1642 | Expression and Function of Muscarinic Sub- type Receptors in Bladder Interstitial Cells of Cajal in Rats Yingbing Wu,1 Chang Shi,2 Jianping Deng,1, Xin Zhang,1 Bo Song,1 Longkun Li1 Corresponding Author: Longkun Li, MD, PhD Department of Urology, Second Affili- ated Hospital, Third Military Medical University, Chongqing 400037, China. Tel: +86 23 6875 5623 E-mail: lilongk@hotmail.com Received July 2013 Accepted April 2014 1 Department of Urology, Second Affiliated Hospital, Third Military Medical Univer- sity, Chongqing, 400037, China. 2 Beijing Institute of Pharma- cology and Toxicology, Beijing, 100850, China. CELLULAR AND MOLECULAR UROLOGY Cellular And Molecular Urology Purpose: To locate the muscarinic (M) M2 and M3 receptors in bladder interstitial cells of Cajal (ICCs) and to determine the effects of M2 and M3 agonists on bladder ICCs. Materials and Methods: A total of 30 adult male Sprague-Dawley rats weighing 225-250 g were used in this study. Double-labeled fluorescence of muscarinic receptors and c-kit was performed for co-localization. To evaluate the effect of muscarinic agents on the excitation of bladder ICCs, we analyzed the inward current of bladder ICCs using the whole-cell patch clamp. The effect of muscarinic agents on the carbachol-induced inward currents was evaluated with the whole-cell patch clamp. Results: M2 and M3 receptors were confirmed in the stroma ICCs in rats’ bladders with double-labeled immunofluorescence. Spontaneous action potential was observed in freshly isolated bladder ICCs. The carbachol-induced inward Ca2+ current in ICCs can be blocked by atropine. The M2 receptor antagonist methoctramine (1 µM) showed a weak inhibitory capability on the inward Ca2+ current [from 74.8 ± 9.6 to 63.3 ± 13.8 Pascal (pA), n = 12, P = .03]. While the M3 receptor antagonist 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) (1 µM) significantly inhibited the inward Ca2+ current (from 78.4 ± 11.2 to 17.3 ± 7.9 pA, n = 12, P < .001). Conclusion: Bladder ICCs express M2 and M3 cholinergic receptors. Most muscarinic cho- linergic receptor antagonists, especially the M3 antagonists, can effectively inhibit the carba- mylcholine-induced inward current of bladder ICCs. Keywords: animals; interstitial cells of Cajal; microscopy; rats; urinary bladder; muscle; smooth; receptors. 1643Vol. 11 | No. 03 | May - June2014 |U R O LO G Y J O U R N A L Muscarinic Subtype Receptors in Bladder ICC | Wu et al INTRODUCTION The excitation and contraction of the urinary bladder are believed to be regulated neurogenically and myogeni-cally. The bladder can autonomously contract, but it can also initiate contraction under voluntary control. Moreover, independent contraction can be observed in isolated detrusor strips.(1) It is unclear how the neurogenic and myogenic interac- tions coordinate. Whether other mechanisms, such as a pace- maker or other regulation center, are involved remains to be clarified. Interstitial cells of Cajal (ICCs) are widely distributed in the visceral smooth muscle organs. They possess the ability to self- excite and produce an action potential. ICCs in the gastrointes- tinal tracts(2) and in the upper urinary tracts(3) are believed to behave as pacemakers. Additionally, they can receive the in- nervation pulse.(4) In the bladder, ICCs have been found among the detrusor smooth muscle bundles and are closely associated with bladder excitation and contraction.(5,6) Changes of bladder ICCs can impact the initiation of spontaneous electrical activ- ity, which may result in homologous functional disturbances in bladder contraction.(7) Our previous studies have found that there were some specific changes in the quantity and physi- ological characteristics of bladder ICCs in bladder instability.(8) It is critical to clarify the interaction between bladder innerva- tion and ICCs. Cholinergic nerves are the main neurogenic factors in bladder function. The muscarinic (M) M2 and M3 receptors are the two main subtypes of muscarinic receptors in the bladder. When stimulated by cholinomimetics in vitro, ICCs generate transient calcium, which is mainly mediated by the M3 receptor agents. (9) However, the expression and function of muscarinic recep- tors in bladder ICC, and its potential value for treatment are still unclear. Based on our previous studies and article reports, we hypothesized that muscarinic receptors were expressed in blad- der ICC, and played important role in the bladder excitation regulation. In this study, the location of M2 and M3 receptors in bladder ICCs were observed with immunofluorescence. The effects of M2 and M3 agonists on bladder ICCs were also re- corded with the patch clamp technique. MATERIALS AND METHODS Animals A total of 30 adult male Sprague-Dawley rats weighing 225- 250 g were used in this study. This study was approved by the Research Council and Animal Care and Use Committee of Southwest Hospital, Third Military Medical University, China (Approval No. SYXK20070002). The experiments conformed to the guidelines for the ethical use of animals, and all efforts were made to minimize animal suffering and to reduce the num- ber of animals used. All animals were similarly fed in a special pathogen-free room, with free access to food and water. Their general states of health and activity were monitored closely during the experiment. The experimental unit for morphology was rat, and for functional study the unit was rat bladder ICCs. All the experimental manipulations were standardized, and ac- complished by an experience experimenter. Double-labeled Fluorescence for Bladder ICCs and M2, M3 Receptors To observe the location of M2 and M3 receptors in bladder ICC, 10 rats were used for this study. Double-labeled fluorescence of muscarinic receptors and c-kit was performed for co-localiza- tion. A 25% urethane (1.1 g/kg) intraperitoneal injection was used for anesthesia. Under sterile conditions, the bladder was exposed through a median abdominal incision, and the bilateral ureters were ligated. Then 4% paraformaldehyde was injected into the bladder through a transurethral epidural catheter, and the bladder neck was ligated after the bladder was filled to 20 cmH2O. The bladder was harvested and immersed into 4% paraformaldehyde solution for 6 to 8 h for continuous fixing. Under a dissecting microscope, the mucous membrane and its substratum were carefully removed. The bladder’s muscular layer was longitudinally cut to 3 × 4 mm and then thoroughly rinsed with 0.01 M phosphate buffered saline (PBS) for 5 min × 3 times, 0.03% H2O2/Methanol sealing endogenous peroxidase at 370C for 30 min, then 0.01 M PBS for 5 min × 3 times. It was then incubated in 1% bovine serum albumin (BSA) for 30 min at room temperature. The incubation solution was removed, and anti-c-Kit displaying ICC (goat anti-rat monoclonal, 1:200, Sigma) and M2 or M3 receptor antibodies (rabbit anti-rat monoclonal, 1:2000C Sigma) were added. The samples were incubated in 40C anti-dilution for 12 h (negative control in 0.01 M PBS as collation), and secondary antibodies marked with Alexa 488 (donkey-anti-goat polyclonal, 1:2000C molecular probes) and Alexa 594 (donkey-anti-rabbit polyclonal, 1:200, molecular probes) was added for 12 h. The samples were then thoroughly rinsed with 0.01 M PBS for 5 min × 3 times and 1644 | Cellular And Molecular Urology incubated with the secondary antibody at 370C for 1 h. Then, 4',6-diamidino-2-phenylindole (DAPI) was added for 10 min for nucleus fluorescence loading. After 3 times of rinses with 0.01 M PBS (30 min each for the first two rinses and 3 h for the last time), the glass was sealed with glycerol and subjected to laser confocal microscopy. The c-kit was labeled in green under an excitation wavelength of 488 nm, and M2 or M3 receptors were labeled in red under a wavelength of 560 nm. Fresh Isolation and Culture of Bladder ICCs To evaluate the effect of muscarinic agents on the excitation of bladder ICCs, we analyzed the inward current of bladder ICCs using the whole-cell patch clamp. According to the sample size for self anterior-posterior contrast study, the number was set at 8 for atropine (effect size/estimated standard deviation = 5.12), and 12 for methoctramine and 4-diphenyl-acetoxy-N-methyl- piperidine methiodide (4-DAMP) (effect size/estimated stand- ard deviation = 0.89), with power of 90%, significance level of 5%. All the variables were continuous variables. Referring to the method of McClosky (McCloskey & Gurney, 2002), the urothelium layer was carefully removed, and the rat bladder was chopped into small pieces of about 1 mm3. The pieces were then incubated in a digestion solution containing (mg/mL) 1.0 Type II collagenase, 2.0 bovine serum albumin, and 1.0 trypsin inhibitor (all from Sigma) at 370C for 30 min. The supernatant was then removed, and the isolated cells were immersed into enzyme-free Hanks’ Balanced Salt Solution (HBS; R and D) and cultured at 370C in 5% CO2 and 95% O2 in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) with 10% fetal bovine serum and 25 ng/mL SCF until proper adherence. ICCs with typical morphology with many branches under visible light positive for c-kit staining were pre- pared for the subsequent patch clamp studies, as reported by McCloskey and colleagues.(10) Effects of Muscarinic Receptor Antagonists on the Carba- chol-induced Inward Current The effect of muscarinic agents on the carbachol-induced in- ward currents was evaluated with the whole-cell patch clamp. Using a one-step method, soft, neutral, hollow glass tubes (Sutter Instrument, America) were drawn by a vertical puller to prepare a microelectrode with a heat value of 56. A micro- electrode with a diameter of approximately 1 μm was selected for the patch clamp studies. The supernatant isolated cells was removed and the adhered recording chamber was constantly perfused with Hanks solution at room temperature and addi- Figure 1. The identification of positive muscarinic (M) receptors in ICCs of rats bladder. A: M2 receptors in rat bladder ICCs; B: M3 recep- tors in rat’s bladder ICCs. Double-labeled immunofluorescence using anti-c-kit (green, A2) for ICC, anti-M2 (red, A3) or anti-M3 (red, B3) for M2 or M3, and DAPI (blue, A1 and B1) for nuclei. Arrows: examples of ICCs double-marked with the c-kit and M2 (A4) or c-kit and M3 (B4) receptors. 1645Vol. 11 | No. 03 | May - June2014 |U R O LO G Y J O U R N A L tionally, the cell under study was continuously superfused by a close-delivery system. Positive pressure was loaded into the microelectrode cavity. The electrode was proceeded to the ICCs to attain negative pressure and create substantial impedance. To record the whole cell current, a pCLAMP (Axon Instruments, America) with a sampling frequency of 4 kHz and a filtering frequency of 2 kHz was used. The 3D manipulator was regulat- ed to seal on contact, and the impedance was set to reach 1 GΩ. The cell membrane was sucked and broken to create a whole- cell recording mode. Whole-cell current recording was con- ducted in voltage clamp mode. All recorded data were analyzed and treated using Clampfit 9.2 at the backstage. The membrane potential was set at -50 mV with the clamp. Carbamylcholine (1 µM and 10 µM, Sigma) was added to induce an ICC in- ward Ca2+ current, and various antagonists, including the M- receptor antagonist atropine (1 µM, Sigma), the M3-receptor antagonist 4-DAMP (1µM, Sigma), and methoctramine, were administered to investigate their functional effect on the carba- chol-induced (10 µM, Sigma) inward Ca2+ current in ICCs. Statistical Analysis All the quantitative data were confirmed to obey the normal distribution with homogeneity of variance by test of normality, and were presented as mean ± SD. Student’s t-test was used for comparison [statistical package for the social science (SPSS Inc, Chicago, Illinois, USA) version 13.0 software)], with P < .05 considered significant. All the data were double-blinded, randomized and analyzed using posteriori tests. RESULTS Double-label fluorescence for M2 or M3 Receptors and Blad- der ICCs ICCs were identified by their c-kit positivity and spindle shape. M2 (Figure 1A) and M3 (Figure 1B) receptors were confirmed in the stroma ICCs in rats’ bladders with double-labeled im- munofluorescence. Effects of Muscarinic Receptor Antagonists on Carbachol- induced Bladder Contraction Spontaneous action potential was observed in freshly isolated bladder ICCs (Figure 2). ICCs can be activated with a dose- dependent inward Ca2+ current induced by carbachol. The peak current values in 1 µM and 10 µM of carbachol are 18.3 ± 8.7 Pascal (pA) and 75 ± 10.9 pA, respectively, with statistical differences (n = 8, P <0.001; Figures 3A and B). The carbachol- induced inward Ca2+ current in ICCs can be blocked by atro- pine. When the carbachol dose was set at 10 µM, 1 µM atropine could significantly reduce the peak current from 77.1 ± 10.0 pA to 5.8 ± 2.7 pA (n = 8, P < .001; Figures 3C and D). The carbachol-induced inward Ca2+ current in ICCs can also be blocked by M2 and M3 receptors antagonists. The M2 recep- tor antagonist methoctramine (1 µM) showed a weak inhibitory capability on the inward Ca2+ current (from 74.8 ± 9.6 to 63.3 ± 13.8 pA , n = 12, P = 0.03; Figures 3E and F), While the M3 receptor antagonist 4-DAMP (1 µM) significantly inhibited the inward Ca2+ current (from 78.4 ± 11.2 to 17.3 ± 7.9 pA, n = 12, P < .001; Figures 3G and H). DISCUSSION Many studies on detrusor myocytes, receptor expression, cell- cell communication and cell phenotypic alteration have been conducted, but the mechanisms responsible for bladder excita- tion and contraction have not been well clarified.(11) ICCs dis- tributing submucously and among the smooth muscle bundles are thought to mediate signal transmission from neurons to smooth muscles.(12,13) In this study, we hypothesized and con- firmed the expression and function of muscarinic cholinergic subtype receptors in ICCs with double-labeled immunofluores- cence. M2 and M3 receptors were confirmed in bladder ICCs. In human and guinea pig bladder, the proportion of M2 and M3 is about 3:1, although the M3 subtype is dominant in the excitation of smooth muscle contraction via Gq/11 activating hydrolysis of phosphoinositide, IP3 production, and elevation of intracellular Ca2+. Conversely, M2 receptors may indirectly enhance M3-mediated contraction, by opposing the normal effects of β-adrenoceptor G protein-coupled activation of ade- nylyl cyclase which lead to cyclic adenosine mono phosphate (cAMP) production and bladder relaxation.(14) The innervation methods of smooth muscles and skeletal muscles are different. Muscarinic Subtype Receptors in Bladder ICC | Wu et al Figure 2. The spontaneous action potential of novel ICC samples isolated from adult rat’s bladders. 1646 | In skeletal muscle organs, neural impulses can directly activate the muscle cells through neuromuscular synapses. In smooth muscle organs, there are no analogous structures connecting the cells and neural terminals. When the innervation impulse reaches the nerve terminal, the secretory vesicle releases neu- rotransmitters, which diffuse throughout the intercellular fluid and act on the target cells. Many factors can affect excitation through this regulatory pathway. Because ICCs in the gastroin- testinal tracts are considered pacemakers, ICCs in the bladder might be presumed to facilitate signal transduction from nerve terminals to detrusor myocytes. Because ICCs express the specific cell membrane surface receptor kit, the c-kit receptor identification companion and its cell shape and phenotype have been acknowledged as the standards for ICCs identification.(15) Yamanishi and colleagues found that ICC in the bladder have close structural relationships with cholinergic nerves. Freshly dispersed detrusor ICCs and ICCs in situ respond to cholinergic stimulation by firing Ca2+ transients.(8) Among the innervation types in the nervous system, choliner- gic nerves - particularly muscarinic cholinergic receptors - are the most important for bladder excitation and contraction. The parasympathetic impulse can trigger the neural transmitter and initiate bladder contraction via the muscarinic cholinergic re- ceptor. Different subtypes of muscarinic cholinergic receptors can be classified by specific selective receptor antagonists. The expression of M2 and M3 subtype receptors has been con- firmed mainly in the bladder. Muscarinic cholinergic receptors with a high affinity with AF-DX 116 are defined as Type M2, while those with a high affinity with 4-DAMP are defined as Type M3. Different blocking agents with relative selectivity for muscarinic receptors have been obtained. Among these agents, triptiramine has been found to possess the highest affinity with M2 receptors, followed by AFDX384 and imbacine. 4-DAMP possesses the highest affinity with M3 receptors, followed by darifenacin.(16) M2 receptors are more abundant than M3 re- ceptors in the bladder, which may indirectly mediate detrusor contraction by deteriorating the detrusor dilatation mediated by the β adrenergic nerve.(17) Although M3 receptors are less prevalent, they are believed to play a central role in mediating bladder contractions.(18) In the gastrointestinal tract, ICCs in the gastric antrum and stomach can be stimulated by cholinergic neural pulses, and they can subsequently increase the slow- wave frequency. This effect can be mediated by M3 receptors, indicating that cholinergic neurotransmitters can regulate ICC excitation of the gastrointestinal tract and subsequently effect smooth muscle contraction.(19) It can be assumed that ICCs in the urinary bladder can influence bladder excitation, which can be regulated by muscarinic cholinergic nerves, especially M3 receptors. In our functional studies, the results might indicate that blad- der ICCs are mainly excited by M3 receptors, as displayed by inward currents and depolarization. It is generally believed that M3 receptors may activate phosphoinositide hydroxylation by coupling with the Gq /11 of G protein to produce IP3 and DAG as second messengers, thereby mediating the increase of the calcium ions in cells. M3 receptors also directly induce detrusor contractions,(20) but the detailed signal pathway still needs to be clarified. The functional studies on the effect of cholinergic antagonists on the carbachol-induced ICC inward current sug- Cellular And Molecular Urology Figure 3. Effects of carbachol, atropine, methoctramine and 4-DAMP to inward Ca2+ currents of bladder ICCs. A and B: Carba- chol-induced inward currents in bladder ICCs. C and D: Atropin’s inhibition of the inward current stimulated by carbachol. E and F: Methoctramine’s inhibition of the inward current stimulated by carbachol. G and H: 4-DAMP’s inhibition of the inward current stimulated by carbachol. 1647Vol. 11 | No. 03 | May - June2014 |U R O LO G Y J O U R N A L REFERENCES 1. Xiao CG, Du MX, Dai C, Li B, Nitti VW, de Groat WC. An artificial somatic-central nervous system-autonomic reflex pathway for con- trollable micturition after spinal cord injury: preliminary results in 15 patients. J Urol. 2003;1701237-41. 2. McCann CJ, Hwang SJ, Bayguinov Y, Colletti EJ, Sanders KM, Ward SM. 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ACKNOWLEDGEMENTS This study was supported by the National Natural Science Foundation of China (No. 81230017, 81170705) and Chong- qing Scientific Fundation (CSTC2010BA5005). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Yingbing Wu and Chang Shi contributed equally. CONFLICT OF INTEREST None declared.