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Some further observations on the stimulation by monensin of the sodium efflux in barnacle muscle fibers
Pergamon Press
Life Sciences Vol . 16, pp .297-304 Printed in the U.S .A .
SOME FURTHER OBSERVATIONS ON THE STIMULATION BY MOtiENSIN OF THE SODIUM EFFLUX IN BARNACLE MUSCLE FIBERS Hanna Walkowiak and Geoffrey Chamber Department of Physiology, University of Wisconsin Madison, Wisconsin 53706, U .S .A .
(Received in final form December 16, 1974) $uRnb I`y
Internal application of 20 y g/m1 monensin causes a large rise in the Na efflux into Na-free Li-substituted artificial sea water (ASW) . A coaperable effect 1s observed in fibers pretreated with ouabaln . External acidification fails to significantly augment the response to annensin of the ouabain-insensitive Na effl ux into LiASW. Fibers cooled to 0°C fail to respond to monensin, when the pH of L1-ASIi 1s adjusted fray 7 .8 to 6 .0 . These results are in keeping with the view that the ion exchange induced by aanensin does not necessarily involve Na for Na . The cooling experiments establish that aionensin can be stopped from acting when the environmental temperature is 0° C, presuaebly as the result of membrane conformational changes . Intmducti on In an earlier paper (1) it was shown that the antibiotic abnensin has the ability to stimulate the Na efflux in barnacle muscle fibers, and to do so, more powerfully in the presence of an inactive Na+-K+ ATPase system or of a low external pH .
One basic question raised by these results is whether monen-
sin stimulates an Ussing type of sodium exchange system and whether its action depends on external Na .
The purpose therefore of the present experiments was
to obtain information about whether aamensin can stimulate the Na efflux when the external sodium is replaced with lithium and whether external acidification modifies the response of the efflux to the antibiotic .
This paper also in-
cludes some experiments carried out at 0° C. Methods Di,assoti.on .
Single muscle fibers measuring 3-5 cas in length and 1-2 mm
in width were dissected from the flexor muscle bundles of the barnacle, Bala nes nubiles or B, uq~ri.Za.
Cannulation was accoaQlished by tying a loop of cot297
Monenein Effect on Na Efflux
29 8
Vol . 16, No . 2
ton thread around each fiber some 2-3 mm below the cut end, and then coating this part with vaseline-parnffin .
To maintain the fiber preparation in a ver-
ti cal position while suspended in artificial sea water, a 50 mg weight was attached to its tendon, Ths mioroinjsotor used.
The microinjector was of the same design as that
used by Hodgkin and Keynes (2) as modified by Caldwell and Nalster (3) . inner capillary of the microinjector was about 110 uin diameter .
The
Since a 1
cm cotuen of test solution was usually discharged in these experiments, and since the fibers used had an intrafiber fluid volume of 25-50 y 1 the diluti on factor owing to the sarcoplasm maybe taken as 250-500 . SoZut~Lone used.
The experiments were done with artificial sea water
having the following composition (mM) :
NaCI 465, KC1 10, CaC1 2 10, MgCl2 10,
NaHC03 10 and pH 7.8 or LiCI 475, CaCl2 10, MgCl2 10, KHC03 10 and pH 7 .8 . In those experiments where the buffer used was HEPES in place of HCOg , the pH was reduced to 6.0 by simply adding HC1 .
Unless otherwise stated, all ex-
periments were done between 23° and 25°C . Radioaati,vity measurements .
Amersham-Searle Co .
22NaC1 in aqueous solution was supplied by
The solution was dried and then made up to a small
volume so that volumes of ca . 0.1 y 1 gave at least 50,000 cpm.
The pro-
cedures used for collecting the wash-out samples at regular intervals of time and counting their activity, as well as the activity remaining in the fiber at the end of each experiment were those described by Bitter (4) and Bitter, Caldwell and Lowe (5) .
It should be noted that in this type of work the
first three or four wash-out samples provide information about the period of
equilibration of the injected 22Na (6) .
A well-type scintillation counter
fitted with a solid phosphor connected to a staler was employed for counting . Estimates of the effects on the Na efflux caused by replacing the external Na with Li, by ouabaln and monensin, were arrived at by taking into account the observed change in rate constant of 22Na efflux .
An effect in each case
was then expressed as a percentage of the immediately preceding rate constant .
Vol. 16, No . 2
Monensin
Effect
on Ntl
Efflux
299
Monensin (lot X370-5~-AD-291) was a gift from Eli Lilly Company, Indianapolis, Indiana; ouabain and HEPES were supplied by Sigma Chenical Company, St, Louts, Missouri . Results Attempts were fi nt made to see whether the microinjectian of 20 u9/ml monensin modifies the Na efflux into Na-free Li-ASW .
Shown in Fig . 1 is
that sudden replacement of external Na with Li results in a fairly large fall in the Na efflux, and that internal application of moner~sin stimulates the loss of Na rather markedly, of 49,6
t
2 .7x (SEM) and 125 .7
Estimates of both effects give average values t
26 .6x (n ~ 8), respectively .
The fall in
aooo M 2
~ 2000
N
1000 W W W 0 2
â
S00 300
FIG . 1 Effects on the Na efflux of substituting external Na with Li, follawed .by internal application of 20 u g/ml ~nonensin (Semi log plot) . Na efflux is not an unexpected result .
Several workers including Keynes and
Swan (7), Beaugé and Sjodin (8) and Horowicz, Taylor and Waggoner (9) found that Na efflux from frog muscle is reduced when external Na is replaced by Li .
Two explanations for this effect suggest thenselves : first, that Ussing
is correct in thinking that Na :Na exchange does exist .
Secondly, that Li
penetrates the plasma membrane, and then inhibits the Na+-K+ ATPese system
30 0
Monensin Effect on Na Efflux
from the inside*.
Vol . 16, No . 2
The second explanation is rendered untenable by unpub-
lished results (R, Schultz and H. Walkowiak) showing that the ouabain-insensitive Na efflux is either reduced or increased when Li is substituted for Na .
With these considerations in mind it is reasonable to assume that the
response to monensin represents an increased activity of the ouabain-insensitive Na efflux .
A value of 125.7X stimulation is to be compared with 70 .SX
reported earlier for the stimulation obtained when the bathing medium contains Na rather than Li .
This difference, however, is not statistically sig-
nificant (P > 0 .2), Having found in earlier work that ouabain-poisoned fibers are more sensitive to monensin than unpoisoned fibers, it seemed natural to test the effect of the antibiotic on the ouabain-insensitive Na efflux into Li-ASW .
As il-
lustrated in Fig. 2, the Na efflux is promptly reduced not only when the external Na is replaced by Li, but also when 10~M ouabain is externally applied
naoo
LI~NUTEs
FI6, 2 Effects an the Na efflux of substituting external Na with Li, external application of 10-4M ouabain for the duration of the experiment, and internal application of 20 u g/ml monensin, *Another explanation would be that L1 inhibits the adenylate cyclase system,
thereafter .
301
Monensin Effect on Na Efflux
Vol. 16, No . 2
Also shown is that Internal application of 20 y g/sl ~nonensin
causes a substantial rise in the rmaining Na efflux . effects are in the order of 47 .8
t
The average inhibitory
2 .9x with Li and 42 .7
while the stimulatory effect is 130
t
28 .8x (n ~ 8) .
t
6.5Z with ouabain,
The size of the stimu-
lation does not really appear to be different from that obtained with ouabaintreated fibers baffled in ASW containing Na, viz 156.1 32 .4x (n ~ 12) reported earlier.
The inference, therefore, is that Li itself is without effect an
the Na+-K+ ATPase system and that it does not interfere with the response to monensin of the ouabain-insensitive Na efflux . Previous experiments revealed that the response of the Na efflux to nanensin is enhanced by reducing the external pH .
Experiments therefore were de-
signed to see whether the same holds for fibers bathed in Li-ASW .
Fig . 3
shows that the Na efflux into HEPES-Na-ASW is reduced when the external Na is replaced with Li, and that external application of 10~M ouabain causes a fur ther fall in the efflux .
In addition, this figure shows that reducing the ex-
ternal pH from 7 .8 to 6,0 results in a fall in the remaining efflux, whereas internal application of 20 y g/sl sonensin markedly stimulates the loss of Na .
FI6 . 3 Effects on th~ Na efflux of substituting external Na with Li, external application of 10-~M ou~ain for the duration of the expert sent, reduction in external pH Eras 7.8 to 6, and internal soplication of 20 y g/el eanensin . In this type of experie~ent where external pH is reduced below 6 .8, the huffier used is HEPES (~) and not HC03-- .
302
Monensin Effect on Na Efflux
Vol . 16, No . 2
The magnitude of these effects average 25,8 t 4 .9X with Li, 29 .08 t 4 .7% with ouabain and 164 t 31 .8% with monensin (n = 8) .
In addition, it is found that
fibers not treated with ouabain show an average stimulation of 147 .2 t 41,5X (n = 8) with monensin when the external pH is 6 .0,
Strictly speaking, the
comparison that needs to be made here is between a monensin effect of 164% obtained with ouabain-poisoned fibers bathed in Li-ASW at pH 6 .0 and a monensin effect of 130X obtained with ouabain-poisoned fibers bathed in Li-ASW at pH 7 .8 .
But these values do not differ (P > 0 .50) . Cooling is one useful means of distinguishing an active process from a Shawn in Fig . 4 is that cooling to 0°C causes a drastic fall
passive process .
in the Na efflux and that internal application of 20 y g/ml monensin is with The magnitude of these effects average 78 .05 ± 4 .6% and
out much effect .
35 .9 t 12 .5X (n - 4), respectively,
Controls carried out with lOX ethyl atco-
200
z loo N H Z
50
W a a
10
140
FIG . 4 Effects on the Na efflux of suddenly lowering the environmental temperature from 24° to 0°C, followed by internal application of 20 v g/ml monensin . hol (this is the solvent used for monensin) show a 25 t 6 .02X stimulation . This means then that the effect observed with monensin can be discounted . The next question was to check whether external acidification would augment
303
Mo~n~nein Effect on Na Efflux
Vol . 16, No . 2
the stimulatory response to monensin . is ineffective.
Shawn in Fig. 5 is that such a maneuver
This is further borne out by the fact that the average effect
caused by injecting 20 v g/ml monensin when the external pH is 6 .0, is only 34 .5
t
6 .9X (n ~ 6) . 3000
100
20
40
60
BO
100
120
140
I60
AAINUTES
FIG. 5
Effects on the Na efflux of suddenly lowering the environmental te~perature from 25° to 0°C, folla+ed by lowering the external pH from 7 .8 to 6 .0 and internal application of 20 y g/ml uonensin . Buffer used in this type of experiment is HEPES (3mf1) . Di scussion The present experiments provide solid evidence that monensin can stimulate the fig efflux when Li rather than Na is present in the bathing median .
This
tells strongly for the argument that soeciflcity in terms of Na for Na exchange is not necessarily a property of the antibiotic .
The possibility that
the exchange stimulated by monensin is Na for Li is not unlikely .
This is sug-
gested by the observation that neither pretreatment of the fibers with ouabain, nor external acidification results in an enhanced response to monensin .
Evi-
dence supporting this view that monensin does not distinguish between Na and Li has been produced by Pressman and Heeb (10) in experiments involving erythrocyte ghosts .
Whether H+ and Li+ compete for transport by monensin remains un-
304
Monensin Effect on Na Efflux
Vol . 16, No . 2
known . The finding that monensin fails to stimulate the Na efflux at 0°C is not wholly surprising .
It can of course be explained by assuming that cooling
causes conformational changes in the membrane, which prevent nanensin frog passing through .
One should not make the mistake however of thinking that
the efflux at 0°C cannot be stimulated by other agents .
This is because
there is good evidence already that substances such as CAMP when injected causes a substantial rise in the Na efflux from fibers cooled down to 0°C (Bittar, Chambers and Schultz, unpublished results) . Acknowledgment Thanks are due to Professor R .C . Wolf for his support, and to Professor E .E . Bittar for guidance, helpful criticism and assistance with the drafting of the manuscript . This work was supported in part by a grant from the Office of Naval Research . H .W . was the holder of a Postdoctoral Fellowship of the Ford Foundation, Endocrinology - Reproductive Physiology Prograr~ . References 1.
H WALKOWIAK, Life Sci . 15, 1353 (1974) .
2.
A . L . HODGKIN 8 R . D . KEYNES, J . Physiol . (London) 131, 592 (1956) .
3.
P . C . CALDWELL b G . E . WALSTER, J . Physiol . (London) 169, 353, (1963) .
4.
E . E . BITTAR, J . Physiol . (London) 187, 81 (1966) .
5.
E . E . BITTAR, P . C . CALDWELL AND A . G . LÖWE, J . mar, biol . Assoc . U .K ., 47, 709 (1967) .
6.
E . E . BITTAR, S . CHEN, B . G . DANIELSON, H . HARTMANN AND E . Y . TONG . J . Physiol . (London) 221, 389 (1972) .
7.
R . D . KEYNES, 5 R . C . SWAN, J . Physiol .
8.
L . A . BEAUGÉ 8 R . A . SJODIN, J . Gen . Physiol . 52, 389 (1968) .
9.
P . HOROWICZ, J . W . TAYLOR AND D . M . WAGGONER, J . Gen . Physiol . 55, 401
(London) 147, 626 (1959) .
(1970) . 10 .
B . C . PRESSMAN 8 M . G . HEER, In : Symposium on Molecular Mechanisms of Antibiotic Actions on Protein Biosynthesis and Membranes . Amsterdam (1972), p . 603 .
Elsevier,
Life Sciences Vol . 16, pp .297-304 Printed in the U.S .A .
SOME FURTHER OBSERVATIONS ON THE STIMULATION BY MOtiENSIN OF THE SODIUM EFFLUX IN BARNACLE MUSCLE FIBERS Hanna Walkowiak and Geoffrey Chamber Department of Physiology, University of Wisconsin Madison, Wisconsin 53706, U .S .A .
(Received in final form December 16, 1974) $uRnb I`y
Internal application of 20 y g/m1 monensin causes a large rise in the Na efflux into Na-free Li-substituted artificial sea water (ASW) . A coaperable effect 1s observed in fibers pretreated with ouabaln . External acidification fails to significantly augment the response to annensin of the ouabain-insensitive Na effl ux into LiASW. Fibers cooled to 0°C fail to respond to monensin, when the pH of L1-ASIi 1s adjusted fray 7 .8 to 6 .0 . These results are in keeping with the view that the ion exchange induced by aanensin does not necessarily involve Na for Na . The cooling experiments establish that aionensin can be stopped from acting when the environmental temperature is 0° C, presuaebly as the result of membrane conformational changes . Intmducti on In an earlier paper (1) it was shown that the antibiotic abnensin has the ability to stimulate the Na efflux in barnacle muscle fibers, and to do so, more powerfully in the presence of an inactive Na+-K+ ATPase system or of a low external pH .
One basic question raised by these results is whether monen-
sin stimulates an Ussing type of sodium exchange system and whether its action depends on external Na .
The purpose therefore of the present experiments was
to obtain information about whether aamensin can stimulate the Na efflux when the external sodium is replaced with lithium and whether external acidification modifies the response of the efflux to the antibiotic .
This paper also in-
cludes some experiments carried out at 0° C. Methods Di,assoti.on .
Single muscle fibers measuring 3-5 cas in length and 1-2 mm
in width were dissected from the flexor muscle bundles of the barnacle, Bala nes nubiles or B, uq~ri.Za.
Cannulation was accoaQlished by tying a loop of cot297
Monenein Effect on Na Efflux
29 8
Vol . 16, No . 2
ton thread around each fiber some 2-3 mm below the cut end, and then coating this part with vaseline-parnffin .
To maintain the fiber preparation in a ver-
ti cal position while suspended in artificial sea water, a 50 mg weight was attached to its tendon, Ths mioroinjsotor used.
The microinjector was of the same design as that
used by Hodgkin and Keynes (2) as modified by Caldwell and Nalster (3) . inner capillary of the microinjector was about 110 uin diameter .
The
Since a 1
cm cotuen of test solution was usually discharged in these experiments, and since the fibers used had an intrafiber fluid volume of 25-50 y 1 the diluti on factor owing to the sarcoplasm maybe taken as 250-500 . SoZut~Lone used.
The experiments were done with artificial sea water
having the following composition (mM) :
NaCI 465, KC1 10, CaC1 2 10, MgCl2 10,
NaHC03 10 and pH 7.8 or LiCI 475, CaCl2 10, MgCl2 10, KHC03 10 and pH 7 .8 . In those experiments where the buffer used was HEPES in place of HCOg , the pH was reduced to 6.0 by simply adding HC1 .
Unless otherwise stated, all ex-
periments were done between 23° and 25°C . Radioaati,vity measurements .
Amersham-Searle Co .
22NaC1 in aqueous solution was supplied by
The solution was dried and then made up to a small
volume so that volumes of ca . 0.1 y 1 gave at least 50,000 cpm.
The pro-
cedures used for collecting the wash-out samples at regular intervals of time and counting their activity, as well as the activity remaining in the fiber at the end of each experiment were those described by Bitter (4) and Bitter, Caldwell and Lowe (5) .
It should be noted that in this type of work the
first three or four wash-out samples provide information about the period of
equilibration of the injected 22Na (6) .
A well-type scintillation counter
fitted with a solid phosphor connected to a staler was employed for counting . Estimates of the effects on the Na efflux caused by replacing the external Na with Li, by ouabaln and monensin, were arrived at by taking into account the observed change in rate constant of 22Na efflux .
An effect in each case
was then expressed as a percentage of the immediately preceding rate constant .
Vol. 16, No . 2
Monensin
Effect
on Ntl
Efflux
299
Monensin (lot X370-5~-AD-291) was a gift from Eli Lilly Company, Indianapolis, Indiana; ouabain and HEPES were supplied by Sigma Chenical Company, St, Louts, Missouri . Results Attempts were fi nt made to see whether the microinjectian of 20 u9/ml monensin modifies the Na efflux into Na-free Li-ASW .
Shown in Fig . 1 is
that sudden replacement of external Na with Li results in a fairly large fall in the Na efflux, and that internal application of moner~sin stimulates the loss of Na rather markedly, of 49,6
t
2 .7x (SEM) and 125 .7
Estimates of both effects give average values t
26 .6x (n ~ 8), respectively .
The fall in
aooo M 2
~ 2000
N
1000 W W W 0 2
â
S00 300
FIG . 1 Effects on the Na efflux of substituting external Na with Li, follawed .by internal application of 20 u g/ml ~nonensin (Semi log plot) . Na efflux is not an unexpected result .
Several workers including Keynes and
Swan (7), Beaugé and Sjodin (8) and Horowicz, Taylor and Waggoner (9) found that Na efflux from frog muscle is reduced when external Na is replaced by Li .
Two explanations for this effect suggest thenselves : first, that Ussing
is correct in thinking that Na :Na exchange does exist .
Secondly, that Li
penetrates the plasma membrane, and then inhibits the Na+-K+ ATPese system
30 0
Monensin Effect on Na Efflux
from the inside*.
Vol . 16, No . 2
The second explanation is rendered untenable by unpub-
lished results (R, Schultz and H. Walkowiak) showing that the ouabain-insensitive Na efflux is either reduced or increased when Li is substituted for Na .
