pH buffering in Musca domestica midguts

Camp. Biochem. Physiol. Vol. I lZA, Nos. 314, pp. 559-564, 1995 Copyright 0 1995 Elsevier Science Inc. Printed in Great Britain. All rights reserved 0300-%29/95 $9.50 + .OO

Pergamon 0300-9629(95)02028-I

pH buffering in Musca domestica

midguts

Walter R. Terra and Rosana Regel Departamento de Bioquimica, 05599-970, Sao Paulo, Brasil

Instituto de Quimica, Universidade

de Sao Paulo, C.P. 26077,

The M. domestica midgut displays three morphological regions with the following luminal pH values: anterior, 6.1; middle, 3.1; posterior, 6.8. Looking for enzymes that might be related to the acidification of middle midguts or to the neutralization of luminal contents in the anterior and posterior midguts, M. domesticu larvae were placed on layers of 10% starch gels containing 0.1% Congo Red or 0.1% lacmoid and one of the following compounds: acetazolamide, vanadate, KSCN, NaF, ouabain, calcium acetate, 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid, or amiloride. None of these compounds induced alteration in anterior or posterior midgut pH, whereas acetazolamide, vanadate, NaF, and ouabain affected luminal pH in middle midgut. Ammonia and phosphate predominate in anterior and posterior midguts, chloride concentrates in middle midgut, and bicarbonate occurs in low concentration along the midgut lumen. Ouabain and vanadate cause a relative decrease of chloride in the middle midgut and an increase of ammonia mainly in the posterior midgut. The results suggest that chloride may follow the protons translocated into middle midgut contents by a type-P ATPase, similar to that found in vertebrate stomachs, which is inhibited by high intracellular concentration of Na+. Ammonia probably neutralizes the contents of the anterior and mainly posterior midguts and is secreted by mechanism probably involving an apical Na + /K +-ATPase. Key words: Buffers; alkalinization. Comp. Biochem.

Flies; Buffering mechanisms;

Midgut pH; Midgut acidification;

Midgut

Physiol. 112A, 559-564,1995.

Introduction The pH of gut contents is one of the important internal environmental properties that affect digestive enzymes. Numerous determinations have been reported of luminal pH values in many insects. These studies led to the finding that there is a correlation between midgut luminal pH and the phylogenetic position of the insect (Terra and Ferreira, 1994). In contrast to pH determinations, few papers deal with midgut pH buffering mechanisms. The early unsuccessful attempts to reCorrespondence

to: Dr. Walter R. Terra, Departamento de Bioqufmica, Instituto de Quimica, Universidade de SPo Paula, C.P. 26077, 05599-970, SBo Paulo, Brasil. FAX 011-815-5579. Received 27 March 1995; revised 5 July 1995; accepted 16 July 1995.

559

late midgut buffering activity to the large amounts of phosphate frequently found in insect midguts, as well as other unsuccessful attempts to describe midgut buffering mechanisms, are reviewed by House (1974). The results of recent research on midgut buffering mechanisms are more encouraging. Dow (1992) showed that the lepidopteran larval midgut transports equal amounts of potassium and alkali from blood to midgut lumen. Based on this and other data, he suggested that protons are pumped into the goblet cell cavity by a V-ATPase (review on ATPases: Pedersen and Carafoli, 1987). Goblet cell carbonic anhydrase forms H,CO,, which dissociates into H + (feeding the VATPase) and HCO; supplying a Cl -/HCO; antiporter. As K+ leaves the cavity, charge

W. R. Terra and R. Regel

560

balance is maintained by HCO? , which looses proton resulting in carbonate ions putatively responsible for the high pH found in Lepidoptera midguts. Terra et al. (1988) showed that along the entire M. domestica larval midgut there is a strong carbonic anhydrase activity, and that in the anterior and posterior regions of the midgut there is a membrane-bound HCO;ATPase that is not present in middle midgut. These data led to the authors to propose the following buffering mechanism, which might be true for all higher (Cyclorrhapha) Diptera. Carbonic anhydrase in midgut cells produces carbonic acid that dissociates into bicarbonate and a proton. In the anterior and posterior midguts, bicarbonate is secreted into the midgut contents through the action of a putative ATP-driven pump. Bicarbonate neutralizes the acidic midgut contents. In middle midgut, protons are actively transported to the midgut lumen, in a manner reminiscent of that of mammals, by cells whose ultrastructure resembles that of mammalian oxyntic (parietal) cells. In this paper, we describe the effect of several compounds on midgut pH and midgut luminal content of chloride, ammonia, bicarbonin Musca domestica ate, and phosphate larvae. The results suggest that chloride may follow the protons translocated by a type-P ATPase in middle midgut, and that ammonia neutralizes the contents of anterior and mainly posterior midguts.

