Changes In Transport Activity Research Articles

Biliary transport of glutathione S-conjugate by rat liver canalicular membrane vesicles.

Transport of S-dinitrophenyl glutathione, a model compound of glutathione S-conjugates, was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited time-dependent uptake of [2-3H]glycine-glutathione conjugate into an osmotically sensitive intravesicular space. Inactivation of vesicle-associated gamma-glutamyltransferase by affinity labeling with L-(alpha-S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole-acetic acid had no effect on the initial rate of transport. Chemical analysis revealed that the intact glutathione conjugate accounted for most vesicle-associated radioactivity, reflecting the low transferase activity in the liver and membrane vesicles. The initial rate of transport followed saturation kinetics with respect to conjugate concentrations; an apparent Km of 1.0 mM and Vmax of 1.7 nmol/mg of protein X 20 s were calculated. These results indicate that transport of the glutathione S-conjugate across the canalicular membranes is a carrier-mediated process. Sodium chloride in the transport medium could be replaced by KCl, LiCl, or choline chloride without any changes in transport activity. The rate of conjugate transport was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). The rate of conjugate uptake was enhanced by replacing KCl in the transport medium with K gluconate, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. These data indicate that conjugate transport is electrogenic and involves the transfer of negative charge. Transport of S-dinitrophenyl glutathione was inhibited by S-benzyl glutathione, oxidized glutathione, or reduced glutathione. This transport system in canalicular membranes may function in biliary secretion of glutathione S-conjugates of xenobiotics whose synthesis in hepatocytes requires glutathione S-transferases.

Altered hepatobiliary transport of taurocholic acid in aged rats

Hepatobiliary transport of taurocholic acid was studied in adult (3 months) and old (2 years) rats using an isolated perfused rat liver technique in order to determine the effect of age on hepatic uptake and secretion of bile acids simultaneously. The results were analyzed using a steady-state compartmental model to estimate the uptake and secretion of taurocholic acid. Hepatic secretion was decreased to a greater extent than the uptake in old rats. These changes in transport activities were associated with increases in perfusate and liver bile acid pool sizes. These results can explain the decrease in total pool size and synthesis rate of bile acids observed previously in old rats using in vivo studies. It has been suggested that the age-dependent decrease in bile acid transport capacity of the liver is secondary to the altered lipid composition of the liver plasma membranes of old rats.

Regulation of amino acid transport in chicken embryo fibroblasts by purified multiplication-stimulating activity (MSA).

Enhanced amino acid transport is observed when quiescent cultures of chicken embryo fibroblasts are stimulated to proliferate by the addition of purified multiplication-stimulating activity (MSA). This increase in amino acid transport is an early event occurring prior to the onset of DNA synthesis in stimulated cells. Results indicate that the changes in transport activity, as measured by alpha-aminoisobutyric acid (AIB) uptake, are due to stimulation of only the Na+-dependent A transport system. There is little or no change in the activities of transport systems ASC, L, or Ly+ upon exposure to MSA. A kinetic analysis shows this increased activity is due to a change in Vmax while Km remains unaltered. Continuous exposure to the stimulus is required to maintain the increased level of transport activity and the presence of inhibitors of RNA and protein synthesis significantly inhibits the response. Results also indicate that a similar specific increase in the A transport system is initiated when RSV tsNY68 infected cells are shifted to the permissive temperature. It appears that the A system of mediation is emerging as a strategic regulatory site for cell function.

Coupling of insulin receptors to glucose transport: A temperature-dependent time lag in activation of glucose transport

We have studied the ability of occupied insulin receptors to activate (or couple to) the glucose transport system in isolated rat adipocytes. Maximal insulin action is seen when only a small proportion (<10%) of the receptors is occupied, and this fraction can be rapidly filled (<5 s) at an insulin concentration of 100 ng/ml. Additionally, control studies show that when the extracellular glucose concentration is tripled, the rate of transport triples within 10 s, indicating that changes in transport activity can be observed nearly instantaneously. Therefore, when cells are exposed to a high insulin concentration (100 ng/ml), any delay in the onset of insulin action beyond this time must be due to the time required for coupling of occupied insulin receptors to the glucose transport system. At 24 °C there is a lag of at least 200 s after insulin addition before a significant stimulation of 2-deoxyglucose transport is seen. The length of this lag phase is temperature dependent, decreasing to 45 s at 37 °C. An Arrhenius plot of the coupling lag is linear, with an activation energy of 25 kcal/mol. After the delay in the onset of initial transport activation the full response appears in a gradual manner, requiring 20 min at 24 °C to attain maximal stimulation. The time required for the full insulin response to appear is also temperature dependent, decreasing to 5 min at 37 °C. Similar results were obtained for the kinetics of insulin activation of 3- O-methyl glucose transport. Thus, the coupling of insulin receptors to the glucose transport system can be divided into two components: an initial absolute time lag followed by a gradual incremental process before the maximal, or full, effect of insulin is achieved. In conclusion, (1) there is an absolute delay in the onset of the insulin's initial action on glucose transport, (2) after an initial delay, activation of transport proceeds in a gradual manner, and (3) the coupling process between insulin receptors and the glucose transport system is temperature dependent and can be described by a linear Arrhenius plot. This suggests that the rate of activation is not limited by membrane fluidity.

Ion selectivity and proximal salt reabsorption.

Electrophysiological techniques were used in isolated perfused superficial (S) and juxtamedullary (JM) rabbit proximal convoluted tubules (PCT) to examine the relative sodium-to-chloride (PNa/PCl) and bicarbonate-to-chloride (PHCO3/PCl) permeability ratios. We found that the great majority of PCT are sodium selective and that PHCO3/PCl depends on the experimental conditions. In the presence of active sodium transport, PHCO3/PCl is high and increases with PNa/PCl. When PHCO3/PCl is determined after inhibition of active sodium transport or at 25 degrees C, PHCO3/PCl approximates the free solution anion mobility ratio of 0.5 and is independent of PNa/PCl. The difference between PHCO3/PCl determined in the presence of and in the absence of active transport suggests that the lowering of bath bicarbonate concentration in the presence of active transport changes both paracellular and transcellular current flow. In addition, we found that during luminal perfusion with high chloride, low bicarbonate, organic solute-free solutions, the transepithelial electrical potential depends on PNa/PCl and PHCO3/PCl. This potential is approximately 4.0 mV in S PCT with low PNa/PCl and falls progressively to zero in JM PCT with high PNa/PCl. From these data we conclude that anion concentration gradients drive an important diffusive flux of sodium chloride through the paracellular pathway only in PCT with low PNa/PCl ratios.

Control by Serum of Amino Acid Transport and Deoxyribonucleic Acid Synthesis in Cultured Avian Fibroblasts

