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Cocaine, amphetamine and cathinone, but not nomifensine and pargyline increase calcium inward current in internally perfused neurons
Life Sciences, Vol. Printed in the USA
52, pp. PL 261-265
Pergamon
Press
PHARMACOLOGY LETTERS Accelerated
Communication
COCAINE, AMPHETAMINE AND CATHINONE, BUT NOT NOMIFENSINE AND PARGYLINE INCREASE CALCIUM INWARD CURRENT IN INTERNALLY PERFUSED NEURONS.* A. I. Vislobokov2, V. V. MantsevI and A. V. Kuzmin~ 1 Pavlov Medical Institute and 2 Physiological Institute of St.-Petersburg University, St.Petersburg, Russia (Submitted June 30, 1992; accepted July 21, 1992; received in final form March 29, 1993)
ABSTRACT: The influence of cocaine, amphetamine, cathinone, pargyline and nomifensine on inward calcium current was studied using internally perfused neurons of the snail Lymnaea Stagnalis. While nomifensine and pargyline inhibited inward calcium current in the concentrations 10-7-10-4 M and did not affect them in the concentrations 10-9-10-8 M, cocaine, amphetamine and cathinone had a biphasic action on inward calcium current, causing activation (10-30 percent) at 10-9-10-7 M, and inhibition at higher concentrations. Only cathinone caused a shift of the I-V characteristics of the membrane along the potential axis. It is suggested that drugs of abuse affect membrane excitability and inward calcium current in neurons directly.
Introduction Reinforcing properties of abused drugs are commonly associated with their ability to increase dopaminergic transmission in the mesoaccumbens system (1). However, numerous drugs that potently increase synaptic dopamine concentrations (e.g. nomifensine, pargyline, mazindole) are not abused (8). Therefore, it is proposed that the direct action of psychoactive substances on neuronal membranes and the functional activity of calcium channels might underlie the addictive drug action. In order to examine whether an activation of calcium current is linked to abused drugs and whether this property distinguishes them from non abused substances that increase synaptic dopamine concentrations, we compared the action on inward calcium currents of well known drugs of abuse (e.g. cocaine, amphetamine and cathinone) with the action of the potent doparnine uptake blocker nomifensine and the MAO inhibitor pargyline, using Corresponding Author: A.V. Kuzmin, Department of Pharmacology, Pavlov Medical Institute, Leo Tolstoy str. 6/8, St.-Petersburg 197089, Russia.
Copyright
0024-3205/93 $6.00 + .00 © 1993 Pergamon Press Ltd All rights
reserved.
PL-262
Cocaine on Inward Calcium Current
Vol.
52, No.
23,
1993
internally perfused snail neurons. Cocaine affects in the CNS have been shown to be calcium dependent (10) while amphetamine and cathinone affect the function of central neurons without any dependence on the extracellular calcium concentration (9). Here we show that typical abused substances, but not nomifensine and pargyline activate inward calcium currents in the isolated neurons. Materials and methods The experiments were carried out on enzymatically-isolated unidentified neurons of snail
Lymnaea stagnalis using the method of intracellular perfusion combined with clamping the membrane potential (for details see 3). To improve frequency characteristics of the voltage clamp, electronic compensation of series resistance was used. High-threshold calcium current was activated by depolarizing shifts of membrane potential to + 10 or +20 mV from the holding level of -70 mV. The intracellular perfusion solution contained 120 mM CsC1 and 10 mM Tris-aspartate (pH 7.4). Extracellular solution contained CaC1z 20 mM, MgCI2 4 mM, Tris-C1 10 mM, CsC1 100 mM (pH 7.5). The system of extracellular medium replacement ensured a multiple (10 times) change of bathing solution in 1-2 min due to forced flow of extracellular solution in the microchamber where the tip of the perfusing pipette with the fixed cell was placed. During the current measurements, the extracellular solution flow was stopped. When measuring concentration-dependence of the effects of studied agents, their applications in increasing concentrations were performed without intermediate washouts with the control solution. Drugs: Cocaine, D-amphetamine, nomifensine and pargyline were obtained from Sigma (U.S.A.), cathinone HC1 was obtained from Institute of Pharmacology (Academy of Sciences, Russia). All drugs were dissolved in extracellular solution to the required concentrations just before the experiment. Results A
~
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,,o t
a ~ t . "
• - ¢athin.
-
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. . . . .
