A Simple Method for Rapid Separation of Endothelial and Smooth Muscle mRNA Reveals Na ⁺/K ⁺-ATPase Alpha-Subunit Distribution in Rat Arteries

Abstract

Background/Aims: The endothelium has been recognized as a key component in the regulation of blood vessels. We designed a simple procedure to separate endothelial and smooth muscle RNA from rat aorta and mesenteric artery and used this method to establish the distribution of Na⁺/K⁺-ATPase α-subunit isoforms (NaKα1, NaKα2 and NaKα3) within the arterial wall. Methods: Rat aorta was perfused with Tripure, a reagent for RNA isolation, yielding 3 successive RNA fractions (E1–E3) and the remaining tissular RNA (Ao[E–]). A similar procedure was applied to the mesenteric artery. Gene expression was studied by semiquantitative reverse-transcription polymerase chain reaction. Results: Compared to unperfused aorta (Ao[E+]), typical endothelial mRNAs were enriched 3- to 5-fold in E1–E3 but almost absent in Ao[E–], whereas smooth muscle mRNAs were low in E1–E3 but similarly expressed in Ao[E–] and Ao[E+]. NaKα1 was uniformly expressed in all fractions, NaKα2 closely followed the expression pattern of smooth muscle markers and NaKα3 expression was weak and attributable to blood contamination. Comparable results were obtained with the mesenteric artery. Conclusion: We conclude that, in aorta and mesenteric artery, Tripure perfusion allows for a rapid and reliable separation of endothelial mRNA from smooth muscle mRNA, and that endothelium only expresses NaKα1, whereas smooth muscle expresses NaKα1 and NaKα2, but not NaKα3.