From the Departments of Medicine and Physiology, Medical College of
Virginia, Richmond, Virginia 23298-0711
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INTRODUCTION |
P2 receptors have been classified recently into
two classes comprising ligand-gated cationic channels or
P2X receptors and G protein-coupled P2Y
receptors (1, 2); P2U and P2T receptors have
been subsumed into the P2Y class of receptors. The term
P2 recognizes the fact that purine and pyrimidine
nucleotides can act as preferential ligands of various receptor
subtypes (2). Up to seven P2X receptor subtypes (3-9) and
eight P2Y receptor subtypes (10-16) have been cloned from
mammalian and avian species. Fuller understanding of the functions
subserved by discrete receptor subtypes is hampered by the organization
of native P2X receptors into homopolymers or heteropolymers
(5) and by the co-existence of P2X and P2Y
receptors on the same cell (17). Earlier classifications based on
agonist potency profiles had been confounded by the paucity of
selective antagonists and radioligands (2), and by the rapid degradation of some nucleotides, mainly ATP and 2-methylthio-ATP, by
ecto-nucleotidases (18), and the interconversion of adenine and uridine
nucleotides by ecto-nucleoside diphosphokinases (19, 20).
P2X1 is the main P2X receptor subtype expressed
in visceral and vascular smooth muscle (21), whereas P2X2
and P2X3 are the main receptor subtypes expressed in
peripheral sensory ganglia (8, 21-23). Both P2X1 and
P2X3 receptors have high affinity for ATP and
AMP-PCP1 and are rapidly
desensitized (23, 24). P2X2, P2X4, and
P2X6 receptors are the predominant receptor subtypes
expressed in the adult brain where they are present in various
heteromeric combinations; these receptor subtypes exhibit lower
affinity for ATP, are insensitive to AMP-PCP, and are not readily
desensitized (7, 8, 22, 23). Their insensitivity to AMP-PCP restricts
the usefulness of this analog as a radioligand for all but the
P2X1 and P2X3 receptor subtypes (24).
P2Y receptors exhibit variable affinity for purine and
pyrimidine nucleotides. P2Y1 are purinoceptors and are
adenine nucleotide-specific (10, 13), whereas P2Y2
receptors (P2U in earlier classifications) have equal
affinity for adenine and uridine nucleotide triphosphates (UTP
ATP) (11, 19). P2Y3, P2Y4, and P2Y6
are pyrimidinoceptors: P2Y4 is UTP selective whereas
P2Y3 and P2Y6 are UDP selective (14, 19). The
functional status of P2Y5 which has low homology to other
P2Y receptors has not been resolved (16, 25), while the
P2Y7 receptor has now been identified as the leukotriene
B4 receptor (26). P2Y receptors are variously
coupled to pertussis toxin-sensitive and -insensitive G proteins which
activate or inhibit various effector enzymes including phospholipase
C-
(PLC-
) (15, 16, 27-30), phospholipase D (31, 32),
phospholipase A2 (33), and adenylyl cyclase (28, 30,
34).
ATP, UTP, and AMP-PCP can mobilize Ca2+ and elicit
contractile responses in vascular and visceral smooth muscle suggesting
that both P2X and P2Y receptors are present
(15, 17, 35-37). Their co-existence raises the question as to which
receptor subtype mediates preferentially the action of the endogenous
ligand, ATP. In the present study, we have used a series of purine and
pyrimdine agonists to characterize P2 receptors in
dispersed gastric smooth muscle cells and identify the signaling
pathways to which they are coupled. Comparative studies characterized
the coupling of P2Y receptors to G proteins in vascular
smooth muscle, heart, liver, and brain. P2X1 and
P2Y2 receptors were shown to co-exist on gastric smooth
muscle cells and to mediate Ca2+ mobilization and muscle
contraction via three distinct pathways. UTP and nanomolar
concentrations of ATP activated exclusively P2Y2 receptors,
whereas micromolar concentrations of ATP activated additionally
P2X1 receptors. The pattern suggests that contraction induced by purine and pyrimidine nucleotides may be preferentially mediated by G protein-coupled receptors.
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EXPERIMENTAL PROCEDURES |
Dispersion of Gastric Smooth Muscle Cells--
Smooth muscle
cells were isolated from the circular muscle layer of rabbit stomach by
sequential enzymatic digestion, filtration, and centrifugation as
described previously (38-40). The cells were resuspended in
enzyme-free medium consisting of 120 mM NaCl, 4 mM KCl, 2.6 mM KH2PO4,
2 mM CaCl2, 0.6 mM
MgCl2, 25 mM HEPES, 14 mM glucose,
and 2.1% Eagle's essential amino acid mixture. The muscle cells were
harvested by filtration through 500-µm Nitex mesh and centrifuged
twice at 350 × g for 10 min.
In some experiments, the muscle cells were reversibly permeabilized
using the Trans.Port reagent (Life Technologies, Inc.) as described
previously (40). The cells were washed in Ca2+- and
Mg2+-free HEPES medium and re-suspended in a medium
containing 10 mM NaCl, 140 mM KCl, 2.4 mM MgCl2, and 10 mM HEPES.
Trans.Port reagent (15 µl/ml) was added with or without GDP
S (10 µM) and the mixture incubated at 31 °C for 20 min.
Permeabilization was terminated by addition of Stop solution (30 µl/ml) and the cell suspension centrifuged for 15 min at 350 × g. The cells were resuspended in control HEPES medium
containing 0.1% bovine serum albumin and incubated at 31 °C for
1 h. The resealed cells were shown to exclude trypan blue and
respond to contractile agonists and depolarizing concentrations of KCl
(20 mM) but not to 2 mM CaCl2 or
inositol 1,4,5-trisphosphate (1 µM) (40). The
effectiveness of GDP
S was tested by measuring its ability to abolish
the contractile response to the contractile agonist, cholecystokinin
octapeptide (40).
Identification of G Protein Subtypes and PLC-
Isozymes
in Gastric Smooth Muscle by Western Blot--
The expression of G
proteins and PLC-
isozymes was determined by Western blot analysis
as described previously (41-43). Homogenates prepared from dispersed
gastric muscle cells were solubilized on ice for 1 h in 20 mM Tris (pH 8.0), 1 mM dithiothreitol, 100 mM NaCl, and 0.5% sodium cholate. The suspension was
centrifuged at 13,000 × g for 5 min. Solubilized
proteins were resolved by SDS-polyacrylamide gel electrophoresis and
electrophoretically transferred to nitrocellulose membranes. The blots
were incubated for 12 h at 4 °C with subtype-specific G protein
or PLC-
antibodies, and then for 1 h with secondary antibody
conjugated with horseradish peroxidase. The bands were identified by
enhanced chemiluminescence.
Selective Protection of P2 Receptors--
A
technique of selective receptor protection previously used to determine
the co-existence and function of various G protein-coupled receptors
(44-48) was used to determine the presence and function of
P2 receptor subtypes. The technique involves protection of one receptor subtype with selective agonists or antagonists followed by
inactivation of all unprotected receptors with a low concentration of
N-ethylmaleimide (5 µM). Freshly dispersed
muscle cells were incubated with one agonist (AMP-PCP, AMP-CPP, UTP, or
ATP) at 31 °C for 2 min followed by addition of 5 µM
N-ethylmaleimide for 20 min. The cells were centrifuged
twice at 150 × g for 10 min and resuspended in control
HEPES medium for 60 min to ensure complete re-sensitization. The
contractile response of cells treated in this fashion was compared with
the response of untreated cells. As previously shown (44-48), muscle
cells incubated with N-ethylmaleimide without protective
agent did not contract in response to receptor-linked agonists, but
they responded fully upon addition of agents that bypass receptors
(e.g. ionomycin, KCl, and forskolin), implying that
post-receptor mechanisms were intact.
Measurement of Contraction in Dispersed Muscle
Cells--
Contraction of dispersed muscle cells was measured by
scanning micrometry as described previously (38-40). The length of 50 muscle cells treated with one concentration of a contractile agent was
measured by scanning micrometry and compared with the length of 50 untreated muscle cells. All measurements were done in the presence of
adenosine A1 and A2 antagonists (1 µM DPCPX and 0.1 µM CGS-15943,
respectively) (47). Time course measurements were done at intervals
ranging from 5 s to 5 min. As with other agonists, peak
contraction was measured at 30 s and the response used to construct concentration-response curves. Contraction was expressed as
the mean decrease in cell length from control in micrometers or as the
percent decrease in cell length (range of control cell length in
various experiments 96 ± 4 to 103 ± 5 µm).
Measurement of Cytosolic Free Ca2+ in Dispersed
Muscle Cells--
Cytosolic free Ca2+
([Ca2+]i) was measured by fluorescence in
suspensions of muscle cells loaded with the fluorescent Ca2+ dye, fura 2, as described previously (40, 45).
Autofluorescence of unloaded cells was determined in each suspension
and subtracted from the fluorescence of fura 2-loaded cells.
Measurements were done in the presence of adenosine A1 and
A2 antagonists. Ca2+ levels were calculated
under basal conditions and upon addition of agonist from the ratios of
observed, minimal and maximal fluorescence (49).
Inositol 1,4,5-Trisphosphate (IP3) Radioreceptor
Assay--
IP3 was measured in dispersed muscle cells by a
radioreceptor assay which utilizes 3H-labeled
D-myo-IP3 and bovine brain
microsomes as described previously (41, 42). Agonists were added for
30 s in the presence of adenosine A1 and
A2 antagonists to 1 ml of muscle cell suspension (106 cells/ml) and the reaction terminated with an equal
volume of ice-cold 10% perchloric acid. The supernatant was extracted
and IP3 content in the aqueous phase was measured. The
results were expressed as picomoles of IP3/106
cells.
Assay of PLC-
Activity in Plasma Membranes--
PLC-
activity was determined in plasma membranes by a modification of the
method of Uhing et al. (50) as described previously (43,
51). The membranes were isolated from dispersed muscle cells labeled
with myo-[3H]inositol. PLC-
assay was
initiated by addition of 0.4 mg of membrane protein to 25 mM Tris-HCl (pH 7.5), 0.5 mM EGTA, 10 mM MgCl2, 300 nM free
Ca2+, 1 µM GTP
S, 5 mM
phosphocreatine, and 50 units/ml creatine phosphokinase in a total
volume of 0.4 ml. After incubation at 31 °C for 60 s, the
reaction was terminated with 0.6 ml of 25% trichloroacetic acid (w/v).
The supernatant was extracted four times with 2 ml of diethyl ether and
the amount of labeled inositol phosphates in the aqueous phase was
counted. All measurements were done in the presence of adenosine
A1 and A2 antagonists. PLC-
activity was
expressed as counts/min/mg protein/min.
Identification of Receptor-activated G Proteins--
G proteins
selectively activated by P2 receptor agonists in muscle
cell membranes were identified by the method of Okomoto et
al. (52) as described previously (41, 42, 48). Muscle cell
homogenates were centrifuged at 27,000 × g for 15 min,
and the crude membranes solubilized for 60 min at 4 °C in 20 mM HEPES medium (pH 7.4) containing 2 mM EDTA,
240 mM NaCl, and 1% CHAPS. The membranes were diluted
10-fold and incubated at 37 °C with 60 nM
[35S]GTP
S in a medium containing 10 mM
HEPES (pH 7.4), 100 µM EDTA, and 10 mM
MgCl2. The reaction was stopped with 10 volumes of 100 mM Tris-HCl medium (pH 8.0) containing 10 mM
MgCl2, 100 mM NaCl, and 20 µM
GTP, and the mixture incubated for 2 h on ice in wells precoated
with specific G protein antibodies. The wells were washed three times
with phosphate buffer solution containing 0.05% Tween 20 and the
radioactivity from each well was counted. Coating with G protein
antibodies (1:1000) was done after the wells were first coated with
anti-rabbit IgG (1:1000) for 2 h on ice. The measurements were
done in the presence of adenosine A1 and A2
antagonists.
In separate experiments on rabbit aortic smooth muscle, heart, liver,
and whole brain, membranes were obtained by homogenization of these
tissues without prior cell isolation. The homogenates were treated as
described above for gastric smooth muscle cells.
Data Analysis--
Results were expressed as means ± S.E.
of n separate experiments and evaluated statistically using
Student's t test for paired or unpaired data.
Concentration-response curves were analyzed using the P.fit 6.0 program.
Materials--
D-myo-Inositol
1,4,5-trisphosphate assay system,
myo-[3H]inositol, and
[35S]GTP
S were obtained from Amersham; fura
2-acetoxymethyl ester from Molecular Probes; U-73122 from Biomol,
Plymouth Meeting, PA; polyclonal antibodies to PLC-
1, PLC-
2,
PLC-
3, PLC-
4, and G
from Santa Cruz Biotechnology,
Santa Cruz, CA; polyclonal antibodies to G
i1,
G
o, G
i1-2, G
i3, G
q/11, and
G
s, and peptide fragments against which antibodies to
G
q/11 (QLNLKEYNLV) and
G
i3 (KNNKECGLY) were raised from Calbiochem;
G
i2 from Chemicon, Temecula, CA; DPCPX, AMP-PCP,
AMP-CPP, and 2-methylthio-ATP from Research Biochemicals, Natick, MA;
CGS-15943 from Ciba-Geigy, Summit, NJ; and all other reagents from
Sigma.
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RESULTS |
Contraction and Ca2+ Mobilization in Dispersed Smooth
Muscle Cells by Purine and Pyrimidine Nucleotides--
Exposure of
muscle cells to 1 µM UTP or ATP caused immediate
contraction that was virtually linear during the first 10 s and attained a peak in 30 s followed by a decline to lower levels (Fig. 1A). The biphasic time
course was identical to that observed with other contractile agonists
(38, 53). The peak response at 30 s was used to construct
concentration-response curves. Prolonged exposure of muscle cells to
purine or pyrimidine agonists resulted in time-dependent
desensitization that was more rapid with P2X receptor
agonists (e.g. AMP-PCP) than with P2Y receptor
agonists (e.g., UTP) (Fig. 1B). With either type
of agonist, however, there was minimal desensitization (<2% of
control response) during the initial 30-s period when peak response was
measured.

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Fig. 1.
Time course and rate of desensitization of
contractile response to purine and pyrimidine nucleotides.
A, time course of contraction in response to 1 µM
ATP and UTP. The initial response detected within 5 s was
virtually linear during the first 20 s and attained a peak at
30 s. Muscle cell contraction was measured by scanning micrometry
and expressed as percent decrease in cell length from control.
B, muscle cells were exposed for various intervals to either
1 µM UTP or 1 µM AMP-PCP
( , -MeATP). The cells were washed and peak response to
the same agonist was measured at 30 s by scanning micrometry. The
results are expressed as percent of the control response before
desensitization.
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UTP, ATP, and ATP analogs caused concentration-dependent
contraction of dispersed smooth muscle cells yielding curves with EC50 values of 33 ± 9 and 34 ± 6 nM
for ATP and UTP, respectively, 78 ± 17 and 85 ± 20 nM for AMP-PCP and AMP-CPP, respectively, and 178 ± 23 nM for 2-methylthio-ATP (Fig.
2). Except for the response to
2-methylthio-ATP, maximal contraction induced by all agonist (29 ± 3 to 30 ± 2% decrease in cell length) was similar to that
elicited by other contractile agonists, such as cholecystokinin octapeptide (30 ± 3%) or acetylcholine (31 ± 4%).

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Fig. 2.
Concentration-response curves for the
contractile effect of purine and pyrimidine nucleotides in dispersed
gastric muscle cells. A, contraction of dispersed gastric
muscle cells in response to ATP, UTP, AMP-PCP
( , -MeATP), AMP-CPP ( , -MeATP), and
2-methylthio-ATP (2-MeSATP) was measured by scanning
micrometry and the response expressed as percent decrease in cell
length from control (mean control cell length: 98 ± 3 µm). All
measurements were done in the presence of adenosine A1
(DPCPX) and A2 (CGS-15943) antagonists. B,
inhibition of ATP-induced contraction by 1 µM U-73122
alone or in the absence of Ca2+ from the medium (0 Ca2+ + 2 mM EGTA). Results are mean ± S.E. of four to seven experiments.
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ATP, UTP, AMP-PCP, and AMP-CPP increased cytosolic Ca2+
([Ca2+]i) in dispersed smooth muscle cells by
1-fold at 10 nM and by 3-fold at 10 µM (Table
II). The increase induced by 2-methylthio-ATP was also
concentration-dependent but significantly lower (Table II).
Contraction and the increase in [Ca2+]i induced
by AMP-PCP, AMP-CPP, and 2-methylthio-ATP were abolished by nifedipine (1 µM) and in Ca2+-free medium but were not
affected by pretreatment of the cells for 1 h with 400 ng/ml PTX,
or for 10 min with the PLC-
inhibitor, U-73122 (1 µM);
insertion of GDP
S into transiently permeabilized muscle cells had no
effect (Tables I and
II). The pattern of response suggested
that contraction and Ca2+ mobilization induced by ATP
analogs with high affinity for P2X receptors was mediated
by Ca2+ influx via dihydropyridine-sensitive
Ca2+ channels.
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Table I
Contraction induced by purine and pyrimidine nucleotides (10 nM and 10 µM) in dispersed smooth muscle
cells
Cell contraction was measured by scanning micrometry and expressed as
percent decrease in cell length from control (control cell length:
98 ± 3 µM). Muscle cells were pretreated for 10 min with U-73122 (1 µM) or 60 min with PTX (400 ng/ml). The
effect of GDP S (10 µM) was determined in reversibly
permeabilized muscle cells. Values are mean ± S.E. of four
experiments.
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Table II
Increase in [Ca2+]i induced by purine and pyrimidine
nucleotides in dispersed smooth muscle cells
[Ca2+]i was measured by fura 2 fluorescence and
expressed as nanomolar above basal level (range 48 ± 4 to 62 ± 5 nM). Cells were pretreated for 10 min with U-73122 (1 µM) or 60 min with PTX (400 ng/ml). The effect of GDP S
(10 µM) was determined in reversibly permeabilized muscle
cells. Values are mean ± S.E. of four experiments.
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In contrast, contraction and the increase in
[Ca2+]i induced by 10 nM or 10 µM UTP, and by 10 nM ATP were not affected by
nifedipine or Ca2+-free medium but were abolished by
GDP
S or U-73122 (Tables I and II). Pertussis toxin partly inhibited
contraction (46 ± 5 to 50 ± 7%) and the increase in
[Ca2+]i (49 ± 6 to 68 ± 7%) induced
by 10 nM and 10 µM UTP and by 10 nM ATP. The pattern suggested that contraction and
Ca2+ mobilization induced by UTP, which has high affinity
for P2Y2 receptors, and by low concentrations of ATP were
mediated by IP3-dependent Ca2+
release resulting from activation of PLC-
via both PTX-sensitive and
-insensitive G proteins.
Contraction induced by 10 µM ATP was not affected by
GDP
S, PTX, U-73122, nifedipine, and Ca2+-free medium
(Table I), while the increase in [Ca2+]i was only
slightly inhibited (13 ± 5 to 28 ± 6%) (Table II).
However, a combination of U-73122 or GDP
S with either nifedipine or
Ca2+-free medium abolished the contraction and the increase
in [Ca2+]i (Table I). Thus, contraction and the
increase in [Ca2+]i induced by high
concentrations of ATP could be independently mediated by
Ca2+ influx and Ca2+ release and appears to
reflect activation of both P2X and P2Y receptors.
The extent of ATP-induced contraction mediated by P2X
receptors was evaluated at different concentrations of ATP in the
presence of 1 µM U-73122. The concentration-response
curve was shifted to the right by U-73122 (Fig. 2), and the
EC50 for ATP acting via P2X receptors was
0.61 ± 0.07 µM (compared with 33 ± 9 nM in the absence of U-73122). In Ca2+-free
medium, the contractile response to all concentrations of ATP was
abolished by U-73122 (Fig. 2).
Stimulation of IP3 Formation in Dispersed Smooth Muscle
Cells by ATP and UTP--
Both ATP and UTP caused a prompt increase in
IP3 formation in dispersed smooth muscle cells (5.1 ± 0.4 and 5.2 ± 0.4 pmol/106 cells above basal level
with 10 µM ATP and UTP, respectively; basal levels:
3.3 ± 0.5 pmol/106 cells) (Fig.
3). IP3 formation induced by
ATP and UTP was partly inhibited by PTX (60 ± 7 and 63 ± 4%), and more potently by GDP
S (82 ± 5 and 80 ± 3%)
(Fig. 3). AMP-PCP, AMP-CPP, and 2-methylthio-ATP did not induce
IP3 formation in muscle cells (0.1 ± 0.2 to 0.7 ± 0.4 pmol/106 cells).

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Fig. 3.
Inhibition of IP3 formation
induced by ATP and UTP in dispersed smooth muscle by PTX and
GDP S. ATP and UTP were added at a concentration of 10 µM. IP3 mass was measured by radioreceptor assay and expressed as picomole/106 cells above basal
levels (control basal level: 3.3 ± 0.5 pmol/106
cells; basal level in reversibly permeabilized muscle cells: 3.4 ± 0.4 pmol/106 cells; basal level in PTX-treated muscle
cells: 3.3 ± 0.3 pmol/106 cells). The effect of
GDP S (10 µM) was determined in reversibly permeabilized muscle cells; the effect of PTX was determined in intact
muscle cells pretreated with 400 ng/ml for 1 h. Values are
mean ± S.E. of three to four experiments. **, significant inhibition, p < 0.01.
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Identification of PLC-
Isozymes and G Proteins Activated by ATP
and UTP in Smooth Muscle--
Western blot analysis disclosed the
presence of PLC-
1, PLC-
3, and PLC-
4 with minimal expression of
PLC-
2 in gastric muscle cell homogenates (Fig.
4). The analysis disclosed the presence also of Gq/11, Gs, Gi1,
Gi2, Gi3, and Go (Fig.
4). The pattern of G protein and PLC-
expression was similar to that
previously reported in intestinal muscle cell homogenates (41-43).

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Fig. 4.
Expression of G proteins and PLC-
isozymes in gastric smooth muscle. Homogenates were prepared from
dispersed gastric circular muscle cells and solubilized with sodium
cholate in Tris buffer. Proteins were resolved by SDS-polyacrylamide
gel electrophoresis, electrophoretically transferred to
nitrocellulose membranes, and probed with specific antibodies to the
PLC- isozymes and -subunits of various G proteins and then with
anti-rabbit IgG conjugated to horseradish peroxidase. The proteins were
identified by enhanced chemiluminescence.
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The isoforms of PLC-
and G proteins activated by ATP and UTP in
smooth muscle were identified by functional blockade with specific
antibodies. PLC-
antibodies and G protein antibodies were used at a
concentration of 10 µg/ml shown previously to be maximally effective
(41-43, 48, 51, 54). PLC-
activity ([3H]inositol
phosphate formation) in membranes derived from dispersed smooth muscle
cells increased by 218 ± 20 and 236 ± 54% with 10 µM ATP and UTP, respectively; AMP-PCP, AMP-CPP, and
2-methylthio-ATP had no effect (3 ± 4 to 9 ± 9%).
Pretreatment of plasma membranes for 1 h with 10 µg/ml PLC-
1
antibody or PLC-
3 antibody inhibited ATP-stimulated PLC-
activity
by 47 ± 5 and 59 ± 8%, respectively (Fig.
5). The effect of a combination of both
antibodies was additive eliciting complete inhibition (91 ± 6%)
(Fig. 5). Pretreatment with PLC-
2 or PLC-
4 antibody had no effect
on ATP-stimulated PLC-
activity (6 ± 5 and 8 ± 4%).
Identical results were obtained for UTP-stimulated PLC-
activity
which was inhibited 41 ± 7% by PLC-
1 antibody, 49 ± 8%
by PLC-
3 antibody, and 93 ± 5% by a combination of PLC-
1
and PLC-
3 antibodies (Fig. 5). The results implied that PI
hydrolysis induced by ATP and UTP was mediated additively by PLC-
1
and PLC-
3.

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Fig. 5.
Inhibition of ATP- and UTP-stimulated PLC-
activity in smooth muscle membranes by PLC- and G protein
antibodies. PLC- activity induced by 10 µM ATP
and UTP in the presence of 1 µM GTP S was measured in
plasma membranes isolated from dispersed smooth muscle cells. The
measurements were repeated in membranes treated for 1 h with 10 µg/ml of each antibody separately. Antibodies to PLC- 2, PLC- 4,
G o, G s, G i1-2, and
G i3 had no effect on ATP- or UTP-stimulated PLC activity and are not depicted. Results are expressed as counts/min/mg of protein/min of inositol phosphates ([3H]IPs) above
basal level (1105 ± 110 cpm/mg/min). Values are mean ± S.E.
of four experiments. **, significant inhibition, p < 0.01 to p < 0.001.
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PLC-
activity stimulated by ATP or UTP was inhibited by
G
q/11 antibody or a common antibody to
G
; the antibodies were used at a concentration of 10 µg/ml previously shown to be maximally effective (41-43, 48, 51,
54). PLC-
activity stimulated by ATP was inhibited 47 ± 8% by
G
q/11 antibody, 56 ± 6% by
G
antibody, and 92 ± 5% by a combination of both
antibodies (Fig. 5). G
i3,
G
i1-2, and G
o antibodies had no
effect on ATP-stimulated PLC-
activity (4 ± 5 to 5 ± 10%). Identical results were obtained for UTP-stimulated PLC-
activity which was inhibited 40 ± 7% by G
q/11
antibody, 57 ± 8% by G
antibody, and
92 ± 4% by a combination of both antibodies (Fig. 5).
The effect of a combination of PLC-
1 antibody and G
antibody was additive, eliciting complete inhibition of PLC-
activity (91 ± 4%), whereas the effect of a combination of
PLC-
3 antibody and G
antibody was not additive
(62 ± 6% inhibition by the combination versus 59 ± 8 and 56 ± 6% inhibition for either antibody alone).
Identical results were obtained for UTP-stimulated PLC-
activity
which was inhibited 90 ± 6% by the combination of PLC-
1 and
G
antibodies and 53 ± 7% by a combination of
PLC-
3 and G
antibodies (Fig. 5).
Pretreatment of the cells for 1 h with 400 ng/ml PTX before
membrane isolation inhibited ATP- and UTP-stimulated PLC-
activity by 59 ± 6 and 60 ± 7%, respectively (Fig.
6). ATP- and UTP-stimulated PLC-
activities were abolished by a combination of PTX with either PLC-
1
or G
q/11 antibody (96 ± 3 to 98 ± 5% inhibition). In contrast, inhibition by a combination of PTX with
either PLC-
3 or G
antibody was not significantly
different from inhibition by PTX, PLC-
3 antibody, or
G
antibody alone (range of inhibition 58 ± 5 to
61 ± 5%) (Fig. 6).

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Fig. 6.
Inhibition of ATP- and UTP-stimulated PLC-
activity in smooth muscle membranes by PLC- and G protein antibodies
in combination with PTX. PLC- activity induced by 10 µM ATP and UTP in the presence of 1 µM
GTP S was measured in plasma membranes isolated from dispersed smooth
muscle cells before and after treatment of the cells with 400 ng/ml PTX
for 1 h. Membranes isolated from cell pretreated with PTX were
incubated for 1 h with 10 µg/ml of each antibody separately.
Results are expressed as counts/min/mg protein/min of inositol
phosphates ([3H]IPs) above basal level (1089 ± 150 cpm/mg/min). Values are mean ± S.E. of four experiments. **,
significant inhibition, p < 0.01 to p < 0.001.
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The results implied that phosphoinositide (PI) hydrolysis induced by
ATP and UTP was mediated by
G
q/11-dependent activation of
PLC-
1, and by PTX-sensitive,
G
-dependent activation of PLC-
3. The
pattern is consistent with PTX-sensitive and -insensitive stimulation
of IP3 formation in dispersed muscle cells by ATP and UTP
(Fig. 3).
Identification of G Proteins Coupled to P2Y
Receptors--
The PTX-sensitive and -insensitive G protein(s)
activated by ATP and UTP in gastric smooth muscle were identified by a
technique that did not involve functional blockade with antibodies.
Solubilized muscle cell membranes were incubated with
[35S]GTP
S (60 nM) with or without ATP or
UTP and added to wells precoated with different G
antibodies; an
increase in the binding of [35S]GTP
S·G
complexes
to a specific G
antibody reflected activation of the corresponding G
protein. Addition of ATP (10 µM) caused a
time-dependent increase in the binding of
[35S]GTP
S to G
q/11 and
G
i3 antibodies (Fig. 7), but not to G
s
G
i1-2 or G
o antibodies (Table
III). An identical pattern was observed
with UTP which stimulated the binding of [35S]GTP
S to
G
q/11 and G
i3 antibodies
but not to G
s, G
i1-2, or
G
o antibodies (Table III). Pretreatment of muscle cells with
400 ng/ml PTX for 1 h before membrane isolation abolished the ATP-
and UTP-stimulated increase in steady-state binding of
[35S]GTP
S to G
i3 antibody, but
not to G
q/11 antibody (Table III).

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Fig. 7.
Time course of binding of
ATP-stimulated GTP S·G complexes in smooth
muscle membranes to G i3 and G q/11 antibody.
Membranes isolated from dispersed smooth muscle cells were solubilized
and incubated with [35S]GTP S in the presence or
absence of 10 µM ATP for various periods of time.
Aliquots were added to wells precoated with G i3 or G q/11 antibody for 2 h and bound
radioactivity measured (counts/min/mg of protein). ATP caused a
significant increase in binding of [35S]GTP S·G
complexes to wells precoated with G i3 antibody (A) or G q/11 antibody (B)
but not to wells precoated with G i1-2,
G s, or G o antibody (see Table III). Values are
mean ± S.E. of four experiments.
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Table III
Binding of GTP S·G complexes stimulated by purine and pyrimidine
nucleotides in smooth muscle membranes to G protein antibodies (Ab)
CHAPS-solubilized membranes were incubated for 20 min with
[35S]GTP S alone or with various agonists and then added to
wells precoated with various G antibodies.
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Peptide I (KNNKECGLY), comprising the G protein sequence against which
the G
i3 antibody was raised, inhibited
selectively ATP- and UTP-stimulated activation of
G
i3 (Table III). Conversely, peptide II
(QLNLKEYNLV), comprising the G protein sequence against which
G
q/11 antibody was raised, inhibited selectively
ATP- and UTP-stimulated activation of G
q/11 (Table III). Peptides I and II inhibited both activation of G proteins by GTP
S as well as the increase in activation induced by ATP or UTP.
Peptides I and II were used at a concentration (1 µM) previously shown to abolish activation of G
i3
and G
q/11, respectively (48). AMP-PCP, AMP-CPP,
and 2-methylthio-ATP did not cause activation of
G
q/11, G
i1-2, G
i3, G
s, and G
o (Table
III).
To determine whether P2Y receptors were invariably coupled
to the same G proteins, similar measurements were done on solubilized membranes from rabbit aortic smooth muscle, heart, liver, and whole
brain (Figs. 8 and
9). The results obtained in heart and vascular smooth muscle membranes were identical to those obtained in
visceral smooth muscle membranes: UTP and ATP activated
Gq/11 and Gi3 but not
Gi1, Gi2, Go, or
Gs (Fig. 8). In liver, ATP and UTP activated
Gq/11 and Gi1 and/or
Gi2, but did not activate Gi3, Go, or Gs (Fig. 9). In contrast to vascular and
visceral smooth muscle, heart, and liver, ATP and UTP activated predominantly Go, as well as Gi3 and
Gi1 and/or Gi2 in brain
membranes, but did not activate Gq/11 or Gs
(Fig. 9). The extent of activation of specific G proteins by ATP and
UTP was similar in all tissues except brain where activation of all
inhibitory G proteins by ATP was more pronounced, suggesting
interaction of ATP with P2Y2 receptors and ATP-preferring
P2Y1 receptors.

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Fig. 8.
Binding of ATP- and UTP-stimulated
GTP S·G complexes to G protein antibodies in solubilized
membranes from aortic muscle and heart. Membranes isolated from
rabbit aortic smooth muscle and heart were solubilized and incubated
with [35S]GTP S in the presence or absence of 10 µM ATP or UTP for 20 min. Aliquots were added to wells
precoated with various G antibodies for 2 h and bound
radioactivity measured (counts/min/mg of protein). ATP and UTP caused
significant (p < 0.01) increases in binding of
[35S]GTP S·G complexes to
G i3 antibody and G q/11 antibody but not to G i1-2, G s, or
G o antibody. Values are mean ± S.E. of five
experiments. **, significant increase above control GTP S binding,
p < 0.01.
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Fig. 9.
Binding of ATP- and UTP-stimulated
GTP S·G complexes to G protein antibodies in solubilized
membranes from liver and brain. Membranes isolated from rabbit
liver and whole brain were solubilized and incubated with
[35S]GTP S in the presence or absence of 10 µM ATP or UTP for 20 min. Aliquots were added to wells
precoated with various G antibodies for 2 h and bound
radioactivity measured (counts/min/mg of protein). ATP and UTP caused
significant (p < 0.01) increases in binding of
[35S]GTP S·G complexes to
G q/11 and G i1-2 antibodies in liver membranes, and to G o,
G i1-2, and Gi3 antibodies
in brain membranes. Values are mean ± S.E. of five experiments.
**, significant increase above control GTP S binding,
p < 0.01. The increase induced by ATP in brain membranes was significantly greater than that induced by UTP, p < 0.02.
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Identification of P2Y and P2X Receptors in
Smooth Muscle Cells by Selective Receptor Protection--
The pattern
of PI hydrolysis, IP3 formation, Ca2+
mobilization and contraction suggested that UTP and ATP interacted with
a common P2Y receptor coupled to PTX-sensitive and
-insensitive G proteins, whereas AMP-PCP, AMP-CPP, and 2-methylthio-ATP
interacted with a distinct ligand-gated P2X receptor; the
latter was also activated by high concentrations of ATP. This notion
was corroborated by selective receptor protection to enrich the muscle
cells with one receptor subtype. After selective receptor protection,
muscle cells were incubated for 60 min in control medium to allow
complete resensitization of the cells (see "Experimental
Procedures").
Receptor protection with 10 nM AMP-PCP preserved completely
the contractile response to 10 nM AMP-PCP (15 ± 3%
decrease in cell length) and AMP-CPP (14 ± 2%) (see Fig. 2 and
Table I for comparison with responses to untreated muscle cells), but
not the responses to ATP or UTP. An identical pattern was obtained by
receptor protection with 10 nM AMP-CPP. In contrast,
receptor protection with 10 nM UTP preserved completely the
responses to 10 nM UTP (15 ± 1%) and ATP (13 ± 2%), but not the responses to AMP-PCP and AMP-CPP. An identical
pattern was obtained by receptor protection with 10 nM ATP.
Receptor protection with a high concentration of ATP (10 µM) preserved completely the responses to UTP and ATP as
well as the responses to AMP-PCP and AMP-CPP, implying that at this
concentration ATP interacted with P2X and P2Y
receptors on muscle cells.
After selective protection of P2Y receptors with UTP, the
contractile response to a high concentration of ATP (10 µM) could be abolished by U-73122 (control response:
28 ± 1% decrease in cell length; with U-73122: 3 ± 2%)
implying that it was exclusively mediated by PI hydrolysis. Following
desensitization of ligand-gated P2X receptors by
preincubation of muscle cells for 30 min with 10 µM
AMP-PCP, the contractile response to 10 µM ATP was
virtually abolished by GDP
S and U-73122 (control, 30 ± 2%
decrease in cell length; GDP
S, 3 ± 1%; U-73122, 4 ± 2%) and partly inhibited by PTX (control, 30 ± 2%; PTX, 13 ± 2%). Thus, after selective desensitization of P2X
receptors or selective protection of P2Y receptors, the response to a high concentration of ATP (10 µM) reflected
exclusively activation of G protein-coupled pathways.
 |
DISCUSSION |
This study demonstrates the co-existence of ligand-gated
P2X and G protein-coupled P2Y receptors on
freshly dispersed gastric smooth muscle cells and suggests that ATP
activates preferentially P2Y receptors to elicit
Ca2+ mobilization and muscle contraction. The
P2Y receptors selectively activated by UTP and ATP were
coupled to PLC-
1 via G
q/11 and to PLC-
3
via G
i3. Concurrent activation of the two
effector enzymes resulted in PTX-sensitive and -insensitive IP3 formation and IP3-dependent
Ca2+ release from sarcoplasmic stores. The high affinity
for UTP and ATP suggested that these were P2Y2 receptors:
UTP-selective P2Y4 receptors and UDP-selective
P2Y6 receptors coupled to PLC-
can be expressed in
smooth muscle but they exhibit low or minimal affinity for ATP (11,
19). The P2X receptors selectively activated by AMP-PCP and
AMP-CPP-mediated Ca2+ influx via dihydropyridine-sensitive,
voltage-gated Ca2+ channels; the Ca2+ channels
were activated by depolarization of the plasma membrane that resulted
from the opening of ligand-gated cation P2X
receptor/channels (22, 23). Their presence on smooth muscle (which
predominantly expresses P2X1 receptors (21)), and their
activation by AMP-CPP (which selectively interacts with P2X
receptors on smooth muscle (36)), and by AMP-PCP (which interacts with
P2X1 and P2X3 receptors (23, 24, 36)),
suggested that the receptors were of the P2X1 subtype.
Although the activity profile in visceral smooth muscle (AMP-PCP = AMP-CPP > 2-methylthio-ATP > ATP) resembled that seen in
vascular smooth muscle (17), it differed from the activity profile
determined in patch-clamp studies of the cloned human and rat
P2X1 receptors where ATP and 2-methylthio-ATP were more
potent than AMP-PCP (4, 5, 23). It seems unlikely that the difference
reflected degradation of ATP or 2-methylthio-ATP by ecto-nucleotidases,
since the measurements of response, particularly those of
[Ca2+]i, in both visceral and vascular smooth
muscle were virtually instantaneous (<2 s), and the ratio of medium to
cell volume (5000:1) was very high. It is possible that the rabbit P2X1 receptor is different from the human and rat homologs
or that its conformation or extent of polymerization is different when
it is expressed in smooth muscle.
The evidence for the co-existence and subtype of P2Y and
P2X receptors in smooth muscle may be summarized as
follows. First, ATP and UTP stimulated IP3 formation,
Ca2+ release, and contraction in dispersed smooth muscle
cells. The responses to UTP and low nanomolar concentrations of ATP
were abolished by GDP
S and the PLC-
inhibitor, U-73122, and
partly inhibited by PTX, implying the participation of PTX-sensitive and -insensitive G proteins in IP3 formation and
IP3-dependent Ca2+ release. In
contrast, ATP analogs with high affinity for P2X receptors
did not stimulate IP3 formation; contraction and the increase in [Ca2+]i were abolished by nifedipine
and in Ca2+-free medium implying that they were mediated by
Ca2+ influx. Higher micromolar concentrations of ATP
stimulated both Ca2+ influx and
IP3-dependent Ca2+ release. A
similar pattern was observed by Pacaud et al. (17) for the
[Ca2+]i response in single aortic smooth muscle
cells.
Second, the interaction of ATP analogs with P2X
receptors, and UTP and ATP with P2Y receptors was
corroborated in experiments using smooth muscle cells enriched with one
receptor subtype. The validity of this approach was previously
established for several agonists (44-48). Selective protection of
P2Y2 receptors with either UTP or ATP preserved the
contractile response to both UTP and ATP, whereas selective protection
of P2X1 receptors with either AMP-PCP or AMP-CPP preserved
the response to both analogs and to high concentrations of ATP.
Third, ATP and UTP activated both Gq/11 and
Gi3 in muscle membranes; activation of
Gi3 was suppressed by pretreatment of muscle cells
with PTX. PI hydrolysis stimulated by ATP and UTP in plasma membranes
was mediated concurrently by the
-subunit of
Gq/11 and the 
-subunits of
Gi3: the
-subunit of Gq/11
activated PLC-
1, whereas the 
-subunits of
Gi3 activated PLC-
3. The activation of PLC-
1
by G
q/11 and PLC-
3 by
G
i3 were independent and additive.
G
q/11 and G
antibodies elicited
partial inhibition of PI hydrolysis separately and complete inhibition in combination; similarly, PLC-
1 and PLC-
3 antibodies elicited partial inhibition separately and complete inhibition in combination. Complete inhibition was also obtained by combining PLC-
1 and G
antibodies, PLC-
1 antibody and PTX, and
G
qq/11 antibody and PTX. It should be emphasized
that PLC-
or G protein antibodies were used at maximally effective
concentrations (41-43, 48, 51, 54), so that when additive effects were
observed with combinations of antibodies, they reflected the
involvement of both G proteins or PLC-
isoforms. The specificities
of these interactions were confirmed by studies with antibodies to
other PLC-
isoforms (PLC-
2 and PLC-
4) and G proteins
(G
s, G
i1-2, and G
o), none
of which inhibited PI hydrolysis.
The activation of PLC-
3 by the 
-subunits of
Gi3 in smooth muscle cells conformed to a pattern
previously established for other agonists (41-43). Thus, PI hydrolysis
induced by somatostatin (via somatostatin type 3 receptors) was
mediated by the 
-subunits of Gi1 and
Go (41), whereas PI hydrolysis induced by opioid agonists (via
µ,
, and
receptors) was mediated by the 
-subunits of
Gi2 and Go (42); PI hydrolysis induced by
adenosine (via A1 receptors) was distinctive in that it
required activation of PLC-
3 by both
- and 
-subunits of
Gi3 (43).
Comparative studies in other tissues to determine whether coupling of
P2Y receptors to Gq/11 and
Gi3 was an invariant characteristic showed
identical coupling to these G proteins in vascular smooth muscle and
heart. Coupling in hepatocytes was to Gq and
Gi1 and/or Gi2. Coupling in
brain was confined to inhibitory G proteins, particularly Go, but also Gi1, Gi2, and
Gi3. The extent of activation of G proteins in
visceral and vascular smooth muscle, heart, and liver was similar for
ATP and UTP, suggesting interaction with P2Y2 receptors.
Activation of inhibitory G proteins in brain was greater for ATP than
UTP, suggesting interaction with P2Y2 receptors and
ATP-preferring P2Y1 receptors (55).
It is noteworthy that ATP exhibited a 200-fold higher affinity for
P2Y2 receptors than for P2X1 receptors in
smooth muscle cells. Ca2+ mobilization and contraction
induced by nanomolar concentrations of ATP were mediated exclusively by
IP3-dependent Ca2+ release. At
higher concentrations (0.1 to 100 µM), ATP interacted additionally with P2X1 receptors eliciting both
Ca2+ influx and IP3-dependent
Ca2+ release. Whether P2X1 receptors
participate in the physiological response of smooth muscle to
endogenous ATP would depend on the ambient concentration of ATP. ATP
released from cells is rapidly metabolized by ecto-nucleotidases and
ecto-nucleoside diphosphokinases (18-20). ATP is co-released with
soluble nucleotidases at neuromuscular junctions where it is rapidly
hydrolyzed to adenosine (56). In the present study, both adenosine
A2 and A1 antagonists were added to the medium
to ensure against adventitious effects resulting from degradation of
ATP. As shown previously (43), adenosine acting on A1
receptors coupled via Gi3 to PLC-
3 can mimic to
some extent the responses mediated by ATP. Recent studies (57) suggest
that UTP like ATP can be released by mechanical stimulation raising the
possibility that P2Y receptors with high affinity for UTP
(P2Y2 and P2Y4) and UDP (P2Y3 and
P2Y6) may have a functional role (14, 19). The protean
nature of purine and pyrimidine nucleotides which can be modified by
ecto-nucleotidases or interconverted by nucleoside diphosphokinases
determines to some extent which specific P2Y receptor
subtypes are activated.