Acta Physiol Scand 1990, 140, 365-372

Functional responses of different muscle types of the female rat urethra in vitro P. 0. ANDERSSON, A. MALMGREN" and B. U V E L I U S t Departments of Physiology, *Clinical Pharmacology and ?Urology, University of Lund, Lund, Sweden

ANDERSON, P.O., MALMGREN, A. & UVELIUS, B. 1990. Functional responses of different muscle types of the female rat urethra in vitro. Acta Physiol Scand 140, 365-372. Received 16 January 1990, accepted 15 June 1990. ISSN 0001-6772. Departments of Physiology, Clinical Pharmacology and Urology, University of Lund, Sweden. The influence of muscle type on functional responses of the female rat urethra was investigated using morphological and functional in-vitro techniques. The urethral submucosa was found to contain longitudinally or obliquely oriented smooth muscle cells. The muscularis layer is composed of circularly oriented muscle cells. Near the bladder orifice smooth muscle fibres dominate, but in the mid-urethra the vast majority is circularly oriented striated muscle cells. Circular preparations responded to electrical field stimulation zn vitro with a rapid contraction. Stimulation with single impulses resulted in a twitch response; frequencies exceeding 5-10 Hz induced a summation and tetanus. The response was unaffected by phenoxybenzamine, propranolol and scopolamine and had a low sensitivity to calciumfree solution but was sensitive to suxamethonium and tetrodotoxin. Using longer impulse trains stimulation evoked also a slow contraction, sensitive to calcium-free solution. In longitudinal preparations stimulation induced a relaxation followed by a contraction, responses much smaller than those seen in the circular preparations. Both preparations relaxed on addition of calcitonin gene-related peptide or capsaicin. The relaxation to calcitonin gene-related peptide was larger than that to capsaicin in longitudinal preparations but equally large in the circular ones. Substance P and 5hydroxytryptamine contracted both preparations. The longitudinal urethra showed a larger contraction to 5-hydroxytryptamine than to substance P, whereas both substances induced similar responses in the circular preparations. The study shows a similar muscle arrangement in the female rat urethra as described in humans and further points to a functional differentiation between the different types of muscle. Key words ; calcitonin gene-related peptide (CGRP), capsaicin, 5-hydroxytryptamine (5-HT), smooth muscle, striated muscle, substance P, urethra.

T h e function of the urethra is to open during micturition to permit the passage of urine but to be closed otherwise, thereby maintaining continence despite large and rapid rises in bladder pressure, i.e. during exercise or coughing. T h e anatomy of the female urethra is complex and consists of smooth as well as striated muscle fibres oriented in a circular as well as a Correspondence : P.O. Andersson, Department of Physiology, Solvegatan 19, S-223 62 Lund, Sweden.

longitudinal direction (e.g. Hutch & Rambo 1967, Droes 1974, Gosling 1985). T h e functional role of these different muscles during continence and micturition is not known. T h e urethra is known to be innervated via both the hypogastric and the pelvic nerves. Activation of a-adrenergic receptors induces a contraction of the urethra, whereas P-adrenergic agonists cause a decrease in urethral pressure (Creed 1979, Slack & Downie 1983). Activation of the pelvic nerves induces a partly cholinergic increase in urethral pressure

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(Creed & Tulloch 1978, Andersson et al. 1989), and choline acetyltransferase activity has been demonstrated in the prosimal rat urethra, indicating a cholinergic innervation of the tissue (Ekstrom & Malmberg 1981). However, activation of both the h!-pogastric and the pelvic innervation also produces non-adrenergic, noncholinergic response (Creed & Tulloch 1978, Creed 1979, Andersson et u / . 1989). T h e neurotransmitter(s) mediating these responses are unknown. However, several putative peptidergic neurotransmitters, i.e. vasoactive intestinal peptide (VIP), substance P, calcitonin generelated peptide (CGRP) and neuropeptide Y (SPY),known to be present in nerves innervating the urethra (-4lm et a / . 1978, 1980, S u et al. 1986) ha\ c been suggested to be putative neurotransmitters. It is further known that the urothelium of the urethra contains endocrine-like cells storing 5-hydroxytryptamine (5-HT) (Hikanson et a / . 1974) and that .i-HT induces an increase in urethral pressure (Creed & Tulloch

preparations were then mounted in the circular or longitudinal direction, between a fixed hook and a Grass force-displacement transducer (FT03). Isometric tension was recorded on a Grass polygraph. The preparations were stretched to a preload of 5 mN and left to equilibrate for 45 min. Electrical field stimulation. Stimulation was performed by means of two longitudinally arranged platinum electrodes and a Grass stimulator (S48) using supramaximal voltage. The impulse duration was varied between 0.005 and 0.8 ms and the train duration between 1 and 5 s with a train interval between 30 s and 2 min. The responses to electrical stimulation were investigated in normal Krebs solution, in Ca"-free solution (for composition see below) and in the presence of propranolol (lo-' M), scopolamine (lo-' M), phenoxybenzamine (lo-' M), noradrenaline ( 5 x lo-' M), substance P (5 x lo-" M), suxamethonium (10 -I to 10P M) or tetrodotoxin M). (TTX; Responsrs t o drugs. The responses to CGRP (1 0-' M), capsaicin M), substance P (5 x M) and 5-IIT (10 ,5 M) were investigated in circular and longitudinal preparations.

1978). T h e aim of the present investigations was to compare the function of the different muscles of the female rat urethra in ritrci and to correlate these findings u-ith morphological investigations. hlL\TERI.4LS AND METHODS ..inrnidls

Solutions

The Krebs solution had the following composition (mLi): NaCl 118, KCI 4.6, MgSO, 1.1-5, NaH,PO, 1.15, NaHCO, 24.9, CaCI, 1.25 and glucose 5.5. High-K' solution (124 mM) was prepared by replacing Na+ with eqimolar amounts of I(+ in the normal Krebs solution. ' Calcium-free' medium was prepared by excluding Ca2+ in the normal Krebs solution and adding EGTA (0.1 mu).

Female Sprague-Dawle) rats (200-225 g) Here used Drugs Prepurutions .4lorphologrcul rniwtigations. The rats were deeply anaesthetized with ether, and a cannula was, via the left ventricle, inserted in the aorta and secured by a ligature around the vessel. The animals were then perfused with j O , glutaraldehyde in 100 mM cacodvlate buffer (pH 7.4). After fixation the urethra was dissected out and cut into six segments, which were embedded in Araldite. Cross- and longitudinal sections &-erecut for light microscopy. The thickness of the urethral muscle layers was measured using an ocular scale. Functional inorstigations. The rats were killed by cervical fracture, and the urethra, together with the bladder, was dissected out. Thereafter the urethra was placed in chilled Krebs solution before transferring it to mantled organ baths (37 "C) containing Krebs solution (composition, see below) and continuousllbubbled with 9 6 O , 0, and 4O, CO,, to give a pH of 7.4. B) means of silk threads the whole urethral

The following drugs were used : capsaicin (Sigma, St Louis, MO, USA) ; cyclic calcitonin gene-related peptide (rat alpha,-CGRP, Sigma); S-hydroxytryptamine hydrochloride (Sigma) ; noradrenaline hydrochloride (Sigma); phenoxybenzamine hydrochloride (Sigma); propranolol hydrochloride (Sigma); scopolamine hydrochloride (Sigma); substance P (Sigma) ; suxamethonium chloride (ACO, Sweden) ; tetrodotoxin (Sigma). Stock solutions of capsaicin (lo-' M) were prepared using 109b v/v ethanol and loo,, v/v Tween 80 (Sigma) in saline and further dilutions were made with saline. (In control experiments this solvent was shown not to have any effect per se.) All other drugs were dissolved in saline. Calculations and statistics

The responses to drugs or electrical stimulation are expressed as a percentage of the response to high-K+ solution.

