This study investigated signal transduction pathways activated by stimulation of I1-imidazoline receptors in PC12 cells. The study found that stimulation of these receptors with moxonidine: 1) Increased the activity of PKC beta II and redistributed the atypical PKC zeta isoform into cell membranes. It did not affect the novel PKC theta isoform. 2) Increased the proportion of active, phosphorylated forms of ERK-1 and ERK-2, as well as increasing JNK enzymatic activity. 3) The effects of moxonidine on ERK activation were blocked by an I1 receptor antagonist and a PC-PLC inhibitor, suggesting PC-PLC mediates I

1. 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 and
Paul 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 and JNK followed similar time courses with peaks at 90 min.
We sought to further elucidate signal transduction pathways The action of moxonidine on ERK activation was blocked by
for the I1-imidazoline receptor in PC12 cells by testing the I1-receptor antagonist efaroxan and by D609, an inhibitor
involvement of protein kinase C (PKC) isoforms (bII, 1, z), of phosphatidylcholine-selective phospholipase C (PC-PLC),
and the mitogen-activated protein kinases (MAPK) ERK and previously implicated as the initial event in I1-receptor
JNK. Stimulation of I1-imidazoline receptor with moxonidine signaling. Inhibition or depletion of PKC blocked activation of
increased enzymatic activity of the classical bII isoform in ERK by moxonidine. Two-day treatment of PC12 cells with the
membranes by about 75% and redistributed the atypical I1/a2-agonist clonidine increased cell number by up to 50% in a
isoform into membranes (40% increase in membrane-bound dose related manner. These data suggest that ERK and JNK,
activity), but the novel isoform of PKC was unaffected. along with PKC, are signaling components of the I1-receptor
Moxonidine and clonidine also increased by greater than pathway, and that this receptor may play a role in cell growth.
two-fold the proportion of ERK-1 and ERK-2 in the phos- Keywords: arachidonic acid metabolism, imidazoline, PC12
phorylated active form. In addition, JNK enzymatic activity cells, pheochromocytoma, phospholipases C, receptors.
was increased by exposure to moxonidine. Activation of ERK J. Neurochem. (2001) 79, 931±940.
The existence of a novel imidazoline receptor was ®rst 2000). The encoded protein contains motifs commonly
proposed to account for differential responses to imidazoline associated with cytokine receptors, including leucine-rich
and phenylethylamine a2-adrenergic agonists (Bousquet et al. repeats and serine-rich regions. When the gene is expressed
1984). Subsequently, binding sites speci®c for imidazolines in Chinese hamster ovary (CHO) cells, high-af®nity
were characterized (Ernsberger et al. 1987). It is now binding sites for imidazolines are induced that show
accepted that there are at least two subtypes of imidazoline nanomolar af®nity for clonidine and moxonidine. Functional
receptors, the I1- and I2-subtypes, and possibly a third I3-
subtype (Eglen et al. 1998). The I1-subtypes are character- Resubmitted manuscript received September 5, 2001; accepted
ized by a high af®nity for a group of agents which act in the September 6, 2001.
brainstem to lower blood pressure, including clonidine, Address correspondence and reprint requests to Dr Paul Ernsberger,
rilmenidine and moxonidine (Ernsberger et al. 1995, 1997; Department of Nutrition, Case Western Reserve University School of
Medicine, Cleveland, OH 44106±4906, USA.
Regunathan and Reis 1996). The I2-subtype shows lower E-mail: pre@po.cwru.edu
af®nity for these antihypertensives with a central nervous Abbreviations used: DAG, diacylglycerides; DMSO, dimethyl
system site of action but higher af®nity for other imidazo- sulfoxide; ERK, extracellular signal-regulated kinase; JNK, c-jun
lines and guanidines, and represents a novel recognition site N-terminal kinase; MAPK, mitogen-activated protein kinases; MTS,
on mitochondrial monoamine oxidase (Limon-Boulez et al. [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium] inner salt; NGF, nerve growth factor; PC12
1996). cells, PC12 pheochromocytoma cell line; PC-PLC, phosphatidyl-
A gene encoding an imidazoline binding protein has choline-selective phospholipase C; PKC, protein kinase C; SDS±
been cloned from a human brain cDNA library (Piletz et al. PAGE, sodium dodecyl sulfate±polyacrylamide gel electrophoresis.
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940 931

2. 932 L. Edwards et al.
