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Prolonged exposure of 3T3 cells to 0.5 osM hypertonic medium induced the accumulation of hsp70 mRNAs. This increase in mRNA levels required active protein synthesis. A weak and transient activation of heat shock factor 1 (HSF1) was noted, but it was temporally uncoupled to the accumulation of the hsp70 mRNAs. Nuclear run-on assay and transfection experiments showed that hsp70 gene transcription was not affected by hypertonicity. ActD chase experiments showed that during hypertonic treatment, degradation of hsp70 mRNAs was markedly reduced. This effect did not appear to be a general phenomenon since the increase in mRNA level of another gene induced by hypertonicity (ATA2 transporter) was scarcely due to RNA stabilization. These findings suggest that hypertonic treatment increases the production of hsp70 protein in 3T3 cells via a stabilization of its corresponding mRNA.

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.

Commercial serum albumins have been found to be able to stimulate the proliferation of Balb/c 3T3 cells transformed by SV40, but not that of the normal counterpart. The effect is most pronounced with crystalline samples of albumin depleted of both globulin and fatty acid components, and depends on conditions used for the attachment and on seeding density. Physical and chemical treatments aimed to remove tightly bound impurities do not abolish the activity of fatty acid free serum albumin, thus supporting the idea that albumin per se is mitogenic towards these cells.

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) 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) Adaptive regulation is restricted to a discrete subgroup of amino acids (L-proline, glycine and the analogue alpha-aminoisobutyric acid) in rat peritoneal macrophages and thymic lymphocytes. (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) 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) 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.

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).

The rate of transport of phenylalanine by System L has been measured in SV40 3T3 cells at various cell densities. When the activity of the L system was determined before any cell depletion of intracellular amino acids, a density-dependent increase in transport paralleled the decrease in cell density. This regulation was lost after cell depletion but reappeared after reloading the cells with pertinent substrates of System L. The phenylalanine transport activity modulated by cell density appeared to be related to the internal level of amino acids capable of exchange up to a definite concentration, beyond which transport activity by System L did not parallel a further increase of internal substrate level. Analysis of the relationship between influx and substrate concentration suggested that two saturable components contribute to entry of phenylalanine and leucine in depleted and in reloaded cells: a low-affinity and a high-affinity component. Both kinetic parameters of the high-affinity component appeared to be modulated by the loading treatment, but only V changed markedly. Activation energies for the high-affinity component of the amino acid transport reaction were calculated from an Arrhenius plot in reloaded cells, and were found to be different for low- and high-density cultures. This result is consistent with the interpretation that cell density modulated the rates at which the amino acid-carrier complex can move within the cell membrane.

The rate of transport of phenylalanine and leucine, pertinent amino acids of System L, has been measured in SV40 3T3 cells as a function of the presence of Na+ ions during the reloading phase that precedes the influx determination. The presence of Na+ ions during the reloading phase resulted in an increase of the subsequent substrate influx through System L. This effect was related to the intracellular Na+ level and was found to be independent by the presence of a chemical sodium gradient outside-inside during influx determination; furthermore, this effect could not be ascribed to a difference between control and Na+-treated cells in the internal levels of those amino acids that participate in the exchange phenomena of transport System L. The transport of phenylalanine appeared to have the ability to accept Li+ for Na+ substitution in the 'trans' position. The presence of Na+ ions in the 'trans' position was not required to optimize the transport of System A-reactive substrates, whose influxes are dependent on the presence of the cation in 'cis' position. Analysis of the relationship between influx and substrate concentration indicated that the Na+-dependent increase of substrate influx was associated with an enlarged capacity of the high-affinity component of transport System L.

Amino acid transport System L in both normal Balb/c 3T3 cells and in those transformed with simian virus 40 (SV 3T3) was analysed kinetically under two different experimental conditions. Under 'zero-trans' conditions the results for both types of cell could be interpreted satisfactorily in terms of System L consisting of two components (L1 and L2) characterized by different Km values. This conclusion is in agreement with previous reports. However, under 'infinite-trans' conditions, the experimental data could not be accounted for in terms of only two components; the introduction of a third component (L3) was necessary to provide a satisfactory fit. Viral transformation affects only the L1 component, either by modification or by replacement, giving it a higher 'affinity' (lower Km) but a lower 'capacity' (lower Vmax).