Free Radical Biology and Medicine
Stroke is the fifth cause of death and a leading cause of disability in the United States. In an ischemic stroke, blood flow to the brain is blocked, leading to lack of oxygen, ATP, membrane damage and eventual neuronal cell death. Therapies to treat ischemic stroke restore blood flow but cause further neuronal damage by generating reactive oxygen species (ROS) and creating oxidative stress. The plasma membrane calcium ATPase (PMCA) is a high-affinity calcium transporter critical for the maintenance of calcium homeostasis and optimal neuronal function. Previous studies from our laboratory have shown a unique sensitivity of PMCA to ROS. PMCA undergoes dramatic inactivation, aggregation and fragmentation when exposed to physiologically relevant ROS. The goal of the present studies was to determine the effects of hypoxia and reperfusion on PMCA. Differentiated SHY5Y cells were exposed to hypoxia (1% O2) followed by reperfusion (21% O2) for various time periods. PMCA activity, protein levels and total ROS were measured under these conditions. Our results show a 50% reduction in PMCA activity under hypoxic conditions (n=3). The maximum decline occurred after 2 hours of hypoxia and remained consistent up to 6 hours (the highest time point tested). Further decrease in PMCA activity was observed during reperfusion. This decline was dependent on both the time of exposure to hypoxia as well as to reperfusion. Six hours of hypoxia followed by 2 hours of reperfusion caused ∼80% decline in PMCA activity. Immunoblot analysis showed a 40% reduction in PMCA protein levels. We also observed a concomitant, time-dependent increase in ROS levels. Decline in neuronal PMCA would create Ca2+-dependent toxicity which could be a major factor influencing cell death in stroke. Interventions to preserve PMCA and boost its activity may be developed as future therapeutic targets for the treatment of patients with ischemic stroke.
Kowalski K, Sarsour E, Zaidi A. Effects of Hypoxia and Reperfusion on the Neuronal Plasma Membrane Calcium Pump. Free Radical Biology and Medicine. 2022; 180(S1).