Introduction

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Esophageal motility disorders, such as achalasia and diffuse esophageal spasm, are characterized by the incomplete passage of swallowed contents into the stomach. Such conditions are often accompanied by incomplete or failure of lower esophageal sphincter (LES) relaxation during swallowing (Castell and Richter, 1999). Exact treatments for such conditions are not known at present. Different therapeutic approaches such as antichlolinergics (Ona and Polintan, 1980; Holloway et al., 1986; Marzio et al., 1994), Ca²⁺-channel blockers (Bortolotti and Labo, 1981; Roman et al., 1985; Traube et al., 1989; Short and Thomas, 1992), nitric oxide donors (Gelfond et al., 1982; Figueiredo et al., 1992), and botulinum toxin (Pasricha et al., 1995; Fishman et al., 1996; Annese et al., 2000) have been suggested. All of these treatments have variable success rates and accompanying untoward effects.

Yet another rational approach may be through the administration of -adrenoceptor agonists, based on their potent relaxant actions in the LES smooth muscle (Goyal and Rattan, 1973). ₂-Adrenoceptor agonists already have been tested in achalasia patients (DiMarino and Cohen, 1982; Wong et al., 1987). The use of such agents in these patients may be limited because of short duration of effect and other systemic effects (Goldberg and Frishman, 1995; Hoffman, 2001). The discovery of ₃-adrenoceptors, with potent relaxant effects of ₃-adrenoceptor agonists in different smooth muscles (Goldberg and Frishman, 1995) offers the possibility of being helpful for treating esophageal motility disorders characterized by hypertensive LES.

₃-Adrenoceptor agonists are known to produce relaxation of different smooth muscles, including those of the gastrointestinal tract (Goldberg and Frishman, 1995; Bardou et al., 1998; Takeda et al., 2000; Horinouchi and Koike, 2001). It is believed that because of selectivity of their action, such agonists may have limited systemic side effects (Takeda et al., 2000). Such agents with protracted action may be of therapeutic interest in the esophageal motility disorders with hypertensive LES.

No information exists on characterization of ₃-adrenoceptor agonists' effects in the spontaneously tonic LES smooth muscle in the appropriate animal model that resembles humans (Christensen and Lund, 1969). The purpose of the present investigation was to determine the presence and role of ₃-adrenoceptors in the LES. The ₃-preferential agonist CL 316243 (Takeda et al., 2000) and antagonist L 748337 (Candelore et al., 1999) were used in the present investigation, and studies were carried out both in smooth muscle strips and smooth muscle cells (SMCs) from the circular smooth muscle layer of the opossum LES.

	Materials and Methods

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Preparation of LES Smooth Muscle Strips. Adult opossums (Didelphis virginiana) of either sex weighing from 2.6 to 4.5 kg were sacrificed after pentobarbital sodium anesthesia (40-50 mg/kg i.p.), by exsanguination. The LES region along with a portion of esophageal body and stomach was dissected out of the animals and transferred immediately into oxygenated (95% O₂ plus 5% CO₂) Krebs' solution, pH 7.4. The composition of the Krebs' solution was as follows: 118.07 mM NaCl, 4.69 mM KCl, 2.52 mM CaCl₂, 1.16 mM MgSO₄, 1.01 mM NaH₂PO₄, 25 mM NaHCO₃, and 11.10 mM glucose. The smooth muscle tissues were cleaned of extraneous connective tissue, blood vessels, and adjoining adventitia. The LES smooth muscle and body of the esophagus were opened flat by an incision along the greater curvature of stomach and the longitudinal axis while placed on a dissecting tray containing oxygenated Krebs' buffer. The tissues were then pinned flat and the mucosa and submucosa were removed by a sharp dissection and circular smooth muscle strips (~1 × 10 mm) of the LES were prepared. The smooth muscle strips were tied at both ends with silk sutures (5-0; Ethicon Inc., Somerville, NJ) for measurements of isometric tension.

Measurement of Isometric Tension. The smooth muscle strips were transferred to 2-ml muscle baths containing oxygenated Krebs' solution, maintained at 35°C. One end of the muscle strip was fixed at the bottom of the muscle bath with the help of a tissue holder and the other end was attached to an isometric force transducer (model FT03; Grass Instruments, Quincy, MA). The smooth muscle tension in the basal state and after different stimuli was amplified by ETH-400 bridge amplifiers (CB Sciences, Inc., Milford, MA) and recorded using a PowerLab/800 analog-to-digital converter (AD Instruments, Mountain View, CA) connected to PC computer. At the start, the smooth muscles strips were stretched at 10-mN tension and allowed to equilibrate for at least 1 h with intermittent washings. During this period, the smooth muscle strips from the LES developed steady-state tension. Baseline and optimal length (L_o) of the smooth muscle strips were determined as described previously (Moummi and Rattan, 1988). Only the smooth muscle strips that developed steady tension and relaxed in response to electrical field stimulation (EFS) were considered sphincteric and were included in the LES studies. The smooth muscle strips from the esophageal body (1 cm above the LES), on the other hand, did not develop any tone and contracted in response to EFS.

Nonadrenergic Noncholinergic (NANC) Nerve Stimulation with EFS. EFS is known to cause relaxation of the LES and contraction of the esophageal body smooth muscles. Both of these responses are primarily mediated by the activation of NANC neurons (Rattan, 1986). EFS was delivered from a Grass stimulator (model S11; Grass Instruments) connected in series to a Med-Lab Stimu-Splitter II (Med-Lab Instruments, Loveland, CO). The Stimu-Splitter was used to amplify and to measure the stimulus intensity by the optimal stimulus parameters known to activate NANC neurons (Rattan and Chakder, 1992) (12-15 V, 0.5-ms pulse duration, 200-400 mA, 4-s train), at varying frequencies of 0.5 to 20 Hz. The electrodes used for EFS were a pair of platinum wires fixed at both sides of the smooth muscle strips of the LES and esophageal body.

Isolation of SMCs and Measurement of Cell Length. SMCs from the circular smooth muscle layer of the opossum LES were isolated as described previously (Rattan and Chakder, 1993). The sphincteric region was identified using water-perfused catheter assembly and marked in situ. The area was marked as high-pressure zone that relaxed in response to swallowing reflex and esophageal distension. The LES smooth muscle tissues were cut into small pieces (1-2-mm cubes) and incubated in oxygenated Krebs' solution containing 0.01% collagenase and 0.01% soybean trypsin inhibitor at 37°C for two successive 1-h periods. After each incubation, the mixture was filtered through a 500-µm Nitex mesh. The tissue trapped on the mesh was rinsed with 25 ml (5 × 5 ml) collagenase-free Krebs' solution. The tissue then was incubated in collagenase-free Krebs' solution at 37°C and dispersion of the cells (0-1 h) was monitored periodically by examining a 10-µl aliquot of the mixture under microscope. SMCs were harvested by filtration through the Nitex mesh. The filtrate containing the cells was centrifuged at 350g for 10 min at room temperature. The cells in the pellet were resuspended in Krebs' solution at a cell density of 3 × 10⁴ cells/ml.

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Drug Responses. To determine the concentration-response curves (CRCs) with CL 316243 and isoproterenol on the basal tone of the LES smooth muscles, the agonists were added in cumulative concentrations to the muscle bath (Rattan and Moummi, 1989). Successive concentrations of the agonists were not added until the response of the previous concentration stabilized. Ten minutes between additions were allowed for lack of effect. In the preliminary studies with a single concentration, we noted that this was an appropriate time to gauge the maximal effect of a given concentration of the agonist. No difference in the results occurred with longer exposures. To determine the effects of antagonists, L 748337 or propranolol (in concentrations from 1 × 10⁸ to 3 × 10⁷ M) were added 30 min before obtaining the CRC of the test agonist. At the end of each experiment, the smooth muscle strips were treated with 1 × 10⁵ M bethanechol followed by 10 mM EGTA to determine the maximum contraction and relaxation of the smooth muscles, respectively (Biancani et al., 1985). Each smooth muscle served as its own control.

Drugs and Chemicals. The following chemicals were used in the study: DL-isoproterenol hydrochloride was from Aldrich Chemical Co. (Milwaukee, WI). Bethanechol chloride and DL-propranolol, disodium (R,R)-5-[2-[2-3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243), a ₃-adrenoceptor agonist (Fletcher et al., 1998; Roberts et al., 1999; Takeda et al., 2000), and 3-(2-ethylphenoxy)-1-[(1S)1,2,3,4-tetrahydronaphth-1-ylaminol]-(2S)-2-propanol oxalate (SR 59230A), a ₃-adrenoceptor antagonist (De Ponti et al., 1996), were from Sigma-Aldrich (St. Louis, MO). Tetrodotoxin (TTX) was from Calbiochem (San Diego, CA). (S)-N-[4-[2-[[3-[-(Acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide (L 748337), a ₃-adrenoceptor antagonist (Candelore et al., 1999), was a generous gift from Merck (Rahway, NJ). (S)-4-[Hydroxy-3-phenoxy-propylamino-ethoxy]-N-(2-methoxyethyl)-phenoxyacetamide (ZD 7114), a ₃-adrenoceptor agonist (Growcott et al., 1993), was obtained from Tocris Cookson (Ballwin, MO). All chemicals except ZD 7114, SR 59230A, and indomethacin (dissolved in dimethyl sulfoxide or dimethyl sulfoxide initially) were dissolved and diluted with Krebs' solution and prepared fresh on each day of experimentation.

Data Analyses. The fall in basal tension of the LES smooth muscle after agonists and EFS was expressed as the percentage of maximal relaxation as explained above. The peak amplitude of esophageal contraction in response to EFS was compared during control experiments versus in the presence of CL 316243. The results were expressed as means ± S.E. of different experiments. The statistical significance between different groups was determined by analysis of variance and by paired or unpaired t test. A p value smaller than 0.05 was considered significant.

	Results

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Effect of CL 316243 on the Basal Tone of LES Smooth Muscle, and on Isolated SMCs. ₃-Adrenoceptor agonist CL 316243 caused a concentration-dependent fall in the basal tension of the LES smooth muscle (Fig. 1). The concentration causing maximal relaxation (EC_max) was 1 × 10⁴ M. The maximal relaxation in different experiments ranged from 76 to 87%. A typical tracing to show the effect of CL 316243 in the LES smooth muscle strips is given in Fig. 1A. The durations of relaxations of CL 316243 and isoproterenol were compared by their single administrations in EC_max concentrations. The smooth muscle relaxation by CL 316243 was significantly longer than with isoproterenol. When left undisturbed, the smooth muscle relaxation by isoproterenol began to recover within 5 min versus 1 h in the case of CL 316243. Such experiments were followed for 2 h, and durations of relaxations calculated for the 50% reversal of the relaxations. Such values in the case of isoproterenol and CL 316243 were 9.1 ± 0.2 and 116.5 ± 5.6 min, respectively. A complete reversal of the tone was observed in both cases after routine washings, and subsequent administration of the agonists caused a reproducible fall in the LES tone.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   A, typical tracing to show concentration-dependent response of CL 316243 on the basal tone of LES. The effect of EFS (10 Hz) is shown on the left side of the tracing that depicts an immediate relaxation of the smooth muscle followed by its recovery to the basal tone. Different concentrations of CL 316243 (added in a cumulative manner) are represented as micromolar. In this experiment, the agonist caused smooth muscle relaxation in a concentration-dependent manner beginning with 0.1 µM and plateauing at 100 µM. The right side of the tracing shows the effect of 10 mM EGTA. B, influence of 3-adrenoceptor antagonist L 748337 on control CRCs with CL 316243 showing percentage of fall (mean ± S.E.; n = 5-8) in the basal LES smooth muscle tension. C, influence of propranolol on CL 316243-induced relaxation of the LES smooth muscle. The values represent mean ± S.E. of five to eight observations. EC50 value of CL 316243 in control experiments was 6.01 × 107 M. CL 316243 CRC under control conditions is significantly shifted (*, p < 0.05) toward right by L 748337 in a concentration-dependent manner. Propranolol on the other hand causes no significant effect on the control CRC of CL 316243 (p > 0.05).

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View this table: [in this window] [in a new window]   TABLE 1 Influence of different neurohumoral antagonists on the LES smooth muscle relaxation by CL 316243 (3 × 105 M)

View larger version (16K): [in this window] [in a new window]   Fig. 2.   CRC showing relaxation by CL 316243 of dispersed SMCs from the opossum LES, before and after L 748337 (3 × 108 M). Relaxation was measured by scanning microscopy and depicted as percentage of maximal inhibition of bethanechol-induced contraction. L 748337 causes a significant antagonism of CL 316243-induced relaxation (*, p < 0.05; n = 4 animals), whereas propranolol has no significant effect (p > 0.05).

