Search

The mechanism of uptake of the sympathomimetic drug broxaterol by Chang liver and HepG2 cell lines was investigated. When cells were incubated in the presence of low concentrations of broxaterol cell cultures take up (t1/2 = 5-10 min) and, depending on the experimental conditions, accumulate the drug such that the intracellular concentration is over 200-1000 times that in the incubation medium. The uptake was saturable and influenced by the presence of sodium and variations in external pH. These data may represent a model for tissue uptake in vivo in an attempt to investigate that selective uptake of broxaterol is involved in withdrawal of the molecule during drug therapy.

The effect of sublethal concentrations of potassium dichromate and fenitrothion on sodium-leucine cotransport in brush border membrane vesicles from Chironomus riparius larvae has been investigated. Exposure to potassium dichromate and fenitrothion caused a dose- and time-dependent inhibition of leucine uptake. Transport inhibition is easily detectable at doses 100-fold lower than LD50. Kinetic experiments showed that inhibition was mainly caused by a decrease of the Vmax (680 ± 53 vs. 382 ± 23 and 555 ± 27 nmol/15s/mg protein in control and exposed larvae to K2Cr2O7 and fenitrothion, respectively). Inhibition is possibly related to a variation of sodium ions permeability as evidenced by increased membrane lipid peroxidation. Appropriate control experiments ruled out that the observed differences could be due to changes in general features of membrane preparations. Transport inhibition observed in larvae exposed to potassium dichromate was accompanied by changes in ascorbate peroxidase and dehydroascorbate reductase activities, whereas those exposed to fenitrothion displayed an increase in transaminase activity. The possible value of leucine uptake as biochemical biomarker is briefly discussed.

We have identified three methyl esters that have a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids in the silkworm Bombyx mori. l-Leucine methyl ester, the most powerful activator, determined a large dose-dependent, K+-independent increase of leucine uptake into midgut brush border membrane vesicles. Kinetic experiments revealed non-essential mixed-type activation, with Ka values of 27±2 and 47±8 micromolar in the presence and in the absence of K+, respectively. The activation increased Km twofold, and Vmax up to 18-fold depending upon the experimental conditions. Leucine uptake mediated by the amino acid uniport appears to be unaffected by the activator.

A number of methyl and ethyl esters of naturally occurring amino acids exert a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids along the midgut of the silkworm Bombyx mori. L-Leucine methyl ester (Leu-OMe), one of the most effective activators, induces a large increase of the initial rate of leucine uptake in midgut brush border membrane vesicles (BBMV) from the anterior-middle (AM) region, and a small effect in BBMV from the posterior (P) region. Nonetheless, the methyl ester causes in both regions a relevant K+-, Deltapsi- and pH-independent increase of the intravesicular accumulation of the amino acid. The activation by Leu-OMe proves that amino acid absorption can be modulated all along the B. mori larval midgut and that the AM region, where the ability to transport and concentrate the substrate is very low, is more susceptible than the P region. Leucine uptake in AM-BMMV can be activated by amino acid methyl esters with definite structural requisites, with the following order of potency: L-leucine > L-phenylglycine > L-methionine> L-phenylalanine > L-norleucine>> L-isoleucine. The activation is stereospecific and occurs also with some ethyl esters (e.g. leucine and phenylalanine). No activation was observed with esters of amino acids with short hydrophobic or polar side-chains. The activation mechanism here described plays a fundamental role in larval growth since silkworms reared on artificial diets supplemented with leucine or methionine methyl esters reach maximum body weight 12-18 h before control larvae and spin cocoons with a larger shell weight. This novel regulatory mechanism of an amino acid transport protein appears to be widespread among lepidopteran larvae. (C) 2002 Elsevier Science Ltd. All rights reserved

Leucine uptake into membrane vesicles from larvae of the midge Chironomus riparius was studied. The membrane preparation was highly enriched in typical brush border membrane enzymes and depleted of other membrane contaminants. In the absence of cations, there was a stereospecific uptake of L-leucine, which exhibited saturation kinetics. Parameters were determined both at neutral (Km 33 ± 5 mM and Vmax 22.6 ± 6.8 pmol/ 7s/mg protein) and alkaline (Km 46 ± 5 mM and Vmax 15.5 ± 2.5 pmol/7s/mg protein) pH values. At alkaline pH, external sodium increased the affinity for leucine (Km 17 ± 1 mM) and the maximal uptake rate (Vmax 74.0 ± 12.5 pmol/7s/mg protein). Stimulation of leucine uptake by external alkaline pH agreed with lumen pH measurements in vivo. Competition experiments indicated that at alkaline pH, the transport system readily accepts most L-amino acids, including branched, unbranched, and a-methylated amino acids, histidine and lysine, but has a low affinity for phenylalanine, b-amino acids, and N-methylated amino acids. At neutral pH, the transport has a decreased affinity for lysine, glycine, and a-methylleucine. Taken together, these data are consistent with the presence in midges of two distinct leucine transport systems, which combine characters of the lepidopteran amino acid transport system and of the sodium-dependent system from lower neopterans.

L-leucine uptake into membrane vesicles from Bombyx mori larval midgut was tested for inhibition by 55 compounds, which included sugars, N-methylated, alpha-, beta-, gamma-, delta-, epsilon-amino acids, primary amines, cc-amino alcohols, monocarboxylic organic acids and alpha-ketoacids, Based on cis-inhibition experiments performed at the high pH (10.8) characteristic of the midgut luminal content in vivo, we find that the carrier binding site interacts with molecules which possess a well-defined set of structural features. Amino acids are preferentially accepted as anions and the ideal inhibitor must have an hydrophobic region and a polar head constituted by a chiral carbon atom bearing two hydrophilic groups, a deprotonated amino-group and a dissociated carboxylic group. Binding is reduced if one of the two hydrophilic groups is removed. Lowering the pH to less alkaline value (8.8) only affects the affinity of delta- and epsilon-amino acids, which are excluded from binding because of their positively charged side-chain. Modifications of the potassium electrochemical gradient increased the affinity constant values of the molecules, but have little effect on the rank of specificity. Physiological implications of the data reported are discussed. (C) 2000 Elsevier Science Ltd. All rights reserved.

The effect of some flavonoids on the K+-dependent and K+-independent leucine uptake into brush border membrane vesicles fromBombyx morilarval midgut was investigated. Among the compounds tested, cyclochampedol, recently purified fromArtocarpus champeden,was able to inhibit in micromolar range the leucine transport. The inhibition occurred both in the absence and in the presence of potassium and was not affected by leucine concentration. The apparentKiwas 0.25 mM. Cyclochampedol represents the first non-competitive inhibitor of an amino acid transport system in Lepidoptera. The relevance of this result is discussed.

The activities of three related Bacillus thuringiensis delta-endotoxins, designated CryIA(a), CryIA(b), and CryIA(c), as inhibitors of K(+)-dependent amino acid transport into membrane vesicles prepared from the anterior and posterior portions of Bombyx mori larval midgut were measured. Under experimental conditions similar to those occurring in vivo (membrane potential approximately -100 mV, inside negative; pH 7.2in/8.8out; an inwardly directed K+ gradient) CryIA(a) toxin produced a clear dose-dependent inhibitory effect on leucine uptake by both the anterior and the posterior gut membrane vesicles, giving half-maximal inhibition constants (IC50) of 2.6 +/- 0.3 and 2.1 +/- 0.2 microgram toxin/mg membrane protein, respectively. The other two delta-endotoxins were practically inactive. A dose-dependent inhibition of amino acid transport by CryIA(a) toxin was also observed on the carrier-mediated K(+)-independent component, i.e., the leucine-only form. This result strongly indicates that the activity of the K+/amino acid cotransporter is directly affected after binding of delta-endotoxin to the brush border membrane. When the extravesicular pH was lowered to pH 7.2, the interaction of CryIA(a) toxin with the brush border appeared more complex, as suggested by the Hill coefficient of the dose-response curves higher than 1. In conclusion, our data indicate that (i) CryIA(a) toxin specifically inhibited K+/leucine symport along the length of the midgut; (ii) the interaction between cotransporters and toxin is affected by the pH of the medium; and (iii) the K+/leucine cotransporter or a strictly associated protein may serve as membrane receptor for CryIA(a) delta-endotoxin in the B. mori larval midgut.

The effect of phenylglyoxylation on the midgut K(+)-dependent leucine transport was studied using lepidopteran brush-border membrane vesicles. The inhibition of leucine uptake by phenylglyoxal (PGO) showed a biphasic inactivation pattern. The second-order rate constant for the slow and fast phases were 0.0020 mM-1 min-1 and 0.0091 mM-1 min-1, respectively. However, substitution of borate buffer for Hepes-Tris buffer produced a mono-exponential inactivation pattern, suggesting modification of a single arginine group. The effect of PGO was dose-dependent and the concentration causing half-maximal inhibition of leucine uptake was 5.1 +/- 0.3 mM. Leucine transport was significantly inhibited also in the absence of a potassium electrochemical gradient (i.e., [K+]in = [K+]out = 100 mM), suggesting that inhibition was not related to a decrease in the driving force. Moreover, intravesicular volume remained unchanged after preincubation with PGO. Kinetic analysis of the interaction of PGO with the leucine cotransporter revealed that (i) inhibition was related to a decrease in the Vmax value and (ii) neither leucine nor K+ were able to prevent the inhibition. Our results suggest an important role for arginine residues in the molecular mechanism of K+/leucine cotransport in lepidopteran larvae midgut

A purified protein fraction of Bacillus thuringiensis subsp. aizawai (HD-133)-activated toxin was subjected to amino acid modification by phenylglyoxal as an arginine-directed reagent. The effect of phenylglyoxylation was assayed on the K (+)-dependent accumulation of leucine into brush border membrane vesicles from Bombyx mori larval midgut. Phenylglyoxal inactivates the toxin in a dose-and time-dependent manner. Loss of inhibitory effect of the toxin followed a pseudo-first-order kinetics. Observed rates of inactivation were linearly dependent upon the square of phenylglyoxal concentration and a second-order constant of 4.17 M(-2) min(-1) was calculated. These results indicate the involvement of highly reactive arginine residues in the formation of specific toxin domains implicated in the interaction with membrane receptor(s)

1. Activated delta-endotoxins from Bacillus thuringiensis strains toxic to lepidopteran larvae inhibit K(+)-dependent accumulation of amino acids into brush border membrane vesicles (BBMV) from the midgut of the susceptible species Bombyx mori. 2. The activated toxins interfere with the K(+)-dependent uptake of histidine into BBMV only if they are effectively active in vivo. 3. To calculate IC50 values (the toxin concentration which determines 50% of the effect), dose-response curves were performed for each toxin. The values obtained correlate well with the LD50 determined by bioassay. 4. This amino acid inhibition test could represent a rapid (3-6 hr, compared to 3-4 days for bioassay) and sensitive method for the screening of larvicidal activity of known or new recombinant delta-endotoxins

1. The delta-endotoxin of Bacillus thuringiensis subsp. aizawai inhibits in a dose-dependent manner the K+-driven accumulation of histidine and lysine as well as their equilibrative uptake into brush border membrane vesicles from the midgut of Bombyx mori larvae. 2. The decrease of uptake in the absence of a K+-gradient is neither due to a leakage of the labelled substrate from the vesicles nor to a reduction of the osmotically active space available, as a result of a detergent-like effect of the toxin. 3. The toxin acts as a non competitive inhibitor of the K+/amino acid cotransporters of the larval midgut of Lepidoptera, with no preference for a specific transport system

The kinetics of K(+)-leucine cotransport in the midgut of lepidopteran larvae was investigated using brush border membrane vesicles. Initial rate (3 s) of leucine uptake was determined under experimental conditions similar to those occurring in vivo, i.e. in the presence of delta psi much greater than 0 (inside negative) and a delta pH of 1.4 units (7.4in/8.8out). Leucine and K+ bind to the carrier according to a sequential mechanism, and the binding of one substrate changed the dissociation constant for the other substrate by a factor of 0.15. Both trans-K+ and trans-leucine were mixed-type inhibitors of leucine uptake. Moreover, a portion of total leucine uptake was K+ independent, and it was competitively inhibited by trans-leucine. We interpret the trans inhibitory effects to mean that the partially loaded K+ only form is virtually unable to translocate across the membrane, whereas the binary complex carrier, leucine, can isomerize from the trans to the cis side of the membrane. However, the K(+)-independent leucine uptake occurs with a Keq greater than 1, i.e. the efflux route through the partially loaded leucine only form is slower than the rate of isomerization of the unloaded carrier from trans to cis side. Taken together, these results suggest a model in which transport occurs by an iso-random Bi Bi system. Since K+ does not act as a pure competitive activator, this model is different from that proposed for most of the Na(+)-linked solutes transport agencies and may be related to the broadening of the cation specificity of the amino acid transporters in lepidopteran larvae

The effect of ethylisopropyl-amiloride (EIPA) and phenamil on sodium uptake in renal brush border membrane vesicles from prehypertensive rats of the Milan strain (MHS) and their normotensive controls (MNS) was investigated. In the presence of both a membrane potential and a pH gradient a differential effect of EIPA and phenamil was evidenced between the two rat strains. In the absence of a pH gradient, but in the presence of a membrane potential, EIPA was about two-fold more potent than phenamil in inhibiting sodium transport in both rat strains, excluding the presence of epithelial sodium channels in our BBMV preparations. Taken together these results support the hypothesis that a structurally different Na+/H+ exchanger located on the brush border membrane may be involved in the increased tubular sodium reabsorption observed in vivo in hypertensive rats

Amino acid accumulation within brush-border membrane vesicles (BBMV) from the larval midgut of Lepidoptera is driven by a K+ gradient. However, it can also be driven by a Na+ gradient, although with reduced efficiency. To examine the possibility that sodium and potassium ions are handled by the same amino acid transporter, glycine uptake into BBMV from Philosamia cynthia Drury was measured in the presence of a pH gradient and of a transmembrane electrical potential difference, i.e. in simulated 'physiological' conditions. The kinetics of glycine uptake at extravesicular saturating Na+ or K+ concentrations discloses a higher affinity of the cotransporter for the amino acid in the presence of Na+ but a maximum transport rate with K+. Glycine uptake at a fixed concentration as a function of external Na+ or K+ concentration yields curves that show saturation but do not fit a rectangular hyperbola, with Hill coefficients less than 1 with Na+ and greater than 1 with K+. These coefficients vary according to glycine concentration. Increasing the concentration of extravesicular Na+ at a saturating external K+ concentration reduced glycine uptake to 70% of the control value. This inhibition curve is compatible with competition between the two cations for the same cotransporter and with the presence of different kinetic constants with Na+ or K+. The data are consistent with a steady-state random two-substrate mechanism for glycine transport, with Na+ and K+ as alternative substrates.

1. The effect of different conditions of preservation of the larval midgut tissue of Bombyx mori and Philosamia cynthia on the efficiency of amino acid transport into brush border membrane vesicles (BBMV) has been studied, 2. Freezing and storage of the tissue in liquid nitrogen followed by rapid thawing at 37-degrees-C allowed the preparation of BBMV which retained fully for B. mori and partially for P. cynthia the ability to perform L-histidine transport and accumulation. 3. BBMV prepared from fresh midguts of B. mori, immediately frozen and preserved in liquid nitrogen for different periods, lost part of their transport efficiency at the moment of freezing, independently of the time of storage. 4. A decrease was also observed for preserved BBMV from P. cynthia midgut. However, the addition of a cryoprotectant such as glycerol to BBMV improved the performance of the reconstituted preparation. 5. The transport capacity of the stored vesicles depended also on their slow thawing on ice

The effect of ethanol on sodium and glucose transport in rabbit renal brush border membrane vesicles was examined. When membrane vesicles were preincubated in the presence of ethanol the sodium-dependent D-glucose uptake was significantly inhibited. This effect, as suggested by O'Neill et al. (1986) FEBS Lett. 194, 183-188, may be due to a faster collapse of the Na+ gradient. As a matter of fact, the amiloride-insensitive sodium pathway was increased by ethanol in our brush border membrane preparation. However, sodium/D-glucose cotransport was inhibited by ethanol, although to a lesser degree, also in the absence of a sodium gradient. In addition, ethanol inhibited glucose-dependent sodium uptake, suggesting that a direct interaction with the translocator was involved. This conclusion was also supported by kinetic measurements showing a decrease of Vmax and an increase in Km for glucose in membrane vesicles treated with ethanol. Moreover, ethanol influenced the interaction of phlorizin with the cotransporter: uptake experiments performed in the presence of the two inhibitors demonstrated that phlorizin and ethanol behave as not mutually exclusive inhibitors of D-glucose transport. These data indicate that in rabbit renal brush border membranes ethanol not only affects the 'passive pathway', i.e. the sodium permeability, but it also directly interferes with carrier functions.

The (Na+,K+)-ATPase activity from the kidney cortex of the Milan hypertensive rat strain (MHS) and the corresponding normotensive control (MNS) was measured both in active solubilized enzyme preparations and in isolated basolateral membrane vesicles. Kinetic analysis of the purified enzyme showed that the Vmax value was significantly higher in MHS rats. The difference between MHS and MNS was not linked to a different number of sodium pumps, but was related to the molecular activity of the enzyme. Using basolateral membrane vesicles, an increased ATP-dependent ouabain-sensitive sodium transport was also demonstrated in MHS rats. These results support the hypothesis that a higher tubular sodium reabsorption may be involved in the pathogenesis of hypertension in this rat strain

Experiments with intestinal brush-border membrane vesicles from lepidopteran larvae disclosed the occurrence of unique cotransporter proteins that use K+ as the driver cation for the transmembrane transfer of amino acids across the luminal border of midgut enterocytes. Six apical membrane amino acid transport systems have been identified. These systems are 1) a neutral amino acid transporter with a broad spectrum of interactions with most neutral amino acids, which is highly concentrative, strongly K+- and electrical potential-dependent, poorly stereospecific, and recognizes histidine, but not proline, glycine, or alpha-(methylamino)isobutyric acid (MeAIB); 2) a specific system for L-proline; 3) a specific system for glycine with a higher affinity for Na+ than for K+; 4) a specific system for L-lysine, which is dependent on membrane potential, is highly sensitive to external K+, and does not interact with L-arginine or neutral amino acids; 5) a specific K+-dependent process for glutamic acid, which does not recognize aspartic acid; and last, 6) an apparently unique K+- driven mechanism for D-alanine, which is potential-dependent and strongly stereospecific

In brush border membrane vesicles from the midgut of Philosamia cynthia larvae (Lepidoptera) the L- and D-alanine uptake is dependent on a potassium gradient and on transmembrane electrical potential difference. Each isomer inhibits the uptake of the other form: inhibition of L-alanine uptake by D-alanine is competitive, whereas inhibition of D-alanine uptake by L-alanine is noncompetitive. Transstimulation experiments as well as the different pattern of specificity to cations suggest the existence of two transport systems. Kinetic parameters for the two transporters have been calculated both when Kout greater than Kin and Kout = Kin. D-alanine is actively transported also by the whole midgut, but it is not metabolized by the intestinal tissue.

Sodium transport was measured in brush-border membrane vesicles prepared from kidney cortex of the Milan hypertensive strain (MHS) rats and the corresponding normotensive controls. In the presence of an outwardly directed proton gradient, 22Na was transiently accumulated in the vesicles. When a transmembrane electrical potential was imposed across membrane vesicles, both the accumulation ratio and the initial uptake were increased, indicating the presence of an electrogenic pathway for sodium in these membranes. The potential-dependent sodium uptake was significantly higher in MHS rats. Kinetic analysis give simple Michaelis Menten curves in the presence and in the absence of a membrane potential. In both conditions Jmax was significantly increased in MHS rats, whereas Km was the same for the two rat strains. Sodium uptake was inhibited by amiloride at concentrations that inhibit Na+-H+ exchange. The presence of the higher, potential-sensitive, sodium uptake in MHS is in agreement with studies on renal physiology which support the hypothesis that an increase in tubular sodium reabsorption may be the primary cause for the development of hypertension in this rat strain.

