The I1 Imidazoline Receptor In Pc12 Pheochromocytoma Cells
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Transcript of The I1 Imidazoline Receptor In Pc12 Pheochromocytoma Cells
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940 931
Journal of Neurochemistry, 2001, 79, 931±940
The I1-imidazoline receptor in PC12 pheochromocytoma cells
activates protein kinases C, extracellular signal-regulated kinase
(ERK) and c-jun N-terminal kinase (JNK)
Lincoln Edwards,* Daniel Fishman,* Peleg Horowitz,* Nicole Bourbon,² Mark Kester² andPaul Ernsberger*
*Departments of Nutrition, Medicine, Pharmacology, and Neuroscience, Case Western Reserve University School of Medicine,
Cleveland, Ohio, USA
²Department of Pharmacology, Pennsylvania State University, Hershey, Pennsylvania, USA
Abstract
We sought to further elucidate signal transduction pathways
for the I1-imidazoline receptor in PC12 cells by testing
involvement of protein kinase C (PKC) isoforms (bII, 1, z),
and the mitogen-activated protein kinases (MAPK) ERK and
JNK. Stimulation of I1-imidazoline receptor with moxonidine
increased enzymatic activity of the classical bII isoform in
membranes by about 75% and redistributed the atypical
isoform into membranes (40% increase in membrane-bound
activity), but the novel isoform of PKC was unaffected.
Moxonidine and clonidine also increased by greater than
two-fold the proportion of ERK-1 and ERK-2 in the phos-
phorylated active form. In addition, JNK enzymatic activity
was increased by exposure to moxonidine. Activation of ERK
and JNK followed similar time courses with peaks at 90 min.
The action of moxonidine on ERK activation was blocked by
the I1-receptor antagonist efaroxan and by D609, an inhibitor
of phosphatidylcholine-selective phospholipase C (PC-PLC),
previously implicated as the initial event in I1-receptor
signaling. Inhibition or depletion of PKC blocked activation of
ERK by moxonidine. Two-day treatment of PC12 cells with the
I1/a2-agonist clonidine increased cell number by up to 50% in a
dose related manner. These data suggest that ERK and JNK,
along with PKC, are signaling components of the I1-receptor
pathway, and that this receptor may play a role in cell growth.
Keywords: arachidonic acid metabolism, imidazoline, PC12
cells, pheochromocytoma, phospholipases C, receptors.
J. Neurochem. (2001) 79, 931±940.
The existence of a novel imidazoline receptor was ®rst
proposed to account for differential responses to imidazoline
and phenylethylamine a2-adrenergic agonists (Bousquet et al.
1984). Subsequently, binding sites speci®c for imidazolines
were characterized (Ernsberger et al. 1987). It is now
accepted that there are at least two subtypes of imidazoline
receptors, the I1- and I2-subtypes, and possibly a third I3-
subtype (Eglen et al. 1998). The I1-subtypes are character-
ized by a high af®nity for a group of agents which act in the
brainstem to lower blood pressure, including clonidine,
rilmenidine and moxonidine (Ernsberger et al. 1995, 1997;
Regunathan and Reis 1996). The I2-subtype shows lower
af®nity for these antihypertensives with a central nervous
system site of action but higher af®nity for other imidazo-
lines and guanidines, and represents a novel recognition site
on mitochondrial monoamine oxidase (Limon-Boulez et al.
1996).
A gene encoding an imidazoline binding protein has
been cloned from a human brain cDNA library (Piletz et al.
2000). The encoded protein contains motifs commonly
associated with cytokine receptors, including leucine-rich
repeats and serine-rich regions. When the gene is expressed
in Chinese hamster ovary (CHO) cells, high-af®nity
binding sites for imidazolines are induced that show
nanomolar af®nity for clonidine and moxonidine. Functional
Resubmitted manuscript received September 5, 2001; accepted
September 6, 2001.
Address correspondence and reprint requests to Dr Paul Ernsberger,
Department of Nutrition, Case Western Reserve University School of
Medicine, Cleveland, OH 44106±4906, USA.
E-mail: [email protected]
Abbreviations used: DAG, diacylglycerides; DMSO, dimethyl
sulfoxide; ERK, extracellular signal-regulated kinase; JNK, c-jun
N-terminal kinase; MAPK, mitogen-activated protein kinases; MTS,
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium] inner salt; NGF, nerve growth factor; PC12
cells, PC12 pheochromocytoma cell line; PC-PLC, phosphatidyl-
choline-selective phospholipase C; PKC, protein kinase C; SDS±
PAGE, sodium dodecyl sulfate±polyacrylamide gel electrophoresis.
I1-imidazoline receptors have been identi®ed in neural and
epithelial cells, including the rostral ventrolateral medulla
oblongata (RVLM) region which mediates sympatholytic
actions of imidazoline agonists (Ernsberger and Haxhiu
1997; Ernsberger et al. 1997), in the eye where they regulate
ocular pressure (Campbell and Potter 1994), and in the
kidney where they promote urinary sodium excretion
(Smyth and Penner 1999). Many ligands active at imidazo-
line receptors also bind to a2-adrenergic receptors. There-
fore, functional studies are typically carried out with prior
blockade of a2-adrenergic receptors. Cellular responses to
I1-imidazoline receptor activation, such as effects on
proliferation, have not been described previously.
