Cpt-cAMP activates human epithelial sodium channels via relieving self-inhibition.

External Na(+) self-inhibition is an intrinsic feature of epithelial sodium channels (ENaC). Cpt-cAMP regulates heterologous guinea pig but not rat αßγ 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 αßγ ENaC in a dose- and external Na(+) concentration-dependent fashion. Intriguingly, cpt-cAMP activated human δßγ more potently than αßγ 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 (ß(ΔV348) and γ(H233R)) 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 (αß(S520C)γ) 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 αßγ and δßγ ENaC channels by relieving self-inhibition.

δ ENaC: a novel divergent amiloride-inhibitable sodium channel.

The fourth subunit of the epithelial sodium channel, termed delta subunit (δ ENaC), was cloned in human and monkey. Increasing evidence shows that this unique subunit and its splice variants exhibit biophysical and pharmacological properties that are divergent from those of α ENaC channels. The widespread distribution of epithelial sodium channels in both epithelial and nonepithelial tissues implies a range of physiological functions. The altered expression of SCNN1D is associated with numerous pathological conditions. Genetic studies link SCNN1D deficiency with rare genetic diseases with developmental and functional disorders in the brain, heart, and respiratory systems. Here, we review the progress of research on δ ENaC in genomics, biophysics, proteomics, physiology, pharmacology, and clinical medicine.

Characterization of a novel splice variant of δ ENaC subunit in human lungs.

Salt absorption via apical epithelial sodium channels (ENaC) is a critical rate-limiting process in maintaining airway and lung lining fluid at the physiological level. δ ENaC (termed δ1 in this article) has been detected in human lung epithelial cells in addition to α, ß, and γ subunits (Ji HL, Su XF, Kedar S, Li J, Barbry P, Smith PR, Matalon S, Benos DJ. J Biol Chem 281: 8233-8241, 2006; Nie HG, Chen L, Han DY, Li J, Song WF, Wei SP, Fang XH, Gu X, Matalon S, Ji HL, J Physiol 587: 2663-2676, 2009) and may contribute to the differences in the biophysical properties of amiloride-inhibitable cation channels in pulmonary epithelial cells. Here we cloned a splicing variant of the δ1 ENaC, namely, δ2 ENaC in human bronchoalveolar epithelial cells (16HBEo). δ2 ENaC possesses 66 extra amino acids attached to the distal amino terminal tail of the δ1 ENaC. δ2 ENaC was expressed in both alveolar type I and II cells of human lungs as revealed by in situ hybridization and real-time RT-PCR. To characterize the biophysical and pharmacological features of the splicing variant, we injected Xenopus oocytes with human ENaC cRNAs and measured whole cell and single channel currents of δ1ßγ, δ2ßγ, and αßγ channels. Oocytes injected with δ2ßγ cRNAs exhibited whole cell currents significantly greater than those expressing δ1ßγ and αßγ channels. Single channel activity, unitary conductance, and open probability of δ2ßγ channels were significantly greater compared with δ1ßγ and αßγ channels. In addition, δ2ßγ and δ1ßγ channels displayed significant differences in apparent Na(+) affinity, dissociation constant for amiloride (K(i)(amil)), the EC(50) for capsazepine activation, and gating kinetics by protons. Channels comprising of this novel splice variant may contribute to the diversities of native epithelial Na(+) channels.

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 αßγ but not δßγ channels. To further elucidate this novel mechanism, we constructed mutants abolishing (ß(ΔV348) and γ(H233R)) or augmenting (α(Y458A) and γ(M432G)) 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 P(o) mutant (ß(S520C)) and plasmin proteolytically cleaved channels. Our data suggest that elimination of self-inhibition of αßγ ENaC may be a novel mechanism for CPT-cGMP to stimulate salt reabsorption in human lungs.

8-pCPT-cGMP stimulates alphabetagamma-ENaC activity in oocytes as an external ligand requiring specific nucleotide moieties.

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 alphabetagamma-ENaC channels expressed in Xenopus oocytes to 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), a cell-permeable cGMP analog. This compound stimulated human alphabetagamma-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 alphabetagamma-ENaC channels. The identical ion selectivity and amiloride sensitivity of the 8-pCPT-cGMP-activated currents to those of alphabetagamma-ENaC channels suggest that the cGMP-activated currents are associated with expressed ENaC. The PKGI activator Sp isomer of beta-phenyl-1,N(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 NH(2)- 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 alphabetagamma-ENaC activity in oocytes by external 8-pCPT-cGMP and 4-chlorothiolphenol-cAMP depends on the para-chlorophenylthiol and the hydroxy groups, and 8-pCPT-cGMP may serve as a novel ENaC ligand in addition to activating PKG signal.

Stem cell therapy for COVID-19 and other respiratory diseases: Global trends of clinical trials.

Respiratory diseases, including coronavirus disease 2019 and chronic obstructive pulmonary disease (COPD), are leading causes of global fatality. There are no effective and curative treatments, but supportive care only. Cell therapy is a promising therapeutic strategy for refractory and unmanageable pulmonary illnesses, as proved by accumulating preclinical studies. Stem cells consist of totipotent, pluripotent, multipotent, and unipotent cells with the potential to differentiate into cell types requested for repair. Mesenchymal stromal cells, endothelial progenitor cells, peripheral blood stem cells, and lung progenitor cells have been applied to clinical trials. To date, the safety and feasibility of stem cell and extracellular vesicles administration have been confirmed by numerous phase I/II trials in patients with COPD, acute respiratory distress syndrome, bronchial dysplasia, idiopathic pulmonary fibrosis, pulmonary artery hypertension, and silicosis. Five routes and a series of doses have been tested for tolerance and advantages of different regimes. In this review, we systematically summarize the global trends for the cell therapy of common airway and lung diseases registered for clinical trials. The future directions for both new clinical trials and preclinical studies are discussed.

Epithelial Sodium Channels in Pulmonary Epithelial Progenitor and Stem Cells.

Regeneration of the epithelium of mammalian lungs is essential for restoring normal function following injury, and various cells and mechanisms contribute to this regeneration and repair. Club cells, bronchioalveolar stem cells (BASCs), and alveolar type II epithelial cells (ATII) are dominant stem/progenitor cells for maintaining epithelial turnover and repair. Epithelial Na(+) channels (ENaC), a critical pathway for transapical salt and fluid transport, are expressed in lung epithelial progenitors, including club and ATII cells. Since ENaC activity and expression are development- and differentiation-dependent, apically located ENaC activity has therefore been used as a functional biomarker of lung injury repair. ENaC activity may be involved in the migration and differentiation of local and circulating stem/progenitor cells with diverse functions, eventually benefiting stem cells spreading to re-epithelialize injured lungs. This review summarizes the potential roles of ENaC expressed in native progenitor and stem cells in the development and regeneration of the respiratory epithelium.

Genes Regulating Epithelial Polarity Are Critical Suppressors of Esophageal Oncogenesis.

Esophageal cancer is an aggressive disease featured by early lymphatic and hematogenous dissemination, and is the sixth leading cause of cancer-related deaths worldwide. The proper formation of apicobasal polarity is essential for normal epithelium physiology and tissue homeostasis, while loss of polarity is a hallmark of cancer development including esophageal oncogenesis. In this review, we summarized the stages of esophageal cancer development associated with the loss or deregulation of epithelial cell apicobasal polarity. Loss of epithelial apicobasal polarity exerts an indispensable role in the initiation of esophageal oncogenesis, tumor progression, and the advancement of tumors from benign to malignant. In particular, we reviewed the involvement of several critical genes, including Lkb1, claudin-4, claudin-7, Par3, Lgl1, E-cadherin, and the Scnn1 gene family. Understanding the role of apicobasal regulators may lead to new paradigms for treatment of esophageal tumors, including improvement of prognostication, early diagnosis, and individually tailored therapeutic interventions in esophageal oncology.