INTRODUCTION

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THE PRESENCE OF AN ALKALINE pH in the lumen content of insect midgut is not a rare event. Scarab beetles, higher termites, and nematoceran Diptera share with lepidopteran larvae midgut fluids with pH values between 9 and 12 (54, 55). But lepidopteran larvae are a singular case, because the morphology and the functional activity of their midgut epithelium are dominated by a peculiar specialized cell, the goblet cell, which belongs exclusively to the larval stage of this order (53, 55).

The lepidopteran larval midgut can be divided into three regions according to the folding of the epithelium (12) and distribution of digestive enzymes (48, 53, 55). The morphology of the goblet cells changes markedly in the posterior region with respect to the anterior and middle regions (11, 47). Although the activity of these cells bestows unique functional properties on the midgut epithelium, the prevalent cell type is columnar cells, which surround each goblet cell in a ratio of of 1:5 (6). Differences in the morphology of these cells in the three midgut regions, although less apparent, can also be observed (47).

The ionic and chemical homeostasis in which the whole midgut is involved results from the cooperative activity of the goblet and columnar cells. A vacuolar-type proton ATPase (57) and a K⁺/2H⁺ antiporter (5, 37, 56) in the membrane lining the goblet cavity (29) generate both the transmembrane electrical potential difference () and the K⁺ luminal concentration that provide the electrochemical gradient for K⁺ uptake into the adjacent columnar cells. The driving force is almost entirely due to the extremely high electrical component, because a voltage of 140 mV or more can be measured at the apical side of columnar cells (40). K⁺ influx across the brush border drives amino acid accumulation (24, 27), because specific K⁺-amino acid symporters (22, 25, 31, 43, 44) are located in the luminal membrane.

The proton pump and the K⁺/2H⁺ antiporter appear to be involved also in the drastic alkalinization of the midgut lumen (5), which requires the ability of the epithelium to secrete an anion, possibly HCO₃ (10, 13, 15). In Lepidoptera, the pH of the lumen contents reaches high values along the entire length of the larval midgut (9, 13, 15, 16, 26, 39), reaching extreme values up to 12 in the middle region (13, 41). The physiological significance of such a drastic alkalinization of the luminal fluids, which imposes a heavy energetic load on the larva (14), has received much attention (7, 38, 51) and is actually under active study (2, 4, 19, 32, 33). The gut represents the boundary between the insect and its environment, so that, as pointed out by Appel (2), it is involved in a complex strategy that aims to reduce toxicity from leaf allelochemicals, limit infections from bacteria, and optimize extraction of nutrients from the ingested food. A high alkaline pH in the lumen appears to be one of the physicochemical characteristics that, together with gut redox conditions (3), contributes to this complex goal. In particular, it increases the solubility of foliar proteins (4, 19), enhancing the efficiency of nutrient extraction from leaves and utilization of dietary amino acids (18).

In the silkworm Bombyx mori, the larval midgut performs a remarkable net absorption of amino acids (24, 45, 46), necessary for the impressive and rapid increase in body weight that occurs during the entire larval age and, in the last instar, for the production of the silk proteins used to spin the cocoon. Amino acid translocation is performed by different K⁺-dependent transport systems (21), and all three midgut regions appear to be involved in the absorption of amino acids in vivo (49). At least for the broad-specificity symporter responsible for the K⁺-dependent transport of leucine and other zwitterionic amino acids, a number of results (21, 22) indicated some dissimilarities between the functional activity of the transport proteins in the different midgut regions.

As in the other species, in silkworms, the alkaline luminal pH increases in the anterior and middle regions of the midgut to reach a value of 11, which drops down to 9 in the posterior region (41). The intracellular pH is presumably near 7.2, as measured in Manduca sexta (10, 16), so that across the brush-border membrane of columnar cells there is a very steep proton gradient. The symporters for neutral amino acids therefore operate in extreme conditions, because they face a severe pH, with one side exposed to an environment of extremely high alkalinity, and they are oriented within a very strong electrical field.

To extend our study of the functional behavior of the K⁺-neutral amino acid symporter expressed along the larval midgut of the silkworm B. mori, we performed experiments with brush-border membrane vesicles (BBMV) prepared from the three midgut regions to answer the following questions. Is the symporter apt to operate in an external high alkalinity, i.e., does it operate at best in such conditions? Is it affected by an alkaline pH at the cytoplasmic side of the membrane? Does the symporter operate in a different manner in the three midgut regions? Does the presence of a pH and of affect to the same extent the magnitude of intravesicular accumulation into BBMV from the three regions? Can the pH alone drive the accumulation of the amino acid?

	MATERIALS AND METHODS

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Materials. L-[4,5-³H]leucine and [¹⁴C]sucrose were purchased from Radiochemical Centre (Amersham International, Amersham, UK). FCCP and all other analytic grade reagents were obtained from Sigma (St. Louis, MO).

Experimental animals. Fifth-instar larvae of B. mori were fed on mulberry leaves or artificial diets (Yakult). The larvae used for the experiments were in the fifth day of the last instar, when their average weight had reached 3.6 ± 0.1 g.

Isolation of midgut, separation of midgut regions, and tissue preservation. The silkworms were kept in crushed ice for 15-20 min and then cut immediately before the first pair of thoracic legs and behind the third pair of abdominal appendages, to exclude the foregut and the hindgut. The integument was cut away, and the exposed midgut, deprived of the peritrophic membrane with intestinal contents and of the Malpighian tubules, was dissected longitudinally and rinsed thoroughly with ice-cold 210 mM sucrose, 45 mM KCl, and 10 mM Tris · HCl at pH 7. The anterior, middle, and posterior regions of the midgut (Fig. 1), easily discernible by eye from the thickness and the color of the tissue, were severed, lightly blotted on filter paper, weighed, and placed in cryotubes. Midguts were immediately frozen in liquid nitrogen and stored (20) for no more than 6 mo. At the moment of use, the cryotubes were kept in a 37°C water bath until the tissue began to melt.

View larger version (115K): [in this window] [in a new window]   Fig. 1.   Bombyx mori larval midgut can be easily divided into 3 different regions from folding and color of tissue. A, anterior; M, middle; P, posterior regions. At the microscopic level, an immediate morphological difference between A-M and P regions is provided by shape of goblet cell (12). Careful examination (47) disclosed several differences in columnar cell morphology in the 3 regions.

Preparation of BBMV. BBMV were prepared from the three midgut regions by Ca²⁺ precipitation and differential centrifugation as indicated elsewhere (24, 27). The final pellets were resuspended by 10 passes through a 22-gauge needle in 100 mM mannitol and 10 mM HEPES-Tris at pH 7.2. In a few experiments, the vesicles were resuspended in alkaline (Tris) or acid (MES) buffers as specified in the legends of Figs. 3, 4 and 10. The membrane protein concentration was assessed with the Coomassie brilliant blue G-250 (Pierce, Rockford, IL) protein assay, using bovine serum albumin as standard, and adjusted to a final concentration of 5 mg/ml.

1

Transport experiments. Transport experiments were performed in triplicate at 23°C by rapid filtration of the vesicle suspension through a prewetted cellulose nitrate filter (27) with a pore size of 0.45 µm. For the determination of the kinetic parameters of leucine transport, incubations were performed in quadruplicate with an automated device at 7 s, because the uptake of leucine (0.1 and 4 mM) in A-, M-, and P-BBMV was linear up to 8 s (data not shown). The uptakes were initiated by mixing 10 µl of the vesicle suspension to 40 µl of the radiolabeled incubation medium. Samples were counted for radioactivity in a scintillation spectrometer (model 300 C, Tri-Carb, Packard). Different buffers were tested to obtain extreme alkaline pH values. The effects of various extravesicular concentrations of 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) and Tris on leucine uptake (0.1 mM) into A-, M-, and P-BBMV were tested in the presence of an inwardly directed K⁺ (100 mM) gradient and (90 mV). Maximum uptakes at the desired pH values were obtained with a concentration of Tris or CAPS of 20 mM. CAPS concentrations exceeding 20 mM caused a consistent reduction of leucine uptake (data not shown). The compositions of the extravesicular solutions are indicated in the legends to Figs. 2, 3, 5, 7, 9, and 10. In the experiments with a near 0 mV and variable extravesicular pH (pH_out), the protonophore FCCP was omitted from the radiolabeled solution. In the experiments with variable , FCCP was added to the extravesicular solution to increase the proton permeability of the vesicle membrane, so that inside negative of different values, according to the transmembrane pH gradient, were obtained by proton diffusion. The impermeant anion SO²₄ was used as a K⁺ counterion to avoid interference in the generation of the membrane potential.

Calculations. For the identification of the kinetic model of leucine transport into A-, M-, and P-BBMV, the best fit of the experimental data to different mathematical models was determined on a statistical basis using an MIR II computer program (8). Kinetic parameters of leucine transport were then routinely calculated using a multiparameter, iterative, nonlinear regression program (Sigma Plot, Jandel, CA).

Uptake measurements in isolated midgut. The midgut, separated from the chilled larvae and deprived of the peritrophic membrane, was mounted as a tube on a suitable apparatus (45). In the experiments reported here, the region of the midgut exposed to the bathing solutions was the middle one, because the anterior region was too short and the posterior one too fragile to be used in the experimental apparatus available. The buffer used had the following composition (in mM): 20 K₂SO₄, 5 KHCO₃, 44 MgSO₄, 9 CaCl₂, 0.1 L-leucine, 124 sucrose, and 5 Tris adjusted to pH 7 or 9. In the experiments without K⁺, K₂SO₄ and KHCO₃ were omitted and substituted with 70 mM sucrose. Salines of luminal and hemolymph compartments were aerated and stirred by bubbling 100% O₂. Leucine uptake was measured by adding 4 µCi/ml [³H]leucine in the luminal compartment. After an incubation of 20 min, the exposed tissue was severed, put into an Eppendorf vial after removal of excess fluid, and weighed to obtain fresh weight. Distilled water in a ratio of 1:10 was added, and then the midgut was frozen and thawed twice. After centrifugation at 10,000 g for 10 min, the supernatant was counted for radioactivity in the scintillation counter. Intracellular uptake values and intracellular pools were calculated after subtraction of the radioactivity in the luminal extracellular space [equal to 15% tissue water, as determined according to Giordana and Sacchi (23) by incubating the midgut for 40 min with [¹⁴C]sucrose in the luminal compartment] by correcting for total extracellular space volume (55% tissue water, as determined with [¹⁴C]sucrose in the lumen and hemolymph compartments).

	RESULTS

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Leucine is an amino acid readily transported by the K⁺-dependent symporter for neutral amino acids. At a concentration of 0.15 mM, near the K_m value of the symporter (Ref. 21 and Table 1), it is transiently accumulated within the vesicles of the three midgut regions under simulated physiological conditions, i.e., in the presence of an intravesicular pH (pH_in) of 7.2, an inwardly directed K⁺ gradient, and an interior negative of ~90 mV (Fig. 2). With an alkaline pH_out of 9, the same K⁺ gradient provides different intravesicular accumulation values (calculated as the ratio of maximum to equilibrium uptakes; values are 5, 9, and 33, respectively, in A-, M-, and P-BBMV in the typical experiment reported in Fig. 2) and uptake curves ("overshoots") of different shape.

View this table: [in this window] [in a new window]   Table 1.   Effect of pHout and on kinetic parameters of leucine transport into BBMV from M and P regions of midgut after subtraction of carrier-mediated K+-independent component

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Time course of leucine uptake into brush-border membrane vesicles (BBMV) prepared from A (), M (), and P () midgut regions. BBMV, resuspended in (in mM) 100 mannitol and 10 HEPES-Tris at pH 7.2, were diluted 1:5 to obtain the following final composition (in mM): 2 HEPES, 20 Tris, 0.15 L-leucine, 40 (A and M) or 15 (P) µCi/ml L-[3H]leucine, 50 K2SO4, and 0.08 FCCP at pH 9. Experiment was performed in triplicate (means ± SE). These comparisons were obtained on vesicles from a single preparation. Similar results were noted in 4 separate membrane preparations.

The value of the pH_in can affect the overshoot curve and the magnitude of the intravesicular accumulation; after 30 min (see Fig. 3 legend) of contact with a pH_in of 8 or 9, the overshoot curves in M- and P-BBMV changed slightly compared with those recorded in vesicles with a pH_in of 7.2 (Fig. 3). Longer incubations with a pH_in of 9 caused a substantial reduction of leucine initial uptake rates (not shown) and maximum uptake values (Fig. 4), more extended in M-BBMV. In these experiments, pH_out was 10.7 and a constant was obtained with 150 mM KSCN.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Time course of leucine uptake into M- (A) and P-BBMV (B) with an intravesicular pH (pHin) of 7.2 (), 8.0 (), or 9.0 (). BBMV were resuspended in (in mM) 100 mannitol and 30 HEPES-Tris at pH 7.2 (), 25 Tris at pH 8 (), or 50 Tris at pH 9 () and then diluted 1:5 to obtain the following final composition (in mM): 6 HEPES (), 5 Tris (), or 10 Tris (), respectively, 0.11 L-leucine, 40 (A) or 15 (B) µCi/ml L-[3H]leucine, 150 KSCN, and 20 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) at pH 10.6. Time 0 indicates start of incubation with radiolabeled buffer. Vesicles had already been in contact with different pH for 30 min, because vesicles were resuspended in suitable buffers before last centrifugation step. Experiment was performed in triplicate (means ± SE) from a single membrane preparation. Similar results were noted in 3 separate preparations.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Leucine maximum uptake values into M- (A) and P-BBMV (B) as a function of duration of preincubation with a pHin of 9. Time 0 refers to uptake measured at end of last centrifugation step. Dashed line represents corresponding uptake value when pHin was 7.2. For experimental conditions, see Fig. 3. Time of incubation of M- and P-BBMV in radiolabeled buffer corresponded to attainment of maximum uptake. Experiment was performed in triplicate (means ± SE) from a single membrane preparation. Similar results were noted in 3 separate preparations.

The dependence on pH_out of leucine initial uptake rates and of the concentrative ability (roughly indicated by maximum uptake values) of the three BBMV preparations was determined in the presence of a K⁺ gradient but in the absence of a (Fig. 5). In the three preparations, leucine uptakes increased with increasing alkalinity of the external medium, with highest values at a pH_out of nearly 11. The effect of the alkaline pH_out on leucine initial uptake rates is less marked in A- and M-BBMV, due to the presence of a remarkable K⁺-independent component in this region (21, 36).

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Effect of extravesicular pH (pHout) value on leucine initial rates (v0) and maximum uptake values into A- (A and B), M- (C and D), and P-BBMV (E and F). BBMV, resuspended in 100 mM mannitol and 10 mM HEPES-Tris at pH 7.2, were diluted 1:5 to obtain the following final composition (in mM): 2 HEPES, 0.20 L-leucine, 40 (A and M) or 15 (P) µCi/ml L-[3H]leucine, 50 K2SO4, and 10 HEPES-Tris at pH 7.2, 20 Tris at pH 8 and 9, or 20 CAPS at pH 10.6. Maximum uptake was measured after an incubation of 2 min for A- (B) and M-BBMV (D) and of 45 s for P-BBMV (F). Experiment was performed in triplicate (means ± SE) from a single membrane preparation. Similar results were noted in 3 separate preparations.

When an aliquot of the BBMV preparations used for the previous experiment was incubated in the presence of the ionophore FCCP, inside negative electrical potentials of different magnitudes, according to the transmembrane pH, were obtained by proton diffusion, due to the increased proton permeability induced by the protonophore. Figure 6 reports total leucine uptake values (initial and maximum uptakes) as a function of . The open part of each bar represents the increase of leucine uptake induced by . Membrane potentials were estimated from the Nernst equation, assuming that protons were the only ions moving freely across the membrane in the experimental condition tested. An increase of leucine initial uptake rates in all three preparations, more remarkable in P-BBMV, was caused by , whereas the concentrative ability of the preparation was markedly affected only in P-BBMV.

View larger version (47K): [in this window] [in a new window]   Fig. 6.   Effect of transmembrane electrical potential difference () on leucine initial uptake rate and maximum uptake values into A- (A and B), M- (C and D), and P-BBMV (E and F ). Experimental conditions were as indicated in Fig. 5, with addition of 80 µM FCCP to radiolabeled incubation buffer. Open part of bar represents increase of uptake due to . Experiment was performed in triplicate (means ± SE) from a single membrane preparation. Similar results were noted in 3 separate preparations.

