Fe2+-induced inhibition of gerbil forebrain microsomal Ca2+-ATPase: effect of stobadine, glutathione and combination of both antioxidants.

The incubation of the gerbil forebrain microsomes in the presence of ferrous sulphate and EDTA for either 30 min or for 60 min at a temperature of 37 degrees C led to the inhibition of Ca2+-ATPase in both a concentration- and time-dependent manner. The concentrations of Fe2+ which led to the inhibition of 50% of the Ca2+-ATPase activity (IC50-value) at these times were 0.59 mM and 0.07 mM, respectively. The preincubation of microsomes with 0.1 mM of stobadine prevented the inhibition of Ca2+-ATPase, however, the effectivity of prevention was dependent on the Fe2+ concentration. The net effect of stobadine was an increase in IC50-value to 0.76 mM. Unlike stobadine, reduced glutathione is a naturally occurring water soluble antioxidant. Glutathione at the concentration of 0.1 mM had no significant protective effect on the inhibition of Ca2+-ATPase. The protective effect of a stobadine-glutathione mixture was also investigated; 0.1 mM of stobadine in combination with 0.1 mM of glutathione was more potent in prevention of Fe2+-induced inhibition of Ca2+-ATPase than stobadine alone (IC50=1. 31 mM). In addition, we have investigated the effect of various stobadine-glutathione molar ratios (the total concentration of both antioxidants being 0.2 mM) on Fe2+-induced inhibition of Ca2+-ATPase. The results indicated that the best stobadine-glutathione ratio was close to 1 : 1. The effect of 0.04 mM stobadine in combination with 0.16 mM glutathione was comparable to the effect of 0.2 mM of stobadine alone, whereas 0.2 mM glutathione was almost ineffective. These results may suggest a possible role of membrane in Fe2+-induced inhibition of Ca2+-ATPase.