Copper-induced calcium release from ER involves the activation of ryanodine-sensitive and IP₃-sensitive channels in Ulva compressa

Abstract

The marine alga Ulva compressa (Chlorophyta) showed a triphasic release of intracellular calcium with maximal levels at 2, 3 and 12 h and a biphasic accumulation of intracellular hydrogen peroxide with peaks at 3 and 12 h when cultivated with copper excess. Intracellular hydrogen peroxide originated exclusively in organelles. In this work, we analyzed the intracellular origin of calcium release and the type of calcium channels activated in response to copper excess. U. compressa was treated with thapsigargin, an inhibitor of endoplasmic reticulum (ER) calcium ATPase, ryanodine, an inhibitor of ryanodine-sensitive channels and xestospongin C, an inhibitor of inositol 1, 4, 5-triphosphate (IP₃)-sensitive channels. Thapsigargin induced the depletion of calcium stored in ER at 75 min and completely inhibited calcium release at 2, 3 and 12 h of copper exposure indicating that calcium release originated in ER. In addition, ryanodine and xestospogin C inhibited calcium release at 2 and 3 h of copper exposure whereas the peak at 12 h was only inhibited by ryanodine. Thus, copper induced the activation of ryanodine-sensitive and IP₃-sensitive calcium channels in ER of U. compressa.

Plants showed common responses to biotic and abiotic stresses, mainly the accumulation of reactive oxygen species (ROS), in particular hydrogen peroxide, and the release of intracellular calcium.^1,2 Regarding abiotic stress, it has been shown that ozone triggers a NADPH oxidase-dependent biphasic oxidative burst in Arabidopsis thaliana that activates antioxidant and defense enzymes.^3,4 In addition, cadmium induced a NADPH oxidase-dependent monophasic accumulation of extracellular hydrogen peroxide in tobacco cells.⁵ On the other hand, ozone as well as absicic acid treatment, dessication, cold, heat, salinity, UV light and anoxia induce intracellular calcium release and the activation of antioxidant enzymes.^6–8 Regarding abiotic stress in algae, copper induced a monophasic increase of intracellular hydrogen peroxide at 2 h of copper exposure in the brown seaweeds Lessonia nigrecsens and Scytosiphon lomentaria.⁹ On the other hand, strontium induced calcium release in the green microalga Eremosphaera viridis as did osmotic stress in the zygote of the brown macroalga Fucus serratus.^10,11 U. compressa is a cosmopolitan marine macroalga (Chlorophyta) growing in copper-impacted coastal areas in northern Chile.¹² U. compressa cultivated in seawater with copper excess (10 µM) showed co-occuring increases of intracellular calcium and hydrogen peroxide.¹³ Copper induced a triphasic release of calcium with maximal levels at 2, 3 and 12 h and a biphasic production of hydrogen peroxide with peaks at 3 and 12 h. Interestingly, the production of hydrogen peroxide occurred exclusively in organelles, i.e., mitochondria and chloroplasts. In addition, calcium and hydrogen peroxide act as signals in the differential activation of antioxidant and defense enzymes.¹³ In this work, we analyzed the intracellular origin of copper-induced calcium release and the type of calcium channels activated in response to copper excess in U. compressa.

Copper Induced the Release of Intracellular Calcium from Endoplasmic Reticulum

In order to determine the intracellular origin of copper-induced calcium release, U. compressa was treated with thapsigargin, an inhibitor of ER-calcium ATPase that induced the depletion of calcium stored in ER.¹⁴ U. compressa was incubated with 5 µM of thapsigargin and the increase in intracellular calcium was detected using Fluo-3AM and confocal microscopy¹³ and monitored every 15 min (Fig. 1A and B). The alga treated with thapsigargin showed an increase in intracellular calcium that reached a maximal level at 45 min and then decreased to reach the control level at 75 min (Fig. 1C). The alga treated with thapsigargin for 90 min and then incubated without copper (control) or with 10 µM copper for 2, 3 or 12 h did not show an increase in intracellular calcium (Fig. 1D–F) suggesting that copper-induced calcium release at 2, 3 or 12 h originated in ER. It is interesting to note that thapsigargin inhibited the ER-calcium ATPase in U. compressa as it has been shown mammalian cells.¹⁷ These results contrast with data obtained in terrestrial plants where the ER-calcium-ATPase is not inhibited by thapsigargin, with the exception of a heavy metal/calcium ATPase detected in the Golgi apparatus of pea seedlings¹⁵ and a similar enzyme recently cloned in Arabidopsis thaliana.¹⁶ In addition, time required to reach the maximal level of calcium depletion induced by thapsigargin i.e., 45 min is similar to that observed in mammalian cells, i.e., 30 min.¹⁷ Thus, it is possible that marine algae ER-calcium ATPase share functional and structural properties with homologous enzymes in mammalian cells.

Figure 1.

Visualization of intracellular calcium in U. compressa incubated in seawater in control conditions (A) or with 5 µM thapsigargin for 45 min (B). Level of intracellular calcium in the alga incubated in control conditions (open circles) or with 5 µM thapsigargin for 90 min (black circles) (C). Level of intracellular calcium in the alga cultivated in control conditions (control), with 10 µM copper (copper) and treated with 5 µM thapsigargin (thap) for 90 min and then cultivated with 10 µM copper for 2 h (D), 3 h (E) and 12 h (F). Levels of intracellular calcium were detected with Fluo-3AM using confocal microscopy and are expressed as fluorescence intensity. Bars and symbols represent mean values of three independent replicates ±SD. Different letters indicate significant differences (p < 0.05).

Copper Induced the Activation of Ryanodine-Sensitive and IP₃-Sensitive Channels

In order to determine the type of calcium channels involved in copper-induced calcium release, U. compressa was incubated with ryanodine, an inhibitor of ryanodine-sensitive calcium channels and xestospongin C, an inhibitor of IP₃-sensitive calcium channels. The alga was incubated with 100 µM ryanodine or with 5 µM xestospongin C for 60 min and then cultivated without copper (control) or with 10 µM copper for 2, 3 and 12 h. Ryanodine and xestospongin C inhibited calcium release at 2 and 3 h of copper exposure (Fig. 2A and B) and only ryanodine inhibited calcium release at 12 h of copper treatment (Fig. 2C). These results indicate that ryanodine-sensitive and IP₃-sensitive channels are involved in copper-induced calcium release from ER in U. compressa. Thus, ryanodine-sensitive and IP₃-sensitive channels might exist in ER of U. compressa as it has been shown in ER of mammalian cells.¹⁸ The activation of ryanodine-sensitive channels in response to copper excess indicates that copper may trigger an initial influx of extracellular calcium that activates ryanodine-sensitive channels in ER. In this sense, we have detected that EGTA, a calcium chelating agent, added to the extracellular medium inhibited the first peak of calcium release (i.e., 2 h) suggesting that calcium influx is required for calcium release in ER (González A, unpublished). In addition, the involvement of IP₃-sensitive channels indicates that copper may induce the activation phospholipase C that releases IP₃ from fatty acids of the plasma membrane. In this sense, it has been shown that cadmium induced the release of intracellular calcium in tobacco cells and that calcium release decreased with phospholipase C inhibitors suggesting that IP₃-sensitive channels are activated in response to cadmium.⁵ It is important to mention that the third peak of intracellular calcium in U. compressa was only inhibited by ryanodine indicating that IP₃ production is no longer occurring at 12 h of copper exposure or that IP₃-sensitive channels are inactivated by IP₃ in a time-dependent manner, as it has been shown in mammalian cells.¹⁹

Figure 2.

Level of intracellular calcium in U. compressa cultivated in control conditions (control), with 10 µM copper (copper), treated with 100 µM ryanodine (Rya) or 5 µM xestospongin C (Xes) for 60 min and then cultivated with 10 µM copper for 2 h (A), 3 h (B) and 12 h (C). Levels of calcium were detected with Fluo-3AM using confocal microscopy and are expressed as fluorescence intensity. Bars represent mean values of three independent replicates ±SD. Different letters indicate significant differences (p < 0.05).

Addendum to: González A, Vera J, Castro J, Dennett G, Mellado M, Morales B, et al. Co-occuring increases of calcium and organellar reactive oxygen species determine differential activation of antioxidant and defense enzymes in Ulva compressa (Chlorophyta) exposed to copper excess. Plant Cell Environ. 2010;33:1627–1640. doi: 10.1111/j.1365-3040.2010.02169.x.