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):xan- thine 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.