Muthalif MM, Benter IF, Karzoun N, Fatima S, Harper J, Uddin MR, Malik KU.20-Hydroxyeicosatetraenoic acid mediates calcium/calmodulin-dependent protein kinase II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. Proc Natl Acad Sci USA 95:12701-12706

Abstract

Norepinephrine (NE) and angiotensin II (Ang II), by promoting extracellular Ca2+ influx, increase Ca2+/calmodulin-dependent kinase II (CaMKII) activity, leading to activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2), resulting in release of arachidonic acid (AA) for prostacyclin synthesis in rabbit vascular smooth muscle cells. However, the mechanism by which CaMKII activates MAPK is unclear. The present study was conducted to determine the contribution of AA and its metabolites as possible mediators of CaMKII-induced MAPK activation by NE, Ang II, and epidermal growth factor (EGF) in vascular smooth muscle cells. NE-, Ang II-, and EGF-stimulated MAPK and cPLA2 were reduced by inhibitors of cytochrome P450 (CYP450) and lipoxygenase but not by cyclooxygenase. NE-, Ang II-, and EGF-induced increases in Ras activity, measured by its translocation to plasma membrane, were abolished by CYP450, lipoxygenase, and farnesyltransferase inhibitors. An AA metabolite of CYP450, 20-hydroxyeicosatetraenoic acid (20-HETE), increased the activities of MAPK and cPLA2 and caused translocation of Ras. These data suggest that activation of MAPK by NE, Ang II, and EGF is mediated by a signaling mechanism involving 20-HETE, which is generated by stimulation of cPLA2 by CaMKII. Activation of Ras/MAPK by 20-HETE amplifies cPLA2 activity and releases additional AA by a positive feedback mechanism. This mechanism of Ras/MAPK activation by 20-HETE may play a central role in the regulation of other cellular signaling molecules involved in cell proliferation and growth.

Full text

Proc. Natl. Acad. Sci. USA
Vol. 95, pp. 12701–12706, October 1998
Pharmacology
20-Hydroxyeicosatetraenoic acid mediates calcium
y
calmodulin-
dependent protein kinase II-induced mitogen-activated protein
kinase activation in vascular smooth muscle cells
M. M. MUTHALIF,I.F.BENTER,N.KARZOUN,S.FATIMA,J.HARPER,M.R.UDDIN, AND K. U. MALIK*
Department of Pharmacology, College of Medicine, The University of Tennessee Center for Health Sciences, Memphis, TN 38163
Communicated by Philip Needleman, Monsanto Company, St. Louis, MO, August 19, 1998 (received for review February 18, 1998)
ABSTRACT Norepinephrine (NE) and angiotensin II (Ang
II), by promoting extracellular Ca
21
influx, increase Ca
21
y
cal-
modulin-dependent kinase II (CaMKII) activity, leading to ac-
tivation of mitogen-activated protein kinase (MAPK) and cyto-
solic phospholipase A
2
(cPLA
2
), resulting in release of arachi-
donic acid (AA) for prostacyclin synthesis in rabbit vascular
smooth muscle cells. However, the mechanism by which CaMKII
activates MAPK is unclear. The present study was conducted to
determine the contribution of AA and its metabolites as possible
mediators of CaMKII-induced MAPK activation by NE, Ang II,
and epidermal growth factor (EGF) in vascular smooth muscle
cells. NE-, Ang II-, and EGF-stimulated MAPK and cPLA
2
were
reduced by inhibitors of cytochrome P450 (CYP450) and lipoxy-
genase but not by cyclooxygenase. NE-, Ang II-, and EGF-induced
increases in Ras activity, measured by its translocation to plasma
membrane, were abolished by CYP450, lipoxygenase, and farne-
syltransferase inhibitors. An AA metabolite of CYP450, 20-
hydroxyeicosatetraenoic acid (20-HETE), increased the activities
of MAPK and cPLA
2
and caused translocation of Ras. These
data suggest that activation of MAPK by NE, Ang II, and EGF
is mediated by a signaling mechanism involving 20-HETE, which
is generated by stimulation of cPLA
2
by CaMKII. Activation of
Ras
y
MAPK by 20-HETE amplifies cPLA
2
activity and releases
additional AA by a positive feedback mechanism. This mecha-
nism of Ras
y
MAPK activation by 20-HETE may play a central
role in the regulation of other cellular signaling molecules
involved in cell proliferation and growth.
