Developmental changes in glutamine transport by rat jejunal basolateral membrane vesicles

The ontogeny of glutamine uptake by jejunal basolateral membrane vesicles (BLMV) was studied in suckling and weanling rats and the results were compared with adult rats. Glutamine uptake was found to represent a transport into an osmotically active space and not mere binding to the membrane surface. Temperature dependency indicated a carrier-mediated process with optimal pH of 7.0. Transport of glutamine was Na+ (out greater than in) gradient dependent with a distinct "overshoot" phenomenon. The magnitude of the overshoot was higher in suckling compared with weanling rats. The uptake kinetics and inhibition profile indicated the existence of two major transport pathways. A Na(+)-dependent system correlated with System A showed tolerance to System N and System ASC substrates, and a Na(+)-independent system similar to the classical L system that favors leucine and BCH. The Vmax for the Na(+)-dependent system was higher in suckling compared with weanling and adult rats. The Vmax for the Na(+)-dependent system was 0.86 +/- 0.17, 0.64 +/- 0.8, and 0.41 +/- 0.9 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. The Vmax for the Na(+)-independent system was 0.68 +/- 0.08, 0.50 +/- 0.03, and 0.24 +/- 0.03 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. We conclude that glutamine uptake undergoes developmental changes consistent with more activity and/or number of glutamine transporters during periods of active cellular proliferation and differentiation.     

A role for membrane transport in modulation of intramuscular free glutamine turnover in streptozotocin diabetic rats.

We wished to examine the effects of diabetes on muscle glutamine kinetics. Accordingly, female Wistar rats (200 g) were made diabetic by a single injection of streptozotocin (85 mg/kg) and studied 4 days later; control rats received saline. In diabetic rats, glutamine concentration of gastrocnemius muscle was 33% less than in control rats: 2.60 +/- 0.06 mumol/g vs. 3.84 +/- 0.13 mumol/g (P < 0.001). In gastrocnemius muscle, glutamine synthetase activity (Vmax) was unaltered by diabetes (approx. 235 nmol/min per g) but glutaminase Vmax increased from 146 +/- 29 to 401 +/- 94 nmol/min per g; substrate Km values of neither enzyme were affected by diabetes. Net glutamine efflux (A-V concentration difference x blood flow) from hindlimbs of diabetic rats in vivo was greater than control values (-30.0 +/- 3.2 vs. -1.9 +/- 2.6 nmol/min per g (P < 0.001)) and hindlimb NH3 uptake was concomitantly greater (about 27 nmol/min per g). The glutamine transport capacity (Vmax) of the Na-dependent System Nm in perfused hindlimb muscle was 29% lower in diabetic rats than in controls (820 +/- 50 vs. 1160 +/- 80 nmol/min per g (P < 0.01)), but transporter Km was the same in both groups (9.2 +/- 0.5 mM). The difference between inward and net glutamine fluxes indicated that glutamine efflux in perfused hindlimbs was stimulated in diabetes at physiological perfusate glutamine (0.5 mM); ammonia (1 mM in perfusate) had little effect on net glutamine flux in control and diabetic muscles. Intramuscular Na+ was 26% greater in diabetic (13.2 mumol/g) than control muscle, but muscle K+ (100 mumol/g) was similar. The accelerated rate of glutamine release from skeletal muscle and the lower muscle free glutamine concentration observed in diabetes may result from a combination of: (i), a diminished Na+ electrochemical gradient (i.e., the net driving force for glutamine accrual in muscle falls); (ii), a faster turnover of glutamine in muscle and (iii), an increased Vmax/Km for sarcolemmal glutamine efflux.     

Ontogeny of glutamine transport by rat liver plasma membrane vesicles.

Glutamine metabolism in the liver is essential for gluconeogenesis and ureagenesis. During the suckling period there is high hepatic protein accretion and the portal vein glutamine concentration is twice that in the adult, whereas hepatic vein glutamine concentration is similar between adult and suckling rats. Therefore, we hypothesized that glutamine uptake by the liver could be greater in the suckling period compared to the adult period. The present studies were, therefore, designed to investigate the transport of glutamine by plasma membranes of rat liver during maturation (suckling--2-week old, weanling--3-week old and adult--12-week old). Glutamine uptake by the plasma membranes of the liver represented transport into an osmotically sensitive space in all age groups. Inwardly directed Na+ gradient resulted in an "overshoot" phenomenon compared to K+ gradient. The magnitude of the overshoot was greater in suckling rats plasma membranes compared to adult membranes. Glutamine uptake under Na+ gradient was electrogenic and maximal at pH 7.5, whereas uptake under K+ gradient was electroneutral. Glutamine uptake with various concentrations of glutamine under Na+ gradient was saturable in all age groups with a Vmax of 1.5 +/- 0.1, 0.7 +/- 0.1 and 0.5 +/- 0.06 nmoles/mg protein/10 seconds in suckling, weanling and adult rats, respectively (P < 0.01). Km values were 0.6 +/- 0.1, 0.5 +/- 0.1 and 0.5 +/- 0.1 mM respectively. Vmax for Na(+)-independent glutamine uptake were 0.6 +/- 0.1, 0.55 +/- 0.07 and 0.54 +/- 0.06 nmoles/mg protein with Km values of 0.54 +/- 0.2, 0. +/- 0.1 and 0.5 +/- 0.2 mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of halothane on transport of 5-hydroxytryptamine by platelet membranes.

Na+-dependent uptake of 5-HT (5-hydroxytryptamine) into plasma membrane vesicles derived from bovine blood platelets and ATP-dependent 5-HT uptake into storage vesicles in platelet lysates were measured. Na+-dependent uptake was temperature-dependent, inhibited by imipramine and exhibited Michaelis-Menten kinetics (apparent Km, 0.12 +/- 0.02 microM; Vmax. 559 +/- 54 pmol/min per mg of protein. Halothane had no effect on Na+-dependent transport of 5-HT in plasma-membrane vesicles. ATP-dependent 5-HT transport into storage granules also exhibited Michaelis-Menten kinetics (apparent Km 0.34 +/- 0.03 microM; Vmax. 34.3 +/- 1.7 pmol/min per mg of protein) and was inhibited by noradrenaline (norepinephrine), but not by imipramine. Exposure of the granules to halothane resulted in a progressive decrease in Vmax. The results demonstrate a possible site for disruption of platelet function by anaesthetics.     

L‐leucine transport in rat heart under normal conditions and effects of a simulated hypoxia

L-leucine plays a central role in the regulation of protein metabolism in heart and has been implicated in myocardial protection, but little is known about the relationship between these phenomena and leucine transport across the cardiac sarcolemma. In this study we used sarcolemmal vesicles and ventricular myocytes isolated from rat heart to characterise L-leucine transport under normal conditions and to investigate the effect of simulated hypoxia or inhibition of protein synthesis. The Km and Vmax of leucine uptake were 5.24+/-0.65 mM and 1.43+/-1.84 nmol min(-1) mg(-1) protein in vesicles compared to 2.17+/-0.13 mM and 1.7+/-0.76 nmol min(-1) microl(-1) intracellular space in cells. Transport was not dependent on Na+ or H+ gradients. In vesicles L-leucine uptake was increased by trans-stimulation, whilst inhibition was observed with classical system L substrates including 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid (BCH) suggesting that this system mediated L-leucine transport in heart. L-Leucine uptake into isolated cardiac myocytes was inhibited after 20, 30 and 60 min of simulated hypoxia. This was not caused by reduced cell viability, although the cells underwent a rigor contracture. Inhibition of protein synthesis did not affect L-leucine transport.     

The transport of glutamine and alanine into rat colonocytes.

The transport of glutamine and alanine into isolated rat colonocytes was studied. The transport of both amino acids appears to be dependent on a Na+ gradient. The apparent Km values for the transport of glutamine and alanine were 2.56 +/- 0.84 and 5.35 +/- 1.20 mM respectively, but with similar Vmax. values. Glutamine and alanine transport were mutually competitive, and the transport of both amino acids was competitively inhibited by 2-methylaminoisobutyrate. In contrast, histidine inhibited the transport of both glutamine and alanine non-competitively. It is concluded that glutamine and alanine are transported into rat colonocytes by a common carrier system similar to System A of other cells. It is suggested that the metabolic function of this transport system in rat colonocytes might be the partial exchange of extracellular glutamine for intracellular alanine.     

Na+-dependent transport of alpha-aminoisobutyrate in isolated basolateral membrane vesicles from rat parotid glands

Basolateral plasma membranes were prepared from rat parotid gland after centrifugation in a self-orienting Percoll gradient. K+-dependent phosphatase [Na+ + K+)-ATPase), a marker enzyme for basolateral membranes, was enriched 10-fold from tissue homogenates. Using this preparation, the transport of alpha-aminoisobutyrate was studied. The uptake of alpha-aminoisobutyrate was Na+-dependent, osmotically sensitive, and temperature-dependent. In the presence of a Na+ gradient between the extra- and intravesicular solutions, vesicles showed an 'overshoot' accumulation of alpha-aminoisobutyrate. Sodium-dependent alpha-aminoisobutyrate uptake was saturable, exhibiting an apparent Km of 1.28 +/- 0.35 mM and Vmax of 780 +/- 170 pmol/min per mg protein. alpha-Aminoisobutyrate transport was inhibited considerably by monensin, but incubating with ouabain was without effect. These results suggest that basolateral membrane vesicles, which possess an active amino acid transport system (system A), can be prepared from the rat parotid gland.     

Characterization of basolateral membrane Na/H antiport in rat jejunum

Na uptake studies were performed in order to examine the activity of a Na/H exchanger in basolateral membrane vesicles isolated from rat jejunum. Experiments were carried out under voltage-clamped conditions in order to avoid electrodiffusional ionic movements. 1 mM Na uptake was found to be enhanced by an outward proton gradient and its initial rate was further increased by the presence of monensin or nigericin. The pH gradient-driven Na uptake was inhibited by 2 mM amiloride and unaffected by 0.1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The initial rate of the proton gradient-induced Na uptake was saturable with respect to external Na, with a Km of 13.6 +/- 1.4 mM and a Vmax of 35.4 +/- 2.2 nmol/mg protein per min. Li competed with Na for the exchange process, whereas K, Rb, Cs, tetramethylammonium had no effect. We conclude that rat jejunal basolateral membrane contains a Na/H exchanger whose properties are similar to those of the antiporter identified in the brush-border membrane.     

Effects of corticosteroid on the transport and metabolism of glutamine in rat skeletal muscle.

Intramuscular glutamine falls with injury and disease in circumstances associated with increases in blood corticosteroids. We have investigated the effects of corticosteroid administration (0.44 mg/kg dexamethasone daily for 8 days, 200 g female rats) on intramuscular glutamine and Na+, muscle glutamine metabolism and sarcolemmal glutamine transport in the perfused hindlimb. After dexamethasone treatment intramuscular glutamine fell by 45% and Na+ rose by 25% (the respective muscle/plasma distribution ratios changed from 8.6 to 4.5 and 0.12 to 0.15); glutamine synthetase and glutaminase activities were unchanged at 475 +/- 75 and 60 +/- 19 nmol/g muscle per min. Glutamine output by the hindlimb of anaesthetized rats was increased from 31 to 85 nmol/g per min. Sarcolemmal glutamine transport was studied by paired-tracer dilution in the perfused hindlimb: the maximal capacity (Vmax) for glutamine transport into muscle (by Na(+)-glutamine symport) fell from 1058 +/- 310 to 395 +/- 110 nmol/g muscle per min after dexamethasone treatment, accompanied by a decrease in the Km (from 8.1 +/- 1.9 to 2.1 +/- 0.4 mM glutamine). At physiological plasma glutamine concentration (0.75 mM) dexamethasone appeared to cause a proportional increase in sarcolemmal glutamine efflux over influx. Addition of dexamethasone (200 nM) to the perfusate of control rat hindlimbs caused acute changes in Vmax and Km of glutamine transport similar to those resulting from 8-day dexamethasone treatment. The reduction in muscle glutamine concentration after dexamethasone treatment may be primarily due to a reduction in the driving force for intramuscular glutamine accumulation, i.e., in the Na+ electrochemical gradient. The prolonged increase in muscle glutamine output after dexamethasone treatment (which occurs despite a reduction in the size of the intramuscular glutamine pool) appears to be due to a combination of (a) accelerated sarcolemmal glutamine efflux and (b) increased intramuscular synthesis of glutamine.     

Uptake and incorporation of inositol by preimplantation mouse embryos.

The uptake of myo-inositol by preimplantation mouse embryos was investigated using [3H]myo-inositol. Uptake increased about 12-fold between one- and two-cell stages and increased again at the blastocyst stage (> 6-fold compared with the two-cell stage). Uptake at the blastocyst stage was time and temperature dependent; it was stimulated by sodium, inhibited by glucose and appeared to take place mainly via a saturable mechanism. Uptake in the presence of 6.25 mmol inositol l-1 was 1424 fmol inositol per blastocyst per h. About 10% of the [3H]inositol taken up by blastocysts during 8 h in culture was incorporated into lipid. Thin layer chromatography of the lipid showed that most of this inositol was incorporated into lipid material co-migrating with phosphatidylinositol with a small proportion co-migrating with phosphatidylinositol 4-phosphate.     

Characterization of threonine transport into a kidney epithelial cell line (BSC-1). Evidence for the presence of Na(+)-independent system asc [corrected

The transport routes for threonine in a primate kidney epithelial cell line (BSC-1) grown as monolayer in continuous cell culture were studied. We discovered at least four different transport systems for threonine uptake. The Na(+)-dependent route shows biphasic kinetics with a low and high affinity parameter. The apparent kinetic constants for Km1 and Km2 were 0.3 and 36 mM with apparent Vmax values of 6.3 and 90 nmol/mg protein/min, respectively. The high affinity, low Km component resembles system ASC activity, with respect to substrate selectivity. The Na(+)-independent route also exhibits biphasic kinetics. A high affinity component (apparent Km of 1.0 mM, and apparent Vmax of 7.2 nmol/mg protein/min) is sensitive to inhibition by leucine and the aminoendolevo-rotatory isomer of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, suggesting participation by system L. The low affinity component (apparent Km of 10.2 mM, and apparent Vmax of 71 nmol/mg protein/min) was specifically inhibited by threonine, serine, and alanine and could be assigned to system asc. The discrimination between system L and asc is based upon differences in pH sensitivity, trans stimulation, and Ki values. In addition, the effects of harmaline, a suspected sodium transport site inhibitor, have been studied. Harmaline noncompetitively inhibited Na(+)-dependent threonine uptake but had no effect on Na(+)-independent transport of threonine. This report is the first to present evidence for the presence of system asc in renal epithelial cells. The physiological and biochemical significance of our findings are discussed.     

Dietary protein reduction in sheep and goats: different effects on <Emphasis Type="SmallCaps">l</Emphasis>-alanine and <Emphasis Type="SmallCaps">l</Emphasis>-leucine transport across the brush-border membrane of jejunal enterocytes

It was the aim of this study to examine the potential regulatory effects of a long-term low dietary protein supply on the transport capacity of the jejunal brush-border membrane for amino acids. For this purpose, we used the neutral amino acids L-alanine (representative for nonessential amino acids) and L-leucine (representative for essential amino acids) as model substances. Ten sheep lambs, 8 weeks of age and 19-27 kg body weight, were allotted to two dietary regimes with either adequate or reduced protein supply which was achieved by 17.9% and 9.7% of crude protein in the concentrated feed, respectively. The feeding periods were 4-6 weeks in length. Similarly, eight goat kids of 5-7 weeks of age and 8-14 kg body weight were allotted to either adequate (crude protein 20.1%, feeding period 9-12 weeks) or reduced protein supply (10.1%, feeding period 17-18 weeks). Dietary protein reduction in lambs caused a significant body weight loss of 0.6 +/- 0.7 kg, whereas the body weight in control animals increased by 1.9 +/- 0.7 kg (P<0.05). Plasma urea concentrations decreased significantly by 60% (low protein 2.3 +/- 0.1 versus control 5.7 +/- 0.2 mmol l(-1), P<0.001). In kids, reduction of dietary protein intake led to significant decreases of the daily weight gain by 48% from 181 +/- 8 g to 94 +/- 3 g (P<0.001) and daily dry matter intake by 27% from 568 +/- 13 g to 417 +/- 6 g (P<0.01). Respective urea concentrations in plasma were reduced by 77% from 5.2 +/- 0.4 to 1.2 +/- 0.2 mmol l(-1) (P<0.01). Kinetic analyses of the initial rates of alanine uptake into isolated jejunal brush-border membrane vesicles from sheep and goats as affected by low dietary protein supply yielded that the apparent Km was neither significantly different between the species nor significantly affected by the feeding regime thus ranging between 0.12 and 0.16 mmol.l(-1). Reduction of dietary protein, however, resulted in significantly decreased Vmax values of the transport system by 25-30%, irrespective of the species. Kinetic analyses of the initial rates of leucine uptake into jejunal brush-border membrane vesicles from sheep and goats yielded that leucine uptake was mediated by Na+-dependent as well as Na+-independent processes. Similar to alanine, apparent Km values of leucine uptake were neither different between the species nor affected due to low dietary protein and ranged between 0.08 and 0.15 mmol l(-1). In contrast to the alanine transport mechanism, dietary protein reduction resulted in increased Vmax values of Na+-dependent leucine transport by 53% in sheep and 230% in goats. Similarly, Na+-independent leucine uptake was stimulated by 85% and 200% in sheep and in goats, respectively. This study shows adaptation of amino acid absorption at the brush-border membrane level of jejunal enterocytes of small ruminants due to dietary protein reduction. Whereas the transport capacity for the nonessential amino acid alanine was reduced due to low dietary protein, the transport capacity for the essential amino acid leucine was markedly stimulated. From this, the involvement of rather different feedback mechanisms in adaptation of intestinal amino acid transport mechanisms has to be discussed.     

alpha-aminoisobutyrate transport into cells from R3230AC mammary adenocarcinoma. Evidence for sodium ion-dependent and -independent carrier-mediated entry and effects of diabetes.

In the presence of Na+, alpha-aminoisobutyrate was transported by saturable and non-saturable processes into R3230AC mammary tumour cells isolated by enzymic treatment. Eadie-Hofstee analysis for the saturable process gave a curvilinear plot, suggesting that transport occurred by more than one carrier. In the absence of Na+, alpha-aminoisobutyrate was also transported by both saturable and non-saturable processes. This Na+-independent saturable process gave a linear plot according to Eadie-Hofstee analysis: V, 708 +/- 105 pmol/min per 5 X 10(6) cells; Km, 0.36 +/- 0.33 mM (mean +/- S.E.M.). Subtracting alpha-aminoisobutyrate entry in the absence of Na+ from total alpha-aminoisobutyrate uptake (in the presence of Na+) showed the presence of another saturable process (Na+-dependent), accounting for 75% of total alpha-aminoisobutyrate uptake. This component gave a linear Eadie-Hofstee plot: V, 2086 +/- 213; Km, 1.75 +/- 0.16 alpha-(Methylamino)isobutyrate, a substrate specifically taken up by the A system, inhibited 80% of alpha-aminoisobutyrate entry. The presence of both alhpa-(methylamino)isobutyrate and phenylalanine inhibited alpha-aminoisobutyrate entry completely. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate, an analogue specifically taken up by the Na+-independent system, inhibited completely the Na+-independent entry of alpha-aminoisobutyrate. In the presence of Na+, the distribution ratio, which is defined as the amino acid concentration in the intracellular space divided by that in the incubation medium for alpha-aminoisobutyrate, at 90 min was 19, and in the absence of Na+ at 60 min was 5. These concentrative processes were sensitive to the metabolic inhibitor pentachlorophenol. The Na+-dependent, but not the Na+-independent, alpha-aminoisobutyrate uptake was increased in cells from diabetic rats. This was primarily due to an increase in the V for the Na+-dependent component (164%) with no effect on the Km. We conclude, therefore, that alpha-aminoisobutyrate entry into cells from this mammary tumour is mediated by two transport systems, one Na+-dependent and another Na+-independent. Furthermore, the Na+-dependent component of alpha-aminoisobutyrate is sensitive to alterations of insulin in vivo.     

Basolateral amino acid transport systems in the perfused exocrine pancreas: sodium-dependency and kinetic interactions between influx and efflux mechanisms

Basolateral amino acid transport systems have been characterized in the perfused exocrine pancreas using a high-resolution paired-tracer dilution technique. Significant epithelial uptakes were measured for L-alanine, L-serine, alpha-methylaminoisobutyric acid, glycine, methionine, leucine, phenylalanine, tyrosine and L-arginine, whereas L-tryptophan and L-aspartate had low uptakes. alpha-Methylaminoisobutyric acid transport was highly sodium dependent (81 +/- 3%), while uptake of L-serine, L-leucine and L-phenylalanine was relatively insensitive to perfusion with a sodium-free solution. Cross-inhibition experiments of L-alanine and L-phenylalanine transport by twelve unlabelled amino acids indicated overlapping specificities. Unidirectional L-phenylalanine transport was saturable (Kt = 16 +/- 1 mM, Vmax = 12.3 +/- 0.4 mumol/min per g), and weighted non-linear regression analysis indicated that influx was best described by a single Michaelis-Menten equation. The Vmax/Kt ratio (0.75) for L-phenylalanine remained unchanged in the presence of 10 mM L-serine. Although extremely difficult to fit, L-serine transport appeared to be mediated by two saturable carriers (Kt1 = 5.2 mM, Vmax1 = 7.56 mumol/min per g; Kt2 = 32.8 mM, Vmax2 = 22.9 mumol/min per g). In the presence of 10 mM L-phenylalanine the Vmax/Kt ratio for the two L-serine carriers was reduced, respectively, by 79% and 50%. Efflux of transported L-[3H]phenylalanine or L-[3H]serine was accelerated by increasing perfusate concentrations of, respectively, L-phenylalanine and L-serine, and trans-stimulated by other amino acids. In the pancreas neutral amino acid transport appears to be mediated by Na+-dependent Systems A and ASC, the classical Na+-independent System L and another Na+-independent System asc recently identified in erythrocytes. The interactions in amino acid influx and efflux may provide one of the mechanisms by which the supply of extracellular amino acids for pancreatic protein synthesis is regulated.     

Sodium-dependent phosphate and alanine transports but sodium-independent hexose transport in type II alveolar epithelial cells in primary culture

Inorganic phosphate, amino acids and sugars are of obvious importance in lung metabolism. We investigated sodium-coupled transports with these organic and inorganic substrates in type II alveolar epithelial cells from adult rat after one day in culture. Alveolar type II cells actively transported inorganic phosphate and alanine, a neutral amino acid, by sodium-dependent processes. Cellular uptakes of phosphate and alanine were decreased by about 80% by external sodium substitution, inhibited by ouabain (30 and 41%, respectively) and displayed saturable kinetics. Two sodium-phosphate cotransport systems were characterized: a high-affinity one (apparent Km = 18 microM) with a Vmax of 13.5 nmol/mg protein per 10 min and a low-affinity one (apparent Km = 126 microM) with a Vmax of 22.5 nmol/mg protein per 10 min. Alanine transport had an apparent Km of 87.9 microM and a Vmax of 43.5 nmol/mg protein per 10 min. By contrast, cultured alveolar type II cells did not express sodium-dependent hexose transport. Increasing time in culture decreased Vmax values of the two phosphate transport systems on day 4 while sodium-dependent alanine uptake was unchanged. This study demonstrated the existence of sodium-dependent phosphate and amino acid transports in alveolar type II cells similar to those documented in other epithelial cell types. These sodium-coupled transports provide a potent mechanism for phosphate and amino acid absorption and are likely to play a role in substrate availability for cellular metabolism and in regulating the composition of the alveolar subphase. The decrease in phosphate uptake with time in culture is parallel to decrease in surfactant synthesis reported in cultured alveolar type II cells, suggesting that phosphate availability for surfactant synthesis may be accomplished by a sodium-dependent phosphate uptake.     

Transport of carnosine by mouse intestinal brush-border membrane vesicles

The characteristics of carnosine (beta-alanyl-L-histidine) transport have been studied using purified brush-border membrane vesicles from mouse small intestine. Uptake curves did not exhibit any overshoot phenomena, and were similar under Na+, K+ or choline+ gradient conditions (extravesicular greater than intravesicular). However, uptake of histidine showed an overshoot phenomenon in the presence of a Na+-gradient. There was no detectable hydrolysis of carnosine during 15 min of incubation with membrane vesicles under conditions used for transport experiments. Analysis of intravesicular contents further showed the complete absence of the constituent free amino acids of carnosine, and indicates that intact carnosine is transported. Studies on the effect of concentration on peptide uptake revealed that transport occurred by a saturable process conforming to Michaelis-Menten kinetics with a Km of 9.6 +/- 1.4 mM and a Vmax of 2.9 +/- 0.2 nmol/mg protein per 0.4 min. Uptake of carnosine was inhibited by both di- and tripeptides with a maximum inhibition of 68% by glycyl-L-leucyltyrosine. These results clearly demonstrate that carnosine is transported intact by a carrier-mediated, Na+-independent process.     

Uptake of L-[14C]Proline by Isolated Rat Brain Capillaries

Rat brain capillaries exhibit concentrative uptake of L-proline. The uptake is mediated by two saturable systems, one with a Km of 0.11 mM and another with a Km of 5.9 mM. Entry also occurred by diffusion, especially at high substrate concentrations. The saturable high-affinity system is sodium-dependent, with a Km for sodium of 36 mM. Proline uptake is not inhibited by lysine, but is inhibited by phenylalanine, glycine, and leucine. alpha-Methylaminoisobutyric acid (MeAIB), a model for sodium-requiring transport systems, is a competitive inhibitor of the low-Km system. b-2-Aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH), a model for nonsodium-dependent transport, however, also inhibited proline uptake.     

Transport of bile acids in a human intestinal epithelial cell line, Caco-2

The transport of taurocholic acid (TA) across Caco-2 cell monolayers was dependent on time in culture and reached a plateau after 28 days, at which time the apical (AP)-to-basolateral (BL) transport was 10-times greater than BL-to-AP transport. The amounts of TA inside the cells following application of 10 nM [14C]TA to the AP or BL side of the monolayers (30 min) were approximately equal (54.4 +/- 2.7 and 64.6 +/- 2.8 fmol/mg protein, respectively). AP-to-BL transport of TA was saturable and temperature-dependent. Vmax and Km for transport were 13.7 pmol/mg protein per min and 49.7 microM, respectively. The transport of TA had an activation energy of 13.2 kcal.mol-1, required Na+ and glucose. AP-to-BL transport of [14C]TA was inhibited by the co-administration (on the AP side) of either unlabeled TA or deoxycholate, but it was not reduced by the presence of unlabeled TA on the BL side.     

The effect of 3-sulphation and taurine conjugation on the uptake of chenodeoxycholic acid by rat hepatocytes

The hepatic uptake of chenodeoxycholic acid, taurochenodeoxycholic acid, chenodeoxycholic acid 3-sulphate and taurochenodeoxycholate acid 3-sulphate by isolated rat hepatocytes was examined. Taurochenodeoxycholic acid, taurochenodeoxycholic acid 3-sulphate and chenodeoxycholic acid 3-sulphate uptake occurred by a saturable, energy-dependent process while chenodeoxycholic acid uptake was predominantly non-saturable, possibly simple diffusion. Apparent Km (mumol/l) and Vmax (nmol/mg protein per min) values (mean +/- S.D.), respectively, were: chenodeoxycholic acid (saturable component), 33 +/- 6.4 and 4.8 +/- 0.6; taurochenodeoxycholic acid, 11.1 +/- 2.0 and 3.1 +/- 0.5; chenodeoxycholic acid 3-sulphate, 6.1 +/- 0.9 and 2.3 +/- 0.4; and taurochenodeoxycholic acid 3-sulphate, 5.0 +/- 0.7 and 0.9 +/- 0.15. Both conjugation with taurine and sulphation at the 3 position resulted in a reduction in the values of Km and Vmax. Uptake of each of the bile acids taurochenodeoxycholic acid, taurochenodeoxycholic acid 3-sulphate and chenodeoxycholic acid 3-sulphate was competitively inhibited by the other two, with taurochenodeoxycholic acid a potent inhibitor of both taurochenodeoxycholic acid 3-sulphate and chenodeoxycholic acid 3-sulphate uptake. Other bile acids also inhibited. Uptake was inhibited by albumin in the order chenodeoxycholic acid 3-sulphate greater than taurochenodeoxycholic acid 3-sulphate greater than taurochenodeoxycholic acid and was dependent on the extent of bile acid binding to albumin.     

Derecruitment in cat liver: extension of undistributed parallel tube model to effects of low hepatic blood flow on ethanol uptake

Previous studies showed two deviations from the predictions of the undistributed parallel tube model for hepatic uptake of substrates: a small deviation at high flows and a large deviation at low flows. We have examined whether these deviations could be described by a single correction factor. In cats anesthetized with pentobarbital, a hepatic venous long-circuit technique with an extracorporeal reservoir was used to vary portal flow and hepatic venous pressure, and allow repeated sampling of arterial, portal, and hepatic venous blood without depletion of the cat's blood volume. Hepatic uptake of ethanol was measured over a wide range of blood flows and when intrahepatic pressure was increased at low flows. This uptake could be described by the parallel tube model with a correction for hepatic blood flow: Uptake = Vmax max.(1 - e-kF).c/(Km + c). In 22 cats, Vmax max = 90 +/- 5 mumols/(min.100 g liver), k = 0.021 +/- 0.0015 when flow (F) was in millilitres per minute per 100 g liver, and Km = 150 +/- 20 microM when c is the log mean sinusoidal concentration. (1 - e-kF) represents the proportion of sinusoids perfused and metabolically active. A dynamic interpretation of this proportion is related to intermittency (derecruitment) of sinusoidal flow. Half the sinusoids were perfused at a flow of 33 mL/(min.100 g liver) and the liver was essentially completely perfused (greater than 95%) at the normal flow of 150 mL/(min.100 g liver). Derecruitment was not changed by raising hepatic venous pressure, and it was not related to hepatic venous resistance.