2 Research Service, Malcom Randall Department of Veterans Affairs Medical Center, and 1 Department of Medicine, University of Florida College of Medicine, Gainesville, Florida 32608
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ABSTRACT |
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Angiotensin (ANG) IV stimulation of pulmonary artery (PA) endothelial cells (PAECs) but not of PA smooth muscle cells (PASMCs) resulted in significant increased production of cGMP in PASMCs. ANG IV receptors are not present in PASMCs, and PASMC nitric oxide synthase activity was not altered by ANG IV. ANG IV caused a dose-dependent vasodilation of U-46619-precontracted endothelium-intact but not endothelium-denuded PAs, and this response was blocked by the ANG IV receptor antagonist divalinal ANG IV but not by ANG II type 1 and 2 receptor blockers. ANG IV receptor-mediated increased intracellular Ca2+ concentration ([Ca2+]i) release from intracellular stores in PAECs was blocked by divalinal ANG IV as well as by the G protein, phospholipase C, and phosphoinositide (PI) 3-kinase inhibitors guanosine 5'-O-(2-thiodiphosphate), U-73122, and LY-294002, respectively, and was regulated by both PI 3-kinase- and ryanodine-sensitive Ca2+ stores. Basal and ANG IV-mediated vasorelaxation of endothelium-denuded PAs was restored by exogenous PAECs but not by exogenous PAECs pretreated with the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. These results demonstrate that ANG IV-mediated vasodilation of PAs is endothelium dependent and regulated by [Ca2+]i release through receptor-coupled G protein-phospholipase C-PI 3-kinase signaling mechanisms.
smooth muscle cells; guanosine 3',5'-cyclic monophosphate; nitric oxide synthase; angiotensin IV receptor; signal transduction
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INTRODUCTION |
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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 (11, 20, 21, 23, 33). The presence of ANG IV-specific binding sites has been identified in several mammalian tissues and cells including the brain (11, 20), heart membranes (16), kidney (14, 15), human collecting duct cells (6), and cultured bovine aortic vascular smooth muscle cells (12) as well as in cultured coronary microvascular (13), aortic (1, 19, 20, 29), and lung endothelial (17, 25, 26) cells. The mechanism of ANG IV-mediated regulation of blood flow is not well characterized, and this is especially true in the lung. Patel et al. (26) reported that ANG IV receptor-mediated activation of the endothelial cell isoform of nitric oxide (NO) synthase (ecNOS) increases NO release and cGMP production, leading to NO/cGMP-mediated vasorelaxation of porcine pulmonary artery (PA) segments. A more recent report (25) from our group demonstrated that ANG IV stimulation increases intracellular Ca2+ concentration ([Ca2+]i) release in PA endothelial cells (PAECs). Although ANG IV causes vasorelaxation in porcine PAs, it is not known whether this response is endothelium dependent, is mediated through the presence of ANG IV receptors in PA smooth muscle cells (PASMCs), or is coordinated by both PAECs and PASMCs.
The catalytic activity of ecNOS is Ca2+ and calmodulin dependent and is transiently activated by agonist-mediated signaling pathways that increase [Ca2+]i mobilization (7, 18). Receptor-mediated activation of signal transduction pathways can result in direct and/or G protein-coupled activation of phospholipase (PL) C and PLD, leading to a rapid increase in [Ca2+]i (2, 27, 30). Increased [Ca2+]i is regulated by multiple mechanisms including one of the most ubiquitous pathways involving the PLC-phosphatidylinositol (PI) 3-kinase pathway, which releases Ca2+ from intracellular stores, namely the endoplasmic reticulum (ER) (2, 30). Alternatively, increased [Ca2+]i can also be regulated through ryanodine-sensitive Ca2+ stores (5, 31, 32). The role of specific signaling events in the regulation of ANG IV-mediated [Ca2+]i mobilization remains to be determined. Thus the purpose of the present study was to define the mechanism responsible for ANG IV-mediated regulation of pulmonary blood flow. To do this, we have 1) examined the role of the endothelium in ANG IV-mediated vasorelaxation using an in situ model of endothelium-denuded PA segments as well as a PAEC-PASMC coculture system, 2) determined which intracellular Ca2+ store (PI 3-kinase versus ryanodine sensitive or both) mediates the ANG IV-induced [Ca2+]i release, and 3) evaluated whether signaling mechanisms involving G proteins, PLC, and PI 3-kinase are associated with the ANG IV-induced [Ca2+]i release in PAECs.
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MATERIALS AND METHODS |
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Cell culture.
PAECs and PASMCs were obtained from the main PAs of 6- to 7-mo-old
pigs. Endothelial cells were propagated in monolayers as previously
described (24). Third- to fifth-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 in all experiments. PASMCs were
isolated from middle media explants after removal of the endothelial,
subendothelial, and adventitial layers of arterial segments as
previously described (9). Tissue explants were cultured
for 2 wk in MEM- containing 15% fetal bovine serum. After 2 wk,
individual cell colonies grown from the explants were isolated and
subcultured (9, 10). All studies involving PAECs and
PASMCs were carried out with cells at passages
3-5. In each experiment, PAECs and/or PASMCs were
studied 1 or 2 days after confluence and were matched for cell line,
passage number, and days after confluence.
ANG IV-mediated cGMP production in PASMCs. To determine whether ANG IV-stimulated activation of ecNOS results in the increased endothelial cell release of NO that is responsible for increased production of cGMP in PASMCs, a Transwell coculture system (Costar, Cambridge, MA) was used. PAECs were seeded on the microporous surface of the removable upper chamber, and PASMCs were cultured on the lower chamber. Transwell units containing PAECs and PASMCs in RPMI 1640 medium without serum were preincubated for 30 min at 37°C. After a 30-min incubation, ANG IV (1 µM) or RPMI 1640 medium only (control) was added to the upper chamber. The Transwell units containing both cell lines were incubated for 10-60 min at 37°C. Basal as well as ANG IV-stimulated cGMP levels in PASMCs and in medium from the PASMCs were measured. In some experiments, PAECs were preincubated with 50 µM N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of ecNOS, before ANG IV stimulation. The direct effect of ANG IV on cGMP production in PASMCs was independently monitored. A cGMP enzyme immunoassay system kit (Amersham) was used to quantitate cGMP according to the manufacturer's instructions. In brief, before and after ANG IV stimulation, PASMCs and medium from the PASMCs were collected. The medium was rapidly frozen, and the cells were suspended in ice-cold ethanol to give a final volume of 65% (vol/vol) ethanol. After centrifugation, the supernatants were collected and dried under a stream of nitrogen at 60°C and dissolved in assay buffer. Cell extracts and medium samples (1 ml each) were used to measure cGMP content by an acetylation assay as described by the manufacturer (Amersham, Arlington Heights, IL). cGMP was not detectable in the medium in any experiment.
ANG IV receptor binding in PASMCs. Specific binding of 125I-ANG IV to PASMC membrane receptors was determined as previously described (26). In brief, cells in 35-mm culture dishes 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), and 10 µM each losartan and PD-123177, pH 7.4]. The cells were then incubated at 24°C for up to 90 min in a total volume of 0.5 ml of buffer A containing 0.1% heat-inactivated 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. At 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. The protein contents of each sample were determined with the method of Lowry et al. (22).
NOS activity in PASMCs. Cells were incubated in RPMI 1640 medium with and without the presence of ANG IV (1 µM) for 2 h at 37°C. In some experiments, the time-dependent (30 min to 6 h) effect of ANG IV (1 µM) on NOS activity was examined. After incubation, NOS activity was measured by monitoring the formation of L-[3H]citrulline from L-[3H]arginine in the total membrane and cytosol fractions as previously reported by Patel et al. (26) and Zhang et al. (39). Total membranes (80 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 EDTA and EGTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/l of leupeptin, 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-[3H]citrulline formation were carried out as previously described (28). The specific activity of NOS is expressed as picomoles of L-citrulline per 30 min per milligram of protein.
Vasorelaxation of PA rings. PA segments (2- to 3-mm diameter × 5- to 6-mm length) were isolated from the lungs of 6- to 7-mo-old pigs as previously described (26). The endothelium of some segments was denuded by gently rubbing the lumen of the vessel with a roughened metal rod. Isolated vessels were preserved in a buffer solution (composition in mM: 128 NaCl, 4.7 KCl, 1.9 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 10.0 HEPES, and 11.1 glucose, pH 7.4) containing 2% Ficoll at 4°C before use. To measure the vasorelaxation response, arterial segments were suspended in individual organ bath chambers with 20 ml of Krebs buffer (composition in mM: 118 NaCl, 4.7 KCl, 1.9 CaCl2, 1.2 MgCl2, 1.2 K2HPO4, 25.0 NaHCO3, and 11.1 glucose, pH 7.4) containing 50 µM L-arginine and oxygenated with 95% O2-5%CO2 at 37°C. An initial resting force of 3 g was applied to the arterial rings. After equilibration, the rings were precontracted with 100 nM U-46619 (a thromboxane A2 mimetic). To standardize the data, U-46619-induced stable vascular tone was set as 100%, and the rings were treated with varying concentrations (0.01-10 µM) of ANG IV. Endothelium integrity or lack of response in endothelium-denuded segments was confirmed by monitoring acetylcholine (25 µM)-mediated vasodilation before further experimentation. In some experiments, ANG IV-mediated vasodilation was monitored in the presence of the ANG IV receptor antagonist divalinal ANG IV (5 µM) (12-14, 37), the ANG II type 1 (AT1) receptor antagonist losartan (10 µM), the ANG II type 2 (AT2) receptor antagonist PD-123319 (10 µM), or the ecNOS inhibitor L-NAME (50 µM). We also monitored the vasorelaxation effect of ANG IV (1 µM) in U-46619-precontracted endothelium-denuded rings after the addition of exogenous PAECs (2 × 106) cultured on Metricel membrane filters (pore size 0.2 µm; Gelman Sciences, Ann Arbor, MI) and pretreated with the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (50 µM in DMSO for 30 min) or DMSO only (control). The vasodilatory response was continuously monitored with an isometric force transducer (Harvard Apparatus, Holliston, MA) and the PO-NE-MATH WIN-SMT smooth muscle tissue system (Gould Instrument System, Valley View, OH). At the end of each experiment, endothelial integrity was reconfirmed by monitoring the response to acetylcholine (1 µM).
ANG IV-mediated [Ca2+]i regulation.
To examine the ANG IV receptor-mediated pathways involved in
[Ca2+]i mobilization, indo 1 was used to
measure [Ca2+]i release by confocal
fluorescent microscopy. In brief, PAEC monolayers were loaded with indo
1-AM by incubation with 10 µM indo 1-AM (Molecular Probes, Eugene,
OR) in 2 ml of Ca2+-free buffer solution (composition in
mM: 140 NaCl, 3.9 KCl, 1.5 KH2PO4, 5.5 glucose,
10.0 HEPES, and 2.0 EDTA, pH 7.4) at 37°C for 30 min. After
incubation, the cell monolayers were washed and placed in a model
PDMI-2 open perfusion microincubator (Medical System, Greenvale, NY)
connected to a temperature controller (Harvard Apparatus, South Natick,
MA) to maintain the system at 37°C. To monitor the fluorescent
digital images of indo 1, the culture dishes together with the
microincubator were placed on a confocal laser scanning system (Zeiss
510 LSM confocal microscope, Zeiss, Thornwood, NY) equipped with an
ultraviolet argon-ion laser (Enterprise Ion Laser, Coherent, Santa
Clara, CA). The fluorescence emitted after indo 1-Ca2+
binding was recorded in two-dimensional dual images with 475-nm and
380- to 430-nm filters in a time-dependent manner. The ratio of 380- to
430-nm to 475-nm recorded emission fluorescence from three to seven
cells in focused areas of the observing dish was used to determine
Ca2+ concentration with the accessory time-course software
of the confocal system. After a stable basal signal was established, the cells were stimulated with ANG IV (10 µM). To determine the roles
of 1) ANG IV-specific receptors, 2) intracellular
Ca2+ stores (PI 3-kinase vs. ryanodine sensitive or both),
and 3) specific signaling pathways involving G proteins,
PLC, and PI 3-kinase in the regulation of
[Ca2+]i release, the cells were separately
pretreated with 1) the ANG IV antagonist divalinal ANG IV
(10 µM), the ANG II AT1 receptor antagonist losartan (10 µM), and the ANG II AT2 receptor antagonist PD-123319 (10 µM); 2) thapsigargin (5 µM) and cyclopiazonic acid (10 µM), both of which block Ca2+ entry into the ER, causing
the ER to empty, and caffeine (10 µM) and ryanodine (10 µM), which
cause Ca2+ release from the ER by blocking the
Ca2+ release channel of the ER; and 3)
pharmacological concentrations of inhibitors of G proteins [250 and
500 µM guanosine 5'-O-(2-thiodiphosphate) (GDPS)]
(4), PLC (5 and 10 µM U-73122) (38), and PI
3-kinase (8 and 16 µM LY-294002) (34). After a 10-min
preincubation with these agents, the cells were stimulated with ANG IV
(10 µM), and the fluorescence intensity of more than nine randomly
focused cells was measured as described above. A single dose of ANG IV was selected from the ANG IV dose-response curve (0.01-10 µM) to
demonstrate the maximal effect. Fluorescence intensities in cells
pretreated with GDP
S, U-73122, and LY-294002 for 10 min were
comparable to control values (data not shown).
Statistical analysis. Significance for the effects of ANG IV, ANG IV receptor antagonists, inhibitors of signaling pathways, and BAPTA-AM on cGMP content; NOS activity; and [Ca2+]i release in PAECs and/or PASMCs as well as for the effects of ANG IV in mediating the vasodilatory responses in endothelium-intact and endothelium-denuded PAs was determined with ANOVA and Student's paired t-test (36). Values are means ± SE for n experiments.
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RESULTS |
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ANG IV-stimulated cGMP production in PASMCs is endothelium
dependent.
Basal and ANG IV-stimulated levels of cGMP in PASMCs were 4.3 ± 0.8 and 4.9 ± 0.5 pmol/mg protein, respectively
(n = 8 in each set). As shown in Fig.
1, after a 30-min preincubation of PASMCs
and PAECs in Transwell coculture units, the basal level of cGMP in
PASMCs was increased to 11.8 ± 3.4 pmol/mg protein (n = 6) and remained at a comparable level for up to
2 h (data not shown). However, after stimulation of PAECs with ANG
IV (1 µM), cGMP levels in PASMCs were significantly increased in a
time-dependent manner (P < 0.01 vs. basal level at all
time points). Pretreatment of PAECs with 50 µM L-NAME
abolished the endothelium-ANG IV-mediated cGMP stimulatory effect on
PASMCs. No cGMP was detected in the medium in any experiment.
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ANG IV does not bind to or activate NOS in PASMCs.
As shown in Fig. 2A, total and
nonspecific binding of 125I-ANG IV were comparable in
PASMCs, indicating the lack of ANG IV-specific binding sites in these
cells. However, under similar conditions, 125I-ANG
IV-specific binding in PAECs was 78 ± 7.5 fmol · h1 · mg protein
1
(n = 6). Nonspecific binding was <5% of specific
binding in PAECs. Figure 2B shows NOS activity in total
membrane and cytosol fractions of control and ANG IV-stimulated PASMCs.
NOS activity in total membrane and cytosol fractions from ANG
IV-stimulated cells was comparable to the activity in the respective
fractions from unstimulated cells.
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ANG IV-mediated vasorelaxation is dose dependent and regulated by
ANG IV receptors.
As shown in Fig. 3, ANG IV caused
vasorelaxation of U-46619-precontracted PAs (5- to 6-mm diameter) in a
dose-dependent manner. ANG IV-mediated vasodilatory responses at
concentrations of 1 and 10 µM were maximal and comparable. Addition
of the ANG IV antagonist divalinal ANG IV (12-14,
37), but not of the ANG II antagonists losartan and
PD-123177, blocked the ANG IV-mediated vasodilatory response in PAs.
The addition of acetylcholine (25 µM) resulted in 80% relaxation.
ANG IV-mediated vasodilatory responses in smaller vessels (2- to 3-mm
diameter) were comparable to those observed in 5- to 6-mm diameter
vessels (data not shown).
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ANG IV-mediated vasorelaxation in PAs is endothelium dependent.
To confirm the role of endothelium in ANG IV-mediated relaxation in
PAs, endothelium-intact and endothelium-denuded PAs were used in situ.
We also examined the effect of exogenous PAECs on ANG IV-mediated
vasorelaxation in endothelium-denuded PAs. As shown in Fig.
4A, ANG IV and acetylcholine
caused vasodilation of U-46619-precontracted endothelium-intact PAs but
not of endothelium-denuded PAs. Figure 4B shows that the
addition of exogenous PAECs (2 × 106) grown on
Metricel membrane filters to the organ bath chamber resulted in ANG IV
(500 nM and 10 µM)-mediated relaxation of U-46619-precontracted endothelium-denuded PAs. In the absence of exogenous PAECs, ANG IV
failed to cause relaxation of U-46619-precontracted endothelium-denuded PAs. In all experiments, preincubation of PAs with 50 µM
L-NAME abolished the ANG IV-mediated vasorelaxation (data
not shown).
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ANG IV receptor-mediated signaling is associated with
[Ca2+]i release.
Patel et al. (25) previously reported that in the presence
and absence of extracellular Ca2+, ANG IV stimulation
resulted in [Ca2+]i release in a
dose-dependent manner in PAECs. Here we determined the specificity for
the ANG IV receptor as well as the role of specific signaling events in
ANG IV-mediated [Ca2+]i release in PAECs. As
shown in Fig. 5A, in the
absence of extracellular Ca2+, ANG IV-stimulated increased
[Ca2+]i release in a dose-dependent fashion.
The ANG IV-specific receptor antagonist divalinal ANG IV, but not the
ANG II AT1 and AT2 receptor antagonists
losartan and PD-123319, respectively, diminished ANG IV-mediated
[Ca2+]i mobilization (P < 0.01 vs. ANG IV and ANG IV plus losartan plus PD-123319; Fig.
5B).
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ANG IV-mediated [Ca2+]i release in PAECs
is regulated by at least two major mechanisms.
To determine which intracellular Ca2+ store (PI 3-kinase
versus ryanodine sensitive or both) mediates the ANG IV-stimulated [Ca2+]i release, the effects of ANG IV were
examined in the presence of modulators of PI 3-kinase- and
ryanodine-sensitive pathways. As shown in Fig.
6, ANG IV stimulated
[Ca2+]i above basal (control) levels, and the
stimulation was significantly inhibited by pretreatment with caffeine
and ryanodine, which cause Ca2+ release from the ER by
blocking the Ca2+ release channel of the ER
(P < 0.01 vs. ANG IV for both). Similarly, pretreatment with thapsigargin and cyclopiazonic acid, which block Ca2+-ATPase of the ER, significantly reduced the ANG
IV-mediated increase in [Ca2+]i
(P < 0.01 vs. ANG IV for both). Despite a reduction in
ANG IV-mediated increased Ca2+ levels by these modulators,
[Ca2+]i remained significantly elevated above
the basal level (P < 0.01 vs. control for all
modulators), possibly due to limited inhibitory effects of these
modulators. Thus ANG IV-mediated [Ca2+]i
release in PAECs is regulated by two major ER pathways.
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ANG IV-mediated [Ca2+]i release in PAECs
is critical for vasorelaxation in PAs.
To evaluate the importance of endothelial Ca2+ release to
vasorelaxation in PAs, we examined the effects of ANG IV-mediated vasorelaxation in U-46619-precontracted endothelium-denuded vessels with and without the presence of exogenous cultured untreated PAECs and
PAECs pretreated with the intracellular Ca2+ scavenger
BAPTA-AM. As shown in Fig. 7, the
addition of cultured PAECs that were pretreated with and without
(control) BAPTA-AM in an organ bath chamber caused relaxation in
U-46619-contracted endothelium-denuded PA rings. Addition of ANG IV to
the chamber of control but not of BAPTA-AM-treated PAECs caused a
further relaxation in U-46619-contracted endothelium-denuded PA rings. This ANG IV-mediated vasodilation was blocked by the NOS inhibitor L-NAME (data not shown).
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DISCUSSION |
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A recent report demonstrated that ANG IV-mediated PA vasorelaxation is causally linked to activation of the constitutively expressed lung ecNOS by a receptor-mediated pathway, leading to increases in [Ca2+]i and NO release, production of cGMP, and activation of NO/cGMP-mediated signaling mechanisms (26). The present study represents the first report that addresses the mechanisms responsible for ANG IV-mediated regulation of blood flow in the pulmonary circulation. Using cultured porcine PASMCs, PAEC-PASMC cocultures, and isolated porcine PA segments, we demonstrated that 1) ANG IV receptors are not present in PASMCs and PASMC NOS activity is not altered by ANG IV, 2) ANG IV-mediated endothelial stimulation and activation of ecNOS are critical for increased cGMP levels in PASMCs in a coculture system, 3) ANG IV receptor-mediated vasorelaxation in U-46619-contracted, endothelium-denuded PAs is endothelium dependent, 4) ANG IV receptor-mediated mobilization of [Ca2+]i is regulated through G protein, PLC, and PI 3-kinase signaling pathways and the mobilized Ca2+ is derived from both PI 3-kinase- and ryanodine-sensitive ER Ca2+ stores, and 5) [Ca2+]i in PAECs is critical for vasorelaxation of U-46619-contracted endothelium-denuded PA segments.
ANG IV-mediated increased cGMP in PASMCs and vasorelaxation of U-46619-contracted endothelium-intact PA segments are clearly endothelium dependent and regulated by ANG IV-specific receptors. The lack of 125I-ANG IV-specific binding in PASMCs as well as the lack of ANG IV-mediated stimulation of NOS in PASMCs further confirms that the ANG IV-mediated responses in lung vascular cells and PAs are endothelium dependent. A previous study by Hall et al. (12) reported the presence of ANG IV binding sites in cultured bovine aortic smooth muscle cells. However, the ANG IV-mediated functional response was not examined by these authors. The lack of ANG IV binding sites in porcine PASMCs in the present study may be due to differences in species and/or heterogeneity in vascular bed dynamics, including site and size of the vasculature.
Our results also demonstrate that ANG IV-mediated
[Ca2+]i mobilization in PAECs is critical for
the vasodilatory function of PAs inasmuch as this response is abrogated
by the intracellular Ca2+ chelator BAPTA-AM.
Agonist-mediated Ca2+ mobilization from internal stores is
associated with receptor-linked signaling pathways. One of the most
ubiquitous pathways is receptor-G protein-coupled activation of PLC,
leading to increased production of PI 3-kinase, which is known to
mobilize Ca2+ from intracellular Ca2+ pools,
namely the ER (2, 5, 32). The results of the present study
are consistent with this receptor-mediated signaling mechanism because
1) ANG IV-mediated
[Ca2+]i mobilization occurs in
the absence of extracellular free Ca2+ and is blocked by
ANG IV receptor-specific but not by ANG II AT1 and
AT2 receptor-specific antagonists and 2) ANG
IV-mediated [Ca2+]i
mobilization was attenuated by GDPS, U-73122, and LY-294004, selective inhibitors of G protein, PLC, and PI 3-kinase, respectively.
The ER in mammalian cells contains two distinct intracellular Ca2+ stores, namely PI 3-kinase- and ryanodine-sensitive Ca2+ stores (2, 5, 31, 32). Our results with agents that block the uptake of Ca2+ in the ER (thapsigargin and cyclopiazonic acid) or agents that release Ca2+ from ryanodine-sensitive stores (caffeine and ryanodine) demonstrate that the ANG IV-mediated mobilization of [Ca2+]i is attenuated by these modulators of PI 3-kinase- and ryanodine-sensitive intracellular Ca2+ stores in PAECs. This suggests that the ANG IV-mediated increased [Ca2+]i release is dependent on both PI 3-kinase- and ryanodine-sensitive intracellular Ca2+ stores in porcine PAECs. Although similar observations involving agonist-mediated increased cytosolic free Ca2+ from both PI 3-kinase- and ryanodine-sensitive stores in diverse mammalian cells have been previously reported (31, 35), the mechanistic differences in the roles of PI 3-kinase- and ryanodine-sensitive Ca2+ stores leading to a selective physiological response by ANG IV or other agonists remain to be determined. Irrespective of the origin of Ca2+ release, our results demonstrate that ANG IV-induced endothelial cell [Ca2+]i mobilization is critical for vasorelaxation of PAs. This is consistent with previous reports by Patel and colleagues that the intracellular Ca2+ chelator BAPTA-AM abolishes ANG IV-mediated activation of ecNOS (25), which is critical for NO/cGMP-mediated vasorelaxation of PAs (26).
ANG IV receptor-mediated activation of signaling events leading to increased [Ca2+]i is a new signaling pathway that may prove to be physiologically significant for vascular regulation through the NO/cGMP mechanism. Agonists, including ANG IV-stimulated [Ca2+]i release from ER stores, have been shown to facilitate a process that results in increased synthesis of Ca2+ binding proteins such as calreticulin, which is located within the lumen of the ER (3, 8). This is particularly important because Patel et al. (25) recently reported that ANG IV-mediated sustained activation of ecNOS may be associated with the interaction of ecNOS and calreticulin proteins in PAECs. Thus ANG IV-mediated vasorelaxation in PAs appears to be regulated by at least two major mechanisms through the modulation of Ca2+ homeostasis.
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ACKNOWLEDGEMENTS |
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We thank Bert Herrera for tissue culture assistance, Janet Wootten for excellent editorial help, Addie Heimer for secretarial assistance, and Weihong Han for technical assistance.
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FOOTNOTES |
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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.
Address for reprint requests and other correspondence: J. M. Patel, Research Service (151), VA Medical Center, 1601 S.W. Archer Road, Gainesville, FL 32608-1197 (E-mail: pateljm{at}medicine.ufl.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.
Received 9 March 2000; accepted in final form 13 June 2000.
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