--1 1
4--
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--10
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--7
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Fig. 1 Influence of different drugs on the inward calcium current in isolated neurons. Data as means (n = 10-12). The range of inward calcium current varied from 18 to 34 hA.
--4
)
Vol.
52, No.
23,
1993
Cocaine
on I n w a r d C a l c i u m
Current
PL-263
Dose-effect relationships for cocaine, amphetamine and cathinone exhibited an unusual biphasic shape over a wide range of concentrations (from 101° to 10-4 M). Low concentrations of these drugs produced an increase in inward calcium current amplitude, whereas large concentrations inhibited it (Fig. 1). The increase of inward current was significant (p < 0.05, Student t-test) in the range of concentrations from 10 9 tO 10 7 for cocaine and amphetamine and from 10-8 to 10.7 for cathinone. The maximal increase in the current amplitude was 114.+6, 131.+7 and 110.+4 percent of basal level for cocaine, amphetamine and cathinone respectively (Mean _+ s.e.m., n=8-12). An inhibition of calcium inward current was seen in the 10-6-10-4 M concentration range. In contrast nomifensine and pargyline failed to increase basal current in the concentrations 101°-10-8 M and inhibited calcium current in higher concentrations. Effects of cocaine, amphetamine, pargyline and nomifensine were observed in all cells investigated. Effects of cathinone were observed in the majority of cells investigated (more than 75%), but not in all. C~>O~lne -,,to
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V-A characteristics for inward calcium current before and after application of drugs. Data represent the recordings from individual neurons. All drugs were added to external solution in the concentration 10E-7 M. Figure 2 exemplifies the I-V characteristics for calcium current under basal conditions, and after introduction of a particular concentration of cocaine, amphetamine or cathinone into the extracellular medium. An increase in calcium current amplitude was observed across a wide range of membrane potential values. Only cathinone caused a shift of the I-V characteristics of the membrane along the potential axis. Both the activating and the inhibiting actions of cocaine, amphetamine and cathinone occured rapidly after application (considering the high speed of shifting the bathing solutions). The most interesting finding is that after washout of drugs with control solution, the post-drug inward calcium current levels became larger than initial levels when cocaine, amphetamine or cathinone were applied, but not after nomifensine or pargyline. The post-drug levels of calcium current were 110 _+ 4, 117 _+ 7, 111 _+ 3, 82 _+ 7 and 86 _+ 3 for cocaine, amphetamine, cathinone, pargyline and nomifensine respectively (percent of initial current, means _+ s.e.m., n=8-12).
PL-264
Cocaine on Inward Calcium Current
Vol.
52, No.
23, 1993
Discussion In the present study we compared effects of cocaine, amphetamine and cathinone, drugs known for their high addictive potential, with the effect of the potent dopamine uptake blocker nomifensine and the MAO inhibitor pargyline on calcium inward current in the membrane of nerve cells. The results show that upon extracellular application only the drugs of abuse but not nomifensine and pargyline are capable of affecting the inward calcium current byphasically. Moreover, the prolonged activation of calcium current was noticed after the wash- out of cocaine, amphetamine and cathinone, but not after nomifensine and pargyline. The present results do not allow for identification of the type of calcium channels involved. It has been shown that L-type calcium channels have the highest density in cell bodies in comparison with neuron terminals (5). In our experiments we dealt with cell bodies. Therefore it is likely that L-type calcium channels play a major role in the observed effects of cocaine, amphetamine and cathinone. Moreover, additional experiments have been performed in order to identify the type of calcium channels which predominate in snail neurons. In these experiments, gadolinium ion (as a chloride salt), which has been shown to inhibit N-type calcium channels selectively (2), was added to external solution in a wide range of concentrations (108-10 -3 M). Gadolinium ion failed to inhibit inward calcium current in the snail neurons (data not shown) in all the concentrations tested. Therefore, it might be concluded that in the isolated neurons of Limnaea Stagnalis L-type-like calcium channels predominate, and that the observed effects of investigated drugs reflect their interaction with L-type-like calcium channels. Our conclusion is in agreement with other studies (6) which show that channels which are similar to neuronal L-type channels of mammals in neurophysiological and pharmacological characteristics predominate in snail neurons. Considering that a) only typical drugs of abuse exhibited a biphasic effect on calcium inward current, b) nomiphensine and pargyline, being potent in increasing synaptic dopamine concentration but not in being abused by humans, failed to increase calcium current in isolated neurones, and c) L-type calcium channel blockers are able to inhibit drug-induced reinforcement (4, 7), we propose that altered calcium influx through L-type calcium channels is essential for realisation of the addictive properties of drugs like cocaine, cathinone and amphetamine. Moreover, it is proposed that alteration of inward calcium current might be the particular characteristic of cocaine, cathinone and amphetamine that distinguishes these drugs of abuse from non-abused drugs which have a similar effect on synaptic dopamine concentrations. Therefore centrally acting blockers of L-type calcium current might be potent inhibitors of drug-induced reward. References . .
