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1. Transport of l-arginine and the nitric oxide synthase inhibitors NG-monomethyl-l-arginine and NG-nitro-l-arginine was investigated in human erythrocytes from healthy donors and uraemic patients on haemodialysis. 2. Although Km values for total l-arginine influx were not significantly different in erythrocytes freshly isolated from controls or uraemic patients, uraemia was associated with an increase in the Vmax for transport (826 compared with 1176 μmol h−1 l−1 of cells) which was reduced to control values after dialysis. 3. Saturable influx of l-arginine was mediated by the classical cationic amino acid transport system y+ and system y+L, known to transport cationic and neutral amino acids with higher affinity. 4. Under zero-trans conditions, the Vmax for l-arginine transport via system y+ increased from 271 to 700 μmol h−1 l−1 of cells in uraemia, while Km values increased from 44 to 94 μmol/l. Dialysis had no significant effect on the kinetic parameters altered by uraemia. 5. Under zero-trans conditions, and with system y+ inhibited by N-ethylmaleimide (0.2 mmol/l), transport of l-arginine via system y+L was unaffected by uraemia. 6. Saturable influx of NG-monomethyl-l-arginine was also mediated by systems y+ (Km = 56 μmol/l, Vmax = 353 μmol h−1 l−1 of cells) and y+L (Km = 17 μmol/l, Vmax = 51.3 μmol h−1 l−1 of cells) and, as with l-arginine, uraemia increased the transport capacity for NG-monomethyl-l-arginine. 7. Influx of the neutral nitric oxide synthase inhibitor NG-nitro-l-arginine was not readily saturable. 8. Intracellular concentrations of l-arginine and NG-monomethyl-l-arginine were significantly increased in erythrocytes from uraemic patients when compared with controls, consistent with an increased transport capacity for l-arginine and NG-monomethyl-l-arginine. 9. The present study provides evidence that system y+ mediates the increased transport of l-arginine and NG-monomethyl-l-arginine in human erythrocytes from patients with chronic renal failure. Our findings may have implications for the activity of the l-arginine—nitric oxide signalling pathway in vascular endothelial and smooth-muscle cells in uraemia.

The effects of exogenous insulin, glucagon and streptozotocin-diabetes on influx (15 s) of L-lysine via a cationic amino acid transporter resembling system y+ were investigated in the isolated perfused rat pancreas. In non-diabetic pancreata, transport of L-lysine was saturable with an apparent Km of 2.11 +/- 0.29 mM and Vmax of 2.21 +/- 0.20 mumol min-1 g-1 (n = 6). Bovine insulin (100 mu u ml-1) increased the maximal transport rate (Vmax = 3.49 +/- 0.30 mumol min-1 g-1, n = 4, P < 0.05) for L-lysine 1.6-fold without altering the Km. L-Lysine transport was not elevated significantly in diabetic pancreata, although insulin (100 mu u ml-1) enhanced transport to values measured in non-diabetic preparations. Human glucagon (1.5 x 10(-9) M) had no stimulatory effect on L-lysine transport. These findings provide the first evidence that exogenous insulin stimulates cationic amino acid transport activity in the exocrine pancreatic epithelium. Activation of the cationic pancreatic amino acid transporter may provide a mechanism to enhance the supply of L-arginine and thus sustain nitric oxide-mediated pancreatic secretion in response to islet hormones and secretagogues.

1. The roles of the gamma-glutamyl cycle and the anionic amino acid transport system xc- in mediating L-cystine uptake were investigated in cultured human pancreatic duct PaTu 8902 cells. This cell line exhibits morphological features of normal pancreatic duct cells and expresses gamma-glutamyl transpeptidase (gamma-GT, EC 2.3.2.2), an enzyme involved in the metabolism and regulation of intracellular glutathione (GSH). 2. Uptake of L-cystine (10 microM) was linear for up to 10 min, temperature dependent, Na+ independent, saturable (Michaelis-Menten constant (Km), 86 +/- 25 microM; maximal velocity (Vmax), 109 +/- 33 nmol (mg protein)-1 h-1) and reduced by 80-90% by a 50-fold excess concentration of L-glutamate and L-homocysteic acid, but not L-aspartate. These transport properties resemble those described for system xc-, which exchanges cystine for intracellular glutamate. 3. Acivicin, a known inhibitor of gamma-GT, decreased gamma-GT activity from 2.58 +/- 0.96 to 0.97 +/- 0.11 mU (mg protein)-1 and decreased the initial rates of L-cystine and L-glutamine uptake by 41-55%. Anthglutin (1-gamma-L-glutamyl-2-(2-carboxyphenylhyl)hydrazine), a structurally different inhibitor of gamma-GT, also caused a concentration-dependent (0.01-1 mM) decrease in gamma-GT activity and L-cystine uptake. 4. Neither acivicin nor anthglutin inhibited the uptake of L-glutamate, a poor substrate for gamma-GT. 5. In the presence of a 500-fold excess concentration of glutamate, which should abolish entry of cystine via system xc-, the remaining fraction of cystine transport was inhibited by 50% by acivicin, suggesting that transport is, in part, dependent on the activity of gamma-GT. 6. Cystine transport was also 60-80% inhibited by a series of gamma-glutamyl amino acids (5 mM) including gamma-glutamyl-glutamate, gamma-glutamyl-glutamine and gamma-glutamyl-glycine. alpha-Dipeptides inhibited cystine transport by only 6-22%. 7. These findings demonstrate that in human pancreatic duct PaTu 8902 cells, cystine uptake is mediated by system xc- (50-60%) and the gamma-glutamyl cycle. Our results provide the first evidence linking gamma-GT with cystine transport in human epithelial cells and are of relevance in view of the importance of cystine as a sulphur amino acid source for GSH synthesis in cells exposed to oxidative stress.

1. Transport of l-arginine was investigated under zero-trans conditions in human erythrocytes from healthy donors and patients with heart failure. 2. Saturable influx of l-arginine was mediated by the classical cationic amino acid transport systems y+ and y+L. 3. The Vmax for l-arginine transport via system y+ increased from 292 to 490 μmol h−-1 l−-1 of cells in heart failure. 4. With system y+ inhibited by N-ethylmaleimide (0.2 mmol/l), the Vmax for the transport of l-arginine via system y+L was unaffected in erythrocytes from patients with heart failure. 5. The inhibition of l-arginine and l-leucine influx by NG-monomethyl-l-arginine was similar in erythrocytes from control and heart failure patients. 6. Plasma l-arginine levels were reduced in patients with heart failure (59 μmol/l) compared with controls (125 μmol/l). Plasma from patients with heart failure also contained the endogenous l-arginine analogue NG-monomethyl-l-arginine, which was undetectable in plasma from controls. 7. Intracellular concentrations of l-arginine and NG-monomethyl-l-arginine were significantly elevated in erythrocytes from patients with heart failure compared with controls, consistent with an increased transport capacity for l-arginine and NG-monomethyl-l-arginine. 8. The present study provides the first evidence that system y+ mediates the increased transport of l-arginine in human erythrocytes from patients with chronic heart failure. These findings are similar to our previous results obtained in patients with chronic renal failure. Since both pathologies seem to present with an increased synthesis of nitric oxide, studies of l-arginine transport in erythrocytes may provide a valuable paradigm to study abnormalities of the l-arginine-nitric oxide signalling pathway.

Vascular endothelial cells synthesize nitric oxide (NO) from the terminal guanidine-nitrogen atom of the cationic amino acid L-arginine (Palmer, R et al, 1988). Although basal NO synthesis is not limited by arginine availability, under conditions of stimulated NO production or following substrate deprivation exogenous L-arginine appears to be rate-limiting (Palmer, R. et al., 1988). We have now investigated whether bradykinin, an agonist which is known to release NO release, modulates the endothelial cell L-arginine transporter.

