Coexpression of Ligand-gated P2X and G Protein-coupled P2Y Receptors in Smooth Muscle
PREFERENTIAL ACTIVATION OF P2Y RECEPTORS COUPLED TO PHOSPHOLIPASE C (PLC)-beta 1 VIA Galpha q/11 AND TO PLC-beta 3 VIA Gbeta gamma i3*

Karnam S. Murthy and Gabriel M. MakhloufDagger

From the Departments of Medicine and Physiology, Medical College of Virginia, Richmond, Virginia 23298-0711

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

P2 receptor subtypes and their signaling mechanisms were characterized in dispersed smooth muscle cells. UTP and ATP stimulated inositol 1,4,5-triphosphate formation, Ca2+ release, and contraction that were abolished by U-73122 and guanosine 5'-O-(3-thio)diphosphate, and partly inhibited (50-60%) by pertussis toxin (PTX). ATP analogs (adenosine 5'-(alpha ,beta -methylene)triphosphate, adenosine 5'-(beta ,gamma -methylene)triphosphate, and 2-methylthio-ATP) stimulated Ca2+ influx and contraction that were abolished by nifedipine and in Ca2+-free medium. Micromolar concentrations of ATP stimulated both Ca2+ influx and Ca2+ release.

ATP and UTP activated Gq/11 and Gi3 in gastric and aortic smooth muscle and heart membranes, Gq/11 and Gi1 and/or Gi2 in liver membranes, and Go and Gi1-3 in brain membranes. Phosphoinositide hydrolysis stimulated by ATP and UTP was mediated concurrently by Galpha q/11-dependent activation of phospholipase (PL) C-beta 1 and Gbeta gamma i3-dependent activation of PLC-beta 3. Phosphoinositide hydrolysis was partially inhibited by PTX or by antibodies to Galpha q/11, Gbeta , PLC-beta 1, or PLC-beta 3, and completely inhibited by the following combinations (PLC-beta 1 and PLC-beta 3 antibodies; Galpha q/11 and Gbeta antibodies; PLC-beta 1 and Gbeta antibodies; PTX with either PLC-beta 1 or Galpha q/11 antibody).

The pattern of responses implied that P2Y2 receptors in visceral, and probably vascular, smooth muscle are coupled to PLC-beta 1 via Galpha q/11 and to PLC-beta 3 via Gbeta gamma i3. These receptors co-exist with ligand-gated P2X1 receptors activated by ATP analogs and high levels of ATP.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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-beta (PLC-beta ) (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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 GDPbeta 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 GDPbeta 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-beta Isozymes in Gastric Smooth Muscle by Western Blot-- The expression of G proteins and PLC-beta 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-beta 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-beta Activity in Plasma Membranes-- PLC-beta 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-beta 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 GTPgamma 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-beta 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]GTPgamma 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]GTPgamma S were obtained from Amersham; fura 2-acetoxymethyl ester from Molecular Probes; U-73122 from Biomol, Plymouth Meeting, PA; polyclonal antibodies to PLC-beta 1, PLC-beta 2, PLC-beta 3, PLC-beta 4, and Gbeta from Santa Cruz Biotechnology, Santa Cruz, CA; polyclonal antibodies to Galpha i1, Galpha o, Galpha i1-2, Galpha i3, Galpha q/11, and Galpha s, and peptide fragments against which antibodies to Galpha q/11 (QLNLKEYNLV) and Galpha i3 (KNNKECGLY) were raised from Calbiochem; Galpha 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.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 (alpha ,beta -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.

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 (alpha ,beta -MeATP), AMP-CPP (beta ,gamma -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.

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-beta inhibitor, U-73122 (1 µM); insertion of GDPbeta 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 GDPbeta 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 GDPbeta S (10 µM) was determined in reversibly permeabilized muscle cells. Values are mean ± S.E. of four experiments.

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 GDPbeta 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-beta via both PTX-sensitive and -insensitive G proteins.

Contraction induced by 10 µM ATP was not affected by GDPbeta 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 GDPbeta 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 GDPbeta 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 GDPbeta 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 GDPbeta 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.

Identification of PLC-beta Isozymes and G Proteins Activated by ATP and UTP in Smooth Muscle-- Western blot analysis disclosed the presence of PLC-beta 1, PLC-beta 3, and PLC-beta 4 with minimal expression of PLC-beta 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-beta 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-beta 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-beta isozymes and alpha -subunits of various G proteins and then with anti-rabbit IgG conjugated to horseradish peroxidase. The proteins were identified by enhanced chemiluminescence.

The isoforms of PLC-beta and G proteins activated by ATP and UTP in smooth muscle were identified by functional blockade with specific antibodies. PLC-beta 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-beta 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-beta 1 antibody or PLC-beta 3 antibody inhibited ATP-stimulated PLC-beta 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-beta 2 or PLC-beta 4 antibody had no effect on ATP-stimulated PLC-beta activity (6 ± 5 and 8 ± 4%). Identical results were obtained for UTP-stimulated PLC-beta activity which was inhibited 41 ± 7% by PLC-beta 1 antibody, 49 ± 8% by PLC-beta 3 antibody, and 93 ± 5% by a combination of PLC-beta 1 and PLC-beta 3 antibodies (Fig. 5). The results implied that PI hydrolysis induced by ATP and UTP was mediated additively by PLC-beta 1 and PLC-beta 3.


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Fig. 5.   Inhibition of ATP- and UTP-stimulated PLC-beta activity in smooth muscle membranes by PLC-beta and G protein antibodies. PLC-beta activity induced by 10 µM ATP and UTP in the presence of 1 µM GTPgamma 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-beta 2, PLC-beta 4, Galpha o, Galpha s, Galpha i1-2, and Galpha 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.

PLC-beta activity stimulated by ATP or UTP was inhibited by Galpha q/11 antibody or a common antibody to Gbeta ; the antibodies were used at a concentration of 10 µg/ml previously shown to be maximally effective (41-43, 48, 51, 54). PLC-beta activity stimulated by ATP was inhibited 47 ± 8% by Galpha q/11 antibody, 56 ± 6% by Gbeta antibody, and 92 ± 5% by a combination of both antibodies (Fig. 5). Galpha i3, Galpha i1-2, and Galpha o antibodies had no effect on ATP-stimulated PLC-beta activity (4 ± 5 to 5 ± 10%). Identical results were obtained for UTP-stimulated PLC-beta activity which was inhibited 40 ± 7% by Galpha q/11 antibody, 57 ± 8% by Gbeta antibody, and 92 ± 4% by a combination of both antibodies (Fig. 5).

The effect of a combination of PLC-beta 1 antibody and Gbeta antibody was additive, eliciting complete inhibition of PLC-beta activity (91 ± 4%), whereas the effect of a combination of PLC-beta 3 antibody and Gbeta 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-beta activity which was inhibited 90 ± 6% by the combination of PLC-beta 1 and Gbeta antibodies and 53 ± 7% by a combination of PLC-beta 3 and Gbeta antibodies (Fig. 5).

Pretreatment of the cells for 1 h with 400 ng/ml PTX before membrane isolation inhibited ATP- and UTP-stimulated PLC-beta activity by 59 ± 6 and 60 ± 7%, respectively (Fig. 6). ATP- and UTP-stimulated PLC-beta activities were abolished by a combination of PTX with either PLC-beta 1 or Galpha q/11 antibody (96 ± 3 to 98 ± 5% inhibition). In contrast, inhibition by a combination of PTX with either PLC-beta 3 or Gbeta antibody was not significantly different from inhibition by PTX, PLC-beta 3 antibody, or Gbeta 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-beta activity in smooth muscle membranes by PLC-beta and G protein antibodies in combination with PTX. PLC-beta activity induced by 10 µM ATP and UTP in the presence of 1 µM GTPgamma 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.

The results implied that phosphoinositide (PI) hydrolysis induced by ATP and UTP was mediated by Galpha q/11-dependent activation of PLC-beta 1, and by PTX-sensitive, Gbeta gamma -dependent activation of PLC-beta 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]GTPgamma S (60 nM) with or without ATP or UTP and added to wells precoated with different Galpha antibodies; an increase in the binding of [35S]GTPgamma S·Galpha complexes to a specific Galpha antibody reflected activation of the corresponding G protein. Addition of ATP (10 µM) caused a time-dependent increase in the binding of [35S]GTPgamma S to Galpha q/11 and Galpha i3 antibodies (Fig. 7), but not to Galpha s Galpha i1-2 or Galpha o antibodies (Table III). An identical pattern was observed with UTP which stimulated the binding of [35S]GTPgamma S to Galpha q/11 and Galpha i3 antibodies but not to Galpha s, Galpha i1-2, or Galpha 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]GTPgamma S to Galpha i3 antibody, but not to Galpha q/11 antibody (Table III).


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Fig. 7.   Time course of binding of ATP-stimulated GTPgamma S·Galpha complexes in smooth muscle membranes to Galpha i3 and Galpha q/11 antibody. Membranes isolated from dispersed smooth muscle cells were solubilized and incubated with [35S]GTPgamma S in the presence or absence of 10 µM ATP for various periods of time. Aliquots were added to wells precoated with Galpha i3 or Galpha q/11 antibody for 2 h and bound radioactivity measured (counts/min/mg of protein). ATP caused a significant increase in binding of [35S]GTPgamma S·Galpha complexes to wells precoated with Galpha i3 antibody (A) or Galpha q/11 antibody (B) but not to wells precoated with Galpha i1-2, Galpha s, or Galpha o antibody (see Table III). Values are mean ± S.E. of four experiments.

                              
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Table III
Binding of GTPgamma S·Galpha 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]GTPgamma S alone or with various agonists and then added to wells precoated with various Galpha antibodies.

Peptide I (KNNKECGLY), comprising the G protein sequence against which the Galpha i3 antibody was raised, inhibited selectively ATP- and UTP-stimulated activation of Galpha i3 (Table III). Conversely, peptide II (QLNLKEYNLV), comprising the G protein sequence against which Galpha q/11 antibody was raised, inhibited selectively ATP- and UTP-stimulated activation of Galpha q/11 (Table III). Peptides I and II inhibited both activation of G proteins by GTPgamma 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 Galpha i3 and Galpha q/11, respectively (48). AMP-PCP, AMP-CPP, and 2-methylthio-ATP did not cause activation of Galpha q/11, Galpha i1-2, Galpha i3, Galpha s, and Galpha 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 GTPgamma S·Galpha complexes to Galpha 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]GTPgamma S in the presence or absence of 10 µM ATP or UTP for 20 min. Aliquots were added to wells precoated with various Galpha 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]GTPgamma S·Galpha complexes to Galpha i3 antibody and Galpha q/11 antibody but not to Galpha i1-2, Galpha s, or Galpha o antibody. Values are mean ± S.E. of five experiments. **, significant increase above control GTPgamma S binding, p < 0.01.


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Fig. 9.   Binding of ATP- and UTP-stimulated GTPgamma S·Galpha complexes to Galpha protein antibodies in solubilized membranes from liver and brain. Membranes isolated from rabbit liver and whole brain were solubilized and incubated with [35S]GTPgamma S in the presence or absence of 10 µM ATP or UTP for 20 min. Aliquots were added to wells precoated with various Galpha 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]GTPgamma S·Galpha complexes to Galpha q/11 and Galpha i1-2 antibodies in liver membranes, and to Galpha o, Galpha i1-2, and Gi3 antibodies in brain membranes. Values are mean ± S.E. of five experiments. **, significant increase above control GTPgamma S binding, p < 0.01. The increase induced by ATP in brain membranes was significantly greater than that induced by UTP, p < 0.02.

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 GDPbeta S and U-73122 (control, 30 ± 2% decrease in cell length; GDPbeta 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
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Abstract
Introduction
Procedures
Results
Discussion
References

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-beta 1 via Galpha q/11 and to PLC-beta 3 via Gbeta gamma 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-beta 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 GDPbeta S and the PLC-beta 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 alpha -subunit of Gq/11 and the beta gamma -subunits of Gi3: the alpha -subunit of Gq/11 activated PLC-beta 1, whereas the beta gamma -subunits of Gi3 activated PLC-beta 3. The activation of PLC-beta 1 by Galpha q/11 and PLC-beta 3 by Gbeta gamma i3 were independent and additive. Galpha q/11 and Gbeta antibodies elicited partial inhibition of PI hydrolysis separately and complete inhibition in combination; similarly, PLC-beta 1 and PLC-beta 3 antibodies elicited partial inhibition separately and complete inhibition in combination. Complete inhibition was also obtained by combining PLC-beta 1 and Gbeta antibodies, PLC-beta 1 antibody and PTX, and Galpha qq/11 antibody and PTX. It should be emphasized that PLC-beta 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-beta isoforms. The specificities of these interactions were confirmed by studies with antibodies to other PLC-beta isoforms (PLC-beta 2 and PLC-beta 4) and G proteins (Galpha s, Galpha i1-2, and Galpha o), none of which inhibited PI hydrolysis.

The activation of PLC-beta 3 by the beta gamma -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 beta gamma -subunits of Gi1 and Go (41), whereas PI hydrolysis induced by opioid agonists (via µ, delta , and kappa  receptors) was mediated by the beta gamma -subunits of Gi2 and Go (42); PI hydrolysis induced by adenosine (via A1 receptors) was distinctive in that it required activation of PLC-beta 3 by both alpha - and beta gamma -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-beta 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.

    FOOTNOTES

* This work was supported by Grant DK-28300 from the NIDDK, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: P. O. Box 980711, Medical College of Virginia, Richmond, VA 23298-0711. Tel.: 804-828-9601; Fax: 804-828-2500.

1 The abbreviations used are: AMP-PCP, adenosine 5'-(alpha ,beta -methylene)triphosphate; AMP-CPP, adenosine 5'-(beta ,gamma -methylene)triphosphate; CHAPS, 3-[(cholamidopropyl)dimethylammonio]-1-propane sulfonic acid; IP3, inositol 1,4,5-trisphosphate; GDPbeta S, guanosine 5'-O-(3-thio)diphosphate; DPCPX, cyclopentyl-1,3-dipropylxanthine; CGS- 15943, 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine; PTX,pertussis toxin; GTPgamma S, guanosine 5'-O-(3-thio)triphosphate; PLC, phospholipase C; [Ca2+]i, intracellular Ca2+; PI, phosphoinositol.

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Abstract
Introduction
Procedures
Results
Discussion
References

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