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Vol. 52, Issue 4, 639-672, December 2000
Medical Research Council of Canada Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Quebec, Canada
Abstract
I. Introduction
II. Physiological Actions of Angiotensin II in Vascular Smooth Muscle Cells
A. The Renin Angiotensin SystemProduction of Angiotensin II
B. Angiotensin Receptors
C. Vascular Actions of Angiotensin II
D. Angiotensin II-Dependent Signaling Pathways
E. Immediate Signaling Events Stimulated by Angiotensin II
1. Stimulation of Phospholipase C and Phosphatidylinositol Hydrolysis.
2. Increased Intracellular Free Calcium Concentration.
3. Activation of Protein Kinase C.
4. Stimulation of Na+/H+ Exchange.
5. Angiotensin II Increases Intracellular Free Concentrations of Na+ and Decreases Intracellular Free Concentrations of Mg2+.
6. Activation of Src Family Kinases.
F. Early Signaling Events Mediated by Angiotensin II
1. Activation of Tyrosine Kinases.
a. Janus family kinases.
b. Focal adhesion kinase and proline-rich tyrosine kinase 2.
c. p130Cas.
d. Phosphatidylinositol 3-kinase.
2. Mitogen-Activated Protein Kinase Pathways.
a. Upstream events.
b. Downstream events.
c. Angiotensin II and the mitogen-activated protein kinase pathway in cardiovascular cells.
3. Activation of Phospholipase A2 and Arachidonic Acid Metabolism.
4. Phospholipase D activation.
5. Angiotensin II Effects on Cyclic Nucleotides.
G. Long-Term Effects Mediated by Angiotensin II
1. Generation of Reactive Oxygen Species.
2. Angiotensin II-Induced Expression of Proto-Oncogenes and Growth Factors.
H. Why the Special Role for Angiotensin II Signaling in Vascular Smooth Muscle Cells?
II. Altered Angiotensin II Signaling in Vascular Smooth Muscle Cells in Cardiovascular DiseasesSpecial Reference to Hypertension
A. Introduction
B. Vascular Changes
C. Vascular Angiotensin Receptors
D. Short-Term Signaling Events
1. Angiotensin II Stimulation of the Phospholipase C-IP3-Diacylglycerol Pathway Is Augmented.
2. Angiotensin II-Stimulated Effects on Vascular [Mg2+]i and [Na+]i.
3. Vascular Eicosanoids, Angiotensin II, and Hypertension.
4. Angiotensin II Increases Activity of Phospholipase D.
5. Cyclic Nucleotides and Angiotensin II.
E. Long-Term Signaling Events
1. Angiotensin II-Induced Generation of Reactive Oxygen Species.
2. Angiotensin II, Tyrosine Kinases, and Hypertension.
3. Angiotensin II-Mediated Mitogen-Activated Protein Kinase Signaling Is Increased.
4. Indirect Effects of Angiotensin II on the Vasculature.
F. Mechanisms Underlying Enhanced Angiotensin II Vascular Responsiveness
IV. Conclusions
Acknowledgments
References
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Abstract |
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Until recently, the signaling events elicited in vascular smooth muscle cells by angiotensin II (Ang II) were considered to be rapid, short-lived, and divided into separate linear pathways, where intracellular targets of the phospholipase C-diacylglycerol-Ca2+ axis were distinct from those of the tyrosine kinase- and mitogen-activated protein kinase- dependent pathways. However, these major intracellular signaling cascades do not function independently and are actively engaged in cross-talk. Downstream signals from the Ang II-bound receptors converge to elicit complex and multiple responses. The exact adapter proteins or "go-between" molecules that link the multiple intracellular pathways await clarification. Ang II induces a multitude of actions in various tissues, and the signaling events following occupancy and activation of angiotensin receptors are tightly controlled and extremely complex. Alterations of these highly regulated signaling pathways in vascular smooth cells may be pivotal in structural and functional abnormalities that underlie vascular pathological processes in cardiovascular diseases such as hypertension, atherosclerosis, and post-interventional restenosis.
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I. Introduction |
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The vascular wall is an active, pliable and integrated organ made
up of cellular (endothelial cells, vascular smooth muscle cells, and
fibroblasts) and noncellular (extracellular matrix) components. It is
not a static organ; the components dynamically change
shape, increase, decrease, or reorganize, in response to physiological
and pathological stimuli (Dubey, 1997
). In the intact arterial media,
smooth muscle cells and matrix are responsible for structural and
functional characteristics of the vessel wall, including
contraction-relaxation, growth, development, remodeling, and repair,
and for the pathogenesis of
cardiovascular disease, such as atherosclerosis, restenosis and
hypertension (Mulvany and Aalkjaer, 1990
; Schiffrin, 1992
; Katoh and
Periasamy, 1996
; Bornfeldt, 1996
). Many local and systemic factors
regulate vascular smooth muscle cell function, including vasoactive
peptides, such as Ang2 II and endothelin-1
(ET-1), that stimulate vasoconstriction and growth and vasorelaxing
factors, such as nitric oxide, prostacyclin, and C-type natriuretic
peptide that induce vasodilation by increasing levels of cyclic
nucleotides (Rubanyi, 1991
; Lüscher, 1993; Lüscher and
Barton, 1997
; Stein and Levin, 1998
).
Ang II is a multifunctional peptide that has numerous actions on
vascular smooth muscle
it modulates vasomotor tone, it regulates cell
growth and apoptosis, it influences cell migration and
extracellular matrix deposition, it is proinflammatory, and it
stimulates production of other growth factors [e.g., platelet-derived
growth factor (PDGF)] and vasoconstrictors (e.g., ET-1). Accordingly,
Ang II plays a fundamental role in controlling the functional and
structural integrity of the arterial wall and may be important in
physiological processes regulating blood pressure and in pathological
mechanisms underlying vascular diseases. The multiple actions of Ang II
are mediated via specific, highly complex intracellular signaling pathways that are stimulated following an initial binding of the peptide to its cell-surface receptors (Matsusaka and Ichikawa, 1997
).
The term "intracellular signaling pathway" includes the interconnected molecular cascades that transmit information from the
cell membrane receptor to the intracellular proteins that regulate cell
activities such as contraction, cell growth, mitogenesis, apoptosis,
differentiation, migration, and other specialized functions. Identification of such signal transduction processes is essential for
understanding mechanisms that regulate vascular smooth muscle cell
function, both physiologically and pathophysiologically. This review
focuses on Ang II-mediated signaling in vascular smooth muscle cells
and implications of altered Ang II-induced signal transduction in
vascular pathological processes, concentrating specifically on
hypertension. The molecular and cellular mechanisms of Ang II in
cardiac and renal diseases have recently been reviewed and will not be
discussed in detail here (Kim and Iwao, 2000
).
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II. Physiological Actions of Angiotensin II in Vascular Smooth Muscle Cells |
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A. The Renin Angiotensin System
Production of Angiotensin II
Ang II, an octapeptide hormone, is the active component of the
renin-angiotensin system (RAS). It regulates blood pressure, plasma
volume via aldosterone-regulated sodium excretion, sympathetic nervous
activity, and thirst responses. It also plays a fundamental role in
pathological adaptation, as manifested in myocardial remodeling after
myocardial infarction and in vascular remodeling in hypertension. Ang
II is produced systemically via the classical or renal RAS, and locally
via tissue RAS. In the classical RAS, circulating renal-derived renin
cleaves hepatic-derived angiotensinogen at the N terminus to form the
decapeptide, angiotensin I, which is converted by the dipeptidyl
carboxypeptidase, angiotensin-converting enzyme (ACE), in the lungs, to
the active Ang II (Skeggs et al., 1967
; Dorer et al., 1972
; Phillips et
al., 1993
; Inagami, 1998
) (Fig. 1). Ang I
can also be processed into the heptapeptide Ang-(1-7) by three tissue
endopeptidases, neutral endopeptidase (NEP) 24.11, NEP 24.15, and NEP
24.26 (Ferrario et al., 1997
). Ang II is degraded by aminopeptidases to
Ang III and Ang IV (Fig. 1).
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The RAS was originally regarded as a circulating system. However, many
of its components are localized in tissues indicating the existence of
a local tissue RAS as well (Dzau, 1989
; Danser, 1996
). ACE exists in
plasma (as the circulating hormone), in the interstitium and
intracellularly. Tissue ACE is present in all major organs, heart,
brain, blood vessels, adrenals, kidney, liver, and reproductive organs
(Hollenberg, 1998
), and is already functional in utero (Schutz et al.,
1996
; Esther et al., 1997
). Tissue ACE activity seems to peak during
the phase of major organ development and declines thereafter (Esther et
al., 1997
). All components of the RAS, except renin, have been
demonstrated to be produced in the vasculature. ACE is found in high
concentrations in the adventitia, as well as in cultured vascular
smooth muscle and endothelial cells (Dzau, 1989
; Ekker et al., 1989
;
Naftilan, 1994
). Angiotensinogen mRNA and protein have been detected in
vascular smooth muscle, endothelium, and perivascular fat (Naftilan et al., 1991
; Naftilan, 1994
; Morgan et al., 1996
). Since vascular renin
is absent, local generation of Ang II in the interstitium is regulated
by tissue ACE that is probably dependent on circulating renin (Fig.
