Elimination of calcium ions from the medium of undifferentiated
cell cultures of Digitalis thapsi increased
cardenolide production and induced extracellular H2O2
accumulation, as measured by the quenching of pyranine
fluorescence. The addition of catalase reduced the response and the inclusion
of superoxide dismutase enhanced
the loss of fluorescence. This suggested that, besides H2O2,
the superoxide anion was also formed before
dismutating to H2O2. Additionally, exogenous H2O2
or superoxide dismutase stimulated cardenolide 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 observed.
Catalase activity was extremely low in control
cultures and remained unaltered upon calcium elimination. Ascorbate peroxidase
activity was not modified 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 glutathione
biosynthesis inhibitor buthionine
sulfoximine and by ethyleneglycol-bis-β-aminoethyl ether and LaCl3.
All these results indicate that the depletion
of extracellular calcium induces changes in the redox state of cells and
suggest that this alteration stimulates
cardenolide formation in D. thapsi cultures.