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The effects of endogenous and exogenous nitric oxide (NO) on chloride currents were analyzed in A549 alveolar epithelial cells via whole-cell patch-clamp recordings. Whole-cell current measurements of epithelial ion transport in A549 alveolar epithelial cells indicated the dominant role of Cl- channels in generating membrane currents. Furthermore, the high permeability of Cl- indicated the role of non-cystic fibrosis conductance regulator Cl- channels in mediating chloride ion transport.

The protein tyrosine kinase Syk is critically involved in immunoreceptor signaling in hematopoietic cells. Recent studies demonstrate Syk expression in nonhematopoietic cells, including fibroblasts, endothelial cells, hepatocytes, and breast epithelium. However, the role of Syk in these cells is uncertain. We hypothesized that Syk is expressed in respiratory epithelial cells (EC) and that it functions as a signaling molecule involved in inflammatory responses in the epithelium. With the use of immunohistochemistry, Western blot, PCR, and laser scanning confocal microscopy, Syk was detected in human, rat, and mouse bronchial epithelium in situ and in cultured human bronchial EC in primary cells and the cell lines HS-24 and BEAS-2B. Syk-dependent signaling pathways in EC were initiated by engagement of beta1-integrin receptors. Stimulation of beta1-integrin receptors by fibronectin or antibody cross-linking caused redistribution of Syk from a cytoplasmic to plasma membrane localization. In stimulated cells, Syk and beta1-integrin colocalized. In addition, following beta1-integrin receptor engagement, tyrosine phosphorylation of Syk was observed. Expression of the intercellular adhesion molecule-1 (ICAM-1) and production of IL-6, both important molecules in lung inflammation, was downregulated in EC treated with Syk small interfering RNA or Syk inhibitor piceatannol. We propose that Syk is involved in signaling pathways induced by integrin engagement in airway EC. Syk-mediated signaling regulates IL-6 and ICAM-1 expression and may be important in the pathophysiology of lung inflammation.

The regulatory actions of adenosine on ion channel function are mediated by four distinct membrane receptors. The concentration of adenosine in the vicinity of these receptors is controlled, in part, by inwardly directed nucleoside transport. The purpose of this study was to characterize the effects of adenosine on ion channels in A549 cells and the role of nucleoside transporters in this regulation. Ion replacement and pharmacological studies showed that adenosine and an inhibitor of human equilibrative nucleoside transporter (hENT)-1, nitrobenzylthioinosine, activated K(+) channels, most likely Ca(2+)-dependent intermediate-conductance K(+) (I(K)) channels. A(1) but not A(2) receptor antagonists blocked the effects of adenosine. RT-PCR studies showed that A549 cells expressed mRNA for I(K)-1 channels as well as A(1), A(2A), and A(2B) but not A(3) receptors. Similarly, mRNA for equilibrative (hENT1 and hENT2) but not concentrative (hCNT1, hCNT2, and hCNT3) nucleoside transporters was detected, a result confirmed in functional uptake studies. These studies showed that adenosine controls the function of K(+) channels in A549 cells and that hENTs play a crucial role in this process.

Nitric oxide (NO) is continuously produced and released in human airways, but the biological significance of this process is unknown. In this study, we have used Calu-3 cells to investigate the effects of NO on transepithelial anion secretion. An inhibitor of NO synthase, NG-nitro-L-arginine methyl ester, reduced short- circuit current (I(sc)), whereas an NO donor, S-nitrosoglutathione (GSNO), increased I(sc), with an EC50 approximately 1.2 microM. The NO-activated current was inhibited by diphenylamine-2-carboxylate, clotrimazole, and charybdotoxin. Selective permeabilization of cell membranes indicated that NO activated both apical anion channels and basolateral potassium channels. An inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, prevented activation of I(sc) by NO but not by 8-bromo-cGMP, suggesting that NO acts via a cGMP-dependent pathway. Sequential treatment of cells with forskolin and GSNO or 1-ethyl-2-benzimidazolinone and GSNO showed additive effects of these chemicals on I(sc). Interestingly, GSNO elevated intracellular Ca2+ concentration ([Ca2+]i) but had no effect on I(sc) activated by thapsigargin. These results show that NO activates transepithelial anion secretion via a cGMP-dependent pathway that involves cross talk between NO and [Ca2+]i.