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-induced contraction of cat esophageal and
lower esophageal sphincter circular smooth muscle
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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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Lower esophageal sphincter (LES)
tone depends on PGF2
and thromboxane A2
acting on receptors linked to Gi3 and Gq to activate phospholipases and produce second messengers resulting in
muscle contraction. We therefore examined PGF2 signal transduction in circular smooth muscle cells isolated by enzymatic digestion from cat esophagus (Eso) and LES. In Eso,
PGF2-induced contraction was inhibited by antibodies
against the 
-subunit of G13 and the monomeric G proteins
RhoA and ADP-ribosylation factor (ARF)1 and by the C3 exoenzyme of
Clostridium botulinum. A [35S]GTP
S-binding
assay confirmed that G13, RhoA, and ARF1 were activated by
PGF2. Contraction of Eso was reduced by propranolol, a
phospholipase D (PLD) pathway inhibitor and by chelerythrine, a PKC
inhibitor. In LES, PGF2-induced contraction was
inhibited by antibodies against the -subunit of Gq and
Gi3, and a [35S]GTPS-binding assay
confirmed that Gq and Gi3 were activated by
PGF2. PGF2-induced contraction of LES was
reduced by U-73122 and D609 and unaffected by propranolol. At low
PGF2 concentration, contraction was blocked by
chelerythrine, whereas at high concentration, contraction was blocked
by chelerythrine and CGS9343B. Thus, in Eso, PGF2
activates a PLD- and protein kinase C (PKC)-dependent pathway through
G13, RhoA, and ARF1. In LES, PGF2 receptors
are coupled to Gq and Gi3, activating phosphatidylinositol- and phosphatidylcholine-specific phospholipase C. At low concentrations, PGF2 activates PKC. At high
concentration, it activates both a PKC- and a calmodulin-dependent pathway.
smooth muscle contraction; prostaglandins; G proteins; phospholipases
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE SIGNAL-TRANSDUCTION
PATHWAYS mediating the esophagus (Eso) and lower esophageal
sphincter (LES) contraction in response to ACh have been previously
described (43) in our laboratory. ACh-induced
contraction of cat Eso is mediated by muscarinic M2 receptors coupled
to pertussis toxin-sensitive Gi3-type G protein activation
of phosphatidylcholine-specific phospholipase C (PI-PLC) and D
(PI-PLD), resulting in the production of diacylglycerol (DAG) and
activation of a protein kinase C (PKC)
-dependent pathway.
In contrast, two distinct contractile signal-transduction pathways are present in LES muscle cells. A PI-PLC, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], 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, resulting in the formation of Ins(1,4,5)P3 and DAG. Ins(1,4,5)P3 causes the release of Ca2+ from intracellular stores, producing a calcium-calmodulin complex, myosin light chain phosphorylation, and contraction (3). This pathway is PKC independent, because maximal activation of calmodulin inhibits PKC activity (3, 24, 49).
A distinct PKC-dependent pathway is activated by submaximal doses of ACh or during maintenance of LES tone (3, 20). In this pathway, contraction is mediated by low levels of PLC activity, resulting in low levels of Ins(1,4,5)P3, which cause the release of low levels of Ca2+ from intracellular stores. These low Ca2+ levels are insufficient to activate a calmodulin-dependent contraction, which requires micromolar Ca2+ concentrations (3). In addition, concurrent activity of a phosphatidylcholine-specific (PC-PLC) in the LES contributes to the production of DAG. Low levels of Ins(1,4,5)P3 act synergistically with DAG to activate a PKC-dependent pathway (20). Thus the amount of Ca2+ available for contraction determines which pathway will be followed, with low Ca2+ levels activating a PKC-dependent pathway and high levels activating a calmodulin-dependent pathway.
In addition, LES tone may be maintained by a low molecular mass (14 kDa) group I-like secreted phospholipase A (PLA)2, which produces arachidonic acid (AA) and AA metabolites, such as
PGF2 and thromboxanes A2 and B2, which maintain
activation of the G proteins coupled to PC-PLC and PI-PLC
(11).
In the current study, we found that in LES, PGF2-induced
contraction was coupled to the heterotrimeric G proteins Gq and Gi3 and linked to activation of PI-PLC and PC-PLC.
Contraction of LES in response to threshold PGF2 was
mediated by PKC activation, and, at high doses, by both PKC and
calmodulin activation. In the Eso, the signal-transduction pathway
mediating PGF2a-induced contraction differs from
ACh-stimulated signaling. PGF2-induced contraction of
Eso was coupled to the heterotrimeric G protein G13 and the
monomeric G proteins RhoA and ADP-ribosylation factor (ARF)1 and linked
to activation of PLD. In this respect, PGF2-induced contraction activates a signaling pathway similar to the one activated by prolonged (10 min) exposure to CCK in circular smooth muscle cells
from the rabbit small intestine (33). In the Eso, however, the physiological significance of prolonged contraction is uncertain, because the Eso contracts only briefly in response to swallowing.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals and preparation of circular smooth muscle tissue.
Adult male cats weighing between 3.5 and 5.5 kg were initially
anesthetized with ketamine (Aveco, Fort Dodge IA), then euthanized with
an overdose of pentobarbital sodium (Schering, Kennilworth, NJ). The
chest and abdomen were opened with a midline incision exposing the Eso
and stomach. The Eso and LES were isolated and excised as previously
described (4, 10). The Eso and LES circular muscle layers
were cut into muscle strips (10 mm long × 2 mm wide) with razor
blades held in a metal block 2 mm apart. This circular smooth muscle
tissue was used for GTPS binding studies and Western blot analysis
and to obtain isolated smooth muscle cells.
Cell isolation and permeabilazation. Circular smooth muscle tissue (1-2 mm wide) was digested in HEPES-buffered solution to obtain isolated smooth muscle cells, as described previously (4, 10). The collagenase solution contained 0.5 mg/ml collagenase Sigma type F, 1 mg/ml papain, 1 mg/ml bovine serum albumin, 1 mM 1,4-dithiothreitol (DTT), 1 mM CaCl2, 0.25 mM EDTA, 10 mM glucose, 10 mM HEPES (sodium salt), 4 mM KCl, 125 mM NaCl, 1 mM MgCl2, and 10 mM taurine. The solution was oxygenated (100% O2) at 31°C and pH adjusted to 7.2. The tissue was put into cold enzyme solution and kept in a refrigerator overnight. The following day, the tissue was brought to room temperature for 30 min and incubated in a water bath at 31°C for an additional 30 min. 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 was poured over a 200-µm Nitex mesh (Tetko, Elmsford, NY), rinsed in collagenase-free HEPES-buffered solution to remove any trace of collagenase, and incubated in this solution at 31°C and gassed with 100% O2. Collagenase-free HEPES-buffered solution (pH 7.4) contained (in mM) 112.5 NaCl, 5.5 KCl, 2.0 KH2PO4, 10.8 glucose, 24.0 HEPES (sodium salt), 1.9 CaCl2, and 0.6 MgCl2, with BME amino acid supplement, 0.3 mg/ml, and 0.08 mg/ml soybean trypsin inhibitor. Gentle trituration was used to release single cells. All the glassware used in this procedure was siliconized with a 0.05% silicon solution (Sigma, St. Louis, MO) to prevent the cells from adhering to the glass.
Cells were permeabilized, when necessary, to allow the use of G protein antibodies, which do not diffuse across the intact cell membrane. After completion of the enzymatic phase of the digestion process, the partly digested muscle tissue was washed with a cytosolic buffer of the following composition (in mM): 20 NaCl, 25 NaHCO3, 100 KCl, 5.0 MgSO4, and 0.96 NaH2PO4, with 2% bovine serum albumin. The cytosolic buffer also contained 0.61 mM CaCl2 and 1.0 mM EGTA, yielding ~0.36 µM free Ca2+ (16). Sohn et al. (41) previously showed that the maximal contractile response of permeabilized esophageal muscle cells to ACh requires the presence of 0.36 µM Ca2+ in the cytosolic buffer. The cytosolic buffer was equilibrated with 95% O2-5% CO2 to maintain a pH of 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 saponin (75 µg/ml). After exposure to saponin, the cell suspension was centrifuged at 200 g and the resulting pellet was resuspended in a saponin-free modified cytosolic buffer. The modified cytosolic buffer (pH 7.2) contained (in mM) 20 NaCl, 25 NaHCO3, 100 KCl, 5.0 MgSO4, 0.96 NaH2PO4, 0.61 CaCl2, 1.0 EGTA, and 1.5 ATP, with antimycin A, 10 µM, 2% bovine serum albumin, and an ATP-regenerating system consisting of 5 mM creatine phosphate and 10 U/ml creatine phosphokinase. After the cells were washed free of saponin, they were resuspended in the modified cytosolic buffer.Agonist-induced contraction of isolated muscle cells.
The cells were contracted by exposure to PGF2 for
30 s. In addition, LES and Eso circular smooth muscle cells were
contracted with a maximally effective concentration of
PGF2 (10
8 M) in the absence or presence of
PC-PLC inhibitor D609 (104 M), PLD inhibitor propanolol
(104 M), PI-PLC inhibitor U-73122 (106 M),
PKC inhibitor chelerythrine (105 M), or calmodulin
inhibitor CGS9343B (105 M). Propranolol and D609 are used
at high concentration to be effective and thus may be not entirely
specific. The efficacy and selectivity of these inhibitors in Eso and
LES, however, have been previously demonstrated (9, 20).
When C3 was used, permeabilized LES and Eso cells were incubated in the
exoenzyme at the indicated concentration for 2 h before the
addition of PGF2 (108 M). When G protein
antibodies were used, permeabilized LES and Eso cells were incubated in
the antiserum at a 1:200 dilution for 1 h before the addition of
PGF2 (108 M).
Cell measurements. Thirty consecutive cells from each slide were observed through a phase-contrast microscope (Carl Zeiss, Thornwood, NY) and a CCTV camera (model WV-CD51; Panasonic, Secaucus, NJ) was connected to a Macintosh computer (Apple, Cupertino, CA). The Image software program [National Institutes of Health (NIH), Bethesda, MD] was used to acquire images and measure cell length. The average length of 30 cells, measured in the absence of agonists, was taken as the "control" length and compared with length measured after addition of test agents. Shortening was defined as percent decrease in average length after agonists compared with the control length.
[35S]GTPS binding experiments.
Circular smooth muscle squares were homogenized in chilled buffer
containing (in mM) 20 HEPES (sodium salt; pH 7.4), 2 MgCl2, 1 EDTA, and 2 DTT. Homogenization consisted of 2- to 10-s
bursts with a tissue tearer (Biospec, Racine, WI) followed by
40-60 strokes with a Dounce tissue grinder (Wheaton, Melville,
NJ). Samples were centrifuged at 40,000 rpm for 30 min at
4oC (80Ti Rotor, Beckman Ultracentrifuge, Palo Alto, CA).
The pellet was resuspended in solubilizing buffer and homogenized in a
Dounce tissue grinder (20 strokes). The solubilizing buffer contained (in mM) 20 HEPES (sodium salt; pH 7.4), 240 NaCl, 2 EDTA, and 2 phenylmethylsulfonyl fluoride, with 20 µg/ml aprotinin, 20 µM leupeptin, and 1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Samples remained in the solubilizing buffer for 1 h at 4oC.
S binding was assayed with the method of
Murthy et al. (32). The crude membranes (2.5 mg
protein/ml) were incubated at 37°C with 30 nM
[35S]GTPS in a solution containing (in mM) 10 HEPES
(sodium salt; pH 7.4), 0.1 EDTA, and 10 MgCl2 for 1 min.
The stimulation of binding was assayed in the presence or absence of a
maximally effective concentration of PGF2
(106M), with a total volume of 300 µl. The reaction was
stopped with 10 vol of chilled 100 mM Tris · HCl (pH 8.0)
containing 10 mM MgCl2, 100 mM NaCl, and 20 µM GTP. The mixtures (200 µl) were added to enzyme-linked immunosorbent assay wells that had
been coated initially with a rabbit or goat immunoglobulin antibody (1:1000, 2 h) and subsequently coated with specific G protein antibodies (1:1,000, 2 h). After 2-h incubation on ice, the wells were washed three times with phosphate buffer solution containing 0.05% polyoxyethylenesorbitan monolaurate (Tween 20). The
radioactivity from each well was counted using a Tri-Carb 1900 CA
liquid-scintillation analyzer (Packard Instruments, Meriden, CT).
Triplicate measurements were carried out for each experiment. Data were
expressed as percent increase from basal levels.
Western blot.
For RhoA and G13 purification, LES and Eso circular
muscle squares were homogenized in chilled buffer containing (in mM) 20 HEPES (sodium salt; pH 7.4), 2 MgCl2, 1 EDTA, and 2 mM DTT.
Homogenization consisted of 2- to 10-s bursts with a tissue tearer
(Biospec) followed by 40-60 strokes with a Dounce tissue grinder
(Wheaton). Samples were centrifuged at 40,000 rpm for 30 min at
4oC (80Ti Rotor, Beckman Ultracentrifuge). The pellet was
resuspended in solubilizing buffer and homogenized in a Dounce tissue
grinder (20 strokes). The solubilizing buffer contained (in mM) 20 HEPES (sodium salt; pH 7.4), 240 NaCl, 2 EDTA, and 2 phenylmethylsulfonyl fluoride, with 20 µg/ml aprotinin, 20 µM
leupeptin, and 1% CHAPS. Samples remained in the solubilizing buffer
for 1 h at 4oC.
13, or ARF antibody (1:500) for 1 h with shaking
followed by three washes with antibody-free buffer. This was followed
by a 60-min incubation in horseradish peroxidase (HRP)-conjugated goat
anti-rabbit antibody (Amersham, Arlington Heights, IL) for RhoA and
G13 and in HRP-labeled protein A for ARF1. Detection was
achieved with an enhanced chemiluminescence agent (Amersham). Molecular
weight was estimated by comparison of sample bands with prestained
molecular weight marker (Amersham).
Drugs and chemicals.
G protein antibodies (Gq, Go,
Gi1-Gi2, Gi3) were purchased from
Calbiochem-Novabiochem International (San Diego, CA);
G13, RhoA, and ARF1 were from Santa Cruz Biotechnology
(Santa Cruz, CA); [35S]GTPS was from New England
Nuclear (Boston, MA); and soybean trypsin inhibitor was from
Worthington Biochemicals (Freehold, NJ). Collagenase type F, papain,
saponin, BME amino acid supplement, EGTA, HEPES, creatine phosphate,
creatine phosphokinase, ATP, antimycin A, and other reagents were
purchased from Sigma.
Statistical analysis. Data are expressed as means ± SE. Statistical differences between means were determined by Student's t-test. Differences between multiple groups were tested using ANOVA for repeated measures and checked for significance using the Sheffé's F-test.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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G proteins in PGF2-induced contraction.
PGF2 induced a concentration-dependent contraction of
LES and Eso smooth muscle cells (P < 0.01, ANOVA; Fig.
1). Maximal response occurred at a
108 M concentration, producing 23 ± 1% shortening
in both Eso and LES circular smooth muscle.
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-induced contraction, we used antibodies raised
against synthetic peptides corresponding to the amino acid sequence of
the COOH-terminal of the -subunit of heterotrimeric G proteins.
Table 1 shows that
PGF2-induced contraction of LES cells was significantly inhibited by Gq and Gi3 antibodies
(P < 0.001, ANOVA) and not by Gi1/i2 or
Go antibodies. These data have been previously published (11) and are shown here for the readers' convenience.
PGF2-induced contraction of Eso muscle cells, however,
was unaffected by the same antibodies (against the -subunit of
Gi3, Gq, Gi1/i2, or Go
G proteins; Table 1). PGF2-induced contraction of Eso muscle cells was reduced in a concentration-dependent manner by G13 antibodies (Fig. 2;
P < 0.001, ANOVA), which did not affect LES
contraction. In permeabilized Eso cells, G13 antibodies (5 µg/ml) reduced shortening from 22.4 ± 0.2 to 0.4 ± 2%.
These data suggest that PGF2-induced contraction may be
mediated by activation of Gq and Gi3 in LES and
by G13 in Eso smooth muscle.
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-induced contraction of Eso
and LES is mediated by monomeric G proteins such as RhoA. C3 is an exoenzyme of Clostridium botulinum, which has been shown to
ADP ribosylate and inhibit the Rho-p21 family of monomeric G proteins. Permeabilized cells were incubated in C3 for 2 h before exposure to a maximally effective dose of PGF2 (108
M). C3 produced a concentration-dependent decrease in
PGF2-induced contraction of Eso cells (P < 0.001, ANOVA) but had no effect on LES smooth muscle cells (Fig.
3). Shortening of permeabilized Eso cells
was reduced from 22.3 ± 0.3% in control cells to 6.8 ± 1.1% in cells incubated in C3 (250 ng/ml). In addition,
PGF2 (108 M)-induced contraction of
permeabilized Eso cells was reduced by 1-h exposure to antibodies
against ARF1 and RhoA (P < 0.001, ANOVA). ARF1 and
RhoA antibodies, used in combination, reduced shortening from 22.4 ± 0.2 to 4.0 ± 1.2% (Fig.
4A). The inhibition of
PGF2-induced contraction by ARF1 was additive to that elicited by RhoA, suggesting that these two monomeric G proteins mediate contraction via different contractile signaling pathways. To
determine whether G13 G proteins are linked to RhoA and/or ARF1, permeabilized Eso cells were incubated for 1 h in buffer containing G13 antibodies in combination with ARF (Fig.
4B) or RhoA (Fig. 4C) antibodies. Figure
4B shows that the inhibition of PGF2-induced
contraction by G13 antibodies was additive to that elicited
by ARF1 antibodies, suggesting that G13 activity is not
linked to ARF1. However, G13 antibodies in combination with
RhoA antibodies did not cause a greater inhibition of contraction than
either antibody used alone, suggesting that G13 may be
linked to RhoA and that these two G proteins may mediate contraction by
activation of the same signal-transduction pathway.
PGF2-induced contraction of LES was unaffected by ARF1
and RhoA antibodies (Fig. 5).
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-induced contraction of Eso
may be mediated by monomeric G proteins, whereas LES contraction is
mediated by the trimeric G proteins Gq and Gi3,
with no contribution of ARF or RhoA.
To confirm this hypothesis, we examined G protein activation in
response to PGF2 by measuring [35S]GTPS
binding to PGF2-activated smooth muscle membranes. Figure 6 shows that in Eso muscle
membranes, PGF2 (106 M) caused significant
activation of RhoA, ARF1, and G13. After PGF2 stimulation, RhoA, ARF1, and G13
binding increased 24.6 ± 2.0, 25.7 ± 3.5, and 32.5 ± 8.0%, respectively. In LES, exposure to PGF2
(106 M) caused significant activation of Gq
and Gi3 but not of RhoA, ARF1, and G13 (Fig.
7). After PGF2
stimulation, Gq and Gi3 binding increased
25.9 ± 5.3 and 16.5 ± 3.9%, respectively. For LES muscle,
the data on PGF2-induced activation of Gi3,
Gq, Gi1/i2, or Go have been
previously reported (11) and are shown here for the
readers convenience. [35S]GTPS binding data are in
agreement with the inhibition of PGF2-induced contraction by selective antibodies for both LES and Eso muscle.
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-induced contraction in
Eso are present in Eso circular smooth muscle. In Eso circular smooth
muscle, RhoA (21 kDa), ARF1 (21 kDa), and G13 (45 kDa) were
detected by Western blot as thick bands (Fig.
8).
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Phospholipases involved in PGF2-induced contraction.
In Eso, PGF2-induced contraction was significantly
reduced by the PLD pathway inhibitor propranolol but was unaffected by
the PI-PLC inhibitor U-73122 (106 M) or the PC-PLC
inhibitor D609 (104 M; Fig.
9). Shortening of Eso cells was reduced
from 22.9 ± 2.6% in controls to 7.2 ± 1.3% after
treatment with propranolol. In LES, PGF2-induced
contraction was significantly reduced by U-73122 and D609
(P = 0.01, ANOVA) and was unaffected by propranolol (Fig. 10). U-73122 and D609 reduced
shortening of LES cells from 21.4 ± 0.4% in controls to 9.0 ± 1.6 and 16.2 ± 0.3%, respectively. These data suggest that
PGF2-induced contraction is mediated by PI-PLC and
PC-PLC in the LES and by PLD in Eso.
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PKC and calmodulin in PGF2-induced contraction.
In Eso smooth muscle cells, contraction in response to a maximally
effective concentration of PGF2 was significantly reduced by the PKC inhibitor chelerythrine (105 M;
P = 0.01, ANOVA) but not by the calmodulin inhibitor
CGS9343B (105 M; Fig.
