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A comparative study of electrical field stimulation of the guinea-pig, ferret and marmoset urinary bladder
European Journal of Pharmacology, 114 (1985) 311-316 Elsevier
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A C O M P A R A T I V E S T U D Y O F E L E C T R I C A L F I E L D S T I M U L A T I O N OF T H E G U I N E A - P I G , F E R R E T AND M A R M O S E T URINARY B L A D D E R H.E. MOSS and G. BURNSTOCK * Department of Anatomy and Embryology, and Centrefor Neuroscience, University College London, GowerStreet, London WCIE 6BT, United Kingdom Received 28 February 1985, revised MS received 21 May 1985, accepted 4 June 1985
H.E. MOSS and G. BURNSTOCK, A comparative study of electrical fieM stimulation of the guinea-pig, ferret and marmoset urinary bladder, European J. Pharmacol. 114 (1985) 311-316. The spontaneous and electrically evoked activity was examined in guinea-pig, ferret and marmoset urinary bladder. Electrical field stimulation of detrusor strips in vitro induced a rapid, frequency-dependent contraction with a maximum response at 40 Hz. This contraction was partly decreased by either atropine (0.29 #M) or desensitisation to a,fl-methylene ATP (a,fl-MeATP) (5 #M), and was totally blocked by a combination of the two. Atropine blocked responses to carbachol (30 /~M) but not to ATP (80 #M), whereas desensitisation to a,fl-MeATP blocked those to ATP but not to carbachol. The nature of the excitatory neurotransmission mechanism in the bladder of those species examined in the present experiments was shown to be similar to that of other species described previously. Non-cholinergic neurotransmission
a,/3-Methylene ATP
1. Introduction The excitatory autonomic innervation of the mammalian urinary bladder is thought to be comprised of a cholinergic component and a nonadrenergic, non-cholinergic component. ATP may be the non-cholinergic transmitter in the guinea-pig detrusor muscle since this component of the response to field stimulation can be blocked by desensitisation of the P2-purinoceptor using a,flmethylene-ATP (a, fl-MeATP), a slowly degradable analogue of ATP (Kasakov and Burnstock, 1983) or by selective P2-purinoceptor antagonism using arylazido aminopropionyl ATP (ANAPP 3), a photoaffinity analogue of ATP (Westfall et al., 1983). Further, MacKenzie et al. (1982) showed that botulinum neurotoxin type A, a neurotoxin selective for cholinergic nerves, totally abolished the excitatory response of the bladder to electrical field stimulation, suggesting that the postgan-
* To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V,
Guinea-pig
Marmoset
Ferret
Bladder
glionic parasympathetic nerves might corelease ATP and acetylcholine. In this study, the spontaneous and electrically evoked activity of detrusor strips of the urinary bladder from three different species, the guinea-pig (rodent), ferret (carnivore) and marmoset (primate), have been compared quantitatively in vitro.
2. Materials and methods 2.1. Pharmacology Albino guinea-pigs of either sex, 300-500 g, were stunned by a blow to the head and exsanguinated. The bladder was removed and mucosa-free detrusor strips were prepared. Bladders from ferrets (650-1100 g) and common marmosets (Callithrix jacchus, 186-354 g) were taken from urethane-anaesthetized animals and prepared in the same way. The strips were mounted in 10 ml organ baths containing Krebs solution of the following composition (mM): NaCI 133.0, KCI 4.7,
312
NaH2PO 4 1.3, N a H C O 3 16.3, MgSO 4 0.6, glucose 7.8, CaC12 2.5. The solution was maintained at 37°C and bubbled with 95% 02/5% CO 2. The tissues were placed under an initial resting tension of 0.5 g and allowed to equilibrate for 1 h. Changes in isometric tension were recorded by means of a force-displacement transducer (Grass FT03C) and displayed on a Grass polygraph (Model 7D). Electrical field stimulation was elicited using two platinum ring electrodes that were placed 1 cm apart. Trains of pulses (0.5 ms duration, 60 V) were delivered using a Digitimer D4030 and isolated stimulator DS2. The detrusor strips were stimulated over a range of frequencies (2-40 Hz) for 5 s with a 5 min interval between each stimulation. This was then repeated after either preincubation with atropine (0.29/~M) for 30 min or Pz-purinoceptor desensitisation by a,fl-MeATP (5 /zM), or a combination of the two treatments. Desensitisation to a, fl-MeATP was achieved by successive additions without washout of a,/3MeATP (5 #M) to the organ bath at 2-3 min intervals until no further response was elicited. The responses to carbachol (30 /~M) and ATP (80/~M) were also tested before and after exposure to atropine or desensitisation to a,fl-MeATP. Guanethidine (2 /~M) was continually present in all the experiments.
2.2. Statistical methods The contractions to electrical stimulation were expressed as a percentage of the maximum contraction where the maximum contraction was that to 40 Hz stimulation, and results were plotted as means _+S.E.M. Means were compared using Student's paired t-test and were considered significantly different if P < 0.05.
2.3. Drugs Drugs were obtained from the following sources: Carbachol (carbamylcholine chloride), ATP (adenosine 5'-triphosphate), a,fl-methylene ATP, tetrodotoxin (TTX), Sigma; Guanethidine monosulphate, Ciba; Atropine sulphate, Antigen.
3. Results
3.1. Spontaneous activity In the guinea-pig, 67% of detrusor strips (n = 40) showed no detectable activity while the remainder showed spontaneous contractions after 10 min equilibration with a frequency of 3.0 + 0.3 contractions per min. In comparison, 43% of ferret detrusor strips (n = 28) and 70% of marmoset detrusor strips (n = 30) displayed patterns of spontaneous activity which occurred immediately after the tissues were placed under the initial 0.5 g load. The remainder were quiescent. The average frequency of spontaneous contractions was 2.7 _+ 0.1 contractions per min for the ferret and 2.9 _+ 0.1 contractions per rain for the marmoset. The spontaneous activity was unaffected by the addition of 0.29 ~tM atropine to the organ bath or desensitisation to 5 ~tM a, fl-MeATP. The different levels of spontaneous activity did not appear to affect the responses to electrical stimulation.
3.2. Electrically evoked activity Field stimulation (2-40 Hz for 5 s, 0.5 ms duration, 60 V) induced rapid, frequency-dependent contractions in the detrusor strips of the guinea-pig, marmoset and ferret bladder (fig. 1). The electrically induced responses of the bladder strips were TTX-sensitive. Since a short stimulation period was used (5 s), only the initial phasic contraction was seen, and this had a similar timecourse in all three species. In each case, the maximum contraction in this frequency range was seen at 40 Hz, Consecutive frequency-response curves obtained in the absence of antagonists did not differ significantly. Exposure to 0.29/~M atropine for 30 rain caused a significant decrease (P < 0.05) in the response at all frequencies in all three species, and showed that at frequencies > 16 Hz,. much of the control phasic response was cholinergic (figs. 1 and 2). Desensitisation to a,fl-MeATP caused a significant decrease (P < 0.05) in the response to field stimulation at all frequencies tested in the guineapig and ferret but in the marmoset this decrease was significant (P < 0.05) only at frequencies < 8
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Fig. 1. (A) Contractile response of guinea-pig detrusor strips to field stimulation: (i) before (O) and after ( © ) preincubation with atropine (n = 6); and (ii) before (O) and after (©) a,fl-MeATP desensitisation (n = 7). Asterisks denote significant (p < 0.05) changes from control at the frequencies indicated. (B) Contractile response of marmoset detrusor strips to field stimulation: (i) before (A) and after (zx) preincubation with atropine (n = 6); and (ii) before (A) and after (zx) a,fl-MeATP desensitisation (n = 6). (C) Contractile response of ferret detrusor strips to field stimulation: (i) before (It) and after ([3) preincubation with atropine (n = 7); and (ii) before (It) and after ([3) a,fl-meATP desensitisation (n = 6).
~x,B-Me-ATP
Hz (figs. 1 and 2). Contractions were abolished by atropine followed by desensitisation to a, flMeATP (fig. 2). When the inhibitory actions of atropine or a, fl-MeATP desensitisation were expressed as a percentage decrease of the control response at each frequency in the guinea-pig, it could be seen that desensitisation to a,/~-MeATP caused a significantly greater decrease (P < 0.05) at the lower frequencies than at the higher frequencies, whereas after atropine, the converse was true (fig. 3). While this was clearly seen in the guinea-pig, the difference was not so apparent in the marmoset and ferret.
atropine + o¢,B-Me-ATP
Fig. 2. (a) Contractile response of marmoset detrusor strips to field stimulation at 2-40 Hz for 5 s before and after preincubation with atropine. (b) Contractile response of marmoset detrusor strips to field stimulation at 2-40 Hz for 5 s before and after a,fl-MeATP desensitisation. (c) Contractile response of guinea-pig detrusor strips to field stimulation at 2-40 Hz before and after atropine preincubation and a,/~-MeATP desensitisation.
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atropine but not after desensitisation to a,/3MeATP, whereas those to 80 /~M ATP were abolished after desensitisation to a,/3-MeATP but not after atropine. Responses to both carbachol and ATP were abolished by a combination of the two treatments (n = 6 in each case for all three species) (fig. 4).
o reduction of response 80-
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Fig. 3. Percentage reduction of control responses to field stimulation in guinea-pig detrusor strips either after preincubation with atropine (O) (n = 6) or a,/~-MeATP desensitation (O) (n = 7).
3.3. Responses to carbachol and A TP
In all three species, contractile responses to 30 /~M carbachol were abolished after exposure to a) carbachol
I lg A
t
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lmin atropine
b)ATP ~k
....
,~
t
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atropine
1lg
,m,o
Fig. 4. (a) Contractile response of guinea-pig detrusor strips to carbachol (30 t~M); control response, response after a,/3MeATP and response after atropine. (b) Contractile response of guinea-pig detrusor strips to ATP (80 #M); control response, responseafter a,/~-MeATP, and response after atropine.
Spontaneous activity was present to a variable extent in detrusor strips from the guinea-pig, marmoset and ferret urinary bladder. In comparison, Sibley (1984) found that while all rabbit detrusor strips elicited spontaneous contractions, only 20% of human detrusor strips were capable of doing so. In the present study, a, fl-MeATP desensitisation and exposure to atropine were without effect on spontaneous activity. In addition, hexamethonium and TTX have been shown not to diminish spontaneous contractions (Sibley, 1984). Spontaneous activity was measured previously in rabbit detrusor strips using electrophysiological techniques and was found to consist of simple or multiple action potentials that occurred with a frequency of 5-30 per min (Creed et al., 1983). In this study, exposure to atropine and desensitisation of the Pz-purinoceptor by a,fl-MeATP were used to block the contraction to field stimulation in the tissues examined, a,fl-MeATP has been found to be specific for purinergic receptors since it does not block responses to carbachol and has previously been shown to have no effect on histamine-induced contractions of guinea-pig bladder at higher doses than used in this study (Kasakov and Burnstock, 1983). Both atropine and a,fl-MeATP desensitisation were shown to block contractions to field stimulation in all three species. While the effect of a,fl-MeATP has been shown in the guinea-pig (Kasakov asnd Burnstock, 1983) this has not been previously shown in the ferret or marmoset. Atropine has been found to partially inhibit bladder contractions in the baboon (Brindley and Craggs, 1975) and monkey (Johns and Paton, 1977). In comparison, contractions elicited in human detrusor strips stimulated over a ~imilar range of frequencies were shown to be
315
totally blocked by atropine (Sibley, 1984). However, the human bladder strips did not show any response to stimulation below 5 Hz, whereas in the present study, control responses in the marmoset were greatly reduced at the same frequency after a, fl-MeATP blockade. This lack of response in the human bladder strips may thus be due to the absence of a non-cholinergic component in the excitatory response to electrical stimulation. It has been established that atropine has a greater blocking effect on the phasic component of the neurogenic response at higher frequencies and that the non-cholinergic component is more prominent at lower frequencies in the guinea-pig (Krell et al., 1981; MacKenzie and Burnstock, 1984; Sibley, 1984). The present study has confirmed this since a, fl-MeATP had a greater antagonistic effect at lower frequencies, reducing the response of the guinea-pig detrusor strips to 37% of control at 2 Hz compared with 74% of control at 40 Hz. In all species studied to date, atropine has been shown to decrease the contraction to electrical stimulation to varying degrees, for example by 45-50% at 40-50 Hz in the guinea-pig (this study; MacKenzie and Burnstock, 1984) and by 100% at 50 Hz in man (Sibley, 1984). While the existence of a non-cholinergic component is widely accepted, the identity of this transmitter is still contentious. Several candidates have been examined, including ATP, VIP, substance P, 5-HT, histamine and prostaglandins (Ambache and Zar, 1970; Burnstock et al., 1972, 1978; Choo and Mitchelson, 1980; Johns, 1981; MacKenzie and Burnstock, 1984), but most recent studies indicate that ATP is the most likely candidate for the non-cholinergic transmitter. Evidence has been provided for a purinergic component in the neurogenic response of the urinary bladder in the rabbit (Dean and Downie, 1978), cat (Theobald, 1982; 1983) and guinea-pig (Kasakov and Burnstock, 1983; Westfall et al., 1983), and from the results of the present study it seems probable that a 'purinergic' component also exists in the ferret and marmoset. Thus it is likely that ATP and acetylcholine act as excitatory transmitters in the urinary bladder of these species, and this may represent a general pattern of autonomic control of this organ in other
species. Evidence provided using botulinum toxin on the guinea-pig bladder suggests that acetylcholine and ATP may be coreleased by the parasympathetic neurons supplying the smooth muscle of the bladder (MacKenzie et al., 1982).
Acknowledgements H.E.M. was in receipt of an MRC scholarship. We would like to thank Wyeth Laboratories, Maidenhead for the gift of the marmosets to St. George's Hospital, London and Dr. P.L.R. Andrews for donating the bladders from the ferrets and marmosets.
References Ambache, N. and M.A. Zar, 1970, Non-cholinergic transmission by postganglionic motor neurons in the mammalian bladder, J. Physiol. (London) 210, 761. Brindley, G.S. and M.D. Craggs, 1976, The effect of atropine on the urinary bladder of the baboon and of man, J. Physiol. (London) 256, 55P. Burnstock, G., T. Cocks, R. Crowe and L. Kasakov, 1978, Purinergic innervation of the guinea-pig urinary bladder, Br.J. Pharmacol. 63, 125. Burnstock, G., B. Dumsday and A. Smythe, 1972, Atropine resistant excitation of the urinary bladder: the possibility of transmission via nerves releasing a purine nucleotide, Br. J. Pharmacol. 44, 451. Choo, L.K. and F. Mitchelson, 1980, The effect of indomethacin and adenosine 5'-triphosphate on the excitatory innervation of the rat urinary bladder, Can. J. Physiol. Pharmacol. 58, 1042. Creed, K.E., S. Ishikawa and Y. Ito, 1983, Electrical and mechanical activity recorded from rabbit urinary bladder in response to nerve stimulation, J . Physiol. (London) 338, 149. Dean, D.M. and J.W. Downie, 1978, Contribution of adrenergic and 'purinergic' neurotransmission to contraction in rabbit detrusor, J. Pharmacol. Exp. Ther. 207, 431. Johns, A., 1981, The effect of indomethacin and substance P on the guinea-pig urinary bladder, Life Sci. 29, 1803. Johns, A. and D.M. Paton, 1977, Effect of indomethacin on atropine-resistant transmission in rabbit and monkey urinary bladder: evidence for involvement of prostaglandins in transmission, Prostaglandins 13, 245. Kasakov, L. and G. Burnstock, 1983, The use of the slowly degradable analog, a,fl-methylene ATP, to produce desensitisation of the Pz-purinoceptor: effect on non-adrenergic, non-cholinergic responses of the guinea-pig urinary bladder, European J. Pharmacol. 86, 291.
316 Krell, R.D., J.L. McCoy and P.T. Ridley, 1981, Pharmacological characterization of the excitatory innervation to the guinea-pig urinary bladder in vitro: evidence for both cholinergic and non-adrenergic, non-cholinergic neurotransmission, Br. J. Pharmacol. 74, 15. MacKenzie, I. and G. Burnstock, 1984, Neuropeptide action on the guinea-pig bladder, a comparison with the effects of field stimulation and ATP, European J. Pharmacol. 105, 85. MacKenzie, I., G. Burnstock and J.O. Dolly, 1982, The effects of purified botulinum neurotoxin type A on cholinergic, adrenergic and non-adrenergic, atropine-resistant autonomic neuromuscular transmission, Neuroscience 4, 997.
Sibley, G.N.A., 1984, A comparison of spontaneous and nerve-mediated activity in bladder muscle from man, pig and rabbit, J. Physiol. (London) 354, 431. Theobald, R.J., 1982, Arylazido aminopropionyl ATP (ANAPP3) antagonism of cat urinary bladder contractions, J. Auton. Pharmacol. 3, 175. Theobald, R.J., 1983, The effect of arylazido aminopropionyl ATP on atropine resistant contractions of the cat urinary bladder, Life Sci. 32, 2479. Westfall, D.P., J.S. Fedan, J. Colby, G.K. Hogaboom and J.P. O'Donnell, 1983, Evidence for a contribution by purines to the neurogenic response of the guinea-pig urinary bladder, European J. Pharmacol. 87, 415.
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A C O M P A R A T I V E S T U D Y O F E L E C T R I C A L F I E L D S T I M U L A T I O N OF T H E G U I N E A - P I G , F E R R E T AND M A R M O S E T URINARY B L A D D E R H.E. MOSS and G. BURNSTOCK * Department of Anatomy and Embryology, and Centrefor Neuroscience, University College London, GowerStreet, London WCIE 6BT, United Kingdom Received 28 February 1985, revised MS received 21 May 1985, accepted 4 June 1985
H.E. MOSS and G. BURNSTOCK, A comparative study of electrical fieM stimulation of the guinea-pig, ferret and marmoset urinary bladder, European J. Pharmacol. 114 (1985) 311-316. The spontaneous and electrically evoked activity was examined in guinea-pig, ferret and marmoset urinary bladder. Electrical field stimulation of detrusor strips in vitro induced a rapid, frequency-dependent contraction with a maximum response at 40 Hz. This contraction was partly decreased by either atropine (0.29 #M) or desensitisation to a,fl-methylene ATP (a,fl-MeATP) (5 #M), and was totally blocked by a combination of the two. Atropine blocked responses to carbachol (30 /~M) but not to ATP (80 #M), whereas desensitisation to a,fl-MeATP blocked those to ATP but not to carbachol. The nature of the excitatory neurotransmission mechanism in the bladder of those species examined in the present experiments was shown to be similar to that of other species described previously. Non-cholinergic neurotransmission
a,/3-Methylene ATP
1. Introduction The excitatory autonomic innervation of the mammalian urinary bladder is thought to be comprised of a cholinergic component and a nonadrenergic, non-cholinergic component. ATP may be the non-cholinergic transmitter in the guinea-pig detrusor muscle since this component of the response to field stimulation can be blocked by desensitisation of the P2-purinoceptor using a,flmethylene-ATP (a, fl-MeATP), a slowly degradable analogue of ATP (Kasakov and Burnstock, 1983) or by selective P2-purinoceptor antagonism using arylazido aminopropionyl ATP (ANAPP 3), a photoaffinity analogue of ATP (Westfall et al., 1983). Further, MacKenzie et al. (1982) showed that botulinum neurotoxin type A, a neurotoxin selective for cholinergic nerves, totally abolished the excitatory response of the bladder to electrical field stimulation, suggesting that the postgan-
* To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V,
Guinea-pig
Marmoset
Ferret
Bladder
glionic parasympathetic nerves might corelease ATP and acetylcholine. In this study, the spontaneous and electrically evoked activity of detrusor strips of the urinary bladder from three different species, the guinea-pig (rodent), ferret (carnivore) and marmoset (primate), have been compared quantitatively in vitro.
2. Materials and methods 2.1. Pharmacology Albino guinea-pigs of either sex, 300-500 g, were stunned by a blow to the head and exsanguinated. The bladder was removed and mucosa-free detrusor strips were prepared. Bladders from ferrets (650-1100 g) and common marmosets (Callithrix jacchus, 186-354 g) were taken from urethane-anaesthetized animals and prepared in the same way. The strips were mounted in 10 ml organ baths containing Krebs solution of the following composition (mM): NaCI 133.0, KCI 4.7,
312
NaH2PO 4 1.3, N a H C O 3 16.3, MgSO 4 0.6, glucose 7.8, CaC12 2.5. The solution was maintained at 37°C and bubbled with 95% 02/5% CO 2. The tissues were placed under an initial resting tension of 0.5 g and allowed to equilibrate for 1 h. Changes in isometric tension were recorded by means of a force-displacement transducer (Grass FT03C) and displayed on a Grass polygraph (Model 7D). Electrical field stimulation was elicited using two platinum ring electrodes that were placed 1 cm apart. Trains of pulses (0.5 ms duration, 60 V) were delivered using a Digitimer D4030 and isolated stimulator DS2. The detrusor strips were stimulated over a range of frequencies (2-40 Hz) for 5 s with a 5 min interval between each stimulation. This was then repeated after either preincubation with atropine (0.29/~M) for 30 min or Pz-purinoceptor desensitisation by a,fl-MeATP (5 /zM), or a combination of the two treatments. Desensitisation to a, fl-MeATP was achieved by successive additions without washout of a,/3MeATP (5 #M) to the organ bath at 2-3 min intervals until no further response was elicited. The responses to carbachol (30 /~M) and ATP (80/~M) were also tested before and after exposure to atropine or desensitisation to a,fl-MeATP. Guanethidine (2 /~M) was continually present in all the experiments.
2.2. Statistical methods The contractions to electrical stimulation were expressed as a percentage of the maximum contraction where the maximum contraction was that to 40 Hz stimulation, and results were plotted as means _+S.E.M. Means were compared using Student's paired t-test and were considered significantly different if P < 0.05.
2.3. Drugs Drugs were obtained from the following sources: Carbachol (carbamylcholine chloride), ATP (adenosine 5'-triphosphate), a,fl-methylene ATP, tetrodotoxin (TTX), Sigma; Guanethidine monosulphate, Ciba; Atropine sulphate, Antigen.
3. Results
3.1. Spontaneous activity In the guinea-pig, 67% of detrusor strips (n = 40) showed no detectable activity while the remainder showed spontaneous contractions after 10 min equilibration with a frequency of 3.0 + 0.3 contractions per min. In comparison, 43% of ferret detrusor strips (n = 28) and 70% of marmoset detrusor strips (n = 30) displayed patterns of spontaneous activity which occurred immediately after the tissues were placed under the initial 0.5 g load. The remainder were quiescent. The average frequency of spontaneous contractions was 2.7 _+ 0.1 contractions per min for the ferret and 2.9 _+ 0.1 contractions per rain for the marmoset. The spontaneous activity was unaffected by the addition of 0.29 ~tM atropine to the organ bath or desensitisation to 5 ~tM a, fl-MeATP. The different levels of spontaneous activity did not appear to affect the responses to electrical stimulation.
3.2. Electrically evoked activity Field stimulation (2-40 Hz for 5 s, 0.5 ms duration, 60 V) induced rapid, frequency-dependent contractions in the detrusor strips of the guinea-pig, marmoset and ferret bladder (fig. 1). The electrically induced responses of the bladder strips were TTX-sensitive. Since a short stimulation period was used (5 s), only the initial phasic contraction was seen, and this had a similar timecourse in all three species. In each case, the maximum contraction in this frequency range was seen at 40 Hz, Consecutive frequency-response curves obtained in the absence of antagonists did not differ significantly. Exposure to 0.29/~M atropine for 30 rain caused a significant decrease (P < 0.05) in the response at all frequencies in all three species, and showed that at frequencies > 16 Hz,. much of the control phasic response was cholinergic (figs. 1 and 2). Desensitisation to a,fl-MeATP caused a significant decrease (P < 0.05) in the response to field stimulation at all frequencies tested in the guineapig and ferret but in the marmoset this decrease was significant (P < 0.05) only at frequencies < 8
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Fig. 1. (A) Contractile response of guinea-pig detrusor strips to field stimulation: (i) before (O) and after ( © ) preincubation with atropine (n = 6); and (ii) before (O) and after (©) a,fl-MeATP desensitisation (n = 7). Asterisks denote significant (p < 0.05) changes from control at the frequencies indicated. (B) Contractile response of marmoset detrusor strips to field stimulation: (i) before (A) and after (zx) preincubation with atropine (n = 6); and (ii) before (A) and after (zx) a,fl-MeATP desensitisation (n = 6). (C) Contractile response of ferret detrusor strips to field stimulation: (i) before (It) and after ([3) preincubation with atropine (n = 7); and (ii) before (It) and after ([3) a,fl-meATP desensitisation (n = 6).
~x,B-Me-ATP
Hz (figs. 1 and 2). Contractions were abolished by atropine followed by desensitisation to a, flMeATP (fig. 2). When the inhibitory actions of atropine or a, fl-MeATP desensitisation were expressed as a percentage decrease of the control response at each frequency in the guinea-pig, it could be seen that desensitisation to a,/~-MeATP caused a significantly greater decrease (P < 0.05) at the lower frequencies than at the higher frequencies, whereas after atropine, the converse was true (fig. 3). While this was clearly seen in the guinea-pig, the difference was not so apparent in the marmoset and ferret.
atropine + o¢,B-Me-ATP
Fig. 2. (a) Contractile response of marmoset detrusor strips to field stimulation at 2-40 Hz for 5 s before and after preincubation with atropine. (b) Contractile response of marmoset detrusor strips to field stimulation at 2-40 Hz for 5 s before and after a,fl-MeATP desensitisation. (c) Contractile response of guinea-pig detrusor strips to field stimulation at 2-40 Hz before and after atropine preincubation and a,/~-MeATP desensitisation.
