Department of Medicine, Rhode Island Hospital, and Brown University School of Medicine, Providence, Rhode Island 02903
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ABSTRACT |
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The signal transduction that mediates
CCK-induced contraction of gallbladder muscle was investigated in the
cat. Contraction was measured by scanning micrometry in single muscle
cells isolated enzymatically with collagenase. Production of
D-myo-inositol
1,4,5-trisphosphate (IP3) and
sn-1,2-diacylglycerol (DAG) was
quantitated using HPLC and TLC, respectively. Protein kinase C (PKC)
activity was determined by measuring the phosphorylation of a specific
substrate peptide from myelin basic protein, Ac-MBP-(414).
CCK-induced contraction was blocked by incubation in strontium medium,
pertussis toxin (PTx), and antibodies against
Gi
3
or
-subunits but was not blocked by
Ca2+-free medium or by antibodies
against Gq/11
,
Gi
1-2,
or Go
. The contraction induced
by CCK was inhibited by the phospholipase C (PLC) inhibitor U-73122,
anti-PLC-
3 antibody, and the
IP3 receptor antagonist heparin
but was not inhibited by the the phospholipase D inhibitor propranolol
or antibodies against PLC-
1 or PLC-
2. Western blot analysis of
gallbladder muscle revealed the presence of PLC-
2 and PLC-
3 but
not PLC-
1. CCK caused a 94% increase in
IP3 generation and an 86%
increase in DAG generation. A low dose of CCK caused PKC translocation,
and CCK-induced contraction was blocked by the PKC inhibitor H-7. A
high dose of CCK, however, caused no PKC translocation, and its
contraction was blocked by the calmodulin antagonist CGS9343B. In
conclusion, CCK contracts cat gallbladder muscle by stimulating
PTx-sensitive Gi 3 protein coupled with PLC-
3, producing
IP3 and DAG. Low doses activate PKC, whereas high doses activate calmodulin.
G proteins; phospholipase C; inositol 1,4,5-trisphosphate; protein kinase C
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INTRODUCTION |
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CCK IS A MAJOR physiological hormone that regulates
gallbladder contraction and emptying of bile during the intestinal
phase of the postprandial state. CCK-induced gallbladder contraction is
partly myogenic and is mediated by direct action on receptors located
on smooth muscle cells (2, 15, 44). The contractile mechanisms of
gallbladder muscle cells in response to CCK, however, are not well
understood. Most studies have shown that CCK activates Gq/11 protein. In the circular
muscle of guinea pig ileum, CCK-induced contraction is mediated by
activation of the pertussis toxin (PTx)-insensitive Gq/11 subunit, which results in
stimulation of phospholipase C-
1 (PLC-
1) (26). However, it has
also been shown that CCK can activate
Gi and
Gs proteins (37).
We have previously shown that in muscle strips CCK causes gallbladder contraction by utilizing intracellular Ca2+ (21). Intracellular Ca2+ can be released by a second messenger, D-myo-inositol 1,4,5-trisphosphate (IP3), in gastric and intestinal smooth muscle cells, as well as other nonmuscle cells (3-4, 8, 25). Released Ca2+ then binds and activates calmodulin (12, 20). The Ca2+-calmodulin complex is capable of activating myosin light chain kinase (MLCK), which causes myosin light chain phosphorylation and muscle contraction (17, 23). IP3 is the product of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by PLC (13, 25, 27). The other product of PIP2 hydrolysis is sn-1,2-diacylglycerol (DAG). DAG can also be formed through a phospholipase D (PLD)-mediated pathway. PLD cleaves phosphatidylcholine to phosphatidic acid, and the latter can be further hydrolyzed to DAG by phosphatidic acid phosphohydrolase (5, 14). DAG is an activator of protein kinase C (PKC), which has also been proposed to play a role in mediating smooth muscle contraction (25, 36). It is not known whether such mechanisms are present in gallbladder muscle. In the present study we therefore investigated the signal-transduction pathways mediating CCK-induced contraction of gallbladder muscle cells in the cat.