Leucine kinetics as a function of external leucine concentration was measured in M- and P-BBMV (Fig. 7, A and C, respectively) at a pH_out of 7.2 or 10.6 with a near 0 mV, and at pH_out 10.6 with a of ~200 mV. The Eadie-Hofstee plots of the data, reported in Fig. 7B for M-BBMV and Fig. 7D for P-BBMV, are curvilinear in both preparations in the three experimental conditions considered.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Leucine kinetics in M- (A) and P-BBMV (C) at pHout 7.2 (), 10.6 without (), and 10.6 with a of ~200 mV (). B and D show Eadie-Hofstee plots of data. BBMV, resuspended in 100 mM mannitol and 10 mM HEPES-Tris at pH 7.2, were diluted 1:5 to obtain the following final composition (in mM): 2 HEPES, 0.05-1 L-leucine, 60 µCi/ml L-[3H]leucine for M-BBMV and 40 µCi/ml for P-BBMV, 50 K2SO4, and 10 HEPES-Tris at pH 7.2 or 20 CAPS at pH 10.6.  was obtained by addition of 0.08 FCCP. Incubations lasted 7 s. Experiment was performed in quadruplicate (means ± SE) from a single membrane preparation. Similar results were noted in 3 separate preparations. [Leu], leucine concentration.

Recently, the activity of a low-affinity, high-capacity uniporter responsible for a large K⁺-independent absorption of leucine along B. mori midgut has been identified (36). Leucine uptake mediated by this transport agency appears to be insensitive to pH_out and (36). Therefore, leucine kinetics reported in Fig. 7, A and C, are in all instances the result of the sum of two carrier-mediated components, as confirmed by the curvilinear relationships in the Eadie-Hofstee plot. Table 1 reports the kinetic parameters of K⁺-dependent leucine uptakes into BBMV after subtraction of the component of leucine transport due to the activity of the uniporter (see Table 1 legend). The presence of an extreme alkaline pH_out caused a twofold increase of V_max in both BBMV preparations that was further enhanced by the presence of a membrane potential. Conversely, the affinity of the symporter for the amino acid does not seem to be markedly affected.

Next we examined how the modification of the kinetic parameters induced by an alkaline pH_out and by affected the concentrative ability of the three BBMV preparations in the presence of an inwardly directed K⁺ gradient. Uptakes of 0.2 mM leucine were measured in the three BBMV preparations at a pH_out of 7.2 or 9, with or without the addition of FCCP, i.e., with or without a (Fig. 8). Leucine was transiently accumulated within the vesicles from anterior, middle, and posterior midgut regions in all three experimental conditions, as expected, because K⁺, the driver cation, was present. However, leucine accumulation in the vesicles (Fig. 8, insets) was markedly increased with an alkaline pH_out, especially in P-BBMV, and the overshoot phenomenon was clearly anticipated. Maximum uptake values were reached even earlier in the presence of , which also caused a striking increase of leucine accumulation in P-BBMV (Fig. 8C, inset).

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Time course of K+-driven 0.2 mM leucine uptake in absence () or presence of a pH () or both pH and () in A- (A), M- (B), and P-BBMV (C). Experimental conditions as in Fig. 5 for pHout 7.2 or pHout 9 with or without FCCP. Experiment was performed in triplicate (means ± SE) from a single membrane preparation. Similar results were noted in 3 separate preparations. Insets report accumulation values at a pHout of 7.2 (a), 9 (b), and 9 with (c).

Then we investigated the uptake with time of 0.2 mM leucine in the absence of K⁺ (Fig. 9). pH_out was 9, the maximal buffer alkalinity that can be reached in our experimental conditions in the absence of alkali metal cations. We expected no accumulation of leucine within the vesicles, as was indeed the case in all three BBMV preparations in the absence of a pH across the vesicle membrane (i.e., when pH_out = pH_in = 7.2). Instead, a consistent intravesicular accumulation of leucine occurred with a pH (Fig. 9, insets). The accumulation values did not decrease in the presence of the protonophore FCCP (Fig. 9, B and C), which should induce a more rapid dissipation of the outwardly directed proton gradient.

View larger version (25K): [in this window] [in a new window]   Fig. 9.   Time course of K+-independent 0.2 mM leucine uptake in absence () or presence of pH () or of pH and FCCP (). Experimental conditions as in Fig. 5 for pHout 7.2 and 9. Fifty millimolar tetramethylammonium sulfate [(TMA)2SO4] was substituted for fifty millimolar K2SO4. Experiment was performed in triplicate (means ± SE) from a single membrane preparation. Similar results were noted in 4 separate preparations. Insets report accumulation values at pHout 7.2 (a), 9 (b), and 9 with FCCP (c). A, A-BBMV; B, M-BBMV; C, P-BBMV.

Figure 10 reports the time course of leucine uptake measured again at pH_out 7.2 but in the presence of a pH obtained by resuspending the vesicles in 20 mM MES at pH 5.4. This experiment differs from that reported in the previous figure in that a pH gradient is still present when the pH_out is 7.2. Despite the pH gradient, the accumulation values were drastically reduced both in M- and P-BBMV (Fig. 10, insets). The addition of the protonophore FCCP slighlty affected the overshoot curves in both preparations.

View larger version (26K): [in this window] [in a new window]   Fig. 10.   Time course of K+-independent 0.2 mM leucine uptake at pHout 7.2 in presence of pH () or of pH and FCCP (). BBMV were resuspended in 100 mM mannitol and 20 mM MES-Tris at pH 5.4 and diluted 1:5 to obtain the following final composition (in mM): 5 MES, 0.20 L-leucine, 40 (M, A) or 15 (P, B) µCi/ml L[3H]leucine, 50 (TMA)2SO4, and 20 Tris at pH 7.2 with or without 0.08 FCCP. Experiment was performed in triplicate (means ± SE) from a single membrane preparation. Similar results were noted in 2 separate preparations. Insets report accumulation values in absence (a) and presence (b) of FCCP.

Leucine kinetics in the absence of external K⁺, at a pH_in of 7 and a pH_out of 7 or 9, was then measured in M- and P-BBMV, and the kinetic parameters for the symporter were calculated (Table 2). Determination of reliable kinetic parameters in M-BBMV was difficult, as might have been expected, because the uptake of leucine through the symporter in its binary form in this midgut region is a minor part of total leucine uptake (Fig. 11). Conversely, in P-BBMV, where leucine is predominantly taken up by the symporter (Refs. 21 and 36 and Fig. 11), a consistent increase of V_max was observed with an alkaline pH_out. The kinetic parameters were not altered by the addition of the protonophore FCCP.

View larger version (23K): [in this window] [in a new window]   Fig. 11.   Relative contribution of symporter and uniporter to total leucine uptake in M- (A and B) and P-BBMV (C and D) within range of symporter's activity in absence (A and C) or presence (B and D) of K+. Kinetic parameters used are those reported in Tables 1 and 2. Note different scales for y-axes.

To verify if the data obtained with the vesicle preparations comply with events occurring in vivo, we performed experiments with intact B. mori midgut in vitro. The midgut part used was the middle region, isolated as a tube so that the epithelium separated a luminal from a hemolymph compartment. TEP was monitored throughout the time lapse of 20 min, during which the uptake of 0.1 mM leucine from the lumen into midgut cells was measured. Uptake values are reported in Table 3, as well as radiolabeled leucine intracellular pools, calculated as indicated in MATERIALS AND METHODS.

When the luminal pH was 7.2, K⁺ driving force was able to induce a twofold intracellular accumulation of leucine; removal of K⁺ from the perfusion fluids caused a reduction of the uptake, and leucine accumulation did not occur. With an alkaline luminal pH and in the presence of K⁺, an increase of the uptake and a threefold accumulation of the amino acid was observed. More interesting is the effect in the absence of luminal potassium; with a pH across the luminal membrane, the uptake of the amino acid was significantly enhanced and leucine was accumulated within midgut cells. It should be taken into account that TEP approaches 0 mV in the absence of K⁺.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The K⁺-dependent system for neutral amino acids has been well characterized in the different regions of B. mori larval midgut (21). It is a high-affinity system that is saturated at 1-2 mM leucine (Ref. 21 and Table 1). It is similar to the broad specificity B⁰-type system identified in the brush border of mammalian absorbing epithelia (17, 50). Its specificity for the different amino acids follows this rank of preference: Leu > Val >> Ala > His > Ser (P. Parenti, M. Casartelli, M.G. Leonardi, and B. Giordana, unpublished observations). Leucine cotransport with K⁺ seems to be characterized by the highest affinity and translocation rates (M. G. Leonardi and B. Giordana, unpublished observations), so leucine is a good test amino acid to study the symporter's properties along the midgut. Leucine accumulation is driven by the K⁺ electrochemical gradient in BBMV of all three midgut regions (Figs. 2 and 8). The ability of the BBMV from the anterior region to accumulate the amino acid proves that this midgut region is involved not only in the enzymatic digestion of ingested proteins but also in the absorption of their ultimate products, as found in vivo by Shinbo et al. (49). Besides, the three overshoot curves show different shapes, maximal uptake is reached at different times, and the ability to concentrate the substrate is different in the three preparations. These features provide evidence for remarkable kinetic differences between the transport proteins and/or the characteristics of the luminal membrane (1, 30, 35).

An alkaline pH_in affects the efficiency of leucine transport into the vesicles (Fig. 3). Intravesicular accumulations (Fig. 4) and initial rates decrease with increasing time of contact with a pH_in of 9. A similar result was observed for glycine uptake into midgut BBMV from Philosamia cynthia (28). We cannot say if this is due to an alteration of the cytoskeletal architecture associated with the brush-border membrane, which would in turn affect the concentrative ability of the system, or to a modification of the transport protein itself. However, SDS-PAGE of the vesicles incubated for 40 min with a pH_in of 9 shows a band pattern markedly different from that of vesicles incubated for the same time lapse with a pH_in of 7.2 (not shown). Some proteins appear to be released or partly degraded. Vesicles might open in these conditions, as observed for rabbit small intestinal BBMV (34). We performed all the subsequent experiments with a pH_in of 7.2, which is the value of the intracellular milieu.

We tested the dependence of leucine initial rate and maximum uptake (an approximate but reliable indication of the accumulation ability) on the pH_out (Fig. 5). To avoid the interference of , which affects to a different extent leucine uptakes into A-, M-, and P-BBMV (Figs. 6 and 8), its value was kept close to 0 mV. Leucine uptake and its accumulation increased steadily with the alkalinity of the external solution in all three BBMV preparations, with the highest values at a pH_out of nearly 11. As expected (21), leucine initial rate and accumulation were much higher in P-BBMV compared with those recorded in A- and M-BBMV (Fig. 2). The effect of the alkaline pH_out on leucine initial uptake rates is less marked in A- and M-BBMV; similarly, appears to affect to a lesser extent leucine accumulation in these midgut regions (Fig. 6). This can be ascribed to the presence of a remarkable K⁺-independent component of leucine transport (21), mediated by a low-affinity, high-capacity uniporter (36), that can be responsible for 30% or more of total leucine uptake when the external leucine concentration is saturating for the symporter (Figs. 7 and 11). When leucine kinetics are determined in M-BBMV, this cation-independent, carrier-mediated component can be clearly detected as a linear component at concentrations >1 mM (21). The uniporter is insensitive to both pH_out and (36), and its activity masks that of the K⁺-amino acid symporter, which is poorly expressed in B. mori anterior and middle regions, especially when pH_out is nearly neutral (Fig. 11).

So, the K⁺ symporter for leucine expressed along the midgut can operate efficiently in the extreme alkaline conditions of the lumen, optimum activity being reached at a pH_out of 11. Moreover, the extremely high transapical voltage created across the brush-border membrane of columnar cells by the activity of the "K⁺ pump" provides the best conditions for the functional activity of the transport protein (Figs. 6-8 and 11).

The kinetic parameter of leucine transport that changes when the external pH is turned from neutral to alkaline was the V_max. The presence of a >150 mV, negative inside the vesicle, further increased the turnover number of the symporter (Table 1), with minor effects, if any, on its affinity for the amino acid.

Both pH and contribute to the ability of K⁺-leucine symporter to perform the concentrative uptake of leucine (Fig. 8). In A- and M-BBMV, the K⁺ gradient alone (curves with a pH_in = pH_out = 7.2) can drive a threefold accumulation of leucine (Fig. 8, insets). A pH increases amino acid accumulation even in the absence of a voltage across the membrane, and with the addition of the accumulation value is only slightly increased, but the overshoot is reached in a shorter time (within 2 instead of several minutes). In P-BBMV, the accumulation values are much higher in all conditions, and not only causes the maximum uptake to be attained more rapidly (from 90 to 45 s) but also causes a large increase of the intravesicular accumulation.

The differences in leucine uptake in A- and M-BBMV compared with P-BBMV (Figs. 8 and 9) could be due to the expression of isoforms of the K⁺-amino acid symporter with different functional properties, in particular, the rate of cycling of the transport protein across the membrane. Alternatively, the same protein could be unevenly distributed along the midgut, with the largest number of proteins per unit membrane expressed in the posterior region. This alternative cannot be resolved definitely by the study of the functional properties in the native membrane but requires the isolation and structural determination of the symporter(s).

With BBMV from P. cynthia midgut, Parenti et al. (43) have shown, with a kinetic approach, that the symporter can translocate the amino acid across the membrane in its binary form. We have examined the symporter's behavior in B. mori in the absence of the driver cation (Fig. 9) by measuring the time course of leucine uptake at a concentration (0.2 mM) in which the contribution of the activity of the uniporter to total leucine transport is minimized (Fig. 11). With a pH across the membrane similar to that present in vivo, leucine is accumulated within the vesicles, and the addition of FCCP, which should induce a more rapid dissipation of the proton gradient, did not reduce the overshoot curves (Fig. 9).

Two possible explanations for leucine intravesicular accumulation with pH in the absence of K⁺ can be advanced. First, the accumulation is achieved through a countertransport of H⁺ for leucine, but FCCP fails to collapse the proton gradient either because of the low anion conductance or the high buffering capacity in the experimental conditions tested. Alternatively, the accumulation is a form of ion trapping, i.e., the zwitterionic form of leucine is trapped within the vesicle. In fact, leucine uptakes into BBMV increase with the alkalinity of the external buffer (Fig. 5), closely following the deprotonation with pH of the -amino group (pK₂ 9.74) and thus the percent increase of the anionic over the zwitterionic form of the amino acid. The alkaline pH_out increased the turnover number of the symporter but did not modify the affinity of the transporter for its substrate (Tables 1 and 2). Recent studies on the characteristics of the amino acid structure relevant for recognition at the binding site (Ref. 22 and P. Parenti, M. Casartelli, M. G. Leonardi, and B. Giordana, unpublished observations) have indeed shown that the integrity of the carboxylic group (i.e., of the negative charge) is determinant, whereas a number of leucine analogs modified in the -amino group are relatively tolerated. If the presence of a positive charge at the -amino group affected the translocation step negatively, this would produce the accumulation of leucine in the lack of a driver but in the presence of a pH gradient. If this hypothesis is correct, the intravesicular accumulation of leucine should be drastically reduced if the uptake is measured in the range of pH values in which leucine is exclusively in the zwitterionic form, i.e., with a pH_in of 5.4 and a pH_out of 7.2. Figure 10 shows that this is indeed the case; despite the large pH gradient, leucine accumulation almost disappeared in M-BBMV and was significantly reduced in P-BBMV. Moreover, leucine uptakes in the presence of FCCP were slightly reduced, suggesting a possible role of the proton gradient as driving force. This role will be confirmed by an extended and more careful experimental design specifically devised to clarify how fast and to what extent the proton gradient is effectively influenced by the addition of the ionophore.

The net absorption of amino acids performed in vivo by B. mori midgut is a selective and energy-dependent extraction of molecules from the environment that requires an "active" step responsible for the intracellular accumulation of the amino acid. Figure 8 shows that three elements combine to this goal. Two of these are well known (22, 24): the high concentration of K⁺ in the midgut lumen and the voltage across the luminal membrane of columnar cells generated by the activity of the electrogenic proton pump provide the driving force for the K⁺-dependent amino acid secondary active absorption. In this paper, we present evidence that the third element is represented by the extremely high proton gradient across the luminal membrane. This gradient favors leucine uptake and accumulation into BBMV (Figs. 8 and 9) as well as into midgut cells (Table 3) not only in the presence of K⁺ and but also in their absence.

In conclusion, in anterior and middle midgut regions, where the luminal pH reaches values of 11 or 12, pH, in addition to K⁺ electrochemical gradient, may be important in driving the accumulation and thus the absorption of amino acids. From the data determined in vivo by Shinbo et al. (49), it appears that almost 70% of the absorption of dietary amino acids occurs in the anterior and middle regions. At least for leucine, this absorption takes place mainly via the uniport insofar as luminal leucine concentration exceeds 1-2 mM, but the activity of the K⁺-dependent symporter could be of importance during the intervals between the feeding periods, when the larva does not ingest food. Besides, the final removal of amino acids from the lumen contents occurs in the posterior region, where the luminal amino acid concentration is extremely low (49), so that the presence of a strong concentrative mechanism is imperative. This is the midgut region where the K⁺-driving force is fully exploited (Fig. 11). Therefore, the ultimate absorption of amino acids is indeed strongly dependent on the activity of the proton pump and remains highly effective although the pH of the luminal content decreases to a value of 8-9 (41).