eATP levels had been suppressed in chondrocytes treated with ANK siRNA compared to these treated with a scramble handle, with out alter ations of ecto enzyme activities or cell viability, ANK mRNA and protein levels have been considerably reduced in ANK siRNA treated chondrocytes. To ensure that reductions in eATP in ANK silenced cells were not indirectly resulting from decreases in ePPi levels, we added back 10 to one hundred uM NaPPi towards the media of ANK silenced cells and measured eATP levels. NaPPi did not alter the pH from the media, which remained at pH 7. four. As shown within the representa tive experiment in Figure 3D, the presence of exogenous PPi did not restore eATP levels in ANK silenced cells to wards levels noticed inside the scramble manage. Nonetheless, there were compact increases in eATP levels within the pres ence of added ePPi observed across groups, which were not statistically substantial.
These data suggest that the re duction in eATP seen with ANK silencing is not mediated by adjustments in ePPi concentrations. Probenecid, which has been shown to inhibit ANK mediated PPi transport, decreased eATP levels inside a dose dependent manner, experienced ANK might act selleck to straight transport ATP or regulate other ATP transport mechanisms We also created experiments to test for the presence of classic ATP egress pathways by investigating the effects of inhibitors of those pathways. None in the pharmaco logic inhibitors lowered basal eATP levels together with the excep tion of probenecid, Table 1 summarizes the effects of those pharmacologic inhibitors on eATP levels measured after a hypotonic challenge. Results are expressed as the fold transform in eATP levels just after a hypo tonic challenge in the presence from the inhibitor compared to the absence with the inhibitor. Despite the expression of hemichannels, such as pannexin 1 and connexin 43, by chondrocytes in the protein and mRNA levels, numerous pharmacological inhibitors known to target hemichannels failed to suppress osmotically induced chondrocyte ATP. The impact of 10panx1, a tiny pep tide inhibitor of pannexin 1 hemichannels, was indistinguishable from its manage peptide at concen trations from one hundred to 400 uM. Flufenamic acid and carbenoxolone also failed to substantially suppress hypotonically induced eATP production.