Naunyn-Schmiedeberg's Arch Pharmacol (1992) 346:496-503
Pharmacology © Springer-Verlag 1992
A component of 5-HT-evoked depolarization of the rat isolated vagus nerve is mediated by a putative 5-HT 4 receptor Keith F. Rhodes, James Coleman, and Norman Lattimer Department of Biomedical Research, Wyeth Research (UK) Ltd., Huntercombe Lane South, Taplow, Maidenhead, Berkshire SL60PH, UK Received March 17, 1992/Accepted July 7, 1992
Summary. This study describes a c o m p o n e n t o f 5-HTevoked depolarization o f the rat isolated vagus nerve which was unaffected by the 5-HT 3 receptor antagonist ondansetron. A grease-gap extracellular recording technique was used. Ondansetron ( 1 0 - 1 0 0 nmol/1) displaced the 5-HT concentration-response curve to the right yielding a p A 2 value o f 8.6 (8.5-8.8), consistent with 5-HT 3 receptor antagonism, and revealing a c o m p o n e n t o f the 5 - H T response which was resistant to ondansetron blockade. In the presence o f ondansetron (100 nmol/1) the maxi m u m depolarization in the resistant phase was 15.5 (12.6-19.2)°70 o f the initial m a x i m u m response to 5-HT and the pECs0 value was 7.0 (6.7-7.3). The mechanism o f the ondansetron-resistant c o m p o n e n t o f the 5-HT response resembled a 5-HT4 -receptor-effect in being absent in preparations equilibrated with 5-methoxytryptamine (10 jxmol/1) and antagonised by ICS 205930 (tropisetron, PA2 6.4). 5-Methoxytryptamine alone was an agonist in the vagus nerve with a m a x i m u m response similar to that o f the ondansetron resistant phase o f the 5-HT response. Similarly renzapride alone evoked small depolarizations o f this preparation but antagonized the ondansetron resistant phase o f the 5-HT response (PA2 7.3-7.4). These effects o f 5-methoxytryptamine and renzapride are also consistent with a 5-HT 4 receptor mechanism. Ketanserin (1 gmol/1) and methysergide (1 ~tmol/1) had little effect on responses to 5-HT. The depolarization evoked by this putative 5-HT 4 receptor mechanism was small but prolonged and appears to mask and after-hyperpolarizing phase o f the 5-HT response in this tissue. Key words: 5 - H T 3 receptor - 5 - H T 4 receptor - Vagus nerve -
Introduction T h e depolarizations evoked by 5-hydroxytryptamine (5-HT) in isolated preparations o f m a m m a l i a n cervical Correspondence to: K. E Rhodes at the above address
vagus nerves are k n o w n to be mediated in the main by 5 - H T 3 receptors (Azami et al. 1985; Ireland and Tyers 1987). More complex effects o f 5 - H T have been reported in the rat superior cervical ganglion where two receptormediated depolarization phases and a hyperpolarizing response have been described (Newberry and Gilbert 1989). I n the course o f investigating the effects o f 5-HT3 receptor antagonists in the rat vagus nerve using a "greaseg a p " extracellular recording technique (Marsh et al. 1987) we have observed biphasic concentration-response curves to 5 - H T in the presence o f 5-HT3 antagonists indicative o f the presence o f two 5 - H T receptor-mediated mechanisms. We describe here some o f the characteristics o f the n o n - 5 - H T 3 depolarizing mechanism and its possible contribution to the net response to 5-HT in this preparation. A preliminary a c c o u n t o f this work was presented at the Serotonin 1991 Symposium, Birmingham, UK (Rhodes and C o l e m a n 1991).
Materials and methods Male Sprague-Dawley rats (180-300 g) were killed by a blow to the head and cervical dislocation. The cervical vagus nerves were dissected free and 10-20 mm lengths (without the nodose ganglion) excised and placed in Krebs solution at room temperature. The connective tissue sheath was removed from around the vagus nerve. Within 30 min of dissection the desheathed vagus nerves were transferred to two compartment perspex baths for extracellular recording of 5-HT-induced membrane potential changes (Ireland and Tyers 1987). Each vagus nerve was positioned so that approximately half the length lay in one compartment whilst the remainder projected through a grease-filled hole in the dividing wall into the second compartment. The grease (Dow-Corning high vacuum grease) served to insulate one compartment from the next. The potential difference between the two compartments was recorded using silver-silver chloride electrodes mounted in Pasteur pipettes containing 4°70 agar in saline (0.9070). Potential changes were displayed on Grass polygraphs (Model 7D with DC 7PIG preamps) or Watanabe potentiometric records (Type MC 6601). One compartment of each bath was perfused with Krebs solution at a rate of 5 ml/min. The second compartment was filled with Krebs solution but was not perfused. Bath temperature was maintained at 27 °C. The Krebs solution reservoir was gassed with 5070CO2 in oxygen.
Agonists. Concentration-response curves to 5-HT (10nmol/1 to 1 mmol/1) or in separate experiments to 2-methyl-5-hydroxytryptamine (2-methyl-5-HT, 0.1-100 ~tmol/1) or 5-methoxytryptamine (5-MeO-T, 0.1 - 3 0 ~tmol/1) were obtained by including the agonist in the perfusing Krebs solution. Agonist contact time was 3 min with a 10-15 min washout period between agonist exposures.
Table 1. Analysis of concentration-response curves for agonist-evoked depolarizations of the rat vagus nerve. The pECs0 (-log10 agonist ECs0 ) and maximum response (gV) results are the arithmetic mean values derived from curve-fitting of concentration-response curves of individual tissues. 95% confidence limits are shown in brackets Agonist
6.3 (6.2 - 6.4) 7.0 (6.7 - 7.3) 5.7 (5.65 - 5.8) 6.1 (5.4 - 6.7) 7.0 a
402 (364 72 (52 410 (324 112 (77 26 a
Antagonists. In general an initial concentration-response curve to 5-HT was constructed in order to establish the maximum response to 5-HT in that tissue. The tissue was then washed for 1 h during which time antagonists were added to the perfusing Krebs solution and equilibrated for 40 min. A second agonist concentration-response curve was then constructed. Control preparations were treated in the same way but were not exposed to antagonist. The data presented here are based on analyses of the second concentration-response curve. The comparison of the effects on ondansetron (100 nmol/1) and tropisetron (ICS 205930, 3 gmol/1) on 5-HT responses was carried out in paired preparations from the same animals as were the comparisons of the effects of ondansetron in the absence and presence of ketanserin (1 gmol/1) and ondansetron (100 nmol/1) in the absence and presence of 5-MeO-T (10 gmol/1). Other 5-HT groups were unpaired. The effects of ondansetron (10 nmol/1) on responses to 2-methyl-5-HT and to 5-MeOT were carried out in paired preparations. Where possible, results from individual tissues were analysed using the Allfit logistic curve fitting program (DeLean et al. 1978). Quoted in the text are arithmetic mean values of pECso ( - l o g w of the concentration giving half the calculated maximum response) and midpoint slope of the curve. A geometric mean of the ECs0 value and of the calculated maximum response (when expressed as a percentage of the initial maximum response to 5-HT) is used. Where maximum response values are in gV an arithmetic mean value is quoted. 95% confidence limits indicate error. Where concentration-response curves were clearly biphasic individual components of the curve were analysed separately using AUfit with the minimum of each phase constrained to zero. In experiments where the "first phase" of the 5-HT response was too variable for concentration-response curves of individual tissues to be analysed the average response of the group at each concentration of 5-HT was calculated and a single Allfit analysis carried out. In this situation it was not possible to estimate confidence limits. The pA z value for ondansetron for the second phase of the 5-HT concentration-response curve was calculated using the method of Arunlakshana and Schild (1959), pooled data being analysed by linear regression. Other pA 2 values (where only 1 or 2 antagonist concentrations were studied) were calculated for individual experiments, assuming a Schild-plot slope of unity. Confidence limits were calculated from the SE values.
Solutions and drugs. Krebs solution was of the following composition (mmol/1): NaC1, 118.4; KC1, 4.8; NaHCO 3, 25.0; MgSO4, 1.2; glucose, 11.1; CaC12, 2.5; KH2PO4, 1.2. Drugs used were 5-hydroxytryptamine hydrochloride and 5-methoxytryptamine hydrochloride (Sigma, Poole, UK), BRL 24924 hydrochloride (renzapride, Beecham Pharmaceuticals, Harlow, UK), ketanserin tartrate (Janssen Pharmaceuticals, Wantage, UK), methysergide maleate (Sandoz Products, Feltham, UK), ondansetron hydrochloride, ICS 205930 hydrochloride (tropisetron) and 2-methyl-5-hydroxytryptamine dihydrobromide. Agonists were dissolved directly into Krebs solution. Antagonists were dissolved in distilled water to give I0 mmol/1 solutions which were then diluted in Krebs solution.
5-HT-first phase ( + ondansetron, 100 nmol/1) 2-methyl-5-HT 5-MeO-T Renzapride
440) 8 92) 4 497) 3 163) 9
a Results from fitting of the mean concentration-response data of 9 tissues
a n d 410 ( 3 2 4 - 4 9 7 ) g V , n = 4 w i t h r e s p e c t i v e g e o m e t r i c m e a n ECs0 v a l u e s o f 0.53 ( 0 . 4 0 - 0 . 7 0 ) a n d 1.82 ( 1 . 5 1 - 2 . 2 0 ) g m o l / 1 (Fig. 1, Table 1). T h e s e results are in g o o d a g r e e m e n t w i t h t h o s e r e p o r t e d b y I r e l a n d a n d Tyers (1987) f o r t h e 5 - H T 3 r e c e p t o r a g o n i s m o f t h e s e c o m p o u n d s in t h e rat v a g u s nerve. T h e c o n c e n t r a t i o n - r e sponse curves for both these agonists appeared monophasic. In comparison, 5-MeO-T (100nmol/1 to 30 Ixmol/1) e v o k e d small, c o n c e n t r a t i o n - r e l a t e d d e p o l a r i z a t i o n s w i t h a m a x i m u m o f 112 ( 7 6 . 9 - 1 6 2 . 9 ) ~tV, n = 3 w i t h a g e o m e t r i c m e a n ECs0 o f 0.82 ( 0 . 1 8 - 3 . 7 4 ) g m o l / 1 (Fig. 1, Table 1).
The effects of ondansetron on depolarizations evoked by 5-HT, 2-methyl-5-HT and 5-MeO-T C o n c e n t r a t i o n - r e s p o n s e c u r v e s t o 5 - H T were d i s p l a c e d t o t h e r i g h t by o n d a n s e t r o n ( 1 0 - 1 0 0 n m o l / l ) a n d were b i p h a s i c w i t h 5 - H T ( 1 0 - 300 n m o l / 1 ) e v o k i n g d e p o l a r i z a t i o n s w h i c h were r e s i s t a n t to t h e a n t a g o n i s t a c t i o n o f o n d a n s e t r o n (Fig. 2). I n t h e p r e s e n c e o f 10, 30 a n d 1 0 0 n m o l / 1 o n d a n s e t r o n t h e ECs0 v a l u e s o f this first p h a s e o f t h e 5 - H T c o n c e n t r a t i o n - r e s p o n s e c u r v e s were 63, 100 a n d 1 0 0 n m o l / l r e s p e c t i v e l y ( T a b l e 2 ) a n d t h e m a x i m u m d e p o l a r i z a t i o n s in t h e first p h a s e were 15.2°70,
300 200 100-
Agonists actions 5 - H T ( 1 0 n m o l / 1 to 3 0 g m o l / 1 ) and 2-methyl-5-HT (100 n m o l / 1 - 1 0 0 g m o l / 1 ) e v o k e d c o n c e n t r a t i o n - r e l a t e d d e p o l a r i z a t i o n s w i t h m a x i m a o f 402 ( 3 6 4 - 4 4 0 ) , n = 11
A g o n i s t (log mol/I)
Fig. 1. Concentration-response curves for the depolarization of the rat isolated vagus nerve by 5-HT (o), n = 11; 2-methyl-5-HT (©), n = 4; 5-MeO-T (), n = 3 and renzapride (A), n = 9. Mean values+__SEM are shown
498 Table 2. The effects o f antagonists on the two phases of the concentration-response curve to 5-HT in the rat isolated vagus nerve. NO, not observed; NT, not tested. Arithmetic and geometric m e a n s of pECs0 and m a x i m u m response values respectively are shown. 95% confidence limits are shown in brackets Antagonist
10 n m o l / l
Renzapride + Methysergide
300 nmol/1 1 gmol/1
Ondansetron + Ketanserin
Phase 1 pEC50
Phase 2 pECs0
NO 15.2 (11.3 13.8 (10.415.5 (12.613.6 (5.9 12.8 (7.7 -
90.7 (85.0 - 96.4) 80.7 (64.9 - 100.5) 69.5 ( 6 3 . 5 - 76.0) 78.5 (70.0-88.1) NT
7.2 (7.1 - 7.4) 7.0 ( 6 . 4 - 7.6) 7.0 (6.7-7.3) 7. I (6.8 - 7.3) 6.1 (5.8 - 6.4)
6.3 (6.2 - 6.4) 5.4 (5.35 - 5.5) 5.0 (4.8 - 5.2) 4.3 (4.2-4.4) NT NT
6.7 (6.6 - 6.9) 6.4 a 6.5 a
20.2) 18.3) 19.2)
6 6 8 6
17.9) 6.3 (6.1 4.8 (4.6 4.3 (4.24.1 (3.9 -
13.6 (10.5 - 17.7) 12.3 a l 3.4a
88.3 (82.8 85.5 (74.3 79.1 (56.9 74.8 (71.7 -
6.5) 5.0) 4.5) 4.3)
9 7 7
98.6) 6 109.6) 4 78.9)
15.4 (10.3 - 23.1) 13.8 (9.9 - 19.4)
4.4 ( 4 . 2 - 4.5) 4.5 (4.3 - 4.7)
85.8 ( 7 6 . 7 - 95.9) 76.0 (65.6 - 88.1)
100 nmol/1 1 ~tmol/l
7.1 ( 6 . 6 - 7.6) 6.8 (6.3 - 7.3)
70.9 (65.9 - 76.2) 95.0 ( 8 0 . 0 - 113.5)
6.2 (6.1 - 6.3) 6. I ( 5 . 9 - 6.4)
a Results calculated from the curve fitting of the averaged concentration-response data of groups of tissues. No confidence limits calculated
5-HT (log mol/I)
-7 -6 -5 -4 2-methyl-5-HT (log mol/I)
5-MeO-T (log tool/I)
Fig. 2. The antagonist actions of ondansetron against depolarizations of the rat vagus nerve evoked by A 5-HT, n = 6 - 1 1 ; B 2-methyl-5-HT, n = 4 and C 5-MeO-T, n = 3. Responses are shown in the absence ( e ) o f ondansetron or in the presence o f 10 ( o ) , 30 (E~) or 100 (Z&) nmol/1 ondansetron. Mean values 4-SEM are shown. Antagonists were equilibrated with the tissues for 40 min before beginning non-cumulative concentration-response curves. The m e a n m a x i m u m depolarizations (the 100070 values) were 402, 410 and 112 IxV for 5-HT, 2-methyl-5-HT and 5-MeO-T respectively
499 13.8% and 15.5°70 of the initial 5-HT maximum response in the same tissues (Fig. 2, Table 2). The first phase maximum depolarization in the presence of 100 nmol/1 ondansetron was 72.1 (52.1-92.1) btV, n = 8. A second phase of the concentration-response curve to 5-HT was displaced to the right by ondansetron (Fig. 2) giving a pA2 value for ondansetron of 8.6 (8.5-8.8) with a Schild plot slope of 1.2 (1.0-1.4), n = 20, consistent with a 5-HT 3 receptor mediated effect. In the presence of ondansetron (100nmol/1) the depolarization evoked by an exposure to 5-HT (1 Ixmol/1) was very much reduced but the duration of the residual response was prolonged, persisting for many minutes after washout of 5-HT (Fig. 3). Ondansetron (10 nmol/1) antagonized 2-methyl-5-HT (100 nmol/1 to 100 Ixmol/1) depolarizations with a mean pA2 value of 8.9 (8.8-9.0), n = 4. Concentration-response curves to 2-methyl-5-HT were monophasic (Fig. 2). The concentration-response curve for 5-MeO-T-evob ed depolarizations was little changed by ondansetron (10 nmol/l, Fig. 2), the geometric mean agonist concentration-ratio being 0.81 (0.23-2.86), n = 3 when paired preparations in the absence and presence of ondansetron were compared.
The depolarizing action of 5-MeO-T at a concentration of 10 Ixmol/1 is shown in Fig. 3. In the presence of 5-MeO-T (10 gmol/1) depolarizations evoked by 5-HT were characteristically followed by a prolonged hyperpolarization (Fig.3) an effect not normally seen in this preparation with 5-HT alone and probably a consequence of the electrogenic extrusion of Na + . The effect o f renzapride on responses to 5-HT After equilibration with renzapride (100 and 300 nmol/1) concentration-response curves to 5-HT were displaced to the right and were biphasic, the responses to low concentrations of 5-HT (10 nmol/l to 3 gmol/1) resembling the first phase of the concentration-response curve in the presence of ondansetron (Fig. 4). The maximum of the first phase was 13.6 (10.5-17.7)°70 of the initial 5-HT maximum (Table 2) and the geometric mean ECs0 for 5-HT in this phase was 199.5 (125.9-251.2)nmol/1, n = 7 after equilibration with 100 nmol/1 renzapride. Increasing the concentration of renzapride to 300 nmol/1 resulted in displacement of the first phase of the 5-HT curve to the right, the ECs0 calculated from the mean concentration response curve being 398.1 nmol/1 with a maximum of 12.3°70 of the initial 5-HT maximum.
The effects o f 5-MeO-T on responses to 5-HT in the presence and absence of ondansetron
In the presence of both 5-MeO-T (10 btmol/1) and ondansetron (100nmol/1), 5-HT (10nmol/1 to 3 gmol/1) evoked small hyperpolarizations of the isolated vagus nerve, the effect being greatest at a 5-HT concentration of 300 nmol/l when a mean hyperpolarization of 40.0 (26.7-53.3) gV, n = 6 was observed (Fig. 4). In paired tissues equilibrated with ondansetron (100 nmol/1) alone, 5-HT (10 nmol/1 to 3 gmol/1) evoked a depolarization similar to that seen with the separate group of tissues described above for the effects of ondansetron. In this case the maximum depolarization reached in this first phase o f the 5-HT response was 13.7 (11.6-15.8)°70 of the 5-HT maximum and the geometric mean ECs0 for this phase was 95.7 (82.4-111.1)nmol/1, n = 6.
t~ 0e~ £3
-20 -9 100-
-'8 -'7 '6 -'~ -'4 ;3 -'2 
-20 -8 1O0 E I-.I
. -6. .
Ondansetron (100 nmol/I)
0"5mY I lOmin
Fig. 3. The effects of 5-MeO-T and ondansetron on depolarization of the rat vagus nerve evoked by 5-HT (1 gmol/1). Solid lines below the trace indicate 5-HT (1 Ixmol/1) contact times
-6 -5 -4 5-HT (log mol/I)
Fig. 4. The effects of antagonists on 5-HT-evoked depolarization of the rat vagus nerve. A ( e ) ondansetron (100nmol/1), ( o ) ondansetron (100 nm91/1) together with 5-MeO-T (10 ~tmol/1), n = 5; B ( • ) control tissues in the absence of antagonists, ( o ) renzapride (100 nmol/1) and (D) renzapride (300 nmol/1), n = 6; C ( • ) ondansetron (100 nmol/1), ( o ) tropisetron (3 lxmol/1), n = 6. Mean values+SEM are shown. Antagonists were equilibrated with the tissues for 40 min before beginning non-cumulative concentration-response curves
500 Comparison o f ECs0 values of the first phase of the 5-HT concentration-response curve in the presence of renzapride with those in the presence o f ondansetron (10 nmol/1) show renzapride (100 and 300 nmol/1) to be an antagonist of this response with pA 2 values of 7.4, n = 7 and 7.3, n = 6 at these concentrations respectively. These values are derived from curve-fitting of the mean concentration-response results in each group as the variability o f 5-HT responses in the presence of renzapride made curve-fitting of results from individual tissues unreliable. The second phase of the 5-HT concentration-response curve was displaced to the right in the presence of renzapride, the mean pA 2 values being 8.4 (8.1-8.7), n = 7 and 8.5 (8.3-8.7), n = 6 with 100 and 300 nmol/l renzapride respectively. Renzapride alone in the concentration range 30nmol/1 to 3 ~tmol/1 produced a weak and variable depolarizing effect (Fig. 1). Curve-fitting of concentration-response curves from individual tissues was not possible. Analysis of the mean results of 9 preparations gave a calculated maximum depolarization with renzapride of 26 ~tV with an ECs0 value o f 91.6mmol/1 (Table 1, Fig. 1). The effect o f tropisetron on responses to 5 - H T In the presence o f tropisetron (3 ~tmol/1) depolarizations of the vagus nerve were observed to 5-HT in the concentration-range 300 nM to 1 mmol/1. The maximum 5-HT response was 12.8 (7.7-17.9)%, n = 6, of the initial 5-HT maximum response and the ECs0 was 0.79 (0.40-1.58) ~tmol/1 (n = 6, Fig. 4, Table 2). At lower concentrations of 5-HT (30 and 100 nmol/1) there was a small hyperpolarization of unknown mechanism in 3/6 preparations in the presence of tropisetron. In these three preparations the mean hyperpolarization observed to 30nmol/1 5-HT was 28.3_+6.0~tV. In paired tissues equilibrated with ondansetron (100 nmol/1) the first phase of the 5-HT concentration-response curve was positioned to the left of that in the presence o f tropisetron and had a calculated maximum o f 13.6 (5.9-21.4)% of the initial 5-HT maximum and a geometric mean ECs0 value of 79.4 (50.1 - 158.4) nmol/1 (Table 2). Comparison of the individual ECs0 values for the first phase of the 5-HT response in paired tissues in the presence of either ondansetron (100nmol/1) or tropisetron (3 ~tmol/1) were consistent with a p A 2 value of 6.4 (5.9-6.9), n = 6 for tropisetron at the receptor mediating the first phase of the 5-HT concentration-response curve. No evidence of a second phase in the 5-HT concentration-response curve was observed in tissues exposed to tropisetron (3 ~tmol/1), up to a concentration of 1 mmol/1 5-HT, presumably due to the profound 5-HT3 antagonist activity of this concentration o f tropisetron. The effect o f methysergide on responses to 5 - H T Methysergide (1 ~mol/l) had no effect on responses to 5-HT (Table2). Similarly, responses to 5-HT in the presence of both methysergide (1 ~mol/1) and renzapride
