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, 2000). We combined 8-PIP-cAMP together with 8-HA-cAMPS tuclazepam to preferentially activate PKA-1 (reported to result in a 5:1 affinity over PKA-2), and we combined 6-MBC-cAMP and Sp-5,6-DCl-cBIMPS to preferentially activate PKA-2 (reported to result in a 60:1 affinity over PKA-1). PKA-1 activation did not cause any significant inhibition of budding. Activation of PKA-2, in contrast, completely arrested the budding process (Fig.?2A). Immunofluorescent staining for the regulatory subunit of PKA-2 revealed its existence along the WD in both the WDctrl (Fig.?2D) and WDPKA+ (data not shown). That PKA-2 is observed in both the budded and unbudded sections of the WD raises the possibility that while all WD cells have some level of non-activated PKA-2 component and, as a result, the capability of transducing the upstream cAMP signaling necessary to limit UB formation, actual activation of PKA-2 in the unbudded region of the WD resulting in the localized increase in PKA activity may be mediated by upstream factors that would increase cAMP regionally. To determine whether the inhibitory effects of PKA on Wolffian duct budding resulted in changes in cellular growth rate, cellular proliferation was determined using BrdU (Fig. 3A�CC). Budded and unbudded portions were examined separately. Quantitative data from over 20 cultures indicated a significant difference selleckchem in the rate of cellular proliferation between the budded and unbudded portions of the WDctrl (p?Tenofovir chemical structure is not statistically different from the basal level of proliferation in the unbudded portions of the WDctrl would indicate that either PKA may be blocking budding independent of any effect on proliferation, or that PKA may in fact be blocking the local increase in proliferation that is expected in the budding region. Moreover, immunohistochemical staining for ZO-1, E-cadherin, and Connexin-26 in the non-budding PKA-activated WD revealed no effect on localization of tight, adherens, and gap junctions respectively ( Supplementary Data: Figures?2A�CC, E�CG). cAMP is known to move through gap junctions and could play a role in recruitment of cells for the budding process ( Kanaporis et al., 2008).
