SEARCH

SEARCH BY CITATION

Keywords:

  • bladder neck;
  • stimulation;
  • physiology

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

OBJECTIVE

To evaluate differing methods of stimulation on strips of human bladder neck smooth muscle and compare muscle taken from the anterior and posterior aspects.

MATERIALS AND METHODS

Samples of adult human male bladder neck muscle were obtained from patients undergoing open radical prostatectomy. Muscle was taken from either the anterior or posterior (nine and six patients, respectively) aspects of the bladder neck. Muscle strips dissected from these samples were suspended in the Brading-Sibley organ bath. The strips were superfused with 100 mm KCl-enriched Krebs’ solution for 4 min to determine viability. This allowed experimentation on 17 strips from the anterior aspect of the bladder neck and 13 from the posterior bladder neck. These remaining strips were then superfused either with various concentrations (×10−7 to ×10−3m) of carbachol or noradrenaline in Krebs’ solution, for 15 s. A further set of strips (eight from anterior, six from posterior) was suspended and responses to electrical field stimulation (EFS) with varying parameters were measured. Each EFS experiment was repeated after a 15 min exposure to 10−3m atropine, and again after a 15 min exposure 10−7m tetrodotoxin (TTX). Tension responses produced in these series of experiments were measured using strain gauges and analysed using data acquisition software. Student’s t-test was used for the statistical analysis.

RESULTS

All muscle strips included in the study responded to EFS. The magnitude of this contraction is frequency dependent. The contractions were abolished by superfusion of the muscle strips with atropine. There was no further suppression of the contractile response on addition of TTX. Posterior bladder neck samples had a greater mean contractile response per unit mass than anterior strips at all frequencies of >1 Hz, and significantly more at 20 and 30 Hz. There was a concentration-dependent response in bladder neck contraction to carbachol but only in the strips from the anterior bladder neck at concentrations of <10−3m. Posterior bladder neck strips did not significantly contract upon application of carbachol. Similarly, there was a concentration-dependent response to noradrenaline. Responses to noradrenaline were not uniform around the bladder neck, but not significantly different. Carbachol was the more ‘potent’ stimulator in anterior smooth muscle strips, but again the differences between agonists were not statistically significant.

CONCLUSION

These experiments show physiological variability around the circumference of the human male bladder neck. The posterior bladder neck shows significantly stronger contraction to α-adrenergic agonists compared with cholinergic agonists; the anterior bladder neck does not have a similarly significant differential response. The uniform response to noradrenaline may underlie the bladder neck’s role in the prevention of retrograde ejaculation. The differential responses to carbachol may reflect differences in the embryological derivation of the anterior and posterior bladder neck fibres or in their innervation. Some of these differences may have clinical importance through the action of therapeutic agents.


Abbreviations
EFS

electrical field stimulation

TTX

tetrodotoxin.

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

The bladder neck is an anatomical area of increasing urological and pharmacological interest. Our understanding of the physiology of the human bladder neck is incomplete. The normal function of the bladder neck in the male is widely thought to be prevention of retrograde ejaculation [1–3]. Its function in women is less clear [4], but it may serve to shorten and widen the urethra on micturition [5].

Normal voiding, at least in the male, involves co-ordination of the contraction of the detrusor with a simultaneous relaxation of the bladder neck. Abnormalities in this function of the bladder neck can cause BOO in some individuals independently of prostatic enlargement [6–9]. Blockade of α-adrenoceptors can relax the bladder neck and prostatic smooth muscle, but has significant cardiovascular side-effects [10,11]. Other agents, such as inhibitors of 5-α reductase, are widely used for BOO but have more action on the prostate than the bladder neck and have no place in the treatment of females. Moreover, they have a slow onset of action (measured in months) and some troublesome side-effects, such as erectile dysfunction, ejaculatory disorders, and gynaecomastia, making them a less than ideal treatment for BOO. Failure of these pharmacological therapies often requires surgical intervention.

The bladder neck is not a simple circumferential sphincter. The anatomy and innervation appears to differ around the circumference. The bladder neck overall has a rich supply of cholinergic and noradrenergic nerve fibres [12], but these appear not to be uniformly distributed. Anterior bladder neck fibres appear to be closely related to the detrusor muscle, with posterior fibres probably having a trigonal origin. These posterior fibres appear to have a more ‘trigonal-type’ innervation, rich in noradrenergic supply [13]. The anterior fibres relate more closely to the detrusor muscle, from which they are histologically indistinct [4], and have a similar innervation to the detrusor.

Most of the research comparing differences in muscle from around the circumference of the bladder neck is histological observation of normal and pathological tissue. The authors could only find one previous study comparing physiological responses of bladder neck muscle taken from different places [14]. The differences in the structure and innervation around the circumference may be important in understanding the role of the bladder neck in BOO. Initial experiments have suggested considerable variation in responses seen to physiological agonists around the bladder neck circumference [15].

Our null hypothesis is that there are no discernible differences in smooth muscle responses to physiological stimulation between muscle sourced from the anterior bladder neck compared with posterior bladder neck. If differences do exist, they may be important in the development of other pharmacological therapies for BOO as well as our basic understanding of the anatomy and physiology of the human bladder neck.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

Due to the physiological, pathological and anatomical differences in the male and female bladder neck, the subject population in these experiments was limited to adult males. As we have previously reported [16], laparoscopic resection specimens were not included as they were found to be less representative in physiological study.

Strips of adult human male bladder neck muscle were obtained at open radical prostatectomy. The experimenter was present at the operations and care was taken to identify the exact anatomical site of origin of the tissue. Muscle was taken from either the anterior or posterior aspect of the bladder neck, as the surgical conditions at the time of resection allowed. A tie was used to allow later orientation of the tissue in the laboratory. Muscle samples were kept in Krebs’ solution at 4 °C until experimentation and all samples were experimented on within 16 h. Preliminary studies (unpublished) showed no alteration in responses of muscle used in experiments within 24 h of resection.

Before experimentation, muscle strips were prepared from the samples. Any mucosa was removed from the samples. With the aid of a magnifying lens, muscle was then dissected in line with its fibres into strips ≈8 mm long. The strips were then suspended in the Brading-Sibley superfusion apparatus [17], pretensioned to 0.5 g and allowed to equilibrate for 60 min while continually superfused with buffered oxygenated Krebs’ solution at 37 °C at pH 7–7.2. The strips were then superfused with 100 mm KCl-enriched Krebs’ solution for 4 min to determine viability; nonresponsive strips were discarded. A total of 54 strips were assessed and 10 were discarded. Of the viable strips, 30 were used for chemical stimulation studies with noradrenaline and carbachol and 14 were used for electric field stimulation (EFS) studies.

For the chemical stimulation studies 17 strips from the anterior aspect of the bladder neck and 13 from the posterior bladder neck were superfused either with various concentrations (×10−7 to ×10−3m) of carbachol or noradrenaline in Krebs’ solution, for 15 s.

A further 14 strips (eight from the anterior bladder neck and six from the posterior bladder neck) were suspended and responses to EFS measured while the strips were superfused with modified Krebs’ solution. Each strip was exposed to 5 s trains of 50 V, 0.05 ms EFS at different frequencies (1–50 Hz), as these parameters have been previously shown with this apparatus to be nerve selective [18]. Contractile responses were measured. At least 3 min were allowed between each stimulation to permit recovery. Atropine (10−3m) was added to the superfusate for 15 min, and the experiment repeated to provide assessment of the cholinergic nerve-mediated contribution to the contractile response. Tetrodotoxin (TTX) was then added (10−7m) and superfused for a further 15 min to determine if there was any additional inhibition of atropine-resistant responses. The experiments were repeated to show any non-nerve mediated contractile responses.

At the end of each experiment, strips were carefully dissected at the points of ligature. The segment of muscle between points of ligature was then weighed after being gently blotted dry with tissue paper to estimate wet mass of effective muscle.

Tension responses produced in these series of experiments were measured using Pioden UF1 transducers and acquired using the MacLab Data Acquisition system. For statistical analysis the two-tailed Student’s t-test was used, with P ≤ 0.05 being considered statistically significant. Where two measurements were made of the same muscle strip (e.g. before and after the addition of atropine), the analysis was paired. Otherwise, t-tests were unpaired.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

The strip masses were 6.6–20.2 mg. Therefore, to allow comparison between strips, the results of the agonist studies were expressed as tension per unit mass (milligrams of tension per milligram mass). In the EFS study, results were also expressed as a percentage of each strips’ maximal response, to allow each strip to act as its own control when comparing responses to different receptor agonists and antagonists.

