Vol. 284, Issue 3, H779-H789, March 2003
ANG II AT2 receptor modulates AT1
receptor-mediated descending vasa recta endothelial
Ca2+ signaling
Kristie
Rhinehart,
Corey A.
Handelsman,
Erik P.
Silldorff, and
Thomas L.
Pallone
Division of Nephrology, University of Maryland School of
Medicine, Baltimore 21201-1595; and Department of Biology, Towson
University, Towson, Maryland 21252
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ABSTRACT |
We tested whether the respective
angiotensin type 1 (AT1) and 2 (AT2) receptor
subtype antagonists losartan and PD-123319 could block the descending
vasa recta (DVR) endothelial intracellular calcium concentration
([Ca2+]i) suppression induced by ANG II. ANG
II partially reversed the increase in [Ca2+]i
generated by cyclopiazonic acid (CPA; 10
5 M),
acetylcholine (ACh; 10
5 M), or bradykinin (BK;
10
7 M). Losartan (10
5 M) blocked that
effect. When vessels were treated with ANG II before stimulation with
BK and ACh, concomitant AT2 receptor blockade with
PD-123319 (10
8 M) augmented the suppression of
endothelial [Ca2+]i responses. Similarly,
preactivation with the AT2 receptor agonist CGP-42112A
(10
8 M) prevented AT1 receptor stimulation
with ANG II + PD-123319 from suppressing endothelial
[Ca2+]i. In contrast to endothelial
[Ca2+]i suppression by ANG II, pericyte
[Ca2+]i exhibited typical peak and plateau
[Ca2+]i responses that were blocked by
losartan but not PD-123319. DVR vasoconstriction by ANG II was
augmented when AT2 receptors were blocked with PD-123319.
Similarly, AT2 receptor stimulation with CGP-42112A delayed
the onset of ANG II-induced constriction. PD-123319 alone
(10
5 M) showed no AT1-like action to
constrict microperfused DVR or increase pericyte
[Ca2+]i. We conclude that ANG II suppression
of endothelial [Ca2+]i and stimulation of
pericyte [Ca2+]i is mediated by
AT1 or AT1-like receptors. Furthermore,
AT2 receptor activation opposes ANG II-induced endothelial
[Ca2+]i suppression and abrogates ANG
II-induced DVR vasoconstriction.
kidney; vasoconstriction; vasodilation; medulla; blood flow
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INTRODUCTION |
BLOOD FLOW TO THE
MEDULLA of the kidney is maintained through the tonic and
stimulated release of nitric oxide (NO). Intrarenal infusion of
vasoconstrictors such as norepinephrine and angiotensin II (ANG II)
cause vasoconstriction and a reduction of renal blood flow. In response
to vasoconstrictor infusions, compensatory release of NO helps to
preserve the small fraction of the total renal blood flow that reaches
the medulla of the kidney (38, 43, 44). An important
physiological role for the production of NO in the control of medullary
blood flow has also been proposed based on the observation that NO
synthase (NOS) inhibition leads to reduction of medullary blood flow,
sodium retention, and hypertension (16, 17, 23). Calcium
sensitive isoforms of NOS are expressed in a variety of structures
within the renal medulla including the medullary thick ascending limb
(mTAL), outer medullary collecting duct (OMCD), and descending vasa
recta (DVR) endothelia (17, 23). ANG II causes a
compensatory increase in medullary NO generation (44) but
reduces DVR endothelial intracellular calcium concentration ([Ca2+]i) and inhibits increases in
[Ca2+]i by bradykinin (BK) (25,
31).
Previous studies have shown that ANG II type 1 (AT1) and 2 (AT2) receptors are expressed in outer medullary vascular
bundles (19, 39). Motivated by this, we sought to
determine which receptor subtype mediates the unusual suppressive
effect of ANG II on DVR endothelial calcium signaling. We found that
the AT1 receptor blocker losartan inhibits the ANG II
effect on DVR endothelia, but a high concentration of losartan is
required. Interestingly, the ability of ANG II to reduce endothelial
[Ca2+]i elevation by BK and acetylcholine
(ACh) is markedly enhanced when AT2 receptors are blocked
with PD-123319, implying an important role for AT2 receptor
activation to facilitate endothelial [Ca2+]i
signaling by vasodilators. We also examined the
[Ca2+]i transients induced by ANG II in the
cytoplasm of isolated DVR smooth muscle/pericytes. In contrast to
effects on endothelia, ANG II-induced pericyte
[Ca2+]i transients exhibited classical peak
and plateau responses that were blocked by losartan but not PD-123319.
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METHODS |
In vitro isolation of DVR.
Studies were performed in 267 vessels from Sprague-Dawley rats
(80-150 g body wt, Harlan) anesthetized before nephrectomy by an
intraperitoneal bolus of thiopental (50 mg/kg body wt). DVR were
microdissected as previously described from outer medullary vascular
bundles and mounted on glass pipettes using a standard apparatus (ITM;
San Antonio, TX) (26). The following buffer was used for
the dissection, bath, and loading of fura 2 (in mM): 5 HEPES, 140 NaCl,
10 Na-acetate, 5 KCl, 1.2 MgCl2, 1.71 Na2HPO4, 0.29 NaH2PO4,
1 CaCl2, 5 alanine, and 5 glucose with 0.5 g/dl albumin;
pH = 7.4.
Measurement of vasoreactivity in microperfused DVR.
To evaluate the effects of vasoactive agents on DVR diameters,
microperfusion experiments were recorded on videotape using a Panasonic
model AG 1980 videocassette recorder with a microphone for audio
recording of experimental events. The inverted microscope was equipped
with a beam splitter and a side port for the attachment of a
videocamera (model 72 CCD, Dage-MTI). DVR were observed with a ×40
objective to yield a final magnification of approximately ×1,300 on
the video screen. During playback, vessel diameters were measured with
calipers at the point of greatest constriction. Changes are expressed
as follows: %constriction = (1
D/D0) × 100, where D
is the internal diameter and D0 is the diameter
before the introduction of hormones and reagents into the bath.
Measurement of endothelial intracellular calcium.
DVR were loaded with fura 2 by exposing them to bath containing 2 µM
fura 2-AM (Molecular Probes; Eugene, OR). At the time the bath was
exchanged to contain fura 2-AM, the feedback controller was turned on,
gradually warming the vessel to 37°C over ~5 min. Total loading
time was 20 min. We (24) have previously shown that fura 2 preferentially loads into endothelial cells rather than pericytes. For
measurement of [Ca2+]i, fura 2-loaded DVR
were excited using 350/380-nm dual-wavelength combinations. The
background-subtracted ratio of the fluorescent emission was calculated
for conversion to the equivalent [Ca2+]i
assuming a dissociation constant for fura 2 at 37°C of 224 nM. The
respective Ca2+-free and Ca2+-saturated
350-to-380-nm fluorescence ratios were measured as previously described
by exposing vessels to buffer containing 5 or 0 mM CaCl2
and 0.5 mM EGTA, respectively, along with 10 µM ionomycin and
converted to [Ca2+]i as previously described
(24, 25). A photon-counting photomultiplier assembly (PMT) was employed to measure fluorescent emission at 510 nm
from fura 2-loaded DVR endothelia. The light for the excitation of fura
2 was provided by a 75-W xenon arc lamp directed through a
computer-controlled shutter. The excitation wavelengths were isolated
using a computer-controlled monochrometer (PTI; Lawrenceville, NJ). DVR
were observed through a 1.3 numerical aperture, Nikon CF fluor ×40 oil
immersion objective. Fluorescent emission was isolated with a 510WB40
filter (Omega Optical; Brattleboro, VT) and directed to the PMT. PMT
output was monitored with commercial hardware and software (PTI).
Measurement of Mn2+ influx into DVR
endothelia using fura 2.
Mn2+ quench of fura 2 fluorescence was used to measure
Mn2+ influx into DVR endothelia as previously described
(25). For these protocols, DVR were loaded with fura 2 and
excited at the isosbestic (calcium insensitive) wavelength (360 nm)
while the fluorescent emission (F360) was monitored at 510 nm with a PMT. After MnCl2 (500 µM) was added to the
bath, the rate of decline of F360 provided a measure of
plasmalemmal Mn2+ influx.
Isolation of DVR pericytes and loading of fura 2 into pericyte
cytoplasm.
To isolate pericytes from individual DVR, small wedges of the renal
medulla were separated from kidney slices by dissection and transferred
to CaCl2-free physiological saline solution (PSS) containing collagenase 1A (0.45 mg/ml, Sigma), protease XIV (0.4 mg/ml,
Sigma), and albumin (1.0 mg/ml) as previously described (31). These were incubated at 37°C for 22 min and then
transferred back to CaCl2 (1 mM)-containing PSS and held at
4°C in a petri dish. At intervals, vessels were isolated from the
digested renal tissue by microdissection and transferred to the
perfusion chamber. We have previously shown that this enzymatic
digestion can be used to enable gigaohm seal formation on pericytes for
electrophysiological recording (22).
To separate the pericytes from the enzymatically digested vessels, DVR
were aspirated into a microperfusion-style collection pipette whose
opening was 5-10 µm. During the aspiration into the pipette,
pericytes strip from the abluminal surface of the vessel and are
retained on the pipette tip (31). Once the vessel has been
completely drawn into the pipette, the pericytes remain isolated as a
group of cells suspended in the bath on the pipette tip. Probenecid has
a marked effect to prevent leak of fura 2 from the pericyte cytoplasm.
For this reason, pericytes were loaded with fura 2 at 37°C for 45 min
in the presence of probenecid (1 mM). Probenecid was also included in
the bath during measurement of pericyte
[Ca2+]i transients.
Reagents.
BK, ACh, ANG II, cyclopiazonic acid (CPA), PD-123319, CGP-42112A, and
ionomycin were purchased from Sigma. Losartan was a gift from Merck.
These agents were dissolved in water at
10
1-10
5 M and stored frozen at
20°C. Aliquots were thawed on the day of the experiment, and excess
was discarded at the end of each day. Calcium ionophore was dissolved
in ethanol at 10 mM. CPA and fura 2-AM (Molecular Probes) were stored
frozen in anhydrous DMSO.
Statistical analysis.
Experimental results are reported as means ± SE. In the figures,
most error bars have been suppressed for clarity of presentation. Plateau [Ca2+]i values quoted in the text are
means ± SE at the end of periods in designated protocols.
Statistical comparisons employed a paired t-test, an
unpaired t-test, ANOVA, or repeated-measures ANOVA as
appropriate. For ANOVA, significance was determined by the Student-Newman-Keuls test. P < 0.05 was used for significance.
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RESULTS |
Inhibition of CPA- and ACh-stimulated
[Ca2+]i by ANG II.
As previously described, ANG II reduces basal endothelial
[Ca2+]i, but the effect is difficult to study
because resting [Ca2+]i is low and only small
changes occur after ANG II application. When
[Ca2+]i is increased by treatment with an
endothelium-dependent vasodilator or a sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA) pump inhibitor, the ability of ANG II
to lower endothelial [Ca2+]i is dramatic
(25). In this study, we used CPA and ACh to stimulate increases in endothelial [Ca2+]i. The effect
of ANG II (10
8 M) on endothelial
[Ca2+]i in vessels treated with CPA or ACh is
shown in Figs. 1 and 2. After application of CPA
(10
5 M), endothelial [Ca2+]i
increased from basal values of <100 to ~600 nM. After 5 min, the
bath was exchanged to contain either vehicle or ANG II
(10
8 M). In the presence of ANG II, endothelial
[Ca2+]i fell from 592 ± 125 to 328 ± 105 nM (Fig. 1; P < 0.05). The effect of ACh on DVR
endothelial [Ca2+]i has not been previously
described. For that reason, we examined its concentration dependence.
Between 10
8 and 10
5 M, successively
increasing concentrations of ACh led to additional [Ca2+]i elevation (Fig. 2A).
Threshold effects were observed at 10
8 M; endothelial
[Ca2+]i increased from 45 ± 9 to
67 ± 13 nM (P < 0.05). Significant increases
were observed throughout the concentration range, and endothelial
[Ca2+]i reached 331 ± 34 and 305 ± 37 nM at 10
5 and 10
4 M, respectively. As
shown in Fig. 2B, a peak and plateau response to ACh was
observed. The plateau value of 306 ± 61 nM fell to 154 ± 20 nM (P < 0.05) after ANG II (10
8 M) was
introduced into the bath.

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Fig. 1.
ANG II inhibition of cyclopenzoic acid (CPA)-stimulated
endothelial intracellular calcium concentration
([Ca2+]i). Basal endothelial
[Ca2+]i was monitored for 1 min, after which
CPA (10 5 M) was introduced into the bath for 5 min to
elevate [Ca2+]i. Subsequent addition of ANG
II (10 8 M) reversed the CPA-induced increase in
[Ca2+]i (n = 7 in each
group).
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Fig. 2.
ANG II inhibition of ACh-stimulated endothelial
[Ca2+]i. A: descending vasa recta
(DVR) endothelial [Ca2+]i was measured at
baseline and at 5-min intervals as ACh was introduced into the bath at
log molar increments from 10 8 to 10 4 M
(n = 7). B: DVR endothelial
[Ca2+]i was measured at baseline and for 15 min after exposure to ACh at 10 4 M. After 10 min of ACh,
ANG II (10 8 M) was also introduced into the bath along
with ACh, resulting in a reduction of the plateau
[Ca2+]i response (n = 7).
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Effect of losartan and PD-123319 on ANG II suppression of DVR
endothelial [Ca2+]i.
We next examined whether the specific ANG II AT1 and
AT2 receptor antagonists losartan and PD-123319 could block
the ability of ANG II to reduce DVR endothelial
[Ca2+]i. In the protocol illustrated in Fig.
3, [Ca2+]i was
measured for 1 min (baseline) and then during 5 min of treatment with
CPA. Subsequently, either losartan (10
6 M; Fig.
3A) or PD-123319 (10
6 M; Fig. 3B)
was introduced for 5 min, followed by a concomitant addition of ANG II
(10
8 M) for another 5 min. In a final period, the blocker
was removed from the bath so that the vessels were exposed to ANG II
alone. When applied alone, neither losartan nor PD-123319 affected
CPA-stimulated endothelial [Ca2+]i. At
10
6 M, neither agent prevented the suppression of
endothelial [Ca2+]i by ANG II. As shown in
Fig. 3A, CPA-stimulated [Ca2+]i
fell from 633 ± 113 to 304 ± 54 nM in the presence of ANG
II and losartan (P < 0.01). As shown in Fig.
3B, it fell from 498 ± 137 to 226 ± 33 nM
(P < 0.01). Given the surprising inability of either
the AT1 or AT2 receptor antagonists to block
the effects of ANG II, we repeated the examination but with a higher
concentration of losartan and PD-123312 (10
5 M) and a
lower concentration of ANG II (10
9 M). Under these
conditions, blockade by losartan but not PD-123312 was obtained (Fig.
4). As shown in Fig. 4A,
CPA-stimulated [Ca2+]i changed from 701 ± 82 to 707 ± 71 nM in the presence of ANG II and losartan
[P = not significant (NS)]. As shown in Fig.
4B, it fell from 797 ± 193 to 326 ± 160 nM
(P < 0.01). The need for such a high concentration of
losartan to achieve blockade is atypical of the AT1
receptor (3, 28). For this reason, we verified the result
using an intragroup comparison (Fig. 5).
In this experiment, losartan was introduced into the bath of
CPA-treated vessels at 10
5 M, after which ANG II was
added at 10
9 M. The losartan concentration was
subsequently lowered in log molar increments. Consistent with the
findings of Figs. 3 and 4, loss of losartan blockade of endothelial
[Ca2+]i inhibition occurred between
10
5 and 10
6 M as CPA-stimulated endothelial
[Ca2+]i fell from 481 ± 97 to 214 ± 29 nM ( P < 0.01).

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Fig. 3.
Effect of losartan (Los; 10 6 M) and PD-123319 (PD;
10 6 M) on ANG II (10 8 M) endothelial
[Ca2+]i suppression. A: DVR
endothelial [Ca2+]i was measured at baseline,
after which CPA (10 5 M) was added to the bath to elevate
[Ca2+]i. At 5-min intervals after CPA, the
bath was exchanged to contain CPA + Los (10 6 M), Los
(10 6 M) + ANG II (10 8 M), and then ANG
II alone. At these concentrations, Los failed to block the effect of
ANG II to reduce DVR endothelial [Ca2+]i
(n = 6). B: protocol in A was
repeated, substituting PD (10 6 M) for Los. PD also failed
to block the effect of ANG II to lower DVR endothelial
[Ca2+]i (n = 7).
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Fig. 4.
Effect of Los (10 5 M) and PD (10 5 M) on
ANG II (10 9 M) endothelial
[Ca2+]i suppression. A: DVR
endothelial [Ca2+]i was measured at baseline,
after which CPA (10 5 M) was added to the bath to elevate
[Ca2+]i. At 5-min intervals after CPA, the
bath was exchanged to contain CPA + Los (10 5 M), Los
(10 5 M) + ANG II (10 9 M), and then ANG
II alone. At these concentrations, Los successfully blocked the effect
of ANG II to inhibit DVR endothelial [Ca2+]i
(n = 10). B: protocol in A was
repeated, substituting PD (10 5 M) for Los. PD failed to
block the effect of ANG II to lower DVR endothelial
[Ca2+]i (n = 6).
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Fig. 5.
