0022-3565/99/2913-1242$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 291:1242–1249, 1999
Vol. 291, No. 3
Printed in U.S.A.
Characterization and Function of the Bovine Kidney Epithelial
Angiotensin Receptor Subtype 4 Using Angiotensin IV and
Divalinal Angiotensin IV as Receptor Ligands1
RAJASH K. HANDA, JOSEPH W. HARDING, and STEVEN M. SIMASKO
Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Washington State
University, Pullman, Washington
Accepted for publication July 26, 1999
This paper is available online at http://www.jpet.org
The renin-angiotensin system is composed of a cascade of
biochemical reactions that have a pivotal role in the homeostatic regulation of the internal environment of organisms.
The numerous actions of the renin-angiotensin system
throughout the body are likely due to the formation of angiotensin (Ang) II and several shorter Ang II fragments [e.g.,
Ang(2-8), Ang(1-7), and Ang(3-8)] that can act on Ang II
receptor subtype 1 (AT1), AT2, or atypical (non-AT1, non-AT2)
AT receptor subtypes to elicit biological responses (Ardaillou
and Chansel, 1998). One such atypical AT receptor is the AT4
receptor, which demonstrates high affinity and specificity for
Ang(3-8) [Ang IV]. This novel AT receptor is distributed in
organs throughout the body, present in a plethora of species
ranging from crab to human (Swanson et al., 1992; Wright et
al., 1995; Delorenzi et al., 1997), and appears to be involved
in diverse functions, including learning and memory (Wright
et al., 1995; Delorenzi et al., 1997; Pederson et al., 1998), cell
growth (Hall et al., 1995; Wang et al., 1995; Moeller et al.,
Received for publication March 17, 1999.
1
This work was supported by the American Heart Association, Washington
Affiliate (to R.K.H.).
nM to 1 mM) caused a concentration-dependent rise in intracellular calcium concentration levels with a reduced calcium
response observed with Ang IV at micromolar concentrations.
These results indicate that Ang IV and divalinal Ang IV bind with
high affinity to the same receptor and that the MDBK AT4
receptor is not coupled to a classic G protein, nor are sulfhydryl
bonds important in regulation of receptor affinity. The MDBK
AT4 receptor appears to be pharmacologically similar to that
described in nonrenal tissues. Functional studies suggest that
AT4 receptor activation can increase intracellular calcium concentration levels in MDBK cells and that divalinal Ang IV possesses agonist activity with respect to this particular intracellular signaling system.
1996; Pawlikowski and Kunert-Radek, 1997), angiogenesis
(J. W. Harding, unpublished results), fibrinolysis (Kerins et
al., 1995), extracellular matrix remodeling (Kakinuma et al.,
1998), anti-apoptosis (Kakinuma et al., 1997), blood flow
regulation (Harberl et al., 1991; Krebs et al., 1996; Coleman
et al., 1998; Patel et al., 1998), and solute transport (Handa
et al., 1998). These findings reveal an evolutionary conserved
AT4 receptor that is likely to be physiologically important.
Elucidation of some of these AT4-dependent functions has
relied on the ability of the putative partial nonpeptide AT4
receptor antagonist divalinal Ang IV to interact with the
same receptor sites as Ang IV (Krebs et al., 1996; Handa et
al., 1998) and to selectively block the actions of Ang IV
(Kerins et al., 1995; Coleman et al., 1998; Handa et al., 1998;
Patel et al., 1998). Despite the growing use of divalinal Ang
IV as a tool to elucidate the physiology of the AT4 receptor
system, no study has yet characterized both the binding and
functional properties of divalinal Ang IV in the same tissue
or cell type.
AT4 receptors are abundant in the kidney and have been
shown to be expressed in cultured rat mesangial cells
ABBREVIATIONS: Ang, angiotensin; MDBK, Mardin-Darby bovine kidney; AT receptor, angiotensin receptor; GTPgS, guanosine-59-O-(3thio)triphosphate; [Ca21]i, intracellular calcium concentration.
1242
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ABSTRACT
125
I-Angiotensin (Ang) IV and 125I-divalinal Ang IV [AT receptor
subtype 4 (AT4)] receptor agonist and putative antagonist, respectively] were used to characterize the AT4 receptor in Mardin-Darby bovine kidney epithelial cells (MDBK cell line). Both
125
I-Ang IV and 125I-divalinal Ang IV bound to a single highaffinity site (KD 5 1.37 and 1.01 nM, respectively) and to a
comparable density of binding sites (Bmax 5 1335 and 1407
fmol/mg protein, respectively). Competition of either radiolabeled ligand with several Ang related peptides demonstrated
similar displacement affinities in the following affinity order: Ang
IV 5 divalinal Ang IV . Ang III . Ang II . losartan 5 PD 123177.
Guanosine-59-O-(3-thio)triphosphate or sulfhydryl reducing
agents did not affect the binding of either radiolabeled ligand.
Brief exposure of MDBK cells to Ang IV or divalinal Ang IV (0.1
1999
Bovine Kidney Epithelial AT4 Receptor
Materials and Methods
Cell Culture. MDBK cells are a distal tubular-like epithelial cell
line (Ishikawa et al., 1978; Gagnon et al., 1994). The cells were grown
in an atmosphere of 95% air/5% CO2 at 37°C in Dulbecco’s modified
Eagle’s medium supplemented with 5% FBS, 5% calf bovine serum,
50 IU/ml penicillin, 50 mg/ml streptomycin, and 5 mg/ml amphotericin B. Cultures were re-fed with fresh media every 2 days. All the
experiments presented in this study were performed at passages 3 to
12 on confluent cells that had been cultured for 5 to 7 days.
Cell Membrane Preparation. Confluent MDBK cells grown in
75-cm2 flasks were washed once with ice-cold PBS followed by the
addition to the flask of 2 ml of ice-cold isotonic buffer [150 mM NaCl,
50 mM tris(hydroxymethyl)aminomethane, 50 mM Plummer’s inhibitor (carboxypeptidase inhibitor), 20 mM bestatin (aminopeptidase
inhibitor), 5 mM EDTA, 1.5 mM 1,10-phenanthroline (divalent ion
chelators), and 0.1% heat-treated BSA at pH 7.4]. The cells were
dislodged by scraping with a rubber policeman, collected in a centrifuge tube, and homogenized in 10 ml of isotonic buffer for ;10 s. The
homogenate was centrifuged at 40,000g for 30 min at 4°C. The
supernatant was discarded, the pellet was rehomogenized in 10 ml of
isotonic buffer, and the high-speed centrifugation was repeated. The
final pellet was resuspended in isotonic buffer to a working concentration of 1 mg protein/ml.
Liver and adrenal medulla tissues were obtained from decapitated
adult male Sprague-Dawley rats. The tissues were immediately homogenized in 10 ml of hypotonic buffer (50 mM Tris, 1 mM EDTA, pH
7.4, at 4°C) for ;10 s. The homogenates were then centrifuged at
500g for 10 min at 4°C, the supernatant saved on ice, the pellet
resuspended in 10 ml of hypotonic buffer, rehomogenized, and recen-
trifuged. The supernatants were combined and centrifuged at
40,000g for 30 min at 4°C. The resulting pellet was then resuspended
in 10 ml of isotonic buffer, homogenized, and the high-speed centrifugation was repeated. The final pellet was resuspended in isotonic
buffer to a working concentration of 1 mg protein/ml.
Radioreceptor Assays. All MDBK experiments were performed
on freshly isolated cell membranes because freezing of membranes at
220°C or 270°C for 24 h resulted in $30% loss in specific binding to
the AT4 receptor (not shown). MDBK cell membranes (25 mg of
protein unless stated otherwise) were incubated in a total volume of
250 ml of assay buffer (isotonic buffer). Incubations were performed
at 37°C for 60 min with 0.6 nM 125I-Ang IV or 90 min with 0.6 nM
125
I-divalinal Ang IV, and nonspecific binding was assessed in the
presence of 1 mM unlabeled Ang IV or divalinal Ang IV, respectively.
Bound and free radioligands were separated by vacuum filtration in
a cell harvester using 32 glass fiber filters and washed with 5 3 4 ml
of PBS (pH 7.2 at room temperature). Radioactivity retained by the
protein-bound filters was counted using an ICN 10/880 gamma
counter (77% efficiency).
To investigate whether the AT4 receptor was G protein linked, the
membrane preparations were first preincubated at 22°C for 60 min
in the assay buffer supplemented with 5 mM MgCl2 and containing
increasing concentrations of a nonhydrolyzable analog of GTP
[guanosine-59-O-(3-thio)triphosphate (GTPgS), 1 nM to 1 mM]. Pretreated membranes were then studied in equilibrium binding experiments. The effect of a sulfhydryl-reducing agent on 125I-peptide
binding was examined by incubating the membranes in the assay
buffer containing increasing concentrations of dithiothreitol (0.1 mM
to 0.1 M).
