| IntroductionMesothelial cells are specialized epithelial cells that line the serous cavities (pleural,pericardial,and peritoneal) and internal organs.Only over the past 20 years has it been realized that mesothelial cells play a critical role in maintaining the integrity and function of the serosal cavities.Pleural effusions are commonly encountered clinical disorders,resulting from the imbalance between pleural fluid turnover and re-absorption.Increasing evidence suggests that active electrolyte transport is involved in the transmesothelial fluid absorption.The epithelial Na channel(ENaC),which participates in sodium movement across the apical membrane of epithelial cells,has been cloned and characterized.Five ENaC subunits have been cloned to date,namely,α,β,γ,δ,andεENaC.The biophysical properties of ENaC channels depend on subunit composition.The diuretic amiloride inhibits Na+ transport when added to the solution bathing the apical plasma membrane, at a concentration less than 10-6 M.Indirect evidence supporting the presence of amiloride-sensitive sodium channels in human and sheep peritoneal membrane has been presented.A significant increment in the transmesothelial electrical resistance was observed after addition of amiloride, indicating an amiloride-sensitive ion transport pathway.It has been proposed that this ion transport system may play a role in the ultrafiltration process and sodium removal during peritoneal dialysis.By comparison with polarized epithelial cells,it is conceivable that ENaC proteins are expressed on the mesothelial surfaces that line the peritoneal cavity.While it has been demonstrated that ENaC in lung epithelial cells are the predominant pathway for alveolar fluid clearance,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 ceils. In the present study,we have examined ENaC expression in human and mouse mesothelial cells.Our data demonstrate for the first time that the ENaC channels are biochemically and functionally expressed in human pleural mesothelial cells,and can be up-regulated by cell cAMP and cGMP.It is conceivable to speculate that ENaC may play a crucial role in governing pleural fluid transport in the pleural cavity and may thereby contribute to the pathogenesis and physiologic resorption of pleural effusions.MethodsCell Culture-NCI-H441 cells were obtained from the American Type Culture Collection(ATCC).The human pleural mesothelial M9K cell line and human primary pleural mesothelial cells were provided by Dr.Steven Idell(University of Texas Health Center at Tyler).The human pleural mesothelial cells were obtained from pleural effusions of patients with congestive heart failure under the auspices of a protocol approved by the Human Subjects Institutional Review Board of The University of Texas Health Science Center at Tyler.H441 and human primary mesothelial cells were grown in RPMI medium(ATCC) supplemented with 10%fetal bovine serum,and antibiotics.M9K cells were grown in Medium 199(Invitrogen).Reverse Transcription-PCR:Confluent M9K and human primary mesothelial cells were scraped into ice-cold PBS.Cells were snap-frozen in a dry ice-methanol mixture and stored at -80℃post pelleting in a microcentrifuge.Mouse parietal pleural tissues were surgically isolated from fully anesthetized BALB/c male mice(Jackson Laboratory).Total RNA was extracted using TRIzol reagent(Invitrogen)according to the manufacturer's instructions.RT-PCR was performed using the OneStep RT-PCR kit (Qiagen) in a Mastercycler gradient thermocycler(Eppendorf Scientific).Authenticity of PCR products in the pCR2.1 vector were verified by automated DNA sequencing (DNA Sequencing Core,UAB).Western Blot assays-Following separation by SDS-PAGE(6%gels),proteins were transblotted onto PVDF membrane(Bio-Rad).After a 1-h incubation in a blocking solution containing 20 mM Tris-C1,pH 7.5,0.5M NaC1,5%non-fat dried milk,the membrane was incubated with anti-ENaC antibodies at 4℃overnight. Incubation at room temperature for 30 min was used for secondary antibodies.ECL kit (Amersham) was used to develop the image.Antibodies against ENaC subunits were diluted 1:5000 when used.Indirect Immunofluorescence and confocal Microscopy-Cells on filters or coverslips were fixed in 3%formaldehyde(EM Sciences) for 45 min,followed by incubation with 0.5%Triton X-100 for 3 min.Cells were incubated with anti-α,β,γ, andδENaC polyclonal antibodies from Santa Cruz,Chemicon,and ABR at 20μg/mL for 2 h at room temperature.The cells were then incubated with Alexa Fluor 488 goat anti-rabbit IgG(H+L) 10μg/mL(Invitrogen) for 2 h at room temperature in dark.Cell nuclei were stained with 300 nM DAPI(Invitrogen) for 5 min.Images were photographed with a high resolution camera(model S97809,Olympus America Inc) attached to an inverted epifluorescence microscope(model BX50,Olympus).Sideview images were digitized using a confocal microscopy.Merged images were processed using Adobe Photoshop CS3 extended.Ussing Chamber Assays-Excised pleural tissues were kept in the physiological salt solution prior to be mounted in the Ussing chambers(Physiologic Instruments). The transmesothelial short-circuit currents(Isc) were measured with 3 M KCl,4%agar bridges placed 3 mm on either side of the membrane,which were connected on either side to Ag-AgCl electrodes.M9K monolayers or mouse pleural tissues were short-circuited to 0 mV,and Isc was measured with a VCC-MC8 voltage clamp amplifier(Physiologic Instruments).Data were collected using the Acquire and Analyse program(version 2.3,Physiologic Instruments).Oocyte Preparation and Voltage Clamp Analysis-Oocytes were surgically removed from appropriately anesthetized adult female Xenopus laevis(Xenopus Express).Defolliculated oocytes were cytosolically injected with hENaC cRNAs(25 ng) per oocyte in 50 nl of RNase free water with a ratio of 1α:1β:1γ,:1δsubunit and incubated in half-strength L-15 medium at 18℃.The two-electrode voltage clamp technique was used to record whole-cell currents 48 h post injection.A TEV-200 voltage clamp amplifier(Dagan) was used to clamp oocytes with concomitant recording of currents.Experiments were controlled by pCLAMP 10.1 software (Molecular Devices),and currents at -40mV,-100mV,and +80 mV were continuously monitored with an interval of 10s.Patch Clamp Recordings-Immediately before each experiment,a coverslip bearing M9K cells was removed from the culture plate and put into a recording chamber,which was mounted on the stage of an inverted fluorescent microscope(Leica DM IRB).Series resistance and capacitance transients were compensated with an Axopatch 200B amplifier(Molecular Devices).Inward and outward whole-cell currents were elicited by employing a step-pulse protocol from -100 to +100 mV in 10 mV increments every 10 s for 500 ms duration from a holding potential of-40 mV.For single-channel recordings,the cell-attached mode was used.Data were sampled at 1-2 kHz and filtered at 0.3 kHz.Data Analysis-All results are presented as means±S.E.One-way analysis of mean and variance computation was used to analyze data with unequal variance between each group.A probability level of 0.05 was considered significant.ResultsOur RT-PCR results showed that four ENaC subunits,namely,α,β,γ,and two 5 ENaC subunits are expressed in human primary pleural mesothelial cells,a human mesothelioma cell line(M9K),and mouse pleural tissue.In addition,Western blot and immunofluorescence microscopy studies revealed thatα-,β-,γ-,andδ-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 21μM 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 cell permeable cGMP increased the short-circuit currents in mouse pleural tissues.ConclusionWe detected the expression ofα,β,γ,andδENaC subunits at the mRNA and protein levels in M9K cells,a human pleural mesothelioma cell line,and mouse pleural tissues.An amiloride-sensitive,ENaC-like short-circuit current was recorded in mouse pleural tissues and confluent M9K monolayers mounted in Ussing chamber. Furthermore,an amiloride-sensitive,highly Na+ permeable channel was characterized using whole-cell and single channel patch clamp techniques.These results clearly demonstrate that the ENaC channel,composed of four subunits,is both biochemically and functionally expressed in mesothelial cells,and can be up-regulated by cell cAMP and cGMR... |
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