Intragroup comparison of the concentration dependence of
Los blockade of ANG II (10 9 M) endothelial
[Ca2+]i suppression. DVR endothelial
[Ca2+]i was measured at baseline, after which
CPA (10 5 M) was added to the bath to elevate
[Ca2+]i. Los was added to the bath at
10 4 M, followed by ANG II (10 9 M). At 5-min
intervals, the concentration of Los was reduced in log molar
increments. Below 10 5 M, Los failed to block the effect
of ANG II to lower DVR endothelial [Ca2+]i
(n = 7).
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CPA-induced elevation of [Ca2+]i is due to
inhibition of the SERCA pump and does not represent a biological
response to an endogenous agonist. For this reason, we reexamined the
ability of losartan (10
5 M) and PD-123319
(10
5 M) to block [Ca2+]i
inhibition by ANG II (10
9 M) when DVR endothelial
[Ca2+]i was elevated by pretreatment with ACh
(10
4 M). ACh-stimulated endothelial
[Ca2+]i fell from 330 ± 95 to 140 ± 33 nM and from 337 ± 75 to 142 ± 32 nM in the presence
of ANG II or ANG II + PD-123319, respectively (P < 0.05 for each group). In the presence of ANG II + losartan, endothelial [Ca2+]i fell from 321 ± 55 to 273 ± 48 nM (P = NS) and then dropped to
154 ± 58 nM after the removal of losartan (P < 0.05). Thus losartan but not PD-123319 blocked the effect of ANG II
(Fig. 6).

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Fig. 6.
Effect of Los and PD (10 5 M) on ANG II
(10 9 M) endothelial [Ca2+]i
suppression. Endothelial [Ca2+]i was
increased by preexposing vessels to ACh (10 4 M).
Subsequently, Los (10 5 M), PD (10 5 M), or
vehicle (n = 6 in each group) was exchanged into the
bath, followed by ANG II (10 9 M). Los, but not PD,
blocked ANG II suppression of endothelial
[Ca2+]i. After the removal of Los, the
blockade was reversible.
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Modulation of endothelial
[Ca2+]i signaling by
AT2 receptors.
When ANG II receptors are stimulated before the induction of
endothelial [Ca2+]i transients, we observed
that AT2 receptor blockade with PD-123319 led to an
augmentation of ANG II inhibition of endothelial responses to BK
(10
7 M) and ACh (10
4 M). To increase our
confidence that PD-123319 was selectively blocking AT2
receptors, we reduced its concentration to 10
8 M for
these studies. Figure 7 shows a
comparison of BK-induced [Ca2+]i signaling in
DVR endothelia under four conditions. BK was introduced into the bath
by itself or with ANG II (10
8 M), ANG II + PD-123319
(10
8 M each), or PD-123319 (10
8 M). As
shown in Fig. 7A, blockade of the AT2 receptor
enhanced the ability of ANG II to inhibit BK-induced
[Ca2+]i signaling. Upon stimulation with BK,
BK + ANG II, or BK + ANG II + PD-123319, the peak
[Ca2+]i achieved was 842 ± 224, 482 ± 114 (P = NS vs. BK), and 204 ± 51 nM
(P < 0.05 vs. BK), respectively. BK-induced
[Ca2+]i responses tended to wane markedly,
and we could not demonstrate a significant difference in plateau
[Ca2+]i at 15 min. By itself, PD-123319 did
not inhibit BK-induced [Ca2+]i signaling
(Fig. 7B). BK + PD-123319 achieved peak and plateau [Ca2+]i of 687 ± 126 and 171 ± 42 nM, respectively. The peak but not the plateau was significantly higher
(P < 0.01) than that achieved by the combined action
of BK + ANG II + PD-123319.

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Fig. 7.
Effect of angiotensin type 2 (AT2) receptor blockade on
ANG II inhibition of bradykinin (BK)-induced
[Ca2+]i signaling. A: after 1 min
of baseline recording, BK (10 7 M, n = 9),
BK + ANG II (10 8 M, n = 9), or
BK + ANG II + PD (10 8 M, n = 8)
was added to the bath for 15 min and then washed out. When
AT2 receptors were blocked, the ability of ANG II to
inhibit BK-induced [Ca2+]i signaling was
markedly enhanced. B: PD, when applied without ANG II, had
no effect on BK [Ca2+]i signaling
(n = 8).
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For additional confidence, we also examined the ability of ANG II
receptor subtype stimulation to affect ACh-induced endothelial [Ca2+]i signaling (Fig.
8). This is especially desirable because
endothelial [Ca2+]i responses to ACh exhibit
higher plateaus and have less tendency to wane than do BK-induced
responses. In the first series, AT1 or AT2
receptors were selectively stimulated before ACh with either ANG
II + PD-123319 or ANG II + losartan. Selective
AT1 receptor stimulation but not AT2 receptor
stimulation markedly inhibited ACh-induced endothelial
[Ca2+]i transients. Peak
[Ca2+]i reached 572 ± 106 and 146 ± 18 nM (P < 0.01) in the ANG II + losartan and
ANG II + PD-123319 groups, respectively (Fig.
8A). In the second series, the ability of ANG II
(AT1 + AT2 receptor stimulation) to block
ACh-stimulated endothelial [Ca2+]i was
compared with that of ANG II + PD-123319 (selective
AT1 receptor stimulation). Vehicle, ANG II, or ANG II + PD-123319 was exchanged into the bath at the same time as ACh
(time = 1 min; Fig. 8B). Selective AT1
receptor stimulation blocked the peak phase of the
[Ca2+]i response (841 ± 186 vs.
126 ± 45 nM, ACh vs. ACh + ANG II + PD-123319,
P < 0.01). The plateau phase of ACh was also
blocked (244 ± 42 vs. 60 ± 9 nM, P < 0.01).

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Fig. 8.
Effect of AT2 receptor blockade on ANG II inhibition of
ACh-induced [Ca2+]i signaling. A:
after 1 min of baseline recording, PD (10 5 M,
n = 7) or Los (10 5 M, n = 6) was added to the bath. Subsequently, ANG II (10 9 M)
was added (at 3 min), followed by ACh (10 5 M, at 8 min).
The ACh-induced [Ca2+]i transient was
markedly inhibited by preexposure to ANG II/PD. B: ACh
(10 5 M, n = 6), ACh + ANG II
(10 8 M, n = 6), or ACh + ANG II + PD (10 8 M, n = 5) was added to the bath
from 2 to 12 min and then washed out. When AT2 receptors
were blocked, ANG II inhibition of the ACh-induced
[Ca2+]i transient was markedly enhanced.
C: endothelial [Ca2+]i transients
were measured after simultaneous introduction of ACh (10 5
M), ANG II (10 8 M), and PD (10 8 M) to the
bath. This was examined in the absence (n = 9) or
presence of AT2 receptor preactivation with CGP-42112A
(CGP; 10 8 M, n = 10). AT2
receptor preactivation prevented the inhibition of ACh-induced
[Ca2+]i responses.
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In a final series, we tested whether prestimulation of AT2
receptors with CGP-42112A (10
8 M) could prevent
subsequent selective AT1 receptor stimulation from
inhibiting ACh-induced endothelial [Ca2+]i
transients. After 5 min of prestimulation with CGP-42112A, selective
AT1 receptor stimulation with ANG II + PD-123319
failed to inhibit [Ca2+]i signaling (Fig.
8C). The differences in peak (591 ± 84 vs. 147 ± 37 nM) and plateau phases (306 ± 23 vs. 104 ± 24 nM) were both highly significant (P < 0.01). Taken together,
the results shown in Figs. 7 and 8 lead to the conclusion that
AT2 receptor stimulation modulates the ability of
AT1 receptors to mediate suppression of DVR endothelial
[Ca2+]i responses.
Blockade of ANG II suppression of
Mn2+ entry into DVR endothelia.
We have previously shown that the ability of ANG II to suppress
endothelial [Ca2+]i is at least partially
explained by an inhibition of divalent cation influx (25).
When Mn2+ enters fura 2-loaded cells, it quenches fura 2 fluorescence. The rate of Mn2+ entry can therefore be
gauged from the rate of decline of fura 2 fluorescence when fura 2 is
excited at its Ca2+-insensitive isosbestic point, 360 nm.
As shown in Fig. 9, ANG II inhibits
Mn2+ quench, an effect that is blocked by losartan but not
PD-123319.

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Fig. 9.
Inhibition of
Mn2+ influx into DVR endothelia by ANG II. A:
fluorescence of fura 2 was measured during excitation at 360 nm
(F360) and normalized to its value at time 0 (F0; ordinate). The addition of 500 µM MnCl2
to the bath resulted in an ~20% reduction of fluorescence over
120 s. The addition of ANG II (10 9 M) blocked the
rate of fura 2 quench attributable to influx of Mn2+ into
the endothelia. B: for purposes of statistical analysis, the
average of the last 5 s of fluorescence was computed for each
vessel from recordings such as those depicted in A. Bars
show means ± SE of that averaged final fluorescence for five
groups in which Mn2+ was not added (vehicle,
n = 6), Mn2+ was added alone
(n = 8), Mn2+ was added along with ANG II
(10 9 M, n = 8), or Mn2+ was
added with ANG II plus either Los (10 5 M,
n = 9) or PD (10 5 M, n = 7). Los, but not PD, blocked the ability of ANG II to inhibit
Mn2+ of fura 2. *P < 0.05.
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Blockade of the ANG II-induced increase in pericyte
[Ca2+]i.
The reduction of DVR endothelial [Ca2+]i by
ANG II contrasts with the expected effect of stimulation of the
AT1 receptor to increase [Ca2+]i
via inositol (1,4,5)-trisphosphate
[Ins(1,4,5)P3]-mediated signaling (2,
40). Because ANG II is a potent vasoconstrictor of DVR,
it is anticipated that it would increase smooth muscle/pericyte [Ca2+]i through AT1 receptor
stimulation. Pericytes were isolated and loaded with fura 2 in the
presence of probenicid to prevent fura 2 leakage from the cytoplasm
(31). Pericyte [Ca2+]i was
measured during exposure to either ANG II (10
8 M), ANG
II + losartan (10
5 M), or ANG II + PD-123319
(10
5 M) (Fig. 10). As
expected for smooth muscle, ANG II increased pericyte
[Ca2+]i from 56 ± 46 nM to a peak of
639 ± 145 nM. A plateau of 146 ± 30 nM was achieved 10 min
after ANG II stimulation. In the presence of losartan, contemporary
values for pericyte [Ca2+]i were 54 ± 21 and 74 ± 26 nM, respectively (P < 0.01, peak
and plateau phases). AT1 receptor blockade completely
eliminated the ANG II response. In contrast, PD-123319 had no
significant effect to alter either the peak (473 ± 100 nM) or
plateau (144 ± 55 nM) phase pericyte
[Ca2+]i. PD-123319 alone had no effect on
pericyte [Ca2+]i (Fig. 10).

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|
Fig. 10.
Los inhibits ANG II-induced increases in pericyte
[Ca2+]i. Pericytes were isolated by stripping
them from the abluminal surface of microdissected DVR and loading them
with fura 2 in the presence of probenecid (1 mM) (31).
Baseline [Ca2+]i was recorded for 1 min,
after which ANG II (10 8 M), ANG II + Los
(10 5 M), or ANG II + PD (10 5 M) was
added to the bath for 10 min (n = 6 in each group). ANG
II induced a peak and plateau [Ca2+]i
response that was blocked by Los but not PD. In a separate group, the
bath was exchanged to include PD alone (10 5 M,
n = 4). In the absence of ANG II, PD did not increase
pericyte [Ca2+]i.
|
|
Role of AT1 and AT2 receptor activation in
DVR vasoconstriction.
We examined the ability of AT1 and AT2 receptor
blockade to influence vasoconstriction (Fig.
11). In one series, losartan
(10
6 M), PD-123319 (10
6 M), or vehicle was
introduced into the bath of microperfused DVR followed by ANG II
(10
8 M). As shown in Fig. 11A, losartan
blocked and PD-123319 augmented constriction. These results demonstrate
that AT1 receptor activation mediates and AT2
receptor activation modulates vasoconstriction of DVR. In separate
experiments, microperfused DVR were sequentially exposed to PD-123319
at 10
8 M and then 10
6 M concentrations
(5-min intervals each). At the end of each period, %constriction
reached 0.7 ± 0.9 and 2.4 ± 3.3, respectively (not significantly different from zero, data not shown). We also tested whether AT2 receptor preactivation with CGP-42112A
(10
8 M) could modulate subsequent ANG II-induced
constriction. During a 5-min preactivation period, CGP-42112A did not
affect basal vasomotor tone. Compared with controls, vessels
preactivated with CGP-42112A exhibited a delayed response to ANG II
(Fig. 11B).

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Fig. 11.
AT2 receptor activation modulates vasoconstriction.
A: microperfused DVR were exposed to Los (10 6
M, n = 8), PD (10 6 M, n = 10), or vehicle (n = 9) at time 0. After the
baseline was recorded for 1 min, ANG II (10 8 M) was
introduced into the bath. After 10 min, the bath was exchanged to
remove the blocker. AT1 receptor blockade with Los
inhibited and AT2 receptor blockade augmented ANG
II-induced vasoconstriction. B: vessels were preexposed to
the AT2 receptor agonist CGP (10 8 M,
n = 8) or vehicle (n = 6) for 5 min,
after which ANG II (10 8 M) was introduced into the bath.
Preactivation of AT2 receptors delayed ANG II-induced
vasoconstriction. (*P < 0.05 vs. control).
|
|
 |
DISCUSSION |
We (25) previously demonstrated that ANG II reduces
both basal DVR endothelial [Ca2+]i and BK- or
thapsigargin-stimulated [Ca2+]i responses of
DVR endothelium. That effect occurred when ANG II was applied to either
the luminal or abluminal surface of the vessel and when pericytes were
separated from the endothelium (31). Taken together, those
findings implicate both an endothelial receptor and an endothelial
signal transduction cascade through which ANG II modulates
[Ca2+]i. In this study, we extended the prior
work by investigating the ability of AT1 and
AT2 receptor blockade to mediate and modulate the
endothelial [Ca2+]i response to ANG II. Two
principal findings emerged. First, the AT1 receptor blocker
losartan inhibits ANG II-induced suppression, but a high concentration
of losartan (10
5 M) is required (Figs. 3-5). A
second major finding is that AT2 receptor blockade with
PD-123319 enhances the ability of ANG II to suppress endothelial
[Ca2+]i responses. This is true provided that
AT2 receptors are activated at the same time as, or prior
to, the [Ca2+]i-stimulating agent (Figs. 7
and 8). The latter effect is likely to be AT2 receptor
specific because it is achieved at a low PD-123319 concentration
(10
8 M).
As previously discussed (25), based on the known signal
transduction pathways of the G protein-coupled AT1
receptor, the finding that DVR endothelial
[Ca2+]i is suppressed rather than stimulated
by ANG II is unexpected. ANG II stimulation of cultured aortic
endothelial cells by Pueyo and colleagues (29, 30)
resulted in a rise in [Ca2+]i, phospholipase
C activation, phospholipase A2 activation, and peroxynitrite production. The opposite response that we observed in
acutely isolated DVR might be explained by phenotypic alteration of
cultured aortic cells or an effect of acute isolation on DVR endothelia. It seems more likely that the difference in
[Ca2+]i signaling is related to a true
regional difference between macrovascular aortic and microvascular DVR
endothelia. We hypothesized that ANG II inhibits
[Ca2+]i signaling in DVR endothelia to reduce
vasodilator production by these cells, thereby enabling the adjacent
thick ascending limbs of Henle to control vasomotion in vascular
bundles (25, 31).
Measurement of endothelial
[Ca2+]i in isolated DVR.
The DVR wall is comprised of smooth muscle/pericytes and endothelial
cells. In this study, we loaded fura 2 into DVR freshly isolated by
microdissection from the renal medulla. We (24) have
previously shown that this yields a strong endothelial fluorescent signal with near exclusion of fura 2 from the pericyte cytoplasm. Recently, we devised a method for removing pericytes from the abluminal
surface of collagenase-treated DVR and used this to show that selective
endothelial loading of fura 2 is partially explained by export of
deesterified fura 2 from pericytes by a pathway that is blocked by
probenecid. We also found that the ability of ANG II to suppress basal
and CPA-stimulated endothelial [Ca2+]i
persists even when pericytes have been removed. In contrast, isolated,
fura 2/probenecid-loaded pericytes respond to ANG II with a classical
peak and plateau [Ca2+]i increase (Fig. 10)
(31). Because ANG II can exert its opposite effects to
raise and lower [Ca2+]i in DVR pericytes and
endothelia, respectively, even after these cells have been separated
from one another, we conclude that ANG II receptors exist on both cell types.
The concentrations at which ANG II exerts maximal effects on
vasoconstriction and endothelial calcium signaling differ.
Microperfused DVR constrict at a threshold ANG II concentration of
~10
11 M and exhibit an EC50 of 5 × 10
10 M. In contrast, threshold and 50% maximal
inhibition of endothelial calcium occurred at 10-fold greater ANG II
concentrations (25). Such concentrations of ANG II are
high relative to circulating levels, but ANG II concentrations in
proximal tubule fluid and star vessel plasma downstream of glomeruli
have been found to exceed circulating levels by 1,000-fold (21,
34).
Inhibition of DVR endothelial Ca2+
influx.
Although the signaling within DVR endothelia that is responsible for
ANG II-induced [Ca2+]i inhibition remains
unknown, a role for inhibition of Ca2+ influx has been
established. We have previously shown that the rate of refilling of
Ca2+ into Ca2+-depleted endothelia is reduced
by pretreatment with ANG II. Similarly, Mn2+ entry, as
gauged by the quench of fura 2 in Ca2+-replete cells, is
blocked by ANG II (25). With the use of the latter
technique, we have now shown that losartan but not PD-123319 can
reverse the ANG II inhibition of Mn2+ entry (Fig. 9). The
pathway through which Ca2+ enters DVR endothelia is
unknown. Endothelia are generally thought to lack voltage-operated
Ca2+ channels and instead to rely on nonselective cation
channels and changes in membrane potential to augment or reduce the
driving force for divalent cation influx by hyperpolarization or
depolarization, respectively (1, 12). According to that
hypothesis, ANG II depolarization of pericytes would be expected to
favor a [Ca2+]i increase (Fig. 10), whereas
ANG II depolarization of DVR endothelium would favor a
[Ca2+]i decrease (Figs. 1-8). In recent
studies, we verified that ANG II depolarizes both cell types and showed
that BK hyperpolarizes DVR endothelia (22, 31).
Modulation of ANG II
[Ca2+]i suppression by
losartan and PD-123319.
ANG II exerts its effects through AT1 (AT1A and
AT1B) and AT2 receptors (13).
AT1A and AT1B receptors are 7 membrane-spanning G protein-coupled receptors with 95% amino acid sequence homology and
are widely localized to vascular and tubular elements of the kidney
(10). AT1 receptor activation signals
largely via phospholipase C-mediated
Ins(1,4,5)P3 generation and
[Ca2+]i responses. Those actions have
been observed in a variety of cell types including smooth muscle and
endothelia (2, 30, 31, 40). Losartan is a nonpeptide,
highly selective antagonist of angiotensin AT1A and
AT1B receptors. This compound inhibits ANG II binding to
AT1 receptors at concentrations between 10
9
and 10
8 M and has a >10,000-fold selectivity for
AT1 over AT2 receptors (3, 28).
Given this sensitivity and the high degree of selectivity, it is
somewhat surprising that [Ca2+]i suppression
by ANG II was only blocked when losartan was used at 10
5
M (Figs. 3-5). Possibly, isolation of vessels alters receptor
affinity or a separate population of low-affinity receptors exists on
DVR endothelia. In any case, it cannot be firmly concluded that
AT1 receptors mediate the suppressive DVR endothelial
[Ca2+]i response to ANG II. Studies with ANG
II receptor blockers alone will probably not suffice to confidently
identify the receptor that mediates DVR endothelial ANG II
[Ca2+]i suppression.
In contrast to the uncertainty concerning the receptor that mediates
ANG II [Ca2+]i suppression, the low
concentration at which PD-123319 augments suppression lends confidence
to a role for AT2 receptors to modulate the effect.
PD-123319 has been described to block ANG II binding to COS7 cells
expressing AT2 receptors with an IC50 = 1.7 nM (14). When we used this compound at
10
8 M, ANG II [Ca2+]i
suppression was markedly augmented (Figs. 7 and 8). Additional confidence that AT2 receptors modulate endothelial
[Ca2+]i suppression is afforded by the
observation that their stimulation with CGP-42112A prevents
AT1 receptor-induced effects (Fig. 8C). Sensitive measurements using RT-PCR and immunochemistry have verified both AT1 and AT2 receptor expression in DVR
(19, 39).
It should be noted that the order of activation of ANG II receptor
subtypes appears to be of importance in the demonstration of the
ability of AT2 receptor stimulation to facilitate DVR
endothelial [Ca2+]i signaling. In Figs.
3B and 4B, PD-123319 failed to block the ability
of ANG II to reduce endothelial [Ca2+]i, and,
in contrast to the effects shown in Figs. 7 and 8, washout of PD-123319
to expose the AT2 receptor to ANG II stimulation did not
lead to an increase in endothelial [Ca2+]i.
Similarly, as shown in Fig. 6, the addition of PD-123319 along with ANG
II had no greater effect to lower plateau phase endothelial calcium
than did ANG II alone. The difference between those classes of
experiments is the order of application of agonists. When
AT2 receptors were activated before or at the same time as
BK or ACh (Figs. 7 and 8), a strong modulation of
[Ca2+]i signaling occurred. In that case,
activation of the AT2 receptor by ANG II or by CGP-42112A
prevented AT1 receptor stimulation from blunting the
ACh-induced endothelial [Ca2+]i transient.
It should also be recognized that ANG II was more effective to inhibit
peak than plateau [Ca2+]i responses (Figs. 7
and 8). BK [Ca2+]i plateaus fall off
dramatically with time so that ANG II-induced effects are difficult to
study near the end of the BK response (Fig. 7). ACh, however, yields a
more robust and sustained plateau so that the effects of ANG II
inhibition on that phase of the response were more easily observed
(Fig. 8B). Additionally, to lend confidence to the efficacy
of preactivation of AT2 receptors to sustain DVR
endothelial [Ca2+]i responses, we compared
the endothelial calcium response to ACh + ANG II + PD-123319
when vessels had been either pretreated with the AT2
receptor agonist CGP-42112A or vehicle. The results were dramatic and
consistent. Prestimulation of AT2 receptors prevented
AT1 receptor-induced inhibition of ACh-mediated calcium transients (Fig. 8C).
In a recent study, Ruan et al. (32) examined the ability
of ANG II antagonists to inhibit intrarenal vasoconstriction in AT1A receptor null mice. Interestingly, coadministration of
PD-123319 with ANG II blocked residual vasoconstriction in
AT1A knockouts. One interpretation of those data is that
PD-123319 exhibits AT1B receptor antagonist activity in
that species. Our data show that PD-123319 enhances vasoconstriction
(Fig. 11) and fails to block pericyte [Ca2+]i
transients (Fig. 10). Both of these findings mitigate against AT1 receptor antagonist activity in the rat. In contrast,
PD-123319 ANG II-induced vasoconstriction, a finding that is consistent with the effects of AT2 receptors to oppose AT1
receptor-mediated effects in other systems (2, 5, 8, 11, 14,
35-37).
Interplay between AT1 and AT2 receptors.
It is generally accepted that AT2 receptor activation
exerts effects that oppose and modulate the activation of
AT1 receptors (5, 8, 11, 14, 35-37).
AT2 receptor expression is markedly reduced after fetal
development but remains in the kidney into adult life
(18). AT2 receptors are inducible by ANG II
infusion and a low-salt diet and are localized to vascular structures
and glomeruli (2, 40). AT1 receptor activation
induces cell proliferation, vasoconstriction, and activation of protein
kinases, whereas AT2 receptor stimulation is vasodilatory,
antiproliferative, and generally increases phosphatase activity. The
findings of this study are entirely compatible with those notions.
AT2 receptor inhibition with PD-123319 augments the ability
of ANG II to suppress endothelial Ca2+ responses to BK and
ACh (Figs. 7 and 8). On the basis of this, we conclude that
AT2 receptor activation should favor vasodilation through
production of NO and prostaglandins via Ca2+-dependent
isoforms of NOS (NOS1 and NOS3) and phospholipase A2, respectively. In particular, NOS3 (endothelial NOS) activity increases over the range of endothelial [Ca2+]i
modulated by ANG II (Figs. 6-8). A steep dependence of NOS
activity on [Ca2+]i exists between 100 and
500 nM (33). It cannot be concluded, however, that
inhibition of DVR vasoconstriction due to AT2 receptor activation (Fig. 11) is entirely attributable to effects on endothelial release of NO. Although PD-123319 did not affect ANG II-induced pericyte [Ca2+]i transients (Fig. 10),
AT2 receptor activation of phosphatases could modulate the
phosphorylation of contractile proteins and their sensitivity to
increases in [Ca2+]i (27).
Renal effects of AT2 receptor stimulation.
In recent years, a number of studies have been performed to examine the
physiological role that AT2 receptor stimulation serves in
the kidney (9, 15, 19, 35, 36, 39, 42). The data support a
role for AT2 receptor stimulation to function in a
counterregulatory manner to oppose AT1 receptor-mediated
vasoconstriction. With the use of intrarenal tissue microdialysis,
Siragy and colleagues (5, 35-37) found that ANG II
tonically stimulates BK production leading to cGMP formation and NO
release. Those observations were corroborated in AT2
receptor null mice. Those animals exhibit sodium retention, a failure
to generate intrarenal BK, and exaggerated hypertension in response to
chronic ANG II infusion (36). Conversely, overexpression
of AT2 receptors leads to vasodilation through kinin
activation (42). Gross et al. (9) examined
the relationship of AT2 receptor activation with medullary
blood flow and sodium excretion. Natriuresis and changes in medullary
blood flow that accompany increased renal perfusion pressure were
blunted in AT2 receptor knockout mice (9).
Molecular machinery for generation of kinins is present in the renal
medulla. Kallikreins, the enzymes that release kinins from kininogen
precursors, are expressed in the connecting tubule of the rat, portions
of the outer medulla, and papillary collecting ducts. Both
high-molecular-weight kininogen and low-molecular-weight kininogen are
expressed by the distal nephron in close proximity to cells that
express tissue kallikrein (7). On the basis of the
molecular weight of kinins and the permeability characteristics of the
vasa recta, it seems likely that these peptide hormones are trapped in
the medulla by countercurrent exchange to act as DVR vasodilators. We
consistently observe brisk [Ca2+]i responses
of DVR endothelia to BK (Fig. 7) (24, 25). The results of
this study expand our understanding of interactions between ANG II and
BK by demonstrating that, in addition to facilitating BK generation
within the kidney, AT2 receptors exert a permissive action
to favor BK-stimulated [Ca2+]i increases
within DVR endothelia (Fig. 7). Evidence suggests that the latter
should favor vasodilation, enhancement of medullary perfusion, and
saliuresis (4).
Effect of ANG II on renal blood flow and NO production.
Perfusion of the renal medulla is sensitive to NO inhibition. NOS
blockade reduces medullary blood flow, favors sodium retention, and
induces hypertension (17, 20). Ca2+-sensitive
isoforms of NOS (NOS1 and NOS3) are expressed in thick ascending limb,
vasa recta, and collecting ducts (17, 23). Endothelial NOS (NOS3) is stimulated by elevation of
[Ca2+]i. Thus ANG II suppression of
endothelial [Ca2+]i should favor rather than
oppose vasoconstriction and tend to reduce blood flow through DVR. In
contrast to this reasoning, past studies have pointed to a reciprocal
relationship between ANG II stimulation and intrarenal NO production
(41, 44). Infusion of ANG II into the medullary
interstitium leads to an increase in intrarenal NO generation, implying
a role for NO to prevent ANG II constriction of medullary resistance
vessels (44). In contrast to this, we found that ANG II
reduces DVR endothelial [Ca2+]i and inhibits
Ca2+ signaling by the endothelium-dependent vasodilator BK.
To explain this apparent paradox, we (25) have noted that
ANG II raises [Ca2+]i in OMCD and mTAL. DVR
on the periphery of outer medullary vascular bundles lie adjacent to
these structures. On the basis of this proximity, we speculated that
ANG II-induced Ca2+ activation of NOS1 and NOS3 within the
cytoplasm of mTAL and OMCD stimulates NO that diffuses to the vascular
bundle periphery to preferentially dilate the vessels that supply them
with oxygen and nutrients. Suppression of endothelial
[Ca2+]i would facilitate that process to
provide a feedback mechanism that transfers control of vasomotion away
from endothelium to transporting epithelia. The latter may be of
importance in the relatively hypoxic renal medulla to prevent
ischemic insult to metabolic active NaCl-transporting nephron
segments (6).
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
DK-42495, HL-68686, and HL-62220.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
T. L. Pallone, Div. of Nephrology, N3W143, Univ. of
Maryland-Baltimore, Baltimore, MD 21201-1595 (E-mail:
tpallone{at}medicine.umaryland.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published November 7, 2002;10.1152/ajpheart.00317.2002
Received 10 April 2002; accepted in final form 5 November 2002.
 |
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