Abstract
Serine proteases play important physiological roles through their activity at G protein-coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C (PKC) isoforms play in the regulation of PAR(2)-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short-circuit current (I(sc)) responses to basolateral application of the selective PAR(2) activating peptide, SLIGRL-NH(2), were monitored as a measure of net electrogenic ion transport caused by PAR(2) activation. SLIGRL-NH(2) induced a transient I(sc) response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLCbeta and PLCgamma following PAR(2) activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKCalpha/betaI (Gö6976), and PKCdelta (rottlerin), but not PKCzeta (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKCbetaI, PKCdelta, and PKCepsilon, but not PKCalpha or PKCzeta, in membrane fractions following PAR(2) activation. Pretreatment of the cells with U73122, ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKCdelta translocation. Immunoblots revealed that PAR(2) activation induced phosphorylation of both cRaf and ERK1/2 via PKCdelta. Inhibition of PKCbetaI and PI3K had only a partial effect on this response. We conclude that basolateral PAR(2)-induced chloride secretion involves activation of PKCbetaI and PKCdelta via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Biological Transport / physiology*
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Cell Line
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Cell Membrane / metabolism
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Chlorides / metabolism*
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Dogs
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Enzyme Inhibitors / pharmacology
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Epithelial Cells / drug effects
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Epithelial Cells / metabolism
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ErbB Receptors / antagonists & inhibitors
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Extracellular Signal-Regulated MAP Kinases / antagonists & inhibitors
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Extracellular Signal-Regulated MAP Kinases / metabolism
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Intestinal Mucosa / cytology
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Intestinal Mucosa / metabolism*
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Isoenzymes / antagonists & inhibitors
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Isoenzymes / metabolism
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Models, Biological
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Oligopeptides / pharmacology
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphoinositide-3 Kinase Inhibitors
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Phospholipase C beta / metabolism
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Phospholipase C gamma / metabolism
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Protein Kinase C / antagonists & inhibitors
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Protein Kinase C / metabolism*
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Protein Kinase C beta
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Protein Kinase C-delta / antagonists & inhibitors
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Protein Kinase C-delta / metabolism
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Protein Kinase C-epsilon / metabolism
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Protein Kinase Inhibitors / pharmacology
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Protein Transport / drug effects
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Protein Transport / physiology
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Proto-Oncogene Proteins c-akt / antagonists & inhibitors
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Proto-Oncogene Proteins c-akt / metabolism
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Proto-Oncogene Proteins c-raf / antagonists & inhibitors
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Proto-Oncogene Proteins c-raf / metabolism
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Receptor, PAR-2 / antagonists & inhibitors
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Receptor, PAR-2 / physiology*
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Signal Transduction / drug effects
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Signal Transduction / physiology*
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Type C Phospholipases / antagonists & inhibitors
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Type C Phospholipases / metabolism*
Substances
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Chlorides
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Enzyme Inhibitors
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Isoenzymes
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Oligopeptides
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Phosphoinositide-3 Kinase Inhibitors
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Protein Kinase Inhibitors
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Receptor, PAR-2
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seryl-leucyl-isoleucyl-glycyl--arginyl-leucinamide
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ErbB Receptors
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Proto-Oncogene Proteins c-akt
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Proto-Oncogene Proteins c-raf
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Protein Kinase C
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Protein Kinase C beta
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Protein Kinase C-delta
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Protein Kinase C-epsilon
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Extracellular Signal-Regulated MAP Kinases
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Type C Phospholipases
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Phospholipase C beta
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Phospholipase C gamma