High Glucose Conditions Increase Detrusor Cholinergic Excitability by Reducing Caveolae-Mediated Inhibition of Muscarinic M2-Receptor Activation in the Urinary Bladder

Cristofaro V1, Carew J2, Dasari S1, Sullivan M1

Research Type

Pure and Applied Science / Translational

Abstract Category

Overactive Bladder

Best in Category Prize: Overactive Bladder
Abstract 87
Physiology and Pharmacology
Scientific Podium Short Oral Session 12
Wednesday 27th September 2023
18:05 - 18:12
Room 104CD
Overactive Bladder Basic Science Pathophysiology Animal Study Pharmacology
1. Division of Urology, VA Boston Healthcare System, Harvard Medical School. Boston MA, United States., 2. Department of Medicine, VA Boston Healthcare System, Harvard Medical School. Boston MA, United States.
Presenter
Links

Abstract

Hypothesis / aims of study
Caveola are cholesterol-enriched membrane microdomains important for the regulation of specific receptor-activated signaling events. In the urinary bladder, the loss of smooth muscle (BSM) caveolae, or the alteration in the expression of caveolins (Cav), the constitutive proteins of caveolae, has been associated with detrusor overactivity. The early compensatory phase of diabetic bladder dysfunction (DBD) is characterized by a hyperreactive phenotype, but the pathophysiologic mechanisms underlying the changes in smooth muscle contractility with hyperglycemia remain unclear. Although alterations in caveolar elements have been demonstrated with diabetes in several organs/systems, including the bladder, whether hyperglycemia affects the caveolae mediated regulation of BSM contraction has never been explored.  Therefore, the purpose of this study was to evaluate the effects of caveolar depletion on muscarinic receptor-mediated contractile responses in the bladder, and to investigate whether these responses are altered by hyperglycemia.
Study design, materials and methods
BSM tissue from C57 mice were mounted in organ bath for isometric tension studies.  Contractions induced by carbachol (CCh, 1µM) were generated before and after caveolar depletion (achieved by methyl-β-cyclodextrin, mβCD, 10mM, 1 hour), in the presence of either 4-DAMP (10nM) or AFDX (0.1µM), muscarinic-M3 and M2 receptor antagonists respectively. In other experiments, BSM tissue was exposed to high glucose (HG, 23mM), or normal glucose (NG 11.5mM) Krebs solution. CCh contractions were measured at baseline (in NG) and repeated after 2 hours of either hyperglycemic or euglycemic conditions. Responses to CCh after HG exposure were also evaluated after mβCD, or in the presence of AFDX. The effect of hyperglycemia on actin polymerization in BSM tissue was analyzed by F/G actin assay and phalloidin staining. The cellular localization and molecular interaction between M2 and M3 receptors with caveolin proteins were determined by western blotting and immunoprecipitation (IP) experiments.
Results
Under baseline conditions, the experimental depletion of caveolae by mβCD did not affect BSM contractions induced by CCh. In contrast, when the M3-mediated component of CCh contraction was blocked by 4-DAMP, the subsequent depletion of caveolae enhanced the response to CCh. The M3R mediated contraction (in the presence of AFDX) was not augmented by mβCD. IP experiments indicate that muscarinic M3 and M2 receptors interact with Cav-1 and Cav-3. Although exposure of BSM to HG had no effect on muscarinic receptor expression, a significant increase in CCh responses occurred, in parallel with an augmented BSM F/G actin ratio. This HG mediated increase in contractions generated by CCh was prevented by mβCD.  Moreover, after exposure to HG, CCh stimulation induced the translocation of caveolin proteins from membrane to the cytosol, along with the molecular dissociation between Cav-3 and M2 receptor. Finally, the increase in CCh-induced contractions and the increase in actin polymerization observed in BSM tissue exposed to HG were both prevented by AFDX.
Interpretation of results
The enhanced response to CCh observed in BSM tissue after treatment with mβCD in the presence of M3R antagonist indicates that M2 receptor-mediated cholinergic contractions in the bladder are negatively regulated by caveolae. The increased polymerization of actin and the enhanced CCh responses after the acute exposure of BSM tissue to HG suggest that hyperglycemia contributes to a hypercontractile smooth muscle phenotype during the compensatory phase of DBD. The translocation of Cav-3 to the cytosol after stimulation in HG as well as its dissociation from M2 receptor reveal that hyperglycemia alters the molecular interaction between Cav-3 and M2 receptors. Moreover, the effect of the M2 receptor antagonist AFDX in preventing both the HG-induced hypercontractility in response to CCh and the increase inF/G actin ratio suggest that hyperglycemia enhances cholinergic BSM excitability by disrupting the inhibitory regulation of M2 receptors imparted by Cav-3, and promoting M2R-mediated actin polymerization.
Concluding message
The present data describe for the first time a mechanism in the bladder by which M2-mediated cholinergic responses are restrained by the inhibitory interaction of this muscarinic receptor with Cav-3. Exposure of BSM to a hyperglycemic environment appeared to be detrimental for this regulation leading to changes in actin cytoskeletal dynamics in BSM and increased detrusor contractility. These findings identify new molecular partners that could potentially be therapeutically targeted for the management of diabetes-induced bladder hyperreactivity.
Figure 1 Figure 1
Disclosures
Funding Department of Veterans Affairs, Research Service, Washington DC. USA Clinical Trial No Subjects Animal Species Mouse Ethics Committee VA Boston Healthcare System IACUC
Citation

Continence 7S1 (2023) 100805
DOI: 10.1016/j.cont.2023.100805

13/11/2024 22:32:42