11). Percent shortening of Eso cells
was reduced from 22.9 ± 2.6 to 8.3 ± 0.9% after treatment
with chelerythrine.
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(1011 M) was selectively reduced by chelerythrine
(P = 0.001, ANOVA). The response to a maximally
effective dose of PGF2 (108 M) was
significantly reduced by both CGS9343B and chelerythrine (P = 0.01, ANOVA; Fig.
12).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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LES tone may be maintained by a low molecular mass (14 kDa)-secreted PLA2, which produces AA and results in formation of AA metabolites, such as PGF2 and thromboxanes A2 and B2,
and activation of Gq and Gi3 G proteins
(11). Because PGF2 may thus play
an important physiological role, we examined the associated
signal-transduction pathway in LES and Eso circular muscle.
PGF2-induced contraction of LES.
In LES circular muscle, Gq is linked to PI-PLC, and
Gi3 is linked to PC-PLC, which are the phospholipases
responsible for maintenance of LES tone (3, 20,
43). Gq and Gi3 are the same G
proteins that are spontaneously active (bound to GTP in the absence of
exogenous neurotransmitter) and that are activated by
PGF2 and linked to LES tone (11). In the
present study, we showed that in LES, PGF2-induced
contraction, similar to contraction induced by other agonists, is
mediated by PI-PLC (42, 43) and by PC-PLC, because the
PI-PLC antagonist U-73122 and the PC-PLC antagonist D609 reduce
PGF2-induced LES contraction. These data, which are
entirely consistent with the previous data, support the view that
PGF2, through PI-PLC and PC-PLC activation, plays a role
in the maintenance of LES tone.
(1011 M) was significantly reduced by
chelerythrine, whereas a maximally effective concentration of
PGF2 (108 M) was significantly reduced by
chelerythrine and CGS9343B, suggesting the activation of a
PKC-dependent pathway at low PGF2 concentration and of
both a calmodulin- and a PKC- dependent pathway at higher doses of
PGF2.
This concentration-related shift in PGF2-induced
contraction from a PKC-dependent to a calmodulin-dependent pathway is similar to the shift observed with ACh-induced contraction of LES
(3) and may depend on the different calcium requirements of the PKC- or calmodulin-dependent contractile pathways. Biancani et
al. (3) previously showed that in LES calmodulin-mediated contraction requires higher cytosolic Ca2+ levels than
PKC-dependent contraction. At low calcium concentration, a
PKC-dependent pathway is activated. When cytosolic calcium reaches a
level sufficient to activate calmodulin, however, as may occur with a
maximally effective concentration of ACh, LES contraction is mediated
by a calmodulin-dependent pathway (3) and the
PKC-dependent pathway is inhibited (3, 12, 13, 24, 49).
Calmodulin-induced inhibition of PKC activity may be localized to the
second and fourth calcium binding domains of calmodulin
(24).
With PGF2-induced contraction, the concentration-related
shift of PKC dependence toward calmodulin dependence is not as clear
cut as with ACh, because at the maximally effective concentration of
PGF2, contraction is inhibited by both PKC and
calmodulin inhibitors. Our data suggest that low concentrations of
PGF2 activate the same PKC-dependent pathway activated
by low concentrations of ACh. However, high concentrations of
PGF2 may not be as effective (in causing
Ca2+ release, full activation of calmodulin, and complete
inhibition of PKC) as high doses of ACh, and, therefore, a partial
activation of a PKC-dependent pathway may still be present.
PGF2-activated G proteins in Eso contraction.
In Eso circular smooth muscle, ACh-induced contraction is nearly
abolished by antibodies directed against the -subunit of Gi3 (43). PGF2, however, was
not linked to activation of the heterotrimeric G proteins
Gi3, Gq, Gi1/i2, or Go,
as measured by [35S]GTPS binding, and
PGF2-induced contraction was not affected by
Gi3, Gq, Gi1/i2, or Go antibodies.
-induced
contraction. G13 is a member of the G12-G13 family of heterotrimeric G proteins
that was first identified by the DNA cloning of its -subunit.
G13 is expressed in most cell lines and tissues and is
especially abundant in human platelets (28).
PGF2 stimulated G13 activation, as measured
by [35S]GTPS binding, and G13 antibodies
reduced PGF2-induced contraction of Eso, supporting the
view that PGF2-induced contraction is mediated by
G13.