Serum has been reported to contain factors that seem to display a physiological role in the control of cell proliferation (Temin, 1971). When cells in culture are deprived of serum they cease growing and enter a quiescent state, often referred to as Go-phase. On restoration of serum, the cells return to the proliferative state as revealed by a quasisynchronous initiation of DNA synthesis (S-phase) after a definite lag period. Internal mechanisms by which the cell re-entry into the S-phase is triggered are not clear and many factors may converge at some common site. I t has been suggested that alterations in transport activities for small nutrients may be ofcentral importance in the control of cell growth and in the performance of the cell cycle (Holley & Kiernan, 1974; Pardee, 1975). Indeed, changes in transport activity for sugars, amino acids and nucleosides are among the earliest events associated with reinitiation of cell growth in serumdeprived cell cultures when serum is added (Baserga, 1976). In particular, Isselbacher (1972) reported that a stimulation of a-aminoisobutyric acid (2-amino-2-methylpropanoic acid) uptake took place within 90min after serum addition to serum-deprived cultured BHK cells. This model amino acid is a preferential substrate of the transport A-system, a Na+-dependent agency formally characterized in a variety of epithelial and mesenchymal cells (Christensen, 1969; Gazzola et a/ . , 1972). The experiments in the present paper were conducted: (a) to explore the temporal relationship between occurrence of amino acid-transport changes and initiation of DNA synthesis after serum re-addition to serum-deprived cultured cells (chick-embryo fibroblasts); (b) to identify the system(s) of mediation involved in serum-stimulated amino acid transport; ( c ) to define the kinetic parameter(s) of the rate of change in amino acid transport and their sensitivity to inhibitors of protein synthesis. Fibroblasts wereobtained from 1 1-day chickembryos. Allexperiments wereperformed with secondary cultures grown in plastic culture flasks in medium 199 (Gibco, Grand Island, NY, U.S.A.) (with Hanks salts) containing 2% (v/v) tryptose phosphate broth and 2% (v/v) chicken serum. Enough NaHCO, was added to the medium to maintain the pH at 7.5 in an atmosphere of C02/air ( I : 19) at 37°C. Exponentially growing cultures were stopped by serum deprivation for 20h. The resulting quiescent cultures were induced to grow by re-addition of serum (2% chicken serum). The relative rates and the pattern of synchronous DNA synthesis were measured by [jHIthymidine incorporation into acid-insoluble material and by radioautography. Initial rates of amino acid uptake were measured at the desired intervals by incubating the cell monolayers (thoroughly rinsed) for 2-3min at 37°C in Krebs-Ringer bicarbonate buffer containing the labelled amino acid under study. The means for determining intracellular accumulation of the tracer amino acid and forevaluating the proper corrections to be introduced wereessentially similar to those described previously (Guidotti et al., 1971). Serum-deprived cultured avian fibroblasts (20h of deprivation) had a very low rate of DNA synthesis. This rate increased abruptly 4-6h after serum exposure reaching a maximum at 1Oh (Fig. 1). At the latter time, approx. 55% of the cells showed labelled nuclei (as measured by conventional radioautography) when exposed to a 20min [3H]thymidine pulse. Fig. 2 shows that amino acid-transport activity, assayed by measurements of L-proline uptake (under conditions of initial velocity for this amino acid substrate of the A-system mediation), doubles between 30 and 90min after serum addition. This change in transport activity appears to be dependent on a definite increase in transport maximum ( VmaX.) without substantial changes in substrate concentration for half-maximal transport velocity (K,,,) and it is prevented by continuous exposure to cycloheximide. Definite increases in transport activity upon serum restoration were also observed with a-aminoisobutyric acid and, when transported by the A-system

Changes in the erythrocyte sodium pump with age.

Various estimations of the erythrocyte sodium pump were determined in 18 female and 11 male adult subjects. In the female group, but not in the male group, the sodium concentration increased with age; the erythrocyte active transport decreased and passive transport increased with age and could account for the observed change in erythrocyte sodium concentration with age. The erythrocyte Na-K ATPase decreased with age in the females and this could account for the observed change in active transport.

Developmental changes in plasma membrane fluidity in chick embryo heart

1. 1. Decreases in the rate of transport of sugars (facilitated transport), amino acids (active transport), and urea (simple diffusion) occur in chick embryo heart during development. This work considers the possibility that changes in the plasma membrane fluidity during development contribute to the observed changes in transport activities. 2. 2. Techniques were developed for subcellular fractionation of chick embryos and adult chickens. 3. 3. The depolarization of the fluorescence of 1.6-diphenylhexatriene was used to estimate the fluidity of the lipid portion of plasma membrane enriched fractions of heart from chick embryos at various stages of development and from adult hearts. 4. 4. There is a pattern of decreasing membrane viscosity as development proceeds. Between 5–6 days and 10 days of embryonic life a 20% decrease in viscosity of the plasma membrane-enriched fraction occurs. Between 10 and 20 days of embryonic life there is no significant change in viscosity. Between 20 days of development (1 day before hatching) and adulthood there is a further 55% decrease in plasma membrane viscosity. 5. 5. It is proposed that the changes in membrane fluidity observed may contribute to developmental changes in membrane transport activities, but other factors must also be involved.

Changes in active transport, intracellular adenosine 5'-triphosphate levels, macromolecular syntheses, and glycolysis in an energy-uncoupled mutant of Escherichia coli.

The temperature-sensitive Escherichia coli mutant ecfts metC (Lieberman and Hong, 1974), previously shown to be defective in the coupling of metabolic energy to active transport, is also altered in a wide variety of cellular activities at the nonpermissive temperature. These alterations include a lowering of intracellular adenosine 5'-triphosphate levels, an alteration of glucose metabolism such that large quantities of pyruvate and dihydroxyacetone phosphate are excreted into the medium, excretion of accumulated potassium ions, and a cessation of deoxyribonucleic acid, ribonucleic acid, and phospholipid synthesis. Since these effects closely mimic the action of colicins E1 and K on E. coli cells, the possibility that the ecf gene product is the primary biochemical target for these colicins is discussed.

Adaptive regulation of amino acid transport across the cell membrane in avian and mammalian tissues

The regulation of amino acid transport across the cell membrane by adaptive mechanisms has been studied in a variety of mesenchymal and epithelial cells and tissues of avian and mammalian origin. Changes in transport activity as a function of time under various in vitro conditions (amino acid dependence, active and inhibited protein synthesis) have been evaluated by measurements of initial entry rates with representative amino acids. Results and conclusions based on the adopted experimental approach include the following. 1. (1)|An adaptive control mechanism for the transport of neutral amino acids corresponding to the typical substrates of the A mediation is operative in (a) mesenchymal cells (fibroblasts, chondroblasts, osteoblasts and myoblasts) from embryonic tissues of avian (chick embryo) origin and (b) mesenchymal cells from immature rat uterus (fibroblasts and smooth muscle cells) and other mammalian tissues (cardiac cells from newborn mouse and rat heart). 2. (2)|Adaptive regulation is restricted to a discrete subgroup of amino acids ( l-proline, glycine and the analogue α-aminoisobutyric acid) in rat peritoneal macrophages and thymic lymphocytes. 3. (3)|Adaptive regulation is absent in erythroid cells (human erythrocytes, rabbit erythrocytes and reticulocytes, avian erythrocytes) which lack the A mediation and are incapable of active gene transcription. 4. (4)|Adaptive regulation is absent in the epithelial kidney cortex tissue and possibly absent in the epithelial component of liver tissue from adult rats: it is fully operative in the chick embryo crystalline lens, i.e. an epithelial preparation of embryonic origin. 5. (5)|These observations indicate that adaptive control mechanisms of amino acid transport across the cell membrane are quite common among tissues and species and emphasize their broad biological significance in eukaryotes.

Regulation of amino acid transport in chick embryo heart cells. IV. Site and mechanism of insulin action

The regulation of amino transport by insulin in chick embryo heart cells has been studied. Experiments were designed to identify the system(s) of mediation involved in this regulation and to investigate the mechanisms of the hormone action. Results and conclusions based on the adopted experimental approach include the following: 1. 1. Among the four systems (A, ASC, L, Ly +) found to be operative in cardiac cells for the transport of neutral and basic amino acids (Gazzola, G. C., Franchi-Gazzola, R., Ronchi, P. and Guidotti, G. G. (1973) Biochim. Biophys. Acta 311, 292–301), only the A mediation is responsive to insulin. 2. 2. The hormone enhances the activity of this mediation by increasing the maximal velocity ( V) of transport (without substantial changes in K m ) even under conditions of inhibited protein synthesis. 3. 3. The rate of degradation of protein components of the A mediation (as estimated by measurements of transport activity under conditions of inhibited protein synthesis) is decreased when cells are incubated in the presence of insulin. The hormone does not affect this rate for systems ASC, L and Ly +. 4. 4. Cells previously incubated in a medium containing cycloheximide (phase of inhibited translation) subsequently exhibit a net increase of transport activity (A mediation) when transferred into a medium containing actinomycin D (phase of inhibited transcription). The presence of insulin during pre-incubation in cycloheximide has no effect on the subsequent rate of change in transport activity; when added to the second phase, the hormone definitely enchances transport activity. 5. 5. These observations have been interpreted to indicate that insulin modulates the activity of the A transport system by acting at two different sites; at the cell membrane by protecting specific transport proteins against degradation and at a post-transcriptional level by increasing the rate of synthesis of these transport proteins.