All muscle strips included in the study responded to EFS (Fig. 1a shows a typical response curve of bladder neck to EFS stimulation). As can be seen from Fig. 1b, the magnitude of this contraction was frequency dependent. Maximal contractions were seen at 50 Hz. The contractions were largely abolished by superfusion of the muscle strips with atropine, indicating that they were mediated by cholinergic nerves. There was no further suppression of the contractile response on addition of TTX (not shown), showing that there was no measurable direct myocyte stimulation.

image

Figure 1. a, Typical tension response of a strip of bladder neck smooth muscle to 50 Hz, 50 V, 0.05 ms pulses of EFS in a 5-s train (commencing at arrow). Time bar, 1 s. b, The mean percentage maximal contractile responses of all bladder neck strips studied to EFS, before and after atropine (error bars, sem). *statistically significant differences between the two groups. N = 14.

Download figure to PowerPoint

When the results for these strips were separated into those from the anterior and posterior bladder neck, it can be seen in Fig. 2 that posterior bladder neck samples had a greater mean contractile response per unit mass at all frequencies of >1 Hz. As shown, this difference was statistically significant at 20 and 30 Hz.

image

Figure 2. The mean contractile response at each frequency of EFS per unit mass of strips from the anterior and posterior bladder neck. *significant differences between the two groups.

Download figure to PowerPoint

There was a concentration-dependent response in bladder neck contraction to carbachol (Fig. 3a shows a typical example of the contractile response of anterior bladder neck muscle to carbachol stimulation). Figure 3b compares the responses of anterior and posterior bladder neck strips to carbachol stimulation. From this, it can be seen that only the strips from the anterior bladder neck showed any contraction to carbachol at concentrations of <10−3m.

image

Figure 3. a, A typical response of anterior bladder neck muscle to 10−4m carbachol stimulation for 15 s. Time bar, 1 min. b, The responses of muscle strips from the anterior and posterior parts of the bladder neck to differing concentrations of carbachol (error bars, sem). *statistically significant differences between the two groups. Nine anterior and five posterior strips.

Download figure to PowerPoint

As with the carbachol studies, there was a concentration-dependent response to noradrenaline in the bladder neck strips studied; a typical response is shown in Fig. 4a.

image

Figure 4. a, A typical response of bladder neck (in this case, posterior) smooth muscle to 15 s exposure to 10−3 m noradrenaline. Time bar, 1 min. b, The differing responses of the anterior and posterior bladder neck muscle strips to noradrenaline stimulation. The differences were not statistically significant. Eight strips in both groups.

Download figure to PowerPoint

Similar to the results seen with carbachol, responses to noradrenaline were not uniform around the bladder neck, as shown in Fig. 4b, but the slightly larger contractile response per unit mass in anterior compared with posterior muscle was not statistically significant.

The anterior bladder neck muscle contracted in response to exposure to both carbachol (cholinergic stimulation) and noradrenaline (α-adrenergic stimulation). Carbachol was a more ‘potent’ stimulator, producing a stronger contraction per unit mass of tissue at any given concentration (Fig. 5a), but the differences in potency of the agonists was not significant in these anterior muscle strips.

image

Figure 5. a, The responses of bladder neck muscle to two physiological agonists. The differences were not statistically significant. Nine strips tested with carbachol and eight with noradrenaline. b, The differing responses of posterior bladder neck muscle strips to carbachol and noradrenaline. *statistically significant differences. Five strips tested with carbachol and eight with noradrenaline.

Download figure to PowerPoint

The posterior bladder neck strips had significantly different responses to carbachol and noradrenaline, as shown in Fig. 5b. The increased ‘potency’ of noradrenaline was significant at agonist concentrations of 10−4m and 10−3m.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

These studies examine and compare the responses of smooth muscle taken from the anterior and posterior aspects of the bladder neck. As mentioned above, there are described anatomical differences between anterior and posterior bladder neck smooth muscle and its innervation [5,19].

The present results show that nerve-selective EFS is an effective method of inducing bladder neck smooth muscle contraction (Fig. 1a shows the typical contractile response seen). The magnitude of the contraction using this method is small compared with cholinergic receptor agonism, with a maximum of ≈23 milligrams of tension per milligram mass (Fig. 2) compared with carbachol-induced contractions of ≈82 milligrams tension per milligram mass (Fig. 3b). EFS does allow stimulation of the nerve pathways rather than direct myocyte stimulation however, and thus possibly more accurately reproduces the natural physiology. These EFS-induced contractions are predominately mediated by cholinergic nerve activity as evidenced by the near ablation of contractile response to EFS of muscle superfused with atropine (Fig. 1b). This inhibition of response after the addition of atropine was seen with muscle strips from both anterior and posterior bladder neck.

As shown in Fig. 2, the responses of anterior and posterior bladder neck muscle to EFS differ. It can be seen that posterior bladder neck strips contract more per unit mass than anterior strips (by a magnitude of 2.4 times at 50 Hz), statistically significantly so at 20–30 Hz. The ‘loss’ of statistical significance at higher frequencies is possibly due to intersample variation, as it is statistically significant at middle frequencies, where the absolute difference in means is less.

Carbachol (carbamyl choline) caused a contractile response in bladder neck muscle in these studies. A typical response of bladder neck muscle to 15 s carbachol exposure is shown in Fig. 3a.

These studies also show differing responses of anterior and posterior bladder neck muscle strips to carbachol stimulation. Figure 3b shows that anterior bladder neck strips contract strongly to carbachol (to 82.8 milligrams of tension per milligram mass at 10−3m carbachol); in comparison, there is minimal response in posterior bladder neck strips under the same conditions. This difference is significant at carbachol concentrations of ≥10−5m.

Interestingly, there was some contraction in posterior bladder neck strips at the highest concentration of carbachol used. This may be due to some true cholinergic activity of this muscle at this concentration. However, the muscle at the posterior bladder neck and trigone can be functionally divided into a ‘superficial’ and a ‘deep’ layer. The deep layer has been shown to be akin to detrusor muscle in structure, innervation and anatomy [20]. Thus, ‘contamination’ of some samples with muscle fibres originating from deep to the bladder neck might explain this response to carbachol.

Finding that the posterior bladder neck contracted poorly to carbachol makes it difficult to interpret the EFS study results seen using posterior bladder neck muscle. If, as shown in Fig. 2, the contractile responses in all strips were almost completely abolished by the addition of atropine to the superfusate, then this would suggest a cholinergic mechanism of contraction to EFS. Despite this, Fig. 3b shows that carbachol was ineffective at inducing contractions in posterior bladder neck muscle. If the posterior bladder neck does not contract to exposure to carbachol, then it is surprising that atropine suppresses the EFS-induced contractions.

If EFS was stimulating preganglionic cholinergic nerve pathways that synapsed with noncholinergic postganglionic nerve fibres (presumably in intramuscular ganglia within the sample), then this might explain the paradox. Superfusing carbachol may fail to cause contraction, while EFS may depolarize these preganglionic nerve fibres to stimulate contraction in posterior bladder neck muscle through noncholinergic, perhaps sympathetic, nerve pathways. This effect might be blocked by atropine at the ganglion level. It has long been known that there are variations in innervation around the human bladder neck, with a preponderance of noradrenergic nerves in the posterior part [21]. However, if these postganglionic nerves are sensitive to atropine, it would be logical that carbachol would activate them as well, so this remains speculative.

An alternative explanation might be that there was contamination of the posterior bladder neck strips with detrusor muscle fibres in the samples used for the EFS studies. This would allow the strips to contract to EFS in an atropine-sensitive manner. If the strips used in the agonist studies were not similarly contaminated, this might explain the differences seen. However, it is difficult to see how this contamination, if present, would affect one set rather than another as all the samples were harvested in a similar manner by the same group of surgeons.

Noradrenaline also caused concentration-dependent contractions in the muscle strips. A typical contractile response is shown in Fig. 4a.

