Fast relaxation and desensitization of angiotensin II contraction in the pulmonary artery via AT1R and Akt-mediated phosphorylation of muscular eNOS
- 92 Downloads
Angiotensin II (AngII) triggers a transient contraction of pulmonary arteries (PAs) followed by protracted desensitization. Based on the unconventional eNOS expression in PA smooth muscle cells (PASMCs), we hypothesized that activation of smooth muscle eNOS by AngII might be responsible for fast relaxation and tachyphylaxis. Using dual-wire myograph, mechanically endothelium-denuded rat PA [E(−)PA] showed AngII concentration–dependent transient contractions (ΔTAngII, 95% decay within 1 min), which were abolished by losartan (AT1R antagonist). Neither PD123319 (AT2R antagonist) nor A779 (MasR antagonist) affected ΔTAngII. When the vessels were pretreated with L-NAME (NOS inhibitor), ODQ (guanylate cyclase inhibitor), or KT5823 (PKG inhibitor), ΔTAngII of E(−)PA became larger and sustained, whereas nNOS or iNOS inhibitors had no such effect. Immunoblotting of human PASMCs (hPASMCs) also showed eNOS expression, and AngII treatment induced activating phosphorylations of Ser1177 in eNOS and of Ser473 in Akt (Ser/Thr protein kinase B), an upstream signal of eNOS phosphorylation. In addition, L-NAME co-treatment promoted AngII-induced Ser19 phosphorylation of myosin light chain. In hPASMCs, AngII abolished plasma membrane expression of AT1R, and recovery by washout took more than 1 h. Consistent with the data from hPASMCs, the second application of AngII to E(−)PA did not induce contraction, and significant recovery of ΔTAngII required prolonged washout (> 2 h) in the myography study. L-NAME treatment before the second application facilitated recovery of ΔTAngII. Muscular eNOS plays an auto-inhibitory role in ΔTAngII of PAs. The molecular changes investigated in hPASMCs revealed eNOS phosphorylation and internalization of AT1R by AngII. We propose that the rat PA smooth muscle eNOS-induced lusitropy and slow recovery of AT1R from tachyphylaxis might counterbalance the excessive contractile response to AngII, contributing to the distinctive low-pressure pulmonary circulation.
KeywordseNOS Angiotensin II AT1R Pulmonary artery Smooth muscle
We greatly appreciate the experimental contribution by Mr. Rany Vorn (Chung-Ang University College of Nursing) in the wire myography study.
Source of funding
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2018R1A5A2025964 and NRF-2018R1D1A1B07048998) to S.J.K.
- 2.Barnett SF, Defeo-Jones D, Fu S, Hancock PJ, Haskell KM, Jones RE, Kahana JA, Kral AM, Leander K, Lee LL, Malinowski J, McAvoy EM, Nahas DD, Robinson RG, Huber HE (2005) Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors. Biochem J 385:399–408. https://doi.org/10.1042/bj20041140 CrossRefGoogle Scholar
- 3.Bkaily G, Sleiman S, Stephan J, Asselin C, Choufani S, Kamal M, Jacques D, Gobeil F Jr, D’Orleans-Juste P (2003) Angiotensin II AT1 receptor internalization, translocation and de novo synthesis modulate cytosolic and nuclear calcium in human vascular smooth muscle cells. Can J Physiol Pharmacol 81:274–287CrossRefGoogle Scholar
- 6.Cacanyiova S, Dovinova I, Kristek F (2013) The role of oxidative stress in acetylcholine-induced relaxation of endothelium-denuded arteries. J Physiol Pharmacol 64:241–247Google Scholar
- 10.Cui T, Nakagami H, Iwai M, Takeda Y, Shiuchi T, Tamura K, Daviet L, Horiuchi M (2000) ATRAP, novel AT1 receptor associated protein, enhances internalization of AT1 receptor and inhibits vascular smooth muscle cell growth. Biochem Biophys Res Commun 279:938–941. https://doi.org/10.1006/bbrc.2000.4055 CrossRefGoogle Scholar
- 11.de Man FS, Tu L, Handoko ML, Rain S, Ruiter G, Francois C, Schalij I, Dorfmuller P, Simonneau G, Fadel E, Perros F, Boonstra A, Postmus PE, van der Velden J, Vonk-Noordegraaf A, Humbert M, Eddahibi S, Guignabert C (2012) Dysregulated renin-angiotensin-aldosterone system contributes to pulmonary arterial hypertension. Am J Respir Crit Care Med 186:780–789. https://doi.org/10.1164/rccm.201203-0411OC CrossRefGoogle Scholar
- 14.Feng YH, Ding Y, Ren S, Zhou L, Xu C, Karnik SS (2005) Unconventional homologous internalization of the angiotensin II type-1 receptor induced by G-protein-independent signals. Hypertension 46:419–425. https://doi.org/10.1161/01.hyp.0000172621.68061.22 CrossRefGoogle Scholar
- 24.Huang ZM, Gao E, Fonseca FV, Hayashi H, Shang X, Hoffman NE, Chuprun JK, Tian X, Tilley DG, Madesh M, Lefer DJ, Stamler JS, Koch WJ (2013) Convergence of G protein-coupled receptor and S-nitrosylation signaling determines the outcome to cardiac ischemic injury. Sci Signal 6:ra95. https://doi.org/10.1126/scisignal.2004225 Google Scholar
- 29.Kim J, Ahn S, Ren XR, Whalen EJ, Reiter E, Wei H, Lefkowitz RJ (2005) Functional antagonism of different G protein-coupled receptor kinases for beta-arrestin-mediated angiotensin II receptor signaling. Proc Natl Acad Sci U S A 102:1442–1447. https://doi.org/10.1073/pnas.0409532102 CrossRefGoogle Scholar
- 30.Kim HJ, Yoo HY, Jang JH, Lin HY, Seo EY, Zhang YH, Kim SJ (2016) Wall stretch and thromboxane A(2) activate NO synthase (eNOS) in pulmonary arterial smooth muscle cells via H2O2 and Akt-dependent phosphorylation. Pflugers Arch 468:705–716. https://doi.org/10.1007/s00424-015-1778-1 CrossRefGoogle Scholar
- 31.Kim HJ, Yoo HY, Lin HY, Oh GT, Zhang YH, Kim SJ (2016) Role of muscular eNOS in skeletal arteries: endothelium-independent hypoxic vasoconstriction of the femoral artery is impaired in eNOS-deficient mice. Am J Phys Cell Physiol 311:C508–C517. https://doi.org/10.1152/ajpcell.00061.2016 CrossRefGoogle Scholar
- 33.Kumar S, Sud N, Fonseca FV, Hou Y, Black SM (2010) Shear stress stimulates nitric oxide signaling in pulmonary arterial endothelial cells via a reduction in catalase activity: role of protein kinase C delta. Am J Phys Lung Cell Mol Phys 298:L105–L116. https://doi.org/10.1152/ajplung.00290.2009 Google Scholar
- 36.Lumb AB, Nunn JF (2005) Nunn’s applied respiratory physiology, 6th edn. Elsevier Butterworth Heinemann, EdinburghGoogle Scholar
- 39.Morinelli TA, Walker LP, Velez JC, Ullian ME (2015) Clathrin-dependent internalization of the angiotensin II AT(1)A receptor links receptor internalization to COX-2 protein expression in rat aortic vascular smooth muscle cells. Eur J Pharmacol 748:143–148. https://doi.org/10.1016/j.ejphar.2014.12.018 CrossRefGoogle Scholar
- 47.Su KH, Tsai JY, Kou YR, Chiang AN, Hsiao SH, Wu YL, Hou HH, Pan CC, Shyue SK, Lee TS (2009) Valsartan regulates the interaction of angiotensin II type 1 receptor and endothelial nitric oxide synthase via Src/PI3K/Akt signalling. Cardiovasc Res 82:468–475. https://doi.org/10.1093/cvr/cvp091 CrossRefGoogle Scholar