Regulatory Roles of ERK1/2, Rho-kinase and NADPH Oxidase-derived Reactive Oxygen Species in Excitation-Contraction Coupling in Vascular Smooth Muscle
博士 === 國立成功大學 === 基礎醫學研究所 === 94 === Smooth muscle contraction is regulated by increase in cytoplsmic Ca2+ and Ca2+ sensitivity of the contractile apparatus. The former activates myosin light chain kinase (MLCK) whereas the later mainly involves the inhibition of myosin light chain phosphatase (MLCP...
Main Authors: | , |
---|---|
Other Authors: | |
Format: | Others |
Language: | en_US |
Published: |
2006
|
Online Access: | http://ndltd.ncl.edu.tw/handle/26160015207638919245 |
id |
ndltd-TW-094NCKU5325009 |
---|---|
record_format |
oai_dc |
collection |
NDLTD |
language |
en_US |
format |
Others
|
sources |
NDLTD |
description |
博士 === 國立成功大學 === 基礎醫學研究所 === 94 === Smooth muscle contraction is regulated by increase in cytoplsmic Ca2+ and Ca2+ sensitivity of the contractile apparatus. The former activates myosin light chain kinase (MLCK) whereas the later mainly involves the inhibition of myosin light chain phosphatase (MLCP). In this study, the signaling mechanisms involved in the regulation of contraction activated by a1-adrenoceptor (a1-AR) agonsit phenylephrine (PE) and thromboxane A2 (TXA2) mimetic U46619 in rat tail artery (RTA) were investigated. The first part of this thesis explored the roles of extracellular signal-regulated kinase 1/2 (ERK1/2) in modulating [Ca2+]i, Ca2+ senstivity and MLC20 phosphorylation activated by both agonists. The results indicated that ERK1/2 modulate mainly Ca2+ sensitivity and, to a smaller extent, Ca2+ mobilization in smooth muscle contraction. In addition, ERK1/2 does not modulate Ca2+ sensitivity through changes in MLC20 phosphorylation. Furthermore, ERK1/2 activation induced by either agonist does not involve Rho-kinase or genistein-sensitive tyrosine kinases in RTA.
The second part of this thesis was aimed to clarify the relative contribution of MYPT1 and CPI-17 phosphorylation, as well as actin polymerization in Rho kinase-mediated contraction. The effects of Rho kinase inhibition on the time course of MLCP subunit MYPT1 phosphorylation at Thr697 and Thr855, CPI-17 phosphorylation at Thr38, and on actin polymerization during vasocontraction were examined. Rho-kinase inhibitor Y27632 suppressed force activated by PE or U46619 with concomitant decreases in MLC20 phosphorylation. Both agonists significantly increased MYPT1Thr855 phosphorylation that was abolished by Y27632 pretreatment but not MYPT1Thr697 phosphorylation. PE, but not U46619, transiently increased CPI-17Thr38 phosphorylation that was not inhibited by Y27632 but was abolished by a protein kinase C inhibitor. Both agonists increased actin polymerization that was attenuated by Y27632 under PE but not U46619 stimulation. These results demonstrated a temporal correlation between MYPT1Thr855 phosphorylation, MLC20 phosphorylation and contraction in a Rho-kinase-dependent manner for both agonists stimulation, suggesting that Rho-kinase regulates MLCP activity through MYPT1Thr855 phosphorylation during RTA smooth muscle contraction. Moreover, Rho-kinase regulates actin polymerization activated by a1-AR but not TXA2 receptor stimulation.
The third part of this thesis examined the role of NADPH oxidase-derived reactive oxygen species (ROS) in PE-induced contraction. Two different NADPH oxidase inhibitors, DPI and apocynin, dose-dependently inhibited PE-activated force. Using lucigenin-enhanced chemiluminescence assay, PE stimulated superoxide production, which was abolished by the pretreatment of a selective a1-AR antagonist prazosin, apocynin or superoxide scavenger tiron, but not by allopurinol or indomethacin. Concurrently, NADPH oxidase activity increased within 1 min upon PE treatment and was reduced by DPI and apocynin, but not by allopurinol or rotenone. Furthermore, MLC20 phosphorylation was dose-dependently decreased by apocynin and DPI at initial phase and by apocynin at sustained phase. Similar pattern was observed in MYPT1Thr855 phosphorylation. Finally, RhoA activation and the phosphorylation of MLCP inhibitor CPI-17 induced by PE were prevented by NADPH oxidase inhibition. This finding offers a new concept that NAD(P)H oxidase-derived ROS regulate a1-AR-activated vasoconstriction by, at least in part, modulating MLCP-mediated MLC20 phosphorylation through both RhoA/Rho kinase- and CPI-17-dependent pathways. In summary, results from this thesis provide new insight into the molecular mechanisms by which vasoconstrictors evoke smooth muscle contraction, and may help to better evaluate the potential targets for future antihypertensive therapy.
|
author2 |
Meei Jyh Jiang |
author_facet |
Meei Jyh Jiang Ming-Ho Tsai 蔡明和 |
author |
Ming-Ho Tsai 蔡明和 |
spellingShingle |
Ming-Ho Tsai 蔡明和 Regulatory Roles of ERK1/2, Rho-kinase and NADPH Oxidase-derived Reactive Oxygen Species in Excitation-Contraction Coupling in Vascular Smooth Muscle |
author_sort |
Ming-Ho Tsai |
title |
Regulatory Roles of ERK1/2, Rho-kinase and NADPH Oxidase-derived Reactive Oxygen Species in Excitation-Contraction Coupling in Vascular Smooth Muscle |
title_short |
Regulatory Roles of ERK1/2, Rho-kinase and NADPH Oxidase-derived Reactive Oxygen Species in Excitation-Contraction Coupling in Vascular Smooth Muscle |
title_full |
Regulatory Roles of ERK1/2, Rho-kinase and NADPH Oxidase-derived Reactive Oxygen Species in Excitation-Contraction Coupling in Vascular Smooth Muscle |
title_fullStr |
Regulatory Roles of ERK1/2, Rho-kinase and NADPH Oxidase-derived Reactive Oxygen Species in Excitation-Contraction Coupling in Vascular Smooth Muscle |
title_full_unstemmed |
Regulatory Roles of ERK1/2, Rho-kinase and NADPH Oxidase-derived Reactive Oxygen Species in Excitation-Contraction Coupling in Vascular Smooth Muscle |
title_sort |
regulatory roles of erk1/2, rho-kinase and nadph oxidase-derived reactive oxygen species in excitation-contraction coupling in vascular smooth muscle |
publishDate |
2006 |
url |
http://ndltd.ncl.edu.tw/handle/26160015207638919245 |
work_keys_str_mv |
AT minghotsai regulatoryrolesoferk12rhokinaseandnadphoxidasederivedreactiveoxygenspeciesinexcitationcontractioncouplinginvascularsmoothmuscle AT càimínghé regulatoryrolesoferk12rhokinaseandnadphoxidasederivedreactiveoxygenspeciesinexcitationcontractioncouplinginvascularsmoothmuscle AT minghotsai erk12rhokinasejínadphoxidaseyǎnshēngzhīhuóxìngyǎngwùzhìzàixuèguǎnpínghuájīxìngfènshōusuōliánjiézhīdiàokòngjiǎosè AT càimínghé erk12rhokinasejínadphoxidaseyǎnshēngzhīhuóxìngyǎngwùzhìzàixuèguǎnpínghuájīxìngfènshōusuōliánjiézhīdiàokòngjiǎosè |
_version_ |
1718284905293545472 |
spelling |
ndltd-TW-094NCKU53250092016-05-30T04:21:58Z http://ndltd.ncl.edu.tw/handle/26160015207638919245 Regulatory Roles of ERK1/2, Rho-kinase and NADPH Oxidase-derived Reactive Oxygen Species in Excitation-Contraction Coupling in Vascular Smooth Muscle ERK1/2,Rho-kinase及NADPHoxidase衍生之活性氧物質在血管平滑肌興奮-收縮聯結之調控角色 Ming-Ho Tsai 蔡明和 博士 國立成功大學 基礎醫學研究所 94 Smooth muscle contraction is regulated by increase in cytoplsmic Ca2+ and Ca2+ sensitivity of the contractile apparatus. The former activates myosin light chain kinase (MLCK) whereas the later mainly involves the inhibition of myosin light chain phosphatase (MLCP). In this study, the signaling mechanisms involved in the regulation of contraction activated by a1-adrenoceptor (a1-AR) agonsit phenylephrine (PE) and thromboxane A2 (TXA2) mimetic U46619 in rat tail artery (RTA) were investigated. The first part of this thesis explored the roles of extracellular signal-regulated kinase 1/2 (ERK1/2) in modulating [Ca2+]i, Ca2+ senstivity and MLC20 phosphorylation activated by both agonists. The results indicated that ERK1/2 modulate mainly Ca2+ sensitivity and, to a smaller extent, Ca2+ mobilization in smooth muscle contraction. In addition, ERK1/2 does not modulate Ca2+ sensitivity through changes in MLC20 phosphorylation. Furthermore, ERK1/2 activation induced by either agonist does not involve Rho-kinase or genistein-sensitive tyrosine kinases in RTA. The second part of this thesis was aimed to clarify the relative contribution of MYPT1 and CPI-17 phosphorylation, as well as actin polymerization in Rho kinase-mediated contraction. The effects of Rho kinase inhibition on the time course of MLCP subunit MYPT1 phosphorylation at Thr697 and Thr855, CPI-17 phosphorylation at Thr38, and on actin polymerization during vasocontraction were examined. Rho-kinase inhibitor Y27632 suppressed force activated by PE or U46619 with concomitant decreases in MLC20 phosphorylation. Both agonists significantly increased MYPT1Thr855 phosphorylation that was abolished by Y27632 pretreatment but not MYPT1Thr697 phosphorylation. PE, but not U46619, transiently increased CPI-17Thr38 phosphorylation that was not inhibited by Y27632 but was abolished by a protein kinase C inhibitor. Both agonists increased actin polymerization that was attenuated by Y27632 under PE but not U46619 stimulation. These results demonstrated a temporal correlation between MYPT1Thr855 phosphorylation, MLC20 phosphorylation and contraction in a Rho-kinase-dependent manner for both agonists stimulation, suggesting that Rho-kinase regulates MLCP activity through MYPT1Thr855 phosphorylation during RTA smooth muscle contraction. Moreover, Rho-kinase regulates actin polymerization activated by a1-AR but not TXA2 receptor stimulation. The third part of this thesis examined the role of NADPH oxidase-derived reactive oxygen species (ROS) in PE-induced contraction. Two different NADPH oxidase inhibitors, DPI and apocynin, dose-dependently inhibited PE-activated force. Using lucigenin-enhanced chemiluminescence assay, PE stimulated superoxide production, which was abolished by the pretreatment of a selective a1-AR antagonist prazosin, apocynin or superoxide scavenger tiron, but not by allopurinol or indomethacin. Concurrently, NADPH oxidase activity increased within 1 min upon PE treatment and was reduced by DPI and apocynin, but not by allopurinol or rotenone. Furthermore, MLC20 phosphorylation was dose-dependently decreased by apocynin and DPI at initial phase and by apocynin at sustained phase. Similar pattern was observed in MYPT1Thr855 phosphorylation. Finally, RhoA activation and the phosphorylation of MLCP inhibitor CPI-17 induced by PE were prevented by NADPH oxidase inhibition. This finding offers a new concept that NAD(P)H oxidase-derived ROS regulate a1-AR-activated vasoconstriction by, at least in part, modulating MLCP-mediated MLC20 phosphorylation through both RhoA/Rho kinase- and CPI-17-dependent pathways. In summary, results from this thesis provide new insight into the molecular mechanisms by which vasoconstrictors evoke smooth muscle contraction, and may help to better evaluate the potential targets for future antihypertensive therapy. Meei Jyh Jiang 江美治 2006 學位論文 ; thesis 143 en_US |