Opioid-like action of eseroline on micturition reflex in rats

Gen. Pharmac. Vol. 20, No. 1, pp. 17-22, 1989

0306-3623/89 $3.00+0.00 Copyright © 1989 Pergamon Press plc

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OPIOID-LIKE ACTION OF ESEROLINE ON MICTURITION REFLEX IN RATS CARLO ALBERTO MAGGI, 1. SANDRO GIULIANI,1 RICCARDO PATACCHINI,l PAOLO SANTICIOLI,l BRUNO CONTE,2 ALESSANDRO BARTOLINi3 and ALBERTO MELI j ~Pharmacology Department, A. Menerini Pharmaceuticals, Via Sette Santi 3, 50131 Florence [Tel. (055) 56801] 2Menarini Sud, Via Tito Speri, Pomezia, Rome, and 3Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Morgagni 65, Florence, Italy (Received 18 April 1988)

Abstract--1. Eseroline (3-10 mg/kg s.c.), the first product of metabolic breakdown of eserine (physostigrnine) markedly increased bladder capacity and decreased voiding efficiency of reflex micturition in rats. 2. Eserine had an opposite effect on bladder function in vivo, while morphine (1-3 mg/kg s.c.) reproduced the action of eseroline. The effects of eseroline or morphine were prevented by naloxone which per se reduced bladder capacity. 3. In vitro both eserine and eseroline potentiated the cholinergic component of the twitch response to field stimulation of the rat bladder and this effect was atropine-sensitive while morphine had no effect. 4. These findings provide further support to the notion that eseroline possesses both eserine- and opioid-like actions, but this latter predominates in determining its actions on bladder function in vivo.

1. INTRODUCTION

In recent years, much attention was directed to explore the role of exogenous and endogenous opiods on the reflex regulation of vesicourethral motility. Multiple sites of action for opioids have been described at both central nervous system and pelvic ganglia level which account for the ability of these substances to inhibit vesicourethral motility (Dray and Metsch, 1984a~i; De Groat and Kawatani, 1985; see also Maggi and Meli, 1986, for review). On the other hand, one would expect that anti-ACh-ase substances facilitate bladder voiding (Maggi et al., 1988), in view of the postganglionic cholinergic excitatory innervation of the detrusor muscle (Carpenter, 1977; Maggi et al., 1984, 1985). In view of the above, it appeared worthwhile to assess the effects of eseroline as compared to eserine and morphine on reflexly-activated bladder motility in rats, to evaluate the relative role of the anti-ACh-ase and opioid-like action of eseroline at this level.

Eseroline [(3aS, 8aR)-l,2,3,3a,8,8a-hexahydro-l,3a, 8-trimethylpyrrolo[2,3,b]indol-5ol)] is the first product of metabolic breakdown of eserine (physostigmine), the prototype of inhibitors of acetyicholinesterase (ACh-ase; Ellis, 1943). Eseroline possesses a marked antinociceptive activity in various animal models of experimentally-induced pain at somatic level (tail flick and tail pressure in rats; hot plate and writhing test in mice; Bartolini et al., 1981a, b; 1982). Further, electrophysiological data indicate that eseroline pretreatment reduced the response of nociceptive thalamic and dorsal horn (lamina V) neurons to noxious stimuli (Braga et al., 1984, 1986). Bartolini et al. (1982) reported that eseroline possesses an opioid-like activity both in vivo and in vitro. Indeed, eseroline binds specifically to opioid receptors in rat brain (IC50 0.32 and 6/zM in the absence and presence of Na ÷ at 25°C, respectively; Galli et al., 1979) and inhibits, in a naloxone-sensitive manner, the twitch contractions of the guinea-pig isolated ileum at concentrations below 5 # M (Furst et al., 1982; Galli et al., 1982). At the same time, eseroline still possesses some anti-ACh-ase activity and could be estimated to be at least 1/100 times as potent as eserine (Galli et al., 1982). Although eserine per se possesses antinociceptive activity (Bartolini et al., 1982) the analgesic action of eseroline seems largely independent upon anti-AChase activity and was ascribed to an opioid-like action of the drug, in that was prevented by naloxone pretreatment (Bartolini et al., 1981a, b, 1982; Braga et al., 1984, 1986).

2. METHODS

2.1. In vivo experiments Male albino rats Wistar-Morini strain weighing 34(~360 g were anaesthetized with subcutaneous urethane (1.2 g/kg). The left jugular vein was cannulated for drug injection. Body temperature was kept constant by means of a heating pad maintained at 37°C. The trachea was cannulated. In other experiments, threshold for initiating reflex micturition was measured by the non-stop infusion of saline (0.046 ml/min) through a needle inserted in the bladder dome, as described previously (transvesical recording of intravesical pressure; Maggi et al., 1986a-c). Intravesical pressure signals were delivered to a Hewlett Packard (H.P.) 8805B carrier amplifier and displayed on a H.P. four channel polygraph.

*To whom correspondence should be addressed. 17 G,P 20/I--B

CARLO ALBERTO MAGGI et al.

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From each cystometrogram (CMG), three parameters were calculated: volume threshold for reflex micturition, maximal amplitude of micturition contraction and residual volume, as described previously (Maggi et al., 1986a-c, 1987). Before the start of saline filling the bladders were manually emptied. Saline infusion was stopped after the second micturition contraction and volume threshold (VT) of the first and second voiding cycle (termed VT1 and VT2, respectively) were calculated. Difference between VT1 and VT2 was expected to reflect residual volume (RV) and consequently its modification by drugs may indicate changes in voiding efficiency. On these assumptions, RV was calculated as a percentage from the formula RV = (I - VT2/VTI) (Maggi et al., 1987).

2.2. In vitro experiments Male albino rats were killed by a blow to the back of the head and exsanguinated. The whole urinary bladder was removed and a strip (about 1 cm long and 2 mm wide of detrusor muscle excised and placed in an organ bath (5 ml) containing oxygenated (96% 02 and 4 % C O J standard Krebs solution at 37°C (Maggi et al., 1985). The strips were connected to an isometric transducer under a resting tension of 10 mN and electrically stimulated by means of two wire platinum electrodes placed at the top and the bottom of the organ bath (single pulses automatically delivered at 0.1 Hz, 60 V, 0.5 msec pulse width by means of GRASS SI 1 stimulator). Eseroline was added cumulatively to the organ bath, the next concentration being tested when the effects of the preceding one had reached a steady state, either in the absence or presence of atropine (1 pM, 15 min before). 2.3. Statistical analysis All data in the text are means _ SE. Statistical analysis of the data was performed by means of Student's t-test for paired or unpaired data or by means of analysis of variance, when applicable. Regression analysis was performed by means of the least squares method. ECs0 and 95% confidence limits were calculated accordingly. Statistical analysis of non-parametric data was made by means of the Z2 test. 2.4. Drugs Drugs used were: eseroline salicylate (mol. wt 356.4), morphine HCI (mol. wt 321.8, Carlo Erba), atropine HC1 (Serva), eserine sulphate (Sigma), naloxone HCI (Sigma). Eseroline salicylate was prepared by Professor G. Renzi, Dipartimento di Scienze Farmaceutiche, Universit~ di Firenze. Control rats received an equivalent amount of sodium salicylate. 3. RESULTS

3. I. Effect o f eseroline on micturition reflex Transvesical saline infusion in anesthetized rats activated at threshold volume a supraspinal vesicovesical excitatory reflex leading to a consistent voiding response, as s h o w n elsewhere (Maggi et al., 1986a-c). In rats receiving s.c. eseroline (3 mg/kg, 3 0 m i n before, N = 14) bladder capacity was increased by a b o u t 48% (filling rate 0.046ml/min), amplitude o f micturition contraction was slightly reduced and voiding efficiency was markedly impaired as judged by the significant increase in residual volume and reduced amplitude o f micturition contraction (Table 1; Fig. 1). A voiding c o n t r a c t i o n < 2 0 m m Hg was observed in 8 out o f 12 cases, and this effect was significantly different ( P < 0.05) from vehicle-treated animals in which amplitude o f micturition contraction was above 20 m m Hg in all cases.

Table 1. Effect of eseroline and naloxone on the bladder response to transvesical saline filling in urethane-anesthetizedrats (supraspinal vesico-vesicalmicturition reflex)

Treatment Controls Naloxone Eseroline Naloxone plus eseroline

Urodynamic parameters Amplitude of Volume micturition Residual No. of threshold contraction volume animals (ml) (mm Hg) (%) 8 0.864 _+0.1 24 _+3 33 _+6 8 0.574_+0.1" 22_+3 16_+8 12 1.278_+0.1" 18.1 _+ 1" 57-+6* 8

0.667 _+0.2t

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Each value is mean _+SE. Naloxone (0.2 mg/kg) was administered intravenously, 15 min before the start of saline infusion. Eseroline (3 mg/kg) was administered subcutaneously, 30min before the start of saline infusion. *Significantly different from controls, P < 0.05. tSigniflcantly different from eseroline alone, P < 0.05.

In 2 out o f 14 cases eseroline abolished the micturition reflex: after 15-25 min from the start o f saline infusion an hypertone ensued during which d r o p s o f fluid were expelled (overflow incontinence) a pattern similar to that observed following high doses o f i.v. h e x a m e t h o n i u m or topical application o f tetrodotoxin on the bladder d o m e (Maggi et al., 1986a-c). W h e n C M G s were run at a higher filling rate (0.25ml/min) the volume threshold o f eseroline ( 3 m g / k g s.c.) treated animals was still higher ( 0 . 8 9 5 + 0 . 2 m l , N = 6 ) than in controls ( 0 . 5 0 + 0 . 1 m l , N = 6 ) but this difference did not reach the significance level (0.10 < P < 0.5). A higher (10 mg/kg s.c.) dose ofeseroline produced a pattern o f overflow incontinence in all four rats tested: in two o f these animals, increasing the filling rate to 0.25 ml/min elicited a functional voiding response at a threshold volume o f 0.62 + 0.1 ml. The same pattern (reversal o f overflow incontinence) was observed in the two animals treated with the lower dose o f eseroline which developed overflow incontinence at an infusion rate o f 0.046ml/min. The overflow incontinence induced by s.c. eseroline was rapidly reverted by intravenous administration o f naloxone (0.2 mg/ml i.v., N = 4).

Table 2. Effect of eserine or morphine on the bladder response to transvesical saline filling in urethane-anesthetizedrats (supraspinal vesico-vesical micturition reflex) Urodynamic parameters Amplitude of Volume micturition Residual No. of threshold contraction volume animals (ml) (mm Hg) (%) 10 0.801 _+0.1 26 _+2 32 + 5

Treatment Controls Eserine 0.2 mg/kg 10 0.458_+0.1" 48_+4* 5_+3* Morphine 1mg/kg 6 1.094 _+0.2 25 _+3 52 _+6 Morphine 3 mg/kg 12 2.037-+0.3* 20+_2* 76-+ 10" Each value is mean+SEM. Eserine was administered intravenously, 15 min before the start of saline infusion. Morphine was administered subcutaneously, 30 min before the start of saline infusion. *Significantly different from controls, P < 0.05.

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Fig. 1. Typical tracings showing the effects of naloxone, eseroline or naloxone plus eseroline on the bladder response to saline filling (0.046 ml/min) in urethane-anesthetized rats. In the lower panel an example is shown of an eseroline-treated animal which failed to micturate in response to filling at 0.046 ml/min. An hypertone along with overflowincontinence ensued. A series of voiding cycleswas observed when stimulus to void was increased by augmenting the fillingrate to 0.25 ml/min. The arrows indicate the start of saline filling.

3.2. Effect of eserine The effects of intravenous eserine (0.2mg/kg, 15 min before) on micturition reflex were opposite to those of eseroline. Bladder capacity was decreased while amplitude of micturition contraction was enhanced. Almost no residual volume was observed (Table 2). All the effects of eserine were prevented by i.v. atropine (1 mg/kg, N = 6; data not shown). Similar observations were made in rats receiving s.c. eserine (0.2 mg/kg, 30 min before, N = 4).

N = 12) was somehow greater than that of eseroline (57 + 6%, N = 12). In four animals, morphine (3 mg/kg s.c.) abolished the micturition reflex during the 40 min observation period. In these animals a picture of overflow incontinence ensued (cf. Maggi et al., 1986a-c, 1988), but the i.v. administration of naioxone (0.2 mg/kg) produced within 30--60 sec a prompt, efficient and repetitive micturition response. Likewise, naloxone pretreatment (15min before the start of saline filling, N = 4) prevented the effects of morphine on micturition reflex (data not shown).

3.3. Effect of morphine The effect of morphine (1-3 mg/kg s.c., 30min before) on the micturition reflex were qualitatively similar to those produced by eseroline, e.g. an increase in bladder capacity and decrease in voiding efficiency (Table 2). At 3 mg/kg morphine increased bladder capacity by 154% as compared to controls (Table 2) while the corresponding effect of eseroline (3 mg/kg) was 47.9% increase (Table 1). Also the effect of morphine on residual volume (76 __. 10%,

3.4. Antagonism by naloxone of the effects of eseroline Intravenous naloxone (0.2mg/kg) produced a small (about 35% reduction) but significant effect on bladder capacity (Table 1). The other parameters were almost unaffected although residual volume was slightly reduced (Table 1). All the effects of eseroline on micturition reflex were prevented by naloxone pretreatment (Table 1, Fig. 1).

20

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Fig. 2. Typical tracings showing the effects of eseroline on "early" and "late" component of the twitch response to field stimulation of the rat isolated bladder before and after addition of atropine to the organ bath. 3.4. In vitro experiments

Single-pulse field stimulation (FS) of the rat isolated bladder strips produced a biphasic contractile response (Maggi et al., 1985). The "early" twitch response was largely ( > 9 0 % ) atropine (3/~M) resistant but completely tetrodotoxin (1/~M) sensitive. The "late" response is atropine- and tetrodotoxinsensitive and can also be potentiated selectively by eserine, in an atropine-sensitive manner (Maggi et al., 1985). Eseroline (0.1-100/~ M) produced a concentrationrelated potentiation of the response to FS, the effect being much more evident on the "late" than "early" twitch response (Figs 2 and 3). Before eseroline, the "late" component averaged 32 + 6% of the "early" response (N = 5). In the presence of eseroline, the

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"late" component became larger (113 _+ 8%). In percentage of basal values the "early" and "late" responses were increased by 21 +_6 and 397 _ 126% by eseroline (100 pM), respectively. The time course of this effect was quite rapid as compared to eserine: after each concentration of eseroline steady state potentiation was observed within 2 ~ , m i n while 15-25 min were required to attain steady state after eserine. Likewise, the potentiating effect of eseroline was rapidly reversed by washing out while that of eserine was much more presistent. In the presence of atropine (3/~m) the potentiating effect of eseroline on the "late" component of twitches was significantly reduced, while the small effect on the "early" component of twitches was unaffected. In the presence of atropine, the "late"

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Fig. 3. Potentiation of twitch response to field stimulation by eseroline (rat isolated bladder) in the absence or presence of atropine (3 t~M). Each value is mean + SE of at least 5 experiments. *Significantlydifferent from the effect observed in the absence of atropine, P < 0.05.

Opioid-like action of eseroline component of twitches averaged 18-t-5% of the "early" response, and after addition of eseroline this value rose to 5 4 _ 5%. Eseroline (10 # M) produced a marked potentiation of the contractile response to acetylcholine (0.1 p M) on the unstimulated rat isolated urinary bladder. The response to acetylcholine averaged 0 . 7 _ 0.2 and 1.5 + 0.3 mN in the absence and presence of eseroline, respectively (N = 4, P < 0.01). Morphine (3 p M , N = 6) had no effect on twitches (cf. Carpenter, 1986) even when parameters of stimulation were lowered to submaximal values (N = 4). 4. DISCUSSION Present findings indicate that in vivo the predominant effect of eseroline on micturition reflex is most likely ascribable to its opioid-like action (Bartolini et al., 1981a, b, 1982) rather than to eserine-like activity. When studying the action of this drug on pain perception it is somehow difficult to separate the relative contribution of the two activities in the genesis of the overall antinociceptive effect since eserine per se has some analgesic action (Furst et al., 1982). However, the antinociceptive action of eseroline was largely ascribed to an opioid-like action of the drug (Bartolini et al., 1981a, b, 1982), as was naloxone-sensitive. In the case of the rat bladder, eserine (present findings) and opioids (Dray and Metsch, 1984a-d) had opposite effects on reflexly-activated bladder motility. The action of eserine could occur either centrally or peripherally, whenever a cholinergic synapse was present in the micturition reflex pathway. However, it seems conceivable that some part of the facilitatory action on micturition observed after in vivo ACh-ase inhibition involved a potentiation of postganglionic cholinergic neuromuscular transmission at the level of detrusor muscle, as indicated by in vitro experiments (Carpenter, 1977; Maggi et al., 1985). The inhibitory action of morphine on reflexly-activated bladder motility in rats involves most likely a central site of action (Dray and Metsch, 1984a-d). Although morphine did not affect postganglionic excitatory transmission to the rat bladder (cf. Carpenter, 1986), some peripheral action could not be excluded. De Groat and Kawatani (1985) showed an inhibitory action of opioids on neurotransmission in cat pelvic ganglia which could be a target for the effects observed in this study. Indeed, intravenous morphine (0.5-1.0 mg/kg i.v.) had some (5-15% reduction) inhibitory effect on contractions of the rat bladder produced by preganglionic (ventral sacral roots) stimulation (Santicioli, unpublished data). Both eseroline and morphine produced, at high doses, a suppression of reflex micturition and overflow incontinence ensued thereby. At lower doses, both drugs increased bladder capacity, decreased amplitude of micturition contraction and reduced voiding efficiency. Therefore, an action on the afferent and efferent arm of reflex micturition is suggested by present findings. While the increase in bladder capacity is probably a primary effect of opioids (as also indicated by the effect of naloxone,

21

which reduced bladder capacity) it is difficult to establish at which extent the action on voiding contraction and efficiency might (or not) be secondary to the first effect. In fact, an increased sensory discharge from the bladder can modulate voiding efficiency (Maggi et al., 1986c). Previous studies in rats indicated that endogenous opioids might influence bladder capacity in rats by acting at both spinal and supraspinal sites (Dray and Metsch, 1984; see also Maggi and Meli, 1986) which could also be involved in the effects of eseroline, morphine and naioxone observed in this study. The inhibitory action of eseroline on reflex micturition was somehow less intense than that of morphine. However, after s.c. administration, eseroline was slightly more potent analgesic than morphine in rats. These slight differences could well be attributed to pharmacokinetic factors. However, it should be noted that in one system (pain perception), both cholinergic and opioidergic transmission exert the same final effect (analgesia) while they have opposite actions on reflexly-activated bladder motility. Thus, the inhibitory action of eseroline on micturition reflex, due to its opioid-like action, might have been counteracted by some anti-ACh-ase activity of the drug. However, we found no sign of facilitation or potentiation of the reflex by.eseroline in naloxonepretreated animals. Indeed, the anti-ACH-ase action of eseroline seems weaker than the opioid-like action as judged by the opposite effects on motility of the guinea-pig ileum which can be observed at different concentrations (Furst et al., 1982). In this case, eseroline reduced twitches in a naloxone-sensitive manner at concentrations below 5/~M, while at higher concentrations a potentiation of twitches and of responses to exogenous acetylcholine was observed (Furst et al., 1982). In the rat isolated bladder, due to apparent lack of prejunctionai inhibition of transmitter release through opioid receptors, only the eserine-like activity of eseroline was evident. Interestingly, both eserine and eseroline increased, at some extent, also the "early" response to FS and this action was totally atropine-resistant. No indication could be drawn from our data as to whether this action might occur at pre- or postjunctional level nor if it is related in some way to ACh-ase inhibition. Further studies are needed to explore this point. In conclusion present findings indicate that eseroline is capable of producing a consistent inhibitory action on micturition in rats and this action seems largely ascribable to its opioid-like properties, similarly as described for its analgesic activity. Acknowledgements--The authors wish to thank Professor

G. Renzi for the kind gift of eseroline salicylate. This work was supported in part by IMI, Rome, Italy, Progetto di Ricerca: Farmaci per il trattamento a lungo termine della incontinenza urinaria, VES Grant No. 46287. REFERENCES

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