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EfEects of diphenylhydantoin and phenobarbital on voltage-clamped myelinated nerve1 W. S. N ~ e r n AND ~ a ~C~. B. FRANK Deparfrnenr of Pharmacology, Unioersity of Albertu, Edrnonron, Alta., Crarradu TBG 2H7 Received July 23, 1946 NEUMAN. K.S., and FRANK, G . B. 1974. Effects of diphenylhydantoin and phenobarbital on voltage-clamped n~yelilnatednerve. Can. J. Physiol. Pharmacol. 55, 42-47. Diphenylhydantoln (DPH) and phenobarbital (PB) have a selective action in blocking spontaneous activity in nerves made hyperexcitable by lowering the calcium concentration of the bathing medium (Rosenberg, P. and Bartels, E. 1967. J. Pharmacol. Exp. Ther. 155, 532-544.). To investigate this further, we examined the action of DPH and PB on voltageclamped single myelinated nerves at two difrerent calcium concentrations. In I.$ mM calcium Ringer, DPH reduced the sodium permeability (P,,) without affecting the potassium conductance (GI()or the voltage-dependent time constants of sodium activation (7,) and inactivation ( T ~ )and , potassium activation (7,). PB was similar to DPH except that in addition to reducing P,,, it shifted 7, in the direction of depolarization. When the calcium concentration was lowered to 0.36 m M , the curves relating T , , and T , to membrane potential were shifted in the direction of hyperpolarization, as expected. However, the addition of DPH or PB reduced or abolished these shifts. It is suggested that both DPH and PB stabilize hyperexcitable membranes by an action on the parameter nm, and that this may contribute to their antiepileptic action. NEUMAN, W. S. et FRANK,G . B. 1977. Effects of diphenylhydantoin and phenobarbital on voltage-clamped nlyelinated nerve. Can. J. Physiol. Pharmacol. 55, 42-47. La diphenylhydantoi'ne (DPH) et le phenobarbital (PB) ont une action sdlective dans le blocage de l'activite spontanke des nerfs rendus excitables par abaissement de la concentration en calcium du milieu d9incubation(Rosenberg, P. et Bartels, E. 1967. J . Pharmacol. Exp. Ther. 155, 532-544.). Dans un but d'investigations complementaires, nous etudions l'effet de la DPH et du PB sur les fibres myeliniskes isolkes dans des condition$ de 'voltage clamp', 21 deux concentrations diflkrentes en calcium. Dan5 un milieu de Riinger contenant 1 .$ m h l de calcium, la DPH djminue la perrntkbilite au sodium ( P N a )sans affecter la conductance au potassium (GK9 ou les constantes de temps dependantes du voltage pour les cystkmes d'activation (7,) et inactivation ( r h ) de Na+ et activation (7,) de Kf. L'effet du PS est semblable B celui de la DPH, except6 pour un deplacement de T,, dans la direction de la dtipolarisation, en plus de la diminution de P,,. Lorscque la concentration en calcium est abaisstie A 0.36 mM, les courbes rapportant 7,,, et T~ au potentiel de membrane soint diplackes dans la direction de l'hyperpolarisation. Cependant, l'addition de DPH ou de PB diminue ou abolit ces dtiplacements. I1 est suggerd que la BPH ou le PB stabilise les membranes hyperexcitables par action sur le paramktrr: m et que cela peut contribuer B leur action antikpileptique. [Traduit par le journal]
Introduction PB can spontaneous activity of giant axons (induced by lowering the external calcium and magnesium concentrations) at concentrations considerably lower f-T{ and &, respectively) than those necessary to block the action potential (Rosenberg and Bartels 196'7). Other drugs such as procaine and &lorprornazine can also abolish spontaneous activity DPH
ABBREVIATIONS: DPH, diphenylhydantoin; PB, phenobarbital. 1Assisted financially by grants from the MRCC. 2Present address: Faculty of Medicine, Memorial University of Newfoundland, St. John's, Nfid., Canada A 1 6 5S4.
but only at concentrations Gr of those necessary to block the action potential, suggesting that the a n t i c o n v ~ l ~ a nare t ~ rather specific in reducing Spontaneous . -,-, activity (Rosenberg and Bartels N6/).
Lipicky el a!. (1972) have suggested that DPH may be "1 effective anticonvul~antbecause it ~dlectivelyreduces the maximum sodium Conductance (EN,) without affecting the sodium system kinetics or the potassium system. Although a reduction in ffNs could account for the block of spontaneous activity by DPH, this does not explain the selectivity of DPH compared with 0 t h drugs such as procaine, which not only reduces gNabut also shifts the sodium activation
43
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NEUMAN AND FRANK
variable m in the direction of depolarization, tending to make the axon even less excitable (Taylor 1959). T o clarify the action of the anticonvulsants on nerve, we examined the actions of DPl-I and PB on voltage-clamped myelinated nerve fibres. Frog nodes d o not show spontaneous activity when the external calcium concentration is reduced as do squid axons (Fran kenhaeuser 1957), but the shifts of the voltage-dependent parameters which give rise to the squid axons spontaneous activity are present in the frog node (Hille 1968), making this a suitable nod el for investigation.
Methods Nerue Fibre Preparation A large single myelinated nerve fibre was isolated from the sciatic nerve of the frog Rana pipiem and one node was voltage-clamped using a procedure similar to that described by Noimner (1969). The temperature of the fibre was set between 2 and 6 "C,and held constant by cooling the nerve chamber. The node to be clamped was bathed in a small compartment (0.1 ml) to which different solutions could be added via a 2-cni3 syringe embedded in a heat exchanger (at the same temperature as the nerve chamber) so that the temperature of the incoming solution was similar to that ,of the node. Solutions
The Ringer solutio~lused had the following composition (in milliimolar): NaCI ( I 1I .8); KC1 (2.5); CaCI2 (1.8); glucose ( I I . I); tris(hydroxymethy1)aminonlethane free base (1). Isotonic KC1 (120 mM) was used for the end compartments of the nerve chamber. Preceding this treatment, the end compartments were filled with a 1;: formalin solution in isotonic KC1 for 30 s to prevent resistance shifts in these nodes (Hille 1967). The pH of all Ringer solutions was adjusted to between 7.1 and 7.2 at 20 "C by addition of hydrochloric acid, yielding a pH of 7.4-7.5 at 2-6 "C. Drug solutions were made by dissolving the drug to be used in Ringer's solution (while it was being made) and adjusting the pH to the above range. Drug solutions made up in this way were, therefore, hypertonic to normal Ringer's solution. DPH was made up by dissolving the drug in the solvent supplied by the manufacturer (propylene glycol and sodium hydroxide at pH 11.5) and then adding the required amount of this solution to the Ringer's solution. Solutions containing lower concentrations of calciutn were made by adding less 6aCI2 to the Ringer. The osnlolarity of all solutions was checked using a freezing-point depression osmometer (Advanced Instruments Inc., Newton Highlands, MA) and were found to lie between 220 and 250 mosmol. The following drugs were used for the research reported in this study: DPH, Park Davis; PB, British Drug House. Recording T o simplify the handling of large amounts of voltageclamp data, a PDP-8 E computer (Digital Equipment Corp., Maynard, MA) was used to record, process, and
store the voltage-clamp data for later analysis. A system similar to that described by Hille (1967) was used and is given in greater detail elsewhere (Neuman 1973). For each run, a series of 20 voltage pulses (test pulses) of 30 ms duration were applied to the node under investigation. Each test pulse varied from -75 mV (the holding potential) to +77 mV in amplitude, and was preceded by a 40-ms prepulse of 125 mV to remove all sodium inactivation (Dodge 1963). It should be noted that all voltages are on the E scale (inside voltage minus the outside voltage). After 20 pulses, the solution bathing the node was changed and 3-4 min later a new run was started. The computer was used to sample the applied voltages and resultant currents from the clamp apparatus and convert the analogue signals to digital values for storage on a digital magnetic tape recorder. During the prepulse, the voltage and current signals were each sampled 50 times and the averaged value of each signal was stored. During the test pulse, the current signal was sampled once every 30 ps for a period of 3 nit;, and thereafter both the voltage and current signals were sampled once every 300 ps. The reason for this is that the early current (the sodium current) changes very rapidly when the node is first depolarized, but after a few milliseconds the rate of change slows considerably so that the slower sampling rate is sufficient. After each test pulse, the digitized data are put onto magnetic tape and the computer resets and waits for the next start pulse. Anulysis Analysis of the data was carried out by graphical means in a sequence of several steps. The procedures used were similar to those of Dodge (1963) and Hille (1967). Early peak currents (sodium currents) and late steadystate currents (potassium currents) were obtained from plots of the membrane current versus time after the capacitive transients and leakage currents had been subtracted from the records. The capacitive transients were removed by taking the transient obtained when the test pulse was equal to the holding potential and scaling this to the magnitude of the test pulse. Because noise is also scaled up with this procedure, the transient was first digitally filtered. The leakage conductance was assumed to be ohmic and was obtained from the voltage and current records during the prepulse. The leakage current was then given by the product of the leakage coilductance and the test-pulse voltage. After carrying out the above subtractions and correcting for the 'attenuation artefact' (Dodge and Frankenhauser 1958) which varied between 10 and 30(x, the current-voltage relations could be drawn using the peak and steady-state currents. Separation of the sodium and potassium currents was carried out mathematically using the relation found by Dodge ( 1963): IK = A[1
- exp (-
t/?,)I4,
where IK is the potassium current, A is the amplitude factor, and 7, is the potassiuin system tinae constant. For each test pulse. A and T, can be found graphically from a log-log plot of the membrane current versus time after first subtracting the capacitive transient and the leakage current. I , can then be calculated and subtracted from the total current leaving the sodium current (I,,).
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CAN. J. PHYSHOE. PHAWMACOL. V O k . 55, 1977
FIG. 1. Plot of the peak (I,) and steady-state (I,,) currents versus membrane potential ( E ) before, during, and after 88 &f DPH. T = 4 "C.Node 83.
Ro. 2. Plot of the peak (I,) and steady-state (I,,) currents versus membrane potential ( E ) before, during, and after 18 ,A4 DPH. Note that for all three runs, the caciuna consentration had been reduced from 1.8 to 0.3fjmM. T = 6 ' ~Node . $3.
Fro. 3. Analysis of the sodium system kinetic parameters rh and 7m in DPH. The curves are plots of 7 , and a, versus membrane potential ( E ) .Note that the calcium concentration u7as 1.8 rnlW for the upper curves. The lower curves show the Ringer control (before), the 0.36 ml"k9 calcium cor~trol(before), and the test curves with 18 ,M DPH in 0.36 mM calciun-a Ringer. T = 6 "C. Node 83.
an'"."ted. Higher concentrations DpH mhf, two nodes) reduced both the steadv-state currents (SO'j;, of control) and peak currents If the sodium current obtained above is plotted semilog, (25(;/, of contro1). The leakage currents remain the kinetic parameter 71, (the time constant for sodium unchanged at all concentrations of DPH up t o inactivatio~~) can be obtained from the falling slope, and including 0. B 8 rn M . Because DPH was made whereas r V , (the time constant for the acti\lation of the sodiu~.a-n system) is obtained from the intersection of the trp with the commercial solvent which is supplied rising phase with a line parallel to, but log (0.5) below, the with the drug, control runs were carried out falling phase. The 7, and ah were used instead s f ~ i aand h using the solvent alone at three times the maxibecause they are easier to obtain while still reflecting mum concentration used in the experiments changes in the parameters. This completes the analysis sf the data to be reported in this paper. The errors have been with DHW0 However, no difference was found dealt with elsewhere (Dodge 1963; Hille 1967; Neun~nn between these results and those with Ringer without the solvent. 1973). To determine whether lowering the external calcium concentration would modify the response s f BPH, 18 pM DPH was applied to BPH The typical current-voltage relations (three nodes in 0.36 m M calcium Ringer. The currentnodes) before, during, and after 18 p M DIPWare voltage relations for one node Lire shown in shown in Fig. 1. As can be seen, only the peak Fig. 2. As with 1.8 mbf calcium Ringer, only the sodium currents (I,) are reduced to $07; s f peak sodium current is reduced (78Yhof control), control while potassium currents (B,,) are un- whereas the steady-state currents remain Ian-
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NEUMAN AND FRANK
FIG.4. Peak (1,) and steady-state (I,,) currents versus membrane potential ( E ) before, during, and after 0.5 mhf PB. T = 5 "6.Node 67.
changed. The poor recovery after drug treatment in 0.34 rn1ai.I calcium Ringer was ansually observed (three nodcs). The voltage-dependent sodium time constants are shown in Fig. 3. The upper curves show that B P H in B .S r n M calcium Ringer has no eEect on either T , or ~ 7,. A higher concentration of DPH (0.18 m!V) did appear to shift T, to the right, but these results were difficult to analyze because of the vcry small currents. Sincc r,, and r,, reflcct the values of m and h, and since the sodium current is a function of only rn, h, pNa(the maximum sodium permeability). and the driving potential (Dodge 1963), it may be concluded that only pKais reduced by DI'H (18 yM) in 1.8 m M calcium Ringer. This is in agreement with the findings of Lipicky et al. (1972). FTowever, BPH has a very difTerent action when the calcium concentration is reduced t o 0.36 I ~ MNodes . bathed in solutions of varied calcium concentration show shifts of 7, and 7, of about 20 mV per 10-fold change in calcium conccntrativn ( H i k 1968). In the experimcnts reportcd here, the shifts of T~ and T, varied from 8 to 12 rrlV for a fivefold reduction in calcium concentration. In the presence of 9 8 pM DIW, however, those shifts were reduced t o 5 mV. That is, DPf4 reduced the normal shifts of 7 h and T, in the direction of hyperpolarization. The T, (not shown) was not af-r'ected by DPH either in 1.8 or 0.36 m M calcium Ringer.
PB The effects of PB on thc node are similar in many ways to the action of pentobarbital on lobster and squid axorls (Blaustein 1968; Warahashi ed ol. 1949). Figure 4 is an example of the
e
R~nger( before l
tt
5x16'~PB
o Rlnqer i after I
FIG. 5. Analysis of sodium (P, and
-
yV1)
and potassium
(a,) system kinetics. Note the shift of s,, to the right in the
presence of 0.5 mha PB. T
5 "C. Node 67.
current-volta_~erelations whcre the peak and steady-state currents are given. The peak sodiun~ current was always redeiced by PB (75-8504 of control at 5 >( los4 M , and 50-70(yi1 of control at 1 x IOe3 M ) , while the potassiu~n current remaincd unchanged (three nodcs) or was reduced slightly (two nodes). The leakage current was unchanged. Unlike DBH but like pentobarbital (Blaustein 1968 ; Narahashi et (11. 19491, PB shifted 7 , t o the right, as seen in Fig. 5. The efi-ect of this is t o reduce the excitability of the node. Neither T~ nor T, was afl'ected, even at I rnM PB concentration. Tlre current-voltage relations for PB-treated nodes in 0.36 mM calcium Ringer were similar t o those in 1.8 m M calcium Ringer, as seen in %;if. 6. The kinetic tznalysis on this same node is shown in Fig. 7. With 1.8 mMcalcium Ringer a s control, there is no difference between the test solutio~land the controls. However, when this same node was bathed iri 0.34 m M calciun-a Ringer without drugs (not shown), the curves for both r h and 7,, were shifted along the voltage axis to the left by 9 and 10 mV, respectively. Thus, the shifts cxpested t o occur in the lower calcium solutio~lare abolished by 0.5 mM FB
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CAN. J. PHYSIOL. PHARMACOL. VOL. 55, 1977
FIG.6. Peak (I,) and steady-state (I,,) currents versus membrane potential. Note the Ringer controls (before and after) 1.8 mlT4 calcium. T 5 "C. Node 67.
-
(three nodes). Lower concentrations of PB (0.1 mM) which do not shift T , in 1.8 m M calcium Ringer did not abolish the shifts produced by low calcium. Additional experiments with intermediate concentrations of PI3 were tried, but far a number of reasons these experiments were unsuccessful and lack of time precluded further experilnents to obtain doseresponse curves.
Discussion Spontaneous activity occurs in nerve when there is a net inward flow of positive current at the resting membrane potential (Noble 1966). In squid axons and frog nodes, this occurs when gNam3h (E - Ex,) (where ENa is the sodium equilibrium potential) is greater in magnitude than the sum of the leakage and potassium currents (Woodbury 1969). Jn squid axons, this net inward current can be produced by holding the axon slightly depolarized or by reducing the calciulll concentration, which is functionally equivalent to depolarization (Frankenhaeuser and Hodgkiil 1957; Woodbury 1969). At the node, reducing the external calcium concentration will only produce a net inward positive current if tetraethylammoniunl is added to reduce the potassium currents (Bergman st al. 1968). Without tetraethylammonium, however, the threshold for inward current is still shifted to the left (compare Ringer before in Fig. 1 with 0.36 mM Ca2+ before in Fig. 2). Anticonvulsant drugs might, therefore, reduce spontaneous activity by any mechanism which would reduce gNam3h (E - ENa) to a value less
k
6.0 ,
-25
0
*25
+%
474
EImVI
FIG.7. Sodium ( T and ~ 7,) and potassium (7,) system kinetics versus membrane potential (E). These were obtained from the same suns shown in Fig. 6. Note the lack of a shift to the left by rl, and T, in the presence of PB. T = 6 "C. Node 67. 0.36 mM calcium
+
than the sum of the leakage and potassium currents. The results of the experiments reported here indicate that the parameter modified by drugs which block spontaneous firing is m. DPH or PB either reduced or prevented the expected shifts along the voltage axis in low external calcium, thereby producing a more stable resting condition. Although PNa was also reduced by both drugs, the efiect of this would be quite small compared with a change in m. The expected shift to thc left of rl,in low calciuln was also prevented by the drugs, but this would favor increased activity since the sodium system would be less inactivated. Assumiilg that these drugs act in a similar fashion on squid axons, their ability to selectively block spontaneous activity is probably due to an action on the parameter in and not gNa,as suggested by Lipicky et ul. (1972). The selectivity may arise from their ability to modify m before they have significant effects on Furthermore, since the coilcentration of DPH used (18 p M ) is similar to the plasma concentration reported for effective therapeutic control of epileptic seizures (Seeman 1975), it may be that BPH stabilizes
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NEUMAN AND FRANK
hyperexcitable neurons in the central nervous system by a similar mechanism. Since adenosine triphosphate can block spontaneously firing nerves and, in addition, is known to form a complex with calcium (Abood 1969), it has been proposed by Seenlan (1972) that 'negative anesthetics' such as DBI-I and PB might block spontaneously firing axons in a manner similar to that of ATP. That is, by forming crosslinks between the membrane and calcium, they could electrically stabilize the membrane. The shifts in the curves of peak sodiun~conductance versus membrane potential, and the slowing of the time to peak of the sodium current observed with pentobarbital could be explained in a similar manner (Blaustein 1968). It was postulated that the charged part of the pentobarbital molecule might bind calcium, whereas the lipid-soluble portion of the molecule would dissolve in the membrane lipids. The increased surface concentration of calcium would then produce shifts in kinetic parameters similar to those produced when the calciumconcentration is increased. There are, however, several difficulties with this explanation. First. pentobarbital does not appear to increase the binding of calcium t o membranes (Seeman t.t a!. 1971). Second, adenosine monophosphate and diphosphate are also able t o block spontaneous activity and yet are not very effective at complexing calcium (Kuperman e t ul. 1967). Third, if BBH and PB were to increase the degree of calcium binding near the external surface of the membrane, then both rh and 7 , should be shifted t o the right by these drugs in 1.8 m M calcium Ringer. In the experiments reported here, only 7, was altered by PB in 1.8 m M calcium Ringer and by very high concentrations of DPH. Thus, it would appear rather unlikely that an increase in calcium binding is the mechanism responsible for the abolition of spontaneous firing by these drugs. ABOOD,I. G., 1969. Calcium - adenosine triphosphate interaction and their significance in the excitatory membrane. In Neurosciences research. Vol. 2. Edited by S. Ehrenpreis and 0. 6 . Solnitzky. Academic Press Inc., New York. pp. 42-70. BERGMAN, C., NONNER,W., and STAMPFLI,R. 1968. Sustained spontaneous activity of Ranvier nodes induced by the combined action of TEA and lack of calcium. Pfluegers Arch. 302,2637. BLAUSTEIN. M. P. 1968. Barbiturates block sodium and potassium conductance increases in voltage-clamped lobster axons. J. Gen. Physiol. 51,293-307.
47
DODGE,F. A. 1963. A study of ionic permeability changes underlying excitation in myelinated nerve fibres of the frog. Ph.D. thesis, The Rockefeller Institute, Ann Arbor, University microfilms. DODGE.F. A., and FRANKENHAEUSER, B. 1958. Membrane currents in isolated frog nerve fibre under voltage clamp conditions. J. Physiol. (London), 143, 76-98. FRANKENHAEUSER, B. 1957. The effect of calcium on the myelinated nerve fibre. J. Physiol. (London), 137,245-260. B., and HODGKIN, A. L. 1957. The FRANKENHAEUSER, action of calcium on the electrical properties of squid axons. J. Physiol. (London), 137,2 18-244. HILLE, B. 1967. A pharmacological analysis of the ionic channels of nerve. Ph.D. thesis, The Rockefeller Institute, Ann Arbor, University microfilms. 1968. Charges and potentials at the nerve surfaces divalent ions and p H . J . Gen. Physiol. 51, 221-236. KUPERMAN, A. S., OKAMOTO, M., and GALLIN, E. 1967. Nucleotide action on spontaneous electrical activity of calcium deficient nerve. J. Cell. Physiol. 70, 257. LIPICKY.R. J., GILBERT,D. L., and STILLMAN, I. M. 1972. Diphenylhydantoin inhibition of sodium conductance in squie giant axon. Proc. Natl. Acad. Sci. U.S.A. 69, 1758-1760. NARAHASHI, T., MOORE,J. W., and POSTON,R. N. 1969. Anesthetic blocking of nerve membrane conductance by internal and external application. J. Neurophysiol. 1, 3-22. NEUMAN, R. S. 1973. Drug stabilization of excitable membranes. Ph.D. thesis, The University of Alberta, Edmonton, Alta. NOBLE, D. 1966. Applications of Hodgkin-Huxley equations to excitable tissues. Physiol. Rev. 74, 150. NONNER,W. 1969. A new voltage clamp method for Ranvier nodes. Pfluegers Arch. 309, 176-192. ROSENBURG, P., and BARTELS, E. 1967. Drug effects on the spontaneous electrical activity of the squid giant axon. J. Pharmacol. Exp. Ther. 155, 532-544. SEEMAN, P. 1972. The membrane actions of anesthetics and tranquilizers. Pharmacol. Rev. 24,582-655. 1975. Anticonvulsants. In Principles of medical pharmacology Toronto. Edited by P. Seeman and E. M. Sellars. University of Toronto Press, Toronto, Ont. pp. 239-241. SEEMAN, P., CHAU,M., GOLDBERG, M., SUAKS,T., and SAX,L. 1971. The binding of Ca2+to the cell membrane increased by volatile anesthetics (alcohol, acetone, ether) which induce sensitization of nerve or muscle. B.B.A. (Biochim. Biophys. Acta) Libr. 225,185-193. TAYLOR, R. E. 1958. Effects of procaine on electrical properties of squid giant axon. Am. J. Physiol. 196, 107 1-1078. WOODBURY, J. W. 1969. Biophysics of nerve membranes. i n Basic mechanisms of the epilepsies. Edited bv H. H . Jasper, A. A. Ward, and A. Pope. Little, Brown, and Co., Boston. pp. 41-70.
