α₁-Antitrypsin Inhibits Epithelial Na⁺ Transport In Vitro and In Vivo

Abstract

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...

Regulation of Alveolar Epithelial Na+ Channels by ERK1/2 in Chlorine-Breathing Mice

“…Trypsin activates silent channels by cleaving a-ENaC and g-ENaC extracellular domains (19,25). As shown in Figures 3A-3D, the inclusion of trypsin (2 mM) in the patch pipettes increased the number of active channels and their P o of air-breathing mice, without affecting their unitary conductance.…”

“…Single-channel activity in both ATII and ATI cells was recorded using the cellattached mode of the patch clamp technique (19,20). ATII cells were identified by the presence of scattered green fluorescence after incubation with Lysotracker Green (catalogue number DND-26; Invitrogen, Eugene, OR).…”

Inhibition of Lung Fluid Clearance and Epithelial Na+ Channels by Chlorine, Hypochlorous Acid, and Chloramines

“…To facilitate the detection of exogenously expressed ␣-, ␤-, and ␥-hENaC in Western blotting studies, in some cases ␣, ␤, and ␥ cDNAs were tagged with FLAG, Myc, and V5 epitopes at their C termini, respectively, and taggedcRNAs were generated as above. All procedures have been described in detail previously (34).…”

“…Expression of ENaC in Xenopus Oocytes-Ovarian tissue containing oocytes from adult female Xenopus laevis toads was dissected under anesthesia, then digested in 2 mg/ml collagenase (type 1A, Roche Applied Science) in Ca 2ϩ -free OR-2 medium, containing 82.5 mM NaCl, 2.0 mM KCl, 1.0 mM MgCl 2 , and 5 mM HEPES, pH 7.4, under rotation at room temperature for 2 h as previously described (34). Defolliculated oocytes were washed 3 times in Ca 2ϩ -free OR-2 medium followed by OR-2 medium with 1.0 mM CaCl 2 .…”

“…Determination of Membrane Protease Enzymatic ActivitySerine protease activity in the oocytes membranes was monitored by measuring the release of a fluorescent probe (7-amido-4-methyl coumarin (AMC)) from a peptide substrate t-butoxycarbonyl (Boc)-Gln-Ala-Arg-AMC-HCl; R&D Systems, Minneapolis, MN) using a fluorescence plate reader (FLUOstar OPTIMA, BMG Labtech; Durham, NC) (34). Briefly, five oocytes each, incubated with either HOCl (2 mM) or vehicle in ND96 for 10 min, were added to a cuvette containing 200 l of ND96 medium, and the reaction was started by adding Boc-Gln-Ala-Arg-AMC-HCl (50 M final concentration).…”