Cross Talk among Calcium, Hydrogen Peroxide, and Nitric Oxide and Activation of Gene Expression Involving Calmodulins and Calcium-Dependent Protein Kinases in Ulva compressa Exposed to Copper Excess

To analyze the copper-induced cross talk among calcium, nitric oxide (NO), and hydrogen peroxide (H₂O₂) and the calciumdependent activation of gene expression, the marine alga Ulva compressa was treated with the inhibitors of calcium channels, ned-19, ryanodine, and xestospongin C, of chloroplasts and mitochondrial electron transport chains, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and antimycin A, of pyruvate dehydrogenase, moniliformin, of calmodulins, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide, and of calcium-dependent protein kinases, staurosporine, as well as with the scavengers of NO, 2-(4-carboxypheny1)-4,4,5,5-tetramethylimidazoline-1-oxy1-3-oxide, and of H₂O₂, ascorbate, and exposed to a sublethal concentration of copper (10 µm) for 24 h. The level of NO increased at 2 and 12 h. The first peak was inhibited by ned-19 and 3-(2,3-dichloropheny1)-1, 1-dimethylurea and the second peak by ned-19 and antimycin A, indicating that NO synthesis is dependent on calcium release and occurs in organdíes. The level of H₂O₂ increased at 2, 3, and 12 h and was inhibited by ned-19, ryanodine, xestospongin C, and moniliformin, indicating that H₂O₂ accumulation is dependent on calcium release and Krebs cycle activity In addition, pyruvate dehydrogenase, 2-oxoxglutarate dehydrogenase, and isocitrate dehydrogenase activities of the Krebs cycle increased at 2, 3,12, and/or 14 h, and these increases were inhibited in vitro by EGTA, a calcium chelating agent. Calcium release at 2, 3, and 12 h was inhibited by 2-(4-carboxypheny1)-4,4,5,5-tetramethylimidazoline-1-oxy1-3-oxide and ascorbate, indicating activation by NO and H₂O₂. In addition, the level of antioxidant protein gene transcripts decreased with N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide and staurosporine. Thus, there is a copper-induced cross talk among calcium, H₂O₂, and NO and a calcium-dependent activation of gene expression involving calmodulins and calcium-dependent protein kinases.