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The effect of diphenylhydantoin on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibres
EUROPEAN JOURNALOF PHARMACOLOGY19 (1972) 94-97. NORTH-HOLLANDPUBLISHINGCOMPANY
T H E E F F E C T O F D I P H E N Y L H Y D A N T O I N ON T H E E L E C T R O G E N I C C O M P O N E N T O F T H E S O D I U M PUMP IN M A M M A L I A N N O N - M Y E L I N A T E D N E R V E FIBRES
A. DEN HERTOG Department of Pharmacology, University of Groningen, Bloemsingel 1, The Netherlands
Received 3 May 1971
Accepted 1 March 1972
A. DEN ttERTOG, The effect of diphenylhydantoin on the electrogenic component of the sodhtm pump in mammalian non-myelinated nerve fibres, European J. Pharmacol. 19 (1972) 94-97. A study has been made of the effect of diphenylhydantoin on the electrogenic component of the sodium pump in non-myelinated fibres of the desheathed rabbit vagus nerve. Acute or chronic administration of this drug did not affect the excitability, nor the membrane permeability, nor the activity of the sodium pump of the nerve fibres. Diphenylhydantoin
C-fibres
1. INTRODUCTION The mechanism of action of the anticonvulsant drug, diphenylhydantoin (DPH), is still obscure although the results of some detailed studies have been reported. DPH intracellular sodium concentration braincells, as well as skeletal and cardiac muscle (Woodbury, 1955). Enhancement of the active sodium extrusion from brain cells by stimulation of the metabolic sodium pump might account for this effect (Woodbury and Kemp, 1970; Goodman and Gilman, 1970). Another significant finding concerning DPH was its ability to depress responses to repetitive stimulation, such as post-tetanic repetitive afterdischarges (PTR) in the motor nerve (Parisi and Raines, 1963) and post-tetanic potentiation (PTP) of the ventral root (Esplin, 1957). The potential reversal of the dorsal root IV (DRIVR) in the spinal cord of the cat was also reduced by DPH (Raines and Standaert, 1967). It was suggested that these phenomena (PTR, PTP and DRIVR) were related to the longlasting post-tetanic hyperpolarization (PTH) evoked after repetitive stimulation of nerves. Therefore the action of DPH on PTR, PTP or DRIVR nright be due to an action of DPH on PTH.
Electrogenicsodium pump
Post-tetanic hyperpolarization was also observed in mammalian non-myelinated nerve fibres (Ritchie and Straub, 1957; Rang and Ritchie, 1968). This hyperpolarization, following activity of the nerve fibres, appeared to reflect activity of an electrogenic sodium pump (Rang and Ritchie, 1968). Since the electrogenic component of the sodium pump can be activated with potassium, the method of eliciting potassium-activated responses in this preparation provides an opportunity to study the effects of drugs on the sodium pump, independently of their effect on the conduction mechanism itself (Rang and Ritchie, 1968; Den Hertog and Ritchie, 1969). The potassium-activated response (PAR) and the post-tetanic hyperpolffrization (PTH) were used to investigate the effects of diphenylhydantoin on the activity of the sodium pump, on the membrane permeability and on the excitability of mammalian nonmyelinated nerve fibres.
2. MATERIALS AND METHODS A desheathed cervical vagus of the rabbit was mounted in a sucrose-gap apparatus for measuring changes in the membrane potential (Stampfli, 1954;
A. Den Hertog, Diphenylhydantoin and the electrogenic sodium pump in C-fibres Straub, 1957). The preparation was perfused with a modified Locke solution (Den Hertog and Ritchie, 1969). Diphenylhydantoin (DPH; 1 0 g / l ) w a s dissolved in a sodium hydroxide solution (pH 11.0). The pH of the DPH containing Locke solution was brought to 7.3 with sulphuric acid. A saturated DPH Locke solution was found to contain 50 mg/l DPH 1 hr after preparation (pH 7.3; temp. 2 1 - 2 3 ° C ) and 20mg/1 after 100hr. The DPH content was determined using the method of Thurkow et al. (1972). The concentration of DPH used in the present in vitro experiments was 20 mg/l. In chronically treated rabbits, diphenylhydantoin (dissolved in a phosphate buffer, pH 11.0) was injected (i.p.) 3 times daily for 5 days prior to the experiment. The control animals were treated with the solvent alone, following the same procedure. The post-tetanic hyperpolarization and the potassium activated response were evoked and recorded as described by Den Hertog and Ras (1970). Submaxireal stimulation was performed by decreasing the stimulus intensity in such a manner that the posttetanic hyperpolarization was reduced to 60% of the amplitude observed after supra-maximal stimulation.
95
D.P.H K
I
K
10 mV1
10 rain.
10
5
E
2
1 r
i
1
i
i
2 3 time (rain.)
3. RESULTS
4
5
Fig. 1 3.1. Diphenylhydantoin and the potassium-activated response Amplitude and decay time constant of the PAR obtained in the presence of DPH were compared with the corresponding values obtained before exposure to the drug. Two responses taken at an interval of about 20 min are shown in fig. 1. When DPH, 20 rag/1, was added to the Locke solution (1 min after the PTH was elicited), no change in the membrane potential was observed. Addition of potassium, 2 mM, 11 min after the PTH, elicited a PAR; the exponential decay of the PAR and the amplitude before and during exposure to the drug are shown in the lower part of fig. 1. The amplitude and time constant of the PAR were identical in both cases (fig. 1, table 1). It was reported by Korey (1951) that DPH equilibrated in nerve fibres within 1 hr. Furthermore, Julien and Halpern (1970) observed a shortening in "the recovery period" after repetitive stimulation of sheathed C-fibres of rabbits during chronic exposure
Table 1 Effect of diphenylhydantoin, 20 mg/1, on the potassium-acti. vated response after a 10-min exposure. Experiment number
Amplitude a of 2mM K-activated response b
Time constant c of the response
8N 7A 25 N 7 A 1D7A 2D7A
0.99 0.99 1.00 0.98
1.00 0.95 0.96 0.95
Mean ± S.E.M.
0.99±0.01
0.97~ 0.02
a 1.00 = 6.1 mV. b Relative to values obtained before exposure to DPH. c 1.00 = 2.38 min.
96
A. Den Hertog, Diphenylhydantoin and the electrogenie sodium pump in C.fibres
to DPH (administered three times daffy for 5 days). This shortening in the "recovery period" was assumed to be correlated with the time course of decay o f the PTH. The behaviour of the nerve fibres during chronic exposure to DPH was investigated in rabbits treated with DPH following the procedure of Julien and Halpern (1970). After 5 days treatment with DPH, 20 mg/kg, the vagus nerves were removed and bathed in Locke solution with DPH, 20 mg/l throughout the experiment. The PAR was elicited 1 l rain after PTH by adding 2 mM potassium. In four experiments no difference was detected between the time constants of the response in DPH-treated (1.98 +- 0.08 rain) and non-treated animals (1.85 -+ 0.27 rain).
II
t
lOmVI --
D.P.H
Experiment number
Amplitude a of the Time constant c post-tetanic hyper- of the response polarization b
24 N 7 A 24 N 7 B 2D7A 8D7A 8D7B
1.05 0.99 1.03 0.95 1.05
1.07 1.00 1.02 0.98 0.96
Mean ± S.E.M.
1.01 +- 0.04
!.06 ± 0.04
a 1.00 -- 5.7 inV. b Relative to values obtained before exposure to DPH. c 1.00 --- 1.55 rain
3.2. Diphenylhydantoin and post-tetanic hyperpolarization The amplitude and the rate constant of the PTH elicited after repetitive stimulation of the nerve in 2 mM potassium Locke solution varied with the stimulus intensity. This was due to activation of only a proportion of the fibres by submaximal stimulation. It is only in these activated fibres that the sodium pump has to extrude excess sodium entering the fibres during the action potential. In the case o f submaximal stimulation, a change in the threshold of excitability of the fibres or in the action potential in one or more fibres, together with a changed sodium influx, would be reflected in a changed amplitude and time constant of the PTH (Den Hertog and Ritchie, 1969). To study the effect of DPH on these phenomena the nerve was exposed to DPH, 20 nag/l, 60 m i n . The PTH elicited by submaximal stimulation, before and after a 60-,rain exposure to DPH, is shown in fig. 2; the results of 5 experiments are given in table 2. No change in the amplitude or in the decay time constant was found.
j
#
Table 2 Effect of diphenylhydantoin, 20 mg/1, on the submaximal post-tetanic hyperpolarization after a 60-min exposure.
10 min. 15 10 ~
>
E
o" e~
E o
4. DISCUSSION !
0
1
l
w
2 3 time(rain.) t"ig. 2
i
l
/,
5
The amplitude and time constant o f the PAR reflect activity of the sodium pump whereas the amplitude of the PAR and the membrane potential are linked to membrane permeability (Den Hertog and
A. Den Hertog, Diphenylhydantoin and the electrogenic sodium pump in C-fibres Ritchie, 1969). Neither these parameters of the PAR nor the membrane potential were changed after acute or chronic treatment with DPH in the present experiments (fig. 1, table 1). From these results, it is concluded that DPH did not affect the activity of the sodium pump or the membrane permeability of the non-myelinated fibres of the desheathed vagus nerve. These observations are consistent with the unchanged intracellular sodium and potassium concentration in lobster nerve during treatment with DPH, as reported by Pincus et al. (1970). The properties of the Na/K ATP-ase system have been determined for many different tissues (Bonting, 1970) and found to be similar; the sodium pump is thought to be linked with this Na/K ATP-ase system (Skou, 1968). It is therefore unlikely, in view of the present observations, that the action of DPH and the stimulation of the sodium pump (Woodbury and Kemp, 1970; Julien and Halpern, 1970) are directly related. An indirect action of DPH on the sodium pump cannot be excluded from our experiments. It is possible that DPH changes the permeability of the sheath in sheathed nerves. The changed ionic environments of the fibres, produced after generation of the action potential, might stimulate the sodium pump. This indirect stimulation of the ionic transport system by DPH might explain the observations made by Esplin (1957), Parisi and Raines (1963), Raines and Standaert (1967) and Julien and Halpern (1970). A decrease in the membrane threshold would increase the number of fibres activated by submaximal stimulation, and accordingly the amplitude of the PTH would be increased and the decay time constant might also be changed. However, the lack of effect of DPH on the PTH makes is unlikely that the membrane threshold or the action potential are affected, which is in agreement with the findings of Korey (1951). ACKNOWLEDGEMENT I am much indebted to Miss I. Thurkow for the determination of the diphenylhydantoin concentrations. REFERENCES Bonting, S.L., 1970, Sodium-potassium activated adenosine triphosphatase and cation transport, in: Membranes and
97
Ion Transport, Vol. 1, ed. E.E. Bittar (Wiley-lnterscience, London) p. 257. Den Hertog, A. and R. Ras, 1970, The effect of some diuretics on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibres, European J. Pharmacol. 10, 249. Den Hertog, A. and J.M. Ritchie, 1969, The effect of some quaternary ammonium compounds and local anaesthetics on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibres, European J. Pharmacol. 6, 138. Den Hertog, A., P. Greengard and J.M. Ritchie, 1969, On the metabolic basis of nervous activity, J. Physiol. 204, 511. Esplin, D.W., 1957, Effects of diphenylhydantoin on synaptic transmission in cat spinal cord and stellate ganglion, J. Pharmacol. Exptl. Therap. 120, 301. Goodman, L.S. and A. Gilman, 1970, The Pharmacological Basis of Therapeutics (Macmillan, New York) p. 208. Julien, R.M. and L.M. Halpern, 1970, Stabilization of excitable membrane by chronic administration of diphenylhydantoin, J. Pharmacol. Exptl. Therap. 175,206. Korey, S.R., 1951, Effects of dilantin and mesantoin on the giant axon of the squid, Proc. Soc. Exptl. Biol. Med. 76, 297. Parisi, A.F. and A. Raines, 1963, Diphenylhydantoinsupression of repetitive activity generated in nerve endings, Federation Proc. 22, 390. Pincus, J.H., I. Grove, B.B. Marino and G.E. Glaser, 1970, Studies on the mechanism of action of diphenylhydantoin, Arch. Neurol. 22,566. Raines, A. and F.G. Standaert, 1967, An effect of diphenylhydantoin on post-tetanic hyperpolarization on intermedullary nerve terminals, J. Pharmacol. Exptl. Therap. 156, 591. Rang, H.P. and J.M. Ritchie, 1968, On the electrogenic sodium pump in mammalian non-myelinated nerve fibres and its activation by various external cations, J. Physiol. 196, 183. Ritchie, J.M. and R.W. Straub, 1957, The hyperpolarization which follows activity in mammalian non-medullated fibres, J. Physiol. 136, 80. Skou, J.C., 1968, Enzymatic basis for active transport of Na÷ and K÷ across cell membrane, Physiol. Rev. 45,596. Stampfli, R., 1954, A new method for measuring membrane potentials with external electrodes, Experienta 10, 508. Straub, R.W., 1957, Sucrose-gap apparatus for studying the resting potentials in mammalian non-medullated fibres, J. Physiol. 135, 2P. Thurkow, I., H. Wesseling and D.K.F. Meijer, 1972, Estimation of phenytoin in body fluids in the presence of sulphonylurea compounds. Clin. Chim. Acta, in the press. Woodbury, D.M., 1955, Effect of diphenylhydantoin on electrolytes and radiosodium turnover in brain and other tissues of normal, hyponatremic and postictal rats, J. Pharmacol. Exptl. Therap. 115, 74. Woodbury, D.M. and J.W. Kemp, 1970, Some possible mechanism of action of anti-epileptic drugs, Pharmakopsychiat. 3,201.
T H E E F F E C T O F D I P H E N Y L H Y D A N T O I N ON T H E E L E C T R O G E N I C C O M P O N E N T O F T H E S O D I U M PUMP IN M A M M A L I A N N O N - M Y E L I N A T E D N E R V E FIBRES
A. DEN HERTOG Department of Pharmacology, University of Groningen, Bloemsingel 1, The Netherlands
Received 3 May 1971
Accepted 1 March 1972
A. DEN ttERTOG, The effect of diphenylhydantoin on the electrogenic component of the sodhtm pump in mammalian non-myelinated nerve fibres, European J. Pharmacol. 19 (1972) 94-97. A study has been made of the effect of diphenylhydantoin on the electrogenic component of the sodium pump in non-myelinated fibres of the desheathed rabbit vagus nerve. Acute or chronic administration of this drug did not affect the excitability, nor the membrane permeability, nor the activity of the sodium pump of the nerve fibres. Diphenylhydantoin
C-fibres
1. INTRODUCTION The mechanism of action of the anticonvulsant drug, diphenylhydantoin (DPH), is still obscure although the results of some detailed studies have been reported. DPH intracellular sodium concentration braincells, as well as skeletal and cardiac muscle (Woodbury, 1955). Enhancement of the active sodium extrusion from brain cells by stimulation of the metabolic sodium pump might account for this effect (Woodbury and Kemp, 1970; Goodman and Gilman, 1970). Another significant finding concerning DPH was its ability to depress responses to repetitive stimulation, such as post-tetanic repetitive afterdischarges (PTR) in the motor nerve (Parisi and Raines, 1963) and post-tetanic potentiation (PTP) of the ventral root (Esplin, 1957). The potential reversal of the dorsal root IV (DRIVR) in the spinal cord of the cat was also reduced by DPH (Raines and Standaert, 1967). It was suggested that these phenomena (PTR, PTP and DRIVR) were related to the longlasting post-tetanic hyperpolarization (PTH) evoked after repetitive stimulation of nerves. Therefore the action of DPH on PTR, PTP or DRIVR nright be due to an action of DPH on PTH.
Electrogenicsodium pump
Post-tetanic hyperpolarization was also observed in mammalian non-myelinated nerve fibres (Ritchie and Straub, 1957; Rang and Ritchie, 1968). This hyperpolarization, following activity of the nerve fibres, appeared to reflect activity of an electrogenic sodium pump (Rang and Ritchie, 1968). Since the electrogenic component of the sodium pump can be activated with potassium, the method of eliciting potassium-activated responses in this preparation provides an opportunity to study the effects of drugs on the sodium pump, independently of their effect on the conduction mechanism itself (Rang and Ritchie, 1968; Den Hertog and Ritchie, 1969). The potassium-activated response (PAR) and the post-tetanic hyperpolffrization (PTH) were used to investigate the effects of diphenylhydantoin on the activity of the sodium pump, on the membrane permeability and on the excitability of mammalian nonmyelinated nerve fibres.
2. MATERIALS AND METHODS A desheathed cervical vagus of the rabbit was mounted in a sucrose-gap apparatus for measuring changes in the membrane potential (Stampfli, 1954;
A. Den Hertog, Diphenylhydantoin and the electrogenic sodium pump in C-fibres Straub, 1957). The preparation was perfused with a modified Locke solution (Den Hertog and Ritchie, 1969). Diphenylhydantoin (DPH; 1 0 g / l ) w a s dissolved in a sodium hydroxide solution (pH 11.0). The pH of the DPH containing Locke solution was brought to 7.3 with sulphuric acid. A saturated DPH Locke solution was found to contain 50 mg/l DPH 1 hr after preparation (pH 7.3; temp. 2 1 - 2 3 ° C ) and 20mg/1 after 100hr. The DPH content was determined using the method of Thurkow et al. (1972). The concentration of DPH used in the present in vitro experiments was 20 mg/l. In chronically treated rabbits, diphenylhydantoin (dissolved in a phosphate buffer, pH 11.0) was injected (i.p.) 3 times daily for 5 days prior to the experiment. The control animals were treated with the solvent alone, following the same procedure. The post-tetanic hyperpolarization and the potassium activated response were evoked and recorded as described by Den Hertog and Ras (1970). Submaxireal stimulation was performed by decreasing the stimulus intensity in such a manner that the posttetanic hyperpolarization was reduced to 60% of the amplitude observed after supra-maximal stimulation.
95
D.P.H K
I
K
10 mV1
10 rain.
10
5
E
2
1 r
i
1
i
i
2 3 time (rain.)
3. RESULTS
4
5
Fig. 1 3.1. Diphenylhydantoin and the potassium-activated response Amplitude and decay time constant of the PAR obtained in the presence of DPH were compared with the corresponding values obtained before exposure to the drug. Two responses taken at an interval of about 20 min are shown in fig. 1. When DPH, 20 rag/1, was added to the Locke solution (1 min after the PTH was elicited), no change in the membrane potential was observed. Addition of potassium, 2 mM, 11 min after the PTH, elicited a PAR; the exponential decay of the PAR and the amplitude before and during exposure to the drug are shown in the lower part of fig. 1. The amplitude and time constant of the PAR were identical in both cases (fig. 1, table 1). It was reported by Korey (1951) that DPH equilibrated in nerve fibres within 1 hr. Furthermore, Julien and Halpern (1970) observed a shortening in "the recovery period" after repetitive stimulation of sheathed C-fibres of rabbits during chronic exposure
Table 1 Effect of diphenylhydantoin, 20 mg/1, on the potassium-acti. vated response after a 10-min exposure. Experiment number
Amplitude a of 2mM K-activated response b
Time constant c of the response
8N 7A 25 N 7 A 1D7A 2D7A
0.99 0.99 1.00 0.98
1.00 0.95 0.96 0.95
Mean ± S.E.M.
0.99±0.01
0.97~ 0.02
a 1.00 = 6.1 mV. b Relative to values obtained before exposure to DPH. c 1.00 = 2.38 min.
96
A. Den Hertog, Diphenylhydantoin and the electrogenie sodium pump in C.fibres
to DPH (administered three times daffy for 5 days). This shortening in the "recovery period" was assumed to be correlated with the time course of decay o f the PTH. The behaviour of the nerve fibres during chronic exposure to DPH was investigated in rabbits treated with DPH following the procedure of Julien and Halpern (1970). After 5 days treatment with DPH, 20 mg/kg, the vagus nerves were removed and bathed in Locke solution with DPH, 20 mg/l throughout the experiment. The PAR was elicited 1 l rain after PTH by adding 2 mM potassium. In four experiments no difference was detected between the time constants of the response in DPH-treated (1.98 +- 0.08 rain) and non-treated animals (1.85 -+ 0.27 rain).
II
t
lOmVI --
D.P.H
Experiment number
Amplitude a of the Time constant c post-tetanic hyper- of the response polarization b
24 N 7 A 24 N 7 B 2D7A 8D7A 8D7B
1.05 0.99 1.03 0.95 1.05
1.07 1.00 1.02 0.98 0.96
Mean ± S.E.M.
1.01 +- 0.04
!.06 ± 0.04
a 1.00 -- 5.7 inV. b Relative to values obtained before exposure to DPH. c 1.00 --- 1.55 rain
3.2. Diphenylhydantoin and post-tetanic hyperpolarization The amplitude and the rate constant of the PTH elicited after repetitive stimulation of the nerve in 2 mM potassium Locke solution varied with the stimulus intensity. This was due to activation of only a proportion of the fibres by submaximal stimulation. It is only in these activated fibres that the sodium pump has to extrude excess sodium entering the fibres during the action potential. In the case o f submaximal stimulation, a change in the threshold of excitability of the fibres or in the action potential in one or more fibres, together with a changed sodium influx, would be reflected in a changed amplitude and time constant of the PTH (Den Hertog and Ritchie, 1969). To study the effect of DPH on these phenomena the nerve was exposed to DPH, 20 nag/l, 60 m i n . The PTH elicited by submaximal stimulation, before and after a 60-,rain exposure to DPH, is shown in fig. 2; the results of 5 experiments are given in table 2. No change in the amplitude or in the decay time constant was found.
j
#
Table 2 Effect of diphenylhydantoin, 20 mg/1, on the submaximal post-tetanic hyperpolarization after a 60-min exposure.
10 min. 15 10 ~
>
E
o" e~
E o
4. DISCUSSION !
0
1
l
w
2 3 time(rain.) t"ig. 2
i
l
/,
5
The amplitude and time constant o f the PAR reflect activity of the sodium pump whereas the amplitude of the PAR and the membrane potential are linked to membrane permeability (Den Hertog and
A. Den Hertog, Diphenylhydantoin and the electrogenic sodium pump in C-fibres Ritchie, 1969). Neither these parameters of the PAR nor the membrane potential were changed after acute or chronic treatment with DPH in the present experiments (fig. 1, table 1). From these results, it is concluded that DPH did not affect the activity of the sodium pump or the membrane permeability of the non-myelinated fibres of the desheathed vagus nerve. These observations are consistent with the unchanged intracellular sodium and potassium concentration in lobster nerve during treatment with DPH, as reported by Pincus et al. (1970). The properties of the Na/K ATP-ase system have been determined for many different tissues (Bonting, 1970) and found to be similar; the sodium pump is thought to be linked with this Na/K ATP-ase system (Skou, 1968). It is therefore unlikely, in view of the present observations, that the action of DPH and the stimulation of the sodium pump (Woodbury and Kemp, 1970; Julien and Halpern, 1970) are directly related. An indirect action of DPH on the sodium pump cannot be excluded from our experiments. It is possible that DPH changes the permeability of the sheath in sheathed nerves. The changed ionic environments of the fibres, produced after generation of the action potential, might stimulate the sodium pump. This indirect stimulation of the ionic transport system by DPH might explain the observations made by Esplin (1957), Parisi and Raines (1963), Raines and Standaert (1967) and Julien and Halpern (1970). A decrease in the membrane threshold would increase the number of fibres activated by submaximal stimulation, and accordingly the amplitude of the PTH would be increased and the decay time constant might also be changed. However, the lack of effect of DPH on the PTH makes is unlikely that the membrane threshold or the action potential are affected, which is in agreement with the findings of Korey (1951). ACKNOWLEDGEMENT I am much indebted to Miss I. Thurkow for the determination of the diphenylhydantoin concentrations. REFERENCES Bonting, S.L., 1970, Sodium-potassium activated adenosine triphosphatase and cation transport, in: Membranes and
97
Ion Transport, Vol. 1, ed. E.E. Bittar (Wiley-lnterscience, London) p. 257. Den Hertog, A. and R. Ras, 1970, The effect of some diuretics on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibres, European J. Pharmacol. 10, 249. Den Hertog, A. and J.M. Ritchie, 1969, The effect of some quaternary ammonium compounds and local anaesthetics on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibres, European J. Pharmacol. 6, 138. Den Hertog, A., P. Greengard and J.M. Ritchie, 1969, On the metabolic basis of nervous activity, J. Physiol. 204, 511. Esplin, D.W., 1957, Effects of diphenylhydantoin on synaptic transmission in cat spinal cord and stellate ganglion, J. Pharmacol. Exptl. Therap. 120, 301. Goodman, L.S. and A. Gilman, 1970, The Pharmacological Basis of Therapeutics (Macmillan, New York) p. 208. Julien, R.M. and L.M. Halpern, 1970, Stabilization of excitable membrane by chronic administration of diphenylhydantoin, J. Pharmacol. Exptl. Therap. 175,206. Korey, S.R., 1951, Effects of dilantin and mesantoin on the giant axon of the squid, Proc. Soc. Exptl. Biol. Med. 76, 297. Parisi, A.F. and A. Raines, 1963, Diphenylhydantoinsupression of repetitive activity generated in nerve endings, Federation Proc. 22, 390. Pincus, J.H., I. Grove, B.B. Marino and G.E. Glaser, 1970, Studies on the mechanism of action of diphenylhydantoin, Arch. Neurol. 22,566. Raines, A. and F.G. Standaert, 1967, An effect of diphenylhydantoin on post-tetanic hyperpolarization on intermedullary nerve terminals, J. Pharmacol. Exptl. Therap. 156, 591. Rang, H.P. and J.M. Ritchie, 1968, On the electrogenic sodium pump in mammalian non-myelinated nerve fibres and its activation by various external cations, J. Physiol. 196, 183. Ritchie, J.M. and R.W. Straub, 1957, The hyperpolarization which follows activity in mammalian non-medullated fibres, J. Physiol. 136, 80. Skou, J.C., 1968, Enzymatic basis for active transport of Na÷ and K÷ across cell membrane, Physiol. Rev. 45,596. Stampfli, R., 1954, A new method for measuring membrane potentials with external electrodes, Experienta 10, 508. Straub, R.W., 1957, Sucrose-gap apparatus for studying the resting potentials in mammalian non-medullated fibres, J. Physiol. 135, 2P. Thurkow, I., H. Wesseling and D.K.F. Meijer, 1972, Estimation of phenytoin in body fluids in the presence of sulphonylurea compounds. Clin. Chim. Acta, in the press. Woodbury, D.M., 1955, Effect of diphenylhydantoin on electrolytes and radiosodium turnover in brain and other tissues of normal, hyponatremic and postictal rats, J. Pharmacol. Exptl. Therap. 115, 74. Woodbury, D.M. and J.W. Kemp, 1970, Some possible mechanism of action of anti-epileptic drugs, Pharmakopsychiat. 3,201.