Moreover, a specific PAR1 agonist also GW786034 VEGFR/PDGFR inhibitor elicited MIF mRNA upregulation establishing that thrombin-mediated MIF effects are due to its well-described affinity for PAR1 receptors. PAR receptors, although not studied in extensive detail in the urogenital tract, have been described in primary human urothelial cells and urothelial cancer cells in vitro. In addition, PAR1-4 receptors were described in mouse urothelium. Given that urothelial cells express PAR1 receptors and also constitutively synthesize MIF and release MIF in response to inflammatory stimuli, we hypothesized that thrombin would elicit MIF release from urothelial cells. Therefore, as part of our investigation of MIFmediated bladder inflammation, we examined whether: 1) Transformed normal human urothelial cells expressed PAR receptors in general, and PAR1 receptor specifically and also whether they express MIF; 2) the location of PAR1 receptors and MIF in rat urothelium; 3) whether thrombin stimulation elicits MIF release from human urothelial cells in vitro and from rat urothelial cells in vivo and 4) whether thrombin stimulation elicits MIF upregulation in human urothelial cells in vitro and from rat urothelial cells in vivo. The present study shows that: 1) urothelial cells express PAR1 receptors and MIF; 2) Thrombin stimulation of urothelial cells evokes MIF-release in vitro and in vivo and 3) Thrombin stimulation also induces MIF upregulation in urothelial cells in vitro and in vivo. These results indicate that activation of PAR1 receptors mediates MIF release from urothelial cell which can then mediate MIF-mediated bladder inflammation, given MIF’s pro-inflammatory role in the bladder. Therefore, thrombin-induced MIF release represents another mechanism to initiate MIF release from the urothelium, aside from nervemediated release which has already been described. Expression of PAR1 and PAR2 receptors was described for normal human urothelial cells and an urothelial cancer cell line and these receptors were shown to be functional since they respond to agonist stimulation. Expression of PAR1-4 receptors has also been reported for an additional human urothelial cancer cell line however, receptor functionality was not investigated. In the current study we document expression of PAR receptors 1 through 4 in normal transformed human urothelial cells . In addition, we document that UROtsa cells express MIF and we show, using dual-immunofluorescence that UROtsa cells can express PAR1 and MIF simultaneously. We observed heterogeneity in PAR1 immunostaining in most of these cells with approximately 13% not displaying immunoreactivity for either PAR1 or MIF. Similarly, only approximately 30% of J82 cells were reported to be positive for PAR1 immunostaining. Differences in PAR1 immunostaining may be due to differences in the cell cycle, differences between normal and transformed cell lines or differences in immunostaining protocols and antibodies. In rat urothelium, we also detected MIF and PAR1 immunostaining.