Effects of anions, acetazolamide, thiocyanate and amiloride on fluid secretion by the Malpighian tubules of Locusta migratoria L.

Pergamon

0022-1910(94)00062-X

J. Insect fh~~siol. Vol. 40, No. 12, pp. 1093-1099, 1994 Copyright Q 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910/94 $7.00 + 0.00

Effects of Anions, Acetazolamide, Thiocyanate and Amiloride on Fluid Secretion by the Malpighian Tubules of Locusta migratoria L. HOSSEIN FATHPOUR,* DOUGLAS L. DAHLMANTS Received 30 April 1993; revised 10 May 1994

The effect of Cl-, HCO;, Br-, acetazolamide, thiocyanate and amiloride on urine formation in Locusta migrutoriu Malpighian tubules have been determined. The rate of Iluid secretion depends markedly on the concentration of Cl- in the bathing solution with concentrations less than 90 mM resulting in reduced fluid secretion. Substitution of Br- for Cl- had no significant effect on the rate of the fluid secretion. Replacement of NaHCO, with NaCl in Hepes buffered Ringer solution reduced the rate of urine production by 23%. Fluid secretions were reduced in the presence of 10-4-10-2 M acetazolamide, a carbonic anhydrase inhibitor. The combined effect of acetazolamide in the absence of HCO; appears to be additive. A 1 mM concentration of thiocyanate, an ionic inhibitor, reduced fluid secretion by 35%. Amiloride interferes with the electrogenic entry of Na+ into the cell and a 1 mM solution reduced fluid secretion by 94% with secretion completely inhibited in 80% of the tubules tested. Locusta

Malpighian tubules Urine production

Inhibitors

INTRODUCTION Renal function in the majority of insect species involves two organ systems, the Malpighian tubules and the rectum. The major function of Malpighian tubules is the production of a “primary urine”, which is subsequently modified before excretion by selective resorption of ions and water in the rectum (Bradley, 1985; Petzel and Stanley-Samuelson, 1992). Primary urine contains many of the low molecular weight components present in the hemolymph; however, the proportion of these solutes in the urine and hemolymph can differ markedly (Morgan and Mordue, 1981). The Malpighian tubules of Locusta, like those of many other species, are able to transport K+ against a chemical gradient over a wide range of external K+ concentrations (Maddrell and Klunsuwan, 1973; Anstee et al., 1979; Maddrell, 1980; Morgan and Mordue, 1981; Baldrick et al., 1988; Fogg et al., 1991). However, in Rhodnius the tubules are able to secrete at maximum rate in ‘sodium only’ solutions (Maddrell, 1969) and in Glossina Na+ is the prime mover (Gee, 1975). Despite the role of K+ (or Na+) as the ‘prime mover’ in fluid secretion, a number of workers have shown that *Department of Biology, University of Esfahan, Esfahan, Iran. TDepartment of Entomology, University of Kentucky, Lexington, KY 40546-0091, U.S.A. fTo whom correspondence should be addressed.

these cations alone will not support water transport across the tubule wall, unless accompanied by an appropriate anion. The mechanism of transporting anions along with the active secretion of K+ (or Na+) may differ among species. For example, in Calliphora, Cl- transport occurs by passive diffusion down an electrochemical gradient created by active K+ transport (Berridge, 1969). In contrast, Cl- is thought to be actively transported across the basal cell membrane of Rhodnius Malpighian tubules cells (Maddrell, 1977; Phillips, 198 1; Bradley, 1985) and Locusta Malpighian tubules. However, Clmovement from the cell to lumen is passive. Alternatively, Baldrick et al. (1988) proposed that Cl- might cross the basal membrane by a cation co-transport mechanism. Cation secretion across the Malpighian tubules of Locusta migratoria has been extensively studied (Anstee et al., 1979; Morgan and Mordue, 1981,1983; Fathpour et al., 1983; Baldrick et al., 1988). However, except for studies by Morgan and Mordue (1981,1983) and Baldrick et al. (1988), information about anion secretion in the Malpighian tubules of Locusta is limited. This study evaluated the effect of different anions on urine production across the Malpighian tubules of Locusta. In addition, the effect of three compounds with different modes of action on urine production were investigated. In mammals acetazolamide is a diuretic which inhibits carbonic anhydrase and thus influences

1093

HOSSEIN FATHPOUR

1094

and DOUGLAS L. DAHLMAN

the concentration of HCO;, thiocyanate is an ionic inhibitor and amiloride interferes with the electrogenic entry of sodium into the cell (Gilman et al., 1985). Tests on the effect of these compounds on fluid secretion will provide further information concerning possible mechanisms that regulate anion transport.

MATERIALS

Reagents

All solutions were made in glass-distilled, deionized water. All inorganic salts were AR grade or the best commercially available. Sodium acetazolamide (Diamox) was obtained from Lederle (American Cyanamid Company, Pearl River, NY, U.S.A.).

AND METHODS

Mature adult locusts, Locusta migratoria L., were taken from a population maintained under crowded conditions at 28 f 0.5”C. The relative humidity was about 60%, but depended on the time since the last daily addition of grass. The photoperiodic regime was 12 h light, 12 h dark. Malpighian tubule secretion rates In vitro measurements of fluid secretion by the Malpighian tubules were carried out using essentially the same method as that described by Maddrell and Klunsuwan (1973). The major modification introduced was that the tubules were not completely severed at their point of entry into the alimentary canal. Instead, individual Malpighian tubules were drawn out of the Ringer solution into liquid paraffin and looped around small stainless steel pegs. A fine tungsten needle was used to partially severe the tubule along its length between two pegs. Temperature was maintained at 30 f 0S”C by placing the dish containing the dissected preparation inside a water-heated chamber. At the end of a 15 min equilibration period, any secreted ‘urine’ droplet was removed and discarded. The secretion rate (Rate 1) for each tubule was then determined by measuring the diameter of the secreted ‘urine’ droplet at 5 min intervals for 30 min. The volume of the secreted droplet in nl was calculated with the assumption that the droplet was a sphere. At the end of the Rate 1 test period, the Ringer solution was replaced with a fresh solution of either a different (experimental) or the same composition (control) and equilibrated for 5-30 min, depending on the experimental conditions. The secreted fluid was then removed and the rate of secretion was determined for an additional 30 min (Rate 2). The effect of the treatment was estimated by comparing the rates of secretion over the two periods (Rate 1 and Rate 2) on the basis of a paired t-test. In all experiments, Rate 2 from the control solution was used to indicate the extent of any change in the rate of secretion over the two periods of measurement that may have resulted from the aging of the preparation.

RESULTS

Eflect of Cl- concentration on jluid secretion by the Malpighian tubules

The rate of fluid secretion over the initial period of 30 min in ‘normal’ Hepes Ringer solution was determined. This solution was then replaced with modified Ringer in which varying concentrations of NO; were substituted for Cl-. After a 15 min equilibration period, the effects of different concentrations of Cl- on the rate of fluid secretion by Malpighian tubules were measured (Fig. 1). In the absence of Cl-, fluid secretion was inhibited approx. 60%. Furthermore, the results showed that fluid secretion was sensitive to the concentration of Cl- in the bathing medium; 75-90 mM Cl- was necessary for normal tubule secretion rates to be maintained. Eflect of HCO;-free Ringer solution on fluid secretion by the Malpighian tubules

Following the initial determination of Rate 1 and a subsequent 20 min equilibration period, the rate of fluid secretion in HCO;-free Hepes Ringer solution was measured (Table 1). The NaHCO, of Hepes Ringer Solution was replaced with NaCl. In the absence of HCO; the mean rate of fluid secretion was reduced 23%. Effect of Br - on fluid secretion by the Malpighian tubules

After the initial rate of fluid secretion was determined over a period of 30 min in the presence of ‘normal’ Hepes Ringer solution, the tubules were immersed in a Cl--free

100 t

E

I

Insect Ringer solution

A Ringer solution buffered with Hepes (pH 7.2) was used throughout. The composition of this Ringer solution was (mM): NaCl (lOO), KC1 (8.6), CaCl, (2), MgCl, (8.5), NaHPO, (4), NaHCO, (4), glucose (34), Hepes (25), NaOH (11). The total concentration of Clin the Ringer solution was 119.1 mM.

