L-alanine Uptake Research Articles

Depolarization of the liver cell membrane by metformin

Metformin (1,1-dimethylbiguanide; MET) is used in the treatment of type 2 diabetes mellitus. MET’s antihyperglycemic action depends at least in part on its inhibitory effect on hepatic gluconeogenesis. As to gluconeogenesis from amino acids (e.g. l-alanine), this is associated with an inhibition of l-alanine uptake into hepatocytes. Since this uptake is mediated by an electrogenic transport mechanism, the aim of the present study was to investigate whether MET has an influence on the liver cell membrane potential which might explain its inhibitory effect on l-alanine uptake. The experiments were performed in vivo in anesthetized rats and in vitro using superfused mouse liver slices with the conventional microelectrode technique. In vivo, MET (160 mg/kg intraperitoneally (i.p.)) significantly depolarized (d V) the liver cell membrane by 6 mV. MET (1 mmol/l) also depolarized the liver cell membrane in vitro (e.g. 15 min after start of superfusion: d V=8 mV). MET’s effect was at least partly reversible. Glucagon (10 −7 mol/l), which hyperpolarized the liver cell membrane, abolished MET’s effect. Further, the MET-induced depolarization was completely absent during superfusion with low Cl − ([Cl −]=27 mmol/l) medium, and significantly attenuated by the Cl − channel blocker NPPB (25 μmol/l). While MET’s effect was only somewhat attenuated by blockade of the Na +/K +/2Cl − cotransporter or by superfusion with (HCO − 3-free) HEPES buffer, the carboanhydrase blocker acetazolamide (1 mmol/l) or blockade of the HCO − 3/Cl − exchanger by DIDS (100 μmol/l), which, however, also blocks Cl − channels, abolished its effect. The depolarization of the liver cell membrane by MET was unaffected by a blockade of K + channels with Ba 2+, a blockade of the Na +/K + pump or superfusion with low Na + medium ([Na +]=26 mmol/l). According to these results, the MET-induced depolarization of the liver cell membrane could be due to an activation of the Cl −/HCO − 3 exchanger and thus depend on intracellular HCO − 3 formation. This activation could then lead to a disturbance of the equilibrium between intra- and extracellular Cl − and therefore to an enhanced Cl − efflux via Cl − channels. It is plausible that the depolarizing effect induced by MET is associated with its inhibitory effect on gluconeogenesis by inhibiting uptake of l-alanine and other amino acids into hepatocytes.

L-leucine and L-alanine uptake by trout ( Salmo trutta ) cardiomyocytes: the effect of IGF-I and insulin

The uptake of L-leucine and L-alanine by trout cardiomyocytes was analysed to identify the carriers involved in their incorporation into cells. The sodium independent systems L and asc, as well as the sodium dependent system ASC, were found to be present. The Vmax of these carriers was 33.0, 40.1 and 19.4 pmol amino acid mg−1 prot min−1 for L, asc and ASC, respectively, and the Km values obtained for these transport systems were 296, 269 and 349 μM amino acid. The L system had as its main substrates the large apolar branched-chain amino acids and showed stereospecificity. The asc and ASC systems carried the short-chain neutral amino acids and were not inhibited by Me-AIB. The ASC transporter also showed adaptive regulation. The effect of IGF-I and insulin on L-alanine uptake, as well as the incorporation of L-leucine into protein, were also studied. Low IGF-I concentrations (below 2.5 ng ml−1) stimulated the uptake of L-alanine and protein synthesis but this effect reverted at high IGF-I concentrations. Insulin depressed protein synthesis and did not affect L-alanine uptake.

Enantioselective uptake of amino acid with overoxidized polypyrrole colloid templated with L-lactate.

