© 1995 American Heart Association, Inc.
Articles |
Inhibition of Vascular Smooth Muscle Cell K+ Currents by Tyrosine Kinase Inhibitors Genistein and ST 638
From the Department of Pharmacology, United Medical and Dental Schools of Guy's and St Thomas's Hospitals, London, England.
Correspondence to P.I. Aaronson, Department of Pharmacology, United Medical and Dental Schools of Guy's and St Thomas's Hospitals, St Thomas's Campus, Lambeth Palace Rd, London SE1 7EH, England.
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Abstract |
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Abstract The whole-cell patch-clamp technique was used to characterize the effects of several tyrosine kinase inhibitors on the voltage-gated K+ current (IK) in rat and rabbit pulmonary artery cells. IK was blocked in a dose-dependent manner by genistein (20 to 100 µmol/L) and ST 638 (0.5 to 40 µmol/L) but not by the inactive genistein analogue diadzein (100 µmol/L). This inhibition was not significantly altered when ATP was excluded from the patch pipette or when it was replaced by the poor tyrosine kinase substrate ATP-
-S. The inhibition was also unaffected
by inclusion of the tyrosine phosphatase inhibitor orthovanadate in
either the bath (0.5 mmol/L) or pipette (0.2 mmol/L) solutions. In the
rat, IK ordinarily inactivated negligibly over 300 ms. In
the presence of 10 µmol/L ST 638, however, IK reached a
peak 
5 ms after depolarization (to +60 mV) and then decayed
markedly. In the rabbit, IK demonstrated a prominent
rapidly decaying initial component that was only slightly inhibited by
ST 638, which preferentially blocked the sustained current; genistein
showed the opposite selectivity. These observations indicated that
IK blockade by genistein and ST 638 was not mediated by an
inhibition of tyrosine kinase activity and further suggested that in
both types of cells genistein and ST 638 preferentially blocked rapidly
and slowly inactivating components of IK,
respectively.
Key Words: pulmonary artery • vascular smooth muscle • ion channels • K+ channels • protein tyrosine kinase inhibitors
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although protein tyrosine kinases (PTKs) are most notable for their role in regulating long-term processes such as cellular growth, division, and metabolism,1 2 tyrosine kinases can also mediate rapidly occurring events, such as phosphatidylinositol turnover3 and vascular smooth muscle contraction.4 Pharmacological inhibitors of PTKs (PTKIs) inhibit contractions induced by the stimulation of G protein–linked receptors in vascular and intestinal smooth muscle,5 6 and it has recently been proposed that an intermediary nonreceptor tyrosine kinase may mediate contractions elicited by many types of agonists.6 In vascular smooth muscle cells of the rabbit ear artery, several PTKIs diminished the Ca2+ current, implying the existence of tonic regulation of this current by a tyrosine kinase.7 In the present study, we have examined the effect of several PTKIs on the voltage-gated K+ current (IK) in rat8 9 and rabbit10 pulmonary artery cells to determine whether this current might also be regulated by a tyrosine kinase. Our results indicate that although the PTKIs genistein and ST 638 potently and reversibly inhibit the voltage-gated IK in these smooth muscle cells, these responses do not appear to involve PTK inhibition. Our data also suggest that genistein and ST 638 are selective for different IKs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Wistar rats (250 to 300 g) were killed by cervical dislocation, and male New Zealand White Rabbits were killed by injection of 80 mg/kg phenobarbitone into the ear artery. After excision of the heart and lungs, pulmonary arteries were rapidly dissected out. Single vascular smooth muscle cells were enzymatically isolated, as previously described for the rat8 9 and the rabbit,10 and conventional whole-cell patch-clamp recordings were made, as described in detail previously.8 9 The control patch pipette solution contained (mmol/L) KCl 110, MgCl2 0.5, Na2ATP 5, HEPES 10, EGTA 10, and CaCl2 0.5 (pKCa=8 nmol/L); the solution was set to pH 7.2 with KOH. The bathing solution contained (mmol/L) NaCl 130, KCl 5, MgCl2 1.2, CaCl2 1.5, HEPES 10, and glucose 10, pH 7.2 to 7.4. Tetraethylammonium chloride (10 mmol/L), which we have previously shown produces a selective abolition of the Ca2+-activated IK in these cells,8 9 was also added to all bath solutions. All experiments were carried out at room temperature. In some cases, cells were preincubated in glucose-free or orthovanadate-containing solutions at 4°C before the experiments (see "Results"). Genistein, diadzein, and tyrphostins 1 and 23 (all from Calbiochem) and ST 638 were dissolved in dimethyl sulfoxide and stored at -20°C until just before use. ST 638 is a 4-hydroxycinnamamide derivative that inhibits PTK activity in vitro by competing with the substrate protein for the PTK.11 12 13 Genistein is an isoflavone derivative that inhibits PTK activity by competing with ATP for its binding to PTKs.14 15 Means were compared by using Student's paired or unpaired t test; differences were deemed significant at P<.05 (unless otherwise stated). All data are presented as mean±SD.
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Results |
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Fig 1
illustrates that both 10 µmol/L ST 638 (Fig 1a and 1b) and 100 µmol/L genistein (Fig 1c and 1d) rapidly and
reversibly blocked the tetraethylammonium-insensitive voltage-gated
IK in rat pulmonary artery smooth muscle cells. The current
amplitude measured at 100 ms was significantly reduced to 0.09±0.03
(n=14) of control by ST 638. IK normally showed negligible
inactivation over 300 ms; however, in the presence of ST 638, the
current decayed rapidly (Fig 1b). Genistein reduced the current
amplitude to 0.47±0.13 (n=15) of control. Although genistein had no
consistent effect on the decay of IK, it did
significantly slow its activation (Fig 1d). The average time to
half-maximal activation of IK increased by about two times,
from 3.6±0.8 ms (n=15) in the control bathing solution to 6.7±2.2 ms
(n=13) in the presence of 100 µmol/L genistein (P<.0001).
Diadzein, an analogue of genistein, which is inactive as a
PTKI,16 had no effect on IK at a concentration
of 100 µmol/L (Fig 1c); the amplitude of IK in diadzein
was 0.96±0.03 (n=4) of the control. Tyrphostin 23, a member of a
different chemical family of PTKIs, also had no effect on the amplitude
of IK (0.95±0.01, n=5); however, a minor inhibition was
observed with its analogue tyrphostin 1 (0.86±0.06, n=4).
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If these inhibitory effects of genistein and ST 638 were due to a
PTKI-mediated decrease in tyrosine phosphorylation of the
IK channel(s), the rapid onset and offset of the responses
to these drugs would imply that basal turnover of phosphorylation must
also be rapid. If so, removal of cellular ATP should reduce the
response to the drugs (by reducing basal tyrosine phosphorylation and
therefore the capacity of IK to be inhibited by
dephosphorylation) and would block rephosphorylation-mediated recovery
of the current after drug removal. However, the Table
shows that the exclusion of ATP from the patch pipette or its
replacement with ATP--S, an ATP analogue that is a poor substrate
for PTKs17 and should thus competitively block their
utilization of any residual cellular ATP, had no effect on the ability
of genistein to block IK. Similar results were obtained
when we used ATP-free and ATP--S-containing pipette solutions under
glucose-free conditions, where cells had also been preincubated without
glucose for 95 to 330 minutes. Current blockade by ST 638 was also
generally unaffected by these maneuvers. There was some attenuation of
the ST 638–mediated current blockade in 0.5 mmol/L ATP--S in the
presence of glucose and in ATP-free conditions in the absence of
glucose, but these effects were very small (7% and 4% reduction of
the response to ST 638, respectively) and are unlikely to be of
mechanistic significance. The recovery of IK on drug
removal was also not significantly altered by ATP exclusion (not
shown).
