We have used in situ hybridization histochemistry to analyze the subceilular distribution of mRNAs encoding Na,K-ATPase a-and /3-subunit isoforms in the rat central nervous system. Substantial differences in the cellspecific pattern ofexpression were found for the genes encoding three isoforms of the a subunit. Transcripts of al-subunit genewere detected in virtually all cell types and structures examined. Expression of a2-subunit mRNA was characteristic of glia, whereas a3-subunit transcripts were predominant in neurons. Transcripts encoding the /31 subunit were detected in neurons, whereas /32-subunit mRNA expression was characteristic of glia. mRNA encoding both /3-subunit isoforms was present in choroidal epithelial cells. The distribution pattern of a-and /8-subunit mRNAs in structures throughout the central nervous system is consistent with the possibility of six structurally distinct Na',K+-ATPase isoenzymes.Na',K+-ATPase is the enzymatic activity responsible for active transport of Na' and K+ in most animal cells (1). The enzyme plays a central role in mediating electrical activity of the brain. In neurons, Na',K+-ATPase maintains the Na'and K+ gradients essential for nerve-impulse generation (2). Uptake of neurotransmitters and efflux of calcium from neurons are also coupled to activity ofthe enzyme (3). In glia, Na',K+-ATPase plays a critical role in potassium uptake during periods of neuronal activity (4-6).Na',K+-ATPase has been shown to consist of two subunits. The a subunit is a polypeptide of Mr -100,000 that contains the catalytic site for ATP hydrolysis and serves as the receptor for cardiac glycosides, such as ouabain and digitalis (1). The /3 subunit is a polypeptide ofMr =55,000 the function of which has yet to be established. Molecular cloning has revealed that Na',K+-ATPase a and / subunits are encoded by multigene families. Three a-and two /-subunit genes have been localized to different chromosomes in the mouse (7,8), and cDNA clones encoding three rat a-(al, a2, a3) and two /8-(J1, /32) subunit isoforms have been characterized (9-12). Substantial differences in the tissue and developmental specificity of expression have been found for each a and /3 subunit. al subunits have been detected in virtually all rat tissues (13). In contrast, a2 subunits are expressed predominantly in brain, heart, and lung (13), whereas a3 subunits are expressed in brain (13), pineal gland (14), and retinal photoreceptors (15). Expression of 81 subunits has been detected in brain, heart, and kidney (13), whereas /2 subunits are expressed primarily in brain (16), pineal gland (14), and photoreceptor cells (15). However, the physiological significance for a-and /-subunit isoform diversity has not been clearly explained.Analysis of the cellular distribution of Na',K+-ATPase expression should provide important clues to the normal physiological function of distinct Na',K+-ATPase isoenzymes. Recent studies have described the anatomical distribution of Na',K+-ATPase mRNAs (17-19) and polypeptides (20) within s...

Abstract. We have characterized cDNAs coding for three Na,K-ATPase tx subunit isoforms from the rat, a species resistant to ouabain. Northern blot and Sl-nuclease mapping analyses revealed that these a subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the al isoform, --~-4.5 kb long, is expressed in all fetal and adult rat tissues examined. The a2 mRNA, also ---4.5 kb long, is expressed predominantly in brain and fetal heart. The ix3 cDNA detected two mRNA species: a ---4.5 kb mRNA present in most tissues and a ---6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the a subunits are encoded by a multigene family. Structural analysis of the a subunits from rat and other species predicts a polytopic protein with seven membranespanning regions. Isoform diversity of the a subunit may provide a biochemical basis for Na,K-ATPase functional diversity.

We deduced the complete amino acid sequence of the rat brain Na,K-ATPase ,-subunit from cDNA. The rat brain "-subunit exhibits a high degree of primary sequence and secondary structural homology with the human and Torpedo (i-subunit polypeptides. Analysis of rat tissue RNA reveals that the ,B-subunit gene encodes four separate mRNA species which are expressed in a tissue-specific fashion. In ouabain-resistant HeLa C+ cells, ,B-subunit DNA sequences are amplified (-20-fold) and "-subunit mRNAs are overproduced relative to levels in parental HeLa cells. These results suggest that the (-subunit plays an important role in Na,K-ATPase structure-function and in the mechanism underlying cellular resistance to the cardiac glycosides.

TG, TPO, and RET/PTC1 mRNA are detectable in the peripheral blood of patients with thyroid disease, which correlates with a diagnosis of carcinoma.

The molecular basis for the marked difference between primate and rodent cells in sensitivity to the cardiac glycoside ouabain has been established by genetic techniques. A complementary DNA encoding the entire alpha 1 subunit of the mouse Na+- and K+-dependent adenosine triphosphatase (ATPase) was inserted into the expression vector pSV2. This engineered DNA molecule confers resistance against 10(-4) M ouabain to monkey CV-1 cells. Deletion of sequences encoding the carboxyl terminus of the alpha 1 subunit abolish the activity of the complementary DNA. The ability to assay the biological activity of this ATPase in a transfection protocol permits the application of molecular genetic techniques to the analysis of structure-function relationships for the enzyme that establishes the internal Na+/K+ environment of most animal cells. The full-length alpha 1 subunit complementary DNA will also be useful as a dominant selectable marker for somatic cell genetic studies utilizing ouabain-sensitive cells.