OL I

0

I 30

Concentration

I

I

I

60

90

120

of Cl ions in bathing medium (mM/I)

FIGURE 1. The e&t of various concentrations of Cl- on the rate of fluid secretion (% of original rate). The vertical lines represent f SE and the numbers in parenthesis indicate the number of determinations.

L. MIGRATORIA TABLE

1. Effect of HCO;-free

FLUID

Ringer solution on the secretion tubules of L. migratoria Mean

N

Control Experimental (HCOC-free Ringer present for Rate 2)

Rate

of fluid by the Malpighian

rate of fluid secretion

Expressed Treatment

1095

SECRETION

f SE

in nl/min Rate 2

1

% Original rate

P >O.l

12

3.47 k 0.74

3.53 + 0.70

104.6 f 6.3

63

3.86 + 0.30

3.17 + 0.29

81.4 + 4.3


Rate 1 and Rate 2 were each determined over 30 min. In each case, Rate 1 was determined with Ringer solution while Rate 2 used either Ringer (control) or modified Ringer (experimental) solution. % Original was determined by Rate 2/Rate 1 x 100 for each preparation and the mean was determined. For this reason, % Original values may be different from the value obtained when the Rate 1 and Rate 2 means are used to calculate the same estimate. P values were obtained by comparing Rate 1 and Rate 2 by paired ‘I’ test

Ringer solution in which all the Cl- was replaced by the equivalent Br- salt. Following an equilibration period of 15 min, the rate of fluid secretion was redetermined over a 30 min period. The replacement of Cl- with Br- did not significantly reduce the rate of fluid secretion by the Malpighian tubules (P > 0.05) (Table 2). Eflect of sodium acetazolamide on fluid secretion by the Malpighian tubules

approx. 55% inhibition of fluid secretion (Table 3). This inhibition was greater than that observed in the presence of either 1 mM sodium acetazolamide (40% reduction) or HCO;-free-Ringer solution (23%) and the combined effects of acetazolamide in the absence of HCO, appear to be additive. Eflect of sodium thiocyanate on fluid secretion by the Malpighian tubules

Experiments were carried out as described above. Various concentrations of sodium acetazolamide (3 x 10-6-10-2M) were added to the Hepes Ringer solution and Rate 2 was determined following a 20 min equilibration period. Results clearly show that acetazolamide inhibits fluid secretion over the concentration range 1 x lO-4-lO-2 M (Fig. 2). The presence of 1 mM inhibitor significantly decreased the mean rate of fluid secretion by approx. 40% (P < 000.1).

The experimental procedure used NaSCN (l-10 mM) included in Hepes Ringer solution to determine Rate 2. This was achieved by substitution for NaCl, thus ensuring that the cation concentration remained unchanged. It is clear that SCN- inhibits fluid secretion by Malpighian tubules of Locusta (Table 4) although a 10mM solution was only slightly more effective than 1 mM.

Eflect of HCO ;-free Ringer containing sodium acetazolamide on &id secretion by the Malpighian tubules

Effect of amiloride on fluid secretion by the Malpighian tubules

To determine whether the effects of sodium acetazolamide and HCO;-free Ringer solution are cumulative, the initial rate of secretion was determined in Hepes Ringer solution, and then the tubule was immersed in either the HCO;-free-Ringer containing 1 mM acetazolamide (experimental) or standard Hepes Ringer solution (control). Following an equilibration period of 20 min, the rate of secretion was redetermined. The presence of 1 mM acetazolamide in the absence of HCO; resulted in

Experiments were carried out in which either 0.1 or 1 mM amiloride was present in Hepes Ringer solution for the Rate 2 determination. A 1 mM amiloride solution almost totally inhibited (94%) secretion of ‘urine’ by the Malpighian tubule of Locusta (Table 5). Indeed, out of 22 tubules examined, 18 tubules failed to secrete any fluid over the second 30 min period (Rate 2). Similarly, substantial inhibition of fluid secretion (ca. 76%) was observed in the presence of 0.1 mM amiloride.

TABLE

2. Effect of Br

on the secretion

of fluid by the Malpighian Mean

rate of fluid secretion

Expressed N

Treatment Control Experimental substituted See Table

(120mM Br- was for Cl- for Rate 2) 1 for details.

Rate

tubules

1

of L. migratoria

k SE

in nl/min Rate 2

% Original rate

P

14

1.8OkO.28

1.71 kO.36

101.2 k 10.6

>O.l

29

3.60 k 0.44

3.13 + 0.38

92.6 & 7.4

io.05

1096

HOSSEIN FATHPOUR

r

and DOUGLAS L. DAHLMAN

(48)

20

1

OL04/

’

6

I

I

5

4

I 3

I 2

-log [ acetazolamide] FIGURE 2. The effect of various concentrations of sodium acetazolamide on fluid secretion (% of original rate). The vertical lines represent k SE and the numbers in parentheses indicate the number of determinations.

DISCUSSION

The rate of fluid secretion by the Malpighian tubules of Locusta depends markedly on the concentrations of Cl- in the bathing solution. This agrees with Bradley (1985) who stated that Cl- is the predominate anion in secretions produced under physiological conditions in all Malpighian tubules examined to date; e.g. Berridge (1969) examined 15 separate anions on the rate of fluid secretion in the Malpighian tubules of Calliphoria erythrocephala placed in bathing media with other anions substituted for Cll . He found that secretion rates were inversely proportional to the hydrated radius of the predominate anions in the bath with Cl-, Br-, I- and NO; all supporting rapid flow. Fluid transport was not supported by SO:-, proprionate or citrate. In fact, in Ringer solutions in which NO; replaced Cl-, the secretory rate was reduced to 38% of that observed in ‘normal’ Ringer solution; the rate of fluid secretion increased as the concentration of Cl- increased from zero to approx. 90 mM. Similarly, Morgan and Mordue (1981) observed the rate of fluid secretion in Locusta Malpighian tubules in SO:- Ringer solution was re-

duced to approx. 10% of that observed in Cl- Ringer solution. Kerkhove et al. (1989) observed that substitution of either SOi- or NO; for Cl- in Ringer solution completely inhibited fluid secretion in Malpighian tubules of Formica. Similar results were reported elsewhere (Maddrell, 1969; Kaufman and Phillips, 1973; Peacock, 1986). The major role of Cl- in fluid secretion and transport across the Malpighian tubules is somewhat obscure. Morgan and Mordue (1981, 1983) working on L. migratoria, found that Cl- is essential for basal secretion and a high level of Cl- is required for maximum secretion. They suggested that Cl- entry into the cell at the basal surface is by an active process while movement from the cell to the lumen is passive. A basal Cl- entry mechanism probably involves an electroneutral cotransport with Na+ (Baldrick et al., 1988), whereas the movement of Cl- across the apical membrane is probably passively aided by the large favorable electrical gradient of an apicial cation pump (Wieczorek et al., 1991; Nicolson, 1993) with only a modest adverse Clconcentration gradient (Morgan and Mordue, 1983; Gupta et al., 1976). Similarly, Kerkhove et aE. (1989), working on Formica Malpighian tubules, proposed that while Cl- transport seems to be passive, at low K+ and Cl- concentrations Cl- can be actively transported. Some component in a corpora cardiaca extract modifies Cl- movement across both the basal and the apical cell membranes of L. migratoria Malpighian tubule (Fogg et al., 1989). The subcellular distribution of cation and anion ATPase activity in Malpighian tubules of Locusta confirmed the presence of HCO;-stimulated ATPase activity (Fogg et al., 1991) which was reported in microsomal preparations of this tissue (Anstee and Fathpour, 1979, 1981). This enzyme might affect Clentry into the cell across the basal cell membrane which appears to be relatively impermeable to this anion (Baldrick et al., 1988). However, a similar anionstimulated ATPase has been reported in the apical cell membrane of locust rectum where it is suggested that it may be responsible for effecting active Cl- transport (Lechleitner and Phillips, 1988). More recently, Leyssens et al. (1992), working on Formica polyctena Malpighian tubules, concluded that no evidence is available to decide the relative importance of cellular versus paracellular pathways for Cl- transfer. If Cl- uptake occurs through

TABLE 3. Effect of aeetazolamide in HCO;-free Ringer solution on the secretion of fluid by the Malpighian tubules of L. migratoria Mean rate of fluid secretion f SE Expressed in nl/min Rate 2

% Original rate

P

3.69kO.74

3.64k0.67

102.7&11.5

>O.l

3.73 + 0.46

1.72 + 0.23

Treatment

N

Rate 1

Control Experimental (1 mM acetazolamide present in HCO;-free Ringer for Rate 2)

11

32

See Table 1 for details.

46.8 + 4.1


L. MIGRATORIA TABLE

FLUID

SECRETION

4. Effect of different concentration of thiocyanate on the secretion Malpighian tubules of L. migratoriu Mean rate of fluid secretion Expressed

Cont. of NaSCN (mM) 0 (Control) 5 10 See Table

N 23 21 55 21

1

Rate 2.90 6.01 4.30 2.95

+ + + +

of fluid by the

+ SE

in nl/min

1

% Original rate

Rate 2

0.36 0.64 0.36 0.38

2.72 3.88 2.77 1.48

k 0.34 & 0.49 i 0.29 f 0.19

100.5 66.8 64.8 55.7

+ f f +

P

6.9 5.2 4.6 6.5

>O.l
1 for details.

a cation/anion symporter at the basolateral side and the cell remains in steady state, the same quantity of Clions has to leave the cell. Thus, part of the net KC1 transport may occur transcellularly. The importance of the shunt versus the cellular pathway for Cl- transport will have to be investigated in luminally perfused tubules, where the composition of the luminal fluid can be controlled. On the other hand, in a model for anion and cation transport in a Malpighian tubule cell, Nicolson (1993) postulated that Cl- secretion by Malpighian tubules on the basal membrane is cotransported by Na+; application of furosemide and/or bumetanide inhibit fluid secretion in isolated tubules of different insects including Locusta (Baldrick et al., 1988). He suggested that although a substantial basal Cl- permeability is so far apparent only in Onymacris and Glossina, it is low for Locusta. He proposed that there is no apical pump or apical antiporter for Cl-, and the only apical ion channels so far identified in Malpighian tubules are Clchannels in Aedes (Wright and Beyenback, 1987). The electrical gradient favors passive exit of Cl- across the apical membrane in all species. Whether the main pathway for Cl- movement is transcellular or paracellular is not yet clear, however, Leyssens et al. (1992) suggested, on the basis of the very high apical membrane resistance in Formica, that much of the Cl- current may pass through a paracellular shunt rather than through apical channels. In Aedes the Cl- pathway appears to be either paracellular or via a second cell type (Pannabecker et al., 1993). Our observation that Br- could replace Cl- was

TABLE

1097

5. Effect

of amiloride

similar to that reported by Berridge (1969) with the Malpighian tubules of Calliphora. In contrast, substitution of Br- for Cl- increased diuresis by approx. 23% by Glossina morsitans adults with smaller hydrated radii of anions generally supporting a higher rate of diuresis (Peacock, 1986). Thus, not all insect species respond to this substitution in a similar manner. The 23% reduction in secretion in HCO;-free Ringer from our data agreed with the 22% inhibition in fluid absorption with rabbit proximal tubule when Cl- replaced HCO; in the perfused fluid.(McKinney and Burg, 1977). As a carbonic anhydrase inhibitor, acetazolamide might further reduce availability of HCO; to the Malpighian tubule. Thus, application of 1 mM acetazolamide in HCO;-free Ringer caused 55% inhibition of the rate of fluid secretion, a value similar to the sum of inhibitions we observed with each individual treatment. McKinney and Burg (1978) observed increased inhibition (30%) in fluid absorption by rabbit proximal tubules when 10m4M acetazolamide was included in HCO;-free Ringer. Our demonstration that fluid secretion by the Malpighian tubules of Locusta was inhibited by sodium acetazolamide was in agreement with the results of Gooding (1975) and Peacock (1986) who reported that acetazolamide inhibited diuresis in Glossina morsitans. Cooper et al. (1989), working on fluid and ion secretion in the Malpighian tubules of Cenocorixa blaisdeli, proposed that the decrease in fluid secretion following addition of acetazolamide must have resulted from a decrease in the rate of ion secretion and thus indicated an effect of acetazolamide on ion transporting mechan-

on the secretion of fluid L. migratoria Mean

Malpighian

rate of fluid secretion

Expressed

of

+ SE

in nl/min Rate 2

% Original rate

N

Control Experimental (0.1 mM amiloride present for Rate 2) (1 mM amiloride present for Rate 2)

14

1.80 & 0.28

1.71 If: 0.36

101.2 + 10.6

25

3.71 & 0.49

0.98 + 0.22

23.4 + 3.9


22

3.62 k 0.40

0.13 f 0.06

6.0 f 2.9


1 for details.

1

tubules

Treatment

See Table

Rate

by the

P >O.l

1098

HOSSEIN FATHPOUR

and DOUGLAS L. DAHLMAN

isms. It might be possible that HCO;-free Ringer alone and/or acetazolamide influence an apical proton pump by changing intracellular pH or rate of H+ production, thus reducing fluid secretion in the Malpighian tubules of Locusta. Acetazolamide inhibited the transepithelial potential across the rectum of Schistocerca gregaria (Herrera et al., 1977, 1978; Williams et al., 1978) and the transepithelial potential across the Malpighian tubules of Locusta (M. Fathpour, unpublished results). Burg and Green (1977) have shown that fluid absorption by rabbit renal proximal tubules is inhibited 2240% by acetazolamide and Cheung et al. (1977) found that acetazolamide caused a rapid and dramatic fall in the secretory rate to about 10% of the control in isolated seminiferous tubules of the rat. Acetazolamide also inhibits net secretion of HCO; in dog renal tubules (Mathisen et al., 1978). In contrast, Berridge (1968) and Maddrell (1969) reported that acetazolamide did not inhibit fluid secretion by the Malpighian tubules of Calliphora or Rhodnius.

We found the effect of SCN- on ‘urine’ production by tubules to be similar to that reported by other workers. Replacement of Cl- by SCNinhibited the rate of fluid secretion by 25% in Calliphora Malpighian tubules (Berridge, 1969) and gastric mucosal acid secretion was completely inhibited by 10 mM SCN(Sachs et al., 1972). In contrast, fluid secretion by the Malpighian tubules of the house fly, Musca domestica, was stimulated by NaSCN and increased the concentration of Na+ and Cl-, but not K+ , in the secreted fluid (Dalton and Windmill, 1980). Amiloride, which is thought to inhibit electrogenic entry of Na+ into the transporting cells (O’Donnell and Villereal, 1982; Fathpour et al., 1983; Palmer and Frindt, 1986; Kleyman and Cragoe, 1988; Hegarty et al., 1992) markedly inhibited (95% by 1 mM) ‘urine’ production by the Malpighian tubules of Locusta. Similar results have been reported with the Malpighian tubules of Glossina morsitans (Gee, 1976) and Aedes aegypti (Hegarty et al., 1992) as well as the salivary gland of Calliphora (Berridge et al., 1976). The sensitivity of Malpighian tubules to amiloride indicates that they require the basal cell membrane to be permeable to Na+ for fluid secretion (Gee, 1976). For example, amiloride did not effect fluid secretion by Culliphora salivary glands in high K+ Ringer solution (120 mM K+ ,55 mM Na+) whereas saliva production fell dramatically in ‘normal’ Ringer solution (20 mM K+, 155 mM Na+) (Berridge et al., 1976). They concluded that “the effect of amiloride is apparently specific for Na+ transport.” In the case of Locusta Malpighian tubules, where K+ is the ‘prime mover’ (Morgan and Mordue, 1981; Fogg et al., 1989; Baldrick et al., 1988), the action of amiloride might be explained on the basis of inhibition of entry of Na+ into the cell across the cell membrane. The resulting reduction in the intracellular Na+ level would be expected to affect normal functioning of the proposed Na+-K+ exchange ‘pump’. This would ultimately reduce the supply to the proposed electrogenic cation pump Locusta Malpighian

on the apical cell membrane. On the other hand, it might be possible that amiloride, an inhibitor of apical antiporters, causes acid secretion when H+/cation exchange by the antiporter does not keep pace with the proton pump (Nicolson, 1993). Thus, fluid secretion in the Malpighian tubules of insects is slowed or stopped (Bertram, 1989; Maddrell and O’Donnell, 1992).

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Baldrick P., Hyde D. and Anstee J. H. (1988) Microelectrode studies on Malpighian tubule cells of Locusta migratoriu: Effects of external ions and inhibitors. .I. Insect Physiol. 34, 963-975. Berridge M. J. (1968) Urine formation by the Malpighian tubules of Culliphoru. I. Cations. J. exp. Biol. 48, 159-174. Berridge M. J. (1969) Urine formation by the Malpighian tubules of Culliphoru. II. Anions. J. exp. Biol. SO, 15-28. Berridge M. J., Lindley B. D. and Prince W. T. (1976) Studies on the mechanism of fluid secretion by isolated salivary gland of Culliphora. J. exp. Biol. 64, 311-322. Bertram G. (1989) Fluid secretion of Malpighian tubules of Drosophila hydei affected by amiloride-is there a K+/H+ antiporter? Verh. dr. Zool. Ges. 82, 2033204.

Bradley T. J. (1985) The excretory system: structure and physiology. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Eds Kerkut G. A. and Gilbert L. I.), Vol. 4, pp. 421465. Pergamon Press, Oxford. Burg M. and Green N. (1977) Bicarbonate transport by isolated perfused rabbit proximal convoluted tubules. Am. J. Physiol. 223, F307-F314. Cheung Y. M., Hwang J. C. and Wong P. Y. D. (1977) In vitro measurement of rate of fluid secretion in rat isolated seminiferous tubules. Effects of metabolic inhibitors and ions. J. Physiol., Land. 269, l-15. Cooper P. D., Scudder G. G. E. and Quamme G. A. (1989) Segmental differences in secretion by the Malpighian tubules of the fresh water dwelling corixid, Cenoeorixu bluisdelh (Hung) (Corixidae, Hemiptera). J. Insect Physiol. 35, 531-536. Dalton T. and Windmill D. M. (1980) Fluid secretion by isolated Malpighian tubules of the housefly Muscu domestica. J. Insect Physiol. 26, 281-286.

Fathpour H., Anstee J. H. and Hyde D. (1983) Effect of Na+, K+, ouabain, amiloride and ethacrynic acid on the transepithelial potential across Malpighian tubules of Locustu. J. Insect Physiol. 29, 773-778.

Fogg K. E., Hyde D. and Anstee J. H. (1989) Microelectrode studies on Malpighian tubule cells of Locusru: Effect of cyclic AMP, IBMX and corpora cardiaca extract. J. Insect Physiof. 35, 387-392. Fogg K. E., Anstee J. H. and Hyde D. (1991) Studies on the subcellular distribution of (Na+ + K+)-ATPase, K+-stimulated ATPase and HCO;-stimulated ATPase activities in Malpighian tubules of Locusta migratoria L. Insect Biochem. 21, 749-758. Gee J. D. (1975) Diuresis in the tsetse fly Glossinu austeni. J. exp. Biol. 63, 381-390. Gee J. D. (1976) Fluid secretion by the Malpighian tubules of the tsetse fly Glossinu morsituns. The effects of ouabain, ethacrynic acid, and amiloride. .I. exp. Biol. 65, 323-332.

L. MIGRATORIA

FLUID SECRETION

Gilman A. G., Goodman L. S., Rail T. W. and Murad F. (Eds) (1985) The Pharmacological Basis of Therapeutics, 7th Edn. Macmillan, New York. Gooding R. H. (1975) Inhibition of diuresis in tsetse fly (Gfossina morsitans) by oubain and acetazolamide. Experientia 31, 938-939. Gupta B. L., Hall T. A., Maddrell S. H. P. and Moreton R. B. (1976) Distribution of ions in a fluid-transporting epithelium detected by electron probe X-ray. Nature Lond. 264, 284-287. Hegarty J. L., Zhang B., Carroll M. C., Cragoe E. J. Jr and Beyenbach K. W. (1992) Effects of amiloride on isolated Malpighian tubules of the yellow fever mosquito (Aedes aegypri). J. Insect Physiol. 38, 329-337.

Herrera L., Jordana R. and Ponz F. (1977) Effect of inhibitors on chloride-dependent transmural potential in the rectal wall of Schisiocerca gregaria. J. Insect Physiol. 23, 677682.

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Acknowledgements-H.

Fathpour expresses his gratitude to the authority of the University of Esfahan for financial support during his sabbatical leave. This is paper 93-07-62 of the Kentucky Agriculture Experiment Station, Lexington, KY 40546-0091.