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Role of tyrosine phosphorylation in excitation–contraction coupling in vascular smooth muscle
Transcript of Role of tyrosine phosphorylation in excitation–contraction coupling in vascular smooth muscle
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Role of tyrosine phosphorylation in excitation±contraction
coupling in vascular smooth muscle
A . D . H U G H E S and S . W I J E T U N G E
Clinical Pharmacology, National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, St Mary's Hospital,
South Wharf Road, London, UK
ABSTRACT
Increasingly it is recognized that tyrosine phosphorylation plays an important part in the regulation
of function in differentiated contractile vascular smooth muscle. Tyrosine kinases and phosphatases
are present in large amounts in vascular smooth muscle and have been reported to influence a
number of processes crucial to contraction, including ion channel gating, calcium homeostasis and
sensitization of the contractile process to [Ca2+]i. This review summarizes current understanding
regarding the role of tyrosine phosphorylation in excitation±contraction coupling in blood vessels.
Keywords calcium channels, calcium, excitation±contraction coupling, tyrosine kinase, tyrosine
phosphatase, vascular smooth muscle.
Received 22 May 1998, accepted 13 July 1998
The role of tyrosine phosphorylation in proliferation
and chemotaxis of cultured vascular smooth muscle
cells in response to activators is now well recognized
(e.g. see recent reviews by Bobik & Campbell 1993,
Bornfeldt et al. 1995, Schieffer et al. 1997) and will not
be dealt with in this article. The object of this paper is
to consider the possible role of tyrosine phosphoryla-
tion in excitation±contraction coupling in smooth
muscle. Consequently this review will focus almost
entirely on work in differentiated, contractile vascular
smooth muscle. Although it will not be covered in
detail here, it should be noted that there is also sub-
stantial evidence that tyrosine phosphorylation plays an
important role in excitation±contraction coupling in
non-vascular smooth muscle (see Hollenberg 1994, Di
Salvo et al. 1997).
TYROSINE PHOSPHORYLATION AND
CELL FUNCTION
The notion that tyrosine phosphorylation, a process
originally identi®ed in the context of growth and on-
cogenesis, might play a role in smooth muscle con-
traction is a relatively recent one (Hollenberg 1994).
Originally tyrosine kinase activity was identi®ed in cells
transformed by oncogenic viruses (Collet & Erikson
1978, Hunter & Sefton 1980) and many viral oncogenes
have proved to code for tyrosine kinases. Subsequently
a number of cellular counterparts, proto-oncogenes
were identi®ed (Patarca 1996) and the majority of re-
ceptors for growth factors are now recognized to be
tyrosine kinases (Schlessinger & Ullrich 1992). Al-
though initial studies focussed on the potential role of
tyrosine kinase in growth and proliferation, it is now
clear that tyrosine phosphorylation plays an important
role in many cellular processes.
TYROSINE KINASES
Numerous tyrosine kinases have now been described
and the superfamily of enzymes has been subdivided
into receptor and non-receptor classes with numerous
subfamilies (Courtneidge 1994). Some tyrosine kinases
are found in many tissues, while others show much
more restricted distribution.
Receptor tyrosine kinases are transmembranous pro-
teins possessing intrinsic tyrosine kinase activity, which
is regulated by an extracellular ligand, such as a growth
factor (Schlessinger & Ullrich 1992). In contrast, non-
receptor tyrosine kinases generally lack extracellular
recognition domains for ligands. Nevertheless, the ac-
tivity of these enzymes may be regulated at an intra-
cellular level by receptors for growth factors or other
receptor systems (e.g. G proteins, cytokine receptors,
Correspondence: A. D. Hughes, Clinical Pharmacology, National Heart and Lung Institute, Imperial College of Science, Technology and
Medicine, St Mary's Hospital, South Wharf Road, London W2 1NY, UK.
Acta Physiol Scand 1998, 164, 457±469
Ó 1998 Scandinavian Physiological Society 457
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GPI-linked receptors) which may themselves lack in-
trinsic tyrosine kinase activity (Thomas & Brugge 1997).
The mechanisms involved in this cross-talk between
signalling systems are only just beginning to be eluci-
dated. In the case of G proteins it seems that activation
of non-receptor tyrosine kinases, such as pp60c±src (src
kinase) may play a key role, at least in some cells. In this
paradigm the non-receptor tyrosine kinase may act as a
signalling molecule itself (Thomas & Brugge 1997) or
induce transactivation of growth factor receptors such
as the EGFR (Luttrell et al. 1997).
Some tyrosine kinases may be activated as a result of
a rise in [Ca2+]i. This mechanism contributes to tyrosine
kinase activation by angiotensin II (AII) in cultured
vascular smooth muscle cells (Huckle et al. 1992) and
endothelin-1 in mesangial cells (Coroneos et al. 1997),
although its importance in differentiated vascular
smooth muscle is uncertain.
Other receptors involved in cell±matrix or cell±cell
contact, such as integrins, cadherins and CAMS also
increase tyrosine phosphorylation in many cell types
(Clark & Brugge 1995). The possibility that similar in-
teractions induce tyrosine phosphorylation in differen-
tiated vascular smooth muscle (possibly accounting for
the basal levels of tyrosine phosphorylation seen in
some studies) or contribute to responses to mechanical
stresses appears not to have been examined.
TYROSINE PHOSPHATASES
Although many tissues have high levels of tyrosine ki-
nase activity in vitro, phosphotyrosine residues in cells
generally amount to less than 0.1% of phosphorylated
serine or threonine (Glenney 1992). This presumably
re¯ects restricted levels of substrates and high tyrosine
phosphatase (PTPase) activity. In addition to the family
of tyrosine kinases, an increasingly large family of
PTPases is now recognized (Hunter 1995, Neel &
Tonks 1997). As yet this group of enzymes has been
less studied and their regulation is less well understood
than tyrosine kinases.
TYROSINE KINASES AND PHOSPHA-
TASES IN VASCULAR SMOOTH MUSCLE
Numerous studies have demonstrated the existence of
multiple tyrosine phosphorylated proteins in vascular
smooth muscle under unstimulated conditions (Lani-
yonu et al. 1994a, b, Ward et al. 1995, Jin et al. 1996,
Watts et al. 1996b, Ohanian et al. 1997, Rembold &
Weaver 1997). However, the enzyme(s) responsible for
this basal level of tyrosine phosphorylation and their
regulation in the absence of receptor stimulation re-
mains undetermined.
Although there is functional evidence for the pres-
ence of several growth factor receptors (see below)
there are relatively few studies exploring the distribution
of receptor tyrosine kinases in uninjured blood vessels.
mRNA for the PDGF receptor was identi®ed in the
media of human carotid and coronary artery specimens,
although considerably less frequently than in intimal
regions. The high levels of mRNA for PDGF receptor
in these regions appeared to be associated with me-
senchyme-like cells, presumably dedifferentiated
smooth muscle cells (Wilcox et al. 1988). Similarly, an-
other study of human coronary arteries only detected
mRNA for PDGF receptor (b isoform) in association
with regions of repair, and in adult kidney mRNA for
PDGF receptor (a isoform) was rarely detected in
vascular smooth muscle cells (Floege et al. 1997).
mRNA for PDGF receptors (a and b isoforms) has also
been seen in rat aorta but at lower levels than in aorta
taken from spontaneously hypertensive (SHR) animals
(Sarzani et al. 1991). Another study using antibodies for
the PDGF receptor (b isoform) found minimal im-
munoreactivity in association with vascular smooth
muscle in normal human kidney or in the arterial media
or intima of undiseased vessels (Rubin et al. 1988).
Smooth muscle, unlike cardiac or skeletal muscle,
possesses high levels of tyrosine kinase activity under
unstimulated conditions ± presumably re¯ective of
non-receptor tyrosine kinase activity (Di Salvo et al.
1988, 1989, Elberg et al. 1995). Src kinase activity has
been detected in bovine coronary artery and aorta (Di
Salvo et al. 1988, 1989), and c-src has also been iden-
ti®ed using anti-src antibodies in rabbit ear artery
smooth muscle cells (unpublished data). In pig coro-
nary tissue Laniyonu et al. (1994a, b) detected tyrosine
kinase activity using both poly (Glu Tyr) and cdc2
peptide (a putative src family selective substrate). In a
subsequent study using the same tissue, tyrosine kinase
activity was found to be predominantly in a membrane
fraction. Following chromatographic puri®cation, two
peaks of activity were separated, both of which were
recognized by an antibody directed against src family
kinases. Interestingly, these partially puri®ed enzymes
showed differential sensitivity to tyrosine kinase in-
hibitors suggesting that they are different members of
the src family (Laniyonu et al. 1995). It was also note-
worthy that the concentrations of genistein and
tyrphostin 25 required to inhibit the activity of either
fraction were considerably higher than those required
to inhibit agonist-induced contraction in the same tis-
sue implying a role for other tyrosine kinases in these
responses. Non-receptor tyrosine kinase activity was
also studied in a number of tissues by Elberg et al.
(1995). The src family kinases, Lyn and Fyn, (the latter
in large amounts) were identi®ed in smooth muscle
cytoplasm by immunoblotting techniques. Intriguingly
in this study immunoprecipitation of these src-related
kinases had negligible effect on total tyrosine kinase
Role of tyrosine phosphorylation � A D Hughes and S Wijetunge Acta Physiol Scand 1998, 164, 457±469
458 Ó 1998 Scandinavian Physiological Society
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activity, again suggesting the existence of other im-
portant, and unidenti®ed, tyrosine kinases in smooth
muscle.
The possibility that some tyrosine kinases in blood
vessels are activated by [Ca2+]i has received relatively
little attention. A Ca2+-induced increase in tyrosine
phosphorylation was seen in permeabilized smooth
muscle (Steusloff et al. 1995), and Rembold & Weaver
(1997) reported that removal of extracellular Ca2+
markedly attenuated tyrosine phosphorylation induced
by histamine in pig carotid artery. However, Ward et al.
(1995) reported that inhibition of Ca2+ entry had no
effect on noradrenaline-induced increases in tyrosine
phosphorylation in the presence of sodium orthov-
anadate.
A number of PTPases including PTP-1D (Ali et al.
1997), MKP-1 (Lai et al. 1996), RPTPa (Daum et al.