I1-imidazoline receptors have been identi®ed in neural and that are divided into three classes, namely the extracellular
epithelial cells, including the rostral ventrolateral medulla regulated protein kinase (ERK), c-jun kinase or JNK (also
oblongata (RVLM) region which mediates sympatholytic known as stress-activated protein kinase or SAPK) and the
actions of imidazoline agonists (Ernsberger and Haxhiu p38 family. Activated MAPKs phosphorylate several sub-
1997; Ernsberger et al. 1997), in the eye where they regulate strates in PC12 cells including various transcription factors
ocular pressure (Campbell and Potter 1994), and in the (Cowley et al. 1994). In the present study, we sought to
kidney where they promote urinary sodium excretion determine whether activation of the I1-imidazoline receptor
(Smyth and Penner 1999). Many ligands active at imidazo- by moxonidine leads to activation of one or more PKC
line receptors also bind to a2-adrenergic receptors. There- isoforms or MAPK species, and further whether an increase
fore, functional studies are typically carried out with prior in cellular proliferation might therefore result from stimula-
blockade of a2-adrenergic receptors. Cellular responses to tion of I1-imidazoline receptors.
I1-imidazoline receptor activation, such as effects on
proliferation, have not been described previously.
The predominant cellular model for investigation of Materials and methods
I1-imidazoline receptor signaling pathways has been PC12 Materials
pheochromocytoma cells. These adrenal tumor cells express RPMI medium and horse serum were obtained from GIBCO
I1-imidazoline receptors but lack a2-adrenergic receptors, as (Gaithersburg, MD, USA). Fetal bovine serum, rat tail collagen and
shown by radioligand binding as well as molecular approaches anti-ERK af®nity puri®ed antibodies were obtained from Upstate
(Separovic et al. 1996). Stimulation of the I1-imidazoline Biotechnology (Lake Placid, NY, USA). Moxonidine was kindly
receptor in PC12 cells with the agonist moxonidine leads to provided by Kali-Chemie (Hannover, Germany). Efaroxan and
activation of phosphatidylcholine selective phospholipase C clonidine were purchased from Research Biochemicals Inter-
(PC-PLC) (Separovic et al. 1996, 1997; Ernsberger 1999). national (Natick, MA, USA). The enzyme inhibitors D609 and
H-7 were purchased from Biomol (Plymouth Meeting, PA).
Activation of PC-PLC is characteristic of the signaling
nPKC1, nPKCz, cPKCb11 and JNK goat polyclonal af®nity
pathways coupled to certain cytokine receptors, including
puri®ed antibodies were obtained from Santa Cruz Biotechnology
some of the interleukins receptors (Cobb et al. 1996; Ho (Santa Cruz, CA, USA). Anti-active ERK antibody and donkey
et al. 1994), and also mediates some of the actions of anti-rabbit horseradish peroxidase antibody were purchased from
thromboxanes in astrocytes (Kobayashi et al. 2000). Activa- Promega (Madison, WI, USA). Nerve growth factor (NGF) was
tion of PC-PLC by imidazoline agonists results in increased obtained from Austral Biologicals (San Ramon, CA, USA). Protein
formation of the second messenger diacylglyceride (DAG) assay reagents and the colorimetric PKC assay kit were obtained
from phosphatidylcholine, and the release of phospho- from Pierce (Rockford, IL, USA). All other chemicals were from
choline. These effects can be blocked by both efaroxan, an Sigma Chemical Co. (St Louis, MO, USA) or Fisher (Pittsburgh,
I1-imidazoline receptor antagonist, and by D609, an inhibi- PA, USA) and were of analytical grade.
tor of PC-PLC. Cell signaling steps subsequent to the PC12 cell culture
accumulation of DAG have not been characterized for PC12 cells were cultured as previously reported (Separovic et al.
I1-imidazoline receptor signaling, but DAG commonly 1996). Brie¯y, PC12 cells were grown on 75 cm2 ¯asks coated with
activates several isoforms of PKC. rat tail collagen at 5% CO2 in RPMI 1640 supplemented with 10%
At least 11 isoforms comprise the PKC family (Liu and (v/v) heat-inactivated horse serum, 5% (v/v) fetal bovine serum
Heckman 1998) and these differ according to structure, (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (com-
substrate speci®city, cofactor requirement and subcellular plete medium). Cells were subcultured at a plating density of 1 : 6
localization. The PKC isoforms can be classi®ed as classi- once per week and medium was refreshed every two days. Because
previous studies showed that the response to I1-imidazoline
cal, novel and atypical. The classical PKC isoforms (cPKC,
receptor stimulation was enhanced following differentiation of
a, b1, b11, g) are calcium-dependent and activated by DAG
PC12 cells with NGF, for most experiments PC12 cells were
derived from phosphatidylinositol or phosphatidylcholine. treated with NGF (50 ng/mL) in RPMI 1640 medium supplemented
The novel PKC isoforms (nPKC, d, 1, h, u) are also with 1% FBS for 2 days in order to initiate neuronal differentiation.
sensitive to DAG but are calcium independent owing to the
absence of a calcium binding domain. Finally, the atypical Preparation of cell fractions for assay of PKC activity
PKC isoforms (aPKC, i, l, z) are insensitive to DAG or PC12 cells were pre-incubated in RPMI 1640 medium with 10 ng/
mL NGF for 30 min. Cells were then exposed to the following
calcium and may be activated by other cellular signals.
treatments for 10 min: 1.0 mm moxonidine, or 200 nm phorbol-12-
Because I1-imidazoline receptors trigger the accumulation
myristate-13-acetate (PMA), or 0.02% DMSO as vehicle control.
of DAG, we hypothesized that classical and novel PKC All treatments were made up in RPMI medium supplemented with
isoforms might be activated by imidazoline agonists. 10 ng/mL NGF. After treatment, cells were washed with ice-cold
Possible downstream targets for PKC in PC12 cells are RPMI containing 5 mm EGTA, and then removed from the ¯ask by
the family of mitogen-activated protein kinases (MAPKs) scraping. All subsequent steps were carried out at 48C, and each
(Cowley et al. 1994). MAPKs are intracellular mediators ¯ask of cells was processed separately. Cells were pelleted by
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3. I1-Imidazoline, PKC and MAPK 933
centrifugation at 2000 g for 5 min at 48C. Cell pellets were Tris-HCl pH 7.4,10 mm MgCl2, 2 mm ATP, 0.1 mm CaCl2,
homogenized with a polytron (Tekmar Tissumizer; setting 6 for 0.002% Triton X-100 detergent, and 0.2 mg/mL phosphatidyl-l-
30 s) in 1.0 mL of homogenization buffer containing Tris-HCl, serine. Negative controls were treated identically, but contained
pH 7.4, 50 mm NaF, 0.2 mm Na3VO4, 2.1 mm EDTA, 6.0 mm 10 mL of Tris-HCl buffer at pH 7.4 containing 50% glycerol in
2-mercaptoethanol, 2 mm EGTA, and a cocktail of protease the place of cell fraction. Antibodies and agarose were included in
inhibitors (0.06 mg/mL anti-pain-HCl, 0.01 mg/mL bestatin, the negative controls. The assay mixture also contained 200 nm
0.02 mg/mL chymostatin, 0.06 mg/mL E-64 {N-[N-(l-3-trans- phorbol myristate acetate, except for assays of preactivated PKC
carboxirane-2-carbonyl-l-leucyl]agmatine}, 0.01 mg/mL leupeptin, where this was omitted. After the incubation, a 20 mL aliquot was
0.01 mg/mL pepstatin, 0.06 mg/mL phosphoramidon, 0.4 mg/mL applied to a ferrite af®nity ®lter (Toomik et al. 1993) and washed
pefabloc, and 0.01 mg/mL aprotinin). The homogenate was with three times by vacuum ®ltration with 250 mL of wash buffer,
centrifuged at 106 000 g for 1 h. The resulting supernatant was consisting of 0.5 m NaCl and 0.1 m sodium acetate at pH 5.0.
retained as the cytosolic fraction. Membrane fractions were Phosphopeptide was eluted with 15% formic acid. Absorbance of
obtained by homogenizing the particulate fraction (setting 6 for the eluate was measured at 570 nm in a Rainbow plate reader with
30 s) in 1.5 mL of solublization buffer (homogenization buffer rhodamine-chromagranin as standard. Protein was assayed by the
containing 1% Triton X-100), bath sonication on ice for 15 min, bicinchoninic acid method (Smith et al. 1985). A signi®cant
mixing by slow rotation for 30 min, and then centrifugation at increase in the phosphorylation of rhodamine-chromagranin
15 000 g for 10 min. The resulting supernatant was kept as the substrate, relative to blanks containing buffer and immunocomplex
membrane fraction. alone, was found for each of the three immunoprecipitated PKC
isoforms.
Immunoprecipitation and assay of PKC activity
Immunoprecipitation was carried out on the cytosolic and mem- Assay of ERK activation
brane fractions as previously described (Mandal et al. 1997). Differentiated PC12 cells in 75 cm2 culture ¯asks were treated with
Aliquots of each fraction (15 mL containing 2±5 mg of protein) various doses of moxonidine (0.1 nm21 mm) or clonidine (100 nm)
were treated with 10 mL of the appropriate isozyme speci®c for 0±180 min. In some experiments, cells were pretreated with
antibody (cPKCb11, nPKC1, aPKCz) then incubated with mixing inhibitors (efaroxan, D609 or H-7) or vehicle (0.1 mm acetic acid
for 18 h at 48C. The immunoprecipitates were captured by adding in RPMI) alone for 10 min before the addition of moxonidine. In
25 mL of agarose conjugated to donkey anti-rabbit secondary other experiments, cells were pretreated with 200 nm phorbol
antibodies to each sample, followed by overnight incubation. myristate acetate for 20 h to deplete PKC. After treatment, cells
Precipitates were isolated by centrifugation at 2000 g for 5 min, were washed with ice-cold calcium-free Hank's buffer, removed
washed twice by resuspension and centrifugation with homo- from the ¯ask by scraping, and then collected by centrifugation.
genization buffer and ®nally resuspended in 100 mL of Tris-HCl Cells were subsequently homogenized in lysis buffer (1% Triton
buffer at pH 7.4 containing 50% glycerol. X-100, 0.5% NP-40, 150 mm NaCl, 10 mm Tris pH 7.4, 1 mm
The ef®ciency of immunoprecipitation was determined by Western EDTA, 1 mm EGTA pH 8.0, 0.2 mm sodium ortho-vanadate,
blot analysis of the supernatant and immunoprecipitated fractions. 0.2 mm PMSF, and protease inhibitor cocktail (Boehringer
The immunoprecipitating antibody was used as the primary anti- Mannheim GmbH, Mannheim, Germany) with a polytron (Tecmar
body for western blot analysis. Following immunoprecipitation of Tissuemizer, 15 s at setting 60) followed by centrifugation
either cytosol or membrane fractions with the cPKCb11 antibody, (16 000 g, 48C) for 10 min. Equal amounts of protein (20 mg)
the supernatants contained immunoreactivity for nPKC1 and from the resulting supernatants were subjected to SDS±PAGE on a
aPKCz, but cPKCb11 could not be detected. Similar results were 10% gel and proteins were electrophoretically transferred to a
obtained following immunoprecipitation of cytosol and membrane nitrocellulose membrane for immunodetection with anti-Active
fractions with nPKC1 and aPKCz antibodies. Thus, the ef®ciency MAPK and anti-MAPK antibodies. with a polytron (Tecmar
of immunoprecipitation by each PKC isozyme antibody approached Tissuemizer, 15 s at setting 6) followed by centrifugation at
100%, within the limits of detection of western blot methods. 97 000 g at 48C for 1 h. Aliquots (10 mg protein as assayed by
Immunoprecipitated PKC activity in both membrane and the bicinchoninic acid method) from the resulting supernatants
cytosolic fractions were assayed using a Pierce PKC Colorimetric were subjected to SDS±PAGE on a 10% acrylamide gel and
Assay Kit employing the eight-well strip format. Dye-coupled proteins were electrophoretically transferred to a nitrocellulose
chromagranin (Lissamine Rhodamine B at the N-terminal) was membrane for immunodetection with anti-active ERK and
used as the substrate because this chromaf®n granule protein is an anti-ERK antibodies.
endogenous PKC substrate in PC12 cells. The assay was carried out A dual antibody method was used to quantitate activation of
according to the manufacturer's instructions with two exceptions. ERK as the ratio of active to total ERK. The anti-active antibody
First, the incubation period was lengthened from 30 to 120 min as recognizes the dually phosphorylated activated forms of ERK-1 and
pilot experiments with both cytosolic and membrane fractions ERK-2, whereas anti-ERK recognizes all forms of ERK-1 and
indicated that four times more reaction product was obtained with a ERK-2. A donkey anti-rabbit secondary antibody coupled to horse-
120-min incubation compared to 30 min. Second, an additional radish peroxidase was utilized to visualize protein bands by chemi-
wash step was added prior to the ®nal elution of phosphopeptide luminescence using Hyper ®lm ECL (Amersham, Buckinghamshire,
with formic acid to reduce background absorbance at 570 nm. UK). Film images were quanti®ed by using a scanning densito-
Aliquots (10 mL) of PC12 cell membranes or cytosol were incu- meter (United States Biochemical, Cleveland, OH, USA). Results
bated 120 min at 378C in a total volume of 25 mL of assay were expressed as a ratio of arbitrary density times area units
buffer containing 5 mm rhodamine-chomagranin substrate, 20 mm between anti-active and anti-ERK blots and then normalized to
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940

4. 934 L. Edwards et al.
vehicle-treated controls run in parallel. Because identical results Results
were obtained for ERK-1 and -2, the data presented here represent
the combined ERK-1 and -2 bands.
Effect of moxonidine and phorbol myristate acetate on
the activity of three PKC isoforms
We ®rst determined whether the selected PKC isoforms
Assay of c-jun kinase activity
Assay of immunoprecipitated c-jun kinase (JNK) was conducted as could be detected in PC12 cells using dye-coupled chromo-
previously described (Coroneos et al. 1996). Cell lysates were granin substrate. The absolute activities for cPKCbII in
immunoprecipitated with rabbit polyclonal IgG directed against untreated control PC12 cells were: cytosol 1.2 ^ 0.2, and
JNK overnight at 48C, and the resulting immunocomplexes were membrane 0.86 ^ 0.1 mg of phosphorylated substrate per
captured with goat anti-rabbit IgG agarose for 8 h at 48C. The ¯ask. For nPKC absolute activities were: cytosol 0.59 ^ 0.1,
agarose complexes were collected by centrifugation and washed and membrane 1.0 ^ 0.1 mg per ¯ask. The activity of aPKCz
twice with PBS. The pellets were then incubated at 378C for 20 min was: cytosol 0.72 ^ 0.1, and membrane 1.0 ^ 0.2 mg of
with 1 mg rat c-jun, 3 mL ATP (cold, ®nal concentrated 25 mm), phosphorylated substrate per ¯ask. Thus, membrane-bound
1 mL [32P]ATP (speci®c activity . 4500 Ci/mmol) in a kinase PKC activity was comparable for the three isoforms, in
buffer (25 mL) as previously described (Coroneos et al. 1996). The
agreement with previous reports (Wooten et al. 1994).
