Group I secreted PLA2 and
arachidonic acid metabolites in the maintenance of cat LES
tone
W. B.
Cao1,
K. M.
Harnett1,
Q.
Chen1,
M. K.
Jain2,
J.
Behar1, and
P.
Biancani1
1 Department of Medicine, Rhode
Island Hospital and Brown University, Providence, Rhode Island
02902; and 2 Department of
Chemistry and Biochemistry, University of Delaware, Newark,
Delaware 19716
 |
ABSTRACT |
Spontaneous tone of in vitro lower esophageal
sphincter (LES) circular muscle is associated with elevated levels of
arachidonic acid (AA), PGF2
,
and increased
[35S]guanosine
5'-O-(3-thiotriphosphate)
(GTP
S) binding to Gq-, Gi3-, and
Gi1/i2-like G proteins. Tone and
AA levels were reduced by inhibitors of a pancreatic-like (group I)
secreted phospholipase A2
(sPLA2), by the cyclooxygenase
inhibitor indomethacin, and by the thromboxane
A2 antagonist SQ-29548. In
addition, pertussis toxin (PTX) reduced LES tone, confirming a role of
PTX-sensitive G proteins in maintenance of LES tone.
PGF2
contracted LES smooth
muscle (strips and cells) and increased
[35S]GTP
S binding
to Gq and
Gi3 in solubilized LES circular
muscle membranes. PGF2
-induced
contraction of LES permeable muscle cells was inhibited by
Gq and
Gi3 but not by
Gi1/i2 and
Go antibodies. The thromboxane
A2 analog U-46619 contracted LES
smooth muscle and increased Gq
binding. U-46619-induced contraction was inhibited by
Gq but not by
Gi3,
Gi1/i2, and
Go antibodies. LES tone and [35S]GTP
S binding
were significantly reduced by indomethacin. We conclude that group I
sPLA2 may mediate
"spontaneous" LES tone by producing AA, which is metabolized to
PGF2
and thromboxane A2. These AA metabolites activate
receptors linked to Gi3 and Gq to maintain LES contraction.
esophagus; lower esophageal sphincter; smooth muscle contraction; phospholipases; prostaglandins; thromboxanes
 |
INTRODUCTION |
THE LOWER ESOPHAGEAL SPHINCTER (LES) circular muscle is
a major determinant of LES tone. Although the relative neurogenic contribution may vary with the animal species, a significant component of tone is thought to be myogenic, as it is not affected by neural antagonists, including TTX (6, 16, 35). Functionally, this muscle is
specialized, with muscle strips from this region developing higher
total and active forces than esophageal strips (11, 16, 17). We have
previously reported that LES tone is maintained by the spontaneous,
low-level activities of phosphatidylinositol-specific phospholipase C
(PI-PLC), and phosphatidylcholine-specific phospholipase C (PC-PLC),
which produce threshold levels of the second messengers diacylglycerol
(DAG) and inositol 1,4,5-trisphosphate
(IP3), which act synergistically
to activate protein kinase C (PKC), and maintain a PKC-dependent basal
tone (8, 41). We now propose that activation of these phospholipases
may be maintained by a low-molecular-mass (14 kDa) group I secreted
phospholipase A2
(sPLA2), which produces arachidonic acid (AA), and AA metabolites, such as
PGF2
and thromboxanes
A2/B2,
which maintain activation of the G proteins coupled to PC-PLC and
PI-PLC (85, 86).
The PLA2 members are a growing
family of enzymes that catalyze the hydrolysis of glycerolphospholipids
at the sn-2 position, producing free
fatty acids and lysophospholipids (24, 25, 49) (Fig.
1). Mammalian
PLA2 enzymes function in the
digestion of dietary lipid, microbial degradation, and regulation of
phospholipid acyl turnover for membrane repair or for the production of
AA. AA is an important regulator of specific cellular processes,
including regulation of PKC and PLC-
, and modulation of calcium
transients. AA is also the precursor to biologically active lipids,
including prostaglandins, hydroxy fatty acids, leukotrienes,
thromboxanes, and platelet activation factor.

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Fig. 1.
Membrane phospholipids are acted on by phospholipases
A2
(PLA2), a family of enzymes that
catalyze the hydrolysis of glycerolphospholipids at the
sn-2 position, producing free fatty
acids and lysophospholipids (24, 25, 49).
PLA2 enzymes are divided into two
major classes: intracellular or cytosolic (high molecular mass,
80-85 kDa) (cPLA2) and
secretory or secreted (low molecular mass, ~14 kDa)
(sPLA2).
cPLA2 enzymes include the 85-kDa
calcium-sensitive cPLA2 and the
80-kDa calcium-insensitive cPLA2
(iPLA2).
sPLA2 enzymes are divided into
different groups (I, II, III, V, VII, VIII, IX) according to their
molecular structure and the localization of their disulfide bridges
(22, 40). Mammalian PLA2 enzymes
produce arachidonic acid (AA), an important regulator of specific
cellular processes and precursor to biologically active lipids,
including prostaglandins, leukotrienes, thromboxanes, and
prostacyclins. NDGA, nordihydroguaiaretic acid; PAF,
platelet-activating factor.
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|
The PLA2 family is divided into
two major classes (Fig. 1): intracellular or cytosolic [high
molecular mass (80-85 kDa)]
PLA2 (cPLA2) and secretory or
secreted [low molecular mass (~14 kDa)] PLA2
(sPLA2).
Some cPLA2 members have been
characterized, such as the 85-kDa calcium-sensitive
cPLA2 and the 80-kDa
calcium-insensitive cPLA2 (24). We
have previously shown that this calcium-sensitive cPLA2 participates in ACh-induced
contraction of the esophagus (87).
The sPLA2 members are divided into
different groups (i.e., I, II, III, V, VII, VIII, IX) according to
their molecular structure and the localization of their disulfide
bridges (23, 40). sPLA2 have an
absolute catalytic requirement for millimolar concentrations of calcium
and a broad specificity for phospholipids with different polar head
groups and fatty acyl chains. Many
sPLA2 function extracellularly, but some have also been localized within mitochondria (91, 95). sPLA2 have been purified from
mammalian sources, including pancreas, spleen, lung, platelets, and
extracellular fluid, and from bee and snake venom. Group I, II, and III
are represented by pancreatic, inflammatory, and bee venom
sPLA2. Group I
sPLA2 was originally identified in
pancreatic juice and then identified and cloned in tissues, including,
spleen, lung, ovary, and kidney (49, 58, 80, 93). It functions in lipid
digestion, cell proliferation, acute lung injury, and smooth muscle
contraction (3, 61, 89).
In this investigation, we examine the role of
sPLA2 in the maintenance of LES
tone. We find that AA production, through an sPLA2 of group I, participates in
maintenance of LES tone. Most likely AA is metabolized to
prostaglandins, such as PGF2
, and to thromboxanes (thromboxanes
A2 and/or
B2), which activate specific G
proteins that contribute to the maintenance of LES tone.
 |
METHODS |
Animals.
Adult male cats weighing between 3.5 and 5.5 kg were initially
anesthetized with ketamine (Aveco, Fort Dodge, IA) and then euthanized
with an overdose of phenobarbital (Schering, Kennilworth, NJ). The
chest and abdomen were opened with a midline incision exposing the
esophagus and stomach. The esophagus and LES were isolated and excised
as previously described (9, 11).
Measurements of in vitro LES tone.
LES strips (2 mm) were mounted in separate 1-ml muscle chambers and
equilibrated for 2 h with continuous perfusion of oxygenated physiological salt solution (PSS) as previously described in detail (7-11, 41). During this time, the tension in LES strips increased, attaining a steady level at 2 h. The PSS contained the following (in
mM): 116.6 NaCl, 21.9 NaHCO3, 1.2 NaH2PO4,
3.4 KCl, 2.5 CaCl2, 5.4 glucose,
and 1.2 MgCl2. The solution was
equilibrated with a gas mixture containing 95%
O2 and 5%
CO2 at pH 7.4 and 37°C.
After equilibration, LES strips were incubated for 30 min in solution
containing the appropriate concentrations of the cyclooxygenase inhibitors indomethacin and acetylsalicylic acid (aspirin), the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), the
PLA2 inhibitor 4-bromophenacyl
bromide (BPB), the cPLA2 inhibitor
AACOCF3, the
sPLA2 inhibitors MJ33, MJ45, AM5,
and X1, and the thromboxane A2
antagonist SQ-29548. In some studies, agents were added together with
TTX (10
4 M). When strips
were treated with TTX, electrical stimulation (square-wave pulses of
supramaximal voltage, 0.2 ms, 1 Hz, 10-s trains) was used to document
inhibition of neurotransmitter release, before addition of inhibitors.
Electrical stimulation was delivered by a stimulator (model S48, Grass
Instruments, Quincy, MA) through platinum wire electrodes placed
longitudinally on either side of the strip. In addition, LES strips
were incubated with different concentrations of pertussis toxin (PTX)
for 1 h and in the indicated concentrations of group I, II, III
sPLA2, thromboxane
B2, or
PGF2
for 15 min. Indomethacin
and SQ-29548 were dissolved in ethanol. The concentrations of ethanol
used in the concentration-response curves (0.005-0.05%) did not
affect LES tone, as illustrated in Fig.
10B.
Smooth muscle tension was recorded on a chart recorder (Grass
Instruments). Passive force was obtained at the end of the experiment by completely relaxing the strips with excess EDTA until no further decrease in resting force was observed. Basal LES tone is the difference between resting and passive force. Percent increase in basal
tone was defined by the ratio between the increase in force after drug
administration and basal LES tone. Percent basal LES tone was
calculated by the ratio between the force after using the drugs and the
basal LES tone.
Preparation of circular smooth muscle tissue.
The LES was excised, the circular muscle layer was cut into
0.5-mm-thick slices with a Stadie Riggs tissue slicer (Thomas Scientific Apparatus, Philadelphia, PA), and tissue squares were made
by cutting twice with a 2-mm blade block, the second cut at right
angles to the first. This circular smooth muscle tissue was used for AA
release, PGF2
measurements, and
guanosine 5'-O-(3-thiotriphosphate)
(GTP
S) binding studies and to obtain isolated smooth muscle cells.
Cell isolation and permeabilization.
Isolated smooth muscle cells were obtained by enzymatic digestion, as
previously described (7-9, 46, 47, 76, 83-88). Briefly, LES
circular smooth muscle was digested in HEPES-buffered physiological
solution containing 150 U/ml collagenase (type II, Worthington
Biochemicals, Freehold, NJ) for 2 h. The HEPES solution contained 114.7 mM NaCl, 5.7 mM KCl, 2.1 mM
KH2PO4,
11 mM glucose, 24.5 mM HEPES, 1.9 mM
CaCl2, 0.57 mM
MgCl2, 0.3 mg/ml basal medium Eagle amino acid supplement (M.A. Bioproducts, Walkersville, MD), and
0.08 mg/ml soybean trypsin inhibitor (Worthington Biochemicals). The
HEPES solution was oxygenated (100%
O2) at 31°C, and the pH was
adjusted to 7.4. At the end of the digestion period, the tissue was
rinsed and then incubated in collagenase-free HEPES buffer. The cells
dissociate freely in collagenase-free solution.
When permeable cells were required to allow the use of G protein
antibodies that do not diffuse across the intact plasma membrane, the
partly digested muscle tissue was washed with a "cytosolic" enzyme-free PSS (cytosolic buffer) of the following composition (in
mM): 20 NaCl, 100 KCl, 25 NaHCO3,
5.0 MgSO4, 0.96 NaH2PO4, 1.0 EGTA, and 0.48 CaCl2. The
cytosolic buffer contained 2% BSA and was equilibrated with 95%
O2-5%
CO2 to maintain pH of 7.2 at
31°C. Muscle cells dispersed spontaneously in this medium. A low
concentration of calcium was present in the cytosolic buffer, to avoid
spontaneous contraction of the cells in the absence of agonists after
the membrane became permeable. 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 spun at
low g, and the resulting pellet was
resuspended in saponin-free modified cytosolic buffer containing
antimycin (10 µM), ATP (1.5 mM), and an ATP-regenerating system
consisting of creatine phosphate (5 mM) and creatine phosphokinase (10 U/ml) (12).
Agonist-induced contraction of isolated muscle cells.
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. Intact esophageal circular smooth muscle cells were
contracted with a maximal concentration (0.1 U/ml) of purified group I,
group II, or group III sPLA2, in
the absence or presence of the
PLA2 inhibitors
(10
5 M) AM5, X1, MJ33, BPB,
or MJ45. Permeabilized LES cells were exposed to a maximally effective
dose of PGF2
(10
8 M) or the thromboxane
A2 mimetic U-46619
(10
8 M) for 30 s. When G
protein antibodies were used, cells were incubated in the antiserum at
a 1:200 dilution for 1 h before the addition of agonist (13).
After exposure to agonist, the cells were fixed in acrolein at a final
1.0% concentration. A drop of the cell-containing medium was placed on
a glass slide and covered by a coverslip. The edges of the coverslip
were sealed with clear nail enamel to prevent evaporation. Slides so
prepared, if refrigerated, could be kept for several days.
Cell measurements.
Thirty consecutive cells from each slide were observed through a
phase-contrast microscope (Carl Zeiss) and a CCTV camera (model
WV-CD51, Panasonic, Secaucus, NJ) connected to a Macintosh computer
(Apple, Cupertino, CA). The Image 1.59 software program (National
Institutes of Health, Bethesda, MD) was used to measure cell length and
was used 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. Shortening was defined as percent decrease in average length
after agonist addition, compared with control length.
[3H]AA release or content in
LES and esophageal circular smooth muscle.
LES circular smooth muscle was incubated in Krebs solution containing
[3H]AA (3 µCi/ml)
for 4 h to allow uptake into cell membrane (45, 56, 68, 72, 78). After
4 h, the tissue was washed twice with 200 ml Krebs solution, and then
incubated with 1 ml of Krebs solution alone (control) or Krebs solution
containing 10
4 M AM5, MJ33,
or BPB. After 30 or 60 min, a 0.2-ml aliquot of the supernatant was
removed and the radioactivity was measured. The remaining fluid and
tissue were frozen and kept at
70°C. Samples were
subsequently thawed and homogenized. Homogenization consisted of 3- to
10-s bursts with a Tissue Tearer (Biospec, Racine, WI) followed by
40-60 strokes with a Dounce tissue grinder (Wheaton, Melville,
NJ). A 0.2-ml aliquot of the homogenate was used to measure tissue
radioactivity. The remaining homogenate was used to measure protein
content. The percent of AA release was calculated as the ratio of
activity of [3H]AA in
the supernatant to
[3H]AA in the
homogenate (87).
PGF2
measurement.
LES and esophageal circular smooth muscle was incubated with 1 ml Krebs
solution at 37°C for 2 h. Aliquots of LES tissue were treated with
Krebs solution alone (control) or Krebs solution containing
indomethacin and incubated for 45 min. The final concentration of
indomethacin was 10
5 M. Circular smooth muscle tissue (100 mg) was homogenized in acetate
buffer (0.2 M, pH 4.5, 4°C). Homogenization consisted of 3- to 10-s
bursts with a Tissue Tearer (Biospec) followed by 40-60 strokes
with a Dounce tissue grinder (Wheaton). An aliquot of homogenate was
taken for protein measurement.
PGF2
was extracted according to
the method of Saksena and Harper (79) as follows. Homogenates were
extracted twice with three volumes of ethyl acetate. Fractions of ethyl
acetate were pooled, washed with 5 ml of distilled water, and brought
to dryness by a stream of nitrogen. The resulting extracts were kept at
70°C. The extracts were redissolved in ethanol and purified
by passage over a Sep-Pak C-18 reverse-phase cartridge. The
PGF2
concentration was quantified by using PGF2
enzyme
immunoassay kit (Cayman Chemical).
[35S]GTP
S
binding experiments.
To compare the binding of
[35S]GTP
S to
activated G proteins in LES with
[35S]GTP
S binding
in esophagus, LES and esophagus muscle squares were incubated in Krebs
solution at 37°C for 2 h. To examine the inhibition of
[35S]GTP
S binding
by indomethacin, aliquots of LES tissue were incubated for 60 min in
Krebs solution alone (control) or in Krebs solution containing
indomethacin. The final concentration of indomethacin was
10
5 M. After treatment, all
tissues were kept in liquid nitrogen until the binding assay was performed.
LES and esophagus circular smooth muscle was homogenized in ice-cold
buffer containing 20 mM HEPES (pH 7.4), 2 mM
MgCl2, 1 mM EDTA, and 2 mM
1,4-dithiothreitol. 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 4°C (80 Ti 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
20 mM HEPES (pH 7.4), 240 mM NaCl, 2 mM EDTA, 2 mM
phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 µM
leupeptin, and 1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Samples remained in the solubilizing buffer for 1 h at
4°C.
[35S]GTP
S binding
was assayed with the method of Okamoto et al. (66) and Murthy et al.
(60). The crude membranes (2.5 mg protein/ml) were incubated for 1 min
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 maximal concentration
(10
6 M) of
PGF2
or the thromboxane
A2-mimetic U-46619
(10
6 M) in a total volume
of 300 µl. The reaction was stopped with 10 volumes of ice-cold 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 ELISA wells that had been
coated initially with an anti-rabbit immunoglobulin antibody (1:1,000)
and subsequently coated with specific G protein antibodies (1:1,000).
After a 2-h incubation on ice, the wells were washed three times with
phosphate buffer solution containing 0.05% Tween 20. The radioactivity
from each well was counted using 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.
Protein determination.
The homogenates of LES and esophageal tissues were solubilized by
addition of 6 ml of 0.1 N NaOH and heating the sample at 80°C for
30 min. The amount of protein present was determined by colorimetric
analysis (Bio-Rad, Melville, NY) according to the method of Bradford
(14).
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 Scheffé's F test.
Drugs and chemicals.
AACOCF3 and G protein antibodies
(Gq,
Gi3,
Gi1/i2) were purchased from
Calbiochem; Sep-Pak C-18 cartridges and ethyl acetate were from Fisher
Scientific (Pittsburgh, PA);
PGF2
enzyme immunoassay kit was
from Cayman Chemical (Ann Arbor, MI);
PGF2
, SQ-29548, and U-46619
were from Biomol (Plymouth Meeting, PA); [35S]GTP
S and
[3H]AA were from NEN
(Boston, MA). Goat anti-rabbit immunoglobulin G Fc antibody was from
Pierce (Rockford, IL); groups I, II, and III
sPLA2, acetylsalicylic acid, NDGA,
indomethacin, thromboxane B2, BPB,
TTX, PTX, hexane, ethanol, and 0.2 M acetate buffer (pH 4.5) and other
reagents were purchased from Sigma (St. Louis, MO).
AM5, MJ33, MJ45, and X1 were synthesized by M. K. Jain.
 |
RESULTS |
Group I sPLA2 and LES tone.
The selectivity of novel cytosolic and
sPLA2 inhibitors was examined by
testing their effectiveness against
PLA2-induced contraction of
isolated esophageal cells (Fig. 2).

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Fig. 2.
Effect of novel PLA2 inhibitors
(10 5 M) MJ33, MJ45, AM5,
4-bromophenacyl bromide (BPB), and X1 on the contraction of isolated
smooth muscle cells from the esophagus. Cells were contracted with a
maximal dose (0.1 U/ml) of group I
(A), group II
(B), or group III
(C)
sPLA2 in the absence (control) or
presence of PLA2 inhibitors. BPB
is a nonselective PLA2 inhibitor.
AM5 and X1 are inhibitors selective for
sPLA2, MJ33 is a group I
sPLA2 inhibitor, and MJ45 is a
group II sPLA2 inhibitor.
A: group I
sPLA2-induced contraction of
isolated esophageal cells was significantly reduced by BPB, AM5, X1,
and by MJ33. MJ45 had no effect on group I
sPLA2-induced contraction.
B: group II
sPLA2-induced contraction of
isolated esophageal cells was significantly reduced by AM5, X1, and
MJ45. BPB and MJ33 had no effect on group II
sPLA2-induced contraction.
C: group III
sPLA2-induced contraction was
reduced only by X1. Values are means ± SE of 3 animals with 30 cells counted for each animal.
** P < 0.001.
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Contraction of isolated esophageal cells in response to group I
sPLA2 was significantly reduced by
the nonselective PLA2 inhibitor BPB, the nonselective sPLA2
inhibitors AM5 and X1, and by the selective group I
sPLA2 inhibitor MJ33 (ANOVA,
P < 0.001). It was not affected by
the selective group II sPLA2
inhibitor MJ45 (Fig. 2A).
Contraction of isolated esophageal cells in response to group II
sPLA2 was significantly reduced by
AM5 and X1 and by the selective group II
sPLA2 inhibitor MJ45 (ANOVA,
P < 0.001; Fig. 2B). BPB and MJ33 had no effect on
group II sPLA2-induced contraction.
Figure 2C shows that group III
sPLA2-induced contraction was
reduced only by X1 (ANOVA, P < 0.001) and not by any of the other inhibitors.
Because group III sPLA2 is not
found in mammalian cells, we examined the effect of group I and II
sPLA2 inhibitors on spontaneous LES tone. AM5 and X1 caused a concentration-dependent reduction of in
vitro LES tone (ANOVA, P < 0.01),
whereas the cytosolic PLA2
inhibitor AACOCF3 had no effect
(Fig.
3A).
Figure 3, B and C, shows that MJ33 and BPB dose
dependently decreased resting LES tone (ANOVA,
P < 0.001) but MJ45 had no effect.
In addition, TTX (10
4 M)
had no effect on the decrease in LES tone induced by MJ33 and BPB.
These data suggest that group I
sPLA2 may play a role in the
maintenance of LES tone and that
PLA2-induced LES tone is not
neurally mediated.

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Fig. 3.
Effect of PLA2 inhibitors on lower
esophageal sphincter (LES) basal tone.
A: LES circular muscle strips were
incubated in the indicated concentrations of
PLA2 inhibitors. AM5 and X1 caused
a concentration-dependent reduction of in vitro LES tone, whereas the
cytosolic PLA2 inhibitor
AACOCF3 had no effect. Values are
means ± SE of 4 animals. B and
C: LES circular muscle strips were
incubated in indicated concentrations of
PLA2 inhibitors alone or in the
presence of TTX (10 4 M).
When strips were treated with TTX, electrical stimulation was used to
document inhibition of neurotransmitter release, before addition of
inhibitors. MJ33 (B) and BPB
(C) dose dependently decreased
resting LES tone, but MJ45 (B) had
no effect. In addition, TTX had no effect on the decrease in LES tone
induced by MJ33 and BPB. These data suggest that group I
sPLA2 may play a role in the
maintenance of LES tone and that this
PLA2 may not come from nerve
terminals. Values are means ± SE of 3-6 animals.
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Next, we examined the effect of exogenously added
sPLA2 on LES resting tone. Figure
4 shows that group I
sPLA2, purified from Naja naja venom (0.01 and 0.1 U/ml),
significantly increased in vitro LES tone (ANOVA,
P < 0.05). In contrast, group II
sPLA2, purified from rattle snake
venom, and group III sPLA2,
purified from bee venom, had no effect on in vitro LES tone. These data suggest that LES muscle, unlike esophageal circular muscle, responds only to group I sPLA2.

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Fig. 4.
Effect of exogenous group I, II, and III
PLA2 on LES basal tone. LES
circular muscle strips were incubated in indicated concentrations of
either group I, II, or III sPLA2.
Group I sPLA2 caused a
concentration-dependent increase of in vitro LES tone. In contrast,
group II sPLA2 and group III
sPLA2 had no effect on in vitro
LES tone. Values are means ± SE of 3-6 animals.
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AA production in LES and esophagus.
Data suggest that group I sPLA2
may participate in maintenance of LES tone. We therefore measured AA
accumulation in LES muscle and its release into the medium surrounding
the muscle.
AA tissue levels were significantly higher in the unstimulated LES
circular muscle than in esophageal circular muscle, both at 30 and 60 min (paired t-test, P < 0.05). In addition, AA released by LES circular muscle into the
medium was significantly higher than that by the esophagus both at 30 and 60 min (paired t-test, P < 0.05; Fig.
5B). AA
release was significantly reduced by BPB, AM5, and MJ33 (ANOVA,
P < 0.05; Fig.
6). These data suggest that the elevated
levels of AA in the LES may be maintained by a spontaneously active
group I sPLA2.

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Fig. 5.
[3H]AA release or
content in LES and esophageal (ESO) circular smooth muscle. LES
circular muscle squares were incubated for 4 h in Krebs solution
containing [3H]AA (3 µCi/ml) to allow uptake into the muscle. After 4 h, muscle squares
were washed with radioactive-free Krebs solution. After 30 or 60 min,
radioactivity released into the medium was measured
(B) and compared with the
radioactivity present in tissue homogenates
(A).
A: AA levels were significantly higher
in unstimulated LES circular muscle than in esophageal circular muscle
after 30 and 60 min. B: LES circular
muscle released significantly higher concentrations of AA after 30 and
60 min than the esophagus. Values are mean ± SE of 5-7
animals. * P < 0.05.
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Fig. 6.
Effect of sPLA2 inhibitors on
[3H]AA release. LES
circular muscle squares were incubated in Krebs solution containing
[3H]AA (3 µCi/ml) to
allow uptake into the muscle. After 4 h, muscle squares were washed and
incubated for 30 min in Krebs alone (control) or in Krebs containing
AM5, MJ33, or BPB (10 4 M).
Radioactivity released into the medium was measured and compared with
the radioactivity present in tissue homogenates. Percent release of
[3H]AA into medium was
calculated as 100 × [radioactivity in the medium (cpm/mg
protein)/radioactivity in tissue homogenates (cpm/mg protein)].
[3H]AA release was
significantly reduced by BPB, AM5, and by MJ33. These
data suggest that elevated levels of AA in the LES may be maintained by
a spontaneously active group I
sPLA2. Values are mean ± SE of
5 animals. * P < 0.05.
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Cyclooxygenase inhibitors reduce LES tone.
Because AA may be metabolized to leukotrienes, prostaglandins, or
thromboxanes, we examined whether these AA metabolites may contribute
to maintenance of LES tone. Figure 7 shows
that the cyclooxygenase inhibitors indomethacin and aspirin caused a
concentration-dependent reduction (ANOVA,
P < 0.001) in basal tone of LES
circular muscle strips, whereas the lipoxygenase inhibitor NDGA had no
effect. In addition, TTX
(10
4 M) had no effect on
the reduction of LES tone induced by indomethacin. These data suggest
that prostaglandins and/or thromboxanes (but not leukotrienes) may play
a role in resting LES tone and that indomethacin-induced reduction of
LES tone is not neurally mediated.

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Fig. 7.
Effect of cyclooxygenase or lipoxygenase inhibitors on LES basal tone.
LES circular muscle strips were incubated in the indicated
concentration of NDGA, indomethacin (Indo), or aspirin alone, or in the
presence of TTX (10 4 M).
When strips were treated with TTX, electrical stimulation was used to
document inhibition of neurotransmitter release before addition of
inhibitors. Cyclooxygenase inhibitors indomethacin
(A) and aspirin
(B) caused a concentration-dependent
reduction in LES basal tone. Lipoxygenase inhibitor NDGA
(A) had no effect. In addition, TTX
had no effect on the reduction of LES tone induced by indomethacin
(A). These data suggest that
prostaglandins and not leukotrienes play a role in LES basal tone and
that prostaglandins may not come from nerve terminals. Values are mean ± SE of 3-7 animals.
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PGF2
and/or thromboxanes
and LES tone.
Because prostaglandins and/or thromboxanes may participate in
maintenance of LES tone, we measured the
PGF2
content in the esophagus
and LES circular smooth muscle and examined the effect of the
thromboxane A2 antagonist SQ-29548
on LES tone.
The PGF2
content of
unstimulated LES circular muscle was significantly higher than in
esophageal muscle (paired t-test, P < 0.05; Fig.
8A).
Indomethacin significantly reduced
PGF2
formation in LES smooth
muscle (paired t-test,
P < 0.05; Fig. 8B). Furthermore,
PGF2
dose dependently increased
LES tone (ANOVA, P < 0.05; Fig.
9A) and
reversed the reduction in LES tone induced by indomethacin (ANOVA,
P < 0.001; Fig.
9B). These data suggest that
PGF2
may participate in the
maintenance of LES resting tone.

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Fig. 8.
A:
PGF2 content in LES and
esophageal (ESO) smooth muscle.
PGF2 was extracted from tissue
squares of LES and esophageal circular smooth muscle according to the
method of Saksena and Harper (79).
PGF2 content was significantly
higher in LES smooth muscle than in esophageal smooth muscle. Values
are mean ± SE of 3 animals. B:
aliquots of LES tissue squares were treated for 45 min with Krebs alone
(control) or in Krebs solution containing indomethacin
(10 5 M) before measurement
of PGF2 content.
PGF2 content was significantly
decreased by indomethacin. Values are mean ± SE of 3-4
animals. * P < 0.05.
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Fig. 9.
Role of PGF2 on LES basal tone.
LES circular muscle strips were incubated in indicated concentrations
of PGF2 alone (control) or in
the presence of indomethacin
(10 5 M).
A:
PGF2 produced a
concentration-dependent increase in LES basal tone.
B:
PGF2
(10 4 M) reversed the
reduction in LES basal tone produced by indomethacin
(10 5 M). Values are mean ± SE of 3-4 animals.
** P < 0.001.
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SQ-29548 dose dependently decreased resting LES tone (ANOVA,
P < 0.001; Fig.
10B).
In addition, thromboxane B2 dose
dependently increased LES tone (ANOVA,
P < 0.01; Fig.
10A) and reversed the reduction of
LES tone induced by indomethacin (ANOVA, P < 0.001; Fig. 10C). These data suggest
that thromboxane A2 and/or
B2 may also contribute to
maintaining LES tone.

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Fig. 10.
Role of thromboxane B2
(TXB2) on LES basal tone. LES
circular muscle strips were incubated in indicated concentrations of
TXB2 alone (control) or in the
presence of indomethacin
(10 5 M).
A:
TXB2 produced a
concentration-dependent increase in LES basal tone.
B: thromboxane antagonist SQ-29548
produced a concentration-dependent reduction in LES basal tone.
C:
TXB2
(10 5 M) reversed the
reduction in LES basal tone produced by indomethacin
(10 5 M). Values are mean ± SE of 3-4 animals.
** P < 0.001.
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G protein activation and LES tone.
In LES smooth muscle, basal or unstimulated
[35S]GTP
S binding
to Gq,
Gi3, or
Gi1/i2 was significantly higher
(ANOVA, P < 0.01) than in esophagus
smooth muscle (Fig. 11), suggesting that,
in the absence of any exogenously added agonist, these G proteins are
at a significantly higher level of activation in LES muscle.

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Fig. 11.
Unstimulated
[35S]guanosine
5'-O-(3-thiotriphosphate)
(GTP S) binding in LES and esophageal (ESO) smooth muscle membranes.
Purified membranes were incubated in
[35S]GTP S for 1, 5, 10, and 20 min. Basal, or unstimulated, activation of specific G
proteins was reflected by the amount of
[35S]GTP S bound to
wells, which were precoated with antibodies raised against the
-subunit of Gq,
Gi3, or
Gi1/i2. G protein activation was
measured as cpm
[35S]GTP S per µg
membrane protein. Values are means ± SE of 4 animals, with each
sample performed in triplicate.
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Spontaneous activity of Gi-type G
proteins was confirmed by examining the effect of PTX on LES tone.
Figure 12 shows that PTX concentration
dependently reduced LES tone (ANOVA, P < 0.001). The highest concentration of PTX did not have a
nonselective effect on LES muscle, since it did not affect contraction
induced by a maximally effective dose of ACh, which is mediated by the
PTX-insensitive Gq (86). These
data suggest that in the LES there is spontaneous activation of G
proteins and that a PTX-sensitive G protein may be involved in tone.

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Fig. 12.
A: role of pertussis toxin (PTX) on
LES basal tone. LES circular muscle strips were incubated in Krebs
containing 0.1% BSA (control) or in Krebs containing 0.1% BSA and
indicated concentrations of PTX for 1 h. PTX produced a
concentration-dependent decrease in LES basal tone.
B: LES circular muscle strips were
incubated in ACh (10 5 M)
alone (control) or in the presence of PTX (5 mg/ml). PTX did not affect
the increase in LES tone produced by ACh. Values are means ± SE of
3 animals (controls) or 7 animals (PTX).
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|
Because PGF2
and thromboxanes
A2 and
B2 contribute to maintenance of
LES tone that is associated with G protein activation, we next examined
the G proteins activated by these AA metabolites.
PGF2
(10
6 M) caused significant
activation of Gq (ANOVA,
P < 0.01) and
Gi3 (ANOVA,
P < 0.05) but had no effect on
Gi1/i2, or
Go (Fig.
13A).

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Fig. 13.
A:
[35S]GTP S binding
to PGF2 -activated membranes of
LES. Purified membranes were exposed to
PGF2
(10 6 M) in the presence of
[35S]GTP S for 1 min. PGF2 -induced activation of
specific G proteins was reflected by the amount of
[35S]GTP S bound to
wells, which were precoated with antibodies raised against the
-subunit of Gq,
Gi3,
Gi1/i2, or
Go. G protein activation was
measured as %increase in
[35S]GTP S binding
in membranes exposed to PGF2
compared with unstimulated membranes.
PGF2
(10 6 M) caused significant
activation of Gq and
Gi3. Values are means ± SE of
4 animals, with each sample performed in triplicate.
B: LES muscle cells were permeabilized
by brief exposure to saponin to allow diffusion of antibodies into the
cytosolic side of the cell membrane. Cells were contracted with
PGF2
(10 8 M) alone (control) or
after a 60-min preincubation in cytosolic medium containing antibodies
raised against the -subunit of G proteins (1:200 dilution).
PGF2 -induced contraction of LES
cells was significantly inhibited by
Gq and
Gi3 and not by
Gi1/i2 and
Go antibodies. Values are means ± SE of 3 animals with each sample performed in triplicate.
* P < 0.05;
** P < 0.001;
P < 0.01.
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To determine which G proteins mediate
PGF2
-induced contraction, we
used G protein antibodies raised against synthetic peptides
corresponding to the amino acid sequence of the COOH terminus of the G
protein
-subunit. Figure 13B shows
that PGF2
-induced contraction
of LES smooth muscle cells was significantly reduced by antibodies
raised against the COOH terminus of the
-subunit of
Gq and
Gi3 (ANOVA,
P < 0.001) and unaffected by
Gi1/i2 or
Go antibodies.
Figure
14A
shows that the thromboxane
A2-mimetic U-46619 stimulates
[35S]GTP
S binding
to Gq (ANOVA,
P < 0.001). Similarly,
U-46619-induced contraction of permeabilized LES muscle cells was
significantly reduced by antibodies raised against the COOH terminus of
the
-subunit of Gq (ANOVA,
P < 0.001) and unaffected by
Gi3,
Gi1/i2, or
Go antibodies (Fig.
14B).

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Fig. 14.
A:
[35S]GTP S binding
to U-46619-activated membranes of LES. Purified membranes were exposed
to U-46619 (10 6 M) in the
presence of
[35S]GTP S for 1 min. U-46619-induced activation of specific G proteins was reflected by
the amount of
[35S]GTP S bound to
wells, which were precoated with antibodies raised against the
-subunit of Gq,
Gi3,
Gi1/i2, or
Gs. G protein activation was
measured as %increase in
[35S]GTP S binding
in membranes exposed to U-46619 compared with unstimulated membranes.
U-46619 (10 6 M) caused
significant activation of Gq.
Values are means ± SE of 3 animals with each sample performed in
triplicate. B: LES muscle cells were
permeabilized by brief exposure to saponin to allow diffusion of
antibodies into the cytosolic side of the cell membrane. Cells were
contracted with U-46619
(10 8 M) alone (control) or
after a 60-min preincubation in cytosolic medium containing antibodies
raised against the -subunit of G proteins (1:200 dilution).
U-46619-induced contraction of LES cells was significantly inhibited by
Gq and not by
Gi3,
Gi1/i2, or
Go antibodies. Values are means ± SE of 3 animals, with each sample performed in triplicate.
** P < 0.001.
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Indomethacin (10 µM) significantly reduced
[35S]GTP
S binding
to Gq,
Gi3, and
Gi1/i2 (ANOVA,
P < 0.01) in LES circular smooth muscle (Fig. 15), supporting the
hypothesis that the cyclooxygenase products
PGF2
and thromboxane
A2 and
B2 may be involved in G protein
activation and maintenance of LES tone.

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Fig. 15.
Indomethacin decreased basal
[35S]GTP S binding
of Gq,
Gi3, or
Gi1/i2 in LES smooth muscle
membranes. LES circular smooth muscle was incubated in Krebs solution
at 37°C for 2 h, and then aliquots of LES tissue were incubated for
another 60 min in Krebs solution alone (control) or in Krebs solution
containing indomethacin
(10 5 M). Purified membranes
were incubated in
[35S]GTP S for 1 min. Basal, or unstimulated, activation of specific G proteins was
reflected by the amount of
[35S]GTP S bound to
wells, which were precoated with antibodies raised against the
-subunit of Gq,
Gi3, or
Gi1/i2. G protein activation was
measured as cpm
[35S]GTP S per µg
membrane protein. Percent inhibition was calculated as 100 × [radioactivity in the control (cpm/µg protein) radioactivity in indomethacin group (cpm/µg protein)/radioactivity in
control (cpm/µg protein)]. Values are means ± SE of 3 animals, with each sample performed in triplicate.
P < 0.01.
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|
 |
DISCUSSION |
Group I sPLA2 contributes to basal LES
tone.
Spontaneous LES tone may be mediated by the activity of a group I
sPLA2 because LES tone was
concentration dependently reduced by inhibitors selective for group I
sPLA2. The selectivity of the
PLA2 inhibitors was demonstrated
in Fig. 2 in which isolated cells from the body of the esophagus were
contracted with either purified group I, group II, or group III
sPLA2. AM5 and X1 are thought to
be nonselective inhibitors for
sPLA2; MJ33 (44) selectively inhibits group I sPLA2; and MJ45
is selective for group II sPLA2 (43). BPB is thought to be a nonselective inhibitor for
PLA2 (30, 57, 59); however, in our
preparation, BPB inhibited only group I
sPLA2 purified from cobra venom.
Group I sPLA2-induced contraction
of isolated esophageal muscle cells was selectively reduced by MJ33,
AM5, X1, and BPB, and these inhibitors dose dependently reduced LES
tone in a TTX-independent manner. The maximal fall in basal LES tone
induced by the PLA2 inhibitors was
40%, whereas indomethacin reduced tone by 80% from basal levels. The
indomethacin-induced reduction in tone may indicate AA release from
different phospholipases, such as PC-PLC (26), which participate in the
maintenance of LES tone (41). TTX was added to the
PLA2 inhibitor to abolish axonal
transmission, eliminating the possibility that indomethacin and the
sPLA2 inhibitors may be acting on
neurons. Even at the high concentrations used in the present study, TTX
did not modify, in any way, the reduction in resting tone induced by
these agents.
The greater AA tissue content and AA release in LES smooth muscle, when
compared with esophageal smooth muscle, which does not maintain basal
tone (Fig. 5), supports the view that spontaneous group I
sPLA2 activity may play a role in
maintaining LES tone. The same group I
sPLA2-selective inhibitors that
reduced LES tone also reduced the spontaneous AA release in LES smooth
muscle (Fig. 6). The concentration of
sPLA2 inhibitors that reduced LES
tone and AA release were similar to those reported for the cytosolic PLA2 inhibitor
AACOCF3. The
IC50 for the concentration of
AACOCF3 required for a twofold
reduction in the amount of AA liberated from 4 × 108 platelets/ml is 8-10
µM. The concentrations of MJ33 and MJ45 for 50%
inhibition are ~0.01 mol fraction, the molar amount of inhibitor that
the enzyme sees at the interface of the substrate (5). For interfacial
enzymology, the relevant inhibitory concentrations are in mole fraction
because there is no direct way to express inhibition in moles per liter
(32) for biological systems because the true lipid concentration is unknown.
LES circular muscle differed from esophagus circular muscle because it
contracted only in response to group I
sPLA2 purified from cobra venom,
whereas esophagus circular muscle contracted in response to all three
sPLA2 enzymes. The
sPLA2 enzymes used in the present
studied are purified from the various venoms. Human pancreatic
sPLA2 is secreted as an inactive
zymogen, which is activated by trypsin in the gastrointestinal lumen to
its active enzyme by the removal of seven amino acid residues from the
NH2 terminus (27). It is likely
that the purified enzymes used in the present study are already active.
The selectivity of the group I
sPLA2 in contracting LES muscle
may be conferred by the specific interaction of
sPLA2 with cell surface receptors.
Specific membrane receptors for neuronal group and muscle group
sPLA2 enzymes have been identified
with snake venom sPLA2 (50,
52-55). One of these sPLA2
receptors, the 180-kDa muscle group, has been cloned in rabbit (50) and humans (2) and has been shown to have very high affinity for mammalian
sPLA2. This receptor has a large
extracellular domain of 1394 amino acids composed of an
NH2-terminal cysteine-rich domain,
a fibronectin-like group II domain, eight carbohydrate recognition
domains (CRDs), and a transmembrane and an intracellular COOH terminus
(50). The CRD5 on the sPLA2
receptor (63) and the residues within or near the calcium binding loop
on sPLA2 (51) appear to be
necessary for phospholipase-receptor interaction. Selective
sPLA2 binding is responsible for
some of the physiological effects of mammalian
sPLA2, including vascular smooth
muscle contraction, cell proliferation, and internalization of
sPLA2 (3, 61, 89). For example,
antigen stimulation results in the selective binding of group I
sPLA2 to bone marrow mast cells,
which contain the mRNA for the group I
PLA2 receptor (29), causing
release of AA.
PGF2
and/or thromboxanes
may mediate LES tone by activation of G proteins.
The AA produced by sPLA2 in the
LES may be metabolized to prostaglandins and thromboxanes because the
cyclooxygenase inhibitors aspirin and indomethacin reduce LES basal
tone (Fig. 7). Leukotrienes are not likely to play a role in this
system because the lipoxygenase inhibitor NDGA had no effect on LES tone.
Our data are consistent with previously reported in vitro data.
However, there is a difference between the effects of AA and indomethacin in in vivo and in vitro preparations. In vitro LES contracts in response to AA (20) and relaxes in response to indomethacin (19, 20). In vivo LES relaxes in response to AA (21, 73).
In vivo indomethacin has no effect on LES resting tone (73). We have
reported that AA contracts in vitro LES muscle strips (75), and in the
present study we demonstrate that indomethacin relaxes them.
The reason for differences between in vitro and in vivo preparations is
not clear. It is possible that injected AA in the in vivo preparations
may be metabolized to prostaglandins in nerve terminals as well as in
muscle. Daniel et al. (20) reported that indomethacin inhibited
relaxations to electric field stimulation, that inhibition of
relaxation preceded loss of muscle tone, and that inhibition of tone
was complete. This may explain why in vitro indomethacin first
contracts and then relaxes LES. The fact that inhibition of relaxation
preceded loss of muscle tone suggests that perhaps inhibitory neurons
are more sensitive to the effect of indomethacin than the muscle. In
vivo indomethacin was administered at doses sufficient to inhibit
AA-induced relaxation, which may be neurally mediated. It is possible
that higher doses of indomethacin may be required to have an effect on
muscle tone. The effect of TTX on AA-induced relaxation was not tested
in any of the previous studies, so that the site of action of in vivo
AA has not been pinpointed.
Similarly, PGF2
has been
reported to contract LES in vitro (19, 20), whereas, in vivo,
PGF2
most often contracts LES
and sometimes relaxes it (74). In vivo,
PGF2
, the stable epoxymethano
derivatives of PGH2, and
thromboxane B2 have been reported
to contract LES smooth muscle strips (19). We therefore examined the
role of PGF2
and thromboxanes
A2 and
B2 in LES tone. We find that
PGF2
produced a
concentration-dependent increase in LES basal tone and reversed the
reduction in tone produced by indomethacin (Fig. 9).
Although other cyclooxygenase products such as
PGE2 have been reported to relax
the LES (21, 34, 36, 74), we find more
PGF2
production in the LES
compared with the esophagus (Fig. 8). Formation of different
prostaglandins depends on the presence of the appropriate synthetic
enzymes required to make the particular prostaglandin. For example, in
the rat ocular system, steady-state levels of
PGE2,
PGF2
, and
PGD2 correlate well with the
enzymatic activities of their respective synthetase enzymes, namely,
glutathione (GSH)-dependent and membrane-bound E, soluble F, and GSH-independent and soluble D synthetase (33). It is possible
that LES may have a higher content/activity of soluble F synthetase or
more 9-ketoreductase, which catalyzes the conversion of
PGE2 to
PGF2
(64, 96). Different
activities of synthetase enzymes will result in different prostaglandin
distributions in different organs. For example, in fetal lamb brain,
lung, liver, spleen, ductus arteriosus, aorta, and pulmonary vein,
different amounts of PGE2,
PGF2
,
PGD2,
6-keto-PGF1
, and thromboxane B2 are present (67). In blood
vessels of rat brain,
6-keto-PGF1
is present in
40-fold higher concentrations than in brain tissue; conversely,
PGD2 levels are high in brain
tissue and below detection levels in brain blood vessels (1).
Thromboxane A2 is an unstable
arachidonate metabolite, with a half-life of 30 s, and rapidly decays
nonenzymatically to the stable thromboxane
B2, which has weak biological
activity (62). We find that thromboxane
B2 also produced a
concentration-dependent increase in LES basal tone and reversed the
reduction in tone induced by indomethacin. In addition, the selective
thromboxane A2 antagonist SQ-29548
(38) dose dependently reduced LES basal tone (Fig. 10). These data
suggest that thromboxanes A2 and
B2 may also play a role in
maintaining LES basal tone.
The action of these prostanoids is mediated by distinct receptors. The
classification of prostanoid receptors in platelets and smooth muscle
is based on the pattern of effects and the relative potencies of
natural and synthetic agonists (18) and substantiated by ligand binding
studies, receptor cloning, and selective antagonists (18, 39). The
receptors are named after their endogenous prostaglandin ligand and are
divided into five main types: DP (PGD), FP (PGF), IP
(PGI2), TP (thromboxane
A2), and EP (PGE). The EP
receptors have been further subdivided into EP1, EP2, EP3, and EP4, on
the basis of physiological activity and molecular cloning (18, 92). The
cDNAs encoding representatives of each of these groups of receptors
have been cloned (69). Heterologous expression of receptor cDNAs
confirmed that they are all G protein-coupled receptors that contain
seven transmembrane domains, an extracellular
NH2 terminus, and an intracellular
COOH terminus. Functional expression of the cloned receptors is
consistent with a single subunit structure containing a ligand binding
site and the determinants required for second messenger coupling. The
DP, IP, EP2, and EP4 receptors are coupled to stimulation of adenylyl
cyclase, and the EP1, FP, and TP receptors are coupled to
phosphatidylinositol hydrolysis (70).
Because LES tone is myogenic, and may be mediated by production of
prostanoids that are coupled to G protein effector mechanisms, G
proteins may be activated in LES smooth muscle. We have previously shown, by Western blot, that Gq,
Gi3, and
Gi1/i2 are all present in
esophageal and LES circular muscle (84). In the current study, we show
that in the LES these G proteins are active, i.e., bound to GTP in the
absence of exogenous stimuli (Fig. 11). The same G proteins that are
spontaneously active are stimulated by
PGF2
and thromboxane
B2. In addition, LES basal tone
can be concentration dependently reduced by PTX, suggesting that basal
Gi activation may contribute to
LES basal tone.
The PGF2
receptor is reported
to be coupled to phosphoinositide metabolism and calcium mobilization
via stimulation of PTX-insensitive
Gq proteins (42). In LES smooth
muscle membranes, PGF2
significantly activates Gq and
Gi3 and slightly activates Gi1/i2 and has no effect on
Go activation. In addition,
stimulation of the FP receptor on LES smooth muscle cells with
PGF2
produces a maximal
contraction (21.3 ± 0.4% shortening) that is selectively reduced
by antibodies raised against the COOH terminus of the
-subunit of
Gq and
Gi3. These data suggest that, in
the LES, FP receptor stimulation results in contraction that is
mediated by both Gq- and
Gi3-like G proteins.
Several studies have demonstrated that stimulation of the platelet TP
receptor results in the activation of a PTX-insensitive PLC with
stimulation of phosphoinositide metabolism and subsequent increase in
intracellular calcium (39, 71, 77, 82). The TP receptor may be coupled
to Gq because thromboxane
A2 agonist-stimulated GTPase
activity was blocked by an antibody raised against the COOH terminus of
the
-subunit of Gq in human
platelet membranes (81). In addition, thromboxane
A2 stimulation has been shown to
inhibit adenylate cyclase and to reduce cAMP-mediated inhibition of
ADP-evoked response in platelets. Therefore, in platelets, the TP
receptor may be coupled to two different G proteins
(Gq- and a
Gi-like G protein) and two
different signal transduction pathways. The TP receptor has also been
shown to be coupled to Gi2,
G12,
G13 and to an unidentified 85-kDa
G protein (48, 65, 94). The prostanoid second messenger system in
smooth muscle cells has not been extensively studied; however,
thromboxane mimetics have been reported to increase intracellular free
calcium and calcium fluxes in vascular tissue and in smooth muscle
cells (15, 28, 31, 37).
In the current study, we show that the thromboxane
A2-mimetic U-46619 (4, 90)
significantly activates Gq and has
a small stimulatory effect on Gi3
and Gi1/i2 but no effect on
Gs activation. In addition,
stimulation of the TP receptor on LES smooth muscle cells with U-46619
produced a maximal contraction (22.4 ± 1.2% shortening) that was
selectively reduced by antibodies raised against the COOH terminus of
the
-subunit of Gq. These data
suggest that, in the LES, TP receptor stimulation results in
contraction that is mediated by
Gq-like G proteins, similar to the
reports of Gq activation by
stimulation of the TP platelet receptor.
These data show that the prostanoids
PGF2
and thromboxane
A2/B2
can account for the activation of
Gq,
Gi3, and
Gi1/i2, which are found to be
spontaneously active in the LES in its basal state. A role for
Gq and
Gi3 is supported by Figs. 13 and
14, which show that PGF2
- and
thromboxane-induced contraction is reduced by
Gq and
Gi3 and not by
Gi1/i2 antibodies. Although
Gi1/i2 is present in the LES (84)
and is active (Fig. 11), it is not coupled to contraction (Figs.
13B and
14B) or maintenance of LES basal
tone (Fig. 15), and its function remains to be determined.
The activity of these G proteins is reduced by indomethacin, supporting
the view that spontaneous activation of these G proteins is maintained
by cyclooxygenase-catalyzed AA products. Activation of these G proteins
may play a role in LES basal tone because the same concentration of
indomethacin (10
5 M) (Fig.
7) that reduced tone by 50% also significantly reduced the level of
activity of Gq,
Gi3, and
Gi1/i2 present in the unstimulated LES circular muscle (Fig. 15). Because basal LES tone is reduced by PTX
and Gi1/i2 has no role in
prostanoid-mediated LES contraction, Gi3 is likely to be the
Gi-type G protein involved in
maintenance of LES tone.
We propose the following hypothesis: Spontaneous activation of a group
I sPLA2 causes production of AA
and AA metabolites such as PGF2
and thromboxanes A2 and
B2, which maintain activation of G
proteins such as Gi3,
Gi1/i2, and
Gq.
Gi3 and
Gq activate the phospholipases
PC-PLC and PI-PLC, which in turn produce DAG and
IP3. DAG and
IP3 synergistically activate
PKC-
and produce LES tone (8, 41, 88). The origin of the group I
sPLA2 remains to be found;
however, preliminary Western blot studies using monoclonal antibodies
of human sPLA2 group I indicate
the presence of sPLA2 in human LES
circular smooth muscle.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants RO1
HD-20054, RO1 DK-42876, and RO1 DK-28614.
 |
FOOTNOTES |
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 and other correspondence: P. Biancani, GI
Motility Research Lab., SWP5 Rhode Island Hospital and Brown Univ., 593 Eddy St., Providence RI 02903 (E-mail:
piero_biancani{at}brown.edu).
Received 8 March 1999; accepted in final form 8 June 1999.
 |
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