The predominant cellular model for investigation of
I1-imidazoline receptor signaling pathways has been PC12
pheochromocytoma cells. These adrenal tumor cells express
I1-imidazoline receptors but lack a2-adrenergic receptors, as
shown by radioligand binding as well as molecular approaches
(Separovic et al. 1996). Stimulation of the I1-imidazoline
receptor in PC12 cells with the agonist moxonidine leads to
activation of phosphatidylcholine selective phospholipase C
(PC-PLC) (Separovic et al. 1996, 1997; Ernsberger 1999).
Activation of PC-PLC is characteristic of the signaling
pathways coupled to certain cytokine receptors, including
some of the interleukins receptors (Cobb et al. 1996; Ho
et al. 1994), and also mediates some of the actions of
thromboxanes in astrocytes (Kobayashi et al. 2000). Activa-
tion of PC-PLC by imidazoline agonists results in increased
formation of the second messenger diacylglyceride (DAG)
from phosphatidylcholine, and the release of phospho-
choline. These effects can be blocked by both efaroxan, an
I1-imidazoline receptor antagonist, and by D609, an inhibi-
tor of PC-PLC. Cell signaling steps subsequent to the
accumulation of DAG have not been characterized for
I1-imidazoline receptor signaling, but DAG commonly
activates several isoforms of PKC.
At least 11 isoforms comprise the PKC family (Liu and
Heckman 1998) and these differ according to structure,
substrate speci®city, cofactor requirement and subcellular
localization. The PKC isoforms can be classi®ed as classi-
cal, novel and atypical. The classical PKC isoforms (cPKC,
a, b1, b11, g) are calcium-dependent and activated by DAG
derived from phosphatidylinositol or phosphatidylcholine.
The novel PKC isoforms (nPKC, d, 1, h, u) are also
sensitive to DAG but are calcium independent owing to the
absence of a calcium binding domain. Finally, the atypical
PKC isoforms (aPKC, i, l, z) are insensitive to DAG or
calcium and may be activated by other cellular signals.
Because I1-imidazoline receptors trigger the accumulation
of DAG, we hypothesized that classical and novel PKC
isoforms might be activated by imidazoline agonists.
Possible downstream targets for PKC in PC12 cells are
the family of mitogen-activated protein kinases (MAPKs)
(Cowley et al. 1994). MAPKs are intracellular mediators
that are divided into three classes, namely the extracellular
regulated protein kinase (ERK), c-jun kinase or JNK (also
known as stress-activated protein kinase or SAPK) and the
p38 family. Activated MAPKs phosphorylate several sub-
strates in PC12 cells including various transcription factors
(Cowley et al. 1994). In the present study, we sought to
determine whether activation of the I1-imidazoline receptor
by moxonidine leads to activation of one or more PKC
isoforms or MAPK species, and further whether an increase
in cellular proliferation might therefore result from stimula-
tion of I1-imidazoline receptors.
Materials and methods
Materials
RPMI medium and horse serum were obtained from GIBCO
(Gaithersburg, MD, USA). Fetal bovine serum, rat tail collagen and
anti-ERK af®nity puri®ed antibodies were obtained from Upstate
Biotechnology (Lake Placid, NY, USA). Moxonidine was kindly
provided by Kali-Chemie (Hannover, Germany). Efaroxan and
clonidine were purchased from Research Biochemicals Inter-
national (Natick, MA, USA). The enzyme inhibitors D609 and
H-7 were purchased from Biomol (Plymouth Meeting, PA).
nPKC1, nPKCz, cPKCb11 and JNK goat polyclonal af®nity
puri®ed antibodies were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). Anti-active ERK antibody and donkey
anti-rabbit horseradish peroxidase antibody were purchased from
Promega (Madison, WI, USA). Nerve growth factor (NGF) was
obtained from Austral Biologicals (San Ramon, CA, USA). Protein
assay reagents and the colorimetric PKC assay kit were obtained
from Pierce (Rockford, IL, USA). All other chemicals were from
Sigma Chemical Co. (St Louis, MO, USA) or Fisher (Pittsburgh,
PA, USA) and were of analytical grade.
PC12 cell culture
PC12 cells were cultured as previously reported (Separovic et al.
1996). Brie¯y, PC12 cells were grown on 75 cm2 ¯asks coated with
rat tail collagen at 5% CO2 in RPMI 1640 supplemented with 10%
(v/v) heat-inactivated horse serum, 5% (v/v) fetal bovine serum
(FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (com-
plete medium). Cells were subcultured at a plating density of 1 : 6
once per week and medium was refreshed every two days. Because
previous studies showed that the response to I1-imidazoline
receptor stimulation was enhanced following differentiation of
PC12 cells with NGF, for most experiments PC12 cells were
treated with NGF (50 ng/mL) in RPMI 1640 medium supplemented
with 1% FBS for 2 days in order to initiate neuronal differentiation.
Preparation of cell fractions for assay of PKC activity
PC12 cells were pre-incubated in RPMI 1640 medium with 10 ng/
mL NGF for 30 min. Cells were then exposed to the following
treatments for 10 min: 1.0 mm moxonidine, or 200 nm phorbol-12-
myristate-13-acetate (PMA), or 0.02% DMSO as vehicle control.
