Effect of Na+, K+, ouabain, amiloride and ethacrynic acid on the transepithelial potential across Malpighian tubules of Locusta

J. Insect Phl/stol.Vol. 29, No. 10. pp. 773-778, 1983 Printed in Great Britain

Copyright 0

0022-1910/83 $3.00 +O.OO 1983 Pergamon Press Ltd

EFFECT OF Na+, K +, OUABAIN, AMILORIDE AND ETHACRYNIC ACID ON THE TRANSEPITHELIAL POTENTIAL ACROSS MALPIGHIAN TUBULES OF LOCUSTA H. FATHPOUR, J. H. ANSTEE* and D. HYDE Department of Zoology, University of Durham, Science Laboratories, South Road, Durham, DHl 3LE, England (Received 21 February 1983; revised3 Muy 1983) Abstract-A transepithelial potential of +8.74 + 0.29 mV (n = 85) has been recorded across the Malpighian tubules of Locusta. The effect of varying the Na+ and K+ concentration in the bathing medium on the transepithelial potential has been determined. The data show that the transepithelial potential does not obey the Nemst equation for K+. Ouabain, ethacrynic acid and amiloride all inhibit the transepithelial potential. The results are discussed in relation to the nature of the mechanisms of cation transport across the Malpighian tubules. Key Word Index: Malpighian tubules, Locusta, inhibitors, transepithelial potential

INTRODUCTION potentials have been measured across the Malpighian tubules of a number of different species of insect (Ramsay, 1953 ; Coast, 1969; Irvine, 1969; Pilcher, 1970; Maddrell, 1971; Maddrell and Klunsuwan, 1973; Anstee et al., 1980). From such studies it appears that, in the majority of insects which have been examined, the lumen is positive with respect to the bathing medium. Ramsay (1953) showed that, in eight different species of insect, the transepithelial potentials across the tubules did not obey the Nemst equation for K+. On this basis, he concluded that in these insects K+ is actively secreted into the lumen, whereas transport of Na+ is apparently brought about by passive diffusion. In contrast, Pilcher (1970) and Maddrell (1971) working with Carausius and Rho&us respectively, reported that a lo-fold increase in K* in the bathing medium increased the transepithelial potential in a manner similar to that predicted by the Nemst equation. It is widely agreed that active K+ transport occurs in the majority of insect species which have been studied; notably Carausius, Calliphora, Rhorinius, Locusta and Schistocerca (Ramsay, 1953, 1955; Berridge, 1967, 1968; Berridge and Oschman, 1969; Pilcher, 1970; Maddrell, 1971, 1972, 1977; Maddrell and Klunsuwan, 1973; Anstee et al., 1979). On the basis of such studies, Maddrell(l971) has proposed a hypothetical model to explain the mechanism of Malpighian tubule function in Carausius and Calliphora. The main features of this mode1 are that active ion transport occurs both at the basal and apical cell membranes. It is suggested that K+ is actively transported into the cell by an electrogenic ‘pump’ which is stimulated by Na+ and is situated on the basal cell membrane, whereas Na+ and Cl- enter the cell passively. On the apical cell surface, Na+ and K+ Transepithelial

* To whom any correspondence

should

be addressed. 773

are transported into the lumen by electrogenic ‘pumps’, whilst the transport of Cl- is, once again, considered to be passive. More recently, Maddrell (1977) has suggested a model which attempts to explain how one basic mechanism can account for the secretion of Na + rather than K + in different species of insect. In this model it is suggested that an electrogenic cation pump is situated on the membrane facing the tubule lumen and that it has a higher affinity for Na+ than K+. This pump acts to maintain the intracellular level of Na+ ions lower than that of K+ and the actual rate at which cations are pumped across the tubules from the bathing medium into the lumen is dependent on both the affinity of the pump for the two cations and on how fast these ions enter the cell. It is proposed that K+, Na+ and Cl- enter the cells passively at a rate depending on the permeability of the membrane to these ions and on the electrochemical gradients across the membrane. Numerous investigators have studied the effects of different inhibitors on the transepithelial potential across a variety of insect epithelia in an attempt to establish the mechanism of ion and fluid transport. For example, the cardiac glycoside, ouabain, a specific inhibitor of Na+-K+ ATPase (Skou, 1969) has been shown to decrease the transepithelial potential across cockroach intestine (O’Riordan, 1969; Datta, 1971), the midgut of larval Sarcophaga bullata (Prusch, 1978) and the Malpighian tubules of Locusta (Anstee et al., 1980 ; see also review on ouabain-sensitivity of insect epithelia by Anstee and Bowler, 1979). In contrast, Pilcher (1970) failed to demonstrate an effect of ouabain on the potential difference across the Malpighian tubules of Carausius. Some researchers, having failed to obtain an effect with ouabain have used other inhibitors in an attempt to elucidate the nature of the ion transport mechanisms involved in insect epithelia. For example, Berridge et al. (1976) reported that CaNiphora salivary glands were insensitive to ouabain but that ethacrynic acid, which is a less specific inhibitor

