Amino acid transport by aggregates of cultured chicken heart cells. Effect of insulin.

Single heart cells dissociated from 14-day-old chicken embryos were reagregated into spheroidal clusters on a gyratory shaker and centrifuged to form cohesive discs of approximately 400 aggregates. These cultured cells accumulated 2-amino[1-14C]isobutyric acid against a gradient. When incubated for 3 hours in a protein-free, buffered, balanced salt solution, concentrative transport decreased to a stable basal level. Incubation in the presence of sodium bovine insulin prevented this fall in transport activity and resulted in increased 2-aminoisobutyric acid uptake to concentrations 40 time sthat in the medium during a subsequent 3-hour transport assay. Intracellular accumulation of 2-aminoisobutyric acid was linear during the initial 15 min of transport in the presence and absence of added insulin. Basal transport of 2-aminoisobutyric acid was temperature-dependent, requied extracellular sodium greater than 125 meg/liter, and demonstrated saturation with an apparent Vmax of 3.4 mmol/liter/10 min and an apparent Km of 2.6 mM. Basal transport activity was not reduced by cycloheximide or puromycin even after 3 hours of exposure...     

Anaerobic transport of serine and 2-aminoisobutyric acid by Staphylococcus epidermidis.

A membrane-bound ATPase detected in extracts of anaerobically grown Staphylococcus epidermidis was inhibited by a variety of compounds which inhibit ATPases in other organisms. Serine and 2-aminoisobutyric acid (AIB) were shown to enter the organism via the same transport system. The transport of AIB, the membrane potential and the transmembrane pH gradient were partially or completely abolished by the same inhibitors and also by uncoupling agents and lipid-soluble ions. It is proposed therefore that this ATPase generates and maintains an electrochemical gradient of protons across the cytoplasmic membrane of S. epidermidis capable of driving AIB uptake. Studies of AIB-induced proton movements suggested that AIB enters via a proton symport mechanism.     

Characterization of 2-aminoisobutyric acid transport in Neurospora crassa: a general amino acid permease-specific substrate.

We report the characterization of an amino acid 2-aminoisobutyric acid was transported solely by the general amino acid permease and not by the neutral amino acid permease. Furthermore, this substrate was not metabolized after transport. The potential for a system-specific nonmetabolizable substrate as a tool in the analysis of amino acid transport and its regulation is discussed.     

Kinetic analysis of insulin action on amino acid uptake by isolated chick embryo heart cells

1. Isolated chick embryo heart cells were used to investigate the mode of action of insulin on the transport of three naturally occurring amino acids: l-proline, l-serine and glycine. Initial velocities of uptake were measured over a period of 5min with an 80-fold range of amino acid concentration. Corrections for amino acid diffusion, incorporation into protein and conversion into carbon dioxide were introduced. 2. The uptake processes approximated Michaelis-Menten kinetics within definite ranges of amino acid concentrations. A single transport system for proline and at least two transport systems for serine and glycine were detected. 3. The kinetic effects of insulin on transport systems for the amino acids tested were consistent with an acceleration of the maximal velocity of the process, without substantial changes in substrate concentration for half-maximal transport velocity. 4. These hormonal effects were not essentially altered by the corrections for amino acid incorporation into protein and conversion into carbon dioxide.     

Active transport of amino acids by gamma-glutamyl transpeptidase through Caco-2 cell monolayers.

The possible role of the gamma-glutamyl cycle in the transport of amino acids, using the Caco-2 cell monolayer as an in vitro model of the small intestine, has been investigated. The transport of [2-3H]glycine and [2-3H]glycylglycine through the Caco-2 monolayer has been shown to occur by two modes of action. Active transport is unidirectional from apical to basolateral region and is a carrier mediated system. The enzyme gamma-glutamyl transpeptidase seems to be involved in this process, since when the enzyme is inhibited, the active transport is also inhibited. However transport still takes place, and this occurs by a slower non-active process, which is bidirectional and is mediated by passive diffusion. The rate of transport of [2-3H]glycylglycine and [2-3H]glycine were 585 (+/- 24) and 287 (+/- 16) pmolcm-2min-1 respectively, while the non-active transport takes place at 87 (+/- 6) pmolcm-2min-1. Thus, amino acid translocation in Caco-2 cells is shown to occur by two methods, one of which involves the gamma-glutamyl cycle.     

Phase resetting of embryonic chick atrial heart cell aggregates. Experiment and theory.

The influence of brief duration current pulses on the spontaneous electrical activity of embryonic chick atrial heart cell aggregates was investigated experimentally and theoretically. A pulse could either delay or advance the time of the action potential subsequent to the pulse depending upon the time in the control cycle at which it was applied. The perturbed cycle length throughout the transition from delay to advance was a continuous function of the time of the pulse for small pulse amplitudes, but was discontinuous for larger pulse amplitudes. Similar results were obtained using a model of the ionic currents which underlie spontaneous activity in these preparations. The primary ion current components which contribute to phase resetting are the fast inward sodium ion current, INa, and the primary, potassium ion repolarization current, IX1. The origin of the discontinuity in phase resetting of the model can be elucidated by a detailed examination of the current-voltage trajectories in the region of the phase response curve where the discontinuity occurs.     

Enhanced insulin action on glucose transport and insulin signaling in 7-day unweighted rat soleus muscle.

Hindlimb suspension (HS), a model of simulated weightlessness, enhances insulin action on glucose transport in unweighted rat soleus muscle. In the present study, we tested the hypothesis that these changes in glucose transport in 3- and 7-day HS soleus of juvenile, female Sprague-Dawley rats were due to increased functionality of insulin signaling factors, including insulin receptor (IR), IR substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3-kinase), and Akt. Insulin-stimulated (2 mU/ml) glucose transport was significantly (P < 0.05) enhanced in 3- and 7-day HS soleus by 59 and 113%, respectively, compared with weight-bearing controls. Insulin-stimulated tyrosine phosphorylation of IR and Ser(473) phosphorylation of Akt was not altered by unweighting. Despite decreased (34 and 64%) IRS-1 protein in 3- and 7-day HS soleus, absolute insulin-stimulated tyrosine phosphorylation of IRS-1 was not diminished, indicating relative increases in IRS-1 phosphorylation of 62 and 184%, respectively. In the 7-day HS soleus, this was accompanied by increased (47%) insulin-stimulated IRS-1 associated with the p85 subunit of PI3-kinase. Interestingly, the enhanced insulin-stimulated glucose transport in the unweighted soleus was not completely inhibited (89-92%) by wortmannin, a PI3-kinase inhibitor. Finally, protein expression and activation of p38 MAPK, a stress-activated serine/threonine kinase associated with insulin resistance, was decreased by 32 and 18% in 7-day HS soleus. These results indicate that the increased insulin action on glucose transport in the 7-day unweighted soleus is associated with increased insulin signaling through IRS-1 and PI3-kinase and decreased p38 MAPK protein expression. However, PI3-kinase-independent mechanisms must also play a small role in this adaptive response to HS.     

Characteristics of transport of selenoamino acids by epithelial amino acid transporters

Selenoamino acids are the main form of organic selenium derived from the diet. They are efficiently absorbed in the intestine and reabsorbed in kidney, but the transporter proteins that mediate their cellular uptake have not yet been identified. We here describe the transport pathways of selenoamino acids and derivatives, including selenomethionine, methylselenocysteine, selenocystine, selenobetaine and selenocystamine. Transport studies employed the Xenopus laevis oocyte system expressing the amino acid transporters SIT1, b^0,+rBAT, B⁰ or PAT1 and intestinal Caco-2 and renal OK cell lines that possess a multitude of amino acid transporters. Our results suggest that the major route for the uptake of selenomethionine is the system b^0,+ rBAT in Caco-2 cells and B⁰ in OK cells. Affinity of selenomethionine or methionine for these transporters did not differ, but for SIT1 selenomethionine shows a higher affinity than methionine. Methylselenocysteine displayed a higher affinity than cysteine for all transporters tested and in both OK and Caco-2 cells, system B⁰ seems to be the primary uptake route. Selenocystine is taken up well by the b^0,+ rBAT system, while selenobetaine is a low-affinity substrate only for SIT1 and PAT1. Selenocystamine was not transported by any of the transport systems investigated. When cells were exposed to selenoamino acids, intracellular selenium levels in OK cells considerably exceeded those in Caco-2 cells, indicating effective renal reabsorption capacity. In conclusion, selenoamino acids but not the seleno-derivatives selenobetaine and selenocystamine, are effectively transported by various intestinal and renal amino acid transporters and are thus available for selenium metabolism and therapeutic approaches.     

Control by amino acids of the activity of system A-mediated amino acid transport in isolated rat hepatocytes.

The effect of amino acids, in concentrations corresponding to those found in the portal vein of rats given a high-protein diet, was investigated on the activity of system A amino acid transport in hepatocytes from fed rats. Amino acids counteracted the induction of system A by insulin or glucagon. This effect was observed at all concentrations of hormones tested, up to 1 microM. Amino acids did not affect the basal cyclic AMP concentration in hepatocytes, or the large rise in cyclic AMP elicited by glucagon. The reversal of system-A induction was observed at relatively low concentration of amino acids, corresponding to plasma values reported in rats given a basal diet. Amino acids were separately tested: substrates of system A were particularly efficient, but so were glutamine and histidine. Non-metabolizable substrates of system A, such as 2-aminoisobutyrate, were also inhibitory, suggesting that a part of the effect of amino acids is independent of their cellular metabolism. Provision of additional energy substrates such as lactate and oleate did not affect induction of system A or the inhibitory effects of amino acids. Thus amino acids do not act by serving as an energy source and by maintaining the integrity of hepatocytes. Inhibition of mRNA synthesis by actinomycin practically abolished the effect of amino acids on the induction of system A by glucagon. The results suggest that amino acids may promote the synthesis of protein(s) affecting the activity of system A either directly at the carrier unit or at an intermediate stage of its emergence.