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Inhibition of phosphatase-mediated tyrosine dephosphorylation would
also be expected to block current inhibition by the PTKIs, since
dephosphorylation in the presence of the PTKIs would require ongoing
phosphatase activity. However, Fig 2 shows that
orthovanadate, which blocks tyrosine phosphatases,18 19 20
did not significantly affect the responses to ST 638 (Fig 2a and 2c)
and genistein (Fig 2b and 2d). This was true whether cells were
incubated in 0.5 mmol/L extracellular orthovanadate before (for 75 to
200 minutes) and during experiments (Fig 2a and 2b) or whether
orthovanadate was included in the pipette solution at a concentration
of 0.2 mmol/L (Fig 2c and 2d). The recovery of IK from
blockade was also not significantly affected by orthovanadate (not
shown).
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Figs 3 and 4 illustrate additional
aspects of the blockade of IK by ST 638. To define the
potential dependence of the effect of ST 638, cells were held at -60
mV and depolarized for 100 ms at a frequency of 0.1 Hz, with each
successive depolarization being incremented by +10 mV. The currents
elicited at -20, 0, +20, and +40 mV in the absence and presence of 10
µmol/L ST 638 are shown superimposed in Fig 3a. The current decayed
quickly in the presence of ST 638 over a range of membrane potentials.
This decay was well fitted by a single monoexponential function with
time constants of 33.5±7.3 ms at 0 mV, 25±4 ms at +20 mV, 22.9±6.1
ms at +40 mV, and 21.2±7.3 ms at +60 mV (n=5 for 0 mV, n=6 for other
potentials). The amplitudes of IK measured at the end of
each depolarizing step in the presence and absence of ST 638 are shown
in the current-voltage (I-V) plot, as is the amplitude measured at the
peak of the current in the presence of ST 638 (Fig 3b). The I-V
relation indicates that the inhibition of IK by ST 638
showed little dependence on the magnitude of depolarization. Fig 3c
shows the effects of three concentrations of ST 638 on IK.
It is apparent that at each concentration of the drug, the extent of
current inhibition increased with time during depolarization. As shown
in Fig 3d, this was manifested as a significant difference between the
apparent potency of ST 638 when its inhibition of the current was
measured 4 to 6 ms after the initiation of depolarization (when the
current resistant to 10 µmol/L ST 638 reached its peak) and when it
was measured 100 ms into the depolarization. The IC50 for
the inhibition of the IK measured at the end of a 100 ms
step to +60 mV was 2 µmol/L, which is 4.3-fold less than the
IC50 obtained if the current was measured at 4 to 6 ms.
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One possible explanation for this time-dependent increase in current
inhibition by ST 638 is that the drug has a use-dependent effect. To
examine for this, cells were subjected to a train of 20 depolarizing
steps of 100-ms duration applied at a frequency of 0.5 Hz. Application
of this train led to a progressive diminution of IK under
control conditions, possibly due to accumulation of current
inactivation (Fig 4a
). The amplitude of IK was completely
restored during a subsequent 1-minute stimulation-free interval, and a
second pulse train caused a similar diminution of IK. When
2 µmol/L ST 638 was then added at the beginning of a second 1-minute
rest period, a typical inhibition of the current amplitude in response
to the first pulse was observed. The current then declined further
during the rapid pulse train (Fig 4a). However, the extent and time
course of this decline were not different from those observed in the
absence of drug (Fig 4b), thus suggesting that ST 638 was not having a
use-dependent effect. Similar experiments carried out in two cells
using a higher frequency of test pulses (20 applied at 1 Hz) also
revealed no apparent use-dependent effect (not shown).
To investigate the effect of ST 638 on the potential dependence of
IK inactivation, the availability of IK was
measured in the absence and presence of drug, using a 100-ms test pulse
to +60 mV, which followed a 10-s step to a conditioning potential
between -90 or -120 and +30 mV. The amplitude of IK was
measured at the end of the test pulse and normalized to that measured
after the conditioning step to -90 mV. Fig 4c illustrates a typical
experiment where the availabilities of IK in the control
solution (open circles) and in the presence of 2 µmol/L ST 638
(triangles) were compared. This concentration of the drug, which
inhibited IK by 50%, produced no apparent shift in its
availability. In a number of similar experiments, the potential of
half-inactivation (V0.5) was -23.9±6.8 mV (n=7) in
controls and -29.6±6.5 mV (n=6) in 2 µmol/L ST 638; the slope
factors (k) were 7.8±2.4 mV and 7.4±2 mV, respectively. Fig 4c also
shows that in 10 µmol/L ST 638, the inactivation of the transient
current (at its peak, 4 to 6 ms after depolarization was initiated)
occurred over a much more negative potential range than did that of the
control current. In this case, V0.5 was -58.2±7.3 mV, and
k was 18.7±2.3 mV in five cells studied (P<.001 compared
with control or 2-µmol/L values).
Fig 5a illustrates the effect of genistein (100
µmol/L) on IK at several test potentials; the resulting
I-V relation is illustrated in Fig 5b. No obvious effect of potential
on IK inhibition was apparent. The inhibition of
IK and of the ST 638–resistant current by 20 and 100
µmol/L genistein is shown in Fig 5c and 5d, respectively. It was
apparent that genistein was a more effective blocker of the ST
638–resistant current than it was of IK itself. This
difference is more easily visualized when the data are normalized, as
shown in Fig 5e. It is clear, for example, that 20 µmol/L genistein
blocked the ST 638–resistant current at least as much as 100 µmol/L
genistein blocked IK itself.
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Fig 6 shows the effects of ST 638 and genistein in
another type of smooth muscle cell, that of the rabbit pulmonary
artery. Here, IK has a particularly prominent A-like
component.10 Panels a and b demonstrate that this rapidly
decaying current component was almost insensitive to 10 µmol/L ST
638, which, however, blocked most of the current measured at 100 ms.
Conversely, 100 µmol/L genistein blocked most of the A-like current,
while having little effect on the current measured at 100 ms (Fig 6c and 6d).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although many PTKs exist as plasmalemmal receptors that are activated directly by the binding of growth factors, there are a large number of types of nonreceptor PTKs that may also serve as key intermediates in mediating signal transduction via cytokine and G protein–linked receptors.3 21 A number of recent reports have suggested that contractions of smooth muscle elicited by agonists such as angiotensin II and noradrenaline, which stimulate G protein–linked receptors, are mediated in part by as-yet-unidentified PTKs.5 6 It is also the case that orthovanadate, which blocks tyrosine phosphatases, induces smooth muscle contraction in parallel with tyrosine phosphorylation of as-yet-unidentified cellular proteins20 and that several growth factors that are known to bind to receptor tyrosine kinases cause tension development.4 It is noteworthy that evidence for the role of putative tyrosine kinases in the contractile responses of smooth muscle to G protein–linked receptor stimulation and to orthovanadate rests largely on the finding that such responses are blocked by pharmacological PTKIs, especially genistein.6 20
Our results indicate, however, that genistein and ST 638 both block IK in concentration ranges similar to those used to inhibit PTKs,11 12 13 14 15 16 although their effects on IK are not mediated by PTK inhibition. Therefore, it appears that two structurally dissimilar PTKIs, which act via different mechanisms to inhibit PTKs, both share the ability to block voltage-gated K+ channels in vascular smooth muscle cells. It has previously been shown that genistein and tyrphostins 1 and 23 block the L-type Ca2+ currents in vascular smooth muscle cells.7 This effect was not shared by diadzein, which is often used with genistein as a control, since it is structurally similar but inactive as a PTKI.16 These data therefore suggested that the effects of these agents were due to some type of effect on a PTK. However, our present results show that diadzein is also ineffective in mimicking an action of genistein that is not mediated by inhibition of a PTK. Therefore, it appears that genistein and ST 638 are somewhat nonselective in their actions and that diadzein may not always be an appropriate control agent. Observations that tyrosine kinase inhibitors, including genistein, cause a selective suppression of agonist- but not depolarization-induced contractions underpin the emerging concept that tyrosine kinases may play an important role in excitation-contraction coupling in vascular and visceral smooth muscle.5 6 However, the present results indicate that observations made in isolated tissues may reflect effects that are independent of tyrosine kinase inhibition. The contribution of such additional actions of tyrosine kinase inhibitors to the inhibition of agonist-mediated contractions remains an important subject for future investigation.
ST 638 not only reduced the amplitude of IK but caused it to decay much more rapidly than was observed in the absence of drug. One explanation for this might be that ST 638 was producing some type of use-dependent block, possibly by interacting preferentially with open or inactivated states of the channel mediating IK. We did not observe, however, any evidence for use dependence when cells were stimulated at high frequencies, although we cannot exclude entirely the possibility of a fast open-channel blockade. There was also no indication that the increased channel opening (and likely inactivation) associated with progressive depolarization promoted the inhibition of IK.
A second explanation for the effects of ST 638, which would explain all of the results obtained in the rat pulmonary artery cells, is that IK is composed of two components that are differentially blocked by ST 638. One is a relatively small A-like current that inactivates rapidly during depolarization and is also inactivated over a fairly negative range of conditioning potentials, as it was described in other types of smooth muscle cells.10 22 23 24 25 This component would be relatively insensitive to ST 638. A second, larger component would be more sensitive to ST 638 and would give rise to a current that inactivated more slowly during depolarization and over a more positive range of conditioning potentials. This arrangement would explain the apparent lower potency of ST 638 against the current measured 5 ms after the initiation of depolarization compared with that measured after 100 ms. This assumption would also be consistent with the observations that the rapidly decaying current observed in the presence of 10 µmol/L ST 638 inactivated over a more negative potential range than did the current in the absence of drug, even though a concentration of drug that inhibited the current by 50% (2 µmol/L) had no effect on the availability of the current measured at 100 ms. The latter observation suggests that ST 638 itself, at a concentration sufficient to markedly diminish the current, did not affect its inactivation-potential relation. Conversely, in the former case, the availability of the ST 638–resistant current would mostly reflect that of the small rapidly inactivating component of current.
The response of the rabbit pulmonary artery cells to ST 638 is also consistent with this model. These cells have previously been shown to demonstrate a prominent A-like current that inactivates over a negative potential range.10 The rapidly decaying component of current in these cells was only slightly blocked by ST 638, whereas the current measured at 100 ms was very markedly reduced.
If this model of two components of current is correct, it also suggests that genistein might have a greater action on the A-like current than on the slowly inactivating component of current. This possibility is supported by the finding that genistein slowed current activation (consistent with the blockade of a rapidly activating and inactivating component of current) and also by our observation that genistein had a greater effect on the ST 638–resistant current than on IK measured 100 ms after depolarization, at which time IK should consist almost entirely of the slowly inactivating component. The effect of genistein on the K+ current in the rabbit pulmonary artery cells, in which it essentially abolished the A-like component of current yet had little effect on the current measured at 100 ms, provides additional support for this concept.
Although our present observations appear to be entirely consistent with this model, they do not rule out the possibility that these compounds are interacting differentially with one type of K+ channel via mechanisms not considered above. Confirmation of the mechanisms of action of these agents must therefore await a more detailed scrutiny, possibly at the single-channel level.
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Acknowledgments |
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We thank the British Heart Foundation, the Wellcome Trust, and the Special Trustees for St Thomas' Hospital for financial support and Dr O. Osipenko for the isolation of rabbit pulmonary artery cells. ST 638 was kindly supplied by T. Shiraishi of the Kanegafuchi Chemical Industry Co, Japan.
Received June 27, 1994; accepted October 18, 1994.
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B. H. Choi, J.-S. Choi, D.-J. Rhie, S. H. Yoon, D. S. Min, Y.-H. Jo, M.-S. Kim, and S. J. Hahn Direct inhibition of the cloned Kv1.5 channel by AG-1478, a tyrosine kinase inhibitor Am J Physiol Cell Physiol, June 1, 2002; 282(6): C1461 - C1468. [Abstract] [Full Text] [PDF]
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