Hypothesis / aims of study
Overactive bladder (OAB) is mostly caused by detrusor uninhibited contraction. Our previous study has demonstrated the important role of chloride channels on the regulation of urinary bladder smooth muscle tone. Application of chloride transport inhibitors or chloride channel blockers could significantly suppress the agonist-induced contraction of bladder smooth muscle. In a study using rat model of bladder outlet obstruction, increased expression of calcium activated chloride channel (CaCC) in detrusor overactivity myocytes has been demonstrated . Our recent study further revealed significantly increased expressions of CLC-3 and CLCA4 chloride channels at both protein and mRNA levels on OAB rat bladder tissue using a cyclophosphamide (CYP) induced OAB model, suggesting that chloride channels may play important roles in the pathogenesis of OAB. We therefore conduct this study to investigate the effect of intravesical instillation of various CaCC blockers on voiding function evaluated by continuous infusion cystometry (CMG) in CYP-induced OAB rats.
Study design, materials and methods
A total of 64 adult male Sprague-Dawley rats (10–12 weeks) were divided into two groups (CYP-OAB and control). CYP-induced OAB was provoked by four i.p. injections in 7 days (CYP-OAB: 80 mg/kg and control: saline). Continuous infusion cystometry (CMG) was performed under anesthesia to record the basal pressure, maximum bladder voiding pressure (MBVP), intercontraction interval (ICI) and episodes of spontaneous contraction. At the end of baseline CMG, the infused physiological saline solution (PSS) was replaced by one CaCC blocker solution for 30 minutes. Then the infusion solution was replaced again by the same reagent at a higher concentration for another 30 minutes. This protocol was repeated with a concentration from 10^-6M to 10^-1M (niflumic acid, NFA and 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, DIDS) or 10^-7M to 10^-2M (T16inh-A01 and N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid, MONNA) to construct the concentration-response curve of each CMG parameter and determine the inhibitory concentration 50 (IC50) of every reagent (N=8 for each reagent).
As expected, the results of CMG showed significant increase of the basal pressure, decrease of the MBVP, decrease of the ICI and increase of episodes of spontaneous contraction after CYP injection, demonstrating the characteristics of OAB. The increased basal pressure in OAB group could be significantly ameliorated by intravesical administration of NFA (≧1X10-3M), DIDS (≧1X10-4M), T16inh-A01 (≧1X10-5M) and MONNA (≧1X10-4M) in a concentration dependent manner. The reduced MBVP in OAB group could also be significantly recovered by intravesical treatment of NFA (≧1X10-3M), DIDS (≧1X10-4M), T16inh-A01(≧1X10-5M) and MONNA (≧1X10-5M) in a concentration dependent manner. The shortened ICI in OAB rats could be significantly lengthened by intravesical infusion of NFA (≧1X10-5M), DIDS (≧1X10-5M), T16inh-A01 (≧1X10-6M) and MONNA (≧1X10-6M) in a concentration dependent manner. The increased episodes of spontaneous contraction in OAB rats could be significantly normalized by intravesical infusion of NFA (≧1X10-2M), DIDS (≧1X10-3M), T16inh-A01 (≧1X10-5M) and MONNA (≧1X10-5M) in a concentration dependent manner. On the contrary, intravesical administration of CaCC blockers via replacement of infused PSS solution could not interfere with the CMG parameters in control group. Generally, the IC50s of TMEM16A channel blockers (T16inh-A01 and MONNA) for all CMG parameters were lower than those of traditional CaCC blockers (NFA and DIDS).
Interpretation of results
The change of CMG parameters including basal pressure, MBVP, ICI and episodes of spontaneous contraction in OAB rats could be "reversed" by intravesical administration of different traditional CaCC and TMEM16A channel blockers in OAB rats. However, the CMG parameters could not be affected by all the reagents in control group. IC50s of TMEM16A channel blockers for all CMG parameters were lower than those of traditional CaCC blockers, indicating that TMEM16A channel blockers are more potent in treating OAB.