INTRODUCTION

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THE MIDGUT OF LEPIDOPTERAN LARVAE represents an extraordinary biological model, owing to the singular features of its epithelium, which combines the functional activity of a unique specialized cell, the goblet cell (11, 17, 18), with the expression in the brush-border membrane of columnar cells of an apparently exclusive class of cotransport proteins, the K⁺-dependent amino acid symporters (6-8, 10, 13, 15, 19).

It has long been known that neutral amino acids can also be translocated across the brush-border membrane vesicles (BBMV) of the midgut in the absence of K⁺ (8, 14), and Parenti et al. (13) developed a kinetic model for K⁺-dependent leucine transport in Philosamia cynthia that included the ability of the symporter to move across the membrane in its binary form with the amino acid. This model has been considered correct without further specific investigation for all larval species, including the silkworm Bombyx mori, for which intestinal amino acid absorption has been studied in detail.

In the course of a recent careful investigation of the characteristics of amino acid absorption along the silkworm midgut and the functional properties of the K⁺-leucine symporter (4), we realized, and here present the experimental evidence, that the middle region of the midgut was able to perform the uptake of a number of neutral amino acids into midgut BBMV through a low-affinity, high-capacity uniporter with broad specificity. This transporter represents an important pathway for neutral amino acid absorption in the anterior and middle regions of the midgut when amino acid concentration in the lumen exceeds 1 mM, whereas the concentrative K⁺-dependent system, which is entirely dependent on the activity of the K⁺ pump (12), i.e., of the vacuolar H⁺-ATPase and K⁺/2H⁺ antiporter (18), is active along the entire length of the midgut, but especially in the posterior region (6), and ensures amino acid absorption at luminal concentrations between 1 and 1,000 µM.

	MATERIALS AND METHODS

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Materials. L-[4,5-³H]leucine was purchased from Radiochemical Centre (Amersham International, Amersham, UK). 2-Amino-2-norbornane-carboxylic acid (BCH), 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 fourth or 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, from which the peritrophic membrane with intestinal contents and the Malpighian tubules were removed, was dissected longitudinally and rinsed thoroughly with ice-cold 210 mM sucrose, 45 mM KCl, and 10 mM Tris · HCl at pH 7. The middle and posterior regions of the midgut were severed, lightly blotted on filter paper, weighed, and placed in cryotubes. Midguts were immediately frozen in liquid nitrogen and stored 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.

Preparation of BBMV and transport experiments. BBMV were prepared from the three midgut regions by Ca²⁺ precipitation and differential centrifugation as reported by Hanozet et al. (10) and Giordana et al. (7). The final pellets were resuspended by 10 passes through a 22-gauge needle in a buffer (100 mM mannitol, 10 mM HEPES-Tris) at pH 7.2. 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. Purity and characteristics of middle and posterior BBMV (M- and P-BBMV, respectively) preparations are reported elsewhere (5). Transport experiments were performed in triplicate or quadruplicate at 23°C by rapid filtration of the vesicle suspension through a prewetted cellulose nitrate filter (10) with a pore size of 0.45 µm. For the determination of the kinetic parameters of leucine transport, incubations were performed with an automated device at 7 s. 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). BBMV were diluted 1:5 in the extravesicular solutions, the compositions of which are reported in the legends of Table 1 and Figs. 1-3. The buffer used was (final concentration in mM): 50 tetramethylammonium sulfate or K₂SO₄, 10 HEPES-Tris or 20 Tris-OH at pH 7.2 or 9, respectively, and radiolabeled leucine at the suitable concentration. To obtain a transmembrane electrical potential difference (), 0.08 mM FCCP was added to the extravesicular buffer at pH 9.0.

Calculations. Kinetic parameters of leucine transport were calculated from the Eadie-Hofstee plot using a multiparameter, iterative regression program (Sigma Plot, Jandel, CA) and on a statistical basis using MIR II computer program (1) for the Michaelis-Menten equation for two membrane carriers.

	RESULTS

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Figure 1 reports leucine uptake into BBMV from the middle region of the silkworm midgut as a function of a wide range of amino acid concentrations at pH 9 and in the presence of an inwardly directed K⁺ gradient. The curve, obtained with a large number of experimental points (each determined in quadruplicate), displays a two-component process. The Eadie-Hofstee plot of the data (Fig. 1B) is curvilinear and compatible with two carrier-mediated components, one with a high affinity and low capacity and another with a low affinity and high capacity. The kinetic parameters [Michaelis constant (K_m) expressed as mM and maximal rate of transport (V_max) expressed as nmol · 7 s¹ · mg protein¹; values ± SE] of leucine transport through the high-affinity, low-capacity and the low-affinity, high-capacity component were K_m 0.085 ± 0.007 and V_max 1.31 ± 0.05, close to the values found for the K⁺-leucine symporter (4), and K_m 4.71 ± 0.83 and V_max 4.33 ± 0.12, respectively.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Leucine initial uptake as a function of leucine concentrations ([leu]) in presence of a K+ gradient. Brush-border membrane vesicles (BBMV) from the middle region were incubated for 7 s in a buffer of the following final composition (in mM): 50 K2SO4, 20 Tris at pH 9, 0.03-16 [3H]leucine (120 µCi/ml), and 0.08 FCCP. A, uptake values; B, Eadie-Hofstee plot of data. Mean ± SE of a typical experiment carried out in quadruplicate. v0, Initial rate.

Analogously, when leucine uptake was measured over a large range of external concentrations in the absence of K⁺, the curvilinear Eadie-Hofstee plot of uptakes suggests the presence of high-affinity, low-capacity and low-affinity, high-capacity systems (Fig. 2B). The parameters calculated, fitting the data to the Michaelis-Menten equation for two carriers, were K_m 0.081 ± 0.013 mM and V_max 0.22 ± 0.02 nmol · 7 s¹ · mg protein¹ for the first component, values similar to those determined for the symporter (4). The kinetic parameters of the low-affinity, high-capacity component were K_m 10.77 ± 0.18 mM and V_max 2.29 ± 0.03 nmol · 7 s¹ · mg protein¹.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Leucine initial uptakes as a function of leucine concentrations in absence of K+. BBMV from middle region were incubated for 7 s in a buffer of the following final composition (in mM): 50 tetramethylammonium sulfate [(TMA)2SO4], 20 Tris-OH at pH 9, and 0.03-9 [3H]leucine (160 µCi/ml). A, uptake values; B, Eadie-Hofstee plot of data. Mean ± SE of a typical experiment carried out in triplicate.

A statistical analysis of leucine kinetics was also performed using the MIR II computer program (1). Three different mathematical models were tested: the Michaelis-Menten equation, the Michaelis-Menten equation with a linear component, and the Michaelis-Menten equation for two carriers; for both the data obtained in the presence and absence of K⁺, the best fit was the Michaelis-Menten equation for two carriers. The mean value of the kinetic parameters of leucine uptake through the uniport as determined in five different preparations was K_m 7.12 ± 1.04 mM and V_max 4.48 ± 0.68 nmol · 7 s¹ · mg protein¹.

The low-affinity, high-capacity K⁺-independent transport protein does not need an extravesicular alkaline pH for its optimum activity, and it is insensitive to the (Table 1).

View this table: [in this window] [in a new window]   Table 1.   Effect of pHout and on kinetic parameters of low-affinity, high-capacity transport system

Moreover, this transporter appears to be able to bind, with equal efficiency, a wide range of neutral amino acids, such as leucine, histidine, valine, serine and phenylalanine, at the amino acid binding site; it does recognize lysine and arginine, amino acids with a positive charge, but accepts to a lower extent alanine and BCH, as can be inferred by the extent of inhibition of 5 mM labeled leucine uptake induced by a 10-fold excess (50 mM) of the chosen amino acids in the extravesicular buffer (Fig. 3).

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Inhibition of 5 mM leucine uptake by a 10-fold excess of indicated amino acids. BBMV from middle region were incubated for 7 s in a buffer of the following final composition (in mM): 50 (TMA)2SO4, 20 Tris-OH at pH 9, 5 [3H]leucine (160 µCi/ml), and 50 inhibitor or mannitol. Each bar represents %inhibition ± SE calculated with respect to control condition (50 mM mannitol) after subtraction of residual uptake with 50 mM leucine. BCH, 2-amino-2-norbornane-carboxylic acid.

To evaluate if the low-affinity, high-capacity K⁺-independent transport protein is also expressed in the posterior region of the midgut, we measured the kinetics of leucine uptake as a function of leucine concentration up to 16 mM into P-BBMV in the absence of K⁺. In that condition, two carrier-mediated components were identified. The low-affinity and high-capacity one, which is pH and independent (Table 1), presents kinetic constants similar to those identified in the midgut middle region (K_m 9.16 ± 1.38 mM and V_max 5.08 ± 0.49 nmol · 7 s¹ · mg protein¹, mean ± SE of 3 different determinations).

	DISCUSSION

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On the basis of in vivo and in vitro studies, amino acid absorption seems to be an important function of all the three midgut regions: anterior, middle, and posterior (5, 16). Neutral amino acids are translocated along the entire length of B. mori larval midgut with a well-characterized K⁺-dependent system highly expressed in the posterior tract (4-6).

This K⁺-dependent symporter is saturated at a substrate concentration of 1.5 mM (4). However, leucine kinetics measured into BBMV isolated from the middle region of the larval midgut display, over 1-2 mM, a quasi-linear increase of the uptake. A detailed analysis of the kinetics of leucine influx as a function of external substrate concentration up to 16 mM led to the identification of a low-affinity, high-capacity carrier acting in parallel with the K⁺-dependent, high-affinity, low-capacity system (Fig. 1).

The presence of the two carrier-mediated components (Fig. 2) is evident also when the initial rate of leucine uptake is measured in the absence of the driver cation. Therefore, as previously observed (13), the K⁺-dependent symporter is able to translocate the amino acid both in the ternary (carrier-amino acid-K⁺) and the binary (carrier-amino acid) form. Moreover, the presence of K⁺ does not affect the translocation or the affinity of this low-affinity, high-capacity K⁺-independent uniport.

An extremely high alkalinity of the midgut lumen contents as well as a high across the apical membrane of midgut columnar cells is a characteristic feature of lepidopteran larvae (3). The K⁺-dependent symport activity is maximal when the experimental conditions resemble the physiological ones, i.e., when an external alkaline pH and a are present (4). Conversely, the uniport is insensitive to both these factors, as no difference was observed between the kinetic parameters determined in the presence of a pH or of a pH plus a (Table 1).

Finally, the low-affinity, high-capacity component is far less selective than the K⁺-dependent component, because the amino acid binding site seems to be able to accept cationic amino acids as well as a large number of neutral ones. Conversely, BCH, a model substrate for the cation-independent system L in mammals (2), is poorly recognized by the uniport.

Leucine kinetics in the absence of K⁺ over a wide range of external amino acid concentrations disclosed the presence of a low-affinity, high-capacity K⁺-independent component, otherwise barely perceptible, also in the posterior midgut region. This component shares the same kinetic characteristics with the transport expressed in the middle region (Table 1). Nevertheless, in the physiological conditions of feeding larvae (9), when a high luminal K⁺ concentration, an alkaline pH, and a are present, the contribution of the uniport to the total amount of leucine uptake is remarkably different in the two midgut regions considered (Fig. 4).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Relative contribution of symporter and uniporter to total leucine uptake. Kinetic parameters used for uniport were mean values reported in text, and values for symport were those reported in Table 1 of Ref. 4. A, middle region; B, posterior region.

The results presented here indicate the presence along B. mori larval midgut of two different amino acid transport systems acting in parallel: the high-affinity, low-capacity K⁺-dependent symporter and the low-affinity, high-capacity K⁺-independent uniporter. The uniporter is present along the entire length of the midgut with similar expression and functional properties but different physiological significance. In particular, it represents the primary pathway for amino acid absorption in the middle region of the midgut (see Fig. 11 in Ref. 4) when the activity of the vacuolar H⁺-ATPase is suppressed, e.g., in starved larvae (9). Therefore, the physiological role of this transport agency is under further investigation.