Study On The Alterative Modulation Of Ca～(2+) Activated K～+ And Cl～- Channel And Its Relationship To DI

Background: Detrusor instability (DI), which causes urinary frequency, urgency, andincontinence, is the most common bladder dysfunctions of people, especially of aging. Theetiological factors of DI are usually referred as bladder outlet obstruction (BOO),neurological dysfunction, and specialized factors. But the pathophysiology is still unclearand the pathogenesis is still intact and inaccurate. In the past years, neurological factorswere thought to be the key role of DI. But no satisfactory results were able to reach whiletreatment aimed to these factors in clinical works. Special attention was given to themyogenic basis for DI nowadays. Detrusor produce spontaneous action potentials andmechanical activity involved the participation of calcium, the most important factors tocontrol the smooth muscle excitability and contractility, influx extracelluar or releasedintracellular. In the past years, a great number of works emphasized on how to diminish thecalcium influx, but with a little effect on the control of DI. So, it is reasonable to speculatethat calcium released intracellular and calcium dependent mechanisms also play a role inthe modulation of detrusor excitability. Up to now, the most important channels related tocalcium are calcium activated potassium channel (KCa) and chloride channel (ClCa) insmooth muscle, which act as negative and positive feedback to control the smooth musclerespectively. To our knowledge, there are a few reports about the modulation of KCa ondetrusor. The objective of this study is to investigate the expression and modulation oftheses channels in detrusor, and to access the relationship of the alterative modulations andDI. Materials and methods: Bladder outlet obstruction was created in female Wistar ratsby ligating the proximal urethra in the perineum for 6 weeks. Cystometries were performedin conscious rats 7 days after suprapubic bladder catheter implantation. The bladdercapacity and pressure was measured and the incidence of detrusor instability was counted.The mRNA was extracted from the detrusor of control and DI rats. BKca, SKca2, SKca3, 3第三军医大学博士学位论文and ClCa were detected by the means of RT PCR. Immunohistochemisty of BKca, SKca2,and SKca3 channels were also performed in bladder slice of control and DI rats. Theexpression differentia was identified. Enzymatically dispersed detrusor cells of control andDI rats were incubated with fluorescence dye FLIPR to detect the membrane potentialunder laser confocal microscope. The alterative ratio of fluorescence intensity wascalculated undergo the actions of Iberiotoxin (selective BKca blocker), NS1619 (selectiveBKca opener), Apamin (selective SKca blocker), Chlorzoxazone (selective SKca opener),and NFA (niflumic acid, selective ClCa blocker). Hypo-osmolar solution was used to mimica tension state, and the performance of the channels was also studied. Detrusor strips wereused to study the spontaneous contraction under a preload tension of 0.5g. The frequencyand MI (motility index), referred as the area under the curve (AUC), was counted, and themodulations of channel blockers and openers were investigated. Results and discussion: 1. Reflex micturition remained in conscious rats during invivo cystometric evaluation. There are 76.17% obstructed rats developed DI. Bladderpressure and capacity were significantly increased in DI rats. Detrusor cells were alsodispersed effectively and identified as smooth muscle cells. 2. KCa and ClCa mRNAexpression can be detected in detrusor. It was confirmed that the expression of BKca,SKca2, and SKca3 were significantly decreased, whereas ClCa was significantly increasedin DI rats. The BKca, SKca2, and SKca3 immuno-reactivity were detected in detrusor, andthe decreased immuno-reactivity was indicated in DI detrusor. These results suggested thealterative role of KCa and ClCa in DI. 3. The alterative fluorescence intensity (△F) of thedetrusor smooth m...