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Effects of piperazine derivatives on the activity of frog skeletal muscle fibers
Cm.
Pergamon
Vol. 26, No. 6, pp. 1431-1439, 1995 Elsevier Science Ltd. Printed in Great Britain
Pharmac.
0306-3623(94)00235-5
Effects of Piperazine Derivatives on the Activity of Frog Skeletal Muscle Fibers N. RADICHEVA,*
M. VYDEVSKA
Institute of Biophysics, Bulgarian Academy
of Sciences,Acad.
and K. MILEVA
G. Bontchev St., bl. 21, 1113 Sojia, Bulgaria
(Received 26 July 1994)
Abstract-l. This study was undertaken to characterize the effects of some piperazine derivatives on excitable cell membranes. Three original Bulgarian compounds with favorable effects on cardiovascular and nervous system-piperazine derivatives with code names P-l 1 (N’-[3-oxo-3-phenyl-2-methyl-propyllN4-[trans-3-hydroxy-1,2,3,4-tetrahydro-2-naphthyl]-piperazine dihydrochloride), AS, (N’-benzhydryl-N4ally1 piperazine dihydrochloride) and 35-M (Schiff’s base of N’-benzhydryl-N4-aminopiperazine with triacetonamine, dioxalate salt) were tested in experiments with conventional microelectrode technique on isolated frog muscle fibers. 2. After 30-min treatment with tested drugs at concentrations of lo-100 PM the recorded intra-(ICAP) and extracellular action potentials (ECAPs) showed an amplitude decrease and duration increase. The total ionic current (I,) decreased as the outward phase was almost abolished by P- 11. The propagation velocity (PV) of excitation and the twitch amplitude also decreased. These changes were agent- and concentration-dependent. 3. The effect potency of the agents diminished in the following order: P-l 1 > AS, > 35-M. 4. Concentrations higher than 100 PM for all agents completely, but reversibly, inhibited membrane excitability. 5. The results demonstrate compound- and concentration-induced modulation of Ca*+ current with blockade of Ca2*-dependent K+ and Cl- membrane channels of muscle fiber treated with the compound tested. Key Words: Piperazine derivatives, skeletal muscle fiber, action potentials, ionic currents
INTRODUCTION Many piperazine derivatives are biologically active substances with a favorable effect on the cardiovascular and central nervous systems. Some of them are widely used in medical practice as antidepressants and anxiolytics. The original Bulgarian compound N’-[3-0x0-3phenyl-2-methyl-propyl]-N4-[trans-3-hydroxy-1,2,3, 4-tetrahydro-2.naphthyll-piperazine dihydrochloride with the code name of P-11 (synthesized in the Pharmaceutical Faculty at High Medical Institute, Sofia, Ivanov et al., 1977; Fig. 1) has manifested vasodilating hypotensive activity and GI- and fladrenoblocking properties (Rainova et al., 1978; Mutafova-Yambolieva et al., 1985; MutafovaYambolieva and Staneva-Stoycheva, 1988a,b). In experiments on smooth muscles from taenia coli P-l 1 and Aligeron (AS,) have shown different adreno*To whom all correspondence should be addressed.
receptor-blocking action from those of the typical Ca*+ antagonists (Mutafova-Yambolieva et al., 1989). The piperazine derivative Aligeron (AS,; Fig. l), N’-benzhydril-N4-ally1 piperazine dihydrochloride (synthesized in Chemico-Pharmaceutical Research Institute (NIHF), Sofia), is a structural analogue of the Bulgarian drug cinnarizine. The advantage of AS, over cinnarizine is its water solubility allowing parenteral application. AS, has twice the cerebral and coronary vasodilatation action of cinnarizine without direct effect on the arterial blood pressure (Nikolov, 1978). These effects of AS2 are probably due to its direct myorelaxant action on the vessels (Kovalyov et al., 1976). On the basis of triacetonamine a number of compounds were synthesized. The presence of amino and carbonyl groups in the chemical structure of triacetonamine makes the synthesis of new products easier. It is known that the involvement of the piperazine cycle in the structure of some cyclic systems with psychotropic action has a favorable effect. Thus, a
1431
1432
N. Radicheva et al.
.2HCl CH,
0
CH
CH,
P-11
2 HCl
AS, (Aligeron)
H,C
-
35-M Fig. 1. Chemical structures of P-l 1, AS, (Aligeron) and 35-M.
of new Schiff’s bases of triacetonamine with N’-substituted-N4-aminopiperazines with low toxicity, versatile biological activity and water soluble salts have been synthesized in the Chemico-Pharmaceutical Research Institute (NIHF), Sofia (Zikolova et al., 1978). At low doses these compounds affect the excitability and potential motor activity but at higher doses they show a depressive myorelaxant effect. The aminopiperazine derivatives exert a hypotensive and central nicotinolytic effect. The most active compound of this group 35-M (Schiff’s base of N’benzhydryl-N4-aminopiperazine with triacetonamine, dioxalate salt; Fig. 1) also has central holynolytic and analgetic effects. The most important is its potentiating influence on noradrenaline effects. The increased sensitivity of a-adrenoreceptors does not mean however, that 35-M increases blood pressure and cerebral level of cathecholamine (Taskov et al., 1977). There are no data about the direct effect of the above mentioned piperazine derivatives on the ionic processes in the cell membrane. The present work was undertaken to characterize the influence of the piperazine derivatives with code names P-11, Aligeron (AS,) and 35-M on the membrane processes. The changes in the muscle fiber potentials and ionic currents were used for evaluation of the effects of these compounds. group
MATERIALS
AND METHODS
Animals and isolated preparations
The experiments were carried out on 180 isolated muscle fibers from m. gastrocnemius of Rana ridibunda. The fibers (in situ length) were immersed in a double wall chamber (Fig. 2). The terminal parts of the isolated fibers were cleaned since parts of the tendon and the fascia were left attached to the ends and were used to fix the fibers. Solutions and drugs
The Ringer’s solution used contained (mM): NaCl 115.0, KC1 2.0, CaCl, 1.8, NaH,PO, 0.68 and Na, HP04 1.3 (pH 7.3-7.4) at a constant temperature 20°C. The compounds investigated (P-11, ASI and 35-M) were added separately to the bathing solution in concentration of 10-100 PM after control recordings of the potentials in standard Ringer’s solution. Experimental procedures
Extracellular stimulation by 0.5-l .Omsec rectangular electrical pulses of suprathreshold intensity was applied. The intracellular action potentials (ICAPs) were recorded by a glass micropipette filled with 3 M solution of KC1 with 1 pm tip diameter and a
Muscle fiber and piperazine derivatives
Fig. 2. Experimental set. (1) Muscle fiber, immersed in a Ringer’s solution-filled chamber. (2) Stimulating electrode. (3) Microelectrode and ICAP with its characteristic time interval: Rf-rising time (interval between 10% and peak in depolarization phase); rdeclining time (interval between pick in depolarization phase and 50% in amplitude of the depolarization phase); AMP-ICAP amplitude. (4) Extracellular electrodes with fixed interelectrode distance d and two ECAPs; t-time interval between negative maxima for PV measurement. (5) Thermometer. (6) Reference electrode. (7) Mechanoelectrotron.
of 15-20 MQ. The pipette was connected ing system (705 Microprobe
to a record-
system) by means of an
Ag/AgCl electrode. We adopted the approach of measuring the time intervals between characteristic points of the ICAP (Wallinga-De Jonge et al., 1985). To avoid errors in depolarization onset the rising time (Rt) was measured at a level of 10% in ICAP amplitude. Simultaneous extracellular recordings were made by two glass-coated tungsten electrodes with a tip of about 10 pm in diameter and a resistance of 150 kfL The pair of electrodes with fixed interelectrode distance (d) was oriented parallel to the fiber axis. The extracellular action potentials (ECAPs) at different axial distances, i.e. over the cylindrical part of the fiber far from the stimulation site, over the transition to the conical area and at the very end, as well as at different radial distances (from 0 pm-visually observed contact, to 2000 pm) were recorded by a pair of electrodes attached to the hydraulic microdriver with an accuracy of shift of 1 pm. The ECAPs recorded at large radial distances (the maximal distance being 2000 pm) were averaged over 256 potentials. By means of an analogue-to-digital converter with sampling interval of 36 psec the signals (ICAPs and
ECAPs) were displayed on an oscilloscope for visual control and illustration and transmitted to a personal computer for further analyses. The propagation velocity (PV) of action potential was calculated dividing the interelectrode distance d into the time interval t described in Fig. 2. The mechanogram (contraction curve, twitch) of the muscle fibers was registered by displacement of a mechanoelectrotronic rod, which was stuck in the tendon between the preparation attaching hook and fiber end. The total ionic current (I,) during action potential of a muscle fiber was calculated by the cable equation for excitable fibers in the form given by Tassaki and Hagiwara (1957). The first and second time derivatives of the experimentally recorded ICAPs were calculated by Fourier transformation. The 1, was computed as a difference between the transmembrane and the capacitive current (Radicheva et al., 1985). Parameters observed and statistics
ICAPs-amplitude, duration: Rt and r ; ECAPsamplitude, duration; PV; J-inward and outward phases; twitch-mechanogram. The results reported as mean f SD were assessed for statistical significance using the Student’s t-test at
N. Radicheva et al.
1434
p c 0.05. All statistical procedures and the plotting of graphs were made by computer programs (Statgraphits 1.2; SlideWrite Plus 3.0). Control RESULTS
Changes in the action potentials and propagation velocity
The overshoot and membrane potential gradually decreased with increase of the compound concentration. Hence, the ICAP amplitude decrease was concentration- and substance-dependent: the amplitude decrease by 1@45% vs controls with P-11; by 5-25% vs controls with AS, and by 15-20% vs controls with 35-M (Figs 3,4 and 5, upper rows). The increase of the ICAP duration (Rt and r) was pro-
P-11
Ii f
50 VM
ii
P
ICAP
The potentials were recorded in standard Ringer’s solution and after 30min treatment of the muscle fiber preparations with a given concentration of the agents. More prolonged treatment at a concentration of 100pM accelerated the changes in membrane processes and was enough to suppress the membrane excitability which led to complete inhibition of the electrical and mechanical activity. The effect of the piperazine derivatives studied on the muscle fiber activity was reversible. The effect potency of the compounds tested on the studied parameters decreased in the following order: P-l 1 > AS2 > 35-M.
COllhl
10PM
100 PM
IO pM
50 pM
f
y‘ Is
1:
ECAP
1
Ii
r
f
f
Fig. 4. Changes in ICAPs, ECAPs and Ii’s calculated (in AU-arbitrary units) from the corresponding ICAP after 30 min treatment with AS, in concentrations of 10, 50 and 100PM. portional to the concentration increase. The slope of the approximating lines of the r duration were steeper than those of the Rt approximating lines (Fig. 6) i.e. the z duration was strongly prolonged. Most pronounced and statistically significant were the ICAP changes induced by P-l 1 (Table 1). ECAPs recorded in the vicinity of the fiber membrane reflected changes in the ICAP (Figs 3, 4 and 5, bottom rows). The ECAP amplitude decreased (by 50-75% with P-11, by 2565% with AS2 and by 1560% with 35-M) and the duration increased (up to 2.5 times by 100 p M P- 11 compared to the controls) with the Rt increase. ECAPs in treated muscle fibers recorded at different axial and radial distances to the membrane were similar in shape but with reduced amplitude and increased duration compared to the control potentials (Fig. 7). Hence the extracellular potential field should be weaker and long ranged. The PV of excitation measured between two ECAPs (PV = d/t, see Fig.2) decreased with a concentration increase. The extent of the PV decrease was substance-dependent with statistical significance only for P-l 1 and AS2 (Table 1, Fig. 6). Changes in the total ionic current and mechanical
5 ms
Fig. 3. Changes in ICAPs, ECAPs and I,‘s calculated (in AU-arbitrary units) from the corresponding ICAP after 30 min treatment with P-l 1 in concentrations of 10, SOand 100PM.
activity
The Z, was also affected in treated muscle fibers. P-l 1 gradually suppressed the outward current (amplitude decreased and duration increased) with a concentration increase. The highest concentration
1435
Muscle fiber and piperazine derivatives Table
Compound
1. Effect of the compounds
Rising time (RI)
(fiM) Control P-l 1 IO 20 30 40 50 100 Control
on the ICAP
parameters and PV Propagation velocity, (m/se@
tmsec)
Declining time (T) cm=)
1.056 f 0.269
I .544 f 0.382
1.257 + 0.249
1.368 1.303 1.296 1.591 1.696 2.239
2.282 2.653 2.310 2.696 3.423 5.144
0.962 f 0.168’
*0.152* + 0.291* f 0.365 f 0.279*** k 0.580*** f 0.660’”
+ f f k + *
0.466*** 0.533*** 0.613*** 0.661*** 1.612*** 1.917***
(PV)
0.679 + 0.225** 0.672 f 0.161** 0.286 f 0.026***
1.084iO.138
1.590 * 0.359
1.112~0.131
1.203 i 0.180 1.281 + 0.219*
1.807 _+0.275 1.898 i 0.496
0.977 i_ 0.222 -
1.296 1.494 1.493 2.122 1.238
1.890 2.275 2.005 4.446 2.180
kO.184 + 1.222 i 0.329’ _t 2.342** + 0.530
0.907 * 0.102**
1.951 2.097 2.430 3.004 3.020
f 0.462 i_ 0.495 + 0.408 * 0.666” k 0.650**
I.149 * 0.173
AS* IO 20 30 40 50 100 Control 35-M 10 20 30 50 100
k 0.158’ & 0.478’ i: 0.236’ + 0.788” _+0.214
1.066 * 0.202 1.280 _t 0.191 1.15O_tO.209 1.741 * 0.534” 1.544 f 0.200**
Values are meansfSD;
n = 9 - 15.
*p < 0.05;
**p < 0.01;
***p
0.763 k 0.076*** 0.405 k 0.124*** 1.096 + 0.250
1.093 + 0.076 0.933 i 0. I36 0.818 * 0.200 by Student’s
~-test in comparison
with
control values.
(100 PM) abolished the outward current and affected the inward current-amplitude decrease and prolonged duration were observed (Fig. 3). The AS, effect on the outward current was similar to that of P-l 1 but less pronounced on inward current (Fig. 4). The effect of 35-M on both inward and outward currents was weaker (Fig. 5).
35-M Control
50 pM
100 PM
ICAP 1
Ii f
i\
r
ECAP + Fig. 5. Changes in the ICAPs, ECAPs and I,‘s calculated (in units) from the corresponding ICAP after 30 min treatment with 35-M in concentrations of 10, 50 and 100 PM.
AU-arbitrary
The excitability threshold increased, the twitch amplitude decreased, contraction and relaxation time increased with increase of the compound concentration. These changes were substance-dependent, similar to the above described parameters. DISCUSSION In the present study we used the biophysical approaches (changes in ICAPs, ECAPs, I,, PV of excitation and contractile properties) to assess the functional state of the muscle fiber membrane influenced by some piperazine derivatives. These substances are known to have favorable effects on very important physiological functions and it is of interest to understand how they change the kinetics of the membrane currents and hence, how they would interact with other biologically active substances. The changes were concentration- and substancedependent. The effect potency of the compounds tested on the studied parameters decreased in the following order: P-l 1 > AS2 > 35-M. The steepness of the lines approximating the changes in Rt and T (ICAP duration) showed a more pronounced increase in T duration indicative of a decrease in the activity of the channels responsible for the repolarization in treated fibers. The observed differences in SD for values of the parameters compared at various concentrations of the substances tested may be related to structural and functional characteristics of the fibers. They were not previously selected on their type. The different properties of the slow and fast fibers would be more clearly expressed
1436
N. Radicheva et al.
in the case of changed functional state similar to that observed in our experiments. The repolarization is attributed to the K+-delayed rectifier current (Kao and Stanfield, 1970; Hille, 1984; Radicheva, 1986; Martin and Drayer, 1989) as well as to the large-conductance Ca*+-activated K+- (maxiKC channels) and to Ca*+-activated Cl- channels (Francilini and Petris, 1990; McManus, 1991). In contrast to the alterations in muscle potentials in-
A
A
10
duced by compounds tested, the blocking of the K+-delayed rectifier current led to the typical “plateau’‘-like ICAPs without changes in the depolarization phase (Gillespie, 1977; Radicheva, 1986). The ECAPs corresponding to those changed ICAPs close to the membrane are usually unchanged while the ECAPs recorded at large radial distances possessed typical shape (abrupt front of the terminal positive phase) attributed to the blocking of the
Rt
A
10
A y=1.104+0.011X; r=O.O72 ~=i.868to.o34x;
10
r=0.072
A 0
Rt
07
y=1.061+0.010x; r=O.O60 ~=i.203+0.027~;
r=0.027
1
6-
”
b
$0
Concentration
Oi
160
(IAM)
Concentration
D
C 1).
10
h E
Rt
oAS,
(j,d)
A35-M
?? y=l.lOO-0.000x; r=O.O45 0 y=l.OOO-0.007x; r=0.005 A y=l.l43-0.003x:r=0.045
G2 2
6
,R ‘i;
6_
0
@ b
2 3 Z g
DP-11
?? r
A y=1.167tO.O06x; r=0.661 ?? y=2.050+0.011x; rm0.665
100
1
cl :
{&_;-_i--------i
0
1 0
$
H 100
50
Concentration
(di)
Z a
0
7
0
I
50
Concentration
t
100 (MM)
Fig. 6. Dependence of rising time (Rt) and declining time (5) duration of the ICAF on the concentration of P-l 1 (A), AS, (B) and 35-M (C) as well as the dependence of PV of action potentials on the substance concentration (D).
1437
Muscle fiber and piperazine derivatives
2000pm
-
I
z-5 pY
+
1goopm
I
--
6 PV
-!-
OP +
++-
I
100 pY
1OOOpm --
I
2000pm
1
5P
2.5 pY
10 ma
Fig. 7. Scheme of the muscle fiber ECAPs recorded at different axial distances (from left to right--over the cylindrical part of the muscle fiber, the transition to the conical part and the fiber end) and at different radial distances (from Oy, 1000~ and 2000~ to the fiber membrane). Potentials above the fiber were recorded before treatment and those below the fiber-after treatment with AS2 at .50pM.
K+-delayed rectifier current (Radicheva and Kolev, 1993). Such alterations in muscle potentials were not observed in the present experiments. It is obvious that the ECAP changes reflect the ionic conductance of the muscle fiber membrane. The alterations in the parameters of the ECAPs as treated by compound tested fibers recorded at large radial distances led to a weaker and long-range extracellular potential field. It wouldbeofinteresttoelectromyographicstudiessince it is known that ECAPs at large radial distances are similar to the extraterritorial motor unit potentials.
That would be important in patients with muscular disorders taking piperazine-derivative drugs. Concentrationand compound-induced proportional decrease in membrane excitability may be due to the influence on the large conductance Ca2+dependent K+ channels which provide a pathway for cell repolarization subsequent to membrane depolarization and on the small conductance Ca2+dependent K+ channels possessing properties necessary to account for afterhyperpolarization in myotube (Garcia et al., 1991). The amplitude of the
1438
N. Radicheva et al.
depolarization afterpotential (affected by substances tested) is also Ca2+-dependent (Harada and Takahashi, 1983). Approximately lO-15% of the total repolarization (outward) current flowing during the action potential is attributed to chloride (Hutter and Nobel, 1960). The similarity of the ICAPs changes induced by the agents (especially P-l 1) to that induced by ZnCl,, known to be a blocker of Cl- channels (Stanfield, 1970; Woll et al., 1987; our unpublished data) showed that the piperazine derivatives may block these channels. Valkanov (1993), using the patch clamp technique, has found a block of Ca*+-gated Clchannels of Ascaris suum muscle vesicles caused by P-11 at a concentration of 10pM. All these findings suggest that the investigated compound blocked Ca*+-activated K+ and Cl- currents and that the extent of the blockade was agent-dependent. P- 11 and AS2 most probably affected the slow Ca2+ current similar to the effect of Zn*+ at higher concentrations (Hagiwara and Takahashi, 1966). The amplitude and duration alterations in the ICAPs observed in our experiments were in agreement with the data of Krishtal (1978) regarding such ICAP changes when the slow Ca*+ current was decreased. This effect was the most weakly manifested by 35-M. The established decrease of the ICAP amplitude could also be attributed to a partial blockade of the sodium current. Some piperazine derivatives are considered to be less “specific blockers” of the Ca*+ channels pointing out the possibility for some of them to act also through blocking of certain Na+ channels (Scriabine, 1991; Pauwels et al., 1992). This might be due to the similarity in structure and kinetics of Na+ and Ca*+ channels (Krishtal, 1978; Tanabe et al., 1987). The above described inhibition of membrane excitability, propagation velocity and muscle contraction depending on the agent, concentration and duration of treatment also results from blockade of the Ca2+activated K+ channels which normally link intracellular free Ca*+ to membrane excitability (McManus, 1991). This process regulates action potential frequency and duration and plays a crucial role in excitation-contraction coupling. The ionic imbalance could explain the myorelaxant effect of the tested agents shown at the highest concentration. All these findings characterize piperazine derivatives as agents whose mechanism of Ca*+ antagonistic action is different from that of the typical Ca*+ antagonists. In extensive pharmacological experiments these compounds were found to manifest multireceptor activity (Rainova 1978; et al., Mutafova-Yambolieva et al., 1985; MutafovaYambolieva and Staneva-Stoycheva, 1988a,b). In agreement with these findings, our results suggest that
the alterations in muscle potentials were related to the alterations in the different Z,components indicative of multichannel activity of the agents. In conclusion, the aminopiperazine derivatives (P11, AS2 and 35-M), being less specific Ca*+ blockers, modulated Ca*+ current blocking predominantly Ca*+-dependent K+ and Cl- channels of muscle fiber membrane. Acknowledgement-This work was supported by Grant K-20/91 from National Science Fund, Ministry of Sciences and Education, Republic of Bulgaria. REFERENCES Francilini F. and Petris A. (1990) Chloride channels of biological membranes. Biochem. biophys. Acta 1031(2), 241-259.
Garcia M. L., Galves A., Garcia-Calvo M., King V. F., Vazquez J. and Kaczorowski G. J. (1991) Use of toxins to study potassium channels. J. Bioenerg. Biomem. 23(4), 615-646.
Gillespie J. I. (1977) Voltage-dependent blockage of the delayed potassium current in skeletal muscle by 4aminopyridine. J. Physiol. 273, 64P-65P. Hagiwara S. and Takahashi K. (1966) Surface density of calcium spikes in the barnacle muscle fiber membrane. J. gen. Physiol. W(3), 583601. Harada Y. and Takahashi T. (1983) The calcium component of the action potential in spinal motoneurons of the rat. J. Physiol. 335, 89-100. Hille B. (1984) Ionic Channels of Excitable Membranes. Sinaner Associates Inc., Sunderland, Mpcsachusetts. Hutter 0. F. and Nobel D. (1960) The chloride conductance of frog skeletal muscle. J. Physiol. 151, 89-102. Ivanov I., Troyanska G., Christova K., Dantchev D., Sulay P. and Waltchanova R. (1977) Synthese einiger N’-substituierter-N-(trans-3-hydroxy-l,2,3,4-tetrahydro2-naphthyl)-piperazine. Arch. Pharm., Weinhein 310, 925-93 1. Kao C. Y. and Stanfield P. R. (1970) Actions of some cations on the electrical properties and mechanical threshold of frog sartorius muscle fibers. J. gen. Physiol. 55, 620-639.
Kovalyov G., Nikolov R., Tyurenkov I. and Taskov M. (1976) Effects of the analogue of cinnarizine AS, on the cardio-vascular system. Acta Physiol. Pharmac. bulg. 2(4), 27-3 I. Krishtal A. (1978) Modulation of Ca++ channels in nerve cell by EGTA. Dokladi Acad. Nauki SSR 238,482485 (in Russian). Martin A. R. and Drayer S. E. (1989) Potassium channels activated by sodium. J. exp. Physiol. 74, 1013-1041. McManus B. (1991) Calcium-activated potassium channels: regulation by calcium. J. Bioenerg. Biomem. 23(4), 537-560. Mutafova-Yambolieva V. and Staneva-Stoytcheva D. (1988a) Pharmacodynamic effects of a f-piperazinotetralin (P- 1I)-combined and 3-adrenorece$orblocking drug with hypotensive activity. Menr. Find. exp. clin. Pharmac. lo(g), 551-557. Mutafova-Yambolieva V. and Staneva-Stoytcheva D. (1988b) Involvement of peripheral dopaminergic mechanisms in the action of a 2-piperazinotetralin (P-11) with hypotensive activity. Ment. Find. exp. clin. Pharmac. lo(g), 559-562.
Mutafova-Yambolieva V., Staneva-Stoytcheva D. and Ivanov D. (1985) Effects of a new 2-piperazinotetralin on a-l- and a-2-adrenergic receptors in isolated rat vas deferens. Meth. Find exp. clin. Pharmac. 7(6), 291-296.
Muscle fiber and piperazine derivatives Mutafova-Yambolieva V., Staneva-Stoytcheva D. and Nikolov R. (1989) Comparative evaluation of the Ca*+antagonist activity of some Bulgarian vasodilating agents and of known Ca2+-antagonists on K+-depolarized smooth muscle from Guinea-pig taenia coli. Acta Physiol. Pharmac. bulg. H(2), 40-46.-
-
Nikolov R. (1978) Pharmacological studies on the influence of the compound Aligeron-As, on the brain blood circulation. Ph.D. Thesis, Sofia. Pauwels P. J., Van Assouw H. P.. Peeters L. and Levsen J. E. (1992) Neurotoxic action of veratridine in rat drain neuronal cultures: mechanism of neuroprotection by Ca2+-antagonists nonselective for slow Ca2+-channels. J. Pharmac. exp. Ther. 255(3), 1117-l 122. Radicheva N. (1986) Intracellular and extracellular action potentials in frog muscle fiber upon blocking the potassium conductivity. Acta physiol. Pharmac. bulg. 12(2), 35-39.
Radicheva N. and Kolev V. (1993) Changes in the muscle fiber extracellular action potentials induced by 4-aminopyridine. Compt. Rend. I’Acad. Bulg. Sci. 45(7), 89-92. Radicheva N. I., Trayanova N. A., Gydikov A., Kostov K. G. and Kossev A. R. (1985) Changes in the total ionic current of frog muscle fiber during action potential under continuous activity. Compt. Rend. I’Acad. Bulg. Sci. 3tX@), 1085-1088. Rainova L., Georgieva A. and Staneva-Stoytcheva D. (1978) On the hypotensive and antihypertensive activity of a new derivative of 2-aminotetraline. Expt. Med. Morph. 17, 3743 (in Bulgarian).
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Scriabine A. (1991) Comparative pharmacology of 1,4dihydropyridines and other calcium channel ligands. J. Cardovasc. Pharmac. 9(Suppl. l), 3-7.
Stanfield P. (1970) The differential effects of tetraethylammonium and zinc ions on the resting conductance of frog skeletal muscle. J. Physiol. 209, 231-256. Tanabe T.. Takeshima H.. Mikani A.. Flockerzi. V.. Takehashi H., Kagnawa K., Kojima M., Matsuo K.; Hirosa T. and Numa S. (1987) Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature 328, 313-318. Taskov M., Velichkova S. and Tjutjulkova N. (1977) On some pharmacological effects of a piperazine derivative. Pharmacia 27(3), 19-23 (in Bulgarian). Tassaki I. and Hagiwara S. (1957) Capacity of muscle fiber membrane. Am. J. Physiol. 188, 423429. Valkanov M. (1993) Voltage-dependence of Cl--channel block by piperazine derivative, P-l 1. Abstracts, 11th International Biophysics Congress, Budapest, Hungary. Wallinga-De Jonge W., Gielen F., Wirtz P., De Jong P. and Broenink J. (1985) The different intracellular action potentials of fast and slow muscle fibers. Electroencephal. clin. Neurophysiol. 60, 539-547.
Woll K. H., Leibowitz M. D., Neumcke B. and Hille B. (1987) A high conductance anion channel in adult amphibian skeletal muscle. Pfliigrs Arch. 410(6), 632-640. Zikolova S., Konstantinova R., Daleva L. and Taskov M. (1978) Synthesis and pharmacological study of new Schiff’s bases of N’-substituted-N4-aminopiperazines with triacetonamine. Pharmaciu 28(3), l-9 (in Bulgarian).
Pergamon
Vol. 26, No. 6, pp. 1431-1439, 1995 Elsevier Science Ltd. Printed in Great Britain
Pharmac.
0306-3623(94)00235-5
Effects of Piperazine Derivatives on the Activity of Frog Skeletal Muscle Fibers N. RADICHEVA,*
M. VYDEVSKA
Institute of Biophysics, Bulgarian Academy
of Sciences,Acad.
and K. MILEVA
G. Bontchev St., bl. 21, 1113 Sojia, Bulgaria
(Received 26 July 1994)
Abstract-l. This study was undertaken to characterize the effects of some piperazine derivatives on excitable cell membranes. Three original Bulgarian compounds with favorable effects on cardiovascular and nervous system-piperazine derivatives with code names P-l 1 (N’-[3-oxo-3-phenyl-2-methyl-propyllN4-[trans-3-hydroxy-1,2,3,4-tetrahydro-2-naphthyl]-piperazine dihydrochloride), AS, (N’-benzhydryl-N4ally1 piperazine dihydrochloride) and 35-M (Schiff’s base of N’-benzhydryl-N4-aminopiperazine with triacetonamine, dioxalate salt) were tested in experiments with conventional microelectrode technique on isolated frog muscle fibers. 2. After 30-min treatment with tested drugs at concentrations of lo-100 PM the recorded intra-(ICAP) and extracellular action potentials (ECAPs) showed an amplitude decrease and duration increase. The total ionic current (I,) decreased as the outward phase was almost abolished by P- 11. The propagation velocity (PV) of excitation and the twitch amplitude also decreased. These changes were agent- and concentration-dependent. 3. The effect potency of the agents diminished in the following order: P-l 1 > AS, > 35-M. 4. Concentrations higher than 100 PM for all agents completely, but reversibly, inhibited membrane excitability. 5. The results demonstrate compound- and concentration-induced modulation of Ca*+ current with blockade of Ca2*-dependent K+ and Cl- membrane channels of muscle fiber treated with the compound tested. Key Words: Piperazine derivatives, skeletal muscle fiber, action potentials, ionic currents
INTRODUCTION Many piperazine derivatives are biologically active substances with a favorable effect on the cardiovascular and central nervous systems. Some of them are widely used in medical practice as antidepressants and anxiolytics. The original Bulgarian compound N’-[3-0x0-3phenyl-2-methyl-propyl]-N4-[trans-3-hydroxy-1,2,3, 4-tetrahydro-2.naphthyll-piperazine dihydrochloride with the code name of P-11 (synthesized in the Pharmaceutical Faculty at High Medical Institute, Sofia, Ivanov et al., 1977; Fig. 1) has manifested vasodilating hypotensive activity and GI- and fladrenoblocking properties (Rainova et al., 1978; Mutafova-Yambolieva et al., 1985; MutafovaYambolieva and Staneva-Stoycheva, 1988a,b). In experiments on smooth muscles from taenia coli P-l 1 and Aligeron (AS,) have shown different adreno*To whom all correspondence should be addressed.
receptor-blocking action from those of the typical Ca*+ antagonists (Mutafova-Yambolieva et al., 1989). The piperazine derivative Aligeron (AS,; Fig. l), N’-benzhydril-N4-ally1 piperazine dihydrochloride (synthesized in Chemico-Pharmaceutical Research Institute (NIHF), Sofia), is a structural analogue of the Bulgarian drug cinnarizine. The advantage of AS, over cinnarizine is its water solubility allowing parenteral application. AS, has twice the cerebral and coronary vasodilatation action of cinnarizine without direct effect on the arterial blood pressure (Nikolov, 1978). These effects of AS2 are probably due to its direct myorelaxant action on the vessels (Kovalyov et al., 1976). On the basis of triacetonamine a number of compounds were synthesized. The presence of amino and carbonyl groups in the chemical structure of triacetonamine makes the synthesis of new products easier. It is known that the involvement of the piperazine cycle in the structure of some cyclic systems with psychotropic action has a favorable effect. Thus, a
1431
1432
N. Radicheva et al.
.2HCl CH,
0
CH
CH,
P-11
2 HCl
AS, (Aligeron)
H,C
-
35-M Fig. 1. Chemical structures of P-l 1, AS, (Aligeron) and 35-M.
of new Schiff’s bases of triacetonamine with N’-substituted-N4-aminopiperazines with low toxicity, versatile biological activity and water soluble salts have been synthesized in the Chemico-Pharmaceutical Research Institute (NIHF), Sofia (Zikolova et al., 1978). At low doses these compounds affect the excitability and potential motor activity but at higher doses they show a depressive myorelaxant effect. The aminopiperazine derivatives exert a hypotensive and central nicotinolytic effect. The most active compound of this group 35-M (Schiff’s base of N’benzhydryl-N4-aminopiperazine with triacetonamine, dioxalate salt; Fig. 1) also has central holynolytic and analgetic effects. The most important is its potentiating influence on noradrenaline effects. The increased sensitivity of a-adrenoreceptors does not mean however, that 35-M increases blood pressure and cerebral level of cathecholamine (Taskov et al., 1977). There are no data about the direct effect of the above mentioned piperazine derivatives on the ionic processes in the cell membrane. The present work was undertaken to characterize the influence of the piperazine derivatives with code names P-11, Aligeron (AS,) and 35-M on the membrane processes. The changes in the muscle fiber potentials and ionic currents were used for evaluation of the effects of these compounds. group
MATERIALS
AND METHODS
Animals and isolated preparations
The experiments were carried out on 180 isolated muscle fibers from m. gastrocnemius of Rana ridibunda. The fibers (in situ length) were immersed in a double wall chamber (Fig. 2). The terminal parts of the isolated fibers were cleaned since parts of the tendon and the fascia were left attached to the ends and were used to fix the fibers. Solutions and drugs
The Ringer’s solution used contained (mM): NaCl 115.0, KC1 2.0, CaCl, 1.8, NaH,PO, 0.68 and Na, HP04 1.3 (pH 7.3-7.4) at a constant temperature 20°C. The compounds investigated (P-11, ASI and 35-M) were added separately to the bathing solution in concentration of 10-100 PM after control recordings of the potentials in standard Ringer’s solution. Experimental procedures
Extracellular stimulation by 0.5-l .Omsec rectangular electrical pulses of suprathreshold intensity was applied. The intracellular action potentials (ICAPs) were recorded by a glass micropipette filled with 3 M solution of KC1 with 1 pm tip diameter and a
Muscle fiber and piperazine derivatives
Fig. 2. Experimental set. (1) Muscle fiber, immersed in a Ringer’s solution-filled chamber. (2) Stimulating electrode. (3) Microelectrode and ICAP with its characteristic time interval: Rf-rising time (interval between 10% and peak in depolarization phase); rdeclining time (interval between pick in depolarization phase and 50% in amplitude of the depolarization phase); AMP-ICAP amplitude. (4) Extracellular electrodes with fixed interelectrode distance d and two ECAPs; t-time interval between negative maxima for PV measurement. (5) Thermometer. (6) Reference electrode. (7) Mechanoelectrotron.
of 15-20 MQ. The pipette was connected ing system (705 Microprobe
to a record-
system) by means of an
Ag/AgCl electrode. We adopted the approach of measuring the time intervals between characteristic points of the ICAP (Wallinga-De Jonge et al., 1985). To avoid errors in depolarization onset the rising time (Rt) was measured at a level of 10% in ICAP amplitude. Simultaneous extracellular recordings were made by two glass-coated tungsten electrodes with a tip of about 10 pm in diameter and a resistance of 150 kfL The pair of electrodes with fixed interelectrode distance (d) was oriented parallel to the fiber axis. The extracellular action potentials (ECAPs) at different axial distances, i.e. over the cylindrical part of the fiber far from the stimulation site, over the transition to the conical area and at the very end, as well as at different radial distances (from 0 pm-visually observed contact, to 2000 pm) were recorded by a pair of electrodes attached to the hydraulic microdriver with an accuracy of shift of 1 pm. The ECAPs recorded at large radial distances (the maximal distance being 2000 pm) were averaged over 256 potentials. By means of an analogue-to-digital converter with sampling interval of 36 psec the signals (ICAPs and
ECAPs) were displayed on an oscilloscope for visual control and illustration and transmitted to a personal computer for further analyses. The propagation velocity (PV) of action potential was calculated dividing the interelectrode distance d into the time interval t described in Fig. 2. The mechanogram (contraction curve, twitch) of the muscle fibers was registered by displacement of a mechanoelectrotronic rod, which was stuck in the tendon between the preparation attaching hook and fiber end. The total ionic current (I,) during action potential of a muscle fiber was calculated by the cable equation for excitable fibers in the form given by Tassaki and Hagiwara (1957). The first and second time derivatives of the experimentally recorded ICAPs were calculated by Fourier transformation. The 1, was computed as a difference between the transmembrane and the capacitive current (Radicheva et al., 1985). Parameters observed and statistics
ICAPs-amplitude, duration: Rt and r ; ECAPsamplitude, duration; PV; J-inward and outward phases; twitch-mechanogram. The results reported as mean f SD were assessed for statistical significance using the Student’s t-test at
N. Radicheva et al.
1434
p c 0.05. All statistical procedures and the plotting of graphs were made by computer programs (Statgraphits 1.2; SlideWrite Plus 3.0). Control RESULTS
Changes in the action potentials and propagation velocity
The overshoot and membrane potential gradually decreased with increase of the compound concentration. Hence, the ICAP amplitude decrease was concentration- and substance-dependent: the amplitude decrease by 1@45% vs controls with P-11; by 5-25% vs controls with AS, and by 15-20% vs controls with 35-M (Figs 3,4 and 5, upper rows). The increase of the ICAP duration (Rt and r) was pro-
P-11
Ii f
50 VM
ii
P
ICAP
The potentials were recorded in standard Ringer’s solution and after 30min treatment of the muscle fiber preparations with a given concentration of the agents. More prolonged treatment at a concentration of 100pM accelerated the changes in membrane processes and was enough to suppress the membrane excitability which led to complete inhibition of the electrical and mechanical activity. The effect of the piperazine derivatives studied on the muscle fiber activity was reversible. The effect potency of the compounds tested on the studied parameters decreased in the following order: P-l 1 > AS2 > 35-M.
COllhl
10PM
100 PM
IO pM
50 pM
f
y‘ Is
1:
ECAP
1
Ii
r
f
f
Fig. 4. Changes in ICAPs, ECAPs and Ii’s calculated (in AU-arbitrary units) from the corresponding ICAP after 30 min treatment with AS, in concentrations of 10, 50 and 100PM. portional to the concentration increase. The slope of the approximating lines of the r duration were steeper than those of the Rt approximating lines (Fig. 6) i.e. the z duration was strongly prolonged. Most pronounced and statistically significant were the ICAP changes induced by P-l 1 (Table 1). ECAPs recorded in the vicinity of the fiber membrane reflected changes in the ICAP (Figs 3, 4 and 5, bottom rows). The ECAP amplitude decreased (by 50-75% with P-11, by 2565% with AS2 and by 1560% with 35-M) and the duration increased (up to 2.5 times by 100 p M P- 11 compared to the controls) with the Rt increase. ECAPs in treated muscle fibers recorded at different axial and radial distances to the membrane were similar in shape but with reduced amplitude and increased duration compared to the control potentials (Fig. 7). Hence the extracellular potential field should be weaker and long ranged. The PV of excitation measured between two ECAPs (PV = d/t, see Fig.2) decreased with a concentration increase. The extent of the PV decrease was substance-dependent with statistical significance only for P-l 1 and AS2 (Table 1, Fig. 6). Changes in the total ionic current and mechanical
5 ms
Fig. 3. Changes in ICAPs, ECAPs and I,‘s calculated (in AU-arbitrary units) from the corresponding ICAP after 30 min treatment with P-l 1 in concentrations of 10, SOand 100PM.
activity
The Z, was also affected in treated muscle fibers. P-l 1 gradually suppressed the outward current (amplitude decreased and duration increased) with a concentration increase. The highest concentration
1435
Muscle fiber and piperazine derivatives Table
Compound
1. Effect of the compounds
Rising time (RI)
(fiM) Control P-l 1 IO 20 30 40 50 100 Control
on the ICAP
parameters and PV Propagation velocity, (m/se@
tmsec)
Declining time (T) cm=)
1.056 f 0.269
I .544 f 0.382
1.257 + 0.249
1.368 1.303 1.296 1.591 1.696 2.239
2.282 2.653 2.310 2.696 3.423 5.144
0.962 f 0.168’
*0.152* + 0.291* f 0.365 f 0.279*** k 0.580*** f 0.660’”
+ f f k + *
0.466*** 0.533*** 0.613*** 0.661*** 1.612*** 1.917***
(PV)
0.679 + 0.225** 0.672 f 0.161** 0.286 f 0.026***
1.084iO.138
1.590 * 0.359
1.112~0.131
1.203 i 0.180 1.281 + 0.219*
1.807 _+0.275 1.898 i 0.496
0.977 i_ 0.222 -
1.296 1.494 1.493 2.122 1.238
1.890 2.275 2.005 4.446 2.180
kO.184 + 1.222 i 0.329’ _t 2.342** + 0.530
0.907 * 0.102**
1.951 2.097 2.430 3.004 3.020
f 0.462 i_ 0.495 + 0.408 * 0.666” k 0.650**
I.149 * 0.173
AS* IO 20 30 40 50 100 Control 35-M 10 20 30 50 100
k 0.158’ & 0.478’ i: 0.236’ + 0.788” _+0.214
1.066 * 0.202 1.280 _t 0.191 1.15O_tO.209 1.741 * 0.534” 1.544 f 0.200**
Values are meansfSD;
n = 9 - 15.
*p < 0.05;
**p < 0.01;
***p
0.763 k 0.076*** 0.405 k 0.124*** 1.096 + 0.250
1.093 + 0.076 0.933 i 0. I36 0.818 * 0.200 by Student’s
~-test in comparison
with
control values.
(100 PM) abolished the outward current and affected the inward current-amplitude decrease and prolonged duration were observed (Fig. 3). The AS, effect on the outward current was similar to that of P-l 1 but less pronounced on inward current (Fig. 4). The effect of 35-M on both inward and outward currents was weaker (Fig. 5).
35-M Control
50 pM
100 PM
ICAP 1
Ii f
i\
r
ECAP + Fig. 5. Changes in the ICAPs, ECAPs and I,‘s calculated (in units) from the corresponding ICAP after 30 min treatment with 35-M in concentrations of 10, 50 and 100 PM.
AU-arbitrary
The excitability threshold increased, the twitch amplitude decreased, contraction and relaxation time increased with increase of the compound concentration. These changes were substance-dependent, similar to the above described parameters. DISCUSSION In the present study we used the biophysical approaches (changes in ICAPs, ECAPs, I,, PV of excitation and contractile properties) to assess the functional state of the muscle fiber membrane influenced by some piperazine derivatives. These substances are known to have favorable effects on very important physiological functions and it is of interest to understand how they change the kinetics of the membrane currents and hence, how they would interact with other biologically active substances. The changes were concentration- and substancedependent. The effect potency of the compounds tested on the studied parameters decreased in the following order: P-l 1 > AS2 > 35-M. The steepness of the lines approximating the changes in Rt and T (ICAP duration) showed a more pronounced increase in T duration indicative of a decrease in the activity of the channels responsible for the repolarization in treated fibers. The observed differences in SD for values of the parameters compared at various concentrations of the substances tested may be related to structural and functional characteristics of the fibers. They were not previously selected on their type. The different properties of the slow and fast fibers would be more clearly expressed
1436
N. Radicheva et al.
in the case of changed functional state similar to that observed in our experiments. The repolarization is attributed to the K+-delayed rectifier current (Kao and Stanfield, 1970; Hille, 1984; Radicheva, 1986; Martin and Drayer, 1989) as well as to the large-conductance Ca*+-activated K+- (maxiKC channels) and to Ca*+-activated Cl- channels (Francilini and Petris, 1990; McManus, 1991). In contrast to the alterations in muscle potentials in-
A
A
10
duced by compounds tested, the blocking of the K+-delayed rectifier current led to the typical “plateau’‘-like ICAPs without changes in the depolarization phase (Gillespie, 1977; Radicheva, 1986). The ECAPs corresponding to those changed ICAPs close to the membrane are usually unchanged while the ECAPs recorded at large radial distances possessed typical shape (abrupt front of the terminal positive phase) attributed to the blocking of the
Rt
A
10
A y=1.104+0.011X; r=O.O72 ~=i.868to.o34x;
10
r=0.072
A 0
Rt
07
y=1.061+0.010x; r=O.O60 ~=i.203+0.027~;
r=0.027
1
6-
”
b
$0
Concentration
Oi
160
(IAM)
Concentration
D
C 1).
10
h E
Rt
oAS,
(j,d)
A35-M
?? y=l.lOO-0.000x; r=O.O45 0 y=l.OOO-0.007x; r=0.005 A y=l.l43-0.003x:r=0.045
G2 2
6
,R ‘i;
6_
0
@ b
2 3 Z g
DP-11
?? r
A y=1.167tO.O06x; r=0.661 ?? y=2.050+0.011x; rm0.665
100
1
cl :
{&_;-_i--------i
0
1 0
$
H 100
50
Concentration
(di)
Z a
0
7
0
I
50
Concentration
t
100 (MM)
Fig. 6. Dependence of rising time (Rt) and declining time (5) duration of the ICAF on the concentration of P-l 1 (A), AS, (B) and 35-M (C) as well as the dependence of PV of action potentials on the substance concentration (D).
1437
Muscle fiber and piperazine derivatives
2000pm
-
I
z-5 pY
+
1goopm
I
--
6 PV
-!-
OP +
++-
I
100 pY
1OOOpm --
I
2000pm
1
5P
2.5 pY
10 ma
Fig. 7. Scheme of the muscle fiber ECAPs recorded at different axial distances (from left to right--over the cylindrical part of the muscle fiber, the transition to the conical part and the fiber end) and at different radial distances (from Oy, 1000~ and 2000~ to the fiber membrane). Potentials above the fiber were recorded before treatment and those below the fiber-after treatment with AS2 at .50pM.
K+-delayed rectifier current (Radicheva and Kolev, 1993). Such alterations in muscle potentials were not observed in the present experiments. It is obvious that the ECAP changes reflect the ionic conductance of the muscle fiber membrane. The alterations in the parameters of the ECAPs as treated by compound tested fibers recorded at large radial distances led to a weaker and long-range extracellular potential field. It wouldbeofinteresttoelectromyographicstudiessince it is known that ECAPs at large radial distances are similar to the extraterritorial motor unit potentials.
That would be important in patients with muscular disorders taking piperazine-derivative drugs. Concentrationand compound-induced proportional decrease in membrane excitability may be due to the influence on the large conductance Ca2+dependent K+ channels which provide a pathway for cell repolarization subsequent to membrane depolarization and on the small conductance Ca2+dependent K+ channels possessing properties necessary to account for afterhyperpolarization in myotube (Garcia et al., 1991). The amplitude of the
1438
N. Radicheva et al.
depolarization afterpotential (affected by substances tested) is also Ca2+-dependent (Harada and Takahashi, 1983). Approximately lO-15% of the total repolarization (outward) current flowing during the action potential is attributed to chloride (Hutter and Nobel, 1960). The similarity of the ICAPs changes induced by the agents (especially P-l 1) to that induced by ZnCl,, known to be a blocker of Cl- channels (Stanfield, 1970; Woll et al., 1987; our unpublished data) showed that the piperazine derivatives may block these channels. Valkanov (1993), using the patch clamp technique, has found a block of Ca*+-gated Clchannels of Ascaris suum muscle vesicles caused by P-11 at a concentration of 10pM. All these findings suggest that the investigated compound blocked Ca*+-activated K+ and Cl- currents and that the extent of the blockade was agent-dependent. P- 11 and AS2 most probably affected the slow Ca2+ current similar to the effect of Zn*+ at higher concentrations (Hagiwara and Takahashi, 1966). The amplitude and duration alterations in the ICAPs observed in our experiments were in agreement with the data of Krishtal (1978) regarding such ICAP changes when the slow Ca*+ current was decreased. This effect was the most weakly manifested by 35-M. The established decrease of the ICAP amplitude could also be attributed to a partial blockade of the sodium current. Some piperazine derivatives are considered to be less “specific blockers” of the Ca*+ channels pointing out the possibility for some of them to act also through blocking of certain Na+ channels (Scriabine, 1991; Pauwels et al., 1992). This might be due to the similarity in structure and kinetics of Na+ and Ca*+ channels (Krishtal, 1978; Tanabe et al., 1987). The above described inhibition of membrane excitability, propagation velocity and muscle contraction depending on the agent, concentration and duration of treatment also results from blockade of the Ca2+activated K+ channels which normally link intracellular free Ca*+ to membrane excitability (McManus, 1991). This process regulates action potential frequency and duration and plays a crucial role in excitation-contraction coupling. The ionic imbalance could explain the myorelaxant effect of the tested agents shown at the highest concentration. All these findings characterize piperazine derivatives as agents whose mechanism of Ca*+ antagonistic action is different from that of the typical Ca*+ antagonists. In extensive pharmacological experiments these compounds were found to manifest multireceptor activity (Rainova 1978; et al., Mutafova-Yambolieva et al., 1985; MutafovaYambolieva and Staneva-Stoycheva, 1988a,b). In agreement with these findings, our results suggest that
the alterations in muscle potentials were related to the alterations in the different Z,components indicative of multichannel activity of the agents. In conclusion, the aminopiperazine derivatives (P11, AS2 and 35-M), being less specific Ca*+ blockers, modulated Ca*+ current blocking predominantly Ca*+-dependent K+ and Cl- channels of muscle fiber membrane. Acknowledgement-This work was supported by Grant K-20/91 from National Science Fund, Ministry of Sciences and Education, Republic of Bulgaria. REFERENCES Francilini F. and Petris A. (1990) Chloride channels of biological membranes. Biochem. biophys. Acta 1031(2), 241-259.
Garcia M. L., Galves A., Garcia-Calvo M., King V. F., Vazquez J. and Kaczorowski G. J. (1991) Use of toxins to study potassium channels. J. Bioenerg. Biomem. 23(4), 615-646.
Gillespie J. I. (1977) Voltage-dependent blockage of the delayed potassium current in skeletal muscle by 4aminopyridine. J. Physiol. 273, 64P-65P. Hagiwara S. and Takahashi K. (1966) Surface density of calcium spikes in the barnacle muscle fiber membrane. J. gen. Physiol. W(3), 583601. Harada Y. and Takahashi T. (1983) The calcium component of the action potential in spinal motoneurons of the rat. J. Physiol. 335, 89-100. Hille B. (1984) Ionic Channels of Excitable Membranes. Sinaner Associates Inc., Sunderland, Mpcsachusetts. Hutter 0. F. and Nobel D. (1960) The chloride conductance of frog skeletal muscle. J. Physiol. 151, 89-102. Ivanov I., Troyanska G., Christova K., Dantchev D., Sulay P. and Waltchanova R. (1977) Synthese einiger N’-substituierter-N-(trans-3-hydroxy-l,2,3,4-tetrahydro2-naphthyl)-piperazine. Arch. Pharm., Weinhein 310, 925-93 1. Kao C. Y. and Stanfield P. R. (1970) Actions of some cations on the electrical properties and mechanical threshold of frog sartorius muscle fibers. J. gen. Physiol. 55, 620-639.
Kovalyov G., Nikolov R., Tyurenkov I. and Taskov M. (1976) Effects of the analogue of cinnarizine AS, on the cardio-vascular system. Acta Physiol. Pharmac. bulg. 2(4), 27-3 I. Krishtal A. (1978) Modulation of Ca++ channels in nerve cell by EGTA. Dokladi Acad. Nauki SSR 238,482485 (in Russian). Martin A. R. and Drayer S. E. (1989) Potassium channels activated by sodium. J. exp. Physiol. 74, 1013-1041. McManus B. (1991) Calcium-activated potassium channels: regulation by calcium. J. Bioenerg. Biomem. 23(4), 537-560. Mutafova-Yambolieva V. and Staneva-Stoytcheva D. (1988a) Pharmacodynamic effects of a f-piperazinotetralin (P- 1I)-combined and 3-adrenorece$orblocking drug with hypotensive activity. Menr. Find. exp. clin. Pharmac. lo(g), 551-557. Mutafova-Yambolieva V. and Staneva-Stoytcheva D. (1988b) Involvement of peripheral dopaminergic mechanisms in the action of a 2-piperazinotetralin (P-11) with hypotensive activity. Ment. Find. exp. clin. Pharmac. lo(g), 559-562.
Mutafova-Yambolieva V., Staneva-Stoytcheva D. and Ivanov D. (1985) Effects of a new 2-piperazinotetralin on a-l- and a-2-adrenergic receptors in isolated rat vas deferens. Meth. Find exp. clin. Pharmac. 7(6), 291-296.
Muscle fiber and piperazine derivatives Mutafova-Yambolieva V., Staneva-Stoytcheva D. and Nikolov R. (1989) Comparative evaluation of the Ca*+antagonist activity of some Bulgarian vasodilating agents and of known Ca2+-antagonists on K+-depolarized smooth muscle from Guinea-pig taenia coli. Acta Physiol. Pharmac. bulg. H(2), 40-46.-
-
Nikolov R. (1978) Pharmacological studies on the influence of the compound Aligeron-As, on the brain blood circulation. Ph.D. Thesis, Sofia. Pauwels P. J., Van Assouw H. P.. Peeters L. and Levsen J. E. (1992) Neurotoxic action of veratridine in rat drain neuronal cultures: mechanism of neuroprotection by Ca2+-antagonists nonselective for slow Ca2+-channels. J. Pharmac. exp. Ther. 255(3), 1117-l 122. Radicheva N. (1986) Intracellular and extracellular action potentials in frog muscle fiber upon blocking the potassium conductivity. Acta physiol. Pharmac. bulg. 12(2), 35-39.
Radicheva N. and Kolev V. (1993) Changes in the muscle fiber extracellular action potentials induced by 4-aminopyridine. Compt. Rend. I’Acad. Bulg. Sci. 45(7), 89-92. Radicheva N. I., Trayanova N. A., Gydikov A., Kostov K. G. and Kossev A. R. (1985) Changes in the total ionic current of frog muscle fiber during action potential under continuous activity. Compt. Rend. I’Acad. Bulg. Sci. 3tX@), 1085-1088. Rainova L., Georgieva A. and Staneva-Stoytcheva D. (1978) On the hypotensive and antihypertensive activity of a new derivative of 2-aminotetraline. Expt. Med. Morph. 17, 3743 (in Bulgarian).
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Scriabine A. (1991) Comparative pharmacology of 1,4dihydropyridines and other calcium channel ligands. J. Cardovasc. Pharmac. 9(Suppl. l), 3-7.
Stanfield P. (1970) The differential effects of tetraethylammonium and zinc ions on the resting conductance of frog skeletal muscle. J. Physiol. 209, 231-256. Tanabe T.. Takeshima H.. Mikani A.. Flockerzi. V.. Takehashi H., Kagnawa K., Kojima M., Matsuo K.; Hirosa T. and Numa S. (1987) Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature 328, 313-318. Taskov M., Velichkova S. and Tjutjulkova N. (1977) On some pharmacological effects of a piperazine derivative. Pharmacia 27(3), 19-23 (in Bulgarian). Tassaki I. and Hagiwara S. (1957) Capacity of muscle fiber membrane. Am. J. Physiol. 188, 423429. Valkanov M. (1993) Voltage-dependence of Cl--channel block by piperazine derivative, P-l 1. Abstracts, 11th International Biophysics Congress, Budapest, Hungary. Wallinga-De Jonge W., Gielen F., Wirtz P., De Jong P. and Broenink J. (1985) The different intracellular action potentials of fast and slow muscle fibers. Electroencephal. clin. Neurophysiol. 60, 539-547.
Woll K. H., Leibowitz M. D., Neumcke B. and Hille B. (1987) A high conductance anion channel in adult amphibian skeletal muscle. Pfliigrs Arch. 410(6), 632-640. Zikolova S., Konstantinova R., Daleva L. and Taskov M. (1978) Synthesis and pharmacological study of new Schiff’s bases of N’-substituted-N4-aminopiperazines with triacetonamine. Pharmaciu 28(3), l-9 (in Bulgarian).