This proposal is a competing continuation of a four year grant which seeks to expand upon the role of mast cells and mast cell products, substance P (SP), and LPS in bladder inflammation and further elucidate the molecular pathways resulting in up-regulation of inflammatory genes. The original specific aims were to determine the role of neutral metalloendopeptidase (NEP), SP receptors, and mast cells in the development of cystitis, utilizing NEP -/- mice, NK-1R -/- mice, and mast cell deficient mice (kit[w]/kit[w-v]), and those which have been reconstituted with mast cells by adoptive transfer from normal mice and NK-1R -/- mice. In the present proposal, we seek to expand upon the hypothesis that regardless of the stimuli there is a common intracellular activation pathway of bladder inflammation. We now include the possible role of Protease Activated-Receptors (PAR) as a signaling mechanism owing to tryptase release following mast cell degranulation. There are 6 specific aims in this proposal seeking to determine the role of PARs in the development of acute and chronic bladder inflammation. We will employ methodology and animal models published by this laboratory, including determination of cellular pattern of the 4 different PAR proteins and their gene expression by immunohistochemistry and in situ hybridization. Results will be confirmed by western blotting and quantifiable RNase protection assay developed by this laboratory. The role of PAR in inflammation will be confirmed in PAR -/- mice. In addition, the role of PARs 1 and 2 on mast cells will be determined by examining kit[w]/kit[w-v] mice, which are deficient in mast cells, after they receive bone-marrow mast cells from PAR1-/- or PAR2-/- mice. This study seeks to determine the molecular pathway of acute and chronic bladder inflammation. Information generated by this study should elucidate whether bladder inflammatory responses, regardless of etiology, occur via a common mechanism or whether the endpoints are specific for each stimulus.