Up-regulation of L-type voltage-dependent calcium channels after long term exposure to nicotine in cerebral cortical neurons.

Effects of long term (72-h) exposure to low concentration (0.1 mum) of nicotine on various types of voltage-dependent Ca(2+) channels (VDCCs) and neuronal nicotinic acetylcholine receptors (nnAChRs) were examined using primary cultures of mouse cerebral cortical neurons. High potassium (30 mm KCl)-stimulated (45)Ca(2+) influx into the neurons increased with increasing the duration of nicotine exposure and its concentrations. The maximal increase of the KCl-stimulated (45)Ca(2+) influx was found 24 h after the initiation of exposure and thereafter maintained up to 72 h. This enhancement of KCl-induced (45)Ca(2+) influx after 72-h exposure to 0.1 mum nicotine was completely abolished by concomitant exposure with mecamylamine, an inhibitor for nnAChRs. Only the component of the KCl-induced (45)Ca(2+) influx observed after long term exposure to nicotine, which was sensitive to nifedipine, an inhibitor of L-type VDCCs, was facilitated, while the (45)Ca(2+) influx through P/Q- and N-type VDCCs showed no changes. Moreover, enhanced immunoreactivity against antibody for the alpha(1C) subunit of L-type VDCCs was recognized, whereas no changes in immunoreactivities against antibodies for alpha(1A) and alpha(1B) subunits of other types of VDCCs were noted. In addition, a Western blot analysis showed an increase of immunoreactivities against antibodies for alpha(1D) and alpha(2)/delta(1), and expression of mRNA for L-type VDCC subunit, alpha(1F), was also enhanced, although beta(4) mRNA expression was not changed. Whole cell patch clamp analysis revealed that the increase of the amplitude of Ba(2+) currents was also recognized in the neurons exposed to nicotine, and nicardipine reduced this increased amplitude to the level of the amplitude detected in nontreated neurons with nicardipine. The up-regulation of alpha(4) and beta(2) subunits, but not the alpha(3) subunit of nnAChRs, was also noted after the nicotine exposure when examining by the Western blot analysis. Taken together, these results indicate that the long term exposure of the neurons to a low concentration of nicotine induces both increased (45)Ca(2+) influx through up-regulated L-type VDCCs and nnAChR up-regulation.

Neuronal nicotinic acetylcholine receptors (nnAChRs) 1 are ligand-gated cation channels activated by endogenous neurotransmitter acetylcholine (ACh) and exogenous drugs, such as nicotine (1,2). nnAChRs consist of five subunits (3), and expressed subunits of nnAChRs are numerous, which gives rise to the potential for enormous functional diversity of nnAChRs (2). nnAChRs in the central nervous system are a target of nicotine and mediate fast depolarization of neuronal membrane (4) to increase the permeability to Ca 2ϩ above that of their counterparts at the neuromuscular junction (5). This high permeability of neuronal membrane to Ca 2ϩ produces, in turn, many effects of nicotine in the central nervous system including memory facilitation, increase in spontaneous activity, convulsions, suppression of irritability and appetite, and stimulation of local glucose utilization (6,7). In addition, nnAChRs participate in alterations of central nervous functions through facilitating the release of several neurotransmitters, such as ACh, dopamine, ␥-aminobutyric acid, glutamate, and serotonin, by its activation, and this potential of nnAChRs to stimulate neurotransmitter release is suppressed by various inhibitors for voltagedependent calcium channels (VDCCs), suggesting that VDCCs play a critical role in neurotransmitter release evoked by nnAChR activation and that the opening of VDCCs via neuronal membrane depolarization is important for alterations in neuronal functions induced by nnAChRs (8). The latter suggestion is also, in part, supported by evidence that the expression of an endogenous neuroactive peptide, diazepam binding inhibitor (DBI), possessing the activity to cause anxiety (9), is enhanced by chronic stimulation of nnAChRs with low doses of nicotine in mouse cerebral cortex and primary cultures of mouse cerebral cortical neurons, and this stimulatory effect is mediated through L-type VDCCs (10).
The effects of both acute and chronic nicotine application on the activity of different nnAChR subtypes may be relevant to tolerance, dependence, and withdrawal syndrome associated with nicotine addiction (11,12). Chronic exposure to low doses of nicotine induces the up-regulation of high affinity ␣ 4 /␤ 2 subunit of nnAChRs possessing [ 3 H]nicotine binding sites in the neurons (13)(14)(15), although chronic nicotine exposure also leads to association with long lasting down-regulation in responsiveness to nicotine (16 -18). This up-regulation of nnAChRs is supposed to be a compensatory reaction against agonist-induced desensitization (16,19). From these data, it is reasonable to suppose that decreased function of nnAChRs after chronic exposure to low doses of nicotine is predominantly due to functional alterations of the ␣ 4 /␤ 2 subunit of nnAChRs.
In our previous reports, we have demonstrated that chronic exposure of cultured neurons to nicotine is reported to induce increase of 45 Ca 2ϩ influx when examining by the application of 30 mM KCl (10), which leads to an assumption that functional changes of nnAChRs after continuous activation may be accompanied with those of VDCCs functionally coupled to nnAChRs. However, pharmacological characteristics of such functional alterations in VDCCs have not been fully investigated, and few available investigations were reported. In the present study, we attempted to investigate this possibility by examining VDCC functions using mouse cerebral cortical neurons in primary culture after long term treatment with low doses of nicotine.

EXPERIMENTAL PROCEDURES
Isolation and Primary Culture of Mouse Cerebral Cortical Neurons-Isolation and primary culture of cerebral cortical neurons were carried out according to the method described previously (20). In brief, the neopallium free of the meninges was removed from the 15-day-old fetus of a ddY strain mouse under anesthesia with diethyl ether, minced with scissors, dispersed by trypsin, and centrifuged. Thereafter, the isolated cells were inoculated on a poly-L-lysine-treated culture dish with Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum and cultured at 37°C in humidified 95% air, 5% CO 2 for 3 days. After the treatment of the cells with 10 M cytosine arabinoside in Dulbecco's modified Eagle's medium containing 10% horse serum for 24 h to suppress the proliferation of nonneuronal cells, the culture medium was exchanged to Dulbecco's modified Eagle's medium with 10% horse serum, and the incubation was continued under the same conditions described above. The culture medium was exchanged to fresh Dulbecco's modified Eagle's medium with 10% horse serum every 4 days. More than 95% of the primary cultured cells used here have been confirmed to be neurons by an immunohistochemical approach (20).
Long Term Treatment of Nicotine-For studying the effects of long term exposure to nicotine on VDCC functions, nicotine was diluted with Hanks' solution and was directly added into the culture medium. To avoid the degradation of nicotine because of its high light sensitivity, the handling, such as the addition of nicotine into the medium and culture of cells with culture medium containing nicotine, was carried out under dark conditions. In addition, nicotine-containing culture medium was exchanged every day during nicotine exposure. Cells cultivated in the absence of nicotine were used as control. Mecamylamine (1 M) was added into the culture medium 5 min before the addition of nicotine.
Under the experimental conditions used here, the exposure of nicotine (0.1 M) to the neurons for 72 h showed no increase of lactic dehydrogenase leakage from the neurons and of the number of cells staining with trypan blue dye (data not shown), indicating that the exposure of nicotine with the concentration used in the present study was not neurotoxic.
To prepare nicotine-dependent mice, male ddY strain mice weighing 30 g purchased from Japan SLC (Hamamatsu, Japan) were used after feeding them laboratory chow (Oriental Yeast, Chiba, Japan) and tap water ad libitum for 1 week. Mice were subcutaneously injected nicotine (three times a day; each dose was 1 mg/kg) for 7 days. Four hours after the last dose of nicotine, mice were killed by decapitation, and the cerebral cortices were dissected to prepare the particulate fractions used for [ 3 H]nicotine and [ 3 H]verapamil bindings described below (21). Mice after continuous treatments with nicotine showed weight loss, increased defecation, and increased locomotor activity, indicating that mice are clearly dependent on nicotine.
Measurement of 45 Ca 2ϩ Influx- 45 Ca 2ϩ influx into the neurons was measured according to the previously reported method (22). In short, the neurons were incubated in Ca 2ϩ -free and 20 mM Hepes-containing Krebs-Ringer bicarbonate buffer (KRB-Hepes; 137 mM NaCl, 4.8 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 ⅐6H 2 O, 25 mM NaHCO 3 , and 10 mM glucose, pH 7.4) at 37°C for 10 min, and the incubation buffer was discarded to change to fresh and warm (37°C) Ca 2ϩ -free KRB-Hepes. The reaction was initiated by the addition of 2.7 mM CaCl 2 ⅐H 2 O (1.0 Ci of [ 45 Ca 2ϩ ]Cl 2 /dish). After the incubation of the neurons at 37°C for 2 min, the radiolabeled Ca 2ϩ -containing incubation buffer was discarded followed by five washes with ice-cold KRB-Hepes containing 2.7 mM CaCl 2 ⅐H 2 O (total volume 7.5 ml), and the neurons were scraped off from a culture dish with 0.5 M NaOH. An aliquot of the alkaline-digested neurons was neutralized with equimolar acetic acid and then used to measure radioactivity accumulated in the neurons by liquid scintillation spectrometry.
KCl (30 mM) and nicotine were simultaneously added into the incubation buffer with [ 45 Ca 2ϩ ]Cl 2 . The addition of Bay k 8644 was also simultaneously added with [ 45 Ca 2ϩ ]Cl 2 into the incubation buffer. To examine the effects of inhibitors for VDCCs on the 30 mM KCl-and nicotine-induced alterations in [ 45 Ca 2ϩ ]influx, these agents were added into the incubation buffer 15 s before the addition of 30 mM KCl, nicotine, and Bay k 8644.

Measurement of [ 3 H]Nicotine and [ 3 H]Verapamil
Binding-The receptor binding assay was carried out using the particulate fractions from the mouse cerebral cortical neurons. For the preparation of the particulate fractions, the neurons were washed three times with icecold phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 , pH 7.4), scraped off from the dishes with ice-cold 50 mM Tris-HCl buffer (pH 7.4) and homogenized with a Polytron homogenizer and centrifuged (48,000 ϫ g, 4°C, 20 min). The washing procedure was carried out four times before stocking at Ϫ80°C for at least 24 h. Before the binding experiment, the frozen pellet was thawed, suspended with the same buffer, and washed three times as described above.
Procedures similar to preparing the particulate fractions from the neurons were employed to prepare those of the cerebral cortex from the nicotine-dependent mice. The techniques for measuring bindings of [ 3 H]nicotine and [ 3 H]verapamil to the particulate fractions obtained from the cerebral cortex were similar to those for the particulate fractions of the neurons described below.
The [ 3 H]nicotine binding assay was carried out according to the method of Zhang and Nordberg (23) with a minor modification. Briefly, the particulate fractions were incubated with 50 mM Tris-HCl buffer (pH 8.0) containing 5 nM [ 3 H]nicotine in a final volume of 1 ml at 2°C for 60 min. The reaction was terminated by adding 2 ml of the ice-cold buffer, and the reaction mixture was filtered through Whatman GF/C glass filters presoaked with 0.3% polyethyleneimine solution for 5 h. The filters were washed three times with the ice-cold buffer and then subjected to measure radioactivity retained on the filters by liquid scintillation spectrometry. Nonspecific binding was determined in the presence of 1 M cystine. For Scatchard analysis, the particulate fractions were incubated with various concentrations of [ 3 H]nicotine (0.2-20 nM) under the same conditions described above.
The [ 3 H]verapamil binding assay was carried out according to the method previously reported (24) with minor modifications. Briefly, the particulate fractions were incubated in 50 mM Tris-HCl buffer (pH 7.4) containing 0.2 nM [ 3 H]verapamil in a final volume of 1 ml at 2°C for 60 min. The reaction was terminated by adding 2 ml of the ice-cold buffer into the assay system, and the reaction mixture was then filtered through Whatman GF/B glass filters presoaked with 0.3% polyethyleneimine solution for 5 h. The filters were washed three times with the ice-cold buffer and then subjected to measurement of radioactivity retained on the filters by liquid scintillation spectrometry. Nonspecific binding was determined in the presence of 10 M nonlabeled verapamil. For Scatchard analysis, the particulate fractions were incubated with various concentrations of [ 3 H]verapamil (0.01-8 nM) under the same conditions described above.
Protein Electrophoresis and Immunoblots for nnAChRs and VD-CCs-The extraction of protein from the neurons was carried out as follows. The neurons attached to a culture dish were washed five times with ice-cold 0.15 M NaCl and fixed with 0.15 M NaCl containing 6% trichloroacetic acid at 4°C. The neurons were scraped off from the dishes and centrifuged (10,000 ϫ g, 5 min, 4°C). After washing the pellet with ice-cold 50 mM Tris-HCl buffer (pH 7.5), the sample buffer (100 mM Tris-HCl (pH 6.8) containing 4% sodium lauryl sulfate, 12% ␤-mercaptoethanol, and 20% glycerol) was added to the pellet in a final volume of 0.2 ml and well mixed. The suspension thus obtained was sonicated (1 min), boiled (3 min), and centrifuged (10,000 ϫ g, 60 min, 4°C), and the supernatant was stored at Ϫ80°C until use.
The relative intensity of immunoreactive bands for each subunit for nnAChRs and VDCCs was calculated using ImageMaster 1D Elite software (Amersham Biosciences), and the data were estimated as percentages of each control (without treatment of nicotine).
Amplification of cDNA for ␣ 1F and ␤ 4 Subunits-cDNA for ␣ 1F and ␤ 4 subunits of L-type VDCCs was amplified from mouse brain cDNA by polymerase chain reaction using a set of oligonucleotides (␣ 1F , TTC-GACTCTTGTTGGGTCTTG and TCAAAGCGGGAAAGAATAGA; ␤ 4 , CTATAAACTCTCATCATTTCAC and TCATAACGGGTTGCACATAC) specific for the cDNA sequence of mouse ␣ 1F and ␤ 4 subunits of L-type VDCC (26,27). After the amplification, the cDNA was ligated with EcoRI adapter and inserted into the EcoRI site of pUC18.
Purification of mRNA and RNA Blot Hybridization-After washing the neurons with ice-cold phosphate-buffered saline (pH 7.4), the neurons were scraped off from a culture dish. Poly(A) ϩ RNAs were isolated from these cells using FASTTrack™ (10). The lysates obtained from the neurons were applied to oligo(dT)-cellulose affinity chromatography to purify poly(A) ϩ RNA.
RNA blot hybridization was performed as previously described (10). Denatured poly(A) ϩ RNA (1 g) was electrophoresed on 1.1% agarose gel containing formaldehyde. Gels were stained with ethidium bromide and photographed under UV illumination to assess the integrity of the RNA by visual inspection. RNA was transferred to a nitrocellulose membrane (NitroPure; Osminics Inc., Westborough, MA) using 20ϫ standard saline citrate (SSC) buffer by the capillary blotting method, and the completeness of transfer was confirmed by examining gels for ethidium fluorescence. The cDNA fragment from mouse brain was labeled by the Klenow fragment with random primer and [␣-32 P]dCTP. Antisense oligomer (AGGTCTCAAACATGATCTGGGTCA) for ␤-actin (28) prepared by an Applied Biosystems DNA synthesizer was endlabeled by terminal deoxynucleotidyl transferase with [␣-32 P]dCTP. These denatured DNA were used as probes in the hybridization. The baked filter was hybridized with 50 mM sodium phosphate buffer (pH 7.0), 5ϫ SSC, 50 mg/ml salmon sperm DNA, 1ϫ Denhardt's solution, 30% formamide, 10% dextran sulfate, and 32 P-labeled probe at 42°C for 24 h. The filters were washed with 0.2ϫ SSC and 0.1% sodium lauryl sulfate at 50°C and autoradiographed. The radioactive intensities of the bands were represented as arbitrary units determined by a Fujix BAS 2000 System (Fuji-Film Co., Ltd., Tokyo, Japan).
The contents of poly(A) ϩ RNAs isolated from the mouse cerebral cortical neurons after the treatments with nicotine were similar to those from the neurons not treated with nicotine (data not shown).
In the present study, the levels of mRNA for subunits of L-type VDCCs were corrected according to the message of ␤-actin signal, and the levels of ␤-actin mRNA in the nicotine-treated neurons were the same as those in the nontreated neurons with the drugs.
Electrophysiological Experiments-The whole-cell configuration of the patch clamp technique was used to perform electrophysiological experiments according to the method (29) previously reported with a minor modification. Patch pipettes were made from a barosilicate glass tube (1.5-mm outer diameter, 0.86-mm inner diameter; A-M System Inc., Pangbourne, UK) in a micropipette puller (model 87; Sutter Instruments). Pipette resistances ranged from 5 to 8 megaohms when filled with a solution (pH 7.4; 117 mM tetraethylammonium chloride, 9 mM Hepes, 9 mM EGTA, 4.5 mM MgCl 2 , 1 mM GTP, 4 mM ATP, and 14 mM phosphocreatine; osmolarity 330 mosmol/liter). Whole-cell currents were amplified with a patch clamp amplifier system (EPC-8; HEKA Elektronik), filtered at 3 kHz, and digitized at 10 kHz with a Digidata 1200A acquisition board (Axon Instruments, Foster City, CA) for subsequent storage on a personal computer. The data acquisition and analysis were carried out with the pClamp 8.0 software package (Axon Instruments, Foster City, CA). The external solution consisted of 25 mM BaCl 2 , 145 mM tetraethylammonium chloride, 10 mM Hepes, 0.1 mM EGTA, and 10 mM D-glucose (pH 7.4; osmolarity 340 mosmol/liter adjusted with sucrose). Leakage currents were subtracted using the P/4 protocol, and the series resistance was compensated by 50 -60%.
Measurement of Protein Content-The protein content in the neurons was determined by the method of Lowry et al. (30) with bovine serum albumin as a standard.
Statistical Analysis-Each value of the data was expressed as the mean Ϯ S.E. The statistical significance was assessed by the methods as described in each legend of figures following the application of the one-way analysis of variance.
Chemicals-PerkinElmer Life Sciences was a source of 45 CaCl 2 (specific activity 0.3511 GBq/mg), [ 3 H]nicotine (specific activity 3.02 TBq/ mmol), and [ 3 H]verapamil (specific activity 2.81 TBq/mmol). Antibodies against ␣ 3 , ␣ 4 , and ␤ 2 subunits of nnAChRs and anti-goat IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and antibodies against VDCCs were products of Almone Labs Ltd. (Jerusalem, Israel). Anti-rabbit IgG as a part of picoBlue™ immunoscreening kit was obtained from Stratagene. Nifedipine was obtained from Wako Pure Chemical Industries (Osaka, Japan). -Conotoxin GIVA (-CTX) and -agatoxin VIA (-ATX) were purchased from the Peptide Institute, Inc. (Osaka, Japan). Gradient gels (5/20%) were products of Atto Co. Ltd. (Tokyo, Japan). Other reagents used were commercially available and of analytical grade. 45 Ca 2ϩ Influx into the Neurons-The effects of the duration of nicotine (0.1 M) exposure on 30 mM KCl-evoked 45 Ca 2ϩ influx were examined. The increase in 30 mM KClinduced 45 Ca 2ϩ influx was found to be dependent on the duration of exposure to 0.1 M nicotine (Fig. 1A). The influx attained its plateau 24 h after the exposure, and a similar extent of influx was thereafter maintained up to 72 h (Fig. 1A). On the other hand, the basal 45 Ca 2ϩ influx was not altered during continuous exposure to nicotine (Fig. 1A).

Effect of Long Term Exposure to Nicotine on 30 mM KClinduced
In addition, we examined effects of 72-h exposure to various concentrations of nicotine on 30 mM KCl-evoked 45 Ca 2ϩ influx. As shown in Fig. 1B, the increase of the 30 mM KCl-induced 45 Ca 2ϩ influx dependent on nicotine concentration was noticed, and its plateau was observed at nicotine concentrations of 0.1-1 M, although a further remarkable increase of 30 mM KCl-evoked 45 Ca 2ϩ influx was detected after 72-h exposure to 3 M nicotine. The latter increase was considered to be due to injury of neuronal membrane function, because leakage lactic dehydrogenase activity was significantly larger than that of nontreated neurons. Moreover, the leakage of lactic dehydrogenase activity tends to increase, but not significantly, after 72-h exposure to 1 M nicotine (data not shown). Taking these data together, we employed 0.1 M nicotine for long term exposure of neurons in the following experiments.
Whether the significant increase of the 30mM KCl-induced 45 Ca 2ϩ influx into the neurons after exposure to 0.1 M nicotine for 72 h was attributable to continuous nnAChR activation by nicotine was examined by measuring 30 mM KCl-stimulated 45 Ca 2ϩ influx after concomitant exposure of the neurons to nicotine and mecamylamine, an antagonist specific for nnAChRs. As shown in Fig. 1C, this manipulation completely abolished the increase of the KCl-induced 45 Ca 2ϩ influx. These results indicate that the increased influx by 30 mM KCl after 72-h exposure to 0.1 M nicotine is mediated through nnAChR activation. Such data also suggest that the increased extent of 30 mM KCl-induced 45 Ca 2ϩ influx found after 72-h exposure to 0.1 M nicotine may be due to functional up-regulation of VD-CCs, because 30 mM KCl is known to produce neuronal membrane depolarization, and the 45 Ca 2ϩ influx observed in the presence of 30 mM KCl occurs through opening VDCCs in the neurons used here as reported previously (22).
Effects of VDCC Inhibitors on 30 mM KCl-induced 45 Ca 2ϩ Influx after 72-h Exposure to 0.1 M Nicotine-In a previous experiment, the neurons used in the present study have been determined to possess at least four types of VDCCs (22). Therefore, which type of VDCC was involved in the enhanced increase of 30 mM KCl-induced 45 Ca 2ϩ influx after the long term (72-h) exposure to 0.1 M nicotine was examined. The concentration of each VDCC inhibitor used in this experiment was confirmed to show their maximal inhibitory effects on the 30 mM KCl-induced 45 Ca 2ϩ influx into the neurons, and there was a significant difference in the degree of inhibitory actions of -ATX on the KCl-induced 45 Ca 2ϩ influx at concentrations between 0.1 and 1 M (22), since previous reports have revealed that -ATX at concentrations lower than 200 nM suppresses only P-type VDCCs and at higher concentrations inhibits both P-and Q-type VDCCs (31)(32)(33)(34). Therefore, we conclude that both P-and Q-type VDCCs are present in the neurons.
Thirty millimolar KCl-increased 45 Ca 2ϩ influx was significantly suppressed by nifedipine (1 M), -CTX (1 M), and -ATX (1 M), inhibitors for L-, N-, and P/Q-type VDCCs, respectively (Fig. 2), which is in good agreement with the data previously reported by our laboratory (22). The inhibitory ratio by nifedipine, -CTX, and -ATX was about 40, 25, and 35%, respectively (Fig. 2). In the neurons exposed to 0.1 M nicotine for 72 h, these VDCC inhibitors significantly reduced the KClinduced 45 Ca 2ϩ influx as in the case of nontreated neurons (Fig.  2). It was noted that the extent of inhibition of the KCl-induced 45 Ca 2ϩ influx by -CTX in nicotine-exposed neurons (1717 Ϯ 108 dpm/mg of protein/2 min, n ϭ 4) was as same as that in nontreated neurons (1633 Ϯ 159 dpm/mg of protein/2 min, n ϭ 4). Similarly, no significant differences in the inhibitory degrees by -ATX of the KCl-induced 45 Ca 2ϩ influx were noticed between nontreated and nicotine-exposed neurons (nontreated: 1895 Ϯ 118 dpm/mg of protein/2 min, n ϭ 4; nicotine-exposed: 2019 Ϯ 123 dpm/mg of protein/2 min, n ϭ 4). On the other hand, nifedipine reduced the KCl-induced 45 Ca 2ϩ influx into the nicotine-treated neurons to the level of the KCl-induced 45 Ca 2ϩ influx into the nontreated neurons observed in the presence of nifedipine (Fig. 2). These results suggest that the increase of the KCl-induced 45 Ca 2ϩ influx in the neurons exp Ͻ 0.01, compared with the control value (without treatment of nicotine, Dunnett's test). C, effect of mecamylamine on 30 mM KCl-stimulated 45 Ca 2ϩ influx. The neurons were cultured with nicotine and mecamylamine at 37°C for 72 h. **, p Ͻ 0.01, Bonferroni's test. Effect of 72-h Exposure to Nicotine on Bay k 8644-induced 45 Ca 2ϩ Influx-As described above, the present study demonstrated the possibility that the long term exposure to low concentration (0.1 M) of nicotine causes the enhancement of Ltype VDCC function. To confirm this possibility, we examined effect of the long term (72-h) exposure to nicotine on 45 Ca 2ϩ influx stimulated by Bay k 8644, an activator specific for L-type VDCCs (35). In the neurons without nicotine treatment, Bay k 8644 dose-dependently increased the entry of 45 Ca 2ϩ into the neurons, and the maximal stimulatory effect was obtained at 1-10 M. The maximal extent of 45 Ca 2ϩ influx evoked by Bay k 8644 was about 40% of the 30 mM KCl-induced 45 Ca 2ϩ influx (Fig. 3), which is in agreement with the extent of reduction of the 30 mM KCl-induced 45 Ca 2ϩ influx by nifedipine (Fig. 2).