samples were then boiled with Laemmli buffer for 2 min followed
The effect on PKC activity of treatment with either
by SDS±PAGE. After transfer to nitrocellulose, the blots were
exposed to Kodak OMAT ®lm for 24 h at 2808C. Protein bands moxonidine or phorbol myristate acetate is shown in Fig. 1.
were quanti®ed by scanning densitometry as described for ERK. 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
Cell proliferation assays cPKCbII showed increased activity in solubilized membrane
Cell proliferation was measured by using the Cell Titer system ( p , 0.05, paired t-test), whereas cytosolic activity was
(Promega; Madison, WI, USA) as speci®ed in the manufacturers unchanged (Fig. 1). Treatment with 200 nm phorbol myri-
instructions. PC12 cells were plated at one-quarter of their normal state acetate for 10 min induced a nearly identical response.
density in 96-well plates in low-serum medium (1% horse serum In contrast, nPKC showed no signi®cant response to either
and 0.5% fetal calf serum). Cells were treated with increasing doses treatment in membrane or cytosolic fractions. The activity of
of clonidine or with 0.1% DMSO vehicle for 48 h. The number of the atypical isoform, aPKC1 showed translocation from the
viable cells was estimated by incubating the cells for 2 h at 37 8C cytosol to the membrane, as indicated by a decrease in the
with the metabolic dye [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
former and an increase in the latter (both p , 0.05, paired
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] inner salt (MTS;
t-test). As expected, there was no in¯uence of phorbol
Owen's reagent) (Cory et al. 1991). Metabolically oxidized
formazan product was read from an absorbance plate reader at
myristate acetate on the activity of aPKCz, a DAG-
490 nm, with the absorbance in cell-free control wells subtracted. insensitive isoform.
Results were expressed as corrected absorbance relative to vehicle-
treated controls run on the same plate.
Pilot experiments indicated that moxonidine had signi®cant
Effect of moxonidine on ERK activation in extracts from
proliferative action only when added every 12 h, consistent with differentiated PC12 cells
the short half life of this compound in vivo (Ernsberger et al. 1993). The activation of ERK-1 and ERK-2 was determined as the
Clonidine, an analog with similar I1-imidazoline receptor af®nity, ratio of the amount of dually phosphorylated active form to
was found to be effective when added once for up to 48 h, so total ERK immunoreactivity. A representative blot is shown
subsequent experiments were carried out with clonidine. This agent in Fig. 2, illustrating the time course of the response to
has a greater activity at a1- and a2-adrenergic receptors than 100 nm moxonidine. An increase in the amount of immuno-
moxonidine, but this was not thought relevant because PC12 cells reactivity to the anti-active antibody is apparent at the later
lack both a1- and a2-adrenergic receptors (Jinsi-Parimoo and Deth time points. The lower blot shows that the amount of ERK-2
1997; Berts et al. 1999). Indeed, PC12 cells have been used for
immunoreactivity was constant between lanes, indicating
transfection studies of these receptors speci®cally because they lack
equal loading. Mean data from four experiments showed
endogenous expression.
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±
Data analysis
Keuls test) was detected at 30 min, and the peak activation of
Statistical comparisons were performed by t-test for two groups or
analysis of variance for multiple comparisons, with Newman± ERK occurred at 90 min, with a decline towards baseline
Keuls post hoc tests. Dose±responses data were ®tted to logistic after 2 h.
equations (Motulsky and Ransnas 1987) using the Prism data The dose-dependence for the action of moxonidine on
analysis package (GraphPad software, San Diego, CA, USA) to ERK is illustrated in Fig. 4. The immunoreactivity to the
obtain EC50 values. anti-active antibody increased with the concentration of
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5. I1-Imidazoline, PKC and MAPK 935
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.
