Elevated venous glutamate levels in (pre)catabolic conditions result at least partly from a decreased glutamate transport activity

Abnormally high postabsorptive venous plasma glutamate levels have been reported for several diseases that are associated with a loss of body cell mass including cancer, human/simian immunodeficiency virus infection, and amyotrophic lateral sklerosis. Studies on exchange rates in well-nourished cancer patients now show that high venous plasma glutamate levels may serve as a bona fide indicator for a decreased uptake of glutamate by the peripheral muscle tissue in the postabsorptive period and may be indicative for a precachectic state. High glutamate levels are also moderately correlated with a decreased uptake of glucose and ketone bodies. Relatively high venous glutamate levels have also been found in non-insulin-dependent diabetes mellitus and to some extent also in the cubital vein of normal elderly subjects, i.e., in conditions commonly associated with a decreased glucose tolerance and progressive loss of body cell mass.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus     

Muscle pH regulation: role of training

In most cell types, including resting skeletal muscle fibers, internal pH (pH_i) is kept constant at a relatively alkaline level. The high pH_i is obtained in spite of a chronic acid load resulting from cellular metabolism and passive influx of protons driven by electrochemical forces. Regulation of pH_i depends on continuous activity of membrane transport systems that mediate an outflux of H⁺ (or bicarbonate influx), whereby the acid load is counterbalanced. The transporters involved in muscle pH regulation at rest are the Na⁺/H⁺ exchange system as well as the Na⁺-dependent and Na⁺-independent Cl^–bicarbonate transport systems. The Na⁺/H⁺ exchanger seems to be active at resting pH_i levels in skeletal muscle. Therefore, pH homeostasis in skeletal muscle most likely involves an equilibrium between counter-directed H⁺ fluxes. A minor fraction of H⁺ release during intense exercise is mediated by the Na⁺/H⁺ exchanger. The capacity of this system is increased with training and hypoxia in rat skeletal muscle. The dominant acid extruding system associated with intense exercise is the lactate/H⁺ co-transporter. It has been demonstrated that the capacity of the lactate/H⁺ co-transporter of rat skeletal muscle is upregulated with training and chronic electrical stimulation, and that it is reduced upon denervation and hindlimb unweighting. Moreover, athletes can have an elevated lactate/H⁺ co-transport capacity, whereas the thigh muscle of spinal cord-injured individuals has a lower transport capacity than the one of healthy untrained subjects. Thus, it appears that the capacity of the lactate/H⁺ transporter is affected by the level of muscle activity in both rats and humans. In addition, the rate of H⁺ release from muscle may also be influenced by capillarization and local blood flow. Finally the resulting pH displacement during acid accumulation is determined by the cellular buffer capacity, which may also undergo adaptive changes.     

Transport of l-leucine hydroxy analogue and l-lactate in rabbit small-intestinal brush-border membrane vesicles

Substitution of the -amino group of amino acids by hydroxyl groups yields hydroxy analogues (HA), which have been ascribed beneficial effects in nitrogensparing diets for uremic patients. In this study, intestinal uptake of l-leucine HA (l-LeuHA) and l-lactate into rabbit jejunal brush-border membrane vesicles was investigated. An inward-directed H⁺ or Na⁺ gradient stimulated uptake of both labelled substrates in a voltageclamped assay. The H⁺ gradient was the major driving force of uptake as compared with the Na⁺ gradient, and it led to a transient accumulation of both l-LeuHA and l-lactate. The proton ionophore carbonylcyanide p trifluoromethoxyphenylhydrazone (FCCP) reduced the initial H⁺-gradient-driven uptake rates of both substrates, but was without effect on Na⁺-gradient-driven uptakes. The H⁺-gradient-driven l-LeuHA uptake was saturable (apparent K_t = 15.4 mM). -HA of l-leucine, l-isoleucine, l-valine, d-leucine, d-valine or l-lactate inhibited the H⁺-gradient-driven l-LeuHA or l-lactate uptakes whereas free branched-chain amino acids had no effect. Preloading the vesicles with one of the l-or d-HA of branched-chain amino acids or with l-lactate stimulated tracer l-LeuHA and also tracer l-lactate uptakes in the presence of a H⁺ gradient. It is concluded that H⁺-gradient-driven transport of l- and d-stereoisomeric HA of branched-chain amino acids as well as of l-lactate across rabbit intestinal brush-border membranes is mediated by the same carrier. Furthermore, there exists a Na⁺gradient-driven l-lactate transport system in the rabbit intestinal brush-border membrane.     

Lysine transport in lactating rat mammary tissue: evidence for an interaction between cationic and neutral amino acids

The transport of lysine by the lactating rat mammary gland has been examined to determine whether there is an interaction between cationic and neutral amino acids. Lysine uptake was time dependent and unaffected by replacing Na⁺ with choline. In the presence of Na⁺, lysine influx was inhibited by cationic amino acids (arginine, homoarginine, ornithine and lysine) and by a range of neutral amino acids (methionine, glutamine, leucine, phenylalanine, alanine, asparagine, α-aminoisobutyric acid (AIB), 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH), proline and tryptophan). Leucine and glutamine also inhibited lysine influx in the absence of Na⁺ but phenylalanine and proline did not. Lysine efflux from mammary tissue was trans-accelerated by various cationic amino acids (lysine, arginine, homoarginine and ornithine). In addition, leucine and glutamine were capable of trans-stimulating lysine efflux in the presence and absence of Na⁺. It appears that cationic and neutral amino acids stimulated lysine efflux at a single locus.     

Creatine uptake in isolated soleus muscle: kinetics and dependence on sodium,but not on insulin

The increased use of creatine by athletes as a dietary supplement to improve their physical performance assumes that increased serum creatine levels will increase intracellular skeletal muscle creatine. Despite this common assumption, skeletal muscle creatine uptake awaits full characterization. Consequently, we have investigated ¹⁴C-labelled creatine uptake in isolated, incubated rat soleus (type I) muscle preparations at 37 °C. We found that the apparent K_m for creatine uptake was 73 μM and the V_max was 77 nmol h^–1 gww^–1. Creatine uptake was 82% inhibited by 2 m M β-guanidinopropionic acid, the structural analogue of creatine. In addition, a decrease in buffer Na⁺ concentration, from 145 to 25 m M , reduced the rate of ¹⁴C-labelled creatine uptake by 77%, indicating that uptake is largely Na⁺-dependent in soleus muscle. Insulin had no effect on the rate of creatine uptake in vitro. The total creatine content was 34% lower, but the rate of creatine uptake in the presence of 100 μM extracellular creatine was 45% higher, in soleus than in extensor digitorum longus (type II) muscle. However, at 1 m M extracellular creatine, the maximal rate of uptake was not significantly different for the two muscle types, implying that soleus muscle has a lower K_m for creatine uptake. We suggest that intracellular creatine levels may play a role in the regulation of skeletal muscle creatine uptake.     

Na+ and pH dependence of proline and beta-alanine absorption in rat small intestine

Aims: Early characterization of intestinal absorption of imino acids in mammals has demonstrated the existence of a Na⁺‐dependent, Cl^?‐independent transport system in rat small intestine, which is the only carrier for β‐alanine. Based on the substrate selectivity, it was proposed that the Proton Amino Acid Transporter 1 (PAT1) could be the same as this imino acid carrier. The present study characterizes the pH and Na⁺ dependence of proline and β‐alanine uptake in rat small intestine. Methods: Intestinal uptake of radiolabelled l ‐proline or β‐alanine was measured in brush border membrane vesicles and everted intestinal rings, in the presence and absence of Na⁺ and at different pH values. Results: The existence of an inwardly directed H⁺ gradient in the absence of Na⁺ enhanced the initial entry of proline and β‐alanine in brush border membrane vesicles, that reached a transient overshoot with maximal value around 30 s. In the absence of pH gradient, no overshoot was shown. In entire tissue, there was an increase of proline and β‐alanine uptake at acidic pH that was higher in the presence of Na⁺ than in its absence. This ion dependence and pH effect of the amino acids uptake also increased with the incubation period. Substrate inhibition studies confirmed that intestinal proline absorption in rat occurs mainly by system B and PAT1‐like transporter. Conclusions: There is a Na⁺‐independent, H⁺‐dependent transporter of amino acids at the apical membrane of the rat enterocytes.     

