Chapter 1 - Role of the Gα13-PI3Kγ-PLD signaling axis in stimulating NADPH oxidase-derived O2•− production by urotensin II in pulmonary artery smooth muscle cells

Abstract

We have investigated the role of the Gα13-PI3Kinase (PI3K) axis on PLD activation via the Arf6-cytohesin1 signaling axis for a marked increase in NADPH oxidase activity by urotensin II (UT-II) in bovine pulmonary artery smooth muscle cells (BPASMCs). UT-II treatment to the cells stimulated PI3Kγ, PLD, and NADPH oxidase activities in the cell membrane. Pretreatment of the cells with chemical and genetic inhibitors of PLD2, but not PLD1, attenuated PLD and NADPH oxidase activities without a discernible alteration of PI3K activity. Pretreatment with the chemical and genetic inhibitors of NADPH oxidase (p47phox, a component of NADPH oxidase complex, is used as a genetic inhibitor of NADPH oxidase) inhibited UT-II-mediated stimulation of NADPH oxidase activity without significantly altering PI3Kγ and PLD2 activities. Preincubation of the cells with chemical and genetic inhibitors of urotensin receptor (UT-R), Gα13, and PI3Kγ attenuated, respectively, UT-II-mediated increase in PI3Kγ, PLD2, and NADPH oxidase activities. Co-immunoprecipitation study suggested association of UT-R with Gα13 in the cell membrane. Preincubation of the cells with chemical and genetic inhibitors of UT-R and Gα13 markedly inhibited UT-II-induced increase in the association between UT-R and Gα13 in the cell membrane. Pretreatment of the cells with the chemical and genetic inhibitors of PI3Kγ did not show a discernible change in the association of UT-R with Gα13 in the cell membrane. Pretreatment of the cells with chemical and genetic inhibitors of Gα13 and PI3Kγ, but not PLD, attenuated UT-II-induced increase in the translocation of Arf-6 and cytohesin-1, and their association in the cell membrane. Transfection of the cells with Arf-6 and cytohesin-1 siRNA inhibited UT-II-induced stimulation of PLD and NADPH oxidase activities without significantly altering PI3Kγ activity. Pretreatment of the cells with chemical and genetic inhibitors of PLD2 attenuated UT-II-induced increase in PKCζ and NADPH oxidase activities, but not PI3Kγ activity, in the cell membrane. Pretreatment of the cells with chemical and genetic inhibitors of PKC-ζ attenuated the increase in NADPH oxidase activity by UT-II without a noticeable change in PLD activity. UT-II-mediated stimulation of p47phox phosphorylation and NADPH oxidase activity were attenuated by the chemical and genetic inhibitors of PKC-ζ. Overall, the UTR-Gα13-PI3Kγ-Arf6-cytohesin1signaling axis plays a crucial role in UT-II caused stimulation of PLD2 activity and subsequently increases PKCζ activity, resulting in stimulation of p47phox phosphorylation for a marked increase in NADPH oxidase-derived O2•− generation in the cells.