All treatments were made up in RPMI medium supplemented with
10 ng/mL NGF. After treatment, cells were washed with ice-cold
RPMI containing 5 mm EGTA, and then removed from the ¯ask by
scraping. All subsequent steps were carried out at 48C, and each
¯ask of cells was processed separately. Cells were pelleted by
932 L. Edwards et al.
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
centrifugation at 2000 g for 5 min at 48C. Cell pellets were
homogenized with a polytron (Tekmar Tissumizer; setting 6 for
30 s) in 1.0 mL of homogenization buffer containing Tris-HCl,
pH 7.4, 50 mm NaF, 0.2 mm Na3VO4, 2.1 mm EDTA, 6.0 mm
2-mercaptoethanol, 2 mm EGTA, and a cocktail of protease
inhibitors (0.06 mg/mL anti-pain-HCl, 0.01 mg/mL bestatin,
0.02 mg/mL chymostatin, 0.06 mg/mL E-64 {N-[N-(l-3-trans-
carboxirane-2-carbonyl-l-leucyl]agmatine}, 0.01 mg/mL leupeptin,
0.01 mg/mL pepstatin, 0.06 mg/mL phosphoramidon, 0.4 mg/mL
pefabloc, and 0.01 mg/mL aprotinin). The homogenate was
centrifuged at 106 000 g for 1 h. The resulting supernatant was
retained as the cytosolic fraction. Membrane fractions were
obtained by homogenizing the particulate fraction (setting 6 for
30 s) in 1.5 mL of solublization buffer (homogenization buffer
containing 1% Triton X-100), bath sonication on ice for 15 min,
mixing by slow rotation for 30 min, and then centrifugation at
15 000 g for 10 min. The resulting supernatant was kept as the
membrane fraction.
Immunoprecipitation and assay of PKC activity
Immunoprecipitation was carried out on the cytosolic and mem-
brane fractions as previously described (Mandal et al. 1997).
Aliquots of each fraction (15 mL containing 2±5 mg of protein)
were treated with 10 mL of the appropriate isozyme speci®c
antibody (cPKCb11, nPKC1, aPKCz) then incubated with mixing
for 18 h at 48C. The immunoprecipitates were captured by adding
25 mL of agarose conjugated to donkey anti-rabbit secondary
antibodies to each sample, followed by overnight incubation.
Precipitates were isolated by centrifugation at 2000 g for 5 min,
washed twice by resuspension and centrifugation with homo-
genization buffer and ®nally resuspended in 100 mL of Tris-HCl
buffer at pH 7.4 containing 50% glycerol.
The ef®ciency of immunoprecipitation was determined by Western
blot analysis of the supernatant and immunoprecipitated fractions.
The immunoprecipitating antibody was used as the primary anti-
body for western blot analysis. Following immunoprecipitation of
either cytosol or membrane fractions with the cPKCb11 antibody,
the supernatants contained immunoreactivity for nPKC1 and
aPKCz, but cPKCb11 could not be detected. Similar results were
obtained following immunoprecipitation of cytosol and membrane
fractions with nPKC1 and aPKCz antibodies. Thus, the ef®ciency
of immunoprecipitation by each PKC isozyme antibody approached
100%, within the limits of detection of western blot methods.
Immunoprecipitated PKC activity in both membrane and
cytosolic fractions were assayed using a Pierce PKC Colorimetric
Assay Kit employing the eight-well strip format. Dye-coupled
chromagranin (Lissamine Rhodamine B at the N-terminal) was
used as the substrate because this chromaf®n granule protein is an
endogenous PKC substrate in PC12 cells. The assay was carried out
according to the manufacturer's instructions with two exceptions.
First, the incubation period was lengthened from 30 to 120 min as
pilot experiments with both cytosolic and membrane fractions
indicated that four times more reaction product was obtained with a
120-min incubation compared to 30 min. Second, an additional
wash step was added prior to the ®nal elution of phosphopeptide
with formic acid to reduce background absorbance at 570 nm.
Aliquots (10 mL) of PC12 cell membranes or cytosol were incu-
bated 120 min at 378C in a total volume of 25 mL of assay
buffer containing 5 mm rhodamine-chomagranin substrate, 20 mm
Tris-HCl pH 7.4,10 mm MgCl2, 2 mm ATP, 0.1 mm CaCl2,
0.002% Triton X-100 detergent, and 0.2 mg/mL phosphatidyl-l-
serine. Negative controls were treated identically, but contained
10 mL of Tris-HCl buffer at pH 7.4 containing 50% glycerol in
the place of cell fraction. Antibodies and agarose were included in
the negative controls. The assay mixture also contained 200 nm
phorbol myristate acetate, except for assays of preactivated PKC
where this was omitted. After the incubation, a 20 mL aliquot was
applied to a ferrite af®nity ®lter (Toomik et al. 1993) and washed
with three times by vacuum ®ltration with 250 mL of wash buffer,
consisting of 0.5 m NaCl and 0.1 m sodium acetate at pH 5.0.
