Ca2+-induced contraction of cat esophageal circular smooth muscle cells

W. Cao1, Q. Chen1, U. D. Sohn2, N. Kim3, M. T. Kirber1, K. M. Harnett1, J. Behar1, and P. Biancani1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACh-induced contraction of esophageal circular muscle (ESO) depends on Ca2+ influx and activation of protein kinase Cepsilon (PKCepsilon ). PKCepsilon , however, is known to be Ca2+ independent. To determine where Ca2+ is needed in this PKCepsilon -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 PKCepsilon (but not by PKCbeta II or PKCgamma antibodies), by a calmodulin inhibitor, by MLCK inhibitors, or by GDPbeta 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 PKCepsilon , which may indirectly cause phosphorylation of MLC. In addition, direct G protein activation by GTPgamma 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 PKCepsilon -dependent mechanisms to cause contraction.

calcium; smooth muscle; protein kinase C; phospholipase C; phospholipase D; myosin phosphorylation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Cepsilon (PKCepsilon ) (61). PKCepsilon 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).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.  

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 PKCepsilon -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 PKCepsilon -dependent pathway, resulting from Ca2+-induced activation of phospholipases, production of second messengers, and phosphorylation of myosin light chains.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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) (GTPgamma S), or guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S), which do not diffuse across the membrane. After completion of the enzymatic phase of the digestion process, the partly digested muscle tissue was washed with an enzyme-free cytosolic physiological salt solution of the following composition (in mM): 20 NaCl, 100 KCl, 25 NaHCO3, 5.0 MgSO4, 0.96 NaH2PO4, 1.0 EGTA, and 0.48 CaCl2, as well as 2% bovine serum albumin. The cytosolic buffer was equilibrated with 95% O2-5% CO2 to maintain pH 7.2 at 31°C. Muscle cells dispersed spontaneously in this medium. After dispersion, the cells were permeabilized by incubation for 3 min in cytosolic buffer containing 75 µg/ml saponin. After exposure to saponin, the cell suspension was spun at 200 g, and the resulting pellet was resuspended in saponin-free modified cytosolic solution containing 10 µM antimycin A, 1.5 mM ATP, and an ATP-regenerating system consisting of 5 mM creatine phosphate and 10 U/ml creatine phosphokinase (10).

Contraction of isolated muscle cells. The cells were contracted by exposing them to the test agonists, i.e., Ca2+ or GTPgamma S. 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 GTPgamma 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 GTPgamma S.

When inhibitors (AACOCF3, chelerythrine, CGS9343B, D609, ML-7, propranolol, quercetin) were used, the cells were incubated in appropriate concentrations of the inhibitors for 1 min before addition of agonist. Inhibitors were used at their maximally effective doses, as previously determined in pilot studies. When PKC antibodies or MLCK antibodies were used, permeabilized cells were incubated with the antibody at a 1:200 dilution for 60 min before addition of Ca2+. Thirty seconds after the agonist was added, the cells were fixed in acrolein at a final 1.0% concentration. A drop of the cell-containing medium was placed on a glass slide, covered by a coverslip, and the edges were sealed with nail enamel to prevent evaporation.

The length of 30 consecutive intact cells, encountered at random, in each slide was measured with a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany), and a closed-circuit video camera (model WV-CD51; Panasonic, Seacaucus, NJ) connected to a Macintosh computer (Apple, Cupertino, CA) with an image analysis software program (NIH Image 1.6; National Institutes of Health, Bethesda, MD; http://128.231.98.16/nih-image/download.html). For each experiment, contraction was expressed as percent shortening of the average of 30 consecutive cells compared with the average of 30 untreated (control) cells. The average cell length of unstimulated cells was 70-75 µm. We have previously shown that permeabilization did not affect initial cell length or response to agonists (58).

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 (10-4 M), propranolol (10-4 M), or U-73122 (10-6 M). The cells were stimulated with calcium (1 µM) or GTPgamma 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.

DAG measurements were performed using the Amersham DAG assay. Briefly, DAG was determined by incubating the samples with sn-1,2-diacylglycerol kinase in the presence of [32P]ATP to form [32P]phosphatidic acid. Lipids were then extracted with chloroform-methanol (1:2 vol/vol) and separated by high-performance thin-layer chromatography (HPTLC; Silica Gel 60; EM Science, Gibbstown, NJ) using a solvent system containing chloroform-methanol-acetic acid (5, 64). The presence of phosphatidic acid was determined by exposure of the HPTLC plate to X-ray film (X-OMat, XAR-2; Sigma, St. Louis, MO) overnight. Each HPTLC plate contained a control lane with standard DAG incubated with DAG kinase to document the precise location of phosphatidic acid. Quantitation of DAG was made by preparation of a standard curve with known concentrations of DAG. The spots containing [32P]phosphatidic acid were cut from the HPTLC plate and placed into scintillation vials and assayed for radioactivity.

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 (beta II, gamma , and epsilon ) 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, GDPbeta S, GTPgamma 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (10-5 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.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 1.   Ca2+-induced shortening of esophageal circular muscle (ESO) cells permeabilized by saponin. ESO cells were permeabilized by saponin to allow diffusion of extracellular Ca2+ into the cytoplasm. 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). A: the cells shortened in a dose-dependent manner in response to increasing Ca2+ concentrations (ANOVA, P < 0.001). The maximally effective concentration was 800-900 nM Ca2+. Ca2+-induced shortening was almost abolished by the protein kinase C (PKC) inhibitor chelerythrine (10-5 M; ANOVA, P < 0.001). Values are means ± SE of 4 animals, with 30 cells counted for each data point. B: at all Ca2+ concentrations shortening was not affected by the calmodulin inhibitor CGS9343B (10-5 M). C: Ca2+-induced contraction was not affected by the myosin light chain kinase (MLCK) inhibitors ML-7 (10-5 M), quercetin (10-5 M), or antibodies raised against MLCK (1:200).The "control" curve is the same as in A, as these experiments were performed in the same cell samples. Values are means ± SE of 3 animals, with 30 cells counted for each data point.

To determine the specific PKC isozyme mediating Ca2+-induced contraction, we examined the effect of antibodies raised against the beta II, gamma , and epsilon  PKC isozymes (61). Figure 2 shows that Ca2+-induced contraction of permeable ESO cells was not affected by PKCbeta II and PKCgamma antibodies, but was significantly reduced by PKCepsilon antibodies (ANOVA, P < 0.001), suggesting that, in ESO, Ca2+ activates a PKCepsilon -dependent contractile pathway.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 2.   Ca2+-induced shortening of permeable cells is mediated through a PKCepsilon -dependent pathway. Permeable cells were incubated for 1 h in a 1:200 dilution of isozyme-selective PKC antibodies, before induction of contraction by a maximally effective Ca2+ concentration (800 nM). Ca2+-induced contraction was reduced by PKCepsilon -selective antibodies (ANOVA, P < 0.001) but not by PKCbeta II or PKCgamma antibodies. Values are means ± SE of 4 animals, with 30 cells counted for each data point.

The PKCepsilon -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.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3.   Ca2+-induced myosin light chain (MLC) phosphorylation. Addition of 1 µM Ca2+ to permeable cells results in a significant increase in MLC phosphorylation (ANOVA, P < 0.001), which was antagonized by the PKC antagonist chelerythrin, by the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609, and by the phospholipase D (PLD) pathway inhibitor propranolol (Prop; ANOVA, P < 0.001). These data suggest that Ca2+-induced phosphorylation of MLC depends on phospholipase-mediated production of diacylglycerol (DAG) and activation of PKC, and not on activation of a calmodulin-dependent pathway.

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 (10-4 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.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   Ca2+-induced contraction depends on activation of PC-PLC and PLD. Ca2+-induced contraction was significantly reduced by the PC-PLC inhibitor D609 (10-4 M) (ANOVA, P < 0.001) and by propranolol (10-4 M) (ANOVA, P < 0.001). Propranolol in high concentrations has been shown to inhibit phosphatidic acid phosphohydrolase, preventing formation of DAG through a PC-PLD-mediated pathway. 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. The "control" curve is the same as in Fig. 1, A and C, as these experiments were performed in the same cell samples Values are means ± SE of 4 animals, with 30 cells counted for each data point.

Figure 5 shows the effect of the phospholipase inhibitors D609 (10-4 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.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of phospholipase inhibitors on Ca2+-induced DAG production. Values are the percent increase in DAG levels when the intracellular Ca2+ concentration is increased from 0 to 1 µM. In the absence of phospholipase inhibitors (control), DAG production was significantly increased by 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 significantly lower in the presence of D609 (10-4 M) and propranolol (10-4 M) than in control samples (ANOVA, P < 0.05). Propranolol and D609, in combination, completely abolished the Ca2+-induced increase in DAG. U-73122 (10-6 M) did not significantly alter the Ca2+-induced effect on DAG production. These data support the hypothesis that elevations in cytosolic Ca2+ are capable of activating PLD and PC-PLC, resulting in production of DAG. Values are means ± SE of 4 animals.

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.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   Ca2+-induced contraction depends on activation of cytosolic phospholipase A2 (cPLA2). The cPLA2 inhibitor AACOCF3 (10-5 M) significantly reduced Ca2+-induced contraction (ANOVA, P < 0.001), suggesting that cPLA2 contributes to Ca2+-induced contraction of ESO cells. Values are means ± SE of 4 animals, with 30 cells counted for each data point.

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 PKCepsilon -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 GDPbeta S. The effectiveness of GDPbeta 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 GDPbeta S (ANOVA, P < 0.001). GDPbeta 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 GDPbeta 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.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 7.   Ca2+-induced shortening of permeable cells does not depend on activation of G proteins. Left: the nonhydrolyzable GDP analog guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S) dose dependently reduced cell shortening (ANOVA, P < 0.001), in response to a maximally effective dose of ACh (10-9 M), which is known to activate muscarinic receptors linked to heterotrimeric G proteins. Values are means ± SE of 3 animals, with 30 cells counted for each animal. Right: a 10-4 M concentration of GDPbeta S, which reduced ACh-induced contraction by 79%, had no effect on Ca2+-induced contraction. Values are means ± SE of 3 animals, with 30 cells counted for each data point.

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 GTPgamma S 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 GTPgamma S (10-8, 10-7, and 10-6 M). When cytosolic Ca2+ levels reach 100 nM and higher, GTPgamma 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 GTPgamma S-induced contraction, suggesting that G protein activation results in activation of phospholipases.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 8.   Potentiation of Ca2+-induced contraction by guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S). A: contraction of permeable ESO cells in response to increasing concentrations of Ca2+ alone (circles) is dose dependently increased by increasing concentrations of GTPgamma S (10-8, 10-7, and 10-6 M). In Ca2+-free cytosolic medium GTPgamma S caused no contraction, but, at 100 nM and higher cytosolic Ca2+ levels, GTPgamma S augmented Ca2+-induced contraction in a dose-dependent manner. Values are means ± SE of 3 animals, with 30 cells counted for each data point. B: in the presence of 0.36 µM Ca2+, contraction of ESO cells in response to increasing concentrations of GTPgamma S is abolished by propranolol (10-4 M) and D609 (10-5 M), in combination, suggesting that activation of G proteins may lead to PC-PLC and PLD stimulation. Values are means ± SE of 3 animals, with 30 cells counted for each data point. C: effect of GTPgamma S on DAG production. Values are the percent increase in DAG levels in the presence of GTPgamma S (10-6 M). The intracellular Ca2+ concentration was 180 nM. In the absence of phospholipase inhibitors (control), DAG production increased 38.94 ± 5.2% in the presence of GTPgamma S. The GTPgamma S-induced increase in DAG level was significantly lower in the presence of D609 (10-4 M) and propranolol (10-4 M) than in control samples (ANOVA, P < 0.0010). Propranolol and D609, in combination, completely abolished the GTPgamma S-induced increase in DAG. U-73122 (10-6 M) did not alter the GTPgamma S-induced effect on DAG production (ANOVA, P = 0.14). These data support the hypothesis that elevations in GTPgamma S are capable of activating PLD and PC-PLC, resulting in production of DAG. Values are means ± SE of 4 animals.

Figure 8C shows the effect of the phospholipase inhibitors D609 (10-4 M), propranolol (10-4 M), and U-73122 (10-6 M) on DAG production induced by GTPgamma 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 GTPgamma S. The GTPgamma 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 GTPgamma S-induced DAG production.

These data suggest that both G protein activation by GTPgamma S 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 GTPgamma S. 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 GTPgamma S, by 490 nM Ca2+ with 10-7 M GTPgamma S, by 600 nM Ca2+ with 10-8 M GTPgamma 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.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 9.   Measurements of Ca2+ during ACh-induced contraction of intact ESO cells. Ca2+ measurements were obtained with a dual-wavelength Ca2+ imaging system. The images at the top show sequential shortening and Ca2+ levels in a single ESO cell at different times after exposure to ACh. The numbers below each image represent elapsed time. Application of ACh (20 µM) by pressure ejection micropipette as indicated by the horizontal bar began at ~1 s and lasted 5 s. The figure shows that the first detectable Ca2+ rise occurs ~1.5 s after the beginning of application of ACh. Visible shortening of the cell occurs after the peak Ca2+ signal is past, and is maximal at 15-30 s, after cytosolic free Ca2+ has returned to near resting levels. A comparison of the pseudocolors observed in the cell with the scale on the right suggests that the highest concentration of the Ca2+ may be between 500 and 600 nM. Bottom: the graph represents the average Ca2+ concentration, as a function of time, in a small window shown in the image in the right upper corner in the figure. The graph shows a peak 600 nM Ca2+ concentration. After the peak, the Ca2+ concentration decreases and returns approximately to prestimulation values at 29 s, the time of maximum shortening.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The data presented show that in ESO smooth muscle Ca2+ directly activates phospholipases, causing production of second messengers and activation of a PKCepsilon -dependent contractile pathway. G protein activation by GTPgamma 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 GTPgamma 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 GTPgamma S (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 PKCepsilon (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 PKCepsilon , 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 beta II, gamma , and epsilon  PKC isozymes are present in ESO circular muscle (61) and that only PKCepsilon 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 PKCepsilon and not by antibodies raised against the beta II or gamma  PKC isozymes (ANOVA, P < 0.01). Inhibition of Ca2+-induced contraction by the PKCepsilon antibody was concentration dependent and reversed by addition of the PKCepsilon -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 PKCepsilon -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 (alpha , beta I, beta II, gamma ) are Ca2+ and phospholipid dependent; the new PKC isozymes (delta , epsilon , eta , theta , µ) are Ca2+ independent and phospholipid dependent; and the atypical PKC isozymes (zeta  and lambda ) are Ca2+ and phospholipid independent (45). The new PKC isozymes (i.e., PKCepsilon ) 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 PKCepsilon .

We have shown that ACh-induced contraction of ESO, which is PKCepsilon 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 PKCepsilon -dependent contractile pathway. In addition, activation of PKCepsilon results in phosphorylation of MLC, which is not calmodulin dependent. It is unlikely that PKCepsilon 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 (10-5 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 GDPbeta S 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 GDPbeta S. Figure 7 shows that GDPbeta 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 GTPgamma S 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 GTPgamma S. In the absence of Ca2+, ESO cells do not contract in response to GTPgamma S (58). When cytosolic Ca2+ levels reach 100 nM and higher, GTPgamma S augments Ca2+-induced contraction in a dose-dependent manner (ANOVA, P < 0.0010). The additional contraction induced by GTPgamma S depends on G protein-induced activation of phospholipases, because propranolol and D609, in combination, almost completely abolish GTPgamma S-induced contraction (Fig. 8B). Similarly, these phospholipase inhibitors (but not U-73122) reduced GTPgamma S-induced production of DAG. The GTPgamma 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 GTPgamma S 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 GTPgamma 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 GTPgamma S. At any Ca2+ concentration, the contraction may be augmented by GTPgamma 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 PKCepsilon . Once PKCepsilon 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 GTPgamma S. Under physiological conditions ACh-induced contraction may use a combination of both mechanisms to activate a contractile pathway.


    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).


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   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].

2.   Adelstein, RS, Conti MA, and Hataway DR. Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosine 3'5'-monophosphate-dependent kinase. J Biol Chem 253: 8347-8350, 1978[Abstract].

3.   Allen, BG, and Walsh MP. The biochemical basis of the regulation of smooth muscular contraction. Trends Biochem Sci 19: 362-368, 1994[ISI][Medline].

4.   Bartoli, F, Lin H-K, Ghomashchi F, Gelb MH, Jain MK, and Apitz-Castro R. Tight binding inhibitors of 85-kDa phospholipase A2 but not 14-kDa phospholipase A2 inhibit release of free arachidonate in thrombin stimulated human platelets. J Biol Chem 269: 15625-15630, 1994[Abstract/Free Full Text].

5.   Bayer, G, Telford D, Giampa L, Coggsshall KM, Baier-Bitterlich G, Isakov N, and Altman A. Molecular cloning and characterization of PKCtheta a novel member of the protein kinase C (PKC) family expressed predominantly in hematopoietic cells. J Biol Chem 268: 4997-5004, 1993[Abstract/Free Full Text].

6.   Biancani, P, Harnett KM, Sohn UD, Rhim BY, Behar J, Hillemeier C, and Bitar KN. Differential signal transduction pathways in LES tone and response to ACh. Am J Physiol Gastrointest Liver Physiol 266: G767-G774, 1994[Abstract/Free Full Text].

7.   Biancani, P, Hillemeier C, Bitar KN, and Makhlou 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].

8.   Billah, MM, and Anthes JC. The regulation and cellular functions of phosphatidylcholine hydrolysis. Biochem J 269: 281-291, 1990[ISI][Medline].

9.   Billah, MM, Eckel S, Mullmann TJ, Egan RW, and Siegel MI. Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diacylglyceride levels in chemotactic peptide-stimulated human neutrophils. J Biol Chem 264: 17069-17077, 1989[Abstract/Free Full Text].

10.   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].

11.   Bleasdale, JE, Bundy GL, Bunting S, Fitzpatric FA, Huff RM, Sun FF, and Pike JE. Inhibition of phospholipase C dependent processes by U-73,122. In: Advances in Prostaglandin, Thromboxane and Leukotriene Research, edited by Samuelson B, Wong P Y-K, and Sun FF.. New York: Raven, 1989, p. 590-593.

12.   Cao, WB, Sohn UD, Behar J, and Biancani P. MAP kinase mediates PKC-dependent contraction of cat LES and esophageal circular smooth muscle (Abstract). Gastroenterology 114: A731, 1998[ISI].

13.   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].

14.   Dennis, EA, Rhee SG, Billah MM, and Hannun YA. Role of phospholipases in generating lipid second messengers in signal transduction. FASEB J 5: 2068-2077, 1991[Abstract/Free Full Text].

15.   Exton, JH. Signaling through phosphatidylcholine breakdown. J Biol Chem 265: 1-4, 1990[Abstract/Free Full Text].

16.   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.

17.   Fujiwara, T, Itoh T, Kubota Y, and Kuriyama H. Effects of guanosine nucleotides on skinned smooth muscle tissue of the rabbit mesenteric artery. J Physiol (Lond) 408: 535-547, 1989[Abstract].

18.   Gong, MC, Iizuka K, Nixon G, Browne JP, Hall A, Eccleston JF, Sugai M, Kobayashi S, Somlyo AV, and Somlyo AP. Role of guanine nucleotide-binding proteins---ras-family or trimeric proteins or both---in Ca2+ sensitization of smooth muscle. Proc Natl Acad Sci USA 93: 1340-1345, 1996[Abstract/Free Full Text].

19.   Gong, MC, Kinter MT, Somlyo AV, and Somlyo AP. Arachidonic acid and diacylglycerol release associated with inhibition of myosin light chain dephosphorylation in rabbit smooth muscle. J Physiol (Lond) 486: 113-122, 1995[Abstract].

