2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/659231摘要：研究指出血管平滑肌的收縮增強會增加總週邊阻力而造成高血壓。而血管平滑肌收縮主要受其細胞(VSMCs)內鈣離子濃度變化調控，但不完全取決於胞內鈣離子濃度的高低，肌原絲(myofilaments)對鈣離子的敏感性(Ca2+ sensitivity)扮演了另一個輔助角色。先前研究發現，VSMCs 若胞內Rho A/Rho kinase 訊息傳遞路徑活化增加，會因增加胞內Ca2+ sensitization 而導致細胞收縮增強。在自發性高血壓鼠(SHR)其血管平滑肌細胞內Rho A/Rho kinase 訊息傳遞路徑，相較於正常血壓鼠(WKY)對於Ca2+ sensitization 有明顯較多作用，因此造成VSMCs 收縮加強而增高血壓。Rho A 的上游是經由大G 蛋白連接之接受器(heterotrimeric Gprotein-coupled receptors) (GPCRs)，透過RhoGEFs 來使Rho A 活化。GPCRs 的agonists 透過Rho/Rho kinase-mediated Ca2+-independent signaling pathways，使VSMCs 內其他基因表現和DNA synthesis，造成蛋白合成，產生Cell migration以及Cell proliferation。血管張力素(Ang II)被認為跟許多心血管方面的疾病有所牽連，例如動脈硬化(atherosclerosis)和高血壓(hypertension)。原因被認為是Ang II透過作用在心臟及血管組織的細胞上之AT1 接受器，再經由heterotrimetric Gproteins，包括Gq/11，Gi，G12 或G13 等的作用，最後造成細胞及組織的重塑(remodeling)。其中，若作用在VSMCs，會造成VSMCs 的hypertrophy、proliferation、和migration，這些都將導致動脈硬化或高血壓。研究顯示Ang II也透過刺激VSMCs 內的小G 蛋白(small G proteins)，例如Ras、Rac、Cdc42 或Rho，藉由intracellular Ca2+-dependent 或-independent signaling pathways 兩類路徑作用，導致VSMCs hypertrophy，但能否不經由新生成蛋白而是直接造成VSMCs的Ca2+ sensitization 而增加收縮力(contractility)並不清楚。本實驗室在材料方法上將使用血壓相互對照的大白鼠品系SHR及WKY，其血管組織的primary cultured VSMCs來進行實驗。其好處在於比cell line接近於真實生物個體的細胞生理，可以直接測試Ang II在VSMCs中對於Rho A蛋白的作用情形。至於細胞膜內調控Rho A蛋白的上游路徑，先前用不同組織細胞的研究指出是PKCδ/PYK2/RhoGEF/Rho A，另外的研究結果卻認為是heterotrimeric Gprotein/ RhoGEF/Rho A；那麼RhoA其上游的調控在primary cultured VSMCs中的情形將是本實驗室欲探討的問題。使用此材料亦可就上述研究比較兩品系間的差異，了解本態性高血壓和正常血壓的生物個體其血管平滑肌細胞內細胞生理在本質上的差異。本實驗室初步結果顯示，Ang II造成Rho A蛋白的活性增加。另外 Rho A 的上游目前被認為可能有3 種RhoGEF，即p115-RhoGEF、LARG以及PDZ-RhoGEF，至於在primary cultured VSMCs 中，Rho A 的上游究是哪一種RhoGEF，其受Ang II 的調控為何？SHR 及WKY 兩品系間，RhoGEF 在細胞中作用的情形有否差異，甚或其基因啟動子功能(promoter function)有否差異？這也將是本實驗室欲探討的課題。希冀藉一連串的探索，讓Rho A/Rho kinase 訊息傳遞路徑上下游真實面貌更為明朗，對於造成原發性高血壓遺傳方面的諸多原因提出多一種可能性，以提供治療或控制血壓上的多一些參考建議。<br> Abstract: Studies have shown that vascular smooth muscle contraction increases totalperipheral resistance and result in hypertension. Then smooth muscle contraction isregulated by the cytosolic Ca2+ concentration and by the Ca2+ sensitivity of myofilamentsin the vascular smooth muscle cells (VSMCs). The small GTPase Rho and its target,Rho-associated kinase, participate in this latter mechanism. In spontaneouslyhypertensive rats (SHR), the contribution of RhoA/Rho kinase signaling pathway in Ca2+sensitivity of myofilaments inVSMCs is more than that in Wistar Kyoto rats (WKY).Angiotensin II (Ang II) is implicated in various cardiovascular diseases, such asatherosclerosis and hypertension. There are more than two G protein-coupled receptors(GPCRs) known to mediate Ang II function, namely, the AT1 and AT2 receptors (AT1R& AT2R). The AT1R has been shown to mediate most of the physiological actions of AngII, and it is predominantly expressed in cardiovascular cells, such as VSMCs. Throughthis receptor, Ang II activates a number of cytoplasmic signaling pathways. AT1Rinteracts with multiple heterotrimetric G proteins, including Gq/11, Gi, G12, and G13,lending to hypertrophy, migration and proliferation of VSMCs. It has also reported thatRhoA/Rho kinase involve in Ang II-induced hypertrophy, not due to Ca2+ sensitization, ofrat VSMCs. But the certain molecule that bind to and activates RhoA and associate withthe Ca2+ sensitivity of myofilaments in primary cultured VSMCs remain to bedetermined.The purpose of the present study is to identify the molecular expression of majorcomponents of induction of RhoA activity induced by Ang II in primary cultured VSMCsfrom WKY and SHR.