The effects of betamethasone (BM) on endothelial nitric oxide synthase (eNOS) expression in adult baboon femoral arterial endothelial cells.
Glucocorticoids have significant effects on endothelium mediated vascular function throughout life. The baboon model has been used extensively to study cellular responses to glucocorticoids at several stages of the lifespan. Endothelial nitric oxide synthase (eNOS) is a major regulator of endothelium dependent arterial vasodilation. We have previously demonstrated that synthetic glucocorticoids down regulate eNOS in the baboon placenta. We have now conducted studies to determine whether glucocorticoids would alter eNOS expression in adult systemic vascular endothelial cells in this important animal model. We explored this potential mechanism in endothelial cells from femoral arteries of adult baboons at necropsy and cultured to the fourth passage. Endothelial cells were treated with 10-100nM betamethasone for 24h at 37 degrees C. Vascular endothelial growth factor (VEGF) was used as a positive control and medium as negative controls. The role of glucocorticoid receptor mediation in betamethasone-induced eNOS changes was investigated with the glucocorticoid receptor antagonist mifepristone. RNA (real-time quantitative RT-PCR) and protein (ELISA) were extracted and measured for eNOS. Expression and subcellular distribution of glucocorticoid receptor were detected with fluorescence labeled antibody microscopy. eNOS mRNA and protein in baboon endothelial cells were downregulated 25% by betamethasone treatment. This effect was attenuated by pre-incubation with mifepristone (P < 0.01). VEGF upregulated eNOS transcription and translation (P < 0.001), medium did not alter eNOS expression. We observed that mifepristone and VEGF increased glucocorticoid receptor cytoplasmic accumulation by fluorescence microscopy. We conclude that betamethasone can downregulate eNOS in cultured baboon endothelial cells via the glucocorticoid receptor pathway.
Aida
Keiko
K
Center of Women's Health, New York University, New York, USA.
Shi
Qiang
Q
Wang
Jian
J
VandeBerg
John L
JL
McDonald
Thomas
T
Nathanielsz
Peter
P
Wang
Xing Li
XL
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D001623:Betamethasone / Q000494:pharmacology*; D003593:Cytoplasm; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005263:Femoral Artery; D005269:Femur / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D006727:Hormone Antagonists / Q000494:pharmacology; D008297:Male; D008856:Microscopy, Fluorescence; D015735:Mifepristone / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010215:Papio; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
D000893:Anti-Inflammatory Agents; D006727:Hormone Antagonists; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D042461:Vascular Endothelial Growth Factor A; D015735:Mifepristone; D001623:Betamethasone; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III