3. 4.
.
G. DI CHIARA and A. IMPERATO, Proc. Natl. Acad. Sci. USA, 85 5274-5278, (1988). R.J. DOCHERTY, J. Physiol. (London), 398 33-47, (1988) P. KOSTYUK and O.A. KRISHTAL, J. Physiol. (London), 270 569-580, (1977) A. KUZMIN, E. ZVARTAU, G.L. GESSA, M.C. MARTELLOTTA and W. FRA'I-TA, Pharmacol. Biochem.& Behav., 41/3 497-500, (1992) R.J. MILLER, Science, 235 46-52, (1987)
Vol.
6. 7. 8. 9. 10.
52, No.
23,
1993
C o c a i n e on I n w a r d C a l c i u m C u r r e n t
PL-265
P. NOVALES-LI, X. PING SUN and H. TAKEUCHI, Eur. J. Pharmacol., 178 161-170, (1990) L. PANI, A. KUZMIN, M.C. MARTELLOTI'A, G.L. GESSA and W. FRATI'A, Brain Res.Bull. 26/3 445-447, (1991) R.B. ROTHMAN, Life Sci. 46 PL17-PL21, (1990) N. WATERS, L. LOFBERG, K. SVENSSON and A. CARLSSON, Eur. J. Pharmacol., 187 425-434, (1990) B.H.C. WESTERINK, R.M. HOFSTEEDE, J. TUNTLER and J.B. DE VRIES, J. Neurochem., 52 722-729, (1989)
52, pp. PL 261-265
Pergamon
Press
PHARMACOLOGY LETTERS Accelerated
Communication
COCAINE, AMPHETAMINE AND CATHINONE, BUT NOT NOMIFENSINE AND PARGYLINE INCREASE CALCIUM INWARD CURRENT IN INTERNALLY PERFUSED NEURONS.* A. I. Vislobokov2, V. V. MantsevI and A. V. Kuzmin~ 1 Pavlov Medical Institute and 2 Physiological Institute of St.-Petersburg University, St.Petersburg, Russia (Submitted June 30, 1992; accepted July 21, 1992; received in final form March 29, 1993)
ABSTRACT: The influence of cocaine, amphetamine, cathinone, pargyline and nomifensine on inward calcium current was studied using internally perfused neurons of the snail Lymnaea Stagnalis. While nomifensine and pargyline inhibited inward calcium current in the concentrations 10-7-10-4 M and did not affect them in the concentrations 10-9-10-8 M, cocaine, amphetamine and cathinone had a biphasic action on inward calcium current, causing activation (10-30 percent) at 10-9-10-7 M, and inhibition at higher concentrations. Only cathinone caused a shift of the I-V characteristics of the membrane along the potential axis. It is suggested that drugs of abuse affect membrane excitability and inward calcium current in neurons directly.
Introduction Reinforcing properties of abused drugs are commonly associated with their ability to increase dopaminergic transmission in the mesoaccumbens system (1). However, numerous drugs that potently increase synaptic dopamine concentrations (e.g. nomifensine, pargyline, mazindole) are not abused (8). Therefore, it is proposed that the direct action of psychoactive substances on neuronal membranes and the functional activity of calcium channels might underlie the addictive drug action. In order to examine whether an activation of calcium current is linked to abused drugs and whether this property distinguishes them from non abused substances that increase synaptic dopamine concentrations, we compared the action on inward calcium currents of well known drugs of abuse (e.g. cocaine, amphetamine and cathinone) with the action of the potent doparnine uptake blocker nomifensine and the MAO inhibitor pargyline, using Corresponding Author: A.V. Kuzmin, Department of Pharmacology, Pavlov Medical Institute, Leo Tolstoy str. 6/8, St.-Petersburg 197089, Russia.
Copyright
0024-3205/93 $6.00 + .00 © 1993 Pergamon Press Ltd All rights
reserved.
PL-262
Cocaine on Inward Calcium Current
Vol.
52, No.
23,
1993
internally perfused snail neurons. Cocaine affects in the CNS have been shown to be calcium dependent (10) while amphetamine and cathinone affect the function of central neurons without any dependence on the extracellular calcium concentration (9). Here we show that typical abused substances, but not nomifensine and pargyline activate inward calcium currents in the isolated neurons. Materials and methods The experiments were carried out on enzymatically-isolated unidentified neurons of snail
Lymnaea stagnalis using the method of intracellular perfusion combined with clamping the membrane potential (for details see 3). To improve frequency characteristics of the voltage clamp, electronic compensation of series resistance was used. High-threshold calcium current was activated by depolarizing shifts of membrane potential to + 10 or +20 mV from the holding level of -70 mV. The intracellular perfusion solution contained 120 mM CsC1 and 10 mM Tris-aspartate (pH 7.4). Extracellular solution contained CaC1z 20 mM, MgCI2 4 mM, Tris-C1 10 mM, CsC1 100 mM (pH 7.5). The system of extracellular medium replacement ensured a multiple (10 times) change of bathing solution in 1-2 min due to forced flow of extracellular solution in the microchamber where the tip of the perfusing pipette with the fixed cell was placed. During the current measurements, the extracellular solution flow was stopped. When measuring concentration-dependence of the effects of studied agents, their applications in increasing concentrations were performed without intermediate washouts with the control solution. Drugs: Cocaine, D-amphetamine, nomifensine and pargyline were obtained from Sigma (U.S.A.), cathinone HC1 was obtained from Institute of Pharmacology (Academy of Sciences, Russia). All drugs were dissolved in extracellular solution to the required concentrations just before the experiment. Results A
~
!
,,o t
a ~ t . "
• - ¢athin.
-
"----.
. . . . .
--1 1
4--
_,c'.--=----r._2-2-.___ Z .
e,
2t
-D- nomif.
--10
Drug
--9
-8
Concentration
--7
--6
(
10E
¢oca.
- ~ - pargyl,
I
-
90
" -
;"-122 -5
M
-4
)
-1 1
.
-10
Drug
.
.
.
-9
.
.
.
.
.
.
.
.
4
--6
--£$
10E
M
..
.
--8
--7
Concentratiot~
(
Fig. 1 Influence of different drugs on the inward calcium current in isolated neurons. Data as means (n = 10-12). The range of inward calcium current varied from 18 to 34 hA.
--4
)
Vol.
52, No.
23,
1993
Cocaine
on I n w a r d C a l c i u m
Current
PL-263
Dose-effect relationships for cocaine, amphetamine and cathinone exhibited an unusual biphasic shape over a wide range of concentrations (from 101° to 10-4 M). Low concentrations of these drugs produced an increase in inward calcium current amplitude, whereas large concentrations inhibited it (Fig. 1). The increase of inward current was significant (p < 0.05, Student t-test) in the range of concentrations from 10 9 tO 10 7 for cocaine and amphetamine and from 10-8 to 10.7 for cathinone. The maximal increase in the current amplitude was 114.+6, 131.+7 and 110.+4 percent of basal level for cocaine, amphetamine and cathinone respectively (Mean _+ s.e.m., n=8-12). An inhibition of calcium inward current was seen in the 10-6-10-4 M concentration range. In contrast nomifensine and pargyline failed to increase basal current in the concentrations 101°-10-8 M and inhibited calcium current in higher concentrations. Effects of cocaine, amphetamine, pargyline and nomifensine were observed in all cells investigated. Effects of cathinone were observed in the majority of cells investigated (more than 75%), but not in all. C~>O~lne -,,to
--20
-
--30
~
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.
-
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.
~ Fig. 2
V-A characteristics for inward calcium current before and after application of drugs. Data represent the recordings from individual neurons. All drugs were added to external solution in the concentration 10E-7 M. Figure 2 exemplifies the I-V characteristics for calcium current under basal conditions, and after introduction of a particular concentration of cocaine, amphetamine or cathinone into the extracellular medium. An increase in calcium current amplitude was observed across a wide range of membrane potential values. Only cathinone caused a shift of the I-V characteristics of the membrane along the potential axis. Both the activating and the inhibiting actions of cocaine, amphetamine and cathinone occured rapidly after application (considering the high speed of shifting the bathing solutions). The most interesting finding is that after washout of drugs with control solution, the post-drug inward calcium current levels became larger than initial levels when cocaine, amphetamine or cathinone were applied, but not after nomifensine or pargyline. The post-drug levels of calcium current were 110 _+ 4, 117 _+ 7, 111 _+ 3, 82 _+ 7 and 86 _+ 3 for cocaine, amphetamine, cathinone, pargyline and nomifensine respectively (percent of initial current, means _+ s.e.m., n=8-12).
