Division of Physiology, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The integrity of
F-actin and its association with the activation of a
Cl current
(ICl) in
cultured chick cardiac myocytes subjected to hyposmotic challenge were
monitored by whole cell patch clamp and fluorescence confocal
microscopy. Disruption of F-actin by 25 µM cytochalasin B augmented
hyposmotic cell swelling by 51% (from a relative volume of 1.54 ± 0.10 in control to 2.33 ± 0.21), whereas stabilization of F-actin
by 20 µM phalloidin attenuated swelling by 15% (relative volume of
1.31 ± 0.05). Trace fluorochrome-labeled (fluorescein
isothiocyanate or tetramethylrhodamine isothiocyanate) phalloidin
revealed an intact F-actin conformation in control cells under
hyposmotic conditions despite the considerable changes in cell volume.
Sarcoplasmic F-actin was very disorganized and occurred only randomly
beneath the sarcolemma in cells treated with cytochalasin B, whereas no
changes in F-actin distribution occurred under either isosmotic or
hyposmotic conditions in cells treated with phalloidin.
Swelling-activated
ICl (68.0 ± 6.0 pA/pF at +60 mV) was suppressed by both cytochalasin B (22.7 ± 5.1 pA/pF) and phalloidin (22.5 ± 3.5 pA/pF). On the basis of these
results, we suggest that swelling of cardiac myocytes initiates dynamic changes in the cytoarchitecture of F-actin, which may be involved in
the volume transduction processes associated with activation of
ICl.
cell volume; cytoskeleton; cytochalasin B; phalloidin; whole cell patch clamp; confocal microscopy
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CARDIAC CELLS, like many other cell types, volume
regulate when challenged by a reduction of extracellular osmolarity
(30). Regulatory volume decrease (RVD) activated under hyposmotic
conditions is commonly accomplished by a net loss of intracellular
inorganic and organic osmolytes along with osmotically obliged water
(14, 30, 31). Activation of a
Cl-selective conductance
has been associated with volume regulatory processes in response to
cardiac cell swelling (33, 34, 40). Little information is currently
available regarding the signaling mechanisms responsible for the
activation of these Cl
channels. A number of second messenger systems are known to activate membrane transporters involved in cell volume regulation (1, 7, 14,
31). Our previous studies also indicate that intracellular Ca2+ and adenosine
3',5'-cyclic monophosphate (cAMP) levels determine the
activation of the swelling-induced
Cl
current
(ICl) in
cultured chick cardiac myocytes (10, 38). However, mechanisms that
sense changes in cell volume and initiate intracellular second
messengers are still largely unknown.
Several lines of evidence have implicated the cytoskeleton in cell
volume regulation. Cell swelling is associated with changes in F-actin
conformation in a variety of cell types (27). Disruption of F-actin
with cytochalasin B, an inhibitor of actin polymerization, has been
shown to abolish RVD (5, 8, 9, 22), and, therefore, an intact F-actin
network was considered to be essential for a normal volume regulatory
response. Identification of possible cytoskeletal determinants of
volume regulation has been complicated by the observation that
stabilization of F-actin with phalloidin, a compound that prevents
F-actin depolymerization, also inhibits RVD (32). In addition, F-actin
conformation in many cell types appeared to undergo dynamic changes
with a transient disappearance of F-actin at the onset of cell swelling
followed by a gradual reorganization coincident with RVD (4, 12, 26,
41). F-actin is also known to modulate a number of membrane
transporters activated by cell swelling (27). However, diverse results
were reported from different cell types. In P12 pheochromocytoma cells
(5) and shark rectal gland cells (26), disruption of F-actin under isosmotic conditions activated
Cl channels similar to
those identified with hyposmotic swelling, whereas a volume-regulated
ICl in myeloma
cells was only enhanced by F-actin disruption under mild hyposmotic
conditions (19). By contrast, the swelling-activated
ICl in human
endothelial cells was not affected by F-actin disruption (29). In
cardiac myocytes, we have reported an early transient current
(Iswell)
associated with hyposmotic swelling and volume regulation (38). Cells
treated with cytochalasin B displayed an absence of
Iswell (11). The functional relationship between F-actin modulation and the
swelling-activated ICl is yet to be
established.
The present study was conducted to explore the role of F-actin in the volume regulatory processes associated with activation of ICl during cardiac cell swelling. F-actin was modulated using either depolymerizing or stabilizing reagents, and the swelling-induced changes in membrane conductance and F-actin architecture were monitored by whole cell patch-clamp and fluorescence confocal microscopy, respectively. Our results demonstrate that swelling of cultured chick cardiac myocytes initiates signal transduction mechanisms that involve F-actin reorganization. The dynamic disassembly and reassembly of F-actin in response to cell swelling appear to be a component of the volume transduction processes that regulate the activation of ICl.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell preparation. Single myocytes were isolated from 11-day-old embryonic chick hearts by enzymatic dissociation as described previously (15). The resultant myocyte-enriched supernatant was seeded at a density of ~0.5 × 106 cells on 35-mm untreated culture dishes (Corning 25050) or on 12-mm microscope cover glasses (Fisher Scientific, Pittsburgh, PA) and was incubated overnight at 37°C in the absence of antibiotics. Single spherical myocytes (~15-20 µm in diameter) were used for patch-clamp studies and simultaneous cell volume measurement. Cells prepared on the cover glasses were used for F-actin staining and subsequent microscopic studies.
Solutions. The control external
solution was a
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES)-buffered salt solution of the following composition (in
mM): 142 NaCl, 5.4 KCl, 0.8 NaH2PO4, 0.8 MgSO4, 2.0 CaCl2, 5.6 dextrose, and 10 HEPES,
adjusted to pH 7.4 with NaOH and with an osmolarity of 290 mosmol/l.
Solutions were rendered hyposmotic by reduction of the NaCl
concentration. Hyposmotic solutions used in fluorescence studies
contained 25% of total NaCl concentration and had an osmolarity of
~100 mosmol/l, whereas hyposmotic solutions used for cell volume and
electrophysiological studies contained 75% of total NaCl concentration
with an osmolarity of ~230 mosmol/l. In electrophysiological
experiments,
N-methyl-D-glucamine (NMDG) and L-aspartic acid were used as partial substitutes
for NaCl in isosmotic solution, such that NaCl concentration remained constant between changes in external osmolarity. The solution used to
block K+ currents was prepared by
adding 1 mM BaCl2 and replacing
NaH2PO4 and MgSO4 with equimolar
concentrations of NaCl and MgCl2,
respectively. Cl-free
solution was prepared by replacing
Cl
salts with aspartate
salts. The pipette solution for whole cell patch-clamp studies
contained (in mM) 95 L-aspartic acid, 100 NMDG, 2 MgCl2, 0.5 CaCl2, 1.0 ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, 30 tetraethylammonium chloride, 10 HEPES, and 5.0 KATP, adjusted
to pH 7.2 with NMDG. The osmolarity of all solutions was measured with
a vapor pressure osmometer (model 5500; Wescor, Logan, UT). The
Cl
channel blocker
5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; Research Biochemicals,
Natick, MA) and cytochalasin B (Sigma, St. Louis, MO) were dissolved in
dimethyl sulfoxide (DMSO) as stock solutions and were diluted to their
final concentrations of 100 and 25 µM, repectively, in the bath
solutions or culture medium before use. The maximum concentration of
DMSO (0.1%) does not affect the electrical or contractile activity of
cultured chick cardiac myocytes (20, 40). In experiments involving cytochalasin B, cells were preincubated with the same concentration of
cytochalasin B for ~16 h to achieve maximum F-actin depolymerization. Phalloidin (20 µM; Molecular Probes, Eugene, OR) was included directly in the culture media or salt solutions and was loaded into the
cells by preincubation for up to 20 h (24). All of the experiments were
performed at 37°C.
Laser scanning confocal microscopy.
For experiments in which F-actin was perturbed, cells were incubated
with cytochalasin B (25 µM) or fluorescein isothiocyanate
(FITC)-phalloidin (20 µM; Sigma) for 10-20 h as required. During
experiments, cells were immersed in isosmotic solution for 30 min
before being subjected to hyposmotic swelling. The same concentrations
of cytochalasin B or phalloidin were included in all solutions
throughout the experiments. Control cells were incubated either in the
absence of any additives or in the presence of DMSO at the final
concentration used for cytochalasin B-treated cells. After 3 or 20 min
of exposure to the hyposmotic solution (100 mosmol/l), the cells were
quick-frozen in liquid nitrogen-cooled isopentane. Cells quick-frozen
before the exposure to hypotonicity served as controls for the
osmotically induced swelling experiments. The temperature of the frozen
specimens was then gradually increased, and the cells were chemically
stabilized with 3.7% formaldehyde in 0.1 M phosphate-buffered saline
(PBS, pH 7.4) at 4°C. In some cases, cells were fixed with a
mixture of 70% methanol and 30% acetone (vol/vol) at 20°C
or, alternatively, were fixed with 4% paraformaldehyde in a
cytoskeleton stabilizing piperazine-N,N'-bis(2-ethanesulfonic
acid) buffer at 37°C before being stained (18). However, no
difference in F-actin staining was revealed for the various fixation
protocols. After fixation, the cells were rinsed in 0.1 M PBS, and then
the control and cytochalasin B-treated cells were exposed to
tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (4 µg/ml;
Sigma; see Ref. 37). The cells were rinsed and mounted on glass slides
using a fluorescence-preserving medium (VECTASHIELD H1000; Vector
Laboratories, Burlingame, CA). Cells treated with FITC-phalloidin
before fixation were mounted directly on glass slides after being
chemically stabilized and subsequently were rinsed with 0.1 M PBS. The
fluorochrome-labeled cells were examined with a laser scanning
confocal microscope (MRC 1000; Bio-Rad Laboratories, Richmond,
CA) equipped with an argon-krypton laser.
Cell volume measurement. Myocyte volume was determined by video microscopy, using the JAVA image analysis system (Jandel Scientific, Corte Madera, CA). The circumference of the cell image was traced to determine the average cell diameter, which then was converted to the volume of a single myocyte. These morphological measurements were shown to correlate well with measured changes in cell water (30). The changes in cell volume were normalized to the control values just before volume perturbation.
Electrophysiological recording.
Membrane currents were recorded using the patch-clamp technique in the
whole cell configuration (13). Patch pipettes were fabricated from
borosilicate glass capillary tubing (7052; Garner Glass, Claremont,
CA) and were fire polished just before use. The pipette resistance was
3-5 M when filled with pipette solution. Current recordings
were obtained using an Axopatch-1D patch-clamp amplifier (Axon
Instruments, Foster City, CA). Currents were low-pass filtered at 2 kHz
by a four-pole Butterworth filter and were acquired by a Gateway 2000 486DX computer using a Digidata 1200 data acquisition system (Axon
Instruments). pCLAMP-6 software (Axon Instruments) was used to generate
voltage protocols and to digitize and analyze the whole cell currents.
Whole cell currents were elicited by voltage ramps every 20 s, over the
voltage range from 90 to +60 mV at a rate of ±0.5 V/s, from
a holding potential of
40 mV. The distributed capacitance was
compensated immediately after the formation of a gigaohm seal. Cell
membrane capacitance was estimated by integrating the transient current
response to a 5-mV hyperpolarizing step and dividing by that voltage
step. Cell capacitance and series resistance were not compensated
during the experiments. All currents were normalized to cell membrane
capacitance (pA/pF).
Data analysis and statistics. All data are presented as digitized recordings or, in the case of a series of measurements, as means ± SE; n represents the number of experiments. Statistical analysis was made by Student's t-test for paired or unpaired data, and a significant difference was assumed at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of F-actin modulators on cell volume. Conformational changes induced in the cytoskeletal network by cell swelling (3, 41) prompted us to examine the relationship between cytoskeletal integrity and cardiac cell volume control. Cells pretreated with cytochalasin B (25 µM) for ~16 h were superfused in isosmotic bath solution containing the same concentration of cytochalasin B. After 1 day of incubation with cytochalasin B, most cells remained spherical but demonstrated an increase in volume [3.2 ± 0.6 × 103 µm3 (n = 30) to 3.9 ± 0.3 × 103 µm3 (n = 18); P < 0.01]. This increase in cell size coincided with an increase in cell membrane capacitance [7.9 ± 0.3 pF (n = 30) to 11.5 ± 0.6 pF (n = 18); P < 0.01], consistent with the growth of embryonic cardiac myocytes during prolonged incubation in culture medium. After establishment of the whole cell patch-clamp configuration whereby the cytoplasm was dialyzed by the pipette solution, the cells were exposed to a hyposmotic solution (230 mosmol/l). The effect of cytochalasin B on cell volume is illustrated in Fig. 1. When challenged by external hyposmolarity, cells treated by cytochalasin B started to swell at the rate similar to that of the control group. After ~10 min, the volume of control cells already reached a plateau of 1.54 ± 0.10 (n = 5) times the volume in isosmotic solution, whereas cells treated with cytochalasin B continued to swell for ~20 min in hyposmotic solution and approached a plateau of 2.33 ± 0.21 (n = 6; P < 0.01) times the volume in isosmotic solution. The absence of cell volume regulation in whole cell patch-clamp experiments, as observed in many cell types (19, 34, 40), is likely due to dialysis of the cell interior with an infinite pool of the pipette solution, which prevents changes in intracellular osmolarity during hyposmotic challenge (40).
|
Potentiation of the cell volume increase by cytochalasin B suggests that F-actin integrity is critical in cell volume homeostasis, and dissociation of the cell membrane from the cytoskeleton may contribute a further increase in cell volume when hyposmotically challenged. To test this conclusion, experiments were performed with phalloidin, which stabilizes F-actin. Cells were treated with phalloidin (20 µM) for ~20 h, and the same concentration of phalloidin was included in the pipette solution during experiments. The rate of cell swelling among phalloidin-treated cells was much slower than control or cytochalasin B-treated cells (Fig. 1). After ~10 min of superfusion in hyposmotic solution, cell volume reached a plateau of 1.31 ± 0.05 (n = 6; P < 0.05) times the volume in isosmotic solution (Fig. 1).
F-actin distribution during hyposmotic challenge. To determine how F-actin distribution changes during hyposmotic cell swelling, similar experiments were carried out by visualizing F-actin with TRITC-labeled phalloidin. After 1 day in culture under normal conditions, a considerable proportion of chick cardiac myocytes was symmetrically spherical and contained a centrally localized nucleus. As shown in Fig. 2, F-actin staining revealed a characteristic sarcomere-like pattern in the central areas of the sarcoplasm surrounding the nucleus, i.e., bands of F-actin were organized in a repetitive fashion consistent with the cross-striation of myofibrillar sarcomeres localized in the perinuclear areas of cardiac myocytes. F-actin was also located in the periphery of the sarcoplasm as a thin layer in close apposition to the sarcolemma. In some instances, finely stained filaments bridged the central and peripheral sites of F-actin. Considerable changes in cell morphology and F-actin staining were observed when cells were treated with cytochalasin B. Compared with DMSO controls (Fig. 3A), cytochalasin B-treated cells were binucleated and asymmetrically spherical with an irregular surface appearance and an increase in cell volume. F-actin was randomly distributed in the perinuclear space, and subsarcolemmal F-actin staining was either significantly reduced or completely absent (Fig. 3D). In contrast, cells treated with phalloidin showed a normal differentiation in cell morphology, and F-actin distribution in these cells was comparable to the control cells (Fig. 3G).
|
|
Changes in F-actin staining were studied further under hyposmotic conditions. When chick cardiac myocytes were exposed to hyposmotic solution, a peak level of swelling was attained within ~3 min followed by a RVD (30, 40). Therefore, we examined F-actin distribution after 3 min (Fig. 3, B, E, and H) and 20 min (Fig. 3, C, F, and I) of exposure to hyposmotic solution (100 mosmol/l). During the first 3 min of cell swelling, the cell volume increase was mainly localized in the sarcoplasm between centrally localized sarcomeres and the sarcolemma (Fig. 3, B, E, and H). Fluorescence staining revealed that F-actin remained in perinuclear and subsarcolemmal areas in DMSO (Fig. 3B)- and phalloidin (Fig. 3H)-treated cells, and sarcomere-like organization of staining was observed. In contrast, F-actin in cytochalasin B-treated cells was randomly organized in the perinuclear areas (Fig. 3E). After 20 min of exposure to hyposmotic solution, cell swelling in cytochalasin B-treated cells was pronounced (Fig. 3F). No further changes in cell volume or morphology were observed in DMSO (Fig. 3C)- and phalloidin (Fig. 3I)-treated cells; F-actin organization appeared normal, and some stained F-actin could still be observed connecting the central core of F-actin with subsarcolemmal F-actin. These results indicate that the F-actin network in cardiac myocytes undergoes dynamic reorganization in response to changes in cell volume to maintain its structural and functional integrity.
F-actin conformation was shown to recover quickly upon removal of cytochalasin B from neuronal cells (6). We examined the ability of cardiac myocytes to recover from cytochalasin B under hyposmotic conditions. Cells were first exposed to cytochalasin B containing hyposmotic solution for 5 min followed by recovery in cytochalasin B-free hyposmotic solution. Recovery of F-actin staining was observed after 15 min of incubation in the absence of cytochalasin B (Fig. 4). Although an organized staining pattern was not observed, fragments of F-actin appeared throughout the sarcoplasm and in the subsarcolemmal space. These results indicate an early recovery of F-actin conformation in cultured chick cardiac myocytes after removal of cytochalasin B from the bath solutions.
|
F-actin modulation and the swelling-activated
ICl.
Hyposmotic swelling of cultured chick cardiac myocytes is known to
activate an outwardly rectifying
ICl (40). To
determine the role of F-actin in
ICl activation,
we studied the effect of F-actin disruption and stabilization on
ICl during cell
swelling. Cells were pretreated with either 25 µM cytochalasin B or
20 µM phalloidin as described above. Under control conditions,
swelling of chick cardiac myocytes in response to a hyposmotic
challenge activated (3 min) a time-independent
Cl
-selective current (Fig.
5). The current-voltage relationship, as
plotted in Fig. 5C, displayed
characteristics similar to those demonstrated in mammalian cardiac
myocytes (33, 34). The peak current amplitude measured at the voltage
of +60 mV was 68.0 ± 6.0 pA/pF
(n = 8).
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Swelling-induced conformational changes in the cytoskeletal network have been documented for many cell types (for review, see Ref. 27). Disruption of F-actin is associated with a loss of RVD during cell swelling (5, 22, 26). The dependence of cell volume regulation on F-actin integrity suggests a critical role for the cytoskeleton in the signal transduction process that initiates volume regulation. This study investigates whether activation of a swelling-induced ICl in cultured chick cardiac myocytes is associated with a signal transduction pathway that involves structural changes in F-actin. Our data indicate that an intact linkage between the F-actin and the cell membrane is important for activation of ICl. Disruption of the F-actin network is associated with an alteration of steady-state cell volume and excessive cell swelling under hyposmotic conditions. Our results also demonstrate that hyposmolarity induces dynamic changes in the F-actin cytoarchitecture of cardiac myocytes along with an increase in cell volume. Such reorganization is important to maintain a normal actin-membrane connection during cell swelling and may contribute to the signaling mechanisms that sense changes in cell volume and initiate the regulatory cellular response. Maneuvers that either disrupt or stabilize F-actin impede the dynamic changes of F-actin and suppress the swelling-activated ICl.
Effect of cytochalasin B and phalloidin on F-actin in cardiac myocytes. Cytochalasin B and phalloidin were used to modulate the F-actin architecture in this study. Cytochalasin B is a fungal toxin that permeates the cell membrane and inhibits actin polymerization (2). Early studies demonstrated that cardiac myocytes exposed to cytochalasin B experienced myofibril disruption and loss of spontaneous contractility (20, 23, 36). Unlike other cell types (3, 4, 19), a pronounced effect of cytochalasin B on cardiac myocytes was only observed after prolonged treatment (20, 23). In addition to the proposed decrease in sensitivity to cytochalasin B during myocardial development (23), myofibrillar-associated F-actin and the dense layer of the submembranous F-actin network in these cells may also contribute to the prolonged period of preincubation. Although cytochalasin D is a more specific disrupter of F-actin cytoskeleton, our previous studies of cytochalasin B in cultured embryonic chick cardiac myocytes (20) and the parallel use of fluorescence confocal microscopy in this study were able to assure the proper disruption of F-actin structure with cytochalasin B and to provide appropriate physiological correlates. In our experiments, incubation of cells with cytochalasin B for 16 h almost completely eliminated the submembranous F-actin network and caused extensive changes in cell morphology. These results are in good agreement with previous observations in cardiac myocytes and other cell types (3, 6, 19, 29). Phalloidin, on the other hand, is a fungal toxin from poisonous mushrooms that stabilizes F-actin by strengthening monomer-monomer interactions (2). Although phalloidin is effectively taken up by pinocytosis in certain cell types (2, 19, 24), a similar mechanism for phalloidin uptake has never been reported in cardiac myocytes. Our results indicate that, after incubation with FITC-phalloidin for ~20 h, F-actin staining with phalloidin was observed in the morphologically intact cardiac myocytes. These structural observations provided the rationale for us to study the relationship between F-actin modulation and the swelling-activated ICl.
During hyposmotic swelling, the submembranous F-actin was still present, indicative of a dynamic process that maintains the structural connection between F-actin and the sarcolemmal membrane. This observation is unlike early reports obtained in myeloma cells (19), in which changes in the membrane-associated F-actin ring did not accompany the increase in cell volume. These results implied a swelling-induced dissociation of the cortical F-actin network from the plasma membrane. However, hyposmotic swelling in shark rectal gland cells was accompanied by the transient disappearance of F-actin fluorescence, followed by a gradual reconstitution of F-actin in parallel with a RVD (41). Such reorganization of the F-actin architecture has been implicated in the volume regulatory processes of shark rectal gland cells. Lack of dissociation between F-actin and the cell membrane, as observed when cultured chick cardiac myocytes undergo a volume change, could be attributed to the rapid changes in polymerization/depolymerization of the submembranous F-actin as well as the bridge-like strands of F-actin in cardiac myocytes.