2). Although the function of tissue ACE
is currently unclear, it may contribute to regulation of regional blood
flow as recently demonstrated in the human forearm where in situ
generated Ang II is more important for vasoconstriction than
circulating Ang II (Saris et al., 2000
).
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In addition to ACE-dependent pathways of Ang II formation, non-ACE
pathways, which could be particularly important in pathological states,
have been demonstrated. Chymotrypsin-like serine protease (chymase) may
represent an important pathway for conversion of Ang I to Ang II in the
human heart (Urata et al., 1990
, 1996
) and kidney (Hollenberg, 1998
).
Functional chymase and a non-ACE pathway have also been demonstrated in
human vascular tissue (Hollenberg et al., 1998
; Takai et al., 1998
) and
in dog carotid artery (Shiota et al., 1999
).
B. Angiotensin Receptors
In mammalian cells, Ang II mediates its effects via at least two
high-affinity plasma membrane receptors, AT1 and
AT2. Both receptor subtypes have been cloned and
pharmacologically characterized (Murphy et al., 1991
; Sasaki et al.,
1991
; Kambayashi et al., 1993
; Mukoyama et al., 1993
). Two other Ang
receptors have been described, AT3 and
AT4 subtypes. The AT3
receptor subtype, initially described in the neuro 2A neuroblastoma
cell line (Chaki and Inagami, 1992
) is peptide-specific recognizing
mainly Ang II. This subtype does not bind nonpeptide ligands such as
losartan (selective AT1 receptor antagonist) or
PD123319 (selective AT2 receptor antagonist), and
has only been observed in cell lines. The AT4
receptor, which is distributed in heart, lung, kidney, brain, and
liver, binds Ang IV (Swanson et al., 1992
) but not losartan or
PD123319. Since the pharmacology of AT3 and
AT4 receptors has not been fully characterized, these receptors are not yet included in a definitive classification of
mammalian AT receptors as defined by the International Union of
Pharmacology Nomenclature Subcommittee for Angiotensin Receptors (de
Gasparo, 1995
).
The AT1 receptor belongs to the seven
membrane-spanning G protein-coupled receptor family and typically
activates phospholipase C through the heterotrimeric Gq protein (de
Gasparo et al., 1995
; Inagami, 1995
) (Table
1). Human AT1
receptor gene is mapped to chromosome 3. To date,
AT1 receptors have been shown to mediate most of
the physiological actions of Ang II, and this subtype is predominant in
the control of Ang II-induced vascular functions (Sadoshima, 1998
). In
the vasculature, AT1 receptors are present at
high levels in smooth muscle cells and relatively low levels in the
adventitia and are undetectable in the endothelium (Zhuo et al., 1998
;
Allen et al., 2000
). Two AT1 receptor subtypes
have been described in rodents, AT1A and
AT1B, with greater than 95% amino acid sequence
identity (Iwai and Inagami, 1992
). AT1A and AT1B receptor genes in rats are mapped to
chromosome 17 and 2, respectively. Based on the cDNA sequence, the
AT1 receptor is composed of 359 amino acids
(Sandberg, 1994
). It is a glycoprotein and contains extracellular
glycosylation sites at the amino terminus (Asn4)
and the second extracellular loop (Asp176 and
Asn188) (Desarnaud et al., 1993
). The
transmembrane domain at the amino-terminal extension and segments in
the first and third extracellular loops are responsible for G protein
interactions with the receptor (Hjorth et al., 1994
). Internalization
of G protein-coupled receptors involves receptor phosphorylation, which
may be mediated, in part via caveola (Berk and Corson, 1997
; Ishizaka
et al., 1998
). Although G protein-coupled receptors do not contain
intrinsic kinase activity, they are phosphorylated on serine and
threonine residues by members of the G protein receptor kinase (GRK)
family. AT1 receptors are phosphorylated both in
the basal state and in response to Ang II stimulation (Kai et al.,
1994
). Threonine and serine residues between
Thr332 and Ser338 of the
cytoplasmic tail are essential for receptor internalization (Hunyady et
al., 1994
). The AT1 receptor is also
phosphorylated at tyrosine residues. Potential tyrosine phosphorylation
sites within the AT1 receptor include amino acids
302, 312, 319, and 339 within the carboxyl terminus (Berk and Corson,
1997
). Tyrosine at position 319 is important as it is part of the motif
Tyr-Ile-Pro-Pro, which is analogous to a Src homology 2 (SH2) binding
motif in the PDGF receptor (Tyr-Ile-Pro) and in the epidermal growth
factor (EGF) receptor (Tyr-Leu-Pro-Pro) (Fantl et al., 1993
). In EGF and PDGF receptors, these motifs are target sequences for tyrosine phosphorylation. Various tyrosine kinases, including Janus kinases (JAK
and TYK), Src family kinases, and focal adhesion kinase (FAK) can
tyrosine phosphorylate AT1 receptors.
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The second major isoform of the Ang receptor,
AT2, is normally expressed at high levels in
fetal tissues and decreases rapidly after birth (Nahmias and Strosberg,
1995
). The AT2 receptor gene is localized as a
single copy on the X chromosome. In adults, AT2
receptor expression is detectable in the pancreas, heart, kidney,
adrenals, brain, and vasculature (Viswanathan and Saavedra, 1994
; Touyz
et al., 1999a
). In the vasculature, AT2 receptors predominate in the adventitia and are detectable in the media (Zhuo et
al., 1998
). AT2 receptors are also expressed in
several cell lines, including PC12W, R3T3, and N1E115 (Inagami, 1995
). The AT2 receptor is a seven transmembrane-type, G
protein-coupled receptor, comprising 363 amino acids. It has low amino
acid sequence homology (~32%) with AT1A or
AT1B receptors (Mukoyama et al., 1993
). Although
the exact signaling pathways and the functional roles of
AT2 receptors are unclear, these receptors, which
appear to be regulated by intracellular cations, particularly
Na+ (Tamura et al., 1999
), may antagonize, under
physiological conditions, AT1-mediated effects
(Ciuffo et al., 1998
; Yamada et al., 1998
) by inhibiting cell growth,
and by inducing apoptosis and vasodilation (Hayashida et al., 1996
;
Horiuchi, 1997a
,b
; Gallinat et al., 2000
; Unger, 1999
; Siragy,
2000
). The exact role of AT2 receptors in cardiovascular disease remains to be defined.
C. Vascular Actions of Angiotensin II
Ang II promotes its effects by acting directly through Ang II
receptors present on vascular cells, indirectly through the release of
other factors, and possibly via cross-talk with intracellular signaling
pathways of other vasoactive agents and growth factors. Although the
principal function of smooth muscle cells is vasoconstriction, it has
become evident that vascular smooth muscle cells have important synthetic properties during development and vascular remodeling (Table
2) and are the major source of
extracellular matrix components of the vascular media (Katoh and
Periasamy, 1996
). During blood vessel development, immature smooth
muscle cells are in a dynamic state of growth and differentiation
characterized by proliferation and migration (Glukhova et al., 1991
).
In the adult vessel, they become quiescent and assume a fibroblast-like
appearance, and become filled with contractile fibers (Gordon et al.,
1990
). Although mature smooth muscle cells remain quiescent until
injury or insult occurs, they undergo physiological hypertrophy in
response to increased load (Bucher et al., 1982
; Katoh and Periasamy,
1996
). Ang II plays a role in these developmental processes, acting via AT1 and AT2 receptors,
which are differentially expressed in vascular smooth muscle cells
during normal development and during pathological processes. In
vascular disease, smooth muscle cells undergo hyperplasia and/or
hypertrophy as an adaptive or reactive response (Table 2). (Geisterfer
et al., 1988
; Berk et al., 1989
; Paquet et al., 1990
; Stouffer and
Owens, 1992
; Dubey, 1997
; Touyz and Schiffrin, 1997a
; Touyz et al.,
1999b
) and may be critical in vascular remodeling associated with
hypertension, atherosclerosis, or neointimal formation. Both Ang II
receptor subtypes appear to be necessary for a complete vascular smooth
muscle cell response to injury (Zahradka et al., 1998
).
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Integrated vascular responses to Ang II are the result of combined
AT1- and AT2-mediated
actions, as well as effects of bioactive end products of the RAS, such
as Ang-(1-7). Whereas Ang II induces vasoconstriction, growth,
migration, production of extracellular matrix components, and
inflammation via AT1 receptors, it promotes apoptosis, and inhibits proliferation and hypertrophy via
AT2 receptors (Allen et al., 2000
; Siragy, 2000
).
Ang-(1-7) has been described as a naturally occurring competitive
inhibitor of Ang II, as it has potent vasodepressor and
antihypertensive effects. It can stimulate release of vasopressin, act
as an excitatory neurotransmitter, augment synthesis and release of
vasodilator prostaglandins, potentiate the actions of bradykinin and
release nitric oxide (Ferrario et al., 1997
). In addition, ACE
inhibitors augment circulating levels of the vasodilator peptide, which
may contribute to the antihypertensive effects associated with ACE inhibitors (Chappel et al., 1998
; Iyer et al., 1998
). The receptor mediating the vascular actions of Ang-(1-7) has been tentatively characterized as a
non-AT1/AT2 subtype
(Ferrario et al., 1997
). Although the exact role of this peptide in the
physiological and pathophysiological regulation of vascular function
awaits clarification, its potential to antagonize
AT1-mediated actions suggests that Ang-(1-7)
could modulate vascular tone by promoting vasodilation.
D. Angiotensin II-Dependent Signaling Pathways
Ang II elicits complex highly regulated cascades of intracellular
signal transduction that lead to short-term vascular effects, such as
contraction, and to long-term biological effects, such as cell growth,
migration, extracellular matrix deposition, and inflammation.
Ligand-receptor binding on the external cell membrane surface induces
the interaction between the receptor and effector protein on the
internal cell membrane surface via G proteins (heterotrimeric proteins
comprised of
,
and
subunits). Intracellular signaling via
the AT2 receptor subtype will not be discussed in
detail here, as progress in Ang II type 2 receptor research in the
cardiovascular system has recently been reviewed (Csikos et al., 1998
;
Horiuchi et al., 1999
; Unger, 1999
). Unless otherwise indicated,
signaling events described in the present review are mediated via
AT1 receptors. AT1
receptors are coupled to multiple, distinct signal transduction processes, leading to diverse biological actions. The signaling processes are multiphasic with distinct temporal characteristics (Fig.
3). Immediate, early, and late signaling
events occur within seconds, minutes, and hours, respectively (Fig. 3).
Ang II-induced phospholipase C (PLC) phosphorylation and Src activation
occur within seconds and constitute immediate signaling events,
activation of phospholipase A2
(PLA2), phospholipase D (PLD), tyrosine kinases and mitogen-activated protein kinases (MAPKs) occurs within minutes and
are early signaling processes, whereas generation of oxidative stress,
proto-oncogene expression, and protein synthesis, which occur within
hours, make up late signaling events.
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E. Immediate Signaling Events Stimulated by Angiotensin II
Ang II-elicited vascular contraction is rapid and utilizes various signaling mechanisms that occur within seconds of Ang II binding to its receptor. These immediate signal transduction processes include: a) G protein-mediated activation of PLC, leading to phosphatidylinositol hydrolysis and formation of inositol trisphosphate (IP3) and diacylglycerol accumulation (DAG); b) increase in cytosolic free calcium concentration ([Ca2+]i) by increasing Ca2+ influx and mobilizing intracellular Ca2+; c) activation of protein kinase C (PKC); d) changes in intracellular pH (alkalinization) via stimulation of the Na+/H+ exchanger; e) changes in intracellular free concentrations of Na+ ([Na+]i) and Mg2+ ([Mg2+]i); and f) activation of the Src family of kinases (Fig. 4).
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1. Stimulation of Phospholipase C and Phosphatidylinositol
Hydrolysis.
One of the earliest detectable events resulting from
Ang II stimulation of vascular smooth muscle cells is a rapid,
PLC-dependent hydrolysis of phosphatidylinositol-4,5-bisphosphate
(PtdInsP2) to yield water soluble
IP3 and membrane bound DAG (Alexander, 1985
;
Griendling et al., 1985
; Berk et al., 1987a
; Griendling et al.,
1989
). PLC is a family of at least three related genes: PLC-
,
PLC-
, and PLC-
(Rhee and Choi, 1992
). PLC-
isoforms are
regulated by
and 
subunits of G proteins (Smrcka et al., 1991
), whereas PLC-
isoforms are regulated by tyrosine
phosphorylation (Rhee, 1991
; Homma et al., 1993
; Marrero et al.,
1995b
). PLC-
regulation is unclear, but may involve intracellular
Ca2+. PLC-
1, PLC-
1, and PLC-
1 have been
identified in vascular smooth muscle cells (Marrero et al., 1994
;
Ushio-Fukai et al., 1998b
). The AT1 receptor
sequentially couples to PLC-
1 via a heterotrimeric G protein and to
PLC-
1 via a tyrosine kinase (Ushio-Fukai et al., 1998b
;
Venema et al., 1998
). The initial AT1
receptor-PLC-
1 coupling is mediated by
G
q/11
and
G
12
. The 
dimer acts as a signal
transducer for activation of PLC (Touhara et al., 1995
; Ushio-Fukai et
al., 1998b
). Both PLC-
1 and PLC-
isoforms play a role in
IP3 formation. PLC-
1 appears to be important
in the rapid generation of IP3 (within 15 s), whereas PLC-
seems to play a role in the later phase of
IP3 formation (Ushio-Fukai et al., 1998b
). Ang
II-stimulated IP3 generation may also be
mediated, in part, via tyrosine kinase-dependent pathways (Goutsouliak
and Rabkin, 1997
). Ang II induces a dose-dependent increase in
phosphatidylinositol turnover resulting in rapid transient
IP3 formation (Griendling et al., 1989
) and
biphasic and sustained DAG generation (Alexander et al., 1985
;
Griendling et al., 1985
). Losartan, the selective AT1 receptor blocker, inhibits Ang II-induced
hydrolysis of PtdInsP2, indicating that Ang II
stimulation of the PLC pathway is mediated exclusively via
AT1 receptors. IP3
stimulates release of Ca2+ from
sarcoplasmic/endoplasmic reticular stores and DAG, with cofactors
phosphatidylserine and Ca2+, activates PKC. Ang
II-elicited IP3 signal slightly precedes a rapid
increase in cytoplasmic free calcium concentration
([Ca2+]i), which is in
large part independent of calcium influx. These events correlate
temporally with initiation of contraction in isolated vascular smooth
muscle cells, as well as in intact small resistance arteries, and most
likely constitute the early signaling pathway for initiation of the
calcium-dependent, calmodulin-activated phosphorylation of the myosin
light chain, which leads to cellular contraction (Lassegue et al.,
1993
; Walsh et al., 1995
; Savineau and Marthan, 1997
; Touyz and
Schiffrin, 1997a
; Touyz et al., 1999c
). DAG can also be formed by the
PLD-mediated hydrolysis of other phospholipids such as
phosphatidylcholine and phosphatidylethanolamine.