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MATERIALS AND METHODS |
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Animals and tissue preparation. Adult cats of both sexes, weighing between 3 and 5 kg, were purchased from Liberty Research Laboratory (Waverly, NY). Their use was approved by the Animal Welfare Committee of Rhode Island Hospital. After an overnight fast, cats were anesthetized with an intramuscular administration of ketamine hydrochloride (30 mg/kg) followed by pentobarbital sodium (30 mg/kg ip). The gallbladder was exposed and the cystic duct was clamped. The bile was carefully removed from the gallbladder, and the gallbladder cavity was rinsed thoroughly with ice-cold oxygenated Krebs buffer. The Krebs buffer contained the following (in mM): 116.6 NaCl, 3.4 KCl, 21.9 NaHCO3, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2, and 5.4 glucose. The gallbladder was then removed from the liver bed and placed in a dissecting pan containing Krebs buffer, which was continuously aerated with 95% O2-5% CO2. Under a dissecting microscope, the mucosa and serosa were peeled off. The smooth muscle layer was carefully cleaned by removing the remaining connective tissue and small blood vessels. The tissue was then minced to squares of about 2 × 2 mm for single cell preparation and biochemical measurements.
Isolation of single muscle cells and measurement of cell contraction. Single muscle cells were isolated from the gallbladder as previously described (48). Briefly, the muscle squares were digested in a collagenase solution and gassed with 100% O2 for 2.5-3 h at 31°C. The collagenase solution contained 150 U of type II collagenase per milliliter of HEPES-buffered nutrient salt solution. The HEPES buffer solution contained (in mM) 112.5 NaCl, 5.5 KCl, 2.0 KH2PO4, 24 HEPES, 1.9 CaCl2, 0.6 MgCl2, and 10.8 glucose, as well as 0.08 mg/ml soybean trypsin inhibitor and 2% (vol/vol) basal medium Eagle (50×) amino acids without L-glutamine. The partly digested tissue was then washed over a Nitex mesh with collagenase-free HEPES buffer and incubated in the same buffer for 30-60 min to allow muscle cells to disperse freely. Cells were then harvested by filtering through a Nitex mesh, and the filtrate (cell suspension) was equilibrated for 20 min before the experiment was started. For some experiments, cells were permeabilized with a brief exposure to saponin (75 µg/ml for 4 min) and equilibrated in a modified cytosolic buffer, which consisted of (in mM) 20 NaCl, 100 KCl, 25 NaHCO3, 5.0 MgSO4, 0.96 NaH2PO4, 0.48 CaCl2, 1.0 EGTA, 0.01 antimycin A, 1.5 ATP, and an ATP-regenerating system consisting of creatine phosphokinase (10 U/ml) and 5 mM creatine phosphate (48).
Aliquots of 0.25 ml cell suspension were added to the test tubes containing 0.1 ml buffer with appropriate concentrations of agonists or antagonists. Antagonists were preincubated with muscle cells for 60 s before cells were exposed to agonists for 30 s. The response was stopped by the addition of acrolein at a final concentration of 1%. A few drops of the fixed cells were placed on a microscope slide and covered with a coverslip. The lengths of 30 undamaged cells from control and experimental samples were measured with a Zeiss phase-contrast microscope (Carl Zeiss, Oberkochen, Germany), a Panasonic CCTV camera (model WV-CD51; Matsushita Communication Industrial, Osaka, Japan), and a Macintosh IIci computer (Apple Computer, Cupertino, CA), using a computer software program, Image 1.33 (National Institutes of Health, Bethesda, MD). Contraction was expressed as percent shortening from initial control length.Preparation of gallbladder muscle membranes. Gallbladder muscle squares were homogenized with a Tissue Tearor (Biospec, Racine, WI) for three bursts of 20 s each at setting 5 in 20 mM ice-cold HEPES-homogenized buffer (pH 7.4) and again with 60 strokes of a Dounce Grinder (Wheaton, Millville, NJ). The homogenates were centrifuged at 600 g for 2 min. The supernatant was collected and the pellet was rehomogenized and filtered through two layers of 200-µm Nitex mesh. The pooled supernatant was then ultracentrifuged at 40,000 g for 30 min at 4°C. The pellet was resuspended and solubilized for 1 h at 4°C in a buffer containing (in mM) 20 HEPES (pH 7.4), 240 NaCl, 2 EDTA, 2 phenylmethylsulfonyl fluoride (PMSF), and 20 leupeptin, with 20 mg/ml aprotinin and 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized membrane suspension was measured for protein content and was used for the following binding studies.
