The expression of renin and the formation of angiotensin II in bovine aortic endothelial cells.
One controversy in the field of vascular angiotensin generation has surrounded the nature and particularly the source of vascular renin. This study investigated the expression of renin protein and its mRNA in aortic endothelial cells using immunocytochemistry, Western blotting, in situ hybridization and reverse transcription PCR (RT-PCR). Using a monoclonal antibody against human renin, immunocytochemical analysis revealed positive immunoreactivity in the cytoplasm of cultured bovine aortic endothelial cells. Immunoblotting of solubilized proteins separated by SDS-PAGE from cultured aortic endothelial cells identified two immunoreactive species with molecular masses of approximately 37-40 kDa. In situ hybridization showed that renin mRNA was localized in the cytoplasm of these cells. Using RT-PCR of RNA extracted from bovine aortic endothelial cells with primers specific for human renin, a clear single band was detected, which had the predicted size of 142 bp for (pro)renin. Angiotensin II (Ang II) was assayed in conditioned medium (CM) from cultured bovine aortic endothelial cells, and in addition, the effects of Ang II and CM on the proliferation of aorta smooth muscle cells (ASMC) were also studied. The results showed that CM contained Ang II equivalent to 15.05+/-4.67 pg/10(6) cells. Assay of smooth muscle cell proliferation by cell number, and by tritiated thymidine uptake, showed that proliferative responses in the presence of Ang II at a concentration of 10(-6)M were evident within 1 day of subculture, and cell numbers were nearly twice those of controls after 2 days. Thymidine incorporation into ASMC was also increased by Ang II in a dose-dependent manner and by endothelial cell CM. In both cases, stimulated proliferation was inhibited by the Ang II type 1 (AT1) receptor selective antagonist, losartan. These findings suggest that these vascular endothelial cells are a source of locally synthesized renin that may thus be involved in vascular Ang II generation. They also suggest that Ang II produced by the endothelial cells may be secreted and stimulate ASMC proliferation via the AT1 receptor.
Xiao
F
F
Molecular and Cellular Biology, Division of Biomedical Sciences, Queen Mary and Westfield College, London E1 4NS, UK.
Puddefoot
J R
JR
Vinson
G P
GP
D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D002455:Cell Division; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D015151:Immunoblotting; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D051381:Rats; D012083:Renin / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013936:Thymidine / Q000493:pharmacokinetics
D000804:Angiotensin II; D012083:Renin; D013936:Thymidine