Several lines of evidence suggest that part of the defect occurring during bladder inflammation is located at the interface of urine in the bladder lumen with transitional epithelial cells lining the bladder. Not only is the subapical endosomal pathway of transitional epithelial cells disrupted structurally with vacuolization during inflammation, but in some animal models of bladder inflammation endosomal fusion reconstituted in vitro is inhibited. The most quantitatively important mediators of bladder inflammation known, neurokinin-1 (substance P) and bradykinin, both have seven transmembrane domain G-protein linked receptors present in the disrupted endosomal pathway. These receptors may be functionally important mediators of bladder inflammation as receptor antagonists largely restore the defect in endosomal fusion associated with bladder inflammation. The current application aims to fully develop or refute the hypothesis that part of the defect occurring during bladder inflammation is inhibition of endosomal fusion in the subapical endosomal pathway of transitional epithelial cells. The application seeks to define the molecular components that modulate changes in endosomal fusion in the subapical endosomal pathway. We propose to determine whether effects on endosomal fusion are a general phenomenon of bladder inflammatory models, and whether the peptides neurokinin-1 and bradykinin can reproduce these effects. The molecular mechanisms by which changes in fusion are mediated will be examined at several points in the signal transduction cascade: effects of neurokinin-1 and bradykinin-2 receptors on endosomal fusion, G-protein mediation of fusion effects, and the identity and role of the final common pathway fusion proteins such an syntaxins and VAMP's. Finally, analogs of another neurally derived peptide, somatostatin, are potently anti-inflammatory in a number of clinical settings including interstitial cystitis. We will determine if this agent with clinical potential reverses the inhibition of endosomal fusion reconstituted in vitro associated with models of bladder inflammation.
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