Anticonvulsant and related effects of U-54494A in various seizure tests.

The anticonvulsant activity of (+-)-cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benz am ide monohydrochloride (U-54494A), a benzamide derivative chemically related to kappa opioid receptor agonists, was investigated in three selected seizure models of experimental epilepsy. In the maximal electroshock seizure test in mice, U-54494A (ED50 28 mg/kg i.p.) was effective, with a potency somewhat less than phenobarbital. In combination with clinically used antiepileptics, especially phenobarbital and carbamazepine, the anticonvulsant activity of the latter was significantly increased. More detailed studies with phenobarbital showed additive anticonvulsant effects. The anticonvulsant activity of U-54494A was partially antagonized by naloxone. On the other hand, this compound did not elevate the pentylenetetrazol seizure threshold (at high doses a tendency of proconvulsant action was seen). Furthermore, in unrestrained rats with chronically implanted electrodes, U-54494A (> or = 10 mg/kg) significantly reduced the duration of electrically evoked hippocampal afterdischarges. However, the focal stimulation threshold was not markedly increased. With respect to the possible mode of action, whole-cell voltage-clamp experiments on cultured neonatal rat cardiomyocytes showed that U-54494A depressed the fast sodium inward current in a concentration- and frequency-dependent manner. In summary, our results agree with earlier reports that demonstrated marked anticonvulsant effects of U-54494A in grand mal-analogous seizure tests. Moreover, in combination with some standard antiepileptics, additive effects can be found. It is suggested that, in addition to kappa opioid and excitatory amino acid receptor related effects, modulations of Na+ membrane currents may contribute to the mechanisms of action.

Anticonvulsant and sodium channel blocking effects of ralitoline in different screening models.

Ralitoline, a thiazolidinone derivative chemically distinct from known antiepileptic drugs, possesses remarkable anticonvulsant properties as demonstrated in various animal models of epilepsy. The efficacy of this compound seems to be comparable or even better than that of conventional antiepileptics. In the present study, the activity of ralitoline was investigated in four seizure models in rodents in order to characterize the anticonvulsant profile of action further. In the maximal electroshock seizure test (mice), this compound showed marked anticonvulsant effects (ED50 2.8 mg/kg i.p.). The efficacy of clinically established anti-epileptics was significantly increased when ralitoline was given as co-medication. In the strychnine seizure test (mice), ralitoline (5 and 10 mg/kg) prolonged the latency of tonic seizures as well as the survival time. On the other hand, in the subcutaneous pentylenetetrazol seizure threshold test (mice), this drug revealed limited protective actions at higher doses and increased the effectiveness of ethosuximide. In unrestrained rats with chronically implanted electrodes, ralitoline (5 mg/kg) significantly reduced the duration of electrically-evoked hippocampal discharges and raised the focal stimulation threshold (10 mg/kg). In the rotorod ataxia test (mice), a TD50 value of 14.5 mg/kg i.p. was determined for ralitoline (protective index TD50/MES-ED50 5.2). With regard to the possible mode of action, whole-cell voltage-clamp experiments on cultured neonatal rat cardiomyocytes showed that ralitoline may act specifically on voltage-sensitive sodium channels. The compound inhibited the fast sodium inward current in a frequency- and voltage-dependent manner. In conclusion, the findings confirm the potent anticonvulsant effects of ralitoline, especially against generalized tonic-clonic and complex partial seizures. Moreover, in combination with antiepileptics, an additive synergism can be found at lower concentrations. Regarding the mode of action, this drug was capable of depressing the fast sodium inward current in cultured heart ventricular cells, suggesting that the local anesthetic properties may be important for the anticonvulsant activity of ralitoline.

The cardiotonic bipyridine AWD 122-60 inhibits adenosine triphosphate-sensitive potassium channels of mouse skeletal muscle.

The inhibition of ATP-sensitive potassium channels in mouse skeletal muscle by the cardiotonic bipyridine AWD 122-60 was investigated with the patch-clamp technique. In excised patches of the inside-out configuration, internally applied AWD 122-60 (10(-6)-10(-3) mol/l) reversibly reduced the open-probability of single ATP-sensitive potassium channels. The agent shortened the periods of channel activity but did not affect the channel conductance. At positive membrane potentials channel inhibition by AWD 122-60 was more pronounced than at negative potentials, the drug concentrations producing 50% channel inhibition were 11 mumol/l at +40 mV and 29 mumol/l at -40 mV. The Hill coefficients of the concentration-response curves were in the range between 0.5 and 0.6 for both potentials. Internal application of another cardiotonic bipyridine, milrinone (10(-4) mol/l), had no effects on ATP-sensitive potassium channels in skeletal muscle. Possible effects of the inhibition of ATP-sensitive potassium channels in heart muscle by AWD 122-60 are discussed.

Characterization of the calcium channel state transitions induced by the enantiomers of the 1,4-dihydropyridine Sandoz 202 791 in neonatal rat heart cells. A nonmodulated receptor model.

The actions of the optical enantiomers of Sandoz 202 791 were studied in barium inward currents recorded from single cultured neonatal rat ventricular heart cells, using the whole-cell configuration of the patch clamp technique. The enantiomers were applied by bath perfusion or rapidly by the technique of concentration jumps during single voltage clamp steps. (1) (-)-202 791 reduced the barium current in response to depolarizations positive to 0 mV. The peak current amplitude in the threshold range (-40 to 0 mV) was either not affected or slightly increased by the substance. (2) The agonist enantiomer (+)-202 791 increased the inward current over the whole voltage range, where the increase in peak inward current amplitude was most prominent in the voltage range from -40 mV to 0 mV. (3) The antagonist enantiomer (10(-6) M) induced a 18.2 +/- 2.1 mV (n = 6) shift of the midpoint of the steady state inactivation curve in the hyperpolarizing direction; in contrast (+)-202 791 at the same concentration did cause only a small but not significant shift of the Ca-channel availability curve (n = 5). (4) Rapid extracellular application of (-)-202 791 (10(-6) M), during the sustained current component at a test potential of 0 mV was followed by a sudden acceleration in barium current decay. The drug-induced barium current block developed with a mean time constant of 214.7 +/- 20.6 ms (n = 5). (5) (+)-202 791 (10(-6) M) rapidly applied during test pulses to 0 and -20 mV caused an increase in barium current with a mono- or biexponential time course. The estimated mean time constant of the drug activated Ba2+ current at 0 mV membrane potential was 617.3 +/- 49.3 ms (n = 4). (6) The interaction of Sandoz 202 791 with the Ca-channels is discussed in terms of a "nonmodulated receptor" model.

[Calcium current effects in the presence and absence of propranolol on cloned neuroblastoma and glioma hybrid cells]. / Wirkung von (-)- und (+)-Propranolol auf den Kalzium-Einwärtsstrom klonierter Neuroblastom x Gliom-Hybridzellen.

The action of the (-)- and (+)-enantiomers of the beta-adrenoceptor blocking drug propranolol on the inward calcium current (ICa) was studied in single mouse neuroblastoma x rat glioma hybrid cells of clone 108CC5 by suction pipette technique for intracellular perfusion and voltage clamp. ICa was recorded after internal cell perfusion with Tris phosphate buffer and suppression of sodium and potassium currents in Na+-free external solution. Extracellularly applied (-)- and (+)-propranolol (10(-7) to 10(-3) M) inhibited ICa in a similar dose-dependent manner. The IC50 values for both substances were approximately 5 . 10(-6) to 10(-5) M. Two other beta-blockers, alprenolol and talinolol, investigated as reference compounds, also depressed the ICa, but in a significantly higher dose-range of 10(-4) to 10(-3) M. The results provide further evidence that propranolol, besides its known effect on sodium inward current, also possesses marked inhibitory actions on the ICa in mammalian nerve cell membranes at relatively low concentrations.

Cell-attached patch clamp measurement of macroscopic rapid inward sodium current in cultured heart cell reaggregates.

A megaohm seal, cell-attached patch clamp technique utilizing plastic suction pipettes for measuring macroscopic rapid inward sodium current (INa) was applied to cell membrane patches 30 to 100 micron2 in area at the periphery of reaggregates of myocardial cells from newborn rats cultured for 3 to 7 days. The reaggregates were composed of about 20 to several thousand of electrically well-coupled cells, the latter forming spheroidal reaggregates with a diameter of maximally 300 microns. This system was shown to guarantee satisfactory voltage-clamp control during ionic current measurements. The time and voltage dependence of the INa recorded during periods of up to 90 min was similar to that observed in single cardiac cell preparations from adult rats. INa inactivation was described by two time constants (tau h1, and tau h2). For maximal INa tau h1 and tau h2 had values of 1.5 +/- 0.25 ms and 8.7 +/- 3.9 ms (n = 4), respectively. The time course for recovery of INa from inactivation at the reaggregate resting potential exhibited also two time constants (tau re1 = 9.8 +/- 3 ms, tau re2 = 193 +/- 50 ms, n = 5). Estimated current density was about 10 pA/micron2. The concentration of tetrodotoxin needed to reduce maximal INa by 75% was 85 times lower in the reaggregates than it was in freshly isolated heart muscle cells from adult rats. The present system offers a combination of features that should make it well-suited for the study of both short- and long-term effects on the sodium channels in neonatal heart cells: Easy handling of the object, great electric stability, high time and amplitude resolution during ionic current measurements, and viability for at least one week.

Inhibition of calcium channels in neuroblastoma x glioma hybrid cells by verapamil, D600, diltiazem and nisoldipine.

The action of verapamil, /+/-D600 and nisoldipine on the inward calcium current (ICa) was studied in mouse neuroblastoma x rat glioma hybrid cells of the line 108CC5 under voltage clamp conditions by means of a suction pipette method.

A patch clamp study of the action of ethacyzine on the rapid inward sodium current in single rat heart muscle cells.

The effect of ethacyzine (a diethylamine analogue of ethmozine) on the rapid inward sodium current (INa) was studied in single rat ventricular muscle cells by a patch clamp technique. Extracellular application of 3 microM ethacyzine depressed the INa in a use-dependent fashion. Rest recovery from the use-dependent block by ethacyzine followed an exponential time course with a time constant of about 215 +/- 40 s (n = 4, mean +/- S.E.).

[Frequency-dependent blockade of sodium channels in isolated rat myocardial cells by the anti-arrhythmia agent ethmozine]. / Chastotnozavisimaia blokada Na-kanalov izolirovannykh kletok miokarda krysy antiaritmikom étmozinom.

A patch-clamp method was used to study the effects of the phenotiazine antiarrhythmic drug ethmozine (E) on the fast sodium inward current (INa) in freshly isolated heart muscle cells of adult rats. At a concentration of 10(-5) M E caused INa inhibition that could be enhanced by increasing the frequency of depolarization. This inhibition was reversible. After the termination of repetitive depolarization the amplitude of INa recovered with a time constant of about 10 sec. These findings may help to explain the therapeutic efficiency of E in high frequency cardiac rhythm disturbances.

Use- and voltage-dependent depression by ethmozine (moricizine) of the rapid inward sodium current in single rat ventricular muscle cells.

Effects of ethmozine (moricizine) on the rapid inward sodium current (INa) were studied in freshly isolated single cells of rat ventricular myocardium. INa was measured by means of a patch clamp method for observing integral ionic currents. Ethmozine was applied extracellularly to a small cell membrane patch at concentrations of 10, 20, and 40 microM. At a stimulation frequency of 0.1 Hz the drug decreased the peak INa without producing a shift of the current-voltage curve, but shifted the V0.5 of the steady-state inactivation curve by -6 mV. At frequencies of 2-5 Hz the ethmozine-induced block exhibited a prominent use dependence, with trains of depolarizing clamp pulses 5-50 ms in duration eliciting maximal INa from holding potentials at which the steady-state inactivation variable h infinity was close to 1. The use-dependent inhibition of INa became more pronounced with an increase in both stimulation rate and pulse duration. In contrast to what has been observed in the node of Ranvier of the frog, the present results indicate that ethmozine binds to both inactivated and open Na+ channels, but that the contribution of the open channel block to the overall block at depolarizing clamp step durations of several hundred milliseconds is small in comparison with the contribution of the block of inactivated channels.

Sodium and calcium currents in neuroblastoma x glioma hybrid cells before and after morphological differentiation by dibutyryl cyclic AMP.

Sodium and calcium inward currents (INa and ICa) were measured in neuroblastoma X glioma hybrid cells of clones 108CC5 and 108CC15 by a single suction pipette method for internal perfusion and voltage clamp. Morphologically undifferentiated, exponentially growing cells were compared with cells differentiated by cultivation with 1 mmol/l dibutyryl cyclic AMP. Outward currents were eliminated by perfusing the cells with a K+-free solution. Voltage dependence and ion selectivity as well as steady state inactivation characteristics of INa and ICa resembled those of differentiated mouse neuroblastoma cells, clone N1E-115 (Moolenaar and Spector 1978, 1979). These parameters were identical in undifferentiated and differentiated cells of both clones. After differentiation the average density of the peak sodium and calcium currents was increased two and four-fold, respectively, in both cell lines. Our data indicate that exponentially growing, morphologically undifferentiated 108CC5 and 108CC15 neuroblastoma X glioma hybrid cells possess functional Na+ and Ca2+ channels undistinguishable from those of non-proliferating cells of these clones differentiated morphologically by treatment with dibutyryl cyclic AMP. That Na+ and Ca2+ spikes were not detected by other authors in these cells prior to morphological differentiation by dibutyryl cyclic AMP may be attributed to the fact that at the low resting membrane potential measured the Na+ and Ca2+ channels are inactivated.

A kinetic analysis of the inward calcium current in 108CC15 neuroblastoma x glioma hybrid cells.

The kinetics of activation and inactivation of the inward calcium current (ICa) in morphologically undifferentiated and differentiated neuroblastoma X glioma hybrid cells of the clone 108CC15 were studied by the suction pipette technique for internal perfusion and voltage clamping. Potassium currents were eliminated by internal perfusion of the cells with a K+-free solution. Activation of ICa followed a sigmoidal time course and could reasonably be fitted by a m2 relation. The kinetics of ICa inactivation were studied by analyzing the current inactivation during long depolarizing steps and by measuring the peak ICa as a function of the length of a prepulse. Both methods gave comparable results indicating that the ICa inactivation cannot be fitted by a single exponential. The ICa inactivation was fitted by a biexponential function. Neither the activation nor the inactivation of ICa were changed after morphological cell differentiation induced by treatment with dibutyryl cyclic AMP.

Calcium channel block by phenytoin in neuroblastoma x glioma hybrid cells.

The action of phenytoin on the inward calcium current (ICa) was studied in cells of the clonal mouse neuroblastoma X rat glioma hybrid line 108CC5 by the suction pipette technique for internal perfusion and voltage clamp. The ICa was recorded after suppression of Na+ and K+ currents. Phenytoin, applied externally in concentrations of 50 to 500 microM, depressed the ICa in the investigated potential range of -60 to +30 mV in a concentration-dependent manner. When the cells were stimulated by depolarizing clamp steps, the extent of the ICa depression increased with the frequency and duration of the activating pulses. ICa was also inhibited on intracellular application of phenytoin.

Trapidil and other 5-triazolo-(1, 5-alpha)-pyrimidine derivatives as calcium channel blockers in 108CC5 cells.

The action of trapidil (RocornalR) and its derivatives AR 12-456 and AR 12-160 on the inward calcium current (ICa) was studied in mouse neuroblastoma x rat glioma hybrid cells of the line 108CC5 under voltage clamp conditions by means of a suction pipette method. A dissociation constant of the calcium channel-trapidil complex of 277 microM was estimated for the initial inhibition of ICa by trapidil. Half maximal block of ICa was produced by 80 +/- 20 microM AR 12-456 and 500 +/- 150 microM AR 12-160.

Sodium current in freshly isolated and in cultured single rat myocardial cells: frequency and voltage-dependent block by mexiletine.

Effects of mexiletine on the rapid inward sodium current (INa) were studied in freshly isolated single cells of the ventricular myocardium of adult rats and in single cultured ventricular muscle cells of newborn rats. The current was measured in internally perfused, voltage-clamped cells by a single suction pipette technique. Mexiletine was applied extracellularly. INa was reduced by the drug in both preparations when the membrane was depolarized to -20 mV by short (8 ms) pulses delivered at a frequency of 0.1 Hz from a holding potential of -100 mV. Mexiletine in a concentration of 50 microM diminished the INa under this condition by 70 +/- 8% (mean +/- S.D.) in the adult myocardial cells. A nearly equal reduction of the current (65 +/- 10%) was caused in the neonatal myocardial cells by 15 microM mexiletine. A use-dependent block of INa was produced in the presence of 10 and of 20 to 30 microM mexiletine, respectively, in the neonatal and the adult myocardial cells by repetitive depolarizing test pulses applied at frequencies between 1 and 7 Hz. Prolongation of the pulse duration from 10 to 100 ms enhanced the use-dependent block of INa in both preparations. The frequency-dependent action of mexiletine could be modulated by 100-ms hyperpolarizing prepulses from -80 to -140 mV. The time course of the use-dependent block (prepulse off) and unblock (prepulse on) was monitored. The slope of the inactivation curve of INa in the neonatal heart cells was reduced in the presence of mexiletine and the midpoint of the curve was shifted in the hyperpolarizing direction. These findings are interpreted as suggesting that binding of mexiletine to the sodium channel of the rat myocardial cells studied is enhanced when the cell membrane becomes depolarized.

[Mineralocorticoid-induced changes of vascular reactivity to noradrenaline and angiotensin II in baboons]. / Mineralokortikoidbedingte Veränderung der vaskulären Reaktivität auf Noradrenalin und Angiotensin II bei Pavianen.

In experiments on normotensive baboons of both sex the vascular reactivity on angiotensin II and noradrenaline was studied after an 18-week administration of DOCA (2 mg/b.w./week i. m.) and of common sal (5 g/day with meals). Dosage and duration of DOCA administration was chosen to prevent hypertensive haemodynamic alterations. 40 to 240 seconds after an angiotensin II injection (0.5 micrograms/kg b.w.) the pressor, especially diastolic response in both narcotized and non-narcotized animals intensified markedly. Infusion of noradrenaline (1.0 micrograms/kg b.w./min for 5 min) called forth after 120 min a significant rise in diastolic BP. Described abnormal pressor response is considered to represent a suitable model of a vascular predisposition to hypertension.

Characterization of the fast sodium current in isolated rat myocardial cells: simulation of the clamped membrane potential.

1. The fast sodium inward current of freshly isolated single rat myocardial cells was studied by means of the internal perfusion-voltage clamp method. 2. The voltage dependence of this current did not differ from the current-voltage characteristics of the fast sodium inward current described for other excitable cells and tissues. 3. The time constant of inactivation of the Na+ current of the isolated myocardial cells ranged between 5.2 msec at -58 mV and 0.5 msec at +18 mV. The activation time constant ranged from 0.3 msec at -55 mV to 70 microseconds at +10 mV. 4. The reactivation time constant of the maximum sodium current at a holding potential of -100 mV was found to be 21 +/- 5 msec. 5. A mathematical model was developed for the simulation and analysis of the influence of the series and shunt resistances on the time response of the membrane potential. The results of the modelling make it clear that control of the series and shunt resistances in any given experiment is a conditio sine qua non for a valid analysis of the kinetic parameters of the sodium inward current. 6. Sodium currents with delayed activation kinetics must be regarded as an indication of insufficient control of the membrane potential.

[Arterial hypertensive dysregulations on the basis of a cerebro-visceral stimulus constellation in baboons]. / Arteriell-hypertone dysregulationen auf der Grundlage einer zerebroviszeralen Reizkonstellation bei Pavianen.

Basing on the hypothesis that disturbances of cerebral information processing on the basis of acute or chronic stress situations or profound neurotic alterations are being directed to the cardiovascular system only by predisposition to hyperreactivity, the influence of a psycho-nervous-humoral-hormonal stepped load schedule upon central nervous and vegetative functions was studied in baboons. Stochastic interventions into the natural day-night rhythm and application of NaCl and DOCA doses not per se causing a blood pressure rise, either single or in combination for altogether 3 years were used as disturbing factors. It has been revealed that experimental disturbance of the light-dark phases led to lasting deviations of the conditional-reflectory activity in the sense of a predominance of irritation processes. With motor response time, initially unchanged but from the second year of experiment significantly shortened by 35%, the failure rates at differentiation increased, on the average, from 6 to 45% and the intersignal responses by 100%. Even after exposure for several months, no disorders of the cardiovascular system occurred. It was only the coupling with an experimentally induced disturbance of electrolyte distribution that provoked a significant increase in mean arterial pressure, on the average, by 24% of the pre-control value with moderate increase of the circulating blood volume. The increases in free fatty acids and blood glucose concentrations by 14 and 23%, respectively, can be interpreted as additional hypertension-favouring factors. Despite an application of mineral corticoids for more than 1 year, it has been impossible to alter the contraction behaviour of the vascular smooth muscle cell in the sense of an experimentally induced predisposition to arteriolar hyperreactivity outlasting the discontinuation of disturbing factors. With higher nervous activity being clearly disturbed as before, the pressure got back to normal; testing the vascular reactivity to noradrenaline (1.0 microgram/kg b.w/min i.v., for 5 min) or angiotensin II (0.5 microgram/kg b.w. i.v.) at the end of the investigation period gave no enhanced pressure responses. By contrast, animals exposed exclusively to the described combination load for 18 weeks, showed a still normal system pressure and sensitivity to the applied noradrenaline and angiotensin II increased by 75-120% of the pre-control response. A liability of the cardiovascular system at acute stress situations (multiple partial immobilization) in long-term neurotically predamaged monkeys in the 24-h experiment was impressive by a cardiodepression during the nightly regeneration phase, reduced on the average by 35 beats/min against the control group. Thus, our results support the hypothesis of a cerebro-visceral pathoconstellation as the etiological principle of certain forms of the inhomogeneous clinical picture of primary hypertension.