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At least 793 records · Page 44Linked to original sources
PubMed1996

Is gamma-actin a regulator of amino acid transport?

A mutated yeast cell line incapable of growth in minimal medium with proline as the sole nitrogen source was restored to normal growth by transfection with a cDNA from mouse Ehrlich cells. The cloned cDNA (E51) was found to be 90% homologous to gamma-actin. Immediately after transfection with E51 cDNA, both alpha-aminoisobutyric acid (AIB) and proline uptake in the mutated yeast were increased, particularly at pH 5. The expression of the same E51 cDNA also enhanced amino acid uptake in Xenopus laevis oocytes after injection into the Xenopus nuclei. A mutated mammalian lymphocyte cell line (GF-17), deficient in system A transport, also showed increased Na(+)-dependent transport after transfection with E51 cDNA. Whereas the mock transfected GF-17 cells failed to grow in the selection medium, the transfectants with E51 cDNA grew better than the untransfected cells. The data are consistent with the conclusion that expression of E51 cDNA can modify inactive, endogenous amino acid transporters, permitting substantial amino acid uptake in cells deficient in amino acid transporter(s) and permitting rapid cell growth. The data suggest that the gamma-actin-like protein coded for by E51 cDNA may play a significant regulatory role in amino acid transport.

Actins↗
PubMed1986

System A transport activity in normal rat hepatocytes and transformed liver cells: substrate protection from inactivation by sulfhydryl-modifying reagents.

The transport of amino acids by normal rat hepatocytes and several hepatoma cell lines has been examined for inactivation by various protein-modifying reagents, including the sulfhydryl-preferring reagents N-ethylmaleimide (NEM) and p-chloromercuribenzene sulfonate (PCMBS). Uptake of 2-aminoisobutyric acid (AIB), a specific probe for hepatic System A-mediated transport, was equally sensitive to inhibition by the organic mercurial PCMBS in each of the cell types tested. In contrast, the sensitivity of System A to inactivation by NEM was substantially different among the five cell types. Normal hepatocytes showed the greatest sensitivity, while the hepatoma cells varied in their responsiveness from moderate to no inhibition. PCMBS inactivated greater than 85% of the System A activity in rat H4 hepatoma cells within 10 min (t1/2 = 3 min). The inhibition by PCMBS was rapidly reversed by treatment of the cells with dithiothreitol. Amino acids showing a high affinity for System A protected the transport system from inactivation, whereas non-substrates produced little or no protection. Amino acid-dependent protection was stereospecific and system-specific. L-norleucine competitively inhibited AIB uptake (Ki = 1.9 +/- 0.1 mM) in H4 cells and also protected System A from PCMBS-dependent inactivation (half-maximal protection occurred at an amino acid concentration of 0.6 +/- 0.1 mM). N-bromosuccinimide was completely ineffective as an inhibitor of System A activity in hepatocytes, whereas treatment of H4 rat hepatoma cells with this reagent resulted in greater than 95% inhibition.

4-Chloromercuribenzenesulfonate↗
PubMed2000

Epidermal growth factor regulates amino acid transport in chick embryo hepatocytes via protein kinase C.

System A-mediated amino acid transport, activation of different steps of signal transduction and involvement of different isoforms of protein kinase C (PKC) have been investigated in chick embryo hepatocytes after epidermal growth factor (EGF) stimulation. EGF rapidly (10 min) increased the rate of aminoisobutyric acid (AIB) uptake in chick embryo hepatocytes freshly isolated on the 19th day of embryonic life, while no change was detectable at other embryonal stages. The growth factor stimulation was abolished by PKC and tyrosine kinase inhibitors and was mimicked by 4-phorbol-12-myristate-13-acetate, dimethyl-2 (PMA). EGF treatment did not modify the phosphorylation of the isoform of phospholipase C (PLC-), and inositol trisphosphate (IP3) and intracellular calcium levels, but it induced an increase in PKC activity. Our data show that EGF regulates amino acid uptake, via PKC and without PLC- activation, only in the last period of chick embryo hepatocyte development. The effects of growth factor on PKC activity suggest the involvement of PKC- and - isoforms in EGF modulation of amino acid transport.

Amino Acids↗
PubMed1987

Effect of antifungal agents on lipid biosynthesis and membrane integrity in Candida albicans.

Eight antifungal agents were examined for effects on lipid biosynthesis and membrane integrity in Candida albicans. Lipids were labeled in vivo or in vitro with [14C]acetate and analyzed by thin-layer and gas chromatography. Membrane integrity was measured by a recently developed [14C]aminoisobutyric acid radiolabel release assay. The imidazole antifungal agents miconazole, econazole, clotrimazole, and ketoconazole, at concentrations inhibiting ergosterol biosynthesis (0.1 microM), decreased the ratio of unsaturated to saturated fatty acids in vivo but not in vitro. Similarly, naftifine, tolnaftate, and the azasterol A25822B, at concentrations inhibiting ergosterol biosynthesis (10, 100, and 1 microM, respectively), decreased the ratio of unsaturated to saturated fatty acids in vivo only. This suggests that the effect on fatty acids observed with ergosterol biosynthesis inhibitors may be secondary to the effect on ergosterol. With imidazoles, oleic acid antagonized inhibition of cell growth but not inhibition of ergosterol. This suggests that, with the C-14 demethylase inhibitors, decreased unsaturated fatty acids, rather than decreased ergosterol, are responsible for growth inhibition. Cerulenin, previously reported to be a potent inhibitor of both fatty acid and ergosterol biosynthesis, was found in the present study to inhibit the former (at 5 microM) but not the latter (up to 100 microM). Of the antifungal agents tested, econazole and miconazole (at 100 microM) produced complete release of [14C]aminoisobutyric acid, which is consistent with membrane damage.

Allylamine↗
PubMed1992

Efflux of potassium (86Rb+) attenuates the volume-restorative effect of sodium-amino acid cotransport in rat renal inner medullary cells shrunken by exposure to hyperosmotic media.

When the osmolality of the bathing medium was increased from 710 to 2000 mosmol/kg H2O, cells in incubated slices of rat renal inner medulla lost water and K+, and the rate of efflux of preloaded 86Rb+ (a tracer for K+) was significantly depressed. Addition of 2-aminoisobutyric acid (AIB, 10 mmol/l) partly restored cell water content but without re-accumulation of K+; the rate of 86Rb+ efflux was greatly increased. The presence of Ba2+ (1 mmol/l) or trifluoperazine (50 mumol/l) led to complete recovery of cell volume and K+ contents, with markedly reduced efflux of 86Rb+. Neither additive had any significant effect upon these variables in the absence of AIB or in media of 710 mosmol/kg. Efflux of 86Rb+ was pH-sensitive within the physiological range, and was depressed when external AIB was reduced below approx. 5 mmol/l. When external Na+ was increased from 145 to 500 mmol/l (total osmolality 350 to 2500 mosmol/kg) efflux was retarded only slightly if AIB was present, but markedly if AIB was omitted. Inner medullary cells may contain a class of Ba(2+)-inhibitable, calmodulin-dependent K+ conductive pathway which is activated in strongly hyperosmotic media by the operation of an inwardly-directed Na(+)-amino acid symport (cf. Law, R.O. (1988) Pflügers Arch. 413, 43-50) and which serves to moderate the volume-restorative effect of this membrane mechanism.

Amino Acids↗
PubMed1986

Alterations in amino acid transport in Na,K-ATPase amplified HeLa cells.

Amino acid transport was studied in C1 cells which contain amplified levels of sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase), in C4 cells which are ouabain-sensitive revertants, and in parental HeLa S3. Sodium-dependent uptake of aminoisobutyric acid and alanine was increased 2-fold in the amplified C1 cells. After a 6 h amino acid starvation period, the rate of sodium-dependent uptake of methylaminoisobutyric acid was 70-90% greater for C1 than for C4 and HeLa. This uptake was inhibitable by ouabain and the apparent Km values for high affinity uptake were similar in all three lines. Overall, neutral amino acid uptake through Systems A, ASC, and L was 2-fold higher in the Na,K-ATPase amplified C1 cells relative to C4 or HeLa. The induction of System A uptake of methylaminoisobutyric acid after starvation was more rapid in both the amplified C1 cells and the revertant C4 when compared to HeLa, which suggests that the selection for amplification of the Na,K-ATPase produced membrane alterations affecting the adaptive regulation of System A.

