Neuronal-specific endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration in mixed primary hippocampal culture homogenates

• Published in: Analytical biochemistry Vol. 330; no. 1; pp. 130 - 139
• Main Authors: Parsons, J Travis, Sun, David A, DeLorenzo, Robert J, Churn, Severn B
• Format: Journal Article
• Language: English
• Published: United States 01.07.2004
• Subjects: Animals; Brain Chemistry; Calcium - metabolism; Calcium-Transporting ATPases - analysis; Cells, Cultured; Endoplasmic Reticulum - enzymology; Endoplasmic Reticulum - metabolism; Hippocampus - cytology; Hippocampus - enzymology; Hippocampus - metabolism; Isoenzymes - analysis; Neuroglia - cytology; Neuroglia - enzymology; Neuroglia - metabolism; Rats; Rats, Sprague-Dawley
• ISSN: 0003-2697
• DOI: 10.1016/j.ab.2004.03.025

Endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration is crucial for maintenance of neuronal Ca(2+) homeostasis. The use of cell culture in conjunction with modern Ca(2+) imaging techniques has been invaluable in elucidating these mechanisms. While imaging protocols evaluate endoplasmic reticulum Ca(2+) loads, measurement of Mg(2+)/Ca(2+) ATPase activity is indirect, comparing cytosolic Ca(2+) levels in the presence or absence of the Mg(2+)/Ca(2+) ATPase inhibitor thapsigargin. Direct measurement of Mg(2+)/Ca(2+) ATPase by isolation of microsomes is impossible due to the minuscule amounts of protein yielded from cultures used for imaging. In the current study, endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration was measured in mixed homogenates of neurons and glia from primary hippocampal cultures. It was demonstrated that Ca(2+) uptake was mediated by the endoplasmic reticulum Mg(2+)/Ca(2+) ATPase due to its dependence on ATP and Mg(2+), enhancement by oxalate, and inhibition by thapsigargin. It was also shown that neuronal Ca(2+) uptake, mediated by the type 2 sarco(endo)plasmic reticulum Ca(2+) ATPase isoform, could be distinguished from glial Ca(2+) uptake in homogenates composed of neurons and glia. Finally, it was revealed that Ca(2+) uptake was sensitive to incubation on ice, extremely labile in the absence of protease inhibitors, and significantly more stable under storage conditions at -80 degrees C.