Neutrophil elastase increases airway epithelial nonheme iron levels.

Alpha-1-antitrypsin (A1AT) deficiency is characterized by increased neutrophil elastase (NE) activity and oxidative stress in the lung. We hypothesized that NE exposure generates reactive oxygen species by increasing lung non-heme iron. To test this hypothesis, we measured bronchoalveolar lavage (BAL) iron and ferritin levels, using inductively coupled plasma (ICP) optical emission spectroscopy and an ELISA respectively, in A1AT-deficient patients and healthy subjects. To confirm the role of NE in regulating lung iron homeostasis, we administered intratracheally NE or control buffer to rats and measured BAL and lung iron and ferritin. Our results demonstrated that A1AT-deficient patients and rats post-elastase exposure have elevated levels of iron and ferritin in the BAL. To investigate the mechanism of NE-induced increased iron levels, we exposed normal human airway epithelial cells to either NE or control vehicle in the presence or absence of ferritin, and quantified intracellular iron uptake using calcein fluorescence and ICP mass spectroscopy. We also tested whether NE degraded ferritin in vitro using ELISA and western analysis. We demonstrated in vitro that NE increased intracellular non-heme iron levels and degraded ferritin. Our results suggest that NE digests ferritin increasing the extracellular iron pool available for cellular uptake.

An intact connexin N-terminus is required for function but not gap junction formation.

The cytoplasmic N-termini of connexins have been implicated in protein trafficking, oligomerization and channel gating. To elucidate the role of the N-terminus in connexin37 (CX37), we studied mutant constructs containing partial deletions of its 23 N-terminal amino acids and a construct with a complete N-terminus in which residues 2-8 were replaced with alanines. All mutants containing nine or more N-terminal amino acids form gap junction plaques in transiently transfected HeLa cells, whereas most of the longer deletions do not. Although wild-type CX37 allowed intercellular transfer of microinjected neurobiotin in HeLa cells and formed conducting hemichannels in Xenopus oocytes, none of the mutant constructs tested show evidence of channel function. However, in coexpression experiments, N-terminal mutants that formed gap junction plaques potently inhibit hemichannel conductance of wild-type CX37 suggesting their co-oligomerization. We conclude that as much as half the length of the connexin N-terminus can be deleted without affecting formation of gap junction plaques, but an intact N-terminus is required for hemichannel gating and intercellular communication.