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

Abstract

To analyze the copper-induced cross talk among calcium, nitric oxide (NO), and hydrogen peroxide (H(2)O(2)) and the calcium-dependent 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-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and of H(2)O(2), 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-dichlorophenyl)-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 organelles. The level of H(2)O(2) increased at 2, 3, and 12 h and was inhibited by ned-19, ryanodine, xestospongin C, and moniliformin, indicating that H(2)O(2) 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-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and ascorbate, indicating activation by NO and H(2)O(2). 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, H(2)O(2), and NO and a calcium-dependent activation of gene expression involving calmodulins and calcium-dependent protein kinases.

Figure 1.

A, Level of NO in U. compressa cultivated in control condition (white circles) and exposed to 10 μm copper (black circles) for 24 h. NO level is expressed as the ratio between DAF-2 fluorescence and chloroplast autofluorescence. B, Activity of NO synthase in extracts of U. compressa cultivated in control condition (white circles) and exposed to 10 μm copper (black circles) for 24 h. NO synthase activity is expressed in micromoles per minute per milligram of protein. Level of NO in the alga cultivated in control condition (control), with 10 μm copper (copper) or treated with 1 mm l-NMMA, 10 μm ned-19, 100 μm ryanodine (rya), 10 μm xestospongin C (xes), 1 mm ASC, 10 μm AA, and 20 μm DCMU, and exposed to 10 μm copper for 2 h (C) and 12 h (D). Symbols and bars represent mean values of three independent replicates ± sd. Different letters indicate significant differences (P < 0.05).

Figure 2.

Level of H₂O₂ (A) and superoxide anions (B) in U. compressa cultivated in control condition (white circles) and exposed to 10 μm copper (black circles) for 24 h. Level of H₂O₂ is expressed as the ratio between dichlorofluorescein fluorescence and chloroplast autofluorescence. Visualization by confocal microscopy of H₂O₂ in U. compressa cultivated in control condition (C), exposed to 10 μm copper (D), or treated with 300 μm moniliformin and exposed to 10 μm copper (E) for 12 h. The white bar represents 20 μm. Level of H₂O₂ in the alga cultivated in control condition (control), exposed to 10 μm copper (copper), or treated with 300 μm moniliformin (mon), 10 μm ned-19, 100 μm ryanodine (rya), 10 μm xestospongin C (xes), 10 μm AA, 20 μm DCMU, and 1 mm cPTIO, and exposed to 10 μm copper for 2 h (F), 3 h (G), and 12 h (H). Symbols and bars represent mean values of three independent replicates ± sd. Different letters indicate significant differences (P < 0.05). [See online article for color version of this figure.]

Figure 3.

Activities of Krebs cycle enzymes PDH (A), IDH (B), and OGDH (C) in extracts of U. compressa cultivated in control condition (white circles) and exposed to 10 μm copper (black circles) for 48 h. Activities are expressed as micromoles per minute per milligram of protein. Symbols represent mean values of three independent experiments ± sd. Different letters indicate significant differences (P < 0.05).

Figure 4.

Activities of Krebs cycle enzymes PDH (A–C), IDH (D–F), and OGDH (G–I) in extracts of U. compressa cultivated in control condition (control) and exposed to 10 μm copper (copper) or in extracts of the alga cultivated with 10 μm copper for 2 h (D and G), 3 h (A), 12 h (B, E, and H), or 14 h (C, F, and I) and supplemented with 0.4 mm EGTA. Activities are expressed as micromoles per minute per milligram of protein. Bars represent mean values of three independent experiments ± sd. Different letters indicate significant differences (P < 0.05).

Figure 5.

Level of intracellular calcium in U. compressa (A) cultivated in control condition (white circles) and with 10 μm copper (black circles). Calcium level is expressed as the ratio of Fluo 3 fluorescence and chloroplast autofluorescence. Level of calcium in the alga cultivated in control condition (control), exposed to 10 μm copper (copper), or treated with 10 μm ned-19, 100 μm ryanodine (rya), 10 μm xestospongin C (xes), 1 mm ASC, or 0.1 mm cPTIO and exposed to 10 μm copper for 2 h (B), 3 h (C), and 12 h (D). Bars represent mean values of three independent experiments ± sd. Different letters indicate significant differences (P < 0.05).

Figure 6.

Relative level of transcripts encoding AP (A), PRX (B), TRX (C), GST (D), and MET (E) in U. compressa exposed to 10 μm copper (copper) and treated with 100 μm W-7 and 10 μm staurosporine (stau) for 3 d (AP, MET) or 5 d (PRX, TRX, GST). The relative level of transcripts is expressed as 2^−ΔΔCT. Bars represent mean values of three independent experiments ± sd. Different letters indicate significant differences (P < 0.05).

Figure 7.

Schematic representation of the cross talk among calcium, NO, and H₂O₂ and the calcium-dependent activation of gene expression involving CaMs and CDPKs in U. compressa exposed to copper excess.