Materials and Methods Animals

Larvae of M. domestica were reared in a mixture of fermented commercial pig food and rice hull (1: 2, v/v). The larvae used in this study were actively feeding third larval instars. Effect of compounds

in midgut pH

Groups of 30 larvae were rinsed in distilled water, blotted with filter paper, and placed on layers (10 ml) of 10% corn starch gel in beakers maintained at 24°C in the dark. The gels contained 0.1% Congo Red (middle midgut pH observations) or 0.1% lacmoid (anterior and posterior midgut pH observations) and one of several different compounds. The following compounds (final concentrations) were used: 1 mM acetazolamide (carbonic anhydrase inhibitor, Turbeck and Foder, 1970), 1 mM ouabain (Na+ /K’-ATPase inhibitor, Pedersen and Carafoli, 1987; Nicolson, 1993), 5 mM vanadate (P-type ATPase inhibitor, Pedersen and Carafoli, 1987; Bertram et al., 1991), 5 mM KSCN (V-type ATPase inhibitor, Pe-

dersen and Carafoli, 1987; Lechleitner and Phillips, 1988), 5 mM NaF (vertebrate gastric H + /K ‘-ATPase inhibitor, Forte et al., 1980), 5 mM amiloride (Na+ /H + antiporter inhibitor, Sundaram et al., 1991; Nicolson, 1993), 1 mM 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS) (Cl - /HCO; antiporter inhibitor, Sundaram et al., 1991), and 0.1 mM N-ethylmaleimide (V-type ATPase inhibitor, Pedersen and Carafoli, 1987; Bertram et al., 1991). Effect of compounds in the midgut content of ammonia, bicarbonate, chloride, and phosphate

Groups of 30 larvae were rinsed in distilled water, blotted with filter paper, and placed on layers (10 ml) of 10% corn starch gel in the absence or presence of 1 mM ouabain or 5 mM vanadate. After standing on the gels for 150 min at 24°C in the dark, the larvae were immobilized by placing them on ice and dissected in cold 300 mM sucrose solution. The small midgut caeca were discarded and the remaining midgut was divided in the following sections: anterior, middle, and posterior midguts (Terra et al., 1988). The peritrophic membrane contents corresponding to each of the midgut sections were collected with the aid of a capillary, dispersed in a known volume of double distilled water, and homogenized in a Potter-Elvehjem homogenizer. The homogenates were centrifuged at 10,000 g for 10 min at 4°C and the supernatants used in the chemical determinations. Chloride was titrated according to Shales and Shales (1941) using 0.2 ml samples. Bicarbonate was enzymatically determined as total CO* according to Fort-ester et al. (1976), following the instructions of kit no 132-A (Sigma Chemical Company, St. Louis, MO). Ammonia was determined with the Nessler’s reagent (Oser, 1965) and phosphate according to Baginski et al. (1967). Molar concentrations were calculated taking into account that the volume of midgut sections were (mean and SEM, n = 3): anterior midgut, 2.5 t 0.3 ul; middle midgut, 0.60 & 0.06 ~1; posterior midgut, 3.5 ? 0.5 p.1. The volumes were calculated from drawings prepared with the help of a camera lucida mounted in a stereoscopical microscope. The volume of the epithelial layer was disregarded in the calculations. Thus, the volume of gut contents was assumed to be identical to that of the corresponding gut section. Glutaminase

assays

M. domestica larvae were rinsed in distilled water, blotted in filter paper, immobilized by placing them on ice, and dissected in 50 mM

Midgut pH buffering

Tris-HCl buffer pH 8.0 containing 200 mM sucrose. The midguts, after being divided into anterior, middle, and posterior sections, were homogenized in the dissection medium with a Potter-Elvehjem homogenizer at a ratio of 20 midgut sections/ml. The homogenates were passed through a nylon mesh of 45 p,rn pore size. The filtrates were centrifuged at 600 g for 10 min and the resulting supernatants centrifuged at 100,000 g for 60 min. Glutaminase assays were performed in the final supernatants using a method modified from Bergmeyer et al. (1974). Assays were accomplished with 40 mM glutamine in 50 mM sodium acetate buffer pH 4.9 or in 50 mM citrate-sodium phosphate buffer pH 6.0. After 30 min (or 160 min) at 30°C (or 37”C), the samples were boiled for 3 min and ammonia enzymatically liberated from glutamine was determined as described above.

