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Am J Physiol Gastrointest Liver Physiol 285: G1189-G1197, 2003. First published August 22, 2003; doi:10.1152/ajpgi.00200.2003
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LIVER AND BILIARY TRACT

PAF-like lipids- and PAF-induced gallbladder muscle contraction is mediated by different pathways in guinea pigs

Michele P. L. Guarino, Zuo-Liang Xiao, Piero Biancani, and Jose Behar

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

Submitted 1 May 2003 ; accepted in final form 16 July 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
H2O2 stimulates gallbladder muscle contraction and scavengers of free radicals through the generation of PGE2. Oxidative stress causes lipid peroxidation and generation of platelet-activating factor (PAF) or PAF-like lipids. The present studies therefore were aimed at determining whether either one induced by H2O2 mediates the increased generation of PGE2. Dissociated muscle cells of guinea pig gallbladder were obtained by enzymatic digestion. Both PAF-like lipids and PAF-induced muscle contraction was blocked by the PAF receptor antagonist CV-3988. This antagonist also blocked the increased PGE2 production caused by PAF-like lipids or PAF. Actions of PAF-like lipids were completely inhibited by indomethacin, but those of PAF were only partially reduced by indomethacin or by nordihydroguaiaretic acid and completely blocked by their combination. PAF-like lipids-induced contraction was inhibited by AACOCF3 (cystolic phospholipase A2 inhibitor), whereas the actions of PAF were blocked by MJ33 (secretory phospholipase A2 inhibitor). Receptor protection studies showed that pretreatment with PAF-like lipids before N-ethylmaleimide protected the contraction induced by a second dose of PAF-like lipids or PGE2 but not by PAF. In contrast, pretreatment with PAF protected the actions of PAF and PGE2 but not that of PAF-like lipids. Both PAF-like lipids and PAF-induced contractions were inhibited by anti-G{alpha}q/11 antibody and by inhibitors of MAPK and PKC. In conclusion, PAF-like lipids seem to activate a pathway different from that of PAF probably by stimulating a different PAF receptor subtype.

oxidative stress; PGE2 production; receptor protection

REACTIVE OXYGEN SPECIES (ROS) such as H2O2 are increased during acute inflammatory processes (9, 25). The initial injury in the gallbladder muscle caused by ROS appears to be confined to the plasma membrane (27). These free radicals induce nonenzymatic oxidation of phospholipids (lipid peroxidation) and damage membrane protein constituents by oxidizing transmembrane receptors and calcium channels (3, 9). H2O2 impairs the contraction induced by receptor-dependent agonist CCK-8 and ACh as well as KCl, which is receptor-G protein independent (25).

H2O2 causes muscle cell contraction and increases PGE2 formation (27). The pathways responsible for these cellular responses are not known. Lipid peroxidation results in the generation of isoprostanes, platelet-activating factor (PAF)-like lipids (10), and oxidized byproducts. Isoprostanes do not appear to be plausible candidates of stimulating the pathways that mediate the contraction and cytoprotective responses, because they tend to behave like prostaglandin receptor antagonists; particularly against PGF2{alpha} (12-14, 16). Other oxidation products of phosphatidylcholine bind and activate PAF receptors (19). These PAF-like lipids are potent, selective inflammatory mediators formed by nonenzymatic mechanisms resulting in an unregulated accumulation in the plasma membranes (10, 20). The precursors of PAF synthesis are susceptible to oxidative attack resulting in the fragmentation of the esterified polyunsaturated fatty acid residue at the sn-2-position (11). Fragmentation of the sn-2 residue is the result of the oxidation of phospholipid byproducts that structurally resemble PAF. Some of the oxidized phospholipids have the ether bond at the sn-1-position of the glycerol backbone that is an important determinant for recognition by the PAF receptor (30). Thus the oxidative byproducts complete the molecular mimicry of PAF by creating a residue that in some cases is only a little longer than the acetyl residue of PAF.

Therefore, the present studies were aimed at determining whether H2O2 induced muscle cell contraction and PGE2 formation are mediated by PAF or PAF-like lipids acting on PAF receptors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals. Adult male guinea pigs were purchased from Elm Hill breeding Laboratory (Chelmsford, MA). The Animal Welfare Committee of Rhode Island Hospital approved their use. Animals were housed in thermoregulated rooms and had free access to food and water. After an overnight fast, the animals were anesthetized with an intramuscular injection of ketamine hydrochloride (30 mg/kg), followed by pentobarbital (30 mg/kg ip). The gallbladder was removed and rinsed with ice-cold, oxygenated Krebs solution (in mM: 116.6 NaCl, 3.4 KCl, 21.9 NaHCO3, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2, and 5.4 glucose). Mucosa and serosa were carefully peeled off under a dissecting microscope. The muscle layer was further cleaned by gently removing the remaining connective tissue.

Isolation of single cells from gallbladder muscle. Gallbladder muscle cells were prepared as previously described (24, 25, 27, 28). After the gallbladder was removed from the animal, it was immediately placed in ice-cold preoxygenated Krebs buffer solution, washed, and transferred to a dissecting pan containing the same buffer continuously aerated with 95% O2-5% CO2. The Krebs buffer solution contained (in mM) 116.6 NaCl, 3.4 KCl, 21.9 NaHCO3, 1.2 NaH2PO4, 2.5 CaC12, 1.2 MgCl2, and 5.4 glucose. Under a dissecting microscope, the mucosa and then the serosa were carefully peeled off. The muscle layer was further cleaned by removing the remaining connective tissue and blood vessels. It was minced to 2 x 2-mm squares and digested in HEPES and in a buffered nutrient solution containing 150 units of type II collagenase per milliliter for 2.5-3.5 h at 31°C. HEPES buffer solution contained (in mM) 112.5 NaCl, 5.5 KCl, 2.0 KH2PO4, 24 HEPES, 1.9 CaCl2, 0.6 MgCl2, and 10.8 glucose, as well as 0.08 mg/ml soybean trypsin inhibitor and 2% (vol/vol) basal Eagle's medium (50x) amino acids without L-glutamine. The pH of the solution was adjusted to 7.4 at 31°C. The solution was gently gassed with 100% O2 during the period of digestion. At the end of digestion, the tissue was placed over a 450-nm Nitex mesh, rinsed with 80 ml of collagenase-free HEPES solution to remove any trace of collagenase, and then incubated in collagenase-free HEPES solution at 31°C for 30-60 mm to allow the cells to disperse freely. Cells were then harvested by filtering through a 450-nm Nitex mesh, and the cell suspension was equilibrated at 31°C for 20 min before the beginning of the experiment.

Permeabilization of smooth muscle cells. Muscle cells were permeabilized with saponin (2, 24, 25, 27, 28). After completion of the enzymatic phase of the digestion process, the partly digested muscle tissue was washed and incubated in a collagenase-free medium resembling cytosol (cytasolic buffer), which had the following composition (in mM): 20 NaCl, 100 KCl, 25 NaHCO3, 5.0 MgSO4, 0.96 NaH2PO4, 0.48 CaCl2, and 1.0 EGTA. Free-calcium concentrations in the cytosolic buffer were controlled by using Ca-to-EGTA ratio. With 0.48 mM CaCl2 and 1.0 mM EGTA, free-calcium concentration was 0.18 nM, which is dosed to the physiological calcium concentration in the cytosol of smooth muscle cells as well as other cells. However, because higher concentrations of calcium are required for the activation of exogenous calmodulin, the free-calcium concentration in the cytosolic buffer was raised to 1.32 nM when calmodulin was to be used by increasing CaCl2 to 0.875 mM without changing the concentration of EGTA. The cytosolic buffer also contained 2% bovine serum albumin and was equilibrated with 95% O2-5% CO2 to maintain a pH of 7.2 at 31°C.

When cells were dispersed, they were harvested by filtering through a 450-nm Nitex mesh. The filtrates (cell suspension) were then incubated with saponin (75 µg/ml) followed by centrifugation at 200 g for 3 min. After removal of the supernatant, the cell pellet was washed with 5 min of modified cytosolic buffer and centrifuged for 3 more minutes. The total permeabilization time from the addition of saponin to washing was 4 min. Modified cytosolic buffer contained, in addition to the above, 10 nM antimycin A, 1.5 mM ATP, and an ATP-regenerating system consisting of creatine phosphate (5 mM) and creatine phosphokinase (10 U/ml). Cells were then equilibrated in modified cytosolic buffer with continuous aeration with 95% O2-5% CO2 at 31°C for 20 min before the beginning of the experiment. Permeabilization with saponin (see above) has been shown to allow the large molecules such as inositol 1,4,5-trisphosphate to diffuse across the plasma membrane freely, whereas it does not affect the intracellular contractile mechanisms.

Experimental procedure and cell measurements. After muscle cells were equilibrated for 20 min, 0.25-ml aliquots of cell suspension were added to test tubes containing 0.1 ml of buffer with appropriate concentrations of an agonist (24, 25, 27, 28). Receptor protection studies were performed in muscle cells first incubated with PAF or H2O2 for 2 min, followed by a 15-min incubation with 5 µM of N-ethylmaleimide (NEM) (5). The cells were then washed by superfusion with HEPES buffer for 20 min. Their control cell length was measured before the contraction studies were performed with the same or different agonist. Cells were then exposed to agonists for 30 s, and their response was stopped by the addition of acrolein at a final concentration of 1%. A drop of fixed cells was placed on a microscope slide and covered with a coverslip. Edges of the coverslip were sealed with nail enamel to prevent evaporation. Lengths of 30 consecutive cells from control and experimental samples were measured. They were measured by using a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany) and a Panasonic CCTV camera (model WV-CD51; Matsushita Communication, Osaka, Japan) that transfers the image from the microscope to a Macintosh Power PC computer (Apple Computer, Cupertino, CA) and utilizes a computer software program (Image version 1.33; National Institutes of Health, Bethesda, MD). Contraction was expressed as percent shortening from initial control cell length.

Preparation of enriched plasma membranes. Enriched plasma membranes from muscle squares were prepared and purified by sucrose gradient centrifugation (17, 23). Muscle squares were homogenized in sucrose-HEPES buffer by using a tissue tearer (Biospec Products, Racine, WI). This sucrose-HEPES buffer contains 0.25 M sucrose, 10 mM HEPES (pH 7.4), 0.01% soybean trypsin inhibitor, 0.1 nM PMSF, 0.1 nM 1,10-phenanthroline, and 1 mM 2-mercaptoethanol. The homogenates were centrifuged at 600 g for 5 min; the supernatant was collected in a clean tube and again centrifuged at 150,000 g for 45 min. The pellet was resuspended in sucrose-HEPES buffer, layered over a linear 9-60% sucrose gradient, and centrifuged at 90,000 g for 3 h. The plasma membranes were collected at ~24% sucrose and stored at 70°C.

[35S]GTP{gamma}S binding and immunoprecipitation. [35S]GTP{gamma}S binding was assayed by immunoprecipitation (22). Enriched plasma membranes were solubilized for 1 h at 4°C with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in HEPES buffer (in mM: 10 HEPES, pH 7.4, 0.1 EDTA, and 10 MgCl2). Solubilized membranes at a concentration of 2.5 mg protein/ml were incubated with 60 nM [35S]GTP{gamma}S in the presence or absence of agonists at 37°C for 10 min (total volume, 300 µl). For nonspecific binding, 6 µM GTP was used. The reaction was stopped with 10 volumes of ice-cold Tris buffer (in mM: 100 Tris · HCl, pH 8.0, 10 MgCl2, 100 NaCl, and 20 GTP). Aliquots (200 ml) of the reaction mixture were added to pretreated ELISA wells. The ELISA wells were prepared by initially coating with an anti-rabbit immunoglobulin antibody (1:2,000) at 4°C for 1 h, by washing out the trace of the antibody, and then by coating with specific G protein subunit antibodies (1:2,000) at 4°C for another 1 h. The specific G protein subunit antibodies of anti-G{alpha}i1-2, -G{alpha}i3, -G{alpha}q/11, and -G{alpha}s were used. After incubation with the reaction mixture in ELISA wells at 4°C for 2 h, the wells were washed three times with phosphate buffer (in mM: 1 KH2PO4, pH 7.4, 10 Na2HPO4, 137 NaCl, and 2.7 KCl) containing 0.05% Tween 20. The radioactivity of each well was counted by a scintillation counter. Data were expressed as percent increase over basal levels (without agonist stimulation).

Measurement of PGE2 levels. The content of PGE2 was measured by using a radioimmunoassay kit from New England Nuclear Life Science Products (Boston, MA) (4, 28). Muscle cells were homogenized and the suspension was centrifuged at 10,000 g for 15 min. PGE2 was extracted from the supernatant (8) and PGE2 was converted into its methyl oximate derivative by using the methyl oximation reagent. The determination of PGE2 content was achieved by following the kit's protocol. Briefly, samples or different concentrations of purified PGE2 (standard curve) were incubated with 125I-labeled PGE2 and PGE2 antiserum overnight at 4°C. Separation of bound form was achieved by adding precipitating reagent and centrifuged for 30 min at 4°C. Samples were counted in a gamma counter and the content of PGE2 was calculated from the standard curve and expressed as nanograms per milligrams protein.

Protein determination. Protein content of muscle membranes was measured by using a protein assay kit (Bio-Rad Laboratories, Hercules, CA). Values for each sample were means of triplicate measurements.

Drugs and chemicals. H2O2 was obtained from Fisher Scientific (Pittsburgh, PA); CV-3988 was obtained from Bio-Mol Research Laboratories (Plymouth Meeting, PA); antibodies against G protein subunits [G{alpha}i1-2 (COOH terminus 345-354 and 346-355), G{alpha}i3 (COOH terminus 345-354), G{alpha}q/11 (internal 283-300), G{alpha}s (COOH terminus 385-394)] were purchased from CytoSignal (Irvine, CA); [35S]GTP{gamma}S and PGE2 radioimmunoassay kit were obtained from New England Nuclear Life Science Products; type F collagenase, papain, NEM, and other reagents were purchased from Sigma (St. Louis, MO).

Statistics. One- and two-factorial repeated ANOVA or Student's t-tests (unless otherwise noted) were used for statistical analysis. P values of <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Enzymatically dissociated muscle cells were treated with PAF receptor antagonist CV-3988 (10-5 M) for 15 min, followed by agonist for 30 s. Buffered, treated muscle cells contract an average of 20% in response to agonists (25). Increasing concentrations of PAF16 and PAF18 (10-13-10-8 M) induced a similar dose-dependent muscle contraction (Fig. 1). Maximal contractions of 20.4 ± 1.5% (PAF16) and 21.3 ± 1.4% (PAF18) were reached with a dose of 10-9 M. PAF16- and PAF18-induced contractions were completely blocked by the PAF receptor antagonist CV-3988 (10-5 M) to 3.1 ± 0.7 and 3.5 ± 0.1% (*P < 0.001 and #P < 0.001 by ANOVA).



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  		Fig. 1. Effect of specific platelet-activating factor (PAF) receptor antagonist CV-3988 on PAF (10-13 to 10-8 M)-induced muscle contraction. Both PAF16 and PAF18 (two isoforms of PAF) caused a dose-dependent muscle contraction of <=20%. Their effects were blocked by pretreatment with CV-3988 (10-5 M) (*P < 0.001, #P < 0.001 by ANOVA). Values are means ± SE of 3 experiments.

 

H2O2 also induced a dose-dependent muscle contraction with a maximal contraction of 13.3 ± 1.9% achieved with a dose of 70 µM (Fig. 2). H2O2-induced muscle contraction was also blocked by pretreatment with the PAF receptor antagonist CV-3988 (10-5 M) (*P < 0.001 by Student's t-test). H2O2 generates PAF-like lipids by acting on plasma membrane phospholipids (10). These data suggest that PAF-like lipids are one of the byproducts of lipid peroxidation acting on membrane receptors.



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  		Fig. 2. Effect of PAF receptor (PAF-F) antagonist CV-3988 (10-5 M) on muscle contraction induced by increasing concentrations of H2O2. H2O2 (17.5 to 140 µM) induced a dose-dependent increase with a maximal contraction achieved at a dose of 70 µM. This effect was blocked by pretreatment with CV-3988 (*P < 0.001 by Student's t-test). Values are means ± SE of 3 experiments.

 

To determine whether PAF and PAF-like lipids are generated by lipid peroxidation, the signal transduction responsible for muscle contraction caused by exogenous PAF and by PAF-like lipids was examined. Muscle cells were pretreated with indomethacin (cyclooxygenase inhibitor) and nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor) for 15 min before agonist stimulation (Fig. 3). PAF-induced contractions were partially inhibited by either indomethacin (20.2 ± 2.4 to 8.4 ± 1.3%; *P < 0.001 by Student's t-test) or NDGA (20.2 ± 2.4 to 12.2 ± 0.9%; **P < 0.001 by Student's t-test) and completely blocked by both (20.2 ± 2.4 to 1.5 ± 1.0%; ***P < 0.001 by Student's t-test). However, H2O2-induced contractions were only blocked by indomethacin (14.6 ± 1.2 to 8.4 ± 1.3%; #P < 0.001, ##P < 0.001 by Student's t-test) and unaffected by NDGA. These data suggest that prostaglandins are involved in the pathways that mediate PAF-like lipid-induced muscle cell contractions. In contrast, the PAF-induced contraction is mediated by prostaglandins and probably also by leukotrienes. The finding that PAF and PAF-like lipids stimulate different phospholipases (PLAs) supports these results. In these studies, the inhibitors of cytosolic PLA2 (cPLA2; AACOCF3) and secretory PLA2 type I (sPLA2; MJ33) were tested against the contraction induced by these agonists (Fig. 4). The highly selective sPLA2 type 1 inhibitor MJ33 was used in permeablized muscle cells. PAF-induced contractions were only blocked by MJ33 (20.2 ± 2.4 to 4.1 ± 0.9%; *P < 0.001 by Student's t-test). H2O2-induced contractions were only inhibited by AACOCF3 (14.2 ± 1.2 to 0.9 ± 0.3%; #P < 0.001 by Student's t-test).



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  		Fig. 3. Effect of inhibitors of cyclooxygenase [indomethacin (Indo) 5 µM] and lypoxygenase [nordihydroguoiaretic acid (NDGA); 10 µM] on PAF (10-9 M)- and H2O2 (70 µM)-induced muscle contraction. Pretreatment of muscle cells with indomethacin or NDGA for 15 min was followed by PAF or H2O2 for 30 s. PAF-induced contraction was partially inhibited by either indomethacin (*P < 0.001 by Student's t-test) or NDGA (**P < 0.001 by Student's t-test) but was completely blocked by their combination (***P < 0.001 by Student's t-test). However, H2O2-induced contractions were completely blocked by indomethacin alone (#P < 0.001, ##P < 0.001 by Student's t-test) and not affected by NDGA. Values are means ± SE of 3 experiments.

 


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  		Fig. 4. Effect of inhibitors of cystolic phospholipase A2 (cPLA2; AACOCF3; 30 µM) and sPLA2 (MJ33; 0.1 µM) on PAF (10-9 M)- or H2O2 (70 µM)-induced muscle contraction. PAF-induced contraction was only blocked by sPLA2 type I inhibitor MJ33 (*P < 0.001 by Student's t-test). H2O2-induced contraction was only inhibited by cPLA2 inhibitor AACOCF3 (#P < 0.001 by Student's t-test). Values are means ± SE of 3 experiments.

 

Levels of PGE2 production were then measured by radioimmunoassay in muscle homogenates stimulated with PAF or H2O2 pretreated with buffer alone or with PAF receptor antagonist CV-3988 (10-5 M) (Fig. 5). PAF and H2O2 increased PGE2 production from 7.5 ± 0.8 to 16.1 ± 1.4 ng/mg protein (*P < 0.001 by Student's t-test) or to 14.7 ± 0.6 ng/mg protein (#P < 0.001 by Student's t-test), respectively. The rise in PGE2 levels was prevented by PAF receptor antagonist CV-3988 (10-5 M) pretreatment from 16.1 ± 1.4 to 7.4 ± 0.6 ng/mg protein (**P < 0.001 vs. PAF alone by Student's t-test) and to 8.1 ± 0.9 ng/mg protein (##P < 0.001 vs. H2O2 alone by Student's t-test). These data suggest that PAF receptors are involved in the increased PGE2 levels induced by PAF and H2O2.



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  		Fig. 5. Effect of PAF receptor antagonist CV-3988 (10-5 M) on PAF (10-9 M)- or H2O2 (70 µM)-induced PGE2 production in gallbladder muscle. PAF or H2O2 increased the levels of PGE2 production (*P < 0.001, #P < 0.001 vs. control by Student's t-test) that was blocked by pretreatment with CV-3988 (10-5 M) (**P < 0.05, ##P < 0.001 vs. PAF or H2O2 alone, respectively, by Student's t-test). Values are means ± SD of 3 experiments.

 

Because PAF and PAF-like lipids stimulate different PLAs, we examine whether PAF and PAF-like lipids act on the same PAF receptor, by performing experiments with NEM, a nonspecific membrane receptor inactivator (Fig. 6). This compound inactivates all transmembrane receptors unprotected by their specific ligands. Agonists are capable of protecting their own receptors if muscle cells are treated with them before NEM (5). Pretreatment of muscle cells with half-maximal doses of PAF (10-11 M) before NEM protected the actions of a second dose of PAF and the functions of PGE2, but it did not protect those receptors stimulated by H2O2. PAF and PGE2 induced a full contraction in muscle cells treated with PAF and followed by NEM. However, the H2O2-induced contraction was significantly reduced (*P < 0.001 by Student's t-test). In contrast, pretreatment of muscle cells with H2O2 (that induces lipid peroxidation and formation of PAF-like lipids) before NEM (Fig. 7) protected the actions of a second dose of H2O2 and the functions of PGE2, but it did not protect that of PAF because H2O2- and PGE2-induced contractions were similar to those of controls, whereas the PAF-induced contraction was reduced (*P < 0.001 by Student's t-test). The PGE2 receptors are protected, because both H2O2 and PAF increased the generation of this prostaglandin (18, 27). Thus these data suggest that PAF-like lipids activate a receptor subtype that is different from that of PAF.



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  		Fig. 6. Protection of receptors by agonists from the actions of N-ethylmaleimide (NEM). Agonists are capable of protecting receptors from the inactivating of NEM. Pretreatment of muscle cells with a low dose of PAF (10-11 M) for before NEM (5 µM) protected the actions of a second dose of PAF and PGE2 on muscle cell contraction, but it did not protect that of H2O2. PAF and PGE2 induced a full contraction in muscle cells pretreated with PAF before NEM but H2O2-induced contractions were significantly reduced (*P < 0.001 by Student's t-test). Values are means ± SE of 3 experiments.

 


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  		Fig. 7. Receptor protection by PAF-like lipids from the actions of NEM. Pretreatment of muscle cells with H2O2 (70 µM) (induction of PAF-like lipids) before NEM (5 µM) protected the actions of H2O2 and PGE2 but not that of PAF, because H2O2- and PGE2-induced contractions were similar to those of controls. In contrast, PAF (10-9 M)-induced contractions were reduced (*P < 0.001 by Student's t-test). Values are means ± SE of 3 experiments.

 

Binding of an agonist to membrane receptors activates specific G proteins and can be blocked by specific G protein subunit antibodies (29). Permeablized muscle cells were pretreated with antibodies against different G protein subunits for 15 min before PAF (Fig. 8). Antibodies themselves had no effect on muscle cell contraction. However, pretreatment of muscle cells with anti-G{alpha}q/11 antibody inhibited the PAF-induced contractions from 19.7 ± 1.2 to 3.1 ± 0.2% (*P < 0.001 by Student's t-test). All other antibodies had no effect on PAF-induced contractions. Similarly, muscle contraction induced by H2O2 was also inhibited by pretreatment with anti-G{alpha}q/11 antibody (*P < 0.001 by Student's t-test) (Fig. 9). These data suggest that receptors that mediate the actions of PAF and PAF-like lipids couple to the same G proteins.



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  		Fig. 8. Effects of specific antibodies against G protein subunits on PAF-induced muscle contraction. Antibodies themselves had no effect on muscle contraction. However, pretreatment of muscle cells with anti-G{alpha}q/11 antibody (1:400 titer) inhibited PAF-induced contraction (*P < 0.001 by Student's t-test). All other antibodies had no effect on PAF-induced muscle contractions. Values are means ± SE of 3 experiments.

 


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  		Fig. 9. Effects of G protein antibodies on H2O2-induced muscle contraction. H2O2-induced muscle contraction was only inhibited by pretreatment with anti-G{alpha}q/11 antibody (1:400 titer) (*P < 0.001 by Student's t-test). Values are means ± SE of 3 experiments.

 

GTP{gamma}S binding studies further supported these data (Fig. 10). Basal levels (without receptor activation) for the [35S]GTP{gamma}S binding to G{alpha}i1-2, G{alpha}i3, G{alpha}q/11, and G{alpha}s were 2,064, 2,075, 2,030, and 2,106 counts/min, respectively. PAF caused a significant increase in [35S]GTP{gamma}S binding to G{alpha}i1-2 (68.0 ± 2.5%) and G{alpha}q/11 (79.0 ± 4.5%). Pretreatment with the cyclooxygenase inhibitor indomethacin before PAF blocked the [35S]GTP{gamma}S binding to G{alpha}i1-2 (68.0 ± 2.5 to 17.0 ± 1.3%; *P < 0.001 by Student's t-test) but not that to G{alpha}q/11. PAF receptor antagonist CV-3988 (10-5 M) abolished the [35S]GTP{gamma}S binding to both G{alpha}i1-2 (68.0 ± 2.5 to 12.0 ± 0.9%; #P < 0.001 by Student's t-test) and G{alpha}q/11 (79.0 ± 4.5 to 12.0 ± 0.2%; ##P < 0.001 by Student's t-test). H2O2 alone (Fig. 11) caused a significant increase in [35S]GTP{gamma}S binding to G{alpha}i1-2, G{alpha}i3 and G{alpha}q/11. Pretreatment with indomethacin before H2O2 blocked [35S]GTP{gamma}S binding to G{alpha}i1-2 (75.0 ± 3.5 to 15.0 ± 1.3%; *P < 0.001 by Student's t-test), G{alpha}i3 (28.0 ± 6.5 to 12.0 ± 0.2%; **P < 0.001 by Student's t-test) but not that to G{alpha}q/11. PAF receptor antagonist CV-3988 (10-5 M) abolished [35S]GTP{gamma}S binding to G{alpha}q/11 (56.0 ± 8.2 to 6.0 ± 2.4%; #P < 0.001 by Student's t-test) but not that to G{alpha}i1-2 and G{alpha}i3.



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  		Fig. 10. PAF (10-9 M) induced [35S]GTP{gamma}S binding to specific G proteins in gallbladder muscle. It caused a significant increase in [35S]GTP{gamma}S binding to G{alpha}i1-2 and G{alpha}q/11. Pretreatment with the cyclooxygenase inhibitor Indo (5 µM) before PAF blocked the [35S]GTP{gamma}S binding to G{alpha}i1-2 (*P < 0.001 by Student's t-test) but not to G{alpha}q/11. PAF receptor antagonist CV-3988 (10-5 M) abolished [35S]GTP{gamma}S binding to both G{alpha}i1-2 (#P < 0.001 by Student's t-test) and G{alpha}q/11 (##P < 0.001 by Student's t-test). Values are means ± SE of 3 experiments.

 


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  		Fig. 11. [35S]GTP{gamma}S binding to specific G proteins in gallbladder muscle stimulated by H2O2 (70 µM). It caused a significant increase in [35S]GTP{gamma}S binding to G{alpha}i1-2, G{alpha}i3, and G{alpha}q/11. Pretreatment with Indo (5 µM) before H2O2 blocked [35S]GTP{gamma}S binding to G{alpha}i1-2 (*P < 0.001 by Student's t-test) and G{alpha}i3 (**P < 0.001 by Student's t-test) but not to G{alpha}q/11. PAF receptor antagonist CV-3988 (10-5 M) abolished [35S]GTP{gamma}S binding to G{alpha}q/11 (#P < 0.001 by Student's t-test) but not to G{alpha}i1-2 and G{alpha}i3. Values are means ± SE of 3 experiments.

 

Furthermore, pretreatment of muscle cells with inhibitors of PKC (0.1 µM chelerythrine) or MAP kinase (10 µM PD-98059) before PAF or H2O2 blocked their contraction (Fig. 12). PAF-induced muscle cell contraction was reduced by chelerythrine (21.2 ± 3.5 to 5.1 ± 1.2%; **P < 0.001 by Student's t-test) and by PD-98059 (21.2 ± 3.5 to 2.1 ± 1.0%; *P < 0.001 by Student's t-test). Similarly, H2O2-induced contractions were also blocked by chelerythrine (16.7 ± 2.4 to 3.1 ± 1.5%; ##P < 0.001 by Student's t-test) and by PD-98059 (16.7 ± 2.4 to 0.8 ± 0.7; #P < 0.001 by Student's t-test). These data suggest that PAF and PAF-like lipids share a common final pathway.



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  		Fig. 12. Effects of inhibitors of MAP kinase (PD-98059; 10 µM) and PKC (0.1 µM chelerythrine) on PAF (10-9 M)- or H2O2 (70 µM)-induced muscle contraction. Both PAF- and H2O2-induced contractions were blocked by PD-98059 (*P < 0.001, #P < 0.001 by Student's t-test) and chelerythrine (**P < 0.001, ##P < 0.001 by Student's t-test). Values are means ± SE of 3 experiments.

 

On the basis of these results, possible pathways that mediate PAF- and H2O2-induced muscle cell contractions in guinea pig gallbladder muscle are illustrated in Fig. 13. Stimulation of muscle cells with PAF (through PAF receptors) or H2O2 (through possible PAF-like lipids receptors) activates G{alpha}q/11 proteins, leading to activation of secreted PLA2 (sPLA2, for PAF) and cytosolic PLA2 (cPLA2; for H2O2) that mediate the hydrolysis of arachidonic acid to produce prostaglandins. Prostaglandins act on their membrane receptors to induced muscle cell contraction.



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  		Fig. 13. Model of the signaling and effector pathways that mediates the actions of PAF and H2O2 in guinea pig gallbladder muscle. Stimulation of muscle cells with PAF (through PAF receptors) or H2O2 (through PAF-like lipids receptors, unidentified) activates G{alpha}q/11 proteins, leading to activation of secreted PLA2 (sPLA2, for PAF) to likely produce prostaglandins and leukotrienes and cPLA2 (for H2O2) to produce PGs. PGs act on their membrane receptors to induced muscle cell contraction. AA, arachidonic acid.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The present studies show that ROS (H2O2) stimulates PGE2 synthesis in gallbladder muscle by increasing the formation of PAF-like lipids acting on receptor subtypes that couple to G{alpha}q/11, which activates cPLA2. This appears to be a different receptor subtype from those stimulated by PAF, because its receptors are not protected by PAF-like lipids and activate a different signal transduction pathway, which although it couples to G{alpha}q/11, it includes sPLA2 and subsequent stimulation of COX and lipoxygenase.

Acute inflammatory processes affect the gallbladder muscle cells and impair the contraction induced by receptor-dependent agonists CCK and ACh and that of the receptor-independent KCl (25). These muscle cells showed evidence of oxidative stress characterized by increased generation of ROS and lipid peroxidation as well as synthesis of PGE2 and of scavengers of free radicals (25). Increased generation of ROS affect cell functions by oxidizing phospholipids and transmembrane proteins. ROS may also increase the formation of PAF (20) and PAF-like lipids (10), and, by generating PGE2, they may stimulate the synthesis of scavengers of free radicals (27). However, the mechanisms by which ROS stimulate PGE2 synthesis resulting in muscle contraction and increase scavengers of free radicals are unknown. PAF and PAF-like lipids have been suggested as potential candidates to mediate these cell responses.

PAF is a potent phospholipid that is known to mediate several biological actions. PAF stimulates various effector functions in eosinophils, such as degranulation, production of superoxide anion and leukotriene C4, and formation of lipid bodies (7). A specific receptor for PAF has been identified in a number of cell types (7). Human and guinea pig PAF receptors are single polypeptides composed of 342 amino acids (6) present as seven transmembrane domains that couple to G proteins. PAF-like lipids are oxidized phospholipids induced by ROS by nonenzymatic mechanisms whose actions are blocked by specific PAF antagonists.

Although a single receptor is currently thought to mediate their effects (1), our data suggest that the actions of PAF and PAF-like lipids are linked to two distinct pathways although the specific PAF receptor antagonist blocked the muscle contraction and increased PGE2 levels induced by both PAF and PAF-like lipids induced by H2O2. This conclusion is supported by the observations made from studies with PLA2 inhibitors and with receptor protection studies against the actions of the nonspecific receptor inactivator NEM. PAF-like lipids induced by H2O2 stimulate cytosolic PLA2, because the specific inhibitor AACOCF3 blocks their actions. This enzyme releases arachidonic acid that seems to be exclusively hydrolyzed by cyclooxygenase, resulting in the formation of prostaglandins, particularly PGE2, because their actions are almost completely antagonized by indomethacin. PGE2 causes gallbladder muscle contraction by acting on EP1 receptors (unpublished observations) and stimulates the formation of catalase and SOD (26). In contrast, the actions of PAF are antagonized by sPLA2-specific blocker MJ33 and unaffected by the cPLA2 inhibitor. Moreover, the PAF-induced contraction was only partially blocked by either COX or lipoxygenase antagonists and was only completely inhibited by their combination.

These results suggest the existence of two receptor subtypes, one activated by PAF and the other by PAF-like lipids, an assumption that is supported by studies with NEM. NEM inactivates all free transmembrane receptors (5). Receptors can be protected against the actions of NEM if they are occupied by their specific ligands. PAF-like lipids generated by the actions of H2O2 in gallbladder muscle cells protected the receptors of PAF-like lipids but were unable to protect PAF receptors against the actions of NEM. In contrast, the contraction induced by PAF was blocked by NEM although the cells had been pretreated with H2O2. Likewise, pretreatment with PAF did not protect the PAF-like receptors, because NEM blocked the contraction induced by H2O2. Thus each compound appears to stimulate a different receptor subtype although the same antagonist blocks the actions of both PAF and PAF-like lipids.

Present studies also present evidence that indicates that PAF-like lipids generated by ROS (H2O2) mediate the increase in PGE2 synthesis that results in contraction and formation of scavengers of free radicals (26), because the specific PAF antagonist blocked the muscle contraction and increased PGE2 induced by both PAF and by H2O2.

Both receptor subtypes, however, couple to the same G protein, because the specific G{alpha}q/11 antibody blocks the contraction induced by PAF and PAF-like lipids. This receptor-G protein coupling was also demonstrated by determining the increase in [35S]GTP{gamma}S binding to G proteins in response to both agonists. Both PAF and PAF-like lipids increased GTP binding to G{alpha}q/11 and to Gis. However, after the cells were pretreated with indomethacin, the [35S]GTP{gamma}S binding was increased only to G{alpha}q/11, suggesting that the increased binding to Gi proteins is due to the generation of lipid peroxides other than PAF-like lipids that stimulate the formation of prostaglandins. Moreover, a similar pathway that includes PKC and MAPK receptors stimulated by prostaglandins (21, 26) and probably by leukotrienes also mediates the muscle contraction induced by PAF and PAF-like receptors.

Activation of different PLA2s by the same G protein (G{alpha}q/11) cannot be explained at this time, because there are no previous reports on this subject. These findings, however, are supported by two sets of experiments using antagonists. First, the contraction induced by PAF-like lipids is blocked by cPLA2 antagonist and by COX inhibitors. Second, the contraction induced by PAF is only blocked by sPLA2 antagonists and by the combined use of cyclooxygenase and lipooxygenase inhibitors. This new finding deserves further investigation. However, it should not be surprising, because a receptor is able to couple to different G proteins either sequentially or simultaneously as Murthy and coworkers (15) have repeatedly shown. It is conceivable that this may be due to the known fact that different receptors may have access to different sets of signaling molecules preassembled in caveolae structures that may include similar G proteins activating different PLAs.

Thus these studies demonstrate that ROS generate PAF-like lipids that appear to act on PAF receptor subtypes and establish the link between lipid peroxidation and PGE2 generation that contributes to the gallbladder muscle contraction and increased synthesis of scavengers of free radicals.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-27389.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Behar, Division of Gastroenterology, APC 421, 593 Eddy St., Providence, RI 02903 (E-mail address: Jose_Behar{at}brown.edu).

These data were partially presented at the Annual Meeting of the American Gastroenterological Association in May 2001, Atlanta, GA.

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.


   REFERENCES
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 RESULTS
 DISCUSSION
 DISCLOSURES
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