Pharmacologic estimations of the relative contributions of the parallel pathways to total ATP release are necessarily approximate, given possible cross-target inhibitions. However, our previous results obtained with 21 inhibitors and PX1 knockdown suggest that these estimations are likely correct for human TM cells.
20 An alternative strategy based on mimetic peptides has proved unsuccessful in generating selective inhibitors.
36 In contrast, probenecid is relatively selective in blocking PX1 and not Cx hemichannels,
37 and CBX also preferentially blocks PX1 hemichannels in low concentrations
38 with a reported IC
50 value 5- to 20-fold lower than that required to block Cx hemichannels. We found that 0.1 mM PRO and 3 μM CBX produced identical inhibition (∼36%) of ATP release by TM5 cells, consistent with an inhibition of 31% ± 2% produced by partial knockdown of PX1.
20 Heptanol is a relatively selective blocker of Cx hemichannels at 1 mM,
39 but it also blocks PX1 hemichannels at 3 mM.
40 External Ca
2+ selectively blocks Cx hemichannels.
41 Once again, the two relatively selective blockers of Cx hemichannels produced similar inhibitions of ATP release, 1 mM heptanol inhibiting by 44% ± 3% and 2.5 mM Ca
2+ inhibiting by 36% ± 7%.
20 Blocking PX1 and Cx hemichannels concurrently produced an additive inhibition.
20 We have assayed the contribution of P2X
7 receptors with the blocker KN-62,
42 which also inhibits calcium/calmodulin-dependent protein kinase II (Ca/CaMKII). However, other P2RX
7 inhibitors, brilliant blue G (BBG)
42 and A438079,
43 produced the same inhibition of ATP release by TM5 cells, and the selective inhibitor of Ca/CaMKII lavendustin C
44 was ineffective.
20 Simultaneously blocking PX1, Cx, and P2RX
7 pathways inhibited ATP release from TM5 cells by >90%.