S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Biomedical Sciences (대학원 의과학과) Theses (Ph.D. / Sc.D._의과학과)
Activation mechanisms of eNOS expressed in pulmonary artery smooth muscle cells
폐동맥 평활근세포에 발현된 내피세포형 산화질소 합성효소의 활성화 기전
- Hae Jin Kim
- 의과대학 의과학과
- Issue Date
- 서울대학교 대학원
- 폐동맥 평활근 세포
- 학위논문 (박사)-- 서울대학교 대학원 : 의과학과, 2016. 8. 김성준.
- Pulmonary arteries (PAs) have a high degree of compliance, which is critical for buffering the wide ranges of blood flow. Here, I aimed to address a hypothesis that PA smooth muscle cells (PASMCs) express endothelial nitric oxide synthases (eNOS) that might be activated by mechanical stress and vasoactive agonists. In the myograph study of endothelium-denuded rat PAs, NOS inhibition (L-NAME, 100 M) induced strong contraction (96 % of 80 mM KCl-induced contraction (80K)) in the presence of 5 nM U46619 (thromboxane A2 (TXA2) analogue) with relatively high basal stretch (0.3 g, S(+)). With lower basal stretch (0.1 g, S(-)), however, L-NAME application following U46619 (TXA2/L-NAME) induced weak contraction (27 % of 80K). Inhibitors of nNOS and iNOS had no such effect in S(+) PAs. In endothelium-denuded S(+) mesenteric and renal arteries, TXA2/L-NAME-induced contraction was only 18 % and 21 % of 80K, respectively. Expression of endothelial-type NOS (eNOS) in rat PASMCs was confirmed by RT-PCR and immunohistochemistry. Even in S(-) PAs, pretreatment with H2O2 (0.1 – 10 M) effectively increased the sensitivity to TXA2/L-NAME (105 % of 80K). Patch clamp experiment showed that H2O2 facilitated membrane stretch-activated cation channels (SACs) in rat PASMCs. Consistently, mechanosensitive cation channel inhibitors (GxMTx4 and DIDS) effectively eliminated SAC activity in PASMCs and also TXA2/L-NAME contraction in S(+) PAs. Vice versa, NADPH oxidase inhibitors, reactive oxygen species scavengers (Tiron and PEG-catalase), or an Akt inhibitor (SC-66) suppressed TXA2/L-NAME-induced contraction in S(+) PAs. In a human PASMC line (PCS-100-023, ATCC), immunoblot analysis showed the following: 1) eNOS expression, 2) Ser1177 phosphorylation by U46619 and H2O2, and 3) Akt activation (Ser473 phosphorylation) by U46619. Taken together, the muscular eNOS in PAs can be activated by TXA2 and mechanical stress via H2O2 and Akt-mediated signaling, which may keep those balance to the contractile signals from TXA2 and mechanical stimuli. Interestingly, both in monocrotaline (MCT)- and chronic hypoxia (CH) -induced pulmonary arterial hypertensive rats, TXA2/L-NAME contraction of the S(+) PAs were largely decreased. The eNOS activated in PASMCs could counterbalance an excessive vasoconstriction, lowering the workload to the right heart.