Physiological Function And Regulatory Mechanism Of Hyperpolarization-Activated Cyclic Nucleotide-Gated Cation Channel And Its Subtypes On Bladder Excitation In Rat Urinary Bladder

Background and Objective:Change of bladder excitation was the most important reason for the generation anddevelopment of micturational dysfunctions. Obstruction, imflammations and nervousdiseases were candidate for the alteration of excitability of urinary bladder. Overactivebladder(OAB) secondary to the partial bladder outlet obstruction (PBOO) and otherdiseases of central and peripheral nervous system is one of the most common micturationaldysfunction in urology which was represented by urinary frequency, urinary urgency, andeven stress urinary incontinence. The exact pathophysiologic mechanism is unknown untilnow. Cholinergic receptors were chose as pharmacol target in treatment in the clinic aspectstoward OAB. Anti-adrenonergic receptor agents and calcium channel blocker are used alsoas assistance and show effective results. However, it's hard to solve the idiopathic OABcaused by non-neurogenic factors and the treatment for it is not effective enough. There isno doubt that researches on bladder excitability will become a hot spot in this field.Urinary bladder is innervatied by nervous system, while self-excitation without thecontrol of nerve is also a special function of vesicles. Two theories were wide-spreaded inthe world on the changes of bladder excitation in several pathologic statuses."Neurogenictheory" proposed by W.C. de Groat emphasized that all activities of bladder were controlledby central and peripheral nervous system. Series of excitatory and contractile changes wereclosely associatied with nerves. Disturbance of afferent and efferent nerve fibers,neurotransmitter secretion problems and neuroreceptor alterations were candidate for it.Nevertheless,"myogenic theory" proposed by A.F. Brading indicated that changes ofdetrusor excitation, cell-to-cell communication and the physiological functions of chemicaland mechanical stretch reception cells participate in the regulation of bladder excitation. "Myogenic" theories were widely accepted by scientists all over the world as a bestcomplementary for "neurogenic" theories in the field of bladder excitation regulations andget more and more concerns."Myogenic/cystogenic" factors in the controlling of bladder excitation gain much moreinterests right after the charactization of interstitial cells of Cajal in bladder which was firstfound in guinea-pig bladder by a British scientists McCloskey. However, the physiologicalfunctions of bladder ICCs are still controversial and gain many attentions of researchers.From the initiation of bladder activity, integration and transmission of neurosigmals toupload the informations as stimulation receptors, there are still a long way to go. Ionchannels, which were considered as direct controlling module in bladder, especially onbladder smooth muscle cells and interstitial cells of Cajal, were investigated by severalresearch groups as a vital component.Maintain and operation of functions in bladder ICCs rely on the various ion channelslocated on the cellular membrane and intracellular aspects. Lots of ion channels have beenidentified in bladder ICCs, including voltage-gated calcium channels, BK channel,ATP-sensitive potassium channel, rapid sodium channel and chloride channels which wereassociated with cellular excitation. Hyperpolarization-activated cyclic nucleoted-gated(HCN) channel, first found in sinoatrial node and neurons, become more and more"popular" to the scientists because it was associated closely with autorhythmicity. Studiesof HCN channels on excitatory cells were just at the beginning stage and most of theapproaches were applied in neurons and sinoatrial node. Experiments on other excitatorycells expected more investigations and may explore some important significance.HCN channel was a special ion channel firstly found in heart and neurons. Unlike theactivation mode of normal channels which opened in the depolarization period, HCNchannels opened in the hyperpolarization stage and generated an inward cation current. Theunique properties such as complicatied dual-gate mechanism, permeation of admixture ionsand weak single channel conductance were different from most of the other channels. Thecurrent Ih generated by HCN channels was considered to be a pacemaker current inpacemaking activities. In addition, Ih also plays an important role in several basicphysiological functions such as stabilizing the resting membrane potential, regulating theautorhythmicity and controlling the release of neurotransmitters in excitatory cells. Wether Ih current form the slow wave in the gastrointestinal tract is a good and complicatiedquestion.Generally speaking, excitation changes of bladder have a close connection with seriesof clinical symptons secondary to the bladder diseases. The newly found bladder ICC was aspecial interstitial cell and functions a lot in urinary tract and was considered to be a keyfactor in modulation of bladder excitation. It is interesting that we hypothesis themulti-functional HCN channels was located on multi-functional ICCs in bladder, and selectHCN channel and its subtypes as a breakpoint to understand the bladder excitation. So far,few studies have focused on the HCN channels located in the urinary bladder, especiallytheir biophysical properties and their role in cell excitation. Researches about HCNchannels on bladder ICCs may reveal a new pathophysiological theory in urology andprovide new ideas on excitation investigations.Materials and Methods:Female Sprague–Dawley rats (weighing180-220g) were used in studies. RT-PCR andWestern blotting were taken to investigate the transcripts and protein expression of HCN1-4.Double labeled immunofluorescent study was performed then to identify the location ofHCN channel and its subtypes in bladder. Change of cytoplasmic Ca2+and K+combinedwith fluorescent molecular probes was observed to understand the effects of HCN channelblocker on ICCs. Bladder smooth muscle was studied as isolated strips and specific channelinhibitor ZD7288was then applied to characterized the effect of HCN channel and itsmechanism in regulating bladder contraction. Last but no the least, patch clamp techniquewas used in enzymed-dispersed bladder ICCs and Ih current can be recorded in thewhole-cell mode. HCN channel blocker was also applied to explore the changes of thecurrent.Results and Discussion:1. We detected mRNA corresponding to all four of the HCN channel subtypes in theurinary bladder of the rat. The heart and intestine were used as positive controls. All HCNchannel isolations protein can be detected in bladder tissue using Western blotting. Relativeexpression level of the HCN channel subtypes showed the expression of HCN1issignificantly higher than that of other subtypes.2. Within the individual cell bodies, the HCN isoform and kit immunostaining were uniformly present throughout the cytoplasm, but no co-labeling of HCN1-4and smoothmuscle was observed.3. There was no apparent staining for the HCN3subtype in the interstitial regions.HCN1, HCN2and HCN4were the prominent isolations locatied on bladder ICCs.4. Specific HCN channel blocker ZD7288raise the concentration of intracellular Ca2+in an inapparent level of bladder ICCs at resting state, but reduced the concentration ofintracellular Ca2+induced by a higher concentration of extracellular K+significantly.5. Concentration of cytoplasmic potassium can be depressed by ZD7288at restingstate. But ZD7288reduce the concentration of K+induced by a higher extracellular K+wasinapparent.6. The amplitude of the detrusor strips can be reduced by selective HCN channelblocker ZD7288, while contractile frequency was raised by it. Application of Glivec, whichwas a significant ICC inhibitor, makes no difference in contraction followed up withZD7288.7. Bladder ICCs can be isolated by enzyme and used in electrophysiological studies.Typical Ih current traces were recorded and ZD7288can significantly reduce the currentamplitude.Conclusion:1. HCN channel and its subtypes were located on bladder ICCs, neither on BSMCs.HCN3subtype was not expressed in native rat bladder. Other isolations can be found in allregions of urinary bladder.2. HCN channel was closely associated with cellular excitation of bladder ICCs.Intracellular Ca2+can be reduced by using HCN channel blocker when ICCs were activatedand HCN channel participate in stabilizing resting membrane potentials. Influx ofpotassium was not carried out by HCN channel.3. HCN channel can affect the excitation and contraction of bladder, presumably viabladder ICCs and the regulation of series cellular functions probably was carried outthrough Ih current.