[35S]GTPS
binding.
35S-labeled guanosine
5'-O-(3-thiotriphosphate)
([35S]GTP
S) binding
was assayed according to the method of Okamoto et al. (11, 28, 33). The
crude membranes, at a concentration of 2.5 mg protein/ml, were
incubated at 37°C with 30 nM
[35S]GTP
S in a
solution containing 10 mM HEPES (pH 7.4), 0.1 mM EDTA, and 10 mM
MgCl2. The stimulation of binding
was assayed in the presence or absence of a maximally effective
concentration of CCK-8 for tissue squares (1 µM) with a total volume
of 300 µl. The reaction was stopped with 10 vol of ice-cold 100 mM
Tris · HCl (pH 8.0) containing 10 mM
MgCl2, 100 mM NaCl, and 20 µM GTP. Mixtures of 200 µl of each were added to enzyme-linked
immunosorbent assay wells that had been coated initially with an
anti-rabbit immunoglobulin antibody (1:1,000) and were subsequently
coated with specific G protein antibodies (1:1,000). After 2-h
incubation on ice, the wells were washed three times with PBS
containing 0.05% Tween-20. The radioactivity from each well was
counted with the use of a Tri-Carb 1900 CA liquid scintillation
analyzer (Packard Instrument, Meriden, CT). Triplicate measurements
were carried out for each experiment. Data were expressed as percent
stimulation from basal levels.
Immunoblot analysis of PLC- isoenzymes in
gallbladder muscle.
Western blot analysis of PLC-
isoenzymes was performed as described
previously (26, 28). Gallbladder muscle squares were homogenized in
medium containing 50 mM Tris · HCl, pH 7.5, 1 mM EGTA, 1% Triton X-100, 2 mM PMSF, 0.1 mM dithiothreitol (DTT), and 2 µg/ml leupeptin and centrifuged at 12,000 g for 5 min. The supernatant was
loaded and subjected to 8% SDS-PAGE (Mini-PROTEAN II cell; Bio-Rad,
Hercules, CA). Prestained molecular weight markers were run in an
adjacent lane to allow molecular weight to be determined. The separated
proteins were electrically transferred at 4°C to a nitrocellulose
membrane (Bio-Rad, Melville, NY) in 25 mM Tris (pH 8.3), 192 mM
glycine, and 20% methanol. The nitrocellulose membranes were blocked
with 5% dried milk in PBS containing 0.2% Tween-20 at room
temperature for 1 h, followed by incubation with different anti-PLC-
isoenzyme-specific antibodies (1 µg/ml) for 1 h. After unbound
antibodies were removed by washing three times with PBS, the
nitrocellulose membranes were incubated for 1 h with horseradish
peroxidase-conjugated protein A. The PLC-
isoenzyme bands were
identified with the use of enhanced chemiluminescence reagents.