Amino acid transport in the isolated perfused exocrine pancreas is mediated by at least four parallel transporters, exhibiting differential sensitivity to islet hormones, diabetes, fasting and dietary composition. Insulin and streptozotocin-induced diabetes stimulate amino acid influx via the Na+-independent Systems asc (L-serine) and y+ (L-lysine). Moreover, activation of transport by insulin is dependent upon extracellular Ca2+ and inhibited by somatostatin-14. Elevation of transport induced by diabetes may not be mediated by glucagon, as exogenous glucagon fails to stimulate transport. Although system L (L-phenylalanine) is unaffected by insulin and diabetes, fasting for 72 h activates transport and refeeding for 24 h restores transport to fed levels. Adaptive responses in the activities of systems L and y+ induced by fasting and changes in dietary protein may involve changes in intracellular amino acid levels which are known to influence counter-transport. Elucidating the mechanisms regulating uptake and incorporation of amino acids into pancreatic enzymes is of clinical relevance, since 11 C-labelled amino acids are currently used as PET imaging probes for regional tissue metabolism in pancreatitis.

Transport of short-chain and long-chain neutral amino acids across the basolateral membrane of the epithelium in the perfused cat salivary gland has been studied using a rapid (less than 30 s) single circulation paired-tracer dilution technique. Amino acid uptake was measured by comparing the venous dilution profiles for a tritiated amino acid and D-[14C]mannitol (extracellular reference) following a bolus intra-arterial injection of a mixture containing both molecules. Unidirectional influx (v) was estimated from the maximal tracer uptake (Umax), the perfusate flow (F) and the perfusate amino acid concentration (Ca): v = [-F . ln (1-Umax)] . Ca. L-alanine influx was saturable and apparently mediated by a single entry system (Km = 0.83 +/- 0.11 mM and Vmax = 655 +/- 32 nmol/min . g). These kinetic constants were considerably lower than our previously reported values for L-phenylalanine: Km = 6.4 mM and Vmax = 1719 nmol/min . g. In cross-inhibition experiments performed over a wide range of concentrations (0.05-24 mM), influx of L-alanine and L-phenylalanine could be further discriminated, since both L-phenylalanine (Ki = 22 mM) and L-alanine (Ki = 19 mM) behaved as poor competitors. Removal of Na+ from the perfusate resulted in a selective inhibition of L-alanine and L-serine influx, whereas influx of the long-chain neutral amino acids L-leucine, L-phenylalanine and L-tryptophan remained unaffected. Although prolonged perfusion of glands with dinitrophenol (0.8 mM for 20-30 min) caused a variable but net inhibition of unidirectional uptake, it markedly enhanced the tracer efflux of L-leucine, L-phenylalanine, L-tyrosine and the basic amino acid L-lysine. It appears that at least two separate neutral amino acid transport systems are operative at the blood-tissue interface of the salivary epithelium: (i) a Na+-dependent alanine-serine-cysteine preferring type of carrier exhibiting a high affinity for amino acids with short, polar or linear side chains and (ii) a Na+-independent leucine preferring type of carrier selective for large neutral amino acids.

We have applied the paired-tracer dilution method to the study of transport processes in a mixed mammalian muscle preparation, the perfused rat hindlimb. The method is suitable for the characterization of the kinetic parameters of sugar and amino acid transport and its regulation by hormones, contractile activity, hypoxia, etc. Insulin stimulates sugar transport by increasing the Vmax. of the process 2-3 fold, but its affinity is unchanged. Starvation increases the affinity of sugar transport in perfused skeletal muscle.

The specificity and kinetics of L-lysine influx across the basolateral surface of the cat salivary epithelium have been investigated in the perfused cat submandibular gland using a high-resolution, paired-tracer dilution technique. L-lysine influx was measured at several different perfusate concentrations (0.05-2.5 mM) and was found to be saturable. A Michaelis-Menten analysis based on a single entry site gave a Km of 0.49 +/- 0.08 mM and a Vmax of 231 +/- 20 nmol/min X g. The uptake of L-lysine was highly stereospecific and markedly inhibited by L-arginine (0.25-2.5 mM). The inhibitor constant (Ki) was 0.23 mM, suggesting that the carrier had a greater affinity for L-arginine than L-lysine. When the inhibitory effects of L-histidine (0.5-10 mM) were examined the Ki, estimated at 10 mM, was 4.6 mM. Nine other neutral amino acids (L-alanine, L-serine, L-cysteine, glycine, L-proline, L-homoserine, L-leucine, L-phenylalanine and L-glutamine), and an acidic amino acid (L-aspartate) were also tested at 10 mM and, although several caused inhibition, the Ki was always at least 20 times higher than the measured Km for L-lysine. It is concluded the carrier is highly specific for the L-form of the basic amino acids. The sodium dependence of L-lysine influx was investigated over a range of L-lysine concentrations (0.05-1 mM), and total removal of sodium from the perfusate had no effect on L-lysine influx. In the presence of sodium, L-homoserine, an amino acid not normally present in animal tissues, inhibited L-lysine influx (Ki = 13 mM). This inhibition was not observed in the absence of sodium, and contrasts with the observation that the inhibitory action of L-histidine was sodium independent. The present data suggest that a specific cationic amino acid transport system is operative in the basolateral membrane of the cat salivary epithelium. The properties of this system appear to be similar to the system y+ which has been described in several other cell types.

1. The unidirectional influx of amino acids into the guinea-pig syncytiotrophoblast was measured using a single circulation paired-tracer dilution technique which allows separate characterization of both fetal and maternal interfaces. An in situ preparation perfused through the fetal circulation was used to examine the fetal side, while an isolated preparation perfused through both the fetal and maternal circulations was used to study both interfaces simultaneously. 2. On the fetal side the maximal uptake (Umax) determined at tracer concentrations was high for the short-chain neutral amino acid alanine (76%) and the long-chain neutrals, leucine (75%), phenylalanine (90%) and tyrosine (82%) and for the basic amino acid lysine (65%). In contrast, Umax was negligible for α-methylaminoisobutyric acid and taurine, a β-amino acid. 3. The uptake of alanine and phenylalanine on the fetal side was inhibited by both short-chain (alanine, serine, cysteine) and long-chain (phenylalanine, methionine, leucine) neutral amino acids. d-alanine had no effect on l-alanine uptake whereas d-phenylalanine significantly inhibited that of l-phenylalanine. Diaminobutyric acid, lysine and arginine were effective inhibitors of alanine uptake but had no effect on phenylalanine uptake. 4. On the maternal side uptake of alanine, phenylalanine and lysine was measured. Over a wide range of concentrations self-inhibition of alanine influx was similar to the cross-inhibition observed with phenylalanine. In contrast, the influx of phenylalanine, which was strongly self-inhibited, was only partially cross-inhibited by alanine. 5. Influx of alanine and phenylalanine was measured at various perfusate concentrations and was found to be saturable on both maternal and fetal sides. The data were fitted to a single hyperbola and, on the maternal side, the Km for alanine (10.3±2.7 m m, mean± s.e., n = 3) was three-fold higher than the value measured for phenylalanine (3·1±0·8 m m). On the fetal side the Km values for alanine (8·4±1·4 m m, n = 4) and phenylalanine (11·9±1·9 m m, n = 3) were similar. 6. The uptake of alanine, phenylalanine and lysine appeared to be highly sodium-dependent accounting for 40-70% of the total influx. However, the inhibited fractions were found to be different on the two sides of the placenta. 7. The results of uptake, cross-inhibition and Na+-dependency experiments suggest the presence of an alanine-serine-cysteine (ASC) type system and a leucine (L) type system with markedly overlapping specificities at both the fetal and maternal interfaces. Separate kinetic characterization of a two carrier system was not possible under the conditions of these experiments. However, kinetic parameters for the over-all transport of alanine and phenylalanine were measured.

Light-microscopic autoradiography was used to localize the cellular sites for neutral amino acid uptake in submandibular and sublingual salivary gland epithelia. The vasculature of isolated glands was perfused for 3–5 min with either L-(3-3H)serine or L-(4-3H)phenylalanine and then fixed by perfusion with buffered glutaraldehyde. In the submandibular gland the small neutral amino acid L-serine and the aromatic amino acid L-phenylalanine were localized to central acinar cells, demilunar cells and ductal cells. In the sublingual gland silver grains associated with each of these tritiated amino acids were localized to central acinar and ductal cells. Perfusion of both submandibular and sublingual glands with unlabelled L-serine (25 mM) or L-phenylalanine (30 mM) resulted in a significant decrease in the silver grain density associated with each labelled amino acid. The absence of silver grains in the lumina of acinar and ductal cells and the presence of tight junctions near the apical surface of the epithelium strongly suggest that the initial uptake of these amino acids was mediated by basolateral plasma membrane carriers.