Copper-induced intracellular calcium release requires extracellular calcium entry and activation of L-type voltage-dependent calcium channels in Ulva compressa

Abstract

The marine alga Ulva compressa exposed to 10 µM copper showed a triphasic increase of intracellular calcium with maximal levels at 2, 3 and 12 h involving the activation of ryanodine-, Ins(1,4,5)P3- and NAADP-sensitive calcium channels. In order to analyze the requirement of extracellular calcium entry for intracellular calcium release as well as the activation of voltage-dependent calcium channels (VDCC) and phospholipase C, U. compressa was treated with EGTA, a non-permeable calcium chelating agent, with verapamil, nipfedipine and diltiazem, inhibitors of L-type VDCC, and with neomycin and U731222, inhibitors of phospholipase C. The release of intracellular calcium was partially inhibited with EGTA at 2 and 3 h and completely inhibited at 12 h of copper exposure and decreased with inhibitors of L-type VDCC and phospholipase C. Thus, copper-induced intracellular calcium release depends on calcium entry and activation of L-type VDCC and phospholipase C. An integrative model of copper-induced cellular responses in U. compressa is presented.

Figure 1. Level of intracellular calcium in U. compressa cultivated in control condition and with 10 µM copper for 2, 3 and 12 h or treated with 1 mM EGTA added 30 min before 2, 3 and 12 h and with 10 µM copper. Calcium level is expressed the ratio of fluorescence of Fluo 3 and autofluorescence of chloroplasts. Bars represent mean values of three independent experiments ± SD. Different letters indicate significant differences (p < 0.05).

Figure 2. Level of intracellular calcium in U. compressa cultivated in control condition, with 10 µM copper or treated with 100 µM nifedipine (nif), 100 µM verapamil (ver) and 100 µM diltiazem (dil) and 10 µM copper for 2, 3 and 12 h. Calcium level is expressed the ratio of fluorescence of Fluo 3 and autofluorescence of chloroplasts. Bars represent mean values of three independent experiments ± SD. Different letters indicate significant differences (p < 0.05).

Figure 3. Level of intracellular calcium in U. compressa cultivated in control condition, with 10 µM copper or treated with 100 µM neomycin (neo) and 30 µM U73112 and 10 µM copper for 2, 3 and 12 h. Calcium level is expressed the ratio of fluorescence of Fluo 3 and autofluorescence of chloroplasts. Bars represent mean values of three independent experiments ± SD. Different letters indicate significant differences (p < 0.05).

Figure 4. Integrative model of copper-induced cellular responses in U. compressa. Voltage-dependent calcium channel, VDCC; phospholipase C, PLC; inositol 1, 4, 5 triphosphate, Ins(1,4,5)P₃; Ins(1,4,5)P₃-sensitive calcium receptor, Ins(1,4,5)P₃R; ryanodine-sensitive calcium receptor, RyaR; NAADP-sensitive calcium receptor, NAADPR; endoplasmic reticulum ER; mitochondrion, Mit; mitochondrial transition pore (MTP); chloroplast, Chl; calmodulins, CaMs; calcium-dependent protein kinases, CDPKs. Putative direct activation of VDCC by copper (1), activation of phospholipase C by calcium entry (2), activation of ER-calcium channels by calcium entry (3), release of Ins(1,4,5)P₃ by phospholipase C (4), release of calcium from ER (5), entry of calcium into the mitochondrion (6), exit of hydrogen peroxide and nitric oxide from mitochondrion through MTP (7), entry of calcium into chloroplast (8), exit of hydrogen peroxide and nitric oxide from chloroplast (9), activation of ER-calcium channels by hydrogen peroxide and nitric oxide (10), activation of CAMs and CDPKs by calcium and activation of gene expression in the nucleus (11).