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The effect of troxypyrrolidinium, a choline uptake inhibitor, on the excitatory innervation of the rat urinary bladder
European Journal of Pharmacology, 61 (1980) 293--301
293
© Elsevier/North-Holland Biomedical Press
THE EFFECT OF TROXYPYRROLIDINIUM, A CHOLINE UPTAKE INHIBITOR, ON THE EXCITATORY INNERVATION OF THE RAT URINARY BLADDER L.K. CHOO and F. MITCHELSON
Department of Pharmacology, Victorian College of Pharmacy, Park ville, Victoria, 3052 Australia Received 2 October 1979, accepted 6 November 1979
L.K. CHOO and F. MITCHELSON, The effect of troxypyrrolidinium, a choline uptake inhibitor, on the excitatory innervation o f the rat urinary bladder, European J. Pharmacol. 61 (1980) 293--301. Troxypyrrolidinium, a choline uptake inhibitor, reduced but failed to abolish responses of the rat urinary bladder to electrical stimulation at 1-100 Hz although it reduced acetylcholine output during stimulation at 10 Hz to a level similar to that of spontaneous release. Inhibition of the response to stimulation was more complete at faster rates of stimulation and was partially reversed by choline. Troxypyrrolidinium produced a greater inhibition of the 'tonic' component of the response to electrical stimulation than of the 'phasic' component. Hemicholinium-3 or hyoscine produced a similar selective effect on the 'tonic' component. Hemicholinium-3 also reduced acetylcholine o u t p u t during electrical stimulation to a similar extent as troxypyrrolidinium but hyoscine increased transmitter output. The results support the concept of a second transmitter in the excitatory innervation of the bladder. Troxypy~rolidinium
Rat urinary bladder
Cholinergic innervation
I. Introduction
Troxypyrrolidinium tosylate (N-ethyl-N-2(3,4,5-trimethoxybenzoyloxy)-ethyl pyrrolidinium tosylate, FWH 428) is a choline uptake inhibitor, structurally different to the hemicholiniums (Bhatnager et al., 1964; Chdnier et al., 1969) and in avian muscle has been suggested to act more selectively than hemicholinium-3 at prejunctional sites (Marshall, 1969). An investigation of the effect of troxypyrrolidinium on the responses to electrical stimulation of the urinary bladder of the rat was undertaken to determine whether the excitatory innervation is wholly cholinergic (Carpenter, 1977; Elmdr, 1978) or may also contain a non-cholinergic component (Dumsday, 1970). In the rat bladder hemicholinium-3 inhibits the response to nervous stimulation (Hukovid et al., 1965; Dhattiwala et al., 1970) although there is no reduction in the amount
Hemicholinium-3
of acetylcholine present in the tissue (Hukovid et al., 1965).
2. Materials and methods 2.1. Isolated tissue experiments Isolated urinary bladders from GlaxoWistar rats (200-350g) were set up for electrical stimulation according to the method of Hukovi~et al. (1965) under a resting tension of 0.5-1 g in a 10 ml organ bath in McEwen's solutton maintained at 37°C. Responses were recorded on a polygraph via a Grass force
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were measured as a percentage o f the control response in the absence of any drug at each corresponding frequency unless otherwise stated. In control experiments it was demonstrated that preparations could be stimulated for at least 3 h with no decline in responsiveness. Thus, after 1, 2 or 3 h of stimulation at 50 Hz (2 msec pulses for 10 sec every 2 min) responses were 98.5 + 6.1% (4) (mean + S.E.M. (n)), 101.1 + 8.6% (4) and 94.5 + 8.5% (4) of the control response respectively. Agonists were added to the bath in a 6 min dose cycle and remained in contact with the tissue for 60 sec (acetylcholine) or 90--120 sec (carbachol). For the determination of dose ratios the tissue was incubated with troxypyrrolidinium for 20 min to allow equilibrium to be attained before re-determining responses to agonists and was re-added to the bathing fluid immediately following each washing of the tissue. Some experiments were conducted after dyflos pretreatment, the tissue being incubated with dyflos (5.4 pmol/1) for 15 min, then washed several times to remove excess anticholinesterase. Responses to agonists were then re
2.2. Determination of acetylcholine release In these experiments bladders were set up as described above except that b o t h ureters were dissected o u t and a longitudinal cut was made from the urethral orifice upwards along three-quarters of the length of the bladder wall. This procedure enabled the 2 ureters to be drawn together through a single bipolar electrode and allowed ready diffusion of the released transmitter from both sides of the bladder wall into the bathing fluid. The preparation was placed in an organ bath conraining 4 ml of Tyrode solution of the following composition (mmol/1): NaC1 137, KC1 2.7, CaC12 1.8, MgC12 1.0, NaH2PO4 0.4, NaHCO3 11.9, dextrose 5.6. Physostigmine (10 ~mol]l) was added and the tissue equilibrated for 60 min, the physostigmine being replaced
L.K. CHOO, F. MITCHELSON
every 15-20 min throughout the course of the experiment unless an assay sample was being collected. Spontaneously released acetylcholine was allowed to accumulate in the bathing fluid for 30 min before collection for assay. Release during electrical stimulation was determined b y collection of the bathing fluid following continuous stimulation of the preparation for 10 min at 10 Hz with 2 msec pulses at supramaximal voltage. To determine the effect of troxypyrrolidinium or hemicholinium-3 on the release of transmitter, the preparation was incubated with the choline uptake inhibitors for 60 min during which time the preparation was stimulated with 2 msec pulses at supramaximal voltage at a frequency of 50 Hz for 10 sec every 2 min. The preparation was then washed several times and again stimulated at 10 Hz for 10 min and the bathing fluid collected for assay. In control experiments, with no drugs added the acetylcholine o u t p u t in the second collection period was 81.0 + 13.7% (4) of that in the initial collection period and did not differ significantly ( P < 0.05) from it. In experiments with the choline uptake inhibitots, during the second period of stimulation troxypyrrolidinium or hemicholinium-3 was not present in the bath as preliminary experiments demonstrated that the high concentration of troxypyrrolidinium or hemicholinium-3 interfered with the subsequent gas chromatographic estimation of acetylcholine output, the chromatograms of extracts containing the uptake inhibitors showing numerous additional peaks which tailed into the peaks for demethylated acetylcholine and propionylcholine. Furthermore, removal of the choline uptake inhibitor does n o t produce any recovery of the responses to electrical stimulation within 30 min using either hemicholinium-3 (Dhattiwala et al., 1970) or troxypyrrolidinium (see Results 3.3.). At the end o f the experiment, bladders were trimmed free of the ureters, blotted dry on filter paper and weighed. After collection of the bathing fluid into chilled glass centrifuge tubes, 0.1 ml of a solution of propionylcholine iodide (Sigma)
T R O X Y P Y R R O L I D I N I U M ON THE R A T U R I N A R Y BLADDER
(1.5/~g/ml) was added as an internal standard and 0.1 ml of a solution of tetraethylammonium chloride (British Drug Houses) (200 pg/ ml) was added as co-precipitant. Quaternary compounds were precipitated as enneaiodides by addition of 0.3 ml of a potassium iodide : iodine solution (20% KI ( A j a x ) : 1 6 % I2 (Ajax)). The mixture was mixed thoroughly on a vortex mixer and then allowed to sit on ice for 20 min. The supernatant was removed by Pasteur pipette following centrifugation at 3 0 0 0 0 g for 30 rain at --4°C in a Sorval RC2-B refrigerated centrifuge. The precipitate was then dissolved in 0.3 ml of acetonitrile (Merck) and drawn through a small glass column containing an anion exchange resin (BIORAD AG 3-X4A, 100-200 mesh, chloride form) to remove excess iodine and convert iodides of choline esters to chlorides. A 50 pl aliquot of sample was plated onto a platinum ribbon of a pyrolysis probe (Pyroprobe 190, Chemical Data Systems), the solvent being evaporated at 100°C by fixing the ribbon over a hot plate mounted beneath an exhaust hood. The sample was analysed by pyrolysis gas chromatography using a nitrogen detector (Perkin Elmer) operating on a duo
295
chloride (BDH), dyflos (Sigma), hemicholiniurn-3 (Aldrich), hyoscine hydrobromide (Drug Houses Australia), physostigmine sulphate (Sigma), troxypyrrolidinium tosylate (N~thyl-N-2-(3,4,5-trimethoxybenzoyloxy) ethyl pyrrolidinium tosylate) (FWH 428) (Homer).
3. Results
3.1. Effect on spontaneous activity In two thirds of the preparations troxypyrrolidinium did not affect spontaneous activity but in the remainder, amplitude and frequency of spontaneous activity increased up to twofold following 1 h in the presence of the choline uptake inhibitor. In control experiments with no drug present or in preparations treated with hemicholinium-3 no change in the level of spontaneous activity was observed over 1 h. 3.2. Effect of troxypyrrolidinium and hemicholinium-3 on electrical stimulation Troxypyrrolidinium (40-1000 pmol/1) inhibited responses to electrical stimulation at frequencies of 1-100 Hz. Maximal inhibition was obtained at 500/~mol/1, no further reduction being achieved by doubling the concentration of troxypyrrolidinium. With stimulation at a frequency of 50 Hz, troxypyrrolidinium (500/zmol/1) produced a greater inhibition of responses the higher the rate of stimulation. Thus responses produced using a pulse width of 2 msec at a rate of 10 sec every 2 rain were inhibited to a significantly (P < 0.05) greater degree than those produced at 5 sec every 4 rain (table 1). Varying the duration of pulses did not affect the degree of inhibition, the amount of reduction obtained at 0.5 msec duration being not statistically different from that obtained with a pulse width of 2 msec when similar rates of stimulation (10 sec every 2 rain) were compared (table 1) (P < 0.05).
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L.K. CHOO, F. M I T C H E L S O N
TABLE 1 T h e effects o f 1-3 h i n c u b a t i o n w i t h t r o x y p y r r o l i d i n i u m ( 5 0 0 flmol/1) o n t h e r e s p o n s e s t o electrical stimul a t i o n a t 5 0 Hz, w i t h a pulse d u r a t i o n o f e i t h e r 0.5 or 2.0 msec a n d a t d i f f e r e n t rates o f s t i m u l a t i o n . Duration (msec)
Rate (n)
0.5
Responses I after (h)
10 sec/ 2 rain (3) 5 sec/ 4 rain (4) 10 sec/ 4 m i n (4) 10 sec/ 2 m i n (3)
2.0
1
2
3
47.0 -+ 8.0 67.7 -+ 6.3 56.5 + 7.6 40.5 2 + 5.2
38.8 -+ 16.6 65.0 + 5.9 51.9 -+ 6.4 33.4 + 2.0
36.5 -+ 11.0 65.8 -+ 6.0 --37.72 -+ 4.7
I M e a n r e s p o n s e (-+S.E.M.) e x p r e s s e d as % o f c o n t r o l response. All values are significantly d i f f e r e n t (P < 0.05) from controls. 2 Choo and Mitchelson (1977).
%
l~lh
control
r-leh IZI3h
trox
I00' 7550 25.
0 I00,
HC-3
75 50, 25
0
I
I0
50
I00
Hz
Fig. 1. T h e e f f e c t o f t r o x y p y r r o l i d i n i u m a n d hemio c h o l i n i u m - 3 o n t h e r e s p o n s e s t o various f r e q u e n c i e s o f s t i m u l a t i o n o f t h e rat u r i n a r y b l a d d e r over 3 h. D u r i n g i n c u b a t i o n w i t h e i t h e r of t h e c h o l i n e u p t a k e i n h i b i t o r s t h r o u g h o u t t h e 3 h period, p r e p a r a t i o n s were s t i m u l a t e d a t 50 Hz (0.5 m s e c ) for 10 sec every 2 rain. A t t h e e n d o f each h, r e s p o n s e s t o s t i m u l a t i o n at 1, 10, 50 a n d 1 0 0 Hz were established.
When the preparation was stimulated at 50 Hz throughout the incubation period with either troxypyrrolidinium (500pmol/1) or hemicholinium-3 (500#mol/1), and a frequency-response relationship was established after 1, 2 and 3 h, it was found that both antagonists tended to produce a greater degree of inhibition at the higher frequencies of stimulation although the inhibition obtained at 1 Hz and 100 Hz was not statistically significant ( P < 0 . 0 5 ) (fig. l). The degree of inhibition produced by troxypyrrolidinium at any particular frequency was maximal within 1 h, whereas with hemicholinium-3, at the higher frequencies of stimulation (50 and 100 Hz) the inhibition produced was maximal only after 3 h (fig. 1). 3.3. E f f e c t o f choline Responses to electrical stimulation (0.5 msec, 50 Hz, 5 sec every 4 m i n following inhibition by troxypyrrolidinium (500 #mol/1 for 1 h) were reduced to 51.5 + 4.2% (5) of control responses (mean + S.E.M. (n)) and showed a significant ( P < 0.01) reversal of inhibition 1 h after addition of choline (500#mol/1), the response recovering to 74.9 + 2.4% of the control response despite the continued presence of troxypyrrolidinium. In other experiments it was demonstrated that choline (500/~mol/1 for 1 h) did not produce any significant increase in the response to electrical stimulation, the response being 101.1 + 10.8% (3) of the control value. Repeated washing of preparations after removal of troxypyrrolidinium and without addition of choline did not result in any reversal of the inhibition of the response to electrical stimulation in 3 experiments. 3.4. Effect o f troxypyrrolidinium and hemicholinium-3 on the time course o f the response to electrical stimulation Electrical stimulation for 10 sec usually resulted in an immediate increase in tension
TROXYPYRROLIDINIUM ON THE RAT URINARY BLADDER
IOsec
g
cont
I
I0
50
I00
Hz
Fig. 2. Responses of the rat urinary bladder to electrical stimulation for 10 sec at various frequencies (0.5 msec pulse duration), in the absence (cont) or presence of troxypyrrolidinium (trox) (500 /~mol/l for 1 h). During incubation with troxypyrrolidinium the preparation was stimulated continually at 50 Hz for 10 sec every 2 rain.
which rapidly reached a peak height. The peak height was either maintained or showed some degree of waning before the end of the stimulation period, the amount of waning being usually greater at the higher frequencies of stimulation (fig. 2). Thus, at a frequency of 1 Hz. (2 msec pulse duration) the response
TABLE 2 Effect of troxypyrrolidinium (500 /~mol/1) on the 2 parameters of the response of the bladder, Ep and E E at various frequencies of stimulation following electrical stimulation at 50 Hz (0.5 msec) for 20 sec every 2 min for 1 h. Parameter
EE p 3
at the end of a 10 sec period of stimulation was 87.8 + 1.9% (10) (mean + S.E.M. (n)) of its peak response whereas at 5 0 H z the response had waned to 19.1 + 6.9% (10) of its peak height. Thus it is possible to evaluate 2 parameters of the response following electrical stimulation; (i) the peak height obtained designated Ep and (ii) the height of the response at the end of stimulation, EE. At the end of 1 h troxypyrrolidinium (500 #mol/1) exhibited a trend to inhibit the response Ep at the high frequencies of stimulation to a greater extent than the response at the low frequencies although the difference between the values of Ep at 1 and 100 Hz was not significant (P > 0.05) (table 2). Similarly, there was a trend for EE to be inhibited to a greater extent at higher frequencies, the difference between the degree of inhibition at 1 and 100 Hz being significant (P ,= 0.05). It was also noted that troxypyrrolidinium altered the time course of the response to electrical s t i m u l a t i o n , E E was inhibited more than Ep at any given frequency, the difference being significant (P < 0.05) at frequencies of 1 to 100 Hz (table 2, fig. 2). Similar trends were observed with hemicholinium-3 (500 ~mol/1) in that E E w a s inhibited to a significantly greater extent (P < 0.01) than Ep at frequencies of stimulation ranging from 10 to 100 Hz.
