Salt and fluid is continuously secreted into and cleared from the pleural space in mammals. The present, widely accepted concept with regard to pleural fluid is that the volume of pleural liquid results from a balance of liquid in- and outflow, governed by Starling forces acting in the presence and absence of inflammation. Lymphatic drainage through amiloride-sensitive parietal pleura stomas, and electrolyte-coupled liquid absorption through the mesothelium of both sides, are major pathways for pleural fluid clearance (4
). A similar scenario exists in the context of malignant pleural mesothelioma, which is characterized by extravascular fibrin deposition, consistent with the aberrant fluid transport that characterizes most forms of solid neoplasia (38
). As abnormal fluid transport is implicated in the pathogenesis of pleural inflammation and neoplasia, we designed this study to incorporate analyses of both pleural mesothelial tissues and cultures of pleural mesothelial and malignant mesothelioma cells. Our results, for the first time, clearly show that epithelial Na+
channels are expressed in pleural mesothelial and malignant mesothelioma cells at the steady-state mRNA and protein levels. Furthermore, the biophysical properties of the channel in these cells, including ion permeability, amiloride sensitivity, Na+
dependence, unitary conductance, and responses to cyclic nucleotides, conform to those of amiloride-inhibitable Na+
channels. These observations suggest that ENaC in mesothelial or malignant mesothelioma cells may be a rate-limiting step for removing pleural fluid or perineoplastic fluids, respectively.
The expression pattern in human and mouse mesothelial cells is identical to that in H441 cells, a human Clara epithelial cell line (10
). Our RT-PCR and immunoassays, including Western blot and immunofluorescence studies, show that four ENaC subunits—namely, α, β, γ, and δ ENaC—are expressed in human primary mesothelial cells and M9K cell lines. We were unable to examine β ENaC protein expression in cell lysates, due to the lack of an anti–β ENaC antibody suitable for Western blotting. Our data, demonstrating ENaC expression at the mRNA and protein levels, prompted us to examine the functional expression of ENaC in mesothelial cells. For the majority of the electrophysiological experiments, we used M9K cells as a model, as these cells are easy to grow and have stable, consistent biophysical properties.
The differences in amiloride sensitivity observed between pleural tissues and single M9K cells can be explained by the voltage dependence of amiloride blockade, as described by several groups (14
). Amiloride carries a positive charge, and its binding site is located at the outer mouth of the channel pore, within the electrical field. It has been confirmed that the inhibitory efficiency of amiloride is less at depolarized Vm
). The holding potential for pleural tissues and M9K monolayer cells was 5 mV, whereas −100 mV was used for patch clamping single M9K cell. The difference in Vm
between these two sets of experiments may result in differences in amiloride sensitivity. On the other hand, the value of IC50
for amiloride in single M9K cells is similar to that in oocytes expressing αβγδ hENaC and in H441 cells when clamped at the same potential, as previously described (10
Although culture conditions influence polarization of epithelial cells and, in turn, ion selectivity and amiloride inhibition (45
), this was not the case in our studies. Assuming mesothelial cells in pleural tissues and confluent M9K monolayers were well polarized, whereas M9K cells grown on coverslips were not, the IC50
value for amiloride in single M9K cells on coverslips should be greater. Contrary to this expectation, a smaller IC50
was obtained. It is intriguing that amiloride only exhibits its inhibitory effects on the native Na+
channels in mesothelial cells when applied to the luminal side (equal to apical membrane of epithelial cells). These results strongly suggest that ENaC channels are expressed at the luminal side of mesothelial cells. Additional morphological studies dedicated to a separate project are needed to further locate their subcellular distribution.
In addition to the amiloride sensitivity, the results for external Na+
affinity, single-channel conductance for Li+
ions, and ion selectivity provide additional lines of evidence that ENaC-like channels are functionally expressed in pleural mesothelial and malignant mesothelioma cells. These biophysical features are consistent with the well characterized ENaC channels in H441 cells (29
Convincing evidence shows that native ENaC channels are regulated by PKA, PKC, and other kinases (25
). In this study, Ussing chamber assays showed that both forskolin and cell-permeable cGMP increased the amiloride inhibitable Isc
s in mouse pleural tissues. These results provide an intrinsic mechanism underlying the up-regulation of pleural or perineoplastic transmesothelial salt transport by cyclic nucleotides.
The combination of our biochemical, RT-PCR, and electrophysiological observations provides the first direct evidence of ENaC expression in pleural mesothelial cells/tissues and in malignant mesothelioma cells. Although we cannot completely exclude the association of amiloride-sensitive current with other amiloride-sensitive transport mechanisms in mesothelial or malignant mesothelioma cells (i.e., nucleotide-gated ion channels and electrogenic Na+/Ca2+ exchangers), the ion selectivity, single-channel conductance, and amiloride sensitivity argue against this possibility.
Pleural effusions are commonly encountered in inflammatory and neoplastic clinical disorders, and result from the imbalance between pleural liquid turnover and reabsorption (48
). Pleural effusion occurs in many diseases, including parapneumonic effusions, tuberculosis, and malignant pleural mesothelioma (49
). Nucleotide-stimulated ENaC channels in human mesothelial cells may govern pleural fluid transport in the pleural cavity in normalcy, inflammatory disorders, and in pleural malignancy, and thereby contribute to homeostatic control of pleural fluid or the pathogenesis of pleural effusions. Whether impairment of ENaC occurs in pleural injuries awaits follow-up studies.