| Background and purposes:Overactive bladder (OAB) is a class of the abnormal voiding dysfunction syndromes,with urgency as main complaint, with or without complaint to urge incontinence, often withfrequent urination or nocturia. This disease can occur independently, but also is associatedwith a variety of urological diseases, such as urinary tract infections, benign prostatichyperplasia, senile voiding dysfunction, neurogenic bladder, urinary tract obstruction,interstitial cystitis/painful bladder syndrome and lower urinary tract tumors, urinary stones,unexplained male and female urinary frequency and other symptoms. If not timelyterminate the course of the disease, severe cases will also cause renal-hydroureter andimpaired renal function. The incidence of the disease increases with age and growth. Itseriously affect the quality of life of the patients. Detrusor excitability abnormalities arecommon pathogenesis of these diseases and the course of the diseases pathway. It isnecessary to research and clarify the abnormal excitability regulation and the mechanism ofsuch diseases, for basic, clinical and social significance.Bladder is controlled by both autonomic nerve and somatic nerve, which makes themechanism of excitability change more complex during the urine storage. The role andmechanism of excited dysfunction disease are not yet fully elucidated. The two majordoctrines, neurogenic/myogenic, are the main theories. The neurogenic theory is long-termdominant. It considers that the the bladder excitement regulation, as well as the majorphysiological activities are from the nerve. Nerves play a decisive dominant role in thephysiological and pathological mechanisms of bladder. And the clinical treatment ofabnormal excitability is mainly focus on the drugs controlling nerve receptors, such as theM-receptor blocking agent, but the efficacy is not satisfied in many patients. At the sametime, the presence of spontaneous contraction of the bladder or detrusor muscle stripshighly suggested there are other regulatory factors addition to neural factors. On this basis, the myogenic theory came into being. The doctrine thinks that the detrusor cells themselveshave pacing and excited function, and advocate the use of inhibition of cell excitabilitydrugs for clinical treatment. The limitations of this theory are that it can not explain the "allor none phenomenon" of bladder contraction, and the clinical treatment have no happyeffect. Therefore, to explore the the bladder endogenous excitatory complete mechanism, toclarify its origin, regulation, and changes in bladder excitability disorders, have a veryimportant significance for clinical diagnosis and treatment.The discovery and identification of the existence and function of the bladder pacingsystem is an important part of endogenous excitatory regulation of the bladder. Reported inthe literature and our preliminary studies have confirmed:①Bladder pacemaker-like cells(Interstitial cells of Cajal, ICC) play an important role in the origin of spontaneouscontractile activity of the bladder.②Bladder ICCs have the Ih characteristics of current aspacemaker cells, with special biological characteristics of pacemaker cells.③Theconnection and signal transduction between detrusor cells confirmed that bladder ICCs arethe hub of excited regulation.In excitatory ion channel regulation, calcium channels have pivotal positions. Ca2+isimportant ion in regulation of muscle cell excitability and contraction. Calcium channelactivation voltage is divided into high (L-type) and low (T-type). L-type calcium channelsare high-voltage activated Ca2+channels, commonly found in various types of cells, mainlyinvolved in the contraction of muscle cells. In detrusor, its open voltage is-30/-25mv, andthe maximum the open voltage is-10/0mv, to form the action potential overshoot. T-typecalcium channel is mainly involved in the regulation of cell excitability, having thefollowing characteristics:①low membrane potential activation;②rapid inactivation andslow closure dynamics;(3) low single-channel conductance;④strong Ni2+selectiveblockade;⑤Ca2+, Ba2+relative infiltration. Since the T-type Ca2+channel has activationcharacteristics of low membrane potential, so that it plays an important role in the initialstage of the action potential.The successful identification and isolation of bladder ICCs, suggesting that it may bethe center of the origin and regulation of excitibility. T-type calcium channel has beenconfirmed as the main channel in the bladder excitatory regulation. In theory, there shouldbe some connection between T-type calcium channel and ICCs, pursuant to which we confirmed the presence of T-type calcium channel in bladder ICCs by pre-experimental. Itprovides the possibility for further study. How the T-type calcium channels regulate theexcitability of bladder ICCs? What is the difference between the various subtypes of Tchannel expression in normal and DI bladder ICCs? Can upward or block the T-typecalcium channels change excitability of the bladder and bladder ICCs? as well as theregulation of T-type calcium channel in bladder ICCs function, is still a lack of in-depthresearch.In order to confirm that the expression of T-type calcium channels in rat bladder ICCs,and to explore the role of T-type calcium channel subtypes in regulation the ICCs andbladder excitability, this study conducted a preliminary exploration.Materials and Methods:We used200-240g size SD rats, and made bladder outlet obstruction (partial bladderoutlet obstruction, PBOO) induced detrusor instability (the detrusor instability DI) ratmodels. Using double immunofluorescence staining technique, both specific marker ofICCs c-kit receptor antibody and antibodies of T-type calcium channel subtypes (α1G, α1H,α1I) were stained to observe T type calcium channel subtype expression in ICCs. Further,we detected the expression of T-type calcium channel subtypes in rat normal and DI bladderby RT-PCR and Western blotting, and observed changes of T-type calcium channel isoformsbetween normal and DI bladder. To investigate the functional role of T-type calciumchannel on spontaneous action potential of ICCs, the changes of intracellular calcium([Ca2+]i) were detected after isolated ICCs loading fluo-3AM, using mibefradil(T-typecalcium channel elective blocker) and glivec mesylate (Glivec, a c-kit tyrosine kinaseinhibitor) to treat the cells. To investigate role of T-type calcium channels on mediatingphasic contractions in ICCs, bladder strips were isolated from adult rats and mounted intissue baths. Strips were stimulated with low concentration of the muscarinic receptoragonist carbachol (CCH;1μM) to upgrade phasic contractions and the effect of mibefradiland glivec both at a certain concentration (1μM) were then investigated. Finally, wedetected the T-type calcium current on acute isolated bladder ICCs by patch clamptechnique, and recorded the impact of Mibefradil on T-type calcium channel current.Results:1. We successfully established DI models. Immunofluorescence double staining displayed T-type calcium channel subtypes (α1G, α1H, α1I) co-expression in the bladderICCs, RT-PCR and WB detection further confirmed this result; DI when ICCs The subtypeexpression volume increase on α1G, α1H, α1I α1G increased the most obvious.2. ICCs were loaded with the intracellular Ca2+indicator fluo-3AM and imaged using aconfocal microscope. The ICCs spontaneously producted a Ca2+wave that appeared to startat a focus in the cell and spread axially in2directions and along the branches. Mibefradil(1μM) significantly inhibited [Ca2+]i(p<0.01). Then glivec(1μM) was added to act on thesame ICCs. Glivec decreased the [Ca2+]iof ICCs further(p<0.01).(n=50from ten animals).3. Bladder strips appearanced phasic contractions and CCH could upgrade them.Mibefradil(1μM) significantly inhibited the amplitude of CCH-upgraded phasic activity ofthe detrusor strips but not the frequency(p<0.05). Glivec(1μM) significantly inhibited theamplitude (p<0.05)of phasic activity in tissue incubated with CCH and mibefradil but notthe frequency. Another group of bladder strips, glivec was used firstly. Glivec significantlyinhibited the amplitude (p<0.05) of CCH-upgraded phasic activity of the detrusor strips butstill not the frequency. However, mibefradil did not significantly inhibite the amplitude andfrequency of phasic activity in tissue incubated with CCH and glivec.4. We got acute enzyme isolated rat bladder ICCs. We recorded the typical T-typecalcium current in ICCs by patch clamp. The calcium current enhanced in DI bladder ICCsand can be significantly inhibited by Mibefradil.Conclusion:1. T-type calcium channel α1G α1H, and α1I subtypes were expressed in the bladderICCs. The expression levels of them were increased in DI bladder, and there was significantchange of expression level of α1G.2. T-type calcium channels are involved in the regulation of ICCs spontaneousexcitability and detrusor excited contraction.3. T-type calcium channel-specific blockers could inhibit the excitability of ICCs,which provides new therapies for the treatment of overactive bladder. |
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