Phosphopeptide was eluted with 15% formic acid. Absorbance of
the eluate was measured at 570 nm in a Rainbow plate reader with
rhodamine-chromagranin as standard. Protein was assayed by the
bicinchoninic acid method (Smith et al. 1985). A signi®cant
increase in the phosphorylation of rhodamine-chromagranin
substrate, relative to blanks containing buffer and immunocomplex
alone, was found for each of the three immunoprecipitated PKC
isoforms.
Assay of ERK activation
Differentiated PC12 cells in 75 cm2 culture ¯asks were treated with
various doses of moxonidine (0.1 nm21 mm) or clonidine (100 nm)
for 0±180 min. In some experiments, cells were pretreated with
inhibitors (efaroxan, D609 or H-7) or vehicle (0.1 mm acetic acid
in RPMI) alone for 10 min before the addition of moxonidine. In
other experiments, cells were pretreated with 200 nm phorbol
myristate acetate for 20 h to deplete PKC. After treatment, cells
were washed with ice-cold calcium-free Hank's buffer, removed
from the ¯ask by scraping, and then collected by centrifugation.
Cells were subsequently homogenized in lysis buffer (1% Triton
X-100, 0.5% NP-40, 150 mm NaCl, 10 mm Tris pH 7.4, 1 mm
EDTA, 1 mm EGTA pH 8.0, 0.2 mm sodium ortho-vanadate,
0.2 mm PMSF, and protease inhibitor cocktail (Boehringer
Mannheim GmbH, Mannheim, Germany) with a polytron (Tecmar
Tissuemizer, 15 s at setting 60) followed by centrifugation
(16 000 g, 48C) for 10 min. Equal amounts of protein (20 mg)
from the resulting supernatants were subjected to SDS±PAGE on a
10% gel and proteins were electrophoretically transferred to a
nitrocellulose membrane for immunodetection with anti-Active
MAPK and anti-MAPK antibodies. with a polytron (Tecmar
Tissuemizer, 15 s at setting 6) followed by centrifugation at
97 000 g at 48C for 1 h. Aliquots (10 mg protein as assayed by
the bicinchoninic acid method) from the resulting supernatants
were subjected to SDS±PAGE on a 10% acrylamide gel and
proteins were electrophoretically transferred to a nitrocellulose
membrane for immunodetection with anti-active ERK and
anti-ERK antibodies.
A dual antibody method was used to quantitate activation of
ERK as the ratio of active to total ERK. The anti-active antibody
recognizes the dually phosphorylated activated forms of ERK-1 and
ERK-2, whereas anti-ERK recognizes all forms of ERK-1 and
ERK-2. A donkey anti-rabbit secondary antibody coupled to horse-
radish peroxidase was utilized to visualize protein bands by chemi-
luminescence using Hyper ®lm ECL (Amersham, Buckinghamshire,
UK). Film images were quanti®ed by using a scanning densito-
meter (United States Biochemical, Cleveland, OH, USA). Results
were expressed as a ratio of arbitrary density times area units
between anti-active and anti-ERK blots and then normalized to
I1-Imidazoline, PKC and MAPK 933
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
vehicle-treated controls run in parallel. Because identical results
were obtained for ERK-1 and -2, the data presented here represent
the combined ERK-1 and -2 bands.
Assay of c-jun kinase activity
Assay of immunoprecipitated c-jun kinase (JNK) was conducted as
previously described (Coroneos et al. 1996). Cell lysates were
immunoprecipitated with rabbit polyclonal IgG directed against
JNK overnight at 48C, and the resulting immunocomplexes were
captured with goat anti-rabbit IgG agarose for 8 h at 48C. The
agarose complexes were collected by centrifugation and washed
twice with PBS. The pellets were then incubated at 378C for 20 min
with 1 mg rat c-jun, 3 mL ATP (cold, ®nal concentrated 25 mm),
1 mL [32P]ATP (speci®c activity . 4500 Ci/mmol) in a kinase
buffer (25 mL) as previously described (Coroneos et al. 1996). The
samples were then boiled with Laemmli buffer for 2 min followed
by SDS±PAGE. After transfer to nitrocellulose, the blots were
exposed to Kodak OMAT ®lm for 24 h at 2808C. Protein bands
were quanti®ed by scanning densitometry as described for ERK.
Cell proliferation assays
Cell proliferation was measured by using the Cell Titer system
(Promega; Madison, WI, USA) as speci®ed in the manufacturers
instructions. PC12 cells were plated at one-quarter of their normal
density in 96-well plates in low-serum medium (1% horse serum
and 0.5% fetal calf serum). Cells were treated with increasing doses
of clonidine or with 0.1% DMSO vehicle for 48 h. The number of
viable cells was estimated by incubating the cells for 2 h at 37 8C
with the metabolic dye [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] inner salt (MTS;
Owen's reagent) (Cory et al. 1991). Metabolically oxidized
formazan product was read from an absorbance plate reader at
490 nm, with the absorbance in cell-free control wells subtracted.
Results were expressed as corrected absorbance relative to vehicle-
treated controls run on the same plate.
Pilot experiments indicated that moxonidine had signi®cant
proliferative action only when added every 12 h, consistent with
the short half life of this compound in vivo (Ernsberger et al. 1993).
Clonidine, an analog with similar I1-imidazoline receptor af®nity,
was found to be effective when added once for up to 48 h, so
subsequent experiments were carried out with clonidine. This agent
has a greater activity at a1- and a2-adrenergic receptors than
moxonidine, but this was not thought relevant because PC12 cells
lack both a1- and a2-adrenergic receptors (Jinsi-Parimoo and Deth
1997; Berts et al. 1999). Indeed, PC12 cells have been used for
transfection studies of these receptors speci®cally because they lack
endogenous expression.
Data analysis
Statistical comparisons were performed by t-test for two groups or
analysis of variance for multiple comparisons, with Newman±
Keuls post hoc tests. Dose±responses data were ®tted to logistic
equations (Motulsky and Ransnas 1987) using the Prism data
analysis package (GraphPad software, San Diego, CA, USA) to
obtain EC50 values.
Results
Effect of moxonidine and phorbol myristate acetate on
the activity of three PKC isoforms
We ®rst determined whether the selected PKC isoforms
could be detected in PC12 cells using dye-coupled chromo-
granin substrate. The absolute activities for cPKCbII in
untreated control PC12 cells were: cytosol 1.2 ^ 0.2, and
membrane 0.86 ^ 0.1 mg of phosphorylated substrate per
¯ask. For nPKC absolute activities were: cytosol 0.59 ^ 0.1,
and membrane 1.0 ^ 0.1 mg per ¯ask. The activity of aPKCz
was: cytosol 0.72 ^ 0.1, and membrane 1.0 ^ 0.2 mg of
phosphorylated substrate per ¯ask. Thus, membrane-bound
PKC activity was comparable for the three isoforms, in
agreement with previous reports (Wooten et al. 1994).
The effect on PKC activity of treatment with either
moxonidine or phorbol myristate acetate is shown in Fig. 1.
Data are expressed as a net increase above untreated control
values determined in parallel. In response to 10 min of
treatment with 1.0 mm moxonidine, immunoprecipitated
cPKCbII showed increased activity in solubilized membrane
( p , 0.05, paired t-test), whereas cytosolic activity was
unchanged (Fig. 1). Treatment with 200 nm phorbol myri-
state acetate for 10 min induced a nearly identical response.
In contrast, nPKC showed no signi®cant response to either
treatment in membrane or cytosolic fractions. The activity of
the atypical isoform, aPKC1 showed translocation from the
cytosol to the membrane, as indicated by a decrease in the
former and an increase in the latter (both p , 0.05, paired
t-test). As expected, there was no in¯uence of phorbol
myristate acetate on the activity of aPKCz, a DAG-
insensitive isoform.
Effect of moxonidine on ERK activation in extracts from
differentiated PC12 cells
The activation of ERK-1 and ERK-2 was determined as the
ratio of the amount of dually phosphorylated active form to
total ERK immunoreactivity. A representative blot is shown
in Fig. 2, illustrating the time course of the response to
100 nm moxonidine. An increase in the amount of immuno-
reactivity to the anti-active antibody is apparent at the later
time points. The lower blot shows that the amount of ERK-2
immunoreactivity was constant between lanes, indicating
equal loading. Mean data from four experiments showed
that moxonidine treatment of PC12 cells increased ERK
activation by about 160% relative to vehicle-treated controls
(Fig. 3). Signi®cant activation of ERK ( p , 0.05 Newman±
Keuls test) was detected at 30 min, and the peak activation of
ERK occurred at 90 min, with a decline towards baseline
after 2 h.
The dose-dependence for the action of moxonidine on
ERK is illustrated in Fig. 4. The immunoreactivity to the
anti-active antibody increased with the concentration of
934 L. Edwards et al.
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
moxonidine tested, whereas the total amount of ERK-2
protein was constant. Summary data from four separate
experiments show that moxonidine's effect on ERK was
dose-dependent up to 100 nm, with an EC50 of 1.3 nm
(Fig. 5). A higher concentration of moxonidine, 1.0 mm,
activated ERK to a lesser extent than 100 nm. Comparable
biphasic dose±response relationships have been reported for
DAG accumulation (Separovic et al. 1996).
In order to test whether the effect of moxonidine on ERK
stimulation was mediated by the I1-imidazoline receptor and
through its known transmembrane signaling pathways, we
treated the cells with efaroxan, a selective I1-imidazoline
receptor antagonist, or with D609, an inhibitor of phospha-
tidylcholine-selective phospholipase C and I1-imidazoline
receptor signaling in PC12 cells (Fig. 6). Efaroxan (10 mm)
abolished ERK activation by 100 nm moxonidine treatment,
but had no signi®cant effect when given alone. The PC-PLC
inhibitor D609 (1.0 mm) also effectively abolished the effect
of moxonidine.
Fig. 2 Western blot illustrating the time course of ERK-2 activation
by moxonidine. The band labeled `phospho-ERK-2 MAPK' was from
a blot labeled with anti-active ERK antibody. The band labeled `pan-
ERK-2 MAPK' was stained for total ERK immunoreactivity. Each
lane was obtained from different ¯asks of PC12 cells incubated with
100 nM moxonidine for increasing amounts of time. Data were ana-
lyzed by determining the ratio of optical density between the ®rst
and second blot.
Fig. 3 Time course of ERK and JNK activation in PC12 cells follow-
ing moxonidine (100 nM) treatment. The relative activation of ERK-1
and ERK-2 is de®ned by the ratio of total enzyme to the dually phos-
phorylated form, as illustrated in Fig. 2. JNK activity was measured
as immunoprecipitated kinase activity. For both kinases, the data
were expressed relative to vehicle treated controls run in parallel.
Data are presented as mean percentage change ^ SE from four
separate experiments run in duplicate.
Fig. 4 Western blot illustrating the dose-dependence of ERK-2 acti-
vation by moxonidine. Bands are labeled as in Fig. 2. Each lane was
obtained from different ¯asks of PC12 cells incubated with increas-
ing concentrations of moxonidine for 90 min.
Fig. 1 Activity of PKC isoforms in fractions from PC12 cells treated
with moxonidine or phorbol myristate acetate. Shown are relative
rates of dye-labeled substrate phosphorylation activity of immuno-
precipitated PKC isoforms. Three representative PKC isozymes
expressed in PC12 cells were isolated: cPKCbII, nPKC1, and
aPKCz. The effects of phorbol myristate acetate and moxonidine are
represented by their percentage change ^ SE relative to controls
run in parallel in the same experiment. Data represent the
mean ^ SE from 12 75-cm2 ¯asks of cells. Asterisks mark statisti-
cally signi®cant increases ( p , 0.05, paired t-test).
I1-Imidazoline, PKC and MAPK 935
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
We next sought to test whether the activation of ERK
was mediated through PKC (Figs 7 and 8). Treatment with
the non-selective PKC inhibitor H-7 [1-(5-isoquinoline-
sulfonyl)-2-methylpiperazine] blocked the action of moxon-
idine. Treatment with H-7 alone had no effect on ERK
activation. In order to down-regulate DAG-sensitive iso-
forms of PKC, we pretreated PC12 cells with 200 nm
phorbol myristate acetate for 20 h prior to exposure to
either phorbol or moxonidine for 90 min. Depletion of PKC
by prolonged treatment with phorbol myristate acetate
abolished the response to short-term phorbol, con®rming
that the prolonged treatment eliminated responsiveness of
ERK to PKC. In this series of experiments, treatment
with 200 nm moxonidine for 90 min roughly tripled the proportion of ERK in the active dually phosphorylated state
(Fig. 8). This action of moxonidine was eliminated by
depletion of PKC by chronic treatment with phorbol
myristate acetate.
We next sought to determine whether another I1-imidazo-
line agonist, clonidine, would elicit similar effects as moxo-
nidine. Flasks of PC12 cells were treated in parallel for
90 min with 100 nm moxonidine, 100 nm clonidine, or
vehicle. The ratio of activated ERK was 272 ^ 36% of
control in cells treated with moxonidine and 273 ^ 35% of
control in cells treated with clonidine. Thus, moxonidine
and clonidine induced similar activation of ERK, consistent
with their similar binding af®nities for the I1-imidazoline
receptor in PC12 cells (Separovic et al. 1996).
Effect of moxonidine on JNK activity in PC12 cell
extracts
In addition to the ERKs, an independently regulated
kinase cascade in PC12 cells involves JNKs. Moxonidine
dose-dependently increased cellular activity of JNK up to
two-fold (Fig. 9). Peak effects were observed at 300 nm
moxonidine. In the presence of 10 mm efaroxan, 100 nm
moxonidine did not increase JNK activity (data not shown).
Fig. 6 Effects of a receptor blocker and an enzyme inhibitor on
ERK activation. PC12 cells were incubated with or without moxoni-
dine (100 nM) in the presence or absence of the I1-imidazoline
antagonist efaroxan (10 mM) or the PC-PLC inhibitor D609 (10 mM)
for 90 min. Efaroxan or D609 were also present during a 10-min
pre-incubation. ERK activation was then determined as described
above. Values are expressed as a percentage of vehicle treated
controls. Each value represents the mean ^ SE of at least nine
separate experiments. The effect of moxonidine alone was signi®-
cant ( p , 0.01, paired t-test) but no other treatment or combination
of treatments had any signi®cant effect ( p . 0.10, paired t-test).
Fig. 7 Western blot showing abrogation of the ERK activation
response to moxonidine by PKC depletion or inhibition. Bands are
labeled as in Fig. 2. Each lane was obtained from different ¯asks of
PC12 cells incubated with various for 20 h or 90 min prior to har-
vesting. First and last lanes are from vehicle-treated control cells.
The second lane shows the response to 100 nM moxonidine relative
to the vehicle control lane. The third lane is from a ¯ask of PC12
cells that was processed in parallel but was pretreated with 200 nM
phorbol-12-myristate-13-acetate overnight to deplete PKC. The fourth
lane shows the response to short-term treatment with phorbol ester.
The ®fth lane shows that results treatment with the PKC inhibitor
H-7 during the 10 min pre-incubation and during moxonidine treat-
ment, while the next lane illustrates the lack of effect of H-7 alone.
The seventh lane shows that the response to phorbol ester is lost
after 20 h exposure to 200 nM phorbol-12-myristate-13-acetate.
Fig. 5 Dose dependence of the activation of ERK by moxonidine
treatment. PC12 cells were treated with increasing concentrations of
moxonidine for 90 min and then analyzed for total and activated
ERK as illustrated in Fig. 4. Data are presented as mean percentage
change ^ SE from four separate experiments run in duplicate.
936 L. Edwards et al.
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
The time course of JNK activation is indicated in Fig. 3
(squares). The increase in JNK activity tended to parallel
the activation of ERK, with both peaking around 90 min
and declining by 120 min. The increase in JNK activity
was evident earlier, and reached signi®cance at 15 min
( p , 0.05, Newman±Keuls' test), whereas ERK was not
increased until 30 min of moxonidine treatment.
Proliferative response of PC12 cells to imidazoline
agonists
The activity of ERK and possibly JNK as well is linked to
cell proliferation, particularly in transformed cell lines such
as PC12 cells (Cowley et al. 1994). Therefore, we tested the
effect of an I1-imidazoline receptor agonist on PC12 cell
number during 2 day treatment (Fig. 10). We used clonidine
rather than moxonidine because of its longer metabolic half-
life in vivo (Ernsberger et al. 1993) and because these two
I1-agonists showed similar activation of ERK (see above).
PC12 cells were seeded at one-fourth normal density in 96
well plates in low-serum medium in order to reduce
background levels of proliferation. The ®nal number of
viable PC12 cells after 2 days of treatment, as determined
with a metabolic dye, was increased by about 20% at the
lowest dose tested (0.1 nm), and by 50% at the highest
dose (1.0 mm), as shown in Fig. 10. Thus, stimulation of
I1-imidazoline receptors appears to induce a small but
consistent increase in PC12 cell number, suggesting an
increase in the number of proliferating cells.
Discussion
The present study identi®es several downstream cell
signaling events that are coupled to the stimulation of
I1-imidazoline receptors in PC12 rat pheochromocytoma
cells. A common and an atypical isoform of PKC each
showed increased enzymatic activity (cPKCbII and aPKCz),
whereas nPKC1 was not affected. The stimulation of
cPKCbII by the I1-imidazoline agonist moxonidine was
comparable to that induced by treatment with a phorbol
Fig. 9 Concentration-dependent stimulation of JNK activity by
moxonidine. PC12 cells were incubated with or without varying
doses of moxonidine (0.1 nM21 mM) or vehicle for 90 min and then
lysates were assayed for JNK phosphorylation. Each value repre-
sents the mean ^ SEM of at least four experiments.
Fig. 10 Concentration-dependent increase in PC12 cell proliferation
by clonidine. PC12 cells were grown in low-serum medium in the
presence of increasing concentrations of clonidine for 48 h, and then
the density of viable metabolically active cells was determined by
using the MTT metabolic dye. Values are expressed as a percen-
tage of vehicle treated controls. Each value represents the mean ^
SEM of 18 separate wells.
Fig. 8 Effects of PKC depletion or inhibition on ERK activation by
moxonidine. PC12 cells were incubated with vehicle alone, moxoni-
dine (100 nM) alone, moxonidine in the presence of the PKC inhibitor
H-7 (1.0 mM), H-7 alone, moxonidine following overnight exposure to
200 nM phorbol-12-myristate-13-acetate in order to deplete PKC, or
the response to short-term treatment with phorbol ester with and
without overnight exposure to phorbol-12-myristate-13-acetate. ERK
activation was determined as described above. Values are
expressed as a percentage of vehicle treated controls. Each value
represents the mean ^ SE of at least six separate experiments. The
effect of moxonidine alone and phorbol-12-myristate-13-acetate
alone were signi®cant ( p , 0.01, paired t-test), but no other treat-
ment or combination of treatments had any signi®cant effect
( p . 0.10, paired t-test).
I1-Imidazoline, PKC and MAPK 937
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
ester. In addition, aPKCz showed clear subcellular relocal-
ization, with activity in the cytosol decreasing and that in the
membrane fraction increasing. Two members of the MAPK
family of kinase cascades were also activated in response to
moxonidine: ERK and JNK. These kinases showed roughly
parallel activation with a peak effect occurring around
90 min of treatment. In PC12 cells stimulated with the
I1-imidazoline agonist moxonidine, the proportion of ERK
in its active dually phosphorylated form was increased
150%, whereas JNK activity was elevated nearly two-fold.
The activation of both kinases was dose-dependent, and
in the case of ERK the EC50 for moxonidine was in
close agreement with the binding af®nity of the drug
for I1-imidazoline receptors [Ki � 7.8 nm; (Separovic et al.
1996)]. Finally, a modest but concentration-dependent
increase in cell number was elicited by 2-day treatment of
PC12 cell cultures with the I1-imidazoline agonist clonidine.
This result implies a weak mitogenic action of I1-imidazo-
line receptors, consistent with their apparent activation of
MAPK cascades.
In the present study, the activation of ERK was apparently
receptor-mediated, because it could be blocked by cotreat-
ment with the I1-imidazoline antagonist efaroxan. Moreover,
the concentration range wherein moxonidine was effective
in activating ERK and JNK was consistent with its binding
af®nity for I1-imidazoline receptors, and the dose±response
curves for ERK and JNK activation closely resembled pre-
viously reported dose±response relationships for I1-imidazo-
line receptor activation of arachidonic acid release (Ernsberger
1998), prostaglandin production (Ernsberger et al. 1995),
and DAG accumulation (Separovic et al. 1996).
The I1-imidazoline receptor has been previously shown to
be coupled to activation of PC-PLC in PC12 cells, which
leads to formation of DAG from phosphatidylcholine and an
increased total cellular mass of this second messenger
(Separovic et al. 1996). We therefore hypothesized that PKC
may be activated by I1-imidazoline receptor stimulation.
The PKC multigene family of enzymes is involved in the
control of many biological events and is a major transducer
of receptor-mediated stimuli. In PC12 cells the I1-imidazo-
line receptor agonist moxonidine has been shown to activate
at least two isoforms of PKC (cPKCbII and aPKCz). PC12
cells are known to express the following PKC isotypes: a,
bI, bII, d, 1, h and z (Hundle et al. 1995) and differentiation
of PC12 cells to a neuronal phenotype by treatment with
NGF induces increased expression of bII, d, and z, and the
appearance of PKC 1 and h within the nucleus (Borgatti
et al. 1996). This suggests that the I1-imidazoline receptor
might modulate cell proliferation or neuronal differentiation
through activation of key PKC isoforms. In addition, the
atypical z-PKC is required for neuronal differentiation
and neurite outgrowth of PC12 cells in response to NGF
(Coleman and Wooten 1994), and thus the activity of this
isoform in PC12 cell membrane fractions was increased by
nearly 125% upon exposure to moxonidine. The possible
modulation of PC12 cell neuronal differentiation by
I1-imidazoline receptors remains to be determined.
Cellular DAG are known to regulate the activity of
cPKCbII. Stimulation of I1-imidazoline receptors in PC12
cells with moxonidine elevates total cellular mass of DAG
(Separovic et al. 1996). Moreover, in the present study, the
effects of moxonidine closely resembled those of phorbol
ester, a diglyceride analog. Thus, the activation of cPKCbII
by moxonidine might plausibly be the result of increased
diglyceride levels. The mechanisms behind the activation of
aPKCz are not as clear. Arachidonic acid activates atypical
PKC isoforms in isolated brain membranes (Huang et al.
1993).
The ERK family of MAPK were also activated in
response to I1-imidazoline receptor stimulation. The MAPK
family members, including ERK and JNK, typically mediate
responses to mitogenic stimuli and promote cell prolifera-
tion (Marshall 1995). Sustained activation of the MAPK
signaling pathway is reportedly both necessary and suf®-
cient to induce neuronal differentiation of PC12 cells
(Cowley et al. 1994). We have reported a two-fold increase
in ERK-activation by the I1-imidazoline agonists moxon-
idine and clonidine which can be blocked by efaroxan, an
I1-imidazoline receptor antagonist, and by D609, an inhibi-
tor of PC-PLC. These data imply that activation of MAPK is
receptor-mediated and is downstream from phospholipid
hydrolysis pathways associated with the I1-imidazoline
receptor. Efaroxan can also acts as an a2-adrenergic antag-
onist in some cells in the dose range used in the present
study, but these receptors are not present in PC12 cells.
Efaroxan has negligible af®nity for the mitochondrial
I2-imidazoline subtype (Lione et al. 1996) which are present
in these cells. The activation of ERK and JNK by moxo-
nidine was not sustained, but rather peaked around 90 min
and declined substantially within 120 min. This pattern
resembles the response to epidermal growth factor and other
agonists that activate ERK in PC12 cells, but stands in
contrast to NGF-activation of ERK, which is sustained for
many hours (Marshall 1995).
The activation of ERK in response to imidazoline agonists
appears to be downstream of PKC activation. Thus, deple-
tion of classical and novel isoforms of PKC by prolonged
exposure to a phorbol ester blocked the response to moxon-
idine. Furthermore, a non-selective PKC inhibitor blocked
the response to moxonidine as well. These results implicate
the stimulation of PKC in the activation of ERK by
imidazoline agonists. The subtype of PKC responsible may
be the bII isoform, as this form was activated in response
to moxonidine. While the aPKCz isoform was also
activated, this atypical subtype of PKC is not known to
be depleted by prolonged stimulation with phorbol esters.
The isoform may participate in other downstream signaling
events.
938 L. Edwards et al.
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
In the present study, a small but persistent and dose-
dependent increase in the number of viable cells was
noted in cultures of serum-starved PC12 cells treated with
I1-imidazoline agonist clonidine. An increase in total cell
number is a strong indicator that increased cell proliferation
has occurred, although the number of actively dividing cells
was not measured. The activation of MAPK cascades may
have been too transient to induce a dramatic proliferative
response. The mitogenic effect was not altered when serum
levels in the media were increased or the initial seeding
density of the cells were changed systematically (data not
shown), implying that the effect of clonidine was not
strongly dependent upon culture conditions.
In conclusion, we have extended our model of the signal-
ing pathway for I1-imidazoline receptor in PC12 cells to
include coupling to PKC and MAPK. One possible function
of these intermediates may be in promoting cell proliferation
or possibly the modulation of neuronal differentiation.
Acknowledgements
This work was supported by HL44514 (to PE and MK) and
DK53715 (to MK) from the National Institutes of Health. We
acknowledge the technical assistance of Kathryn Zalovcik, BS
and David Bedol, BS.
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