Amino acid transport systems in animal cells: Interrelations and energization

After summarizing the discrimination of the several transport systems of neutral amino acids in the cell of the higher animal, I discuss here the ways in which 2 dissimilar transport systems interact, so that one tends to run forward for net entry and the other backwards for net exodus. An evaluation of the proposals for energization shows that uphill transport continues when neither alkali-ion gradients nor ATP levels are favorable. Evidence is presented that under these conditions a major contribution is made by another mode of energization, which may depend on the fueling of an oxidoreductase in the plasma membrane. This fueling may involve the export by the mitochondrion of the reducing equivalents of NADH by one of the known shuttles, e.g., the malate-aspartate shuttle. After depletion of the energy reseves in the Ehrilich cell by treating it with dinitrophenol plus iodoacetate concentrative uptake of test amino acids is restoration by pyruvate but in poor correlation with the restoration of alkali-ion gradients and ATP levels. This restoration by pyruvate but not by glucose is highly senstitive to rotenone. A combination of phenazine methosulfate and ascorbate will also produce transport restoration, before either the alkali-ion gradients or ATP levels have begun to rise. The restoration of transport applies to a model amino acid entering by the Na⁺-independent system, as well as to one entering by the principal Na⁺-dependent system, restoration being blocked by ouabain, despite the weak effect of ouabain on the alkali-ion gradients in the Ehrlich cell. Quinacrine terminates very quickly the uptake of model amino acids, before the alkali-ion gradients have begun to fall and before the ATP level has been halved. Quinacrine is also effective in blocking restoration of uphill transport by either pyruvate or the phenazine reagent. Preliminary results show that vesicles prepared from the plasma membrane of the Ehrlich cell quickly reduce cytochrome c or ferricyanide in the presence of NADH, and that the distribution of a test amino acid between the vesicle and its environment is influenced by NADH, quinacrine, and an uncoupling agent in ways consistent with the above proposal, assuming that a majority of the vesicles are everted.     

D-glucose transport in decapod crustacean hepatopancreas

Physiological mechanisms of gastrointestinal absorption of organic solutes among crustaceans remain severely underinvestigated, in spite of the considerable relevance of characterizing the routes of nutrient absorption for both nutritional purposes and formulation of balanced diets in aquaculture. Several lines of evidence attribute a primary absorptive role to the digestive gland (hepatopancreas) and a secondary role to the midgut (intestine). Among absorbed organic solutes, the importance of D-glucose in crustacean metabolism is paramount. Its plasma levels are finely tuned by hormones (crustacean hyperglycemic hormone, insulin-like peptides and insulin-like growth factors) and the function of certain organs (i.e. brain and muscle) largely depends on a balanced D-glucose supply. In the last few decades, D-glucose absorptive processes of the gastrointestinal tract of crustaceans have been described and transport mechanisms investigated, but not fully disclosed. We briefly review our present knowledge of D-glucose transport processes in the crustacean hepatopancreas. A discussion of previous results from experiments with hepatopancreatic epithelial brush-border membrane vesicles is presented. In addition, recent advances in our understandings of hepatopancreatic D-glucose transport are shown, as obtained (1) after isolation of purified R-, F-, B- and E-cell suspensions from the whole organ by centrifugal elutriation, and (2) by protein expression in hepatopancreatic mRNA-injected Xenopus laevis oocytes. In a perspective, the applicability of these novel methods to the study of hepatopancreatic absorptive function will certainly improve our knowledge of this structurally complex organ.     

Specific D-glucose transport in sarcolemma vesicles.

The sarcolemmal fraction prepared from rat skeletal muscle consists of osmotically active vesicles that accumulate D-glucose in preference to L-glucose, apparently by facilitated diffusion into intravesicular space. Stereospecific D-glucose uptake by these vesicles is a saturable rpocess, inhibited by phloridzin, by cytochalasin B, and by certain sugars, and enhanced by counterflow. An additional leak pathway permits entry of both D- and L-glucose into the vesicles. Stereospecific D-glucose transport by sarcolemmal vesicles is enhanced to a small extent by insulin, provided the hormone is administered prior to cell disruption. In membranes prepared from insulin-pretreated muscle, Ca2+ produces a small further enhancement. Local anesthetics preferentially inhibit stereospecific D-glucose transport. Apparent uptake of both D- and L-glucose is greater when vesicles are suspended in salt solutions rather than sucrose, an effect attributed to increased functional vesicular volume.     

Temperature dependence of D-glucose transport in reconstituted liposomes

Sodium-dependent D-glucose uptake into proteoliposomes reconstituted from dimyristoylphosphatidylcholine (DMPC) and hog kidney brush border membrane extract is strongly affected by temperature and the physical state of the membranes. This dependence is defined by a nonlinear Arrhenius plot with a break point at 23 degrees C, a temperature not significantly different from the phase transition temperature of the pure lipid (24 degrees C). The transport process is characterized by different activation energies: 35.1 kcal/mol below and 5.5 kcal/mol above the transition temperature. The shift in the break point for the D-glucose transport activity from 15 degrees C, in the brush border membranes, to 23 degrees C in the reconstituted system leads us to conclude that the lipids surrounding the sodium/D-glucose cotransport system can exchange readily with the bulk lipid used for reconstitution. The results thus provide no evidence for the presence of an annulus of specific lipids surrounding the transport system.     

Phorbolesters enhance basal D-glucose transport but inhibit insulin stimulation of D-glucose transport and insulin binding in isolated rat adipocytes

The C-kinase activating phorbolester TPA (12-O-Tetradecanoyl-beta-phorbol-13-acetate) and PdBu (4-beta-Phorbol-12,13,dibutyrate) stimulated D-glucose transport twofold in isolated rat adipocytes but inhibited high affinity insulin binding and the responsiveness of D-glucose transport to insulin stimulation by about 30%. Phorbolesters have therefore insulin-like effects but antagonize insulin on receptor and postreceptor level.     

Interactions of sodium pentobarbital with D-glucose and L-sorbose transport in human red cells.

Pentobarbital acts as a mixed inhibitor of net D-glucose exit, as monitored photometrically from human red cells. At 30 degrees C the Ki of pentobarbital for inhibition of Vmax of zero-trans net glucose exit is 2.16+/-0.14 mM; the affinity of the external site of the transporter for D-glucose is also reduced to 50% of control by 1. 66+/-0.06 mM pentobarbital. Pentobarbital reduces the temperature coefficient of D-glucose binding to the external site. Pentobarbital (4 mM) reduces the enthalpy of D-glucose interaction from 49.3+/-9.6 to 16.24+/-5.50 kJ/mol (P<0.05). Pentobarbital (8 mM) increases the activation energy of glucose exit from control 54.7+/-2.5 kJ/mol to 114+/-13 kJ/mol (P<0.01). Pentobarbital reduces the rate of L-sorbose exit from human red cells, in the temperature range 45 degrees C-30 degrees C (P<0.001). On cooling from 45 degrees C to 30 degrees C, in the presence of pentobarbital (4 mM), the Ki (sorbose, glucose) decreases from 30.6+/-7.8 mM to 14+/-1.9 mM; whereas in control cells, Ki (sorbose, glucose) increases from 6.8+/-1.3 mM at 45 degrees C to 23.4+/-4.5 mM at 30 degrees C (P<0.002). Thus, the glucose inhibition of sorbose exit is changed from an endothermic process (enthalpy change=+60.6+/-14.7 kJ/mol) to an exothermic process (enthalpy change=-43+/-6.2 7 kJ/mol) by pentobarbital (4 mM) (P<0.005). These findings indicate that pentobarbital acts by preventing glucose-induced conformational changes in glucose transporters by binding to 'non-catalytic' sites in the transporter.     

Choline transport specificity in animal cells and tissues

1. Beta carbolines inhibit choline transport in rat brain. 2. The aziridinium ring on the nitrogen of mustard analogs of choline causes irreversible binding to the carrier in rat brain. 3. The uptake system in rat brain is stereoselective, requires a quaternary nitrogen, and prefers analogs with a nitrogen-oxygen distance of about 3.26 A. 4. In mouse brain troxonium derivatives inhibit choline transport. 5. In cuttlefish optic lobes and torpedo electric organ pyrene derivatives potently inhibit choline transport. 6. In guinea pig placenta, the affinity of the choline carrier remains high even when this molecule lacks one or two methyl groups.     

An evaluation of intra-cage ventilation in three animal caging systems

Although temperature and relative humidity have been quantitated and their effects on research data studied, few studies have measured the air turnover rates at cage level. We evaluated the air distribution and air turnover rates in unoccupied shoe-box mouse cages, filter-top covered cages and shoe-box mouse cages housed in a flexible film isolator by using discontinuous gas chromatography/mass spectrometry and smoke. Results showed that air turnover was most rapid in the unoccupied shoe-box mouse cage and slowest in the filter-top covered cage. Placing mice in the filter-top covered cage did not significantly improve the air turnover rate. Although filter-top covered cages reduce cage-to-cage transmission of disease, the poor airflow observed within these cages could lead to a buildup of gaseous pollutants that may adversely affect the animal's health.     

