Synthetic deoxynojirimycin derivatives bearing a thiolated, fluorinated or unsaturated N-alkyl chain: identification of potent α-glucosidase and trehalase inhibitors as well as F508del-CFTR correctors.

The synthesis of eleven 1-deoxynojirimycin (DNJ) derivatives presenting either a monofluoro, difluoro, thiolated or unsaturated N-alkyl chain of various length is described. Exploiting the unsaturated moiety on the nitrogen, fluorine has been introduced through a HF/SbF5 superacid catalysed hydrofluorination and thiol-ene click chemistry allowed introduction of sulfur. The synthetic derivatives have been tested for their ability to inhibit glycosidases and correct F508del-CFTR. Two of the unsaturated iminosugars exhibited potency similar to Miglustat as F508del-CFTR correctors. The thioalkyl iminosugars as well as the corresponding alkyl iminosugars demonstrated low micromolar α-glucosidases and trehalases inhibition. Introduction of fluorine abolished F508del-CFTR correction and trehalase inhibition.

Leucine methyl ester is a powerful allosteric activator of the neutral amino acid cotransport system in Bombyx mori larval midgut.

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 K(a) values of 27+/-2 and 47+/-8 microM in the presence and in the absence of K(+), respectively. The activation increased K(m) twofold, and V(max) up to 18-fold depending upon the experimental conditions. Leucine uptake mediated by the amino acid uniport appears to be unaffected by the activator.

Role of specific activators of intestinal amino acid transport in Bombyx mori larval growth and nutrition.

Nutrient absorption and its modulation are critical for animal growth. In this paper, we demonstrate that leucine methyl ester (Leu-OMe) can greatly increase the activity of the transport system responsible for the absorption of most essential amino acids in the larval midgut of the silkworm Bombyx mori. We investigated leucine uptake activation by Leu-OMe in brush border membrane vesicles and in the apical membrane of epithelial cells in the midgut incubated in vitro. Moreover, the addition of this strong activator of amino acid absorption to diet significantly affected larval growth. Silkworms fed on artificial diet supplemented with Leu-OMe reached maximum body weight 12-18 h before control larvae, and produced cocoon shells up to 20% heavier than those of controls. The activation of amino acid absorption plays an essential role in larval development so that larval growth and cocoon production similar to controls reared on an artificial diet with 25% of dry mulberry leaf powder were observed in silkworms fed on an artificial diet with only 5% of mulberry powder. Arch.

Leucine transport in membrane vesicles from Chironomus riparius larvae displays a mélange of crown-group features.

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 microM and Vmax 22.6 +/- 6.8 pmol/7s/mg protein) and alkaline (Km 46 +/- 5 microM 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 microM) 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 alpha-methylated amino acids, histidine and lysine, but has a low affinity for phenylalanine, beta-amino acids, and N-methylated amino acids. At neutral pH, the transport has a decreased affinity for lysine, glycine, and alpha-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.

Multiple transport pathways for dibasic amino acids in the larval midgut of the silkworm Bombyx mori.

The transport pathways for dibasic amino acids were investigated in brush border membrane vesicles (BBMV) from the anterior-middle (AM) and posterior (P) regions of Bombyx mori midgut. In the absence of K(+), a low-affinity saturable transport of arginine in both AM- and P-BBMV (K(m) 1.01 mM, V(max) 4.07 nmol/7s/mg protein and K(m) 1.38 mM, V(max) 2.26 nmol/7s/mg protein, respectively) was detected. Arginine influx was dependent on the membrane electrical potential (Deltapsi) and increased raising the alkalinity of the external medium from pH 7.2 to 10.6. Competition experiments indicated the following order of substrate affinity: arginine, homoarginine, N(G)-monomethylarginine, N(G)-nitroarginine>lysine>>ornithine>cysteine>methionine. Leucine, valine and BCH (2-amino-2-norbornanecarboxylic acid) did not inhibit arginine influx. In the presence of external K(+), the influx of arginine as a function of arginine concentration fitted to a complex saturation kinetics compatible with both a low-affinity and a high-affinity component. The latter (K(m) 0.035 mM, V(max) 2.54 nmol/7s/mg protein) was fully characterized. The influx rate had an optimum at pH 8.8, was strongly affected by Deltapsi and was homogeneous along the midgut. The substrate affinity rank was: homoarginine>arginine, N(G)-monomethylarginine>>cysteine, lysine>>N(G)-nitroarginine>ornithine>methionine. Leucine and amino acids with a hydrophobic side chain were not accepted. This system is also operative in the absence of potassium, with the same order of specificity but a very low activity. Lysine influx is mediated by two more transport systems, the leucine uniport and the K(+)/leucine symport specific for amino acids with a hydrophobic side chain that recognizes lysine at extravesicular pH values (pH(out)) exceeding 9. Both the uniport and the symport differ from the cationic transport systems so far identified in mammals because they are unaffected by N-ethylmaleimide, have no significant affinity for neutral amino acids in the presence of the cation and show a striking difference in their optimum pH.

