Role of tyrosine phosphorylation in excitation–contraction coupling in vascular smooth muscle

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

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

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


Acta Physiol Scand 1998, 164, 457±469 A D Hughes and S Wijetunge � Role of tyrosine phosphorylation

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.



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



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



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



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



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.

REFERENCES

Abebe, W. & Agrawal, D.K. 1995. Role of tyrosine kinases in

norepinephrine-induced contraction of vascular smooth

muscle. J Cardiovasc Pharmacol 26, 153±159.

Adachi, M., Iwaki, H., Shindoh, M., Akao, Y., Hachiya, T.,

Ikeda, M., Hinoda, Y. & Imai, K. 1997. Predominant

expression of the src homology 2-containing tyrosine

phosphatase protein SHP2 in vascular smooth muscle cells.

Virchows Arch 430, 321±325.

Adam, L.P., Franklin, M.T., Raff, G.J. & Hathaway, D.R.

1995. Activation of mitogen-activated protein kinase in

porcine carotid arteries. Circ Res 76, 183±190.

Akiyama, T., Ishida, J., Nakagawa, S. et al. 1987. Genistein, a

speci®c inhibitor of tyrosine-speci®c protein kinases.

J Biol Chem 262, 5592±5595.

Ali, M.S., Schieffer, B., Delafontaine, P., Bernstein, K.E.,

Ling, B.N. & Marrero, M.B. 1997. Angiotensin II

stimulates tyrosine phosphorylation and activation of

insulin receptor substrate 1 and protein-tyrosine

phosphatase 1D in vascular smooth muscle cells.

J Biol Chem 272, 12373±12379.

Bassett, J.E., Bowen-Pope, D.F., Takayasu, M. & Dacey, R.G.

1988. Platelet-derived growth factor does not constrict rat

intracerebral arterioles in vitro. Microvasc Res 35, 368±373.

Beretz, A., Anton, R. & Stoclet, J.C. 1978. Flavanoid

compounds are potent inhibitors of cyclic AMP

phosphodiesterase. Experientia 34, 1054±1055.

Berk, B.C., Alexander, R.W., Brock, T.A. & Gimbrone Jr.,

M.A. &. Webb, R.C. 1986. Vasoconstriction: a new activity

for platelet-derived growth factor. Science 232, 87±90.

Berk, B.C., Brock, T.A., Webb, R.C., et al. 1985. Epidermal

growth factor, a vascular smooth muscle mitogen, induces

rat aortic contraction. J Clin Invest 75, 1083±1086.

Birukov, K.G., Lehoux, S., Birukova, A.A., Merval, R.,

Tkachuk, V.A. & Tedgui, A. 1997. Increased pressure

induces sustained protein kinase C-independent herbimycin

A-sensitive activation of extracellular signal-related kinase 1/

2 in the rabbit aorta in organ culture. Circ Res 81, 895±903.

Block, L.H., Emmons, L.R., Vogt, E., Sachinidis, A., Vetter,

W. & Hoppe, J. 1989. Ca2+-channel blockers inhibit the

action of recombinant platelet- derived growth factor in

vascular smooth muscle cells. Proc Natl Acad Sci USA 86,

2388±2392.

Bobik, A. & Campbell, J.H. 1993. Vascular derived growth

factors: cell biology, pathophysiology, and pharmacology.

Pharmacol Rev 45, 1±42.

Bolton, T.B. 1979. Mechanisms of action of transmitters and

other substances on smooth muscle. Physiol Rev 59, 606±

718.

Borges, L.G., Seifert, R.A., Grant, F.J., et al. 1996. Cloning

and characterization of rat density-enhanced phosphatase-

1, a protein tyrosine phosphatase expressed by vascular

cells. Circ Res 79, 570±580.

Bornfeldt, K.E., Raines, E.W., Graves, L.M., Skinner, M.P.,

Krebs, E.G. & Ross, R. 1995. Platelet-derived growth

factor. Distinct signal transduction pathways associated

with migration versus proliferation. Ann N Y Acad Sci 766,

416±430.

Brunton, V.G., Carlin, S. & Workman, P. 1994. Alterations in

EGF-dependent proliferative and phosphorylation events

in squamous cell carcinoma cell lines by a tyrosine kinase

inhibitor. Anticancer Drug Des 9, 311±329.

Burger, A.M., Kaur, G., Alley, M.C. et al. 1995. Tyrphostin

AG17 [3,5-ditert-butyl-4-hydroxybenzylidene)-

malononitrile], inhibits cell growth by disrupting

mitochondria. Cancer Res 55, 2794±2799.

Childs, T.J. & Mak, A.S. 1993. Smooth-muscle mitogen-

activated protein (MAP) kinase: puri®cation and

characterization, and the phosphorylation of caldesmon.

Biochem J 296, 745±751.

Clark, E.A. & Brugge, J.S. 1995. Integrins and signal

transduction pathways: the road taken. Science 268, 233±239.

Collett, M.S. & Erikson, R.L. 1978. Protein kinase activity

associated with the avian sarcoma virus src gene product.

Proc Natl Acad Sci USA 75, 2021±2024.



Coroneos, E.J., Kester, M., Maclouf, J., Thomas, P. & Dunn,

M.J. 1997. Calcium-regulated protein tyrosine

phosphorylation is required for endothelin-1 to induce

prostaglandin endoperoxide synthase-2 mRNA expression

and protein synthesis in mesangial cells. J Am Soc Nephrol 8,

1080±1090.

Courtneidge, S.A. 1994. Protein tyrosine kinases, with

emphasis on the Src family. Semin Cancer Biol 5, 239±246.

Daum, G., Regenass, S., Sap, J., Schlessinger, J. & Fischer,

E.H. 1994. Multiple forms of the human tyrosine

phosphatase RPTP alpha. Isozymes and differences in

glycosylation. J Biol Chem 269, 10524±10528.

Di Salvo, J., Gifford, D. & Kokkinakis, A. 1988. pp. 60c-src

kinase activity in bovine coronary extracts is stimulated by

ATP. Biochem Biophys Res Commun 153, 388±394.

Di Salvo, J., Gifford, D. & Kokkinakis, A. 1989. ATP- and

polyphosphate-mediated stimulation of pp. 60c-src kinase

activity in extracts from vascular smooth muscle. J Biol

Chem 264, 10773±10778.

Di Salvo, J., Nelson, S.R. & Kaplan, N. 1997. Protein tyrosine

phosphorylation in smooth muscle: a potential coupling

mechanism between receptor activation and intracellular

calcium. Proc Soc Exp Biol Med 214, 285±301.

Di Salvo, J., Semenchuk, L.A. & Lauer, J. 1993a. Vanadate-

induced contraction of smooth muscle and enhanced

protein tyrosine phosphorylation. Arch Biochem Biophys 304,

386±391.

Di Salvo, J., Steusloff, A., Semenchuk, L., Satoh, S., Kolquist,

K. & P®tzer, G. 1993b. Tyrosine kinase inhibitors suppress

agonist-induced contraction in smooth muscle. Biochem

Biophys Res Commun 190, 968±974.

Duarte, J., Ocete, M.A., Perez, V.F., Zarzuelo, A. & Tamargo,

J. 1997. Effect of tyrosine kinase and tyrosine phosphatase

inhibitors on aortic contraction and induction of nitric

oxide synthase. Eur J Pharmacol 338, 25±33.

Elberg, G., Li, J., Leibovitch, A. & Shechter, Y. 1995. Non-

receptor cytosolic protein tyrosine kinases from various rat

tissues. Biochim Biophys Acta 1269, 299±306.

Epstein, A.M., Throckmorton, D. & Brophy, C.M. 1997.

Mitogen-activated protein kinase activation: an alternate

signaling pathway for sustained vascular smooth muscle

contraction. J Vasc Surg 26, 327±332.

Erdmann, E., Werdan, K., Krawietz, W., Schmitz, W. &

Scholz, H. 1984. Vanadate and its signi®cance in

biochemistry and pharmacology. Biochem Pharmacol 33, 945±

950.

Erlinge, D., Heilig, M. & Edvinsson, L. 1996. Tyrphostin

inhibition of ATP-stimulated DNA synthesis, cell

proliferation and fos-protein expression in vascular smooth

muscle cells. Br J Pharmacol 118, 1028±1034.

Filipeanu, C.M., Brailoiu, E., Huhurez, G., Slatineanu, S.,

Baltatu, O. & Branisteanu, D.D. 1995. Multiple effects of

tyrosine kinase inhibitors on vascular smooth muscle

contraction. Eur J Pharmacol 281, 29±35.

Floege, J., Hudkins, K.L., Seifert, R.A., Francki, A., Bowen,

P.D. & Alpers, C.E. 1997. Localization of PDGF alpha-

receptor in the developing and mature human kidney.

Kidney Int 51, 1140±1150.

Florian, J.A. & Watts, S.W. 1998. Integration of mitogen-

activated protein kinase kinase activation in vascular 5-

hydroxytryptamine2A receptor signal transduction.

J Pharmacol Exp Ther 284, 346±355.

Gan, B.S. & Hollenberg, M.D. 1990. Distinct coronary artery

receptor systems for epidermal growth factor-urogastrone.

J Pharmacol Exp Ther 252, 1277±1282.

Garcha, R.S., Sever, P.S. & Hughes, A.D. 1998. Role of Ca2+

channels, tyrosine kinase and MAP/ERK kinase kinase

(MEK) in angiotensin II-induced contraction in human

isolated resistance arteries. J Vasc Res 35(suppl 1)

Glenney Jr., J. 1992. Tyrosine-phosphorylated proteins:

mediators of signal transduction from the tyrosine kinases.

Biochim Biophys Acta 1134, 113±127.

Gould, E.M., Rembold, C.M. & Murphy, R.A. 1995.

Genistein, a tyrosine kinase inhibitor, reduces Ca2+

mobilization in swine carotid media. Am J Physiol 268,

C1425±C1429.

Hamm, H.E. 1998. The many faces of G protein signaling.

J Biol Chem 273, 669±672.

Hanks, S.K. & Polte, T.R. 1997. Signaling through focal

adhesion kinase. Bioessays 19, 137±145.

Hollenberg, M.D. 1994. Tyrosine kinase pathways and the

regulation of smooth muscle contractility. Trends Pharmacol

Sci 15, 108±114.

Horowitz, A., Menice, C.B., Laporte, R. & Morgan, K.G.

1996. Mechanisms of smooth muscle contraction. Physiol

Rev 76, 967±1003.

Hsu, C.Y., Persons, P.E., Spada, A.P., Bednar, R. A., Levitzki,

A. & Zilberstein, A. 1991. Kinetic analysis of the inhibition

of the epidermal growth factor receptor tyrosine kinase by

Lavendustin-A and its analogue. J Biol Chem 266, 21105±

21112.

Hu, X.Q., Singh, N., Mukhopadhyay, D. & Akbarali, H.I.

1998. Modulation of voltage-dependent Ca2+ channels in

rabbit colonic smooth muscle cells by c-Src and focal

adhesion kinase. J Biol Chem 273, 5337±5342.

Huckle, W.R., Dy, R.C. & Earp, H.S. 1992. Calcium-

dependent increase in tyrosine kinase activity stimulated by

angiotensin II. Proc Natl Acad Sci USA 89, 8837±8841.

Hughes, A.D. 1995a. Calcium channels in vascular smooth

muscle cells. J Vasc Res 32, 353±370.

Hughes, A.D. 1995b. Increase in tone and intracellular Ca2+

in rabbit isolated ear artery by platelet-derived growth

factor. Br J Pharmacol 114, 138±142.

Hunter, T. 1995. Protein kinases and phosphatases: the yin

and yang of protein phosphorylation and signaling. Cell 80,

225±236.

Hunter, T. & Sefton, B.M. 1980. Transforming gene product

of Rous sarcoma virus phosphorylates tyrosine. Proc Natl

Acad Sci USA 77, 1311±1315.

Inoue, R., Waniishi, Y., Yamada, K. & Ito, Y. 1994. A

possible role of tyrosine kinases in the regulation of

muscarinic receptor-activated cation channels in guinea pig

ileum. Biochem Biophys Res Commun 203, 1392±1397.

Jayaraman, T., Ondrias, K., Ondriasova, E. & Marks, A.R.

1996. Regulation of the inositol 1,4,5-trisphosphate

receptor by tyrosine phosphorylation. Science 272, 1492±

1494.

Jerius, H., Beall, A., Woodrum, D., Epstein, A. & Brophy, C.

1998. Thrombin-induced vasospasm: cellular signaling

mechanisms. Surgery 123, 46±50.



Jin, N., Siddiqui, R.A., English, D. & Rhoades, R. A. 1996.

Communication between tyrosine kinase pathway and

myosin light chain kinase pathway in smooth muscle.

Am J Physiol 271, H1348±H1355.

Jinsi, A. & Deth, R.C. 1995. a2-adrenoceptor-mediated

vasoconstriction requires a tyrosine kinase. Eur J Pharmacol

277, 29±34.

Jinsi, A., Paradise, J. & Deth, R.C. 1996. A tyrosine kinase

regulates alpha-adrenoceptor-stimulated contraction and

phospholipase D activation in the rat aorta. Eur J Pharmacol

302, 183±190.

Khalil, R.A., Menice, C.B., Wang, C.-L.A. & Morgan, K.G.

1995. Phosphotyrosine-dependent targeting of mitogen-

activated protein kinase in differentiated contractile

vascular cells. Circ Res 76, 1101±1108.

Khalil, R.A. & Morgan, K.G. 1993. PKC-mediated

redistribution of mitogen-activated protein kinase during

smooth muscle cell activation. Am J Physiol 265, C406±

C411.

Kitazono, T., Ibayashi, S., Nagao, T., Kagiyama, T., Kitayama,

J. & Fujishima, M. 1998. Role of tyrosine kinase in

serotonin-induced constriction of the basilar artery in vivo.

Stroke 29, 494±497.

Klohs, W.D., Fry, D.W. & Kraker, A.J. 1997. Inhibitors of

tyrosine kinase. Curr Opin Oncol 9, 562±568.

Lai, K., Wang, H., Lee, W.S., Jain, M.K., Lee, M.E. & Haber,

E. 1996. Mitogen-activated protein kinase phosphatase-1 in

rat arterial smooth muscle cell proliferation. J Clin Invest 98,

1560±1567.

Laniyonu, A., Eto, S., Wang, J.H. & Hollenberg, M.D. 1995.

Detection of sarcoma virus family tyrosine kinase activity in

coronary arterial tissue. Can J Physiol Pharmacol 73, 1552±

1560.

Laniyonu, A., Saieddine, M., Ahmad, S. & Hollenberg, M.D.

1994a. Regulation of vascular and gastric smooth muscle

contractility by pervanadate. Br J Pharmacol 113, 403±410.

Laniyonu, A.A., Saifeddine, M., Yang, S.-G. & Hollenberg,

M.D. 1994b. Tyrosine kinase inhibitors and the contractile

action of G protein-linked vascular agonists. Can J Physiol

Pharmacol 72, 1075±1085.

Large, W.A. & Wang, Q. 1996. Characteristics and

physiological role of the Ca 2�± activated Cl± conductance

in smooth muscle. Am J Physiol 271, C435±C454.

Levitzki, A. & Gazit, A. 1995. Tyrosine kinase inhibition: an

approach to drug development. Science 267, 1782±1788.

Liu, H., Li, K. & Sperelakis, N. 1997a. Tyrosine kinase

inhibitor, genistein, inhibits macroscopic L-type calcium

current in rat portal vein smooth muscle cells. Can J Physiol

Pharmacol 75, 1058±1062.

Liu, H. & Sperelakis, N. 1997b. Tyrosine kinases modulate the

activity of single L-type calcium channels in vascular

smooth muscle cells from rat portal vein. Can J Physiol

Pharmacol 75, 1063±1068.

Liu, C.Y. & Sturek, M. 1996. Attenuation of endothelin-1-

induced calcium response by tyrosine kinase inhibitors in

vascular smooth muscle cells. Am J Physiol 270, C1825±

C1833.

Low, A.M. 1996. Role of tyrosine kinase on Ca2+ entry and

re®lling of agonist-sensitive Ca2+ stores in vascular

smooth muscles. Can J Physiol Pharmacol 74, 298±304.

Luttrell, L.M., Della, R.G., van, B.T., Luttrell, D.K. &

Lefkowitz, R.J. 1997. G betagamma subunits mediate Src-

dependent phosphorylation of the epidermal growth factor

receptor. A scaffold for G protein-coupled receptor-

mediated Ras activation. J Biol Chem 272, 4637±4644.

Lyall, R.M., Zilberstein, A., Gazit, A., Gilon, C., Levitzki, A. &

Schlessinger, J. 1989. Tyrphostins inhibit epidermal growth

factor (EGF)-receptor tyrosine kinase activity in living cells

and EGF-stimulated cell proliferation. J Biol Chem 264,

14503±14509.

Malloy, L.G. & Sauro, M.D. 1996. Tyrosine kinase inhibition

suppresses angiotensin contraction in hypertensive and

normotensive small resistance arteries. Life Sci 58, L317±

L324.

Marrero, M.B., Paxton, W.G., Duff, J.L., Berk, B.C. &

Bernstein, K.E. 1994. Angiotensin II stimulates tyrosine

phosphorylation of phospholipase C-gamma 1 in vascular

smooth muscle cells. J Biol Chem 269, 10935±10939.

Marrero, M.B., Schieffer, B., Paxron, W.G. et al. 1995. Direct

stimulation of Jak/STAT pathway by the angiotensin II

AT1 receptor. Nature 375, 247±250.

Masumoto, N., Nakayama, K., Oyabe, A. et al. 1997. Speci®c

attenuation of the pressure-induced contraction of rat

cerebral artery by herbimycin A. Eur J Pharmacol 330, 55±63.

Merkel, L.A., Rivera, L.M., Colussi, D.J. & Perrone, M.H.

1993. Inhibition of EGF-induced vasoconstriction in

isolated rabbit aortic rings with the tyrosine kinase inhibitor

RG50864. Biochem Biophys Res Commun 192, 1319±1326.

Minami, K., Fukuzawa, K. & Inoue, I. 1994. Regulation of a

non-selective cation channel of cultured porcine coronary

artery smooth muscle cells by tyrosine kinase. P¯ugers Arch

426, 254±257.

Morgan, K.G., DeFeo, T.T., Wenc, K. & Weinstein, R. 1985.

Alterations of excitation-contraction coupling by platelet-

derived growth factor in enzymatically isolated and cultured

vascular smooth muscle cells. P¯ugers Arch 405, 77±79.

Muramatsu, I., Hollenberg, M.D. & Lederis, K. 1985.

Vascular actions of epidermal growth factor-urogastrone:

possible relationship to prostaglandin production.

Can J Physiol Pharmacol 63, 994±999.

Nakanishi, S., Kakita, S., Takahashi, I. et al. 1992.

Wortmannin, a microbial product inhibitor of myosin light

chain kinase. J Biol Chem 267, 2157±2163.

Neel, B.G. & Tonks, N.K. 1997. Protein tyrosine

phosphatases in signal transduction. Curr Opin Cell Biol 9,

193±204.

Neer, E.J. 1994. G proteins: critical control points for

transmembrane signals. Protein Sci 3, 3±14.

Nelson, M.T. & Quayle, J.M. 1995. Physiological roles and

properties of potassium channels in arterial smooth muscle.

Am J Physiol 268, C799±C822.

Nilsson, H., Jensen, P.E. & Mulvany, M.J. 1994. Minor role

for direct adrenoceptor-mediated calcium entry in rat

mesenteric small arteries. J Vasc Res 31, 314±321.

Nobes, C., Hawkins, P.T., Stephens, L. & Hall, A. 1995.

Activation of the small GTP-binding proteins rho and rac

by growth factor receptors. J Cell Sci 108, 225±233.

O'Dowd, B.F., Lefkowitz, R.J. & Caron, M.G. 1989. Structure

of the adrenergic and related receptors. Annu Rev Neurosci

12, 67±83.



Ogata, R., Kitamura, K., Ito, Y. & Nakano, H. 1997.

Inhibitory effects of genistein on ATP-sensitive K+

channels in rabbit portal vein smooth muscle. Br J

Pharmacol 122, 1395±1404.

Ohanian, J., Ohanian, V., Shaw, L., Bruce, C. & Heagerty,

A.M. 1997. Involvement of tyrosine phosphorylation in

endothelin-1-induced calcium-sensitization in rat small

mesenteric arteries. Br J Pharmacol 120, 653±661.

Osol, G., Laher, I. & Kelley, M. 1993. Myogenic tone is

coupled to phospholipase C and G protein activation in

small cerebral arteries. Am J Physiol 265, H415±H420.

Ozaki, H. & Urakawa, N. 1980. Effects of vanadate or

mechanical responses and Na-K pump in vascular smooth

muscle. Eur J Pharmacol 68, 339±347.

Ozaki, H., Satoh, T., Karaki, H. & Ishida, Y. 1988. Regulation

of metabolism and contraction by cytoplasmic calcium in

the intestinal smooth muscle. J Biol Chem 263, 14074±14079.

Patarca, R. 1996. Protein phosphorylation and

dephosphorylation in physiologic and oncologic processes.

Crit Rev Oncog 7, 343±432.

Pavalko, F.M., Adam, L.P., Wu, M.F., Walker, T.L. & Gunst,

S.J. 1995. Phosphorylation of dense-plaque proteins talin

and paxillin during tracheal smooth muscle contraction.


Posner, B.I., Faure, R., Burgess, J.W. et al. 1994.

Peroxovanadium compounds. A new class of potent

phosphotyrosine phosphatase inhibitors which are insulin

mimetics. J Biol Chem 269, 4596±4604.

Ramdas, L., McMurray, J.S. & Budde, R.J. 1994. The degree

of inhibition of protein tyrosine kinase activity by

tyrphostin 23 and 25 is related to their instability. Cancer Res

54, 867±869.

Rembold, C.M. & Weaver, B.A. 1997. Tyrosine

phosphorylation and regulation of swine carotid artery

contraction. J Vasc Res 34, 1±10.

Robinson, M.J. & Cobb, M.H. 1997. Mitogen-activated

protein kinase pathways. Curr Opin Cell Biol 9, 180±186.

Ross, R. 1993. The pathogenesis of atherosclerosis: a

perspective for the 1990s. Nature 362, 801±809.

Rubin, K., Tingstrom, A., Hansson, G.K. et al. 1988.

Induction of B-type receptors for platelet-derived growth

factor in vascular in¯ammation: possible implications for

development of vascular proliferative lesions. Lancet 1,

1353±1356.

Sachinidis, A., Locher, R., Hoppe, J. & Vetter, W. 1990. The

platelet-derived growth factor isomers, PDGF-AA, PDGF-

AB and PDGF-BB, induce contraction of vascular smooth

muscle cells by different intracellular mechanisms. FEBS

Lett 275, 95±98.

Saifeddine, M., Laniyonu, A., Ahmad, S. & Hollenberg, M.D.

1994. Bi-directional control of smooth muscle tension:

regulation by tyrosine kinase and tyrosine phosphatase. Proc

West Pharmacol Soc 37, 21±24.

Sanchez Ferrer, C.F., Marin, J., Lluch, M., Valverde, A. &

Salaices, M. 1988. Actions of vanadate on vascular tension

and sodium pump activity in cat isolated cerebral and

femoral arteries. Br J Pharmacol 93, 53±60.

Sarzani, R., Arnaldi, G. & Chobanian, A.V. 1991.

Hypertension-induced changes of platelet-derived growth

factor receptor expression in rat aorta and heart.

Hypertension 17, 888±895.

Satoh, S., Rensland, H. & P®tzer, G. 1993. Ras proteins

increase Ca (2+)-responsiveness of smooth muscle

contraction. FEBS Lett 324, 211±215.

Sauro, M.D. & Thomas, B. 1993a. Decreased sensitivity of

aorta from hypertensive rats to vasorelaxation by

tyrphostin. Life Sci 53, L371±L376.

Sauro, M.D. & Thomas, B. 1993b. Tyrphostin attenuates

platelet-derived growth factor-induced contraction in aortic

smooth muscle through inhibition of protein tyrosine

kinase (s). J Pharmacol Exp Ther 267, 1119±1125.

Sauro, M.D., Sudakow, R., Burns, S., 1996. In vivo effects of

angiotensin II on vascular smooth muscle contraction and

blood pressure are mediated through a protein tyrosine-

kinase-dependent mechanism. J Pharmacol Exp Ther 277,

1744±1750.

Schieffer, B., Drexler, H., Ling, B.N. & Marrero, M.B. 1997.

G protein-coupled receptors control vascular smooth

muscle cell proliferation via pp. 60c-src and p21ras.


Schlessinger, J. & Ullrich, A. 1992. Growth factor signalling

by receptor tyrosine kinases. Neuron 9, 383±391.

Schwartz, S.M., Heimark, R.L. & Majesky, M.W. 1990.

Developmental mechanisms underlying the pathology of

arteries. Physiol Rev 70, 1177±1209.

Shimada, T., Shimamura, K. & Sunano, S. 1986. Effects of

sodium vanadate on various types of vascular smooth

muscles. Blood Vess 23, 113±124.

Small, J.V. 1995. Structure-function relationships in smooth

muscle: the missing links. Bioessays 17, 785±792.

Small, J.V., Furst, D.O. & Thornell, L.E. 1992. The

cytoskeletal lattice of muscle cells. Eur J Biochem 208, 559±

572.

Smirnov, S.V. & Aaronson, P.I. 1995. Inhibition of vascular

smooth muscle cell K+ currents by tyrosine kinase

inhibitors genistein and ST 638. Circ Res 76, 310±316.

Somlyo, A.P. & Somlyo, A.V. 1994. Signal transduction and

regulation in smooth muscle. Nature 372, 231±236.

Somlyo, A.P. & Somlyo, A.V. 1998. From

pharmacomechanical coupling through G-proteins to

myosin phosphatase. Acta Physiol Scand 164, 437±448.

Steusloff, A., Paul, E., Semenchuk, L.A., Di Salvo, J. &

P®tzer, G. 1995. Modulation of Ca2+ sensitivity in smooth

muscle by genistein and protein tyrosine phosphorylation.

Arch Biochem Biophys 320, 236±242.

Swarup, G., Cohen, S. & Garbers, D.L. 1982. Inhibition of

membrane phosphotyrosyl-protein phosphatase activity

by vanadate. Biochem Biophys Res Commun 107, 1104±

1109.

Tanizawa, S., Ueda, M., van-der, L.C., van-der, W.A. &

Becker, A.E. 1996. Expression of platelet derived growth

factor B chain and beta receptor in human coronary arteries

after percutaneous transluminal coronary angioplasty: an

immunohistochemical study. Heart 75, 549±556.

Tay, U.J., Poon, M.C., Ahmad, S. & Hollenberg, M.D. 1995.

Contractile actions of thrombin receptor-derived polypeptides

in human umbilical and placental vasculature: evidence for

distinct receptor systems. Br J Pharmacol 115, 569±578.



Thomas, S.M. & Brugge, J.S. 1997. Cellular functions

regulated by Src family kinases. Ann Rev Cell Dev Biol 13,

513±609.

Toma, C., Jensen, P.E., Prieto, D., Hughes, A.D. & Aalkjaer,

C. 1995. Effects of tyrosine kinase inhibitors on the

contractility of rat mesenteric resistance arteries.

Br J Pharmacol 114, 1266±1272.

Touyz, R.M. & Schiffrin, E.L. 1997. Angiotensin II regulates

vascular smooth muscle cell pH, contraction, and growth

via tyrosine kinase-dependent signaling pathways.

Hypertension 30, 222±229.

Ui, M., Okada, K. & Hazeki, O. 1995. Wortmannin as a

unique probe for an intracellular signalling protein,

phosphoinositide 3-kinase. T I B S 20, 303±307.

Van Breemen, C. & Saida, K. 1989. Cellular mechanisms

regulating Ca2+ in smooth muscle. Annu Rev Physiol 51,

315±329.

Waen-Safranchik, V.I. & Deth, R.C. 1994. Effects of

wortmannin on alpha-1/alpha-2 adrenergic receptor-

mediated contractile responses in rabbit vascular tissues.

Pharmacology 48, 349±359.

Ward, D.T., Ohanian, J., Heagerty, A.M. & Ohanian, V. 1995.

Phospholipase D-induced phosphatidate production in

intact small arteries during noradrenaline stimulation:

involvement of both G-protein and tyrosine-

phosphorylation-linked pathways. Biochem J 307, 451±456.

Watts, S.W. 1996a. Serotonin activates the mitogen-activated

protein kinase pathway in vascular smooth muscle: use of

the mitogen-activated protein kinase kinase inhibitor

PD098059. J Pharmacol Exp Ther 279, 1541±1550.

Watts, S.W., Yeum, C.H., Campbell, G. & Webb, R.C. 1996b.

Serotonin stimulates protein tyrosyl phosphorylation and

vascular contraction via tyrosine kinase. J Vasc Res 33, 288±

298.

Wijetunge, S., Aalkjaer, C., Schachter, M. & Hughes, A.D.

1992. Tyrosine kinase inhibitors block calcium channel

currents in vascular smooth muscle cells. Biochem Biophys Res

Commun 189, 1620±1623.

Wijetunge, S. & Hughes, A.D. 1995a. Effect of platelet-

derived growth factor on voltage-operated calcium

channels in isolated rabbit ear artery cells. Br J Pharmacol

115, 534±538.

Wijetunge, S. & Hughes, A.D. 1995b. pp60src increases

voltage-operated calcium channel currents in vascular

smooth muscle cells. Biochem Biophys Res Commun 217,

1039±1044.

Wijetunge, S. & Hughes, A.D. 1996. Activation of

endogenous c-Src or a related tyrosine kinase by

intracellular (pY) EEI peptide increases voltage-operated

calcium channel currents in rabbit ear artery cells. FEBS

Lett 399, 63±66.

Wijetunge, S., Lymn, J.S. & Hughes, A.D. 1998. Effect of

inhibition of tyrosine phosphatases on voltage-operated

calcium channel currents in rabbit ear artery cells. Br J

Pharmacol 124, 307±316

Wilcox, J.N., Smith, K.M., Williams, L.T., Schwartz, S. M. &

Gordon, D. 1988. Platelet-derived growth factor mRNA

detection in human atherosclerotic plaques by in situ

hybridization. J Clin Invest 82, 1134±1143.

Wolbring, G., Hollenberg, M.D. & Schnetkamp, P. P. 1994.

Inhibition of GTP-utilizing enzymes by tyrphostins. J Biol

Chem 269, 22470±22472.

Xiong, Z., Burnette, E. & Cheung, D.W. 1995. Modulation of

Ca2+-activated K+ channel activity by tyrosine kinase

inhibitors in vascular smooth muscle cell. Eur J Pharmacol

290, 117±123.

Young, S.W., Poole, R.C., Hudson, A.T., Halestrap, A.P.,

Denton, R.M. & Tavare, J.M. 1993. Effects of tyrosine

kinase inhibitors on protein kinase-independent systems

[published erratum appears in FEBS Lett 22 March 1993;

319 (3), 292]. FEBS Lett 316, 278±282.

Zhou, Q., Satake, N., & Shibata, S. 1997. The contractile

mechanism of sodium metavanadate in isolated rat aortae.

J Cardiovasc Pharmacol 30, 84±89.



Role of tyrosine phosphorylation in excitation–contraction coupling in vascular smooth muscle

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