title: 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.
authors.affiliation: Department of Pediatrics/Division of Neonatology, University of Texas Health Science Center, San Antonio, Texas 78229, USA. mustafa@uthscsa.edu
authors.forename: Shamimunisa B
authors.initials: SB
authors.lastname: Mustafa
authors.forename: Robert
authors.initials: R
authors.lastname: Castro
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Falck
authors.forename: Jean A
authors.initials: JA
authors.lastname: Petershack
authors.forename: Barbara M
authors.initials: BM
authors.lastname: Henson
authors.forename: Yvonne M
authors.initials: YM
authors.lastname: Mendoza
authors.forename: Ahsan
authors.initials: A
authors.lastname: Choudary
authors.forename: Steven R
authors.initials: SR
authors.lastname: Seidner
mesh_terms: D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D053503:Epithelial Sodium Channels; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D003994:Bucladesine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases