|dc.description.abstract||The renin-angiotensin-aldosterone system (RAAS) is one of the major
regulators of blood pressure. The actions and generation of RAAS components at the
tissue level are less well appreciated. This work was designed to investigate the
vascular wall not only as a target of the RAAS, but also as one of its sources.
Immunocytochemical and immunoblotting analysis revealed positive renin
immunoreactivity in the cytoplasm of cultured bovine aortic endothelial cells. Two
immunoreactive bands of molecular mass approximately 37,000 and 40,000 dalton
were identified. In situ hybridization confirmed that renin mRNA was localized in the
same cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) using primers
specific for human renin gave a clear single band with the predicted size for
(pro)renin. These findings suggest that these vascular endothelial cells are a source of
local synthesised renin.
Conditioned medium from cultured bovine aortic endothelial cells (BAECCM)
and rat aortic smooth muscle cells (RASMCCM) were shown to contain
immunoreactive angiotensin II (Ang II) equivalent to 15.05 ± 4.67 pg/106 cells and
1 1.16 ± 1.8 pg/ 106 cells, respectively. Tritiated thymidine incorporation into aortic
smooth muscle cells (ASMC) was increased by Ang II and by BAECCM. In both
cases, this stimulated proliferation was inhibited by the Ang II type I (AT, ) receptor
selective antagonist, losartan. Although tritiated thymidine uptake by rat aortic
smooth muscle cells (RASMC) was not significantly enhanced by RASMCCM, it was
significantly decreased by losartan in the presence of RASMCCM or of serum-free
medium. Assay of RASMC proliferation by cell counting showed that the number of
cells in the presence of Ang II (10'6M) were nearly twice that in control cultures after r
2 days. These findings suggest that Ang II produced by ASMC locally may regulate
ASMC growth in an autocrine or/and paracrine fashion, via the AT, receptor.
RASMC was also shown to produce immunoreactive aldosterone. Ang II
significantly enhanced aldosterone formation by RASMC. but not in the presence of
losartan. Ang II stimulated 3H-thymidine uptake into RASMC was further enhanced
by aldosterone, but inhibited by the aldosterone antagonist. spironolactone. and the
3ß-hydroxysteroid-dehydogenase inhibitor trilostane. These results suggest that the
presence of locally generated aldosterone is essential for the stimulatory effects of
Ang II, acting via the AT, receptor. on RASMC proliferation.
Amplified products corresponding to transcripts of the CYP 11 BI gene were
obtained by RT-PCR on RNA extracted from RASMC, using primers chosen from
homologous parts of the exon I and exon 2 regions of CYP IIBI and CYP II B2
genes. Sequencing showed the presence of CYP 11 B1 transcript, but gave no evidence
for CYP 11 B2 gene transcription.
RT-PCR also gave a band corresponding to the 770 bp fragment from bases -
486 (upstream) to + 284 (exon 2) bp of the CYP 1lB1 gene. Furthermore, the present
experiments demonstrated the transcription of the sequences 183-480 bp upstream
from the CYP 11 B1 gene, and the use of competitive RT-PCR showed this was
regulated by Ang 11. Thus. cultured RASMC may be the site of Ang 11 regulated
transcription of a longer fragment of the CYP 11 B1 gene than generally expected.
Finally, use of immunofluorescence and immunoblotting demonstrated the
presence of an apparent binding carrier for 18-OH-DOC in cultured RASMC similar
to that found in the rat adrenal.||en_US