| Background and Objective：The main function of the bladder is the storage and discharge of urine, as a result oflower urinary tract symptoms in the important organs of the lower urinary tract, bladderdysfunction, the incidence of urinary frequency and urgency was high. And the incidence ratewill further increasing with the growth of age that reduce the older’s quality of life andbecome an social problem. Bladder function disorders are getting more and more attention,but now scientists not yet well explain the causes and find a solution. Among the bladderdysfunction diseases, the most common is a class described as urinary frequency, urgency, andurge incontinence called overactive bladder syndrome(overactive bladder, OAB). Found thatchronic obstruction, inflammation and other incentives caused the bladder abnormalexcitability, may be an important cause of the OAB. Clinically used cholinergic-blockers, caneliminate the impact of the nerves of the bladder, the treatment supplemented adrenergicreceptor and the calcium channel blocker with treatment, not yet a good deal with OABsymptoms. Bladder own excitement exception may be the real reason for the pathogenesis ofsome patients with OAB, the bladder exciting research has also become a focus in the urinarybladder function disorders.Although the current understanding of bladder was mainly dominated by theparasympathetic and sympathetic nervous system to complete urine storage and discharge.But have found that the bladder has a similar gastrointestinal vitro spontaneous contractilefunction in a period, therefore the source of the excitement control the bladder respectivedevelopment two theories, WC de Groat on behalf of the neurogenic theory and to AFBrading represented myogenic theory. Both unable to explain the drawbacks of the otherphenomena, looking for new excitement origins to become one of the main directions of academic research. Especially in terms of the doctrine of myogenic. The bladder spontaneousexcitement study of the origin of the long-standing failure to get substantial breakthrough"myogenic" theory only as an important complement to the traditional "neurogenic" doctrine,the bladder excitability regulation play the role of self-regulation.Until2002, British scientists, McCloskey found a special kinds of cells in the guinea pigbladder which has similar shape of gastrointestinal tract interstitial cells of Cajal (interstitialcell of Cajal, ICC) and positive staining its marker c-kit."Myogenic" theory so as to inject aburst of stimulants, and open a new chapter of myogenic theory, more and more studies onbladder ICC-like cells and in the renal pelvis, ureter, urethra, prostate, penis multiple organsof the urinary system found c-kit positive staining cells, collectively referred to as theICC-like cells of the urinary tract, the role of these cells in the respective organs havegradually been demonstrated. Where in the renal pelvis, urethra ICC-like cells have beenconfirmed to have a spontaneous excitatory function, play an important role in the urineoutput; bladder ICC-like cells was first discovered, but there is still a lack of deepunderstanding of their physiological and pathological roles. Especially, the cells can be usedas the "pacemaker" cells of the bladder is still controversial. And the the spontaneousexcitatory important physiological basis transmembrane ion flow cell, where calcium ion asexcited ions is widely recognized, not only involved in signal transduction, but also play amajor role in bladder smooth muscle cell contraction. There are also sufficient evidence toprove that the calcium imbalance is one of the important reasons in OAB and urinary bladderfunction disorders. Thus, calcium as the object of study and explains its role in the ICC-likecells in the bladder, will play an important role in resolving the current bladder spontaneousexcitatory.Are known such as myocardial spontaneous excitatory cell membrane, there is a lot ofcalcium ion channels, such as L-type and T-type voltage-dependent calcium ion channels,which are the mainly influx channel of calcium ions, and the efflux of calcium ions mainlyrely on a transmembrane protein of the sodium-calcium exchanger (NCX). NCX because ofits characteristics of two-way transit calcium ions play an important stabilizing intracellularcalcium ion balance including cardiac muscles, neurons and other cells. Urethra ICC-like cellsexpressing only NCX isoform3, and the NCX reverse mode elevated urethral ICC-like cellsintracellular calcium, thus affecting existing urethral ICC-like cells display spontaneous excitatory role. NCX also play an important role in diseases, such as heart failure, arrhythmias,ischemia reperfusion. Therefore bladder voiding dysfunction disease and the role of the NCXin ICC-like cells in the bladder has not been reported yet. Therefore, we speculate that theNCX may also affect the balance of the bladder ICC-like cells, intracellular calcium, therebyregulating excitability.If our hypothesis is true, not only enriched the theory of calcium and ICC-like cell inregulation of bladder excitability. And added a new evidence in the bladder myogenic theory,and provides a target for the clinical treatment of urinary function disorders.Based on the above about NCX in bladder ICC-like cell excitability regulation reasoning,this study first make through molecular biology and morphology of NCX and its subtypes inbladder. Further functional experiment were tested to find the role of NCX and its subtypes inbladder and the mechanism of regulation cell excitability. At last, preliminary study of thebasic physiological characteristics of NCX channels on the ICC-like cells of the bladder.Materials and Methods：In this study, the2-3month rats were used. At first, the expression of the NCX1-3in ratbladder were verified by RT-PCR and Western blotting. Then, the distribution of NCX1-3were elaborated by immunofluorescence technique. The ion fluorescent probe loaded incultured ICC-like cells, intracellular calcium ion concentration in the NCX positive changesin forward and reverse mode, as well as blockers were detected by LSCM. Isometricexperiment were used to in the isolated bladder muscle strips to detect NCX forward, reversemode in bladder excitement-contraction process. Finally, the diaphragm clamp technique onacute isolated ICC-like was used to record NCX current changes in the specific blockerKB-R7943. The ICC-like cells have been verified by single-cell PCR identification.Results and Discussion：1. RT-PCR showed NCX1-3mRNA expressed in bladder tissue, and the brain as control.Western Blot also detected NCX1-3protein expressed in bladder tissue. NCX1subtype wasthe highest proportion and NCX2was the lowest value.2. Laser confocal microscope NCX3positive and c-kit-positive cells in the normal ratbladder coexistence, NCX1-2coexistence of myosin-positive cells.3. NCX forward mode can reduce the concentration of calcium in the bladder ICC-likecells, blocker KB-R7943at high concentrations (30μM) reduces calcium ion outflow speed, but could not stop the decline of the intracellular calcium amplitude.4. The reverse mode of the NCX elevated bladder ICC-like cell intracellular calciumconcentration. Blocker KB-R7943can reduce the speed of intracellular calcium ionconcentration at low concentrations (5μM), but the magnitude was not statistic significantly.5. NCX forward mode can lead to a decline in vitro smooth muscle contractionamplitude and frequency increased, blocker KB-R7943at high concentrations (30μM) canfurther reduce the contraction of the muscle strips, but the frequency change no statisticallysignificant.6. NCX reverse mode can cause the decline in amplitude of the smooth musclecontraction of the muscle strips in vitro, and the frequency increases, KB-R7943blockers atlow concentrations (5μM) can be further reduced the contraction amplitude of the muscle strip,but the frequency was no significantly change.7. Successful acute enzyme isolated rat bladder ICC-like cells, typical NCX current wasrecorded in the cell was voltage-dependent.8. KB-R7943can significantly reduce the strength of the current, and KB-R7943showedthe voltage-dependent and concentration dependent.9. The single-cell PCR confirmed the c-kit positive cells after the patch-clamp test.Conclusion：1. Normal rat bladder NCX1-3, NCX1is main.2. Smooth muscle cells express NCX1-2, bladder ICC-like cells express the NCX3.3. NCX3with the excitability of the bladder ICC-like cells is closely related to the NCX3forward and reverse mode, which can influence the intracellular calcium ion concentration.Blockers KB-R7943at different concentrations display different effects of inhibition, retardelevated intracellular calcium and decrease in speed, but not the magnitude of the changes.4. NCX forward and reverse mode affects muscle strips in vitro smooth muscleexcitation-contraction, but whether ICC-like cells of smooth muscle cells in the regulationrequires further experiments.5. NCX3typical NCX current in bladder ICC-like cells could affecting cell excitability. |
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