Challah M, Nicoletti A, Arnal J F, Philippe M, Laboulandine I, Allegrini J, Alhenc-Gelas F, Danilov S, Michel J B
U-367 INSERM, Paris, France.
Cardiovasc Res. 1995 Aug;30(2):231-9.
Angiotensin converting enzyme (ACE) activity in the plasma does not change significantly with hypertension in two-kidney, one-clip hypertensive (2K-1C) rats. However, heart ACE activity and mRNA increase with hypertension. We measured the ACE activity and mRNA in hypertrophied hearts at different times after clipping, and determined the cellular distribution of its increase in the left ventricle of 2K-1C hypertensive rats.
Cardiac ACE activity was quantified in left and right ventricles using a radiolabeled synthetic ACE substrate, and ACE mRNA steady-state level was quantified by ribonuclease protection assay. Tissue localization of ACE in normal and hypertrophied hearts was determined by measuring ACE activity in isolated ventricular cells. In situ hybridization with a rat ACE cDNA and immunohistochemistry with a monoclonal anti-ACE antibody were used to identify tissue compartments producing ACE mRNA and protein.
The left ventricle was hypertrophied 2 weeks after clipping and remained hypertrophied at 12 weeks. Left ventricular ACE activity was significantly increased 2 and 4 weeks (3.2 +/- 0.3 in 2K-1C vs. 1.7 +/- 0.1 pmol/mg prot/min in sham-operated rat) after renal artery clipping, but not at 12 weeks. The right ventricle was slightly hypertrophied 4 weeks after clipping and remained hypertrophied at 12 weeks. Right ventricular ACE activity was significantly increased at 4 (6.7 +/- 0.6 in 2K-1C vs. 3.1 +/- 0.3 pmol/mg prot/min in sham-operated rat) and 12 weeks. ACE activity was not detectable in cardiomyocytes isolated by Percoll gradient. Neither was ACE mRNA detected in isolated cardiomyocytes, even after ACE mRNA amplification by RT-PCR. In contrast, ACE activity and mRNA were detected in pooled non-cardiomyocytic cells. Thus the increase in cardiac ACE activity associated with hypertension must be due to an increase in ACE expression by non-cardiomyocytic cells. In situ hybridization showed an autoradiographic signal for ACE mRNA over the endothelial cells of coronary arteries and over the interstitial spaces including pericoronary and fibrosis areas. Immunohistochemistry confirmed these data, showing ACE on endothelial cells and in pericoronary spaces with an increased signal in pericoronary and fibrosed areas in hypertensive hypertrophied left ventricle.
Besides its usual endothelial expression, ACE is absent from cardiomyocytes and present in interstitial tissue, in the pericoronary spaces in normal tissue and more markedly in hypertrophied ventricles.
在二肾一夹高血压（2K-1C）大鼠中，血浆中的血管紧张素转换酶（ACE）活性不会随高血压而发生显著变化。然而，心脏ACE活性和mRNA会随高血压而增加。我们测量了夹闭后不同时间点肥厚心脏中的ACE活性和mRNA，并确定了2K-1C高血压大鼠左心室中其增加的细胞分布。
使用放射性标记的合成ACE底物对左、右心室的心脏ACE活性进行定量，并通过核糖核酸酶保护试验对ACE mRNA稳态水平进行定量。通过测量分离的心室细胞中的ACE活性来确定正常和肥厚心脏中ACE的组织定位。使用大鼠ACE cDNA进行原位杂交以及使用抗ACE单克隆抗体进行免疫组织化学，以鉴定产生ACE mRNA和蛋白质的组织区域。
夹闭后2周左心室肥厚，并在12周时仍保持肥厚。肾动脉夹闭后2周和4周时左心室ACE活性显著增加（2K-1C组为3.2±0.3，假手术大鼠为1.7±0.1 pmol/mg蛋白/分钟），但在12周时未增加。夹闭后4周右心室轻度肥厚，并在12周时仍保持肥厚。右心室ACE活性在4周（2K-1C组为6.7±0.6，假手术大鼠为3.1±0.3 pmol/mg蛋白/分钟）和12周时显著增加。通过Percoll梯度分离的心肌细胞中未检测到ACE活性。即使通过RT-PCR扩增ACE mRNA后，在分离的心肌细胞中也未检测到ACE mRNA。相反，在汇集的非心肌细胞中检测到了ACE活性和mRNA。因此，与高血压相关的心脏ACE活性增加必定是由于非心肌细胞中ACE表达增加所致。原位杂交显示冠状动脉内皮细胞以及包括冠状动脉周围和纤维化区域在内的间质空间中有ACE mRNA的放射自显影信号。免疫组织化学证实了这些数据，显示内皮细胞和冠状动脉周围空间中有ACE，在高血压肥厚左心室的冠状动脉周围和纤维化区域信号增强。
除了其通常在内皮细胞中的表达外，ACE在心肌细胞中不存在，存在于间质组织、正常组织的冠状动脉周围空间中，在肥厚心室中更为明显。
