Localization of electrogenic Na/bicarbonate cotransporter NBCe1 variants in rat brain.

ABSTRACT

The activity of HCO(3)(-) transporters contributes to the acid-base environment of the nervous system. In the present study, we used in situ hybridization, immunoblotting, immunohistochemistry, and immunogold electron microscopy to localize electrogenic Na/bicarbonate cotransporter NBCe1 splice variants (-A, -B, and -C) in rat brain. The in situ hybridization data are consistent with NBCe1-B and -C, but not -A, being the predominant NBCe1 variants in brain, particularly in the cerebellum, hippocampus, piriform cortex, and olfactory bulb. An antisense probe to the B and C variants strongly labeled granule neurons in the dentate gyrus of the hippocampus, and cells in the granule layer and Purkinje layer (e.g. Bergmann glia) of the cerebellum. Weaker labeling was observed in the pyramidal layer of the hippocampus and in astrocytes throughout the brain. Similar, but weaker labeling was obtained with an antisense probe to the A and B variants. In immunoblot studies, antibodies to the A and B variants (alphaA/B) and C variant (alphaC) labeled approximately 130-kDa proteins in various brain regions. From immunohistochemistry data, both alphaA/B and alphaC exhibited diffuse labeling throughout brain, but alphaA/B labeling was more intracellular and punctate. Based on co-localization studies with antibodies to neuronal or astrocytic markers, alphaA/B labeled neurons in the pyramidal layer and dentate gyrus of the hippocampus, as well as cortex. alphaC labeled glia surrounding neurons (and possibly neurons) in the neuropil of the Purkinje cell layer of the cerebellum, the pyramidal cell layer and dentate gyrus of the hippocampus, and the cortex. According to electron microscopy data from the cerebellum, alphaA/B primarily labeled neurons intracellularly and alphaC labeled astrocytes at the plasma membrane. In summary, the B and C variants are the predominant NBCe1 variants in rat brain and exhibit different localization profiles.