Introduction

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Stimulation of parasympathetic nerves or administration of ACh stimulates prostaglandin (PG) synthesis in various tissues, including the heart (Junstad and Wennmalm, 1974; Jaiswal and Malik, 1988). PGI₂, the principal prostanoid synthesized in the heart in response to ACh, may contribute to its cardioprotective effects by inhibiting free radical production and ventricular arrhythmias (Kesckemeti et al., 1973), by producing coronary vasodilation (Dusting et al., 1978), inhibiting platelet aggregation (Moncada et al., 1976) and by release of norepinephrine from sympathetic fibers (norepinephrine release causes ventricular contractility) (Khan and Malik, 1982; Lanier and Malik, 1985). Prostacyclin is synthesized in the heart in various cell types, including ventricular myocytes (Ahumada et al., 1980; Bolton et al., 1980) and coronary vascular endothelial cells (Gerritsen and Cheli, 1983; Revtyak et al., 1988). ACh elicits prostacyclin synthesis by activation of muscarinic ACh receptors (mAChR) M₂ and M₃ in the rabbit heart (Jaiswal et al., 1988), M₂ and M₃ mAChR in ventricular myocytes, and M₃ mAChR in CEC (Kan et al., 1996). All subtypes of mAChR (m₁-m₅) are coupled to multiple G proteins and regulate several effector systems, including the activity of adenylyl cyclase, Ca⁺⁺ and K⁺ channels (Jones et al., 1991; Jones et al., 1988; Fukuda et al., 1988) and phospholipases (Peralta et al., 1988; Sandmann et al., 1991; Felder et al., 1990). m₁, m₃ and m₅ mAChR are in general coupled to mobilization of intracellular Ca⁺⁺, whereas m₂ and m₄ mAChR are coupled to inhibition of adenylyl cyclase (Jones et al., 1991). Other cellular events influenced by m₁, m₃, and m₅ mAChR include activation of phospholipase A₂, C and D (Peralta et al., 1988; Sandmann et al., 1991) and of tyrosine kinase (Huang et al., 1993) and Ca⁺⁺ influx (Felder et al., 1990). However, most of these studies have been conducted in cells transfected with muscarinic receptors. Whether these mechanisms operate with endogenously expressed mAChR remains to be established. The purposes of our study were to investigate the contributions of extra- and intracellular Ca⁺⁺ in prostacyclin synthesis and of G proteins in the rise in cytosolic Ca⁺⁺ and prostacyclin production mediated by M₃ mAChR in cultured CEC of the rabbit heart. This study has been published in an abstract form (Kan et al., 1994).

	Methods

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Preparation of Coronary Endothelial Cells

Endothelial cells were isolated from rabbit coronary vessels by modification of the method of Nees et al. (1981). Briefly, the isolated rabbit heart was initially perfused with BSS via the aorta for 15 min; then the perfusion solution was changed to BSS without Ca⁺⁺. When the heart stopped beating, it was immersed in 20 ml HBSS containing 20% sucrose in a 50-ml conical tube. Dispersion of the endothelial lining of the coronary vessels was initiated by infusing into the heart 5 ml HBSS containing 0.15% of collagenase (Boehringer Mannheim Biochemicals, Indianapolis, IN), soybean trypsin inhibitor (type I-S; Sigma, St. Louis, MO), and bovine serum albumin and then maintaining the coronary vascular system in this solution (which had been retained in the vascular space) for 10 min. CEC were then flushed out of the heart by perfusing it with 40 ml of BSS. The cell suspension that overlayed the sucrose medium was aspirated and centrifuged. The sedimented cells were resuspended in 10 ml M199 medium supplemented with 20% of fetal bovine serum in 100-mm Falcon tissue culture dishes and incubated at 37°C in 5% CO₂, 95% humidified air for 1.5 hr. Then the incubation medium was replaced with fresh culture medium and thereafter was changed every 2 to 3 days. The cells obtained by this method consisted of a > 95% homogenous population of CEC. These cells have been well characterized by previous investigators (Nees et al., 1981; Gerristen and Cheli, 1983). Primary cultured cells were passed to 48-well plates for experiments. The cells were divided into three batches from each heart, and three to four hearts were used for each group of experiments. Nine to 12 wells of cells from different hearts were used for each experiment.

Experimental Protocols

Series 1. The first series of experiments was performed to determine the contribution of extracellular Ca⁺⁺ to 6-keto-PGF₁ production elicited by ACh in CEC. Subconfluent CEC cultured in 48-well plates were washed twice with 1 ml of HBSS without Ca⁺⁺ (pH 7.4) and then incubated for 10 min with 1 ml of BSS containing ACh (3 µM) in the presence of different concentrations of extracellular Ca⁺⁺ (0, 0.45, 0.9, 1.8, 3.6 and 5.4 mM) or in the absence of extracellular Ca⁺⁺ plus 10 mM EGTA. The basal and ACh-induced accumulations of 6-keto-PGF₁ in the medium were measured by radioimmunoassay. The cells were extracted with 1 ml of 1 M NaOH for protein assay.

Series 2. To determine the effect of guanine nucleotides on 6-keto-PGF₁ production, CEC cultured in 48-well plates were permeabilized with 10 µg/ml saponin at 37°C for 10 min in a cytoplasmic buffer solution comprised of (in mM): 20 NaCl, 0.5 MgCl₂^.6H₂O, 102 KCl, 2.5 NaHCO₃, 0.96 NaH₂PO₄, 1 EGTA, 10 HEPES, 0.46 CaCl₂ and 0.2% albumin (pH 7.4). After permeabilization, the cells were washed three times with 1 ml of HBSS without Ca⁺⁺. Then the cells were incubated for 10 min with BSS containing different concentrations of GTP--S, GDP--S or GTP--S plus GDP--S for 10 min in the presence or absence of extracellular Ca⁺⁺. The cells were then exposed to ACh or its vehicle for another 10 min, and the medium was collected for measurement of 6-keto-PGF₁.

Series 3. To determine the effect of pertussis toxin (100 ng/ml), which inactivates Go and G_i, on 6-keto-PGF₁ synthesis elicited in CEC by ACh (3 µM), the noncholinergic agonist ATP (100 µM), or the nonreceptor agent Ca⁺⁺ ionophore A23187 (1 µM), cells were washed with HBSS three times and incubated with pertussis toxin for 4 hr at 37°C in M199 culture medium containing 0.2% fetal bovine serum. The cells were then washed and incubated for 10 min with BSS containing the agents and their vehicle.

Series 4. Because ACh is known to stimulate NO and cGMP production (Castoldi et al., 1993) and NO has been reported to stimulate cyclooxygenase activity (Salvemini et al., 1993), we examined the possible role of NO in 6-keto-PGF₁ production in response to ACh by examining the effect of the NO synthase inhibitor N^G-monomethyl-L-arginine on 6-keto-PGF₁ accumulation in CEC. CEC were exposed to different concentrations of N^G-monomethyl-L-arginine (1-100 µM) for 10 min followed by an additional 10-min incubation with ACh at 37°C. cGMP in the incubation medium was measured as an index of changes in NO production by ACh as described (Jaiswal et al., 1991).

Series 5. To examine the contribution of PLC to the ACh induced rise in cytosolic Ca⁺⁺ and 6-keto-PGF₁ synthesis, CEC were preincubated with PLC inhibitor D609 (20 µM) for 20 min and washed three times with 1 ml HBSS. The cells were then incubated for 10 min with 1 ml BSS containing ACh (3 µM) and D609 (20 µM), and the medium was collected for 6-keto-PGF₁ measurement. The effect of D609 (20 µM) on the rise in intracellular Ca⁺⁺ level elicited by ACh was also examined. After fura-2AM loading, CEC were incubated with D609 (20 µM) for 20 min and the cover slip was then placed in the cuvette and perfused with D609 (20 µM) and ACh (3 µM).

Intracellular Ca⁺⁺ Measurement

The cells were prepared as described by Cornwell and Lincoln (1989). Briefly, cover slips (9 × 33 mm) seeded with endothelial cells were loaded with 2 µM fura-2AM in BSS with 0.025% bovine serum albumin and then incubated for 30 min at 37°C; cover slips were then placed into plastic cuvettes and perfused with BSS at the rate of 1 ml/min until the basal fluorescence level became stable. Fluorescence measurements were made using two excitation wavelengths, 340 and 380 nm, with emission measured at 510 nm. Cytosolic Ca⁺⁺ level was determined using the equation: where K_D for Fura-2AM = 133 nM (intracellular pH 7.18; 37°C), F = experimental fluorescence value, Fmax = the maximal fluoresence in the presence of saturating Ca⁺⁺ and Fmin = minimal fluorescence in the presence of low Ca⁺⁺.

Radioimmunoassay of 6-Keto-PGF₁ Synthesis

6-Keto-PGF₁ synthesis and release were determined in the incubation medium by radioimmunoassay as described by Shaffer and Malik (1982). Briefly, 100 µl of sample were mixed with 3000 to 4000 cpm of [³H]6-keto-PGF₁ tracer (0.925 MBq/0.025 mCi) and the appropriate amount of antibody. The tracer and antibody were prepared in a buffer consisting of (in g/liter): 1.0 NaN₃, 9.0 NaCl, 6.8 KH₂PO₄, 26.1 K₂HPO₄ and 2.0 gelatin. The tubes were vortexed and incubated overnight at 4°C. Bound and free tracer were separated by adding 1 ml of dextran-coated charcoal to each tube, and radioactivity was determined by liquid scintillation spectroscopy. The antibody for 6-keto-PGF₁ was provided by Dr. C. Leffler (Department of Physiology, University of Tennessee, Memphis, TN). Cross-reactivity of the 6-keto-PGF₁ antibody was <1% with thromboxane B₂ and 13,14-dihydro-15-keto-PGE₂ and <0.5% with PGE₂ and PGF₁. The minimum detection level of the radioimmunoassay for 6-keto-PGF₁ was 1.95 pg.

Assay of PLA₂

Subconfluent CEC cultured in 100 mm dishes were exposed to ACh (3 µM) in the presence of SK&F96365 (1 µM) or its vehicle were scraped and sonicated in HEPES buffer (pH 7.4) containing: 340 mM sucrose, 1 mM EGTA, 100 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 20 µg/ml soybean trypsin inhibitor. The concentration of protein in the lysate was determined by Lowry's assay and adjusted for the hydrolysis of L-3-phosphatidylcholine, 1-steroyl-2-(1-¹⁴C) arachidonyl as described previously (Kan et al., 1996) by the method of Leslie (1990).

Assay of cGMP

cGMP was assayed as described by Bruckner et al. (1985). Subconfluent CEC were incubated with various drugs for 10 min at 37°C, in the presence of IBMX (100 µM) to minimize cGMP degradation by phosphodiesterases. Thereafter, the medium was aspirated, and the reaction was terminated by addition of 1 ml of sodium acetate (50 mM, pH 4). The cells were frozen in dry ice and then boiled for 3 min; cGMP was measured in duplicate 50-µl samples. Samples and standards were acetylated with acetic anhydride and triethylamine (2:3) to increase the sensitivity of the assay. To 50 µl of acetylated sample or standard were added 50 µl of ¹²⁵I-labeled cGMP (3000 counts/min) in 50 mM acetate buffer (pH 6.2) and 200 µl antibody in 50 mM acetate buffer (pH 6.2) containing 5 mg/ml BSA. The mixture was vortexed vigorously and incubated at 4°C overnight. The next day, 150 µl of phosphate buffer containing 1% -globulin and 2 ml of polyethylene glycol (25%) were added to the tubes, which were then centrifuged at 3000 revolution/min for 30 min. The supernatant was removed, and the radioactivity determined by gamma counter.

Drugs

Acetylcholine, verapamil, nifedipine, SK&F96365, D609 and thapsigargin were purchased from Research Biochemicals International (Natick, MA); L-3-phosphatidylcholine and 1-stearoly-2-(1-¹⁴C) arachidonyl from Amersham Life Science (Arlington Heights, IL); dioleoylglycerol from Avanti Polar Lipids Inc. (Alabaster, AL); and ATP, pertussis toxin, GTP--S, GDP--S, -escin, N^G-monomethyl-L-arginine, M199, fetal bovine serum and cGMP antibody from Sigma Chemical Co. (St. Louis, MO).

Data Analysis

The results are expressed as means ± S.E. The data were analyzed by one-way analysis of variance; the Newman-Keuls multiple range test was applied to determine the difference among multiple groups and the unpaired Student's t test for difference between two groups. The null hypothesis was rejected at P < .05. Basal 6-keto-PGF₁ is expressed as picograms of immunoreactive 6-keto-PGF₁ per microgram of protein. Because basal 6-keto-PGF₁ production was variable in different batches of cells and the increase in basal 6-keto-PGF₁ production elicited by ACh was consistent as compared to its vehicle control within the same batch of cells and independent of basal levels, the changes in ACh-induced 6-keto-PGF₁ synthesis produced by various experimental interventions are expressed as percent above basal.

	Results

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Effect of alterations in extracellular Ca⁺⁺ concentrations on 6-keto-PGF₁ synthesis elicited by ACh in CEC. The basal production of 6-keto-PGF₁ was variable among different batches of cells, but the effect of ACh to increase 6-keto-PGF₁ production was independent of basal synthesis. Figure 1 shows 6-keto-PGF₁ synthesis elicited by ACh in CEC in the presence and absence of different concentrations of extracellular Ca⁺⁺. In the absence of extracellular Ca⁺⁺, and in the presence of 10 mM EGTA, the effect of ACh to increase 6-keto-PGF₁ production was abolished. Increasing extracellular Ca⁺⁺ concentration enhanced the effect of ACh on PG synthesis. The basal 6-keto-PGF₁ accumulation was significantly increased in the presence of 3.6 mM Ca⁺⁺. Alterations in the extracellular concentration of Ca⁺⁺ (0.45, 0.9, 1.8, 3.6, mM) did not alter the conversion of added AA (1 µM) to 6-keto-PGF₁ (3.3 ± 0.1, 3.2 ± 0.1, 3.2 ± 0.1 and 3.3 ± 0.1 pg/µg protein, respectively). In the absence of extracellular Ca⁺⁺ the conversion of AA (1 µM) to 6-keto-PGF₁ (2.7 ± 0.1 pg/µg) was not altered and also remained unchanged by the addition of 10 mM EGTA.

View larger version (K): [in this window] [in a new window]   Fig. 1.   Effect of alteration in Ca++ concentration on 6-keto-PGF1 synthesis stimulated by ACh in CEC of rabbit heart. Data are expressed as mean ± S.E. of nine wells of cells prepared from three different hearts, three batches of cells from each. The asterisk denotes a value significantly different from vehicle; the dagger denotes a significant difference from the vehicle values obtained in 0 to 0.9 mM Ca++ (P < .05).

Effect of K⁺ on 6-keto-PGF₁ synthesis and cytosolic Ca⁺⁺ in CEC. Exposure of CEC to high K⁺ for 10 min failed to alter either the production of 6-keto-PGF₁ (basal: 3.2 ± 0.4 pg/µg protein vs. high potassium: 3.6 ± 0.5 pg/µg protein, P > .05) or the cytosolic Ca⁺⁺ ([Ca⁺⁺]i basal: 98 ± 8 nm vs. high potassium: 98 ± 8 nm, P > .05).

Effect of Ca⁺⁺ channel blockers on ACh-induced 6-keto-PGF₁ synthesis and the rise in cytosolic Ca⁺⁺. Figure 2A shows that the voltage-dependent calcium channel blockers verapamil (1 µM) and nifedipine (1 µM) failed to reduce ACh-stimulated 6-keto-PGF₁ synthesis in CEC, whereas it was inhibited by SK&F96365 (1 µM), a ROCC blocker. At high concentration (10 µM), SK&F96365 abolished the action of ACh on 6-keto-PGF₁ synthesis (fig. 2B). SK&F96365 (1 µM) inhibited the rise in intracellular Ca⁺⁺ elicited by ACh, but verapamil (1 µM) and nifedipine (1 µM) did not, as shown in figure 3. The Ca⁺⁺ channel blockers did not alter the conversion of AA (1 µM) to 6-keto-PGF₁, 2.0 ± 0.3 pg/µg protein in the absence vs. 2.0 ± 0.4, 2.0 ± 0.4 and 2.3 ± 0.3 pg/µg protein in the presence of SK&F96365 (1 µM), verapamil (1 µM) and nifedipine (1 µM), respectively. The effect of SK&F96365 (1 µM) on PLA₂ activity in cell lysates prepared from CEC exposed to ACh (3 µM) was also measured. SK&F96365 did not alter the activity of PLA₂ in cell cysates prepared from cells exposed to ACh (amount of radioactivity released from L-3-phosphatidylcholine, 1-stearoyl-2-(1-¹⁴C) arachidonyl was: basal: 1530 ± 103 cpm vs. ACh: 3000 ± 83 cpm, and ACh: 3433 ± 365 cpm in the presence of SK&F96365, P > .05).

View larger version (K): [in this window] [in a new window]   Fig. 2.   Effects of verapamil, nifedipine and SK&F96365 (A) and of different concentrations of SK&F96365 (B) on 6-keto-PGF1 production in response to ACh in CEC of rabbit heart. Data are expressed as mean ± S.E. of nine wells of cells prepared from three different hearts, three batches of cells from each. The asterisk denotes a value significantly different from basal; the dagger denotes a value significantly different from that obtained in the presence of vehicle (ACh) (P < .05).

View larger version (K): [in this window] [in a new window]   Fig. 3.   Effects of verapamil, nifedipine, and SK&F96365 on cytosolic Ca++ level elicited by ACh. Each value is expressed as mean ± S.E. of four cover slips seeded with endothelial cells from three different hearts. Basal levels of [Ca++]i (nM): ACh, 96 ± 15.3; verapamil, 106 ± 21.0; nifedipine, 92 ± 16.9; SK&F96365, 109 ± 11.5. The asterisk denotes value significantly different from the basal; the dagger denotes a value significantly different from the corresponding value obtained in the presence of vehicle (P < .05).

Effect of thapsigargin on ACh-enhanced cytosolic Ca⁺⁺ level. In the presence of extracellular Ca⁺⁺, thapsigargin increased cytosolic Ca⁺⁺ by 15 to 20% over a period of 20 min (data not shown) and infusion of ACh at 8 to 20 min further increased the cytosolic Ca⁺⁺ level in the presence of thapsigargin (fig. 4A). In the absence of extracellular Ca⁺⁺ thapsigargin increased cytosolic Ca⁺⁺ by 18 to 28% over a 20-min period (data not shown) and ACh infusion at 8 to 20 min failed to increase further cytosolic Ca⁺⁺ level above that caused by thapsigargin (fig. 4B).

View larger version (K): [in this window] [in a new window]   Fig. 4.   Effect of ACh on cytosolic Ca++ level in CEC incubated with thapsigargin in the presence (A) or absence (B) of extracellular Ca++. Each value is expressed as mean ± S.E. of four cover slips seeded with endothelial cells from three different hearts. The asterisk denotes value significantly different from the basal; the dagger denotes a value significantly different from the corresponding value obtained in the presence of ACh (P < .05).

Effects of guanine nucleotides on ACh-induced 6-keto-PGF₁ production in CEC. In CEC permeabilized with saponin, a nonhydrolysable guanine nucleotide, GTP--S (1-100 µM), increased 6-keto-PGF₁ synthesis in a dose-dependent manner (fig. 5A). However, in the absence of extracellular Ca⁺⁺, GTP--S failed to stimulate 6-keto-PGF₁ synthesis (basal: 0.8 ± 0.1 pg/µg protein vs. GTP--S: 0.9 ± 0.06 pg/µg protein, P > .05). Moreover, GTP--S had no effect on 6-keto-PGF₁ synthesis in nonpermeabilized CEC (basal: 1.1 ± 0.12 pg/µg protein vs. GTP--S: 1.2 ± 0.1 pg/µg protein, P > .05). ACh alone (3 µM) failed to increase 6-keto-PGF₁ production in permeabilized CEC. ACh, in the presence of GTP--S (10 µM) increased 6-keto-PGF₁ synthesis above that elicited by GTP--S alone (10 µM) (fig. 5A). GDP--S (100 µM) reduced the effect of GTP--S alone or in the presence of ACh (fig. 5B).

View larger version (K): [in this window] [in a new window]   Fig. 5.   Effects of GTP--S (A) and of GDP--S (B) on 6-keto-PGF1 production elicited by GTP--S or GTP--S plus ACh in CEC permeabilized with 10 µg/ml saponin (pH 7.4). Data are expressed as mean ± S.E. of nine wells of cells prepared from three different hearts, three batches of cells from each. The asterisk denotes a value significantly different from basal; the dagger indicates value significantly different from that obtained in the presence of GTP--S alone (A) or that obtained in the presence of GTP--S alone, and GTP--S + ACh (B) (P < .05).

Effects of guanine nucleotides on the ACh- and ATP-induced rise in cytosolic Ca⁺⁺ in reversibly permeabilized CEC. In CEC that were permeabilized with -escin and resealed, ACh or ATP failed to increase cytosolic Ca⁺⁺ in the absence of extracellular Ca⁺⁺. In cells loaded with GTP--S (100 µM), ACh or ATP increased the cytosolic Ca⁺⁺ level in the absence of extracellular Ca⁺⁺; however, in cells loaded with GTP--S (100 µM) plus GDP--S (200 µM), the effect of ACh or ATP to increase cytosolic Ca⁺⁺ was inhibited (fig. 6, A and C). In contrast, in the presence of extracellular Ca⁺⁺ (1.8 mM), in CEC permeabilized with -escin and incubated with the vehicle of GTP--S and resealed, ACh or ATP increased cytosolic Ca⁺⁺ level; and this was unaffected by loading the cells with either GTP--S or GDP--S (fig. 6, B and D).

View larger version (K): [in this window] [in a new window]   Fig. 6.   Effects of guanine nucleotides on the rise in the level of cytosolic Ca++ elicited by ACh or ATP in CEC permeabilized with -escin and resealed. Data are expressed as mean ± S.E. of four cover slips seeded with endothelial cells obtained from three different hearts. Basal levels of [Ca++]i (nM): (Ca++ free) ACh, 117 ± 4.0; GTP--S, 116 ± 4.5; GTP--S + GDP--S, 114 ± 5.0; ATP, 111.0 ± 2.0; GTP--S, 108 ± 6.0; GTP--S + GDP--S, 112 ± 3.0; (Ca++ 1.8 mM) ACh, 118 ± 7.5; GTP--S, 112 ± 4.0; GTP--S + GDP--S, 117 ± 4.7; ATP, 109 ± 5.9; GTP--S, 106 ± 4.7; GTP--S+GDP--S, 112 ± 10.0. The asterisk denotes a value significantly different from the vehicle; the dagger denotes a value significantly different from that obtained in the presence of GTP--S (P < .05).

Effects of pertussis toxin on 6-keto-PGF₁ synthesis and on the rise in cytosolic Ca⁺⁺ elicited by ACh, ATP and A23187. Incubation of pertussis toxin (100 ng) for 4 hr with CEC reduced the ATP- but not the ACh- or Ca⁺⁺ ionophore A23187-induced 6-keto-PGF₁ production (fig. 7). Treatment of CEC with pertussis toxin also reduced the ATP- but not the ACh-induced rise in cytosolic Ca⁺⁺ level in the absence of extracellular Ca⁺⁺ (fig. 8A); however, in the presence of extracellular Ca⁺⁺ (1.8 mM), pertussis toxin did not alter the ACh- or ATP-induced increase in cytosolic Ca⁺⁺ level (fig. 8B).

View larger version (K): [in this window] [in a new window]   Fig. 7.   Effect of pertussis toxin (PT) on 6-keto-PGF1 production elicited by ACh, A23187 and ATP. Data are expressed as mean ± S.E. of 12 wells of cells prepared from four different hearts, three batches of cells from each. The asterisk denotes a value significantly different from basal; the dagger denotes a value significantly different from that obtained in the presence of vehicle (ATP) (P < .05).

View larger version (K): [in this window] [in a new window]   Fig. 8.   Effect of pertussis toxin on the rise in cytosolic Ca++ elicited by ACh or ATP. Data are expressed as mean ± S.E. of four cover slips seeded with endothelial cells obtained from three different hearts. Basal levels of [Ca++]i (nM): (Ca++ free) ACh, 114 ± 3.1; ACh + PT, 113 ± 4.1; ATP, 111 ± 6.0; ATP + PT, 115 ± 4.0; (Ca++ 1.8 mM) ACh, 114 ± 4.4; ACh + PT, 114 ± 5.0; ATP, 124 ± 8.0; ATP + PT, 113 ± 4.3. The asterisk denotes a value significantly different from basal; the dagger denotes a value significantly different from that obtained in the presence of vehicle of PT (P < .05).

Effects of NO synthase inhibitor N^G-monomethyl-L-arginine on ACh- induced 6-keto-PGF₁ synthesis and cGMP accumulation. N^G-monomethyl-L-arginine (1-100 µM), which inhibited ACh-induced cGMP accumulation in CEC (fig. 9A), failed to alter the effect of ACh on 6-keto-PGF₁ synthesis (fig. 9B).

View larger version (K): [in this window] [in a new window]   Fig. 9.   Effect of NG-monomethyl-L-arginine on cGMP accumulation (A) and on 6-keto-PGF1 production (B) stimulated by ACh. Data are expressed as mean ± S.E. of 12 wells of cells prepared from 4 different hearts, 3 batches of cells from each. The asterisk denotes a value significantly different from vehicle (A) or basal (B); the dagger denotes a value significantly different from that obtained in the presence of ACh alone (P < .05).

Effects of the phospholipase C inhibitor D609 on the ACh-induced rise in cytosolic Ca⁺⁺ and 6-keto-PGF₁ synthesis. D609 (20 µM) significantly reduced the rise in cytosolic Ca⁺⁺ in the absence of extracellular Ca⁺⁺ (fig. 10A) without altering 6-keto-PGF₁ production elicited by ACh (fig. 10B).

View larger version (K): [in this window] [in a new window]   Fig. 10.   Effect of D609 (20 µM), a phospholipase C inhibitor, on 6-keto-PGF1 production (A) and on the rise in cytosolic Ca++ elicited by ACh or ATP in the absence of extracellular Ca++ (B). Data are expressed as mean ± S.E. of 12 wells of cells prepared from 4 different hearts, 3 batches of cells from each. Basal levels of [Ca++]i (nM): ACh, 119 ± 6.0, ACh+D609, 118 ± 6.3, ATP, 109 ± 4.4, ATP+D609, 111 ± 7.5. The asterisk denotes a value significantly different from basal; the dagger denotes a value significantly different from that obtained in the presence of ACh + vehicle or ATP + vehicle (P < .05).

	Discussion

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We have previously reported that ACh stimulates prostacyclin production in CEC of the rabbit heart via activation of M₃ mAChR and inhibits cyclic adenosine 3,5 monophosphate accumulation through M₂ mAChR (Kan et al., 1996). Our study provides the following new information: 1) ACh promotes prostacyclin production in CEC from rabbit heart by activating a pertussis toxin-insensitive G protein and by increasing the influx of extracellular Ca⁺⁺ through a receptor-operated Ca⁺⁺ channel by a G protein- independent mechanism and 2) ACh-induced release of intracellular Ca⁺⁺ which is mediated by a pertussis toxin insensitive G protein, is not involved in prostacyclin production. These conclusions are based in part on our findings that ACh-induced 6-keto-PGF₁ production was enhanced by increasing the extracellular concentration of Ca⁺⁺ from 0.45 to 5.6 mM and was abolished by depletion of extracellular Ca⁺⁺. However, ACh increased cytosolic Ca⁺⁺ to a similar degree in the presence or absence of extracellular Ca⁺⁺. Evidently, ACh, which produces a transient rise in cytosolic Ca⁺⁺ in the absence of extracellular Ca⁺⁺, is either insufficient or is compartmentalized and inaccessible to the lipase involved in the release of AA for PG synthesis.

SK&F96365, a ROCC blocker, but not two voltage-dependent Ca⁺⁺ channel blockers, verapamil and nifedipine, inhibited ACh-induced 6-keto-PGF₁ synthesis and the rise in cytosolic Ca⁺⁺. The effect of SK&F96365 to reduce ACh-induced 6-keto-PGF₁ synthesis was not due to a decrease in cyclooxygenase activity or PLA₂ because SK&F96365 did not alter the conversion of exogenous AA to 6-keto-PGF₁ or PLA₂ activity in CEC. Exposure of CEC to high concentrations of K⁺ (55 mM), which activate voltage-dependent Ca⁺⁺ channels, failed, as expected, to increase either 6-keto-PGF₁ or cytosolic Ca⁺⁺. Therefore, it appears that the M₃ mAChR in CEC of rabbit heart (Kan et al., 1996) is coupled to a receptor-operated Ca⁺⁺ channel. This conclusion is further confirmed by our study that thapsigargin, which inhibits Ca⁺⁺-ATPase and prevents the refilling of myo-inositol trisphosphate-sensitive Ca⁺⁺ pools (Inesi and Sagara, 1992), attenuated the ACh-induced rise in cytosolic Ca⁺⁺ in the absence, but not in the presence, of extracellular Ca⁺⁺. Because ACh also increased cytosolic levels of Ca⁺⁺ in the absence of extracellular Ca⁺⁺, the M₃ mAChR is also coupled to intracellular Ca⁺⁺ release, presumably through the action of myo-inositol trisphosphate, generated consequent to activation of PLC. m1, m3 and m5 of mAChR are known to couple to polyphosphoinostide hydrolysis in cells transfected with these receptors (Hosey, 1992). In addition, the PLC inhibitor D609 also attenuated the ACh-induced rise in cytosolic Ca⁺⁺ in the absence of extracellular Ca⁺⁺ in CEC of the rabbit heart.

Muscarinic receptors transduce their signals by coupling with G proteins, which then influence the activity of effector enzymes and/or ion channels (Hosey, 1992). Our findings that in rabbit heart CEC permeabilized with saponin: 1) GTP--S, a nonhydrolyzable analogue of GTP, but not ACh, enhanced 6-keto-PGF₁ production in the presence and not in the absence of extracellular Ca⁺⁺; 2) ACh in the presence of GTP--S increased 6-keto-PGF₁ production to a greater degree than GTP--S alone and 3) the effect of GTP--S and ACh on 6-keto-PGF₁ was attenuated by GDP--S, suggest that 6-keto-PGF₁ synthesis induced by activation of M₃ mAChR in CEC by ACh is mediated in part by a G protein. However, the influx of extracellular Ca⁺⁺ that is required for 6-keto-PGF₁ synthesis does not appear to involve G proteins for the following reasons. First, in CEC permeabilized with -escin to deplete endogenous GTP and loaded with or without GTP--S and resealed, ACh produced similar increases in cytosolic Ca⁺⁺ in the presence of extracellular Ca⁺⁺. Second, GDP--S loaded with GTP--S failed to inhibit the effect of ACh to increase cytosolic Ca⁺⁺ in the presence of extracellular Ca⁺⁺. Interestingly, in the absence of extracellular Ca⁺⁺, ACh increased cytosolic Ca⁺⁺ in CEC that were permeabilized with -escin and resealed after loading with GTP--S but failed to do so in those loaded with the vehicle of GTP--S. Moreover, the increase in the cytosolic Ca⁺⁺ produced by ACh in CEC loaded with GTP--S in the absence of extracellular Ca⁺⁺ was minimized in CEC that were loaded with GTP--S plus GDP--S. From these observations, it follows that ACh-induced mobilization of intracellular Ca⁺⁺, but not influx of extracellular Ca⁺⁺, is mediated by a G protein.

This conclusion is supported by the elegant studies of Felder et al. (1992) in A9 fibroblasts cells stably transfected with m2 and m3 or m2/m3 chimeric mAChR in which the third cytoplasmic loop (the primary determinant of G protein-coupling) was exchanged. These authors demonstrated that in cells expressing mAChR m3 (coupled to increased cellular Ca⁺⁺) but not m2 (coupled to adenylyl cyclase inhibition), carbachol increased the influx of extracellular Ca⁺⁺ and myo-inositol trisphosphate-mediated mobilization of intracellular Ca⁺⁺. However, in cells expressing the chimeric m2 mAChR with the m3 loop, carbachol generated a typical myo-inositol trisphosphte-mediated Ca⁺⁺ response but not the sustained response dependent on influx of extracellular Ca⁺⁺. In contrast, in cells expressing m3 mAChR with the m2 loop construct, carbachol produced a sustained Ca⁺⁺ response but did not cause a myo-inositol trisphosphate mediated one. Determinants of activation of Ca⁺⁺ influx by m3 mAChR are thus distinct from determinants of G protein interaction.

Guanine nucleotides modulated the mobilization of intracellular Ca⁺⁺ elicited by ACh as well as by the noncholinergic agent ATP. The ATP-induced increase in cytosolic Ca⁺⁺ in the absence of extracellular Ca⁺⁺ has also been shown to be decreased by GDP--S in osteoclasts (Yu and Ferrier, 1994). However, it has been reported that both the influx of extracellular Ca⁺⁺ and the release of intracellular Ca⁺⁺ from IP₃-sensitive stores by alpha-1 and alpha-2 adrenergic receptors are mediated by a pertussis toxin-sensitive G protein. Because the influx of Ca⁺⁺ induced by alpha-1 and alpha-2 adrenergic receptors in rabbit aortic smooth muscle cells (Nebigil and Malik, 1993) but not by ACh or ATP in CEC was inhibited by voltage-dependent Ca⁺⁺ channel blockers, it would appear that voltage- but not receptor-operated influx of extracellular Ca⁺⁺ is regulated by a G protein. Because the release of internal Ca⁺⁺ by IP₃ is known to activate the entry of Ca⁺⁺ from the extracellular fluid to replenish the depleted stores (Putney and Bird, 1993), it is possible that ACh and ATP, by releasing intracellular Ca⁺⁺ via G protein-activated PLC stimulation and IP₃ generation, promote the influx of extracellular Ca⁺⁺ via capacitative Ca⁺⁺ entry channels (Zhu et al., 1996). ATP-induced influx of extracellular Ca⁺⁺, like that produced by ACh, was not affected by pertussis toxin in CEC. However, in contrast to the results with ACh, the mobilization of intracellular Ca⁺⁺ elicited by ATP in the absence of extracellular Ca⁺⁺ was inhibited by pertussis toxin. Therefore, it appears that a pertussis toxin-sensitive G protein is involved in the action of ATP and a pertussis-toxin insensitive G protein, presumably a G_q- or G₁₂-type G protein, in the action of ACh on the release of intracellular Ca⁺⁺ in CEC of the rabbit heart. Alternatively, it is possible that the PLCs coupled to mAChR and ATP receptors in these cells are distinct.

Although both ACh- and ATP-induced 6-keto-PGF₁ production was dependent on the influx of extracellular Ca⁺⁺ via a G protein-independent mechanism, the effect of ATP as shown also by others (Gerritsen and Mannix, 1989), but not that of ACh, to stimulate 6-keto-PGF₁ was attenuated by pertussis toxin. It appears that ATP- but not ACh-induced 6-keto-PGF₁ synthesis is mediated in part by G_i and/or G_o protein.

It is well established that ACh increases NO synthase activity and NO production in endothelial cells by increasing Ca⁺⁺ influx (Busse and Mulsch, 1990). Because NO has been reported to stimulate cGMP production and cyclooxygenase activity (Salvemini et al., 1993), we examined the possible involvement of NO in ACh-induced 6-keto-PGF₁ synthesis. Our demonstration that the NO synthase inhibitor N-mono ethyl arginine, which abolished ACh-induced cGMP accumulation, did not alter 6-keto-PGF₁ synthesis implies that NO is not involved in ACh-induced 6-keto-PGF₁ production in CEC of the rabbit heart.

ACh is known to stimulate PLC activity and to promote breakdown of polyphosphoinositides into inositol phosphates and diacylglycerol (De George et al., 1987). The latter could be a source of AA via activation of diacylglycerol lipase (Zahler et al., 1986). However, this appears unlikely in the present case since the selective PLC inhibitor D609, which attenuated the ACh-induced rise in intracellular Ca⁺⁺, failed to alter 6-keto-PGF₁ synthesis. Two types of Ca⁺⁺-dependent phospholipase A₂ have been implicated in the release of AA in response to various stimuli (Leslie, 1990; Lin et al., 1992; Murakami et al., 1993). Our recent study indicates that AA release from CEC for prostacyclin synthesis in response to ACh is mediated predominantly via cytosolic phospholipase A₂ activation (Kan et al., 1996), although other phospholipases may also be partly involved, for example, phospholipase D (Sandmann et al., 1991).

In conclusion, our study demonstrates that ACh stimulates prostacyclin production in CEC of the rabbit heart by increasing the influx of extracellular Ca⁺⁺ through a receptor-operated Ca⁺⁺ channel via activation of, most likely, cPLA₂. Although ACh-induced 6-keto-PGF₁ production was mediated by a pertussis toxin-insensitive G protein, the influx of extracellular Ca⁺⁺ required for PG synthesis was not directly dependent on G protein. However, the ACh-induced release of intracellular Ca⁺⁺ not required for prostacyclin production was mediated through a pertussis toxin-insensitive G protein. Further studies utilizing electrophysiological techniques are required to establish the requirement of G protein for the release of intracellular but not the influx of extracellular Ca⁺⁺ elicited by ACh in CEC of the rabbit heart.