Substrate specificity of the brush border K+-leucine symport of Bombyx mori larval midgut.

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, alpha-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.

K(+)-neutral amino acid symport of Bombyx mori larval midgut: a system operative in extreme conditions.

The K(+)-dependent symporter for leucine and other neutral amino acids expressed along the midgut of the silkworm Bombyx mori operates with best efficiency in the presence of a steep pH gradient across the brush-border membrane, with external alkaline pH values up to 11, and an electrical potential difference (delta psi) of approximately 200 mV. Careful determinations of leucine kinetics as a function of external amino acid concentrations between 50 and 1,000 microM, performed with brush-border membrane vesicles (BBMV) obtained from the middle and posterior midgut regions, revealed that the kinetic parameter affected by the presence of a delta pH was the maximal rate of transport. The addition of delta psi caused a further marked increase of the translocation rate. At nonsaturating leucine concentrations in the solution bathing the external side of the brush-border membrane, leucine accumulation within BBMV and midgut cells was not only driven by the gradient of the driver cation K+ and delta psi but occurred also in the absence of K+. The ability of the symporter to translocate the substrate in its binary form allows the intracellular accumulation of leucine in the absence of K+, provided that a pH gradient, with alkaline outside, is present. The mechanisms involved in this accumulation are discussed.

Evidence for a low-affinity, high-capacity uniport for amino acids in Bombyx mori larval midgut.

We investigated the kinetics of leucine influx as a function of external substrate concentration between 0.03 and 16 mM in brush-border membrane vesicles (BBMV) prepared from the middle region of Bombyx mori larval midgut. A detailed kinetic analysis of leucine uptake led to the identification, in parallel with the K(+)-dependent symporter for neutral amino acids, of a K(+)-independent, low-affinity, high-capacity system. The parameter values of the Michaelis constant (7.12 mM) and maximal rate of transport (4.48 nmol.7 s-1.mg protein-1) were not influenced by an external alkaline pH nor by a transmembrane electrical potential difference. The uniporter is poorly specific, as it displayed the following rank of preference: Leu, His, Val, Ile, Phe, Ser > Lys, Arg, Gln > Pro, 2-amino-2-norbornane-carboxylic acid, Ala, Gly. The kinetic analysis performed in BBMV prepared from the posterior midgut portion indicates that the low-affinity, high-capacity uniporter is present along the entire length of the silkworm larval midgut with similar expression and functional properties.

A new prenylated flavone from Artocarpus champeden inhibits the K(+)-dependent amino acid transport in Bombyx mori midgut.

The effect of some flavonoids on the K(+)-dependent and K(+)-independent leucine uptake into brush border membrane vesicles from Bombyx mori larval midgut was investigated. Among the compounds tested, cyclochampedol, recently purified from Artocarpus 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 apparent Ki was 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.

Bacillus thuringiensis CrylAa delta-endotoxin affects the K+/amino acid symport in Bombyx mori larval midgut.

We have examined the type of inhibition exerted by an activated preparation of the Bacillus thuringiensis delta-endotoxin CrylAa on K+-dependent leucine transport into midgut brush border membrane vesicles or epithelial cells of the isolated midgut from Bombyx mori to study its possible interaction with the amino acid symporter. K+ permeability and the cation-dependent amino acid translocation into brush border membrane vesicles were evaluated by monitoring the fluorescence of the voltage-sensitive cyanine dye 3, 3'-dipropylthiadicarbocyanine iodide. The symporter ability to accept Na+ instead of K+ was exploited and the dissipation of an imposed inside-negative potential (K+ gradient in>out and valinomycin) was registered in the presence of a Na+ gradient (out>in) and of the amino acid. The fluorescence quenching dissipated more rapidly when the amino acid was present. Preincubation of brush border membrane vesicles with CrylAa caused a significant decrease of the amino acid-dependent recovery of fluorescence, whereas K+ permeability was sparely affected. In the isolated midgut, CrylAa inhibits leucine uptake as well as the transepithelial electrical potential difference. The strong inhibition exerted by the delta-endotoxin was observed also in the absence of potassium and the transepithelial electrical potential difference. The results obtained strongly suggest a direct interaction of CrylAa delta-endotoxin with the K+/amino acid symporter.

Renal Na,K-ATPase in genetic hypertension.

Milan hypertensive rats (MHS) develop hypertension because of a primary renal alteration. Both apical and basolateral sodium transport are faster in membrane vesicles derived from renal tubules of MHS than in those of Milan normotensive control rats (MNS). These findings suggest that the increased renal sodium retention and concomitant development of hypertension in MHS may be linked to an altered transepithelial sodium transport. Since this transport is mainly under the control of the Na-K pump, we investigated whether an alteration of the enzymatic activity and/or protein expression of the renal Na,K-ATPase is detectable in prehypertensive MHS. We measured the Na,K-ATPase activity, Rb+ occlusion, turnover number, alpha 1- and beta 1-subunit protein abundance, and alpha 1 and beta 1 mRNA levels in microsomes from renal outer medulla of young (prehypertensive) and adult (hypertensive) MHS and in age-matched MNS. In both young and adult MHS, the Na,K-ATPase activity was significantly higher because of an enhanced number of active pump sites, as determined by Rb+ occlusion maximal binding. The higher number of pump sites was associated with a significant pretranslational increase of alpha 1 and beta 1 mRNA levels that preceded the development of hypertension in MHS. Since a molecular alteration of the cytoskeletal protein adducin is genetically associated with hypertension in MHS and is able to affect the actin-cytoskeleton and Na-K pump activity in transfected renal cells, we propose that the in vivo upregulation of Na-K pump in MHS is primary and linked to a genetic alteration of adducin.

Interaction of the insecticidal crystal protein CryIA from Bacillus thuringiensis with amino acid transport into brush border membranes from Bombyx mori larval midgut.

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.

Effect of arginine modification on K(+)-dependent leucine uptake in brush-border membrane vesicles from the midgut of Philosamia cynthia larvae.

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.

DETERMINANTS FOR THE ACTIVITY OF THE NEUTRAL AMINO ACID/K+ SYMPORT IN LEPIDOPTERAN LARVAL MIDGUT

The columnar cells of lepidopteran larvae express, in their apical brush-border membrane, a class of symporters which in vivo couple the intracellularly directed amino acid and K+ fluxes. An analysis of the functional properties of the symporter for neutral amino acids along the anterior, middle and posterior regions of the larval midgut of Bombyx mori demonstrated the ability of a K+ gradient to drive leucine accumulation into brush-border membrane vesicles (BBMV) in all three preparations. However, marked differences are evident between the posterior (P) and the anterior­middle (AM) regions. In P-BBMV, much higher intravesicular accumulations were observed, Vmax was six- to eightfold higher than in AM-BBMV, a lowering of external pH (pHe) from 8.7 to 7.2 caused a tenfold increase of Km, and the absence of a potential difference (delta psi) caused a threefold decrease of Vmax. In contrast, leucine uptake in AM-BBMV was poorly sensitive to both pH and delta psi. The kinetics of leucine uptake as a function of cis K+ concentration were hyperbolic in P-BBMV and sigmoidal in AM-BBMV. More than 50 amino acids and analogues were used in inhibition experiments to characterize the amino acid binding site. Branched-chain amino acids modified on the carboxyl moiety were recognized only by the P-BBMV symporter. In AM-BBMV, substrate affinity was increased by the presence of a heterocyclic sidechain, even in the presence of a modified carboxyl- or alpha-amino group. Together, these results suggest that isoforms of the neutral amino acid/K+ symporter are present. A natural inhibitor of amino acid symport has not yet been identified. However, several lines of evidence suggest that strong interactions exist between the amino acid/K+ symporter and the receptor for the lepidopteran-specific Bacillus thuringiensis delta-endotoxins. CryIA(a) toxin, highly toxic for B. mori larvae, produced a dose-dependent inhibition of leucine uptake into both BBMV populations. The toxin was able to block the symporter in its ternary and leucine-only forms.

Relationships among alterations in renal membrane sodium transport, renin and aminopeptidase M activities in genetic hypertension.

Rats of the Milan Hypertensive Strain (MHS) may be considered a useful model for understanding the genetic molecular mechanism underlying a primary form of hypertension in at least a subgroup of patients. Many differences between MHS and its normotensive control strain (MNS) were found at the organ, cellular and biochemical level. In the present investigation renal cell membrane proteins (BBMV) were analysed by two-dimensional electrophoresis and a difference between MHS and MNS was shown in a polypeptide of 32 kDa, subsequently identified as the C-terminal fragment of aminopeptidase M (APM). The activity of the enzyme was higher in MHS. Genetic relationships between this enzyme and the other biochemical cellular abnormalities of MHS, namely sodium transport in BBMV and renin activity in kidney cortex were investigated in MHS, MNS and in two inbred recombinant strains. This analysis showed that faster sodium transport, low kidney levels of renin and hypertension, but not differences in two-dimensional electrophoretic pattern and in aminopeptidase M activity, cosegregated in recombinant strains. These results are consistent with the hypothesis that the faster sodium transport can be considered a primary cellular abnormality responsible for hypertension in MHS and that the aminopeptidase difference is not involved in the cellular abnormalities.

A rapid and sensitive in vitro assay for the activity of Bacillus thuringiensis delta-endotoxins.

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.

Uptake of broxaterol by cultured human cells.

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.

Na+,2Cl-,K+ cotransport system as a marker of antihypertensive activity of new torasemide derivatives.

A series of compounds related to torasemide, a loop diuretic, were synthesized and examined for their diuretic potency and inhibitory activity on the erythrocyte and renal medullary thick ascending limb vesicle Na+,2Cl-,K+ cotransport in Milan hypertensive (MHS) and normotensive (MNS) rat strains, where previous studies had demonstrated an alteration of the cotransport system genetically related to hypertension. From the results of the screening, structure-activity relationships were drawn and two compounds, JDL 961 and C 2921 were selected. Their IC50 on renal vesicle cotransport were similar in the two strains (JDL 961: MHS = 1.8 microM; MNS = 1.2 microM; C 2921: MHS = 4 microM; MNS = 3.8 microM), and were 4-8 times lower than those of torasemide (MHS = 13 microM; MNS = 31 microM, P less than 0.01) and 50-60 times lower than those of bumetanide (MHS = 145 microM; MNS = 206 microM, P less than 0.05) taken as reference compounds. Their ability to reduce the development rate of hypertension was tested both in MHS and in Okamoto spontaneously hypertensive rats (SHR) strain, in which cotransport alterations are opposite to those of MHS. Both torasemide derivatives (7.5 mg.kg-1 os per day) prevented development of hypertension in the two strains. The time course of this hypotensive activity was faster and the percentage of blood pressure fall greater in MHS (20-25%) than in SHR rats (12-15%), even though the absolute value of blood pressure fall was similar in MHS (JDL 961 = -17 mm Hg; C 2921 = -30 mm Hg) and SHR (JDL 961 = -25 mm Hg; C 2921 = -20 mm Hg). A superimposable effect of bumetanide was observed in the two strains, but at 8 times higher daily dose (60 mg.kg-1). These results suggest that new loop diuretics can be selected for their antihypertensive activity on the basis of their in vitro potency in inhibiting the Na+,2Cl-,K+.