Substance P dependence of endosomal fusion during bladder inflammation

Urinary bladder instillation of ovalbumin into presensitized guinea pigs st imulates rapid development of local bladder inflammation. Substance P is an important mediator of this inflammatory response, as substance P antagonis ts largely reverse the process. Vacuolization of the subapical endosomal co mpartment of the transitional epithelial cells lining the bladder suggests that changes in endosomal trafficking and fusion are also part of the infla mmatory response. To test directly for substance P mediation of changes in endosomal fusion, we reconstituted fusion of transitional cell endosomes in vitro using both cuvette-based and flow cytometry energy transfer assays. Bladders were loaded with fluorescent dyes by a hypotonic withdrawal protoc ol before endosomal isolation by gradient centrifugation. Endosomal fusion assayed by energy transfer during in vitro reconstitution was both cytosol and ATP dependent. Fusion was confirmed by the increase in vesicle size on electron micrographs of fused endosomal preparations compared with controls . In inflamed bladders, dye uptake was inhibited 20% and endosomal fusion w as inhibited 50%. These changes are partly mediated by the neurokinin-1 (NK 1) receptor (NK1R), as 4 mg/kg of CP-96,345, a highly selective NK1 antagon ist, increased fusion in inflamed bladders but had no effect on control bla dders. The receptor-mediated nature of this effect was demonstrated by the expression of substance P receptor mRNA in rat bladder lumen scrapings and by the detection of the NK1R message in guinea pig subapical endosomes by W estern blot analysis. The NK1Rs ware significantly upregulated following in duction of an inflammatory response in the bladder. These results demonstra te that 1) in ovalbumin-induced inflammation in the guinea pig bladder, in vitro fusion of apical endosomes is inhibited, showing endocytotic processe s are altered in inflammation; 2) pretreatment in vivo with an NK1R antagon ist blocks this inhibition of in vitro fusion, demonstrating a role for NK1 R in this process; and 3) the NK1R is present in higher amounts in apical e ndosomes of inflamed bladder, suggesting changes in translation or traffick ing of the NK1R during the inflammatory process. This suggests that NK1R ca n change the fusion properties of membranes in which it resides.