过刊浏览

蛋白激酶C--细胞外信号调节激酶1/2通路介导精氨酸升压素对成年大鼠心肌成纤维细胞的促增殖作用

何燕萍, 赵连友, 郑强荪, 刘少伟, 赵晓燕, 陆晓龙, 牛晓琳

第四军医大学唐都医院心血管内科.陕西,西安 710038

摘要

精氨酸升压素(arginine vasopressin, AVP)是高血压和心力衰竭时激活的神经体液和血流动力学因子，同时，它还具有直接的生长刺激作用。作者以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖。该研究旨在进一步观察AVP是否对成年大鼠CFs增殖具有促进作用及其机制。采用组织块法培养成年大鼠CFs，用[~(3)H]--TdR掺入法和流式细胞仪方法观察AVP作用下CFs的DNA合成和细胞周期分布。根据特异性底物髓磷脂基质蛋白(myelin basic protein, MBP)的磷酸化水平测定细胞外信号调节激酶1/2 (extracellular signal--regulated kinase 1/2, ERK1/2)的活性。用Western blot检测ERK1/2的磷酸化和p27~(Kip1)、细胞周期蛋白D1、A、E的表达。结果显示AVP (0.1 #mu#mol/L)可促进成年大鼠CFs的DNA合成，该作用可被V1受体拮抗剂d(CH_(2))_(5)[Tyr~(2)(Me), Arg~(8)]--vasopressin (0.1 #mu#mol/L)阻断，而不受V2受体拮抗剂desglycinamide--[d(CH_(2))_(5), D-Ile~(2), Ile~(4), Arg~(8)]--vasopressin (0.1 #mu#mol/L)的影响。AVP可激活ERK1/2，用蛋白激酶C (protein kinase C, PKC)激动剂佛波酯(phorbol 12--myristate 13--acetate, PMA, 30 nmol/L, 5 min)急性刺激可模拟该作用，而PMA持续慢性作用(2.5 #mu#mol/L, 24 h)耗竭PKC后则抑制AVP对ERK1/2的激活。AVP可抑制p27~(Kip1)的蛋白表达，升高细胞周期蛋白D1、A和E的表达，同时促进细胞周期由G_(0)/G_(1)期进入S期。ERK1/2抑制剂PD98059 (30 #mu#mol/L)阻断AVP对DNA合成、p27~(Kip1)、细胞周期蛋白D1、A和E的作用，并抑制细胞周期进程。以上结果表明，AVP可促进成年大鼠CFs增殖，该作用由PKC--ERK1/2通路介导。AVP可通过ERK1/2调控p27~(Kip1)、细胞周期蛋白D1、A和E的表达，从而促进成年大鼠CFs的细胞周期进程。

关键词： 精氨酸升压素; V1受体; 细胞外信号调节激酶1/2; 蛋白激酶C; p27~(Kip1); 细胞周期蛋白; 心肌成纤维细胞

Arginine vasopressin stimulates proliferation of adult rat cardiac fibroblasts via protein kinase C--extracellular signal--regulated kinase 1/2 pathway

He Yanping, Zhao Lianyou, Sun Qiangsun, Liu Shaowei, Zhao Xiaoyan, Lu Xiaolong, Niu Xiaolin

Department of Cardiology,Tangdu Hospital,the Fourth Military Medical University.Xi'an 710038,Shaanxi

Abstract

Arginine vasopressin (AVP), a neurohormone and hemodynamic factor, is implicated in the pathophysiology of hypertension and congestive heart failure, can also act as a growth-stimulating factor. Our previous work demonstrated that AVP is a mitogen for neonatal rat cardiac fibroblasts (CFs). In the present study, we extended our investigations to adult rat CFs to explore whether AVP could induce adult rat CF proliferation and, if so, to identify the mechanism involved. Adult rat CFs were isolated, cultured and subjected to AVP treatment. DNA synthesis and cell cycle distribution were analyzed by [~(3)H]-thymidine incorporation and flow cytometry. Cellular extracellular signal-regulated kinase 1/2 (ERK1/2) activity was measured by in vitro kinase assay using myelin basic protein (MBP) as a substrate. Protein expressions of total- and phospho- ERK1/2, p27~(Kip1), cyclin D1, A, E were assessed by Western blot. Results showed that AVP stimulated DNA synthesis in adult rat CFs, an effect abolished by the V1 receptor antagonist, d(CH_(2))_(5)[Tyr~(2)(Me), Arg~(8)]-vasopressin (0.1 #mu#mol/L), but not by the V2 receptor antagonist, desglycinamide-[d(CH_(2))_(5), D-Ile~(2), Ile~(4), Arg~(8)]-vasopressin (0.1 #mu#mol/L). AVP induced an activation of ERK1/2, which could be mimicked by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA, 30 nmol/L, 5 min), but abolished by depletion of PKC via chronic PMA incubation (2.5 #mu#mol/L, 24 h). In addition, AVP down-regulated protein expression of p27~(Kip1), increased expressions of cyclins D1, A and E, and induced cell cycle progression from G_(0)/G_(1) into S stage. Inhibition of ERK1/2 activation by PD98059 (30 #mu#mol/L) abolished the effect of AVP on DNA synthesis, protein expressions of p27~(Kip1), cyclins D1, A and E as well as cell cycle progression. These results suggest that AVP is also a growth factor for adult rat CFs. The mitogenic effect of AVP is mediated via the type 1 vasopressin receptor and PKC-ERK1/2 pathway. Moreover, AVP modulates the expressions of cell cycle regulatory proteins p27~(Kip1) and cyclins D1, A and E, which lie downstream of ERK1/2 activation, and induces cell cycle progression in adult rat CFs.

Key words: Arginine vasopressin;V1 receptor;Extracellular signal-regulated kinase 1/2;Protein kinase C;p27~(Kip1);cyclin;Cardiac fibroblast

收稿日期：　　录用日期：

通讯作者：　　E-mail:

引用本文：

何燕萍, 赵连友, 郑强荪, 刘少伟, 赵晓燕, 陆晓龙, 牛晓琳. 蛋白激酶C--细胞外信号调节激酶1/2通路介导精氨酸升压素对成年大鼠心肌成纤维细胞的促增殖作用[J]. 生理学报 2008; 60 (3): 333-340.

He Yanping, Zhao Lianyou, Sun Qiangsun, Liu Shaowei, Zhao Xiaoyan, Lu Xiaolong, Niu Xiaolin. Arginine vasopressin stimulates proliferation of adult rat cardiac fibroblasts via protein kinase C--extracellular signal--regulated kinase 1/2 pathway. Acta Physiol Sin 2008; 60 (3): 333-340 (in Chinese with English abstract).