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Vol. 283, Issue 3, 1293-1304, 1997
Department of Pharmacology, Chung Ang University Pharmacy College, Seoul 156-756, Korea (U.D.S.), Department of Medicine, Rhode Island Hospital and Brown University School of Medicine, Providence, Rhode Island (K.M.H., W.C., J.B., P.B), Department of Medicine, VA Medical Center and Brown University School of Medicine, Providence, Rhode Island (H.R.) and Kangnam General Hospital, Public Corporation, Seoul, 135-090, Korea (N.K.)
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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In single cells, isolated by enzymatic digestion from the circular
muscle layer of the lower esophageal sphincter (LES), acute experimental esophagitis (AE) alters signal transduction in response to
a maximally effective dose of acetylcholine. In normal LES contraction
was inhibited by M3 
M1 or M2
antagonists. In AE inhibition by M2 antagonists increased
significantly so that contraction was inhibited by M3 > M2 > M1 antagonists. In normal cells
permeabilized by saponin, contraction was antagonized by antibodies
against Gq/11, by the phosphatidylinositol-specific
phospholipase C (PI-PLC) antagonist U 73122, but not by the
phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609,
or by the phospholipase D pathway inhibitor propranolol. In AE
contraction was reduced by Gq/11 and Gi3
antibodies and by U73122, propranolol and D609. After thapsigargin
treatment of normal cells to reduce intracellular Ca++
stores, contraction was inhibited by M2 and M3
antagonists, by antibodies against Gq/11 and
Gi3, by U73122, D609 and propranolol, suggesting that
depletion of Ca++ stores reproduces the changes induced by
AE. We conclude that in normal LES smooth muscle cells
acetylcholine-induced contraction is mediated by M3
receptors linked to Gq/11 and PI-PLC, whereas in AE,
contraction through this pathway is reduced, perhaps because of
reduction in Ca++ stores, and a second pathway is activated
by M2 receptors linked to Gi3, PC-PLC and
phospholipase D.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Two
distinct contractile signal transduction pathways are present in LES
muscle cells. A PI-PLC, 1,4,5-IP3,
calmodulin-dependent pathway is activated by stimulation with a
maximally effective dose of ACh. In this pathway
M3 muscarinic receptors, linked to Gq/11-type G-proteins, stimulate PLC, which
results in the formation of 1,4,5-IP3 and DAG.
1,4,5-IP3 causes the release of
Ca++ from intracellular stores producing a
calcium-calmodulin complex, myosin light chain phosphorylation and
contraction (Biancani et al., 1994
). This pathway
is PKC-independent, because activated calmodulin may inhibit PKC
activity (Biancani et al., 1994; Chakravarthy et
al., 1995a, b; Kraft and Anderson, 1983; Yu et al.,
1993; Zhao et al., 1991).
A second PKC-dependent pathway is activated by submaximal doses of ACh
or during maintenance of LES tone. In this pathway, submaximal doses of
ACh or spontaneous tone are linked to low levels of PLC activity,
resulting in low levels of 1,4,5-IP3 which causes
the release of low levels of Ca++ from
intracellular stores. These low Ca++ levels are
insufficient to activate calmodulin but can act synergistically with
DAG to activate PKC (Biancani et al., 1994). Thus the amount of Ca++ available for contraction determines
which pathway will be followed, with low Ca++
levels activating a PKC-dependent pathway, and high levels activating a
calmodulin-dependent pathway.
In a model of AE, repeated perfusion of the esophagus with 0.1 N hydrochloric acid causes a reduction in resting in
vivo LES pressure, in vitro spontaneous tone and levels
of 1,4,5-IP3 (Biancani et al., 1984).
These data suggest that AE affects spontaneous production of
1,4,5-IP3 and intracellular
Ca++ stores (Rich et al., 1997). In
addition, AE causes a shift in the intracellular pathway mediating the
response to a maximally effective dose of ACh from a
calmodulin-dependent to a PKC-dependent pathway.
In the present study we show that the shift may be related to the amount of Ca++ that is present in and releasable from intracellular stores. After AE, or depletion of Ca++ stores by thapsigargin, a PKC-dependent pathway is activated, which depends on different receptors, G-proteins and effector enzymes localized to the membrane of isolated smooth muscle cells of the LES.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Animals. Experimental procedures were approved by the animal welfare committee of Rhode Island Hospital. Adult cats of either sex, weighing 3 to 5 kg, were used in this study. After an overnight fast, they were anesthetized with ketamine (10 mg/kg), and maintenance doses of ketamine (2.5-5 mg/kg) were administered as needed. To determine LES position, esophageal pressure was measured by a repeated station pull-through technique, 1 to 2 mm at the time, with a multilumen catheter having three proximal openings 3 cm apart and a distal perfused sleeve. The sleeve device was used to measure LES pressure, whereas the three proximal openings measured amplitude of contraction in the body of the esophagus. With the sleeve placed across the LES, the most proximal opening, which was 9 cm proximal to the LES, was used to perfuse acid in the esophagus when needed.
In each experiment two different groups of animals were examined: the first group consisted of normal animals, whereas animals from the second group (esophagitis animals), after initial measurement of LES pressure, had their esophagus perfused with 0.1 N HCl at a rate of 1 ml/min for 45 min during 3 consecutive days. These animals were placed on a slant board at a 30° angle during the perfusion to avoid aspiration. This protocol has been shown to produce inflammatory changes in the esophageal mucosa and submucosa. Our previous histologic studies with this model of experimental esophagitis show no evidence of inflammatory infiltrate in the circular muscle layer, which appears normal under light microscopy, and under electron microscopy exhibits changes (enlargement of mitochondria and slight disruption of endoplasmic reticulum) characteristic of an early inflammatory phase (Biancani et al., 1994). There is a concurrent reduction in
the LES in vivo resting pressure and in vitro
spontaneous tone, whereas esophageal perfusion with distilled water had
no effect on mucosal appearance or LES resting pressure (Biancani
et al., 1984; Eastwood et al., 1976).
Tissue preparation.
The animals were anesthetized with
sodium pentobarbital (Nembutal, Abbott Laboratories, N. Chicago, IL)
(50 mg/kg) and the chest and abdomen were opened with a midline
incision, exposing the stomach, esophagus and esophagogastric junction.
A suture was placed on the esophagogastric junction, and a second
suture was placed on the esophageal body, 5 cm proximal to it. Stomach, esophagogastric junction and esophagus were removed together and pinned
on a wax block, with the distance between the sutures held at 5 cm.
This insured that the specimen was stretched to its in vivo
length. The esophagus and stomach were opened along the lesser curvature. The location of the squamocolumnar junction was identified, and the mucosa and submucosa were removed by sharp dissection under
microscope. The high-pressure zone of the LES is characterized by a
visible thickening of the circular muscle layer at the squamocolumnar junction, and immediately proximal to the sling fibers of the stomach.
We previously showed that a 5- to 8-mm band of tissue coinciding with
the thickened area constitutes the LES and has distinct characteristics
when examined in vivo in the organ bath, or as single cells
after enzymatic digestion (Biancani et al., 1982,1987).
After surgically isolating the LES, it was placed, serosal side down,
in a Stadie Riggs tissue slicer (Thomas Scientific Apparatus,
Philadelphia, PA) and cut into 0.5-mm-thick slices. The last slices
containing the myenteric plexus, longitudinal muscle and serosa were
discarded. This method provided approximately 600 to 800 mg of
relatively pure circular smooth muscle of LES. The slices of smooth
muscle were placed flat on a wax surface and tissue squares were made
by cutting twice with a 2-mm blade block, the second cut at right
angles to the first. LES circular smooth muscle squares were used for
measurement of PKC activity and 1,4,5-IP3
formation, or further digested to isolated single cells for
contractility studies.
Cell dispersion.
Isolated smooth muscle cells were obtained
by enzymatic digestion, as described previously (Biancani et
al., 1987). LES smooth muscle squares were digested in
HEPES-buffered physiologic solution, containing collagenase 150 U/ml
(CLS type II, Worthington Biochemicals, Freehold, NJ) for 2 h. The
HEPES solution contained: NaCl, 114.7 mM; KCl, 5.7 mM;
KH2PO4, 2.1 mM; glucose, 11 mM; HEPES, 24.5 mM; CaCl2, 1.9 mM;
MgCl2, 0.57 mM, BME amino acid supplement (M.A. Bioproducts, Walkersville MD) 0.3 mg/ml, and soybean trypsin inhibitor (Worthington Biochemicals, Freehold, NJ) 0.08 mg/ml. The HEPES solution
was oxygenated (100% O2) at 31°C, and the pH
was adjusted to 7.4. During the digestion period the gas flow rate was
kept low to avoid agitating the tissue. At the end of the digestion period, the tissue and digestion medium were poured out over 500-µm Nitex mesh (Tetko, Inc., Elmsford, NY). The tissue on the mesh was
rinsed with 150 ml of collagenase-free HEPES solution to remove any
trace of collagenase and then incubated in collagenase-free HEPES
solution at 31°C. The cells were allowed to dissociate freely in the
collagenase-free solution for 10 to 20 min. Care was taken not to
agitate the fluid to avoid cell contraction in response to mechanical
stress.
Permeabilization of single cells.
Isolated LES smooth muscle
cells were permeabilized, when required, to allow the use of G-protein
antibodies, which usually do not diffuse across the intact plasma
membrane, or to control the intracellular calcium concentration. After
completion of the enzymatic phase of the digestion process, the partly
digested muscle tissue was washed with a "cytosolic" enzyme-free
physiologic salt solution (cytosolic buffer) of the following
composition (mM): NaCl, 20; KCl, 100; MgSO4, 5.0;
NaH2PO4, 0.96; EGTA, 1.0; and CaCl2, 0.48. The cytosolic buffer contained
2% bovine serum albumin and was equilibrated with 95%
O2/5% CO2 to maintain a pH
of 7.2 at 31°C. Muscle cells dispersed spontaneously in this medium.
Permeabilization was accomplished by incubation of the freely dispersed
cells for 3 min in cytosolic buffer containing saponin (75 µg/ml).
When this incubation was complete, the cell suspension was spun at
500 × g and the pellet resuspended in saponin-free modified cytosolic buffer containing antimycin A (10 µM), ATP (1.5 mM) and an ATP-regenerating system consisting of creatine phosphate (5 mM) and creatine phosphokinase (10 U/ml) (Bitar et al.,
1986). The procedure was repeated twice to ensure complete removal of
saponin. After the cells were washed free of saponin, they were
resuspended in the modified cytosolic buffer.
Experimental procedure.