Results and Discussion Effect of compounds in M. domestica midgut PH

Control larvae fed with starch gels containing 0.1% Congo Red have red colored anterior and posterior midguts (pH over 5.0) and blue colored middle midgut (pH under 4.0). Table 1 shows the percentage of larvae that have had their middle midgut pH affected by different compounds. Middle midgut pH is much affected by ouabain, vanadate, and NaF and by acetazolamide in a smaller extension. The midgut of control larvae fed starch gels containing 0.1% lacmoid acquired the following colors: anterior midgut, light purple (pH between 5 and 6); middle midgut, dark red (pH under 4.4); posterior midgut, from dark blue to light blue (pH above 6.4). None of the compounds described in Table 1 affected the colors of anterior and posterior midguts of larvae feeding lacmoid; that is, their pH values remained constant under 1 pH unit. Also, no effect was observed in the presence of the following compounds: 0.1 mM N-ethylmaleimide, 0.1 mM DIDS, and 5 mM amiloride. Table 1. Percentage of larvae with middle midgut pH over 5.0 in the presence of different compounds Compound (mM)

% larvae

Acetazolamide Ouabain (1) Vanadate (5) KSCN (5) NaF (5)

26 43 40 11 41

(1)

k 3 f 8 ‘- 8 -+ 4 * 10

Values (means and SEM) were calculated subtracting the % of larvae with middle midgut pH over 5.0 (red midguts) in the experimental groups from the corresponding % in control groups. Five groups with 30 larvae were observed in each experimental condition.

561

Effect of compounds in the M. domestica midgut contents of ammonia, bicarbonate, chloride, and phosphate In an attempt to identify the buffers occurring along the larval midgut, several compounds were determined in luminal contents. Bicarbonate occurs in low concentrations along the whole midgut (Table 2), discounting an important role as a buffer. Chloride, in spite of the large deviations found, seems to occur in middle midgut in high concentrations in control larvae and in low concentrations in larvae treated with ouabain and vanadate (Table 2). This suggests that chloride may follow the protons putatively pumped by an ATPase in middle midgut. Ammonia and phosphate occur in high concentrations in anterior and posterior midguts (Table 2), and may, thus, be involved in luminal neutralization. Ouabain increases the luminal ammonia concentration along the whole midgut, and vanadate does the same in posterior midgut (Table 2). Ouabain and vanadate seem to increase the phosphate concentration a little in anterior midgut (Table 2). The maintenance of midgut pH values The pH changes remarkably along the midgut of M. domestica. The anterior midgut contents display a pH of 6.1, the middle midgut contents a pH of 3.1, and the posterior midgut contents a pH of 6.8 (Espinoza-Fuentes and Terra, 1987). The low pH observed in the middle midgut of M. domestica, and of other cyclorrhaphous flies, deserves special attention because those insects are the only animals, other than vertebrates, to display such an acidic region in their guts (Vonk and Western, 1984). M. domestica middle midgut contains cupshaped (oxyntic) cells rich in mitochondria and with particles in their microvillar membranes that might be ion pumps (Terra et al., 1988). The M. domestica oxyntic cells are morphologically like the oxyntic (also called parietal) cells from the mammalian stomach. The oxyntic cells from the mammalian stomach are responsible for the secretion of protons, resulting from the dissociation of carbonic acid (originated through the action of carbonic anhydrase upon carbon dioxide), and of chloride ions; thus, forming hydrochloric acid in the stomach lumen (review: Forte et al., 1980). The data suggest that oxyntic cells m the midgut of M. domestica (and other cyclorrhaphous flies) also secrete hydrochloric acid, maintaining low pH in middle midgut lumen (Fig. 1). Several facts support this hypothesis. The first is the observed increase of middle

562

W. R. Terra and R. Regel

midgut pH in larvae treated with vanadate and NaF, which are inhibitors of the mammalian stomach proton pump (Forte et al., 1980), and with acetazolamide, which inhibits carbonic anhydrase. The increase in pH following treatment with ouabain suggests an indirect role for the Na+ /K+-ATPase. This may result from Na+-inhibition of the proton pump, as observed in mammals (Sachs et al., 1982). Other evidence favoring the abovementioned hypothesis is the cytochemical finding of a large concentration of chloride in oxyntic cells (Dimitriadis, 1991) and in chloride middle midgut contents, which is decreased parallel to the inhibition of the putative proton pump (Table 2). It has been pointed out earlier that the pH of the contents of the M. domestica digestive tract rise suddenly from about 3.1 to 6.8 as the contents pass from the middle midgut into the posterior midgut. A good guess as to the cause of this rise in pH is the secretion of bicarbonate ions by posterior midgut cells, in a way reminiscent to what occurs in the mammalian duodenum. In mammals, stomach contents are alkalinized by bicarbonate ions secreted into the intestine through the action of an ATP-driven pump or a Cl - /HCO; antiporter located on the plasma membrane covering the enterocyte microvilli (Sachs et al., 1982). A similar phenomenon was thought to occur in M. domestica posterior midgut based on the finding that, in this region, there is a plasma membrane-bound ATPase that is activated by bicarbonate ions and is not inhibited by olygomycin (Terra et al., 1988). Nevertheless, bicarbonate is present in uniformly low concentration along the whole midgut contents (Table 2), discounting an important role as a buffer. Another evidence against a bicarbonate role in M. domestica midgut luminal buffering, and which passed unaware until now, was the fact that carbonic anhydrase in posterior midgut is as low as only one third of that found in middle midgut (Terra et al., 1988). Finally, the possibility of bicarbonate being secreted into M. domestica midguts by a Cl- IHCO; antiporter was discounted by the finding that DIDS does not change M. domestica midgut pH values; although, in this case, the possibility remains that DIDS is ineffective in our conditions. The origin and role of the bicarbonate-activated ATPase found in M. domestica midgut cell membranes (Terra et al., 1988) remain unknown. Although phosphate could have a neutralization role in the anterior and posterior midguts, this was dismissed by the finding that 5 mM calcium acetate (which should precipitate phosphate as calcium phosphate) added to the

563

Midgut pH buffering

A

Lumen

I+20 + coz---) CA

H2CO3 7’

Lumm

H+

NH4+7

HCOs-

Memolymph

Hemolvmnh

H+

NH3

I + H+

Fig. 1. Diagrammatic representation of ion movement supposed to be responsible for the maintenance of the pH of the larval midgut contents of M. domestica. Carbonic anhydrase (CA) in cup-shaped oxyntic cells in middle midgut (A) produces carbonic acid that dissociates into bicarbonate and a proton. The bicarbonate is transported into the hemolymph, whereas the proton is actively translocated into the midgut lumen acidifying its contents. Chloride ions follow the movement of protons. NHs diffuses from anterior and posterior midgut cells (B) into the midgut lumen, becoming protonated and alkalinizing their contents. NH: is then exchanged for Na+ by a microvillar Na+/K’-ATPase. Inside the cells, NH,,? forms NH,, which diffuses into midgut lumen, and proton that is transferred into the hemolymph.

starch gels causes no pH changes in those regions (not shown). Ammonia is, thus, the more probable buffer in anterior and posterior M. domestica midguts. This may be accomplished as follows: NH3 from midgut cells diffuses through the cell membranes into the midgut lumen where it becomes protonated, increasing the luminal pH, The resulting NH: is then exchanged for Na + by a Na+ /K +-ATPase localized in the microvillar membranes of anterior and posterior midgut cells. Inside the cells, NH,’ may dissociate in H + to be directed to the hemolymph and NH3, which will diffuse out of the cells again (Fig. 1). Evidence supporting this model is: (1) animal cells are usually highly permeable to NH3 (Kleiner, 1981); (2) it is well established in several organisms that the Na+ site of Na+ /K +-ATPase is very specific, whereas a number of monovalent cations can replace K + , among them NH: (Kleiner, 1981); (3) inhibitors of Na’/K+-ATPase. such as ouabain and vanadate, cause an increase in ammonia concentration in M. domestica midgut lumen (see Table 2). In accordance with the postulated model, inhibitors of V-type ATPases, such as KSCN and N-ethylmaleimide, do not affect luminal pH, whereas P-type ATPase inhibitors, such

as vanadate as well as ouabain, should change at least posterior midgut pH. Nevertheless, as vanadate and ouabain cause an increase in middle midgut pH, it was not possible to verify the postulated fail in posterior midgut content buffering. Taking into account ammonia concentrations (Table 2), ammonia buffering should be more efficient in posterior midgut, in agreement with the physiological necessity to neutralize the food bolus coming from the acidic middle midgut. Ammonia to be excreted is frequently derived from glutamine through the action of glutaminase both in mammals and in insects (Thomson et al., 1988). Nevertheless, no glutaminase could be found in M. domestica midgut homogenates. Thus, NH, may not originate from glutamine in M. domestica midguts or, alternatively, our glutaminase assays were not sensitive enough to detect this enzyme. In any case, the formation of NH, in M. domestica posterior midgut is not necessarily great (this is even more true for anterior midgut) because ammonia seems to be recycled in the process of pH buffering. Fig. 1 presents the secretion of ions proposed to be responsible for the maintenance of midgut pH values in M. domestica larvae midguts.

564

W. R. Terra amd R. Regel

Acknowledgments-We

are much indebted to Dr. C. Ferreira for heloful discussion and to Miss L. Y. Nakabayashi for technical assistance. This work was supported by the Brazilian Research Agencies FAPESP, CNPq, and FINEP. R. Regel is an undergraduate fellow of CNPq and W. R. Terra is a staff member of the Biochemistry Department and research fellow of the CNPq.

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