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1.  Correlation of Apical Fluid-Regulating Channel Proteins with Lung Function in Human COPD Lungs 
PLoS ONE  2014;9(10):e109725.
Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD) are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC), cystic fibrosis transmembrane conductance regulator (CFTR), and aquaporin 5 (AQP5) proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI) and II (ATII)-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3) was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.
doi:10.1371/journal.pone.0109725
PMCID: PMC4201481  PMID: 25329998
2.  Ectopic TSH-secreting pituitary tumor: a case report and review of prior cases 
BMC Cancer  2014;14(1):544.
Background
Ectopic TSH-secreting pituitary adenoma (TSH-oma) is a very unusual disorder. To date, there are only four cases reported. It is difficult to distinguish ectopic cases from both regular TSH-omas and resistance to thyroid hormone (RTH).
Case presentation
A newly identified case of ectopic TSH-oma arising from the nasal pharynx was described, and reports of four prior cases were reviewed. The patient was a 41-year-old male who developed what appeared to be typical hyperthyroidism and atrial fibrillation in 2009. Thyroid function tests showed elevated basal levels of free T3 (FT3, 24.08 pmol/L), free T4 (FT4, 75.73 pmol/L), and serum TSH (7.26 μIU/ml). Both TSH-oma and resistance to thyroid hormone syndrome were considered. TRH stimulating test was negative, whereas octreotide inhibition test showed a reduction in TSH by 30.8%. Furthermore, a large space-occupying lesion located at the nasopharynx was found by computed tomography and magnetic resonance imaging (MRI). A normal pituitary was visualized. Ectopic TSH-oma was preliminarily established. Using an endoscopic endonasal approach, the tumor was resected. Histological features and immunophenotypes were consistent with those of TSH-secreting tumor. The levels of both free thyroxine and TSH returned to normal ranges the day after surgery and remained within normal range for 48 months.
Conclusions
Although exceedingly rare, ectopic TSH-oma should be considered for patients with inappropriate secretion of TSH with hyperthyroidism and pituitary tumor undetectable by computed tomography and MRI. To our knowledge, this is the first case followed up more than 4 years. The characteristics and successful interventions summarized in this report provide a guideline for clinicians.
doi:10.1186/1471-2407-14-544
PMCID: PMC4125694  PMID: 25069990
Ectopic TSH-secreting pituitary adenoma; Resistance to thyroid hormone (RTH); TRH stimulating test; Octreotide inhibition test; Hyperthyroidism
3.  8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na+ Self-Inhibition of Epithelial Na+ Channels 
Salt absorption via alveolar epithelial Na+ channels (ENaC) is a critical step for maintaining an airspace free of flooding. Previously, we found that 8-(4-chlorophenylthio)-guanosine-3′,5′-cyclic monophosphate-Na (CPT-cGMP) activated native and heterologous ENaC. To investigate the potential pharmacological relevance, we applied this compound intratracheally to human lungs and found that ex vivo alveolar fluid clearance was increased significantly. Furthermore, this compound eliminated self-inhibition in human lung H441 cells and in oocytes expressing human αβγ but not δβγ channels. To further elucidate this novel mechanism, we constructed mutants abolishing (βΔV348 and γH233R) or augmenting (αY458A and γM432G) self-inhibition. The mutants eliminating self-inhibition lost their responses to CPT-cGMP, whereas those enhancing self-inhibition facilitated the stimulatory effects of this compound. CPT-cGMP was unable to activate a high Po mutant (βS520C) and plasmin proteolytically cleaved channels. Our data suggest that elimination of self-inhibition of αβγ ENaC may be a novel mechanism for CPT-cGMP to stimulate salt reabsorption in human lungs.
doi:10.1165/rcmb.2011-0004OC
PMCID: PMC3262684  PMID: 21562313
lung fluid reabsorption; amiloride-sensitive sodium channel; CPT-cGMP; ENaC self-inhibition
4.  Cpt-cAMP activates human epithelial sodium channels via relieving self-inhibition 
Biochimica et biophysica acta  2011;1808(7):1818-1826.
External Na+ self-inhibition is an intrinsic feature of epithelial sodium channels (ENaC). Cpt-cAMP regulates heterologous guinea pig but not rat αβγ ENaC in a ligand-gated manner. We hypothesized that cpt-cAMP may eliminate the self-inhibition of human ENaC thereby open channels. Regulation of self-inhibition by this compound in oocytes was analyzed using the two-electrode voltage clamp and Ussing chamber setups. External cpt-cAMP stimulated human but not rat and murine αβγ ENaC in a dose- and external Na+ concentration-dependent fashion. Intriguingly, cpt-cAMP activated human δβγ more potently than αβγ channels, suggesting that structural diversity in ectoloop between human α, δ, and those ENaC of other species determines the stimulating effects of cpt-cAMP. Cpt-cAMP increased the ratio of stationary and maximal currents. Mutants having abolished self-inhibition (βΔV348 and γH233R) almost completely eliminated cpt-cAMP mediated activation of ENaC. On the other hand, mutants both enhancing self-inhibition and elevating cpt-cAMP sensitivity increased the stimulating effects of the compound. This compound, however, could not activate already fully opened channels, e.g., degenerin mutation (αβS520Cγ) and the proteolytically cleaved ENaC by plasmin. Cpt-cAMP activated native ENaC to the same extent as that for heterologous ENaC in human lung epithelial cells. Our data demonstrate that cpt-cAMP, a broadly used PKA activator, stimulates human αβγ and δβγ ENaC channels by relieving self-inhibition.
doi:10.1016/j.bbamem.2011.03.004
PMCID: PMC3091966  PMID: 21419751
5.  Structural and Functional Characterization of Two Alternative Splicing Variants of Mouse Endothelial Cell-Specific Chemotaxis Regulator (ECSCR) 
Endothelial cells (ECs) that line the lumen of blood vessels are important players in blood vessel formation, and EC migration is a key component of the angiogenic process. Thus, identification of genes that are specifically or preferentially expressed in vascular ECs and in-depth understanding of their biological functions may lead to discovery of new therapeutic targets. We have previously reported molecular characterization of human endothelial cell-specific molecule 2 (ECSM2)/endothelial cell-specific chemotaxis regulator (ECSCR). In the present study, we cloned two mouse full-length cDNAs by RT-PCR, which encode two putative ECSCR isoform precursors with considerable homology to the human ECSCR. Nucleotide sequence and exon-intron junction analyses suggested that they are alternative splicing variants (ECSCR isoform-1 and -2), differing from each other in the first and second exons. Quantitative RT-PCR results revealed that isoform-2 is the predominant form, which was most abundant in heart, lung, and muscles, and moderately abundant in uterus and testis. In contrast, the expression of isoform-1 seemed to be more enriched in testis. To further explore their potential cellular functions, we expressed GFP- and FLAG-tagged ECSCR isoforms, respectively, in an ECSCR deficient cell line (HEK293). Interestingly, the actual sizes of either ECSCR-GFP or -FLAG fusion proteins detected by immunoblotting are much larger than their predicted sizes, suggesting that both isoforms are glycoproteins. Fluorescence microscopy revealed that both ECSCR isoforms are localized at the cell surface, which is consistent with the structural prediction. Finally, we performed cell migration assays using mouse endothelial MS1 cells overexpressing GFP alone, isoform-1-GFP, and isoform-2-GFP, respectively. Our results showed that both isoforms significantly inhibited vascular epidermal growth factor (VEGF)-induced cell migration. Taken together, we have provided several lines of experimental evidence that two mouse ECSCR splicing variants/isoform precursors exist. They are differentially expressed in a variety of tissue types and likely involved in modulation of vascular EC migration. We have also defined the gene structure of mouse ECSCR using bioinformatics tools, which provides new information towards a better understanding of alternative splicing of ECSCR.
doi:10.3390/ijms13044920
PMCID: PMC3344256  PMID: 22606020
ECSCR/ECSM2; alternative splicing; isoform; gene structure; exon-intron boundary; cDNA cloning and expression; endothelial cell migration
6.  Expression and Regulation of Epithelial Na+ Channels by Nucleotides in Pleural Mesothelial Cells 
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, α, β, γ, and two δ 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 α, β, γ, and δ 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 (Km: 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.
doi:10.1165/rcmb.2008-0166OC
PMCID: PMC2677435  PMID: 18927349
M9K mesothelioma cells; Ussing chamber; protein kinase A; protein kinase G; human primary mesothelial cells
7.  Identification of novel splice variants and exons of human endothelial cell-specific chemotaxic regulator (ECSCR) by bioinformatics analysis 
Recent discovery of biological function of endothelial cell-specific chemotaxic regulator (ECSCR), previously known as endothelial cell-specific molecule 2 (ECSM2), in modulating endothelial cell migration, apoptosis, and angiogenesis, has made it an attractive molecule in vascular research. Thus, identification of splice variants of ECSCR could provide new strategies for better understanding its roles in health and disease. In this study, we performed a series of blast searches on the human EST database with known ECSCR cDNA sequence (Variant 1), and identified additional three splice variants (Variants 2–4). When examining the ECSCR gene in the human genome assemblies, we found a large unknown region between Exons 9 and 11. By PCR amplification and sequencing, we partially mapped Exon 10 within this previously unknown region of the ECSCR gene. Taken together, in addition to previously reported human ECSCR, we identified three novel full-length splice variants potentially encoding different protein isoforms. We further defined a total of twelve exons and nearly all exon-intron boundaries of the gene, of which only eight are annotated in current public databases. Our work provides new information on gene structure and alternative splicing of the human ECSCR, which may imply its functional complexity. This undoubtedly opens new opportunities for future investigation of the biological and pathological significance of these ECSCR splice variants.
doi:10.1016/j.compbiolchem.2012.10.003
PMCID: PMC3513626  PMID: 23147565
ECSCR (ECSM2); alternative splicing; data mining; EST alignment; sequence homology; exon-intron boundary
8.  LOX-1 Deletion Improves Neutrophil Responses, Enhances Bacterial Clearance, and Reduces Lung Injury in a Murine Polymicrobial Sepsis Model ▿ 
Infection and Immunity  2011;79(7):2865-2870.
Inflammatory tissue injury and immunosuppression are the major causes of death in sepsis. Novel therapeutic targets that can prevent excessive inflammation and improve immune responses during sepsis could be critical for treatment of this devastating disease. LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), a membrane protein expressed in endothelial cells, has been known to mediate vascular inflammation. In the present study, we demonstrated that LOX-1 deletion markedly improved the survival rate in a murine model of polymicrobial sepsis. Wild-type (LOX-1+/+) and LOX-1 knockout (LOX-1−/−) mice were subjected to cecal ligation and puncture (CLP) to induce sepsis. LOX-1 deletion significantly reduced systemic inflammation and inflammatory lung injury during sepsis, together with decreased production of proinflammatory cytokines and reduced lung edema formation. Furthermore, LOX-1 deletion improved host immune responses after the induction of sepsis, as indicated by enhanced bacterial clearance. Interestingly, we were able to demonstrate that LOX-1 is expressed in neutrophils. LOX-1 deletion prevented neutrophil overreaction and increased neutrophil recruitment to infection sites after sepsis induction, contributing at least partly to increased immune responses in LOX-1 knockout mice. Our study results indicate that LOX-1 is an important mediator of inflammation and neutrophil dysfunction in sepsis.
doi:10.1128/IAI.01317-10
PMCID: PMC3191957  PMID: 21576343
9.  α1-Antitrypsin Inhibits Epithelial Na+ Transport In Vitro and In Vivo 
A variety of studies have shown that Na+ reabsorption across epithelial cells depends on the protease–antiprotease balance. Herein, we investigate the mechanisms by which α1-antitrypsin (A1AT), a major anti-serine protease in human plasma and lung epithelial fluid and lacking a Kunitz domain, regulates amiloride-sensitive epithelial Na+ channel (ENaC) function in vitro and in vivo. A1AT (0.05 mg/ml = 1 μM) decreased ENaC currents across Xenopus laevis oocytes injected with human α,β,γ-ENaC (hENaC) cRNAs, and human lung Clara-like (H441) cells expressing native ENaC, in a partially irreversible fashion. A1AT also decreased ENaC single-channel activity when added in the pipette but not in the bath solutions of ENaC-expressing oocytes patched in the cell-attached mode. Incubation of A1AT with peroxynitrite (ONOO−), an oxidizing and nitrating agent, abolished its antiprotease activity and significantly decreased its ability to inhibit ENaC. Intratracheal instillation of normal but not ONOO−-treated A1AT (1 μM) in C57BL/6 mice also decreased Na+-dependent alveolar fluid clearance to the same level as amiloride. Incubation of either H441 cells or ENaC-expressing oocytes with normal but not ONOO−-treated A1AT decreased their ability to cleave a substrate of serine proteases. A1AT had no effect on amiloride-sensitive currents of oocytes injected with hENaC bearing Liddle mutations, presumably because these channels remain at the surface longer than the wild-type channels. These data indicate that A1AT may be an important modulator of ENaC activity and of Na+-dependent fluid clearance across the distal lung epithelium in vivo by decreasing endogenous protease activity needed to activate silent ENaC.
doi:10.1165/rcmb.2008-0384OC
PMCID: PMC2742747  PMID: 19131639
alveolar fluid clearance; serine proteases; H441 cells; Xenopus oocytes; ENaC
10.  K+ channel openers restore verapamil-inhibited lung fluid resolution and transepithelial ion transport 
Respiratory Research  2010;11(1):65.
Background
Lung epithelial Na+ channels (ENaC) are regulated by cell Ca2+ signal, which may contribute to calcium antagonist-induced noncardiogenic lung edema. Although K+ channel modulators regulate ENaC activity in normal lungs, the therapeutical relevance and the underlying mechanisms have not been completely explored. We hypothesized that K+ channel openers may restore calcium channel blocker-inhibited alveolar fluid clearance (AFC) by up-regulating both apical and basolateral ion transport.
Methods
Verapamil-induced depression of heterologously expressed human αβγ ENaC in Xenopus oocytes, apical and basolateral ion transport in monolayers of human lung epithelial cells (H441), and in vivo alveolar fluid clearance were measured, respectively, using the two-electrode voltage clamp, Ussing chamber, and BSA protein assays. Ca2+ signal in H441 cells was analyzed using Fluo 4AM.
Results
The rate of in vivo AFC was reduced significantly (40.6 ± 6.3% of control, P < 0.05, n = 12) in mice intratracheally administrated verapamil. KCa3.1 (1-EBIO) and KATP (minoxidil) channel openers significantly recovered AFC. In addition to short-circuit current (Isc) in intact H441 monolayers, both apical and basolateral Isc levels were reduced by verapamil in permeabilized monolayers. Moreover, verapamil significantly altered Ca2+ signal evoked by ionomycin in H441 cells. Depletion of cytosolic Ca2+ in αβγ ENaC-expressing oocytes completely abolished verapamil-induced inhibition. Intriguingly, KV (pyrithione-Na), K Ca3.1 (1-EBIO), and KATP (minoxidil) channel openers almost completely restored the verapamil-induced decrease in Isc levels by diversely up-regulating apical and basolateral Na+ and K+ transport pathways.
Conclusions
Our observations demonstrate that K+ channel openers are capable of rescuing reduced vectorial Na+ transport across lung epithelial cells with impaired Ca2+ signal.
doi:10.1186/1465-9921-11-65
PMCID: PMC2889873  PMID: 20507598
11.  Electrolyte and Fluid Transport in Mesothelial Cells 
Mesothelial cells are specialized epithelial cells, which line the pleural, pericardial, and peritoneal cavities. Accumulating evidence suggests that the monolayer of mesothelial cells is permeable to electrolyte and fluid, and thereby govern both fluid secretion and re-absorption in the serosal cavities. Disorders in these salt and fluid transport systems may be fundamental in the pathogenesis of pleural effusion, pericardial effusion, and ascites. In this review, we discuss the location, physiological function, and regulation of active transport (Na+-K+-ATPase) systems, cation and anion channels (Na+, K+, Cl−, and Ca2+ channels), antiport (exchangers) systems, and symport (co-transporters) systems, and water channels (aquaporins). These secretive and absorptive pathways across mesothelial monolayer cells for electrolytes and fluid may provide pivotal therapeutical targets for novel clinical intervention in edematous diseases of serous cavities.
doi:10.2174/1875044300801010001
PMCID: PMC2629602  PMID: 19169368
mesothelioma; ion channel; permeability; effusion; filtration; ENaC

Results 1-11 (11)