Efficiency of amino acid transport into preserved brush border membrane vesicles from lepidopteran larval midgut

Camp. Biochem. Physiol. Vol. I03A. No. Printedin Great Britain

I, pp.65-71,

1992 0

0300-9629/92 $5.00 + 0.00 1992 PergamonPressLtd

EFFICIENCY OF AMINO ACID TRANSPORT INTO PRESERVED BRUSH BORDER MEMBRANE VESICLES FROM LEPIDOPTERAN LARVAL MIDGUT 3. GIORDANA,*

P. BELGIOJOSO,* G. M. HANOZET,~ M. TASCA*

and P.

PARENTI?

+Dipartimento

di Biologia and tDipartimento di Fisiologia e Biochimica Generali, Universita’ di Milano, via Celoria 26, 20133 Milano, Italy. Tel.: (39) 02-266-04440, Fax: (39) 02-236-1070 (Received 22 January 1992)

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

Abstract-l.

Midgut isolation and preservation

INTRODUCTION

Larvae were chilled on ice for 15-20min and then cut immediately behind the last pair of thoracic legs and behind the second pair of abdominal appendages. The integument was cut away and the exposed midgut, deprived of the ~ritrophic membrane and of the Malpi~ian tubules, was dissected lon~tudinally and thoroughly rinsed with ice-cold 300 mM sucrose, 17 mM Tris-HCl, pH 7.4. The isolated midguts were then gently blotted, weighed and either used immediately for the preparation of brush border membrane vesicles or placed in cryotubes and frozen by immersing the vial in liquid nitrogen. The cryotubes were then stored in liquid nitrogen or at -80°C. In some instances glycerol at a final concentration of 5% was added to the buffer used for isolation; the tissue was then frozen in liquid nitrogen and kept at -80°C.

The possibility of obtaining brush border membrane vesicles (BBMV) with a well preserved ability to perform solute transport from stored tissue is extremely important when the model tissue is provided by animals, like some larvae of lepidoptera, which are only available during a limited period of the year. Moreover, several advantages come from the storage of BBMV from a unique preparation, which upon reconstitution may exhibit the same qualitative and possibly quantitative transport properties shown by the original fresh preparation. These advantages (use of a same vesicle population for different kinds of experiments and reduction of the time necessary to perform the experiment) have been well stressed by those authors who have devised specific methods of preservation for vesicles obtained from vertebrate epithelia (Hittelman et uf., 1978; Stevens et al., 1982; Reshkin ef al., 1988; Karl et al., 1991). In this paper we report the effect of freezing and storage of either the entire midgut or directly of the BBMV preparation, on a well characterized amino acid carrier system of the lepidopteran larval enterocyte, i.e. the brush border neutral amino acid transporter (Hanozet et al., 1989; Giordana et al., 1989), using r.,-histidine as model amino acid.

Preparation of brush border membrane uesicles (BBMV)

BBMV were prepared from fresh or frozen tnidguts, rapidly thawed by immersing the cryotube in a 37°C water bath, by the differential calcium pr~ipi~tion method (Schmitz et al., 1973; Kessler ef al., 1978) as described by Giordana et al. (1982). The final pellet was resuspended in the buffer suitable for the transport experiments (100mM mannitol, 1OmM Hepes-Tris at pH 7.4) to provide a final vesicle protein concentration of 2-1 mg/ml. The protein concentration in BBMV and, when necessary, in the homogenate, was assessed using the BioRad (Richmond, CA) protein assay, with bovine serum albumin as standard. Conditions of preservation, storage and reclaiming of BBMV

Aliquots of a vesicle suspension prepared from fresh midguts were transferred to cryotubes and rapidly frozen in liquid nitrogen. Alternatively, trehalose was added to the buffer used to resuspend the vesicles at a final concentration of 1 mg trehalose per mg of membrane protein (Reshkin et al., 1989). Alternatively, glycerol to a final con~ntration of 15% was added to the resuspended vesicles (at a protein

MATERIALS AND METHODS

Fifth instar larvae of Bombyx mori or Philosamta Cynthia, fed respectively on mulberry and ailanthus leaves, were used. The larvae were in their fourth day of the last instar, when their average weight had reached 3.6f 0.1 and 5.81 Ifc0.32 g, respectively. 65

66

B.

