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Alveolar type 2 epithelial (AT2) cells synthesize surfactant protein C (SPC) and repair an injured alveolar epithelium. A mutated surfactant protein C gene (SftpcL184Q, Gene ID: 6440) in newborns has been associated with respiratory distress syndrome and pulmonary fibrosis. However, the underlying mechanisms causing Sftpc gene mutations to regulate AT2 lineage remain unclear. We utilized three-dimensional (3D) feeder-free AT2 organoids in vitro to simulate the alveolar epithelium and compared AT2 lineage characteristics between WT (C57BL/6) and SftpcL184Q mutant mice using colony formation assays, immunofluorescence, flow cytometry, qRT-PCR, and Western blot assays. The AT2 numbers were reduced significantly in SftpcL184Q mice. Organoid numbers and colony-forming efficiency were significantly attenuated in the 3D cultures of primary SftpcL184Q AT2 cells compared to those of WT mice. Podoplanin (PDPN, Alveolar type 1 cell (AT1) marker) expression and transient cell count was significantly increased in SftpcL184Q organoids compared to in the WT mice. The expression levels of CD74, heat shock protein 90 (HSP90), and ribosomal protein S3A1 (RPS3A1) were not significantly different between WT and SftpcL184Q AT2 cells. This study demonstrated that humanized SftpcL184Q mutation regulates AT2 lineage intrinsically. This regulation is independent of CD74, HSP90, and RPS3A1 pathways. [ABSTRACT FROM AUTHOR]

Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at ((177)GR↓KR(180)) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically "silent") channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions.

Epithelial sodium channels (ENaC) are regulated by protein kinase A, in addition to a broad spectrum of other protein kinases. It is not clear whether cGMP/PKG signaling might regulate ENaC activity. We examined the responses of [alpha][beta][gamma]-ENaC channels expressed in Xenopus oocytes to 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), a cell-permeable cGMP analog. This compound stimulated human [alpha][beta][gamma]-ENaC activity in a dose-dependent fashion, but cell-impermeable cGMP had no effect. Similar stimulatory effects of cGMP were observed in oocytes expressing either mouse or rat [alpha][beta][gamma]-ENaC channels. The identical ion selectivity and amiloride sensitivity of the 8-pCPT-cGMP-activated currents to those of [alpha][beta][gamma]-ENaC channels suggest that the cGMP-activated currents are associated with expressed ENaC. The PKGI activator Sp isomer of 13-phenyl1, [N.sup.2]-etheno-8-bromo-cGMP did not elicit a rise in ENaC current and that the 8-pCPT-cGMP-induced activation of ENaC channels was blocked by incubating oocytes with a PKG inhibitor, but not with other cGMP-sensitive kinase inactivators for PKA, MEK, MAP, and PKC. Surprisingly, both site-directed mutation of putative consensus PKG phosphorylation sites and truncation of entire cytosolic NH2- and COOH-terminal tails did not alter the response to 8-pCPT-cGMP. The ENaC activity was activated to the same extent by 8-pCPT-cGMP in cells in which PKGII expression was knocked down using small interfering RNA. Analog to 8-CPT-cAMP, 8-pCPT-cGMP was capable of activating ENaC in the identical manner in cell-free outside-out patches. We conclude that the rapid upregulation of human [alpha][beta][gamma]-ENaC activity in oocytes by external 8-pCPT-cGMP and 4-chlorothiolphenol-cAMP depends on the parachlorophenylthiol and the hydroxy groups, and 8-pCPT-cGMP may serve as a novel ENaC ligand in addition to activating PKG signal. amiloride; epithelial [Na.sup.+] channel; guanosine 3',5'-cyclic monophosphate; domain mapping doi: 10.1152/ajprenal.00307.2009

Among the multiple organ disorders caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), acute lung failure following atypical pneumonia is the most serious and often fatal event. We hypothesized that two of the hydrophilic structural coronoviral proteins (S and E) would regulate alveolar fluid clearance by decreasing the cell surface expression and activity of amiloride-sensitive epithelial sodium ([Na.sup.+]) channels (ENaC), the rate-limiting protein in transepithelial [Na.sup.+] vectorial transport across distal lung epithelial cells. Coexpression of either S or E protein with human [alpha]-, [beta]-, and [gamma]-ENaC in Xenopus oocytes led to significant decreases of both amiloride-sensitive [Na.sub.+] currents and [gamma]-ENaC protein levels at their plasma membranes. S and E proteins decreased the rate of ENaC exocytosis and either had no effect (S) or decreased (E) rates of endocytosis. No direct interactions among SARS-CoV E protein with either [alpha]- or [gamma]-ENaC were indentified. Instead, the downregulation of ENaC activity by SARS proteins was partially or completely restored by administration of inhibitors of PKC[alpha]/[beta]1 and PKC[zeta]. Consistent with the whole cell data, expression of S and E proteins decreased ENaC single-channel activity in oocytes, and these effects were partially abrogated by PKC[alpha]/[beta]1 inhibitors. Finally, transfection of human airway epithelial (H441) cells with SARS E protein decreased whole cell amiloride-sensitive currents. These findings indicate that lung edema in SARS infection may be due at least in part to activation of PKC by SARS proteins, leading to decreasing levels and activity of ENaC at the apical surfaces of lung epithelial cells. Xenopus oocytes; voltage clamp; cell-attached patches; amiloride-sensitive currents; severe acute respiratory syndrome coronavirus; surface epithelial sodium channels; H441 cells

Cation selectivity and inhibition of malignant glioma [Na.sup.+] channels by Psalmotoxin 1. Am J Physiol Cell Physiol 287: C1282-C1291, 2004. First published July 14, 2004; doi:10.1152/ajpcell. 00077.2004.--Psalmotoxin 1 (a component of the venom of a West Indies tarantula) is a 40-amino acid peptide that inhibits cation currents mediated by acid-sensing ion channels (ASIC). In this study we performed electrophysiological experiments to test the hypothesis that Psalmotoxin 1 (PcTX1) inhibits [Na.sup.+] currents in high-grade human astrocytoma cells (glioblastoma multiforme, or GBM). In whole cell patch-clamped cultured GBM cells, the peptide toxin quickly and reversibly inhibited both inward and outward current with an I[C.sub.50] of 36 [+ or -] 2 pM. The same inhibition was observed in freshly resected GBM cells. However, when the same experiment was performed on normal human astrocytes, the toxin failed to inhibit the whole cell current. We also determined a cationic selectivity sequence for inward currents in three cultured GBM cell lines (SK-MG-1, U87-MG, and U251-MG). The selectivity sequence yielded a unique biophysical fingerprint with inward [K.sup.+] conductance approximately fourfold greater than that of [Na.sup.+], [Li.sup.+], and [Ca.sup.2+]. These observations suggest that PcTX1 may prove useful in determining whether GBM cells express a specific ASIC-containing ion channel type that can serve as a target for both diagnostic and therapeutic treatments of aggressive malignant gliomas. patch clamp; amiloride; ion channels: acid-sensing ion channels

The hypothesis that there is a highly conserved, positively charged region distal to the second transmembrane domain in [alpha]-ENaC (epithelial sodium channel) that acts as a putative receptor site for the negatively charged COOH-terminal [beta]- and [gamma]-ENaC tails was tested in mutagenesis experiments. After expression in Xenopus oocytes, [alpha]-ENaC constructs in which positively charged arginine residues were converted into negatively charged glutamic acids could not be inhibited by blocking peptides. These observations provide insight into the gating machinery of ENaC. Liddle mutant; amiloride; inside-out patch; voltage clamp; post-M2 region