Quantitative analysis of vascular prostaglandin H synthase mRNA levels by competitive polymerase chain reaction.
Prostaglandin H synthase (PGHS) occupies a central position in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. Expression of this enzyme is enhanced by inflammatory cytokines and phorbol esters. Because of the low abundance of PGHS, PGHS mRNA levels in tissues and cells were difficult to measure accurately. In this study, we quantified vascular PGHS-1 mRNA levels by a competitive polymerase chain reaction assay. The mean basal PGHS-1 level in cultured human umbilical vein endothelial cells (HUVECs) was 24.3 +/- 10.6 amol/microgram RNA. Treatment of the HUVECs with recombinant human interleukin-1 beta at 37 degrees C for 4 hours increased the mRNA to 49.1 +/- 9.1 amol/microgram RNA, a 2.1-fold increase. Treatment of the cells with phorbol 12-myristate 13-acetate at 37 degrees C for 4 hours caused a 1.7-fold increase in the mRNA level. This procedure was utilized to measure PGHS-1 mRNA levels in rabbit abdominal aorta, inferior vena cava, liver, kidney, and lung. The aortic mRNA level (9.2 +/- 1.9 amol/microgram RNA) was about 5 times higher than the inferior vena caval level (2.4 +/- 1.3 amol/microgram RNA) but only slightly higher than the level of other organs. These results reveal a significant difference in PGHS-1 mRNA levels between arterial and venous tissues. The synthase mRNA levels in organs such as liver, kidneys, and lungs do not differ significantly. The PCR assay should be valuable for studying the role of PGHS expression in disease states.
Zyglewska
T
T
Vascular Biology Research Center, University of Texas Health Science Center at Houston.
Wu
L C
LC
Xu
X M
XM
Conlan
M G
MG
Wang
L H
LH
Wu
K K
KK
D000818:Animals; D001011:Aorta / Q000737:chemistry* / Q000166:cytology; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology; D006801:Humans; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014682:Vena Cava, Inferior / Q000737:chemistry* / Q000166:cytology
D012333:RNA, Messenger; D011451:Prostaglandin-Endoperoxide Synthases; D013755:Tetradecanoylphorbol Acetate