Induction of amino acid transport activity in chick embryo fibroblasts by replacement of extracellular sodium chloride with disaccharide.

The activity of amino acid transport System A in avian fibroblasts was increased following incubation of the cells in a medium in which most of the NaCl normally present had been isoosmotically replaced by sucrose. This increase was detectable after 2 h of incubation, reached a maximum at about 4 h, and remained constant thereafter. Transfer of treated cells back to a normal medium resulted in decay of the induced transport activity, with a half-life of less than 2 h. Kinetic analysis revealed that the increase in transport activity arose from an increase in Vmax, with little change in Km. This induction of System A activity did not occur if an inhibitor of either RNA or protein synthesis was present in the modified medium. The use of various different solutes as replacements for NaCl in the incubation medium showed that, although each replacement caused a decrease in both cellular Na+ content and protein synthesis, only disaccharides produced the increase in amino acid transport activity. In addition, estimates of cell volume indicated that, even under iso-osmotic conditions, incubation in the sucrose-containing medium caused initial cell shrinkage, followed by swelling. It is concluded that this induction of System A activity is associated with a volume regulatory process and that this process probably accounts for the parallel responses previously observed when cells were incubated in hyperosmolar media. Induction of amino acid transport activity by this process is distinct from adaptive regulation, caused by amino acid starvation; but the two processes are not strictly additive, and so appear to converge at some step.

Control of protein synthesis by extracellular Na+ in cultured fibroblasts.

In chick embryo fibroblasts (CEFs), a partial substitution of extracellular Na+ with other cations or carbohydrates decreased the intracellular Na+ content without altering the K+ level. Concomitantly, a significant decrease in the serum-dependent rate of protein synthesis occurred. This phenomenon appeared to be quickly reversible upon reconstitution of the correct extracellular Na+ concentration in the culture medium. The presence of a transcriptional inhibitor such as actinomycin D during the treatment did not inhibit the reversibility of the phenomenon. The presence in the culture medium of K+ in such excess as to dissipate the membrane potential did not alter the observed relationship between the protein synthesis rate and the internal Na+ content. Analysis of the amino acid pool indicated that the observed inhibition of the rate of protein synthesis in CEFs incubated in low Na+ medium was not caused by an unbalanced availability of intracellular amino acids. In addition, intracellular pH, as estimated by the measurement of the equilibrium distribution of benzoic acid, did not show any significant alteration in cells incubated in the presence of bicarbonate buffer and in low extracellular Na+. Moreover, the relationship between the rate of protein synthesis and the internal Na+ content was still observed in CEFs cultured in bicarbonate-containing media, but at lower or higher than physiological pH. Analysis by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of the proteins synthesized by CEFs cultured at a reduced extracellular Na+ concentration showed that specific alterations of gene expression occurred.

Hyperosmolarity-induced stress proteins in chick embryo fibroblasts.

The effects of a short exposure of chick embryo fibroblasts to a hyperosmolar medium on monovalent cation content, rate of protein synthesis, and polypeptide pattern expression were studied. The hyperosmolar shock gave an immediate and pronounced inhibition of the protein-synthesis rate temporally related to a marked alteration of the intracellular Na+ content. Following the return of the cells to an osmolar environment, the internal Na+ content quickly resumed its previous level, while the recovery of the protein-synthesis rate was more gradual. During the recovery period, there was enhanced expression of at least 12 proteins. The 4 major induced proteins exhibited apparent molecular weights of 96, 87, 70, and 48 kDa. A reduction in the synthesis of five protein bands including three large polypeptides of 220, 160, and 140 kDa was also observed. A comparison with the 3 major proteins induced by a 44 degrees C heat shock indicated an apparent similarity with only two of the hyperosmolarity-inducible polypeptides. Moreover, evidence has been also obtained of the close similarity between the 96 and 75 kDa glucose-regulated proteins and the 96 and 75 kDa proteins inducible by a hyperosmolar shock or by a continuous hyperosmolar treatment, respectively. The kinetics of the stress-proteins appearance indicated nonsimultaneous induction. The presence of actinomycin D during the exposure of the cells to the stress and the recovery period suggested that the expression of some hyperosmolarity-enhanced proteins is regulated at the transcriptional level.

Adaptive response of cultured fibroblasts to hyperosmolarity.

Raising to 0.4 osM the osmolarity of the medium in which chick embryo fibroblasts are incubated quickly increases the internal Na+ concentration, inhibits protein synthesis and also stimulates amino acid transport. On extending the incubation time, cells appear to adapt to the altered environment, as the Na+ content declines toward control values within few hours. Protein synthesis resumes its normal rate within 12-14 h of treatment. Experimental alteration of the monovalent cation content by substituting extracellular Na+ with other osmolites or by using ouabain or the ionophore monensin reveals an impairment of protein synthesis. Analysis by SDS-PAGE reveals an alteration of the polypeptide pattern expressed by hyperosmolarity-exposed cells, resulting in an enhanced synthesis of the 87, 75 and 53 kD proteins and inhibition of a 125 kD band. The previously increased amino acid transport activity also reverts to its normal level, but only after 40-50 h of incubation. The growth rate of these cells does not appear to be significantly affected during the first 3 days of the hyperosmolar treatment. Results presented in this publication identify the alteration of the protein synthesis rate, the change in the intracellular cation content and the increase in amino acid transport activity as plausible parameters of adaptive response, and suggest that the modulation of gene expression observed in cells exposed continuously to hyperosmolarity may be a consequence of the alteration of the intracellular monovalent cation concentration.

Induction of stress proteins by hyperosmolarity in normal and transformed cells.

The synthesis of at least three proteins, with molecular weights of approximately 87, 70, and 53 kd, was enhanced following the exposure of chick embryo fibroblasts to hyperosmolar shock of 30 min at 0.6 osM. Two of these proteins, the 87 and 70 kd, comigrated on one-dimensional gel electrophoresis with the stress proteins induced by heat shock after 30 min at 44 degrees C. In 3T3 cells, the hyperosmolar shock enhanced the expression of two proteins of 88 and 52 kd, whereas the heat shock increased the synthesis of several new polypeptides including the 88 and 52 kd mw. In SV40-transformed 3T3 cells the synthesis of two proteins of 72 and 69 kd was enhanced by heat shock, but no change of the protein pattern was recorded after the hyperosmolar shock.

Effect of hyperosmolarity on the activity of amino acid transport system L in avian fibroblasts.

The transport of selected neutral amino acids known as good substrates of amino acid transport System L has been studied in chick embryo fibroblasts exposed for 4 hours to hyperosmolar culture medium. The activity of the L system, as measured by initial rates of L-phenylalanine uptake, increased in hyperosmolarity treated cells when determined before any cell depletion of intracellular amino acids. This effect was lost after depletion but reappeared after reloading the cells with pertinent substrates of System L. This transport activity appeared to be related to the internal level of amino acids capable of exchange through System L. In hyperosmolarity-treated chick embryo fibroblasts a higher level of System L substrates was obtained during the reloading phase in comparison to control cells. This expanded amino acid pool reflected an increased activity of transport System A, an agency of amino acid mediation known to enlarge its capacity following a hyperosmolar treatment of chick embryo fibroblasts (see Tramacere et al., 1984). L-Methionine, a preferred substrate of both A and L systems, appeared to be involved in the coupling between the activity of amino acid transport Systems A and L in these cells.

Osmoregulation of amino acid transport activity in cultured fibroblasts.

The effect of exposure of chick embryo cells to increasing concentrations of Na+ in the culture medium on the subsequent amino acid transport as determined at physiological osmolarity was investigated in detail. It was found that the hyperosmolar treatment stimulated amino acid transport in a dose-dependent manner up to 200 mM Na+. Changes were measurable as early as 1 h after altering Na+ and reached a maximum after 4 h, remaining constant thereafter. The maintenance of this effect required continuous exposure of the cell to high Na+ in the culture medium. Hyperosmolarity-mediated increases in amino acid transport activity by system A have been detected with L-proline and L-alanine. Transport activities of systems ASC and L did not change appreciably after exposure of the cells to high Na+. Inhibition of protein synthesis by cycloheximide or RNA synthesis by actinomycin D (actD) prevented these uptake changes. Kinetic analysis indicated that the stimulation of the activity of transport system A by high Na+ treatment occurred through a mechanism affecting Vmax rather than Km.

Amino acid transport in pig lymphocytes. Enhanced activity of transport system asc following mitogenic stimulation.

Changes in neutral amino acid transport activity caused by addition of phytohaemagglutinin-P to quiescent peripheral pig lymphocytes have been evaluated by measurements of 14C-labelled neutral and analogue amino acids under conditions approaching initial entry rates. Utilizing methylaminoisobutyric acid, the best model substrate of System A, we confirmed our previous report (Borghetti, A.F., Kay, J.E. and Wheeler, K.P. (1979) Biochem. J. 182, 27-32) on the absence of this transport system in quiescent cells and its emergence following stimulation. Furthermore, we demonstrated the presence in quiescent cells of an Na+-dependent transport system for neutral amino acids that has been characterized as System ASC by several criteria including intolerance to methylaminoisobutyric acid, strict Na+-dependence, the property of transtimulation and specificity for pertinent substrates such as alanine, serine, cysteine and threonine. Analysis of the relationship between influx and substrate concentration revealed that two independent saturable components contribute to entry of alanine in quiescent cells: a low affinity (Km = approximately 4 mM) and a high affinity (Km = approximately 0.2 mM) component. The high affinity component could be inhibited in a competitive way by serine, cysteine and threonine, but methylaminoisobutyric acid did not change appreciably its constants. The enhanced activity of alanine transport through the ASC system observed in activated cells resulted from a large increase in the capacity (V) of the high affinity component without any substantial change in the apparent affinity constant (Km).

Cell density and amino acid transport in 3T3, SV3T3, and SV3T3 revertant cells.

The transport of selected neutral and cationic amino acids has been studied in Balb/c 3T3, SV3T3, and SV3T3 revertant cell lines. After properly timed preincubations to control the size of internal amino acid pools, the activity of systems A, ASC, L, and Ly+ has been discriminated by measurements of amino acid uptake (initial entry rate) in the presence and absence of sodium and of transport-specific model substrates. L-Proline, 2-aminoisobutyric acid, and glycine were primarily taken up by system A; L-alanine and L-serine by system ASC; L-phenylalanine by system L; and L-lysine by system Ly+ in SV3T3 cells. L-Proline and L-serine were also preferential substrates of systems A and ASC, respectively, in 3T3 and SV3T3 revertant cells. Transport activity of the Na+-dependent systems A and ASC decreased markedly with the increase of cell density, whereas the activity of the Na+-independent systems L and Ly+ remained substantially unchanged. The density-dependent change in activity of system A occurred through a mechanism affecting transport maximum (Vmax) rather than substrate concentration for half-maximal velocity (Km). Transport activity of systems A and ASC was several-fold higher in transformed SV3T3 cells than in 3T3 parental cells at all the culture densities that could be compared. In SV3T3 revertant cells, transport activity by these systems remained substantially similar to that observed in transformed SV3T3 cells. The results presented here add cell density as a regulatory factor of the activity of systems A and ASC, and show that this control mechanism of amino acid transport is maintained in SV40 virus-transformed 3T3 cells that have lost density-dependent inhibition of growth, as well as in SV3T3 revertant cells that have resumed it.

Insulin regulation of amino acid transport in mesenchymal cells from avian and mammalian tissues.

Insulin regulation of amino acid transport across the cell membrane was studied in a variety of mesenchymal cell directly isolated from avian and mammalian tissues or collected from confluent cultures. Transport activity of the principal systems of mediation in the presence and absence of insulin was evaluated by measuring the uptake of representative amino acids under conditions approaching initial entry rates. Insulin enhanced the transport rate of substrate amino acids from the A system(alpha-aminoisobutyric acid, L-proline, glycine, L-alanine and L-serine) in fibroblasts and osteoblasts from chick-embryo tissues, in mesenchymal cells (fibroblasts and smooth muscle cells) from immature rat uterus, in thymic lymphocytes from young rats and in chick-embryo fibroblasts from confluent secondary cultures. In these tissues, the uptake of amino acid substrates of transport systems L and Ly+ (L-leucine, L-phenylalanine, L-lysine) was not affected by the presence of the hormone. No insulin control of amino acid transport was detected in chick-embryo chondroblasts and rat peritoneal macrophages. These observations identify the occurrence of hormonal regulatory patterns of amino acid transport for different mesenchymal cells types and indicate that these properties emerge early during cell differentiation.

[Regulation of amino acid transport in avian fibroblasts from growing and quiescent cell cultures (author's transl)]. / Regolazione del trasporto degli aminoacidi in fibroblasti da colture in crescita e quiescenti

The regulation of amino acid transport across the cell membrane by adaptive mechanisms has been studied in chick embryo fibroblasts obtained from growing and quiescent cell cultures. 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.