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

Abstract

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 1 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, a, b, g, and two d 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 a, b, g, and d 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 mM in M9K cells. This cation channel has a high affinity for extracellular Na 1 ions (K m : 53 mM). The ion selectivity of this channel to cations follows the same order as ENaC: Li 1 . Na 1 . K 1 . The unitary Li 1 conductance was 15 pS in on-cell patches. Four ENaC subunits form a functional Na 1 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.

Permeability of the arachnoid and pia mater. The role of ion channels in the leptomeningeal physiology

“…Thus, the sides of arachnoid and pia mater cells facing the subarachnoid space (inwards) do not come in contact with the applied substances. The proposed explanation is consistent with studies that identify the polarized presence of ENaC channels in mesothelial tissues responsible for fluid turnover, at the side of a biological cavity that contains fluid [16,24,25,[42][43][44]. In this case the biological cavity that contains fluid (here CSF) is the subarachnoid space and the cells that define and line this structure are the arachnoid and pia mater cells that could possibly contribute to CSF turnover due to their polarized ion channel presence.…”

“…In mesothelial tissues, e.g., pleura or tissues that produce CSF, e.g., choroid plexus, the presence of Na + -K + -ATPase and ENaC is important in regulating the production of biological fluids, e.g., pleural fluid or CSF [5,18,37,44]. Changes that affect the function of these channels, e.g., mutations, cellular localization, or recycling could affect the ability of a tissue to regulate fluid turnover and thus lead to disease.…”

The δ-Subunit of the Epithelial Sodium Channel (ENaC) Enhances Channel Activity and Alters Proteolytic ENaC Activation

“…Genes corresponding to human ␦-ENaC have been identified in chimpanzee, dog, chicken, and rabbit (19). Although it has previously been thought that ␦-ENaC is absent in rat and mouse (20,21), recent reports suggest that ␦-ENaC is expressed in mouse sperm (22) and in mouse pleural tissue (23).…”

Dexamethasone acutely accelerates pleural fluid absorption in mice hydrothoraces

“…/H ? exchanger) [7][8][9][10][11], which, in turn, have been implicated in pleural fluid absorption [12,13]. We thus hypothesized that corticosteroids increase the absorption rate of isosmotic hydrothoraces in mice.…”