(300nmol/1) were similar to those with renzapride (300 nmol/1) alone (Table 2). The effect o f ketanserin on responses to 5 - H T Some reduction in the calculated maximum response of the 5-HT concentration-response curve was apparent in the presence of ketanserin (1 ~tmol/1), the maximum being 70.9 (65.9-76.2)% of the initial 5-HT maximum (n = 6, Table 2). However, the 5-HT geometric mean EDs0 value was similar to that of control preparations being 660.7 nmol/1 (n = 6). Concentration-response curves to 5-HT were monophasic in the presence of ketanserin (1 ~tmol/1). The effect o f ketanserin on responses to 5 - H T in the presence o f ondansetron In 2 groups (n = 5) of paired tissues, one exposed to ondansetron (100nmol/1) and the other to ondansetron (100 nmol/l) and ketanserin (1 ~mol/1) biphasic concentration-response curves to 5-HT were obtained. In the group exposed to ondansetron alone concentration-response curves to 5-HT were like those of other similarly treated groups (Table 2). In the group equilibrated with both antagonists there was a small shift to the right in the first phase consistent with a pA2 value of 6.0 for ketanserin. The ECs0 value for 5-HT in the second phase of the concentration-response curve was the same in both groups (41.7 ~mol/1) consistent with a pA2 value for ondansetron of 8.9 in the absence and presence of ketanserin. The calculated maximum of the second phase was somewhat reduced in the presence of both antagonists being 76.0°70 of the initial 5-HT maximum in contrast to a value of 85.8°70 in the presence of ondansetron alone (Table 2).
Discussion In the presence of the 5-HT 3 antagonist ondansetron, concentration-response curves for 5-HT-evoked depolarization of the rat isolated vagus nerve were biphasic, with a first phase of the curve resistant to blockade by ondansetron. In 5 groups of ondansetron treated preparations (Table 2) the mean maximum of the first phase was in the range 13.6°70 to 15.5°70 of the initial maximum response to 5-HT in the same tissues with mean pECs0 values in the range 7.0 to 7.2. A second phase of the 5-HT concentration-response curve was displaced to the right by ondansetron (Table 2, Fig. 2) with a pA 2 value of 8.6 (8.5 to 8.8), consistent with the reported 5-HT 3 antagonist action of this compound (Ireland and Tyers 1987). We were impressed by the similarity between the 5-HT concentration-response curves obtained in the vagus nerve preparation, in the presence of ondansetron, with those reported by Butler et al. (1988) for 5-HT-evoked contraction of the guinea-pig ileum longitudinal musclemyenteric plexus preparation in the presence of this antagonist. These authors demonstrated a two phase re-
501 sponse to 5-HT in the presence of ondansetron with a "lower phase" (in the 5-HT concentration range 3 to 300 nmol/1) contributing about 20% of the 5-HT maximum response and being resistant to ondansetron blockade. This first phase of the 5-HT concentration-response curve in the guinea-pig has been attributed to a 5-HT 4 receptor mechanism (Craig et al. 1990) and was first described by Buchheit et al. (1985). We have attempted here to characterize the first phase of the 5-HT response in the rat vagus nerve using ondansetron, reported to have little affinity for the 5-HT 4 receptor (Dumuis et al. t989), to displace the 5-HT 3 component of the response and unmask the high affinitiy phase of the 5-HT-evoked depolarization. In experiments with agonists, 2-methyl-5-HT evoked depolarizations of the vagus nerve of similar maximum to those of 5-HT (Table 1, Fig. 1). In the presence of ondansetron the concentration-response curve to 2-methyl-5-HT was displaced to the right (Fig. 2) with an apparent pA2 value of 8.9 (8.8 to 9.0), consistent with a 5-HT3 receptor-mediated effect (Butler et al. 1988). There was no evidence of a biphasic response to 2-methyl-5-HT in the presence of ondansetron which may exclude a 5-HT~p mechanism for the first phase of the 5-HT response as 2-methyl-5-HT is said to be equipotent with 5-HT as an agonist at this site (Mawe et al. 1986). However, 2-methyl-5-HT lacks marked 5-HT 4 affinity (Craig and Clarke 1990) leaving open the possibility that this receptor may mediate the first phase of the 5-HT concentration-response curve in the rat vagus nerve. In contrast 5-MeO-T alone, evoked a maximum depolarization of the rat vagus nerve of 112 ~V (Table 1, Fig. 1) comparable to the magnitude of the first phase of the 5-HT response (72 ~V in the presence of ondansetron, 100 nmol/1, Table 1). The response to 5-MeO-T was not antagonized by ondansetron (10 nmol/1, Fig. 2). It is known that 5-MeOT has 5-HT4 agonist actions in, for instance, the guineapig ileum (Craig and Clarke 1990) and in rat hippocampal pyramidal neurones, where a slow 5-HT 4 -mediated excitatory response has been described (Andrade and Chaput 1991). Fozard (1985) and Craig et al. (1990) have described the use of 5-MeO-T to discriminate between the two phases of the 5-HT response of the guinea-pig ileum where it acts to desensitize the 5-HT 4 component of the 5-HT response. In the present study in the presence of both 5-MeO-T (10 ~mol/l) and ondansetron (100 nmol/1) the first phase of the 5-HT concentration-response curve was absent and a small hyperpolarization was evoked by low concentrations (30 nmol/1-3 ~mol/1) of 5-HT (Fig. 4). The mechanism of this hyperpolarization is not known. The 5-HT 3 antagonist renzapride (Sanger 1987) has been extensively used to characterize 5-HT 4 responses at which it may have agonist (Dumuis et al. 1989; Craig and Clarke 1990; Baxter et al. 1991) or partial agonist (Kaumann 1990; Andrade and Chaput 1991) actions, depending on the tissue studied. In the rat vagus nerve, renzapride had weak agonist actions. Depolarizations were observed with renzapride (100nmol/l to 3 ~tmol/1) but the calculated maximum response was only 26 ~tV (Table 1, Fig. 1). The agonist potencies of renzapride and
5-HT (in the first phase) are similar to those reported by Bockaert et al. (1991) for 5-HT 4 receptor mediated stimulation of adenylate cyclase in mouse colliculi neurons. The ECs0 value for 5-MeO-T was, however, 8 fold less than that reported by these authors. When renzapride (100 and 300 nmol/1) was used as an antagonist, biphasic concentration-response curves to 5-HT were obtained (Fig. 4) with the first phase displaced to the right of that seen in the presence of ondansetron. Comparison of pECs0 values for the first phase of the 5-HT response in the presence of ondansetron with those in the presence of renzapride indicate mean pA 2 values for renzapride (100 and 300 nmol/1) of 7.4 and 7.3 at the two concentrations respectively. The concentration of renzapride active in blocking the first phase of the rat vagus nerve response to 5-HT are similar to those shown to block the first phase of the guinea-pig ileum contraction to 5-HT (Sanger 1987) and to have 5-HT 4 agonist effects in that preparation (Craig and Clarke 1990). The results suggest a possible similarity between the mechanism of action of renzapride at the 5-HT 4 site in the guinea-pig ileum and in the first phase of the 5-HT concentration-response curve of the rat vagus nerve. Tropisetron has been shown to have antagonist action at 5-HT 4 receptors in various tissues with pA 2 values in the range 6.01 (Dumuis et al. 1989) to 6.89 (Kaumann 1990). In the present study 5-HT (300 nmol/1-1 mmol/1) in the presence of tropisetron (3 ~mol/1) evoked small depolarizations of the rat vagus nerve, the maximum response being 12.8% of the initial 5-HT maximum response with an pEC50 value of 6.1 (Table 2, Fig. 4). It seems likely that this response corresponds to the first phase of the 5-HT concentration-response curve observed in the presence of ondansetron. Comparison with paired tissues in the presence of ondansetron (100nmol/l) showed the first phase of the 5-HT concentration-response curve was displaced to the right in the presence of tropisetron (Fig. 4) with a pA2 value for tropisetron of 6.4, in keeping with a 5-HT4-receptor-mediated response. No evidence of a second phase in the concentration-response curve to 5-HT (30 nmol/1 to 1 mmol/1) was obvious, presumably due to intense 5-HT 3 antagonism by the concentration of tropisetron employed. In general, the antagonist effects of renzapride and tropisetron on the ondansetron-resistant component of the 5-HT response were small and difficult to quantify. An indication of a real effect can perhaps be most easily seen if the lack of depolarizing response to 100 nmol/1 5-HT in the presence of renzapride (300 nmol/1, Fig. 4B) or tropisetron (3 ~mol/l, Fig. 4C) is compared to the depolarization evoked in ondansetron-treated tissues (Figs. 2A, 4C) where 100nmol/1 is the approximate EDs0 value for the first phase of the 5-HT concentration-response curve. In the presence of tropisetron, 5-HT (30 nmol/1 and 100nmol/1) evoked small hyperpolarizations in 3/6 preparations (Fig. 4). The mechanism of these hyperpolarizing responses is unknown, but together with the similar observations made in tissues in the presence of both 5-MeO-T and ondansetron (Fig. 4), suggest 5-HT may have a hyperpolarizing action normally masked by
