Russell Chess-Williams

Urothelium-derived inhibitory factor(s) influences on detrusor muscle contractility in vitro

The function of the bladder urothelium in modulating contractile responses of the underlying detrusor smooth muscle to muscarinic stimulation has been examined in the pig bladder. Saturation curves for [3H]‐QNB binding demonstrated a greater muscarinic receptor density in the urothelium than in the detrusor smooth muscle. The presence of an intact urothelium on isolated bladder strips inhibited contractions induced by carbachol but not KCl. Contractions of a urothelium‐denuded muscle strip were inhibited in the presence of a second bladder strip with an intact urothelium, but not if the second strip was denuded. The urothelium‐induced inhibition of contractions was not prevented in the presence of L‐NOARG, methylene blue, indomethacin, propranolol, suramin, TEA or apamin. The data suggest the presence of a diffusable, urothelium‐derived inhibitory factor, which could not be identified but appears to be neither nitric oxide, a cyclo‐oxygenase product, a catecholamine, adenosine, GABA nor an EDHF sensitive to apamin.

Muscarinic receptor subtypes and management of the overactive bladder

Anticholinergic agents are the most widely used therapy for urge incontinence despite exerting adverse effects, such as constipation, tachycardia, and dry mouth, that limit their use. These adverse effects result from a lack of selectivity for the bladder over other organs. Although M2-muscarinic receptors are the predominant cholinoreceptor present in urinary bladder, the smaller population of M3-receptors appears to be the most functionally important and mediates direct contraction of the detrusor muscle. M2-receptors modulate detrusor contraction by several mechanisms and may contribute more to contraction of the bladder in pathologic states, such as bladder denervation or spinal cord injury. Prejunctional inhibitory M2-receptors or M4-receptors and prejunctional facilitatory M1-muscarinic receptors in the bladder have also been reported, but their relevance to the clinical effectiveness of muscarinic antagonists is unknown. In clinical studies, tolterodine, a nonselective muscarinic antagonist, has been reported to be equally effective to oxybutynin but to induce less dry mouth. Controlled-release and intravesical, intravaginal, and rectal administrations of oxybutynin have all been reported to cause fewer adverse effects. Conversely, darifenacin, a new M3-selective antagonist, has been reported to have selectivity for the bladder over the salivary gland in vivo. Whether M3-selective or nonselective muscarinic antagonists will be the most clinically effective for the overactive bladder-preserving the best balance between efficacy and tolerability-has yet to be established, and comparative clinical trials between compounds, such as darifenacin (M3 selective) and tolterodine (nonselective) will be required.

Bladder afferent sensitivity in wild-type and TRPV1 knockout mice

Understanding bladder afferent pathways may reveal novel targets for therapy of lower urinary tract disorders such as overactive bladder syndrome and cystitis. Several potential candidate molecules have been postulated as playing a significant role in bladder function. One such candidate is the transient receptor potential vanilloid 1 (TRPV1) ion channel. Mice lacking the TRPV1 channel have altered micturition thresholds suggesting that TRPV1 channels may play a role in the detection of bladder filling. The aim of this study was therefore to investigate the role of TRPV1 receptors in controlling bladder afferent sensitivity in the mouse using pharmacological receptor blockade and genetic deletion of the channel. Multiunit afferent activity was recorded in vitro from bladder afferents taken from wild‐type (TRPV+/+) mice and knockout (TRPV1−/−) mice. In wild‐type preparations, ramp distension of the bladder to a maximal pressure of 40 mmHg produced a graded increase in afferent activity. Bath application of the TRPV1 antagonist capsazepine (10 μm) caused a significant attenuation of afferent discharge in TRPV1+/+ mice. Afferent responses to distension were significantly attenuated in TRPV1−/− mice in which sensitivity to intravesical hydrochloric acid (50 mm) and capsaicin (10 μm) were also blunted. Altered mechanosensitivity occurred in the absence of any changes in the pressure–volume relationship during filling indicating that this was not secondary to a change in bladder compliance. Single‐unit analysis was used to classify individual afferents into low‐threshold and high‐threshold fibres. Low threshold afferent responses were attenuated in TRPV1−/− mice compared to the TRPV1+/+ littermates while surprisingly high threshold afferent sensitivity was unchanged. While TRPV1 channels are not considered to be mechanically gated, the present study demonstrates a clear role for TRPV1 in the excitability of particularly low threshold bladder afferents. This suggests that TRPV1 may play an important role in normal bladder function.