EfEects of diphenylhydantoin and phenobarbital on voltage-clamped myelinated nerve1 W. S. N ~ e r n AND ~ a ~C~. B. FRANK Deparfrnenr of Pharmacology, Unioersity of Albertu, Edrnonron, Alta., Crarradu TBG 2H7 Received July 23, 1946 NEUMAN. K.S., and FRANK, G . B. 1974. Effects of diphenylhydantoin and phenobarbital on voltage-clamped n~yelilnatednerve. Can. J. Physiol. Pharmacol. 55, 42-47. Diphenylhydantoln (DPH) and phenobarbital (PB) have a selective action in blocking spontaneous activity in nerves made hyperexcitable by lowering the calcium concentration of the bathing medium (Rosenberg, P. and Bartels, E. 1967. J. Pharmacol. Exp. Ther. 155, 532-544.). To investigate this further, we examined the action of DPH and PB on voltageclamped single myelinated nerves at two difrerent calcium concentrations. In I.$ mM calcium Ringer, DPH reduced the sodium permeability (P,,) without affecting the potassium conductance (GI()or the voltage-dependent time constants of sodium activation (7,) and inactivation ( T ~ )and , potassium activation (7,). PB was similar to DPH except that in addition to reducing P,,, it shifted 7, in the direction of depolarization. When the calcium concentration was lowered to 0.36 m M , the curves relating T , , and T , to membrane potential were shifted in the direction of hyperpolarization, as expected. However, the addition of DPH or PB reduced or abolished these shifts. It is suggested that both DPH and PB stabilize hyperexcitable membranes by an action on the parameter nm, and that this may contribute to their antiepileptic action. NEUMAN, W. S. et FRANK,G . B. 1977. Effects of diphenylhydantoin and phenobarbital on voltage-clamped nlyelinated nerve. Can. J. Physiol. Pharmacol. 55, 42-47. La diphenylhydantoi'ne (DPH) et le phenobarbital (PB) ont une action sdlective dans le blocage de l'activite spontanke des nerfs rendus excitables par abaissement de la concentration en calcium du milieu d9incubation(Rosenberg, P. et Bartels, E. 1967. J . Pharmacol. Exp. Ther. 155, 532-544.). Dans un but d'investigations complementaires, nous etudions l'effet de la DPH et du PB sur les fibres myeliniskes isolkes dans des condition$ de 'voltage clamp', 21 deux concentrations diflkrentes en calcium. Dan5 un milieu de Riinger contenant 1 .$ m h l de calcium, la DPH djminue la perrntkbilite au sodium ( P N a )sans affecter la conductance au potassium (GK9 ou les constantes de temps dependantes du voltage pour les cystkmes d'activation (7,) et inactivation ( r h ) de Na+ et activation (7,) de Kf. L'effet du PS est semblable B celui de la DPH, except6 pour un deplacement de T,, dans la direction de la dtipolarisation, en plus de la diminution de P,,. Lorscque la concentration en calcium est abaisstie A 0.36 mM, les courbes rapportant 7,,, et T~ au potentiel de membrane soint diplackes dans la direction de l'hyperpolarisation. Cependant, l'addition de DPH ou de PB diminue ou abolit ces dtiplacements. I1 est suggerd que la BPH ou le PB stabilise les membranes hyperexcitables par action sur le paramktrr: m et que cela peut contribuer B leur action antikpileptique. [Traduit par le journal]
Introduction PB can spontaneous activity of giant axons (induced by lowering the external calcium and magnesium concentrations) at concentrations considerably lower f-T{ and &, respectively) than those necessary to block the action potential (Rosenberg and Bartels 196'7). Other drugs such as procaine and &lorprornazine can also abolish spontaneous activity DPH
ABBREVIATIONS: DPH, diphenylhydantoin; PB, phenobarbital. 1Assisted financially by grants from the MRCC. 2Present address: Faculty of Medicine, Memorial University of Newfoundland, St. John's, Nfid., Canada A 1 6 5S4.
but only at concentrations Gr of those necessary to block the action potential, suggesting that the a n t i c o n v ~ l ~ a nare t ~ rather specific in reducing Spontaneous . -,-, activity (Rosenberg and Bartels N6/).
Lipicky el a!. (1972) have suggested that DPH may be "1 effective anticonvul~antbecause it ~dlectivelyreduces the maximum sodium Conductance (EN,) without affecting the sodium system kinetics or the potassium system. Although a reduction in ffNs could account for the block of spontaneous activity by DPH, this does not explain the selectivity of DPH compared with 0 t h drugs such as procaine, which not only reduces gNabut also shifts the sodium activation
43
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NEUMAN AND FRANK
variable m in the direction of depolarization, tending to make the axon even less excitable (Taylor 1959). T o clarify the action of the anticonvulsants on nerve, we examined the actions of DPl-I and PB on voltage-clamped myelinated nerve fibres. Frog nodes d o not show spontaneous activity when the external calcium concentration is reduced as do squid axons (Fran kenhaeuser 1957), but the shifts of the voltage-dependent parameters which give rise to the squid axons spontaneous activity are present in the frog node (Hille 1968), making this a suitable nod el for investigation.
Methods Nerue Fibre Preparation A large single myelinated nerve fibre was isolated from the sciatic nerve of the frog Rana pipiem and one node was voltage-clamped using a procedure similar to that described by Noimner (1969). The temperature of the fibre was set between 2 and 6 "C,and held constant by cooling the nerve chamber. The node to be clamped was bathed in a small compartment (0.1 ml) to which different solutions could be added via a 2-cni3 syringe embedded in a heat exchanger (at the same temperature as the nerve chamber) so that the temperature of the incoming solution was similar to that ,of the node. Solutions
The Ringer solutio~lused had the following composition (in milliimolar): NaCI ( I 1I .8); KC1 (2.5); CaCI2 (1.8); glucose ( I I . I); tris(hydroxymethy1)aminonlethane free base (1). Isotonic KC1 (120 mM) was used for the end compartments of the nerve chamber. Preceding this treatment, the end compartments were filled with a 1;: formalin solution in isotonic KC1 for 30 s to prevent resistance shifts in these nodes (Hille 1967). The pH of all Ringer solutions was adjusted to between 7.1 and 7.2 at 20 "C by addition of hydrochloric acid, yielding a pH of 7.4-7.5 at 2-6 "C. Drug solutions were made by dissolving the drug to be used in Ringer's solution (while it was being made) and adjusting the pH to the above range. Drug solutions made up in this way were, therefore, hypertonic to normal Ringer's solution. DPH was made up by dissolving the drug in the solvent supplied by the manufacturer (propylene glycol and sodium hydroxide at pH 11.5) and then adding the required amount of this solution to the Ringer's solution. Solutions containing lower concentrations of calciutn were made by adding less 6aCI2 to the Ringer. The osnlolarity of all solutions was checked using a freezing-point depression osmometer (Advanced Instruments Inc., Newton Highlands, MA) and were found to lie between 220 and 250 mosmol. The following drugs were used for the research reported in this study: DPH, Park Davis; PB, British Drug House. Recording T o simplify the handling of large amounts of voltageclamp data, a PDP-8 E computer (Digital Equipment Corp., Maynard, MA) was used to record, process, and
store the voltage-clamp data for later analysis. A system similar to that described by Hille (1967) was used and is given in greater detail elsewhere (Neuman 1973). For each run, a series of 20 voltage pulses (test pulses) of 30 ms duration were applied to the node under investigation. Each test pulse varied from -75 mV (the holding potential) to +77 mV in amplitude, and was preceded by a 40-ms prepulse of 125 mV to remove all sodium inactivation (Dodge 1963). It should be noted that all voltages are on the E scale (inside voltage minus the outside voltage). After 20 pulses, the solution bathing the node was changed and 3-4 min later a new run was started. The computer was used to sample the applied voltages and resultant currents from the clamp apparatus and convert the analogue signals to digital values for storage on a digital magnetic tape recorder. During the prepulse, the voltage and current signals were each sampled 50 times and the averaged value of each signal was stored. During the test pulse, the current signal was sampled once every 30 ps for a period of 3 nit;, and thereafter both the voltage and current signals were sampled once every 300 ps. The reason for this is that the early current (the sodium current) changes very rapidly when the node is first depolarized, but after a few milliseconds the rate of change slows considerably so that the slower sampling rate is sufficient. After each test pulse, the digitized data are put onto magnetic tape and the computer resets and waits for the next start pulse. Anulysis Analysis of the data was carried out by graphical means in a sequence of several steps. The procedures used were similar to those of Dodge (1963) and Hille (1967). Early peak currents (sodium currents) and late steadystate currents (potassium currents) were obtained from plots of the membrane current versus time after the capacitive transients and leakage currents had been subtracted from the records. The capacitive transients were removed by taking the transient obtained when the test pulse was equal to the holding potential and scaling this to the magnitude of the test pulse. Because noise is also scaled up with this procedure, the transient was first digitally filtered. The leakage conductance was assumed to be ohmic and was obtained from the voltage and current records during the prepulse. The leakage current was then given by the product of the leakage coilductance and the test-pulse voltage. After carrying out the above subtractions and correcting for the 'attenuation artefact' (Dodge and Frankenhauser 1958) which varied between 10 and 30(x, the current-voltage relations could be drawn using the peak and steady-state currents. Separation of the sodium and potassium currents was carried out mathematically using the relation found by Dodge ( 1963): IK = A[1
- exp (-
t/?,)I4,
where IK is the potassium current, A is the amplitude factor, and 7, is the potassiuin system tinae constant. For each test pulse. A and T, can be found graphically from a log-log plot of the membrane current versus time after first subtracting the capacitive transient and the leakage current. I , can then be calculated and subtracted from the total current leaving the sodium current (I,,).
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CAN. J. PHYSHOE. PHAWMACOL. V O k . 55, 1977
FIG. 1. Plot of the peak (I,) and steady-state (I,,) currents versus membrane potential ( E ) before, during, and after 88 &f DPH. T = 4 "C.Node 83.
Ro. 2. Plot of the peak (I,) and steady-state (I,,) currents versus membrane potential ( E ) before, during, and after 18 ,A4 DPH. Note that for all three runs, the caciuna consentration had been reduced from 1.8 to 0.3fjmM. T = 6 ' ~Node . $3.
Fro. 3. Analysis of the sodium system kinetic parameters rh and 7m in DPH. The curves are plots of 7 , and a, versus membrane potential ( E ) .Note that the calcium concentration u7as 1.8 rnlW for the upper curves. The lower curves show the Ringer control (before), the 0.36 ml"k9 calcium cor~trol(before), and the test curves with 18 ,M DPH in 0.36 mM calciun-a Ringer. T = 6 "C. Node 83.
an'"."ted. Higher concentrations DpH mhf, two nodes) reduced both the steadv-state currents (SO'j;, of control) and peak currents If the sodium current obtained above is plotted semilog, (25(;/, of contro1). The leakage currents remain the kinetic parameter 71, (the time constant for sodium unchanged at all concentrations of DPH up t o inactivatio~~) can be obtained from the falling slope, and including 0. B 8 rn M . Because DPH was made whereas r V , (the time constant for the acti\lation of the sodiu~.a-n system) is obtained from the intersection of the trp with the commercial solvent which is supplied rising phase with a line parallel to, but log (0.5) below, the with the drug, control runs were carried out falling phase. The 7, and ah were used instead s f ~ i aand h using the solvent alone at three times the maxibecause they are easier to obtain while still reflecting mum concentration used in the experiments changes in the parameters. This completes the analysis sf the data to be reported in this paper. The errors have been with DHW0 However, no difference was found dealt with elsewhere (Dodge 1963; Hille 1967; Neun~nn between these results and those with Ringer without the solvent. 1973). To determine whether lowering the external calcium concentration would modify the response s f BPH, 18 pM DPH was applied to BPH The typical current-voltage relations (three nodes in 0.36 m M calcium Ringer. The currentnodes) before, during, and after 18 p M DIPWare voltage relations for one node Lire shown in shown in Fig. 1. As can be seen, only the peak Fig. 2. As with 1.8 mbf calcium Ringer, only the sodium currents (I,) are reduced to $07; s f peak sodium current is reduced (78Yhof control), control while potassium currents (B,,) are un- whereas the steady-state currents remain Ian-
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NEUMAN AND FRANK
FIG.4. Peak (1,) and steady-state (I,,) currents versus membrane potential ( E ) before, during, and after 0.5 mhf PB. T = 5 "6.Node 67.
changed. The poor recovery after drug treatment in 0.34 rn1ai.I calcium Ringer was ansually observed (three nodcs). The voltage-dependent sodium time constants are shown in Fig. 3. The upper curves show that B P H in B .S r n M calcium Ringer has no eEect on either T , or ~ 7,. A higher concentration of DPH (0.18 m!V) did appear to shift T, to the right, but these results were difficult to analyze because of the vcry small currents. Sincc r,, and r,, reflcct the values of m and h, and since the sodium current is a function of only rn, h, pNa(the maximum sodium permeability). and the driving potential (Dodge 1963), it may be concluded that only pKais reduced by DI'H (18 yM) in 1.8 m M calcium Ringer. This is in agreement with the findings of Lipicky et al. (1972). FTowever, BPH has a very difTerent action when the calcium concentration is reduced t o 0.36 I ~ MNodes . bathed in solutions of varied calcium concentration show shifts of 7, and 7, of about 20 mV per 10-fold change in calcium conccntrativn ( H i k 1968). In the experimcnts reportcd here, the shifts of T~ and T, varied from 8 to 12 rrlV for a fivefold reduction in calcium concentration. In the presence of 9 8 pM DIW, however, those shifts were reduced t o 5 mV. That is, DPf4 reduced the normal shifts of 7 h and T, in the direction of hyperpolarization. The T, (not shown) was not af-r'ected by DPH either in 1.8 or 0.36 m M calcium Ringer.
PB The effects of PB on thc node are similar in many ways to the action of pentobarbital on lobster and squid axorls (Blaustein 1968; Warahashi ed ol. 1949). Figure 4 is an example of the
e
R~nger( before l
tt
5x16'~PB
o Rlnqer i after I
FIG. 5. Analysis of sodium (P, and
-
yV1)
and potassium
(a,) system kinetics. Note the shift of s,, to the right in the
presence of 0.5 mha PB. T
5 "C. Node 67.
current-volta_~erelations whcre the peak and steady-state currents are given. The peak sodiun~ current was always redeiced by PB (75-8504 of control at 5 >( los4 M , and 50-70(yi1 of control at 1 x IOe3 M ) , while the potassiu~n current remaincd unchanged (three nodcs) or was reduced slightly (two nodes). The leakage current was unchanged. Unlike DBH but like pentobarbital (Blaustein 1968 ; Narahashi et (11. 19491, PB shifted 7 , t o the right, as seen in Fig. 5. The efi-ect of this is t o reduce the excitability of the node. Neither T~ nor T, was afl'ected, even at I rnM PB concentration. Tlre current-voltage relations for PB-treated nodes in 0.36 mM calcium Ringer were similar t o those in 1.8 m M calcium Ringer, as seen in %;if. 6. The kinetic tznalysis on this same node is shown in Fig. 7. With 1.8 mMcalcium Ringer a s control, there is no difference between the test solutio~land the controls. However, when this same node was bathed iri 0.34 m M calciun-a Ringer without drugs (not shown), the curves for both r h and 7,, were shifted along the voltage axis to the left by 9 and 10 mV, respectively. Thus, the shifts cxpested t o occur in the lower calcium solutio~lare abolished by 0.5 mM FB
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CAN. J. PHYSIOL. PHARMACOL. VOL. 55, 1977
FIG.6. Peak (I,) and steady-state (I,,) currents versus membrane potential. Note the Ringer controls (before and after) 1.8 mlT4 calcium. T 5 "C. Node 67.
-
(three nodes). Lower concentrations of PB (0.1 mM) which do not shift T , in 1.8 m M calcium Ringer did not abolish the shifts produced by low calcium. Additional experiments with intermediate concentrations of PI3 were tried, but far a number of reasons these experiments were unsuccessful and lack of time precluded further experilnents to obtain doseresponse curves.
Discussion Spontaneous activity occurs in nerve when there is a net inward flow of positive current at the resting membrane potential (Noble 1966). In squid axons and frog nodes, this occurs when gNam3h (E - Ex,) (where ENa is the sodium equilibrium potential) is greater in magnitude than the sum of the leakage and potassium currents (Woodbury 1969). Jn squid axons, this net inward current can be produced by holding the axon slightly depolarized or by reducing the calciulll concentration, which is functionally equivalent to depolarization (Frankenhaeuser and Hodgkiil 1957; Woodbury 1969). At the node, reducing the external calcium concentration will only produce a net inward positive current if tetraethylammoniunl is added to reduce the potassium currents (Bergman st al. 1968). Without tetraethylammonium, however, the threshold for inward current is still shifted to the left (compare Ringer before in Fig. 1 with 0.36 mM Ca2+ before in Fig. 2). Anticonvulsant drugs might, therefore, reduce spontaneous activity by any mechanism which would reduce gNam3h (E - ENa) to a value less
k
6.0 ,
-25
0
*25
+%
474
EImVI
FIG.7. Sodium ( T and ~ 7,) and potassium (7,) system kinetics versus membrane potential (E). These were obtained from the same suns shown in Fig. 6. Note the lack of a shift to the left by rl, and T, in the presence of PB. T = 6 "C. Node 67. 0.36 mM calcium
+
than the sum of the leakage and potassium currents. The results of the experiments reported here indicate that the parameter modified by drugs which block spontaneous firing is m. DPH or PB either reduced or prevented the expected shifts along the voltage axis in low external calcium, thereby producing a more stable resting condition. Although PNa was also reduced by both drugs, the efiect of this would be quite small compared with a change in m. The expected shift to thc left of rl,in low calciuln was also prevented by the drugs, but this would favor increased activity since the sodium system would be less inactivated. Assumiilg that these drugs act in a similar fashion on squid axons, their ability to selectively block spontaneous activity is probably due to an action on the parameter in and not gNa,as suggested by Lipicky et ul. (1972). The selectivity may arise from their ability to modify m before they have significant effects on Furthermore, since the coilcentration of DPH used (18 p M ) is similar to the plasma concentration reported for effective therapeutic control of epileptic seizures (Seeman 1975), it may be that BPH stabilizes
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NEUMAN AND FRANK
hyperexcitable neurons in the central nervous system by a similar mechanism. Since adenosine triphosphate can block spontaneously firing nerves and, in addition, is known to form a complex with calcium (Abood 1969), it has been proposed by Seenlan (1972) that 'negative anesthetics' such as DBI-I and PB might block spontaneously firing axons in a manner similar to that of ATP. That is, by forming crosslinks between the membrane and calcium, they could electrically stabilize the membrane. The shifts in the curves of peak sodiun~conductance versus membrane potential, and the slowing of the time to peak of the sodium current observed with pentobarbital could be explained in a similar manner (Blaustein 1968). It was postulated that the charged part of the pentobarbital molecule might bind calcium, whereas the lipid-soluble portion of the molecule would dissolve in the membrane lipids. The increased surface concentration of calcium would then produce shifts in kinetic parameters similar to those produced when the calciumconcentration is increased. There are, however, several difficulties with this explanation. First. pentobarbital does not appear to increase the binding of calcium t o membranes (Seeman t.t a!. 1971). Second, adenosine monophosphate and diphosphate are also able t o block spontaneous activity and yet are not very effective at complexing calcium (Kuperman e t ul. 1967). Third, if BBH and PB were to increase the degree of calcium binding near the external surface of the membrane, then both rh and 7 , should be shifted t o the right by these drugs in 1.8 m M calcium Ringer. In the experiments reported here, only 7, was altered by PB in 1.8 m M calcium Ringer and by very high concentrations of DPH. Thus, it would appear rather unlikely that an increase in calcium binding is the mechanism responsible for the abolition of spontaneous firing by these drugs. ABOOD,I. G., 1969. Calcium - adenosine triphosphate interaction and their significance in the excitatory membrane. In Neurosciences research. Vol. 2. Edited by S. Ehrenpreis and 0. 6 . Solnitzky. Academic Press Inc., New York. pp. 42-70. BERGMAN, C., NONNER,W., and STAMPFLI,R. 1968. Sustained spontaneous activity of Ranvier nodes induced by the combined action of TEA and lack of calcium. Pfluegers Arch. 302,2637. BLAUSTEIN. M. P. 1968. Barbiturates block sodium and potassium conductance increases in voltage-clamped lobster axons. J. Gen. Physiol. 51,293-307.
47
DODGE,F. A. 1963. A study of ionic permeability changes underlying excitation in myelinated nerve fibres of the frog. Ph.D. thesis, The Rockefeller Institute, Ann Arbor, University microfilms. DODGE.F. A., and FRANKENHAEUSER, B. 1958. Membrane currents in isolated frog nerve fibre under voltage clamp conditions. J. Physiol. (London), 143, 76-98. FRANKENHAEUSER, B. 1957. The effect of calcium on the myelinated nerve fibre. J. Physiol. (London), 137,245-260. B., and HODGKIN, A. L. 1957. The FRANKENHAEUSER, action of calcium on the electrical properties of squid axons. J. Physiol. (London), 137,2 18-244. HILLE, B. 1967. A pharmacological analysis of the ionic channels of nerve. Ph.D. thesis, The Rockefeller Institute, Ann Arbor, University microfilms. 1968. Charges and potentials at the nerve surfaces divalent ions and p H . J . Gen. Physiol. 51, 221-236. KUPERMAN, A. S., OKAMOTO, M., and GALLIN, E. 1967. Nucleotide action on spontaneous electrical activity of calcium deficient nerve. J. Cell. Physiol. 70, 257. LIPICKY.R. J., GILBERT,D. L., and STILLMAN, I. M. 1972. Diphenylhydantoin inhibition of sodium conductance in squie giant axon. Proc. Natl. Acad. Sci. U.S.A. 69, 1758-1760. NARAHASHI, T., MOORE,J. W., and POSTON,R. N. 1969. Anesthetic blocking of nerve membrane conductance by internal and external application. J. Neurophysiol. 1, 3-22. NEUMAN, R. S. 1973. Drug stabilization of excitable membranes. Ph.D. thesis, The University of Alberta, Edmonton, Alta. NOBLE, D. 1966. Applications of Hodgkin-Huxley equations to excitable tissues. Physiol. Rev. 74, 150. NONNER,W. 1969. A new voltage clamp method for Ranvier nodes. Pfluegers Arch. 309, 176-192. ROSENBURG, P., and BARTELS, E. 1967. Drug effects on the spontaneous electrical activity of the squid giant axon. J. Pharmacol. Exp. Ther. 155, 532-544. SEEMAN, P. 1972. The membrane actions of anesthetics and tranquilizers. Pharmacol. Rev. 24,582-655. 1975. Anticonvulsants. In Principles of medical pharmacology Toronto. Edited by P. Seeman and E. M. Sellars. University of Toronto Press, Toronto, Ont. pp. 239-241. SEEMAN, P., CHAU,M., GOLDBERG, M., SUAKS,T., and SAX,L. 1971. The binding of Ca2+to the cell membrane increased by volatile anesthetics (alcohol, acetone, ether) which induce sensitization of nerve or muscle. B.B.A. (Biochim. Biophys. Acta) Libr. 225,185-193. TAYLOR, R. E. 1958. Effects of procaine on electrical properties of squid giant axon. Am. J. Physiol. 196, 107 1-1078. WOODBURY, J. W. 1969. Biophysics of nerve membranes. i n Basic mechanisms of the epilepsies. Edited bv H. H . Jasper, A. A. Ward, and A. Pope. Little, Brown, and Co., Boston. pp. 41-70.