Mean response (% of control) 1 Frequency (Hz)
Ep
297
p 2
1
10
50
100
71.7 ±7.6 52.3 ±8.1
69.3 ±0.6 33.3 ±9.3
53.8 ±9.5 11.6 ±6.3
55,2 ±9.8 7.7 ±3.2
<0.05
<0.05
<0.05
<0.05
>0.05 <0.05
I Mean response ± S.E.M. (n = 5), expressed as % of corresponding values for control responses. 2 Significance of the difference between the values at 1 Hz and 100 Hz (paired t-test). 3 Significance of the difference b e t w e e n the values of Ep and E E (paired t-test).
3.5. Effect o f hyoscine on the time course o f the response to electrical stimulation
The peak response Ep exhibited a trend towards a greater inhibition in the presence of hyoscine (25 ~molfl for 1 h) at the higher frequencies of stimulation. Thus at 2 Hz the response in presence of hyoscine was 91.5 + 5.9% of control (4) (mean ± S.E.M. (n)) and at 50 Hz was 73.9 ± 6.0% control (4) although the difference was not significant (P ~> 0.05). The maintained phase of the response Ev showed a similar trend, at 2 Hz the response was 66.4 + 12.7% of control (4) whereas at
298
L.K. CHOO, F. MITCHELSON
TABLE 3 Effect of troxypyrrolidinium (20--1000 pmol/l) on the responses to acetylcholine alone or after dyflos pretreatment (5.4 pmol/l) and to carbachol.
Agonist
Geometric mean dose ratio (95% confidence limits) (n) Concentration of troxypyrrolidinium (pmol/1) 20
100
500
1000
2.57 (4) (1.95--3.40) 2.29 (4) (1.76--2.29)
1.50 (4) (0.50--4.20) 4.65 (4) (4.14--5.23) 7.15 (4) (3.99--12.81)
0.98 (3) (0.89--1.09) 8.30 (4) (6.96--9.91) 25.21 1 (4) (11.93--53.28)
13.83 (4) (10.13--18.89) 47.04 I (4) (20.31--108.95)
Acetylcholine Acetylcholine after dyflos Carbachol
1 p < 0.01: significance of the difference in dose ratio compared to corresponding value for acetylcholine after dyflos pretreatment.
50 Hz the response was abolished, the difference being significant (P < 0.01). As with troxypyrrolidinium the reduction in the magnitude of EE was significantly greater (P < 0.05) than Ep over the frequency range 2-50 Hz. 3.6. Effect o f troxypyrrolidinium on responses to agonists Troxypyrrolidinium (20-1000 pmol/1) shifted the dose-response curves to carbachol rightwards without depression of its maximum but the dose-response curve to acetylcholine was only slightly affected (table 3). However, following dyflos pretreatment (5.4 #mol/1 for 15 min) the sensitivity to acetylcholine was increased 20-fold and the doseresponse curves to the agonist were shifted rightwards in a parallel fashion by troxypyrrolidinium without depression of the maximum response (table 3). The difference in the dose-ratios for troxypyrrolidinium using carbachol and acetyl' choline after dyflos was signficant (P < 0.01) at the higher concentrations of the antagonist (table 3). The slope of the ArunlakshanaSchild plots (Arunlakshana & Schild, 1959) for carbachol was 0.91 and for acetylcholine
after dyflos was 0.52 both being significantly different from a slope of 1 (P < 0.05 and 0.001 respectively). 3.7. Effect o f troxypyrrolidinium, hemicholinium-3 and hyoscine on acetylcholine output During electrical stimulation at 10 Hz, the amount of acetylcholine released (353.9 + 51.1 pmol/g tissue/min: 14) (mean + S.E.M., 14 experiments) was increased 5.7-fold above the amount released spontaneously (61.9 + 7.5 pmol/g tissue/min; 14). Electrical stimulation (50 Hz, for 10 sec every 2 min) in the presence of either troxypyrrolidinium (500pmol/1) or hemicholinium-3 (500 pmolfl) for 1 h caused significant (P < 0.05) reductions in acetylcholine output at 10 Hz to levels similar to that of spontaneous release (table 4). In these experiments in the presence of physostigmine (10 pmol/1), responses to stimulation (Ep) at 10 Hz were reduced to 32.6 + 5.1% of control (4) (mean + S.E.M.) (n)) by troxypyrrolidinium (500 #mol/1) and 35.3 + 8.4% (4) by hemicholinium-3. At the end of an hour's incubation with hyoscine (25 #mol/1), there was a 2.8-fold increase in transmitter output following
TROXYPYRROLIDINIUM ON THE RAT URINARY BLADDER TABLE 4 Effect of troxypyrrolidinium (500 pmol/l), hemicholinium-3 (500 ~/mol/l) and hyoscine (25 pmol/1) on acetylcholine release at 10 Hz. In the experiments with choline uptake inhibitors electrical stimulation for 1 h was carried out with the following parameters; 2 msec, 50 Hz for 10 sec every 2 min. Drug (n)
Acetylcholine release during nerve stimulation (pmol/g tissue/rain)
Before
After drug
pI
65.7 -+ 14.1 34.8 -+ 20.8 1013
<0.05
Hyoscine
502 -+92.2 232.8 + 55.1 357.8
(6)
-+ 80.5
+ 191.1
Troxypyrrolidinium (4) Hemicholinium-3 (4)
<0.05 <0.01
i Significance of the difference before and after the drug (paired t-test).
electrical stimulation (10 Hz), this latter value being significantly different (P < 0.01) from control (table 4).
4. Discussion Troxypyrrolidinium has been shown to be a potent inhibitor of choline uptake in mouse brain with a similar order of potency to hemicholinium-3, the concentration required for 50% inhibition, being 10 and 4 gmol/1 respectively (Bhatnagar et al., 1964). In the rat bladder troxypyrrolidinium (500 gmol/1) produced inhibition of responses to electrical stimulation consistent with an inhibitory action on choline uptake. The blockade developed to a greater extent at faster rates of stimulation and the inhibition was reversed by choline. Similar findings have been made with hemicholinium-3 in the rat bladder (Hukovid et al., 1965; Dhattiwala et al., 1970). At higher frequencies of stimulation (50 and 100 Hz) the inhibition of the response produced by hemicholinium-3 developed more slowly
299
than t h a t produced b y troxypyrrolidinium. The reason f o r this difference is not known. Hemicholinium-3 is a more effective inhibitor of butyrylcholinesterase than of acetylcholinesterase whereas the reverse is found with troxypyrrolidinium (Hemsworth, 1971; Choo and Mitchelson, 1978). The possibility that inhibition of butyrylcholinesterase by hemicholinium-3 and n o t by troxypyrrolidinium partly counteracted the inhibition o f nervous stimulation appears unlikely as iso~)MPA, a selective inhibitor of butyrylcholinesterase, does not potentiate responses of the rat bladder to nervous stimulation or to acetylcholine (Hecker and Mitchelson, 1976). Desensitization of the receptor due to the inhibitory action of troxypyrrolidinium on acetylcholinesterase causing persistence of large amounts of acetylcholine in the receptor region after high frequency stimulation also appears untenable as responses to exogenous acetylcholine were not inhibited by troxypyrrolidinium. An antimuscarinic action o f troxypyrrolidinium is exhibited when carbachol is used as the agonist and in the presence o f dyflos some weak antimuscarinic activity was observed using acetylcholine as agonist. Similar findings have been made with hemicholinium-3 in rat bladder (GySrgy et al., 1970; Hecker and Mitchelson, 1976). Both troxypyrrolidinium and hemicholinium-3 inhibit carbachol to a greater extent than acetylcholine in other tissues and the antimuscarinic effect has been suggested to involve non-competitive inhibition (Bieger et al., 1968; Choo and Mitchelson, 1978). The finding that Arunlakshana~qchild plots have slopes significantly less than u n i t y would suggest t h a t the antimuscarinic action o f troxypyrrolidinium in the bladder also does n o t involve competitive inhibition. The response to electrical stimulation is neuronally mediated as responses are inhibited by cinchocaine (Choo and Mitchelson, 1977) and yet troxypyrrolidinium or hemicholiniurn-3 failed to produce complete inhibition of the response to electrical stimulation and had the greatest effect on the late phase of the
300 response to a 10 sec period of stimulation. Measurement of the response at the end of the stimulation period provided some measure of what is generally described as the 'tonic component' of the response (De Groat and Saum, 1976; Ticku and Triggie, 1976) as opposed to the immediate or 'phasic component'. Hyoscine also caused selective inhibition of the tonic phase in agreement with previous findings using atropine in rat bladder (Carpenter, 1977) or cat bladder (De Groat and Saum, 1976). Carpenter (1977) explained the effect of atropine solely in terms of a cholinergic innervation. With prolonged stimulation particularly at a high frequency, diffusion of transmitter to receptors lying some distance away from the nerve endings is more likely and if these receptors are also more accessible to atropine and exogenous acetylcholine, then atropine-resistance of the 'phasic' c o m p o n e n t of the response is explainable as this would involve receptors immediately adjacent to the nerve ending. This explanation which is also supported b y Elmer (1978), implies that there is a limited access of atropine and other drugs applied exogenously to the receptors activated b y nerve stimulation, particularly at low frequency. De Groat and Saum (1976) suggested that a second transmitter could be responsible for the phasic c o m p o n e n t of the response as the 'tonic' phase was selectively blocked b y relatively low doses o f atropine. The findings with troxypyrrolidinium or hemicholinium-3 were similar to those obtained with hyoscine and as the 2 quaternary compounds acted on nerve endings to reduce responses to stimulation the possibility of some physical barrier inhibiting access of exogenous drugs would appear to be eliminated. Downie and Dean (1977) also suggested the existence of a non-cholinergic c o m p o n e n t in the excitatory innervation of the rabbit detrusor on the basis of experiments with hemicholinium-3. Further evidence in favour of a non-cholinergic excitatory innervation being present was that release of transmitter b y stimulation at
L.K. CHOO, F. MITCHELSON 10 Hz was also inhibited b y troxypyrrolidinitun or hemicholinium-3 to less than that obtained b y spontaneous release and yet responses to nervous stimulation were still present. Kilbinger (1977) has recently demonstrated the presence of inhibitory muscarinic receptors on parasympathetic nerve endings in guinea-pig ileum and that atropine can increase the o u t p u t of transmitter in this tissue. In the bladder, hyoscine markedly increased the output of transmitter, a finding which indicates that hyoscine does have access to the nerve ending, a region which is in close proximity to the receptor region in bladder as every muscle cell is innervated (E1-Badawi and Schenk, 1966). The nature of other excitatory transmitters in the rat bladder is u n k n o w n b u t Burnstock et al. (1978) have suggested that ATP is an excitatory transmitter in the guinea-pig urinary bladder and ATP may have a similar role in the rat bladder (Brown et al., 1979). Spontaneous activity in the rat bladder is not inhibited b y hyoscine b u t is inhibited b y indomethacin and restored b y prostaglandins (Hills, 1976; Choo and Mitchelson, 1977). The finding that troxypyrrolidinium or hemicholinium-3 did not inhibit spontaneous activity provides further support for the lack of any cholinergic involvement in this phenomenon in the rat in contrast to findings in the cat and rabbit where spontaneous activity has been reported to be reduced b y atropine (De Groat and Saum, (1976) or hemicholinium-3 (Bultitude et al., 1976) respectively.
Acknowledgements A gift of troxypyrrolidinium tosylate supplied by F.W. Homer, Montreal, is gratefully acknowledged.
References Arunlakshana, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonists, Br. J. Pharmacol. Chemother. 14, 48.
TROXYPYRROLIDINIUM ON THE RAT URINARY BLADDER Bie.g.er, D.H., H. Liillmann and O. Wasserman, 1968, Uber eine akute Anti-Acetylcholin-Wirkung yon Hemicholinium No 3 am Vorhof des Meerschweinchens, Naunyn-Schmiedeb. Arch. Pharmakol. Exp. Pathol. 259, 386. Bhatnagar, S.P., A. Lam and J.D. McColl, 1964, Inhibition of synthesis of acetylcholine by some esters of trimethoxybenzoic acid, Nature 204, 485. Brown, C., G. Burnstock and T. Cocks, 1979, Effects of adenosine-5t-triphosphate (ATP) and ~-7-memethylene ATP on the rat urinary bladder, Br. J. Pharmacol. 65, 97. Bultitude, M.I., N.H. Hills and K.E.D. Shuttleworth, 1976, Clinical and experimental studies on the action of prostaglandins and their synthesis inhibitors on detrusor muscle in vitro and in vivo, Br. J. Urol. 4 8 , 6 3 1 . Burnstock, G., T. Cocks, R. Crowe and L. Kasakov, 1978, Purinergic innervation of the guinea-pig urinary bladder, Br. J. Pharmacol. 6 3 , 1 2 5 . Carpenter, F.G., 1977, Atropine resistance and muscarinic receptors in the rat urinary bladder, Br. J. Pharmacol. 59, 43. Ch~nier, L.P., A. Lam et J.D. McCoU, 1969, Effet du troxypyrrolidinium sur la teneur en ac~tylcholine de divers muscles du chat, Rev. Can. Biol. 28, 223. Choo, L.K. and F. Mitchelson, 1977, The role of prostaglandins in the excitatory innervation of the rat urinary bladder, Prostaglandins 13, 917. Choo, L.K. and F. Mitchelson, 1978, Antagonism of cholinomimetics b y troxypyrrolidinium in guineapig atria and longitudinal ileal muscle: comparison with hemicholinium-3, European J. Pharmacol. 52,313. De Groat, W.C. and W.R. Saum, 1976, Synaptic transmission in parasympathetic ganglia in the urinary bladder of the cat, J. Physiol. 2 5 6 , 1 3 7 . Dhattiwala, A.S., M.N. Jindal and V.V. Kelkar, 1970, Effect of some drugs on the responses of the rat isolated innervated urinary bladder to indirect electrical stimulation, Br. J. Pharmacol. 39,738.