An overoxidized polypyrrole colloid, which can recognise enantiomers of amino acids has been prepared by a newly developed molecular imprinting technique. A polypyrrole colloid, which had been polymerised from a mixture of pyrrole (monomer), polyvinylpyrrolidone (steric stabiliser), peroxodisulfate (oxidant) and L-lactate (dopant), was overoxidized to create a dopant-complementary cavity. The enantioselectivity of the overoxidized colloid was evaluated by comparing the uptake of L-alanine and L-cysteine with that of the respective D-enantiomers. The L/D uptake ratios for these amino acids were about 2, while phenylalanine showed suppressed uptake for both the enantiomers. The absence of phenylalanine uptake can be explained in terms of the molecular size, which is too large to be accommodated by the cavity created by L-lactate. In contrast, a colloid templated with L-phenyllactate took up L-phenylalanine with a higher enantioselectivity of about 7. A colloid templated with L-lactate was applied to surface chirality analysis through enantioselective adsorption on cysteine-modified gold surfaces. Quartz microbalance experiments and scanning electron microscope observation of the gold surface revealed that the colloidal particle has higher affinity to a surface modified with L-cysteine than to one modified with D-cysteine.

Characteristics of transport systems of L-alanine in mouse mammary gland and their regulation by lactogenic hormones: evidence for two broad spectrum systems.

The characteristics of the transport systems of L-alanine in lactating mouse mammary gland and their regulation by lactogenic hormones have been studied. L-alanine uptake was mediated by three Na(+)-dependent and one Na(+)-independent systems. The 2-(methylamino)isobutyric acid-sensitive component of Na(+)-dependent uptake exhibited the usual characteristics of system A. Cl- dependency has been established for system A. The other two Na(+)-dependent systems, which we have named BCl(-)-dependent and BCl(-)-independent, are described for the first time. These are systems with broad specificity and were distinguished on the basis of inhibition analysis, Cl- dependency and the effect of preloading mammary tissue with amino acids. The Na(+)-independent route was identified as system L, which operates independent of Cl-. The A, L and BCl(-)-independent transport systems were upregulated in pregnant mouse mammary tissue cultured in vitro in the presence of lactogenic hormones (insulin plus cortisol plus prolactin). Insulin alone also upregulated systems A and L to some extent in pregnant mouse mammary tissue. BCl(-)-dependent activity was not detected in pregnant mouse mammary tissue and was not induced by lactogenic hormones in vitro.

Na +-dependent and Na +-independent transport of l-arginine and l-alanine across dog intestinal brush border membrane vesicles

We prepared intestinal brush border membrane vesicles (BBMVs) from beagle dogs fed a commercial diet (protein content: 24–26%), and investigated the characteristics of transport for basic and neutral amino acids across the intestinal BBMVs. To determined the kinetic parameters for l-arginine and l-alanine uptake, their total uptake was resolved into three routes: (1) Na +-dependent carrier-mediated transport; (2) Na +-independent carrier-mediated transport; and (3) simple diffusion. We could observe subtle, but clear-cut, Na +-dependent basic amino acid transport for the first time among studies with intestinal BBMVs prepared from mammals fed a normal diet. The Na +-dependent system for l-arginine transport can be best characterized as ‘low affinity, low capacity’, in contrast to that for l-alanine transport, which is ‘low affinity, high capacity’. Maximal velocities of the Na +-dependent carrier-mediated transport are estimated to be higher for both l-arginine and l-alanine in dog intestinal BBMVs than in rabbit intestinal BBMVs reported previously. These results suggest that food habit of mammals is an important factor to decide the characteristic of system B 0,+, a Na +-dependent carrier-mediated transport system common to basic and neutral amino acids across intestinal brush border membranes, as is protein content of the diet.

Characteristics of L-alanine transport in cardiac sarcolemmal vesicles and into isolated cardiac myocytes.

During cardiac insults, heart cells synthesise and accumulate alanine as a part of the anaerobic energy production pathway. The transport of alanine presumably influences this pathway, making it important to characterise the L-alanine transporter in the heart. In this study, we have investigated the transport of L-alanine across the sarcolemma using a novel approach, namely utilisation of two preparations: cardiac sarcolemmal vesicles and cardiac myocytes. Both preparations were isolated from the heart of the same mammalian species. L-Alanine uptake in both preparations was sodium dependent. In the sarcolemmal vesicles, the sodium dependent component was electrogenic and saturated with an estimated Michaelis-Menten constant (Km) and maximal reaction velocity (Vmax) of 0.48+/-0.18 mM and 279.97+/-64.17 pmol/mg per min respectively at room temperature. In the isolated myocytes, L-alanine uptake was linear in sodium-containing media, with an estimated Km and Vmax of 9.65+/-0. 76 mM and 169.81+/-13.22 pmol/ microl per min respectively at 10 degreesC for the sodium-dependent component. Inhibition of cotransport by a variety of substrates indicated that L-alanine uptake in the heart is mediated by an A- or ASC-like system. These characteristics of L-alanine transport suggest that under ischaemic conditions, L-alanine efflux will be activated, thus allowing for the continuous utilisation of other amino acids for energy production.

L-Alanine uptake by frog ( Rana esculenta) red blood cells

l-Alanine uptake has been studied in frog red blood cells. The present study shows the presence of different carriers for this amino acid in these cells. In the physiological concentration range, most l-alanine is taken up through the Na +-dependent system ASC, although the sodium-independent systems asc and L are also active. The competitive inhibition data obtained makes difficult to differentiate the two Na +-independent activities in a clear contrast with data from fish or mammalian erythrocytes, indicating that despite its widespread occurrence in vertebrates, these carriers show characteristics that are species specific.

Blood-endothelial cell and blood-brain transport of L-proline, alpha-aminoisobutyric acid, and L-alanine.

We report the application of multiple time regression analysis with the in situ brain perfusion technique to measure the rates of passage between blood and brain for [14C] L-proline, [14C] L-alanine, and [14C] alpha-aminoisobutyric acid (AIB) and their rapidly reversible volumes following perfusion of these amino acids from 10 to 60 seconds. We also report on their mechanism of transport. Proline diffused through the blood-brain barrier with a transfer coefficient (Kin) of 0.55 +/- 0.15 x 10(-4) ml/s/g and had no reversible compartment. AIB had a low Kin of 0.68 +/- 0.14 x 10(-4) ml/s/g and a significant reversible volume of 4.34 +/- 0.51 x 10(-3) ml/g in parietal cortex. L-alanine had the highest transfer coefficient, 3.11 +/- 0.26 x 10(-4) ml/s/g, and a reversible volume of 10.03 +/- 0.93 x 10(-3) ml/g in the same cerebral region. Postwash procedures which remove any radiotracer in the vasculature and capillary depletion were performed for alanine and AIB, as they had significant reversible compartments, to test the possibility of rapid efflux from the endothelial cells. Results obtained from wash and capillary depletion procedures suggest that a rapid efflux could occur from endothelial cells after entry of alanine and AIB. Mechanisms of transport for L-alanine and AIB were investigated using amino acids (5 mM) as substrates and inhibitors of different amino acid transport systems. AIB transport was reduced by plasma and L-leucine and unchanged by sodium-free buffer, confirming its passage by the L1 system. L-alanine uptake was sodium-independent and not reduced by plasma. L-serine, L-cysteine, L-leucine and L-phenylalanine produced similar inhibition (66%) while L-alanine produced a lower inhibition (41%). L-arginine increased alanine uptake in cortex and thalamus. Adding L-serine to L-phenylalanine reduced the uptake only in cortex and hippocampus. These data suggest that L-alanine is transported by another L transport system different from the L1 system at the luminal membrane.

Interactions of alkylglucosides with the renal sodium/ d-glucose cotransporter

To study the nature of the glucose binding pocket of the renal Na +/ d-glucose cotransporter, we have evaluated the inhibitory potency of various alkylglucosides (AG) on sodium-dependent D-glucose uptake into hog kidney brush border membrane vesicles (BBMV). Inhibition at 0.1 mM AG level was found to be strongly dependent on the anomeric configuration, on the length and on the flexibility of the side chain. β-n-AG inhibited transport significantly more effectively compared to the corresponding α-anomer ( n-octylglucoside: α-anomer 15%, (β-anomer 84%) and AG with an unsaturated n-alkenyl side chain were significantly less effective inhibitors than the corresponding saturated compound (cis/trans 3-n-β-hexenylglucoside 53% and 32%, β-n-hexylglucoside 76%). A series of β-n-AG increasing in side chain length from 1 to 13 carbon atoms revealed a global maximum in the inhibition pattern when β-AG with side chains ranging from 8 to 11 carbon atoms were used, thus β-methylglucoside inhibited glucose transport by 13%, β-n-nonylglucoside by 92%, and (β-n-tridecylglucoside showed no effect. Kinetic analysis of inhibition by β-n-octylglucoside revealed a fully competitive type of inhibition with an apparent K i of 10 ± 2 μM. n-Octylglucoside at 0.1 mM did not inhibit sodium-dependent l-alanine uptake; similarly, n-octylmannoside at 0.1 mM level did not affect D-glucose uptake. These results suggest that the inhibition of sodium-dependent d-glucose uptake was, at least in the concentration range tested (up to 0.1 mM), not due to a detergent effect of AG, but due to interaction with the carrier. Optimum interaction requires a β-anomer with a glycosidic bond that places the alkyl chain into an equatorial position with regard to the d-glucose molecule and the two main determinants of the sugar recognition site C2 and C3. In addition, the alkyl chain has to be highly flexible. The alkyl chains thus apparently interact with hydrophobic sites at the carrier in a slightly coiled conformation, thereby AG with a chain length up to 6 carbon atoms interact only with one hydrophobic site, AG with higher chain length probably with two sites.

Cloning and Functional Characterization of a System ASC-like Na+-dependent Neutral Amino Acid Transporter

A cDNA was isolated from mouse testis which encodes a Na+-dependent neutral amino acid transporter. The encoded protein, designated ASCT2, showed amino acid sequence similarity to the mammalian glutamate transporters (40-44% identity), Na+-dependent neutral amino acid transporter ASCT1 (57% identity; Arriza, J. L., Kavanaugh, M. P., Fairman, W. A., Wu, Y.-N., Murdoch, G. H., North, R. A., and Amara, S. G.(1993) J. Biol. Chem. 268, 15329-15332; Shafqat, S., Tamarappoo, B. K., Kilberg, M. S., Puranam, R. S., McNamara, J. O., Guadano-Ferraz, A., and Fremeau, T., Jr. (1993) J. Biol. Chem. 268, 15351-15355) and a mouse adipocyte differentiation-associated gene product AAAT (94% identity; Liao, K., and Lane, D.(1995) Biochem. Biophys. Res. Commun. 208, 1008-1015). When expressed in Xenopus laevis oocytes, ASCT2 exhibited Na+-dependent uptakes of neutral amino acids such as L-alanine, L-serine, L-threonine, L-cysteine, and L-glutamine at high affinity with Km values around 20 microM. L-Methionine, L-leucine, L-glycine, and L-valine were also transported by ASCT2 but with lower affinity. The substrate selectivity of ASCT2 was typical of amino acid transport system ASC, which prefers neutral amino acids without bulky or branched side chains. ASCT2 also transported L-glutamate at low affinity (Km = 1.6 mM). L-Glutamate transport was enhanced by lowering extracellular pH, suggesting that L-glutamate was transported as protonated form. In contrast to electrogenic transport of glutamate transporters and the other ASC isoform ASCT1, ASCT2-mediated amino acid transport was electroneutral. Na+ dependence of L-alanine uptake fits to the Michaelis-Menten equation, suggesting a single Na+ cotransported with one amino acid, which was distinct from glutamate transporters coupled to two Na+. Northern blot hybridization revealed that ASCT2 was mainly expressed in kidney, large intestine, lung, skeletal muscle, testis, and adipose tissue. Functional characterization of ASCT2 provided fruitful information on the properties of substrate binding sites and the mechanisms of transport of Na+-dependent neutral and acidic amino acid transporter family, which would facilitate the structure-function analyses based on the comparison of the primary structures of ASCT2 and the other members of the family.

Effects of insulin on the uptake of amino acids by hepatocytes and red blood cells from trout ( Salmo trutta) are opposite

Insulin provoked a rise in the uptake of L-alanine by isolated trout hepatocytes. This increase was dose-dependent and partially inhibited by actinomycin D and cycloheximide, suggesting that protein synthesis was involved in the hormonal action. There was an increase in the V max of the ASC system as well as in K m, but no expression of a different membrane carrier was observed. Although insulin was effective in cells from fed animals, its action was markedly higher in cells from fasted animals. Insulin had the opposite effect on red blood cells, making the incorporation of amino acids into the cell difficult as a result of a fall in the maximal capacity of uptake. This action was characteristically observed in incorporations through the ASC system, while the activity of another Na +-dependent carrier, the Gly system, was unaffected. Further, when the amino acid content of erythrocytes was in equilibrium with the extracellular concentration, insulin promoted a transient release of alanine from the cells. The physiological role of these actions of insulin may be related to the postprandial flux of amino acids from the gut to the liver.

The transport of vitamin C in the isolated human near-term placenta

We investigated the specific uptake (reference: [H 3]- l-glucose) as a measure of membrane transfer of [C 14]-labeled l-ascorbic acid (AA), l-dehydroascorbic acid (DHA) and diketogulonic acid using the single injection, double tracer dilution method in the artificially perfused lobe of the near-term human placenta. The uptake of DHA (40–60%) on both the fetal and the maternal side was 3–6 times higher than the uptake of AA, whereas an uptake of diketogulonic acid was not detected. AA transport was slightly higher on the maternal side. Uptake of DHA was suppressed by phloretin and cytocholasin B, whereas AA transfer was not affected. Low sodium concentrations inhibited the uptake of DHA. d-glucose (> 20 mmol/l) inhibited the DHA uptake, and DHA inhibited d-glucose uptake but not l-alanine uptake. The K m value (self inhibition) for DHA was 6–14 mmol/l. Vitamin C enters the trophoblast predominantly as l-dehydroascorbic acid. Its transfer through the microvillous and basal membrane might use the glucose transporter, however, a specific sodium-dependent pathway is not ruled out. Our transfer data suggest an intracellular pool of vitamin C which fills up with increasing plasma DHA-concentrations.

Ontogeny of L-alanine uptake in plasma membrane vesicles from rat liver.

Alanine uptake into liver plasma membrane vesicles was studied at different stages of postnatal rat development. Before weaning, alanine hepatic uptake showed lower values for the global KM than after weaning (0.34, 0.77, 1.45, and 1.61 mM for 1-, 15-, and 28-d-old and adult rats, respectively). Alanine uptake capacity increased progressively until reaching maximum values in the adult state (values for Vmax: 0.078, 0.199, 0.317, and 0.613 nmol alanine/mg protein/3 s for 1-, 15-, and 28-d-old and adult rats, respectively). These results seem to point to a prevalence of a high affinity, low capacity alanine transport component (traditionally assumed to be attributable to system A) in newborn and suckling rats, in agreement with our previous results on isolated hepatocytes (Martínez-Mas JV, Casado J, Felipe A, Marin JJG, Pastor-Anglada M: Biochem J 293: 819-824, 1993). The suckling-weaning developmental transition seems to play a role in establishing the pattern of adult hepatic alanine transport characterized by a higher capacity but a lower affinity (because most alanine is taken up by system ASC) inasmuch as KM values show a 100% increase after weaning, although Vmax values continue to increase steadily until the adult age.

Effect of olsalazine on sodium-dependent bile acid transport in rat ileum

Olsalazine (OLZ), a relatively new form of 5-aminosalicylic acid (5-ASA), is being used for the treatment of colitis. A major side effect of olsalazine is diarrhea, reported in 12-25% of patients. One suggested mechanism for this side effect is enhanced ileal water and electrolyte secretion. We propose that OLZ may also inhibit ileal bile acid (BA) transport, resulting in choleretic diarrhea. This would result in excess BAs reaching the colon, with consequent BA-induced secretory diarrhea. Therefore, we studied the effect of OLZ on rat ileal absorption of taurocholate. BA uptake was determined in rat ileal segments, everted sacs, brush border membrane vesicles (BBMV), and Xenopus laevis oocytes. Segments and everted sacs were treated with 5 mM OLZ for 30 min prior to and throughout 10-min taurocholate (Tc) uptake. Terminal ileal BBMV were used to study the effect of OLZ on sodium-dependent bile acid uptake independent of cellular metabolism. Direct effects on the bile acid carrier were examined using Xenopus laevis oocytes expressing the cloned apical rat ileal BA transporter. In ileal segments 5 mM OLZ inhibited 10-min Tc uptake by 69.4 +/- 8.8% (P < 0.01) (N = 10 animals). Increasing concentrations of OLZ resulted in a dose-dependent inhibition of Tc uptake. Ten-minute Tc uptake with 0.5, 1.0, 2.0, 2.5, and 5 mM OLZ was inhibited by 13.5, 39.6, 49.7, and 70.5%, respectively. In BBMV, OLZ inhibited 45-sec Tc uptake in a dose-dependent manner but did not effect Na-dependent L-alanine uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of a Novel Insulin-Activated Amino Acid Transporter Gene During Differentiation of 3T3-L1 Preadipocytes into Adipocytes

