title: Rotenone-stimulated superoxide release from mitochondrial complex I acutely augments L-type Ca2+ current in A7r5 aortic smooth muscle cells.
abstract: Voltage-gated L-type Ca(2+) current (ICa,L) induces contraction of arterial smooth muscle cells (ASMCs), and ICa,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 ICa,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. ICa,L was recorded with 5 mM BAPTA in the pipette solution. Rotenone administration (10 nM to 10 μM) resulted in a greater ICa,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 ICa,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 ICa,L without a negative shift in I-V, and rotenone produced an additional increase with a shift. H2O2 (0.3 mM) inhibited ICa,L by 13%, and additional rotenone induced an increase with a negative shift. Sustained treatment with Tempol (4-hydroxy tempo) led to a significant ICa,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 ICa,L preceding its dismutation to H2O2 The removal of nitric oxide is a likely mechanism for the increase in ICa,L.
authors.affiliation: Department of Pharmacology, New York Medical College, Valhalla, New York; and rikuoochi@yahoo.com.
authors.forename: Rikuo
authors.identifier: 0000-0002-8790-4496
authors.initials: R
authors.lastname: Ochi
authors.affiliation: Department of Pharmacology, New York Medical College, Valhalla, New York; and.
authors.forename: Vidhi
authors.initials: V
authors.lastname: Dhagia
authors.affiliation: Department of Pharmacology, New York Medical College, Valhalla, New York; and.
authors.forename: Anand
authors.initials: A
authors.lastname: Lakhkar
authors.affiliation: Department of Physiology, New York Medical College, Valhalla, New York.
authors.forename: Dhara
authors.initials: D
authors.lastname: Patel
authors.affiliation: Department of Physiology, New York Medical College, Valhalla, New York.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Wolin
authors.affiliation: Department of Pharmacology, New York Medical College, Valhalla, New York; and.
authors.forename: Sachin A
authors.initials: SA
authors.lastname: Gupte
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001013:Aorta, Thoracic / Q000187:drug effects* / Q000201:enzymology; D020746:Calcium Channels, L-Type / Q000187:drug effects* / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D042967:Electron Transport Complex I / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000633:toxicity*; D008564:Membrane Potentials; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000201:enzymology; D009569:Nitric Oxide / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D012402:Rotenone / Q000633:toxicity*; D013481:Superoxides / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000975:Antioxidants; D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D012402:Rotenone; D013481:Superoxides; D009569:Nitric Oxide; D042967:Electron Transport Complex I
keywords: L-type calcium channel; mitochondria; rotenone; smooth muscle; superoxide