2. Increased Intracellular Free Calcium
Concentration.
Ang II-stimulated Ca2+
signaling is complex and occurs via multiple pathways to elicit an
integrated Ca2+ signal. Ang II typically mediates
a biphasic [Ca2+]i
response comprising a rapid initial transient phase and a sustained plateau phase (Dostal, 1990
; Touyz et al., 1994
; Assender et al., 1997
;
Touyz and Schiffrin, 1997b
). Both AT1A and
AT1B receptors have been shown to mediate calcium
signaling in rodent vascular smooth muscle cells (Zhu et al.,
1998b
). The first
[Ca2+]i transient is
generated primarily by IP3-induced mobilization of intracellular Ca2+ and to a lesser extent by
Ca2+-induced Ca2+ release
(Touyz and Schiffrin, 1997b
). The second
[Ca2+]i phase, which
appears to contribute to the sustained Ang II-induced vasoconstriction,
is dependent on external Ca2+ and is the result
of transmembrane Ca2+ influx (Rembold, 1992
; Ruan
and Arendshorst, 1996a
; Inscho et al., 1997
; Iverson and Arendshorst,
1998
; Touyz et al., 1999c
). Exact mechanisms whereby Ang II stimulates
Ca2+ influx are unclear but may involve
voltage-dependent calcium channels, which are directly or indirectly
activated by Ang II, Ca2+-permeable, nonspecific
dihydropyridine-insensitive cation channels, receptor-gated
Ca2+ channels,
Ca2+-activated Ca2+ release
channels, and activation of the
Na+/Ca2+ exchanger
(Arnaudeau et al., 1996
; Lu et al., 1996
). In addition to
IP3-mediated mobilization of intracellular
Ca2+ and influx of extracellular
Ca2+, tyrosine kinase-dependent increases in
[Ca2+]i have been
demonstrated in vascular smooth muscle cells (Hughes and Bolton, 1995
;
Touyz and Schiffrin, 1996a
; Di Salvo et al., 1998
).
3. Activation of Protein Kinase C.
Ang II-induced DAG
production, together with Ca2+ and
phosphatidylserine, activate PKC, a serine/threonine kinase that is a member of a multigene family consisting of at least 11 isoenzymes (Hug
and Sarre, 1993
; Newton, 1997
). Ang II stimulates the translocation of
cytosolic PKC to the plasma membrane where the activated enzyme phosphorylates specific proteins associated with vascular function (Walsh et al., 1996
; Damron et al., 1998
). PKC is implicated in Ang
II-induced vascular contraction as well as in vascular smooth muscle
cell growth (Rasmussen et al., 1987
; Ruan and Arendshorst, 1996b
; Orjii
and Keiser, 1997
; Kiron and Loutzenhiser, 1998
; Bauer, 1999). These
effects are mediated via activation of the
Na+/H+ exchanger leading to
intracellular alkalinization, an important modulator of actin-myosin
interaction, and of contraction (Aalkjaer and Peng, 1997
). In addition,
Ang II-stimulated PKC induces its actions through phosphorylation of
tyrosine kinases, such as proline-rich tyrosine kinase (PYK2) (Sabri et
al., 1998
), p130Cas (Sayeski et al.,
1998
), and Src family tyrosine kinases (Zou et al., 1998
), and by
stimulating MAP kinase signaling pathways (Zou et al., 1996
; Wilkie et
al., 1997
; Kudoh, 1997
; Li et al., 1998a
). The PKC isoform that
activates ERK-1 and ERK-2 (extracellular signal-regulated kinases) in
vascular smooth muscle cells has been identified as PKC-
(Liao et
al., 1997
). Some studies failed to demonstrate that Ang II effects are
PKC-dependent and others reported only a partial dependence on PKC
(Berk et al., 1987b
, 1989
; Assender et al., 1997
). Thus both
PKC-dependent and -independent mechanisms are involved in Ang
II-stimulated vascular contraction and growth. In addition to its
second messenger function, PKC has been implicated in the
rapid-agonist-induced desensitization of AT1
receptors (Balmforth et al., 1997
).
4. Stimulation of Na+/H+
Exchange.
Ang II elicits a biphasic change in intracellular pH
(pHi), comprising an initial acidification
followed by a sustained alkalinization (Griendling et al., 1989
; Touyz
and Schiffrin, 1997a
; Touyz et al., 1999d
). The rapid acidification is
associated with Ca2+-ATPase-regulated
Ca2+ mobilization (Berk et al., 1987b
). Ang
II-stimulated alkalinization is entirely dependent on activation of the
Na+/H+ exchanger (Berk et
al., 1987b
; Touyz and Schiffrin, 1997a
; Touyz et al., 1999d
), which is
modulated by PKC-dependent and PKC-independent mechanisms (Berk et al.,
1987b
). MAPKs also play a role in Ang II-stimulated activation of the
Na+/H+ exchanger.
ERK-1/ERK-2 and p38 activate the
Na+/H+ exchanger in
vascular smooth muscle cells (Kusuhara, 1998
; Touyz et al., 1999d
) and
p90rsk has been identified as a putative potent
Na+/H+ kinase (Takahashi et
al., 1997a
). Activation of the
Na+/H+ exchanger and
akalinization induce vasoconstriction in various vascular beds by
increasing [Na+]i and
[Ca2+]i and by
sensitizing the contractile machinery to Ca2+
(Grinstein et al., 1989
; Carr et al., 1995
; Ye, 1996
; Tepel et al.,
1998b
; Touyz et al., 1999
). In addition, increased intracellular pHi is a potent stimulus for DNA synthesis
(Sachinidis et al., 1996
). Thus alkalinization is an important
mechanism whereby Ang II modulates vascular smooth muscle function by
stimulating both contraction and growth.
5. Angiotensin II Increases Intracellular Free Concentrations of
Na+ and Decreases Intracellular Free Concentrations of
Mg2+.
In addition to increasing
[Ca2+]i and
pHi, Ang II raises
[Na+]i and reduces
[Mg2+]i in a
concentration-dependent fashion in vascular smooth muscle cells
(Johnson et al., 1991
; Ye et al., 1996
; Touyz and Schiffrin, 1999
).
These effects are rapid and maximal responses occur within 40 to
60 s (Touyz and Schiffrin, 1998).
[Na+]i is regulated by
the Na+/H+ exchanger, the
Na+/Ca2+ exchanger, the
Na+/K+ ATPase pump, and
Na+ channels (Shigekawa et al., 1996
; Juhaszova
and Blaustein, 1997
; Cox et al., 1998
). The cellular mechanisms
regulating [Mg2+]i are
unknown, but we and others have shown that a putative
Na+/Mg2+ exchanger
regulates [Mg2+]i by
inducing Mg2+ efflux and by stimulating
Na+ influx (Touyz and Schiffrin, 1996b
; Touyz and
Schiffrin, 1999a
; Murphy, 2000
). Ang II-stimulated increase in
[Na+]i and reduction in
[Mg2+]i influence
vascular smooth muscle contraction directly or indirectly by modulating
[Ca2+]i.
6. Activation of Src Family Kinases.
The Src family of
protein tyrosine kinases that characteristically interact with
transmembrane tyrosine kinase receptors, also interact functionally
with G protein-coupled receptors, such as AT1
(Paxton et al., 1994
; Marrero et al., 1995b
; Parsons and Parsons, 1997
;
Thomas and Brugge, 1997
; Ishida et al., 1998
). To date, at least 14 Src-related kinases have been identified, of which the 60-kDa c-Src is
the best characterized (Thomas and Brugge, 1997
). The Src family
kinases are subdivided into three groups based on their pattern of
expression. Src, Fyn, and Yes are expressed ubiquitously, Blk, Fgr,
Hck, Lck, and Lyn are found primarily in hematopoietic cells and
Frk-related kinases (Frk/Rak and Iyk/Bsk) are expressed predominantly
in epithelial-derived cells (Thomas and Brugge, 1997
). Src family
kinases share a high degree of structural similarity, with common
domain architecture and regulatory mechanisms. They consist of one or
more amino-terminal acylation sites (required for membrane
localization), a unique domain (which defines the individual members),
an SH3 domain, an SH2 domain, a catalytic domain, and a
carboxyl-terminal noncatalytic domain. Regulation of Src activity is
complex. Phosphorylation of Tyr527 by Csk
inhibits Src activity, whereas dephosphorylation of this residue
activates Src. Activation may also occur by autophosphorylation of
Tyr419 in the catalytic domain, by displacement
of the intramolecular interactions of the SH2 or SH3 domains by
high-affinity ligands or modification of certain residues (Erpel and
Courtneidge, 1995
). Src family kinases are activated in response to
various stimuli in many cell types and have been suggested to play an
important role in signal transduction pathways that control growth and
cellular architecture.
and IP3
formation. We reported that Ang II-stimulated
[Ca2+]i responses in
human vascular smooth muscle cells are mediated, in part, via
Src-dependent mechanisms (Touyz et al., 1999e
, caveolin, and the adapter protein, Shc (Li et al., 1996bF. Early Signaling Events Mediated by Angiotensin II
In addition to rapid signaling events associated with contraction, the AT1 receptor couples to multiple intracellular transduction pathways that are linked to long-term regulation of vascular smooth muscle cell function, such as growth, migration, deposition of extracellular matrix, and production of growth factors. These processes are initiated by signaling pathways that are stimulated by Ang II within minutes and include: a) phosphorylation of tyrosine kinases; b) activation of MAPKs; c) activation of PLA2 and arachidonic acid metabolism; d) activation of PLD; and e) modulation of cyclic nucleotides (Fig. 5).
|
1. Activation of Tyrosine Kinases.
Ang II stimulates
phosphorylation of a tyrosine residue of many vascular smooth muscle
cell proteins. These include the AT1 receptor
itself, PLC-
1 and Src family kinases (activated within seconds), as
well as JAK and TYK, FAK, Pyk2, p130Cas (a
Crk-associated substrate), and phosphatidylinositol 3-kinase (PI3K),
all of which are activated within minutes (Fig.
6). The role of tyrosine kinases in Ang
II-mediated signal transduction pathways in cardiovascular cells was
extensively reviewed in 1997 (Marrero et al., 1995a
; Berk et al., 1997
;
Berk and Corson, 1997
; Dostal et al., 1997
; Griendling et al., 1997
).
Only recent developments relating to Ang II signaling and tyrosine
kinases will be discussed in detail here.
|
(Sasaki et al., 1995
-actin
expression, cellular proliferation, migration, and cell adhesion
(Nojima et al., 1995
, Rac1,
and JNK and the protein Ser/Thr kinase Akt/protein kinase B (PKB)
(Wymann and Pirola, 19982. Mitogen-Activated Protein Kinase Pathways.
MAP kinases
constitute a superfamily of serine/threonine protein kinases involved
in the regulation of a number of intracellular pathways. Mammalian
MAPKs are grouped into six major subfamilies: a) ERK-1/ERK-2; b)
JNK/stress-activated protein kinases (SAPK); c) p38; d) ERK-6, p38-like
MAPK; e) ERK-3; and f) ERK-5 (also called Big MAP kinase 1) (Robinson
and Cobb, 1997
) (Fig. 7). MAP kinase-dependent signaling pathways have been associated with cellular
growth and apoptosis, with cellular differentiation and transformation
and with vascular contraction (Mii et al., 1996
; Force and Bonventre,
1998
; Touyz et al., 1999b
,c
). The ERKs are activated in response to
growth and differentiation factors, whereas JNKs and p38 are usually
activated in response to inflammatory cytokines and cellular stress
(Robinson and Cobb, 1997
; Force and Bonventre, 1998
; Morinville et al.,
1998
; New and Han, 1998
; Ip and Davis, 1998
). Ang II activates the
three major members of the MAP kinase family, ERKs, JNKs, and p38
(Leduc and Meloche, 1995
; Kudoh et al., 1997
; Touyz et al., 1999d
). MAP
kinase pathways comprise a three-component protein kinase cascade
consisting of a serine/threonine protein kinase (MAPKKK), which
phosphorylates and activates a dual-specificity protein kinase (MAPKK),
which in turn phosphorylates and activates another protein kinase
(MAPK) (Cobb and Goldsmith, 1995
; Robinson and Cobb, 1997
). In the
Ras/Raf/MEK/ERK pathway, Raf corresponds to MAPKKK, MEK corresponds to
MAPKK, and ERK corresponds to MAPK (Fig. 7).
|

subunits, by a
receptor-associated tyrosine kinase or by tyrosine phosphorylation of a
linker protein, such as Shc (Crespo et al., 1994
|
|