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Effects of clonixin on the electrical activity of cardiac pacemaker cells
Gen. Pharmac. Vol. 23, No. 3, pp. 515-521, 1992 Printed in Great Britain. All rights reserved
0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
EFFECTS OF CLONIXIN ON THE ELECTRICAL ACTIVITY OF CARDIAC PACEMAKER CELLS M. A. MORALES,* L. INOSTROZA, T. SALAZARand C. PAEILE Department of Pharmacology, Faculty of Medicine, University of Chile, P.O. Box 70.000, Santiago 4, Chile (Received 15 August 1991)
Abstract--1. The electrophysiological effects of clonixin, a non-steroidal analgesic, on cardiac pacemaker cells of spontaneously beating frog sinus venosus, were studied by intracellular recording of transmembrane potentials. 2. Results show that clonixin (Clx) between 1 × 10 6 M and 3 x 10-4 M, decreases the OS, APA, Fma~ and frequency of primary and subsidiary cells, however pacemaker cells differ in their sensitivity to Clx. 3. At 2 x 10 -6 M, Clx completely blocked the spontaneous beating of primary cells. It is necessary to increase the Clx concentration about two orders of magnitude in order to attain a similar degree of blockade of subsidiary cells. 4. Previous or simultaneous superfusion with atropine does not modify Clx effects, thus a probable cholinergic mechanism of action for Clx is discarded. 5. When Clx concentrations were lower than 5 x 10 -4 M, their effects on both types of cells were partially reversed by a 100% increase of external calcium concentration. 6. BAY K-8644 which stimulates calcium influx through calcium L-type channels, reverted Clx effects on pacemaker cells. 7. It is suggested that Clx blocks calcium inward current which generates all or part of the upstroke of primary cells and subsidiary ones, respectively.
INTRODUCTION Clinical evidence has demonstrated that clonixin (Clx), widely used in Latin America, is a good analgesic (Finch and De Kornfeld, 1971; Paredes et al., 1986). This drug of the fenamate group is similar to mefenamic acid and is more potent as an analgesic than as an anti-inflammatory. This finding suggests that Clx may act on C.N.S. (Pellisier et al., 1984). This central action is not mediated by opiate receptors but seems to be similar to the action of indoprofen, propionic derivatives, aspirin and others compounds (Paeile et al., 1989; Willier et al., 1989; Ferreira et al., 1978; Jurna and Brune, 1990). Recently, it has been reported that the i.v. administration of high doses of Clx (100mg/kg) induces bradycardia and hypotension (Bustamante et al., 1988). The action induced by Clx (10-6-10 -3 M) was investigated in isolated atria preparations, recording negative chronotropic and inotropic effects. Based on the above-mentioned findings, it was decided to study the electrophysiological effects of Clx on cardiac pacemaker cells. Frog sinus venosus is a suitable preparation which facilitates the microelectrode impalement of pacemaker cells and it is a useful experimental model in order to determine putative calcium antagonist properties of any drug (Hernfindez et al., 1987; Morales et al., 1988, 1989). Pacemaker cells generate a spontaneous action potential, whose phase 0 and the last third of phase 4, are fully dependent (primary cells) or partially dependent (subsidiary cells) on slow inward calcium
current (Brown et al., 1977; Molivdas and Sperelakis, 1986; Hern~indez et al., 1987; Morales et al., 1988; Hagiwara et al., 1988). The effects of verapamil on both types of cells have been widely described (Morales et al., 1988; Koild et al., 1986; Hernfindez et al., 1987) and it is known that verapamil action is more pronounced on primary cells. These cells are completely blocked, with disappearance of their electrical activity as a result of calcium current blockade. On the other hand, verapamil at moderate concentrations partially diminish the overshoot and potential amplitude of subsidiary cells. It is clear then that both cellular types present different sensitivity to calcium antagonists and this obeys to the different degree of contribution that calcium inward current plays in their respective electrical activities. In a previous report (Bustamante et al., 1988) we stated that Clx depresses the action potentials of cardiac pacemaker cells acting like a calcium antagonist, however, the possibility of a cholinergicagonist mechanism of action that would also explain the bradicardic effect of Clx was not discarded. In the present work we report that Clx effects on overshoot, action potential amplitude and frequency are not of a cholinergic nature and these effects are partially reverted by increasing external calcium to 3.6 m M and fully reverted by using BAY K-8644. MATERIALS AND METHODS
Frogs from Caudiverbera caudiverbera species weighing between 200 and 350 g were used throughout these experiments. Animals were sacrificed by spinal cord transection.
*To whom all correspondence should be addressed. 515
M. A. MORALESet al.
516
Table 1. Electrophysiologicaleffects of Clx on primary cells of the frog heart APA Vmax TP MDP OS DPA60 Frequency N (mV) (V/sec) (mV) (mV) (mV) (msec) (beats/min) 16 62.9_+0.8 2.9+0.2 48.7_+0.9 61.8_+0.9 13.9_+0.4 639.2_+4.3 28.9_+0.1 11 60.8_+1.3 2.7_+0.1 46.3_+1.4 59.7_+1.7 14.4_+0.3 649.5_+3.9 27.1_+0.1" 11 62.5_+1.3 2.8_+0.1 44.4_+0.9 59.0_+0.4 18.1 _+1.4 634.7_+21.4 29.4_+0.8 9 54.0+2.3* 1.8_+0.1" 46.8-+1.9 57.3-+2.1 7.2_+1.3" 713.5_+12.9 25.4_+0.2*
Control Clx 1 × 10-6M Control Clx2x 10 6M
N: number of cells. Each value is the mean _+SEM. *P < 0.005 with respect to control. The heart was dissected and transferred to a dish containing oxygenated Ringer solution. Under a dissection microscope the ventricles were removed and the sinus venosus separated from the atria. Preparations were pinned to the bottom of a 12 ml organ bath and superfused continuously with oxygenated (100% 02) Ringer. Experiments were done at room temperature (19 21°C). The composition of the Ringer's solution employed was (mM): Na + 120.1; K + 2.5; Ca 2+ 1.8; C1- 121; H2PO42 0.85; HPO~- 2.15; glucose 11.0. pH was adjusted to 7.2 with diluted NaOH. Ringer's solution with high calcium contained Ca 2+ 3.6 mM; Tris buffer replaced phosphate buffer and the osmolarity was corrected in basis to sodium ion. The flow rate of the superfusion was 3 ml/min. Action potentials from primary and subsidiary cells were recorded differentially with glass microelectrodes filled with 3 M KCI and a reference saline bridge (Ag/AgCl-agar Ringer). The tip resistance of the electrodes ranged from 20 to 40 MfL The recording system consists mainly of a differential preamplifier providing capacity compensation with a time constant of 40/~sec. The output of the d.c. amplifier was displayed in a dual beam oscilloscope and simultaneously displayed, analyzed and recorded using an IBM compatible XT (640 kbyte) computer (Hercules high resolution graphics card) supplied with an analog to digital converter and ad hoc software devised by us to calculate the action potential parameters and Vm~x. The following electrophysiological parameters were analyzed: maximum diastolic potential (MDP); action potential amplitude (APA); overshoot lOS); maximum rate of depolarization [Vm~~ or (dV/dt)max]; threshold potential (TP); action potential duration to 60 and 90% of repolarization (APD60 and APD90 ) and frequency of potential firing (or rate). All these parameters have been defined elsewhere (Guerrero et al., 1987; Morales et al., 1988). Preparations were equilibrated in Ringer solution for 45 rain after surgery and bath installation procedures; then control recordings were taken. Drugs assayed were added to the Ringer solution and each preparation was exposed to only one drug concentration. Data are expressed as the means of clonixin steady state effects observed 20-40 rain after drug exposure of different sinus venosus preparations under the same experimental conditions. Each preparation was explored for the area most indicative of pacemaker activity and primary and subsidiary cells were impaled. Criteria for identifying both types of cells in the Chilean frog, C. caudiverbera, have been previously published (Hern~indez et al., 1987). Drugs used. Clonixinate of L-lysine, provided by Pharma Investi Laboratory, Santiago, Chile. L-lysine (British House A
-
~
,
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B
Ltd, England). BAY K-8644 (Bayer AG, Fed. Rep. Germany) Atropine sulfate (Sigma Co., St Louis, Mo., U.S.A.). The mean value + the standard error of the mean (SEM) was calculated for each parameter measured. The statistical analysis was done using Student's t-test. Significance was accepted at P-values less than 0.05. RESULTS C l x effects on action potential p a r a m e t e r s P r i m a r y cells. The different p a r a m e t e r s studied A P A , TP, M D P , OS a n d APD60 were not significantly modified by Clx 1 x 10 6M, however the firing rate diminished approximately 6 % ( P < 0.005) as can be observed in Table 1. W h e n primary cells were exposed to Clx 2 x 10 6 M, a notorious decrease of A P A , OS a n d Fma× was observed, a b o u t 60, 14 a n d 50% respectively. These a n d other variations are depicted in Table 1 a n d are according with an increased negative c h r o n o t r o p i c effect (see also Fig. 1). By elevating the time of superfusion to 4 0 r a i n or the Clx c o n c e n t r a t i o n above 2.5 × l0 6, primary cells action potentials were completely suppressed. Subsidiary cells. Clx effects on subsidiary pacem a k e r cells were studied with c o n c e n t r a t i o n s from 1 x 10 -6 to 1.5 x l0 4 M. CIx induced a conc e n t r a t i o n - d e p e n d e n t decrease of OS, APA, Vmax a n d frequency of beating. Moreover, T P a n d M D P were slightly displaced to less negative potential values, suggesting a depolarizing effect. These results are depicted in Table 2 and illustrated in Fig. 2. With higher c o n c e n t r a t i o n s of Clx, above 2.5 x 10 -4 M, the electrical activity of subsidiary cells was completely suppressed (see Fig. 3). Before complete blockade of the s p o n t a n e o u s activity m e m b r a n e potential, oscillations were observed, with amplitudes not exceeding 2-3 m V (not shown). After a few minutes, the impalement o f subsidiary p a c e m a k e r cells showed a stable m e m b r a n e potential a r o u n d - 4 0 _+ 0.7 mV. Partial recuperation of bioelectrical activity was attained after superfusing the p r e p a r a t i o n s during almost 3 hr (3 ml/min). C
BO
5 0 0 rns
Fig. 1. Effect of 2 x 10-6 M Clx on the action potential of a primary pacemaker cell. (A) control; (B) and (C) 45 and 70 min after Clx respectively.
Clonixin effects on pacemaker cells
517
Table 2. Electrophysiological effects of CIx on subsidiary pacemaker cells of the frog heart N
APA (mV)
Vma~ (V/see)
TP (mV)
MDP (mV)
OS (mV)
APD60 (msec)
Frequency (beats/min)
Control Clx 1 × 1 0 6M
14 11
72.7_+0.9 71.6_+1.4
5.5+0.1 4.9+0.1"
54.9+0.8 53.2_+1.2
69.6_+0.8 67.0_+1.3
18.0+0.4 18.5_+0.7
641.8+3.9 659.2_+3.7*
28.9_+0.1 27.1_+0.1"
Control CIx l x l 0
SM
18 22
79.9_+1.5 69.8_+1.3"
6.5_+0.1 5.6_+1.1"
60.2+0.7 55.6_+1.1"
71.0_+0.9 63.4+1.3"
20.2_+1.1 14.2_+0.6"
523.1_+4.3 545.1_+5.1"
22.5_+0.1 21.2-+0.1"
Control CIx 1 x 10 4M
23 14
77.2_+ 1.1 65.2_+0.2
6.6_+0.1 5.4_+0.2*
58.7-+0.9 55.6_+ 1.6
70.6_+0.7 65.7_+ 1.6"
18.5_+0.7 9.7_+ 1.4"
576.7_+3.8 551.1 +5.5*
32.0_+0.1 29.5_+0.5*
Control Clx 1.5x 10 4M
12 22
83.6+_0.5 68.0_+2.1"
6.9_+0.1 5.3_+0.2*
59.9_+0.5 54.5_+1.5"
67.8_+0.6 64.6_+1.4"
23.7_+0.6 13.5_+1.2"
508.9_+3.8 509.2_+5.8
32.6_+0.1 30.2_+0.3*
N: number of impaled cells. Each value is the mean + SEM. *P < 0.005 with respect to control.
Influence of atropine on the effects of Clx
Clx effects and calcium concentration
In order to investigate an eventual role of atrial muscarinic receptors on Clx effects, a series of experiments were carried out in atropinetreated preparations. The data is summarized in Table 3. It has been previously described that atropine, 1.4 x 10-SM, prevents acetylcholine activation of muscarinic receptors of frog heart (Guerrero, 1982; Guerrero et al., 1987) and its consequent bradicardic effects. As shown in Table 3, in sinus venosus exposed to 2.8 x 10-SM atropine sulphate, this drug significantly increased APA, TP, MDP, OS, frequency and the duration of action potential. When Clx 2 x 10 . 6 M was added, APA, Vmax,OS and frequency of primary cells were reduced in a similar percentage to that shown in Table 1, for preparations untreated with atropine. Therefore it appeared that Clx was acting at other sites, different from muscarinic receptors.
When the calcium concentration of Ringer's solution was elevated to 3.6 mM (100%), Clx induced a lower depressor effect on subsidiary cells than that observed with normal Ringer solution, as can be seen in Fig. 4. The effect on APA and OS was reduced by not more than 50% of the effect observed with normal calcium concentration. However, the I/max was depressed about 20% in both experimental conditions. The rate of firing was more affected in high Ca 2÷ Ringer solution. It is known that in sinus venosus preparations superfused with increased calcium concentrations a significant decrease in sinus rate can be observed (Guerrero, 1982; West, 1972). In summary, Clx effects were only partially reversed in the presence of elevated (100%) external calcium concentration. When Clx concentrations high enough to obtain blockade of spontaneous action potential of both types of pacemaker cells were used, i.e. above 2.5 x 10 -4 M , C a 2+ increase up to
A 0 mV
B
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
?"
-70 mV
500ms
I
I
I
t
t
I
Fig. 2. Effect of 1.5 x 10 . 4 M Ch on the action potential of a subsidiary pacemaker cell. Lower trace is (dV/dt) signal. (A) control; (B) 40 rain after Clx.
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500ms
Fig. 3. Effect of 5 x 10-4 M Clx on the action potential of a subsidiary pacemaker cell. (A) control; (B) and (C) after 20 and 40 min of Clx, respectively. GP 23/3--0
M. A. MORALESet al.
518
Table 3. Clonixin effects on primary pacemaker cells treated with atropine APA Vm~x TP MDP OS N (mV) (V/sec) (mV) (mV) (mV) 17 60.2_+0.9 2.9_+0.1 44.8_+0.7 63.7_+0.8 15.4_+0.5 14 64.6_+0.6**~ 3.1+0.1 47.7_+0.4**~ 66.5_+0.7**~ 16.8_+0.6*~
Control Atropine 2.8 × 10 5M Atropine 2.8 x 10 5M +clonixin 2 x 10 6M 11 56.9-+1.3**~ 1.9-+0.1**~ 48.4-+ 1.2 N: number of impaled cells. Each value is the mean -+ SEM. Significance level with respect to control (~), with respect to atropine (2). *P < 0.05; **P < 0.005. 100% was unable to prevent the complete disappearance of electrical activity.
Modification o f Clx effects by B A Y K-8644 An intent to localize the probable Clx site of action was done using BAY K-8644, which is a " L " type calcium channel agonist. As can be seen in Fig. 5, for primary pacemaker cells, after 15 min of superfusion with Clx 2.5 × 10 6M all parameters were significantly modified, with the exception of A P D . When OS and frequency decayed to 40 and 16% respectively, superfusion with BAY K-8644, 1 × 10 7M, with Clx at the same concentration as before was started. After about 20 min, a complete recuperation of the initial values of action potential parameters was observed (see also Fig. 6). A similar recuperation was attained in subsidiary cells treated with Clx when BAY K-8644 was added (Fig. 7). Some electrophysiological parameters were even increased above the control values, suggesting a clear enhancing effect of the agonist. F r o m these results it was concluded that BAY K-8644 reverted the depressor effect of Clx on both primary and subsidiary pacemaker cells. Clx effects on action potential o f f r o g sciatic nerve To rule out the possible effect of Clx on sodium conductance, Clx (5 × 10 4 M) was superfused in a frog-isolated sciatic nerve preparation electrically stimulated. After 120 min the action potential amplitude, Vm,x and relative refractory period were not changed. These results suggest a certain specificity for the mechanism of action of clonixine.
62.9-+1.8*~
Frequency APD60 (beats/min) (msec) 27.5_+0.1 595.0_+6.7 28.8_+0.1**~ 622.3-+5.4**
8.5_+ 1.2**: 24.0_+0.1**: 639.4_+8.9
DISCUSSION
The results of the present work provide evidence of the concentration-dependent effects of Clx on the spontaneous electrical activity of cardiac pacemaker cells. This is clearly manifested by the Clx-induced depression of APA, OS, Vmax and frequency of primary and subsidiary pacemaker cells. It is known that both types of pacemaker cells exhibit some electrophysiological differences in relation to the parameters which characterize their respective action potentials. In this way, primary cells Vmax, A P A and OS are significatively lower than those of subsidiary cells and their values of TP and M D P are less negative (Brown et al., 1977; Hernfindez et al., 1987; Noble, 1985). These differential characteristics arise from the composition of ionic currents which underly the genesis of the action potential of both types of pacemakers cells. As a fact, primary cells upstroke is exclusively dependent on calcium entry and it is verapamil-sensitive (Hagiwara et al., 1988; Molivdas and Sperelakis, 1986; Morales et al., 1988) and TTX-insensitive (Brown et al., 1977; Hernfindez et al., 1987). On the contrary, action potentials of subsidiary cells exhibit two different inward currents causing the upstroke, a fast TTXsensitive (sodium inward current) and a slow verapamil-sensitive current (calcium inward current). These cells are less readily and less efficiently blocked by verapamil (Morales et al., 1988, 1989). Although Clx induced a progress reduction and then a total blockade of the electrical activity of both types of
20
OS
APA
z
RATE
Vmax
-20
N\\N ,x\x-~
-40
N\\'¢,
1
I$ -60
k~
CLX + 1.8 m.lll Ca++
~
CI,X+ 3.6 ~
Ca++
Fig. 4. Modification of the effects of 1 x 10 -4M Clx on APA, OS, RATE and Vm~x of subsidiary pacemaker cells by calcium concentration increase. Each column indicates a percentage change (mean + SEM) from the drug-free control values. **P < 0.005 compared to control.
519
Clonixin effects on pacemaker cells 20
LU t.9 Z -IC.)
APA
RATE
Vmax
OS
-20
-40 Ill
CLX
~
c~
÷ ~ Y K-~B44
Fig. 5. Modification of the effects of 2.5 × 10 6 M Clx on APA, OS, Vmaxand RATE of primary pacemaker cells by 1 × 10 7M BAY K-8644. Each column indicates a percentage change (mean _ SEM) from the drug-free control values. **P < 0.005 compared to control. pacemaker cells, primary cells showed a greater sensitivity. It was necessary to increment Clx concentration about two orders of magnitude in order to obtain a similar degree of blockade of subsidiary cells. As it is known, the depression of APA, OS and Vmax indirectly reflects the modification of the currents involved in the upstroke (Li and Sperelakis, 1983; Hondeghem and Cotner, 1978; Tung and Sperelakis, 1982; Woods and West, 1985). According to this, results obtained in the present work clearly indicate that Clx, like verapamil, acts by decreasing calcium entry. It seems probable that Clx through this mechanism decreases the frequency of spontaneous firing of pacemaker cells and it could explain the bradicardic effects observed in rats after a Clx i.v. injection and the negative chronotropic effect that is observed in preparations of rat isolated atria (Bustamante et al., 1988). The experiments with atropine indicate that Clx does not act through muscarinic receptors. It was necessary to discard this possibility because it is known that acetylcholine and muscarinic agonists, induce a negative chronotropic effect on pacemaker cells by decreasing the slow inward calcium current and increasing the outward potassium current (Nargeot and Garnier, 1982; Del Castillo and Katz, 1955; Hutter and Trautwein, 1956; Giles and Shibata, 1981; Iijima et al., 1984). The data presented in this report suggest that Clx acts by blocking the calcium entry which originates A
-70
the phase zero of the action potential of primary cells and the overshoot of subsidiary cells in a similar form to that observed for verapamil-treated sinus venosus preparations (Morales et al., 1988). This calcium inward current is carried through calcium channels of long duration or "L" type which have been found in heart, neurons and other tissues (Hagiwara et al., 1988; Fozzard, 1988; Schwartz et al., 1988; Nowycky et al., t985). As it has been determined that BAY K-8644 specifically increases L-channel conductance, it is a useful tool to determine if a calcium antagonist acts on this calcium channel. From our results it can be observed that the Clx depressor effect on cardiac pacemaker cells was almost completely reversed by BAY K-8644. These results allow to establish a certain resemblance between Clx, verapamii and other calcium channel blockers regarding their cardio-depressant activities and calcium antagonist properties. On the other hand, the increase of external calcium concentration induced a partial reversion of Clx effect on pacemaker cells. Different studies have shown that the calcium antagonism of verapamil, nifedipine and diltiazem is not readily reversed by increasing Ca 2+ concentration (Ishii et al., 1985), and that an increment of 200% of external calcium is needed to displace membrane-bound calcium channel antagonists (Hulth6n et al., 1982) or their effects (Sperelakis, 1987). The results of experiments with frog sciatic nerve show that Clx does not affect sodium conductance B
C
mV
500 ms
Fig. 6. Reversion of Clx effects on action potential of primary pacemaker cells by BAY K-8644. (A) Control; (B) after 2.5 x 10.6 M Clx; (C) same impaled cell of (B) after addition of 1 x l0 -7 M BAY K-8644.
