Cell-type specific expression of Na+, K(+)-ATPase catalytic subunits in cultured neurons and glia: evidence for polarized distribution in neurons.
Na+,K(+)-ATPase (the sodium pump) is a family of proteins consisting of catalytic (alpha) and glycoprotein (beta) subunit isoforms which are differentially expressed in excitable tissue. To gain insight into the cell-type distribution of sodium pump protein, we determined the expression pattern of fetal rat telencephalic cultures, of telencephalic cultures depleted of neurons, and of pure astrocyte cultures. Isoform-specific antibodies were used for immunoblotting and immunohistochemistry, with supplemental [3H]ouabain binding to assess levels of functional alpha 2/alpha 3 protein. The results show that neurons of mixed telencephalic cultures uniquely express alpha 3 and high levels of alpha 1. The marked similarity in the distribution of microtubule-associated protein-2 and alpha 1 immunocytochemical staining strongly suggests that alpha 1 subunits are enriched in dendrites. Further, highly correlative growth cone-associated protein-43 and alpha 3 staining is consistent with a preferential expression of alpha 3 subunits in axons, which are also characterized by low levels of alpha 1 and no alpha 2 immunoreactivity. Process-bearing glia are intimately associated with neuronal aggregates and express high levels of both alpha 1 and alpha 2 protein, as well as GFAP. Interestingly, polygonal, flat glia not within neuronal aggregates are weakly immunopositive only for alpha 1 and GFAP. Pure astrocytic cultures possess appreciable alpha 1 protein and GFAP, but lack both alpha 2 and alpha 3 immunoreactivity. As predicted by the immunohistochemical findings, [3H]ouabain binding was low in pure astrocytic cultures, and much higher in the neuron-enriched mixed cultures. These observations confirm that neurons express all three catalytic isoforms of the sodium pump. They also suggest that specific alpha-isoforms may be polarized to targeted membrane regions of neurons. Further, glia intimately associated with neurons express alpha 2, bind significant amounts of [3H]ouabain, and possess much higher levels of alpha 1 and GFAP compared to glia not near neurons. Thus, neurons may regulate glial sodium pump expression.
Brines
M L
ML
Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510.
Robbins
R J
RJ
D000818:Animals; D001253:Astrocytes / Q000201:enzymology; D015153:Blotting, Western; D002384:Catalysis; D016764:Cell Polarity; D002478:Cells, Cultured; D007150:Immunohistochemistry; D009457:Neuroglia / Q000201:enzymology*; D009474:Neurons / Q000201:enzymology*; D010042:Ouabain / Q000378:metabolism; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013687:Telencephalon / Q000166:cytology / Q000201:enzymology; D014018:Tissue Distribution
D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase