Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois and West Side Veterans Affairs Medical Center, Chicago, Illinois 60612
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ABSTRACT |
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The basally located actin cytoskeleton has been demonstrated
previously to regulate Cl
secretion from intestinal epithelia via its effects on the
Na+-K+-2Cl
cotransporter (NKCC1). In nontransporting epithelia, inhibition of
myosin light chain kinase (MLCK) prevents cell-shrinkage-induced activation of NKCC1. The aim of this study was to investigate the role
of myosin in the regulation of secretagogue-stimulated Cl
secretion in intestinal
epithelia. The human intestinal epithelial cell line T84 was used for
these studies. Prevention of myosin light chain phosphorylation with
the MLCK inhibitor ML-9 or ML-7 and inhibition of myosin ATPase with
butanedione monoxime (BDM) attenuated cAMP but not
Ca2+-mediated
Cl
secretion. Both ML-9 and
BDM diminished cAMP activation of NKCC1. Neither apical
Cl
channel activity,
basolateral K+ channel activity,
nor
Na+-K+-ATPase
were affected by these agents. Cytochalasin D prevented such
attenuation. cAMP-induced rearrangement of basal actin microfilaments was prevented by both ML-9 and BDM. The phosphorylation of mosin light
chain and subsequent contraction of basal actin-myosin bundles are
crucial to the cAMP-driven activation of NKCC1 and subsequent apical
Cl
efflux.
sodium-potassium-chloride cotransporter; myosin light chain kinase; chloride secretion; myosin phosphorylation
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INTRODUCTION |
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THE SECRETION OF CHLORIDE from transporting
epithelia is the end result of the coordinated activities
of several different individual transporters. This event requires not
only the activation of the apical
Cl channel but also of
three basolateral transporters:
Na+-K+-ATPase,
K+ channels, and
Na+-K+-Cl
cotransporter (NKCC1). The regulation of NKCC1 activity has received increased attention over the past few years. Several different aspects
of regulation have been identified (reviewed in Ref. 9). These include
direct phosphorylation of NKCC1, alterations in cell volume,
intracellular Cl
concentration ([Cl
]i), and the
cytoskeleton. Shapiro et al. (25) have elegantly demonstrated a
regulatory role for the actin cytoskeleton in cAMP-mediated Cl
secretion from
intestinal epithelial cells. Further investigation revealed the
regulatory end point to be at the level of the NKCC1 (22).
Specifically, stimulation of these cells with forskolin induces
rearrangement of the basally located stress fibers (25). Stabilization
of the actin cytoskeleton with phalloidin prevents such rearrangement
and inhibits NKCC1 activation (22). Interestingly, although
secretagogues whose effects are mediated by increasing intracellular
Ca2+ concentration
([Ca2+]i),
such as carbachol, obviously stimulate
Cl
secretion as well, no
regulatory role for the actin cytoskeleton can be demonstrated (25).
In intestinal epithelial cells (23), endothelial cells (15), and Ehrlich ascites tumor cells (16), hypertonicity-elicited cell shrinkage has been shown to stimulate NKCC1 activity. The regulation of NKCC1 activity in this setting appears quite complex. In both endothelial and Ehrlich cells, inhibition of myosin light chain kinase (MLCK) by enzymatic inhibitors ML-7 and/or ML-9 blocked NKCC1 activity, suggesting a regulatory role for the cytoskeleton in this model as well. It has been proposed that changes in cell volume, i.e., cell shrinkage, would alter tension within the cytoskeletal filaments to which MLCK is localized (27) and thus activate this enzyme. In fact, activation of MLCK would serve to contract the cell, thus altering cell shape in response to changes in cell volume. Such a link between cell volume-induced activation of MLCK and NKCC1 would explain the orchestration of cellular responses to volume changes.
The regulatory mechanisms governing NKCC1 activity in transporting
epithelia appear, however, to be different. Matthews et al. (23) have
shown that, in intestinal epithelial T84 cells, NKCC1 activation by
hypotonicity, but not hypertonicity, is attenuated by stabilization of
actin microfilaments with phalloidin. In contrast, the actin
destabilizer cytochalasin D diminished NKCC1 activation in response to
hypertonicity but not hypotonicity. These findings suggest that
hypertonicity-induced NKCC1 activity requires an intact cytoskeleton
but not microfilament rearrangement. In fact, this study demonstrated
that hypotonic, but not hypertonic, states induced actin rearrangements
similar to that seen in response to cAMP-mediated secretagogues such as
forskolin. It should be noted, however, that, although both
hypertonicity and hypotonicity activated NKCC1 in T84 cells, active
Cl secretion did not occur,
highlighting the fact that NKCC1 serves at least a dual role for the
cell, including regulation of cell volume and active secretion of
Cl
.
Although a clear role of the actin cytoskeleton in cAMP-mediated
Cl secretion has been
demonstrated, the exact nature of involvement remains undefined.
Similarly, the participation of MLCK in cell-shrinkage-elicited NKCC1
activation in endothelial and Ehrlich ascites cells has been shown, but
the mechanism is not fully understood. Although inhibition of MLCK
prevents both myosin light chain phosphorylation and NKCC1 activation,
cotransporter phosphorylation is not disturbed (15). It has been
suggested that cell-volume-induced cytoskeleton reorganization is the
signal that activates NKCC1 in this particular model.
The involvement of myosin in the regulation of
Cl secretion has not been
examined. In general, the movement of actin microfilaments is dependent
on interactions with myosin. Specifically, actin associates with only
the phosphorylated form of myosin light chain. This interaction then
results in the hydrolysis of ATP by actin-activated myosin ATPase and
filament movement (1). The aim of this study was to determine whether
myosin is involved in regulating
Cl
secretion from
intestinal epithelial cells. For these studies, the well-characterized
intestinal epithelial cell line T84 was used. The action of myosin was
interrupted in two ways. First, the phosphorylation of myosin light
chain was prevented by MLCK inhibitors ML-9 and ML-7. Second, the most
distal step in myosin-based actin microfilament movement was blocked by
inhibition of actin-activated myosin II ATPase with butanedione
monoxime (BDM). The effects of these interventions on
secretagogue-stimulated Cl
secretion were examined.
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METHODS |
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T84 cell culture. T84 cells, a generous gift from Dr. Kim Barrett (University of California, San Diego, CA), were grown as previously described (21) in a 1:1 (vol/vol) mixture of Dulbecco-Vogt modified Eagle's medium and Ham's F-12 plus 6% newborn calf serum. For electrophysiological studies, cells were grown to confluence on 0.33-cm2 collagen-coated permeable supports (Transwell; Costar, Cambridge, MA). For radioisotope studies, 0.6-cm2 collagen-coated permeable supports were used.
Storage and use of BDM. BDM (Sigma Chemical, St. Louis, MO) was stored in powder form at room temperature in the dark. On the day of the experiment, a fresh 0.5 M stock was made in water by vigorously vortexing. Just before experimentation, a 10 mM dilution was made in tissue culture medium. BDM has been shown to remain stable at 37°C in tissue culture medium for at least 60 min (6).
Measurement of short-circuit current.
Short-circuit current
(Isc) was
determined using a simplified technique for measuring electrical
parameters of cultured monolayers as published by Madara et al. (20). A
voltage clamp (Bioengineering Department, University of Iowa, Iowa
City, IA) was interfaced with a pair of calomel electrodes immersed in
saturated KCl with a pair of Ag-AgCl electrodes immersed in Ringer
solution. Agar bridges connected the electrodes and the medium
surrounding the cultured monolayers. The tips of the bridges were
cleaned with 70% ethanol and rinsed in sterile PBS before contact with
the tissue culture medium. Under voltage-clamped conditions,
Isc was measured
in response to either carbachol
(104 M) or forskolin
(10
6 M) in the presence or
absence of ML-9, ML-7, or BDM.
cAMP extraction. Cells were grown to confluence on 24-well plates. Medium was removed and replaced with PBS containing forskolin alone or forskolin plus BDM for the appropriate time periods at room temperature. PBS was removed and 200 µl of ice-cold 65% (vol/vol) ethanol were added. The supernatant was collected on ice, centrifuged at 2,000 g for 15 min at 4°C, transferred into new tubes, and dried in a vacuum oven. cAMP concentrations were assessed utilizing the nonacetylation assay protocol for the cAMP enzyme immunoassay system (Amersham, Arlington Heights, IL).
Fluorescent staining of actin. Fluorescent staining of F-actin was performed using rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR). Monolayers were rinsed in PBS, fixed for 10 min at room temperature in 3.7% formaldehyde, and permeabilized for 5 min in ice-cold acetone. They were stained at room temperature in the dark for 30 min with rhodamine phalloidin, rinsed, and mounted in Slow Fade (Molecular Probes). Stained monolayers were examined and photographed using epifluorescence microscopy. Photographs were scanned by using DeskScan II and the images were compiled in PowerPoint.
125I and 86Rb
efflux studies.
T84 cell monolayers were grown to confluence on
0.6-cm2 collagen-coated permeable
supports (Transwell; Costar, Cambridge, MA). Monolayers were loaded
with 2 µCi of either 125I or
86Rb by incubating
for 3 h. Monolayers were then rapidly washed four times with
HEPES-phosphate-buffered Ringer solution (HPBR) composed of (in mM) 135 NaCl, 5 KCl, 3.33 NaH2PO4,
0.83 Na2HPO4, 1 CaCl2, 1 MgCl2, 5 HEPES, and 10 glucose. A
"sample/replace" technique (28) was used to determine the rate
constants of 125I and
86Rb efflux. These studies
estimate the secretagogue-stimulated activation of
Cl and
K+ channels, respectively (28).
Four baseline samples were obtained before addition of secretagogue.
Samples were collected every 2 min following activation with
secretagogue. Residual intracellular radioactivity was determined by
extracting cells with 1 ml of 0.1 N NaOH and by counting samples in a
scintillation counter. The efflux rate constant was calculated as
[ln(R2)
ln(R1)]/(t2
t1),
where Rx is percent of
radioactivity remaining in the monolayer at time
tx (28).
86Rb uptake.
The secretagogue-stimulated uptake of
86Rb was used to assess NKCC1
activity. T84 monolayers grown on permeable supports were treated or
not with forskolin in the presence and absence of bumetanide (20 µM
for 20 min) and/or BDM and ML-9. HPBR containing 2 µCi/ml of
86Rb was then added to the
basolateral reservoir of monolayers, and uptake was allowed to proceed
over 3 min. Uptake was halted by placing the monolayers in ice-cold
buffer composed of 100 mM MgCl2
and 10 mM Tris · HCl, pH 7.5. Radioactivity was
extracted from monolayers with 0.1 N NaOH and counted in a
scintillation counter. Protein concentration was determined by the
Bradford assay.
Na+-K+-2Cl
cotransporter activity was defined as the bumetamide-sensitive portion
of 86Rb uptake.
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RESULTS |
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Inhibition of myosin light chain phosphorylation or myosin II ATPase
attenuates cAMP-mediated
Isc.
Confluent T84 monolayers grown on collagen-coated permeable supports
were stimulated by adding either the
Ca2+-mediated secretagogue
carbachol (104 M) or the
cAMP-mediated secretagogue forskolin
(10
6 M) to the basal
reservoir. The
Isc response was
measured every minute in the presence or absence of either ML-9 or BDM.
The peak responses, 2 min for carbachol and 10 min for forskolin, are
shown in Fig. 1. Neither ML-9
nor BDM attenuated the
Isc response to carbachol. In fact, BDM significantly increased the secretory response.
In contrast, both ML-9 and BDM significantly diminished the
forskolin-induced
Isc. This
differential response is reminiscent of findings observed with
phalloidin stabilization of F-actin (25).
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Neither apical Cl nor basolateral
K+ channel
activity is blocked by BDM.
The majority of
Isc from
stimulated T84 cell monolayers is attributable to apical
Cl
secretion (8). The
coordinated activities of several transporters are required for
vectorial Cl
secretion to
ensue. To determine whether myosin-altering agents interfere with
apical Cl
channel or basal
K+ channel activity, apical
125I and basolateral
86Rb efflux studies were
performed. This approach has been validated in T84 cell monolayers (28)
and is routinely used as a method to examine the activities of these
specific channels (22, 23). Figure 4 shows
that neither Cl
channel
activity (A) in response to
forskolin nor basolateral K+
channel activity (B) is altered by
BDM.
