Quantification of α-subunit isoforms of Na,K-ATPase in rat resistance vessels
Rat mesenteric resistance vessels (RV) were characterized with respect to concentration of individual α-subunit isoforms of Na,K-ATPase. Methods:
Total vessel homogenates were used to avoid any loss or subfractionation of membranes. They were applied to sodium dodecyl sulphate gels and, for calibration, in parallel lanes were run purified rat Na,K-ATPase preparations with known isoform distribution and content. The capacity per mg protein for Na+-dependent 32P-phosphorylation of Na,K-ATPase isolated from rat kidney was used for α1 calibration and that for high-affinity (3H)ouabain binding of Na,K-ATPase isolated from rat brain was used for (α2 + α3) calibration. Western blots containing homogenate proteins and reference enzyme were incubated with isoform-specific antibodies and radiolabelled secondary antibodies. The signals from adjacent α spots were used for qualitative and quantitative characterization of rat vessels. Results:
A concentration of 100.7 ± 14.4 pmol (n = 11) per g wet weight of the α1-isoform containing Na,K-ATPase was found in RV from 12–14-week rats. A much lower and more unreliable content of α2- and α3-isoforms was found. These ouabain-sensitive isoforms seem to represent a maximum of 5–10% each compared with the ouabain-insensitive rat α1-isoform. Conclusions:
The isoform pattern in RV, in which the isoform with high/intermediate Na+-affinity is the absolutely dominating one representing nearly all sodium pumps in this tissue, is very different from that seen in rat skeletal muscles. Due to the high content of the ouabain-insensitive α1-isoform in rat RV this species would seem a less relevant model in studies addressing a role of cardiac glycosides and putative endogenous ouabain-like factors in hypertension.