Polar distribution of sodium-dependent and sodium-independent transport system forl-lactate in the plasma membrane of rat enterocytes

The uptake ofl-lactate by rat small intestinal brush-border and basal-lateral plasma membrane vesicles has been studied.l-Lactate uptake by the isolated membrane vesicles is osmotically sensitive and represents predominantly transport into an intravesicular space and not binding to the membranes.The transport ofl-lactate across the brush-border membrane is stimulated by sodium, whereas the transport across the basal-lateral plasma membrane is sodium-independent. In both types of membrane vesiclesl-lactate is transported faster thand-lactate andl-lactate transport is inhibited by -cyano-cinnamic acid.l-Lactate transport across basal-lateral membranes is inhibited byd-lactate and pyruvate and transstimulated byl-lactate and pyruvate.The polar distribution of transport system forl-lactate in the plasma membrane of rat enterocytes—a Na⁺/l-lactate cotransport system in the brush-border membrane and a facilitated diffusion system in the basal-lateral membrane — can explain the fact that in the intact epitheliuml-lactate produced by cell metabolism is preferentially released on the serosal side and could enable the cell to perform vectorial, secondary active transport ofl-lactate from the intestinal lumen to the serosal compartment.     

Transport ofl-lysine by rat renal brush border membrane vesicles

l-³H-lysine uptake into brush border membrane vesicles was measured by a rapid filtration technique. A significant binding ofl-lysine at the vesicle interior was observed. Extrapolating initial linear uptake to zero incubation time did not indicate binding of the amino acid to the external membrane surface.Sodium stimulated thel-lysine uptake specifically. Experiments in the presence of potassium/valinomycin induced diffusion potentials, and experiments with a potential sensitive fluorescent dye documented an electrogenic uptake mechanism forl-lysine only in the presence of sodium. Sodium independent uptake proceeds via an electroneutral pathway. Transstimulation experiments show carrier mediated uptake in the presence and absence of sodium. An outwardly directed proton-gradient stimulatedl-lysine uptake in the presence and absence of sodium.Saturation ofl-lysine uptake was observed in the presence and absence of sodium. In the absence of sodium,l-lysine uptake was inhibited byl-arginine,l-cystine,l-phenylalanine andl-methionine. The sodium dependent uptake was inhibited byl-arginine andl-cysteine; small inhibition byl-phenylalanine was observed. In the presence or absence of sodium,l-lysine uptake was inhibited neither byd-lysine nor byl-glutamic acid.These results document carrier mediated transport ofl-lysine via (a) transport mechanism(s) not obligatory requiring sodium.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - Tris Tris(hydroxymethy)aminomethane - EGTA ethyleneglycol-bis-(-aminoethyl-ether)-N,N-tetraacetic acid - diamide azodicarboxylic acid[bisdimethylamide] - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - MES 2-(N-morpholino) ethanesulfonic acid     

l-Lactate andd-lactate carriers on the fetal and the maternal side of the trophoblast in the isolated guinea pig placenta

The transfer of¹⁴C-labelledd- orl-lactate (test substance) relative to³H-l-glucose (control substance, extracellular marker) into the trophoblast of the isolated guinea pig placenta was determined during an open loop perfusion on both sides. Using a single passage, paired tracer dilution technique, the maximal lactate uptake (U _max) was derived from the venous concentration ratio of lactate tol-glucose.A significant metabolism ofl-lactate was not observed. The lactate uptake, which occurred in all placentas studied, was not significantly different on the fetal and maternal side. Within one placenta thel-lactate uptake was always higher than thed-lactate uptake. The uptake of bothl- andd-lactate could be inhibited by phloretin. The lactate uptake was inversely correlated with the pH of the perfusate fluid within the range from 6.2–8. A first order saturation kinetic (Hofstee-plot) was used to approximate the relationship between thel-lactate uptake and the chemicall-lactate concentration. We conclude that similar lactate carriers exist in the membranes on both the maternal and the fetal side of the trophoblast.Supported by the Deutsche Forschungsgemeinschaft     

Demonstration of sodium-dependent,electrogenic substrate transport in rat small intestinal brush border membrane vesicles by a cyanine dye

The cyanine dye DiS-C₂(5) was tested as an indicator for changes in membrane potential of subfractionated rat jejunal brush border membrane vesicles. The fluorescence of this dye increased with inside positive and decreased with inside negative potentials. The sensitivity to inside negative potentials was greater than to inside positive potentials. The addition ofl-alanine,l-phenylalanine,l-methionine,d-galactose andd-glucose in the presence of sodium provoked a transient fluorescence increase indicating an inside positive membrane potential due to electrogenic, sodium-coupled transport. Besides the sodium-dependence, the dye reflected stereo-specificity and saturability ofd-glucose transport. Whend-glucose loaded vesicles were incubated ind-glucose-free medium, a decrease in fluorescence was observed indicating thatd-glucose efflux is also electrogenic.Abbreviations Dis-C₂(5) 3,3-diethylthiadicarbocyanine iodide - DiS-C₃(5) 3,3-dipropylthiadicarbocyanine iodide - HEPES N-2-hydroxyethylpiperazine-N-ethane sulfonic acid - Tris tris(hydroxymethyl)aminomethane - EGTA ethyleneglycol-bis-(-aminoethyl-ether)-N,N-tetraacetic acid     

The effect of harmaline on intestinal sodium transport and on sodium-dependentd-glucose transport in brush-border membrane vesicles from rabbit jejunum

Harmaline inhibition of sodium uptake and of sodium-dependentd-glucose transport was investigated using brush-border membrane vesicles from frozen rabbit jejunum. Under sodium-gradient conditions, initiald-glucose uptake (20 s) was inhibited by harmaline at concentrations above 0.5 mM, but at lower harmaline concentrationsd-glucose uptake was stimulated by 10–15%. When a similar potassium gradient was used, harmaline had no effect. At concentrations upt to 2 mM, harmaline did not alter the equilibrium uptake ofd-glucose ord-mannitol. After pre-equlibration with sodium (25 mM),d-glucose uptake was inhibited at harmaline concentrations ranging from 0.1 to 2 mM. Sodium (10 mM) uptake was also inhibited by harmaline. Increasing the sodium concentration reduced the inhibitory effect of harmaline on tracer sodium uptake as well as on sodium-dependentd-glucose uptake. Similar to phlorizin, harmaline (1 mM) was able to prevent glucose-induced sodium influx across the brush-border membrane.Sodium uptake into brush-border membrane vesicles seems to be inhibited at lower harmaline concentrations than sodium-dependentd-glucose uptake. At high (2 mM) inhibitor concentrations, however, sodium-dependent glucose uptake is more strongly inhibited than sodium uptake. These results suggest that harmaline inhibits both sodium and sodium-dependent transport across intestinal brush-border membranes by interacting with specific sodium-binding sites.     

Facilitated diffusion of lactic acid in the guinea-pig placenta

In the guinea-pig placenta which was artificially perfused on the fetal side while maternal placental blood flow was controlled, the placental transfer per mean transplacental concentration difference (the transfer coefficient TC) was determined for lactate. TC forl-lactate (TC_LL) was compared to that ford-lactate (TC_DL) and measured for various concentrations ofl-lactate, bicarbonate, pyruvate and CO₂. Applying a closed circuit perfusion technique,l-lactate and proton concentrations on both sides of the placenta were followed during infusion of HCl and sodiuml-lactate into the fetal circulation.It was found that TC_LL is 3 times TC_DL. TC_LL is depressed by increasing concentrations ofl-lactate while TC for Cl-36 is not. TC_LL is also depressed by 50 mmol·l^–1 pyruvate. Concentration changes of glucose do not affect TC_LL. TC_LL rises with the proton concentration, independently of the concomitant changes of the bicarbonate concentration. Transplacentral proton concentration gradients producel-lactate concentration gradients and vice versa.It is concluded that (1) facilitated diffusion ofl-lactate occurs in the placenta and that (2)l-lactate transfer is coupled with proton transfer. Beside the well-known placental transport system for glucose this is the second passive transport system found in a placenta.Supported by the Deutsche Forschungsgemeinschaft (Mo 105/8)Partly presented at the Frühjahrstagung der Deutschen Physiologischen Gesellschaft, 1977     

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.     