Measurements of Cytosolic Calcium. All studies were performed using either a standard bath solution containing 140 mM
NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 6
mM glucose (pH 7.4 with NaOH) or a Ca21-deficient bath solution
containing 0 mM CaCl2 and 3 mM MgCl2. Fluorometric determination of cytosolic calcium used subconfluent monolayers of MDBK
cells grown on glass coverslips. Cells were loaded with 2 mM Fura
2-acetoxymethyl ester and pluronic acid for 30 min and then washed
for 30 min in standard bath solution at room temperature. Coverslips
were placed in a temperature-regulated chamber that sat on the
stage of an inverted microscope that was mounted with a micropipette perfusion system. Studies were performed at 37°C on groups of
two to six cells whose fluorescence was delimited by an adjustable
window. Fura 2 was alternatively excited at wavelengths of 340 and
380 nm, and the emission at 510 nm was measured with a photomultiplier tube using a Nikon Photoscan-2 fluorescence photometry
system. To assess whether all cells responded to Ang IV, we examined individual cell intracellular Ca21 concentration ([Ca21]i) measurements in groups of cells with digital imaging fluorescent microscopy using Metafluor Imaging system software. We confirmed that
Ang peptide-induced alteration in [Ca21]i was due to 340- and
380-nm emission intensities changing in reciprocal directions and
that the peptides did not exhibit autofluorescence. The ratio of the
340/380-nm emission intensity was converted to an actual calcium
concentration using a Fura 2 calibration table without determination
of minimum and maximum values in individual cells. Consequently,
the cytosolic calcium values must be interpreted as approximate
concentrations.
Iodination of Peptides. Ang IV and divalinal Ang IV were
iodinated by incubating each peptide (50 mg) in 0.2 M sodium phosphate buffer (pH 7.2), containing 80 mg of chloramine T and ;2 mCi
of Na125I for 25 s at room temperature (total volume of 120 ml). The
reaction was terminated by the addition of 500 mg of Na2S2O5 in 50
ml of sodium phosphate buffer. Monoiodinated peptides were separated from unlabeled and diiodinated peptides by HPLC using a
reversed phase C18 column and a linear acetonitrile (solvent A)
gradient of 9 to 26% over 90 min. Solvent B was 83 mM triethylamine
phosphate at pH 3.0.
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(Ardaillou and Chansel, 1996; Chansel et al., 1998), rat and
rabbit proximal tubules (Dulin et al., 1994; Handa et al.,
1998), cultured opossum proximal tubule cells (Dulin et al.,
1995), and cultured SV-40 transformed human collecting
duct cells (Czekalski et al., 1996). To date, functions associated with the renal Ang IV AT4 receptor system include
increased cortical renal blood flow (Coleman et al., 1998),
modulation of mesangial cell contractility (Ardaillou and
Chansel, 1996), and inhibition of energy-dependent solute
transport in the proximal tubule (Handa et al., 1998). The
renal epithelial AT4 receptor has been partially characterized in opossum proximal tubule (OK7A) cells and SV-40
transformed human collecting duct cells and has yielded
conflicting results on ligand/receptor-coupling mechanisms
and intracellular signal-transduction pathways (Dulin et al.,
1995; Czekalski et al., 1996). In the present study, we used a
Mardin-Darby bovine kidney (MDBK) epithelial cell line to
explore the properties of the renal epithelial AT4 receptor
because most of the known facts on ligand binding pharmacology, physiology, and protein analysis of the AT4 receptor
has been performed in bovine tissues (e.g., Swanson et al.,
1992; Kerins et al., 1995; Bernier et al., 1998; Zhang et al.,
1998). In addition, preliminary results revealed that MDBK
cells expressed abundant amounts of the AT4 receptor with
no detectable AT1 or AT2 receptors. Consequently, the aims
of this study were 3-fold: first, to pharmacologically characterize the AT4 receptor in a bovine renal epithelial cell line
(MDBK cells); second, to provide data demonstrating that the
putative AT4 receptor antagonist divalinal Ang IV binds exclusively and with high affinity to the renal epithelial AT4
receptor; and third, to examine functional responses to Ang
IV and divalinal Ang IV in the same cell line used to characterize the AT4 receptor.
1243
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Handa et al.
Vol. 291
Drugs. We received gifts of losartan (DuP 753) from DuPont/
Merck Pharmaceuticals and PD 123177 and PD 123319 from ParkeDavis. Norleucine1 Ang IV, D-Val1 Ang IV, divalinal Ang IV
[Vc(CH2NH)YVc(CH2NH)HPF], and norleucine-Tyr-Ile-(6)-hexamide were prepared in our laboratory (J.W.H.). Fura 2-acetoxymethyl
ester was purchased from Molecular Probes (Eugene, OR). Ang IV,
Ang II, GTPgS, dithiothreitol, and other reagents were acquired
from Sigma-Aldrich Co. (St. Louis, MO), Bachem California (Torrance, CA), or Peninsula Laboratories Inc. (Belmont, CA).
Statistics. All values represent mean 6 S.E. One- and two-way
ANOVAs and an appropriate post hoc test were used to analyze
multiple groups. Differences between mean values were taken to be
significant at the .05 level. CRunch Interactive Statistical Package
(CRISP), SigmaStat, and InPlot4 computer software programs were
used to analyze results.
Results
Fig. 1. Specific binding of 0.6 nM 125I-peptide to MDBK cell membranes
as a function of protein concentration. Data are from three experiments,
each performed in duplicate. dpm, disintegrations per minute.
Fig. 2. Time course for the association and dissociation of 125I-Ang IV and
125
I-divalinal Ang IV to MDBK cell membranes. The pseudo-first order
association rate constant (kobs) for Ang IV and divalinal Ang IV was found
to be 0.05266 and 0.04167 min21, respectively (each n 5 3), with a
dissociation rate constant (k21) of 0.00383 and 0.00186 min21, respectively (each n 5 3). The calculated association rate constant {k1 5 (kobs 2
k21)/[ligand]} for Ang IV and divalinal Ang IV was determined to be
7.284 3 107 M21 min21 and 7.265 3 107 M21 min21, respectively. Based
on these rate constants, the apparent kinetic kd (k21/k1) values for Ang IV
and divalinal Ang IV were 52.6 and 25.7 pM, respectively.
results from saturation binding isotherms and Scatchard
plots are shown in Fig. 3 and demonstrate that both radioligands bound to a single class of high-affinity binding sites
(apparent Kd: Ang IV, 1.37 6 0.37 nM; divalinal Ang IV,
1.01 6 0.01 nM; n 5 6 each) and to a similar maximum
number of receptors (Bmax: Ang IV, 1335 6 148 fmol/mg
protein; divalinal Ang IV, 1407 6 161 fmol/mg protein; n 5 6
each).
The similarity seen in kinetic experiments and saturation
isotherms for 125I-Ang IV and 125I-divalinal Ang IV was
mirrored by nearly identical rank order affinities and Ki
Fig. 3. Saturation binding and Scatchard transformation (inset) for 125IAng IV and 125I-divalinal Ang IV in MDBK cell membranes. These data
best describe a single high-affinity site for 125I-Ang IV (Kd 5 2.22 nM,
Bmax 5 1049 fmol/mg protein) and 125I-divalinal Ang IV (Kd 5 1.10 nM,
Bmax 5 1077 fmol/mg protein). Data presented represent the results from
a typical experiment.
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Radioligand Binding Studies. Figure 1 illustrates the
comparative binding of 125I-Ang IV, 125I-divalinal Ang IV
(both AT4 receptor ligands), and 125I-Sar1,Ile8 Ang II (AT1/
AT2 receptor ligand) to MDBK cell membranes as a function
of protein concentration. Both 125I-Ang IV and 125I-divalinal
Ang IV displayed high specific binding to MDBK cell membranes, suggesting the possible presence of AT4 receptors,
whereas 125I-Sar1,Ile8 Ang II bound negligibly and likely
reflected the absence or low density of AT1/AT2 receptors in
this renal epithelial cell line. Specific binding of 125I-Ang IV
and 125I-divalinal Ang IV to MDBK membranes increased as
a function of time, and apparent steady state was reached
between 60 and 90 min for both 125I-peptides and remained
stable until 120 to 150 min, after which time we generally
found an upward drift in binding. Consequently, incubation
times of .90 min were not performed. Under these radioreceptor binding conditions, the amount of nonspecific 125Ipeptide bound to the tissue was #10% of total binding. Specific binding of 125I-peptide was reversible after the addition
of 1 mM unlabeled peptide to steady-state conditions (Fig. 2).
Calculation of the Kd value from association and dissociation
rate constants indicated that 125I-Ang IV and 125I-divalinal
Ang IV bound with high affinity to the membrane receptor
with kinetic Kd values of 52.6 and 25.7 pM, respectively. The
1999
Bovine Kidney Epithelial AT4 Receptor
1245
TABLE 1
Binding constants for Ang-related compounds derived from competition
curves for MDBK cell membrane binding sites defined by 125I-Ang IV
and 125I-divalinal Ang IV
Competitor
125
I-Ang IV Ki
M
125
I-divalinal Ang IV Ki
M
n 5 3 or 4.
N.D., not done; Ang II, Ang(1-8); Ang III, Ang(2-8); Ang IV, Ang(3-8).
values for several competing Ang-related peptides for 125IAng IV and 125I-divalinal Ang IV binding (Table 1). All competition curves were best fit with a single-site binding model.
Ligand structure-binding studies revealed that amino-terminal extensions or deletions of Ang IV produced peptides with
less affinity for the binding protein than native Ang IV. We
also obtained a reduction in binding affinity for carboxyl
terminal-extended or -deleted analogs of Ang IV. In agreement with previous reports (Sardinia et al., 1993), Val-TyrHis [Ang(3-5)] appeared to be the minimal molecular sequence of Ang IV that demonstrated high affinity for the AT4
receptor. Also, L-valine at the carboxyl terminal of Ang IV
was critical for high-affinity binding as its removal [Ang(4-8)]
or substitution with its inactive isomer, D-valine, produced a
dramatic loss of binding. Although substitution of L-valine
with L-norleucine in the number 1 position of Ang IV greatly
enhances the binding affinity of the peptide for the bovine
adrenal AT4 receptor (Wright et al., 1995), we found no
improvement in binding to the MDBK AT4 receptor. 125I-Ang
IV binding to MDBK cell membranes was largely unaffected
by losartan (AT1 receptor antagonist), PD 123177 (AT2 receptor antagonist), Sar1,Thr8 Ang II and Sar1,Ile8 Ang II
(both AT1/AT2 receptor antagonists), or D-Ala7 Ang(1-7) [putative Ang(1-7) receptor antagonist], whereas divalinal Ang
IV and NleYI-6-hexamide (both putative AT4 receptor antagonists) competed with high affinity for the 125I-Ang IV binding site. Similar rank order affinities for competing peptides
were found using 125I-divalinal Ang IV as the receptor ligand.
Preincubation of MDBK cells with GTPgS (uncouples G
protein-linked receptors) did not affect 125I-Ang IV or 125Idivalinal Ang IV binding to the membrane AT4 receptor yet
decreased .70% of 125I-Ang II binding to Ang AT1 receptors
Fig. 4. Effect of increasing concentrations of GTPgS (A, n 5 4) and
dithiothreitol (B, n 5 3) on 125I-Ang IV and 125I-divalinal Ang IV binding
to MDBK cell membranes and 125I-Ang II binding to rat liver membranes.
in rat liver membranes (Fig. 4A). Only the 125I-Ang II binding to rat liver membranes could be inhibited further by
exposing membranes to GTPgS for longer periods. Binding of
125
I-Ang IV or 125I-divalinal Ang IV to MDBK cells was also
insensitive to the presence of the sulfhydryl-reducing agent
dithiothreitol at concentrations that abolished 125I-Ang II
binding to rat liver Ang AT1 receptors (Fig. 4B). Similar
responses were observed with a second sulfhydryl-reducing
agent, mercaptoethanol (not shown, n 5 3), except that its
potency to inhibit 125I-Ang II binding to rat liver AT1 receptors was one order of magnitude less than that of dithiothreitol (mercaptoethanol: IC50 5 6.94 mM; dithiothreitol: IC50 5
0.88 mM). These results suggested that the radioligandbound receptor protein studied in MDBK cell membranes
was distinct from the AT1 receptor and that the binding site
of the AT4 receptor did not require G protein or sulfhydryl
bonds for binding affinity.
We also examined whether Ang IV and divalinal Ang IV
demonstrated high affinity for the Ang II type AT1 or AT2
receptors as defined, respectively, by 125I-Sar1,Ile8 Ang II
binding to rat liver membranes treated with 10 mM PD
123177 and 125I-Sar1,Ile8 Ang II binding to rat adrenal medulla membranes treated with 10 mM losartan. As shown in
Fig. 5, A and B, AT1 receptor ligands (Ang II and losartan)
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Amino-terminal extension and deletion of Ang IV
Ang II
4.11 6 2.12 3 1027
5.81 6 1.00 3 1027
Ang III
6.32 6 0.30 3 1028
3.68 6 1.07 3 1028
Ang IV
5.54 6 1.23 3 1029
3.58 6 1.01 3 1029
Ang(4-8)
.1026
N.D.
Carboxyl-terminal extension and deletion of Ang IV
Ang(3-11)
1.17 6 0.19 3 1027
N.D.
Ang(3-10)
1.88 6 0.10 3 1028
N.D.
29
Ang(3-9)
7.17 6 0.46 3 10
N.D.
29
Ang IV
5.54 6 1.23 3 10
3.58 6 1.10 3 1029
28
Ang(3-7)
1.05 6 0.02 3 10
N.D.
Ang(3-6)
1.44 6 0.09 3 1028
N.D.
Ang(3-5)
9.91 6 0.04 3 1029
N.D.
Ang(3-4)
.1026
N.D.
Angiotensin receptor antagonists
Divalinal Ang IV
6.39 6 2.50 3 1029
4.07 6 0.35 3 1029
NleYI-6-hexamide
1.37 6 0.04 3 1028
N.D.
Sar1,Ile8 Ang II
.1026
.1026
1
8
26
Sar ,Thr Ang II
.10
.1026
7
D-Ala Ang(1-7)
.1026
.1026
Losartan
.1024
.1024
PD 123177
.1024
.1024
Other angiotensin peptide analogs
Ang(1-7)
.1026
.1026
Ang(2-7)
3.35 6 0.06 3 1028
N.D.
Nor1 Ang IV
8.49 6 0.27 3 1029
3.25 6 1.90 3 1029
1
26
D-Val Ang IV
.10
N.D.
1246
Handa et al.
Vol. 291
Fig. 5. Competitive inhibition of binding to Ang II type AT1 and AT2
receptors as defined by 125I-Sar1,Ile8 Ang II binding to rat liver membranes treated with 10 mM PD 123177 (A, n 5 3) and 125I-Sar1,Ile8 Ang II
binding to rat adrenal medulla membranes treated with 10 mM losartan
(B, n 5 3), respectively.
and AT2 receptor ligands (Ang II, PD 123177, and PD
123319) displayed high affinity for their respective receptors,
whereas Ang IV and divalinal Ang IV exhibited either no
affinity or extremely low affinity for the AT1 and AT2 receptor.
Intracellular Calcium Studies. [Ca21]i response experiments were performed at 37°C in MDBK cells that were
briefly exposed (;20 s) to a concentration of Ang IV or divalinal Ang IV that was varied from 0.1 nM to 1 mM. Temperature appeared to be one critical factor in these studies 1)
because Ca21 responses to Ang IV were observed in 38% of
experiments performed at 22°C and increased to 75% of experiments when performed at 37°C and 2) because in Ang
IV-responsive cells, where temperature of the bath solution
was randomly ramped-up or ramped-down between 22°C and
37°C, we found that the [Ca21]i response to Ang IV at 22°C
was ;60% less than the corresponding responses recorded at
37°C. As shown in Figs. 6 and 7, both Ang IV and divalinal
Ang IV were capable of producing a rise in [Ca21]i. Although
the concentration-response curves (0.1–10 nM) for Ang IV
and divalinal Ang IV were not significantly different from
each other (as shown in Fig. 7), they diverged at the highest
concentration tested, with 1 mM Ang IV eliciting a dramatic
diminished [Ca21]i response compared with an enhanced
[Ca21]i response with 1 mM divalinal Ang IV (P , .01). The
small but detectable [Ca21]i response to 1 mM Ang IV was not
due to receptor desensitization because it occurred even if the
Fig. 7. Percent change in [Ca21]i from basal value in response to increasing concentration of Ang peptides in MDBK cells. Estimated basal [Ca21]i
immediately before exposure to Ang IV or divalinal Ang IV was 115 6 4
(n 5 23) and 112 6 5 (n 5 15) nM, respectively. Each point represents the
mean of five to nine separate measurements.
highest concentration of Ang IV was administered first, followed immediately by lower concentrations that produced
greater [Ca21]i responses (not shown), or interspersed between repeated 10 nM concentrations that produced maximal increases in [Ca21]i (as shown in Fig. 6A). Although
elevated concentrations of Ang IV metabolites could poten-
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Fig. 6. Representative tracings of [Ca21]i responses in MDBK cells exposed to Ang IV (A) and divalinal Ang IV (B). Digital imaging analysis of
[Ca21]i in a group of nine cells perfused with 1 nM Ang IV (C). Each line
represents the [Ca21]i of an individual cell contained within the group.
1999
Bovine Kidney Epithelial AT4 Receptor
Discussion
In the present study, we identified the presence of a single,
high-affinity AT4 receptor in a bovine renal epithelial cell
line (MDBK), a line that expresses distal tubule cell characteristics (Ishizuka et al., 1978; Ganong et al., 1994). MDBK
cells appear to exclusively express the AT4 receptor subtype
with concentrations that are the highest yet reported for
renal epithelial membranes. These traits, coupled with rapid
growth and a high protein yield, made MDBK cells an attractive cell line in which to characterize renal epithelial AT4
receptors. We used a radiolabeled AT4 receptor agonist and
putative antagonist (125I-Ang IV and 125I-divalinal Ang IV,
respectively) to characterize the MDBK AT4 receptor. Both
ligands bound to the membrane-binding site in a specific,
saturable, reversible, and time-dependent manner. Competition of ligand binding with related Ang peptides characterized the binding site as the AT4 receptor. The affinity of the
receptor for Ang IV and divalinal Ang IV was in the picomolar range (based on kinetic Kd values) and was at least one
order of magnitude greater than the affinity calculated from
saturation binding isotherms (apparent Kd values). The kinetic Kd is generally regarded as a good reflection of the true
Kd because the association and dissociation rate constants
are calculated under steady-state conditions for a single concentration of ligand. These results emphasize the extremely
high affinity of Ang IV and divalinal Ang IV for the AT4
receptor. Coupled with the likelihood that only a fraction of
the cell AT4 receptors need to be occupied for biological activity, it is perhaps not surprising that picomolar concentra-
tions of Ang IV have been reported to elicit changes in renal
epithelial transport function (Handa et al., 1998).
The MDBK AT4 receptor has similar pharmacological and
coupling characteristics as the AT4 receptor described in
immortalized human collecting duct cells and in nonrenal
tissues (e.g., receptor was unlikely to be coupled to a “classic”
G protein, did not require sulfhydryl bonds for binding affinity, and had similar structural requirements for the Ang IV
molecule to bind to the AT4 receptor; Bernier et al., 1995;
Wright et al., 1995; Czekalski et al., 1996). However, 125IAng IV binding to the opossum proximal tubule (OK7A cells)
AT4 receptor was inhibited by GTPgS and dithiothreitol (Dulin et al., 1995), suggesting the possible existence of an AT4
receptor subtype that was clearly distinct from the AT4 receptor described in more distal epithelial segments of the
nephron (Czekalski et al., 1996; present study). Several investigators have estimated that the molecular mass of the
major AT4 receptor expressed in several bovine tissues (hippocampus, thymus, adrenals, kidney, aorta, cultured aortic
endothelial cells) under dithiothreitol-reducing conditions is
165 to 186 kDa with ;20 to 30% of the receptor protein
N-glycosylated (Bernier et al., 1998; Zhang et al., 1998,
1999). The AT4 receptor appears to have a long extracellular
domain (Bernier et al., 1998) and is associated with additional protein subunits in its nonreduced or native form
(Bernier et al., 1998; Zhang et al., 1998). There also is the
possibility that multiple AT4 receptor isoforms may exist
(Zhang et al., 1999). Analysis of Scatchard plots, as well as
Ang-related compounds competing for 125I-Ang IV or 125Idivalinal Ang IV binding sites on MDBK cell membranes,
indicated that the data best fit a one-binding-site model.
These results would be consistent with the presence of a
single high-affinity AT4 receptor subtype on MDBK cells and
is supported by the observation that SDS-polyacrylamide gel
electrophoresis analysis (under reducing conditions) of 125Ibenzoylphenylalanine-Ang IV cross-linked to the solubilized
AT4 receptor from bovine kidney tissue (Zhang et al., 1998,
1999) or MDBK cell membranes (R.K.H., unpublished results) resulted in the labeling of a single protein band.
