Involvement of piezo-1 ion channel in the regulation of acetylcholine release in the mouse urinary bladder

Bashir S1, Sana-Ur-Rehman H1, Noreen M2, Ahsan H1

Research Type

Basic Science / Translational

Abstract Category

Pharmacology

Abstract 48
Basic Science: Pharmacology
Scientific Podium Short Oral Session 5
Wednesday 29th August 2018
11:07 - 11:15
Hall B
Cell Culture Pharmacology Molecular Biology Overactive Bladder
1. College of Pharmacy, University of Sargodha, 2. Tehsil Headquater Hospital Hazro
Presenter
S

Sajid Bashir

Links

Abstract

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 [1]. 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 [2], 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).
Results
RT-PCR revealed abundant piezo-1 mRNA expression in mouse bladder urothelial cells. Piezo-1 immunoreactivity was highly expressed in the cytoplasm and at the plasma membrane of mouse bladder urothelial tissue and in primary cultured urothelial cells (Figure 1 A). Piezo-1 siRNA intervention has reduced piezo-1 mRNA expression and piezo-1 immunoreactivity by ~72% compared to the naïve urothelial cells (Figure 1 A and B). 
Hypotonic solution induced swelling triggered a transient ACh release from confluent urothelial cell monolayers, which was consistently maximum at around 10 min. However, ACh release from piezo-1 knock down urothelial cells was significantly reduced at 10 min compared to control siRNA treated and naive urothelial cells. When confluent urothelial cells were pre-incubated with GsMTX4 (5µM) for 1 hr, the amount of stretch-induced ACh release was significantly decreased (Figure 2).
Interpretation of results
Piezo-1 has previously been implicated an ATP release channel in different cell types including urothelial cells [2]. 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.
Concluding message
Here, we demonstrated for the first time that piezo-1 channel function as a mechanosensor to regulate the non-neuronal ACh release in mouse bladder urothelium. Based on the findings of the present study it can be concluded that pharmacological inhibition of piezo-1 channel mediated ACh release may contribute to the actions of anti-muscarinic drugs and may lead to development of new therapeutics for the clinical management of lower urinary tract storage disorders.
Figure 1
Figure 2
References
  1. Kuteesa et al. (2006). Anticholinergic drugs for overactive bladder. Australian Prescriber, 29(1), 22-4.
  2. Miyamoto et al. (2014). Functional role for Piezo1 in stretch-evoked Ca2+ influx and ATP release in urothelial cell cultures. Journal of Biological Chemistry, 289(23), 16565-16575.
  3. Michishita et al. (2016). Piezo1 expression increases in rat bladder after partial bladder outlet obstruction. Life sciences, 166, 1-7.
Disclosures
<span class="text-strong">Funding</span> None <span class="text-strong">Clinical Trial</span> No <span class="text-strong">Subjects</span> Animal <span class="text-strong">Species</span> Mouse <span class="text-strong">Ethics Committee</span> UOS animal ethics committee