Role of PKCα-p38 MAPK-Giα axis in peroxynitrite-mediated inhibition of β-adrenergic response in pulmonary artery smooth muscle cells.

In the context of cross-talk between transmembrane signaling pathways, we studied the loci within the β-adrenergic receptor/G protein/adenyl cyclase system at which PKC exerts regulatory effects of peroxynitrite (ONOO(-)) on isoproterenol stimulated adenyl cyclase activity in pulmonary artery smooth muscle cells. Treatment of the cells with ONOO(-) stimulated PKC-α activity and that subsequently increased p(38)MAPK phosphorylation. Pretreatment with Go6976 (PKC-α inhibitor) and SB203580 (p(38)MAPK inhibitor) eliminated ONOO(-) caused inhibition on isoproterenol stimulated adenyl cyclase activity. Pretreatment with Go6976, but not SB203580, prevented ONOO(-) induced increase in PKC-α activity. Studies using genetic inhibitors of PKC-α (PKC-α siRNA) and p(38)MAPK (p(38)MAPK siRNA) also corroborated the findings obtained with their pharmacological inhibitors in eliminating the attenuation of ONOO(-) effect on isoproterenol stimulated adenyl cyclase activity. This inhibitory effect of ONOO(-) was found to be eliminated upon pretreatment of the cells with pertussis toxin thereby pointing to a G(i) dependent mechanism. This hypothesis was reinforced by G(i)α phosphorylation as well as by the observation of the loss of the ability of Gpp(NH)p (a measure of G(i) mediated response) to stimulate adenyl cyclase activity upon ONOO(-) treatment to the cells. We suggest the existence of a pertussis toxin sensitive G protein (G(i))-mediated mechanism in isoproterenol stimulated adenyl cyclase activity, which is regulated by PKCα-p(38)MAPK axis dependent phosphorylation of its α-subunit (G(i)α) in the pulmonary artery smooth muscle cells.