PGF2alpha -induced contraction of cat esophageal and lower esophageal sphincter circular smooth muscle

Weibiao Cao, Karen M. Harnett, Jose Behar, and Piero Biancani

Department of Medicine, Rhode Island Hospital and Brown University School of Medicine, Providence, Rhode Island 02903


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lower esophageal sphincter (LES) tone depends on PGF2alpha and thromboxane A2 acting on receptors linked to Gi3 and Gq to activate phospholipases and produce second messengers resulting in muscle contraction. We therefore examined PGF2alpha signal transduction in circular smooth muscle cells isolated by enzymatic digestion from cat esophagus (Eso) and LES. In Eso, PGF2alpha -induced contraction was inhibited by antibodies against the alpha -subunit of G13 and the monomeric G proteins RhoA and ADP-ribosylation factor (ARF)1 and by the C3 exoenzyme of Clostridium botulinum. A [35S]GTPgamma S-binding assay confirmed that G13, RhoA, and ARF1 were activated by PGF2alpha . Contraction of Eso was reduced by propranolol, a phospholipase D (PLD) pathway inhibitor and by chelerythrine, a PKC inhibitor. In LES, PGF2alpha -induced contraction was inhibited by antibodies against the alpha -subunit of Gq and Gi3, and a [35S]GTPgamma S-binding assay confirmed that Gq and Gi3 were activated by PGF2alpha . PGF2alpha -induced contraction of LES was reduced by U-73122 and D609 and unaffected by propranolol. At low PGF2alpha concentration, contraction was blocked by chelerythrine, whereas at high concentration, contraction was blocked by chelerythrine and CGS9343B. Thus, in Eso, PGF2alpha activates a PLD- and protein kinase C (PKC)-dependent pathway through G13, RhoA, and ARF1. In LES, PGF2alpha receptors are coupled to Gq and Gi3, activating phosphatidylinositol- and phosphatidylcholine-specific phospholipase C. At low concentrations, PGF2alpha activates PKC. At high concentration, it activates both a PKC- and a calmodulin-dependent pathway.

smooth muscle contraction; prostaglandins; G proteins; phospholipases


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE SIGNAL-TRANSDUCTION PATHWAYS mediating the esophagus (Eso) and lower esophageal sphincter (LES) contraction in response to ACh have been previously described (43) in our laboratory. ACh-induced contraction of cat Eso is mediated by muscarinic M2 receptors coupled to pertussis toxin-sensitive Gi3-type G protein activation of phosphatidylcholine-specific phospholipase C (PI-PLC) and D (PI-PLD), resulting in the production of diacylglycerol (DAG) and activation of a protein kinase C (PKC)epsilon -dependent pathway.

In contrast, two distinct contractile signal-transduction pathways are present in LES muscle cells. A PI-PLC, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], calmodulin-dependent pathway is activated by stimulation with a maximally effective dose of ACh. In this pathway, M3 muscarinic receptors linked to Gq/11-type G proteins stimulate PLC, resulting in the formation of Ins(1,4,5)P3 and DAG. Ins(1,4,5)P3 causes the release of Ca2+ from intracellular stores, producing a calcium-calmodulin complex, myosin light chain phosphorylation, and contraction (3). This pathway is PKC independent, because maximal activation of calmodulin inhibits PKC activity (3, 24, 49).

A distinct PKC-dependent pathway is activated by submaximal doses of ACh or during maintenance of LES tone (3, 20). In this pathway, contraction is mediated by low levels of PLC activity, resulting in low levels of Ins(1,4,5)P3, which cause the release of low levels of Ca2+ from intracellular stores. These low Ca2+ levels are insufficient to activate a calmodulin-dependent contraction, which requires micromolar Ca2+ concentrations (3). In addition, concurrent activity of a phosphatidylcholine-specific (PC-PLC) in the LES contributes to the production of DAG. Low levels of Ins(1,4,5)P3 act synergistically with DAG to activate a PKC-dependent pathway (20). Thus the amount of Ca2+ available for contraction determines which pathway will be followed, with low Ca2+ levels activating a PKC-dependent pathway and high levels activating a calmodulin-dependent pathway.

In addition, LES tone may be maintained by a low molecular mass (14 kDa) group I-like secreted phospholipase A (PLA)2, which produces arachidonic acid (AA) and AA metabolites, such as PGF2alpha and thromboxanes A2 and B2, which maintain activation of the G proteins coupled to PC-PLC and PI-PLC (11).

In the current study, we found that in LES, PGF2alpha -induced contraction was coupled to the heterotrimeric G proteins Gq and Gi3 and linked to activation of PI-PLC and PC-PLC. Contraction of LES in response to threshold PGF2alpha was mediated by PKC activation, and, at high doses, by both PKC and calmodulin activation. In the Eso, the signal-transduction pathway mediating PGF2a-induced contraction differs from ACh-stimulated signaling. PGF2alpha -induced contraction of Eso was coupled to the heterotrimeric G protein G13 and the monomeric G proteins RhoA and ADP-ribosylation factor (ARF)1 and linked to activation of PLD. In this respect, PGF2alpha -induced contraction activates a signaling pathway similar to the one activated by prolonged (10 min) exposure to CCK in circular smooth muscle cells from the rabbit small intestine (33). In the Eso, however, the physiological significance of prolonged contraction is uncertain, because the Eso contracts only briefly in response to swallowing.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and preparation of circular smooth muscle tissue. Adult male cats weighing between 3.5 and 5.5 kg were initially anesthetized with ketamine (Aveco, Fort Dodge IA), then euthanized with an overdose of pentobarbital sodium (Schering, Kennilworth, NJ). The chest and abdomen were opened with a midline incision exposing the Eso and stomach. The Eso and LES were isolated and excised as previously described (4, 10). The Eso and LES circular muscle layers were cut into muscle strips (10 mm long × 2 mm wide) with razor blades held in a metal block 2 mm apart. This circular smooth muscle tissue was used for GTPgamma S binding studies and Western blot analysis and to obtain isolated smooth muscle cells.

