produced slight depolarization. Oestrogens are used in the treatment of urinary incontinence in post-menopausal

Transcription

1 J. Phy8iol. (1985), 358, pp With 6 text-figures Printed in Great Britain THE EFFECTS OF OESTROGENS ON SPONTANEOUS ACTIVITY AND RESPONSES TO PHENYLEPHRINE OF THE MAMMALIAN URETHRA BY S. M. CALLAHAN AND K. E. CREED From the School of Veterinary Studies, Murdoch University, Western Australia 6150 (Received 4 July 1984) SUMMARY 1. The electrical and mechanical activity of strips of urethral smooth muscle from female ovariectomized rabbits were studied. Results were compared with strips from guinea-pigs, dogs and Tammar wallabies and with anaesthetized rabbits. 2. From pressure recordings in intact animals and contractile responses of strips it was concluded that in the urethra of the rabbit both cholinergic and a-adrenergic receptors were excitatory. 3. Urethral strips from rabbits and wallabies had continuous spontaneous mechanical activity that was reduced by pre-treatment with oestrogen. In dogs spontaneous activity was transient and in guinea-pigs was usually absent. 4. The a-adrenergic agonist, phenylephrine, produced a dose-related contraction of urethral strips from all species. Pre-treatment with oestrogen produced no significant change in the rabbit and guinea-pig but increased sensitivity in the dog and wallaby. 5. Electrical activity was recorded with micro-electrodes from smooth muscle cells of rabbit and guinea-pig urethra. In both species pre-treatment with oestrogen produced slight depolarization. 6. In rabbits regular spike activity was recorded from all animals but there was a tendency for double spikes and reduced amplitude following oestrogen treatment. In guinea-pigs bursts of spikes occurred in control animals; after hormone treatment there was often incomplete recovery of spikes within the burst. 7. The results indicate that oestrogens can influence the smooth muscle of the urethra by modifying both spontaneous activity and the responses to stimulation of a-adrenoceptors. INTRODUCTION Oestrogens are used in the treatment of urinary incontinence in post-menopausal women and spayed bitches (Salmon, Walter & Geist, 1941; Osborne, Low & Finco, 1972). Their probable action is to increase closure of the urethra by preventing atrophy of the mucous membrane, maintaining turgor of blood vessels in the wall and modifying activity of smooth muscle cells (Salmon et al. 1941; Smith, 1972; Raz, Zeigler & Caine, 1973). In the uterus, which is embryologically related to the urethra, much attention has been paid to the smooth muscle cells and their innervation and oestrogens have been found to affect many processes including basic properties of 2-2

2 36 S. M. CALLAHAN AND K. E. CREED smooth muscle cells, neuronal influences on the muscle and transmitter metabolism especially in adrenergic nerves (Marshall, 1981; Gibson, 1981). Little work has been done on the lower urinary tract but it has been shown, in anaesthetized dogs, that oestrogens produce a marked increase in sensitivity (median effective dose; ED50) of the urethra to the a-adrenergic agonist phenylephrine (Creed, 1983), but in isolated urethra from rabbits the change in ED50 was not significant (Hodgson, Dumas, Bolling & Heesch, 1978). Some properties of smooth muscle cells can be studied with micro-electrodes and this technique has revealed changes in spontaneous and evoked activity induced in the uterus by oestrogens (Bfilbring, Casteels & Kuriyama, 1968; Kuriyama & Suzuki, 1976). Normal activity has been recorded in bladder and urethra of guinea-pig (Creed, 1971 a; Callahan & Creed, 1981) and rabbit (Ursillo, 1961; Creed, Ishikawa & Ito, 1983) but there are no reports of urethral changes induced by hormones. In the present experiments rabbits were used to investigate the effect of oestrogens on mechanical responses to phenylephrine of isolated urethra and to record any electrical changes in membrane activity produced by oestrogens. Since no marked changes could be recorded, the effects of oestrogens on isolated tissues from three other species, guinea-pig, dog and Tammar wallaby, were investigated. In addition, the responses in intact anaesthetized rabbits to nerve stimulation and some drugs were compared with those in dogs to ascertain whether there is a basic difference between the species. METHODS Pressure changes were recorded from female and male rabbits weighing kg. Anaesthesia was induced by intravenous injection of Nembutal (30 mg/kg body weight) and maintained with further Nembutal as necessary. Bladder and urethral pressures were measured through a double-bore polythene tube with the two openings separated by 30 mm. In the females the opening of the urethra from the vagina could not be located from the exterior. The tube was therefore introduced through the urethral meatus by passing it over a wire which had been inserted from the abdomen into the bladder and along the urethra. The hole in the bladder was closed with a purse-string ligature. In the male the tube could be introduced through the penis. The tube was connected via two Statham transducers (P23) to a Grass polygraph and perfused continuously with normal saline (Creed & Tulloch, 1978). The distal ends of the divided hypogastric or pelvic nerves were stimulated supramaximally with trains of pulses at 10 Hz. Drugs were either injected directly into the local blood supply through a cannula tied into the internal iliac artery or into the systemic circulation through a cannula in the jugular vein. Electrical and mechanical activity of isolated strips of lower urinary tract of rabbits, guinea-pigs, dogs and Tammar wallabies was studied. After killing the animal, the lower urinary tract was removed and opened by a mid-dorsal incision. For mechanical recording the urethra was divided into two equal parts by a mid-ventral cut and the mucous membrane removed. The strips were mounted in a bath at C containing modified Krebs solution (Callahan & Creed, 1981). The Krebs solution had the following composition (mm): NaCI, 120; KCl, 5-0; CaCl2, 2-5; NaH2P04, 1 0; glucose, 11 0; equilibrated with 5% CO2 in 02. For low-ca Krebs half the CaCl2 was omitted. The tension was measured with a force transducer and displayed on a Grass polygraph. Drugs were added directly to the bath. For electrical recording, longitudinal strips running from the dome of the bladder to the urethra were dissected out and the mucous membrane and superficial connective tissue was removed. The strips were normally mounted with the serosal side uppermost and bathed in modified Krebs solution made hypertonic by addition of 15 g sucrose to 100 ml Krebs to abolish movement. The electrical properties of the smooth muscle cells were recorded with glass microelectrodes filled with 3 M-KCl and photographed from a cathode ray oscilloscope. The tissue was stimulated by application of current between two silver plates 10 mm apart through which one end of the tissue passed (Abe & Tomita, 1968). Drugs were added to the bath.

3 OESTROGENS AND THE URETHRA The effect of oestrogen was investigated by comparing tissues from ovariectomized animals with those from either entire or ovariectomized animals treated with oestrogen. Rabbits were given four subcutaneous injections of400 jag stilboestrol in peanut oil on alternate days; guinea-pigs were given 50 jag on alternate days; dogs (20-30 kg) received 1000 jag daily for 1 week; wallabies were given daily injections of 100 Bag oestradiol. These doses produced a significant increase in the mean weight ofthe uterus in rabbits (3-5 to 13.8 g) and in guinea-pigs (I 0 to 2-2 g). In dogs and wallabies the uterus was removed at the same time as the ovaries. 37 RESULTS Mechanical responses of bladder and urethra in anaesthetized rabbits to nerve stimulation and drugs It had been hoped to record the effects of oestrogens on the intact urethra of anaesthetized rabbits. However, in female rabbits the urethra is short and it was difficult to separate it anatomically from the bladder base which together form a cone extending about 20 mm from the ureter openings to the vagina. Although care was taken to position the catheter within the urethra, movements of the region sometimes caused the tip to move into the vagina or towards the bladder. Furthermore, as the catheter could not be introduced through the urethral meatus, animals could not be used more than once, so that each animal could not act as its own control. The effects of oestrogen were therefore only studied on isolated tissues but the effects of nerve stimulation and drugs were observed in anaesthetized rabbits with the abdomen open. Results in the female were identical to those in the male in which the urethra is up to 100 mm long. In the absence of stimulation the pressure of the bladder ranged from 5 to 17-5 mmhg (mean+ S.D. = mmhg, n = 12); the urethral pressure ranged from 6 to 17-5 mmhg ( mmhg, n = 12) and there was usually some superimposed spontaneous activity. Hypogastric nerve stimulation always increased the bladder pressure, by up to 6 mmhg, and the increase was maintained as long as stimulation continued (Fig. 1). The urethral pressure response was variable, the most usual response being an increase unless the pressure was already elevated, when stimulation often resulted in a transient relaxation. The contractile response of the bladder was only partially blocked by phentolamine (1 mg/kg body weight) though the urethral response was completely abolished. Pelvic nerve stimulation produced a small increase in pressure in both bladder and urethra. The bladder response levelled off after about 5 s but the smaller urethral response tended to be biphasic with an initial increase followed by a decrease in pressure. The responses were reduced to about half by atropine (2 mg/kg) and were unaffected by phentolamine (1 mg/kg). Close arterial injection of acetylcholine (100 jug), noradrenaline (10 jug) or phenylephrine (10,sg) increased pressure in both bladder and urethra (Fig. 1). The bladder response to acetylcholine ( mmhg, n = 12) was similar to the urethral response ( mmhg, n = 13) whereas the contraction produced by phenylephrine was much larger in the urethra ( mmhg, n = 15) than in the bladder ( mmhg, n = 15). Isoprenaline (10 jug) was without effect unless the pressure was already raised. It then produced relaxation and abolished spontaneous activity. ATP (1 mg) consistently increased the bladder pressure but responses of the urethra were variable even in one animal. Recordings of mechanical responses of strips taken

4 38 S. M. CALLAHAN AND K. E. CREED from rabbits confirmed in vivo responses but ATP consistently produced relaxation of the urethra. The contractile response of bladder strips to phenylephrine was always small. The responses obtained from strips of bladder and urethra from non-oestrous Tammar wallabies were similar to those of the rabbit. B U*. 0-5 min B. ~~~~~* * 0 r20mmhg H. NA PhE Iso 1 min Fig. 1. Pressures recorded in the bladder (B.) and urethra (U.) of two anaesthetized rabbits in response to hypogastric nerve stimulation (H.) at 20 Hz and injection of noradrenaline (NA), phenylephrine (PhE) and isoprenaline (Iso) into the internal iliac artery. The top records are from male and the bottom from female rabbits. From these experiments and from the effects of specific antagonists it was concluded that in both rabbits and wallabies, the bladder has predominantly excitatory cholinergic and inhibitory fl-adrenergic receptors, whereas in the urethra cholinergic and az-adrenergic receptors are both excitatory; in addition noncholinergic, non-adrenergic fibres are present in both hypogastric and pelvic nerves. The results are therefore similar to those recorded in the dog and other species. The effects of oestrogens on the mechanical activity of isolated urethral strips Rabbits. Strips of urethra from control, ovariectomized rabbits showed continuous spontaneous mechanical activity reaching g and occurring at /min. Pre-treatment with stilboestrol reduced the amplitude of the contraction (to less than 0-2 g) but increased the frequency from 1-5 to 6-0 contractions/min. In some preparations all activity was abolished. The contractions of urethral strips from control animals, produced by application of the a-adrenergic agonist, phenylephrine, were compared with those of oestrogen-treated animals. The threshold concentration for contraction, determined from dose-response curves, was about 2 x 10-8 M for both groups and the maximum contraction occurred at 1.0 x 1O-4 M-phenylephrine (Fig. 2). The maximum force of contraction varied widely with values ranging from 3-0 to 8-4 g for control animals (Table 1). Oestrogen pre-treatment produced a slight decrease in the mean maximum force but this was not significant. The mean ED50 to phenylephrine, calculated from individual dose-response curves, was reduced by stilboestrol from 3-2 x 10-6 M (n = 8) for tissues from control animals to 2-2 x 10-6 M (n = 8) (P= 0-1) (Fig. 2). Acetylcholine produced contraction of urethral strips but responses had not

5 OESTROGENS AND THE URETHRA reached a maximum at 2-5 x l0-2 M. Because responses to these high doses may result from stimulation of ganglion cells, the experiments were repeated in the presence of hexamethonium (I1 4 x IO-5 M). However, this produced no reduction and contractions produced by 2'5 x 10-2 M were chosen for comparison. Responses ranged from 3-3 to 39 ~ 0 0 U Fig l-, 10o Phenylephrine (M) Mean dose-response curves to phenylephrine of urethral strips taken from rabbits (left) and wallabies (right). Pre-treatment of animals with oestrogens for 1 week had no effect in the rabbit but there was a significant shift to the left in wallabies. x, ovariectomized controls; 0, oestrogen treated. Bars indicate standard deviations g with mean values of g (n = 10) for treated animals and 10-5 ± 3 0 g (n = 7) for controls (P = ). For each strip this response to acetylcholine was % of the maximum response to phenylephrine with no difference between strips from control ( %) and treated animals ( %). Other species. Oestrogen also modified spontaneous activity recorded from urethral strips of Tammar wallabies. Strips from non-oestrous or ovariectomized wallabies showed regular contractions of 05-1 g at a frequency of about 3/min (Fig. 3). After treatment with stilboestrol, long quiescent periods were seen with bursts of contractions lasting up to 1 min. A similar pattern was seen in late pregnancy. Strips taken from dogs showed some spontaneous activity in the form of bursts of contractions occurring initially every few minutes but these tended to disappear during the course of an experiment, especially in strips from oestrogen-treated animals. Spontaneous activity was rarely seen in urethra from either control or treated guinea-pigs. Phenylephrine produced contraction of urethral strips from all species (Table 1). However, maximum contraction of strips from control wallabies were always less than 300 mg, so that it was not always possible to measure ED50 accurately. Stilboestrol produced a large effect on maximum contractions which always reached over 500 mg (Fig. 3). In this species the ED50 was significantly reduced from 45-5 to 119 x 10-6 M (P = O001). In the dog the mean ED50 was reduced from 4-5 x 10-6 M (n = 10) in controls to 2-6 x 10-6 M (n = 10). This was probably significant (P = ). The

6 40 S. M. CALLAHAN AND K. E. CREED maximum force of contraction was also increased (Table 1). In the guinea-pig, stilboestrol produced a small non-significant increase in the mean ED50 (3*1 to 3-5 x 10-6 M) and in the maximum contraction. The urethral response of the dog to acetylcholine varied from slight relaxation to A 1 min 5 min PhE * S. [i PhE 1 min Fig. 3. Effects of oestrogens on spontaneous mechanical activity (A) and responses to phenylephrine (PhE) (B) of strips of urethra from ovariectomized wallabies. In the control (top) regular spontaneous contractions occurred at about 3/min; after treatment with oestrogen (bottom) long quiescent periods occurred between bursts. The response to 50 psg PhE was markedly enhanced by oestrogen. contraction. At a dose of 5 x 10-3 M the response to acetylcholine was always smaller than the maximum response to phenylephrine with no value greater than 33 %. There was no significant difference between responses in control and treated animals. The effects of oestrogens on electrical activity recorded with intracellular micro-electrodes Rabbits. Spontaneous electrical activity could be recorded with micro-electrodes from all parts of the lower urinary tract of female rabbits and was in the form of regular, single or (occasionally) compound spikes occurring at a frequency of9-30/min (Creed et al. 1983). No clear distinction could be seen in electrical properties of the detrusor muscle, bladder base and urethra although recordings were obtained from cells up to 20 mm caudal and rostral of the ureter openings. In cells with single spikes, there was often an overshoot of up to 20 mv and an after-hyperpolarization of mv lasting up to 1 s (Figs. 4 and 5). In records taken caudal to the ureter openings (bladder base/urethra) a depolarization lasting about 200 ms was superimposed on the hyperpolarization in about two-thirds of cells. The peak of this late depolarization was more negative than the resting membrane potential in about 50 % of these cells; in the remainder it ranged from 0 to 20 mv above the resting potential

7 1-1 4 OESTROGENS AND THE URETHRA r 41 mv I 1. I -1 I I n s O05s Fig. 4. Intracellular records taken with a micro-electrode from a smooth muscle cell of rabbit urethra. The top record shows spontaneous action potentials at two different sweep speeds. The lower record was obtained from the same cell after addition of phenylephrine which increased the frequency and enlarged the late depolarization. TABLE 1. Effect of oestrogen on the response of urethral strips to phenylephrine Control Maximum Rabbit Guinea-pig Wallaby Dog contraction (g) Range Means+s.D ED50 ( X 10-6 M) Range Mean n Oestrogen Maximum contraction (g) Range (05-3 Mean+S.D ED60 ( X 10-6 M) Range A Mean, P 2-2 n.s. 3-5 n.s. 1j.9** 2.6* n For ED60, the means were estimated from log values. The values for control and treated tissues were compared with Student's t test. Probability (P) ** = < 0.01, * = < 0-05, n.s. = > (Fig. 4). In the bladder base/urethra it was only occasionally possible to record the spread of evoked spikes through the tissue though in the detrusor muscle evoked spikes could be recorded more than 10 mm from the point of stimulation. Addition of noradrenaline or phenylephrine to the bath (final concentration 10-6 M) increased the frequency of spontaneous spikes without significantly changing the membrane potential (Fig. 4). The amplitude of the late depolarization was also increased or it

8 42 S. M. CALLAHAN AND K. E. CREED now occurred in cells where it was previously absent. Acetylcholine (10-8 M) also increased the frequency of spikes and this was accompanied by a slight depolarization. Following treatment with oestrogen, regular spontaneous spikes could still be A_ 2s 02s Fig. 5. The effects of low-ca Krebs (A) and oestrogens (B) on the membrane potential recorded from smooth muscle cells of rabbit urethra. The two records in A were taken from the same cell. The reduction in extracellular Ca2+ concentration to half (bottom) produced depolarization, increased frequency and increased spike duration compared with normal Krebs (top). In another rabbit (B) treated with oestrogen for 1 week, there was also an increase in spike duration and the membrane was depolarized compared with control animals. 50 recorded from all regions, though in a few preparations no activity was seen. The mean resting membrane potential of cells of the bladder base/urethra was slightly reduced ( mv, n = 63 in ovariectomized animals compared with mv, n = 16 after treatment with stilboestrol) and this was probably significant (P = ). There was a tendency for spikes to be reduced in amplitude, many spikes having no overshoot, and more compound action potentials were seen. There

9 OESTROGENS AND THE URETHRA was also a marked increase in spike duration (Fig. 5B). There was no obvious difference in the frequency or in the late potential changes between strips from control and stilboestrol-treated animals but the wide range in values made it difficult to analyse quantitatively any effect produced by oestrogens. Guinea-pig8. In young female guinea-pigs, the normal electrical activity of the detrusor muscle was regular spiking at 6-30/min. When impalements were made [ s Fig. 6. Bursts of spontaneous action potentials recorded from smooth muscle cells of the guinea-pig urethra with micro-electrodes. The top record, taken from an ovariectomized control, has discrete spikes, whereas there was incomplete recovery of individual spikes within each burst in an animal with elevated hormone levels (bottom). between the ureter openings, cells were frequently found which had bursts of spikes superimposed on the regular pattern. In the urethra bursts alone were the usual pattern of activity and they contained fifteen to thirty spikes (Fig. 6) and were separated by up to 7 min (Callahan & Creed, 1981). Additional bursts were initiated by phenylephrine. Following treatment with oestrogen the predominant activity of the detrusor muscle was still regular spikes. In the trigone and urethra there was a tendency for the duration of bursts to increase together with the frequency of spikes within each burst. In these animals, and also in pregnant animals when both oestrogen and progesterone levels are elevated, spikes within the burst sometimes showed incomplete recovery so that a plateau at about 0 mv occurred (Fig. 6). Such bursts were rarely seen in control animals. The mean resting potential in the quiescent period was mv (n = 17) compared with mv (n = 19) in control females (P = ). Wallabie8 and dog8. Regular spike activity was seen in the cells of dog and wallaby bladder. However, it was not possible to obtain satisfactory impalements of urethral cells, so that changes produced by oestrogen were not investigated. The effects of reducing Ca2+ concentration on electrical activity As it has been proposed that oestrogens reduce the availability of Ca2+ to smooth muscle cells, the effects on the membrane potential of reducing [Ca2+] in the extracellular fluid were observed. Once a satisfactory impalement was obtained, the

