Transport Of L-lysine Research Articles

Fucoidan from Undaria pinnatifida mitigates intestinal inflammation in Atlantic salmon (Salmo salar)

Using functional feed ingredients to increase the intestinal health of Atlantic salmon (Salmo salar) could allow greater variation and higher inclusion of plant-based proteins in the feed. This is beneficial both for the fish feed industry and fish welfare. Components with strong bioactive properties, such as fucoidans from marine macroalgae, have a great potential as a functional feed ingredient. In the present trial, we challenged Atlantic salmon (Salmo salar) post-smolts with 17.8% soya HP48, a diet that has been shown to cause low-level intestinal inflammation. Fucoidan from Undaria pinnatifida was included at 0, 1, or 3% (Control, FUC1, and FUC3, respectively) to study its ability to alleviate this low-level inflammation . The fish were fed the diets in triplicate tanks for 59 days. At the end of the experiment, the fish were euthanised and the mid- and hind intestine were sampled for morphological-, integrity- (Ussing chamber) and gene expressional- (RNAseq) assessments. Addition of 3% fucoidan lowered the protein digestibility of the feed, however, this did not result in significant differences in the growth parameters. The paracellular permeability of the hydrophilic marker 14C-mannitol tended to decrease with the addition of fucoidan in both tissue segments. The active transport of l-lysine in the mid intestine was significantly reduced in a dose-dependent manner of fucoidan. An anti-inflammatory effect of fucoidan was observed in the hind intestine with significant reductions in the stratum granulosum width and lamina propria height of fish fed FUC1 and FUC3 compared to the Control. This suggests that U. pinnatifida fucoidan can have positive effect on both intestinal integrity and immune regulation of Atlantic salmon (Salmo salar).

The Transport of Lysine across Monolayers of Human Cultured Intestinal Cells (Caco-2) Depends on Na+-Dependent and Na+-Independent Mechanisms on Different Plasma Membrane Domains

To characterize the mechanisms involved in the intestinal absorption of the essential amino acid L-lysine from the diet, the transepithelial transport of L-lysine was studied in monolayers of cultured human intestinal cells (Caco-2) grown and differentiated on microporous membrane supports. L-lysine was transported mainly in the apical (AP) to basolateral (BL) direction and the BL to AP transport was ∼ one order of magnitude lower at all concentrations tested. Nonlinear regression analysis of the transport in the AP to BL and the BL to AP direction identified, in both cases, single saturable components with similar Km but different Vmax and a nonsaturable diffusional component. The AP to BL L-lysine transport was highly energy- and sodium-dependent and was unaffected by an unfavorable concentration gradient. Selective replacement of sodium ions in the AP or the BL compartment and determination of both AP to BL transport and the intracellular soluble lysine pool showed that uptake occurs via a sodium-independent mechanism, not significantly influenced by membrane potential, whereas efflux is a sodium-dependent process. Competition experiments showed that L-lysine uptake is highly stereospecific and is shared by cationic and large neutral amino acids. This study demonstrates the presence of a sodium-dependent mechanism of lysine efflux across the BL membrane of intestinal cells, which may be essential for lysine transport into the blood circulation. Overall, these results support the use of the Caco-2 cell model for studies of intestinal nutrient transport.

The role of environmental salinity on Na+-dependent intestinal amino acid uptake in rainbow trout (Oncorhynchus mykiss)

Na+/K+-ATPases (NKA) in the basolateral membrane of the intestinal enterocytes create a Na+-gradient that drives both ion-coupled fluid uptake and nutrient transport. Being dependent on the same gradient as well as on the environmental salinity, these processes have the potential to affect each other. In salmonids, L-lysine absorption has been shown to be higher in freshwater (FW) than in seawater (SW) acclimated fish. Using electrophysiology (Ussing chamber technique), the aim was to explore if the decrease in L-lysine transport was due to allocation of the Na+-gradient towards ion-driven fluid uptake in SW, at the cost of amino acid transport. Intestinal NKA activity was higher in SW compared to FW fish. Exposure to ouabain, an inhibitor of NKA, decreased L-lysine transport. However, exposure to bumetanide and hydrochlorothiazide, inhibitors of Na+, K+, 2Cl−-co-transporter (NKCC) and Na+, Cl−-co-transporter (NCC) respectively, did not affect the rate of intestinal L-lysine transport. In conclusion, L-lysine transport is Na+-dependent in rainbow trout and the NKA activity and thus the available Na+-gradient increases after SW acclimation. This increased Na+-gradient is most likely directed towards osmoregulation, as amino acid transport is not compromised in SW acclimated fish.

