Hong Zhang1, Geng-Yun Sun. 1. Department of Respiratory Medicine, Department of Emergency Medicine, First Affiliated Hospital of Anhui Medical University, Anhui, Hefei, China.
Abstract
BACKGROUND: The renin-angiotensin system is thought to be involved in the development and progression of vascular endothelium inflammation, thereby contributing to vascular endothelium injury. To clarify the role of angiotensin II (Ang II) in rat pulmonary microvascular endothelial cells (RPMVECs), we examined the expression and functional significance of angiotensin II (Ang II) receptors in normal and lipopolysacchride (LPS) treated RPMVECs. METHODS: The expressions of Ang II type 1(AT(1)) and Ang II type 2 (AT(2)) receptors in cultured RPMVECs were identified by the reverse transcription-polymerase chain reaction (RT-PCR) technique, Western blot and (125)I-labeled [Sar(1),Ile(8)] Ang II binding assays. The RPMVECs were treated with LPS (0.1-100 microg/ml) and Ang II (10(-8)-10(-5) M) for 24 h, respectively. Next, RPMVECs were treated with 10 microg/ml LPS or 10(-7) M Ang II for various times (3, 6, 12, and 24 h). The mRNA and protein levels of, AT(1) and AT(2) receptors, were evaluated at 3, 6, 12, and 24 h, respectively. RESULTS: The presence of specific Ang II binding sites in RPMVECs was found by Ang II saturated assays. RT-PCR revealed that only the AT(1) receptor mRNA is presented in RPMVECs. Western blot analysis of the RPMVECs protein extracts showed only one prominent band of the protein at approximately 41 KDa when probed with anti-AT(1) antibody and anti-AT(2) antibody. No AT(2) receptor mRNA and protein was detected. LPS treated cells resulted in an increase in the mRNA and protein levels of AT(1) receptor, whereas, Ang II treated cells showed a decrease in the mRNA and protein levels of AT(1) receptor. CONCLUSIONS: We found that primary cultured RPMVECs expressed only AT(1) receptor, but not AT(2) receptor. LPS up-regulated the transcriptional and post-transcriptional expression of AT(1) receptor in RPMVECS; in contrast, Ang II treatment caused a reduction in the mRNA and protein of AT(1) receptor in a time-dependent manner.
BACKGROUND: The renin-angiotensin system is thought to be involved in the development and progression of vascular endothelium inflammation, thereby contributing to vascular endothelium injury. To clarify the role of angiotensin II (Ang II) in rat pulmonary microvascular endothelial cells (RPMVECs), we examined the expression and functional significance of angiotensin II (Ang II) receptors in normal and lipopolysacchride (LPS) treated RPMVECs. METHODS: The expressions of Ang II type 1(AT(1)) and Ang II type 2 (AT(2)) receptors in cultured RPMVECs were identified by the reverse transcription-polymerase chain reaction (RT-PCR) technique, Western blot and (125)I-labeled [Sar(1),Ile(8)] Ang II binding assays. The RPMVECs were treated with LPS (0.1-100 microg/ml) and Ang II (10(-8)-10(-5) M) for 24 h, respectively. Next, RPMVECs were treated with 10 microg/ml LPS or 10(-7) M Ang II for various times (3, 6, 12, and 24 h). The mRNA and protein levels of, AT(1) and AT(2) receptors, were evaluated at 3, 6, 12, and 24 h, respectively. RESULTS: The presence of specific Ang II binding sites in RPMVECs was found by Ang II saturated assays. RT-PCR revealed that only the AT(1) receptor mRNA is presented in RPMVECs. Western blot analysis of the RPMVECs protein extracts showed only one prominent band of the protein at approximately 41 KDa when probed with anti-AT(1) antibody and anti-AT(2) antibody. No AT(2) receptor mRNA and protein was detected. LPS treated cells resulted in an increase in the mRNA and protein levels of AT(1) receptor, whereas, Ang II treated cells showed a decrease in the mRNA and protein levels of AT(1) receptor. CONCLUSIONS: We found that primary cultured RPMVECs expressed only AT(1) receptor, but not AT(2) receptor. LPS up-regulated the transcriptional and post-transcriptional expression of AT(1) receptor in RPMVECS; in contrast, Ang II treatment caused a reduction in the mRNA and protein of AT(1) receptor in a time-dependent manner.