Age-Associated Changes in Urothelium-Dependent ATP Release Advancing Understanding of Overactive Bladder Syndrome (OAB) Pathophysiology Ayse Gurpinar, Maxwell Roberts, Lisiane Meira, Patrizia Camelliti and Changhao Wu Department of Biochemistry, Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH,UK Normal bladder function relies on release of ATP from the urothelium (epithelial lining of bladder) [1]. This ATP communicates the degree of bladder fullness to the central nervous system. As a key regulator of urinary bladder sensation, disturbed release of this ATP is believed to contribute to pathophysiology of OAB [1]. Introduction Documented Key Evidence Prevalence increase with age for both sexes [1] Affects 30% of people over 65 years worldwide [2] Costs the NHS nearly £2 billion every year [3] Patients exhibit increased stretch-induced urothelial ATP release [4] Patients exhibit increased urothelial secretion of neuropeptides associated with inflammation [4] Methodology Hypothesis: Inflammatory mediators can modulate urothelial function via specific pathways during bladder ageing, contributing to aetiology of OAB. Animal model: Young (12-33 weeks) and aged (>80weeks) male C57BL/6 wild type mice. 1. Longitudinal full-thickness tissue strips were prepared via microdissection and fixed within organ bath with continuous perfusion 2. Superfusate samples were collected at specific times and tested for ATP content via Luciferin-Luciferase assay 3. Interventions used: Substance P (SP: inflammatory neuropeptide), Interleukin- 1α (IL-1α: pro-inflammatory cytokine), Thapsigargin (TG: raises intracellular Ca2+) and Staurosporine (STS: Protein kinase C (PKC) inhibitor) Results  Aged bladders exhibit variations in some pathways  Inflammation (IL-1α and SP) can induce urothelial ATP release in both young and aged bladders  PKC signalling dysfunction (STS) can be associated with augmented ATP release in aged bladders  Aged bladders might be associated with dysregulation of intracellular Ca2+ (TG) for ATP release Conclusions 1. Silva-Ramos⁎, M., Silva⁎, I., Oliveira, O., Ferreira, S., Reis, M., Oliveira, J. and Correia-de-Sá, P., 2013. Overactive bladder syndrome: Evidence that urinary ATP is a dynamic biomarker of detrusor overactivity. Autonomic Neuroscience, 177(1), pp.21- 22. 2. Puckering, G., Walker, D., Xu, L., Congdon, P. and Gooch, K., 2019. The Prevalence and Forecast Prevalence of Overactive Bladder in the Medicare Population. Clinical Medicine Insights: Urology, 12, p.117956111984746. 3. Ganz ML, Smalarz AM, Krupski TL, Anger JT, Hu JC, Wittrup- Jensen KU, et al. Economic Costs of Overactive Bladder in the United Kingdom. Urology [Internet]. 2010 Mar [cited 2020 May 5];75(3):526–32, 532-18. 4. Nässel, D., Zandawala, M., Kawada, T. and Satake, H., 2019. Tachykinins: Neuropeptides That Are Ancient, Diverse, Widespread and Functionally Pleiotropic. Frontiers in Neuroscience, 13. References Acknowledgements This study is University of Surrey, NIH [1R01AG049321- 01A1] and BBSRC [BB/P004695/1] supported. All the experiments mentioned above fulfil the ethical requirements. Figure 2.Illustrates the experimental set up. The arrow indicates orientation of the tissue strip in the organ bath. Figure 1. Depicts OAB phenomena [1]. Young Wild Type Figure 3. Data are expressed as median±interquartile range and tested by Wilcoxon’s. (SP[n=10]: con - 8[5,19] SP - 11[7,27] *p<0.05, IL-1α[n=14]: con- 12[5,26] IL-1α- 14[6,43] **p≤0.01,TG[n=13]: con- 11[8,14] TG- 15[7,20] * p<0.05, STS[n=15]: con- 11[6,19] STS - 13[6,26] NS p>0.05). Aged Wild Type Figure 4. Data are expressed as median±interquartile range and tested by Wilcoxon’s. (SP[n=9]: con - 6[4,19] SP - 15[8,39] **p≤0.01, IL-1α[12]: con- 6[4,15] IL-1α- 18[4,31] *p<0.05,TG [n=11] con-12[4,15] TG-9[6,20] NS p>0.05, STS[n=14]: con- 7[4,10] STS- 12[6,14] *p≤0.05). SHAPING FUTURES : This is the first project to compare the effect of inflammation on ATP release in