A cDNA encoding a novel insulin-activated adipocyte amino acid transporter (designated AAAT) was cloned from a mouse 3T3-L1 adipocyte library. The deduced amino acid sequence of the cDNA corresponds to a protein of 553 amino acids that possesses 56% amino acid sequence identity to the human neutral amino acid transporter and 42% identity to the rat brain glutamate transporter. Transient transfection of 3T3-L1 preadipocytes with an AAAT expression vector led to insulin-dependent uptake of L-serine and to a lesser extent, uptake of L-alanine and L-glutamate. Expression of the AAAT message is tissue-specific, with the highest level occurring in mouse adipose tissue and a lower level in lung. Unlike other sodium-dependent amino acid transporter mRNAs, the AAAT message is not expressed in brain, kidney, liver or heart and only traces are detected in spleen, thymus and skeletal muscle. Consistent with its high level in adipose tissue, expression of the AAAT message is markedly increased when 3T3-L1 preadipocytes are induced to differentiate into adipocytes.

Effect of protein malnutrition on neutral amino acid transport by rat hepatocytes during development.

Hepatocytes from suckling rats whose mothers were fed a low-protein diet (9% protein) showed a lower capacity for Na(+)-dependent L-alanine uptake [due to a decrease in maximal uptake rate (Vmax) of a low-affinity component of transport] and were not able to respond to insulin or glucagon, whereas those from suckling pups whose mothers were fed the control diet (17% protein) had already developed the ability to upregulate L-alanine transport after hormone treatment. When animals from low-protein-fed mothers were weaned onto a hypoprotein diet, the overall capacity for Na(+)-dependent L-alanine uptake (apparent Vmax) and its responsiveness to pancreatic hormones were restored. Hepatocytes from these animals showed a lower response to glucocorticoid treatment. Amino acid availability was dramatically decreased in suckling and weanling rats fed a low-protein diet. These results support the hypothesis that nutrient supply is an important factor in the proper development of hepatic transport functions during the suckling-weaning transition.

Coordinate induction of Na +-dependent transport systems and Na +,K +-ATPase in the liver of obese Zucker rats

Solute uptake into liver plasma membrane vesicles from either lean or obese Zucker rats was monitored. d-Glucose and l-leucine uptakes at physiological concentrations of the substrate were not different in lean and obese Zucker rats. In agreement with a previous report (Ruiz et al. (1991) Biochem. J. 280, 367–372) l-alanine uptake was significantly enhanced in those preparations from obese animals. Na +-coupled uridine transport was markedly enhanced also in obese rats. The effect was due to an increase in V max ( 5.5 ± 0.6 vs. 2.1 ± 0.2 pmol/mg protein per 3 s, P < 0.01) without any significant change in K m ( 11.0 ± 2.8 vs. 9.0 ± 2.7 μM for obese and lean rats, respectively). Na +,K +-ATPase activity was also higher in liver plasma membrane vesicles from rat liver and it correlated with a higher amount of α 1 - subunit protein in both, plasma membrane vesicles and homogenates from obese rat livers. In summary, in the hypertrophic liver of obese Zucker rats a coordinate induction of several Na +-dependent transport systems occurs and, in order to sustain the metabolic pressure associated with this adaptation, a significant induction of the Na +,K +-ATPase expression is also found. These data also provide new evidence for regulation of the recently characterized Na +-dependent nucleoside transporter.

