Chronic Nicotine Treatment Up-Regulates 3 and 7
Acetylcholine Receptor Subtypes Expressed by the Human
Neuroblastoma Cell Line SH-SY5Y
XIAO PENG, VOLODYMYR GERZANICH, REN
´
E ANAND, FAN WANG, and JON LINDSTROM
Departments of Neuroscience (X.P., V.G., F.W., J.L.) and Pharmacology (R.A.), University of Pennsylvania Medical School, Philadelphia,
Pennsylvania 19104-6074
Received September 5, 1996; Accepted January 20, 1997
SUMMARY
Chronic exposure to nicotine has been reported to increase the
number of nicotinic acetylcholine receptors (AChRs) in brain.
The mechanism of up-regulation for the 42 AChR subtype,
which accounts for the majority of high affinity nicotine binding
in mammalian brain, has previously been shown to involve a
decrease in the rate of 42 AChR turnover. Here, we report an
investigation of the extent and mechanism of nicotine-induced
up-regulation of 3 AChRs and 7 AChR subtypes expressed
in the human neuroblastoma cell line SH-SY5Y. Up-regulation
of human 3 AChRs and 7 AChRs, unlike 42 AChRs, re-
quires much higher nicotine concentrations than are encoun-
tered in smokers; the extent of increase of surface AChRs is
much less; and the mechanisms of up-regulation are different
than with 42 AChRs. The mechanisms of up-regulation may
be different for 3 AChRs or 7 AChRs. Chronic treatment with
nicotine or carbamylcholine, but not d-tubocurarine, mecamyl-
amine, or dihydro--erythroidine, induced a 500 – 600% in-
crease in the number of 3 AChRs but only a 30% increase in
7 AChRs. Chronic nicotine treatment did not increase affinity
for nicotine or increase the amount of RNA for 3 or 7 sub-
units. The effect of nicotine on up-regulation of 7 AChRs was
partially blocked by either d-tubocurarine or mecamylamine.
The effect of nicotine treatment on the number of 3 AChRs
was only slightly blocked by the antagonists d-tubocurarine,
mecamylamine, or dihydro--erythroidine at concentrations
that efficiently block 3 AChR function. Most of the nicotine-
induced increase in 3 AChRs was found to be intracellular.
The 3 AChRs, which accumulate intracellularly, were shown to
have been previously exposed on the cell surface by their
susceptibility to antigenic modulation. The data suggest that
chronic exposure to nicotine may induce a conformation of cell
surface 3 AChRs that at least in this cell line are consequently
internalized but not immediately destroyed.
It is well established that chronic nicotine exposure results
in increased binding of [
3
H]nicotine and
125
I-Bgt in brain
(1– 8). AChRs composed of 4 and 2 subunits have high
affinity for nicotine and ACh and account for most of the high
affinity nicotine binding in rat brain (9 –11). AChRs com-
posed of 3 subunits in combination with 2, 4, and/or 5
subunits have lower affinity for ACh and nicotine than do
42 AChRs and account for a small amount of high affinity
nicotine binding in brain (12). Flores et al. (4) showed that
42 AChRs are increased in the cortex of rats chronically
treated with nicotine. In addition, Collins et al. (5, 6) reported
that chronic exposure to nicotine or the antagonist
mecamylamine increased mouse brain [
3
H]nicotine binding
in numerous regions to various extents without increasing
the levels of 4 or 2 AChR subunit mRNAs. We found that
chronic treatment of Xenopus laevis oocytes expressing 42
AChRs or a mouse fibroblast cell line permanently trans-
fected with chicken 42 AChRs with nicotine or
mecamylamine caused a 2-fold increase in 42 AChRs
(13). The nicotine concentration dependence, time course,
and extent of 42 AChR up-regulation are similar to those
reported for 42 AChRs in mammalian brains. The nico-
tine-induced increase in 42 AChRs is due to a decrease in
the rate of 42 AChR turnover (13). This induction mecha-
nism does not seem to require cation flow through 42
AChRs because the channel blocker mecamylamine causes
up-regulation (6, 13). Nicotine and mecamylamine are syn-
ergistic in causing up-regulation (6, 13) because
mecamylamine preferentially blocks open channels and nic-
otine is an agonist, so together they are more effective at
accumulating the inactive conformation of 42 AChR,
J.L. is supported by grants from the National Institute of Neurological and
Communicative Disorders and Stroke (NS11323), Muscular Dystrophy Asso-
ciation, Council for Tobacco Research, USA, Inc., and Smokeless Tobacco
Research Council, Inc., and R.A. is supported by Grant NS33625 from the
National Institute of Neurological and Communicative Disorders and Stroke.
ABBREVIATIONS: Bgt, -bungarotoxin; AChR, acetylcholine receptor; mAb, monoclonal antibody; DHE, dihydro--erythroidine; EGTA,
ethylene glycol bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; SSPE, standard
saline/phosphate/EDTA; MOPS, 3-(N-morpholino)propanesulfonic acid.
0026-895X/97/050776-09$3.00/0
Copyright © by The American Society for Pharmacology and Experimental Therapeutics
All rights of reproduction in any form reserved.
MOLECULAR PHARMACOLOGY, 51:776 –784 (1997).
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