- Email: [email protected]
Localization of M2 and M3 Muscarinic Receptors in Human Bladder Disorders and Their Clinical Correlations
Localization of M2 and M3 Muscarinic Receptors in Human Bladder Disorders and Their Clinical Correlations Gaurav Mukerji, Yiangos Yiangou, Joanna Grogono, Jenny Underwood, Sanjiv K. Agarwal, Vikram Khullar* and Praveen Anand† From the Peripheral Neuropathy Unit (GM, YY, JG, SKA, PA) and Department of Urology (GM, SKA), Hammersmith Hospital and Imperial College London and Urogynaecology Unit, St. Mary’s Hospital and Imperial College (JG, JU, VK), London, United Kingdom
Purpose: We studied the cellular localization of muscarinic receptor subtypes 2 and 3 in the human bladder and related any changes in overactive and painful bladder syndromes to measures of clinical dysfunction. Materials and Methods: Bladder specimens obtained from patients with painful bladder syndrome (11), idiopathic detrusor overactivity (12) and from controls with asymptomatic microscopic hematuria (16) were immunostained using specific antibodies to muscarinic receptor subtypes 2 and 3, and to vimentin, which is a marker for myofibroblasts. Immunostaining results were quantified with computerized image analysis and correlated with clinical dysfunction using frequency and urgency scores. Results: Muscarinic receptor subtype 2 and 3 immunoreactivity was observed in the urothelium, nerve fibers and detrusor layers. In addition, strong myofibroblast-like cell staining, similar to vimentin, was present in the suburothelial region and detrusor muscle. A significant increase in suburothelial myofibroblast-like muscarinic receptor subtype 2 immunoreactivity was seen in patients with painful bladder syndrome (p ⫽ 0.0062) and idiopathic detrusor overactivity (p ⫽ 0.0002), and in muscarinic receptor subtype 3 immunoreactivity in those with idiopathic detrusor overactivity (p ⫽ 0.0122) with a trend in painful bladder syndrome. Muscarinic receptor subtype 2 and 3 immunoreactivity significantly correlated with the urgency score (p ⫽ 0.0002 and 0.0206, respectively) and muscarinic receptor subtype 2 immunoreactivity correlated with the frequency score (p ⫽ 0.0029). No significant difference was seen in urothelial and detrusor muscarinic receptor subtypes 2 and 3 or vimentin immunostaining. Conclusions: To our knowledge this is the first study to show the cellular localization of muscarinic receptor subtypes 2 and 3 in the human bladder. The increase in muscarinic receptor subtypes 2 and 3 immunostaining in myofibroblast-like cells in clinical bladder syndromes and its correlation with clinical scores suggests a potential role in pathophysiological mechanisms and the therapeutic effect of anti-muscarinic agents. Key Words: bladder; receptors, muscarinic; pain; urinary incontinence
which increases bladder compliance via -adrenoceptor mediated relaxation of the bladder. During the voiding phase acetylcholine is released from parasympathetic nerves. This is thought to activate M2 and produce the inhibition of -adrenoceptor mediated relaxation of the bladder. The result is the reversal of relaxed bladder tone which, concomitant with M3 mediated contraction of the detrusor, provides efficient and complete bladder emptying. Binding and subtype selective immunoprecipitation studies have demonstrated that, while the majority of muscarinic receptors in the bladder are of the M2 subtype, M3 is believed to be the most important for detrusor contraction.2,3 KO mice lacking M3 show prominent bladder distention and have greatly decreased detrusor contraction in vitro.1 The small, direct contractile response that persists in the bladder of M3 KO mice is completely lost in mice lacking M2 and M3, demonstrating that M2 is capable of mediating small contractions and muscarinic receptors other than M2 and M3 do not seem to mediate any direct bladder contraction.4 However, in vivo M3 KO mice were able to empty the bladder and they showed little changes in urodynamic parameters compared to WT controls, illustrating that in rodents the
uscarinic acetylcholine receptors belong to a group of 7 transmembrane spanning receptors that are distributed widely in the central and peripheral nervous systems, where they have key physiological roles.1 Five muscarinic receptor subtypes (M1 to M5) have been cloned and defined pharmacologically.2 Bladder contractions in humans are normally mediated mainly via the stimulation of muscarinic receptors expressed by bladder smooth muscle cells. The bladder has 2 functions, that is storing and emptying urine. During the filling or storage phase the bladder is inhibited by the sympathetic stimulation mediated by noradrenaline released from sympathetic nerves,
M
Submitted for publication August 10, 2005. Study received local ethics committee approval. Supported by GlaxoSmithKline, Harlow, United Kingdom (GM) and Roche Bioscience, Palo Alto, California (YY). * Financial interest and/or other relationship with Pfizer, Yamanouchi, Novartis, Schwarz Pharmaceuticals, Takeda and AstraZeneca. † Correspondence: Peripheral Neuropathy Unit, Area A, Hammersmith Hospital, Du Cane Rd., London, United Kingdom W12 0NN (telephone: 0044 208 383 3309; FAX: 0044 208 383 3363; e-mail: [email protected]).
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noncholinergic component of bladder contraction seems sufficient for voiding contraction.5 Although M2 is thought to have only a minor role in smooth muscle contraction despite its prevalence, there is evidence to suggest that M2 may be an active participant in detrusor contraction in certain pathological states.6,7 Hypertrophied bladders induced by denervation and spinal cord injury in rats showed an increase in total and M2 density, and a change in muscarinic receptor subtype mediating bladder contraction from M3 to M2.7 Similar changes were later demonstrated in human bladder with neurogenic bladder dysfunction.8 PBS is a debilitating, chronic bladder hypersensitivity disorder that typically presents with suprapubic pain related to bladder filling, accompanied by other symptoms, such as increased daytime and nighttime frequency in the absence of a definable etiology.9 OAB is characterized by urinary urgency with or without urge incontinence, usually with frequency and nocturia.9 Determining the distribution and subtype specific functions has been a matter of considerable interest because selective therapeutic agents may have a narrower side effect profile. The presence of muscarinic receptors in the bladder has been shown mainly through indirect techniques, such as reverse transcriptase-polymerase chain reaction, Northern blot, radioligand binding and immunoprecipitation studies.2,3 We report the distribution of M2 and M3 in the human bladder using immunohistochemistry in control subjects, and in patients with OAB and PBS. In addition, we describe the correlation of these receptors with clinical symptoms.
MATERIALS AND METHODS Bladder tissue specimens were obtained from 16 control subjects under investigation for asymptomatic microscopic hematuria, 12 patients with idiopathic detrusor overactivity and 11 with PBS who met National Institute for Diabetes and Digestive and Kidney Diseases research criteria for interstitial cystitis.10 Mean age in controls, patients with IDO and patients with PBS was 53.1 (range 21 to 89), 51.2 (range 32 to 73) and 47.4 years (range 22 to 76), respectively. Approval was given by the local ethics committee and informed consent was obtained from patients and controls. Clinical assessments included history and clinical examination, followed by a midstream urine specimen, culture and cytology, and urodynamics. Symptom scores were recorded on the PUF symptom scale.11 Patients with IDO presented with overactive bladder symptoms, including urgency with or without urge incontinence, frequency and nocturia, and they showed involuntary detrusor contractions during the filling phase of urodynamics.9 All patients with PBS complained of frequency (greater than 5 voids in 12 hours), nocturia (greater than 2 voids), urgency and suprapubic/ pelvic pain without any signs of detrusor overactivity on urodynamics. Flexible or rigid cystoscopic bladder biopsies were obtained from a consistent site just above and lateral to the ureteral orifices. All patients had sterile urine cultures at cystoscopy and biopsy. Human dorsal root ganglion specimens were collected after death (Netherlands Brain Bank, Amsterdam, The Netherlands) with less than an 8-hour delay and stored at ⫺80C until use.
Antibodies Muscarinic receptor subtypes M2 (rabbit polyclonal, H-170) and M3 (rabbit polyclonal, H-210) antibodies (Santa Cruz Biotechnology, Santa Cruz, California) were used at a dilution of 1:300 and 1:500, respectively. Monoclonal antibodies to the human vimentin intermediate filament subunit (NovoCastra, Newcastle upon Tyne, United Kingdom) was used at a dilution of 1:6,000. Immunohistochemistry Frozen sections (12 m) were post-fixed in freshly prepared 4% weight per volume paraformaldehyde in phosphate buffered saline for 20 minutes and dehydrated for a further 20 minutes (hydrogen peroxide). Rabbit and mouse primary antibodies were diluted in goat and horse serum, respectively, (1:30) and incubated overnight. Sites of primary antibody attachment were revealed using the nickel enhanced avidin-biotin peroxidase method. Nuclei were counterstained with 0.1% weight per volume aqueous neutral red. Image Analysis Computerized image analysis was performed to quantify immunoreactivity. Images were captured using an Olympus DP70 camera mounted to an Olympus BX50 microscope and analyzed using analySIS, version 5.0 software (Olympus, Tokyo, Japan). Positive immunostaining was highlighted by setting the gray level detection limits to threshold and the area of highlighted immunoreactivity obtained as percent area of the field scanned. The threshold was kept constant throughout all specimens analyzed. Five fields per tissue section were scanned and the mean value was used for subsequent statistical analysis. The mean values of readings obtained by 2 blinded, independent observers were used for final analysis. The Mann-Whitney test was used for statistical analysis with p ⬍0.05 considered statistically significant. The ratio between M2 and M3 in serial tissue sections was calculated. The results of immunohistochemistry were correlated with frequency and urgency scores using Spearman’s correlation, including all specimens studied. The frequency score was obtained from the PUF questionnaire11 and rated as 0 —3 to 6, 1—7 to 10, 2—11 to 14, 3—15 to 19 and 4 —20 or greater voids daily. Similarly the urgency score was obtained from the PUF questionnaire11 and graded as 0 —no urgency, 1—mild, 2—moderate and 3—severe. RESULTS M2 M2 and M3 immunoreactivity was seen in the urothelium, detrusor layer and nerve fiber bundles (fig. 1, A to C). In addition, strong myofibroblast-like staining, consisting of cell bodies associated with long fiber processes, was present in the suburothelium (figs. 1, A and 2, A to C). M2 staining appeared more frequent and intense in IDO and PBS specimens than in controls (fig. 2, A to C). Image analysis showed a significant increase in suburothelial myofibroblast-like M2 immunoreactivity in the PBS and IDO groups vs controls (mean immunoreactive area ⫾ SEM 2.35% ⫾ 0.55% vs 0.68% ⫾ 0.08%, p ⫽ 0.0062 and 2.21% ⫾ 0.37% vs 0.68% ⫾ 0.08%, p ⫽ 0.0002, respectively, fig. 3, A). No statistically significant change in M2 immunoreactivity was seen in the
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FIG. 1. Representative M2 immunoreactive staining in human bladder section showing staining in detrusor and myofibroblast-like staining (arrows) in suburothelium (A). Representative urothelial M2 immunoreactivity (B). Representative M3 immunoreactive staining reveals detrusor and rare nerve fiber bundle (arrow) (C). Representative M3 immunoreactive staining in human dorsal root ganglion demonstrates staining in subset of small and medium sensory neurons (D). Reduced from ⫻20 (A), ⫻40 (B and D) and ⫻10 (C).
control, IDO and PBS groups in the urothelium (mean immunoreactive area 0.633% ⫾ 0.168%, 0.615% ⫾ 0.234% and 1.641% ⫾ 0.631%) or in the detrusor (1.464% ⫾ 0.431%, 2.333% ⫾ 0.333% and 2.00% ⫾ 0.393%, respectively). Myofibroblast-like cells immunostained in the detrusor were sporadic and could not be image analyzed separately. M3 M3 immunoreactivity was seen in the urothelium and detrusor layer (fig. 1, C). Myofibroblast-like staining similar to M2, was also seen with M3 antibody (fig. 2, E to G). A significant increase in suburothelial myofibroblast-like M3 immunoreactivity was seen in the IDO group vs controls (mean immunoreactive area 1.38% ⫾ 0.35% vs 0.61% ⫾ 0.24%, p ⫽ 0.0122, fig. 3, B). No difference was seen in M3 staining between the control and PBS groups (mean immunoreactive area 0.61% ⫾ 0.24% and 0.76% ⫾ 0.18%, respectively, p ⫽ 0.0717, fig. 3, B). There was no statistically significant difference in M3 immunoreactivity among the control, IDO and PBS groups in the urothelium (mean immunoreactive area 1.20% ⫾ 0.515%, 0.971% ⫾ 0.344% and 1.79% ⫾ 0.889%) or in the detrusor (1.429% ⫾ 0.455%, 1.833% ⫾ 0.358% and 1.643% ⫾ 0.261%, respectively). In addition to urothelial, detrusor and myofibroblast-like staining, M2 and M3 immunoreactivity was also observed in
nerve bundles in some specimens (fig. 1, C). However, it was not observed or it was weak in fine nerve fibers in the suburothelial region. Thus, it was not possible to perform image analysis between the groups. Sensory neurons in human dorsal root ganglia also showed M2 and M3 immunoreactivity (fig. 1, D). There was no significant difference in the M2-to-M3 staining ratio in serial sections among the control, IDO and PBS groups (2.81:1, 3.68:1 and 3.63:1, respectively, control vs IDO p ⫽ 0.8709 and vs PBS p ⫽ 0.3613, fig. 3, C).
Vimentin Staining Vimentin positive cells were characteristic of the description of myofibroblasts as stellate cells with ramifying processes. They were observed in the suburothelium and detrusor layers in all control, IDO and PBS specimens (fig. 2, H). The staining pattern appeared similar to staining seen using muscarinic receptor antibodies (fig. 2, D). On analysis a 2-fold increase in suburothelial vimentin immunoreactivity was seen in the PBS group compared to controls but this did not achieve statistical significance (mean immunoreactive area 7.71% ⫾ 1.32% and 3.93% ⫾ 0.81%, respectively, p ⫽ 0.1014, fig. 3, D). Similarly no significant difference was seen in vimentin staining between the control and IDO
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FIG. 2. Representative myofibroblast-like, M2 immunoreactive staining in suburothelium of control (A), IDO (B) and PBS (C) bladder, and M3 immunoreactive staining in suburothelium of control (E), IDO (F) and PBS (G) bladder. Comparison of M3 staining (D) with vimentin staining (H) shows myofibroblast-like structures in suburothelium. Reduced from ⫻40.
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FIG. 3. Relative mean percent area ⫾ SEM of M2 (A), and M3 (B) immunoreactive myofibroblasts, M2-to-M3 ratio (C) and vimentin staining (D) in 16 control, 12 IDO and 11 PBS preparations.
groups (mean immunoreactive area 3.93% ⫾ 0.81% and 5.22% ⫾ 1.19%, respectively, p ⫽ 0.5350, fig. 3, D). Correlation With Clinical Scores The frequency score correlated significantly with the M2 immunoreactive area (r ⫽ 0.5325, p ⫽ 0.0029, fig. 4, A). However, no correlation was found between M3 staining and
FIG. 4. Correlation of frequency score with M2 (A) and M3 (B) receptor density, and urgency score with M2 (C) and M3 (D) receptor density.
the frequency score (r ⫽ 0.3340, p ⫽ 0.0766, fig. 4, B). The urgency score correlated significantly with M2 and M3 immunoreactive staining (r ⫽ 0.6355, p ⫽ 0.0002 and r ⫽ 0.4279, p ⫽ 0.0206, respectively, fig. 4, C and D).
DISCUSSION In this study M2 and M3 immunoreactivity was seen in the urothelium, nerve fibers and detrusor muscle. In general agreement with other studies3,12 the overall M2-to-M3 immunoreactive staining ratio was 3:1 in the normal (control) bladder, which was preserved in IDO and PBS specimens. However, this ratio was not maintained when the urothelium and detrusor were individually analyzed, which may reflect different affinity and/or avidity of the antibodies or methods used. Stronger suburothelial myofibroblast-like staining was also seen with M2 and M3 receptor antibodies, and it appeared similar to vimentin staining. The suburothelial myofibroblast-like staining seen in this study was in agreement with that noted by Gillespie et al, who reported a similar pattern of M3 immunoreactive spindle-shaped cells in the suburothelium of guinea pig bladder.13 They have an immunochemical feature of staining to vimentin and ␣-smooth muscle actin, and can be identified by their characteristic cell bodies with spindle-like cytoplasmic processes.14,15 Colocalization and ultrastructural studies15 would be helpful to further confirm the cell type expressing the muscarinic receptors. Myofibroblasts have been observed in the upper urinary tract and urethra,16 where they may have a pacemaker role analogous to interstitial cells of Cajal in the gut. Recently the presence of a layer of myofibroblasts has been reported below the urothelium in close proximity to afferent and
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efferent nerve varicosities.15 Due to their location and association with nerve varicosities it has been proposed that these myofibroblasts with associated axonal varicosities could collectively function as a bladder stretch receptor.14 This proposal is supported by our finding of cholinergic receptor expression in these myofibroblasts. It could also at least partly explain why anticholinergic medication increases bladder capacity and decreases the sensation of urgency without impairing detrusor contraction during voiding,17 an observation that is difficult to account for only by the action of anticholinergics on detrusor muscle parasympathetic innervation.15 Further studies are required to assess any immunostaining in sensory nerve fibers that may be relevant in this regard. Immunostaining was seen in large nerve fiber bundles but it was faint or absent in suburothelial fine fibers using these antibodies and, thus, it did not enable quantitative image analysis. M2 expression has previously been noted in sensory neurons of rat dorsal root ganglia and their peripheral endings in skin. In our studies of human dorsal root ganglion sensory neurons using these antibodies we observed M2 and M3 immunoreactivity in a subset of small and medium sensory neurons, suggesting a potential role in sensory mechanisms. We noted a significant increase in suburothelial myofibroblast-like cell M2 immunoreactivity in the IDO and PBS groups, and of M3 immunoreactivity in IDO cases with a trend in PBS cases. The pattern of immunostaining M2 and M3 appeared similar. M3 findings in PBS could reflect differences in pathological conditions. The increase in M2 in myofibroblast-like cells in IDO suggests that they may significantly contribute to involuntary detrusor contractions. The role of these myofibroblasts in PBS is still unclear. In other tissues myofibroblasts are important in the inflammatory responses since they are prolific producers of chemokines and cytokines.18 The etiology and pathophysiology of PBS remain obscure. The increase in suburothelial myofibroblast-like cell M2 immunoreactivity may be a part of a larger complex of transmitters that may be involved in the pathogenesis of PBS. An interesting finding in the study was the significant correlation of the urgency score with M2 and M3 immunoreactivity, and of the frequency score with M2 but not with M3 immunoreactivity. Urinary frequency is used as a clinical surrogate of maximum bladder capacity, which anticholinergic drugs have been found to increase.19 Thus, the relationship between M2 and urinary frequency is important because anticholinergic drugs consistently have an effect on this symptom. Urinary urgency is a sensory symptom that has unknown origins.20 It has been proposed that urgency may result from an involuntary detrusor contraction but this is not the case in patients with PBS. This raises the possibility of M2 and M3 being expressed by the sensory innervation of the bladder. These preliminary correlations with clinical scores must be confirmed in further studies with more accurate methods of clinical measurement, including voiding diaries and urodynamic results.
ther investigation of a role in pathophysiology. Clinical studies using selective M2 and M3 antagonists singly and in combination may be necessary to establish the contribution of each receptor to symptomatology and therapeutic effects.
Abbreviations and Acronyms IDO KO M2 M3 OAB PBS PUF
idiopathic detrusor overactivity knockout muscarinic receptor subtype 2 muscarinic receptor subtype 3 overactive bladder syndrome painful bladder syndrome pain, urgency and frequency
REFERENCES 1.
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CONCLUSIONS M2 and M3 have a similar distribution in the human bladder. The increase in M2 and M3 immunostaining in myofibroblast-like cells in clinical bladder syndromes and their correlation with frequency and urgency scores suggest fur-
⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽
13.
Matsui, M., Motomura, D., Karasawa, H., Fujikawa, T., Jiang, J., Komiya, Y. et al: Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M3 subtype. Proc Natl Acad Sci USA, 97: 9579, 2000 Scarpero, H. M. and Dmochowski, R. R.: Muscarinic receptors: what we know. Curr Urol Rep, 4: 421, 2003 Chess-Williams, R.: Muscarinic receptors of the urinary bladder: detrusor, urothelial and prejunctional. Auton Autacoid Pharmacol, 22: 133, 2002 Matsui, M., Motomura, D., Fujikawa, T., Jiang, J., Takahashi, S., Manabe, T. et al: Mice lacking M2 and M3 muscarinic acetylcholine receptors are devoid of cholinergic smooth muscle contractions but still viable. J Neurosci, 22: 10627, 2002 Igawa, Y., Zhang, X., Nishizawa, O., Umeda, M., Iwata, A., Taketo, M. M. et al: Cystometric findings in mice lacking muscarinic M2 or M3 receptors. J Urol, 172: 2460, 2004 Braverman, A. S., Luthin, G. R. and Ruggieri, M. R.: M2 muscarinic receptor contributes to contraction of the denervated rat urinary bladder. Am J Physiol, 275: 1654, 1998 Braverman, A. S. and Ruggieri, M. R., Sr.: Hypertrophy changes the muscarinic receptor subtype mediating bladder contraction from M3 toward M2. Am J Physiol Regul Integr Comp Physiol, 285: 701, 2003 Pontari, M. A., Braverman, A. S. and Ruggieri, M. R., Sr.: The M2 muscarinic receptor mediates in vitro bladder contractions from patients with neurogenic bladder dysfunction. Am J Physiol Regul Integr Comp Physiol, 286: 874, 2004 Abrams, P., Cardozo, L., Fall, M., Griffiths, D., Rosier, P., Ulmsten, U. et al: The standardisation of terminology in lower urinary tract function: report from the Standardisation Sub-Committee of the International Continence Society. Urology, 61: 37, 2003 Payne, C. K., Terai, A. and Komatsu, K.: Research criteria versus clinical criteria for interstitial cystitis. Int J Urol, suppl., 10: S7, 2003 Parsons, C. L., Dell, J., Stanford, E. J., Bullen, M., Kahn, B. S., Waxell, T. et al: Increased prevalence of interstitial cystitis: previously unrecognized urologic and gynecologic cases identified using a new symptom questionnaire and intravesical potassium sensitivity. Urology, 60: 573, 2002 Mansfield, K. J., Liu, L., Mitchelson, F. J., Moore, K. H., Millard, R. J. and Burcher, E.: Muscarinic receptor subtypes in human bladder detrusor and mucosa, studied by radioligand binding and quantitative competitive RT-PCR: changes in ageing. Br J Pharmacol, 144: 1089, 2005 Gillespie, J. I., Harvey, I. J. and Drake, M. J.: Agonist- and nerve-induced phasic activity in the isolated whole bladder
M2 AND M3 IN BLADDER DISORDERS of the guinea pig: evidence for two types of bladder activity. Exp Physiol, 88: 343, 2003 14. Fry, C. H., Ikeda, Y., Harvey, R., Wu, C. and Sui, G. P.: Control of bladder function by peripheral nerves: avenues for novel drug targets. Urology, 63: 24, 2004 15. Wiseman, O. J., Fowler, C. J. and Landon, D. N.: The role of the human bladder lamina propria myofibroblast. BJU Int, 91: 89, 2003 16. Sergeant, G. P., Hollywood, M. A., McCloskey, K. D., Thornbury, K. D. and McHale, N. G.: Specialised pacemaking cells in the rabbit urethra. J Physiol, 526: 359, 2000 17. Hirani, K., Rahmanou, P., Lamba, A., Dostonova, A., Chaliha, C. and Khullar, V.: Does the bladder contractility change
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with anticholinergic therapy in women with detrusor overactivity? Neurourol Urodyn, 23: 594, 2004 Powell, D. W.: Myofibroblasts: paracrine cells important in health and disease. Trans Am Clin Climatol Assoc, 111: 271, 2000 Herbison, P., Hay-Smith, J., Ellis, G. and Moore, K.: Effectiveness of anticholinergic drugs compared with placebo in the treatment of overactive bladder: systematic review. BMJ, 326: 841, 2003 Chapple, C. R., Artibani, W., Cardozo, L. D., Castro-Diaz, D., Craggs, M., Haab, F. et al: The role of urinary urgency and its measurement in the overactive bladder symptom syndrome: current concepts and future prospects. BJU Int, 95: 924, 2005
Purpose: We studied the cellular localization of muscarinic receptor subtypes 2 and 3 in the human bladder and related any changes in overactive and painful bladder syndromes to measures of clinical dysfunction. Materials and Methods: Bladder specimens obtained from patients with painful bladder syndrome (11), idiopathic detrusor overactivity (12) and from controls with asymptomatic microscopic hematuria (16) were immunostained using specific antibodies to muscarinic receptor subtypes 2 and 3, and to vimentin, which is a marker for myofibroblasts. Immunostaining results were quantified with computerized image analysis and correlated with clinical dysfunction using frequency and urgency scores. Results: Muscarinic receptor subtype 2 and 3 immunoreactivity was observed in the urothelium, nerve fibers and detrusor layers. In addition, strong myofibroblast-like cell staining, similar to vimentin, was present in the suburothelial region and detrusor muscle. A significant increase in suburothelial myofibroblast-like muscarinic receptor subtype 2 immunoreactivity was seen in patients with painful bladder syndrome (p ⫽ 0.0062) and idiopathic detrusor overactivity (p ⫽ 0.0002), and in muscarinic receptor subtype 3 immunoreactivity in those with idiopathic detrusor overactivity (p ⫽ 0.0122) with a trend in painful bladder syndrome. Muscarinic receptor subtype 2 and 3 immunoreactivity significantly correlated with the urgency score (p ⫽ 0.0002 and 0.0206, respectively) and muscarinic receptor subtype 2 immunoreactivity correlated with the frequency score (p ⫽ 0.0029). No significant difference was seen in urothelial and detrusor muscarinic receptor subtypes 2 and 3 or vimentin immunostaining. Conclusions: To our knowledge this is the first study to show the cellular localization of muscarinic receptor subtypes 2 and 3 in the human bladder. The increase in muscarinic receptor subtypes 2 and 3 immunostaining in myofibroblast-like cells in clinical bladder syndromes and its correlation with clinical scores suggests a potential role in pathophysiological mechanisms and the therapeutic effect of anti-muscarinic agents. Key Words: bladder; receptors, muscarinic; pain; urinary incontinence
which increases bladder compliance via -adrenoceptor mediated relaxation of the bladder. During the voiding phase acetylcholine is released from parasympathetic nerves. This is thought to activate M2 and produce the inhibition of -adrenoceptor mediated relaxation of the bladder. The result is the reversal of relaxed bladder tone which, concomitant with M3 mediated contraction of the detrusor, provides efficient and complete bladder emptying. Binding and subtype selective immunoprecipitation studies have demonstrated that, while the majority of muscarinic receptors in the bladder are of the M2 subtype, M3 is believed to be the most important for detrusor contraction.2,3 KO mice lacking M3 show prominent bladder distention and have greatly decreased detrusor contraction in vitro.1 The small, direct contractile response that persists in the bladder of M3 KO mice is completely lost in mice lacking M2 and M3, demonstrating that M2 is capable of mediating small contractions and muscarinic receptors other than M2 and M3 do not seem to mediate any direct bladder contraction.4 However, in vivo M3 KO mice were able to empty the bladder and they showed little changes in urodynamic parameters compared to WT controls, illustrating that in rodents the
uscarinic acetylcholine receptors belong to a group of 7 transmembrane spanning receptors that are distributed widely in the central and peripheral nervous systems, where they have key physiological roles.1 Five muscarinic receptor subtypes (M1 to M5) have been cloned and defined pharmacologically.2 Bladder contractions in humans are normally mediated mainly via the stimulation of muscarinic receptors expressed by bladder smooth muscle cells. The bladder has 2 functions, that is storing and emptying urine. During the filling or storage phase the bladder is inhibited by the sympathetic stimulation mediated by noradrenaline released from sympathetic nerves,
M
Submitted for publication August 10, 2005. Study received local ethics committee approval. Supported by GlaxoSmithKline, Harlow, United Kingdom (GM) and Roche Bioscience, Palo Alto, California (YY). * Financial interest and/or other relationship with Pfizer, Yamanouchi, Novartis, Schwarz Pharmaceuticals, Takeda and AstraZeneca. † Correspondence: Peripheral Neuropathy Unit, Area A, Hammersmith Hospital, Du Cane Rd., London, United Kingdom W12 0NN (telephone: 0044 208 383 3309; FAX: 0044 208 383 3363; e-mail: [email protected]).
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noncholinergic component of bladder contraction seems sufficient for voiding contraction.5 Although M2 is thought to have only a minor role in smooth muscle contraction despite its prevalence, there is evidence to suggest that M2 may be an active participant in detrusor contraction in certain pathological states.6,7 Hypertrophied bladders induced by denervation and spinal cord injury in rats showed an increase in total and M2 density, and a change in muscarinic receptor subtype mediating bladder contraction from M3 to M2.7 Similar changes were later demonstrated in human bladder with neurogenic bladder dysfunction.8 PBS is a debilitating, chronic bladder hypersensitivity disorder that typically presents with suprapubic pain related to bladder filling, accompanied by other symptoms, such as increased daytime and nighttime frequency in the absence of a definable etiology.9 OAB is characterized by urinary urgency with or without urge incontinence, usually with frequency and nocturia.9 Determining the distribution and subtype specific functions has been a matter of considerable interest because selective therapeutic agents may have a narrower side effect profile. The presence of muscarinic receptors in the bladder has been shown mainly through indirect techniques, such as reverse transcriptase-polymerase chain reaction, Northern blot, radioligand binding and immunoprecipitation studies.2,3 We report the distribution of M2 and M3 in the human bladder using immunohistochemistry in control subjects, and in patients with OAB and PBS. In addition, we describe the correlation of these receptors with clinical symptoms.
MATERIALS AND METHODS Bladder tissue specimens were obtained from 16 control subjects under investigation for asymptomatic microscopic hematuria, 12 patients with idiopathic detrusor overactivity and 11 with PBS who met National Institute for Diabetes and Digestive and Kidney Diseases research criteria for interstitial cystitis.10 Mean age in controls, patients with IDO and patients with PBS was 53.1 (range 21 to 89), 51.2 (range 32 to 73) and 47.4 years (range 22 to 76), respectively. Approval was given by the local ethics committee and informed consent was obtained from patients and controls. Clinical assessments included history and clinical examination, followed by a midstream urine specimen, culture and cytology, and urodynamics. Symptom scores were recorded on the PUF symptom scale.11 Patients with IDO presented with overactive bladder symptoms, including urgency with or without urge incontinence, frequency and nocturia, and they showed involuntary detrusor contractions during the filling phase of urodynamics.9 All patients with PBS complained of frequency (greater than 5 voids in 12 hours), nocturia (greater than 2 voids), urgency and suprapubic/ pelvic pain without any signs of detrusor overactivity on urodynamics. Flexible or rigid cystoscopic bladder biopsies were obtained from a consistent site just above and lateral to the ureteral orifices. All patients had sterile urine cultures at cystoscopy and biopsy. Human dorsal root ganglion specimens were collected after death (Netherlands Brain Bank, Amsterdam, The Netherlands) with less than an 8-hour delay and stored at ⫺80C until use.
Antibodies Muscarinic receptor subtypes M2 (rabbit polyclonal, H-170) and M3 (rabbit polyclonal, H-210) antibodies (Santa Cruz Biotechnology, Santa Cruz, California) were used at a dilution of 1:300 and 1:500, respectively. Monoclonal antibodies to the human vimentin intermediate filament subunit (NovoCastra, Newcastle upon Tyne, United Kingdom) was used at a dilution of 1:6,000. Immunohistochemistry Frozen sections (12 m) were post-fixed in freshly prepared 4% weight per volume paraformaldehyde in phosphate buffered saline for 20 minutes and dehydrated for a further 20 minutes (hydrogen peroxide). Rabbit and mouse primary antibodies were diluted in goat and horse serum, respectively, (1:30) and incubated overnight. Sites of primary antibody attachment were revealed using the nickel enhanced avidin-biotin peroxidase method. Nuclei were counterstained with 0.1% weight per volume aqueous neutral red. Image Analysis Computerized image analysis was performed to quantify immunoreactivity. Images were captured using an Olympus DP70 camera mounted to an Olympus BX50 microscope and analyzed using analySIS, version 5.0 software (Olympus, Tokyo, Japan). Positive immunostaining was highlighted by setting the gray level detection limits to threshold and the area of highlighted immunoreactivity obtained as percent area of the field scanned. The threshold was kept constant throughout all specimens analyzed. Five fields per tissue section were scanned and the mean value was used for subsequent statistical analysis. The mean values of readings obtained by 2 blinded, independent observers were used for final analysis. The Mann-Whitney test was used for statistical analysis with p ⬍0.05 considered statistically significant. The ratio between M2 and M3 in serial tissue sections was calculated. The results of immunohistochemistry were correlated with frequency and urgency scores using Spearman’s correlation, including all specimens studied. The frequency score was obtained from the PUF questionnaire11 and rated as 0 —3 to 6, 1—7 to 10, 2—11 to 14, 3—15 to 19 and 4 —20 or greater voids daily. Similarly the urgency score was obtained from the PUF questionnaire11 and graded as 0 —no urgency, 1—mild, 2—moderate and 3—severe. RESULTS M2 M2 and M3 immunoreactivity was seen in the urothelium, detrusor layer and nerve fiber bundles (fig. 1, A to C). In addition, strong myofibroblast-like staining, consisting of cell bodies associated with long fiber processes, was present in the suburothelium (figs. 1, A and 2, A to C). M2 staining appeared more frequent and intense in IDO and PBS specimens than in controls (fig. 2, A to C). Image analysis showed a significant increase in suburothelial myofibroblast-like M2 immunoreactivity in the PBS and IDO groups vs controls (mean immunoreactive area ⫾ SEM 2.35% ⫾ 0.55% vs 0.68% ⫾ 0.08%, p ⫽ 0.0062 and 2.21% ⫾ 0.37% vs 0.68% ⫾ 0.08%, p ⫽ 0.0002, respectively, fig. 3, A). No statistically significant change in M2 immunoreactivity was seen in the
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FIG. 1. Representative M2 immunoreactive staining in human bladder section showing staining in detrusor and myofibroblast-like staining (arrows) in suburothelium (A). Representative urothelial M2 immunoreactivity (B). Representative M3 immunoreactive staining reveals detrusor and rare nerve fiber bundle (arrow) (C). Representative M3 immunoreactive staining in human dorsal root ganglion demonstrates staining in subset of small and medium sensory neurons (D). Reduced from ⫻20 (A), ⫻40 (B and D) and ⫻10 (C).
control, IDO and PBS groups in the urothelium (mean immunoreactive area 0.633% ⫾ 0.168%, 0.615% ⫾ 0.234% and 1.641% ⫾ 0.631%) or in the detrusor (1.464% ⫾ 0.431%, 2.333% ⫾ 0.333% and 2.00% ⫾ 0.393%, respectively). Myofibroblast-like cells immunostained in the detrusor were sporadic and could not be image analyzed separately. M3 M3 immunoreactivity was seen in the urothelium and detrusor layer (fig. 1, C). Myofibroblast-like staining similar to M2, was also seen with M3 antibody (fig. 2, E to G). A significant increase in suburothelial myofibroblast-like M3 immunoreactivity was seen in the IDO group vs controls (mean immunoreactive area 1.38% ⫾ 0.35% vs 0.61% ⫾ 0.24%, p ⫽ 0.0122, fig. 3, B). No difference was seen in M3 staining between the control and PBS groups (mean immunoreactive area 0.61% ⫾ 0.24% and 0.76% ⫾ 0.18%, respectively, p ⫽ 0.0717, fig. 3, B). There was no statistically significant difference in M3 immunoreactivity among the control, IDO and PBS groups in the urothelium (mean immunoreactive area 1.20% ⫾ 0.515%, 0.971% ⫾ 0.344% and 1.79% ⫾ 0.889%) or in the detrusor (1.429% ⫾ 0.455%, 1.833% ⫾ 0.358% and 1.643% ⫾ 0.261%, respectively). In addition to urothelial, detrusor and myofibroblast-like staining, M2 and M3 immunoreactivity was also observed in
nerve bundles in some specimens (fig. 1, C). However, it was not observed or it was weak in fine nerve fibers in the suburothelial region. Thus, it was not possible to perform image analysis between the groups. Sensory neurons in human dorsal root ganglia also showed M2 and M3 immunoreactivity (fig. 1, D). There was no significant difference in the M2-to-M3 staining ratio in serial sections among the control, IDO and PBS groups (2.81:1, 3.68:1 and 3.63:1, respectively, control vs IDO p ⫽ 0.8709 and vs PBS p ⫽ 0.3613, fig. 3, C).
Vimentin Staining Vimentin positive cells were characteristic of the description of myofibroblasts as stellate cells with ramifying processes. They were observed in the suburothelium and detrusor layers in all control, IDO and PBS specimens (fig. 2, H). The staining pattern appeared similar to staining seen using muscarinic receptor antibodies (fig. 2, D). On analysis a 2-fold increase in suburothelial vimentin immunoreactivity was seen in the PBS group compared to controls but this did not achieve statistical significance (mean immunoreactive area 7.71% ⫾ 1.32% and 3.93% ⫾ 0.81%, respectively, p ⫽ 0.1014, fig. 3, D). Similarly no significant difference was seen in vimentin staining between the control and IDO
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FIG. 2. Representative myofibroblast-like, M2 immunoreactive staining in suburothelium of control (A), IDO (B) and PBS (C) bladder, and M3 immunoreactive staining in suburothelium of control (E), IDO (F) and PBS (G) bladder. Comparison of M3 staining (D) with vimentin staining (H) shows myofibroblast-like structures in suburothelium. Reduced from ⫻40.
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FIG. 3. Relative mean percent area ⫾ SEM of M2 (A), and M3 (B) immunoreactive myofibroblasts, M2-to-M3 ratio (C) and vimentin staining (D) in 16 control, 12 IDO and 11 PBS preparations.
groups (mean immunoreactive area 3.93% ⫾ 0.81% and 5.22% ⫾ 1.19%, respectively, p ⫽ 0.5350, fig. 3, D). Correlation With Clinical Scores The frequency score correlated significantly with the M2 immunoreactive area (r ⫽ 0.5325, p ⫽ 0.0029, fig. 4, A). However, no correlation was found between M3 staining and
FIG. 4. Correlation of frequency score with M2 (A) and M3 (B) receptor density, and urgency score with M2 (C) and M3 (D) receptor density.
the frequency score (r ⫽ 0.3340, p ⫽ 0.0766, fig. 4, B). The urgency score correlated significantly with M2 and M3 immunoreactive staining (r ⫽ 0.6355, p ⫽ 0.0002 and r ⫽ 0.4279, p ⫽ 0.0206, respectively, fig. 4, C and D).
DISCUSSION In this study M2 and M3 immunoreactivity was seen in the urothelium, nerve fibers and detrusor muscle. In general agreement with other studies3,12 the overall M2-to-M3 immunoreactive staining ratio was 3:1 in the normal (control) bladder, which was preserved in IDO and PBS specimens. However, this ratio was not maintained when the urothelium and detrusor were individually analyzed, which may reflect different affinity and/or avidity of the antibodies or methods used. Stronger suburothelial myofibroblast-like staining was also seen with M2 and M3 receptor antibodies, and it appeared similar to vimentin staining. The suburothelial myofibroblast-like staining seen in this study was in agreement with that noted by Gillespie et al, who reported a similar pattern of M3 immunoreactive spindle-shaped cells in the suburothelium of guinea pig bladder.13 They have an immunochemical feature of staining to vimentin and ␣-smooth muscle actin, and can be identified by their characteristic cell bodies with spindle-like cytoplasmic processes.14,15 Colocalization and ultrastructural studies15 would be helpful to further confirm the cell type expressing the muscarinic receptors. Myofibroblasts have been observed in the upper urinary tract and urethra,16 where they may have a pacemaker role analogous to interstitial cells of Cajal in the gut. Recently the presence of a layer of myofibroblasts has been reported below the urothelium in close proximity to afferent and
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efferent nerve varicosities.15 Due to their location and association with nerve varicosities it has been proposed that these myofibroblasts with associated axonal varicosities could collectively function as a bladder stretch receptor.14 This proposal is supported by our finding of cholinergic receptor expression in these myofibroblasts. It could also at least partly explain why anticholinergic medication increases bladder capacity and decreases the sensation of urgency without impairing detrusor contraction during voiding,17 an observation that is difficult to account for only by the action of anticholinergics on detrusor muscle parasympathetic innervation.15 Further studies are required to assess any immunostaining in sensory nerve fibers that may be relevant in this regard. Immunostaining was seen in large nerve fiber bundles but it was faint or absent in suburothelial fine fibers using these antibodies and, thus, it did not enable quantitative image analysis. M2 expression has previously been noted in sensory neurons of rat dorsal root ganglia and their peripheral endings in skin. In our studies of human dorsal root ganglion sensory neurons using these antibodies we observed M2 and M3 immunoreactivity in a subset of small and medium sensory neurons, suggesting a potential role in sensory mechanisms. We noted a significant increase in suburothelial myofibroblast-like cell M2 immunoreactivity in the IDO and PBS groups, and of M3 immunoreactivity in IDO cases with a trend in PBS cases. The pattern of immunostaining M2 and M3 appeared similar. M3 findings in PBS could reflect differences in pathological conditions. The increase in M2 in myofibroblast-like cells in IDO suggests that they may significantly contribute to involuntary detrusor contractions. The role of these myofibroblasts in PBS is still unclear. In other tissues myofibroblasts are important in the inflammatory responses since they are prolific producers of chemokines and cytokines.18 The etiology and pathophysiology of PBS remain obscure. The increase in suburothelial myofibroblast-like cell M2 immunoreactivity may be a part of a larger complex of transmitters that may be involved in the pathogenesis of PBS. An interesting finding in the study was the significant correlation of the urgency score with M2 and M3 immunoreactivity, and of the frequency score with M2 but not with M3 immunoreactivity. Urinary frequency is used as a clinical surrogate of maximum bladder capacity, which anticholinergic drugs have been found to increase.19 Thus, the relationship between M2 and urinary frequency is important because anticholinergic drugs consistently have an effect on this symptom. Urinary urgency is a sensory symptom that has unknown origins.20 It has been proposed that urgency may result from an involuntary detrusor contraction but this is not the case in patients with PBS. This raises the possibility of M2 and M3 being expressed by the sensory innervation of the bladder. These preliminary correlations with clinical scores must be confirmed in further studies with more accurate methods of clinical measurement, including voiding diaries and urodynamic results.
ther investigation of a role in pathophysiology. Clinical studies using selective M2 and M3 antagonists singly and in combination may be necessary to establish the contribution of each receptor to symptomatology and therapeutic effects.
Abbreviations and Acronyms IDO KO M2 M3 OAB PBS PUF
idiopathic detrusor overactivity knockout muscarinic receptor subtype 2 muscarinic receptor subtype 3 overactive bladder syndrome painful bladder syndrome pain, urgency and frequency
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CONCLUSIONS M2 and M3 have a similar distribution in the human bladder. The increase in M2 and M3 immunostaining in myofibroblast-like cells in clinical bladder syndromes and their correlation with frequency and urgency scores suggest fur-
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