A simple method for rapid separation of endothelial and smooth muscle mRNA reveals Na/K+ -ATPase alpha-subunit distribution in rat arteries.
OBJECTIVE
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 alpha-subunit isoforms (NaKalpha1, NaKalpha2 and NaKalpha3) 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+]. NaKalpha1 was uniformly expressed in all fractions, NaKalpha2 closely followed the expression pattern of smooth muscle markers and NaKalpha3 expression was weak and attributable to blood contamination. Comparable results were obtained with the mesenteric artery.


CONCLUSIONS
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 NaKalpha1, whereas smooth muscle expresses NaKalpha1 and NaKalpha2, but not NaKalpha3.
Krenek
Peter
P
Laboratoire de Pharmacologie, Université catholique de Louvain, Brussels, Belgium.
Hamaide
Marie-Christine
MC
Morel
Nicole
N
Wibo
Maurice
M
D000818:Animals; D001011:Aorta / Q000201:enzymology; D004730:Endothelium, Vascular / Q000201:enzymology*; D008297:Male; D008638:Mesenteric Arteries / Q000201:enzymology; D008967:Molecular Biology / Q000379:methods*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D021122:Protein Subunits / Q000235:genetics; D012333:RNA, Messenger / Q000302:isolation & purification*; D051381:Rats; D011921:Rats, Inbred WKY; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*
D021122:Protein Subunits; D012333:RNA, Messenger; C499075:Atp1a1 protein, rat; C498532:Atp1a2 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase