Abstract
We have recently reported an important role of Connexin 43 (Cx43) hemichannels in the pathogenesis of lethal sepsis through facilitating ATP efflux to potentiate the double-stranded RNA-activated protein kinase R (PKR)-dependent macrophage activation. Here we further elucidated the possible role of Pannexin 1 (Panx1) hemichannel in lethal sepsis by assessing its expression along with the impact of a Panx1-specific mimetic inhibitory peptide, 10Panx, on macrophage hemichannel activity in vitro and animal sepsis lethality in vivo. Both crude bacterial lipopolysaccharide (LPS) and purified serum amyloid A (SAA) effectively induced the expression and extracellular release of Panx1 by macrophages or monocytes as judged by Western blotting and immunocytochemistry assays. In animal model of lethal sepsis, Panx1 expression levels were significantly elevated in the heart, but reduced in the kidney, lung, spleen, and blood. At relatively lower doses (10, 50, and 100 mg/kg), the Panx1 mimetic peptide, 10Panx, reproducibly exacerbated the sepsis-induced animal lethality, reducing survival rates from 60–70% to 0–10%. Consistently, 10Panx did not inhibit, but rather promoted, the LPS-induced elevation of Lucifer Yellow dye uptake, ATP release, and Nitric Oxide (NO) production. Collectively, these findings suggested that elevated macrophage Panx1 expression and hemichannel activation contribute to the pathogenesis of lethal sepsis.
Highlights
To determine whether extracellular Panx[1] or HMGB1 was associated with membrane-containing cell debris or microvesicles, the macrophage-conditioned culture medium was subjected to differential centrifugations (Fig. 2a), and each fraction was immunoblotted with Pannexin 1 (Panx1)- or HMGB1-specific antibodies
Monocytes originate from bone marrow hematopoietic stem cells, and continuously circulate in the blood to search for invading pathogens or damaged tissues
The pathogenesis of sepsis is partly attributable to dys-regulated inflammation propagated by both macrophages and monocytes in response to pathogen-associated molecular patterns (PAMPs, e.g., bacterial endotoxin) or host-derived inflammatory mediators[8]
Summary
The expression profile of another class of hemichannel protein, Pannexin 1 (Panx1), during lethal sepsis remains poorly understood. Pannexins were first discovered in flatworm by Panchin et al as a class of innexin-like membrane channels that form gap junctions in the invertebrate animals. They were named as pannexins (‘pan’, meaning ‘all’ in Greek) to reflect their widespread expression in many vertebrate species[21]. Unlike their invertebrate counterparts, the vertebrate pannexins assemble into hexameric hemichannels that allow ATP release from many types of cells[22,23]. Here we sought to examine whether Panx[1] protein was inducible in macrophage cultures, and how 10Panx influences macrophage hemichannel activation in vitro and affects the sequelae of lethal sepsis in vivo
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