Gating of Pannexin‐1 Channels by P2X7R‐Induced Inflammation or Apoptosis in Macrophages

Abstract

Pannexin‐1 hemichannels (Panx1) function as ATP efflux or dye influx channels and in inflammatory cascades initiated by P2X7 ATP receptors. Caspase‐mediated cleavage of the Panx1 C‐terminus is a gating mechanism for this channel in apoptotic cells. This study is testing whether Panx1 channel activity is similarly triggered by the delayed caspase‐3‐dependent apoptotic response to P2X7R activation in naïve macrophages versus the immediate caspase‐1‐dependent inflammatory response to P2X7R activation in endotoxin‐primed macrophages. Naïve or endotoxin‐treated J774 murine macrophages were transiently (10 min) stimulated with ATP to activate P2X7R and then incubated in ATP‐free medium for varying times prior to analysis of ethidium+ influx as an index of gated hemichannels. Increased ethidium+ influx was observed in endotoxin‐primed cells after 10 min of P2X7R stimulation followed by 10 min of ATP‐free incubation. In contrast, enhanced ethidium+ influx in naïve cells required more prolonged ATP‐free incubation following the 10 min P2X7R stimulation. Current experiments are comparing these responses in primary macrophages from control, Panx1‐knockout mice, and caspase‐1 knockout mice. Secondary activation of macrophage Panx1 hemichannels by P2X7R‐triggered apoptotic or inflammatory caspases may facilitate ATP‐induced ATP release as a feed forward loop for amplifying innate immune responses.Support: R01‐GM36387