Activation of phospholipase A
2
(PLA
2
) liberates arachidonic acid
(AA) from phospholipids. AA metabolites, including prostaglan-
dins, leukotrienes, lipoxins, and hydroxy derivatives, have been
implicated in numerous physiological and pathophysiological
processes (1–5). Recent studies using ‘‘knock-out’’ mice or inhib-
itors indicate that AA-generating cytosolic PLA
2
(cPLA
2
) plays
a role in macrophage production of inflammatory mediators,
reproductive physiology, allergic responses, postischemic brain
injury, cell proliferation, and cancer (6–9). Neurotransmitters,
hormones, and growth factors activate cPLA
2
and protein kinases
in many cell types, including vascular smooth muscle cells
(VSMCs) (10–14). The adrenergic transmitter norepinephrine
(NE) and angiotensin II (Ang II), by promoting extracellular
Ca
21
influx, increases Ca
21
ycalmodulin-dependent kinase II
(CaMKII) activity, leading to activation of mitogen-activated
protein kinase (MAPK) and cPLA
2
, resulting in release of AA for
prostacyclin synthesis (15, 16). This pathway of MAPK activation
by CaMKII is mediated through stimulation of MAPK kinase
(MEK). CaMKIV expressed in PC-12 cells has also been shown
to activate MAPKs (17). MAPKs are also stimulated by
bg
subunits of heterotrimeric G proteins (18, 19). However, the
mechanism by which CaMK activates MAPK is not known.
AA is metabolized by cyclooxygenase into prostaglandins and
thromboxane A
2
, by lipoxygenase into leukotrienes and hydroxy-
eicosatetraenoic acids (HETEs) [5-, 12(S)-, and 15-HETE], and
by cytochrome P450 (CYP450) into epoxyeicosatrienoic acid and
12(R),19- and 20-HETE (1, 3). AA and some of its lipoxygenase
products (12- and 15-HETE) stimulate MAPK activity (20–22).
Moreover, 5-HETE has been shown to increase cPLA
2
activity in
human neutrophils (23). These observations led us to hypothesize
that the NE-induced increase in MAPK activity is caused by AA
or its metabolites generated through activation of cPLA
2
by
CaMKII. To test this hypothesis, we investigated the effects of
NE, Ang II, and epidermal growth factor (EGF) on Ras, MAPK,
CaMKII, and cPLA
2
activity in the presence and absence of
inhibitors of CYP450, lipoxygenase, and cyclooxygenase in rabbit
aortic VSMCs. We have found that NE, Ang II, and EGF activate
the RasyMAPK pathway through generation of a CYP450 me-
tabolite of AA, 20-HETE, after initial activation of cPLA
2
by
CaMKII. Activation of MAPK by 20-HETE amplifies cPLA
2
activity and releases additional AA by a positive feedback mech-
anism.
MATERIALS AND METHODS
Preparation of VSMCs. Aortae were rapidly removed from
male New Zealand White rabbits and the VSMCs were isolated
as described (24). Cells between passages 2 and 8 were plated in
12- or 24-well plates or 100-mm plates. Cells were maintained
under 5% CO
2
in M-199 medium (Sigma) with penicillin, strep-
tomycin, and 10% fetal bovine serum.
Experimental Protocol. VSMCs that were arrested for 48 h
with medium containing 0.05% fetal bovine serum were used for
all studies. Cells were incubated with inhibitors of cPLA
2
(methy-
larachidonylfluorophosphonate, MAFP) (25); MEK (PD-98059)
(26); CaMKII (KN-93) (27); cyclooxygenase (indomethacin)
(28); lipoxygenase (baicalein, BACL) (29); CYP450 (17-
octadecynoic acid, 17-ODYA) (30); farnesyltransferase (FPT III)
(31); or their respective vehicles and exposed to NE (10
m
M); AA
(1–20
m
M); Ang II (100 nM); EGF (100 nM); 5-, 12(R)-, 12(S)-,
15-, or 20-HETEs (1–250 nM); or their vehicles for an additional
5–15 min. The concentrations of various inhibitors used in our
study have been reported to be effective in blocking the activity
of these enzymes in other cell systems (25–31). MAFP and
HETEs were obtained from Cayman Chemicals (Ann Arbor,
MI). NE, myelin basic protein, and indomethacin were from
Sigma. Ang II was from Bachem. AA was from Nu Chek Prep
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1998 by The National Academy of Sciences 0027-8424y98y9512701-6$2.00y0
PNAS is available online at www.pnas.org.
Abbreviations: Ang II, angiotensin II; AA, arachidonic acid; BACL,
baicalein; CaM, calmodulin; CaMKII, Ca
21
yCaM-dependent protein
kinase II; cPLA
2
, cytosolic phospholipase A
2
; CYP450, cytochrome
P450; EGF, epidermal growth factor; HETE, hydroxyeicosatetraenoic
acid; ERK, extracellular regulated kinase; MAFP, methylarachido-
nylfluorophosphonate; MAPK, mitogen-activated protein kinase;
MEK, MAPK kinase; NE, norepinephrine; 17-ODYA, 17-octade-
cynoic acid; VSMC, vascular smooth muscle cell.
*To whom reprint requests should be addressed. e-mail: kmalik@
utmem1.utmem.edu.
12701

(Elysian, MN). PD-98059 was from New England Biolabs. Indo-
methacin, 17-ODYA, and BACL were from Biomol (Plymouth
Meeting, PA). FPT III and KN-93 were from Calbiochem.
Enzyme Assays. In vitro MAPK assays: The activity of MAPK
was determined in VSMC lysates with a Biotrak kit (Amersham)
using a peptide substrate KRELVEPLTPAGEAPNQALLR as
directed by the manufacturer’s instructions.
In-Gel MAPK Assay. MAPK activity was measured in VSMC
homogenates by renaturation assay in polyacrylamide gels con-
taining myelin basic protein as described (32).
In Vitro CaMKII Assay. CaMKII activity was assayed in cell
lysates by using CaMKII assay kits (Upstate Biotechnology) with
a peptide substrate KKALRRQETVDAL by the manufacturer’s
recommendations.
cPLA
2
Assay. cPLA
2
activity in VSMC lysates (20–30
m
gof
protein per assay) was measured by using
L-[1-
14
C]phosphatidyl-
choline (57 mCiymmol, American Radiolabeled Chemicals; 1
Ci 5 37 GBq) as substrate as described (33).
[
3
H]AA Release. VSMCs were labeled with [
3
H]AA (0.25
m
Ciyml; 100 Ciymmol; Dupont-New England Nuclear) for 16 h,
and AA release was measured as described (15).
CYP450 Metabolism of AA in Rabbit VSMCs. VSMC (pas-
sages 2–4), cultured in 150-mm plates, were permeabilized with
Tween 40 (0.1%) for 15 min, washed twice with Krebs buffer, and
incubated with [
14
C]AA (40–60 mCiymmol; 0.5
m
Ciyml; NEN)
at 37°C for1hin0.1Mpotassium phosphate (pH 7.4) containing
10 mM MgCl
2
, 1 mM EDTA, 1 mM NADPH, and an NADPH-
regenerating system containing 20 mM isocitrate and 0.1 M
isocitrate dehydrogenase. Cells were incubated with 20
m
MAA
for 5 min and then stimulated with NE (10
m
M) for 30 min. The
aqueous medium was collected, and AA metabolites were ex-
tracted and separated with a binary gradient HPLC system
(Waters Associates) using a Nucleosil C
18
column (5
m
M) with a
two-solvent gradient elution as described (34). The column eluate
was monitored at 235 nm, and radioactivity in the samples was
measured by liquid scintillation spectrometry.
Western Blotting of CYP450 4A. Lysate and microsomal pro-
teins of VSMCs were separated on an SDSypolyacrylamide gel
(10%), transferred to a nitrocellulose membrane, and incubated
FIG. 1. NE-induced activation of MAPK and cPLA
2
but not
CaMKII is attenuated by inhibitors of cPLA
2
, CYP450, and lipoxy-
genase. Rabbit VSMCs were treated with the vehicle dimethyl sulfox-
ide (VEH) alone or with inhibitors of cPLA
2
(MAFP, 50
m
M),
CaMKII (KN-93, 20
m
M), or MEK (PD-98059, 20
m
M)for4horwith
inhibitors of CYP450 (17-ODYA, 5
m
M), lipoxygenase (BACL, 5
m
M),
or cyclooxygenase (indomethacin, IND, 5
m
M) for 30 min and then
stimulated with NE (10
m
M) for 10 min. Cells were harvested, and cell
lysates were analyzed. (A) Effect of MAFP on MAPK activity
measured by the in-gel MAPK assay. Arrows indicate 44- and 42-kDa
bands identified as ERK1 and ERK2. (B) Effect of MAFP on cPLA
2
,
MAPK, and CaMKII activities as measured by in vitro kinase assay.
