Fig 1.
(A) Expression of the mRNAs of the nicotinic receptor subunits in SH-SY5Y cells. The type of nicotinic receptor expressed in SH-SY5Y cells was examined by real-time PCR. The expression of mRNA for the α3, α5, α7, β2, and β4 nicotinic receptor subunits were confirmed. Data represent the mean ± standard error of 3 independent experiments. (B) Nicotine-induced intracellular Ca2+ ([Ca2+]i) elevation in SH-SY5Y cells. Left panel: The [Ca2+]i increase induced by nicotine was observed for 15 min, and ionomycin was administered at the end of observation. The fluorescence before nicotine administration was assumed to be 100%, and the change in [Ca2+]i after application of nicotine at each concentration is shown. Data represent the average of the experiments shown in right panel. Right panel: The ratio of peak [Ca2+]i elevation induced by nicotine to the [Ca2+]i elevation induced by ionomycin was evaluated as an index of nicotine-induced [Ca2+]i elevation. A concentration-dependent nicotine-induced [Ca2+]i increase was observed. The data represent the means ± standard error.
Fig 2.
The origin of the Ca2+ involved in the nicotine-induced [Ca2+]i elevation in SH-SY5Y cells.
(A) Effect of eliminating extracellular Ca2+. A Ca2+-free buffer in which calcium was replaced by EGTA was used. The [Ca2+]i elevation after 500 μM nicotine treatment was observed when extracellular Ca2+ was eliminated. (B) In the presence of extracellular Ca2+, 5 μM thapsigargin was incubated for 15 min to deplete the intracellular Ca2+ stores. Then, 500 μM nicotine was applied. The [Ca2+]i elevation, which is thought to be the result of extracellular influx, was observed (purple line). [Ca2+]i elevation was also observed when nicotine was administered without thapsigargin (black line). (C) In the presence of the Ca2+ -free buffer, thapsigargin was incubated for 15 min to eliminate intracellular and extracellular Ca2+. The subsequent application of nicotine did not induce [Ca2+]i increases (purple line). The [Ca2+]i elevation was observed when nicotine was administered without thapsigargin, which is thought to mobilize Ca2+ from intracellular organelles (black line). A representative example of more than 5 cases is shown in Fig 2A–2C.
Fig 3.
Effects of tubocurarine, a nonselective nAChR antagonist, on nicotine-induced intracellular [Ca2+]i elevation and current.
(A) The representative time plot of leak currents at -70 or -60 mV. Leak currents were sampled every ten seconds. Left panel: The bath application of nicotine (500 μM) evoked the large inward current in a SH-SY5Y cell. Nicotine was applied during a period indicated by a black line. Right panel: Pretreatment with 100 μM tubocurarine (white line) completely suppressed the nicotine-induced current. Representative examples from 8 independent experiments are presented. (B) Quantitative analysis of the effects of tubocurarine on nicotine (500 μM)-induced currents. The average of three consecutive data just prior to drug administration was set at 100% and the inward current under each condition was normalized. Abbreviations, nic and tub, indicate nicotine and tubocurarine, respectively. **p<0.01, ns p>0.05, one-way ANOVA followed by Dunnett’s posttest, n = 8. (C) Left panel: We investigated the temporal changes in nicotine (250 μM)-induced [Ca2+]i elevation following 15 min of pretreatment with the nonselective nAChR antagonist tubocurarine (100 μM). Black line: 250 μM nicotine alone, Blue line: 15 min pretreatment with 100 μM tubocurarine. Data represent the average of the six experiments shown in right panel. Right panel: Ca2+ increases in the 250 μM nicotine alone-treated group and the 15 min pretreatment with 100 μM tubocurarine groups are shown. Data indicate the means ± standard error. * p < 0.05, unpaired t-test vs the nicotine-alone-treated group.
Fig 4.
Involvement of various nAChR subtypes and voltage-gated calcium channels in nicotine (250 μM)-induced [Ca2+]i elevation.
