Determination of xanthine dehydrogenase mRNA by a reverse transcription-coupled competitive quantitative polymerase chain reaction assay: regulation in rat endothelial cells by hypoxia and hyperoxia.
The enzyme system xanthine dehydrogenase (XD):xanthine oxidase, which generates the superoxide anion as a by-product of action on endogenous substrates, is believed to play a role in mediating pathophysiological changes through its contribution to total superoxide production. To aid with analysis of factors that regulate XD, we have developed a reverse transcription (RT)-coupled competitive quantitative polymerase chain reaction (PCR) assay which enables XD mRNA to be determined from small amounts of cultured cells where constitutive XD levels are low. A homologous insertion mutant of wild-type XD cDNA was prepared and used as an internal standard to normalize intersample PCR efficiency differences. XD mRNA levels determined by RT-PCR also were normalized to tubulin mRNA to compensate for RT differences and loading effects among samples. We report that XD mRNA levels, determined by RT-PCR, were increased twofold in hypoxic (3% oxygen) rat pulmonary microvascular endothelial cells relative to normoxic controls (20% oxygen). Conversely, XD mRNA was decreased threefold within 24 h under hyperoxic (95% oxygen) conditions. These data support the hypothesis that XD is regulated by oxygen tension in the pulmonary vasculature.
New England Medical Center, Pulmonary and Critical Care Division, Boston, Massachusetts 02111, USA.
J J
JJ
Lanzillo
F S
FS
Yu
J
J
Stevens
P M
PM
Hassoun
D000818:Animals; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D018496:Hyperoxia / Q000235:genetics*; D000860:Hypoxia / Q000235:genetics*; D008168:Lung / Q000166:cytology; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014968:Xanthine Dehydrogenase / Q000235:genetics*
D012333:RNA, Messenger; D014968:Xanthine Dehydrogenase