Introduction

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Z-338 [N-(N',N'-diisopropylaminoethyl)-[2-(2-hydroxy-4,5-dimethoxy-benzoylamino)-1,3-thiazole-4-yl] carboxyamide monohydrochloride trihydrate] (Fig. 1) is a newly synthesized gastroprokinetic agent that enhances gastrointestinal motility in conscious dogs and gastric emptying in rats and dogs (Ueki et al., 1998). In an in vitro study, Z-338 inhibited the activity of acetylcholinesterase derived from human erythrocyte membranes and produced a contraction of antrum preparations from guinea pig stomach (Kurimoto et al., 1998). The most widely used gastrointestinal prokinetic benzamides interact with 5-hydroxytryptamine (5-HT) receptors, especially the 5-HT₄ receptor subtype to facilitate acetylcholine (ACh) release from enteric neurons (Taniyama et al., 1991). Z-338 does not possess binding affinity for 5-HT receptors (Kurimoto et al., 1998), and the mechanism underlying its gastroprokinetic action has not been elucidated. Thus, this study was attempted to determine whether Z-338 stimulates the release of ACh from strips isolated from the guinea pig stomach and interacts with muscarinic receptors with membrane receptor-binding assay and Xenopus oocytes heterologously expressing cloned receptors.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Chemical structure of Z-338.

	Materials and Methods

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Preparation of Stomach Strips. Male Hartley guinea pigs weighing 300 to 500 g (Kyudo Inc., Kumamoto, Japan) were decapitated. The whole stomach was dissected out and placed in Krebs-Henseleit solution (118 mM NaCl, 4.8 mM KCl, 1.18 mM KH₂PO₄, 1.19 mM MgSO₄, 2.5 mM CaCl₂ 2.5, 25 mM NaHCO₃, and 11 mM d-glucose). The antrum and corpus of the stomach were immediately cut into circular strips ~10 × 2 mm. The mucosa was rapidly removed from the tissue and the muscle layers and intramural plexus were left intact.

Measurement of Mechanical Activity. The strips were mounted in a superfusion apparatus with resting tension of 0.5 g and superfused at a constant rate of 1.2 ml/min with Krebs-Henseleit solution gassed with 95% O₂ and 5% CO₂ at 37°C for 80 min. The tension was kept constant by readjustment during the equilibration period. The strips were stimulated with two parallel platinum electrodes for 2 min of 1-ms duration, 10-V intensity, and a frequency of 1 Hz successively three times (S1, S2, S3) at 30-min intervals. When the effect of Z-338 on electrically stimulated contractions was evaluated, Z-338 was applied to the superfusion solution 10 min before the third stimulation (S3). The contractile activity was analyzed quantitatively by measuring the area under the contractile waves with Flexi Trace (Tree Star, Inc., San Carlos, CA). The ratio of S3/S2 calculated from the S2 and S3 without Z-338 was used as a control, and the effect of Z-338 on the electrically stimulated contraction was evaluated by the ratio of S3/S2 calculated from the S3 in the presence of Z-338.

Measurements of Tritium Outflow. The methods of incubation and superfusion were as described by Kusunoki et al. (1985) and Takeda et al. (1991). The strips were incubated with [³H]choline (2997 GBq/mmol; DuPont-NEN, Boston, MA) at a final concentration of 90 nM in oxygenated Krebs-Henseleit solution at 37°C for 60 min. At the end of the labeling period, the strips were mounted in a superfusion apparatus and washed out by superfusion with Krebs-Henseleit solution gassed with 95% O₂ and 5% CO₂ at a constant rate of 0.6 ml/min for 60 min at 37°C. Hemicholinium-3 (10 µM) was present in the superfusion solution to prevent the uptake of [³H]choline. Two parallel platinum electrodes were used to stimulate intramural nerves. The strips were stimulated electrically at parameters of 1-ms duration, 15-V intensity, and a frequency of 5 Hz for 30 s. The strip was stimulated successively four times (S1, S2, S3, and S4) at 34-min intervals, at 6 min (S1), 40 min (S2), 74 min (S3), and 108 min (S4) after the end of washout. The superfusate was collected every 2 min and the radioactivity of the sample was determined by counting in a liquid scintillation spectrometer (Packard Instrument Co., Downers Grove, IL). The radioactivity of the tissue dissolved in Soluene-350 (Packard Instrument Co.) at the end of the release experiment was measured in a liquid scintillation spectrometer.

Receptor-Binding Assay. The methods of receptor-binding assays for muscarinic M₁, M₂, and M₃ were carried out according to the methods of Wang et al. (1987; M₁ muscarinic receptor) and Delmendo et al. (1989; M₂ and M₃ muscarinic receptors), respectively. The cerebral cortex, heart, and submaxillary gland were dissected from male Sprague-Dawley rats (250-350 g). The tissues were homogenized separately in 40 volumes (cerebral cortex), 20 volumes (heart), and 30 volumes (submaxillary gland) of ice-cold buffer (50 mM sodium/potassium phosphate buffer, pH 7.4, for the cortex and 50 mM Tris-HCl buffer with 5 mM EDTA, pH 7.4, for the heart and submaxillary gland) with an Ultra-Turrax homogenizer (Janke and Kunkel, Staufen, Germany) for 30 s, three times (cerebral cortex), and 20 sec, two times (heart and submaxillary gland). The homogenates of heart and submaxillary gland were passed through a double layer of cheesecloth. The homogenates were centrifuged at 1,000g for 5 min (cerebral cortex) and 500g for 10 min (heart and submaxillary gland) at 4°C, and then the supernatants were centrifuged at 40,000g for 20 min (cerebral cortex) and 30,000g for 15 min (heart and submaxillary gland). The pellets were washed by resuspension in 30 volumes of ice-cold buffer and subsequently centrifuged at 40,000g for 20 min (cerebral cortex) and 30,000g for 15 min (heart and submaxillary gland). The final pellets were resuspended in 40 ml (cerebral cortex) and 30 ml (heart and submaxillary gland) of ice-cold buffer and frozen at 80°C until assay.

Expression of Muscarinic M₁ and M₂ Receptors in Xenopus oocytes. The cRNAs for rat M₁ muscarinic receptor and human M₂ muscarinic receptor were synthesized in vitro with T7 polymerase with Ambion's MEGAscrip kits (Austin, TX) from linearized cDNAs with HindIII (Matsumoto et al., 1998; Uezono et al., 1998). Xenopus were anesthetized by hypothermia. Small incisions were made on Xenopus's abdomen, and portions of the ovary removed (Dascal et al., 1985). Stage V-VI (Dascal, 1987) Xenopus oocytes were isolated and deffoliculated by gently shaking them at room temperature (21-23°C) for 60 min in Ca²⁺-free solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 10 mM HEPES, pH 7.4) containing 0.5 mg/ml collagenase (Yakult, Tokyo, Japan). cRNAs (5 ng) for M₁ or M₂ receptor were injected into the oocytes with a Picospritzer II (General Valve Co., Fairfield, NJ); oocytes were then incubated at 19°C in modified Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 0.82 mM MgSO₄, 0.41 mM CaCl₂, and 10 mM HEPES, pH 7.4) containing 2.5 mM sodium pyruvate and 20 mg/ml gentamycin for 2 to 4 days. The oocytes were incubated in modified Barth's solution that did not contain gentamycin >3 h before electrophysiological study. For the experiment on the M₂ receptor, 5 ng of cRNA for the G protein-gated inward rectifying K⁺ channel (GIRK1) was injected into the oocytes together with cRNA for M₂ receptor. GIRK1 was synthesized in vitro with T3 polymerase with Ambion's MEGAscrip kits from linearized cDNA for rat GIRK1 with SalI (Uezono et al., 1998). The Ca²⁺-free solution and modified Barth's solution were sterilized before use.

Drugs and Chemicals. The drugs and chemicals used were as follows: Z-338 and AF-DX 116 [11,2-(diethylamino)methyl-1-piperidinyl-acetyl-5,11-dihydro-6H-pyrido-2,3b-1,4-benzodiazepine-6-one] (a gift from Zeria Pharmaceutical Co., Ltd., Tokyo, Japan); [³H]choline chloride, [³H]pirenzepine, and [³H]NMS (DuPont-NEN); acetylcholine chloride, hemicholinium-3, and pirenzepine dihydrochloride (Sigma, St Louis, MO); tetrodotoxin (TTX; Wako Pure Chemical, Osaka, Japan); Soluene-350 (Packard Instrument Co.); and EGTA (Nacarai Tesque, Kyoto, Japan). Drugs were dissolved in distilled water, then diluted with buffer to the required concentration.

Statistical Analysis. All data are represented as the mean ± S.E. A statistical analysis between the control and substance-treated group was made with Dunnett's test or the Mann-Whitney U test (MUSCOT Statistical Analysis Program; Yukms Co., Ltd., Tokyo, Japan). A P value <.05 was considered statistically significant.

	Results

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Effect of Z-338 on Electrically Stimulated Contractions of Strips of Stomach. Electrical transmural stimulation (1 Hz, 10 V, 1 ms) for 2 min caused contraction of the strips. This contraction was prevented by application of 1 µM atropine or 0.3 µM TTX to the superfusion solution 10 min before the stimulation. When Z-338 at concentrations of 3 to 100 µM was applied to the superfusion solution 10 min before the electrical stimulation, the contraction was enhanced in a concentration-dependent manner (Fig. 2).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Concentration-response curve of the enhancement of the electrically stimulated contraction by Z-338. Electrical stimulation was applied to the strips for 2 min (1 Hz, 10 V, 1 ms). Z-338 was added to the superfusion solution 10 min before and during S3. Each point represents the mean ± S.E. of six animals. , vehicle and , Z-338.

Effect of Z-338 on Electrically Stimulated Outflow of Tritium. The outflow of tritium was studied in the presence of TTX, or after calcium had been omitted from the Krebs-Henseleit solution. TTX (0.3 µM, pretreatment for 10 min) and omission of calcium (pretreatment for 20 min) completely and reversibly depressed the electrically stimulated outflow of tritium, as noted by Kusunoki et al. (1985) and Takeda et al. (1991), thereby indicating that the outflow of tritium is neuronal in origin. Z-338 (30 µM) significantly enhanced the electrically stimulated outflow of tritium (Fig. 3).

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Effect of Z-338 on the electrically stimulated outflow of tritium from antral strips. Z-338 was applied 10 min before and during the stimulation (5 Hz, 1 ms, 15 V for 30 s). Each point represents the mean ± S.E. of 6 to 11 animals. *, significantly different from the value in the absence of Z-338 (control; P < .05).

5

Effects of Pirenzepine and AF-DX 116 on Electrically Stimulated Outflow of Tritium. Pretreatment with pirenzepine at 0.5 µM for 10 min enhanced the electrically stimulated outflow of tritium (Fig. 4). Pretreatment with AF-DX 116 at concentrations of 1 and 10 µM also enhanced the electrically stimulated outflow of tritium (Fig. 4).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Effects of pirenzepine (A) and AF-DX 116 (B) on the electrically stimulated outflow of tritium from the strips. Pirenzepine and AF-DX 116 were applied 10 min before and during the stimulation (5 Hz, 1 ms, 15 V for 30 s). Each column represents the mean ± S.E. of 6 to 11 animals. *, significantly different from the value in the absence of substance (control; P < .05).

Receptor-Binding Assay. Z-338 displaced the specific binding of [³H]pirenzepine to rat cortex membrane (M₁ receptor) and [³H]NMS to rat heart membrane (M₂ receptor), but did not displace the specific binding of [³H]NMS to rat submaxillary gland membrane (M₃ receptor; Fig. 5). The K_i values of M₁ and M₂ receptors were 8.4 and 9.4 µM, respectively.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Competitive effects of Z-338 on the binding of [3H]pirenzepine to membrane preparations from rat cortex (), and of [3H]NMS to membrane preparations from rat heart () and submaxillary gland ().

Effect of Z-338 on Muscarinic M₁ and M₂ Receptors Expressed in Xenopus Oocytes. In oocytes expressing the M₁ receptor, ACh at 1 µM produced a Ca²⁺-activated Cl current. Reapplication of the same concentration of ACh 30 min later produced a similar magnitude of current (data not shown). Z-338 did not produce any currents, whereas pretreatment with Z-338 for 30 s attenuated the ACh-induced Ca²⁺-activated Cl current in oocytes expressing the M₁ receptors, in a dose-dependent manner (Fig. 6A).

View larger version (10K): [in this window] [in a new window]   Fig. 6.   A typical pattern of the effect of Z-338 on the ACh-induced inward currents in the oocyte expressing the muscarinic M1 (A) and M2 (B) receptors. The effects of various concentrations of Z-338 on the M1 receptor-mediated and M2 receptor-mediated responses were respectively examined in the same oocyte.

	Discussion

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Z-338 enhanced the electrically stimulated contractions of strips isolated from guinea pig stomach, in a concentration-dependent manner. The contractions were sensitive to TTX and atropine, therefore the enhancement by Z-338 is probably due to an increase in ACh release. This concept is supported by the finding that Z-338 enhanced the electrically stimulated outflow of tritium. The electrically stimulated outflow of tritium was TTX sensitive and extracellular Ca²⁺ dependent, and the validity of assuming total tritium as a measure of [³H]ACh release has been documented in our previous studies with stomach strips (Kusunoki et al., 1985; Takeda et al., 1991).

Z-338 does not possess binding affinity for the serotonin 5-HT₄ receptor (Kurimoto et al., 1998); however, the receptor-binding assay revealed that Z-338 bound to muscarinic M₁ and M₂ receptors but not to the muscarinic M₃ receptor. Whether Z-338 is an agonist or antagonist for muscarinic M₁ and M₂ receptors was examined in Xenopus oocytes expressing one or the other of these receptors. Z-338 alone did not produce any currents in the oocytes expressing either M₁ or M₂ receptors, but inhibited the ACh-induced inward currents mediated by stimulation of M₁ and M₂ receptors. Thus, Z-338 acts as an antagonist at M₁ and M₂ receptors.

In gastrointestinal tissues, M₁, M₂, and M₃ receptors are reported to be present in the neurons and/or smooth muscle cells. The muscarinic receptors present in the neurons appear to be located on the cholinergic nerve terminals and to operate as autoreceptors. The release of ACh from cholinergic nerves is modulated by a negative feedback mechanism that is triggered by stimulation of presynaptic muscarinic receptors. Such an autoinhibition of ACh release has been detected in many tissues (Starke et al. 1989). In the enteric nervous system, previous studies have indicated that the release of ACh from guinea pig myenteric and submucous plexus neurons is inhibited by the presynaptic M₁ receptor (Kawashima et al., 1988; Schwörer and Kilbinger, 1988; Kilbinger et al., 1993; Dietrich and Kilbinger, 1995) and M₂ receptor in rat antral mucosal/submucosal neurons (Ren and Harty, 1994). The effects of pirenzepine (M₁ antagonist) and AF-DX 116 (M₂ antagonist) on release of ACh were examined. These selective muscarinic antagonists enhanced the electrically stimulated release of ACh. Thus, the facilitation of ACh release in the presence of pirenzepine and AF-DX 116 may be attributed to a blockade of the presynaptic M₁ and M₂ autoreceptors that are activated by ACh released from the nerve terminals. It has been reported that pirenzepine accelerates gastrointestinal transit time (Jaup et al., 1985) and increases the frequency of antral contraction (Stacher et al., 1982) in humans.

Because Z-338 inhibits the activity of cholinesterase (Kurimoto et al., 1998), the enhancing effect of Z-338 on ACh release was examined under the conditions of inhibition of cholinesterase activity by physostigmine. Z-338 enhanced the electrically stimulated release of ACh to a greater extent when physostigmine was present in the superfusion solution. Thus, Z-338 apparently does not increase the apparent outflow of tritium simply by inhibiting acetylcholinesterase. Similarly, Mike (1994) suggested that the effect of atropine on the stimulated release of ACh was much greater in the presence of physostigmine than in its absence. In the guinea pig ileum, scopolamine has been shown to greatly facilitate the stimulated release of ACh in the presence of cholinesterase inhibitor (Kilbinger and Wessler, 1980). Thus, ACh release is clearly enhanced by inhibition of muscarinic autoreceptors with muscarinic antagonists when the cholinesterase activity is inhibited.

Although Z-338 antagonized M₁ and M₂ receptors it had no effect on the M₃ receptor, and had the same effect on the release of ACh as did other antagonists for M₁ and M₂ receptors. Thus, facilitation of ACh release from cholinergic nerve terminals of guinea pig stomach by blockade of presynaptic muscarinic autoreceptors may be one mechanism by which Z-338 facilitates gastric motility. A substance such as Z-338 that is an antagonist for muscarinic autoreceptors, but not for the muscarinic M₃ receptor, may soon be available for clinical use as a gastroprokinetic agent possessing a new mechanism of action.