Gen. Pharmac. Vol. 23, No. 4. pp. 649~58, 1992
0306-3623/92 $5.00 + 0.00 Copyright ~ 1992Pergamon Press Ltd
Printed in Great Britain. All rights reserved
PHARMACOLOGICAL PROFILE OF THE 5-HYDROXYTRYPTAMINE RECEPTOR THAT MEDIATES RELAXATION OF RAT OESOPHAGEAL SMOOTH MUSCLE S. E. OHIA,*Y.-D. CHEUNG,'["D. BIEGER~.and C. R. TRIGGLE§ Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St John's, Newfoundland, Canada A IB 3V6 (Received 24 December 1991)
Abstract--I. The pharmacological profile of the inhibitory 5-hydroxytryptamine (5-HT) receptor in rat oesophageal smooth muscle has been characterized by means of a series of agonists active at 5-HTj-, 5-HT2-, 5-HT3- and 5-HT4-receptor sites, and a broad range of antagonists. The possible involvement of cyclic nucleotides in the 5-HT response was also examined. 2. Under conditions of tone induced by muscarinic receptor activation, the upper two-thirds (proximal segment) of the oesophageal smooth muscle tunic was more sensitive to the inhibitory effects of 5-HT receptor agonists when compared with the distal region. 3.The inhibitory response to 5-HT was blocked by MDL 72222 (5-HT 3 antagonist) and ICS 205-930 (5-HT3/5-HT4 antagonist) but not by antagonists active at 5-HT~- or 5-HT2-receptors. 4. The phosphodiesterase inhibitor, 3-isobutyl-methyl-xanthine (IBMX) enhanced oesophageal smooth muscle inhibitory response to 5-HT, isoprenaline and forskolin, but not that elicited by the potassium channel opener, BRL 34915. 5. 5-HT increased tissue cyclic AMP content over basal levels in proximal and distal segments of oesophageal smooth muscle. However, 5-HT had no significant effect on basal cyclic GMP levels in both segments. 6. We conclude that the inhibitory 5-HT receptor in rat oesophageal smooth muscle may represent a high affinity subtype which is sensitive to 5-HT3/5-HT( antagonists and is coupled to the cyclic AMP pathway.
INTRODUCTION
It is well established that 5-hydroxytryptamine (5HT) has both direct and indirect actions on smooth muscle of the alimentary tract (Gaddum and Picarelli, 1957; Gershon, 1967). Thus, the 5-HT contraction of the smooth muscle of the guinea-pig oesophagus results from the release of acetyicholine (Kamikawa and Shimo, 1983), whereas 5-HT contractions of the rat stomach fundus (Clineschmidt et al., 1985; Cohen and Colbert, 1986) and relaxations of the guinea-pig ileum (Feniuk et al., 1983) represent direct actions. Neither the binary classifications offered by Gaddum and Picarelli (1957) and Peroutka and Snyder (1979) nor the current extended classification of 5-HT receptors (Bradley et al., 1986; Richardson and Engel, 1986; Fozard, 1987; Hoyer, 1988; Peroutka, 1988) have provided a satisfactory taxonomy for these and other intestinal or vascular effects of 5-HT. For instance, contraction of the rat stomach fundus is held to be mediated by the 5-HT~-like receptor *Present address: Department of Pharmaceutical Sciences, School of Pharmacy and Allied Health Professions, Creighton University, Omaha, NE 68178, U.S.A. [Tel. (402) 280-2950]. tPresent address: Glaxo Canada Inc., 1025 The Queen's Way, Toronto, Ontario, Canada M8Z 5S6. :~To whom reprint requests should be addressed. §Present address: Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
(Clineschmidt et al., 1985; Buchheit et al., 1986; Peroutka, 1988), or by an unclassified tryptamine receptor (Cohen and Wittenauer, 1985; Cohen and Colbert, 1986; Cohen and Fludzinski, 1987). As regards the inhibitory effects of 5-HT, several 5-HT~ receptor subtypes appear to be involved which generally share a high affinity for 5-carboxamidotryptamine, metitepine and methysergide (Feniuk et al., 1984; Humphrey and Feniuk, 1987; Left et al., 1987). Nevertheless, evidence exists which shows that 5-HT-induced relaxations of sheep (Eyre, 1975) and goat (Chand, 1981) pulmonary veins and the circular muscle of human colon (Burleigh, 1977) are methysergide-insensitive. Likewise, in rat oesophageal smooth muscle, inhibitory responses to 5-HT under conditions of induced tone have also been found to be insensitive to several putative 5-HT~- and 5-HT 2receptor antagonists (Bieger and Triggle, 1985; Akbarali et al., 1986, 1987). In a preliminary study, we reported that 5-HT-induced relaxation of rat and mouse oesophageal smooth muscle was antagonized by some reputedly selective 5-HTrreceptor blockers and that inhibitory 5-HT receptors in the two murid species may share the same pharmacological properties (Triggle et al., 1988). Since then other workers have attempted to classify inhibitory 5-HT receptors in the distal portion of the whole oesophagus (Reeves et al., 1991) and in the lower thoracic segment of the oesophageal tunica muscularis mucosae (Baxter et al., 1991) of the rat. Although there are discrepancies between the two studies, these workers
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have classified the inhibitory 5-HT receptor in the rat oesophagus as belonging to the 5-HT,-receptor subtype based: (1) on the weak antagonism (noncompetitive, Reeves et al., 1991; competitive, Baxter et al., 1991) caused by ICS 205-930, and (2) agonism displayed by benzamides. The nature of the cell messenger system coupling 5-HT receptor activation to cellular metabolism is currently a subject o f intensive investigation. The evidence available suggests that 5-HTtA, 5-HTt, (De Vivo and Maayani, 1986; Bockhaert et al., 1987; Fozard, 1987) and 5-HTID (Hoyer and Schoeffter, 1988) receptor subtypes are negatively linked to adenylate cyclase, whereas 5-HT,-receptors (Dumuis et al., 1989) are positively coupled to the cyclic A M P pathway. 5-HT~c and 5-HT3-receptors are coupled to the activation o f phospholipase C (Conn et al., 1986; Hoyer, 1988) and to a ligand-gated monovalent cation channel (Fozard, 1987; Kilpatrick et al., 1990), respectively. Evidence available shows that the current classification of 5 - H T receptors is incomplete and there is a need to initiate further studies on the nature of 5-HT receptor and its signal transduction pathway in the rat oesophagus. We have, therefore, attempted to classify the inhibitory 5-HT receptor in both proximal and distal segments of the rat oesophageal smooth muscle: (a) by determining the potency ratios and efficacy of a series of tryptamine analogs and other agonists acting on 5-HT receptors, and (b) by examining the effects of a broad range of antagonists. We have also investigated the effect of 5-HT on cyclic nucleotide levels in both proximal and distal portions o f the oesophageal smooth muscle. Pa~ts of this study have been presented in preliminary form (Ohia et al., 1987, 1988; Bieger et al., 1989; Triggle et al., 1989). MATERIALS AND METHODS
Male Sprague-Dawley rats weighing 225-280 g were used in all experiments. Animals were sacrificed by stunning and exsanguination. The isolated oesophageal smooth muscle preparation The oesophageal smooth muscle was isolated from rats as described previously (Bieger and Triggle, 1985). 2cm lengths of proximal (cervical) and distal (infradiaphragmatic) segments of the muscle were set up in organ baths filled with Tyrode solution which was aerated (95% 02, 5% CO2) and maintained at 37~C. The Tyrode solution had the following composition (raM): NaCI, 137; KCI, 2.7; CaCI2, 1.8; MgCI2, 0.88; NaH2PO,, 0.26; NaHCO3, 12.0; and glucose, 5.5 (pH 7.4). Longitudinal isometric tension was recorded via Grass FTO3 force~tisplacement transducers and displayed on a Grass Model 7D polygraph. An initial load of 300 mg was placed on each tissue which was then allowed to equilibrate for I hr. Since the smooth muscle of the rat oesophageal body does not possess intrinsic tone, inhibitory response produced by 5-HT and its analogs, l-(3-trifluoromethylphenyl) piperazine (TFMPP), phenylbiguanide and the tetralin derivative, 8-hydroxy-2(di- n-propylamino)-tetraline (8-OH-DPAT) and other agonists such as isoprenaline, forskolin and BRL 34915 were examined under conditions of tone induced with a submaximal concentration (EC7o-80) of the muscarinic receptor agonist, c~s-2-methyl-4-dimethylamino-methyl-I, 3-dioxolane methiodide (cis-dioxolane, 10-7-3 x 10 -7 M). This concentration of cis-dioxolane elicited a sustained tonic response which lasted for more than 1 hr enabling
cumulative concentration-response curves (CRCs) to be constructed for the 5-HT receptor agonists. The relaxant effect of the agonists was expressed as percentage inhibition of cis-dioxolane-induced tone. In all experiments, controls response to cis-dioxolane and the 5-HT agonists under investigation were obtained before addition of antagonists. Generally, tissues were exposed to antagonists for at least 30 rain before the effect of agonists were re-examined. Cyclic nucleotide assays 2cm lengths of the appropriate segment of the oesophageal smooth muscle were incubated in aerated (95% 02, 5% CO 2) Tyrode solution containing theophylline (I0 -3 M) at 37¢C for I hr. After incubation, tissues were immediately frozen in liquid nitrogen. Tissues were thawed in 1 ml of ice-cold trichloroacetic acid (TCA) and were left in a cold room (2-4°C) for 24 hr. 0.5 ml portions of the acid-soluble extract were then extracted four times with 2 ml of water-saturated diethyl ether. Any remaining ether was evaporated by placing sample tubes in a water bath at 80°C. The pH of the samples was adjusted to 7.0 and both cyclic AMP and cyclic GMP were measured by means of a radioimmunoassay kit (Amersham Corporation). Cyclic nucleotide concentrations were expressed as pmol/g wet weight of tissue. Effect o f 5 - H T on cyclic nucleotide levels Tissues were incubated in Tyrode solution at 37°C for 1 hr as described above. After incubation, tissues were transferred to an aerated Tyrode solution containing concentrations of 5-HT from 10 -j° to 10 -s M for 1 rain, and then rapidly frozen in liquid nitrogen. Cyclic nucleotides were extracted and assayed as reported above. Data analysis Equipotent molar concentration ratios. In experiments on the oesophageal smooth muscle, the concentration of agonists which produced 50% inhibition of the sustained c/sdioxolane tone (i.e. IC~0 was determined in each tissue). Equipotent molar concentration ratios (EMCRs) were calculated according to the expression: (IC50 test agonist)/(ICs0 5-HT). pk a ~,alues. The negative logarithm of the apparent dissociation constant for an antagonist (Pka) was estimated by calculating the mean of the individual results, pk B = log (dose ratio - 1 ) - l o g (antagonist concentration). Statistics. Results are expressed as means + SEM. Student's t-test (two tailed) was used to compare means of unpaired observations. Results were considered to be significant at P ~ 5 - methoxytryptamine > 5 - methoxy - Nmethyltryptamine > 5-carboxamidotryptamine > 5methoxy-N,N-dimethyltryptamine > 8-hydroxy-2(di - n - propylamino) tetralin (8 - OH - DPAT) > tryptamine > 2-methyl°5-hydroxytryptamine > l(3-trifluoromethylphenyl)piperazine (TFMPP) > 5,7dihydroxytryptamine and in the distal segment, 5 - HT > 5 - methoxytryptamine > 5 - methoxy - Nmethyitryptamine > 5-carboxamidotryptamine > 8-
5-HT receptor in rat oesophageal smooth muscle OH-DPAT > 5-methoxy-N,N-dimethyltryptamine= 2- methyl- hydroxytryptamine = T F M P P > tryptamine > 5,7- dihydroxytryptamine. N' - acetyl- 5- hydroxytryptamine and phenylbiguanide neither relaxed ocsophageal smooth muscle nor did they inhibit 5-HT-induced relaxations. Antagonists at 5-HT~ and or 5-HT2 receptors had not effect on the 5-HT-induced relaxation. However, the 5-HT3 receptor antagonist, MDL 7222 and the reputedly selective 5-HT3/5-HT4-receptor antagonist, ICS 205-930 blocked the inhibitory 5-HT response. On the other hand, quipazine, another 5-HT3 antagonist and d-tubocurarine in concentrations up to 10 -S M, were inactive. The phosphodiesterase inhibitor, 3-isobutylmethyl-xanthine (IBMX, 10-6-10-5 M) enhanced oesophageal smooth muscle inhibitory responses to 5-HT, isoprenaline and forskolin, but not that elicited by the potassium channel opener, BRL 34915. 5-HT (10-~°-10-6 M) increased tissue cyclic AMP content over basal levels in proximal segments of ocsophageal smooth muscle. A corresponding increase in tissue cyclic AMP levels was also observed in distal segments at high 5-HT concentrations ( > 10-s M). In general, 5-HT produced greater effects on tissue cyclic AMP levels in the proximal than in the distal segments. However, 5-HT had no significant effect on basal cyclic GMP levels in both segments. We conclude that the inhibitory 5-HT receptor in rat oesophageal smooth muscle may represent a high affinity subtype which is sensitive to 5-HT3/5-HT4 antagonists and is coupled to the cyclic AMP pathway. Acknowledgements--This project was supported by a grantin-aid from the Medical Research Council of Canada (MA 9141). The authors wish to acknowledge the excellent secretarial assistance of Ms Betty Granter (Memorial University of Newfoundland) and Thelma Cornelius (Creighton University).
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April. Bockhaert J., Dumuis A., Bouhelal R., Sceben M. and Cory R. N. (1987) Piperazine derivatives including the putative anxiolytic drugs, buspirone and isapirone, are agonists at 5-HT~, receptors negatively coupled with adenylate
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Ohia S. E., Bieger D. and Triggle C. R. (1987) Pharmacological properties of the 5-hydroxytryptamine receptor that mediates relaxation of rat oesophageal smooth muscle. Abstract of the 30th Canadian Federation o f Biological Societies Meeting, No. 56. Winnipeg, 22-26 June. Ohia S. E., Bieger D. and Triggle C. R. (1988) Is there a non-neuronal 5-HT 3 mediating oesophageal smooth muscle relaxation in the rat? Abstract of the 31st Canadian Federation o f Biological Societies Meeting. No. 167. Quebec City, 15-18 June. Peroutka S. J. (1988) 5-hydroxytryptamine receptor subtypes. A. Re~'. Neurosci. 11, 45-60. Peroutka S. J. and Hamik A. (1988) [3HI Quipazine labels 5-HT 3 recognition sites in rat cortical membranes. Eur. J. Pharmac. 148, 297-299. Peroutka S. J. and Snyder S. H. (1979) Multiple serotonin receptors: Differential binding of [3H] 5-hydroxytryptamine, [3HI lysergic acid diethylamide and [3H] spiroperidol. Molec. Pharmac. 16, 687-699. Reeves J. J., Bunce K. T. and Humphrey P. P. A. (1991) Investigation of the 5-hydroxytryptamine receptor mediating smooth muscle relaxation in the rat oesophagus. Br. J. Pharmac. 103, 1067-1072. Richardson B. P. and Engel G. (1989) The pharmocology and function of 5-HT 3 receptors. Trends Neurosci. 7, 42~428. Richardson B. P., Engel G., Donatsch P. and Stadler P. A. (1986) Identification of serotonin M-receptor subtypes and their specific blockade by a new class of drugs. Nature 336, 126-131. Torphy T. J., Fine C. F., Burman M., Barnette M. S. and Osmsbee H. S. II1 (1986) Lower oesophageal sphincter relaxation is associated with increased cyclic nucleotide content. Am. J. Physiol. 251, G786-G793. Trevethick M. A., Feniuk W. and Humphrey P. P. A. (1984) 5-Hydroxytryptamine-induced relaxation of neonatal porcine vena cava in vitro. Life Sci. 35, 477-486. Trevethick M. A., Feniuk W. and Humphrey P. P. A. (1986) 5-Carboxamidotryptamine: a potent agonist mediating relaxation and elevation of cyclic AMP in the isolated neonatal porcine vena cava. Life Sci. 38, 1521-1528. Triggie C. R., Ohia S. E. and Bieger D. (1987) Pharmacological properties of the 5-hydroxytryptamine receptor which mediates relaxation of rat oesophageal smooth muscle. Abstract of the Satellite Symposium on Serotonin, IUPHAR Congress, 39P. Sydney, Australia, 4-6 September. Triggle C. R., Ohia S. E. and Bieger D. (1988) 5-Hydroxytryptamine-induced relaxation of rat and mouse oesophageal smooth muscle. Pharmacologist 30, A126. Triggle C. R., Ohia S. E. and Bieger D. (1989) A comparison of the properties of the 5-HT receptor that mediates relaxation of the rat oesophageal tunica muscularis mucosae (TMM). Int. Syrup. Serotonin: From Cell Biology to Pharmacology and Therapeutics, p. 117. Florence, Italy, 29 March-I April, Weir S. W. and Weston A. H. (1986) Effects of apamin on responses to BRL 34915, nicorandil and other relaxants in the guinea-pig taenia caeci. Br. J. Pharmac. 88, I 13-120. Wells J. N. and Kramer G. L. (1981) Phosphodiesterase inhibitors as tools in cyclic nucleotide research: a precautionary comment. Molec. cell. Endocr. 23, I-9.
0306-3623/92 $5.00 + 0.00 Copyright ~ 1992Pergamon Press Ltd
Printed in Great Britain. All rights reserved
PHARMACOLOGICAL PROFILE OF THE 5-HYDROXYTRYPTAMINE RECEPTOR THAT MEDIATES RELAXATION OF RAT OESOPHAGEAL SMOOTH MUSCLE S. E. OHIA,*Y.-D. CHEUNG,'["D. BIEGER~.and C. R. TRIGGLE§ Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St John's, Newfoundland, Canada A IB 3V6 (Received 24 December 1991)
Abstract--I. The pharmacological profile of the inhibitory 5-hydroxytryptamine (5-HT) receptor in rat oesophageal smooth muscle has been characterized by means of a series of agonists active at 5-HTj-, 5-HT2-, 5-HT3- and 5-HT4-receptor sites, and a broad range of antagonists. The possible involvement of cyclic nucleotides in the 5-HT response was also examined. 2. Under conditions of tone induced by muscarinic receptor activation, the upper two-thirds (proximal segment) of the oesophageal smooth muscle tunic was more sensitive to the inhibitory effects of 5-HT receptor agonists when compared with the distal region. 3.The inhibitory response to 5-HT was blocked by MDL 72222 (5-HT 3 antagonist) and ICS 205-930 (5-HT3/5-HT4 antagonist) but not by antagonists active at 5-HT~- or 5-HT2-receptors. 4. The phosphodiesterase inhibitor, 3-isobutyl-methyl-xanthine (IBMX) enhanced oesophageal smooth muscle inhibitory response to 5-HT, isoprenaline and forskolin, but not that elicited by the potassium channel opener, BRL 34915. 5. 5-HT increased tissue cyclic AMP content over basal levels in proximal and distal segments of oesophageal smooth muscle. However, 5-HT had no significant effect on basal cyclic GMP levels in both segments. 6. We conclude that the inhibitory 5-HT receptor in rat oesophageal smooth muscle may represent a high affinity subtype which is sensitive to 5-HT3/5-HT( antagonists and is coupled to the cyclic AMP pathway.
INTRODUCTION
It is well established that 5-hydroxytryptamine (5HT) has both direct and indirect actions on smooth muscle of the alimentary tract (Gaddum and Picarelli, 1957; Gershon, 1967). Thus, the 5-HT contraction of the smooth muscle of the guinea-pig oesophagus results from the release of acetyicholine (Kamikawa and Shimo, 1983), whereas 5-HT contractions of the rat stomach fundus (Clineschmidt et al., 1985; Cohen and Colbert, 1986) and relaxations of the guinea-pig ileum (Feniuk et al., 1983) represent direct actions. Neither the binary classifications offered by Gaddum and Picarelli (1957) and Peroutka and Snyder (1979) nor the current extended classification of 5-HT receptors (Bradley et al., 1986; Richardson and Engel, 1986; Fozard, 1987; Hoyer, 1988; Peroutka, 1988) have provided a satisfactory taxonomy for these and other intestinal or vascular effects of 5-HT. For instance, contraction of the rat stomach fundus is held to be mediated by the 5-HT~-like receptor *Present address: Department of Pharmaceutical Sciences, School of Pharmacy and Allied Health Professions, Creighton University, Omaha, NE 68178, U.S.A. [Tel. (402) 280-2950]. tPresent address: Glaxo Canada Inc., 1025 The Queen's Way, Toronto, Ontario, Canada M8Z 5S6. :~To whom reprint requests should be addressed. §Present address: Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
(Clineschmidt et al., 1985; Buchheit et al., 1986; Peroutka, 1988), or by an unclassified tryptamine receptor (Cohen and Wittenauer, 1985; Cohen and Colbert, 1986; Cohen and Fludzinski, 1987). As regards the inhibitory effects of 5-HT, several 5-HT~ receptor subtypes appear to be involved which generally share a high affinity for 5-carboxamidotryptamine, metitepine and methysergide (Feniuk et al., 1984; Humphrey and Feniuk, 1987; Left et al., 1987). Nevertheless, evidence exists which shows that 5-HT-induced relaxations of sheep (Eyre, 1975) and goat (Chand, 1981) pulmonary veins and the circular muscle of human colon (Burleigh, 1977) are methysergide-insensitive. Likewise, in rat oesophageal smooth muscle, inhibitory responses to 5-HT under conditions of induced tone have also been found to be insensitive to several putative 5-HT~- and 5-HT 2receptor antagonists (Bieger and Triggle, 1985; Akbarali et al., 1986, 1987). In a preliminary study, we reported that 5-HT-induced relaxation of rat and mouse oesophageal smooth muscle was antagonized by some reputedly selective 5-HTrreceptor blockers and that inhibitory 5-HT receptors in the two murid species may share the same pharmacological properties (Triggle et al., 1988). Since then other workers have attempted to classify inhibitory 5-HT receptors in the distal portion of the whole oesophagus (Reeves et al., 1991) and in the lower thoracic segment of the oesophageal tunica muscularis mucosae (Baxter et al., 1991) of the rat. Although there are discrepancies between the two studies, these workers
649
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have classified the inhibitory 5-HT receptor in the rat oesophagus as belonging to the 5-HT,-receptor subtype based: (1) on the weak antagonism (noncompetitive, Reeves et al., 1991; competitive, Baxter et al., 1991) caused by ICS 205-930, and (2) agonism displayed by benzamides. The nature of the cell messenger system coupling 5-HT receptor activation to cellular metabolism is currently a subject o f intensive investigation. The evidence available suggests that 5-HTtA, 5-HTt, (De Vivo and Maayani, 1986; Bockhaert et al., 1987; Fozard, 1987) and 5-HTID (Hoyer and Schoeffter, 1988) receptor subtypes are negatively linked to adenylate cyclase, whereas 5-HT,-receptors (Dumuis et al., 1989) are positively coupled to the cyclic A M P pathway. 5-HT~c and 5-HT3-receptors are coupled to the activation o f phospholipase C (Conn et al., 1986; Hoyer, 1988) and to a ligand-gated monovalent cation channel (Fozard, 1987; Kilpatrick et al., 1990), respectively. Evidence available shows that the current classification of 5 - H T receptors is incomplete and there is a need to initiate further studies on the nature of 5-HT receptor and its signal transduction pathway in the rat oesophagus. We have, therefore, attempted to classify the inhibitory 5-HT receptor in both proximal and distal segments of the rat oesophageal smooth muscle: (a) by determining the potency ratios and efficacy of a series of tryptamine analogs and other agonists acting on 5-HT receptors, and (b) by examining the effects of a broad range of antagonists. We have also investigated the effect of 5-HT on cyclic nucleotide levels in both proximal and distal portions o f the oesophageal smooth muscle. Pa~ts of this study have been presented in preliminary form (Ohia et al., 1987, 1988; Bieger et al., 1989; Triggle et al., 1989). MATERIALS AND METHODS
Male Sprague-Dawley rats weighing 225-280 g were used in all experiments. Animals were sacrificed by stunning and exsanguination. The isolated oesophageal smooth muscle preparation The oesophageal smooth muscle was isolated from rats as described previously (Bieger and Triggle, 1985). 2cm lengths of proximal (cervical) and distal (infradiaphragmatic) segments of the muscle were set up in organ baths filled with Tyrode solution which was aerated (95% 02, 5% CO2) and maintained at 37~C. The Tyrode solution had the following composition (raM): NaCI, 137; KCI, 2.7; CaCI2, 1.8; MgCI2, 0.88; NaH2PO,, 0.26; NaHCO3, 12.0; and glucose, 5.5 (pH 7.4). Longitudinal isometric tension was recorded via Grass FTO3 force~tisplacement transducers and displayed on a Grass Model 7D polygraph. An initial load of 300 mg was placed on each tissue which was then allowed to equilibrate for I hr. Since the smooth muscle of the rat oesophageal body does not possess intrinsic tone, inhibitory response produced by 5-HT and its analogs, l-(3-trifluoromethylphenyl) piperazine (TFMPP), phenylbiguanide and the tetralin derivative, 8-hydroxy-2(di- n-propylamino)-tetraline (8-OH-DPAT) and other agonists such as isoprenaline, forskolin and BRL 34915 were examined under conditions of tone induced with a submaximal concentration (EC7o-80) of the muscarinic receptor agonist, c~s-2-methyl-4-dimethylamino-methyl-I, 3-dioxolane methiodide (cis-dioxolane, 10-7-3 x 10 -7 M). This concentration of cis-dioxolane elicited a sustained tonic response which lasted for more than 1 hr enabling
cumulative concentration-response curves (CRCs) to be constructed for the 5-HT receptor agonists. The relaxant effect of the agonists was expressed as percentage inhibition of cis-dioxolane-induced tone. In all experiments, controls response to cis-dioxolane and the 5-HT agonists under investigation were obtained before addition of antagonists. Generally, tissues were exposed to antagonists for at least 30 rain before the effect of agonists were re-examined. Cyclic nucleotide assays 2cm lengths of the appropriate segment of the oesophageal smooth muscle were incubated in aerated (95% 02, 5% CO 2) Tyrode solution containing theophylline (I0 -3 M) at 37¢C for I hr. After incubation, tissues were immediately frozen in liquid nitrogen. Tissues were thawed in 1 ml of ice-cold trichloroacetic acid (TCA) and were left in a cold room (2-4°C) for 24 hr. 0.5 ml portions of the acid-soluble extract were then extracted four times with 2 ml of water-saturated diethyl ether. Any remaining ether was evaporated by placing sample tubes in a water bath at 80°C. The pH of the samples was adjusted to 7.0 and both cyclic AMP and cyclic GMP were measured by means of a radioimmunoassay kit (Amersham Corporation). Cyclic nucleotide concentrations were expressed as pmol/g wet weight of tissue. Effect o f 5 - H T on cyclic nucleotide levels Tissues were incubated in Tyrode solution at 37°C for 1 hr as described above. After incubation, tissues were transferred to an aerated Tyrode solution containing concentrations of 5-HT from 10 -j° to 10 -s M for 1 rain, and then rapidly frozen in liquid nitrogen. Cyclic nucleotides were extracted and assayed as reported above. Data analysis Equipotent molar concentration ratios. In experiments on the oesophageal smooth muscle, the concentration of agonists which produced 50% inhibition of the sustained c/sdioxolane tone (i.e. IC~0 was determined in each tissue). Equipotent molar concentration ratios (EMCRs) were calculated according to the expression: (IC50 test agonist)/(ICs0 5-HT). pk a ~,alues. The negative logarithm of the apparent dissociation constant for an antagonist (Pka) was estimated by calculating the mean of the individual results, pk B = log (dose ratio - 1 ) - l o g (antagonist concentration). Statistics. Results are expressed as means + SEM. Student's t-test (two tailed) was used to compare means of unpaired observations. Results were considered to be significant at P ~ 5 - methoxytryptamine > 5 - methoxy - Nmethyltryptamine > 5-carboxamidotryptamine > 5methoxy-N,N-dimethyltryptamine > 8-hydroxy-2(di - n - propylamino) tetralin (8 - OH - DPAT) > tryptamine > 2-methyl°5-hydroxytryptamine > l(3-trifluoromethylphenyl)piperazine (TFMPP) > 5,7dihydroxytryptamine and in the distal segment, 5 - HT > 5 - methoxytryptamine > 5 - methoxy - Nmethyitryptamine > 5-carboxamidotryptamine > 8-
5-HT receptor in rat oesophageal smooth muscle OH-DPAT > 5-methoxy-N,N-dimethyltryptamine= 2- methyl- hydroxytryptamine = T F M P P > tryptamine > 5,7- dihydroxytryptamine. N' - acetyl- 5- hydroxytryptamine and phenylbiguanide neither relaxed ocsophageal smooth muscle nor did they inhibit 5-HT-induced relaxations. Antagonists at 5-HT~ and or 5-HT2 receptors had not effect on the 5-HT-induced relaxation. However, the 5-HT3 receptor antagonist, MDL 7222 and the reputedly selective 5-HT3/5-HT4-receptor antagonist, ICS 205-930 blocked the inhibitory 5-HT response. On the other hand, quipazine, another 5-HT3 antagonist and d-tubocurarine in concentrations up to 10 -S M, were inactive. The phosphodiesterase inhibitor, 3-isobutylmethyl-xanthine (IBMX, 10-6-10-5 M) enhanced oesophageal smooth muscle inhibitory responses to 5-HT, isoprenaline and forskolin, but not that elicited by the potassium channel opener, BRL 34915. 5-HT (10-~°-10-6 M) increased tissue cyclic AMP content over basal levels in proximal segments of ocsophageal smooth muscle. A corresponding increase in tissue cyclic AMP levels was also observed in distal segments at high 5-HT concentrations ( > 10-s M). In general, 5-HT produced greater effects on tissue cyclic AMP levels in the proximal than in the distal segments. However, 5-HT had no significant effect on basal cyclic GMP levels in both segments. We conclude that the inhibitory 5-HT receptor in rat oesophageal smooth muscle may represent a high affinity subtype which is sensitive to 5-HT3/5-HT4 antagonists and is coupled to the cyclic AMP pathway. Acknowledgements--This project was supported by a grantin-aid from the Medical Research Council of Canada (MA 9141). The authors wish to acknowledge the excellent secretarial assistance of Ms Betty Granter (Memorial University of Newfoundland) and Thelma Cornelius (Creighton University).
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