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Fig. 1. Representative sections from (left to right) segments 1, 4 and 6 (see Materials and Methods) of the female rat. Scattered longitudinal bundles (arrow) of smooth muscle are found in the submucosa. The muscularis layer consists of both circular muscle (triangle), predominantly in the proximal part (left) and striated muscle (asterisk), predominantly in the middle part of the urethra (centre). The amount of muscle in the distal part of the urethra (right) is small. Bar indicates 0.1 mm.

+

Data are given as mean values SEM with number of preparations given in brackets. Statistical analyses were peformed using the Student’s t-test. Unpaired comparisons were performed between groups and paired comparisons between treatments. P-values less than 0.05 were considered to be significant.

RESULTS Morphological investigations

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The rat urethra was found to be composed of a mucosa, a submucosa, and a muscularis layer. The submucosa contained a large number of scattered, mostly longitudinally or obliquely oriented, smooth muscle cells (Figs. 1 and 2). I t was not possible to quantify the amount of smooth muscle cells in the submucosa. The muscularis layer was found to be composed of muscle bundles with a circular or near-circular orientation. Near the bladder only smooth muscle cells were observed, mostly outside, but often also between the smooth muscle bundles and the submucosa. The smooth muscle component decreased in proportion in the distal direction and was replaced by circularly oriented striated fibres (Fig. 1, centre). These striated fibres were the dominating muscle component in the midurethra (Fig. 2 ) . The cross-sectional diameter of the striated muscle cells, measured in longi-

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Fig. 2. Quantification of the smooth (0) and striated ( 0 )components of the circular muscle layer of the female rat urethra (mean$-SD, n = 3). Note the predominance of smooth muscle in the proximal urethra and of striated muscle in the mid-urethra.

tudinal urethral sections, was found to be 13.1 k 1.0 p m ( n = 30). Functional investigations Responses to electrical field stimulation. Figure 3 shows the frequency-response relations of 15-2

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contraction, starting 1-2 s after the initiation of the stimulation. Upon cessation of the stimulation the rapid component disappeared immediately whereas the slow component persisted for 7 s to several minutes at a stimulation frequency of 60 Hz. Addition of propranolol ( M), scopolamine (lo-' M) and phenoxybenzamine ( M) depressed the rapid contractile component slightly but not significantly, whereas no effect was observed on the slow one. T h e responses of the longitudinal preparation (n = 10) to electrical stimulation are shown in Fig. 3. I n most preparations (n = 6) stimulation induced a marked initial relaxation. T h i s response had reached a maximum at 2.5 Hz and then amounted to - 32.1 7.1 Q0 of the response to high-K' solution. I n most preparations ( n = -130 J I I I I 1 I 1 I I I 05 1 2 3 5 10 20 LO6080 9) the relaxation was followed by a small Frequency ( H z ) contractile response (Fig. 3). This contraction Fig. 3. Frequenc>--response relations for circular (a) was significantly smaller than the rapid conand longitudinal ( 0 )preparations of the rat urethra in tractile component seen in the circular prepararesponse to electrical field stimulation. The responses tions. I n 60°b (n = 6) of the preparations it are related to the contractile effect of a high-Kappeared during stimulation, but in some (n = solution (124 m v ) . Note the two-phasic response of 3) not until after the stimulation was disthe longitudinal urethra. Solid lines depict the continued, as shown in inset (b) of Fig. 3. One contractile and broken lines the relaxatorl; response. Insets represent original recordings of circular (a) and preparation exhibited no relaxant response at all, longitudinal (b) preparations obtained at a stimulation whereas one showed no contraction at all. I n order to investigate the two components of frequency of60 H z . Note the difference in time scales. the contractile response seen in the circular preparations, stimulation was performed using a circular ( N = 11) and longitudinal ( n = 10) frequency inducing a response approximately -urethral preparations to electrical field stimu- 1 3 " ~of~ the maximal one with a train duration of lation in ritro with 5-s trains of impulses at 5 s and a train interval of 2 min (Fig. 4). T h e 2-min intervals. contraction had a rapid and a slow component as The circular preparations showed a large previously described. Noradrenaline (5 x M) contractile response to stimulation. Already at induced an increase in basal tone but did not 0.5 H z it exceeded the response to high- influence either of the components of a superpotassium solution, and a maximal response vi-as imposed electrical stimulation (Fig. 4). Substance reached at a stimulation f r e q u e n q exceeding P (5 x lo-' M) also increased basal tone without 80 1-12,T h e contractile response consisted of two affecting the response to field stimulation (not components as seen from inset (a) in Fig. 3 shown). When changing to calcium-free solution showing an original recording obtained at 60 Hz. the slow component of the electrically induced At all frequencies a rapid component M-as seen contraction decreased rapidly in magnitude to initially during stimulation and the magnitude of disappear after approximately 10 min. Also the that response is described in the frequent!-- rapid component was gradually curtailed in response curve in Fig. 3. L1t frequencies ex- calcium-free solution. However, this decrease ceeding 1 €32 a slow component appeared. This was slower, the contractions being abolished first contraction was significantly smaller than the after more than 30 min of exposure to Ca"-free rapid contraction and was present in approxi- solution. Neither noradrenaline nor substance P mately 90", of the preparations. Using a high- had any effect on basal tone or on the response speed recording (Fig. 3a) the slow component to electrical stimulation after 1&15 min of was shown to be superimposed on the rapid exposure to CaP+-free solution (Fig. 4).

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Fig. 4. Representative original recording of the effects of noradrenaline (NA, 5 x lo-‘ M), substance P (SP, 5 x lo-‘ M) and calcium-free solution on the contractile responses of circular preparations of the rat urethra elicited by electrical field stimulation (impulse duration 0.5 ms, train duration 5 s). ‘w’ indicates washout. T o be able to characterize further the rapid component in circular preparations, experiments with shorter impulse and train duration were performed. An original recording of the responses elicited in a circular preparation to stimulation with increasing frequencies and with an impulse duration of 0.03 ms, a train duration of 1 s and a train interval of 30 s is depicted in Fig. 5 . As can be seen a single impulse induced a twitch response with a duration of 50-100 ms. Stimulation frequencies exceeding 5-10 H z led to summation, and a smooth tetanus was reached at 120 Hz. This response was approximately four times the twitch response in magnitude. Another series of experiments was performed in order to evaluate the effect of neuromuscular blockade on the responses of the circular urethra to single impulses delivered at 30-s intervals. T h e threshold duration was found to be 0.010.02 ms. When the impulse duration was decreased from 0.5 to 0.03 ms the response was reduced by 63.3_+8.3% ( n = 5 ; Fig. 6). T h i s response was abolished by suxamethonium M). When the impulse duration was increased again to 0.5 ms the contractile response reappeared but was somewhat smaller than in the absence of suxamethonium. In the present of TTX ( M) the contractions were abolished (Fig. 6). Responses to exogenously applied drugs. In one series of experiments the effects of maximal doses of CGRP, capsaicin, substance P and 5HT on circular and longitudinal preparations of the rat urethra were investigated, and the collected data from these experiments are shown in Fig. 7. CGRP ( M) and capsaicin ( M) induced a relaxation in both types of preparations. T h e longitudinal urethra showed a relaxation to CGRP that was significantly larger than

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Fig. 5. Representative original recording of the contractile responses of a circular preparation of the rat urethra to electrical field stimulation with increasing frequencies (impulse duration 0.03 ms, train duration 1 s). Note the twitch response during stimulation with single impulses and summation and tetanus at higher frequencies. The effect of a high-K’ solution (124 KIM) from the same preparation is shown for comparison. ‘w ’ indicates washout. duration (ms) 0.5 0.03 5 mN

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Fig. 6. Representative original recording of the effects of suxamethonium ( M) and tetrodotoxin (TTX, M) on the contractile response of a circular preparation of the rat urethra to electrical field stimulation (single impulses, duration 0.03 or 0.5 ms). the one to capsaicin (86.9 f7.1 and 36.6 6.7 % of the contraction to high-K+ solution respectively, P < 0.001, n = 6). T h e responses of

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Fig. 7 . Collected data on the contractile and relaxator! responses of longitudinal (open bars) and circular preparations (hatched bars) of the rat urethra on exposure to C G R P ( l o - ' M), capsaicin ( 1 0 ~ 'M), substance P (SP, 5 x XI) and 5-HT (lo-" Ji). ,411 responses are related to the contractile effect o f a highIC- solution (1 24 rnxi).

CGRP and capsaicin in the circular urethra were, on the other hand, similar (7.7 2.7 and 1 1 . I *7.9'),) of K- respectively, n = 6). Substance P (5 x 11) and 5-HT (10 ,' XI) contracted both circular and longitudinal preparations. T h e longitudinal urethra showed a larger contraction to 5-HT than to substance P (53..5+ 18.3 and 4.3F0.9°,0 of K' respectively, P < 0.05, n = 6). Contrary to this, in the circular preparations the contraction to 5-HT was somewhat, but not significantly, smaller than to substance P (2.3f0.5 and 4 . 9 1 1 . 2 0 0 of K' respectively, n = 6).

DISCUSSION T h e urethra of the female rat is composed of roughly longitudinally oriented smooth muscle cells in the submucosa, and a circular layer which is composed of smooth muscle in the proximal urethra, but in the mid-urethra striated muscle cells predominate. A similar arrangement has been described in the female human urethra (Hutch & Rambo 1967). These authors described an inner longitudinal smooth muscle layers as a continuation of the bladder musculature. Then a proximal, circular smooth muscle layer and, from mid-urethra distally, circular or obliquely oriented striated muscle fibres. T h e present study shows clear-cut functional differences between these different muscle types found in the female rat urethra. T h e circular preparations responded to electrical field stimulation with a very rapid contractile response followed, at train durations exceeding 2 s, by a slo\dy developing after-contraction. T h e characteristics of the rapid contractile component, a fast (50-100 ms) twitch response, summation and a smooth tetanus at > 100Hz, makes it highlj- likely that this response is due to the contraction of the circularly oriented striated muscle fibres seen morphologically. Further indications are the relative resistance of the twitch response to depletion of extracellular Ca" (Fig. 4) and the resistance to autonomic receptor blockade. T h e experiments on the circular preparation using suxamethonium and T T X (Fig. 6) indicate that the electrical field stimulation employed might stimulate not only the nerves but also the muscle directly and, further, that the threshold for stimulation is different. It is most likely that the slow response to electrical stimulation of the circular preparations is the result of contraction of the circularly oriented smooth muscle. This response was found to be highly sensitive to depletion of extracellular Ca". T h e finding that the contractile responses to noradrenaline and substance P were also rapidly abolished in Ca2+-free solution makes it most likely that these substances act on the smooth but not on the striated muscle fibres. T h e finding of striated circular muscle fibres is in accordance with observations- in humans (Hutch & Rambo 1967, von Hayek 1969, Gosling 1985). T h e fibres were in the present study

Rat urethra found to be unusually small, 13.1 1.0 pm, and similar fibres diameters, 15-20 pm, have been observed in humans (von Hayek 1969). A sparse occurrence of skeletal muscle fibres concomitant with small, fast contractile responses to electrical field stimulation has been reported in the rabbit (Mattiasson et al. 1990). The response to electrical stimulation of longitudinal preparations of the urethra was found to be clearly different from that seen in the circular ones. In the vast majority of these preparations no rapid contraction was evident. This indicated that the striated muscle fibres act almost purely in the circular direction. The response to electrical stimulation of the longitudinally oriented urethra was found to be variable, with some preparations showing a relaxation, some a contraction and others a combined response. The present study does not provide any clear explanation for this variability. However, the intrinsic tone of the preparations may vary and thereby influence the magnitude of a relaxatory response. It has previously been shown that substance P and neurokinin A contract circular preparations from the urethra of the male rat (Maggi et al. 1987a). In the same preparation these authors also showed that capsaicin caused a small contractile response in some preparations and that CGRP relaxed precontracted preparations and depressed the response to electrical stimulation (Maggi et al. 1987a, b, 1989). The present study shows that substance P also induces a contraction of the circular muscle layer of the urethra in the female rat. This effect seems to be confined to the smooth muscle, since the contractile response of substance P was highly susceptible to abolition of Caz+from the medium (Fig. 4). In contrast to the effects shown in the male rat, both CGRP and capsaicin relaxed the basal tone in vitro of the circular preparation of the female urethra. This could be the result of different degrees of intrinsic tone in our preparations and in the preparations obtained by Maggi and co-workers from the male rat. On the other hand, those authors found an inhibition of electrically induced contractions by CGRP and capsaicin (Maggi et al. 1989), whereas no such effect was observed in the present study. CGRP and capsaicin apparently act on the urethral smooth muscle. The smooth muscle component of the response to electrical stimulation was small in our circular preparations. It might

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therefore be difficult to detect any effect of CGRP or capsaicin on this response with the technique used. Both in the circular and in the longitudinal preparations of the female rat urethra, substance P and 5-HT induced contractions whereas capsaicin and CGRP caused a relaxation. However, although it is not possible to make a comparison in absolute terms, there was a clearcut difference between the responses in circular and longitudinal urethra. In the circular urethra capsaicin and CGRP produced a relaxation of similar magnitude, whereas in the longitudinal strips CGRP was clearly more potent than capsaicin. Similarly, in the circular strips substance P and 5-HT induced approximately equally large contractions, while in the longitudinal ones 5-HT was far more potent than substance P. The present study shows a similar muscle arrangement in the female rat urethra as that previously described in humans, and it further points to a functional differentiation between the different types of muscles. The occurrence of both a slow smooth muscle and a faster, special type of striated muscle in the circular layer may indicate that the smooth muscle is tonically active to maintain the normal closed state of the urethra. The fast striated muscle, on the other hand, might be activated especially during rapid events, i.e. in response to sudden changes in abdominal pressure. The longitudinal smooth muscle can possibly be seen as a continuation of the bladder smooth muscle and, if activated simultaneously with the bladder, might create a shortening of the urethra, thereby decreasing urethral resistance during voiding. The technical assistance of Mrs Gull-Britt Ask and Mrs Agneta Bengtsson is gratefully acknowledged. The study was supported by grants from the Swedish Medical Research Council and the Medical Faculty, University of Lund, Sweden. REFERENCES ALM, P., ALUMETS, J., BRODIN, E., HKKANSON, R., NILSSON, G., SJOBERG, N.-0. & SUNDLER, F. 1978. Peptidergic (substance P) nerves in the genitourinary tract. Neuroscience 3, 419425. ALM,P., ALUMETS, J., H~KANSON, R., OWMAN, CH., SJOBERC, N.-O., SUNDLER, F. & WALLES, B. 1980. Origin and distribution of VIP (vasoactive intestinal polypeptide)-nerves in the genito-urinary tract. Cell Tissue Res 205, 337-347.

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~ D E R S S OP.O., N , SJOGREN, C., Lvxis, B. & C v s i s ~ I O B E RK. G , 1990. Urinary bladder and urethral responses to pelvic and hypogastric nerve stimulation and their relation to vasoacti\-e intestinal polypeptide in the anaesthetized dog. ,4cra Ph,ysiol Scand 138, 409-416. CREED,K.E. 1979. The role of the hypogastric nerve in the bladder and urethral activit!- of the dog. Br 3 Pharmacol 65, 367-375. CREF.II,K.E. & TLLLOCH,-1.G.S. 1978. The effect of pelvic n e n e stimulation and some drugs on the urethra and bladder of the dog. BY 3 Crol 50, 398405. DROES,J.TH. P.51. 19’74. Observations on the musculature of the urinarl- bladder and the urethra in the human foetus. B r 3 Crol 46, 179-185. EKSTROM,J. & ~IALMBERG, L.. 1984. On a cholinergic motor innervation of the rat urethra. A r t a Ph.ysrol Scand 120, 237-242. GOSLIW,J..I 1985. T h e structure of the female lower urinar! tract and pelvic floor. 1-rol Clin I’.4m 12, 207-214. H~~ANSO R.,S ,LARSSOZ, L.-I., SJOBERG,N.-0. & SL-NDLER, F. 1974. .4mine-producing endocrinelike cells in the epithelium of urethra and prostate of the guinea-pig. A chemical, fluorescence histochemical, and electron microscopic study. Histor.hemrstrj~38, 259-270. \ O N H.4YF,K, 14. 1969. Die hluskulatur des Beckenbodens. I n : C.E. Alken, V.W. Dix, W.E. Goodwin & E. \Vildbolz (eds.) Handbitch der Li-dogie. vol. 1. Springer, Berlin. HLTCH, J.A. & RAMBO,JR, 0.3. 1967. A new theor!of the anatomy of the internal urinar!- sphincter and the physiologj- of micturition. 111. Anatomy of the urethra. 3 l , r o l 9 7 , 696-704.

C...\., SANTICIOLI, P., ABELL.1, I>.,PARLANI, M., CONTE,B., GIULIANI, S. & MELI, M., C.ASAPPO, -1.1987a. Regional differences in the effects of capsaicin and tachykinins on motor activity and vascular permeability of the lower urinary tract. .~aun,)in-Schmiedrberg’s Arch Pharmacol 335, 636645. X ~ G G I C..\., , GIULIANI, S., SANTICIOLI, P., ABELLI, L. & MELI, A. I987 b. Visceromotor responses to calcitonin gene-related peptide (CGRP) in the rat lower urinary tract: evidence for a transmitter role in capsaicin-sensitive nerves of the ureter. Eur f Pharmacol 143, 73-82. >lAGGl, c..4., SASTICIOLI, P., MANZINI, s.,CONTI, s., GILLIANI, s., PATACCHINI, P. & MELI,A. 1989. Functional studies on the cholinergic and sympathetic innervation of the rat proximal urethra: effects of pelvic ganglionectomj or experimental diabetes. 3 Auton Pharmacol 9, 231-242. \I.ATTI.4SSON, -I., ANDERSSON,K.-E., ANDERSON, C. & UVELIUS, B. 1990. P.-O., LARSSON, B . , SJOGREN, Nerve-mediated functions in the circular and longitudinal muscle layers of the proximal female rabbit urethra. 3 Irrol 143 (in press). SL-ACK, B. & DOWNIE, J.W. 1983. Pharmacological analpis of the responses, of the feline urethra to autonomic nerve stimulation. 3 Auton Nervnus System 8, 141-160. Sv, II.C., WHARTON,J., POLAK, J.M., MULDERRY, P.K., GHATEI,MA., GIBSON, S.J., TERENGHI, G., MORRISON, J.F.B., BALLESTA, J. & BLOOM,S.R. 1986. Calcitonin gene-related peptide immunoreactivity in afferent neurons supplying the urinary tract: combined retrograde tracing and immunohistochemistry. Neuroscience 3, 127-747.

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