• Edith Dalgaard posted an update 1 year, 11 months ago

    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. We therefore explored the possibility that NTPD2 positive cells were fibroblasts, myofibroblasts or neuronal in origin by co-staining with antibodies for fibroblast-specific protein-1, a-smooth muscle actin and calcitonin gene related peptide respectively. The merged images shown in Fig. 5 clearly illustrate that NTPD2 does not colocalize with any of these three cell markers. FSP1- and aSMA-positive cells were predominantly found in the region of the lamina propria proximate to the urothelium. aSMA staining also indicates blood vessels within the lamina propria. Coimmunostaining of neurons revealed that NTPD2 expressing cells are distinct, however in Fig. 5c it can be seen that there is a close association between a well defined neuron and surrounding NTPD2 positive cells. Endothelia did not express NTPD2, in contrast to the expression pattern seen for NTPD1. The urothelium is a major source of ATP released in bladder therefore we were interested to know if any of the NTPDases were expressed by these cells. Fig. 6 shows that NTPD3 is specifically expressed in the urothelium and is differentially localized to the plasma membranes of intermediate and basal cells. The presence of lateral actin staining and corresponding tight junctions in the superficial umbrella cells can be seen in the top middle panel. However, there is little evidence for colocalization of NTPD3 at lateral borders of the umbrella cells, indicating that the superficial cells of the urothelium are unlikely to express this enzyme. Antibody staining within the lamina propria is localized to cells within blood vessels and detrusor shows no evidence for NTPD3. A different primary antibody to NTPD3 confirmed the intermediate and basal cell distribution of NTPD3 by precise colocalization with aquaporin 3, a marker of these cell membranes. NTPD8 immunostaining of bladder showed a diffuse relatively non-differentiated signal throughout several regions and there was little evidence for a concentration at cell boundaries. There is however a suggestion from the merged images that NTPD8 may be present in the superficial cells of the urothelium but the lack of clear membrane localization for this surface enzyme requires caution in interpretation. To demonstrate the efficacy of the antibody, liver sections were immunostained as a positive control. Mouse liver showed specific and higher intensity staining patterns with appropriate cell border localization to canaliculi. A different primary antibody to NTPD8 was also tried but gave identical staining patterns on both bladder and liver sections. We conclude that expression of NTPD8 may be low and that antibody staining is not sufficiently sensitive to define its location with certainty. RT-PCR supports this conclusion with NTPD8 signal lower than for other family members. NT5E which is WZ4002 inquirer responsible for the conversion of AMP to adenosine was clearly absent from urothelium and from lamina propria but was present throughout detrusor smooth muscle in a pattern very similar to that seen for NTPD1 The importance of purinergic signaling for urinary bladder function has become clear, with a broad spectrum of bladder pathologies now known to exhibit aberrant purinergic metabolism. ATP release from the urothelium is significantly elevated in aging, interstitial cystitis, in spinal cord injury, during inflammation and in syndromes of detrusor overactivity resulting in urgency and/or incontinence. Furthermore overactive bladder has been shown to broadly downregulate the expression of P2X receptors in detrusor while conversely P2X3 was upregulated in sensory nerve fibers from patients with neurogenic detrusor overactivity. P2X3 is also upregulated in a model of outlet obstruction in rats while human patients with outlet obstruction had elevated P2X1 and P2X2 receptors in their bladder smooth muscle. P2X2/X3 are also elevated in urothelium of patients with interstitial cystitis. Furthermore, visceral pain originating from tube and sac-like organs is now thought to be critically dependent on ATP signaling between epithelia and adjacent sensory neurons. Therefore painful bladder syndromes of mysterious etiology might occur through mechanisms in which nucleotide signaling is dysregulated or accentuated. The existence of ATP/ADP degrading enzymes on the surface of cells had been recognized for decades, but molecular identification of the first member of the NTPDase family was not elucidated until the mid-1990s. It is now understood that these enzymes modulate purinergic signaling through effects on ligand availability to P1 and P2 receptors in virtually every tissue of the body and have been shown to play important functional roles in vasculature and the immune and nervous systems. The experiments presented here, are therefore intended to define the expression and localization patterns of NTPDs. We believe this is the first systematic attempt to catalog and describe the location of ectonucleotidases within the mammalian urinary bladder. We successfully amplified specific mRNA for all eight members of the NTPD family as well as for NT5E, thus confirming the likely importance of modulating nucleotide concentrations within bladder tissue elements. Our goal in this study was to characterize the distribution of nucleotide-hydrolyzing enzymes which could modulate the signaling of secreted ATP/UTP. Therefore we focused in more detail on the four cell surface localized enzymes known to specifically catabolize extracellular ATP as well as NT5E. Western blotting confirmed that all five were expressed in bladder, but using immunofluorescence we were only able to unequivocally confirm the localization of four, since NTPD8 exhibited low expression levels.