Pharmacological characterization of the 5-hydroxytryptamine

Transcription

1 British Journal of Pharmaology (1996) 119, Stokton Press ll rights reserved /96 $12. M Pharmaologial haraterization of the 5-hydroxytryptamine reeptor mediating relaxation in the rat isolated ileum 1B.R. Tuladhar, B. Costall & R.J. Naylor Postgraduate Studies in Pharmaology, The Shool of Pharmay, University of Bradford, Bradford BD7 ldp 1 The aim of the present study was to investigate a 5-HT4 reeptor involvement in the mediation of a 5- HT-indued relaxation response in the rat isolated ileum in vitro. 2 Ileal segments were taken at regular intervals from the ileo-aeal juntion to duodenum. 5-HT (1,Mm) indued a relaxation or ontration response in segments taken from the terminal ileum: the relaxation dereased and finally disappeared as ontrations dominated in the proximal tissues. The 5- HT-indued relaxations were enhaned in the terminal segments and the ontrations attenuated in both terminal and proximal segments, in the presene of methysergide (1 gim) and atropine (.1 um). 3 In the presene of methysergide (1 pm) and atropine (.1 Mm), a umulative addition of 5-HT (.1-1 gm) indued a onentration-dependent relaxation in the terminal (1-2 m from the ileo-eaal juntion) ileal segments whih at higher onentrations of 5-HT (3-3 gm) reverted to ontration. 4 The rank order of poteny of indole agonists in induing a onentration-related relaxation response in tissues of the terminal ileum (pretreated with pargyline (1 gm) and in the presene of methysergide (1 or 1 gm) and atropine (.1 gm)) was 5-hydroxytryptamine ( ), 5-methoxytryptamine ( ), a-methyl-5-hydroxytryptamine ( ), 5-arboxamidotryptamine ( ) and 2- methyl-5-hydroxytryptamine (<5), the pc, values (mean+s.e.mean) being shown in parentheses. 5 Pretreatment of tissues with pargyline (1 ym) seletively enhaned the poteny of 5-methoxytryptamine by a fator of 19 but failed to modify the poteny of the other indole agonists. 6 The 5-HT4 reeptor antagonists, tropisetron, SDZ and GR antagonized the relaxation response to 5-HT (in the presene of methysergide (1 or 1 Mm) and atropine (.1 gm)) with pkb values (95% CL) of 6.9 ( ), 7. ( ) and 8.95 ( ) respetively. pparent pkb values estimations for tropisetron (1 gm) and GR (1 nm) using the agonists 5- methoxytryptamine and 5-arboxamidotryptamine were , and , respetively. 7 Tropisetron (1 Mm), SDZ (3 MM) and GR (1-1 nm) aused an inrease in basal tone of the rat terminal ileum when administered in the presene of methysergide and atropine. 8 The relaxation response to 5-HT in the rat terminal ileum was not antagonized by ritanserin (1 gm), ondansetron (1 um) or Nw-nitro-L-arginine methyl ester (1 gm) and with only a twofold dextral shift of the onentration-response urve by tetrodotoxin (1 gm). 9 It is onluded that the relaxant response to 5-HT in the terminal region of the ileum is mediated diretly at the smooth musle; a ranked indole agonist poteny and seletive antagonism by 5-HT4 reeptor antagonists tropisetron, SDZ and GR indiate a 5-HT4 reeptor involvement in the relaxation response. Keywords: 5-Hydroxytryptamine; 5-HT4 reeptor; rat ileum; relaxation; tropisetron; SDZ ; GR Introdution The 5-HT4 reeptor belongs to the seven transmembrane domain G protein oupled reeptor superfamily, ativation of whih results in the stimulation of adenylyl ylase and elevation of adenosine 3': 5'-yi monophosphate (ylimp) (Hoyer et al., 1994). In the gut, ativation of the 5-HT4 reeptor in in vitro experiments auses ontration of guinea-pig ileum (Craig & Clarke, 199) and olon (lswood et al., 1991) by the release of aetylholine from the enteri neurones. 5-HT4 reeptor stimulation also inreases peristalti reflex sensitivity in isolated intat preparations taken from the guinea-pig ileum (Craig & Clarke, 1991; Buhheit & Buhl, 1991; Costall et al., 1993) and marmoset (Tuladhar et al., 1996) and the failitation of the peristalti reflex may explain the prokineti effet of benzamide derivatives whih are agonists at the 5-HT4 reeptor (Dumuis et al., 1989). Furthermore, 5-HT4 reeptor stimulation has been shown to inrease gastri emptying in rats (Hedge et al., 1995) and to be involved in the 5-hydroxytryptophan-in- 1 uthor for orrespondene. dued defaeation and diarrhoea in mie (Banner et al., 1993; Hedge et al., 1994). In addition to enhaning gut motility, an inrease in the seretion from the rat and human gastrointestinal muosa (Bune et al., 1991; Borman & Burleigh, 1993; Burleigh & Borman, 1993) has also been shown to be mediated by the 5-HT4 reeptor. However, a physiologial role of the 5-HT4 reeptor in gut motility has remained unertain sine 5-HT4 reeptor antagonists suh as SB 247 when administered alone fail to redue faeal pellet output (Banner et al., 1993) and the intestinal transit of radioative mirospheres administered into the duodenum of the onsious rat is not altered by the administration of either 5-HT4 reeptor agonists or antagonists (Clayton & Gale, 1996). In addition to enhaning gut motility, stimulation of the 5- HT4 reeptor loated on smooth musle auses a relaxation of the rat oesophagus and terminal ileum (Reeves et al., 1991; Baxter et al., 1991; Tuladhar et al., 1991), human intestinal smooth musle (Kuemmerle et al., 1995) and a derease of spontaneous ativity of the human olon (Tam et al., 1993). relaxation potential mediated via a 5-HT4 reeptor ould potentially attenuate a ontratile effet. The aim of the present

2 34 B.R. Tuladhar et a! study was to investigate the involvement of the 5-HT4 reeptor in the mediation of the relaxation response in the rat ileum. preliminary aount of some of these findings has been presented at meetings of The British Pharmaologial Soiety, July 1991 (Tuladhar et al., 1991) and Deember 1991 (Tuladhar et al., 1992). Methods nimals and housing onditons Female Hooded Lister rats (Bradford strain) weighing 2-3 g were housed in groups of 5-6 in a age (56 m x 38 m x 18 m) and allowed food and water ad libitum. The animals were maintained in a onstant environment of 18-19'C on a 14 h/i h light dark yle. Preparation of tissue nimals were stunned by a blow to the head and killed by ervial disloation. The abdomen was opened, a length of small intestine from a designated region was removed and transferred to a 5 ml organ bath ontaining Krebs-Henseleit solution (omposition: NaCl 118, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, NaHCO3 25., gluose 1. mm) kept at 37C and oxygenated with 95% 2 and 5% CO2. The ileum was removed from the bath for a minimum time neessary during further preparation of the tissue and kept oxygenated and warm at all times. The mesentery was ut away, segments of the intestine (2-3 m in length) were removed and the intraluminal ontent was flushed out using a 5 ml syringe filled with Krebs-Henseleit solution. The tissues were then mounted in 1 ml organ baths ontaining Krebs-Henseleit solution kept at 37C and oxygenated with 95% 2 and 5% CO2. Changes in tissue tension were measured isometrially using Grass Fore Displaement transduers (FT3C, Grass Instrument Co., Mass., U.S..) and reorded on a multihannel Grass reorder. The tissues were washed 4-6 times and equilibrated further for at least 45 min with washout every 15 min. Tension was then slowly applied to the tissues to a maximum of 1 g: the tension was inreased or dereased as neessary until a stable baseline tension of.6-1 g was obtained. This proedure almost always produed quiesent tissues with little (less than 1 mg in amplitude) or no spontaneous ativity. If higher spontaneous ativity was present then the tension was removed, the tissues washed 2-3 times and the tension slowly reapplied after 1-15 min to attain a slightly lower value. Tissues from any animal still exhibiting high spontaneous ativity were disarded. Constrution of onentration-effet urves Non umulative onentration-ontration/relaxation response urves were established by inreasing the onentration of 5-HT added to the organ baths at intervals of 1 min. ah onentration was left in ontat for 1 min. Cumulative onentration-relaxation response urves to 5-HT and other agonists were obtained by addition of the agonists with a 2 min ontat time. In the experiments examining the variation in the relaxation response to 5-HT in the different regions of the small intestine, segments from different regions were hallenged with a single onentration of 1,UM 5-HT. xperimental protool for studying the effets of S-HT reeptor agonists