Once the cells had dissociated,
0.5-ml aliquots of the cell-containing fluid were added to tubes for
exposure to agonists and measurement of contraction. Cells were exposed
to a maximally effective dose of ACh
(10
10-109 M)
for 30 s (Hillemeier et al., 1991). When antagonists
were used, cells were incubated in appropriate concentration of the antagonist for 1 min before the addition of ACh. When G-protein antibodies were used, the permeable cells were incubated in the antiserum at a 1:200 dilution for 1 h before the addition of ACh (Bitar et al., 1991). To assess the effect of
thapsigargin-induced reduction in intracellular
Ca++ stores, cells were preincubated in
106 M thapsigargin for 30 min before the
addition of ACh and/or antagonists.
Cell measurements. Thirty consecutive cells from each slide were observed through a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany), and a CCTV camera (model WV-CD51, Panasonic, Secaucus, NJ) connected to a Macintosh IICi Computer (Apple, Inc., Cupertino, CA). The Image 1.33 software program (NIH, Bethesda, MD) was used to measure cell length and for data accumulation. The average length of 30 cells, measured in the absence of agonists, was taken as "control" length. In addition, average cell length was measured after addition of test agents. Control cells underwent the same manipulations as stimulated cells. Shortening was defined as the percent decrease in average length after agonists, when compared with control length.
Measurement of PKC activity.
Measurement of PKC activity in
the cytosolic and membrane fractions was performed by colorimetric
assay. The cytosolic and membrane fraction of LES circular smooth
muscle were prepared from normal cats and from cats after the induction
of AE as follows. LES smooth muscle squares (600-800 mg) obtained from
one cat were equilibrated in 400 µl Krebs' solution and gassed with
95% O2-5% CO2 at 37°C
for 20 min. For measurement of agonist-stimulated PKC activity LES
smooth muscle squares were divided into 150-mg aliquots. One was used
for PKC measurement of a control (untreated) sample, a second was
exposed to ACh (107 M) and a third was
exposed to ACh (105 M); then PKC activity
was measured. ACh (105 M) was previously
determined to produce maximal contraction in LES smooth muscle strips.
After 30 s the reaction was stopped with 10 volumes of ice-cold
Krebs' solution. Muscle squares were collected and homogenized in 20 mM Tris buffer, pH 7.4, containing: EDTA, 0.5 M; EGTA, 0.5 M;
leupeptin, 10 mg/ml; aprotinin, 10 mg/ml; and 
-mercaptoethanol, 10 mM. Homogenization consisted of 2- to 10-s bursts with a Tissue Tearer
(Biospec, Racine, WI) followed by 40 to 60 strokes with a Dounce tissue
grinder (Wheaton, Melville, NJ). Samples were centrifuged at
100,000 × g for 40 min at 4°C (50 Ti rotor, Beckman
Ultracentrifuge, Palo Alto, CA). The supernatant was collected, and
after the addition of 30 µl of phenylmethylsulfonyl fluoride (10 mg/ml) and a 30-min incubation, it was used as the cytosolic fraction.
The pellet was resuspended in 3 ml of homogenizing buffer containing
0.1% Triton X-100 and rehomogenized by 20 strokes with a Dounce tissue
grinder. The resuspended sample was mixed well by means of a tube
rotator for 30 to 40 min at 4°C, and centrifuged at 100,000 × g for 50 min at 4°C. The supernatant of this
centrifugation was collected as the membrane fraction.
II
isozyme (Sohn et al., 1997). Five micrograms of antibodies raised against PKCII were added to 400 µl of cytosolic or membrane fraction, and incubated for 1 h at 4°C. Forty microliters of
protein A/G PLUS-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added, and samples were incubated at 4°C with rocking. After 1 h
samples were microcentrifuged (Microcentrifuge, Fisher Scientific, Pittsburgh, PA) for 15 to 20 s at 4°C. The pellet was suspended in 20 µl RIFA buffer and solubilized. The supernatant was removed, and the pellet was suspended in 20 µl RIFA buffer containing: KH2PO4, 1 mM;
Na2HPO4, 10 mM; NaCl, 137 mM; KCl, 2.7 mM; 1% tergitol, 0.5% sodium deoxycholate, 0.1% SDS, pH
7.4 and solubilized. Ten microliters of solubilized sample was used in
the PKC colorimetric assay.
PKC activity of immunoprecipitated smooth muscle of the LES was
measured by the Pierce Colorimetric PKC Assay No. 29510 (Pierce, Rockford, IL). A peptide substrate, labeled with a brightly colored fluorescent dye, was incubated with the kinase-containing sample. The
reaction mixture was applied to an affinity column that binds phosphorylated peptides. The phosphorylated product was eluted from the
column and quantitated by measurement of its absorbance at 570 nm.
Measurements of inositol phosphates.
The method for the
measurement of inositol phosphates, by HPLC has been described
previously (Biancani et al., 1992; Szewczak et
al., 1990). LES circular smooth muscle squares were incubated for
4.5 h (37°C) in 1.5 ml Krebs' solution containing 60 µCi/ml myo-(2-3H)inositol and gassed with 95%
O2-5% CO2. After
incubation, the smooth muscle squares were poured out over 360-µ
Nitex mesh (Tetko Inc, Elmsford NY) and rinsed with 50 ml of Krebs'
solution to remove excess [2-3H]inositol.
The muscle squares were evenly divided into the experimental tubes
containing 0.5 ml of gassed Krebs' solution at 37°C and allowed to
equilibrate for 30 min at 37°C. Krebs' solution (control; 0.5 ml) or
Krebs' solution containing 105 M ACh was
then added and the experiment stopped at the appropriate time points by
the addition of 0.6 ml of chloroform/methanol/HCl (100:50:1) and 25 µl phytate (100 mg/ml) (Hughes et al., 1988). Samples were
collected at zero time and at 1 and 5 s. Zero time levels were
obtained by adding the vehicle only (0.5 ml of Krebs' solution) and
immediately quenching the tissue. The aqueous, cytosolic phase from
each sample was separated by centrifugation and 0.5 ml was removed. The
remaining aqueous phase was washed twice with ice-cold water and a
total 1.5 ml of aqueous extract was collected and neutralized to pH 6.5 to 7.5 with 0.5 N NaOH. The extract was stored at 
70°C for later
analysis. The remaining tissue-containing aqueous and organic phases
were stored at 70°C for protein determination.
; Szewczak et al., 1990). After 2.5 min of water, the concentration of
ammonium formate was increased to 3% for 21 min, 23% for 9 min, 50%
for 9 min followed by the final gradient increase to 100% ammonium formate for 15 min.
Radioactivity was determined by continuous flow liquid scintillation
counting with a FLO-ONE/Beta Radiochromatography Detector Series A-200
(Radiomatic Instruments and Chemicals Co., Inc., Tampa, FL) with
FLO-SCINT IV scintillator (Packard Instrument Company, Inc., Downer's
Grove, IL). The data were converted to dpm with use of the counting
efficiency of an
[2-3H]1,4,5-IP3 standard
(Amersham, Arlington Heights, IL). This efficiency averaged 37.4% and
was constant throughout the elution gradient.
1-IP1, 1,4-IP2 and
1,4,5-IP3 were identified by use of standard
[2-3H]inositol phosphate metabolites. In all
cases, the inositol phosphate metabolites eluted as sharp peaks. The
data were expressed as dpm/mg protein.
Protein determination.
Protein content was obtained after
hydrolysis by 0.1 N NaOH at 80°C to solubilize the protein, followed
by neutralization with HC1. The amount of protein present was
determined by colorimetric analysis (BioRad Protein Assay; Bio Rad
Laboratories, Richmond CA) according to the method of Bradford (1976).
Drugs and chemicals.
G-protein antibodies
(Gq/11, Gi3,
Go, Go-i3,
Gi1-2) raised against synthetic peptides
corresponding to the amino acid sequence of the COOH-terminal of the
G-protein 
subunits were purchased from New England Nuclear (Boston,
MA); methoctramine HCl, pirenzepine 2 HCl and pF-HSD from Research
Biochemical Inc.(Natick, MA); collagenase type II and soybean trypsin
inhibitor from Worthington Biochemicals (Freehold, NJ); D609 from
Kamiya Biochemical Co (Thousand Oaks, CA); H-7
(1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) from
Seikagaku America Inc. (St. Petersburg FL) and U73122 from Biomol
(Plymouth Meeting, PA). Myo-[2-3H]inositol and
[2-3H]inositol phosphate metabolites were
purchased from Amersham Corp.(Arlington Heights, IL). CGS9343B was a
gracious gift of Dr. M. Crettaz from Zyma SA, NYON, Switzerland.
Statistical analysis. Data are expressed as the mean ± S.E. Statistical differences between multiple groups were tested by analysis of the variance (ANOVA) for repeated measures and checked for significance with use of the Scheffé's F-test.
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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|---|
AE causes a switch in the intracellular contractile pathway
mediating ACh-induced contraction.
We examined PKC translocation
from the cytosol to the membrane in response to stimulation of LES
circular smooth muscle squares by ACh. Translocation of the enzyme from
the cytosol to the membrane fraction is thought to be a measure of PKC
activation (Khalil and Morgan, 1991; Kraft and Anderson, 1983;
Nishizuka, 1986, 1989). Figure
1A shows that in normal
LES tissue PKC activity (measured by membrane/cytosolic PKC activity
ratio) increases significantly at first (ANOVA, P < .05), then
decreases when a maximally effective concentration
(105 M) of ACh is used. In contrast, after
induction of AE, PKC activity increases dose-dependently with ACh to a
maximum 3-fold increase at 105 M ACh
(ANOVA, P < .001). Thus it is likely that, at least at maximally
effective ACh concentration (105 M), PKC
may mediate contraction in AE but not in normal LES.
|
). To test whether stimulated 1,4,5-IP3
levels are similarly reduced, we measured inositol phosphate production
in LES circular smooth muscle squares in response to ACh with HPLC. One
and five seconds after the administration of ACh,
1-IP1, 1,4-IP2 and
1,4,5-IP3 increased significantly in the normal
LES (ANOVA, P < .05); but not in AE muscle (fig. 1B). After 1 and
5 s exposure to ACh, 1,4,5-IP3 increased
59.4% and 82.8%, respectively, above the unstimulated value in normal
LES. However, 1-IP1,
1,4-IP2 and 1,4,5-IP3
levels of AE LES did not increase with maximal ACh stimulation. This finding supports the view that ACh-induced stimulation is associated with a significant increase in inositol phosphate production in normal
LES, but not in AE.
The data presented in figure 1 suggest that, in the normal and AE LES
muscle, different intracellular pathways may be activated in response
to ACh. In the normal LES, ACh causes production of 1,4,5-IP3; in contrast in AE, ACh causes PKC
activation. The data are consistent with previous findings (Rich
et al., 1997) that contraction induced by a maximally
effective dose of ACh is mediated by a calmodulin-dependent pathway in
normal LES and a PKC-dependent pathway after induction of AE. We
therefore investigated whether the transduction mechanisms localized to
the membrane, such as the receptors, G-proteins and effector enzymes,
are affected by the induction of AE.
Different muscarinic receptors mediate ACh-induced contraction in
normal LES cells and AE cells.
Figure
2 shows the effect of the
M1, M2,
M3 antagonists pirenzepine, methoctramine and
pF-HSD, respectively, on the contraction induced by a maximally
effective dose of ACh (109 M) in normal
LES smooth muscle cells and in AE cells. It should be noted that for
single cell experiments the maximally effective ACh concentration is
lower than when intact tissue is used. Cells were exposed for 1 min to
106 M antagonist before contraction with
ACh. Figure 2 shows in normal smooth muscle cells ACh-induced
contraction was reduced by all antagonists, but the
M3 antagonist pF-HSD caused the largest
inhibition, reducing contraction by two thirds (ANOVA, ***P < .001). Table 1 shows that % inhibition
of contraction after methoctramine pretreatment was significantly
greater in AE than in normal LES (unpaired t test, P < .05). After induction of AE the M3 antagonist still caused a two-thirds reduction in contraction (fig. 2, ANOVA, ***P < .001).
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Different G-proteins mediate ACh-induced contraction in normal LES
cells and AE cells.
Because AE affects the receptors
responsible for ACh-induced contraction, it is reasonable to expect
that the G-proteins linked to these receptors may be similarly
affected. To identify the specific G-proteins, we used G-protein
antibodies raised against synthetic peptides corresponding to the amino
acid sequence of the COOH-terminal of the G-protein subunit. The
cells were permeabilized by brief exposure to saponin to allow
diffusion of the antibodies into the cytoplasm, as described previously
(Sohn et al., 1993, 1995).
|
Different phospholipases mediate ACh-induced contraction in normal
and AE LES cells.
The difference in G-proteins activated in normal
and AE LES cells may cause differences in the phospholipases activated
in response to maximally effective ACh. To test this hypothesis, cells
from normal cats and from cats with esophagitis were contracted with
ACh (109 M) in the presence of the
phospholipase inhibitors, U73122 (106 M),
D609 (104 M) and propranolol
(104 M). U73122 inhibits PI-PLC (Bleasdale
et al., 1989), D609 inhibits PC-PLC (Schutze et
al., 1992) and propranolol in high concentrations inhibits
phosphatidic acid phosphohydrolase, preventing formation of DAG through
a PLD-mediated pathway (Billah et al., 1989; Qian and
Drewes, 1990).
|
M3 receptors are linked to Gq/11 and PI-PLC; M2 receptors are linked to Gi3 and PC-PLC and PLD. After induction of AE, contraction induced by a maximally effective dose of ACh is mediated by M2 and M3 receptors, activating Gq/11, Gi3 and three phospholipases, PI-PLC, PC-PLC and PLD. To test whether two separate pathways exist, permeabilized smooth cells from esophagitis cats were incubated in a maximally effective concentration of antibodies raised against either Gq/11 or Go-i3 for 1 h before contraction with ACh. In addition to the antibodies, cells were exposed to muscarinic or phospholipase antagonists 1 min before ACh administration.
Figure 5A shows that in the presence of a maximally effective concentration of Gq/11 antibodies (Sohn et al., 1993), ACh-induced contraction was
not further reduced by the M3 antagonist pF-HSD or by the PI-PLC inhibitor U73122, which suggests that these
antagonists inhibit the same signal transduction pathway that is linked
to Gq/11. In contrast, the
M2 antagonist methoctramine, the PC-PLC inhibitor
D609 and the PLD pathway inhibitor propranolol caused additional
relaxation (ANOVA, P < .01), which suggests that the pathway
inhibited by these antagonists is not linked to
Gq/11.
|
Depletion of intracellular calcium stores in normal LES cells
mimics changes induced by AE.
To test whether a reduction in
intracellular calcium stores is sufficient to reproduce the changes in
signal transduction observed in AE, normal LES cells were exposed to
106 M thapsigargin (fig.
6). Thapsigargin causes
depletion of intracellular calcium stores by inhibiting ATP-dependent
calcium uptake.
|
), ACh-induced contraction of
thapsigargin-treated normal LES cells was not further reduced by the
M3 antagonist pF-HSD or by the PI-PLC inhibitor U73122, which suggests that these antagonists inhibit the same signal
transduction pathway that is linked to Gq/11. In
contrast, the M2 antagonist methoctramine (ANOVA,
P < .05), the PC-PLC inhibitor D609 (ANOVA, P < .01) and
the PLD pathway inhibitor propranolol (ANOVA, P < .01) caused
additional relaxation, which suggests that the pathway inhibited by
these antagonists is not linked to Gq/11.
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| |
Discussion |
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|
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
AE causes a switch in the intracellular contractile pathway
mediating ACh-induced contraction.
We have previously demonstrated
that ACh-induced contraction of LES smooth muscle cells can be mediated
by either calmodulin or PKC, according to the calcium requirements of
these two distinct intracellular pathways. When cytosolic levels of
calcium are low, as during the maintenance of spontaneous tone, LES
smooth muscle contraction is mediated via a PKC-dependent
pathway. When cytosolic calcium reaches a level sufficient to activate
calmodulin, as may be achieved with a maximally effective concentration
of ACh, LES smooth muscle contraction is mediated by a
calmodulin-dependent pathway (Biancani et al., 1994) and the
PKC-dependent pathway is inhibited (Biancani et al., 1994;
Chakravarthy et al., 1995a, b; Kraft and Anderson, 1983; Yu
et al., 1993; Zhao et al., 1991). The mechanism
of calmodulin-induced inhibition of PKC activity has not been
investigated extensively. Kruger et al. (1990) examined tryptic fragments of calmodulin and found that two PKC-inhibitory sequences where localized to the second and fourth calcium binding domains of calmodulin and that calmodulin-induced PKC inhibition was
not affected by calmodulin antagonists.
) and damages intracellular
calcium stores in the LES (Rich et al., 1997), we
hypothesized that maximal ACh-induced contraction may be mediated by a
PKC-dependent pathway after the induction of AE. We found that after
the induction of AE, ACh-induced contraction was inhibited by the PKC
antagonist H7 but not by the calmodulin antagonist CGS9343B, and these
changes were mimicked in normal cells by acutely depleting calcium
stores by exposure to thapsigargin (Rich et al., 1997).
Thapsigargin causes release of intracellular Ca++
and inhibits ATP-dependent Ca++ uptake, which
results in depletion of intracellular Ca++
stores. These data support the view that changes in signal transduction pathways may be related to the Ca++ levels
available to support contraction. In the present investigation, we
therefore examined in some detail the characteristics of the pathway
mediating contraction in response to a maximally effective dose of ACh
in AE. Because AE causes a switch from an
1,4,5-IP3- and calmodulin-dependent to a
PKC-dependent pathway, we measured PKC activity and
1,4,5-IP3 production in response to ACh
stimulation in normal and AE LES muscle.
To test the functional significance of PKC in normal and AE LES, we
examined PKC translocation from cytosol to membrane in response to ACh
stimulation of intact muscle squares. Activation of PKC is associated
with the translocation of the enzyme from the cytosol to the membrane
fraction (Khalil and Morgan, 1991; Kraft and Anderson, 1983; Nishizuka,
1986). The dose of ACh (105 M) used was
previously shown to produce a maximal contraction in LES smooth muscle
strips (Biancani et al., 1987) and is different from the
maximally effective concentration of ACh used in studies of isolated
smooth muscle cells.
We found that in normal LES muscle tissue ACh-induced PKC activation
was maximal at an intermediate ACh concentration and that a maximally
effective dose of ACh, which causes contraction through a
calmodulin-dependent pathway, causes little increase in PKC activity
when compared with unstimulated muscle. In contrast, after induction of
AE, PKC activity increased dose-dependently with increasing ACh
concentrations and was 3-fold greater than control values in response
to maximal ACh. These results are consistent with calmodulin-induced
inhibition of PKC.
To determine whether 1,4,5-IP3 plays a role in
ACh-induced contraction in AE, we examined formation of inositol
phosphates, in response to a maximally effective dose of ACh. Rapid
breakdown of phosphatidylinositol 4,5-bisphosphate by PI-PLC results in the formation of 1,4,5-IP3, which is metabolized
to 1,4-IP2 and 1-IP1.
1,4-IP2 and 1-IP1 are also
produced by PI-PLC-induced hydrolysis of phosphatidylinositol
4-phosphate (Berridge, 1987; Berridge and Irvine, 1984; Michell, 1975;
Murthy and Makhlouf, 1991, 1995). In our system, as reported previously
(Sohn et al., 1993), 1-IP1, 1,4-IP2 and 1,4,5-IP3 were
increased by ACh stimulation in normal LES muscle at 1 and 5 s
after administration of ACh, i.e., in a time frame
consistent with contraction induced by ACh (Hillemeier et
al., 1991). In contrast, after AE none of the inositol phosphates were increased by ACh stimulation in LES muscle. These data support the
view that 1,4,5-IP3 plays a role in ACh-induced
contraction of LES muscle in normal but not in AE animals. Because
1,4,5-IP3-induced calcium release is thought to
activate a calmodulin-myosin light chain kinase pathway these data also
support our previous findings that contraction of normal LES, but not
of AE LES, is affected by the calmodulin antagonist CGS9343B (Rich
et al., 1997).
Because AE causes a switch from an 1,4,5-IP3- and
calmodulin-dependent to a PKC-dependent pathway we examined the
transduction mechanisms in LES smooth muscle cells. We found that AE
produced a change in the muscarinic receptors, G-proteins and
phospholipases activated by stimulation with a maximally effective dose
of ACh.
Different muscarinic receptors mediate ACh-induced contraction in
normal LES cells and AE cells.
The pharmacologic classification of
muscarinic receptors is based on different receptor affinities for
selective antagonists. In the present investigation we examined the
effect of pirenzepine, methoctramine and pF-HSD on ACh-induced
contraction. Pirenzepine is a tricyclic drug with higher selectivity
for M1 relative to M2 or
M3 muscarinic receptors (Caulfield, 1993; Dorje
et al., 1991). Methoctramine is the prototype of the
polymethylene tetramine class of muscarinic receptor antagonists which
have been reported to display selectivity toward cardiac
M2 muscarinic receptors (Giraldo et
al., 1988; Melchiorre, 1988; Melchiorre et al., 1987a, b). pF-HSD is an hexahydro-sila-difenidol analog which has been shown
to possess considerable selectivity for the smooth muscle M3 muscarinic receptor in guinea pig ileum
(Lambrecht et al., 1988, 1989).
).
In the present study we show (table 1) that inhibition of contraction
by pirenzepine in normal and AE LES, and by methoctramine in normal LES
were relatively low and not different form each other. This inhibition
may be related to nonselective binding of antagonists to muscarinic
receptors.
Methoctramine-induced inhibition was significantly greater in AE than
in normal LES (unpaired t test, P < .05), and pF-HSD caused the greatest inhibition in normal and esophagitis animals.
These data suggest that when intracellular Ca++
storage or release mechanisms are damaged by AE, ACh-induced
contraction is mediated not only thorough M3, as
in the normal LES, but also by M2 receptors.
Different G-proteins mediate ACh-induced contraction in normal LES
and AE cells.
G-proteins transduce ligand binding to a cell
surface receptor into intracellular signals. Antibodies, raised against
synthetic peptides corresponding to the amino acid sequence of the
COOH-terminal of specific G-protein subunits, have been used as
effective probes of G-protein structure and function (Goldsmith
et al., 1988; Gutowski et al., 1991; Shenker
et al., 1991; Simonds et al., 1989; Sohn et
al., 1993; Spiegel et al., 1990). We used these antibodies to identify the G-proteins coupled to the muscarinic receptors in LES circular muscle.
). In normal
LES smooth muscle only Gq/11 antibodies inhibited
ACh-induced contraction, which suggests that M3
receptors are coupled to G-proteins of the Gq/11 type (Sohn et al., 1993). After induction of AE, ACh-induced
contraction was inhibited by both M2 and
M3 receptors and by antibodies raised against
both the Gq/11- and the
Gi3-type G-protein. This suggests that in
addition to a M3-Gq/11
coupled pathway, a second
M2/Gi3-dependent pathway is
activated after induction of AE.
Different phospholipases mediate ACh-induced contraction in normal
LES and AE cells.
We have previously shown that in normal LES,
contraction in response to a maximally effective dose of ACh results
from PI-PLC activation, because it is associated with an increase in
inositol phosphates and is inhibited by the PI-PLC inhibitor U-73122
(106 M) (Biancani et al., 1994;
Sohn et al., 1993). U-73122 is a amphipathic cation which
reversibly competes with calcium for the binding site on PLC that
regulates expression of the phospholipase activity (Bleasdale et
al., 1989), and is capable of reducing spontaneously elevated
1,4,5-IP3 levels in LES circular muscle (Biancani
et al., 1994) without affecting contraction of normal
esophageal smooth muscle cells, which is PKC-dependent and mediated by
PC-PLC, PLD and PLA2 (Sohn et al.,
1993,1994a,b, 1995). In the current study, we show that after induction
of AE, LES contraction is inhibited not only by U73122, but also by
D609 and propranolol, which suggests that other phospholipases mediate
ACh-induced contraction of LES besides PI-PLC.
). Propranolol at high concentrations is thought to
inhibit PLD-mediated production of DAG from phosphatidylcholine (Billah
and Anthes, 1990; Exton, 1990; Lassegue et al., 1991; Welsh
et al., 1990). Phosphatidylcholine hydrolysis by PC-specific PLC and PLD has been shown to be an alternative source of DAG (Billah
and Anthes, 1990; Exton, 1990; Lassegue et al., 1991; Welsh
et al., 1990). PC-specific PLC produces DAG and
phosphocholine, whereas PC-specific PLD produces choline and
phosphatidic acid, which is metabolized to DAG by phosphatidic acid
phosphohydrolase (Billah and Anthes, 1990; Dennis et al.,
1991). D609 blocks PC-specific PLC activity derived from Bacillus
cereus, without affecting PLA2, PLD and
PIP2-specific PLC activity (Schutze et
al., 1992). High concentrations (0.1-1 mM) of propranolol have
been shown to reduce DAG production by inhibition of phosphatidic acid
phosphohydrolase without affecting phosphatidylcholine-specific PLD
activity or PI-PLC activity (Billah et al., 1989; Qian and
Drewes, 1990, 1991).
ACh-induced contraction of LES after the induction of AE was
antagonized by D609 and by the phosphatidic acid phosphohydrolase inhibitor propranolol, which had no effect on contraction of normal LES
muscle. This finding is consistent with the view that after the
induction of AE LES contraction may be mediated by activation of
PC-specific PLC and PLD, resulting in hydrolysis of PC rather than of
PI.
We conclude that after induction of AE, contraction induced by a
maximally effective dose of ACh is mediated by M2
and M3 receptors, activating
Gq/11, Gi3 and the
phospholipases PI-PLC, PC-PLC and PLD.
M3 receptors are linked to Gq/11 and PI-PLC; M2 receptors are linked to Gi3 and PI-PLC and PLD. To test whether the multiple muscarinic receptors, G-proteins and phospholipases are organized in distinct pathways, we examined the effect of muscarinic antagonists and phospholipase inhibitors in the presence of a maximally effective dose of selective G-protein antibodies. Permeabilized smooth cells from esophagitis cats were incubated in either Gq/11 or Go-i3 antibodies, then exposed to muscarinic or phospholipase antagonists before ACh administration. The antibodies were used at a maximally effective concentration to maximally reduce the pathway activated by a specific G-protein, before using additional inhibitors. When the pathway blocked by a selective G-protein antibody is maximally inhibited, addition of muscarinic receptor antagonists or phospholipase inhibitors can cause additional inhibition only when linked to a pathway different from the one inhibited by the G-protein antibody.
We found that, in the presence of Gq/11 antibodies, no additional inhibition was produced by either pF-HSD or by U73122, which suggests that Gq/11-type G-proteins participated in the pathway containing M3 muscarinic receptors and PI-PLC. In contrast, methoctramine, propranolol and D609 caused additional and almost complete inhibition in the presence of Gq/11 antibodies, which suggests that the pathway inhibited by these agents was not coupled Gq/11. Conversely, in the presence of Go-i3 antibodies, no additional inhibition was produced by either methoctramine, propranolol and D609, which suggests that Gi3-type G-proteins participated in the pathway containing M2 muscarinic receptors, PC-PLC and PLD. In contrast, pF-HSD and U73122 caused additional and almost complete inhibition in the presence of Go-i3 antibodies, which suggests that the pathway inhibited by these agents was not coupled Gi3. The results of the current study demonstrate that, in contrast to the normal LES in which ACh, at a maximally effective dose, activates only M3, Gq/11 and PI-PLC, after induction of esophagitis, a second pathway is activated. This pathway, which is similar to the one that has been previously reported for the esophageal body (Sohn et al., 1993), consists of
M2 muscarinic receptors linked to
Gi3-type G-proteins and, PC-PLC and PLD-type
phospholipases.
Depletion of intracellular calcium stores in normal LES cells
mimics changes induced by AE.
We have previously reported that AE
reduces resting 1,4,5-IP3 levels and
intracellular Ca++ stores (Rich et
al., 1997). In the current study, we demonstrate that a reduction
in the intracellular calcium stores of normal LES smooth muscle cells
by 30 min incubation in thapsigargin is sufficient to reproduce the
changes in signal transduction observed in AE. We show that ACh-induced
contraction of thapsigargin-treated normal LES cells is mediated by
M2 and M3 receptors,
activating Gq/11, Gi3 and
the phospholipases PI-PLC, PC-PLC and PLD and these muscarinic
receptors, G-proteins and phospholipases are organized in distinct
pathways. After pharmacologic depletion of intracellular Ca++ stores with thapsigargin, ACh-induced
contraction of LES smooth muscle cells is mediated not only by
M3 muscarinic receptors linked to
Gq/11-type G-proteins and PI-PLC, but also by
M2 muscarinic receptors linked to
Gi3-type G-proteins and PC-PLC and PLD-type phospholipases, as occurs after induction of AE.
, 1994a, b,
1995). In addition, U73122 inhibited spontaneous tone and
1,4,5-IP3 formation in normal LES muscle
(Biancani et al., 1994; Hillemeier et al.,
1996). These findings demonstrate that in our system U73177 is
selective enough to inhibit PI-PLC in LES muscle without affecting
PC-PLC, PLD or PLA2 in the esophageal muscle. In
the present study, we show that inhibiting Gq/11,
which is known to be linked to PI-PLC, has the same effect as
inhibiting PI-PLC, and the antibodies used to inhibit
Gq/11 are reasonably selective (Sohn et
al., 1995; Sohn et al., 1993). Therefore, the fact that
inhibition of PI-PLC by U73122, and inhibition of
Gq/11 by the antibodies significantly reduces LES
contraction in response to ACh, suggests that PI-PLC contributes to
ACh-induced contraction.
An alternative hypothesis that may be considered is that the inositol
phospholipids, which are the substrates for PI-PLC and constitute only
a small percent of membrane phospholipids (Billah and Anthes, 1990),
may be depleted as a consequence of AE. Numerous phospholipids are
present in cell membranes, and in the absence of inositol
phospholipids, it is possible that other substrates may be hydrolyzed
by one or more of the almost 20 PI-PLC isozymes discovered to date.
This hypothesis is not entirely consistent with previous
characterizations of PI-PLCs, however, which have been reasonably
selective for inositol phospholipids (Exton, 1996; Lee and Rhee, 1995;
Späth et al., 1991). We can only speculate that there
may be a U73122-sensitive phospholipase, which in some circumstances
may hydrolyze non-inositol-containing membrane phospholipids.
We conclude that in normal LES cells contraction is
calmodulin-dependent and mediated by M3 receptors
linked to Gq/11 and PI-PLC, whereas in AE,
contraction through this pathway is reduced because
1,4,5-IP3 production and
Ca++ stores are damaged. In AE a second
PKC-dependent pathway is activated, which is mediated by
M2 receptors linked to Gi3,
PC-PLC and PLD.
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Footnotes |
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Accepted for publication August 1, 1997.
Received for publication March 14, 1997.
1 Supported by NIH grant DK 28614, and KOSEF hacsim 961-0704-042-2.
Send reprint requests to: P. Biancani, G.I. Motility Research Lab., SWP5, Rhode Island Hospital & Brown University, 593 Eddy Street, Providence RI 02903.
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Abbreviations |
|---|
ACh, acetylcholine;
AE, acute experimental
esophagitis;
ANOVA, analysis of the variance;
ATP, adenosine
triphosphate disodium salt;
DAG, diacylglycerol;
EGTA, ethylene
glycol-bis(-amino ethyl ether)N,N,N
,N-tetraacetic acid;
G protein, guanine nucleotide-binding protein;
Gq/11, Gi3,
Go, Go-i3, Gi1-2 antibody:
antibody raised against terminal peptide to respective subfamily of G
proteins subunit ;
HEPES, lN-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid);
HPLC, high-performance liquid chromatography;
1-IP1, inositol
1-monophosphate;
1, 4-IP2, inositol 1,4-bisphosphate;
1, 4,5-IP3, inositol 1,4,5-trisphosphate;
LES, lower
esophageal sphincter;
PKC, protein kinase C;
PI-PLC, phosphatidylinositol-specific phospholipase C;
PC-PLC, phosphatidylcholine-specific phospholipase C;
PLD, phospholipase D;
PLA2, phospholipase A2;
pF-HSD, p-fluoro-hexahydro-sila-difenidol.
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References |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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