Materials and Methods

Animals.

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. Animals were housed in an American Association for Laboratory Animal Science-approved facility, and the study was approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University.

Drugs.

Mecamylamine hydrochloride and dihydro-β-erythroidine hydrobromide were supplied as a gift from Merck, Sharp and Dohme & Co. (West Point, PA). Both (+)- and (−)-epibatidine (hemioxalate salt) were supplied by Dr. S. Fletcher (Merck, Sharp and Dohme & Co., Essex, UK). Cytisine, hexamethonium hydrochloride, nifedipine, and lobeline were purchased from Sigma Chemical Co. (St. Louis, MO). Atropine sulfate, BAY K 8644, MLA, riluzole, N^G-nitro-l-arginine (L-NNA),N^G-nitro-d-arginine (D-NNA),N^G-nitro-l-arginine methyl ester HCl (L-NAME),N^G-nitro-d-arginine methyl ester HCl (D-NAME), (±)-CPP, (+)-MK801, and α-BGTX were purchased from Research Biochemicals Inc. (Natick, MA). Nudicauline was purchased from Calbiochem (San Diego, CA). Nimodipine was a gift from Miles, Inc. (West Haven, CT). Nicotine enantiomers were synthesized and converted to the ditartrate salt as described by Aceto et al. (1979). Other drugs that were synthesized are 6-chloronicotine (Dukat et al., 1996), (±)-isonicotine (Glassco et al., 1994), AMP-MP [3-(N-methyl-N-n-propylaminomethyl)pyridine], AMP-ME [3-(N-ethyl-N-n-methylaminomethyl)pyridine] (Glennon et al., 1993), and N-MNP [1,2,3,4,-tetrahydro-N-methyl)-1,6-naphhyridine] (Dukat et al., 1996). Metanicotine was synthesized as described byAcheson et al. (1980). All drugs were dissolved in physiological saline (0.9% sodium chloride) and administered in a total volume of 1 ml/100 g b.wt. for s.c. injections. Nimodipine, nifedipine, and BAY K 8644 was prepared in emulphor/ethanol/saline (1:1:18). Emulphor (EL620) was obtained from Rhone Poulenc (Crambury, NJ). All doses are expressed as the free base of the drug.

Intraventricular Injections.

Intraventricular injections were performed according to the method of Pedigo et al. (1975). Mice were lightly anesthetized with ether, and an incision was made in the scalp such that the bregma was exposed. Injections were performed using a 26-gauge needle with a sleeve of PE 20 tubing to control the depth of the injection. Mice were administered each drug in an injection volume of 5 μl at a site 2 mm rostral and 2 mm caudal to the bregma at a depth of 2 mm.

Seizure Testing.

After the injection of nicotinic agonists, each animal was placed in a 30 × 30-cm² Plexiglas cage and observed for 3 min. Whether a clonic seizure occurred within a 3-min time period was noted for each animal after the s.c. administration of different agonists. This time was chosen because seizures occur very quickly after nicotinic agonist administration. The percentage of animals exhibiting a seizure was calculated, and dose-response curves were constructed for each agonist. Antagonism studies were carried out by pretreating the mice s.c. with either saline or different antagonists 10 min before nicotinic agonists. For the i.c.v. experiments, mice were pretreated i.c.v. with either saline or nicotinic antagonists; 5 min later, mice received nicotine s.c. at a dose of 9 mg/kg.

Statistical Analysis.

Data were analyzed statistically with ANOVA followed by the Fisher protected least significant difference multiple comparison test. The null hypothesis was rejected at the .05 level. ED₅₀ and AD₅₀ values with 95% CL determined according to the method of Litchfield and Wilcoxon (1949).

Results

Seizure Induction by Different Nicotinic Agonists after s.c. Administration.

Nicotine and other nicotine agonists administered s.c. induced seizures in a dose-dependent manner (Fig.1). The (+)-enantiomer of nicotine also produced seizures with a potency (ED₅₀ = 103 mg/kg) less than that of (−)-nicotine (ED₅₀ = 4.8 mg/kg). Table 1 summarizes the pharmacological potency of different nicotinic ligands in inducing seizures after s.c. administration. The enantiomers of epibatidine and 6-chloronicotine were the most potent agonists tested. AMP-MP and AMP-ME were the least potent nicotinic agonists in producing seizures. On the other hand, s.c. administration of metanicotine, isonicotine, cotinine, and N-MNP elicited no convulsive responses at the doses tested (Fig. 1). In addition, cytisine and lobeline elicited partial effects with a response of 33 and 50%, respectively, after s.c. injection. It was not possible to obtain complete dose-response curves due to the lethality of higher doses of these drugs. No significant deviation from parallelism among the different dose-response functions after s.c. injection was found.

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Figure 1

Dose-response relationship of nicotinic ligands for inducing seizures after s.c. administration in mice. ■, (−)-nicotine; ▪, (+)-nicotine; ○, (+)-epibatidine; ●, (−)-epibatidine; ⊞, lobeline; ⋄, cytisine; ▴, 6-chloronicotine; ⊕, (±)-isonicotine; ┌, metanicotine; ✚, N-MNP; ▹, AMP-ME; ⬗, AMP-MP; and ♦, cotinine. The mice were observed for seizures for 3 to 5 min after drug injection. Each point represents the average for six to eight mice.

Antagonism of Nicotine-Induced Seizures by Different Nicotinic Antagonists after s.c. Administration.

The s.c. injection of 9 mg/kg nicotine produced a reliable loss of righting reflexes in mice followed by seizures. Pretreatment with mecamylamine, a noncompetitive nicotinic antagonist, administered s.c. inhibited the seizure responses of s.c. nicotine in a dose-dependent manner (Fig.2A). An AD₅₀ value of 0.1 mg/kg was calculated for mecamylamine (Table2). In contrast, dihydro-β-erythroidine, a competitive nicotinic antagonist, failed to significantly prevent the effects of nicotine up to an s.c. dose of 5 mg/kg (Fig. 2B). The administration of higher doses of dihydro-β-erythroidine was associated with toxicity and lethality in mice. Similarly, the peripheral nicotinic antagonist hexamethonium failed to block the effect of nicotine up to the dose of 1 mg/kg. The plant alkaloid α7 antagonist MLA produced a dose-dependent inhibition of nicotine-induced seizures, with an AD₅₀ value of 1.9 mg/kg (Fig. 2C and Table 2) after s.c. pretreatment. The effect of MLA seems to be specific to the convulsive effects of nicotine because it failed to significantly block other effects of nicotine, such as hypothermia, antinociception, and hypomotility, after s.c. administration (Table 3). Indeed, in mice pretreated with MLA at a dose that is three times higher (6 mg/kg s.c.) than the AD₅₀ value for blocking seizures, nicotine did not elicit significantly reduce effects compared with control animals.

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Figure 2

Effects of mecamylamine (A), dihydro-β-erythroidine (B), and MLA (C) on the nicotine-induced seizures after s.c. administration. Mice were pretreated s.c. with different doses of nicotinic antagonists 5 min before nicotine (9 mg/kg) and observed for seizures. Each point represents the average for six to eight mice.

Antagonism of Nicotine-Induced Seizures by Different Nicotinic Antagonists after i.c.v. Administration.

Similar to the results observed after s.c. administration, i.c.v. injection of mecamylamine prevented the seizures elicited by nicotine (9 mg/kg s.c.) in a dose-dependent manner (Fig. 3A) with an AD₅₀ value of 0.45 μg/animal (Table 2). Interestingly, MLA injected i.c.v. prevented the seizures caused by nicotine (Fig. 3B) with a higher potency than that obtained after s.c. administration. Indeed, the difference in potency between mecamylamine and MLA observed after s.c. injection (with mecamylamine being 20 times more potent than MLA) is much smaller after central administration (with mecamylamine being 2.5 times more potent than MLA only). Furthermore, nudicauline, a potent α7 antagonist, significantly blocked nicotine-induced seizures at a dose of 1 μg/animal. Higher doses were not tested due to a lack of supplies. Finally, dihydro-β-erythroidine, similar to the results of s.c. injection, failed to significantly prevent the convulsive effects of nicotine up to an i.c.v. dose of 10 μg/animal. By themselves, these antagonists did not cause seizures at the indicated doses and times. In addition, no convulsions were observed when they were given at doses up to 60 μg/animal.

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Figure 3

Effects of i.c.v. administration of mecamylamine (A) and MLA (B) on the nicotine-induced seizures after s administration. Mice were pretreated i.c.v. with different doses of nicotinic antagonists 5 min before nicotine (9 mg/kg s.c.) and observed for seizures. Each point represents the average for six to eight mice.

Modulation of Nicotine-Induced Seizures by Non-nicotinic Agents.

A number of non-nicotinic drugs were tested for their ability to influence nicotine-induced seizures. Pretreatment with atropine, a muscarinic antagonist, at the dose of 10 mg/kg failed to significantly decrease the seizures caused by nicotine after s.c. administration (Table 2).

In the first series of experiments, we studied the effects of modulation of voltage-dependent calcium channels (L-type channel) on the effects of nicotine. The seizure effects of nicotine alone (4 mg/kg) and in combination with BAY K 8644, a calcium channel activator, at different doses are shown in Fig. 4B. BAY K 8644 pretreatment resulted in seizures at a dose of nicotine that is otherwise inactive when administered alone. For example, nicotine (4 mg/kg) alone produced no seizures, whereas BAY K 8644 pretreatment at 1 mg/kg increased the number of animals exhibiting seizures to 83%. BAY K 8644 potentiation of nicotine-induced seizures was dose related with an ED₅₀ value of 0.70 mg/kg. By itself, BAY K 8644 did not cause seizures at the indicated doses and times. The effects were examined of i.p. administration of the calcium channel blockers nifedipine, nimodipine, and verapamil on nicotine-induced seizures administered s.c. By themselves, those agents did not cause seizures at the indicated doses and times. Verapamil (10 mg/kg) produced a partial blockade (33%) on seizures produced by a 9 mg/kg dose of nicotine (Table 2). Doses of verapamil of more than 10 mg/kg were not tested. However, nifedipine (Fig. 4A) and nimodipine significantly blocked the effect of nicotine. Indeed, the administration of nifedipine and nimodipine, at 10 min before nicotine, produced a blockade of nicotine-induced seizures in a dose-dependent manner with AD₅₀ values of 0.5 and 0.6 mg/kg, respectively (Table 2).

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Figure 4

Effects of the calcium channel antagonist nifedipine (A) and calcium channel activator BAY K 8644 (B) on the nicotine-induced seizures after s.c. administration. For the nifedipine studies, mice were pretreated i.p. with different doses of the channel antagonist 10 min before nicotine (9 mg/kg) and tested for seizures. For the BAY K 8644 studies, mice were pretreated i.p. with different doses of the channel activator 10 min before an inactive dose of nicotine (4 mg/kg) and tested for seizures. Each point represents the average for six to eight mice.

In another series of experiments, we investigated the effects of riluzole, a glutamate release inhibitor, and NMDA receptor antagonists. As shown in Fig. 5B, the noncompetitive NMDA receptor antagonist MK-801, administered 10 min before nicotine, dose-dependently suppressed the development of seizures with an AD₅₀ value of 0.2 mg/kg. Similarly, (±)-CPP, a competitive NMDA antagonist, blocked nicotine-induced seizures with an AD₅₀ value of 0.74 mg/kg (Fig. 5A and Table 2). Finally, nicotine-induced seizures were dose-dependently abolished by pretreatment with riluzole with a potency of 15.5 mg/kg (Fig. 5C).

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Figure 5

NMDA receptor antagonists and glutamate release inhibitor prevent nicotine-induced seizures. Mice were pretreated s.c. with different doses of (±)-CPP (A), MK-801 (B), and riluzole (C) 5 min before nicotine (9 mg/kg) and tested for seizures. Each point represents the average for six to eight mice.

The final series of experiments was concerned with investigation of the effects of NO synthase inhibitors on nicotine-induced seizures. The NO synthase inhibitors L-NAME and L-NNA, administered 15 min before nicotine, significantly suppressed the development of seizures (Fig. 6).L-NAME and L-NNA blocked nicotine-induced seizures in a dose-related manner with AD₅₀values of 550 and 14.5 mg/kg, respectively. However, D-NNA (50 mg/kg), an inactive isomer, did not affect the development of seizures.

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Figure 6

Effects of NO synthase inhibitors L-NNA (A) and L-NAME (B) on nicotine-induced seizures. Mice were pretreated s.c. with different doses of NO synthase inhibitors 5 min before nicotine (9 mg/kg) and tested for seizures. Each point represents the average for six to eight mice.

Discussion

Consistent with previous reports (Miner et al., 1985; Miner and Collins, 1989), we showed that peripheral administration of nicotine produced seizures in a stereospecific and mecamylamine-sensitive manner. The fact that hexamethonium, a nicotinic antagonist that poorly penetrates the blood-brain barrier, showed little blockade of the effect of nicotine confirms previous reports that nicotinic seizure is centrally mediated (Dixit et al., 1971; Caulfield and Higgins, 1983). In assessment of the involvement of different nicotinic receptor subunits, our data suggest that α7-nicotinic receptor subtypes are involved in nicotinic seizures. Indeed, MLA administered systemically or centrally significantly blocked nicotine-induced convulsions. MLA binds potently (K_i = 4 nM) to¹²⁵I-α-BGTX-binding sites (α7-subunits), in contrast to its weak interactions with other neuronal nicotinic receptors (micromolar range). Moreover, it has been classified as a competitive antagonist at α7-nicotinic receptors (Ward et al., 1990). It also possesses the ability to cross the blood-brain barrier after peripheral injection and to achieve pharmacologically relevant concentrations in rat brain (Turek et al., 1995). In our model, MLA was surprisingly 20 times less potent than mecamylamine in blocking the effects of nicotine after s.c. injection. However, this difference in potency between the two drugs was much smaller (2.5 times) after i.c.v. administration, suggesting differences in their ability to penetrate the blood-brain barrier. In addition, nudicauline, an α7-antagonist with higher affinity than MLA (5 times higher) to¹²⁵I-α-BGTX-binding sites (Hardick et al., 1996), blocked the seizures caused by nicotine with higher potency than MLA when given i.c.v. Although these results strongly support the involvement of α7-subunits in nicotine-induced seizures, the relative potency of MLA and mecamylamine in blocking this effect suggests that in addition to α7-subunits, other nicotinic subunits may be involved. Indeed, α7-homo-oligomers expressed in oocytes are 1000 times more sensitive to blockade by MLA than mecamylamine (Briggs and Mc- Kenna, 1996). This difference between the in vitro concentrations and the in vivo doses suggests that nAChRs mediating the seizures caused by nicotine may not be equivalent to the α7-receptor expressed in oocytes. These α7 containing nAChRs may include other subunits that influence MLA sensitivity to the α7-subunit.

The extent of α4β2-receptor subtype involvement in nicotine-induced seizures was difficult to assess. On one hand, we observed that dihydro-β-erythroidine, a competitive nicotinic antagonist, failed to block nicotine-induced seizures after both peripheral and central administration. It is reported that the sensitivity of dihydro-β-erythroidine in blocking α4β2-receptors expressed in oocytes was 180 times higher than that of α7-expressed nAChRs (Chavez-Noriega et al., 1997). Furthermore, a poor correlation was found between binding affinity for³H-nicotine-labeled sites (predominantly α4β2) and seizure potency for several nicotinic agonists. Indeed, compounds such as lobeline, anabasine, (±)-isonicotine, metanicotine, and N-MNP bind with high affinity to³H-nicotine-binding sites; yet they failed to produce seizures after s.c. injection. In contrast, AMP-MP, an aminomethylpyridine derivative that binds with very low affinity to the³H-nicotine-labeled sites (Damaj et al., 1998), produced seizures in a dose-related manner with an ED₅₀ value similar to that of AMP-ME, a high-affinity nicotinic agonist. On the other hand, some of our results argue for a more active involvement of α4β2- receptors. For example, cytisine, a full α7 and a partial α4β2-agonist, exhibited only a partial effect in producing seizures. Furthermore, the results with mecamylamine, a noncompetitive antagonist, are difficult to interpret. Indeed, mecamylamine blocked nicotine-induced seizures in the same general dose range of other nicotinic effects that are not α7 mediated, such as antinociception and hypothermia (Damaj et al., 1995). It is generally thought that mecamylamine has good selectivity toward α4β2 nAChRs. However, no large differences were found in the sensitivities to the drug between α4β2- and α7-expressed nAChRs (Briggs and McKenna, 1996; Chavez-Noriega et al., 1997). In fact, the potency of mecamylamine in blocking α4β2 nAChRs has not been fully established. Most studies did not report full dose-response concentrations. Generally, its estimated that IC₅₀ values appear to fall in the range of 0.2 to 2 μM (Bertrand et al., 1990; Connolly et al., 1992; Briggs et al., 1996; Chavez-Noriego et al., 1997). These estimated IC₅₀ values are similar to that found at α7 receptors (IC₅₀ = 1.8–2.0 μM; Briggs and McKenna, 1996; Meyer et al., 1997). The very low potency of mecamylamine observed by Couturier et al. (1990) at the α7-receptors may be underestimated by not preapplying mecamylamine (Meyer et al., 1997). Furthermore, mecamylamine at low doses blocked the behavioral effects of DMXB, an α7-agonist (Meyer et al., 1997). In summary, mecamylamine does not seem to discriminate well between α4β2 and α7 nAChRs. However, significant differences in the sensitivity of mecamylamine between some other nAChRs (α4β2, α3β2, and α3β4 nAChRs, for example) were reported (Connolly et al., 1992;Chavez-Noriega et al., 1997). Although mecamylamine blockade of nicotine-induced seizures likely involves α7-receptors, other receptor subtypes have not been ruled out. Although our results do not eliminate the involvement of α4β2-receptors, they suggest that this receptor subtype does not play a major role in nicotine-induced seizures.

The present results indicate that dihydropyridine derivatives are able to modulate nicotine-induced seizures. Similar results were reported for other effects of nicotine, such as antinociception and hypomotility (Damaj et al., 1993; Damaj and Martin, 1993). The blockade of calcium channels by nifedipine and nimodipine decreased the potency of nicotine in the tail-flick test. On the contrary, BAY K 8644, a calcium channel agonist, potentiated the activity of nicotine on locomotor activity and tail-flick. Similar results are observed with nicotine-induced seizures. Nifedipine and nimodipine decreased the potency of nicotine and BAY K 8644 potentiated the activity of nicotine. Thus, alterations in intracellular Ca²⁺ concentration levels directly by allowing entry of calcium through the receptor or indirectly by activation of voltage-gated calcium channels have a profound influence on nicotine-induced seizures. The reason for the failure of verapamil, a phenylalkylamine calcium antagonist, to block nicotine-induced seizures is not clear. However, there are several differences between verapamil and nifedipine that may be relevant. Verapamil interacts at a site on calcium channels that is distinct from the dihydropyridine site at which nifedipine and BAY K 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 (Hitchott et al., 1992). Finally, Little (1991) found dihydropyridines superior to verapamil against ethanol withdrawal-induced seizures.

In the present study, the noncompetitive NMDA receptor antagonist MK-801 suppressed the development of nicotine-induced seizures. Similar results were also observed with the competitive NMDA antagonist (±)-3-(RS)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP). It should be noted that MK-801 was reported to inhibit α7-expressed nAChRs at high concentrations (Briggs and McKenna, 1996). However, to our knowledge there is no report of (±)-CPP-blocking nAChRs. This specificity strengthens the hypothesis of NMDA receptor involvement. In addition, riluzole, a glutamate release inhibitor, blocked nicotinic seizures in a dose-related manner. It was also reported that MK-801 and (±)-CPP attenuate the development of sensitization to the locomotor-stimulating effects of nicotine (Shoaib et al., 1994). These findings and those of the present study suggest that NMDA receptors are involved with the development of seizures caused by nicotine.

Our findings also suggest that NO formation is involved in the mechanisms underlying the development of seizures induced by nicotine. Indeed, the NO synthase inhibitor L-NAME prevented the development of seizures when administered before nicotine.L-NNA, a more potent NO synthase inhibitor, also inhibited nicotine-induced seizures at higher potency than L-NAME. In addition, the inactive isomer D-NNA did not inhibit the development of seizures. L-NAME not only inhibits NO synthase but also blocks muscarinic receptors (Buxton et al., 1993). However, it is unlikely that the muscarinic system is involved in the development of seizures, because nicotine-induced seizures was not antagonized by the muscarinic antagonist atropine. This notion is supported by our finding that the NO synthase inhibitorL-NNA, which is devoid of antimuscarinic activity (Buxton et al., 1993), attenuated the development of seizures.

In summary, a proposed model for the involvement of calcium and calcium-mediated events in nicotine-induced seizures is presented in Fig. 7. We hypothesize that the administration of nicotine either directly (nAChR permeable to calcium) or indirectly (membrane depolarization that opens L-type calcium channels, classified as voltage-dependent calcium channels) produces a rise in intracellular free calcium. This rise in intracellular calcium activates calcium-dependent events (calmodulin, calmodulin-dependent protein kinase, and so on), leading to the release of glutamate. The released glutamate can activate multiple postsynaptic receptors, of which the NMDA type is known to be involved in seizures processes. In addition, the influx of calcium through NMDA ion channels stimulates NO synthase to produce NO (Synder and Bredt, 1991). In other words, by acting at the presynaptic level, nicotine would enhance the release of glutamate, which in turn stimulates NMDA receptors and triggers the cascade of events leading to NO formation and seizure production. Another possibility is that the increase in intracellular calcium through nicotinic receptors is directly involved in the activation of NO synthase and the subsequent production of NO. This possibility was recently described in hippocampus when rats were administered nicotine peripherally (Fedele et al., 1998). Finally, the blockade of nicotine-induced seizures by diazepam and haloperidol, as previously reported (Aceto, 1975), suggests the involvement of other neurotransmitter receptors, such as γ-aminobutyric acid_A and dopamine receptors.

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Figure 7

Putative model by which calcium channels, NMDA receptors, and NO formation are involved in nicotine-induced seizures in mice. It is proposed that the activation of nicotinic receptors produces an increase in intracellular calcium directly (channel permeable to Ca²⁺) and/or indirectly [through voltage-dependent calcium channel (VGCC)]. This increase will enhance glutamate release, which in turn stimulates NMDA receptors, thus triggering the cascade of events leading to NO formation and seizures production. GABA, γ-aminobutyric acid.