Inhibition of Ca²⁺-pump ATPase and the Na⁺/K⁺-pump ATPase by iron-generated free radicals: Protection by 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine sulfate (U-89843D), a potent, novel, antioxidant/free radical scavenger

Preincubation of red blood cell (RBC) membranes with a model system known to generate reactive oxygen species (ROS) and free radicals (200 μM ferrous sulfate and 200 μM EDTA, Fe²⁺/EDTA) resulted in inhibition of the Na⁺/K⁺-pump ATPase, the basal Ca²⁺-pump ATPase, and the calmodulin-activated Ca²⁺-pump ATPase. Inhibition of the ion pump ATPases was also associated with membrane protein cross-linking and lipid peroxidation, the latter as monitored by the formation of thiobarbituric acid reactive substances (TBARS). Inhibition of the ion transport ATPases, protein cross-linking and formation of TBARS were prevented by U-89843D in a concentration-dependent manner, with half-maximal protection seen at 0.3 μM. U-89843D was more potent than the classical antioxidant butylated hydroxytoluene. Neither U-89843D nor the solvent DMSO had any effect on the assay of TBARS. U-89843D exerted only minimal inhibitory activity on ATPase activities. Thus, U-89843D was potent in vitro in preventing a variety of membrane-damaging reactions mediated by ROS. It is suggested that protection of membranes from ROS-mediated damage is of potential usefulness in the prevention and treatment of certain disease processes.