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M3 Muscarinic Receptor-Like Immunoreactivity in Sham Operated and Obstructed Guinea Pig Bladders
M3 Muscarinic Receptor-Like Immunoreactivity in Sham Operated and Obstructed Guinea Pig Bladders Simone Grol, Christopher John Nile, Piluca Martinez-Martinez, Gommert van Koeveringe,* Stefan de Wachter, Jan de Vente and James I. Gillespie† From the Department of Urology, University Hospital Maastricht (SG) and Department of Psychiatry and Neuropsychology, European Graduate School of Neuroscience, Maastricht University (JdV, JIG), Maastricht (GAvK, SDW), The Netherlands, Uro-physiology Research Group, Medical and Dental School, Newcastle University (JIG), Newcastle upon Tyne and Infection and Immunity Research Group, University of Glasgow Dental School (CJN), Glasgow, United Kingdom
Purpose: Type 3 muscarinic receptors, which are present in the bladder wall, are important for bladder function. However, their role in the context of the urothelium is not well defined. Understanding the role of type 3 muscarinic receptors has been limited by the lack of specific type 3 muscarinic receptor antibodies. Thus, we identified a specific type 3 muscarinic receptor antibody and investigated the site of type 3 muscarinic receptors in sham operated and obstructed guinea pig bladders. Materials and Methods: The specificity of 4 commercially available type 3 muscarinic receptor antibodies was determined. Immunohistochemistry was then done in bladder tissue from sham operated and obstructed guinea pig bladders. Results: One of the 4 antibodies examined had the needed specificity in terms of blocking peptide and Western blot characterization. Using this antibody type 3 muscarinic receptor immunoreactivity was associated with muscle cells, nerves and interstitial cells. Four types of interstitial cells were identified, including suburothelial, lamina propria, surface muscle and intramuscular interstitial cells. In the obstructed model the bladder wall was hypertrophied and there was nerve fiber loss. The number of lamina propria, surface muscle and intramuscular interstitial cells was increased but not the number of suburothelial interstitial cells. Also, surface muscle interstitial cells appeared to form clusters or nodes with type 3 muscarinic receptor immunoreactivity. Conclusions: Nerve loss and the up-regulation of interstitial cells with type 3 muscarinic receptor immunoreactivity may underlie major functional changes in the pathological bladder. This indicates that type 3 muscarinic receptor specific anticholinergic drugs may affect not only the detrusor muscle, as previously thought, but also interstitial cells and nerve fibers.
Abbreviations and Acronyms cGMP ⫽ cyclic guanosine monophosphate IC ⫽ interstitial cell IR ⫽ immunoreactivity LP-IC ⫽ lamina propria IC M3 ⫽ type 3 muscarinic receptor NO ⫽ nitric oxide PGP ⫽ protein gene product SM-IC ⫽ smooth muscle IC SU-IC ⫽ suburothelial IC Submitted for publication April 12, 2010. Study received Maastricht University institutional animal care and use committee approval. Supported by the British Journal of Urology International Collaborative Research Award 2007 (JIG, GAvK). * Financial interest and/or other relationship with Astellas and Allergan. † Correspondence: Uro-physiology Research Group, Medical and Dental School, Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom (telephone: 44 191 222 6988; FAX: 44 191 222 6988; e-mail: j.i.gillespie@ ncl.ac.uk).
See Editorial on page 1562.
Key Words: urinary bladder; urothelium; urinary bladder neck obstruction; receptors, muscarinic; guinea pigs BLADDER dysfunction was thought to be associated with the M3 receptor activation of involuntary bladder contractions. On this basis M3 specific anticholinergic drugs were developed
to treat bladder symptoms. These drugs are clinically effective1 but at therapeutic doses they do not affect bladder contraction.2– 4 This has led to the suggestion that they may act on
0022-5347/11/1855-1959/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
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sensory mechanisms operating during the filling phase. To develop this study we need to know the cellular locations of M3 receptors in normal and pathological bladders. Recent studies using antibodies to locate types 2 and 3 muscarinic receptors described different patterns of staining. Some groups suggested that they are found on ICs in the lamina propria,5,6 muscle,7 urothelium8 or smooth muscle.9 An explanation for such different findings may be the specificity of the antibodies used. Muscarinic antibodies are problematic10 –12 and, thus, all observations must be regarded with caution. This has led to some debate in regard to the hard criteria that must be applied to ensure antibody specificity.10 We determined the specificity of 4 commercial M3 receptor antibodies, including goat anti-M3 antibody SC7474 (1:300), rabbit anti-M3 antibody SC9108 (Santa Cruz Biotechnology, Santa Cruz, California) (1:300), rabbit anti-M3 antibody 13063 (Abcam®) (1:300) and rabbit anti-M3 antibody A5259 (LifeSpan BioSciences, Seattle, Washington) (1:300). Using the most specific of this panel (goat anti-M3 antibody SC7474) the cellular locations of M3 receptors were studied in the bladders of sham operated and obstructed guinea pigs.
MATERIALS AND METHODS Animals Studies were performed in 14 male Hartley guinea pigs weighing between 266 and 299 gm. All procedures were approved by the Maastricht University institutional animal care and use committee, and were in line with European Commission guidelines.
Surgical Procedures The procedures used to create bladder obstruction were previously reported in detail.13 In 8 animals partial outflow obstruction was induced while 6 underwent sham operation. Four weeks postoperatively the guinea pigs were sacrificed by cervical dislocation.
Immunohistochemistry Immunohistochemistry was done as described previously.5 The primary antibodies used were goat anti-M3 antibody SC7474 (1:300), rabbit anti-M3 antibody SC9108 (1:300), rabbit anti-M3 antibody 13063 (1:300), rabbit anti-M3 antibody A5259 (1:300), rabbit anti-PGP antibody (Biogenesis®) (1:1,000), rabbit anti-choline acetyltransferase antibody (1:100), mouse anti-vimentin antibody (1:1,000) and calcitonin gene-related peptide (Chemicon®) (1:200). Goat primary antibodies were visualized using donkey anti-goatCY3 conjugate IgG (Jackson ImmunoResearch, West Grove, Pennsylvania) (1:100). Rabbit primary antibodies were visualized with Alexa Fluor® 594 donkey anti-rabbit IgG conjugate (1:100). Anti-M3 antibodies were pre-absorbed by incubating the antibody overnight at 4C with 10 g/ml of the appropriate peptide (SC7474P and SC9108P). Sections were analyzed and photographed using an AX70 microscope equipped with a cooled charged coupled device digital video camera (Olympus®).
Cell Culture, Transfection and Preparation of Protein Extracts HEK-293 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 2 mM L-glutamine, 10% (volume per volume) fetal bovine serum and penicillin (100 U/ml)/streptomycin sulfate (0.1 mg/ml) at 37C in a humidified 5% CO2 environment. Transfection was performed for 15 hours using the ProFection® Mammalian Transfection System-Calcium Phosphate according to manufacturer recommendations. The DNA used was open reading frame clone of Homo sapiens cholinergic receptor, muscarinic 3 (OriGene, Rockville, Maryland). To obtain cell extracts the tissue or cell suspensions were rinsed with ice-cold phosphate buffered saline and homogenized on an ice bed with 25 mM tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride and 10 g/ml leupeptin. Mixtures were cleared by centrifugation at 500 ⫻ gravity for 10 minutes and stored at ⫺70C.
Western Blot Western blots were made under reducing conditions according to standard procedures using the Odyssey® Infrared Imaging System. Primary antibodies used for Western blot were goat anti-M3 antibody SC7474 (1:500), rabbit anti-M3 antibody SC9108 (1:500) and rabbit anti-M3 antibody 13063 (1:500). Secondary antibodies used were donkey anti-goat 926-32214 IRdye® 800 (1:10,000), and donkey anti rabbit 611-732-127 IRdye 800 (1:10,000).
Tissue Preparation The bladder was removed and placed in ice-cold KrebsHenseleit buffer (Sigma-Aldrich®). The lateral wall was dissected and incubated in Krebs solution containing 1 mM of the nonspecific phosphodiesterase inhibitor isobutylmethyl-xanthine (Sigma-Aldrich) at 36C for 30 minutes. Incubation was terminated by immersing in an ice-cold fixative solution of 4% freshly prepared depolymerized paraformaldehyde for 120 minutes at 4C. The tissues were then washed at 4C in 0.1 M phosphate buffer containing 10%, 20% and 30% sucrose, each for 24 hours, and snap frozen with CO2 in Tissue-Tek® O.C.T.™ compound. Cryostat sections (10 m) were cut before thawing on chromealum-gelatin coated slides.
RESULTS Antibody Specificity and M3 IR To identify a specific antibody for M3 receptor we investigated 4 commercially available M3 receptor antibodies. Rabbit anti-M3 antibody A5259 revealed no staining (data not shown). However, goat anti-M3 antibody SC7474, rabbit anti-M3 antibody SC9108 and rabbit anti-M3 antibody 13063 showed M3 staining in the guinea pig bladder urothelium, suburothelium, lamina propria and inner muscle. Figure 1, a shows an example of staining with goat anti-M3
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Figure 1. M3 IR localization in normal guinea pig bladder lamina propria and inner muscle. Luminal surface section of bladder wall stained with SC7474 goat anti-M3 antibody (a). uro, urothelium. M3 IR in suburothelium at higher magnification using SC7474 goat anti-M3 antibody (b), 13063 rabbit anti-M3 antibody (d) and SC9108 rabbit anti-M3 antibody (f ). SU-IC processes and somata stained for M3 receptor. M3 IR in inner muscle bundles stained with SC7474 goat anti-M3 antibody (c), 13063 rabbit anti-M3 antibody (e) and SC9108 rabbit anti-M3 antibody (g). Scale bars indicate 40 (a) and 10 (b to g) m.
antibody. At higher magnification and gain all 3 antibodies revealed M3 IR in the stomata and processes of SU-ICs (Fig. 1, b, d and f). M3 IR was also observed in cells in the inner muscle bundle (Fig. 1, c, e and g). Vimentin, a general marker for cells in the interstitium, was used to identify these cells, which are a heterogeneous population and were formally described as fibroblasts. However, approaches such as this allow descriptions of the possible functional, better subclassification of individual cells of the interstitium, that is ICs. Of the 3 antibodies that showed M3 IR goat anti-M3 antibody SC7474 and rabbit anti-M3 antibody SC9108 showed no M3 staining when preincubated with the appropriate blocking peptide (fig. 2, a and b). Since the antigenic peptide of the third antibody, 13063, was not commercially available and the sequence was not made public by the manufacturer, pre-absorption studies were not done. As a control, immunohistochemical analysis using rabbit anti-vimentin antibodies pre-incubated with M3 blocking peptides showed normal vimentin staining (data not shown). Therefore, the antigenic peptides were specific for the M3 antibodies. The 3 antibodies showing staining were
further characterized by Western blot analysis of M3 transfected HEK-293 cell homogenates (figs. 1 and 2, c). Rabbit anti-M3 antibody 13063 did not reveal any bands upon Western blot analysis of transfected HEK-293 protein homogenates. Rabbit anti-M3 antibody SC9108 showed 2 bands with a molecular weight of about 45 and about 65 kDa, respectively. However, goat anti-M3 antibody SC7474 showed a single band of about 102 kDa, which is the molecular weight of the M3 receptor reported in previous Western blot studies.14,15 Untransfected HEK-293 revealed no band with any tested antibody. Notably not all antibodies are equally suitable for Western blot analysis and immunohistochemistry. For complete antibody characterization we accept that sophisticated technologies must be used, such as knockout animals.10 However, blocking peptide studies and Western blot analysis of M3 receptor transfected HEK-293 cells provided enough evidence to validate the adequate specificity of goat anti-M3 antibody Sc7474. Thus, we used this antibody to study M3 receptor localization in the guinea pig bladder.
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Animals with bladder neck damage. The mean ⫾ SD weight gain in sham operated and obstructed animals was 190 ⫾ 52 and 112 ⫾ 56 gm, respectively. Mean bladder weight in sham operated and obstructed animals was 400 ⫾ 73 and 1,568 ⫾ 500 mg, respectively. Obstructed bladders showed increased wall thickness (fig. 6). There were also major differences in the number and distribution of ICs, and increased IC density in the lamina propria and surrounding the muscle bundles, particularly the outer muscle layers. These cells demonstrated increased M3 IR (figs. 6 to 8). Little difference was noted in the SU-IC population but there was a higher density of LP-ICs. LP-ICs also showed M3 IR and appeared to extend complex bifurcating processes that formed a continuous network down to the SM-ICs (fig. 7). In obstructed bladders there was high IC density in the outer muscle layer (fig. 8). Cell bodies of these
Figure 2. Specificity of 3 commercially available anti-M3 receptor antibodies. Sections lacked M3 IR after staining with SC9108 rabbit anti-M3 antibody (a) and SC7474 goat anti-M3 antibody (b) after pre-incubation with respective blocking peptides. Western blot analysis of protein homogenates from control untransfected HEK-293 cells (C) and HEK-293 cells transfected with M3 receptor (T) (c). Blots were stained with SC7474 goat anti-M3 antibody, 13063 rabbit anti-M3 antibody and SC9108 rabbit anti-M3 antibody. SC, Santa Clara Biotechnology. Abc, Abcam.
Bladders Sham operated controls. M3 IR was located in the urothelium, suburothelium, lamina propria and the muscle layers of sham operated guinea pig bladders. M3 IR was associated with a population of cells termed SU-ICs in 2 isolated sham operated guinea pig bladders (fig. 3, a and d). SU-ICs formed a dense cell layer in the suburothelium. M3 IR was also associated with a less sparse population of cells termed LP-ICs. In different bladder regions the number of M3 IR cells varied (fig. 3, b and c). Studying ICs at higher magnification revealed M3 IR located specifically in the region of IC bodies (fig. 3, b, c, e and f ). Two nerve fiber populations were identified in the suburothelial space, including M3 IR nerves and nerves showing no M3 IR (fig. 4, a and b). In the muscle layers M3 IR was associated with smooth muscle cells, some nerve fibers and ICs (fig. 4, c to e). M3 IR was also found on SM-ICs (figs. 4 and 5). In addition, in 3 of the 7 sham operated control bladders studied these SM-ICs were in close apposition to muscle bundles in collections of the cell bodies that de Jongh et al first termed nodes (fig. 5).16
Figure 3. M3 IR localization in guinea pig bladder suburothelium and lamina propria (lp) in sections double labeled with antibodies to M3 receptor (red areas) and vimentin (vim) (green areas). Lumen, urothelium (uro), and suburothelial and inner muscle (iml) layers were identified (a). In same regions M3 IR (b) and vimentin staining (c) reveal variations in M3 IR density in nearby bladder wall regions with fewer (b) vs more (c) M3 IR cells. Section shows urothelium from different bladder (d). Note regions (d) at higher magnification as merged individual M3 IR and vimentin images (e and f ). Scale bars indicate 120 (a and d), 75 (b, c and e), and 15 (f ) m.
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conflicting results.5– 8 Previous attempts to characterize commercially available M3 receptor antibodies failed to identify any as providing specific staining.10 –12 Using the rabbit anti-M3 receptor antibody SC9108 used in our study Jositsch et al noted nonspecific staining in tissue from M3 receptor knockout mice.10 Although we observed what we may call a specific pattern of staining with the same antibody and the disappearance of a signal in pre-absorption experiments (fig. 1), our Western blot analysis revealed more than 1 band of the incorrect molecular weight (fig. 2). Thus, in agreement with Jositsch et al, this calls into question the specificity of this antibody. It also confirms that pre-absorption studies alone are not suitable to characterize specificity.
Figure 4. M3 IR localization in guinea pig bladder lamina propria and muscle layers. Sections of different bladders were stained with antibodies to M3 receptor (red areas) and nonspecific nerve marker PGP 9.5 (green areas) (a and b). Note M3 IR (asterisks) and PGP 9.5 gray value images, including lamina propria nerve fibers on PGP 9.5 staining. Pound sign indicates fiber that is not M3 IR. Section from outer muscle layer shows easily recognized nerve fibers (asterisks) and M3 IR associated with smooth muscle cells (pound signs) (c). Regions (c) are shown with M3 IR and PGP 9.5 results (d and e). Scale bars represent 120 (a and b), 40 (c) and 50 (d and e) m.
SM-ICs are found in nodes and show M3 IR.16 ICs were also apparent in the muscle bundles. There are regions of the bladder wall where trabeculae can be identified, which usually comprise a high density of ICs. Such trabeculae were often found in close opposition to others with few ICs (fig. 8). Intramuscular ICs showed M3 IR and extended processes to SM-ICs (fig. 8).
DISCUSSION The basic problem using an immunohistochemical approach for M3 receptor localization is the lack of well characterized antibodies. A number of studies of the M3 receptor location in the bladder provide
Figure 5. M3 IR localization in guinea pig bladder muscle ICs. Section of bladder wall inner layer immunostained with antibodies to vimentin (vim) (green areas) and M3 receptor (red areas) (a). M3 IR staining was noted on muscle cells in punctuate manner (asterisk). uro, urothelium. Two regions at higher magnification show vimentin and M3 images (b and c). Section from bladder wall outer layer (d) demonstrates 2 regions. Two cells (arrows) lying on muscle bundle surface show M3 IR and vimentin staining at higher magnification (e). Note small IC cluster or node (arrows) with M3 IR located in central regions of vimentin positive cells (f ). Scale bars indicate 60 (a and d), 30 (b) and 15 (c, e and f ) m.
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smooth muscle cells was to be expected. For nerve fibers M3 IR appears to be associated with a nerve population in the suburothelial space and the muscle layer. At least 2 types of suburothelial sensory fibers have been identified in the guinea pig bladder, including choline acetyltransferase positive fibers and calcitonin gene-related peptide/substance P/neurofilament positive fibers.17 The presence of nerves expressing M3 IR indicates possible regulation by cholinergic stimuli. To our knowledge the physiological source of the acetylcholine that may activate these nerves is not known to date. It may come from the cholinergic nerves as a form of auto-excitation or inhibition. Alternatively the acetylcholine may orig-
Figure 6. M3 receptor localization in full-thickness sections of sham operated guinea pig bladder (a) and bladder with previous surgical intervention to restrict bladder neck (b). Sections were immunostained with antibodies against M3 receptor (red areas) and vimentin (vim) (green areas). Note individual images of M3 IR and vimentin staining. Arrows indicate lumen, serosa, urothelium (uro), SU-ICs and lamina propria (lp). Note inner (iml) and outer (oml) muscle layers in bladder neck of operated animals. Scale bars indicate 110 (a) and 100 (b) m.
Therefore, we used 2 criteria, including the combination of a lack of signal after pre-incubation with a blocking peptide and the presence of a single band of the correct molecular weight after Western blot analysis of homogenates from HEK-293 cells transfected with M3 receptor. Using these criteria only goat anti-M3 receptor antibody SC7474 showed specific M3 IR. In pre-absorption studies with its blocking peptide any staining from this antibody was abolished and Western blot analysis revealed a single band with a molecular weight of about 102 kDa. Although the amino acid backbone of the human M3 receptor has a theoretical molecular weight of 66 kDa according to the National Center for Biotechnology Information UniGene database, the protein is highly modified post-translationally with an actual molecular weight of 102 kDa.14,15 This was confirmed by labeling studies using the muscarinic ligand [3H]propylbenzilycholine mustard14 and in a previous report using goat anti-M3 receptor antibody SC7474.15 Although we accept that our specificity validations were not as complete as previously reported methods10 and not all antibodies are equally suitable for Western blot and immunohistochemistry, based on these results we decided that goat anti-M3 antibody was specific and suitable for studying M3 receptor localization in the guinea pig. In the normal bladder M3 IR is associated with the smooth muscle cells, nerves and ICs. M3 IR on
Figure 7. M3 IR localization in obstructed guinea pig bladder urothelium (uro), suburothelium, lamina propria and inner muscle (iml). Sections were immunostained with antibodies to vimentin (vim) (green areas) and M3 receptor (red areas). Note gray value images of individual staining results. M3 IR was seen in structures within suburothelial cell layer and in lamina propria (a). Note region of lamina propria from different bladder (b) and same region at higher magnification (c). Network of vimentin positive cells was lying over surface of muscle bundles in mid muscle layer (d). Vimentin and M3 IR were also seen in structures within muscle bundles (plus signs). Regions (d) at higher magnification (e) and (f ) show vimentin and M3 IR images, including clear M3 IR localization to cell bodies of vimentin positive structures (pound signs). Pound signs indicate M3 IR positive structures. Scale bars indicate 90 (a and d), 50 (b and e), 20 (c) and 15 (f ) m.
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Figure 8. M3 IR localization in obstructed guinea pig bladder outer muscle layer. Sections were double immunostained using antibodies against vimentin (vim) (green areas) and M3 receptor (red areas) (a, b and e). Note regions (a, b and e) at higher magnification with individual images of vimentin and M3 IR results (c, d, f and g). There were 2 categories of muscle bundle, including those with numerous intramuscular ICs (plus signs) and those with few intestinal cells (pound signs) (e). Note examples of intramuscular ICs that were M3 IR (f and g). Scale bars indicate 80 (a, b and e) and 15 (c, d, f and g) m.
inate from other structures, such as the urothelium. Indeed, upon stretch the human bladder urothelium releases acetylcholine.18 Thus, acetylcholine released from the urothelium upon stretch may modulate afferent nerve activity. However, perhaps the most unexpected observation in the sham operated bladder was M3 IR associated with SU-ICs and SM-ICs. To our knowledge the functional role of SU-ICs is not known. These cells demonstrate an NO dependent increase in cGMP.16,19,20 The basal layer of the urothelium expresses neuronal NO synthase and so can produce NO.21 The finding of M3 IR on SU-IC-like cells suggests that these cells may respond to urothelial derived signals. In the normal bladder there is a network of SM-ICs.22,23 In the outer muscle layer these cells respond to NO with a increase in cGMP.6,20,24
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The presence of M3 IR on this network suggests the possibility of a cholinergic regulation of their function. Whole bladder experiments revealed cholinergic mechanisms involved in the activation of phasic activity, which may be controlled by a cholinergic driven pacemaker mechanism.4,20 M3 IR on ICs may represent the pacemaker element or regulation of the distributive element. Phasic activity occurs in the normal bladder in vivo as nonmicturition activity. Such activity was suggested to have a central role in a motor/sensory system involved in the generation and regulation of bladder afferent nerve activity and sensation.25,26 Evidence is accumulating to link cholinergic driven ICs with phasic activity and phasic activity with the generation of sensation. Thus, ICs may have a key role in bladder sensory mechanisms. Obstructed bladders showed distinct differences compared to sham operated control bladders. There was little change in the SU-IC network but the number of LP-ICs and muscle ICs increased dramatically in the obstructed bladder model. The finding of M3 IR allows us to hypothesize that these cells can respond to acetylcholine released from the urothelium, which suggests that cellular cross-talk can occur between the urothelium and SU-ICs. In addition, the network of LP-ICs that lie more deeply and extend inward from the SU-IC layer and the SM-ICs also showed M3 IR and underwent hypertrophy in the obstructed bladder model. Therefore, we propose that signals derived from the urothelium in response to an insult such as over distention activate a signaling cascade through the interlinked network of ICs, resulting in the pathological changes observed in the obstructed bladder model. To our knowledge the exact signaling mechanisms involved in this response to danger remain unknown. However, preliminary data show that ICs express cytokines and growth factors such as tumor necrosis factor-␣, interleukin-1 and TGF- as well as their respective receptors (Nile and Gillespie, unpublished data). In the obstructed animals there was an increase in the number of ICs. These cells showed M3 IR and, thus, may be regulated by cholinergic mechanisms. Recently de Jongh et al described cGMP positive ICs in the outer muscle layers of obstructed bladders and in some cases these cells were seen in nodes associated with nerve fibers.16 Thus, it is reasonable to hypothesize that these nodes are innervated and possibly activate the cholinergic receptors on SM-ICs. The altered number and distribution of M3 IR muscle ICs may be linked to functional changes. Recently it was reported that isolated obstructed bladders demonstrate large phasic contractions and have increased sensitivity to cholinergic stimuli.27 Since ICs may be involved in
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phasic activity generation and sensation modulation, they may have a role in the increased sensation of urgency and frequency.
CONCLUSIONS Observations indicate that ICs may be regulated by cholinergic mechanisms. This increased activity could contribute to increased motor/sensory
activity. Evidence is also beginning to accumulate for a secondary role of ICs in coordinating urothelial derived signals, showing cellular cross-talk and responding to dangerous insults such as partial bladder flow output obstruction. Since the 2 systems have a cholinergic component that is upregulated by pathological conditions, it is reasonable to hypothesize that this is a therapeutic target for anticholinergic drugs.
REFERENCES 1. Abrams P and Andersson KE: Muscarinic receptor antagonists for overactive bladder. BJU Int 2007; 100: 987. 2. Andersson KE and Yoshida M: Antimuscarinics and the overactive detrusor–which is the main mechanism of action? Eur Urol 2003; 43: 1. 3. Andersson KE and Arner A: Urinary bladder contraction and relaxation: physiology and pathophysiology. Physiol Rev 2004; 84: 935. 4. Finney SM, Andersson KE, Gillespie JI et al: Antimuscarinic drugs in detrusor overactivity and the overactive bladder syndrome: motor or sensory actions? BJU Int 2006; 98: 503. 5. Grol S, Essers PB, van Koeveringe GA et al: M(3) muscarinic receptor expression on suburothelial interstitial cells. BJU Int 2009; 104: 398. 6. Gillespie JI, Markerink-van Ittersum M and De Vente J: Interstitial cells and cholinergic signalling in the outer muscle layers of the guinea-pig bladder. BJU Int 2006; 97: 379. 7. Mukerji G, Yiangou Y, Grogono J et al: Localization of M2 and M3 muscarinic receptors in human bladder disorders and their clinical correlations. J Urol 2006; 176: 367. 8. Bschleipfer T, Schukowski K, Weidner W et al: Expression and distribution of cholinergic receptors in the human urothelium. Life Sci 2007; 80: 2303. 9. Grol S, van Koeveringe GA, de Vente J et al: Regional differences in sensory innervation and suburothelial interstitial cells in the bladder neck and urethra. BJU Int 2008; 102: 870. 10. Jositsch G, Papadakis T, Haberberger RV et al: Suitability of muscarinic acetylcholine receptor antibodies for immunohistochemistry evaluated on tissue sections of receptor gene-deficient
mice. Naunyn Schmiedebergs Arch Pharmacol 2009; 379: 389.
triphosphate release during aging. Urology 2004; 63: 17.
11. Pradidarcheep W, Labruyere WT, Dabhoiwala NF et al: Lack of specificity of commercially available antisera: better specifications needed. J Histochem Cytochem 2008; 56: 1099.
19. Gillespie JI, Markerink-van Ittersum M and De Vente J: Endogenous nitric oxide/cGMP signalling in the guinea pig bladder: evidence for distinct populations of sub-urothelial interstitial cells. Cell Tissue Res 2006; 325: 325.
12. Pradidarcheep W, Stallen J, Labruyere WT et al: Lack of specificity of commercially available antisera against muscarinergic and adrenergic receptors. Naunyn Schmiedebergs Arch Pharmacol 2009; 379: 397. 13. Mostwin JL, Karim OM, van Koeveringe G et al: The guinea pig as a model of gradual urethral obstruction. J Urol 1991; 145: 854. 14. Tobin AB and Nahorski SR: Rapid agonist-mediated phosphorylation of m3-muscarinic receptors revealed by immunoprecipitation. J Biol Chem 1993; 268: 9817. 15. Dawson LJ, Allison HE, Stanbury J et al: Putative anti-muscarinic antibodies cannot be detected in patients with primary Sjögren’s syndrome using conventional immunological approaches. Rheumatology (Oxford) 2004; 43: 1488. 16. de Jongh R, van Koeveringe GA, van Kerrebroeck PE et al: Alterations to network of NO/cGMPresponsive interstitial cells induced by outlet obstruction in guinea-pig bladder. Cell Tissue Res 2007; 330: 147. 17. Gillespie JI, Markerink-van Ittersum M and de Vente J: Sensory collaterals, intramural ganglia and motor nerves in the guinea-pig bladder: evidence for intramural neural circuits. Cell Tissue Res 2006; 325: 33. 18. Yoshida M, Miyamae K, Iwashita H et al: Management of detrusor dysfunction in the elderly: changes in acetylcholine and adenosine
20. Gillespie JI, Markerink-van Ittersum M and de Vente J: cGMP-generating cells in the bladder wall: identification of distinct networks of interstitial cells. BJU Int 2004; 94: 1114. 21. Gillespie JI, Markerink-van Ittersum M and de Vente J: Expression of neuronal nitric oxide synthase (nNOS) and nitric-oxide-induced changes in cGMP in the urothelial layer of the guinea pig bladder. Cell Tissue Res 2005; 321: 341. 22. Brading AF and McCloskey KD: Mechanisms of disease: specialized interstitial cells of the urinary tract—an assessment of current knowledge. Nat Clin Pract Urol 2005; 2: 546. 23. Drake MJ, Fry CH and Eyden B: Structural characterization of myofibroblasts in the bladder. BJU Int 2006; 97: 29. 24. Lagou M, Drake MJ, Markerink-Van Ittersum M et al: Interstitial cells and phasic activity in the isolated mouse bladder. BJU Int 2006; 98: 643. 25. Starling E: Elements of Human Physiology, 7th ed. London: Spottiswoode 1905. 26. Gillespie JI: A developing view of the origins of urgency: the importance of animal models. BJU Int, suppl., 2005; 96: 22. 27. de Jongh R, van Koeveringe GA, van Kerrebroeck PE et al: Damage to the bladder neck alters autonomous activity and its sensitivity to cholinergic agonists. BJU Int 2007; 100: 919.
Purpose: Type 3 muscarinic receptors, which are present in the bladder wall, are important for bladder function. However, their role in the context of the urothelium is not well defined. Understanding the role of type 3 muscarinic receptors has been limited by the lack of specific type 3 muscarinic receptor antibodies. Thus, we identified a specific type 3 muscarinic receptor antibody and investigated the site of type 3 muscarinic receptors in sham operated and obstructed guinea pig bladders. Materials and Methods: The specificity of 4 commercially available type 3 muscarinic receptor antibodies was determined. Immunohistochemistry was then done in bladder tissue from sham operated and obstructed guinea pig bladders. Results: One of the 4 antibodies examined had the needed specificity in terms of blocking peptide and Western blot characterization. Using this antibody type 3 muscarinic receptor immunoreactivity was associated with muscle cells, nerves and interstitial cells. Four types of interstitial cells were identified, including suburothelial, lamina propria, surface muscle and intramuscular interstitial cells. In the obstructed model the bladder wall was hypertrophied and there was nerve fiber loss. The number of lamina propria, surface muscle and intramuscular interstitial cells was increased but not the number of suburothelial interstitial cells. Also, surface muscle interstitial cells appeared to form clusters or nodes with type 3 muscarinic receptor immunoreactivity. Conclusions: Nerve loss and the up-regulation of interstitial cells with type 3 muscarinic receptor immunoreactivity may underlie major functional changes in the pathological bladder. This indicates that type 3 muscarinic receptor specific anticholinergic drugs may affect not only the detrusor muscle, as previously thought, but also interstitial cells and nerve fibers.
Abbreviations and Acronyms cGMP ⫽ cyclic guanosine monophosphate IC ⫽ interstitial cell IR ⫽ immunoreactivity LP-IC ⫽ lamina propria IC M3 ⫽ type 3 muscarinic receptor NO ⫽ nitric oxide PGP ⫽ protein gene product SM-IC ⫽ smooth muscle IC SU-IC ⫽ suburothelial IC Submitted for publication April 12, 2010. Study received Maastricht University institutional animal care and use committee approval. Supported by the British Journal of Urology International Collaborative Research Award 2007 (JIG, GAvK). * Financial interest and/or other relationship with Astellas and Allergan. † Correspondence: Uro-physiology Research Group, Medical and Dental School, Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom (telephone: 44 191 222 6988; FAX: 44 191 222 6988; e-mail: j.i.gillespie@ ncl.ac.uk).
See Editorial on page 1562.
Key Words: urinary bladder; urothelium; urinary bladder neck obstruction; receptors, muscarinic; guinea pigs BLADDER dysfunction was thought to be associated with the M3 receptor activation of involuntary bladder contractions. On this basis M3 specific anticholinergic drugs were developed
to treat bladder symptoms. These drugs are clinically effective1 but at therapeutic doses they do not affect bladder contraction.2– 4 This has led to the suggestion that they may act on
0022-5347/11/1855-1959/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
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sensory mechanisms operating during the filling phase. To develop this study we need to know the cellular locations of M3 receptors in normal and pathological bladders. Recent studies using antibodies to locate types 2 and 3 muscarinic receptors described different patterns of staining. Some groups suggested that they are found on ICs in the lamina propria,5,6 muscle,7 urothelium8 or smooth muscle.9 An explanation for such different findings may be the specificity of the antibodies used. Muscarinic antibodies are problematic10 –12 and, thus, all observations must be regarded with caution. This has led to some debate in regard to the hard criteria that must be applied to ensure antibody specificity.10 We determined the specificity of 4 commercial M3 receptor antibodies, including goat anti-M3 antibody SC7474 (1:300), rabbit anti-M3 antibody SC9108 (Santa Cruz Biotechnology, Santa Cruz, California) (1:300), rabbit anti-M3 antibody 13063 (Abcam®) (1:300) and rabbit anti-M3 antibody A5259 (LifeSpan BioSciences, Seattle, Washington) (1:300). Using the most specific of this panel (goat anti-M3 antibody SC7474) the cellular locations of M3 receptors were studied in the bladders of sham operated and obstructed guinea pigs.
MATERIALS AND METHODS Animals Studies were performed in 14 male Hartley guinea pigs weighing between 266 and 299 gm. All procedures were approved by the Maastricht University institutional animal care and use committee, and were in line with European Commission guidelines.
Surgical Procedures The procedures used to create bladder obstruction were previously reported in detail.13 In 8 animals partial outflow obstruction was induced while 6 underwent sham operation. Four weeks postoperatively the guinea pigs were sacrificed by cervical dislocation.
Immunohistochemistry Immunohistochemistry was done as described previously.5 The primary antibodies used were goat anti-M3 antibody SC7474 (1:300), rabbit anti-M3 antibody SC9108 (1:300), rabbit anti-M3 antibody 13063 (1:300), rabbit anti-M3 antibody A5259 (1:300), rabbit anti-PGP antibody (Biogenesis®) (1:1,000), rabbit anti-choline acetyltransferase antibody (1:100), mouse anti-vimentin antibody (1:1,000) and calcitonin gene-related peptide (Chemicon®) (1:200). Goat primary antibodies were visualized using donkey anti-goatCY3 conjugate IgG (Jackson ImmunoResearch, West Grove, Pennsylvania) (1:100). Rabbit primary antibodies were visualized with Alexa Fluor® 594 donkey anti-rabbit IgG conjugate (1:100). Anti-M3 antibodies were pre-absorbed by incubating the antibody overnight at 4C with 10 g/ml of the appropriate peptide (SC7474P and SC9108P). Sections were analyzed and photographed using an AX70 microscope equipped with a cooled charged coupled device digital video camera (Olympus®).
Cell Culture, Transfection and Preparation of Protein Extracts HEK-293 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 2 mM L-glutamine, 10% (volume per volume) fetal bovine serum and penicillin (100 U/ml)/streptomycin sulfate (0.1 mg/ml) at 37C in a humidified 5% CO2 environment. Transfection was performed for 15 hours using the ProFection® Mammalian Transfection System-Calcium Phosphate according to manufacturer recommendations. The DNA used was open reading frame clone of Homo sapiens cholinergic receptor, muscarinic 3 (OriGene, Rockville, Maryland). To obtain cell extracts the tissue or cell suspensions were rinsed with ice-cold phosphate buffered saline and homogenized on an ice bed with 25 mM tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride and 10 g/ml leupeptin. Mixtures were cleared by centrifugation at 500 ⫻ gravity for 10 minutes and stored at ⫺70C.
Western Blot Western blots were made under reducing conditions according to standard procedures using the Odyssey® Infrared Imaging System. Primary antibodies used for Western blot were goat anti-M3 antibody SC7474 (1:500), rabbit anti-M3 antibody SC9108 (1:500) and rabbit anti-M3 antibody 13063 (1:500). Secondary antibodies used were donkey anti-goat 926-32214 IRdye® 800 (1:10,000), and donkey anti rabbit 611-732-127 IRdye 800 (1:10,000).
Tissue Preparation The bladder was removed and placed in ice-cold KrebsHenseleit buffer (Sigma-Aldrich®). The lateral wall was dissected and incubated in Krebs solution containing 1 mM of the nonspecific phosphodiesterase inhibitor isobutylmethyl-xanthine (Sigma-Aldrich) at 36C for 30 minutes. Incubation was terminated by immersing in an ice-cold fixative solution of 4% freshly prepared depolymerized paraformaldehyde for 120 minutes at 4C. The tissues were then washed at 4C in 0.1 M phosphate buffer containing 10%, 20% and 30% sucrose, each for 24 hours, and snap frozen with CO2 in Tissue-Tek® O.C.T.™ compound. Cryostat sections (10 m) were cut before thawing on chromealum-gelatin coated slides.
RESULTS Antibody Specificity and M3 IR To identify a specific antibody for M3 receptor we investigated 4 commercially available M3 receptor antibodies. Rabbit anti-M3 antibody A5259 revealed no staining (data not shown). However, goat anti-M3 antibody SC7474, rabbit anti-M3 antibody SC9108 and rabbit anti-M3 antibody 13063 showed M3 staining in the guinea pig bladder urothelium, suburothelium, lamina propria and inner muscle. Figure 1, a shows an example of staining with goat anti-M3
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Figure 1. M3 IR localization in normal guinea pig bladder lamina propria and inner muscle. Luminal surface section of bladder wall stained with SC7474 goat anti-M3 antibody (a). uro, urothelium. M3 IR in suburothelium at higher magnification using SC7474 goat anti-M3 antibody (b), 13063 rabbit anti-M3 antibody (d) and SC9108 rabbit anti-M3 antibody (f ). SU-IC processes and somata stained for M3 receptor. M3 IR in inner muscle bundles stained with SC7474 goat anti-M3 antibody (c), 13063 rabbit anti-M3 antibody (e) and SC9108 rabbit anti-M3 antibody (g). Scale bars indicate 40 (a) and 10 (b to g) m.
antibody. At higher magnification and gain all 3 antibodies revealed M3 IR in the stomata and processes of SU-ICs (Fig. 1, b, d and f). M3 IR was also observed in cells in the inner muscle bundle (Fig. 1, c, e and g). Vimentin, a general marker for cells in the interstitium, was used to identify these cells, which are a heterogeneous population and were formally described as fibroblasts. However, approaches such as this allow descriptions of the possible functional, better subclassification of individual cells of the interstitium, that is ICs. Of the 3 antibodies that showed M3 IR goat anti-M3 antibody SC7474 and rabbit anti-M3 antibody SC9108 showed no M3 staining when preincubated with the appropriate blocking peptide (fig. 2, a and b). Since the antigenic peptide of the third antibody, 13063, was not commercially available and the sequence was not made public by the manufacturer, pre-absorption studies were not done. As a control, immunohistochemical analysis using rabbit anti-vimentin antibodies pre-incubated with M3 blocking peptides showed normal vimentin staining (data not shown). Therefore, the antigenic peptides were specific for the M3 antibodies. The 3 antibodies showing staining were
further characterized by Western blot analysis of M3 transfected HEK-293 cell homogenates (figs. 1 and 2, c). Rabbit anti-M3 antibody 13063 did not reveal any bands upon Western blot analysis of transfected HEK-293 protein homogenates. Rabbit anti-M3 antibody SC9108 showed 2 bands with a molecular weight of about 45 and about 65 kDa, respectively. However, goat anti-M3 antibody SC7474 showed a single band of about 102 kDa, which is the molecular weight of the M3 receptor reported in previous Western blot studies.14,15 Untransfected HEK-293 revealed no band with any tested antibody. Notably not all antibodies are equally suitable for Western blot analysis and immunohistochemistry. For complete antibody characterization we accept that sophisticated technologies must be used, such as knockout animals.10 However, blocking peptide studies and Western blot analysis of M3 receptor transfected HEK-293 cells provided enough evidence to validate the adequate specificity of goat anti-M3 antibody Sc7474. Thus, we used this antibody to study M3 receptor localization in the guinea pig bladder.
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Animals with bladder neck damage. The mean ⫾ SD weight gain in sham operated and obstructed animals was 190 ⫾ 52 and 112 ⫾ 56 gm, respectively. Mean bladder weight in sham operated and obstructed animals was 400 ⫾ 73 and 1,568 ⫾ 500 mg, respectively. Obstructed bladders showed increased wall thickness (fig. 6). There were also major differences in the number and distribution of ICs, and increased IC density in the lamina propria and surrounding the muscle bundles, particularly the outer muscle layers. These cells demonstrated increased M3 IR (figs. 6 to 8). Little difference was noted in the SU-IC population but there was a higher density of LP-ICs. LP-ICs also showed M3 IR and appeared to extend complex bifurcating processes that formed a continuous network down to the SM-ICs (fig. 7). In obstructed bladders there was high IC density in the outer muscle layer (fig. 8). Cell bodies of these
Figure 2. Specificity of 3 commercially available anti-M3 receptor antibodies. Sections lacked M3 IR after staining with SC9108 rabbit anti-M3 antibody (a) and SC7474 goat anti-M3 antibody (b) after pre-incubation with respective blocking peptides. Western blot analysis of protein homogenates from control untransfected HEK-293 cells (C) and HEK-293 cells transfected with M3 receptor (T) (c). Blots were stained with SC7474 goat anti-M3 antibody, 13063 rabbit anti-M3 antibody and SC9108 rabbit anti-M3 antibody. SC, Santa Clara Biotechnology. Abc, Abcam.
Bladders Sham operated controls. M3 IR was located in the urothelium, suburothelium, lamina propria and the muscle layers of sham operated guinea pig bladders. M3 IR was associated with a population of cells termed SU-ICs in 2 isolated sham operated guinea pig bladders (fig. 3, a and d). SU-ICs formed a dense cell layer in the suburothelium. M3 IR was also associated with a less sparse population of cells termed LP-ICs. In different bladder regions the number of M3 IR cells varied (fig. 3, b and c). Studying ICs at higher magnification revealed M3 IR located specifically in the region of IC bodies (fig. 3, b, c, e and f ). Two nerve fiber populations were identified in the suburothelial space, including M3 IR nerves and nerves showing no M3 IR (fig. 4, a and b). In the muscle layers M3 IR was associated with smooth muscle cells, some nerve fibers and ICs (fig. 4, c to e). M3 IR was also found on SM-ICs (figs. 4 and 5). In addition, in 3 of the 7 sham operated control bladders studied these SM-ICs were in close apposition to muscle bundles in collections of the cell bodies that de Jongh et al first termed nodes (fig. 5).16
Figure 3. M3 IR localization in guinea pig bladder suburothelium and lamina propria (lp) in sections double labeled with antibodies to M3 receptor (red areas) and vimentin (vim) (green areas). Lumen, urothelium (uro), and suburothelial and inner muscle (iml) layers were identified (a). In same regions M3 IR (b) and vimentin staining (c) reveal variations in M3 IR density in nearby bladder wall regions with fewer (b) vs more (c) M3 IR cells. Section shows urothelium from different bladder (d). Note regions (d) at higher magnification as merged individual M3 IR and vimentin images (e and f ). Scale bars indicate 120 (a and d), 75 (b, c and e), and 15 (f ) m.
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conflicting results.5– 8 Previous attempts to characterize commercially available M3 receptor antibodies failed to identify any as providing specific staining.10 –12 Using the rabbit anti-M3 receptor antibody SC9108 used in our study Jositsch et al noted nonspecific staining in tissue from M3 receptor knockout mice.10 Although we observed what we may call a specific pattern of staining with the same antibody and the disappearance of a signal in pre-absorption experiments (fig. 1), our Western blot analysis revealed more than 1 band of the incorrect molecular weight (fig. 2). Thus, in agreement with Jositsch et al, this calls into question the specificity of this antibody. It also confirms that pre-absorption studies alone are not suitable to characterize specificity.
Figure 4. M3 IR localization in guinea pig bladder lamina propria and muscle layers. Sections of different bladders were stained with antibodies to M3 receptor (red areas) and nonspecific nerve marker PGP 9.5 (green areas) (a and b). Note M3 IR (asterisks) and PGP 9.5 gray value images, including lamina propria nerve fibers on PGP 9.5 staining. Pound sign indicates fiber that is not M3 IR. Section from outer muscle layer shows easily recognized nerve fibers (asterisks) and M3 IR associated with smooth muscle cells (pound signs) (c). Regions (c) are shown with M3 IR and PGP 9.5 results (d and e). Scale bars represent 120 (a and b), 40 (c) and 50 (d and e) m.
SM-ICs are found in nodes and show M3 IR.16 ICs were also apparent in the muscle bundles. There are regions of the bladder wall where trabeculae can be identified, which usually comprise a high density of ICs. Such trabeculae were often found in close opposition to others with few ICs (fig. 8). Intramuscular ICs showed M3 IR and extended processes to SM-ICs (fig. 8).
DISCUSSION The basic problem using an immunohistochemical approach for M3 receptor localization is the lack of well characterized antibodies. A number of studies of the M3 receptor location in the bladder provide
Figure 5. M3 IR localization in guinea pig bladder muscle ICs. Section of bladder wall inner layer immunostained with antibodies to vimentin (vim) (green areas) and M3 receptor (red areas) (a). M3 IR staining was noted on muscle cells in punctuate manner (asterisk). uro, urothelium. Two regions at higher magnification show vimentin and M3 images (b and c). Section from bladder wall outer layer (d) demonstrates 2 regions. Two cells (arrows) lying on muscle bundle surface show M3 IR and vimentin staining at higher magnification (e). Note small IC cluster or node (arrows) with M3 IR located in central regions of vimentin positive cells (f ). Scale bars indicate 60 (a and d), 30 (b) and 15 (c, e and f ) m.
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smooth muscle cells was to be expected. For nerve fibers M3 IR appears to be associated with a nerve population in the suburothelial space and the muscle layer. At least 2 types of suburothelial sensory fibers have been identified in the guinea pig bladder, including choline acetyltransferase positive fibers and calcitonin gene-related peptide/substance P/neurofilament positive fibers.17 The presence of nerves expressing M3 IR indicates possible regulation by cholinergic stimuli. To our knowledge the physiological source of the acetylcholine that may activate these nerves is not known to date. It may come from the cholinergic nerves as a form of auto-excitation or inhibition. Alternatively the acetylcholine may orig-
Figure 6. M3 receptor localization in full-thickness sections of sham operated guinea pig bladder (a) and bladder with previous surgical intervention to restrict bladder neck (b). Sections were immunostained with antibodies against M3 receptor (red areas) and vimentin (vim) (green areas). Note individual images of M3 IR and vimentin staining. Arrows indicate lumen, serosa, urothelium (uro), SU-ICs and lamina propria (lp). Note inner (iml) and outer (oml) muscle layers in bladder neck of operated animals. Scale bars indicate 110 (a) and 100 (b) m.
Therefore, we used 2 criteria, including the combination of a lack of signal after pre-incubation with a blocking peptide and the presence of a single band of the correct molecular weight after Western blot analysis of homogenates from HEK-293 cells transfected with M3 receptor. Using these criteria only goat anti-M3 receptor antibody SC7474 showed specific M3 IR. In pre-absorption studies with its blocking peptide any staining from this antibody was abolished and Western blot analysis revealed a single band with a molecular weight of about 102 kDa. Although the amino acid backbone of the human M3 receptor has a theoretical molecular weight of 66 kDa according to the National Center for Biotechnology Information UniGene database, the protein is highly modified post-translationally with an actual molecular weight of 102 kDa.14,15 This was confirmed by labeling studies using the muscarinic ligand [3H]propylbenzilycholine mustard14 and in a previous report using goat anti-M3 receptor antibody SC7474.15 Although we accept that our specificity validations were not as complete as previously reported methods10 and not all antibodies are equally suitable for Western blot and immunohistochemistry, based on these results we decided that goat anti-M3 antibody was specific and suitable for studying M3 receptor localization in the guinea pig. In the normal bladder M3 IR is associated with the smooth muscle cells, nerves and ICs. M3 IR on
Figure 7. M3 IR localization in obstructed guinea pig bladder urothelium (uro), suburothelium, lamina propria and inner muscle (iml). Sections were immunostained with antibodies to vimentin (vim) (green areas) and M3 receptor (red areas). Note gray value images of individual staining results. M3 IR was seen in structures within suburothelial cell layer and in lamina propria (a). Note region of lamina propria from different bladder (b) and same region at higher magnification (c). Network of vimentin positive cells was lying over surface of muscle bundles in mid muscle layer (d). Vimentin and M3 IR were also seen in structures within muscle bundles (plus signs). Regions (d) at higher magnification (e) and (f ) show vimentin and M3 IR images, including clear M3 IR localization to cell bodies of vimentin positive structures (pound signs). Pound signs indicate M3 IR positive structures. Scale bars indicate 90 (a and d), 50 (b and e), 20 (c) and 15 (f ) m.
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Figure 8. M3 IR localization in obstructed guinea pig bladder outer muscle layer. Sections were double immunostained using antibodies against vimentin (vim) (green areas) and M3 receptor (red areas) (a, b and e). Note regions (a, b and e) at higher magnification with individual images of vimentin and M3 IR results (c, d, f and g). There were 2 categories of muscle bundle, including those with numerous intramuscular ICs (plus signs) and those with few intestinal cells (pound signs) (e). Note examples of intramuscular ICs that were M3 IR (f and g). Scale bars indicate 80 (a, b and e) and 15 (c, d, f and g) m.
inate from other structures, such as the urothelium. Indeed, upon stretch the human bladder urothelium releases acetylcholine.18 Thus, acetylcholine released from the urothelium upon stretch may modulate afferent nerve activity. However, perhaps the most unexpected observation in the sham operated bladder was M3 IR associated with SU-ICs and SM-ICs. To our knowledge the functional role of SU-ICs is not known. These cells demonstrate an NO dependent increase in cGMP.16,19,20 The basal layer of the urothelium expresses neuronal NO synthase and so can produce NO.21 The finding of M3 IR on SU-IC-like cells suggests that these cells may respond to urothelial derived signals. In the normal bladder there is a network of SM-ICs.22,23 In the outer muscle layer these cells respond to NO with a increase in cGMP.6,20,24
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The presence of M3 IR on this network suggests the possibility of a cholinergic regulation of their function. Whole bladder experiments revealed cholinergic mechanisms involved in the activation of phasic activity, which may be controlled by a cholinergic driven pacemaker mechanism.4,20 M3 IR on ICs may represent the pacemaker element or regulation of the distributive element. Phasic activity occurs in the normal bladder in vivo as nonmicturition activity. Such activity was suggested to have a central role in a motor/sensory system involved in the generation and regulation of bladder afferent nerve activity and sensation.25,26 Evidence is accumulating to link cholinergic driven ICs with phasic activity and phasic activity with the generation of sensation. Thus, ICs may have a key role in bladder sensory mechanisms. Obstructed bladders showed distinct differences compared to sham operated control bladders. There was little change in the SU-IC network but the number of LP-ICs and muscle ICs increased dramatically in the obstructed bladder model. The finding of M3 IR allows us to hypothesize that these cells can respond to acetylcholine released from the urothelium, which suggests that cellular cross-talk can occur between the urothelium and SU-ICs. In addition, the network of LP-ICs that lie more deeply and extend inward from the SU-IC layer and the SM-ICs also showed M3 IR and underwent hypertrophy in the obstructed bladder model. Therefore, we propose that signals derived from the urothelium in response to an insult such as over distention activate a signaling cascade through the interlinked network of ICs, resulting in the pathological changes observed in the obstructed bladder model. To our knowledge the exact signaling mechanisms involved in this response to danger remain unknown. However, preliminary data show that ICs express cytokines and growth factors such as tumor necrosis factor-␣, interleukin-1 and TGF- as well as their respective receptors (Nile and Gillespie, unpublished data). In the obstructed animals there was an increase in the number of ICs. These cells showed M3 IR and, thus, may be regulated by cholinergic mechanisms. Recently de Jongh et al described cGMP positive ICs in the outer muscle layers of obstructed bladders and in some cases these cells were seen in nodes associated with nerve fibers.16 Thus, it is reasonable to hypothesize that these nodes are innervated and possibly activate the cholinergic receptors on SM-ICs. The altered number and distribution of M3 IR muscle ICs may be linked to functional changes. Recently it was reported that isolated obstructed bladders demonstrate large phasic contractions and have increased sensitivity to cholinergic stimuli.27 Since ICs may be involved in
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phasic activity generation and sensation modulation, they may have a role in the increased sensation of urgency and frequency.
CONCLUSIONS Observations indicate that ICs may be regulated by cholinergic mechanisms. This increased activity could contribute to increased motor/sensory
activity. Evidence is also beginning to accumulate for a secondary role of ICs in coordinating urothelial derived signals, showing cellular cross-talk and responding to dangerous insults such as partial bladder flow output obstruction. Since the 2 systems have a cholinergic component that is upregulated by pathological conditions, it is reasonable to hypothesize that this is a therapeutic target for anticholinergic drugs.
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mice. Naunyn Schmiedebergs Arch Pharmacol 2009; 379: 389.
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11. Pradidarcheep W, Labruyere WT, Dabhoiwala NF et al: Lack of specificity of commercially available antisera: better specifications needed. J Histochem Cytochem 2008; 56: 1099.
19. Gillespie JI, Markerink-van Ittersum M and De Vente J: Endogenous nitric oxide/cGMP signalling in the guinea pig bladder: evidence for distinct populations of sub-urothelial interstitial cells. Cell Tissue Res 2006; 325: 325.
12. Pradidarcheep W, Stallen J, Labruyere WT et al: Lack of specificity of commercially available antisera against muscarinergic and adrenergic receptors. Naunyn Schmiedebergs Arch Pharmacol 2009; 379: 397. 13. Mostwin JL, Karim OM, van Koeveringe G et al: The guinea pig as a model of gradual urethral obstruction. J Urol 1991; 145: 854. 14. Tobin AB and Nahorski SR: Rapid agonist-mediated phosphorylation of m3-muscarinic receptors revealed by immunoprecipitation. J Biol Chem 1993; 268: 9817. 15. Dawson LJ, Allison HE, Stanbury J et al: Putative anti-muscarinic antibodies cannot be detected in patients with primary Sjögren’s syndrome using conventional immunological approaches. Rheumatology (Oxford) 2004; 43: 1488. 16. de Jongh R, van Koeveringe GA, van Kerrebroeck PE et al: Alterations to network of NO/cGMPresponsive interstitial cells induced by outlet obstruction in guinea-pig bladder. Cell Tissue Res 2007; 330: 147. 17. Gillespie JI, Markerink-van Ittersum M and de Vente J: Sensory collaterals, intramural ganglia and motor nerves in the guinea-pig bladder: evidence for intramural neural circuits. Cell Tissue Res 2006; 325: 33. 18. Yoshida M, Miyamae K, Iwashita H et al: Management of detrusor dysfunction in the elderly: changes in acetylcholine and adenosine
20. Gillespie JI, Markerink-van Ittersum M and de Vente J: cGMP-generating cells in the bladder wall: identification of distinct networks of interstitial cells. BJU Int 2004; 94: 1114. 21. Gillespie JI, Markerink-van Ittersum M and de Vente J: Expression of neuronal nitric oxide synthase (nNOS) and nitric-oxide-induced changes in cGMP in the urothelial layer of the guinea pig bladder. Cell Tissue Res 2005; 321: 341. 22. Brading AF and McCloskey KD: Mechanisms of disease: specialized interstitial cells of the urinary tract—an assessment of current knowledge. Nat Clin Pract Urol 2005; 2: 546. 23. Drake MJ, Fry CH and Eyden B: Structural characterization of myofibroblasts in the bladder. BJU Int 2006; 97: 29. 24. Lagou M, Drake MJ, Markerink-Van Ittersum M et al: Interstitial cells and phasic activity in the isolated mouse bladder. BJU Int 2006; 98: 643. 25. Starling E: Elements of Human Physiology, 7th ed. London: Spottiswoode 1905. 26. Gillespie JI: A developing view of the origins of urgency: the importance of animal models. BJU Int, suppl., 2005; 96: 22. 27. de Jongh R, van Koeveringe GA, van Kerrebroeck PE et al: Damage to the bladder neck alters autonomous activity and its sensitivity to cholinergic agonists. BJU Int 2007; 100: 919.