G~~RDANA

et

al.

concentration of 9 mg/ml), which were then frozen in liquid nitrogen 10 min after the addition, as described by Hittelman Edal. (1978). All the samples were then stored in liquid nitrogen. For the transport experiments, BBMV were slowly thawed on ice, resuspended by seven passes through a 22 gauge needle and immediately used. The samples stored with glycerol were diluted immediately after thawing with 500

volumes of ice-cold transport buffer, centrifuged for 30 min at 27,OOOg,resuspended by passing through the needle as described above and used. Alternatively, some samples were rapidly thawed by immersing the cryotube in a 37°C water bath. Enzyme assays

--_--,~/,&I

The relative purity of the membrane preparation was tested by determining the specific activities in the homogenate and in the final preparation of leucine amino-peptidase (EC 3.4. 11.2)and of NADPH-cytochrome c reductase (EC 1.6.99.3). The activity of the first enzyme was measured with the Boehringer kit no. 124869 (Mannheim, D) and of the second one according to Masters et al. (1967). Transport experiments

Transport experiments were performed in triplicate at 22°C by the rapid ~ltration technique, as described by Hanozet et al. (1980). For the determination of the kinetic parameters of L-histidine transport, short time incubations (7 set) were performed with the automated device previously described (Giordana et af., 1985) which allowed the automatic control of the incubation time up to 1 sec. Transport was initiated by mixing 10 ~1 of vesicle suspension to 10 ~1 of the radiolabelled incubation medium containing (mM): 100 mannitol, 10 Hepes-Tris at pH 7.4, 100 KSCN or NaSCN and L-[2,5-jH]histidine according to the legends to figures and tables. Radioactive amino acids were purchased from Radiochemical Centre (Amersham International, Amersham, U.K.). RESULTS

Figures 1 and 2 report typical experiments of histidine uptake versus time into BBMV prepared

0

1

2

3

4

5

6

60

minutes

Fig. 1. Time course of histidine uptake into BBMV from B. mori midguts preserved in different conditions.

BBMV were resuspended in (mM): 100 mannitol, 10 Hepes-Tris at pH 7.4 then incubated in a medium of the following final composition (mM): 100 mannitol, 10 Hepes-Tris at pH 7.4, 100 KSCN, 0.S L-[2,5-3H]histidine (10 @/ml). Each curve represents a typical experiment performed in triplicate. Symbols represent the mean + SE of uptake values at the different times. Standard error bars, when not given, were smailer than the symbols.

0

1

2

3

4

5

6

60

minutes Fig. 2. Time course of histidine uptake into BBMV from P. Cynthia midguts preserved in different conditions, Experimental conditions as reported in Fig. 1. Each curve represents a typical experiment performed in triplicate. Symbols represent the mean + SE of uptake values at the different times. Standard error bars, when not given, were smaller than the symbols.

from B. mori (Fig. I) or P. cy~f~~u (Fig. 2) midguts immediately after isolation or after freezing in liquid nitrogen and storage in either liquid nitrogen or at - 80°C. The time course of the amino acid uptake in the presence of an inwardly directed K+-gradient was chosen as a test experiment to verify the preservation of the transport activity. This is because the uptake values and the shape of the expected curve, i.e. the overshoot phenomenon, depend both on the kinetic parameters and the energy conversion efficiency of the cotransport system, which means its ability to concentrate the amino acid in the intravesicular space. Therefore, this experimental design allows an immediate evaluation of the possible alterations induced by cryopreservation. While freezing in liquid nitrogen and storage of the tissue at -80°C drastically affected, although to a different extent, amino acid uptake and accumulation into BBMV from both species, the preservation of midguts in liquid nitrogen gave rise to vesicles with different transport efficiency in the two larvae. This can be readily appreciated from Tables 1 and 2, which report the mean values of different experiments. Storage of midguts of B. mori (Table 1) in liquid nitrogen provided BBMV which fully retained the ability to perform histidine transport and accumulation: preservation at -80°C caused instead a reduction of the uptake values and a significant decrease of the total intravesicular space accessible for the amino acid. The addition of 5% glycerol to the tissue at the moment of isolation, prior to storage at -8O”C, did not improve BBMV performances. Table 2 shows the freezing of P. cynthia midguts in liquid nitrogen drastically affected the transport capacity of the BBMV whether the tissue was stored in liquid nitrogen or stored at -8O”C, although the latter condition was again less suitable for the preparation of vesicles with a good transport ability. The absolute values of histidine uptake, the intravesicular amino acid accumulation and the vesicular volume were all remarkably reduced in BBMV prepared from frozen midguts.

Amino

acid transport

into preserved

67

BBMV

Table 1. Time course of histidine uptake into BBMV from B. mori midguts preserved in different conditions Uptake at different times of incubation (nmol~mg protein) Condition of preservation

IOsec

1min

3 min

6 min

60 min

Accumulation ratio

Vesicular volume

2.78 + 0.30 4.08 Ir 0.43 1.39 f0.15 3.9 f 0.4 4.61 f 0.33 5.26 * 0.40 1.79~0.12 (4) 2.64 & 0.46 4.84 ?r:0.78 I .32 f 0.23 4.4 f 0.6 4.39 f 0.16 5.49 f 0.51 Liquid nitrogen 1.46&0.10 (4) I .63 rt 0.24’ 2.31 _t:0.46* 0.81 k 0.12’ 4.4 + 0.3 3.53 + 0.56 3.24 f 0.63. -80°C I .32 cf:0.20 (4) 2.07 -1_0.79 2.62 i: I .43” I .04 f 0.40 3.4 f 0.4 2.78 f 0.36* 3.10 + 1.23* - 80°C + glycerol I .02 + 0.09’ (3) Histidine uptake was measured in the experimental conditions reported in the legend to Fig. 1. Accumulation is the ratio between 3 min uptake values and 60 min uptake values. Vesicular volumes were calculated from 60 min uptake values and are expressed as ~~l/mg of protein. Values are means i: SE; number of experiments is in parentheses. Asterisks indicate values significantly different from those obtained in BBMV from fresh tissue (P < 0.05: r-test).

Fresh tissue

These results were further supported by the comparison of the kinetic parameters of the carrier-mediated histidine transport in B. mori and P. Cynthia BBMV, measured in the presence of a potassium or a sodium gradient. Table 3 reports K, and V,,,,,values determined in BBMV prepared from fresh and frozen midguts stored in liquid nitrogen: the values of the kinetic parameters were the same for B. mori, while a relevant decrease of V,,,,, was observed for P. Cynthia.

To verify if the low uptakes measured in BBMV from frozen midguts in P. cynthiu couId be due to a different response of the substrate to the purification procedure, the purity of the BBMV suspension obtained from the midguts stored in liquid nitrogen was assayed by measuring the specific activities, in homogenate and final preparation, of the marker enzyme for the brush border leucine amino peptidase (LAP), and that for the microsomal fraction NADPH cytochrome c reductase. Table 4 shows that in both larvae for the control preparations as for those from frozen midguts LAP specific activity increased several fold in the BBMV suspension, while the activity of the microsomal marker enzyme was greatly reduced relative to the homogenate. In B. mot-i, at variance with P. Cynthia, the values of LAP and NADPH cytochrome c reductase specific activity in the homogenate and BBMV were consistently reduced for the midguts stored in liquid nitrogen, as observed also for other substrates (Reshkin et al., 1988; Eisen et al., 1989). Nonetheless, LAP enrichment factors of the preparations obtained from frozen midguts remained in B. mori as in P. cynthia similar to the control values. These preparations are, however, more contaminated by microsomal membranes than those

obtained from fresh midguts, since a consistent increase of the enrichment factors, albeit still lower than one, is observable in Table 4. This increased contamination could in part be the reason for the slightly higher yield (mg protein/g of midguts) of the final preparation obtained from frozen midguts (2.36 k 0.21 instead of 1.52 f 0.10, 10 experiments, in B. mori; 1.76 + 0.07 instead of 1.11 + 0.10, 4 experiments in P. Cynthia). However, since LAP enrichment factors are not modified, the higher yield seem to be essentially represented by brush border proteins. Altogether, the results reported in Table 4 suggest a simitar behaviour of the midguts of the two different species subjected to the purification procedure and therefore do not provide an explanation for the low amino acid transport rates measured in BBMV from P. Cynthia. Figure 3 reports the time courses of histidine uptake for Bombyx mori BBMV prepared from fresh tissue and immediately used and also for vesicles after 24 hr, 96 hr and 110 days of storage in liquid nitrogen. The vesicles had been rapidly frozen by immersion of the cryotube in liquid nitrogen and had been thawed slowly on ice, according to Reshkin et nl. (1988). The figure shows that the frozen vesicles lost immediately part of their transport ability upon freezing, but that subsequently the amino acid transport remained unchanged even after very long periods of storage. Table 5 allows a more detailed analysis of the results, as well as a comparison with the situation found for BBMV prepared from P. Cynthia midguts. Uptake values in the frozen vesicles are in both cases 70-80% of those measured in the fresh vesicles. A slight reduction of the overshoot value and of the vesicular volume is noticeable for B. mori BBMV,

Table 2. Time course of histidine uptake into BBMV from P. cynrhia midguts preserved in different conditions

Condition of preservation

IOsec

Uptake at different times of incubation fnmolimg protein) .~_~_ I min 3 min 6 min

60 min

Accumulation ratio

Vesicular volume

14.92 5 1.51 6.02 + 0.51 17.31 t 1.82 14.61 S 1.63 2.1 I to.29 8.3 k 0.2 4.23 & 0.58 (3) Liquid nitrogen 3.41 I 0.38* 9.19 k 0.93+ 10.13 k 0.87’ 9.25 rt 1.21’ 1.57+0.21 6.4 f 0.5* 3.14 F 0.42 (4) -80°C 1.16*0.26* 2.71 + 0.83. 3.31 f 0.15* 3.43 f 0.55. 0.88 t O.lO* 3.9 k 0.2* I .76 * 0.20* (3) Histidine uptake was measured in the experimental conditions reported in the legend to Fig. I. Accumulation is the ratio between 3 min uptake values and 60 min uptake values. Vesicular volumes were calculated from 60 min uptake values and are expressed as pI/mg of protein. Values are means 2 SE; number of experiments is in parentheses. Asterisks indicate values significantly different from those obtained in BBMV from fresh tissue (P <0.05, f-test). Fresh tissue

68

B. GIORDANAet

al.

Table 3. Kinetic parameters of cation-dependent histidine uptake into BBMV prepared from E. mori and P. cynrhio midguts preserved in liquid nitrogen KSCN

NaSCN

K

Vmsr

K,

Fresh tissue Frozen tissue

0.64 & 0. IO 0.59 + 0.09

2.23 + 0.31 2.45 f 0.14

0.34 If:0.04 0.43 + 0.05

1.19+0.08 0.95+0.11

F. cymhia Fresh tissue Frozen tissue

0.35 + 0.04 0.27 * 0.02

6.77 + 0.21 3.00 + 0.02;

0.30 f 0.03 0.26 + 0.04

4.84f0.12 1.54+0.06*

---!!-

V --- max

B. mori

IiBMV resuspended in (mM): 100 mannitol, 10 Hepe-Tris at pH 7.4 were incubated in a medium of the following final composition (mM): 1M) mannitol, IO Hepes-Tris at pH 7.4, 100 KSCN or NaSCN, 0.1-5.0 L-jZ,S-‘Hlhistidine (25 pCi/ml). Experiments were performed in quadrupiicate at eight different amino acid concentrations and uptakes were terminated after 7 set of incubation. Parameters were determined from the experimental data using a nonlinear regression computer program. K, is expressed as mM + SE, V,,,,, as nmol/l sec/mg of protein + SE. A nonsaturable component was also present in ff. mod, its value ranging between 2.24.8 x 10~81/min/mg of protein. Asterisks indicate values significantly different from those obtained in BBMV from fresh tissue (P < 0.05, r-test).

while a consistent decrease (30%) of the concentrating capability is present for P. cynthiu frozen vesicles. However, the addition of the cryoprot~tive substrate glycerol at a final concentration of 15% to the BBMV suspension from P. cynthiu 10 min before freezing, gave rise, upon thawing on ice, to a preparation which maintained the histidine transport and accumulation characteristics found in the fresh preparation (Table 5). Table 6 reports the kinetic parameters of the cation-dependent transport of the amino acid, measured in frozen vesicles with and without the addition of glycerol: K,,, values, in comparison to control values, were not influenced by storage in either condition while V,,, were slightly decreased, to a greater extent for vesicles with glycerol. Thawing conditions proved to be important: when BBMV (P. cynthiu) were thawed by immersing the cryotuhe for 30 set in a water bath at 37”C, in time course experiments histidine uptake values were reduced to 70% of control as welt as the vesicular volume (to 60%), despite the presence of glycerol during refrigeration (data not shown). Trehalose has been successfully used as a storage protective agent for the preservation of glucose transport properties in lyophilized BBMV from fish intestine (Reshkin et al., 1988): besides, the addition of this sugar to the BBMV suspension (B. mori, Table 4) before freezing did not improve the performance of the preparation.

DISCUSSION

Different transport systems for the translo~ation of amino acids are present in the luminal membrane of the midgut enterocytes of Iepidopteran larvae (Giordana et al., 1989). The best characterized is the system responsible for the intake of neutral amino acids, which are transferred across the luminal membrane with high efficiency. This system recognizes and translocates also histidine, amino acid transported at a high rate, especially in P. Cynthia BBMV where it is concentrated up to eightfold. Moreover, histidine aspecific binding to the membranes is very low, less than 10% of total initial uptake rate in our experimental conditions (Giordana et al., 1985). For these reasons, histidine has been chosen as a test amino acid to verify if the preservation techniques used were suitable for the maintenance of the transport properties of midgut luminal carriers or, at least, of those responsible for the transiocation of neutral amino acids. As shown in Figs 1 and 2 and Tables 1 and 2, the midgut tissue of the two larvae, after being rapidly frozen in liquid nitrogen, gives rise to BBMV whose transport abilities are, partially for B. mori and drastically for P. Cynthia, reduced when the tissue is stored at -8O”C, which therefore proves to be an inadequate system of preservation. It should be noted that this way of storage has been the only one used to date for lepidopteran midguts, by ourselves

Table 4. Enzyme activities in the homogenate and in BBMV from 8. mori and P. cynthia midguts preserved in liquid nitrogen Leucine aminopeptidase Homogenate S. mori Fresh tissue Frozen tissue

0.36 f 0.04 (3) 0.18 + 0.01’ (4)

-~-..-. BBMV

Enrichment

NADPH cytochrome L’reductase __________ _-. Enrichment BBMV

Homogenate _-

4.04 f 0.19

11.3 f 1.3

14.06 & 3.23

1.86~0.15’

10.2 f I.1

5.01 + 0.52’

0.08 4 0.01

0.058 T 0.030

I .06 + 0.24’

