Dr N Moriyama, Department of Urology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Email: JCF00714@nifty.ne.jp
Background: α1-Adrenoceptors are highly concentrated in the urethral smooth muscles and may play an important role in the contraction of this area. However, detailed examinations of age-related changes of the properties of urethral smooth muscle have rarely been undertaken.
Methods: The contractile properties of urethras from young non-parous and old parous female beagles were determined with a urethral function study, macroscopic autoradiography for urethras using [3H]-labeled tamsulosin and morphometry of the urethral muscles.
Results: The antagonistic effect (pA2) of prazosin for norepinephrine was 7.76 ± 0.13 in young dogs and 7.62 ± 0.06 in aged dogs. The specific binding of [3H]-tamsulosin (a relatively selective α1A-adrenoceptor antagonist) was recognized diffusely in proximal urethras with in vitro autoradiography. The density of binding in smooth muscles was approximately 60 and 40% in circular longitudinal layers, respectively, for both dogs.
Conclusions: The female canine urethra had α1A- and α1L-adrenoceptors. No age-related changes were seen in the function of the proximal urethra, distribution of α1-adrenoceptor binding sites and smooth muscle densities.
Stress urinary incontinence is a common disease in elderly women and severely decreases their quality of life. Women with this condition can suffer from a lack of energy, emotional disturbance, sleep disturbance, social isolation and mobility problems. 1,2
Stress incontinence may be caused by an interaction of various complex factors. In this condition, the urethrovesical angle increases due to physical strain and this may inhibit urethral contraction. In contrast, it is well known that α1-adrenoceptors are highly concentrated in the urethra 3 and may play an important role in the contraction of the urethra and bladder neck. Thus, in the pharmacological treatment of stress urinary incontinence, α1-adrenoceptor agonists, which increase urethral pressure, are expected to be useful drugs. In a previous study on the urodynamics of the urethra of young non-parous dogs and aged parous dogs, we concluded that one of the etiologies of detrusor instability in the older dogs may be an age-related increase in responsiveness of α1-adrenoceptors in the bladder and proximal urethra. 4 We also demonstrated, using [3H]-JTH 601 (a novel α1-adrenocetor antagonist), that the α1L-adrenoceptor is functionally important in urethral smooth muscle contraction in both young non-parous and aged parous dogs. 5 Furthermore, we have demonstrated that, in the human female urethra, phenylephrine-induced contractions are mediated through α1L-adrenoceptors. 6 However, detailed examinations of age-related changes of the properties of urethral smooth muscle have rarely been undertaken.
In the present study, we used urethras isolated from both young non-parous and aged parous beagles. To study urethral function, the pharmacological response of the urethra in vitro was analyzed by concentration–response curves. Macroscopic auto-radiography for urethras using [3H]-labeled tamsulosin (a relatively selective α1A-adrenoceptor antagonist) and morphometry of the urethral muscles were also performed to examine age-related changes in young non-parous and aged parous beagles.
Whole urethras were obtained from four young non-parous female dogs (mean age ± SEM 16.5 ± 5.0 months; mean weight ± SEM 11.6 ± 1.9 kg) and six aged parous female dogs (mean age ± SEM 95.0 ± 6.7 months; mean weight ± SEM 11.3 ± 0.5 kg). The use of canine urethral tissues for experiments was approved by the local ethics committee. The urethras were prepared in ice-cold oxygenated Krebs–Henseleit solution (KHS) for measuring tension and were suspended in an organ bath filled with KHS. After equilibration, cumulative concentration–response curves for phenylephrine and norepinephrine (NE) were obtained.
Functional study of canine urethral muscle
Transverse muscle strips, 10~20 × 2~3 × 2~3 mm, of the proximal urethra were prepared in ice-cold KHS (composition (in mmol/L): NaCl 118; KCl 4.7; CaCl2 2.54; MgSO4 1.17; KH2PO4 1.19; NaHCO3 25.0; glucose 11.0). The KHS also contained 2 μmol/L propranolol to block β-adrenoceptors, 0.1 μmol/L yohimbine to block α2-adrenoceptors and 0.1 μmol/L desipramine and 10 μmol/L corticosterone to inhibit intra- and extraneuronal uptake, respectively. Yohimbine was not added to the organ baths during the construction of the concentration–response curves to clonidine. Muscle strips were suspended vertically under 1.0 g resting tension in an organ bath filled with 10 mL KHS at 37°C. Tensions were measured by a force-displacement transducer (TB-611T; Nihon Kohden, Tokyo, Japan) and recorded with a pen recorder (Recti-Horiz, 8K-23; NEC-Sannei, Tokyo, Japan). Each bath was continuously bubbled with a gas mixture consisting of 95% O2 and 5% CO2. The KHS was replaced every 20 min during the equilibration period.
After 1 h equilibration, cumulative concentrations of NE, as an α1-adrenoceptor agonist, were added to produce a concentration–response curve twice. The second concentration–response curve was regarded as the control and, next, concentration–response curves to several agonists were constructed to compare with the control curves. Various concentrations of prazosin, as a specific α1-adrenoceptor antagonist, were added 1 h prior to the construction of a third concentration– response curve to NE.
Responses in developed tension to various concentrations of NE are expressed as a percentage of the maximal contraction. The EC50 value of each agent was defined as the concentration that produced 50% of the maximal contraction. Individual EC50 values were determined. The dissociation constant (KB) of each antagonist was determined from the following equation:
where CR is the concentration ratio between EC50 values of the control curves and those of curves constructed in the presence of an antagonist. The pA2 values for young non-parous and aged parous dogs, which are expressed as a negative logarithm of KB, were estimated from Schild plots made by plotting log(CR – 1) against the log molar concentration of the antagonist. 7 In non-parous dog urethras, because time-dependent decreases in EC50 values of concentration– response curves to NE were observed (see Results), the difference of mean EC50 values between the second and third concentration–response curves was subtracted from each value of log(CR – l) in the case of non-parous dogs.
Urethras were isolated and embedded in OCT compound, quickly frozen in liquid nitrogen and stored at −80°C before the experiment. Specimens were sliced into 10 μm sections in a cryostat at −20°C and mounted on polylysine-coated glass slides. After drying at 25°C, all specimens were pre-incubated with 1 mmol/L phosphate-buffered saline (PBS; pH 7.4) at 25°C for 30 min. To assess specimens for the amount of total binding, specimens were pre-incubated with PBS at 25°C for 30 min and incubated with 5 nmol/L [3H]-tamsulosin (an α1A-adrenoceptor antagonist) at 25°C for 60 min. To assess specimens for the amount of non-specific binding, specimens were pre-incubated with PBS and 100 μmol/L phentolamine (pH 7.4) at 25°C for 30 min and incubated with 5mmol/L radioligands, including 100μmol/L phentolamine, at 25°C for 60 min. Specimens were then washed with ice-cold PBS and distilled water. The dried sections were exposed on a [3H]-sensitive imaging plate with a [3H] microscale (Amersham Pharmacia Biotech, Uppsala, Sweden) for 1 week at room temperature. The autoradiograms were processed using a computerized image analysis system (Bio-imaging Analyzer BAS3000; Fuji Photo Film, Tokyo, Japan). The amount of total and non-specific binding was registered and the images were color coded according to the [3H] microscale density. The amount of specific binding was determined by subtracting the non-specific binding from total binding. The images for specific binding were also color coded in the same manner.
A morphometric study was performed in a separate series of experiments. Proximal urethral strips were first fixed in 10% of buffered formalin (pH 7.4) and then embedded in paraffin. Specimens were sliced into 5 μm sections and were then stained with hematoxylin– eosin and the Mallory–Azan method. Quantitative image analysis was performed with a SP-500 system (Olympus, Tokyo, Japan). Sections were viewed under the microscope and the image was captured using a change-coupled device vision camera module onto a computer. The images were then digitized by the image analysis instrument. The area densities corresponding to muscle were calculated per full screen of the monitor for two directions (longitudinal and circumferential) at random in 10 areas for 10 cases.
