Immunologic localization and kinetic characterization of a Na+/Ca2+ exchanger in neuronal and non-neuronal cells.
The plasma membrane Na+/Ca2+ exchanger is believed to play a role in the regulation of Ca2+ fluxes in neurons, though the lack of specific inhibitors has limited the delineation of its precise contribution. We recently reported the development of antibodies against a 36-kDa brain synaptic membrane protein which immunoprecipitated exchanger activity from solubilized membranes. In the present study we examined the kinetics of the Na+/Ca2+ exchanger in primary neurons in culture, in a neuronal hybrid cell line (NCB-20), and in a fibroblast-like cell line (CV-1) to see whether the level of exchanger activity correlated with the degree of immunostaining produced by our antibodies. The Vmax was determined for each cell type and found to be highest in primary neurons. Exchanger activity increased in primary neurons between days 1 and 6 in culture, but no such time-dependent change occurred in either of the cell lines. Immunoblot analysis of the three cell types probed with the anti-36-kDa protein antibodies revealed significantly greater immunostaining in the primary neurons compared with the other two cell types. Intensity of staining of neurons also increased significantly between days 1 and 6 in culture. Immunocytochemistry showed significant labelling of the primary neurons on the neuritic processes and points of contact between cells. The NCB-20 and CV-1 cells showed considerably lower levels of immunoreactivity. The antibodies immunoextracted approximately 90% of the exchanger activity in the primary neurons and approximately 70 and 50% of the activity in NCB-20 and CV-1 cells respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Michaelis
M L
ML
Center for Biomedical Research, University of Kansas, Lawrence 66047.
Walsh
J L
JL
Pal
R
R
Hurlbert
M
M
Hoel
G
G
Bland
K
K
Foye
J
J
Kwong
W H
WH
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004622:Embryo, Mammalian; D015151:Immunoblotting; D007150:Immunohistochemistry; D007668:Kidney; D007700:Kinetics; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019831:Sodium-Calcium Exchanger; D013997:Time Factors
D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; D002118:Calcium