In the case of the neurons exposed to 0.1 M nicotine for 72 h, the 45 Ca 2ϩ influx induced by 10 M Bay k 8644 was significantly larger than that into the nontreated neurons, and this increased influx of 45 Ca 2ϩ was inhibited by nifedipine in a dose-dependent manner (Fig. 3), indicating that the continuous exposure to nicotine certainly enhances 45 Ca 2ϩ influx via Ltype VDCCs.
Changes of L-type VDCCs after Long Term Exposure to Nicotine-To investigate mechanisms for the changes of L-type VDCC function accompanying with continuous activation of nnAChRs by nicotine, the Scatchard analysis on [ 3 H]verapamil binding to the particulate fractions prepared from the neurons exposed to nicotine was carried out. The binding of [ 3 H]verapamil is saturable, and only high affinity binding sites were recognized in the preparations obtained from both nicotinetreated and nontreated neurons (data not shown). In addition, the long term exposure to 0.1 M nicotine significantly increased the B max value with no changes in the K d value (Table   I). This increase of [ 3 H]verapamil binding sites was not observed when incubating the neurons with both nicotine and mecamylamine (Table I).
The immunoblotting analysis was carried out to confirm the up-regulation of L-type VDCCs in association with continuous activation of nnAChRs with nicotine. The immunoreactivity against the ␣ 1C subunit of L-type VDCCs in membrane proteins obtained from the nicotine-exposed neurons was found at the band with a molecular mass of 240 kDa, and this reactivity was significantly higher than that from the nontreated neurons, whereas no significant alterations in expressions of immunoreactivities against ␣ 1A and ␣ 1B subunits of P/Q-and N-type VDCCs, respectively (Fig. 4A). Similarly, significant increase in expressions of ␣ 1D and ␣ 2 /␦ 1 subunits of L-type VDCCs was observed in the neurons exposed to nicotine for 72 h (Fig. 4B). In addition, we also checked expressions of mRNAs for ␣ 1F and ␤ 4 subunits of L-type VDCCs. As shown in Fig. 4C, a Northern blot analysis revealed increased expression of ␣ 1F subunit mRNA, whereas expression of mRNA for the ␤ 4 subunit of L-type VDCCs did not show any changes after the exposure of the neurons to nicotine for 72 h. These data certainly establish that the up-regulation of L-type VDCCs following the long term exposure of the neurons to a low concentration of nicotine occurs.
Effect of Long Term Exposure to Nicotine on Ba 2ϩ Currents-Ba 2ϩ currents evoked by depolarizing voltage steps in increments of 5 mV ranging from Ϫ80 to ϩ50 mV and maximum currents were obtained at 0 mV in both nicotine-treated and nontreated neurons (Fig. 5, A-C). The peak Ba 2ϩ current amplitudes in membrane potentials between Ϫ25 and ϩ35 mV in the nicotine-treated neurons were significantly greater than those in the nontreated neurons (Fig. 5C). The maximum current amplitudes at 0 mV were Ϫ268.3 Ϯ 27.2 pA (n ϭ 18) and Ϫ124.9 Ϯ 27.2 pA (n ϭ 17) in the nicotine-treated and nontreated neurons, respectively. There were no significant differences in size (diameter) or capacitance in the cerebral cortical neurons between the nicotine-treated and nontreated groups.
Effect of Nicardipine on Voltage-dependent Ba 2ϩ Currents after Long Term Exposure to Nicotine-To evoke Ba 2ϩ currents, 100-ms voltage steps to Ϫ20 mV from a holding potential of Ϫ90 mV in both nicotine-treated and nontreated neurons. Nicardipine (1 M), an selective antagonist for L-type VDCCs, was applied to the bath for 2 min. In the nontreated neurons, 1 ]Cl 2 /dish) were simultaneously added into the incubation buffer, and the neurons were incubated at 37°C for 2 min. The reaction was terminated by aspiration of the incubation buffer followed by five washes of the neurons with ice-cold incubation buffer containing 2.7 mM CaCl 2 , and the 45 Ca 2ϩ influx was measured as described in the legend to Fig. 1  M nicardipine significantly reduced the peak amplitude of Ba 2ϩ currents from 98.3 Ϯ 10.9 pA (n ϭ 24) to 50.7 Ϯ 6.9 pA (n ϭ 24) in the neurons without the exposure to nicotine (p Ͻ 0.01, Bonferroni's test) (Fig. 6, A and B). Moreover, the peak amplitude of Ba 2ϩ currents in the neurons exposed to nicotine for 72 h was significantly reduced by 1 M nicardipine (without nicardipine, 160.3 Ϯ 21.1 pA, n ϭ 18; with nicardipine, 69.6 Ϯ 8.5 pA, n ϭ 24; p Ͻ 0.01 by Bonferroni's test) (Fig. 6, A and C).
In the presence of nicardipine, Ba 2ϩ currents in the both types of neurons were at similar level (Fig. 6A). These results indicate that increased Ba 2ϩ current found in the neurons after chronic exposure to nicotine is produced by up-regulated L-type VDCCs.
Changes of Pharmacological Characteristics of nnAChRs after Long Term Exposure to Nicotine-Previous studies have revealed that continuous activation of nnAChR with nicotine produces an increase in nnAChR number with desensitization (19, 36 -39). Therefore, whether such modification of pharmacological characteristics occurs in the neurons exposed to 0.1 M nicotine for 72 h was investigated by measuring [ 3 H]nicotine to the particulate fractions prepared from the neurons exposed to nicotine. The Scatchard analysis revealed that the long term exposure to nicotine caused the increased B max value with no changes of K d value (Table I).
The expressions of ␣ 3 , ␣ 4 , and ␤ 2 subunits of nnAChRs were also examined in the nicotine-exposed neurons using Western blot analysis. As shown in Fig. 7, significant increased expression of ␣ 4 and ␤ 2 subunits was observed, whereas the expression of ␣ 3 subunit did not show any changes. Based on the data obtained by the Scatchard and the Western blot analyses, the up-regulation of nnAChRs consisting of ␣ 4 and ␤ 2 subunits certainly appeared after the long term exposure to 0.1 M nicotine.
As mentioned above, the conditions in which nnAChRs were continuously activated produce the reduction in nnAChR functions, although the numbers of nnAChRs increased (19, 36 -39). Accordingly, in the following experiments, we checked whether the alterations in the nicotine-induced 45 Ca 2ϩ influx into the neurons alter the exposure to nicotine for 72 h. In a preliminary experiment, 1 M nicotine linearly stimulated 45 Ca 2ϩ influx into the nontreated neurons up to 3 min and also nicotine dose-dependently increased 45 Ca 2ϩ influx for 2 min. The maximal stimulation was found at 1-3 M, which was completely inhibited by 0.1 M mecamylamine (data not sonicated, boiled, and centrifuged. An aliquot of the resultant supernatant was used for SDS-PAGE electrophoresis using a 5/20% gradient gel. The separated proteins were transferred onto a nitrocellulose filter. Thereafter, the nitrocellulose filter was incubated at 4°C overnight with antibodies against each subunit of VDCCs as indicated in the figure. The separated antigenic proteins were stained with anti-gout IgG antibody conjugated with alkaline phosphatase. Representative immunoblotting analysis was indicated in each image. Each value represents the mean Ϯ S.E. obtained from four separate experiments run in triplicate. **, p Ͻ 0.01, compared with each control value (without treatment of nicotine, Bonferroni's test). Cont, control (nontreated neurons); Nic, nicotine-treated neurons. L, N, and P/Q represent L-, N-, and P/Q-type VDCCs, respectively. C, expression of mRNA for subunits (␣ 1F and ␤ 4 of L-type VDCCs) after nicotine exposure for 3 days. The neurons were cultured with 0.1 M nicotine at 37°C for 72 h, and poly(A) ϩ RNA was isolated. Denatured poly(A) ϩ RNA was electrophoresed on 1.1% agarose gel. Separated mRNA was transferred to a nitrocellulose membrane and hybridized with 32 P-labeled oligonucleotides for ␣ 1F and ␤ 4 . The radioactive intensities of the band for each subunit mRNA were represented as arbitrary units determined by the Fujix BAS 2000 system. Each value represents the mean Ϯ S.E. obtained from four separate experiments run in triplicate. **, p Ͻ 0.01, compared with each control value (without treatment of nicotine, Bonferroni's test). Cont, control (nontreated neurons); Nic, nicotine-treated neurons. L, L-type VDCCs.

FIG. 4. Effect of nicotine exposure on expression of VDCC subunits in cerebral cortical neurons.
A and B, expression of proteins of VDCC subunits after nicotine exposure for 3 days. The neurons were cultured with 0.1 M nicotine at 37°C for 72 h, and the expressed proteins were measured as described below. After the neurons were fixed with 6% trichloroacetic acid in 0.15 M NaCl, they were centrifuged. The resultant pellet was mixed with the buffer described in the text, shown). Based on these results obtained from the preliminary experiment, we used 1 M nicotine and 2 min as the concentration of nicotine and the reaction time to measure 45 Ca 2ϩ influx in the presence of nicotine, respectively. Fig. 8 shows that 1 M nicotine increases 45 Ca 2ϩ influx into the neurons as well as 30 mM KCl does in the neurons exposed to 0.1 M nicotine for 72 h (Fig. 2). In addition, the inhibitory pattern of 1 M nicotine-induced 45 Ca 2ϩ influx by various inhibitors for VDCCs was the same as that of 30 mM KCl-induced 45 Ca 2ϩ influx. These data therefore indicate that the potential of nnAChRs to induce 45 Ca 2ϩ influx into the neurons is maintained even after its chronic exposure of nicotine inducing nnAChR up-regulation.

Changes in [ 3 H]Nicotine and [ 3 H]Verapamil Binding Prepared from the Cerebral Cortices of Nicotine-dependent
Mice-In the present study, whether long term treatment with nicotine produces up-regulation of L-type VDCCs and nnAChRs has been examined by measuring [ 3 H]verapamil and [ 3 H]nicotine binding, respectively, to the particulate fractions derived from nicotine-dependent mice. Fig. 9 shows an increase of [ 3 H]verapamil binding to the particulate fractions prepared from the cerebral cortices of nicotine-dependent mice. Similarly, [ 3 H]nicotine binding was found to increase. These results indicated that the up-regulation of [ 3 H]verapamil binding as well as that of [ 3 H]nicotine binding occurs in the cerebral cortices of nicotine-dependent mice. DISCUSSION In the present study, we have attempted to clarify mechanisms for behavioral alterations in Ca 2ϩ entry observed after long term exposure to low concentrations of nicotine using a primary culture of mouse cerebral cortical neurons possessing nnAChRs and VDCCs (22,40), because our previous report reveals that long term exposure of the neurons to nicotine FIG. 5. Calcium current-voltage relationship in cerebral cortical neurons exposed to nicotine. The neurons were exposed to 0.1 M nicotine at 37°C for 72 h. Superimposed traces of high voltage-gated Ca 2ϩ channel currents detected in nontreated neurons (control) (A) and neurons exposed to nicotine (B) are shown. The currents were elicited by 100-ms voltage steps in the range of Ϫ80 to 50 mV from a holding potential of Ϫ90 mV at a 5-s interval. C, peak calcium current-voltage relationship was obtained by plotting the peak current amplitudes in the control neurons (open circle) and nicotine-treated neurons (filled circle) as a function of the test pulse potential. Each value shown in C represents the mean Ϯ S.E. obtained from 17-18 separate cells. *, p Ͻ 0.05; **, p Ͻ 0.01, compared with each control value (Bonferroni's test).

FIG. 6. Effect of nicardipine on high voltage-gated Ca 2؉ channel currents.
The neurons were exposed to 0.1 M nicotine at 37°C for 72 h. A, superimposed trace of time course of effect of nicardipine on high voltage-gated Ca 2ϩ channel currents. Nicardipine (1 M) was applied for 2 min while currents were evoked by 100-ms step potentials to Ϫ20 mV from a holding potential of Ϫ90 mV at a 10-s interval. Each value represents the mean Ϯ S.E. obtained from 19 -24 separate neurons. During the experiments, currents were measured for 60 s after the initiation of the experiments and then measured in the presence of nicardipine (1 M). Thereafter, currents were recorded in the absence of nicardipine. B and C, effects of nicardipine (1 M) on high voltage-gated Ca 2ϩ channel currents in nontreated (control) (B) and nicotinetreated (C) neurons. In B and C, control and washout represent the phases before the application of nicardipine and after nicardipine was washed out from the incubation buffer, respectively. enhances the expression of DBI, an endogenous anxiogenic peptide with a pharmacological property as an inverse agonist for benzodiazepine receptors associated with the increase in Ca 2ϩ influx (10).
As shown in this study, high potassium (30 mM KCl)-evoked 45 Ca 2ϩ influx into the neurons was dependent on the duration of nicotine exposure and the nicotine concentration. This enhancement of the 30 mM KCl-induced 45 Ca 2ϩ influx after long term exposure to nicotine is considered to be mediated through continuous interaction between nnAChRs and nicotine, which is supported by the inhibition of the influx with mecamylamine, an antagonist for nnAChRs. In addition, continuous exposure of nnAChRs to nicotine for less than 6 h is demonstrated not to cause an increase in the KCl-induced 45 Ca 2ϩ influx as demonstrated in this study.
