oligomycins and diphenyleneiodonium

NADPH oxidase is the major source of superoxide production in cardiovascular tissues. We and others reported that PG (prostaglandin) F2alpha, PDGF (platelet-derived growth factor) and angiotensin II cause hypertrophy of vascular smooth muscle cells by induction of NOX1 (NADPH oxidase 1), a catalytic subunit of NADPH oxidase. We found DPI (diphenylene iodonium), an inhibitor of flavoproteins, including NADPH oxidase itself, almost completely suppressed induction of NOX1 mRNA by PGF2alpha or PDGF in a rat vascular smooth muscle cell line, A7r5. Exploration into the site of action of DPI using various inhibitors suggested the involvement of mitochondrial oxidative phosphorylation in PGF2alpha- or PDGF-induced increase in NOX1 mRNA. In a luciferase reporter assay, activation of the CRE (cAMP-response element)-dependent gene transcription by PGF2alpha was attenuated by oligomycin, an inhibitor of mitochondrial F(o)F1-ATPase. Oligomycin and other mitochondrial inhibitors also suppressed PGF2alpha-induced phosphorylation of ATF (activating transcription factor)-1, a transcription factor of the CREB (CRE-binding protein)/ATF family. Silencing of the ATF-1 gene by RNA interference significantly reduced the induction of NOX1 by PGF2alpha or PDGF, while overexpression of ATF-1 recovered NOX1 induction suppressed by oligomycin. Taken together, ATF-1 may play a pivotal role in the up-regulation of NOX1 in rat vascular smooth muscle cells.

Topics: Activating Transcription Factor 1; Animals; Catalytic Domain; Cell Line; Cyclic AMP Response Element-Binding Protein; Dinoprost; DNA-Binding Proteins; Electron Transport Chain Complex Proteins; Enzyme Induction; Enzyme Inhibitors; Free Radical Scavengers; Gene Silencing; Mitochondria; NADH, NADPH Oxidoreductases; NADPH Oxidase 1; NADPH Oxidases; Oligomycins; Onium Compounds; Phosphorylation; Rats; Reactive Oxygen Species; RNA, Messenger; Transcription Factors; Transcriptional Activation

Epididymal sperm maturation culminates in the acquisition of functional competence by testicular spermatozoa. The expression of this functional state is dependent upon a redox-regulated, cAMP-mediated signal transduction cascade that controls the tyrosine phosphorylation status of the spermatozoa during capacitation. Analysis of superoxide anion (O2(-.)) generation by rat epididymal spermatozoa has revealed a two-component process involving electron leakage from the sperm mitochondria at complexes I and II and a plasma membrane NAD(P)H oxidoreductase. Following incubation in a glucose-, lactate-, and pyruvate-free medium (-GLP), O2(-.) generation was suppressed by 86% and 96% in caput and cauda spermatozoa, respectively. The addition of lactate, malate, or succinate to spermatozoa incubated in medium -GLP stimulated O2(-.) generation. This increase could be blocked by rotenone and oligomycin (R/O) in the presence of malate or lactate but not succinate. Stimulation with all three substrates, as well as spontaneous O2(-.) production in +GLP medium, was blocked by the flavoprotein inhibitor, diphenylene iodonium. Diphenylene iodonium, but not R/O, suppressed NAD(P)H-induced lucigenin-dependent chemiluminescence. This NAD(P)H-dependent enzyme resided in the sperm plasma membrane and its activity was regulated by zinc and uncharacterized cytosolic factors. Reverse transcription-polymerase chain reaction analysis indicated that the sperm NAD(P)H oxidoreductase complex is quite distinct from the equivalent leukocyte system.

Topics: Animals; Cell Membrane; Epididymis; Lactic Acid; Leukocytes; Malates; Male; Mitochondria; NAD; NADP; NADPH Oxidases; Oligomycins; Onium Compounds; Oxidation-Reduction; Rats; Reactive Oxygen Species; Rotenone; Spermatozoa; Succinic Acid; Superoxides; Uncoupling Agents; Zinc