Cytoarchitectural relationships between [3H]ouabain binding and mRNA for isoforms of the sodium pump catalytic subunit in rat brain.
We examined the cell type-specific expression of the alpha 1, alpha 2, and alpha 3 subunits of the sodium pump in rat brain using in situ hybridization and [3H]ouabain autoradiography. These techniques allowed us to colocalize mRNA and functional alpha 2/alpha 3 pumps on adjacent sections. The perikarya of many neurons possessed high levels of alpha 1 and/or alpha 3 transcripts, while alpha 2 mRNA appeared to be present in only a few neuronal types. [3H]Ouabain binding in general paralleled the distribution of alpha 3 mRNA-positive neurons. The regional variation of alpha 1 and alpha 3 transcripts was complex and varied. Large neurons of the olfactory bulb and piriform cortex expressed high levels of alpha 3 transcripts, but low levels of alpha 1 mRNA. In frontal cortex, neurons of layers II-III were enriched in alpha 1 mRNA, while those in layer V exhibited high levels of alpha 3 transcripts. In the hippocampus, principal neurons expressed all three alpha subunit mRNAs. CA subfield pyramidal neurons exhibited a high alpha 3/alpha 1 ratio, while dentate granule cells and hilar pyramidal neurons expressed approximately equal levels of alpha 1 and alpha 3. In the cerebellum, Purkinje and Golgi cells were rich in alpha 3 mRNA, while the granule cells appeared to express only alpha 1 transcripts. The distribution of functional sodium pump protein, as localized by [3H]ouabain binding, was highest in the neuropil of the hippocampus and cerebral cortex, and lowest over perikarya and white matter. [3H]ouabain did not bind to alpha 1 pump units, as confirmed by the complete absence of labeling over the choroid plexus, a tissue expressing only alpha 1 mRNA. In the cerebellum, regions of dense [3H]ouabain binding were localized to the granule cell layer, the inner third of the molecular layer in the basket region, and the deep cerebellar nuclei. Surprisingly, the dense neuropil in the outer 2/3 of the molecular layer lacked high [3H]ouabain binding. Thus, functional alpha 3 sodium pump units appear distributed to the axon terminals and not to apical dendrites of Purkinje, Golgi and basket cells. A similar pattern of increased [3H]ouabain binding in axonal but not dendritic fields of alpha 3-enriched neurons was present in the cerebral cortex and the hippocampus. Considering that many alpha 3-enriched neurons are of the Golgi I type with long axons, the alpha 3 isoform may be preferentially directed into axons to function in presynaptic membranes.
Brines
M L
ML
Neuroendocrine Program, Yale University School of Medicine, New Haven, CT 06510.
Gulanski
B I
BI
Gilmore-Hebert
M
M
Greene
A L
AL
Benz
E J
EJ
Robbins
R J
RJ
D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000033:anatomy & histology / Q000166:cytology / Q000201:enzymology*; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000201:enzymology; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D009928:Organ Specificity; D010042:Ouabain / Q000378:metabolism*; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism; D013462:Sulfur Radioisotopes
D007527:Isoenzymes; D015345:Oligonucleotide Probes; D015347:RNA Probes; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase