On the mode of action of ethacrynic acid, using the barnacle muscle fiber as a model

Lüe Sciences Yol. 11, Part Printed in Great Britain

Pergamon Press

I, pp . 19-21, 1972 .

ON THE MODE OF ACTION OF ETHACRYNIC ACID, USING THE BARNACLE MUSCLE FIBER AS A MODEL Bo G. Danielson ; E . Edward Bittar, Stephen S. Chen and Eànund Y. Tong** Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706, U.S .A .

(Received il October

1971 ;

in final form

22

November

1971)

Sua~aar~ External application of 10~ M ethacrynic acid at pH 7.8 is usually without effect on the Na efflux into a 10 nt~l HCO3 - solution . At an external pH of 6.8, however, 10-3 M ethacrynic acid causes a gradual rise in the loss of Na into a solution the initial HCp~ concentration of which is 5 nM . Lowering the external pH of this solution to 5.8 results in reversal of the slow rise in Na loss . External application of 10-~ M ethacrynic at pH 6 .9 and pH 6 .3 is unaccaapanied by a rise in the Na efflux into a Cat+-free solution the initial HC0~ concentration of which is 5 sM . External application of T0- M ethacrynic acid following stimulation of the Na efflux into a Cat+-free solution by reducing the external pH fros 7 .8 to 6.3 results in a large fall in the rate of loss of Na . These results indicate that the COp-sensitive Na efflux is the principal site at which ethacrynic aciâ acts . Little is known of the mechanism whereby ethacrynic acid acts as a potent diuretic agent.

One indirect but experimental approach which A1ght shad sage

light on the question whether ethacrynic acid is a genuine inhibitor of Na reabsorption in the nephron would be to esploy a variety of single cells as nodels .

For example, it has been found feasible to carry out experiments on

single giant muscle fibers fron the crab Haia equdnado and frao the barnacle

azZmsr4a nubitua or B. aquila .

As already shown by usl extrene sensitivity to a

raised pC02 is a characteristic property of barnacle muscle fibers, the

* Visiting Scientist of the A+aerican Heart Association. Permanent address: Institute of Physiology and Medical Biophysics, University of Uppsala, Uppsala, Sweden . **Postdoctoral

Fellow of the Wisconsin Heart Association. 19

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Mode of Action of Ethacrynic Acid

Vol. 11, No . 1

threshold C02 tension for stimulation of the Na efflux being approximately 40 mm Hg .

Knowing that the ascending limb of Henle's loop is a region in which

the pC02 is high, 2 as well as the prime target of ethacrynic acid, 3 1t seemed a matter of obvious interest to discover if ethacrynic acid is able to inactivate the CO2-sensitive component of the Na efflux in barnacle muscle fibers .

The

purpose of this communication is to report the results obtained with ethacrynic acid and to particularly show that the main point of action of this diuretic agent is the CO2-sensitive component of the Na efflux . Methods Single muscle fibers measuring roughly 5 cm in length and 1 .5 mm in width were dissected from specimens of Batanua nubiZue and B. aqui.ta, cannulated in the same way as squid axons or crab muscle fibers, and then loaded with 2 2 Na by microinjection .

The microinjector was basically the same as that used by

Hodgkin and Keynes 4 as modified by Caldwell and Walster . 5

The microlnjector

discharged ca . 0 .1 ul of fluid per 1 cm excursion of the micromanipulator .

The

artificial sea water used as the bathing medium had the same composition as that reported by

Bittar and Tong . 6

Sharp and Doime, Rahway, N .J .

Ethacrynic acid was a gift from Merck

.The 22 NaC1 in aqueous solution supplied by

Amersham-Searle Corp, (SKS .1) was evaporated and then made up in distilled water so that volumes of about 0 :1 u1 gave more

than 50,000 cpm .

Counting of

22ya activity in the wash-out samples and the fiber was done according to the methods described by Bittar g and ßittar, Caldwell and Lowe .B

The well-type

counter with a solid phosphor was a Panax model CSNP ; this was connected to a Nuclear-Chicago Analyzer .

All experiments were done between 20 and 23°C . Results

The first group of experiments (63 fibers) was designed to see whether ethacrynic acid at pH 7 .8 affects the Na efflux .

The results of these trials

indicated that only two of the fibers tested were sensitive .

A typical

experiment recorded in Fig . 1 shows that external application of 10- 4 M

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Mode of Action od Ethacrgnic Acid

15

+oo

200

X

100

W

â

I

20

I

I

40

80

I

80

nme cMiN~

1

1

100

120

FIG. 1 Lack of effect of 10~ M ethacrynic acid at pH 7.8 on the Na efflux from a barnacle muscle fiber, plotted semilogarithnically. ethacrynic acid at pH 7.8 failed to modify the Na efflux .

Since the pK value

for ethacrynlc acid 1s 3 .5, the next step was to lower the external presence of ethacrynic acid .

pH in the

This was done after halving the HCO3'

concentration of the bathing solution, the threshold pH for stimulatiog the Na efflux in this case being 6.4 and not 6.8 (as wlth a 10 mM NC03 - solution) . Fit's. 2a and b clearly show that external application of 10'3 M ethacrynic acid at pH 6.8 resulted 1n a gradual rise in the loss of Na .

When, however, the

external pH was further reduced to 5.8, there was a marked reversal of this effect, suggesting

that an increase in the- undissociated fraction of the

ethacrynic acid led to abolition of the phase of stimulation .

Importantly,

fibers poisoned with ethacrynic acid at pH 6.8 were found to shorten.

This

however was not true of unpoisoned fibers when the external pH was lowered from 6.8 to 5.8, as illustrated by Fig. 3 (10 fibers) .

From these results there

emerged the possibility that stimulation of the Na efflux by ethacrynlc acid was associated with a raised internal

free Caz+, resulting from an Increase in

18

Mode od Action ad >sthacrynic Acid

I

800

u

Vol . il, No . 1

10-3 N EA

400

X W W W

N

200

~1 0

5mM HC03

I 20

1 40

1 60

1 80

I 100

1 120

1 140

I 160

TIME (MIN)

FIG . 2a The effect of 10' 3 M ethacrynic acid at pH 6 .8 and 5 .8 on the Na effl ux into artificial sea water initially containlng 5 mM HC03" .

pH 6.8

pH 5 .B

Ib O 2 N N 5mM HCO~

QI O

I 20

I 40

I 60

I I 80 100 TIMé (MINI

I 120

I NO

I 160

FIG . 2b The rate constant curve for Na efflux, as recorded in this experiment .

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Mode of Action od Ethacrynic Acid

n H s.e

17

s.e

nH

z

a_U X W W W O z

a a

FIG . 3 Stimulation of the Na effl ux into artificial sea water initially containing 5 mM HC03 - by lowering the external pH from 6 .8 to 5 .8 . Cat+ influx .

This led to additional experiments involving the use of a Cat +-

free bathing

median .

Preliminary control experiments revealed that Cat+

removal in itself does not significantly alter the Na efflux (Bitter, Chen, Tong and Danielson, unpublished results) .

They also revealed that the response

of fibers to external acidification is unaffected by the absence of external Cat+, as illustrated 1n Fig . 4 . The importance of omitting Cat+ from the bathing solution is strikingly brought out in Fig . 5, which shows that ethacrynic acid in as low a concentration as 10-5 M virtually halted the Na efflux from responding to external acidification (16 fibers) .

Two points should be noted :

first that this fiber

was isolated from the same muscle bundle as that used 1n the experiment recorded in Fig . 4, and second that these fibers did not contract when treated with ethacrynic acid .

It thus became clear from both types of experiments that

18

Mode ad Action of Ethacrynic Acid

pH 8 .9

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pH G3

FIG . 4 Stimulation of the Na efflux into a Caz+-free artificial sea water solution initially containing 5 mM HC03- by lowering the external pH from 6 .9 to 6,3 .

eoo a

W e Z N N

0 CO

200

zo

4o

so so TIIiAE (MIN)

ioo

izo

FIG . 5 Abolition of the sensitivity of the Na efflux into a Cat+-free solution to external acidification by applying 10- 5 M ethacrynlc acid at pH 6 .3 .

Mode of Action of Ethacrynic Acid

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19

ethacrynic acid is a powerful inhibitor of the Na efflux into a Cat+-free solution .

Evidence confirming this conclusion was provided by experiments in

which 10 -3 M ethacrynic acid was externally applied following stimulation of the tJa efflux into a Cat+-free solution by reducing the pH from 7 .8 to 6 .3 .

As

illustrated by Fig . 6, external application of 10-3 M ethacrynic acid brought about a sharp reversal of the stimulating effect produced by adding protons to the bathing median .

The magnitude of this effect as calculated on the basis of

the change in the rate constant averaged 82X (n ~ 6) .

A comparison of this

value with that calculated from the rise in the rate constant caused by external acidification indicates that ethacrynic acid exerts an additional inhibitory effect, which is estimated to be 65~ of the original Na efflux .

z

x

4 W W 20 N N

0

FIG . 6 The inhibitory effect of 10 -3 M ethacrynic acid on the Na efflux into a Cat+free solution following its stimulation by lowering the external pH from 7 .8 to 6 .3 .

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Mode od Action of Etha,crynfc Acid

Vol. 11, No . 1

Discussion The results as presented here demonstrate that the Na efflux into a Cat+containing or Cat+-free solution is sensitive to ethacrynic acid when the external pH is tow and that the main site of action of the diuretic is the COp-sensitive Na efflux .

Failure of ethacrynic acid at pH 7 .8 to modify the

Na efflux strengthens the view that it 1s not an'inhibitor of the Na+-K} ATPase system, and that Its action is pH-dependent .

The slow rise in the rate

constant for Na loss observed to take place at pH 6 .8 is not very different from that reported by Erlij and Leblanc9 who worked with frog muscle .

However,

these workers omitted giving any information about the pH of the solutions used .

The argument that lowering the external pH allows ethacrynic acid to

cross more rapidly the fiber membrane, leading to a reduction in the Na efflux, is supported by experiments carried out on crab muscle fibers

showing

inhibition at pH 7 .0, and by the present results showing (i) reversal of the effect of ethacrynic acid in a Cat+-containing solution at pH 6 .8 by lowering the external pH to 5 .8, and (11) occurrence of an extra-effect when ethacrynic acid is introduced into a Cat +-free bathing medium whose pH is 6 .3 . The finding that fibers poisoned with ethacrynic acid at pH 6 .8 (using 5 mm and not 10 m~a HCOg- as buffer) shorten has provided a clue as to why the rate constants for Na loss rise .

That the rise in rate constant is probably

related to an increased internal free Cat+ concentration resulting wholly or in part froe~ an increased Cat+ influx is strongly suggested by the experiments done with Cat+-free solutions .

It is worth remembering here that

microinjection of CaC12 into barnacle fibers causes a rise in the Na effl ux, 10,11 a fact which favors the idea that fibers poisoned with ethacrynic acid at low pH have a raised internal free Cat+ concentration . It is equally obvious from the above results that in the absence of external Cat+ the action of ethacrynic acid at low pH is not restricted to the COp-sensitive component of the Na efflux .

Whether this action is the result of

direct involve~aent of the Na+-IC+ ATPase l2 or of interruption of mitochondrial

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Mode of Acrion od Ethacrynic Acid

respirationl3,l4 or of glycolysisl 5 is far from certain.

21

But what appears

fairly clear is that ethacrynic acid behaves as an inhibitor of more than one system, which perhaps explains why its mode of action has eluded discovery (see Peters and Roch-Rame1 16 ), Ackrrowledgment - This work was supported in part by grants from the Medical School Research Committee, the Graduate School Research Committee, the Office of Naval Research, the National Science Foundation, and the Swedish Medical School Research Council . We are grateful to Mr . Hens Ôstling for his invaluable technical assistance during this investigation . References 1.

B. G . DANIELSON, E . E. BITTAR, S . CHEN and E. TONG, Life Sci . (in press) .

2.

E . UHLICH, C. A. BALDAMUS and K. J . ULLRICH, Pflügers Arch . 303, 21

3.

M. GOLDBERG, Ann . N.Y . Acad . Sci . 139 443 (1966) . Also : D. K. McCURDY, E . L . FOLTZ a . . .°, 7. Clin . Invest . 43, 201 (1964) .

4.

A. L . HODGKIN and R. D . KEYNES, J. Physiol . (Lord .),

5.

P . C . CALDWELL and G . E. WALSTER, J. Physiol . (Lond.) 169, 353 (1963) .

6.

E . E. BITTAR and E. TONG, Life Sci . ~, 43 (1971) .

7.

E . E . BITTAR, J . Physiol . (Lord .) 187, 81

8.

E . E . BITTAR, P. C . CALDWELL and A . G. LOTIE, J, mar, biol . Assoc. U.K . 4~7 709 (1967) .

9.

D . ERLIJ and 6. LEBLANC, J . P

10 .

E . E. BITTAR, B . G. DANIELSON, E. TONG and S. CHEN, Experientia (in press) .

11 .

B . G . DANIELSON, E. E. BITTAR, S. CHEN and E . TONG, Life Sci . 10, 833 (1971) . -

12 .

D. E. DUGGAN and R. M. NOLL, Arch . Biachem. Biophys . 1~ 388 (1965) .

13 .

E. J. LANDON and Y. D. JONES, Pharniacologlst 8, 209 (1966) .

14 .

E. E. GORDON, Blochem. Phanmscol .

15 .

E. E. GORDON and M. DeHART06, J . Gen . Physiol . 54, 650 (1969) .

16 .

G . PETERS and F . ROCH-RAMEL, In : (1969) .

1 J1 .

(1968) .

592 (1956) .

(1966) .

siol . (Lond .) 2~, 327 (1971) .

7, 1237 (1968) .

Hand . Exptl . Pharmacol . Yol . 24, 406