Angiotensin IV receptor-mediated activation of lung
endothelial NOS is associated with vasorelaxation
Jawaharlal M.
Patel1,2,
Jeffrey
R.
Martens3,
Yong D.
Li2,
Craig H.
Gelband3,
Mohan K.
Raizada3, and
Edward R.
Block1,2
1 Research Service, Department
of Veterans Affairs Medical Center, and Departments of
2 Medicine and
3 Physiology, University of
Florida College of Medicine, Gainesville, Florida 32608-1611
 |
ABSTRACT |
The
hexapeptide angiotensin (ANG) IV, a metabolic product of ANG II, has
been reported to play a functional role in the regulation of blood flow
in extrapulmonary tissues. Here, we demonstrate that ANG IV-specific
(AT4) receptors are present in
porcine pulmonary arterial endothelial cells (PAECs) and that the
binding of ANG IV to AT4 receptors
can be blocked by its antagonist divalinal ANG IV but not by the ANG
II-, AT1-, and
AT2-receptor blockers [Sar1,Ile8]ANG
II, losartan, and PD-123177, respectively. ANG IV significantly increased endothelial cell constitutive nitric oxide synthase (ecNOS)
activity (P < 0.05) as well as
cellular cGMP content (P < 0.001).
Western blot analysis revealed that ecNOS protein expression was
comparable in control and ANG IV-stimulated cells. Divalinal ANG IV but
not
[Sar1,Ile8]ANG
II, losartan, or PD-123177 inhibited the ANG II- and ANG IV-stimulated
increases in ecNOS activity and cGMP content in PAECs. Incubation in
the presence of
N-nitro-L-arginine
methyl ester (L-NAME) or
methylene blue but not of indomethacin significantly diminished ANG
IV-stimulated as well as basal levels of cGMP
(P < 0.001). Similarly, in situ
studies with precontracted porcine pulmonary arterial rings showed that
ANG IV caused an endothelium-dependent relaxation that was blocked by
L-NAME or methylene blue.
Collectively, these results demonstrate that ANG IV binds to
AT4 receptors, activates ecNOS by
posttranscriptional modulation, stimulates cGMP accumulation in PAECs,
and causes pulmonary arterial vasodilation, suggesting that ANG IV
plays a role in the regulation of blood flow in the pulmonary circulation.
nitric oxide synthase; endothelium
 |
INTRODUCTION |
VASCULAR ENDOTHELIUM is a metabolically active tissue
that regulates pulmonary and systemic vascular tone through metabolism and/or generation and release of vasoactive mediators such as angiotensin (ANG) peptides and nitric oxide (NO) (6, 9, 12, 16). NO
plays a key role in the regulation of vasodilation (6, 16), whereas
other endothelium-derived bioactive mediators, including ANG II, play a
critical role as vasoconstrictors in the systemic and pulmonary
vasculatures (9, 12, 16). In contrast to ANG II, it has been suggested
that ANG IV, a biologically active metabolic product of ANG II, plays a
critical role as a vasodilator (13, 17, 34). For example, several
reports (13, 17, 21, 34) showed that ANG IV acts as a vasodilator and increases blood flow in the kidney and the brain through a novel ANG
IV-specific (AT4)
receptor-mediated pathway. However, a recent report (22) suggested that
ANG IV administered in pharmacological doses is a weak vasoconstrictor
in the pulmonary circulation of the rat and that this vasoconstrictor
action is modulated by NO release.
Vascular endothelial cells generate NO from the metabolism of
L-arginine via an oxidative
catabolic reaction mediated by an endothelial cell constitutively
expressed isoform of NO synthase (ecNOS) (16, 23). The catalytic
activity of ecNOS is calcium/calmodulin, tetrahydrobiopterin, and NADPH
dependent, and several reports (10, 29, 31) indicated that endothelial
NOS is localized to plasma membrane caveolae in endothelial cells. The
physiological action of NO is dependent on the activation of smooth
muscle cell-soluble guanylate cyclase and the formation of cGMP, which
is mediated by ecNOS-derived NO (6, 20). Endothelial cell release of NO
is enhanced by receptor-mediated agonists such as acetylcholine, bradykinin, histamine, serotonin, and substance P via signal
transduction-mediated activation of ecNOS (6, 16).
The vascular endothelium also processes a variety of biologically
active substances in the circulation, including ANG I, a product of the
renin-ANG system. ANG-converting enzyme, located on the luminal surface
of vascular endothelium, metabolizes ANG I to ANG II (9). ANG II is
enzymatically cleaved by aminopeptidase A (AA) to form ANG III, which
is further cleaved by aminopeptidase M (AM) to form ANG IV in various
tissues (34). A number of recent studies (3, 5, 14, 15, 27) identified
and characterized AT4 receptors in
a variety of tissues except the lung in a number of species including
humans. At present, nothing is known about the formation of ANG IV, its
receptors, or its function in the pulmonary circulation. The present
study examined whether ANG IV-specific receptors are present in
pulmonary arterial endothelial cells (PAECs), whether ANG IV mediates
the activation of ecNOS, and whether ecNOS activation is linked to
increased cGMP production and pulmonary arterial vasodilation.
 |
MATERIALS AND METHODS |
Cell culture and treatment.
Endothelial cells were obtained from the main pulmonary artery of 6- to
7-mo-old pigs and propagated in monolayers as previously described
(25). Fifth- to seventh-passage cells in postconfluent monolayers
maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY)
with 4% fetal bovine serum (HyClone Laboratories, Logan, UT) were used
for monitoring AA and AM activities, for
125I-labeled ANG IV-receptor
binding assays, and for assessing the stimulatory effects of various
ANG fragments on ecNOS activity and cGMP production. Cell monolayers
were incubated in RPMI 1640 medium with and without the presence of
either ANG II or ANG IV (1 µM) for 2 h at 37°C. In some
experiments, the time-dependent effects of ANG IV (1 µM) on ecNOS
activity (30 min to 4 h) or protein expression (4-12 h) were
examined, and in other experiments, the concentration dependence (100 nM to 1 µM) of the effects of incubation with ANG IV for 2 h on ecNOS
activity was examined. We also determined the effect of the AA and AM
inhibitors amastatin and bestatin, respectively (18), on ANG
II-mediated ecNOS activity. In these experiments, cell monolayers were
pretreated with 50 µM each amastatin or bestatin in RPMI 1640 medium
for 30 min followed by 1 µM ANG II for 2 h at 37°C. To examine
the effects of ANG II and ANG IV antagonists, cell monolayers were
preincubated in RPMI 1640 medium containing 10 µM each
[Sar1,Ile8]ANG
II (an ANG II-receptor antagonist), losartan (an ANG II
AT1-receptor antagonist),
PD-123177 (an ANG II AT2-receptor
antagonist), or divalinal ANG IV (an
AT4-receptor antagonist) for 15 min followed by a 2-h incubation in the presence of 1 µM
each ANG II or ANG IV at 37°C. After incubation, the cells were
used to measure total membrane fraction ecNOS activity and cGMP
production. To determine the potential effect of ANG IV on
translocation of ecNOS, we also examined ecNOS activity in the cytosol.
In some experiments, the effects of
N-nitro-L-arginine
methyl ester (L-NAME),
methylene blue, and indomethacin, inhibitors of ecNOS, soluble
guanylate cyclase, and prostacyclin
(PGI2) production, respectively,
on ANG IV-stimulated cGMP generation were examined. In these
experiments, cell monolayers were preincubated in RPMI 1640 medium with
and without either 50 µM
L-NAME, 10 µM methylene blue,
or 10 µM indomethacin for 15 min followed by a 2-h incubation with
and without 1 µM ANG IV at 37°C.
Measurement of AA and AM activities.
Total membrane fractions from untreated cells were isolated as
previously described (26), and AA and AM activities were assayed with
L-aspartyl-
-naphthylamide and
L-alanyl-
-napthylamine,
respectively, as substrates as previously reported (18). Specific
activity is expressed as fluorescence units per minute per milligram of protein.
ANG IV-receptor binding. Specific
binding of 125I-ANG IV to membrane
receptors was determined with the method described by Hall et al. (14).
Briefly, cell monolayers in 24-well cluster trays were washed twice
with isotonic buffer A [50 mM
Tris · HCl, 150 mM NaCl, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 10 µM bestatin, and 50 µM
DL-2-mercaptomethyl-3-guandinoethylthiopropanoic acid
(Plummer's inhibitor), pH 7.4]. Cell monolayers were then incubated at 24°C for up to 90 min in a total volume of 250 µl of
buffer A containing 0.1% bovine serum
albumin and 1 nM 125I-ANG IV
(Amersham, Arlington Heights, IL) to determine total binding.
Nonspecific binding was determined by the addition of 10 µM unlabeled
human sequence ANG IV in the incubation mixture. After specific time
intervals, the cultures were washed three times with ice-cold
phosphate-buffered saline, pH 7.4. The cells were dissolved with 0.5 ml
of 0.2 N NaOH, and radioactivity was determined in a Beckman 5500 gamma
counter. Specific binding of 125I-ANG IV to PAECs was
determined by subtracting nonspecific binding from total binding.
Competition of binding of 125I-ANG
IV by the antagonist was carried out essentially as described above but
in the presence of 1 or 5 µM divalinal ANG IV (Hedral Therapeutics,
Portland, OR) (17, 34). In some experiments, the effects of 10 µM each
[Sar1,Ile8]ANG
II, losartan, and PD-123177 on
125I-ANG IV binding were also
examined. The protein contents of each sample were determined by the
method of Lowry et al. (19).
ecNOS activity. ecNOS activity was
measured by monitoring the formation of
L-[3H]citrulline from
L-[3H]arginine in total cell
membrane and cytosol fractions (26, 35). Total membranes (100-200
µg of protein) and cytosol (120 µg of protein) were incubated
(total volume 0.4 ml) in buffer (50 mM
Tris · HCl, 0.1 mM each of EDTA and EGTA, 1 mM
phenylmethylsulfonyl fluoride, and 1.0 mg of leupeptin/l, pH
7.4) containing 1 mM NADPH, 100 nM calmodulin, 10 µM
tetrahydrobiopterin, and 5 µM combined L-arginine and purified
L-[3H]arginine for 30 min at
37°C. Purification of
L-[3H]arginine and measurement of
L-citrulline formation were carried out as previously
described (24). The specific activity of ecNOS is expressed as
picomoles of L-citrulline per
minute per milligram of protein.
Western analysis of ecNOS. Control and
ANG IV-induced cells were washed twice with phosphate-buffered saline
and then lysed in boiled SDS-PAGE sample buffer (0.06 M
Tris · HCl, 2% SDS, and 5% glycerol, pH 6.8). The
cell lysate protein (15 µg) was fractionated on a 7.5% SDS-PAGE gel
and blotted onto a polyvinylidene difluoride membrane (Bio-Rad). The
blot was incubated in blocking solution and then hybridized with
anti-ecNOS monoclonal antibody as previously described (35). The
immunoreactive bands were visualized by enhanced chemiluminescence
reagent (Amersham) with Kodak X-OMAT film. The blots were scanned by
laser densitometry to quantify ecNOS protein content (24).
Measurement of cGMP production. Cell
monolayers were incubated with ANG II or ANG IV with and without ANG
II- or ANG IV-receptor antagonists as well as with and without
inhibitors of ecNOS, soluble guanylate cyclase, and
PGI2 as described in
Cell culture and
treatment. After incubation, the
monolayers were washed, and the cells were used to measure cGMP
content. A cGMP enzyme immunoassay system kit (Amersham) was used to
quantitate cGMP according to the manufacturer's instructions. No cGMP
was detected in the medium in any experiment.
Vasorelaxation of pulmonary arterial rings by ANG
IV. Fifth- to sixth-generation pulmonary arterial
segments were isolated from 6- to 7-mo-old pigs. Pulmonary arterial
rings (3 mm in diameter) prepared from these segments were
suspended in individual organ chambers with 25 ml of Krebs buffer
(composition in mM: 118 NaCl, 4.7 KCl, 1.9 CaCl2, 1.2 MgSO4, 1.0 K2HPO4,
25.0 NaHCO3, and 11.1 glucose)
containing 50 µM L-arginine
and were precontracted with 500 nM U-46619 (a thromboxane
A2 mimetic). After stable
contraction, the rings were washed several times, and ACh (1 µM) was
added to assess the endothelium-dependent vasorelaxation response
attributed to agonist-stimulated NO production. The chambers were
maintained at 37°C, and a resting force of 1.5 g was applied to the
arterial rings. In a separate set of experiments, U-46619-contracted
pulmonary arterial rings were incubated in the presence of the ANG II
AT1-receptor antagonist losartan
(10 µM) or the ANG II
AT2-receptor antagonist PD-123319
(10 µM), and a response to ANG IV (1 µM) was monitored. In some
experiments, the effects of the ecNOS inhibitor
L-NAME (100 µM) or the soluble
guanylate cyclase inhibitor methylene blue (10 µM) on ANG IV-mediated
vasorelaxation were assessed.
Statistical analysis. Significance for
the effects of ANG II, ANG IV, and their receptor antagonists on ecNOS
activity, cGMP production, and/or receptor binding in cultured
cells and the effects of ecNOS and soluble guanylate cyclase inhibitors
on ANG IV-mediated vasorelaxation of pulmonary arterial rings was
determined by analysis of variance and Student's
t-test (32).
 |
RESULTS |
AA and AM activities are present in membranes of
porcine PAECs. AA and AM activities in the total
membrane fraction of PAECs were 3.7 ± 0.5 and 32.4 ± 3.8 fluorescence
units · min
1 · mg
protein
1, respectively
(n = 6 for both). Although both AA and
AM activities are present in PAEC membranes, these results suggest that
AM activity is 10-fold greater than AA activity. These results also
indicate that PAECs are capable of metabolizing ANG II to ANG IV.
AT4-specific receptors are present on
porcine PAECs.
To demonstrate that ANG IV-specific binding sites are present on PAECs,
we measured binding of 125I-ANG IV
in the presence and absence of ANG IV- and ANG II-specific receptor
antagonists. As shown in Fig.
1A,
125I-ANG IV binding to PAECs was
time dependent. The total binding increased linearly and then reached a
plateau by 45 min, with a half-life of association of 24 min. Specific
binding was >90% of the total binding, and 76 and 89% of the
specific binding were blocked by 1 and 5 µM divalinal ANG IV,
respectively (Fig. 1B), whereas
<5% of 125I-ANG IV-specific
binding was blocked by
[Sar1,Ile8]ANG
II, losartan, or PD-123177 (data not shown).

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Fig. 1.
Time course of 125I-angiotensin
(ANG) IV association (A) and
antagonist competition of binding of
125I-ANG IV
(B) in porcine pulmonary arterial
endothelial cells (PAECs). Cells were incubated with 1 nM
125I-ANG IV at 24°C for up to
90 min with and without presence of 10 µM unlabeled ANG IV (for
nonspecific binding). ANG IV-specific binding ( ) was determined by
subtracting nonspecific binding ( ) from total binding
(A). For antagonist competition, cells were incubated in
presence of 125I-ANG IV at 24°C for 60 min with and
without presence of 1 or 5 µM divalinal ANG IV (B). Data
are means ± SE; n = 4 cells/point
in A and n = 6 cells in
B.
* P < 0.001 vs. control
cells.
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ANG IV-mediated activation of ecNOS is time and
concentration dependent. To determine whether ANG
IV-mediated activation of ecNOS is time dependent, ecNOS activity was
monitored from 30 min to 4 h in the presence and absence of 1 µM ANG
IV. As shown in Fig. 2, ecNOS
activity increased after 30 min of incubation, and the maximal
activation was observed at 1 h. This ANG IV-mediated activation of
ecNOS remained unchanged with more prolonged incubation for up to 4 h.
To examine the effect of ANG IV concentration, ecNOS activity was
monitored after 2 h of incubation in the presence of 100 nM to 1 µM
ANG IV. ecNOS activity in control monolayers (6.3 ± 0.5 pmol
L-citrulline · min
1 · mg
protein
1) increased to
7.0 ± 0.4, 8.6 ± 0.2, 8.8 ± 0.4, 8.3 ± 0.1,
and 9.0 ± 0.3 pmol
L-citrulline · min
1 · mg
protein
1 with 100 nM, 200 nM, 500 nM, 750 nM, and 1 µM ANG IV, respectively (P < 0.05 for 200 nM to 1 µM ANG
IV vs. control value). ecNOS activity in the cytosolic fraction was
<5% of the activity in the total membrane fraction and was
comparable in control and ANG IV-stimulated cells (data not shown).
These results suggest that ANG IV-induced activation of membrane
fraction ecNOS is associated with its receptor binding, and this
increased activity is not causally linked to translocation from the
cytosol.

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Fig. 2.
Time-dependent activation of endothelial cell constitutive nitric oxide
synthase (ecNOS) by ANG IV. Porcine PAEC monolayers were incubated in
RPMI 1640 medium alone (control; ) and with 1 µM ANG IV
( ) for 4 h at 37°C. After incubation, total membrane fraction
ecNOS activity was measured as described in MATERIALS
AND METHODS. Data are means ± SE;
n = 6 monolayers/time point.
* P < 0.05 vs. control
monolayers for all time points.
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|
ANG IV does not alter expression of ecNOS
protein. To examine whether ANG IV-induced activation
of ecNOS is associated with increased expression of ecNOS protein,
porcine PAECs were incubated with and without 1 µM ANG IV
for 4, 6, and 12 h, and ecNOS expression was analyzed by Western blot.
As shown in Fig. 3, the expression of ecNOS
protein in 4-, 6-, and 12-h ANG IV-stimulated cells was comparable to
that in control PAECs. This suggests that ANG IV-stimulated activation
of ecNOS is not mediated through increased expression of ecNOS protein.

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Fig. 3.
Western blot analysis of ecNOS protein in ANG IV-stimulated PAECs.
Porcine PAECs in RPMI 1640 medium alone [control (con)] and
with 1 µM ANG IV were incubated for 4, 6, and 12 h at 37°C. Cell
lysate protein (15 µg) was fractionated on a 7.5% SDS-PAGE gel,
blotted onto polyvinylidene difluoride membranes, and then hybridized
with anti-ecNOS monoclonal antibody. Blots were scanned with a laser
densitometer to quantify ecNOS protein content.
A: representative data from 1 of 3 independent experiments. Nos. at left,
molecular mass. B: results of
densitometric analysis of blots from the 3 independent experiments.
Values are means ± SE.
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ANG II-receptor antagonists fail to block ANG II- and
ANG IV-induced activation of ecNOS. As shown in
Fig. 4, ANG II- and ANG
IV-induced stimulation of ecNOS activity was blocked by the ANG
IV-specific antagonist divalinal ANG IV but not by ANG II and ANG II
AT1- or
AT2-specific receptor antagonists.
This suggests that activation of ecNOS by ANG II and ANG IV is mediated
through a novel AT4 receptor in
PAECs.

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Fig. 4.
Effect of ANG II (A) or ANG IV
(B) on ecNOS activity in porcine
PAECs. Cell monolayers were incubated with and without 10 µM each
[Sar1,Ile8]ANG
II (an ANG II-receptor antagonist), losartan (an ANG II
AT1-receptor antagonist),
PD-123177 (an ANG II AT2-receptor
antagonist), or divalinal ANG IV (an ANG-IV receptor antagonist) for 15 min followed by a 2-h incubation in presence of 1 µM each ANG II or
ANG IV at 37°C. After incubation, total membrane fraction ecNOS
activity was measured. 1, Control;
2, ANG II or ANG IV;
3, ANG II or ANG IV + [Sar1,Ile8]ANG
II; 4, ANG II or ANG IV + losartan;
5, ANG II or ANG IV + PD-123177;
6, ANG II or ANG IV + divalinal ANG
IV. Values are means ± SE; n = 8 monolayers. * P < 0.05 vs. control monolayers. ecNOS activity in monolayers incubated
with ANG II- or ANG IV-receptor antagonists alone was comparable to
that in control monolayers (data not shown).
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AA and AM activity inhibitors block ANG II-induced
activation of ecNOS. To examine whether metabolic
conversion of ANG II to ANG IV is critical for the activation of ecNOS,
the effects of the AA and AM inhibitors amastatin and bestatin,
respectively, on ANG II-induced ecNOS activity were determined. ecNOS
activity was increased by a 2-h incubation in the presence of 1 µM
ANG II (9.8 ± 0.5 vs. 6.5 ± 0.4 pmol
L-citrulline · min
1 · mg
protein
1;
P < 0.05;
n = 6 monolayers). Pretreatment with
amastatin and bestatin reduced ANG II-induced ecNOS activity from 9.8 ± 0.5 to 7.6 ± 0.3 pmol
L-citrulline · min
1 · mg
protein
1
(P < 0.05;
n = 6 monolayers). ecNOS activity in
cells treated with amastatin and bestatin in the absence of ANG II was
comparable to that in control cells. These results suggest that
metabolism of ANG II to ANG IV is critical for activation of ecNOS in PAECs.
ANG II and ANG IV increase cGMP production in
PAECs. In the vascular bed, ecNOS-generated NO
activates soluble guanylate cyclase, an enzyme that promotes conversion
of GTP to cGMP. This NO-cGMP signaling system is critical for
maintaining vascular tone (2, 4, 14). As shown in Fig.
5, both ANG II and ANG IV significantly increased cGMP production compared with control PAECs. In addition, incubation in the presence of the
AT4-receptor antagonist divalinal ANG IV but not in the presence of the ANG II-receptor antagonist [Sar1,Ile8]ANG
II blocked ANG II- and ANG IV-stimulated cGMP production. Thus both the
ANG II- and ANG IV-induced increases in cGMP appear to be mediated
through an AT4 receptor-linked
activation of ecNOS.

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Fig. 5.
Effect of ANG II and ANG IV on cGMP production in porcine PAECs. Cell
monolayers were incubated in RPMI 1640 medium containing 1 µM each
ANG II or ANG IV with and without 10 µM each ANG II- or ANG
IV-receptor antagonists
[Sar1,Ile8]ANG
II or divalinal ANG IV, respectively, for 2 h at 37°C. After
incubation, cGMP content of cells was measured.
1, Control;
2, ANG II;
3, ANG II + [Sar1,Ile8]ANG
II; 4, ANG II + divalinal ANG IV;
5, ANG IV;
6, ANG IV + [Sar1,Ile8]ANG
II; 7, ANG IV + divalinal ANG IV.
Values are means ± SE; n = 6 monolayers. * P < 0.001 vs.
control monolayers.
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ANG IV-induced cGMP production is blocked by
L-NAME and methylene blue but not by
indomethacin.
To determine whether ANG IV-induced cGMP production is associated with
activation of ecNOS and the generation of NO, the effects of the ecNOS
inhibitor L-NAME and the soluble
guanylate cyclase inhibitor methylene blue were examined. We also
determined the specificity of NO-mediated cGMP production by using an
inhibitor of PGI2 production. As
shown in Fig. 6, ANG IV
significantly increased cGMP production in PAECs
(P < 0.001). Incubations in the
presence of L-NAME or methylene
blue but not of indomethacin significantly diminished ANG IV-stimulated
as well as basal levels of cGMP in PAECs
(P < 0.001). These results suggest
that ANG IV-induced cGMP production for the most part is causally
linked to the activation of ecNOS and subsequent release of NO but not
to PGI2 production by porcine
PAECs.

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Fig. 6.
Effects of
N-nitro-L-arginine
methyl ester (L-NAME), methylene
blue (MB), and indomethacin (Indo) on basal and ANG IV-stimulated cGMP
production in porcine PAECs. Cell monolayers were preincubated in RPMI
1640 medium with and without 50 µM
L-NAME or 10 µM each MB or
Indo for 15 min followed by a 2-h incubation with and without 1 µM
ANG IV at 37°C. After incubation, cGMP content of cells was
measured. Values are means ± SE; n = 6 monolayers. Basal cGMP content in MB-treated cells was below
detectable levels. * P < 0.001 vs. control monolayers. ** P < 0.001 vs. respective basal or ANG IV control monolayers.
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ANG IV causes endothelium-dependent vasorelaxation of
porcine pulmonary arterial rings. As shown in Fig.
7, addition of U-46619 to porcine pulmonary
arterial rings elicited significant vasoconstriction. The addition of 1 µM ACh resulted in >70% relaxation. Similarly, the addition of ANG
IV elicited significant relaxation in U-46619-contracted arterial
rings. The ANG IV-mediated vasodilatory effects on arterial rings were
blocked by L-NAME and methylene
blue. This ANG IV-mediated vasodilation in porcine pulmonary arterial
rings appears to be regulated through activation of ecNOS and NO
release.

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Fig. 7.
ANG IV-mediated vasorelaxation of porcine pulmonary arterial rings.
Top: representative recordings of
thromboxane A2 mimetic U-46619
(500 nM)-induced contraction and effects of ACh (1 µM), losartan (10 µM), PD-123319 (10 µM), and ANG IV (1 µM).
Bottom: effects of
L-NAME (100 µM) and MB (10 µM) on ANG IV-mediated vasorelaxation. All data are means ± SE;
n = 4 monolayers.
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 |
DISCUSSION |
We demonstrate here for the first time that ANG IV causes
concentration- and time-dependent activation of ecNOS in lung
endothelial cells and that this activation is mediated through a novel
AT4 receptor in these cells.
Time-dependent 125I-ANG
IV-specific binding, which was blocked by an
AT4-specific receptor antagonist
but not by ANG II-specific receptor antagonists, further confirms the
presence of AT4 receptors on
PAECs. The AT4 receptor-mediated
activation of ecNOS is physiologically important because endogenous NO
produced from L-arginine via an
oxidative catabolic reaction mediated by ecNOS has been shown to reduce pulmonary hypertension (6, 9, 13, 16). The catalytic activity of ecNOS
is directly linked to NO production and activation of soluble guanylate
cyclase, and this leads to modulation of vascular tone through an
NO-cGMP signaling mechanism (6, 20). The results of the present study
are consistent with this physiological mechanism and demonstrate that
ANG IV-induced cGMP production is causally linked to the activation of
ecNOS as an inhibitor of ecNOS-blocked cGMP production. Near-complete
inhibition of basal and ANG IV-mediated cGMP production by
L-NAME and methylene blue and
the failure of indomethacin to block ANG IV-induced cGMP further
confirmed the specificity of the NO-cGMP signaling mechanism in PAECs. These in vitro results are consistent with the observation that ANG IV induced vasodilation in porcine pulmonary arterial rings
that was blocked by L-NAME and
methylene blue, suggesting that NO release is most likely responsible
for this in vivo response. The presence of a novel
AT4 receptor has recently been
reported in rat brain, kidney, and heart tissues (13, 17, 21, 34), and
these reports suggested that increased blood flow is associated with
ANG IV, at least in the brain and kidney (17, 21). The results of the
present study demonstrating the presence of
AT4 receptors in PAECs are
consistent with these reports, but they also identify a specific
mechanism involving activation of ecNOS, resulting in NO-cGMP-linked
vasodilation. ANG IV-induced activation of ecNOS in PAECs is not
associated with increased expression of ecNOS protein or increased
translocation of ecNOS from the cytosol to the membranes. Although this
suggests that posttranscriptional modulation of ecNOS is responsible
for ANG IV-induced activation of ecNOS, the precise mechanism of
activation remains to be determined.
Our results also indicate that ANG II-induced activation of ecNOS and
generation of cGMP in PAECs are mediated through ANG IV. This appears
to be linked to the presence of AA and AM activities in lung
endothelial cell membranes, which are known to actively metabolize ANG
fragments. ANG II is converted to ANG III by AA, which is primarily
responsible for cleaving aspartic acid from the
NH2 terminus of ANG II, whereas
ANG III is further converted to the hexapeptide ANG IV by AM, which
cleaves arginine from the NH2
terminus of ANG III in various tissues (33). The fact that inhibitors
of AA and AM activities prevented ANG II-mediated activation of ecNOS
further confirms that metabolism of ANG II is critical for activation
of ecNOS. The ability of the
AT4-receptor antagonist divalinal
ANG IV but not the ANG II-receptor antagonists
[Sar1,Ile8]ANG
II, losartan, and PD-123177 to block ANG II- and ANG IV-mediated activation of ecNOS and production of cGMP confirms that these ANG
II-induced responses are mediated through the formation of ANG IV.
Although our data clearly demonstrate that ANG IV-induced activation of
ecNOS results in NO-cGMP-mediated vasorelaxation in porcine pulmonary
arterial rings, a few recent reports suggested that ANG IV is a weak
vasoconstrictor in the pulmonary circulation of the rat (22) and the
cat (4) and that this vasoconstrictor action is modulated by NO release
(22). Although it is not clear how ANG IV acts as a weak
vasoconstrictor, several possibilities exist. For example, species
differences in the catalytic activities of AA and AM may influence
endogenous levels of ANG II and ANG IV and, therefore, the vasomotor
response. Similarly, the physiological response to ANG II and ANG IV
may be dependent on the density of their specific receptors in a given
tissue in a species. Endogenous levels of ANG IV and the presence of
AT4 receptors may be critical in
counterbalancing the vasoconstrictive effects in the pulmonary circulation. Differences in the catalytic activities of AA and AM
and/or ANG II- and ANG IV-receptor densities between conduit vessels and resistance vessels may also be playing a role. However, this latter possibility seems less likely in view of the congruence between our results in cultured cells derived from conduit pulmonary vessels and our results in arterial rings derived from resistance vessels. Finally, differences in the levels of extracellular
L-arginine may explain why ANG
IV acts as a vasodilator in our system and as a weak vasoconstrictor in
other experimental models.
L-Arginine added to the
extracellular medium results in the generation and release of NO from
vascular preparations even in the presence of saturating levels of
intracellular L-arginine (1, 8, 28, 30). Therefore, NO production may depend on the presence of
adequate amounts of extracellular
L-arginine. The incubation mixture used in the present organ bath studies contains a physiological concentration (50 µM) of
L-arginine (2). Although the
L-arginine content in the
isolated perfused lung experiments describing a weak vasoconstrictive
action for ANG IV was not reported, it is likely that this
concentration was less than physiological because the perfusate in
these studies was blood that had been diluted by a factor of 3 (4, 21).
As such, the vasoconstrictive response of ANG IV may be due to an
inadequate amount of L-arginine
in the perfusate.
In conclusion, our results reveal the presence of novel
AT4 receptors in porcine lung
endothelial cells that activate ecNOS and generate cGMP, resulting in
vasodilation in porcine pulmonary arterial rings. This ANG IV-mediated
vasodilatory response may be important in the management of pulmonary
hypertension, especially when the pulmonary hypertension is the primary
manifestation of lung vascular dysfunction such as occurs in patients
with primary pulmonary hypertension and chronic obstructive lung
disease (7, 11). Moreover, a number of other factors such as drug- or
chemical-induced endothelial cell injury as well as stress, aging, and
dietary changes may influence endothelial cell-mediated
vasoconstrictive or vasodilatory responses in the pulmonary
circulation. For example, injury or species-related differences in AA
and AM activities in lung endothelial cells will result in changes in
the relative proportions of ANG II and ANG IV in the pulmonary
circulation that may dictate the pulmonary vascular response.
Similarly, the absolute and relative densities of ANG II and
AT4 receptors in the pulmonary
vascular bed will affect its vasomotor response to physiological and
pathophysiological stimuli. Increasing pulmonary levels of ANG IV or
increasing the density of AT4
receptors in the pulmonary circulation or both may offer new
therapeutic avenues for the management of pulmonary hypertension and
the pulmonary vascular complications accompanying a variety of acute
and chronic lung disorders.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Joseph W. Harding (Washington State University and
Hedral Therapeutics, Portland, OR) for providing divalinal angiotensin
IV. We thank Bert Herrera for tissue culture assistance, Janet Wootten
for excellent editorial help, Denise Christian for secretarial
assistance, and Weihong Han for technical assistance.
 |
FOOTNOTES |
This work was supported by the Medical Research Service of the
Department of Veterans Affairs and National Heart, Lung, and Blood
Institute Grant HL-58679.
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. §1734 solely to indicate this fact.
Address for reprint requests: J. M. Patel, Research Service (151), VA
Medical Center, 1601 S.W. Archer Road, Gainesville, FL 32608-1197.
Received 13 April 1998; accepted in final form 26 August 1998.
 |
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