20.   Gronich, J, Konieczkowski M, Gelb MH, Nemenhoff RA, and Sedor JR. Interleukin 1alpha causes rapid activation of cytosolic phospholipase A2 by phosphorylation in rat mesangial cells. J Clin Invest 93: 1224-1233, 1994[ISI][Medline].

21.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

22.   Hagiwara, M, Inoue S, Tanaka T, Nunoki K, Ito M, and Hidaka H. Differential effects of flavinoids as inhibitors of tyrosine protein kinases and serine/threonine protein kinases. Biochem Pharmacol 37: 2987-2992, 1988[ISI][Medline].

23.   Herbert, JM, Augereau JM, Glyeye J, and Maffrand JP. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 172: 993-999, 1990[ISI][Medline].

24.   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].

25.   Hill, TD, Campos-Gonzalez T, Kindmark H, and Boynton AL. Inhibition of inositol trisphosphate-stimulated calcium mobilization by calmodulin antagonists in rat liver epithelial cells. J Biol Chem 263: 16479-16484, 1988[Abstract/Free Full Text].

26.   Hillemeier, AC, Bitar KB, 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].

27.   Himpens, B, Kitazawa P, and Somlyo AP. Agonist-dependent modulation of Ca2+-sensitivity in rabbit pulmonary artery smooth muscle. Pflügers Arch 417: 21-29, 1990[ISI][Medline].

28.   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].

29.   Johannes, F-J, Prestle J, Eis S, Oberhagamann P, and Pfizenmaier K. PKCµ is a novel, atypical member of the protein kinase C family. J Biol Chem 269: 6140-6148, 1994[Abstract/Free Full Text].

30.   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].

31.   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].

32.   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].

33.   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].

34.   Kim, NY, Sohn UD, Mangannan V, Rich H, Behar J, and Biancani P. Leukotrienes in ACh-induced contraction of esophageal circular smooth muscle in experimental esophagitis. Gastroenterology 112: 1548-1558, 1997[ISI][Medline].

35.   Kitazawa, T, Kobayashi S, Horiuti T, Somlyo AV, and Somlyo AP. Receptor coupled, permeabilized smooth muscle: role of the phosphatidylinositol cascade, G-proteins and modulation of the contractile response to Ca2+. J Biol Chem 264: 5339-5342, 1989[Abstract/Free Full Text].

36.   Kitazawa, T, Masuo M, and Somlyo AP. G-protein-mediated inhibition of myosin light chain phosphatase in vascular smooth muscle. Proc Natl Acad Sci USA 88: 9307-9310, 1991[Abstract].

37.   Kitazawa, T, and Somlyo AP. Modulation of Ca2+ sensitivity by agonists in smooth muscle. Adv Exp Med Biol 304: 97-109, 1991[Medline].

38.   Kubota, Y, Nomura M, Kamm KE, Mumby MC, and Stull JT. GTPgamma S-dependent regulation of smooth muscle contractile elements. Am J Physiol Cell Physiol 262: C405-C410, 1992[Abstract/Free Full Text].

39.   Lassegue, B, Alexander RW, Clark M, and Griendling KK. Angiotensin II-induced phosphatidylcholine hydrolysis in cultured vascular smooth-muscle cells. Regulation and localization. Biochem J 276: 19-25, 1991[ISI][Medline].

40.   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].

41.   Mennice, B, Hulvershorn J, Adam LP, Wang C-LA, and Morgan KG. Calponin and mitogen-activated protein kinase signaling in differentiated vascular smooth muscle. J Biol Chem 272: 25157-25161, 1997[Abstract/Free Full Text].

42.   Morgan, JP, and Morgan KG. Stimulus-specific patterns of intracellular calcium levels in smooth muscle of the ferret portal vein. J Physiol (Lond) 351: 155-167, 1984[Abstract].

43.   Morgan, KG, and Leinweber BD. PKC-dependent signalling mechanisms in differentiated smooth muscle. Acta Physiol Scand 164: 495-505, 1998[ISI][Medline].

44.   Nishimura, J, Kolber M, and van Breemen C. Norepinephrine and GTPgamma S increase myofilament Ca2+ sensitivity in alpha -toxin permeabilized arterial smooth muscle. Biochem Biophys Res Commun 157: 677-683, 1988[ISI][Medline].

45.   Nishizuka, Y. Protein kinase C and lipid signaling for sustained responses. FASEB J 9: 484-496, 1995[Abstract/Free Full Text].

46.   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].

47.   Osadas, S, Mizuno K, Saido TC, Suzuki K, Kuroki T, and Ohno S. A new member of the protein kinase family, nPKCtheta , predominantly expressed in skeletal muscle. Mol Cell Biol 12: 3030-3938, 1992.

48.   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].

49.   Prozialeck, WC, and Weiss B. Inhibition of calmodulin by phenothiazines and related drugs: structure-activity relationships. J Pharmacol Exp Ther 222: 509-516, 1982[ISI][Medline].

50.   Qian, Z, and Drewes LR. Cross-talk between receptor-regulated phospholipase D and phospholipase C in brain. FASEB J 5: 315-319, 1991[Abstract/Free Full Text].

51.   Qian, Z, and Drewes LR. A novel mechanism for acetylcholine to generate diacylglycerol in brain. J Biol Chem 265: 3607-3610, 1990[Abstract/Free Full Text].

52.   Rembold, CM, and Murphy RA. Myoplasmic [Ca2+] determines myosin phosphorylation in agonist-stimulated swine arterial smooth muscle. Circ Res 63: 593-603, 1988[Abstract].

53.   Riendeau, D, Guay J, Weech PK, Laliberte F, Yergey JLC, Desmarais S, Perrier H, Liu S, Nicoll-Griffith D, and Street IP. Arachidonoyl trifluoromethyl ketone, a potent inhibitor of 85 kDa phospholipase A2, blocks production of arachidonate and 12-hydroxyeicosatetranoic acid by calcium ionophore-challenged platelets. J Biol Chem 269: 1-6, 1994[Free Full Text].

54.   Saitoh, M, Ishikawa Y, Matsushima S, Naka M, and Hidaka H. Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase. J Biol Chem 262: 7796-7780, 1987[Abstract/Free Full Text].

55.   Schutze, S, Potthoff K, Machleidt T, Berkovic D, Wiegmann K, and Kronke M. TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell 71: 765-776, 1992[ISI][Medline].

56.   Sims, S, Vivadou M, Hillemeier C, Biancani P, Walsh J, and Singer J. Membrane currents and cholinergic regulation of K+ current in esophageal smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 258: G794-G802, 1990[Abstract/Free Full Text].

57.   Smith, JB. Calcium homeostasis in smooth muscle cells. New Horiz 4: 2-18, 1996[Medline].

58.   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].

59.   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].

60.   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].

61.   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].

62.   Somlyo, A. Modulation of the Ca2+ switch: by G proteins, kinases, and phosphatases. News Physiol Sci 8: 2, 1993[ISI].

63.   Street, IP, Lin KH, Laliberté F, Ghomashchi F, Wang Z, Perrier H, Tremblay NM, Huang Z, Weech PK, and Gelb MH. Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2. Biochemistry 32: 5935-5940, 1993[ISI][Medline].

64.   Tang, DC, Stull JT, Kubota Y, and Kamm KE. Regulation of the Ca2+ dependence of smooth muscle contraction. J Biol Chem 267: 11839, 1992[Abstract/Free Full Text].

65.   Tansey, MG, Word RA, Hidaka H, Singer A, Schworer CM, Kamm KE, and Stull JT. Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells. J Biol Chem 267: 12511-12516, 1992[Abstract/Free Full Text].

66.   Welsh, CJ, Schmeichel K, Cao HT, and Chabbott H. Vasopressin stimulates phospholipase D activity against phosphatidylcholine in vascular smooth muscle cells. Lipids 25: 675-684, 1990[ISI][Medline].

67.   Whitney, G, Throckmorton D, Isales C, Takuwa Y, Yeh J, Rasmussen H, and Brophy C. Kinase activation and smooth muscle contraction in the presence and absence of calcium. J Vasc Surg 22: 37-44, 1995[ISI][Medline].


Am J Physiol Cell Physiol 280(4):C980-C992
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society