Myosin light chain kinase- and PKC-dependent contraction of
LES and esophageal smooth muscle
U. D.
Sohn1,
Weibiao
Cao2,
Da-Chun
Tang3,
J. T.
Stull3,
J. R.
Haeberle4,
C.-L. A.
Wang5,
K. M.
Harnett2,
J.
Behar2, and
P.
Biancani2
1 Department of Pharmacology, College of Pharmacy, Chung Ang
University, Seoul 156-756, Korea; 2 Department of Medicine,
Rhode Island Hospital and Brown University, Providence, Rhode
Island 02903; 3 Department of Physiology, University of Texas
Southwestern Medical Center, Dallas, Texas 75235;
4 Department of Molecular Physiology and Biophysics,
University of Vermont, Burlington, Vermont 05405; and
5 Department of Muscle Research, Boston Biomedical Research
Institute, Boston, Massachusetts 02114
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ABSTRACT |
In smooth muscle cells enzymatically
isolated from circular muscle of the esophagus (ESO) and lower
esophageal sphincter (LES), ACh-induced contraction and myosin light
chain (MLC) phosphorylation were similar. Contraction and
phosphorylation induced by purified MLC kinase (MLCK) were
significantly greater in LES than ESO. ACh-induced contraction and MLC
phosphorylation were inhibited by calmodulin and MLCK inhibitors in LES
and by protein kinase C (PKC) inhibitors in ESO. Contraction of LES and
ESO induced by the PKC agonist 1,2-dioctanoylglycerol (DG) was
unaffected by MLCK inhibitors. Caldesmon and calponin
concentration-dependently inhibited ACh-induced contraction of ESO and
not LES. In ESO, caldesmon antagonist GS17C reversed caldesmon- but not
calponin-induced ACh inhibition. GS17C caused contraction of
permeabilized ESO but had much less effect on LES. GS17C-induced
contraction was not affected by MLCK inhibitors, suggesting that MLCK
may not regulate caldesmon-mediated contraction. DG-induced contraction of ESO and LES was inhibited by caldesmon and calponinin, suggesting that these proteins may regulate PKC-dependent contraction. We conclude
that calmodulin and MLCK play a role in ACh-induced LES contraction,
whereas the classical MLCK may not be the major kinase responsible for
contraction and phosphorylation of MLC in ESO. ESO contraction is PKC
dependent. Caldesmon and/or calponin may play a role in PKC-dependent contraction.
calcium stores; caldesmon; calmodulin; calponin; cat; second
messenger system
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INTRODUCTION |
IN SMOOTH MUSCLE CELLS
ISOLATED by enzymatic digestion from the circular layer of the
lower esophageal sphincter (LES), contraction in response to a
maximally effective concentration of ACh
(10
10-10
9 M) is mediated through a
calmodulin-dependent pathway (9). Calmodulin in the
presence of calcium is known to activate myosin light chain kinase
(MLCK), resulting in myosin phosphorylation and contraction (5,
17, 18, 31, 42, 56, 67, 81, 86, 87). Phosphorylation of MLC by
the calmodulin-MLCK pathway is thought to be responsible for coupling
increased calcium concentration with contraction in smooth muscle
(40). Accordingly, it has been proposed (43)
that MLCK plays an essential role in the activation process in the
smooth muscle cell and that activation of this enzyme is both necessary
and sufficient for the initiation of contraction.
ACh-induced contraction of circular muscle of the esophagus (ESO),
however, is calmodulin independent and mediated through a protein
kinase C (PKC)-dependent pathway (70), involving a calcium-insensitive PKC-
(75). In this pathway, calcium
is required for activation of phospholipases and production of the second messengers diacylglycerol (DAG) and arachidonic acid (AA) (74). When the second messengers are present, contraction
can proceed even in the absence of intracellular calcium
(70). The precise mechanisms responsible for mediation of
this PKC-dependent contraction are not well established (5, 12,
48, 49, 82), but because calcium and calmodulin are not required
to support the PKC-
-mediated contraction of ESO muscle, we propose that MLCK may not play a major role in this pathway.
Caldesmon and calponin have been implicated in the regulation of smooth
muscle contraction as a result of their ability to inhibit
actin-activated Mg2+-ATPase of smooth muscle myosin
(59, 89). This inhibitory effect is abolished by
phosphorylation of caldesmon and/or calponin, by
calcium-calmodulin-dependent protein kinase II or PKC (38, 90), and restored by dephosphorylation by a type 2A protein phosphatase (88).
In the present investigation, we examined the role of MLCK and the thin
filament-associated regulatory proteins caldesmon and calponin in
contraction of LES and ESO. We find that in ESO, contraction in
response to agonists or direct PKC activation by L-
-1,2-dioctanoylglycerol (DG) is calmodulin independent
and not regulated by MLCK, even though ACh-induced contraction of ESO
is associated with MLC phosphorylation. In contrast in LES, activation
by a maximally effective concentration of ACh results in calmodulin-
and MLCK-dependent contraction. However, LES contraction in response to
direct PKC activation by DG is similar to contraction of ESO and is
calmodulin independent and not affected by MLCK inhibitors. In
addition, caldesmon and calponin do not play a role in MLCK-dependent
contraction of LES but may exert a regulatory effect on PKC-dependent
contraction of ESO and LES.
We conclude that in these two types of muscle there are two distinct
types of contractions that depend on distinct regulatory mechanisms. A
PKC-dependent pathway, which may be regulated by caldesmon and
calponin, is present in ESO and LES. A calmodulin and MLCK-dependent
pathway is present only in LES muscle. MLC phosphorylation occurs in
both pathways.
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METHODS |
Animals.
Adult cats of either sex, weighing between 2.5 and 5 kg, were used. The
animals were initially anesthetized with ketamine (Aveco, Fort Dodge,
IA) then euthanized with an overdose of phenobarbital (Schering,
Kennilworth, NJ). The chest and abdomen were opened with a mid-line
incision exposing the ESO and stomach. The ESO and stomach were removed
together, opened along the lesser curvature, and pinned on a wax block
at their in vivo dimensions. The location of the squamocolumnar
junction was identified, and the mucosa was peeled. The high-pressure
zone of the LES is characterized by a visible thickening of the
circular muscle layer in correspondence to the squamocolumnar junction
and immediately proximal to the sling fibers of the stomach.
Preparation of tissue squares.
After opening the ESO and stomach and identifying the LES, we removed
the mucosa and submucosal connective tissue by sharp dissection. The
LES was excised, and the circular muscle layer was cut into 0.5-mm
thick slices with a Stadie Riggs tissue slicer (Thomas Scientific
Apparatus, Philadelphia, PA). The last slices containing the myenteric
plexus, longitudinal muscle, and serosa were discarded. The slices of
circular muscle were placed flat on a wax surface, and tissue squares
were made by cutting twice with a 2-mm blade block, with the second cut
at right angles to the first. Tissue squares were used in the
measurement of myosin phosphorylation or enzymatically digested to
obtain isolated smooth muscle cells.
Dispersion of smooth muscle cells.
Isolated smooth muscle cells were obtained by enzymatic digestion, as
previously described (10). LES and ESO tissue squares were
digested in HEPES-buffered physiological salt solution (PSS), containing 0.1% collagenase type II (Worthington Biochemicals, Freehold, NJ) for 2 h. The HEPES solution contained 115 mM NaC1, 5.8 mM KC1, 2 mM KH2PO4, 10.8 mM glucose, 25 mM
HEPES, 2 mM CaC12, 0.6 mM MgC12, 0.3 mg/ml
basal medium Eagle amino acid supplement (Sigma, St. Louis, MO), and
0.09 mg/ml soybean trypsin inhibitor (Worthington Biochemicals). The
solution was gently gassed with 100% O2. At the end of the
digestion period, the tissue was placed over a 200-µm Nitex mesh,
rinsed in collagenase-free PSS to remove any trace of collagenase, and
then incubated at 31°C in collagenase-free PSS gassed with 100%
O2. The cells were allowed to dissociate freely in the PSS
for 10-20 min.
Preparation of permeable smooth muscle cells.
Cells were permeabilized to control cytosolic calcium concentration and
to allow diffusion of calmodulin, MLCK, caldesmon, and calponin across
the cell membrane. After completion of the enzymatic phase of the
digestion process, the partly digested muscle tissue was washed with a
cytosolic enzyme-free medium (cytosolic buffer) of the following
composition (in mM): 20 NaCl, 100 KCl, 25 NaHCO3, 5 MgSO4, 0.96 NaH2PO4, 1 EGTA, and
0.48 CaCl2. The medium contained 2% BSA and was
equilibrated with 95% O2-5%CO2 to maintain a
pH of 7.2 at 31°C. Muscle cells dispersed spontaneously in this medium.
Isolated cells were permeabilized by a 3-min incubation in cytosolic
buffer containing saponin (75 µg/ml). Permeabilized cells were washed
in saponin-free cytosolic buffer containing 0.48 mM CaCl2
and 1 mM EGTA, yielding 180 nM free calcium, as described previously by
Fabiato and Fabiato (19). When different calcium concentrations were required, calcium concentrations were changed as
needed, and free calcium levels were similarly calculated. The modified
cytosolic buffer also contained antimycin (10 µM), ATP (ATP disodium
salt, 1.5 mM), and an ATP-regenerating system consisting of creatine
phosphate (5 mM) and creatine phosphokinase (10 U/ml)
(11).
Agonist-induced contraction of isolated muscle cells.
Cells were contracted by 30-s exposure to ACh, DG, GS17C, or MLCK. The
concentration of ACh required to produce a maximal contraction of
isolated muscle cells is 10
10-10
9 M
(see RESULTS). When inhibitors were used, the cells were
incubated in their presence for 1 min before addition of agonists.
Cells were equilibrated in each calcium concentration for several
minutes during the permeabilization process and before calmodulin
addition. When MLCK was used, cells were incubated in threshold
concentrations of calcium and calmodulin. At the appropriate time
interval, cells were fixed in acrolein at a final 1% concentration.
The length of 30 consecutive intact cells from each slide was measured
through a phase-contrast microscope (Carl Zeiss) and a digital
charge-coupled television camera (model WV CD-51, Panasonic, Secaucus,
NJ). The camera was connected to a Macintosh IICi computer (Apple,
Cupertino, CA). A software program, Image 1.59 (National Institutes of
Health, Bethesda, MD), was used to obtain a measurement of cell length.
Intact or viable cells are distinguished by a membrane that is bright,
smooth, and shiny, with the appearance of a halo around the periphery
of the cell. These cells will contract when placed on a shallow muscle
chamber under an inverted microscope and "spritzed" with ACh using
a pressure ejection micropipette system (13). Data were
expressed as cell shortening defined as percent decrease in cell length
from control.
Determination of myosin phosphorylation.
For measurement of myosin phosphorylation, intact circular smooth
muscle tissue (see Figs. 4 and 5) and permeabilized circular smooth
muscle cells (see Fig. 6) were used. For ACh-induced myosin phosphorylation, tissue samples were equilibrated in oxygenated Krebs
PSS at 37°C for 2 h and then exposed to a concentration of ACh
(10
5 M) used to obtain a maximal contractile response. We
(8) previously found that this is the maximally effective
ACh concentration in intact circular muscle. The reaction was stopped
by freezing the samples in a slurry of acetone and dry ice, after
7 s for ESO and 10 s for LES. These are the times required
for these muscles to achieve two-thirds of maximal contraction in
response to ACh (8).
For MLCK-induced phosphorylation, permeabilized smooth muscle cells
were equilibrated in the indicated concentration of calcium and
calmodulin for 30 min. The concentration of calcium and calmodulin used
produces a maximal contractile response (9, 70).
Permeabilized smooth muscle cells were stimulated with the indicated
concentration of MLCK for 30 s, and the reaction was stopped by
freezing the samples in a slurry of acetone and dry ice.
Nonphosphorylated and phosphorylated forms of MLC were separated by
electrophoresis and localized with antibodies against MLC. The relative
amounts of phosphorylated and nonphosphorylated MLC were quantitated by
densitometry (15, 41). Briefly, protein was extracted in
an 8 M urea buffer and processed for urea/glycerol-PAGE as described
previously by Persechini et al. (64). Nonphosphorylated and phosphorylated forms of the light chain were separated after electrophoresis at 20°C and 400 V for 12-18 h. Proteins were
electrophoretically transferred from glycerol gels onto nitrocellulose
paper. MLC were localized on nitrocellulose paper with antibodies
against MLC. Relative amounts of phosphorylated and nonphosphorylated MLC were quantitated from densitometry scans of the immunostained nitrocellulose blots. Myosin phosphorylation was expressed as the
percentage of total MLC (15, 41).
Calponin and caldesmon determination.
The relative calponin and caldesmon contents of ESO and LES were
measured by immunostaining Western blots and normalized to either actin
or dry tissue weight. Tissue slices were prepared from the circular
smooth muscle layer of the ESO and LES, as described for single cell
digestion. Each slice was carefully dropped into liquid nitrogen and
stored at
70°C until use. Tissue was homogenized under liquid
nitrogen, dehydrated in acetone on ice, and dried to a powder. All
tissue weights reported are tissue dry weights. Proteins were
solubilized by incubation for 2 min at 100°C in buffer containing
65.5 mM Tris base, 3% SDS, 20% glycerol, and 40 mM dithiothreitol.
LES (15 µg) and ESO (12 µg) samples were processed by 5% SDS-PAGE
using the buffer system of Porzio and Pearson (66) and
electrophoretically transferred onto nitrocellulose. The nitrocellulose
blots were immunostained using anti-rat uterus caldesmon and calponin
polyclonal antisera, and audioradiographic and densitometric scans were
performed. The linearity of the audioradiographic and
densitometric scans was tested using a dilution series of purified
chicken gizzard calponin on the same gel.
Because we did not know the relative staining intensity of calponin and
caldesmon from chicken gizzard and from our LES and ESO samples, we
could not calculate absolute amounts of calponin and caldesmon present
in our tissues. Instead, we have reported the relative amounts of
calponin and caldesmon normalized to either tissue dry weight or actin
content. The actin content was determined by running a series of
loadings on a 7.5% Porzio and Pearson (66) SDS-PAGE (from
2.5 to 25 µg of LES and ESO dry weight) along with several lanes of
BSA (2 µg) to establish linearity of actin measurements. Gels
containing the actin and BSA bands were stained for 2 h with a
stain containing 0.1% Coomassie blue R-250, 25% isopropyl alcohol, and 10% acetic acid and destained overnight in destaining solution containing 10% acetic acid and 30% methanol. Stained bands were quantified by densitometric gel scanning, and the amount of actin per
dry tissue weight of ESO and LES was calculated. Corrections were made
for the difference in staining intensity of actin and BSA by Coomassie
blue (actin/BSA , 0.78:1).
Peptide and protein preparation.
Caldesmon-binding peptides GS17C and XGS17C were synthesized as
previously described (91). Caldesmon and calponin were
prepared from frozen chicken gizzards in the laboratory of J. R. Haeberle, according to the methods of Lynch and Bretcher
(53) and Abe et al. (1). MLCK was isolated in
the laboratory of J. T. Stull as previously reported (4,
52).
Drugs and chemicals.
The following agents were used: collagenase type II and soybean trypsin
inhibitor (Worthington Biochemicals);
1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7; Seikagaku America, St. Petersburg, FL); calmodulin (Calbiochem, San Diego, CA); DG (Avanti Polar Lipids, Alabaster, AL);
1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7, Seikagaku, Rockville, MD); SDS (Bio-Rad, Hercules, CA); polyacrylamide (BDH Chemicals, Poole, UK); acetic acid
(Mallinckrodt Specialty Chemicals, Paris, KY); and Coomassie blue
(R-250; Schwartz/Mann Biotech). CGS-9343B was a gift from Dr. M. Crettaz (Zyma SA, Nyon, Switzerland). ACh, L-thyroxine, quercetin, saponin, thapsigargin, basal medium Eagle amino acid supplement, creatine phosphate, creatine phosphokinase, ATP, antimycin A, and other reagents were purchased from Sigma.
Data analysis.
Estimates of the dose giving half-maximal response were determined by
interpolation from graphs of log concentration vs. logit values of
percent shortening for MLCK and GS17C (28). Data are expressed as means ± SE. Statistical differences between means were determined by Student's t-test. Differences between
multiple groups were tested using ANOVA for repeated measures and
checked for significance using Scheffe's F test.
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RESULTS |
MLCK-dependent LES contraction.
We (9, 33, 70) have previously shown, in smooth muscle
cells isolated by enzymatic digestion from the circular layer of the
LES, that ACh-induced contraction in the LES is mediated through a
calmodulin-dependent pathway and contraction of ESO circular muscle is
mediated through a PKC-dependent pathway. Because calmodulin is known
to activate MLCK and ESO contraction is calmodulin independent, we
investigated whether MLCK was involved in ESO contraction. We exposed
LES and ESO circular smooth muscle cells to MLCK purified from chicken
gizzard, in the presence of calcium and calmodulin. LES and ESO smooth
muscle cells were permeabilized and equilibrated in the presence of 1.3 µM calcium and 1 nM calmodulin to facilitate activation of the
kinase. Figure 1 shows that MLCK caused a
concentration-dependent contraction of LES cells (ANOVA, P < 0.01). The half-maximal response, calculated by
logit transformation, was seen at 0.2 nM. MLCK-induced contraction of
ESO was significantly less than that of LES under the same experimental
conditions (ANOVA, P < 0.01), and a statistically
significant relationship between MLCK concentration and ESO contraction
could not be established (ANOVA, P > 0.05).

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Fig. 1.
Effect of purified myosin light chain kinase (MLCK) on
smooth muscle cells isolated by enzymatic digestion from the circular
layer of the lower esophageal sphincter (LES) and esophagus (ESO).
Cells were permeabilized to allow diffusion of the kinase into the
cytoplasm. Before exogenous MLCK was added, the cells were equilibrated
in the presence of calcium (1.3 µM) and calmodulin (1 nM) to
facilitate activation of the kinase. These concentrations caused a
small initial shortening in the absence of MLCK (control), compared
with cell length in standard cytosolic medium with no calmodulin.
%Shortening was calculated with reference to the length in standard
cytosolic medium with no calmodulin. In permeable LES cells, MLCK
caused a concentration-dependent contraction (ANOVA, P < 0.01). Under the same experimental conditions, ESO cells contracted
much less. Values are means ± SE of 3 animals, with 30 cells
counted for each animal.
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To confirm that MLCK plays a greater role in contraction of LES than of
ESO circular smooth muscle, we tested the effect of MLCK inhibitors on
ACh-induced contraction of smooth muscle cells (Fig.
2). Muscle cells were contracted by a
maximally effective concentration of ACh. The MLCK inhibitors
quercetin, L-thyroxine, and ML-7 concentration-dependently
inhibited ACh-induced contraction of LES muscle (ANOVA,
P < 0.01) but had no effect on ESO muscle contraction.

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Fig. 2.
Effect of MLCK inhibitors on contraction of smooth muscle cells
isolated by enzymatic digestion from the circular layer of the LES and
ESO. Muscle cells were contracted by a maximally effective
concentration of ACh (10 10 M). Quercetin,
L-thyroxine, and ML-7 concentration-dependently inhibited
ACh-induced contraction of LES muscle (ANOVA, P < 0.01) but had no effect on ESO muscle contraction. Values are
means ± SE of 3 animals, with 30 cells counted for each animal.
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PKC-dependent contraction of ESO and LES.
These results suggest that ACh-induced ESO contraction, which is PKC
dependent, may be mediated through a pathway not regulated by
calmodulin and MLCK. To test the possibility that PKC-induced contraction may not be regulated by calmodulin and MLCK, LES and ESO
smooth muscle cells were contracted by a maximally effective concentration of the PKC agonist DG, i.e., 10
7 M for ESO
(70) and 10
6 M for LES muscle cells
(9). We have previously shown that at these concentrations
DG-induced contraction of LES (34) and ESO cells
(70) is inhibited by the PKC inhibitor H-7 and not affected by the calmodulin inhibitor CGS-9343B, supporting the view
that DG is a selective PKC agonist. Figure
3 shows that this PKC-mediated
contraction of ESO and LES muscle cells is not affected by the MLCK
inhibitors quercetin, L-thyroxine, and ML-7.

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Fig. 3.
1,2-Dioctanoylglycerol (DG) contracts LES and ESO muscle
cells through protein kinase C (PKC)-dependent pathways. Muscle cells
were contracted by a maximally effective concentration of DG, which was
10 7 M for ESO and 10 6 M for LES muscle
cells. Quercetin (10 5 M), L-thyroxine
(10 5 M), and ML-7 (10 6 M) had no effect on
contraction of ESO and LES muscle cells. Values are means ± SE of
3 animals (except n = 4 animals for ML-7), with 30 cells counted for each animal.
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MLC phosphorylation in ACh-induced contraction of LES and ESO.
Because ACh-induced contraction of ESO and DG-induced contraction of
ESO and LES, which are PKC dependent, were not affected by MLCK
inhibitors, we tested whether ACh-induced contraction of both ESO and
LES was associated with MLC phosphorylation. MLC phosphorylation
increased concentration dependently in response to ACh in both LES and
ESO circular muscle (ANOVA, P < 0.01; Fig. 4). Phosphorylation of LES but
not ESO circular muscle was significantly reduced in the presence of
the calmodulin inhibitor CGS-9343B (ANOVA, P < 0.01;
Fig. 4). To confirm that ACh-induced MLC phosphorylation is calmodulin
dependent in LES but not in ESO muscle, we used the structurally
unrelated calmodulin inhibitor
N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W-7).
Figure 5A shows that W-7
nearly abolished ACh-induced MLC phosphorylation in LES circular muscle
(ANOVA, P < 0.01). In addition, we examined the effect
of the PKC inhibitor chelerythrine on LES MLC phosphorylation and found
that PKC inhibition had no effect. Conversely, ESO phosphorylation was
significantly reduced by chelerythrine (ANOVA, P < 0.01) and unaffected by W-7 (Fig. 5B).

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Fig. 4.
MLC phosphorylation in ESO and LES circular muscle in
response to ACh. Nonphosphorylated and phosphorylated forms of MLC were
separated by electrophoresis and localized with antibodies against MLC.
The relative amounts of phosphorylated and nonphosphorylated MLC were
quantitated by densitometry. Myosin phosphorylation was expressed as
%total MLC. A: MLC phosphorylation increased
concentration-dependently in response to ACh in both LES (ANOVA,
P < 0.01) and ESO circular muscle (ANOVA,
P < 0.01). Values are means ± SE of 5 animals,
with 3 measurements for each animal. B: phosphorylation of
LES but not of ESO circular muscle was reduced by the calmodulin
inhibitor CGS-9343B (10 5 M) (ANOVA, P < 0.01). Values are means ± SE of 2 animals, with 3 measurements
for each animal.
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Fig. 5.
ACh-induced MLC phosphorylation in LES (A) and
ESO (B) in the presence of PKC inhibitor chelerythrine or
calmodulin inhibitor W-7. MLC phosphorylation is presented as the
%increase in phophorylation in response to a maximally effective
concentration of ACh (10 5 M) compared with unstimulated
smooth muscle. A: W-7 (10 5 M) nearly abolished
ACh-induced MLC phosphorylation in LES circular muscle (ANOVA,
P < 0.01), but the PKC inhibitor chelerythrine
(10 5 M) had no effect. B: ESO phosphorylation
was significantly reduced by chelerythrine (ANOVA, P < 0.01) and unaffected by W-7. A and B: values are
means ± SE of 5 animals.
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To test whether exogenous MLCK phosphorylates myosin, we measured
phophorylation in permeabilized smooth muscle cells in calcium-free medium, after adding calcium and calmodulin but not MLCK, after adding
calcium, calmodulin, and MLCK, or after adding MLCK alone. The data
were expressed as percent phosphorylation of myosin, calculated as
phosphorylated MLC/(phosphorylated + unphosphorylated MLC). Figure
6A shows that LES
phosphorylation of myosin was low under all of the conditions examined
except in the combined presence of calcium, calmodulin, and MLCK where
phosphorylation increased from 6 ± 4% in calcium-free medium to
25 ± 5% (ANOVA, P < 0.05) in the presence of
calcium, calmodulin, and MLCK. In ESO, myosin phosphorylation increased
from 11 ± 8% in calcium-free medium to 18 ± 2% in the
combined presence of calcium, calmodulin, and MLCK (Fig.
6B). Although this slight increase in phosphorylation was
not statistically significant, it mirrors the slight contraction induced by MLCK in permeable ESO cells (Fig. 1).

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Fig. 6.
MLCK-induced MLC phosphorylation in LES (A)
and ESO permeabilized smooth muscle cells (B). MLC
phosphorylation was determined in permeabilized smooth muscle cells to
control cytosolic calcium concentration and to add the impermeant
calmodulin (CaM, 1 nM) and MLCK (10 8 M). Data are
expressed as %phosphorylation of myosin calculated as phosphorylated
MLC/(phosphorylated + unphosphorylated MLC). Representative
Western blots are shown for each experiment. A: LES
phosphorylation of myosin was low under all of the conditions examined,
except in the combined presence of calcium, calmodulin, and MLCK where
phosphorylation increased from 6 ± 4% in calcium-free medium to
25 ± 5% (ANOVA, P < 0.05). B: in
ESO, myosin phosphorylation increased from 11 ± 8% in
calcium-free medium to 18 ± 2% in the combined presence of
calcium, calmodulin, and MLCK. A and B: values
are means ± SE of 3 animals.
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Caldesmon, calponin, and regulation of PKC-induced contraction.
These data suggest that MLCK-dependent MLC phosphorylation may play a
lesser role in the ESO than in LES. In ESO, contraction and myosin
phosphorylation may be preferentially mediated, directly or indirectly,
by PKC activation.
Calponin and caldesmon are regulatory proteins associated with actin
filaments in smooth muscle and are thought to be either direct or
indirect targets of PKC. Calponin and caldesmon bind to actin,
inhibiting Mg2+-ATPase of phosphorylated smooth muscle
myosin, thereby preventing cross-bridge cycling and smooth muscle
contraction. It has been proposed (38, 59, 89, 90) that
when calponin and caldesmon are phosphorylated by kinases such as PKC,
actin binding and ATPase inhibition are abolished, restoring
cross-bridge cycling and smooth muscle contraction. We examined the
role of these regulatory proteins in contraction of LES and ESO.
Table 1 demonstrates that LES and ESO
contain approximately the same amount of calponin, but ESO contains
~50% more caldesmon than LES. These values have been normalized to
either actin or tissue dry weight.
To examine the role of caldesmon and calponin in contraction of LES and
ESO, we exposed smooth muscle cells to caldesmon and calponin purified
from chicken gizzard. Muscle cells were contracted by a maximally
effective concentration of ACh (10
9 M). ACh-induced
contraction of ESO cells was concentration-dependently inhibited by
caldesmon and calponin (ANOVA, P < 0.01; Fig.
7). However, caldesmon and calponin did
not concentration-dependently inhibit ACh-induced contraction of LES
cells (ANOVA, P > 0.5; Fig. 7).

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Fig. 7.
Effect of calponin and caldesmon on contraction of LES
(A) and ESO smooth muscle cells (B) isolated by
enzymatic digestion and permeabilized by brief exposure to saponin.
Muscle cells were contracted by a maximally effective concentration of
ACh (10 9 M). Caldesmon and calponin concentration
dependently inhibited ACh-induced contraction of ESO smooth muscle
(ANOVA, P < 0.01) but had no effect on LES
contraction. Values are means ± SE of 3 animals; with 30 cells
counted for each animal.
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In ESO cells, the caldesmon peptide GS17C, which acts as a caldesmon
antagonist (44), reversed caldesmon- but not
calponin-induced inhibition of ACh (Fig.
8). GS17C is a 18-amino-acid peptide that contains the calmodulin- and actin-binding sequence of caldesmon. Exposure of ESO cells to GS17C alone produced a concentration-dependent contraction (Fig. 9). The half-maximal
response, calculated by logit transformation, was seen at 1.3 µM.
Contraction induced by GS17C was significantly less in LES cells than
in ESO cells (ANOVA, P < 0.01; Fig. 9). XGS17C, a
peptide containing the same amino acid residues as GS17C, but with
a randomized sequence, did not cause contraction (Fig.
9).

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Fig. 8.
Effect of GS17C, a caldesmon (CD) antagonist, on
ACh-induced contraction of ESO smooth muscle cells isolated by
enzymatic digestion from the circular layer of the ESO and
permeabilized by brief exposure to saponin. GS17C is an 18-residue
peptide that contains the calmodulin- and actin-binding sequence of
caldesmon but does not inhibit actomyosin ATPase activity. Muscle cells
were contracted by a maximally effective concentration of ACh
(10 9 M) alone, or in the presence of GS17C, caldesmon
(10 5 M), caldesmon (10 5 M) plus GS17C,
calponin (CP; 10 5 M), or calponin plus GS17C. Caldesmon
and calponin significantly inhibited ACh-induced contraction of ESO
smooth muscle cells (ANOVA, P < 0.01). GS17C
(10 5 M) abolished the inhibition produced by caldesmon
(ANOVA, P < 0.01) but had no effect on calponin
inhibition. Values are means ± SE of 3 animals, with 30 cells
counted for each animal.
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Fig. 9.
GS17C, an caldesmon inhibitor, produces a
concentration-dependent contraction of ESO cells. LES (A)
and ESO smooth muscle cells (B), isolated by enzymatic
digestion and permeabilized by brief exposure to saponin, were exposed
to two peptide segments of caldesmon, GS17C and XGS17C. GS17C is an
18-residue peptide that contains the calmodulin- and actin-binding
sequence of caldesmon but does not inhibit actomyosin ATPase activity.
XGS17C contains the same amino acid residues as GS17C but with a
randomized sequence. GS17C concentration-dependently contracted ESO
cells but not LES smooth muscle cells (ANOVA, P < 0.01). The randomized peptide XGS17C had no effect on LES or ESO smooth
muscle cells. These data suggest a regulatory role for endogenous
caldesmon in ESO contraction. Values are means ± SE of 4 animals,
with 30 cells counted for each animal.
|
|
These data suggest a regulatory role for caldesmon in ESO contraction.
Because ESO contraction in response to ACh was not affected by MLCK
inhibitors, we tested whether MLCK inhibitors affected
caldesmon-dependent contraction of ESO smooth muscle cells. Cells were
exposed to a maximally effective concentration of GS17C
(10
5 M; Fig. 8). GS17C-induced contraction of ESO or LES
smooth muscle cells was not inhibited by the MLCK inhibitors quercetin,
L-thyroxine, and ML-7, suggesting that contraction induced
through inhibition of caldesmon may not be regulated by MLCK (Fig.
10).

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Fig. 10.
Effect of MLCK inhibitors on GS17C-induced contraction
of LES and ESO smooth muscle cells isolated by enzymatic digestion and
permeabilized by brief exposure to saponin. Smooth muscle cells were
exposed to GS17C (10 5 M) alone (control) or in the
presence of the MLCK inhibitors quercetin (10 5 M),
L-thyroxine (10 5 M), or ML-7
(10 5 M). GS17C-induced contraction of ESO smooth muscle
was unaffected by MLCK inhibition, suggesting that contraction induced
through inhibition of caldesmon may not be regulated by MLCK. Values
are means ± SE of 4 animals, with 30 cells counted for each
animal.
|
|
Because caldesmon and calponin can affect ACh-induced contraction of
ESO, which is PKC dependent, but not of LES, which is calmodulin
dependent, we tested the role of caldesmon and calponin in DG-induced
contraction of LES and ESO (Fig. 11).
LES and ESO were contracted by a maximally effective
concentration of the PKC agonist DG, in the presence of caldesmon and
calponin. Figure 11 shows that DG-induced contraction was inhibited by
caldesmon and calponin in both LES (ANOVA, P < 0.01)
and ESO (ANOVA, P < 0.01), suggesting that these
proteins regulate PKC-induced contraction.

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Fig. 11.
Effect of calponin and caldesmon on contraction of LES
(A) and ESO smooth muscle cells (B) isolated by
enzymatic digestion and permeabilized by brief exposure to saponin.
Muscle cells were contracted by a maximally effective concentration of
DG (10 7 M for ESO and 10 6 M for LES).
Caldesmon and calponin concentration dependently inhibited DG-induced
contraction of both LES and ESO smooth muscle (ANOVA, P < 0.01). Values are means ± SE of 3 animals, with 30 cells
counted for each animal.
|
|
 |
DISCUSSION |
Calmodulin and MLCK- and PKC-dependent contraction.
Phosphorylation of MLC by calcium and calmodulin-dependent MLCK is
believed to be the primary determinant of smooth muscle contractility.
However, a dissociation of the relationship between the intracellular
calcium concentration and tension and myosin phosphorylation has been
reported (57, 76). Therefore, other mechanisms may act in
concert with or without the participation of MLC phosphorylation.
Recently, much attention has been focused on the role of the
thin-filament, actin-binding proteins calponin and caldesmon in smooth
muscle contraction (35, 36, 65). In the present
study, we demonstrate that ACh-induced contraction of LES smooth muscle
cells depends on calcium and calmodulin-dependent MLCK activation.
Contraction of ESO smooth muscle cells is PKC dependent and may be
mediated by activation of calponin and caldesmon.
We (9) have previously shown that LES contraction in
response to a maximally effective concentration of ACh is mediated by
M3 muscarinic receptors linked to a calmodulin-dependent pathway and
spontaneous tone or contraction in response to low ACh level depends on
PKC. In contrast, ACh-induced contraction of ESO muscle depends on M2
muscarinic receptor-induced activation of a calcium-independent PKC-
(72, 73). In the ESO, ACh-induced contraction is inhibited by the PKC inhibitors H-7, calphostin C, and chelerythrine
(70). In addition, in ESO smooth muscle, ACh stimulates
the translocation of the PKC-
isozyme, but not of other isozymes,
from the cytosol to the membrane. This pathway does not involve
calmodulin, because ESO muscle cells contract relatively little in
response to exogenous calmodulin under the same conditions that cause
maximal contraction of LES cells and the calmodulin inhibitors
CGS-9343B and W-7 do not inhibit ACh-induced contraction of ESO cells
(70). In addition, permeabilized ESO cells contract in
response to DAG even in calcium-free medium containing 2 mM EGTA
(70). This finding excludes participation of the classical
calmodulin-activated MLCK in the PKC-induced contractile process,
because of the absolute requirement for calcium to activate calmodulin.
Nevertheless, exogenous calmodulin and MLCK may still cause
contraction, but this pathway is less efficient or sensitive in the ESO
than in LES. The reduced calmodulin-MLCK sensitivity of ESO
contraction is consistent with this view (Fig. 1). In LES cells
permeabilized by saponin, addition of MLCK in the presence of calcium
and calmodulin caused concentration-dependent contraction, with a
maximal response at 10
8 M MLCK. When ESO cells are
exposed to MLCK, under the same conditions in which LES cells contract,
relatively little contraction occurs. The calcium concentration used in
this experiment was the threshold amount required in our system to
cause calcium and calmodulin-dependent contraction. The calmodulin
concentration used (1 nM) is the dissociation constant of calmodulin
binding to MLCK (4). Although the mode of action of
exogenous MLCK is not entirely clear, it is plausible that MLCK, in the
presence of appropriate calcium and calmodulin concentrations, may
phosphorylate MLC and cause contraction even if ACh-induced contraction
of ESO cells depends on PKC activation and does not utilize this
pathway. This view was confirmed in Fig. 6, A and
B, in which MLCK-induced myosin phosphorylation was
examined. The MLCK-induced phosphorylation data are in agreement with
MLCK-induced contraction data. In the ESO, exogenously added MLCK, in
the presence of sufficient calcium and calmodulin, causes lower levels
of MLC phosphorylation than in the LES and causes only a slight
contraction in ESO circular smooth muscle. In contrast, MLC
phosphorylation and contraction in response to ACh are comparable in
LES and ESO, as shown in Fig. 4.
Why MLCK fails to fully contract ESO muscle cells or phosphorylate ESO
myosin is unclear. It is unlikely that the lack of effect of purified
chicken gizzard MLCK on ESO cells may depend on a different MLCK
structure in ESO. MLCK has been isolated from several smooth muscle
tissues and characterized (45, 82). The amino acid
sequences of MLCK from chicken gizzard (62), rabbit
uterine (21), and bovine stomach smooth muscles
(51) have been deduced from the corresponding cDNA
sequences. These sequences are highly conserved even between different
species (81). MLCK exhibits a high degree of substrate
specificity (46), and there is no difference in substrate
recognition properties of avian or mammalian MLCK (15). On
the other hand, it is possible that some difference may exist between
the MLC of ESO and LES, allowing recognition of LES but not of ESO MLC
by chicken gizzard MLCK.
Our data suggest that contraction of ESO muscle depends less on MLCK
than contraction of LES muscle. Several putative MLCK inhibitors, which
inhibit the maximal LES contractile response to ACh, have no effect on
ACh-induced contraction of ESO smooth muscle. Quercetin, a flavonoid,
and ML-7 are two structurally different compounds that selectively and
potently inhibit MLCK by binding hydrophobically at or near the
ATP-binding site at the active center of the enzyme (32,
68). L-Thyroxine binds to the calmodulin-binding
site of MLCK, inhibiting the binding of the calcium-calmodulin complex
to the enzyme (29).
Different levels of phosphatase activity may be present in ESO and LES.
If ESO muscle is heavily regulated by phosphatase activity, it may be
less sensitive to activation of MLCK than to inactivation of
phosphatase. Activation of PKC, and production of AA in response to
ACh, may result in inactivation of MLC phosphatase (16),
causing contraction. In permeabilized smooth muscle obtained from the
rabbit femoral artery, PKC activation by phorbol esters and short-chain
synthetic DAGs significantly increases force development and myosin
phosphorylation of serine-19, the site phosphorylated by MLCK. In
addition, the effect of phorbol ester is enhanced by the phosphatase
inhibitor microcystin LR, suggesting that PKC activation increases MLC
phosphorylation and force development through inhibition of MLC
phosphatase (54). However, in our system, PKC activation
by DG produced a contraction in both ESO and LES cells that was
unaffected by MLCK inhibitors, suggesting that MLC phosphorylation
induced by MLCK does not play a major role in PKC-mediated contraction.
These data are in agreement with Horowitz et al. (36) who
report that stimulation of ferret aortic smooth muscle cells with a
constitutively active form of PKC-
results in contraction that is
reversed by a selective PKC inhibitor but not by an MLCK inhibitor.
MLC is phosphorylated in calmodulin and MLCK- and PKC-dependent
contraction.
Because MLCK activation is not directly responsible for PKC-dependent
contraction, we examined whether ACh-induced contraction of ESO is
associated with MLC phosphorylation. Phosphorylation of the 20-kDa MLC
during contraction of cat ESO and LES muscle was previously reported by
Weisbrodt and Murphy (85), who found that phosphorylation
increased in both LES and ESO during development of contraction.
Phosphorylation reached a maximum before contraction, with maximum
phosphorylation occurring at approximately two-thirds of the time
required to achieve maximum contraction in both ESO and LES muscle
(85). After reaching a peak, phosphorylation declined even
though contraction persisted (85), and this behavior is
consistent with data in other smooth muscles (6, 22, 30, 55,
80). Our data are in agreement with these findings. We find that
contraction of ESO muscle peaks at 10 s after injection of ACh
into the muscle chamber, whereas LES contraction peaks at 15 s. We
measured myosin phosphorylation at 7 s for ESO and 10 s for
LES, i.e., at two-thirds of the time required for contraction to reach
its maximal value (85). At these times, we found that phosphorylation of the 20-kDa MLC increased concentration-dependently in both ESO and LES muscle. In addition, we report that LES, but not
ESO, phosphorylation was antagonized by the calmodulin inhibitors CGS-9343B and W-7. The selective inhibitory effect of CGS-9343B shown
in the current study correlates well with data previously reported in
which CGS-9343B selectively inhibits the contractile response to a
maximally effective concentration of ACh (10
9 M) in the
LES (9) and not in the ESO (70). CGS-9343B
selectively inhibits calmodulin by binding to the hydrophobic region of
the calmodulin molecule; it does not inhibit PKC and only weakly
affects PKA (61). In contrast, in ESO, phosphorylation was
almost abolished by the PKC inhibitor chelerythrine and not by W-7.
MLC phosphorylation was measured under free-floating (auxotonic)
conditions. We (9) previously found that under these
conditions LES tissue produces inositol 1,4,5-trisphosphate
(IP3) in basal conditions and in response to ACh
(73), suggesting that, although IP3 levels may
be higher when measured in conditions of isometric stretch, a
measurable level is produced in auxotonic conditions. We measured MLC
phosphorylation under isometric stretch, by tying muscle strips to a
Plexiglas rod, and found that under these conditions phosphorylation
was slightly higher than under free-floating conditions (data not
shown). Percent changes in ACh-induced phosphorylation, however, were
the same under either condition.
The phosphorylation studies show that ACh-induced MLC phosphorylation
(similar to ACh-induced contraction) is the same in LES and ESO. When
purified MLCK is used, however, MLCK-induced phosphorylation (and
MLCK-induced contraction) is significantly greater in LES than ESO.
These findings suggest that the classical MLCK may not be the major
kinase responsible for phosphorylation of MLC (and contraction) in ESO
and raise a question about the identity of the kinase responsible for
phosphorylating ESO muscle MLC. Because ESO contraction and MLC
phosphorylation (Fig. 5B) are PKC dependent and mediated
through a calcium-independent PKC-
isoform (70),
activation of PKC-
may result in MLC phosphorylation through a
process that may not involve an increase in activity of calcium and
calmodulin-dependent MLCK. Although PKC may directly phosphorylate MLC,
PKC phosphorylation sites of the 20-kDa MLC (serine-1, serine-2,
threonine-3) are different from the MLCK phosphorylation sites
(serine-18, serine-19) and inhibit rather than stimulate contraction
(7, 37, 60, 77). Thus it is likely that PKC-induced muscle
contraction may not result from direct PKC-induced phosphorylation of
MLC (15), but that intermediate mechanisms may be
activated in response to PKC (47-49). A chicken gizzard calcium-independent MLCK has been recently separated from MLCK
by differential extraction from myofilaments and calmodulin affinity
chromatography (84). This calcium-independent
kinase associated with the myofilaments and distinct from MLCK has been shown to mediate contraction induced by the phosphatase inhibitor microcystin. Microcystin-induced contraction correlated with
phosphorylation of MLC at serine-19 and threonine-18 (84).
In ESO circular muscle, however, phosphatase inhibitors cause
contraction by activating the calcium-independent PKC-
(50). Thus it is probable that a different kinase may
produce MLC phosphorylation in PKC-dependent contraction of ESO and LES
circular muscle.
Calponin and caldesmon regulation of PKC-induced contraction.
There is accumulating evidence for a secondary pathway of contraction
that is mediated by PKC activation of calponin and caldesmon, two
regulatory proteins associated with actin filaments in smooth muscle
(35, 36, 65). Calponin and caldesmon bind to actin and
inhibit the Mg2+-ATPase of phosphorylated smooth-muscle
myosin, thereby preventing cross-bridge cycling and smooth muscle
contraction. Actin binding and ATPase inhibition are abolished by
phosphorylation of these thin-filament proteins by PKC and calcium and
calmodulin-dependent protein kinase II and restored by
dephosphorylation (5).
Caldesmon was first described in 1981 (69) as a major
protein component of chicken-gizzard smooth muscle that interacts with
calmodulin in a calcium-dependent manner. Caldesmon interacts in a
calcium-independent manner with actin, tropomyosin, and myosin (26, 39, 69). Caldesmon colocalizes in situ with
actin, particularly with the contractile actin domain,
consistent with a role in the regulation of contraction
(20). The tissue content of caldesmon is variable, with
tonic vascular smooth muscle containing less caldesmon (1 caldesmon:
205 actin monomers) than phasic smooth muscle (1 caldesmon: 22-28
actin monomers; Ref. 27). Our data are consistent with
this finding: LES contains 50% less caldesmon than ESO smooth muscle
that does not maintain tone.
Calponin is a 34-kDa, smooth muscle-specific protein that shares many
properties with caldesmon. Shared properties are as follows:
1) calponin binds to actin and tropomysin in a
calcium-independent manner and to calmodulin in a calcium-dependent
manner (78, 79); 2) calponin colocalizes with
actin and tropomyosin in isolated smooth muscle cells
(89), is found on thin filaments in situ, and is recovered
in native thin-filament preparations (58, 83); 3) calponin inhibits myosin Mg2+-ATPase in a
reconstituted contractile system (90); and 4)
the inhibitory effects of calponin are abolished by phosphorylation.
In the current study, we demonstrate a possible role of caldesmon and
calponin in PKC-dependent contraction, as it occurs in ESO in response
to ACh or DG (as either one results in activation of PKC; Refs.
13, 70, and 75) or in LES in response
to DG. LES contraction in response to a maximally effective dose of ACh (10
9 M) is calmodulin- and MLCK dependent and PKC
independent (9) and is not affected by either caldesmon or calponin.
