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 investigated in the present study. Exposure of isolated endothelium-intact and denuded 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-induced 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)], Gö6976 (a PKCalpha and PKCbeta1 selective antagonist), genistein (an antagonist of protein tyrosine kinase), PD-98059 (an antagonist of mitogen-activated protein kinase), wortmannin [an antagonist of phosphatidylinositol 3 (PI3)-kinases], and LY-294002 (an antagonist of PI3-kinases). These agents were also found to relax arteries precontracted by H2O2. Removal of extracellular Ca2+ or pretreatment of the vessels with 5.0 microM verapamil markedly attenuated the contractions. Complete inhibition of the contractile response was obtained after buffering intracellular Ca2+ with BAPTA-AM. A variety of specific pharmacological antagonists of several known vasoconstrictors neither inhibited nor attenuated the H2O2-induced contractions. Exposure of smooth muscle cells to H2O2 (4.4x10(-6) M to 4.4x10(-4) M) significantly raised intracellular Ca2+ ([Ca2+]i) within 20 s. This was abolished in the absence of extracellular Ca2+ or after application of 5.0 microM verapamil. Pretreatment of the cells with low concentrations of staurosporine, bisindolylmaleimide I, Gö6976, genistein, PD-98059, wortmannin, and LY-294002 markedly suppressed the H2O2-mediated [Ca2+]i elevation. The present findings suggest that hydrogen peroxide, in vitro, produces endothelium-dependent and independent contractions of canine basilar arteries, which are clearly Ca2+-dependent and are not associated with release of endogenous vasoconstrictors. Several intracellular signal transduction systems, such as PKC (both Ca2+-dependent and Ca2+-independent), protein tyrosine phosphorylation, IP3, mitogen-activated protein kinase and PI3 kinase appear to play a role in the H2O2-induced contractions in cerebral arterial muscle.