174

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FATHPOURet al.

of membrane-bound ATPase, inhibited fluid secretion and abolished the positive phase of the transepithelial potential which has been attributed to pump activity. Similarly, these workers showed that the diuretic agent, amiloride, which is thought to inhibit passive movement of Na +, was capable of inhibiting fluid secretion and of causing the transepithelial potential of stimulated salivary glands to return towards the resting value. The present study has been carried out to determine the effect of varying the Na+ and K + composition of the bathing medium on the transepithelial potential across the Malpighian tubules of Locusta. In addition, the effect of ouabain, amiloride and ethacrynic acid on the transepithelial potential has been studied to provide further information concerning the likely nature of the ion transport processes.

MATERIALS AND METHODS

Mature adult locusts, Locusta migratoria L., were used and these were taken from a population maintained under crowded conditions at 28 kO.YC and 60% r.h. The transepithelial potential (PD) of actively secreting tubules was measured with KCl/agar bridges connected via 3 M KC1 to two Calomel half cells. A high input impedance amplifier with a gain of 10 x was used. The PD was measured by placing the recording electrode in contact with the lumen and the reference electrode in contact with the outside of the tubule (see Anstee et al., 1980). The amplifier output

was adjusted to zero with the KCl/agar bridges in the same Ringer pool. The PD was displayed and recorded on a flat-bed recorder (Servoscribe, Goerz Electra). initially, the PD was measured with the tubules bathed in ‘normal’ Ringer solution. Continuous recordings were taken for 15 min to ensure that the PD was stable. The ‘normal’ Ringer solution was then changed for a fresh solution of the same (control) or different (experimental) composition. The PD was then recorded continuously over the next 45 min. Control experiments were carried out in which the same procedure was followed except that ‘normal’ Ringer solution was used throughout. The temperature was maintained at 30°C at all times. The ‘normal’ Ringer solution had the following composition (mM): NaCl 100; KC1 8.6; CaCl, 2; MgClz 8.5; NaH*PO, 4; NaHCO, 4; glucose 34;

HEPES (N.2-hydroxyl ethyl piperazine N’.2-ethanesulphonic acid) 25 ; NaOH 11 (pH 7.2). However, an alternative Ringer solution was used in studying the effect of Na+ and K+ on the PD. The composition of this second Ringer solution was as follows (mM): NaCl 129; KC1 8.6; CaCl, 2; MgC& 8.5; NaHCO, 10.2 ; NaH2P0, 4.3 ; glucose 34. The pH was adjusted to 1.2. All solutions were made up in glass distilled, deionized water. All inorganic salts were Analar grade or the best commercially available. Ouabain was obtained from the Sigma Chemical Co. and amiloride and ethacrynic acid were a gift from Merck, Sharpe and Dohme Ltd. RESULTS

The e#ect of Na + and K+ on the transepithelialpotential

A series of experiments was carried out in which the PD across Malpighian tubules bathed in the ‘alternative’ Ringer solution were compared to those obtained with Ringer solution containing different concentrations of Na+ and K +. The bathing solutions were adjusted to maintain the total concentration of Na + and K + at 152 mM and only the relative concentrations of the two cations were altered. The results are shown in Table 1. In the absence of K+. the PD was rapidly reduced and the lumen became negative with respect to the bathing medium in less than 4 min. Increasing the concentration of K + in the bathing medium increased the PD such that in the presence of Na+-free Ringer solution (i.e. in the presence of 152 mM K ‘) the potential increased from + 11.1 mV in the ‘alternative’ Ringer solution to a new stable PD of + 35.6 mV. The relationship between the external K+ concentration and the increase in lumen positivity is non-linear. The effect ofouabain on the transepithelialpotential

When ‘normal’ Ringer solution surrounding the gut was replaced by ‘normal’ Ringer solution containing 1 mM ouabain, the PD decreased; the initial value of ~9.1 + 0.7 mV reaching a new stable potential of + 1.8 f 0.6 mV in 27.6 + 1.0 min. The results of these experiments are shown in Table 2. No significant change in PD was observed in control experiments. The eflect of amiloride on the transepithelial potential

The presence of 1 mM amiloride in ‘normal’ Ringer solution rapidly reduced the PD. The initial potential

Table 1. The effect of varying Na+ and K+ concentrations

in the bathing medium on the transepithelial the Malpighian tubules

Concentration (mM)

n

K+ 0 8.6 38 76 114 152

13 22 14 15 17 I5

Naf 152 143.4 114 76 38 0

Initial potential f SEM (mV)

Final potential ?SEM (mV)

+ 10.6 f 0.8 +10.1 _+1.2 +8.4 + 0.8 f11.3 + 1.0 +10.8 ? 1.1 +11.1 +0.9

-5.3 + 1.1 -+9.6 _+1.3 +23.1 + 1.4 -t 29.0 + 2.5 +35.1 +2.7 +35.6 k 2.4

potential of

Mean difference f SEM (mV)

Mean time in min to establish new stable PD

-16.0 + 1.2 -0.2 + 0.5 +14.7 + 1.3 +17.7 + 1.9 mt24.3 _+2.3 f24.5 + 1.9

3.4 + 0.4 4.2 f 0.2 2.1 f 1.6 1.4 *0.2 2.1 f0.3 1.8 +0.3

The transepithelial Table 2. The effect of ouabain,

Treatment Control 1 mM 1 mM 1 mM 1 mM 1 mM

Ouabain Amiloride Ouabain Amiloride Ethacrynic acid

?I

amiloride,

and ethacrynic

Initial potential f SEM (mV)

11 21 16

+7.6 f9.1 +9.7

f 0.7 kO.7 + 0.9

1+'2 12

to.5 +9.6

12

f9.2

P were obtained by comparing ouabain. the Ringer solution

potential

Malpighian

tubules

acid on the transepithelial

potential

Mean difference f SEM (mV)

Final potential + SEM (mV)

77s of the Malpighian

tubules

Mean time in min taken to establish new stable PD

P

+ 1.1 1.3

+7.4 +I.8 -4.5 +0.5 -4.6

+ 0.8 kO.6 +I.6 * 1.3 f 1.2

-0.5 -7.3 -13.8 -9.1 -5.1

* 0.4 kO.8 +I.4 k3.9 + 1.0

27.6 8.8 20.3 9.9

1.1 0.4 1.7 1.1

>O.l
k 0.7

-8.4

f 1.7

-17.6

f 1.8

26.3 + 1.6


the initial containing

The effect of ethacrynic acid on the transepitheliaf potential When 1 mM ethacrynic acid was included in ‘normal’ Ringer solution bathing the tubules, the PD decreased dramatically and the lumen became negative (-~ 8.4 t 1.7 mV) with respect to the bathing medium.

potential

Species Rho&k prolixus Rhoctrius proiixus Dixippus (Carausius) morosus Aedes aegypti Dyriscus marginalis Pieris brassicae Tenebrio molitor Locusra migratoria Locusta migratoria Loeusta migratoria Schistocerca gregaria Tipula paludosa Calpodes ethlius

f f + f

PD and final PD values by a ‘t’-test for paired data. *After treatment ouabain was replaced by Ringer solution containing amiloride.

of +9.7 f 0.9 mV dropped to a new stable value of -4.5 + 1.6 mV in less than 10 min (Table 2). In another series of experiments, the initial PD was determined in the presence of ‘normal’ Ringer solution. This was then replaced by ‘normal’ Ringer solution containing 1 mM ouabain and left until a new stable PD was established. The ouabain-Ringer solution was then replaced with ‘normal’ Ringer solution containing 1 mM amiloride and again time was allowed for a new stable PD to be established. The results are shown in Table 2. It can be seen that the reduced PD obtained following treatment with ouabain (+ 0.5 t 1.3 mV) was further reduced by the application of amiloride to -4.6 + 1.2 mV. However, the pretreatment with ouabain did not change the extent to which amiloride alone affected the PD. It is clear, therefore, that the inhibitory effect of amiloride is greater than that of ouabain and that both effects are not additive.

Table 3. Transepithelial

across

with

The difference between the initial and final values for PD was 17.6 mV (see Table 2), indicating a greater effect than that obtained with either ouabain or amiloride. The time taken for the establishment of a new stable potential was greater than for amiloride but similar to that taken with ouabain (Table 2).

