0022-3565/99/2901-0028$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 290:28 –37, 1999
Vol. 290, No. 1
Printed in U.S.A.
a1-Adrenergic Receptor Stimulation of Mitogenesis in Human
Vascular Smooth Muscle Cells: Role of Tyrosine Protein
Kinases and Calcium in Activation of Mitogen-Activated
Protein Kinase1
ZHUO-WEI HU, XIAO-YOU SHI, RICHARD Z. LIN, JIN CHEN, and BRIAN B. HOFFMAN
Department of Medicine, Stanford University School of Medicine, and Veterans Affairs Palo Alto Health Care System, Palo Alto, California
Accepted for publication March 18, 1999
This paper is available online at http://www.jpet.org
Protein tyrosine phosphorylation, stimulated by mitogens
such as peptide growth factors, plays an important role in
regulation of development, growth, differentiation, and other
biological functions in many cells including vascular smooth
muscle cells (VSMCs; Srivastava, 1995; Seger and Krebs,
1995). Signaling pathways involved in sequential activation
of Ras, Raf-1, and ultimately the protein kinase cascade
termed mitogen-activated protein kinase (MAPK) have been
Received for publication September 10, 1998.
1
This work was supported in part by National Institutes of Health Grant
HL41315 and a Grant-in-Aid from the American Heart Association, California
Affiliate. R.Z.L. and J.C. were supported by a Pharmaceutical Research and
Manufacturers of America Foundation Fellowship for Careers in Clinical Pharmacology during the course of this work.
noxy)ethane-N,N,N9,N9-tetraacetic acid tetra(acetoxymethyl)ester], completely blocked norepinephrine stimulation of phosphorylation of tyrosine proteins, suggesting that intracellular
Ca21 plays a critical role in a1 receptor stimulation phosphorylation of tyrosine proteins. Of the tyrosine-phosphorylated
proteins, the results suggest that two of them are PLCg1 and
adapter protein Shc. Also, a1 receptor stimulation caused a
time-dependent increase in MAPK activity due to increased
phosphorylation of p42/44ERK1/2. The a1 receptor-mediated
activation of MAPK was also attenuated by TPK inhibitors and
intracellular Ca21 chelator [1,2-bis(o-aminophenoxy)ethaneN,N,N9,N9-tetraacetic acid tetra(acetoxymethyl)ester]. These
results suggest that phosphorylation of tyrosine proteins and
intracellular Ca21 plays a critical role in a1 receptor-stimulated
MAPK signaling pathways, potentially contributing to increased
DNA synthesis and cell proliferation.
shown to be very important in mediating receptor tyrosine
kinase (RTK) regulation of cell growth and differentiation
(Schlessinger and Ullrich, 1992). Recent work suggests that
tyrosine kinase-Ras-MAPK signaling pathways may be also
utilized by G protein-coupled receptors (GPCRs), including
those for angiotensin II, endothelin, thrombin, and others
(for review, see Malarkey et al., 1995; Post and Brown, 1996).
Modulation of activity of multiple protein kinases is a key
control step in regulation of pathophysiological processes of
blood vessels such as growth and vascular remodeling.
a1-Adrenergic receptors (ARs) are members of the class of
GPCRs and mediate many of the important physiological
effects of catecholamines such as epinephrine. a1-ARs play a
particularly important role in control of cardiovascular re-
ABBREVIATIONS: 2-AP, 2-aminopurine; AR, adrenergic receptor; p125FAK, focal adhesion kinase; IGF-I, insulin-like growth factor I; IP3,
inositol-1,4,5-triphosphate; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; PDBu, 4b-phorbol 12,13-dibutyrate; PDGF-BB,
platelet-derived growth factor BB; PI-3 kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; PYK2, proline-rich tyrosine kinase 2; RTK,
receptor tyrosine kinase; TPK, tyrosine protein kinase; VSMC, vascular smooth muscle cell; HVSMC, human vascular smooth muscle cell; PAGE,
polyacrylamide gel electrophoresis; GPCR, G protein-coupled receptor; DMEM, Dulbecco’s modified Eagle’s medium; BAPTA-AM, [1,2-bis(oaminophenoxy)ethane-N,N,N9,N9-tetraacetic acid tetra(acetoxymethyl)ester].
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ABSTRACT
Signaling pathways of many G protein-coupled receptors overlap with those of receptor tyrosine kinases. We have found
previously that a1-adrenergic receptors stimulate DNA synthesis and cell proliferation in human vascular smooth muscle
cells; these effects were attenuated by the tyrosine protein
kinase (TPK) inhibitor genistein and the mitogen-activated protein kinase (MAPK) antagonist 2-aminopurine. Experiments
were designed to determine if activation of a1 receptors directly
stimulated TPKs and MAPKs in human vascular smooth muscle
cells. Norepinephrine stimulated time- and concentration-dependent tyrosine phosphorylation of multiple proteins, including p52-, 75-, 85-, 120-, and 145-kDa proteins. Increased TPK
activity was demonstrated in proteins precipitated by an antiphosphotyrosine antibody, both in autophosphorylation assays and with a peptide substrate. These effects of norepinephrine were completely blocked by a1 receptor antagonists. A
membrane-permeable Ca21 chelator [1,2-bis(o-aminophe-
Protein Kinases and a1-AR Receptor Signaling
1999
Experimental Procedures
Materials. Control antiserum (anti-rabbit serum and anti-IgG),
genistein, H7, myelin basic protein (MBP), norepinephrine, and 4bphorbol 12,13-dibutyrate (PDBu) were purchased from Sigma (St.
Louis, MO); [g-32P]ATP (2000 Ci/mmol), [32P]orthophosphate (370
MBq/ml), and an enhanced chemiluminescence Western Detection
System were obtained from Amersham Corp. (Arlington, IL). Immobilon-P transfer membranes were purchased from Millipore Corp.
(Bedford, MA); cell culture medium, fetal bovine serum, and human
recumbent PDGF-AB and -BB were obtained from Gibco-BRL
(Grand Island, NY). Wortmannin was purchased from Worthington
Biochemical Co. (Freehold, NJ); antibodies against TPKs, ERK1/2,
PLCg1, Shc, TPK substrates, and PKC/PKA inhibitors were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphorylated MAPK and anti-ERK antibodies were obtained from New England BioLabs, Inc. (Beverly, MA). All other chemicals were reagent
or molecular biology grade and were obtained from standard commercial sources.
Preparation of Cultured Human Aortic Smooth Muscle
Cells. Human aortic VSMCs were purchased from Clonetics Corp.
(San Diego, CA). Cells were grown in smooth muscle growth medium-2 with 5% fetal bovine serum obtained from Clonetics Corp. or
maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% (v/v)
heat-inactivated fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2/95% air. The cells were harvested for passaging at
confluence with trypsin-EDTA and plated in 100-mm dishes at a
density about 5 3 105, with a 80 to 90% confluence being reached
about 10 days later. The medium was replaced every 2 days. Cells
were generally used for studies at 8 to 10 days after seeding. To
examine effects of norepinephrine-stimulated changes, cells were
incubated with DMEM without serum for the indicated time after
achieving confluence. Throughout the course of these experiments,
cells from the fifth through seventh passage were used. The cells
were treated with agonists or vehicle solution (as control) starting
from the longest time point and the cells were harvested at the same
time.
a1 Agonist-Induced DNA Synthesis and Cell Proliferation.
Stimulation of a1 receptors induces DNA synthesis (Nakaki et al.,
1990) and cell proliferation (Kuriyama et al., 1988). HVSMCs were
cultured in DMEM containing 5% fetal bovine serum to near confluence. The medium was replaced with medium containing 0.4% serum
for 48 h. Norepinephrine (1 mM) plus the b-AR antagonist timolol (1
mM) and a2-AR antagonist idazoxan (1 mM) were added to the medium and, 20 h later, [3H]thymidine (0.1 mCi/dish) was added. The
incorporation of [3H]thymidine was determined 4 h later. To examine
if a1-receptor agonist-induced increases in [3H]thymidine incorporation occurred via activation of TPKs and MAPKs or through pertussis toxin-sensitive G proteins, cells were preincubated with inhibitors of TPKs and MAPKs for 2 h or with pertussis toxin for 12 h
before each experiment.
To measure cell proliferation (Tsai et al., 1995) HVSMCs were
grown to 70% confluence and the medium was replaced with medium
containing 0.4% serum for 48 h. The medium was then changed to
DMEM containing 0.4% fetal calf serum and various agonists as
indicated. Cells were incubated for an additional 72 h in the presence
of a b-AR antagonist timolol (1 mM) and an a2-AR antagonist idazoxan (1 mM). Inhibitors were added into cell dishes 1 h before
addition of agonists. At the end of incubation, medium was removed
and cells were treated with 0.25 ml of 0.05% trypsin-0.53 mM EDTA
(Gibco, Grand Island, NY) for 5 min and diluted to 10 ml with a
balanced electrolyte solution. Cell number was determined.
Immunoprecipitation and Immunodetection. Cultures on
100-mm plates were rinsed with ice-cold PBS containing 1 mM
sodium orthovanadate. Cells were incubated with cell lysis buffer
[1% nonide P-40, 25 mm HEPES (pH 7.5), 50 mm NaCl, 50 mM NaF,
5 mM EDTA, 10 nM okadaic acid, 1 mM sodium orthovanadate, 1
mM phenylmethylsulfonyl fluoride, and 10 mg/ml of aprotinin, leupeptin] for 10 to 15 min on ice. Insoluble material was removed by
centrifugation at 12,100g for 20 min. The amount of cell lysate was
normalized by protein content in each experiment. Lysates were
incubated with various antibodies as described for 2 h and then with
20 ml of protein A/G plus agarose for 1 h. The beads containing the
immunoprecipitates were washed 3 times with cell lysis buffer, once
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sponses such as regulation of blood pressure via activation of
smooth muscle contraction (Graham et al., 1996). Activation
of a1-AR also stimulates cardiac and vascular smooth muscle
growth and hypertrophy (Jackson and Schwartz, 1992; Milano et al., 1994). Human vascular smooth muscle cells
(HVSMCs) and cardiac myocytes express at least three subtypes of a1-ARs, namely, a1A, a1B, and a1D receptors (Price et
al., 1994; Hieble et al., 1995). It has been generally accepted
that activation of all three subtypes of a1 receptors increases
hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-triphosphate (IP3) and diacylglycerol via Gq, a family of pertussis toxin-insensitive G proteins leading to activation of protein kinase C (PKC) and raising intracellular
Ca21. Recent evidence demonstrates that a1 receptors also
stimulate production of IP3 and diacylglycerol via activation
of phospholipase Cb via bg subunits released from G proteins
(Muller and Lohse, 1995). Furthermore, a1 receptors activate
phospholipase D in brain and promote the release of arachidonic acid by activation of phospholipase A2 via pertussis
toxin-sensitive G proteins (Perez et al., 1993).
Traditionally, a1-ARs have been thought of as mainly utilizing PKC and Ca21 to mediate their effects in cells. Indeed,
substantial evidence indicates that activation of PKC is involved in a1-AR induction of mitogenic effects in cardiac
myocytes and VSMCs (Kariya et al., 1994). On the other
hand, although Ca21 plays an important role in the regulation of smooth muscle contraction, there is little evidence
demonstrating a mitogenic role of intracellular Ca21. Additionally, it has become clear in the past several years that
activation of PKC or/and raising Ca21 are not sufficient to
initiate cell cycle progression (Malarkey et al., 1995; Nishizuka, 1995). Consequently, there likely exist additional signaling mechanisms that contribute to a1-AR stimulation of
growth-related nuclear events. Recent evidence suggests that
a1-AR-stimulated mitogenic responses in neonatal myocytes
involve activation of tyrosine protein kinases (TPKs) and
activation of the MAPK pathway because inhibitors of tyrosine kinase and MAPK block a1-AR agonist stimulation of
myocyte hypertrophy (Thorburn et al., 1994). However, it is
not clear how activation of TPKs leads to MAPK activation
and mitogenic responses in these cells. Also, to what extent
tyrosine kinases and Ras-MAPK signaling pathways are utilized to mediate a1-AR effects in VSMCs is largely unknown.
In the present study, we have found a1-AR activated mitogenic responses such as DNA synthesis and increased cell
proliferation; these effects were attenuated by inhibitors of
TPKs and MAPK, suggesting that activation of a1-AR in
HVSMCs activates protein kinase cascades, leading to promotion of HVSMC growth. Further experiments demonstrated that a1-AR agonists directly stimulate phosphorylation and increase in activities of TPKs and MAPKs in these
cells. Interestingly, we found that intracellular calcium and
calcium-linked TPKs play a critical role in mediating catecholamine stimulation of MAPK signaling pathways.
29
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Hu et al.
(2 mg/mg protein) and washed as
with antibody against p42
above. The washed immunocomplexes were resuspended in 50 ml of
kinase buffer (25 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 0.5 mM EGTA, 40 nM ATP, 1 mCi of [g-32P]ATP, and MBP
1 mg/ml as a substrate). The reaction mixture was incubated for 10
min at 30°C. Preliminary experiments suggested that the phosphorylation of MBP is linear for 20 to 30 min. The reaction was stopped
by spotting 10 ml of reaction mixture onto p-81 phosphocellulose
paper (Whatman), which was then washed in 75 mM phosphoric acid
for 1 h and transferred to another washing overnight. The paper was
then washed with acetone for 5 min and dried. 32P was quantitated
by scintillation counting. Alternatively, reaction mixtures were
loaded and separated on 14% SDS-PAGE, and the dried gels were
exposed to Kodak XAR-5 film at 270°C with an intensifying screen
for 16 to 24 h or were visualized after development with a PhosphorImager System.
[Ca21]i Measurement. HVSMCs were plated on coverslips to
form a monolayer and loaded with 1.5 mM Fura-2 pentaacetoxymethyl in HBSS containing 0.1% BSA at room temperature for 30
min. Cytosolic free Ca21([Ca21]i) was determined as described previously (Chen and Giri, 1997). Cell Ca21 responses are expressed as
the ratio (F340/F380) of fluorescence intensity at excitation of 340
and 380 nm. For testing chelation of BATPA, the cells were pretreated with 10 mM BAPTA/AM or vehicle DMSO for 30 min at 37°C
before fluorescence measurement.
Data Analysis. Data are presented as mean 6 S.E.M., and treatment effects were compared by one-way ANOVA or Student’s paired
t test (two-tailed). P , .05 was used as level of significance.
Results
Inhibitors of TPKs Block Norepinephrine-Induced
DNA Synthesis and Cell Proliferation in Human
Smooth Muscle Cells. Activation of a1-AR in rat or human
myocytes and VSMCs stimulates DNA synthesis, protein
synthesis, and growth-related gene expression (Nakaki et al.,
1990; Okazaki et al., 1994; Chen et al., 1996). We wondered
if norepinephrine-stimulated DNA synthesis and cell proliferation could be blocked by inhibitors of TPKs and MAPK.
Stimulation of HVSMCs with norepinephrine (1 mM) resulted a 90% increase in DNA synthesis (Fig. 1A). This effect
of norepinephrine was blocked by a1-AR antagonist terazosin
(1 mM) but not by b-AR antagonist timolol (1 mM) or by a2-AR
antagonist idazoxan (1 mM), suggesting that norepinephrine
stimulated DNA synthesis via activation of a1-AR (Fig. 1A).
The TPK inhibitor genistein, the MAPK inhibitor 2-aminopurine (2-AP), and the PKC inhibitor H7, completely or partially blocked norepinephrine-stimulated DNA synthesis
(Fig. 1A). To investigate potential actions of these inhibitors
in norepinephrine stimulation of cell proliferation, partial
confluent HVSMCs (70%) were treated with norepinephrine
for 3 days in the presence of a b-AR antagonist timolol (1 mM)
and an a2-AR antagonist idazoxan (1 mM). Norepinephrine
treatment resulted a 50% increase in cell number; TPK, PKC,
and MAPK inhibitors, and a1-AR antagonist terazosin
blocked norepinephrine stimulation of cell proliferation
(Fig. 1B).
a1 Receptors Stimulate Phosphorylation of Tyrosine
Proteins. To determine whether incubation of HVSMCs
with norepinephrine stimulated phosphorylation of tyrosine
proteins, cells were metabolically labeled with 32Pi for 12 h
and stimulated with norepinephrine (10 mM) for the indicated times in the presence of timolol and idazoxan as illustrated in Fig. 2. Norepinephrine rapidly stimulated a time-
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with washing buffer (0.1 M NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH
7.5), once with kinase buffer, and subjected to MAPK assays. For
immunodetection, immunoprecipitates were washed three times
with lysis buffer, twice with distilled water, and analyzed by SDSpolyacrylamide gel electrophoresis (PAGE). Resolved proteins were
transferred to membrane and detected using the enhanced chemiluminescence western blotting detection system (Amersham) with the
indicated primary antibody and an appropriate horseradish peroxidase conjugated secondary antibody.
Analysis of Phosphotyrosine Phosphorylation. Analysis of
the phosphorylation of phosphotyrosine proteins followed a method
described by Siegel (1994). Confluent cultures of cells were serumstarved for 12 h and then labeled with 0.1 mCi/(1 Ci 5 37.5 GBq)/ml
of [32P]Pi in phosphate-free DMEM for 12 h. Cells were then stimulated with or without norepinephrine, phenylephrine, or PDGF for
the indicated times. In experiments using a1 antagonists or inhibitors of PKC and tyrosine kinases, the respective compounds were
added to cells 60 min before stimulation with agonists and growth
factors. After indicated times (1–30 min), cells were washed with
ice-cold PBS buffer and lysed on ice in 0.5 ml of cell lysis buffer.
Equal protein aliquots (1 mg) were precipitated with 2 mg/mg
antiphosphotyrosine antibodies and washed as described above. Immunoprecipitates were solubilized in SDS-PAGE sample buffer and
subsequently heated to 95°C for 5 min. Supernatants were resolved
by 8% SDS-PAGE. Gels were dried and followed by autoradiography
using a PhosphorImager or exposed to Kodak XAR-5 film at 270°C
with an intensifying screen for the indicated times.
Autophosphorylation of TPKs. Quiescent HVSMCs were stimulated with the indicated agents for the indicated times, and whole
cell lysates were prepared as described above. Equal protein aliquots
(1 mg) were immunoprecipitated for 2 h at 4°C using antibodies
against phosphotyrosine proteins (2 mg/mg protein) in the absence or
presence of phosphotyrosine (0.2 mM). Immune complexes were recovered as described above and subjected to an additional final rinse
with tyrosine kinase buffer (50 mM HEPES, pH 7.4, 10 mM MnCl2,
1 mM ATP). Immune complexes were resuspended in 25 to 50 ml of
kinase buffer containing 50 mCi of [g-32P]ATP. Protein kinase reactions were carried out for 15 min at 37°C. Reactions were stopped by
addition of SDS-PAGE sample buffer and subsequently heated to
95°C for 5 min. Labeled phosphoproteins were resolved by 10%
SDS-PAGE and visualized by autoradiography as described previously (Burkhardt, 1994).
In Vitro Assays of TPK Activity. For assay of TPK activity,
whole cell lysates (400 mg) were immunoprecipitated with antiphosphotyrosine antibody (2 mg/mg protein) as described above. The
washed immunocomplexes were resuspended in 50 ml of kinase reaction buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 0.5 mM EGTA, 1
mM dithiothreitol, 40 nM ATP, 1 mCi of [g-32P]ATP, and 10 mg of
TPK peptide substrate specific for Src-family kinases (cdc2:8 –20;
Santa Cruz Biotechnology) (Burkhardt, 1994). The reaction mixture
was incubated for 10 min at 30°C because preliminary experiments
suggested that the TPK activity is linear for at least 30 min. The
reaction was stopped by spotting 10 ml of reaction mixture onto p-81
phosphocellulose paper (Whatman), which was then washed in 75
mM phosphoric acid for 1 h and transferred to another washing
overnight. The papers were washed with acetone for 5 min and dried.
32
P was quantitated by scintillation counting.
In Vitro Assay of MAPK Phosphorylation and Activity. To
determine phosphorylation of MAPK, cells were incubated in the
absence of serum for 18 h and cells were treated with norepinephrine
or other agonists for various times. The cells were lysed in 0.4 ml of
lysis buffer. After a 30-min centrifugation (500g) at 4°C, cell lysates
(100 mg of protein) were loaded on 12% SDS-PAGE and transferred
to membranes as described above for Western blotting. The membrane was probed with an antiphosphorylated MAPK antibody or
with an anti-p44ERK1 as control.
Assay of MAPK activity was conducted as described previously
(Hu et al., 1996a). Cell lysates (250 mg of protein) were incubated
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ERK2
1999
dependent phosphorylation of several tyrosine proteins,
including p145, p125, p85, p75, and p52 (Fig. 2A). Increased
phosphorylation of tyrosine proteins occurred at 2 min and
was sustained for about 60 min. Norepinephrine induced a
similar tyrosine phosphorylation of several protein molecules
as platelet-derived growth factor BB (PDGF-BB) and insulinlike growth factor I (IGF-I). Figure 2B illustrates the dose-
31
response for norepinephrine-induced phosphorylation of tyrosine proteins. Concentrations of norepinephrine as low as
100 nM stimulated tyrosine phosphorylation of several protein molecules. Figure 2C illustrates the results of inhibitory
effects of a a1 receptor antagonist, inhibitors of TPKs, PKC,
and an L-type Ca21 channel blocker. Terazosin, an antagonist of a1-ARs, inhibited norepinephrine-induced phosphorylation of tyrosine proteins in these cells. Genistein, a TPK
inhibitor, partially inhibited norepinephrine-stimulated
phosphorylation of some of the TPKs. However, H7, a
PKC inhibitor and nifedipine, an L-type Ca21 channel
blocker, had little if any effect on norepinephrine-stimulated
phosphorylation of tyrosine proteins. This conclusion was
further supported by experiments demonstrating that
down-regulating PKC by preincubation cells with 4b-phorbol
12-myristate 13-acetate (10 mM) for 24 h did not inhibit
norepinephrine-stimulated tyrosine phosphorylation (data
not shown). Interestingly, a membrane-permeable Ca21 chelator [1,2-bis(o-aminophenoxy)ethane-N,N,N9,N9-tetraacetic
acid tetra(acetoxymethyl)ester] (BAPTA-AM) (10 mM) completely inhibited norepinephrine-induced tyrosine phosphorylation of multiple protein molecules (Fig. 2C), suggesting
that the rise in intracellular [Ca21] stimulated by norepinephrine is critical for a1-AR-mediated tyrosine protein phosphorylation in HVSMCs. Specificity of the antiphosphotyrosine protein antibody was confirmed by experiments in
which tyrosine-phosphorylated proteins were not precipitated by control antiserum such as anti-rabbit serum or
anti-IgG in the 32P-labeled HVSMCs (Fig. 2D).
We measured a1 agonist phenylephrine stimulation of intracellular free Ca21 ([Ca21]i) mobilization in HVSMCs pretreated with DMSO (vehicle; Fig. 3A) or 10 mM BAPTA-AM
(Fig. 3B). Results indicated that BAPTA-AM pretreatment of
HVSMCs completely lost Ca21 responses but did not interfere with the Ca21/Fura-2 signal and cell viability.
a1 Receptors Increase Tyrosine Kinase Phosphorylation and Activity. The capability of a1 receptor activation
to induce phosphorylation of TPKs in HVSMCs was investigated by measurements of phosphorylation in vitro of proteins immunoprecipitated by an antiphosphotyrosine antibody in norepinephrine-treated HVSMCs (Fig. 4). In these
experiments, HVSMCs were treated with or without norepinephrine (10 mM) for 10 min, cell lysates were immunoprecipitated with antiphosphotyrosine antibody, and in vitro
kinase activity was determined as described in Experimental
Procedures. The results of these experiments demonstrated
that norepinephrine stimulated phosphorylation of some of
these proteins (autophosphorylation; Fig. 4). Precipitation of
cell lysates with antiphosphotyrosine antibody in the presence of 2 mM tyrosine phosphate significantly inhibited autophosphorylation of these phosphotyrosine proteins except
for a protein of p85 kDa (Fig. 4A, lanes 3 and 5), which may
relate to serine/threonine phosphorylation of a1 receptorstimulated phospholipase A2 (Xing and Insel, 1996) or p85a
subunit of phosphatidylinositol 3-kinase (PI-3-kinase; Hu et
al., 1996).
To determine if stimulation of a1 receptors led to changes
in activity of specific TPKs, a defined substrate of TPKs was
used to measure tyrosine kinase activity. Figure 5 illustrates
the results of assays of TPK activity in cell lysates immunoprecipitated by antiphosphotyrosine antibody using a synthetic TPK peptide specific for Src family kinases (cdc2,
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Fig. 1. Effect of inhibitors on DNA synthesis and cell proliferation. A,
HVSMCs were grown to near confluence in 24-well dishes. The quiescent
cells were incubated with DMEM containing 0.4% fetal calf serum plus
norepinephrine (1 mM) for 20 h in the presence of timolol (1 mM) and
idazoxan (1 mM). Cells were then incubated with [3H]thymidine (0.1 mCi)
for another 4 h. Inhibitors were added to cell dishes 1 h before addition of
norepinephrine. The inhibitor H7 (1 mM), 2-AP (1 mM), or genistein (0.1
mM) was added as indicated. Incorporation of [3H]thymidine into cells
was performed as described in Experimental Procedures. The data are
average 6 S.E.M of three experiments performed in triplicate. *, comparison to control, p , .05. B, HVSMCs were grown to 70% confluence and
incubated with 0.4% fetal calf serum for 48 h. Then medium was changed
to DMEM containing 0.4% fetal calf serum plus norepinephrine (1 mM) or
PDBu (50 nM), and cells were incubated for 72 h in the presence of timolol
(1 mM) and idazoxan (1 mM). Genistein (0.1 mM), 2-AP (1 mM), and H7 (1
mM) were added as indicated. These antagonists were added to cell dishes
1 h before the addition of agonists. At the end of incubation, cells were
counted as described in Experimental Procedures. The data are average 6
S.E.M of three experiments performed in triplicate. *, comparison to
control, p , .05.
Protein Kinases and a1-AR Receptor Signaling
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Hu et al.
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Fig. 2. Norepinephrine stimulated timeand concentration-dependent phosphorylation of protein tyrosine kinases. A, [32P]Pilabeled cells were treated with vehicle or
norepinephrine (10 mM), PDGF-BB (1 nM),
or IGF-I (10 nM) for the indicated times.
Cell lysates (1 mg of protein) were subjected
to immunoprecipitation with a monoclonal
antibody against phosphotyrosine proteins.
Phosphorylation of tyrosine proteins in immunoprecipitates from control or agonisttreated cells was determined as described in
Experimental Procedures. The autoradiogram of film was exposed for 20 h. Experiments were repeated three times with essentially identical results. B, cells were
treated with vehicle or indicated concentrations of norepinephrine for 10 min and cell
lysates were prepared and immunoprecipitated as described above. The autoradiogram of film was exposed for 24 h. Experiments were repeated three times with
essentially identical results. C, cells were
metabolically labeled with [32P]Pi and
treated with norepinephrine (10 mM) as described above. Various inhibitors including
terazosin (1 mM), genistein (50 mM), H7 (10
mM), nifedipine (10 mM), and BAPTA-AM
(10 mM) were added to cell dishes 1 h before
addition of agonists. Data is a representative of three experiments. D, cell lysate (1
mg of protein) was subjected to immunoprecipitation with an anti-rabbit serum or an
anti-IgG antibody respectively. Phosphorylated tyrosine proteins in immunoprecipitates from control or agonist-treated cells
were determined. The autoradiogram of
film was exposed for 22 h. Experiments
were repeated three times with essentially
identical results.
1999
amino acids 8 –20) as kinase substrate. There was a timedependent rapid increase followed by a rapid decline in phosphorylation of this substrate in immunoprecipitates of cells
stimulated by norepinephrine in the presence of a2 and b
antagonists in HVSMCs. Taken together, these results suggest that activation of a1 receptors stimulates increases in
phosphorylation and activity of TPKs in HVSMCs.