and antagonists in the terminal ileum (1-2 m from the ileo-eaal juntion) Conentration-response urves were onstruted by the umulative addition of 5-HT or other agonists. The buffer routinely ontained atropine (.1 gm) and methysergide (1 gm) exept in the experiments involving GR when the methysergide onentration in the bathing medium was 1 gm 5-HT4 reeptor-mediated relaxation in rat ileum and in experiments involving a-methyl-5-ht when the bathing medium ontained 1 gm methysergide. In experiments designed to determine the rank order of agonist poteny, tissues were treated for 3 min with pargyline (1 pm) followed by washout before the appliation of tension. The antagonist drugs were added either to the reservoir itself or added before the appliation of tension and equilibrated for at least 3 min before onstrution of the onentration-response urves to the test agonist. When the effets of the antagonists were being determined on the baseline tension, the antagonists were added after a stable baseline tension was obtained ( g). The omparisons were made using paired preparations and in most experiments with dupliate tissues from the same animal in eah group. The relaxation response was expressed as the perentage of the maximum response in the response urve giving the greatest response. nalysis of results The ability of the agonists to relax the ileum was expressed both in absolute terms, as pc5 values (relative to individual maxima) and in terms of their relative poteny versus 5-HT. Poteny relative to 5-HT was obtained in the experiments in whih two onentration-effet urves were obtained from the tissues of the same animal and expressed as an C5 ratio. pc5 values were determined graphially. ll results are expressed as arithmeti mean together with s.e.mean or 95% onfidene limits exept the C5 ratios whih are expressed as the geometri mean. The antagonist dissoiation onstants were determined in two ways. Firstly, in the experiments employing multiple onentrations of the antagonists, p2 values were alulated by the method of runlakshana & Shild (1959) at 5% response. The slope of the resulting straight line was determined by linear regression. If the slope was not signifiantly different from 1 the pkb value was estimated restriting the slope to 1. Seondly in the experiments where a single onentration of antagonist was used, an apparent pkb value was alulated using the formula: apparent pkb = loglo (onentration - ratio -1) -loglo(antagonist onentration) assuming a ompetitive interation. The signifiane of differenes between the values was determined at P<.5 using Student's unpaired two-tailed t test. Drugs 5-Hydroxytryptamine maleate, 5-methoxytryptamine hydrohloride, Nw-nitro-L-arginine methyl ester hydrohloride, tetrodotoxin, pargyline hydrohloride, atropine sulphate (Sigma), ondansetron hydrohloride dihydrate, GR ([1-[2-methylsulphonyl)amino] ethyl]-4-piperidinyl]methyl-1- methyl-lh-indole-3-arboxylate) maleate (Glaxo), SDZ (2-methoxy-4-amino-5-hloro-benzoi aid 2-(diethylamino)ethyl ester) and tropisetron hydrohloride (Sandoz), 2- methyl-5-hydroxytryptamine maleate, x-methyl-5-hydroxytryptamine maleate and 5-arboxamidotryptamine maleate (RBI) were dissolved in distilled water and diluted in Krebs- Henseleit solution. Methysergide hydrogen maleate (Sandoz) was dissolved by soniation. The drugs were added in a volume not exeeding 1 p1 to a bath volume of 1 ml. Results ffet of a non-umulative addition of 5-HT In the initial studies using tissues taken from the terminal ileum (taken 1-2 m from the ileo-eaal juntion), a nonumulative addition of 5-HT produed small relaxations up to

3 B.R. Tuladhar et a! 5-HT4 reeptor-mediated relaxation in rat ileum 35C a onentration of.3 pm of 5-HT. ddition of higher onentrations of 5-HT (1-1 gm) produed only ontrations whih were aompanied by an inrease in the spontaneous ativity (Figure la). The addition of methysergide (1 pm) and atropine (.1 M) in the Krebs-Henseleit solution enhaned the relaxation and attenuated the ontratile response. The maximum relaxation to 5-HT ( mg, n = 5) was obtained at 1 gm in the presene of methysergide and atropine (Figure lb). The pc5 ± s.e.mean value for the relaxation response to 5- HT was t higher onentrations of 5-HT (1-3 gm), there was a redution in the relaxation response and the development of ontration. the relaxation response to 5-HT (I gum) in Variations in different regions of the rat small intestine In the ileal segments taken from the terminal portion of the intestine (<2 m from ileo-aeal juntion), tissues taken from two out of four animals tested showed lear relaxations to 5-HT (1 pm). The terminal ileal segments from the remaining two animals exhibited ontrations of small magnitude with the tissues from one of these animals failing to show any relaxation. ll the segments of ileum in all animals derived from the portion proximal to 2 m distane from the ileo-aeal juntion produed only ontrations (Figure 2a). The relaxation responses to 5-HT (1 gm) in the segments obtained from the terminal part of the ileum were enhaned and onsistent in the presene of methysergide (1 gm) and atropine (.1 pm), with no ontrations observed in any tissue obtained from the terminal part of the ileum (Figure 2b). In the presene of methysergide and atropine, the ontratile responses were also greatly redued in tissues from the proximal parts of the ileum. ffet of a umulative addition of 5-HT The umulative addition of 5-HT allowed a muh more rapid study of 5-HT onentration-relaxation response urves than using non-umulative additions. In tissues taken from the terminal ileum, onsistent and onentration-dependent relaxations were observed following the umulative addition of 5-HT (.1-1 UM) in the presene of methysergide (1 gm) and atropine (.1 M) but not in their absene. With a umulative addition of higher onentrations of 5-HT (3-3 gm) no further relaxations were observed and indeed dereased and even reverted to ontration in some tissues. The 5-HT-indued relaxation had no effet on the spontaneous ativity (if present) of the tissue. The ontration was usually aompanied by a high degree of spontaneous ativity whih persisted in most tissue even after washing out of 5-HT. This made the onstrution of a seond onentration-response urve on the same tissue diffiult and hene subsequent omparisons were made with paired preparations. typial relaxation response to 5-HT and the onentration-relaxation response urves of four tissues from the terminal ileum is shown in Figure 3. Suh tissues were taken: (a) nearest to the ileo-aeal juntion and (b) adjaent to the first, () seond and (d) third tissues and their response was indistinguishable. Hene suh tissues were subsequently used to assess and ompare agonist and antagonist potenies. n inrease in the methysergide onentration to 1 or 1 gm or atropine to 1 pm had no effet on the onentration-relaxation response urves to 5-HT, data not shown. The response of the rat terminal ileum to 5-HT reeptor agonists In the tissues pretreated with pargyline, umulative onentration-relaxation response urves to a range of indole agonists gave a rank order of agonist poteny 5-HT > 5- methoxytryptamine> a-methyl-5-ht > 5-arboxamidotryptamine > 2-methyl-5-HT (Figure 4, Table 1). The pretreatment of the tissues with pargyline was essential as it was shown to shift the response urve of 5-methoxytryptamine seletively to the right by approximately 19 fold (pc5 values with and without pretreatment with pargyline and respetively, n = 6). The pc5 values obtained in the absene of pargyline (5-HT ( , n = 5), 5-arboxamidotryptamine ( , n=9), a-methyl-5-ht (5.59±.8, n=6) and 2- methyl-5-ht (< 5, n = 5)) were not hanged signifiantly by the pretreatment of the tissues with pargyline but the pargyline treatment dereased the maximum response to 5-HT by 32% (data not shown). The onstrution of a relaxation-response a W h "4% L w ]FS is 25 mg b 1 3 ~[11m 1mm gim 5-HT jim 5-HT ) ) ) CD._ log [5-HTJ M log [5-HT] M Figure 1 Representative traes and onentration-ontration () and-relaxation () response urves to 5-HT in the rat terminal ileum (taken 1-2 m from the ileo-aeal juntion) to the non-umulative addition of 5-HT at 1 min intervals (a) in absene of any antagonists and (b) in the presene of methysergide (1 gm) and atropine. (.1 um). Values shown in the onentration-response urves are the means with s.e.mean in tissues taken from 3-5 animals. rrows in the traing indiate the addition of 5-HT.

4 36 B.R. Tuladhar et al urve to a high onentration of a-methyl-5-ht required the use of 1 gm methysergide. This onentration of methysergide had no effet on the onentration-relaxation response indued by 5-HT but bloked a slow loss of tone previously seen at or above 1 JIM a-methyl-5-ht when used in the presene of 1 gm methysergide. The latter effet was always aompanied by abolition of spontaneous ativity (if present) in the tissue and eventually aused a omplete loss of the tissue tone. In some tissues there was an apparent biphasi relaxation response to 5-arboxamidotryptamine. However, the initial relaxation (at onentrations below.3 gm) observed in these tissues was very small and diffiult to reprodue in all experiments. The response urve to 5-arboxamidotryptamine at onentrations higher than.3 JM (1-1 gm) was, however, essentially parallel to that of 5-HT. The effet of tropisetron, SDZ and GR on the relaxation response indued by 5-HT reeptor agonists Tropisetron (1-1 ym), SDZ (.3-3 JM) and GR (.1-.1 gm) aused parallel dextral shifts of the onentration-response urve to 5-HT (Figure 5). lower a 1-8-._en 6- UDO r -2 - C C m C1 C) -4 - b :. r-j. I I I ' I I' I I I' I 'I I ' I I ' I I ' Duodonum Distane from ileo-aeal juntion (m) 25 mg L 1>_min.4u,_,WO 416_1 I & 2m 4m 6m 2m 22m 4m 42m Duodenum Duodenum Distane from ileo-aeal juntion (m) Figure 2 The ontratile (open olumns) and relaxant (stippled olumns) response of different regions of the rat ileum and duodenum to 5-HT (1 gm) in (a) absene of any antagonist and (b) in the presene of methysergide (1 gm) and atropine (.1 gm) in the bathing medium. Values shown in the graphs are mean with s.e.mean in tissues taken from 4-6 animals. n example traing of the responses to 5-HT in the presene of methysergide and atropine is also presented. rrows in the traing indiate the addition of 5-HT (1 gm). 5-HT4 reeptor-mediated relaxation in rat ileum onentration of tropisetron (.1 Mm) had no effet, data not shown. Shild analysis revealed slopes not signifiantly different from unity. The p2 values taken as the interept on the x- axis on the Shild plot, slopes and the pkb values estimated onstraining the slope to unity are presented in Table 2. Both tropisetron (1 gm) and GR (.1 gm) aslo aused an essentially parallel dextral shift of the onentrationresponse urves to 5-methoxytryptamine and 5-arboxamidotryptamine. The apparent pkb estimates are presented in Table 3. ) Co C( Co ) x o a [5mg min 3 gm 5-HT b j log [5-HTJ M Figure 3 (a) n example trae of the hanges in baseline tension on umulative addition of 5-HT at 2 min intervals in the rat terminal ileum in the presene of methysergide (1 gm) and atropine (.1 gm). rrows indiate the addition of 5-HT. (b) Comparison of the relaxation-response urves to 5-HT in four terminal tissues: () nearest to the ileo-eaal juntion; (@) adjaent to the first tissue; (l) adjaent to the seond tissue and () adjaent to the third tissue. Values shown are the means with s.e.mean in tissues taken from 6 animals. Results are presented as a perentage of the maximum response in the tissue giving the largest relaxation O- x m Co -6_ - o log [gonist] M I Figure 4 Conentration-relaxation response urves to 5-hydroxytryptamine and other indole 5-HT reeptor agonists in the rat terminal ileum pretreated with pargyline (1 gm for 3 min). (a) Relaxation response urves to 5-HT (O n = 15), 5-methoxytryptamine (, n = 5), 5-arboxamidotryptamine (l, n = 5), 2-methyl-5-HT (-, n = 3) and a-methyl-5-ht (, n = 5). Buffer routinely ontained atropine (.1 Mm) and methysergide (1 gm) exept in the experiment with a-methyl-5-ht when it ontained atropine (.1 Mm) and methysergide (1 gm). Values shown are the means with s.e.mean from a number of animals indiated by n.

5 B.R. Tuladhar et al The effet of tropisetron, SDZ and GR on quiesent tissues The three 5-HT4 reeptor antagonists, tropisetron (1 gum), SDZ (3 gm) and GR (.1-.1 ym) when administered alone inreased the tone of the quiesent tissues (by approximately 1 to 2 mg, n = 6, data not shown). With all of these antagonists, there was a time lag of about 3 min before the inrease in the tone beame evident. GR a 5-HT4 reeptor-mediated relaxation in rat ileum (.3 gm) ould also reverse the relaxation due to 5-HT (.3 gm). The stable baseline obtained after the reversal of the relaxant effet of 5-HT by GR was also above ( mg, n = 2) the initial baseline tension. The effet of ondansetron, ritanserin, tetrodotoxin and Nw-nitro-L-arginine methyl ester (L-NM) on the relaxation response to 5-HT 37 The inlusion of ondansetron (1 gm), tetrodotoxin (.1 Mm), ritanserin (.1 Mm) or L-NM (3 or 1 UM) in the bathing medium had no effet on the onentration-relaxation response urve to 5-HT. The fade of the relaxation at higher b X -2- x xco 4- I I I I Table 1 Poteny of the 5-HT reeptor agonists in ausing a relaxation response in the rat terminal ileum pretreated with 1 gm pargyline for 3 min followed by washout pc5 gonists (+ s.e.mean) n 5-Hydroxytryptamine Methoxytryptamine a-methyl-5-ht* * 5 5-Carboxamido tryptamine 2-Methyl-5-HT <5 3 C5 ratio,,,,, I11** 3 17 The bathing medium routinely ontained methysergide (1 ym) and atropine (.1 gm). pc5o values shown are the olletive data from the maximum number of animals used where the C5 ratio and ma,,a values for agonists are alulated by omparison of the response with the effet of 5-HT in tissues from the same animal. *In the presene of 1 UM methysergide; **the response to 3uM x-methyl-5-ht, the highest onentration employed. n R I I ' Table 2 Poteny of the 5-HT4 reeptor antagonists in antagonizing the 5-HT indued relaxation in the rat terminal ileum ntagonists p2* Slope (95% CL) pkb**(95% CL) Tropisetron SDZ GR ( ) 6.9( ) ( ) 7. ( ) ( ) 8.95( ) The buffer routinely ontained atropine (.1 Mm) along with either 1 gum (in experiments using tropisetron or SDZ ) or 1 gm methysergide (in the experiments using GR 11388). *Slope not restrited to 1; **Calulated by restriting the slope to unity I ll log [5-HT1 M Figure 5 ntagonism of the relaxation response to 5-HT in the rat terminal ileum by tropisetron, SDZ and GR Conentration-relaxation response urves to 5-HT (a) in the absene (-, n= 18) and presene of 1 (, n=7), 3 (-, n=6) and 1 gm (, n = 6) tropisetron; (b) in the absene (-, n = 18) and presene of.3 (Dl, n=6), 1 (a, n=6) and 3 gm (, n=6) SDZ ; () in the absene (, n=18) and presene of.1 (, n=6),.3 (M, n=6) and.1 gm (O, n=6) GR Buffer routinely ontained atropine (.1 Mm) and methysergide (1 gm) exept in the experiment with GR when it ontained atropine (.1 gm) and methysergide (1 gm). Values shown are the means with s.e.mean from a number of animals indiated by n. Table 3 pparent pkb estimations with tropisetron and GRI1388 using a single onentration of the antagonists gonists 5-Methoxytryptamine 5-Carboxamido tryptamine 5-Methoxytryptamine 5-Carboxamido tryptamine pparent ntagonists pkb (onentration) (±s.e.mean) Tropisetron (1 PM) Tropisetron (1 MM) GR (1 nm) GR (1 nm) n The buffer routinely ontained atropine (.1 Mm) along with either 1 Mm (in experiments using tropisetron) or 1 ym methysergide (in the experiments using GR 11388).

6 38 Table 4 B.R. Tuladhar et al The poteny of 5-HT in ausing relaxant response in the rat terminal ileum in the presene of 5-HT reeptor antagonists, tetrodotoxin and L-NM Relaxant responses to 5-HT (pc5±s.e.mean) Conentration Without With ntagonists tested antagonist antagonist Ondansetron Ritanserin Tetrodotoxin Tetrodotoxin L-NM L-NM I gm.1 gm.1 gm 1 /M 3 M 1 PM * ** Methysergide (1 gm) and atropine (.1 Mm) were routinely inluded in the bathing medium unless otherwise mentioned. *In the absene of methysergide and atropine in the bathing medium; **differene statistially signifiant: Students t test, unpaired two tail. onentrations of 5-HT was also not affeted by the presene of the above antagonists. n approximate two fold shift of the onentration-response urve to 5-HT was, however, seen in the presene of 1 gm tetrodotoxin. The pc5 values for 5-HT in the presene and absene of the above antagonists are presented in Table 4. Disussion The dominant effet of submiromolar onentrations of 5-HT in the rat terminal ileum (defined as tissues taken 1-2 m from the ileo-aeal juntion) was a relaxation response but onentrations higher than 1 M indued ontration. Regionally a onentration of 1 gm 5-HT indued variable ontration or relaxation when tissues were taken 2 m or more from the ileo-aeal juntion, but the ontratile response began to dominate until this refleted the sole omponent in the more proximal tissues. The appearane of the ontration is probably related to two distint fators. Firstly, a diret effet of 5-HT ating at ontratile-induing 5-HT reeptors sensitive to methysergide and also those reeptors mediating ontration via a holinergi (atropine sensitive) mehanism; using 1 gm 5-HT a ombined treatment with methysergide plus atropine abolished the ontration response seen in some tissues from the terminal ileum and attenuated ontrations in more proximal tisses. Seondly it is also possible that a desensitization of the 5-HT reeptor mediating the relaxation response was indued by higher onentrations of 5-HT in the terminal ileum. Subsequent studies foused on a haraterization of the 5-HT reeptor mediating the relaxation response in the terminal ileum. The routine inlusion of methysergide (up to 1 gm) in the buffer medium, exluded the relaxant response in the rat terminal ileum being mediated by the 5-HTI and 5-HTIF reeptors (mlaiky et al., 1992; dham et al., 1993); the 5-HT2a/ b/ reeptors (Hoyer et al., 1994; Baxter et al., 1994) or the 5- HT5a, 5-HT5, and 5-HT7 reeptors (rlander et al., et al., 1993; Ruat 1993). The lower poteny of 5-arboxamidotryptamine as ompared to 5-HT in ausing a relaxation response also indiates that the response is unlikely to be mediated by the 5- HTI, 5-HTIB, 5-HTID or 5-HTI-like reeptors (Bradley et al., 1986; Hoyer et al., 1994) or the 5-HT6 reeptor (Monsma et al., 1993). The involvement of the putative 5-HT1p reeptor desribed by Mawe et al. (1986) an also be disounted sine tropisetron (even at higher onentrations) and 5-methoxytryptamine were reported to be inative (Branhek et al., 1988). The involvement of the 5-HT3 reeptor in the relaxation response to 5-HT is also unlikely, due to the failure of ondansetron (1 gm) and a lower onentration of tropisetron (.1 gm) to antagonize the response. These onentrations of n HT4 reeptor-mediated relaxation in rat ileum ondansetron and tropisetron are at least 1 and 1 times respetively their reported affinity at the 5-HT3 reeptors (Humphrey et al., 1993). Diret evidene for the involvement of the 5-HT4 reeptor in the relaxation response to 5-HT in the rat terminal ileum omes from the antagonism of the relaxation response in a surmountable manner with all three 5-HT4 reeptor antagonists tested, namely tropisetron, SDZ and GR In the present study the pkb value alulated for tropisetron with the slope restrited to 1 for 5-HT is 6.9 (95% CL ), whih is omparable to the potenies of tropisetron in antagonizing 5-HT4 reeptors in the mouse olliuli neurones (pki value ) (Dumuis et al., 1988a), rat oesophagus tunia musularis muosa (p ) (Baxter et al., 1991) and guinea-pig ileum (p ) (Craig & Clarke, 199). The apparent pkb values obtained for tropisetron using 5-methoxytryptamine and 5-arboxamidotryptamine (6.37 and 5.91 respetively) were similar to the values obtained with 5-HT. This agonist-independene of the pkb values further supports the single site of agonist ation with these ompounds. Tropisetron is not a seletive antagonist of 5-HT4 reeptors. It has a higher poteny at 5-HT3 reeptors (Rihardson et al., 1985) and at high onentrations it an affet potassium, sodium and alium urrents (Sholtysik et al., 1988). However, there is no evidene of suh nonseletive effets of tropisetron hampering the haraterization of the 5-HT4 reeptor in the present study. Furthermore the more seletive 5-HT4 reeptor antagonists, SDZ (Buhheit et al., 1992) and GR (Grossman et al., 1993) shifted the onentrationrelaxation response urves to 5-HT to the right in a parallel and surmountable manner. The pkb value of 7 (95% CL ) obtained for SDZ in the rat ileum is omparable with the antagonist potenies of SDZ in other 5-HT4 systems, the quiesent and eletrially stimulated guinea-pig ileum (p2 7.4 and 7.3 respetively) (Buhheit et al., 1992), rat tunia musularis muosa (p2 7.3) (glen et al., 1993) and guinea-pig hippoampal adenylyl ylase (p2 7.5) (glen et al., 1993). The pkb value obtained for GR was 8.95 (95% CL ) and is in agreement with the previously obtained value for the 5-HT4 system in the human atrial appendages (pkb value 8.8) (Kaumann, 1993), guinea-pig proximal olon (p2 9.2) and rat oesophagus (p2 9.5). gonistindependene of the pkb values was again demonstrated with GR using 5-methoxytryptamine (8.83) and 5-arboxamidotryptamine (8.82). Support for a 5-HT4 reeptor involvement in the relaxation response to 5-HT in the rat terminal ileum also omes from the rank order of poteny of the indole agonists obtained when metaboli interferene due to monoamine oxidase enzymes was prevented. The rank order thus obtained: 5-HT> 5-methoxytryptamine> a-methyl-5-ht > 5-arboxamidotryptamine > 2- methyl-5-ht is the same as the rank of poteny for the indole 5-HT reeptor agonists to stimulate the 5-HT4 reeptor system in the longitudinal musle myenteri plexus of the guinea-pig ileum (Craig & Clarke, 199), the rat oesophagus musularis muosa (Baxter et al., 1991) and the mouse olliuli neurones (Dumuis et al., 1988b). The seletive breakdown of 5-methoxytryptamine but not other indole agonists may explain some of the disrepant results on the rank order of agonist potenies obtained in different tissues. Thus in the rat oesophagus Reeves et al. (1991) found 5-methoxytryptamine to be less potent than 5-arboxamidotryptamine whereas using the tunia musularis muosa treated with pargyline, other investigators have found 5-methoxytryptamine to be nearly equipotent to 5- HT and more potent than 5-arboxamidotryptamine (Baxter et al., 1991; glen et al., 1992). It is interesting to note that suh lower poteny estimates of 5-methoxytryptamine are observed in the gastrointestinal tissues obtained from the rat (Vane, 1959; Reeves et al., 1991; Baxter et al., 1994) but not the guinea-pig (Craig & Clarke, 199; lswood et al., 1991; glen et al., 1992; Costall et al., 1993). It should be noted that the use of a-methyl-5-ht required a higher onentration of methy-

7 B.R. Tuladhar et al 5-HT4 reeptor-mediated relaxation in rat ileum 39 sergide. The slow loss of tone aompanied by abolition of spontaneous ativity observed with a-methyl-5-ht in the presene of a lower onentration of 1 gum methysergide in the buffer is probably a 5-HT2 reeptor-mediated effet; it is observed only with a-methyl-5-ht whih is known to have higher affinity at 5-HT2 reeptors (Hoyer et al., 1994) and the effet was bloked by a higher onentration of methysergide (1 gm). The relaxant response to 5-HT in the rat terminal ileum was not abolished by tetrodotoxin at either.1 or 1 gm, onentrations suffiient to blok the ation potential in the nerve ells (Gershon, 1967). This indiates that the reeptor mediating the relaxation response in the rat terminal ileum is probably loated on the smooth musle ells. The signifiane of a small and an approximate two fold shift of the 5-HT onentration-response urve to the right with 1 M tetrodotoxin is unertain. Nevertheless, this may have resulted from some antagonism of 5-HT4 reeptor funtion sine a slight shift in the response urves has also been noted in other 5-HT4 systems loated on smooth musle suh as human oloni irular musle (Tam et al., 1993). In the present study the exat ileal musular layer responsible for mediating the observed relaxation is not known. lthough the tissues were mounted in the longitudinal axis, the relaxation annot be simply dedued to be due to relaxation of the longitudinal musle as even the ontration of the irular musle may also passively elongate the tissues (Wood & Perkins, 197), giving an apparent effet of relaxation. Furthermore, the possibility of an involvement of other musle layers suh as the musularis muosa annot be ignored. The inativity of L-NM indiates the non-involvement of nitri oxide in the 5-HT-indued relaxations. The ourrene of the 5-HT4 reeptor-mediated relaxant response to 5-HT varied in different regions of the rat small intestine, with a lear and onsistent relaxation to 5-HT ourring only in the segments obtained from the terminal region (approximately 2 m) of the intestine. This variation in the relaxation response to 5-HT may reflet the variation in 5-HT4 reeptor density or an inreasing dominane of the ontraile funtion. It would be of interest to assess 5-HT4 reeptor density throughout the GI trat using an autoradiographi study. Suh studies would also further an understanding of the exat loation of the reeptor in the rat ileum. The ability of endogenous 5-HT to influene the 5-HT4 reeptor was shown in two ways. Firstly, the inrease in the basal tone of the tissues due to the administration of the 5-HT4 reeptor antagonists, tropisetron, SDZ and GR alone is hypothesized to be due to the antagonism of an endogenous ativation of the 5-HT4 reeptor mediating a relaxant tone. The omparable maximum inrease in tone irrespetive of the antagonists used makes it unlikely that the inrease in the tone is a speifi property of the individual antagonists. Seondly, desensitization of the 5-HT4 reeptor using higher onentrations of exogenous 5-HT auses an inrease in tone. The ability of endogenous 5-HT to stimulate 5- HT4 reeptors has previously been shown to exist in the rat tunia musularis muosa (Waikar et al., 1993). The endogenous 5-HT onentration at the reeptor site after pargyline treatment might be inreased after prevention of metabolism by monoamine oxidase enzymes. This might have resulted in partial desensitization resulting in the lower maximum response seen with 5-HT after the pargyline treatment. In summary, the present study has demonstrated a role for the 5-HT4 reeptor in mediating a relaxation response in the rat ileum. 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