Electrophysiologic and voltage clamp analysis of the effects of sotalol on isolated cardiac muscle and Purkinje fibers

The effect of dl-, d- and I-sotalol on electrophysiologic characteristics of guinea-pig papillary muscles, sheep and rabbit Purkinje fibers was studied. Standard electrophysiologic and voltage clamp techniques were used. At concentrations between 10(-6) and 10(-4) M, the main effect of sotalol consisted of prolongation of the action potential duration. In voltage clamp experiments this effect correlated with a substantial reduction of the time-dependent K current activated during the plateau of the action potential and a small reduction of the background K current. At concentrations above 10(-4) M, a secondary shortening of the action potential concomitant with a fall in maximal rate of depolarization was seen. In voltage clamp experiments this effect correlated with a decrease of a slowly inactivating Na current. In the absence of catecholamines d- and I-sotalol exerted identical effects on action potentials and voltage clamp currents.     

Adenosine and prostacyclin independent electrophysiological effects of dipyridamole in guinea-pig papillary muscles and canine cardiac Purkinje fibers

Dipyridamole was initially introduced as a coronary vasodilator. The exact mechanism of action of dipyridamole on the coronary vasculature is unknown, but proposed mechanisms of action include inhibition of adenosine uptake, increased myocardial prostacyclin production and inhibition of phosphodiesterase activity. The purpose of our study was to examine the electrophysiological effects of dipyridamole on guinea-pig papillary muscles and canine cardiac Purkinje fibers to determine whether similar mechanisms might account for the electrophysiological effects of this compound. Conventional microelectrode techniques were used to record transmembrane action potentials from either guinea-pig papillary muscles or canine cardiac Purkinje fibers. Dipyridamole produces a dose-dependent prolongation of action potential duration with a threshold concentration of approximately 5 X 10(-7) M in tissues from either species. Dipyridamole (10(-5) M) increases action potential amplitude (124 +/- 1 to 127 +/- 1 mV), increases action potential duration (119 +/- 6 to 146 +/- 5 msec) and produces hyperpolarization of the resting potential (-85 +/- 1 to -87 +/- 1 mV) in guinea-pig papillary muscles (n = 27, P less than .05). Dipyridamole (10(-5) M) increases action potential duration (276 +/- 5 to 293 +/- 5 msec) in canine cardiac Purkinje fibers (n = 21, P less than .05). The effects of dipyridamole (5 X 10(-7) M) are neither accentuated by adenosine (10(-4) M) nor attenuated by adenosine deaminase (1 U/ml) Pretreatment with indomethacin (10(-5) M) does not block these effects. Dipyridamole (10(-5) M) produces a negative chronotropic response in canine Purkinje fibers, increases mean escape intervals from 4.9 +/- 0.9 to 7.8 +/- 1.4 sec (n = 8, P less than .05) and fails to suppress slow response action potentials in 22 mM K+ depolarized tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of verapamil on rapid Na channel-dependent action potentials of K+-depolarized ventricular fibers.

We studied the effects of 1 to 3 micrograms/ml of verapamil on Vmax and plateau duration in guinea-pig papillary muscles depolarized from -90 to -55 mV by increasing extracellular potassium from 5.4 to 20 mM. Under the conditions of our experiments, we found that verapamil did not influence the steady-state or recovery characteristics of Vmax at any of the studied K levels even when Vmax was less than 20 V/sec. Thus in the absence of rapid sodium channel blockade, the value of Vmax cannot be used to identify slow channel-dependent action potentials. Verapamil caused no shortening of plateau or total action potential duration when potassium was less than 7.5 mM. Above this level, verapamil caused progressive shortening of plateau and total action potential duration, due to the increase in potassium and not the associated decrease in resting membrane potential. Increasing extracellular calcium shortened plateau duration at all levels of potassium before verapamil but lengthened plateau duration in the high K, verapamil-treated fibers. These results, which can be explained by known effect of verapamil on the slow outward as well as slow inward currents, provide a mechanism whereby verapamil may increase Vmax of K-depolarized but rapid sodium current-dependent premature action potentials.     

Ibutilide, a new compound with potent class III antiarrhythmic activity, activates a slow inward Na+ current in guinea pig ventricular cells.

Ibutilide, a class III antiarrhythmic compound under clinical development, was tested for specific activity on inward currents in guinea pig ventricular cells. Current clamp experiments showed a bell-shaped, dose-dependent effect of the compound on action potential duration and plateau height at concentrations beginning at 10(-9) M. In voltage clamp experiments in which the cells were bathed in K(+)-free solutions that contained external Na+0 and Ca++0, ibutilide, at 10(-8) M, increased a late inward current without affecting the fast inward Na+ current. The late inward current amplitudes, measured at 60 and 600 msec into a +20-m V step from -80 mV, were increased, respectively, from 0.73 +/- 0.07 to 1.1 +/- 0.06 nA, and from 0.02 +/- 0.02 to 0.15 +/- 0.03 nA, with a corresponding Kd of 2.3 x 10(-9) and 1.3 x 10(-9) M. However, if Na+0 was removed via appropriate ion substitution, the effect of ibutilide on the late inward current disappeared. On the other hand, when inward currents were maximized by the drug in Na+0 and Ca++0 containing external solution, removal of Na+0 reversibly removed a prominent late inward current which, when isolated from the L-type Ca++ current by current subtraction method, displayed a peak current potential of +30 mV and two decaying time constants of 38.2 +/- 3.8 and 200.8 +/- 43.3 msec at +20 mV. We concluded that ibutilide prolonged action potential duration through activation of a slow inward Na+ current. The identity of this current was discussed.     

Electrophysiological study of cibenzoline in voltage-clamped rabbit sinoatrial node preparations

Effects of cibenzoline (0.1-30 microM) on membrane potentials and currents were investigated in rabbit sinoatrial node cells using conventional microelectrode and double microelectrode voltage clamp methods. Cibenzoline reduced the heart rate, the maximum rate of rise and the action potential amplitude in a dose-dependent manner. At the same time, the action potential duration at half-amplitude was prolonged, and the slope of phase 4 depolarization was decelerated. The voltage clamp experiment revealed that cibenzoline (7-30 microM) depressed the slow inward current, the time-dependent potassium outward current and the hyperpolarization activated current. Neither the kinetic variable of the time-dependent potassium outward current nor the decay time of the outward current tail was altered by the drug perfusion. The results suggest that cibenzoline exerts a depressant effect on the electrical activity of sinoatrial node cells and that the depression of the currents induced by cibenzoline is due to a reduction in conductances of the current systems.     

Stimulation of cardiac slow Ca++ current by high concentration of cimetidine

Electrophysiological studies on the cardiac effects of a H2-antagonist, cimetidine, were examined in three kinds of preparations: guinea-pig papillary muscles, rat left atria and perfused chick hearts. Cimetidine (10(-5) to 10(-4) M) depressed or abolished the slow action potentials (APs) induced by histamine (10(-5) to 10(-4) M) in hearts whose fast Na+ channels had been inactivated by 25 mM K+. Higher concentrations of cimetidine (10(-3) to 5 X 10(-3) M) increased myocardial cyclic AMP level and allowed the generation of slow APs in such inexcitable tissues. These cimetidine-induced slow APs were not prevented by propranolol (10(-6) to 10(-5) M) or pretreatment with 6-hydroxydopamine (50 mg/kg). These results suggest that cimetidine, in doses higher than that required to block cardiac H2-receptors, may have a cardio-stimulating action mediated through increase of inward Ca++ current.     

Voltage and current clamp studies of muscarinic and nicotinic excitation of the rat adrenal chromaffin cells

Characteristics of the muscarinic and nicotinic excitation of chromaffin cells that had been freshly isolated from the rat adrenal medullae were analyzed using voltage and current clamp techniques. A dose-dependent increase in the extracellularly recorded firing of cells was observed when 10(-6) to 10(-4) M acetylcholine (ACh) were locally applied to the cells in the vicinity of the target cell being recorded using a microinflow method. During voltage clamp recording at the resting membrane potential, ACh induced two different sequential inward currents: a transient current with a rapid rising phase (fast response) and an apparent inward current with a slow rising phase (slow response). The membrane conductance increased during the ACh-induced fast response, and it subsequently decreased during the slow response. The amplitude of the fast response decreased when the holding potential was shifted to depolarized levels, whereas the amplitude of the slow response increased with depolarization. Nicotine produced fast depolarization and a transient inward current that was reduced by the membrane depolarization. In contrast, muscarine induced a slow depolarization and an apparent inward current that increased with depolarization. Muscarine also reduced the inward K+ current that had been induced by the application of a high K+ medium to the outside of the cell at the resting membrane potential. It is suggested that muscarinic excitation is triggered by the suppression of K+ channels that are open at potentials near the resting membrane potential. The present results indicate that ACh-induced excitation of adrenal chromaffin cells involves two separate mechanisms mediated by nicotinic and muscarinic receptors.     

Activation of ATP-sensitive outward K+ current by nicorandil (2-nicotinamidoethyl nitrate) in isolated ventricular myocytes

The vasodilatory agent, nicorandil (2-nicotinamidoethyl nitrate) activates an outward K+ current in cardiac and vascular smooth muscle. This current was studied with the patch clamp technique using isolated guinea pig and rabbit ventricular myocytes. Nicorandil (10(-5) and 10(-4) M) shortened the action potential duration without any significant change in the resting membrane potential. Under voltage clamp, nicorandil increased the time-independent outward current at potentials positive to -80 mV, and decreased the inward current at potentials negative to -90 mV. The drug did not affect Ca++ current activated upon depolarization from the holding potential of -30 mV, or did it influence delayed outward K+ current on repolarization. In rabbit myocytes, nicorandil did not increase the Ca++-sensitive and -insensitive transient outward K+ currents. When the ATP concentration of the pipette solution was reduced from 5 to 2 to 3 mM, nicorandil produced a large increase in outward current, which decreased slightly with time. The increased outward current was antagonized by raising the intracellular ATP concentration. Nicorandil increased the probability of opening of the ATP-sensitive single channel current without affecting its unitary amplitude. These results indicate that nicorandil activates the ATP-sensitive K+ current, which is responsible for shortening of the action potential duration.     

Effects of diacetyl monoxime on cardiac excitation-contraction coupling

Diacetyl monoxime (DAM) is a negative inotropic agent. To identify the mechanism of its actions, electrical and mechanical studies with various cardiac tissues were carried out. DAM (0.2-20 mM) inhibited the contractile force in both normal and 22 mM KCl-depolarized (in presence of 10(-6) M isoproterenol) guinea-pig papillary muscles in a concentration-dependent manner. In general, there was a lack of major effects of DAM on sarcolemmal electrical properties. The fast action potentials were somewhat depressed and the slow action potentials were slightly enhanced. In chemically skinned pig ventricular muscles, the myofibrillar contraction induced in 6.25 pCa was inhibited by DAM in a similar concentration range. DAM also produced an apparent decrease in sensitivity toward Ca++ in this preparation. Myofibrillar adenosine triphosphatase assay showed similar results as in the skinned muscles. All DAM effects were reversible upon washout and could be partially antagonized by raising [Ca++]. Taken together, the negative inotropic effect of DAM cannot be ascribed to an inhibitory effect on the slow inward current, as suggested previously. An inhibitory effect at the myofibril level is a distinct possibility. Additional effects of DAM on the sarcoplasmic reticulum cannot be ruled out.     

Effects of lidocaine and on slow response and depressed fast response action potentials of canine cardiac Purkinje fibers

Disease may decrease resting potential of cardiac fibers, thereby depressing the upstroke velocity of the action potential, causing slow conduction and reentry. A decrease in resting potential may also cause automaticity. We studied the effects of lidocaine (5 and 20 mg/l) on canine Purkinje fibers with reduced membrane potentials with either depressed Na+-dependent upstrokes (depressed fast responses) or with slow inward (Ca++) current-dependent upstrokes (slow responses). Depressed fast responses were produced by elevating [K+]0 in the perfusate, reducing membrane potential to around -60 mV, without abolishing excitability. Slow responses were produced by either perfusing fibers with a Na+-free, Ca++-rich solution, or by perfusing them with a high [K+]0 Tyrode's solution containing norepinephrine. Lidocaine had a marked depressant effect on depressed fast response action potentials. The drug markedly decreased Vmax and conduction velocity. It sometimes decreased action potential amplitude and caused conduction block. Resting potential was not changed. On the other hand, lidocaine had little effect on slow response action potentials. Resting potential, Vmax and action potential amplitude were not altered nor was conduction changed. The rate of spontaneous impulse initiation was slightly reduced by 5 mg/l of lidocaine but not by 20 mg/l. We conclude that lidocaine does not exert its antiarrhythmic effect by directly depressing the slow inward current but may be antiarrhythmic because it depresses an already depressed fast inward current and can cause conduction block.     

Muscarine depolarizes rat substantia nigra zona compacta and ventral tegmental neurons in vitro through M1-like receptors

Intracellular recordings were made from presumed dopamine-containing neurons in slices of rat mesencephalon. Muscarine (3-100 microM) increased the rate of spontaneous action potentials; it also caused a membrane depolarization and, in voltage-clamp, an inward current. Concentration-effect curves to muscarine were shifted rightwards by pirenzepine (0.03-1 microM) with an estimated KD of 14 nM. The inward current caused by muscarine was voltage-dependent. Between about -50 and (-)-65 mV it was associated with a decrease in membrane conductance, but between -70 and -110 mV it was unaccompanied by any change in membrane conductance. Muscarine was without effect on the action potential afterhyperpolarization, or on a slowly developing inward current evoked by step hyperpolarizations of up to 20 mV from -45 mV. Muscarinic depolarizations or inward currents were reduced reversibly or abolished by a low calcium (0.25 mM)/high magnesium (10 mM) solution. It is concluded that muscarinic excitation of dopaminergic neurons is mediated by M1-like receptors.     

Alpha adrenoceptor stimulation reduces outward currents in rat ventricular myocytes

The effects of alpha adrenoceptor stimulation with noradrenaline were investigated in rat ventricular myocytes after blockade of beta receptors with propranolol (1 microM). At room temperature and low stimulation frequency (0.5 Hz), noradrenaline evoked a phentolamine-sensitive increase in contraction amplitude by 22%. The action potentials of myocytes were prolonged. When the sodium current was inactivated by depolarization in whole-cell voltage clamp experiments, noradrenaline caused a small, but highly variable increase in net inward current and shifted the current-voltage relation between -30 and -5 mV to the hyperpolarizing direction. These effects were absent when K+ currents were inhibited by Cs+ substitution. After inhibition of the Ca++ current with Cd++ (0.1 mM), noradrenaline decreased the peak transient outward current; it reduced the steady-state outward current in a concentration-dependent manner (pD2 value, 6.9), but had no effect on the amplitude of the transient component of outward current. Noradrenaline reduced holding current at -40 mV. The inward branch of the inward rectifier was not affected. The noradrenaline-induced changes in membrane currents were significantly smaller in the presence of phentolamine (10 microM). They are therefore considered to be mediated by alpha adrenoceptor stimulation. The reduction in outward currents can explain the prolongation in action potential duration which could contribute to the increase in contractility.     

Time- and voltage-dependent block of the delayed K+ current by quinidine in rabbit sinoatrial and atrioventricular nodes

The modes in which quinidine blocks the delayed K+ current (IK) of rabbit sinoatrial and atrioventricular nodes were investigated by voltage clamp experiments using small preparations. Depolarizing pulses were applied from a holding potential of -50 mV and resultant IK current was evaluated. At a concentration of 2 x 10(-6) M, quinidine blocked 52 +/- 5% of IK with a 1000-msec test pulse of 0 mV, whereas it inhibited the slow inward current by only 5 to 10%. IK inhibition was enhanced with increasingly larger depolarizations. The activation curve obtained with the use of 1000-msec test pulses shifted toward hyperpolarization by 3.0 mV and its slope factor increased from 7.3 to 8.8, suggesting a voltage-dependent mechanism for IK blockage. Short and small depolarizing pulses from the holding potential of -50 mV hardly affected II. The activation of IK in the presence of quinidine did not show any delay. These results indicated a low affinity of quinidine to the closed (deactivated) channels. The deactivation time constant of IK was significantly prolonged from 120 to 145 msec at -50 mV. By applying the modulated receptor model to the K+ current, the tail current in the presence of quinidine was well reconstructed when the time constant of drug-channel interactions was 300 msec at -50 mV and 2000 msec at -30 mV. It is suggested that the unblocking of quinidine slows the decay of the IK tail current.     

Mechanism of inotropic action of xestoquinone, a novel cardiotonic agent isolated from a sea sponge.

Xestoquinone (XQN) isolated from the sea sponge Xestospongia sapra produced dose-dependent cardiotonic effects on guinea pig left and right atria. A direct action of XQN (1-30 microM) on the contractile machinery of cardiac myofilaments was demonstrated in chemically skinned fiber preparations from guinea pig papillary muscles. In atrial preparations, the XQN-induced inotropic effect was markedly inhibited by verapamil or nifedipine, but was not affected by practolol, chlorpheniramine, cimetidine, tetrodotoxin or reserpine. The Ca++ dependence curve for the contractile response of the atria was substantially shifted to the left by XQN (10 microM), and this XQN-induced shift was reversed by verapamil. The time-to-peak tension and relaxation times of the atrial contractions were shortened by XQN, and the action potential duration was markedly prolonged. Whole-cell patch clamp recordings in left atrial strips confirmed that XQN (30 microM) increased the slow inward current. However, there was a temporal dissociation between altered tension development and prolongation of the action potential duration. Cyclic AMP phosphodiesterase activity was inhibited and tissue cyclic AMP content of guinea pig left atria was increased by XQN (0.3-10 microM) in a concentration-dependent manner, but increases in cyclic AMP content did not occur in parallel with increases in contractile response. These observations suggest that an enhancement of intracellular cyclic AMP content and Ca++ influx across the cell membrane contribute to the late phase of XQN-caused cardiotonic responses, whereas the early phase may largely be elicited through direct activation of contractile elements. XQN may provide a novel leading compound for valuable cardiotonic agents.     

Characterization of the block of sodium channels by phenytoin in mouse neuroblastoma cells

The interaction of phenytoin (DPH) with membrane ionic channels of cultured N1E-115 neuroblastoma cells was studied. The single suction pipette technique was used for voltage clamp and intracellular perfusion. When the cells were held at -80 mV for periods of 1 min or more, DPH (20-100 microM) inhibited inward sodium current in a dose-dependent manner (resting block); resting block was relieved by hyperpolarizing cells to -100 mV for 1 min. A hyperpolarizing shift of the slow inactivation curve for the Na current was induced by DPH and can explain the effect of holding potential on the resting block. The fast Na inactivation curve, however, was not affected. During repetitive pulsing the DPH-induced inhibition of Na current was enhanced (conditioned block). Conditioned block was both voltage- and frequency-dependent. Conditioning pulses to potentials which do not appreciably open Na channels also produced conditioned block; prolongation of conditioning pulses even to durations longer than the time for maximal steady-state inactivation of the Na current progressively increased the extent of conditioned block, suggesting that DPH can interact with inactivated and closed Na channels. The time course of recovery from voltage-dependent inactivation of sodium current during conditioned block was both slowed and exhibited voltage dependence. Recovery occurred faster when membrane potential during the recovery period was more negative. We conclude that DPH blocks Na channels both by increasing the fraction of channels in an inactivated state and by delaying the transition from inactivated to closed but available channels. This effect is enhanced by depolarizing membrane potential and increasing the frequency of stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of chlorpromazine block of sodium channels in single guinea pig cardiac myocytes

Block of sodium current by chlorpromazine in single ventricular myocytes isolated from guinea pigs was studied using the whole cell patch clamp technique. Chlorpromazine in micromolar concentrations reduced the amplitude of peak sodium current associated with step depolarizations from a holding potential of -140 mV. Concentration-response curves obtained with a holding potential of -140 mV were best fit by a 2:1 stoichiometry, and were shifted in the direction of lower concentrations when a holding potential of -100 mV was used. In agreement with this observation, the steady-state inactivation curve was shifted to more negative potentials by chlorpromazine. The block was not associated with any change in the time course of sodium current activation or inactivation during a depolarizing step. Chlorpromazine also produced marked use-dependent block as demonstrated by a cumulative increase in the block during a train of depolarizing pulses. This use dependence was due to a higher affinity of chlorpromazine for the inactivated state of sodium channels than for the resting state and to a very slow repriming of the drug-bound sodium channels from inactivation. These blocking actions could contribute to the antiarrhythmic effects of chlorpromazine at low concentrations and to the cardiotoxic effects at high concentrations.     

Voltage- and time-dependent block of the delayed K+ current in cardiac myocytes by dofetilide.

The delayed K+ current (ik) and its change by dofetilide was studied in single myocytes from the guinea pig and rabbit heart using the two-electrode voltage clamp technique. In rabbit myocytes, ik consisted of only one component (Kr), which developed for moderate depolarizations and with a fast time course. In guinea pig myocytes, activation consisted of a rapid and a slow component, and the latter (Ks) only became manifest for depolarizations positive to 0 mV. Ks was resistant to block by dofetilide. Kr, however, was very sensitive: Kd 3.9 x 10(-9) M, Hill coefficient 2.0 (n = 5). The effect was voltage-dependent block increasing at depolarized levels. Block development was time dependent and occurred in two phases: a first fast and voltage-dependent phase was followed by a second much slower phase (time constant of 4.4 +/- 0.48 sec (n = 11). Recovery from block was slower as the membrane potential became more negative. This resulted in the absence of a steady-state frequency-dependent effect at negative membrane potentials. It is concluded that dofetilide is an efficient blocker of the fast component of ik. The block, as well as recovery, are voltage and time dependent. Block is greater at more depolarized levels, recovery is slower at more hyperpolarized levels.     

Effect of palytoxin on membrane and potential and current of frog myelinated fibers.

Palytoxin is a highly toxic compound isolated form several zoanthid Palythoa species. The effects of palytoxin on the nodal membrane of frog myelinated fiber have been studied under current clamp and under voltage clamp conditions. Under current clamp conditions, palytoxin (0.1 microng/ml, 3 x 10(-8)M) induces a depolarization which is not reversed by washing. The resting potential reaches a value of -35 mV after 10 minutes. During the same period, the evoked action potential shows a gradual decline and finally disapears after about 30 minutes. The membrane depolarization is suppressed by removal of Na ions from the external medium, but only slightly diminished when tetrodotoxin (10(-6)M) is subsequently added to the external medium. When the potential of the nodal membrane is maintained at -70 mV, palytoxin (0.1 microng/ml) causes the appearance of an inward current that increases in magnitude during 30 minutes before attaining a steady-state value. The kinetics of development of that current is modified in the presence of tetrodotoxin or saxitoxin. Voltage clamp analysis shows that palytoxin causes an increase of the resting sodium permeability that is accompanied by a shift of the voltage dependence of the transient sodium permeability in the direction of membrane hyperpolarization. The shift in the voltage dependence of the transient permeability is accompanied by a decrease of the peak transient permeability. A similar shift in the potential dependence of the sodium inactivation is observed. During and after the application of palytoxin, the internal sodium concentration increases. The steady-state (potassium) conductance is also decreased at the same time as the leak current is increasing.     

Block of Na+ channels by imipramine in guinea-pig cardiac ventricular cells.

The effects of imipramine on the Na+ current of guinea-pig ventricular myocytes were examined by the whole-cell clamp method. Imipramine inhibited the Na+ current with a dissociation constant value of 25 microM at a -130 mV holding potential. At 1 microM, imipramine caused a negative shift of the channel availability curve by 4.0 +/- 1.03 mV with its steepness unaffected. The inactivation time constants were not changed by 30 microM imipramine. Paired pulse experiments revealed that imipramine binds to the inactivated Na+ channels with time constants of 3.7 +/- 0.27 sec at -65 mV and 2.4 +/- 0.58 sec at -20 mV, and that it dissociates from the channels with time constants of 5.9 +/- 1.05 sec at -90 mV and 2.0 +/- 0.87 sec at -130 mV. From these paired pulse experiments, the dissociation constant for the interactions between imipramine and inactivated channels was calculated to be 0.67 microM, a value within its therapeutic plasma concentration. These slow interactions of imipramine with inactivated Na+ channels resulted in a slow onset of the frequency-dependent extrablock in the effects of imipramine on the Na+ current. Consequently, the imipramine-induced extrablock sufficient to terminate re-entrant tachyarrhythmias would not develop shortly after their initiation. Short depolarizations of 1- to 3-msec duration sustained appreciable extra blockage when a high concentration of 10 microM imipramine was used, or they were repeatedly applied at a high frequency. However, access of imipramine to the open channels seems to play a minor role in the drug-channel interactions.     

Block of sodium channel current by anticonvulsant U-54494A in mouse neuroblastoma cells.

(+/- ) -cis-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzamide (U-54494A), structurally related to a kappa opioid agonist U-50448H, is a potent anticonvulsant without analgesic or sedative effects of the opioid agonist in intact animal studies (VonVoigtlander et al., 1987). To explore the mechanism of its anticonvulsant action, we investigated the interaction of U-54494A with the voltage-gated sodium channel using the whole cell patch clamp technique in mouse neuroblastoma cells (NIE-115). The drug reversibly and dose-dependently reduced the tetrodotoxin-sensitive inward Na current without affecting its activation or inactivation kinetics or the reversal potential. Nearly half of this resting block by 50 microM U-54494A at a holding potential of -80 mV was reversed upon further hyperpolarization to -120 mV. We also observed a hyperpolarization shift (9.3 mV) of the steady-state slow inactivation curve in the presence of 50 microM drug with no shift in the steady-state activation or the fast inactivation curves. These results indicate that the drug interacts with the resting and the slowly inactivated channels. The drug appears not to interact with the open state, judging from the absence of a time-dependent block in chloramine-T-treated cells. The recovery rate of the inactivated channel was markedly delayed by the drug, and apparently is responsible for its use-dependent block upon repetitive depolarizations. Our results suggest that voltage- and use-dependent block of the Na channel by U-54494A may be an important pharmacological basis for its anticonvulsant action.