Hypothesis / aims of study
Urothelial cells have the ability to sense changes in their extracellular environment and respond to various stimuli by releasing numerous neurotransmitters such as ATP and acetylcholine (ACh). Non-neuronal release of ACh from urothelial cells has been demonstrated to play an important role in the urinary bladder physiology and pathophysiology by the activation of cholinergic receptors on urothelial cells, myofibroblasts, detrusor muscle cells and nearby bladder nerves. Currently, anticholinergics have widely been used for overactive bladder treatment, which suggests an essential role of ACh in the aetiology of the bladder overactivity . Characterising mechanisms involved in ACh release in the urinary bladder could be interesting and potentially important as a part of multidrug approach, in the treatment of disorders associated with increased ACh release, particularly in aged patients who exhibit a greater incidence of bladder overactivity. In the present study we aimed to investigate for the first time the role of piezo-1, a mechanosensitive ATP release channel , to mediate ACh release in the urinary bladder.
Study design, materials and methods
Immunohistochemistry was conducted on the cross sections of mouse bladder tissue as well as on cultured urothelial cells using a primary anti-piezo-1 antibody (PA5-72974) and secondary Alexa Fluor488 antibody (ab150077 Abcam) for florescence staining. Isolated urothelial cells were cultured and then transfected after 3 hrs with 10 nM siRNA by using Lipofectamine RNAi MAX (Invitrogen). Piezo-1 expression was measured by immunocytochemistry and quantitative RT-PCR after 48 and 72 hrs of cultivation. Stretch experiments were performed after 72-84 hrs of cultivation using hypotonic solution induced swelling and ACh was measured using an Amplex®Red ACh/acetyl cholinesterase assay kit (Invitrogen, A12217).
Interpretation of results
Piezo-1 has previously been implicated an ATP release channel in different cell types including urothelial cells . This study has established for the first time that piezo-1 channel could play an important role in urothelial derived ACh release in the urinary bladder to mediate various cell functions. Piezo-1 was predominantly expressed in the mouse bladder urothelium and its expression was more prominent in the cytoplasm and on the plasma membrane in cultured urothelial cells which is consistent with the previous reports [2,3]. Augmented ACh release from cultured urothelial cells in response to hypotonic solution induced cell swelling, was significantly reduced in piezo-1 knock down cells, and naive urothelial cells in the presence of piezo-1 channel blocker GsMTX4. This suggests that piezo-1 channel release ACh upon mechanical stretch in primary urothelial cell cultures and may regulate mechanosensory transduction in the urinary bladder. Pharmacological inhibition of piezo-1 channel in the presence of GsMTX4 may implicate piezo-1 a novel therapeutic drug target that might improve urine storage disorders such as overactive bladder and/or interstitial cystitis.