Handling of oxalate by the rat kidney

Uptake ofd- andl-glucose, and fructose by purified brush border membrane vesicles isolated from human small intestine was studied using a rapid filtration technique. The uptake ofd-glucose by the vesicles was osmotically sensitive and represented transport into an intravesicular space and not binding to the membranes. Transport of both,d- andl-glucose was inhibited by phlorizin. Uptake ofd-fructose into the brush border vesicles was not stimulated by sodium. In the presence of a sodium gradientd-glucose was taken up 5 times faster thanl-glucose. The amount ofd-glucose transported into the vesicles in the presence of a sodium gradient was transiently higher than the amount ofd-glucose taken up at equilibrium (overshoot).d-Glucose transport was stimulated only by a sodium gradient; other monovalent cations had no effect. In the presence of a sodium gradientd-glucose transport was increased by the lipophilic anion thiocyanate and decreased by the nearly impermeable anion sulfate as compared with uptake ofd-glucose in the presence of a sodium chloride gradient. This indicates an influence of the electrical membrane potential on the sodium coupled non-electrolyte transport.     

Reabsorption of monocarboxylic acids in the proximal tubule of the rat kidney

The transport ofd-lactate across the epithelium of the late proximal convolution was investigated by two methods: 1. by measuring the zero net flux transtubular concentration difference (c _tt,45s) and the permeability (P) ofd-lactate and calculating from both the transtubular active transport rate (J _lac ^act ). 2. By measuring the 3.5 s efflux ofd-lactate from the tubular lumen, while blood was flowing through the capillaries. The 3.5 s efflux comprises two components, one going through the brush border (J _lac ^bb ) and one going the paracellular pathway (J _lac ^paracell =P _lac·c _{lac lumen}). Both,J _lac ^act andJ _lac ^bb ofd-lactate gave the sameK _m 1.9 and 1.7 mmol/l and the same maximal transport rate 3.2 and 2.9 pmol cm^–1 s^–1. TheK _i ofl-lactate tested againstJ _lac ^act andJ _lac ^bb ofd-lactate was also the same: 1.1 and 1.0 mmol/l. These data indicate that under our experimental conditions only the flux through the brush border seems to be rate limiting and thatd-lactate uses the same transport system asl-lactate.When Na⁺ was omitted from the perfusatesJ _lac ^act disappeared completely, whileJ _lac ^bb was reduced by 64%. These data reflect the Na⁺ dependence of thed-lactate transport through the brush border. Variation of intra-and extracellular pH by raisingpCO₂, omitting HCO ₃ ^– from the perfusates or adding acetazolamide had no effect on the transport ofd-lactate when -ketoglutarate was used as fuel. However, when acetate was used as fuel, intracellular acidosis brought the reducedJ _lac ^act back to the values obtained with -ketoglutarate as fuel. It is suggested that this is an effect on a contraluminal transport step.Probenecid (5 mmol/l) and phloretin (0.25 mmol/l) inhibitedJ _lac ^act significantly.J _lac ^bb , however, was only inhibited by probenecid when acetate was used as fuel. These data indicate that both compounds act on thed-lactate exit at the contraluminal cell side, but that probenecid acts in addition at the luminal cell side. SITS (1 mmol/l) augmentedJ _lac ^bb when acetate was used as fuel and is similar to the effect of lowering intracellular pH as described above. The SH reagents mersalyl (1.0 mmol/l) and maleolylglycine (1 mmol/l) did not influenceJ _lac ^bb .     

Sugar transport in isolated rat kidney papillary collecting duct cells

d-Glucose is an important substrate of energy metabolism and osmolyte synthesis in the renal papillary collecting duct. In order to characterize the cellular entry ofd-glucose in this tubular segment, collecting duct cells were isolated from rat kidney papilla and the rate ofd-glucose uptake was measured indirectly by monitoring thed-glucose-dependent O₂ uptake in the presence of the uncoupler CCCP.d-Glucose uptake was found to be sodium-independent and not sensitive to phlorizin even at a concentration of 10^–3 M. Uptake was, however, completely inhibited by 10^–5 M cytochalasin B and 10^–4 M phloretin. The apparentK _i for cytochalasin B was 1.5×10^–6 M and for phloretin 2.0×10^–5 M. Studies on the substrate specificity revealed that at 1 mMd-mannose is taken up and metabolized to the same extent asd-glucose. A 50-fold higher concentration of 2-deoxy-d-glucose and 2-amino-2-deoxy-d-glucose inhibitedd-glucose uptake completely whereas -methyl-d-glucoside,d-allose, andd-galactose were without effect. Under conditions whered-glucose utilization was maximally stimulated an apparentK _m of 1.2 mM and aV _max of 1 mmold-glucose/g protein hour was found ford-glucose uptake.These results indicate that thed-glucose uptake into papillary collecting duct cells is probably mediated by a transport system similar to the one found in basal-lateral membranes of pelarized renal, intestinal, and liver cells as well as in nonpolarized fat cells and erythrocytes.Supported by grant DFG Gr 916/1-1     

Substrate utilization in the isolated perfused cortical thick ascending limb of rabbit nephron

Isolated segments of cortical thick ascending limbs (cTAL) of rabbit kidney were perfused in vitro and the equivalent short circuit current (Isc) was measured. In a first series all substrates were removed on either side. Isc fell rapidly to 50±12% after 3 min and to 27±6% (n=5) after 10 min. This indicates that in cTAL segments Isc is strictly dependent on the presence of substrates. In series two it was tested what substrates can be utilized by the cTAL segment, and from which epithelial side [bath (b) or lumen (l)] the substrates are taken up. From the l-side only butyrate (10 mmol · l^–1) sustained the Isc at 95±2% (n=7). All other tested substrates (10 mmol · l^–1): pyruvate, acetate, -OH-butyrate,d-glucose, andl-lactate lead to a marked decline in Isc. From the b-side several substrates (5–10 mmol · l^–1) sustained the Isc:d-glucose,d-mannose, butyrate, -OH-butyrate, acetoacetate,l-lactate, acetate and pyruvate. Other compounds (1–10 mmol · l^–1): citrate, -ketoglutarate, succinate, glutamate, glutamine, propionate, caprylate and oleate did not sustain Isc. In the third series the mechanism of substrate utilization from the basolateral cell side was studied. It was shown that the Isc is a saturable function of thed-glucose,l-lactate, acetate, pyruvate or -OH-butyrate concentration with apparentK _m's between 0.05–1.0 mmol · l^–1. Several known inhibitors of sugar and of anion transport were tested at the bath side: phlorrhizin was without effect. Phloretin (500 mol · l^–1) inhibited Isc by 96%, yet its effect was not dependent on the presence of substrates on the b-side since inhibition ocurred also if the b-perfusate contained no substrate and Isc was driven by luminal butyrate. Also SITS (5 mmol · l^–1) exerted only a small inhibitory effect which was not specific since it was also observed with luminal butyrate. -Cyano-m-OH-cinnamate (10 mmol · l^–1) inhibited the Isc specifically whenl-lactate was the bath substrate. Probenecid (1 mmol · l^–1) had a similar yet less marked inhibitory effect. Thed-glucose uptake from the b-side was specifically inhibited by cytochalasin B at 5 · 10^–6 mol · l^–1. We conclude that the cTAL segment of the rabbit utilizesd-glucose and/or small anions such as pyruvate orl-lactate or acetate to energize salt reabsorption. The link between substrate availability and salt reabsorption is extremely close in this nephron segment. Substrate uptake occurs from the blood side. Sugar uptake can be inhibited by cytochalasin B andl-lactate uptake by probenecid and -cyano-m-OH-cinnamat. These data suggest that substrate uptake at the basolateral cell side occurs probably via carrier systems.Parts of this study have been presented at the 57th and 58th Tagung Deutsche Physiologische Gesellschaft, 17th Meeting Europ. Soc. Clin. Investigation, and XXIX Int. Congress Physiol. Sciences. This study was supported by Deutsche Forschungsgemeinschaft Gr 480/5-7     

Transport of 3-O-methyl-d-glucose into mammalian pancreatic β-cells

Summary The transport and oxidation of 3-O-methyl-d-(U-¹⁴C)glucose was studied in microdissected pancreatic islets of obese-hyperglycemic mice. There was no significant production of¹⁴CO₂ during incubation for 2 h. A comparison with the uptake of sucrose and mannitol indicated that 3-O-methyl-d-glucose was uniformly distributed across the -cell plasma membrane. Externald-glucose inhibited the entry of 3-O-methyl-d-glucose and caused a significant net loss of 3-O-methyl-d-glucose from islets equilibrated with this compound. The transport of 3-O-methyl-D-glucose was also markedly reduced in the presence of phlorizin or phloretin, whereasd-mannoheptulose ord-glucosamine exerted a slight inhibition. The results support our hypothesis that the transport ofd-glucose into the pancreatic -cells is carriermediated, and indicate that 3-O-methyl-d-glucose is a non-metabolizable substrate for this carrier in the pancreatic islets. Since in contrast tod-glucose 3-O-methyl-d-glucose does not stimulte insulin release from the type of islets used, the secretagoric recognition system ford-glucose is probably not identical with the membrane transport system.     