Effect of feeding a high protein diet on amino acid uptake into rat intestinal brush border membrane vesicles

Uptake of the neutral amino acidl-leucine into isolated rat intestinal brush border membrane (=BBM) vesicles and into a jejunal mucosa preparation as affected by the protein content of the diet was investigated. Adult rats fed either a high carbohydrate (HC) diet (11% protein) or a high protein (HP) diet (77% protein) for several weeks were used for the experiments.The time course ofl-leucine uptake into BBM vesicles prepared from the small intestine of HC-or HP-rats was studied under conditions of an inwardly directed Na⁺-gradient and under Na⁺-equilibrium conditions. Furthermore, in one series of experiments the Na⁺-equilibrium was replaced by a K⁺-equilibrium. l-leucine uptake under Na⁺-gradient conditions displayed the overshoot phenomenon typically associated with Na⁺-gradient-dependent active transport processes in BBM vesicles and the overshoot in group HP exceeded that in group HC significantly.Under both Na⁺-and K⁺-equilibrium conditionsl-leucine uptake into the BBM-vesicles also was faster in group HP.Finallyl-leucine uptake into jejunal mucosa in group HP exceeded that in group HC, too.The results therefore indicate that Na⁺-dependent and Na⁺-independent transport of neutral amino acids across the intestinal brush border membrane adapts to the dietary protein level.Some of the results were reported in a preliminary form [16]     

Transport of inorganic and organic substances in the renal proximal tubule

Summary The transport through the epithelial cell layer of the renal proximal tubule proceeds in principle by passive paracellular and active transcellular transport. The active transcellular transport is mostly secondary active. This means it proceeds coupled with the flux of Na⁺ ions, where-by the transcellular gradient of sodium, created by the (Na⁺+K⁺)-ATPase, located at the contraluminal cell side, provides the main driving force. Once in the cell the substances leave the other cell side by a Na⁺-independent, but carrier-mediated transport system. Using microperfusion and electrophysiological techniques as well as brush border membrane vesicle preparation the Na⁺-H⁺ countertransport and the Na⁺-cotransport of amino acids, phosphate, sulfate, thiosulfate, bile acids, aliphatic-aromatic monocarboxylic acids (lactate) and dicarboxylic acids was studied. Special emphasis will be given to the bidirectional transport of thiosulfate as well as to the specificity of the monocarboxylic acid and dicarboxylic acid transport system.     

Skeletal muscle Na+/H+ exchange in rats: pH dependency and the effect of training

Skeletal muscle Na⁺/H⁺ exchange was studied using giant sarcolemmal vesicles obtained from rat hind limb muscle. Experiments with either the ²²Na tracer technique or with the Na⁺ sensitive fluorescent probe SBFI were conducted to determine the activity of the Na⁺/H⁺ exchanger, which was quantified from the amiloride or amiloride derivative 5-N-ethyl-N-propylamiloride (EIPA) sensitive Na⁺ influx. At a constant external pH of 7.4 the exchange system was close to half-activation at an internal pH of 7.2. A further activation was observed at lower internal pH values. The activity of the muscle Na⁺/H⁺ exchanger was elevated after 6 weeks of high-intensity treadmill training. In contrast, the activity of the system was unaffected by endurance training. The enhanced initial rate of amiloride-sensitive Na⁺/H⁺ exchange appears to be involved in the elevated in vivo (dynamical) buffer capacity reported for trained rats and human subjects, indicating that adaptive changes in the exchange system are of importance for pH regulation in association with high-intensity exercise.     

Effect of starvation on neutral amino acid transport in isolated small-intestinal cells from guinea pigs

The effects of starvation on neutral amino acid transport were examined in isolated enterocytes. Starvation stimulated L-alanine transport by the Na⁺-dependent system A and the Na⁺-independent system L without producing any changes in either the Na⁺-dependent systems ASC or the passive non-mediated uptake. Starvation produces a twofold increase in V _max of system A without any change in K _t. Starvation produces an increase in V _max of system L of 1.7 times without any change in K _t. Activation of systems A and L by starvation was reversible with subsequent refeeding. The effects of a series of amino acids on systems A and L were evaluated. A different inhibition pattern was found in starved animals as compared to controls. Starvation increases Na⁺-dependent L-alanine uptake and Na⁺-independent cycloleucine uptake by small-intestinal brushborder membrane vesicles. These results suggest that starvation stimulates amino acid transport across the apical plasma membrane of the enterocytes by inducing specific carrier units.     

Identification of sodium-dependent and sodium-independent dicarboxylate transport systems in rat liver basolateral membrane vesicles

The mechanisms involved in the hepatocellular uptake of Krebs-cycle intermediates were investigated in isolated basolateral (sinusoidal and lateral) rat liver plasma membrane (blLPM) vesicles. An inwardly directed Na⁺ gradient markedly stimulated uptake of 2-oxoglutarate and succinate into voltage- and pH-clamped blLPM vesicles. This Na⁺-dependent portion of the dicarboxylate uptake was characterized by (a) saturability with increasing substrate concentrations (K _m= 6.4–10 mM; V _max0.2 nmol min^–1 mg protein^–1), (b) cisinhibition by lithium (10 mM), other Krebs-cycle dicarboxylates (1 mM) and DIDS (4,4-diisothiocyanostilbene-2,2-disulfonic acid; 1 mM) but not by sulphate, monocarboxylates, oxalate, acidic amino acids, bile salts and probenecid, (c) stimulation by an intravesicular negative K⁺-diffusion potential indicating electrogenic [(Na⁺) _n>2-succinate] cotransport, and (d) a pH optimum for transport between 7.0 and 7.5. In the absence of Na⁺, an inside alkaline pH gradient also markedly stimulated 2-oxoglutarate uptake. This pH-gradient-driven 2-oxoglutarate uptake was insensitive to lithium, but could also be inhibited by DIDS and succinate. Furthermore, saturation kinetics demonstrated K _m ( 34 mM) and V _max ( 0.8 nmol min^–1 mg protein^–1) values that were clearly different from those of the Na⁺-dependent uptake system. These results indicate the occurrence of two separate dicarboxylate transport systems along the sinusoidal border of hepatocytes, one being a Na⁺-dicarboxylate symporter and the other representing an anion-exchange system.     

Uptake of galactose by the epithelial syncytium of Schistosoma mansoni

A steady-state compartmental analytical solution to radiolabeled galactose fluxes into and out of S. mansoni using a three compartment model is presented. Experiments included phlorizin inhibition, Na⁺-free incubations and inhibition experiments with glucose and 3-O-methylglucose. The inward rate constants exceeded the outward exchange rates by a factor of 1.55 in females and 1.85 in males. Phlorizin, Na⁺-free conditions and the other hexoses reduced the inward exchange rates such that net secretion of galactose would be favored since the inward to outward exchange rates were less than unity. In the Na⁺-free incubations, the outward exchange rate was increased but not in the other experiments. The steady-state K_t values were increased in Na⁺-free incubations and during inhibitions by glucose and 3-O-methylglucose, but not in response to phlorizin. The size of the exchangeable tissue pool of galactose was decreased in a predictable manner when the exchange rates across the surface epithelial syncytium were reduced by glucose, 3-O-methylglucose, phlorizin and Na⁺-free incubations. The results were generally consistent with previous findings of stereospecific Na⁺-coupled uptake of galactose by S. mansoni.     

The Na+, K+ pump in skeletal muscle: quantification,regulation and functional significance

In skeletal muscle, the Na⁺, K⁺ pump is predominantly situated in the sarcolemma (1000–3500 pumps per μm²). The total concentration can be determined in fresh or frozen biopsies (1–5 mg) using a ³H-ouabain binding assay. The values obtained have been confirmed by measurements of maximum ouabain suppressible Na⁺, K⁺-transport capacity in intact muscles as well as Na⁺, K⁺-ATPase-related enzyme activity in muscle homogenates. In the mature organism, the concentration of Na⁺, K⁺ pumps varies with muscle type and species in the range 150–600 pmol (g wet wt)^-1. In rat and human muscle, the concentration increases markedly with thyroid status. Semi-starvation and untreated diabetes reduce the concentration by 20–48%. K⁺ deficiency leads to a downregulation of up to 75%. Both in animals and in humans, training increases the concentration of Na⁺, K⁺ pumps in muscle, and inactivity leads to a downregulation. High-frequency stimulation elicits up to a 20-fold increase in the net efflux of Na⁺ within 10 s This is the major activation mechanism for the Na⁺, K⁺ pump, utilizing its entire capacity and possibly represents a drive on de novo synthesis of Na⁺, K⁺ pumps. A variety of hormones (insulin, insulin-like growth factor I, adrenaline, noradrenaline, calcitonin gene-related peptide, calcitonin, amylin) increase the rate of active Na⁺, K⁺ transport by 60–120% within a few minutes. This leads to a decrease in intracellular Na⁺ and hyperpolarization. In isolated muscles, where contractility is inhibited by high extracellular K⁺, such agents produce rapid force recovery, which is entirely suppressed by ouabain and closely correlated to the stimulation of K⁺ uptake and the decline in intracellular Na⁺. The observations support the conclusion that the Na⁺, K⁺ pump plays a central role in the acute recovery and maintenance of excitability during contractile activity.     

Activation of the Na-K pump by intracellular Na in rat slow- and fast-twitch muscle

Experiments were performed on isolated rat soleus (slow-twitch) and extensor digitorum longus (EDL) (fast-twitch) muscle of 4-week-old rats. In soleus muscle, electrical simulation at 2 Hz for 5 min increased the ouabain-suppressible ⁸⁶Rb⁺uptake by 138%, without significant changes in intracellular Na⁺content or Na⁺/K⁺ratio. In EDL muscle, the ouabain-suppressible ⁸⁶Rb⁺uptake was stimulated by only 58%, whereas intracellular Na⁺content and Na⁺/K⁺ratio were increased by around 70%. Na⁺-loading of the muscles by exposure to K⁺-free or K⁺-Ca²-Mg²⁺-free buffer stimulated the ouabain-suppressible ⁸⁶Rb⁺uptake in the two muscles to roughly the same extent, but in EDL muscle this was associated with a more than twofold larger increase in Na⁺/K⁺ratio. When the Na⁺influx was increased by exposure to veratridine similar results were obtained. Graded variation in intracellular Na⁺content was achieved by exposure to monensin. In soleus muscle, a 25% increase in intracellular Na⁺/K⁺ratio resulted in a doubling of the ouabain-suppressible ⁸⁶Rb⁺uptake, whereas a doubling of the Na⁺–K⁺transport rate in EDL muscle required a 140% increase in Na⁺/K⁺ratio. The results indicate that in soleus muscle the Na⁺/K⁺pump is much more sensitive to changes in intracellular Na⁺content than in EDL muscle. This might explain the larger activation of the Na⁺–K⁺pump in slow-twitch muscle during electrical stimulation and might be of significance for the activation of the Na⁺-K⁺pump in vivo during work.     

A role for the sodium, potassium adenosine triphosphatase (Na+, K+ ATPase) enzyme in degranulation of rat basophilic leukaemia cells

Background A circulating inhibitor of the sodium, potassium adenosine triphosphatase (Na⁺, K⁺ ATPase) enzyme has been described in allergic subjects. Recent studies have suggested that the Na⁺, K⁺ ATPase enzyme may be involved in the signal transduction pathways of various cell types and that inhibition of its activity can modulate histamine release from basophils and mast cells. Objective The purpose of this study was to determine if modulation of Na⁺, K⁺ ATPase activity alters degranulation in the 2H3 subline of rat basophitic leukaemia cells (RBL-2H3), a mucosal mast cell model bearing high-affinity Fc receptors for IgE. Methods Degranulation was measured by the release of both exogenous serotonin and endogenous histamine. Na⁺, K⁺ ATPase activity was assessed by ouabain-sensitive [⁸⁶rubidium] uptake ([⁸⁶Rb] uptake) and ex situ enzyme activity. Results Ouabain-sensitive [⁸⁶Rb] uptake and degranulation increased in parallel and in a dose–response fashion with increasing Fc receptor cross-linking. Additionally, incubation with ouabain, a known inhibitor of Na⁺, K⁺ ATPase activity, decreased both anti-IgE and calcium ionophore-induced degranulation, but increased spontaneous degranulation, each in a dose-response manner. Moreover, the effect of ouabain on degranulation was reversed by rinsing and mimicked by other known inhibitors of Na⁺, K⁺ ATPase activity. Finally, in the absence of anti-IgE or calcium ionophore, stimulation of ouabain-sensitive [⁸⁶Rb] uptake by the sodium (Na⁺) ionophore monensin was associated with a corresponding dose–response increase in ouabain-sensitive degranulation. These experiments demonstrate that ouabain-sensitive [⁸⁶Rb] uptake increases following IgE receptor cross-linking in RBL-2H3, and that factors which modulate Na⁺, K⁺ ATPase activity in these cells may also regulate degranulation. Conclusion The results of this study suggest an important role for Na⁺, K⁺ ATPase activation in the signal transduction pathway of stimulated RBL-2H3.     

Stimulation of Na+, K+-pump activity in skeletal muscle by methylxanthines: evidence and proposed mechanisms

Evidence is presented to support the hypothesis that submillimolar concentrations of methylxanthines stimulate Na⁺, K⁺-ATPase activity in skeletal muscle. Administration of methylxanthines to skeletal muscle results in plasma membrane hyperpolarization and increased rates of K⁺ uptake and Na⁺ efflux. These effects are both dose- and time-dependent and inhibited by blockers of the Na⁺, K⁺ ATPase. The mechanisms for stimulation of Na⁺, K⁺-ATPase activity and the signal transduction pathways are not known. The methylxanthine concentrations required for stimulation of Na⁺, K⁺-ATPase activity are less than those required to cause a 50% inhibition of phosphodiesterase activity, and therefore increases in cyclic AMP due to inhibition of the enzyme are not involved. Possible mechanisms by which methylxanthines may increase Na⁺, K⁺-ATPase activity include: (1) a role for increased intracellular [Ca²⁺]; (2) Ca²⁺ or adenosine-receptor-mediated increases in intracellular cyclic AMP; and (3) a direct action of methylxanthines on the Na⁺, K⁺ ATPase.     

The regulation of L-proline transport by insulin-like growth factor-I in human osteoblast-like SaOS-2 cells

The effect of insulin-like growth factor-I on amino acid transport was studied by measuring the uptake of tritiated L-proline in the cultured human osteoblast-like SaOS-2 cells. The uptake of L-proline was supported by both transport system A, ASC and Gly and by Na⁺-dependent amino acid transport system A, and by Na⁺-independent system L. The initial rate of total L-proline uptake as a function of concentration showed saturation and obeyed Michaelis-Menten kinetics with Michaelis constant (K _m) and maximum velocity (V _max) values of 1.87 mM and 8.89 nmol⋅(mg protein)⁻¹⋅(3 min)⁻¹, respectively. Na⁺-dependent L-proline uptake was significantly stimulated by insulin-like growth factor-I in a time- and concentration-dependent manner. Kinetic analysis showed that insulin-like growth factor-I enhanced transport activity by increasing the V _max of transport without significant changes in the affinity (K _m) of the carrier for the substrate. The increase in transport activity was significantly reduced by cycloheximide. The stimulated increment above basal L-proline uptake was completely inhibited by α-(methylamino) isobutyric acid, suggesting that only system A was affected by insulin-like growth factor-I. Na⁺-dependent L-proline uptake was also stimulated by insulin-like growth factor-II and insulin-like growth factor-I analogues. The insulin-like growth factor-I-stimulated L-proline uptake was inhibited by one of its binding protein, insulin-like growth factor binding protein-4, in a concentration-dependent manner. Received: 15 January 1996/Accepted 21 February 1996     

Increased insulin-stimulated glucose uptake in athletes: the importance of GLUT4 mRNA,GLUT4 protein and fibre type composition of skeletal muscle

In the present study the expression of GLUT4 and fibre type composition were examined in biopsies from skeletal muscle in seven male athletes and eight male sedentary subjects. Estimated maximal oxygen uptake was increased in the trained group when compared with the sedentary group (74.0 ± 3.9 vs. 42.9±5.1 ml kg^-1 min^-1; P < 0.01). A biopsy of vastus lateralis muscle was taken in the fasting state, 36 h after the last bout of exercise. A second muscle biopsy was obtained following 4 h of a hyperinsulinaemic (2 mU kg^-1 min^-1), euglycaemic clamp. The rate of insulin-stimulated glucose uptake was increased in the trained subjects (17.34±0.53 vs. 13.53±0.79 mg kg^-1 min^-1, P < 0.01). In parallel, the steady state levels of GLUT4 protein and mRNA per DNA were higher in muscle biopsies obtained in the basal state from athletes than in sedentary controls, 21 and 71% respectively (P < 0.05). In the total group of participants, GLUT4 protein per DNA in the basal state and insulin-stimulated glucose uptake rate correlated positively, (r = 0.51, P = 0.05). In the insulin-stimulated state we did not find any significant correlation between GLUT4 protein per DNA and glucose uptake rate (r = 0.13, n.s.). No significant relationships between GLUT4 protein abundance per DNA and muscle fibre type distribution were observed. A significantly negative correladon was found between type 2B fibre area and insulin-stimulated glucose uptake (r =–0.63, P < 0.05). In conclusion, the abundance of GLUT4 protein and mRNA, respectively, is increased in skeletal muscle from endurance trained subjects compared to sedentary subjects. However, factors other than GLUT4 immunoreactive protein abundance seem to be determinant for the increased insulin-stimulated whole body glucose uptake in endurance trained subjects.     

Alpha-aminoadipic acid blocks the Na+-dependent glutamate transport into acutely isolated Müller glial cells from guinea pig retina

The effect of the glial toxin α-aminoadipic acid (AAA) upon theNa⁺/glutamate cotransporter of acutely isolated guinea pig retinal glial cells was studied using the whole-cell voltage-clamp technique. Glutamate evoked an in ward current in these cells at negative holding potentials dependent on the presence of extracellular Na⁺ and intracellular K⁺. A reversal potential could not be found for the current. L-trans-Pyrrolidine-2.4-dicarboxylic acid (PDC), a blocker of Na⁺-dependent glutamate uptake, diminished the glutamate current also in our cells. Application of L-AAA also generated an inward current at negative holding potentials, without a reversal potential, being suppressed if extracellularNa⁺ or intracellular K⁺ was removed. The glutamate uptake blocker, PDC (200 μM), blocked the L-AAA (1 mM) current. Thus, L-AAA proved to be transported by the Na⁺/glutamate transporter of Müller cells. Hence, glutamate currents were diminished by L-AAA competitively with a K_m of 499 μM at a glutamate concentration of 10 μM. The Na⁺/glutamate uptake was less sensitive to DL- and D-AAA block. It is suggested that the blocking effect of AAA on Na⁺-dependent glutamate uptake into glial cells might be involved in the well known glia toxicity of this compound.