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SPHINGOSINE‐1‐PHOSPHATE INDUCED NAD(P)H OXIDASE ACTIVATION IN SMOOTH MUSCLE CELL MIGRATION REQUIRES Gα12/13 PROTEIN MEDIATED PHOSPHOLIPASE C ACTIVATION.
Author(s) -
Davies Mark G,
Roztocil Elisa,
Nicholl Suzanne
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a69
Subject(s) - nad+ kinase , pertussis toxin , nad(p)h oxidase , gq alpha subunit , phospholipase d , phospholipase c , microbiology and biotechnology , nadph oxidase , g protein , chemistry , oxidase test , sphingosine , nicotinamide adenine dinucleotide phosphate , phospholipase , biochemistry , signal transduction , biology , receptor , enzyme
Background:S‐1‐P is a bioactive sphingolipid that stimulates migration of smooth muscle cells (VSMC) through Gαi, Gαq and Gα12/13 coupled receptors and has been shown to activate NAD(P)H oxidase. Objective: To examine the role of the phospholipase C signaling during NAD(P)H oxidase activation in response to sphingosine‐1‐phosphate (S‐1‐P). Methods: Rat arterial VSMCs were cultured in vitro. Oxygen free radical generation was measured by fluorescent redox indicator assays in response to S‐1‐P in the presence and absence of the active PLC inhibitor (U73122; U7) or its inactive analog U773343 (InactU7). Activation of PLC was assessed both by immunoprecipitation and western blotting for the phosphorylated isozymes (β and γ). siRNA to the G‐proteins Gαi, Gαq and Gα12/13 was developed commercially and cells transfected by a superlipofectamine method with appropriate downregulation of targets. Statistics were by one‐way ANOVA. Results: S‐1‐P induced time dependent activation of PLCβ but not PLCγ; PLCβ activation was blocked by U7 but not by InactU7. This activation was Gαi independent (not blocked by Pertussis toxin a Gαi inhibitor or Gαi2 and Gαi3 siRNA) and Gαq independent (not blocked by GP‐2A a Gαq inhibitor or Gαq siRNA). siRNA to Gα12/13 blocked PLCβ activation. S‐1‐P induced NAD(P)H oxidase activation as measured by DHE staining was significantly inhibited by U7 but not by InactU7. VSMC migration in response to S‐1‐P was inhibited by both PLC and NAD(P)H oxidase inhibition. Conclusion: S‐1‐P induces NAD(P)H oxidase activation through a Gα12/13, PLCβ mediated mechanism which facilitates VSMC migration. This is the first description of PLC‐mediated NAD(P)H oxidase as a mediator of S‐1‐P VSMC.