We investigated whether some a1 receptor-stimulated tyrosine-phosphorylated proteins were recognized by antibodies directed against proteins known to be activated by other
GPCRs. Norepinephrine-stimulated cell lysates were first
immunoprecipitated with antiphosphotyrosine antibody and
separated on gels for Western blotting. The blots were probed
by several specific anti-TPK antibodies including anti-insulin
substrate-1, anti-EGF, antiphospholipase Cg1, anti-GAP,
and anti-Shc. We found that protein of 145 kDa was recognized by antiphospholipase Cg1 (Fig. 6A) and proteins of 52
kDa and 46 kDa were recognized by anti-Shc (Fig. 6B) in the
antiphosphotyrosine antibody-precipitated complex, respectively. These experiments suggest that stimulation of a1 receptors leads to phosphorylation of phospholipase Cg1 and
adapter protein Shc.
a1 Receptor Stimulation of MAPK Partially Involves
a Pertussis Toxin-Sensitive G Protein. The MAPK cascade plays an important role in mediating TPK signaling
pathways stimulated by many growth factors, as well as
ligands for GPCRs (Malarkey et al., 1995). We had found that
norepinephrine-stimulated tyrosine phosphorylation included two protein molecules at the position of 44/42 kDa
(Fig. 2). Because this doublet is compatible in size with
ERK1/2, we further determined the profiles of MAPK activation in norepinephrine-treated HVSMCs. Figure 7 illustrates
these results. Norepinephrine (1 mM) stimulated a rapid
increase in phosphorylation of p44/42ERK1/2 (Fig. 7A) without
a change in the amount of proteins (Fig. 7B). Also, norepinephrine stimulated an increase in MAPK activity in the
presence of timolol and idazoxan (Fig. 7C). Norepinephrinestimulated increase in MAPK activity was time-dependent
33
and the increased activity was maintained above basal for 30
min (Fig. 7, C and D). The norepinephrine-stimulated increase in activity of MAPK could be almost completely
blocked by a1 receptor antagonist terazosin (1 mM; Fig. 8).
Interestingly, pertussis toxin (100 ng/ml) partially inhibited
activation of MAPK, suggesting that norepinephrine-stimulated activation of MAPK may involve a pertussis toxinsensitive G protein. Additionally, norepinephrine-stimulated
activation of MAPK could be partially blocked by the PTK
inhibitor genistein or by Ca21 chelator BAPTA-AM (10 mM;
Fig. 8), suggesting that TPK and the intracellular Ca21 are
critical for a1-AR-mediated MAP activation in HVSMCs.
Norepinephrine-stimulated MAPK activity was also partially
inhibited by PKC inhibitor H7 (data not shown). IGF-I receptors are RTKs known to activate MAPK (Hansson and
Thoren, 1995) and PI-3 kinase (Karenberg et al., 1994). IGFI-increased MAPK activity in these cells was not inhibited by
pertussis toxin (Fig. 8). These results suggest that pertussis
toxin was not having nonspecific effects on hormonal activation of MAPK.
Discussion
Regulation of tyrosine protein phosphorylation by various
mitogens plays a central role in the control of cell growth and
Fig. 4. Autophosphorylation of norepinephrine-stimulated TPKs.
HVSMCs were incubated with serum-free DMEM for 24 h and then
treated with vehicle, 10 mM norepinephrine, or 1 nM PDGF-BB for 10
min. Cell lysates (1 mg of protein) were prepared, immunoprecipitated,
and subjected to autophosphorylation of TPKs as described in Experimental Procedures. The autoradiogram of film was exposed for 10 h. Experiments were repeated three times with similar results.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 3. Intracellular Ca21 chelator BAPTA-AM attenuated a1-adrenergic
agonist-stimulated Ca21 mobilization. Fura2-loaded cells were incubated
with DMSO control (A) or intracellular Ca21 chelator BAPTA-AM (10
mM; B) for 30 min at 37°C. A normal Ca21 response to 10 mM phenylephrine (PE) is shown in the DMSO-pretreated cells with Ca21 influx after
addition of Ca21 (2 mM). In cells pretreated with BAPTA-AM, PE did not
significantly increase in [Ca21 ]i until ionomycin (Ion) was added, suggesting that pretreatment with BAPTA attenuated PE-stimulated Ca21
mobilization but did not interfere with the Ca21/Fura2 signal and cell
viability. Data is representative of three experiments.
Protein Kinases and a1-AR Receptor Signaling
34
Hu et al.
Vol. 290
differentiation. In the present study, our results demonstrate
that inhibitors of TPKs and MAPK attenuate catecholaminestimulated DNA synthesis and cell proliferation in HVSMCs.
Norepinephrine was found to stimulate time- and concentration-dependent tyrosine phosphorylation of multiple proteins. Tyrosine-phosphorylated proteins had TPK activity in
autophosphorylation assays, suggesting that some of these
proteins may function as TPKs. This observation was further
supported by experiments that directly demonstrated increased TPK activity in norepinephrine-stimulated
HVSMCs. These effects of norepinephrine were completely
blocked by a TPK inhibitor genistein. A PKC inhibitor and an
L-type calcium channel blocker did not attenuate norepinephrine-stimulated phosphorylation of tyrosine proteins.
Interestingly, an intracellular calcium chelator, BAPTA-AM,
completely attenuated norepinephrine-stimulated phosphorylation of tyrosine proteins, suggesting that intracellular
Ca21 plays a critical role in mediating a1 receptor stimulation of phosphorylation of tyrosine proteins. a1 receptor stimulation caused a time-dependent increase in MAPK activity
due to increased phosphorylation of p42/44ERK1/2. Our study
demonstrates that a1 receptor-mediated activation of MAPK
was also attenuated by TPK inhibitors and the intracellular
Ca21 chelator BAPTA-AM.
There is substantial evidence indicating that stimulation
of a1 receptors enhances growth-related gene expression and
cell growth in cardiac myocytes and VSMCs. In cardiac myocytes, stimulation of a1 receptors produces long-term changes
in the cardiac structure and function, including an increase
in cell size and an increase in the expression of the cardiac
structural genes such as b-myosin heavy chain gene (Simpson et al., 1991; Morgan and Baker, 1991). In vascular
smooth muscle, it has long been known that adrenergic ago-
Fig. 6. Norepinephrine stimulated tyrosine-phosphorylated proteins included PLCg1 and adapter protein Shc. Quiescent HVSMCs were treated
with or without norepinephrine (10 mM) for indicated times in the presence of timolol (1 mM) and idazoxan (1 mM). Terazosin (1 mM; T) or
genistein (50 mM; G) was added into dishes 1 h before norepinephrine
treatment. Cell lysates were subjected to immunoprecipitation with an
antiphosphotyrosine or antiphospholipase Cg1 antibodies and Western
blotting with anti-phospholipase Cg1 antibody (A). These results show
increased tyrosine phosphorylation of phospholipase Cg1, inhibited by T
and G with similar total amounts of phospholipase Cg1 in each group of
cells. B, illustrates similar experiments involving immunoprecipitation
with an antiphosphotyrosine antibody and Western blotting with antiShc antibody. The data are a representative of three experiments.
nists lead to the growth of arterial smooth muscle cells in
vitro. Catecholamines stimulate the proliferation of rat and
bovine smooth muscle cells (Jackson and Schwartz, 1992) in
primary culture via activation of a1 receptors. In intact animals, activation of a1 receptors may contribute to atherosclerosis by enhancing proliferation of VSMCs (Hauss et al.,
1990). Indeed, the a1 receptor antagonists prazosin and doxazocin inhibits smooth muscle hyperplasia induced in experimental models of endothelial injury (Fingerle et al., 1991;
Vashisht et al., 1992). Activation of a1 receptors on cardiac
myocytes and VSMCs leads to stimulation of DNA and protein synthesis, either directly or indirectly via stimulation
activation of growth factor expression such as platelet-derived growth factor (Majesky et al., 1990). Moreover, we
recently found that a1 agonists markedly increase expression
of early and delayed proto-oncogenes in vitro in intact aorta
(Okazaki et al., 1994). a1 agonist stimulation of mitogenesis
in HVSMCs is associated with activation of PI-3 kinase (Hu
et al., 1996b). In the present study, we found that mitogenic
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 5. Activities of TPKs in norepinephrine-stimulated HVSMCs. Confluent HVSMCs were incubated with serum-free DMEM for 24 h and cells
were treated with or without norepinephrine (10 mM) for indicated times
in the presence of timolol (1 mM) and idazoxan (1 mM). Cell lysates (400
mg) were subjected to immunoprecipitation with antiphosphotyrosine.
TPK activities in the immunocomplexes were determined using a synthetic TPK peptide (10 mg) specific for Src family kinases as kinase
substrate as described in Experimental Procedures. Relative kinase activity was calculated using control activity at 0 min as 100%. The data are
average 6 S.E.M. of four experiments.
1999
Protein Kinases and a1-AR Receptor Signaling
35
Fig. 7. Norepinephrine stimulated phosphorylation and increase in activity of MAPK. A, norepinephrine stimulated phosphorylation of p42/
44ERK1/2. Quiescent HVSMCs were treated with or without norepinephrine (10 mM) for indicated times in the presence of timolol (1 mM) and
idazoxan (1 mM). Cells were lysed by direct addition of SDS-PAGE sample
buffer and subjected to SDS-PAGE and Western blotting. The blots were
probed with an antiphosphorylated MAPK described in Experimental
Procedures. The data is a representative of three experiments. B, same
blot was probed with an anti-p44ERK1, indicating that similar amounts of
these proteins were in each lane. C, norepinephrine stimulated a timedependent increase in activity of MAPK. Quiescent HVSMCs were
treated with or without norepinephrine (10 mM) for indicated times. Cell
lysates (250 mg) were prepared and subjected to immunoprecipitation
with an anti-p42ERK2 antibody. Washed immunocomplexes were resuspended in a kinase buffer and subjected to kinase activity assay using
MBP as substrate as described in Experimental Procedures. D, data are
average 6 S.E.M of three experiments.
responses of HVSMCs to a1 receptor stimulation could be
blocked by inhibition of TPKs. Moreover, stimulation of
VSMCs with a1 receptor agonists results in tyrosine phosphorylation of several proteins, suggesting that phosphorylation of tyrosine proteins plays an important role in a1
receptor signal transduction.
It is not clear how TPKs contribute to the overall signal
transduction of specific GPCRs; recent evidence suggests
that these kinases play an important role in GPCR signaling
in a number of different cells (Malarkey et al., 1995). In
contrast to peptide growth factor receptors, which possess
endogenous TPK activities, GPCRs have not been demonstrated to posses endogenous TPK activity. Agonist stimulation of these receptors leads to activation of several different
G proteins that dissociate into a and bg subunits. There is
evidence indicating that these subunits may activate TPKs,
leading to tyrosine phosphorylation of target proteins. The
phosphorylated proteins may then serve as links between the
receptors or G proteins and various adapter proteins such as
Grb2, SOS1, or Shc (Downward, 1994). These adapter proteins all possess SRC homology 2 or 3 (SH2 or SH3) domains
and some are TPKs that subsequently dock and activate
downstream effectors such as p21Ras leading to activation of
protein kinase cascades (Smithgall, 1995). In the present
study, we have demonstrated that activation of a1 receptors
stimulates tyrosine phosphorylation of several protein molecules and activates MAPK. The results suggest that two
tyrosine-phosphorylated proteins are phospholipase Cg1 and
adapter protein Shc.
It is unlikely that PKC plays a major role in mediating a1
receptor activation of TPKs in HVSMCs because neither
enzyme inhibition nor down-regulation of PKC inhibited
TPK activation. Similarly, an L-type calcium channel blocker
did not inhibit a1 receptor stimulation of tyrosine protein
phosphorylation. On the other hand, the intracellular Ca21
chelator BAPTA-AM completely blocked norepinephrine
stimulation of tyrosine protein phosphorylation in these
cells. Recent studies suggest that an increase in intracellular
Ca21 concentration may be an important early event in
GPCR-mediated TPK/Ras-MAPK signaling pathways. For
example, angiotensin II activation of several TPKs and downstream MAPK in cultured rat neonatal myocytes is Ca21dependent.
A major question is how intracellular Ca21 participates in
activation of TPKs by GPCR agonists such as angiotensin II
and norepinephrine. Stimulation of PLCg1 by many growth
factors increases intracellular Ca21 mobilization via release
of IP3 (Carpenter et al., 1992), indicating that activation of
PLCg1 may function as an early mediator of GPCR-stimulated TPK/MAPK signaling pathway (Rusanescu et al.,
1995). Activation of members of cytosolic TPKs such as the
SRC family may also serve as mediator of GPCR-stimulated
intracellular Ca21 signaling to activate MAPK (Dikic et al.,
1996). Additionally, focal adhesion kinase (p125FAK) has
been suggested to mediate GPCR-stimulated Ca21-dependent signaling. Tyrosine phosphorylation of p125FAK is de-
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Fig. 8. Effect of inhibitors on norepinephrine-stimulated activation of
MAPK. Cells were treated as described in the legend of Fig. 7. Inhibitors
(1 mM terazosin, 50 mM genistein, or 10 mM BAPTA-AM) were added to
dishes 1 h before norepinephrine (10 mM) or IGF-I (10 nM) treatment.
PTx (100 ng/ml) was added 12 h before agonist treatment. Cell lysates
(250 mg) were prepared and subjected to immunoprecipitation with an
anti-p42ERK2 antibody. Washed immunocomplexes were resuspended in a
kinase buffer and kinase activity was measured using MBP as substrate.
Data are average 6 S.E.M. of four experiments. *, comparison to control,
p , .05.
36
Hu et al.
pendent on intracellular Ca21 (Shattil et al., 1994). In this
context, proline-rich tyrosine kinase 2 (PYK2), a member of
the p125FAK family, has attracted particular attention. PYK2
has been found to be rapidly phosphorylated on tyrosine
residues in response to various stimuli including GPCR ligands that elevate intracellular calcium concentrations.
Phosphorylated PYK2 then in turn activates Ras-MAPK activity (Lev et al., 1995). In the present study, we have found
that activation of a1 receptors likely stimulates tyrosine
phosphorylation of PLCg1, which could lead to Ca21 mobilization that may in turn activate the Ras-MAPK cascade. The
detailed interactions between PLCg1, Ca21, and TPKs such
as P125FAK or PYK2 as mediators of norepinephrine-stimulated MAPK activation requires further detailed study.
It is now known that the MAPK cascade is not restricted to
RTK signaling pathways but is also utilized by phorbol esters, heat shock, and GPCR ligands to induce mitogenesis
(Malarkey et al., 1995; Post and Brown, 1996). Activation of
P21Ras and then Raf-1 are key steps not only for the understanding of growth factor signal transduction but also for
GPCR activation of MAPK pathways. Generally, growth factors activate p21Ras, which stimulates Raf-1, leading to activation of MAPK via phosphorylated RTKs. On the other
hand, GPCR agonists stimulate MAPK via two independent
pathways depending on G protein-receptor coupling. For receptors coupled to pertussis toxin-sensitive G proteins such
as thrombin and lysophosphatidic acid, activation of MAPK
is via activation of p21Ras (LaMorte et al., 1994). This pathway is PKC-independent. For receptors coupled to pertussis
toxin-insensitive G proteins such as the Gq family, stimulation of these receptors leads activation of PKC. Activated
PKC in turn directly activates Raf-1 and thereby activates
MAPK. Indeed, there is evidence that demonstrates that
PKC phosphorylates Raf-1 upon serine residues in vitro
(Kolch et al., 1993); a1B receptors activate MAPK via stimulation of pertussis toxin-insensitive G proteins, PKC, and
Raf-1 (Hawes et al., 1995). However, muscarinic M1 receptors have been found to activate MAPK via pertussis toxininsensitive G proteins using Ras-dependent pathways (Crespo et al., 1994), indicating that p21Ras may also be utilized
by pertussis toxin-insensitive GPCRs to activate MAPK. We
have recently demonstrated that a1 receptors activate p21Ras
protein in HVSMCs (Hu et al., 1996). In the present study,
we provide evidence to show that a1 receptor-activated
MAPKs are attenuated by pertussis toxin and a TPK inhibitor, suggesting that pertussis toxin-sensitive G protein and
TPKs are involved in a1 receptor-activated MAPK signaling
pathways in HVSMCs. These results indicate that TPK/RasMAPK signaling pathways are utilized by a1 receptors to
mediate mitogenic actions of catecholamines in HVSMCs.
In summary, we have characterized phosphorylation of
tyrosine proteins as an early event in the a1 receptor-induced
protein kinase signaling cascade, which may be responsible
for regulation of catecholamine stimulation of mitogenic effects in HVSMCs. Activation of tyrosine phosphorylation by
a1 receptors was independent in the activation of PKC but
dependent of the intracellular Ca21 in these cells, suggesting
that the intracellular Ca21 plays a critical role in a1 receptorsignaling pathways not only for smooth muscle contraction
but also for cell growth (Scheme 1). HVSMCs provide an
important model system for the further elucidation of a1
adrenergic signaling mechanisms, particularly relating to
cell growth and division, which is important for vascular
changes in diseases such as hypertension and atherosclerosis.
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