0022-3565/97/2831-0131$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 283:131–137, 1997
Vol. 283, No. 1
Printed in U.S.A.
Effects of Tyrosine Kinase Inhibitors on Antigen Challenge of
Guinea Pig Lung In Vitro1
W. S. FRED WONG, DIANA S. K. KOH, ALAN H. M. KOH, W. L. TING and PETER T. H. WONG
Department of Pharmacology, Faculty of Medicine, National University of Singapore, Republic of Singapore
Accepted for publication June 2, 1997
Antigen-stimulated mast cell degranulation is known to be
associated with increased intracellular Ca11 concentration
and protein kinase C activity (Beaven and Metzger, 1993;
Ozawa et al., 1993). Recently, cumulating evidence obtained
from rat basophilic mast cell line (RBL-2H3) and bone marrow-derived mast cells showed that activation of non-transmembrane PTKs is the earliest detectable signaling response
to FceRI cross-linking. This is followed by downstream signaling events such as activation of PLCg (Li et al., 1992;
Jouvin et al., 1994) and mitogen-activated protein kinase
(Fukamachi et al., 1993), increase in inositol 1,4,5-trisphosphate and intracellular Ca11 levels and enhanced protein
kinase C activity, and it eventually leads to mast cell degranulation (Beaven and Metzger, 1993; Scharenberg and Kinet,
1994). Specific tyrosine kinases such as src-related kinase
Lyn (Eiseman and Bolen, 1992), 72-kDa Syk (Hutchcroft et
al., 1992; Benhamou et al., 1993), 94-kDa Fer (Penhallow et
al., 1995) and 77-kDa Btk (Kawakami et al., 1994) have been
shown to be activated rapidly after FceRI aggregation. Inhibitors of PTK have been shown to block antigen-induced activation of PTK, related downstream signaling events (e.g.,
Received for publication April 3, 1997.
1
This work was supported by grant NMRC/0169/1996 of the National
Medical Research Council of Singapore (W.S.F.W.).
genistein, did not alter OA-induced anaphylactic contraction.
However, it slightly reduced bronchial contraction to leukotriene D4 and the OA-stimulated release of peptidoleukotrienes.
The inhibitory effects were significantly weaker than those of
genistein. Taken together, our results show that tyrphostin 47
inhibited anaphylactic contraction mainly by preventing mast
cell degranulation, whereas genistein exerted inhibitory effects
partly by blocking mast cell degranulation and partly by attenuating leukotriene D4-induced bronchial contraction. These
findings suggest that protein tyrosine kinase inhibitors have a
therapeutic potential as mast cell stabilizers in the treatment of
allergic diseases such as bronchial asthma.
inositol 1,4,5-trisphosphate production) and histamine release from mast cells (Kawakami et al., 1992; Lavens et al.,
1992; Oliver et al., 1994). Because mast cell degranulation is
the hallmark of immediate-type hypersensitivity reaction,
which is also the major mechanism for a variety of allergic
diseases such as bronchial asthma, it is logical to examine
the effects of PTK inhibitors on an in vitro model of allergic
asthma.
The Schultz-Dale reaction (Schultz, 1910; Dale, 1913;
Chand and Eyre, 1978) has been used extensively to study
anaphylactic contraction of airway tissue preparations such
as trachea, bronchi and lung parenchymal strips. Among a
wide array of mast cell-derived inflammatory mediators,
such as thromboxane A2 and platelet-activating factor, peptidoleukotrienes and histamine have been shown to be the
major mediators responsible for the anaphylactic contraction
of the airways (Ro et al., 1991; Bjorck and Dahlen, 1993;
Jonsson and Dahlen, 1994). A combination of histamine (H1)
receptor antagonist and peptidoleukotriene receptor antagonist has been shown to block substantially the anaphylactic
contraction of airway tissue preparations (e.g., bronchi and
lung parenchymal strips) from both human and guinea pig
(Regal, 1985; Bjorck and Dahlen, 1993; Jonsson and Dahlen,
1994). In guinea pigs, both IgE and IgG are able to sensitize
mast cells to specific antigen (Regal, 1985; Undem et al.,
ABBREVIATIONS: OA, ovalbumin; LTD4, leukotriene D4; HNMT, histamine N-methyltransferase; PTK, protein tyrosine kinase; FceRI, high affinity
IgE-binding Fc receptor; FcgR, IgG-binding Fc receptor; [3H]-t-MHm, tritiated N-t-methylhistamine; PLCg, phospholipase Cg.
131
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
ABSTRACT
The present study was conducted to examine the effects of two
protein tyrosine kinase inhibitors, genistein and tyrphostin 47,
on an in vitro model of allergic asthma. Guinea pigs were
sensitized with purified IgG raised against ovalbumin (OA). Isolated sensitized bronchial rings contracted in response to OA in
a concentration-dependent manner, maximum contraction being achieved at 1 mg/ml. Genistein and tyrphostin 47 concentration-dependently (10 –100 mM) inhibited OA-induced anaphylactic contraction of the bronchi, as well as release of
histamine and peptidoleukotrienes from chopped lung preparations. Genistein, but not tyrphostin 47, significantly suppressed bronchial contraction to leukotriene D4 at 50 mM and to
histamine at 100 mM. Daidzein, an inactive congener of
132
Wong et al.