Prazosin HCl, (–)-NE bitartrate, desipramine HCl and corticosterone were purchased from Sigma Chemical Co. (St Louis, MO, USA). Clonidine HCl was purchased from Tokyo Chemical Industry (Tokyo, Japan). Propranolol HCl was purchased from Wako Pure Chemical Industries (Osaka, Japan) and yohimbine HCl was purchased from Funakoshi (Tokyo, Japan).
All other chemicals were purchased from New England Nuclear (Boston, MA, USA). Prazosin HCl was dissolved in dimethyl sulfoxide and diluted with physiological saline (0.9% NaCl solution) to the appropriate concentrations. Other antagonists were dissolved with physiological saline to the appropriate concentrations.
The EC50 value and maximal contraction produced by each agent were determined for each urethral sample. Morphometric data are expressed as percentages. All results are expressed as the mean±SEM. Significant differences for the data of this study were tested by unpaired t-test, with the level of significance set at P < 0.05.
Results of studies on urethral function
Norepinephrine produced contraction of the muscle sample and the degree of contraction was dependent on the concentration of NE in both young non-parous and old parous dogs. Figure 1 shows the second and third concentration–response curves as control and time control, respectively, for NE in both young non-parous and aged parous dogs. A slight time-dependent depression of NE-induced contractions was seen only in non-parous dogs ( Fig. 1a). No time-dependent depression was observed in aged parous dogs ( Fig. 1b).
Phenylephrine, an α1-adrenoceptor agonist, also produced muscle contraction and the degree of contraction increased as the concentration of the agonist increased in both groups of dogs. However, phenylephrine was slightly less potent than NE in both groups ( Fig. 2). Figure 3 shows a comparison of the muscle contractions produced by various concentrations of NE and clonidine (an α2-adrenoceptor agonist). Although clonidine induced contraction of the urethral muscle from both non-parous and aged parous dogs, the responses to clonidine were much smaller than those to NE.
When both NE and prazosin, an α1-adrenoceptor antagonist, were both added to the organ bath, the degree of NE-induced contraction was decreased in both non-parous and aged parous dogs. The decrease in muscle contraction was greater at higher concentrations of prazosin ( Fig. 4). Prazosin is a potent and competitive antagonist for α1-adrenoceptors. To calculate the log(CR – 1) values for prazosin, time-dependent depression was subtracted in the urethra of non-parous dogs with a value of 0.30. The pA2 values of non-parous and aged parous dogs did not differ significantly, with the pA2 values being 7.76 ± 0.13 and 7.62 ± 0.06 for non-parous and aged parous dogs, respectively. The slopes of Schild plots for non-parous and aged parous dogs did not differ significantly from unity and were 1.11 and 1.08, respectively.
Quantitative autoradiography in the urethra of young and aged dogs revealed that non-specific binding of [3H]-tamsulosin was extremely rare. Specific binding sites, which are determined by the subtraction of non-specific binding from total binding, were demonstrated diffusely in the urethral wall corresponding to urethral smooth muscles. Quantitated mean (±SEM) specific binding of [3H]-tamsulosin was 40.7 ± 3.7 and 40.7 ± 11.1 Bq/mg in young non-parous and aged parous dogs, respectively (n = 3). There were no differences in specific binding sites and the amount between the two groups ( Fig. 5).
The percentage area densities of inner longitudinal smooth muscle were 38.9 ± 17.6 and 33.2 ± 14.3% in young non-parous and aged parous dogs, respectively. The percentage area densities of outer circular smooth muscles were 59.8 ± 13.7 and 57.8 ± 18.2% in young non-parous and aged parous dogs, respectively. There were no differences in the above two parameters in specimens from young and old dogs ( Fig. 6; Table 1).
