [P(i)]-to-[PCr] ratio (1.21 +/- 0.30 vs. 1.30 +/- 0.30 W) were observed in Alk. No differences in total [H(+)], [P(i)], or [Lac(-)](pl) accumulation were detected. In conclusion, NaHCO(3) ingestion was shown to increase plasma pH at rest, which resulted in a delayed onset of intracellular acidification during incremental exercise. Conversely, NaHCO(3) was not associated with increased [Lac(-)](pl) accumulation or PCr breakdown.     

Transport activity of the high-affinity monocarboxylate transporter MCT2 is enhanced by extracellular carbonic anhydrase IV but not by intracellular carbonic anhydrase II

The ubiquitous enzyme carbonic anhydrase isoform II (CAII) has been shown to enhance transport activity of the proton-coupled monocarboxylate transporters MCT1 and MCT4 in a non-catalytic manner. In this study, we investigated the role of cytosolic CAII and of the extracellular, membrane-bound CA isoform IV (CAIV) on the lactate transport activity of the high-affinity monocarboxylate transporter MCT2, heterologously expressed in Xenopus oocytes. In contrast to MCT1 and MCT4, transport activity of MCT2 was not altered by CAII. However, coexpression of CAIV with MCT2 resulted in a significant increase in MCT2 transport activity when the transporter was coexpressed with its associated ancillary protein GP70 (embigin). The CAIV-mediated augmentation of MCT2 activity was independent of the catalytic activity of the enzyme, as application of the CA-inhibitor ethoxyzolamide or coexpressing the catalytically inactive mutant CAIV-V165Y did not suppress CAIV-mediated augmentation of MCT2 transport activity. Furthermore, exchange of His-88, mediating an intramolecular H(+)-shuttle in CAIV, to alanine resulted only in a slight decrease in CAIV-mediated augmentation of MCT2 activity. The data suggest that extracellular membrane-bound CAIV, but not cytosolic CAII, augments transport activity of MCT2 in a non-catalytic manner, possibly by facilitating a proton pathway other than His-88.     

Monocarboxylate transporters, blood lactate removal after supramaximal exercise, and fatigue indexes in humans.

The present study investigated whether muscular monocarboxylate transporter (MCT) 1 and 4 contents are related to the blood lactate removal after supramaximal exercise, fatigue indexes measured during different supramaximal exercises, and muscle oxidative parameters in 15 humans with different training status. Lactate recovery curves were obtained after a 1-min all-out exercise. A biexponential time function was then used to determine the velocity constant of the slow phase (gamma(2)), which denoted the blood lactate removal ability. Fatigue indexes were calculated during 1-min all-out (FI(AO)) and repeated 10-s (FI(Sprint)) cycling sprints. Biopsies were taken from the vastus lateralis muscle. MCT1 and MCT4 contents were quantified by Western blots, and maximal muscle oxidative capacity (V(max)) was evaluated with pyruvate + malate and glutamate + malate as substrates. The results showed that the blood lactate removal ability (i.e., gamma(2)) after a 1-min all-out test was significantly related to MCT1 content (r = 0.70, P < 0.01) but not to MCT4 (r = 0.50, P > 0.05). However, greater MCT1 and MCT4 contents were negatively related with a reduction of blood lactate concentration at the end of 1-min all-out exercise (r = -0.56, and r = -0.61, P < 0.05, respectively). Among skeletal muscle oxidative indexes, we only found a relationship between MCT1 and glutamate + malate V(max) (r = 0.63, P < 0.05). Furthermore, MCT1 content, but not MCT4, was inversely related to FI(AO) (r = -0.54, P < 0.05) and FI(Sprint) (r = -0.58, P < 0.05). We concluded that skeletal muscle MCT1 expression was associated with the velocity constant of net blood lactate removal after a 1-min all-out test and with the fatigue indexes. It is proposed that MCT1 expression may be important for blood lactate removal after supramaximal exercise based on the existence of lactate shuttles and, in turn, in favor of a better tolerance to muscle fatigue.     

Intracellular and Extracellular Carbonic Anhydrases Cooperate Non-enzymatically to Enhance Activity of Monocarboxylate Transporters

Proton-coupled monocarboxylate transporters (MCTs) are carriers of high-energy metabolites such as lactate, pyruvate, and ketone bodies and are expressed in most tissues. It has previously been shown that transport activity of MCT1 and MCT4 is enhanced by the cytosolic carbonic anhydrase II (CAII) independent of its catalytic activity. We have now studied the influence of the extracellular, membrane-bound CAIV on transport activity of MCT1/4, heterologously expressed in Xenopus oocytes. Coexpression of CAIV with MCT1 and MCT4 resulted in a significant increase in MCT transport activity, even in the nominal absence of CO₂/HCO₃⁻. CAIV-mediated augmentation of MCT activity was independent of the CAIV catalytic function, since application of the CA-inhibitor ethoxyzolamide or coexpression of the catalytically inactive mutant CAIV-V165Y did not suppress CAIV-mediated augmentation of MCT transport activity. The interaction required CAIV at the extracellular surface, since injection of CAIV protein into the oocyte cytosol did not augment MCT transport function. The effects of cytosolic CAII (injected as protein) and extracellular CAIV (expressed) on MCT transport activity, were additive. Our results suggest that intra- and extracellular carbonic anhydrases can work in concert to ensure rapid shuttling of metabolites across the cell membrane.     

Effects of live high, train low hypoxic exposure on lactate metabolism in trained humans.

We determined the effect of 20 nights of live high, train low (LHTL) hypoxic exposure on lactate kinetics, monocarboxylate lactate transporter proteins (MCT1 and MCT4), and muscle in vitro buffering capacity (betam) in 29 well-trained cyclists and triathletes. Subjects were divided into one of three groups: 20 consecutive nights of hypoxic exposure (LHTLc), 20 nights of intermittent hypoxic exposure [four 5-night blocks of hypoxia, each interspersed with 2 nights of normoxia (LHTLi)], or control (Con). Rates of lactate appearance (Ra), disappearance (Rd), and oxidation (Rox) were determined from a primed, continuous infusion of l-[U-14C]lactic acid tracer during 90 min of steady-state exercise [60 min at 65% peak O2 uptake (VO(2 peak)) followed by 30 min at 85% VO(2 peak)]. A resting muscle biopsy was taken before and after 20 nights of LHTL for the determination of betam and MCT1 and MCT4 protein abundance. Ra during the first 60 min of exercise was not different between groups. During the last 25 min of exercise at 85% VO(2 peak), Ra was higher compared with exercise at 65% of VO(2 peak) and was decreased in LHTLc (P < 0.05) compared with the other groups. Rd followed a similar pattern to Ra. Although Rox was significantly increased during exercise at 85% compared with 65% of VO(2 peak), there were no differences between the three groups or across trials. There was no effect of hypoxic exposure on betam or MCT1 and MCT4 protein abundance. We conclude that 20 consecutive nights of hypoxia exposure decreased whole body Ra during intense exercise in well-trained athletes. However, muscle markers of lactate metabolism and pH regulation were unchanged by the LHTL intervention.     

Forearm muscle metabolism studied using (31)P-MRS during progressive exercise to fatigue after Acz administration.

The effects of acetazolamide (Acz)-induced carbonic anhydrase inhibition (CAI) on muscle intracellular thresholds (T) for intracellular pH (pH(i)) and inorganic phosphate-to-phosphate creatine ratio (P(i)/PCr) and the plasma lactate (La(-)) threshold were examined in nine adult male subjects performing forearm wrist flexion exercise to fatigue. Exercise consisted of raising and lowering (1-s contraction, 1-s relaxation) a cylinder whose volume increased at a rate of 200 ml/min. The protocol was performed during control (Con) and after 45 min of CAI with Acz (10 mg/kg body wt iv). T(pH(i)) and T(P(i)/PCr), determined using (31)P-labeled magnetic resonance spectroscopy (MRS), were similar in Acz (722 +/- 50 and 796 +/- 75 mW, respectively) and Con (855 +/- 211 and 835 +/- 235 mW, respectively). The pH(i) was similar at end-exercise (6.38 +/- 0.10 Acz and 6.43 +/- 0.22 Con), but pH(i) recovery was slowed in Acz. In a separate experiment, blood was sampled from a deep arm vein at the elbow for determination of plasma lactate concentration ([La(-)](pl)) and T(La(-)). [La(-)](pl) was lower (P < 0.05) in Acz than Con (3.7 +/- 1.7 vs. 5.0 +/- 1.7 mmol/l) at end-exercise and in early recovery, but T(La(-)) was higher (1,433 +/- 243 vs. 1,041 +/- 414 mW, respectively). These data suggest that the lower [La(-)](pl) seen with CAI was not due to a delayed onset or rate of muscle La(-) accumulation but may be related to impaired La(-) removal from muscle.     

Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast

Monocarboxylate transporters (MCTs) comprise a group of highly homologous proteins that reside in the plasma membrane of almost all cells and which mediate the 1:1 electroneutral transport of a proton and a lactate ion. The isoform MCT3 is restricted to the basal membrane of the retinal pigment epithelium where it regulates lactate levels in the neural retina. Kinetic analysis of this transporter poses formidable difficulties due to the presence of multiple lactate transporters and their complex interaction with MCTs in adjacent cells. To circumvent these problems, we expressed both the MCT3 gene and a green fluorescent protein-tagged MCT3 construct in Saccharomyces cerevisiae. Since L-lactate metabolism in yeast depends on the CYB2 gene, we disrupted CYB2 to study the MCT3 transporter activity free from the complications of metabolism. Under these conditions L-lactate uptake varied inversely with pH, greater uptake being associated with lower pH. Whereas the V(max) was invariant, the K(m) increased severalfold as the pH rose from 6 to 8. In addition, MCT3 was highly resistant to a number of "classical" inhibitors of lactate transport. Last, studies with diethyl pyrocarbonate and p-chloromercuribenzenesulfonate set limitations on the locus of potential residues involved in the critical site of lactate translocation.     

Hematological and acid-base changes in men during prolonged exercise with and without sodium-lactate infusion.

An emerging technique used for the study of metabolic regulation is the elevation of lactate concentration with a sodium-lactate infusion, the lactate clamp (LC). However, hematological and acid-base properties affected by the infusion of hypertonic solutions containing the osmotically active strong ions sodium (Na(+)) and lactate (Lac(-)) are a concern for clinical and research applications of LC. In the present study, we characterized the hematological and plasma acid-base changes during rest and prolonged, light- to moderate-intensity (55% Vo(2 peak)) exercise with and without LC. During the control (Con) trial, subjects were administered an isotonic, isovolumetric saline infusion. During LC, plasma lactate concentration ([Lac(-)]) was elevated to 4 meq/l during rest and to 4-7 meq/l during exercise. During LC at rest, there were rapid and transient changes in plasma, erythrocyte, and blood volumes. LC resulted in decreased plasma [H(+)] (from 39.6 to 29.6 neq/l) at the end of exercise while plasma [HCO(3)(-)] increased from 26 to 32.9 meq/l. Increased plasma strong ion difference [SID], due to increased [Na(+)], was the primary contributor to decreased [H(+)] and increased [HCO(3)(-)]. A decrease in plasma total weak acid concentration also contributed to these changes, whereas Pco(2) contributed little. The infusion of hypertonic LC caused only minor volume, acid-base, and CO(2) storage responses. We conclude that an LC infusion is appropriate for studies of metabolic regulation.     

Lactate transport in rat adipocytes: identification of monocarboxylate transporter 1 (MCT1) and its modulation during streptozotocin-induced diabetes

We have characterised L-lactate transport in rat adipocytes and determined whether these cells express a carrier belonging to the monocarboxylate transporter family. L-Lactate was taken up by adipocytes in a time-dependent, non-saturable manner and was inhibited (by approximately 90%) by alpha-cyano-4-hydroxycinnamate. Lactate transport was stimulated by 3.7-fold upon lowering extracellular pH from 7.5 to 6.5 suggesting the presence of a lactate/proton-cotransporter. Antibodies against mono carboxylate transporter 1 (MCT1) reacted positively with plasma membranes (PM), but not with intracellular membranes, prepared from adipocytes. MCTI expression was down-regulated in PM of adipocytes from diabetic rats, which also displayed a corresponding loss (approximately 64%) in their capacity to transport lactate. The data support a role for MCT1 in lactate transport and suggest that changes in MCT1 expression are likely to have important implications for adipocyte lactate metabolism.     

Comparison of Quasielastic Light Scattering and Laser Diffractometry as Nondestructive Probes into the Structure of Bacillus sphaericus Spores Produced at Different Temperatures

Volume 62, no. 5, p. 1702, column 2, equation 3: the equation should read as follows. g(sup1)((tau)) = [g(sup2)((tau)) - 1](sup1/2) = exp[-K(sup2)(D(inf1) cos(sup2)(alpha) + D(inf2) sin(sup2)(alpha))(tau)] (3) [This corrects the article on p. 1699 in vol. 62.].     

Direct extracellular interaction between carbonic anhydrase IV and the human NBC1 sodium/bicarbonate co-transporter

Sodium/bicarbonate co-transporters (NBC) are crucial in the regulation of intracellular pH (pH(i)) and HCO(3)(-) metabolism. Electrogenic NBC1 catalyzes HCO(3)(-) fluxes in mammalian kidney, pancreas, and heart cells. Carbonic anhydrase IV (CAIV), which is also present in these tissues, is glycosylphosphatidyl inositol-anchored to the outer surface of the plasma membrane where it catalyzes the hydration-dehydration of CO(2)/HCO(3)(-). The physical and functional interactions of CAIV and NBC1 were investigated. NBC1 activity was measured by changes of pH(i) in NBC1-transfected HEK293 cells subjected to acid loads. Cotransfection of CAIV with NBC1 increased the rate of pH(i) recovery by 44 +/- 3%, as compared to NBC1-alone. In contrast, CAIV did not increase the functional activity of G767T-NBC1 (mutated on the fourth extracellular loop (EC4) of NBC1), and G767T-NBC1, unlike wild-type NBC1, did not interact with CAIV in glutathione-S-transferase pull-down assays. This indicates that G767 of NBC1 is directly involved in CAIV interaction. NBC1-mediated pH(i) recovery rate after acid load was inhibited by 40 +/- 7% when coexpressed with the inactive human CAII mutant, V143Y. V143Y CAII competes with endogenous CAII for interaction with NBC1 at the inner surface of the plasma membrane, which indicates that NBC1/CAII interaction is needed for full pH(i) recovery activity. We conclude that CAIV binds EC4 of NBC1, and this interaction is essential for full NBC1 activity. The tethering of CAII and CAIV close to the NBC1 HCO(3)(-) transport site maximizes the transmembrane HCO(3)(-) gradient local to NBC1 and thereby activates the transport rate.     

Effects of high-intensity training on muscle lactate transporters and postexercise recovery of muscle lactate and hydrogen ions in women

The purpose of this study was to investigate the effects of high-intensity interval training (3 days/wk for 5 wk), provoking large changes in muscle lactate and pH, on changes in intracellular buffer capacity (betam(in vitro)), monocarboxylate transporters (MCTs), and the decrease in muscle lactate and hydrogen ions (H+) after exercise in women. Before and after training, biopsies of the vastus lateralis were obtained at rest and immediately after and 60 s after 45 s of exercise at 190% of maximal O2 uptake. Muscle samples were analyzed for ATP, phosphocreatine (PCr), lactate, and H+; MCT1 and MCT4 relative abundance and betam(in vitro) were also determined in resting muscle only. Training provoked a large decrease in postexercise muscle pH (pH 6.81). After training, there was a significant decrease in betam(in vitro) (-11%) and no significant change in relative abundance of MCT1 (96 +/- 12%) or MCT4 (120 +/- 21%). During the 60-s recovery after exercise, training was associated with no change in the decrease in muscle lactate, a significantly smaller decrease in muscle H+, and increased PCr resynthesis. These results suggest that increases in betam(in vitro) and MCT relative abundance are not linked to the degree of muscle lactate and H+ accumulation during training. Furthermore, training that is very intense may actually lead to decreases in betam(in vitro). The smaller postexercise decrease in muscle H+ after training is a further novel finding and suggests that training that results in a decrease in H+ accumulation and an increase in PCr resynthesis can actually reduce the decrease in muscle H+ during the recovery from supramaximal exercise.     

Transport activity of the sodium bicarbonate cotransporter NBCe1 is enhanced by different isoforms of carbonic anhydrase

Transport metabolons have been discussed between carbonic anhydrase II (CAII) and several membrane transporters. We have now studied different CA isoforms, expressed in Xenopus oocytes alone and together with the electrogenic sodium bicarbonate cotransporter 1 (NBCe1), to determine their catalytic activity and their ability to enhance NBCe1 transport activity. pH measurements in intact oocytes indicated similar activity of CAI, CAII and CAIII, while in vitro CAIII had no measurable activity and CAI only 30% of the activity of CAII. All three CA isoforms increased transport activity of NBCe1, as measured by the transport current and the rate of intracellular sodium rise in oocytes. Two CAII mutants, altered in their intramolecular proton pathway, CAII-H64A and CAII-Y7F, showed significant catalytic activity and also enhanced NBCe1 transport activity. The effect of CAI, CAII, and CAII mutants on NBCe1 activity could be reversed by blocking CA activity with ethoxyzolamide (EZA, 10 μM), while the effect of the less EZA-sensitive CAIII was not reversed. Our results indicate that different CA isoforms and mutants, even if they show little enzymatic activity in vitro, may display significant catalytic activity in intact cells, and that the ability of CA to enhance NBCe1 transport appears to depend primarily on its catalytic activity.     

Metabolic costs induced by lactate in the toad Bufo marinus: new mechanism behind oxygen debt?

The mechanism of an increase in metabolic rate induced by lactate was investigated in the toad Bufo marinus. Oxygen consumption (Vo(2)) was analyzed in fully aerobic animals under hypoxic conditions (7% O(2) in air), accompanied by measurements of catecholamines in the plasma, and was measured in isolated hepatocytes in vitro under normoxia by using specific inhibitors of lactate proton symport [alpha-cyano-4-hydroxycinnamate (CHC)] and sodium proton exchange (EIPA). The rise in metabolic rate in vivo can be elicited by infusions of hyperosmotic (previous findings) or isosmotic sodium lactate solutions (this study). Despite previous findings of reduced metabolic stimulation under the effect of adrenergic blockers, the increase in Vo(2) in vivo was not associated with elevated plasma catecholamine levels, suggesting local release and effect. In addition to the possible in vivo effect via catecholamines, lactate induced a rise in Vo(2) of isolated hepatocytes, depending on the concentration present in a weakly buffered Ringer solution at pH 7.0. No increase was found at higher pH values (7.4 or 7.8) or in HEPES-buffered Ringer solution. Inhibition of the Lac(-)-H(+) transporter with alpha-CHC or of the Na(+)/H(+) exchanger with EIPA prevented the increase in metabolic rate. We conclude that increased Vo(2) at an elevated systemic lactate level may involve catecholamine action, but it is also caused by an increased energy demand of cellular acid-base regulation via stimulation of Na(+)/H(+) exchange and thereby Na(+)-K(+)-ATPase. The effect depends on entry of lactic acid into the cells via lactate proton symport, which is likely favored by low cellular surface pH. We suggest that these energetic costs should also be considered in other physiological phenomena, e.g., when lactate is present during excess, postexercise Vo(2).     

Structural and functional characterization of haemocyanin from the anemone hermit crab Dardanus calidus

Oxygen-binding to haemocyanin (Hc) is generally an exothermic process, with overall enthalphy of oxygenation varying from species to species. A number of crustacean Hcs showed a null or reduced enthalphy of oxygenation, among others, the anomuran Pagurus bernhardus and Paralithodes camtscaticae possess a completely temperature-independent oxygen-binding in a wide range of temperature and pH. Functional analysis performed on purified native, hexameric and dodecameric Hc forms of the anemone hermit crab Dardanus calidus allowed to calculate the enthalphy of oxygenation values that resulted equal to -36.2, -33.8 and -26.8 kJ/mol, respectively. Thus, the temperature sensitivity of oxygen binding of D. calidus Hc is in contrast with the temperature independence reported for P. bernhardus and P. camtscaticae, suggesting a high Hc functional heterogeneity within Anomura. Functional characterization also evidenced a strong oxygen affinity modulation by protons (DeltalogP(50)/DeltapH = -0.97) and lactate [DeltalogP(50)/Deltalog(lactate) = -0.38], and a significant decrease in cooperativity by physiological concentration of lactate (n(50) from 2.8 to 1.7 at pH 7.5).     

Enthalpy changes in the formation of the proton electrochemical potential and its components

Enthalpy changes in the formation of a proton electrochemical potential (Delta mu H+) and its components, DeltapH (proton gradient) and Deltapsi (electrical potential), across two types of E. coli membrane vesicles were investigated. Flow dialysis experiments showed that in 0.1 M KPi, pH 6.6, E. coli GR19N membrane vesicles coupled with d-lactate exhibited 57 mV for DeltapH, 70 mV for Deltapsi, and 127 mV for Delta mu H+. Microcalorimetric measurements revealed that the corresponding enthalpy changes (DeltaH(pH), DeltaH(psi) and DeltaHm) were 3.5, 3.3 and 6.9 kcal/mole, respectively. Moreover, in E. coli ML 308-225 membrane vesicles across which 120mV of Delta mu H+ was generated, values of DeltaH(pH) and DeltaH(psi) were determined as 7.0 and 6.6 kcal/mole, as compared with the previously reported 14.1 kcal/mole for DeltaH(m). Comparisons of these enthalpy data revealed that component enthalpies (DeltaH(pH) and DeltaH(psi)) essentially added up to the total enthalpy (DeltaHm), providing a self-consistent test for the obtained data. In both membranes, the ratio ofDeltaH(psi) to Deltapsi was comparable to that of DeltaH(pH) to DeltapH in the formation of Delta mu H+. These observations indicated that the process of the movement of H+ across the membranes was the major contributor to the observed energetic changes. Moreover, the enthalpy change in the formation of Delta mu H+ was compared with the membranes derived from GR19N and ML 308-225 and coupled with NADH and d-lactate. The results were discussed in terms of trans-membrane phenomena.     

Sodium and sulfate ion transport in yeast vacuoles

The intra-luminal acidic pH of endomembrane organelles is established by a proton pump, vacuolar H(+)-ATPase (V-ATPase), in combination with other ion transporter(s). The proton gradient (DeltapH) established in yeast vacuolar vesicles decreased and reached the lower value after the addition of alkaline cations including Na(+). As expected, the uptake of (22)Na(+) was coupled with DeltapH generated by V-ATPase. Disruption of NHX1 or NHA1, encoding known Na(+)/H(+) antiporters, did not result in the loss of (22)Na(+) uptake or the alkaline cation-dependent DeltapH decrease. Upon the addition of sulfate ions, the V-ATPase-dependent DeltapH in the vacuolar vesicles increased, but the membrane potential (DeltaPsi) decreased. Consistent with this observation, radioactive sulfate was transported into the vesicles with a K(m) value of 0.07 mM. The transport activity was unaffected upon disruption of the putative genes coding for homologues of plasma membrane sulfate transporters. These results indicate that the vacuoles exhibit unique Na(+)/H(+) antiport and sulfate transport, which regulate the luminal pH and ion homeostasis in yeast.     

Carbonic anhydrase inhibition delays plasma lactate appearance with no effect on ventilatory threshold.

The effect of carbonic anhydrase (CA) inhibition with acetazolamide (Acz, 10 mg/kg body wt iv) on exercise performance and the ventilatory (VET) and lactate (LaT) thresholds was studied in seven men during ramp exercise (25 W/min) to exhaustion. Breath-by-breath measurements of gas exchange were obtained. Arterialized venous blood was sampled from a dorsal hand vein and analyzed for plasma pH, PCO(2), and lactate concentration ([La(-)](pl)). VET [expressed as O(2) uptake (VO(2)), ml/min] was determined using the V-slope method. LaT (expressed as VO(2), ml/min) was determined from the work rate (WR) at which [La(-)](pl) increased 1.0 mM above rest levels. Peak WR was higher in control (Con) than in Acz sutdies [339 +/- 14 vs. 315 +/- 14 (SE) W]. Submaximal exercise VO(2) was similar in Acz and Con; the lower VO(2) at exhaustion in Acz than in Con (3.824 +/- 0. 150 vs. 4.283 +/- 0.148 l/min) was appropriate for the lower WR. CO(2) output (VCO(2)) was lower in Acz than in Con at exercise intensities >/=125 W and at exhaustion (4.375 +/- 0.158 vs. 5.235 +/- 0.148 l/min). [La(-)](pl) was lower in Acz than in Con during submaximal exercise >/=150 W and at exhaustion (7.5 +/- 1.1 vs. 11.5 +/- 1.1 mmol/l). VET was similar in Acz and Con (2.483 +/- 0.086 and 2.362 +/- 0.110 l/min, respectively), whereas the LaT occurred at a higher VO(2) in Acz than in Con (2.738 +/- 0.223 vs. 2.190 +/- 0.235 l/min). CA inhibition with Acz is associated with impaired elimination of CO(2) during the non-steady-state condition of ramp exercise. The similarity in VET in Con and Acz suggests that La(-) production is similar between conditions but La(-) appearance in plasma is reduced and/or La(-) uptake by other tissues is enhanced after the Acz treatment.     

Effects of acute and chronic exercise on sarcolemmal MCT1 and MCT4 contents in human skeletal muscles: current status

Two lactate/proton cotransporter isoforms (monocarboxylate transporters, MCT1 and MCT4) are present in the plasma (sarcolemmal) membranes of skeletal muscle. Both isoforms are symports and are involved in both muscle pH and lactate regulation. Accordingly, sarcolemmal MCT isoform expression may play an important role in exercise performance. Acute exercise alters human MCT content, within the first 24 h from the onset of exercise. The regulation of MCT protein expression is complex after acute exercise, since there is not a simple concordance between changes in mRNA abundance and protein levels. In general, exercise produces greater increases in MCT1 than in MCT4 content. Chronic exercise also affects MCT1 and MCT4 content, regardless of the initial fitness of subjects. On the basis of cross-sectional studies, intensity would appear to be the most important factor regulating exercise-induced changes in MCT content. Regulation of skeletal muscle MCT1 and MCT4 content by a variety of stimuli inducing an elevation of lactate level (exercise, hypoxia, nutrition, metabolic perturbations) has been demonstrated. Dissociation between the regulation of MCT content and lactate transport activity has been reported in a number of studies, and changes in MCT content are more common in response to contractile activity, whereas changes in lactate transport capacity typically occur in response to changes in metabolic pathways. Muscle MCT expression is involved in, but is not the sole determinant of, muscle H(+) and lactate anion exchange during physical activity.     

Kinetic analysis and design of experiments to identify the catalytic mechanism of the monocarboxylate transporter isoforms 4 and 1

Transport of lactate, pyruvate, and other monocarboxylates across the sarcolemma of skeletal and cardiac myocytes occurs via passive diffusion and by monocarboxylate transporter (MCT) mediated transport. The flux of lactate and protons through the MCT plays an important role in muscle energy metabolism during rest and exercise and in pH regulation during exercise. The MCT isoforms 1 and 4 are the major isoforms of this transporter in skeletal and cardiac muscle. The current consensus on the mechanism of these transporters, based on experimental measurements of labeled lactate fluxes, is that monocarboxylate-proton symport occurs via a rapid-equilibrium ordered mechanism with proton binding followed by monocarboxylate binding. This study tests ordered and random mechanisms by fitting experimental measurements of tracer exchange fluxes from MCT1 and MCT4 isoforms to theoretical predictions derived using relationships between one-way fluxes and thermodynamic forces. Analysis shows that: 1), the available kinetic data are insufficient to distinguish between a rapid-equilibrium ordered and a rapid-equilibrium random-binding model for MCT4; 2), MCT1 has a higher affinity to lactate than does MCT4; 3), the theoretical conditions for the so-called trans-acceleration phenomenon (e.g., increased tracer efflux from a vesicle caused by increased substrate concentration outside the vesicle) do not necessarily require the rate constant for the lactate and proton bound transporter to reorient across the membrane to be higher than that for the unbound transporter; and finally, 4), based on model analysis, additional experiments are proposed to be able to distinguish between ordered and random-binding mechanisms.