Changes In HCN Channel Expression And Electrophysiological Characteristics In Bladder ICCs From Rats With Detrusor Overactivity

Background and objective:Overactive bladder(OAB) is a common disease caused by detrusor overactivity(DO) in the department of urology, which the pathogenesis of DO development has not been elucidated. Detrusor excitability abnormalities are the direct cause of DO; And nerve disease, bladder outlet obstruction, and urinary system infection can lead to enhancement of detrusor excitability, which the clinical clinical features show frequent micturition, urgency and urgency incontinence. In severe cases, people can die from serious kidney, ureter and renal function damage eventually. The bladder has dual autonomic innervation / neurons, therefore the occurrence mechanism of detrusor excitability changes are complex. So far, the two main theories including neurogenic and myogenic theory. However, the single theory of neurogenic or myogenic can not explain regulation mechanism of bladder excitation. In 1996, Smet et al. found ICC-like cells in guinea pig and human bladder, the characteristics of which were similar with the gastrointestinal ICC, and the bladder ICCs are distributed in bladder submucosa and smooth muscle bundles. In organizational structure, these cells were close with bladder ICC cell, nerve and detrusor smooth cells. Previous studies also confirmed that ICC cells and cholinergic submucosal plexus are linked with each other closely, and cholinergic receptors of M2, M3 are expressed in ICC cells. The electron microscopy revealed that there were exist desmosomes and gap junction between ICC cells and bladder detrusor cells. All these evidences indicated a pivotal role of bladder ICCs in the regulation of bladder excitation. The discovery of bladder ICCs provided a common pathway of bladder excitation regulation mechanism for neurogenic/myogenic theory may help us to find excitement of bladder regulation, and these findings also elucidated the regulatory mechanism of ICC cells will help understand the excitement of regulation and OAB pathogenesis of bladder.HCN channel is cation channel and activated at condition of hyperpolarization, which consists of 6 transmembrane segments(S1-S6), an amino acid sequence part between S5 and S6 formed the unique GYG symbol sequence of K+ ion selective channels. Ih current is based on HCN channel which now is considered as an important feature of pacemaker cells. Ih current play an important role in the physiological functions including the heart and brain pacemaker activity. The study found that the HCN1-4 channel subtypes were expressed on bladder ICC cell, and the inward hyperpolarizing Ih current generated by HCN channels were also found in bladde ICCs. The HCN channel blocker ZD7288 could reduced the current of T type calcium channel in the bladder ICC cell, and could significantly reduce the frequency and contraction amplitude of bladder detrusor. These findings showed that the HCN channel could regulatie the calcium influx of bladder ICCs. Therefore, HCN channel regulated excition of ICC cells play an important role in bladder excitated regulation. In many states of diseases, the activation and physiological characteristics of HCN channel changed. Previous study found that over activation HCN channels existed in ventricular muscle cells of heart failure. So, we supposed that DO may be related with changes HCN channel of bladder ICCs. The changes of expression and electrophysiological of HCN channel in DO bladder ICCs are beneficial to elucidate the pathogenesis of DO, and provide a new target for the treatment of DO in the future.While the causes of DO is most common in PBOO disease, of which BPH is most reason. A large number of studies show that the change of quantity and physiological function of ICC cells of bladder after bladder outlet obstruction play an important role in the occurrence and development of PBOO induced by DO. In summary, the regulation role of HCN derived ICC cells was important in bladder excitation. Our study used method of PBOO to construct the model of DO rats, and the sham operation group rats regarded as the normal control group. We also detected changes of expression and electrophysiological of HCN channel in bladder ICC cell with the methods of molecular biology and immunofluorescence; we used physiological method to detect electrophysiological properties of the HCN channel; and we used bladder muscle contraction experiments to detect the impact of systolic function in normal and DO rat bladders.Method:The first part: establishment and identification of DO rat modelThe DO model of rats were established by PBOO method. We used the lower abdominal incision, abdominal postoperative suture ligate the urethra in rats to simulate the model of rat partial bladder outlet obstruction. Sham operation group rats were cut and sutured, which the rats with a free next urethral ligation. Eight weeks after operation completion, the rats were tested with bladder filling pressure experiment, and we observed rat bladder voiding contraction is normal or not. In the operation group rats, if the bladder filling of manometric experiments the rats showed abnormal bladder contractions which were recorded as DO model rats; sham operation group rats were recorded as normal control group.The second part: the changes in expression of HCN channel in ICCs of DO bladder1. The method of RT-PCR was used to detect changes in expression of the mRNA of HCN1-4 channel subtype in control group and DO model group rats bladder;2. The method of WB was used to detect the changes in expression of the protein of HCN1-4 channel subtype in control group and DO model group rats bladder;3. The method of immunofluorescence double staining was used to detect the changes in expression of protein of HCN1-4 channel subtypes in ICC cells of control group and DO model group rats bladder.The third part: the changes in electrophysiology of HCN channel in DO rat bladder ICC cells and the effect of HCN blocker ZD7288 on bladder contraction1. The patch clamp method was used to detect the physiological changes in of HCN channel current of Ih in ICCs isolated from control group and DO model group rats bladder;2. The living cells workstation method was used to detect the influence of HCN channel specific blocker ZD7288 on calcium signaling in ICCs isolated from control group and DO model group rats bladder;3 The rats bladder muscle strip experiment was used to detect the effect of HCN channel specific blocker ZD7288 on the function of contraction of the bladder muscle source in control group and DO model group rats bladder.Results:The first part: establishment and identification of DO rat modelBladder filling pressure experiments showed that DO rats bladder micturition reflex were not showed a filling- micturition- filling activity, which showed an involuntary bladder contraction. These evidences suggested that DO rat model had been successfully construced by PBOO operation.The second part: the changes in expression of HCN channel in DO ICCs in rat bladder1. The RT-PCR results showed the expression of HCN1-4 channel subtype mRNA in DO bladder on ICC cells significantly increased compared with the control group;2. The WB results showed the expression of HCN1-4channel subtype protein in bladder DO on ICC cells was significantly increased compared with the control group;3. The immunofluorescence double staining showed the expression of HCN1-4 channel subtype protein in bladder DO on ICC cells was increased obviously compared to the normal group.The third part: the changes in HCN channel electrophysiology in DO rat bladder ICCs and the effect of HCN blocker ZD7288 on bladder contraction1. The results of patch clamp show that Ih current amplitude of ICCs was significantly increased in the DO group compared to the normal control gorup, and the maximum half activation voltage of V1/2 of Ih current was obviously reduced;2. The results of viable cells workstation showed that 10 μmol/L ZD7288 could significantly inhibit the calcium ion concentration of ICC cells in control group, while the same 10 μmol/L ZD7288 can not inhibit calcium ion concentration of ICC cells in DO group. When the concentration of ZD7288 increased to 50 μmol/L, all calcium ion concentration of ICC cells in the DO group and normal group will significantly inhibited;3. The results of bladder detrusor strip experiments showed that 10 μmol/L ZD7288 could significantly inhibit the contraction of detrusor strips in control group, while the same 10 μmol/L ZD7288 cannot inhibit the contraction amplitude of DO group of detrusor strip. When the concentration of ZD7288 increased to 50 μmol/L, all detrusor strip contraction in the DO group and normal control group will significantly inhibited;Conclusions:This study aims to explore the changes in expression and physiological of HCN channel in ICCs of DO and normal control rat bladder.Firstly, All HCN channel isoforms were highly expressed in bladders from the DO group, which was correlated with increased Ih currents in DO ICCs. Secondly, the increased of HCN expression enhanced the bladder ICC intracellular calcium signal and contraction of bladder function. These results suggested that HCN channels may be involved in the pathogenesis of DO, and HCN channel may be as the potential targets for the treatment of DO.