Cell isolation and permeabilazation. Circular smooth muscle tissue (1-2 mm wide) was digested in HEPES-buffered solution to obtain isolated smooth muscle cells, as described previously (4, 10). The collagenase solution contained 0.5 mg/ml collagenase Sigma type F, 1 mg/ml papain, 1 mg/ml bovine serum albumin, 1 mM 1,4-dithiothreitol (DTT), 1 mM CaCl2, 0.25 mM EDTA, 10 mM glucose, 10 mM HEPES (sodium salt), 4 mM KCl, 125 mM NaCl, 1 mM MgCl2, and 10 mM taurine. The solution was oxygenated (100% O2) at 31°C and pH adjusted to 7.2. The tissue was put into cold enzyme solution and kept in a refrigerator overnight. The following day, the tissue was brought to room temperature for 30 min and incubated in a water bath at 31°C for an additional 30 min. During the digestion period, the gas flow rate was kept low to avoid agitating the tissue. At the end of the digestion period, the tissue was poured over a 200-µm Nitex mesh (Tetko, Elmsford, NY), rinsed in collagenase-free HEPES-buffered solution to remove any trace of collagenase, and incubated in this solution at 31°C and gassed with 100% O2. Collagenase-free HEPES-buffered solution (pH 7.4) contained (in mM) 112.5 NaCl, 5.5 KCl, 2.0 KH2PO4, 10.8 glucose, 24.0 HEPES (sodium salt), 1.9 CaCl2, and 0.6 MgCl2, with BME amino acid supplement, 0.3 mg/ml, and 0.08 mg/ml soybean trypsin inhibitor. Gentle trituration was used to release single cells. All the glassware used in this procedure was siliconized with a 0.05% silicon solution (Sigma, St. Louis, MO) to prevent the cells from adhering to the glass.

Cells were permeabilized, when necessary, to allow the use of G protein antibodies, which do not diffuse across the intact cell membrane. After completion of the enzymatic phase of the digestion process, the partly digested muscle tissue was washed with a cytosolic buffer of the following composition (in mM): 20 NaCl, 25 NaHCO3, 100 KCl, 5.0 MgSO4, and 0.96 NaH2PO4, with 2% bovine serum albumin. The cytosolic buffer also contained 0.61 mM CaCl2 and 1.0 mM EGTA, yielding ~0.36 µM free Ca2+ (16). Sohn et al. (41) previously showed that the maximal contractile response of permeabilized esophageal muscle cells to ACh requires the presence of 0.36 µM Ca2+ in the cytosolic buffer. The cytosolic buffer was equilibrated with 95% O2-5% CO2 to maintain a pH of 7.2 at 31°C. Muscle cells dispersed spontaneously in this medium. After dispersion, the cells were permeabilized by incubation for 3 min in cytosolic buffer containing saponin (75 µg/ml). After exposure to saponin, the cell suspension was centrifuged at 200 g and the resulting pellet was resuspended in a saponin-free modified cytosolic buffer. The modified cytosolic buffer (pH 7.2) contained (in mM) 20 NaCl, 25 NaHCO3, 100 KCl, 5.0 MgSO4, 0.96 NaH2PO4, 0.61 CaCl2, 1.0 EGTA, and 1.5 ATP, with antimycin A, 10 µM, 2% bovine serum albumin, and an ATP-regenerating system consisting of 5 mM creatine phosphate and 10 U/ml creatine phosphokinase. After the cells were washed free of saponin, they were resuspended in the modified cytosolic buffer.

Agonist-induced contraction of isolated muscle cells. The cells were contracted by exposure to PGF2alpha for 30 s. In addition, LES and Eso circular smooth muscle cells were contracted with a maximally effective concentration of PGF2alpha (10-8 M) in the absence or presence of PC-PLC inhibitor D609 (10-4 M), PLD inhibitor propanolol (10-4 M), PI-PLC inhibitor U-73122 (10-6 M), PKC inhibitor chelerythrine (10-5 M), or calmodulin inhibitor CGS9343B (10-5 M). Propranolol and D609 are used at high concentration to be effective and thus may be not entirely specific. The efficacy and selectivity of these inhibitors in Eso and LES, however, have been previously demonstrated (9, 20). When C3 was used, permeabilized LES and Eso cells were incubated in the exoenzyme at the indicated concentration for 2 h before the addition of PGF2alpha (10-8 M). When G protein antibodies were used, permeabilized LES and Eso cells were incubated in the antiserum at a 1:200 dilution for 1 h before the addition of PGF2alpha (10-8 M).

Cell measurements. Thirty consecutive cells from each slide were observed through a phase-contrast microscope (Carl Zeiss, Thornwood, NY) and a CCTV camera (model WV-CD51; Panasonic, Secaucus, NJ) was connected to a Macintosh computer (Apple, Cupertino, CA). The Image software program [National Institutes of Health (NIH), Bethesda, MD] was used to acquire images and measure cell length. The average length of 30 cells, measured in the absence of agonists, was taken as the "control" length and compared with length measured after addition of test agents. Shortening was defined as percent decrease in average length after agonists compared with the control length.

[35S]GTPgamma S binding experiments. Circular smooth muscle squares were homogenized in chilled buffer containing (in mM) 20 HEPES (sodium salt; pH 7.4), 2 MgCl2, 1 EDTA, and 2 DTT. Homogenization consisted of 2- to 10-s bursts with a tissue tearer (Biospec, Racine, WI) followed by 40-60 strokes with a Dounce tissue grinder (Wheaton, Melville, NJ). Samples were centrifuged at 40,000 rpm for 30 min at 4oC (80Ti Rotor, Beckman Ultracentrifuge, Palo Alto, CA). The pellet was resuspended in solubilizing buffer and homogenized in a Dounce tissue grinder (20 strokes). The solubilizing buffer contained (in mM) 20 HEPES (sodium salt; pH 7.4), 240 NaCl, 2 EDTA, and 2 phenylmethylsulfonyl fluoride, with 20 µg/ml aprotinin, 20 µM leupeptin, and 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Samples remained in the solubilizing buffer for 1 h at 4oC.

[35S]GTPgamma S binding was assayed with the method of Murthy et al. (32). The crude membranes (2.5 mg protein/ml) were incubated at 37°C with 30 nM [35S]GTPgamma S in a solution containing (in mM) 10 HEPES (sodium salt; pH 7.4), 0.1 EDTA, and 10 MgCl2 for 1 min. The stimulation of binding was assayed in the presence or absence of a maximally effective concentration of PGF2alpha (10-6M), with a total volume of 300 µl. The reaction was stopped with 10 vol of chilled 100 mM Tris · HCl (pH 8.0) containing 10 mM MgCl2, 100 mM NaCl, and 20 µM GTP. The mixtures (200 µl) were added to enzyme-linked immunosorbent assay wells that had been coated initially with a rabbit or goat immunoglobulin antibody (1:1000, 2 h) and subsequently coated with specific G protein antibodies (1:1,000, 2 h). After 2-h incubation on ice, the wells were washed three times with phosphate buffer solution containing 0.05% polyoxyethylenesorbitan monolaurate (Tween 20). The radioactivity from each well was counted using a Tri-Carb 1900 CA liquid-scintillation analyzer (Packard Instruments, Meriden, CT). Triplicate measurements were carried out for each experiment. Data were expressed as percent increase from basal levels.

Western blot. For RhoA and Galpha 13 purification, LES and Eso circular muscle squares were homogenized in chilled buffer containing (in mM) 20 HEPES (sodium salt; pH 7.4), 2 MgCl2, 1 EDTA, and 2 mM DTT. Homogenization consisted of 2- to 10-s bursts with a tissue tearer (Biospec) followed by 40-60 strokes with a Dounce tissue grinder (Wheaton). Samples were centrifuged at 40,000 rpm for 30 min at 4oC (80Ti Rotor, Beckman Ultracentrifuge). The pellet was resuspended in solubilizing buffer and homogenized in a Dounce tissue grinder (20 strokes). The solubilizing buffer contained (in mM) 20 HEPES (sodium salt; pH 7.4), 240 NaCl, 2 EDTA, and 2 phenylmethylsulfonyl fluoride, with 20 µg/ml aprotinin, 20 µM leupeptin, and 1% CHAPS. Samples remained in the solubilizing buffer for 1 h at 4oC.

For ARF1 purification, LES and Eso circular muscle squares were homogenized in 1 ml chilled buffer containing 10% (wt/vol) sucrose and (in mM) 1 EDTA, 1 mM DTT, 1 sodium azide, 1 benzamidine, and 20 Tris (pH 8.0). Homogenization consisted of 3- to 20-s bursts with a tissue tearer (Biospec) followed by 50 strokes with a Dounce tissue grinder (Wheaton). Samples were centrifuged at 14,000 rpm for 15 min at 4°C (Micro-Centrifuge, Fisher Scientific, Pittsburgh, PA). The pellet containing tissue squares was discarded, and the supernatent was run on SDS-PAGE.

Purified samples were subjected to SDS-PAGE using an acrylamide concentration of 15% in the separating gel for RhoA and ARF and 10% acrylamide concentration for G13. The separated proteins were electrophoretically transferred to a nitrocellulose (NC) membrane (Bio-Rad, Melville, NY) at 30 V overnight. Transfer of proteins to the NC membrane was confirmed with Ponseau S staining reagent (Sigma). To block nonspecific binding, the NC membrane was incubated in 5% nonfat dry milk in phosphate-buffered saline for 60 min followed by three rinses in milk-free buffer. Samples were incubated with RhoA, Galpha 13, or ARF antibody (1:500) for 1 h with shaking followed by three washes with antibody-free buffer. This was followed by a 60-min incubation in horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Amersham, Arlington Heights, IL) for RhoA and Galpha 13 and in HRP-labeled protein A for ARF1. Detection was achieved with an enhanced chemiluminescence agent (Amersham). Molecular weight was estimated by comparison of sample bands with prestained molecular weight marker (Amersham).

Drugs and chemicals. G protein antibodies (Gq, Go, Gi1-Gi2, Gi3) were purchased from Calbiochem-Novabiochem International (San Diego, CA); Galpha 13, RhoA, and ARF1 were from Santa Cruz Biotechnology (Santa Cruz, CA); [35S]GTPgamma S was from New England Nuclear (Boston, MA); and soybean trypsin inhibitor was from Worthington Biochemicals (Freehold, NJ). Collagenase type F, papain, saponin, BME amino acid supplement, EGTA, HEPES, creatine phosphate, creatine phosphokinase, ATP, antimycin A, and other reagents were purchased from Sigma.

Statistical analysis. Data are expressed as means ± SE. Statistical differences between means were determined by Student's t-test. Differences between multiple groups were tested using ANOVA for repeated measures and checked for significance using the Sheffé's F-test.


    RESULTS
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ABSTRACT
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G proteins in PGF2alpha -induced contraction. PGF2alpha induced a concentration-dependent contraction of LES and Eso smooth muscle cells (P < 0.01, ANOVA; Fig. 1). Maximal response occurred at a 10-8 M concentration, producing 23 ± 1% shortening in both Eso and LES circular smooth muscle.


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Fig. 1.   PGF2alpha induced a concentration-dependent contraction of lower esophageal sphincter (LES) and esophagus (Eso) smooth muscle cells (P < 0.01, ANOVA). Intact circular smooth muscle cells from the body of the Eso and LES were exposed to the indicated concentration of PGF2alpha for 30 s. Values are means ± SE of 3 samples, with 30 cells counted at random for each data point.

To identify the specific G proteins mediating PGF2alpha -induced contraction, we used antibodies raised against synthetic peptides corresponding to the amino acid sequence of the COOH-terminal of the alpha -subunit of heterotrimeric G proteins. Table 1 shows that PGF2alpha -induced contraction of LES cells was significantly inhibited by Gq and Gi3 antibodies (P < 0.001, ANOVA) and not by Gi1/i2 or Go antibodies. These data have been previously published (11) and are shown here for the readers' convenience. PGF2alpha -induced contraction of Eso muscle cells, however, was unaffected by the same antibodies (against the alpha -subunit of Gi3, Gq, Gi1/i2, or Go G proteins; Table 1). PGF2alpha -induced contraction of Eso muscle cells was reduced in a concentration-dependent manner by G13 antibodies (Fig. 2; P < 0.001, ANOVA), which did not affect LES contraction. In permeabilized Eso cells, G13 antibodies (5 µg/ml) reduced shortening from 22.4 ± 0.2 to 0.4 ± 2%. These data suggest that PGF2alpha -induced contraction may be mediated by activation of Gq and Gi3 in LES and by G13 in Eso smooth muscle.

                              
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Table 1.   PGF2alpha -induced contraction of LES and Eso cells



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Fig. 2.   G13 antibodies inhibited PGF2alpha -induced contraction of Eso and not of LES permeabilized smooth muscle cells. LES and Eso 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 PGF2alpha (10-8 M) alone (control) or after 60 min preincubation in cytosolic medium containing the indicated concentration of antibody. PGF2alpha -induced contraction of Eso cells was concentration-dependently reduced by antibodies against the alpha -subunit of the heterotrimeric G13 G protein (P < 0.001, ANOVA). LES cells were unaffected by G13 antibodies. Values are means ± SE of 3 samples, with 30 cells counted at random for each data point.

Because the heterotrimeric G protein G13 is often linked to RhoA, we tested whether PGF2alpha -induced contraction of Eso and LES is mediated by monomeric G proteins such as RhoA. C3 is an exoenzyme of Clostridium botulinum, which has been shown to ADP ribosylate and inhibit the Rho-p21 family of monomeric G proteins. Permeabilized cells were incubated in C3 for 2 h before exposure to a maximally effective dose of PGF2alpha (10-8 M). C3 produced a concentration-dependent decrease in PGF2alpha -induced contraction of Eso cells (P < 0.001, ANOVA) but had no effect on LES smooth muscle cells (Fig. 3). Shortening of permeabilized Eso cells was reduced from 22.3 ± 0.3% in control cells to 6.8 ± 1.1% in cells incubated in C3 (250 ng/ml). In addition, PGF2alpha (10-8 M)-induced contraction of permeabilized Eso cells was reduced by 1-h exposure to antibodies against ARF1 and RhoA (P < 0.001, ANOVA). ARF1 and RhoA antibodies, used in combination, reduced shortening from 22.4 ± 0.2 to 4.0 ± 1.2% (Fig. 4A). The inhibition of PGF2alpha -induced contraction by ARF1 was additive to that elicited by RhoA, suggesting that these two monomeric G proteins mediate contraction via different contractile signaling pathways. To determine whether G13 G proteins are linked to RhoA and/or ARF1, permeabilized Eso cells were incubated for 1 h in buffer containing G13 antibodies in combination with ARF (Fig. 4B) or RhoA (Fig. 4C) antibodies. Figure 4B shows that the inhibition of PGF2alpha -induced contraction by G13 antibodies was additive to that elicited by ARF1 antibodies, suggesting that G13 activity is not linked to ARF1. However, G13 antibodies in combination with RhoA antibodies did not cause a greater inhibition of contraction than either antibody used alone, suggesting that G13 may be linked to RhoA and that these two G proteins may mediate contraction by activation of the same signal-transduction pathway. PGF2alpha -induced contraction of LES was unaffected by ARF1 and RhoA antibodies (Fig. 5).


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Fig. 3.   C3 exoenzyme of Clostridium botulinum inhibited PGF2alpha -induced contraction of Eso and not of LES permeabilized smooth muscle cells. LES and Eso cells were permeabilized by brief exposure to saponin to allow diffusion of C3 into the cytosolic side of the cell membrane. Cells were contracted with PGF2alpha (10-8 M) alone (control) or after 2 h preincubation in cytosolic medium containing the indicated concentration of C3. PGF2alpha -induced contraction of Eso cells was concentration-dependently reduced by C3 (P < 0.001, ANOVA), an inhibitor of the Rho-p21 family of monomeric G proteins, suggesting that Eso contraction may be mediated by monomeric G proteins. PGF2alpha -induced contraction of LES cells was unaffected by C3. Values are the means ± SE of 3 samples, with 30 cells counted at random for each data point.



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Fig. 4.   PGF2alpha -induced contraction of permeabilized smooth muscle cells from the Eso was inhibited by ADP-ribosylation factor (ARF)1, RhoA, and G13 antibodies (Ab). Eso 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 PGF2alpha (10-8 M) alone (control) or after 60 min preincubation in cytosolic medium containing the indicated concentration of antibody. A: PGF2alpha -induced contraction of Eso cells was concentration dependently reduced by antibodies against ARF1 and RhoA (P < 0.001, ANOVA). ARF1 and RhoA antibodies used in combination nearly abolished PGF2alpha -induced contraction of Eso. Values are means ± SE of 3 samples, with 30 cells counted at random for each data point. B: PGF2alpha -induced contraction of Eso cells was abolished by antibodies against ARF1 and G13 in combination. To demonstrate the effect of the antibodies used in combination, the G13 data shown in Fig. 2 and the ARF1 data shown in Fig. 4A are presented again in this figure. The inhibition of PGF2alpha -induced contraction by G13 was additive to the inhibition by ARF1, suggesting that these two G proteins mediate contraction by activation of separate signal-transduction pathways. Values are means ± SE of 3 samples, with 30 cells counted at random for each data point. C: PGF2alpha -induced contraction of Eso cells in the presence of antibodies against RhoA and G13. To demonstrate the effect of the antibodies used in combination, the G13 data shown in Fig. 2 and the RhoA data shown in Fig. 4A are presented again in this figure. RhoA and G13 antibodies in combination caused no greater inhibition of PGF2alpha -induced contraction than either alone, suggesting that these two G proteins mediate contraction by activation of the same signal-transduction pathway. Values are means ± SE of 3 samples, with 30 cells counted at random for each data point.



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Fig. 5.   PGF2alpha -induced contraction of permeabilized smooth muscle cells from the LES was unaffected by ARF1 and RhoA antibodies. 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 PGF2alpha (10-8 M) alone (control) or after 60-min preincubation in cytosolic medium containing the indicated concentration of antibody. Values are means + SE of 3 samples, with 30 cells counted at random for each data point.

These data suggest that PGF2alpha -induced contraction of Eso may be mediated by monomeric G proteins, whereas LES contraction is mediated by the trimeric G proteins Gq and Gi3, with no contribution of ARF or RhoA.

To confirm this hypothesis, we examined G protein activation in response to PGF2alpha by measuring [35S]GTPgamma S binding to PGF2alpha -activated smooth muscle membranes. Figure 6 shows that in Eso muscle membranes, PGF2alpha (10-6 M) caused significant activation of RhoA, ARF1, and G13. After PGF2alpha stimulation, RhoA, ARF1, and G13 binding increased 24.6 ± 2.0, 25.7 ± 3.5, and 32.5 ± 8.0%, respectively. In LES, exposure to PGF2alpha (10-6 M) caused significant activation of Gq and Gi3 but not of RhoA, ARF1, and G13 (Fig. 7). After PGF2alpha stimulation, Gq and Gi3 binding increased 25.9 ± 5.3 and 16.5 ± 3.9%, respectively. For LES muscle, the data on PGF2alpha -induced activation of Gi3, Gq, Gi1/i2, or Go have been previously reported (11) and are shown here for the readers convenience. [35S]GTPgamma S binding data are in agreement with the inhibition of PGF2alpha -induced contraction by selective antibodies for both LES and Eso muscle.


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Fig. 6.   [35S]GTPgamma S binding to PGF2alpha -activated membranes of Eso smooth muscle. Purified membranes were exposed to PGF2alpha (10-6 M) in the presence of [35S]GTPgamma S for 5 min. PGF2alpha (10-8 M)-induced activation of specific G proteins was reflected by the amount of [35S]GTPgamma S bound to wells that were precoated with Gq, Gi3, Gi1/i2, Go, RhoA, ARF1, or G13 antibodies. G protein activation was measured as %increase in [35S]GTPgamma S binding in membranes exposed to PGF2alpha compared with unstimulated membranes. Exposure to PGF2alpha (10-6 M) caused significant activation of RhoA, ARF1, or G13 G proteins (**P = 0.001, dagger P = 0.01, ANOVA) in Eso muscle membranes. Values are means ± SE of 4 cats, with each sample performed in triplicate.



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Fig. 7.   [35S]GTPgamma S binding to PGF2alpha -activated membranes of LES smooth muscle. Purified membranes were exposed to PGF2alpha (10-6 M) in the presence of [35S]GTPgamma S for 5 min. PGF2alpha (10-8 M)-induced activation of specific G proteins was reflected by the amount of [35S]GTPgamma S bound to wells that were precoated with Gq, Gi3, Gi1/i2, Go, RhoA, ARF1, or G13 antibodies. G protein activation was measured as %increase in [35S]GTPgamma S binding in membranes exposed to PGF2alpha compared with unstimulated membranes. Exposure to PGF2alpha (10-6 M) caused significant activation of Gq and Gi3 (*P = 0.05, dagger P = 0.01, ANOVA) G proteins in LES muscle membranes. Values are means ± SE of 4 cats, with each sample performed in triplicate.

By Western blot, Gq, Gi3, Gi1/i2, and Go are all present in esophageal and LES circular muscle Sohn et al. (42). In the current study, we showed that the monomeric G proteins RhoA, ARF1, and the heterotrimeric G13 that mediate PGF2alpha -induced contraction in Eso are present in Eso circular smooth muscle. In Eso circular smooth muscle, RhoA (21 kDa), ARF1 (21 kDa), and G13 (45 kDa) were detected by Western blot as thick bands (Fig. 8).


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Fig. 8.   Identification of RhoA, ARF1, and G13 by Western blot. Solubilized Eso circular smooth muscle samples were loaded into an SDS-PAGE system, transferred to nitrocellulose membrane, and incubated with G protein antibody at a 1:500 concentration for 1 h with shaking. RhoA (21 kDa), ARF1 (21 kDa), and G13 (45 kDa) were detected as a thick bands in Eso circular smooth muscle.

Phospholipases involved in PGF2alpha -induced contraction. In Eso, PGF2alpha -induced contraction was significantly reduced by the PLD pathway inhibitor propranolol but was unaffected by the PI-PLC inhibitor U-73122 (10-6 M) or the PC-PLC inhibitor D609 (10-4 M; Fig. 9). Shortening of Eso cells was reduced from 22.9 ± 2.6% in controls to 7.2 ± 1.3% after treatment with propranolol. In LES, PGF2alpha -induced contraction was significantly reduced by U-73122 and D609 (P = 0.01, ANOVA) and was unaffected by propranolol (Fig. 10). U-73122 and D609 reduced shortening of LES cells from 21.4 ± 0.4% in controls to 9.0 ± 1.6 and 16.2 ± 0.3%, respectively. These data suggest that PGF2alpha -induced contraction is mediated by PI-PLC and PC-PLC in the LES and by PLD in Eso.


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Fig. 9.   Phospholipase D inhibitor reduced PGF2alpha -induced contraction of Eso smooth muscle cells. Cells were contracted with a maximally effective dose of PGF2alpha (10-8 M) in the absence (control) or presence of the phosphatidylinositol-specific phospholipase C inhibitor U-73122 (10-6 M), the phosphatidylcholine-specific phospholipase C inhibitor D609 (10-4 M),or an inhibitor of a phospholipase D-dependent pathway propranolol (10-4 M). PGF2alpha -induced contraction was significantly reduced by proparanolol (dagger P = 0.01, ANOVA). Values are the means ± SE of 3 samples, with 30 cells counted at random for each data point.



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Fig. 10.   Phosphatidylinositide-specific and phosphatidylcholine-specific phospholipase C inhibitors reduced PGF2alpha -induced contraction of LES smooth muscle cells. Cells were contracted with a maximally effective dose of PGF2alpha (10-8 M) in the absence (control) or presence of the phosphatidylinositol-specific phospholipase C inhibitor U-73122 (10-6 M), the phosphatidylcholine-specific phospholipase C inhibitor D609 (10-4 M), or an inhibitor of a phospholipase D-dependent pathway propranolol (10-4 M). PGF2alpha -induced contraction of LES was significantly reduced by U-73122 and D609 (dagger P = 0.01, ANOVA). Values are means ± SE of 3 samples, with 30 cells counted at random for each data point.

PKC and calmodulin in PGF2alpha -induced contraction. In Eso smooth muscle cells, contraction in response to a maximally effective concentration of PGF2alpha was significantly reduced by the PKC inhibitor chelerythrine (10-5 M; P = 0.01, ANOVA) but not by the calmodulin inhibitor CGS9343B (10-5 M; Fig. 11). Percent shortening of Eso cells was reduced from 22.9 ± 2.6 to 8.3 ± 0.9% after treatment with chelerythrine.


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Fig. 11.   PKC inhibitor reduced PGF2alpha -induced contraction of Eso smooth muscle cells. Cells were contracted with a maximally effective dose of PGF2alpha (10-8 M) in the absence (control) or presence of the protein kinase C inhibitor chelerythrine (10-5 M) or the calmodulin inhibitor CGS9343B (10-5 M). PGF2alpha -induced contraction of Eso was significantly reduced by chelerythrine (dagger P = 0.01, ANOVA) and unaffected by CGS9343B. Values are means ± SE of 3 samples, with 30 cells counted at random for each data point.

In the LES, the response to low concentrations of PGF2alpha (10-11 M) was selectively reduced by chelerythrine (P = 0.001, ANOVA). The response to a maximally effective dose of PGF2alpha (10-8 M) was significantly reduced by both CGS9343B and chelerythrine (P = 0.01, ANOVA; Fig. 12).


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Fig. 12.   Effect of CGS9343B and chelerythrine on the contractile response of intact LES muscle cells to low and high concentration of PGF2alpha . Cells were incubated with indicated concentrations of PGF2alpha in the absence (control) or presence of chelerythrine (10-5 M), or CGS9343B (10-5 M). Threshold response to PGF2alpha (10-11 M) was selectively reduced by chelerythrine (**P = 0.001, ANOVA), whereas the response to maximally effective dose of PGF2alpha (10-8 M) was reduced by CGS9343B and chelerythrine (dagger P = 0.01, ANOVA). Values are means ± SE of 3 samples, with 30 cells counted at random for each data point.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

LES tone may be maintained by a low molecular mass (14 kDa)-secreted PLA2, which produces AA and results in formation of AA metabolites, such as PGF2alpha and thromboxanes A2 and B2, and activation of Gq and Gi3 G proteins (11). Because PGF2alpha may thus play an important physiological role, we examined the associated signal-transduction pathway in LES and Eso circular muscle.

PGF2alpha -induced contraction of LES. In LES circular muscle, Gq is linked to PI-PLC, and Gi3 is linked to PC-PLC, which are the phospholipases responsible for maintenance of LES tone (3, 20, 43). Gq and Gi3 are the same G proteins that are spontaneously active (bound to GTP in the absence of exogenous neurotransmitter) and that are activated by PGF2alpha and linked to LES tone (11). In the present study, we showed that in LES, PGF2alpha -induced contraction, similar to contraction induced by other agonists, is mediated by PI-PLC (42, 43) and by PC-PLC, because the PI-PLC antagonist U-73122 and the PC-PLC antagonist D609 reduce PGF2alpha -induced LES contraction. These data, which are entirely consistent with the previous data, support the view that PGF2alpha , through PI-PLC and PC-PLC activation, plays a role in the maintenance of LES tone.

