Differential calcium regulation by hydrogen peroxide and superoxide in vascular smooth muscle cells from spontaneously hypertensive rats

We investigated the role of reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2) and superoxide anion ( .O2-) in the regulation of vascular smooth muscle cell (VSMC) Ca2+ concentration ([Ca2+]i) and vascular contraction and assessed whether redox-dependent Ca2+ signaling and contraction are altered in hypertension. VSMCs and mesenteric arteries from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were studied. Cells were stimulated with H2O2 (10-4 mol/l) or LY83583 (.O2- generator, 10 -5 mol/l). [Ca2+]i and cytosolic .O2- were measured by fura2AM and tempo-9-AC fluorescence respectively. L-type and T-type Ca2+ channels were assessed using verapamil/diltiazem and mibefradil respectively and mRNA and protein expression of these channels was assessed by real-time PCR and immunoblotting respectively. H2O2 time-dependently increased [Ca2+]i and contraction with significantly greater effects in SHR versus WKY (P < 0.001). LY83583 increased [Ca 2+]i in both strains, but responses were blunted in SHR. Removal of extracellular Ca2+ abrogated [Ca2+]i responses to H2O2 and .O2-. Verapamil and diltiazem, but not mibefradil, significantly decreased H2O2-induced [Ca2+]i responses with greater effects in SHR (P < 0.01). L-type and T-type Ca 2+ channel inhibition reduced LY83583-mediated [Ca2+] i increase only in WKY cells. Both types of Ca2+ channels were expressed (mRNA and protein) in VSMCs from WKY and SHR, with greater abundance in SHR than WKY (2- to 3-fold). These results demonstrate that ROS increase vascular [Ca2+]i and contraction, primarily via extracellular Ca2+ influx. Whereas responses to H2O 2 are enhanced, .O2--mediated actions are blunted in SHR. These effects may relate to differential activation of Ca2+ channels by H2O2 and .O 2-. Enhanced activation of L-type Ca2+ channels and increased Ca2+ influx by H2O2 may contribute to increased Ca2+ signaling in VSMCs from SHR