Uptake Of Α-aminoisobutyric Acid Research Articles

Functional and molecular characteristics of system l in human breast cancer cells

The functional and molecular properties of system l in human mammary cancer cells (MDA-MB-231 and MCF-7) have been examined. All transport experiments were conducted under Na +-free conditions. α-Aminoisobutyric acid (AIB) uptake by MDA-MB-231 and MCF-7 cells was almost abolished by BCH (2-amino-2-norbornane-carboxylic acid). AIB uptake by MDA-MB-231 cells was also inhibited by l-alanine (83.6%), l-lysine (75.6%) but not by l-proline. Similarly, l-lysine and l-alanine, respectively, reduced AIB influx into MCF-7 cells by 45.3% and 63.7%. The K m of AIB uptake into MDA-MB-231 and MCF-7 cells was, respectively, 1.6 and 8.8 mM, whereas the V max was, respectively, 9.7 and 110.0 nmol/mg protein/10 min. AIB efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH, l-glutamine, l-alanine, l-leucine, l-lysine and AIB (all at 2 mM). In contrast, l-glutamate, l-proline, l-arginine and MeAIB had no effect. The interaction between l-lysine and AIB efflux was one of low affinity. The fractional release of AIB from MDA-MB-231 cells was trans-accelerated by d-leucine and d-tryptophan but not by d-alanine. MDA-MB-231 and MCF-7 cells expressed LAT1 and CD98 mRNA. MCF-7 cells also expressed LAT2 mRNA. The results suggest that AIB transport in mammary cancer cells under Na +-free conditions is predominantly via system l which acts as an exchange mechanism. The differences in the kinetics of AIB transport between MDA-MB-231 and MCF-7 cells may be due to the differential expression of LAT2.

A STUDY OF THE EFFECT OF THEOPHYLLINE ON THE ANTI-INFLAMMATORY POTENCY OF DEXAMETHASONE USING α-AMINOISOBUTYRIC ACID UPTAKE IN RAT SKIN FIBROBLASTS: A POSSIBLE MECHANISM THROUGH THE GLUCOCORTICOID RECEPTOR

The action of theophylline on the uptake of α-aminoisobutyric acid (AIB; an indicator of anti-inflammatory potency) stimulated by the glucocorticoid, dexamethasone, in cultured rat fibroblast monolayers was evaluated. Theophylline alone (0.1mm) did not show significant activity (3314±27cpm) compared with the baseline level (3186±130cpm), but in the presence of 10nmdexamethasone the stimulation of AIB uptake was increased to 5263±100cpm, approximately to the same extent as with 100nmdexamethasone alone (5397±28cpm). Activation of glucocorticoid-receptor complexes in rat fibroblast cytosol was studied by assessing the extent of their binding to DNA-cellulose. Activated and non-activated forms of glucocorticoid-receptor complexes were analysed by DEAE-Sephadex chromatography. Theophylline (1mm) was found to have a direct effect (0°C), similar to that of heat (25°C) on DNA-cellulose binding, that is approximately 64.5% and 68.7%, respectively, thus indicating that theophylline promotes activation of glucocorticoid receptors at low temperature. The effect of theophylline on the stimulation of AIB uptake by dexamethasone when considered in the light of its activation of GR receptors in the fibroblast cytosol indicates that this effect may be mediated by GR activation.

Insulin signaling and its regulation of system A amino acid uptake in cultured rat vascular smooth muscle cells.

Hyperinsulinemia has been recognized as an independent risk factor for atherosclerosis. However, its exact mechanisms are still unclear. In our previous work, we showed that 10 nmol/L insulin stimulated neither mitogen-activated protein kinase (MAP kinase) activity nor [3H]thymidine incorporation but did stimulated S6 kinase through the specific insulin receptors in cultured rat vascular smooth muscle cells (VSMCs). In this study, we observed that > or = 1 nmol/L insulin stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and activated IRS-1-dependent phosphatidylinositol 3'-kinase (PI 3'-kinase) and p70 S6 kinase (p70S6K) but not MAP kinase (extracellular signal-regulated kinase 2) and p90 S6 kinase (p90RSK). However, 10 nmol/L insulin-like growth factor I stimulated all these pathways. Finally, 10 nmol/L insulin stimulated alpha-amino-isobutyric acid (AIB) uptake, and wortmannin (100 nmol/L) completely inhibited insulin-stimulated AIB uptake, whereas rapamycin (20 nmol/L) had no such effect. Furthermore, cycloheximide (10 micrograms/mL) completely inhibited insulin-stimulated AIB uptake, but actinomycin D (5 micrograms/mL) failed to inhibit this. Thus, we reached the following conclusions: (1) Insulin (1 nmol/L) induced phosphorylation of IRS-1 and activated the PI 3'-kinase and p70S6K pathways in VSMCs, even though 10 nmol/L insulin did not significantly stimulate MAP kinase or p90RSK. (2) Stimulation of AIB uptake by insulin was regulated at the translational level via wortmannin-sensitive pathways but not p70S6K pathways.

Effects of benfluorex metabolites on membrane fluidity and insulin-related processes

As little work has dealt with the antihyperglycemic property of benfluorex at the hepatocyte level, we studied the effects of its main metabolites, S422 and S1475, on membrane fluidity and on insulin binding, internalization and action in healthy rat hepatocytes. Both metabolites were effective fluidizing agents. Neither one affected insulin binding. Only S422 favored the bound insulin-receptor internalization process. The metabolites produced no change in basal α-aminoisobutyric acid uptake. Only S422 promoted the insulin-stimulated α-aminoisobutyric acid uptake in a dose-dependent way. Therefore, our study demonstrated that: (i) the effects of S422 on insulin-related processes in isolated hepatocytes were direct, specific and not due to any membrane fluidizing mechanism; (ii) S422 improved hepatocyte response to insulin at a post-binding level. These results in vitro give an additional explanation, at the cellular level, of the benefit of benfluorex treatment for non insulin-dependent diabetes patients.

A comparative study on the uptake of α-aminoisobutyric acid by normal and immortalized human embryonic kidney cells from proximal tubule

We investigated whether an immortalized human kidney epithelial cell line (IHKE), compared with normal embryonic cells (NHKE), can be used as a representative system with which to characterize the transport of neutral amino acids in the proximal tubule of the human kidney. The IHKE cell line, immortalized by treatment with NiSO 4, exhibited microvilli and enzyme markers specific for highly specialized tubule cells. The Na +-dependent uptake of α-aminoisobutyric acid (AIB) by IHKE and NHKE cells occurred by means of a single transport system with identical half-saturation constants, but the capacity for uptake was higher in the immortalized cells. Proton-dependent influx of AIB was also mediated by a single transport component with similar uptake characteristics in both types of cells. Imposition of an H +-gradient to a Na +-gradient reduced the sodium dependent uptake of AIB with the exception of short incubation time (1 min), where addition of a proton gradient produced a marked increase in the Na +-dependent influx of AIB in NHKE but not in IHKE cells. Competition experiments revealed that the Na +-dependent uptake at 50 μM AIB was reduced by neutral α-amino acids in the two cell lines. L-Glutamate, L-aspartate, L-arginine and the β-amino acid taurine had no effect. Only in the IHKE cell line did addition of 5 mM L-lysine produce a slight inhibition. Except for L-proline all of the neutral and acidic amino acids tested reduced the H +-dependent uptake of AIB in the IHKE cell line. By contrast, addition of L-aspartate did not influence the transport of AIB in NHKE cells, L-Arginine, but not L-lysine decreased the influx in both cell lines. We conclude that the IHKE cell line has retained the capability to accumulate AIB by transport protein(s) similar to those present for neutral α-amino acids in NHKE cells.

Amino acid uptake by the cultured human placental trophoblast: Exogenous regulation

Summary Amino acid uptake by the human placenta is known to occur via several transport mechanisms. However, regulation by extracellular factors has received relatively little attention. A recent report by this laboratory had demonstrated that α-(methylamino)isobutyric acid (MeAIB)-sensitive, α-aminoisobutyric acid (AIB) uptake by the cultured human placental trophoblast cells is stimulated by insulin. This study evaluated insulin like growth factor-1 (IGF-1) stimulation of AIB uptake in cultured human placental trophoblast cells. Na + -dependent AIB uptake was significantly stimulated by IGF-I in a time dependent manner, as early as 30 minutes. The maximum effect was at 2–4 hours of continuous exposure to IGF-I. Stimulation was dependent upon IGF-1 concentration. AIB uptake was inhibited by increasing concentrations of MeAIB. Approximately 75% of basal (unstimulated) AIB uptake was inhibited by MeAIB. The IGF-1 stimulated increment above basal AIB uptake was completely inhibited by MeAIB. At 200 ng/ml, stimulation was greater than IGF-1 than with IGF-1. The IGF-1 effect was inhibited by anti-IGF-1 receptor antibody, confirming mediation via IGF-1 receptor. In addition to the IGFs, treating the cultured trophoblast cells with 8-br-cAMP or PMA temporally stimulated AIB uptake. Therefore, Na + -dependent AIB uptake appears to be stimulated by factors which function via receptor tyrosine kinase and/or serine/threonine protein kinases (protein kinases A and C).

In cultured human placental trophoblasts, ethanol inhibits uptake of α-amino isobutyric acid (AIB) following stimulation by insulin (Ins) and insulin-like growth factor-1 (IGF-1)

In cultured human placental trophoblasts, ethanol inhibits uptake of α-amino isobutyric acid (AIB) following stimulation by insulin (Ins) and insulin-like growth factor-1 (IGF-1)

Regional differences in insulin receptor function in Werner's syndrome

Werner's syndrome is a genetic disease characterized by premature aging and is often associated with glucose intolerance due to insulin resistance. The clinical manifestations in this syndrome are preferentially expressed in the face and acral regions without apparent involvement of the trunk. We compared insulin receptor binding and amino acid uptake of fibroblasts derived from the forearm that had sclerodermoid features, and from the abdomen that was apparently normal in a patient with Werner's syndrome. In normal controls, specific insulin binding was not different in forearm and abdomen-derived fibroblasts (10.72 ± 2.11%, 10.40 ± 1.27%, respectively). In the patient, however, specific insulin binding was reduced in the fibroblasts derived from the forearm compared with those derived from the abdomen (3.55%, 8.16%, respectively). Scatchard analysis revealed that the reduction in insulin binding of the forearm fibroblasts from the patient was due to a reduction in receptor number with no change in receptor affinity. The dose-response curve for insulin of α-aminoisobutyric acid (AIB) uptake is shifted to the right in the fibroblasts derived from the acral area. The results show that in a patient with Werner's syndrome, regional differences occur in fibroblast insulin receptor binding and function. This suggests early phenotypic expression of the genetic abnormality of insulin receptor function in these patients.

In vitro effect of insulin on the uptake of glucose and α-aminoisobutyric acid in the thyroid gland of the turtle ( Chrysemys dorbigni)

The presence of specific insulin binding sites in the thyroid gland of the turtle Chrysemys dorbigni has been previously reported. The purpose of the present work was to investigate the probable action of insulin on the uptake of [ 14C]deoxy- d-glucose ([ 14C]DG) and [ 14C]α-aminoisobutyric acid ([ 14C]AIB) in turtle ( C. dorbigni) thyroid glands in vitro. Thyroid fragments (±40 mg) were incubated at 25° in Krebs-Ringer-bicarbonate buffer containing 0.2 μCi of [ 14C]DG or [ 14C]AIB without or with bovine insulin at different periods of time. The uptake of [ 14C]DG and [ 14C]AIB increased with incubation time. The presence of insulin (7 × 10 −6 M) in the incubation medium during 240 min did not modify the [ 14C]DG uptake. However if the thyroid fragments were previously incubated with insulin (60 min) and then incubated (240 min) with the same concentration of the hormone, the [ 14C]DG uptake was markedly increased. This stimulatory effect of insulin was dose-dependent. In similar experimental conditions, insulin also produced a significant increase in the uptake of [ 14C]AIB. Therefore, these findings strongly support the hypothesis that insulin might exert a direct action on the thyroid function.

