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Calcium agonists and antagonists of the dihydropyridine type: effect on nicotine-induced antinociception and hypomotility
73
Drug and Alcohol Dependence, 32 (1993) 73 - 79
Elsevier Scientific Publishers
Ireland Ltd.
Calcium agonists and antagonists of the dihydropyridine type: effect on nicotine-induced antinociception and hypomotility M.I. Damaj and B.R. Martin Department
of Pharmacology
and Toxicology, Medical College of Virginia, VA 23298-0613 (USA)
(Accepted September
Virginia Commonwealth University, Richmond,
22nd, 1992)
The influence of a calcium agonist (BAYK 8644) and several calcium channel blockers on nicotine-induced antinociception was investigated in mice. The effect of nicotine was sharply increased by BAYK 8644. This potentitation by BAYK 8644 was blocked by pretreating the animals with nifedipine at 2 and 10 mglkg. The calcium channel antagonists, nifedipine and nimodipine at doses that had no effect on tail-flick time, reduced significantly the antinociception induced by nicotine (1.5 mg/kg, s.c.). However, the effect of verapamil on nicotine was not significant. These results indicate that DHP calcium channels (type L-channel) play a role in some of the pharmacological effects of nicotine, particularly, locomotor activity and antinociception. Key words: calcium antagonists;
BAYK 8644; nicotine; antinociception;
Introduction The nicotinic acetylcholine receptor (nAChR) is one of the most widely studied ligand-gated ion channels in neurobiology. Despite the voluminous literature regarding nAChR in muscle, Torpedo and ganglia, studies pertaining to the mammalian central nervous system are limited mostly to ligand-binding studies. Unlike the nAChR found at the neuromuscular junction, those found in the CNS exhibit considerable diversity in their subunit composition (Deneris et al., 1991). Recent molecular biology studies further suggest the existence of several subtypes of nAChR in the brain based upon the discovery of several genes encoding for both alpha and beta subunits (Nef et al., 1988). However, the physiological properties, pharmacological activities and the transduction to: Billy R. Martin, Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 2329% 0613, USA. Correspondence
mice
mechanisms associated with these nAChR are largely unknown. The involvement of calcium in the signalling process of the peripheral nAChR is well established. Apart from depolarizing the cell membrane, activation of the nAChR on muscle also leads to significant Ca2+ influx into the cell (Decker et al., 1990). Similarly, in chromaffin cells, activation of nAChR causes an influx of Ca2+ leading to an increase in the intracellular calcium [Ca2+]i (Kilpatrick et al., 1982; Baumgold et al., 1989; O’Sullivan et al., 1989; Noronha-Blob et al., 1989) which is thought to result in great part from local depolarization and opening of voltage-dependent Ca2+ channels. In the CNS, nicotine-evoked neurotransmitter release is calcium dependent (Westfall, 1974; Rowe11 et al., 1984; Rapier et al., 1988; Kubo et al., 1990). Recently, by using Patch-clamp recordings, Vermin0 et al. (1992) showed that changes in extracellular calcium modulate neuronal nAChR in a dose-dependent manner and that the activation of the neuronal nAChR produces a significant influx of calcium. Further-
0376-8716/93/$06.00 0 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
more, Mulle et al. (1992a) showed that neuronal nAChR located on the soma of rat medial habenular nucleus neurons are permeable to Ca2+ leading to an increase of [Ca’+]i up to the micrdmolar range, a level largely sufficient for the activation of Ca2 +-dependent regulatory process. Although there is evidence that calcium likely plays an important role in the central action of nicotine, there is little documentation that alterations in intracellular calcium influences the pharmacological effects of nicotine. Therefore, the objective of the present investigation was to determine whether modulation of voltage-dependent calcium channels (L-type channel) alters the pharmacological effects of nicotine. For these studies we have chosen to determine the effects of the dihydropyridine receptor agonist and antagonists on nicotineinduced antinociception and depression of spontaneous activity.
Measurement
Modulation Methods Animals
and drugs
Male ICR mice (20-25 g) obtained from Harlan Laboratories (Indianapolis, IN) were used throughout the study. They were housed in groups of six and had free access to food and water. Nicotine was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI) and converted to the ditartrate salt as described previously (Aceto et al., 1979). (+)-Verapamil hydrochloride and nifedipine were obtained from Sigma (St. Louis, MO), (&)-BAKY 8644 from RBI (Natick, MA) whereas nimodipine was a gift from Miles, Inc. (West Haven, CT). Nicotine ditartrate and verapamil hydrochloride were dissolved in physiological saline (0.9% sodium chloride). Nifedipine, nimodipine and BAKY were emulphor: 8644 prepared in ethanol:saline (1:1:18). Emulphor (EL620) was obtained from Rhone-Poulenc (Cranbury, NJ). Solutions of nifedipine, nimodipine and BAKY 8644 were refrigerated in foil-lined containers. All doses are expressed as the free base of the drug.
of antinociception
Nicotine-induced antinociception was measured by the tail-flick method of D’Amour and Smith (1941) as modified by Dewey et al. (1970). Groups of six mice were used for each dose and for each treatment. A control response (2 - 4 s) was determined for each animal before treatment and test latencies were assessed at various times after drug administration. A maximum latency of 10 s was imposed if no response occurred within that time. Antinociceptive response was calculated as % MPE, where % MPE = [(test-control)/(lO-control) x 1001. The time course of antinociception was determined by pretreating animals with either vehicle or BAYK 8644 (0.75 mglkg, i.p.) 10 min before nicotine (1.75 mg/kg, s.c.) and measuring tail-flick response at 5, 10, 20, 30, 60 and 120 min after the nicotine injection. A separate group of mice was used for each time point. of nicotine antinociception
Mice were pretreated i.p. or S.C. with either the appropriate vehicle or verapamil, nifedipine, nimodipine 30 min before administration of nicotine (1.5 mglkg). Mice were treated with BAKY 8644 10 min before nicotine. Mice were tested for the antinociceptive response 5 min after nicotine administration. The treatment protocol for BAYK 8644 and calcium antagonists was based on the time of peak effect of these drugs. Locomotor
activity assays
Mice were placed into individual photocell activity cages (28 x 16.5 cm) immediately after S.C. administration of either 0.9% saline or nicotine. They were allowed to acclimate for 10 min. Interruptions of the photocell beams were recorded for the next 10 min. Data were expressed as percentage of depression where % depression = [l - (counts from nicotine-treated animals/counts from vehicle-treated animals)] x 100. Mice were pretreated with either saline, emulphor:ethanol:saline or BAKY 8644 (0.5 mg/kg, i.p.) 10 min before nicotine. Doseresponse curves were determined for nicotine in the presence of BAKY 8644.
75
Statistical
analysis
Data were analyzed statistically by an analysis of variance (ANOVA) followed by Fisher PLSD multiple comparison test. The null hypothesis was rejected at the 0.05 level. Results BAKY 8644. and nicotine-induced
hypomotility
BAYK 8644 produced a 5- to lo-fold enhancement in the hypomotility induced by nicotine. For example, nicotine (0.05 mg/kg) alone produced no significant depression of spontaneous activity (7%) whereas BAYK 8644 pretreatment increased this depression to 80% (Fig. 1). The slopes of the nicotine dose-response curves with and without BAYK 8644 were not statistically different ((t95yo) = 2.517). The dosecurve for nicotine-induced deresponse pression was shifted to the left by BAKY 8644. The ED5,, of nicotine was decreased from 0.65 mglkg to 0.15 mglkg by 0.5 mg/kg of BAKY 8644. In order to determine whether BAKY 8644 is acting through DHP channels to potentiate nicotine, mice were pretreated with nifedipine, a DHP calcium channel antagonist, at a dose (2 mglkg) known to block the behavioral effects of BAKY 8644 in the mouse (Bolger et al., 1985). This dose of nifedipine given 10 min before
100
1
Nicotine
(mgikg)
Fig. 1. Effects of nicotine with (closed circles) and without (open circles) BAKY 8644 on spontaneous activity. The results are presented as the mean * S.E.M. obtained in 12 or more mice.
BAKY 8644 blocked the hypomotility induced in mice by the combination of BAKY 8644(0.50 mg/kg) and nicotine (0.05 mglkg). (see Fig. 2) BAKY 86~ ciception
and nicotine-induced
antino-
The antinociceptive effects of nicotine alone and in combination with BAKY 8644 (0.75 mglkg, i.p.) are shown in Fig. 3. BAKY 8644 pretreatment resulted in antinociceptive effects of nicotine at doses which are otherwise inactive when given alone. For example, nicotine (0.05 mg/kg) alone produced no significant effect (%MPE = 5), whereas BAYK 8644 pretreatment increased the %MPE to 74. At higher doses of nicotine (0.25, 0.5 and 1.5 mg/kg), the enhancement by BAKY 8644 was only slight and %MPE reached after these doses are 76, 81 and 100, respectively. The pattern of shift for nicotine dose-response curves in producing antinociception by BAKY 8644 was markedly different from that for depression of spontaneous activity. The dose response curve for nicotineinduced antinociception in the presence of BAKY 8644 appears to be biphasic. BAKY 8644 potentiation of nicotine-induced antinociception was dose responsive with an EDs0 of 0.65 (0.45 - 1.00) mg/kg as can be seen in Fig. 4. Nifedipine, at 2 mglkg and 10 mg/kg, given 10 min before BAKY 8644 blocked the antinociception induced in mice by the combination of BAKY 8644 (0.75 mg/kg) and nicotine (0.05 mglkg). The blockade of BAKY 8644’s effect by nifedipine confirms that BAYK 8644 is acting through DHP channels to potentiate nicotine’s antinociception (see Fig. 5) A comparison of the time course of nicotine antinociception administered alone and in combination with BAKY 8644 showed that the calcium agonist pretreatment had relatively little effect on nicotine’s duration of action (data not shown). Although there was a trend toward an increase in antinociceptive effects at all time points with BAYK 8644 pretreatment, none was significantly enhanced. Nicotine
The
and calcium antagonists
effects
of
calcium
channel
blockers
76
Fig. 2. Nifedipine (2 mg/kg, i.p.) reversal of BAKY 8644 (0.50 mg/kg, i.p.) enhancement of nicotine-induced hypomotility. The results are presented as the mean + S.E.M. obtained in 6 or more mice. *P < 0.05 as compared to the vehicle-treated group.
