大鼠厚升支的管腔膜表达AT1受体和氨肽酶活性。

Poumarat Jean-Stéphane, Houillier Pascal, Rismondo Claude, Roques Bernard, Lazar George, Paillard Michel, Blanchard Anne

Laboratoire de Physiologie et Endocrinologie Cellulaire Rénale, Université Pierre et Marie Curie, Faculté de Médecine Broussais-Hotel Dieu, Paris, France.

Kidney Int. 2002 Aug;62(2):434-45. doi: 10.1046/j.1523-1755.2002.00453.x.

BACKGROUND

Endogenous intratubular angiotensin II (Ang II) supports an autocrine tonic stimulation of NaCl absorption in the proximal tubule, and its production may be regulated independently of circulating Ang II. In addition, endogenous Ang II activity may be regulated at the brush border membrane (BBM), by the rate of aminopeptidase A and N (APA and APN) activities and the rate of Ca2+-independent phospholipase A2 (PLA2-dependent endocytosis and recycling of the complex Ang II subtype 1 (AT1) receptor (AT1-R). The aim of the present study was to look for subcellular localization of AT1-R, and APA and APN activities in the medullary thick ascending limb of Henle (mTAL), as well as search for an asymmetric coupling of AT1-R to signal transduction pathways.

METHODS

Preparations of isolated basolateral membrane (BLMV) and luminal (LMV) membrane vesicles from rat mTAL were used to localize first, AT1-R by 125I-[Sar1, Ile8] Ang II binding studies and immunoblot experiments with a specific AT1-R antibody, and second, APA and APN activities. Microfluorometric monitoring of cytosolic Ca2+ with a Fura-2 probe was performed in mTAL microperfused in vitro, after apical or basolateral application of Ang II.

RESULTS

AT1-R were present in both LMV and BLMV, with a similar Kd (nmol/L range) and Bmax. Accordingly, BLMV and LMV preparations similarly stained specific AT1-R antibody. APA and APN activities were selectively localized in LMV, although to a lesser extent than those measured in BBM. In the in vitro microperfused mTAL, basolateral but not apical Ang II induced a transient increase in cytosolic [Ca2+].

CONCLUSIONS

Besides the presence of basolateral AT1-R in mTAL coupled to the classical Ca2+-dependent transduction pathways, AT1-R are present in LMV, not coupled with Ca2+ signaling, and co-localized with APA and APN activities. Thus, apical APA and APN may play an important role in modulating endogenous Ang II activity on NaCl reabsorption in mTAL.

背景

内源性肾小管内血管紧张素II（Ang II）支持近端小管中氯化钠重吸收的自分泌性紧张性刺激，其产生可能独立于循环中的Ang II进行调节。此外，内源性Ang II活性可能在刷状缘膜（BBM）处，通过氨肽酶A和N（APA和APN）的活性以及不依赖钙离子的磷脂酶A2（PLA2）的活性，以及Ang II亚型（AT1）受体复合物（AT1-R）的非钙离子依赖性内吞和再循环速率进行调节。本研究的目的是寻找AT1-R在亨氏髓袢升支粗段（mTAL）中的亚细胞定位，以及APA和APN的活性，并寻找AT1-R与信号转导通路的不对称偶联。

方法

使用从大鼠mTAL分离的基底外侧膜囊泡（BLMV）和管腔膜囊泡（LMV）制剂，首先通过125I-[Sar1，Ile8] Ang II结合研究和使用特异性AT1-R抗体的免疫印迹实验来定位AT1-R，其次定位APA和APN的活性。在体外微灌注的mTAL中，在从顶端或基底外侧应用Ang II后，使用Fura-2探针进行细胞溶质Ca2+的微荧光监测。

结果

AT1-R存在于LMV和BLMV中，具有相似的解离常数（nmol/L范围）和最大结合容量。因此，BLMV和LMV制剂对特异性AT1-R抗体的染色相似。APA和APN活性选择性地定位于LMV中，尽管程度低于在BBM中测得的活性。在体外微灌注的mTAL中，基底外侧而非顶端的Ang II诱导细胞溶质[Ca2+]短暂增加。