Production of Inhibitor of Insulin-Receptor Tyrosine Kinase in Fibroblasts From Patient With Insulin Resistance and NIDDM

Although non-insulin-dependent diabetes mellitus (NIDDM) is associated with defects in insulin action, the molecular basis of this resistance is unknown. We studied fibroblasts from a markedly insulin-resistant patient with NIDDM but without acanthosis nigricans. Her fibroblasts were resistant to insulin when alpha-aminoisobutyric acid uptake was measured. Fibroblasts from this patient demonstrated normal insulin-receptor content as measured by both insulin-receptor radioimmunoassay and by Scatchard analysis. However, when compared with nondiabetic control subjects, insulin-receptor kinase assays of wheat-germ-purified receptors prepared from her fibroblasts showed very low basal and no insulin-stimulated tyrosine kinase activity. The insulin receptor was then removed from the wheat-germ fraction by monoclonal antibody affinity chromatography. This insulin-receptor-deficient fraction inhibited both basal and insulin-stimulated tyrosine kinase activity of highly purified insulin receptors. When the specificity of this inhibition was tested, less inhibition was seen with insulinlike growth factor I-receptor tyrosine kinase, and even less inhibition was seen with the proto-oncogene p60c-src tyrosine kinase. Thus, these studies indicate that fibroblasts from an insulin-resistant patient with NIDDM produce a relatively specific glycoprotein inhibitor of insulin-receptor tyrosine kinase. Therefore, these studies raise the possibility that this inhibitor may play an important role in the insulin resistance seen in this patient.

Effect of sepsis on amino acid transport system A and its response to insulin in incubated rat skeletal muscle

The effect of sepsis on neutral amino acid transport systems A, ASC, and L, was studied in incubated rat soleus (SOL) muscles. We also examined the effects of plasma from septic rats and of varying concentrations of insulin (10 to 10 5 μU/mL), added in vitro to incubated muscles, on system A amino acid transport. Sepsis was induced by cecal ligation and puncture (CLP) in rats weighing 40 to 60 g. Control rats were sham-operated. System A activity was assessed by determining uptake of 2-(methylamino)isobutyrate (MeAIB) 16 hours after CLP or sham-operation. System ASC was studied by measuring uptake of α-aminoisobutyric acid (AIB) in the presence of 25 mmol/L MeAIB and 25 mmol/L 2-amino-2-norbornane carboxylic acid (BCH) to inhibit uptake by systems A and L. System L activity was defined as sodium-independent uptake of cycloleucine. MeAIB uptake was reduced by 28% in muscles of septic rats, while amino acid transport by systems ASC and L was almost identical in muscles from control and septic rats. Addition of plasma from septic rats to incubated normal SOL muscles inhibited MeAIB uptake by 31%. Addition of insulin to the incubation medium resulted in increased uptake of MeAIB, both in nonseptic and septic muscle. The lowest hormone concentration tested that significantly enhanced MeAIB uptake in nonseptic muscle was 10 2 μU/mL and in septic muscle 10 μU/mL. The results suggest that sepsis in rats specifically inhibits amino acid transport system A and that reduced muscle amino acid uptake may be caused by a circulating factor in sepsis. Physiological concentrations of insulin stimulate system A amino acid transport in incubated skeletal muscle of both nonseptic and septic rats.