The effects of tamsulosin, a high affinity antagonist at functional α1A- and α1D-adrenoceptor subtypes

The actions of the α1‐adrenoceptor antagonist tamsulosin have been examined at functional α1‐adrenoceptor subtypes and compared with those at the human prostate receptor. At the α1D‐adrenoceptors of the rat aorta, tamsulosin acted as a competitive antagonist with a high affinity (pKB=10.1). At the α1B‐adrenoceptor of the rat spleen and rabbit corpus cavernosum penis, tamsulosin again acted as a competitive antagonist but with a significantly lower affinity (pKB=8.9–9.2). Tamsulosin acted as an unsurmountable antagonist of the α1A‐adrenoceptor‐mediated responses of the rat and human vas deferens, reducing maximal responses to phenylephrine by 20% and 50%, respectively, at an antagonist concentration of 1 nm. Responses of depolarized (100 mm KCl) rat vas deferens preparations were unaffected by 10 nm tamsulosin but this concentration reduced maximal responses to 5‐hydroxytryptamine (5‐HT) in this tissue. When longer antagonist incubation periods (60 min) were used, tamsulosin behaved as a competitive antagonist on the human prostate with a significantly higher affinity (pKB=10.0) than obtained at the α1B‐adrenoceptor. The data demonstrate that tamsulosin is a high affinity antagonist at functional α1‐adrenoceptors with a selectivity α1Dα1A>α1B. In some tissues the compound exhibits an additional unsurmountable antagonist action, the clinical significance of which is unknown.

Which muscarinic receptor is important in the bladder

Abstract Antimuscarinic agents are the most widely used therapy for urge incontinence, but have side effects such as constipation, tachycardia and dry mouth, resulting from a lack of selectivity for the bladder. M2 receptors are the predominant cholinoceptors present in urinary bladder, but mainly the minor population of M3 receptors mediate its contraction. M2 receptors modulate detrusor contraction by several mechanisms, and may contribute more to contraction of the bladder in pathological states such as bladder denervation or spinal cord injury. Prejunctional inhibitory M2 or M4 receptors and prejunctional facilitatory muscarinic M1 receptors in the bladder have all been reported. In clinical studies, tolterodine, a non-selective muscarinic antagonist, has been reported to be as effective as oxybutynin but inducing less dry mouth. Thus, although it is not certain which antimuscarinic drugs have the better efficacy and tolerability, the non-selective antimuscarinic drugs seem to be better than M3-selective antagonists in their clinical efficacies. However, controlled release, or intravesical, intravaginal, or rectal administrations of oxybutynin have been reported to cause fewer side effects. Darifenacin, a new M3 selective antagonist, has been reported to have selectivity for the bladder over the salivary gland in vivo. To verify which antimuscarinic drugs selective for the muscarinic subtypes have the best efficacy and tolerability, comparative clinical trials between M3 selective antagonists and non-selective compounds, such as olterodine, are required in the future.

In vitroα1‐adrenoceptor pharmacology of Ro 70–0004 and RS‐100329, novel α1A‐adrenoceptor selective antagonists

It has been hypothesized that in patients with benign prostatic hyperplasia, selective antagonism of the α1A‐adrenoceptor‐mediated contraction of lower urinary tract tissues may, via a selective relief of outlet obstruction, lead to an improvement in symptoms. The present study describes the α1‐adrenoceptor (α1‐AR) subtype selectivities of two novel α1‐AR antagonists, Ro 70‐0004 (aka RS‐100975) and a structurally‐related compound RS‐100329, and compares them with those of prazosin and tamsulosin. Radioligand binding and second‐messenger studies in intact CHO‐K1 cells expressing human cloned α1A‐, α1B‐ and α1D‐AR showed nanomolar affinity and significant α1A‐AR subtype selectivity for both Ro 70‐0004 (pKi 8.9: 60 and 50 fold selectivity) and RS‐100329 (pKi 9.6: 126 and 50 fold selectivity) over the α1B‐ and α1D‐AR subtypes respectively. In contrast, prazosin and tamsulosin showed little subtype selectivity. Noradrenaline‐induced contractions of human lower urinary tract (LUT) tissues or rabbit bladder neck were competitively antagonized by Ro 70‐0004 (pA2 8.8 and 8.9), RS‐100329 (pA2 9.2 and 9.2), tamsulosin (pA2 10.4 and 9.8) and prazosin (pA2 8.7 and 8.3 respectively). Affinity estimates for tamsulosin and prazosin in antagonizing α1‐AR‐mediated contractions of human renal artery (HRA) and rat aorta (RA) were similar to those observed in LUT tissues, whereas Ro 70‐0004 and RS‐100329 were approximately 100 fold less potent (pA2 values of 6.8/6.8 and 7.3/7.9 in HRA/RA respectively). The α1A‐AR subtype selectivity of Ro 70‐0004 and RS‐100329, demonstrated in both cloned and native systems, should allow for an evaluation of the clinical utility of a ‘uroselective’ agent for the treatment of symptoms associated with benign prostatic hyperplasia.

The role of M2-muscarinic receptors in mediating contraction of the pig urinary bladder in vitro

In urinary bladder, M2‐muscarinic receptors predominate, but it is the smaller population of M3‐receptors which mediate detrusor contraction. This study examines the M2 : M3 ratio and the role of M2‐receptors in contraction of pig urinary bladder. Competition experiments with [3H]‐QNB determined the ratio of M2 : M3. In functional studies, affinity values (pKB) for 4‐DAMP, darifenacin and methoctramine were calculated. Similar experiments were performed on tissues following selective M3‐inactivation (incubation with 40 nM 4‐DAMP mustard in the presence of 1 μM methoctramine to protect M2‐receptors), precontraction with 50 mM KCl and relaxation with isoprenaline (30 μM) or forskolin (1 μM). In competition binding, displacement of [3H]‐QNB by 4‐DAMP, darifenacin and methoctramine best fitted a two‐site model suggesting a predominant (70–80%) population of M2‐receptors. On normal detrusor in vitro, 4‐DAMP and methoctramine caused surmountable antagonism of responses to carbachol with pKB values of 9.37±0.07 and 6.05±0.05 respectively. Darifenacin caused unsurmountable antagonism, the apparent pKB value being 8.61±0.10. In tissues where the M3‐receptors had been inactivated and cyclic AMP levels elevated, 4‐DAMP and darifenacin were less potent, with apparent pKB values of 8.72±0.08 and 6.74±0.07. In contrast, methoctramine was more potent, the apparent pKB value increasing significantly to 6.86±0.06. These data suggest that the pig bladder possesses a similar muscarinic receptor population to the human bladder and that the M3‐receptor subtype mediates contraction of the normal detrusor muscle. However an involvement of M2‐receptors in contraction can be observed following pharmacological manipulation of the receptor population.

UROTHELIUM DERIVED INHIBITORY FACTOR AND CROSS-TALK AMONG RECEPTORS IN THE TRIGONE OF THE BLADDER OF THE PIG

PURPOSE In the dome of the bladder of the pig muscarinic receptor stimulation has been shown to release a factor from the urothelium that exerts an inhibitory effect on the underlying smooth muscle. We examined whether the urothelium in the trigone of the bladder also releases this factor, identified which receptors stimulate its release and investigated possible cross-talk among these receptor systems in the trigone. MATERIALS AND METHODS Paired longitudinal strips of pig bladder were isolated, the urothelium was removed from 1 strip per pair and tissues were set up in gassed Krebs solution at 37C. Cumulative concentration-response curves to carbachol, phenylephrine or histamine were constructed. In some tissues a second phenylephrine curve was constructed in the presence of 1 microM. carbachol or 1 microM. histamine. In a further group of tissues the second phenylephrine curve was constructed in the presence of 1 microM. carbachol and 1 microM. atropine or 1 microM. histamine and 100 nM. mepyramine. RESULTS In the presence of an intact urothelium contractile responses to carbachol and histamine but not to phenylephrine were depressed. In the presence of 1 microM. carbachol or 1 microM. histamine the responses of intact urothelium strips to phenylephrine were significantly depressed. This effect was absent in the presence of atropine and mepyramine, respectively. CONCLUSIONS Carbachol and histamine induce the release of a urothelium derived inhibitory factor in the bladder trigone. The factor appears to mediate cross-talk between these systems and the alpha-adrenoceptor system in this region of the bladder.

In vitro release of adenosine triphosphate from the urothelium of human bladders with detrusor overactivity, both neurogenic and idiopathic.

BACKGROUND There is increased evidence to suggest a role for nonadrenergic-noncholinergic neurotransmission in the pathogenesis of bladder dysfunction. OBJECTIVE In this set of experiments, we have assessed the contribution of the urothelium to purinergic activity by quantifying the amount of adenosine triphosphate (ATP) released from the urothelium of patients with idiopathic detrusor overactivity (IDO) and with neurogenic detrusor overactivity (NDO) and comparing these releases to those of controls. DESIGN, SETTING, AND PARTICIPANTS Bladder tissue with urodynamically and clinically proven NDO (n=8) and IDO (n=8) were included in this study. The carefully dissected urothelium was stimulated by mechanically stretching as well as electrically stimulating and the ATP; thus, release was quantified. MEASUREMENTS We used a Lucy Anthos 1 luminometre (Anthos Labtec Instruments GmBH, Wals, Austria) to perform the assay. The results were analysed using Stingray software (Dazdaq Ltd, Brighton, UK). RESULTS AND LIMITATIONS Both mechanical stretch and electric field stimulation (EFS) led to increased ATP release in both sets of tissues with overactivity compared to the controls; this rise was even more significant for the IDO urothelium (2416.7±479.8 pmol/g [p<0.005]) than for the NDO urothelium (133.1±22.4 pmol/g [p<0.01]); values for the controls were 77.6±16.2 pmol/g. ATP release following mechanical stretch was more sensitive to tetrodotoxin in bladders with NDO compared to those with IDO as well as to the controls, with ATP levels falling from 233.5±20.7 pmol/g to 107.2±11.6 pmol/g, expressed as percentage of basal levels (p<0.002). The experiments were performed in vitro, and the female patients were a mix of peri- and postmenopausal states. CONCLUSIONS These experiments suggested a significant rise in ATP release from the urothelium of bladders with NDO as well as those with IDO in comparison to controls. Most of the ATP released from bladders with NDO is primarily from neuronal sources.

The role of the urothelium in mediating bladder responses to isoprenaline.

To investigate whether the responses of the pig bladder to isoprenaline (a nonselective β‐adrenoceptor agonist) are influenced by the presence of an intact urothelium and whether any influence might be attributed to the release of nitric oxide (NO), since stimulation of β‐adrenoceptors induces a direct relaxation of detrusor smooth muscle and β‐adrenoceptors are also present on the urothelium.