The Cellular Excitability Alteration Of Detrusor Myocytes From A Detrusor Overactivity Rat Model Induced By Partial Bladder Outflow Obstruction And Its Underlying Ionic Mechanisms

Background and ObjectiveDetrusor overactivity (DO) is a very common urinary dysfunction which could give rise to a variety of irritating symptoms such as urgency ,frequency with or without urge incontinence .DO could seriously affect the suffers'life quality or even mental health and it now apparently becomes a heavy burden to the social medical system.When in the filling stage,the healthy urinary bladder is kept in"quiescence"and low intravesical pressure is maintained due to the excellent compliance of the urinary bladder which is of help to the urine transmission from the upper urinary tract to the bladder and in preventing vesico-ureteric reflux .This physiological process is under complex control of central nervous system,body humour and consciousness along with the low urinary organ themselves.However,whatever reason,if the above harmonious interactions are interrupted ,the urinary bladder could exhibit uninhibited involuntary contractions during the filling stage which in turn induces the above mentioned symptoms complex.This symptoms complex is termed as the overactive bladder syndrome (OAB) ,whilst the involuntary urinary bladder contraction is referred to as detrusor overactivity (DO) which could be recorded and diagnosed by a cystometry assay.In accordance with the latest clinical epidemiology report,the incidence of DO is very high and it is among the top 10 most common chronic diseases in western world .Though the corresponding investigation is lacking regarding Chinese population ,it is however indeed occupy a big portion of urological clinic workload suggesting similarly high incidentce in China. Unfortunately,there is few effective therapeutic means available to treat DO patients .The anticholinergic agents are now the first line drug,however,because of their seriously unpleasant side-effects such as dry mouth and constipation ,also mild and limited effectiveness in relieving OAB symptoms,few patients can comply with this drug for long period of time,therefore , more effective strategies with less side effects must be come up with in a short time in order to deal with this trying situation.Though a lot have been done regarding the pathophysiology of DO,unfortunately, it is still largely unknown.The currnt opinions are that DO is caused by multifaceted etiologies and the responsible mechanisms might vary among DO with distinctive origins but the only thing with certainty is that the exact pathological mechanism is not uncovered yet.If they could be summed up ,the mainstay theories can be attributable to two main categories ,ie,one is the neurogenic displine and the other is myogenic one with strikingly contrasting arguments stressing on nerve drive or musle drive respectively.The newest definition about DO from the authoritative ICS is that this disease entity could follow into two categories.One is the neurogenic DO if the suffers are demonstrated with a related neurological impairment and however,if not it is called the idiopathetic DO which is commonly secondary to BOO with the BPH as the most frequent reason. Accumulating evidence has now indicate that the morphological and functional detrusor remolding after BOO play a pivotal role in the DO's pathophysiology .Given that DO with distinctive etiology may vary in pathological mechanisms and the DO caused by BOO is the most common type ,we decided to choose the DO arising from BOO as our working model. According to myogenic displine of DO,the hyperexcitability of detrusor myocytes is closely associated with DO but however,the direct evidence is lacking after our painstaking literature reviewing,so it is reasonable to choose this myth as our focus because this is an extremely important issue and might bear fruitful results by resolving it.Our study has 3 parts.In the first part ,we firstly establish the PBOO rat model and then measured the cellular excitability index by the aid of the perforated patch clamp recording which is the best one in the physiologists'disposal when addressing the excitability investigation.In the second part,we investigate the L and T type VDCC channels change by using a combined methods,ie.the perforated whole cell patch clamp and calcium concentration measurement under LSCM using the fluo-4AM calcium sensitive fluorescence dye in the hope to reflect the VDCC channel activities change in DO detrusor myocytes from different perspectives.In the last part,the BKca channel activity changes were measured by patch clamp technique and its protein expression changes were assayed by western blot . Materials and methods1. The PBOO animal model was established by proximal urethral ligation in the female SD rats.2. Between the sixth and eighth week after animal model ,the validation of DO was performed by a cystometry assay and based on results of this investigation,the rats were grouped as DO and control(healthy rat with matching age but without DO)3. The detrusor myocytes were dispersed by enzymatic digestion.4. The resting membrane potential ,input resistance ,action potential voltage etc were measured by the whole cell perforated patch clamp configuration under the current clamp mode.5. The L and T type VDCC currents were recorded by perforated patch clamp and peak current density and channel kinetic curve were obtained .6. The modulation roles of L and T VDCC on the resting detrusor free calcium concentration were probed by the VDCC antagonists through LCSM calcium concentration measurement method.7. The whole cell BKca currents and STOCs were recorded by perforated patch clamp.8. The single BKca channel activities were measured by patch clamp under inside-out patch configuration.9. The protein level of BKcaα,βsubunits were assayed by western blot .respectively.Results1.The DO rat model induced by PBOO is successfully established and the incidence is as high as 82.3%.2 . A simple and highly reproducible detrusor myocytes dispersion method is successfully established and they are proved to be suitable for patch clamp study.3.The perforated patch clamp configuration which is the most physiological method for cellular electrophysiological study is successfully applied in recording the single detrusor membrane potential4.The results by comparing excitability index between DO and normal detrusor myocytes indicate that the DO detrusor myocytes have much higher excitability than the normal control ,which is a possible reason for DO .5.The first perforated patch recording of voltage gated calcium currents on detrusor myocytes is successfully done .6.The L type calcium channels have changed in DO myocytes as follows(1) The current density increase significantly which will result in more calcium influx after activation(2) The inactivation curve shifts to the depolarization direction and the percentage of channels in inactivating state decrease implying that it needs much higher concentration of calcium channel blockers to completely inhibit the channel opening .7.The T type calcium channels have changed in DO myocytes as follows(1) The current density increase significantly .(2) The window currents of the T channels widen as a result of shift of both activation and inactivation curve.,which would increase the resting free calcium concentration.(3) The activation curve shifts to the hyperpolarization direction which might underly the hyperexcitability of DO detrusor myocytes.(4) T type calcium channels play a vital role in modulating the resting free calcium concentration of both normal and DO detrusor myocytes through the window currents pathway.The change ratio of RCFI is higher in DO detrusor than that in normal after adding the T channel antagonist mibefradil suggesting that the window currents are up-regulated in DO state .8.The whole cell BKca current density decrease in DO detrusor.9.The single channel activities also decrease in DO detrusor myocytes significantly manifested by decrease of both open probability and mean open time.10.Both the frequency and amplitude of detrusor STOCs decrease significantly.11.The protein expression level of bothαandβsubunites of BKca channels decreases significantly.Conclusions1.The detrusor from DO model exhibit hyperexcitability which maybe a key reason for DO .2.Both the L and T type calcium channels have profound changes such as increase of current density and shift of kinetic curve which will facilitate the action potential firing and may be one of the important ionic mechanisms underlying DO detrusor hyperexcitability. 3.Both the protein expression and channel activities of BKca channels decrease significantly in DO detrusor which will impair its capacity to antagonize the excitability and may be one of the important ionic mechanisms underlying DO detrusor hyperexcitability.