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Downie, J.W. and D.M. Dean, 1977, The contribution of cholinergic postganglionic neurotransmission to contractions of rabbit detrusor, J. Pharmacol. Exp. Ther. 203,417. Dumsday, B., 1970, Atropine-resistance of the urinary bladder innervation, J. Pharm. Pharmacol. 23, 222. EI-Badawi, A. and E.A. Schenk, 1966, Dual innervation of the mammalian urinary bladder. A histochemical study of the distribution of cholinergic and adrenergic nerves, Am. J. Anat. 1 1 9 , 4 0 5 . Elmer, M., 1978, Cholinergic mechanisms in the rat detrusor muscle, Acta Phsrmacol. Toxicol. 43, SII, 63. Gy6rgy, L., A.K. Pfeifer and J. Kenyeres, 1970, The interaction of hemicholinium-3 a n d oxotremorine in isolated organ preparations, J. Pharm. Pharmacol. 22, 96. Hecker, S.E. and F. Mitchelson, 1976, The effect of cholinesterase inhibitors on the antimuscar/nic effect of hemicholinium-3 (HC-3) in the rat, J. Pharm. Pharmacol. 2 8 , 4 4 1 . Hemsworth, B.A., 1971, The effect of a hemicholinium analogue, HC-15 on neuromuscular transmission, European J. Pharmacol. 15, 91. Hills, N.H., 1976, Prostaglandins and tone in isolated strips of mammalian bladder, Br. J. Pharmacol. 57, 464 P. Hukovi~, S., M.J. Rand and S. Vanov, 1965, Observations on an isolated innervated preparation of rat urinary bladder, Br. J. Pharmacol. 2 4 , 1 7 8 . Kilbinger, H., 1977, Modulation by oxotremorine and atropine of acetylcholine release evoked by electrical stimulation of the myenteric plexus of the guinea-pig ileum, Naunyn-Schmiedeb. Arch. Pharmacol. 300, 145. Marshall, I.G., 1969, The effect o f some hemicholiniurn-like substances on the chick biventer cervicis muscle preparation, European J. Pharmacol. 8, 204. Ticku, M.K. and D.J. Triggle, 1976, Calcium and the muscarinic receptor, Gen. Pharmacol. 7 , 1 3 3 .
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© Elsevier/North-Holland Biomedical Press
THE EFFECT OF TROXYPYRROLIDINIUM, A CHOLINE UPTAKE INHIBITOR, ON THE EXCITATORY INNERVATION OF THE RAT URINARY BLADDER L.K. CHOO and F. MITCHELSON
Department of Pharmacology, Victorian College of Pharmacy, Park ville, Victoria, 3052 Australia Received 2 October 1979, accepted 6 November 1979
L.K. CHOO and F. MITCHELSON, The effect of troxypyrrolidinium, a choline uptake inhibitor, on the excitatory innervation o f the rat urinary bladder, European J. Pharmacol. 61 (1980) 293--301. Troxypyrrolidinium, a choline uptake inhibitor, reduced but failed to abolish responses of the rat urinary bladder to electrical stimulation at 1-100 Hz although it reduced acetylcholine output during stimulation at 10 Hz to a level similar to that of spontaneous release. Inhibition of the response to stimulation was more complete at faster rates of stimulation and was partially reversed by choline. Troxypyrrolidinium produced a greater inhibition of the 'tonic' component of the response to electrical stimulation than of the 'phasic' component. Hemicholinium-3 or hyoscine produced a similar selective effect on the 'tonic' component. Hemicholinium-3 also reduced acetylcholine o u t p u t during electrical stimulation to a similar extent as troxypyrrolidinium but hyoscine increased transmitter output. The results support the concept of a second transmitter in the excitatory innervation of the bladder. Troxypy~rolidinium
Rat urinary bladder
Cholinergic innervation
I. Introduction
Troxypyrrolidinium tosylate (N-ethyl-N-2(3,4,5-trimethoxybenzoyloxy)-ethyl pyrrolidinium tosylate, FWH 428) is a choline uptake inhibitor, structurally different to the hemicholiniums (Bhatnager et al., 1964; Chdnier et al., 1969) and in avian muscle has been suggested to act more selectively than hemicholinium-3 at prejunctional sites (Marshall, 1969). An investigation of the effect of troxypyrrolidinium on the responses to electrical stimulation of the urinary bladder of the rat was undertaken to determine whether the excitatory innervation is wholly cholinergic (Carpenter, 1977; Elmdr, 1978) or may also contain a non-cholinergic component (Dumsday, 1970). In the rat bladder hemicholinium-3 inhibits the response to nervous stimulation (Hukovid et al., 1965; Dhattiwala et al., 1970) although there is no reduction in the amount
Hemicholinium-3
of acetylcholine present in the tissue (Hukovid et al., 1965).
2. Materials and methods 2.1. Isolated tissue experiments Isolated urinary bladders from GlaxoWistar rats (200-350g) were set up for electrical stimulation according to the method of Hukovi~et al. (1965) under a resting tension of 0.5-1 g in a 10 ml organ bath in McEwen's solutton maintained at 37°C. Responses were recorded on a polygraph via a Grass force
294
were measured as a percentage o f the control response in the absence of any drug at each corresponding frequency unless otherwise stated. In control experiments it was demonstrated that preparations could be stimulated for at least 3 h with no decline in responsiveness. Thus, after 1, 2 or 3 h of stimulation at 50 Hz (2 msec pulses for 10 sec every 2 min) responses were 98.5 + 6.1% (4) (mean + S.E.M. (n)), 101.1 + 8.6% (4) and 94.5 + 8.5% (4) of the control response respectively. Agonists were added to the bath in a 6 min dose cycle and remained in contact with the tissue for 60 sec (acetylcholine) or 90--120 sec (carbachol). For the determination of dose ratios the tissue was incubated with troxypyrrolidinium for 20 min to allow equilibrium to be attained before re-determining responses to agonists and was re-added to the bathing fluid immediately following each washing of the tissue. Some experiments were conducted after dyflos pretreatment, the tissue being incubated with dyflos (5.4 pmol/1) for 15 min, then washed several times to remove excess anticholinesterase. Responses to agonists were then re
2.2. Determination of acetylcholine release In these experiments bladders were set up as described above except that b o t h ureters were dissected o u t and a longitudinal cut was made from the urethral orifice upwards along three-quarters of the length of the bladder wall. This procedure enabled the 2 ureters to be drawn together through a single bipolar electrode and allowed ready diffusion of the released transmitter from both sides of the bladder wall into the bathing fluid. The preparation was placed in an organ bath conraining 4 ml of Tyrode solution of the following composition (mmol/1): NaC1 137, KC1 2.7, CaC12 1.8, MgC12 1.0, NaH2PO4 0.4, NaHCO3 11.9, dextrose 5.6. Physostigmine (10 ~mol]l) was added and the tissue equilibrated for 60 min, the physostigmine being replaced
L.K. CHOO, F. MITCHELSON
every 15-20 min throughout the course of the experiment unless an assay sample was being collected. Spontaneously released acetylcholine was allowed to accumulate in the bathing fluid for 30 min before collection for assay. Release during electrical stimulation was determined b y collection of the bathing fluid following continuous stimulation of the preparation for 10 min at 10 Hz with 2 msec pulses at supramaximal voltage. To determine the effect of troxypyrrolidinium or hemicholinium-3 on the release of transmitter, the preparation was incubated with the choline uptake inhibitors for 60 min during which time the preparation was stimulated with 2 msec pulses at supramaximal voltage at a frequency of 50 Hz for 10 sec every 2 min. The preparation was then washed several times and again stimulated at 10 Hz for 10 min and the bathing fluid collected for assay. In control experiments, with no drugs added the acetylcholine o u t p u t in the second collection period was 81.0 + 13.7% (4) of that in the initial collection period and did not differ significantly ( P < 0.05) from it. In experiments with the choline uptake inhibitots, during the second period of stimulation troxypyrrolidinium or hemicholinium-3 was not present in the bath as preliminary experiments demonstrated that the high concentration of troxypyrrolidinium or hemicholinium-3 interfered with the subsequent gas chromatographic estimation of acetylcholine output, the chromatograms of extracts containing the uptake inhibitors showing numerous additional peaks which tailed into the peaks for demethylated acetylcholine and propionylcholine. Furthermore, removal of the choline uptake inhibitor does n o t produce any recovery of the responses to electrical stimulation within 30 min using either hemicholinium-3 (Dhattiwala et al., 1970) or troxypyrrolidinium (see Results 3.3.). At the end o f the experiment, bladders were trimmed free of the ureters, blotted dry on filter paper and weighed. After collection of the bathing fluid into chilled glass centrifuge tubes, 0.1 ml of a solution of propionylcholine iodide (Sigma)
T R O X Y P Y R R O L I D I N I U M ON THE R A T U R I N A R Y BLADDER
(1.5/~g/ml) was added as an internal standard and 0.1 ml of a solution of tetraethylammonium chloride (British Drug Houses) (200 pg/ ml) was added as co-precipitant. Quaternary compounds were precipitated as enneaiodides by addition of 0.3 ml of a potassium iodide : iodine solution (20% KI ( A j a x ) : 1 6 % I2 (Ajax)). The mixture was mixed thoroughly on a vortex mixer and then allowed to sit on ice for 20 min. The supernatant was removed by Pasteur pipette following centrifugation at 3 0 0 0 0 g for 30 rain at --4°C in a Sorval RC2-B refrigerated centrifuge. The precipitate was then dissolved in 0.3 ml of acetonitrile (Merck) and drawn through a small glass column containing an anion exchange resin (BIORAD AG 3-X4A, 100-200 mesh, chloride form) to remove excess iodine and convert iodides of choline esters to chlorides. A 50 pl aliquot of sample was plated onto a platinum ribbon of a pyrolysis probe (Pyroprobe 190, Chemical Data Systems), the solvent being evaporated at 100°C by fixing the ribbon over a hot plate mounted beneath an exhaust hood. The sample was analysed by pyrolysis gas chromatography using a nitrogen detector (Perkin Elmer) operating on a duo
295
chloride (BDH), dyflos (Sigma), hemicholiniurn-3 (Aldrich), hyoscine hydrobromide (Drug Houses Australia), physostigmine sulphate (Sigma), troxypyrrolidinium tosylate (N~thyl-N-2-(3,4,5-trimethoxybenzoyloxy) ethyl pyrrolidinium tosylate) (FWH 428) (Homer).
3. Results
3.1. Effect on spontaneous activity In two thirds of the preparations troxypyrrolidinium did not affect spontaneous activity but in the remainder, amplitude and frequency of spontaneous activity increased up to twofold following 1 h in the presence of the choline uptake inhibitor. In control experiments with no drug present or in preparations treated with hemicholinium-3 no change in the level of spontaneous activity was observed over 1 h. 3.2. Effect of troxypyrrolidinium and hemicholinium-3 on electrical stimulation Troxypyrrolidinium (40-1000 pmol/1) inhibited responses to electrical stimulation at frequencies of 1-100 Hz. Maximal inhibition was obtained at 500/~mol/1, no further reduction being achieved by doubling the concentration of troxypyrrolidinium. With stimulation at a frequency of 50 Hz, troxypyrrolidinium (500/zmol/1) produced a greater inhibition of responses the higher the rate of stimulation. Thus responses produced using a pulse width of 2 msec at a rate of 10 sec every 2 rain were inhibited to a significantly (P < 0.05) greater degree than those produced at 5 sec every 4 rain (table 1). Varying the duration of pulses did not affect the degree of inhibition, the amount of reduction obtained at 0.5 msec duration being not statistically different from that obtained with a pulse width of 2 msec when similar rates of stimulation (10 sec every 2 rain) were compared (table 1) (P < 0.05).
296
L.K. CHOO, F. M I T C H E L S O N
TABLE 1 T h e effects o f 1-3 h i n c u b a t i o n w i t h t r o x y p y r r o l i d i n i u m ( 5 0 0 flmol/1) o n t h e r e s p o n s e s t o electrical stimul a t i o n a t 5 0 Hz, w i t h a pulse d u r a t i o n o f e i t h e r 0.5 or 2.0 msec a n d a t d i f f e r e n t rates o f s t i m u l a t i o n . Duration (msec)
Rate (n)
0.5
Responses I after (h)
10 sec/ 2 rain (3) 5 sec/ 4 rain (4) 10 sec/ 4 m i n (4) 10 sec/ 2 m i n (3)
2.0
1
2
3
47.0 -+ 8.0 67.7 -+ 6.3 56.5 + 7.6 40.5 2 + 5.2
38.8 -+ 16.6 65.0 + 5.9 51.9 -+ 6.4 33.4 + 2.0
36.5 -+ 11.0 65.8 -+ 6.0 --37.72 -+ 4.7
I M e a n r e s p o n s e (-+S.E.M.) e x p r e s s e d as % o f c o n t r o l response. All values are significantly d i f f e r e n t (P < 0.05) from controls. 2 Choo and Mitchelson (1977).
%
l~lh
control
r-leh IZI3h
trox
I00' 7550 25.
0 I00,
HC-3
75 50, 25
0
I
I0
50
I00
Hz
Fig. 1. T h e e f f e c t o f t r o x y p y r r o l i d i n i u m a n d hemio c h o l i n i u m - 3 o n t h e r e s p o n s e s t o various f r e q u e n c i e s o f s t i m u l a t i o n o f t h e rat u r i n a r y b l a d d e r over 3 h. D u r i n g i n c u b a t i o n w i t h e i t h e r of t h e c h o l i n e u p t a k e i n h i b i t o r s t h r o u g h o u t t h e 3 h period, p r e p a r a t i o n s were s t i m u l a t e d a t 50 Hz (0.5 m s e c ) for 10 sec every 2 rain. A t t h e e n d o f each h, r e s p o n s e s t o s t i m u l a t i o n at 1, 10, 50 a n d 1 0 0 Hz were established.
When the preparation was stimulated at 50 Hz throughout the incubation period with either troxypyrrolidinium (500pmol/1) or hemicholinium-3 (500#mol/1), and a frequency-response relationship was established after 1, 2 and 3 h, it was found that both antagonists tended to produce a greater degree of inhibition at the higher frequencies of stimulation although the inhibition obtained at 1 Hz and 100 Hz was not statistically significant ( P < 0 . 0 5 ) (fig. l). The degree of inhibition produced by troxypyrrolidinium at any particular frequency was maximal within 1 h, whereas with hemicholinium-3, at the higher frequencies of stimulation (50 and 100 Hz) the inhibition produced was maximal only after 3 h (fig. 1). 3.3. E f f e c t o f choline Responses to electrical stimulation (0.5 msec, 50 Hz, 5 sec every 4 m i n following inhibition by troxypyrrolidinium (500 #mol/1 for 1 h) were reduced to 51.5 + 4.2% (5) of control responses (mean + S.E.M. (n)) and showed a significant ( P < 0.01) reversal of inhibition 1 h after addition of choline (500#mol/1), the response recovering to 74.9 + 2.4% of the control response despite the continued presence of troxypyrrolidinium. In other experiments it was demonstrated that choline (500/~mol/1 for 1 h) did not produce any significant increase in the response to electrical stimulation, the response being 101.1 + 10.8% (3) of the control value. Repeated washing of preparations after removal of troxypyrrolidinium and without addition of choline did not result in any reversal of the inhibition of the response to electrical stimulation in 3 experiments. 3.4. Effect o f troxypyrrolidinium and hemicholinium-3 on the time course o f the response to electrical stimulation Electrical stimulation for 10 sec usually resulted in an immediate increase in tension
TROXYPYRROLIDINIUM ON THE RAT URINARY BLADDER
IOsec
g
cont
I
I0
50
I00
Hz
Fig. 2. Responses of the rat urinary bladder to electrical stimulation for 10 sec at various frequencies (0.5 msec pulse duration), in the absence (cont) or presence of troxypyrrolidinium (trox) (500 /~mol/l for 1 h). During incubation with troxypyrrolidinium the preparation was stimulated continually at 50 Hz for 10 sec every 2 rain.
which rapidly reached a peak height. The peak height was either maintained or showed some degree of waning before the end of the stimulation period, the amount of waning being usually greater at the higher frequencies of stimulation (fig. 2). Thus, at a frequency of 1 Hz. (2 msec pulse duration) the response
TABLE 2 Effect of troxypyrrolidinium (500 /~mol/1) on the 2 parameters of the response of the bladder, Ep and E E at various frequencies of stimulation following electrical stimulation at 50 Hz (0.5 msec) for 20 sec every 2 min for 1 h. Parameter
EE p 3
at the end of a 10 sec period of stimulation was 87.8 + 1.9% (10) (mean + S.E.M. (n)) of its peak response whereas at 5 0 H z the response had waned to 19.1 + 6.9% (10) of its peak height. Thus it is possible to evaluate 2 parameters of the response following electrical stimulation; (i) the peak height obtained designated Ep and (ii) the height of the response at the end of stimulation, EE. At the end of 1 h troxypyrrolidinium (500 #mol/1) exhibited a trend to inhibit the response Ep at the high frequencies of stimulation to a greater extent than the response at the low frequencies although the difference between the values of Ep at 1 and 100 Hz was not significant (P > 0.05) (table 2). Similarly, there was a trend for EE to be inhibited to a greater extent at higher frequencies, the difference between the degree of inhibition at 1 and 100 Hz being significant (P ,= 0.05). It was also noted that troxypyrrolidinium altered the time course of the response to electrical s t i m u l a t i o n , E E was inhibited more than Ep at any given frequency, the difference being significant (P < 0.05) at frequencies of 1 to 100 Hz (table 2, fig. 2). Similar trends were observed with hemicholinium-3 (500 ~mol/1) in that E E w a s inhibited to a significantly greater extent (P < 0.01) than Ep at frequencies of stimulation ranging from 10 to 100 Hz.
Mean response (% of control) 1 Frequency (Hz)
Ep
297
p 2
1
10
50
100
71.7 ±7.6 52.3 ±8.1
69.3 ±0.6 33.3 ±9.3
53.8 ±9.5 11.6 ±6.3
55,2 ±9.8 7.7 ±3.2
<0.05
<0.05
<0.05
<0.05
>0.05 <0.05
I Mean response ± S.E.M. (n = 5), expressed as % of corresponding values for control responses. 2 Significance of the difference between the values at 1 Hz and 100 Hz (paired t-test). 3 Significance of the difference b e t w e e n the values of Ep and E E (paired t-test).
3.5. Effect o f hyoscine on the time course o f the response to electrical stimulation
The peak response Ep exhibited a trend towards a greater inhibition in the presence of hyoscine (25 ~molfl for 1 h) at the higher frequencies of stimulation. Thus at 2 Hz the response in presence of hyoscine was 91.5 + 5.9% of control (4) (mean ± S.E.M. (n)) and at 50 Hz was 73.9 ± 6.0% control (4) although the difference was not significant (P ~> 0.05). The maintained phase of the response Ev showed a similar trend, at 2 Hz the response was 66.4 + 12.7% of control (4) whereas at
298
L.K. CHOO, F. MITCHELSON
TABLE 3 Effect of troxypyrrolidinium (20--1000 pmol/l) on the responses to acetylcholine alone or after dyflos pretreatment (5.4 pmol/l) and to carbachol.
Agonist
Geometric mean dose ratio (95% confidence limits) (n) Concentration of troxypyrrolidinium (pmol/1) 20
100
500
1000
2.57 (4) (1.95--3.40) 2.29 (4) (1.76--2.29)
1.50 (4) (0.50--4.20) 4.65 (4) (4.14--5.23) 7.15 (4) (3.99--12.81)
0.98 (3) (0.89--1.09) 8.30 (4) (6.96--9.91) 25.21 1 (4) (11.93--53.28)
13.83 (4) (10.13--18.89) 47.04 I (4) (20.31--108.95)
Acetylcholine Acetylcholine after dyflos Carbachol
1 p < 0.01: significance of the difference in dose ratio compared to corresponding value for acetylcholine after dyflos pretreatment.
50 Hz the response was abolished, the difference being significant (P < 0.01). As with troxypyrrolidinium the reduction in the magnitude of EE was significantly greater (P < 0.05) than Ep over the frequency range 2-50 Hz. 3.6. Effect o f troxypyrrolidinium on responses to agonists Troxypyrrolidinium (20-1000 pmol/1) shifted the dose-response curves to carbachol rightwards without depression of its maximum but the dose-response curve to acetylcholine was only slightly affected (table 3). However, following dyflos pretreatment (5.4 #mol/1 for 15 min) the sensitivity to acetylcholine was increased 20-fold and the doseresponse curves to the agonist were shifted rightwards in a parallel fashion by troxypyrrolidinium without depression of the maximum response (table 3). The difference in the dose-ratios for troxypyrrolidinium using carbachol and acetyl' choline after dyflos was signficant (P < 0.01) at the higher concentrations of the antagonist (table 3). The slope of the ArunlakshanaSchild plots (Arunlakshana & Schild, 1959) for carbachol was 0.91 and for acetylcholine
after dyflos was 0.52 both being significantly different from a slope of 1 (P < 0.05 and 0.001 respectively). 3.7. Effect o f troxypyrrolidinium, hemicholinium-3 and hyoscine on acetylcholine output During electrical stimulation at 10 Hz, the amount of acetylcholine released (353.9 + 51.1 pmol/g tissue/min: 14) (mean + S.E.M., 14 experiments) was increased 5.7-fold above the amount released spontaneously (61.9 + 7.5 pmol/g tissue/min; 14). Electrical stimulation (50 Hz, for 10 sec every 2 min) in the presence of either troxypyrrolidinium (500pmol/1) or hemicholinium-3 (500 pmolfl) for 1 h caused significant (P < 0.05) reductions in acetylcholine output at 10 Hz to levels similar to that of spontaneous release (table 4). In these experiments in the presence of physostigmine (10 pmol/1), responses to stimulation (Ep) at 10 Hz were reduced to 32.6 + 5.1% of control (4) (mean + S.E.M.) (n)) by troxypyrrolidinium (500 #mol/1) and 35.3 + 8.4% (4) by hemicholinium-3. At the end of an hour's incubation with hyoscine (25 #mol/1), there was a 2.8-fold increase in transmitter output following
TROXYPYRROLIDINIUM ON THE RAT URINARY BLADDER TABLE 4 Effect of troxypyrrolidinium (500 pmol/l), hemicholinium-3 (500 ~/mol/l) and hyoscine (25 pmol/1) on acetylcholine release at 10 Hz. In the experiments with choline uptake inhibitors electrical stimulation for 1 h was carried out with the following parameters; 2 msec, 50 Hz for 10 sec every 2 min. Drug (n)
Acetylcholine release during nerve stimulation (pmol/g tissue/rain)
Before
After drug
pI
65.7 -+ 14.1 34.8 -+ 20.8 1013
<0.05
Hyoscine
502 -+92.2 232.8 + 55.1 357.8
(6)
-+ 80.5
+ 191.1
Troxypyrrolidinium (4) Hemicholinium-3 (4)
<0.05 <0.01
i Significance of the difference before and after the drug (paired t-test).
electrical stimulation (10 Hz), this latter value being significantly different (P < 0.01) from control (table 4).
4. Discussion Troxypyrrolidinium has been shown to be a potent inhibitor of choline uptake in mouse brain with a similar order of potency to hemicholinium-3, the concentration required for 50% inhibition, being 10 and 4 gmol/1 respectively (Bhatnagar et al., 1964). In the rat bladder troxypyrrolidinium (500 gmol/1) produced inhibition of responses to electrical stimulation consistent with an inhibitory action on choline uptake. The blockade developed to a greater extent at faster rates of stimulation and the inhibition was reversed by choline. Similar findings have been made with hemicholinium-3 in the rat bladder (Hukovid et al., 1965; Dhattiwala et al., 1970). At higher frequencies of stimulation (50 and 100 Hz) the inhibition of the response produced by hemicholinium-3 developed more slowly
299
than t h a t produced b y troxypyrrolidinium. The reason f o r this difference is not known. Hemicholinium-3 is a more effective inhibitor of butyrylcholinesterase than of acetylcholinesterase whereas the reverse is found with troxypyrrolidinium (Hemsworth, 1971; Choo and Mitchelson, 1978). The possibility that inhibition of butyrylcholinesterase by hemicholinium-3 and n o t by troxypyrrolidinium partly counteracted the inhibition o f nervous stimulation appears unlikely as iso~)MPA, a selective inhibitor of butyrylcholinesterase, does not potentiate responses of the rat bladder to nervous stimulation or to acetylcholine (Hecker and Mitchelson, 1976). Desensitization of the receptor due to the inhibitory action of troxypyrrolidinium on acetylcholinesterase causing persistence of large amounts of acetylcholine in the receptor region after high frequency stimulation also appears untenable as responses to exogenous acetylcholine were not inhibited by troxypyrrolidinium. An antimuscarinic action o f troxypyrrolidinium is exhibited when carbachol is used as the agonist and in the presence o f dyflos some weak antimuscarinic activity was observed using acetylcholine as agonist. Similar findings have been made with hemicholinium-3 in rat bladder (GySrgy et al., 1970; Hecker and Mitchelson, 1976). Both troxypyrrolidinium and hemicholinium-3 inhibit carbachol to a greater extent than acetylcholine in other tissues and the antimuscarinic effect has been suggested to involve non-competitive inhibition (Bieger et al., 1968; Choo and Mitchelson, 1978). The finding that Arunlakshana~qchild plots have slopes significantly less than u n i t y would suggest t h a t the antimuscarinic action o f troxypyrrolidinium in the bladder also does n o t involve competitive inhibition. The response to electrical stimulation is neuronally mediated as responses are inhibited by cinchocaine (Choo and Mitchelson, 1977) and yet troxypyrrolidinium or hemicholiniurn-3 failed to produce complete inhibition of the response to electrical stimulation and had the greatest effect on the late phase of the
300 response to a 10 sec period of stimulation. Measurement of the response at the end of the stimulation period provided some measure of what is generally described as the 'tonic component' of the response (De Groat and Saum, 1976; Ticku and Triggie, 1976) as opposed to the immediate or 'phasic component'. Hyoscine also caused selective inhibition of the tonic phase in agreement with previous findings using atropine in rat bladder (Carpenter, 1977) or cat bladder (De Groat and Saum, 1976). Carpenter (1977) explained the effect of atropine solely in terms of a cholinergic innervation. With prolonged stimulation particularly at a high frequency, diffusion of transmitter to receptors lying some distance away from the nerve endings is more likely and if these receptors are also more accessible to atropine and exogenous acetylcholine, then atropine-resistance of the 'phasic' c o m p o n e n t of the response is explainable as this would involve receptors immediately adjacent to the nerve ending. This explanation which is also supported b y Elmer (1978), implies that there is a limited access of atropine and other drugs applied exogenously to the receptors activated b y nerve stimulation, particularly at low frequency. De Groat and Saum (1976) suggested that a second transmitter could be responsible for the phasic c o m p o n e n t of the response as the 'tonic' phase was selectively blocked b y relatively low doses o f atropine. The findings with troxypyrrolidinium or hemicholinium-3 were similar to those obtained with hyoscine and as the 2 quaternary compounds acted on nerve endings to reduce responses to stimulation the possibility of some physical barrier inhibiting access of exogenous drugs would appear to be eliminated. Downie and Dean (1977) also suggested the existence of a non-cholinergic c o m p o n e n t in the excitatory innervation of the rabbit detrusor on the basis of experiments with hemicholinium-3. Further evidence in favour of a non-cholinergic excitatory innervation being present was that release of transmitter b y stimulation at
L.K. CHOO, F. MITCHELSON 10 Hz was also inhibited b y troxypyrrolidinitun or hemicholinium-3 to less than that obtained b y spontaneous release and yet responses to nervous stimulation were still present. Kilbinger (1977) has recently demonstrated the presence of inhibitory muscarinic receptors on parasympathetic nerve endings in guinea-pig ileum and that atropine can increase the o u t p u t of transmitter in this tissue. In the bladder, hyoscine markedly increased the output of transmitter, a finding which indicates that hyoscine does have access to the nerve ending, a region which is in close proximity to the receptor region in bladder as every muscle cell is innervated (E1-Badawi and Schenk, 1966). The nature of other excitatory transmitters in the rat bladder is u n k n o w n b u t Burnstock et al. (1978) have suggested that ATP is an excitatory transmitter in the guinea-pig urinary bladder and ATP may have a similar role in the rat bladder (Brown et al., 1979). Spontaneous activity in the rat bladder is not inhibited b y hyoscine b u t is inhibited b y indomethacin and restored b y prostaglandins (Hills, 1976; Choo and Mitchelson, 1977). The finding that troxypyrrolidinium or hemicholinium-3 did not inhibit spontaneous activity provides further support for the lack of any cholinergic involvement in this phenomenon in the rat in contrast to findings in the cat and rabbit where spontaneous activity has been reported to be reduced b y atropine (De Groat and Saum, (1976) or hemicholinium-3 (Bultitude et al., 1976) respectively.
Acknowledgements A gift of troxypyrrolidinium tosylate supplied by F.W. Homer, Montreal, is gratefully acknowledged.
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