Mechanisms For The Generation And Regulation Of Spontaneous Ca²⁺ Transients Recorded From DSMCs And ICCs In The Rat Bladder

Bladder is a hollow muscular organ collects urine excreted by the kidneys prior to disposal by urination. The detrusor muscle is a layer of the urinary bladder wall made of smooth muscle fibers arranged in spiral, longitudinal, and circular bundles. As a speical smooth muscle tissue, the detrusor muscle is double controlled by splanchnic nerve and somatic nerves. Whether the detrusor muscle can generate spontaneous contraction like heart and gastrointestinal tract do was ever a mysterious question.Our previous research have demonstrated several interesting phenomenons:first, all the strips isolated from bladder exhibit spontaneous contraction activity under certain tension load, and the unstable strips will develop spontaneous contraction under little tension load; second, when under the same tension load, the frequency of unstable detrusor strips increases more significantly than that of normal detrusor; third, the spontaneous contraction is little affected by neural factors. These phenomenons indicated that the spontaneous contraction of detrusor strips may not be neurogenic, and the detrusor should have the ability of generating spontaneous excitability itself.It is also notable that the detrusor muscle exhibited significant increased activity of spontaneous contraction in a urological condition termed as DI(Detrusor instability) induced by bladder outlet obstruction or abnormal nerve function. Compared with normal detrusor strips, Smaller tension load is sufficient to induced spontaneous contractions in unstable detrusor strips.When under the same tension load, the frequency of unstable detrusor strips increases more significantly than that of normal detrusor strips.The enhanced spontaneous contraction also were observed in patients with overactive bladder (OAB)who suffered nearogenic bladder dysfunction and partial bladder outlet obstruction.The etiology of OAB is unclear, and indeed there may be multiple possible causes.In present,two different mechanism were made to explanation cause and development of OAB.â‘  neurogenic mechanism:de Groat is one of supporter of neurogenic mechanism which emphasized that enhance spontaneous contraction all induced by abnormal nerve fuction(for example:up-regulation of number and function of neuroreceptors)and its subsequent effects.â‘¡Myogenci mechanism:Brading and other research insisted that besides neurogenic dysfunction, myogenic factors such as increased excitability of detrusor smooth muscle, enhaced communication between detusor smooth cells,ganglionic cells and up-regulation of the number and function of "pacemaker cells".As for myogenic mechanism of OAB, it is important for subsequent research to confirm whether pacemarker cells exist in bladder.ICCs(Interstitial cells of cajal) possesses different shape and function from smooth muscle cells.It has been accepted widely that as a pacemaker cell of gastrointestinal tract,ureter and other smooth muscle organs, spontaneous excitation of smooth muscle cells orginated from ICCs.With the help of immunofluorescence, ICCs(c-kit positive cells) were found distributed in bladder of guinea pig and human.ICCs, with Fusiform appearance, were showed to have a close relationship with smooth muscle cells and nerve ending and can generate spontaneous Ca²⁺ transients which suggested that ICCs may be the candidate of pacemaker cell of bladder. Subsequently,ICCs were found to drive spontaneous transient outward currents(STOCs) and Glivec, a specific c-kit inhibitot, were showed to significantly inbibit spontaneous action potentials(APs) of smooth muscle cells.These research data prompted that ICCs do have the ability of autodepolarization which confirmed the presence functionally and suggested that similiar gastrointestinal tract and ureter,ICCs may be the pacemaker cell in bladder.However, there are evidences points to the conclusion that APs can also be recorded in smooth muscle cells freshly isolated from bladder which prompted that smooth muscle cells may be another candidate of pacemaker cell of bladder spontaneous contraction .Then is ICCs or DSMCs the pacemaker cell of bladder? Research at tissue level should be taken to confirm since research data at single cell level are not sufficient to answer the question.Release of calcium from intracellular stores, endoplasmic/sarcoplasmic reticulum (ER/SR), is one of the key signal transduction mechanisms that play a pivotal role in the regulation of numerous cellular functions. In smooth myocytes, Ca²⁺ release from intracellular Ca²⁺ stores to cytoplasm are controlled by ryanodine Ca²⁺ release receptors subtype 2 (RYR2), which can occur in form of spontaneous SR Ca²⁺ release events, or Ca²⁺ sparks, and Ca²⁺ release that is triggered by the influx of Ca²⁺ through sarcolemmal ion channels, often termed CICR.Intracellular Ca²⁺-mediated excitation-contraction coupling in smooth muscle cells (DSMCs) is primarily controlled by the influx of extracellular Ca²⁺ and the mobilization of Ca²⁺ from intracellular stores. Ca²⁺ transients represent Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCCs) during action potentials, the influx of extracellular Ca²⁺ produces smooth muscle constriction in part by stimulating Ca²⁺ release from the sarcoplasmic reticulum (SR) through Ca²⁺-induced Ca²⁺ release (CICR).Cyclic adenosine diphosphate ribose (cADPR) is a naturally occurring cyclic nucleotide and represents a novel class of endogenous Ca²⁺ messengers implicated in the regulation of the gating properties of ryanodine receptors (RyRs).In smooth muscle, cADPR can regulate Ca²⁺ transient by modulating Ca²⁺ release through RyR2 from SR. Alteration of gating properties of RyR2 induced by cADPR can regulate depolarization/repolarization and characteristic of Ca²⁺ transients of DSMCs by modulate STOCs(spontaneous transient outward current), STICs(spontaneous transient outward currents) and CICR. Howerver,the role and mechanism of cADPR regulation of Ca²⁺ release remain unclear and very controversial and investigating of the mechanism will help us to further comprehend the procedure that cADPR modulate Ca²⁺ transients of DSMCs.Since there is a close relationship between Ca²⁺ transients and action potentials(APs) in smooth muscle tissue and cADPR is an important Ca²⁺ mobilizer which can modulate Ca²⁺ transients of DSMCs by regulating Ca²⁺ release from SR, we plan to do some works as following:First, immunofluorescence and immunofluorescence double staining protocol were used to obeserve the distribution of ICCs and its relationship with smooth muscle cells in the rat bladder;Second, spontaneous Ca²⁺ transients of ICCs and DSMCs were viewed in detrusor strips loaded with Fluo-4 AM by confocal microscopy, then amplitude and frequecy of spontanoues Ca²⁺ transients in ICCs and DSMCs were compared to initially investigate the function of ICCs;Third, a series of pharmacologic experiments were taken to explore the mechanism for generation and regulation of spontaneous Ca²⁺ transients in ICCs and DSMCs; finally, FKBP12.6 gene knockout mice were used to examine the effect and mechanism of cADPR on Ca²⁺ release in mouse bladder smooth muscle.Materials and methods: Female SD rats of 2-3 months,weighing from 220-280g were used. Immunofluorescence(anti-cKit)and immunofluorescence double staining (anti-ckit and anti-myosin) protocol were used to obeserve the distribution of ICCs and its relationship with smooth muscle cells in the rat bladder. By referencing to methods estabilished by hashitani,we recorded spontaneous Ca²⁺ transients of ICCs and smooth muscle cells with confocal microscopy.Serosa and mucosa removed bladder strips were prepared and superfused with warmed physiological saline(PSS) to recover and maintain their activity. After spontaneous muscle movements of the muscle layers were visually detected, the tissues were loaded with the fluo-4 AM and then spontaneous Ca²⁺ transients of ICCs in the muscle layers were observed with confocal microscopy. Then L type Ca²⁺ channel blocker Nimodipine, T type Ca²⁺ channel blockers Nicl2 ,Ca²⁺ free and High Ca²⁺ physiological saline and several reagents which can interfere SR Ca²⁺ release were added to investigate the mechanism of generation of spontaneous Ca²⁺ transients in ICCs and DSMCs.Then we sought to examine the effect and mechanism of cADPR on Ca²⁺ release in mouse bladder smooth muscle. We have used FKBP12.6 null mice to test directly if cADPR action on Ca²⁺ sparks is through FKBP12.6 disassociation from the RyR2 complex. Genetype of mice were tested by conducting a PCR (Polymerase Chain Reaction) on DNA samples taken from the mouse tail. Effects of cADPR on STOCs(spontaneous transient out currents) and Ca²⁺ sparks were recorded by patch clamp and confocal microscopy. Thapsigargin(A SERCA blocker) was used to detect whether SERCA activity is necessary for cADPR-induced increase in Ca²⁺ sparks.To further confirm that the effect of cADPR on Ca²⁺ spark properties is directly dependent on the dissociation of FKBP12.6 from the RyR2 complex, Western blotting analysis was performed on SR microsomes from mouse bladder smooth muscle. Previous studies demonstrated that the amount of FKBP12.6 bound to RYR2 can influence SR Ca²⁺ load. To determine whether cADPR affected SR Ca²⁺ content, caffeine (10 mM) was used to estimate SR Ca²⁺ load in myocytes of WT and FKBP12.6-null mice before and after cADPR treatment.Results:1. There are three categories ICCs in rat bladder strips, namely ICCs tracking smooth muscle boundary, ICCs between the bundles and ICCs in the bundles.There is a close relationship between ICCs and smooth muscle cells and neuro ending; 2. Properties of spontaneous Ca²⁺ transients recorded from DSMCs in detrusor strips(1) Asynchronous Ca²⁺ transients were obeserved right after the ending of Fluo-4 loading protocol.Some 30 min after superfusion with dye free physiological saline, synchronous Ca²⁺ transients generated in smooth muscle bundles;(2) Mostly, spontaneous Ca²⁺ transients of DSMCs originated along a boundary of each smooth muscle bundle and then spread to the other boundary.Some Ca²⁺ transients originated from the middle of the bundles and travelled to two boundaries.(3) Delay of propagation can be observed along the transverse direction but not along the axial direction;(4) Propagations of spontaneous Ca²⁺ transients between smooth muscle bundles were also observed by confocal laser scanning microscopy;3. Frequency of spontaneous Ca²⁺ transients recorded from DSMCs were significantly faster than frequency of that recorded from ICCs which suggested that ICCs tracking smooth muscle bundles may not be the pacemaker cell of bladder spontaneous contraction;4. Research data of subsequent pharmalogical experiments:(1) Nimodipine can abolish spontaneous Ca²⁺ transients in DSMCs. Frequency but not amplitude of spontaneous Ca²⁺ transients in DSMCs was significantly reduced by Nicl2. No alteration induced by Nimodipine and Nicl2 in spontaneous Ca²⁺ transient were observed in ICCs.(2) Ryanodien(RyR receptor antagonist),Caffeine(RyR receptor agonist),2-APB(IP3 receptor antagonist) and Thapsigargin(SERCA blocker) can significantly inhibite but can not abolish spontaneous Ca²⁺ transients of DSMCs.(3) Contrary to findings in DSMCs, spontaneous Ca²⁺ transients of ICCs can be abolished by Ryanodien,Caffeine,2-APBand Thapsigargin.(4) Ca²⁺ free physiological saline also are found can abolish spontaneous Ca²⁺ transients of ICCs.Frequncy of spontaneous Ca²⁺ transients of DSMCs can be increased significantly by high Ca²⁺ physiological solutions.5. Mice that are from heterozygous (one wildtype allele and one knockout) knockout parents were tested for their genotype by PCR. Three genetype were found in mice:Wildtype(FKBP12.6 +/+),Knockout(FKBP12.6 -/-) and heterozygous (FKBP12.6 +/-).6. On dialysis of 5μM cADPR into bladder smooth muscle cells via patch pipettes, both the frequency and amplitude of STOCs were significantly altered in wildtype cells.The frequency and amplitude of STOCs increased significantly in wildtype cells. In contrast to wildtype cells, the cells from FKBP12.6 null mice did not respond to cADPR stimulation.7. Ca²⁺ sparks are due to the concerted opening of multiple RyRs and STOCs were generated by Ca²⁺ Sparks.Patch clamp and confocal microscopy combined study showed that on dialysis of wildtype cells with cADPR, both STOCs and Ca²⁺ sparks were markedly altered. In contrast, dialysis of FKBP12.6 knockout cells with cADPR resulted in a negligible change in the properties of STOCs and Ca²⁺ sparks.8. After dialysis of cADPR, marked alterations were observed in WT cells but not in FKBP12.6 KO cells. Ca²⁺ spark frequency was increased significantly in wildtype but not in FKBP12.6 null cells. Similarly, on dialysis of wildtype cells with cADPR, peak Ca²⁺ (F/F0),FWHM increased remarkably; rise timeand half-time decay were both prolonged markedly. However, cADPR did not alter Ca²⁺ spark properties in FKBP12.6 knockout cells.9. After dialysis of cADPR, Ca²⁺ spark frequency increased significantly and the alteration in Ca²⁺ spark frequency was not affected by thapsigargin. Similarly, increases in F/F0, FWHM, rise time and half-time decayinduced by cADPR were not markedly affected by thapsigargin.10. The endogenous FKBP12.6 was released from SR microsomes by incubation with cADPR and practically all of the FKBP12.6 was bound to SR microsomes in the pellet.11. It was notable that the FKBP12.6 protein deletion caused a reduction in Ca²⁺ content of SR in wildtype but not in FKBP12.6 null smooth muscle cells.Conclusion:1. Mechanism of spontaneous Ca²⁺ transients' propogation along the transverse direction may be different from that along the axial direction and spontaneous Ca²⁺ transients' propagate between smooth muscle bundles may have some relationship with etiology of detrusor instability;2. Asynchronous Ca²⁺ transients were transformed to synchronous Ca²⁺ transients by superfusion with dye free physiological saline in smooth muscle bundles suggested pacemaker cell did exist in bladder and candidate of the pacemaker may include:â‘ DSMCs at the boundary of smooth bundles;â‘¡ICCs tracking smooth muscle boundary and ICCs in the bundles. However some Ca²⁺ transients also were found originated from the middle of the bundles suggested that different distribution of pacemaker cells may existed in distinct muscle bundles;3. Frequency of spontaneous Ca²⁺ transients recorded from DSMCs were significantly faster than that recorded from ICCs, which suggested that ICCs tracking smooth muscle bundles may not be the pacemaker cell of bladder spontaneous contraction. However, it is far from making a decision that ICCs tracking smooth muscle bundles are not the pacemake cell since we still have some technological problem(for example the limitation from focal length of confocal microscopy) and some ICCs tracking smooth muscle may have different functions. Besides, ICCs in the bundles may be another candidate of pacemaker cell of bladder sponteneous contraction;4. DSMCs generated spontaneous Ca²⁺ transients via extracellular Ca²⁺ entry through L type Ca²⁺ channels and the spontanoue Ca²⁺ transients were partly supported by Ca²⁺ release from Ca²⁺ release from intracellular Ca²⁺ stores; T-type Ca²⁺ channels may contribute to the preceding depolarization to activate the L-type Ca²⁺ channels and maintain the resting potential of DSMCs and thus determine the action potential frequency;5. spontaneous Ca²⁺ transients of ICCs rely on both Ca²⁺ release from intracellular Ca²⁺ stores and extracellular Ca²⁺ entry through non L type Ca²⁺ channels. Candidate of the non L type Ca²⁺ channels may include:â‘ NCX(Na+/Ca²⁺ exchanger)â‘¡T type Ca²⁺ channels andâ‘¢TRP channels;6. cADPR significantly alters Ca²⁺ spark properties and Ca²⁺ content in SR and that the underlying mechanism of cADPR action is related to the dissociation of FKBP12.6 proteins from the RYR2 complex in mouse bladder smooth muscle.