DISCUSSION The transepithelial potential across the Malpighian tubules of Locusta, reported in the present study, was + 8.74 f 0.29 mV (n = 85) with the lumen being positive with respect to the bathing medium. This agrees with the value of + 10.8 mV reported previously (Anstee et al., 1980) and the value of + 16.6 mV reported for Malpighian tubules of Schistocerca (Maddrell and Klunsuwan, 1973). However, Ramsay (1953) recorded a PD of - 16 mV across the Malpighian tubules of Locusta in vivo. More recently, Morgan and Mordue (1981) reported that there was considerable variation in potential between different tubules (from + 10 to -44 mV) in their studies on Locusta, but that in general the lumen was negative relative to the bathing medium. However, more recently these workers have observed transepithelial

values reported

for Malpighian

Potential with respect to the haemolymph/bathing medium (mV) --35 -30 +21 +21 f22 f28 f45 -16 + 10.8 +8.7 +16.6 f32 +25

tubules

of different

species

Reference Ramsay, 1953 Maddrell, 1971 Ramsay, 1953 Ramsay, 1953 Ramsay, 1953 Ramsay, 1953 Ramsay, 1953 Ramsay, 1953 Anstee et al., 1980 present study Maddrell and Klunsuwan. Coast, 1969 Irvine, 1969

1973

H. FATHPOURet a/.

176

potentials of similar sign and size to those reported by Anstee et al. (1980) (Morgan and Mordue, personal communication). In Table 3 the transepithelial potential observed in the present study is compared with data from other studies involving a variety of different species. Figure 1 shows that for a IO-fold increase in K+ concentration in the bathing medium, the PD increased by only 19.2 mV and not the 60. I mV predicted by the Nernst equation. This agrees well with the previous studies of Ramsay (1953) and Anstee et al. (1980) which showed that the PD across the Malpighian tubules of Locustu was considerably more positive than would be predicted by the Nernst equation, for K+. One might conclude from this that the transepithelial potential is not the result of passive K+ permeability. In the present study, the PD was shown to increase in lumen positivity with increasing concentration of K + in the bathing medium. However, there was not a significant increase in PD associated with increasing the K+ concentration above 114mM. It may be that this can be explained in terms of saturation of the pump at high K+ levels or alternatively that Na+ concentration may have become rate limiting. These findings contrast with those reported for Caruusius (Pilcher. 1970) and Rhodnius (Maddrell, 1971) where the PD across the Malpighian tubules responded to a 1O-fold increase in K + concentration in the bathing medium in a manner similar to that predicted by the Nernst equation. Berridge et al. (1976) studied the effect of varying the external K+ concentration on the potential across the basal cell membrane of Calliphoru salivary glands. They showed that the slope.of the Nemst plot WaS 44 mV in the absence and 53 mV in the presence of 5-HT and concluded that the latter agent may increase K + permeability. The reduction in the transepithelial potential observed in the presence of I mM ouabain. taken in conjunction with the deviation from the Nemst equation discussed above, indicates that in Malpighian tubules of Locusta active ion transport is responsible

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Fig. 1. Effect of K+ concentration on the transepithelial potential of Malpighian tubules. The dotted line represents a 60.1 mV change in potential for a IO-fold change in external K+ concentration as predicted by the Nernst equation. Ordinate: Mean difference in the PD ( +SEM) (mV). Abscissa: log,, K+ concentration (mM) in bathing medium.

