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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-beta -naphthylamide and L-alanyl-beta -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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (bullet ) 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.

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; black-triangle) 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.

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.

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).

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.

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.

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.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(6):L1061-L1068