Sodium transport across luminal membranes of proximal tubules isolated from the kidney cortex of young, prehypertensive rats of the Milan hypertensive strain (MHS) and their corresponding normotensive controls, the Milan normotensive strain (MNS), was measured. A higher sodium uptake was observed in vesicles from MHS, although membrane preparations from both strains behaved similarly as far as enzyme profile and sodium-dependent glucose transport were concerned. In the presence of an inwardly directed sodium gradient, sodium uptake depended on the relative permeability of the counterion: in the presence of 100 mM NaCl, sodium transport in MHS was 26% higher than that in normotensive controls (p less than 0.05). Also, a significantly faster sodium uptake by membrane vesicles from MHS was observed when a pH gradient and an electrical potential difference (inside-negative) were imposed across the membrane. In this condition, sodium uptake by membrane vesicles from MHS was up to 39% higher than that in control MNS (p less than 0.01). Therefore, the difference in sodium transport observed between preparations of luminal membrane from the proximal tubule of MNS and MHS seems to be due to a higher rheogenic sodium pathway in the MHS. The present results are in keeping with previous data showing an increased sodium transport across renal tubules of the MHS and support the hypothesis that the abnormality in sodium and water handling by kidneys from MHS can be related to an alteration in sodium transport across the luminal membrane of the proximal tubule cells.

Modification by pyridoxal-5-phosphate of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) purified from Saccharomyces cerevisiae produces an inactivation effect, partially reversible by dilution in the presence of substrates. Spectroscopic analysis of the enzyme pyridoxal-5-phosphate complex reduced with NaBH4 provides the values expected for the binding of the aldehydic group to Lys residue. One Lys residue appears to be responsible for the observed enzyme inactivation, and the presence of the phosphate group is required for the effect. Besides the change of activity, the binding of pyridoxal-5-phosphate to the enzyme causes an increase in susceptibility to degradation by the intracellular yeast proteinase A at pH 7.6.

The luminal membrane of larval midgut cells is the site of action of insecticidal delta-endotoxin from Bacillus thuringiensis. At concentrations that correspond to normal effective doses in vivo, the toxin inhibits the uptake of amino acids by brush border membrane vesicles prepared from midguts of Pieris brassicae larvae. The toxin does not interact with the K+-amino acid symport but rather increases the K+ permeability of the membrane. The toxin does not increase the permeability of lepidopteran midgut brush border membrane to either Na+ or H+ nor does it increase the K+ permeability of brush border membrane vesicles prepared from mammalian small intestine,. © 1986.

A net absorption of sodium ions and of l-phenylalanine, in the absence of chemical gradients, occurs across the isolated midgut of the cockroach Blabera gigantea. Both sodium and amino acid net fluxes were abolished by the haemolymphatic addition of the Na-K ATPase inhibitor ouabain, at a concentration 1 mM. A purified fraction of brush border membranes, prepared from the midgut tissue by Ca-precipitation, was used to investigate the occurrence of a cotransport system in the luminal membrane of the cockroach enterocyte. An inwardly directed Na gradient (100 mM outside the vesicles, 0 mM inside) drives the uphill movement of phenylalanine into the vesicular space, whilst other monovalent cations fail to induce the concentrative uptake of the amino acid. Moreover, the amino acid uptake seems to be dependent on the transmembrane potential, since inwardly directed gradients of different sodium salts determine a decreasing rate of phenylalanine uptake in agreement with the presumptive permeabilities of Na counterions. These data suggest the presence of a Na-phenylalanine cotransport system located on the brush border membrane of B. gigantea midgut. © 1986 Springer-Verlag.