The presence of ectonucleotidases in bladder has not been studied systematically; however their existence was inferred, since the half-life of ATP is very different depending on which side of the urothelium it is released from. In Ussing chamber studies, Lewis and Lewis showed that both constitutive and stretchinduced ATP release from the luminal surface of rabbit bladders increase ATP concentration in a linear fashion with continuous accumulation, whereas serosal ATP rises and then plateaus �C the kinetics of which are consistent with its initial release and then subsequent consumption. Our long term goal is to develop an in-depth understanding of the regulation of purinergic signaling in bladder and its importance to normal and abnormal bladder function. Since secreted nucleotides are potent stimulators which may exert both autocrine and paracrine TC-I 2000 effects, our focus in these experiments was to determine the expression and location of cell-surface ectohydrolytic members of the NTPD family. Furthermore, as this group is not capable of completing the final phosphohydrolysis step which results in production of adenosine �C another important signaling molecule, we also characterized tissue distribution of NT5E in bladder. Our findings suggest specific and synergistic mechanisms for the control of nucleotide availability throughout the stratified layers of the bladder. Immunostaining of frozen bladder sections was performed for all five proteins of interest. By counterstaining actin with rhodamine-phalloidin we are able to clearly identify cell layers and tissue boundaries throughout the bladder. Confocal immunofluorescent laser scanning microscopy revealed that NTPD1 is expressed at high levels in endothelium of vascular elements occurring prominently within the lamina propria and is also present throughout the detrusor RQ 00203078 smooth muscle. There was no evidence for NTPD1 in the urothelium which is typically three cell layers deep. Merged panels on the right show that the protein is in or near plasma membranes as expected. NTPD2 was also absent from the urothelium but was distributed differentially in the lamina propria. The merged images in Fig. 4a and 4b show a region immediately subjacent to urothelium which is actin positive but NTPD2- negative. In the more distal region of the lamina propria, dispersed but interlinked cells with non-uniform morphology are NTPD2-positive. This positive staining pattern extends deep into the detrusor in an organized filamentous pattern which clearly delineates and surrounds smooth muscle bundles. These cells exhibit narrow elongated and branched cell processes.