Neuronal-specific endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration in mixed primary hippocampal culture homogenates.
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.
Parsons
J Travis
JT
Department of Neurology, Virginia Commonwealth University, PO Box 980599 (USPS), 1217 E Marshall St, MSB-619 (UPS, Fed-Ex), Richmond, VA 23298, USA. jparsons@hsc.vcu.edu
Sun
David A
DA
DeLorenzo
Robert J
RJ
Churn
Severn B
SB
D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000032:analysis*; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D007527:Isoenzymes / Q000032:analysis; D009457:Neuroglia / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D007527:Isoenzymes; D000252:Calcium-Transporting ATPases; D002118:Calcium