Differentiation induces up-regulation of plasma membrane Ca(2+)-ATPase and concomitant increase in Ca(2+) efflux in human neuroblastoma cell line IMR-32.
Precise regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is achieved by the coordinated function of Ca(2+) channels and Ca(2+) buffers. Neuronal differentiation induces up-regulation of Ca(2+) channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca(2+)-ATPase (PMCA), the principal Ca(2+) extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca(2+)](i) was monitored in single cells using indo-1 microfluorimetry. When the Ca(2+)-ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca(2+)](i) recovery after small depolarization-induced Ca(2+) loads was governed primarily by PMCAs. [Ca(2+)](i) returned to baseline by a process described by a monoexponential function in undifferentiated cells (tau = 52 +/- 4 s; n = 25). After differentiation for 12-16 days, the [Ca(2+)](i) recovery rate increased by more than threefold (tau = 17 +/- 1 s; n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR-32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up-regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca(2+) influx and efflux pathways may enable differentiated neurons to precisely localize Ca(2+) signals in time and space.
Usachev
Y M
YM
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455, USA. usach001@tc.umn.edu
Toutenhoofd
S L
SL
Goellner
G M
GM
Strehler
E E
EE
Thayer
S A
SA
D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002454:Cell Differentiation; D002462:Cell Membrane / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009447:Neuroblastoma; D011189:Potassium Chloride / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007211:Indoles; D007527:Isoenzymes; D011189:Potassium Chloride; D000252:Calcium-Transporting ATPases; C048960:indo-1; D002118:Calcium; C000543:cyclopiazonic acid