0.2 I I i 0.045*

P. cynthia

24.48 f I .70 3.16 + 0.25 0.129 f 0.006 8.7 + 0.5 0.20 * 0.02 1.72 It 0.15 (4) 20.47 _?rI .OI 9.29 rt 2.14* 0.447 ? 0.089’ 0.18 + 0.02 I .57 + 0.25 8.8 * I.7 Frozen tissue (5) Leucine aminopeptidase and NADPH cytochrome c reductase activities are expressed in ~mol/m~n/mg of protein and nmoljmin~mg of protein, respectively. Values are means i SE of assays performed on the number of different preparations indicated in parentheses. Enrichment factors are the mean a SE of the ratios between specific activities in the vesicles and in the midgut homogenate of each preparation. Asterisks indicate values significantly different from those obtained in BBMV from fresh tissue (P < 0.05, t-test). Fresh tissue

69

Amino acid transport into preserved BBMV

nitrogen. Conversely, storage of midgut tissue from P. Cynthia in the same manner does not preserve the

z4\ 3z E c

2=a

l-

0

PI 0

1

2

3

4

5

6

60

minutes

Fig. 3. Time course of histidine uptake into B. mori BBMV stored in liquid nitrogen for different periods. Experimental conditions as reported in Fig. 1. Each curve represents a typical experiment performed in triplicate. Symbols represent the mean + SE of uptake values at the different times. Standard error bars, when not given, were smaller than the symbols.

(Wolfersberger et al., 1987; Hanozet et al., 1989) and others (Eisen et al., 1989). However, the midgut of the two larvae responds in a different way to storage in liquid nitrogen since, upon purification, BBMV preparations were obtained with different characteristics in comparison to the corresponding fresh vesicles. For B. mori, histidine uptake values into BBMV are identical to the control values (Fig. 1A) and so it is the intravesicular accumulation of the amino acid (Table 1). These results are confirmed by the values of the kinetic constants measured in vesicles from fresh and frozen B. mori midguts, in the presence of a K+- or a Na+-gradient (Table 3). The transport system for the translocation of neutral amino acids in the brush border of lepidopteran enterocytes is activated by both potassium and sodium (Hanozet et al., 1980; Giordana et al., 1989): it can be seen that the specificity of the carrier for the cotransported cation remains also unaffected after preservation in liquid

transport characteristics in the final preparation, since a consistent reduction of all histidine uptake values and of the intravesicular accumulation is observed (Fig. 2, Table 2). Accordingly, V,,,, values with potassium and sodium decrease by 60 and 70%, respectively (Table 3), while K,,, values are almost unaffected. However, Fig. 2 shows that the shape of the overshoot curve in BBMV from liquid nitrogen stored midguts is not different from that obtained with fresh vesicles. Apparently, freezing of the midgut affects the tissue in a different manner in the two lepidopteran species, so that a different final BBMV preparation is obtained: besides, the analysis of the enrichment factors of LAP and NADPH cytochrome c reductase for the BBMV preparation (Table 4) rules out the possibility that the difference should be ascribed to a more relevant presence of membranes other than luminal ones in P. Cynthia than in B. mori. Alternatively, the carrier protein itself could be affected in a different way in the two larvae during the freezing procedure. This possibility is also ruled out by the results obtained with vesicles from both larvae frozen in liquid nitrogen and then slowly thawed on ice. Table 5 shows that in both cases a part (15-30%) of the amino acid transport ability is lost by preserved BBMV: this is more evident for P. Cynthia vesicles where, however, histidine uptake is very high, nearly threefold higher than in B. mori. In particular, the concentrating ability of the vesicles is markedly reduced in P. Cynthia. Besides, the addition of 15% glycerol to the BBMV suspension before immersing the cryotube into liquid nitrogen preserves almost completely the capability of the transport system to transfer and accumulate histidine into the vesicles. Since V,,,,, values of histidine carrier-mediated transport are decreased in the vesicles stored without glycerol less than in those with glycerol (Table 6), the cryoprotective effect should be exerted mainly on the lipid bilayer, possibly preserving the characteristics of the conductive pathways for the actions and thus preventing a more rapid dissipation of the K+ and

Table 5. Time course of histidine uptake into midgut BBMV frozen and stored in liquid nitrogen Uptake at different times of incubation (nmol/mg protein)

B.

-

Accumulation ratio

Vesicular volume

IOsec

1min

3 min

6 min

60 min

1.79_+0.12 (4) I .54 + 0.08 (5) 1.29 + 0.16’ (3)

4.61 + 0.33

5.26 f 0.40

4.08 * 0.43

1.39*0.15

3.9 f 0.4

2.78 rt 0.30

3.92 f 0.13

4.00 k 0.08.

2.95 + 0.14’

l.l6+0.07

3.6 f 0.2

2.33 + 0.15

3.36 f 0.47

3.26 f 0.10’

2.37 f 0.13’

0.91 + 0.05*

3.7 * 0.74

I.81 fO.ll*

mori

Fresh BBMV Frozen BBMV Frozen BBMV + trehalose P. Cynthia

6.02 + 0.51 14.92 + I.51 17.31 + 1.82. 14.61 k I.63 2. I I +_0.29 8.3 f 0.2 4.23 f 0.58 (3) Frozen BBMV 4.44 + 0.69’ 10.72 f I .42* 12.54 f 0.60’ IO.61 + 0.15’ 2.19 + 0.23 5.7 f 0.3’ 4.39 f 0.47 (3) Frozen BBMV 6.64 + 0.10 14.63 f 0.28 15.83 rt 0.12 12.05 f 0.95 1.86 _+0.09 8.5 + 0.4 3.72 f 0.10 + glycerol (3) Histidine uptake was measured in the experimental conditions reported in the legend to Fig. I. Accumulation is the ratio between 3 min uptake values and 60 min uptake values. Vesicular volumes were calculated from 60 min uptake values and are expressed as /rl/mg of protein. Values are means + SE; number of experiments is in parentheses. Asterisks indicate values significantly different from those obtained in fresh BBMV (P i 0.05, r-test). Fresh BBMV

B. GIORDANAet al.

70

Table 6. Kinetic parameters of cation-dependent histidine uptake into P. cynfhia BBMV stored in liouid nitrogen KSCN

-. Fresh BBMV Frozen BBMV Frozen BBMV

&I 0.30 + 0.04 0.41 i: 0.05 0.25 _t 0.04

NaSCN --_._..~

VIWPX 6.39 2 0.32 5.70 + 0.16 4.88 k O.IO*

Ktn 0.48 f 0.09 0.26 * 0.05 0.45 f 0.03

V mar

5.38 i: 0.27 4.45 i: 0.12’ 3.18+0.10*

in a medium of the following final composition (mM): 100 mannitol, 10 Hepes-Tris at pH 7.4, 100 KSCN or NaSCN, 0.1-5.0 L-[2,S-3H]histidine (25~Ci/ml). Experiments were performed in quadruphcate at eight different amino acid concentrations and uptakes were terminated after 7 set of incubation. Parameters were determined from the experimental data using a nonlinear regression computer program. K,,, is expressed as mM + SE, ym,, as nmol/7 sec/mg of protein rt SE. Asterisks indicate values significantly drfferent from those obtained in fresh BBMV (P c 0.05, r-test).

Naf gradients across the vesicle, which would affect the intravesicular accumulation of histidine (Table 5). A tentative explanation for the diminished histidine uptakes in BBMV prepared from frozen midguts in P. cynthia should take into account a number of reports upon the importance of the cytoskeletal elements which forms the architecture of the brush border for a correct transport of nutrients across the vesicle membrane (Klip et al., 1979; Hopfer et al., 1983; Karl et al., 1991). Removal of intravesicular cytoskeleton by incubating the vesicles with an alkaline medium or with chaotropic agents caused an increase of the enrichment of brush border marker enzymes (Hopfer et al., 1983; Karl et al., 1991) associated with a strong reduction of Na+-dependent glucose or amino acid uptakes and overshoots, and of the total intravesicular volume (Klip et al., 1979; Karl et al., 1991). If freezing of midguts in liquid nitrogen would determine in P. qmthia the preparation of BBMV in which the cytoskeletal elements were poorly preserved, this could explain the decrease of histidine transport. It would also provide an explanation for the presence of a same LAP enrichment factor despite a higher contamination (Table 4) as well as for the reduction of the intravesicular volume (Table 2), absent in B. mori BBMV (Table 1). In other words, a larger quantity of brush border membranes is recovered, but the alteration of the cytoskeleton

would reduce the number of closed membrane fragments. Besides, the midgut of lepidopteran larvae is, whatever the preservation t~hnique used, a substrate in which the efficiency of its transport proteins is maintained to a noticeable extent. In particular, stored BBMV proved a very effective and stable preparation in comparison to mammalian preserved BBMV: Hittelman et al. (1978) found the total disappearance of the intravesicular glucose accumulation for renal BBMV, Karl et al. (1991) found for MeAIB a marked decrease of the total uptake and no overshoot in stored human placental microvillous membrane vesicles, while Stevens et al. (1982) had variable results for intestine and kidney vesicles stored in liquid nitrogen. It should also be considered that the preserved BBMV from the larval lepidopteran midguts maintain their properties unchanged over long periods of storage (Fig. 2), and were still efficient after 455 days.

In conclusion, for the two lepidopteran species considered two opportunities are available to obtain amino acid transporting BBMV perfectly similar to the freshly prepared ones: the storage in liquid nitrogen of midguts for B. mori and the storage in liquid nitrogen of BBMV after the addition of glycerol for P. qmthia. However, it should be kept in mind that this holds true for the K+-dependent neutral brush border amino acid transport system, by which histidine is readily transported (Giordana et al., 1989). Other transport systems of the same brush border could react in a different manner to freezing and storage in liquid nitrogen, as it happened for different Na+-dependent amino acid transport systems in membrane vesicles from human placenta (Karl et al., 1991). Acknowledgement-This 60% research grant.

work was supported by a MURST

REFERENCES

Eisen N. S., Fernandes V. F., Harvey W. R., Spaeth D. D. and Wolfersberger M. G. (1989) Comparison of brush border membrane vesicles prepared by three methods from the larval Manduca .sexta midgut. Insect Biochem. 19, 331-342.

Giordana B., Sacchi V. F. and Hanozet G. M. (1982) Intestinal amino acid absorption in lepidopteran larvae. Biochim. biophys. Acta 692, 81-88. Giordana B., Parenti P., Hanozet G. M. and Sacchi V. F. (1985) Electrogenic K’-basic amino-acid cotransport in the midgut of lepidopteran larvae. J. Membrarze Biol. 88, 45-53. Giordana B., Sacchi V. F., Parenti P. and Hanozet G. M. (1989) Amino acid transport systems in intestinal brushborder membranes from lepidopferan larvae. Am. 1. Physiol. 257, R494-R500. Hanozet G. M., Giordana B. and Sacchi V. F. (1980) K+-dependent phenylalanine uptake in membrane vesicles isolated from the midgut of Philosamia cvnthia larvae. Biochim. biophys. Act: 596, 481-486. . Hanozet G. M.. Giordana B.. Sacchi V. F. and Parenti P. (1989) Amino acid transport systems in brush-border membrane vesicles from lepidopteran enterocytes. J. exp. Biof. 143, 87-100. Hittelman K., Mamelok R. D. and Prusiner S. B. (1978) Preservation by freezing of glucose and alanine transport into kidney membrane vesicles. Analyt. Biochem. 89, 32433 1. Hopfer U., Crowe T. D. and Tandler B. (1983) Purification of brush border membrane by thi~yanate treatment. Anaiyt. Biochem. 134, 447-452.

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Wolfersberger M. G., Luthy P., Maurer A., Parenti P., Sacchi V. F., Giordana B. and Hanozet G. M. (1987) Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Camp. Biochem. Physiol. 86A, 301-308.