In addition, LES contraction induced by a threshold concentration of PGF2alpha (10-11 M) was significantly reduced by chelerythrine, whereas a maximally effective concentration of PGF2alpha (10-8 M) was significantly reduced by chelerythrine and CGS9343B, suggesting the activation of a PKC-dependent pathway at low PGF2alpha concentration and of both a calmodulin- and a PKC- dependent pathway at higher doses of PGF2alpha .

This concentration-related shift in PGF2alpha -induced contraction from a PKC-dependent to a calmodulin-dependent pathway is similar to the shift observed with ACh-induced contraction of LES (3) and may depend on the different calcium requirements of the PKC- or calmodulin-dependent contractile pathways. Biancani et al. (3) previously showed that in LES calmodulin-mediated contraction requires higher cytosolic Ca2+ levels than PKC-dependent contraction. At low calcium concentration, a PKC-dependent pathway is activated. When cytosolic calcium reaches a level sufficient to activate calmodulin, however, as may occur with a maximally effective concentration of ACh, LES contraction is mediated by a calmodulin-dependent pathway (3) and the PKC-dependent pathway is inhibited (3, 12, 13, 24, 49). Calmodulin-induced inhibition of PKC activity may be localized to the second and fourth calcium binding domains of calmodulin (24).

With PGF2alpha -induced contraction, the concentration-related shift of PKC dependence toward calmodulin dependence is not as clear cut as with ACh, because at the maximally effective concentration of PGF2alpha , contraction is inhibited by both PKC and calmodulin inhibitors. Our data suggest that low concentrations of PGF2alpha activate the same PKC-dependent pathway activated by low concentrations of ACh. However, high concentrations of PGF2alpha may not be as effective (in causing Ca2+ release, full activation of calmodulin, and complete inhibition of PKC) as high doses of ACh, and, therefore, a partial activation of a PKC-dependent pathway may still be present.

PGF2alpha -activated G proteins in Eso contraction. In Eso circular smooth muscle, ACh-induced contraction is nearly abolished by antibodies directed against the alpha -subunit of Gi3 (43). PGF2alpha , however, was not linked to activation of the heterotrimeric G proteins Gi3, Gq, Gi1/i2, or Go, as measured by [35S]GTPgamma S binding, and PGF2alpha -induced contraction was not affected by Gi3, Gq, Gi1/i2, or Go antibodies.

Because other heterotrimeric G proteins, particularly G12 and G13, regulate cell function (15, 17), we examined the effect of G13 on PGF2alpha -induced contraction. G13 is a member of the G12-G13 family of heterotrimeric G proteins that was first identified by the DNA cloning of its alpha -subunit. G13 is expressed in most cell lines and tissues and is especially abundant in human platelets (28). PGF2alpha stimulated G13 activation, as measured by [35S]GTPgamma S binding, and G13 antibodies reduced PGF2alpha -induced contraction of Eso, supporting the view that PGF2alpha -induced contraction is mediated by G13.

Because G13 is linked to monomeric G proteins, such as Rho and possibly ARF, we tested Rho as a possible mediator in the PGF2alpha -G13-dependent contractile pathway in Eso. Small G proteins of the Rho family are thought to regulate various cell functions in which the actomyosin system is involved, including cell morphology, membrane ruffling, cell motility, cell aggregation, and smooth muscle contraction (34, 45). Ten different mammalian Rho GTPases have been identified including Rho (A,B,C isoforms) Rac (1,2,3 isoforms), Cdc42 (Cdc42Hs, G25K isoforms), Rnd1/Rho6, Rnd2/Rho7, Rnd3/RhoE, RhoD, RhoG, and TC10 (38). In smooth muscle, the Rho p21 and smg p21/rap1 p21 families are perhaps the most abundant of the monomeric or small G proteins, and RhoA p21 may be involved in GTPgamma S-induced contraction of permeabilized vascular smooth muscle (21).

To study the role of RhoA in PGF2alpha -induced contraction, we examined the effect of the C3 exoenzyme of C. botulinum, which has been shown to ADP ribosylate and inhibit the Rho-p21 family of monomeric G proteins. C3 reduced PGF2alpha -induced contraction of Eso in a concentration-dependent manner but had no effect on LES contraction, suggesting that monomeric G proteins mediate PGF2alpha -induced contraction of Eso smooth muscle but not of LES. These data are similar to those reported in vascular smooth muscle from the mesenteric artery (21), in which contraction was abolished by C3 and by an exoenzyme of Staphilococcus aureus called EDIN, which also ADP rabosylates and inhibits the Rho-p21 family. In addition, RhoA antibodies inhibited PGF2alpha -induced contraction of Eso and PGF2alpha caused activation of RhoA, as measured by [35S]GTPgamma S binding.

In Eso, G13 may be linked to RhoA, because antibodies against G13 in combination with RhoA antibodies did not cause a greater inhibition of contraction than either G protein used alone, suggesting that these two G proteins mediate contraction by activation of the same signal-transduction pathway.

ARF is a second G protein, which, similar to RhoA, is a member of the Ras superfamily of monomeric 20- to 30-kDa GTP-binding proteins. ARFs are 20-kDa ADP-ribosylation factors that were originally recognized and purified because of their ability to stimulate the ADP-ribosyltransferase activity of the cholera toxin A subunit (30). Mammalian ARFs are divided into three classes based on size, amino acid sequence, gene structure, and phylogenic analysis: ARF1 and ARF2 are in class I; ARF4 and ARF5 are in class II; and ARF6 is in class 6 (31).

In Eso, PGF2alpha -induced contraction may be mediated by ARF1, because PGF2alpha significantly activates ARF1 and antibodies directed against ARF1 G proteins significantly reduced contraction. The contractile pathway mediated by ARF1 may be separate from the G13-RhoA-linked pathway because the inhibition of contraction by ARF1 antibodies was additive to the inhibition by G13 and RhoA.

PGF2alpha -activated phospholipases in Eso. Cao et al. (9) and Sohn and co-workers (42, 43) previously demonstrated that ACh-, substance P-, and bombesin-induced contraction of Eso depend on the production of DAG by PC-PLC and PLD, because the PC-PLC antagonist D609 and the PLD pathway inhibitor propranolol reduce DAG production and agonist-induced contraction.

We currently report that PGF2alpha -induced contraction of Eso was reduced by propranolol, suggesting that phospholipid metabolism by PLD may be the main signaling pathway mediating contraction of esophageal circular muscle in response to PGF2alpha .

PLD hydrolyses membrane phospholipids to produce phosphatidic acid and the free polar head group of the phospholipid substrate. Phosphatidic acid is then dephosphorylated by a phosphatase to yield DAG (14). High concentrations of propranolol (0.1-1 mM) have been shown to reduce DAG production by inhibiting the phosphatidic acid phosphatase (5, 37).

Our data suggest that in the Eso, PGF2alpha -induced contraction is mediated by G13, ARF, and RhoA G proteins, which activate PLD. This view is consistent with numerous reports indicating that intracellular PLD activators include the Rho (6, 26, 33, 39) and ARF (7, 8, 33) families of monomeric G proteins and the trimeric G protein G13 (33, 35). PLD interacts with specific amino acid residues in the activation loop (switch I) region of Rho (1) and interacts with the NH2-terminal 73 amino acids of ARF (48).

Rho stimulates PLD activity in the presence of GTPgamma S in membranes from rat liver, neutrophils, and HL60 cells (6, 27, 39). Studies with constitutively active and dominant negative forms of RhoA indicate that RhoA controls PLD activity in intact cells and mediates the effect of various agonists (19, 35).

ARF stimulation of PLD activity has been detected in plasma membranes (29, 36, 47), nuclei (2, 36), Golgi (25, 36, 47), and cytosol (36, 39). ARF has been reported to mediate M2 muscarinic receptor activation of PLD in human embryonic kidney cells. The activation of PLD by RhoA can be amplified synergistically by ARF (39, 40). In addition, G13 has been reported to activate rat brain PLD in a RhoA-dependent fashion (35).

Recent reports indicate that G13 may link receptor-mediated activation of monomeric G proteins to activation of PLD (33). G13 is capable of directly activating a guanine nucleotide exchange factor (GEF) for Rho (18, 23), which facilitates the exchange of GDP for GTP (22, 46). GTPases must be in their active (GTP or GTPgamma S ligand) form to stimulate their effectors such as PLD. Members of the regulators of the G protein signaling (RGS) family stimulate the intrinsic GTPase activity of the alpha -subunit of certain heterotrimeric G proteins. One Rho-specific GEF, p115 RhoGEF, serves as a direct link between Rho GTPases and heterotrimeric G proteins, because it has an NH2-terminal region that contains a domain characteristic of RGS proteins and specifically stimulates the GTPase activity of the alpha -subunit of G13 (23). Therefore, one pathway for the hormonal stimulation of PLD via G protein-coupled receptors may be through G13 and its direct regulation of the exchange activity of p115 Rho GEF for Rho proteins. For example, CCK-stimulated PLD activity of intestinal smooth muscle may be mediated by G13-dependent RhoA (33). Our data support a role of PLD activation by the monomeric G proteins RhoA and ARF1 and by the heterotrimeric G13. It is possible that PGF2alpha -induced contraction of Eso is mediated by activation of one or more of these G proteins that can work alone or cooperatively to stimulate the activity of PLD. Our data suggest that two signal-transduction pathways mediate PGF2alpha -induced contraction of Eso: one a G13-RhoA-linked pathway, and a second pathway mediated by activation of ARF1. Activation of PLD results in the production of DAG, which stimulates PKC and PKC-dependent contraction of the Eso.

PKC and calmodulin in PGF2alpha -induced contraction in Eso. Eso contraction induced by a maximally effective dose of PGF2alpha (10-8 M) was inhibited by the PKC antagonist chelerythrine and not affected by the calmodulin antagonist CGS9343B, as occurs with other agonists such as ACh, substance P, and bombesin (42).

In conclusion, these data suggest that in Eso, PGF2alpha activates G13. G13 may then regulate the exchange activity of GDP/GTP exchange proteins, causing GTP binding to RhoA (and possibly ARF). These monomeric G proteins, which, in their GDP-bound state, are inactive and cytosolic, translocate to the membrane and may act separately or cooperatively to activate PLD and a PKC-dependent pathway.

In LES, PGF2alpha receptors are coupled to Gq and Gi3 G proteins, which activate PI- and PC-PLC. At low concentrations, PGF2alpha activates a PKC-dependent pathway and, at maximally effective concentration, activates both a PKC- and a calmodulin-dependent pathway.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-28614.


    FOOTNOTES

These data were presented in part at the 1999 meeting of the American Gastroenterological Association.

Address for reprint requests and other correspondence: P. Biancani, Gastrointestinal Motor Function Research Laboratory, SWP5 Rhode Island Hospital and Brown University, 593 Eddy St., Providence RI 02903 (Piero_Biancani{at}brown.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpgi.00357.2001

Received 9 August 2001; accepted in final form 18 February 2002.


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Am J Physiol Gastrointest Liver Physiol 283(2):G282-G291
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