The luminal membrane of rat thick limb expresses AT1 receptor and aminopeptidase activities.
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.
Poumarat
Jean-Stéphane
JS
Laboratoire de Physiologie et Endocrinologie Cellulaire Rénale, Université Pierre et Marie Curie, Faculté de Médecine Broussais-Hotel Dieu, Paris, France.
Houillier
Pascal
P
Rismondo
Claude
C
Roques
Bernard
B
Lazar
George
G
Paillard
Michel
M
Blanchard
Anne
A
D015059:1-Sarcosine-8-Isoleucine Angiotensin II / Q000378:metabolism / Q000494:pharmacology; D000626:Aminopeptidases / Q000378:metabolism*; D000804:Angiotensin II / Q000378:metabolism; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000918:Antibody Specificity; D018826:CD13 Antigens / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D016764:Cell Polarity / Q000502:physiology; D043384:Glutamyl Aminopeptidase; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008138:Loop of Henle / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000276:immunology / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism
D000806:Angiotensin-Converting Enzyme Inhibitors; D007457:Iodine Radioisotopes; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D012965:Sodium Chloride; D015059:1-Sarcosine-8-Isoleucine Angiotensin II; D000626:Aminopeptidases; D018826:CD13 Antigens; D043384:Glutamyl Aminopeptidase; D002118:Calcium