Alanine↗
PubMed1975

Biphasic kinetic plots and specific analogs distinguishing and describing amino acid transport sites in S37 ascites tumor cells.

Curve-fitting procedures indicated that exo-2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) modified V and Km for one of two systems serving for histidine transport into the S37 ascites tumor cells. When this system was obliterated by leucine in the medium, BCH had no effect on histidine transport. Curve-fitting procedures similarly suggest N-methyl-alpha-aminoisobutyric acid affected the Km and V values for the other histidine-transporting system and that carboxymethylhistidine (His(Cm)) inhibited both transport systems. His(Cm) further inhibited histidine uptake into leucine-inhibited cells. Km and V values were altered simultaneously in the presence of several inhibitory analogs. Alanine methyl ester markedly inhibited high-concentration histidine uptake, whereas leucine methyl ester markedly inhibited low-concentration histidine uptake. The present results confirm earlier suggestions that our high c system is Christensen's A system and our low c system his L system. We also confirm a very high degree of specificity of N-methyl-alpha-aminoisobutyric acid for the A or high c system, and of BCH for the L or low c system. We suggest the utility of combining two approaches to the study of transport system properties; use of specific analogs and modification of biphasic plots. We demonstrate that the carboxyl group is not a prerequisite molecular feature for inhibitory interaction with the A or L system.

Alanine↗
PubMed1986

Maintenance of glucagon-stimulated system A amino acid transport activity in rat liver plasma membrane vesicles.

Plasma membrane vesicles prepared from intact rat liver or isolated hepatocytes retain transport activity by systems A, ASC, N, and Gly. Selective substrates for these systems showed a Na+-dependent overshoot indicative of energy-dependent transport, in this instance, driven by an artificially-imposed Na+ gradient. Greater than 85% of Na+-dependent 2-aminoisobutyric acid (AIB) uptake was blocked by an excess of 2-(methylamino)isobutyric acid (MeAIB) with an apparent Ki of 0.6 mM. Intact hepatocytes obtained from glucagon-treated rats exhibited a stimulation of system A activity and plasma membrane vesicles isolated from those same cells partially retained the elevated activity. Transport activity induced by substrate starvation of cultured hepatocytes was also evident in membrane vesicles prepared from those cells. The membrane-bound glucagon-stimulated system A activity decays rapidly during incubation of vesicles at 4 degrees C (t1/2 = 13 h), but not at -75 degrees C. Several different inhibitors of proteolysis were ineffective in blocking the decay of transport activity. Hepatic system N transport activity was also elevated in plasma membrane vesicles from glucagon-treated rats, whereas system ASC was essentially unchanged. The results indicate that both glucagon and adaptive regulation cause an induction of amino acid transport through a plasma membrane-associated protein.

Amino Acids↗
PubMed1976

Placental amino acid uptake. III. Transport systems for neutral amino acids.

The human placenta is known to concentrate nearly all amino acids intracellularly for transfer to the fetus. To clarify the mechanism and regulation of this process we have determined the specificity of the principal placental transport systems for neutral amino acids. With the use of competitive inhibition techniques, three transport systems of overlapping specificity have been elucidated. These correspond approximately to the "A", "L", and "ASC" systems of Christensen and associates. In the placenta the specificity of these systems is as follows: A system - alpha aminoisobutyric acid (AIB), glycine, proline, N-methylalanine, alanine, serine, threonine, and glutamine; L system - isoleucine, valine, phenylalanine, BCH, alanine, serine, threonine, and glutamine; and ASC system - alanine, serine, threonine, and glutamine. Placental AIB uptake previously has been shown to increase with preincubation of tissue in vitro. This increase has now been found to be limited to the A system. Activity of the other two systems is essentially unaffected, demonstrating that the transport pathways are separately regulated.

Amino Acids↗
PubMed1982

Neutral amino acid transport in hepatocytes isolated from streptozotocin-induced diabetic rats.

In the present study, transport by Systems A, ASC, and N was shown to be elevated in hepatocytes isolated from diabetic rats. After the cells were placed in primary culture, the System ASC activity declined rapidly, while the decay of Systems A and N was slower and dependent on protein synthesis. The elevated 2-aminoisobutyric acid uptake was the result of an increase in the Vmax of a single, high affinity system, presumably System A. The stimulation of System A could not be accounted for by an increase in the affinity for Na+; in fact, the apparent Km for the ion was actually greater in the experimental cells. Release from trans-inhibition was also eliminated as a possible explanation. The data suggest that during the development of the diabetic state the liver is triggered to induce the activity of System A by synthesizing the necessary protein components. Treatment of the cultured hepatocytes with insulin could partially reverse the stimulation due to diabetes, indicating that the induction of System A may be the result of the hyperglucagonemia associated with the disease. In support of this hypothesis, the cells from the diabetic rats were resistant to further stimulation of System A by glucagon, yet they did respond to high levels of insulin or to amino acid starvation. Glucagon does not appear to be involved in the induction of System N in the diabetic animal because this system is not responsive to either glucagon or insulin when tested in vitro.

Adrenalectomy↗
PubMed1980

Incorporation and Degradation of C and H-labeled Thymidine by Sugarcane Cells in Suspension Culture.

Sugarcane cells growing in suspension culture degrade exogenous thymidine, releasing thymine. Thymine is not utilized for DNA synthesis. Thymine is rapidly catabolized to beta-aminoisobutyric acid which is found within the cell. Thymidine in the medium is used for DNA synthesis. The label of [2-(14)C]thymidine is lost as (14)CO(2), but the label of [(3)H]methylthymidine is found in the cell as [(3)H]beta-aminoisobutyric acid, some of which is used for the synthesis of other cell components. The degradation of thymidine can be partially inhibited by addition of certain substituted pyrimidines.

Journal Article↗
PubMed1993

Effect of platelet-activating factor on basal and insulin-mediated system A amino acid transport in rat soleus muscle.

We investigated the effect of platelet-activating factor (PAF) on basal and insulin-stimulated sodium-dependent neutral amino acid transport by system A in rat soleus muscle. Fasted male Sprague-Dawley rats (75-100 g) were given an intravenous injection of saline or PAF (4 micrograms/kg) and killed 1 hr later. Isolated soleus muscles were incubated in media containing 1-14C-alpha-aminoisobutyric acid (AIB; system A amino acid analog), 3H-inulin, and 0 (basal) or 100 mU/ml insulin. Na(+)-free media were assessed by Na(+)-independent AIB uptake. The rate of AIB uptake by muscle was corrected for uptake into extracellular (inulin) space. Basal cellular AIB uptake by soleus muscles from PAF-treated rats was 30.2% lower than in controls. Insulin increased the absolute rate of cellular AIB uptake above basal levels to the same extent in muscles of control and PAF-treated rats, although the rate of maximal insulin-stimulated AIB uptake was 20% lower in muscles of PAF-treated rats. Na(+)-independent AIB uptake (measured in Na(+)-free media) was the same in muscles of control and PAF-treated rats. The fact that AIB uptake in Na(+)-free media was the same in muscles of control and PAF-treated rats indicates that the reduction in basal and insulin-stimulated AIB uptake in muscles of PAF-treated rats was due to an alteration of system A Na(+)-dependent amino acid transport.

Amino Acids↗
PubMed1977

Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake.

Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.

Amino Acids↗
PubMed1990

Insulin-induced phospho-oligosaccharide stimulates amino acid transport in isolated rat hepatocytes.

The ability of the insulin-induced phospho-oligosaccharide to stimulate amino acid transport was studied in isolated rat hepatocytes. At low alpha-aminoisobutyric acid concentrations (0.1 mM), both 100 nM-insulin and 10 microM-phospho-oligosaccharide doubled amino acid uptake after 2 h of incubation. This stimulation was prevented by 0.1 mM-cycloheximide or 5 micrograms of actinomycin D/ml, indicating that the phospho-oligosaccharide, like insulin, was acting via the synthesis of a high-affinity transport component. The effects of the phospho-oligosaccharide and of insulin were blocked by Ins2P (2.5 mM), but not by myo-inositol, inositol hexaphosphoric acid or several monosaccharides such as mannose, glucosamine and galactose. Both the temporal effect on amino acid entry and the extent of stimulation of this process by the phospho-oligosaccharide indicate that this molecule mimics, and may mediate, some of the long-term actions of insulin. However, the effects of phospho-oligosaccharide and insulin were not exactly the same, since the effect of insulin, but not of the phospho-oligosaccharide, was additive with that of glucagon.

Amino Acids↗
PubMed1987

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.

Amino Acids↗
PubMed1977

Effects of Osmotic Shock on Some Membrane-regulated Events of Oat Coleoptile Cells.

Oat coleoptile sections (Avena sativa L. cv. "Garry") were osmotically shocked with 0.5 m mannitol followed by 1 mm Na-phosphate (pH 6.4) at 4 C. This treatment reduced uptake of alpha-aminoisobutyric acid, 3-o-methyl glucose, and leucine by 75 to 90% but inhibited (36)Cl(-) uptake only 30%. Some recovery was observed 1 to 3 hours later. Respiration rates were unaffected by osmotic shock and protein synthesis was reduced 11%.Osmotic shock also stimulated efflux of alpha-aminoisobutyric acid and K(+) and led to an increase in conductivity of the solution bathing shocked sections. The transmembrane electropotential of 75% of the shocked cells fell to -20 mv to -45 mv compared with the majority of unshocked cells at -80 mv to -120 mv.We concluded that osmotic shock selectively modifies the plasma membrane. The inhibitions of uptake could be due to removal of specific components of the plasma membrane and/or to the lowered electropotential.

Journal Article↗
PubMed1977

Blood flow and uptake of glucose and amino acids in ischemic muscle.

In order to examine muscle ischemia, muscle blood flow in the rat hindlimb was decreased by vessel ligation. Amino acid uptake, studied with [14C]alpha-aminoisobutyric acid, was decreased in ischemic Type I (soleus) muscle. Glucose uptake, studied with [14C]deoxyglucose, was increased in Type I muscle. These changes were temporally associated with histologic changes of ischemia in soleus muscle. Denervation, atrophy, and hypertrophy also produced uptake changes with these techniques, and although more prominent in soleus, the changes were also seen in Type II muscle.

Absorption↗
PubMed1995

In vivo release of neuroactive amino acids from the inferior colliculus of the guinea pig using brain microdialysis.

Microdialysis techniques were used to measure in vivo release of neuroactive amino acids from the central nucleus of the inferior colliculus (ICC) in anesthetized guinea pigs. Concentric dialysis probes were implanted in the ICC and perfused with Ringer solution of various compositions at a flow rate of 2.0 microliters/min. Consecutive 10-min fractions of the dialysate were collected for up to 3 h under different experimental conditions, frozen and assayed for amino acid content by high performance liquid chromatography (HPLC). There was an initial high outflow of amino acids which declined to stable baseline levels after 2 h. Following this stabilization period, perfusion with a medium containing 100 mM KCl produced an increase in the extracellular levels of aspartate (Asp), glutamate (Glu), gamma-aminobutyric acid (GABA) and glycine (Gly). Only the increases in GABA and Gly were statistically significant. None of the increases occurred in the presence of 2.0 mM cobalt suggesting the release of amino acids is calcium dependent. Histological examination revealed that tissue damage was minimal and largely confined to the immediate vicinity of the probes. We were also able to show that the blood brain barrier (BBB) appeared to heal 2 h after probe implantation. Thus, following intravenous injection of [3H]alpha-aminoisobutyric acid (AIB), which does not cross the intact BBB, no isotope was recovered in the dialysate. These results demonstrate that microdialysis is a unique and suitable method to monitor changes in the extracellular levels of amino acid neurotransmitters in a central auditory structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