Polarized rabbit type 1 angiotensin ii receptors expressed in llc
-pk cells manifest differential rates of endocytosis and
recycling.
Becker, B. N., H-F Cheng, K. D. Burns, and R. C. Harris.
Department of Medicine, Vanderbilt University School of Medicine,
and the Department of Veterans Affairs Medical Center, Nashville, TN
37232 and the Departments of Medicine and Physiology, University of
Ottawa, Ottawa, Ontario, Canada K1H 8M5
APStracts 2:0216C, 1995.
Receptor-mediated endocytosis and receptor recycling have been
described for extra-renal angiotensin II (Ang II) receptors. However,
in proximal tubule epithelial cells, in which Ang II receptors have
been identified at brush border (BBM) and peritubular membranes, Ang
II receptor endocytosis and recycling have not been examined as
thoroughly. We utilized a proximal tubule cell model to investigate
these receptor-associated properties. LLCPKCl4 cells stably
transfected with rabbit type 1 Ang II receptor cDNA (LLCPK-AT1R)
expressed type 1 Ang II receptors (AT1R), as demonstrated by reverse
transcription polymerase chain reaction and Northern blot analysis.
LLCPK-AT1R cells, grown on permeable supports, displayed losartan
-inhibitable specific 125I-Ang II binding at apical and basolateral
membranes. Assays for internalized 125I-Ang II demonstrated that
apical (AP) AT1R internalized 125I-Ang II more rapidly than
basolateral (BL) AT1R (AP 95 +/- 7% vs BL 37.5 +/- 7% at 10 min; n=6
-8; p < 0.005). Phenylarsine oxide (PAO) treatment inhibited AP AT1R
internalization (83 +/- 12%; n=7; p < 0.025 vs. apical control);
whereas PAO had no significant effect on basolateral 125I-Ang II
internalization (33 +/- 20%; n=6; p > 0.375 vs. basolateral
control, N.S.). Pertussis toxin treatment also had no effect on AP or
BL AT1R endocytosis. In addition, AP AT1R completely recovered
specific 125I-Ang II binding after Ang II pre-treatment (a measure of
receptor recycling) (127 +/- 26% at 40 min; n=4; p < 0.005 vs. AP
time 0). In contrast, BL AT1R demonstrated little recovery of
specific 125I-Ang II binding (53 +/- 13 % at 40 min; n=3; p < 0.2
vs. BL time 0, N.S.). These data suggested that AP AT1R enter
endocytic/endosomal pathways. Phospholipase A2 (PLA2) activity has
been linked to endosomal fusion in other preparations. In addition,
proximal tubule BBM AT1R also have been associated with PLA2
activity. Therefore, LLCPK-AT1R cells were treated with quinacrine
(quin), a non-specific PLA2 inhibitor, or Compound I (CI), a
selective inhibitor of a calcium-independent PLA2, to determine if
PLA2 activity was involved in AT1R recycling. With treatment, AP AT1R
recycling decreased (control: 68.2 +/- 12.2%; quin: 24.2 +/- 11%; CI:
12 +/- 6.5%; n=4-5; p < 0.025). BL AT1R recycling however did not
change significantly vs. control (n=3-4 for each). Polarized AT1R
expressed in LLCPKCl4 cells display differential rates of endocytosis
and recycling. PLA2 activity appears to be preferentially associated
with AP AT1R recycling. In addition, the inhibition of AP AT1R
recycling suggests the possible involvement of calcium-independent
PLA2 activity in this process.
Received 15 December 1994; accepted in final form 4 May 1995.
APS Manuscript Number C718-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 30 May 1995.