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The effect of diphenylhydantoin on sodium efflux from single barnacle muscle fibers
Lüe Sciences Vol . 10, Part I, pp. 497-443, 1971. Printed in Great Britain
Pergamon Press
THE EFFECT OF DIPHENYLHrDAMTOIM OM SODIUM EFFLUX FROM SIM6LE BJIRWICLE MUSCLE FIBERS Bo 6 . Danlelson,* E . Edward Bittar, Stephen Chen and Edeund
r.
Tong
Department of Physiology, University of Wisconsin, Madison, Wisconsin, 53706, U .S .A .
(Received 21 January 1971 ; in final form 28 February 1971) Susa~ Diphenylhydantoin is shown to reduce the Ma efflux in single barnacle muscle fibers . The iaagnitude of the inhibiting effect is 38X, which is slightly wore than half that of ouabaln . The results are explained by supposing that the action of dipher~yllgMantoin on the transport enryee is lncoaplete or that its main point of action is the T-systes . Little 1s known of the chesical cowponents of the sodlun pump of a single cell preparation and the precise correlation between the Na+-K+ ATPase and the sodiu~ transport system . done on red cells .
Much of the published work consists of experiments
These have shown a parallelism between the activity of the
Na+-K+ ATPase and sodiun navenents (see for example 6arrahan, 1, for a review), but the significance or relevance of this information with respect to ~nen~brane transport in skeletal e:uscle 1s not yet certain . The present paper 1s concerned with active sodium transport 1n single ~uscle fibers from the barnacle Eiatmoca ~wbiZwa, in particular with the inhibiting action of dipher~ylhydantoin (DPH) .
This anti-epileptic agent has aroused our
interest mainly because it has been found to inhibit the Ma+-K} ATPase isolated from brain and skeletal nwscle (2, 3, 4) .
The results with barnacle fibers
show that DPH hag the ability to reduce the sodium efflux, but not as much as ouabain .
*Visiting Scientist of the Aserican Heart Association . Peneanent address : Institute of Physiology and Medical Biophysics, University of Uppsala, Uppsala, Sweden .
497
Diphen~ylhydantoin and Sodium Efflux
438
Vol. 10, No. 8
Methods Single muscle fibers isolated from the depressor muscle bundles of the barnacle &~Zunua aqu{Za or ~wbiZue were used .
These were cannulated and loaded
with 22Na in the same way as described by Bittar (5) and Bittar, Caldwell and Love (6) .
A modified form of ~nicroinjector described by Caldwell and Walster
(7) was used .
The injector discharged 0 .1 u1 of fluid per 1 an excursion of
the micrananipulator .
Carwposltlon of the artificial sea water (ASN) was the
same as that reported by Bitter and Tong (8) .
All experiments were performed
at pH 7 .8 . A [22Na]C1 solution was obtained frosr An~ersham-Searle Corporation (spec . act. 164 mCi/mg Na), evaporated and then node up 1n distilled meter.
Volumes
of ca . 0.1 yl gave counts of about 50,000 cpm, and this was found sufficient for experiments of this type carried out for periods of 120 min. Diphegylhydantoin (Parke, Davis and Co .) was a gift from Dr . W. E. Stone of this department .
Afresh solution of DPH in ASY was prepared each time to
glue a finial concentration of 10~ M, Ca~apany .
Ouabaln was obtained from Sigma Chemical
A11 the experiments were carried out at roam temperature, 20-23° C .
Counting of 22 Na activity in the effluent and fiber was done using the methods described by Bitter (5) .
The well-type counter with a solid phosphor
was a Panax model C5MP ; this was connected to a Nuclear-Chicago Ganwa Count Analyser . Results Fig . 1 shows the results of a control experiment in which a 1 cs column of distilled water was injected into the fiber 35 min after loading it with 22Na . It will be seen that the radiosodiuar leaving the fiber obeys simple exponential kinetics and that insertion of the microinjector, as well as ejection of a 1 am column of distilled water produced ro significant charge in the behavior of the effi ux . Shown in Fig. 2 is a typical experiment (7 fibers) in which the application
Vol . 10, No. 8
Diphe>r~ylhydantoin aad Sodium Sülux
439
TIME (MIN)
FI6 . 1 Seai-log plot showing the efflux of 22Na in artlficial sea water at pH 7 .8 and the sleall, transitory change 1n Na efflux caused by injecting a 1 a coluen of distilled water. (n - 10)
io-' r orn
FI6 . 2 The effect of external application of 10-4 M diphergrlhydantoin on the rate of loss of tgjected 22Na . (n ~ 7)
440
Diphenylhyda.ntoin and Sodium Efflua
of 10'4 M DPH resulted in a marked fall in the efflux .
Vol. 1 0, No. 8 Fu11 effect is observed
to take place about 40 min after application of the agent .
Estimates of the
size of the Inhibition produced by 10-4 M DPH were made on the basis of the change in rate constant .
These showed an average fall of 38X, the range being
30-56x . Atteapts were made to see whether the remaining efflux is sensitive to ouabaln .
Fig . 3 illustrates the extent to which 5 " 10- 5 M ouabain abolishes the
efflux remaining after complete DPH action, a concentration known to exert nbxlmal inhibition in barnacle fibers (Dlare~t~tann and Btt~", u~rbZ{ahwd r~eZi;a) .
In view of the marked Inhibiting effect of ouabain on the DPH-
insensitive component of the sodium efflux, it was a matter of special Interest
u ~ .O
The effect of 10'4 M dlpherq~lhydantoln followed by the application of ouabain (5 " 10- 5 M) on the rate of loss of Na . (n ~ 5) to see whether there is a similar pattern of inhibition when ouabain, and not DPH, 1s first added to the bathing medium .
In each of the five experiments
done the size of the inhibition caused by both ouabain and DPH was not very different from that already observed by applying DPH before ouabain .
This 1s
illustrated by Fig . 4a and b . As reported by Bittar, Caldwell and Lowe (6) the injection of ouabaln into IIAaia fibers 1s without effect on Na efflux .
This is also true of the response
Vol. 10, No . 8
Diphenylhydantoia and Sodium EYflua
FIG . 4a The effect on Na efflux of 5 " 10-5 M ouabain and 10~ M dlphegylhydantoln . (n - 5)
FIG . 4b The rate constant curve for the preceding experi~ent .
441
442
Diphe~lhyda.ntoin and Sodium Efflux
Vol. 10, No. 8
of fia ettlux 1n barnacle fibers to injected ouabain (DianLetaon and urrpwbtiehwd rrarctte) .
jected DPH.
Biota,
The question then is whether this 1s the case with in-
The result presented in Fig . 5 illustrates that like ouabaln, DPH
1s ineffective (4 expts .) .
FI6 . 5 The lack of effect of j~nternal application of DPH (> 10 -4 M, saturated solution) on the rate of 2 fra loss . (n - 4) Discussion The results of the experiments with DPH clearly show that this agent 1s an 1~,h1bi+~r of fia efflux and that 1t acts only when applied externally . that DPH 1s known to inhibit the
The fact
ra+-K+ ATPase of various tissues, e .g . rat
brain (2) and skeletal muscle (4) raises the possibility that DPH acts on the Na efflux 1n barnacle fibers by slowing down the transport enryme .
The onset
of maximal inhibition 40 min after application of DPH suggests that the site of action of DPH .ay be the inner side of the plasma menbrane or the outer side of the T-system .
That 1t does not act on the loner side of the md~brane is indi-
cated by Its (allure to modify the fla efflux following injection into the fiber. The finding of a large reduction 1n the DPH-insensitive component of the Ma efflux is consistent with the concept that inhibition of the Ha+-K+ ATPase
Vol . 10, No. 8 by DPH is incomplete .
Diphe~lhydantoin and Sodium Efflua
449
However, another possibility is that DPH acts at two
different sites in the barnacle fiber - the plas~aa ~ebrane and the surface of the T-system .
This explanation is reasonable not only because 1t implies
differences in the properties of the two sites but because it may lead to a more accurate and meaningful analysis of the sodium efflux . Acknowledgment - This work was supported in part by grants from the Medical School Research Committee, the Graduate School Research Committee, the Office of Naval Research and the Swedish Medical Research Council (grant B70-2329-04A) . We wish to thank Mr . Hens Ostlang for excellent technical assistance . References 1.
P . J . GARRAHAN, In : Membranes and Ion Trans ort, ed . E. E. Bittar, Vol . 2, W11ey - Intersc ente, on on, .
2.
J . H . PINCUS and N. J . GIARMAN, Baochem. Phaneacol . ~, 600 (1967) .
3.
M. D . RAWSON and J . H. PINCUS, Biochem . Phan~acol . ~, 573 (1968) .
4.
J . B . PETER, Biochea. B1o
5.
E. E. BITTAR, J . P
6.
E. E. BITTAR, P . C . CALDWELL and A. G. LÖWE, J . mar. baol . Assoc . U .K . 47, 709 (1967) .
7.
P. C . CALDWELL and G. E. WILSTER, J . Physiol ., London 169, 353 (1963) .
8.
E. E. BITTAR and E . Y. TONG, Life Sciences 2, 1971
s. Res. Carom.
siol ., London 1~ 81
1
6, p. 1362 (1970) .