520
M. A. MORALESet al. 40
APA
0S
Vrnax
*o
20
@@
t.9 Z -r t_)
RATE
j *o
*o
-Z0
oo
-40
CI~
~
CLX + BkY K - 8 6 4 4
Fig. 7. Modification of the effects of 1 x 10 5 M Clx on APA, OS, Vmax and RATE of subsidiary pacemaker cells by 1 x 10 -7 M BAY K-8644. Each column indicates a percentage change (mean + SEM) from the drug-free control values. **P < 0.005 compared to control.
which originates the phase zero of the nerve action potential. It suggests a certain specificity in the m e c h a n i s m of action of Clx. A l t h o u g h with low Clx c o n c e n t r a t i o n s the action potential d u r a t i o n was slightly modified, as the blockade of cardiac p a c e m a k e r activity led to a stable potential o f a b o u t - 4 0 mV, it seems plausible that this depolarization to a stable resting potential can be due to a Clx-induced lowering o f K ÷ conductance. This would be a n o t h e r characteristic shared by Clx and organic calcium antagonists (Molivdas a n d Sperelakis, 1986; Morales et al., 1988, 1989), a l t h o u g h this blockade of K ÷ c o n d u c t a n c e is only seen with high c o n c e n t r a t i o n s of the latter (Hume, 1985). It is still necessary to carry out more studies with improved techniques, such as single channel recording, for a n accurate determination of Clx m e c h a n i s m o f action o n calcium conductance. However, from the results presented here we consider plausible t h a t this drug m a y be acting like a calcium channel blocker o n cardiac p a c e m a k e r cells. It is n o t e w o r t h y to m e n t i o n here t h a t previous work from o u r l a b o r a t o r y has shown that Clx inhibits n e u r o t r a n s m i t t e r release from the rat vas deferens ( B u s t a m a n t e et al., 1989b), an event which is associated with calcium entry t h r o u g h " L " type channels in nerve terminals. On the other h a n d , it is k n o w n that the analgesic action of Clx is also exerted at C N S level and it is not antagonized by n a l o x o n e ( B u s t a m a n t e et al., 1989a; Ci6falo et al., 1972). Discarding a m e c h a n i s m of action t h r o u g h opioid receptors and with the antecedent of a great resemblance between Clx and opiates analgesic effect, it is possible to assume that Clx could be acting on a calcium channel associated to an opioid receptor. It has been d e m o n s t r a t e d that m o r p h i n e reduces depolarization-dependent calcium uptake in n e u r o n s ( K o n n o a n d Takayanagi, 1982) and there are studies which have d e m o n s t r a t e d that the n u m b e r of brain calcium channels is m o d u l a t e d by chronic use of m o r p h i n e ( R a m k u n a r and E l - F a k a h a n y , 1984). These findings open the
possibility of Clx calcium a n t a g o n i s t action to C N S level as a good working hypothesis for further research. Acknowledgements--This research was supported by Proyecto de Investigaci6n B2897-9033 from DTI, University of Chile. The authors are grateful to Dr Gianni Pinardi for his criticism during the manuscript redaction and to Dr Jaime Zacharias for AD-converter design. REFERENCES
Brown H. D., Giles W. and Noble S. J. (1977) Membrane currents underlying activity in frog sinus venosus. J. Physiol. 271, 783 816. Bustamante D., Miranda H. F., Paeile C. and Wolstenholme W. W. (1988) Effects of clonixin, a non-steroidal analgesic, on the neurotransmission of the vas deferent of the rat. Gen. Pharmac. 19, 755-758. Bustamante D., Miranda H., Pelissier T. and Paeile C. (1989a) Analgesic action of clonixin, nifedipine and morphine using the formaline test. Gen. Pharmac. 20, 319 322. Bustamante D., Morales M., Pellisier T., Saavedra H., Miranda H. F. and Paeile C. (1989b) Experimental cardio-depressant effect of clonixin. Gen. Pharmac. 20, 605~508. Ci6falo V. B., Patel J. and Taber R. I. (1972) Antinociceptive activity of clonixin in Rhesus monkeys. Jap. J. Pharmac. 22, 749-753. Del Castillo J. and Katz B. (1955) Production of membrane potential changes in the frog's heart by inhibitory nerve impulses. Nature 175, 1035. Ferreira S. G., Lorenzetti B. B. and Correa F. M. A. (1978) Central and peripheral antialgesic actions of aspirin-like drugs. Eur. J. Pharmae. 53, 3948. Finch J. S. and De Kornfeld T. (1971) Clonixin: a clinical evaluation of a new oral analgesic. J. Clin. Pharmac. 11, 371 377. Fozzard H. (1988) Cellular basis for inotropic changes in the heart. Am. Heart. J. 116, 230~35. Giles W. and Shibata E. (1981) Autonomic transmitter actions on cardiac pacemaker tissue: a brief review. Fedn Proc. 40, 2618 2624. Guerrero S. (1982) Effects of 4-aminoquinoline on action potentials of the frog sinus venosus. Archs Int. Pharmacodyn. ThOr. 259, 271-283.
Clonixin effects on pacemaker cells Guerrero S., Morales M. A. and Hernhndez D. (1987) Electrophysiological effects of bupivacaine on transitional pacemaker cells of the frog heart. Archs Int. Pharmacodyn. ThOr. 288, 59-70. Hagiwara N., Irisawa H. and Kameyama M. (1988) Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J. Physiol. 395, 233-253. Hernfindez D., Guerrero S. and Morales M. A. (1987) Electrophysiological characteristics of cardiac pacemaker cells of the frog Caudiverbera caudiverbera. Comp. Biochem. Physiol. 87, 649~i56. Hondeghem L. and Cotner C. (1978) Measurement of Vmax of the cardiac action potential with sample/hold peak detector. Am. J. Physiol. 234, H312 H314. Hulth~n U. L., Landmann R., Burgisser E. and Buhler F. R. (1982) High affinity binding sites for 3H' verapamil in cardiac membranes. J. Cardiovasc. Pharmac. 4, $291-$293. Hume J. (1985) Comparative interactions of organic Ca ++ channels antagonists with myocardial Ca ++ and K + channels. J. Pharmac. Exp. Ther. 234, 134-140. Hutter O. F. and Trautwein W. (1956) Vagal and sympathetic effects on the pacemaker fibres in the sinus venosus of the heart. J. Gen. Physiol. 39, 715-733. Iijima T., Irisawa H. and Kameyama M. (1984) Membrane currents and their modification by acetylcholine in isolated single atrial cells of the guinea-pig. J. Physiol. 359, 485-501. Iishii K., Taira N. and Yanagisawa T. (1985) Differential antagonism by BAY K-8644, a dihydropiridine calcium agonist, of the negative inotropic effects of nifedipine, verapamil, diltiazem and manganese ions in canine ventricular muscle. Br. J. Pharmac. 84, 577-584. Jurna L. and Brune K. (1990) Central effect of the nonsteroid anti-inflammatory agents, indometacin, ibuprofen and diclofenac, determined in C fibre-evoked activity in single neurones of the rat thalamus. Pain 41, 71-80. Koild V., Tritthart H. A. and MacLeod R. S. (1986) Different effects of calcium-antagonist on automaticity in single pacemaker cells in synchronized networks of cultured embrionic heart muscle cells. J. Molec. Cell. Cardiol. 18, 207-217. Konno F. and Takayanagi I. (1982) Effect of morphine on the stimuli-induced calcium uptake into sinaptosomes isolated from morphine-tolerant rats. Jap. J. Pharmac. 32, 625-~532. Li T. and Sperelakis N. (1983) Calcium antagonist blockade of slow action potentials in cultured chick heart cells. Can. J. Physiol. Pharmac. 61, 957-966. Molivdas P. A. and Sperelakis N. (1986) Effect of calcium antagonistic drugs on the electrical activity of rabbit sinoatrial node. Br. J. Pharmac. 88, 249-258.
521
Morales M. A., Hern~.ndez D., Ladr6n de Guevara R. and Guerrero S. (1988) Electrophysiological effects of verapamil on primary and transitional pacemaker cells of the frog heart. Gen. Pharmac. 19, 425-430. Morales M. A., Gallardo R., Martinez J. L., Puebla R. and Hern~.ndez D. (1989) Effects of 7-0-demethylisothalicberine, a bisbenzyl-isoquinoline alkaloid of Berberis chilensis, on electrical activity of frog cardiac pacemaker cells. Gen. Pharmac. 20, 641~546. Nargeot J. and Garnier D. (1982) The action of muscarinic agonists and antagonists on frog atrial fibers. J. Pharmac. 13, 431-445. Noble D. (1985) Ionic basis of rhythmic activity in the heart. In Cardiac Eleetrophysiology and Arrhytmias (Edited by Zipes D, F. and Jalife J.), pp. 3-9. Grune & Stratton, Florida. Nowycky M., Fox A. and Tsien R. (1985) Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature 316, 440-443. Paeile C., Lombard M. C., Saavedra H., Bustos E. and Pelissier T. (1989) Evidence of a central component in the analgesic effect of indoprofene. Gen. Pharmac. 20, 519-524. Paredes H., Solis R. m Palacios J., Rocha M., Montecinos A., Ramos H. and Hern~.ndez A. (1986) The use of intravenous lysine clonixinate for rapid pain relief. An open clinical study. Curr. Ther. Res. 40, 86-91. Pellisier T., Bustos E. and Paeile C. (1984) Effects of the anti-inflammatory drug mefenamic acid on the electrophysiological activity of some cerebral structures. Res. Commun. Psychiat. Behav. 9, 221 226. Ramkumar V. and El-Fakahany E. (1984) Increase in 3H nitrendipine binding sites in the brain in morphinetolerant mice. Eur. J. Pharmac. 102, 371-372. Schwartz A., McKenna E. and Vaghy P. (1988) Receptors for calcium antagonists. Am. J. Cardiol. 62, 3~5. Sperelakis N. (1987) Electrophysiology of calcium antagonists. J. Molec. Cell Cardiol. 19 (Suppl. II), 19-47. Tung L. and Sperelakis N. (1982) Calcium antagonist blockade of slow potentials in cultured chick heart cell. Can. J. Physiol. Pharmae. 61, 957-966. West T. C. (1972) Electrophysiology of the sinoatrial node. In Electrical Phenomena in the Heart (Edited by De Mello W. C.), pp. 191-217. Academic Press, New York. Willier J. C., De Brucker B., Bussel A., Roby-Branfi and Harrewyn J. M. (1989) Central analgesic effect of ketoprofene in humans: electrophysiological evidence for a supraspinal mechanism in a double blind and cross-over study. Pain 38, 1-8. Woods J. P. and West T. C. (1985) Frequency-dependence of Vmax in K-depolarized guinea pig ventricle: effects of nifedipine and verapamil. J. Cardiovasc. Pharmac. 7, 197-204.
0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
EFFECTS OF CLONIXIN ON THE ELECTRICAL ACTIVITY OF CARDIAC PACEMAKER CELLS M. A. MORALES,* L. INOSTROZA, T. SALAZARand C. PAEILE Department of Pharmacology, Faculty of Medicine, University of Chile, P.O. Box 70.000, Santiago 4, Chile (Received 15 August 1991)
Abstract--1. The electrophysiological effects of clonixin, a non-steroidal analgesic, on cardiac pacemaker cells of spontaneously beating frog sinus venosus, were studied by intracellular recording of transmembrane potentials. 2. Results show that clonixin (Clx) between 1 × 10 6 M and 3 x 10-4 M, decreases the OS, APA, Fma~ and frequency of primary and subsidiary cells, however pacemaker cells differ in their sensitivity to Clx. 3. At 2 x 10 -6 M, Clx completely blocked the spontaneous beating of primary cells. It is necessary to increase the Clx concentration about two orders of magnitude in order to attain a similar degree of blockade of subsidiary cells. 4. Previous or simultaneous superfusion with atropine does not modify Clx effects, thus a probable cholinergic mechanism of action for Clx is discarded. 5. When Clx concentrations were lower than 5 x 10 -4 M, their effects on both types of cells were partially reversed by a 100% increase of external calcium concentration. 6. BAY K-8644 which stimulates calcium influx through calcium L-type channels, reverted Clx effects on pacemaker cells. 7. It is suggested that Clx blocks calcium inward current which generates all or part of the upstroke of primary cells and subsidiary ones, respectively.
INTRODUCTION Clinical evidence has demonstrated that clonixin (Clx), widely used in Latin America, is a good analgesic (Finch and De Kornfeld, 1971; Paredes et al., 1986). This drug of the fenamate group is similar to mefenamic acid and is more potent as an analgesic than as an anti-inflammatory. This finding suggests that Clx may act on C.N.S. (Pellisier et al., 1984). This central action is not mediated by opiate receptors but seems to be similar to the action of indoprofen, propionic derivatives, aspirin and others compounds (Paeile et al., 1989; Willier et al., 1989; Ferreira et al., 1978; Jurna and Brune, 1990). Recently, it has been reported that the i.v. administration of high doses of Clx (100mg/kg) induces bradycardia and hypotension (Bustamante et al., 1988). The action induced by Clx (10-6-10 -3 M) was investigated in isolated atria preparations, recording negative chronotropic and inotropic effects. Based on the above-mentioned findings, it was decided to study the electrophysiological effects of Clx on cardiac pacemaker cells. Frog sinus venosus is a suitable preparation which facilitates the microelectrode impalement of pacemaker cells and it is a useful experimental model in order to determine putative calcium antagonist properties of any drug (Hernfindez et al., 1987; Morales et al., 1988, 1989). Pacemaker cells generate a spontaneous action potential, whose phase 0 and the last third of phase 4, are fully dependent (primary cells) or partially dependent (subsidiary cells) on slow inward calcium
current (Brown et al., 1977; Molivdas and Sperelakis, 1986; Hern~indez et al., 1987; Morales et al., 1988; Hagiwara et al., 1988). The effects of verapamil on both types of cells have been widely described (Morales et al., 1988; Koild et al., 1986; Hernfindez et al., 1987) and it is known that verapamil action is more pronounced on primary cells. These cells are completely blocked, with disappearance of their electrical activity as a result of calcium current blockade. On the other hand, verapamil at moderate concentrations partially diminish the overshoot and potential amplitude of subsidiary cells. It is clear then that both cellular types present different sensitivity to calcium antagonists and this obeys to the different degree of contribution that calcium inward current plays in their respective electrical activities. In a previous report (Bustamante et al., 1988) we stated that Clx depresses the action potentials of cardiac pacemaker cells acting like a calcium antagonist, however, the possibility of a cholinergicagonist mechanism of action that would also explain the bradicardic effect of Clx was not discarded. In the present work we report that Clx effects on overshoot, action potential amplitude and frequency are not of a cholinergic nature and these effects are partially reverted by increasing external calcium to 3.6 m M and fully reverted by using BAY K-8644. MATERIALS AND METHODS
Frogs from Caudiverbera caudiverbera species weighing between 200 and 350 g were used throughout these experiments. Animals were sacrificed by spinal cord transection.