Influence of L 748337 on CL 316243-Induced Relaxation of LES Smooth Muscle. The fall in LES tension caused by CL 316243 in vitro smooth muscle strips was antagonized significantly and in a concentration-dependent manner (1 × 10⁸ to 1 × 10⁷ M) by ₃-adrenoceptor antagonist L 748337 (*p < 0.05; n = 5-8; Fig. 1B). In control experiments, EC₅₀ value for CL 316243 in causing fall in the LES tension was 6.01 × 10⁷ M. In the presence of 1 × 10⁸, 3 × 10⁸, and 1 × 10⁷ M L 748337, the calculated EC₅₀ values for CL 316243 were 7.0 × 10⁶, 3.0 × 10⁵, and 1.0 × 10⁴ M, respectively. pA₂ value for L 748337 in antagonizing the effect of CL 316243 was 7.9. Data showing antagonism and Schild plot of log concentrations of L 748337 versus log(r  1) are shown in Fig. 3. Propranolol had no significant effect on the fall in LES tension by CL 316243 (p > 0.05; n = 5-8; Fig. 1C).

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Schild plot showing different concentrations of L 748337 versus log(r  1) of CL 316243. The pA2 value of L 748337 in antagonizing CL 316243-induced fall in the basal tone of the LES smooth muscle is 7.9.

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Influence of L 748337 and Propranolol on Fall in Basal LES Tone by Isoproterenol. Isoproterenol also caused a concentration-dependent fall in the basal tone of LES with EC_max of 1 × 10⁶ M. The fall in the basal tension of LES smooth muscle by isoproterenol was not significantly affected by ₃-adrenoceptor antagonist L 748337 (1 × 10⁸ and 3 × 10⁸ M) (p > 0.05; n = 5-8; Fig. 4A). The higher concentration of the antagonist caused a small rightward shift in the CRC. Propranolol caused significant and concentration-dependent antagonism of isoproterenol-induced relaxation of the LES smooth muscle (*p < 0.05; n = 5-8; Fig. 4B).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Influence of L 748337 (A) and propranolol (B) on fall in LES tension caused by isoproterenol. Comparison of curves revealed that L 748337 has no significant effect on the control isoproterenol CRC (p > 0.05; n = 5-8). In contrast to CL 316243, isoproterenol CRC is significantly shifted toward right by propranolol in a concentration-dependent manner (*, p < 0.05; n = 5-8).

Effect of ZD 7114 on the Basal LES Tone before and after SR 59230A. Another ₃-agonist ZD 7114 also caused a concentration-dependent decrease in the basal tone of the LES, with EC₅₀ and EC_max concentrations of 9.9 × 10⁸ and 3 × 10⁷ M, respectively. The fall in LES tension by ZD 7114 was antagonized significantly by ₃-antagonist SR 59230A (1 × 10⁷ M) (*p < 0.05; n = 6; Fig. 5), with pK_B value of 6.8. Conversely, the LES smooth muscle relaxation by the ₃-agonist was not modified by propranolol (1 × 10⁷ M) (p > 0.05; n = 6; Fig. 5).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Influence of SR 59230A (3-antagonist) on LES smooth muscle relaxation by ZD 7114 (3-agonist). The agonist caused concentration-dependent relaxation of the LES smooth muscle that was antagonized significantly by SR 59230A (*, p < 0.05; n = 4) but not by propranolol.

Effect of CL 316243 versus Isoproterenol on LES Smooth Muscle Relaxation and Esophageal Contraction by NANC Nerve Stimulation by EFS. EFS produced a frequency-dependent relaxation of the LES smooth muscle and contraction of the esophageal smooth muscle. Against the background of a low concentration of CL 316243 (1 × 10⁸ M), EFS caused a frequency-dependent increase in the relaxation of LES smooth muscle (*p < 0.05; n = 5-8; Fig. 6). Similar experiments carried out in the presence of isoproterenol (1 × 10⁹ or 3 × 10⁹ M) showed no significant augmentation of EFS-induced relaxation of the LES smooth muscle. The concentrations of CL 316243 and isoproterenol used in these experiments had minimal effect on the basal tone of the LES smooth muscle.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effect of CL 316243 on percentage of fall in LES tension by different frequencies of EFS. EFS-induced relaxation of LES smooth muscle is significantly augmented in the presence of CL 316243 (*, p < 0.05; n = 5-8).

	Discussion

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The studies show the pharmacological characterization of inhibitory ₃-adrenoceptors in the spontaneously tonic smooth muscle in vitro in the appropriate animal model in which the anatomy and physiology of the esophagus and LES resemble humans. The studies show a potent and direct effect of CL 316243 on the LES smooth muscle. The agonist causes an augmentation of LES relaxation after NANC nerve stimulation with no adverse effect on the amplitude of esophageal contraction. These data suggest the presence of ₃-adrenoceptors, physiological significance, and therapeutic potential of ₃-adrenoceptors' agonists in gastrointestinal motility disorders.

The inhibitory effects of CL 316243 in the LES smooth muscle are characteristic of selective ₃-adrenoceptor activation. The agonist causes concentration-dependent full relaxation of the LES by its action directly at the smooth muscle. The direct effect of CL 31643 on the LES smooth muscle cells was demonstrated by the lack of influence of TTX, hexamethonium, a combination of phentolamine and guanethidine, atropine, haloperidol, indomethacin, and a combination of pyrilamine and cimetidine on the agonist-induced relaxation of the LES smooth muscle relaxation. The agonist also causes relaxation of the SMCs isolated from the circular smooth muscle layer of the LES. ₃-Adrenoceptor antagonist L 748337 causes a significant and concentration-dependent rightward shift in the control CRC of smooth muscle relaxation by CL 316243.

Additional data with another ₃-adrenoceptor agonist ZD 7114 and antagonist SR 59230A further support the concept of the presence and actions of ₃-adrenoceptor activation in the LES. ZD 7114 causes a concentration-dependent and full relaxation of the LES smooth muscle that is selectively antagonized by SR 59230A.

L 748337 has been shown to be a selective antagonist of ₃-adrenoceptors in different systems (Weber et al., 1998; Candelore et al., 1999). The antagonist was developed via heterologously expressed cloned human receptors. The effect of CL 316243 and L 748337 in the spontaneously tonic smooth muscles of the gastrointestinal smooth muscle has not been examined before. ₃-Adrencoceptor antagonist L 748337, especially in the lower concentrations, was found to be selective in antagonizing the effect of CL 316243. Such concentrations of L 748337 have no significant effect on the LES smooth muscle relaxation by isoproterenol. Conversely, the lower concentrations of propranolol that cause significant antagonism of LES smooth muscle relaxation by isoproterenol have no significant effect on CL 316243. Propranolol in the concentrations used is considered to be primarily an antagonist of ₁- and ₂-adrenoceptors (Hoffman, 2001).

The inhibitory effects of CL 316243 are similar to those in the rat stomach (Cohen et al., 2002). In that study, the selectivity of action of CL 316243 at the ₃-adrenoceptor was confirmed by investigating the effects in ₃-receptor knockout. Using a similar knockout animal model, the significance of ₃-adrenoceptor was determined by an increase in the overall gastrointestinal transit with selective ₃-agonists such as CL 316243 in wild-type versus knockout animals (Fletcher et al., 1998). The presence of ₃-adrenoceptors has also been shown by the receptor binding, autoradiographic localization, and mRNA studies using reverse transcription-polymerase chain reaction in the rat small intestine (Roberts et al., 1999) and human colon (Krief et al., 1993).

In causing the LES smooth muscle relaxation, ₃-agonist CL 316243 was found to be ~50 times less potent than isoproterenol (EC₅₀ values of 6.01 × 10⁷ versus 2.9 × 10⁸ M, respectively). These potencies with CL 316243 and other selective ₃-agonists are similar to those in vivo studies in the LES (DiMarino et al., 2002), in the cardiovascular smooth muscles (Gauthier et al., 2000), in vitro studies in human detrusor smooth muscle (Igawa et al., 1999), and in rat esophageal muscularis mucosa (de Boer et al., 1993). The effects of certain ₃-selective agonists in other smooth muscles may be either of comparable or of higher potency than of ₁- and ₂-agonists (Lezama et al., 1996; Roberts et al., 1999). It is noteworthy that the smooth muscles examined in the previous studies were not spontaneously tonic.

The potency differences may be either related to tissue or species specificity, differences in receptor density, or receptor affinity. Although there is a good correlation between the effects of activation and receptor density of ₃-adrenoceptors in certain tissues, it may not hold for others. For example, in rat ileum the relaxation of smooth muscle with the selective agonists is in general agreement with ₁-, ₂-, and ₃-receptors expression (Roberts et al., 1999). The exact reason for the differences in potency of the ₃-adrencoceptor agonist activation in the LES is not known. However, the possibilities include lower levels of ₃-receptors sites, receptor affinity for the agonist used in the present studies, or that the full expression of ₃-activation may be masked by the simultaneous binding with ₁- and ₂-adrenoceptors. ₁- and ₂-Adrenoceptors agonists may have unknown inhibitory effect on ₃-adrenoceptors. Future studies on -adrenoceptors density distribution combined with functional data with selective agonists and antagonists could resolve these issues.

The advent of newer and more selective ₃-agonists targeted toward the tonic smooth muscles may lead to more potent and selective agonists for such tissues. These agents with prolonged smooth muscle relaxation without untoward systemic effects may be of considerable importance in gastrointestinal motility disorders. Our in vivo studies suggest that ₃-agonist causes selective and prolonged relaxation of the LES smooth muscle without significant effects on the cardiovascular system (DiMarino et al., 2002), unlike ₁- and ₂-agonists (Goldberg and Frishman, 1995; Takeda et al., 2000; Hoffman, 2001). The systemic effects combined with the short-lived actions limit the potential use of ₁- and ₂-adrenoceptor agonists in the gastrointestinal motility dysfunction. Whether short-lived action of ₁- and ₂-activation (compared with that of ₃) is related to cAMP-dependent protein kinase A-associated phosphorylation sites at ₁- and ₂-adrenceptors (Goldberg and Frishman, 1995; Strosberg, 1997; Gauthier et al., 2000) and/or receptor desensitization and internalization, remains to be determined.

The potencies of ₃-agonists CL 316243 and ZD 7114 and antagonists L 748337 and SR 59230A are in general agreement with the potencies reported in the literature. In human and rat detrusor smooth muscles (Igawa et al., 1999; Woods et al., 2001), CL 316243 has been shown to be a full agonist as in the present studies. In causing relaxation of human detrusor smooth muscle, the CRC of CL 316243 was similar to that in the LES (Igawa et al., 1999). Likewise, ZD 7114 produced relaxation of guinea pig ileum with EC₅₀ value of 2 × 10⁷ M. Candelore et al. (1999) reported pA₂ values of 8.5 for L 748337 in inhibiting increased cAMP accumulation in chicken hamster ovary cells. pK_B and pA₂ values of 6.41 and 7.26 for SR 59230A in rat brown adipocytes and human colon, respectively, have been reported (Nisoli et al., 1996; Bardou et al., 1998). Higher potencies of CL 316243 in rat ileum (Roberts et al., 1999) and SR 59230A (De Ponti et al., 1996; Bardou et al., 1998) in human colon also have also been reported. Although, an exact reason for the disparity in potencies is not known, it may be related to the differences in the agonist, animal species, tissues, or regional variation in the same tissue. In one study (Tomiyama et al., 1998), it was determined that ₃-agonist CL 316243 was most potent in dog ureter smooth muscle but had negligible effect in the same tissue from other species. Interestingly, in certain preparations ZD 7114, rather than being a ₃-agonist, behaved as an antagonist (MacDonald and Lamont, 1993; Roberts et al., 1999).

In contrast to the effects of other conventional -adrenoceptor agonists, the ₃-adrenoceptor agonist lacks negative effects on the amplitude of peristaltic contraction in the smooth muscle of the esophagus. Isoproterenol causes inhibition of the amplitude of esophageal contraction, whereas CL 316243 does not have such an effect. In addition, the present studies show that the ₃-adrenoceptor agonist causes an augmentation of the NANC relaxation in the LES and suggest direct involvement of ₃-adrenoceptors in the facilitatory modulation of the NANC relaxation. The role of ₃-agonists in the NANC relaxation is suggested by indirect studies in rat LES by the use of nonspecific agents (Oriowo, 1998). Because of the lack of spontaneous tone, and the use of nonselective agents, it is difficult to examine the relevance of those studies. It is noteworthy that NANC relaxation in the gut may be tissue-specific. Although in most of the regions it is nitrergic in nature, the inhibitory neurotransmission may be noradrenergic in other regions that use -adrenoceptors (Chen et al., 1998). The exact mechanisms underlying gastrointestinal smooth muscle relaxation, and augmentation of NANC relaxation caused by ₃-adrenoceptor agonists remain to be determined.

We conclude that ₃-adrenoceptor agonists, because of their selective and prolonged effects in the tonic smooth muscle of the LES, may have a role in the management of esophageal and other spastic gastrointestinal motility disorders.