The Roles Of Muscarinic Receptors In Excitability Of ICCs And Contraction Of Rat Bladder

Background and objective:Bladder, an empty organ with high compliance, always requires a coordinated contraction of the detrusor and relaxation of the urethra. Bladder dysfunctions are very common in clinic. Patients suffer from unbearable symptoms and there are no effective therapies to them in some patients because the regulation of bladder contractile function is complex and the pathogenesis of these diseases is not clear.Physiological contraction and relaxation of the urinary bladder are predominantly controled by central nervous system(CNS). The bladder contraction is mainly regulated by cholinergic nerve and the relaxation is mainly regulated by adrenergic nerve combining with the assistance of non-adrenergiccholinergic nerve. Unfortunately, satisfactory therapeutic effect is seldom obtained when treating the diseases of bladder constriction disorders using several kinds of antagonists to nerve regulation, which suggests that the theory based on neuroregulation can't explain the exact mechanisms underling bladder contractile function disoders. Previous studies have demonstrated that the isolated bladder had localized micromotion or generalized contraction,and the generalized contraction induced the change of intravesical pressure. It was also found that detrusor muscle strip could generate spontaneous contraction. Then we supposed a hypothesis based on intrinsic bladder changed to explain the disorders besides nerve regulation, and tried to find the pathogenesis of them for many years in our reseach center.Recently, some specialized cells with various morphological, electrophisiological and immunohistochemical characteristics of interstitial cells of Cajal (ICC) have been located in many regions of ureter, bladder, prostate and so on. ICC, the pacemaker cells of the gastrointestinal tract, play important roles in the formation of basic electric rhythm of gastrointestinal tract, the regulation of neural conduction, signal integration and so on. The normal structures, function and distribution are important to maintain the gastrointestinal motility and contractile function. The number of ICC decreaseing or damage of the cellular network may induce some gastrointestinal motility disorders. McCloskey et al found ICC may play important roles in the regulation of bladder contraction. Moreover, the location of bladder ICC have connections with intramural nerves and associated with smooth muscle cells. Then, there may be some special relationships between ICC and nerve in bladder.Cholinergic nerve play the predominant role in nerve regulation in bladder via released acetylcholine combining muscarinic receptor to induce detrusor contraction. Muscarinic receptors are related with many abnormalities of bladder contraction. Braverman et al found that total amount of M receptor or density of M₂ receptor increasing were associated with the contractility ability of bladder and those changes were independent with pelvic nerve, which suggests that M₂ receptor may be the main causative factor to this disease. Instability of detrusor muscle is the most common urination dysfunction; its pathogenesis may be related with the up-regulation of subtype of M receptor and disproportion of M₂ and M₃ receptor subtypes.Recent studies demonstrated that ICC in gastroinstestinal tract expresses several neuroceptors, such as purinergic receptors, bradykinin receptors, cholinergic and prostaglandin receptors. Kim et al found that the cholinergic stimulus mediated by M3 receptor increased the pacing frequency of ICC in gastic antrum and body, which suggests that cholinergic transmitter regulates the excitory of ICC in gastrointestinal tract to control the contractile function of smooth muscle in gastrointestinal tract. Our previous studies found that the ICC in bladder express purinergic receptors and ICC in bladder can be activated under the stimulation of purinergic neurotransmitter ATP. Then we are interested in the correlation between activation of M receptor and excitability of ICC in bladder, and whether ICC play roles in the regulation of bladder contraction underlying cholinergic nerve? There are no data on this subject reported by researchs of internal and abroad.This study will investigate the distribution of M receptor subtypes in bladder ICC, the relations of activation of M receptor and the excitory of ICC, and the contractile function of bladder when ICC were inhibited by Glivec, a specialized inhibitor of c-kit tyrosine kinase. The mechanism of regulation underling bladder contraction will be investigated by a firenew point, which is a worthy theory to offer new therapies for abnormalities of bladder contraction. Materials and MethodsIn this study, the roles of M receptors in bladder contractile function were observed in adult SD rats.â‘´We identified the area of ICCs and the expression of the five muscarinic Cholinergic Receptors (M₁-M₅) on ICCs in bladder of rats using couple immunofluorescences and observing with confocal laser microscopy.⑵We investigated the contribution of M receptors to the function of ICCs in mixed primary culture of ICCs and detrusor muscle cells. Then the changes of [Ca²⁺]_i in ICCs were observed under confocal laser microscopy after using Fluo-3 AM Calcium fluorescence indicator, Cholinergic receptor agonist and subtypes antagonist to examine how M receptors regulate the excitability of ICCs.⑶The role of ICCs in bladder contractile function was researched by comparing the changes of contractile functions using Carbachol in normal rat bladder in vitro, M₂ and M₃ receptors hypospecificity blocking agents in bladder strips. Furthermore the bladder strips contractile properties induced by Carbachol application were detected after been exposed to Glivec to block c-kit and the normal function of ICCs.Results1. C-kit positive ICCs were found in bladder of SD rats under confocal laser microscopy.2. We found that M₂ and M₃ but not M1, M4 and M5 receptor subtypes were expressed in bladder ICCs of SD rat by double lable of immunofluorescence.3. We successfully identified bladder ICC in mixed primary culture.4. In vitro application of Carbachol, an agonist of M receptor, significantly enhanced [Ca²⁺]_i in cultured ICC.5. The Carbachol-induced excitability in cultured ICCs was mildly inhibited by Methoctramine, the antagonist of M₂ receptor, while it was significantly inhibited by 4-DAMP, the antagonist of M₃ receptor.6. The bladder strips showed spontaneous contractions under a fixed tention(0.75g). Gradually increasing the concentration of Carbachol could induce significant increasing in the amplitude of whole contractions and spontaneous contractions. Moreover, the frequency of spontaneous contractions showed high frequency in first and then down later.7. The amplitude/frequency of spontaneous contraction and the tention of the strips induced by Carbachol could be significantly inhibited by 4-DAMP but mildly inhibited by Methoctramine.8. The contraction of bladder strips could be inhibited by concentration dependent Glivec. Glivec could relax the bladder strips which were in contraction state induced by Carbachol.9. Glivec could significantly decrease the amplitude of spontaneous contraction and contractile effect of bladder strips induced by Carbachol.Conclutions1. ICCs express M₂ and M₃ receptors in bladder of rats.2. Cholinergic nerve stimulate can excite ICCs in bladder mainly via M₃ muscarinic receptors.3. ICCs in bladder might play roles in the regulation of detrusor contraction by the interaction with cholinergic nerve.