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Effect of ML-9 and BDM on
Na+-K+-2Cl
cotransport activity.
NKCC1 activity can be assessed by measuring the bumetanide-sensitive,
basolateral uptake of 86Rb. Both
ML-9 and BDM (Fig. 5) significantly
decreased forskolin-stimulated, bumetanide-sensitive uptake of
86Rb, whereas the
bumetanide-insensitive component, representing primarily
Na+-K+-ATPase
activity, remained unchanged. The decrease in forskolin-stimulated Isc by bumetanide
was identical to that seen with inhibition of myosin ATPase by BDM
(Fig. 6).
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An intact actin cytoskeleton is required for myosin-perturbing
agents to inhibit cAMP-induced Cl
secretion.
If the effects of the agents used herein are truly acting by
interference with actomyosin interactions, one would predict that
disruption of the actin cytoskeleton would prevent their ability to
diminish cAMP-stimulated Cl
secretion. To test this prediction, monolayers were incubated with
cytochalasin D (20 µM) for 10 min before stimulation with forskolin
in the presence or absence of ML-9 or BDM. Cytochalasin completely
prevented the effects of ML-9 on forskolin-elicited Isc and nearly
ablated the inhibitory impact of BDM (Fig.
7). These findings suggest that an intact
actin cytoskeleton is required for ML-9 and BDM to interfere with
cAMP-driven Cl
secretion.
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DISCUSSION |
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The activity of the
Na+-K+-2Cl
cotransporter is crucial for maintaining cellular homeostasis. This
transporter is key in regulating the cell volume of numerous cell
types. In transporting epithelia, NKCC1 is important for regulating
intracellular salt concentrations in the face of dramatic changes. In
view of the critical functions that NKCC1 serves, it should not be
surprising to find that the regulation of the activity of this
transporter is complex and perhaps variable between cell types. Several
general mechanisms of regulation have been identified and include
direct phosphorylation of NKCC1 (18, 19), alterations in
[Cl
]i
(10, 13), and cytoskeletal-dependent mechanisms (13, 22, 23, 29).
Understanding the role of the latter presents a challenge, yet elegant
studies published previously have provided key insights. Matthews et
al. (22) were the first to show that stabilization of the actin
cytoskeleton of intestinal epithelial cells attenuated cAMP-driven
Cl
secretion by inhibiting
NKCC1 activity (22). Whether NKCC1 activation occurred via direct
effects of cAMP or in response to diminished
[Cl
]i was not
known. By comparing cAMP-induced activation of NKCC1 in cells
expressing, or not, the apical
Cl
channel cystic fibrosis
transmembrane conductance regulator, these investigators concluded that
NKCC1 can be activated by pathways independent of apical
Cl
efflux, although
mechanisms dependent on and independent from [Cl
]i
may work in tandem (24). It was determined, however, that cytoskeletal
remodeling was required for ion uptake by NKCC1.
The signaling event(s) that induces cytoskeletal rearrangement and subsequent NKCC1 activation to occur remains elusive. It is plausible that alterations in cell volume are the initiating factor. Endothelial cells challenged by hypertonic conditions demonstrated an increase in the phosphorylation of myosin light chain (15). Inhibition of MLCK, however, with ML-7 blocked both myosin light chain phosphorylation and NKCC1 activation. NKCC1 phosphorylation was not affected by MLCK inhibition. These data led the investigators to conclude that cell volume status, volume-regulating transporters such as NKCC1, and cytoskeletal contraction are closely linked.
The effect of hypertonic challenge on active transporting epithelia
may, however, be different. Using the intestinal epithelial T84 cell
model, Matthews et al. (23) have shown that hypertonicity-induced activation of NKCC1 was not altered by the actin-stabilizing agent phalloidin. This suggests that cytoskeletal movement is not induced under these conditions. One would predict, therefore, that
hypertonicity does not cause phosphorylation of myosin light chain in
these actively transporting epithelia. Interestingly, although NKCC1 was activated by hypertonicity, active
Cl efflux was not
stimulated. These data indicate that activation of basolateral NKCC1
can, under certain circumstances, be uncoupled from the apical membrane
transport events, namely Cl
efflux.
Because the cytoskeleton appears to play such a key role in one of the
major physiological functions provided by intestinal epithelia, namely
vectorial ion transport, we wanted to investigate whether the
previously described cAMP-induced, actin-mediated activation of NKCC1
was myosin based. Actin microfilament movement in nonmuscle cells, such
as epithelia, is regulated in the manner as has been described for
smooth muscle cells (5). Actin interacts only with the phosphorylated
form of myosin light chain. This protein is phosphorylated by the
specific enzyme MLCK. The interaction of actin with phosphorylated
myosin light chain activates myosin ATPase. It is the energy from this
reaction that leads to microfilament movement. In the present study, we
interrupted this process in two ways. First, MLCK activity was blocked
with an enzyme inhibitor, ML-9 or ML-7, thus preventing myosin light
chain phosphorylation. Second, the terminal step in regulating actin
microfilament movement was prevented by inhibiting myosin ATPase
activity with BDM. Interruption at either of these steps resulted in
the same outcome: attenuation of NKCC1 activity in response to cAMP and
decreased apical Cl efflux.
These findings confirm that active contraction of actin microfilaments,
through interactions with myosin, is required for NKCC1 activation and
Cl
secretion stimulated by cAMP.
The signal by which MLCK is activated remains unidentified. Increased [Ca2+]i is the sole identified stimulator of MLCK. Shrinkage-induced activation of MLCK in endothelial cells, however, appears to not result from increased [Ca2+]i (15). In addition, increased [Ca2+]i could not be demonstrated to correlate with myosin light chain phosphorylation in hypertonicity-challenged mesangial cells (27), suggesting that a novel mechanism may be responsible. Previous studies have demonstrated that [Ca2+]i and the level of myosin light chain phosphorylation do not always correlate (2). However, a GTP-dependent mechanism that enhances myosin light chain phosphorylation and muscle contraction at a fixed concentration of Ca2+ has been identified (26). This has been termed "GTP-induced increase in Ca2+ sensitivity." The small GTPase Rho is responsible (12) for this event. In fact, Rho has been found to regulate myosin light chain phosphorylation by two separate pathways. First, activated Rho kinase can phosphorylate the myosin-binding subunit of myosin phosphatase, thereby inactivating myosin phosphatase (14). Second, Rho kinase itself has been shown to phosphorylate myosin light chain at the same site as MLCK and subsequently activate myosin ATPase (3). In many contractile models, both pathways have been demonstrated to be required to increase myosin light chain phosphorylation (17). Interestingly, the binding of halides to heterotrimeric G proteins has been shown to alter GTPase activity (11). Whether activated Rho is involved in the regulation of NKCC1 and ultimately vectorial ion transport is not known. It is intriguing to speculate, in view of the role of myosin light chain phosphorylation in NKCC1 activation yet in the absence of increased [Ca2+]i, that Rho GTPases may control the cytoskeletal regulation of NKCC1.
In summary, this study demonstrates that phosphorylation of myosin
light chain and subsequent contraction of actin-myosin bundles are
crucial to the cAMP activation of NKCC1 and subsequent apical
Cl efflux. These data serve
to highlight the importance of this regulatory pathway in transporting
epithelia, much as has been shown previously for shrinkage-induced
NKCC1 activation in endothelial cells. Additional studies will allow
the deciphering of the signals involved in stimulating the cytoskeletal
changes that so closely govern these crucial cellular events.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50694 (to G. Hecht) and a grant from the Department of Veterans Affairs (Merit Award to G. Hecht).
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FOOTNOTES |
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A portion of this work was presented in abstract form at the Annual Meeting of the American Gastroenterological Association in New Orleans, LA, in May 1998.
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.
Address for reprint requests and other correspondence: G. Hecht, Univ. of Illinois, Dept. of Medicine, Digestive and Liver Diseases (M/C 787), 840 South Wood St., CSB Rm. 704, Chicago, IL 60612 (E-mail: gahecht{at}uic.edu).
Received 17 November 1998; accepted in final form 6 May 1999.
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262 (Renal Fluid Electrolyte Physiol. 31):
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1992