Variations between G protein-coupled receptor-mediated signalling pathways in the urinary bladder

Abstract

One of the most common causes of lower urinary tract symptoms are a result of dysfunction to the bladder’s ability to contract. In normal micturition, the detrusor smooth muscle responds to acetylcholine to facilitate voiding and as a result, the main pharmaceutical treatments target the muscarinic receptors. However, other receptors may also be involved in bladder contractions. Aside from the muscarinic receptors, stimulation of a variety of other receptors and systems in the urinary bladder tissue are known to cause strong and sustained contractions. These include the histamine (H1), 5-hydroxytryptamine (5-HT2A, serotonin), neurokinin (NK2), prostaglandin (PGE2), and angiotensin-II (ATII) receptors. Any link to contractions provides a potential association with contractile disorders, such as overactive and underactive bladder. However, the mechanisms underlying contraction, including the second messengers and associated receptor pathways, are not fully understood. As such, investigating the contractile-mediating receptors and their downstream signalling pathways presents an area of interest.

This thesis aimed to investigate the G protein-coupled receptor systems (GPCRs) which are known to mediate strong contractions in urinary bladder tissue: muscarinic; histamine; 5-HT; NK; PGE2; and ATII, and to determine the signalling pathways underlying their identified responses. As age is often correlated with increased urinary tract symptoms, age-related alterations to the signalling pathways underlying receptor-mediated contractions within the detrusor smooth muscle were also investigated. To achieve these aims, isolated tissue baths containing adjacent strips of urothelium with lamina propria (U&LP), or detrusor smooth muscle, were used to observe the contractile activity in response to receptor activation with various agonists. The influence of extracellular calcium (Ca2+), intracellular Ca2+, and Rho kinase in receptor-mediated contractions of the urinary bladder was also assessed.

In response to agonist stimulation, muscarinic, histamine, 5-HT, NK, PGE2, and ATII receptors in both the U&LP and detrusor, demonstrated increases in baseline tension and spontaneous phasic contractile activity. The magnitude of influence of Ca2+ from extracellular sources varied between the receptors but was responsible for approximately 20-50% of the contractile activity of the six GPCRs in the U&LP. A prominent role of L-type Ca2+ channels in the cell membrane was identified in the U&LP for receptor-mediated contractions, due to a lack of any differences identified between responses after directly removing Ca2+ from the extracellular fluid or using an L-type Ca2+ channel antagonist, nifedipine. The contribution of Ca2+ from intracellular stores was also investigated. In the U&LP, histamine exhibited the only significant dependence on intracellular Ca2+, which was responsible for 40% of the contraction. Alternatively, in the detrusor smooth muscle, intracellular Ca2+ was responsible for 38% of contractions to NKA and 35% of contractions to PGE2. Contractions induced by stimulating the muscarinic, histamine, 5-HT and ATII receptors were not significantly affected by inhibiting intracellular Ca2+. The influence of Rho kinase was also investigated for this pathway’s contribution to GCPR-mediated contractions. Both the U&LP and detrusor of juvenile and adult porcine urinary bladders had a strong dependence on Rho kinase for contraction, and the Rho kinase inhibitor Y-27632 abolished contractions for all six GPCRs assessed by approximately 50%.

Uncovering insights into the mechanisms underlying novel receptor systems in the urinary bladder may present novel therapeutic targets for future pharmacological therapies in the management of bladder disorders. This may also provide further understanding of the pathophysiology underlying bladder contractile disorders, such as underactive and overactive bladder.

Date of Award	6 Jun 2024
Original language	English
Supervisor	Christian Moro (Supervisor) & Russ Chess-Williams (Supervisor)