Differing temporal roles of
Ca2+ and cAMP in
nicotine-elicited elevation of tyrosine hydroxylase mRNA
Volodia D.
Gueorguiev1,
Richard
J.
Zeman2,
Bhargava
Hiremagalur1,
Ana
Menezes1, and
Esther L.
Sabban1
Departments of 1 Biochemistry
and Molecular Biology and 2 Cell
Biology and Anatomy, New York Medical College, Valhalla, New York
10595
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ABSTRACT |
The involvement of cAMP- and
Ca2+-mediated pathways in the
activation of tyrosine hydroxylase (TH) gene expression by nicotine was
examined in PC-12 cells. Extracellular
Ca2+ and elevations in
intracellular Ca2+ concentration
([Ca2+]i)
were required for nicotine to increase TH mRNA. The nicotine-elicited rapid rise in
[Ca2+]i
was inhibited by blockers of either L-type or N-type, and to a lesser
extent P/Q-, but not T-type, voltage-gated
Ca2+ channels. With continual
nicotine treatment,
[Ca2+]i
returned to basal levels within 3-4 min. After a lag of
~5-10 min, there was a smaller elevation in
[Ca2+]i
that persisted for 6 h and displayed different responsiveness to
Ca2+ channel blockers. This second
phase of elevated
[Ca2+]i
was blocked by an inhibitor of store-operated
Ca2+ channels, consistent with the
observed generation of inositol trisphosphate.
1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), when added before or 2 h after nicotine,
prevented elevation of TH mRNA. Nicotine treatment significantly raised cAMP levels. Addition of the adenylyl cyclase inhibitor
2',5'-dideoxyadenosine (DDA) prevented the
nicotine-elicited phosphorylation of cAMP response element binding
protein. DDA also blocked the elevation of TH mRNA only when added
after the initial transient rise in [Ca2+]i
and not after 1 h. This study reveals that several temporal phases are
involved in the induction of TH gene expression by nicotine, each of
them with differing requirements for
Ca2+ and cAMP.
adenylyl cyclase; voltage-gated calcium channels; adenosine
3',5'-cyclic monophosphate response element binding protein
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INTRODUCTION |
EXPOSURE TO NICOTINE, a major component of cigarette
smoke, produces many physiological changes and increases the risk of coronary and peripheral vascular disease. As a potent agonist of
nicotinic acetylcholine receptors, nicotine triggers rapid secretion of
catecholamines. The nicotine-triggered elevations in plasma
catecholamine levels from the sympathetic nerve endings and the adrenal
medulla are associated with alterations in heart rate and arterial
pressure (13). Nicotine treatment also increases catecholamine
biosynthesis by phosphorylation and rapid activation of tyrosine
hydroxylase (TH), the first and major rate-limiting enzyme in
catecholamine biosynthesis (23). In addition, prolonged exposure to
nicotine for several days was found to elicit elevations in gene
expression of rat adrenomedullary catecholamine biosynthetic enzymes as
well as of several neuropeptides (neuropeptide Y and enkephalin) and
other constituents of chromaffin vesicles that can be coreleased with
the catecholamines (17, 18, 20). In the rat adrenal medulla,
transcriptional mechanisms were shown to be involved in the induction
of TH gene expression (9).
Cultured cells of adrenomedullary origin (bovine chromaffin and PC-12
cells) have been used to examine the underlying mechanisms by which
nicotine activates gene expression. In these cells, as was found in
vivo, nicotine increased the levels of mRNA for TH, as well as for
dopamine
-hydroxylase, proenkephalin, preproneuropeptide Y, and
several soluble proteins of chromaffin granule cores (4, 16, 36, 40).
However, the precise mechanism for nicotine-driven gene expression is
still unclear, and conflicting results have been reported. Several
signaling pathways have been implicated in mediating the effect of
nicotine on gene expression in cells of adrenomedullary origin. These
pathways include the activation of protein kinase C (PKC),
Ca2+/calmodulin-dependent protein
kinases, and/or protein kinase A (PKA), which can phosphorylate
cAMP response element binding protein (CREB) and lead to its
transactivation. Several transcription factors also respond to nicotine
treatment. Nicotine not only elicits the phosphorylation of CREB but
also rapidly enhances c-fos
transcription, which precedes a slower rise in
c-jun and junB mRNA levels (11, 36, 40).
C-fos has been proposed to induce
nicotine-stimulated proenkephalin transcription (40). However, the
nicotine induction of TH gene transcription is reportedly independent
of c-fos gene activation (5).
Experiments using transient transfection of PC-12 cells with reporter
constructs of the TH promoter mapped the nicotine response to the
cAMP/Ca2+ response element
(CRE/CaRE) (16). Similarly, CRE sites in the chromogranin A and
proenkephalin promoters also mediated the nicotine-induced activation
of these genes (36, 40).
Upon nicotinic stimulation, an influx of extracellular
Ca2+ and
Na+ occurs via nicotinic
receptors, resulting in membrane depolarization and the recruitment of
voltage-gated Ca2+ channels that
promote Ca2+ entry, leading to a
rapid increase in intracellular
Ca2+ concentration
([Ca2+]i)
(33). Studies by Craviso et al. (4, 5) suggested that the influx of
extracellular Ca2+ is necessary
for the effect of nicotine on TH gene expression, since nitrendipine,
an L-type Ca2+ channel blocker,
prevented the elevation of c-fos and
TH mRNA levels in bovine chromaffin cells treated with the nicotinic
agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP). The extent of
induction depended on the extracellular
Ca2+ concentration.
In addition to nicotine, a number of treatments that raise
[Ca2+]i,
either from extracellular or intracellular sources, activate TH gene
expression (31). Thus elevated levels of
K+, veratridine, ionomycin, and
bradykinin activate TH transcription (29, 30, 32). However, previous
studies have also indicated discrepancies between the activating
mechanisms of these compounds and that triggered by nicotine. Like
nicotine, ionomycin and elevated K+ were found to increase TH
promoter activity via the CRE/CaRE site (21, 31). The
ionomycin-elicited induction of TH promoter activity and the
phosphorylation of CREB were observed in normal and in PKA-deficient
PC-12 cells (31). In contrast, the nicotine-triggered activation of TH
gene expression did not occur in the PKA-deficient cell lines,
suggesting that PKA is needed for the induction by nicotine. Consistent
with this report, cAMP analogs and nicotinic receptor agonists exhibit
nonadditive effects on TH mRNA levels (5, 34) despite exerting additive
effects on chromogranin A promoter activity (35).
In this study, we explored the involvement of cAMP-mediated events and
increased
[Ca2+]i
in the nicotine-triggered induction of TH gene expression. The
elevation of TH mRNA by nicotine was prevented by either chelation of
extracellular Ca2+ or adenylyl
cyclase inhibition. The types of channels involved were examined with
selective antagonists. The rise in
[Ca2+]i
is biphasic, with a second prolonged but moderate increase after the
initial transient rise. Our results indicate that this second rise in
[Ca2+]i
is necessary for activation of TH gene expression and suggest that
several temporal phases with different requirements for
Ca2+ and cAMP are involved in the
induction of TH gene expression by nicotine.
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MATERIALS AND METHODS |
Materials were obtained as follows: DMEM, streptomycin, penicillin, and
the Select-Amine kit were obtained from GIBCO BRL (Gaithersburg, MD),
tissue culture dishes were from Falcon (Lincoln Park, NJ), and Calcium
Green-1-AM, fura 2-AM,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, and EGTA-AM were purchased from Molecular Probes (Eugene, OR).
-Conotoxins (GVIA, MVIIA, and MVIIC) were from Alomone
Labs (Jerusalem, Israel). The primary antibodies specific for CREB and
phosphorylated CREB (P-CREB) (10) were purchased from Upstate
Biotechnology (Lake Placid, NY). Alkaline phosphatase-conjugated secondary antibody was obtained from Promega (Madison, WI), and the
enhanced 3,3'-diaminobenzidine tetrahydrochloride (DAB) substrate was purchased from Pierce (Rockford, IL). Fetal bovine serum and donor
horse serum were obtained from JRH Biosciences (Lenexa, KS). W-7 and
2',5'-dideoxyadenosine (DDA) were from Calbiochem (San
Diego, CA),
[
-32P]dCTP and
myo-[3H] inositol were
from DuPont NEN Research Products (Boston, MA), and nicotine
bi-D-tartrate was from RBI
(Natick, MA). All other reagents were purchased from Sigma Chemical
(St. Louis, MO) and were of reagent grade unless specified.
Treatment of cells.
PC-12 cells were maintained in DMEM supplemented with 10% fetal bovine
serum, 5% heat-inactivated donor horse serum, 50 µg/ml streptomycin,
and 50 IU/ml penicillin in a humidified atmosphere at 37°C and 7%
CO2, as described previously (16).
Cells were treated at a medium density (~3 × 105
cells/cm2). For
nicotine treatment, nicotine solution in sterile water was added to a
final concentration of 200 µM. For elevated
K+ treatment, osmotically balanced
medium with 50 mM K+ was prepared
using the Select-Amine kit (GIBCO BRL) as previously described (21,
29). In some experiments, cells were pretreated with EGTA (5 mM),
EGTA-AM or BAPTA-AM (10 µM), nifedipine (10 µM), econazole (100 nM,
5 µM, or 10 µM), DDA (10 or 100 µM), calciseptine (300 nM) or
-conotoxins (GVIA, MVIIA, or MVIIC; 500 nM), or flunarizine (1 µM)
for 10 min. For experiments with medium without
Ca2+, the medium was prepared by
using the Select-Amine kit (GIBCO BRL) with all the components except
the Ca2+ salts. At least three or
four duplicate cell culture plates were used in each experiment. All
experiments were performed at least twice.
Northern blot analysis.
At the times indicated, cells were washed once with PBS and pelleted.
Total RNA was isolated, and Northern blot analysis was performed as
previously described (16). Briefly, total RNA (15 µg) was
fractionated through 1.3% agarose gels containing 2.2 M formaldehyde
and 1× MOPS buffer [20 mM MOPS (pH 7.0), 5 mM sodium acetate, and 1 mM EDTA], transferred to GeneScreen Plus (NEN), and baked for 2 h at 80°C in a vacuum oven. Filters were
prehybridized in a mixture of 50% formamide, 5× Denhardt's
solution, 5× SSPE (1× SSPE is 0.15 M NaCl, 10 mM
NaH2PO4,
and 1 mM EDTA), and 0.4% SDS at 42°C for 4 h. Hybridizations were
then performed consecutively using a 1.1-kb
EcoR I fragment from the rat TH cDNA
and a DNA probe for 18S rRNA (as previously described in Ref. 16)
labeled with
[
-32P]dCTP by using
the random primer method (Megaprime, Amersham). The labeled probes were
heat denatured (90°C, 5 min) and
~105 dpm/ml were added to the
prehybridization solution and hybridized at 42°C for 18 h. After
hybridization, the filters were washed twice with 2× SSPE and
once with 0.2× SSPE and 1% SDS at room temperature for 30 min.
The filters were then exposed to X-ray films for various times. In
addition, the autoradiographic images were captured with a
charge-coupled device (CCD) camera (Datavision), and the ratio of TH
mRNA to 18S rRNA was quantified by performing densitometric analyses
within the linear range of each captured signal by using the Image Pro
Plus software (Media Cybernetics, Silver Spring, MD).
[Ca2+]i
measurements.
PC-12 cells were grown in 25-mm glass coverslip chambers (Nunc)
previously coated with collagen. The cells were loaded with 3 µM fura
2-AM or 15 µM Calcium Green-1-AM for 30 min at 37°C. Alterations
in
[Ca2+]i
were measured by analyzing the ratio of fura 2 fluorescence (>480 nm)
excited at 340 and 380 nm. Fluorescent images of fura 2-loaded PC-12
cells were captured with a Nikon Diaphot fluorescence microscope
equipped with a Quantex QX-7 CCD camera and a digital imaging system,
as previously described (31). The
[Ca2+]i
of individual cells was calculated as described by Grynkiewicz et al.
(12) after the average values of pixels overlying each cell in ratioed
(340 nm/380 nm) images were obtained. A value of 224 nM was used for
the dissociation constant of fura
2-Ca2+. For confocal images,
Calcium Green-1-loaded cells were visualized with a Bio-Rad MRC-1000
confocal microscope. Calcium Green-1 loaded cells were illuminated with
an argon ion laser at a wavelength of 488 nm, the resulting
fluorescence (>515 nm) was imaged, and the average pixel value of
each cell was obtained. As an indication of changes in
[Ca2+]i,
the fluorescence of nicotine-treated cells was expressed relative to
untreated cultures. At least four microscopic fields in two or three
separate culture dishes were analyzed for each treatment.
Analysis of inositol phosphates.
Measurement of inositol phosphates was performed as previously
described (29). PC-12 cells were prelabeled with
myo-[3H]inositol
(6 µCi/ml) for 48 h at 37°C. Cells were incubated for 10 min in
medium containing 10 mM LiCl to inhibit inositol phosphatases and were
exposed at different time points to 200 µM nicotine, 50 mM
K+, or 1 µM bradykinin. The
supernatants after homogenization in 10% ice-cold TCA were extracted
with diethyl ether, neutralized with NaOH (pH 6.5-7.5), and
applied to a Dowex AG1-8X column to isolate the inositol monophosphate,
inositol bisphosphate, and inositol trisphosphate
(IP3) by a step gradient. The
amount of newly synthesized inositol phosphates was determined by
scintillation counting.
Immunocytochemistry.
Cells were plated in triplicate on 24-well tissue culture plates and
allowed to attach overnight. After treatment with nicotine or DDA for
10 min, the cells were rinsed once with PBS and fixed in 0.7 ml of 4%
paraformaldehyde in PBS at room temperature for 45 min. The cells were
then given three 5-min washings in PBS containing 10 mM glycine and
permeabilized by incubation in freshly prepared PBS with 0.5% NP-40 at
room temperature for 30 min. After a rinsing with PBS containing 5 mM
sodium fluoride and 1 mM ammonium molybdate, cells were incubated at
room temperature for 2 h in a PBS-based blocking solution containing
3% BSA. Subsequently, the cells were incubated with 0.7 µg/ml
anti-P-CREB antibody for 24 h at 4°C. After the incubation, the
cells were washed three times (5 min each wash) in PBS and incubated in
excess secondary antibody [goat anti-rabbit antibody at a 1:200
(vol/vol) dilution] for 2 h at room temperature. After three
washes of 5 min each in PBS at room temperature, incubations in
enhanced DAB substrate for 5-10 min were performed. Finally, the
cells were washed with tap water, mounted, and observed for nuclear
staining. The anti-P-CREB antibodies used in this study were raised
against a phosphopeptide corresponding to amino acids 123-136 of
CREB (10).
cAMP determination.
The cAMP content of the cells was measured as follows: individual PC-12
cell cultures were treated with 200 µM nicotine for 15 min and 24 h,
the media were removed by aspiration, and 1 ml of ice-cold 10% TCA was
added to each sample. The TCA extracts were then washed five times with
3 ml of ether, and the aqueous phases were dried under a stream of
nitrogen gas and reconstituted in 1 ml of 0.005 M sodium acetate (pH
5.8). Subsequently, cAMP was acetylated and the levels were quantitated
by an enzyme immunoassay using the Biotak dual-range enzyme immunoassay
kit (Amersham Corp, IL) according to the manufacturer's specifications.
Statistical analysis.
Statistical significance was determined by Student's
t-test for experiments with two groups
or by an ANOVA followed by Fisher's least significant difference test
for experiments with more than two groups. Levels of
P < 0.05 were accepted as
statistically significant.
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RESULTS |
Increased
[Ca2+]i
is required for nicotine-stimulated elevations of TH mRNA levels.
Previous experiments revealed that treatment of PC-12 cells with 10 µM to 1 mM nicotine elicited rapid rises in
[Ca2+]i.
Concentrations of 50-200 µM nicotine caused maximal increases in
the amounts of TH, chromogranin A, and
c-fos mRNAs (11, 16, 36). To ascertain
whether extracellular Ca2+ and
increased
[Ca2+]i
were required for nicotine-triggered upregulation of TH mRNA, extracellular Ca2+ was reduced by
using media either prepared without added
Ca2+ or containing 5 mM EGTA. Both
of these conditions prevented the induction of TH mRNA expression by
nicotine (Fig. 1), indicating a requirement
for extracellular
Ca2+.

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Fig. 1.
Effect of reduction in extracellular or intracellular
Ca2+ on nicotine-induced increases
in intracellular Ca2+
concentration
([Ca2+]i)
and tyrosine hydroxylase (TH) gene expression. PC-12 cells were
incubated for 6 h in absence or presence of 200 µM nicotine alone or
in several conditions that reduce extracellular or intracellular
Ca2+. EGTA was at 5 mM, and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA)-AM and EGTA-AM were at 10 µM.
Ca2+-free medium was prepared as
described in MATERIALS AND METHODS. In
all cases, cells were preincubated for 10 min before addition of
nicotine and further incubated for 6 h. RNA was isolated, and levels of
TH mRNA were determined by Northern blot analysis. Data are means ± SE. * P < 0.01 compared with
control group. Inset: PC-12 cells were
first loaded with 3 µM fura 2 for 30 min.
[Ca2+]i
was then measured in control cells without BAPTA-AM treatment and in
cells pretreated with 10 µM BAPTA-AM before and at 1-min intervals
after addition of 200 µM nicotine.
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Next, the effects of intracellular
Ca2+ chelators were examined. In
these experiments, pretreatment with 10 µM BAPTA-AM (Fig. 1) or
EGTA-AM (not shown) prevented the nicotine-induced rise in
[Ca2+]i.
These concentrations of EGTA-AM or BAPTA-AM had no significant effect
on basal TH mRNA levels. However, both agents completely prevented the
elevation of TH mRNA levels in response to nicotine treatment,
indicating that the rise of
[Ca2+]i
is necessary for the induction of TH mRNA in response to nicotine.
Ca2+ channels
involved in nicotine-triggered rise in
[Ca2+]i.
In PC-12 cells, as well as in adrenal chromaffin cells, membrane
depolarization by nicotine leads to an influx of extracellular Ca2+ via voltage-gated
Ca2+ channels and the nicotinic
channel (33). We determined whether activation of voltage-gated
Ca2+ channels was required for TH
induction as well as which type of channel was involved. PC-12 cells
were treated with nicotine in the presence of two different L-type
Ca2+ channel blockers. Results
revealed that either the dihydropyridine blocker nifedipine (10 µM)
or the inhibitory peptide calciseptine (300 nM) (8) prevented the
nicotine-induced rise in
[Ca2+]i
(Figs. 2 and
3A).
Furthermore, Northern blot analysis showed that nifedipine prevented
the rise in TH mRNA levels in the presence of nicotine, without
affecting basal levels (Fig. 2).

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Fig. 2.
Effect of L-type Ca2+ channel
blocker nifedipine on nicotine-elicited elevation in
[Ca2+]i
and TH mRNA levels. PC-12 cells preloaded with 15 µM Calcium
Green-1-AM were pretreated with 10 µM nifedipine for 20 min, followed
by treatment with 200 µM nicotine.
[Ca2+]i
was then monitored by confocal microscopy.
Inset: representative Northern blot
showing TH mRNA levels. PC-12 cells were treated with nifedipine (Nf),
nicotine (N), or nifedipine and nicotine (N + Nf) or were untreated
(C). Equivalent amounts of total RNA loading were ensured by
hybridization with a probe for 18S rRNA (not shown).
* P < 0.01 compared with
controls.
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Fig. 3.
Effects of different types of Ca2+
channel blockers on
[Ca2+]i
in nicotine-treated PC-12 cells. Cells were loaded with 3 µM fura 2 for 30 min and then incubated with or without respective
Ca2+ channel blockers for 10 min.
Resulting
[Ca2+]i
was measured at 15-s intervals before and after addition of 200 µM
nicotine. A: L-type
Ca2+ channel blocker calciseptine
(300 nM). B: N-type channel blockers
-conotoxin GVIA and -conotoxin MVIIA (500 nM).
C: T-type channel blocker flunarizine
(500 nM). D: P/Q-type
Ca2+ channel blocker -conotoxin
MVIIC (500 nM). Data are means ± SE. Nic., nicotine.
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The effects of other voltage-sensitive
Ca2+ channel blockers on the rise
of
[Ca2+]i
triggered by nicotine were also examined. Pretreatment of cells with
the N-type channel blocker
-conotoxin GVIA (500 nM) also prevented
the rise in
[Ca2+]i
in the presence of 200 µM nicotine. Similarly, another N-type channel
blocker,
-conotoxin MVIIA (500 nM), greatly reduced the rise in
[Ca2+]i
caused by nicotine (Fig. 3B).
However, the T-type channel blocker flunarizine (1 µM) had little
effect on the extent of the rise, although the time course of the decay
was more rapid than that seen in the control cells (Fig.
3C). A P/Q-type
Ca2+ channel blocker,
-conotoxin MVIIC (500 nM), did not completely prevent the rise in
[Ca2+]i
but led to a substantial reduction of ~65% (Fig.
3D). These results indicate that
blockage of L-type, N-type, and to some extent P/Q-type voltage-gated
Ca2+ channels can eliminate or
greatly reduce the rise in
[Ca2+]i
elicited by nicotine. For comparison, the effects of some of these
inhibitors on the previously reported rapid rise in
[Ca2+]i
induced by 50 mM K+ (29) were
examined (Fig. 4). In contrast to their
blockade of the nicotine-elicited rise, the same concentrations of
calciseptine or
-conotoxin GVIA only partially prevented the
elevation of [Ca2+]i
in response to depolarization with elevated
K+. On the other hand, the effect
of
-conotoxin MVIIC was similar for both treatments.

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Fig. 4.
Effect of several voltage-dependent
Ca2+ channel blockers on rise in
[Ca2+]i
induced by nicotine (Nic) or elevated
K+. Fura 2-loaded PC-12 cultures
were pretreated for 10 min with 500 nM -conotoxin MVIIC, 500 nM
-conotoxin GVIA, or 300 nM calciseptine alone or together with 500 nM -conotoxin MVIIC. Then either 50 mM
K+ or 200 µM nicotine was added,
and changes in
[Ca2+]i
were measured. Values are presented as % of maximal increase from
average basal level (50 nM) to 120 nM for elevated
K+ and to 350 nM for nicotine.
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Time course of the elevation of
[Ca2+]i.
To examine the long-term effect of continuous nicotine treatment on
[Ca2+]i,
we treated PC-12 cells with 200 µM nicotine for up to 6 h (Fig.
5A).
Nicotine treatment elevated the
[Ca2+]i
within seconds of its addition, from a basal level of ~50 nM to a
level between 200 and 450 nM (Figs. 1, 3,
A-D,
and 5, A and C). These increments were followed
by a rapid decrease within several minutes. After a lag of ~5-10
min, a second elevation to 100-150 nM was observed. This second
elevation was stable for relatively long periods of time, and
[Ca2+]i
remained elevated at 80-110 nM (Fig.
5A) after 6 h of continuous exposure
to nicotine.

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Fig. 5.
Changes in
[Ca2+]i
with prolonged nicotine treatment. PC-12 cells were loaded with 3 µM
fura 2. A:
[Ca2+]i
was measured before and up to 6 h after addition of 200 µM nicotine.
B: cells treated with nicotine for 6 h
were exposed to either 300 nM calciseptine, 500 nM -conotoxin GVIA,
or 10 µM econazole. C: PC-12 cells
were pretreated with 100 nM or 5 µM econazole 10 min before addition
of nicotine. Data are means ± SE.
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The dependence of this prolonged rise in
[Ca2+]i
on Ca2+ channels was examined
(Fig. 5B). In contrast to its
inhibition of the initial rise in
[Ca2+]i
induced by nicotine, calciseptine did not alter the
[Ca2+]i
under these conditions. However, addition of the N-type channel blocker
(500 nM
-conotoxin GVIA) to cells treated with nicotine for 6 h
reduced the
[Ca2+]i
to basal levels. This inhibition was transient, and after several minutes the
[Ca2+]i
returned to the previous elevated levels. The reduced effectiveness of
these Ca2+ channel blockers at
these later times suggested that other
Ca2+ channels, such as
store-operated Ca2+ (SOC)
channels, may contribute to the rise at 6 h. Therefore, econazole was
added at a concentration (10 µM) that inhibits SOC channels (24).
Econazole elicited a rapid and more sustained reduction in
[Ca2+]i.
Because econazole was effective in dissipating the sustained rise in
Ca2+ with prolonged nicotine
treatment, we examined its effects on the initial nicotine-triggered
rise in
[Ca2+]i
(Fig. 5C). This inhibitor was
partially effective at 100 nM, and at 5 µM it essentially prevented
the initial rise in
[Ca2+]i.
Because capacitative influx via SOC channels is stimulated by depletion
of IP3-sensitive intracellular
Ca2+ stores, we examined whether
nicotine treatment generated IP3. Nicotine was found to elicit a prolonged elevation of
IP3, which peaked at 15 min (Fig.
6). This rise in
IP3 is about one-fourth of that
generated by bradykinin (not shown). In contrast, depolarization with
50 mM K+ did not generate
IP3.

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Fig. 6.
Inositol trisphosphate (IP3)
synthesis in PC-12 cells treated with nicotine.
IP3 was measured for up to 60 min
in PC-12 cells treated with 200 µM nicotine or 50 mM
K+ as described in
MATERIALS AND METHODS.
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Temporal requirement of
Ca2+ and role of
adenylyl cyclase in nicotine-elicited elevation of TH mRNA.
To determine the temporal requirement for increased
[Ca2+]i
in the induction of TH mRNA by nicotine, we treated the PC-12 cells with 10 µM BAPTA-AM at different time points before and after the
addition of nicotine (Fig. 7). BAPTA-AM
blocked the induction of TH mRNA by nicotine when added before or after
the initial elevation of
[Ca2+]i.
Even when added 2 h after nicotine, BAPTA-AM still prevented the rise
in TH mRNA levels. These data indicate that a sustained rise in
[Ca2+]i
is required for the elevation of TH mRNA by nicotine.

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Fig. 7.
BAPTA-AM added at different times prevents nicotine-triggered elevation
of TH mRNA levels. PC-12 cells were untreated (Control) or incubated in
presence of 10 µM BAPTA-AM, 200 nM nicotine, or nicotine + 10 µM
BAPTA-AM added either 15 min before ( 15) or 5 or 15 min or 1 or
2 h after addition of nicotine. RNA was isolated, and levels of TH mRNA
relative to untreated controls were determined by Northern blots. Data
are means ± SE from 3 separate experiments.
* P < 0.01 compared with
control.
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Previous results with PKA-deficient cells suggested that activation of
PKA was required for nicotine-elicited gene transcription in PC-12
cells (16). To investigate this possibility, we measured the cAMP
concentrations in untreated and nicotine-treated cells to examine
whether inhibition of adenylyl cyclase affected the TH mRNA levels.
Statistically significant increases in cAMP concentrations were
observed in PC-12 cells exposed to nicotine for 15 min (means ± SE
in pmol/3 × 105 cells:
control, 13 ± 0.4; nicotine, 16.4 ± 0.8;
P < 0.05). The concentration of cAMP
remained significantly elevated in cells exposed to nicotine for 24 h
(means ± SE in pmol/3 × 105 cells: control, 13.8 ± 0.4; nicotine, 15.2 ± 0.2; P < 0.05).
Cells were also exposed to 10 and 100 µM DDA, an adenylyl cyclase
inhibitor, before and during the nicotine treatment. Northern blot
analyses of RNA prepared from these cells are presented in Fig.
8. The addition of 10 µM DDA inhibited,
but 100 µM DDA prevented, the nicotine-induced rise in TH mRNA
levels. In contrast, 100 µM DDA did not prevent the phorbol
ester-elicited induction of TH mRNA (Fig. 8), consistent with the
specificity of DDA. These results suggest that cAMP is critically
involved in the induction of TH gene expression by nicotine.

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Fig. 8.
Inhibition of adenylyl cyclase activity prevents induction of TH mRNA
by nicotine. PC-12 cells were untreated (Control), were treated with
nicotine alone, or were pretreated with 10 or 100 µM
2',5'-dideoxyadenosine (DDA) for 20 min before treatment
with 200 µM nicotine (Nic + DDA10 and Nic + DDA100, respectively). Treatments
with 100 µM
12-O-tetradecanoylphorbol 13-acetate
(TPA) or with TPA + 100 µM DDA (TPA + DDA100) were carried out for 4 h. TH mRNA and 18S rRNA levels were analyzed by Northern blotting, and
a representative blot is presented.
|
|
One of the steps mediating cAMP-induced activation of gene expression
is phosphorylation of CREB, a transcription factor that binds the TH
CRE/CaRE. Thus we further examined the phosphorylation of CREB in
nicotine-treated cells (200 µM) in the presence or absence of DDA.
Immunocytochemistry with antisera to P-CREB showed increased
immunoreactivity in the nucleus of nicotine-treated cells (Fig.
9). Pretreatment with the
adenylyl cyclase inhibitor DDA greatly reduced the immunoreactivity to
P-CREB, indicating that the nicotine-elicited phosphorylation of CREB
requires adenylyl cyclase activity.

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Fig. 9.
Nicotine stimulates adenylyl cyclase-dependent phosphorylation of cAMP
response element binding protein (CREB). PC-12 cells were untreated
(A) or were treated with 200 µM
nicotine (B) or with nicotine + DDA
(C) for 10 min before
immunocytochemistry with antisera to phosphorylated CREB (P-CREB), as
described in MATERIALS AND METHODS.
|
|
Next, DDA was added at various times before and after nicotine addition
to determine any temporal effects this inhibition may have (Fig.
10). DDA prevented the rise of TH mRNA
after 6 h of treatment with nicotine when it was added 15 min before or 15 min after nicotine. However, when added 1 h after nicotine, DDA was
no longer inhibitory. These results suggest that the temporal requirement for adenylyl cyclase activity differs from that of Ca2+.

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Fig. 10.
Effect of DDA on elevation of TH mRNA levels induced by nicotine. PC-12
cells were untreated (Control) or were treated with 50 µM DDA, 200 µM nicotine, or with 50 µM DDA added 15 min before
( 15'), 15 min after (15'), or 60 min after
(60') addition of nicotine. RNA was isolated, and levels of TH
mRNA relative to those in untreated controls were determined by
Northern blot analysis, as described. Data are means ± SE.
* P < 0.05, ** P < 0.01 compared with
control.
|
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 |
DISCUSSION |
Extracellular
Ca2+.
The present study investigated the involvement of
Ca2+ and cAMP in the induction of
TH mRNA expression caused by nicotine. We have shown that several
distinct temporal phases exist in the nicotine-triggered elevation of
TH mRNA levels that possess different requirements for cAMP or
Ca2+. Nicotine elicited a rapid
rise in
[Ca2+]i
in PC-12 cells, and the absence of extracellular
Ca2+ prevented the
nicotine-stimulated induction of TH mRNA. This is in contrast to the
induction of TH mRNA caused by bradykinin, which occurred in
Ca2+-free medium or in the
presence of EGTA (29). Upon nicotinic stimulation of PC-12 cells, an
influx of extracellular Ca2+ and
Na+ occurs via nicotinic receptors
and results in membrane depolarization and the activation of
voltage-gated Ca2+ channels. This
promotes Ca2+ entry and leads to a
rapid increase in
[Ca2+]i
(33). Our results indicate that the influx of extracellular Ca2+ is essential for the
induction of TH mRNA. In addition, we found that nifedipine blocked
nicotine's ability to increase TH mRNA levels, consistent with its
inhibition of TH gene transcription in nifedipine-treated cultured
bovine adrenal chromaffin cells treated with the nicotinic agonist DMPP
(5). Dihydropyridines were also found to inhibit nicotine-stimulated
activation of the chromogranin A promoter and expression of the
proenkephalin gene (34). Collectively, these data imply that
Ca2+ channels are critically
involved in the nicotine-induced increase of TH gene expression.
The blockage of more than one type of voltage-gated
Ca2+ channel was found to
eliminate, or greatly reduce, the nicotine-elicited rise in
[Ca2+]i.
Inhibitors of either L-type (nifedipine or calciseptine) or N-type
(
-conotoxins GVIA or MVIIA)
Ca2+ channels essentially
prevented any increase in
[Ca2+]i.
This is in contrast to the only partial inhibition of the rise in
[Ca2+]i
elicited by depolarization with elevated
K+ with the same concentrations of
these N-type or L-type voltage-dependent Ca2+ channel blockers.
There is some evidence that L-type
Ca2+ channel blockers,
specifically the 1,4-dihydropyridines, may directly inhibit the
nicotine receptor (25). In such a situation, these inhibitors would
prevent all the downstream effects of nicotine, including the
activation of N-type Ca2+ channels
and SOC channels. Such an effect would not alter the changes in
[Ca2+]i
by depolarization with elevated
K+. The same N-type channel
blockers used in our study (
-conotoxins GVIA and MVIIA) were
previously found to inhibit nicotine-stimulated 45Ca2+
entry into bovine adrenal chromaffin cells by ~25-30%. However, L-type blockers reduced a much larger percentage (53-89%) of the nicotine-triggered
45Ca2+
entry (39). This is despite the somewhat smaller
Ca2+ currents attained with
dihydropyridine-sensitive voltage-gated Ca2+ currents compared with the
N-type or P-type voltage-gated
Ca2+ channels (2). This would be
consistent with an additional inhibitory effect of nifedipine on
acetylcholine receptors. Various dihydropyridine inhibitors of L-type
Ca2+ channels were found to
prevent
45Ca2+
uptake or elevation in
[Ca2+]i
in fura 2-loaded bovine chromaffin cells (25) as well as to block
DMPP-evoked catecholamine release. However, this nonspecific inhibition
by L-type channel blockers is less likely to explain the findings in
the present study, since calciseptine had an effect similar to that of
nifedipine and is not a dihydropyridine (8). Therefore, it is unlikely
that the prevention of the rise in
[Ca2+]i
by this L-type channel blocker is due to a nonspecific action on the
nicotinic receptor.
The results indicate that nicotinic stimulation activates several
different types of Ca2+ channels
in PC-12 cells, with blockage of the L-type and N-type and, to some
extent, the P/Q-type, each affecting the rise in Ca2+. Such a coordinated blockage
may indicate that the inhibition of one type (N-type or L-type) is
sufficient to allow the efflux and intracellular buffering of
Ca2+ to overcome the influx of
extracellular Ca2+ from the
remaining activated channels and therefore may explain the complete
inhibition by either L-type or N-type channel blockers alone. Another
possibility is that there are interactions among the channels. For
example, cAMP generated via activation of one channel may activate
another type of Ca2+ channel.
Consistent with this possibility, recruitment of
dihydropyridine-sensitive, voltage-gated
Ca2+ currents by cAMP has been
observed in chromaffin cells (2).
However, the effect of the channel antagonists on the nicotine-elicited
rise of
[Ca2+]i,
in contrast to the effect of elevated
K+, is likely influenced by
concurrent desensitization of the nicotinic receptors. Because of
nicotinic receptor desensitization, the initial rise in
Ca2+ does not achieve a steady
level. This dynamic situation may magnify the effectiveness of blockade
of specific Ca2+ channels.
Several temporal phases of elevation of
[Ca2+]i.
Many studies have confirmed the ability of nicotine to elicit rapid
elevations in
[Ca2+]i,
however, the long-term effects are not well studied. Our experiments demonstrated that several minutes after the initial transient rise in
[Ca2+]i,
there was a second smaller elevation that was sustained for at least
several hours. Evidence from a variety of sources indicates that
nicotinic receptors exist in a number of functional states, including a
closed resting state that is briefly converted to an open state upon
agonist binding. The receptor can be converted to its desensitized or
inactive state, remaining unresponsive to agonists, for extended times
(6). The desensitization of nicotine receptors and the development of
tolerance to catecholamine secretion have been examined in chromaffin
cell cultures (3). These authors demonstrated that catecholamine
release exhibited both acute and chronic tolerance to nicotine.
Interestingly, the majority of the tolerance occurred within the first
10 min of nicotine exposure, the time frame of the first peak of
elevated [Ca2+]i.
The smaller, but sustained, subsequent rise in
[Ca2+]i
observed in the present study may be consistent with such
desensitization and is consistent with the depression (but not
abolition) of catecholamine release in chromaffin cells preexposed for
several days to nicotine (3).
The second elevation of
[Ca2+]i
appears to be a necessary event, since the elevation in TH mRNA levels
was inhibited by BAPTA-AM even when added 2 h after nicotine. The
second sustained peak of elevated
[Ca2+]i
differed from the first initial transient rise in the effect of
voltage-gated Ca2+ channel
blockers. Thus pretreatment with calciseptine prevented the initial
elevation of
[Ca2+]i.
However, when added at later times, after 6 h of nicotine treatment, it
was no longer effective. An N-type channel blocker,
-conotoxin GVIA,
reduced the long-term rise in
[Ca2+]i,
but its effect was not sustained after several minutes. However, a
sustained inhibition of the second as well as of the initial rise in
[Ca2+]i
in response to nicotine was observed with the imidazole-type blocker
econazole (10 µM). At these concentrations, econazole inhibits SOC
channels (24) as well as voltage-dependent
Ca2+ channels (38). Because the
effectiveness of calciseptine and
-conotoxin GVIA was lost or
reduced after the early phase, it is likely that the effect of
econazole on the long-term rise indicates a contribution of SOC
channels. These results are consistent with the occurrence of
time-dependent changes in the relative contribution of different types
of Ca2+ channels to the elevation
in
[Ca2+]i.
The L-type voltage-dependent Ca2+
channels appear to be involved only in the initial rise, whereas the
SOC and N-type channels may contribute to the longer term effect,
although the inhibition of the N-type blocker was transient, perhaps
overcome by leak channels as well.
Although a prolonged rise of
[Ca2+]i
is needed for induction of TH mRNA by nicotine, such a sustained
elevation was not required for induction by elevated
K+ or bradykinin. TH mRNA
induction by membrane depolarization with elevated
K+ was blocked when EGTA was added
within the first 10 min, but not after 30 min or longer (29). With
bradykinin treatment, even the transient rise in
[Ca2+]i
within 5 min of exposure in the presence of EGTA was sufficient to
elevate TH mRNA (29). Bradykinin, which mainly elevates
[Ca2+]i
by generating IP3, may more
directly elevate nuclear Ca2+
concentration via IP3 receptors,
some of which are known to be located on the inner nuclear membrane.
This study found that nicotine, but not depolarization by elevated
K+, also generated
IP3. Activation of phospholipase C
in nicotine-treated cells would lead to generation of
IP3 and activation of PKC.
Nicotine-simulated activation of PKC in PC-12 has been previously
observed (35). Nevertheless, the amount of
IP3 seen with nicotine treatment
is a fraction of that generated with bradykinin. The requirement for
prolonged exposure to nicotine for elevation of TH mRNA levels, compared with these more rapidly acting agents (bradykinin and elevated
K+) may involve a relatively
lower ability to elevate nuclear
Ca2+. Elevated
K+ also increases cytosolic and
nuclear Ca2+ by activating
voltage-gated Ca2+ channels.
However, its elevation of nuclear
Ca2+ may be more sustained, since,
in this case, Ca2+ channel
activation does not involve acetylcholine receptors, which desensitize
following stimulation with nicotine.
Nicotine receptor subtypes.
There are a number of nicotinic receptor subtypes on PC-12 cells that
may respond differently to prolonged exposure to nicotine, and the
short- and long-term effects of nicotine observed here may be mediated
by a different subset of receptors. The neuronal nicotinic
acetylcholine receptors are diverse cationic ion channel complexes
composed of two different types of subunits (
and
). Recently, at
least eight
-subunits
(
2-
9)
and three
-subunits (
2-
4)
have been identified (13). The PC-12 cells were shown to express genes
for nicotinic receptor subunits
3,
5,
7,
2,
3, and
4 (15, 19). The expression of
nicotinic receptor subunits is reportedly regulated by cAMP and nerve
growth factor (15, 27). However, there is conflicting evidence
regarding the ability of nicotine to alter the expression of its
receptors in PC-12 cells. Nicotine is reported to reduce the mRNA
levels of
3 and slightly
increase those for
2 in
wild-type, but not in PKA-deficient, PC-12 cells (28). Conversely,
another study failed to find significant changes in the expression
patterns of any of the nicotinic acetylcholine receptor mRNAs in PC-12 cells in response to long-term nicotine treatments, which elevated TH
mRNA levels (19).
Involvement of cAMP.
The crucial involvement of the PKA pathway in the elevation of TH mRNA
levels was further supported by our results. A modest but significant
rise in cAMP levels was observed in the nicotine-treated PC-12 cells.
Pretreatment with the adenylyl cyclase inhibitor DDA prevented both the
nicotine-elicited phosphorylation of CREB and the subsequent induction
of TH mRNA. DDA appeared to act specifically, since it did not prevent
the induction of TH mRNA by phorbol esters. We speculate that the
activation of adenylyl cyclase by nicotine may be caused by
microdomains of elevated Ca2+ near
the membrane, in the proximity of the voltage-gated
Ca2+ channels, since these
cyclases are associated with sites of
Ca2+ entry (37). Alternatively,
activation of adenylyl cyclase may be coupled to the influx of
Ca2+ through nicotinic receptors,
leading to phosphorylation and activation of voltage-gated
Ca2+ channels. There are eight
isoforms of adenylyl cyclases, of which five (I, III, V, VI, and VIII)
are reported to be Ca2+ sensitive
based on in vitro assays (37). The type I adenylyl cyclase is a
neural-specific, Ca2+-stimulated
enzyme that couples
[Ca2+]i
to cAMP increases, and could be involved in the observed responses.
The inhibition of the rise in TH mRNA levels by DDA is consistent with
other studies that suggested the involvement of cAMP-mediated pathways
in nicotine-driven gene activation. Cholinergic regulation of cAMP
pathways in bovine adrenal medullary cells has been reported by
Anderson et al. (1). We found that the inhibition of TH gene expression
was effective when DDA was added 15 min, but not 60 min, after
nicotine. These results further demonstrate that the initial elevation
of
[Ca2+]i
within the first few minutes is not sufficient to lead to the induction
of TH mRNA, because adding DDA after the first peak of
Ca2+ still prevented the induction
of TH mRNA. However, after 1 h of nicotine treatment, DDA was no longer
inhibitory, indicating that a requirement for cAMP exists within the
first 1 h.
PKA-deficient cells treated with nicotine were unable to support many
of the alterations in gene expression observed in normal cells treated
similarly, including the elevation of TH mRNA levels (16, 28). However,
surprisingly, PKA-deficient cells reportedly support the induction of
chromogranin A promoter activity by nicotine, despite a CRE element
being involved in this promoter's activation (35). It is possible that
the nicotine-driven activation of chromogranin A may utilize a
signaling pathway different from that for TH. Detailed studies of the
mechanism of nicotine-stimulated transcription of chromogranin A
revealed that its transcription depended on PKC activation (35). This
difference may be related to the finding that the CRE in chromogranin A
(TCACGTAA) is not identical to the consensus CRE/CaRE (TGACGTCA) of the
TH and somatostatin promoters.
Although we found that DDA inhibited the phosphorylation of CREB, this
may not be sufficient for nicotine to induce chromogranin A promoter
activity. Previous experiments with dominant-negative CREB are
confusing because, although dominant-negative CREB completely inhibited
the activation of the chromogranin A promoter by cAMP, it reduced the
induction of TH by phorbol esters or nicotine by ~70% (35). We can
speculate that different CRE-like elements may utilize different
signaling pathways and transcription factors in response to nicotine.
In this regard, it is interesting to note that the CRE-2 element in the
enkephalin promoter, which is also transcriptionally activated by
nicotine, binds primarily activator protien-1-like factors
in chromaffin cells and CREB family members in the striatum (22, 26).
Further experiments are needed to ascertain whether phosphorylation of
CREB is directly involved in nicotine-triggered induction of TH gene
transcription. We observed that the TH CRE/CaRE also forms complexes in
PC-12 cells with other transcription factors, such as activating
transcription factor-1 and Jun (31, 32).
The findings of this study indicate that there are several temporal
phases involved in the induction of TH mRNA levels by nicotine, each
with different requirements for cAMP and
Ca2+. There is an early phase of
cAMP formation, and there is a late phase that is not inhibited by DDA
but requires Ca2+ in order to lead
to elevated TH mRNA levels. In this regard, studies with DMPP
stimulation in bovine chromaffin cells distinguished an early phase of
transcriptional activation that peaked at ~30 min and then declined,
although mRNA levels continued to accumulate and were maximal at
8-18 h (4). One explanation for these differing temporal
requirements for cAMP and Ca2+ in
the present study is that adenylyl cyclase may only be required for an
early transcriptional phase. Alternatively, different intracellular sites containing elevated Ca2+ may
be involved in promoting CREB phosphorylation and perhaps activation of
other transcription factors involved in CRE-dependent gene expression.
The role of different intracellular sites of elevated
Ca2+ in activation of gene
expression has been shown in hippocampal neurons, where BAPTA, which is
selective compared with EGTA for submembranous microdomains, blocks the
phosphorylation of nuclear CREB after
N-methyl-D-aspartate
receptor activation (7). In these hippocampal cells, calmodulin that is
in close proximity to the postsynaptic
Ca2+ channels is thought to be
responsible for calmodulin kinase-induced CREB phosphorylation. In
contrast, it was found that elevated nuclear, but not cytosolic,
Ca2+ is required for CRE-dependent
gene expression by depolarization in AtT-20 cells, a pituitary cell
line (14).
In the case of nicotine-induced TH gene expression, elevations in
[Ca2+]i
at several subcellular locations (Fig.
11) may be required at different times.
For example, after receptor desensitization to nicotine,
[Ca2+]i
falls nearly to basal levels. Under these conditions, submembranous microdomains containing both concentrated amounts of
Ca2+ as well as
Ca2+/calmodulin-sensitive adenylyl
cyclase could lead to activation of PKA and CREB phosphorylation.
Later, when Ca2+ levels rise
again, the activation of nuclear calmodulin kinase(s) may occur,
thereby modulating TH gene expression. Consistent with this scheme, we
have shown that the induction of TH mRNA was blocked by chelating
intracellular Ca2+ with BAPTA, as
well as with EGTA, which allows microdomains of highly concentrated
elevated Ca2+ to persist within
the cell due to slower binding kinetics. This biphasic mechanism would
occur if nicotine-induced TH gene expression required localized (both
nuclear and cytosolic) increases in
Ca2+ levels. A more detailed study
of the time course of alterations in
Ca2+ levels in different
subcellular cytoplasmic and nuclear locations is required for further
elucidation of the diverse mechanisms by which neuronal activation
leads to Ca2+-mediated gene
expression.

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Fig. 11.
Proposed signal transduction pathways for elevation of TH mRNA levels
by nicotine in PC-12 cells. Binding of nicotine to nicotinic
acetylcholine receptors leads to influx of
Na+ and
Ca2+. Resulting depolarization of
cell membrane stimulates Ca2+
influx via L-type, N-type, P/Q-type, and T-type voltage-dependent
Ca2+ channels, leading to further
accumulation of
[Ca2+]i.
This accumulation can be further amplified by several mechanisms,
including nicotine-stimulated IP3
formation, with subsequent release of
Ca2+ from
IP3-sensitive stores, and
capacitative Ca2+ influx via
store-operated channels. Submembranous microdomains of
Ca2+ from nicotinic receptors can
lead to formation of cAMP by
Ca2+-sensitive forms of adenylyl
cyclase (AC). Subsequent activation of protein kinase A (PKA) can
further phosphorylate and activate dihydropyridine-sensitive
Ca2+ influx (2). Calmodulin (CaM)
kinase (activated by elevation of
[Ca2+]i)
and PKA can both phosphorylate CREB, which as a homodimer or
heterodimer with other transcription factors [such as activating
transcription factor-1 (ATF-1) or Jun] can transactivate TH
transcription at its cAMP/Ca2+
response element (CRE/CaRE) site. After desensitization of nicotinic
receptors,
[Ca2+]i
attains a smaller but steady elevation in which contribution of
store-operated Ca2+ channels is
greater, whereas that of PKA and L-type channels is diminished. AP1,
activator protein-1.
|
|
 |
ACKNOWLEDGEMENTS |
We thank Dr. Bistra Nankova for useful suggestions.
 |
FOOTNOTES |
This work was supported by National Institute of Neurological Disorders
and Stroke Grant NS-28869, Smokeless Tobacco Research Council Grant
251, and postdoctoral fellowships from the American Heart Association
(to V. D. Gueorguiev and A. Menezes).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: E. L. Sabban, Dept. of Biochemistry and
Molecular Biology, New York Medical College, Valhalla, NY 10595.
Received 15 May 1998; accepted in final form 17 September 1998.
 |
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