| Summary: | 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 I 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.
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