Acid Transport Mechanisms Research Articles

On the Mechanism of Long Chain Fatty Acid Transport in Cardiomyocytes as Facilitated by Cytoplasmic Fatty Acid-binding Protein

Fatty acid-binding protein (FABP) is abundantly present in the cytoplasm of the cardiomyocyte, i.e. the cell which causes the contractile activity of the heart. Although FABP is thought to act as an intracellular long chain fatty acid (FA) carrier, definite experimental proof on this putative function has yet to be obtained. In the present study, experimental results from several authors were combined in an attempt to elucidate the precise physiological function of heart-type FABP in cardiac FA transport. It was calculated that, under normal conditions, the major part of FA in the cardiomyocyte is dissolved in lipid bilayers and that the presence of FABP in the heart enhances the aqueous solubility of FA more than 700-fold despite the fact that only a minor part (<2%) of the total FABP content is then complexed with FA. Moreover, it is shown that, as a result of the enhanced cytoplasmic solubility, the FA flux from sarcolemma (the cellular membrane of the cardiomyocyte) to mitochondria is increased at least 17-fold in the presence of physiological amounts of FABP compared with the hypothetical situation in which FABP is absent. These calculations indicate the involvement of FABP in the transport of FA from the sarcolemma to those mitochondria lying in the innermost region of the cardiomyocyte. The extent to which FABP facilitates FA trafficking through the cytoplasm of the cardiomyocyte under physiological circumstances remains, however, to be established.

How amino acids get into cells: mechanisms, models, menus, and mediators.

The bloodstream provides a readily available pool of amino acids, which can be taken up by all cells of the body to support the myriad of biochemical reactions that are essential for life. The transport of amino acids into the cytoplasm occurs via functionally and biochemically distinct amino acid transport systems that have been defined on the basis of their amino acid selectivities and physico-chemical properties. Each system presumably relates to a discrete putative membrane-bound transporter protein that resides within the cell membrane and functions to translocate the amino acid from the extracellular environment into the cytoplasm. Many of these transporters require sodium for maximal activity. The sodium-dependent model presented is consistent with "preferred random" kinetics, with sodium binding preferentially before the amino acid. The transporter acts as an enzyme that catalyzes the movement of its bound amino acid (and sodium) into the cell. In this review, the authors provide a conceptual view of the mechanism of carrier-mediated amino acid transport as well as an overview of the various models that can be used in the laboratory to study this process. In addition, the known agencies that accomplish transport and their regulation by nutrition, hormones, and other mediators of critical illness are discussed.

Mechanism of arachidonic acid transport across rabbit distal colonic mucosa

The initial rate of [1-14C]arachidonic acid (AA) entry in the serosal side of rabbit distal colonic mucosa mounted in Ussing-type chambers is linear and independent of intracellular metabolism. When the maximal AA uptake was plotted as a function of medium AA concentration in ranges between 50 and 500 nM, saturation of the AA uptake with increasing concentrations was observed. The time course of the uptake of oleic acid and palmitic acid was similar to that observed with AA, and their separate addition to incubation medium strongly reduced the AA uptake. The influx of arachidonate was largely inhibited by ouabain and by incubation with mucosal sodium-free solution and amiloride, while it was increased when colonic mucosa was exposed to luminal amphotericin B. However, voltage-clamp studies showed that the AA entry rate appeared to be linearly related (r = 0.99) to transepithelial potential difference (PD) and suggested that the sodium dependence of AA translocation is an indirect effect of the changes in transepithelial PD induced by sodium transport shifts. These features provide evidence that there is a common entry pathway for AA and other long-chain free fatty acids mediated by a mechanism of facilitated diffusion driven by transmembrane PD.

