Paolo Parenti

Preparation and partial characterization of amino-acid transporting brush-border membrane-vesicles from the larval midgut of the cabbage butterfly (pieris-brassicae)

Abstract 1. 1. Brush border membrane vesicles (BBMV) were prepared from midguts of Pieris brassicae larvae by Mg/EGTA precipitation and differential centrifugation. 2. 2. Their morphology, polypeptide composition, and marker enzyme enrichment was similar to BBMV from other larval lepidopteran midguts. 3. 3. In the presence of an inwardly directed KSCN gradient, these BBMV transiently accumulated alanine, phenylalanine, histidine, lysine, or gultamic acid. 4. 4. Initial uptake rates for neutral and basic amino acids were similar in the presence of either a potassium or a sodium gradient. 5. 5. Initial uptake of glutamic acid was much more efficient in the presence of a sodium gradient.

Bacillus thuringiensis toxin inhibits K+‐gradient‐dependent amino acid transport across the brush border membrane of Pieris brassicae midgut cells

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,

Na+/K+/Cl--cotransporter mediated Rb+ fluxes in membrane vesicles from kidneys of normotensive and hypertensive rats

This paper describes experiments to examine Rb+ fluxes via the Na+/K+/Cl- cotransporter in membrane vesicles from renal outer medulla of three strains of rat: (A) Wistar (B) Milan hypertensive (MHS) and normotensive (MNS), and (C) Sabra salt-sensitive hypertensive (SBH) and salt-resistant (SBN). Initially, Na(+)-dependent furosemide- or bumetanide-inhibited 86Rb+ fluxes were characterised using Wistar rat microsomes. The latter were partially purified on a metrizamide cushion, and assay conditions were optimized for use with microsomes from the other rats. The major result is that in microsomes from adult Milan hypertensive (MHS) rats the rate of the Na+/K+/Cl(-)-cotransporter mediated 86Rb flux at sub-saturating concentrations of Rb, appears to be significantly greater than in the normotensive (MNS) controls. The effect reflects an increased apparent Rb affinity of the cotransporter in MHS microsomes. There is no difference in maximal rate or in the apparent Na+ activation affinity of the 86Rb+ flux. In addition bumetanide appears to be a somewhat more effective inhibitor in MHS compared to MNS microsomes. The 86Rb+ flux result is compatible with a previous finding that in red cells, Na+/K+ -cotransporter mediated fluxes are increased in MHS compared to MNS. It supports the notion that the Na+/K+/Cl(-)-cotransporter in in both red cells and kidney is a genetic marker for hypertension. It is of interest that apparently more than one Na+ transport system is affected in MHS hypertensive kidneys (a) the Na+/K+/Cl- cotransporter in the thick ascending limb of Henle and (b) the Na+/H+ exchanger and/o a conductive Na(+)-pathway in brush-border membranes from proximal tubule. It is conceivable that in the hypertensive animals a common regulatory pathway (e.g., phosphorylation) or protein (e.g., cytoskeleton) is affected along the length of the nephron. In Sabra SBH and SBN rat microsomes, no difference was found for the 86Rb+ flux via the Na+/K+/Cl- cotransporter (or via a K+ channel).

Presence of a potential-sensitive Na+ transport across renal brush-border membrane vesicles from rats of the Milan hypertensive strain

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.

Effects of the topical application of an insect growth regulator (fenoxycarb) on some physiological parameters in the fifth instar larvae of the silkworm Bombyx mori

Abstract The effects of topical applications of the commercial insecticide Insegar ( 25% fenoxycarb) on the fifth instar larvae of Bombyx mori fed on an artificial diet were investigated. Clear dose-response relationships were established with respect to frass production as well as to the percentage of dauer larvae, whereas less evident was the correlation to the growth rate and the percentage of pupation. The Insegar concentration, giving 50% of dauer larvae, was 60 pg per larva, but effects on the parameters tested were evident also with concentrations as low as 100 fg per larva. Apparently, this insect growth regulator (IGR) causes a reduced intake of food as well as a reduction in growth rate. The absorption of amino acids carried out by the K + -dependent symporter in the anterior-middle and the posterior regions of the midgut was evaluated in vitro from leucine uptake into brush border membrane vesicles obtained from control and treated larvae. IGR in a dose of 10 fg per larva causes a significant, albeit small, increase of amino acid intake in both midgut regions. At higher doses, no effect of Insegar on leucine uptake is evident in the posterior midgut, whereas a significant decrease of the uptake in the anterior-middle region is apparent at the dose of 10 μg per larva.

L- and D-alanine transport in brush border membrane vesicles from lepidopteran midgut: evidence for two transport systems.

SummaryIn brush border membrane vesicles from the midgut ofPhilosamia cynthia larvae (Lepidoptera) thel- andd-alanine uptake is dependent on a potassium gradient and on transmembrane electrical potential difference. Each isomer inhibits the uptake of the other form: inhibition ofl-alanine uptake byd-alanine is competitive, whereas inhibition ofd-alanine uptake byl-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>Kin and Kout=Kin.d-alanine is actively transported also by the whole midgut, but it is not metabolized by the intestinal tissue.

Electrogenic K+-Basic Amino-Acid Cotransport in the Midgut of Lepidopteran Larvae

SummaryExperiments performed on isolated midgut demonstrate that the model proposed for the absorption of neutral amino acids in the K+-transporting intestinal epithelium of lepidopteran larvae applies also to the transport of the basic amino acids histidine and lysine. The characteristics of these K+-basic amino-acid cotransports have been studied in brush-border membrane vesicles. Histidine and lysine are transported by different transport agencies, which share, to a different degree, a high sensitivity to transmembrane electrical potential difference. Kinetic analysis showed thatKm for histidine and lysine increased 10-fold and three-fold, respectively, whereasVmax was only slightly modified when the electrical potential difference was abolished. The relationship between potassium concentration and histidine uptake indicates a cooperative binding of more than one potassium to the transporter. Countertransport experiments with glutamine as elicitor show that histidine and glutamine are transported through the same system.

The amino acid/K+ symporters for neutral amino acids along the midgut of lepidopteran larvae: Functional differentiations

Abstract The functional properties of the K + -dependent symporter for neutral amino acids have been investigated in brush border membrane vesicles prepared from the anterior, middle and posterior portions corresponding to the three morphologically distinguishable regions of the midgut of Bombyx mori larvae. An intravesicular accumulation of leucine was driven by a K + -gradient in the three preparations, but vesicles from the posterior tract displayed much higher uptake and accumulation values. Kinetic analysis of leucine uptake, performed in experimental conditions which mimic as closely as feasible experimentally those occurring in vivo (Δ ψ = −90 mV, pH in 7.2 pH out 8.7 , [K + ] out 100 mM), evidenced that the affinity for the amino acid was similar along the midgut (150 μM), but V max in the posterior region was more than 11-fold higher than that of the anterior-middle tract (11.3 ± 0.7 and 0.98 ± 0.07 nmol/7s/mg protein, respectively). Leucine uptake was remarkably influenced by extravesicular pH and by Δ ψ only in vesicles from the posterior midgut: a lowering of pH to 7.2 caused a sevenfold increase of K m , whereas in the absence of Δ ψ , V max decreased threefold. The selectivity sequence for the alkali cations was somewhat different in the two midgut regions, but K + remained the most effective. In the posterior midgut, the selectivity for K + was greatly enhanced when a transmembrane electrical potential was present. Leucine kinetics as a function of external potassium concentration was hyperbolic in the posterior and sigmoidal in the anterior-middle part. Inhibition of leucine uptake induced by a 20-fold excess of different amino acids suggested the presence in both midgut tracts of a broad specificity system for neutral amino acids, with many-but not all-features in common with the B o system of mammal intestinal and renal epithelial brush borders. However, there are differences between the two midgut regions as regard to the ability of the symporters to recognize the different amino acids, which concern the side chain and the presence of the aromatic ring. Altogether these data suggest that two kinds of symporters for neutral amino acids, with different functional properties, are expressed in the anterior-middle and posterior regions of the lepidopteran midgut.

Renal Function of Isolated Perfused Kidneys from Hypertensive (mhs) and Normotensive (mns) Rats of the Milan Strain: Role of Calcium

Abnormal sodium (Na+) and water handling by the kidney may be the cause of hypertension in rats of the Milan hypertensive strain (MHS). In addition, the plasma cell membrane Ca-ATPase at Vmax is lower in MHS than in normotensive controls (MNS). The isolated kidney preparation was used to explore the role of extracellular free calcium (Ca2+) concentrations (1.25, 1.00 and 0.75 mmol/l) in tubular Na+ transport of pre-hypertensive MHS rats. At the Ca2+ concentration of 1.0 mmol/l, the following parameters were significantly higher (P less than 0.01) in MHS: glomerular filtration rate (GFR) and urinary volume (UV), +104.8 and +147.2%, respectively; urinary Na+ excretion (UNa+), +318.0%; tubular Na+ reabsorption (TNa+), +91.0% and oxygen consumption Q O2, +42.9%. Raising the Ca2+ concentration to 1.25 mmol/l increased UV (+167.0%) and UNa+ (+231.0%) in MHS without substantially affecting MNS. The difference between the two strains was therefore considerably larger for these parameters, but disappeared for TNa+ and Q O2. The overall kidney function of MHS was poor as compared wit renal function at Ca2+ 1.0 mmol/l. The differences between the two strains virtually disappeared at the lowest Ca2+ concentration (0.75 mmol/l); only GFR and TNa+ remained slightly greater in MHS mainly because of the poor viability of MNS kidneys in this experimental condition. It thus appears that the lower activity of Ca2+-ATPase of MHS compared with MNS plasma cell membranes influences the differences in Na+ and water handling in the two strains.

Na-dependent uptake of phenylalanine in the midgut of a cockroach (Blabera gigantea)

SummaryA net absorption of sodium ions and ofl-phenylalanine, in the absence of chemical gradients, occurs across the isolated midgut of the cockroachBlabera 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 ofB. gigantea midgut.