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atropine but not after desensitisation to a,/3MeATP, whereas those to 80 /~M ATP were abolished after desensitisation to a,/3-MeATP but not after atropine. Responses to both carbachol and ATP were abolished by a combination of the two treatments (n = 6 in each case for all three species) (fig. 4).
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40
F r e q u e n c y (Hz)
Fig. 3. Percentage reduction of control responses to field stimulation in guinea-pig detrusor strips either after preincubation with atropine (O) (n = 6) or a,/~-MeATP desensitation (O) (n = 7).
3.3. Responses to carbachol and A TP
In all three species, contractile responses to 30 /~M carbachol were abolished after exposure to a) carbachol
I lg A
t
toc,B-Me-A'h°
lmin atropine
b)ATP ~k
....
,~
t
t
~,g-Me-ATP
atropine
1lg
,m,o
Fig. 4. (a) Contractile response of guinea-pig detrusor strips to carbachol (30 t~M); control response, response after a,/3MeATP and response after atropine. (b) Contractile response of guinea-pig detrusor strips to ATP (80 #M); control response, responseafter a,/~-MeATP, and response after atropine.
Spontaneous activity was present to a variable extent in detrusor strips from the guinea-pig, marmoset and ferret urinary bladder. In comparison, Sibley (1984) found that while all rabbit detrusor strips elicited spontaneous contractions, only 20% of human detrusor strips were capable of doing so. In the present study, a, fl-MeATP desensitisation and exposure to atropine were without effect on spontaneous activity. In addition, hexamethonium and TTX have been shown not to diminish spontaneous contractions (Sibley, 1984). Spontaneous activity was measured previously in rabbit detrusor strips using electrophysiological techniques and was found to consist of simple or multiple action potentials that occurred with a frequency of 5-30 per min (Creed et al., 1983). In this study, exposure to atropine and desensitisation of the Pz-purinoceptor by a,fl-MeATP were used to block the contraction to field stimulation in the tissues examined, a,fl-MeATP has been found to be specific for purinergic receptors since it does not block responses to carbachol and has previously been shown to have no effect on histamine-induced contractions of guinea-pig bladder at higher doses than used in this study (Kasakov and Burnstock, 1983). Both atropine and a,fl-MeATP desensitisation were shown to block contractions to field stimulation in all three species. While the effect of a,fl-MeATP has been shown in the guinea-pig (Kasakov asnd Burnstock, 1983) this has not been previously shown in the ferret or marmoset. Atropine has been found to partially inhibit bladder contractions in the baboon (Brindley and Craggs, 1975) and monkey (Johns and Paton, 1977). In comparison, contractions elicited in human detrusor strips stimulated over a ~imilar range of frequencies were shown to be
315
totally blocked by atropine (Sibley, 1984). However, the human bladder strips did not show any response to stimulation below 5 Hz, whereas in the present study, control responses in the marmoset were greatly reduced at the same frequency after a, fl-MeATP blockade. This lack of response in the human bladder strips may thus be due to the absence of a non-cholinergic component in the excitatory response to electrical stimulation. It has been established that atropine has a greater blocking effect on the phasic component of the neurogenic response at higher frequencies and that the non-cholinergic component is more prominent at lower frequencies in the guinea-pig (Krell et al., 1981; MacKenzie and Burnstock, 1984; Sibley, 1984). The present study has confirmed this since a, fl-MeATP had a greater antagonistic effect at lower frequencies, reducing the response of the guinea-pig detrusor strips to 37% of control at 2 Hz compared with 74% of control at 40 Hz. In all species studied to date, atropine has been shown to decrease the contraction to electrical stimulation to varying degrees, for example by 45-50% at 40-50 Hz in the guinea-pig (this study; MacKenzie and Burnstock, 1984) and by 100% at 50 Hz in man (Sibley, 1984). While the existence of a non-cholinergic component is widely accepted, the identity of this transmitter is still contentious. Several candidates have been examined, including ATP, VIP, substance P, 5-HT, histamine and prostaglandins (Ambache and Zar, 1970; Burnstock et al., 1972, 1978; Choo and Mitchelson, 1980; Johns, 1981; MacKenzie and Burnstock, 1984), but most recent studies indicate that ATP is the most likely candidate for the non-cholinergic transmitter. Evidence has been provided for a purinergic component in the neurogenic response of the urinary bladder in the rabbit (Dean and Downie, 1978), cat (Theobald, 1982; 1983) and guinea-pig (Kasakov and Burnstock, 1983; Westfall et al., 1983), and from the results of the present study it seems probable that a 'purinergic' component also exists in the ferret and marmoset. Thus it is likely that ATP and acetylcholine act as excitatory transmitters in the urinary bladder of these species, and this may represent a general pattern of autonomic control of this organ in other
species. Evidence provided using botulinum toxin on the guinea-pig bladder suggests that acetylcholine and ATP may be coreleased by the parasympathetic neurons supplying the smooth muscle of the bladder (MacKenzie et al., 1982).
Acknowledgements H.E.M. was in receipt of an MRC scholarship. We would like to thank Wyeth Laboratories, Maidenhead for the gift of the marmosets to St. George's Hospital, London and Dr. P.L.R. Andrews for donating the bladders from the ferrets and marmosets.
References Ambache, N. and M.A. Zar, 1970, Non-cholinergic transmission by postganglionic motor neurons in the mammalian bladder, J. Physiol. (London) 210, 761. Brindley, G.S. and M.D. Craggs, 1976, The effect of atropine on the urinary bladder of the baboon and of man, J. Physiol. (London) 256, 55P. Burnstock, G., T. Cocks, R. Crowe and L. Kasakov, 1978, Purinergic innervation of the guinea-pig urinary bladder, Br.J. Pharmacol. 63, 125. Burnstock, G., B. Dumsday and A. Smythe, 1972, Atropine resistant excitation of the urinary bladder: the possibility of transmission via nerves releasing a purine nucleotide, Br. J. Pharmacol. 44, 451. Choo, L.K. and F. Mitchelson, 1980, The effect of indomethacin and adenosine 5'-triphosphate on the excitatory innervation of the rat urinary bladder, Can. J. Physiol. Pharmacol. 58, 1042. Creed, K.E., S. Ishikawa and Y. Ito, 1983, Electrical and mechanical activity recorded from rabbit urinary bladder in response to nerve stimulation, J . Physiol. (London) 338, 149. Dean, D.M. and J.W. Downie, 1978, Contribution of adrenergic and 'purinergic' neurotransmission to contraction in rabbit detrusor, J. Pharmacol. Exp. Ther. 207, 431. Johns, A., 1981, The effect of indomethacin and substance P on the guinea-pig urinary bladder, Life Sci. 29, 1803. Johns, A. and D.M. Paton, 1977, Effect of indomethacin on atropine-resistant transmission in rabbit and monkey urinary bladder: evidence for involvement of prostaglandins in transmission, Prostaglandins 13, 245. Kasakov, L. and G. Burnstock, 1983, The use of the slowly degradable analog, a,fl-methylene ATP, to produce desensitisation of the Pz-purinoceptor: effect on non-adrenergic, non-cholinergic responses of the guinea-pig urinary bladder, European J. Pharmacol. 86, 291.
316 Krell, R.D., J.L. McCoy and P.T. Ridley, 1981, Pharmacological characterization of the excitatory innervation to the guinea-pig urinary bladder in vitro: evidence for both cholinergic and non-adrenergic, non-cholinergic neurotransmission, Br. J. Pharmacol. 74, 15. MacKenzie, I. and G. Burnstock, 1984, Neuropeptide action on the guinea-pig bladder, a comparison with the effects of field stimulation and ATP, European J. Pharmacol. 105, 85. MacKenzie, I., G. Burnstock and J.O. Dolly, 1982, The effects of purified botulinum neurotoxin type A on cholinergic, adrenergic and non-adrenergic, atropine-resistant autonomic neuromuscular transmission, Neuroscience 4, 997.
Sibley, G.N.A., 1984, A comparison of spontaneous and nerve-mediated activity in bladder muscle from man, pig and rabbit, J. Physiol. (London) 354, 431. Theobald, R.J., 1982, Arylazido aminopropionyl ATP (ANAPP3) antagonism of cat urinary bladder contractions, J. Auton. Pharmacol. 3, 175. Theobald, R.J., 1983, The effect of arylazido aminopropionyl ATP on atropine resistant contractions of the cat urinary bladder, Life Sci. 32, 2479. Westfall, D.P., J.S. Fedan, J. Colby, G.K. Hogaboom and J.P. O'Donnell, 1983, Evidence for a contribution by purines to the neurogenic response of the guinea-pig urinary bladder, European J. Pharmacol. 87, 415.