The Mechanism of Human Placental Urea Transport: A Study Using Placental Brush Border (Microvillous) Membrane Vesicles

To elucidate the mechanism of placental amino acid transport, the transport of amino acids into brush border membrane vesicles was investigated using brush border membrane vesicles (BBMV) separated from the human full-term placenta. 1. The transport of l-alanine and l-glutamine into the brush border membrane vesicles prepared from either early gestational placenta or full-termed placenta disclosed a typical overshooting phenomenon in the presence of H+ concentration gradient (extravesicular pH greater than intravesicular pH). 2. The H(+)-dependent overshooting transport of urea into the brush border membrane vesicles disappeared in the presence of H+ ionophore. 3. The initial velocity of H+ concentration gradient dependent uptake of urea into the brush border membrane vesicles was regulated by the saturation kinetics determined by the concentration of urea. The values of Km and Vmax, calculated as the kinetic parameters of urea transport by reciprocal plotting of the initial velocity of uptake and the concentration of urea, were 10.8 mM and 410 mumol/mg protein/10 sec, respectively. The above results indicate that a transport system which transports urea in exchange for H+ exists in human full-term placental microvillous membrane.

Hepatocellular bile acid transport and ursodeoxycholic acid hypercholeresis.

This review focuses on mechanisms of bile acid transport across the basolateral and canalicular hepatocyte plasma membranes and on ursodeoxycholic acid (UDCA) hypercholeresis and biotransformation. Conjugated trihydroxy bile acids enter hepatocytes via a sodium-coupled mechanism localized to the basolateral membrane, which is saturable, concentrative, inhibited by other bile acids as well as by furosemide and bumetanide, and exhibits developmental changes in rats and probably also in humans. The stoichiometry of sodium-coupled bile acid uptake has been controversial. Hydrophobic, unconjugated dihydroxy and monohydroxy bile acids, including UDCA, enter hepatocytes more rapidly than does taurocholate, and their uptake is largely nonsaturable and sodium independent. A hydroxyl-exchange mechanism that mediates the uptake of cholic acid has also been reported, but its existence is controversial. Current evidence suggests that a 49-kDa protein mediates Na+-dependent taurocholate uptake and that a 54-kDa protein is involved in Na+-independent bile acid uptake. Studies with canalicular membrane vesicles have demonstrated saturable, sodium-independent taurocholate transport, which is sensitive to electrical potential, exhibits trans-stimulation, and appears to be mediated by a 100-kDa canalicular membrane glycoprotein. Studies in mutant rats with conjugated hyperbilirubinemia suggest the presence of a separate canalicular transport mechanism utilized by sulfated bile acids and organic anions such as bilirubin and sulfobromophthalein. UDCA produces in some species a dramatic hypercholeresis that is greater than expected based on the osmotic effect of the secreted bile acid. The hypercholeresis appears attributable to stimulation of biliary bicarbonate output and is decreased or abolished in the perfused rat liver by amiloride or perfusate Na+ substitution. These same maneuvers dramatically alter UDCA biotransformation (unconjugated UDCA disappears from bile, and UDCA glucuronide becomes a major metabolite) and lower hepatocyte intracellular pH. These and other findings indicate that UDCA hypercholeresis is tightly linked to biliary excretion of the unconjugated species and suggest that UDCA biotransformation may be influenced by intracellular pH.

Mechanisms of integumental amino acid transport in marine bivalves

The marine bivalve integument accumulates amino acids from seawater at rates that can significantly supplement other nutritional strategies. Reviewed here are studies that used both intact bivalve gill tissue and membranes isolated from gills to examine the mechanism of integumental transport. The evidence supports a model for secondary active transport in which an inward flux of Na+ down its electrochemical gradient provides the energy to support uptake of amino acids. Indeed, the Na+-cotransport paradigm appears to be a ubiquitous strategy by which animal cells energize uphill transport of organic solutes. What appears to distinguish integumental processes from others are the conditions under which they must routinely work: exposed to ambient concentrations of substrate of less than 1 microM and to cytoplasmic concentrations that exceed 0.1 M, transport of amino acid occurs against gradients that can exceed 10(6) to 1! Somewhat paradoxically, these transporters, which are characterized by an extremely high affinity for amino acid (Kt values of congruent to 5 microM), work so well because they have a low affinity for Na+; low cytoplasmic Na+ concentrations prevent the system from acting as an avenue for efflux, whereas the very high concentrations of Na+ in seawater are adequate to fully activate the influx mode of operation. The combination of these kinetic characteristics results in a class of transporters that makes efficient use of the scarce nutritional resource represented by dissolved organic material in near-shore waters.

Brush-border amino acid transport mechanisms in carnivorous eel intestine.

Brush-border membrane vesicles (BBMV) were prepared from European eel (Anguilla anguilla) intestinal epithelium by a magnesium-ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) precipitation technique. Amino acid transport by these purified vesicle preparations was investigated using either radiolabeled substrates or the voltage-sensitive fluorescent dye 3,3'-diethylthiadicarbocyanine iodide [DiSC2(5)]. All amino acids tested exhibited carrier-mediated, Na+-dependent and Na+-independent transfer processes plus diffusion. The only exceptions were glutamic acid and proline, which displayed Na+ dependency and diffusion but did not appear to be transported by Na+-independent agencies. Carrier-mediated transport kinetic constants (Kapp and Jmax) for several amino acids are reported. Cis-inhibition experiments suggested the presence of at least four distinct Na+-dependent transport systems in eel intestinal BBMV: 1) an anionic transport process for glutamic and aspartic acids; 2) a cationic mechanism for lysine and arginine; 3) a relatively specific neutral amino acid carrier for proline and alpha-(methylamino)isobutyric acid; and 4) a nonspecific neutral amino acid system for most other substrates of this group. This scheme for carnivorous fish intestine most closely approximates that reported for mammalian gut with minor dissimilarities that may relate to metabolic differences or specific dietary requirements of the two vertebrate groups.

Placental Amino Acid Uptake in Normal and Complicated Pregnancies

Amino acids are the essential substrates for fetal growth and catabolism. The fetus is dependent on the placenta for the provision of amino acids, the first step being concentration of amino acids within the syncytiotrophoblast for subsequent transfer to the fetus. A reliable technique for the isolation of human syncytiotrophoblast plasma membrane has been described, and the suitability of this preparation for the study of amino acid uptake and membrane transport has been well documented. Using this technique, the microvillous vesicle uptake of alpha-aminoisobutyric acid (AIB), a nonmetabolizable amino acid, has been determined over multiple time points for normal (NL) pregnancies and those complicated by pregnancy-induced hypertension (PIH), non-insulin-dependent diabetes mellitus (NIDDM) and those delivering small-for-gestational-age (SGA) neonates. There was no significant difference in AIB uptake between NL pregnancies and those complicated by PIH or NIDDM. Compared to each of the above, AIB uptake by the SGA group was significantly less at each time point. These results suggest that normal placental amino acid transport mechanisms may be altered in SGA pregnancies. If so, such alterations may interfere with the normal provision of nutrients to the fetus and ultimately contribute to impaired growth in utero.

Transport of branched-chain amino acids in membrane vesicles of Streptococcus cremoris.

The kinetics, specificity, and mechanism of branched-chain amino acid transport in Streptococcus cremoris were studied in a membrane system of S. cremoris in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (delta p)-generating system. Influx of L-leucine, L-isoleucine, and L-valine can occur via a common transport system which is highly selective for the L-isomers of branched chain amino acids and analogs. The pH dependency of the kinetic constants of delta p-driven L-leucine transport and exchange (counterflow) was determined. The maximal rate of delta p-driven transport of L-leucine (Vmax) increased with increasing internal pH, whereas the affinity constant increased with increasing external pH. The affinity constant for exchange (counterflow) varied in a similar fashion with pH, whereas Vmax was pH independent. Further analysis of the pH dependency of various modes of facilitated diffusion, i.e., efflux, exchange, influx, and counterflow, suggests that H+ and L-leucine binding and release to and from the carrier proceed by an ordered mechanism. A kinetic scheme of the translocation cycle of H+-L-leucine cotransport is suggested.

Uptake and transport of lipid substrates in the heart.

Between 1955-1960 it was realized that the fatty acids circulating in the blood, after transport into the cardiac cells, then beta-oxidation in the myocyte mitochondria, were the major source of energy behind the impressive hydraulic performance of the heart. Only albumin-bound fatty acids and, to a lesser extent, cholesterol ester and triglyceride fatty acids have access to the cardiac cells. Circulating phospholipid fatty acids are excluded. From experiments with isolated perfused hearts it was concluded that fatty acid uptake by the myocardium was essentially an energy independent process. An important question still pending in the literature concerns the mechanisms of fatty acid transport through the capillary endothelium, through the cardiac cell plasma membrane and then through the intracellular compartments. The most plausible model now emerging considers that specific fatty acid-binding proteins, sequentially disposed along this cascade of barriers, might facilitate and drive the flux of fatty acids entering the cardiac cells.

Renal taurine transport--recent developments.

The renal proximal tubule, with its brush border surface is the site of the active accumulation of all amino acids, including taurine (19). Following filtration at the glomeru-lus, taurine resides in the ultrafiltrate of plasma which passes by the apical or brush border surface of the tubule. As in many other biological membranes, the transfer of taurine across the brush border membrane is Na+-dependent (5,20) and requires external Cl− (6). This transport of taurine can best be studied by employing isolated renal brush border membrane vesicles (BBMV) which permit an evaluation of factors which influence the uptake of taurine both in vitro by changing the chemical composition of the incubation medium and in vivo by exposure of animals to various manipulations (4–5). Our group has been interested in establishing how renal amino acid transport mechanisms adapt to changes in the level of taurine or its precursors in the diet. To carry out these studies, a rat model has been used and the accumulation of taurine by BBMV has been measured.

Ascorbic acid transport in mammalian kidney.

Ascorbic acid is known to circulate free in the plasma of several species and is therefore filtered in the kidney; reabsorption subsequently takes place and prevents urinary loss. However, no specific mechanism of renal ascorbic acid transport has previously been presented. In the present study, rat and guinea pig kidney were incubated as slices or as isolated tubules in vitro in the presence of low concentrations of [14C]ascorbic acid. The kidneys of both species handle ascorbic acid similarly. Ascorbic acid accumulates in the renal tissue to a concentration three to four times that present in the bathing media. Recently absorbed ascorbic acid diffuses freely from the kidney and is predominantly nonmetabolized during absorption. Uptake is reduced following replacement of bathing solution sodium by lithium or cesium, or when incubation is performed in the presence of metabolic inhibitors or at low temperatures. The results indicate that ascorbic acid is reabsorbed in the kidney by a sodium-dependent active transport mechanism that operates by concentrating ascorbic acid in the cellular fluid. Renal slices and tubules both appear to transport ascorbic acid and galactose across the brush-border membrane; this indicates that the tubular lumens in these preparations are not collapsed or sealed off.

The complete nucleotide sequences of the Escherichia coli LIV-BP and LS-BP genes. Implications for the mechanism of high-affinity branched-chain amino acid transport.

The Escherichia coli LIV-I and LS amino acid transport systems are high-affinity, periplasmic, binding protein-dependent systems that utilize the leucine-, isoleucine-, valine-binding protein (LIV-BP) and leucine-specific binding protein (LS-BP), respectively. These two binding proteins (BPs) interact with a common set of membrane proteins to transport branched-chain amino acids into the cytoplasm. The two BP genes are encoded in a regulon at minute 76 of the E. coli chromosome that also contains the genes for the common membrane protein components. We report here the nucleotide and deduced amino acid sequences for the LIV-BP and LS-BP genes and their protein products. Both BPs are encoded with a 23-amino acid signal sequence that is removed when the BPs are secreted into the periplasm. We have examined the pattern of amino acid sequence conservation between the two BP molecules and, by comparison of the predicted secondary structures to the 2.0-A crystal structure of the LIV-BP, have found regions of the BPs that may be involved in membrane protein interaction. We also analyzed the translational efficiency of the two BP mRNAs as determined by their nucleotide sequences.

Characteristics of glutamic acid transport by rabbit intestinal brush-border membrane vesicles. Effects of Na +-, K +-and H +-gradients

In the presence of a Na +-gradient (out > in), l-glutamic acid and l-and d-aspartic acids were equally well concentrated inside the vesicles, while no transport above simple diffusion levels was seen by replacement of Na + by K +. Equilibrium uptake values were found inversely proportional to the medium osmolarity, thus demonstrating uptake into an osmotically sensitive intravesicular space. The extrapolation of these lines to infinite medium osmolarity (zero space) showed only a small binding component in acidic amino-acid transport. When the same experiment was performed at saturating substrate concentrations, linear relationships extrapolating through the origin but showing smaller slope values were recorded, thus indicating that the binding component could be more important than suspected above. However, binding to the membrane was neglected in our studies as it was absent from initial rate measurements. Na +-dependent uphill transport of l-glutamic acid was stimulated by K + present on the intravesicular side only but maximal stimulation was recorded under conditions of an outward K +-gradient (in > out). Quantitative and qualitative differences in the K + effect were noted between pH 6.0 and 8.0. Initial uptake rates showed pH dependency in Na +-(out > in) + K +-(in > out) gradient conditions only with a physiological pH optimum between 7.0 and 7.5. It was also found that a pH-gradient (acidic outside) could stimulate both the Na +-gradient and the Na + + K +-gradient-dependent transport of l-glutamic acid. However, pH- or K +-gradient alone were ineffective in stimulating uptake above simple diffusion level. Finally, it was found that increased rates of efflux were always observed with an acidic pH outside, whatever the conditions inside the vesicles. From these results, we propose a channel-type mechanism of l-glutamic acid transport in which Na + and K + effects are modulated by the surrounding pH. The model proposes a carrier with high or low affinity for Na + in the protonated or unprotonated forms, respectively. We also propose that K + binding occurs only to the unprotonated carrier and allows its fast recycling as compared to the free form of the carrier. Such a model would be maximally active and effective in the intestine in the in vivo physiological situations.

Modulation of amino acid transport in preimplantation mouse embryos by low concentrations of non-ionic and zwitterionic detergents

In 4-cell embryos (but not in blastocysts), Triton X-100, a non-ionic detergent, stimulated leucine, phenylalanine, methionine and glutamic acid transport from 1.6 to 3.2-fold. All of these amino acids were transported exclusively by a sodium-independent mechanism. Triton X-100, however, did not stimulate the transport of other amino acids tested in 4-cell embryos. Furthermore, phenylalanine transport rates were stimulated about 2-fold at the 4-cell stage by all of the non-ionic and zwitterionic detergents tested at concentrations which were approximately one-tenth of the critical micellar concentration for each detergent. These concentrations did not block development, disrupt the cells, or make the cell membranes freely permeable. At the blastocyst stage, Z312, a zwitterionic detergent, inhibited the transport of phenylalanine and alanine and stimulated the transport of lysine, a pattern previously found to be linked to the sodium-dependent amino acid transport mechanism. We suggest that Z312 may be acting upon some component of sodium-dependent amino acid transport in blastocysts. The non-ionic and zwitterionic detergents seemed to have a common effect on amino acid transport in 4-cell embryos but elicited varied transport responses from blastocysts. These differential responses to detergents by blastocysts may reflect intrinsic changes in membrane composition and/or organization which occur during the normal course of preimplantation development.

THE UPTAKE AND METABOLISM OF DISSOLVED AMINO ACIDS BY BIVALVE LARVAE

The rates of uptake and metabolism of 14C-labeled glycine and alanine from sea water into larval oysters, Crassostrea gigas (Thunberg) and mussels, Mytilus edulis L. were determined. Kinetic studies showed that both species have a Kt value of 3-4 µM, indicating that bivalve larvae have amino acid transport mechanisms that function efficiently in natural sea water. The Kt values for larvae are similar to those reported for adult bivalves. However, larvae take up dissolved amino acids at approximately ten times the rate reported for adult bivalves on a gram dry weight basis. This difference in uptake capacity presumably reflects the greater absorptive surface area to volume ratio of a larva. Rates of metabolism of absorbed amino acids by larvae were also rapid. Following a 100 min exposure, oyster larvae incorporated 47% of the glycine into protein and 38% was produced as CO2. In comparison to adults, larval bivalves have a more rapid weight-specific uptake and faster rate of utilizing absorbed amino acids....

Cystine transport is defective in isolated leukocyte lysosomes from patients with cystinosis.

The activity of a cystine transport system in lysosomes prepared from the leukocytes of patients with cystinosis was found to be deficient. In normal subjects, this system was resistant to N-ethylmaleimide and demonstrated saturation kinetics. Lysosomes from individuals heterozygous for cystinosis demonstrated a reduced maximum velocity for cystine egress from lysosomes. The rate of cystine escape from normal lysosomes was enhanced by adenosine triphosphate. The availability of normal and mutant lysosomes provides a means of investigating mechanisms of amino acid transport across lysosomal membranes.

Functional membrane vesicles from the nervous system of insects: I. Sodium- and chloride-dependent γ-aminobutyric acid transport

(1) A synaptosomal fraction obtained from locust nervous tissue has been shown to possess an active γ-aminobutyric acid transport mechanism. This activity is preserved and even enriched by the membrane vesicles derived from osmotically shocked synaptosomes. (2) Electron-microscopy examination indicates that the above membrane vesicles are derived predominantly from the neuronal plasma membrane and are devoid of any internal cellular organelles and components. Active transport of γ-aminobutyric acid into these vesicles has been demonstrated with artificially imposed ion gradients as the sole energy source. (3) γ-Aminobutyric acid transport can be driven by an Na + gradient (out>in) and /or by a gradient of Cl − (out>in). This process is absolutely dependent on the simultaneous presence of both types of ion in the external medium. The stimulation of the process by valinomycin indicates that γ-aminobutyric acid transport is an electrogenic process which is stimulated by a membrane potential (interior negative).

A micro-electrode study of oligopeptide absorption by the small intestinal epithelium of Necturus maculosus.

1. The effects of some amino acids and oligopeptides on the electrical properties of the brush-border membrane of the small intestine of the mudpuppy, Necturus maculosus were studied in vitro using micro-electrodes. 2. A number of amino acids (glycine, L-proline and L-leucine) and small peptides (carnosine, glycyl-L-proline, L-leucyl-L-leucine, glycylglycine and glycylglycylglycine) depolarized the brush-border membrane. This was associated with a reduction of input resistance. Tetraglycine did not appreciably reduce the membrane potential. 3. Evidence is presented that the electrical effects caused by application of the peptides are not solely due to the effects of the products of peptide hydrolysis. Furthermore there appears to be more than a single system available to the peptides. 4. Stereospecificity is found for both amino acids and peptides but appears to be more marked for the peptides. 5. Peptide-induced depolarizations are less markedly reduced in the absence of external Na+ than are the depolarizations caused by the amino acids. 6. These results are discussed with respect to the mechanism and significance of amino acid and peptide transport in the intestinal tract.

Mechanisms of Organic Acid and Monosaccharide Transport in the Kidney of the Brush-tailed Possum, Trichosurus vulpecula

The mechanisms of organic acid and monosaccharide transport in the kidney of T. vulpecula were investigated using the renal cortical slice preparation. The kinetics, the sensitivity to inhibitors of metabolism and sodium transport, and the specificity of the concentrative uptake of p-amino-hippurate and alpha-methyl-D-glucoside were examined and compared with published results from studies in eutherian mammals. Some minor differences between metatherians and eutherians were noted with regard to the specificities of the renal slice uptake systems. Penicillin G, a competitor of organic acid transport in eutherian kidneys, did not interact with the marsupial uptake system, and sodium acetate, which stimulates transport in other mammals, inhibited p-aminohippurate uptake in the slice of the possum kidney. 2-Deoxy-D-glucose, which interacts with the phlorizin-sensitive monosaccharide transport system in the dog, rat and rabbit kidney, had no effect on alpha-methyl-D-glucoside uptake in the possum, and the magnitude of the interaction of D-fructose resembled that reported in the dog and rat but was greater than the inhibition reported in the rabbit. D-Glucuronic acid and D-glucuronic acid lactone inhibited alpha-methyl-D-glucoside uptake in the possum but had no effect on uptake in rat renal slices. In consideration of the reported variability of these parameters between different classes of eutherians, it was concluded that the primary mechanisms involved in organic solute transport by the proximal nephron of metatherians and eutherians were not significantly different.