Relationship of Na+K+-activated ATPase to fluid production by malpighian tubules of Locusta migratoria

J. Insect Physiol., 1975, Vol. 21, pp. 1779 to 1784. Pe*gnmon Press. Printed in Great Britain.

RELATIONSHIP OF Na+-K+-ACTIVATED FLUID PRODUCTION BY MALPIGHIAN

LOCUSTA

ATPASE TO TUBULES OF

MIGRATORIA

J. H. ANSTIZE and D. M. BELL Department of Zoology, University of Durham, Durham, England (Received 22 March 1975) Abstract-The presence of a Na+-K+-activated, Mg *+-dependent ATPase (E.C. 3.6.1.3) has been demonstrated in microsomal preparations from the Malpighian tubules of Locusta. The effects of sodium and potassium ions, and different concentrations of ouabain, have been studied in relation to the activity of this enzyme and the ability of in vitro Malpighian tubule preparations to secrete fluid. From these studies it seems highly likely that a Na+-K+ activated ATF’ase ‘pump’ is involved in fluid transport across the walls of the tubules.

to what extent such an enzyme can be related to fluid secretion by these tubules.

INTRODUCTION THE SECRETION of urine by the Malpighian

tubules of insects has been the subject of numerous studies in the past (BAMSAY, 1953, 1954, 1955, 1956;

MATERIALS

MADDRELL, 1969; PILCHER, 1970). As a result of these and other studies (see review by MADDRELL, 1971) a model has been proposed to explain fluid transport across the walls of the tubules. The main features of this model are that active transport of ions takes place at the basal and apical surfaces of the cells and that water movements are osmotically linked to ion movements. With the exception of Rhodnius tubules (MADDRELL,1969), which function in its absence, potassium is generally considered to be the ‘prime mover’ (BERRIDGE,1968). BERRIDGE and OSCHMAN(1969) have suggested that the apical surface of the cells possesses an electrogenic pump whereas the basal cell membrane possesses a coupled sodium-potassium pump. One of the main objections to the latter has arisen from the failure to show that tubule secretion is inhibited by the cardiac glycoside, ouabain, which is a specific inhibitor of the Na+-K+-activated, Mgr+-dependent ATPase (E.C. 3.6.1.3) (SKOU, 1969). In addition, at this time, the only demonstration of a membrane ATPase in insect Malpighian tubules was confined to the electron histochemical localization of a Mgs+ ATPase in both the basal and apical membranes (SMITH, 1968). More recently, however, PEACOCK et al. (1972) have demonstrated, by biochemical methods, the presence of a Na+-K+-activated, Mgr+-dependent ATFase in microsomal preparations from the hindgut and Malpighian tubules of Schistocerca gregaria F6rsk and the tettigoniid, Jamaicana fiva. The present investigation has been carried out to determine whether such a Na+-K+-activated ATFase occurs in the Malpighian tubules of Locusta migratoria and

AND h%JXI-IODS

Mature adult locusts, Locusta migratoria, were employed and these were taken from a population maintained under crowded condiditions at 28 f O*S”C and 607/o r.h. Preparation

of membrane microsomal fraction

Twelve animals were killed by decapitation and and their Malpighian tubules quickly dissected out and placed in 10 ml of an ice-cold homogenization medium consisting of 250 mM mannitol, 5mM EDTA, O*l”h sodium deoxycholate in 30 mM histidine-HCl, pH 7.2. Homogenization was carried out in a Potter-Elvehjem homogenizer with a Teflon pestle (clearance O-1 to 0.15 mm) with 20 passes of the plunger at 1000 rev/min; the homogenizer was surrounded by ice throughout this procedure. The resulting homogenate was then extracted with sodium iodide as described by NAKAO et al. (1965). This extract was then centrifuged at 50,000 g for 30 mm at 0°C using an MSE Automatic Superspeed 40, head No. 2409. The pellet was discarded and the supematant centrifuged at 100,000 g for 60 mm. The resulting pellet was resuspended in approximately 10 ml of washing medium (5 mM sodium choride and 5 mM EDTA, pH 7.2) and recentrifuged at 100,000 g for 60 min. This washing procedure was repeated twice more. The resulting microsomal pellet was suspended in deionized water. ATPase

activity

Appropriate ionic media (l-5 ml) were thermoequilibrated at 30°C for 15 min in boiling tubes

1779

1780

J. H. ANSTEEANDD. M. BELL

and the reaction started by adding 0.5 ml of the microsomal suspension. The reaction was stopped by adding 4 ml of a 1 : 1 mixture of 1 y0 Lubrol and 1 o/o ammonium molybdate in 1.8 N sulphuric acid (ATKINSONet al., 1973). Enzyme activity was measured by determining the amount of inorganic phosphate released.

The d&rmination fates

of Malpighian

tubule

secretion

In oitro measurements of fluid secretion by the Malpighian tubules were carried out using essentially the same technique as that described by MADDRELL and KIUNSUWAN(1973). The major modification introduced was that the tubules were not completely severed at their point of entry into the alimentary Analysis of inorganic phosphate canal. Instead, each tubule was drawn out of the Following centrifugation to remove any protein Ringer bath into the liquid paraffin and looped which precipitated, tubes were allowed to stand at around a small peg. The tubule was then partially room temperature for exactly 10 min. The intensity severed at a convenient point along its length and of the yellow colour which developed during this the rate of secretion determined by measuring the time was read at 390 nm and is proportional to the rate of increase in the diameter of the approxiamount of inorganic phosphate present (ATKINSON mately spherical droplet secreted from the cut. et al., 1973). The secretion rate for each tubule was determined by measuring the diameter of the secreted Reaction media droplet at 5 min intervals over a period of 40 min. At the end of this time, the Ringer solution (solution Five reaction media were used: (1) 4 mM 1, Table 1) was replaced by a fresh solution which magnesium chloride; (2) 4 mM magnesium chloride had either the same or a different composition. The and 100 mM sodium chloride; (3) 4 mM magnesium rate of secretion was re-determined, after an chloride and 20 mM potassium chloride; (4) 4 mM equilibration period of 20 min for a further 40 min. magnesium chloride, 100 mM sodium chloride, and The temperature throughout was maintained at 20 mM potassium chloride ; (5) 4 mM magnesium 30°C. Table 1 shows the composition of the various chloride, 100 mM sodium chloride, 20 mM potasexperimental solutions employed. sium chloride, and ouabain (concentrations from lo-’ M to lo-* M). Each medium contained 3 mM Reagan ts ATP (Tris salt) and was made up in 50 mM histidineHC1, pH 7.2. All solutions were made up in glass-distilled, deionized water. All inorganic salts were AnalaR Protein determinations grade; hi&line, ATP, and ouabain were obtained These were made by the method of LOWRY et al. from Sigma Chemical Co. ; ATP Tris salt was made (1951), using bovine serum albumen Fraction V from the sodium salt by ion exchange. Lubrol was (Sigma Chemical Co.) as standard. a gift from I.C.I. Dyestuffs Division. Table 1. The composition of the experimental solutions (concentrations 1

2

3

129.0

129.0

137.6

KC1

0.6

8.6

W12

a.5

0.5

N&l

in mM/l.)

4

5

137.6

8.6

0.5

8.5

8.5

2.0

2.0

KEG3

10.2

10.2

TP04

4.3

4.3

CE12 l-3 Nai12P04

2.0

2.0

2.0

10.2

10.2

10.2

4.3

4.3

4.3

CholineCl Glucose Ouabaia

129.0 34.0

34.0 10 . to 10-4

All solutionsveta adjustedto pli 7.2.

34.0

34.0

34.0

Relationship of ATPsse to fluid production by Malpighian tubules of Locusta

1781

Table 2. ATPase activity of rnicrosomal preparations from Malpighian tubules Coepoaition of reaction

EnzyPlcactivity in rimlessPi

media

liberated/mg protcin/mia

29.3

4&l MS'+ a)

4mM MS': 100 r&fNa+

a)

4mM MS': 20 IGUK' 4mM Mg*:

100

~54 No’,

e

31.7

b)

25.i

46.1

b)

31.8

229.8 e

20 ~$4K+

4uM MST+100 &l Na' 20 mM K: 1 IPM ouabrin

4.9)

27.3)

54.5 (,+5.9)

2+

Activity in the presence of Mg , Na+ K+ , minus activity in the presence 0f'ng2+

205.3 (2 27.2)

Na: K+and 1 rd4ouabain. 2+ Activity in the presence of Mg , Na: K+ minus activity in the presence of

200.5 c+ 26.6)

MS2+alone.

ATPase activity is expressed as the mean of five separate determinations (+ S.E. of the mean). The exceptions ( a) and b) above) refer to the individual data obtained for each of two separate determinations.

RESULTS

A TPase actiuity The data obtained with the various reaction media are shown in Table 2. From these it is clear that the microsomal preparations from the Malpighian tubules of L. migratoria are able to hydrolyse ATP. Two distinct components of ATPase activity were determined, viz. that activated by Mg*+ ions alone (the Mgs+-dependent ATPase) and that activated by Naf and K+ ions in addition to Mg*+ (total ATPase). The difference represents the Na+-K+ ATPase and it is this component which is inhibited by the cardiac glycoside, ouabain, and synergistically stimulated by Na+ and K+ ions. The inhibition curve for ouabain (Fig. 1) shows that as the concentration of duabain increases from 10-s to 10e4 M so does the inhibition of the Na+K+ ATPase, At ouabain concentrations of lo-‘ M or higher, inhibition was found to be more or less complete. The negative logarithm of the ouabain concentration causing 50 per cent inhibition of the Na+-Kf ATPase (PI,,) was 6.1. This value compares favourably with the value of 6.8 obtained with the rectal glands of the spiny dogfish (BONTING, 1966) but is somewhat higher than those obtained with certain other tissues, e.g. 3.9 with rat liver (BAKXERENand BONTING,1968) and 5-4 with rabbit pancreas (RIDDERSTRAPand BONTING, 1969). It would seem, therefore, that compared to the latter two tissues the Na+-K* ATPase in Locusta Malpighian tubules is highly sensitive to ouabain.

Fig. 1. Dose-response curve of the inhibition of the Na+-K+-activated ATPase by ouabain. The vertical lines represent + 2 S.E. of the mean.

The effect of alterations in Na and K concentrations on fluid secretion by Malpighian tubules In view of the fact that Na+ and K+ ions are necessary for the Na+-K+ ATPase enzyme to function, it was to be expected that, if thia enzyme was involved in Auid secretion by Locusta tubules, both ions would also be required for normal fluid production to occur. To test this, the effects of the presence or absence of these two cations on fluid

J.H.

1782

D.M. BELL

~STEAND

Table 3. The effect of sodium and potassium, and different concentrations of ouabain on the secretion of fluid by the Malpighian tubules No.

Treaizcent

of

Maan.rate of secretion

tubules

(X original tate + S.E.)

Control (solution 1, Table 1) K

12

88.0 c+ 7.2)

12

12.0 c+ 6.8)

16’

57.5 (2 14.3)

18

28.9 (2

free

(solution 3. Table 1) Na free, high K

(solution 4, Table 1) Na free,

chbline Cl

(solurion 5, Table 1)

6.2)

Solution 2, Table 1 (a) 10e7Mouabain

16

77.6 c+ 12.7)

(b) 10%

ouabain

17

31.1 (2

7.0)

(c) 10m5Mouabain

16

29.7 (2

8.8)

ouabain

17

38.3 (2 10.8)

(e) lo-Sr ouabain

11

Cd) 10%

secretion by the tubules was studied. The results are summarized in Table 3. When tubules were placed in potassium-free Ringer, the rate of secretion diminished to 12.0 per cent of normal within 20 min and soon afterwards stopped altogether. Subsequent attempts to re-start secretion by introducing K+ ions failed. In sodiumfree, high-potassium Ringer, fluid secretion, whilst reduced, still occurred at a fairly substantial rate. In this case, however, the effect was rather more variable than with the other solutions used. By contrast, when sodium was replaced by choline chloride there was a marked decrease in the rate of fluid secretion to 28.9 per cent of the original rate, as measured in normal Ringer. Whilst the possibility remains that both choline chloride and high levels of potassium adversely affect fluid secretion per se, it does seem clear that both sodium and potassium ions are necessary for the normal functioning of the tubules.

6.7 e

4.9)

BOUMENDIL-PODEVIN, 1972) the critical test for its involvement here was to determine whether fluid secretion by the tubules was ouabain sensitive. Fig. 2 shows some representative examples of the results obtained with measurements of secretion by

60

1

Control

1 ‘“-‘M 1 l”-‘Mi

The effect of ouubain on fluid secretion by the Ma&high&-m tubules As was demonstrated above, and elsewhere on numerous occasions (SKOU, 1965 ; BOWLER and DUNCAN, 1968), the cardiac glycoside ouabain inhibits the Na+-K+ ATPase. Since this enzyme has been implicated in ion and water transport across various epithelia in a variety of different species (SKOU, 1969; PROVERBIO et al., 1970; WHIT-TAM and WHEELW, 1970; PODEVIN and

I

’

0

’

’ ’ ’ 20 40

1’

0

1.1

’ ’ ’ a0 40 TIME IMinsl

’ 0

’

’

10

’

L 40

Fig. 2. The effect of various concentrations of ouabain on fluid production by representative individual tubules over two 40 min periods. Fluid production was initially determined in normal Ringer solution (0) and subsequently in either fresh Ringer solution in the case of the control (m) or in fresh Ringer solution containing the indicated concentration of ouabain ((I).

Relationship of ATPase to fluid production by Malpigbisn tubules of Locusta tubules over two 40 min periods (the fust in normal and the second in one of the experimental solutions 2, Table 1). From these and the data contained in Table 3 it is clear that ouabain inhibits fluid secretion by the Malpighian tubules of Locustu. Comparisons between the rates observed in the first and second 40 min periods, by means of a paired t-test, confirm the view that within the range lops to low6 M, ouabain inhibition is significant (P< 0.05). The slight reduction in the rate of secretion apparent with control tubules and those subjected to a ouabain concentration of lo-’ M was not significant. Thus it would seem that the threshold concentration for the response lies between 10m6 and lo-’ M which is comparable to the concentrations effective in the inhibition of other fluid transporting tissues (SKOU, 1960; GLYNN, 1964; FARQUARSON, 1974). DISCUSSION Cardiac glycosides, such as ouabain, have been known for some time to inhibit ion transport across cell membranes and more recently their specific effect on the Naf-K+ ATPase has been demonstrated (NAKAO, 1974). In the present study, ouabain has been shown to inhibit a microsomal Na+-K+ ATPase preparation from the Malpighian tubules of Locustu; pIso = 6.1. Ouabain has also been shown to inhibit fluid secretion by the isolated tubule preparations and in this the tubules of Locusta differ from other insects which have been studied. BERRIDGE(1968), MADDRELL(1969), and PILCHER (1970) have failed to show an inhibitory effect of ouabain on fluid production by Malpighian tubules from Calliphora, Rhodnius, and Carausius at concentrations of 10ea and 2.5 x lo-* M. More recently, FARQUARSON (1974) has shown that fluid secreted by the Malphighian tubules of the pill millipede, Glomtis marginata, is sensitive to ouabain at concentrations as low as 5 x 10m6 M. These observations are far more in line with the present ones for Locusta. Here, there is a significant reduction in the rate of fluid secretion at 10d6 M ouabain but not at a concentration of lo-’ M; so as with Glomeris, the threshold concentration for Locusta tubules lies between these two concentrations. The only previous support for an effect of ouabain on insect Malpighian tubule function comes from the recent work of ATZBACHER et al. (1974) who have shown that the rate of excretion of the two dyes azocarmine and indigocarmine is significantly diminished by this drug in Drosophila hydei. In the present study, it was noticeable that whereas some 14 per cent reduction in the Na+-Kf ATPase was observed at a ouabain concentration of lo-’ M this did not result in a significant decrease in the rate of fluid secretion. From this one might conclude that a somewhat greater reduction of the pump is necessary before fluid secretion is significantly

1783

There is, however, an alternative impaired. explanation, viz. that at the lower concentrations of ouabain (cu. lo-’ M) insufficient glycoside is reaching the site of the Na+-K+ ATPase in the membranes. It is, indeed, possible that the failure of other workers to demonstrate that tubule secretion is ouabain sensitive arises from difficulties in ensuring the ouabain is getting to the site of the ATPase ‘pump’. The main r6le of the Na+-K+-activated ATPase is active exchange transport; for example, in red blood cells, three sodium ions from the inside to outside and two potassium ions from outaide to inside move with the hydrolysis of one ATP (POST and JOLLY, 1957). This being the case, the observations that both sodium and potassium ions am necessary for normal fluid production by the tubules, that a Na+-K+ activated ATPase is present, and that fluid secretion is ouabain sensitive, argue strongly for the physiological r6le of the enzyme in active cation and water transport across the Malpighian tubules of Locusta. REFBRBNCBS ATKINSONA., GATENBY A. D., and Lowe A. G. (1973) The determination of inorganic orthophosphate in biological systems. Biocha’m. biophys. Acta 320,

195-204.

ATZEZACHKR U., HE~ERT F., WEBER-VON GROTTHIJSSE., and W~~~INC A. (1974) The in8uence of ouabain on the elimination of injected and orally applied dyes in

Drosophila hydei. J. Insect Physiol. 20, 1989-1997. BJWCEIENJ. A. J. M. and BONTINGS. L. (1968) Studies on Na-K activated ATPase-XX. Properties of the Na-K activated ATPase in rat liver. Biochim. biophys. Acta 150,460-466. BERRIDGE M. J. (1968) Urine formation by the Malpighian tubules of Calliphora-I. Cations. J. exp. Biol. 48, 159-174. BERRIDGEM. J. and OSCHMAN J. L. (1969) A structural basis for fluid secretion by Malpighian tubules.

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Biochem. Physiol. 24, 1043-1054. FARQUAR~ONP. A. (1974) A study of the Malpighian tubules of the pill millipede, Glomeris margkata (Villers+II. The effect of variations in osmotic pressure and sodium and potassium concentrations on fluid production. J. exp. Biol. 60, 29-39. GLENN I. M. (1964) The action of cardiac glycosides. Pharmac. Rev. 16, 381-407. LOWRY 0. H., ROS~BROUGHN. J., FARR A. L., and RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MADDRELLS. H. P. (1969) Secretion by the Malpighian tubules of Rhodmks. The movements of ions and water. J. exp. Biol. 51, 71-97.

1784

J. H. hSTL03 AND D. M. BELL

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282, 234-249. POST R. L. and JOLLY P. C. (1957) The linkage of sodium, potassium and ammonium active transport across the human erythrocyte membrane. Biochim. biophys. Acta 25, 118-128. PRO~ERBIO F., ROBINSON J. W. L., and WHITIXMBURY G. (1970) Sensitivities of the (Na+-K+)-ATPase and Na+ extrusion mechanisms to ouabain and ethacrynic acid in the cortex of the guinea-pig kidney. B&him.

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ATPase in the of ions. In The Molecular Basis of Membrane Function (Ed. by TOSTESON D. C.), pp. 455-482. Prentice-Hall, New Jersey. SMITH D. S. (1968) Insect Cells-their structure and function, pp. 285-297. Oliver & Boyd, Edinburgh. WHITTAM R. and WHEBLER K. P. (1970) Transport across cell membranes. A. Rev. Physiol. 32, 21-60. active transport