1 Department of Medicine, Rhode Island Hospital and Brown Medical School, Providence, Rhode Island 02903; 2 Department of Pharmacology, College of Pharmacy, Chung Ang University, Seoul 156-756, and 3 Department of Internal Medicine, Kangnam General Hospital, Public Corporation, Seoul 135-090, Korea
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ABSTRACT |
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ACh-induced contraction of esophageal circular
muscle (ESO) depends on Ca2+ influx and activation of
protein kinase C (PKC
). PKC
, however, is known to be
Ca2+ independent. To determine where Ca2+ is
needed in this PKC
-mediated contractile pathway, we examined successive steps in Ca2+-induced contraction of ESO muscle
cells permeabilized by saponin. Ca2+ (0.2-1.0 µM)
produced a concentration-dependent contraction that was antagonized by
antibodies against PKC
(but not by PKC
II or PKC
antibodies),
by a calmodulin inhibitor, by MLCK inhibitors, or by GDP
s. Addition
of 1 µM Ca2+ to permeable cells caused myosin light chain
(MLC) phosphorylation, which was inhibited by the PKC inhibitor
chelerythrine, by D609 [phosphatidylcholine-specific phospholipase C
inhibitor], and by propranolol (phosphatidic acid phosphohydrolase
inhibitor). Ca2+-induced contraction and diacylglycerol
(DAG) production were reduced by D609 and by propranolol, alone or in
combination. In addition, contraction was reduced by
AACOCF3 (cytosolic phospholipase A2
inhibitor). These data suggest that Ca2+ may directly
activate phospholipases, producing DAG and arachidonic acid (AA), and
PKC
, which may indirectly cause phosphorylation of MLC. In addition,
direct G protein activation by GTP
S augmented Ca2+-induced contraction and caused dose-dependent
production of DAG, which was antagonized by D609 and propranolol. We
conclude that agonist (ACh)-induced contraction may be mediated by
activation of phospholipase through two distinct mechanisms (increased
intracellular Ca2+ and G protein activation), producing DAG
and AA, and activating PKC
-dependent mechanisms to cause contraction.
calcium; smooth muscle; protein kinase C; phospholipase C; phospholipase D; myosin phosphorylation
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INTRODUCTION |
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MYOSIN LIGHT CHAIN KINASE (MLCK), in the presence of Ca2+ and calmodulin, phosphorylates the 20-kDa myosin light chain, causing contraction. The relationship between increased Ca2+ concentration, activation of the calmodulin-myosin light-chain kinase pathway, and contraction has been extensively examined. It has been proposed that MLCK plays an essential role in the activation process in the smooth muscle cell, so that activation of this enzyme is both necessary and sufficient for the initiation of contraction (31). Because the magnitude of changes in intracellular Ca2+ is not directly related to the force developed (27, 42, 52), an effort has been made to characterize the "Ca2+ sensitivity" of the contractile process by proposing that Ca2+ sensitivity may be modified by a number of factors, including activation of trimeric G proteins (19, 27, 35, 38, 42, 44, 52) and monomeric or "small" G proteins, and resulting in inhibition of phosphatases (19, 36, 40, 63) or in modulation of MLCK (2, 65, 67).
Although these factors affect the amplitude of contraction, a view of
the contractile process as a MLCK-dependent relationship between
Ca2+ and contraction excludes the possibility that
contraction may occur through calmodulin-MLCK-independent pathways.
Data from our laboratory (Table 1) suggest that
calmodulin and MLCK play a role in ACh-induced lower esophageal
sphincter (LES) contraction but not in contraction of esophageal
circular muscle (ESO) (Table 1), which is mediated by activation of
protein kinase C (PKC
) (61). PKC
is
Ca2+ independent, and diacylglycerol (DAG)-induced
activation of permeable ESO cells can occur in Ca2+-free
medium (58). It is thus unlikely that this ESO contraction may be mediated by MLCK, which requires Ca2+/calmodulin to
be activated. In addition, ACh- or DAG-induced contraction of ESO cells
is not affected by either calmodulin or MLCK inhibitors (Table 1), and
permeable ESO cells do not contract well in response to purified
calmodulin or MLCK at concentrations that cause pronounced contraction
of LES cells (Sohn UD, Tang DC, Stull JT, Haeberle JR, Wang C-LA,
Harnett KM, and Biancani P, unpublished observations).
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To understand this calmodulin-MLCK-independent contraction, we
investigated Ca2+-induced contraction in ESO to identify
the step in the contractile pathway that was dependent on the presence
of Ca2+ to initiate contraction. We have previously shown
that ACh-induced contraction of ESO depends on influx of
Ca2+ but is mediated through a
Ca2+-insensitive PKC-dependent pathway (7,
58, 61). In addition, we have shown that Ca2+ is
required for production of the second messenger DAG. DAG, however, does
not need Ca2+ to contract ESO cells (58).
These data suggest that Ca2+ may be required for activation
of the phospholipases responsible for production of second messengers.
