Research On Mechanism Of EP3 Activation On The Bladder Excitability

Backgrounds and purposes:Overactive bladder (OAB) is a syndrome which is accompanied with many urological diseases in clinical, such as the infection of urinary tract, neurogenic bladder, interstitial cystitis, bladder pain syndrome and so on. Sometimes this disease could occur independently. The OAB is a symptom with urinary urgency accompanied by urinary frequency and nocturia, with or without urinary urgency incontinence. OAB could lead to ureteral hydrops, hydronephrosis and even severe damage to the renal function. The pathogenic factors of OAB remains unknown. In clinical, the treatment for this disease mainly focus on the medicine for neuroreceptors such as M receptor and unfortunately,these treatment could not be satisfied. Therefore, the research on OAB attracts more and more interest. The recent research has found that the level of prostaglandin E2 (PGE2) was increased in the patients’ bladder, which indicated that PGE2 was an important factor to the development of OAB. Moreover, there were researches showing that PGE2 could enhance the frequency and amplitude of bladder detrusor. However, the mechanism of PGE2 function on bladder hyperactivity keep mystery. The receptors of PGE2 have attracted more and more attention. The research has found that EP3 could influence the voiding interval and urine volume which were also demonstrated in the EP3 knockout mice. Intravesical instillation with PGE2 in mice could induce the bladder hyperactivity, while it had no effect on the EP3 knockout mice. These researches indicated the important role of EP3 in the bladder hyperactivity and the mechanism also need more further research.The dysfunction of bladder detrusor excitability usually leads to the detrusor overactivity (DO) which could induce the OAB. The research on the bladder detrusor excitability will be significant for the mechanism of OAB. Researchers were believed that the bladder detrusor excitability was mediated by the nerves for many years. However, it is thought there are limitations in the theory of innervation of bladder detrusor with further research, especially when the research indicated that the bladder detrusor could also contract rhythmically in vitro. The spontaneous excitatory system independent of innervation in bladder was proposed and attracted more and more interest. The first indication that the bladder might contain ICC came from a study by Smet et al.3 who observed cyclic GMP immunopositive cells in guinea-pig and human bladder which had a morphological resemblance to gut ICC. More recently, the presence of ICC in the bladder had been demonstrated by using the special marker c-kit for ICC. ICC could induce excitement and transmit the signal to the smooth cells via the tight junction in gut. In another word, ICC act as the pacemakers and intermediaries in the peristaltic activity throughout the gut. As for the ICC in the bladder, people thought they had the same function as ICC in gut and therefore, the spontaneous contractions of bladder detrusor independent of innervation could be explained to some extent, the number of ICC was increased in the patients’ bladder with OAB,which indicate the significant role of ICC in the bladder excitability. However, the understanding of the physiological functions of ICC in the urinary bladder is comparatively limited and need much research to elucidate the specific roles of these cells in all aspects of bladder function.During the research on the ICC in the bladder, Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels were found in ICC of bladder. HCN channels are composed of 4 subtypes and could be activated in the hyperpolarization of cells. When the HCN channels are activated, the Na+ and K+ become permeable to the cell membrane and therefore,it could induce an inward current which is called "funny current"(If) because of the special activation pattern. If had also been designated as pacemaker current and it played a key role in controlling rhythmic activity of cardia pacemaker cells and spontaneously firing neurons. It was demonstrated that HCN channels were present in the bladder and they only existed in the ICC of bladder. The western blotting has indicated that HCN1 channels were the main subtype in the ICC of bladder, while the other three subtypes had less expression. The existence of HCN channels in the ICC of bladder has not only provided a further evidence that the spontaneous excitability of ICC in the bladder, but also provided a novel target for the research on the mechanism of the ICC excitability.However, the regulator of HCN channels remain more concern.Taken together, EP3 activation plays an important role in the bladder function and the excitability of ICC based on the HCN channels is also related to the regulation of bladderexcitability. It indicates the relationship of the EP3 activation and the ICC excitability in the bladder. Therefore, we will verify the hypothesis that EP3 activation could influence the bladder excitability via the HCN channels in bladder. Our research will illuminate the mechanism of bladder excitability regulation and provide novel target for the treatment to urinary diseases in clinical.Methods and material:Section 1: The expression of EP3 in the ICC of bladderDouble immunofluorescence staining was used in this section to explore the expression of EP3 in the ICC of bladder by the antibodies for EP3 and c-kit marker for ICC.Section 2: The influence of EP3 activation on HCN channels in the ICC(1) Firstly, intravesical instillation with sulprostone (EP3 special agonist) to mice bladder was used to establish the mice model. Then western blotting was used to compare the expression of HCN channels in mice instilled by sulprostone with that in normal mice.(2) Secondly, the ICC of bladder was isolated with acute enzyme isolation. The whole-cell patch clamping technique was then employed to detect the influence of PGE2 and sulprostone on the HCN channels function of ICC.(3) Thirdly, single cell PCR technology was used to demonstrate that the cells used in our whole-cell patch clamping were ICC by the c-kit marker.(4) Fourthly, coimmunoprecipitation was employed to explore the interaction between the EP3 and HCN channels.Section 3: the influence of EP3 activation on the excitability of ICC.The ICC isolated from the mice was cultured and then used to examine the function of PGE2 and sulprostone on the intracellular concentration of calcium in the ICC via confocal microscopy. ZD7288 (antagonist of HCN channels) was also used to demonstrate whether the function was via HCN channels.Section 4: the influence of EP3 activation on the contract of bladder detrusor.The rats and HCN1 knockout (HCN1-/-) mice were also used in this section. The function of EP3 activation on the bladder contraction was tested by detrusor strip contractions in vitro. The influence of HCN channels in the function of EP3 activation was also investigated in this section.Results:(1) As the double immunofluorescence staining revealed, EP3 was existed in the ICC of bladder.(2) Western blotting indicated that the expression of HCN1 and HCN4 in the mice bladder instilled with sulprostone was higher than that in the normal mice, while the expression of HCN2 and HCN3 had no significant difference.(3) The whole-cell patch clamping demonstrated that PGE2 and sulprostone could both enhance the HCN channel current.(4) As coimmunoprecipitation revealed, there was interaction between EP3 and HCN1 or HCN4 channels.(5) Under confocal microscopy, the intracellular concentration of calcium in the ICC was increased with PGE2 and sulprostone administration. However, the function of PGE2 and sulprostone could be interrupted by ZD7288.(6) Just as the detrusor strip contraction indicated, the amplitude of bladder detrusor was increased with PGE2 and sulprostone. These functions were interrupted by the HCN channel antagonist ZD7288.(7) When the L798106 (EP3 special antagonist) was used firstly, the PGE2 had no effect on the bladder detrusor contraction.(8) Both PGE2 and sulprostone could increase the amplitude of the HCN1-/- mice bladder detrusor, while the effects were less than those in wild-type mice.Conclusion:(1) EP3 was existed in the ICC of the bladder.(2) EP3 could interact with HCN channels and therefore, increased the expression of HCN channels and enhanced the HCN current.(3) EP3 activation could facilitate the excitability of the ICC via HCN channels.(4) EP3 activation could facilitate the bladder excitability via HCN channels.Taken together, EP3 activation could facilitate the bladder excitability via HCN channels in ICC.