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L‐type Ca 2+ Current Increase by Rotenone, Stimulant of Mitochondrial Superoxide Release, in A7r5 Arterial Smooth Muscle Cells. Effect of Dithiothreitol, Hydrogen Peroxide, Tempol and Staurosporine
Author(s) -
Ochi Rikuo,
Dhagia Vidhi,
Patel Dhara,
Wolin Michael S.,
Gupte Sachin A.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1224.15
Subject(s) - dithiothreitol , staurosporine , superoxide , rotenone , chemistry , superoxide dismutase , mitochondrial ros , depolarization , reactive oxygen species , mitochondrion , stereochemistry , biochemistry , oxidative stress , biophysics , protein kinase c , biology , kinase , enzyme
L‐type Ca current (I CaL ) is primary Ca 2+ current to initiate contraction in arterial smooth muscle cells. Mitochondrial respiratory chain (MRC) generates and releases superoxide (O 2 − ) in energy metabolism. O 2 − is converted to H 2 O 2 by superoxide dismutase (SOD). Rotenone (Rot), phytogenic toxin, inhibits complex I of MRC and stimulates the release of O 2 − . We recently found that Rot increases I CaL in A7r5 cultured aortic smooth muscle cells. Here, we performed further pharmacological study of the increase by applying several redox‐related agents. Rot (0.01 mM) and other drugs were introduced during repetitive depolarization at 1/20s or recording I–V relationship with 2 min interval. Rot increased I CaL by 25% in 1–2 minute. It increased macroscopic conductance and shifted V 0.5 of activation curve to the hyperpolarizing direction. Dithiothreitol (DTT, 2 mM), thiol‐reducing agent, increased I CaL by 20% without producing the negative shift and, in the presence of DTT, Rot increased I CaL with the negative shift. H 2 O 2 (0.3 mM) inhibited I CaL by 13% in 5 min without shifting I–V and, in the presence of H 2 O 2 , Rot induced 17% increase of I CaL with negative shift. Pretreatment by Tempol, SOD mimetic scavenger of O 2 − , markedly depressed Rot‐induced increase of I CaL . Staurosporine (100 nM), broad‐spectrum protein‐kinase inhibitor, inhibited I CaL with slow time course and completely suppressed Rot‐induced increase of I CaL . Rotenone increases I CaL by increasing the release of O 2 − from mitochondrial complex I and the increase is independent of thiol‐oxidation but is mediated by phosphorylation of proteins that regulates I CaL .