Faster lactate transport across red blood cell membrane in sickle cell trait carriers.

The physical and physiological behavior of sickle cell trait carriers (AS) is somewhat equivocal under strenuous conditions, although this genetic abnormality is generally considered to be a benign disorder. The occurrence of incidents and severe injuries in AS during exercise might be explained, in part, by the lactic acidosis due to a greater lactate influx into AS red blood cells (RBCs). In the present study, the RBC lactate transport activity via the different pathways was compared between AS and individuals with normal hemoglobin (AA). Sixteen Caribbean students, nine AS and seven AA, performed a progressive and maximal exercise test to determine maximal oxygen consumption. Blood samples were obtained at rest to assess haematological parameters and RBC lactate transport activity. Lactate influxes [total lactate influx and monocarboxylate transporter (MCT-1)-mediated lactate influx] into erythrocytes were measured at four external [14C]-labeled lactate concentrations (1.6, 8.1, 41, and 81.1 mM). The two groups had similar maximal oxygen consumption. Total lactate influx and lactate influx via the MCT-1 pathway were significantly higher in AS compared with AA at 1.6, 41, and 81.1 mM. The maximal lactate transport capacity for MCT-1 was higher in AS than in AA. Although AS and AA had the same maximal aerobic physical fitness, the RBCs from the sickle cell trait carriers took up more lactate at low and high concentrations than the RBCs from AA individuals. The higher MCT-1 maximal lactate transport capacity found in AS suggests greater content or greater activity of MCT-1 in AS RBC membranes.     

Does exercise-induced hypoxemia modify lactate influx into erythrocytes and hemorheological parameters in athletes?

This study investigated 1) red blood cells (RBC) rigidity and 2) lactate influxes into RBCs in endurance-trained athletes with and without exercise-induced hypoxemia (EIH). Nine EIH and six non-EIH subjects performed a submaximal steady-state exercise on a cyclo-ergometer at 60% of maximal aerobic power for 10 min, followed by 15 min at 85% of maximal aerobic power. At rest and at the end of exercise, arterialized blood was sampled for analysis of arterialized pressure in oxygen, and venous blood was drawn for analysis of plasma lactate concentrations and hemorheological parameters. Lactate influxes into RBCs were measured at three labeled [U-14C]lactate concentrations (1.6, 8.1, and 41 mM) on venous blood sampled at rest. The EIH subjects had higher maximal oxygen uptake than non-EIH (P < 0.05). Total lactate influx was significantly higher in RBCs from EIH compared with non-EIH subjects at 8.1 mM (1,498.1 +/- 87.8 vs. 1,035.9 +/- 114.8 nmol.ml(-1).min(-1); P < 0.05) and 41 mM (2,562.0 +/- 145.0 vs. 1,618.1 +/- 149.4 nmol.ml(-1).min(-1); P < 0.01). Monocarboxylate transporter-1-mediated lactate influx was also higher in EIH at 8.1 mM (P < 0.05) and 41 mM (P < 0.01). The drop in arterial oxygen partial pressure was negatively correlated with total lactate influx measured at 8.1 mM (r = -0.82, P < 0.05) and 41 mM (r = -0.84, P < 0.05) in the two groups together. Plasma lactate concentrations and hemorheological data were similar in the two groups at rest and at the end of exercise. The results showed higher monocarboxylate transporter-1-mediated lactate influx in the EIH subjects and suggested that EIH could modify lactate influx into erythrocyte. However, higher lactate influx in EIH subjects was not accompanied by an increase in RBC rigidity.     

Inhibition of L-lactate transport and band 3-mediated anion transport in erythrocytes by the novel stilbenedisulphonate N,N,N',N'-tetrabenzyl-4,4'-diaminostilbene-2,2'-disulpho nat e (TBenzDS).

(1) The synthesis of the novel stilbenedisulphonate N,N,N',N'-tetrabenzyl- 4,4'-diaminostilbene-2,2'-disulphonate (TBenzDS) is described, and its interaction with the lactate transporter and band 3 protein of erythrocytes investigated. At 10% haematocrit the IC50 (concn. required for 50% inhibition) for inhibition of transport of 0.5 mM L-lactate into rat erythrocytes at 7 degrees C was approx. 1.6 microM, as low as any other inhibitor of the transporter. In human erythrocytes at 10% haematocrit the IC50 value was increased from approx. 3 microM to 9 microM upon raising the temperature from 7 degrees C to 25 degrees C. (2) TBenzDS inhibited transport of L-lactate into rat erythrocytes in a manner that was competitive with the substrate, as is the case for some other stilbene disulphonate derivatives (Poole, R.C. and Halestrap, A.P. (1991) Biochem. J. 275, 307-312). (3) Increasing the haematocrit from 5 to 20% caused a 3-fold increase in the IC50 value for inhibition of L-lactate transport in rat erythrocytes. (4) TBenzDS was found to bind to erythrocyte membranes, with a partition coefficient (Pm) of 6000-7000 under all conditions tested. (5) TBenzDS also inhibited band 3-mediated sulphate transport in rat erythrocytes; 50% inhibition required approx. 2.5 microM TBenzDS for cells at 10% haematocrit. (6) TBenzDS is fluorescent, and an enhancement of this fluorescence occurs upon addition of BSA or erythrocyte membranes. The fluorescence enhancement caused by erythrocyte membranes is due to binding of the inhibitor to the band 3 protein at the same site as the stilbenedisulphonate 4,4'-diisothiocyanodihydrostilbene-2,2'-disulphonate (H2DIDS).     

L(+)-lactate transport in perfused rat skeletal muscle: kinetic characteristics and sensitivity to pH and transport inhibitors

We have examined lactate uptake (as the rate of net muscle lactate accumulation) and unidirectional inward transport (measured by a paired-tracer dilution method) in muscle of the perfused skinned rat hindlimb. Inhibition of tracer influx (fractional uptake at 1 mM L(+)-lactate, 43.3 +/- 3.1% but only 32.9 +/- 1.8% at 50 mM lactate) suggested some competition between tracer and native forms of the carboxylate for transport. D(-)-lactate (50 mM) did not inhibit uptake of tracer L(+)-lactate. Pyruvate (25 mM), but none of five other monocarboxylates, inhibited uptake of tracer lactate, by 22% (P less than 0.01). Altering perfusate pH from 7.4 to 6.8 caused a 36% increase (P less than 0.001) in the unidirectional L(+)-lactate transport at 1 mM L(+)-lactate, whereas increasing pH to 7.7 reduced transport by 18% (P less than 0.01). Tracer lactate influx was inhibited by 500 microM 4-acetamido-4'-isothiocyanostilbene (SITS) (19%), 5 mM alpha-cyano-4-hydroxycinnamic acid (CIN) (20-30%), 1 mM amiloride (27%) and by a thiol group reagent p-chloromercuribenzenesulphonic acid (pCMBS) (26%). Overall the results indicate that at least two processes are involved in the transfer of lactate: one, saturable, with a Vmax of 0.84 mumol.min-1.g-1 and an apparent Km of 21 mM was sensitive to SITS, CIN, and a thiol group reagent; the other was non-saturable and insensitive to SITS and CIN with an apparent rate constant of 0.1 min-1.     

Features of the inhibition of catalase activity by 3-amino-1,2,4-triazole in erythrocytes and liver of rats with streptozotocin diabetes]

Lipid peroxidation was intensified by streptozotocin induced diabetes mellitus in erythrocytes and liver. Activity of antioxidant enzyme superoxide-dismutase was decreased, activity of catalase was increased. Concentration of lipid peroxidation products was decreased after nicotinamide injections. It was investigated liver- and erythrocyte catalase inhibition in the presence of 3-amino-1,2,4-triazole. Effective inhibitor concentration for liver catalase by streptozotocin induced diabetes mellitus was 10 mM, by control-20 mM. Ascorbic acid induced catalase inhibition in the erythrocytes by diabetes mellitus increased by ascorbic acid concentration from 25 to 150 mM. [DHAA]/[AA]-ratio increased from 0.26 by control to 1.6 by diabetes mellitus and decreased to 0.44 after nicotinamide injections.     

Effects of darbepoetin injections on erythrocyte membrane transport protein expressions in humans.

The present study investigated the effects of injected darbepoetin [novel erythropoietin stimulating protein (NESP)] on the density of three erythrocyte membrane transport proteins: the lactate-H+ cotransporter (monocarboxylate transporter 1), the chloride/bicarbonate exchanger 1 (anion exchanger 1), and the water channel aquaporin 1. Thirteen subjects were injected with NESP once a week for 4 wk. Blood samples were obtained before, during, and after the injection period, and the erythrocyte transport proteins were determined by Western blotting. The NESP injections induced a transient increase in hematocrit, red cell volume, and reticulocyte fraction. The density of aquaporin 1 protein was higher (maximal increase +59%) (P < 0.01) during the injection period compared with the preinjection value and lower (P < 0.01) after the injection period. The density of anion exchanger 1 protein was higher (maximal increase +15%) (P < 0.05) during the injection period compared with the preinjection value and tended (P = 0.06) to be lower after the injection period than before the injection period. The density of the erythrocyte monocarboxylate transporter 1 protein was higher (maximal increase +43%) (P < 0.05) during the injection period than in the preinjection period. Age separation experiments using self-creating Percoll gradients demonstrated a higher density of membrane transport proteins in young red blood cells. These data suggest that the NESP-induced increase in membrane transport proteins is caused by a higher fraction of newly formed erythrocytes (and reticulocytes), which have a higher density of membrane transport proteins. However, increased incorporation of membrane proteins during erythrocyte formation may also be involved. We suggest that NESP improves the quality of erythrocyte membrane transport through these mechanisms.     

Reduced Serum Hydroxyl Radical Scavenging Activity in Erythropoietin Therapy Resistant Renal Anemia

Relation between anemia resistant to recombinant human erythropoietin (rHuEPO) therapy and the oxidative stress in hemodialysis (HD) patients was investigated. Stable HD patients who had consistent hemoglobin concentrations on a constant dose of rHuEPO were studied. Patients were excluded if there were factors that might affect hemopoiesis or administration of antioxidant supplements. Patients were classified into three groups: High (9000 U/week), Low (1500-4500 U/week) and No rHuEPO group. Thiobarbituric acid reactive substances (TBARS) of sera and erythrocyte were examined. Serum superoxide and hydroxyl radical scavenging activities were measured using electron spin resonance. TBARS in the erythrocyte was higher in High rHuEPO group compared with No rHuEPO group, though the serum TBARS were similar. A diminution of serum hydroxyl radical scavenging activity was observed in High rHuEPO group. Hydroxyl radical signal intensity showed a strong correlation with the serum ferritin in High rHuEPO group, although ferritin concentrations were not different among the 3 groups. Superoxide scavenging activity showed no differences. These results indicate that increased lipid peroxidation in erythrocyte, raised by decreased serum hydroxyl radical scavenging activity, is one cause of rHuEPO resistant anemia. Serum ferritin may be involved in this hydroxyl radical production.     

Luminal leptin enhances CD147/MCT-1-mediated uptake of butyrate in the human intestinal cell line Caco2-BBE

In the intestine, butyrate constitutes the major energy fuel for colonocytes. However, little is known about the transport of butyrate and its regulation in the intestine. In this study we demonstrate that the monocarboxylate transporter (MCT-1) is apically polarized in model human intestinal epithelia and is involved in butyrate uptake by Caco2-BBE cell monolayers. The butyrate uptake by Caco2-BBE cell monolayers displayed conventional Michaelis-Menten kinetics and was found to be pH-dependent, Na(+)-independent, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-insensitive, and inhibited by the monocarboxylate transporter inhibitor alpha-cyano-4-hydroxycinnamate and by an excess of unlabeled butyrate. We show that MCT-1 associates with CD147 at the apical plasma membrane in Caco2-BBE cell monolayers. Using antisense CD147, we demonstrate that the association of CD147 with MCT-1 is critical for the butyrate transport activity. Interestingly, we show for the first time hormonal regulation of CD147/MCT-1 mediated butyrate uptake. Specifically, luminal leptin significantly up-regulates MCT-1-mediated butyrate uptake by increasing its maximal velocity (V(max)) without any modification in the apparent Michaelis-Menten constant (K(m)). Finally, we show that luminal leptin up-regulates butyrate uptake in Caco2-BBE monolayers by two distinct actions: (i) increase of the intracellular pool of MCT-1 protein without affecting CD147 expression and (ii) translocation of CD147/MCT-1 to the apical plasma membrane of Caco2-BBE cell monolayers.     

Training intensity-dependent and tissue-specific increases in lactate uptake and MCT-1 in heart and muscle

We investigatedthe effects of 3 wk of moderate- (21 m/min, 8% grade) andhighintensity treadmill training (31 m/min, 15% grade) on1) monocarboxylate transporter 1 (MCT-1) content in rat hindlimb muscles and the heart and2) lactate uptake in isolated soleus(Sol) muscles and perfused hearts. In the moderately trained groupMCT-1 was not increased in any of the muscles [Sol, extensor digitorum longus (EDL), and red (RG) and white gastrocnemius(WG)] (P > 0.05). Similarly,lactate uptake in Sol strips was also not increased(P > 0.05). In contrast, in theheart, MCT-1 (+36%, P < 0.05) andlactate uptake (+72%, P < 0.05)were increased with moderate training. In the highly trained group,MCT-1 (+70%, P < 0.05) and lactateuptake (+79%, P < 0.05) wereincreased in Sol. MCT-1 was also increased in RG (+94%,P < 0.05) but not in WG and EDL(P > 0.05). In the highly trainedgroup, heart MCT-1 (+44%, P < 0.05)and lactate uptake (+173%, P < 0.05) were increased. In conclusion, it has been shown that1) in both heart and skeletal musclelactate uptake is increased only when MCT-1 is increased; 2) training-induced increases inMCT-1 occurred at a lower training intensity in the heart than inskeletal muscle; 3) in the heart, lactate uptake was increased much more after high-intensity training than after moderate-intensity training, despite similar increases inheart MCT-1 with these two training intensities; and4) the increases in MCT-1 occurredindependently of any changes in the heart's oxidative capacity (asmeasured by citrate synthase activity).

     

Lactate transport activity in rat skeletal muscle sarcolemmal vesicles after acute exhaustive exercise.

The effect of a single bout of exhaustive exercise on muscle lactate transport capacity was studied in rat skeletal muscle sarcolemmal (SL) vesicles. Rats were assigned to a control (C) group (n = 14) or an acutely exercised (E) group (n = 20). Exercise consisted of treadmill running (25 m/min, 10% grade) to exhaustion. SL vesicles purified from C and E rats were sealed because of sensitivity to osmotic forces. The time course of 1 mM lactate uptake in zero-trans conditions showed that the equilibrium level in the E group was significantly lower than in the C group (P < 0.05). The initial rate of 1 mM lactate uptake decreased significantly from 2.44 +/- 0.22 to 1.03 +/- 0.08 nmol. min(-1). mg protein(-1) (P < 0.05) after exercise, whereas that of 50 mM lactate uptake did not differ significantly between the two groups. For 100 mM external lactate concentration ([lactate]), exhaustive exercise increased initial rates of lactate uptake (219.6 +/- 36.3 to 465.4 +/- 80.2 nmol. min(-1). mg protein(-1), P < 0.05). Although saturation kinetics were observed in the C group with a maximal transport velocity of 233 nmol. min(-1). mg protein(-1) and a Michealis-Menten constant of 24.5 mM, saturation properties were not seen after exhaustive exercise in the E group, because initial rates of lactate uptake increased linearly with external [lactate]. We conclude that a single bout of exhaustive exercise significantly modified SL lactate transport activity, resulting in a decrease in 1 mM lactate uptake and was associated with alterations in the saturable properties at [lactate] above 50 mM. These results suggest that changes in sarcolemmal lactate transport activity may alter lactate and proton exchanges after exhaustive exercise.     

Protein kinase C activity in erythrocytes in primary hypertension: regulation of cell shape and cation transport

Protein kinase C activity in the lysate of erythrocytes of patients with essential hypertension (EH) and spontaneously hypertensive rats (SHR) was found to be increased by 1.6-2.0 times as compared with normotensive controls. Membrane cytoskeleton alterations observed in the erythrocytes of patients with EH and SHR were revealed in decreased average erythrocyte volume, increase of cup-shaped cell formation, and increase of basal phosphorylation of band 4.9 protein. In addition, the rate of Na(+)-H+ exchange in erythrocytes of EH patients and SHR was increased by 1.9-fold. In vitro treatment of erythrocytes of healthy donors and Wistar-Kyoto rats (WKY) with protein kinase C activator (12-O-tetradecanoylphorbol-13-acetate) leads to similar changes of cell shape, cell volume, band 4.9 protein phosphorylation and Na(+)-H+ exchange, as well as to an increase of diS-C3-(5) fluorescence. It may be assumed that alterations of these parameters revealed in primary hypertension are caused by increased activity of protein kinase C.     

Human erythrocyte band 3 functions as a receptor for the sialic acid-independent invasion of Plasmodium falciparum. Role of the RhopH3–MSP1 complex

Plasmodium falciparum takes advantage of two broadly defined alternate invasion pathways when infecting human erythrocytes: one that depends on and the other that is independent of host sialic acid residues on the erythrocyte surface. Within the sialic acid-dependent (SAD) and sialic acid-independent (SAID) invasion pathways, several alternate host receptors are used by P. falciparum based on its particular invasion phenotype. Earlier, we reported that two putative extracellular regions of human erythrocyte band 3 termed 5C and 6A function as host invasion receptor segments binding parasite proteins MSP1 and MSP9 via a SAID mechanism. In this study, we developed two mono-specific anti-peptide chicken IgY antibodies to demonstrate that the 5C and 6A regions of band 3 are exposed on the surface of human erythrocytes. These antibodies inhibited erythrocyte invasion by the P. falciparum 3D7 and 7G8 strains (SAID invasion phenotype), and the blocking effect was enhanced in sialic acid-depleted erythrocytes. In contrast, the IgY antibodies had only a marginal inhibitory effect on FCR3 and Dd2 strains (SAD invasion phenotype). A direct biochemical interaction between erythrocyte band 3 epitopes and parasite RhopH3, identified by the yeast two-hybrid screen, was established. RhopH3 formed a complex with MSP1₁₉ and the 5ABC region of band 3, and a recombinant segment of RhopH3 inhibited parasite invasion in human erythrocytes. Together, these findings provide evidence that erythrocyte band 3 functions as a major host invasion receptor in the SAID invasion pathway by assembling a multi-protein complex composed of parasite ligands RhopH3 and MSP1.     

Phosphoinositide metabolism and cGMP levels are not coupled to the muscarinic-cholinergic receptor in human erythrocyte

Recently, Tang et al. [BBA 772, 235 (1984)] reported that cholinergic agonists stimulate calcium uptake and cGMP formation in the human erythrocyte. We undertook this investigation since polyphosphoinositide breakdown precedes calcium mobilization and cGMP formation in several tissues. In [32P]-prelabeled erythrocyte ghosts, calcium (0.5 mM) but not carbachol (0.1 mM) caused a 2- and 20-fold increase in the accumulation of IP2 and IP3, respectively. This was accompanied by a 50% decrease in PIP2 and PIP. In intact erythrocytes prelabeled with [32P], 1 microM A23187 but not carbachol (0.1 mM) produced a 300% increase in radioactivity in PA after a 30-min incubation. cGMP levels after a 2-min incubation with saline, A23187 (1 microM), or carbachol (0.1 mM) were 0.27 +/- .03, 0.27 +/- .04, and 0.34 +/- .04 fmol/10(6) cells. Our studies indicate that the muscarinic receptor in the erythrocytes is "non-functional" insofar as its stimulation is not accompanied by phosphoinositide breakdown or cGMP formation.     

The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide

The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1-60 microM and 50-370 microM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2(+)-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2(+)-binding affinity was 1.5-2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2(+)-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2(+)-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2(+)-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 microM, ouabain (0.5-1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2(+)-ATPase activity. Taking this observation into account the Na+/K(+)-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 microM), the addition of 0.3-1 microM Ca2+ increased Na+/K(+)-ATPase activity by 1.5-3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 microM) only Na+/K(+)-ATPase inhibition by Ca2+ was seen. It was found that at [NaCl] less than 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2(+)-free media. This activating effect of furosemide was enhanced with a diminution of [Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K(+)-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes.     

Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) interacts with the band 3 receptor to promote erythrocyte invasion by malaria parasites

Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) is an erythrocyte binding protein known to be involved in malarial parasite invasion. Although anti-GAMA antibodies have been shown to block GAMA attachment to the erythrocyte surface and subsequently inhibit parasite invasion, little is known about the molecular mechanisms by which GAMA promotes the invasion process. In this study, LC-MS analysis was performed on the erythrocyte membrane to identify the specific receptor that interacts with GAMA. We found that ankyrin 1 and the band 3 membrane protein showed affinity for GAMA, and characterization of their binding specificity indicated that both Plasmodium falciparum and Plasmodium vivax GAMA bound to the same extracellular loop of band 3 (loop 5). In addition, we show the interaction between GAMA and band 3 was sensitive to chymotrypsin. Furthermore, antibodies against band 3 loop 5 were able to reduce the binding activity of GAMA to erythrocytes and inhibit the invasion of P. falciparum merozoites into human erythrocytes, whereas antibodies against P. falciparum GAMA (PfGAMA)-Tr3 only slightly reduced P. falciparum invasion. The identification and characterization of the erythrocyte GAMA receptor is a novel finding that identifies an essential mechanism of parasite invasion of host erythrocytes.     

Reduced serum hydroxyl radical scavenging activity in erythropoietin therapy resistant renal anemia

Relation between anemia resistant to recombinant human erythropoietin (rHuEPO) therapy and the oxidative stress in hemodialysis (HD) patients was investigated. Stable HD patients who had consistent hemoglobin concentrations on a constant dose of rHuEPO were studied. Patients were excluded if there were factors that might affect hemopoiesis or administration of antioxidant supplements. Patients were classified into three groups: High (9000 U/week), Low (1500-4500 U/week) and No rHuEPO group. Thiobarbituric acid reactive substances (TBARS) of sera and erythrocyte were examined. Serum superoxide and hydroxyl radical scavenging activities were measured using electron spin resonance. TBARS in the erythrocyte was higher in High rHuEPO group compared with No rHuEPO group, though the serum TBARS were similar. A diminution of serum hydroxyl radical scavenging activity was observed in High rHuEPO group. Hydroxyl radical signal intensity showed a strong correlation with the serum ferritin in High rHuEPO group, although ferritin concentrations were not different among the 3 groups. Superoxide scavenging activity showed no differences. These results indicate that increased lipid peroxidation in erythrocyte, raised by decreased serum hydroxyl radical scavenging activity, is one cause of rHuEPO resistant anemia. Serum ferritin may be involved in this hydroxyl radical production.     

The Effect of Citrulline/Malate on Blood Lactate Levels in Intensive Exercise

The purpose of this study was to examine the effects of Citrulline/Malate supplementation with intensive training on blood lactate level in active handball players. The athletes were subjected to intense training for 4 weeks, 4 days a week, mainly pre-season strength and technique training. In this training period, stimol group (n = 11) athletes were given stimol 3 times a day as 1 g for breakfast, 1 g for lunch, and 1 g for dinner while the placebo group (n = 11) athletes were given only placebo in the same dosage and the same color at the same time. Blood lactate levels in athletes were measured 4 times, prior to and after a 1-month program as follows: rest (R), end effort (EE), recuperation 5 min (R5 m), and recuperation 20 min (R20 m). Blood lactate levels were compared both as intra-group and between the groups. In intra-group comparison, no change was observed in blood lactate levels in placebo group while a significant difference was found in the levels of stimol group as p < 0.05 with a 49.8% decrease in blood lactate level. In the measurements between groups, in the post-test measurements made after the training period, significant differences as p < 0.05 were found with a 60.7% decrease in blood lactate level EE. Considerable decline was seen especially immediately after exercise in blood lactate levels of the athletes being given stimol supplement. In this case, we can say that Citrulline/Malate supplementation may contribute positively to the performance of athletes and may help postpone fatigue at excessive or prolonged activity.     

Studies on toxin of Aspergillus fumigatus. XXII. Fashion of binding of Asp-hemolysin to human erythrocytes and Asp-hemolysin-binding proteins of erythrocyte membranes

The fashion of binding of Asp-hemolysin to human erythrocytes and the isolation of Asp-hemolysin-binding proteins from erythrocyte membranes were investigated by the immunocytochemical technique and affinity chromatography. Asp-hemolysin bound best at a pH range from 5 to 7. The erythrocytes treated with Asp-hemolysin showed diffuse, ring-like or cap-like staining by the peroxidase-labeled antibody method under the light microscope. The distribution of Asp-hemolysin on the erythrocyte surface was clearly observed as patches or caps in the scanning electron microscope. The erythrocyte ghosts were extracted with 1% sodium deoxycholate-0.1 M Tris-HC1 buffer (pH 7.5) containing 0.2 M NaCl and 1 mM EDTA, and the extract was chromatographed on an affinity column consisting of Asp-hemolysin attached to activated thiol-Sepharose 4B. Four proteins present in the membrane extract were retained by activated thiol-Sepharose 4B and eluted with 50 mM cysteine as toxin-membrane components. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the polypeptides correspond to band 2.1, one protein of the 2 region, band 3 and band 7 in the Steck nomenclature system.     

Four weeks of normobaric "live high-train low" do not alter muscular or systemic capacity for maintaining pH and K⁺ homeostasis during intense exercise

It was investigated if athletes subjected to 4 wk of living in normobaric hypoxia (3,000 m; 16 h/day) while training at 800-1,300 m ["live high-train low" (LHTL)] increase muscular and systemic capacity for maintaining pH and K(+) homeostasis as well as intense exercise performance. The design was double-blind and placebo controlled. Mean power during 30-s all-out cycling was similar before and immediately after LHTL (650 ± 31 vs. 628 ± 32 W; n = 10) and placebo exposure (658 ± 22 vs. 660 ± 23 W; n = 6). Supporting the performance data, arterial plasma pH, lactate, and K(+) during submaximal and maximal exercise were also unaffected by the intervention in both groups. In addition, muscle buffer capacity (in mmol H(+)·kg dry wt(-1)·pH(-1)) was similar before and after in both the LHTL (140 ± 12 vs. 140 ± 16) and placebo group (145 ± 2 vs. 140 ± 3). The expression of sarcolemmal H(+) transporters (Na(+)/H(+) exchanger 1, monocarboxylate transporters 1 and 4), as well as expression of Na(+)-K(+) pump subunits-α(1), -α(2), and -β(1) was also similar before and after the intervention. In conclusion, muscular and systemic capacity for maintaining pH and K(+) balance during exercise is similar before and after 4 wk of placebo-controlled normobaric LHTL. In accordance, 30-s all-out sprint ability was similar before and after LHTL.     

Muscle contraction increases lactate transport while reducing sarcolemmal MCT4, but not MCT1

Rates of lactate uptake into giant sarcolemmal vesicles were determined in vesicles collected from rat muscles at rest and immediately after 10 min of intense muscle contraction. This contraction period reduced muscle glycogen rapidly by 37-82% in all muscles examined (P < 0.05) except the soleus muscle (no change P > 0.05). At an external lactate concentration of 1 mM lactate, uptake into giant sarcolemmal vesicles was not altered (P > 0.05), whereas at an external lactate concentration of 20 mM, the rate of lactate uptake was increased by 64% (P < 0.05). Concomitantly, the plasma membrane content of monocarboxylate transporter (MCT)1 was reduced slightly (-10%, P < 0.05), and the plasma membrane content of MCT4 was reduced further (-25%, P < 0.05). In additional studies, the 10-min contraction period increased the plasma membrane GLUT4 (P < 0.05) while again reducing MCT4 (-20%, P < 0.05) but not MCT1 (P > 0.05). These studies have shown that intense muscle contraction can increase the initial rates of lactate uptake, but only when the external lactate concentrations are high (20 mM). We speculate that muscle contraction increases the intrinsic activity of the plasma membrane MCTs, because the increase in lactate uptake occurred while plasma membrane MCT4 was decreased and plasma membrane MCT1 was reduced only minimally, or not at all.