for the maintenance of the transepithelial potential and that a Na+-K+ ATPase is involved in cation secretion. This interpretation is further supported by the fact that fluid secretion by Malpighian tubules of Locusta is inhibited by ouabain (Anstee and Bell, 1975; Anstee et al., 1979; Donkin and Anstee, 1981), that the Na+-K+ ratio (Anstee et ul., 1979) or Na+ content (Mordue and Rafaeli-Bernstein, 1978) of the secreted fluid is affected by ouabain, and that a Na+K+ ATPase enzyme is present in this tissue (Anstee and Bell, 1975 ; Donkin and Anstee, 1981). Ouabain has been shown to decrease the transepithelial potential in other tissues. O’Riordan (1969) and Datta (1971) working on cockroach intestine observed a 50-750/i decrease in the transepithelial potential when 10 -4 M ouabain was applied on the haemolymph side. Wollberg and Cocos (1981) have shown that 5 10m4 M ouabain caused a depolarization of 4.2 t 3.5 mV in the transwall potential of developing oijcytes in Locusta. Berridge and Schlue (1978) reported that 10m4 M ouabain inhibited the basal membrane potential and decreased the intracellular K+ level in unstimulated salivary glands of Calliphora. However, on the addition of 1Oms M 5-HT there was a rapid restoration of both potential and K+ level to a value exceeding that seen in the resting glands. They suggested that, in unstimulated glands, ouabain might Frevent the reaccumulation of K + by depleted glands and that a Na+-K+ exchange pump was responsible for maintaining high intracellular K + levels, whereas a potential-dependent mechanism for K+ entry takes over when large amounts of this cation are required to generate the increased flow of saliva during stimulation by 5-HT. This effect of 5-HT is of particular interest because it may well offer an explanation for the lack of response to ouabain reported for the Malpighian tubules of certain species where a stimulant is included in the bathing medium (see review by Anstee and Bowler. 1979). Tolman and Steele (1980) have shown that extracts prepared from the corpora cardiaca and corpora allata together, partially overcame the inhibitory effect of ouabain on oxygen consumption by cockroach rectum. However, Pilcher (1970) reported that the transepithelial potential across the Malpighian tubules of Curuusius was unaffected by 10e4 M ouabain in the absence of any stimulant such as diuretic hormone or 5-HT. Ethacrynic acid is a diuretic agent which is known to inhibit Na+-K+ ATPase from a variety of tissues (Duggon and Nell, 1965 ; Chamock et a/., 1970; Davis, 1970) including insect excretory systems (Peacock et al.. 1976). This drug blocks phosphorylation of the enzyme, prevents the ADP-ATP exchange reaction and stabilizes the spontaneous disappearance of the phosphorylated intermediate (Schwartz et al., 1975). On this basis, the decrease in the transepithelial potential observed in the presence of 1 mM ethacrynic acid. in the present study, might be taken as further support for Na+-K+ ATPase involvement in Malpighian tubule transport processes. However, the effect of ethacrynic acid appears to be non-specific and it has been shown to affect a variety of cellular processes (Perez-Gonzalez De La Manna et al., 1980). It may be that its effect on the transepithelial potential across the Malpighian tubules of Locusta is due to inhibition of ATP production (both mitochondrial

The transepithelial

potential

and glycolytic) which would affect all energy-requiring processes including the activity of ATPase enzymes. Alternatively, it may be due to direct inhibition of Na+-K+ ATPase itself or a combination of effects. The latter may well explain why ethacrynic acid caused a more dramatic change in the transepithelial potential than either ouabain or amiloride. The inclusion of 1 mM amiloride in the bathing medium caused a fall in the transepithelial potential across the Malpighian tubules of Locusta. Indeed, amiloride effected a greater change in the PD than ouabain and the fact that the action of these two inhibitors wasnot additive (pre-incubation in ouabainRinger solution did not increase the effect of amiloride) suggests that they do not act on different systems. Amiloride is thought to inhibit Na+ transport by blocking the entry of Na+ into the transporting cells (Gee, 1976; O’Donnell and Villereal, 1982) and has been shown to inhibit fluid secretion by the Malpighian tubules of Gloss&~ morsizans (Gee, 1976) and Locusta migrutoriu (Fathpour and Anstee, unpublished), and by the salivary glands of Calliphora (Berridge et al., 1976). This sensitivity of the Malpighian tubules of Locusta to amiloride indicates that they require the basal cell membrane to be permeable to Na+ for cation and fluid secretion. This is supported by the studies of Berridge et al. (1976) which showed that amiloride did not affect fluid secretion by salivary glands of Cdiphora in high K+ Ringer solution (120 mM K+. 55 mM Na+) whereas in normal Ringer solution (20mM K+, 155 mM Na +) the saliva production fell dramatically. They concluded that the effect of amiloride was apparently specific for Na+ transport. Similarly, in toad bladder (Bentley, 1968; Sodou and Hoshi. 1977; Canessa rt al.. 1978) it has been shown that amiloride restricts the entry of Na+ into the cell across the mucosal membrane and thereby reduces the intracellular pool of Na+ available to the sodium pump on the opposite cell membrane. In the case of Locustu Malpighian tubules, where K+ is thought to be the ‘prime mover’ in fluid secretion, the action of amiloride may be explained on the basis of inhibition of entry of Na + into the cell across the basal cell membrane. The resulting reduction in the intracellular Na+ level would be expected to affect the normal functioning of the Na +-K + ATPase pump and thereby reduce the transport of K+ across the tubule wall. In most vertebrate systems the available evidence suggests that ouabain-sensitive Na +-K + ATPase is confined to the basolateral membranes, although in many cases its presence on the apical or luminal membrane has not been rigorously excluded. Recent studies by Gupta and Hall (1983) suggest that a NatK + ATPase may be present on both the apical and basal membranes of cockroach salivary gland cells. The exact location of Na +-K + ATPase in Malpighian tubules of Locusta remains to be determined. REFERENCES

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