5-HT 3 and 5-HT 4 -receptor mediated depolarizations. A hyperpolarizing action of 5-HT has previously been noted in the rat superior cervical ganglion (Newberry and Gilbert 1989). It is unlikely that this small hyperpolarizing response has the same mechanism as that seen following a depolarization (see below) which is believed to involve electrogenic pumping of Na ÷. A direct hyperpolarizing effect of 5-HT seems more likely and it is possible that this may reduce the observed 5-HTa-like depolarization. The lack of effect of methysergide (1 ~xmol/1) on depolarizations evoked by 5-HT either in untreated preparations or those in the presence of renzapride (Table 2) would suggest no involvement of receptors of the 5-HT 2, 5-HT1A, 5-HTlc or 5-HT1D subtypes for which methysergide has good affinity (Schoeffter and Hoyer 1990). Similarly in preparations treated with ondansetron (100 nmol/l), ketanserin (1 Ixmol/1) had little effect on the first or second phase of the 5-HT concentration-response curve, when compared with paired tissues treated with ondansetron alone (Table 2). Ketanserin (1 Ixmol/1) alone did not change the ECs0 value for 5-HT but was associated with a small reduction in the maximum depolarization evoked by 5-HT. The results with ketanserin confirm those with methysergide that no major 5-HT2 receptor component of the 5-HT evoked depolarization exists in this preparation. The effects of selective 5-HT 3 and 5-HT 4 receptor blockade on individual responses to 5-HT are exemplified in Fig. 3. In the presence of ondansetron the amplitude of the observed depolarization was greatly reduced, as expected for a 5-HT3 mediated response, but paradoxically the duration of depolarization was extended for many minutes. In the presence of ondansetron the residual depolarization is presumably evoked by the mechanism mediating the first (5-HT4-1ike) phase of the 5-HT concentration-response curve, which is clearly a prolonged response. It is possible that the prolonged component of d e p o l a r i z a t i o n ( 5 - H T 4 mediated) is not observed with 5-HT alone because of the onset of electrogenic extrusion of Na + known to be initiated following agonist-evoked depolarization in preparations of this type (Brown et al. 1972; Ireland 1987) and probably triggered here by the 5-HT3 component of depolarization. The effect of electrogenic extrusion of Na + is to rapidly repolarize and sometimes hyperpolarize cells following exposure to agonists. This possibility is also supported by the observation of a prolonged hyperpolarization following exposure to 5-HT in the presence of 5-MeO-T (Fig. 3) an effect not normally observed in the rat vagus nerve with 5-HT alone (Ireland 1987). In this preparation the 5-HT 4 -like component of 5-HT depolarization may act to mask the hyperpolarization which would otherwise be a consequence of activity of the electrogenic Na + pump. It is not possible in preparations of this type, where many neurones contribute to the potentials recorded, to be sure that different phases of potential change originate from the same cells. It is difficult therefore to speculate on physiological roles for different components of the 5-HT response. It is, however, of interest that Andrade and Chaput (1991) have described a 5-HT 4 receptor mediated
mechanism for the 5-HT-evoked slow membrane depolarization of rat hippocampal pyramidal neurones using intracellular recording techniques. This slow depolarization evoked by 5-HT is known to reduce the long lasting afterhyperpolarizations which follow spike firing in these cells (Colino and Halliwell 1987). It is tempting to speculate that the prolonged 5 - H T amediated depolarizations unmasked by ondansetron in this tissue may have correlates in the central nervous system related to the cognition enhancing and anxiolytic effects of 5-HT 3 antagonists (Jones et al. 1988; Costall et al. 1989). Perhaps of more direct relevance to this study are the observations of Bhandari and Andrews (1991) providing evidence for a 5 - H T 4 receptor component of the vagally mediated emesis evoked by zacopride and copper sulphate in the ferret.
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