Materials and Methods
Drugs. The following drugs and chemicals were used in this
study: OA (grade V), histamine dihydrochloride, indomethacin, tyrphostin 47 (RG50864), L-cysteine, bovine serum albumin (Sigma
Chemical Co., St. Louis, MO), toluene, isoamyl alcohol, boric acid,
potassium phosphate, dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany), rabbit IgG fraction to chicken egg albumin (OA)
(Organon Teknicka Corp., Durham, NC), genistein and daidzein
(Research Biochemicals International, Natick, MA), LTD4 (Cayman
Chemical Co., Ann Arbor, MI), tritiated S-adenosyl-L-[methyl3
H]methionine (60–85 Ci/mmol), leukotriene C4/D4/E4 radioimmunoassay kit and biodegradable liquid scintillant (Amersham Life Science, Buckinghamshire, U.K.) and HNMT (New England Nuclear,
Boston, MA). Rabbit anti-OA IgG antibody was stored in sterile H2O,
and genistein, tyrphostin 47 and daidzein in DMSO at 220°C. Indomethacin was dissolved in 4.2% (g/ml) NaHCO3 stock solution. OA
and histamine were prepared fresh in deionized H2O. All other
reagents were of analytical grade and were dissolved in deionized
H2O.
Sensitization procedures. Guinea pigs were passively sensitized by a single i.p. injection of 1 mg/kg rabbit IgG antibody against
OA (Wong et al., 1992a). The animals were killed 2 days after
injection.
Preparation of bronchial rings. Male Hartley guinea pigs (Interfauna, U.K. Ltd. England) weighing 350 to 450 g were sacrificed
by CO2 asphyxiation and subsequent decapitation. After thoracotomy, heart and lung were excised en bloc and perfused with 50 ml of
Krebs-bicarbonate solution via the pulmonary artery. Lung lobes
were isolated for studies on the release of histamine and peptidoleukotrienes (see below). Bronchial rings (;3 mm in length) were obtained from the hilar bronchi and cleaned of any parenchyma. Ring
preparations were then suspended isometrically under an optimum
resting load of 2 g in organ baths containing 10 ml of Krebs-bicarbonate solution aerated with 95% O2 and 5% CO2 at 37°C and of the
following composition (mM): NaCl, 118.2; KCl, 4.6; NaHCO3, 24.8;
CaCl2 z 2H2O, 2.5; KH2PO4, 1.2; MgSO4 z 7H2O, 1.2 and dextrose,
10.0. Contractile responses were monitored using force-displacement
transducers (Grass FT-03) coupled to a MacLab/8 data-recording
system (ADInstruments, Castle Hill, Australia). A 90-min equilibration period was allowed before any experimentation was begun, and
during this time the bath fluid was changed every 10 min.
Contractile studies. After equilibration, bronchial rings were
contracted with 60 mM KCl. The contraction was defined as the
maximum tissue response, and all subsequent contractions were
compared to it. For antigen challenge studies, ring preparations were
preincubated with indomethacin (4 mM) for 30 min before addition of
OA. Indomethacin has been shown to reduce the production of relaxant prostanoids (e.g., PGE2 and PGI2) capable of modulating the
anaphylactic contraction (Abela and Daniel, 1994; Bertrand et al.,
1991; Ro et al., 1991). To determine maximum antigen-induced contraction, bronchial rings were exposed to increasing concentrations
of OA (0.001–10 mg/ml). To evaluate the role of PTK in mediating
bronchial smooth muscle anaphylactic contraction, PTK inhibitors
such as genistein, tyrphostin 47 and daidzein, a structural analog of
genistein devoid of tyrosine kinase inhibitory activity (Akiyama et
al., 1987), were preincubated with bronchial rings 30 min before
addition of OA. The effects of PTK inhibitors were compared to those
of their corresponding control ring preparations in the absence of
inhibitor. To confirm that the inhibitory effects of PTK inhibitors on
anaphylactic contraction are mediated by mast cell stabilization, we
examined the effects of genistein and tyrphostin 47 on histamine- or
LTD4-induced bronchial contraction. In the LTD4-induced bronchial
ring contraction study, 4 mM indomethacin and 5 mM L-cysteine
were preincubated with the tissue for 30 min. It has been reported
that very minute amounts of relaxant prostanoids (e.g., PGE2 and
PGI2) were able to mask LTD4-induced canine bronchial contraction
and that the addition of indomethacin restored the LTD4 effect
(Abela and Daniel, 1994). L-cysteine is an inhibitor of an aminopeptidase that converts LTD4 to less potent LTE4. Adding L-cysteine has
been shown to enhance the smooth muscle contractile response to
LTD4 (Abela and Daniel, 1994).
Release of mediators from chopped lung preparations.
Lung lobes obtained from sensitized guinea pigs were cut into approximately 1-mm3 pieces using a McIlwain tissue chopper (Brinkmann Instruments, Westbury, NY). Fragmented lung preparations
were washed thoroughly with oxygenated Krebs-NaHCO3 buffer before incubation. Duplicate aliquots of 200-mg lung fragments were
weighed and placed in plastic scintillation vials containing 2 ml of
oxygenated Krebs solution in the presence of 4 mM indomethacin (for
histamine release) plus 5 mM cysteine (for release of peptidoleukotrienes). Lung samples were then incubated in a shaker bath at 37°C
for 45 min before they were challenged with OA for 3 min (histamine
release) or 10 min (release of peptidoleukotrienes) (Wong et al.,
1992b). To determine the mast cell-stabilizing effects of tyrosine
kinase inhibitors, they were preincubated with the lung preparation
for 30 min before immunologic stimulation. Diffusates were then
collected and stored at 270°C until assay.
Histamine radioenzymatic assay. Histamine release from lung
samples in response to OA was determined via a radioenzymatic
assay as described by Henry et al. (1991), utilizing highly purified
HNMT. Briefly, a total incubation volume of 60 ml was prepared by
sequential addition of 10 ml of biological samples (or H2O for the
blank), 25 ml of H2O (or H2O containing 500 pg of histamine as
internal standard) and 25 ml of reaction reagent in 12 3 75-mm
polypropylene culture tubes. Reaction reagent contained 21 ml of 0.4
M potassium phosphate/0.1% bovine serum albumin, pH 7.8, 2 ml of
HNMT, and 2 ml of tritiated S-adenosylmethionine (80 Ci/mmol).
After a 1-hr incubation in a shaker bath at 2°C, the enzymatic
reaction was terminated by the addition of 75 ml of 2.5 M potassium
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
1985; Ro et al., 1991; Moore and Dannenberg, 1993), and
cross-linking of their corresponding FceRI and FcgR leads to
mast cell degranulation. It has been shown that IgG-sensitized guinea pig airway tissues released more histamine and
peptidoleukotrienes (Undem et al., 1985; Ro et al., 1991) and
that their anaphylactic contractions were more sensitive to
inhibition by combined H1-receptor and peptidoleukotriene
receptor antagonism, results that closely resemble the IgEdependent anaphylactic responses in human (Regal, 1985;
Bjorck and Dahlen, 1993). FcgR (e.g., FcgRIII) and FceRI are
structurally and functionally related, and both belong to a
family of multisubunit antigen receptors (Alber et al., 1992;
Bolen, 1995). It has been shown that engagement of these cell
surface receptors activates PTKs as the initial events for
successful signal propagation (Bolen, 1995).
In this study, we passively sensitized guinea pigs with IgG
raised against OA and studied the effects of two PTK inhibitors, genistein (Akiyama et al., 1987) and tyrphostin 47
(Gazit et al., 1989; Levitzki and Gazit, 1995), on antigeninduced anaphylactic contraction of the bronchi and release
of histamine and peptidoleukotrienes from chopped lung
preparations. Our findings show that genistein and tyrphostin 47 substantially inhibited both antigen-induced release of
mediators and anaphylactic contraction. Tyrphostin 47 acted
mainly by preventing mast cell degranulation, whereas
genistein exerted inhibitory effects partly by blocking mast
cell degranulation and partly by attenuating LTD4-induced
bronchial contraction. Taken together, these data suggest
that PTK inhibitors have a therapeutic potential as mast cell
stabilizers in the treatment of allergic diseases such as bronchial asthma.
Vol. 283
1997
Tyrosine Kinase in Antigen Challenge
133
Results
Antigen challenge. Sensitized guinea pig bronchial rings
contracted in response to 60 mM KCl with an active force of
contraction that amounted to 1.45 6 0.10 g (n 5 19). It also
produced graded contractile responses to increasing concentrations of OA (fig. 1A). The sensitized bronchial rings did not
contract to irrelevant protein such as bovine serum albumin.
The threshold concentration of OA to induce anaphylactic
contraction was 0.01 mg/ml, and maximum response was
achieved at 1 mg/ml. The concentration that caused 50%
maximum contraction was 0.05 mg/ml (fig. 1B). For all subsequent antigen-challenge studies, OA at a concentration of 1
mg/ml was used to induce maximum anaphylactic bronchial
smooth muscle contraction (1.83 6 0.10 g, n 5 19).
Effects of PTK inhibitors on Schultz-Dale reaction.
To determine the effects of PTK inhibitors on the SchultzDale reaction, the inhibitors were preincubated with the ring
preparations for 30 min before OA challenge. Genistein concentration-dependently inhibited OA-induced bronchial contraction by 15%, 60%, 73% and 99% at 10 mM, 30 mM, 50 mM
and 100 mM, respectively (fig. 2A). In contrast, 50 mM daidzein, a structural analog of genistein devoid of tyrosine kinase inhibition activity, did not affect OA-induced bronchial
contraction. On the other hand, tyrphostin 47 significantly
inhibited anaphylactic contraction by 25%, 31%, 73% and
77% at 10 mM, 30 mM, 50 mM and 100 mM, respectively (fig.
2B).
Effects of PTK inhibitors on mediator-induced bronchial contraction. To determine whether the inhibition of
Fig. 1. A) Representative tracing of guinea pig bronchial contraction to
increasing concentrations of OA. Numbers represent cumulative dose
added. B) Log concentration-response curve of guinea pig bronchial
rings to increasing concentrations of OA. Each point represents the
mean 6 S.E.M. of seven experiments.
anaphylactic contraction is mediated by blocking the release
of mast cell-derived mediators or by attenuating bronchial
smooth muscle contraction, we evaluated the PTK inhibitors
in histamine- or LTD4-induced bronchial ring contraction. At
50 mM, a concentration that significantly blocked the OAinduced bronchial contraction, neither genistein nor tyrphostin 47 attenuated bronchial contraction (1.45 6 0.07 g, n 5
26) induced by 30 mM histamine (fig. 3A). At 100 mM,
genistein but not tyrphostin 47 substantially suppressed histamine-induced bronchial contraction by 88%. Diadzein (100
mM) did not exhibit any inhibitory effect on histamine-induced bronchial contraction. Bronchial contraction induced
by 0.1 mM LTD4 (1.45 6 0.06 g, n 5 14) was significantly
reduced by genistein (75%), but not by tyrphostin 47, at a
concentration of 50 mM. Daidzein (50 mM) also inhibited
LTD4-induced contractile response by 44%. Nevertheless, the
inhibitory effect of daidzein was significantly (P , .05) less
than that of genistein (fig. 3B).
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
borate, pH 11; 4 ml of toluene-isoamyl alcohol (3:1, v/v) was then
added to each tube. After centrifugation for 3 min, 3.8 ml of the
organic phase, which contained tritiated N-t-methylhistamine ([3H]t-MHm) formed by the HNMT reaction, was transferred to another
set of tubes containing 500 ml of 0.5 M HCl for back extraction of the
[3H]-t-MHm into the aqueous phase. Tubes were centrifuged for 3
min, and the organic phase was aspirated and discarded. The aqueous phase was mixed with 1.25 ml of toluene-isoamyl alcohol. After
centrifugation and removal of the organic phase, 300 ml of the acidified aqueous phase was transferred to scintillation vials containing
8 ml of biodegradable counting scintillant. Radioactivity was quantitated by liquid scintillation spectrometry (Beckman LS 3801, Beckman Instruments, Inc., Fullerton, CA).
Leukotrienes radioimmunoassay. The release of peptidoleukotrienes from chopped lung preparations in response to OA was
quantitated by radioimmunoassay (Amersham Life Science, Buckinghamshire, U.K.). Briefly, total incubation volumes of 400 ml were
prepared by sequential addition of 100 ml of biological samples or
LTC4 standard, 100 ml of [3H]-LTC4, 100 ml of peptidoleukotrienespecific antiserum (cross-reactivity for LTC4/D4/E4 5 100%/100%/
41%), and 100 ml of assay buffer (pH 7.4) in polypropylene tubes.
Antigen-antibody competition reaction was allowed to take place
overnight at 2°C to 8°C. Dextran-coated charcoal suspension (250 ml)
was then added to each reaction tube to adsorb any unbound leukotrienes. After centrifugation, the supernatant was transferred to
scintillation vials containing 10 ml of scintillant. Radioactivity was
measured using liquid scintillation spectrometry. Samples were assayed in duplicate.
Data analysis. All data are presented as mean 6 S.E.M. Statistical differences in contractile responses to OA challenge, histamine
or LTD4, and in the release of mediators in response to OA, in the
presence and absence of inhibitors were analyzed using ANOVA
followed by Student-Newman-Keuls test (Armitage and Berry,
1987). The critical level for significance was set at P , .05.
134
Wong et al.
Vol. 283
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 2. Effects of genistein (GEN)/daidzein (DZ) (panel A) and tyrphostin
47 (panel B) on OA-induced anaphylactic contraction of guinea pig
bronchi. Each point represents the mean 6 S.E.M. for the number of
experiments indicated in parentheses. * Significant differences from
DMSO vehicle controls, P , .05.
Effects of PTK inhibitors on the release of mediators. Chopped lung preparations released low levels of histamine (75.5 6 21.0 ng/g tissue, n 5 13) and peptidoleukotrienes (1.2 6 0.1 ng/g tissue, n 5 8) spontaneously. Upon 1
mg/ml OA challenge, the release of histamine and that of
peptidoleukotrienes from lung fragments were significantly
increased by 24-fold (1809.0 6 135.2 ng/g tissue, n 5 13) and
60-fold (71.6 6 5.7 ng/g tissue, n 5 8), respectively. Genistein
concentration-dependently inhibited antigen-induced histamine release by 32%, 54% and 67% at 10 mM, 50 mM and 100
mM, respectively (fig. 4A). Likewise, it substantially reduced
OA-induced release of peptidoleukotrienes by 19%, 58% and
Fig. 3. Effects of tyrphostin 47 (T-47), genistein (GEN) and daidzein (DZ)
on guinea pig bronchial contraction induced by 30 mM histamine (panel
A) or 0.1 mM leukotriene D4 (panel B). Each point represents the
mean 6 S.E.M. for the number of experiments indicated in parentheses. * Significant differences from DMSO vehicle controls, P , .05.
74% at 10 mM, 50 mM and 100 mM, respectively (fig. 4B). In
contrast, daidzein (50 mM), failed to block histamine release
but significantly reduced the release of peptidoleukotrienes
from lung fragments by 29%. Nevertheless, the inhibitory
effect of daidzein on the release of peptidoleukotrienes was
significantly (P , .05) weaker than that of genistein. On the
other hand, tyrphostin 47 at concentrations of 10 mM, 50 mM
and 100 mM significantly reduced histamine release from
lung fragments by 22%, 41% and 48%, respectively, and
markedly blocked the release of peptidoleukotrienes by 40%,
92% and 98%, respectively (fig. 4). The inhibitory effect of
tyrphostin 47 on the release of leukotrienes was substantially more pronounced than that of genistein.
1997
Tyrosine Kinase in Antigen Challenge
Discussion
Genistein and tyrphostin 47 are structurally and functionally unrelated inhibitors of PTK (Levitzki and Gazit, 1995).
Genistein is an isoflavone compound that inhibits PTK activity of the epidermal growth factor receptor and pp60v-src
via competitive inhibition at the ATP-binding domain of the
kinases (Akiyama et al., 1987; Di Salvo et al., 1993). Tyrphostin 47 (RG50864) is a derivative of the dihydroxybenzylidene
malononitrile class of PTK inhibitors that acts by competitive
inhibition at the substrate site of the kinase (Gazit et al.,
1989). Genistein is a broad-spectrum PTK inhibitor; the ATP
binding site that it inhibits has been shown to be highly
conserved among PTKs. Tyrphostin 47 exhibits selective inhibition for epidermal growth factor receptor kinase 800
times more potently than for insulin receptor kinase (Lev-
itzki and Gazit, 1995). Their relative potencies against nontransmembrane PTKs (e.g., Jak, Fer, Syk, Btk, and Lyn)
remain to be determined.
Bronchial rings from guinea pigs passively sensitized with
purified IgG raised against OA contracted in response to OA,
but not to bovine serum albumin, in a concentration-dependent manner (fig. 1). This observation is consistent with our
previous study using guinea pig lung parenchymal strips
(Wong et al., 1992a) and with other reports using guinea pig
trachea (Undem et al., 1985; Bertrand et al., 1991; Ro et al.,
1991), which showed that IgG is able to sensitize guinea pig
mast cells to specific antigen. Both IgG-binding FcgR (e.g.,
FcgRIII) and IgE-binding FceRI are expressed on the surface
of human and murine mast cells (Ravetch and Kinet, 1991;
Alber et al., 1992). IgG-mediated aggregation of FcgRIII has
been shown to stimulate the release of arachidonic acid metabolites (Alber et al., 1992) and serotonin (Daeron et al.,
1992) from murine mast cells. In an effect analogous to that
of FceRI, cross-linking of two or more FcgRs triggers nontransmembrane PTK activities as the initial signaling
events, followed by increases in intracellular Ca11 level and
protein kinase C activity, which in turn leads to exocytosis of
mast cell-derived inflammatory mediators (Alber et al., 1992;
Beaven and Metzger, 1993).
OA-induced anaphylactic contraction of the bronchi was
significantly inhibited by either genistein or tyrphostin 47 in
a concentration-dependent manner (fig. 2). At 50 mM, both
genistein and tyrphostin 47 markedly suppressed bronchial
anaphylactic contraction by at least 70%. In contrast, 50 mM
daidzein, an analog of genistein that has no inhibitory activity for PTK (Akiyama et al., 1987), failed to alter the anaphylactic contraction. On the other hand, the OA-induced release
of histamine and peptidoleukotrienes from chopped lung
preparations was also concentration-dependently reduced by
genistein and tyrphostin 47 (fig. 4). Lavens et al. (1992)
previously showed that genistein inhibited anti-IgE-induced
histamine release from human lung mast cells. These findings suggest that PTK is involved in the Schultz-Dale reaction and that the inhibitors of PTK interrupted the early
signaling pathway of FcgR cross-linking in the mast cells
(Alber et al., 1992; Bolen, 1995) and, therefore, attenuated
the anaphylactic contraction by preventing the release of
mast cell-derived inflammatory mediators such as histamine
and peptidoleukotrienes.
In order to ascribe the inhibitory effects of genistein and
tyrphostin 47 on anaphylactic contraction solely to mast cell
stabilization, we must first determine whether the PTK inhibitors themselves have any effects on the bronchial smooth
muscle contraction. Our current understanding of the biological activities of PTK has been derived mainly from studies of
growth factor-associated responses such as mitogenesis, differentiation and proliferation of immune cells (Bolen, 1995;
Levitzki and Gazit, 1995). Lately, evidence has been accumulating that protein tyrosine phosphorylation also participates
in the regulation of smooth muscle contraction (Di Salvo et
al., 1994; Hollenberg, 1994; Semenchuk and Di Salvo, 1995;
Jin et al., 1996). In addition to playing a central role in the
EGF-induced gastric smooth muscle contraction (Hollenberg,
1994), PTK mediates vascular smooth muscle contraction
induced by serotonin or phenylephrine (Semenchuk and Di
Salvo, 1995; Watts et al., 1996) and gastric longitudinal
smooth muscle contraction induced by angiotensin-II (Yang
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 4. Effects of tyrphostin 47 (T-47), genistein (GEN) and daidzein (DZ)
on the release of histamine (panel A) or peptidoleukotrienes (panel B)
from OA-stimulated guinea pig chopped lung preparations. Each point
is the mean 6 S.E.M. for the number of experiments indicated in
parentheses. * Significant differences from DMSO vehicle controls, P ,
.05.
135
136
Wong et al.
tivity of this compound on EGF receptor kinase and pp60v-src
(Akiyama et al., 1987). In addition, 50 mM daidzein reduced
the release of peptidoleukotrienes from lung fragments. Nevertheless, the inhibitory effects of daidzein are significantly
weaker than those of genistein (figs. 3 and 4). Recently,
daidzein has been shown to attenuate serotonin-, norepinephrine- or KCl-induced vascular smooth muscle contraction (Toma et al., 1995; Watts et al., 1996). These recent
findings, together with our results, suggest that daidzein
might possess some activity against PTK (Toma et al., 1995;
Watts et al., 1996). Alternatively, the inhibitory effects of
both genistein and daidzein on LTD4-induced bronchial contraction and peptidoleukotrienes release from lung fragments might be due to certain unidentified nonselective effects of the drugs.
In summary, genistein and tyrphostin 47 substantially
inhibited the Schultz-Dale reaction in the guinea pig airways. Genistein exhibited its modulatory effects partly by
mast cell stabilization and partly by reduction of smooth
muscle contraction in response to LTD4. Tyrphostin 47 acted
primarily as a mast cell stabilizer without having any significant effect on smooth muscle contraction. Because mast cells
play a pivotal role in initiating allergic disorders such as
bronchial asthma (Wasserman, 1994), our findings suggest
that PTK inhibitors may have therapeutic potential as mast
cell stabilizers in the treatment of asthma.
Acknowledgments
The authors thank Professor Matthew C. E. Gwee for his continuous support of this project.
References
ABEBE, W. AND AGRAWAL, D. K.: Role of tyrosine kinases in norepinephrineinduced contraction of vascular smooth muscle. J. Cardiovasc. Pharmacol.
26: 153–159, 1995.
ABELA, A. AND DANIEL, E. E.: Neural and myogenic effects of leukotrienes C4,
D4, and E4 on canine bronchial smooth muscle. Am. J. Physiol. 266: L414–
L425, 1994.
AKIYAMA, T., ISHIDA, J., NAKAGAWA, S., OGAWARA, H., WATANABE, S. I., ITOH, N.,
SHIBUYA, M. AND FUKAMI, Y.: Genistein, a specific inhibitor of tyrosine-specific
protein kinases. J. Biol. Chem. 262: 5592–5595, 1987.
ALBER, G., KENT, U. E. AND METZGER, H.: Functional comparison of FceRI,
FcgRII, and FcgRIII in mast cells. J. Immunol. 149: 2428–2436, 1992.
ARMITAGE, P. AND BERRY, G.: Statistical Methods in Medical Research. Blackwell Scientific Publications, Oxford, 1987.
BEAVEN, M. A. AND METZGER, H.: Signal transduction by Fc receptors: The FceRI
case. Immunol. Today 14: 222–226, 1993.
BENHAMOU, M., RYBA, N. J. P., KIHARA, H., NISHIKATA, H. AND SIRAGANIAN, R. P.:
Protein-tyrosine kinase p72syk in high affinity IgE receptor signaling.
J. Biol. Chem. 268: 23318–23324, 1993.
BERTRAND, C., TSCHIRHART, E. AND LANDRY, Y.: Epithelial modulation of thromboxane A2 and PAF involvement in IgE- and IgG-mediated guinea pig
anaphylaxis. Immunopharmacology 22: 115–126, 1991.
BJORCK, T. AND DAHLEN, S. E.: Leukotrienes and histamine mediate IgEdependent contractions of human bronchi: Pharmacological evidence obtained with tissues from asthmatic and non-asthmatic subjects. Pulm. Pharmacol. 6: 87–96, 1993.
BOLEN, J. B.: Protein tyrosine kinases in the initiation of antigen receptor
signaling. Curr. Opin. Immunol. 7: 306–311, 1995.
CHAND, N. AND EYRE, P.: The Schultz-Dale reaction: A review. Agent Action 8:
171–184, 1978.
DAERON, M., BONNEROT, C., LATOUR, S. AND FRIDMAN, W. H.: Murine recombinant
FcgRIII, but not FcgRII, trigger serotonin release in rat basophilic leukemia
cells. J. Immunol. 149: 1365–1373, 1992.
DALE, H. H.: The anaphylactic reaction of plain muscle in guinea pig. J. Pharmacol. Exp. Ther. 4: 167–223, 1913.
DI SALVO, J., STEULOFF, A., SEMENCHUK, L., SATOH, S., KOLQUIST, K. AND PFITZER,
G.: Tyrosine kinase inhibitors suppress agonist-induced contraction in
smooth muscle. Biochem. Biophys. Res. Comm. 190: 968–974, 1993.
DI SALVO, J., PFITZER, G. AND SEMENCHUK, L. A.: Protein kinase phosphorylation,
cellular Ca21, and Ca21 sensitivity for contraction of smooth muscle. Can.
J. Physiol. Pharmacol. 72: 1434–1439, 1994.
EISEMAN, E. AND BOLEN, J. B.: Engagement of the high-affinity IgE receptor
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
et al., 1993). These findings indicate that PTK is involved in
the signal transduction of neurotransmitters known to act
through G protein-coupled receptors. Inhibitors of PTK such
as genistein and tyrphostins have been shown to inhibit
smooth muscle contraction to carbachol, norepinephrine,
phenylephrine, serotonin and angiotensin-II (Di Salvo et al.,
1993; Yang et al., 1993; Abebe and Agrawal, 1995; Watts et
al., 1996).
At a concentration (50 mM) that significantly inhibited
OA-induced anaphylactic contraction and mediator release,
neither genistein nor tyrphostin 47 altered histamine-induced bronchial contraction. However, as the concentration
was increased to 100 mM, genistein, but not tyrphostin 47,
abolished bronchial contraction induced by histamine. As
expected, 100 mM daidzein had no effect on histamine-mediated contraction (fig. 3A). The inhibitory effect of genistein
might be related to the substantial inactivation of certain
PTKs that participate in the signal transduction of histamine-induced bronchial contraction. It has been shown in
human umbilical vein endothelial cells that histamine induced a delayed tyrosine phosphorylation of the 42/44-kDa
mitogen-activated protein kinase, which suggests that PTK
is a mediator in the histamine signal transduction cascade
(Fleming et al., 1995). However, it remains to be determined
whether tyrosine phosphorylation of the same or other substrates occurs in histamine-treated bronchial smooth muscle
cells. Alternatively, genistein at a concentration of 100 mM
might have exhibited some nonspecific activities against histamine-induced bronchial contraction. Wijetunge et al. (1992)
showed that 100 mM genistein, but not daidzein, inhibited
voltage-operated calcium channel currents in vascular
smooth muscle. However, we still do not know whether the
inhibition of calcium channel currents is a direct blockade by
genistein or a result of PTK inactivation.
On the other hand, LTD4-induced bronchial smooth muscle
contraction was significantly inhibited by genistein, but not
by tyrphostin 47, at 50 mM concentration. Diadzein (50 mM)
also attenuated the LTD4-mediated contraction, but its inhibitory effect was significantly weaker than that of
genistein (fig. 3B). The effect of genistein on LTD4-induced
contraction is unlikely to be associated with nonspecific activity on other serine/threonine kinases. It has been shown
that genistein scarcely inhibited cAMP-dependent protein
kinase, protein kinase C, phosphorylase kinase and phosphodiesterase, even at concentrations above 300 mM
(Akiyama et al., 1987). Another possible explanation for
genistein’s inhibitory effect is blockade of Ca11 channel currents in the bronchial smooth muscle. This does not appear to
be the case, however, because 50 mM genistein failed to block
the histamine-induced bronchial contraction. Thus it is likely
that genistein attenuated LTD4-induced contraction by mitigating the PTK activities stimulated by LTD4 receptor activation. It has been demonstrated in human epithelial cells
that LTD4 induced tyrosine phosphorylation of PLCg and of
two other associated protein substrates (Gronroos et al.,
1995). Activation of PLCg produces inositol 1,4,5-trisphosphate and diacylglycerol, resulting in increased intracellular
Ca11 concentration and protein kinase C activity, which
ultimately regulate smooth muscle contractility (Hollenberg,
1994).
The inhibitory effects of daidzein on LTD4-induced bronchial contraction were unexpected, given the reported inac-
Vol. 283
1997
137
isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat
basophilic RBL-2H3 cells. Reconstitution of secretory responses with Ca12
and purified isozymes in washed permeabilized cells. J. Biol. Chem. 268:
1749–1756, 1993.
PENHALLOW, R. C., CLASS, K., SONODA, H., BOLEN, J. B. AND ROWLEY, R. B.:
Temporal activation of nontransmembrane protein-tyrosine kinases following mast cell FceRI engagement. J. Biol. Chem. 270: 23362–23365, 1995.
RAVETCH, J. V. AND KINET, J. P.: Fc receptor. Ann. Rev. Immunol. 9: 457–492,
1991.
REGAL, J. F.: IgG vs. IgE: Mediators of antigen-induced guinea pig lung parenchymal contraction. Immunopharmacology 10: 137–146, 1985.
RO, J. Y., BUCKNER, C. K., BRENDEL, J. K., FISHLEDER, R. I. AND GRAZIANO, F. M.:
Influence of indomethacin and L-cysteine on histamine and peptidoleukotriene release from superfused tracheas taken from guinea pigs passively
sensitized with IgG1 and IgE antibodies. J. Allergy. Clin. Immunol. 87:
1150–1160, 1991.
SCHARENBERG, A. M. AND KINET, J. P.: Initial events in FceRI signal transduction. J. Allergy Clin. Immunol. 94: 1142–1146, 1994.
SCHULTZ, W. H.: Physiological studies in anaphylaxis. I. The reaction of smooth
muscle of the guinea pig sensitized with horse serum. J. Pharmacol. Exp.
Ther. 1: 549–567, 1910.
SEMENCHUK, L. A. AND DI SALVO, J.: Receptor-activated increases in intracellular calcium and protein tyrosine phosphorylation in vascular smooth muscle
cells. FEBS Lett. 370: 127–130, 1995.
TOMA, C., JENSEN, P. E., PRIETO, D., HUGHES, A., MULVANY, M. J. AND AALKJAER,
C.: Effects of tyrosine kinase inhibitors on the contractility of rat mesenteric
resistance arteries. Br. J. Pharmacol. 114: 1266–1272, 1995.
UNDEM, B. J., BUCKNER, C. K., HARLEY, P. AND GRAZIANO, F. M.: Smooth muscle
contraction and release of histamine and slow-reacting substance of anaphylaxis in pulmonary tissues isolated from guinea pigs passively sensitized
with IgG1 or IgE antibodies. Am. Rev. Respir. Dis. 131: 260–266, 1985.
WASSERMAN, S. I.: Mast cells and airway inflammation in asthma. Am. J.
Respir. Crit. Care Med. 150: S39–S41, 1994.
WATTS, S. W., YEUM, C. H., CAMPBELL, G. AND WEBB, R. C.: Serotonin stimulates
protein tyrosyl phosphorylation and vascular contraction via tyrosine kinase. J. Vasc. Res. 33: 288–298, 1996.
WIJETUNGE, S., AALKJAER, C., SCHACHTER, M. AND HUGHES, A. D.: Tyrosine kinase
inhibitors block calcium channel currents in vascular smooth muscle cells.
Biochem. Biophys. Res. Commun. 189: 1620–1623, 1992.
WONG, W. S. F., BENDELE, A. M. AND FLEISCH, J. H.: Pharmacologic evaluation
of A23187-induced contractions of three distinct preparations of guinea pig
lung parenchymal strips. J. Pharmacol. Exp. Ther. 260: 714–721, 1992a.
WONG, W. S. F., SPAETHE, S. M., HENRY, D. P. AND FLEISCH, J. H.: Contributory
role of lung pleura to release of anaphylactic mediators from guinea pig lung
in response to ovalbumin or A23187. Biochem. Pharmacol. 44: 1609–1615,
1992b.