PL-264
Cocaine on Inward Calcium Current
Vol.
52, No.
23, 1993
Discussion In the present study we compared effects of cocaine, amphetamine and cathinone, drugs known for their high addictive potential, with the effect of the potent dopamine uptake blocker nomifensine and the MAO inhibitor pargyline on calcium inward current in the membrane of nerve cells. The results show that upon extracellular application only the drugs of abuse but not nomifensine and pargyline are capable of affecting the inward calcium current byphasically. Moreover, the prolonged activation of calcium current was noticed after the wash- out of cocaine, amphetamine and cathinone, but not after nomifensine and pargyline. The present results do not allow for identification of the type of calcium channels involved. It has been shown that L-type calcium channels have the highest density in cell bodies in comparison with neuron terminals (5). In our experiments we dealt with cell bodies. Therefore it is likely that L-type calcium channels play a major role in the observed effects of cocaine, amphetamine and cathinone. Moreover, additional experiments have been performed in order to identify the type of calcium channels which predominate in snail neurons. In these experiments, gadolinium ion (as a chloride salt), which has been shown to inhibit N-type calcium channels selectively (2), was added to external solution in a wide range of concentrations (108-10 -3 M). Gadolinium ion failed to inhibit inward calcium current in the snail neurons (data not shown) in all the concentrations tested. Therefore, it might be concluded that in the isolated neurons of Limnaea Stagnalis L-type-like calcium channels predominate, and that the observed effects of investigated drugs reflect their interaction with L-type-like calcium channels. Our conclusion is in agreement with other studies (6) which show that channels which are similar to neuronal L-type channels of mammals in neurophysiological and pharmacological characteristics predominate in snail neurons. Considering that a) only typical drugs of abuse exhibited a biphasic effect on calcium inward current, b) nomiphensine and pargyline, being potent in increasing synaptic dopamine concentration but not in being abused by humans, failed to increase calcium current in isolated neurones, and c) L-type calcium channel blockers are able to inhibit drug-induced reinforcement (4, 7), we propose that altered calcium influx through L-type calcium channels is essential for realisation of the addictive properties of drugs like cocaine, cathinone and amphetamine. Moreover, it is proposed that alteration of inward calcium current might be the particular characteristic of cocaine, cathinone and amphetamine that distinguishes these drugs of abuse from non-abused drugs which have a similar effect on synaptic dopamine concentrations. Therefore centrally acting blockers of L-type calcium current might be potent inhibitors of drug-induced reward. References . .
3. 4.
.
G. DI CHIARA and A. IMPERATO, Proc. Natl. Acad. Sci. USA, 85 5274-5278, (1988). R.J. DOCHERTY, J. Physiol. (London), 398 33-47, (1988) P. KOSTYUK and O.A. KRISHTAL, J. Physiol. (London), 270 569-580, (1977) A. KUZMIN, E. ZVARTAU, G.L. GESSA, M.C. MARTELLOTTA and W. FRA'I-TA, Pharmacol. Biochem.& Behav., 41/3 497-500, (1992) R.J. MILLER, Science, 235 46-52, (1987)
Vol.
6. 7. 8. 9. 10.
52, No.
23,
1993
C o c a i n e on I n w a r d C a l c i u m C u r r e n t
PL-265
P. NOVALES-LI, X. PING SUN and H. TAKEUCHI, Eur. J. Pharmacol., 178 161-170, (1990) L. PANI, A. KUZMIN, M.C. MARTELLOTI'A, G.L. GESSA and W. FRATI'A, Brain Res.Bull. 26/3 445-447, (1991) R.B. ROTHMAN, Life Sci. 46 PL17-PL21, (1990) N. WATERS, L. LOFBERG, K. SVENSSON and A. CARLSSON, Eur. J. Pharmacol., 187 425-434, (1990) B.H.C. WESTERINK, R.M. HOFSTEEDE, J. TUNTLER and J.B. DE VRIES, J. Neurochem., 52 722-729, (1989)