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Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms.
Am J Physiol Gastrointest Liver Physiol. 2009 Jun; 296(6):G1258-66.AJ

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.

Authors+Show Affiliations

Dept. of Physiology, Univ. of Calgary, 3330 Hospital Dr. NW, Calgary, AB T2N 4N1, Canada.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19359428

Citation

van der Merwe, Jacques Q., et al. "Protease-activated Receptor-2 Stimulates Intestinal Epithelial Chloride Transport Through Activation of PLC and Selective PKC Isoforms." American Journal of Physiology. Gastrointestinal and Liver Physiology, vol. 296, no. 6, 2009, pp. G1258-66.
van der Merwe JQ, Moreau F, MacNaughton WK. Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms. Am J Physiol Gastrointest Liver Physiol. 2009;296(6):G1258-66.
van der Merwe, J. Q., Moreau, F., & MacNaughton, W. K. (2009). Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms. American Journal of Physiology. Gastrointestinal and Liver Physiology, 296(6), G1258-66. https://doi.org/10.1152/ajpgi.90425.2008
van der Merwe JQ, Moreau F, MacNaughton WK. Protease-activated Receptor-2 Stimulates Intestinal Epithelial Chloride Transport Through Activation of PLC and Selective PKC Isoforms. Am J Physiol Gastrointest Liver Physiol. 2009;296(6):G1258-66. PubMed PMID: 19359428.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms. AU - van der Merwe,Jacques Q, AU - Moreau,France, AU - MacNaughton,Wallace K, Y1 - 2009/04/09/ PY - 2009/4/11/entrez PY - 2009/4/11/pubmed PY - 2009/7/31/medline SP - G1258 EP - 66 JF - American journal of physiology. Gastrointestinal and liver physiology JO - Am. J. Physiol. Gastrointest. Liver Physiol. VL - 296 IS - 6 N2 - 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. SN - 0193-1857 UR - https://www.unboundmedicine.com/medline/citation/19359428/Protease_activated_receptor_2_stimulates_intestinal_epithelial_chloride_transport_through_activation_of_PLC_and_selective_PKC_isoforms_ L2 - http://www.physiology.org/doi/full/10.1152/ajpgi.90425.2008?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -