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Expression and Regulation of Epithelial Na⁺ Channels by Nucleotides in Pleural Mesothelial Cells

¹ Department of Biochemistry, Texas Lung Injury Institute, University of Texas Health Science Center at Tyler, Tyler, Texas; ² Department of Physiology and Biophysics, and ³ Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama; and ⁴ Department of Pharmacology, School of Pharmaceutical Sciences, China Medical University, Shenyang, China

Correspondence and requests for reprints should be addressed to Hong-Long (James) Ji, M.D., Department of Biochemistry, Texas Lung Injury Institute, The University of Texas Health Science Center at Tyler, 11937 U.S. Highway 271, Tyler, TX 75708. E-mail: James.Ji{at}uthct.edu

Pleural effusions are commonly clinical disorders, resulting from the imbalance between pleural fluid turnover and reabsorption. The mechanisms underlying pleural fluid clearance across the mesothelium remain to be elucidated. We hypothesized that epithelial Na⁺ channel (ENaC) is expressed and forms the molecular basis of the amiloride-sensitive resistance in human mesothelial cells. Our RT-PCR results showed that three ENaC subunits, namely, , β, , and two ENaC subunits, are expressed in human primary pleural mesothelial cells, a human mesothelioma cell line (M9K), and mouse pleural tissue. In addition, Western blotting and immunofluorescence microscopy studies revealed that , β, , and ENaC subunits are expressed in primary human mesothelial cells and M9K cells at the protein level. An amiloride-inhibitable short-circuit current was detected in M9K monolayers and mouse pleural tissues when mounted in Ussing chambers. Whole-cell patch clamp recordings showed an ENaC-like channel with an amiloride concentration producing 50% inhibition of 12 µM in M9K cells. This cation channel has a high affinity for extracellular Na⁺ ions (K_m: 53 mM). The ion selectivity of this channel to cations follows the same order as ENaC: Li⁺ > Na⁺ > K⁺. The unitary Li⁺ conductance was 15 pS in on-cell patches. Four ENaC subunits form a functional Na⁺ channel when coinjected into Xenopus oocytes. Furthermore, we found that both forskolin and cGMP increased the short-circuit currents in mouse pleural tissues. Taken together, our data demonstrate that the ENaC channels are biochemically and functionally expressed in human pleural mesothelial cells, and can be up-regulated by cyclic AMP and cyclic GMP.

Key Words: M9K mesothelioma cells • Ussing chamber • protein kinase A • protein kinase G • human primary mesothelial cells

CLINICAL RELEVANCE This study shows, for the first time, that epithelial sodium channels are expressed in pleural mesothelial cells and govern ion transport.

 

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