Intestinal health in Atlantic salmon post-smolt (Salmo salar) when fed low- and high HUFA diets

It is well established that farmed Atlantic salmon (Salmo salar) need n3-highly unsaturated fatty acids (HUFA) in their diet to thrive and grow. However, the biological functions to the individual HUFAs may differ, implying that future supplementation could require fixed ratios for maximum benefit. The intestinal barrier is essential to fish health, and any disruption of the barrier can have detrimental effects. The current experiment was designed to examine the response of the intestinal hindgut when fed a low HUFA diet with 8.5 g/kg EPA + DHA (4.5% total fatty acid) or two high HUFA diets, with either high DHA (28.7 g/kg and 5.9% total fatty acid) or high EPA (25 g/kg and 14.2% total fatty acid). The diets were fed to Atlantic salmon post-smolt over 10 weeks and thereafter exposed to 3 weeks of chronic stress. After 10 weeks of feeding there were no differences in intestinal permeability and integrity, but intestinal morphology indicated increased intestinal health in the high EPA group. Gene expression also suggest that fish fed the high EPA diet had more regulation of pathways related to protein turnover compared to the high DHA fed fish. There was also indication of lower energy utilization in the low HUFA fed fish than high HUFA. Subjecting fish to 3 weeks of chronic stress led to a reduction in transepithelial resistance, increased ion flux and active l-lysine transport across the intestinal barrier in addition to a decrease in mucosal fold, enterocyte height and supranuclear vacuole density and an increase in thickness of the intestinal muscularis. After stress, the low HUFA group showed signs of inflammation with increased infiltration of MHCII positive cells. Gene expression also showed that low HUFA fed fish had a lower response to chronic stress compared to the high HUFA groups. Comparing fish fed either high DHA or EPA exposed to chronic stress showed few physical effects, but a lower density of supranuclear vacuoles and upregulation in immune-related gene expression indicate inflammation in the high DHA group.

Uptake of Manganese from the Manganese-Lysine Complex in Primary Chicken Intestinal Epithelial Cells.

Simple SummaryManganese (Mn) supplementation is especially necessary to avian species because the absorption of dietary Mn is relatively inefficient in birds. Recently, there has been increasing interest in the use of organic Mn to replace inorganic Mn as dietary Mn supplements in poultry. This study compared the uptake of Mn from Mn-lysine complex (MnLys) and MnSO4 in the primary chicken intestinal epithelial cells when the Fe, N-ethylmaleimide (a transport system y+ inhibitor), or cycloheximide (a transport system b0,+ activator) added in the culture medium. The results revealed that the uptake of Mn from the MnLys complex not only might be transported through the ionized Mn2+ pathway, but also appeared to be transported through the transport systems y+ and b0,+ in the intestine of chickens.Organic manganese (Mn) sources can replace inorganic Mn as dietary Mn supplements in poultry. To compare the uptake of Mn from the Mn-lysine complex (MnLys) and MnSO4, we first established the primary chicken intestinal epithelial cells (IECs) model and used it to determine Mn uptake. The MnLys increased the uptake of Mn compared to MnSO4. The uptake of Mn decreased in the IECs with Fe addition in the medium regardless of the Mn sources. The MnLys decreased the Mn2+ efflux transporter ferroportin 1 (FPN1) mRNA level but did not influence the Mn2+ influx transporter divalent metal transporter 1 (DMT1) mRNA expression when compared to MnSO4. The results above indicated that the increase of Mn accumulation for MnLys at least partly was due to the decrease of Mn efflux by reduced FPN1 expression. The addition of N-ethylmaleimide, an L-lysine transport system y+ inhibitor, decreased the uptake of Mn from MnLys but did not affect the uptake of Mn from MnSO4. The cycloheximide, as an L-lysine transport system b0,+ activator, increased the uptake of Mn from MnLys, whereas they did not influence the uptake of Mn from MnSO4. The MnLys increased the system y+ members cationic amino acid transporter (CAT) 1 and CAT2, and system b0,+ components rBAT and b0,+AT mRNA expression when compared to MnSO4. These results suggested that the uptake of MnLys complex might be transported by CAT1/2 and system b0,+, which was different from the ionized Mn2+ uptake pathway. In conclusion, the uptake of Mn from MnLys complex not only might be uptake through the ionized Mn2+ pathway, but also appeared to be transported through the CAT1/2 and system b0,+ in primary chicken IECs.

