Group I secreted PLA₂ and arachidonic acid metabolites in the maintenance of cat LES tone

¹ Department of Medicine, Rhode Island Hospital and Brown University, Providence, Rhode Island 02902; and ² Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Spontaneous tone of in vitro lower esophageal sphincter (LES) circular muscle is associated with elevated levels of arachidonic acid (AA), PGF₂, and increased [³⁵S]guanosine 5'-O-(3-thiotriphosphate) (GTPS) binding to G_q-, G_i3-, and G_i1/i2-like G proteins. Tone and AA levels were reduced by inhibitors of a pancreatic-like (group I) secreted phospholipase A₂ (sPLA₂), by the cyclooxygenase inhibitor indomethacin, and by the thromboxane A₂ antagonist SQ-29548. In addition, pertussis toxin (PTX) reduced LES tone, confirming a role of PTX-sensitive G proteins in maintenance of LES tone. PGF₂ contracted LES smooth muscle (strips and cells) and increased [³⁵S]GTPS binding to G_q and G_i3 in solubilized LES circular muscle membranes. PGF₂-induced contraction of LES permeable muscle cells was inhibited by G_q and G_i3 but not by G_i1/i2 and G_o antibodies. The thromboxane A₂ analog U-46619 contracted LES smooth muscle and increased G_q binding. U-46619-induced contraction was inhibited by G_q but not by G_i3, G_i1/i2, and G_o antibodies. LES tone and [³⁵S]GTPS binding were significantly reduced by indomethacin. We conclude that group I sPLA₂ may mediate "spontaneous" LES tone by producing AA, which is metabolized to PGF₂ and thromboxane A₂. These AA metabolites activate receptors linked to G_i3 and G_q to maintain LES contraction.

esophagus; lower esophageal sphincter; smooth muscle contraction; phospholipases; prostaglandins; thromboxanes

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

THE LOWER ESOPHAGEAL SPHINCTER (LES) circular muscle is a major determinant of LES tone. Although the relative neurogenic contribution may vary with the animal species, a significant component of tone is thought to be myogenic, as it is not affected by neural antagonists, including TTX (6, 16, 35). Functionally, this muscle is specialized, with muscle strips from this region developing higher total and active forces than esophageal strips (11, 16, 17). We have previously reported that LES tone is maintained by the spontaneous, low-level activities of phosphatidylinositol-specific phospholipase C (PI-PLC), and phosphatidylcholine-specific phospholipase C (PC-PLC), which produce threshold levels of the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃), which act synergistically to activate protein kinase C (PKC), and maintain a PKC-dependent basal tone (8, 41). We now propose that activation of these phospholipases may be maintained by a low-molecular-mass (14 kDa) group I secreted phospholipase A₂ (sPLA₂), which produces arachidonic acid (AA), and AA metabolites, such as PGF₂ and thromboxanes A₂/B₂, which maintain activation of the G proteins coupled to PC-PLC and PI-PLC (85, 86).

The PLA₂ members are a growing family of enzymes that catalyze the hydrolysis of glycerolphospholipids at the sn-2 position, producing free fatty acids and lysophospholipids (24, 25, 49) (Fig. 1). Mammalian PLA₂ enzymes function in the digestion of dietary lipid, microbial degradation, and regulation of phospholipid acyl turnover for membrane repair or for the production of AA. AA is an important regulator of specific cellular processes, including regulation of PKC and PLC-, and modulation of calcium transients. AA is also the precursor to biologically active lipids, including prostaglandins, hydroxy fatty acids, leukotrienes, thromboxanes, and platelet activation factor.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Membrane phospholipids are acted on by phospholipases A2 (PLA2), a family of enzymes that catalyze the hydrolysis of glycerolphospholipids at the sn-2 position, producing free fatty acids and lysophospholipids (24, 25, 49). PLA2 enzymes are divided into two major classes: intracellular or cytosolic (high molecular mass, 80-85 kDa) (cPLA2) and secretory or secreted (low molecular mass, ~14 kDa) (sPLA2). cPLA2 enzymes include the 85-kDa calcium-sensitive cPLA2 and the 80-kDa calcium-insensitive cPLA2 (iPLA2). sPLA2 enzymes are divided into different groups (I, II, III, V, VII, VIII, IX) according to their molecular structure and the localization of their disulfide bridges (22, 40). Mammalian PLA2 enzymes produce arachidonic acid (AA), an important regulator of specific cellular processes and precursor to biologically active lipids, including prostaglandins, leukotrienes, thromboxanes, and prostacyclins. NDGA, nordihydroguaiaretic acid; PAF, platelet-activating factor.

The PLA₂ family is divided into two major classes (Fig. 1): intracellular or cytosolic [high molecular mass (80-85 kDa)] PLA₂ (cPLA₂) and secretory or secreted [low molecular mass (~14 kDa)] PLA₂ (sPLA₂).

Some cPLA₂ members have been characterized, such as the 85-kDa calcium-sensitive cPLA₂ and the 80-kDa calcium-insensitive cPLA₂ (24). We have previously shown that this calcium-sensitive cPLA₂ participates in ACh-induced contraction of the esophagus (87).

The sPLA₂ members are divided into different groups (i.e., I, II, III, V, VII, VIII, IX) according to their molecular structure and the localization of their disulfide bridges (23, 40). sPLA₂ have an absolute catalytic requirement for millimolar concentrations of calcium and a broad specificity for phospholipids with different polar head groups and fatty acyl chains. Many sPLA₂ function extracellularly, but some have also been localized within mitochondria (91, 95). sPLA₂ have been purified from mammalian sources, including pancreas, spleen, lung, platelets, and extracellular fluid, and from bee and snake venom. Group I, II, and III are represented by pancreatic, inflammatory, and bee venom sPLA₂. Group I sPLA₂ was originally identified in pancreatic juice and then identified and cloned in tissues, including, spleen, lung, ovary, and kidney (49, 58, 80, 93). It functions in lipid digestion, cell proliferation, acute lung injury, and smooth muscle contraction (3, 61, 89).

In this investigation, we examine the role of sPLA₂ in the maintenance of LES tone. We find that AA production, through an sPLA₂ of group I, participates in maintenance of LES tone. Most likely AA is metabolized to prostaglandins, such as PGF₂, and to thromboxanes (thromboxanes A₂ and/or B₂), which activate specific G proteins that contribute to the maintenance of LES tone.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals. Adult male cats weighing between 3.5 and 5.5 kg were initially anesthetized with ketamine (Aveco, Fort Dodge, IA) and then euthanized with an overdose of phenobarbital (Schering, Kennilworth, NJ). The chest and abdomen were opened with a midline incision exposing the esophagus and stomach. The esophagus and LES were isolated and excised as previously described (9, 11).

Measurements of in vitro LES tone. LES strips (2 mm) were mounted in separate 1-ml muscle chambers and equilibrated for 2 h with continuous perfusion of oxygenated physiological salt solution (PSS) as previously described in detail (7-11, 41). During this time, the tension in LES strips increased, attaining a steady level at 2 h. The PSS contained the following (in mM): 116.6 NaCl, 21.9 NaHCO₃, 1.2 NaH₂PO₄, 3.4 KCl, 2.5 CaCl₂, 5.4 glucose, and 1.2 MgCl₂. The solution was equilibrated with a gas mixture containing 95% O₂ and 5% CO₂ at pH 7.4 and 37°C.

After equilibration, LES strips were incubated for 30 min in solution containing the appropriate concentrations of the cyclooxygenase inhibitors indomethacin and acetylsalicylic acid (aspirin), the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), the PLA₂ inhibitor 4-bromophenacyl bromide (BPB), the cPLA₂ inhibitor AACOCF₃, the sPLA₂ inhibitors MJ33, MJ45, AM5, and X1, and the thromboxane A₂ antagonist SQ-29548. In some studies, agents were added together with TTX (10⁴ M). When strips were treated with TTX, electrical stimulation (square-wave pulses of supramaximal voltage, 0.2 ms, 1 Hz, 10-s trains) was used to document inhibition of neurotransmitter release, before addition of inhibitors. Electrical stimulation was delivered by a stimulator (model S48, Grass Instruments, Quincy, MA) through platinum wire electrodes placed longitudinally on either side of the strip. In addition, LES strips were incubated with different concentrations of pertussis toxin (PTX) for 1 h and in the indicated concentrations of group I, II, III sPLA₂, thromboxane B₂, or PGF₂ for 15 min. Indomethacin and SQ-29548 were dissolved in ethanol. The concentrations of ethanol used in the concentration-response curves (0.005-0.05%) did not affect LES tone, as illustrated in Fig. 10B.

Smooth muscle tension was recorded on a chart recorder (Grass Instruments). Passive force was obtained at the end of the experiment by completely relaxing the strips with excess EDTA until no further decrease in resting force was observed. Basal LES tone is the difference between resting and passive force. Percent increase in basal tone was defined by the ratio between the increase in force after drug administration and basal LES tone. Percent basal LES tone was calculated by the ratio between the force after using the drugs and the basal LES tone.

Preparation of circular smooth muscle tissue. The LES was excised, the circular muscle layer was cut into 0.5-mm-thick slices with a Stadie Riggs tissue slicer (Thomas Scientific Apparatus, Philadelphia, PA), and tissue squares were made by cutting twice with a 2-mm blade block, the second cut at right angles to the first. This circular smooth muscle tissue was used for AA release, PGF₂ measurements, and guanosine 5'-O-(3-thiotriphosphate) (GTPS) binding studies and to obtain isolated smooth muscle cells.

Cell isolation and permeabilization. Isolated smooth muscle cells were obtained by enzymatic digestion, as previously described (7-9, 46, 47, 76, 83-88). Briefly, LES circular smooth muscle was digested in HEPES-buffered physiological solution containing 150 U/ml collagenase (type II, Worthington Biochemicals, Freehold, NJ) for 2 h. The HEPES solution contained 114.7 mM NaCl, 5.7 mM KCl, 2.1 mM KH₂PO₄, 11 mM glucose, 24.5 mM HEPES, 1.9 mM CaCl₂, 0.57 mM MgCl₂, 0.3 mg/ml basal medium Eagle amino acid supplement (M.A. Bioproducts, Walkersville, MD), and 0.08 mg/ml soybean trypsin inhibitor (Worthington Biochemicals). The HEPES solution was oxygenated (100% O₂) at 31°C, and the pH was adjusted to 7.4. At the end of the digestion period, the tissue was rinsed and then incubated in collagenase-free HEPES buffer. The cells dissociate freely in collagenase-free solution.

When permeable cells were required to allow the use of G protein antibodies that do not diffuse across the intact plasma membrane, the partly digested muscle tissue was washed with a "cytosolic" enzyme-free PSS (cytosolic buffer) of the following composition (in mM): 20 NaCl, 100 KCl, 25 NaHCO₃, 5.0 MgSO₄, 0.96 NaH₂PO₄, 1.0 EGTA, and 0.48 CaCl₂. The cytosolic buffer contained 2% BSA and was equilibrated with 95% O₂-5% CO₂ to maintain pH of 7.2 at 31°C. Muscle cells dispersed spontaneously in this medium. A low concentration of calcium was present in the cytosolic buffer, to avoid spontaneous contraction of the cells in the absence of agonists after the membrane became permeable. The cells were permeabilized by incubation for 3 min in cytosolic buffer containing saponin (75 µg/ml). After exposure to saponin, the cell suspension was spun at low g, and the resulting pellet was resuspended in saponin-free modified cytosolic buffer containing antimycin (10 µM), ATP (1.5 mM), and an ATP-regenerating system consisting of creatine phosphate (5 mM) and creatine phosphokinase (10 U/ml) (12).

Agonist-induced contraction of isolated muscle cells. Once the cells had dissociated, 0.5-ml aliquots of the cell-containing fluid were added to tubes for exposure to agonists and measurement of contraction. Intact esophageal circular smooth muscle cells were contracted with a maximal concentration (0.1 U/ml) of purified group I, group II, or group III sPLA₂, in the absence or presence of the PLA₂ inhibitors (10⁵ M) AM5, X1, MJ33, BPB, or MJ45. Permeabilized LES cells were exposed to a maximally effective dose of PGF₂ (10⁸ M) or the thromboxane A₂ mimetic U-46619 (10⁸ M) for 30 s. When G protein antibodies were used, cells were incubated in the antiserum at a 1:200 dilution for 1 h before the addition of agonist (13).

After exposure to agonist, the cells were fixed in acrolein at a final 1.0% concentration. A drop of the cell-containing medium was placed on a glass slide and covered by a coverslip. The edges of the coverslip were sealed with clear nail enamel to prevent evaporation. Slides so prepared, if refrigerated, could be kept for several days.

Cell measurements. Thirty consecutive cells from each slide were observed through a phase-contrast microscope (Carl Zeiss) and a CCTV camera (model WV-CD51, Panasonic, Secaucus, NJ) connected to a Macintosh computer (Apple, Cupertino, CA). The Image 1.59 software program (National Institutes of Health, Bethesda, MD) was used to measure cell length and was used for data accumulation. The average length of 30 cells, measured in the absence of agonists, was taken as control length. In addition, average cell length was measured after addition of test agents. Shortening was defined as percent decrease in average length after agonist addition, compared with control length.

[³H]AA release or content in LES and esophageal circular smooth muscle. LES circular smooth muscle was incubated in Krebs solution containing [³H]AA (3 µCi/ml) for 4 h to allow uptake into cell membrane (45, 56, 68, 72, 78). After 4 h, the tissue was washed twice with 200 ml Krebs solution, and then incubated with 1 ml of Krebs solution alone (control) or Krebs solution containing 10⁴ M AM5, MJ33, or BPB. After 30 or 60 min, a 0.2-ml aliquot of the supernatant was removed and the radioactivity was measured. The remaining fluid and tissue were frozen and kept at 70°C. Samples were subsequently thawed and homogenized. Homogenization consisted of 3- to 10-s bursts with a Tissue Tearer (Biospec, Racine, WI) followed by 40-60 strokes with a Dounce tissue grinder (Wheaton, Melville, NJ). A 0.2-ml aliquot of the homogenate was used to measure tissue radioactivity. The remaining homogenate was used to measure protein content. The percent of AA release was calculated as the ratio of activity of [³H]AA in the supernatant to [³H]AA in the homogenate (87).

PGF₂ measurement. LES and esophageal circular smooth muscle was incubated with 1 ml Krebs solution at 37°C for 2 h. Aliquots of LES tissue were treated with Krebs solution alone (control) or Krebs solution containing indomethacin and incubated for 45 min. The final concentration of indomethacin was 10⁵ M. Circular smooth muscle tissue (100 mg) was homogenized in acetate buffer (0.2 M, pH 4.5, 4°C). Homogenization consisted of 3- to 10-s bursts with a Tissue Tearer (Biospec) followed by 40-60 strokes with a Dounce tissue grinder (Wheaton). An aliquot of homogenate was taken for protein measurement. PGF₂ was extracted according to the method of Saksena and Harper (79) as follows. Homogenates were extracted twice with three volumes of ethyl acetate. Fractions of ethyl acetate were pooled, washed with 5 ml of distilled water, and brought to dryness by a stream of nitrogen. The resulting extracts were kept at 70°C. The extracts were redissolved in ethanol and purified by passage over a Sep-Pak C-18 reverse-phase cartridge. The PGF₂ concentration was quantified by using PGF₂ enzyme immunoassay kit (Cayman Chemical).

[³⁵S]GTPS binding experiments. To compare the binding of [³⁵S]GTPS to activated G proteins in LES with [³⁵S]GTPS binding in esophagus, LES and esophagus muscle squares were incubated in Krebs solution at 37°C for 2 h. To examine the inhibition of [³⁵S]GTPS binding by indomethacin, aliquots of LES tissue were incubated for 60 min in Krebs solution alone (control) or in Krebs solution containing indomethacin. The final concentration of indomethacin was 10⁵ M. After treatment, all tissues were kept in liquid nitrogen until the binding assay was performed.

LES and esophagus circular smooth muscle was homogenized in ice-cold buffer containing 20 mM HEPES (pH 7.4), 2 mM MgCl₂, 1 mM EDTA, and 2 mM 1,4-dithiothreitol. Homogenization consisted of 2- to 10-s bursts with a Tissue Tearer (Biospec) followed by 40-60 strokes with a Dounce tissue grinder (Wheaton). Samples were centrifuged at 40,000 rpm for 30 min at 4°C (80 Ti rotor, Beckman ultracentrifuge, Palo Alto, CA). The pellet was resuspended in solubilizing buffer and homogenized in a Dounce tissue grinder (20 strokes). The solubilizing buffer contained 20 mM HEPES (pH 7.4), 240 mM NaCl, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 µM leupeptin, and 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Samples remained in the solubilizing buffer for 1 h at 4°C.

[³⁵S]GTPS binding was assayed with the method of Okamoto et al. (66) and Murthy et al. (60). The crude membranes (2.5 mg protein/ml) were incubated for 1 min at 37°C with 30 nM [³⁵S]GTPS in a solution containing 10 mM HEPES (pH 7.4), 0.1 mM EDTA, and 10 mM MgCl₂. The stimulation of binding was assayed in the presence or absence of a maximal concentration (10⁶ M) of PGF₂ or the thromboxane A₂-mimetic U-46619 (10⁶ M) in a total volume of 300 µl. The reaction was stopped with 10 volumes of ice-cold 100 mM Tris · HCl (pH 8.0) containing 10 mM MgCl₂, 100 mM NaCl, and 20 µM GTP. The mixtures (200 µl) were added to ELISA wells that had been coated initially with an anti-rabbit immunoglobulin antibody (1:1,000) and subsequently coated with specific G protein antibodies (1:1,000). After a 2-h incubation on ice, the wells were washed three times with phosphate buffer solution containing 0.05% Tween 20. The radioactivity from each well was counted using a Tri-Carb 1900 CA liquid scintillation analyzer (Packard Instrument, Meriden, CT). Triplicate measurements were carried out for each experiment. Data were expressed as percent stimulation from basal levels.

Protein determination. The homogenates of LES and esophageal tissues were solubilized by addition of 6 ml of 0.1 N NaOH and heating the sample at 80°C for 30 min. The amount of protein present was determined by colorimetric analysis (Bio-Rad, Melville, NY) according to the method of Bradford (14).

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

Drugs and chemicals. AACOCF₃ and G protein antibodies (G_q, G_i3, G_i1/i2) were purchased from Calbiochem; Sep-Pak C-18 cartridges and ethyl acetate were from Fisher Scientific (Pittsburgh, PA); PGF₂ enzyme immunoassay kit was from Cayman Chemical (Ann Arbor, MI); PGF₂, SQ-29548, and U-46619 were from Biomol (Plymouth Meeting, PA); [³⁵S]GTPS and [³H]AA were from NEN (Boston, MA). Goat anti-rabbit immunoglobulin G Fc antibody was from Pierce (Rockford, IL); groups I, II, and III sPLA₂, acetylsalicylic acid, NDGA, indomethacin, thromboxane B₂, BPB, TTX, PTX, hexane, ethanol, and 0.2 M acetate buffer (pH 4.5) and other reagents were purchased from Sigma (St. Louis, MO).

AM5, MJ33, MJ45, and X1 were synthesized by M. K. Jain.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Group I sPLA₂ and LES tone. The selectivity of novel cytosolic and sPLA₂ inhibitors was examined by testing their effectiveness against PLA₂-induced contraction of isolated esophageal cells (Fig. 2).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Effect of novel PLA2 inhibitors (105 M) MJ33, MJ45, AM5, 4-bromophenacyl bromide (BPB), and X1 on the contraction of isolated smooth muscle cells from the esophagus. Cells were contracted with a maximal dose (0.1 U/ml) of group I (A), group II (B), or group III (C) sPLA2 in the absence (control) or presence of PLA2 inhibitors. BPB is a nonselective PLA2 inhibitor. AM5 and X1 are inhibitors selective for sPLA2, MJ33 is a group I sPLA2 inhibitor, and MJ45 is a group II sPLA2 inhibitor. A: group I sPLA2-induced contraction of isolated esophageal cells was significantly reduced by BPB, AM5, X1, and by MJ33. MJ45 had no effect on group I sPLA2-induced contraction. B: group II sPLA2-induced contraction of isolated esophageal cells was significantly reduced by AM5, X1, and MJ45. BPB and MJ33 had no effect on group II sPLA2-induced contraction. C: group III sPLA2-induced contraction was reduced only by X1. Values are means ± SE of 3 animals with 30 cells counted for each animal. ** P < 0.001.

Contraction of isolated esophageal cells in response to group I sPLA₂ was significantly reduced by the nonselective PLA₂ inhibitor BPB, the nonselective sPLA₂ inhibitors AM5 and X1, and by the selective group I sPLA₂ inhibitor MJ33 (ANOVA, P < 0.001). It was not affected by the selective group II sPLA₂ inhibitor MJ45 (Fig. 2A).

Contraction of isolated esophageal cells in response to group II sPLA₂ was significantly reduced by AM5 and X1 and by the selective group II sPLA₂ inhibitor MJ45 (ANOVA, P < 0.001; Fig. 2B). BPB and MJ33 had no effect on group II sPLA₂-induced contraction.

Figure 2C shows that group III sPLA₂-induced contraction was reduced only by X1 (ANOVA, P < 0.001) and not by any of the other inhibitors.

Because group III sPLA₂ is not found in mammalian cells, we examined the effect of group I and II sPLA₂ inhibitors on spontaneous LES tone. AM5 and X1 caused a concentration-dependent reduction of in vitro LES tone (ANOVA, P < 0.01), whereas the cytosolic PLA₂ inhibitor AACOCF₃ had no effect (Fig. 3A). Figure 3, B and C, shows that MJ33 and BPB dose dependently decreased resting LES tone (ANOVA, P < 0.001) but MJ45 had no effect. In addition, TTX (10⁴ M) had no effect on the decrease in LES tone induced by MJ33 and BPB. These data suggest that group I sPLA₂ may play a role in the maintenance of LES tone and that PLA₂-induced LES tone is not neurally mediated.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effect of PLA2 inhibitors on lower esophageal sphincter (LES) basal tone. A: LES circular muscle strips were incubated in the indicated concentrations of PLA2 inhibitors. AM5 and X1 caused a concentration-dependent reduction of in vitro LES tone, whereas the cytosolic PLA2 inhibitor AACOCF3 had no effect. Values are means ± SE of 4 animals. B and C: LES circular muscle strips were incubated in indicated concentrations of PLA2 inhibitors alone or in the presence of TTX (104 M). When strips were treated with TTX, electrical stimulation was used to document inhibition of neurotransmitter release, before addition of inhibitors. MJ33 (B) and BPB (C) dose dependently decreased resting LES tone, but MJ45 (B) had no effect. In addition, TTX had no effect on the decrease in LES tone induced by MJ33 and BPB. These data suggest that group I sPLA2 may play a role in the maintenance of LES tone and that this PLA2 may not come from nerve terminals. Values are means ± SE of 3-6 animals.

Next, we examined the effect of exogenously added sPLA₂ on LES resting tone. Figure 4 shows that group I sPLA₂, purified from Naja naja venom (0.01 and 0.1 U/ml), significantly increased in vitro LES tone (ANOVA, P < 0.05). In contrast, group II sPLA₂, purified from rattle snake venom, and group III sPLA₂, purified from bee venom, had no effect on in vitro LES tone. These data suggest that LES muscle, unlike esophageal circular muscle, responds only to group I sPLA₂.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of exogenous group I, II, and III PLA2 on LES basal tone. LES circular muscle strips were incubated in indicated concentrations of either group I, II, or III sPLA2. Group I sPLA2 caused a concentration-dependent increase of in vitro LES tone. In contrast, group II sPLA2 and group III sPLA2 had no effect on in vitro LES tone. Values are means ± SE of 3-6 animals.

AA production in LES and esophagus. Data suggest that group I sPLA₂ may participate in maintenance of LES tone. We therefore measured AA accumulation in LES muscle and its release into the medium surrounding the muscle.

AA tissue levels were significantly higher in the unstimulated LES circular muscle than in esophageal circular muscle, both at 30 and 60 min (paired t-test, P < 0.05). In addition, AA released by LES circular muscle into the medium was significantly higher than that by the esophagus both at 30 and 60 min (paired t-test, P < 0.05; Fig. 5B). AA release was significantly reduced by BPB, AM5, and MJ33 (ANOVA, P < 0.05; Fig. 6). These data suggest that the elevated levels of AA in the LES may be maintained by a spontaneously active group I sPLA₂.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   [3H]AA release or content in LES and esophageal (ESO) circular smooth muscle. LES circular muscle squares were incubated for 4 h in Krebs solution containing [3H]AA (3 µCi/ml) to allow uptake into the muscle. After 4 h, muscle squares were washed with radioactive-free Krebs solution. After 30 or 60 min, radioactivity released into the medium was measured (B) and compared with the radioactivity present in tissue homogenates (A). A: AA levels were significantly higher in unstimulated LES circular muscle than in esophageal circular muscle after 30 and 60 min. B: LES circular muscle released significantly higher concentrations of AA after 30 and 60 min than the esophagus. Values are mean ± SE of 5-7 animals. * P < 0.05.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Effect of sPLA2 inhibitors on [3H]AA release. LES circular muscle squares were incubated in Krebs solution containing [3H]AA (3 µCi/ml) to allow uptake into the muscle. After 4 h, muscle squares were washed and incubated for 30 min in Krebs alone (control) or in Krebs containing AM5, MJ33, or BPB (104 M). Radioactivity released into the medium was measured and compared with the radioactivity present in tissue homogenates. Percent release of [3H]AA into medium was calculated as 100 × [radioactivity in the medium (cpm/mg protein)/radioactivity in tissue homogenates (cpm/mg protein)]. [3H]AA release was significantly reduced by BPB, AM5, and by MJ33. These data suggest that elevated levels of AA in the LES may be maintained by a spontaneously active group I sPLA2. Values are mean ± SE of 5 animals. * P < 0.05.

Cyclooxygenase inhibitors reduce LES tone. Because AA may be metabolized to leukotrienes, prostaglandins, or thromboxanes, we examined whether these AA metabolites may contribute to maintenance of LES tone. Figure 7 shows that the cyclooxygenase inhibitors indomethacin and aspirin caused a concentration-dependent reduction (ANOVA, P < 0.001) in basal tone of LES circular muscle strips, whereas the lipoxygenase inhibitor NDGA had no effect. In addition, TTX (10⁴ M) had no effect on the reduction of LES tone induced by indomethacin. These data suggest that prostaglandins and/or thromboxanes (but not leukotrienes) may play a role in resting LES tone and that indomethacin-induced reduction of LES tone is not neurally mediated.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Effect of cyclooxygenase or lipoxygenase inhibitors on LES basal tone. LES circular muscle strips were incubated in the indicated concentration of NDGA, indomethacin (Indo), or aspirin alone, or in the presence of TTX (104 M). When strips were treated with TTX, electrical stimulation was used to document inhibition of neurotransmitter release before addition of inhibitors. Cyclooxygenase inhibitors indomethacin (A) and aspirin (B) caused a concentration-dependent reduction in LES basal tone. Lipoxygenase inhibitor NDGA (A) had no effect. In addition, TTX had no effect on the reduction of LES tone induced by indomethacin (A). These data suggest that prostaglandins and not leukotrienes play a role in LES basal tone and that prostaglandins may not come from nerve terminals. Values are mean ± SE of 3-7 animals.

PGF₂ and/or thromboxanes and LES tone. Because prostaglandins and/or thromboxanes may participate in maintenance of LES tone, we measured the PGF₂ content in the esophagus and LES circular smooth muscle and examined the effect of the thromboxane A₂ antagonist SQ-29548 on LES tone.

The PGF₂ content of unstimulated LES circular muscle was significantly higher than in esophageal muscle (paired t-test, P < 0.05; Fig. 8A). Indomethacin significantly reduced PGF₂ formation in LES smooth muscle (paired t-test, P < 0.05; Fig. 8B). Furthermore, PGF₂ dose dependently increased LES tone (ANOVA, P < 0.05; Fig. 9A) and reversed the reduction in LES tone induced by indomethacin (ANOVA, P < 0.001; Fig. 9B). These data suggest that PGF₂ may participate in the maintenance of LES resting tone.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   A: PGF2 content in LES and esophageal (ESO) smooth muscle. PGF2 was extracted from tissue squares of LES and esophageal circular smooth muscle according to the method of Saksena and Harper (79). PGF2 content was significantly higher in LES smooth muscle than in esophageal smooth muscle. Values are mean ± SE of 3 animals. B: aliquots of LES tissue squares were treated for 45 min with Krebs alone (control) or in Krebs solution containing indomethacin (105 M) before measurement of PGF2 content. PGF2 content was significantly decreased by indomethacin. Values are mean ± SE of 3-4 animals. * P < 0.05.

View larger version (14K): [in this window] [in a new window]   Fig. 9.   Role of PGF2 on LES basal tone. LES circular muscle strips were incubated in indicated concentrations of PGF2 alone (control) or in the presence of indomethacin (105 M). A: PGF2 produced a concentration-dependent increase in LES basal tone. B: PGF2 (104 M) reversed the reduction in LES basal tone produced by indomethacin (105 M). Values are mean ± SE of 3-4 animals. ** P < 0.001.

SQ-29548 dose dependently decreased resting LES tone (ANOVA, P < 0.001; Fig. 10B). In addition, thromboxane B₂ dose dependently increased LES tone (ANOVA, P < 0.01; Fig. 10A) and reversed the reduction of LES tone induced by indomethacin (ANOVA, P < 0.001; Fig. 10C). These data suggest that thromboxane A₂ and/or B₂ may also contribute to maintaining LES tone.

View larger version (17K): [in this window] [in a new window]   Fig. 10.   Role of thromboxane B2 (TXB2) on LES basal tone. LES circular muscle strips were incubated in indicated concentrations of TXB2 alone (control) or in the presence of indomethacin (105 M). A: TXB2 produced a concentration-dependent increase in LES basal tone. B: thromboxane antagonist SQ-29548 produced a concentration-dependent reduction in LES basal tone. C: TXB2 (105 M) reversed the reduction in LES basal tone produced by indomethacin (105 M). Values are mean ± SE of 3-4 animals. ** P < 0.001.

G protein activation and LES tone. In LES smooth muscle, basal or unstimulated [³⁵S]GTPS binding to G_q, G_i3, or G_i1/i2 was significantly higher (ANOVA, P < 0.01) than in esophagus smooth muscle (Fig. 11), suggesting that, in the absence of any exogenously added agonist, these G proteins are at a significantly higher level of activation in LES muscle.

View larger version (15K): [in this window] [in a new window]   Fig. 11.   Unstimulated [35S]guanosine 5'-O-(3-thiotriphosphate) (GTPS) binding in LES and esophageal (ESO) smooth muscle membranes. Purified membranes were incubated in [35S]GTPS for 1, 5, 10, and 20 min. Basal, or unstimulated, activation of specific G proteins was reflected by the amount of [35S]GTPS bound to wells, which were precoated with antibodies raised against the -subunit of Gq, Gi3, or Gi1/i2. G protein activation was measured as cpm [35S]GTPS per µg membrane protein. Values are means ± SE of 4 animals, with each sample performed in triplicate.

Spontaneous activity of G_i-type G proteins was confirmed by examining the effect of PTX on LES tone. Figure 12 shows that PTX concentration dependently reduced LES tone (ANOVA, P < 0.001). The highest concentration of PTX did not have a nonselective effect on LES muscle, since it did not affect contraction induced by a maximally effective dose of ACh, which is mediated by the PTX-insensitive G_q (86). These data suggest that in the LES there is spontaneous activation of G proteins and that a PTX-sensitive G protein may be involved in tone.

View larger version (14K): [in this window] [in a new window]   Fig. 12.   A: role of pertussis toxin (PTX) on LES basal tone. LES circular muscle strips were incubated in Krebs containing 0.1% BSA (control) or in Krebs containing 0.1% BSA and indicated concentrations of PTX for 1 h. PTX produced a concentration-dependent decrease in LES basal tone. B: LES circular muscle strips were incubated in ACh (105 M) alone (control) or in the presence of PTX (5 mg/ml). PTX did not affect the increase in LES tone produced by ACh. Values are means ± SE of 3 animals (controls) or 7 animals (PTX).

Because PGF₂ and thromboxanes A₂ and B₂ contribute to maintenance of LES tone that is associated with G protein activation, we next examined the G proteins activated by these AA metabolites.

PGF₂ (10⁶ M) caused significant activation of G_q (ANOVA, P < 0.01) and G_i3 (ANOVA, P < 0.05) but had no effect on G_i1/i2, or G_o (Fig. 13A).

View larger version (24K): [in this window] [in a new window]   Fig. 13.   A: [35S]GTPS binding to PGF2-activated membranes of LES. Purified membranes were exposed to PGF2 (106 M) in the presence of [35S]GTPS for 1 min. PGF2-induced activation of specific G proteins was reflected by the amount of [35S]GTPS bound to wells, which were precoated with antibodies raised against the -subunit of Gq, Gi3, Gi1/i2, or Go. G protein activation was measured as %increase in [35S]GTPS binding in membranes exposed to PGF2 compared with unstimulated membranes. PGF2 (106 M) caused significant activation of Gq and Gi3. Values are means ± SE of 4 animals, with each sample performed in triplicate. B: LES muscle cells were permeabilized by brief exposure to saponin to allow diffusion of antibodies into the cytosolic side of the cell membrane. Cells were contracted with PGF2 (108 M) alone (control) or after a 60-min preincubation in cytosolic medium containing antibodies raised against the -subunit of G proteins (1:200 dilution). PGF2-induced contraction of LES cells was significantly inhibited by Gq and Gi3 and not by Gi1/i2 and Go antibodies. Values are means ± SE of 3 animals with each sample performed in triplicate. * P < 0.05; ** P < 0.001;  P < 0.01.

To determine which G proteins mediate PGF₂-induced contraction, we used G protein antibodies raised against synthetic peptides corresponding to the amino acid sequence of the COOH terminus of the G protein -subunit. Figure 13B shows that PGF₂-induced contraction of LES smooth muscle cells was significantly reduced by antibodies raised against the COOH terminus of the -subunit of G_q and G_i3 (ANOVA, P < 0.001) and unaffected by G_i1/i2 or G_o antibodies.

Figure 14A shows that the thromboxane A₂-mimetic U-46619 stimulates [³⁵S]GTPS binding to G_q (ANOVA, P < 0.001). Similarly, U-46619-induced contraction of permeabilized LES muscle cells was significantly reduced by antibodies raised against the COOH terminus of the -subunit of G_q (ANOVA, P < 0.001) and unaffected by G_i3, G_i1/i2, or G_o antibodies (Fig. 14B).

View larger version (25K): [in this window] [in a new window]   Fig. 14.   A: [35S]GTPS binding to U-46619-activated membranes of LES. Purified membranes were exposed to U-46619 (106 M) in the presence of [35S]GTPS for 1 min. U-46619-induced activation of specific G proteins was reflected by the amount of [35S]GTPS bound to wells, which were precoated with antibodies raised against the -subunit of Gq, Gi3, Gi1/i2, or Gs. G protein activation was measured as %increase in [35S]GTPS binding in membranes exposed to U-46619 compared with unstimulated membranes. U-46619 (106 M) caused significant activation of Gq. Values are means ± SE of 3 animals with each sample performed in triplicate. B: LES muscle cells were permeabilized by brief exposure to saponin to allow diffusion of antibodies into the cytosolic side of the cell membrane. Cells were contracted with U-46619 (108 M) alone (control) or after a 60-min preincubation in cytosolic medium containing antibodies raised against the -subunit of G proteins (1:200 dilution). U-46619-induced contraction of LES cells was significantly inhibited by Gq and not by Gi3, Gi1/i2, or Go antibodies. Values are means ± SE of 3 animals, with each sample performed in triplicate. ** P < 0.001.

Indomethacin (10 µM) significantly reduced [³⁵S]GTPS binding to G_q, G_i3, and G_i1/i2 (ANOVA, P < 0.01) in LES circular smooth muscle (Fig. 15), supporting the hypothesis that the cyclooxygenase products PGF₂ and thromboxane A₂ and B₂ may be involved in G protein activation and maintenance of LES tone.

View larger version (23K): [in this window] [in a new window]   Fig. 15.   Indomethacin decreased basal [35S]GTPS binding of Gq, Gi3, or Gi1/i2 in LES smooth muscle membranes. LES circular smooth muscle was incubated in Krebs solution at 37°C for 2 h, and then aliquots of LES tissue were incubated for another 60 min in Krebs solution alone (control) or in Krebs solution containing indomethacin (105 M). Purified membranes were incubated in [35S]GTPS for 1 min. Basal, or unstimulated, activation of specific G proteins was reflected by the amount of [35S]GTPS bound to wells, which were precoated with antibodies raised against the -subunit of Gq, Gi3, or Gi1/i2. G protein activation was measured as cpm [35S]GTPS per µg membrane protein. Percent inhibition was calculated as 100 × [radioactivity in the control (cpm/µg protein)  radioactivity in indomethacin group (cpm/µg protein)/radioactivity in control (cpm/µg protein)]. Values are means ± SE of 3 animals, with each sample performed in triplicate.  P < 0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Group I sPLA₂ contributes to basal LES tone. Spontaneous LES tone may be mediated by the activity of a group I sPLA₂ because LES tone was concentration dependently reduced by inhibitors selective for group I sPLA₂. The selectivity of the PLA₂ inhibitors was demonstrated in Fig. 2 in which isolated cells from the body of the esophagus were contracted with either purified group I, group II, or group III sPLA₂. AM5 and X1 are thought to be nonselective inhibitors for sPLA₂; MJ33 (44) selectively inhibits group I sPLA₂; and MJ45 is selective for group II sPLA₂ (43). BPB is thought to be a nonselective inhibitor for PLA₂ (30, 57, 59); however, in our preparation, BPB inhibited only group I sPLA₂ purified from cobra venom.

Group I sPLA₂-induced contraction of isolated esophageal muscle cells was selectively reduced by MJ33, AM5, X1, and BPB, and these inhibitors dose dependently reduced LES tone in a TTX-independent manner. The maximal fall in basal LES tone induced by the PLA₂ inhibitors was 40%, whereas indomethacin reduced tone by 80% from basal levels. The indomethacin-induced reduction in tone may indicate AA release from different phospholipases, such as PC-PLC (26), which participate in the maintenance of LES tone (41). TTX was added to the PLA₂ inhibitor to abolish axonal transmission, eliminating the possibility that indomethacin and the sPLA₂ inhibitors may be acting on neurons. Even at the high concentrations used in the present study, TTX did not modify, in any way, the reduction in resting tone induced by these agents.

The greater AA tissue content and AA release in LES smooth muscle, when compared with esophageal smooth muscle, which does not maintain basal tone (Fig. 5), supports the view that spontaneous group I sPLA₂ activity may play a role in maintaining LES tone. The same group I sPLA₂-selective inhibitors that reduced LES tone also reduced the spontaneous AA release in LES smooth muscle (Fig. 6). The concentration of sPLA₂ inhibitors that reduced LES tone and AA release were similar to those reported for the cytosolic PLA₂ inhibitor AACOCF₃. The IC₅₀ for the concentration of AACOCF₃ required for a twofold reduction in the amount of AA liberated from 4 × 10⁸ platelets/ml is 8-10 µM. The concentrations of MJ33 and MJ45 for 50% inhibition are ~0.01 mol fraction, the molar amount of inhibitor that the enzyme sees at the interface of the substrate (5). For interfacial enzymology, the relevant inhibitory concentrations are in mole fraction because there is no direct way to express inhibition in moles per liter (32) for biological systems because the true lipid concentration is unknown.

LES circular muscle differed from esophagus circular muscle because it contracted only in response to group I sPLA₂ purified from cobra venom, whereas esophagus circular muscle contracted in response to all three sPLA₂ enzymes. The sPLA₂ enzymes used in the present studied are purified from the various venoms. Human pancreatic sPLA₂ is secreted as an inactive zymogen, which is activated by trypsin in the gastrointestinal lumen to its active enzyme by the removal of seven amino acid residues from the NH₂ terminus (27). It is likely that the purified enzymes used in the present study are already active.

The selectivity of the group I sPLA₂ in contracting LES muscle may be conferred by the specific interaction of sPLA₂ with cell surface receptors. Specific membrane receptors for neuronal group and muscle group sPLA₂ enzymes have been identified with snake venom sPLA₂ (50, 52-55). One of these sPLA₂ receptors, the 180-kDa muscle group, has been cloned in rabbit (50) and humans (2) and has been shown to have very high affinity for mammalian sPLA₂. This receptor has a large extracellular domain of 1394 amino acids composed of an NH₂-terminal cysteine-rich domain, a fibronectin-like group II domain, eight carbohydrate recognition domains (CRDs), and a transmembrane and an intracellular COOH terminus (50). The CRD5 on the sPLA₂ receptor (63) and the residues within or near the calcium binding loop on sPLA₂ (51) appear to be necessary for phospholipase-receptor interaction. Selective sPLA₂ binding is responsible for some of the physiological effects of mammalian sPLA₂, including vascular smooth muscle contraction, cell proliferation, and internalization of sPLA₂ (3, 61, 89). For example, antigen stimulation results in the selective binding of group I sPLA₂ to bone marrow mast cells, which contain the mRNA for the group I PLA₂ receptor (29), causing release of AA.

PGF₂ and/or thromboxanes may mediate LES tone by activation of G proteins. The AA produced by sPLA₂ in the LES may be metabolized to prostaglandins and thromboxanes because the cyclooxygenase inhibitors aspirin and indomethacin reduce LES basal tone (Fig. 7). Leukotrienes are not likely to play a role in this system because the lipoxygenase inhibitor NDGA had no effect on LES tone.

Our data are consistent with previously reported in vitro data. However, there is a difference between the effects of AA and indomethacin in in vivo and in vitro preparations. In vitro LES contracts in response to AA (20) and relaxes in response to indomethacin (19, 20). In vivo LES relaxes in response to AA (21, 73). In vivo indomethacin has no effect on LES resting tone (73). We have reported that AA contracts in vitro LES muscle strips (75), and in the present study we demonstrate that indomethacin relaxes them.

The reason for differences between in vitro and in vivo preparations is not clear. It is possible that injected AA in the in vivo preparations may be metabolized to prostaglandins in nerve terminals as well as in muscle. Daniel et al. (20) reported that indomethacin inhibited relaxations to electric field stimulation, that inhibition of relaxation preceded loss of muscle tone, and that inhibition of tone was complete. This may explain why in vitro indomethacin first contracts and then relaxes LES. The fact that inhibition of relaxation preceded loss of muscle tone suggests that perhaps inhibitory neurons are more sensitive to the effect of indomethacin than the muscle. In vivo indomethacin was administered at doses sufficient to inhibit AA-induced relaxation, which may be neurally mediated. It is possible that higher doses of indomethacin may be required to have an effect on muscle tone. The effect of TTX on AA-induced relaxation was not tested in any of the previous studies, so that the site of action of in vivo AA has not been pinpointed.

Similarly, PGF₂ has been reported to contract LES in vitro (19, 20), whereas, in vivo, PGF₂ most often contracts LES and sometimes relaxes it (74). In vivo, PGF₂, the stable epoxymethano derivatives of PGH₂, and thromboxane B₂ have been reported to contract LES smooth muscle strips (19). We therefore examined the role of PGF₂ and thromboxanes A₂ and B₂ in LES tone. We find that PGF₂ produced a concentration-dependent increase in LES basal tone and reversed the reduction in tone produced by indomethacin (Fig. 9).

Although other cyclooxygenase products such as PGE₂ have been reported to relax the LES (21, 34, 36, 74), we find more PGF₂ production in the LES compared with the esophagus (Fig. 8). Formation of different prostaglandins depends on the presence of the appropriate synthetic enzymes required to make the particular prostaglandin. For example, in the rat ocular system, steady-state levels of PGE₂, PGF₂, and PGD₂ correlate well with the enzymatic activities of their respective synthetase enzymes, namely, glutathione (GSH)-dependent and membrane-bound E, soluble F, and GSH-independent and soluble D synthetase (33). It is possible that LES may have a higher content/activity of soluble F synthetase or more 9-ketoreductase, which catalyzes the conversion of PGE₂ to PGF₂ (64, 96). Different activities of synthetase enzymes will result in different prostaglandin distributions in different organs. For example, in fetal lamb brain, lung, liver, spleen, ductus arteriosus, aorta, and pulmonary vein, different amounts of PGE₂, PGF₂, PGD₂, 6-keto-PGF₁, and thromboxane B₂ are present (67). In blood vessels of rat brain, 6-keto-PGF₁ is present in 40-fold higher concentrations than in brain tissue; conversely, PGD₂ levels are high in brain tissue and below detection levels in brain blood vessels (1).

Thromboxane A₂ is an unstable arachidonate metabolite, with a half-life of 30 s, and rapidly decays nonenzymatically to the stable thromboxane B₂, which has weak biological activity (62). We find that thromboxane B₂ also produced a concentration-dependent increase in LES basal tone and reversed the reduction in tone induced by indomethacin. In addition, the selective thromboxane A₂ antagonist SQ-29548 (38) dose dependently reduced LES basal tone (Fig. 10). These data suggest that thromboxanes A₂ and B₂ may also play a role in maintaining LES basal tone.

The action of these prostanoids is mediated by distinct receptors. The classification of prostanoid receptors in platelets and smooth muscle is based on the pattern of effects and the relative potencies of natural and synthetic agonists (18) and substantiated by ligand binding studies, receptor cloning, and selective antagonists (18, 39). The receptors are named after their endogenous prostaglandin ligand and are divided into five main types: DP (PGD), FP (PGF), IP (PGI₂), TP (thromboxane A₂), and EP (PGE). The EP receptors have been further subdivided into EP1, EP2, EP3, and EP4, on the basis of physiological activity and molecular cloning (18, 92). The cDNAs encoding representatives of each of these groups of receptors have been cloned (69). Heterologous expression of receptor cDNAs confirmed that they are all G protein-coupled receptors that contain seven transmembrane domains, an extracellular NH₂ terminus, and an intracellular COOH terminus. Functional expression of the cloned receptors is consistent with a single subunit structure containing a ligand binding site and the determinants required for second messenger coupling. The DP, IP, EP2, and EP4 receptors are coupled to stimulation of adenylyl cyclase, and the EP1, FP, and TP receptors are coupled to phosphatidylinositol hydrolysis (70).

Because LES tone is myogenic, and may be mediated by production of prostanoids that are coupled to G protein effector mechanisms, G proteins may be activated in LES smooth muscle. We have previously shown, by Western blot, that G_q, G_i3, and G_i1/i2 are all present in esophageal and LES circular muscle (84). In the current study, we show that in the LES these G proteins are active, i.e., bound to GTP in the absence of exogenous stimuli (Fig. 11). The same G proteins that are spontaneously active are stimulated by PGF₂ and thromboxane B₂. In addition, LES basal tone can be concentration dependently reduced by PTX, suggesting that basal G_i activation may contribute to LES basal tone.

The PGF₂ receptor is reported to be coupled to phosphoinositide metabolism and calcium mobilization via stimulation of PTX-insensitive G_q proteins (42). In LES smooth muscle membranes, PGF₂ significantly activates G_q and G_i3 and slightly activates G_i1/i2 and has no effect on G_o activation. In addition, stimulation of the FP receptor on LES smooth muscle cells with PGF₂ produces a maximal contraction (21.3 ± 0.4% shortening) that is selectively reduced by antibodies raised against the COOH terminus of the -subunit of G_q and G_i3. These data suggest that, in the LES, FP receptor stimulation results in contraction that is mediated by both G_q- and G_i3-like G proteins.

Several studies have demonstrated that stimulation of the platelet TP receptor results in the activation of a PTX-insensitive PLC with stimulation of phosphoinositide metabolism and subsequent increase in intracellular calcium (39, 71, 77, 82). The TP receptor may be coupled to G_q because thromboxane A₂ agonist-stimulated GTPase activity was blocked by an antibody raised against the COOH terminus of the -subunit of G_q in human platelet membranes (81). In addition, thromboxane A₂ stimulation has been shown to inhibit adenylate cyclase and to reduce cAMP-mediated inhibition of ADP-evoked response in platelets. Therefore, in platelets, the TP receptor may be coupled to two different G proteins (G_q- and a G_i-like G protein) and two different signal transduction pathways. The TP receptor has also been shown to be coupled to G_i2, G₁₂, G₁₃ and to an unidentified 85-kDa G protein (48, 65, 94). The prostanoid second messenger system in smooth muscle cells has not been extensively studied; however, thromboxane mimetics have been reported to increase intracellular free calcium and calcium fluxes in vascular tissue and in smooth muscle cells (15, 28, 31, 37).

In the current study, we show that the thromboxane A₂-mimetic U-46619 (4, 90) significantly activates G_q and has a small stimulatory effect on G_i3 and G_i1/i2 but no effect on G_s activation. In addition, stimulation of the TP receptor on LES smooth muscle cells with U-46619 produced a maximal contraction (22.4 ± 1.2% shortening) that was selectively reduced by antibodies raised against the COOH terminus of the -subunit of G_q. These data suggest that, in the LES, TP receptor stimulation results in contraction that is mediated by G_q-like G proteins, similar to the reports of G_q activation by stimulation of the TP platelet receptor.

These data show that the prostanoids PGF₂ and thromboxane A₂/B₂ can account for the activation of G_q, G_i3, and G_i1/i2, which are found to be spontaneously active in the LES in its basal state. A role for G_q and G_i3 is supported by Figs. 13 and 14, which show that PGF₂- and thromboxane-induced contraction is reduced by G_q and G_i3 and not by G_i1/i2 antibodies. Although G_i1/i2 is present in the LES (84) and is active (Fig. 11), it is not coupled to contraction (Figs. 13B and 14B) or maintenance of LES basal tone (Fig. 15), and its function remains to be determined.

The activity of these G proteins is reduced by indomethacin, supporting the view that spontaneous activation of these G proteins is maintained by cyclooxygenase-catalyzed AA products. Activation of these G proteins may play a role in LES basal tone because the same concentration of indomethacin (10⁵ M) (Fig. 7) that reduced tone by 50% also significantly reduced the level of activity of G_q, G_i3, and G_i1/i2 present in the unstimulated LES circular muscle (Fig. 15). Because basal LES tone is reduced by PTX and G_i1/i2 has no role in prostanoid-mediated LES contraction, G_i3 is likely to be the G_i-type G protein involved in maintenance of LES tone.

We propose the following hypothesis: Spontaneous activation of a group I sPLA₂ causes production of AA and AA metabolites such as PGF₂ and thromboxanes A₂ and B₂, which maintain activation of G proteins such as G_i3, G_i1/i2, and G_q. G_i3 and G_q activate the phospholipases PC-PLC and PI-PLC, which in turn produce DAG and IP₃. DAG and IP₃ synergistically activate PKC- and produce LES tone (8, 41, 88). The origin of the group I sPLA₂ remains to be found; however, preliminary Western blot studies using monoclonal antibodies of human sPLA₂ group I indicate the presence of sPLA₂ in human LES circular smooth muscle.

	ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants RO1 HD-20054, RO1 DK-42876, and RO1 DK-28614.

	FOOTNOTES

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

Address for reprint requests and other correspondence: P. Biancani, GI Motility Research Lab., SWP5 Rhode Island Hospital and Brown Univ., 593 Eddy St., Providence RI 02903 (E-mail: piero_biancani{at}brown.edu).

Received 8 March 1999; accepted in final form 8 June 1999.

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