Relationship between active oxygen species and cardenolide production in cell cultures of Digitalis thapsi: effect of calcium restriction

Elimination of calcium ions from the medium of undifferentiated cell cultur es of Digitalis thapsi increased cardenolide production and induced extrace llular H2O2 accumulation, as measured by the quenching of pyranine fluoresc ence. The addition of catalase reduced the response and the inclusion of su peroxide dismutase enhanced the loss of fluorescence. This suggested that, besides H2O2, the superoxide anion was also formed before dismutating to H2 O2. Additionally, exogenous H2O2 or superoxide dismutase stimulated cardeno lide production whereas the addition of catalase markedly reduced it. These results point to a connection between H2O2 and cardenolide formation. The absence of calcium did not alter the levels of lipid peroxidation products; however, changes in the antioxidant system of D. thapsi cells were observe d. Catalase activity was extremely low in control cultures and remained una ltered upon calcium elimination. Ascorbate peroxidase activity was not modi fied in calcium-free cultures. By contrast, calcium deprivation stimulated superoxide dismutase activity and strongly inhibited glutathione reductase activity. Also, a significant decrease in reduced glutathione was observed. These responses were emulated by treatment of the cultures with the glutat hione biosynthesis inhibitor buthionine sulfoximine and by ethyleneglycol-b is-beta-aminoethyl ether and LaCl3. All these results indicate that the dep letion of extracellular calcium induces changes in the redox state of cells and suggest that this alteration stimulates cardenolide formation in D. th apsi cultures.