Because G13 is linked to monomeric G proteins, such as Rho
and possibly ARF, we tested Rho as a possible mediator in the
PGF2-G13-dependent contractile pathway in
Eso. Small G proteins of the Rho family are thought to regulate various
cell functions in which the actomyosin system is involved, including
cell morphology, membrane ruffling, cell motility, cell aggregation,
and smooth muscle contraction (34, 45). Ten different
mammalian Rho GTPases have been identified including Rho (A,B,C
isoforms) Rac (1,2,3 isoforms), Cdc42 (Cdc42Hs, G25K isoforms),
Rnd1/Rho6, Rnd2/Rho7, Rnd3/RhoE, RhoD, RhoG, and TC10
(38). In smooth muscle, the Rho p21 and smg
p21/rap1 p21 families are perhaps the most abundant of the monomeric or
small G proteins, and RhoA p21 may be involved in GTPS-induced
contraction of permeabilized vascular smooth muscle (21).
To study the role of RhoA in PGF2-induced contraction,
we examined the effect of the C3 exoenzyme of C. botulinum,
which has been shown to ADP ribosylate and inhibit the Rho-p21 family of monomeric G proteins. C3 reduced PGF2-induced
contraction of Eso in a concentration-dependent manner but had no
effect on LES contraction, suggesting that monomeric G proteins mediate PGF2-induced contraction of Eso smooth muscle but not of LES. These data are similar to those reported in vascular smooth muscle
from the mesenteric artery (21), in which contraction was
abolished by C3 and by an exoenzyme of Staphilococcus aureus called EDIN, which also ADP rabosylates and inhibits the Rho-p21 family. In addition, RhoA antibodies inhibited
PGF2-induced contraction of Eso and PGF2
caused activation of RhoA, as measured by [35S]GTPS binding.
In Eso, G13 may be linked to RhoA, because antibodies
against G13 in combination with RhoA antibodies did not
cause a greater inhibition of contraction than either G protein used
alone, suggesting that these two G proteins mediate contraction by
activation of the same signal-transduction pathway.
ARF is a second G protein, which, similar to RhoA, is a member of the
Ras superfamily of monomeric 20- to 30-kDa GTP-binding proteins. ARFs
are 20-kDa ADP-ribosylation factors that were originally recognized and
purified because of their ability to stimulate the
ADP-ribosyltransferase activity of the cholera toxin A subunit (30). Mammalian ARFs are divided into three classes based
on size, amino acid sequence, gene structure, and phylogenic analysis: ARF1 and ARF2 are in class I; ARF4 and ARF5 are in class II; and ARF6
is in class 6 (31).
In Eso, PGF2-induced contraction may be mediated by
ARF1, because PGF2 significantly activates ARF1 and
antibodies directed against ARF1 G proteins significantly reduced
contraction. The contractile pathway mediated by ARF1 may be separate
from the G13-RhoA-linked pathway because the inhibition of
contraction by ARF1 antibodies was additive to the inhibition by
G13 and RhoA.
PGF2-activated phospholipases in Eso.
Cao et al. (9) and Sohn and co-workers (42,
43) previously demonstrated that ACh-, substance P-, and
bombesin-induced contraction of Eso depend on the production of DAG by
PC-PLC and PLD, because the PC-PLC antagonist D609 and the PLD pathway
inhibitor propranolol reduce DAG production and agonist-induced contraction.
-induced contraction of Eso
was reduced by propranolol, suggesting that phospholipid metabolism by
PLD may be the main signaling pathway mediating contraction of
esophageal circular muscle in response to PGF2.
PLD hydrolyses membrane phospholipids to produce phosphatidic acid and
the free polar head group of the phospholipid substrate. Phosphatidic
acid is then dephosphorylated by a phosphatase to yield DAG
(14). High concentrations of propranolol (0.1-1 mM) have been shown to reduce DAG production by inhibiting the phosphatidic acid phosphatase (5, 37).
Our data suggest that in the Eso, PGF2-induced
contraction is mediated by G13, ARF, and RhoA G proteins,
which activate PLD. This view is consistent with numerous reports
indicating that intracellular PLD activators include the Rho (6,
26, 33, 39) and ARF (7, 8, 33) families of
monomeric G proteins and the trimeric G protein G13
(33, 35). PLD interacts with specific amino acid residues
in the activation loop (switch I) region of Rho (1) and
interacts with the NH2-terminal 73 amino acids of ARF
(48).
Rho stimulates PLD activity in the presence of GTPS in membranes
from rat liver, neutrophils, and HL60 cells (6, 27, 39).
Studies with constitutively active and dominant negative forms of RhoA
indicate that RhoA controls PLD activity in intact cells and mediates
the effect of various agonists (19, 35).
ARF stimulation of PLD activity has been detected in plasma membranes
(29, 36, 47), nuclei (2, 36), Golgi
(25, 36, 47), and cytosol (36, 39). ARF has
been reported to mediate M2 muscarinic receptor activation of PLD in
human embryonic kidney cells. The activation of PLD by RhoA can be
amplified synergistically by ARF (39, 40). In addition,
G13 has been reported to activate rat brain PLD in a
RhoA-dependent fashion (35).
Recent reports indicate that G13 may link receptor-mediated
activation of monomeric G proteins to activation of PLD
(33). G13 is capable of directly activating a
guanine nucleotide exchange factor (GEF) for Rho (18, 23),
which facilitates the exchange of GDP for GTP (22, 46).
GTPases must be in their active (GTP or GTPS ligand) form to
stimulate their effectors such as PLD. Members of the regulators of the
G protein signaling (RGS) family stimulate the intrinsic GTPase
activity of the -subunit of certain heterotrimeric G proteins. One
Rho-specific GEF, p115 RhoGEF, serves as a direct link between Rho
GTPases and heterotrimeric G proteins, because it has an
NH2-terminal region that contains a domain characteristic
of RGS proteins and specifically stimulates the GTPase activity of the
-subunit of G13 (23). Therefore, one
pathway for the hormonal stimulation of PLD via G protein-coupled receptors may be through G13 and its direct regulation of
the exchange activity of p115 Rho GEF for Rho proteins. For example, CCK-stimulated PLD activity of intestinal smooth muscle may be mediated
by G13-dependent RhoA (33). Our data support a role of PLD
activation by the monomeric G proteins RhoA and ARF1 and by the
heterotrimeric G13. It is possible that
PGF2-induced contraction of Eso is mediated by
activation of one or more of these G proteins that can work alone or
cooperatively to stimulate the activity of PLD. Our data suggest that
two signal-transduction pathways mediate PGF2-induced
contraction of Eso: one a G13-RhoA-linked pathway, and a
second pathway mediated by activation of ARF1. Activation of PLD
results in the production of DAG, which stimulates PKC and
PKC-dependent contraction of the Eso.
PKC and calmodulin in PGF2-induced contraction in
Eso.
Eso contraction induced by a maximally effective dose of
PGF2 (108 M) was inhibited by the PKC
antagonist chelerythrine and not affected by the calmodulin antagonist
CGS9343B, as occurs with other agonists such as ACh, substance P, and
bombesin (42).
activates G13. G13 may then regulate the
exchange activity of GDP/GTP exchange proteins, causing GTP binding to
RhoA (and possibly ARF). These monomeric G proteins, which, in their
GDP-bound state, are inactive and cytosolic, translocate to the
membrane and may act separately or cooperatively to activate PLD and a
PKC-dependent pathway.
In LES, PGF2 receptors are coupled to
Gq and Gi3 G proteins, which activate PI- and
PC-PLC. At low concentrations, PGF2 activates a
PKC-dependent pathway and, at maximally effective concentration,
activates both a PKC- and a calmodulin-dependent pathway.
| |
ACKNOWLEDGEMENTS |
|---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-28614.
| |
FOOTNOTES |
|---|
These data were presented in part at the 1999 meeting of the American Gastroenterological Association.
Address for reprint requests and other correspondence: P. Biancani, Gastrointestinal Motor Function Research Laboratory, SWP5 Rhode Island Hospital and Brown University, 593 Eddy St., Providence RI 02903 (Piero_Biancani{at}brown.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00357.2001
Received 9 August 2001; accepted in final form 18 February 2002.
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TOP
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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P = 0.01, ANOVA) in Eso
muscle membranes. Values are means ± SE of 4 cats, with each
sample performed in triplicate.