Aflatoxin B 1 reduces Na +-P i co-transport in proximal renal epithelium: studies in opossum kidney (OK) cells

In vivo studies indicate that aflatoxin B 1 (AFB 1) may affect the renal regulation of inorganic phosphate (P i), possibly by altering the renal response to parathyroid hormone (PTH). Therefore, the present study utilized opossum kidney (OK) cells, a mammalian renal epithelial cell line, to determine whether AFB 1 exposure alters sodium-phosphate (Na +-P i) co-transport and the hormonal modulation thereof. OK cells are an established renal cell line with many properties analagous to the proximal renal epithelium, including receptors for PTH, insulin, and high levels of Na +-P i co-transport. PTH and insulin have been shown to decrease and increase Na +-P i co-transport, respectively, in OK cells. In the present study, AFB 1-treated cells responded to PTH; however, AFB 1 exposure decreased Na +-P i uptake such that additional decreases in Na +-P i uptake in response to PTH were minimal. In the presence of insulin, AFB 1-treated cells were only able to increase Na +-P i uptake to levels 30% below that of control cells. The net result was that the range of the proximal renal epithelium to adjust Na +-P i co-transport in response to hormonal modulation was reduced by AFB 1 exposure. Sodium-dependent L-alanine uptake was measured and was found not to be affected by the highest concentration of AFB 1; thus, indicating that AFB 1 exposure may have specific effects on Na +-P i uptake and does not generally inhibit Na +-dependent transport. These observations are evidence that AFB 1 exposure may alter key elements of renal function. Such effects raise concern that AFB 1 exposure may have broad physiological impact in addition to its known carcinogenic properties.

L-Alanine transport in small intestine brush-border membrane vesicles of obese rats

Membrane vesicles from the small intestine brush border were obtained and used to determine the possible effects of genetic or nutritional obesity on l-alanine uptake. Membrane vesicles from Zucker fa fa obese rats and cafeteria diet-fed Zucker Fa/? rats showed the same characteristics as those of standard diet-fed lean animals. All preparations showed sodium-dependent transport as the main pathway for l-alanine uptake within the substrate concentration range tested. The apparent substrate affinity constant ( K rmm ) values and the pattern of inhibition of Na +-dependent l-alanine uptake by other amino acids ( l-leucine and l-glutamine), suggests that system B involved in the transport of dipolar amino acids (formerly named Neutral Brush Border System) participates in the Na +-dependent transport of l-alanine. The affinity constant ( K m) for l-alanine was essentially the same for all the groups studied (in the range of 10 mM). However, there was a higher ( P < 0.05) maximal capacity ( V max) in preparations from diet-induced obese animals (cafeteria diet) than that of genetically obese rats. These results indicate that either nutritional or genetic obesity may modify the capacity but not the affinity of transport systems for l-alanine uptake in the brush border of rat small intestine.

Regional differentiation in rat aorta: L-arginine metabolism and cGMP content in vitro.

Sequential segments of rat aorta incubated in vitro exhibit a characteristic activity pattern for the metabolism of L-arginine, the substrate for nitric oxide synthase, and for the content of guanosine 3',5'-cyclic monophosphate (cGMP), the mediator of nitric oxide relaxation of vascular smooth muscle. Highest values were observed just distal to the arch and diminish peripherally. Prior removal of the endothelium, treatment with ouabain, or replacement of ambient medium Na+ decreased L-arginine uptake and metabolism and eliminated the pattern of regional differentiation. Removal of endothelium reduced the cGMP content with loss of the regional pattern. A favorable extracellular/intracellular Na+ gradient is required for the moiety of L-arginine uptake destined for metabolism in the endothelial cell. Replacement of ambient Na+ or treatment with ouabain also decreases markedly the L-arginine metabolism and uptake in cultured rat aortic endothelial cells. When aortic segments were tested with five additional substances, L-leucine uptake alone followed a regional pattern similar to that for L-arginine, and no such pattern was observed for the uptake of L-alanine, alpha-aminoisobutyrate, 3-methylglucose, or Ca2+.