Rotenone-Stimulated Superoxide Release From Mitochondrial Complex I Acutely Augments L-Type Ca2+ Current In A7r5 Aortic Smooth Muscle Cells

Voltage-gated L-type Ca2+ current (I-Ca,I-L) induces contraction of arterial smooth muscle cells (ASMCs), and I-Ca,I-L is increased by H2O2 in ASMCs. Superoxide released from the mitochondrial respiratory chain (MRC) is dismutated to H2O2. We studied whether superoxide per se acutely modulates I-Ca,I-L in ASMCs using cultured A7r5 cells derived from rat aorta. Rotenone is a toxin that inhibits complex I of the MRC and increases mitochondrial superoxide release. The superoxide content of mitochondria was estimated using mitochondrial-specific MitoSOX and HPLC methods, and was shown to be increased by a brief exposure to 10 mu M rotenone. I-Ca,I-L was recorded with 5 mM BAPTA in the pipette solution. Rotenone administration (10 nM to 10 mu M) resulted in a greater I-Ca,I-L increase in a dose-dependent manner to a maximum of 22.1% at 10 mu M for 1 min, which gradually decreased to 9% after 5 min. The rotenone-induced I-Ca,I-L increase was associated with a shift in the current-voltage relationship (I-V) to a hyperpolarizing direction. DTT administration resulted in a 17.9% increase in I-Ca,I-L without a negative shift in I-V, and rotenone produced an additional increase with a shift. H2O2 (0.3 mM) inhibited I-Ca,I-L by 13%, and additional rotenone induced an increase with a negative shift. Sustained treatment with Tempol (4-hydroxy tempo) led to a significant I-Ca,I-L increase but it inhibited the rotenone-induced increase. Staurosporine, a broad-spectrum protein kinase inhibitor, partially inhibited I-Ca,I-L and completely suppressed the rotenone-induced increase. Superoxide released from mitochondria affected protein kinases and resulted in stronger I-Ca,I-L preceding its dismutation to H2O2. The removal of nitric oxide is a likely mechanism for the increase in I-Ca,I-L.