*To whom all correspondence should be addressed. 515
M. A. MORALESet al.
516
Table 1. Electrophysiologicaleffects of Clx on primary cells of the frog heart APA Vmax TP MDP OS DPA60 Frequency N (mV) (V/sec) (mV) (mV) (mV) (msec) (beats/min) 16 62.9_+0.8 2.9+0.2 48.7_+0.9 61.8_+0.9 13.9_+0.4 639.2_+4.3 28.9_+0.1 11 60.8_+1.3 2.7_+0.1 46.3_+1.4 59.7_+1.7 14.4_+0.3 649.5_+3.9 27.1_+0.1" 11 62.5_+1.3 2.8_+0.1 44.4_+0.9 59.0_+0.4 18.1 _+1.4 634.7_+21.4 29.4_+0.8 9 54.0+2.3* 1.8_+0.1" 46.8-+1.9 57.3-+2.1 7.2_+1.3" 713.5_+12.9 25.4_+0.2*
Control Clx 1 × 10-6M Control Clx2x 10 6M
N: number of cells. Each value is the mean _+SEM. *P < 0.005 with respect to control. The heart was dissected and transferred to a dish containing oxygenated Ringer solution. Under a dissection microscope the ventricles were removed and the sinus venosus separated from the atria. Preparations were pinned to the bottom of a 12 ml organ bath and superfused continuously with oxygenated (100% 02) Ringer. Experiments were done at room temperature (19 21°C). The composition of the Ringer's solution employed was (mM): Na + 120.1; K + 2.5; Ca 2+ 1.8; C1- 121; H2PO42 0.85; HPO~- 2.15; glucose 11.0. pH was adjusted to 7.2 with diluted NaOH. Ringer's solution with high calcium contained Ca 2+ 3.6 mM; Tris buffer replaced phosphate buffer and the osmolarity was corrected in basis to sodium ion. The flow rate of the superfusion was 3 ml/min. Action potentials from primary and subsidiary cells were recorded differentially with glass microelectrodes filled with 3 M KCI and a reference saline bridge (Ag/AgCl-agar Ringer). The tip resistance of the electrodes ranged from 20 to 40 MfL The recording system consists mainly of a differential preamplifier providing capacity compensation with a time constant of 40/~sec. The output of the d.c. amplifier was displayed in a dual beam oscilloscope and simultaneously displayed, analyzed and recorded using an IBM compatible XT (640 kbyte) computer (Hercules high resolution graphics card) supplied with an analog to digital converter and ad hoc software devised by us to calculate the action potential parameters and Vm~x. The following electrophysiological parameters were analyzed: maximum diastolic potential (MDP); action potential amplitude (APA); overshoot lOS); maximum rate of depolarization [Vm~~ or (dV/dt)max]; threshold potential (TP); action potential duration to 60 and 90% of repolarization (APD60 and APD90 ) and frequency of potential firing (or rate). All these parameters have been defined elsewhere (Guerrero et al., 1987; Morales et al., 1988). Preparations were equilibrated in Ringer solution for 45 rain after surgery and bath installation procedures; then control recordings were taken. Drugs assayed were added to the Ringer solution and each preparation was exposed to only one drug concentration. Data are expressed as the means of clonixin steady state effects observed 20-40 rain after drug exposure of different sinus venosus preparations under the same experimental conditions. Each preparation was explored for the area most indicative of pacemaker activity and primary and subsidiary cells were impaled. Criteria for identifying both types of cells in the Chilean frog, C. caudiverbera, have been previously published (Hern~indez et al., 1987). Drugs used. Clonixinate of L-lysine, provided by Pharma Investi Laboratory, Santiago, Chile. L-lysine (British House A
-
~
,
.
B
Ltd, England). BAY K-8644 (Bayer AG, Fed. Rep. Germany) Atropine sulfate (Sigma Co., St Louis, Mo., U.S.A.). The mean value + the standard error of the mean (SEM) was calculated for each parameter measured. The statistical analysis was done using Student's t-test. Significance was accepted at P-values less than 0.05. RESULTS C l x effects on action potential p a r a m e t e r s P r i m a r y cells. The different p a r a m e t e r s studied A P A , TP, M D P , OS a n d APD60 were not significantly modified by Clx 1 x 10 6M, however the firing rate diminished approximately 6 % ( P < 0.005) as can be observed in Table 1. W h e n primary cells were exposed to Clx 2 x 10 6 M, a notorious decrease of A P A , OS a n d Fma× was observed, a b o u t 60, 14 a n d 50% respectively. These a n d other variations are depicted in Table 1 a n d are according with an increased negative c h r o n o t r o p i c effect (see also Fig. 1). By elevating the time of superfusion to 4 0 r a i n or the Clx c o n c e n t r a t i o n above 2.5 × l0 6, primary cells action potentials were completely suppressed. Subsidiary cells. Clx effects on subsidiary pacem a k e r cells were studied with c o n c e n t r a t i o n s from 1 x 10 -6 to 1.5 x l0 4 M. CIx induced a conc e n t r a t i o n - d e p e n d e n t decrease of OS, APA, Vmax a n d frequency of beating. Moreover, T P a n d M D P were slightly displaced to less negative potential values, suggesting a depolarizing effect. These results are depicted in Table 2 and illustrated in Fig. 2. With higher c o n c e n t r a t i o n s of Clx, above 2.5 x 10 -4 M, the electrical activity of subsidiary cells was completely suppressed (see Fig. 3). Before complete blockade of the s p o n t a n e o u s activity m e m b r a n e potential, oscillations were observed, with amplitudes not exceeding 2-3 m V (not shown). After a few minutes, the impalement o f subsidiary p a c e m a k e r cells showed a stable m e m b r a n e potential a r o u n d - 4 0 _+ 0.7 mV. Partial recuperation of bioelectrical activity was attained after superfusing the p r e p a r a t i o n s during almost 3 hr (3 ml/min). C
BO
5 0 0 rns
Fig. 1. Effect of 2 x 10-6 M Clx on the action potential of a primary pacemaker cell. (A) control; (B) and (C) 45 and 70 min after Clx respectively.
Clonixin effects on pacemaker cells
517
Table 2. Electrophysiological effects of CIx on subsidiary pacemaker cells of the frog heart N
APA (mV)
Vma~ (V/see)
TP (mV)
MDP (mV)
OS (mV)
APD60 (msec)
Frequency (beats/min)
Control Clx 1 × 1 0 6M
14 11
72.7_+0.9 71.6_+1.4
5.5+0.1 4.9+0.1"
54.9+0.8 53.2_+1.2
69.6_+0.8 67.0_+1.3
18.0+0.4 18.5_+0.7
641.8+3.9 659.2_+3.7*
28.9_+0.1 27.1_+0.1"
Control CIx l x l 0
SM
18 22
79.9_+1.5 69.8_+1.3"
6.5_+0.1 5.6_+1.1"
60.2+0.7 55.6_+1.1"
71.0_+0.9 63.4+1.3"
20.2_+1.1 14.2_+0.6"
523.1_+4.3 545.1_+5.1"
22.5_+0.1 21.2-+0.1"
Control CIx 1 x 10 4M
23 14
77.2_+ 1.1 65.2_+0.2
6.6_+0.1 5.4_+0.2*
58.7-+0.9 55.6_+ 1.6
70.6_+0.7 65.7_+ 1.6"
18.5_+0.7 9.7_+ 1.4"
576.7_+3.8 551.1 +5.5*
32.0_+0.1 29.5_+0.5*
Control Clx 1.5x 10 4M
12 22
83.6+_0.5 68.0_+2.1"
6.9_+0.1 5.3_+0.2*
59.9_+0.5 54.5_+1.5"
67.8_+0.6 64.6_+1.4"
23.7_+0.6 13.5_+1.2"
508.9_+3.8 509.2_+5.8
32.6_+0.1 30.2_+0.3*
N: number of impaled cells. Each value is the mean + SEM. *P < 0.005 with respect to control.
Influence of atropine on the effects of Clx
Clx effects and calcium concentration
In order to investigate an eventual role of atrial muscarinic receptors on Clx effects, a series of experiments were carried out in atropinetreated preparations. The data is summarized in Table 3. It has been previously described that atropine, 1.4 x 10-SM, prevents acetylcholine activation of muscarinic receptors of frog heart (Guerrero, 1982; Guerrero et al., 1987) and its consequent bradicardic effects. As shown in Table 3, in sinus venosus exposed to 2.8 x 10-SM atropine sulphate, this drug significantly increased APA, TP, MDP, OS, frequency and the duration of action potential. When Clx 2 x 10 . 6 M was added, APA, Vmax,OS and frequency of primary cells were reduced in a similar percentage to that shown in Table 1, for preparations untreated with atropine. Therefore it appeared that Clx was acting at other sites, different from muscarinic receptors.
When the calcium concentration of Ringer's solution was elevated to 3.6 mM (100%), Clx induced a lower depressor effect on subsidiary cells than that observed with normal Ringer solution, as can be seen in Fig. 4. The effect on APA and OS was reduced by not more than 50% of the effect observed with normal calcium concentration. However, the I/max was depressed about 20% in both experimental conditions. The rate of firing was more affected in high Ca 2÷ Ringer solution. It is known that in sinus venosus preparations superfused with increased calcium concentrations a significant decrease in sinus rate can be observed (Guerrero, 1982; West, 1972). In summary, Clx effects were only partially reversed in the presence of elevated (100%) external calcium concentration. When Clx concentrations high enough to obtain blockade of spontaneous action potential of both types of pacemaker cells were used, i.e. above 2.5 x 10 -4 M , C a 2+ increase up to
A 0 mV
B
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
?"
-70 mV
500ms
I
I
I
t
t
I
Fig. 2. Effect of 1.5 x 10 . 4 M Ch on the action potential of a subsidiary pacemaker cell. Lower trace is (dV/dt) signal. (A) control; (B) 40 rain after Clx.
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.
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.
.
.
500ms
Fig. 3. Effect of 5 x 10-4 M Clx on the action potential of a subsidiary pacemaker cell. (A) control; (B) and (C) after 20 and 40 min of Clx, respectively. GP 23/3--0
M. A. MORALESet al.
518
Table 3. Clonixin effects on primary pacemaker cells treated with atropine APA Vm~x TP MDP OS N (mV) (V/sec) (mV) (mV) (mV) 17 60.2_+0.9 2.9_+0.1 44.8_+0.7 63.7_+0.8 15.4_+0.5 14 64.6_+0.6**~ 3.1+0.1 47.7_+0.4**~ 66.5_+0.7**~ 16.8_+0.6*~
Control Atropine 2.8 × 10 5M Atropine 2.8 x 10 5M +clonixin 2 x 10 6M 11 56.9-+1.3**~ 1.9-+0.1**~ 48.4-+ 1.2 N: number of impaled cells. Each value is the mean -+ SEM. Significance level with respect to control (~), with respect to atropine (2). *P < 0.05; **P < 0.005. 100% was unable to prevent the complete disappearance of electrical activity.
Modification o f Clx effects by B A Y K-8644 An intent to localize the probable Clx site of action was done using BAY K-8644, which is a " L " type calcium channel agonist. As can be seen in Fig. 5, for primary pacemaker cells, after 15 min of superfusion with Clx 2.5 × 10 6M all parameters were significantly modified, with the exception of A P D . When OS and frequency decayed to 40 and 16% respectively, superfusion with BAY K-8644, 1 × 10 7M, with Clx at the same concentration as before was started. After about 20 min, a complete recuperation of the initial values of action potential parameters was observed (see also Fig. 6). A similar recuperation was attained in subsidiary cells treated with Clx when BAY K-8644 was added (Fig. 7). Some electrophysiological parameters were even increased above the control values, suggesting a clear enhancing effect of the agonist. F r o m these results it was concluded that BAY K-8644 reverted the depressor effect of Clx on both primary and subsidiary pacemaker cells. Clx effects on action potential o f f r o g sciatic nerve To rule out the possible effect of Clx on sodium conductance, Clx (5 × 10 4 M) was superfused in a frog-isolated sciatic nerve preparation electrically stimulated. After 120 min the action potential amplitude, Vm,x and relative refractory period were not changed. These results suggest a certain specificity for the mechanism of action of clonixine.
