Discrimination of parallel neutral amino acid transport systems in the basolateral membrane of cat salivary epithelium.

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SUMMARY1. Nutrient transport in cultured human umbilical vein endothelial cells was characterized using a rapid dual-isotope dilution technique. Microcarrier beads with confluent endothelial cells were perfused in small columns, and uptake and efflux were assessed relative to D-mannitol (extracellular tracer) during a single transit through the column.2. At tracer concentrations significant unidirectional uptakes were measured for L-leucine (53 + 2 %), L-phenylalanine (73 + 2 %), L-serine (40 +4%), L-arginine (42 + 3 %) and L-ornithine (26 + 3 %). Uptake for L-proline, D-glucose, dopamine and serotonin was lower (6-10 %), whereas uptake for the system A analogue 2-methylaminoisobutyric acid (2-MeAIB) was negligible. Uptakes rapidly decreased with time due to tracer efflux. 6. Our results provide the first evidence that cultured human endothelial cells of venous origin express a saturable transport system for large neutral amino acids resembling system L described in brain microvascular endothelium. Detection of Na+-dependent and Na+-independent L-arginine uptake is of interest in view of recent reports that this cationic amino acid may be the physiological precursor for nitric oxide released by endothelium.

Section: Endothelial Cell Removal Of Serotonin and Dopaminementioning

confidence: 58%

Section: Measurement Of Endothelial Cell Transportmentioning

confidence: 99%

Expression of amino acid transport systems in cultured human umbilical vein endothelial cells.

Mann

Pearson

Sheriff

et al. 1989

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“…Previous paired-tracer dilution studies (see of amino acid transport systems present in the basolateral membrane of the cat salivary epithelium have revealed that the influx of small and large neutral amino acids was mediated by two separate carriers (Bustamante, Mann & Yudilevich, 1981;Mann & Yudilevich, 1984). This epithelium readily takes up L-lysine (Mann & Yudilevich, 1984) and earlier cross-inhibition experiments performed in vivo (Bustamante et at.…”

Section: Introductionmentioning

confidence: 99%

“…This epithelium readily takes up L-lysine (Mann & Yudilevich, 1984) and earlier cross-inhibition experiments performed in vivo (Bustamante et at. 1981) showed that L-lysine had no affinity for the neutral transport systems ASC (alanineserine-cysteine preferring) and L (leucine preferring).…”

Section: Introductionmentioning

confidence: 99%

Characteristics of a cationic amino acid transport system in the basolateral membrane of the cat salivary epithelium.

Mann¹,

Wilson²,

Yudilevich³

1984

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SUMMARY1. 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.2. 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. g.3. 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. 4. When the inhibitory effects of L-histidine (0-5-10 mM) were examined the K1, 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 Lglutamine), 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. 5. The sodium dependence ofL-lysine influx was investigated over a range ofL-lysine concentrations (0 05-1 mM), and total removal of sodium from the perfusate had no effect on L-lysine influx.6. 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. 7. 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. E. MANN, S. M. WILSON AND D. L. YUDILEVICH

“…lactate) transport at the sarcolemmal membrane of the rabbit heart , we chose to quantify noradrenaline uptake relative to D-mannitol, an extracellular tracer of similar size and diffusibility. This approach in the heart resembles the analysis used to evaluate cellular uptake in organs with fenestrated capillaries such as the salivary gland (Bustamante et al 1981;Mann, Tradatti & Yudilevich, 1982;Mann & Yudilevich, 1984), in which transcapillary exchange of small solutes was shown to be flow limited (Mann, Smaje & Yudilevich, 1979).…”

Section: Transcapillary Exchange Of D-mannitol and L-noradrenalinementioning

confidence: 99%

Rapid transcapillary exchange and unidirectional neuronal uptake of noradrenaline in the perfused rabbit heart.

Mann¹,

Yudilevich²

1984

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SUMMARY1. Capillary permeability and cellular uptake of noradrenaline by the isolated artificially perfused rabbit heart was measured using rapid (< 30 s) single-circulation tracer-dilution techniques.2. In a single coronary circulation capillary extractions of L-[14C]noradrenaline and D-[3H]mannitol (extracellular reference) relative to an intravascular marker, 1251_ labelled albumin, were similar and above 60 %.3. The 'apparent' volume of distribution for tracer noradrenaline was 2-5-fold larger than that measured for D-mannitol (0-32 ml g-1) suggesting cellular uptake of the amine.4. Unidirectional noradrenaline uptake was estimated by directly comparing coronary sinus dilution profiles ofL-[3H]noradrenaline and D-[14C]mannitol. MichaelisMenten saturation kinetics based on a single-entry system were determined (Km = 2-8 + 1 5 /M, Vmax = 2-1 + 0-5 nmol min' g-1, n = 4) by perfusing hearts with varying concentrations of L-noradrenaline (1-10 ,M).5. Various known inhibitors ofnoradrenaline uptake were investigated to determine whether uptake was mediated by neuronal (uptake,) and/or extraneuronal (uptake2) mechanisms. Desipramine (5 zM), imipramine (5/M) and metaraminol (2 /M) resulted in a 66-940 inhibition of noradrenaline influx. In comparison, the steroids, 17 ,Joestradiol (1 uM) and corticosterone (10 /M), and the noradrenaline metabolite normetanephrine (5/tM) caused virtually no inhibitory effects. The 6-adrenergic antagonist propranolol (5/M) was also relatively ineffective.6. These results together with the kinetic constants estimated suggest that the rapid noradrenaline uptake reflects transport into adrenergic neurones lying in the coronary interstitium. The high resolution of this paired-tracer dilution technique has permitted a 'non-invasive' study of neuronal uptake mechanisms and its application may be of clinical value.