Na, K‐ATPase: Comparison of the cellular localization of α‐subunit mRNA and polypeptide in mouse cerebellum, retina, and kidney

Abstract

A clone encoding mouse brain Na,K-ATPase alpha-subunit was isolated from a mouse brain lambda gt11 cDNA library by using antisera to mouse and bovine brain alpha-subunit. A comparison of the nucleotide sequence of this clone with published sequences of rat brain alpha-subunit isoform clones showed it to be most similar to rat brain alpha 1. An RNA antisense probe prepared from the cDNA insert of the mouse clone detected a single mRNA of approximately 4.5 kb in Northern blots of mouse brain and kidney RNAs. This probe hybridized only to an alpha 1-cDNA insert from rat brain under high stringency conditions on Northern blots. The RNA antisense probe was used for in situ hybridization to sections of mouse kidney, cerebellum, and retina, and the cellular distribution of alpha-subunit mRNA (alpha-mRNA) was compared with that of alpha-subunit polypeptide (alpha-subunit) detected by immunofluorescence in similar sections. In kidney, alpha-mRNA distribution closely paralleled that of the polypeptide with abundant expression in ascending thick limbs and cortical distal tubules and weaker labeling in cortical proximal tubules. The co-distribution of alpha-mRNA and polypeptide in kidney where Na,K-ATPase localization is well established is consistent with the specificity of these probes. In the retina, prominent labeling with both probes was seen in photoreceptor inner segments, inner nuclear layer, and ganglion cell bodies. Plexiform layers and optic fibers expressed abundant alpha-subunit but little mRNA. Light labeling for both was seen in the outer nuclear layer. In cerebellum, alpha-mRNA and alpha-subunit were associated with soma of granule cells, basket cells, and stellate cells. Glomeruli and basket terminals contained abundant alpha-subunit but exhibited little reactivity with the riboprobe. In Purkinje cell bodies, in contrast, the antibody used to identify the cDNA clone did not resolve significant polypeptide in the somal plasmalemma despite abundant somal mRNA expression. Comparison of distribution of the two probes in cerebellum and retina indicates that message accumulation is primarily in cell bodies, while alpha-subunit epitopes may be co-expressed in cell bodies and/or transported to distant sites in cell-specific patterns.

Differential expression of Na,K-ATPase ?-isoform mRNAs in aging rat cerebellum

“…9) of young and old rats. The results in both are similar to previous reports in young animals (Siegel et al, 1984;Hieber et al, 1989;Sheedlo et al, 1989), and there is no appreciable difference in the density or distribution of immunostaining in the young and old animals either in the cerebellum or hippocampus (Table II).…”

“…Sections were washed in a wash solution containing 10 mM Tris-HCl (pH 7.6), 0.5% bovine serum albumin, and 0.87% NaCl, three times for 5 min each. After these washes, incubations were performed in a humid chamber as follows: a) 3% nonimmune goat serum for 60 min; b) rabbit antiserum to Na,K-ATPase total catalytic subunit purified from bovine cerebrum (Hieber et al, 1989;Sheedlo et al, 1989) diluted 1:500 with 1% goat serum for 18-20 hr; c) wash in three changes of wash solution, 5 min each; d) goat antirabbit IgG diluted 1:20 with 1% goat serum for 60 min and washed as in step c; e) rabbit PAP complex diluted 1:100 with 1% goat serum for 60 min and washed as in step c; and f) chromogenic solution containing 0.05% 3,38-diaminobenzidine tetrahydrochloride and 0.01% H 2 O 2 in 50 mM Tris-HCl (pH 7.6). The last reaction was stopped by immersing the sections in water.…”

Heterotrimeric G-protein Gq/11 Localized on Pancreatic Zymogen Granules Is Involved in Calcium-regulated Amylase Secretion

“…Monoclonal antibody against rat pancreatic GP-2 was a gift from Dr. Anson Lowe (Stanford University). Polyclonal antibody (31B) to the ␣ subunit of the sodium pump has been described previously (25). Secondary antibodies included peroxidase-coupled goat anti-rabbit immunogloblin G (IgG, Amersham) and fluorescein isothiocyanate-labeled goat antirabbit IgG (Sigma).…”

Glucose-specific regulation of aldose reductase in capan-1 human pancreatic duct cells In vitro.

“…The effect of elevated glucose on Capan-1 cell Na,K-ATPase and AR protein levels were determined by quantitative immunoblotting using well-characterized antisera raised against the catalytic subunit of Na,K-ATPase (25) and human placental AR (26). 300-l aliquots of Capan-1 cell homogenate prepared as described for Na,K-ATPase assay were mixed with 150 l of SDS-containing sample buffer, heated at 95 Њ C for 5 min, mixed with 50 l of 20% ␤ -mercaptoethanol, and stored at Ϫ 20 Њ C. After thawing, samples corresponding to 10 g of cell protein per lane were electrophoresed on 7.5% linear polyacrylamide mini-gels.…”