In neurons located in the central nervous system, at least four types of VDCCs (i.e. P-, Q-, N-, and L-type) are present (41,42). High potassium stimulation is well known to depolarize the neuronal membrane. Therefore, the increase of 30 mM KCl-induced 45 Ca 2ϩ influx in the neurons exposed to nicotine for the long term may be due to functional acceleration of any and/or all types of VDCCs. On the other hand, the neurons used here have all types of VDCCs mentioned above when examining inhibitory effects of VDCC inhibitors on 45 Ca 2ϩ influx in response to 30 mM KCl stimulation (22). From the results obtained in the experiments measuring the 45 Ca 2ϩ influx in the presence of various inhibitors for VDCCs, it is indicated that the portion of increased 45 Ca 2ϩ influx after the long term exposure to nicotine is attributed to only the increase in the component of 45 Ca 2ϩ influx inhibited by nifedipine. The 45 Ca 2ϩ influx into the neurons induced by an activator specific for L-type VDCCs, Bay k 8644, was also significantly facilitated after chronic nicotine exposure. These data on 45 Ca 2ϩ influx suggest that this increased 45 Ca 2ϩ influx produced by the long term exposure to nicotine is consequent to functional enhancement of L-type VDCCs.
The possibility of up-regulation of L-type VDCCs after continuous nnAChR activation was further explored to examine whether expression of the protein molecule of L-type VDCCs increases by a binding assay and a Western blotting analysis. The binding sites for [ 3 H]verapamil in the particulate fractions obtained from the neurons both treated with and without nicotine consisted of those with only high affinity. Previous investigations have also reported that only a high affinity site of dihydropyridine binding is present in the brain (43)(44)(45). In addition, the present study demonstrated that the exposure to nicotine for the long term significantly increased B max value with no alterations in the K d value of [ 3 H]verapamil binding. Furthermore, the Western and Northern blot analyses revealed significant increase of ␣ 1C , ␣ 1D , ␣ 1F , and ␣ 2 /␦ 1 subunits of L-type VDCCs without any quantitative alterations in ␣ 1A , ␣ 1B , and ␤ 4 subunits of P/Q-, N-, and L-type VDCCs, respectively. These results indicate that the enhancement of 45 Ca 2ϩ influx induced after long term exposure to nicotine is caused by an increased number of ␣ 1 and ␣ 2 /␦ 1 subunits of L-type VDCC molecules.
To further confirm the up-regulated function of L-type VD-CCs, electrophysiological examinations were also carried out. Whole-cell patch cramp analysis showed that the amplitude of Ba 2ϩ currents in the nicotine-exposed neurons was signifi- FIG. 7. Effect of nicotine exposure on expression of nnAChR subunits in cerebral cortical neurons. The neurons were cultured with 0.1 M nicotine at 37°C for 72 h, and the expressed proteins were measured as described below. After the neurons were washed with 0.15 M NaCl, they were fixed with 6% trichloroacetic acid in 0.15 M NaCl. The neurons were centrifuged at 10,000 ϫ g at 4°C for 5 min, and the pellet was mixed with 100 mM Tris-HCl buffer containing 4% sodium lauryl sulfate, 12% ␤-mercaptoethanol, and 20% glycerol, sonicated, boiled, and centrifuged (10,000 ϫ g, 60 min, 4°C). An aliquot of the resultant supernatant was used for SDS-PAGE electrophoresis using a 5/20% gradient gel. The separated proteins were transferred onto a nitrocellulose filter, and the filter was blocked with 20 mM Tris-HCl buffer containing 0.15 M NaCl and 1% bovine serum albumin at room temperature. Thereafter, the nitrocellulose filter was incubated at 4°C overnight with antibodies against each subunit of nnAChR as indicated in the figure. The filter was then washed with 20 mM Tris-HCl buffer containing 0.15 M NaCl and 0.05% Tween 20, and the separated antigenic proteins were stained with anti-rabbit IgG antibody conjugated with alkaline phosphatase. Representative immunoblotting analysis is indicated in each image. Each value represents the mean Ϯ S.E. obtained from four separate experiments run in triplicate. **, p Ͻ 0.01, compared with each control value (without treatment of nicotine, Bonferroni's test). Cont, control (nontreated neurons); Nic, nicotine-treated neurons. 45 Ca 2؉ influx into cerebral cortical neurons in primary culture following exposure to nicotine. The neurons were cultured with 0.1 M nicotine at 37°C for 72 h. After the incubation of the neurons in Ca 2ϩ -free incubation buffer for 10 min, the buffer was exchanged to fresh Ca 2ϩ -free incubation buffer, and nicotine and [ 45 Ca 2ϩ ]Cl 2 (1.0 Ci of [ 45 Ca 2ϩ ]Cl 2 /dish) were simultaneously added into the incubation buffer. The neurons were thereafter incubated at 37°C for 2 min. The reaction was terminated by aspiration of the incubation buffer followed by five washes of the neurons with ice-cold incubation buffer containing 2.7 mM CaCl 2 , and the 45 Ca 2ϩ influx was measured as described in the legend to Fig. 1. The neurons were digested with 0.5 M NaOH, and an aliquot of the alkaline-digested neurons was subjected to measure radioactivity following neutralization with equimolar acetic acid. To examine effects of inhibitors for VDCCs, each inhibitor was added into the incubation buffer 15 s before the simultaneous addition of nicotine and cantly higher than those in nontreated neurons and that this increase was completely abolished in the presence of nicardipine. In addition, these Ba 2ϩ currents in the control and nicotine-exposed neurons are initiated at the membrane potentials at Ϫ60 and Ϫ50 mV. These electrophysiological data indicate that the increased amplitude of Ba 2ϩ currents in the nicotineexposed neurons is mediated through an increase in L-type VDCCs. Accordingly, it has been confirmed that the up-regulation of L-type VDCCs is induced by long term exposure to nicotine.

FIG. 8. Effects of VDCC inhibitors on 1 M nicotine-stimulated
Nicotine is a drug of abuse, and its chronic administration mainly by smoking produces physical dependence. In patients suffering from nicotine dependence, withdrawal of nicotine produces withdrawal syndrome such as anxiety, irritability, disturbance of sleep, tremors, and convulsions. Similarly, other drugs of abuse including ethanol, morphine, and benzodiazepines cause physical dependence, and most of the withdrawal syndrome resemble those in patients with nicotine dependence. Although mechanisms for the formation of drug dependence and emergence of withdrawal syndrome have not adequately been clarified, recent investigations suggest that disturbance of Ca 2ϩ homeostasis in neurons of the central nervous system, especially abnormal influx of Ca 2ϩ into neurons, is partially involved in these phenomena observed in patients with drug dependence (46). In addition, such enhanced increase of Ca 2ϩ entry is reported to be due to up-regulation of L-type VDCCs under the conditions of drug dependence by ethanol, morphine, and benzodiazepine (46 -49), although in the case of nicotine little available data on these events has been reported, and the involvement of P/Q-and N-type VDCCs in these pathophysiological conditions is controversial (46). As demonstrated in the present investigation, the up-regulation of L-type VDCCs formed after long term exposure to nicotine, therefore, may participate in the formation of nicotine dependence and/or the emergence of withdrawal syndrome as in the cases of other drugs of abuse.
There are few available data on up-regulation of L-type VD-CCs in the brains of nicotine-dependent animals. As the present report demonstrates, the up-regulation of L-type VDCCs in the cerebral cortical neurons exposed to nicotine for the long term, whether or not similar alterations in the L-type VDCC function occur in cerebral cortex of nicotine-dependent mice, was examined using the cerebral cortices of nicotine-dependent mice. We employed here the binding assay methods using [ 3 H]verapamil and [ 3 H]nicotine to measure functional alterations in L-type VDCCs and nnAChR, respectively. The binding assay revealed an increase in both [ 3 H]verapamil and [ 3 H]nicotine binding, which indicates that functional increase of L-type VDCCs and nnACh receptors, respectively, is induced in the cerebral cortex under nicotine-dependent conditions. The latter is certified by the increase of [ 3 H]nicotine binding to the particulatefractionsderivedfromthecerebralcorticesofnicotinedependent mice, because previous investigations have demonstrated the increased binding of [ 3 H]nicotine to brain preparations from animals treated with nicotine (38,39). Therefore, the up-regulation of L-type VDCCs in the cerebral cortical neurons induced by long term exposure to nicotine is considered to occur in the brain of nicotine-dependent animals.
Nicotine is a potent factor to stimulate release of several neurotransmitters including ACh, dopamine, serotonin, ␥-aminobutyric acid, and glutamate, which are mediated via stimulation of nnAChRs. VDCC inhibitors also inhibit this stimulatory action of nicotine, suggesting that the role of VDCCs in the release process of neurotransmitter by nicotine is critical (8). Among various types of VDCCs, recent investigations have indicated that L-type (50, 51) as well as N-type VDCCs (52-54) play a role in neurotransmitter release. In addition, Ca 2ϩ moved from extracellular to intracellular space through L-type VDCCs modulates protein function and transcription process through phosphorylation (55)(56)(57). Thus, functional up-regulation of L-type VDCCs accompanying nnAChR up-regulation induced by long term exposure to nicotine may modify a variety of biochemical processes in neurons. Indeed, the findings showing that the long term exposure of the mouse cerebral cortical neurons to nicotine increases DBI mRNA expression in association with increases in [ 3 H]nicotine binding and 45 Ca 2ϩ influx and that this enhancement of DBI mRNA expression is completely abolished by L-type VDCC blockade (10) are assumed to be a case of the phenomenon described above.
The present investigation demonstrates that continuous exposure of the mouse cerebral cortical neurons to nicotine for 72 h causes an increase of [ 3 H]nicotine binding to their particulate fractions, and this increase is attributed to increased [ 3 H]nicotine binding sites with no changes in the affinity of nnAChRs to the radiolabeled ligand. Similarly, the up-regulation of nAChRs is reported to be induced in the central nervous system of experimental animals chronically treated with nicotine (38,39,58) and neural cells exposed to nicotine (13,59) when examined by a receptor binding assay using [ 3 H]nicotine as a radiolabeled ligand.
nnAChRs exist as a variety of subtypes, which is due to the diversity of the genes encoding nnAChRs. Among various regions of the central nervous system, the ␣ 3 , ␣ 4 , ␤ 2 , and ␤ 4 subunits of nnAChRs are unequally distributed in the different layers of the cerebral cortex (60), and several investigations have reported that the up-regulation of the receptors is due to the increased numbers of ␣ 4 /␤ 2 , ␣ 3 /␤ 4 , and ␣ 7 nnAChR subunits in neurons and nonneuronal expression systems (13,17,61). In addition to these data, nnAChRs composed with ␣ 4 /␤ 2 are adequately activated by nicotine at concentrations less than 10 - 100 nM (13) and also have been reported to show up-regulation after long term nicotine treatment (13)(14)(15)18). Based on these data, we attempted to examine which of the ␣ 3 , ␣ 4 , and ␤ 2 subunits showed up-regulation under the conditions used in the present study. The Western blot analysis demonstrated the up-regulation of ␣ 4 /␤ 2 nnAChRs after the long term exposure of the neurons to 0.1 M nicotine. These data are in good agreement with the previous reports using human embryonic kidney cells (14) and Xenopus oocytes (15), although in the latter study the induction of up-regulation of ␣ 3 /␤ 4 nnAChRs required a 10-fold higher nicotine concentration for up-regulating ␣ 4 /␤ 2 nnAChRs (15).
Previous reports indicated that one of the functional changes of up-regulated nAChRs is desensitization, which is confirmed by the electrophysiological method (15) and neurochemical measurement of 86 Rb ϩ efflux from cultured cells (14,62). On the other hand, our data presented here indicate that nicotine itself stimulates 45 Ca 2ϩ influx via up-regulated L-type VDCCs even after nnAChRs are up-regulated. Although electrophysiological investigation demonstrated desensitization as a result of nnAChR up-regulation, the electrophysiological response to nicotine is only significantly reduced and not completely abolished (15). Therefore, the potential of nnAChRs to depolarize the neuronal membrane to open L-type VDCCs may be adequately maintained even under such conditions with up-regulation of nnAChRs, which may explain the increased 45 Ca 2ϩ influx through L-type VDCCs in the neurons possessing upregulated nnAChRs after the long term treatment with 0.1 M nicotine.
In summary, the present study demonstrates that long term exposure of mouse cerebral cortical neurons in primary culture to a low concentration (0.1 M) of nicotine induces the increase of L-type VDCC molecules associated with functional enhancement and the up-regulation of ␣ 4 /␤ 2 nnAChRs. The former may be involved in the formation of nicotine dependence as in the cases of ethanol and morphine dependence.