Expression of the renin-angiotensin system in a human placental cell line

Abstract

Background: The renin-angiotensin system (RAS) is present in human placental tissue and participates in regulation of maternal-fetal blood flow during pregnancy. RAS expression in placental tissue is regulated by various hormones and is altered in various disease conditions. An in vitro system is needed to further investigate regulation of the placental RAS. To this end, we studied RAS expression in the human placenta-derived cell line, CRL-7548.

Methods: CRL-7548 cells were cultured in plastic plates. Total RNA was extracted, reverse transcribed, and amplified by polymerase chain reaction (PCR) with specific primers. Angiotensin II peptide in the culture media was measured by radioimmunoassay. Renin activity was detected by radioimmunoassay measuring angiotensin I generated. Angiotensin receptor type I was detected by Western blot.

Results: Specific mRNA for angiotensin, renin, angiotensin converting enzyme, and angiotensin receptor type I was detected by real-time PCR. Renin activity was detected in the placental cell lysate, and angiotensin II peptide, the final product of the RAS system, was detected in cell culture media by radioimmunoassay. Angiotensin receptor type I was identified as a 41 kDa protein in cell lysates by Western blot.

Conclusions: These results demonstrate that all necessary components of the classic RAS are expressed in the human placental cell line CRL-7548. This cell line may prove useful as an in vitro system for studying RAS regulation in the placenta.

Figure 1

Detection of AGT, renin, ACE and AT1R mRNA expression by real-time PCR. Amplification products of (panel A) AGT (128 bp), renin (121 bp), ACE (164 bp), and (panel B) AT1R (80 bp) and GAPDH (225 bp) obtained from placental cell total RNA were visualized on ethidium bromide stained 2% agarose gels. Reverse transcriptase (RT) was present (+) or absent (-) in the step of reverse transcription. No PCR products were seen in samples when RT was omitted (-). A 50 bp DNA ladder was used as a size marker (M). GAPDH refers to the internal control gene, glyceraldehyde 3-phosphate dehyrodrogenase.

Figure 2

Secretion of immunoreactive Ang II into culture medium by human placental cells. Conditioned media (n=6) was collected from plates with cultured placental cells. Controls are unconditioned media (n=6) from plates without cultured cells. Values are picograms immunoreactive Ang II per milliliter of culture medium.

Figure 3

Representative Western blot experiment showing the expression of the AT1R in human placental cells. Lanes 1 and 2 are placental cell lysates from separate preparations. Approximately 30 μg of lysate proteins were loaded on each lane. A single band of 41 kDa protein was detected using an AT1R-specific antibody.