Energy sources for amino acid transport in animal cells

The existence of an electrogenic Na⁺ pump in Ehrlich cells which substantially contributes to the membrane potential, previously derived from the distribution of the lipid soluble cation tetraphenylphosphonium (TPP⁺), could be confirmed by an independent method based on the quenching of fluorescence of a cyanine dye derivative, after the mitochondrial respiration had been suppressed by appropriate inhibitors. The mitochondrial membrane potential, by adding to the overall potential as measured in this way is likely to cause an overestimation of the membrane potential difference (p.d.). But since this error tends to diminish with increasing pump activity, the true p.d. of the plasma membrane should easily account for the driving force to drive the active accumulation of amino acids in the absence of an adequate Na⁺ concentration gradient. Accordingly, the F₂-aminoisobutyric acid (AIB) uptake rises linearly with the distribution of TPP⁺ at constant Na⁺ concentrations, suggesting that each responds directly to membrane potential. There is evidence that the electrogenic (free) movement of Cl^? is slow, at least at normal p.d., whereas a major part of the Cl^? movement across the cellular membrane appears to occur by an electrically silent Cl^?-base exchange mechanism. By such a mode Cl^?, together with an almost stoichiometric amount of K⁺, may under certain conditions move into the cell against a high adverse electrical potential difference. This “paradoxical” movement of K⁺Cl^? contributing to the deviation of the Cl^? distribution from the electrochemical equilibrium distribution, is not completely understood. It is insensitive towards ouabain but can almost specifically be inhibited by furosemide. As a likely explanation a H⁺–K⁺ exchange pump was previously offered, even though unequivocal evidence of such a pump is so far lacking. According to available evidence the electrogenic movement of free Cl^? is too small, at least at normal orientation of the p.d., to significantly shunt the electrogenic pump potential so that the establishment of such a potential is plausible. The evidence presented is considered strong in favor of the gradient hypothesis since even in the absence of an adequate Na⁺ concentration gradient, the electrogenic Na⁺ pump will contribute sufficient extra driving force to actively transport amino acid into the cells.     

Acidic amino acid transport in animal cells and tissues

1. The occurrence and characterization of acidic amino acid transport in the plasma membrane of a variety of cells and tissues of a number of organisms is reviewed. 2. Several cell types, especially in brain, possess both high- and low-affinity transport systems for acidic amino acids. 3. High-affinity systems in brain may function to remove neurotransmitter amino acid from the extracellular environment. 4. Many cell systems for acidic amino acid transport are energized by an inwardly directed Na+ gradient. Moreover, certain cell types, such as rat brain neurons, human placental trophoblast and rabbit and rat kidney cortex epithelium, respond to an outwardly directed K+ gradient as an additional source of energization. This simultaneous action may account for the high accumulation ratios seen with acidic amino acids. 5. Rabbit kidney has been found to have a glutamate-H+ co-transport system which is subject to stimulation by protons in the medium. 6. Acidic amino acid transport in rat brain neurons occurs with a stoichiometric coupling of 1 mol of amino acid to 2 mol of Na+. For rabbit intestine, one Na+ is predicted to migrate for each mol of amino acid. 7. Uptake in rat kidney cortex and in high-K+ dog erythrocytes is electrogenic. However, uptake in rabbit and newt kidney and in rat and rabbit intestine is electroneutral. 8. Na+-independent acidic amino acid transport systems have been described in the mouse lymphocyte, the human fibroblast, the mouse Ehrlich cell and in rat hepatoma cells. 9. In a number of cell systems, D-acidic amino acids have substantial affinity for transport; D-glutamate, in a number of systems, however, appears to have little reactivity. 10. Acidic amino acid transport in some cell systems appears to occur via the "classical" routes (Christensen, Adv. Enzymol. Relat. Areas Mol. Biol. 49, 41-101, 1979). For example, uptake in the Ehrlich cell is partitioned between the Na+-dependent A system (which transports a wide spectrum of neutral amino acids), the Na+-dependent ASC system (which transports alanine, serine, threonine, homoserine, etc.), and the Na+-independent L system (which shows reactivity centering around neutral amino acids such as leucine and phenylalanine). Also, a minor component of uptake in mouse lymphocytes occurs by a route resembling the A system. 11. Human fibroblasts possess a Na+-independent adaptive transport system for cystine and glutamate that is enhanced in activity by cystine starvation.(ABSTRACT TRUNCATED AT 400 WORDS)