CAJ | 학술논문

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.我们以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖.本研究旨在进一步观察AVP是否对成年大鼠CFs具有促增殖作用,并探计其机制.采用组织块法培养成年大鼠CFs,用[3H]-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p27Kip1、细胞周期蛋白D1、 A、 E的表达.结果显示,AVP(0.1μmol/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(CH2)5[Tyr2(Me),Arg8]-vasopressin (0.1μmol/L)阻断,而不受V2受体拮抗剂desglycinamide [d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin (0.1μmol/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, 30nmol/L, 5min)急性刺激可模拟该作用,而PMA持续慢性作用(2.5μmol/L,24h)耗竭PKC后则抑制AVP对ERK1/2的激活.AVP可抑制p27Kip1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G0/G1期进入S期.ERK1/2抑制剂PD98059 (30μmol/L)阻断AVP对DNA合成、p27Kip1、细胞周期蛋白D1、A和E蛋白表达的作用,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V1受体和PKC-ERK1/2通路介导.AVP可通过ERK1/2调控p27Kip1、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.
정안산승압소(arginine vasopressin, AVP)시고혈압화심력쇠갈시격활적신경체액화혈류동역학인자,동시,타환구유직접적생장자격작용.아문이왕적연구현시AVP가유도신생대서심기성섬유세포(cardiac fibroblasts, CFs)증식.본연구지재진일보관찰AVP시부대성년대서CFs구유촉증식작용,병탐계기궤제.채용조직괴법배양성년대서CFs,용[3H]-TdR참입법화류식세포의방법관찰AVP작용하CFs적DNA합성화세포주기분포.근거특이성저물수린지기질단백(myelin basic protein, MBP)적린산화수평측정세포외신호조절격매1/2 (extracellular signal-regulated kinase 1/2, ERK1/2)적활성.용Western blot검측ERK1/2적린산화화p27Kip1、세포주기단백D1、 A、 E적표체.결과현시,AVP(0.1μmol/L)가촉진성년대서CFs적DNA합성,해작용가피V1수체길항제d(CH2)5[Tyr2(Me),Arg8]-vasopressin (0.1μmol/L)조단,이불수V2수체길항제desglycinamide [d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin (0.1μmol/L)적영향.AVP가격활ERK1/2,용단백격매C(protein kinase C, PKC)격동제불파지(phorbol 12-myristate 13-acetate, PMA, 30nmol/L, 5min)급성자격가모의해작용,이PMA지속만성작용(2.5μmol/L,24h)모갈PKC후칙억제AVP대ERK1/2적격활.AVP가억제p27Kip1적단백표체,승고세포주기단백D1、 A화E적표체,동시촉진세포주기유G0/G1기진입S기.ERK1/2억제제PD98059 (30μmol/L)조단AVP대DNA합성、p27Kip1、세포주기단백D1、A화E단백표체적작용,병억제세포주기진정.이상결과표명,AVP가촉진성년대서CFs증식,해작용유V1수체화PKC-ERK1/2통로개도.AVP가통과ERK1/2조공p27Kip1、세포주기단백D1、A화E적표체,종이촉진성년대서CFs적세포주기진정.
Arginine vasopressin (AVP), a neurohormone and hemodynamic factor 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 [3H]-thymidine incorporation and flow cytometry. Cellular extracellular signal-regulated kinase 1/2 (ERK 1/2) activity was measured by in vitro kinase assay using myelin basic protein (MBP) as a substrate. Protein expressions of total-and phospho-ERK1/2, p27Kip1, cyclins D1, A, E were assessed by Western blot. The results showed that AVP stimulated DNA synthesis in adult rat CFs, and the effect was abolished by a V1 receptor antagonist, d(CH2)5[Tyr2(Me),Arg8-vasopressin (0.1μmol/L), but not by a V2 receptor antagonist, desglycinamide-[d(CH2)5, D-Ile2, Ile4, Arg8-vasopressin (0.1μmol/L). AVP induced an activation of ERK1/2, which could be mimicked by the protein kinase C (PKC) activator, phorbol 12-myfistate 13-acetate (PMA, 30nmol/L, 5min), but abolished by depiction of PKC via chronic PMA incubation (2.5μmol/L,24h). In addition, AVP down-regulated protein expression of p27Kip1, increased protein expressions of cyclins D1, A and E, and induced cell cycle progression from G0/G1 into S stage. Inhibition of ERK1/2 activation by PD98059 (30μmol/L) abolished the effect of AVP on DNA synthesis, protein expressions of p27Klp1, 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 V1 receptors and PKC-ERK 1/2 pathway. Moreover, AVP modulates the expressions of cell cycle regulatory proteins p27Kip1 and cyclins D1, A and E, which lie downstream of ERK 1/2 activation, and induces cell cycle progression in adult rat CFs.