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1、 Received 2000201231 Accepted 20002072153Supported by the Science Foundation of Guangdong Province (No1960124)33Corresponding author. Tel : 020287330639; E2mail : jing gzsums1edu1cn450 Acta Physiologica SinicaDec. 2000 , 52 (6) , 450454Nitric oxide inhibits the expression of proto2oncogen
2、ec2fosinduced by angiotensinand endothelin21 in cardiomyocytes3ZHAN Chang2De ,PAN Jing2Yun133( Key Laboratory of the Ministry of Health on Assisted Circulation ,1Department of Physiology ,Sun Yat2sen University of Medical Sciences , Guangzhou510089)Abstract : The effect of nitric oxide (NO) on the h
3、ypertrophic response and the proto2oncogenec2fosexpression induced by an2giotensin(A) or endothelin21 (ET21) was investigated in the primary culture of neonatal rat cardiomyocytes. T otal proteincontent of cardiomyocytes (used as the index of cardiac myocyte hypertrophy) was determined by the Bradfo
4、rd method. The proto2oncogenec2fosexpression was assessed using reverse transcription2polymerase chain reaction (RT2PCR) standardized with glycer2aldehyde232phosphate dehydrogenase (G APDH) . RT2PCRwas performed in a single tube using gene2specific primers and the Super2Script One2Step RT2PCR System
5、. T otal protein content of cardiomyocytes increased significantly on day 5 after Atreatment or onday 3 after ET21 treatment and the increased protein content was inhibited by SNP (NO donor) . A, ET21 and PMA (protein ki2nase C activator) induced thec2fosgene expression of cardiomyocytes , while L2a
6、rginine inhibited it. The L2arginine effect wasblocked by L2NAME (NOS inhibitor) . SNP inhibited thec2fosgene expression of cardiomyocytes induced by A,ET21 or PMA aswell. These results suggest that NO can inhibit the hypertrophic response and the proto2oncogenec2fosexpression of cardiomyocytesinduc
7、ed by Aor ET21 and the cross2link may be located at the site of protein kinase C.Key words : nitric oxide ; angiotensin; endothelin21;c2fos; protein kinase C一氧化氮抑制血管紧张素 和内皮素21诱导的 心肌细胞原癌基因c2fos表达3詹昌德,潘敬运133(中山医科大学卫生部辅助循环重点实验室;1生理学教研室,广州510089)摘 要: 在原代培养的新生大鼠心肌细胞上,探讨一氧化氮(NO)对血管紧张素 (A)和内皮素21 (ET
8、21)诱导的心肌细胞肥大和原癌基因c2fos表达的影响。用Bradford法测定心肌细胞总蛋白含量(作为心肌细胞肥大的指标) ;用基因特异性引物和SuperScript一步法进行逆转录聚合酶链式反应(RT2PCR) ,检测大鼠心肌细胞原癌基因c2fos的表达(以G APDH为内标)。结果显示, A 和ET21分别作用5 d和3 d后,心肌细胞总蛋白含量显著增加;硝普钠(NO供体)可抑制A 或ET21诱导的心肌细胞总蛋白增加。A,ET21和PMA (蛋白激酶C激动剂)均可诱导心肌细胞原癌基因c2fos的表达; L2精氨酸可抑制A,ET21和PMA诱导心肌细胞原癌基因c2fos的表达, L2NAM
9、E(NOS抑制剂)可抑制L2精氨酸的这一作用;硝普钠对可抑制A,ET21和PMA诱导心肌细胞原癌基因c2fos的表达。结果表明, NO可抑制A 或ET21诱导的心肌细胞肥大和原癌基因c2fos表达,其作用机制可能与蛋白激酶C这一环节有关。关键词:一氧化氮;血管紧张素 ;内皮素21;c2fos;蛋白激酶C学科分类号: Q463; R331136Generally , the initiation of cardiac hypertrophy isclosely related to the abnormal expression of proto2onco2genes1. Cardiacc2fo
10、sgene expression is induced within30 min by pressure overload2, which is involved in theaugmented release of angiotensin(A) and endothe2lin21 (ET21) ; A3and ET214themselves can inducethec2fosgene expression independent of hemodynamicfactors.Our previous investigation showed that nitric ox2 1995-2005
11、 Tsinghua Tongfang Optical Disc Co., Ltd. All rights reserved.ide (NO) played an important role in the cardiac hypertro2phy induced by pressure overload5or A6. The pur2pose of the present study was to investigate the effect ofNO on the hypertrophic response and the proto2oncogenec2fosexpressi
12、on induced by Aor ET21 in the culturedneonatal rat cardiomyocytes.1 MATERIALS AND METHODS111 Primary neonatal rat cardiac myocyte cultures Primary cultures of cardiomyocytes from neonatal rats wereprepared by the method described previously6 ,7. Briefly ,the hearts from 132day2old SD rats were
13、 minced into 1mm3pieces and dissociated with 011 % trypsin. After dis2persion the cells were plated on 1002mm culture dishes for1 h at 37in 5 % CO2, and then the nonattached viablecells were collected and plated into 252ml flask (3106cells/fla) . 52Bromodeoxyuridine (BrdU , 011 mmol/L)was added duri
14、ng the first 2 days to prevent proliferation ofnonmyocytes.Cardiomyocytes were incubated in M199supplemented with 10 % fetal bovine serum for 48 hoursand then replaced by serum2free medium.112 Measurement of protein content Cardiomyocyteswere collected and re2suspended in PBS. 1 ml sampleswere
15、 homogenized and 50l homogenized solutions wereused for the assay of total protein content. T otal proteincontent of cardiomyocytes was determined by the Bradfordmethod , using crystalline bovine serum albumin (Sigma)as standard.113 Total RNA isolation of cardiomyocytes T otalRNA from approxim
16、ately 3106cardiomyocytes was ex2tracted by the single2step method using TRIZOL Reagent(Life Technologies , Inc , USA) . The concentration andpurity of isolated RNA were measured by ultraviolet spec2trophotometer and repeated three times. The isolated RNAhad an OD260/ 280 ratio of 116118.114 Primers Thec2fosprimers for reverse trans2cription2polymerase chain reaction (RT2PCR) were usedpreviously by Yanget al.8(forward , 5 2GGT CAT CGGGG A TCT TGC23; backward , 5 2GGG CTC TCC TG