YANG, S. G., SAIFEDDINE, M., LANIYONU, A. AND HOLLENBERG, M. D.: Distinct
signal transduction pathways for angiotensin-II in guinea pig gastric smooth
muscle: Differential blockade by indomethacin and tyrosine kinase inhibitors. J. Pharmacol. Exp. Ther. 264: 958–966, 1993.
Send reprint requests to: Dr. W. S. Fred Wong, Department of Pharmacology, Faculty of Medicine, National University of Singapore, 10 Kent Ridge
Crescent, Singapore 119260, Republic of Singapore.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
activates src protein-related tyrosine kinases. Nature (Lond.) 355: 78–80,
1992.
FLEMING, I., FISSLTHALER, B. AND BUSSE, R.: Calcium signaling in endothelial
cells involves activation of tyrosine kinases and leads to activation of mitogen-activated protein kinases. Circ. Res. 76: 522–529, 1995.
FUKAMACHI, H., TAKEI, M. AND KAWAKAMI, T.: Activation of multiple protein
kinases including a MAP kinase upon FceRI cross-linking. Int. Arch. Allergy
Immunol. 102: 15–25, 1993.
GAZIT, A., YAISH, P., GILON, C. AND LEVITZKI, A.: Tyrphostins. I. Synthesis and
biological activity of protein kinase inhibitors. J. Med. Chem. 32: 2344–2352,
1989.
GRONROOS, E., SCHIPPERT, A., ENGSTROM, M. AND SJOLANDER, A.: The regulation of
leukotriene D4-induced calcium influx in human epithelial cells involves
protein tyrosine phosphorylation. Cell Calcium 17: 177–186, 1995.
HENRY, D. P., BOWSHER, R. R. AND VERBURG, K. M.: Radioenzymatic assay for
histamine: Development and validation. Scan. J. Gastroenterol. 26: suppl
180; 26–31, 1991.
HOLLENBERG, M. D.: Tyrosine kinase pathways and the regulation of smooth
muscle contractility. Trends Pharmacol. Sci. 15: 108–114, 1994.
HUTCHCROFT, J. E., GEAHLEN, R. L., DEANIN, G. G. AND OLIVER, J. M.: FceRImediated tyrosine phosphorylation and activation of the 72-kDa proteintyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells. Proc. Natl. Acad.
Sci. U.S.A. 89: 9107–9111, 1992.
JIN, N., SIDDIQUI, R. A., ENGLISH, D. AND RHOADES, R. A.: Communication
between tyrosine kinase pathway and myosin light chain pathway in smooth
muscle. Am. J. Physiol. 271: H1348–H1355, 1996.
JONSSON, E. W. AND DAHLEN, S. E.: Interactions between leukotrienes and
histamine in the anaphylactic contraction of guinea pig lung parenchyma.
J. Pharmacol. Exp. Ther. 271: 615–623, 1994.
JOUVIN, M. H. E., ADAMCZEWSKI, M., NUMEROF, R., LETOURNEUR, O., VALLE, A. AND
KINET, J. P.: Differential control of the tyrosine kinases Lyn and Syk by the
two signaling chains of the high affinity immunoglobulin E receptor. J. Biol.
Chem. 269: 5918–5925, 1994.
KAWAKAMI, T., INAGAKI, N., TAKEI, M., FUKAMACHI, H., COGGESHALL, K. M., ISHIZAKA, K. AND ISHIZAKA, T.: Tyrosine phosphorylation is required for mast cell
activation by FceRI cross-linking. J. Immunol. 148: 3513–3519, 1992.
KAWAKAMI, Y., YAO, L., MIURA, T., TSUKADA, S., WITTE, O. N. AND KAWAKAMI, T.:
Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon
FceRI cross-linking. Mol. Cell. Biol. 14: 5108–5113, 1994.
LAVENS, S. E., PEACHELL, P. T. AND WARNER, J. A.: Role of tyrosine kinases in
IgE-mediated signal transduction in human lung mast cells and basophils.
Am. J. Resp. Cell Mol. Biol. 7: 637–644, 1992.
LEVITZKI, A. AND GAZIT, A.: Tyrosine kinase inhibition: An approach to drug
development. Science (Wash. DC) 267: 1782–1788, 1995.
LI, W., DEANIN, G. G., MARGOLIS, B., SCHLESSINGER, J. AND OLIVER, J. M.: FceRImediated tyrosine phosphorylation of multiple proteins, including phospholipase Cg1 and the receptor bg2 complex, in RBL-2H3 rat basophilic leukemia cells. Mol. Cell. Biol. 12: 3176–3182, 1992.
MOORE, K. G. AND DANNENBERG, A. M.: Immediate and delayed (late-phase)
dermal contact sensitivity reactions in guinea pigs. Int. Arch. Allergy Immunol. 101: 72–81, 1993.
OLIVER, J. M., BURG, D. L., WILSON, B. S., MCLAUGHLIN, J. L. AND GEAHLEN, R. L.:
Inhibition of mast cell FceRI-mediated signaling and effector function by the
syk-selective inhibitor, piceatannol. J. Biol. Chem. 269: 29697–29703, 1994.
OZAWA, K., SZALLASI, Z., KAZANIETZ, M. G., BLUMBERG, P. M., MISCHAK, H.,
MUSHINSKI, J. F. AND BEAVEN, M. A.: Ca12-dependent and Ca12-independent
Tyrosine Kinase in Antigen Challenge