After the second messengers are produced, contraction may proceed
through a pathway, which is Ca2+ independent.
The data support this hypothesis: Ca2+ causes
dose-dependent contraction of ESO cells through a PKC-dependent
pathway, resulting from Ca2+-induced activation of
phospholipases, production of second messengers, and phosphorylation of
myosin light chains.
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METHODS |
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Tissue dissection and dispersion of smooth muscle cells. Adult cats of either sex weighing 3-5 kg were euthanized, and esophageal smooth muscle squares from the circumferential muscle layer were prepared as previously described (7). The chest and abdomen were opened with a midline incision exposing the esophagus and stomach. The esophagus and stomach were removed together and pinned on a wax block at their in vivo dimensions and orientation. The esophagus and stomach were opened along the lesser curvature. After opening the esophagus and stomach and identifying the LES, we removed the mucosa and submucosal connective tissue by sharp dissection. The LES was excised, and a 3- to 5-mm-wide strip at the junction of the LES and esophagus was discarded to avoid overlap. The circular muscle layer from the esophagus 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, and slices were cut by hand into 2 × 2-mm tissue squares.
Tissue squares were digested in HEPES buffer, containing 0.1% collagenase type II to isolate smooth muscle cells, as previously described (7). The HEPES-buffered solution contained 112.5 mM NaCl, 5.5 mM KCl, 2 mM KH2PO4, 10.8 mM glucose, 24 mM HEPES sodium salt, 1.87 mM CaCl2, 0.6 mM MgCl2, 0.3 mg/ml basal medium Eagle amino acid supplement, and 0.08 mg/ml soybean trypsin inhibitor. The solution was gently gassed with 100% O2. At the end of the digestion period, the tissue was poured over a 200-µm nylon mesh (Tetko, Elmsford, NY), rinsed in collagenase-free HEPES buffer to remove any trace of collagenase, and incubated in this solution at 31°C, gassed with 100% O2. The cells were allowed to dissociate freely for 10-20 min. Cells were permeabilized, when necessary, to control intracellular Ca2+ concentration or to allow the use of agents such as antibodies, guanosine 5'-O-(3-thiotriphosphate) (GTPContraction of isolated muscle cells.
The cells were contracted by exposing them to the test agonists, i.e.,
Ca2+ or GTPS. For generation of Ca2+
concentration-response curves, cells were permeabilized in cytosolic medium containing 0 CaCl2 and 1 mM EGTA. Cells were
contracted by 30-s exposure to different Ca2+
concentrations calculated by the method of Fabiato and Fabiato (16). For generation of GTP
S concentration-response
curves, cells were permeabilized in the indicated concentration of
Ca2+, and then contracted by 30-s exposure to the indicated
concentration of GTP
S.
Myosin phosphorylation. For measurement of myosin phosphorylation, permeabilized smooth muscle cells of the ESO were prepared as described above and preincubated in modified cytosolic buffer at 31°C for 20 min. Cells were then stimulated with 1 µM Ca2+ for 10 s. The reaction was stopped by freezing the cells in a slurry containing acetone (90%), trichloroacetic acid (10%), 1 mM dithiothreitol, and dry ice.
Nonphosphorylated and phosphorylated forms of myosin light chain were separated by electrophoresis and localized with antibodies against myosin light chain. The relative amounts of phosphorylated and nonphosphorylated myosin light chain were quantitated by densitometry (13, 30). Briefly, protein was extracted in an 8 M urea buffer and processed for urea/glycerol-polyacrylamide gel electrophoresis as described in Ref. 48. Nonphosphorylated and phosphorylated forms of the light chain were separated following electrophoresis at 20°C and 30 mA for 4-6 h. Proteins were electrophoretically transferred from glycerol gels onto nitrocellulose paper. Myosin light chains were localized on nitrocellulose paper with antibodies against myosin light chain. Relative amounts of phosphorylated and nonphosphorylated myosin light chain were quantitated from densitometry scans of the immunostained nitrocellulose blots. Myosin phosphorylation was expressed as percent of total myosin light chain (13, 30).Cytosolic Ca2+ measurements. Freshly digested cells were placed on a shallow muscle chamber mounted on the stage of an inverted microscope (Carl Zeiss). The cells were allowed to settle to the bottom of the chamber. ACh was "spritzed" directly on the cells using a pressure ejection micropipette system.
Ca2+ measurements were obtained using a dual excitation wavelength imaging system (IonOptix Milton, MA). The Ca2+ concentrations were obtained from the ratios of fluorescence elicited by 340 nm excitation to 380 nm excitation using standard techniques (21). Fura 2 calibration was carried out using the potassium salt of fura 2 and solutions containing an excess of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) for determination of minimum ratio (Rmin) and addition of saturating amounts of calcium for determination of maximum ratio (Rmax). The dissociation constant (Kd) used was 224 nM. Rmax and Rmin were adjusted for in-cell conditions. Rmin was measured in loaded cells permeabilized with ionomycin in the presence of excess BAPTA in the extracellular solution. The fractional decrease in Rmin was measured to be 0.65 times the value measured in solution. Rmax was also then corrected by this same factor. Background fluorescence was subtracted by defining a region outside of the cell and averaging the fluorescence in those pixels and subtracting that average from the fluorescence measured in the cell at each of the wavelengths. Additional autofluorescence in the cells was negligible. The ratiometric images were masked in the region outside the borders of the cell, since low photon counts can give unreliable ratios near the edges. An adaptive mask followed the borders of the cell as the Ca2+ changed and the cell contracted. A pseudoisosbestic image (i.e., an image insensitive to Ca2+ changes) was formed in computer memory from a weighted sum of the images generated by 340 nm excitation and 380 nm excitation. This image was then thresholded, and values below a selected level were considered outside the cell and called 0. For each ratiometric image, the outline of the cell was determined and the generated mask was applied to the ratiometric image. This method allowed the simultaneous imaging of both the rapid changes in Ca2+ and cell length. Our algorithm was incorporated into the IonOptix software.DAG measurements.
Permeabilized smooth muscle cells of the ESO were prepared as described
above and preincubated in modified cytosolic buffer at 31°C for 20 min. Cells were then incubated for 1 min in 0.5 ml modified cytosolic
buffer alone, or containing the phospholipase inhibitors D609
(104 M), propranolol (10
4 M), or U-73122
(10
6 M). The cells were stimulated with calcium (1 µM)
or GTP
S (10
6 M) for 40 s. The reaction was
stopped with 3 ml chloroform-methanol (1:2 vol/vol). DAG was
extracted by the addition of 1 ml of 1 M NaCl and 1 ml chloroform. The
upper aqueous phase was discarded, and the lower organic phase was
collected, evaporated under a stream of nitrogen, and frozen for DAG
determination within 1 wk.
Drugs and chemicals.
AACOCF3 was purchased from Calbiochem; chelerythrine
chloride from LC Services (Woburn, MA); collagenase type II and soybean trypsin inhibitor from Worthington Biochemicals (Freehold, NJ); fura 2 and BAPTA tetrasodium salt from Molecular Probes (Eugene, OR);
1-(5-iodonaphthalene-1-sulfonyl)-H-hexahydro-1,4-diazepine hydrochloride (ML-7) from Seikagaku (Rockville, MD); MLCK and MLC
antibodies from Accurate Chemicals and Scientific (Westbury, NY);
[32P]ATP from New England Nuclear (Boston, MA); PKC
antibodies (II,
, and
) from GIBCO BRL (Gaithersburg, MD); SDS
sample buffer from Bio-Rad (Melville, NY); D609 from Kamiya Biomedical
(Thousand Oaks, CA); and U-73122 from Biomol (Plymouth Meeting, PA).
ACh, ATP (disodium salt), antimycin A, BME amino acid supplement,
creatine phosphate, creatine phosphokinase, EGTA, GDP
S, GTP
S
(tetralithium salt), HEPES (sodium salt), propranolol, saponin, and
other reagents were purchased from Sigma.
Statistical analysis. Data are expressed as means ± SE. Statistical differences between multiple groups were tested using ANOVA for repeated measures and checked for significance using Scheffé's F-test.
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RESULTS |
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Esophageal cells were permeabilized by saponin and exposed to
increasing Ca2+ concentrations alone or in the presence of
appropriate inhibitors. Figure
1A shows that Ca2+
caused a dose-dependent contraction (ANOVA, P < 0.001), with maximal contraction occurring at a 0.72 µM
Ca2+. The contraction was almost completely inhibited by
the PKC inhibitor chelerythrine (105 M; ANOVA,
P < 0.001; Fig. 1A) and was not affected by
the calmodulin inhibitor CGS9343B (Fig. 1B), by the putative
MLCK inhibitors quercetin and ML-7, or by antibodies raised against
MLCK (Fig. 1C), suggesting that, in ESO, Ca2+
activates a PKC-dependent, MLCK-independent contractile pathway.
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To determine the specific PKC isozyme mediating
Ca2+-induced contraction, we examined the effect of
antibodies raised against the II,
, and
PKC isozymes
(61). Figure 2 shows that
Ca2+-induced contraction of permeable ESO cells was not
affected by PKC
II and PKC
antibodies, but was significantly
reduced by PKC
antibodies (ANOVA, P < 0.001),
suggesting that, in ESO, Ca2+ activates a PKC
-dependent
contractile pathway.
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The PKC-dependent mechanisms responsible for esophageal contraction
have not been elucidated. Figure 3 shows
that addition of 1 µM Ca2+ to permeable cells results in
a significant increase in myosin light chain phosphorylation (ANOVA,
P < 0.001), which was antagonized by PKC (ANOVA,
P < 0.001) but not by calmodulin inhibitors. In addition, the increase in phosphorylation was antagonized by the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609 (ANOVA, P < 0.001) and by the phospholipase D
(PLD) pathway inhibitor propranolol (ANOVA, P < 0.001)
and not by U-73122, a selective inhibitor of
phosphatidylinositol-specific phospholipase C (PI-PLC) (11, 26,
60). These data suggest that Ca2+-induced
phosphorylation of myosin light chain depends on phospholipase-mediated production of DAG and activation of PKC, and not on activation of a
calmodulin-dependent pathway.
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To confirm that Ca2+-induced contraction is mediated by
activation of selected phospholipases, we used selective phospholipase inhibitors. Figure 4 shows that
Ca2+-induced contraction of ESO cells was significantly
inhibited by propranolol (104 M; ANOVA, P < 0. 01) (9, 40, 49) and by D609 (10
4 M;
Fig. 4; ANOVA, P < 0.01) (55).
Propranolol and D609, in combination, almost completely abolished
Ca2+-induced contraction, suggesting that elevation of
cytosolic Ca2+ may directly induce hydrolysis of
phosphatidylcholine by activating PLD and PLC, resulting in production
of DAG, which in turn may activate PKC.
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Figure 5 shows the effect of the
phospholipase inhibitors D609 (104 M), propranolol
(10
4 M), and U-73122 (10
6 M) on DAG
production induced by 1 µM Ca2+ in permeable cells. In
the absence of phospholipase inhibitors (control), DAG production was
significantly increased (61.53 ± 13.7%) when the intracellular
Ca2+ concentration was increased from 0 to 1 µM (ANOVA,
P < 0.05). The Ca2+-induced increase in
DAG level was reduced by D609 and propranolol (ANOVA, P < 0.05). Propranolol and D609, in combination, completely abolished
the Ca2+-induced increase in DAG. U-73122 did not
significantly alter Ca2+-induced DAG production.
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We have previously shown that ACh-induced esophageal contraction is
mediated by activation of PC-PLC, PLD, and of a 100-kDa cytosolic
phospholipase A2 (cPLA2). cPLA2
produces arachidonic acid (AA) which potentiates DAG-induced activation
of PKC (58). We therefore tested whether cPLA2
may also participate in Ca2+-induced contraction of ESO
cells. We found that the cPLA2 inhibitor AACOCF3 significantly reduced Ca2+-induced
contraction (Fig. 6; ANOVA,
P < 0.001), suggesting that cPLA2
contributes to Ca2+-induced contraction of ESO cells.
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Taken together, these data suggest that an increase in cytosolic
Ca2+ may directly activate PC-PLC, PLD, and
PLA2, resulting in production of DAG and AA and activation
of a PKC-dependent pathway, similar to the contractile pathway
activated by ACh.
To test whether Ca2+-induced activation of phospholipases
involves the activation of G proteins, we used the nonhydrolyzable GDP
analog GDPS. The effectiveness of GDP
S was tested against ACh,
which is known to activate muscarinic receptors linked to trimeric
G proteins. Figure 7 shows that
contraction in response to a maximally effective dose of ACh was dose
dependently reduced by GDP
S (ANOVA, P < 0.001).
GDP
S at a 10
4 M concentration reduced ACh-induced
contraction by 79% and at a 10
3 M concentration by 90%.
A 10
4 M concentration of GDP
S, however, had no
effect on Ca2+-induced contraction (Fig. 7), supporting the
view that Ca2+-induced contraction of ESO cells may result
from directly induced activation of phospholipases, bypassing G
proteins.
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However, ACh binding to muscarinic receptors in ESO (60)
causes not only elevation of cytosolic Ca2+
(56) but also activation of G proteins. We therefore
examined whether the nonhydrolyzable GTP analog GTPS and
Ca2+ could interact in contraction of permeable ESO cells.
Figure 8A compares contraction
of permeable ESO cells in response to increasing concentrations of
Ca2+ alone and in the presence of three concentrations of
GTP
S (10
8, 10
7, and 10
6
M). When cytosolic Ca2+ levels reach 100 nM and higher,
GTP
S augments Ca2+-induced contraction in a
dose-dependent manner (ANOVA, P < 0.0010). Figure
8B shows that propranolol and D609, in combination, almost completely abolish GTP
S-induced contraction, suggesting that G
protein activation results in activation of phospholipases.
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Figure 8C shows the effect of the phospholipase inhibitors
D609 (104 M), propranolol (10
4 M), and
U-73122 (10
6 M) on DAG production induced by GTP
S
(10
6 M) in permeable cells. The intracellular
Ca2+ level was maintained at a threshold concentration of
180 nM. DAG production increased 38.94 ± 5.2% in the presence of
GTP
S. The GTP
S-induced increase in DAG level was almost abolished
by D609 (ANOVA, P < 0.0010) or propranolol (ANOVA,
P < 0.0010) and completely abolished by propranolol
and D609, in combination. The PI-PLC inhibitor U-73122 did not
significantly alter the GTP
S-induced DAG production.
These data suggest that both G protein activation by GTPS and the G
protein-independent contraction induced by Ca2+ alone cause
contraction of esophageal muscle cells through the same intracellular
pathway, i.e., by inducing phospholipase activation and production of
second messengers
Figure 8A demonstrates that the same level of contraction
may result from different combinations of Ca2+ and GTPS.
For example, by interpolating the dose-response curves we find that
20% shortening of ESO cells may be produced by 300 nM Ca2+
in combination with 10
6 M GTP
S, by 490 nM
Ca2+ with 10
7 M GTP
S, by 600 nM
Ca2+ with 10
8 M GTP
S, or by 700 nM
Ca2+ alone. To estimate which combination of cytosolic
Ca2+ and G protein activation occurs when ESO cells
contract in response to the endogenous neurotransmitter ACh, we
examined the Ca2+ concentration after ACh stimulation.
Ca2+ measurements were obtained with a dual wavelength
Ca2+ imaging system. The images at the top of Fig.
9 show sequential shortening and
Ca2+ levels in a single ESO cell at different times after
exposure to ACh. The numbers below each image represent the time
elapsed. Application of ACh by pressure ejection micropipette began at ~1 s and lasted 5 s. Figure 9 shows that the first detectable Ca2+ rise occurs ~1.5 s after the beginning of
application of ACh. Ca2+ levels do not increase uniformly
across the cell, but rather an area of elevated Ca2+ sweeps
from the bottom to the top of the cell in ~1 s, presumably as the
applied ACh engulfs the cell from the bottom to the top. Visible
shortening of the cell is maximal at 15-30 s and occurs after
the peak of the Ca2+ signal is past, when cytosolic free
Ca2+ has returned to near resting levels. A comparison of
the pseudocolors observed in the cell with the scale on the right of
the figure suggests that the highest peak of
Ca2+ concentration that sweeps this cell may
be between 500 and 600 nM. This observation is confirmed by the graph
in the lower panel. The graph represents the average Ca2+
concentration as a function of time in a small window in the cell. A
small window may be reasonably representative of the maximum Ca2+ levels reached in the cell, as the same maximum level
seems to occur throughout the cell, albeit at slightly different times. For the muscle cell shown in Fig. 9, the graph shows a peak 600 nM
Ca2+ concentration occurring at 3-4 s after
application of ACh. When Ca2+ was measured in 20 cells
under identical conditions, the peak was 616 ± 50 nM (means ± SE). The Ca2+ concentration decreases after the peak and
returns to approximately prestimulation values at 29 s, the time
of maximum shortening.
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DISCUSSION |
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The data presented show that in ESO smooth muscle Ca2+
directly activates phospholipases, causing production of second
messengers and activation of a PKC-dependent contractile pathway. G
protein activation by GTP
S (or by muscarinic receptors linked to
trimeric G proteins) activates the same phospholipases and contractile pathway. These findings may help in understanding a possible mechanism responsible for "modulation of Ca2+ sensitivity" by
GTP
S.
Ca2+ is thought to be a universal messenger of intracellular signaling for a wide variety of cell processes. In smooth muscle an increase in cytoplasmic Ca2+ leads to phosphorylation of the 20-kDa myosin light chains (62). However, smooth muscle cells contain numerous proteins that are capable of binding Ca2+, either to buffer changes in ionized Ca2+ or to elicit a cellular response (57), and the intermediate processes initiated by Ca2+ and leading to phosphorylation of the 20-kDa myosin light chains are not well understood.
Several investigators have shown that the magnitude of changes in
intracellular Ca2+ is not directly related to the force
developed (27, 42, 52), and an effort has been made to
characterize the Ca2+ sensitivity of the contractile
process by examining other possible modulators of Ca2+
sensitivity, such as GTPS (17, 18, 35, 38, 44),
phosphatases (19, 36, 37, 40), or modulation of MLCK
(2, 64, 65).
A "Ca2+-centric" view of the contractile process may not be helpful in understanding the sequence of intracellular events occurring between Ca2+ influx, or release, and myosin phosphorylation. Often the sequence of events leading to contraction is complex and not amenable to explanation in terms of Ca2+ sensitivity. This point is well illustrated by reports of Ca2+-independent contraction in some vascular and esophageal muscles (28, 58, 67). Definition of the sequence of events and determination of the precise site of action of Ca2+ in the sequence leading to contraction are needed.
We have previously shown that different intracellular processes mediate contraction in different muscles (59, 60) or even in the same muscle under different conditions (6). For instance, in the LES, contraction in response to a maximally effective dose of ACh is mediated by inositol trisphosphate (IP3)-induced release of Ca2+ from intracellular stores and activation of a calmodulin-MLCK-dependent pathway, but spontaneous tone or contraction in response to a low level of ACh depends on PKC and not on calmodulin (6, 26).
In contrast, contraction of ESO muscle in response to ACh depends on
activation of phospholipases and production of DAG and AA, without any
measurable production of IP3 (58, 60).
Once DAG and AA are produced, they interact to activate a
Ca2+-independent PKC (58, 61), and in
permeable cells DAG-induced contraction can occur even in
Ca2+-free solution. This pathway does not involve
calmodulin or MLCK, which require Ca2+ to be
activated. In addition, ESO muscle cells do not contract in response to
exogenous calmodulin or MLCK under the same conditions that cause
maximal contraction of LES cells, and the calmodulin inhibitor CGS9343B
or the MLCK antibody and inhibitors (quercetin or ML-7) do not inhibit
ACh-induced contraction of ESO cells (Table 1) (Sohn UD, Tang DC, Stull JT, Haeberle
JR, Wang C-LA, Harnett KM, and Biancani P, unpublished observations).
Ca2+ requirements exist even when contraction is mediated
by the Ca2+-independent PKC, since contraction of ESO
cells is almost abolished by prolonged exposure to Ca2+
channel inhibitors or to Ca2+ chelators such as EGTA
(7, 58). In the present study we examined the mechanism
mediating Ca2+-induced contraction of ESO cells, isolated
by enzymatic digestion and permeabilized by brief exposure to saponin,
to allow control of cytosolic Ca2+ levels. We found that
permeable esophageal cells contract in a dose-dependent manner as
cytosolic Ca2+ concentration increases from 200 nM to
micromolar. These data suggest that diffusion of Ca2+ into
the cytoplasm is sufficient to produce contraction of esophageal muscle. In addition, similar to ACh-induced contraction in ESO (7, 58, 61), Ca2+-induced contraction is
mediated through a PKC-dependent pathway as it is almost abolished by
chelerythrine (Table 1). Chelerythrine interacts with the catalytic
domain of PKC and is a potent PKC inhibitor with a half-maximal
inhibition occurring at 0.66 µM (23).
Ca2+-induced contraction was not affected by the calmodulin
inhibitor (25, 46, 49) CGS9343B, by two putative MLCK
inhibitors, or by antibodies raised against MLCK (Fig. 1). Quercetin, a
flavonoid, and ML-7 are two structurally different compounds that
inhibit MLCK by binding hydrophobically at or near the ATP-binding site at the active center of the enzyme (22, 24, 54). We have previously shown that both ML-7 and quercetin cause dose-dependent inhibition of LES but not of ESO muscle cells in response to a maximally effective dose of ACh (Table 1), suggesting that both ML-7
and quercetin are selective enough to cause inhibition of LES but not
of ESO. These data are consistent with the view that Ca2+-induced contraction of ESO may be calmodulin- and
MLCK-independent, and mediated through a PKC-dependent pathway. This
hypothesis is also consistent with the finding that DAG-induced
contraction of ESO-permeable cells is not affected by incubation in
Ca2+-free medium (58), as the presence of
Ca2+ at relatively high concentration is needed to activate
calmodulin and MLCK (6).
We have previously shown that the II,
, and
PKC isozymes are
present in ESO circular muscle (61) and that only PKC
translocates from the cytosol to the membrane in response to ACh (61). In the present investigation we identified the
isozyme activated by cytosolic Ca2+ elevation, by examining
the effect of isozyme-selective PKC antibodies on
Ca2+-induced contraction . Figure 2 shows that contraction
of permeabilized ESO cells was inhibited by antibodies raised against
PKC
and not by antibodies raised against the
II or
PKC
isozymes (ANOVA, P < 0.01). Inhibition of
Ca2+-induced contraction by the PKC
antibody was
concentration dependent and reversed by addition of the PKC
-specific
antibody-binding peptide (Table 1). Thus, as in the case of ACh-induced
contraction of ESO cells, Ca2+-induced contraction results
in activation of a PKC
-dependent contractile pathway. PKC is a
family of homologous serine and threonine protein kinases that can be
divided into three groups based on their Ca2+ and
phospholipid requirements for activation: the classical or conventional
PKC isozymes (
,
I,
II,
) are Ca2+ and
phospholipid dependent; the new PKC isozymes (
,
,
,
, µ)
are Ca2+ independent and phospholipid dependent; and the
atypical PKC isozymes (
and
) are Ca2+ and
phospholipid independent (45). The new PKC isozymes (i.e., PKC
) lack the region that has been implicated in the regulation of
PKC by Ca2+ (5, 29, 47). It is thus unlikely
that Ca2+ may directly activate PKC
.
We have shown that ACh-induced contraction of ESO, which is PKC
dependent, is associated with myosin light chain phosphorylation (Sohn
UD, Tang DC, Stull JT, Haeberle JR, Wang C-LA, Harnett KM, and Biancani
P, unpublished observations), which was inhibited by PKC inhibitors and
not by calmodulin inhibitors. We therefore examined whether an increase
in cytosolic Ca2+ may also result in PKC-dependent
phosphorylation of myosin light chains. Figure 3 shows that
Ca2+-induced myosin light chain phosphorylation was
antagonized by PKC but not by calmodulin inhibitors and by the PC-PLC
inhibitor D609 and by the PLD pathway inhibitor propranolol. These data confirm that, like Ca2+-induced contraction,
Ca2+-induced phosphorylation of myosin light chain depends
on phospholipase-mediated production of DAG and activation of PKC, and
not on activation of a calmodulin-dependent pathway.
Similarly, Ca2+-induced contraction, like Ca2+-induced myosin phosphorylation, was significantly reduced by the phospholipase inhibitors D609 and propranolol, when used separately, and was abolished by D609 and propranolol, when used in combination. We have previously shown that ACh-induced contraction of esophageal muscle is not associated with hydrolysis of phosphatidylinositol 4,5-bisphosphate and production of IP3 (50). Phosphatidylcholine hydrolysis by PC- PLC and by PLD has been shown to be an alternative source of DAG (8, 15, 39, 66). PC-PLC produces DAG and phosphocholine, while PLD produces choline and phosphatidic acid, which is metabolized to DAG by phosphatidic acid phosphohydrolase (8, 14). D609 blocks PC-PLC activity derived from Bacillus cereus, without affecting PLA2, PLD, and PI-PLC activity (55). A high concentration (0.1-1 mM) of propranolol has been shown to reduce DAG production by inhibition of phosphatidic acid phosphohydrolase without affecting PLD activity or PI-PLC activity (9, 50, 51). Ca2+-induced contraction of ESO muscle cells and production of DAG were antagonized by D609 and by propranolol at concentrations that had no effect on contraction of LES muscle (59, 60). The lack of effect of propranolol on contraction of LES muscle cells implies that, at the concentration used, propranolol is not acting nonselectively as a local anesthetic. This finding is consistent with the view that esophageal contraction may be mediated by activation of PC-PLC and PLD. The fact that inhibition by D609 and propranolol is additive, and results in complete abolition of esophageal contraction, suggests that this is the main signaling pathway responsible for contraction of esophageal circular muscle.
In addition, Ca2+-induced production of DAG in response to 1 µM Ca2+, similarly to cell contraction, was reduced by D609 and propranolol, when used alone, and abolished when the inhibitors were present in combination. U-73122, a selective inhibitor of PI-PLC, had no effect. These data confirm that, in the ESO, elevations in cytosolic Ca2+ are capable of activating PLD and PC-PLC selectively.
Taken together, these data show that DAG formation, myosin light chain
phosphorylation, and contraction all depend on Ca2+-induced
activation of PC-PLC and PLD, strongly supporting phospholipases as the
site of action for Ca2+ to activate a PKC-dependent
contractile pathway. In addition, activation of PKC
results in
phosphorylation of MLC, which is not calmodulin dependent. It is
unlikely that PKC
directly phosphorylates MLC. Other kinases, such
as extracellularly regulated kinase (ERK) 1 and ERK2 (12),
and other regulatory proteins, such as caldesmon and/or calponin
(1, 3, 32, 33, 41, 43), are likely to be involved in
PKC-mediated contraction.
A high-molecular-mass (85-110 kDa) cPLA2 participates
in contraction of esophageal but not of LES muscle by producing AA and potentiating DAG-induced activation of PKC (58). We
therefore examined the role of cPLA2 in
Ca2+-induced contraction of ESO circular muscle.
AACOCF3 , an analog of AA in which the COOH group is
replaced with trifluoromethyl ketone (63), has been shown
to selectively inhibit cPLA2 in platelets (4, 20,
53) and mesangial cells (20).
AACOCF3 significantly reduced Ca2+-induced
contraction of ESO circular muscle cells (Fig. 6). Inhibition of
Ca2+-induced contraction by AACOCF3
(105 M) varies between 20% (at high Ca2+)
and 40% at (at 360 nM Ca2+), is statistically significant
and slightly lower than previously reported in response to ACh
(34). It should be noted, however, that ACh causes both
Ca2+ influx (which can directly activate phospholipases)
and G protein activation (which can independently activate the same
phospholipases). When cPLA2 is activated only by
Ca2+, it is reasonable to expect that its
contribution may be less than when activated by ACh, as the effect
arising from G protein activation is absent.
These data suggest that, in ESO circular smooth muscle, Ca2+ functions by activating phospholipases, resulting in production of DAG and AA. Thus the signal transduction pathway activated by Ca2+ elevation is the same as the one activated by ACh (58-60).
To determine whether Ca2+-induced activation of
phospholipases was direct or mediated by activation of G proteins, we
used the GDP analog GDPS to inhibit GTP binding to G proteins, and G
protein activation. ACh is thought to be the endogenous
neurotransmitter mediating contraction of esophageal muscle in
physiological conditions, e.g., in response to swallowing. ACh-induced
contraction of ESO is mediated by M2 muscarinic receptors linked to
Gi3-type G proteins (60) and is inhibited by GDP
S.
Figure 7 shows that GDP
S dose dependently reduced ACh-induced
contraction but had no effect on Ca2+-induced contraction
of ESO cells. These data suggest that Ca2+ may induce
contraction by direct activation of phospholipases, without activation
of G proteins.
Because ACh causes both elevation of cytosolic Ca2+ and
activation of G proteins, we examined the interaction of G protein
activation by GTPS and Ca2+ in contraction of ESO.
Figure 8A compares contraction of permeable ESO cells in
response to increasing concentrations of Ca2+ alone and in
the presence of GTP
S. In the absence of Ca2+, ESO cells
do not contract in response to GTP
S (58). When cytosolic Ca2+ levels reach 100 nM and higher, GTP
S
augments Ca2+-induced contraction in a dose-dependent
manner (ANOVA, P < 0.0010). The additional contraction
induced by GTP
S depends on G protein-induced activation of
phospholipases, because propranolol and D609, in combination, almost
completely abolish GTP
S-induced contraction (Fig. 8B).
Similarly, these phospholipase inhibitors (but not U-73122) reduced
GTP
S-induced production of DAG. The GTP
S-induced increase in DAG
level was almost abolished by D609, by propranolol, and completely
abolished by propranolol and D609 in combination. The PI-PLC inhibitor
U-73122 did not alter DAG production.
Several investigators have observed that GTPS increases
Ca2+ sensitivity of the contractile process (17, 18,
36-38, 44). Our data suggest that the G protein-independent
contraction induced by Ca2+ and the G protein-dependent
contraction induced by GTP
S are both mediated by the same
intracellular pathway, i.e., by inducing phospholipase activation and
production of second messengers.
Figure 8A demonstrates that the same level of contraction
may result from different combinations of Ca2+ and GTPS.
At any Ca2+ concentration, the contraction may be augmented
by GTP
S (i.e., by G protein activation) in a dose-dependent manner.
To estimate which combination of cytosolic Ca2+ and G
protein activation occurs when ESO cells contract in response to ACh,
we measured cytosolic Ca2+ levels in the ESO after ACh.
Ca2+ measurements were obtained with a dual-wavelength Ca2+ imaging system. The images in Fig. 9 show sequential shortening and Ca2+ levels in a single ESO cell at different times after exposure to ACh. Application of ACh by pressure ejection micropipette began at ~1 s and lasted 5 s. Ca2+ levels did not increase uniformly across the cell, but rather an area of elevated Ca2+ swept from the bottom to the top of the cell in ~1 s, presumably as the applied ACh engulfed the cell from the bottom to the top. The highest peak of Ca2+ concentration (500-600 nM) occurred at the beginning of ACh application, and much before contraction began. The mean Ca2+ peak measured in 20 cells under identical conditions was 616 ± 50 nM and was slightly lower than the 700-800 nM Ca2+ concentration required to produce maximal contraction in response to Ca2+ alone. Visible shortening of the cell began at a time when Ca2+ concentration was already decreasing and was maximal at 15-30 s at a time when cytosolic free Ca2+ had returned to near resting levels. Contraction of intact cells in response to application of ACh by pressure ejection may not be directly comparable to contraction of permeable cells exposed to a fixed Ca2+ concentration, and the calculated Ca2+ concentrations used for permeable cells may not be directly comparable to the fura 2-measured concentrations. Nevertheless, analysis of the cell images shows a 26% shortening, which was comparable to the shortening in permeable cells in response to Ca2+ or ACh, and in intact esophageal cells, in response to a variety of agonists. ACh-induced Ca2+ elevation was below the 700-800 nM levels required to produce maximal contraction in isolated esophageal cells, as shown in Figs. 1, 4, 6, and 8. Thus it is possible that ACh-induced contraction of ESO cells may result from interaction of Ca2+ and G proteins and that the relatively low Ca2+ concentration measured in response to ACh may be amplified by concurrent ACh-induced activation of G proteins.
We conclude that Ca2+ and G protein activation in ESO cells
results in activation of the same phospholipases, producing the second
messengers required to activate the Ca2+-insensitive
PKC. Once PKC
is activated, contraction can occur in the absence
of Ca2+. Phospholipase activation by Ca2+ and G
proteins may account in part for previously reported Ca2+
sensitization by GTP
S. Under physiological conditions ACh-induced contraction may use a combination of both mechanisms to activate a
contractile pathway.
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ACKNOWLEDGEMENTS |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-28614 (P. Biancani) and DK-47223 (K. M. Harnett), by Korean Science and Engineering Foundation hacsim 961-0704-042-2 (U. D. Sohn), and by the Non-Directed Research Fund, Korea Research (U. D. Sohn).
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FOOTNOTES |
---|
These data were presented in part at the meeting of the American Motility Society, in Traverse City, MI, in September 1996.
Address for reprint requests and other correspondence: P. Biancani, GI Motility Research, SWP 520, Rhode Island Hospital, 593 Eddy St., Providence, RI 02903.
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 23 June 1998; accepted in final form 20 October 2000.
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