Glyburide but not ciglitazone enhances insulin action in the liver independent of insulin receptor kinase activation

To test the hypothesis that sulfonylureas enhance insulin action by activating the insulin receptor tyrosine kinase, the effects of glyburide, a second generation sulfonylurea, and ciglitazone, a nonsulfonylurea hypoglycemic agent, were determined in primary cultures of rat hepatocytes on insulin action and insulin receptor structure and function. Twenty hours of preincubation with glyburide (1 μg/mL) resulted in increased insulin (1 × 10 −7 mol/L) stimulation of [ 14C] acetate incorporation into lipids and [ 14C] α-aminoisobutyric acid uptake without any change in basal activity. Ciglitazone (1 μg/mL) was without any effect. Glyburide's actions were mediated without altering the following: (1) 125I-insulin binding; (2) the electrophoretic mobility of the affinity labeled α-subunit or the autophosphorylated β-subunit of the insulin receptor; and (3) the insulin-stimulated insulin receptor kinase activity using histone or the β-subunit of the insulin receptor as phosphoacceptors. These data suggest that the action of sulfonylureas is distal to the insulin receptor tyrosine kinase. Ciglitazone in vitro is ineffective in the liver, which suggests the peripheral tissues as the possible site of action.

The Proton Electrochemical Transmembrane Gradients Generated by the Transfer Cells of the Haustorium of Polytrichum formosum and Their Use in the Uptake of Amino Acids

The epidermal cells of the sporophyte haustorium of Polytrichum formosum are modified into transfer cells. These cells are located in a strategic place allowing them to control the exchanges between the two generations. Their plasmalemma creates proton gradients (Deltapsi and DeltapH) which increase during the development of the sporophyte. As the sporophyte grows from 2 to 4 cm long, the pH of the incubation medium of the haustoria decreases from 5.2 to 4.3, and the transmembrane potential difference (PD) hyperpolarizes form -140 to -210 millivolts. These gradients become rapidly larger than that generated by the plasmalemma of the basal cells of the sporophyte. They are used to energize the uptake of the solutes present in the apoplast of the gametophyte, particularly the amino acids. Below 20 micromolar alpha-aminoisobutyric acid uptake in the transfer cells is mediated by a saturable system and is optimal at acidic pH (4.0 and 4.5). It is strongly inhibited by compounds dissipating both Deltapsi and DeltapH (10 micromolar carbonylcyanide-m-chlorophenyl hydrazone) or only Deltapsi (0.1 molar KCl). The absorption of alpha-aminoisobutyric acid and of the other neutral amino acids tested induces an alkalinization of the medium and a depolarization of membrane potential difference which is concentration dependent. These data show that the uptake of amino acids by the transfer cells of the haustorium is a secondary translocation (proton-amino acid symport) energized by a primary translocation (proton efflux). More particularly, they show that transfer cells possess a membrane enzymic equipment particularly efficient to achieve the uptake of the solutes leaked in the apoplast from other cell types.

Different preparations of natural and recombinant human interleukin-6 (ifn-β 2, bsf-2) similarly stimulate acute phase protein synthesis and uptake of α-aminoisobutyric acid by cultured rat hepatocytes

1. 1. Rat hepatocytes were cultured for 2 days in Williams E medium containing l μM insulin and dexamethasone. 2. 2. Production of five plasma proteins was determined by electroimmunoassay in the media, and amino acid uptake was measured by [α- 14C]aminoisobutyric acid accumulation in hepatocytes. 3. 3. Supernatants from rat peritoneal macrophages and IL-6/IFN-β 2/BSF-2 obtained from four different laboratories similarly stimulated synthesis of fibrinogen, α 1-cysteine proteinase inhibitor and α 2-macroglobulin, as well as[ 14C]-accumulation in cultured hepatocytes. 4. 4. It is concluded that IL-6 is the principal hepatocyte stimulating factor responsible for typical features of the acute phase response of liver cells.

Effects of caffeine on amino acid transport in the brain

Chronic administration of caffeine to mice did not alter cellular (low-affinity) transport as measured in brain slices of the amino acids γ-aminobutyric acid, glutamic acid, and glycine. Chronic caffeine administration did, however, increase the long-term (60-min) uptake of α-aminoisobutyric acid and valine into brain slices. A similar tendency, although not statistically significant, towards increased amino acid uptake was also seen in the transport of phenylalanine and lysine across the blood—brain barrier in chronically treated rats. The increase in neutral amino acid uptake seems unrelated to changes in brain protein metabolism, since chronic caffeine administration did not significantly alter brain protein synthesis rates. In vitro, caffeine (0.01–1 mM) did not alter brain slice transport of the amino acids γ-aminobutyric acid, glutamic acid, glycine, valine, and the amino acid analog α-aminoisobutyric acid. Caffeine (1–100 μM) was also ineffective in altering the synaptosomal (high-affinity) transport of the neurotransmitter amino acids γ-aminobutyric acid, glutamic acid, and glycine. These data support the idea that caffeine is a mildly stimulating drug with no significant effect on neurotransmitter amino acid transport systems in rodent brain.

Inhibition of Taurocholate and Ouabain Transport in Isolated Rat Hepatocytes by Cyclosporin A

The use of cyclosporin A in transplantation procedures has been reported to cause hepatotoxicity as evidenced by elevated serum bilirubin and bile salt levels. However, these biochemical abnormalities could also result from interference with hepatic transport processes. This possibility was investigated in the present study in which the effect of cyclosporin A on transport processes was examined in isolated rat liver cells. Taurocholate, ouabain, and α-aminoisobutyric acid were selected as compounds known to enter liver cells by distinct active transport systems and cadmium was selected as a substance taken up by a combination of simple and facilitated diffusion. Cyclosporin A was found to cause a dose-related inhibition of both taurocholate and ouabain uptake. On the other hand, the uptake of α-aminoisobutyric acid and of cadmium were unaffected by cyclosporin A. These findings indicate a substrate-specific effect of cyclosporin A rather than a general effect on cellular transport. Efflux of taurocholate from preloaded hepatocytes was also inhibited by cyclosporin A. Cyclosporin A caused a decrease in maximum velocity for ouabain uptake with no change in Km. Kinetic analysis for both uptake and efflux of taurocholate showed an unchanged maximum velocity and an increased Km. The data indicate that the ability of liver cells to take up and release bile acids is impaired in the presence of cyclosporin A. These findings provide a possible explanation for the finding of increased serum bile acids during cyclosporin A therapy and suggest that hepatic clearance of other compounds could also be impaired.

Effects of Ca 2+ on phytoalexin induction by fungal elicitor in soybean cells

A glucan elicitor from the cell walls of the fungus Phytophthora megasperma f.sp. glycinea caused increases in the activities of the phytoalexin biosynthetic enzymes, phenylalanine ammonia-lyase and chalcone synthase, and induced the production of the phytoalexin, glyceollin, in soybean ( Glycine max) cell suspension cultures when tested in culture medium containing 1.2 mmol/liter Ca 2+. Removal of extracellular Ca 2+ by treatment with ethylene glycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid followed by washing the cells with Ca 2+-free culture medium abolished the elicitor-mediated phytoalexin response. This suppression was largely reversed on readdition of Ca 2+. Elicitor-mediated enhancement of biosynthetic enzyme activities and accumulation of glyceollin was strongly inhibited by La 3+; effective concentrations for 50% inhibition were (μmol/liter) 40 for phenylalanine ammonia-lyase, 100 for chalcone synthase, and 30 for glyceollin. Verapamil caused similar effects only at concentrations higher than 0.1 mmol/ liter, whereas trifluoperazine and 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate did not affect enzyme induction by the elicitor in the concentration range tested. Uptake of α-amino isobutyric acid into soybean cells, which was rapidly inhibited in the presence of the glucan elicitor, was not affected by La 3+ nor was uptake inhibition by the elicitor relieved by La 3+. The Ca 2+ ionophore, A23187, enhanced phytoalexin biosynthetic enzyme activities and glyceollin accumulation in a dose-dependent manner, with 50% stimulation (relative to the elicitor) occurring at about 5 μmol/liter. The results suggest that the glucan elicitor causes changes in metabolite fluxes across the plasma membrane of soybean cells, among which changes in Ca 2+ fluxes appear to be important for the stimulation of the phytoalexin response.

Identification of an insulin fragment produced by an insulin degrading enzyme, neutral thiopeptidase

Previous studies have shown that neutral thiopeptidase (E.C.3.4.22.11, insulinase) degrades (processes) insulin with a high affinity ( K m = 30 × 10 −9 M). In the current studies, insulin was subjected to digestion with a highly purified rat liver neutral thiopeptidase and the peptides generated were separated by HPLC using a C 8 column. With the use of structural analysis (which included the determination of amino terminal residues and amino acid composition), the major product was identified as a peptide containing portions of both chains of insulin, A 1 to A 13 and B 1 to B 9 having two disulfide bonds, an interchain disulfide bond and presumably the intra-A chain disulfide bond as well. Examination of insulin-like biological activity using a primary cultured hepatocyte test system showed that the fragment promoted neither short-term (α-aminoisobutyric acid uptake) nor long-term (glycogen synthesis) bioactivities of insulin.

Effect of delta-9-tetrahydrocannabinol on the in vitro uptake of alpha-amino isobutyric acid by term human placental slices.

Tetrahydrocannabinol (THC), the active component in marijuana smoke, crosses the placenta and is a potential fetotoxic agent. In both human and animal studies, the most consistent fetal effect of THC is intrauterine growth retardation. Since fetal somatic growth is dependent on placental transfer of nutrients, including essential amino acids, we studied the effect of THC upon the in vitro uptake of amino acid by term human placental slices. Uptake of alpha-amino isobutyric acid was inhibited in a dose-dependent fashion, correlating with the log of the dose (1-100 microM THC; r = 0.945; p less than 0.01). Compared to control tissue, significant impairment of alpha-amino isobutyric acid uptake began at 20 microM THC. Similar results were found for valine. The time course (30-120 min) for alpha-amino isobutyric acid uptake showed linearity for both control and THC-(50 microM) treated tissue, but there was a marked reduction in the THC slope. Uptake of alpha-amino isobutyric acid was significantly reduced at all times. The sustained effect of THC was slightly, but significantly, reversed by removal of THC from the medium after 90 min of 50 microM THC exposure. Only partial reversal may have been due to the 15- to 20-fold accumulation of THC in the placental tissue. Uptake kinetics showed noncompetitive inhibition with decreased Vmax: control Vmax = 51.66 +/- 6.26 versus 50 microM THC = 26.96 +/- 6.22 (mmol/liter intracellular water per h) (p less than 0.01); and no change in diffusion constant (Km): control Km = 0.78 +/- 0.08 versus 50 microM THC = 0.80 +/- 0.09 (mM).(ABSTRACT TRUNCATED AT 250 WORDS)

HPLC Analysis of Amino Acids with Ion Exchange Chromatography and O-Phthalaldehyde Post-Column Derivatization: Applications to the Assay of Endogenous Free Amino Acids and Their Transport in Human Placental Villus

Abstract A method using high performance liquid chromatography (HPLC) for the analysis of primary amino acids in human placenta is described. This method involves separation of primary amino acids by high performance ion-exchange chromatography followed by post column derivatization using O-phlthalaldehyde (OPA) and 2-mercaptoethanol and fluorescence (excitation 340 nm and emission 410 nm) detection of derivatives. Waters 840 HPLC Amino Acid System was used for this purpose. For analysis, villus tissue was extracted with acetonitrile, and the recovered amino acids were reconstituted in a sodium diluent (pH 2.2). The complete profile of the primary amino acids in the sample could be constructed in about 90 minutes. Up to 44 samples can be analyzed without special attention. Using this method, essential amino acids (threonine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine) and nonessential amino acids (aspartic acid, serine, glutamic acid, glycine, alanine, arginine) were detected and quantified in human placental villus in pmol quantities. Plots of peak heights (or areas) were linear for several amino acids. The same method was also used for (a) the assay of free primary amino acids in umbilical bloods, (b) the efflux of amino acids from isolated human placental villus, and (c) to study the uptake of α-aminoisobutyric acid (AIB), a non-metabolizable amino acid, by the isolated placental villus.