Role Of Purinergic Signal Mediated By P2X Receptor On ICC-like Cells Of SD Rat Urinary Bladder In Detrusor Neuromodulation

Background and Aim: Up to now, the therapies of many voiding disorders are unspecific and the curative effects are unsatisfactory because the mechanisms of urinary bladder neuromodulation are not elucidated completely. As a smooth muscle organ, bladder not only kept the independence character of smooth muscle organs, such as gastrointestinal tract, but also gained the adlibitum control character of somatic muscles. How this two physiological character cooperate each other are not clear. So bladder was defined as a special smooth muscle organ. The traditional neuromodulation mode of smooth muscle can not be used to explain the complicate detrusor neuroregulation exactly.In the gastrointestinal tract, the interstitial cells of Cajal (ICC) have important physiological functions, including pacemaking, relaying of nerve impulses and propagation of excitation. In the last few years,Several papers have described the presence of ICC-like cells throughout the urinary tract. Bladder ICC-like cells are recognized to lie in two predominant anatomical locations, beneath the urothelium and within the detrusor muscle, in both the human and guinea-pig. While under physiological condition, spontaneous contraction was not observed during urine storage. So it is very intriguing to investigate the functional properties of bladder ICC-like cells. In this study, we speculate that ICC-like cells in bladder are contributed to the propagation of excitation from the nerve terminals to detrusor smooth muscle,just be a'relay station'more than a'pacemaker'. As we know, the motility of bladder is under control of the nervous system.Sympathetic, parasympathetic, somatic and sensory pathways contribute to the control of bladder during urine storage and micturition. In addition to the classical neurotransmitters noradrenaline and acetylcholine, peptides, purines and the enzyme responsible for the synthesis of nitric oxide have all been localized in nerves supplying the bladder. As early as 1972 Burnstock et al postulated that ATP can act as the excitatory co-transmitter with acetylcholine in parasympathetic nerves supplying the urinary bladder. It was later shown that, indeed, ATP is released with acetylcholine (Ach) in response to nerve stimulation and that the ATP response is mediated through ligand-gated cation channels, P2X receptors.In this study, we focused on the role of ICC-like cells in bladder neuromodulation, incisivused with purinergic signal mediated by P2X receptor. The results are helpful to enrich neurophysiology theory of the bladder, important for understanding of the physiology and pathology of micturition and to provide new targets for the therapy of some voiding dysfunctions.Methods: 64 SD rats and 1 guinea pig were sacrificed in this study. Firstly, we investigated the ultrastructure of interstitial cells beneath the urothelium and within the detrusor muscle of SD rat by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).On the second, immunoflurorescence were used to identify ICC-like cells with c-Kit as the markers in SD rat bladder and then immunoflurorescence (double-label) was used to study the relationship between ICC-like cells and nerve terminals. Thirdly, ICC-like cells and detrusor smooth muscle cells (DSMC) were isolated by collagenase digestion and co-cultured in vitro, then the effect of ATP on [Ca2+]i variation of ICC-like cells and DSMC were assessed by confocal laser scanning microscopy (CLSM),then immunohistochemistry and immunoflurorescence (double-label)were used to study the expression of P2X purinoceptor subtypes in DSMC and ICC-like cells respectively. In the third part, to evaluate the role ICC-like cells in urinary bladder sensation, the variation of rat pelvic nerve afferent discharge evoked by filling ATP into the urinary bladder before and after blocking the function of ICC-like cells with Glevic。Main results:1. SEM showed us some interstitial cells beneath the urothelium with the similar morphological features as follow, small round or polygon cell body and several slender ecphyma stretching out. They connected with each other and constructed a cell-net.2. With TEM, we found the morphological features of interstitial cells within the detrusor muscles, such as gap junctions with DSMCs and very close apposition with nerve terminals.3. ICC-like cells were identified to exist in suburothlium layer,along the longititude of smooth muscle bundles and among detrusor smooth muscle in adult SD rat urinary bladder through c-Kit immunofluorescence.4. Double-labelled tissue with c-Kit and PGP9.5 antibodies showed that ICC-like cells are very close apposition with nerve terminals in SD rat bladder.5. Spontaneous contractions were recorded on detrusor smooth muscle strips in vitro under certain preload (0.5g. When ATP at concentrations greater than 10μM was added, a transient contraction followed by long time diastole was recorded on detrusor smooth muscle strips in vitro, the dose-effect relationship of ATP is obviously. The spontaneous contractile amplitude was vanished completely by 1 mmol/L ATP.6. We established the primary culture method of ICC-like cells and DSMCs in vitro by collagenase digestion.7. The concentration of intracellular Ca2+ ion of ICC-like cells and DSMCs were detected in dynamic state by LSCM, the mean intensity of [Ca2+]i of ICC-like cells (55.16±3.23)is higher than that of DSMCs(45.59±2.08)(P<0.05). Continuous application of ATP at concentrations greater than 20μM caused a prompt rise followed by a slow decline of the [Ca2+]i baseline in both ICC-like cells and DSMCs.8. Immunohistochemistry results showed us that all seven subtype of P2X receptor expressed in SD rat urinary bladder, among them, only P2X1 was distributed on DSMCs. P2X2 and P2X5 were detected on ICC-like cells by immunoflurorescence (double-label).9. Filling with 20 mM ATP into the urinary bladder can evoke SD rat pelvic nerve afferent activity. When blocking the function of ICC-like cells by 100μM Glevic in advance , the pelvic nerve afferent activity evoked by ATP was obviously decreased(P<0.01)。Conclusions:1. Morphological-study hinted that ICC-like cells in rat urinary bladder owned the ability of"relay station"between the nerve terminals and DSMCs.2. The effects of ATP on detrusor smooth muscle strips indicated that purinergic signal is very important for detrusor neuromodulation.3. Our results of ATP on intracellular Ca2+ ion of ICC-like cells and DSMCs and the difference expression of P2X receptor on ICC-like cells and DSMCs showed us that ATP can excite ICC-like cells and DSMCs through different P2X receptor subtypes.4. The effects of Glevic on rat pelvic nerve afferent discharge evoked by ATP indicated that ICC-like cells may play very important role in bladder sensation.