J. exp. Biol. 116, 69-78 (1985) 6 9Printed in Great Britain © The Company of Biologists Limited 1985

METABOLIC ACTIVITY RELATED TO THE POTASSIUMPUMP IN THE MIDGUT OF BOMBYXMORI LARVAE

BY P. PARENTI, B. GIORDANA, V. F. SACCHI, G. M. HANOZETAND A. GUERRITORE

Department of General Physiology and Biochemistry, University of Milano, ViaCeloria 26, 20133 Milano, Italy

Accepted 4 October 1984

SUMMARY

The transepithelial electrical potential difference across the isolated midgutof Bombyx mori larvae is dependent on the presence of potassium and isunaffected by the addition of hexoses to perfusion media, whereas it isenhanced by alanine, aspartic acid, glutamic acid and the corresponding 2-oxoacids, glutamine and malate. The midgut enzyme profile indicates that thesubstrates for the tricarboxylic acid cycle are supplied mainly by amino acidmetabolism via transaminases. Accordingly, aminoxyacetate drasticallyreduces the intestinal transepithelial electrical potential difference stimulatedby amino acids. Measurement of the free amino acid concentration in thelumen content, intestinal cells and haemolymph shows that glutamic acid,asparagine and glutamine are accumulated in the cell, whilst the haemolymphis enriched with basic amino acids and with glycine, alanine, serine andtyrosine, the major components of the silk fibroin. Therefore, amino acidmetabolism directly related to the tricarboxylic acid cycle seems to be theprimary source of energy for the potassium pump activity in B. mori midgut.

INTRODUCTION

The midgut of lepidopteran larvae actively transports potassium from haemo-lymph to lumen. The transport is electrogenic and the short-circuit current isaccounted for almost entirely (up to 99 %) by potassium net flux towards the lumen(Harvey, Cioffi, Dow & Wolfersberger, 1983a). The potassium extrusion, and thetransepithelial electrical potential difference (PD) thus generated, are also responsiblefor the secondary active absorption of amino acids (Giordana, Sacchi & Hanozet,1982). The potassium pump is dependent on oxidative metabolism, since nitrogen,2,4-dinitrophenol and potassium cyanide drastically reduce the PD (Haskell,Clemons & Harvey, 1965; P. Parenti & B. Giordana, unpublished results); however,at present very little information is available aboutthe metabolic support of potassiumtransport.

Key words: Insect midgut, enzyme activity, potassium pump, amino acid metabolism, transepithelialelectrical potential.

70 P. PARENTI AND OTHERS

Previous studies on the isolated midgut (Giordana & Sacchi, 1978, 1980) haveshown that in Bombyx mori, L-alanine can counteract the spontaneous PD decay withtime. Chamberlin & Phillips (1982) have demonstrated that alanine, glutamine andglutamic acid stimulate to some extent short-circuit current across locust rectum, butthat proline, as well as glucose, are the major sources of energy fuelling chlorideabsorption.

In the present paper the effect of different metabolites on the PD of B. mori midguthas been tested, and the enzyme activities of some metabolic pathways have beendetermined. A possible link between metabolite effect on PD and enzyme pattern istentatively proposed.

MATERIALS AND METHODS

Animals

The experiments were performed on B. mori larvae in the last instar, supplied bythe Sezione Specializzata per la Bachicoltura, Padova, Italy, and fed on Morus albafresh leaves. B. mori has been chosen as the experimental animal because, unlike otherspecies, e.g. Philosamia cynthia (Giordana & Sacchi, 1980) and Manduca sexta(Cornell & Jungreis, 1983), this species has a potassium pump readily sensitive toadded metabolic substrates.

Transepithelial electrical potential difference measurements

In order to perform the electrical measurements, the midgut was isolated andmounted as a tube as previously described (Giordana & Sacchi, 1977). Unless other-wise stated the bathing solution used (referred to as K-sucrose) was: (in mmolF1)KHCO3, 25; KC1, 20; sucrose, 208; bubbled with 95 % O2, 5 % CO2; pH 7-4.

The PD was recorded by means of calomel electrodes connected via agar-KClbridges to the solution bathing both sides of the isolated midgut. The PD wasmeasured by means of a Keithley 176 microvoltmeter.

The tested substrates were added from concentrated solutions at pH 7-4 to theluminal and/or to the haemolymph side of the tissue 30 min after isolation. Duringthis time the PD declined to about 50 % of the initial value and the midgut could beconsidered sufficiently depleted of endogenous substrates.

Tissue preparation for enzyme assay

Midguts were dissected, cleared of Malpighiantubules and intestinal content, andrinsed with cold lOOmmoir1 mannitol, 10mmoll"1 HEPES-Tris, pH 7-4 [A/-2-hydroxyethylpiperazine-W-2-ethansulphonic acid and tris-(hydroxymethyl)amino-methane]. A 7-4 % homogenate with the same buffer was prepared with a glass TeflonThomas homogenizer; nine strokes at 3000 rev/min. The homogenate was filteredthrough a double layer of cheesecloth and centrifuged at 100 000 £ for 1 h in an I ECultracentrifuge. The supernatant was collected and used for enzyme assays.

Metabolic activity and potassium pump 71

Glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, pyruvatekinase, malate dehydrogenase, glutamate-oxaloacetate transaminase, glutamate-pyruvate transaminase, isocitrate dehydrogenase, glucose-6-phosphate dehydro-genase and glutamate dehydrogenase were assayed according to Bergmeyer, Gawehn& Grassl (1974) except that glutamate dehydrogenase activity was tested at pH 8-0.Fructose-6-phosphate kinase and fructose 1,6-disphosphatase were assayed accordingto Gancedo & Gancedo (1979), except that O^mmoll"1 fructose-1,6 disphosphatewas used. Phosphoenolpyruvate carboxykinase was assayed according to Hansen,Hintze & Holzer (1976) except that 5 mmol I"1 ADP and 2 mmol I"1 MnCl were used.

All enzyme assays were carried out at 30 °C in a final volume of 1 ml and reactionrate was monitored by means of a Varian DMS-90 spectrophotometer at 340 nm.Protein determination was carried out according to Bradford (1976), using a BioRad kit.

Free amino acid analysis

Tissue extract was obtained as follows: the midgut obtained as described forenzyme assay was rapidly homogenized in cold O^moll"1 perchloric acid (4 ml g - 1

fresh weight) with a glass Teflon Thomas homogenizer; nine strokes at 3000 rev/min.The homogenate was kept in an ice-bath for 10 min and then centrifuged at 3000 # for15 min at 4°C. The pH of the supernatant was adjusted to 7-0 by the addition of2-5 mol I"1 K2CO3. After 15 min at 0°C, the sample was centrifuged as before and thesupernatant was collected. Intestinal content, free from leaf fragments, was processedas described above. Aliquots of the supernatants were used for glutamine andasparagine assays according to Lund (1974) and Bergmeyer, Bernt, Mollering &Pfleiderer (1974). The free amino acid analysis was performed by means of an AutoAnalyzer Carlo Erba model 3A27, after hydrolysis of the samples in 6 mol I"1 HC1 at105°C for 24 h under vacuum. Glutamic acid and aspartic acid concentrations werecorrected for the values of glutamine and asparagine measured enzymatically. Cellularamino acid concentrations were calculated by correcting for the extracellular luminaland haemolymph space volumes reported in a previous study (Giordana & Sacchi,1978).

MATERIALS

Reagents for enzyme assays were purchased from Boerhinger (Mannheim, F.R.G.)or from Sigma (St Louis, MO, U.S.A.). Other reagents were analytical grade productfrom Merck (Darmstadt, F.R.G.).

RESULTS AND DISCUSSION

The composition of the bathing solution used for the experiments was restricted togive a potassium concentration similar to that of B. mori haemolymph, together withsucrose to reach the physiological osmolarity. Calcium and magnesium which are

72 P. PARENT! AND OTHERS

actively absorbed by the midgut epithelium (Wood, Jungreis & Harvey, 1975; Wood& Harvey, 1976; Giordana & Sacchi, 1980), were omitted to avoid their possible effecton PD. As discussed in detail by Moffett (1979) for M. sexta, the midgutelectrogenicity requires the presence in the bathing solution of sucrose or othercompound of similar molecular weight. Despite the presence of disaccharidaseactivity in lepidopteran midgut (Hanozet, Giordana & Sacchi, 1980; Sacchi, Hanozet& Giordana, 1984), the improvement of potassium transport in the presence ofsucrose cannot be ascribed to a metabolic effect, since the midgut electrogenicity islost following the replacement of sucrose by its metabolic products, i.e. glucose orfructose (Moffett, 1979).

The PD generated by isolated B. tnori midgut perfused with sucrose and apotassium salt was entirely due to potassium transport from haemolymph to lumen(Fig. 1). In the absence of potassium salts, PD approached zero (Fig. 1A). Theaddition of potassium gluconate at a final concentration of 46 mmol 1~l induced a rapidincrease of the PD to the physiological value of 120 mV, lumen positive. The PD thensteadily declined in 30 min as expected in the absence of any metabolic substrate. Theaddition of 10 mmol I"1 L-alanine to the haemolymph side caused a 50% increase ofPD. The potassium-dependent PD remained constant when L-alanine was addedprior to the addition of potassium gluconate (Fig. IB, upper trace), whereas no effectof the amino acid was evident in the absence of potassium (lower trace). Gluconate isan impermeant anion through membranes, so the effect of L-alanine on PD cannot be

60

Time (min)

Fig. 1. Effect of potassium and L-alanine on the transepitbelial electrical potential difference acrossthe midgut of a Bombyx mori larva. Typical experiment. The midgut was initially perfused with200 mmol I"1 sucrose, pH 7-0, bubbled with 100% O2. The arrows indicate the addition to bothlumen and haemolymph side. K + = 46 mmol 1~' potassium gluconate; L-ala =10 mmol 1~' L-alanine.(A) Effect of L-alanine in the presence of potassium; (B) effect of potassium in the presence ofL-alanine (a); control in the absence of potassium (b).

Metabolic activity and potassium pump 73due to a movement of negative charges through the intestinal epithelium. The sameresults have been obtained with the K-sucrose solution, i.e. with chloride andbicarbonate as counterions. Therefore, the data reported in Fig. 1 give furtherevidence that the potassium pump is not influenced by anions (Harvey, Cioffi &Wolfersberger, 19836) and suggest that chloride in this tissue is poorly permeable.

The effects of different substrates on the maximal enhancement of the PD inisolated midgut perfused with K-sucrose were examined (Table 1). Glucose, ametabolite which classically energizes cellular activities and transports, did not affectthe PD, although the concentration used (10 mmol \~x) greatly exceeded physiologicalglucose concentration in .6. mori haemolymph (less than 0-2 mmol I"1; Wyatt & Kalf,1957). Similarly, no effect was evident for fructose and glycerol.

The substrates affecting the PD can be divided in two groups: organic acids andamino acids. The organic acids and acidic amino acids bear an overall negative chargeat the pH of the saline used, so when added either to the haemolymph or to the lumenside, their eventual electrogenic transport would short-circuit or enhance respectivelythe PD. In most cases, when a substrate elicited an enhancement of the PD, the effectwas the same on either side of the gut (Table 1). Therefore, the reported effectscannot be ascribed to a transfer of charges across the epithelium. Among the organicacids, malate was the most effective. Oxalacetate and acetate also affected the PD to adifferent extent, but only when added to the haemolymph compartment. Pyruvatecaused a small enhancement of PD.

Table 1. Effect of different metabolic substrates on transepithelial electrical potentialdifference (PD) across the midgut o/Bombyx mori larvae

Substanceadded

GlucoseFructoseGlycerolPyruvateMalateEthanolAcetateOxalacetateGlycineL-alanineD-alanineL-phenylalanineL-serineL-prolineL-aspartic acidL-asparagineL-glutamic acidL-glutamineL-histidine

Concentration(mmoir1)

10101010101052-5

1010101010101010101010

% of PD enhancement

Luminaladdition

0(6)0(1)0(1)

6-0±l-7(S)71-3 ±25-0 (3)

0(2)0(2)0(4)0(5)

125-7 ±33-2 (4)0(5)0(8)0(3)0(4)

16-0 ±1-8 (5)0(3)

76-3 ±23-6 (4)89-2 ±21-8 (5)

0(4)

The effect is expressed as maximal percentage enhancement of the PD i

Haemolymphaddition

0(6)0(2)0(2)

7-7 ±0-8 (4)91-7 ±33-7 (3)

0(3)102-0 ± 25-0 (3)27-1 ±7-8 (3)

0(4)100-9 ±12-8 (10)

0(6)0(7)0(3)0(4)

18-7 ±6-4 (5)0(2)

84-8 ±29-3 (5)85-4118-9(6)

0(4)

referred to the PD value prior to theaddition of the substance, i.e. 50 ± 1 mV (190). Mean ± S.E., number of experiments in parenthesis.

74 P. PARENTI AND OTHERS

Among the tested amino acids only L-alanine, L-aspartic acid, L-glutamic acid andL-glutamine exerted a positive effect on PD, L-alanine being the most effective. It hasbeen shown that L-alanine is actively transported and extensively metabolized by theintestinal epithelium (Sacchi & Giordana, 1980), whereas D-alanine, which is activelytransported but not metabolized by lepidopteran midgut (Hanozet, Giordana,Parenti & Guerritore, 1984), failed to affect the PD. None of the other amino acidstested exerted an appreciable effect on PD; neither glycine and phenylalanine, whichare actively transported but not metabolized by the tissue (Sacchi & Giordana, 1980),nor proline, which is known to cause the largest increase of chloride active transportacross locust rectum (Chamberlin & Phillips, 1982). The effect of L-alanine on PDwas remarkably smaller when 9 mmol I"1 Ca2+ and 44 mmol I"1 Mg2"1" were present inthe bathing solution (data not shown). As already suggested (Giordana & Sacchi,1980) this could possibly be related to the presence of specific pumps for these divalentcations (Wood et al. 1975; Wood & Harvey, 1976).

The time courses of the PD changes after addition of different substrates was alsocompared (Fig. 2). Whenever a substrate failed to enhance the PD, the viability of themidgut used in the experiment was checked by the subsequent addition of astimulating metabolite. 10 mmol I"1 amino-oxyacetate, an inhibitor of transaminases

100

>E

50

L-alanine

L-alanine

Amino-oxyacetate

Amino-oxyacetate

L-glutamine

£

D

loo

50Oxalacetate

L-glutamate L-aspartate

20 40 60 20 40 60Time (min)

20 40 60

Fig. 2. Effect of various metabolic substrates and inhibitors on the transepithelial electrical potentialdifference across the midgut of Bombyx mori larvae. Typical experiments. The arrows indicateaddition to the haemolymph side. The concentrations used were those reported in Table 1. (B)-(F)Midgut perfused with K-sucrose solution; (A) All sucrose was substituted with raffinose. Typicalexperiments. Statistical estimate of variance is given in Table 1. The percentage inhibition induced by10 mmol I"1 amino-oxyacetate (B, C) was 67-6 ±3-7, mean±s.E. of five experiments.

Metabolic activity and potassium pump 75(Hopper & Segal, 1962), caused a drop of PD sustained by L-alanine or L-glutamine(Fig. 2B,C).

In Table 2, some enzyme activities of the intestinal tissue are shown. The lowactivity of lactate dehydrogenase is similar to that found in insect flight muscle, atissue in which anaerobic glycolysis does not occur (Crabtree & Newsholme, 1972).This agrees with the well established fact that intestine has a poor capacity foranaerobic metabolism: in nitrogen, the intestinal active transport of ions and aminoacids ceases (Wood & Harvey, 1976; Haskell et al. 1965; Nedergaard, 1983). More-over Table 2 shows that the midgut has a limited capacity for hexose metabolism, bothvia the phosphogluconate pathway and via glycolysis to trioses. A very low activity isalso shown by typical gluconeogenic enzymes such as fructose disphosphatase andphosphoenolpyruvate carboxykinase. From trioses, glycolysis is in contrast fullyactive, as indicated by glyceraldehyde-3-phosphate dehydrogenase and pyruvatekinase activities. However, by far the most important channels of nutrients intoenergy metabolism are amino acid degradation pathways via transaminases, asindicated by their very high activities and by the relevant effect of amino-oxyacetateon PD. In agreement with the relevant effect of malate on PD, malate dehydrogenaseis the enzyme showing the highest specific activity. This can be accounted for bythe multiple metabolic role of this enzyme as a component of the tricarboxylic acidcycle and, together with glutamic oxalacetic transaminase, of the malate-aspartateshuttle for reoxidizing cytoplasmic NADH. Finally, malate dehydrogenase takespart in the acetyl-group shuttle for transfer of acetyl-groups across the mitochon-drial membrane in lipid biosynthesis. This last pathway is probably operative inthe B. mori intestine, as indicated by the activity of NADP-dependent isocitratedehydrogenase.

Luminal and intracellular concentrations of free amino acids were determinedimmediately after excision of the midgut and compared with haemolymphconcentrations calculated from the data reported by Wyatt, Loughheed & Wyatt

Table 2. Enzyme activities of the midgut o/Bombyx mori larvaeEnzyme Activity

Hexokinase 0-1307 ± 0-0049Fructose-6-phosphate kinase 0-0041 ± 0-0018Glucose-6-phosphate dehydrogenase 0-0077 ± 0-0002Lactate dehydrogenase 0-1133 ± 0-0128Fructose-1,6-diphosphatase 0-0135 ± 0-0007Phoaphoenolpyruvate carboxykinase 00159 ± 0-0012Glyceraldehyde-3-phosphate dehydrogenase 0-9479 ± 0-0215Pyruvate kinase 0-6324 ± 0-0601Glutamate-oxalacetate transaminase 0-3709 ± 0-0075Glutamate-pyruvate transaminase 0-8190 ± 0-0440Glutamate dehydrogenase 0-0603 ± 00170Malate dehydrogenase 4-7720 ±0-3651Isocitrate dehydrogenase 0-1796 ±00114

Enzyme activities are expressed as /anolmin^mg" 1 protein at 30°C. Protein content was 50-1 ± 2-8mgg~fresh weight. Mean ± S.E. of three experiments.

76 P. PARENTI AND OTHERS

(1956) for larvae in the fourth day of the last instar (Table 3). Intracellularconcentrations have been calculated assuming that luminal and haemolymphextracellular volumes in irivo are not different from those measured in vitro, i.e. 13• 1and 31-7% tissue water respectively. Those amino acids which are the majorcomponents of the silk fibroin (L-alanine, glycine, L-serine and L-tyrosine) were all,except glycine, accumulated in the haemolymph compartment. Moreover, thehaemolymph was almost lacking in acidic amino acids, whereas it was enriched withbasic amino acids with respect to the luminal fluid. It is noteworthy that glutamic acidand asparagine concentrations are high in the cell compared to the lumen andhaemolymph values. Glutamine is the most concentrated amino acid both in thehaemolymph and in the cell.

In conclusion, unlike in other insect tissues where glucose is readily metabolized(Wyatt, 1967; Chamberlin & Phillips, 1982), amino acid metabolism related to thetricarboxylic acid cycle seems to be the primary source of energy for the potassiumpump activity in B. tnori intestine: the PD is enhanced by pyruvate and tricarboxylicacids cycle intermediates and by those amino acids which can be readily transformedinto these intermediates; and the enzyme pattern is similar to that found in otherinsect tissues which utilize amino acids as fuels for energy production, and differsfrom that found in tissues which utilize carbohydrates (Crabtree & Newsholme, 1970,1972; Chamberlin & Phillips, 1983). This is in agreement with the lack of any effect ofhexoses on PD. Also, hexose permeability in this tissue could be low; in fact, D-glucose uptake in brush border membrane vesicles of lepidopteran larvae intestine is

Table 3. Amino acid concentration in the lumen content, midgut cells and haemo-lymph o/Bombyx mori larvae in vivo

Amino acid

LysineHUtidineArginineAspartic acidAsparagineThreonineSerineGlutamic acidGlutamineProlineGlycineAlanineValineMethionineIsoleucine +

leucineTyrosinePhenylalanine

Lumen

2-17±0-290-93 ±0-130-81 ±0-07110±0163-54 ±0-58218±0163-57 ±0-32l-52±0145-55 ±0-533-05 ± 0-24

14-42 ±0-813-4010-202-17 ±0-200-72 ±0-06

3-94 ±0-201-24 ±0-05l-2S±0-09

Cell

0-004-28 ±0-283-94 ±0-281-61 ±0-516-54 ±1-48

0-001-62 ±0-174-75 ±0-71

13-02 ±0-72ND

6-8710-602-59 ± 0-290-13 ±0-081-46 ±0-07

1-39 ±0-280-00

0-19 ±0-05

Haemolymph

10-8111-671-950-604-004-78

11-130-7S

13-551-567-005-953-76100

4-112-210-79

Lumen and haemolymph concentrations are given as mmol 1 ' fluid. Intracellular concentration is given asmmol I"1 cell water. Haemolymph values were calculated from Wyatt, Loughheed & Wyatt (1956).

ND = not determined.Mean ± s.E. of three experiments.

Metabolic activity and potassium pump 77negligible compared to that of amino acids (Hanozet et al. 1980). The occurrence ofthe suggested metabolic events is ensured in vivo by the availability of the amino acidseffective on PD in the lumen content and by their accumulation within the cell.Actually, on the luminal membrane of the columnar cells of lepidopteran larvaemidgut, amino acid co-transport systems have been demonstrated (Hanozet et al.1980; Giordana et al. 1982; Sacchi ef al. 1984). According to the model proposed forthis absorption, the driving force for the uptake and accumulation of amino acids isprovided by the electrochemical potassium gradient sustained by the potassiumpump. Therefore, the potassium pump burns amino acids and at the same timepromotes their uptake by the cell.

The authors are grateful to Dr Armando Negri and Professor Severino Ronchi(Istituto di Fisiologia Veterinaria e Biochimica, University of Milan) for the aminoacid analysis. The authors are also indebted to Professor V. Capraro for helpful adviceand criticism in the preparation of the paper. This work was supported by grants fromthe Italian Ministero della Pubblica Istruzione, D.P.R. 382/80, art.65, and fromItalian Consiglio Nazionale delle Ricerche, Rome.

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