10 44 S. M. CALLAHAN AND K. E. CREED bathing fluid was changed and the membrane potential recorded in the test solution and during subsequent recovery in normal Krebs. For smooth muscle cells of rabbit urethra, the membrane was depolarized by up to 5 mv and the spike frequency increased after 20 min in Krebs solution with Ca reduced to half (Fig. 5A). The shapes of the spikes were altered so that the duration of individual spikes was increased and double spikes were frequently seen. These effects were reversible, with complete recovery occurring within 10 min of restoring normal Krebs solution. In the guinea-pig urethra, reduction of [Ca2+] to half also produced slight depolarization of the membrane and bursts often led to a sustained depolarization, at about zero membrane potential, lasting several seconds. Similar prolonged depolarizations were recorded in guinea-pig bladder when [Ca2+] was reduced or Mn2+ added to the bathing fluid (Creed, 1971b). DISCUSSION The urethra is a tissue which has been largely neglected, and much work must be carried out to understand its mechanical and electrical characteristics and its innervation. Pharmacological experiments on the whole animal or on strips suggest that in the rabbit and other species there are excitatory a-adrenergic and cholinergic receptors (Taira, 1972; Creed & Tulloch, 1978; Slack & Downie, 1983). In addition, there is evidence in both bladder and urethra for non-cholinergic, non-adrenergic innervation, the nature of which has not been identified. Although no major variations in innervation have been reported, it has become apparent that species differences occur in the amount of spontaneous activity and the shapes and patterns of action potentials recorded from smooth muscle cells (Callahan & Creed, 1981 ; Creed et al. 1983). The present results indicate that oestrogens can influence the smooth muscle by modifying this spontaneous activity and, in some species, by altering the responses to stimulation of a-adrenoceptors. Spontaneous mechanical activity was seen in urethral strips from untreated rabbits, dogs and wallabies but rarely from guinea-pigs. Oestrogen reduced this activity, the effect being most marked in the wallaby in which quiescent periods were seen instead of the continuous activity of control animals. Electrical recording from the guinea-pig urethra indicated that spontaneous activity also occurred in this species and it was suggested that limited spread of activity probably prevents generalized, synchronous contraction (Callahan & Creed, 1981). The reduction in mechanical activity produced by oestrogen in urethral strips from other species observed in the present experiments may indicate uncoupling of smooth muscle cells rather than decreased activity of individual smooth muscle cells. This could produce the decrease in amplitude and increase in frequency seen in rabbit urethral strips. However, even in control rabbits, conduction of evoked action potentials was only occasionally recorded. It may be necessary to record from other layers or cut the strips with a different orientation to observe the spread of activity within this tissue. In both guinea-pig and rabbit urethra the mean resting membrane potential of cells was slightly reduced following oestrogen treatment. However, it has been reported for the myometrium of rabbits, guinea-pigs and rats that oestrogen produces a large increase in resting membrane potential and furthermore in the rat the length constant

11 OESTROGENS AND THE URETHRA was increased suggesting that cells may be more tightly coupled (Goto & Csapo, 1959; Bilbring et al. 1968; Kuriyama & Suzuki, 1976). Oestrogen also produces a change in the pattern of action potentials in the urethra with a tendency for compound spikes in the rabbit and incomplete recovery during bursts in the guinea-pig. In rat myometrium, following oestrogen treatment, bursts of spikes were also superimposed on a maintained depolarization (Kuriyama & Suzuki, 1976). The precise mechanism of action of oestrogens on the myometrium has not been elucidated but it has been suggested that they produce their effects by reducing intracellular Ca2+ available to the smooth muscle cells by inhibiting Ca2+ influx and transport by cell organelles (Batra & Bengtsson, 1978). This would reduce excitation of contractile proteins and modify membrane permeabilities. Preliminary experiments have therefore been carried out on the urethra on changing Ca2+ concentration. Reduced Ca2+ and oestrogen pre-treatment both prolonged the action potential recorded from the rabbit urethra and some depolarization occurred; in the guinea-pig urethra bursts of spikes on a prolonged depolarization were seen with both treatments. This may therefore suggest that oestrogen has an effect through Ca2+. However, for confirmation, further experiments must be carried out. For instance does increased Ca2+ reverse the effects of oestrogen? In the present experiments it was confirmed that phenylephrine produces an acceleration of spike frequency in the rabbit (Creed et al. 1983) and initiation of bursts in the guinea-pig (Callahan & Creed, 1981). It was also shown to produce a dose-dependent contraction of urethral strips from all four species studied. The effect of oestrogen on the dose-response curve ranged from a significant shift to the left in the wallaby, where about one-quarter of the original dose produced the same response, to negligible in the guinea-pig. In the dog the mean ED50 was approximately one-half the control value. This compares with a reduction of one-third to one-sixth for the pressure responses of the urethra in anaesthetized dogs, a more sensitive test since each animal acted as its own control (Creed, 1983). It was not possible to carry out similar experiments on anaesthetized rabbits because of the extreme shortness of the urethra in this species. However, the present in vivo results suggest that oestrogens have little or no effect on the responses to phenylephrine in this species. Hodgson et al. (1978) also saw only a small, non-significant decrease in ED50 for both longitudinal and annular strips of urethra from oestrodiol-treated compared with castrated rabbits. In spite of this, high-affinity oestrogen binding sites have been recorded in both the urethra and bladder of rabbits (Batra & losif, 1983) and oestrogens have been reported to produce a marked increase in the density of adrenergic receptors in the body though not the base of the rabbit bladder (Levin, Shofer & Wein, 1980). It was not possible to estimate the ED50 to acetylcholine of rabbit urethral strips because a maximum could not be obtained at high doses. In general, however, contractions were larger and more variable than those to phenylephrine. In the dog responses were also variable but were much smaller than the phenylephrine responses. The large range of responses may have obscured any differences between control and treated animals of each species. With these methods it is therefore not possible to determine whether oestrogens modify general contractile properties of the urethral strips compared with specific responses to phenylephrine. 45

12 46 S. M. CALLAHAN AND K. E. CREED This work was supported by the Australian Kidney Foundation. REFERENCES ABE, Y. & TOMITA, T. (1968). Cable properties of smooth muscle. Journal of Phy8iology 196, BATRA, S. & BENGTSSON, B. (1978). Effects of diethylstilboestrol and ovarian steroids on the contractile responses and calcium movements in rat uterine smooth muscle. Journal ofphysiology 276, BATRA, S. C. & IOSIF, C. S. (1983). Female urethra: A target for estrogen action. Journal of Urology 129, BtULBRRNG, E., CASTEELS, R. & KURIYAMA, H. (1968). Membrane potential and ion content in cat and guinea pig myometrium and the responses to adrenaline and noradrenaline. British Journal of Pharmacology 34, CALLAHAN, S. M. & CREED, K. E. (1981). Electrical and mechanical activity of the isolated lower urinary tract of the guinea pig. British Journal of Pharmacology 74, CREED, K. E. (1971a). Membrane properties of the smooth muscle membrane of the guinea pig urinary bladder. Pflugers Archiv 326, CREED, K. E. (1971 b). Effects of ions and drugs on the smooth muscle cell membrane of the guinea pig urinary bladder. Pflugers Archiv 326, CREED, K. E. (1983). Effect of hormones on urethral sensitivity to phenylephrine in normal and incontinent dogs. Research in Veterinary Science 34, CREED, K. E., ISHIKAWA, S. & ITO, Y. (1983). Electrical and mechanical activity recorded from rabbit urinary bladder in response to nerve stimulation. Journal of Physiology 338, CREED, K. E. & TuLLoCH, A. G. S. (1978). The effect of pelvic nerve stimulation and some drugs on the urethra and bladder of the dog. British Journal of Urology 50, GIBSON, A. (1981). The influence of endocrine hormones on the autonomic nervous system. Journal of Autonomic Pharmacology 1, GOTO, M. & CSAPO, A. (1959). The effect of ovarian steroids on the membrane potential of uterine muscle. Journal of General Physiology 43, HOWXSON, B. J., DumAs, S., BOLIANG, D. R. & HEESCH, C. M. (1978). Effect of estrogen on sensitivity of rabbit bladder and urethra to phenylephrine. Investigative Urology 16, KURIYAMA, H. & SuzuKi, H. (1976). Changes in electrical properties of rat myometrium during gestation and following hormonal treatments. Journal of Physiology 260, LEvIN, R. M., SHOFER, F. S. & WEIN, A. J. (1980). Estrogen-induced alterations in the autonomic respones of the rabbit urinary bladder. Journal of Pharmacology and Experimental Therapeutics 215, MARSHALL, J. (1981). Effects of ovarian steroids and pregnancy on adrenergic nerves of uterus and oviduct. American Journal of Physiology 240, OSBORNE, C. A., Low, D. G. & FINCO, D. R. (1972). Canine and Feline Urology, p Philadelphia: W. B. Saunders. RAZ, S., ZEIGLER, M. & CAINE, M. (1973). The role of female hormones in stress incontinence. XVI Congress of the socigtg Internationale d'urologie, pp Amsterdam. SALMON, U. J., WALTER, R. I. & GEIST, S. H. (1941). The use of estrogens in the treatment of dysuria and incontinence of postmenopausal women. American Journal of Obstetrics and Gynecology 42, SLACK, B. E. & DOwNIE, J. W. (1983). Pharmacological analysis of the responses of the feline urethra to autonomic nerve stimulation. Journal of the Autonomic Nervowus System 8, SMITH, P. (1972). Age changes in the female urethra. British Journal of Urology 4, TAMA, N. (1972). The autonomic pharmacology of the bladder. Annual Review of Pharmacology 12, UnsILLo, R. C. (1961). Electrical activity of the isolated nerve-urinary bladder strip preparation of the rabbit. American Journal of Physiology 201,