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Dexamethasone produces anti-secretory responses in airway epithelium through the inhibition of basolateral membrane K+ channels [1–3]. We have used the human bronchial epithelial cell line 16HBE14o− to investigate the effects of dexamethasone on the expression of K+ channels and regulatory protein kinases. The data demonstrate the expression of three distinct K+ channel types – KCNQ1:KCNE3, KCNN4 and KATP which are differentially regulated by protein kinase A and protein kinase C. The data also provide evidence for rapid non-genomic actions of dexamethasone on PKC and PKA phosphorylation and their association with the various K+ channel sub-types. Biotinylation experiments provide data on the effects of dexamethasone on membrane expression of the K+ channels. Antibody co-immunoprecipitation, rtPCR and western blotting data are given for the non-genomic dexamethasone transcription-cell signaling pathway involving Gi-protein coupled receptor, PKC, adenylyl cyclase Type IV, cAMP, PKA and ERK1/2 activation. Keywords: Dexamethasone, Non-genomic, Airway epithelium, KCNQ1, KCNN4, KATP channels, Protein kinases

Basolateral membrane K+ channels play a key role in basal and agonist stimulated Cl- transport across airway epithelial cells by generating a favourable electrical driving force for Cl- efflux. The K+ channel sub-types and molecular mechanisms of regulation by hormones and secretagoues are still poorly understood. Here we have identified the type of K+ channels involved in cAMP and Ca2+ stimulated Cl- secretion and uncovered a novel anti-secretory effect of dexamethasone mediated by inhibition of basolateral membrane K+ channels in a human airway cell model of 16HBE14o- cells commonly used for ion transport studies. Dexamethasone produced a rapid inhibition of transepithelial chloride ion secretion under steady state conditions and after stimulation with cAMP agonist (forskolin) or a Ca2+ mobilizing agonist (ATP). Our results show three different types of K+ channels are targeted by dexamethasone to reduce airway secretion, namely Ca2+-activated secretion via KCNN4 (KCa3.1) channels and cAMP-activated secretion via KCNQ1 (Kv7.1) and KATP (Kir6.1,6.2) channels. The down-regulation of KCNN4 and KCNQ1 channel activities by dexamethasone involves rapid non-genomic activation of PKCα and PKA signalling pathways, respectively. Dexamethasone signal transduction for PKC and PKA activation was demonstrated to occur through a rapid non-genomic pathway that did not implicate the classical nuclear receptors for glucocorticoids or mineralocorticoids but occurred via a novel signalling cascade involving sequentially a Gi-protein coupled receptor, PKC, adenylyl cyclase Type IV, cAMP, PKA and ERK1/2 activation. The rapid, non-genomic, effects of dexamethasone on airway epithelial ion transport and cell signalling introduces a new paradigm for glucocorticoid actions in lung epithelia which may serve to augment the anti-inflammatory activity of the steroid and enhance its therapeutic potential in treating airway hypersecretion in asthma and COPD.

Glucocorticoids have been shown to produce rapid nongenomic responses in airway epithelia. By using an intracellular pH (pH(i)) spectrofluorescence imaging system and the NH4Cl acid-loading technique, we have shown that the synthetic glucocorticoid,dexamethasone, accelerated intracellular pH recovery after an acid load in a human bronchial epithelial cell line (16HBE14o- cells). Exposure to NH4Cl (20 mm) elicited an intracellular acidification, followed by a pH(i) recovery. Inhibition of the Na+/H+ exchanger decreased the steady-state pH(i) and antagonized the dexamethasone stimulation of pH(i) regulation. The rapid effect of dexamethasone on pH(i) was neither affected by the inhibitor of transcription, cycloheximide, nor by the classical glucocorticoid and mineralocorticoid receptors antagonists, RU486 and spironolactone, respectively. The dexamethasone effect on pH(i) regulation was reduced by inhibitors of adenylate cyclase, cAMP-dependent protein kinase and mitogenactivated protein kinase (ERK1/2). By using a PepTag assay system and Western blotting, we have shown that dexamethasone stimulated cAMP-dependent protein kinase and mitogen-activated protein kinase activities. Taken together our results provide evidence for the rapid stimulation of Na+/H+ exchange activity by glucocorticoids in bronchial epithelial cells via a nongenomic mechanism involving cAMP-dependent protein kinase and mitogen-activated protein kinase ERK1/2 pathways.