digitonin and 1-1-diethyl-2-hydroxy-2-nitrosohydrazine

We examined the effects of dissolved nitric oxide (NO) gas on cytoplasmic calcium levels ([Ca(2+)](i)) in C6 glioma cells under anoxic conditions. The maximum elevation (27 +/- 3 nM) of [Ca(2+)](i) was reached at 10 microM NO. A second application of NO was ineffective if the first was >0.5 microM. The NO donor diethylamine/NO mimicked the effects of NO. Acute exposure of the cells to low calcium levels was without effect on the NO-evoked response. Thapsigargin (TG) increased [Ca(2+)](i) and was less effective if cells were pretreated with NO. Hemoglobin inhibited the effects of NO at a molar ratio of 10:1. 8-Bromo-cGMP was without effect on the NO-evoked response. If cells were pretreated with TG or exposed chronically to nominal amounts of calcium, NO decreased [Ca(2+)](i). The results suggest that C6 glioma cells have two receptors for NO. One receptor (NO(A)) elevates [Ca(2+)](i) and resides on the endoplasmic reticulum (ER). The other receptor (NO(B)) decreases [Ca(2+)](i) and resides on the plasmalemma or the ER. The latter receptor dominates when the level of calcium within intracellular stores is diminished.

Topics: Aerobiosis; Animals; Calcium; Cell Hypoxia; Cytoplasm; Cytosol; Digitonin; Egtazic Acid; Glioma; Hemoglobins; Homeostasis; Hydrazines; Kinetics; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Thapsigargin; Tumor Cells, Cultured

Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to orotate in the pyrimidine biosynthesis pathway. It is functionally connected to the respiratory chain, delivering electrons to ubiquinone. We report here that inhibition of cytochrome c oxidase by nitric oxide (NO) indirectly inhibits DHODH activity. In digitonin-permeabilized cells, DEA/NO, a chemical NO donor, induced a dramatic decrease in DHO-dependent O(2) consumption. The inhibition was reversible and more pronounced at low O(2) concentration; it was correlated with a decrease in orotate synthesis. Since orotate is the precursor of all pyrimidine nucleotides, indirect inhibition of DHODH by NO may significantly contribute to NO-dependent cytotoxicity.

Topics: Animals; Antioxidants; Ascorbic Acid; Digitonin; Dihydroorotate Dehydrogenase; Electron Transport; Electron Transport Complex IV; Enzyme Inhibitors; Humans; Hydrazines; K562 Cells; Leukemia L1210; Leukemia-Lymphoma, Adult T-Cell; Macrophages; Mice; Mitochondria; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Orotic Acid; Oxidation-Reduction; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Oxygen; Rats; Recombinant Proteins; Tumor Cells, Cultured