With these considerations in mind it is reasonable to assume that the
response to monensin represents an increased activity of the ouabain-insensitive Na efflux .
A value of 125.7X stimulation is to be compared with 70 .SX
reported earlier for the stimulation obtained when the bathing medium contains Na rather than Li .
This difference, however, is not statistically sig-
nificant (P > 0 .2), Having found in earlier work that ouabain-poisoned fibers are more sensitive to monensin than unpoisoned fibers, it seemed natural to test the effect of the antibiotic on the ouabain-insensitive Na efflux into Li-ASW .
As il-
lustrated in Fig. 2, the Na efflux is promptly reduced not only when the external Na is replaced by Li, but also when 10~M ouabain is externally applied
naoo
LI~NUTEs
FI6, 2 Effects an the Na efflux of substituting external Na with Li, external application of 10-4M ouabain for the duration of the experiment, and internal application of 20 u g/ml monensin, *Another explanation would be that L1 inhibits the adenylate cyclase system,
thereafter .
301
Monensin Effect on Na Efflux
Vol. 16, No . 2
Also shown is that Internal application of 20 y g/sl ~nonensin
causes a substantial rise in the rmaining Na efflux . effects are in the order of 47 .8
t
The average inhibitory
2 .9x with Li and 42 .7
while the stimulatory effect is 130
t
28 .8x (n ~ 8) .
t
6.5Z with ouabain,
The size of the stimu-
lation does not really appear to be different from that obtained with ouabaintreated fibers baffled in ASW containing Na, viz 156.1 32 .4x (n ~ 12) reported earlier.
The inference, therefore, is that Li itself is without effect an
the Na+-K+ ATPase system and that it does not interfere with the response to monensin of the ouabain-insensitive Na efflux . Previous experiments revealed that the response of the Na efflux to nanensin is enhanced by reducing the external pH .
Experiments therefore were de-
signed to see whether the same holds for fibers bathed in Li-ASW .
Fig . 3
shows that the Na efflux into HEPES-Na-ASW is reduced when the external Na is replaced with Li, and that external application of 10~M ouabain causes a fur ther fall in the efflux .
In addition, this figure shows that reducing the ex-
ternal pH from 7 .8 to 6,0 results in a fall in the remaining efflux, whereas internal application of 20 y g/sl sonensin markedly stimulates the loss of Na .
FI6 . 3 Effects on th~ Na efflux of substituting external Na with Li, external application of 10-~M ou~ain for the duration of the expert sent, reduction in external pH Eras 7.8 to 6, and internal soplication of 20 y g/el eanensin . In this type of experie~ent where external pH is reduced below 6 .8, the huffier used is HEPES (~) and not HC03-- .
302
Monensin Effect on Na Efflux
Vol . 16, No . 2
The magnitude of these effects average 25,8 t 4 .9X with Li, 29 .08 t 4 .7% with ouabain and 164 t 31 .8% with monensin (n = 8) .
In addition, it is found that
fibers not treated with ouabain show an average stimulation of 147 .2 t 41,5X (n = 8) with monensin when the external pH is 6 .0,
Strictly speaking, the
comparison that needs to be made here is between a monensin effect of 164% obtained with ouabain-poisoned fibers bathed in Li-ASW at pH 6 .0 and a monensin effect of 130X obtained with ouabain-poisoned fibers bathed in Li-ASW at pH 7 .8 .
But these values do not differ (P > 0 .50) . Cooling is one useful means of distinguishing an active process from a Shawn in Fig . 4 is that cooling to 0°C causes a drastic fall
passive process .
in the Na efflux and that internal application of 20 y g/ml monensin is with The magnitude of these effects average 78 .05 ± 4 .6% and
out much effect .
35 .9 t 12 .5X (n - 4), respectively,
Controls carried out with lOX ethyl atco-
200
z loo N H Z
50
W a a
10
140
FIG . 4 Effects on the Na efflux of suddenly lowering the environmental temperature from 24° to 0°C, followed by internal application of 20 v g/ml monensin . hol (this is the solvent used for monensin) show a 25 t 6 .02X stimulation . This means then that the effect observed with monensin can be discounted . The next question was to check whether external acidification would augment
303
Mo~n~nein Effect on Na Efflux
Vol . 16, No . 2
the stimulatory response to monensin . is ineffective.
Shawn in Fig. 5 is that such a maneuver
This is further borne out by the fact that the average effect
caused by injecting 20 v g/ml monensin when the external pH is 6 .0, is only 34 .5
t
6 .9X (n ~ 6) . 3000
100
20
40
60
BO
100
120
140
I60
AAINUTES
FIG. 5
Effects on the Na efflux of suddenly lowering the environmental te~perature from 25° to 0°C, folla+ed by lowering the external pH from 7 .8 to 6 .0 and internal application of 20 y g/ml uonensin . Buffer used in this type of experiment is HEPES (3mf1) . Di scussion The present experiments provide solid evidence that monensin can stimulate the fig efflux when Li rather than Na is present in the bathing median .
This
tells strongly for the argument that soeciflcity in terms of Na for Na exchange is not necessarily a property of the antibiotic .
The possibility that
the exchange stimulated by monensin is Na for Li is not unlikely .
This is sug-
gested by the observation that neither pretreatment of the fibers with ouabain, nor external acidification results in an enhanced response to monensin .
Evi-
dence supporting this view that monensin does not distinguish between Na and Li has been produced by Pressman and Heeb (10) in experiments involving erythrocyte ghosts .
Whether H+ and Li+ compete for transport by monensin remains un-
304
Monensin Effect on Na Efflux
Vol . 16, No . 2
known . The finding that monensin fails to stimulate the Na efflux at 0°C is not wholly surprising .
It can of course be explained by assuming that cooling
causes conformational changes in the membrane, which prevent nanensin frog passing through .
One should not make the mistake however of thinking that
the efflux at 0°C cannot be stimulated by other agents .
This is because
there is good evidence already that substances such as CAMP when injected causes a substantial rise in the Na efflux from fibers cooled down to 0°C (Bittar, Chambers and Schultz, unpublished results) . Acknowledgment Thanks are due to Professor R .C . Wolf for his support, and to Professor E .E . Bittar for guidance, helpful criticism and assistance with the drafting of the manuscript . This work was supported in part by a grant from the Office of Naval Research . H .W . was the holder of a Postdoctoral Fellowship of the Ford Foundation, Endocrinology - Reproductive Physiology Prograr~ . References 1.
H WALKOWIAK, Life Sci . 15, 1353 (1974) .
2.
A . L . HODGKIN 8 R . D . KEYNES, J . Physiol . (London) 131, 592 (1956) .
3.
P . C . CALDWELL b G . E . WALSTER, J . Physiol . (London) 169, 353, (1963) .
4.
E . E . BITTAR, J . Physiol . (London) 187, 81 (1966) .
5.
E . E . BITTAR, P . C . CALDWELL AND A . G . LÖWE, J . mar, biol . Assoc . U .K ., 47, 709 (1967) .
6.
E . E . BITTAR, S . CHEN, B . G . DANIELSON, H . HARTMANN AND E . Y . TONG . J . Physiol . (London) 221, 389 (1972) .
7.
R . D . KEYNES, 5 R . C . SWAN, J . Physiol .
8.
L . A . BEAUGÉ 8 R . A . SJODIN, J . Gen . Physiol . 52, 389 (1968) .
9.
P . HOROWICZ, J . W . TAYLOR AND D . M . WAGGONER, J . Gen . Physiol . 55, 401
(London) 147, 626 (1959) .
(1970) . 10 .
B . C . PRESSMAN 8 M . G . HEER, In : Symposium on Molecular Mechanisms of Antibiotic Actions on Protein Biosynthesis and Membranes . Amsterdam (1972), p . 603 .
Elsevier,