Bernard Lassègue
(Cardiology Div., Emory University,
USA)
Masuko Ushio-Fukai (Cardiology Div., Emory University, USA)
Kathy K. Griendling (Cardiology Div., Emory University, USA)
Contact Person: Bernard Lassègue (medbpl@emory.edu)
Many effects of angiotensin II (AngII)
in vascular disease are ascribed to its potent stimulation of
vascular smooth muscle cells (vsmc). Within seconds of AngII
binding to the AT1 receptor, phospholipase Cß1
is activated by heterotrimeric G protein subunits 
q/11, 12 and ß
. PLC is subsequently
activated by a tyrosine kinase. Transient activation of PLCs
is followed by sustained phospholipase D activation via G protein
subunits 12,
ß,
calcium, protein kinase C and src. Concomitantly, stimulation
of phospholipase A2 initiates arachidonic acid signalling.
Internalization of the AT1 receptor/agonist complex into
membrane domains such as caveolae where effectors are localized
is required for sustained AngII signalling. AngII
also activates members of kinase cascades such as JAK/TYK/STAT,
Src, Ras, and MAPK. Recently, reactive oxygen species have emerged
as crucial components of AngII-mediated signal transduction
in vsmc. Both superoxide and hydrogen peroxide are produced intracellularly
via activation of an NADH oxidase, and these molecules serve to
mediate downstream signalling events such as activation
of p38MAPK and gene transcription. Thus, AngII activates a remarkable
spectrum of signalling pathways, and the integration of these
diverse signals directs and modulates the physiological and
pathophysiological effects of this hormone on vsmc.
Presentation Number: SAlassegue0362
Keywords: angiotensin II, vascular smooth muscle, signalling, phospholipases, G proteins, superoxide.
The AT1 subtype of angiotensin II (AngII) receptors mediates most of the effects of this agonist in the healthy adult cardiovascular system [1, 2], thus we will review here new advances in AT1-mediated signalling.
Protein sequence homologies indicate that AT1
belongs to the vast family of receptors with seven transmembrane
domains, a notion consistent with experiments indicating that
AngII signals via heterotrimeric G-proteins [3]. Phospholipase
C (PLC) is activated by AngII within 5 s in vascular smooth muscle
cells (vsmc), thus generating diacylglycerol (DG) and inositol
trisphosphate (IP3), an activator of intracellular calcium
mobilization [1, 2]. G-proteins usually activate the ß isoform
of PLC [4]. Thus the discovery that AngII activates
PLC via
tyrosine phoshorylation, just like a typical growth factor, appeared
paradoxical [5]. AngII increased PLC1 phosphorylation 4.5 fold at 30 s in rat vsmc.
Electroporation of c-Src antibodies or preincubation with the
tyrosine kinase inhibitors genistein or tyrphostin A inhibited
AngII-induced PLC
phosphorylation, IP3 production and calcium mobilization [5-8].
AngII induced phosphorylation of tyrosine 319 in the cytoplasmic
domain of AT1, thus permitting binding of PLC via its C-terminal SH2
(Src homology 2) domain [9]. Opposite results were obtained in human
vsmc, in which anti PLCß1 antibodies inhibited AngII-induced
IP3 generation. In this system, neither anti PLC1 antibodies, nor incubation
with genistein or herbimycin A had any effect on IP3 formation
or the calcium signal [10]. These discrepancies were reconciled in
a recent study in rat vsmc which showed that both mechanisms
sequentially contributed to AngII-induced IP3 production.
Before 30 s, IP3 generation was insensitive to genistein
and was inhibited by anti PLCß1 antibodies. In contrast,
after 30 s both genistein and anti PLC1 antibodies reduced IP3 accumulation
[11]. The presence of PLCß1 in rat vsmc was confirmed by
RT-PCR and western blotting [10, 11]. In additional experiments,
early coupling of AT1 to G-proteins was demonstrated
by inhibition of AngII-induced IP3 generation at 15
s with anti Gq/11,
anti G12
and Gß antibodies, as well as by overexpression of the
ß binding
domain of an adrenergic receptor kinase [11]. These studies demonstrate
that AngII couples sequentially, first to PLCß1, through
Gß as
well as two different G subunits (Fig. 1, left panel); and second to
PLC1 (Fig.
1, middle panel).
Figure 1. Sequential activation of different phospholipase subtypes by AngII in vsmc
Following transient generation of IP3
and diacylglycerol (DG) by PLC (terminated within minutes), AngII
induced a prolonged activation of phospholipase D (PLD) in vsmc
[12]. This resulted in a long-lasting accumulation of phosphatidic
acid and its dephosphorylation product DG, that accounted for
the sustained stimulation of protein kinase C observed in vsmc
[2, 13]. Coupling of AT1 to PLD activation was dependent
on several pathways in vsmc. The heterotrimeric G protein activator
sodium fluoride almost doubled PLD activity in vsmc. Furthermore,
AngII-induced PLD activation was inhibited by 80% in cells overexpressing
a ß
binding protein and more than 50% by electroporation
of anti G12
or anti Gß antibodies and 75% by anti-Src antibodies [14].
PLD activation by AngII in vsmc also required extracellular calcium,
protein kinase C [13, 15] and sequestration of AT1
in a compartment such as caveolae [16]. Thus, initial activation
of PLD presumably results from AT1-induced dissociation
of G12
and ß
subunits which may activate PLD through a tyrosine kinase pathway.
Sustained activation of PLD may result from a positive feedback loop
in which protein kinase C, stimulated by calcium influx and PLD-generated
DG, phosphorylates G12 [17], thus keeping ß subunits dissociated (Fig.
1, right panel). These recent studies have clarified the earliest
coupling events important for AngII responses, leading to a greater
understanding of AT1 coupling to G proteins at the molecular
level.
Several important AngII-induced signalling
cascades start with activation of the Src family of tyrosine
kinases [18-21]. Src does not bind to AT1, but may
be activated by ß
subunits of G-proteins [14, 19]. In addition to activating PLC and PLD, Src is
probably responsible for phosphorylation of Shc in vsmc [18]
as seen in similar systems [22]. A complex of Shc, GRB2 and SOS
may stimulate the small G-protein Ras [23], leading to activation of
the serine/threonine kinase Raf-1 [24-26]. Raf-1 is one of the activators
of the threonine/tyrosine kinase MEK1 [24, 25, 27] which activates
the serine/threonine MAP kinases ERK1/2 [28]. In vsmc, AngII
activates ERK1/2 from about 2 to 30 min [23, 25, 29-33]. Activation
of ERK1/2 is terminated by the MKP-1 phosphatase [34, 35].
ERK1/2 can phosphorylate a number of proteins, at least in vitro,
such as cytosolic phospholipase A2, myelin basic protein,
transcription factors such as c-jun and p62TCF and may increase
protein synthesis by activation of p90 ribosomal S6 kinase and
the mRNA cap-binding protein eIF-4A [1, 2, 36, 37]. Tyrosine
kinase inhibitors such as genistein or herbimycin A reduced AngII-induced
activation of the ERK1/2 and JNK MAP kinases, vessel contraction and
protein synthesis [1, 20, 38].
AngII-induced ERK1/2 activity in vsmc is dependent on calcium
signalling since it was blocked by inhibition of PLC, calmodulin
or intracellular calcium mobilization [23]. AngII-induced ERK1/2
activity was not affected by PKC inhibition or down-regulation
with phorbol esters [23, 24]. However, antisense inhibition of
the atypical PKC zeta subtype, which is not down-regulated by phorbol
esters, decreased AngII-induced ERK1/2 activity [39].
AngII may also activate the signalling pathways of other typical
tyrosine kinase-coupled receptors. Thus, AngII induced transactivation
of the EGF receptor, without EGF secretion [40], and stimulated
secretion of autocrine mediators such as IGF-I [41]. These pathways
probably contribute to the sustained activation of tyrosine kinases
by AngII.
Reactive oxygen species, such as superoxide
and hydrogen peroxide (H2O2), can chemically
alter many cellular components. However, oxidants once only known
for their toxicity are now being recognized as possible signalling
molecules at moderate concentration [42]. Inside the cell the
superoxide free radical can be quickly metabolized by superoxide
dismutase (SOD) to hydrogen peroxide which is then more slowly converted
to water by catalase.
Oxidants may have both rapid and long-term signalling effects.
Exogenous superoxide generated for example with xanthine/xanthine
oxidase doubled IP3-induced calcium release from the vsmc sarcoplasm.
This effect appeared to be mediated by superoxide since it was
sensitive to SOD, but not to catalase [42, 43]. H2O2
was also capable of inducing rapid and sustained mobilization
of calcium from agonist-sensitive stores in another vsm model
[44]. Exogenous superoxide increased DNA synthesis [45-48] and
cell number [45, 48] in quiescent vsmc. Similarly, incubation with H2O2
increased DNA synthesis [46-50] in the same model. In some instances
the effect of exogenous oxidants may be indirect, since it may
be dependent upon IGF-I, bFGF or the EGF receptor [47, 51, 52].
The physiological relevance of treatments
with exogenous oxidants was recently established by the discovery
of an AngII-activated endogenous superoxide-generating pathway
in vsmc [53]. The source of superoxide is a membrane-bound NADH
or NADPH-dependent oxidase with features similar to the phagocyte
NADPH oxidase [53-58]. This latter enzyme complex is composed of a
catalytic membrane-bound cytochrome b558 heterodimer (gp91- and
p22-phox) that is activated by binding at least three cytosolic components
(p47-, p67-phox and Rac2). p47phox may be activated by protein
kinase C [59, 60] and Rac2 by exchange of GTP for GDP (Fig. 2).
AngII induced almost a 3-fold increase in superoxide production
and a 5-fold accumulation of H2O2 in vsmc [57, 61].
Superoxide was the major source of H2O2 because inhibition
of oxidase activity with diphenylene iodonium (DPI) or p22-phox
antisense mRNA inhibited the formation of both compounds [33,
53, 57, 61, 62]. This pathway was critical to growth in vsmc
since inhibition of the oxidase or SOD, or overexpression of catalase, greatly
reduced AngII-induced protein synthesis [53, 57, 61].
The function of oxidants was confirmed in-vivo. Hypertension
induced by infusion of AngII (but not norepinephrine) in the
rat was accompanied by up-regulation of p22phox mRNA, cytochrome
b558 protein, and NAD(P)H oxidase-dependent activity, as well
as an impairment of endothelium-dependent relaxation. Importantly,
AngII-induced hypertension was corrected by infusion of heparin-binding
SOD. Moreover, the biochemical effects of AngII infusion were
inhibited in vitro by DPI or liposomal SOD [58, 63, 64].
Figure 2. The NAD(P)H signalling cascade in vsmc
Exogenous superoxide increased the activity of ERK1/2, an effect that was inhibited by the superoxide scavenger tiron and protein kinase C down-regulation [48]. The effect of exogenous H2O2 on ERK1/2 activation has been controversial. ERK1, ERK2 or both were phosphorylated by exposure to H2O2 in some studies [51, 65, 66], but not in others [33, 48, 67]. Stimulation of vsmc with AngII increases endogenous H2O2 accumulation and is accompanied by ERK1/ERK2 activation [33, 67]. However, the fact that AngII-induced ERK1/2 phosphorylation was not inhibited by DPI, tiron or by overexpression of catalase suggests that these kinases are not redox-sensitive within cells [33, 48]. Exogenous H2O2 increased phosphorylation and/or activity of other MAP kinases present in vsmc, namely JNK, p38MAPK and BMK1 (ERK5) [33, 66, 67]. AngII induced p38MAPK activation appeared to result from endogenous H2O2 generation via the NAD(P)H oxidase pathway, since it was blocked by DPI or catalase overexpression [33]. It is worth noting that ERK1/2 and p38MAPK control together the major portion of AngII-mediated hypertrophy. While specific inhibitors of either kinase were partially effective, simultaneous incubation with both compounds blocked most of AngII-induced protein synthesis [33]. This may be explained by the fact that these two kinases regulate complementary sets of proteins and transcription factors. For example, p70 and p90 ribosomal S6 kinases are phosphorylated by p38MAPK and ERK1/2, respectively. Similarly, ATF-2 and CHOP-1 are activated by p38MAPK, while Elk-1 and AP-1 are stimulated by ERK1/2 (Fig. 2).
Following an immediate stimulation of vascular smooth muscle contraction, AngII chronically induces the cellular hypertrophy and proliferation observed in vascular diseases. These effects are mediated by a vast array of signals, notably heterotrimeric and small G-proteins, phospholipases, and kinase cascades which control gene transcription. It is interesting to note that the most recently recognized of these mediators, superoxide and H2O2, now fulfill the conditions required for classification as signalling molecules. Both agents are rapidly synthesized in response to agonist stimulation, have specific molecular targets and are rapidly degraded enzymatically. Although formerly known for their toxicity, these oxidants are now gaining acceptance as redox signal mediators. Their complex roles in AngII responsiveness are only beginning to be understood.
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