PLA enabled highly specific detection of M3 receptor expression, demonstration of M3/GP differential coupling and intracellular M3 trafficking in human detrusor smooth muscle cells. This new approach minimized background fluorescence and antibody cross-reactions resulting from single antibody application, and enhanced specificity due to the use of two primary antibodies. Use of subcellular markers allowed visualization of subcellular receptor location. PLA/CLSM allows analyses of muscarinic "receptor - G protein - promiscuity" and intracellular trafficking even in bladder paraffin sections and may give new insights into the etiology and pathology of BPS/IC.

Aims: To explore caveolae-and clathrin-mediated internalization of muscarinic M2 and M3 receptors, recycling and degradation in formalin-fixed paraffin-embedded detrusor sections; to study alterations possibly involved in the pathophysiology of the bladder functional disorder, interstitial cystitis/bladder pain syndrome (IC/BPS). Materials and Methods:Samples of IC/BPS (n = 11) and cystectomy patients (n = 11) were analyzed. Proximity ligation assay (PLA) was used to detect interactions of M2 and M3 with endocytotic regulators (Cav-1, clathrin, Rab7, and Rab11) by Cy3 labeling. Analyses of three-dimensional (3D)-reconstructed z-stacks (63 × Oil 1.4) were done with Huygens software. We determined the object density for quantification and assessed membrane localization.Results: Receptor/protein complexes were detected as well-demarcated 3D objects. Interactions of M2 with Cav-1, clathrin, Rab11, and Rab7 were significantly increased in IC/BPS. M3/clathrin and M3/Rab11 complexes were higher in IC/BPS, while M3/Cav-1 and M3/Rab7 were not. A significant shift of complexes from the membrane to cytoplasm was observed in conjunction with increased internalization via clathrin vesicles or caveolae in IC/BPS. Conclusions: High numbers of M3/clathrin and M3/Rab11 complexes reflect the well-documented clathrin-mediated desensitization of M3 and speak in favor with enhanced receptor protein expression in IC/BPS. Increased amounts of M2/Cav-1, M2/clathrin, and M2/Rab11 complexes represent altered M2 internalization and recycling leading to high abundance in IC/BPS. In this regard, caveolae-localized M2 could be possibly associated with the activation of nitric oxide (NO) synthase and NO production.