Uptake of manganese from manganese-lysine complex in the primary rat intestinal epithelial cells.

This study was conducted to compare the differences of the uptake of Mn from Mn-lysine complex (MnLys) and MnSO4 and to determine the potential mechanisms of the uptake of Mn from MnLys. We first established the primary rat intestinal epithelial cell culture model and used it to determine the uptake of Mn from different Mn sources. The MnLys increased (p<0.001) Mn uptake when compared to MnSO4 . The uptake of Mn decreased (p<0.05) with added Fe concentration increasing in the medium regardless of Mn source. The MnLys decreased (p<0.01) Mn2+ efflux transporter ferroportin 1 (FPN1) mRNA level, but did not influence (p>0.06) Mn2+ influx transporter DMT1 mRNA expression when compared to MnSO4 . The results above indicated that the increase of Mn accumulation for MnLys at least partly was due to the decrease of Mn efflux by reduced FPN1 expression. The N-ethylmaleimide, as an l-lysine transport system y+ inhibitor, decreased (p<0.001) the uptake of Mn from MnLys, but did not affect (p>0.10) the uptake of Mn from MnSO4 . The cycloheximide, as an l-lysine transport system b0,+ activator, increased (p<0.001) the uptake of Mn from MnLys, whereas also did not influence the uptake of Mn from MnSO4 . The MnLys increased (p<0.01) the system y+ member cationic amino acid transporter (CAT) 1, and system b0,+ components rBAT and b0,+ AT mRNA expression when compared to MnSO4 . These results suggested that the uptake of Mn from MnLys complex might be transported by CAT1 and system b0,+ , which was different from ionized Mn2+ uptake pathway. In conclusion, the uptake of MnLys complex not only might be absorbed as Mn2+ , but also appeared to be transported through CAT1 and system b0,+ in the primary rat intestinal epithelial cells.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease from Pseudomonas aeruginosa.

The prokaryotic lysine-specific permease (LysP) belongs to the amino acid-polyamine-organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of L-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP from Pseudomonas aeruginosa was recombinantly expressed in Escherichia coli and purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of L-lysine and the L-lysine analogue L-4-thialysine by vapour diffusion and diffracted to 7.5 Å resolution. The diffraction data were indexed in space group P21, with unit-cell parameters a = 169.53, b = 169.53, c = 290.13 Å, γ = 120°.

Characterization of L-lysine transport across equine and porcine jejunal and colonic brush border membrane

In nonruminant herbivores, microbially derived AA could contribute to whole-body AA homeostasis and thus decrease predicted AA requirements. However, postileal capacity of AA uptake is currently unknown. Therefore, to test the hypothesis that Lys is transported across the large colon mucosal apical membrane with capacity similar to that of the small intestinal mucosa in the pony and pig, we examined Lys transport in vitro using brush border membrane vesicles (BBMV). Mucosa was collected from the distal jejunum (DJ) and proximal large colon (PLC) of growing pigs (n = 3) and ponies (n = 4), flash frozen in liquid nitrogen, and stored at -80°C. Jejunal and colonic BBMV were manufactured by Mg(2+) precipitation and used to determine initial rates and kinetics [the maximal transport rate (V(max)) and the Michaelis constant (K(M))] of l-Lys transport into apical epithelia by rapid filtration technique in Na(+)-gradient incubation buffer. Initial rates of total l-Lys uptake did not differ between the PLC and DJ in either the pig or the pony, or between the pony and the pig, at each l-Lys concentration. In the pig, compared with the DJ, l-Lys transport V(max) in the PLC did not differ (121 ± 26 and 180 ± 26 pmol•mg of protein(-1)•s(-1), respectively; P = 0.14) and l-Lys K(M) in the PLC tended to be greater (0.23 ± 0.22 and 0.89 ± 0.22 mM, respectively; P = 0.09). In the pony, compared with the DJ, l-Lys transport V(max) in the PLC was greater (62 ± 25 and 149 ± 25 pmol•mg of protein(-1)•s(-1), respectively; P = 0.04) and l-Lys K(M) in the PLC was greater (0.08 ± 0.22 and 1.05 ± 0.22 mM, respectively; P = 0.02). l-Lysine diffusion was not different between segments; however, total intestinal diffusion was greater (P = 0.03) in the pony than in the pig (115 ± 10 and 73 ± 10 pmol·mg of protein(-1)•s(-1), respectively). These results demonstrate that the large colon is capable of l-Lys transport across the apical epithelial membrane with greater capacity and less affinity than the jejunum, indicating that the large colon may play a significant role in l-Lys absorption and homeostasis in hindgut fermenters.

Characterization of L-Lysine Transport across Equine Small Intestine and Large Colon Brush Border Membrane

Characterization of L-Lysine Transport across Equine Small Intestine and Large Colon Brush Border Membrane

Functional relationship between cationic amino acid transporters and β-defensins: Implications for dry skin diseases and the dry eye

The ocular surface, constantly exposed to environmental pathogens, is particularly vulnerable to infection. Hence an advanced immune defence system is essential to protect the eye from microbial attack. Antimicrobial peptides, such as β-defensins, are essential components of the innate immune system and are the first line of defence against invaders of the eye. High concentrations of l-arginine and l-lysine are necessary for the expression of β-defensins. These are supplied by epithelial cells in inflammatory processes. The limiting factor for initiation of β-defensin production is the transport of l-arginine and l-lysine into the cell. This transport is performed to 80% by only one transporter system in the human, the y +-transporter. This group of proteins exclusively transports the cationic amino acids l-arginine, l-lysine and l-ornithine and is also known under the term cationic amino acid transporter proteins (CAT-proteins). Various infections associated with l-arginine deficiency (for example psoriasis, keratoconjuctivitis sicca) are also associated with an increase in β-defensin production. For the first time, preliminary work has shown the expression of human CATs in ocular surface epithelia and tissues of the lacrimal apparatus indicating their relevance for diseases of the ocular surface. In this review, we summarize current knowledge on the human CATs that appear to be integrated in causal regulation cascades of β-defensins, thereby offering novel concepts for therapeutic perspectives.

Root uptake of cationic amino acids by Arabidopsis depends on functional expression of amino acid permease 5

* Specific transporters mediate uptake of amino acids by plant roots. Earlier studies have indicated that the lysine histidine transporter 1 and amino acid permease 1 participate in this process, but although plant roots have been shown to absorb cationic amino acids with high affinity, neither of these transporters seems to mediate transport of L-arginine (L-Arg) or L-lysine (L-Lys). * Here, a collection of T-DNA knockout mutants were screened for alterations in Arabidopsis root uptake rates of L-Arg and it was found that only the AAP5 mutant displayed clear phenotypic divergence on high concentrations of L-Arg. A second screen using low concentrations of (15)N-labelled L-Arg in the growth media also identified AAP5 as being involved in L-Arg acquisition. * Momentaneous root uptake of basic amino acids was strongly affected in AAP5 mutant lines, but their uptake of other types of amino acids was only marginally affected. Comparisons of the root uptake characteristics of AAP5 and LHT1 mutants corroborated the hypothesis that the two transporters have distinct affinity spectra in planta. * Root uptake of all tested amino acids, except L-aspartic acid (L-Asp), was significantly affected in double AAP5*LHT1 mutants, suggesting that these two transporters account for a major proportion of roots' uptake of amino acids at low concentrations.

Rickettsial phospholipase A2 as a pathogenic mechanism in a model of cell injury by typhus and spotted fever group rickettsiae.

Phospholipase A2 activity by typhus group rickettsiae causes hemolysis in vitro. Rickettsial phospholipase A2 has been proposed to mediate entry into the host cell, escape from the phagosome, and cause injury to host cells by both typhus and spotted fever group rickettsiae. In a rickettsial contact-associated cytotoxicity model, the interaction of Rickettsia prowazekii or R. conorii with Vero cells caused temperature-dependent release of 51Cr from the cells. Treatment of rickettsiae, but not the cells, with a phospholipase A2 inhibitor (bromophenacyl bromide) or with antibody to king cobra venom inhibited cell injury. Rickettsial treatment with bromophenacyl bromide inhibited the release of free fatty acids from the host cell. Neither the inhibitor nor antivenom impaired rickettsial active transport of L-lysine. Thus, host cell injury was mediated by a rickettsial phospholipase A2-dependent mechanism.

Reaction engineering analysis of L-lysine transport by Corynebacterium glutamicum

To identify potential L-lysine export limitations by Corynebacterium glutamicum in the L-lysine production process, the excretion of L-lysine was studied in continuous and fed-batch operated stirred tank reactors. A structured biochemical model of the L-lysine excretion mechanism was used to determine the activity of the export carrier and to calculate a cell-specific concentration of the export carrier. For the biochemical characterization of this specific carrier concentration a standardized L-lysine efflux test was developed. Carrier activity, cell-specific carrier concentration, and the specific L-lysine export rate were identified as a function of pH value and L-lysine concentration in the reactors. Also, the correlation of these parameters to the metabolic state of C. glutamicum was determined. The pH value in the reactor governs the carrier activity (maximum at pH 6.5) and the specific carrier concentration (maximum at pH 8.0). The specific L-lysine export rate, as the product of carrier activity and specific carrier concentration, revealed a maximum at pH 7.0. Decreasing L-lysine productivities also correlated with decreasing specific carrier concentrations. The L-lysine concentration in the reactor had no effect on the specific carrier concentration but strongly inhibited the carrier activity. The specific export rate was reduced to 50% at 400 mM L-lysine compared to the specific export rate at 80 mM L-lysine. (c) 1996 John Wiley & Sons, Inc.

Sodium-independent lysine uptake by the bewo choriocarcinoma cell line

Transport of L-lysine by a cultured placental trophoblast cell line was investigated by characterization of L-[ 3H]lysine uptake. In the mononuclear form of the BeWo clone b30 choriocarcinoma cell, at least two sodium-independent systems are present. Concentration dependence data were fitted by a two system model with K m values ( ± s.e.) of 2 ± 0.7 and 94 ± 31 μ m and V max values ( ± s.e.) of 0.7 ± 0.3 and 25 ± 6.0 n m/mg DNA/min. A portion of sodium-independent uptake was inhibited by the sulphydryl modifying reagent N-ethylmaleimide (NEM). Following NEM treatment, the data were fitted by a single system with K m=10±2 μ m and V max=5.1 ± 0.8 n m/ mg DNA/min. In the absence of sodium, NEM-resistant uptake was sensitively inhibited by leucine whereas NEM-sensitive uptake was not inhibited by leucine. It is concluded that like placental basal membrane, the mononuclear BeWo cell possesses two sodium-independent L-lysine transport systems. The high-capacity, NEM-sensitive, leucine-insensitive system resembles the widespread system y+. The high-affinity, NEM-resistant, leucine-sensitive system resembles system b 0,+.

Changes in structural and functional properties of rat intestinal brush border membrane during starvation

Changes in surface area of microvilli, fluidity of brush border membrane and transport of L-amino acids through intestinal epithelial cells were studied in wellfed and starved (2,4 and 6 days) rats. The surface area of microvilli per unit area of intestinal epithelial cells increased during starvation. Studies with fluoroprobes — pyrene, 1-anilinonaphthalene-8-sulphonate and 1,6-diphenyl-1,3,5-hexatriene, showed increased fluidity of brush border membrane on progressive starvation. Transport of five amino acids representing five different transport systems was studied during starvation in everted intestinal sleeves. Transport of L-proline, glycine and L-glutamic acid which represent imino, glycine and acidic systems respectively increased significantly in Na +-dependent pathway whereas transport of L-lysine representing basic system increased significantly in Na +-independent pathway during starvation.

The influence of luminal pH on transport of neutral and charged dipeptides by rat small intestine, in vitro

Four hydrolysis-resistant dipeptides ( d-phenylalanyl- l-alanine, d-phenylalanyl- l-glutamine, d-phenylalanyl- l-glutamate and d-phenylalanyl- l-lysine) were synthesized to investigate the effects of net charge on transmural dipeptide transport by isolated jejunal loops of rat small intestine. At a luminal pH of 7.4 and a concentration of 1 mM the two dipeptides with a net charge of −1 and +1 were transported at substantially slower rates (18±1.3 and 8.4±1.3 nmol min −1 (g dry wt.) −1, respectively) than neutral d-phenylalanyl- l-alanine and d-phenylalanyl- l-glutamine (87±0.2 and 197±14 nmol min −1 (g dry wt.) −1, respectively). We investigated the effects of luminal pH on dipeptide transport by varying the NaHCO 3 content of Krebs Ringer perfusate equilibrated with 95%O 2/5% CO 2. The pH changes did not affect water transport, but serosal glucose appearance increased significantly at pH 6.8. Transmural transport of d-phenylalanyl- l-alanine and d-phenylalanyl- l-glutamine at pH 6.8 was stimulated ( P<0.01) by 61% and 49%, respectively, whereas the lower pH increased the rate for negatively charged d-phenylalanyl- l-glutamate by 306% ( P<0.01) and decreased that for positively charged d-phenylalanyl- l-lysine by 46% ( P<0.05). Increasing luminal pH to 8.0 inhibited d-phenylalanyl- l-alanine transport by 60%, whereas d-phenylalanyl- l-lysine transport was 60% faster.

Transport of nitrullin, a new nitrosoalkylurea derivative, into tumor and normal cells

The interaction between the antitumor drug nitrullin and the system providing the transport ofl-lysine into P388 leukemic cells and murine enterocytes is studied. Two types of lysine carriers with low and high affinity for the substrate are identified. Nitrullin competitively inhibits the transport of3H-lysine and shows the same affinity for both carriers. It is similar to that of lysine for the low-affinity carrier and is 80-fold lower than lysine affinity for the high-affinity carrier. Kinetic characteristics of the low-affinity transport of3H-lysine and Ki of nitrullin are similar to those obtained at a reciprocal substrate-inhibitor ratio. Nitrullin does not inhibit active transport of3H-lysine into enterocytes against the background of considerable (70%) passive diffusion.

L-lysine transport in chicken jejunal brush border membrane vesicles.

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (KmA = 2.4 +/- 0.7 micromol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na+ (l-methionine inhibition constant KiA, NaSCN = 21.0 +/- 8.7 micromol/L and KSCN = 55.0 +/- 8. 4 micromol/L); second, a low-affinity transport mechanism (KmB = 164. 0 +/- 13.0 micromol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na+ (KiB for L-methionine, NaSCN = 1.7 +/- 0.3 and KSCN = 3.4 +/- 0.9 mmol/L). L-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b0,+ and y+, respectively.

Insulin stimulates cationic amino acid transport activity in the isolated perfused rat pancreas.

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.

Expression of rat intestinal L-lysine transport systems in isolated oocytes of Xenopus laevis.

The mechanisms by which cationic amino acids are transported across the intestinal epithelium are poorly understood. We show that isolated stage VI oocytes of Xenopus laevis can express lysine transport activity, which is due to the microinjection of mRNA from rat small intestine. L-Lysine transport activity (0.2 mM, 20 degrees C) reaches 400 pmol.oocyte-1.h-1, compared with a typical endogenous rate of 85 pmol.oocyte-1.h-1. Na(+)-dependence and amino acid inhibition studies resolved the expressed transport activity into three components: 1) a Na(+)-dependent transport system that can be inhibited by leucine with high affinity and also by alanine; 2) a Na(+)-independent system that can be inhibited by leucine with high affinity when Na+ is present, but this affinity is reduced in its absence; 3) a Na(+)-independent system that is inhibited by leucine with high affinity only when Na+ is present. Peak arginine-inhibitable lysine influx was found in a mRNA size fraction of 1.5-2.25 (median 2.0) kb.