Volume response to F-actin modulation. Our cell volume measurement was undertaken from the whole cell patch-clamped cells of which the intracellular environment was effectively buffered by the pipette solution. As water enters the cell by external hypotonicity, dilution of the cell contents is compensated by the ionic composition of the pipette solution, which maintains the gradient for water influx and allows the cell to swell beyond the theoretically expected volume change. Such a configuration could contribute, in part, to the lack of cell volume regulation in the whole cell patch-clamped cells (40). Although dilution of the cell contents by external hypotonicity may also stimulate solute entry through membrane transport pathways, this does not seem to occur in cultured chick cardiac myocytes, since a loss of intracellular Na+, K+, and amino acid contents was always observed in the same cell type during hyposmotic swelling (30). Cultured chick cardiac myocytes treated with cytochalasin B undergo a larger than normal increase in cell volume when challenged by external hypotonicity. Insertion of new membrane components into sarcolemma appeared to be unlikely because no significant changes in membrane capacitance were observed during hyposmotic swelling. Cardiac myocytes are known to possess a small volume-to-surface area ratio due to considerable invaginations of sarcolemma (21). Such invaginations have been shown in yeast cells to be surrounded by densely stained F-actin (28). Actin binding proteins tightly connect the F-actin and sarcolemmal invaginations to provide an interface for cytoskeleton-membrane linkage, which may play a unique role in cell volume regulation. Disruption of F-actin by cytochalasin B could remove the mechanical restraint on the sarcolemma and allow the cells to swell without the insertion of new membrane components. On the other hand, cells treated with phalloidin became rigid due to the stabilization of the F-actin network. When phalloidin-treated myocardial cells were subject to a hyposmotic challenge, the tightened cytoskeleton-membrane linkage could have constrained the mechanical distension of the sarcolemma to attenuate cell swelling.
Response of swelling-activated
ICl to F-actin modulation.
Although cytochalasin B and phalloidin have opposite effects on the
F-actin, our data indicate that both compounds inhibit the
swelling-activated
ICl. These
results are distinct from reports in some cell types that disruption of
F-actin by cytochalasin B increased the sensitivity of ion channels to
cell swelling or membrane stretch (17, 19, 32). As a typical example,
the volume-regulated
ICl in myeloma
cells was enhanced by cytochalasin B and was inhibited by phalloidin
(19). No direct evidence, however, has indicated that these channels
were involved in the extrusion of intracellular osmolytes during cell
volume regulation. By contrast, observations from many cell types have
indicated that cytochalasin B inhibits the RVD in response to
hyposmotic cell swelling (4, 5, 8, 22). Our results indicate that maneuvers either disrupting or stabilizing the F-actin cytoskeleton suppress the swelling-activated
ICl without
altering the characteristics of the
Cl channel, as demonstrated
by the effect of external
Cl
removal and the
Cl
channel blocker NPPB.
These data suggest that swelling of cardiac myocytes is associated with
a functionally dynamic change in F-actin that may involve
depolymerization and repolymerization of the F-actin network. As a
consequence, the linkage between the cytoskeleton and the sarcolemma
would be maintained to enable cell volume regulation. Disruption or
stabilization of F-actin accompanies the suppression of the
swelling-activated
ICl and
concomitantly attenuates cell volume regulation.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the technical assistance of R. Boyle, E. Moore, and S. Revels. Our thanks are also given to Dr. L. A. Lobaugh for critical review of the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grants HL-27105 and HL-07063. T. H. Larsen was supported by the Norwegian Research Council and the United States-Norway Fulbright Foundation.
Preliminary results of this study have been presented as an abstract (The Physiologist 37: A10, 1994).
Address for reprint requests: M. Lieberman, Div. of Physiology, Dept. of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710.
Received 4 March 1996; accepted in final form 30 May 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Chamberlin, M. E.,
and
K. Strange.
Anisosmotic cell volume regulation: a comparative view.
Am. J. Physiol.
257 (Cell Physiol. 26):
C159-C173,
1989
2.
Cooper, J. A.
Effects of cytochalasin and phalloidin on actin.
J. Cell Biol.
105:
1473-1478,
1987[Medline].
3.
Cornet, M.,
E. Delpire,
and
R. Gilles.
Study of microfilaments network during volume regulation process of cultured PC 12 cells.
Pflügers Arch.
410:
223-225,
1987[Medline].
4.
Cornet, M.,
I. H. Lambert,
and
E. K. Hoffmann.
Relation between cytoskeleton, hypo-osmotic treatment and volume regulation in Ehrlich ascites tumor cells.
J. Membr. Biol.
131:
55-66,
1993[Medline].
5.
Cornet, M.,
J. Ubl,
and
H.-A. Kolb.
Cytoskeleton and ion movements during volume regulation in cultured PC12 cells.
J. Membr. Biol.
133:
161-170,
1993[Medline].
6.
Forscher, P.,
and
S. J. Smith.
Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone.
J. Cell Biol.
107:
1505-1516,
1988[Abstract].
7.
Foskett, J. K.
The role of calcium in the control of volume-regulatory transport pathways.
In: Cellular and Molecular Physiology of Cell Volume Regulation, edited by K. Strange. Ann Arbor, MI: CRC, 1994, p. 259-277.
8.
Foskett, J. K.,
and
K. R. Spring.
Involvement of calcium and cytoskeleton in gallbladder epithelial cell volume regulation.
Am. J. Physiol.
248 (Cell Physiol. 17):
C27-C36,
1985
9.
Gilles, R.,
E. Delpire,
C. Duchene,
M. Cornet,
and
A. Pequeux.
The effect of cytochalasin B on the volume regulation response of isolated axons of the green crab Carcinus maenas submitted to hypo-osmotic media.
Comp. Biochem. Physiol. A Physiol.
85A:
523-525,
1986.
10.
Hall, S. K.,
J. Zhang,
and
M. Lieberman.
Cyclic AMP prevents activation of a swelling-induced chloride-sensitive conductance in chick heart cells.
J. Physiol. (Lond.)
488:
359-369,
1995[Abstract].
11.
Hall, S. K.,
J. Zhang,
and
M. Lieberman.
An early transient current is associated with hyposmotic swelling and volume regulation in embryonic chick cardiac myocytes.
Exp. Physiol.
82:
43-54,
1997[Abstract].
12.
Hallows, K. R.,
C. H. Packman,
and
P. A. Knauf.
Acute cell volume changes in anisotonic media affect F-actin content of HL-60 cells.
Am. J. Physiol.
261 (Cell Physiol. 30):
C1154-C1161,
1991
13.
Hamill, O. P.,
A. Marty,
E. Neher,
B. Sakmann,
and
F. J. Sigworth.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch.
391:
85-100,
1981[Medline].
14.
Hoffmann, E. K.,
L. O. Simonsen,
and
I. H. Lambert.
Cell volume regulation: intracellular transmission.
In: Interaction of Cell Volume and Cell Function, edited by F. Lang,
and D. Häussinger. New York: Springer-Verlag, 1993, vol. 14, p. 187-247.
15.
Jacob, R.,
M. Lieberman,
and
S. Liu.
Electrogenic sodium-calcium exchange in cultured embryonic chick heart cells.
J. Physiol. (Lond.)
387:
567-588,
1987[Abstract].
16.
Janmey, P. A.
Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly.
Annu. Rev. Physiol.
56:
169-191,
1994[Medline].
17.
Kim, D.
Novel cation-selective mechanosensitive ion channel in the atrial cell membrane.
Circ. Res.
72:
225-231,
1993[Abstract].
18.
Larsen, T. H.,
H. Huitfeldt,
O. Myking,
and
T. Sætersdal.
Microtubule-associated distribution of specific granules and secretion of atrial natriuretic factor in primary cultures of rat cardiomyocytes.
Cell Tissue Res.
272:
201-210,
1993[Medline].
19.
Levitan, I.,
C. Almonte,
P. Mollard,
and
S. S. Garber.
Modulation of a volume-regulated chloride current by F-actin.
J. Membr. Biol.
147:
283-294,
1995[Medline].
20.
Lieberman, M.,
F. J. Manasek,
T. Sawanobori,
and
E. A. Johnson.
Cytochalasin B: its morphological and electrophysiological actions on synthetic strands of cardiac muscle.
Dev. Biol.
31:
380-403,
1973[Medline].
21.
Lieberman, M.,
T. Sawanobori,
J. M. Kootsey,
and
E. A. Johnson.
A synthetic strand of cardiac muscle: its passive electrical properties.
J. Gen. Physiol.
65:
527-550,
1975[Abstract].
22.
Linshaw, M. A.,
C. A. Fogel,
G. P. Downey,
E. W. Y. Koo,
and
A. I. Gotlieb.
Role of cytoskeleton in volume regulation of rabbit proximal tubule in dilute medium.
Am. J. Physiol.
262 (Renal Fluid Electrolyte Physiol. 31):
F144-F150,
1992
23.
Manasek, F. J.,
B. Burnside,
and
J. Stroman.
The sensitivity of developing cardiac myofibrils to cytochalasin-B.
Proc. Natl. Acad. Sci. USA
69:
308-312,
1972[Abstract].
24.
Matthews, J. B.,
C. S. Awtrey,
and
J. L. Madara.
Microfilament-dependent activation of Na+/K+/2Cl cotransport by cAMP in intestinal epithelial monolayers.
J. Clin. Invest.
90:
1608-1613,
1992[Medline].
25.
Mills, J. W.,
and
L. J. Mandel.
Cytoskeletal regulation of membrane transport events.
FASEB J.
8:
1161-1165,
1994
26.
Mills, J. W.,
E. M. Schwiebert,
and
B. A. Stanton.
Evidence for the role of actin filaments in regulating cell swelling.
J. Exp. Zool.
268:
111-120,
1994[Medline].
27.
Mills, J. W.,
E. M. Schwiebert,
and
B. A. Stanton.
The cytoskeleton and cell volume regulation.
In: Cellular and Molecular Physiology of Cell Volume Regulation, edited by K. Strange. Ann Arbor, MI: CRC, 1994, p. 241-258.
28.
Mulholland, J.,
D. Preuss,
A. Moon,
A. Wong,
D. Drubin,
and
D. Botstein.
Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane.
J. Cell Biol.
125:
381-391,
1994[Abstract].
29.
Oike, M.,
G. Schwarz,
J. Sehrer,
M. Jost,
V. Gerke,
K. Weber,
G. Droogmans,
and
B. Nilius.
Cytoskeletal modulation of the response to mechanical stimulation in human vascular endothelial cells.
Pflügers Arch.
428:
569-576,
1994[Medline].
30.
Rasmusson, R. L.,
D. G. Davis,
and
M. Lieberman.
Amino acid loss during volume regulatory decrease in cultured chick heart cells.
Am. J. Physiol.
264 (Cell Physiol. 33):
C136-C145,
1993
31.
Sarkadi, B.,
and
J. C. Parker.
Activation of ion transport pathways by changes in cell volume.
Biochim. Biophys. Acta
1071:
407-427,
1991[Medline].
32.
Schwiebert, E. M.,
J. W. Mills,
and
B. A. Stanton.
Actin-based cytoskeleton regulates a chloride channel and cell volume in a renal cortical collecting duct cell line.
J. Biol. Chem.
269:
7081-7089,
1994
33.
Sorota, S.
Swelling-induced chloride-sensitive current in canine atrial cells revealed by whole-cell patch-clamp method.
Circ. Res.
70:
679-687,
1992[Abstract].
34.
Tseng, G. N.
Cell swelling increases membrane conductance of canine cardiac cells: evidence for a volume-sensitive Cl channel.
Am. J. Physiol.
262 (Cell Physiol. 31):
C1056-C1068,
1992
35.
Watson, P. A.
Accumulation of cAMP and calcium in S49 mouse lymphoma cells following hyposmotic swelling.
J. Biol. Chem.
264:
14735-14740,
1989
36.
Wessells, N. K.,
B. S. Spooner,
J. F. Ash,
J. T. Brandley,
and
K. M. Yamada.
Microfilaments in cellular and developmental processes.
Science
171:
135-143,
1971[Medline].
37.
Wulf, E.,
A. Deboben,
F. A. Fautz,
H. Faulstich,
and
T. Wieland.
Fluorescent phallotoxin, a tool for the visualization of cellular actin.
Proc. Natl. Acad. Sci. USA
76:
4498-4502,
1979[Abstract].
38.
Zhang, J.,
S. K. Hall,
and
M. Lieberman.
An early transient current activates the swelling-induced chloride conductance in cardiac myocytes (Abstract).
Biophys. J.
66:
A442,
1994.
39.
Zhang, J.,
and
M. Lieberman.
Chloride conductance is activated by membrane distention of cultured chick heart cells.
Cardiovasc. Res.
32:
168-179,
1996[Medline].
40.
Zhang, J.,
R. L. Rasmusson,
S. K. Hall,
and
M. Lieberman.
A chloride current associated with swelling of cultured chick heart cells.
J. Physiol. (Lond.)
472:
801-820,
1993[Abstract].
41.
Ziyadeh, F. N.,
J. W. Mills,
and
A. Kleinzeller.
Hypotonicity and cell volume regulation in shark rectal gland: role of organic osmolytes and F-actin.
Am. J. Physiol.
262 (Renal Fluid Electrolyte Physiol. 31):
F468-F479,
1992