(A) Effects of siRNA treatment of nAChR subtypes (α3, α5 and β4) on nicotine-induced [Ca2+]i elevation. (B) Effect of the α3β4nAChR-selective antagonist α-conotoxin AuIB on nicotine-induced [Ca2+]i elevation. (C) The effect of siRNA treatment of the α7nAChR subunit on nicotine-induced [Ca2+]i elevation. (D) The effect of MLA (5 μM), a selective α7nAChR antagonist, on nicotine-induced [Ca2+]i elevation. Cells were pretreated with MLA for 15 min before 250 μM nicotine was added. (E) The effects of 5 μM nifedipine, an L-type Ca2+ channel antagonist, and 1 μM ω-conotoxin GVIA, an N-type Ca2+ channel antagonist, on nicotine (250 μM)-induced [Ca2+]i elevation. The cells were pretreated with inhibitors for 15 min before 250 μM nicotine was added. The data represent the means ± standard error. * p< 0.05, unpaired t-test, vs the nontreated control group or the control siRNA-treated group.
Fig 5.
Properties of acetylcholine-induced [Ca2+]i elevation in SH-SY5Y cells.
(A) Temporal changes in [Ca2+]i elevation after administration of 10 μM acetylcholine alone. The steep [Ca2+]i increase with sustained shape was prominent compared with the nicotine-induced shape. (B) Temporal changes in the [Ca2+]i elevation induced by 10 μM acetylcholine in the presence of 1 μM atropine. It is presumed that increases in [Ca2+]i are mediated though nAChRs. Compared with the outcome of acetylcholine treatment alone, the [Ca2+]i was steeply elevated but was quickly decreased. In both (A) and (B), a representative of 5 independent trials is shown. (C) Involvement of voltage-gated calcium channels in 10 μM acetylcholine and 1 μM atropine-induced [Ca2+]i elevation. Pretreatment with 5 μM nifedipine (+ nifedipine) and 1 μM ω-conotoxin GVIA (+ ω-conotoxin) significantly inhibited the acetylcholine and atropine-induced [Ca2+]i elevation (D) Involvement of α7nAChR in the [Ca2+]i increase induced by 10 μM acetylcholine with 1 μM atropine. Acetylcholine and atropine-induced calcium elevation was significantly suppressed by pretreatment with 5 μM MLA (+MLA). Data indicate the means ± standard error. * p < 0.05, unpaired t-test, vs the acetylcholine + atropine group.
Fig 6.
Fundamental properties of nicotine-induced [Ca2+]i elevation in SH-SY5Y cells.
Nicotine may cause an increase in intracellular calcium through three mechanisms: (1) Nicotine binds to α3 * nAChR, α5 * nAChR or α7nAChR expressed in the plasma membrane, and calcium and sodium ions enter the cell. (2) The influx of cations causes depolarization, opening the voltage-gated calcium channel, and causes increased Ca2+ influx. (3) Lipophilic nicotine penetrates the plasma membrane and enters cells. The internalized nicotine mobilizes Ca2+ from stores, including those in the endoplasmic reticulum, by some mechanism. Acetylcholine, with no cell membrane permeability, binds to nicotinic and muscarinic receptors on the plasma membrane, causing an increase in intracellular calcium. We hypothesize that acetylcholine activates only the a3 * nAChRs and a5 * nAChRs expressed on the plasma membrane when muscarinic receptors, α7nAChR, and voltage-gated calcium channels are blocked by individual antagonists. In this study, therefore, we observed an increase in intracellular calcium induced by acetylcholine and the inhibitor mix as described in the main text.
Fig 7.
Effect of dbcAMP treatment on [Ca2+]i elevation as induced by acetylcholine and the inhibitor mix.
(A) SH-SY5Y cells were treated with 1 mM dbcAMP for 15 min (left panel) or 48 h (right panel), and the [Ca2+]i elevation induced by the acetylcholine and inhibitor mix was examined. Treatment with dbcAMP for 15 min resulted in no changes, but treatment for 48 h significantly inhibited the [Ca2+]i elevation. Data represent the means ± standard error. * p < 0.05, unpaired t-test, vs the nontreated control group. (B) Effects of 48 h-treatment with 1 mM dbcAMP on the mRNA expression of various nAChR subunits. Data represent the means ± standard error. * p < 0.05, ** p<0.01, unpaired t-test, vs the nontreated control group.