Protein kinase A and mitogen-activated protein kinase pathways mediate cAMP induction of alpha-epithelial Na+ channels (alpha-ENaC).
ABSTRACT A major mechanism for Na+ transport across epithelia occurs through epithelial Na+ channels (ENaC). ENaC is a multimeric channel consisting of three subunits (alpha, beta, and gamma). The alpha-subunit is critical for ENaC function. In specific culture conditions, the rat submandibular gland epithelial cell line (SMG-C6) demonstrates minimal Na+ transport properties and exposure to dibutyryl cAMP (DbcAMP) for up to 48 h caused an elevation of alpha-ENaC mRNA and protein expression and amiloride-sensitive short-circuit current (I(SC)). Here we examined the early signaling pathways evoked by DbcAMP which contribute to the eventual increase in Na+ transport is present. Treatment with either of the protein kinase A (PKA) inhibitors KT5720 or H-89 followed by exposure to 1 mM DbcAMP for 24 h markedly attenuated DbcAMP-induced alpha-ENaC protein formation and I(SC). Exposure of SMG-C6 cells to 1 mM DbcAMP induced a rapid, transient phosphorylation of the cAMP response element binding protein (CREB). This response was attenuated in the presence of either KT5720 or H-89. Dominant-negative CREB decreased DbcAMP-induced alpha-ENaC expression. Suppression of the extracellular signal-regulated protein kinase (ERK 1,2) with PD98059 or the p38 mitogen-activated protein kinase (MAPK) pathway with SB203580 reduced DbcAMP-induced alpha-ENaC protein levels in SMG-C6 cells. DbcAMP-induced phosphorylation of CREB was markedly attenuated by PD98059 or SB203580. DbcAMP-induced activation of the either the p38 or the ERK 1,2 MAPK pathways was abolished by either of the PKA inhibitors, H-89 or KT5720. Cross talk between these signaling pathways induced by DbcAMP via the activation of CREB appears to contribute to increased levels of alpha-ENaC observed after 24 h of treatment in SMG-C6 epithelial cells.
Article: Lipopolysaccharide modifies amiloride-sensitive Na+ transport processes across human airway cells: role of mitogen-activated protein kinases ERK 1/2 and 5.[show abstract] [hide abstract]
ABSTRACT: Bacterial lipopolysaccharides (LPS) are potent inducers of proinflammatory signaling pathways via the activation of nuclear factor-kappa B (NF-kappaB) and mitogen-activated protein kinase (MAPK), causing changes in the processes that control lung fluid homeostasis and contributing to the pathogenesis of lung disease. In human H441 airway epithelial cells, incubation of cells with 15 microg ml(-1) LPS caused a significant reduction in amiloride-sensitive I (sc) from 15 +/- 2 to 8 +/- 2 microA cm(-2) (p = 0.01, n = 13) and a shift in IC(50) amiloride of currents from 6.8 x 10(-7) to 6.4 x 10(-6) M. This effect was associated with a decrease in the activity of 5 pS, highly Na(+) selective, amiloride-sensitive <1 microM channels (HSC) and an increase in the activity of approximately 18 pS, nonselective, amiloride-sensitive >10 microM cation channels (NSC) in the apical membrane. LPS decreased alphaENaC mRNA and protein abundance, inferring that LPS inhibited alphaENaC gene expression. This correlated with the decrease in HSC activity, indicating that these channels, but not NSCs, were comprised of at least alphaENaC protein. LPS increased NF-kappaB DNA binding activity and phosphorylation of extracellular signal-related kinase (ERK)1/2, but decreased phosphorylation of ERK5 in H441 cells. Pretreatment of monolayers with PD98059 (20 microM) inhibited ERK1/2 phosphorylation, promoted phosphorylation of ERK5, increased alphaENaC protein abundance, and reversed the effect of LPS on I (sc) and the shift in amiloride sensitivity. Inhibitors of NF-kappaB activation were without effect. Taken together, our data indicate that LPS acts via ERK signaling pathways to decrease alphaENaC transcription, reducing HSC/ENaC channel abundance, activity, and transepithelial Na(+) transport in H441 airway epithelial cells.Pflügers Archiv - European Journal of Physiology 10/2009; 459(3):451-63. · 4.46 Impact Factor