Addition of caldesmon to permeable smooth muscle cells
concentration-dependently inhibits ACh-induced contraction. GS17C
concentration-dependently induced contraction of ESO smooth muscle
cells. GS17C is a peptide containing an 18-residue sequence of
caldesmon (glycine-651 and serine-667) with an artificial cysteine
residue added at the COOH terminus and contains both the calmodulin-
and actin-binding sites of caldesmon. GS17C is a competitive antagonist
of endogenous caldesmon that competes with caldesmon at the strong
binding site for actin without having any effect, by itself, to inhibit
myosin ATPase activity (44). GS17C has been shown to
induce contraction of permeabilized ferret aortic cells with an
EC50 (0.92 µM) similar to our data
(44). ACh-induced contraction of ESO cells may
result from agonist-induced inhibition of endogenous caldesmon
because addition of GS17C to cells precontracted with ACh does not
result in additional contraction. In addition, GS17C reversed
caldesmon-induced inhibition of ACh. Furthermore, GS17C-induced
contraction, similar to PKC-induced contraction of ESO muscle, may be
insensitive to activation of MLCK because it was not affected by MLCK inhibitors.
We (74) have previously demonstrated that in the ESO, ACh
stimulates receptor-mediated activation of phospholipase
A2, phospholipase D (71, 73), and
phosphatidylcholine-specific phospholipase C (71, 73),
producing AA and DAG. AA and DAG act synergistically and
calcium-independently to activate PKC-
(74). It is
possible that PKC-
activation in ESO smooth muscle may result,
directly or indirectly, in the phosphorylation of caldesmon. When
caldesmon is phosphorylated, actin binding and ATPase inhibition are
abolished, restoring cross-bridge cycling and smooth muscle contraction.
Evidence (2, 3, 48) suggests that the mitogen-activated
protein kinase (MAPK) may play a role in caldesmon regulation. Caldesmon is phosphorylated by MAPK in vitro and at the same sites as
intact canine aortic strips treated with phorbol esters. These sites
are near the COOH-terminal domain of caldesmon, which interacts with
tropomysin and actin. Caldesmon can reduce mean actin sliding velocity,
and this inhibition is reversed by phosphorylation of caldesmon by p44
MAPK (23). PKC, Ras, Raf, MAPK kinase, and caldesmon have
all been identified in aortic smooth muscle. Carbachol stimulation of
airway smooth muscle increases caldesmon phosphorylation, and purified
caldesmon is a substrate for activated murine extracellular signal-related kinase 2 (ERK2) MAPK (24). In
gastrointestinal smooth muscle, a protein kinase cascade is activated
by contractile agonists, which activates ERK MAP kinases, leading to
phosphorylation of caldesmon (25). Both PKC-
and MAPK
translocate from the cytosol to the sarcolemma in response to
-adrenergic stimuli; PKC-
remains associated with the sarcolemma
whereas MAPK redistributes to the cytosol coincident with contraction
(47, 48). It is possible that in the ESO, MAPK activation
results in phosphorylation of caldesmon and contraction. This remains
to be tested.
The role of calponin in contraction of ESO smooth muscle is less well
defined. Calponin, similar to caldesmon, concentration-dependently inhibited ACh-induced contraction of ESO smooth muscle cells and PKC-dependent contraction of both LES and ESO. The physiological role
of endogenous calponin was not examined in our investigation. However,
the literature (35) supports a regulatory role of calponin in smooth muscle contraction. Treatment of aortic smooth
muscle cells with a peptide corresponding to leucine-166 and
glycine-194 of calponin produces a concentration-dependent contraction.
This calponin peptide, which includes the actin-binding domain but excludes the actomyosin ATPase inhibitory region, presumably induces contraction by alleviating the inhibitory effect of calponin
(35). Phosphorylation of both calponin and myosin increase
in intact smooth muscle tissue strips when contracted by carbachol or
the phosphatase inhibitor okadaic acid (14). Phenylephrine
stimulation of single cells isolated from ferret portal vein activates
a PKC-dependent pathway, resulting in a redistribution of calponin from
the cytosol to the surface cortex. This agonist-induced redistribution
of calponin was partially inhibited by the PKC inhibitor calphostin, overlapped in time with PKC translocation, and preceded contraction of
these cells (63). In addition, a constitutively active
form of PKC-
has been shown (36) to phosphorylate both
caldesmon and calponin. These studies suggest a possible physiological
role of calponin in mediating agonist-induced PKC-dependent contraction.
We conclude that in LES and ESO muscle there are two distinct types of
contractions, mediated by distinct regulatory mechanisms. A
PKC-dependent pathway, perhaps regulated by caldesmon and calponin, is
present in ESO and LES. A calmodulin and MLCK-dependent pathway is
present only in LES muscle. MLC phosphorylation occurs in both pathways.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-28614 and Korea Science and
Engineering Foundation Grant 2000-1-21400-001-3.
 |
FOOTNOTES |
A portion of this work was presented at the annual meeting of the
American Gastroenterological Association in San Diego, CA, May 1995, and has been previously published in abstract form
(Gastroenterology 108: A692, 1995).
Address for reprint requests and other correspondence: P. Biancani, Gastrointestinal Motility Research Laboratory, SWP5, Rhode Island Hospital and Brown Univ., 593 Eddy St., Providence, RI 02903 (E-mail: Piero-Biancani{at}brown.edu).
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. Section 1734 solely to indicate this fact.
Received 16 November 2000; accepted in final form 16 March 2001.
 |
REFERENCES |
1.
Abe, M,
Takahashi K,
and
Hiwada K.
Simplified co-purification of vascular smooth muscle calponin and caldesmon.
J Biochem (Tokyo)
107:
507-509,
1990[Abstract].
2.
Adam, LP,
Haeberle JR,
and
Hathaway DR.
Phosphorylation of caldesmon in arterial smooth muscle.
J Biol Chem
264:
7698-7703,
1989[Abstract/Free Full Text].
3.
Adam, LP,
and
Hathaway DR.
Identification of mitogen-activated protein kinase phosphorylation sequences in mammalian h-Caldesmon.
FEBS Lett
322:
56-60,
1993[ISI][Medline].
4.
Adelstein, RS,
and
Klee CB.
Purification and characterization of smooth muscle myosin light chain kinase.
J Biol Chem
256:
7501-7509,
1981[Abstract/Free Full Text].
5.
Allen, BG,
and
Walsh MP.
The biochemical basis of the regulation of smooth muscular contraction.
Trends Biochem Sci
19:
362-368,
1994[ISI][Medline].
6.
Bárány, M,
and
Bárány K.
Dissociation of relaxation and myosin light chain dephosphorylation in porcine uterine muscle.
Arch Biochem Biophys
305:
202-204,
1993[ISI][Medline].
7.
Bengur, AB,
Robinson EA,
Appella E,
and
Sellers JR.
Sequence of the sites phosphorylated by protein kinase C in smooth muscle myosin light chain.
J Biol Chem
262:
7613-7617,
1987[Abstract/Free Full Text].
8.
Biancani, P,
Billett G,
Hillemeier C,
Nissenshon M,
Rhim BY,
Sweczack S,
and
Behar J.
Acute experimental esophagitis impairs signal transduction in cat LES circular muscle.
Gastroenterology
103:
1199-1206,
1992[ISI][Medline].
9.
Biancani, P,
Harnett KM,
Sohn UD,
Rhim BY,
Behar J,
Hillemeier C,
and
Bitar KN.
Differential signal transduction pathways in cat lower esophageal sphincter tone and response to ACh.
Am J Physiol Gastrointest Liver Physiol
266:
G767-G774,
1994[Abstract/Free Full Text].
10.
Biancani, P,
Hillemeier C,
Bitar KN,
and
Makhlouf GM.
Contraction mediated by Ca2+ influx in the esophagus and by Ca2+ release in the LES.
Am J Physiol Gastrointest Liver Physiol
253:
G760-G766,
1987[Abstract/Free Full Text].
11.
Bitar, KN,
Bradford P,
Putney JW,
and
Makhlouf GM.
Stoichiometry of contraction and Ca2+ mobilization by inositol 1,4,5-triphosphate in isolated gastric smooth muscle cells.
J Biol Chem
261:
16591-16596,
1986[Abstract/Free Full Text].
12.
Bitar, KN,
Kaminski MS,
Hailat N,
Cease KB,
and
Strahler JR.
HSP27 is a mediator of sustained smooth muscle contraction in response to bombesin.
Biochem Biophys Res Commun
181:
1192-1200,
1991[ISI][Medline].
13.
Cao, W,
Chen Q,
Sohn UD,
Kim NY,
Kirber MT,
Harnett KM,
Behar J,
and
Biancani P.
Ca2+-induced contraction of cat esophageal circular smooth muscle cells.
Am J Physiol Cell Physiol
280:
C980-C992,
2001[Abstract/Free Full Text].
14.
Carmichael, JD,
Winder SJ,
and
Walsh MP.
Calponin and smooth muscle regulation.
Can J Physiol Pharmacol
72:
1415-1419,
1994[ISI][Medline].
15.
Colburn, JC,
Michnoff CH,
Hsu LC,
Slaughter CA,
Kamm KE,
and
Stull JT.
Sites phosphorylated in myosin light chain in contracting smooth muscle.
J Biol Chem
263:
19166-19173,
1988[Abstract/Free Full Text].
16.
Cui Gong, M,
Fuglsang A,
Alessi D,
Kobayashi S,
Cohen P,
Somlyo AV,
and
Somlyo AP.
Arachidonic acid inhibits myosin light chain phosphatase and sensitizes smooth muscle to calcium.
J Biol Chem
267:
21492-21498,
1992[Abstract/Free Full Text].
17.
Dabrowska, R,
Aromatorio D,
Sherry JMF,
and
Hartshorne DJ.
Composition of the myosin light chain kinase from chicken gizzard.
Biochem Biophys Res Commun
78:
1263-1272,
1977[ISI][Medline].
18.
DeFeo, TT,
and
Morgan KG.
Calcium-force relationship as detected with aequorin in two different vascular smooth muscles of the ferret.
J Physiol (Lond)
369:
269-282,
1985[Abstract].
19.
Fabiato, A,
and
Fabiato F.
Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells.
J Physiol (Lond)
75:
463-505,
1979.
20.
Furst, DO,
Cross RA,
DeMey J,
and
Small JV.
Caldesmon is an elongated, flexible molecule localized in the actomyosin domains of smooth muscle.
EMBO J
5:
251-257,
1986[Abstract].
21.
Gallager, PJ,
Herring BP,
Griffin SA,
and
Stull JT.
Molecular characterization of mammalian smooth muscle myosin light chain kinase.
J Biol Chem
266:
23936-23944,
1991[Abstract/Free Full Text].
22.
Gerthoffer, WT.
Dissociation of myosin phosphorylation and active tension during muscarinic stimulation of tracheal smooth muscle.
J Pharmacol Exp Ther
240:
8-15,
1987[Abstract].
23.
Gerthoffer, WT,
and
Pohl J.
Caldesmon and calponin phosphorylation in regulation of smooth muscle contraction.
Can J Physiol Pharmacol
72:
1410-1414,
1994[ISI][Medline].
24.
Gerthoffer, WT,
Yamboliev IA,
Pohl J,
Haynes R,
Dang S,
and
McHugh J.
Activation of MAP kinases in airway smooth muscle.
Am J Physiol Lung Cell Mol Physiol
272:
L244-L252,
1997[Abstract/Free Full Text].
25.
Gerthoffer, WT,
Yamboliev IA,
Shearer M,
Pohl J,
Haynes R,
Dang S,
Sato K,
and
Sellers JR.
Activation of MAP kinases and phosphorylation of caldesmon in canine colonic smooth muscle.
J Physiol (Lond)
495:
597-609,
1996[Abstract].
26.
Graceffa, P.
Evidence for interaction between smooth muscle tropomysin and caldesmon.
FEBS Lett
218:
139-142,
1987[ISI][Medline].
27.
Haeberle, JR,
Hathaway DR,
and
Smith CL.
Caldesmon content of mammalian smooth muscles.
J Muscle Res Cell Motil
13:
81-89,
1992[ISI][Medline].
28.
Hafner, D,
Heinen E,
and
Noack E.
Mathematical analysis of concentration-response relationships. Method for the evaluation of the ED50 and the number of binding sites per receptor molecule using logit transformation.
Arzneim Forsch
27:
1871-1873,
1977[Medline].
29.
Hagiwara, M,
Mamiya S,
and
Hidaka H.
Selective binding of L-thyroxine by myosin light chain kinase.
J Biol Chem
264:
40-44,
1989[Abstract/Free Full Text].
30.
Hai, CM,
and
Murphy RA.
Ca2+ crossbridge phosphorylation and contraction.
Annu Rev Physiol
51:
285-298,
1989[ISI][Medline].
31.
Hartshorne, DJ.
Biochemistry of the contractile process in smooth muscle.
In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by Johnson LR.. New York: Raven, 1987, p. 131-142.
32.
Hidaka, H,
Hagiwara M,
and
Tokumitsu H.
Novel and selective inhibitors of CaM-kinase and other calmodulin-dependent enzymes.
Adv Exp Med Biol
269:
159-162,
1990[Medline].
33.
Hillemeier, C,
Bitar KN,
Marshall JM,
and
Biancani P.
Intracellular pathways for contraction in gastroesophageal smooth muscle cells.
Am J Physiol Gastrointest Liver Physiol
260:
G770-G775,
1991[Abstract/Free Full Text].
34.
Hillemeier, C,
Bitar KN,
Sohn UD,
and
Biancani P.
Protein kinase C mediates spontaneous tone in the cat lower esophageal sphincter.
J Pharmacol Exp Ther
277:
144-149,
1996[Abstract].
35.
Horowitz, A,
Clement-Chomienne O,
Walsh M,
Tao T,
Katsuyama H,
and
Morgan KG.
Effects of calponin on force generation by single smooth muscle cells.
Am J Physiol Heart Circ Physiol
270:
H1858-H1863,
1996[Abstract/Free Full Text].
36.
Horowitz, A,
Clement-Chomienne O,
Walsh MP,
and
Morgan KG.
Epsilon-isozyme of protein kinase C induces a Ca2+-independent contraction in vascular smooth muscle.
Am J Physiol Cell Physiol
271:
C589-C594,
1996[Abstract/Free Full Text].
37.
Ikebe, M,
Hartshorne DJ,
and
Elzinga M.
Phosphorylation of 20,000-dalton light chain of smooth muscle myosin by the calcium-activated, phospholipid-dependent protein kinase.
J Biol Chem
262:
9569-9573,
1987[Abstract/Free Full Text].
38.
Ikebe, M,
and
Hornick T.
Determination of the phosphorylation site of smooth muscle caldesmon by protein kinase C.
Arch Biochem Biophys
288:
538-542,
1991[ISI][Medline].
39.
Ikebe, M,
and
Reardon S.
Binding of caldesmon to smooth muscle myosin.
J Biol Chem
263:
3055-3058,
1988[Abstract/Free Full Text].
40.
Itoh, T,
Ikebe M,
Kargacin GJ,
Hartshorne DJ,
Kemp BE,
and
Fay FS.
Effects of modulators of myosin light-chain kinase activity in single smooth muscle cells.
Nature
338:
164-167,
1989[ISI][Medline].
41.
Kamm, KE,
Hsu LC,
Kubota Y,
and
Stull JT.
Phosphorylation of smooth muscle myosin heavy and light chains.
J Biol Chem
264:
21223-21229,
1989[Abstract/Free Full Text].
42.
Kamm, KE,
and
Stull JT.
Activation of smooth muscle contraction: relation between myosin phosphorylation and stiffness.
Science
232:
80-82,
1986[ISI][Medline].
43.
Kargacin, GJ,
Ikebe M,
and
Fay FS.
Peptide modulators of myosin light chain kinase affect smooth muscle cell contraction.
Am J Physiol Cell Physiol
259:
C315-C324,
1990[Abstract/Free Full Text].
44.
Katsuyama, H,
Wang C-LA,
and
Morgan KG.
Regulation of vascular smooth muscle tone by caldesmon.
J Biol Chem
267:
14555-14558,
1992[Abstract/Free Full Text].
45.
Kemp, B,
and
Stull JT.
Myosin light chain kinases.
In: Peptides and Protein Phosphorylation, edited by Kemp B.. Boca Raton, FL: CRC, 1990, p. 115-133.
46.
Kemp, BE,
Pearson RB,
and
House C.
Role of basic residues in the phosphorylation of synthetic peptides by myosin light chain kinase.
Proc Natl Acad Sci USA
80:
7471-7475,
1983[Abstract].
47.
Khalil, RA,
Menice CB,
Wang C-LA,
and
Morgan KG.
Phosphotyrosine-dependent targeting of mitogen-activated protein kinase in differentiated contractile vascular cells.
Circ Res
76:
1101-1108,
1995[Abstract/Free Full Text].
48.
Khalil, RA,
and
Morgan KG.
PKC-mediated redistribution of mitogen-activated protein kinase during smooth muscle cell activation.
Am J Physiol Cell Physiol
265:
C406-C411,
1993[Abstract/Free Full Text].
49.
Khalil, RA,
and
Morgan KG.
Protein kinase C: a second E-C coupling pathway in vascular smooth muscle?
N Engl J Med
7:
10-15,
1992.
50.
Kim, N,
Song SC,
Kim Y,
Harnett KM,
and
Biancani P.
Contraction induced by phosphatase inhibitors in esophageal and LES smooth muscle (Abstract).
Gastroenterology
114:
A777,
1998.
51.
Kobayashi, H,
Inoue A,
Mikawa T,
Kuwayame H,
Hotta Y,
Masaki T,
and
Ebashi S.
Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity.
J Biochem (Tokyo)
112:
786-791,
1992[Abstract].
52.
Leachman, SA,
Gallager PJ,
Herring BP,
McPhaul MJ,
and
Stull JT.
Biochemical properties of chimeric skeletal and smooth muscle myosin light chain kinases.
J Biol Chem
267:
4930-4938,
1992[Abstract/Free Full Text].
53.
Lynch, W,
and
Bretcher A.
Purification of caldesmon.
Methods Enzymol
134:
37-42,
1986[ISI][Medline].
54.
Masuo, M,
Reardon S,
Ikebe M,
and
Kitazawa T.
A novel mechanism for the Ca2+-sensitizing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase.
J Gen Physiol
104:
265-286,
1994[Abstract].
55.
McDaniel, NL,
Chen XL,
Singer HA,
Murphy RA,
and
Rembold CM.
Nitrovasodilators relax arterial smooth muscle by decreasing [Ca2+] and uncoupling stress from myosin phosphorylation.
Am J Physiol Cell Physiol
263:
C461-C467,
1992[Abstract/Free Full Text].
56.
Miller-Hance, WC,
Miller JR,
Wells JN,
Stull JT,
and
Kamm KE.
Biochemical events associated with activation of smooth muscle contraction.
J Biol Chem
262:
13979-13982,
1988[Abstract/Free Full Text].
57.
Moreland, S,
Nishimura J,
van Breemen C,
Ahn HY,
and
Moreland RS.
Transient myosin phoshorylation at constant Ca2+ during agonist activation of permeabilized arteries.
Am J Physiol Cell Physiol
263:
C540-C544,
1992[Abstract/Free Full Text].
58.
Ngai, PK,
Scott-Woo GC,
Lim MS,
Sutherland C,
and
Walsh MP.
Activation of smooth muscle myosin Mg2+-ATPase by native thin filaments and actin-tropomysin.
J Biol Chem
262:
5352-5359,
1987[Abstract/Free Full Text].
59.
Ngai, PK,
and
Walsh MP.
Inhibition of smooth muscle actin-activated myosin Mg2+-ATPase activity by caldesmon.
J Biol Chem
259:
13656-13659,
1984[Abstract/Free Full Text].
60.
Nishikawa, M,
Hidaka H,
and
Adestein RS.
Phosphorylation of smooth muscle heavy meromyosin by calcium-activated, phospholipid-dependent protein kinase: the effect on actin-activated MgATPase activity.
J Biol Chem
258:
14069-14072,
1983[Abstract/Free Full Text].
61.
Norman, JA,
Ansell J,
Stone GA,
Wennogle LP,
and
Wasley JW.
CGS 9343B, a novel, potent, and selective inhibitor of calmodulin activity.
Mol Pharmacol
31:
535-540,
1987[Abstract].
62.
Olson, NJ,
Pearson R,
Needelman DS,
Hurwitz MY,
Kemp BE,
and
Means AR.
Regulatory and structural motifs of chicken gizzard myosin light chain kinase.
Proc Natl Acad Sci USA
87:
2284-2288,
1990[Abstract].
63.
Parker, CA,
Takahashi K,
Tai T,
and
Morgan KG.
Agonist-induced redistribution of calponin in contractile vascular smooth muscle.
Am J Physiol Cell Physiol
267:
C1262-C1270,
1994[Abstract/Free Full Text].
64.
Persechini, A,
Kamm KE,
and
Stull JT.
Different phosphorylated forms of myosin in contracting tracheal smooth muscle.
J Biol Chem
261:
6293-6299,
1986[Abstract/Free Full Text].
65.
Pohl, J,
Winder SJ,
Allen BG,
Walsh MP,
Sellers JR,
and
Gerthoffer WT.
Phosphorylation of calponin in airway smooth muscle.
Am J Physiol Lung Cell Mol Physiol
272:
L115-L123,
1997[Abstract/Free Full Text].
66.
Porzio, MA,
and
Pearson AM.
Improved resolution of myofibrillar proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Biochim Biophys Acta
490:
27-34,
1977[ISI][Medline].
67.
Rembold, CM.
Regulation of contraction and relaxation in arterial smooth muscle.
Hypertension
20:
129-137,
1992[Abstract].
68.
Saitoh, M,
Ishikawa T,
Matsushima S,
Naka M,
and
Hidaka H.
Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase.
J Biol Chem
262:
7796-7801,
1987[Abstract/Free Full Text].
69.
Sobue, K,
Muramoto Y,
Fujita M,
and
Kakiuchi S.
Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin.
Proc Natl Acad Sci USA
78:
5652-5655,
1981[Abstract].
70.
Sohn, UD,
Chiu TT,
Bitar KN,
and
Hillemeier C.
Calcium requirements for ACh induced contraction of cat esophageal circular muscle cells.
Am J Physiol Gastrointest Liver Physiol
266:
G330-G338,
1994[Abstract/Free Full Text].
71.
Sohn, UD,
Han B,
Tashjian AH, Jr,
Behar J,
and
Biancani P.
Agonist independent, muscle type specific signal transduction pathways in cat esophageal and lower esophageal sphincter (LES) circular smooth muscle.
J Pharmacol Exp Ther
273:
482-491,
1995[Abstract].
72.
Sohn, UD,
Harnett KM,
Cao W,
Rich H,
Kim N,
Behar J,
and
Biancani P.
Acute experimental esophagitis activates a second signal transduction pathway in cat smooth muscle from the lower esophageal sphincter.
J Pharmacol Exp Ther
283:
1293-1304,
1997[Abstract/Free Full Text].
73.
Sohn, UD,
Harnett KM,
De Petris G,
Behar J,
and
Biancani P.
Distinct muscarinic receptors, G-proteins, and phospholipases in esophageal and lower esophageal sphincter circular muscle.
J Pharmacol Exp Ther
267:
1205-1214,
1993[Abstract].
74.
Sohn, UD,
Kim DK,
Bonventre JV,
Behar J,
and
Biancani P.
Role of 100 kDa cytosolic PLA2 in ACh-induced contraction of esophageal circular muscle.
Am J Physiol Gastrointest Liver Physiol
267:
G433-G441,
1994[Abstract/Free Full Text].
75.
Sohn, UD,
Zoukhri D,
Dartt D,
Sergheraert C,
Harnett KM,
Behar J,
and
Biancani P.
Different PKC isozymes mediate lower esophageal sphincter (LES) tone and phasic contraction of esophageal (ESO) circular smooth muscle in the cat.
Mol Pharmacol
51:
462-470,
1997[Abstract/Free Full Text].
76.
Stull, JT,
Gallagher PJ,
Herring BP,
and
Kamm KE.
Vascular smooth muscle contractile elements. Cellular regulation.
Hypertension
17:
723-732,
1991[Abstract].
77.
Sutton, TA,
and
Haeberle JR.
Phosphorylation by protein kinase C of the 20,000-dalton light chain of myosin in intact and chemically skinned vascular smooth muscle.
J Biol Chem
265:
2749-2754,
1990[Abstract/Free Full Text].
78.
Takahashi, K,
Abe M,
Hiwada K,
and
Kokubu T.
A novel troponin T-like protein (calponin) in vascular smooth muscle: interaction with tropomysin paracrystals.
J Hypertension
6 Suppl:
S40-S43,
1988[ISI].
79.
Takahashi, K,
Hiwada K,
and
Kokubu T.
Isolation and characterization of a 34,000 dalton calmodulin- and F actin-binding protein from chicken gizzard smooth muscle.
Biochem Biophys Res Commun
141:
20-26,
1986[ISI][Medline].
80.
Tansey, MG,
Hori M,
Karaki H,
Kamm KE,
and
Stull JT.
Okadaic acid uncouples myosin light chain phosphorylation and tension in smooth muscle.
FEBS Lett
270:
219-221,
1990[ISI][Medline].
81.
Walsh, MP.
Calmodulin and the regulation of smooth muscle contraction.
Mol Cell Biochem
135:
21-41,
1994[ISI][Medline].
82.
Walsh, MP.
Regulation of vascular smooth muscle tone.
Can J Physiol Pharmacol
72:
919-936,
1994[ISI][Medline].
83.
Walsh, MP,
Carmichael JD,
and
Kargacin GJ.
Characterization and confocal imaging of calponin in gastrointestinal smooth muscle.
Am J Physiol Cell Physiol
265:
C1371-C1378,
1993[Abstract/Free Full Text].
84.
Weber, LP,
Van Lierop JE,
and
Walsh MP.
Ca2+-independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments.
J Physiol (Lond)
516:
805-824,
1999[Abstract/Free Full Text].
85.
Weisbrodt, NW,
and
Murphy RA.
Myosin phosphorylation and contraction of feline esophageal smooth muscle.
Am J Physiol Cell Physiol
249:
C9-C14,
1985[Abstract/Free Full Text].
86.
Williams, DA,
Becker PL,
and
Fay FS.
Regional changes in calcium underlying contraction of single smooth muscle cells.
Science
235:
1644-1648,
1987[ISI][Medline].
87.
Williams, DA,
and
Fay FS.
Calcium transients and resting levels in isolated smooth muscle cells as monitored with quin-2.
Am J Physiol Cell Physiol
250:
C779-C791,
1986[Abstract/Free Full Text].
88.
Winder, SJ,
Pato MD,
and
Walsh MP.
Purification and characterization of calponin phosphatase from smooth muscle. Effect of dephosphorylation on calponin function.
Biochem J
286:
197-203,
1992[ISI][Medline].
89.
Winder, SJ,
and
Walsh MP.
Calponin: thin filament-linked regulation of smooth muscle contraction.
Cell Signal
5:
677-686,
1993[ISI][Medline].
90.
Winder, SJ,
and
Walsh MP.
Smooth muscle calponin. Inhibition of actomysin Mg ATPase and regulation by phosphorylation.
J Biol Chem
265:
10148-10155,
1990[Abstract/Free Full Text].
91.
Zhan, Q,
Wong SS,
and
Wang C-LA.
A calmodulin-binding peptide of caldesmon.
J Biol Chem
266:
21810-21814,
1991[Abstract/Free Full Text].
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