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. 1 Activity of PKC isoforms in fractions from PC12 cells treated (Fig. 5). A higher concentration of moxonidine, 1.0 mm,
with moxonidine or phorbol myristate acetate. Shown are relative activated ERK to a lesser extent than 100 nm. Comparable
rates of dye-labeled substrate phosphorylation activity of immuno- biphasic dose±response relationships have been reported for
precipitated PKC isoforms. Three representative PKC isozymes DAG accumulation (Separovic et al. 1996).
expressed in PC12 cells were isolated: cPKCbII, nPKC1, and
In order to test whether the effect of moxonidine on ERK
aPKCz. The effects of phorbol myristate acetate and moxonidine are
stimulation was mediated by the I1-imidazoline receptor and
represented by their percentage change ^ SE relative to controls
run in parallel in the same experiment. Data represent the
through its known transmembrane signaling pathways, we
mean ^ SE from 12 75-cm2 ¯asks of cells. Asterisks mark statisti- treated the cells with efaroxan, a selective I1-imidazoline
cally signi®cant increases ( p , 0.05, paired t-test). 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 Fig. 4 Western blot illustrating the dose-dependence of ERK-2 acti-
100 nM moxonidine for increasing amounts of time. Data were ana- vation by moxonidine. Bands are labeled as in Fig. 2. Each lane was
lyzed by determining the ratio of optical density between the ®rst obtained from different ¯asks of PC12 cells incubated with increas-
and second blot. ing concentrations of moxonidine for 90 min.
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6. 936 L. Edwards et al.
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
Fig. 7 Western blot showing abrogation of the ERK activation
change ^ SE from four separate experiments run in duplicate.
response to moxonidine by PKC depletion or inhibition. Bands are
labeled as in Fig. 2. Each lane was obtained from different ¯asks of
We next sought to test whether the activation of ERK PC12 cells incubated with various for 20 h or 90 min prior to har-
was mediated through PKC (Figs 7 and 8). Treatment with vesting. First and last lanes are from vehicle-treated control cells.
The second lane shows the response to 100 nM moxonidine relative
the non-selective PKC inhibitor H-7 [1-(5-isoquinoline-
to the vehicle control lane. The third lane is from a ¯ask of PC12
sulfonyl)-2-methylpiperazine] blocked the action of moxon-
cells that was processed in parallel but was pretreated with 200 nM
idine. Treatment with H-7 alone had no effect on ERK phorbol-12-myristate-13-acetate overnight to deplete PKC. The fourth
activation. In order to down-regulate DAG-sensitive iso- lane shows the response to short-term treatment with phorbol ester.
forms of PKC, we pretreated PC12 cells with 200 nm The ®fth lane shows that results treatment with the PKC inhibitor
phorbol myristate acetate for 20 h prior to exposure to H-7 during the 10 min pre-incubation and during moxonidine treat-
either phorbol or moxonidine for 90 min. Depletion of PKC ment, while the next lane illustrates the lack of effect of H-7 alone.
by prolonged treatment with phorbol myristate acetate The seventh lane shows that the response to phorbol ester is lost
abolished the response to short-term phorbol, con®rming after 20 h exposure to 200 nM phorbol-12-myristate-13-acetate.
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).
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
Effect of moxonidine on JNK activity in PC12 cell
antagonist efaroxan (10 mM) or the PC-PLC inhibitor D609 (10 mM)
extracts
for 90 min. Efaroxan or D609 were also present during a 10-min
In addition to the ERKs, an independently regulated
pre-incubation. ERK activation was then determined as described
above. Values are expressed as a percentage of vehicle treated
kinase cascade in PC12 cells involves JNKs. Moxonidine
controls. Each value represents the mean ^ SE of at least nine dose-dependently increased cellular activity of JNK up to
separate experiments. The effect of moxonidine alone was signi®- two-fold (Fig. 9). Peak effects were observed at 300 nm
cant ( p , 0.01, paired t-test) but no other treatment or combination moxonidine. In the presence of 10 mm efaroxan, 100 nm
of treatments had any signi®cant effect ( p . 0.10, paired t-test). moxonidine did not increase JNK activity (data not shown).
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940

7. I1-Imidazoline, PKC and MAPK 937
Fig. 10 Concentration-dependent increase in PC12 cell proliferation
Fig. 8 Effects of PKC depletion or inhibition on ERK activation by
by clonidine. PC12 cells were grown in low-serum medium in the
moxonidine. PC12 cells were incubated with vehicle alone, moxoni-
presence of increasing concentrations of clonidine for 48 h, and then
dine (100 nM) alone, moxonidine in the presence of the PKC inhibitor
the density of viable metabolically active cells was determined by
H-7 (1.0 mM), H-7 alone, moxonidine following overnight exposure to
using the MTT metabolic dye. Values are expressed as a percen-
200 nM phorbol-12-myristate-13-acetate in order to deplete PKC, or
tage of vehicle treated controls. Each value represents the mean ^
the response to short-term treatment with phorbol ester with and
SEM of 18 separate wells.