1994), 3CH1334 (Duff et al. 1993) and rat density en-
hanced phosphatase 1 (Borges et al. 1996) have been
identi®ed in cultured vascular smooth muscle. How-
ever, although SHP2 has been identi®ed in blood ves-
sels (Adachi et al. 1997), there is little data regarding the
identity of PTPases present in contractile smooth
muscle. PTPase activity is implied by the ability of in-
hibitors such as sodium orthovanadate or pervanadate
(a mixture of sodium orthovanadate and H2O2) to in-
crease levels of tyrosine phosphorylation in vascular
smooth muscle (Laniyonu et al. 1994a, b, Saifeddine
et al. 1994, Ward et al. 1995, Wijetunge et al. 1998).
However, the major PTPase types responsible for this
activity are unde®ned, and whether they are subject to
physiological regulation is unknown.
INVOLVEMENT OF TYROSINE KINASES
IN THE CONTRACTILE PROCESS
Contractile actions of receptor tyrosine kinases (growth factors)
Epidermal growth factor
Following its initial description as a vasoconstrictor in
vitro (Berk et al. 1985, Muramatsu et al. 1985) EGF has
reported to contract a number of isolated arteries. In
some, such as rat aorta (Berk et al. 1985) and cow
coronary (Gan & Hollenberg 1990) this action appears
to be as a result of a direct effect of EGF on vascular
smooth muscle. In other tissues, such as pig coronary
(Gan & Hollenberg 1990) and rat ileocolic (Muramatsu
et al. 1985) arteries, the action of EGF is blocked by
indomethacin, an inhibitor of cyclo-oxygenase. In rat
aorta the contractile action of EGF was blocked by
RG50864 (tyrphostin 47), a selective inhibitor of tyro-
sine kinase (Merkel et al. 1993) and in pig coronary
artery genistein and tyrphostin 25 (AG82) antagonized
its action (Hollenberg 1994). EGF does not contract all
isolated arteries, and although it potentiates contraction
of rat superior mesenteric artery to KCl, it failed to
induce tone in the absence of other stimuli (Muramatsu
et al. 1985). It has also been reported to have no con-
tractile effect on squirrel aorta and mesenteric artery
and in one study of rat aortic strips (Muramatsu et al.
1985). In dog coronary artery or mesenteric artery not
only did EGF not induce contraction it suppressed
responses to other stimuli (Muramatsu et al. 1985, Gan
et al. 1990).
Platelet-derived growth factor
Berk and colleagues reported that puri®ed human
PDGF induced contraction of rat aortic strips (Berk
et al. 1986). The action of PDGF was unaffected by
endothelial removal, indomethacin, or antagonists of a-
adrenoceptors or 5HT2 receptors. The maximum effect
of PDGF was »30% of tone induced by AII, and
PDGF acted additively with serotonin or PGF2a. In
the same study a smaller (»15%) contraction was also
seen in response to ®broblast growth factor (FGF) and
transforming growth factor-b (TGF-b). PDGF has
also been reported to contract single smooth muscle
cells isolated from rat aorta (Morgan et al. 1985), and
rabbit isolated ear artery rings (Hughes 1995b). The
effect of the various isoforms of PDGF was examined
in rat aortic rings (Block et al. 1989, Sachinidis et al.
1990) and rank order of maximum ef®cacy was found
to be PDGF-BB ³ PDGF-AB > PDGF-AA. In con-
trast, all isoforms of PDGF failed to contract rat iso-
lated intracerebral arteries (Bassett et al. 1988). The
contractile action of PDGF was antagonized by
selective inhibitors of tyrosine kinases in rat aorta
(Sauro & Thomas 1993b) and rabbit ear artery (Hughes
1995b).
TYROSINE KINASES AND CONTRAC-
TION IN RESPONSE TO RECEPTORS
LINKED TO HETEROTRIMERIC G PRO-
TEINS (R 7G) AND OTHER STIMULI
The majority of `classical vasoconstrictors' such as
noradrenaline, serotonin, angiotensin II or vasopressin
are know to act via receptors belonging to the seven
transmembrane domain superfamily (R7G) (O'Dowd
et al. 1989). These receptors couple with heterotrimeric
G proteins (Neer 1994, Hamm 1998) and are known to
activate a number of intracellular signalling systems
including phospholipase C (PLC) which generates IP3
and DAG (Van Breemen & Saida 1989). IP3 and DAG
release intracellular Ca2+ stores and activate protein
kinase C (PKC), respectively. In addition, R7G cause
the opening of voltage- and receptor-operated calcium
channels in vascular smooth muscle (reviewed in
Hughes 1995a). Although R7G lack intrinsic tyrosine
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kinase activity, recently evidence has accumulated to
indicate that activation of tyrosine kinases may con-
tribute to the intracellular actions of R7G in many ar-
teries. Several R7G agonists including noradrenaline
(Ward et al. 1995), phenylephrine (Khalil et al. 1995, Jin
et al. 1996), AII (Laniyonu et al. 1994a, b, Malloy &
Sauro 1996), serotonin (Watts et al. 1996b, Florian &
Watts 1998), endothelin (Ohanian et al. 1997), hista-
mine (Rembold & Weaver 1997) and thrombin (Jerius
et al. 1998) have been shown to increase tyrosine
phosphorylation of cellular proteins in arterial smooth
muscle. In the case of AII, it has been shown that a
tyrosine kinase, JAK2 co-immunoprecipitates with the
AT1 receptor and is tyrosine phosphorylated in re-
sponse to AII in rat aortic smooth muscle cells (Mar-
rero et al. 1995).
Functional studies have also implicated tyrosine ki-
nases in contractile responses to R7G activation. These
studies have generally used putatively selective low
molecular weight inhibitors of tyrosine kinase, such as
genistein, tyrphostins and erbstatin-like compounds
(Levitzki & Gazit 1995, Klohs et al. 1997). Whilst these
agents, especially the tyrphostins are relatively selective
for tyrosine kinases and show little activity against
serine/threonine kinases (Di Salvo et al. 1993a), they
are not without non-speci®c actions. There appears to
have little systematic investigation into whether any of
these agents possesses receptor antagonist properties,
although Watts suggested that genistein and daidzein
might antagonize serotonin receptors (Watts et al.
1996b). Genistein is a ¯avanoid which inhibits tyrosine
kinases via interaction with the ATP-binding site, it also
possesses some inhibitory action at cAMP phosphodi-
esterase (Beretz et al. 1978, Akiyama et al. 1987).
Tyrphostins are generally more selective as most in-
teract with the substrate binding site of tyrosine kinases,
but members of the tyrphostin family have been re-
ported to inhibit GTPases (Young et al. 1993), fatty acid
synthesis, lactate transport, aldehyde dehydrogenase
and mitochondrial function (Wolbring et al. 1994,
Burger et al. 1995). Another problem in interpreting
®ndings with tyrosine kinase inhibitors is that they may
show varying potencies against different tyrosine ki-
nases or the same kinase with a different substrate
(Akiyama et al. 1987, Brunton et al. 1994, Levitzki &
Gazit 1995, Klohs et al. 1997). Whilst the former fea-
ture has been exploited recently to develop selective
antagonists of speci®c tyrosine kinases (Klohs et al.
1997), it also complicates interpretation of results in
systems such as vascular smooth muscle where several
tyrosine kinases may contribute to different extent to
responses to a particular agonist. A further complicat-
ing factor with the tyrphostins and related compounds
relates to the slow onset (Lyall et al. 1989, Hsu et al.
1991), and poor stability of some of these compounds,
with degradation leading to the production of more
potent products under some circumstances (Ramdas
et al. 1994). Nevertheless, despite these problems these
agents have proved to be valuable tools in elucidating
the possible role of tyrosine kinase in cellular functions
in many systems.
Selective tyrosine kinase inhibitors have been re-
ported to inhibit contraction in response to a wide
range of contractile agents (Table 1.). In addition to
inhibiting agonist-induced tone tyrosine kinase inhibi-
tors have been reported to inhibit myogenic tone in rat
cerebral artery (Osol et al. 1993, Masumoto et al. 1997)
with herbimycin being reported to show a selective
action against pressure induced tone. In rabbit aorta
studied using an organ culture technique, an increase in
perfusion pressure was reported to activate MAP kinase
(ERK1/2). This effect was inhibited by herbimycin, but
not by genistein or tyrphostin A48 (Birukov et al. 1997).
In contrast with these inhibitory effects on agonist-
induced tone, contraction in response to some stimuli is
reported to be unaffected by tyrosine kinase inhibitors.
Tyrphostin 25 had no effect on ATP-induced con-
traction in rat aorta at concentrations which inhibited
mitogenesis in cultured aortic smooth muscle cells
(Erlinge et al. 1996). Tyrphostin 23 and genistein were
also reported not to inhibit PDBu-induced tone in rat
aorta (Watts et al. 1996b), and genistein failed to inhibit
PDBu-induced contraction of rat basilar artery (Kit-
azono et al. 1998).
In some of these studies (Di Salvo et al. 1993b,
Laniyonu et al. 1994a, b, Jinsi & Deth 1995, Filipeanu
et al. 1995) it was noted that tyrosine kinase inhibitors
were more effective antagonists of agonist-induced
tone than depolarization (KCl)-induced tone. Fur-
thermore in a detailed study (Laniyonu et al. 1994a,b)
differences in potency of inhibitors were noted using
different agonists and different tissues. In other
studies (Toma et al. 1995, Gould et al. 1995) no
marked differences were seen in the effect of tyrosine
kinase inhibitors on agonist as opposed to KCl-in-
duced tone and Toma et al. (1995) reported that the
purportedly inactive tyrphostin 1 and daidzein also
inhibited contraction. Allowing for methodological
and technical differences, there are two other possible
explanations for differences in potencies of inhibitors
against different agonists or in different tissues.
Firstly, more than one tyrosine kinase may be in-
volved in contraction and different tyrosine kinases
may be recruited to different extent by different
stimulants in different tissues. Secondly, many of the
inhibitors used are now known to be selective in their
potency towards different tyrosine kinases, hence the
apparent differences in potency reported may re¯ect
differential selectivity of inhibitors towards speci®c
tyrosine kinases.
460 Ó 1998 Scandinavian Physiological Society
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TYROSINE PHOSPHATASES AND VAS-
CULAR TONE
Amongst its many other actions (Erdmann et al. 1984),
sodium orthovanadate is an inhibitor of PTPases
(Swarup et al. 1982). Sodium orthovanadate has been
shown to increase tyrosine phosphorylation in arterial
tissue (Laniyonu et al. 1994a, b, Ward et al.. 1995) and
to contract isolated arterial smooth muscle (Shimada
et al. 1986, Ozaki et al. 1988, Sanchez Ferrer et al. 1988).
Results from studies employing Na+-free external so-
lutions (Laniyonu et al. 1994a, b) or ouabain (Ozaki &
Urakawa 1980) suggest the contractile action of vana-
date is not simply as a result of inhibition of Na±K-
ATPase. Moreover, pervanadate, a more potent inhib-
itor of PTPases (Posner et al. 1994) has also been re-
ported to increase tyrosine phosphorylation and
contract vascular smooth muscle more potently than
sodium orthovanadate (Laniyonu et al. 1994a, b). In the
latter study the contractile action of sodium vanadate
and pervanadate was blocked by genistein and
tyrphostin-23, consistent with their effects being
mediated by the unopposed action of tyrosine kinases.
In contrast, in rat aorta (Zhou et al. 1997) sodium
vanadate-induced contraction was not inhibited by ge-
nistein or tyrphostin, but was inhibited by a 5-lipoxy-
genase inhibitor and an inhibitor of PLC suggesting
that in this tissue the action of vanadate was the result
of interference with phosphoinositide metabolism.
HOW DOES TYROSINE PHOSPHORYLA-
TION AFFECT THE CONTRACTILE
PROCESS?
The numerous observations of inhibition of tone with
inhibitors of tyrosine kinases raise the questions, at
what points in the contractile process are tyrosines
kinase involved? And which tyrosine kinases? At
present answers to both questions are incomplete. It is
outside the scope of this review to discuss in detail the
process of contraction in vascular smooth muscle (see
Somlyo & Somlyo 1994, Horowitz et al. 1996, for re-
views), but in brief, smooth muscle contraction in-
volves elevation of [Ca2+]i with resultant activation of
calmodulin±myosin light chain kinase and myosin light
chain (LC20) phosphorylation. Phosphorylation of
LC20 is also regulated by myosin phosphatase and the
actin±myosin interaction may be further in¯uenced by
Table 1 Table 1 lists vascular tissues in which responses to various contractile agonists have been reported to be inhibited by tyrosine kinase
inhibitors
Agonist Species Tissue Reference
Noradrenaline
/Phenylephrine Ferret Aorta Khalil et al. 1995
Guinea-pig Mesenteric artery Di Salvo et al. 1993b
Pig Coronary artery Laniyonu et al. 1994a, b
Rat Aorta Jinisi et al. 1996, Duarte et al. 1997,
Jin et al. 1996, Abebe & Agarwal 1995
Mesenteric artery Toma et al. 1995
Pulmonary artery Jin et al. 1996
Gracilis small artery Malloy & Sauro 1996
UK 14301 Rat Aorta Jinsi & Deth 1995
Serotonin Rat Aorta Watts et al. 1996b, Florian & Watts 1998
Carotid artery Watts et al. 1996b
Basilar artery Kitazono et al. 1998
Angiotensin II Cow Carotid artery Epstein et al. 1997
Dog Coronary artery Saifeddine 1994
Pig Coronary artery Laniyonu et al. 1994a, b, Saifeddine 1994
Rat Aorta Laniyonu et al. 1994a,b, Sauro et al. 1996
Mesenteric artery Touyz & Schiffrin 1997
Vasopressin Pig Coronary artery Laniyonu et al. 1994a, b
Histamine Pig Carotid artery Gould et al. 1995
PGF2a Rat Aorta Laniyonu et al. 1994
Pig Coronary artery Laniyonu et al. 1994a, b
U46619 Rat Basilar artery Masumoto et al. 1997
Endothelin-1 Cow Carotid artery Epstein et al. 1997
Pig Coronary artery cells Liu & Sturek 1996
Rat Mesenteric artery Ohanian et al. 1997
Thrombin Human Umbilical A&V Tay et al. 1995
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thin ®lament proteins such as caldesmon and calponin.
In principle tyrosine kinases could in¯uence any, or all,
of these steps. Evidence for such actions is discussed
below.
MODULATION OF [Ca 2 +] i BY TYROSINE
KINASES
Under physiological conditions contraction of vascular
smooth muscle is almost invariably preceded by a rise in
[Ca2+]i. A rise in [Ca2+]; results from in¯ux of Ca2+ or
release of intracellular Ca2+ stores. In¯ux of Ca2+
through calcium channels represents a major source of
[Ca2+]i in vascular smooth muscle and may be of par-
ticular importance in smaller resistance arteries and ar-
terioles (Hughes 1995a). Two types of Ca2+ entry
pathways have been de®ned in smooth muscle (Bolton
1979); voltage-operated calcium channels and receptor-
operated cation channels. While receptor-operated
channels are by de®nition gated by receptors there is
also extensive evidence that opening of voltage-oper-
ated calcium channels may be affected by receptor ac-
tivation (reviewed in Hughes 1995a).
MODULATION OF ION CHANNELS BY
TYROSINE PHOSPHORYLATION
Calcium channels
As discussed above tyrosine kinase inhibitors have been
demonstrated to inhibit depolarization-induced con-
traction, if with generally lower potency than agonist-
induced tone. This suggests that tyrosine kinases may
in¯uence voltage-operated calcium channel opening.
Further evidence for such an action comes from direct
measurements of [Ca2+]i in blood vessels. In pig carotid
artery genistein inhibited the rise in [Ca2+]i induced by
KCl, as well as that induced by histamine (Gould et al.
1995). Similarly, in rat mesenteric artery 30 lM genistein
inhibited the rise in [Ca2+]i induced by noradrenaline
(Toma et al. 1995), which is largely dependent on Ca2+
entry through voltage-operated calcium channels (Nils-
son et al. 1994). Surprisingly in this study, genistein had
no signi®cant effect on KCl-induced rise in [Ca2+]i al-
though it inhibited KCl-induced tone.
These functional observations have been extended
by voltage clamp studies showing that tyrosine kinase
inhibitors reduce L-type voltage-gated calcium channel
currents in rabbit ear artery cells (Wijetunge et al. 1992)
and rat portal vein cells (Liu et al. 1997a, Ogata et al.
1997). In rat portal vein this action was found to be as a
result of a decrease in single channel availability (Liu &
Sperelakis, 1997b). Further evidence for a modulatory
effect of tyrosine phosphorylation on voltage-operated
calcium channels has been provided by Wijetunge et al.
(1998), who have shown that inhibition of PTPase by
pervanadate, sodium orthovanadate, phenylarsine oxide
or dephostatin increases calcium channel currents in
rabbit ear artery smooth muscle cells. Together these
studies imply that endogenous tyrosine kinases active
under resting conditions regulate the availability of
voltage-operated calcium channels in vascular smooth
muscle cells.
Growth factors have also been shown to affect
voltage-operated calcium channels in vascular smooth
muscle in vitro. In rat aorta and rabbit ear artery PDGF-
induced tone is wholly dependent on in¯ux of Ca2+
(Berk et al. 1986, Hughes 1995b). PDGF-induced
contraction is also profoundly inhibited by calcium
channel antagonists (Block et al. 1989, Hughes 1995b)
and a dihydropyridine calcium channel antagonist,
(-)202791, was also found to abolish the rise in [Ca2+]iin response to PDGF in rabbit ear artery (Hughes
1995b). Electrophysiological studies have provided
more direct evidence linking growth factors to modu-
lation of L-type voltage-operated calcium channels. In
rabbit ear artery cells PDGF was found to increase
voltage-gated calcium channel currents by a tyrosine
kinase-dependent mechanism (Wijetunge & Hughes
1995a). At present the most likely candidate for such
modulation of L-type voltage-operated calcium chan-
nels in vascular smooth muscle appears to be src kinase
or a closely related kinase. Intracellular application of
src kinase has been shown to increase calcium channel
currents in rabbit ear artery cells by a PKC-independent
mechanism (Wijetunge & Hughes 1995b). Similarly a
peptide containing the (P)YEEI motif which activates
src family kinases has also been shown to increase
calcium channel currents in the same cells (Wijetunge &
Hughes 1996), presumably by activation of endogenous
src kinase or a closely related kinase. Interestingly, a
recent study in gastric smooth muscle has shown that
the a1 subunit of the voltage-operated calcium channel
can be co-immunoprecipitated with src kinase and
FAK and that the a1 subunit undergoes tyrosine
phosphorylation following application of PDGF (Hu
et al. 1998). These ®ndings suggest that tyrosine phos-
phorylation of the a1 subunit of the L-type channel may
account, at least in part, for the modulatory effect of src
kinase on calcium channel opening in smooth muscle.
At present there is relatively little data regarding the
possible role of tyrosine kinases in regulation of re-
ceptor-operated cation channels in vascular smooth
muscle. Inoue et al. 1994 reported that genistein in-
hibited, while sodium orthovanadate stimulated a
muscarinic receptor-operated cation conductance in
guinea-pig ileal cells. However, in cultured coronary
artery cells Minami et al. (1994) reported that genistein,
but not daidzein (an analogue of genistein inactive
against tyrosine kinases), caused a marked increase in
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the opening of a large conductance non-selective cation
channel. The functional role of this channel or its
relationship to other receptor-operated cation channels
described in vascular smooth muscle (Hughes 1995a) is
unknown.
K channels
Another means by which tyrosine phosphorylation
could in¯uence Ca2+ entry in vascular smooth muscle is
through effects on membrane potential (Em). K chan-
nels are a major determinant of Em in vascular smooth
muscle (Nelson & Quayle 1995), although Cl channels
also make a contribution (Large & Wang 1996). The
possibility that tyrosine phosphorylation affects Cl
channels in smooth muscle appears not to have been
investigated but there is evidence implicating tyrosine
kinases in regulation of K channel in smooth muscle.
Genistein and lavendustin A, but not tyrphostin 25
or daidzein were reported to increase Ca2+ activated K
channel (KCa) currents in whole cell and single channel
studies of vascular smooth muscle cells isolated from
rat tail artery (Xiong et al. 1995). Neither agent had any
effect when ATP was omitted from the intracellular
solution and the ®ndings were taken to suggest that a
tyrosine kinase might exert an inhibitory effect on KCa
channel opening. In rat portal vein (Ogata et al. 1997)
genistein was reported to inhibit pinacidil-induced KATP
current with an IC50 of 5.5 lM. Although herbimycin,
lavendustin A, tyrphostin 23 and EGF failed to affect
KATP currents, sodium orthovanadate induced a small
increase in KATP current. In this study, although a direct
inhibitory action of genistein was not ruled out, the
authors interpreted their data as suggesting a role for a
tyrosine kinase in regulation of KATP channels. In the
same study genistein was also found to inhibit voltage-
dependent K channel (KV) currents (IC50 75 lM), KV
currents were also increased by sodium orthovanadate
but herbimycin had no effect on KV.
In rat and rabbit pulmonary artery genistein and
ST638 (a putative selective tyrosine kinase inhibitor),
but not daidzein were observed to inhibit KV (Smirnov
& Aaronson 1995). However tyrphostin 23 was inactive
and sodium orthovanadate had no effects on currents.
Furthermore removal of ATP or its replacement by
ATP-c-S had no signi®cant effect of KV inhibition by
these agents and it was concluded that the effects of
genistein and ST638 did not involve inhibition of ty-
rosine kinases.
TYROSINE KINASES AND INTRACEL-
LULAR Ca2 + STORES
In rat thoracic aorta, genistein or quercetin reduced
contraction in response to phenylephrine in conditions
when all extracellular Ca2+ was removed (Ca2+-free
conditions) (Filipeanu et al. 1995). Sodium vanadate
was also found to induce tone in Ca2+ free conditions,
however only 50% of this contraction was inhibited by
genistein and so was probably only partially related to
vanadate's action as a PTPase inhibitor. In pig carotid
artery genistein was also found to inhibit histamine-
induced tone in Ca2+ free conditions and in pig coro-
nary artery cells genistein inhibited the endothelin-1
induced rise in [Ca2+]i under Ca2+ free conditions but
did not affect release of intracellular Ca2+ by IP3 or
caffeine (Liu & Sturek 1996). In contrast, Low (1996)
reported that phenylephrine-induced contraction in
Ca2+-free conditions was not affected consistently by
tyrosine kinase inhibitors. Furthermore, on the basis of
experiments with cyclopiazonic acid, an inhibitor of the
sarcoplasmic Ca2+ ATPase, these authors suggested
that tyrosine kinases were involved in Ca2+ entry in-
duced by store depletion. The mechanism of action of
tyrosine kinase inhibitors in inhibiting store release is
uncertain. The type I IP3 receptor (which is found in
smooth muscle) has been reported to undergo tyrosine
phosphorylation (Jayaraman et al. 1996). But the ®nd-
ings of Liu and Sturek (1996) suggest a more proximal
action of tyrosine kinase inhibitors in the pathway
linking ligand binding to intracellular Ca2+ release. In
cultured vascular smooth muscle cells it has been sug-
gested that PLC-c an isoform of PLC which is regu-
lated by tyrosine phosphorylation plays a role in
agonist-induced IP3 generation (Marrero et al. 1994).
Whether this is also true in contractile vascular smooth
muscle remains to be established.
MODULATION OF Ca2 + SENSITIVITY
OF THE CONTRACTILE APPARATUS BY
TYROSINE PHOSPHORYLATION
In addition to the rise in [Ca2+]i which generally ac-
companies contraction modulation of the sensitivity of
the contractile apparatus to [Ca2+]i is also an important
factor in agonist regulation of vascular tone (see So-
mlyo & Somlyo 1994, Horowitz et al. 1996 for reviews).
The possibility that tyrosine phosphorylation contrib-
utes to modulation of the Ca2+ sensitivity of the con-
tractile apparatus is disputed at present. In intact
isolated pig carotid artery genistein reduced [Ca2+]i but
had no effect on the relationship between [Ca2+]i and
phosphorylation of myosin LC20 or the dependence of
steady state stress on myosin phosphorylation (Gould
et al. 1995). In a-toxin skinned preparations of rat
mesenteric small arteries genistein, tyrphostin 23 and
tyrphostin 47 all relaxed Ca2+-induced tone (Toma et al.
1995). In these studies genistein was found to be a
more potent relaxant of tone induced in the presence of
noradrenaline and GTP, but no comparable difference
in potency was seen with the tyrphostins. In contrast, in
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b-escin skinned guinea-pig mesenteric arteries
tyrphostin partially reversed ras or GTP-c-S induced
tone, but did not interfere with Ca2+-induced activation
(Satoh et al. 1993, Di Salvo et al. 1993b). The difference
between these observations may relate to the loss of key
cytoplasmic constituents during skinning by b-escin
which are preserved in a-toxin skinned preparations.
Although the importance of tyrosine kinases in
modulating Ca2+ sensitivity is uncertain, a number of
potential mechanisms exist, which could link tyrosine
phosphorylation to increased Ca2+ sensitivity. These
include activation of phospholipase D (PLD), activa-
tion of MAP kinases and activation of small GTPases
(e.g. rho).
Stimulation of PLD with resultant activation of
PKC is a possible tyrosine phosphorylation-dependent
mechanism affecting the Ca2+ sensitivity in vascular
smooth muscle. Activation of PLD has been proposed
as a mechanism of a 2-adrenoceptor induced contrac-
tion in rabbit saphenous vein on the basis of the ability
of wortmannin to inhibit a2-adrenoceptor-induced tone
(Waen-Safranchik & Deth 1994). However wortmannin
is a poorly selective agent and may act via inhibition of
phosphatidylinositol 3 kinase (Ui et al. 1995), or inhi-
bition of myosin light chain kinase at higher concen-
trations (Nakanishi et al. 1992). Nevertheless, both
noradrenaline and the a2-adrenoceptor agonist, UK
14304, have been shown to increase PLD activity in rat
aortic smooth muscle. This effect was inhibited by
genistein, which also inhibited tone induced by these
agonists (Jinsi & Deth 1995). In rat mesenteric arteries
noradrenaline and AlF4- (a direct activator of G pro-
teins) have been shown to stimulate PLD activity by a
mechanism inhibited by genistein (Ward et al. 1995).
Furthermore, PLD activity was markedly stimulated
when noradrenaline was applied in the presence of
vanadate to inhibit PTPases.
MAP kinases are a family of �40 kDa serine/
threonine kinases involved in a number of cellular
functions including growth (Robinson & Cobb 1997),
which require to be phosphorylated on tyrosine and
threonine residues for full activation. In the context of
contraction their ability to phosphorylate caldesmon, a
thin ®lament protein regulator of actin±myosin inter-
action is now thought to be particularly relevant (Childs
& Mak 1993, Adam et al. 1995). A number of agonists
including serotonin (Watts 1996a), angiotensin II, ( Jin
et al. 1996) and phenylephrine (Khalil & Morgan 1993)
have been shown to activate or cause redistribution of
MAP kinase. In addition inhibition of MAP kinase ac-
tivation by PD098059, a selective MAP kinase (MEK)
inhibitor has also been shown to reduce contraction in
response to serotonin (Watts 1996a) and angiotensin II
(Garcha et al. 1998). The precise mechanism by which
contractile agonists activate MAP kinase is uncertain,
although it appears to involve tyrosine phosphorylation
and activation of PKC (Khalil et al. 1995).
The regulation of myosin phosphatases by rho in
smooth muscle and its potential role in excitation±
contraction coupling has been reviewed elsewhere
(Somlyo & Somlyo 1998). At present there is no direct
evidence in vascular smooth muscle that tyrosine ki-
nases can activate rho, although growth factors activate
rho in other cell types (Nobes et al. 1995). Further
studies are needed to explore whether such an effect
occurs in vascular smooth muscle.
MODULATION BY TYROSINE
PHOSPHORYLATION OF INTERAC-
TIONS BETWEEN THE CYTOSKELETON
AND CONTRACTILE APPARATUS
Another possible route of interaction between tyrosine
phosphorylation and contraction is at the level of the
actin cytoskeleton. Although the cytoskeleton is gen-
erally considered not to actively contribute to force
generation in smooth muscle (Small et al. 1992), it
clearly provides a framework for contractile ®laments
to exert force and connects cells to matrix and other
cells via dense plaques. The role of tyrosine phos-
phorylation in actin organization is well recognized in
many cell types and tyrosine kinases such as FAK and
src kinase are accepted as key components in the
processes of attachment and migration, playing an
important role in focal adhesion formation and dis-
ruption (Hanks & Polte 1997). A number of proteins
found in focal adhesions are also found in dense
plaques (Small 1995) and the dense plaque proteins,
talin and paxillin have been reported to undergo ty-
rosine phosphorylation during contraction of tracheal
smooth muscle (Pavalko et al. 1995). What role, if any,
this process plays in the contractile process remains to
be determined, but it seems plausible that alterations
in dense plaque structure or changes in the interac-
tions between cytoskeleton and contractile ®laments
could contribute to force production. Possibly, such a
process contributes to sustained force production seen
in the presence of low levels of [Ca2+]i and myosin
phosphorylation.
TYROSINE KINASES AND VASCULAR
PATHOLOGY
The role of growth factors in response to injury, and
atherosclerosis is widely recognized (Schwartz et al.
1990, Ross 1993), and will not be discussed here.
However, it is conceivable that tyrosine kinases con-
tribute to other pathological conditions in the vascu-
lature, such as hypertension, which may be associated
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with alterations in excitation±contraction coupling as
well as growth.
At present, relatively few studies have explored this
possibility. Aorta from spontaneously hypertensive rats
(SHR) has been reported to have elevated levels of
mRNA for PDGF a and b receptors (Tanizawa et al.
1996). Sauro and Thomas (1993a) have also reported
that a particulate (membrane) fraction derived from
SHR aorta showed higher levels of PDGF-stimulated
TK activity than normotensive rats and that aorta from
SHR is hyper-responsive to PDGF and PDGF-induced
tone is less sensitive to inhibition by tyrphostin 25. In a
subsequent study on small gracilis arteries contraction
in response to angiotensin II was also shown to be less
sensitive to tyrphostin 25 in SHR vessels (Malloy &
Sauro 1996).
CONCLUSIONS
There is now substantial evidence that tyrosine phos-
phorylation plays a number of roles in excitation±
contraction coupling in vascular smooth muscle. In
view of the ubiquity of tyrosine kinases in cellular sig-
nalling it seems likely that even more roles await dis-
covery. At present there is convincing evidence that
tyrosine kinases play some part in contractile responses
to growth factors and classical vasoconstrictors. The
ability of tyrosine kinases, particularly src kinase, to
in¯uence voltage-operated calcium channels and pos-
sibly K channels is of particular interest given the im-
portance of these channels to excitation±contraction
coupling in vascular smooth muscle. Future studies
should serve to clarify at which other points in the
contractile process tyrosine kinases in¯uence vascular
tone and may help to explain changes in excitation±
contraction coupling in disease states such as hyper-
tension and atherosclerosis.
The authors' work in this ®eld has been supported by grants from the
British Heart Foundation, Medical Research Council and Wellcome
trust.
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