45Ca2+ uptake by dispersed pancreatic islet cells: Effects ofd-glucose and the calcium probe,chlorotetracycline

Uptake of⁴⁵Ca²⁺ was studied in dispersed pancreatic islet cells from non-inbredob/ob-mice. Like whole islets the dispersed cells responded to 20 mMd-glucose with a markedly increased⁴⁵Ca²⁺-labeling of both the lanthanum-nondisplaceable and the lanthanum-displaceable calcium pools. The pronounced effect ofd-glucose could not be reproduced with 3-O-methyl-d-glucose,l-glucose,d-mannose,l-leucine, ord-leucine; however,⁴⁵Ca²⁺ uptake was greater in the presence ofl-leucine as compared withd-leucine.⁴⁵Ca²⁺ uptake by dispersed cells or whole islets was stimulated severalfold by 100 M or more chlorotetracycline. At the concentration of only 10 M, chlorotetracycline had no effect on whole islets and partially inhibited⁴⁵Ca²⁺ uptake by the dispersed cells. The ability ofd-glucose to stimulate⁴⁵Ca²⁺ uptake by islets or dispersed cells remained in the presence of 10 M chlorotetracyline. Islet cell suspensions apparently represent a valid model for studying how Ca²⁺ interacts with the cells. However, when using chlorotetracycline as fluorescent Ca²⁺ probe, attention must be paid to its potential ionophoric activity. At only 10 M, the drug seems to monitor a peripheral pool of Ca²⁺, some of which may reside in normal transport channels.     

l-Alanine andl-phenylalanine activate Na+ and K+ conductance pathways in the exocrine mouse pancreas

The effects of some amino acids,l-alanine,l-phenylalanine,dl-alanine,d-alanine and -alanine on membrane potential, membrane current, amylase secretion and⁴⁵Ca and⁸⁶Rb fractional efflux in isolated mouse pancreatic segments were investigated. A two micro-electrode voltage clamp technique was applied to study the effects of the amino acids on membrane current. The amino acids evoked dose-dependent (0.05–0.5 mmole) and reversible membrane depolarization and increases in membrane current. The relative potencies of the actions of the amino acids were:l-alanine>dl-alanine>l-phenylalanine>d-alanine>-alanine. A more detailed study of the action ofl-alanine showed that the relationship between thel-alanine-evoked membrane current and membrane potential was virtually linear with reversal of current polarity being observed at a membrane potential of about +30 mV. While thel-alanine-induced increase in membrane conductance was dose-dependent, the reversal potential (E _l-ala) was independent of thel-alanine concentration used. Replacement of the normal Na-rich superfusion fluid by a low Na solution (5 mM) markedly reduced thel-alanine-elicited inward current at the normal resting potential. Thel-alanine-evoked conductance increase was also reduced in low Na solution and the E _l-ala was close to 0 mV. During the exposure of pancreatic segments to Cl free solution (sulphate substitution) E _l-ala was about 12 mV more positive (+43.7±0.8 mV) than during exposure to control solution (+31.5±1.0 mV). Calculations based on the Goldman-Hodgkin-Katz equations taking into account the values of E _l-ala in the different ionic situations, indicate that the relative permeability of the ionic pathway opened up in the presence ofl-alanine isP _Na/P _Cl/P _K=7.5/1.5/1.0 The pathway is therefore Na selective, but with some leak permeability for Cl and K. In the physiological range of membrane potentials (–15 to –40 mV) the current evoked byl-alanine is virtually entirely carried by Na. Measurements of amylase secretion and⁴⁵Ca fractional efflux show that none of the amino acids (all at 10 mM concentrations) had significant effect whereas pentagastrin (Pn; 10^–6 M) elicited marked increases in amylase output and⁴⁵Ca release. Both pentagastrin andl-alanine stimulated the release of⁸⁶Rb from prelabelled tissue whereasdl-alanine,l-phenylalanine,d-alanine and -alanine had little or no effect. The present results indicate that although the amino acids have no effect on either amylase secretion or calcium metabolism, they nevertheless open conductance pathways in the acinar plasma membrane mainly permeable to Na.     

Sodium dependence of the amino acid transport in the proximal convolution of the rat kidney

Summary With the technique of stop flow microperfusion with simultaneous capillary microperfusion the zero net flux transtubular concentration differences (c) of labelled amino acids which are equivalent to their active transport rates were measured. Alll-amino acids tested (phenylalanine, histidine, aminobicycloheptane-carboxylic acid, aminoisobutyric acid; lysine, ornithine, arginine; aspartic acid; proline and glycine) showed a considerable c, i.e. active transport rate. When, however, the ambient sodium was replaced by choline the c values dropped to zero. An analysis of the Na⁺ dependence of the ornithine transport revealed that the sodium-dependence is of the mixed type, i.e. thatK _m decreased andV _max increased with increasing Na⁺ concentration to the same extent.In contrast to other biological systems no mutual interaction between the Na⁺-dependentd-glucose andl-histidine transport could be observed.Incidental to these studies it was observed that the active transport rate ofd-histidine was in the range of 40% of that of thel-isomer while ford-phenylalanine it was only in the range of 10% of the active transport of thel-isomer. Furthermore it was found that thel-aspartic acid transport was already saturated at a luminall-aspartic acid concentration of 0.05 mmol/l while that ofl-phenylalanine was not saturated even at a luminal concentration of 9 mmol/l.     

Contraluminal transport of hexoses in the proximal convolution of the rat kidney in situ

In order to study contraluminal hexose transport, concentration and time-dependent influx of³H-2-deoxy-d-glucose from the interstitium into cortical tubular cells has been measured. The influx curves fit to a two parameter kinetics (K _m 1.3±0.2 mmol/l,J _max 0.67±0.16 pmol/s · cm) plus an additional diffusion term (withP=6·10^–8 cm²/s) and a distribution ratio extracellular to intracellular amount of 2-deoxy-d-glucose of 10.6. Since the extracellular to intracellular free water space as estimated from morphological data was 12, one must conclude that glucose has only free access to 1/3 of the cell water. The intracellularly accessible space was augmented when the tubules were preperfused for 10 s with hypotonic saline. Thereby an increase of the compartment into which diffusion occurs was revealed and a final rupture of this intracellular compartment at 1/4 isotonic solutions was observed. Total replacement of ions in the peritubular perfusate by mannitol did not change 2-deoxy-d-glucose influx, indicating that it is Na⁺-independent. By adding isotonic concentrations of the respective sugars to the capillary perfusate, three degrees of inhibition of 2-deoxy-d-glucose influx could be revealed: strong inhibition byd-glucose, methyl--d-glucoside,d-mannose, 3-O-methyl-d-glucose, 2-deoxy-d-galactose, methyl--d-galactoside and 6-deoxy-d-glucose, moderate inhibition byd-galactose,l-glucose,l-mannose andd-fructose, no or borderline inhibition by methyl -d-glucoside, 2-deoxy-methyl--d-galactoside, 1-thio--d-glucose, 1-thio--d-galactose, 5-thio--d-glucose, myo-inositol and mannitol. The contraluminal 2-deoxy-d-glucose influx was also inhibited by phloretin, chlormerodrin and preperfusion with cytochalasin B. Starvation as well as streptozotocin diabetes has no influence on contraluminal 2-deoxy-d-glucose transport. Thus, in contrast to the luminal hexose transport system the contraluminal system is Na⁺-independent, does not require on OH-group at C-atom 2, acceptsl-glucose and fructose, but not an -methyl group at C-atom 1.     

Evidence for carrier-mediated uptake of sugars at the serosal side of lamb colon mucosa

Sugar uptake through the basolateral membrane into epithelial cells was investigated in lamb colon stripped of serosa and muscle layers. Only the antiluminal surface of the mucosa was exposed to the incubation medium. 2-Deoxy-d-glucose (2-DG) and 3-O-methyl-d-glucose (3-MG) were used as model substrates. Both sugars were taken up by a saturable process. Transport apparently occurred by facilitated diffusion. 2-DG uptake was inhibited byd-glucose and 3-MG, but not byd-galactose and -methyl-d-glucoside and 3-MG uptake was inhibited by 2-DG andd-glucose but not by -methyl-d-glucoside. Thus 2-DG, 3-MG and glucose appear to compete for a common transport mechanism.Carrier-mediated uptake of glucose through the basolateral membranes is probably important for the energy supply of colon epithelium.Supported by the Deutsche Forschungsgemeinschaft     

Molecular specificity of the tubular resorption of “acidic” amino acids

Single sections of superficial proximal convolutions of rat kidney were microperfused in vivo and in situ. The perfusion fluids contained radioactively labelledl- ord-aspartate,l-glutamate,l-pyroglutamate, or N-methyl-d-aspartate.l--Carboxyglutamate as well as the other amino acids were added in the unlabelled from. Results.l- andd-Aspartate (0.073 mmol·1^–1) are quickly resorbed at about the same rate.d-Aspartate resorption was blocked byl-aspartate (5 mmol·1^–1) but not by -alanine (5 mmol·1^–1).l-Aspartate resorption was inhibited byl-glutamate (2 mmol·1^–1) but not byd-glutamate,l-asparagine,l-phenylalanine or by succinate (2 mmol·1^–1, each). The fast resorption ofl-glutamate (0.073 mmol·1^–1) was blocked byd-aspartate,l-cysteate (2 mmol·1^–1), but not by 3-mercaptopicolinic acid (0.15 mmol·1^–1),l-glutamine, 2-oxoglutarate, taurine, N-methyl-l-glutamate or kainic acid (2 mmol·1^–1, each).l--Carboxyglutamate (0.66 mmol·1^–1) and N-methyl-d-aspartate (2mol·1^–1) were found to be resorbed only at an extremely small rate.l-pyroglutamate (0.076 mmol·1^–1) resorption was not influenced byl-glutamate (1 mmol·1^–1). Fractional excretion of -carboxyglutamate was 7–25% (l-from) or 45–70% (d-form) at an artificially elevated plasma level of 12mol·1^–1.It is concluded thatl- andd-aspartate,l-glutamate,l-cysteate and, to a much smaller extent,l--carboxyglutamate, are accepted by the tubular resorption mechanism highly specific for acidic amino acids. N-Substitution, the amidation of the - or -carboxyl group, or the removal of the -amino moiety almost completely abolish the ability of such compounds to be resorbed via this carrier; N-methylated or -carboxylated derivatives of acidic amino acids are not resorbed at all from the proximal tubule. The resorption of glutamate, but not of aspartate, is highly stereospecific.Parts of this work were presented at meetings of the German Physiological Society in 1978 [28] and of the Gesellschaft für Nephrologie in 1980 [29] as well as at the VIIIth International Congress of Nephrology in Athens in 1981 [26]with technical assistance of Angelika Ascher and Gertaud Vetter     

Insulin enhances l-dopa renal proximal tubule uptake: a regulatory mechanism impaired in insulin resistance

A stimulatory role for insulin in the uptake of neutral amino acids has been reported for a variety of tissues. Here we examine the effect of insulin on l-dopa uptake by proximal tubule cells (PT cells) isolated from control and fructose-fed rats (FR-rats, 10% w/v fructose solution in tap water), a model of insulin resistance. Insulin (200 U/ml) increased l-dopa uptake into PT cells by about 50% (705±186 vs.1117±140 pmol l-dopa/mg protein per minute) (p<0.05). The higher uptake correlated with a 40% increase in the number of high-affinity l-dopa transport sites (l-dopa 0.2 M) (0.59±0.05 vs. 0.82±0.09 pmol l-dopa/mg protein per minute), without changing their affinity. The effect of insulin was not modified by ouabain (1 mM), nocodazole (1–10 M) or colchicine (50–100 M), whereas it was abolished by cytochalasin D or latrunculin B (both 1 M). This suggests that the process is independent of Na⁺,K⁺-ATPase activity or the microtubule network but that it requires the integrity of the actin cytoskeleton. l-dopa transport by the low-affinity transport sites (l-dopa 5 M) was not affected by insulin, neither was the effect of insulin observed in PT cells isolated from FR-rats. In line with this, FR-rats showed lower renal l-dopa reabsorption as compared to control animals (81±4 vs. 51±9%). Taken together, our results support the involvement of insulin in the multifactorial regulation of renal l-dopa reabsorption.     

The mechanisms of amino acid uptake byBrugia pahangi in vitro

The transcuticular uptake ofl-glycine andl-arginine by adult femaleBrugia pahangi has been shown to involve both a saturable carrier transport system and diffusion. The uptake ofl-glycine was competitive with regard tol-methionine,l-valine, andl-phenylalanine but noncompetitive withl-arginine. It is suggested that at least two distinct loci are involved in the transcuticular movement of amino acids in this filarial nematode.