(1966) .
(1n press) .
Pergamon Press
THE EFFECT OF DIPHENYLHrDAMTOIM OM SODIUM EFFLUX FROM SIM6LE BJIRWICLE MUSCLE FIBERS Bo 6 . Danlelson,* E . Edward Bittar, Stephen Chen and Edeund
r.
Tong
Department of Physiology, University of Wisconsin, Madison, Wisconsin, 53706, U .S .A .
(Received 21 January 1971 ; in final form 28 February 1971) Susa~ Diphenylhydantoin is shown to reduce the Ma efflux in single barnacle muscle fibers . The iaagnitude of the inhibiting effect is 38X, which is slightly wore than half that of ouabaln . The results are explained by supposing that the action of dipher~yllgMantoin on the transport enryee is lncoaplete or that its main point of action is the T-systes . Little 1s known of the chesical cowponents of the sodlun pump of a single cell preparation and the precise correlation between the Na+-K+ ATPase and the sodiu~ transport system . done on red cells .
Much of the published work consists of experiments
These have shown a parallelism between the activity of the
Na+-K+ ATPase and sodiun navenents (see for example 6arrahan, 1, for a review), but the significance or relevance of this information with respect to ~nen~brane transport in skeletal e:uscle 1s not yet certain . The present paper 1s concerned with active sodium transport 1n single ~uscle fibers from the barnacle Eiatmoca ~wbiZwa, in particular with the inhibiting action of dipher~ylhydantoin (DPH) .
This anti-epileptic agent has aroused our
interest mainly because it has been found to inhibit the Ma+-K} ATPase isolated from brain and skeletal nwscle (2, 3, 4) .
The results with barnacle fibers
show that DPH hag the ability to reduce the sodium efflux, but not as much as ouabain .
*Visiting Scientist of the Aserican Heart Association . Peneanent address : Institute of Physiology and Medical Biophysics, University of Uppsala, Uppsala, Sweden .
497
Diphen~ylhydantoin and Sodium Efflux
438
Vol. 10, No. 8
Methods Single muscle fibers isolated from the depressor muscle bundles of the barnacle &~Zunua aqu{Za or ~wbiZue were used .
These were cannulated and loaded
with 22Na in the same way as described by Bittar (5) and Bittar, Caldwell and Love (6) .
A modified form of ~nicroinjector described by Caldwell and Walster
(7) was used .
The injector discharged 0 .1 u1 of fluid per 1 an excursion of
the micrananipulator .
Carwposltlon of the artificial sea water (ASN) was the
same as that reported by Bitter and Tong (8) .
All experiments were performed
at pH 7 .8 . A [22Na]C1 solution was obtained frosr An~ersham-Searle Corporation (spec . act. 164 mCi/mg Na), evaporated and then node up 1n distilled meter.
Volumes
of ca . 0.1 yl gave counts of about 50,000 cpm, and this was found sufficient for experiments of this type carried out for periods of 120 min. Diphegylhydantoin (Parke, Davis and Co .) was a gift from Dr . W. E. Stone of this department .
Afresh solution of DPH in ASY was prepared each time to
glue a finial concentration of 10~ M, Ca~apany .
Ouabaln was obtained from Sigma Chemical
A11 the experiments were carried out at roam temperature, 20-23° C .
Counting of 22 Na activity in the effluent and fiber was done using the methods described by Bitter (5) .
The well-type counter with a solid phosphor
was a Panax model C5MP ; this was connected to a Nuclear-Chicago Ganwa Count Analyser . Results Fig . 1 shows the results of a control experiment in which a 1 cs column of distilled water was injected into the fiber 35 min after loading it with 22Na . It will be seen that the radiosodiuar leaving the fiber obeys simple exponential kinetics and that insertion of the microinjector, as well as ejection of a 1 am column of distilled water produced ro significant charge in the behavior of the effi ux . Shown in Fig. 2 is a typical experiment (7 fibers) in which the application
Vol . 10, No. 8
Diphe>r~ylhydantoin aad Sodium Sülux
439
TIME (MIN)
FI6 . 1 Seai-log plot showing the efflux of 22Na in artlficial sea water at pH 7 .8 and the sleall, transitory change 1n Na efflux caused by injecting a 1 a coluen of distilled water. (n - 10)
io-' r orn
FI6 . 2 The effect of external application of 10-4 M diphergrlhydantoin on the rate of loss of tgjected 22Na . (n ~ 7)
440
Diphenylhyda.ntoin and Sodium Efflua
of 10'4 M DPH resulted in a marked fall in the efflux .
Vol. 1 0, No. 8 Fu11 effect is observed
to take place about 40 min after application of the agent .
Estimates of the
size of the Inhibition produced by 10-4 M DPH were made on the basis of the change in rate constant .
These showed an average fall of 38X, the range being
30-56x . Atteapts were made to see whether the remaining efflux is sensitive to ouabaln .
Fig . 3 illustrates the extent to which 5 " 10- 5 M ouabain abolishes the
efflux remaining after complete DPH action, a concentration known to exert nbxlmal inhibition in barnacle fibers (Dlare~t~tann and Btt~", u~rbZ{ahwd r~eZi;a) .
In view of the marked Inhibiting effect of ouabain on the DPH-
insensitive component of the sodium efflux, it was a matter of special Interest
u ~ .O
The effect of 10'4 M dlpherq~lhydantoln followed by the application of ouabain (5 " 10- 5 M) on the rate of loss of Na . (n ~ 5) to see whether there is a similar pattern of inhibition when ouabain, and not DPH, 1s first added to the bathing medium .
In each of the five experiments
done the size of the inhibition caused by both ouabain and DPH was not very different from that already observed by applying DPH before ouabain .
This 1s
illustrated by Fig . 4a and b . As reported by Bittar, Caldwell and Lowe (6) the injection of ouabaln into IIAaia fibers 1s without effect on Na efflux .
This is also true of the response
Vol. 10, No . 8
Diphenylhydantoia and Sodium EYflua
FIG . 4a The effect on Na efflux of 5 " 10-5 M ouabain and 10~ M dlphegylhydantoln . (n - 5)
FIG . 4b The rate constant curve for the preceding experi~ent .
441
442
Diphe~lhyda.ntoin and Sodium Efflux
Vol. 10, No. 8
of fia ettlux 1n barnacle fibers to injected ouabain (DianLetaon and urrpwbtiehwd rrarctte) .
jected DPH.
Biota,
The question then is whether this 1s the case with in-
The result presented in Fig . 5 illustrates that like ouabaln, DPH
1s ineffective (4 expts .) .
FI6 . 5 The lack of effect of j~nternal application of DPH (> 10 -4 M, saturated solution) on the rate of 2 fra loss . (n - 4) Discussion The results of the experiments with DPH clearly show that this agent 1s an 1~,h1bi+~r of fia efflux and that 1t acts only when applied externally . that DPH 1s known to inhibit the
The fact
ra+-K+ ATPase of various tissues, e .g . rat
brain (2) and skeletal muscle (4) raises the possibility that DPH acts on the Na efflux 1n barnacle fibers by slowing down the transport enryme .
The onset
of maximal inhibition 40 min after application of DPH suggests that the site of action of DPH .ay be the inner side of the plasma menbrane or the outer side of the T-system .
That 1t does not act on the loner side of the md~brane is indi-
cated by Its (allure to modify the fla efflux following injection into the fiber. The finding of a large reduction 1n the DPH-insensitive component of the Ma efflux is consistent with the concept that inhibition of the Ha+-K+ ATPase
Vol . 10, No. 8 by DPH is incomplete .
Diphe~lhydantoin and Sodium Efflua
449
However, another possibility is that DPH acts at two
different sites in the barnacle fiber - the plas~aa ~ebrane and the surface of the T-system .
This explanation is reasonable not only because 1t implies
differences in the properties of the two sites but because it may lead to a more accurate and meaningful analysis of the sodium efflux . Acknowledgment - This work was supported in part by grants from the Medical School Research Committee, the Graduate School Research Committee, the Office of Naval Research and the Swedish Medical Research Council (grant B70-2329-04A) . We wish to thank Mr . Hens Ostlang for excellent technical assistance . References 1.
P . J . GARRAHAN, In : Membranes and Ion Trans ort, ed . E. E. Bittar, Vol . 2, W11ey - Intersc ente, on on, .
2.
J . H . PINCUS and N. J . GIARMAN, Baochem. Phaneacol . ~, 600 (1967) .
3.
M. D . RAWSON and J . H. PINCUS, Biochem . Phan~acol . ~, 573 (1968) .
4.
J . B . PETER, Biochea. B1o
5.
E. E. BITTAR, J . P
6.
E. E. BITTAR, P . C . CALDWELL and A. G. LÖWE, J . mar. baol . Assoc . U .K . 47, 709 (1967) .
7.
P. C . CALDWELL and G. E. WILSTER, J . Physiol ., London 169, 353 (1963) .
8.
E. E. BITTAR and E . Y. TONG, Life Sciences 2, 1971
s. Res. Carom.
siol ., London 1~ 81
1
6, p. 1362 (1970) .
(1966) .
(1n press) .