ABSTRACT
A histological study of the structure of the urinary bladder of the ringtail possum has been made. The innervation of the bladder has been studied in vitro, using the technique of analytical pharmacology.
The bladder has well-defined inner longitudinal and outer circular muscle layers. Nerves supplying the bladder are found both in the pelvic nerves and in the vesical nerves which run with the vascular supply of the bladder fundus. Ganglia have been demonstrated along the trunks of the vesical nerves and also aggregated at the bladder neck.
The response of the bladder to stimulation of either nerve supply in situ or in vitro is always a simultaneous contraction of both longitudinal and circular muscles. Inhibitory responses to nerve stimulation have never been observed. The optimal frequency for stimulation of these nerves at 30° C. is 50 pulses/sec.
The bladder is contracted by ACh and 5-hydroxytryptamine, but is relaxed by adrenaline, noradrenaline and histamine.
The response to nerve stimulation is reduced by atropine and potentiated by eserine. Adrenergic blocking agents do not affect the nerve-mediated response unless they also affect the response to applied ACh in a similar manner.
Ganglionic blocking agents, in concentrations which do not reduce the response to ACh, cause up to a 40% reduction of the response to stimulation of either the vesical or the pelvic nerves.
It is concluded that the nerve fibres supplying the possum bladder are cholinergic, perhaps 40 % of them being stimulated pre-ganglionically.
The evolutionary significance of these observations is discussed.
Some points of pharmacological interest have been discussed in relation to drug actions on placental mammal preparations.
INTRODUCTION
The work reported in this paper is one of a series of comparative physiological studies, which is designed to clarify the pattern of evolution of the vertebrate autonomic nervous system. There have been many investigations of the anatomy and physiology of the autonomic nervous system of placental mammals. (See reviews by Langley (1921), McSwiney (1931), Gruber (1933) and Hillarp (1960).) Less attention has been given to the physiology of the autonomic nervous systems of lower vertebrates (Nicol, 1952). The autonomic nervous system of marsupial mammals has been particularly neglected.
The present work is concerned with the innervation of the bladder of a common Australian marsupial, the ringtail possum (Pseudocheirus peregrinus). The results are discussed in relation to the innervation of the bladder in other vertebrate groups. A brief preliminary account of this work has been presented previously (Campbell & Burnstock, 1962).
METHOD
Specimens of the ringtail possum were caught in the vicinity of Melbourne, and were used for experiments within 2 weeks of capture. General histological studies were made on bladders fixed in Bouin’s solution and stained with haematoxylin and eosin or with Masson’s triple stain. Nerve fibres were stained by inflating the bladder with 0·05 % methylene-blue solution at pH 6. The bladders were then fixed in 8 % ammonium molybdate and mounted in flattened strips.
In preparing the bladder for physiological experiments the animal was killed and opened on the ventral mid-line and the pubic symphysis was split. The ureters and, in the male, the vasa deferentia were severed close to the bladder wall. The vesical blood vessels and attached nerves were freed from the mesentery and sectioned near their origins from the common iliacs. The urethral blood vessels, together with the pelvic nerves, were tied in situ with a loop of cotton, and then sectioned near the origin of the vessels from the common iliacs. The urethra was then severed near the orifice and removed with the bladder and the nerve supply still attached.
The bladder was tied to a Perspex holder with a cotton looped through the base of the bladder fundus. The holder was then immersed in modified Krebs’s solution (Biilbring, 1953) in an organ bath. A gas mixture containing 95 % O2+ 5 % CO2 was bubbled through the solution in the bath. The bath temperature was maintained at 30 ± 0·5° C. The nerves to be stimulated were looped over two platinum electrodes embedded in the Perspex holder. In most experiments both sets of nerves were stimulated, but in some experiments the nerve supplies were stimulated separately. A Grass stimulator was used to deliver square-wave pulses of variable frequency, duration and strength. Recording was either isometric (with a Grass force-displacement transducer recording with a Grass polygraph) or isotonic (with a frontal-writing lever recording on a smoked kymograph drum).
RESULTS
Morphology of the bladder
The bladder of the ringtail opossum lies above the pelvic cavity, and is borne on a long urethra (Fig. 1). The ureters and the vasa deferentia or the lateral vaginae enter the bladder at the urethro-vesical junction. An artery and vein, arising from the common iliacs, pass up each antero-lateral face of the bladder, supplying the bulk of the bladder fundus. The urethra is also supplied with blood vessels from the iliacs, branches of these vessels extending on to the bladder neck and the lower part of the fundus. For convenience these vessels have been termed ‘bladder’ and ‘urethral’ respectively.
The bladder vessels are accompanied by nerves to the bladder fundus (‘vesical nerves’). Lying just caudal to the urethral vessels are the pelvic nerves which also supply the bladder with fibres. The pelvic nerves appear to arise from the second and third sacral nerves, but it is not clear whether the vesical nerves are branches of the pelvic nerves or whether they have an independent origin. Ganglia can be demonstrated along the trunks of the vesical nerves and a large accumulation of ganglia is also found in the bladder neck.
In about half of the bladders examined, whether from male or female animals, there was a thin strip of longitudinal muscle passing up the mid-dorsal face of the fundus (Fig. 2a). This strip arises at the bladder neck and extends to the apex, where the muscle fibres enter the circular musculature. In section, the musculature is seen to comprise an inner longitudinal layer and an outer circular layer of muscle fibres. In contrast to placentals and amphibians, it is only at the apex of the bladder that these layers merge and become indistinguishable. The muscle fibres are arranged in bundles widely separated by connective tissue. There is a thick submucosa, which is folded into ridges in the contracted bladder, and the bladder cavity is lined by a transitional epithelium (Fig. 2b). The dorsal longitudinal muscle band, where present, consists of separate muscle bundles lying in the thickened serosa (Fig. 2 c). The vesical blood vessels lie in ridges of connective tissue which also contain a few longitudinal muscle bundles. In the areas underlying these ridges, the longitudinal layer of the bladder wall is absent, while the circular layer is thicker than elsewhere (Fig. 2d).
Physiology of the innervation of the bladder
Spontaneous movements occurred in about two-thirds of the preparations used. These movements consisted of small contractions and relaxations of irregular amplitude and rate (Fig. 3 a). The tone of the preparation appeared to be related to the degree of spontaneous activity, i.e. high tone was associated with little or no spontaneous movement.
The response of the bladder to stimulation of either the vesical nerves or the pelvic nerves, when recorded in the longitudinal direction, was always a contraction, followed by a slower relaxation to basal tone (Fig. 3b, c). The response to stimulation of the pelvic nerves was always greater in magnitude than the response to stimulation of the vesical nerves, usually by a factor of 5 to 10. The maximal isometric tension response to stimulation of both nerve supplies was in the order of 10g. (Fig. 3d). Similar responses were recorded from the bladder in situ in recently killed animals.
The response of the circular muscle to stimulation of these nerves was also a Contraction (Fig. 3 e). In some experiments the sympathetic chains were stimulated at various points in situ, but no effect on the bladder was ever seen.
Variations in tone of the preparation did not cause qualitative changes in the response to nerve stimulation. At high tone, however, the size of the response was smaller than at low tone.
The response to stimulation of either vesical or pelvic nerves was tested over the strength range 1–150 V. Maximal contractions were obtained with about 20 V. with the particular electrode arrangement used. The shape of the response did not vary over this range (Fig. 4 a). The threshold for stimulation was below 1 V. At strengths greater than about 40 V. there was a spread of current through the bath, since responses occurred even when the nerves had been cut between the electrodes and the bladder.
The amplitude of the response was maximal at pulse durations of 3–5 msec. (Fig. 4b). Durations greater and less than this caused submaximal responses. Responses could be obtained with stimulation pulses as short as 0·01 msec. The contraction had a latency after the beginning of stimulation of less than 1 sec. Variation of the length of the stimulating burst between 1 and 120 sec. did not alter the response in a qualitative manner. It was shown that, after 25 sec., the muscle began to relax, even while the stimulating pulses were still being applied (Fig. 4 c).
The frequency of stimulation which caused maximal responses at 30° C. was 50 pulses/sec. (Fig. 4 d). This optimum was independent of variation of the other parameters of stimulation (strength, duration, etc.). The optimal frequency and duration were the same whether recorded under isometric or isotonic conditions, although the peak contractions were more clearly demonstrated under isometric suspension (Fig. 4 e,f).
Since the optimal parameters of stimulation were found to be the same for either the vesical or the pelvic nerves, both sets of nerves were stimulated together for pharmacological analysis. Ganglion blocking agents and some of the nerve-muscle blocking agents were tested against the individual nerves.
Actions of possible transmitter substances
Acetylcholine chloride (ACh) had no detectable action on the bladder at concentrations of less than 10−9 g./ml. At higher concentrations of ACh, both the circular and the longitudinal muscles contracted (Fig. 5 a). When ACh was left in the bath, the muscle remained in a contracted state for about 30 min. (Fig. 5b). When the drug was washed out of the bath, the muscle relaxed rapidly.
5-Hydroxytryptamine. (serotonin creatinine sulphate) caused a contraction at concentrations between 10−9 and 10−4 g./ml. (Fig. 5 c). The contraction was sustained in the presence of the drug, but at the higher concentrations relaxation did not follow the washing out of the drug.
Adrenaline tartrate caused relaxation and a cessation of spontaneous movements in the concentration range tested (10−7 to 5 × 10−6 g./ml.). When the drug was left in the bath, movements started spontaneously after some time and there was a gradual return of tone to control levels (Fig. 6 a).
Noradrenaline bitartrate (10−7 and 10−5 g./ml.) caused relaxation and a cessation of spontaneous movements. However, even at subthreshold concentrations (e.g. 10−8 g./ ml.), noradrenaline appreciably reduced the response to nerve stimulation (Fig. 6b). This effect was decreased after the application of dichloro-isoproterenol (DCI).
Dopamine (3,4-dihydroxyphenylethyIamine) had no effect on the tone or on the nerve-mediated response in concentrations of 10−7 to 10− g./ml.
Histamine acid phosphate (10−7 to 10−6 g./ml.) caused a fall in tone and a reduction of the response to nerve stimulation (Fig. 6c). At a concentration of 10−8 g./ml. it completely blocked the nerve-mediated response of the bladder.
Effects of blocking and potentiating agents on the nerve-mediated response
Tests for cholinergic transmission
Atropine sulphate (10−8 to 10−6 g./ml.) reduced the responses of both circular and longitudinal muscles to nerve stimulation and to applied ACh (Fig. 7a, b). The block of the nerve-mediated response was never complete, although these concentrations of atropine completely blocked the action of ACh. No differences were observed in the extent to which the vesical and the pelvic nerve responses were reduced. The concentrations of atropine used had no effect on the tone or the spontaneous movements of the preparation.
Eserine (physostigmine sulphate) usually caused a slight rise in tone at 10−7 and 10−6 g./ml. In some experiments, however, the muscle was greatly contracted by these concentrations. Where the effect on the tone was small, it was clear that eserine increased the amplitude of both the nerve-mediated response and the response to applied ACh to as much as twice the control amplitude, at the same time prolonging the relaxation phase of the responses (Fig. 7 c).
Tests for adrenergic transmission
All the adrenergic blocking agents were tested against the direct action of ACh as well as on the nerve-mediated response, to enable correction for their ACh-blocking actions, demonstrated by Boyd, Burnstock, Campbell, Jowett, O’Shea & Wood (1962, 1963).
Dibenxyline (phenoxybenzamine hydrochloride) caused potentiation of the nerve-mediated response followed by a slight block at concentrations of 10−7 and 10−6 g./ml. At 5 × 10−6 g./ml. the nerve-mediated response was reduced by 50% and the response fo applied ACh was completely blocked (Fig. 8 a).
Tolazoline (Priscol) also caused strong potentiation of the nerve-mediated and ACIP responses in concentrations of 10−7 to 5 × 10−4 g./ml. In concentrations between 10−4 and 10−3 g./ml. the initial potentiation was often followed by a strong block of both nerve-mediated and ACh responses (Fig. 8 b). In these concentrations the latency of the ACh response was increased by as much as tenfold, even when the amplitude of the response was potentiated.
933F (piperoxane hydrochloride) at 8× 10−5g./ml. caused 90% block of the response to ACh, although at this concentration the nerve-mediated response was not decreased in amplitude. At 2 × 10−4 g./ml. the nerve-mediated response was reduced to 70% of the control amplitude and the ACh response was almost completely blocked (Fig. 8c). These concentrations of 933 F caused the appearance of large spontaneous contractions and a general rise in tone.
Yohimbine hydrochloride in a concentration of 5 × 10−5 g./ml. caused a rapid partial block of the nerve-mediated response and of the response to applied ACh (Fig. 8 d). In two experiments this concentration caused complete block of the ACh response within 25 min.
Phentolamine (Regitine methane sulphonate) (10−4 to 5 × 10−4 g./ml.) caused more than 50% reduction of the nerve-mediated response and completely blocked the response to ACh.
Ergotamine tartrate (2 × 10−5 g./ml.) caused a slow partial block of both nervemediated and ACh responses. DCI caused a stronger block of the nerve-mediated response and an almost complete block of the ACh response at concentrationns of to-® and 5 × 10−5 g./ml. (Fig. 9a).
Guanethidine (5 × 10−5 g./ml.) rarely had any effect on the nerve-mediated response, but at 10−4 g./ml. it reduced the response to ACh without affecting the nerve-mediated response (Fig. 9b).
Bretylium tosylate (Darenthin) (10−4 g./ml.) caused a slight potentiation of both nerve-mediated and ACh responses, but at 5 × 10−4 g./ml. it caused a partial block of the nerve-mediated response and a stronger block of the ACh response (Fig. 9 c).
Tests for ganglia
Hexamethonium bromide (5 × 10−5 to 5 × 10−4 g./ml.) caused potentiation of the response to ACh, at the same time reducing the response to either pelvic or vesical nerve stimulation by 10–20% (Fig. 10a). In some cases these concentrations caused potentiation of the nerve-mediated response. At 10−3 g./ml. the nerve-mediated response was reduced by more than 20%, but the response to applied ACh was also reduced.
Pentolinium tartrate, at 10−6 to 10−5 g./ml., had no effect on the response to applied ACh, but at 10−4 g./ml. reduced this response to some extent. At 10−8 g./ml. the response to stimulation of either the vesical or the pelvic nerves was reduced by about 20–25 % 9(Fig.10b).
Mecamylamine hydrochloride potentiated the response to ACh in concentrations between 10−6 and 5 × 10−8 g./ml., while at 10−4 g./ml. (and, in some cases, at 5 × 10−8 g./ ml.) it caused Urge spontaneous contractions which obscured the effect of the drug on the ACh responses. At 5 × 10−5 g./ml. this drug blocked the vesical and pelvic nerve-mediated responses by 30–40% (Fig. 10c). In contrast with pentolinium and hexamethonium, it was difficult to decrease the block after mecamylamine by washing the drug out of the bath.
DISCUSSION
The nerve fibres supplying the bladder of the possum are excitatory. These fibres appear to be cholinergic. A large proportion of the nerve fibres reaching the bladder via either the pelvic nerves or the vesical nerves appear to be postganglionic. There is as yet no evidence for the presence of adrenergic fibres. Supporting evidence for these conclusions is as follows:
The nerve-mediated response is always a contraction. An inhibitory response could not be revealed by any variation of the parameters of the stimulating pulses, nor by treatment with any of the drugs used. Stimulation of the sympathetic chains in situ had no effect on the bladder.
The nerve-mediated response is mimicked by the action of ACh, but not by the actions of adrenaline, noradrenaline or dopamine.
The nerve-mediated response is reduced by atropine and potentiated by eserine, as is the response to ACh.
The adrenergic blocking agents tested affect the nerve-mediated response only when they also affect the ACh response in a similar manner.
Ganglia have been demonstrated along the trunks of the vesical nerves and aggregated at the base of the bladder.
Ganglion blocking agents, at concentrations which do not reduce the response to ACh, block the response to stimulation of either pelvic or vesical nerves by up to 40 %.
There has been no anatomical demonstration of any nerve supply apart from those fibres which were stimulated in the above experiments.
The bladders of placental mammals, reptiles and amphibians are all outgrowths of the alimentary canal (Nelsen, 1953). Some parts of the bladder neck of placentals and reptiles are derived from the mesodermal tissues of the ureters. Buchanan & Fraser (1918) demonstrated a similar dual mode of origin for the marsupial bladder. In amphibians it seems unlikely that there should be any contribution of ureteral tissues to the bladder, since the ureters do not at any stage enter the bladder directly. In teleost and ganoid fish, however, the bladders are simply the swollen ends of the ureters. There is no bladder as such in elasmobranchs and birds.
In placental mammals the pelvic nerves to the bladder are excitatory and cholinergic (Elliott, 1907; Henderson & Roepke, 1934; Ursillo & Clark, 1956; Gyermek, 1961). The cat and the dog are the only placentals in which the sympathetic influence on the bladder has been studied in detail. Many workers have described an excitation followed by inhibition resulting from stimulation of the hypogastric nerve in these animals (Mellanby & Pratt, 1939, 1940; Kuntz & Saccomanno, 1944). The results reported by Edge (1955) suggest that both the contraction and the inhibition of the cat bladder resulting from hypogastric stimulation are mediated by adrenergic nerves. Elliott (1907) studied a series of placentals and concluded that there may be excitatory or inhibitory effects or no effects at all of the hypogastric nerve on the bladder musculature of different species, even within one family, but this has not been confirmed. Similarly, the excitatory nerves to the toad bladder are cholinergic (Burnstock, O’Shea & Wood, 1963). On the other hand, the lizard bladder appears to be innervated by a mixture of excitatory cholinergic and both excitatory and inhibitory adrenergic fibres (Burnstock, Wood & O’Shea, 1961). The innervation of the teleost bladder is excitatory, but the nature of the transmitter used is unknown (Young, 1936).
Thus it would appear that excitatory cholinergic nerve fibres to the urinary bladder are present throughout the vertebrates. On the other hand, adrenergic nerve fibres, where present, exert inhibitory, and possibly also excitatory, influences on the bladder fundus. There is as yet no indication of any evolutionary trend in the presence or absence of adrenergic innervation.
During the course of this work, several features of general pharmacological interest have been noted.
Many of the drugs which reduce the nerve-mediated responses of the possum bladder block the ACh response to a greater extent. This effect is particularly marked with atropine, and seems to be similar to the atropine-resistance found in many placental mammal preparations, and in particular in thebladder (Ursillo, 1961). Ursillo & Clark (1956) showed that the time taken for atropine to cause 50% block of the nerve-mediated response of the rabbit bladder strip/pelvic nerve preparation was eight times longer than the time taken to block the ACh response to the same extent. In common with the placental bladder, the marsupial bladder cannot be completely blocked with atropine. In fact, Henderson & Roepke (1934) took this as evidence for a non-cholinergic process in transmission, but Gyermek (1961) found no evidence to support this view.
The anti-ACh action of the pre- and post-synaptic adrenergic blocking agents used has also been noted in preparations from other animals, and has been made the subject of a more detailed study (Boyd et al. 1962, 1963). The apparent paradox of the possum bladder being blocked by both cholinergic and adrenergic blocking agents is thus explained by the atropine-like action of the anti-adrenergic agents. Similarly, the potentiation of ACh and nerve responses by some of these agents is explicable in terms of their anti-cholinesterase actions, already described for some of these drugs by Boyd, Chang & Rand (1960).
The relaxation of the possum bladder by histamine (10−7 to 10−5 g./ml.) is also pharmacological interest, since very few visceral smooth muscles have been shown to be relaxed by histamine. Gyermek (1961) has shown that the bladders of cats and dogs are contracted in vivo by histamine. The inhibition of the nerve response by histamine and also by adrenaline and noradrenaline is most probably due to a general depression of the excitability of the muscle.
When ganglion-blocking agents are used in pharmacological analysis it is necessary to check the action of the blocking agents against the response to directly applied terminal transmitter. If the concentration of blocking agent used reduces the response to the terminal transmitter, the amount of block of the nerve response is not a reliable indication of the degree of ganglionic blockade, e.g. on the possum bladder, hexamethonium and pentolinium cannot be used at concentrations greater than 5 × 10−4 and 10−5 g./ml. respectively. Similarly, the degree of ganglionic blockade is probably more than would be indicated by the depression of the response if the direct response of the muscle to applied transmitter is potentiated by the ganglionic blocking agent, e.g. hexamethonium at concentrations less than 5 × 10−4 g./ml. and mecamylamine at concentrations between 10−6 and 5 × 10−5 g./ml. potentiate the response to ACh. An additional problem is presented by the action of mecamylamine (10−4 g./ml.) in increasing the spontaneous movements to such an extent that responses can no longer be distinguished clearly. The potentiation of excitatory responses and increase in excitability produced by hexamethonium and mecamylamine on the possum bladder is not easily explained. Zauder (1954) and Bülbring (1955) reported similar effects of hexamethonium on placental mammal preparations, and suggested that they were due to a general increase in excitability of the muscles. However, Sjostrand (1962) showed that, although hexamethomum potentiates the excitatory responses of the guinea-pig vas deferens to noradrenaline, it reduces responses to ACh. Another explanation might be that these agents have an anti-cholinesterase action, but Paton & Zaimis (1949) have reported that hexamethonium has little, if any, anti-cholinesterase property.
The ACh-blocking action of hexamethonium and pentolinium on the possum bladder is similar to the atropine-like actions of these drugs on some placental mammal preparations (Feldberg, 1951; Mason & Wien, 1955). In contrast, Stone, Torchiana, Navarro & Beyer (1956) reported that mecamylamine had no atropine-like action at 2 × 10−5 g./ml., a result which was also true for the possum bladder.
If the amount of blockade of the nerve response produced by these three ganglionic blocking agents is taken as a measure of potency, the drugs may be arranged in the following order of increasing potency: hexamethonium:pentolinium:mecamylamine. However, in the light of the above discussion, the factor of specificity should also be included in any true index of potency.
It would seem that, in the absence of any truly specific blocking agents, the use of analytical pharmacology for the determination of the transmitter substance involved in an autonomic nerve/smooth muscle preparation is a difficult and, in some cases, inconclusive process. It is advisable to use such an analysis in combination with a quantitative biochemical assay of the perfusates from organs after stimulation of the nerve supply.
ACKNOWLEDGEMENTS
This work was supported by a P.H.S. research grant (B-2902) from the National Institute of Neurological Diseases and Blindness, Public Health Service (U.S.A.) and by the National Health and Medical Research Council (Aust.).
We would like to express our thanks to the following pharmaceutical firms for their kind donations of drugs: CIBA, Basle; May and Baker, Dagenham; Merck, Sharp and Dohme; Smith, Kline and French; Wellcome Research Laboratories. We would also like to thank Mr D. Rogers for his valuable assistance with histological material.