When the responses of anterior and posterior bladder neck are compared, it can be seen that carbachol causes significant contraction in anterior but not posterior bladder neck muscle. Noradrenaline will induce similar contractions in muscle strips of both anatomical origins. Anterior bladder neck contracted slightly more strongly to noradrenaline than posterior bladder neck, but the difference was not significant (Fig. 4b).

From Figs 3b and 4b, it can also be seen that anterior bladder neck muscle strips contracted more strongly per unit mass to both agonists than did posterior bladder neck muscle strips to either agonist.

Figure 5a shows that the response of anterior bladder neck strips to carbachol stimulation is stronger than that to noradrenaline, albeit not reaching statistical significance. Figure 5b shows that the opposite is true for posterior bladder neck; noradrenaline is a significantly more potent agonist in these strips. This is in keeping with the findings of previous studies suggesting posterior bladder neck muscle to be functionally devoid of cholinergic receptors but having a strong but variable adrenergic response [14].

However, the posterior muscle strips showed a differential response to the agonists used, with noradrenaline causing statistically significant and about a two-fold greater contractile response than carbachol. This significant difference was not seen in muscle strips from the anterior bladder neck. Thus, anterior bladder neck strips were more responsive to either agonist than posterior bladder neck strips, which showed a significant difference depending on the agonist used.

The response of the whole circumference of the bladder neck to noradrenergic stimulation suggests that the organ would contract to stimulation through sympathetic innervation. As discussed above, the normal role of the bladder neck in the male may be to prevent retrograde ejaculation. Contraction to sympathetic innervation would support this, allowing the bladder neck to close at the time of ejaculation under sympathetic control.

These studies show significant differences in the physiological behaviour of anterior and posterior bladder neck muscle in vitro. These differences probably reflect the differing embryological origins of the opposing parts of the bladder neck, anterior fibres arising from endodermal tissue like the detrusor muscle, and posterior fibres having a mesodermal origin similar to trigone and ureter.

The differing responses may be crucial in the normal functioning of the bladder neck. The contraction of the anterior muscle to cholinergic stimulation may aid opening or shortening of the bladder neck at micturition. Moreover, the adrenergic-mediated contraction of the muscle fibres from around the circumference of the bladder neck may be important in preventing retrograde ejaculation; this role is further suggested by the documented side-effect of retrograde ejaculation in men taking α-adrenergic blockers [22].

The findings of the present study may have other potential pharmaceutical implications. The results show that both anterior and posterior bladder neck muscle strips will contract to noradrenaline, confirming the role of α-adrenergic receptors in bladder neck contraction. This underpins the use of α-adrenergic blocking agents (such as doxazosin, alfuzosin, and tamsulosin) in the clinical treatment of BOO caused by hypertonicity or hypertrophy of the bladder neck. Blockade of these receptors would reduce bladder neck tone, and thereby potentially reduce the BOO.

The use of anticholinergic agents, such as oxybutynin, is associated with an increased risk of urinary retention and incomplete voiding. While this may be largely due to impairment of detrusor contractility, it may also reflect a reduction in the opening of the bladder neck by impairing the contraction of the anterior fibres, thus limiting the shortening and opening of this part of the bladder neck mentioned above.

In the present study, the role of the mucosa is not examined. There is increasing evidence to suggest that neurotransmitter release from nerves originating in the mucosal and submucosal layers may have the potential to alter underlying detrusor smooth muscle function in both animal models [23] and humans [24]. Although we are not aware of any studies reporting these effects specifically at the bladder neck, there is no reason to think that it does not also occur here and thus it cannot as yet be excluded as an important factor in vivo.

The human bladder neck is a complex organ, and its physiology is incompletely understood. The present experiments show important physiological variability around the circumference of the human male bladder neck. The posterior bladder neck had significantly stronger contraction to α-adrenergic agonists compared with cholinergic agonists; the anterior bladder neck did not have a similarly significant differential response. The uniform response to noradrenaline may underlie the bladder neck’s role in the prevention of retrograde ejaculation. The differential responses to carbachol may reflect differences in the embryological derivation of the anterior and posterior bladder neck fibres or in their innervation. Some of these differences may have clinical importance through the action of therapeutic agents.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

We would like to thank Messrs R. Persad and M. Wright of the Department of Urology, Bristol Royal Infirmary, Bristol, UK for the acquisition of tissue from their patients.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES