Effects of paraquat-induced oxidative stress on the neuronal plasma membrane Ca(2+)-ATPase.
Oxidative stress leads to the disruption of calcium homeostasis in brain neurons; however, the direct effects of oxidants on proteins that regulate intracellular calcium ([Ca(2+)](i)) are not known. The calmodulin (CaM)-stimulated plasma membrane Ca(2+)-ATPase (PMCA) plays a critical role in regulating [Ca(2+)](i). Our previous in vitro studies showed that PMCA present in brain synaptic membranes is readily inactivated by a variety of reactive oxygen species (ROS). The present studies were conducted to determine the vulnerability of PMCA to ROS generated in neurons as would probably occur in vivo. Primary cortical neurons were exposed to paraquat (PQ), a redox cycling agent that generates intracellular ROS. Low concentrations of PQ (5-10 microM) increased PMCA basal activity by two-fold but abolished its sensitivity to CaM. Higher concentrations (25-100 microM) inhibited both components of PMCA activity. Immunoblots showed the formation of high-molecular-weight PMCA aggregates. Additionally, PMCA showed evidence of proteolytic degradation. PMCA proteolysis was prevented by a calpain inhibitor, suggesting a role for calpain. Our findings suggest that PMCA is a sensitive target of oxidative stress in primary neurons. Inactivation of this Ca(2+) transporter under prolonged oxidative stress could alter neuronal Ca(2+) signaling.
Zaidi
Asma
A
Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS 66045, USA. Azaidi@kcumb.edu
Fernandes
Denzyl
D
Bean
Jennifer L
JL
Michaelis
Mary L
ML
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D006706:Homeostasis; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D010269:Paraquat / Q000494:pharmacology*; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism
D017382:Reactive Oxygen Species; D053499:Plasma Membrane Calcium-Transporting ATPases; D010269:Paraquat; D002118:Calcium