Semi-quantitation of mRNA by polymerase chain reaction. Levels of oxidative defense enzymes and aldose reductase in rat lenses cultured in hyperglycemic or oxidative medium.
The high sensitivity of reverse transcriptase-polymerase chain reaction for detecting low copy number mRNA transcripts has been standardized to analyze the mRNA profiles of catalase, glutathione peroxidase, CuZn-superoxide dismutase and aldose reductase, with respect to the housekeeping gene cyclophilin, in rat lenses cultured in hyperglycemic (50mM glucose) or oxidative (100 microM H2O2) media for 24, 40 and 60 hr. In response to hyperglycemia mRNA expression of catalase appeared to be inhibited at 24 hr but attained normal levels by 40 hr. On the other hand, mRNA levels of catalase were higher than normal between 40 and 60 hr in the presence of H2O2. Glutathione peroxidase mRNA abundance although enhanced in response to both hyperglycemia as well as H2O2-induced stress, displayed opposite trends with time-an increase from 24-60 hr due to hyperglycemia and a decrease to normal by 60 hr in the presence of H2O2. In contrast, CuZn-superoxide dismutase was inhibited at 50 mM glucose achieving baseline levels by 60 hr, while H2O2 elicited an induction at 24 hr which waned to basal levels by 60 hr. Interestingly, aldose reductase was unaffected by hyperglycemia but showed an appreciable increase with time upon exposure of the lens to H2O2. The role of these enzymes in cataractogenesis with regard to their respective mRNA levels is discussed.
Khanna
P
P
Department of Human Biological Chemistry & Genetics, University of Texas Medical Branch, Galveston 77555-0647, USA.
Wang
L
L
Ansari
N H
NH
D000449:Aldehyde Reductase / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002374:Catalase / Q000378:metabolism*; D003470:Culture Media / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000378:metabolism*; D006943:Hyperglycemia / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D013482:Superoxide Dismutase / Q000378:metabolism*
D003470:Culture Media; D012333:RNA, Messenger; D000449:Aldehyde Reductase; D002374:Catalase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase