Lysine and alanine transport in the perfused guinea-pig placenta

The characteristics of L-lysine transport were investigated at brush-border (maternal) and basal (fetal) sides of the syncytiotrophoblast in the term guinea-pig placenta artificially perfused either through the umbilical vessels in situ or through both circulations simultaneously. Cellular uptake, efflux and transplacental transfer were determined using a single-circulation paired-tracer dilution technique. Unidirectional L-[3H]lysine uptake (%) (perfusate lysine 50 microM) was high on maternal (M = 87 +/- 1) and fetal (F = 73 +/- 2) sides. L-[3H]Lysine efflux back into the ipsilateral circulation was asymmetrical (F/M ratio = 2.3) and transplacental flux occurred in favour of the fetal circulation. Unidirectional lysine influx kinetics (0.05-8.00 mM) gave Km values of 1.75 +/- 0.70 mM and 0.90 +/- 0.25 mM at maternal and fetal sides, respectively; corresponding Vmax values were 1.95 +/- 0.38 and 0.87 +/- 0.10 mumol.min-1.g-1. At both sides, lysine influx (50 microM) could be inhibited (about 60-80%) by 4 mM L-lysine and L-ornithine and less effectively (about 10-40%) by L-citrulline, L-arginine, D-lysine and L-histidine. At the basal side: (i) lysine influx kinetics were greatly modified in the presence of 10 mM L-alanine (Km = 6.25 +/- 3.27 mM; Vmax = 2.62 +/- 0.94 mumol.min-1.g-1), but unchanged by equimolar L-phenylalanine or L-tryptophan; (ii) in the converse experiments, lysine (10 mM) did not affect the kinetic characteristics for either L-alanine or L-phenylalanine; (iii) L-lysine and L-alanine influx kinetics were not dependent on the sodium gradient; (iv) the inhibition of L-[3H]lysine uptake by 4 mM L-homoserine was partially (60%) Na+-dependent. At the maternal side the kinetic characteristics for alanine influx were highly Na+-dependent, while lysine influx was partially Na+-dependent only at low concentrations (0.05-0.5 mM). Bilateral perfusion with 2,4-dinitrophenol (1 mM) reduced L-[3H]lysine uptake into the trophoblast and abolished transplacental transfer. It is suggested that lysine transport in the guinea-pig placenta is mediated by a specific transport system (y+) for cationic amino-acids. The asymmetry in the degree of sodium-dependency at both trophoblast membranes may in part explain the maternal-to-foetal polarity of placental amino-acid transfer in vivo.     

Modulation of lysine transport in cultured rat astrocytes and astrocytoma cells

In the previous paper, it was shown that the transport of lysine into astrocytes and astrocytoma cells obeys the classical enzyme kinetics. Although unmodulated lysine transport into both normal rat astrocytes and rat astrocytoma cells is somewhat slower than needed for observed growth in the culture, it is capable of a large degree of enhancement. Insulin increases the Vmax for lysine influx in astrocytes tenfold and in astrocytoma cells fivefold. Glutathione produces a Vmax enhancement of 80% for astrocytes and 70% for astrocytoma cells. gamma-Glutamyl hydrazide is a weak inhibitor of lysine transport. Diethyl maleate appears to break down the regulation of lysine transport and allows a large increase in lysine influx in both cell types studied. Basic amino acid analogues canaline and S-aminoethylcysteine are not potent inhibitors of lysine transport. Lysine efflux kinetics are slower for C6 cells than for astrocytes; this difference is abolished by diethyl maleate and by dithiothreitol.     

Transport of glucose and fructose in rat hepatocytes at 37 degrees C

The kinetic parameters for transport of the nonmetabolizable glucose analogue 3-O-methyl-D-glucose and the relationship between transport and metabolism of D-glucose and D-fructose were determined in isolated rat hepatocytes at 37 degrees C and pH 7.4. 3-O-Methylglucose at a very low concentration (0.1 mM) equilibrated with the intracellular water with a rate constant of 0.41 s-1. Km for equilibrium exchange entry was 5.5 mM and Vmax was 2.2 mM X s-1 and similar results were obtained when using the zero-trans entry protocol. The rate constant for entry of tracer D-glucose was 0.15 s-1 and Km for glucose was about 20 mM. The phosphorylation rate for D-glucose was much slower than the transport rate. The rate constant for D-fructose entry was about 0.04 s-1, the apparent Km was about 100 mM and Vmax about 5 mM X s-1. The concentration dependence of 3-O-methylglucose inhibition of labelled fructose transport revealed biphasic kinetics indicating that fructose was transferred by both the glucose transporter and a fructose transporter. At concentrations lower than 1 mM, fructose metabolism appeared to be limited by the transport step.     

Evidence for non-uniform distribution of D-glucose within human red cells during net exit and counterflow

The kinetic parameters of net exit of D-glucose from human red blood cells have been measured after the cells were loaded to 18 mM, 75 mM and 120 mM at 2 degrees C and 75 mM and 120 mM at 20 degrees C. Reducing the temperature, or raising the loading concentration raises the apparent Km for net exit. Deoxygenation also reduces the Km for D-glucose exit from red blood cells loaded initially to 120 mM at 20 degrees C from 32.9 +/- 2.3 mM (13) with oxygenated blood to 20.5 +/- 1.3 mM (17) (P less than 0.01). Deoxygenation increases the ratio Vmax/Km from 5.29 +/- 0.26 min-1 (13) for oxygenated blood to 7.13 +/- 0.29 min-1 (17) for deoxygenated blood (P less than 0.001). The counterflow of D-glucose from solutions containing 1 mM 14C-labelled D-glucose was measured at 2 degrees C and 20 degrees C. Reduction in temperature, reduced the maximal level to which labelled D-glucose was accumulated and altered the course of equilibration of the specific activity of intracellular D-glucose from a single exponential to a more complex form. Raising the internal concentration from 18 mM to 90 mM at 2 degrees C also alters the course of equilibration of labelled D-glucose within the cell to a complex form. The apparent asymmetry of the transport system may be estimated from the intracellular concentrations of labelled and unlabelled sugar at the turning point of the counterflow transient. The estimates of asymmetry obtained from this approach indicate that there is no significant asymmetry at 20 degrees C and at 2 degrees C asymmetry is between 3 and 6. This is at least 20-fold less than predicted from the kinetic parameter asymmetries for net exit and entry. None of the above results fit a kinetic scheme in which the asymmetry of the transport system is controlled by intrinsic differences in the kinetic parameters at the inner and outer membrane surface. These results are consistent with a model for sugar transport in which movement between sugar within bound and free intracellular compartments can become the rate-limiting step in controlling net movement into, or out of the cell.     

Determination of kinetic parameters of a carrier-mediated transport in the perfused intestine by two-dimensional laminar flow model: effects of the unstirred water layer

The kinetic parameters of a carrier-mediated transport for D-glucose and for taurocholate were determined from rat in situ intestinal single perfusion experiments. The true parameters were obtained by the two-dimensional laminar flow model, in which the solute concentration at the aqueous-intestinal membrane interface can be calculated numerically without assuming the aqueous diffusion layer, discriminating the effects of the unstirred water layer. The true Michaelis constant was 4.5 mM for D-glucose and 1.5 mM for taurocholate. The true maximal transport velocity was 3.4 nmol/s per cm2 for D-glucose and 0.29 nmol/s per cm2 for taurocholate. The apparent Michaelis constant was raised by the factor of 6.6 for D-glucose and 3.6 for taurocholate due to the effects of the unstirred water layer. The maximal transport velocity was relatively unaffected by the unstirred water layer in both compounds. The values of the effective (operational) thickness of the unstirred water layer were compatible with those reported previously by employing various experimental methods. The kinetic parameters obtained in vitro everted sacs, for comparison, almost coincided with the true ones in situ. Therefore, the two-dimensional laminar flow model is shown to be valid not only for determining the kinetic parameters of a carrier-mediated transport in situ but also for predicting the absorption rate in situ from the uptake rate in vitro.     

Lysine uptake and exchange in Corynebacterium glutamicum.

Resting cells of Corynebacterium glutamicum (ATCC 13032) accumulate [14C]lysine by a transport system with a relatively high affinity (10 microMs) and a low maximum velocity (0.15 nmol/min per mg [dry weight]). Uptake of lysine was not inhibited by uncouplers or by ionophores affecting the ion gradients and the energetic state of the cell. Analysis of intracellular amino acid concentrations during the transport reaction as well as kinetic studies revealed that the observed uptake of lysine in fact represents a homologous antiport between extracellular [14C]lysine and intracellular unlabeled lysine. Intracellular [14C]lysine could only be released by the addition of unlabeled lysine to the bacterial suspension. In contrast to this homologous antiport reaction, we observed net uptake of lysine in lysine-depleted cells of a lysine auxotrophic strain. This net uptake was found to be electrogenic and could also be observed as a heterologous antiport reaction in wild-type cells under particular conditions. In this case exchange was mediated between internal lysine and external alanine, isoleucine, or valine. This antiport was electrogenic, since the substrates differ in charge. The cells can switch between electroneutral homologous exchange and electrogenic heterologous antiport mode during fermentation because of changing metabolic conditions.     

Transport of 2-oxoisocaproate in isolated hepatocytes and liver mitochondria

The transport of 2-oxoisocaproate into isolated hepatocytes and liver mitochondria of rat was studied using [U-14C]2-oxoisocaproate and the silicone oil filtration procedure. 2-Oxoisocaproate uptake by hepatocytes was composed of: rapid adsorption, unmediated diffusion and carrier-mediated transport. The carrier-mediated transport was strongly inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid and p-chloromercuribenzoate, was less sensitive to alpha-cyano-4-hydroxycinnamate and insensitive to p-chloromercuriphenylsulphonate. Other 2-oxo acids: pyruvate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, were also inhibitory. The kinetic parameters of the carrier-mediated transport were Km 30.6 mM and Vmax 23.4 nmol/min per mg wet wt, at 37 degrees C. It is concluded that at its low, physiological, concentration, 2-oxoisocaproate penetrates the hepatocyte membrane mainly by unmediated diffusion. The uptake of 2-oxoisocaproate by isolated liver mitochondria was partly inhibited by alpha-cyano-4-hydroxycinnamate, the inhibitor of mitochondrial monocarboxylate carrier. The remaining uptake was linearly dependent on 2-oxoisocaproate concentration and represented unmediated diffusion. The carrier-mediated transport exhibited the following kinetic parameters: Km 0.47 mM, Vmax 1.0 nmol/min per mg protein at 6 degrees C; and Km 0.075 mM and Vmax about 8 nmol/min per mg protein at 37 degrees C.     

Effects of sugars on leucine and lysine uptake by intestinal cells from rats fed sucrose and stock diets.

The effect of various dietary sugars on the uptake of 1 mM leucine and 1 mM lysine by intestinal cells isolated from stock-fed and sucrose-fed rats was determined. Leucine uptake was activated by 10 mM fructose and inhibited by 10 mM glucose or 20 mM sucrose on both diets. The major dietary effect noted was a significant increase in the inhibition of leucine by glucose in the sucrose-fed rats. The uptake of lysine was minimally affected by the sugars irrespective of the diet fed. These results demonstrate an important dichotomy in the properties of glucose and fructose transport in the intestine and suggest that dietary fructose may increase the transport of certain amino acids.     

Transcellular transport of lipoprotein through arterial endothelial cells in monolayer culture

To study the mechanism of lipoprotein transport through arterial endothelial cells, porcine endothelial cells were cultured on gelated type I collagen supported by a dacron sheet, and the transport of low density lipoprotein (LDL) labeled with rhodamine B isothiocyanate (RB-LDL) through the cells was measured. Light and scanning electron microscopy showed that the cells on the gel were confluent. There was little RB-LDL transport through the endothelial monolayer at 0 degrees C. RB-LDL transport through the monolayer at 37 degrees C was dose-dependent saturable at 0.4 mg protein/ml. The transport was energy-dependent, since its rate was affected by temperature and was inhibited by a combination of 2-deoxyglucose (50 mM) and NaN3 (10 mM). RB-LDL was shown not to be degraded during transport.     

Developmental changes in amino acid transport in the chicken erythrocyte

1. Influx of leucine, lysine and glycine was found to be highest in prehatch (day -1) chicken red blood cells and to diminish during posthatch development when tested at two and four weeks of age. 2. The greatest decline in transport rate during development was seen with leucine; lysine showed a substantial age-related decline only at substrate concentrations greater than Km, the apparent Michaelis constant of transport. 3. Vmax, the maximal transport influx, of each amino acid tested declined during development. 4. Km of glycine and leucine appeared to increase slightly over the test period. 5. In contrast, a 7-fold decrease in Km for lysine transport was seen over the same period. 6. These results are discussed in context of changes in kinetic parameters of amino acid transport during development reported for various animal organs or tissues.     

Reassessment of the translocation hypothesis by kinetic studies on hexose transport in isolated rat adipocytes

The effect of insulin and factors which have insulin-like activity on the kinetic parameters of 3-O-methyl-D-glucose (MeGlc) transport in rat adipocytes were assessed. Carrier-mediated uptake of MeGlc was estimated by the difference in the amounts of [14C]MeGlc and L-[3H]glucose taken up in cells under equilibrium exchange conditions at 37 degrees C. The Km and Vmax values in basal cells were 17.4 mM and 0.24 nmol/10(6) cells/s, respectively. Removal of endogenous adenosine by adenosine deaminase resulted in a 26% decrease in the basal rate due to a slight increase in the Km (19.6 mM) and a decrease in the Vmax value (0.20 nmol/10(6) cells/s). The maximum concentration (10 nM) of insulin decreased the Km to approximately one-half of the basal (7.1 mM) concomitant with an 8.5-fold increase in the Vmax value (2.04 nmol/10(6) cells/s). Submaximal concentrations (50 and 150 pM) of insulin, N6-phenylisopropyladenosine (1 microM), mechanical agitation of cells by centrifugal force (160 x g), low temperature (15 degrees C), 12-O-tetradecanoylphorbol-13-acetate (1 microM), and hydrogen peroxide (10 mM) all decreased the basal Km value to a range of 13.5-7.3 mM, concomitant with a 1.7-7.4-fold increase in the Vmax. A possible explanation for the alterations in the kinetic parameters may be that insulin and other factors cause the translocation of the mobile low-Km glucose transporters from an intracellular site to the cell surface, where the stationary high-Km transporters are located. Thus, when the Km and Vmax values of the hypothetical high-Km transporters were assumed to be 20 mM and 0.20 nmol/10(6) cells/s, respectively, and the Km of the low-Km transporters was assumed to be 7 mM, the theoretical Km decreased from 20 to 7.5 mM as the Vmax of the low-Km transporters increased from near 0 to 2.0 nmol/10(6) cells/s. The relation between empirical Km and Vmax values as affected by several agents and conditions followed closely the relation predicted by the above two-transporter model.     

Adenine transport and binding in cultured mammalian cells deficient in adenine phosphoribosyltransferase.

Rapid kinetic techniques were employed to measure the transport of adenine in adenine phosphoribosyltransferase-deficient L929 and Chinese hamster ovary (CHO) cells in zero-trans entry and exit and equilibrium exchange procedures. The kinetic parameters of transport were computed by fitting appropriate integrated rate equations to time courses of transmembrane equilibration of radiolabeled adenine. Adenine transport conformed to the simple carrier model with directional symmetry and equal mobility of loaded and empty carrier. The Michaelis-Menten constants and maximum velocities for various strains of L929 cells fell between 2.3 and 3.5 mM and 90 and 150 pmol/microliters of cell water per s, respectively, values similar to those previously reported for CHO and Novikoff hepatoma cells. The corresponding values for hypoxanthine transport in L929 cells were 413 microM and 16 pmol/microliters of cell water per s. Adenine transport velocities were directly proportional to adenine concentrations between 0.03 and 50 microM in both CHO and Novikoff cells. The results indicate that adenine is transported in these cells by a single, low-affinity, high-capacity transporter. Adenine transport was inhibited by hypoxanthine in some cell strains, but not in others. Adenine also rapidly bound to L929 cells in a saturable manner (KD = 18 microM), presumably to the cell surface (about 3 X 10(7) sites per cell).     

Glucose transporter in bovine mammary epithelial cells is an asymmetric carrier that exhibits cooperativity and trans-stimulation

Glucose transport kinetics were quantified in isolated bovine mammary epithelial cells using 3-O-methyl-D-glucose. Isolated cells retained satisfactory viability and glucose uptake activity, which was inhibited by cytochalasin B, phloretin, HgCl2, and low temperature. Initial rates of entry were measured over a 15-s interval at 37 degrees C under zero-trans, equilibrium-exchange, high-cis, and high-trans concentrations of 3-O-methyl-D-glucose between 0 and 20 mM. The combined set of rate measurements from all experimental conditions was fit to the fixed-site carrier model by nonlinear regression to estimate parameters of transport. For the regression between predicted and observed initial rates, r2 was 0.97. Forward Vmax was estimated at 18.2 nmol.min-1.mg protein-1, and the Michaelis constant was 8.29 mM. The cooperativity parameter was 1.63, trans-stimulation was 2.13-fold, and asymmetry was 2.06-fold. On the basis of the kinetic parameters, variations in intracellular glucose concentrations are not responsible for the range of glucose uptakes by bovine mammary glands observed in vivo.     

Electrophysiology of L-lysine entry across the brush-border membrane of Necturus intestine

Microelectrode measurements of apical membrane potentials (Va) in absorptive cells of isolated Necturus intestine showed that, in the presence or absence of external Na+, 10 mM lysine added to the mucosal medium caused rapid depolarization followed by slower repolarization of Va. In Na+-free media the effects of 10 mM lysine on Va were abolished by 10 mM leucine which alone had no effect on Va under these conditions. This indicates that uncoupled electrodiffusion of lysine plays little or no role in lysine entry across the brush-border membrane. When external Na+ was greater than 10 mM the maximum depolarization of Va (delta Va') induced by [Lys] ranging from 5 to 30 mM was a simple saturable function of [Lys]. In Na+-free media, the relationship between delta Va' and [Lys] was biphasic. At first, delta Va' increased with increasing [Lys] reaching a maximum at 10 mM lysine. When [Lys] was further increased, delta Va' declined progressively to reach zero or near zero values. A single transport pathway model is proposed to account for rheogenic lysine entry across the brush-border membrane in the presence and absence of Na+. This postulates an amino acid transporter in the membrane with two binding sites. One is an amino acid site specific for the alpha-amino-alpha-carboxyl group. The other is a Na+ site. Neutral amino acids (e.g. leucine) compete with lysine for the amino acid site. The Na+ site has some affinity for the epsilon-amino group of lysine. When external Na+ is high the Na+ site is essentially 'saturated' with Na+ and formation of a mobile complex between an amino acid and the transporter depends in a saturable fashion on amino acid concentration. In Na+-free media or in media containing low [Na+]; at low external [Lys] the epsilon-amino group of a lysine molecule (simultaneously attached to the amino acid site) interacts with the Na+ site to form a mobile complex, as external [Lys] is increased, attachment of different lysine molecules to each site of an increasing number of transporters to form nontransported or poorly transported complexes results in substrate inhibition of the rheogenic lysine transport process.     

A role for ornithine in the regulation of putrescine accumulation and ornithine decarboxylase activity in Reuber H35 hepatoma cells

We investigated the ability of intracellular ornithine to alter both the biosynthesis of putrescine and the activity of ornithine decarboxylase in Reuber H35 hepatoma cells in culture incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA). In confluent cultures of H35 cells, the addition of TPA (1.6 μM) caused the activity of ornithine decarboxylase to increase by more than 100-fold within 4 h. When exogenous ornithine (0.1–1.0 mM) was added to the culture medium with TPA, a marked dose-dependent increase in the production of putrescine was observed. The activity of ornithine decarboxylase in the same cultures incubated with ornithine decreased in a similar dose-dependent manner. The addition of arginine (0.1–1.0 mM) (but not lysine or histidine) to the H35 cells in culture concomitant with TPA also led to a relative increase in putrescine biosynthesis and a decrease in ornithine decarboxylase activity compared to cultures not receiving the amino acids. A similar response to exogenous ornithine and TPA was observed in a series of less confluent rapidly growing cultures which were in culture for a shorter period of time. The confluent cultures possessed a basal level of arginase (55 units/mg protein) which increased approx. 2-fold upon treatment with TPA. The intracellular concentration of ornithine in the unstimulated cells was in the order of 0.02–0.03 mM. Upon incubation of the cells with exogenous ornithine or arginine, the intracellular pools of these amino acids increased 4- to 8-fold.     

Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter.

We present evidence that polyamine uptake into rat liver mitochondria is mediated by a specific polyamine uniporter. Polyamine transport is not mediated by the ornithine, lysine, or Ca2+ transporters of mitochondria. Polyamine transport is a saturable process, with apparent Km values of 0.13 mM for spermine, 0.26 mM for spermidine, and 1 mM for putrescine. These substrates are mutually competitive inhibitors, indicating a common transport system. Polyamine transport is strictly dependent on membrane potential and insensitive to medium pH, showing that these polycations are transported electrophoretically. Spermine, spermidine, and putrescine are taken up by rat liver mitochondria at rates that increase with increasing valence of the transported species. The activation enthalpies for transport were 24, 32, and 59 kJ/mol for putrescine, spermidine, and spermine, respectively. These values, which amount to about 12 kJ/mol per charge transferred, may be compared to a value of 76 kJ/mol observed for monovalent tetraethylammonium cation. Flux-voltage analysis is consistent with the hypothesis that the mitochondrial polyamine transporter catalyzes transport via a channel mechanism.     

Purine and pyrimidine transport and phosphoribosylation and their interaction in overall uptake by cultured mammalian cells. A re-evaluation.

The zero-trans uptake of purines and pyrimidines was measured in suspensions of Novikoff rat hepatoma, mouse L, P388 mouse leukemia, and Chinese hamster ovary cells by a rapid kinetic technique which allows the determination of uptake time points in intervals as short as 1.5 s. Kinetic parameters for purine/pyrimidine transport were determined by measuring substrate influx into cells in which substrate conversion to nucleotides was negligible either due to lack of the appropriate enzymes or to depletion of the cells of ATP (5'-phosphoribosylpyrophosphate), and by computer fitting exact, integrated rate equations derived for various carrier-mediated transport models directly to zero-trans influx data. The results indicate that different carriers function in the transport of hypoxanthine/guanine, adenine, and uracil with substrate:carrier association constants (K) at 24 degrees C of 300 to 400 muM, 2 to 3 mM, and about 14 mM, respectively, for Novikoff cells. K and Vmax for hypoxanthine transport by L and P388 cells are similar to those for Novikoff cells, but the transport capacity of Chinese hamster ovary cells is much lower and K = 1500 muM. All transport systems are completely symmetrical. Hypoxanthine transport is so rapid that an intracellular concentration of free hypoxanthine (90%) close to that in the medium is attained within 20 to 50 s of incubation at 24 degrees C, at least at extracellular concentrations below K. In cells in which conversion to nucleotides is not blocked free hypoxanthine accumulates intracellularly to steady state levels with equal rapidity and thereafter the rate of hypoxanthine uptake into total cell material is strictly a function of the rate of phosphoribosylation. The low Km systems for hypoxanthine (1 to 9 muM) and adenine (0.2 to 40 muM) uptake detected previously in many types of cells reflect the substrate saturation of the respective phosphoribosyltransferases rather than of the transport system.     

Characteristics of lysine uptake by isolated renal cortical tubule fragments from mature and immature dogs

The uptake of L-lysine was examined in isolated renal cortical tubule fragments from adult and 1-week-old dogs. Lysine uptake by adult tubules was initially more rapid than that by the immature tubules. This uptake by mature tubules reached a steady state after 30 min of incubation, while the newborn tubules still had not reached a steady state by 90 min of incubation. Because a steady state of lysine uptake was not attained with the immature tubules, their uptake of lysine exceeded that of the adult after 60 min of incubation. Kinetic studies revealed that lysine was taken up by one saturable transport system with a Km of 0.56 mM and Vmax of 6.18 mmol/liter intercellular fluid per 5 min in the adult and one saturable transport system in the 1-week-old with a Km of 0.38 mM and Vmax of 3.66 mmol/l intracellular fluid per 5 min. Lysine also entered the renal tubule cells in both age groups via a diffusional pathway with a kd of 0.35 min-1 in the adult and 0.30 min-1 in the newborn. Cystine competitively inhibited lysine uptake by adult dog tubules with a Ki of 0.61 mM. The other dibasic amino acids, ornithine and arginine, also inhibited lysine uptake in both the adult and the newborn.     

Sensitivity of larval and adult crickets (Gryllus bimaculatus) to adipokinetic hormone

The transport of lysine has been investigated in epithelial cells isolated from chicken jejunum. The kinetics of lysine transport and the pattern of interaction with zwitterionic amino acids were consistent with system b(0,+) activity, the broad-spectrum and Na(+)-independent amino acid transporter. The half-saturation constant for lysine entry (K(m)+/-S.E.) was 0.029+/-0.002 mM and the flux was not affected significantly by Na(+) replacement with choline. Lysine influx was inhibited by L-leucine both in Na(+) and choline medium with inhibition constants (K(i)+/-S.E.) 0.068+/-0.006 mM (in Na(+)) and 0.065+/-0.009 mM (in choline). Other inhibitory amino acids (K(i)+/-S.E.) were (mM): L-tyrosine (0.073+/-0.018), L-methionine (0.15+/-0.015), L-cystine (0.42+/-0.04), L-cysteine (1.1+/-0.07), L-isoleucine (1.1+/-0.09), L-glutamine (1.8+/-0.16) and L-valine (2.5+/-0.13). Lysine exit was trans-accelerated (approx. 20 fold) by 2 mM L-lysine and L-leucine. The flux was resistant to pretreatment of the cells with p-chloromercuriphenylsulfonate (0.2 mM), which is an inhibitor of system y(+)L, the broad-spectrum and cation-modulated transporter.     

Inhibition of intestinal absorption of 5-methyltetrahydrofolate by fluoxetine

Thein vitro uptake of 5-methyltetrahydrofolate (5-MeTHF) by rat and human intestine is dose-dependently inhibited by the antidepressant drug fluoxetine (FLX). In rat jejunum rings, 0.2 mM FLX inhibited the uptake of 5-MeTHF (0.25 μM) by 32% (15 min) and 49% (45 min). In brush border membrane vesicles (BBMV) from rat jejunum, 0.2 mM FLX inhibited the folate uptake at the overshoot (90 s) by 40 %. Similar inhibition was observed with human Caco-2 cells and duodenal biopsies. FLX action is exerted on the active transport component of the folate uptake, since the drug has no effect when the passive diffusion component becomes prominent by high substrate concentration, or by 0-4 ºC incubation or by addition of the folate transport inhibitor DIDS (1mM). The kinetic analysis with rat BBMV suggests a non-competitive inhibition of the 5-MeTHF transport by FLX, with apparent values for K_M = 0.89 μM, Vmax = 1.89 pmol/mg prot./10 s, and K_I = 0.21 mM. After 21 days of treatment with FLX (10 mg/kg/day), the folate uptake by jejunum rings or by BBMV from the treated rats was diminished, and the folate levels in erythrocytes and serum were also decreased.