Bijan Rafii

Inhibition of amiloride-sensitive sodium-channel activity in distal lung epithelial cells by nitric oxide

Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current ( I sc) without affecting total I sc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the I sc. The effect was reversed by the addition of N-monomethyl-l-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current (Isc) without affecting total Isc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the Isc. The effect was reversed by the addition of N-monomethyl-L-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.

Pulmonary oedema fluid induces non‐α‐ENaC‐dependent Na+ transport and fluid absorption in the distal lung

To determine if pulmonary oedema fluid (EF) alters ion and fluid transport of distal lung epithelium (DLE), EF was collected from rats in acute heart failure. EF, but not plasma, increased amiloride‐insensitive short circuit current (Isc) and Na+‐K+ ATPase protein content and pump activity of DLE grown in primary culture. Inhibitors of Cl− transport or cGMP‐gated cation channels had a significant (P < 0.05), but limited ability to block the increased Isc. EF increased amiloride‐insensitive, but not amiloride‐sensitive, DLE apical membrane Na+ conductance. The level of mRNA encoding epithelial sodium channel (ENaC) subunits was unchanged (α, β), or decreased (γ, P < 0.05) in EF‐exposed DLE. EF also induced an amiloride‐insensitive increase in the potential difference across murine tracheal cysts. Distal lung explants from late gestation wild‐type and α‐ENaC‐deficient fetal mice, which normally expand due to liquid secretion, decreased in size due to liquid absorption when exposed to EF. Trypsin digestion or heat treatment of EF abrogated the ability of EF to increase amiloride‐insensitive Isc in DLE and liquid absorption by distal lung explants. Thus proteins or protein‐dependent factors within cardiogenic EF induce an α‐ENaC‐independent and amiloride‐insensitive apical membrane Na+ conductance and liquid absorption in the distal lung.

Arginine vasopressin and atrial natriuretic peptide do not alter ion transport by cultured fetal distal lung epithelium.

ABSTRACT: Previous studies have shown that i.v. arginine vasopressin (AVP) decreases but does not stop lung fluid secretion in term fetuses not in labor. Although it has been presumed that the response to AVP results from augmented sodium transport, there is controversy whether AVP actually does affect sodium transport in mammalian lung epithelium. To determine if AVP or aldosterone could alone or together augment sodium transport in the perinatal lung, we studied primary cultures of fetal rat distal lung epithelium in Ussing chambers. The short circuit current of these sodium-transporting cells was not affected by the application of either 30 or 300 mU/mL AVP whether or not they were previously exposed to aldosterone (10−6 M). Aldosterone also did not affect the baseline bioelectric properties. Short circuit current increased in response to 8-bromo cAMP (10−4 M) and 3-isobutyl-1-methylxanthine (10−3 M) to levels 169 ± 16 (SEM) and 172 ± 7% of respective baseline values. AVP had no effect in cells pretreated with 3-isobutyl-1-methylxanthine. Monolayers also did not respond to atrial natriuretic peptide (10−11 to 10−8 M). Monolayers of Na-absorbing A6 renal epithelium did increase short circuit current with either aldosterone or AVP. AVP increased endogenous cAMP levels in A6 but not fetal rat distal lung epithelium cells, suggesting that fetal rat distal lung epithelium lacks V2 receptors. These studies demonstrate that AVP does not increase ion transport in cultured fetal distal lung epithelium although these cells possess the necessary second messenger system.

Preventing Endotoxin-Stimulated Alveolar Macrophages from Decreasing Epithelium Na + Channel (ENaC) mRNA Levels and Activity

The acute respiratory distress syndrome is characterized by impairment of the alveolar-capillary barrier. Our laboratory has shown that distal lung epithelial cell (DLEC) amiloride-sensitive Na+ transport is impaired by in vitro coculture with endotoxin (lipopolysaccharide)-stimulated alveolar macrophages (AM) through an l-arginine-dependent mechanism. To investigate the effect of this model on mRNA levels of the rat epithelial Na+ channel, mature fetal rat DLEC monolayers were incubated for 16 h with rat AM (1 × 107) and lipopolysaccharide (10 μg/mL), or the cell-free supernatant of lipopolysaccharide-stimulated rat AM. Such exposure resulted in a profound decrease in mRNA expression for all subunits (α, β, and γ) of the rat epithelial Na+ channel, without affecting 18S RNA levels. This effect was prevented by the antioxidant N-acetylcysteine. In separate experiments, confluent DLEC monolayers were exposed to lipopolysaccharide-stimulated AM supernatant for 16 h with or without N-acetylcysteine and DTT and studied in Ussing chambers. As previously demonstrated in our laboratory, AM supernatant resulted in a significant (p < 0.05) impairment of DLEC Na+ transport, as reflected by a decrease in the amiloride-sensitive component of short-circuit current (control, 3.96 ± 0.18 μA/cm2versus supernatant, 2.34 ± 0.56 μA/cm2;p < 0.05). This effect was significantly reversed by N-acetylcysteine (3.55 ± 0.48 μA/cm2), but not by DTT (1.87 ± 0.21 μA/cm2). N-acetylcysteine, but not DTT, increased DLEC thiol levels. These studies elucidate mechanisms by which activated AM impair alveolar epithelial barrier function in an in vitro model of acute lung injury.

A Human Epithelium-Specific Vector Optimized in Rat Pneumocytes for Lung Gene Therapy

Gene therapy vectors based on mammalian promoters offer the potential for increased cell specificity and may be less susceptible than viral promoters to transcription attenuation by host cytokines. The human cytokeratin 18 (K18) gene is naturally expressed in the lung epithelia, a target site for gene therapies to treat certain genetic pediatric lung diseases. Our original vector based on the promoter and 5′ control elements of K18 offered excellent epithelial cell specificity but relatively low expression levels compared with viral promoters. In the present study, we found that adding a stronger SV40 poly(A) signal boosted primary rat lung epithelial cell expression but greatly reduced cell specificity. Addition of a 3′ portion of the K18 gene to our vector as a 3′ untranslated region (UTR) improved epithelial cell-specific expression by reducing expression in lung fibroblasts. The effect of the 3′ UTR was not related to gross differences in cell-specific splicing. A deletion variant of this UTR further increased lung epithelial cell expression while retaining some cell specificity. These data illustrate the possibilities for using 3′ UTR to regulate cell-specific transgene expression. Our improved K18 vector should prove useful for pediatric lung gene therapy applications.

Protease-activated receptor (Par)1 alters bioelectric properties of distal lung epithelia without compromising barrier function.

Proteinases contribute to the pathogenesis of various lung diseases, partly through activating cell surface receptors by limited proteolytic cleavage. The authors provide evidence that in primary cultures of distal lung epithelia, basolateral protease-activated receptor 1 activation rapidly reduces transepithelial resistance but does not alter paracellular permeability to small uncharged solutes. Changes in transepithelial resistance were partially blocked by ion transport inhibitors and were completely blocked by placing cells in low chloride buffer. In vivo studies did not reveal enhanced lung permeability in response to pulmonary or intravenous administration of protease-activated receptor 1 activators. This information is relevant as strategies to inhibit protease-activated receptor 1 signaling are considered in order to preserve lung epithelial barrier function.