Measurement of IP3 production. Extraction and HPLC separation of IP3 were performed as previously described (43, 48). Freshly obtained gallbladder muscle squares were incubated with 60 µCi/ml myo-[2-3H]inositol at 37°C for 4 h. The tissue was collected and evenly divided into the experimental and control tubes. The experimental groups were exposed to CCK-8 (1 mM) for 10 s, and the control groups were exposed to Krebs solution only. The reaction was quenched by the addition of an ice-cold chloroform-methanol-HCl mixture (100:50:1, vol/vol). Phytic acid (25 µl at 100 mg/ml) was added to each tube to enhance HPLC signals. The aqueous cytosolic phase was separated by centrifugation, collected three times, and neutralized to pH 6.5-7.5. Samples were passed through a Partisil 10 SAX HPLC column (Whatman, Clinton, NJ), which was connected to a HPLC system (Bio-Rad, Richmond, CA). The phosphate-containing inositol phosphate metabolites were selectively eluted in a stepwise fashion with 1.5 M ammonium formate buffer as previously described (43, 48). The HPLC column eluate was pumped into the detector system, where it was mixed with FLO-SCINT IV scintillation fluid (Packard Instrument, Downer's Grove, IL) and pumped through a 1-ml flow cell for radiochemical detection. Radioactivity was determined by continual flow liquid scintillation counting using a FLO-ONE/Beta Radiochromatography Detector Series A-200 (Radiomatic Instruments Chemical, Tampa, FL). Five major radioactive peaks were isolated with the use of the elution pattern listed above. Among them, three could be identified as inositol 1-monophosphate (IP1), inositol 1,4-bisphosphate (IP2), and IP3, according to the standard [3H]inositol phosphate marker (Amersham, Arlington Heights, IL). Values were expressed as disintegrations per minute (dpm) per milligram protein.
Extraction and quantitation of DAG.
DAG was extracted and measured as previously described with some
modifications (34, 48). The gallbladder muscle squares were
equilibrated in 450 µl Krebs solution for 20 min at 37°C. Aliquots of 50 µl of CCK-8 (10 µM) or Krebs solution only (control) were added to each tube, and the reaction was stopped at 15, 30, 60, and 90 s by the addition of 2 ml of ice-cold methanol. Samples were
then homogenized with a Polytron, and 1 ml of chloroform and 0.5 ml of
1 M NaCl were added. After 15-min incubation on ice, another 1 ml of
chloroform and 1 M NaCl were added to separate phases by centrifuging
at 2,000 g for 5 min. The lower
organic layer was collected and dried under nitrogen and stored at
70°C for later measurement.
PKC extraction and partial purification.
PKC was extracted and partly purified according to methods described
previously (1, 16). Muscle squares were equilibrated in 400 µl Krebs
solution in a test tube at 37°C for 20 min. Aliquots (100 µl) of
appropriate concentrations of CCK-8 or Krebs solution (control) were
then added. At certain time points, the reaction was stopped with 10 vol of ice-cold Krebs solution, and test tubes were put on ice
immediately. The muscle squares were collected by filtering through a
450-µm Nitex mesh and transferred to a test tube containing 3 ml
ice-cold buffer
A.
Buffer
A contained (in mM) 20 Tris (pH 7.5),
0.5 EDTA, 0.5 EGTA, and 10 -mercaptoethanol, as well as 25 µg/ml
each of aprotinin and leupeptin. The samples were then homogenized with
a Polytron and 60 strokes of a Dounce Grinder. Aliqots (100 µl) of
homogenates were saved for later protein determination. Samples were
ultracentrifuged at 100,000 g for 30 min at 4°C. The supernatant was retained as the cytosolic fraction.
The pellet was resuspended with 3 ml
buffer
A and homogenized again with 20 strokes, 150 µl of 10% Triton X-100 was added to the homogenates,
and samples were well mixed with a Tube Rotator (Scientific Equipment
Products, Baltimore, MD) for 45 min at 4°C. After centrifuging
again at 100,000 g for 30 min, the
supernatant was collected as the membranous fraction. Both cytosolic
and membranous fractions were passed through 1.0 ml
diethylaminoethyl cellulose columns for partial
purification. The columns were prepared by mixing 0.5 g Whatman DE52
cellulose with 1.0 ml buffer
B
(buffer A without protease inhibitors) and
washing with 1.0 ml buffer B before applying samples. After the
samples passed through, the columns were washed again with 2.5 ml
buffer
B and finally eluted with 2 ml
buffer
C
(buffer
B plus 0.2 M NaCl).
PKC assay.
PKC activity was measured using the GIBCO PKC assay kit (GIBCO, Life
Technologies, Grand Island, NY). The amount of
32P transferred from
[-32P]ATP
(Amersham) to a specific substrate peptide from myelin basic protein,
Ac-MBP-(4
14), on its phosphorylation by PKC in the presence of 10 µM phorbol 12-myristate 13-acetate, 0.28 mg/ml phosphatidylserine, 20 mM MgCl2, 1 mM
CaCl2, 0.5 mM EDTA, and 0.5 mM
EGTA was determined (1, 16, 46). PKC activity was
calculated by subtracting the nonspecific kinase activity obtained in
the presence of a specific PKC inhibitor, the pseudosubstrate
PKC-(19
36) at 20 µM, and in the absence of lipids. Triplicate PKC
determinations were made for each sample. Briefly, 25 µl of partly
purified enzyme (properly diluted with
buffer
C) was incubated with either lipids or peptide PKC-(19
36) at room temperature for 20 min. The reaction was initiated by adding 10 µl of 5×
32P-labeled substrate solution
containing 100 µM Ac-MBP-(4
14), 100 mM ATP, 5 mM
CaCl2, and 100 mM
MgCl2 in 20 mM Tris with 0.5 mM
EDTA and 0.5 mM EGTA (final volume 50 µl). The reaction was allowed
to proceed at 30°C for 5 min. Then, aliquots of 25 µl were
removed and spotted on to a P81 ion-exchange phosphocellulose paper
(Whatman). The phosphocellulose paper was washed twice with 1%
(vol/vol) phosphoric acid and twice with distilled water (5 min each).
The remaining 32P activity on the
phosphocellulose paper was quantitated with the use of the Tri-Carb
1900CA liquid scintilation analyzer. Data were expressed as picomoles
per minute per milligram protein.
Protein determination. Protein content in each tissue sample was measured according to the method of Bradford, using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Melville, NY). Values were means of triplicate measurements for each sample.
Drugs and chemicals.
Type II collagenase and soybean trypsin inhibitor were purchased from
Worthington Biochemicals (Freehold, NJ). Polyclone antibodies to
Gi1-2,
Gi
3,
Gq/11
,
Go
, and
-subunits and PTx
were obtained from Calbiochem (La Jolla, CA); polyclonal antibodies to
PLC-
2 and PLC-
3 were from Santa Cruz Biochemicals (Santa Cruz,
CA); and monoclonal antibody to PLC-
1 was obtained from Upstate
Biotechnology (Lake Placid, NY). Recent studies have demonstrated the
ability of these G protein and PLC-
isoenzyme antibodies to block
activation or inhibition of specific effector enzymes (11, 29, 40).
CCK-8 was purchased from Bachem (Torrance, CA).
[35S]GTP
S was
purchased from DuPont-New England Nuclear (Boston, MA). Horseradish
peroxidase-conjugated protein A, enhanced chemiluminescence reagents,
and rainbow prestained molecular markers were obtained from Amersham.
1-(5-Isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) was purchased
from Seikagaku America (St. Petersburg, FL). 1,3-Dihydro-1(1-{[4-methyl-4H,6H-pyrrolo(1,2-
)-(4,1)benzoxazepin-4-yl]methyl}-4-piperindinyl)2H-benzimidazol-2-1-maleate (CGS9343B) was a gift from Richard A. Lovell of Ciba-Geigy
(Summit, NJ).
1-(6-{[17
-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)1H-pyrrole-2,5-dione (U-73122) was a gift from Dr. John J. Bleasdale (Upjohn, Kalamazoo, MI). Heparin (from porcine intestinal mucosa, mol wt 4,000-6,000), IP3, GTP
S, aprotinin,
leupeptin, and other reagents were purchased from Sigma Chemical (St.
Louis, MO).
Statistics. Paired and unpaired Student's t-tests and one- and two-factorial repeated ANOVA were used for statistical analysis. P < 0.05 was considered significant.
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RESULTS |
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Ca2+ source utilized by CCK. CCK-8 (0.1 pM-10 nM) contracted the intact and permeabilized gallbladder muscle cells in a dose-dependent manner (P < 0.01, by one factorial ANOVA). Contraction reached a plateau at about 26 ± 1% shortening at 10 nM of CCK-8. There was no significant difference in contractility between intact and permeabilized muscle cells. When muscle cells were preincubated in a Ca2+-free medium containing 2 mM EGTA for 60 min, their maximal shortening in response to 10 nM CCK-8 was 26 ± 3% and was not significantly different from that in normal Ca2+ medium. The Ca2+-free medium, however, abolished the contraction caused by the maximally effective dose of KCl (Fig. 1). KCl has been shown to utilize only extracellular Ca2+ by depolarizing the plasma membrane and inducing Ca2+ influx (21). When muscle cells were preincubated in a medium containing 4 mM Sr2+ instead of Ca2+ for 60 min, their response to 10 nM CCK-8 was reduced by 72% from that in a normal Ca2+ medium (Fig. 1). Sr2+ has been shown to replace Ca2+ in the endoplasmic reticulum but cannot be readily released (41, 47), therefore blocking the processes that require Ca2+ release from storage sites. Sr2+, however, can replace the role of extracellular Ca2+ in mediating contraction, since the 4 mM Sr2+ medium did not affect the contraction caused by KCl in both muscle strips (21) and single muscle cells.
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Identification of CCK receptor-activated G protein.
To examine the G protein subtype functionally coupled with the CCK
receptor, we preincubated muscle cells with PTx at 0.5 and 1.6 µg/ml
or with different G protein subunit antibodies (1:400) before exposure
to CCK. The ability of these G protein antibodies to block activation
of specific effector enzymes has been demonstrated in previous studies
(11, 40). The maximal contraction induced by 10 nM CCK-8 in
permeabilized gallbladder muscle cells was significantly inhibited by
pretreating muscle cells with PTx,
anti-Gi3,
and anti-
-subunit-specific antibodies, but not by pretreating
with anti-Gi
1-2-,
Go
-, or
Gq/11
-specific antibodies (Fig. 2). These data suggest that in gallbladder
muscle the G proteins activated by CCK-8 are PTx-sensitive
-
and
-subunits of
Gi 3 protein. To further
confirm this finding
[35S]GTP
S binding
induced by CCK-8 stimulation was performed using specific G protein
antibodies to immunoprecipitate the activated specific G proteins. As
shown in Fig. 3, CCK-8 (1 µM) caused a significant increase in
[35S]GTP
S binding
to
Gi
3
but not to
Gi
1-2
and Gq/11
. This increased
binding was completely blocked by pretreatment of muscle membranes with
PTx.
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Activation of PLC- isoenzymes by CCK.
To examine the roles of the phospholipases, we tested the effects of a
PLC inhibitor, U-73122, and a phosphatidic acid phosphohydrolase (which
produces DAG through the PLD-mediated pathway) inhibitor, propranolol,
on CCK-8-induced contraction. U-73122 at 10 mM inhibited the
contraction caused by the maximally effective dose (10 nM) of CCK-8 by
78%. It also inhibited the contraction induced by a low dose of CCK-8
(0.1 pM) by 77%. On the other hand, 10 µM propranolol had no effect
on the contractions caused by the high and low concentrations of CCK-8
(Fig. 4).
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The role of IP3 in mediating CCK-induced contraction. To examine the role of IP3 in CCK-induced contraction in gallbladder muscle, the effect of low molecular weight heparin, a specific IP3 receptor antagonist, on CCK-8 was tested. Permeabilized cells were preincubated with heparin for 60 s before CCK-8 was added. The maximal contraction caused by 10 nM CCK-8 was reduced by heparin in a concentration-dependent manner (Fig. 7A). Maximal inhibition of 84% was reached at a heparin concentration of 10 µM. The effect of exogenous IP3 on gallbladder muscle contraction was also tested. Like heparin, IP3 does not diffuse across the plasma membrane. Therefore, it was used in permeabilized cells as well. IP3 contracted the permeabilized muscle cells in a dose-dependent manner (0.1 nM-1 µM), and peak contraction (21 ± 1%) was obtained at 1 mM IP3 (Fig. 7B).
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PKC activation in CCK-induced contraction. When Ca2+ is released from intracellular stores by IP3, it can either activate calmodulin-MLCK or potentiate DAG to activate PKC (12, 20, 31-32, 42). To examine the roles of calmodulin and PKC in mediating gallbladder muscle contraction, we tested the effects of the calmodulin antagonist CGS9343B and the PKC inhibitor H-7 on CCK-8-induced contraction and measured PKC activity in the cytosolic and membranous fractions of gallbladder muscle. CGS9343B at 10 mM reduced the maximal contraction caused by 10 nM CCK-8 (23.4 ± 1.4%) to 5.6 ± 2.0%. CGS9343B, however, had no effect on contraction caused by a low dose (0.1 pM) of CCK-8. In contrast, H-7 at 10 µM had no effect on contraction caused by 10 nM CCK-8, but it abolished the contraction caused by 0.1 pM CCK-8 (Fig. 9).
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DISCUSSION |
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The results of the present study support our previous finding (21) that CCK causes gallbladder muscle contraction in muscle strips by utilizing intracellular Ca2+, since the contraction was blocked by Sr2+ replacement for Ca2+ in the medium and was not affected by a Ca2+-free medium. It appears that these two media are able to distinguish the Ca2+ sources utilized by agonists. In both preparations of gallbladder muscle strips and single cells, we were able to show that a Ca2+-free medium abolished the contraction caused by KCl. Potassium contracts the smooth muscle cells by depolarizing plasma membranes and opening Ca2+ channels, resulting in increased Ca2+ influx. Sr2+ displaces Ca2+ in the endoplasmic reticulum but cannot be released from the intracellular stores (41, 47). The finding that Sr2+ does not affect KCl-induced contraction suggests that it can only replace the role of extracellular Ca2+. Indeed, the conductance and permeability of the L-type Ca2+ channel to Sr2+ and Ca2+ are closely similar (38, 45).
It has been shown in most studies that CCK activates
Gq/11 proteins. In the circular
muscle of the guinea pig ileum, CCK-induced contraction is mediated by
activation of a PTx-insensitive
Gq/11 subunit that results in
stimulation of PLC-
1 (26). However, it has also been reported that
CCK can activate Gi and
Gs proteins (37). In the present
study, our data showed that the contraction induced by CCK in
gallbladder muscle was blocked by PTx and by anti-Gi
3-,
anti-
-subunit-, and anti-PLC-
3-specific antibodies, suggesting
that CCK receptors in gallbladder muscle are selectively coupled to
PLC-
3 via both
- and
-subunits of
Gi 3 protein. It thus
appears that different pathways couple to CCK receptors in the
gallbladder and ileal circular muscle. These differences therefore
appear to be tissue specific rather than peptide specific and are
further supported by the immunoblot analysis, which shows only PLC-
2
and PLC-
3 in the gallbladder muscle, whereas in the ileal circular
muscle cells the entire spectrum of PLC-
isoenzymes, including
PLC-
1, PLC-
2, and PLC-
3, is fully expressed. These findings
are in agreement with a previous study showing that A1 adenosine
receptors of intestinal smooth muscle are coupled with PLC-
3 via
- and
-subunits of the
Gi 3 protein (29).
Our findings also suggest that PLC-3 mediates CCK-induced
contraction by hydrolyzing PIP2
into IP3 and DAG. They further showed that 1)
IP3 contracts the permeabilized
gallbladder muscle in a dose-dependent manner, suggesting the existence
of IP3 receptors in the
endoplasmic reticulum. Similar findings have been observed in guinea
pig ileum (26). 2) The actions of
CCK were blocked in a dose-dependent manner by low molecular weight
heparin (3-4, 8, 10, 18). It is known that heparin blocks
IP3 competitively at receptors
that mediate Ca2+ release from
Ca2+ stores.
3) CCK stimulation generates
IP3 and DAG. Direct measurements of IP3 and DAG showed a nearly
twofold increase in these second messengers at 10 s.
IP3 is the exclusive product of
PIP2 hydrolysis by PLC (13, 27).
DAG, however, can also be formed by the hydrolysis of phosphatidylcholine by phospholipase D (PLD), which forms phosphatidic acid. Phosphatidic acid is further hydrolyzed to DAG by phosphohydrolase (5, 14). Therefore, the effects of the PLC and phosphohydrolase antagonists U-73122 and propranolol, respectively, were tested to determine whether the CCK-induced contraction was also mediated by PLD. U-73122 competes with Ca2+ for binding to the site on PLC that must be occupied by Ca2+ for expression of PLC activity, and therefore inhibits agonist-induced IP3 production (9, 39). Propranolol inhibits phosphatidic acid phosphohydrolase, preventing the formation of DAG through the PLD-mediated pathway (7, 35). The finding that U-73122, but not propranolol, inhibited the contraction induced by CCK supports the conclusion that PLC, not PLD, plays a role in the CCK action on gallbladder muscle.
Ca2+ released from the storage sites may activate calmodulin (12, 20, 42) by affecting PKC directly or by its synergistic action with DAG (31, 32). To characterize these final steps in the signal-transduction pathways leading to muscle contraction, we tested the effects of a calmodulin antagonist, CGS9343B, and a PKC inhibitor, H-7, on CCK-induced contraction and measured the PKC activity after CCK stimulation. The data show that low concentrations of CCK (i.e., 0.1 pM for single muscle cells and 10 nM for muscle strips and squares) caused a small contraction, which was blocked by H-7 but not by CGS9343B, and caused a significant PKC translocation from the cytosol to the membrane, an indication of PKC activation (1, 16). High concentrations of CCK (i.e., 10 nM for single cells and 1 mM for muscle strips and squares) caused a contraction that was not affected by H-7 but was blocked by CGS9343B. These CCK concentrations caused no PKC translocation. One possibility is that low doses of CCK utilize an alternative pathway (PLD) to produce DAG, resulting in the activation of PKC, whereas a high dose of CCK activates PLC, forming IP3, releasing Ca2+, and activating calmodulin. However, it is unlikely that PLD is involved in the contraction caused by low doses of CCK, since the contraction was not affected by propranolol but was inhibited by U-73122. Therefore, the factor that determines this switch may be the amount of Ca2+ released from intracellular stores.
Although the intracellular Ca2+ concentrations in these experiments were unknown, it has been shown in other tissues that CCK and IP3 release Ca2+ from intracellular stores in a concentration-dependent manner, which correlates with the dose-dependent contraction in response to the same agonist (22, 26). With a low dose of CCK, low levels of IP3 and DAG are presumably formed by the activation of PLC. When a small amount of Ca2+ is released by low levels of IP3, it could potentiate DAG to activate PKC but may not be sufficient to activate calmodulin by itself. It is known that PKC has a greater affinity for Ca2+ than calmodulin (5, 48). This assumption is also supported by our previous findings that the potentiation with low doses of exogenous IP3 and DAG was blocked by H-7 but was unaffected by CGS9343B (48). With high doses of CCK, larger amounts of Ca2+ are presumably released, which may be able to activate calmodulin, and the calmodulin activation appears to inhibit the activation of PKC. This is supported by the finding that exogenously activated calmodulin inhibited the contraction caused by the PKC activator DAG (48). The inhibition of PKC activity by calmodulin has been shown in several other tissues and at different levels, i.e., at the kinase itself or at the substrate level (19, 30, 49). Our results suggest that activated calmodulin inhibits the activation of PKC in gallbladder muscle.
In summary, in gallbladder muscle cells CCK activates PTx-sensitive
Gi3
protein coupled with PLC-
3 to produce
IP3 and cause intracellular
Ca2+ release. Depending on the
concentration, CCK could activate either PKC or calmodulin, leading to
gallbladder muscle cell contraction; low doses of CCK activate the
PKC-dependent pathway, whereas high doses of CCK activate the
calmodulin-dependent pathway.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-27389.
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FOOTNOTES |
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Portions of this study have been presented previously at the Annual Meeting of the American Gastroenterological Association, May 1997, Washington, DC, and in abstract form (Gastroenterology 112: A711, 1997.)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: J. Behar, Ambulatory Patient Center 421, Division of Gastroenterology, Brown University School of Medicine, 593 Eddy St., Providence, RI 02903.
Received 13 January 1998; accepted in final form 1 April 1998.
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