Hydrogen peroxide induces contraction and raises [Ca2+](i) in canine cerebral arterial smooth muscle: participation of cellular signaling pathways

The effects of hydrogen peroxide (H2O2) on isolated canine basilar arteries , and single smooth muscle cells isolated from these arteries, were investi gated in the present study. Exposure of isolated endothelium-intact and den uded arterial rings to H2O2, at concentrations of 2.2x10(-5) M to 4.4x10(-3 ) M, produced concentration-dependent contractile responses. Removal of the endothelium attenuated, but did not eliminate the contractions. H2O2- indu ced contractions were inhibited, to different degrees, by preincubation of the vessels with low concentrations of staurosporine or bisindolylmaleimide I HCl [antagonists of protein kinase C (PKC)], Go6976 (a PKCalpha and PKCb eta 1 selective antagonist) genistein (an antagonist of protein tyrosine ki nase), PD-98059 (an antagonist of mitogen-activated protein kinase), wortma nnin [an antagonist of phosphatidylinositol 3 (PI3)-kinases], and LY-294002 (an antagonist of PI3-kinases). These agents were also found to relax arte ries precontracted by H2O2. Removal of extracellular Ca2+ or pretreatment o f the vessels with 5.0 mu M verapamil markedly attenuated the contractions. Complete inhibition of the contractile response was obtained after bufferi ng intracellular Ca2+ with BAPTA-AM. A variety of specific pharmacological antagonists of several known vasoconstrictors neither inhibited nor attenua ted the H2O2-induced contractions. Exposure of smooth muscle cells to H2O2 (4.4x10(-6) M to 4.4x10(-6) M) significantly raised intracellular Ca2+ ([Ca 2+](i)) within 20 s. This was abolished in the absence of extracellular Ca2 + or after application of 5.0 mu M verapamil. Pretreatment of the cells wit h low concentrations of staurosporine, bisindolylmaleimide I, Go6976, genis tein, PD-98059, wortmannin, and LY-294002 markedly suppressed the H2O2-medi ated [Ca2+](i) elevation. The present findings suggest that hydrogen peroxi de, in vitro, produces endothelium-dependent and independent contractions o f canine basilar arteries, which are clearly Ca2+-dependent and are not ass ociated with release of endogenous vasoconstrictors. Several intracellular signal transduction systems, such as PKC (both Ca2+-dependent and Ca2+-inde pendent), protein tyrosine phosphorylation, IP3, mitogen-activated protein kinase and PI3 kinase appear to play a role in the H2O2-induced contraction s in cerebral arterial muscle.