1 Department of Molecular and Cellular Physiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0576; and 2 First Department of Physiology, School of Medicine, University of Ryukyus, Nishihara, Okinawa 903-0215, Japan
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ABSTRACT |
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To clarify
interactions between the cytoskeleton and activity of L-type
Ca2+ (CaL) channels in vascular smooth muscle
(VSM) cells, we investigated the effect of disruption of actin
filaments and microtubules on the L-type Ca2+ current
[IBa(L)] of cultured VSM cells (A7r5 cell
line) using whole cell voltage clamp. The cells were exposed to each
disrupter for 1 h and then examined electrophysiologically and
morphologically. Results of immunostaining using anti--actin and
anti-
-tubulin antibodies showed that colchicine disrupted both actin
filaments and microtubules, cytochalasin D disrupted only actin
filaments, and nocodazole disrupted only microtubules.
IBa(L) was greatly reduced in cells that were
exposed to colchicine or cytochalasin D but not to nocodazole.
Colchicine even inhibited IBa(L) by about 40%
when the actin filaments were stabilized by phalloidin or when the
cells were treated with phalloidin plus taxol to stabilize both
cytoskeletal components. These results suggest that colchicine must
also cause some inhibition of IBa(L) due to
another unknown mechanism, e.g., a direct block of CaL
channels. In summary, actin filament disruption of VSM cells inhibits
CaL channel activity, whereas disrupting the microtubules
does not.
cytoskeleton; microtubules; colchicine; cytochalasin D; nocodazole
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INTRODUCTION |
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THE CYTOSKELETON IS MADE
UP of microfilaments (actin), microtubules, and intermediate
filaments (IFs). The microfilaments are composed of subunits of actin
( and
), and the microtubules are composed of tubulin (
and
). The IFs are composed of several classes of IF protein. Changes in
the cytoskeletal network alter the mechanical properties of the cell
that are essential for functions such as locomotion (16)
and cytokinesis (6). The cytoskeletal network also
provides a scaffolding on which motor proteins, such as kinesin,
dynein, and myosin, can translocate to move organelles or generate
internal stress. The transmembrane receptors and intracellular signals
producing cytoskeletal changes in response to extracellular stimuli
have been extensively studied and are the subject
of several recent reviews (13, 30,
39).
The influence of cytoskeletal structures on signaling in vivo is often detected by the effects of specific agents, such as cytochalasin, nocodazole, or acrylamide, that selectively disrupt actin filaments, microtubules, and IFs, respectively. Ion channels and other ion transport molecules (e.g., Na+-K+ pump and Na+/Ca2+ exchanger) are integral to the plasma membrane and are attached to cytoskeletal strands, in particular actin filaments (12, 24). Actin accounts for >20% of the total cell proteins, and actin and actin-binding proteins couple to several ion channels and ion transport molecules (4).
Intracellular Ca2+ is known to play an important role in regulating cell morphology. Early studies showed the Ca2+ sensitivity of actin and actin-binding proteins, which are major cytoskeletal components (20, 33). Moreover, Ca2+ ions regulate the formation of actin bundles and networks (3, 36).
Fukuda et al. (7) obtained indirect evidence that the cytoskeleton may regulate Ca2+ channel kinetics. They showed that colchicine caused a reduction of the upstroke velocity of action potentials, and they inferred that Ca2+ channels interact with the cytoskeleton. Johnson and Byerly (14) have provided direct evidence that microtubules are implicated in the inactivation of snail neuron Ca2+ currents. These authors reported that cytoskeletal disrupters and stabilizers affect the rundown of Ca2+ (CaL) channels. Galli and DeFelice (8) reported that colchicine and taxol strongly influence the kinetics of L-type Ca2+ channels in intact cardiac cells through their action on the cytoskeleton, which, in turn, might regulate the effective concentration of inactivating ions near the channel mouth. It is not clear whether the actin filaments and/or microtubules are involved in this regulation.
We investigated several of the cytoskeletal regulatory mechanisms for CaL channels in a cultured vascular smooth muscle (VSM) cell line (A7r5) derived from rat aorta. The relationship between the cytoskeleton and CaL channel activity has not been clearly defined for VSM cells. To clarify the interactions between the cytoskeleton and CaL channel activity, we used specific disrupters of different components of the cytoskeleton, namely, cytochalasin D for the actin microfilaments, nocodazole for the microtubules, and colchicine for both components. We confirmed the disruption by immunofluorescence staining. The effects of disruption of actin filaments and microtubules on the CaL channel current of cultured VSM cells (A7r5 cell line) were measured using the whole cell voltage clamp. We found that disruption of the actin filaments inhibited activity of the CaL channels, whereas microtubule disruption had no effect.
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MATERIALS AND METHODS |
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Cell culture. A7r5 cells were purchased from American Type Culture Collection (Rockville, MD) and maintained as previously reported (28) under 5% CO2 at 37°C in DMEM (Life Technologies, Grand Island, NY) containing 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 100 U/ml penicillin G, and 100 mg/ml streptomycin sulfate (Life Technologies). After cells were grown to confluence, the cells were trypsinized by treatment for 5 min with 0.05% trypsin (Clonetics, San Diego, CA), plated on microscope glass coverslips (Fisher Scientific, Pittsburgh, PA) coated with poly-L-lysine (Sigma Chemical, St. Louis, MO), and then grown again to confluency. On the day of each experiment, the cells on coverslips were trypsinized for 1 min to isolate the cells and were then incubated for 30 min in serum-free DMEM (SFM) at CO2 incubator. To study the effect of various disrupters (colchicine, cytochalasin D, nocodazole, and the mixture of nocodazole with cytochalasin D), the cells were exposed to SFM containing each drug for 1 h at 37°C. To study the effect of the cytoskeleton stabilizers (phalloidin, taxol), the cells were exposed to SFM containing each drug for 30 min at 37°C. These cells were then exposed to SFM containing cytoskeleton disrupters (plus the stabilizer) for 1 h at 37°C. For immunostaining of cytoskeleton, the cells were fixed immediately after they were rinsed with phosphate-buffered saline (PBS). For electrophysiological study, the cells were kept under 5% CO2 at 37°C in SFM until the start of measuring CaL channel current by whole cell voltage clamp (usually between 1 and 2 h after treatment). That is, all current measurements were made after washout of all drugs. Cells examined by immunostaining 0.5-2 h after wash out of the cytoskeletal disrupters showed continued marked disruption of the cytoskeletal components. Therefore, we completed our electrical measurements within 2 h. The drugs were removed to circumvent the possibility that the drugs might have a direct effect on the ion channels.
Immunofluorescence study.
For immunofluorescence study, the cells were incubated in the absence
(control) or presence of each disrupter (colchicine, cytochalasin D,
nocodazole, or the mixture of nocodazole with cytochalasin D) in SFM
for 1 h in CO2 incubator at 37°C. Control cells were
incubated in the presence of 0.1% DMSO (final concentration) in SFM.
After incubation, the cells were washed with PBS containing 1 mM EGTA
(PBS-EGTA). The cells were washed three times with PBS-EGTA at every
step in this immunofluorescence experiment. The cells were fixed for 30 min with 10% Formalin and permeabilized with 0.5% Triton X-100 in
PBS-EGTA. The cells were incubated with blocking solution (20% normal
goat serum in PBS-EGTA) for 30 min at room temperature. To detect actin
filaments and microtubules, monoclonal antibody against smooth muscle
anti--actin (Sigma) and anti
-tubulin (Sigma) was used as a
primary antibody, respectively. The cells were reacted with primary
antibody for 30 min and reacted with FITC-conjugated goat anti-mouse
IgG (Sigma) for 30 min in a dark room. After the cells were mounted on
the microscope slides with Gel/Mount (Biomed), the cells were
visualized using a fluorescent microscope.
Electrophysiological study.
Whole cell voltage-clamp recordings were made by using a patch-clamp
amplifier (Axopatch-1D; Axon Instruments, Foster City, CA) according to
the standard techniques. All experiments were performed at room
temperature (20-22°C). The recording electrodes (resistance of
2-5 M) were made from borosilicate glass capillary tubing by
using a two-stage puller. Coverslips with attached cells were placed in
a small chamber on an inverted microscope (Diaphoto-TMD; Nikon, Tokyo, Japan).
Experimental solutions. Seal formation was accomplished in a bath solution having the following composition (in mM): 140 NaCl, 6 KCl, 2.5 MgCl2, 10 glucose, and 10 HEPES; the pH was adjusted to 7.4 with Tris. To isolate the Ca2+ channel current in the whole cell configuration, the bath solution was changed to the following Na+- and K+-free solution (in mM): 140 tetraethylammonium (TEA)-Cl, 5 BaCl2, 1 MgCl2, 11 glucose, 10 HEPES, and 5 4-aminopyridine; pH was adjusted to 7.4 with TEA-OH. The pipette solution consisted of (in mM) 100 CsOH, 30 CsCl, 1 MgCl2, 10 EGTA, 10 HEPES, 112 L-glutamate, and 5 ATP·Na2; pH was adjusted to pH 7.2 with CsOH.
Drugs and reagents.
Colchicine, cytochalasin D, nocodazole, phalloidin, and taxol were
purchased from Sigma Chemical. The stock solutions of the disrupters
and stabilizers, which were dissolved in DMSO, were stored at 20°C.
The final concentration of DMSO to which the cells were exposed was
0.01% (0.1% at 100 µM cytochalasin D); this concentration
by itself was shown to have no significant effect on
IBa(L) in 10 cells tested.
Data analysis. All data are given as means ± SE. Statistical analyses were performed by Student's unpaired t-test and one-way ANOVA, followed by Dunnett's multiple comparison by using the software of StatView (version 4.5; Abacus Concepts, Berkeley, CA). A value of P < 0.01 was taken to be statistically significant.
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RESULTS |
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Disruption of actin filaments and microtubules produced by the
various cytoskeletal disrupters was confirmed by immunofluorescence staining using specific antibodies to -actin and
-tubulin. In control cells treated with 1% DMSO (the solvent), a dense array of
actin filaments (top left) and a fine mesh of microtubules (top right) were seen in Fig.
1A (control).
Those typical structures disappeared 1 h after exposure of cells
to 100 µM colchicine with results in diffuse fluorescence in the
cytoplasma (Fig. 1B). Cytochalasin D (100 µM) produced
prominent changes in the actin filaments; however, the fine mesh of
microtubules similar to that in control remained (Fig. 1C).
These results showed that colchicine (100 µM) disrupted both actin
filaments and microtubules, whereas cytochalasin D (100 µM) only
disrupted actin filaments.
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To test the effect of disruption of cytoskeleton on the CaL
channel activity, IBa(L) was measured 1 h
after treatment with the various cytoskeleton disrupters in A7r5 cells.
After the cells were exposed to the different concentrations of
colchicine (1-100 µM) for 1 h, the
IBa(L) was measured under the conditions of 130 mM Cs+ in the pipette solution and 5 mM Ba2+ in
the bath solution. To obtain current-voltage relationships (I-V), IBa(L) was elicited every
15 s by various test potentials (30 to +60 mV, 10-mV increments,
300-ms duration from a holding potential of
40 mV). The control
I-V relationship had a maximal amplitude of
7.88 ± 0.78 pA/pF at a test potential of +10 mV. The peak amplitude of
IBa(L) was inhibited by colchicine in a dose-dependent manner: 1 µM,
5.95 ± 0.73 pA/pF; 10 µM,
4.5 ± 0.37 pA/pF; and 100 µM,
2.62 ± 0.3 pA/pF (Fig.
2). Because the reversal potentials of
colchicine-treated cells were all near to 60 mV, colchicine inhibition
of IBa(L) was not due to stimulation of an
outward current (e.g., ICl). The results
of immunostaining of cytoskeleton showed that 10 µM colchicine also
disrupted both actin filaments and microtubules, whereas 1 µM
colchicine disrupted microtubules but not actin filaments (data not
shown). Therefore, CaL channel activity was inhibited by
colchicine most likely through either disruption of actin filaments or
microtubules (Fig. 2).
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Because we found that colchicine actually disrupted both actin
filaments and microtubules at higher concentrations than 1 µM, a
specific actin filament disrupter, cytochalasin D, was used to test the
effect of disruption of actin filaments on the CaL channel
activity. The I-V relationships were obtained from the cells
without the drug (control) or after exposure to 100 µM cytochalasin D. In control, maximal peak amplitude was 8.21 ± 0.47 pA/pF at +10 mV of a test potential. The peak amplitude of
IBa(L) was inhibited to
2.99 ± 0.3 pA/pF
by 100 µM cytochalasin D (Fig. 3). As
shown in Fig. 1B, only actin filaments were completely
disrupted by 100 µM cytochalasin D. Therefore, CaL
channel activity was inhibited by cytochalasin D likely due to
disruption of actin filaments.
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To compare the degree of inhibition of CaL channel activity
by colchicine and cytochalasin D, various concentrations of these drugs
were tested. Figure 4 shows the
concentration-response relations for the inhibitory effect of these
drugs on IBa(L). The inhibition of
IBa(L) produced by 10 and 100 µM cytochalasin
D was statistically significant compared with control
(P < 0.01 by one-way ANOVA): 10 µM, 5.9 ± 0.4 pA/pF (n = 14); and 100 µM,
4.9 ± 0.6 pA/pF (n = 10). The inhibition of
IBa(L) produced by 10 and 100 µM colchicine was also statistically significant compared with control
(P < 0.01 by one-way ANOVA): 10 µM,
4.5 ± 0.4 pA/pF (n = 14); and 100 µM,
2.6 ± 0.3 pA/pF (n = 12). As can be seen in Fig. 4, colchicine inhibition of IBa(L) was more potent than that
produced by cytochalasin D.
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We examined whether colchicine and cytochalasin D affected the
inactivation of Ca2+ current. The time constants () for
colchicine (100 µM) and cytochalasin D (100 µM) were not
statistically significant compared with control (P = 0.146 by one-way ANOVA; Table 1).
Therefore, colchicine and cytochalasin D did not affect the decay of
CaL channel current.
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The actin filament stabilizer phalloidin was used to further verify the
involvement of actin filaments in the inhibition of the CaL
channel activity produced by cytochalasin D or colchicine. The peak
amplitude of IBa(L) (at +10-20 mV) was (in
pA/pF) phalloidin, 7.18 ± 0.64 (n = 10);
phalloidin plus cytochalasin D,
7.03 ± 0.69 (n = 8); and phalloidin plus colchicine,
4.24 ± 0.64 (n = 8) (Fig. 5, Table
2). Therefore, the cytochalasin D
inhibition of IBa(L) was completely prevented by
phalloidin, whereas the larger colchicine inhibition was only partially
prevented (Fig. 5, Table 2). Immunostaining showed that the actin
filaments were not disrupted by cytochalasin D or by colchicine in the
presence of phalloidin.
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To test the possible involvement of microtubules in the excess
inhibition of IBa(L) produced by colchicine, a
combination of phalloidin and taxol was used to stabilize both
components of the cytoskeleton. As can be seen in Table 2 and Fig.
6, IBa(L) was
inhibited (by ~36%) by colchicine even when the cytoskeleton was
completely stabilized. Therefore, colchicine must exert an effect on
the CaL channels that is independent of the cytoskeleton. Colchicine also failed to disrupt either cytoskeletal component when
the cells were treated with phalloidin plus taxol.
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Because colchicine disrupted both actin filaments and microtubules (as
shown in Fig. 1), it was expected that the difference in inhibition of
IBa(L) between colchicine and cytochalasin D occurred due to the disruption of microtubules. To test this
hypothesis, a specific microtubule disrupter, nocodazole, was used.
Immunostaining of cytoskeleton after exposure to 1.3 µM nocodazole
demonstrates that a dense array of actin filaments can be seen in
similar structure to that in control (Fig.
7A,
left). There was diffuse fluorescence in the
cytoplasma when anti--tubulin was used (Fig. 7A,
right). Therefore, nocodazole disrupted microtubules, but
not actin filaments, even when the concentration was increased to 10 µM (data not shown). The I-V relationships were obtained
from the cells without the drug (control) or after exposure to 1.3 µM
nocodazole. In control, maximal peak amplitude was
7.68 ± 0.78 pA/pF at +10 mV of a test potential. The peak amplitude of
IBa(L) was not inhibited by nocodazole (Fig.
7B).
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Although both actin filaments and microtubules were disrupted after exposure to the mixture of 10 µM nocodazole and 10 µM cytochalasin D (data not shown), there was no further inhibition of IBa(L) compared with inhibition produced by cytochalasin D alone (Table 1). Therefore, the difference in inhibition of IBa(L) between colchicine and cytochalasin D may not be due to the disruption of microtubules.
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DISCUSSION |
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We investigated the effects of disruption of actin filaments and microtubules on the L-type Ca2+ current [IBa(L)] of cultured VSM cells (A7r5 cell line) using whole cell voltage clamp. Immunostaining was used to confirm the degree of disruption. Colchicine disrupted both actin filaments and microtubules (at concentrations >1 µM), whereas cytochalasin D disrupted only actin filaments (even when the concentration was increased up to 100 µM). IBa(L) was greatly reduced in cells that were exposed to 100 µM colchicine or cytochalasin D. In the presence of phalloidin, cytochalasin D (100 µM) did not disrupt actin filaments and did not inhibit IBa(L). Phalloidin alone, which stabilizes actin filaments, did not affect IBa(L). Nocodazole, which only disrupted microtubules, did not affect IBa(L). Therefore, only disruption of actin filaments produced inhibition of CaL channel activity.
Interactions between various ion channel proteins and the cytoskeleton have been implicated in mediating spatial localization of channel proteins and in the regulation of ion channel activity (9, 25). For example, Galli and DeFelice (8) reported that colchicine strongly influences the kinetics of L-type Ca2+ channels in intact cardiac cells. They reported that colchicine (80 µM, 15 min) caused the single CaL channels of chick ventricular myocardial cells to have a greater probability of being in the closed state, and the inactivation time constant was decreased. Thus there would be inhibition of the whole cell ICa(L). Therefore, our results on VSM cells are in essential agreement with those reported for myocardial cells.
Bonfoco et al. (2) found that the intracellular Ca2+ concentration ([Ca2+]i) response to high-K+ depolarization of neuronal cells was increased by colchicine, and the increase was abolished by nifedipine. Therefore, they concluded that disruption of the cytoskeleton stimulated the activity of the CaL channels. These results are opposite to what we have found for VSM cells. However, because they did not measure the CaL channel activity, it is not clear whether their observed increase in [Ca2+]i produced by colchicine was due to stimulation of CaL channel activity. If stimulation of CaL channel activity did occur with colchicine, it could have resulted from stimulation via the cAMP/cAMP-dependent protein kinase (PKA) pathway [because the cytoskeleton affects adenylate cyclase (A-cyclase) activity]. Also, it was reported (32) that disruption of the microtubules (by colchicine or oryzalin) caused a very large increase in voltage-dependent Ca2+ channel activity (and half-life) of carrot cells, whereas the microtubule stabilizer taxol had no effect.
Although, increasing [Ca2+]i produces Ca2+ inhibition of Ca2+ channels (17), because we used 5 mM Ba2+ in the bath solution (without Ca2+) and a Ca2+-free solution (with 10 mM EGTA) in the pipette, an increase of intracellular [Ca2+]i should not occur under these conditions. Therefore, the decrease in ICa(L) we found upon actin-filament disruption cannot be explained by Ca2+ inhibition of Ca2+ channels. In addition, colchicine and cytochalasin D did not affect the voltage-dependent inactivation of CaL channel current.
The tyrosine kinase inhibitor genistein was found to inhibit basal ICa(L) in whole cell voltage clamp of rat ventricular cells (38) and to significantly inhibit single-channel activity in rat portal vein VSM cells (15). A nonreceptor tyrosine protein kinase (Tyr-PK), pp60c-Src, was reported to stimulate Ca2+ channel currents in rabbit ear artery VSM cells (34). These results suggest that Ca2+ channels are modulated (stimulated) by phosphorylation with Tyr-PK. Some nonreceptor Tyr-PKs that are bound to the cytoplasmic face of the plasma membrane may play a role in signaling (22). pp60c-Src was the first protein kinase found to translocate to the cytoskeleton after its activation. The actin filaments are involved in this translocation (5). c-Src moves from the cytosol to the cytoskeleton near the cell membrane when cells are infected with a virus, and the actin-binding protein vinculin is involved (1). In addition, translocation of c-Src to the cytoskeleton of A-172 glioblastoma cells is induced by both platelet-derived growth factor and epidermal growth factor in a process that also activates tyrosine kinase activity (18). Stimulation of platelets by thrombin also translocates c-Src to the actin cytoskeleton (10). In fibroblasts, translocation of c-Src to near the cell membrane could be blocked by cytochalasin D, but not by nocodazole (5). These results suggest that disruption of actin filaments releases the nonreceptor Tyr-PK (c-Src) from the cytoplasmic face of the plasma membrane into the cytoplasm. As a result of this translocation, the nonreceptor Tyr-PK would not be able to activate the CaL channel by phosphorylation. Therefore, CaL channel activity would be inhibited by disruption of actin filaments.
Although 1 µM colchicine dose not disrupt actin filaments, this low dose reduced CaL channel activity (see Fig. 2). In addition, colchicine inhibited IBa(L) by 40% when actin filaments were stabilized by phalloidin. Colchicine still inhibited IBa(L) by 36% even when the cells were treated with phalloidin plus taxol to stabilize both cytoskeletal components (see Table 2). These results suggest that colchicine must also cause some inhibition of IBa(L) due to another unknown mechanism e.g., a direct block of CaL channels. That is, in addition to the disruption of the actin cytoskeleton and resultant partial inhibition of the activity of the CaL channels, because colchicine produced stronger inhibition of CaL channel activity than did cytochalasin D (see Fig. 4), colchicine must exert an additional effect. Therefore, colchicine not only disrupts the cytoskeleton but may also directly inhibit the CaL channels.
Because colchicine also stimulates A-cyclase activity by disruption of the microtubules (21), it should increase cAMP level. Ousterhout and Sperelakis (19) reported that inhibition of CaL channels and depression of membrane excitability may be important factors in the relaxation of aortic smooth muscle produced by agents that increase intracellular levels of the cyclic nucleotides cAMP and cGMP. Xiong et al. (35) showed that intracellular application of PKA (catalytic subunit) caused marked inhibition of ICa(L). Ruiz-Velasco et al. (23) reported that cAMP/PKA stimulation (low) enhanced, whereas high elevation and cGMP/cGMP-dependent protein kinase (PKG) stimulation inhibited CaL channel activity in rabbit portal vein myocytes. We recently also showed that low elevation of cAMP actually stimulated ICa(L) (29, 37). When PKG is added to the patch pipette for diffusion into the cell during whole cell voltage clamp, basal ICa is inhibited markedly (and rapidly) in both embryonic chick ventricular cells (11) and in early neonatal rat ventricular myocytes (27). These findings indicate that the inhibitory effects of cGMP on ICa are mediated by activation of PKG and resultant phosphorylation, and that the basal ICa is inhibited (26). Even though an increase in cAMP might occur due to disruption of microtubules by colchicine, the contribution of this pathway to colchicine inhibition of IBa(L) must be small because the colchicine inhibition of IBa(L) was about the same in the presence of phalloidin (only microtubule disruption) and in phalloidin plus taxol (no disruption). The lack of involvement of the cAMP/PKA pathway is also supported from the fact that nocodazole (which disrupted the microtubules, and thereby should stimulate the A-cyclase) did not inhibit CaL channel activity (Fig. 5). Perhaps the association between A-cyclase and microtubules may be absent in cultured cells.
In conclusion, actin filament disruption of VSM cells inhibits CaL channel activity, whereas disrupting the microtubules does not. Nonreceptor Tyr-PK, which is located on the actin-binding protein linking the ion channel to the actin filament, would be displaced from the cytoplasmic face of the plasma membrane by the disruption of the actin filaments. Therefore, actin-filament disruption may interfere with phosphorylation of the CaL channel by Tyr-PK, thereby removing a basal stimulatory pathway. Alternately, the linking of the ion channel to the actin cytoskeleton by means of a linking protein acts to tether the channel in place, which somehow modulates basal activity of the channel and its regulation (31).
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ACKNOWLEDGEMENTS |
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We acknowledge Drs. K. Fukasawa and M. Okuda for excellent technical help with immunostaining.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-40572.
Address for reprint requests and other correspondence: M. Nakamura, 1st Dept. of Physiology, School of Medicine, Univ. of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan (E-mail: mnaka{at}cosmos.ne.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 22 October 1999; accepted in final form 8 March 2000.
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