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A variety of studies have shown that Na 1 reabsorption across epithelial cells depends on the protease-antiprotease balance. Herein, we investigate the mechanisms by which a 1 -antitrypsin (A1AT), a major anti-serine protease in human plasma and lung epithelial fluid and lacking a Kunitz domain, regulates amiloridesensitive epithelial Na 1 channel (ENaC) function in vitro and in vivo. A1AT (0.05 mg/ml 5 1 mM) decreased ENaC currents across Xenopus laevis oocytes injected with human a,b,g-ENaC (hENaC) cRNAs, and human lung Clara-like (H441) cells expressing native ENaC, in a partially irreversible fashion. A1AT also decreased ENaC singlechannel activity when added in the pipette but not in the bath solutions of ENaC-expressing oocytes patched in the cell-attached mode. Incubation of A1AT with peroxynitrite (ONOO 2 ), an oxidizing and nitrating agent, abolished its antiprotease activity and significantly decreased its ability to inhibit ENaC. Intratracheal instillation of normal but not ONOO 2 -treated A1AT (1 mM) in C57BL/6 mice also decreased Na 1 -dependent alveolar fluid clearance to the same level as amiloride. Incubation of either H441 cells or ENaC-expressing oocytes with normal but not ONOO 2 -treated A1AT decreased their ability to cleave a substrate of serine proteases. A1AT had no effect on amiloride-sensitive currents of oocytes injected with hENaC bearing Liddle mutations, presumably because these channels remain at the surface longer than the wild-type channels. These data indicate that A1AT may be an important modulator of ENaC activity and of Na 1 -dependent fluid clearance across the distal lung epithelium in vivo by decreasing endogenous protease activity needed to activate silent ENaC.Keywords: alveolar fluid clearance; serine proteases; H441 cells; Xenopus oocytes; ENaCThe amiloride-sensitive epithelial Na 1 channels (ENaCs) play an important role in Na 1 and fluid homeostasis across a number of epithelial cells, including those in airway and alveolar spaces (1, 2). ENaC mRNA, protein levels, and ENaC activity are regulated by a variety of hormones (such as aldosterone and dexamethasone), second messengers cAMP/protein kinase A, and reactive intermediates (2-4). In addition, endogenous channelactivating proteases (CAPs), as well as proteases released by inflammatory cells (trypsin, elastase), activate ENaC either by cleaving critical amino acids in a-and g-ENaC subunits, or by activating signaling pathways (1, 2, 5-7).There has been considerable interest in identifying the mechanisms by which endogenous and exogenous proteases activate ENaC. Aprotinin, a potent and reversible Kunitz-type inhibitor of several serine proteases, including trypsin, plasmin, and kallikreins (8), has been reported to inhibit sodium transport among a variety of epithelial cells, including A6 (9), human bronchial epithelial (HBE) cells (10, 11), and rat and mouse lung alveolar epithelial cells (1). Other Kunitz-type serine protease inhibitors, such as hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2 (plac...

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A first research development progress report of the Chromosome 19 Consortium with members from Sweden, Norway, Spain, USA, China and India, a part of the Chromosome-Centric Human Proteome Project (C-HPP) global initiative is presented (http://www.c-hpp.org). From the chromosome 19 peptide-targeted library constituting 6159 peptides, a pilot study was conducted using a sub-set with 125 isotope-labeled peptides. We applied an annotation strategy with triple quadrupole, ESI-Qtrap, and MALDI mass spectrometry platforms, comparing the quality of data within, and in-between these instrumental set-ups. LC-MS conditions were outlined by multiplex assay developments, followed by MRM assay developments. SRM was applied to biobank samples, quantifying kallikrein 3 (prostate specific antigen) in plasma from prostate cancer patients. The antibody production has been initiated for more than 1200 genes from the entire chromosome 19, and the progress developments are presented. We developed a dedicated transcript microarray, to serve as the mRNA identifier by screening cancer cell lines. NAPPA protein arrays were built to align with the transcript data with the Chromosome 19 NAPPA chip, dedicated to 90 proteins, as the first development delivery. We have introduced an IT-infrastructure utilizing a LIMS system that serves as the key interface for the research teams in order to share and explore data generated within the project. The cross-site data repository will form the basis for sample processing, including biological samples, as well as patient samples from national Biobanks.