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Urinary bladder innervation in children
Urinary Bladder Innervation in Children By Carl Magnus Kullendorff, Mats Elm&r, and Per Aim Lund, Sweden
MATERIALS AND METHODS
taken from the fundus and trigone of the bladder and from the distal ureter with the surrounding bladder tissue. All patients were treated with nitrofurantoin and had no urinary tract infection at the time of operation. Some of the specimens were instantly frozen in isopentane. From some of the frozen specimens cryostat sections were cut at a thickness of 10 #. The sections were processed according to a modified copper-thiocholine method 3 to demonstrate acetylcholinesterase (AchE) activity. Prometazine hydrochloride was used to inhibit nonspecific cholinesterase activity. 4 Eosin was used as counter-stain. The other frozen pieces were freeze-dried. Some of these were then exposed to gaseous formaldehyde and further processed for the demonstration of adrenergic nerves. 5 The other freeze-dried tissue specimens were exposed to vapors of diethylpyrocarbonate. 6 Cut sections of 6/~ were processed with the PAP-immunoperoxidase technique 7 for the neuronal demonstration of various peptides (vasoactive intestinal peptide [VIP], substance P, somatostatin, leuenkephaline and met-enkephaline). In the immunohistoehemical procedures, controls with antisera inactivated by excess of antigen were used. The sections were examined with a standard light microscope. The muscle strips, 8 to 10 m m long and 1 to 2 m m wide, were mounted between platinum pins in an organ bath containing 20 m L of Krebs solution at 37 ~ and aerated to maintain pH at 7.4. To obtain a K+-rich solution, NaCI was omitted and substituted with KC1120 m m o l / L . The muscular tension was measured isometrically by means of strain gauge transducers and recorded on a Houston Omniscribe servo recorder. The initial tension was set at 0.5 g. For electrical field stimulation a Grass stimulator (Quincy, MA) giving biphasic square pulses with a duration of 0.5 ms, a frequency of 0.5 to 100 Hz and of supramaximal voltage was used. The muscles were studied either in the resting state or during contraction induced by electrical stimulation, metacholine, or K+-rich solution. After addition of each dose of the various drugs to the bath, the preparation was washed several times and allowed to reach its initial state of tension. The following drugs were added directly to the bath: metacholine, eserine, noradrenaline, ephedrine, phenylephrine, isoprenaline, prealterol, terbutaline, atropine, dihydroergotamine, phenoxybenzamine, prazosin, propranolol, metoprolol, H 3 5 / 2 5 etanol, hexamethonium, tetrodotoxine, imipramine, terodiline, substance P, and VIP. Concentrations given are final bath concentrations of active substance. The results were analyzed statistically using the Student's t test. The probability level of 0.05 was accepted as significant.
Eleven children 0.5 to 10 years of age (mean 5 years) were operated on because of vesicoureteric reflux. Tissue specimens were
RESULTS
9 Muscle strips from the fundus, trigone, and distal ureter obtained from children at operation for vesicoureteric reflux w e r e studied using histochemical and immunohistochemical methods, and electrical nerve stimulation in an organ bath. A rich supply of cholinergic nerves was found and the transmitter causing contraction of the detrusor muscle was regarded as being acetylcholine. The adrenergic innervation was very sparse except around the ureteric orifices. No contractile o~-adrenoceptors could be detected but /~-receptor-mediated relaxation was found. The type was not /~1 or /32, suggesting a third type of /~-receptor. Peptidergic nerves containing vasoactive intestinal peptide (VIP) w e r e demonstrated in a f e w nerve terminals. No nerves containing enkephaline, somatostatin, or substance P w e r e found. VIP affected the detrusor muscle, indicating a possible role as a modulator of transmitter action. Imipramine, used for enuresis, had no anticholinargic effect on the bladder in the doses used clinically. The anticholinergic and calcium antagonistic drug terodiline inhibited all muscle activity, making it suitable for t r e a t m e n t of diurnal enuresis. 9 1987 by Grune & Stratton, Inc. INDEX W O R D S : Urinary bladder innervation; adrenoceptors; peptidergic nerves; calcium antagonist; enuresis.
N CHILDREN the autonomic innervation of the urinary bladder has only been studied pharmacologically. Occurrence of specific receptors is not always accompanied by the corresponding innervation. The fetal urinary tract contains adrenoceptors but no adrenergic nerves can be demonstrated. 1Relaxation of the bladder has been presumed to be mediated by a certain type of/3-adrenoceptor, which differs from/31 or/31 receptors. 2 Morphologic and physiologic studies in children are of importance because of the changes of innervation that occur during development. We have investigated the autonomic innervation of the child's urinary bladder and the effects of drugs usable in the treatment of enuresis.
I
Nerve Histochemistry From the Departments of Pediatric Surgery, Physiology and Pathology, University of Lund, Sweden. Presented at the 33rd Annual Congress of the British Association of Paediatric Surgeons, Birmingham, England, July 16-18, 1986. Address reprint requests to Carl Magnus Kullendorff, MD, Department of Pediatric Surgery, University Hospital, S-221 85 Lund, Sweden. 9 1987 by Grune & Stratton, Inc. 0022-3468/87/2203~014503.00/0 240
The adrenergic innervation appeared as fine varicose nerve terminals along smooth muscle bundles. The nerve density was very sparse in the fundus and trigone of the bladder and in the distal ureter. Around the ureteric orifice in the bladder wall it was slightly denser. There was, however, an abundance of AchE-positive nerves (APN) in the fundus and trigone of the bladder Journal of Pediatric Surgery, Vol 22, No 3 (March), 1987: pp 240-242
URINARY BLADDER INNERVATION
with plexuses of APN terminals around the smooth muscle bundles. The nerve density was less in the distal ureter. APN terminals were also found regularly in the connective tissue of the lamina propria just beneath the bladder epithelium. Moreover, some blood vessels were encircled by APN terminals. Peptidergic nerves generally occurred very sparsely. VIP-immunoreactivity was only found in a few nerve terminals that ran along smooth muscle bundles. No specific immunoreactivity with the other antisera tested could be demonstrated. No intramural ganglion cells, adrenergic, APN, or peptidergic could be detected in any tissue specimen.
Nerve Stimulation Electrical field stimulation elicited frequencydependent contractions of the muscle, increasing from a threshold frequency of 0.5 to 1 Hz to a maximum tension of 50 Hz. The response was completely abolished after the administration of tetrodotoxin, indicating that the short stimulation pulses used had no direct effect on the muscle cells but were nerve-mediated. The response to 20 Hz was reduced by about 80% in the presence of atropine or imipramine at 50 ug/mL. The contractions increased by about 100% after adding eserine (5 ug/mL) to the bath. The responses were not affected by previous addition of hexamethonium (500 ug/mL), dihydroergotamine (50 #g/mL), phenoxybenzamine (50 to 2,500 ~g/mL), and prazosine (0.5 to 500 ~tg/mL) or ephedrine (0.5 to 5 #g/mE). Noradrenaline and isoprenaline (5 ug/mL) caused a further reduction by about 60% of the response remaining after atropine. Prenalterol and terbutaline (5 to 10 ug/mL) had no effect. This inhibiting effect of noradrenaline and isoprenaline was abolished by previous addition of propranolol (50 ug/mL).
Drug Effects Phenylephrine (0.25 to 500 ~tg/mL) had no effect on the bladder muscle from fundus or trigone. Noradrenaline did not affect the muscle in the resting state but caused a dose-dependent relaxation of the contracted preparation. Isoprenaline (0.05 to 5 #g/mL) caused a similar relaxation both in the resting state and during contraction, whereas prenalterol and terbutaline had no effect. The relaxation was totally abolished by previous addition of propranolol (50 #g/mL) but was not affected by the fll blocking agent metoprolol or the f12blocker H35/25. VIP (0.025 to 2.5 ug/mL) elicited a slow dosedependent contraction in the resting muscular strips from the trigone but had no effect on resting strips from the fundus. In the contracted preparation both from trigone and fundus, the response to VIP was a
241
relaxation similar to that caused by noradrenaline. Substance P (0.1 to 2 t~g/mL) had no effect on either the resting or contracted muscle. Terodiline, a drug with both anticholinergic and calcium antagonistic properties, s (0.5 to 5 #g/mL) did not affect the resting muscle but inhibited the contracted strips both from the fundus and the trigone. DISCUSSION
The occurrence and distribution of adrenergic and cholinergic nerves in the child's urinary bladder have been very sparsely investigated.9 The occurrence of peptidergic nerves in the urinary bladder of children has not previously been reported. Accordingly, the present combined nerve-histochemical and physiological investigation appears to be important. The contractile responses to electrical nerve stimulation is exclusive to the child's urinary bladder. The findings are different in animals and in human adults demonstrating diverse combinations of relaxation and contraction. In the rat, contraction is followed by a relaxation phase only when the nerves are stimulated for a very short period, l~ In the pig and in man the responses are either relaxation only, contraction only, or different combinations of the relaxation and the contraction phase.ll No intramural ganglion cells were found morphologically, as is the case in adults. ~2 Functionally, this was confirmed by the lack of effect of a ganglionblocking drug on the responses to nerve stimulation. Ganglia, however, have been found in the bladder wall of many species, the exceptions being the rat and human) 3 The distribution of APN did not diverge from the corresponding bladder parts, which have been investigated in adults. ~2 There existed a rich smooth muscle detrusor supply of probably cholinergic nerves as verified by our physiologic findings. Some APN fibers were encircling some blood vessels indicating a vasomotor function. Some intramucosal APN fibers were ending in the connective tissue beneath the uroepithelium and unrelated to neuroeffector target sites, suggesting a sensory functional role. l* The contractions caused by nerve stimulation were reduced by anticholinergic drugs and potentiated by AchE. These findings indicate that the neuromuscular transmitter is acetylcholine, which is confirmed by the rich supply of APN found in the detrusor muscle. The partial atropine resistance of the response to nerve stimulation in the urinary bladder of animals has been known for a long time. ~5 It has been suggested that acetylcholine is liberated by the nerve endings in such an intimate relationship to the receptor that atropine cannot prevent its access. 16 Imipramine, used for nocturnal enuresis, reduced
242
KULLENDORFF, ELMER, AND ALM
the detrusor contractions to the same degree as atropine. However, the dose required was about a thousand times the expected c o n c e n t r a t i o n when used clinically. Accordingly, the effect of i m i p r a m i n e in bed-wetting children seems to be caused by an action on the central nervous system rather than by a direct anticholinergic effect on the u r i n a r y bladder. A very small n u m b e r of V I P nerves have been found in the adult h u m a n u r i n a r y bladder. 17 T h e results of this study, showing a few V I P nerves distributed along smooth muscle bundles, a n d the physiologic smooth muscle responses in vitro indicate that V I P has a regulatory role on smooth m u s c u l a t u r e in the child's u r i n a r y bladder. This study revealed a generally sparse adrenergic innervation of the bladder and ureteric smooth musculature. T h e distribution p a t t e r n was the same as in adults except in the trigone. I n the present m a t e r i a l this region of the bladder showed a scanty adrenergic nerve supply. This finding is interesting, as in experi-
m e n t a l a n i m a l studies vesicoureteral reflux has been shown to occur after s y m p a t h e c t o m y ) 8 N o contractile a-adrenoceptors could be detected in the fundus or trigone, as in a d u l t s ) 9 Relaxation of the detrusor muscle was mediated by fl-adrenoceptors but the n a t u r e of these receptors was neither fll nor f12. T h e presence of a third type of fl receptor, earlier suggested by Nerg~trdh, 2 was thus confirmed. The adrenergic t r a n s m i t t e r n o r a d r e n a l i n e caused relaxation of the contracted muscle, indicating a m o d u l a t i n g effect on bladder activity of the few a d r e n e r g i c nerves found. T h e adrenergic agonist ephedrine, used clinically in children for t r e a t m e n t of enuresis, had no effect, however. Terodiline inhibited muscle activity induced in every way. The anticholinergic and direct muscle relaxant effects indicate action through c a l c i u m antagonism. These effects m a k e this drug suitable for t r e a t m e n t of d a y t i m e enuretic children with a small functional bladder capacity a n d u n s t a b l e bladder. 2~
REFERENCES
1. Andersson K-E, Persson CGA, Aim P, et al: Effects of acetylcholine, noradrenaline, and prostaglandins on the isolated, perfused human fetal urethra. Acta Physiol Scand 104:394-401, 1978 2. Nerg~irdhA, Boreus LO, Naglo A-S: Characterization of the adrenergic beta-receptor in the urinary bladder of man and cat. Acta Pharmacol Toxico140:14-21, 1977 3. Holmstedt B: A modification of the thiocholine method for determination of cholinesterase II. Histochemical application. Acta Physiol Scand 40:331-337, 1957 4. Matthews AD, Nadler JV, Lynch GS, et al: Development of cholinergic innervation in the hippocampal formation of the rat I. Histochemical demonstration of acetylcholine activity. Dev Biol 36:130-141, 1974 5. Bj~SrklundA, Falck B, Owman C: in Berson SA (ed): Methods of Investigative and Diagnostic Endocrinology.Amsterdam, North Holland Publishing Company, 1972, p 138 6. Pearse AGE, Polak J: Immunocytochemical localization of substance P in mammalian intestine. Histochemistry 41:373-375, 1975 7. Stenberger LA: Immunocytochemistry. Englewood Cliff, NJ, Prentice-Hall, 1974 8. Andersson K-E, Ulmsten U, Sundwall A: Terodiline in the treatment of motor urge incontinence. Acta Pharmacol Toxicol 46: 1980 (suppl I) 9. Oemichen M, Reifferscheid P, Flack A, et al: Histochemische Untersuchungen zur Innervation des distalen Ureters bei prim~iren vesiko-ureteralen Reflux im Kindesalter. Klin P/~diatr 188:30-34, 1976
10. Elm6r M: Stimulation of adrenergic nerve fibres to the urinary bladder of the rat. Acta Physiol Scand 94:517-521, 1975 11. KlarskovP, Gerstenberg TC, Ramirez D, et al: Non-cholinergic, non-adrenergic nerve mediated relaxation of trigone, bladder neck and urethral smooth muscle in vitro. J Urol 129:848-850, 1983 12. Ek A, Aim P, Andersson K-E, et al: Adrenergic and cholinergic nerves of the human urethra and urinary bladder. A histochemical study. Acta Physiol Scand 99:345-352, 1977 13. Aim P, Elm6r M: Adrenergic and cholinergic innervation of the rat urinary bladder. Acta Physiol Scand 94:36~,5, 1975 14. Gosling JA, Dixon JS: Sensory nerves in the mammalian urinary tract. An evaluation using light and electron microscopy. J Anat 177:133-144, 1974 15. Langley JN, Anderson HK: The innervation of the pelvicand adjoining viscera II. The bladder. J Physiol 19:71-84, 1895 16. Elm6rM: Cholinergicmechanismsin the rat detrusor muscle. Acta Pharmacol Toxico143:63-68, 1978 17. Aim P, Alumets J, Ek A, et al: Peptidergic (VIP) nerves in the human urinary tract. Proc Int Cont Soc 9th Ann Meeting 1979, pp 147-150 18. Lenaghan D: Experimental neurogenic vesico-uretetral reflux. Br J Urol 41:606-607, 1969 19. Awad SA, Bruce AW, Carro-Ciampi G, et al: Distribution of a- and fl-adrenoceptors in human urinary bladder. Br J Pharmacol 50:525-529, 1974 20. Elm6r M: Terodiline in children with diurnal enuresis. Scand J Urol Nephrol 87:59-61, 1984 (suppl)
MATERIALS AND METHODS
taken from the fundus and trigone of the bladder and from the distal ureter with the surrounding bladder tissue. All patients were treated with nitrofurantoin and had no urinary tract infection at the time of operation. Some of the specimens were instantly frozen in isopentane. From some of the frozen specimens cryostat sections were cut at a thickness of 10 #. The sections were processed according to a modified copper-thiocholine method 3 to demonstrate acetylcholinesterase (AchE) activity. Prometazine hydrochloride was used to inhibit nonspecific cholinesterase activity. 4 Eosin was used as counter-stain. The other frozen pieces were freeze-dried. Some of these were then exposed to gaseous formaldehyde and further processed for the demonstration of adrenergic nerves. 5 The other freeze-dried tissue specimens were exposed to vapors of diethylpyrocarbonate. 6 Cut sections of 6/~ were processed with the PAP-immunoperoxidase technique 7 for the neuronal demonstration of various peptides (vasoactive intestinal peptide [VIP], substance P, somatostatin, leuenkephaline and met-enkephaline). In the immunohistoehemical procedures, controls with antisera inactivated by excess of antigen were used. The sections were examined with a standard light microscope. The muscle strips, 8 to 10 m m long and 1 to 2 m m wide, were mounted between platinum pins in an organ bath containing 20 m L of Krebs solution at 37 ~ and aerated to maintain pH at 7.4. To obtain a K+-rich solution, NaCI was omitted and substituted with KC1120 m m o l / L . The muscular tension was measured isometrically by means of strain gauge transducers and recorded on a Houston Omniscribe servo recorder. The initial tension was set at 0.5 g. For electrical field stimulation a Grass stimulator (Quincy, MA) giving biphasic square pulses with a duration of 0.5 ms, a frequency of 0.5 to 100 Hz and of supramaximal voltage was used. The muscles were studied either in the resting state or during contraction induced by electrical stimulation, metacholine, or K+-rich solution. After addition of each dose of the various drugs to the bath, the preparation was washed several times and allowed to reach its initial state of tension. The following drugs were added directly to the bath: metacholine, eserine, noradrenaline, ephedrine, phenylephrine, isoprenaline, prealterol, terbutaline, atropine, dihydroergotamine, phenoxybenzamine, prazosin, propranolol, metoprolol, H 3 5 / 2 5 etanol, hexamethonium, tetrodotoxine, imipramine, terodiline, substance P, and VIP. Concentrations given are final bath concentrations of active substance. The results were analyzed statistically using the Student's t test. The probability level of 0.05 was accepted as significant.
Eleven children 0.5 to 10 years of age (mean 5 years) were operated on because of vesicoureteric reflux. Tissue specimens were
RESULTS
9 Muscle strips from the fundus, trigone, and distal ureter obtained from children at operation for vesicoureteric reflux w e r e studied using histochemical and immunohistochemical methods, and electrical nerve stimulation in an organ bath. A rich supply of cholinergic nerves was found and the transmitter causing contraction of the detrusor muscle was regarded as being acetylcholine. The adrenergic innervation was very sparse except around the ureteric orifices. No contractile o~-adrenoceptors could be detected but /~-receptor-mediated relaxation was found. The type was not /~1 or /32, suggesting a third type of /~-receptor. Peptidergic nerves containing vasoactive intestinal peptide (VIP) w e r e demonstrated in a f e w nerve terminals. No nerves containing enkephaline, somatostatin, or substance P w e r e found. VIP affected the detrusor muscle, indicating a possible role as a modulator of transmitter action. Imipramine, used for enuresis, had no anticholinargic effect on the bladder in the doses used clinically. The anticholinergic and calcium antagonistic drug terodiline inhibited all muscle activity, making it suitable for t r e a t m e n t of diurnal enuresis. 9 1987 by Grune & Stratton, Inc. INDEX W O R D S : Urinary bladder innervation; adrenoceptors; peptidergic nerves; calcium antagonist; enuresis.
N CHILDREN the autonomic innervation of the urinary bladder has only been studied pharmacologically. Occurrence of specific receptors is not always accompanied by the corresponding innervation. The fetal urinary tract contains adrenoceptors but no adrenergic nerves can be demonstrated. 1Relaxation of the bladder has been presumed to be mediated by a certain type of/3-adrenoceptor, which differs from/31 or/31 receptors. 2 Morphologic and physiologic studies in children are of importance because of the changes of innervation that occur during development. We have investigated the autonomic innervation of the child's urinary bladder and the effects of drugs usable in the treatment of enuresis.
I
Nerve Histochemistry From the Departments of Pediatric Surgery, Physiology and Pathology, University of Lund, Sweden. Presented at the 33rd Annual Congress of the British Association of Paediatric Surgeons, Birmingham, England, July 16-18, 1986. Address reprint requests to Carl Magnus Kullendorff, MD, Department of Pediatric Surgery, University Hospital, S-221 85 Lund, Sweden. 9 1987 by Grune & Stratton, Inc. 0022-3468/87/2203~014503.00/0 240
The adrenergic innervation appeared as fine varicose nerve terminals along smooth muscle bundles. The nerve density was very sparse in the fundus and trigone of the bladder and in the distal ureter. Around the ureteric orifice in the bladder wall it was slightly denser. There was, however, an abundance of AchE-positive nerves (APN) in the fundus and trigone of the bladder Journal of Pediatric Surgery, Vol 22, No 3 (March), 1987: pp 240-242
URINARY BLADDER INNERVATION
with plexuses of APN terminals around the smooth muscle bundles. The nerve density was less in the distal ureter. APN terminals were also found regularly in the connective tissue of the lamina propria just beneath the bladder epithelium. Moreover, some blood vessels were encircled by APN terminals. Peptidergic nerves generally occurred very sparsely. VIP-immunoreactivity was only found in a few nerve terminals that ran along smooth muscle bundles. No specific immunoreactivity with the other antisera tested could be demonstrated. No intramural ganglion cells, adrenergic, APN, or peptidergic could be detected in any tissue specimen.
Nerve Stimulation Electrical field stimulation elicited frequencydependent contractions of the muscle, increasing from a threshold frequency of 0.5 to 1 Hz to a maximum tension of 50 Hz. The response was completely abolished after the administration of tetrodotoxin, indicating that the short stimulation pulses used had no direct effect on the muscle cells but were nerve-mediated. The response to 20 Hz was reduced by about 80% in the presence of atropine or imipramine at 50 ug/mL. The contractions increased by about 100% after adding eserine (5 ug/mL) to the bath. The responses were not affected by previous addition of hexamethonium (500 ug/mL), dihydroergotamine (50 #g/mL), phenoxybenzamine (50 to 2,500 ~g/mL), and prazosine (0.5 to 500 ~tg/mL) or ephedrine (0.5 to 5 #g/mE). Noradrenaline and isoprenaline (5 ug/mL) caused a further reduction by about 60% of the response remaining after atropine. Prenalterol and terbutaline (5 to 10 ug/mL) had no effect. This inhibiting effect of noradrenaline and isoprenaline was abolished by previous addition of propranolol (50 ug/mL).
Drug Effects Phenylephrine (0.25 to 500 ~tg/mL) had no effect on the bladder muscle from fundus or trigone. Noradrenaline did not affect the muscle in the resting state but caused a dose-dependent relaxation of the contracted preparation. Isoprenaline (0.05 to 5 #g/mL) caused a similar relaxation both in the resting state and during contraction, whereas prenalterol and terbutaline had no effect. The relaxation was totally abolished by previous addition of propranolol (50 #g/mL) but was not affected by the fll blocking agent metoprolol or the f12blocker H35/25. VIP (0.025 to 2.5 ug/mL) elicited a slow dosedependent contraction in the resting muscular strips from the trigone but had no effect on resting strips from the fundus. In the contracted preparation both from trigone and fundus, the response to VIP was a
241
relaxation similar to that caused by noradrenaline. Substance P (0.1 to 2 t~g/mL) had no effect on either the resting or contracted muscle. Terodiline, a drug with both anticholinergic and calcium antagonistic properties, s (0.5 to 5 #g/mL) did not affect the resting muscle but inhibited the contracted strips both from the fundus and the trigone. DISCUSSION
The occurrence and distribution of adrenergic and cholinergic nerves in the child's urinary bladder have been very sparsely investigated.9 The occurrence of peptidergic nerves in the urinary bladder of children has not previously been reported. Accordingly, the present combined nerve-histochemical and physiological investigation appears to be important. The contractile responses to electrical nerve stimulation is exclusive to the child's urinary bladder. The findings are different in animals and in human adults demonstrating diverse combinations of relaxation and contraction. In the rat, contraction is followed by a relaxation phase only when the nerves are stimulated for a very short period, l~ In the pig and in man the responses are either relaxation only, contraction only, or different combinations of the relaxation and the contraction phase.ll No intramural ganglion cells were found morphologically, as is the case in adults. ~2 Functionally, this was confirmed by the lack of effect of a ganglionblocking drug on the responses to nerve stimulation. Ganglia, however, have been found in the bladder wall of many species, the exceptions being the rat and human) 3 The distribution of APN did not diverge from the corresponding bladder parts, which have been investigated in adults. ~2 There existed a rich smooth muscle detrusor supply of probably cholinergic nerves as verified by our physiologic findings. Some APN fibers were encircling some blood vessels indicating a vasomotor function. Some intramucosal APN fibers were ending in the connective tissue beneath the uroepithelium and unrelated to neuroeffector target sites, suggesting a sensory functional role. l* The contractions caused by nerve stimulation were reduced by anticholinergic drugs and potentiated by AchE. These findings indicate that the neuromuscular transmitter is acetylcholine, which is confirmed by the rich supply of APN found in the detrusor muscle. The partial atropine resistance of the response to nerve stimulation in the urinary bladder of animals has been known for a long time. ~5 It has been suggested that acetylcholine is liberated by the nerve endings in such an intimate relationship to the receptor that atropine cannot prevent its access. 16 Imipramine, used for nocturnal enuresis, reduced
242
KULLENDORFF, ELMER, AND ALM
the detrusor contractions to the same degree as atropine. However, the dose required was about a thousand times the expected c o n c e n t r a t i o n when used clinically. Accordingly, the effect of i m i p r a m i n e in bed-wetting children seems to be caused by an action on the central nervous system rather than by a direct anticholinergic effect on the u r i n a r y bladder. A very small n u m b e r of V I P nerves have been found in the adult h u m a n u r i n a r y bladder. 17 T h e results of this study, showing a few V I P nerves distributed along smooth muscle bundles, a n d the physiologic smooth muscle responses in vitro indicate that V I P has a regulatory role on smooth m u s c u l a t u r e in the child's u r i n a r y bladder. This study revealed a generally sparse adrenergic innervation of the bladder and ureteric smooth musculature. T h e distribution p a t t e r n was the same as in adults except in the trigone. I n the present m a t e r i a l this region of the bladder showed a scanty adrenergic nerve supply. This finding is interesting, as in experi-
m e n t a l a n i m a l studies vesicoureteral reflux has been shown to occur after s y m p a t h e c t o m y ) 8 N o contractile a-adrenoceptors could be detected in the fundus or trigone, as in a d u l t s ) 9 Relaxation of the detrusor muscle was mediated by fl-adrenoceptors but the n a t u r e of these receptors was neither fll nor f12. T h e presence of a third type of fl receptor, earlier suggested by Nerg~trdh, 2 was thus confirmed. The adrenergic t r a n s m i t t e r n o r a d r e n a l i n e caused relaxation of the contracted muscle, indicating a m o d u l a t i n g effect on bladder activity of the few a d r e n e r g i c nerves found. T h e adrenergic agonist ephedrine, used clinically in children for t r e a t m e n t of enuresis, had no effect, however. Terodiline inhibited muscle activity induced in every way. The anticholinergic and direct muscle relaxant effects indicate action through c a l c i u m antagonism. These effects m a k e this drug suitable for t r e a t m e n t of d a y t i m e enuretic children with a small functional bladder capacity a n d u n s t a b l e bladder. 2~
REFERENCES
1. Andersson K-E, Persson CGA, Aim P, et al: Effects of acetylcholine, noradrenaline, and prostaglandins on the isolated, perfused human fetal urethra. Acta Physiol Scand 104:394-401, 1978 2. Nerg~irdhA, Boreus LO, Naglo A-S: Characterization of the adrenergic beta-receptor in the urinary bladder of man and cat. Acta Pharmacol Toxico140:14-21, 1977 3. Holmstedt B: A modification of the thiocholine method for determination of cholinesterase II. Histochemical application. Acta Physiol Scand 40:331-337, 1957 4. Matthews AD, Nadler JV, Lynch GS, et al: Development of cholinergic innervation in the hippocampal formation of the rat I. Histochemical demonstration of acetylcholine activity. Dev Biol 36:130-141, 1974 5. Bj~SrklundA, Falck B, Owman C: in Berson SA (ed): Methods of Investigative and Diagnostic Endocrinology.Amsterdam, North Holland Publishing Company, 1972, p 138 6. Pearse AGE, Polak J: Immunocytochemical localization of substance P in mammalian intestine. Histochemistry 41:373-375, 1975 7. Stenberger LA: Immunocytochemistry. Englewood Cliff, NJ, Prentice-Hall, 1974 8. Andersson K-E, Ulmsten U, Sundwall A: Terodiline in the treatment of motor urge incontinence. Acta Pharmacol Toxicol 46: 1980 (suppl I) 9. Oemichen M, Reifferscheid P, Flack A, et al: Histochemische Untersuchungen zur Innervation des distalen Ureters bei prim~iren vesiko-ureteralen Reflux im Kindesalter. Klin P/~diatr 188:30-34, 1976
10. Elm6r M: Stimulation of adrenergic nerve fibres to the urinary bladder of the rat. Acta Physiol Scand 94:517-521, 1975 11. KlarskovP, Gerstenberg TC, Ramirez D, et al: Non-cholinergic, non-adrenergic nerve mediated relaxation of trigone, bladder neck and urethral smooth muscle in vitro. J Urol 129:848-850, 1983 12. Ek A, Aim P, Andersson K-E, et al: Adrenergic and cholinergic nerves of the human urethra and urinary bladder. A histochemical study. Acta Physiol Scand 99:345-352, 1977 13. Aim P, Elm6r M: Adrenergic and cholinergic innervation of the rat urinary bladder. Acta Physiol Scand 94:36~,5, 1975 14. Gosling JA, Dixon JS: Sensory nerves in the mammalian urinary tract. An evaluation using light and electron microscopy. J Anat 177:133-144, 1974 15. Langley JN, Anderson HK: The innervation of the pelvicand adjoining viscera II. The bladder. J Physiol 19:71-84, 1895 16. Elm6rM: Cholinergicmechanismsin the rat detrusor muscle. Acta Pharmacol Toxico143:63-68, 1978 17. Aim P, Alumets J, Ek A, et al: Peptidergic (VIP) nerves in the human urinary tract. Proc Int Cont Soc 9th Ann Meeting 1979, pp 147-150 18. Lenaghan D: Experimental neurogenic vesico-uretetral reflux. Br J Urol 41:606-607, 1969 19. Awad SA, Bruce AW, Carro-Ciampi G, et al: Distribution of a- and fl-adrenoceptors in human urinary bladder. Br J Pharmacol 50:525-529, 1974 20. Elm6r M: Terodiline in children with diurnal enuresis. Scand J Urol Nephrol 87:59-61, 1984 (suppl)