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Publisher Summary This chapter discusses the problem of plasma membrane-bound Mg-adenosine triphosphatase (ATPase) in rat kidney and experimentally proves the existence of an anion-sensitive Mg 2+ - ATPase in brush border membranes from... more
Publisher Summary This chapter discusses the problem of plasma membrane-bound Mg-adenosine triphosphatase (ATPase) in rat kidney and experimentally proves the existence of an anion-sensitive Mg 2+ - ATPase in brush border membranes from rat kidney cortex. It is possible to differentiate between this enzyme and a mitochondrial Mg-ATPase from the same tissue using substances such as atractyloside and filipin that interact exclusively with mitochondria or brush border membranes. Parallel studies on Mg-ATPase activity of mitochondrial and brush border fractions reveal that the enzyme in both fractions is stimulated by oxyanions—such as sulfite and bicarbonate—and is inhibited by aurovertin and oligomycin. The mitochondrial ATPase activity shows a higher sensitivity to the stimulators and inhibitors with a difference in the response of Mg-ATPase activity of the two fractions that has been obtained using atractyloside—an inhibitor of the mitochondrial adenine nucleotide translocator—and filipin—an antibiotic interacting with cholesterol in the brush border membrane. The use of filipin might provide a tool to further investigate the relationship between brush border Mg-ATPase and protein translocation in vitro and in vivo .
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Epithelia are characterized by a morphological polarity of their cells. This polarity consists mainly of a different arrangement of the plasma membranes at the two cell poles. In the renal proximal tubule and in the small intestine, the... more
Epithelia are characterized by a morphological polarity of their cells. This polarity consists mainly of a different arrangement of the plasma membranes at the two cell poles. In the renal proximal tubule and in the small intestine, the apical plasma membrane or brush border is composed of numerous microvilli. At the basal pole of the cell, interdigitations of the cell occur to form the basal infoldings, which are more pronounced in the proximal tubule than in the small intestine.
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Research Interests: Biochemistry, Biology, Immunohistochemistry, Medicine, Biological Sciences, and 12 moreGlucose, Kidney, Animals, Protein Sequence Analysis, Xenopus laevis, Amino Acid Sequence, Base Sequence, Molecular Sequence Data, Sequence homology, Tissue distribution, conserved sequence , and Medical and Health Sciences
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Transport properties of brush border microvilli and basal-lateral plasma membranes isolated from rat kidney cortex were studied by a millipore filtration technique. Brush border microvilli but not basal-lateral plasma membranes contain... more
Transport properties of brush border microvilli and basal-lateral plasma membranes isolated from rat kidney cortex were studied by a millipore filtration technique. Brush border microvilli but not basal-lateral plasma membranes contain sodium dependent stereospecific transport system for D-glucose, L-phenylalanine and inorganic phosphate as indicated by saturability, countertransport and inhibition by structurally related compounds. Reduction of equilbrium uptake by increasing medium osmolarity suggests transport into an osmotically reactive space rather than binding to the membranes. Electrogenecity of the sodium-sugar and sodium-amino-acid cotransport system was established by their dependence on artificially imposed diffusion potentials. Also a NA+/H+ antiport system can be demonstrated in microvilli vesicles by demonstrating counterflow of both ions under short circuit conditions. Basal-lateral plasma membranes contain sodium independent stereospecific transport systems for sugars and amino acids. These results demonstrate a marked functional polarity of the cell membranes in respect to sodium dependent and sodium independent transport systems. This polarity in conjunction with the asymmetrical distribution of sodium between the intra- and extracellular space seems to enable the proximal tubule epithelial cells to perform active transepithelial transport.
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The H+ ion secretion in the proximal tubule as revealed by the reabsorption of the glycodiazine buffer vanishes when the ambient solutions are sodium-free. The same holds for other Na+-dependent transport processes such as Ca++,... more
The H+ ion secretion in the proximal tubule as revealed by the reabsorption of the glycodiazine buffer vanishes when the ambient solutions are sodium-free. The same holds for other Na+-dependent transport processes such as Ca++, phosphate, glucose and amino acid reabsorption. If Na+ transport is blocked by ouabain the latter transport processes are abolished, the secretion of H+ ions, however, remains unchanged suggesting H+ to be not exclusively driven by active Na+ transport. These observations agree with electrical measurements which show an electrogenic component of H+ secretion to exist in rat proximal tubule. In experiments with isolated membrane vesicles an electroneutral Na+/H+-exchange mechanism could be demonstrated in the brush border membrane and an ATP-driven Ca++ pumpt as well as Na+-Ca++ countertransport in the baso-lateral cell membrane. These data suggest that both, the Na+ gradient and ATP, are used to drive H+ ion secretion across the luminal brush border and Ca++ reabsorption across the baso-lateral cell side. The biochemical nature of the various systems and their relative importance for the transepithelial ion movement remain to be elucidated.
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Epithelial cells separate phases of different ionic composition and by transepithelial transport maintain or establish the asymmetric distribution of ions. The epithelia of the small intestine and the proximal tubule are additionally... more
Epithelial cells separate phases of different ionic composition and by transepithelial transport maintain or establish the asymmetric distribution of ions. The epithelia of the small intestine and the proximal tubule are additionally specialized in the (re)absorption of a variety of solutes, such as sugars, amino acids, and phosphate. Such vectorial transports can only be performed if a sidedness of the epithelial cell in respect to the transport properties of the luminal and contraluminal membrane exists. Maintenance of asymmetric distribution of solutes involves active transport processes that are energized by cellular metabolism. The coupling of transepithelial transport to energy can be direct or indirect. In primary active transport the movement of a solute across the cell membrane is directly coupled to an energy-transducing chemical reaction, such as the coupling of sodium transport via the Na+-K+-ATPase to ATP hydrolysis. In secondary active transport the movement of the solute across the membrane is coupled to a flux of a primary actively transported substrate.
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Research Interests: Biology, Medicine, DISTRIBUTION, Biological Sciences, FISH, and 15 moreKidney, Animals, Cloning, Membrane, Urinary System, Vesicle, Distribution, Skate, Amino Acid Sequence, Molecular Identification, Molecular Sequence Data, Convoluted tubule, Nucleotide sequence, Tissue distribution, and Medical and Health Sciences
Abstract 1. 1. Transport of l -glutamic acid by rat renal brush border membrane vesicles is sodium-dependent. 2. 2. Present at the inner membrane surface potassium stimulates sodium-dependent L-glutamic acid uptake. 3. 3. Sodium... more
Abstract 1. 1. Transport of l -glutamic acid by rat renal brush border membrane vesicles is sodium-dependent. 2. 2. Present at the inner membrane surface potassium stimulates sodium-dependent L-glutamic acid uptake. 3. 3. Sodium stimulated l -glutamic acid uptake is potential-sensitive only in the presence of potassium. 4. 4. Transport of l -ornithine can also be stimulated by a sodium gradient. 5. 5. However, in the absence of sodium, l -ornithine seems to cross the membrane by facilitated diffusion.
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Research Interests: Chemistry and Endocytosis
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p-Aminohippuric acid (PAH) transport was investigated in brush border vesicles isolated from renal proximal tubules of the winter flounder. Three characteristics of carrier-mediated transport were demonstrated: 1) unlabeled PAH inhibited... more
p-Aminohippuric acid (PAH) transport was investigated in brush border vesicles isolated from renal proximal tubules of the winter flounder. Three characteristics of carrier-mediated transport were demonstrated: 1) unlabeled PAH inhibited the uptake of [3H]PAH; 2)[3H]PAH efflux from the vesicles was stimulated in the presence of unlabeled PAH in the extravesicular medium; and 3) PAH influx was inhibited by 2,4-dinitrophenol (DNP) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene (SITS). D-Glucose plus a sodium gradient stimulated PAH uptake, as did a K2SO4 gradient plus valinomycin, suggesting that PAH is transported as an anion. In contrast, PAH uptake into a membrane fraction containing mainly basal-lateral plasma membranes exhibited a larger inhibition by probenecid but a smaller inhibition by unlabeled PAH and SITS. Thus, carrier-mediated transfer of PAH driven by the electrochemical potential difference for PAH is demonstrated in the brush border membrane of the f...
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Abstract 1. 1. Viable cells were isolated from the thick ascending limb of Henle's loop (TALH) of rabbit renal medulla. 2. 2. The TALH cells were characterized by high activities of calcitonin-stimulated adenylate cyclase and... more
Abstract 1. 1. Viable cells were isolated from the thick ascending limb of Henle's loop (TALH) of rabbit renal medulla. 2. 2. The TALH cells were characterized by high activities of calcitonin-stimulated adenylate cyclase and Na-K-ATPase and low activities of alkaline phosphatase, γ-glutamyl-transpeptidase and ADH-sensitive adenylate cyclase. 3. 3. The oxygen consumption of the TALH cells was inhibitable in a dose dependent manner by furosemide and bumetanide. 4. 4. Ouabain (10 −4 M) also inhibited oxygen consumption as did replacement of either Na or Cl in the incubation medium. 5. 5. Preliminary experiments with plasma membrane vesicles prepared from the TALH cells indicated the presence of a coupled NaCl transport system.
Research Interests: Biochemistry, Chemistry, Kinetics, Biology, Medicine, and 15 moreGlucose, Animals, Alkaline phosphatase, Sodium, Adenylate Kinase, Calcitonin, Rabbits, Furosemide, Oxygen Consumption, Biochemistry and cell biology, Cell Membrane, Bumetanide, In Vitro Techniques, Arginine Vasopressin, and Kidney Medulla
The effect of two parameters regulating renal phosphate excretion, namely parathyroid hormone application and dietary phosphate intake, on the transport properties of isolated rat renal brush border membrane vesicles was investigated. In... more
The effect of two parameters regulating renal phosphate excretion, namely parathyroid hormone application and dietary phosphate intake, on the transport properties of isolated rat renal brush border membrane vesicles was investigated. In the first set of experiments brush border membrane vesicles from young normal rats injected i.m. with 30 USP parathyroid hormone or i.v. with 1 mg dibutyryl cAMP were compared. PTH and dbcAMP injection decreased specifically the Vmax of the sodium-dependent phosphate transport system by appr. 30%. In a second set of experiments rats were kept on phosphate-rich and phosphate-poor diet and after 6-8 weeks the brush border membranes were isolated. The membranes obtained from phosphate-depleted animals showed a markedly (approximately 100%) higher initial sodium-dependent phosphate uptake than membranes isolated from animals kept on phosphate-rich diet. Again only the sodium-dependent phosphate uptake was affected, sodium-independent phosphate permeabil...
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The uptake of L(+)lactate into rat renal cortical brush border (BBV) and basolateral (BLV) membrane vesicles, isolated through differential centrifugation and free flow electrophoresis, were studied using a rapid filtration technique. In... more
The uptake of L(+)lactate into rat renal cortical brush border (BBV) and basolateral (BLV) membrane vesicles, isolated through differential centrifugation and free flow electrophoresis, were studied using a rapid filtration technique. In contrast to the lactate transport into the BBV, that into the BLV: 1) was found to proceed only towards equilibrium, 2) showed Na+ -independent coupling of the influx of L(+)lactate and the efflux of L(+) but not to the efflux of D(-)lactate, 3) was not inhibited by D(-)lactate, 2-thiolactate or 3-phenyl-lactate, but 4) was inhibited by 3-thiolactate and alpha-hydroxybutyrate and 5) was accelerated by changes in inwardly directed ionic gradients or by increases in cation conductance both of which led to increased intravesicular positivity. The latter changes had the opposite effect on the uptake of L(+)lactate by BBV. Thus, while the L(+)lactate transport system present in BBV showed the characteristics of Na-dependent electrogenic cotransport system, that in the BLV was consistent with a carrier mediated Na-dependent, facilitated diffusion system.
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Research Interests: Microbiology, Chemistry, Kinetics, Medical Microbiology, Membrane Biology, and 15 moreMembrane Proteins, Medicine, Animals, Potassium, Ammonia, Sodium, Rabbits, Plasma Membrane, Biochemistry and cell biology, Chlorides, Cell Membrane, Bumetanide, In Vitro Techniques, Ion transporter, and Kidney Medulla
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Studies of luminal and contraluminal membranes isolated from epithelial cells have contributed markedly to our understanding of the molecular mechanisms, the driving forces and the sequence of events involved in transcellular transport of... more
Studies of luminal and contraluminal membranes isolated from epithelial cells have contributed markedly to our understanding of the molecular mechanisms, the driving forces and the sequence of events involved in transcellular transport of solutes (1). They were extended recently to investigate the properties of phosphate transport systems in renal and intestinal brush border and basal-lateral plasma membrane vesicles (2,3). The investigations revealed that both in the renal proximal tubule and in the small intestine the luminal membranes but not the contraluminal membranes contain a sodium-phosphate cotransport system. This suggests that the driving force for the transcellular phosphate movement is derived from the sodium gradient present and maintained across the brush border membrane. The importance of the sodium cotransport system for the active phosphate reabsorption in the proximal tubule is further indicated by the fact that parathyroid hormone (PTH), which decreases the phosphate reabsorption in the renal proximal tubule also reduces the capacity of the sodium-phosphate cotransport system in isolated renal brush border microvilli (4).
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Studies using intact tissue preparations have documented that transepithelial transport of inorganic phosphate is dependent on the presence of sodium1. Physiological studies also revealed that the inorganic phosphate transport rate in the... more
Studies using intact tissue preparations have documented that transepithelial transport of inorganic phosphate is dependent on the presence of sodium1. Physiological studies also revealed that the inorganic phosphate transport rate in the proximal tubule is subject to regulation by various factors such as parathyroid hormone, dietary intake, chronic administration of diphosphonates and chronic application of 1,25 (OH)2 calciferol2–5.
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Research Interests: Microbiology, Chemistry, Medical Microbiology, Membrane Biology, Medicine, and 15 moreGlucose, Glucose Metabolism, Animals, Brush Border, Membrane, Glucose Transport, Rats, Sodium, Glucose Uptake, Plasma Membrane, Biochemistry and cell biology, Cell Membrane, Chick embryo, stereoisomerism, and Kidney cortex
Until recently our knowledge on the renal handling of lactate was based on clearance experiments [e.g., 9,14,17,25]. These studies suggested that filtered lactate is reabsorbed along the nephron and found only in minor quantities in the... more
Until recently our knowledge on the renal handling of lactate was based on clearance experiments [e.g., 9,14,17,25]. These studies suggested that filtered lactate is reabsorbed along the nephron and found only in minor quantities in the final urine. Studies on the renal transport of lactate, or handling of lactate in almost any tissue, are complicated by the fact that lactate is an important metabolic substrate and/or intermediate. Thus, the metabolic conversion and the endogenous production of lactate during transport experiments create experimental and interpretative obstacles. The present knowledge on the renal transport of lactate is based mainly on recent experiments using electrophysiological, biochemical, as well as microperfusion techniques, designed to overcome the problems related to the metabolism of lactate.
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Peroral nanoparticle-mediated drug absorption was studied using a laser scanning confocal microscope. Additional diffusion studies in side-by-side diffusion cells with radiolabelled polybutylcyanoacrylate (PBCA) nanoparticles were carried... more
Peroral nanoparticle-mediated drug absorption was studied using a laser scanning confocal microscope. Additional diffusion studies in side-by-side diffusion cells with radiolabelled polybutylcyanoacrylate (PBCA) nanoparticles were carried out to confirm the results of this study. Fluorescence-labelled PBCA nanoparticles were incubated in vitro in the lumen of freshly excised intestine. Computer-aided optical sectioning of thick samples with dramatically improved resolution and the possibility of rejecting out-of-focus noise enabled tracking of the fluorescence-labelled PBCA nanoparticles in the intestinal tissue after incubation of the particles in freshly excised porcine small intestine. The results of this study suggest that the nanoparticles are absorbed by the surface of the gut wall, creating a high concentration gradient, thereby enhancing the absorption of drugs that may be loaded to the nanoparticles. A significant amount of particles was found in hot (very fluorescent) spots that were assumed to be Peyer's patches. No particles, however, traversed the entire gut wall over a period of 2 to 4 h. These results were confirmed by the diffusion study. No radioactivity permeated through Peyer's-patch-free intestine within 4 h, whereas the amount of radioactivity that was transported through intestine with Peyer's patches during this time was 1.1% of the total amount in the donor chamber.
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Research Interests: Physiology, Biophysics, Chemistry, Confocal Microscopy, Medicine, and 15 moreLiver, Animals, Male, Microelectrodes, Medical Physiology, Confocal Laser Scanning Microscopy, Physiological Stress Markers, Primary Culture, Cell Volume, General Physiology, Ouabain, Cell Membrane, Amiloride, Osmotic Shock, and Ion transporter
In the present work we have investigated whether the changes in the renal handling of inorganic phosphate (Pi) induced by 1) dietary Pi, 2) removal of parathyroid glands and 3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], are associated with... more
In the present work we have investigated whether the changes in the renal handling of inorganic phosphate (Pi) induced by 1) dietary Pi, 2) removal of parathyroid glands and 3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], are associated with alterations in the Na-dependent Pi uptake by brush border membrane vesicles (BBMV) isolated from renal cortex. Sham-operated (SHAM) or thyroparathyroidectomized (TPTX) rats treated or not with 26 pmol/day of 1,25(OH)2D3 i.p. were fed low (0.2%) or high (1.2%) P diet for 7 days. The results showed that in SHAM, TPTX and TPTX+1,25(OH)2D3 the Pi uptake by BBMV was greater after low than high Pi diet. It was greater in TPTX than in SHAM counterparts fed either diets. In TPTX fed low or high Pi diet 1,25(OH)2D3 decreased the Pi uptake to the level observed in SHAM. A striking parallelism was found between variations in Pi uptake by BBMV and in the tubular Pi reabsorption of the whole kidney. The Na-dependent glucose, the mannitol uptake by BBMV, and the alkaline phosphatase activity in cortical homogenates and BBMV were not affected by the various treatments. Thus, dietary Pi, chronic TPTX and 1,25(OH)2D3 appear to specifically affect the Na-dependent Pi transport system bound to the brush border membranes of renal cortical tubules. The alterations observed at this membrane level could account, at least in part, for the changes induced by these factors on the overall tubular reabsorption of Pi.
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Research Interests: Biochemistry, Biophysics, Materials Science, Data Analysis, Imaging, and 15 moreCell Biology, Atomic Force Microscopy, Medicine, Biological Sciences, Cell line, Animals, CHEMICAL SCIENCES, High Resolution, Biological systems, Biomolecule, Cell Membrane, binding sites, Cricetinae, Medical and Health Sciences, and Cell culture techniques
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Free-flow electrophoresis was used to separate microvilli from the lateral basal plasma membrane of the epithelial cells from rat small intestine. The activities of the marker enzyme for the microvillus membrane, ie alkaline phosphatase... more
Free-flow electrophoresis was used to separate microvilli from the lateral basal plasma membrane of the epithelial cells from rat small intestine. The activities of the marker enzyme for the microvillus membrane, ie alkaline phosphatase (EC 3.1.31), was clearly separated from the marker ...
Research Interests: Chemical Engineering, Chemistry, Medicine, Biological Sciences, Glucose, and 15 moreIntestinal Mucosa, Animals, Male, Alkaline phosphatase, Microvillus, Brush Border, Membrane, Glucose Transport, Electrophoresis, Epithelium, Epithelial cells, Biochemistry and cell biology, Brush, Isomerism, and Cell Membrane
Publisher Summary This chapter describes experiments in which alterations in proximal tubular inorganic phosphate (Pi) reabsorption were achieved in vivo by various means and sodium-dependent transport of Pi across the luminal membrane... more
Publisher Summary This chapter describes experiments in which alterations in proximal tubular inorganic phosphate (Pi) reabsorption were achieved in vivo by various means and sodium-dependent transport of Pi across the luminal membrane was analyzed in vitro with isolated membrane vesicles. Transport of Pi across cells consists of the following: (1) uphill influx across the brush border membrane via a sodium–Pi cotransport system driven by the gradients of sodium and Pi across the brush border membrane and (2) efflux of Pi at the contraluminal cell side driven by the electrochemical potential difference for Pi. The administration of parathyroid hormone slowed down the initial uptake phase and also reduced the intravesicular accumulation of Pi; however, this hormone did not provoke an alteration in the uptake of Pi in the absence of sodium. In the different situations tested earlier, a significant change was observed only in the sodium-dependent transport of Pi and not in the sodium-dependent transport of glucose. A close relationship exists between the estimate of the tubular capacity for Pi reabsorption of the whole kidney and the Pi uptake in the brush border vesicles measured under various conditions. Such a correlation strongly suggests that the translocation step across the brush border membrane is of primary importance in the regulation of the transport of Pi along the renal tubule.
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Publisher Summary Sodium cotransport systems are present not only in the brush border membranes but also in the basolateral membranes and play an important role of sodium in almost all transport processes occurring in the renal proximal... more
Publisher Summary Sodium cotransport systems are present not only in the brush border membranes but also in the basolateral membranes and play an important role of sodium in almost all transport processes occurring in the renal proximal tubule. This chapter describes three aspects of sodium cotransport systems—(1) the intracellular localization of sodium cotransport systems, (2) the stoichiometry of sodium cotransport systems with particular emphasis on sodium anion cotransport systems, and (3) studies on the incorporation of sodium cotransport systems into liposomes. Sodium symport systems in the luminal membrane and sodium antiport systems in the contraluminal membrane (Na + /Ca 2+ ) are involved in tubular reabsorption; sodium antiport systems (Na + /H + ) in the luminal membrane and sodium symport systems in the contraluminal membrane catalyze sodium-dependent secretion. The cellular distribution and nature of the sodium cotransport system determine—in the presence of a given sodium gradient across the membranes—the direction of the sodium-dependent solute flow. However, the stoichiometry of sodium cotransport systems can vary depending on the sodium concentration. Thus, sodium-dependent transport of L-glutamate seems to involve—at least partially—the influx of two sodium ions together with one glutamate anion coupled to the efflux of one potassium ion.
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The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration... more
The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up L-phenylalanine by an Na+-dependent, saturable transport system. The apparent affinity of the transport system for L-phenylalanine is 6.1 mM at 100 mM Na+ and for Na+ 13mM at 1 mM L-phenylalanine. Reduction of the Na+ concentration reduces the apparent affinity of the transport system for L-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K+ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of L-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na+ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na+ and on the electrical potential difference. The exit of L-phenylalanine across the basolateral plasma membranes is Na+-independent and probably involves facilitated diffusion.
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Page 1. 0270-9139/82/0205-0572$02.00/0 HEPATOLOGY Copyright 0 19x2 by the American Association for the Study of Liver Diseases Vol. 2, No. 5, pp. 572-579,1982 Printed in USA Taurocholate Transport by Rat Liver Sinusoidal Membrane... more
Page 1. 0270-9139/82/0205-0572$02.00/0 HEPATOLOGY Copyright 0 19x2 by the American Association for the Study of Liver Diseases Vol. 2, No. 5, pp. 572-579,1982 Printed in USA Taurocholate Transport by Rat Liver Sinusoidal Membrane Vesicles: Evidence of ...