Rotenone-stimulated superoxide release from mitochondrial complex I acutely augments L-type Ca^sup 2+^ current in A7r5 aortic smooth muscle cells

• Published in: American journal of physiology. Heart and circulatory physiology 2016-05, Vol.310 (9), p.H1118
• Main Authors: Ochi, Rikuo, Dhagia, Vidhi, Lakhkar, Anand, Patel, Dhara, Wolin, Michael S, Gupte, Sachin A
• Format: Article
• Language: English
• Subjects: Calcium; Cell culture; Mitochondria; Proteins; Smooth muscle; Toxins
• ISSN: 0363-6135; 1522-1539

Voltage-gated L-type Ca2+ current (ICa,L) induces contraction of arterial smooth muscle cells (ASMCs), and I^sub Ca,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^sub Ca,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 μM rotenone. I^sub Ca,L^ was recorded with 5 mM BAPTA in the pipette solution. Rotenone administration (10 nM to 10 μM) resulted in a greater I^sub Ca,L^ increase in a dose-dependent manner to a maximum of 22.1% at 10 μM for 1 min, which gradually decreased to 9% after 5 min. The rotenone-induced I^sub Ca,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^sub Ca,L^ without a negative shift in I-V, and rotenone produced an additional increase with a shift. H2O2 (0.3 mM) inhibited I^sub Ca,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^sub Ca,L^ increase but it inhibited the rotenone-induced increase. Staurosporine, a broad-spectrum protein kinase inhibitor, partially inhibited ICa,L and completely suppressed the rotenone-induced increase. Superoxide released from mitochondria affected protein kinases and resulted in stronger I^sub Ca,L^ preceding its dismutation to H2O2. The removal of nitric oxide is a likely mechanism for the increase in I^sub Ca,L^.