themselves on the reaction time to a thermal stimulus in mice and on the antinociceptive effects of nicotine are shown in Table I. No antinociceptive responses could be obtained when
the calcium channel antagonists were tested at various doses in tail-flick procedure. However, nicotine-induced antinociception was blocked by relatively large doses of nifedipine and nimodi-
d 0 0.00
0.25
BAYK8644 0.00
0.25
0.50
0.75
1.00
1.25
7
I
0.50
0.75
(mgikg)
1.50
Nicotine (mgikg)
Fig. 3. Effects of nicotine with (closed circles) and without (open circles) BAKY 8644 (0.75 mg/kg i.p.) on tail-flick response. The data are presented as the mean + S.E.M. obtained in 12 or more mice.
Fig. 4. Dose-response relationship of BAKY 8644 on nicotine-induced antinociception (0.05 mklkg, s.c.). Closed circles represent nicotine (0.05 mg/kg, s.c.) and open circles represent pretreatment with different doses of BAYK 8644 prior to administration of nicotine at 0.05 mg/kg. Each point represents the mean f S.E.M. obtained in 6 or more mice.
77
P
Fig. 5. Nifedipine (2 and 10 mgkg, i.p.) reversal of BAKY 8644 (0.75 mgkg, i.p.) enhancement of nicotine-induced antinociception. Each point represents the mean f S.E.M. obtained in 6 or more mice. *P < 0.05 as compared to the vehicle-treated group.
pine. Verapamil attenuated the effects of nicotine, but this blockade was not statistically significant even at the highest dose tested (Table I). Discussion The purpose of this study was to elucidate the possible involvement of Ca2+ entry through LTable I. Effects of calcium channel antagonists tinociceptive response to nicotine in mice.
Drug
Dose mg/kg
Saline Emulphor:ethanol:sahne Nicotine. 1.5
Verapamil
Nifedipine
Nimodipine
2 5 15 2 5 15 2 5 15
on the an-
% MPE 2*1 6+4 72 zt 18a Antagonist alone
Antagonist pius nicotine
13 f 4 3~2 6zt.5 4~2 7zt6 4*3 l*l o*o 2&l
63 44 43 52 25 11 65 42 22
zt 17 zt 18 + 18 zt 17 + 16b + 6b +z 14 f 17 f lob
9ignificantly different from the vehicle, P < 0.05. bSignificantly different from nicotine, P < 0.05.
type channels in the pharmacological effects of nicotine in mice. The present results indicate that dihydropyridine derivatives are able to modulate the antinociception and hypomotility induced by nicotine. The blockade of calcium channels by nifedipine and nimodipine decreased the potency of nicotine in the tail-flick test. On the other hand, BAKY 8644, a calcium channel agonist, potentiated the activity of nicotine on locomotor activity and tail flick. The reason for the failure of verapamil, a phenylalkylamine calcium antagonist, to block nicotine-induced antinociception is not clear. However, there are several differences between verapamil and nifedipine and BAYK 8644 which may be relevant. Verapamil interacts at a site on calcium channels which is distinct from the dihydropyridine site where nifedipine and BAYK 8644 bind. Verapamil inhibits many other neuronal processes, including Na’ and K + channels, a variety of neurotransmitter receptors and enzymes (Miller, 1987). In addition, a non-calcium dependent mechanism for verapamil has been described (Hitchoft et al., 1992). Finally, Little et al. (1986) found dihydropyridines superior to verapamil against ethanol withdrawal-induced seizures. At the doses used in the present study, neither the calcium channel blockers nimodipine, verapamil and nifedipine nor the calcium channel opener BAKY 8644 were able to modify the basal nociceptive threshold of mice in the tail flick test. These results confirm and extend those of Benedick et al. (1984) and Contreras et al. (1988) who showed that calcium channel blockers such verapamil, nifedipine and cinnarizine lack antinociceptive properties in animals. However, Del Pozo et al. (1987) and Ohnishi et al. (1988) reported an antinociceptive effect for calcium antagonists in mice. The conflict between these reports and our own findings could be attributed to the difference in test systems. Indeed, the acetic acid-induced writhing used by these authors to evaluate calcium antagonists is less specific than the tail flick test used in our experiments and the hot plate method used by Benedick et al. (1984) and Contreras et al. (1988)
78
Our results suggest that L-type channels, a high-voltage-activated calcium channel, modulate the activity of the central nicotinic receptors, since the antinociception and hypomotility induced by nicotine are mediated by central mechanisms (Clarke and Kumar, 1983; Martin et al., 1990). The agonistic effect of BAKY 8644 is most probably due to an increase in intracellular calcium and, as a result potentiates the effects of nicotine. Another possibility is that BAYK 8644 alters the pharmacokinetics of nicotine. However, evidence for such an interaction is lacking. The antagonistic effect of calcium channel blockers on nicotine supports the notion that transmembrane movement of calcium plays a crucial role. There is considerable evidence for the involvement of neuronal calcium and its channel in the actions of some drugs of abuse. Calcium channel antagonists have been shown to increase some acute in vivo effects of opiates such as analgesia and hypothermia and to decrease other actions such as respiratory depression and hypermotility (Ben-Sreti et al., 1983; Benedick et al., 1984; Hoffmeister and Tettenborn, 1986; Contreras et al., 1988; Martin et al., 1990). On the other hand, BAKY 8644 potentiated the locomotor inhibitory effects of low-dose ethanol (Turna and Erglu, 1991) and attenuated fentanylinduced antinociception in rats (Hoffmeister and Tettenborn, 1986). Our results show that calcium has a profound influence on nicotine’s central effects and are consistent with the biochemical and electrophysiological observations that calcium plays a crucial role in the pharmacological actions of nicotine. The present findings may provide new insights into the understanding of nicotine-induced antinociception and hypoactivity in mice. Further studies are needed to clarify the impact of calcium involvement in nicotine dependence and tolerance. Acknowledgments
This work DA-05274.
was
supported
by PHS
grant
References Aceto, M.D., Martin, B.R., Uwaydah, I.M., May, E.L., Harris, L.S., Izazola-Conde, C., Dewey, W.L. and Vincek, W.C. (1979) Optically pure (+) nicotine from (*) nicotine and biological comparison with (-) nicotine. J. Med. Chem. 22, 174- 177. Ben-sreti, M.M., Gonzalez, J.P. and Sewell, R.D.E. (1983) Effects of elevated calcium and calcium antagonists on 6,7-benzomorphan-induced analgesia. Eur. J. Pharmacol. 90, 385-391. Benedek, G. and Szikszay, M. (1984) Potentiation of thermoregulatory and analgesic effects of morphine by calcium antagonists. Pharmacol. Res. Commun. 16, 1009 - 1018. Bolger, G.T., Weissman, B.A and Skolnick, P. (1985) The behavioral effects of the calcium agonist BAKY 8644 in the mouse: antagonism by the calcium antagonist nifedipine. Naunyn-Schmiedeberg’s Arch. Pharmacol. 328,373 - 377. Clarke P.B.S. and Kumar R. (1983) The effects of nicotine on locomotor activity in non-tolerant and tolerant rats. Br. J. Pharmacol. 78,329-337. Contreras, E., Tamayo, L. and Amigo, M. (1988) Calcium channel antagonists increase morphine-induced analgesia and antagonize morphine tolerance. Eur. J. Pharmacol. 148, 463- 466. D’Amour, F.E. and Smith, D.L. (1941) A method for determining loss of pain sensation. J. Pharmacol. Exp. Ther. 72, 74 - 79. Decker, E.R. and Dani, J.A. (1990) Calcium permeability of the nicotinic acetylcholine receptor: the single-channel calcium influx is significant. J. Neurosci. 10, 3413- 3420. Del Pozo, E., Caro, G. and Baeyens, J.M. (1987) Analgesic effects of several calcium channel blockers in mice. Eur. J. Pharmacol. 137, 1055 - 1060. Deneris, E.S., Connolly, J., Rogers, S.W. and Duvoisin, R. (1991) Pharmacological and functional diversity of neuronal nicotinic acetylcholine receptors. Trends Pharmacol. Sci. 12, 34-40. Dewey, W.L., Harris, L.S., Howes, J.F. and Nuite, J.A. (1970) The effect of various neurohormonal modulations on the activity of morphine and the narcotic antagonists in tail-flick and phenylquinone test. J. Pharmacol. Exp. Ther. 175, 435-442. Hitchcott, P.K., Zharkovsky, A. and File, S.E. (1992) Concurrent treatment with verapamil prevents diazepam withdrawal-induced anxiety, in the absence of altered calcium flux in cortical synaptosomes. Neuropharmacology 31, 55-60. Hoffmeister, F. and Tettenborn, D. Calcium agonists and antagonists of the dihydropyridine type: antinociceptive effects, interference with opiate-p-receptor agonists and neuropharmacological actions in rodents. Psychopharmacology 90, 299 - 307. Holz, R.W., Senter, R.A. and Frye, R.A. (1982) Relationship between Ca2+ uptake and catecholamine secretion in pri-
79
mary dissociated cultures of adrenal medulla. J. Neurochem. 39, 635 - 646. Kilpatrick D.L., Slepetis, R.J., Corcoran, J.J. and Kirshner, N. (1982) Calcium uptake and catecholamine secretion by cultured bovine adrenal medulla cells. J. Neurochem. 38, 427-435. Martin, MI., Lizasoain, I. and Leza, J.C. (1990) Calcium channels blockers: effect on morphine-induced hypermotility. Psychopharmacology 101, 267 - 270. Martin, T.J., Suchocki, J., May, E.L. and Martin, S.R. (1990) Pharmacological evaluation of the antagonism of nicotine’s central effects by mecamylamine and pempidine. J. Pharmacol. Exp. Ther. 254, 5-51. Miller, R.J. (1987) Multiple calcium channels and neuronal functions. Science 235, 46 - 52. Misu, Y., Goshima, Y., Nakamura, S. and Kubo, T. (1990) Nicotine releases stereoselectively and Ca2+-dependently endogenous DOPA from rat striatal slices. Brain Res. 520, 334-337. Mulle, C., Choquet, D., Korn, H. and Changeux J.-P. (1992) Calcium influx through nicotinic receptor in rat central neurons: its relevance to cellular regulation. Neuron 8, 135 - 143. Nef, P., Oneyser, C., Alliod, C., Couturier, S. and Ballivet, M. (1988) Genes expressed in the brain define three distinct neuronal nicotinic acetylcholine receptors. EMBO J. 7, 595-601. Noronha-Blob, L., Gover, R. and Baumgold, J. (1989) Calcium influx mediated by nicotinic receptors and voltage human channels in SK-N-SH sensitive calcium neuroblastoma cells. Biochem. Biophy. Res. Commun. 162, 1230 - 1235. Ohnishi, T., Saito, K., Matsumoto, K., Kakuda, M. and Inoki, R. (1988) Decrease in analgesic effect of nifedipine following chronic morphine administration. Eur. J. Pharmacol. 158,173- 175.
O’Sullivan, A.J., Cheek, T.R., Moreton, R.B., Berrigde, M.J. and Burgoyne R.D. (1989) Localization and heterogeneity of agonist-induced changes in cytosolic calcium concentration in single bovine adrenal chromaffin cells from video imaging of fura-2. EMBO J. 8, 401-411. Phan, D.V., Doda, M., Bite, A. and Gyorgy, L. (1973) Antinociceptive activity of nicotine. Acta Physiol. Acad. Sci. Hung. 44,85 - 93. Rapier, C., Lunt, G.G. and Wonnacott, S. (1988) Stereoselective nicotinic-induced release of dopamine from striatal synaptosomes: concentration dependence and repetitive stimulation. J. Neurochem. 50, 1123- 1130. Rowell, P.P. and Winkler, D.J. (1984) Nicotinic stimulation of [3H]acetylcholine release from mouse cerebral cortical synaptosomes. J. Neurochem. 43, X93- 1598. Sontag, J.M., Sanderson, P., Klepper, M., Aunis, D., Takeda, K. and Bader, M.F. (1990) Modulation of secretion by dopamine involves decreases in calcium and nicotinic currents in bovine chromaffin cells. J. Physiol. 427, 495- 517. Tripathi, H.L., Martin, B.R and Aceto, M.D. (1982) Nicotine-induced antinoception in rats and mice: correlation with nicotine brain levels. J. Pharmacol. Exp. Ther. 221, 91-96. Tuna, R.K. and Erogly, L. (1991) Effects of BAKY 8644 and nifedipine on locomotor activity and striatal homovanillic acid concentration in acutely ethanol-treated rats. Alcohol Alcoholism 26, 465 - 471. Vernino, S., Amador, M., Luetje, C.W., Patrick, J. and Dani, J.A. (1992) Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors. Neuron 8, 127 - 134. Westfall, T.C. (1974) Effect of nicotine and other drugs on the release of 3H-norepinephrine and 3H-dopamine from rat brain slices. Neuropharmacology 13, 693 - 700.
Drug and Alcohol Dependence, 32 (1993) 73 - 79
Elsevier Scientific Publishers
Ireland Ltd.
Calcium agonists and antagonists of the dihydropyridine type: effect on nicotine-induced antinociception and hypomotility M.I. Damaj and B.R. Martin Department
of Pharmacology
and Toxicology, Medical College of Virginia, VA 23298-0613 (USA)
(Accepted September
Virginia Commonwealth University, Richmond,
22nd, 1992)
The influence of a calcium agonist (BAYK 8644) and several calcium channel blockers on nicotine-induced antinociception was investigated in mice. The effect of nicotine was sharply increased by BAYK 8644. This potentitation by BAYK 8644 was blocked by pretreating the animals with nifedipine at 2 and 10 mglkg. The calcium channel antagonists, nifedipine and nimodipine at doses that had no effect on tail-flick time, reduced significantly the antinociception induced by nicotine (1.5 mg/kg, s.c.). However, the effect of verapamil on nicotine was not significant. These results indicate that DHP calcium channels (type L-channel) play a role in some of the pharmacological effects of nicotine, particularly, locomotor activity and antinociception. Key words: calcium antagonists;
BAYK 8644; nicotine; antinociception;
Introduction The nicotinic acetylcholine receptor (nAChR) is one of the most widely studied ligand-gated ion channels in neurobiology. Despite the voluminous literature regarding nAChR in muscle, Torpedo and ganglia, studies pertaining to the mammalian central nervous system are limited mostly to ligand-binding studies. Unlike the nAChR found at the neuromuscular junction, those found in the CNS exhibit considerable diversity in their subunit composition (Deneris et al., 1991). Recent molecular biology studies further suggest the existence of several subtypes of nAChR in the brain based upon the discovery of several genes encoding for both alpha and beta subunits (Nef et al., 1988). However, the physiological properties, pharmacological activities and the transduction to: Billy R. Martin, Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 2329% 0613, USA. Correspondence
mice
mechanisms associated with these nAChR are largely unknown. The involvement of calcium in the signalling process of the peripheral nAChR is well established. Apart from depolarizing the cell membrane, activation of the nAChR on muscle also leads to significant Ca2+ influx into the cell (Decker et al., 1990). Similarly, in chromaffin cells, activation of nAChR causes an influx of Ca2+ leading to an increase in the intracellular calcium [Ca2+]i (Kilpatrick et al., 1982; Baumgold et al., 1989; O’Sullivan et al., 1989; Noronha-Blob et al., 1989) which is thought to result in great part from local depolarization and opening of voltage-dependent Ca2+ channels. In the CNS, nicotine-evoked neurotransmitter release is calcium dependent (Westfall, 1974; Rowe11 et al., 1984; Rapier et al., 1988; Kubo et al., 1990). Recently, by using Patch-clamp recordings, Vermin0 et al. (1992) showed that changes in extracellular calcium modulate neuronal nAChR in a dose-dependent manner and that the activation of the neuronal nAChR produces a significant influx of calcium. Further-
0376-8716/93/$06.00 0 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
more, Mulle et al. (1992a) showed that neuronal nAChR located on the soma of rat medial habenular nucleus neurons are permeable to Ca2+ leading to an increase of [Ca’+]i up to the micrdmolar range, a level largely sufficient for the activation of Ca2 +-dependent regulatory process. Although there is evidence that calcium likely plays an important role in the central action of nicotine, there is little documentation that alterations in intracellular calcium influences the pharmacological effects of nicotine. Therefore, the objective of the present investigation was to determine whether modulation of voltage-dependent calcium channels (L-type channel) alters the pharmacological effects of nicotine. For these studies we have chosen to determine the effects of the dihydropyridine receptor agonist and antagonists on nicotineinduced antinociception and depression of spontaneous activity.
Measurement
Modulation Methods Animals
and drugs
Male ICR mice (20-25 g) obtained from Harlan Laboratories (Indianapolis, IN) were used throughout the study. They were housed in groups of six and had free access to food and water. Nicotine was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI) and converted to the ditartrate salt as described previously (Aceto et al., 1979). (+)-Verapamil hydrochloride and nifedipine were obtained from Sigma (St. Louis, MO), (&)-BAKY 8644 from RBI (Natick, MA) whereas nimodipine was a gift from Miles, Inc. (West Haven, CT). Nicotine ditartrate and verapamil hydrochloride were dissolved in physiological saline (0.9% sodium chloride). Nifedipine, nimodipine and BAKY were emulphor: 8644 prepared in ethanol:saline (1:1:18). Emulphor (EL620) was obtained from Rhone-Poulenc (Cranbury, NJ). Solutions of nifedipine, nimodipine and BAKY 8644 were refrigerated in foil-lined containers. All doses are expressed as the free base of the drug.
of antinociception
Nicotine-induced antinociception was measured by the tail-flick method of D’Amour and Smith (1941) as modified by Dewey et al. (1970). Groups of six mice were used for each dose and for each treatment. A control response (2 - 4 s) was determined for each animal before treatment and test latencies were assessed at various times after drug administration. A maximum latency of 10 s was imposed if no response occurred within that time. Antinociceptive response was calculated as % MPE, where % MPE = [(test-control)/(lO-control) x 1001. The time course of antinociception was determined by pretreating animals with either vehicle or BAYK 8644 (0.75 mglkg, i.p.) 10 min before nicotine (1.75 mg/kg, s.c.) and measuring tail-flick response at 5, 10, 20, 30, 60 and 120 min after the nicotine injection. A separate group of mice was used for each time point. of nicotine antinociception
Mice were pretreated i.p. or S.C. with either the appropriate vehicle or verapamil, nifedipine, nimodipine 30 min before administration of nicotine (1.5 mglkg). Mice were treated with BAKY 8644 10 min before nicotine. Mice were tested for the antinociceptive response 5 min after nicotine administration. The treatment protocol for BAYK 8644 and calcium antagonists was based on the time of peak effect of these drugs. Locomotor
activity assays
Mice were placed into individual photocell activity cages (28 x 16.5 cm) immediately after S.C. administration of either 0.9% saline or nicotine. They were allowed to acclimate for 10 min. Interruptions of the photocell beams were recorded for the next 10 min. Data were expressed as percentage of depression where % depression = [l - (counts from nicotine-treated animals/counts from vehicle-treated animals)] x 100. Mice were pretreated with either saline, emulphor:ethanol:saline or BAKY 8644 (0.5 mg/kg, i.p.) 10 min before nicotine. Doseresponse curves were determined for nicotine in the presence of BAKY 8644.
75
Statistical
analysis
Data were analyzed statistically by an analysis of variance (ANOVA) followed by Fisher PLSD multiple comparison test. The null hypothesis was rejected at the 0.05 level. Results BAKY 8644. and nicotine-induced
hypomotility
BAYK 8644 produced a 5- to lo-fold enhancement in the hypomotility induced by nicotine. For example, nicotine (0.05 mg/kg) alone produced no significant depression of spontaneous activity (7%) whereas BAYK 8644 pretreatment increased this depression to 80% (Fig. 1). The slopes of the nicotine dose-response curves with and without BAYK 8644 were not statistically different ((t95yo) = 2.517). The dosecurve for nicotine-induced deresponse pression was shifted to the left by BAKY 8644. The ED5,, of nicotine was decreased from 0.65 mglkg to 0.15 mglkg by 0.5 mg/kg of BAKY 8644. In order to determine whether BAKY 8644 is acting through DHP channels to potentiate nicotine, mice were pretreated with nifedipine, a DHP calcium channel antagonist, at a dose (2 mglkg) known to block the behavioral effects of BAKY 8644 in the mouse (Bolger et al., 1985). This dose of nifedipine given 10 min before
100
1
Nicotine
(mgikg)
Fig. 1. Effects of nicotine with (closed circles) and without (open circles) BAKY 8644 on spontaneous activity. The results are presented as the mean * S.E.M. obtained in 12 or more mice.
BAKY 8644 blocked the hypomotility induced in mice by the combination of BAKY 8644(0.50 mg/kg) and nicotine (0.05 mglkg). (see Fig. 2) BAKY 86~ ciception
and nicotine-induced
antino-
The antinociceptive effects of nicotine alone and in combination with BAKY 8644 (0.75 mglkg, i.p.) are shown in Fig. 3. BAKY 8644 pretreatment resulted in antinociceptive effects of nicotine at doses which are otherwise inactive when given alone. For example, nicotine (0.05 mg/kg) alone produced no significant effect (%MPE = 5), whereas BAYK 8644 pretreatment increased the %MPE to 74. At higher doses of nicotine (0.25, 0.5 and 1.5 mg/kg), the enhancement by BAKY 8644 was only slight and %MPE reached after these doses are 76, 81 and 100, respectively. The pattern of shift for nicotine dose-response curves in producing antinociception by BAKY 8644 was markedly different from that for depression of spontaneous activity. The dose response curve for nicotineinduced antinociception in the presence of BAKY 8644 appears to be biphasic. BAKY 8644 potentiation of nicotine-induced antinociception was dose responsive with an EDs0 of 0.65 (0.45 - 1.00) mg/kg as can be seen in Fig. 4. Nifedipine, at 2 mglkg and 10 mg/kg, given 10 min before BAKY 8644 blocked the antinociception induced in mice by the combination of BAKY 8644 (0.75 mg/kg) and nicotine (0.05 mglkg). The blockade of BAKY 8644’s effect by nifedipine confirms that BAYK 8644 is acting through DHP channels to potentiate nicotine’s antinociception (see Fig. 5) A comparison of the time course of nicotine antinociception administered alone and in combination with BAKY 8644 showed that the calcium agonist pretreatment had relatively little effect on nicotine’s duration of action (data not shown). Although there was a trend toward an increase in antinociceptive effects at all time points with BAYK 8644 pretreatment, none was significantly enhanced. Nicotine
The
and calcium antagonists
effects
of
calcium
channel
blockers
76
Fig. 2. Nifedipine (2 mg/kg, i.p.) reversal of BAKY 8644 (0.50 mg/kg, i.p.) enhancement of nicotine-induced hypomotility. The results are presented as the mean + S.E.M. obtained in 6 or more mice. *P < 0.05 as compared to the vehicle-treated group.
themselves on the reaction time to a thermal stimulus in mice and on the antinociceptive effects of nicotine are shown in Table I. No antinociceptive responses could be obtained when
the calcium channel antagonists were tested at various doses in tail-flick procedure. However, nicotine-induced antinociception was blocked by relatively large doses of nifedipine and nimodi-
d 0 0.00
0.25
BAYK8644 0.00
0.25
0.50
0.75
1.00
1.25
7
I
0.50
0.75
(mgikg)
1.50
Nicotine (mgikg)
Fig. 3. Effects of nicotine with (closed circles) and without (open circles) BAKY 8644 (0.75 mg/kg i.p.) on tail-flick response. The data are presented as the mean + S.E.M. obtained in 12 or more mice.
Fig. 4. Dose-response relationship of BAKY 8644 on nicotine-induced antinociception (0.05 mklkg, s.c.). Closed circles represent nicotine (0.05 mg/kg, s.c.) and open circles represent pretreatment with different doses of BAYK 8644 prior to administration of nicotine at 0.05 mg/kg. Each point represents the mean f S.E.M. obtained in 6 or more mice.
77
P
Fig. 5. Nifedipine (2 and 10 mgkg, i.p.) reversal of BAKY 8644 (0.75 mgkg, i.p.) enhancement of nicotine-induced antinociception. Each point represents the mean f S.E.M. obtained in 6 or more mice. *P < 0.05 as compared to the vehicle-treated group.
pine. Verapamil attenuated the effects of nicotine, but this blockade was not statistically significant even at the highest dose tested (Table I). Discussion The purpose of this study was to elucidate the possible involvement of Ca2+ entry through LTable I. Effects of calcium channel antagonists tinociceptive response to nicotine in mice.
Drug
Dose mg/kg
Saline Emulphor:ethanol:sahne Nicotine. 1.5
Verapamil
Nifedipine
Nimodipine
2 5 15 2 5 15 2 5 15
on the an-
% MPE 2*1 6+4 72 zt 18a Antagonist alone
Antagonist pius nicotine
13 f 4 3~2 6zt.5 4~2 7zt6 4*3 l*l o*o 2&l
63 44 43 52 25 11 65 42 22
zt 17 zt 18 + 18 zt 17 + 16b + 6b +z 14 f 17 f lob
9ignificantly different from the vehicle, P < 0.05. bSignificantly different from nicotine, P < 0.05.
type channels in the pharmacological effects of nicotine in mice. The present results indicate that dihydropyridine derivatives are able to modulate the antinociception and hypomotility induced by nicotine. The blockade of calcium channels by nifedipine and nimodipine decreased the potency of nicotine in the tail-flick test. On the other hand, BAKY 8644, a calcium channel agonist, potentiated the activity of nicotine on locomotor activity and tail flick. The reason for the failure of verapamil, a phenylalkylamine calcium antagonist, to block nicotine-induced antinociception is not clear. However, there are several differences between verapamil and nifedipine and BAYK 8644 which may be relevant. Verapamil interacts at a site on calcium channels which is distinct from the dihydropyridine site where nifedipine and BAYK 8644 bind. Verapamil inhibits many other neuronal processes, including Na’ and K + channels, a variety of neurotransmitter receptors and enzymes (Miller, 1987). In addition, a non-calcium dependent mechanism for verapamil has been described (Hitchoft et al., 1992). Finally, Little et al. (1986) found dihydropyridines superior to verapamil against ethanol withdrawal-induced seizures. At the doses used in the present study, neither the calcium channel blockers nimodipine, verapamil and nifedipine nor the calcium channel opener BAKY 8644 were able to modify the basal nociceptive threshold of mice in the tail flick test. These results confirm and extend those of Benedick et al. (1984) and Contreras et al. (1988) who showed that calcium channel blockers such verapamil, nifedipine and cinnarizine lack antinociceptive properties in animals. However, Del Pozo et al. (1987) and Ohnishi et al. (1988) reported an antinociceptive effect for calcium antagonists in mice. The conflict between these reports and our own findings could be attributed to the difference in test systems. Indeed, the acetic acid-induced writhing used by these authors to evaluate calcium antagonists is less specific than the tail flick test used in our experiments and the hot plate method used by Benedick et al. (1984) and Contreras et al. (1988)
78
Our results suggest that L-type channels, a high-voltage-activated calcium channel, modulate the activity of the central nicotinic receptors, since the antinociception and hypomotility induced by nicotine are mediated by central mechanisms (Clarke and Kumar, 1983; Martin et al., 1990). The agonistic effect of BAKY 8644 is most probably due to an increase in intracellular calcium and, as a result potentiates the effects of nicotine. Another possibility is that BAYK 8644 alters the pharmacokinetics of nicotine. However, evidence for such an interaction is lacking. The antagonistic effect of calcium channel blockers on nicotine supports the notion that transmembrane movement of calcium plays a crucial role. There is considerable evidence for the involvement of neuronal calcium and its channel in the actions of some drugs of abuse. Calcium channel antagonists have been shown to increase some acute in vivo effects of opiates such as analgesia and hypothermia and to decrease other actions such as respiratory depression and hypermotility (Ben-Sreti et al., 1983; Benedick et al., 1984; Hoffmeister and Tettenborn, 1986; Contreras et al., 1988; Martin et al., 1990). On the other hand, BAKY 8644 potentiated the locomotor inhibitory effects of low-dose ethanol (Turna and Erglu, 1991) and attenuated fentanylinduced antinociception in rats (Hoffmeister and Tettenborn, 1986). Our results show that calcium has a profound influence on nicotine’s central effects and are consistent with the biochemical and electrophysiological observations that calcium plays a crucial role in the pharmacological actions of nicotine. The present findings may provide new insights into the understanding of nicotine-induced antinociception and hypoactivity in mice. Further studies are needed to clarify the impact of calcium involvement in nicotine dependence and tolerance. Acknowledgments
This work DA-05274.
was
supported
by PHS
grant
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