American Journal of Respiratory Cell and Molecular Biology. 2008 Oct 16. [Epub ahead of print] [Link]
Nie HG, Tucker T, Su XF, Na T, Peng JB, Smith PR, Idell S, Ji HL.
Biochemistry, University of Texas Health Science Center at Tyler, Texas Lung Injury Institute, Tyler, Texas, United States; Pharmacology, China Medical University, School of Pharmaceutical Sciences, Shenyang, China.
Pleural effusions are commonly clinical disorders, resulting from the imbalance between pleural fluid turnover and re-absorption. The mechanisms underlying pleural fluid clearance across the mesothelium remain to be elucidated. We hypothesized that ENaC is expressed and forms the molecular basis of the amiloride-sensitive resistance in human mesothelial cells. Our RT-PCR results showed that four ENaC subunits, namely, [alpha], [beta], [gamma], and two [delta] 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 [alpha], [beta], [gamma], and [delta] 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 IC50 of 12 M in M9K cells. This cation channel has a high affinity for extracellular Na+ ions (Km: 53mM). 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+ channels when co-injected 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 cAMP and cGMP.
Keywords: Ussing chamber, protein kinase, human primary mesothelial cells, amiloride