The intracellular signaling mechanisms associated with
AT4 receptor activation are unknown. Our results suggest
that a rise in [Ca21]i is at least one mechanism by which
ligand binding to the membrane AT4 receptor is transduced
into an intracellular signal. Furthermore, extracellular Ca21
appears to be a major source contributing to the observed
transient and sustained elevation in [Ca21]i in response to
Ang IV and divalinal Ang IV. The pattern and sources of the
[Ca21]i response to Ang IV may be cell specific (e.g., Ang IV
produced only a sustained elevation in [Ca21]i in cultured rat
vascular smooth muscle cells that was related to the influx of
extracellular calcium and possibly the generation of intracellular inositol phosphates; Dostal et al., 1990), whereas only a
rapid, transient [Ca21]i response was observed in cultured
opossum OK7A proximal tubule cells (Dulin et al., 1995) and
cultured rat mesangial cells (Chansel et al., 1998), which was
solely due to an influx of extracellular calcium. In contrast,
there are reports that high concentrations of Ang IV ($0.1
mM) had no effect on [Ca21]i in neuroblastoma cells (Ranson
et al., 1992), chick cardiac myocytes (Baker and Aceto, 1990),
bovine aortic endothelial cells (Briand et al., 1998), or human
collecting duct cells (Czekalski et al., 1996). Because in these
latter studies cells were exposed to only a single, high con-
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tially compete with the parent peptide for the membrane AT4
receptor and result in a diminished [Ca21]i response, the
local concentration of Ang IV metabolites around the cell
would most likely be low because MDBK cells were continuously perfused with Ang IV. Also, smaller peptide fragments
of Ang IV had less affinity than Ang IV for the MDBK AT4
receptor and, therefore, were unlikely to effectively compete
for membrane binding sites (Table 1). Exposure of cells to
Ang IV for longer periods (;3 min) resulted in an initial
transient increase in [Ca21]i that fell to a plateau level and
remained constant until the exposure to Ang IV was terminated (far right response shown in Fig. 6A). Incubation of
cells with a Ca21-free buffer for 5 min resulted in a greatly
diminished (.90%) transient and plateau response to Ang IV
that could be fully restored by incubating cells with the
Ca21-containing bath solution (not shown). Short- or longterm exposure of cells to divalinal Ang IV, in the presence or
absence of extracellular Ca21, produced a similar [Ca21]i
response pattern (except at micromolar concentrations) as
that described for Ang IV (Figs. 6B and 7).
Because the photomultiplier tube averaged the calcium
response from the group of cells under investigation, it was
unclear whether all cells or subgroups of cells responded to
the Ang peptides. Consequently, we used digital imaging
fluorescent microscopy, which allowed simultaneous measurements of [Ca21]i from individual cells within groups of
cells. These experiments could only be performed with
MDBK cells and buffers that were at room temperature and
revealed that all cells within a group responded to Ang IV,
with the magnitude of the response being highly variable
from cell to cell (Fig. 6C).
1247
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Handa et al.
the physiological process and/or cell type that is being examined.
In conclusion, a single high-affinity AT4 receptor subtype is
expressed in the MDBK epithelial cell line and appears to be
pharmacologically similar to the AT4 receptor described in
human collecting duct cells and in nonrenal tissue. Although
divalinal Ang IV has many of the attributes necessary for it
to be an AT4 receptor antagonist, it possesses intrinsic AT4
receptor agonist activity with respect to intracellular calcium
signaling. Consequently, care must be taken to evaluate the
agonist/antagonist activity of divalinal Ang IV in the physiological system under investigation.
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mechanism in these particular cell types or, alternatively,
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AT2 receptors (shown in Fig. 5, A and B). The concentrationdependent biphasic action of Ang IV on [Ca21]i is not restricted to MDBK cells in that it has also been observed in the
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Several investigators have successfully used divalinal Ang
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(Ki) for competing Ang-related peptides. These results are in
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cells revealed that both peptides could significantly elevate
[Ca21]i. Consequently, the apparent physiological activity
(antagonism or agonism) of divalinal Ang IV may depend on
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Send reprint requests to: Rajash K. Handa, Ph.D., Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, College of
Veterinary Medicine; Washington State University, Pullman, WA 99164-6520.
E-mail: [email protected]
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