Identification of anoctamine (Ano1) calcium activated chloride channel in porcine urinary bladder and characterization of its functional role using niflumic acid

DA Bijos1,2, MJ Drake1,2, B Vahabi1,3. 1Bristol Urological Institute, NBT NHS Trust, UK, 2University of Bristol, Faculty of Medicine and Dentistry, UK, 3University of the West of England, Department of Applied Sciences, UK

Introduction: Interstitial cells (ICs), analogous to the interstitial cells of Cajal in the gut, may generate phasic activity (PA) in smooth muscle tissues including the bladder. An established marker of the ICs is c-kit [1]. However, recent studies have shown that Anoctamin-1 (Ano1, encoded by Tmem16a), a calcium activated chloride channel (CaCC), has a fundamental role in generation of pacemaker activity in the ICs of the gut and therefore could be used as a novel marker for these cells [2]. CaCC blocking drugs such as niflumic acid were able to alter the pacemaker activity of the ICs in the gut and thus may be important modulators of these cells in other tissues. Thus, the aim of this study was to investigate whether Ano1 is expressed in the porcine urinary bladder and to explore the role of niflumic acid in modulating the phasic activity of the bladder tissue.

Methods: Female pig (~6months old) bladders were obtained from the local abattoir, hence ethical approval was unnecessary. PCR was carried out on the c-DNA synthesized from total RNA isolated from bladder mucosa (composed of the urothelium, lamina propria and a thin layer of smooth muscle (muscularis mucosa)) and denuded detrusor. Primers were designed for Sus scrofa Ano1 mRNA (XM_003122417.2). PCR products were separated by electrophoresis and sequenced. Longitudinal strips of denuded detrusor (n=7) or mucosa (n=47) were mounted in perspex microbaths and superfused with Krebs’ solution at 37ºC. Isometric tension was measured via UF1 force transducers connected to a Powerlab using LabChart software. Denuded detrusor strips were superfused constantly with 0.1µM carbachol (CCh) solution to induce PA. The effect of increasing concentrations of niflumic acid (1-30µM added cumulatively, 10-min exposure for each concentration) or drug vehicle (DMSO) on spontaneous and CCh-stimulated PA was investigated by measuring the amplitude and frequency of PA. All data are expressed as the mean±SEM. Statistical analysis was carried out by using repeated measure ANOVA followed by Dunett’s post hoc test.

Results: Ano1 mRNA expression was found in both mucosal and detrusor layers of the porcine urinary bladder. Niflumic acid did not have a significant effect on the amplitude or the frequency of CCh-stimulated PA in the denuded detrusor strips at all concentrations. However, the amplitude of basal PA in mucosal strips was significantly reduced (p<0.001) with 10µM (22.3±4.6% inhibition) and 30µM (26.6±4.4% inhibition) niflumic acid. The frequency of basal mucosal contractions was reduced only at 30µM (25.0±7.7% inhibition) niflumic acid (p<0.001). Drug vehicle had no effect on PA.

Discussion: Niflumic acid was able to modulate the basal phasic contractions of mucosal strips which may suggest a role of ANO1 channels in mediating the PA of ICs found in the suburothelial layer of mucosal strips. However, niflumic acid had no effect on the cholinergic-induced PA of the denuded detrusor strips. This may be due to different mechanisms of activation of ICs in the mucosa vs. the detrusor.

1. Torihashi S. et al (1995). Cell Tissue Res. 280, 97-111.

2. Hwang SJ. et al (2009). J Physiol. 587, 4887-904.