Lactate transport by skeletal muscle sarcolemmal vesicles

Recent studies have indicated that lactate traversal of the sarcolemmal membrane of skeletal muscle could be a carrier mediated process. In the present study, the initial rates of L(+)-lactate flux (J_lact) were measured in highly purified rat hindlimb skeletal muscle sarcolemmal vesicles. Fluxes were determined by the vesicle uptake of L(+)-[U-¹⁴C] lactate from the extra-vesicular medium. J_lact was saturable with respect to increasing concentrations of L(+)-lactate. Regression of these data to the Michaelis-Menten equation yielded a Km of 12.5 mM. J_lact was inhibited 81% by 10 mM pyruvate and 83% by 5mM alpha-cyano 4 hydroxycinnamate (p<0.05), but not by D-lactate indicating the presence of a stereoselective monocarboxylate transporter in the sarcolemmal membrane. Preincubation of the vesicles with the protein modifier, N-ethylmaleimide (20mM), inhibited J_lact by 86% (p<0.05). An inhibitor of the inorganic anion exchanger, SITS (1mM), had no effect on J_lact. However, J_lact was markedly sensitive to an inwardly directed proton gradient (p<0.05), and the flux was more closely related to the concentration of external ionic L(+)-lactate than to the protonated (HLa) form. These studies suggest that skeletal muscle sarcolemmal membranes possess a specific transport system for L-lactate and other monocarboxylates, which has similar properties to the lactate carrier described for several other tissues.     

Effect of insulin, prostaglandin E1 and uptake inhibitors on glucose transport in the perfused guinea-pig placenta

The effects of insulin, prostaglandin E1 (PGE1) and uptake inhibitors on unidirectional D-glucose influx at brush border (maternal) and basal (fetal) sides of the guinea-pig syncytotrophoblast were investigated in the intact, perfused guinea-pig placenta by rapid, paired-tracer dilution. Experiments were performed in either an in situ preparation artificially perfused through the umbilical vessels (intact maternal circulation) or in the fully isolated dually-perfused placenta in which both interfaces were studied simultaneously. Kinetic characterization of unidirectional D-glucose influx gave apparent Km values (mean +/- SEM) at maternal and fetal sides of 70 +/- 6 and 87 +/- 16 mM respectively; corresponding Vmax values were 53 +/- 3 and 82 +/- 6 mumol min-1g-1. At the fetal side (singly-perfused placenta) cytochalasin B (50 microM), ethylidene-D-glucose (100 mM) and PGE1 (1 microM) partially inhibited D-glucose uptake whereas cortisol (50 microM) and progesterone (100 microM) had no effect. Abolition of the sodium gradient across the fetal interface did not modulate the kinetics of influx. In the presence of 150 mu units ml-1 insulin (dually-perfused placenta), unidirectional uptake into the trophoblast and transplacental D-[3H]glucose transfer were unaltered. In contrast, prostaglandin E1 (1 microM) markedly reduced the Km and Vmax for D-glucose at both interfaces and the inhibitory effect was reflected in a reduction in specific transplacental D-glucose transfer. Further experiments showed that the isolated placenta releases prostaglandins (PGE; PGF2 alpha) into both circulations. Bilateral insulin perfusion did not affect either lactate release by the placenta or rapid metabolism of D-[14C]glucose to [3H]lactate (usually less than 10% effluent [14C]lactate in 5 min). An asymmetric degradation of exogenous insulin was observed in the dually-perfused placenta: uterine venous samples contained 24 +/- 7 microunits ml-1 immunoreactive insulin when compared to the arterial concentration (151 +/- 3 microU ml-1 perfusate) while no change was measureable in the fetal circulation within the same time period (152 +/- 5 microU ml-1). This asymmetry was confirmed in experiments employing [125I]insulin. These results demonstrate that glucose transport in the intact guinea-pig placenta occurs by a sodium-independent, cytochalasin B-inhibitable system which is insulin-insensitive. Prostaglandin E1 appeared to be a potent transport inhibitor which suggests that prostaglandins may be involved in the 'down' regulation of placental glucose transport in vivo.     

Lysine and alanine transport in the perfused guinea-pig placenta

The characteristics of L-lysine transport were investigated at brush-border (maternal) and basal (fetal) sides of the syncytiotrophoblast in the term guinea-pig placenta artificially perfused either through the umbilical vessels in situ or through both circulations simultaneously. Cellular uptake, efflux and transplacental transfer were determined using a single-circulation paired-tracer dilution technique. Unidirectional L-[3H]lysine uptake (%) (perfusate lysine 50 microM) was high on maternal (M = 87 +/- 1) and fetal (F = 73 +/- 2) sides. L-[3H]Lysine efflux back into the ipsilateral circulation was asymmetrical (F/M ratio = 2.3) and transplacental flux occurred in favour of the fetal circulation. Unidirectional lysine influx kinetics (0.05-8.00 mM) gave Km values of 1.75 +/- 0.70 mM and 0.90 +/- 0.25 mM at maternal and fetal sides, respectively; corresponding Vmax values were 1.95 +/- 0.38 and 0.87 +/- 0.10 mumol.min-1.g-1. At both sides, lysine influx (50 microM) could be inhibited (about 60-80%) by 4 mM L-lysine and L-ornithine and less effectively (about 10-40%) by L-citrulline, L-arginine, D-lysine and L-histidine. At the basal side: (i) lysine influx kinetics were greatly modified in the presence of 10 mM L-alanine (Km = 6.25 +/- 3.27 mM; Vmax = 2.62 +/- 0.94 mumol.min-1.g-1), but unchanged by equimolar L-phenylalanine or L-tryptophan; (ii) in the converse experiments, lysine (10 mM) did not affect the kinetic characteristics for either L-alanine or L-phenylalanine; (iii) L-lysine and L-alanine influx kinetics were not dependent on the sodium gradient; (iv) the inhibition of L-[3H]lysine uptake by 4 mM L-homoserine was partially (60%) Na+-dependent. At the maternal side the kinetic characteristics for alanine influx were highly Na+-dependent, while lysine influx was partially Na+-dependent only at low concentrations (0.05-0.5 mM). Bilateral perfusion with 2,4-dinitrophenol (1 mM) reduced L-[3H]lysine uptake into the trophoblast and abolished transplacental transfer. It is suggested that lysine transport in the guinea-pig placenta is mediated by a specific transport system (y+) for cationic amino-acids. The asymmetry in the degree of sodium-dependency at both trophoblast membranes may in part explain the maternal-to-foetal polarity of placental amino-acid transfer in vivo.     

Pleural surface fluorescence measurement of Na+ and Cl- transport across the air space-capillary barrier.

We developed a pleural surface fluorescence method to measure Na(+) and Cl(-) transport in perfused mouse lungs. The air space was filled with aqueous fluid containing membrane-impermeant fluorescent indicators of Cl(-) (lucigenin) or Na(+) (Sodium Green). After instillation of a Cl(-)-free solution into the air space, an increase in perfusate Cl(-) concentration from 0 to 30 mM produced a decrease in surface lucigenin fluorescence (6.5%/min) corresponding to Cl(-) influx of 1.0 mM/min. Cl(-) influx was increased to 2.1 +/- 0.3 mM/min by forskolin, and the increase was inhibited by glibenclamide. cAMP-stimulated Cl(-) influx was decreased by 57% in CFTR null mice. After instillation of a Na(+)-free solution into the air space, an increase in perfusate Na(+) concentration from 0 to 30 mM gave increased Sodium Green fluorescence (Na(+) influx of 1.2 mM/min), which increased approximately fivefold after cAMP agonists. Cl(-) and Na(+) transport were not affected in lungs from mice lacking aquaporins AQP1 or AQP5. Our results establish a pleural surface fluorescence method to measure unidirectional Cl(-) and Na(+) flux in intact lung and provide evidence for cAMP-stimulated transcellular Cl(-) and Na(+) transport.     

Evidence for a lactate transport system in the sarcolemmal membrane of the perfused rabbit heart: kinetics of unidirectional influx, carrier specificity and effects of glucagon

The kinetics and specificity of L-lactate transport into cardiac muscle were studied during a single transit through the isolated perfused rabbit heart using a rapid (15 s) paired-tracer dilution technique. Kinetic experiments revealed that lactate influx was highly stereospecific and saturable with an apparent Kt = 19 +/- 6 mM and a Vmax = 8.4 +/- 1.5 mumol/min per g (mean +/- S.E., n = 14 hearts). At high perfusate concentrations (10 mM), the inhibitors alpha-cyano-4-hydroxycinnamate (Ki = 7.3 mM), pyruvate (Ki = 6.5 mM), acetate (Ki = 19.4 mM) and chloroacetate (Ki = 28 mM) reduced L-lactate influx, and Ki values were estimated assuming a purely competitive interaction of the inhibitors with the monocarboxylate carrier. The monocarboxylic acids [14C]pyruvate and [3H]acetate were themselves transported, and sarcolemmal uptakes of respectively 38 +/- 1% and 70 +/- 8% were measured relative to D-mannitol. Perfusion of hearts for 10-30 min with 0.15 or 1.5 microM glucagon increased myocardial lactate production and simultaneously inhibited tracer uptake of lactate, pyruvate and acetate. It is concluded that a stereospecific lactate transporter exhibiting an affinity for other substituted monocarboxylic acids is operative in the sarcolemmal plasma membrane of the rabbit myocardium.     

Activation of sodium transport in rat erythrocytes by inhibition of protein phosphatases 1 and 2A

Four structurally different protein phosphatases (PPs) inhibitors - fluoride, calyculin A, okadaic acid and cantharidin--were tested for their ability to modulate unidirectional Na(+) influx in rat red blood cells. Erythrocytes were incubated at 37 degrees C in isotonic and hypertonic media containing 1 mM ouabain and (22)Na in the absence or presence of PP inhibitors. Exposure of the cells to 20 mM fluoride or 50 nM calyculin A for 1 h under isosmotic conditions caused a significant stimulation of Na(+) influx, whereas addition of 200 microM cantharidin or 100 nM okadaic acid had no effect. After 2 h of treatment, however, all these PPs blockers significantly enhanced Na(+) transport in rat erythrocytes. Selective inhibitors of PP-1 and PP-2A types, calyculin A, cantharidin and okadaic acid, produced similar ( approximately 1.2-1.4-fold) stimulatory effects on Na(+) influx in the cells. Activation of Na(+) influx was unchanged with increasing calyculin A concentration from 50 to 200 nM. No additive stimulation of Na(+) influx was observed when the cells were treated with combination of 20 mM fluoride and 50 nM calyculin A. Na(+) influx induced by PPs blockers was inhibited by 1 mM amiloride and 200 muM bumetanide approximately in the equal extent, indicating the involvement of Na(+)/H(+) exchange and Na-K-2Cl cotransport in sodium transport through rat erythrocytes membrane. Activation of Na(+) transport in the cells induced by calyculin A and fluoride was associated with increase of intracellular Na(+) content. Shrinkage of the rat erythrocytes resulted in 2-fold activation of Na(+) influx. All tested PPs inhibitors additionally activated the Na(+) influx by 70-100% above basal shrinkage-induced level. Amiloride and bumetanide have diminished both the shrinkage-induced and PPs-inhibitors-induced Na(+) influxes. Thus, our observations clearly indicate that activities of Na(+)/H(+) exchanger and Na-K-2Cl cotransporter in rat erythrocytes are regulated by protein phosphatases and stimulated when protein dephosphorylation is inhibited.     

Plasma membrane depolarization reduces nitric oxide (NO) production in P388D.1 macrophage-like cells during Leishmania major infection

In the present study, we compare changes in host cell plasma membrane potential (V(m)), K(+) fluxes, and NO production during K(+) channel blockade with those changes that occur during infection with Leishmania major. Infection of P388D.1 cells with L. major promastigotes or treatment with K(+) channel blockers (either 1mM 4-AP, 10mM TEA, or 200 microM quinine) suppressed NO production. Inhibition of NO production correlated with depolarization of the P388D.1 cell V(m). Infection of P388D.1 cells with L. major increased the unidirectional influx of rubidium (86Rb), a tracer for K(+) flux, that was comparable to that induced by K(+) channel blockade by 1mM 4-AP. The similar effects of K(+) channel blockers and L. major on NO production, K(+) influx, and V(m) suggest that K(+) channel activity and the maintenance of V(m) is important for NO production in these cells. We suggest that intracellular parasites employ a strategy to inhibit NO production by disrupting V(m) during the invasion/infection process by altering host cell K(+) channel activity.     

Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target size of system asc in red cells from Przewalski's horse. The transporter's in situ apparent molecular weight was 158,000 +/- 2500 (SE).     

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.     

Expression of rat liver glutamine transporters in Xenopus laevis oocytes.

As a first step in attempting to isolate the Na(+)-dependent System N transporter from rat liver we have investigated the use of prophase-arrested oocytes from Xenopus laevis for the functional expression of rat liver glutamine transporters. Individual oocytes, defolliculated by collagenase treatment, were injected with 50 nl of a 1 mg.ml-1 solution of poly(A)+ RNA (mRNA) isolated from rat liver. 50 microM L-[3H]glutamine uptake was measured 1-5 days post-injection: after 48 h, poly(A)+ RNA-injected oocytes showed a 60 +/- 12% increase in Na(+)-dependent glutamine uptake compared to controls. This increased uptake showed characteristic features of hepatic System N: that is, it tolerated Li(+)-for-Na+ substitution and was inhibited by the System N substrate L-histidine (5 mM) in Li medium, unlike endogenous Na(+)-dependent glutamine transport. In subsequent experiments rat liver poly(A)+ RNA, size-fractionated by density gradient fractionation, was injected into oocytes. Injection of poly(A)+ RNA of 1.9-2.8 kilobases (kb) in size resulted in a significant stimulation of Na(+)-dependent glutamine transport to 0.362 +/- 0.080 pmol.min-1/oocyte from 0.178 +/- 0.060 pmol.min-1/oocyte in vehicle-injected oocytes (p less than 0.01). A lighter fraction, with poly(A)+ RNA of less than 1.9 kilobases size resulted in a similar increase in Na(+)-dependent glutamine uptake which was largely Li(+)-tolerant: Li(+)-stimulated glutamine uptake in oocytes injected with this fraction increased to 0.230 +/- 0.070 pmol.min-1/oocyte from 0.098 +/- 0.029 pmol.min-1/oocyte in controls (p less than 0.05). This enhanced rate of Li(+)-stimulated glutamine uptake was inhibited 28 and 70%, respectively, by 1 and 5 mM L-histidine. Na(+)-independent uptake of glutamine rose by 72 +/- 12% in oocytes injected with poly(A)+ RNA of 2.8-3.6 kb (p less than 0.001). These results demonstrate that glutamine transporters, with characteristics associated with hepatic Systems N, L, and A (or ASC), can be expressed in X. laevis oocytes injected with specific size fractions of rat liver mRNA.     

Utilization of Lactate Isomers by Propionibacterium freudenreichii subsp. shermanii: Regulatory Role for Intracellular Pyruvate

Five strains of Propionibacterium freudenreichii subsp. shermanii utilized the l-(+) isomer of lactate at a faster rate than they did the d-(-) isomer when grown with a mixture of lactate isomers under a variety of conditions. ATCC 9614, grown anaerobically in defined medium containing 160 mM dl-lactate, utilized only 4 and 15% of the d-(-)-lactate by the time 50 and 90%, respectively, of the l-(+)-lactate was used. The intracellular pyruvate concentration was high (>100 mM) in the initial stages of lactate utilization, when either dl-lactate or the l-(+) isomer was the starting substrate. The concentration of this intermediate dropped during dl-lactate fermentation such that when only d-(-)-lactate remained, the concentration was <20 mM. When only the d-(-) isomer was initially present, a similar relatively low concentration of intracellular pyruvate was present, even at the start of lactate utilization. The NAD-independent lactate dehydrogenase activities in extracts showed different kinetic properties with regard to pyruvate inhibition, depending upon the lactate isomer present. Pyruvate gave a competitive inhibitor pattern with l-(+)-lactate and a mixed-type inhibitor pattern with d-(-)-lactate. It is suggested that these properties of the lactate dehydrogenases and the intracellular pyruvate concentrations explain the preferential use of the l-(+) isomer.     

Alleviation of rapid, futile ammonium cycling at the plasma membrane by potassium reveals K+-sensitive and -insensitive components of NH4+ transport

Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 micromol g(-1) h(-1) was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5-40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.     

Na(+) and Cl(-) transport by the urinary bladder of the freshwater rainbow trout (Oncorhynchus mykiss)

Freshwater (FW) rainbow trout (Oncorhynchus mykiss) urinary bladders mounted in vitro under symmetrical saline conditions displayed electroneutral active absorption of Na(+) and Cl(-) from the mucosal side; the transepithelial potential (V(t)) was 0.1 mV, and the short-circuit current was less than 1 microA cm(-2). Removal of Na(+) from mucosal saline decreased Cl(-) absorption by 56% and removal of Cl(-) decreased Na(+) absorption by 69%. However, active net absorption of both Na(+) and Cl(-) was not abolished when Cl(-) or Na(+) was replaced with an impermeant ion (gluconate or choline, respectively). Under physiological conditions with artificial urine (?Na(+) = 2.12 mM, ?Cl(-) = 3.51 mM) bathing the mucosal surface and saline bathing the serosal surface, transepithelial potential (V(t)) increased to a serosal positive approximately +7.6 mV. Unidirectional influx rates of both Na(+) and Cl(-) were 10-20-fold lower but active absorption of both ions still occurred according to the Ussing flux ratio criterion. Replacement of Na(+) with choline, or Cl(-) with gluconate, in the mucosal artificial urine yielded no change in unidirectional influx of Cl(-) or Na(+), respectively. However, kinetic analyses indicated a decrease in maximum Na(+) transport rate (J(max)) of 66% with no change in affinity (K(m)) in the low Cl(-) mucosal solution relative to the control solution. Similarly, there was a 79% decrease in J(max) values for Cl(-), again with no change in K(m), in the low-Na(+) mucosal bathing. The mucosal addition of DIDS, amiloride or bumetanide (10(-4) M) had no effect on either Na(+) or Cl(-) transport, under either symmetrical saline or artificial urine/saline conditions. Addition of the three drugs simultaneously (10(-4) M), or chlorothiazide (10(-3) M), under symmetrical saline conditions also had no effect on Na(+) or Cl(-) transport rates. Cyanide (10(-3) M) addition to mucosal artificial urine caused a slowly developing decrease of Na(+) influx to 59% and Cl(-) influx to 50% in the period after drug addition. Na(+) and Cl(-) reabsorption appears to be a partially coupled process in the urinary bladder of O. mykiss; transport mechanisms are both dependent upon and independent of the other ion.     

Effect of a myocardial volume overload on lactate transport in skeletal muscle sarcolemmal vesicles

This study sought to determine the effect of a myocardial volume overload (MVO) on sarcolemmal (SL) lactate (La(-)) transport and the aerobic profile of skeletal muscle. SL vesicles were obtained from female rats 10 wk after either a MVO was induced by creation of an infrarenal fistula (n = 10), or sham surgeries were performed (n = 11). Influx of (14)C-labeled L(+)-La(-) was measured at various unlabeled La(-) concentrations under zero-trans conditions. La(-) transport kinetics were determined using a Michaelis-Menten equation with an added linear component to discriminate between carrier-mediated and diffusional transport. Although heart and lung weights were significantly increased (P < 0.0001) in the MVO group, left ventricular function was only modestly altered (P < 0.05). A significant reduction in type I myosin heavy chain (MHC) in the soleus and a strong trend (P = 0.06) for a reduced type IIx MHC in the plantaris were observed in MVO rats, but no differences in citrate synthase activity or monocarboxylate transporter proteins (MCT)-1 expression were noted in any muscle. Carrier-mediated La(-) influx into SL vesicles was similar between sham and MVO (K(m) = 12 +/- 1 and 18 +/- 3 mM; apparent V(max) = 772 +/- 99 and 827 +/- 80 nmol. mg(-1). min(-1), respectively). Total influx at 100 mM was lower in MVO, and this was due to a 30% reduction in membrane diffusion. In conclusion, a 10-wk MVO did not alter MCT-mediated La(-) transport or protein expression but was associated with modest changes in myofibrillar proteins and impaired SL diffusive properties.     

Influx and accumulation of Cs(+) by the akt1 mutant of Arabidopsis thaliana (L.) Heynh. lacking a dominant K(+) transport system.

An extensive literature reports that Cs(+), an environmental contaminant, enters plant cells through K(+) transport systems. Several recently identified plant K(+) transport systems are permeable to Cs(+). Permeation models indicate that most Cs(+) uptake into plant roots under typical soil ionic conditions will be mediated by voltage-insensitive cation (VIC) channels in the plasma membrane and not by the inward rectifying K(+) (KIR) channels implicated in plant K nutrition. Cation fluxes through KIR channels are blocked by Cs(+). This paper tests directly the hypothesis that the dominant KIR channel in plant roots (AKT1) does not contribute significantly to Cs(+) uptake by comparing Cs(+) uptake into wild-type and the akt1 knockout mutant of Arabidopsis thaliana (L.) Heynh. Wild-type and akt1 plants were grown to comparable size and K(+) content on agar containing 10 mM K(+). Both Cs(+) influx to roots of intact plants and Cs(+) accumulation in roots and shoots were identical in wild-type and akt1 plants. These data indicate that AKT1 is unlikely to contribute significantly to Cs(+) uptake by wild-type Arabidopsis from 'single-salt' solutions. The influx of Cs(+) to roots of intact wild-type and akt1 plants was inhibited by 1 mM Ba(2+), Ca(2+) and La(3+), but not by 10 microM Br-cAMP. This pharmacology resembles that of VIC channels and is consistent with the hypothesis that VIC channels mediate most Cs(+) influx under 'single-salt' conditions.     

Effect of 2-chloropropionate on initial lactate uptake by rat skeletal muscle sarcolemmal vesicles

Granier, P., H. Dubouchaud, N. Eydoux, J. Mercier, and C. Préfaut. Effect of 2-chloropropionate on initial lactate uptake by rat skeletal muscle sarcolemmal vesicles. J. Appl. Physiol. 81(5): 1973-1977, 1996.2-Chloropropionate (2-CP) is a halogenated monocarboxylic acidgenerally used to decrease blood lactate concentration in variousmetabolic states. To investigate whether it has an inhibitory effect onsarcolemmal lactate transport, we compared the initial rate of lactatetransport in sarcolemmal membrane vesicles purified from 20 male Wistarrats with and without 2-CP. Transport by these vesicles was measured asuptake ofL-(+)-[U-¹⁴C]lactateunder pH gradient-stimulated cisinhibition. The time courses of 1 mML-(+)-lactate uptake intovesicles both with and without 10 mM 2-CP(L- orD-) displayed saturationkinetics. Lactate uptake values were lower with 10 mML-2-CP and 10 mMD-2-CP in comparison to thecontrol values. Both 10 mML-2-CP and 10 mM D-2-CP significantly inhibited 1 mM L-(+)-lactate uptake (55.8 ± 9.1 and 53.5 ± 12.1%, respectively;P < 0.001), whereas a smaller inhibition was observed with a higher lactate concentration of 50 mM(40.2 ± 11.2 and 38.7 ± 12.4%;P < 0.001 andP < 0.05, respectively). However, ahigher D-2-CP concentration (50 mM) increased the inhibition of pH-stimulated 1 mML-(+)-lactate uptake (77.0 ± 9.4%; P < 0.001). D-2-CP had atrans-stimulation effect on theinitial rate of lactate efflux of 1 mML-(+)-lactate compared withbaseline efflux (9.5 ± 0.8 vs. 5.1 ± 0.4 nmol ^· min^{1 ·} mgprotein¹;P < 0.05). 2-CPsignificantly inhibited the initial rate of lactate uptake in skeletalmuscle sarcolemmal membrane vesicles. This result suggests that 2-CP isa nonstereoselective substrate of the lactate musclecarrier that impairs lactate transport.

     

Hepatocyte heterogeneity in glutamate metabolism and bidirectional transport in perfused rat liver

1. The metabolic fate of infused [1-14C]glutamate was studied in perfused rat liver. The 14C label taken up by the liver was recovered to 85 +/- 2% as 14CO2 and [14C]glutamine. Whereas 14CO2 production accounted for about 70% of the [1-14C]glutamate taken up under conditions of low endogenous rates of glutamine synthesis, stepwise stimulation of glutamine synthesis by NH4Cl increased 14C incorporation into glutamine at the expense of 14CO2 production. Extrapolation to maximal rates of hepatic glutamine synthesis yielded an about 100% utilization of vascular glutamate taken up by the liver for glutamine synthesis. This was observed in both, antegrade and retrograde perfusions and suggests an almost exclusive uptake of glutamate into perivenous glutamine-synthetase-containing hepatocytes. 2. Glutamate was simultaneously taken up and released from perfused rat liver. At a near-physiological influent glutamate concentration (0.1 mM), the rates of unidirectional glutamate influx and efflux were similar (about 100 and 120 nmol g-1 min-1, respectively). 3. During infusion of [1-14C]oxoglutarate (50 microM), addition of glutamate (2 mM) did not affect hepatic uptake of [1-14C]oxoglutarate. However, it increased labeled glutamate release from the liver about 10-fold (from 9 +/- 2 to 86 +/- 20 nmol g-1 min-1; n = 4), whereas 14CO2 production from labeled oxoglutarate decreased by about 40%. This suggests not only different mechanisms of oxoglutarate and glutamate transport across the plasma membrane, but also points to a glutamate/glutamate exchange. 4. Oxoglutarate was recently shown to be taken up almost exclusively by perivenous glutamine-synthetase-containing hepatocytes [Stoll, B & H?ussinger, D. (1989) Eur. J. Biochem. 181, 709-716] and [1-14C]oxoglutarate (9 microM) was used to label selectively the intracellular glutamate pool in this perivenous cell population. The specific radioactivity of this intracellular (perivenous) glutamate pool was assessed by measuring the specific radioactivity of newly synthesized glutamine which is continuously released from these cells into the perfusate. Comparison of the specific radioactivities of glutamine and glutamate released from perivenous cells indicates that about 60% of total glutamate release from the liver is derived from the perivenous glutamine-synthetase-containing cell population. Following addition of unlabeled glutamate (0.1 mM), unidirectional glutamate efflux from perivenous cells increased from about 30 to 80 nmol g-1 min-1, whereas glutamate efflux from non-perivenous (presumably periportal) hepatocytes remained largely unaltered (i.e. 20-30 nmol g-1 min-1). 5. It is concluded that, in the intact liver, vascular glutamate is almost exclusively taken up by the small perivenous hepatocyte population containing glutamine synthetase.     

Sodium transport in erythrocytes of the mollusc Anadara broughtonii: Evidence for inhibition by quinine

Sodium transport through the molluscan erythrocyte membrane was examined using ²²Na as a tracer. Incubation of the red cells in standard saline resulted in a rapid ²²Na uptake reaching steady state concentration (about 21.5 mmol/l cells) in the first 60 min. A similar pattern in the time course of ²²Na uptake was seen in the erythrocytes incubated in mantle fluid. The average value of unidirectional Na⁺ influx, measured as a 5-min ²²Na uptake, was 7.76 ± 0.36 mmol/1 cells/5 min or 93 ± 4.3 mmol/1 cells/hr. The initial rate of Na⁺ influx increased in a saturable fashion as a function of external Na⁺ concentration with apparent AT., of 380±12mM and V_max of 14.3 ± 2.4 mmol/1 cells/5 min. Amiloride (1 mM), furosemide (1 mM), and DIDS (0.1 mM) had no effect on either initial Na⁺ influx (5 min ²²Na uptake) or equilibrium Na⁺ concentration (60 min and 120min ²²Na uptake) in the molluscan red cells exposed to standard saline. Quinine (1 mM) caused a significant fall in the initial Na⁺ influx (by 48%) and in 60-min ²²Na uptake (by 32%) as compared with control levels. In the presence of 0.1 mM ouabain, ²²Na uptake into the red cells was enhanced by an average 27% and 44% during 60 min and 120 min of cell incubation, respectively. The ouabain-sensitive Na⁺ accumulation in the red cells reflected a contribution of the Na, K-pump to Na⁺ transport and the mean value was 5.6 ± 1.0 mmol/1 cells/hr.     

Injections of recombinant human erythropoietin increases lactate influx into erythrocytes.

Previous studies showed that erythropoietin not only increases erythrocyte production but is also essential in both the synthesis and the good functioning of several erythrocyte membrane proteins, including band 3. It is still unknown whether anion and/or H(+) fluxes are modified by erythropoietin. This study aimed to evaluate the effect of recombinant human erythropoietin (rHuEPO) injections on lactate transport into erythrocytes via band 3 and H(+)-monocarboxylate transporter MCT-1, two proteins involved in lactate exchange. Nine athletes received subcutaneous rHuEPO (50 U/kg body mass 3 times a week for 4 wk), and seven athletes received a saline solution (placebo group). All subjects were also supplemented with oral iron and vitamins B(9) and B(12). Lactate transport into erythrocytes was studied before and after the rHuEPO treatment at different lactate concentrations (1.6, 8.1, 41, and 81.1 mM). After treatment, MCT-1 lactate uptake was increased at 1.6, 41 (P < 0.01), and 81.1 mM lactate concentration (P < 0.001) although lactate uptake via band 3 and nonionic diffusion were unchanged. MCT-1 maximal velocity increased in the rHuEPO group (P < 0.05), reaching higher values than in the placebo group (P < 0.05) after treatment. Our results show that rHuEPO injections increased MCT-1 lactate influx at low and high lactate concentrations. The increase in MCT-1 maximal velocity suggests that rHuEPO may stimulate MCT-1 synthesis during erythrocyte formation in bone marrow.     

Effect of parathyroid hormone and dietary phosphate on phosphate transport in renal outer cortical and outer medullary brush-border membrane vesicles

Two distinctive sodium-dependent phosphate transport systems have been identified in early and late proximal tubules; a high-capacity process located only in outer cortical tissue, and a high affinity present in both outer cortical and outer medullary brush-border membranes (Km 0.1-0.25 mM). A third, sodium-independent, pH gradient-stimulated system (Vmax 4.7 +/- 0.3 nmol.mg-1.min-1, Km 0.15 +/- 0.002 mM) is present in the outer medulla, but absent in outer cortex. Brush-border vesicles were prepared from outer cortical and outer medullary tissue of pigs maintained on low (less than 0.05%), normal (0.4%), or high (4%) phosphate diets. Sodium-dependent phosphate uptake of the high-capacity system decreased (Vmax, 9.4 to 2.2 nmol.mg-1.min-1) from low to high phosphate diet, whereas uptake rates decreased about 50% in the high-affinity system. There were no changes in the respective Km values. The pH gradient-stimulated uptake also decreased (Vmax, 6.9 to 3.0 nmol.mg-1.min-1) with no change in mean Km value (0.15 +/- 0.001 mM) with dietary manipulation. Administration of 1 U parathyroid hormone prior to study resulted in a decrease in sodium-dependent uptake by 40-50% and in pH-dependent uptake (36%) with no change in the respective Km values. In conclusion, the antecedent dietary phosphate intake and parathyroid hormone administration appropriately alters phosphate uptake across the brush-border membrane of all three systems, sodium-dependent and pH gradient-stimulated phosphate transport.