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


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



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).



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.



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).



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.



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.

References

Berts A., Zhong H. and Minneman K. P. (1999) No role for Ca11 or

protein kinase C in alpha-1A adrenergic receptor activation of

mitogen-activated protein kinase pathways in transfected PC12

cells. Mol. Pharmacol. 55, 296±303.

Borgatti P., Mazzoni M., Carini C., Neri L. M., Marchisio M., Bertolaso

L., Previati M., Zauli G. and Capitani S. (1996) Changes of

nuclear protein kinase C activity and isotype composition in PC12

cell proliferation and differentiation. Exp. Cell Res. 224, 72±78.

Bousquet P., Feldman J., Bloch R. and Schwartz J. (1984) Central

cardiovascular effects of a-adrenergic drugs: Differences between

catecholamines and imidazolines. J. Pharmacol. Exp. Ther. 230,

232±236.

Campbell W. R. and Potter D. E. (1994) Potential role of imidazoline

(I1) receptors in modulating aqueous humor dynamics. J. Ocul.

Pharmacol. 10, 393±402.

Cobb R. R., Felts K. A., Parry G. C. and Mackman N. (1996) D609, a

phosphatidylcholine-speci®c phospholipase C inhibitor, blocks

interleukin-1 beta-induced vascular cell adhesion molecule 1 gene

expression in human endothelial cells. Mol. Pharmacol. 49,

998±1004.

Coleman E. S. and Wooten M. W. (1994) Nerve growth factor-induced

differentiation of PC12 cells employs the PMA-insensitive protein

kinase C-zeta isoform. J. Mol. Neurosci. 5, 39±57.

Coroneos E., Wang Y., Panuska J. R., Templeton D. J. and Kester M.

(1996) Sphingolipid metabolites differentially regulate extra-

cellular signal- regulated kinase and stress-activated protein

kinase cascades. Biochem. J. 316, 13±17.

Cory A. H., Owen T. C., Barltrop J. A. and Cory J. G. (1991) Use of an

aqueous soluble tetrazolium/formazan assay for cell growth assays

in culture. Cancer Commun. 3, 207±212.

Cowley S., Paterson H., Kemp P. and Marshall C. J. (1994) Activation

of MAP kinase kinase is necessary and suf®cient for PC12

differentiation and for transformation of NIH 3T3 cells. Cell 77,

841±852.

Eglen R. M., Hudson A. L., Kendall D. A., Nutt D. J., Morgan N. G.,

Wilson V. G. and Dillon M. P. (1998) `Seeing through a glass

darkly': casting light on imidazoline `I' sites. Trends Pharmacol.

Sci. 19, 381±390.

Ernsberger P. (1998) Arachidonic acid release from PC12 pheochromo-

cytoma cells is regulated by I1-imidazoline receptors. J. Auton.

Nerv. Syst. 72, 147±154.

Ernsberger P. (1999) The I1-imidazoline receptor and its cellular

signaling pathways. Ann. NY Acad. Sci. 881, 35±53.

Ernsberger P. and Haxhiu M. A. (1997) The I1-imidazoline-binding site

is a functional receptor mediating vasodepression via the ventral

medulla. Am. J. Physiol. 273, R1572±R1579.

Ernsberger P., Meeley M. P., Mann J. J. and Reis D. J. (1987) Clonidine

binds to imidazole binding sites as well as a2-adrenoceptors in the

ventrolateral medulla. Eur. J. Pharmacol. 134, 1±13.

Ernsberger P., Elliott H. L., Weimann H.-J., Raap A., Haxhiu M. A.,

Hofferber E., LoÈw-KroÈger A., Reid J. L. and Mest H.-J. (1993)

Moxonidine: a second-generation central antihypertensive agent.

Cardiovasc. Drug Rev. 11, 411±431.

Ernsberger P., Graves M. E., Graff L. M., Zakieh N., Nguyen P., Collins

L. A., Westbrooks K. L. and Johnson G. G. (1995) I1-imidazoline

receptors: De®nition, characterization, distribution and transmem-

brane signaling. Ann. NY Acad. Sci. 763, 22±42.

Ernsberger P., Friedman J. E. and Koletsky R. J. (1997) The

I1-imidazoline receptor: from binding site to therapeutic target

in cardiovascular disease. J. Hypertens. 15, S9±S23.

Ho J. L., Zhu B., He S., Du B. and Rothman R. (1994) Interleukin 4

receptor signaling in human monocytes and U937 cells involves

the activation of a phosphatidylcholine-speci®c phospholipase C:

a comparison with chemotactic peptide, FMLP, phospholipase D,

and sphingomyelinase. J. Exp. Med. 180, 1457±1469.

Huang X. P., Da Silva C., Fan X. T. and Castagna M. (1993)

Characteristics of arachidonic-acid-mediated brain protein kinase

C activation: Evidence for concentration-dependent heterogeneity.

Biochim. Biophys. Acta Mol. Cell Res. 1175, 351±356.

Hundle B., McMahon T., Dadgar J. and Messing R. O. (1995) Over-

expression of epsilon-protein kinase C enhances nerve growth

factor-induced phosphorylation of mitogen-activated protein

kinases and neurite outgrowth. J. Biol. Chem. 270, 30134±30140.

Jinsi-Parimoo A. and Deth R. C. (1997) Reconstitution of alpha2D-

adrenergic receptor coupling to phospholipase D in a PC12 cell

lysate. J. Biol. Chem. 272, 14556±14561.

Kobayashi H., Honma S., Nakahata N. and Ohizumi Y. (2000)

Involvement of phosphatidylcholine-speci®c phospholipase C in

thromboxane A2-induced activation of mitogen-activated protein

kinase in astrocytoma cells. J. Neurochem. 74, 2167±2173.

Limon-Boulez I., Tesson F., Gargalidis-Moudanos C. and Parini A.

(1996) I2-imidazoline binding sites: relationship with different

monoamine oxidase domains and identi®cation of histidine

residues mediating ligand binding regulation by H11. J. Pharma-

col. Exp. Ther. 276, 359±364.

Lione L. A., Nutt D. J. and Hudson A. L. (1996) [3H]2-(2-

benzofuranyl)-2-imidazoline: a new selective high af®nity radio-

ligand for the study of rabbit brain imidazoline I2 receptors. Eur. J.

Pharmacol. 304, 221±229.

Liu W. S. and Heckman C. A. (1998) The sevenfold way of PKC

regulation. Cell Signal. 10, 529±542.

Mandal A., Wang Y., Ernsberger P. and Kester M. (1997) Interleukin-1-

induced ether-linked diglycerides inhibit calcium- insensitive

protein kinase C isotypes. Implications for growth senescence.

J. Biol. Chem. 272, 20306±20311.



Marshall C. J. (1995) Speci®city of receptor tyrosine kinase signaling:

transient versus sustained extracellular signal-regulated kinase

activation. Cell 80, 179±185.

Motulsky H. J. and Ransnas L. A. (1987) Fitting curves to data using

nonlinear regression: a practical and nonmathematical review.

FASEB J. 1, 365±374.

Piletz J. E., Ivanov T. R., Sharp J. D., Ernsberger P., Chang C. H.,

Pickard R. T., Gold G., Roth B., Zhu H., Jones J. C., Baldwin J.

and Reis D. J. (2000) Imidazoline receptor antisera-selected

(IRAS) cDNA: cloning and characterization. DNA Cell Biol. 19,

319±329.

Regunathan S. and Reis D. J. (1996) Imidazoline receptors and their

endogenous ligands. Annu. Rev. Pharmacol. Toxicol. 36, 511±544.

Separovic D., Kester M. and Ernsberger P. (1996) Coupling of I1-

imidazoline receptors to diacylglyceride accumulation in PC12

rat pheochromocytoma cells. Mol. Pharmacol. 49, 668±675.

Separovic D., Kester M., Haxhiu M. A. and Ernsberger P. (1997)

Activation of phosphatidylcholine-selective phospholipase C by

I1-imidazoline receptors in PC12 cells and rostral ventrolateral

medulla. Brain Res. 749, 335±339.

Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H.,

Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J. and

Klenk D. C. (1985) Measurement of protein using bicinchoninic

acid. Anal. Biochem. 150, 76±85.

Smyth D. D. and Penner S. B. (1999) Peripheral and central imidazoline

receptor-mediated natriuresis in the rat. Ann. NY Acad. Sci. 881,

344±357.

Toomik R., Ekman P., Eller M., Jarv J., Zaitsev D., Myasoedov N.,

Ragnarsson U. and Engstrom L. (1993) Protein kinase assay using

tritiated peptide substrates and ferric adsorbent paper for

phosphopeptide binding. Anal. Biochem. 209, 348±353.

Wooten M. W., Zhou G., Seibenhener M. L. and Coleman E. S. (1994)

A role for zeta protein kinase C in nerve growth factor-induced

differentiation of PC12 cells. Cell Growth Differ. 5, 395±403.



Technology

The I1 Imidazoline Receptor In Pc12 Pheochromocytoma Cells