Derepression of amino acid transport by amino acid starvation in rat hepatoma cells.

Amino acid starvation causes an adaptive increase in the initial rate of transport of selected neutral amino acids in an established line of rat hepatoma cells in tissue culture. After a lag of 30 min, the initial rate of transport of alpha-aminoisobutyric acid (AIB) increases to a maximum after 4 to 6 h starvation of 2 to 3 times that seen in control cells. The increased rate of transport is accompanied by an increase in the Vmax and a modest decrease in the Km for this transport system, and is reversed by readdition of amino acids. The enhancement is specific for amino acids transported by the A or alanine-preferring system (AIB, glycine, proline); uptake of amino acids transported by the L or leucine-preferring system (threonine, phenylalanine, tyrosine, leucine) or the Ly+ system for dibasci amino acids (lysine) is decreased under these conditions. Amino acids which compete with AIB for transport also prevent the starvation-induced increase in AIB transport; amino acids which do not compete fail to prevent the enhancement. Paradoxically threonine, phenylalanine, tryptophan, and tyrosine, which do not compete with AIB for transport, block the enhancement of transport upon amino acid starvation. The starvation-induced enhancement of amino acid transport does not appear to be the result of a release from transinhibition. After 30 min of amino acid starvation, AIB transport is either unchanged or slightly decreased even though amino acid pools are already depleted. Furthermore, loading cells with high concentrations of a single amino acid following a period of amino acid starvation fails to prevent the enhancement of AIB transport, whereas incubation of the cells with the single amino acid for the entire duration of amino acid starvation prevents the enhancement; intracellular amino acid pools are similar under both conditions. The enhancement of amino acid transport requires concomitant RNA and protein synthesis, consistent with the view that the adaptive increase reflects an increased amount of a rate-limiting protein involved in the transport process. Dexamethasone, which dramatically inhibits AIB transport in cells incubated in amino acid-containing medium, both blocks the starvation-induced increase in AIB transport, and causes a time-dependent decrease in transport velocity in cells whose transport has previously been enhanced by starvation.     

Hormonal regulation of amino acid transport and cAMP production in monolayer cultures of rat hepatocytes

The effects of insulin, glucagon or Dexamethasone (DEX) and of glucagon with insulin or DEX were examined on the uptake of 2-amino [1-¹⁴C]isobutyric acid (AIB) and N-Methyl-2-amino [1-¹⁴C]isobutyric acid (NMe AIB) in monolayer cultures of rat hepatocytes. Insulin and glucagon stimulated the uptake of both the amino acids and DEX inhibited it, showing that all three of these hormones regulate the A system (the sodium-dependent system that permits the transport of NMe AIB) for amino acid transport in these cultures. Experiments investigating the transport of aminocyclopentane-1-carboxylic acid, 1- [carboxyl-¹⁴C] in the presence of excess AIB or in the absence of sodium showed that insulin had no effect on the activity of the L system (the sodium-independent system that prefers leucine). Experiments on the uptake of AIB in the presence of excess NMe AIB showed insulin had no effect on the transport activity of the ASC system (the sodium-dependent system that does not transport NEe AIB). Insulin concentrations ranging from 0.1 nM to 100 nM did not antagonize the stimulatory effect of optimum or suboptimum concentrations of glucagon on the uptake of either AIB or NMe AIB. Similarly, glucagon did not antagonize the stimulatory effect of optimum or suboptimum concentrations of insulin on the uptake of both the amino acids. The combined effect of insulin and glucagon was additive on the rate as well as the cumulative uptake of both AIB and NMe AIB. DEX alone inhibited the transport of both AIB and NMe AIB by about 25%, while glucagon caused a 2–3-fold increase; however, the addition of glucagon to cultures containing DEX caused a 7–8-fold increase in the uptake of both AIB and NMe AIB when compared to cultures containing DEX alone. The effect of insulin on the levels of cAMP was also investigated. Insulin had no effect on the cAMP levels in cultures treated or untreated with optimum or suboptimum concentrations of glucagon.     

Neutral amino acid transport in human synovial cells: substrate specificity of adaptative regulation and transinhibition

Neutral amino acid transport was characterized in human synovial cells. The amino acids tested are transported by all three major neutral amino acid transport systems, that is, A, L, and ASC. The model amino acid 2-aminoisobutyric acid (AIB) was found to be a strong specific substrate for system A in synovial cells. When cells were starved of amino acids, the activity of AIB transport increased, reaching a maximum within 1 h. The stimulation of transport activity was not blocked by cycloheximide and would thus appear to be related to a release from transinhibition. Similarly, the decrease in the activity of AIB transport observed after the addition of alpha-methyl-aminoisobutyric acid (meAIB) appeared to be related to transinhibition. However, using a different approach, that is, amino acid starvation followed by incubation with 10 mM meAIB and transfer to an amino acid-free medium with or without cycloheximide supplementation, a clear increase in AIB uptake, due both to derepression and a release from transinhibition, was observed. Unlike human fibroblasts, the depression of system A in these synovial cells was not serum-dependent. The process of derepression was observed only after preloading with meAIB. Neither AIB nor alanine produced this phenomenon. Moreover, alanine preloading led to a large increase in AIB transport activity due to a release from transinhibition. These observations indicate that the process of derepression and release from transinhibition are specific to the substrates present in the culture medium prior to amino acid starvation.     

Regulation of polyamine synthesis by dietary alpha-aminoisobutyric acid and ornithine

An experiment was conducted to determine the effect of feeding ornithine in combination with alpha-aminoisobutyric acid (AIB), an inhibitor of arginase, on the regulation of polyamine synthesis in chicks. A total of 48 chicks with genetically elevated renal arginase activity was fed diets containing crystalline amino acids and 1% AIB with or without 2% ornithine. Feeding AIB reduced renal arginase activity, while renal and hepatic ornithine decarboxylase (ODC) activity increased. Feeding AIB plus ornithine caused no further reduction in renal arginase activity compared with that in chicks fed the AIB-supplemented diet. Renal and hepatic ODC activities, however, fell to below control levels. Renal, hepatic, and breast muscle ornithine concentrations increased substantially when ornithine was fed. AIB plus ornithine increased renal putrescine and spermidine concentrations. It was concluded that AIB could partially overcome the ornithine-induced inhibition of ODC activity. These findings support the hypothesis that dietary manipulation of precursor amino acids of polyamines in the presence of metabolites that induce ODC activity can influence tissue polyamine concentrations.     

A Search for Extraterrestrial Amino Acids in Carbonaceous Antarctic Micrometeorites

Antarctic micrometeorites (AMMs) in the 100–400 m size range are the dominant mass fraction of extraterrestrial material accreted by the Earth today. A high performance liquid chromatography (HPLC) based technique exploited at the limits of sensitivity has been used to search for the extraterrestrial amino acids -aminoisobutyric acid (AIB) and isovaline in AMMs. Five samples, each containing about 30 to 35 grains, were analyzed. All the samples possess a terrestrial amino acid component, indicated by the excess of the L-enantiomers of common protein amino acids. In only one sample (A91) was AIB found to be present at a level significantly above the background blanks. The concentration of AIB (280 ppm), and the AIB/isovaline ratio (10), in this sample are both much higher than in CM chondrites. The apparently large variation in the AIB concentrations of the samples suggests that AIB may be concentrated in rare subset of micrometeorites. Because the AIB/isovaline ratio in sample A91 is much larger than in CM chondrites, the synthesis of amino acids in the micrometeorite parent bodies might have involved a different process requiring an HCN-rich environment, such as that found in comets. If the present day characteristics of the meteorite and micrometeorite fluxes can be extrapolated back in time, then the flux of large carbonaceous micrometeorites could have contributed to the inventory of prebiotic molecules on the early Earth.     

Derepression of amino Acid-h cotransport in developing soybean embryos

The uptake of the unnatural amino acid α-aminoisobutyric acid (AIB) and glutamine by developing soybean (Glycine max Merr. cv Chippewa 64) embryos was investigated. In freshly excised embryos, the accumulation ratio (cytoplasmic concentration/external concentration) of AIB did not exceed 1.0. After an 18-hour preincubation in nitrogen-free medium the accumulation ratio of AIB exceeded 4.5 at an external AIB concentration of 10 micromolar. This indicates the derepression of an active amino acid uptake mechanism operative at low external amino acid concentration. The presence of sucrose, NH₄NO₃, or glutamine during a 21-hour preincubation prior to measuring glutamine uptake inhibited the enhancement of uptake by 43%, 51%, and 96%, respectively. The time course of the decline in free amino acids and the time course of enhancement of amino acid uptake was not consistent with enhanced uptake resulting from relief of transinhibition, but suggested instead the derepression of synthesis of new carriers. The time course of enhancement of amino acid uptake was paralleled by an increase in glutamine-induced depolarization of the membrane potential. The kinetics of glutamine uptake indicated the presence of a saturable and a nonsaturable component of uptake. The saturable component of uptake is attributed to a mechanism of amino acid-H⁺ cotransport which is derepressed by nitrogen and/or carbon starvation. At physiological concentrations of amino acids, uptake through the saturable system in freshly excised embryos is negligible. Thus, uptake through the nonsaturable system is of primary importance in the nitrogen nutrition of developing soybean embryos.     

The effect of transport system A and N amino acids and of nerve and epidermal growth factors on the induction of ornithine decarboxylase activity

The induction of ornithine decarboxylase (EC 4.1.1.17) (ODC) by amino acids and by the peptide hormones nerve growth factor (NGF) and epidermal growth factor (EGF) in salts-glucose media has been studied. Only those neutral amino acids taken into the cell via one of the Na+ dependent transport systems stimulate ODC activity. Asparagine and the nonmetabolizable alpha-amino-isobutyric acid (AIB) were used as representatives of this class of inducing amino acids, and their intracellular concentrations were related to the levels of ODC induced. A threshold intracellular concentration of asparagine or AIB has to be attained before ODC can be induced. Further slight increases in intracellular concentrations of asparagine or AIB produce disproportionately large increases of ODC, resulting in a sigmoidal curve of ODC induction. These results, and the fact that the decrease in ODC levels caused by valine is associated with a concurrent decrease in the intracellular level of the inducing amino acid, suggest that the intracellular amino acid level is causally related to the induction of ornithine decarboxylase. Glutamic acid, EGF, and NGF do not induce ODC except in the presence of an inducing amino acid. They act synergistically with the inducing amino acid and produce higher ODC levels at the same intracellular concentration of the inducing amino acid.     

Modulation of cellular heat sensitivity by specific amino acids

When either plateau-phase or exponentially growing Chinese hamster ovary (CHO) cells are incubated in amino acid-free medium, the cells become sensitized to killing by heat. For cells deprived of amino acids for 12 h survival decreases from 1 X 10(-2) for controls to 1 X 10(-6) for the deprived cells, following heating at 45 degrees C for 38 min. The survival of these sensitized cells is rapidly increased by the addition of a single amino acid just prior to heating. Of the 21 amino acids which are added in purified form to make McCoy's 5a medium, 12 show no protective effect, four have a small protective effect, and either alanine, asparagine, glutamine, serine, or theronine raise survival to a level similar to that of the control cells. The nonmetabolizable alanine analogue, 2-aminoisobutyric acid (AIB), increases survival of amino acid-deprived cells as effectively as each member of the group of five listed above, suggesting that metabolic conversion of the amino acids is not required for their protective effect. The data suggest that an increase in the intracellular concentrations of specific amino acids, independent of any change in cellular ATP content or the rate of protein synthesis, enables these cells to become quickly more resistant to killing by heat. We also conclude that the amino acid concentrations in poorly vascularized regions of some tumors should be considered, along with the oxygen, glucose, and proton concentrations, as factors which determine cellular survival following hyperthermia.     

Adaptive enhancement of amino acid uptake and exodus by thymic lymphocytes: influence of pH.

Entry of certain free amino acids (alpha aminoisobutyric acid (AIB), alanine and proline), but not of leucine into rat thymic lymphocytes increased progressively when the cells were incubated in amino acid deficient medium. Actinomycin D, cycloheximide, or a high concentration of AIB abolished the time-related increase in AIB accumulation, whereas exposure to a high concentration of leucine had no effect. This phenomenon could not be attributed to a progressive alteration in the nature of the incubation medium nor to reduced transinhibition of AIB uptake. The exodus of AIB also increased with time, but to a smaller degree than AIB entry. Initial rates of AIB entry and exodus increased with increases in the pH of the incubation medium over the range 6.5-8.0. The effects of pH on entry and exodus were time-related, increasing progressively oveb nullified the magnified time related increments in AIB transport caused by prolonged incubation at pH 8.0. The influence of a given pH on transport of AIB decreased rapidly when the cells were transferred to medium of another pH, but this tendency diminished the longer the cells were exposed to the initial pH. pH influenced the entry of alanine and proline in the same fashion as that of AIB, but did not affect leucine entry. These results indicate that thymic lymphocytes exhibit adaptive enhancement in the accumulation of free amino acids that are transported largley by the A or alanine-preferring system, and that the adaptive process involves both entry and exodus. Moreover, alterations in pH modify entry and exodus of these same amino acids, profoundly affect the magnitude of time-released increases, and may induce fundamental changes in the mechanism(s) serving amino acid transport.     

The catalytic mechanism of 1-aminocyclopropane-1-carboxylic acid oxidase

It has been proposed that 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase catalyzes the oxidation of ACC to ethylene via N-hydroxyl-ACC as an intermediate. However, due to its chemical instability the putative intermediate has never been isolated. Here, we have shown that a purified recombinant ACC oxidase can utilize alpha-aminoisobutyric acid (AIB), an analog of ACC, as an alternative substrate, converting AIB into CO2, acetone, and ammonia. We chemically synthesized the putative intermediate compound, N-hydroxyl-AIB (HAIB), and tested whether it serves as an intermediate in the oxidation of AIB. When [1-(14)C]AIB was incubated with ACC oxidase in the presence of excess unlabeled HAIB as a trap, no labeled HAIB was detected. By comparing the acetone production rates employing HAIB and AIB as substrates, the conversion of HAIB to acetone was found to be much slower than that of using AIB as substrate. Based on these observations, we conclude that ACC oxidase does not catalyze via the N-hydroxylation of its amino acid substrate. ACC oxidase also catalyzes the oxidation of other amino acids, with preference for the D-enantiomers, indicating a stereoselectivity of the enzyme.     

Effects of formamidoxime on macromolecular synthesis in regenerating liver and hepatomas

Formamidoxime caused an inhibition of [³H]thymidine incorporation into DNA in regenerating liver and transplanted hepatomas of different growth rates when administered by i.p. injection to rats. A dose level of formamidoxime (500 mg/kg body weight) which caused at least a 75% inhibition of DNA synthesis in these tissues had little or no effect on the incorporation of [³H]orotate into total RNA. After administration of formamidoxime there was no significant effect on amino acid nitrogen concentration in the tissues. The incorporation of ³H-labeled amino acids into acid-soluble material, cytoplasmic proteins and acid-insoluble nuclear proteins were either unaffected or showed only small changes after treatment of rats with the drug. In regenerating rat liver and Morris hepatomas 7787 and 7777, formamidoxime caused an inhibition of incorporation of ³H-labeled amino acids into both lysine-rich and arginine-rich histones. In the host livers of rats bearing the transplanted hepatomas, histone synthesis was less affected. The data indicated that formamidoxime causes inhibitory effects which are similar in nature and extent to those previously shown for the structurally related compound, hydroxyurea, in the regenerating rat liver and demonstrated that these effects can also be observed in liver tumors.     

Uptake and translocation of 2-aminoisobutyric acid bySchizophyllum commune

The nonnative amino acid, 2-aminoisobutyric acid (AIB), is transported into homokaryotic mycelia ofSchizophyllum commune. However, because it is neither incorporated into protein nor further metabolized by the fungal cells, it may be used as a marker for the free amino acid pool. Colonies grown or maintained under nitrogen-limiting conditions show enhanced uptake and higher concentrations of AIB in the mycelium than controls. Based on growth inhibition and uptake kinetic experiments, the enhanced uptake appears to be the result of decreased competition for a common uptake channel withl-asparagine normally found in the medium. Colonies containing radiolabeled AIB show translocation of the label to growing hyphal apices when they are transferred to unlabeled nitrogen-deficient media. Chases on nitrogen-rich media do not result in translocation of the label. In contrast to colonies chased on nitrogen-rich media, nitrogen deprivation does not lead to release of significant amounts of AIB into the medium by older cells. This supports an intramycelial pathway for translocation of pool amino acids.     

Effects of cyclic AMP and dibutyryl cyclic AMP on amino acid transport in the isolated rat ovary.

Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.     

The effect of prostaglandin E2 (PGE2) on amino acid uptake and protein formation by lung fibroblasts

The effect of prostaglandin E2 (PGE2) on the utilization of extracellular amino acids by fetal lung fibroblasts was examined. PGE2 decreased the uptake of proline and aminoisobutyric acid (AIB) by quiescent fibroblasts in culture. The uptake of AIB by serum-activated cultures was also dramatically decreased by PGE2. The PGE2-induced decrease in the uptake of AIB was first observed at 4 h following the addition of the effector molecule to the cultures. PGE2 did not affect the uptake of leucine. The addition of cycloheximide also resulted in a decrease in the uptake of proline, similar to that induced by PGE2 at 5 X 10(-8) M. The combination of cycloheximide and PGE2 resulted in a further decrease in proline uptake. Kinetic analysis of AIB uptake following a 24-h PGE2 treatment showed an increase in the apparent Km as compared with untreated cultures. The prostaglandin remained active for at least 72 h after the addition of the molecule. Removal of the PGE2 was followed by an influx of proline into the cells. The decrease in proline uptake was associated with a decrease in the amount of intracellular free proline and an overall decrease in the amount of cell-associated protein. While PGE2 is known to increase intracellular protein degradation, the effect of PGE2 on amino acid uptake was not the result of an increase in the intracellular concentration of amino acids (transinhibition).     

Uptake of 14C-α-aminoisobutyric acid by Phaseolus vulgaris

Specific uptake (S.U.) of α-aminoisobutyric acid ([1-¹⁴C]AIB), a non-metabolizable neutral amino acid analog, by dwarf bush bean plants (Phaseolus vulgaris cv Top Crop) demonstrated wide differences in active transport between various plant organs. The kinetic and timed uptake data reported were expressed as S.U. because this corrects for the diffusion of AIB which is part of the total AIB uptake process. Roots accumulated AIB to concentrations up to 18 times and leaf disks to twice those of the incubation medium. Stem tissue showed very little uptake, if any, that could not be accounted for by simple diffusion or water free space. Although initial rate kinetic studies demonstrated the presence of a normal transport system, timed uptake studies revealed greatly decreased transport by etiolated plants, suggesting a relationship between active transport and the lack of photosynthate. The reproducibility of the AIB uptake pattern by mature roots strongly supports the concept that the transport of neutral amino acids is biphasic and suggested one or more carrier systems are inducible by either low intracellular concentrations or repressed by high intracellular concentrations of the amino acid.     

The effects of ionizing radiation on the pulmonary endothelial cell uptake of alpha-aminoisobutyric acid and synthesis of prostacyclin

The effects of gamma irradiation (150-3000 rad) on prostacyclin synthesis (PGI2) and Na+-dependent amino acid uptake (alpha-aminoisobutyric acid, AIB) were assessed in vitro in bovine pulmonary artery endothelial cells grown in plastic culture dishes. A dose-dependent increase in both PGI2 synthesis and AIB was found 24 h after irradiation at exposure levels greater than 600 rad. The increase in PGI2 synthesis [297% of sham-irradiated values at 3000 rad, P less than 0.01] was due to an increase in release of arachidonic acid from plasma membrane stores as well as stimulation of cyclooxygenase and/or prostacyclin synthetase enzymes. The increase in AIB uptake (75% increase at 3000 rad compared to sham-exposure values) correlated with the increased synthesis of PGI2 (r = 0.94). There was also a dose-dependent increase in the number of cells that became detached from the culture dishes during the 24-h period after irradiation. The changes in PGI2 synthesis and AIB uptake induced by gamma irradiation differed if the endothelial cells were grown on cover slips, indicating that the endothelial response to irradiation may be dependent on the interaction between the endothelial cell and its extracellular basement membrane matrix.     

Transport and Compartmentation of 1-Aminocyclopropane-1-Carboxylic Acid and Its Structural Analog, alpha-Aminoisobutyric Acid, in Tomato Pericarp Slices

The uptakes of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor to ethylene, and its structural analog, α-aminoisobutyric acid (αAIB) by tomato pericarp slices were investigated. Both uptakes show a biphasic (saturable-linear) dependence on external concentration of the transported amino acid. At low concentrations, ACC uptake is competitively inhibited by αAIB and vice versa. Both uptakes also are inhibited by other neutral amino acids but not by acidic or basic amino acids. ACC and αAIB uptakes are metabolically dependent and are increased with time of tissue incubation. αAIB efflux patterns from pericarp slices indicated three distinct αAIB compartments having efflux kinetics consistent with those for cell wall, cytoplasm, and vacuole. The bulk of the αAIB taken up by pericarp tissue is sequestered into the vacuole. The ability of pericarp tissue to accumulate αAIB in the vacuole declines with fruit development.     

rBAT is an Amino Acid Exchanger with Variable Stoichiometry

The rBAT protein, when expressed in Xenopus oocytes, was previously shown to reproduce the selectivity of the Na⁺-independent neutral and basic amino acid transport system called b^o,+. More recently, the capacity of rBAT to generate a transmembrane current was demonstrated when addition of neutral amino acids stimulated the efflux of cations (presumably basic amino acids) in rBAT-injected oocytes. In the present paper, aminoisobutyric acid (AIB), a neutral amino acid analogue, was shown to induce outward currents (efflux of basic amino acids) through rBAT similar to those caused by alanine in terms of affinity, maximal currents and I-V curves. Despite generating similar currents, the AIB transport rate was more than 30 times lower than that of alanine, thus challenging the assumption that rBAT functions as a classical exchanger. Experiments using a cut-open oocyte voltage clamp demonstrated that AIB was capable of stimulating rBAT-mediated currents from either side of the membrane. AIB, like alanine, was able to stimulate the efflux of radiolabeled alanine and arginine while no rBAT-mediated efflux was measurable in the absence of external rBAT substrates. These results demonstrate that (i) the presence of amino acids is required on both sides of the membrane for rBAT to mediate amino acid flux and thus rBAT must be some type of exchanger but (ii) rBAT-mediated amino acid influx is not stoichiometrically related to the efflux. A model of a ``double gated pore' is proposed to account for these properties of rBAT, which contravene standard models of exchangers and other transporters. Received: 15 June 1995/Revised: 21 September 1995     

Effect of dibutyryl cyclic adenosine monophosphate on active amino acid transport in Streptomyces hydrogenans

The active uptake of 2-aminoisobutyric acid (AIB) and several other amino acids in resting cells of Streptomyces hydrogenans was found to be stimulated by exogenously added adenosine cyclic monophosphate (cAMP). The uptake of glycerol, sorbose, and pyrimidine nucleosides remained unaffected. Among the various cAMP derivatives tested, the dibutyryl derivative was found to be most effective, followed by monobutyryl cAMP, and cAMP. Dibutyryl cGMP was also found to stimulate AIB transport, and its effectivity was as good as that of dibutyryl cAMP. The effect of dibutyryl cAMP is time dependent and attains its maximum after 40–60 min of incubation at 30°C in K-Na-phosphate buffer. Dibutyryl cAMP-dependent transport stimulation has a high temperature coefficient and is prevented by rifamycin SV or chloramphenicol. The rate of leucine incorporation into protein was rapidly increased upon addition of dibutyryl cAMP. Kinetic studies reveal that the stimulation of AIB transport is characterized by an increase in maximum uptake rate and an unaltered apparent Michaelis constant. Analysis of the unidirectional fluxes show that both influx and efflux are enhanced by dibutyryl cAMP. It is concluded that exogenous dibutyryl cAMP stimulates de novo synthesis of certain protein including the transport catalysts for various amino acids.     

Regulation of lactate production and utilization in rat tumors in vivo

These experiments were performed to determine the factor(s) that regulate lactic acid production and utilization by rat tumors in vivo. Arteriovenous differences for glucose and lactic, pyruvic, 3-OH-butyric, and acetoacetic acids were measured across "tissue-isolated" Walker 256 sarcocarcinomas and Morris 5123C hepatomas in fasted rats anesthetized with sodium pentobarbital. Twenty-six per cent of the sarcocarcinomas (n = 53) and 48% of the hepatomas (n = 29) utilized blood lactic acid. The remainder released lactic acid into the venous blood. The steady-state rate of glucose consumption was similar in both lactate-producing and lactate-utilizing tumors. The range of lactate concentrations in the blood leaving the tumors was narrower than the range of lactate concentrations in the blood entering the tumors. This difference was caused by tumor lactic acid production at low arterial lactate concentrations and tumor lactic acid utilization at high arterial lactate concentrations. Individual tumors changed from lactic acid production to lactic acid utilization in a matter of minutes in response to an increase in the arterial lactic acid concentration. Mean lactic plus pyruvic acid concentrations and lactic/pyruvic acid ratios in the tumor venous blood were 2.15 +/- 0.22 and 23.4 +/- 3.7 mM, respectively, for Walker sarcocarcinoma 256 (n = 18) and 1.28 +/- 0.13 and 48.1 +/- 5.1 mM, respectively, for hepatoma 5123C (n = 11). The results suggest: that a steady-state lactic plus pyruvic acid concentration and lactic/pyruvic acid ratio are maintained in the tumor cell cytoplasm by the active glycolytic pathway and by lactic acid dehydrogenase; that the tumor intracellular concentrations equilibrate with the arterial blood and that the tumor steady state is expressed in the tumor venous blood; and that tumor lactic acid production or utilization results from the equilibration between the variable arterial lactic acid concentration and the more constant tumor intracellular steady-state lactic acid concentration. Since the arterial lactate concentration may be less than, greater than, or equal to the intracellular steady-state concentration, an individual tumor may produce, utilize or neither produce nor utilize lactic acid.