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
Proliferative response of PC12 cells to imidazoline
represents the mean ^ SE of at least six separate experiments. The
agonists
effect of moxonidine alone and phorbol-12-myristate-13-acetate
alone were signi®cant ( p , 0.01, paired t-test), but no other treat-
The activity of ERK and possibly JNK as well is linked to
ment or combination of treatments had any signi®cant effect cell proliferation, particularly in transformed cell lines such
( p . 0.10, paired t-test). 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
The time course of JNK activation is indicated in Fig. 3 rather than moxonidine because of its longer metabolic half-
(squares). The increase in JNK activity tended to parallel life in vivo (Ernsberger et al. 1993) and because these two
the activation of ERK, with both peaking around 90 min I1-agonists showed similar activation of ERK (see above).
and declining by 120 min. The increase in JNK activity PC12 cells were seeded at one-fourth normal density in 96
was evident earlier, and reached signi®cance at 15 min well plates in low-serum medium in order to reduce
( p , 0.05, Newman±Keuls' test), whereas ERK was not background levels of proliferation. The ®nal number of
increased until 30 min of moxonidine treatment. 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
Fig. 9 Concentration-dependent stimulation of JNK activity by
cells. A common and an atypical isoform of PKC each
moxonidine. PC12 cells were incubated with or without varying showed increased enzymatic activity (cPKCbII and aPKCz),
doses of moxonidine (0.1 nM21 mM) or vehicle for 90 min and then whereas nPKC1 was not affected. The stimulation of
lysates were assayed for JNK phosphorylation. Each value repre- cPKCbII by the I1-imidazoline agonist moxonidine was
sents the mean ^ SEM of at least four experiments. comparable to that induced by treatment with a phorbol
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940

8. 938 L. Edwards et al.
ester. In addition, aPKCz showed clear subcellular relocal- nearly 125% upon exposure to moxonidine. The possible
ization, with activity in the cytosol decreasing and that in the modulation of PC12 cell neuronal differentiation by
membrane fraction increasing. Two members of the MAPK I1-imidazoline receptors remains to be determined.
family of kinase cascades were also activated in response to Cellular DAG are known to regulate the activity of
moxonidine: ERK and JNK. These kinases showed roughly cPKCbII. Stimulation of I1-imidazoline receptors in PC12
parallel activation with a peak effect occurring around cells with moxonidine elevates total cellular mass of DAG
90 min of treatment. In PC12 cells stimulated with the (Separovic et al. 1996). Moreover, in the present study, the
I1-imidazoline agonist moxonidine, the proportion of ERK effects of moxonidine closely resembled those of phorbol
in its active dually phosphorylated form was increased ester, a diglyceride analog. Thus, the activation of cPKCbII
150%, whereas JNK activity was elevated nearly two-fold. by moxonidine might plausibly be the result of increased
The activation of both kinases was dose-dependent, and diglyceride levels. The mechanisms behind the activation of
in the case of ERK the EC50 for moxonidine was in aPKCz are not as clear. Arachidonic acid activates atypical
close agreement with the binding af®nity of the drug PKC isoforms in isolated brain membranes (Huang et al.
for I1-imidazoline receptors [Ki ˆ 7.8 nm; (Separovic et al. 1993).
1996)]. Finally, a modest but concentration-dependent The ERK family of MAPK were also activated in
increase in cell number was elicited by 2-day treatment of response to I1-imidazoline receptor stimulation. The MAPK
PC12 cell cultures with the I1-imidazoline agonist clonidine. family members, including ERK and JNK, typically mediate
This result implies a weak mitogenic action of I1-imidazo- responses to mitogenic stimuli and promote cell prolifera-
line receptors, consistent with their apparent activation of tion (Marshall 1995). Sustained activation of the MAPK
MAPK cascades. signaling pathway is reportedly both necessary and suf®-
In the present study, the activation of ERK was apparently cient to induce neuronal differentiation of PC12 cells
receptor-mediated, because it could be blocked by cotreat- (Cowley et al. 1994). We have reported a two-fold increase
ment with the I1-imidazoline antagonist efaroxan. Moreover, in ERK-activation by the I1-imidazoline agonists moxon-
the concentration range wherein moxonidine was effective idine and clonidine which can be blocked by efaroxan, an
in activating ERK and JNK was consistent with its binding I1-imidazoline receptor antagonist, and by D609, an inhibi-
af®nity for I1-imidazoline receptors, and the dose±response tor of PC-PLC. These data imply that activation of MAPK is
curves for ERK and JNK activation closely resembled pre- receptor-mediated and is downstream from phospholipid
viously reported dose±response relationships for I1-imidazo- hydrolysis pathways associated with the I1-imidazoline
line receptor activation of arachidonic acid release (Ernsberger receptor. Efaroxan can also acts as an a2-adrenergic antag-
1998), prostaglandin production (Ernsberger et al. 1995), onist in some cells in the dose range used in the present
and DAG accumulation (Separovic et al. 1996). study, but these receptors are not present in PC12 cells.
The I1-imidazoline receptor has been previously shown to Efaroxan has negligible af®nity for the mitochondrial
be coupled to activation of PC-PLC in PC12 cells, which I2-imidazoline subtype (Lione et al. 1996) which are present
leads to formation of DAG from phosphatidylcholine and an in these cells. The activation of ERK and JNK by moxo-
increased total cellular mass of this second messenger nidine was not sustained, but rather peaked around 90 min
(Separovic et al. 1996). We therefore hypothesized that PKC and declined substantially within 120 min. This pattern
may be activated by I1-imidazoline receptor stimulation. resembles the response to epidermal growth factor and other
The PKC multigene family of enzymes is involved in the agonists that activate ERK in PC12 cells, but stands in
control of many biological events and is a major transducer contrast to NGF-activation of ERK, which is sustained for
of receptor-mediated stimuli. In PC12 cells the I1-imidazo- many hours (Marshall 1995).
line receptor agonist moxonidine has been shown to activate The activation of ERK in response to imidazoline agonists
at least two isoforms of PKC (cPKCbII and aPKCz). PC12 appears to be downstream of PKC activation. Thus, deple-
cells are known to express the following PKC isotypes: a, tion of classical and novel isoforms of PKC by prolonged
bI, bII, d, 1, h and z (Hundle et al. 1995) and differentiation exposure to a phorbol ester blocked the response to moxon-
of PC12 cells to a neuronal phenotype by treatment with idine. Furthermore, a non-selective PKC inhibitor blocked
NGF induces increased expression of bII, d, and z, and the the response to moxonidine as well. These results implicate
appearance of PKC 1 and h within the nucleus (Borgatti the stimulation of PKC in the activation of ERK by
et al. 1996). This suggests that the I1-imidazoline receptor imidazoline agonists. The subtype of PKC responsible may
might modulate cell proliferation or neuronal differentiation be the bII isoform, as this form was activated in response
through activation of key PKC isoforms. In addition, the to moxonidine. While the aPKCz isoform was also
atypical z-PKC is required for neuronal differentiation activated, this atypical subtype of PKC is not known to
and neurite outgrowth of PC12 cells in response to NGF be depleted by prolonged stimulation with phorbol esters.
(Coleman and Wooten 1994), and thus the activity of this The isoform may participate in other downstream signaling
isoform in PC12 cell membrane fractions was increased by events.
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940

9. I1-Imidazoline, PKC and MAPK 939
In the present study, a small but persistent and dose- of MAP kinase kinase is necessary and suf®cient for PC12
dependent increase in the number of viable cells was differentiation and for transformation of NIH 3T3 cells. Cell 77,
841±852.
noted in cultures of serum-starved PC12 cells treated with
Eglen R. M., Hudson A. L., Kendall D. A., Nutt D. J., Morgan N. G.,
I1-imidazoline agonist clonidine. An increase in total cell Wilson V. G. and Dillon M. P. (1998) `Seeing through a glass
number is a strong indicator that increased cell proliferation darkly': casting light on imidazoline `I' sites. Trends Pharmacol.
has occurred, although the number of actively dividing cells Sci. 19, 381±390.
was not measured. The activation of MAPK cascades may Ernsberger P. (1998) Arachidonic acid release from PC12 pheochromo-
cytoma cells is regulated by I1-imidazoline receptors. J. Auton.
have been too transient to induce a dramatic proliferative
Nerv. Syst. 72, 147±154.
response. The mitogenic effect was not altered when serum Ernsberger P. (1999) The I1-imidazoline receptor and its cellular
levels in the media were increased or the initial seeding signaling pathways. Ann. NY Acad. Sci. 881, 35±53.
density of the cells were changed systematically (data not Ernsberger P. and Haxhiu M. A. (1997) The I1-imidazoline-binding site
shown), implying that the effect of clonidine was not is a functional receptor mediating vasodepression via the ventral
strongly dependent upon culture conditions. medulla. Am. J. Physiol. 273, R1572±R1579.
Ernsberger P., Meeley M. P., Mann J. J. and Reis D. J. (1987) Clonidine
In conclusion, we have extended our model of the signal-
binds to imidazole binding sites as well as a2-adrenoceptors in the
ing pathway for I1-imidazoline receptor in PC12 cells to ventrolateral medulla. Eur. J. Pharmacol. 134, 1±13.
include coupling to PKC and MAPK. One possible function Ernsberger P., Elliott H. L., Weimann H.-J., Raap A., Haxhiu M. A.,
of these intermediates may be in promoting cell proliferation È È
Hofferber E., Low-Kroger A., Reid J. L. and Mest H.-J. (1993)
or possibly the modulation of neuronal differentiation. Moxonidine: a second-generation central antihypertensive agent.
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This work was supported by HL44514 (to PE and MK) and brane signaling. Ann. NY Acad. Sci. 763, 22±42.
DK53715 (to MK) from the National Institutes of Health. We Ernsberger P., Friedman J. E. and Koletsky R. J. (1997) The
acknowledge the technical assistance of Kathryn Zalovcik, BS I1-imidazoline receptor: from binding site to therapeutic target
and David Bedol, BS. in cardiovascular disease. J. Hypertens. 15, S9±S23.
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