resiniferatoxin and diphenyleneiodonium

As rat spermatozoa undergo epididymal maturation, they acquire the ability to exhibit a spontaneous burst of luminol-peroxidase-dependent chemiluminescence when released into a simple, defined culture medium. This activity was suppressed by inhibitors of plasma membrane redox systems such as diphenylene iodonium, p-chloromercuribenzenesulfonic acid, and capsaicin, but was resistant to inhibition by resiniferatoxin and rotenone. The luminol-peroxidase signal was dependent on the presence of bicarbonate, enhanced by the substitution of fructose for glucose, and severely suppressed by desferoxamine, superoxide dimutase, and catalase. Both L- and D-arginine were stimulatory, suggesting the involvement of *NO in this spontaneous chemiluminescence activity. The L-arginine-dependent, but not the D-arginine-dependent, activity was significantly suppressed by an inhibitor of nitric oxide synthase (N(G)-nitro-L-arginine methyl ester). L- and D-arginine could also stimulate redox activity observed in immature caput epididymal cells, but only after prolonged incubation. The inhibitory effects of uric acid and ascorbate suggested the chemiluminescence signal might be induced by peroxynitrite. This conclusion was supported by confocal imaging of the cells following treatment with 4-amino-5-methylamino-2',7'-difluorofluorescein. Stimulation or suppression of the redox activity detected by luminol-peroxidase led to corresponding changes in the ability of the spermatozoa to exhibit acrosomal exocytosis, indicating that this pathway is of fundamental biological significance.

Topics: 4-Chloromercuribenzenesulfonate; Acridines; Acrosome Reaction; Animals; Arginine; Bicarbonates; Calcium; Capsaicin; Catalase; Culture Media; Deferoxamine; Diterpenes; Enzyme Inhibitors; Epididymis; Fructose; Glucose; Indicators and Reagents; Iron Chelating Agents; Luminol; Male; Microscopy, Confocal; Microscopy, Fluorescence; Models, Biological; NADH, NADPH Oxidoreductases; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Onium Compounds; Oxidation-Reduction; Peroxidase; Rats; Rats, Wistar; Reactive Oxygen Species; Rotenone; Spermatozoa; Superoxide Dismutase; Temperature; Time Factors; Uncoupling Agents

Mitochondrial gene knockout (rho(0)) cells that depend on glycolysis for their energy requirements show an increased ability to reduce cell-impermeable tetrazolium dyes by electron transport across the plasma membrane. In this report, we show for the first time, that oxygen functions as a terminal electron acceptor for trans-plasma membrane electron transport (tPMET) in HL60rho(0) cells, and that this cell surface oxygen consumption is associated with oxygen-dependent cell growth in the absence of mitochondrial electron transport function. Non-mitochondrial oxygen consumption by HL60rho(0) cells was extensively inhibited by extracellular NADH and NADPH, but not by NAD(+), localizing this process at the cell surface. Mitochondrial electron transport inhibitors and the uncoupler, FCCP, did not affect oxygen consumption by HL60rho(0) cells. Inhibitors of glucose uptake and glycolysis, the ubiquinone redox cycle inhibitors, capsaicin and resiniferatoxin, the flavin centre inhibitor, diphenyleneiodonium, and the NQO1 inhibitor, dicoumarol, all inhibited oxygen consumption by HL60rho(0) cells. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and reduction of the cell-impermeable tetrazolium dye, WST-1, suggest that both systems may share a common tPMET pathway. This is supported by the finding that terminal electron acceptors from both pathways compete for electrons from intracellular NADH.

Topics: Aerobiosis; Capsaicin; Cell Membrane; Cell Survival; Dicumarol; Diterpenes; Electron Transport; Flavins; HL-60 Cells; Humans; Mitochondria; NAD; NADP; Onium Compounds; Oxidation-Reduction; Oxygen Consumption; Tetrazolium Salts; Time Factors; Ubiquinone; Uncoupling Agents