Muscarinic receptor-induced contractility of mouse urinary bladder strips is not influenced by the urothelium

Abstract

The urothelium is thought to exert a direct influence on urinary bladder smooth muscle (UBSM) contractility by releasing active agents such as acetylcholine, ATP, and nitric oxide. Previous studies suggest that urothelial cells express muscarinic receptors, and upon stimulation, factors released from the urothelium may modulate UBSM contractility in response to muscarinic receptor activation on UBSM. This study aimed to determine whether the urinary bladder urothelium affects the contractility of UBSM in response to muscarinic stimulation. We hypothesized that the muscarinic receptor-induced contractile response of UBSM would be attenuated in the presence of the urothelium.Urinary bladders were isolated from male C57Bl6/J mice post-euthanasia. After dissection, bladders were placed in ice-cold nominally Ca2+-free dissection solution. Urinary bladders were opened and cut into strips with and without the urothelium. Strips were placed in myograph tissue baths (DMT) with physiological saline solution for contractility analysis. Each strip was stretched to 1g and was left to equilibrate for 45 minutes (min). Strips were washed 2-3x before applying carbachol (CCh). CCh was applied at concentrations of 0.001 μM, 0.01 μM, 0.1 μM,1 μM, and 10 μM with at least a 5-min waiting period between doses. [GH1] Contractile responses were analyzed using LabChart software and GraphPad Prism. Data were analyzed using a 2-way ANOVA with Tukey’s test for multiple comparisons. P<0.05 was considered statistically significant. CCh induced a biphasic contraction in UBSM strips, which consisted of a rapid initial phasic contraction followed by a smaller stable elevation in tone. In strips without the urothelium (n=16 strips from 8 mice), peak contractions were 0.069 ± 0.104, 0.087 ± 0.102, 0.335 ± 0.302, 0.992 ± 0.606, and 1.197 ± 0.636 g in concentrations 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, and 10 μM of CCh, respectively. In urothelium-denuded strips (n=15 strips from 8 mice), peak contractions were 0.054 ± 0.132, 0.077 ± 0.160, 0.365 ± 0.323, 1.222 ± 0.791, 1.416 ± 1.075 g across the same CCh concentration range. There were no significant differences in peak contraction responses to CCh at any concentration between urothelium-intact and -denuded UBSM strips (P = 0.2809). Similar results were obtained when quantifying steady-state contraction amplitudes. The contractile response to the muscarinic agent CCh in UBSM strips is not altered by the presence of the urothelium. This finding is in contrast with previous studies, and it suggests that muscarinic receptor stimulation on urothelial cells does not alter the contractile response induced by the activation of muscarinic receptors in the UBSM. This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK125543). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.