Full text of DOI:10.1038/sj.bjp.0704508

British Journal of Pharmacology (2002) 135, 1051 ± 1059
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E€ects of chronic drug treatments on increases in intracellular
calcium mediated by nicotinic acetylcholine receptors in SH-SY5Y
cells
1,2Diana L. Ridley, 1,3Jukka Pakkanen & *,1Susan Wonnacott
1Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY
1
SH-SY5Y cells express a7 and a3* subtypes of nicotinic acetylcholine receptors (AChR).
Numbers of these receptors are upregulated by chronic treatment with nicotinic agonists or KCl. In
this study we have examined the functional consequences of these drug treatments on nicotine- or
KCl-evoked increases in [Ca2+]i, in SH-SY5Y cells.
2
In untreated cells, nicotine increased [Ca2+]i (EC50 7.5 mM). Responses to 10 mM nicotine were
abolished by the non-selective nicotinic antagonist mecamylamine and were partially blocked by a7-
selective antagonists, the a3b2*-selective antagonist a-conotoxin-MII, and by cadmium and
verapamil.
3
After treatment for 4 days with nicotinic agonists, nicotine-evoked increases in [Ca2+]i were
signi®cantly decreased by about 25%. Nicotine-evoked responses were paradoxically increased in the
presence of acute methyllycaconitine (MLA; an a7-selective antagonist) although other a7-selective
antagonists were without e€ect, while a-conotoxin-MII gave a partial inhibition. The increase
observed with MLA was abolished by mecamylamine but not by a-conotoxin-MII and was still
observed 24 h after chronic nicotine treatment.
4
After treatment for 4 days with KCl, nicotine-evoked increases in [Ca2+]i were also decreased by
25%, but acute MLA was without e€ect. Responses to 20 mM KCl were unchanged by prior
treatment with nicotine or KCl. Treatment for 4 days with 5 mM verapamil reduced responses to
both nicotine and KCl by about 50%.
5
Multiple nicotinic AChR subtypes contribute to nicotine-evoked increases in [Ca2+]i in SH-SY5Y
cells. Responses to acute nicotine are reduced after chronic nicotine or KCl treatment, with loss of
the component attributed to the a7 subtype. However, in nicotine-treated cells this e€ect is reversed
when nicotine stimulation is applied in the presence of acute MLA. The antagonist may assist in
converting a non-functional a7 nicotinic AChR to a conducting state.
British Journal of Pharmacology (2002) 135, 1051 ± 1059
Keywords: Nicotinic receptor; SH-SY5Y cell line; intracellular Ca2+; nicotine; DMAC; abungarotoxin; a-conotoxin;
methyllycaconitine; upregulation
Abbreviations: a-Bgt, a-bungarotoxin; BSA, bovine serum albumin; [Ca2+]i, concentration of intracellular calcium; CaM-kinase
II, Ca2+-calmodulin dependent kinase II; DMAC, 3, [(4-dimethylamino) cinnamylidene] anabaseine maleate;
DMEM, Dulbecco's modi®ed eagle's medium; DMSO, dimethylsulphoxide; FCS, foetal calf serum; MLA,
methyllycaconitine; NEAAs, non-essential amino-acids; nicotinic AChR, nicotinic acetylcholine receptor; PBS,
phosphate bu€ered saline; TSS, Tyrode's salt solution
Introduction
Nicotinic acetylcholine receptors (AChR) are a family of
pentameric ligand-gated cation channels (Lukas et al.,
1999). In the brain, nicotinic AChR are responsible for
mediating the psychoactive and addictive properties of
nicotine (Dani & Heinemann, 1996), and are also targets
for the pharmaceutical industry, with respect to a portfolio
et al., 1996). The a4b2 nicotinic AChR subtype appears to
be most sensitive to this in¯uence, but other subtypes are
also upregulated at higher concentrations of nicotine (Peng
et al., 1997; Wang et al., 1998). The functional con-
sequences of nicotinic AChR upregulation are controversial.
Chronic nicotine treatments in vivo result in behavioural
stimulation (typically measured as locomotor activity), with
of diverse brain disorders (Decker
& Arneric, 1998).
Chronic exposure to nicotine (and other nicotinic agonists)
upregulates the numbers of neuronal nicotinic AChR
binding sites in vivo and in vitro (Wonnacott, 1990; Lukas
a bell shaped dose-response curve (Ksir et al., 1987),
whereas nicotine-evoked dopamine over¯ow, measured by
in vivo microdialysis, has been found to increase (Benwell &
Balfour, 1992; Benwell et al., 1995; Marshall et al., 1997)
or remain unchanged (Damsma et al., 1989; Nisell et al.,
1994) after various nicotine treatment regimes. In contrast,
nicotine-evoked prolactin release, sampled in vivo, was
abolished after chronic nicotine injections, with a time-
course that mimicked that of the upregulation of [3H]-ACh
*Author for correspondence; E-mail: s.wonnacott@bath.ac.uk
Current addresses: 2Institute for Cardiovascular Research, The
School of Medicine, University of Leeds, Leeds; 3Division of
Pharmacology and Toxicology, Department of Pharmacy,
University of Helsinki, Finland.

1052
D.L. Ridley et al
Nicotinic responses in SH-SY5Y cells
Methods
binding sites (Hulihan-Giblin et al., 1990). Ex vivo
measurements of nicotine-evoked transmitter release, after
chronic nicotine treatment in vivo, have shown decreased
Drugs and reagents
(Marks et al., 1993) and increased responses (Yu
Wecker, 1994).
&
Tissue culture media, serum and plasticware were from Gibco
BRL (Paisley, Renfrewshire, Scotland). Media supplements,
cadmium chloride, (7)-nicotine hydrogen tartrate, (+)-
verapamil hydrochloride, mecamylamine and a-conotoxin-
ImI and a-bungarotoxin (a-Bgt) were purchased from Sigma
Co. (Poole, Dorset, U.K.). Nicotine, KCl and verapamil used
for chronic treatment of SH-SY5Y cells were made up freshly
and directly in Dulbecco's Modi®ed Eagle's Medium
(DMEM):Ham's F12 supplemented medium on day 1 of
drug treatment. (+)-Anatoxin-a fumarate and methyllycaco-
nitine (MLA) were purchased from Tocris Cookson, (Avon-
Interpretation of these studies in relation to nicotinic
AChR upregulation is compromized by the complexity of the
preparation, with many unknown variables in¯uencing the
e€ects of systemically administered nicotine. In vitro cell
systems provide a better de®ned and more easily manipulated
model. Chronic nicotine treatment is reported to decrease, or
completely block, whole cell currents recorded from Xenopus
oocytes in which various nicotinic AChR subunit combina-
tions have been heterologously expressed (Hsu et al., 1996;
Olale et al., 1997). In transfected HEK-293 cells stably
expressing a7 or a4b2 nicotinic AChR, ACh-evoked whole
cell currents were increased after chronic treatment with
nicotine or with the a7-selective antagonist methyllycaconi-
tine (MLA) (Molinari et al., 1998; Buisson & Bertrand,
2001). The a4b2 nicotinic AChR in HEK-293 cells gave
increased responses (86Rb+ e‚ux) after chronic treatment
with low agonist concentrations and decreased responses
after higher concentrations (Gopalakrishnan et al., 1997).
The over-expression of nicotinic AChR in non-neuronal cells
may not provide a suitable model for studying neuronal
mechanisms regulating these receptors. Neuroblastoma cell
lines endogenously express native nicotinic AChR in a more
relevant context, although the low levels and heterogeneity of
nicotinic AChR expression complicate analyses. Short
applications (up to 1 h) of high concentrations of nicotine
induce `persistent functional inactivation' of responses (86Rb+
in¯ux) in the neuroblastoma SH-SY5Y cell line (Ke et al.,
1998). This phenomenon may be the trigger for nicotinic
AChR upregulation; we wished to know the magnitude of
nicotinic responses after nicotinic AChR binding sites have
been upregulated.
mouth,
U.K.).
3,[(4-dimethylamino)
cinnamylidene]
anabaseine maleate (DMAC) was synthesized by Organon
Laboratories Ltd., Newhouse, Lanarkshire, U.K. and was
made up in dimethylsulphoxide (DMSO) to give a stock
solution of 10 mM; the ®nal concentration of DMSO on
cultures did not exceed 0.1%. a-conotoxin-MII was synthe-
sized as previously described (Cartier et al., 1996; Kaiser et
al., 1998). Fura-2 AM and Fluo-3 AM were purchased from
Molecular Probes, Eugene, Oregon, U.S.A. and stored at
7208C as stock solutions in DMSO.
Cell culture of SH-SY5Y cells
Cell culture was carried out as described in detail by Ridley
et al. (2001). In brief, SH-SY5Y human neuroblastoma
stock cultures were routinely maintained in DMEM:Ham's
F12 (1 : 1) modi®ed medium containing 1% non-essential
amino-acids (NEAAs) and supplemented with foetal calf
serum (FCS, 15%), glutamine
(2 mM),
penicillin
(50 iu ml71) and streptomycin (50 mg ml71). Stock cultures
were passaged 1 : 5 weekly and fed twice weekly. For fura-2
assays, cultures were sub-cultured and grown to con¯uency
in 175 cm2 ¯asks containing 25 ml of supplemented medium
and maintained at 378C in 5% CO2/humidi®ed air. For
chronic treatments, drugs were added 3 days after the cells
reached con¯uency. After 4 days of drug treatment, cells
were washed three times over 4 h with warm Ca2+-free
phosphate bu€ered saline (PBS; 150 mM NaCl, 8 mM
K2HPO4, 2 mM KH2PO4, pH 7.4, 378C) to ensure the
removal of nicotine or other drugs taken up by the cells,
We have shown in primary hippocampal cultures and SH-
SY5Y cells that a few days' exposure to nicotine or KCl
upregulates a7 nicotinic AChR binding sites, labelled with
[
125I]-a-Bgt, through di€erent mechanisms (Ridley et al.,
2001), consistent with previous studies on sympathetic
neurones (De Koninck & Cooper, 1995). Upregulation by
KCl appears to occur through activation of L-type Ca2+
channels and activation of Ca2+-calmodulin dependent kinase
(CaM-kinase II), probably leading to an increase in
transcription of a7 RNA, whereas neither L-type Ca2+
channel blockers nor CaM-kinase II inhibitors prevent
nicotine-induced upregulation of [125I]-a-Bgt binding sites. In
SH-SY5Y cells, [3H]-epibatidine binding sites, corresponding
to a3* nicotinic AChR, were also upregulated after nicotine
treatment, but were una€ected by chronic KCl depolarization
(Ridley et al., 2001). The a7-selective agonist 3,[(4-dimethy-
lamino) cinnamylidene] anabaseine maleate (DMAC) (De
Fiebre et al., 1995) also upregulated [125I]-a-Bgt and [3H]-
epibatidine binding sites, in a manner indistinguishable from
that of nicotine (Ridley et al., 2001). In the present studies we
have characterized increases in [Ca2+]i in response to acute
application of either nicotine or KCl, as an index of nicotinic
AChR-mediated responses or general responsiveness respec-
tively, in control SH-SY5Y cells and after chronic drug
treatment regimes that upregulate numbers of nicotinic
AChR binding sites.
before measurement of [Ca2+
] using fura-2. For ¯uo-3
i
assays, cultures were sub-cultured into 96 well plates in
180 ml of modi®ed medium. To avoid the cells becoming
over-con¯uent, chronic drug treatments were initiated
immediately. After 4 days, cells in each well were washed
six times with warm medium (200 ml/well) over 4 h, before
measurement of Ca2+ using ¯uo-3.
Calcium fluorimetry: fura-2 assays on cells in suspension
Changes in [Ca2+]i in suspensions of SH-SY5Y cells were
measured essentially as described by Sharples et al. (2000). In
brief, after extensive washing (above) cultured cells were
removed from ¯asks by incubation for 3 min at 378C in
Ca2+-free PBS. The cell suspension was centrifuged (5006g,
3 min) and the cell pellet was resuspended in 5 ml of Ca2+
free HEPES bu€er (in mM: HEPES 10, NaCl 145, KCl 5,
-
British Journal of Pharmacology vol 135 (4)

D.L. Ridley et al
Nicotinic responses in SH-SY5Y cells
1053
MgCl2-6-hydrate 1, Na2HPO4 0.5 and glucose 5.5) and
recentrifuged (5006g, 3 min). The cells were loaded with
5 mM fura-2 AM by resuspending the pellet in 3 ml HEPES
Data analysis
Changes in [Ca2+
] are expressed as mean+s.e.mean con-
i
bu€er containing 0.25% (w v71
)
BSA, to improve the
centration or as a percentage of the increase in [Ca2+]i (fura-2
experiments) or as a percentage of Max ± Min ¯uorescence
(¯uo-3 experiments) measured in control cells. For concen-
tration response curves, agonist responses were calculated as
solubilization of the dye. After incubating in darkness for
45 min at room temperature, excess dye was removed by
centrifugation (5006g, 3 min), followed by three washes with
5 ml of Ca2+-free HEPES bu€er. When used, a-Bgt was
added to the cells with fura-2 AM for 45 min at room
temperature.
a percentage of the increase in [Ca2+
] or ¯uorescence
i
produced by a maximally e€ective concentration of the same
drug, assayed in parallel. Data points were ®tted to the Hill
equation, using the nonlinear least squares curve ®tting
facility of Sigma Plot V2.0 for Windows. All data were
analysed statistically using an analysis of variance (ANOVA)
for repeated measures using SPSS (Statistics Package for
Social Scientists, PC version).
Cell density was adjusted to 1 ± 26106 cells ml71 and
[Ca2+]i was measured in 2 ml aliquots of cell suspension in
a
constantly stirred cuvette, in a PTI dual-excitation
photo¯uorimeter. The excitation wavelengths were of 340
and 380 nm, with an emission wavelength of 510 nm, 4 nm
slit width, equipped with PTI software version 2.060 (Photon
Technology International Inc., Deerpark Drive, South
Brunswick, NJ, U.S.A.). Before the addition of nicotinic
agonist or KCl, 2 mM CaCl2 was introduced into the cuvette
and the fura-2 ¯uorescence was monitored for 80 s. Where
used, antagonists (mecamylamine (10 mM), MLA (10 nM), a-
conotoxin-ImI (1 mM) or a-conotoxin-MII (112 nM)), or the
L-type Ca2+-channel blocker verapamil (5 mM) were added
5 min before the addition of nicotinic agonist or KCl.
Subsequently, calibration was performed by sequentially
adding 0.5% Triton X-100 to lyse the cells in order to
obtain Rmax (the ¯uorescence of calcium-saturated dye),
followed by 20 mM EGTA to determine Rmin, the ¯uores-
cence signal after the dye is quenched.
Results
Effects of acute nicotine and KCl depolarization on
[Ca2+]i in SH-SY5Y cells
The resting [Ca2+
140+4 nM (n=160). Application of nicotine produced a
rapid increase in ¯uorescence which was followed by a
] in fura-2-loaded SH-SY5Y cells was
i
[Ca2+]i was determined from the ¯uorescence ratio of fura-2
at 340/380 nm excitation (A340/A380; given as R below)
according to the Grynkiewicz Equation (Grynkiewicz et al.,
1985):
Ã
‰Ca2‡Š ˆ KD ‰ꢀR Rmin†=ꢀRmax R†Š à ꢀSf2=Sb2
†
i
where KD is the dissociation constant of fura-2 for Ca2+
binding, 224 nM (Tsien et al., 1982), Rmin is the ¯uorescence
ratio under `zero' Ca2+ conditions, Rmax is the ¯uorescence
ratio under saturating Ca2+ conditions and Sf2/Sb2 is the ratio
of ¯uorescence values of Ca2+-free and Ca2+-saturated fura-2,
measured at the wavelength used to follow Ca2+-free fura-2.
Calcium fluorimetry: fluo-3 assays in 96 well plates
Culture medium was removed from the wells and the cells
were washed twice with Tyrode's salt solution (TSS). Fluo-3
AM (10 mM) and 0.02% pluronic acid were added in TSS
(40 ml per well) and the cells were incubated at room
temperature for 1 h in the dark. After two further washes
in TSS, 80 ml of TSS with or without antagonists were added,
and the plate transferred to a Fluoroscan Ascent (Labsys-
tems, Helsinki, Finland). After 10 min preincubation (or
30 min in the case of a-Bgt) at 208C in the dark, nicotine
(®nal concentration 30 mM unless otherwise stated) was added
(20 ml per well), and ¯uorescence monitored at 538 nM
wavelength. Maximum and minimum ¯uorescence was
determined by the sequential addition of 20 ml Triton 6100
(2%, ®nal concentration) and 20 ml MnCl2 (40 mM, ®nal
concentration). Changes in intracellular calcium were calcu-
lated as a percentage of the di€erence between the minimum
and maximum ¯uorescence.
Figure 1 Increases in [Ca2+]i in SH-SY5Y cells evoked by nicotinic
agonists and KCl. (a) A representative trace from fura-2-loaded SH-
SY5Y cells in suspension, showing the change in [Ca2+]i in response
to 10 mM nicotine (arrow) in the presence (lower trace) and absence
(upper trace) of 10 mM mecamylamine. (b) Increases in [Ca2+]i above
basal levels in response to nicotine (Nic; 10 mM, 100 mM), anatoxin-a
(Ana; 10 mM) and KCl (20, 80 and 100 mM). Values are the
mean+s.e.mean from at least ®ve separate cultures. (c) Concentra-
tion-response curve for nicotine-evoked increases in [Ca2+]i.
Responses to 100 mM nicotine were taken as 100%. Data points are
the mean+s.e.mean of ®ve separate cultures and are ®tted to the Hill
equation giving an EC50 value of 7.5+4 mM. (d) Concentration-
response curve for KCl-evoked increases in [Ca2+]i. Responses to
80 mM KCl were taken as 100%. Data points are the mean+
s.e.mean of ®ve separate cultures and are ®tted to the Hill equation
giving an EC50 value of 26+8 mM.
British Journal of Pharmacology vol 135 (4)

1054
D.L. Ridley et al
Nicotinic responses in SH-SY5Y cells
sustained plateau (Figure 1a). At higher concentrations of
nicotine (100 mM) the plateau slowly declined over time in
some cases. The initial plateau value of the response was used
nicotine-evoked increase in [Ca2+]i to a similar extent (Figure
2): a-Bgt (50 nM), a-conotoxin-ImI (1 mM; Pereira et al.,
1996) and methyllycaconitine (MLA; 10 nM; Alkondon et al.,
1992) blocked the response by 41+8% (n=5), 39+5%
(n=4) and 40+6% (n=4) respectively. The a3b2-selective
antagonist a-conotoxin-MII (112 nM; Cartier et al., 1996;
Kaiser et al., 1998) blocked the response to nicotine by
50+5% (Figure 2). Co-application of MLA (10 nM) and a-
conotoxin-MII (112 nM) inhibited the nicotine-evoked in-
crease in [Ca2+]i by 69+9%, suggesting that their actions are
partially additive, although this did not reach statistical
signi®cance. These results demonstrate that this functional
nicotinic response is mediated by at least two receptor
subtypes: a7 and a3b2-containing nicotinic AChRs.
to derive the [Ca2+]i. Nicotine-evoked increases in [Ca2+
]
i
were concentration-dependent, with a maximum response
produced by 100 mM nicotine, which elevated [Ca2+
by
]
i
102+9 nM (n=29) above its resting level (Figure 1b,c). The
EC50 value for nicotine-evoked increase in [Ca2+
]
i
was
7.5+4 mM. For comparison with previous studies (Sharples
et al., 2000), a maximally e€ective concentration of the
nicotinic agonist anatoxin-a (10 mM) was tested in parallel
with nicotine. It elicited a response comparable in magnitude
to that of 100 mM nicotine (Figure 1b).
KCl also produced a rapid increase in [Ca2+]i followed by
a sustained plateau, similar to the response evoked by
nicotine shown in Figure 1a. KCl-evoked increases in
The increase in [Ca2+
] in response to nicotinic AChR
i
activation may be complicated by the entry of Ca2+ into the
cell by routes other than the nicotinic AChR. Nicotine- and
KCl-evoked increases in [Ca2+]i were totally dependent on the
presence of extracellular Ca2+ (data not shown). CdCl2, a
general blocker of voltage-operated Ca2+ channels that does
not block nicotinic currents at the concentration used
[Ca2+
]
were concentration-dependent, with
a
maximum
]
i
response produced by 80 mM KCl, which increased [Ca2+
i
by 100+12 nM (n=12). The EC50 value was 26+8 mM KCl
(Figure 1b,d). In subsequent experiments, approximate EC50
concentrations of nicotine (10 mM) and KCl (20 mM) were
selected to elicit responses.
(200 mM; Rathouz & Berg, 1994), did not a€ect basal [Ca2+
]
i
As SH-SY5Y cells express a variety of nicotinic AChR
subunit mRNAs (Lukas et al., 1993) and nicotinic AChR
subtypes (Peng et al., 1997), we used a selection of chemically
distinct, subtype-selective nicotinic antagonists to dissect the
contributions of di€erent nicotinic AChR subtypes to the
increase in [Ca2+]i produced by 10 mM nicotine. None of the
antagonists examined in this study had any e€ect on basal
levels of [Ca2+]i. Mecamylamine (10 mM) completely blocked
the response to nicotine (Figures 1a and 2), con®rming that
the response is nicotinic AChR-mediated. Maximally e€ective
concentrations of three a7-selective antagonists inhibited the
when applied for 5 min prior to stimulation with nicotine
(10 mM). However, the nicotine-evoked increase in [Ca2+]i was
inhibited by 76+3% (mean+range; n=2) by CdCl2, whereas
the phenylalkylamine L-type Ca2+-channel blocker, verapamil
(5 mM) partially inhibited nicotine-evoked increases in [Ca2+]i,
by 53+7% (n=4; Figure 2). Thus a major proportion of the
response to nicotine arises from activation of voltage-operated
Ca2+ channels, including L-type channels.
The effect of chronic drug treatments on nicotine- and
KCl-evoked responses
Having characterized the nicotinic AChR-evoked changes in
[Ca2+]i in untreated SH-SY5Y cells, we next investigated if
chronic treatment with nicotinic agonists or KCl, regimes
that upregulate nicotinic binding sites (Ridley et al., 2001),
would modify these functional responses. Chronic exposure
(4 days) to nicotine (10 mM), the a7-selective nicotinic agonist
DMAC (10 mM) and KCl (20 mM) was investigated. After
thorough washing (four washes over 4 h, see Methods),
increases in [Ca2+]i evoked by 10 mM nicotine and 20 mM
KCl were measured (Figure 3). Acute stimulation with 10 mM
nicotine resulted in signi®cantly lower responses (approxi-
mately 25% less than in untreated control cells) after chronic
treatment with nicotinic agonist or KCl. Acute stimulation
with KCl, on the other hand, resulted in no signi®cant
di€erence in the [Ca2+]i response between chronically treated
and control cells, examined in parallel (Figure 3a ± c). DMAC
could not be applied acutely to evoke changes in [Ca2+]i due
to its intense purple colour, even when diluted, which
interfered with the ¯uorescence measurements. Chronic
treatment of cells with 5 mM verapamil for 4 days (a
treatment that has no e€ect on numbers of nicotinic AChR
binding sites, Ridley et al., 2001) decreased responses to both
nicotine and KCl by 50% (Figure 3d).
Figure 2 E€ect of nicotinic antagonists and Ca2+ channel blockers
on nicotine-evoked increases in [Ca2+
] in SH-SY5Y cells. Suspen-
i
sions of fura-2-loaded SH-SY5Y cells were incubated for 5 min with
the nicotinic AChR antagonists mecamylamine (Mec), a-conotoxin-
Iml (aCtx-ImI), MLA, a-conotoxin-MII (aCtx-MII) or blockers of
voltage-operated Ca2+ channels: CdCl2 or verapamil (Ver). a-
Bungarotoxin (aBgt) was incubated with the cells, together with
fura-2 AM, for 45 min. Nicotine (10 mM, Nic) was added and the
increase in [Ca2+]i monitored as in Figure 1. Responses are expressed
as a percentage of the control response measured in parallel in the
absence of antagonist. Values are the mean+s.e.mean of at least four
independent experiments carried out in triplicate (except for CdCl2,
where n=2 independent experiments and values are mean+range).
*Signi®cantly di€erent from control, P50.05, one way ANOVA.
As nicotine and KCl treatments upregulated a7 nicotinic
AChR binding sites in SH-SY5Y cells through di€erent
mechanisms (Ridley et al., 2001), we examined the a7
component of the functional response to nicotine stimula-
tion in control and treated cells. In control cells, acute
British Journal of Pharmacology vol 135 (4)

D.L. Ridley et al
Nicotinic responses in SH-SY5Y cells
1055
Figure 4 E€ect of MLA on nicotine-evoked increases in [Ca2+
]
i
from SH-SY5Y cells chronically treated with nicotine or KCl. SH-
SY5Y cells were treated with 10 mM nicotine (black columns) or
20 mM KCl (hatched columns) for 4 days, thoroughly washed, loaded
with fura-2 and incubated with or without MLA (10 nM) for 5 min
before stimulation with nicotine (10 mM). Responses are expressed as
a percentage of control responses from untreated cells cultured and
assayed in parallel in the absence of MLA. Values are the
mean+s.e.mean of at least four independent experiments. *Sig-
ni®cantly di€erent from untreated control in the absence of MLA,
P50.05, one way ANOVA. #Signi®cantly di€erent from untreated
control in the presence of MLA, P50.05, one way ANOVA.
Figure 3 E€ect of chronic treatment of SH-SY5Y cells with
nicotinic agonists, KCl or verapamil on nicotine- and KCl-evoked
increases in [Ca2+]i. SH-SY5Y cells were treated with (a) 10 mM
nicotine, (b) 10 mM DMAC, (c) 20 mM KCl, or (d) 5 mM verapamil
for 4 days, thoroughly washed, loaded with fura-2 and stimulated
with nicotine (Nic; 10 mM) or KCl (20 mM). Responses from treated
cells (®lled columns) are expressed as
a percentage of control
responses (open columns) from untreated cells cultured and assayed
in parallel. Values are the mean+s.e.mean of at least four
independent experiments. Signi®cantly di€erent from control,
*P50.05, one way ANOVA.
control cells exhibited the same pharmacological pro®le
(Figure 5) as previously observed (Figure 2), with
approximately 30% inhibition by the a7-selective antagonists
MLA, a-Bgt and a-conotoxin-ImI, 50% inhibition by a-
conotoxin-MII and more than 80% inhibition by mecamy-
lamine. In cells cultured in parallel for 4 days with 10 mM
nicotine, followed by thorough washing (six washes over
4 h), the response to nicotine was decreased by 33+4%
(n=4) whereas the response to nicotine in the presence of
acute MLA (10 nM) was 127+15% of control (n=4; Figure
5). This assay therefore reproduces the unexpected increase
in the nicotine-evoked response in the presence of MLA
seen in Figure 4. This result appears to be peculiar to
MLA: nicotine-evoked responses from chronically treated
cells in the presence of other a7-selective antagonists, a-Bgt
and a-conotoxin-ImI, were not di€erent from responses in
the absence of these agents. This suggests that the a7
nicotinic receptor no longer contributes to the nicotine-
stimulated response after chronic nicotine treatment. In
contrast, in the presence of the a3b2*-selective antagonist a-
conotoxin-MII, nicotine-evoked responses were 50+4%
(n=3) and 46+4% (n=3) of the control response from
untreated cells, in untreated and treated cells respectively,
compared with the nicotine-evoked response of 67+4% of
control (n=4) in treated cells (Figure 5). Thus a-conotoxin-
MII inhibits nicotine-evoked responses in treated cells to the
same level as observed in control preparations. Similarly,
mecamylamine reduced responses in treated cells to a level
comparable to that observed in control cells (Figure 5).
MLA (10 nM) inhibited the response to nicotine by 40+8%
(n=5; Figure 4), in agreement with the results presented in
Figure 2. After chronic nicotine treatment, the response to
nicotine stimulation was reduced by 27+13% (n=3)
compared with control cells. Surprisingly, in the presence
of acute MLA, the nicotine-evoked change in [Ca2+]i was
enhanced, and was comparable to or greater than that
elicited from untreated cells in the absence of MLA. After
chronic KCl treatment, the response to nicotine stimulation
was reduced by 26+7% (n=4) in the absence of MLA and
34+5% (n=7) in the presence of MLA (Figure 4). Thus
MLA had no signi®cant e€ect on this response in KCl-
treated cells.
To explore the e€ects of other nicotinic antagonists on
the nicotine-evoked increase in [Ca2+
]
in chronically
i
nicotine-treated SH-SY5Y cells, we employed a 96 well
format higher throughput assay, that uses ¯uo-3 instead of
fura-2 to monitor Ca2+ levels in adherent cells (see
Methods). Using this assay, the dose-response relationship
for
nicotine-evoked
responses
(Figure
5,
insert;
EC50=21 mM) was similar to that derived using fura-2
(Figure 1c). A higher nicotine concentration (30 mM) was
used in subsequent experiments, in order to elicit more
robust and consistent increases in Ca2+. These responses in
British Journal of Pharmacology vol 135 (4)

1056
D.L. Ridley et al
Nicotinic responses in SH-SY5Y cells
Figure 5 Comparison of the e€ects of nicotinic AChR antagonists
on nicotine-evoked increases in [Ca2+]i in SH-SY5Y cells chronically
treated with nicotine. SH-SY5Y cells were grown in 96 well plates
and treated with (black columns) or without (open columns) 10 mM
nicotine for 4 days, thoroughly washed (six times over 4 h) and
loaded with ¯uo-3 AM. Cells were incubated with antagonist: MLA,
a-Bgt, a-conotoxin-ImI (aCtx-ImI), a-conotoxin-MII (aCtx-MII) or
mecamylamine (Mec) for 10 min before stimulation with nicotine
(30 mM, Nic). Changes in Ca2+ in ¯uo-3-loaded SH-SY5Y cells were
monitored using a 96 well format assay (see Methods). Responses are
expressed as a percentage of the response to nicotine in untreated
cells cultured and assayed in parallel, in the absence of antagonist.
The dashed line indicates the response to nicotine in nicotine-treated
cells, assayed in the absence of antagonist. Values are the mean+
s.e.mean of at least three independent experiments. *Signi®cantly
di€erent from untreated control in the absence of MLA, P50.05,
#signi®cantly di€erent from untreated control in the presence of
MLA, P50.05, one way ANOVA. Insert: concentration-response
curve for nicotine-evoked increases in Ca2+ in untreated SH-SY5Y
cells, monitored using the 96 well format assay. Data points are the
mean+range for two independent experiments, each with six
replicates for every concentration, and are ®tted to the Hill equation,
giving an EC50 value of 20.9 mM.
Figure 6 Nicotine-evoked increases in [Ca2+
] in SH-SY5Y cells
i
measured 24 h after chronic treatment with nicotine. SH-SY5Y cells
were treated with (black columns) or without (open columns) 10 mM
nicotine for 4 days, thoroughly washed (six times over 4 h) and
incubated in normal medium for 20 h before loading with ¯uo-3-AM
and measurement of [Ca2+]i. Cells were incubated with MLA, a-Bgt
or mecamylamine (Mec) for 10 min before stimulation with nicotine
(30 mM, Nic). Changes in Ca2+ in ¯uo-3-loaded SH-SY5Y cells were
monitored using a 96 well format assay (see Methods). Responses are
expressed as a percentage of the response to nicotine in untreated
cells cultured and assayed in parallel, in the absence of antagonist.
Results are from one experiment with six replicates for each
condition.
Discussion
In this study we have shown that both a7 and a3* subtypes
of nicotinic AChR contribute to nicotine-evoked increases in
[Ca2+]i in SH-SY5Y cells. We have examined the e€ects of
chronic treatments with nicotinic agonists and KCl on
subsequent nicotine-evoked increases in [Ca2+]i. Functional
responses in nicotine- or DMAC-treated cells were decreased,
compared with responses from untreated control cells. This
appears to re¯ect, predominantly, the loss of the a7 subtype-
mediated component of the response judged by the loss of
antagonism by a-conotoxin-lml and a-Bgt, although the a7-
selective antagonist MLA provoked an unexpected increase
in nicotine-evoked Ca2+ ¯uorescence. This action of MLA
appeared to be nicotinic AChR-mediated as it was prevented
in the presence of mecamylamine. Responses to nicotine were
also diminished in cells treated with KCl for 4 days, but in
this case MLA had no e€ect.
To ascertain if the paradoxical increase observed in the
presence of MLA was nicotinic AChR-mediated, MLA was
applied in combination with mecamylamine. In this case,
nicotine-evoked responses were not signi®cantly di€erent
between treated and control cells, and did not di€er signi®cantly
from nicotine-evoked responses in the presence of mecamyla-
mine alone (Figure 5). However, MLA in combination with a-
conotoxin MII did result in a somewhat higher nicotine-evoked
response in treated cells compared with untreated cells (Figure
5), suggesting that MLA can still facilitate an increased
response when a3b2* nicotinic AChR are blocked.
To examine the reversibility of the e€ects of chronic
nicotine treatment, and the enhancement by MLA of
nicotine-evoked responses, SH-SY5Y cells were treated with
nicotine for 4 days, thoroughly washed as before and then
incubated in normal medium for 20 h before measuring
nicotine-evoked Ca2+ responses (Figure 6). It is clear from
this experiment that the reduced response to nicotine
persisted, and the pharmacology of responses from control
and treated cells was the same as observed in assays carried
out immediately after the removal of chronically applied
nicotine (Figures 4 and 5). In particular, the enhanced
response to nicotine in the presence of MLA in chronically
treated cells was preserved (Figure 6).
The human neuroblastoma SH-SY5Y cell line gave robust
and reproducible increases in [Ca2+]i in response to nicotine
and KCl, in agreement with previous studies (Lambert et al.,
1990). SH-SY5Y cells endogenously express a3,a5,a7,b2 and
b4 nicotinic AChR subunits (Lukas et al., 1993; Peng et al.,
1994), which form a number of receptor subtypes (for
example a3b2, a3a5b2, a3b4, a3a5b4, a3b2b4 and
a3a5b2b4, in addition to a7; Wang et al., 1996). Three a7-
selective antagonists and the a3b2*-selective antagonist a-
conotoxin-MII provide evidence that both of these types of
nicotinic AChR contribute to the observed increase in [Ca2+
]
i
British Journal of Pharmacology vol 135 (4)

D.L. Ridley et al
Nicotinic responses in SH-SY5Y cells
1057
in response to 10 mM nicotine (Figure 2). The incomplete
blockade of the nicotine-evoked response by MLA and a-
conotoxin-MII together (at concentrations that should
maximally inhibit a7 and a3b2* nicotinic AChR respectively,
Alkondon et al., 1992; Kaiser et al., 1998) suggests that
a3b4* receptors also participate. Despite the rapid desensi-
tization of neuronal nicotinic AChR (especially the a7
subtype, Seguela et al., 1993), nicotine-induced increases in
[Ca2+]i were sustained (Figure 1a), as recently observed in
CNS neurones (Dajas-Bailador et al., 2000; Tsuneki et al.,
2000). The long lasting response may re¯ect the fact that
most of the increase in [Ca2+]i could be attributed to voltage-
operated Ca2+ channels (including verapamil-sensitive L-type
channels), activated in response to nicotinic AChR-induced
membrane depolarization. Furthermore, a contribution from
internal Ca2+ stores may also participate in the observed
response (Tsuneki et al., 2000, Dajas-Bailador et al., 2001).
Exposure to 20 mM KCl for 4 days upregulates [125I]-a-Bgt
binding sites in SH-SY5Y cells by about 50% but has no
e€ect on numbers of [3H]-epibatidine binding sites (Ridley et
function are required to clarify the relationship. The
possibility that verapamil may interact with nicotinic AChR
(especially the a3b4* subtype; Herraro et al., 1999) further
complicates the analysis.
Chronic nicotine and DMAC treatments, in contrast to KCl,
upregulate the numbers of both [125I]-a-Bgt and [3H]-epibati-
dine binding sites in SH-SY5Y cells, by a mechanism
independent of voltage-operated Ca2+ channels (Ridley et al.,
2001). After these treatments, responses to acute stimulation
with 10 mM nicotine (but not to KCl) were signi®cantly
decreased by about 25% (Figures 3 and 4), similar to the
decrease seen after chronic KCl treatment. The diminished
response was equivalent to that observed in untreated cells
assayed in the presence of a7 nicotinic AChR antagonists
(Figure 5). Indeed, a-Bgt and a-conotoxin-ImI failed to inhibit
responses from nicotine treated cells, whereas a-conotoxin-MII
still produced some inhibition. These observations are
consistent with the selective loss of the a7 nicotinic AChR-
mediated component of the nicotine-evoked increase in [Ca2+]i.
However, in the presence of acute MLA, nicotine provoked
an unexpected increase in intracellular Ca2+, compared with
responses to nicotine alone, in nicotine-treated cells only
(Figures 4 and 5); this e€ect was observed using two
al., 2001). The upregulation of
[
125I]-a-Bgt sites occurs
through a voltage-operated Ca2+ channel and CaM-kinase
II dependent mechanism (Ridley et al., 2001), and probably
involves an increase in a7 gene transcription (De Koninck &
Cooper, 1995). Despite the increase in numbers of surface
¯uorescence assays to measure Ca2+
. Thus MLA acts
di€erently from the other a7-selective antagonists employed
in this study. MLA does not appear to achieve this unexpected
response through a non-speci®c action (for example, by
interacting directly with voltage-operated ion channels) as its
e€ect was abolished by co-application with the non-selective,
non-competitive nicotinic antagonist mecamylamine (but not
by the a3b2*-selective antagonist a-conotoxin-MII, Figure 5).
This is consistent with an a7 nicotinic AChR-mediated action.
Also, the lack of reversibility (within 24 h after the removal of
chronic nicotine) of the diminished responsiveness to nicotine
alone and its enhancement in the presence of acute MLA
suggests a common process.
Chronic treatment with MLA (albeit at relatively high
concentrations) upregulates numbers of a7 nicotinic AChR
binding sites in hippocampal neurones (Ridley et al., 2001)
and in transfected HEK-293 cells (Molinari et al., 1998). In
the latter study, nicotine-evoked whole cell currents recorded
from the transfected cells were increased after chronic
nicotine or MLA treatment, suggesting that upregulated
nicotinic AChR can be functional. Concentrations of nicotine
(1 mM) and MLA (10 mM) that elicited comparable levels of
upregulation of [125I]a-Bgt binding sites (approximately 3
fold) resulted in di€erent degrees of responsiveness, with 2
fold and 6 fold increases in whole cell currents, respectively
(Molinari et al., 1998). This di€erential modulation by
agonist and antagonist ligands was suggested to re¯ect their
stabilization of di€erent states of the nicotinic AChR. In the
present study, a7 nicotinic AChR upregulated by nicotine
[
125I]-a-Bgt binding sites (Ridley et al., 2001), there was a
signi®cant (26%) decrease in the nicotine-evoked elevation of
[Ca2+
after chronic KCl treatment, whereas KCl-evoked
]
i
responses were unchanged (Figure 3). As nicotine-evoked
responses from KCl-treated SH-SY5Y cells were insensitive
to acute MLA and of the same magnitude as those observed
in untreated cells assayed in the presence of MLA (Figure 4),
the a7 nicotinic AChR-mediated component of the nicotine-
evoked increase in [Ca2+]i appears to have been selectively
lost. This interpretation is consistent with the selective e€ect
of chronic KCl treatment on a7 (but not a3*) nicotinic AChR
binding sites (Ridley et al., 2001). The mechanism whereby
chronic KCl depolarization leads to the elimination of a7
nicotinic AChR-mediated responses (either inactivation of the
a7 receptor itself or of a point in its downstream pathway
that leads to increases in [Ca2+]i), remains to be elucidated.
It is likely that chronic depolarization would a€ect a
number of cellular processes that might in¯uence the
regulation of [Ca2+]i. One candidate is the voltage-operated
Ca2+ channel: in PC12 cells, exposure to elevated extra-
cellular KCl (50 mM) for
4 days caused concomitant
decreases in numbers of [3H]-nitrendipine binding sites and
depolarization-dependent 45Ca2+ entry (De Lorme et al.,
1988), implicating a modi®cation of L-type Ca2+ channels.
Nevertheless, activation of L-type Ca2+ channels underlies
the KCl-induced upregulation of a7 mRNA (De Koninck &
Cooper, 1995) and [125I]-a-Bgt binding sites (Ridley et al.,
2001), as shown by its inhibition by verapamil. In the present
study, KCl-evoked increases in [Ca2+]i were not signi®cantly
changed after chronic KCl treatment. On the other hand,
chronic verapamil treatment (which is anticipated to down-
regulate L-type Ca2+ channels, Panza et al., 1985, but which
has no e€ect on numbers of [125I]-a-Bgt or [3H]-epibatidine
binding sites in SH-SY5Y cells, Ridley et al., 2001), provoked
substantial decreases (50%) in responses to both nicotine and
KCl depolarization (Figure 3). These observations are
dicult to reconcile and more direct measures of ion channel
(but not those upregulated by KCl) may be in
a
conformation which is converted by interaction with MLA
to a conducting state upon agonist binding. The persistence
of the decreased responsiveness to nicotine (in the absence of
MLA) for at least 20 h after removal of the chronically
applied agonist (Figure 6) implies sustained inactivation,
rather than reversible desensitization, of the response. It is
not clear why enhanced responses to nicotine were observed
by Molinari et al. (1998) after chronic nicotine treatment.
Di€erences from the present study include over-expression of
British Journal of Pharmacology vol 135 (4)

1058
D.L. Ridley et al
Nicotinic responses in SH-SY5Y cells
a7 nicotinic AChR in a non-neuronal cell line, and a more
direct measurement of receptor activity using whole cell
recording techniques. The ability of MLA to block nicotine-
evoked currents recorded from the chronically treated HEK-
293 a7-expressing cells was not described, but would be of
interest in the light of the present results: MLA may be a
useful ligand to probe the relationship between di€erent
conformations of the a7 nicotinic AChR.
and KCl treatments abolished the a7 nicotinic AChR-
mediated component of the nicotine-evoked increase in
intracellular Ca2+. The a7-selective antagonist MLA, acutely
applied, was unique in producing a paradoxical enhancement
of nicotine-evoked response, (after chronic nicotine but not
chronic KCl), thus nicotine and KCl treatments exert subtle
di€erential e€ects on nicotinic AChR in SH-SY5Y cells.
In this study we have characterized the functional
responses to stimulation with nicotine and KCl, after chronic
drug treatments previously shown to modulate nicotinic
AChR numbers. None of the treatment regimes that
increased receptor numbers produced any increase in the
[Ca2+]i response stimulated by 10 mM nicotine or 20 mM KCl,
compared with untreated controls. Rather, chronic nicotine
This study was supported by a MRC Collaborative Studentship (to
D. Ridley) in conjunction with Organon Laboratories, Newhouse,
Scotland, and a grant from BAT Co. (to S. Wonnacott). J.
Pakkanen was supported by the Socrates-Erasmus Programme of
the Commission of the European Communities. We are grateful to
Organon Laboratories for the provision of DMAC.
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(Received August 7, 2001
Revised November 7, 2001
Accepted November 15, 2001)
British Journal of Pharmacology vol 135 (4)