Table 1. Morphometric measurements of the two types of smooth muscle layers in the female dog urethra
Data are the mean ± SD.
Percentage area density of muscle = (density of muscle area/density of all tissue area) × 100.
Young, non-parous dogs
59.8 ± 13.7
38.9 ± 17.6
Aged parous dogs
57.8 ± 18.2
33.2 ± 14.3
Analysis of the canine urethra indicates that the proximal urethra is mainly smooth muscle. 8α-Adrenoceptors distributed throughout the urethral smooth muscle increase urethral closing pressure. 3 Contraction of the human urethra has been demonstrated in response to an α-adrenoceptor agonist and relaxation of the muscle has been shown to occur in response to an α-adrenoceptor antagonist. 9 There is a correlation between the urethral pressure profile (UPP) and the anatomical structures in and around the urethra of the dog. 10 In the present studies using in vitro urethral strips, the urethral tension and the response of the urethra to NE and phenylephrine of both young and aged dogs support the previous findings.
It has been reported that the α2-adrenoceptor density in the female rabbit urethra is significantly greater than that in the male rabbit urethra. 11 In contrast, no significant difference was observed in the density of and affinity for the urethral α1-adrenoceptor between male and female rabbits. 12 Agonists of α1- and α2-adrenoceptors cause a similar degree of urethral contraction. 13–15 The contractions in these previous reports may be mediated by post-junctional α2-adrenoceptors. Estrogen treatment has been shown to increase the density of α2-adrenoceptors in various smooth muscle preparations, such as the uterus, 16 bladder 17 and urethra. 11 From the present study, there was no apparent difference in the function of adrenoceptors between non-parous and aged parous dogs. Norepinephrine, phenylephrine and clonidine produced concentration-dependent contractions in both non-parous and aged parous dogs. In Fig. 2, NE appeared to be more potent and had a larger contractile effect than phenylephrine. The EC50 values for both NE and phenylephrine were not significantly different between the non-parous and aged parous groups. In contrast, the maximal contractions induced by clonidine were less than 20% of the response induced by NE in both non-parous and aged parous dog urethras. This is in agreement with the report of Hashimoto et al.18 Although we have not determined the density or number of α2-adrenoceptors on either non-parous or aged parous dog urethras, our study using clonidine showed that an α2-adrenoceptor agonist caused a much smaller contractile response of urethral smooth muscle than an α1-adrenoceptor agonist. The urethral smooth muscle may have fewer post-junctional α2-adrenoceptors than post-junctional α1-adrenoceptors. Therefore, the effect of an agonist on urethral smooth muscle α2-adrenoceptors may differ between species. Similar differences between canine and human urinary systems have to be taken into consideration.
Previous studies on the human female urethra have reported that changes in the urethra due to aging are the result of atrophy. 19 However, reports have suggested that, as a person gets older, there is a decrease in the relative volume of striated muscle and an increase in the relative volume of connective tissue and no change in the smooth muscle components of the urethra. 20,21 Our study showed the ratio of urethral smooth muscles (circumferential and longitudinal) are almost the same.
In the rat prostate, the density of α1-adrenoceptors was significantly lower in older than younger rats. 22 Takahashi et al.4 have reported that midodrine (an α1-adrenoceptor agonist) significantly increased the UPP of the proximal urethra of aged parous dogs to a greater degree than that of young dogs. In previous 5 and present studies, the contractile responsiveness via α1-adrenoceptors on the urethra of young non-parous and aged parous dogs did not show any significant changes. We think this discrepancy occurred because of the differences caused by experiments being performed in vivo and in vitro. We measured the tension specifically in the proximal circumferential smooth muscle. In contrast, in vivo measurement of UPP is influenced by many factors. One factor is the structure of the urethra, which consists of two layers of smooth muscle, three layers of striated muscle, connective tissue and blood vessels, 23 all of which have the ability to respond to an adrenergic agonist. Another factor is the effect of the nervous system on the urethra. α-Adrenoceptor antagonists can influence the central nervous system to effect pudendal nerve-dependent urethral responses. 24 Other differences between in vivo and in vitro experiments on urethral smooth muscle that may affect the results obtained are the presence of anesthesia, dose differences of drugs in the urethra and the kinds of drugs used.
Our present study showed that specific binding sites for [3H]-tamsulosin (relatively selective α1A-adrenoceptor antagonist) were distributed diffusely throughout all smooth muscle layers of the urethra and that there was no significant difference between young non-parous and old parous dogs. In the present study, we used only [3H]-tamsulosin because of the following reasons. This ligand is commercially available, shows little non-specific binding and although it does not strictly selectively bind α1A-adrenoceptors, it mainly binds them. The findings of the present study showed good agreement with findings obtained by autoradiograms in a previous study of dog urethras using [3H]-JTH 601, 5 rat urethral muscles demonstrating diffuse [14C]-midodrine binding (α1-adrenoceptor agonist) 25 and in situ hybridization of human proximal urethra demonstrating diffuse α1a-adrenoceptor subtype mRNA in muscle layers. 26
Native and cloned α1-adrenoceptor subtypes (α1A, α1B and α1D) have been fully characterized based on pharmacological, structural and transductional criteria. 27α1-Adrenoceptors have also been classified into α1H, α1L and α1N subtypes. 28,29 Prazosin has a higher affinity for α1H- than for α1L- and α1N-adrenoceptors. It may be considered that the α1A-, α1B- and α1D-adrenoceptor subtypes are included in the α1H group of the latter classification because of their higher affinity for prazosin (pKi or pA2> 9). The pA2 values for prazosin in non-parous and aged parous dog urethras, which were obtained from the present study, are those typically seen for α1L-adrenoceptor subtypes. In addition, the Schild plots for prazosin revealed a straight line for both the non-parous and aged parous groups and the slopes were not different from unity in both cases, suggesting that α1-adrenoceptors were a single population. There have been some previous reports that contractile responses to NE are maintained by the α1L-adrenoceptor subtype located in the human prostate and prostatic urethra. 30,31 Thus, the smooth muscle contraction of urethra is also considered to be maintained by α1L-adrenoceptor subtypes in female dogs.
Muramatsu et al.30 have demonstrated that the contractile response to NE of human prostatic tissue is mediated through the α1L-adrenoceptor subtype because of its lower affinity for prazosin (the pA2 value was reported to be 8.3). The estimated pA2 for prazosin, which is a measure of the degree of the antagonistic effect of prazosin on NE-induced contraction, obtained from the previous 5 and present studies also supports the theory on the potency for the α1L-adrenoceptor. The pA2 values of prazosin obtained in the present study, which were 7.76 and 7.62 in the young non-parous and aged parous groups, respectively, shows that the female canine urethra contracts through an interaction of NE with the α1L-adrenoceptor in both young and aged dogs. Ford et al.32 have reported that upon functional pharmacological analysis, the cloned α1A-adrenoceptor displays pharmacological recognition properties consistent with those of the α1L-adrenoceptor. In addition, the α1L-adrenoceptor has not been cloned. The presumption that the α1L-adrenoceptor is a reconformation of the α1A-adrenoceptor is very attractive, but the nature of this receptor requires further investigation.
In conclusion, our investigation has shown that: (i) the female canine urethra contracts mainly through the action of agonists and antagonists on the α1A- and α1L-adrenoceptors; and (ii) no significant age-related change in the function of the proximal urethra, distribution of α1L-adrenoceptor binding sites and smooth muscle properties was seen.
This investigation was supported, in part, by the Human Science Foundation and a Grant-in-Aid for Scientific Research (C), no. 10671459, from the Ministry of Education, Science and Culture, Japan.