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     

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     

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.     

Competitive inhibition of phlorizin binding byd-glucose and the influence of sodium: a study on isolated brush border membrane of rat kidney

Summary Glucose binding to the luminal cell membrane was studied in the isolated brush border of rat renal cortex by means of inhibition of phlorizin binding to a specific receptor site. This effect was reversible and stereospecific and fulfilled the criteria of fully competitive inhibition. Thus, the kinetic parameters ofd-glucose binding to the same receptor could be derived. The affinity of the receptor to either substrate, phlorizin as well asd-glucose, increased with rising ambient sodium concentration while the number of binding sites remained unchanged. Other cations (K⁺, Ca⁺⁺, Mg⁺⁺) showed no effect on either parameter. The results of this study are in agreement with a model of transmembrane transport of glucose put forward by Crane.Part of these data were presented at the Spring Meeting 1970 of the German Physiological Society in Erlangen [26].     

The effects of exogenous amino acids on the relaxant responses of pig urethral smooth muscle evoked by stimulation of the inhibitory nitrergic nerves

Inhibitory innervation of urethral smooth muscle is mediated partly through release of NO. We investigated the mechanisms involved in the supply of the substrate l-arginine to NO synthase by examining the relaxant response of the muscle to electrical field stimulation (EFS) and the effects of addition of amino acids to the bathing medium. Relaxant responses persisted during hours of repetitive stimulation but were enhanced rapidly by addition of l-arginine (the arginine paradox). Addition of l-lysine (competes with l-arginine for transport on the y⁺ carrier) and l-glutamine (competing on the y⁺L carrier) attenuated the enhancement. Enhancement persisted after washing but was reversed by application of l-lysine, suggesting that exogenous l-arginine fills an intracellular pool and that l-lysine can trans-stimulate its efflux from the pool. After prolonged depolarization in high-K⁺, Na⁺-free solution the relaxant response became purely nitrergic. Addition of l-arginine during the exposure continued to enhance the subsequent responses but l-glutamine added with l-arginine, could no longer reduce this enhancement. The results show the arginine paradox in inhibitory nerves and suggest the involvement of y⁺ and y⁺L carriers in the transport of l-arginine.     

Amino acid-evoked membrane potential and resistance changes in pancreatic acinar cells

The effect of some neutrall-amino acids, alanine, valine and proline, on the pancreatic acinar cell membrane potential and resistance was investigated. Simultaneous recordings were made with two intracellular microelectrodes on isolated superfused segments of mouse pancreas. Amino acids were applied by inclusion at known concentrations in the superfusion fluid or by microionophoresis from extracellular micropipettes. l-Alanine (10 mmol·l^–1) evoked a maximal membrane depolarization of about 18 mV. A just detectable depolarization was observed at 0.1 mmol·l^–1 (3 mV). Halfmaximal depolarization was observed at 1.6 mmol·l^–1.d-Alanine had virtually no effect.Microionophoretic applications ofl-alanine,l-valine orl-proline evoked depolarization and resistance reduction with a very short delay (<50 ms). The dose response curves for depolarization and resistance reduction were similar.The amplitude of the depolarization evoked byl-alanine,l-valine andl-proline depended linearly on the level of the pre-set membrane potential (membrane potential could be changed by direct current injection). With decreasing intracellular negativity there was a decrease in the size of the amino acid-evoked depolarization. When the membrane potential was inside positive the amplitude became very small. Extrapolation of the linear relations between membrane potential and size of depolarization revealed a null potential of +20 to +45 mV.Thel-alanine-evoked depolarization was acutely reduced but not abolished by replacing extracellular Na by Tris or Li. l-Alanine,l-proline andl-valine exhibited mutual inhibition of evoked depolarization even when the depolarizing effect of the first applied amino acid was balanced by direct current injection.It is concluded that severall-amino acids act on the pancreatic acinar plasma membrane by opening conductance pathways mainly permeable to Na.     

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]     

Fictive locomotion in the adult decerebrate rat

In adult immobilised, decerebrate rats, administration of l-3,4-dihydroxyphenylalanine, stimulation of the mesencephalic locomotor centre, or a combination of the two elicited fictive locomotor patterns in hindlimb muscle nerves. The patterns correspond closely to those observed in decerebrate animals that were free to move.     

The stimulus-secretion coupling of amino acid-induced insulin release

l-Glutamine enhances insulin release evoked byl-leucine in isolated rat pancreatic islets. The enhancing action ofl-glutamine, which is a rapid but steadily increasing and not rapidly reversible phenomenon, is not attributable to any major change in either K⁺ or Ca²⁺ outflow from the islet cells. It coincides with an apparent increase in Ca²⁺ inflow rate and, hence, with Ca accumulation in the islets. The initial ionic response tol-leucine is not qualitatively altered by the presence ofl-glutamine. In their combined capacity to stimulate⁴⁵Ca net uptake in the islets,l-glutamine can be replaced byl-asparagine but not byl-glutamate, whereasl-leucine can be replaced byl-norvaline orl-isoleucine, but not byl-valine, glycine orl-lysine. Such a specificity is identical to that characterizing the effect of these various amino acids upon insulin release. It is postulated that the release of insulin evoked by the combination ofl-leucine andl-glutamine involves essentially the same remodelling of ionic fluxes as that evoked by other nutrient secretagogues with, however, an unusual time course for the functional response tol-glutamine.     

Transport of organic cations in brush border membrane vesicles from rabbit kidney cortex

The transport of three organic cations, tetraethylammonium (TEA), morphine and N¹-methylnicotinamide (NMN) was studied in brush border membrane vesicles from rabbit kidney cortex under voltage clamp conditions. A proton gradient (pH_i=6.0, pH_o=7.4) produced a large stimulation of TEA and morphine uptake, yielding a transient overshoot of 190 and 220% respectively, as compared to equilibrium uptake values. No overshoot was observed under pH equilibrium conditions (pH_i=pH_o=7.4, control). These data suggest the presence of a proton-organic cation exchange mechanism in the rabbit renal cortical brush border membrane. Identical experimental conditions (proton gradient) failed however to stimulate significantly NMN transport above control values measured under pH equilibrium conditions. Proton gradient driven TEA transport showed an inhibition of 21% in the presence of NMN (1 mM) and of 63% in the presence of TEA (1 mM), and TEA transport was stimulated by preloading the vesicles with 1 mM TEA (305%) but not with 1 mM NMN (128%). NMN transport showed an inhibition of 39% in the presence of 1 mM TEA and of 27% in the presence of 1 mM NMN and its transport was stimulated by preloading the vesicles with 1 mM TEA (228%) and 1 mM NMN (178%). Our data suggest that TEA, NMN and morphine are transported by a common transport mechanism for which NMN has only a low affinity.     

The Na+-dependent binding of [3H]l-aspartate in thaw-mounted sections of rat forebrain

Binding of [³H]l-aspartate to thaw-mounted coronal sections of frozen rat forebrain was strong in grey regions of telencephalon (neocortex, hippocampus and neostriatum), but it was weaker and unevenly distributed in diencephalon. At low nanomolar concentrations of ligand used in the present studies, [³H]l-aspartate binding was strongly inhibited by l-threo-3-hydroxyaspartate and l-trans-pyrrolidine-2,4-dicarboxylate, compounds known to be substrate/inhibitors of the high affinity uptake of l-glutamate and l-aspartate. None of the typical ligands for the glutamate and aspartate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), N-methyl-d-aspartate and kainate, produced a strong enough inhibition (only CNQX at 100 M weakly inhibited) of the Na⁺-dependent [³H]l-aspartate binding to suggest that [³H]l-aspartate was bound to the receptor binding sites. Furthermore, the binding was absolutely dependent on the presence of Na⁺ in the incubation medium. It is concluded that [³H]l-aspartate is a ligand suitable for autoradiographic studies of the distribution of Na⁺-dependent, high affinity uptake of acidic amino acids in the central nervous system (CNS). However, feasibility of using [³H]l-aspartate as a specific marker of glutamatergic and/or aspartergic synapses in the CNS requires further investigation.     

Fabrication and evaluation of microgrooved polymers as peripheral nerve conduits

Cell alignment plays an important role in the repair of damaged peripheral nerves. The aligned Schwann cells could direct the axonal outgrowth during nerve reconstruction. One way of aligning Schwann cells is to use surface grooves in micrometric dimensions. In this study, microgrooves on chitosan or poly(d,l-lactide) (PLA) were fabricated and the behaviors of Schwann cells and glial cell line C6 on these surfaces were examined. It was found that Schwann cells and C6 cells could be successfully aligned by the microgrooves, and express the genes related to the production of neurotrophic factors. The polymer conduits with microgrooves on the inner surface were implanted in rats to repair the damaged sciatic nerve. The microgrooved conduits were demonstrated to enhance peripheral nerve regeneration as compared to the smooth conduits.     

Mutation and haplotype analyses of the <Emphasis Type="Italic">MUT</Emphasis> gene in Japanese patients with methylmalonic acidemia

Methylmalonic acidemia (MMA) is caused by a deficiency in the activity of l-methylmalonyl-CoA mutase (MCM), a vitamin B12 (or cobalamin, Cbl)-dependent enzyme. Apoenzyme-deficient MMA (mut MMA) results from mutations in the nuclear gene MUT. Most of the MUT mutations are thought to be private or restricted to only a few pedigrees. Our group elucidated the spectrum of mutations of Japanese mut MMA patients by performing mutation and haplotype analyses in 29 patients with mut MMA. A sequence analysis identified mutations in 95% (55/58) of the disease alleles. Five mutations were relatively frequent (p.E117X, c.385 + 5G > A, p.R369H, p.L494X, and p.R727X) and four were novel (p.M1V, c.753_753 + 5delGGTATA, c.1560G > C, and c.2098_2099delAT). Haplotype analysis suggested that all of the frequent mutations, with the exception of p.R369H, were spread by the founder effect. Among 46 Japanese patients investigated in the present and previous studies, 76% (70/92) of the mutations were located in exons 2, 6, 8, and 13. This finding – that a limited number of mutations account for most of the mutations in Japanese mut MMA patients – is in contrast with results of a previous study in Caucasian patients.     

Virtual elimination of the interference of unstirred water layers on intestinal sugar transport kinetics by use of the tissue accumulation method at appropriate shaking rates

The intestinal transport of sugars and amino acids seems to follow Michaelis-Menten kinetics, but the presence of unstirred water layers at the outer face of the brush border membrane may distort kinetic measurements. According to current theory, the capacity parameter,J _mc ^max would not be affected, but theK _t would be increased to a higher value,K _t , in proportion to the thickness of the unstirred water layer,d.We reasoned that by increasing the shaking rate in the tissue accumulation method,d might drop to such small values thatK _t would fall to a constant level practically equal to the trueK _t .We measuredd-galactose influx into rings of everted hamster intestine as a function of both the substrate concentration and the shaking rate. Our results show that as the circular stirring rate increases from 0.38–6.2 Hz,J _mc ^max remains constant, as expected, butK _t first drops, then levels off to reach a plateau between 2 and 6.2 Hz. We conclude that the averageK _t values in this frequency range (K _t =7.4 mM) represent the true transportK _t . Furthermore, all previous kinetic work performed in our laboratory has been carried out under identical conditions, including shaking rates of 4 Hz. The validity of our preceding results is thus upheld.     

Metabolism of NAD by isolated rat renal brush border membranes

NAD and NADH are metabolized to adenosine, ribosyl nicotinamide and P_i by isolated rat renal brush border membranes (BBM). The similar distribution like the BBM marker enzyme alkaline phosphatase indicates that nucleotide pyrophosphatase and 5-nucleotidase are BBM enzymes.     

Renal handling ofl-histidine studied by continuous microperfusion and free flow micropuncture in the rat

Renal tubular reabsorption ofl-histidine (His) was measured in vivo et situ by continuous microperfusion and free flow micropuncture of single proximal convoluted tubules of the rat kidney. The reabsorption is shown to be saturable. A permeability coefficient (P) of <29 m² · s^–1, a maximum reabsorption rate (J _max) of 2.75±1.05>J _max>1.97±0.86 (SEM) nmol · m^–1 · s^–1 and an affinity constant (K _m) of 13.8±4.2>K _m>10.9±4.0 (SEM) mol · l^–1 (lower values forP=29 m² · s^–1, higher values forP=0) were calculated from the microperfusion data. Using these constants and taking backflux of His and water reabsorption into account a good fit with the concentration profile of His along the proximal tubule — measured by free flow micropuncture — was obtained.Varying the buffered pH-values of the perfusion fluids (5.0 or 7.4) influenced neither the active reabsorption nor passive permeability of His. This indicates that the charge of the imidazol group of His does not play a significant role in His reabsorption. Further experiments showed that the addition of 20 mmol · l^–1 l-arginine — a strong inhibitor of the reabsorption system for dibasic ammino acids — did not have a significant effect on the reabsorption ofl-histidine. It is concluded, therefore, that His is reabsorbed by a system for neutral amino acids. Non ionic diffusion does not play an important role for His reabsorption.Part of this work was presented at the 51st meeting of the German Physiological Society in Kiel, 1979 [15]     

Stimulation of Cl/HCO3 exchange in rat duodenal brush border membrane vesicles by cAMP

Brush border membrane vesicles have been used to study the regulation of rat duodenal HCO₃ secretion. When vesciles were loaded with cAMP and ATP SITS-sensitive Cl/HCO₃ exchange (unidirectional³⁶Cl influx in response to an outward-facing OH/HCO₃ gradient) was stimulated by approximately 25%. In contrast, there was no effect of cAMP upon SITS-insensitive³⁶Cl uptake. The stimulation of Cl/HCO₃ exchange caused by cAMP was abolished in the presence of a specific heat-stable cAMP-dependent protein kinase inhibitor. These results suggest that Cl/HCO₃ exchange, and hence HCO₃ secretion, is regulated by cAMP via phosphorylation of some component of the membrane associated with the transport protein.     

Phosphate transport by isolated renal brush border vesicles

Summary A sodium dependent specific transport system for phosphate is present in the brush border microvilli but absent from the basal-lateral plasma membranes. The apparent affinity of this transport system for phosphate is 0.08 mM at 100 mM sodium and pH 7.4. It is inhibited competitively by arsenate with an apparent inhibitor constant of 1.1 mM (100 mM sodium, pH 7.4). Sodium dependent phosphate uptake is two times higher at pH 8 compared to the uptake observed at pH 6. The apparent affinity of the transport system for sodium is also pH-dependent, half-maximal stimulation of uptake is found at pH 6 with 129 mM sodium, at pH 7.4 with 60 mM sodium and at pH 8 with 50 mM sodium. Under all conditions a nonhyperbolic dependence of phosphate uptake on the sodium concentration is observed. The uptake of phosphate by brush border microvilli vesicles shows a typical overshoot phenomenon in the presence of sodium gradient across the membrane {\text{ }}C_{Na_i } )$$ " align="middle" border="0"> . The amount of phosphate taken up after 2 min is about twice the equilibrium value reached after 2 h of incubation. At pH 7.4 the initial rate of uptake is increased only slightly (12%) by inside negative membrane diffusion potentials and inhibited to the same extent by inside positive membrane diffusion potentials.These results indicate that the entry of phosphate across the brush border membrane into the epithelial cell of the proximal tubule is coupled to the entry of sodium. The transfer of phosphate is dependent on its concentration gradient and on the concentration difference of sodium. The data are best explained by the following hypothesis: Both the primary phosphate as well as the secondary phosphate are transported in cotransport with sodium. The divalent form however seems to be transported preferentially. Its transport occurs electroneutral with 2 sodium ions; the monovalent phosphate also enters the cell together with 2 sodium ions but as a positively charged complex.The exit of phosphate across the contraluminal cell border is sodium independent and is favoured by the high intracellular phosphate concentration and the inside negative membrane potential.Part of these data have been presented at the Spring Meeting of the German Physiological Society at Bochum 1975 (Pflügers Arch.355, R 98, 1975).     

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     

Sodium-dependent adenosine transport is diminished in brush border membrane vesicles from hypothyroid rat kidney

 Studies of the uptake of [3H]adenosine ([³H]ADO) were performed using brush border membrane vesicles (BBMV) from normal (N) and hypothyroid (Tx) rat kidneys, to test if decreased Na⁺ reabsorption in hypothyroidism might be associated with abnormalities in ADO transport. [³H]ADO uptake (1–10 μmol) for both conditions was measured in the presence of Na⁺ (10–150 mmol/l); the effects of dipyridamole (10 μmol/l) and 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX, 10 μmol/l) were also studied. Na⁺-stimulated ADO uptake was decreased in Tx BBMV. Michaelis–Menten constants showed a decreased ADO carrier affinity (K _m 2.46 ± 0.14 in N, vs K _m 4.46 ± 0.88 μmol/l in Tx, P<0.05), with no change in the number of carriers (V _max 295 ± 25 in N, vs 229.2 ± 56 pmol·min^–1·mg protein in Tx). Na⁺ affinity remained unchanged (K _Na+ 11.5 ± 3.5 in N, vs K _Na+ 12.72 ± 0.7 mmol/l in Tx). Inhibition of Na⁺-dependent ADO transport was 50% in N as opposed to 58% in Tx with dipyridamole, and 72% in N versus 47% in Tx with PACPX. These results suggest that decreased Na⁺-dependent ADO cotransport contributes to the diminished tubular reabsorption that occurs in hypothyroidism. Received: 17 June 1996 / Received after revision: 9 September 1996 / Accepted: 16 September 1996