62.9-+1.8*~
Frequency APD60 (beats/min) (msec) 27.5_+0.1 595.0_+6.7 28.8_+0.1**~ 622.3-+5.4**
8.5_+ 1.2**: 24.0_+0.1**: 639.4_+8.9
DISCUSSION
The results of the present work provide evidence of the concentration-dependent effects of Clx on the spontaneous electrical activity of cardiac pacemaker cells. This is clearly manifested by the Clx-induced depression of APA, OS, Vmax and frequency of primary and subsidiary pacemaker cells. It is known that both types of pacemaker cells exhibit some electrophysiological differences in relation to the parameters which characterize their respective action potentials. In this way, primary cells Vmax, A P A and OS are significatively lower than those of subsidiary cells and their values of TP and M D P are less negative (Brown et al., 1977; Hernfindez et al., 1987; Noble, 1985). These differential characteristics arise from the composition of ionic currents which underly the genesis of the action potential of both types of pacemakers cells. As a fact, primary cells upstroke is exclusively dependent on calcium entry and it is verapamil-sensitive (Hagiwara et al., 1988; Molivdas and Sperelakis, 1986; Morales et al., 1988) and TTX-insensitive (Brown et al., 1977; Hernfindez et al., 1987). On the contrary, action potentials of subsidiary cells exhibit two different inward currents causing the upstroke, a fast TTXsensitive (sodium inward current) and a slow verapamil-sensitive current (calcium inward current). These cells are less readily and less efficiently blocked by verapamil (Morales et al., 1988, 1989). Although Clx induced a progress reduction and then a total blockade of the electrical activity of both types of
20
OS
APA
z
RATE
Vmax
-20
N\\N ,x\x-~
-40
N\\'¢,
1
I$ -60
k~
CLX + 1.8 m.lll Ca++
~
CI,X+ 3.6 ~
Ca++
Fig. 4. Modification of the effects of 1 x 10 -4M Clx on APA, OS, RATE and Vm~x of subsidiary pacemaker cells by calcium concentration increase. Each column indicates a percentage change (mean + SEM) from the drug-free control values. **P < 0.005 compared to control.
519
Clonixin effects on pacemaker cells 20
LU t.9 Z -IC.)
APA
RATE
Vmax
OS
-20
-40 Ill
CLX
~
c~
÷ ~ Y K-~B44
Fig. 5. Modification of the effects of 2.5 × 10 6 M Clx on APA, OS, Vmaxand RATE of primary pacemaker cells by 1 × 10 7M BAY K-8644. Each column indicates a percentage change (mean _ SEM) from the drug-free control values. **P < 0.005 compared to control. pacemaker cells, primary cells showed a greater sensitivity. It was necessary to increment Clx concentration about two orders of magnitude in order to obtain a similar degree of blockade of subsidiary cells. As it is known, the depression of APA, OS and Vmax indirectly reflects the modification of the currents involved in the upstroke (Li and Sperelakis, 1983; Hondeghem and Cotner, 1978; Tung and Sperelakis, 1982; Woods and West, 1985). According to this, results obtained in the present work clearly indicate that Clx, like verapamil, acts by decreasing calcium entry. It seems probable that Clx through this mechanism decreases the frequency of spontaneous firing of pacemaker cells and it could explain the bradicardic effects observed in rats after a Clx i.v. injection and the negative chronotropic effect that is observed in preparations of rat isolated atria (Bustamante et al., 1988). The experiments with atropine indicate that Clx does not act through muscarinic receptors. It was necessary to discard this possibility because it is known that acetylcholine and muscarinic agonists, induce a negative chronotropic effect on pacemaker cells by decreasing the slow inward calcium current and increasing the outward potassium current (Nargeot and Garnier, 1982; Del Castillo and Katz, 1955; Hutter and Trautwein, 1956; Giles and Shibata, 1981; Iijima et al., 1984). The data presented in this report suggest that Clx acts by blocking the calcium entry which originates A
-70
the phase zero of the action potential of primary cells and the overshoot of subsidiary cells in a similar form to that observed for verapamil-treated sinus venosus preparations (Morales et al., 1988). This calcium inward current is carried through calcium channels of long duration or "L" type which have been found in heart, neurons and other tissues (Hagiwara et al., 1988; Fozzard, 1988; Schwartz et al., 1988; Nowycky et al., t985). As it has been determined that BAY K-8644 specifically increases L-channel conductance, it is a useful tool to determine if a calcium antagonist acts on this calcium channel. From our results it can be observed that the Clx depressor effect on cardiac pacemaker cells was almost completely reversed by BAY K-8644. These results allow to establish a certain resemblance between Clx, verapamii and other calcium channel blockers regarding their cardio-depressant activities and calcium antagonist properties. On the other hand, the increase of external calcium concentration induced a partial reversion of Clx effect on pacemaker cells. Different studies have shown that the calcium antagonism of verapamil, nifedipine and diltiazem is not readily reversed by increasing Ca 2+ concentration (Ishii et al., 1985), and that an increment of 200% of external calcium is needed to displace membrane-bound calcium channel antagonists (Hulth6n et al., 1982) or their effects (Sperelakis, 1987). The results of experiments with frog sciatic nerve show that Clx does not affect sodium conductance B
C
mV
500 ms
Fig. 6. Reversion of Clx effects on action potential of primary pacemaker cells by BAY K-8644. (A) Control; (B) after 2.5 x 10.6 M Clx; (C) same impaled cell of (B) after addition of 1 x l0 -7 M BAY K-8644.
520
M. A. MORALESet al. 40
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Fig. 7. Modification of the effects of 1 x 10 5 M Clx on APA, OS, Vmax and RATE of subsidiary pacemaker cells by 1 x 10 -7 M BAY K-8644. Each column indicates a percentage change (mean + SEM) from the drug-free control values. **P < 0.005 compared to control.
which originates the phase zero of the nerve action potential. It suggests a certain specificity in the m e c h a n i s m of action of Clx. A l t h o u g h with low Clx c o n c e n t r a t i o n s the action potential d u r a t i o n was slightly modified, as the blockade of cardiac p a c e m a k e r activity led to a stable potential o f a b o u t - 4 0 mV, it seems plausible that this depolarization to a stable resting potential can be due to a Clx-induced lowering o f K ÷ conductance. This would be a n o t h e r characteristic shared by Clx and organic calcium antagonists (Molivdas a n d Sperelakis, 1986; Morales et al., 1988, 1989), a l t h o u g h this blockade of K ÷ c o n d u c t a n c e is only seen with high c o n c e n t r a t i o n s of the latter (Hume, 1985). It is still necessary to carry out more studies with improved techniques, such as single channel recording, for a n accurate determination of Clx m e c h a n i s m o f action o n calcium conductance. However, from the results presented here we consider plausible t h a t this drug m a y be acting like a calcium channel blocker o n cardiac p a c e m a k e r cells. It is n o t e w o r t h y to m e n t i o n here t h a t previous work from o u r l a b o r a t o r y has shown that Clx inhibits n e u r o t r a n s m i t t e r release from the rat vas deferens ( B u s t a m a n t e et al., 1989b), an event which is associated with calcium entry t h r o u g h " L " type channels in nerve terminals. On the other h a n d , it is k n o w n that the analgesic action of Clx is also exerted at C N S level and it is not antagonized by n a l o x o n e ( B u s t a m a n t e et al., 1989a; Ci6falo et al., 1972). Discarding a m e c h a n i s m of action t h r o u g h opioid receptors and with the antecedent of a great resemblance between Clx and opiates analgesic effect, it is possible to assume that Clx could be acting on a calcium channel associated to an opioid receptor. It has been d e m o n s t r a t e d that m o r p h i n e reduces depolarization-dependent calcium uptake in n e u r o n s ( K o n n o a n d Takayanagi, 1982) and there are studies which have d e m o n s t r a t e d that the n u m b e r of brain calcium channels is m o d u l a t e d by chronic use of m o r p h i n e ( R a m k u n a r and E l - F a k a h a n y , 1984). These findings open the
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