(C–F) NE-induced activation of MAPK and cPLA
2
but not CaMKII
is attenuated by inhibitors of CYP450 and lipoxygenase but not
cyclooxygenase. MAPK activity was determined by in vitro kinase
assay (C) and in-gel kinase assay method (D). As a control for protein
loading, an unidentified myelin basic protein kinase band is indicated
by an arrow. (G) Effect of NE on AA release and cPLA
2
activity
during blockade of MAPK activity and AA metabolism. Cells were
incubated with a combination of 17-ODYA, BACL, and PD-98059 and
then stimulated with NE (10
m
M) for 10 min. Basal values of cPLA
2
,
MAPK, and CaMKII activities and AA release are given in text. The
data shown represent the mean 6 SEM of three to six experiments on
three batches of cells. p, Value significantly different from basal;
†
,
value significantly different from that obtained with VEH of inhibi-
tors;
††
, value significantly different from that obtained with either of
the inhibitors alone.
FIG. 2. Production of 20-HETE and expression of CYP450 in
VSMCs. (A) Representative reverse-phase HPLC of products formed
by VSMCs incubated with [
14
C]AA. Solid line, absorbance units full
scale (AUFS); dashed line, radioactivity; arrow, position of authentic
standards. (B) Detection of CYP450 4A in microsomes and lysates of
VSMCs. Approximately 100–200
m
g of proteins from microsomes and
lysates were subjected to SDSyPAGE (10% gel) and detected by
Western blotting using a rat CYP450 4A polyclonal antibody raised in
goats. Lanes (from left to right) are clofibrate-treated liver microsomes
as a standard, VSMC lysates (two lanes), and microsomes isolated
from rabbit VSMCs.
12702 Pharmacology: Muthalif et al. Proc. Natl. Acad. Sci. USA 95 (1998)

for 16 h with rat CYP450 4A antibody (Gentest, Woburn, MA;
1:500 dilution) raised in goats. The immunoblots were developed
by using an enhanced chemiluminescence kit (Amersham).
Raf Phosphorylation. Raf-1 kinase threonine phosphorylation
was determined in cell lysates. The cell lysates were immunopre-
cipitated with anti-phosphothreonine antibody (Sigma). The im-
munoprecipitates were separated in a 7.5% SDSypolyacrylamide
gel and transferred to nitrocellulose membrane; Raf-1 kinase was
detected by the enhanced chemiluminescence method with anti-
Raf-1 kinase antibody (Upstate Biotechnology, Lake Placid,
NY).
Translocation of Ras. Cells were fixed in 4% paraformalde-
hyde in buffer (10 mM Pipesy5 mM EGTAy2 mM MgCl
2
y0.2%
Triton) for 3 min and postfixed in 95% ethanol for 5 min at
220°C. Cells were visualized with anti-Ras (Santa Cruz Biotech-
nology) and Texas red-conjugated horse anti-mouse IgG (Vector
Laboratories) by confocal microscopy (Bio-Rad, MRC-1000,
Laser Scanning Confocal Imaging system using an argonykrypton
lamp) as described (15).
Analysis of Data. The basal values ranged between 916 and
2,387 cpm of [
14
C]AA per 25
m
g of protein per 60 min for cPLA
2
,
11,300 and 26,345 cpm of
32
P per 15
m
g of protein per 10 min for
MAPK, 11,340 and 22,986 cpm of
32
P per 15
m
g of protein per 30
min for CaMKII, and 0.93 and 1.72% fractional AA release in
different batches of cells. The
3
H released into the medium is
expressed as percentage of the total cellular activity and referred
to as fractional AA release. Although the basal values of cPLA
2
,
MAPK, and CaMKII activity and AA release were variable in
different batches of cells, the effect of agonists and inhibitors on
the activity of these enzymes and AA release was consistent
within each batch of cells. Therefore, the changes produced by
agonists and inhibitors have been presented as percent above
basal. The results are expressed as mean 6 SEM. Data were
analyzed with one-way ANOVA. The Newman–Keuls multiple
range test was applied to determine the difference among mul-
tiple groups, and an unpaired Student’s t test was used to
determine the difference between two groups. Differences were
considered significant at P , 0.05.
RESULTS
CYP450 and Lipoxygenase Metabolites of Arachidonic Acid
Mediate NE-Induced MAPK Activity in Rabbit VSMCs. MAFP,
an inhibitor of cPLA
2
(25), attenuated the NE-induced increase
in cPLA
2
and MAPK activity but not in CaMKII activity (Fig. 1
A and B). This raises the possibility that the NE-induced increase
in MAPK activity is caused by AA or its metabolites generated
through activation of cPLA
2
by CaMKII. That an inhibitor of AA
metabolism, 5,8,11,14-eicosatetraynoic acid (35), decreased
MAPK but not CaMKII activity (data not shown) supports this
hypothesis.
If AA metabolites activate MAPK in VSMCs, then inhibition
of the AA-metabolizing enzymes CYP450, lipoxygenase, andyor
cyclooxygenase should attenuate the NE-induced increase in
MAPK and cPLA
2
activities. The inhibitors of CYP450 (17-
ODYA) and, to a lesser degree, lipoxygenase (BACL) attenuated
the NE-induced increase in MAPK and cPLA
2
activity but not
CaMKII activity (Fig. 1C–F). Indomethacin did not affect NE-
induced activation of MAPK or cPLA
2
activity. Exogenous AA
increased MAPK and cPLA
2
activity, and this effect was also
inhibited by 17-ODYA and by BACL. AA also increased
CaMKII activity, but this was not inhibited by 17-ODYA or
BACL (data not shown). The inhibitors of CYP450 (17-ODYA),
lipoxygenase (BACL), and MEK (PD-98059) (Fig. 1G), which
diminished the activities of extracellular regulated kinase (ERK)
1 and ERK2 (Fig. 1D and ref. 15), did not completely block the
NE- and AA-induced increases in cPLA
2
activity.
Metabolism of AA by CYP450 and Lipoxygenase in Rabbit
VSMCs. In VSMCs, AA is metabolized by cyclooxygenase to
prostaglandins and thromboxane A
2
and by lipoxygenase to 5-,
12(S)-, or 15-HETE (36). Since the NE-induced increase in
MAPK activity was not only attenuated by a lipoxygenase inhib-
itor but also by a CYP450 inhibitor, we investigated the produc-
tion of CYP450 metabolites of AA in rabbit VSMCs. We found
by reverse-phase HPLC that AA is metabolized by CYP450 to
hydroxy acid(s), one of which has been tentatively identified as
20-HETE (Fig. 2). The calculated amount of this product was
approximately 0.5
m
gymg of protein. Further studies are needed
to confirm the identity of this peak. Moreover, we demonstrated
that CYP4A protein, which may be involved in the formation of
20-HETE (37), is expressed in these cells (Fig. 2).
12(S), 15-, and 20-HETEs Stimulate MAPK and cPLA
2
Ac-
tivities in Rabbit VSMCs. AA has been shown to increase cPLA
2
activity in human neutrophils through formation of 5-HETE (23)
FIG. 3. Effects of 20-HETE and other HETEs on MAPK (A) and cPLA
2
(B) activities and AA release (C) in rabbit VSMCs. VSMCs were
preincubated with 20
m
M PD-98059 (PD) for 4 h and exposed to 20-, 15-, 12(S)-, and 12(R)-HETEs (each at 0.25
m
M) for 10 min. To measure
AA release, VSMCs were labeled with [
3
H]AA for 18 h and then exposed to HETEs for 15 min. The data shown represent the mean 6 SEM of
triplicate measurements of MAPK and cPLA
2
activities and AA release in three batches of cells. Basal values of cPLA
2
, MAPK activity, and AA
release are given in text. p, Value significantly different from basal;
†
, value significantly different from that obtained with VEH of PD-98059. (D)
Effect of 20-HETE on MAPK activity. Cells were incubated with 20-HETEs (0.25
m
M) for 10 min after pretreatment of cells with or without 5
m
M PD-98059 for4hor5
m
M17-ODYA and 5
m
M BACL for 30 min. This figure is a representation of two experiments.
Pharmacology: Muthalif et al. Proc. Natl. Acad. Sci. USA 95 (1998) 12703

and to increase MAPK activity in rat aortic VSMCs through
formation of 15-HETE (21, 22). In rabbit VSMCs, addition of
12(S)-, 15-, or 20-HETE, but not 12(R)- or 5-HETE (data not
shown), increased MAPK and cPLA
2
activities and AA release
(Fig. 3). 20-HETE also caused activation of MAPK, measured as
ERK1 and ERK2, in the presence of inhibitors of CYP450 and
lipoxygenase (Fig. 3D). 20-HETE increased MAPK activity at
concentrations as low as 0.1
m
M; the maximal effect was pro-
duced at 1
m
M. The effect of 12(S)-, 15-, and 20-HETE of
stimulating MAPK and cPLA
2
was attenuated by the MEK
inhibitor PD-98059 (Fig. 3A), suggesting that these effects could
be mediated by MEK or other signaling elements upstream of
MEK.
20-HETE Mediates NE-Induced Activation of RasyRaf Path-
ways in Rabbit VSMCs. Many agonists stimulate MEK activity by
promoting phosphorylation of proximal kinases, including c-Raf;
Raf is known to be activated by Ras and recruited to the plasma
membrane (38). In the presentstudy, NE and 20-HETE increased
Raf phosphorylation in VSMCs and increased the translocation
of Ras to the plasma membrane. 17-ODYA and BACL blocked
this effect of NE but not of 20-HETE (Fig. 4). Farnesylation of
Ras by farnesyltransferase is required for proper membrane
localization and activity of Ras (39, 40). FPT III, which inhibits
farnesyltransferase activity (31), also blocked the NE- and 20-
HETE-induced translocation of Ras to the plasma membrane
(Fig. 4B). Moreover, FPT III attenuated MAPK and cPLA
2
activities and the AA release elicited by NE (Fig. 4C). Thus,
20-HETE may activate Raf in VSMCs by promoting the trans-
location of Ras to the plasma membrane.
20-HETE Mediates Ang II- and EGF-Induced RasyMAPK
Activation. Ang II and EGF may also stimulate MAPK through
CYP450 and lipoxygenase products of AA released by initial
activation of cPLA
2
by CaMKII. Ang II- and EGF-induced
activation of MAPK was attenuated by inhibitors of CYP450 and
lipoxygenase (Fig. 5A). Moreover, Ang II and EGF caused
translocation of Ras to the plasma membrane, and this was
inhibited by a combination of 17-ODYA and BACL and by FPT
III (Fig. 5B). FPT III also attenuated MAPK activity and AA
release elicited by Ang II and EGF (Fig. 5C). The inhibitory
effect of 17-ODYA and BACL on the Ang II- and EGF-induced
increase in MAPK activity and Ras translocation was reversed by
20-HETE (data not shown).
DISCUSSION
NE, through
a
1
- and
a
2
-adrenergic receptors, stimulates influx of
extracellular Ca
21
(41) and activates CaMKII in rabbit VSMCs
(15). The CaMKII in turn stimulates MAPK and increases cPLA
2
activity, resulting in the release of AA for prostaglandin synthesis
(15). The present study demonstrates a mechanism by which a
CYP450 metabolite of AA, 20-HETE, mediates NE-, Ang II-,
and EGF-stimulated CaMKII-induced activation of the Rasy
MAPK pathway. MAPK then amplifies cPLA
2
activity and
releases further AA for prostaglandin synthesis.
In the present study, the cPLA
2
inhibitor MAFP and the
inhibitor of AA metabolism 5,8,11,14-eicosatetraynoic acid at-
tenuated NE-induced MAPK activity. These results suggest that
a metabolite of AA and not this fatty acid itself is involved in
CaMKII-induced MAPK activation. An important finding is that
inhibitors of CYP450 (17-ODYA) and lipoxygenase (BACL), but
not of cyclooxygenase (indomethacin), attenuated the NE- and
AA-induced increase in MAPK and cPLA
2
and not the CaMKII
activities in VSMCs. From these observations, it follows that
products of AA generated by CYP450 and lipoxygenase but not
by cyclooxygenase contribute to the activation of MAPK and
cPLA
2
elicited by NE. Although the combination of 17-ODYA,
BACL, and the MEK inhibitor PD-98059 abolished the NE-
induced increase in MAPK activity, measured as ERK1 and
ERK2, it only partially reduced cPLA
2
activity. We have reported
that CaMKII mediates activation of cPLA
2
and MAPK in re-
sponse to NE (15). Therefore, it appears that NE-stimulated
CaMKII activates cPLA
2
and releases AA and that products of
AA generated via CYP450 and lipoxygenase stimulate MAPK,
which then amplifies cPLA
2
activity and releases additional AA.
Although CaMKII activates cPLA
2
in VSMCs, it inhibits PLA
2
activity in rat brain synaptosomes (42). This could be due to
differences in the species of PLA
2
; e.g., forskolin enhances cPLA
2
FIG. 4. Involvement of RafyRas in NE-stimulated MAPK path-
way. (A) Effect of NE and 20-HETE on Raf-1 kinase threonine
phosphorylation in VSMCs. VSMCs were pretreated with 17-ODYA
and BACL or vehicle and then stimulated with NE for 5 min, lysed, and
immunoprecipitated with Sepharose-coupled anti-phosphothreonine
antibody. The immunoprecipitates were subjected to SDSyPAGE and
blotted with anti Raf-1 kinase monoclonal antibody and detected by
the enhanced chemiluminescence method. (B) Translocation of Ras in
response to NE (10
m
M) and 20 HETE (0.25
m
M) in the presence of
5
m
M 17-ODYA and 5
m
M BACL (30 min), farnesyltransferase
inhibitor (FPT III, 25
m
M, 24 h), or their vehicle. Cells were visualized
with anti-Ras and Texas red-conjugated horse anti-mouse IgG by
confocal microscopy. This figure is a representation of three experi-
ments. (C) Effect of farnesyltransferase inhibitor (FPT III) on NE-
stimulated MAPK and cPLA
2
activities and AA release. Basal values
of MAPK and cPLA
2
activities and AA release are given in text. The
data shown represent the mean 6 SEM of three to six experiments on
MAPK and cPLA
2
activities and AA release in three batches of cells.
p, Value significantly different from basal;
†
, value significantly
different from that obtained with VEH of inhibitors.
12704 Pharmacology: Muthalif et al. Proc. Natl. Acad. Sci. USA 95 (1998)

activity in intact synaptosomes but does not alter PLA
2
activity in
rabbit VSMCs (41).
In VSMCs, AA is metabolized by lipoxygenase into 5-, 12-, and
15-HETE (36). The present study indicates that rabbit VSMCs
also express CYP450 4A enzyme, which may be involved in the
formation of 20-HETE. 12(S)-, 15-, and 20-HETE increased
MAPK and cPLA
2
activities and AA release in rabbit VSMCs.
However, 20-HETE (a CYP450 product) was more potent than
12(S)- or 15-HETE (lipoxygenase products) in stimulating
MAPK and cPLA
2
activity. Moreover, the CYP450 inhibitor
(17-ODYA) produced a much greater reduction than the lipoxy-
genase inhibitor (BACL) in NE-induced MAPK and cPLA
2
activities. These results raise the possibility that a product(s) of
AA generated via CYP450, most likely 20-HETE, plays a major
role in regulating NE-induced activation of MAPK and, conse-
quently, amplification of cPLA
2
activity.
Our studies also indicate that MEK mediates NE- and 12(S)-,
15-, and 20-HETE-induced MAPK activation because the MEK
inhibitor PD-98059 attenuated these agents’ effect of increasing
MAPK activity. Since PD-98059 selectively inhibits MEK1 (26),
it appears that MAPK stimulation is mediated primarily by
MEK1. However, we cannot exclude the participation of other
MEK family members that stimulate MAPKs, including c-jun
N-terminal kinase and p38 MAPK.
It is well established that Raf activates MEK (43). Our dem-
onstration that the NE-increased phosphorylation of c-Raf was
inhibited by ODYA and BACL is consistent with a role for Raf
in activation of MEK by NE. Also supporting this view is the
finding that 20-HETE increased Raf phosphorylation in VSMCs.
NE stimulates Ras in human VSMCs (44), and Ras stimulates
Raf by promoting its association with the plasma membrane (45).
The finding that translocation of Ras by NE was blocked by
inhibitors of CYP450 and lipoxygenase suggests that metabolites
of AA activate Raf in VSMCs by promoting the translocation of
Ras to the plasma membrane. The findings that 20-HETE
translocated Ras to the plasma membrane and that the farnesyl-
transferase inhibitor FPT III blocked the Ras translocation
elicited by NE and by 20-HETE supports this contention.
The positive feedback mechanism of RasyMAPK activation by
CYP450 and lipoxygenase products of AA may be a general
mechanism in the actions of hormones like Ang II and growth
factors such as EGF. Ang II and EGF are known to stimulate AA
release, MAPK activity, and cell growth (12, 16, 46, 47). EGF
increased CaMKII activity and AA release in rabbit VSMCs; this
effect was inhibited by removal of calcium from the medium or
addition of the CaMKII inhibitor KN-93 (data not shown). In the
present study, both Ang II and EGF increased MAPK activity
and the translocation of Ras to the plasma membrane, and these
effects were inhibited by 17-ODYA and BACL and by the
farnesyltransferase inhibitor FPT III. It would appear that these
agents, like NE, generate AA metabolites through initial activa-
tion of cPLA
2
by CaMKII, which stimulates Ras and activates
MAPK, amplifies cPLA
2
activity, and releases additional AA for
prostanoid synthesis.
FIG. 5. Ang II and EGF stimulate RasyMAPK pathways through
AA metabolites of CYP450 and lipoxygenase. (A) Effects of 17-
ODYA and BACL on Ang II- and EGF-stimulated MAPK activity.
MAPK activity was measured by in-gel MAPK assay. (B) Transloca-
tion of Ras in response to Ang II and EGF in VSMCs. Arrested
VSMCs were exposed to 5
m
M 17-ODYA and 5
m
M BACL (30 min),
FPT III (24 h, 25
m
M), or their vehicle. Cells were visualized by
confocal microscopy. (C) Effects of FPT III on Ang II- and EGF-
stimulated MAPK activity. Basal values of MAPK are given in text. p,
Value significantly different from basal;
†
, value significantly different
from that obtained with VEH of FPT III. The data represent the
mean 6 SEM of three to six experiments on three batches of cells.
FIG. 6. Schematic model illustrating 20, 12(S)-, and 15-HETE as
mediators of MAPK and cPLA
2
activation in response to NE, Ang II,
and EGF. In this model, activation of receptors with NE, Ang II, or
EGF leads to an influx of Ca
21
ions that bind to CaM and activate
CaMKII. CaMKII activates cPLA
2
and releases AA. CYP450 and, to
a lesser, extent lipoxygenase metabolites of AA activate MAPK by the
RasyRafyMEK pathway by a positive feedback mechanism. Activation
of MAPK amplifies cPLA
2
activity and further releases AA. R,
receptor; RTK, receptor tyrosine kinase; G, G protein.
Pharmacology: Muthalif et al. Proc. Natl. Acad. Sci. USA 95 (1998) 12705

In rat VSMCs, Ang II-induced MAPK activation has been
reported to be Ras-independent (47, 48). However, another study
conducted in the same cell system proposed that Ang II-induced
MAPK activation was Ras-dependent and is mediated by an
unidentified Ca
21
yCaM-dependent tyrosine kinase through
transactivation of the EGF receptor (14, 49).
The mechanism by which 20-HETE activates Ras is unknown
and is currently being investigated. HETEs may activate Ras by
hydroxy arachidonylation of Ras, which could promote its binding
to the plasma membrane and subsequent activation. The modi-
fication of
a
subunits of G proteins by myristoylation, arachido-
nylation, andyor palmitoylation (50) and of Ras by farnesylation
(39, 40) is known to be required for anchorage to membrane or
interaction with other proteins. In human platelets,
a
subunits of
G proteins (
a
i
,
a
q
,
a
z,
and
a
13
) have been shown to covalently bind
arachidonate and palmitate but not myristate (50). The present
study proposes a signaling mechanism by which NE, Ang II, and
EGF activate the RasyMAPK pathway through generation of an
AA metabolite of CYP450, 20-HETE (Fig. 6). The activation of
MAPK by 20-HETE amplifies cPLA
2
activity and releases ad-
ditional AA by a positive feedback mechanism (Fig. 6). This
mechanism of RasyMAPK activation by 20-HETE might play a
central role in other signaling processes involved in inflammation
and in cell growth, proliferation, and differentiation.
We thank Drs. Alan H. Stephenson and Andrew J. Lonigro (St. Louis
University) for their generous help in HPLC separation of AA metab-
olites, Anne Estes for technical assistance, Dr. Lauren Cagen for scientific
discussions, and Ms. Jin Emerson-Cobb for editing the manuscript. This
work was supported by National Institutes of Health Grant 19134 from
the National Heart, Lung and Blood Institute (to K.U.M.), an American
Heart Association Tennessee Affiliate Award (I.F.B. and M.M.M.), and
a Center for Neuroscience Fellowship (to S.F.).
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