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₁-Antitrypsin Inhibits Epithelial Na⁺ Transport In Vitro and In Vivo

Ahmed Lazrak¹, Izabella Nita^1,2,*, Devipriya Subramaniyam^1,2,*, Shipeng Wei¹, Weifeng Song¹, Hong-Long Ji¹, Sabina Janciauskiene^2, and Sadis Matalon^1,3,4,

¹ Departments of Anesthesiology and ³ Physiology and Biophysics, and ⁴ Center of Pulmonary Injury and Repair, University of Alabama at Birmingham, Birmingham, Alabama; and ² Lund University, Department of Clinical Sciences, Wallenberg Laboratory, Malmö University Hospital, Malmö, Sweden

Correspondence and requests for reprints should be addressed to Sadis Matalon, Ph.D., Department of Anesthesiology, University of Alabama at Birmingham, 224 BMR II, 901 South 19th Street, Birmingham, AL 35205-3703. E-Mail: sadis{at}uab.edu

A variety of studies have shown that Na⁺ reabsorption across epithelial cells depends on the protease–antiprotease balance. Herein, we investigate the mechanisms by which ₁-antitrypsin (A1AT), a major anti-serine protease in human plasma and lung epithelial fluid and lacking a Kunitz domain, regulates amiloride-sensitive epithelial Na⁺ channel (ENaC) function in vitro and in vivo. A1AT (0.05 mg/ml = 1 µM) decreased ENaC currents across Xenopus laevis oocytes injected with human ,β,-ENaC (hENaC) cRNAs, and human lung Clara-like (H441) cells expressing native ENaC, in a partially irreversible fashion. A1AT also decreased ENaC single-channel 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^–), an oxidizing and nitrating agent, abolished its antiprotease activity and significantly decreased its ability to inhibit ENaC. Intratracheal instillation of normal but not ONOO^–-treated A1AT (1 µM) in C57BL/6 mice also decreased Na⁺-dependent alveolar fluid clearance to the same level as amiloride. Incubation of either H441 cells or ENaC-expressing oocytes with normal but not ONOO^–-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⁺-dependent fluid clearance across the distal lung epithelium in vivo by decreasing endogenous protease activity needed to activate silent ENaC.

Key Words: alveolar fluid clearance • serine proteases • H441 cells • Xenopus oocytes • ENaC

CLINICAL RELEVANCE Our data show that 1-antitrypsin (A1AT) inhibits active Na+ reabsorption and lung fluid clearance across distal lung epithelial cells and highlight a potential mechanism by which A1AT administration may prove beneficial in clinical situations in which increased ENaC activity may contribute to lung pathology (such as cystic fibrosis).

 

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