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目的:研究血管紧张素Ⅱ对心肌细胞Bcl-2和c-myc基因转录的调控作用及其受体机制。方法:分离纯化培养的乳鼠心肌细胞，分别加入血管紧张素Ⅱ或/和氯沙坦（血管紧张素Ⅱ的I型受体特异性拮抗剂）处理，然后提取总RNA，用Northern杂交检测Bcl-2和c-myc基因的转录水平。结果分析时，以正常乳鼠心肌细胞中Bcl-2 mRNA/18S或c-myc mRNA/18S的比值定为100%。结果：血管紧张素Ⅱ10-5 mol·L-1处理24小时使Bcl-2基因转录水平下调至正常的26%(P＜0.01,n=3)。血管紧张素Ⅱ的处理时间短至12小时或血管紧张素Ⅱ浓度为10-7 mol·L-1或10-6 mol·L-1时均无此作用，表明血管紧张素Ⅱ使心肌细胞Bcl-2基因转录下调具有时间和剂量依赖性。氯沙坦10-5mol·L-1能完全取消10-5 mol·L-1血管紧张素Ⅱ对心肌细胞Bcl-2基因转录下调的作用(103±4%,P＜0.01,n=3)，单独用10-6 mol·L-1或10-5 mol·L-1的氯沙坦对心肌细胞Bcl-2基因转录无影响。另一方面，10-5 mol·L-1血管紧张素Ⅱ处理2小时，对心肌细胞c-myc基因转录具有显著上调作用(340±70 %,P＜0.01,n=3 )。其上调作用可为10-5 mol·L-1的氯沙坦阻断(120±20%,P＜0.01,n=3)。结论:血管紧张素Ⅱ主要通过AT1受体使心肌细胞Bcl-2基因转录下调，c-myc基因转录上调。
要点：AngⅡ有使培养心肌细胞Bcl-2基因下调和c-myc基因上调的作用，同时这种作用可被氯沙坦(AT1受体阻滞剂)所阻断，说明AngⅡ是通过AT1受体起作用的。
中图分类号：Q555;Q753;R331.3文献标识码：A
文章编号：1006-2866(1999)02-0272-04
Effects of AngiotensinⅡ on the Regulation of Bcl-2 and c-myc Genes Transcription in Cultured neonatal Rat Cardiac Myocytes*
CHEN Tiehua(陈铁骅),LING Qi(凌琦)
(P.O.Box 5,Department of Molecular Pharmacology,Hunan Medical university,Changsha,410078)
ABSTRACT Aim:To investigate the effects of angiotensin Ⅱ(Ang Ⅱ) on the regulation of Bcl-2 and c-myc genes transcription in cardiac myocytes and its receptor mechanism.Methods:The cultured neonatal rat cardiac myocytes were treated with Ang Ⅱ and losartan (a specific antagonist of AT1 receptor).Then total RNA was isolated,the level of Bcl-2 and c-myc mRNA was detected by Northern blot.The ratio of Bcl-2 mRNA/18S and c-myc mRNA/18S in control group was set to 100 %.Results:Incubation of cultured neonatal rat cardiac myocytes with 10-5 mol·L-1 ang Ⅱ for 24 hours elicited the down-regulation of Bcl-2 gene transcription to26 % of control ( P＜ 0.01,n=3 ),whereas the above down-regulation change was not observed with a treatment period shorter than 12 hours or at a dose lower than 10-6 mol·L-1 Ang Ⅱ.Losartan (10-6 mol·L-1 and 10-5 mol·L-1) had no direct effects on the regulation of Bcl-2 gene transcription in cardiac myocytes,but it can suppress the down-regulation induced by Ang Ⅱ (10-5mol·L-1).After the treatment of cultured neonatal rat cardiac myocytes with 10-5mol·L-1 ang Ⅱfor 2 h,the transcription of c-myc gene was up-regulated by 340± 70%,P＜ 0.01,n= 3.This effect can be abolished by 10-5 mol·L-1 losartan (120± 20 % vs 340 ± 70 %,P＜0.01,n=3).Conclusion:The results indicate that the down-regulation of the Bcl-2 gene transcription and enhancement of c-myc gene transcription by AngⅡwere mainly mediated by AT1 receptor.
Key Words:angiotensinⅡ;cardiac myocytes;rat;Bcl-2 gene;c-myc gene;transcription regulation;losartan
Two subtypes of AngⅡ receptor AT1 and AT2 have been identified in many tissues［1,2］ with specific distribution and different functions.Most of the known functional effects of Ang Ⅱ,including vascular tone and blood pressure regulation and aldosterone release,are mediated by the AT1 ［3］.AngⅡ,via activation of protein kinase C and modulation of cytosolic calcium［4］,activates the immediate-early genes (nuclear proto-oncogene c-fos and c-myc) transcription,which has been postulated to contribute to the regulation of cell proliferation in cardiac muscle［5,6］.However,the role of Ang Ⅱ on the regulation of Bcl-2 gene transcription remains to be defined.The present study was aimed to examine the roles of Ang Ⅱ on the regulation of Bcl-2 and c-myc gene transcription and its receptor mechanism.
METHODS
1 Cardiac myocytes culture:Primary cultures of the neonatal rat cardiac myocytes were prepared by previously published method.In brief,cells were obtained from the hearts of 1-2-d-old neonatal rats by 0.1 % trypsinization,and then incubated on 60 mm culture dishes for 90 min at 37℃ in 5 % CO2,until the non-myocytes attached readily to the bottom of the culture dish.The resultant suspension of myocytes were collected and plated onto 24 well plates (2.5×105 cells/well).Cardiac myocytes were incubated in M199 supplemented with 10 % calf serum and 100 mmol·L-1 bromodeoxyuridine for 48 h,and then replaced with serum-free medium (M199-2) supplemented with insulin 10 mg·L-1,transferrin 10 mg·L-1,hydrocortisone0.05 mg·L-1 ,thyro0.1 mg·L-1,vitamin b12 1.5 mmol·L-1,vitamin C 100 mmol·L-1.All experiments were performed in the serum-free condition 24 h thereafter.
2 RNA isolation：Total cellular RNA was extracted from myocytes with Life-Times total RNA isolation reagent (LTTRI):In brief,1 ml LTTRI was added into 4 wells of the culture dish,transferred to an Eppendorf tube.Then,0.1 ml of chloroform-iso-amyl alcohol mixture (49 :1) was added to the eppendorf tube.Samples were centrifuged at 10 000×g for 10 min.The aqueous phase was transferred to a fresh Eppendorf tube,mixed with 0.5 ml of pre-cooled isopropanol,and then was centrifuged again at 10 000×g for 10 min.The RNA pellet was washed with 75 % ethanol,air dried for 10 min,and dissolved in 20～30μl water (DEPC treated).All procedures were carried out at room temperature.RNA samples were electrophoresed on a 1 % agarose/6 % formaldehyde gel at 50 V for 2 h and transferred to a nylon membrane (Gene Screen Plus Co.).
3 Probe labelling:The oligonucleotide probe was end-labeled with α-32P-dATP at 5' end using T4 polynucleotide kinase according to the method described［7］.
4 Northern hybridization:The membrane was pre-hybridized in a solution containing 50 % formamide,5×Denhart's solution (containing ficoll,polyvinylpyrrolidone,and bovine serum albumin,1 mg·mL-1 each),5×SSPE (containing 0.75 mol·L-1 sodium chloride,50 mmol·L-1 sodium phosphate,and 5 mmol·L-1 eDTA),1 % sodium dodecyl sulfate,30 mg·mL-1 denatured salmon sperm DNA at 42 ℃ for 2 h.Then,the membrane was hybridized in the same solution with32P-labeled oligonucleotide probe at 42 ℃for4 h.The membrane was washed twice with 2×SSPE/0.1 % sodium dodecyl sulfate(SDS) for 5 min at room temperature,and once with 0.1×SSPE/0.1 % SDS for 10 min at 45 ℃.Autoradiography was performed on Kodak omit x-ray film with an intensifying screen at -30 ℃,and then quantitated by densitometry(CS-930,Japan).
5Statistics:The data were expressed as
