Abstract
Abstract. In this study, a nitric oxide (NO) sensor was used to examine the ability of angiotensin II (AngII), AngIV, and bradykinin (Bk) to stimulate NO release from porcine pulmonary artery (PPAE) and porcine aortic endothelial (PAE) cells and to explore the mechanism of the AngII-stimulated NO release. Physiologic concentrations of AngII, but not Bk, caused release of NO from PPAE cells. In contrast, Bk, but not AngII, stimulated NO release from PAE cells. AngII-stimulated NO release from PPAE cells required extracellular L-arginine and was inhibited by L-nitro-arginine methyl ester. AT1 and AT2 receptor inhibition had no affect on AngII-mediated NO release or activation of NO synthase (NOS). AngIV, and AngII metabolite with binding sites that are pharmacologically distinct from the classic AngII receptors, stimulated considerably greater NO release and greater endothelial-type constitutive NOS activity than the same amount of AngII. The AngIV receptor antagonist, divalinal AngIV, blocked both AngII- and AngIV-mediated NO release as well as NOS activation. The results demonstrate that AngIV and the AngIV receptor are responsible, at least in part, for AngII-stimulated NO release and the associated endothelium-dependent vasorelaxation. Furthermore, these results suggest that differences exist in both AngII- and Bk-mediated NO release between PPAE and PAE cells, which may reflect important differences in response to these hormones between vascular beds.
The vascular endothelium is a heterogeneous, metabolically active organ with a multitude of regulatory functions (1) that is capable of synthesizing and releasing a number of vasoactive agents (2). Dysfunction of this organ has been implicated in the pathogenesis of various cardiovascular disease processes such as atherosclerosis and hypertension (3). Since the proposed existence of endothelium-derived relaxing factor or factors (EDRF) (3) by Furchgott and Zawadzki in 1980 (4), later proposed to represent nitric oxide (NO) or a closely related agent (5), the L-arginine-NO pathway has been recognized as a principal regulator of BP (6). Furthermore, endothelium-derived NO has been implicated as an antithrombotic as well as antiproliferative agent (3) that is important in the prevention of vascular remodeling and development of arterial hypertension (3).
NO is generated from a terminal guanidino nitrogen of L-arginine (L-Arg) and catalyzed by a family of enzymes called NO synthases (7,8). One of these enzymes, endothelial-type constitutive NO synthase (NOS-III or ecNOS), is Ca2+-dependent and constitutively present in many cell types, including endothelial cells (8). The endothelium contributes to the control of vascular smooth muscle tone by the release of NO (2,9).
Endothelial cells have not only been demonstrated to mediate the vascular response to vasodilators (4,10), but also to modulate the action of many vasoconstricting agents (11,12). Angiotensin II (AngII), a biologically active component of the renin-angiotensin system, is a potent vasoconstrictor exerting its action on vascular smooth muscle by binding to the AT1 receptor (13). However, depending on the vascular bed and species, AngII-mediated contractions have been shown to be variably inhibited by endothelium, an effect that has been proposed by some investigators to be EDRF-mediated (14,15). AngII has also been reported to cause relaxation of certain blood vessels (16,17,18), an effect shown to be inhibited by the calmodulin antagonist calmidazolium (17) and proposed to occur via stimulation of AT2 receptors (18).
There is evidence that AngII may have some protective function in the development of hypoxia-induced pulmonary hypertension (19). The presence of AngII-binding receptors on pulmonary arterial endothelial cells, as well as their link to activation of phospholipase C- and phospholipase D-activated pathways, has been well documented (20,21). However, a direct link between AngII and NO release has been reported thus far in only two studies (22,23). Therefore, we hypothesized that the protective effects of AngII on the development of hypoxia-induced pulmonary hypertension may occur through the release of NO.
The purpose of this study was to determine the effect of the administration of physiologic concentrations of AngII on NO release and ecNOS activity in cultured porcine pulmonary arterial endothelial (PPAE) and porcine aortic endothelial (PAE) cells. The AngII receptor subtype responsible for NO release and activation of ecNOS was explored using AngII receptor antagonists. Angiotensin IV (AngIV; also known as angiotensin II fragment ([3,4,5,6,7,8])), a metabolite of AngII, was also examined for NO release and ecNOS activation in PPAE cells. Furthermore, the effect of divalinal AngIV, an AngIV receptor antagonist, on both AngII- or AngIV-mediated NO release and ecNOS activation was determined. PPAE and PAE cell cultures were chosen because angiotensin receptors have been demonstrated to be present in these cell cultures (20). NO release was detected using an electrochemical NO sensor designed for cell cultures (22). The ecNOS activity was measured by using the L-arginine/L-citrulline conversion assay.
Materials and Methods
Materials
Collagenase (Worthington, type I CLS, 145 U/mg) was obtained from Worthington Biomedical (Freehold, NJ). RPMI 1640, fetal bovine serum (FBS), penicillin-streptomycin antibiotic mixture, trypsin, and Hanks' balanced saline solution (HBSS) were obtained from Life Technologies (Grand Island, NY). Ca2+-Mg2+-free HBSS was from KC Biological (Lenexa, KS). Zinc protoporphyrin IX, Nafion, and pure NO gas were obtained from Aldrich Chemical Co. (Milwaukee, WI). AngII human acetate salt, AngIV human acetate salt, bradykinin (Bk) acetate salt, calmodulin, CaCl2, calcium ionophore A23187, ethylenediaminetetra-acetic acid (EDTA), ethyleneglycol-bis(β-aminoethyl ether)-N, N′-tetra-acetic acid (EGTA), L-Arg, NADPH, NG-nitro-L-arginine methyl ester (L-NAME), phenylmethylsulfonyl fluoride, [Sar1 Ile8] AngII acetate salt (saralasin), tetrahydrobiopterin, and Tris-HCl were obtained from Sigma Chemical Co. (St. Louis, MO). Losartan was from DuPont Merck (Wilmington, DE). PD123319 and PD123177 were obtained from Parke Davis (Ann Arbor, MI). Divalinal AngIV was from Hedral Therapeutics, Inc. (Portland, OR). [3H] L-Arginine was obtained from New England Nuclear (Boston, MA).
AngII, AngIV, and Bk were stored at -70°C in 1.0 mM aliquots (in distilled water). Divalinal AngIV was stored at -20°C in 2.5 mM aliquots (in distilled water). Losartan, PD123319, PD123177, and saralasin were stored at 4°C in 0.1 mM aliquots (in distilled water). A23187 stock solution (1.0 mM in DMSO) was stored at room temperature. All other solutions were prepared fresh daily. Solutions were diluted in HBSS for NO measurements (in RPMI for L-Arg/L-citrulline conversion assay) and equilibrated to 37°C in a heater just before application.
Tissue Culture
Confluent PPAE and PAE cell cultures (passages 3 to 7) in fibronectin-coated culture dishes were used in all experiments. PPAE and PAE cell cultures were isolated, propagated, and identified according to established techniques (24). Endothelial cells were obtained from the main pulmonary artery or thoracic aorta of 6- to 7-mo-old pigs. In brief, fresh blood vessels were obtained from the slaughterhouse, and cells were enzymatically isolated by filling the lumen of each vessel with 0.3% collagenase in HBSS containing 100 U/ml penicillin, 100 μg/ml streptomycin, 10 μg/ml gentamicin, and 2.5 μg/ml fungizone (1× antimicrobial agents) and incubated at 37°C for 20 min (aortas) or 25 min (pulmonary arteries). At the end of the incubation period, the loosened cell-enzyme mixture was transferred to a centrifuge tube containing RPMI 1640 supplemented with 15% FBS and 1× antimicrobial agents and centrifuged at 160 × g for 5 min at 4°C. The pellet was resuspended in fresh medium, and the suspension was seeded into sterile 35-mm-diameter tissue culture dishes at densities of 1 to 2 × 104 cells/cm2. After 60 min incubation at 37°C and 5% CO2 in air, the nonadherent cell suspension and medium was replaced with fresh medium. Cells were subcultured 4 to 5 d after confluence using 0.1% trypsin in Ca2+-Mg2+-free HBSS. Cells were maintained at 37°C and 5% CO2 in RPMI 1640 containing 4% (PPAE) or 5% (PAE) FBS and 100 U/ml penicillin and 100 μg/ml streptomycin. Confluent cells (passages 3 to 7) in fibronectin-coated culture dishes were used in all experiments. All cell monolayers in culture were initially identified as endothelial cells by phase contrast microscopy. Representative culture dishes were further characterized by electron microscopy or indirect immunofluorescence staining for factor VIII antigen, or both. On the basis of these criteria, monolayer cultures used in these experiments were estimated to be >95% pure endothelial cells.
Porphyrinic NO Sensor Fabrication
An electrochemical sensor designed for tissue culture was used for the in vitro detection of NO release. Fabrication of the sensor back-bone is described in detail elsewhere (22). The sensor was produced by electrochemically depositing metalloporphyrin (zinc protoporphyrin IX) on a reticulated vitreous carbon electrode (approximately 1 cm × 0.5 cm with a thickness of approximately 0.5 mm) by cyclic voltammetry using an EG&G PAR model 264 B voltammetric analyzer/stripping voltammeter (EG&G Instruments Princeton Applied Research [PAR], Princeton, NJ). The polymeric film was deposited from a deoxygenated, 0.5 mM zinc protoporphyrin solution in 0.1 M NaOH, by continuous scan cyclic voltammetry from -0.5 to +1.0 V at 100 mV/s for 26 min, recorded by an EG&G PAR model RE0150 X-Y recorder (EG&G PAR). After porphyrin film formation, the electrodes were dip-coated 3 times for 5 s in a 5% Nafion solution in alcohol and completely air-dried between each application. The porphyrinic NO sensor is used as the working electrode in the electrochemical NO measurements described below.
Electrochemical Methods
A three-electrode system was used for all NO measurements: the porphyrinic NO sensing electrode (described in detail above), a saturated calomel reference electrode, and a platinum wire auxiliary electrode. All electrochemical techniques were performed with an EG&G PAR model 264 B voltammetric analyzer/stripping voltammeter. The potential at which maximum NO oxidation occurs was identified by differential pulse voltammetry from +0.4 to +1.0 V, with a modulation pulse of 50 mV at 0.5-s intervals. Once the optimal potential for NO oxidation was determined (+0.76 V), it was used in amperometry, a more sensitive electrochemical method in which current is monitored versus time at a constant applied potential (50 mV pulse height, 0.5-s time intervals). The current signal was recorded with a strip chart recorder (Soltec Instruments, Encino, CA). The current generated during the catalytic electrochemical oxidation of NO is the analytical signal. Before in vitro electrochemical measurements of NO in cell cultures, calibration curves of NO concentration versus current (Figure 1) were generated for each electrode by adding known amounts of NO standard solution to 5 ml of deoxygenated HBSS. Standard NO solutions (1.8 mM) were prepared by saturating an aqueous solution with pure NO gas.
Calibration curve of a nitric oxide (NO) sensor. The amperogram on the left is current versus time at a constant applied potential of 0.76 V. Known aliquots of a standard NO solution were added to a stirred solution of Hanks' balanced saline solution (HBSS), under nitrogen (in the absence of cells). From the amperogram, a current versus concentration curve was generated.
L-Arginine/L-Citrulline Conversion Assay
Cell monolayers were washed, scraped, and homogenized in buffer (0.1 M Tris-HCl, pH 7.0, containing 1.25 mM CaCl2). The homogenates were centrifuged at 100,000 × g for 60 min at 5°C in an L3-50 Beckman ultracentrifuge (Beckman, Irvine, CA), and the total membrane and cytosolic fractions were collected. Total membrane or cytosol fractions (100 to 120 μg protein) were incubated at 37°C for 30 min in 0.4 ml of 50 mM Tris-HCl buffer, pH 7.4 (containing 0.1 mM EDTA and EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mg/L leupeptin, 2.5 mM CaCl2, 1 mM NADPH, 10 μM tetrahydrobiopterin, 100 nM calmodulin, 5 μM L-arginine, and purified [3H] L-arginine (0.6 μCi; specific activity 69 Ci/mmol). Purification of [3H] L-arginine and measurement of [3H] L-citrulline were carried out as described previously (25). The specific activity of ecNOS was determined by subtracting the activity in the blank (without membrane or cytosolic proteins), which was incubated under identical conditions, and is expressed as pmol L-citrulline/min per mg protein.
Experimental Protocol
Direct Measurements of NO Release. Direct measurements of NO release were performed on confluent PPAE or PAE cell cultures (passages 3 to 7) in 60-mm fibronectin-coated cultures dishes. NO measurements were done in buffer (pH 7.4, 37°C), which consisted of HBSS supplemented with 1 mM L-Arg, with or without inhibitors, unless noted otherwise.
AngII-Mediated NO Release from PPAE Cells. PPAE cells were examined for NO release in response to buffer alone, or with 10-4, 10-6, 10-7, 10-8, 10-10, or 10-12 M AngII. Figure 2 represents a schematic diagram of the experimental apparatus for NO measurements. The NO sensor was approximated to the endothelial cell surface by a Brinkmann model RP-III micromanipulator (Brinkmann Instruments, Westbury, NY). NO concentration was determined from the measured current by comparison with the calibration curve of the NO standard.
A schematic diagram of a porphyrinic NO sensor and the experimental apparatus for NO measurements.
To determine whether NO release was due to NOS activation, cells were preincubated in maintenance media supplemented with 1.0 mM L-NAME, a NOS inhibitor, for 1 h before NO measurements. NO measurements were performed in buffer and 1 mM L-NAME. After AngII addition and amperometric measurements, the HBSS solution was replaced with L-NAME-free maintenance media and incubated at 37°C and 5% CO2. After 1 h, NO release in response to 10-8 M AngII was reexamined in the same cultures in HBSS containing only 1 mM L-Arg. In separate experiments, the response of PPAE cells to 10-8 M AngII after L-Arg depletion was examined. Before NO measurements, PPAE cells were incubated for 2 h in L-Arg-free HBSS, pH 7.4 (essentially L-Arg-starved). NO measurements were performed in fresh L-Arg-free HBSS, pH 7.4.
Effects of AngII Receptor Antagonists on AngII-Mediated NO Release. The role of AT1 and AT2 receptors in AngII-mediated NO release in PPAE cells was explored using various receptor antagonists. For AngII receptor studies, the NO response of PPAE cells to 10-8 M AngII was examined in the presence of 10-7 M losartan, an AT1 receptor blocker, or 10-7 M PD123319, an AT2 receptor antagonist.
AngIV-Mediated NO Release in PPAE Cells. AngIV, a metabolite of AngII, has a binding site pharmacologically distinct from the classic AngII receptors and also affects vascular tone (26). Therefore, the response of PPAE cell cultures to 10-6 M and 10-8 M AngIV was examined. Furthermore, the effects of AngII (10-8 M) or AngIV (10-8 M) on PPAE cells in the presence of 1 μM divalinal AngIV, a partial nonpeptide antagonist of the AngIV receptor, were also evaluated to explore the role of the AngIV receptor on AngII- and AngIV-stimulated NO release. As a positive control, calcium ionophore A23187 (10 μM) was used.
NO Release from PAE Cells. PAE and PPAE cell cultures were compared in their responsiveness to both AngII and Bk. NO measurements were performed after addition of AngII (10-8 and 10-6 M) or Bk (10-8 and 10-6 M) to confluent PAE or PPAE cells. As a positive control, NO measurements on both PPAE and PAE cells were performed with administration of 10 μM A23187.
Measurements of ecNOS Activity by the L-Arg/L-Citrulline Conversion Assay. AngII and AngIV Activation of ecNOS in PPAE Cells. The activities of ecNOS were measured in total membrane and cytosol fractions of PPAE cells by monitoring the conversion of [3H] L-arginine to [3H] L-citrulline as described previously (25). To examine the stimulatory effects of AngII and AngIV on ecNOS, PPAE cells were incubated with 10-6 M AngII or 10-6 M AngIV in RPMI 1640 or RPMI 1640 alone (control) for 2 h at 37°C.
Effects of AngII and AngIV Receptor Antagonists on AngII or AngIV Activation of ecNOS in PPAE Cells. To examine the effects of AngII and AngIV receptor blockers on AngII- or AngIV-stimulated activation of ecNOS, PPAE cells were preincubated in RPMI 1640 containing 10-5 M saralasin (a nonspecific AngII receptor antagonist), 10-5 M losartan, 10-5 M PD123177 (an AT2 receptor blocker), or 5 × 10-5 M divalinal AngIV, for 15 min followed by a 2-h incubation (at 37°C) in the presence of 10-6 M AngII or 10-6 M AngIV. After incubation, cells were used to measure total membrane fraction or cytosolic ecNOS activity.
Statistical Analyses
For statistical analysis, the maximal concentration of NO produced (NO peak height; nmol/L) by PPAE and PAE cells was measured under the various conditions stated above. Data are given as means ± SEM. In each set of experiments, n is the number of culture dishes studied. Statistical analysis was performed using one-way ANOVA for comparison of NO release in response to different concentrations of AngII as well as ecNOS activity data. Unpaired t test was used for the comparison of NO release evoked from AngIV versus AngII. Means were considered significantly different at P < 0.05.
Results
Direct Measurements of NO Release
AngII-Mediated NO Release from PPAE Cells. A typical amperometric (current versus time) curve obtained for in vitro measurement of NO in PPAE cells is shown in Figure 3. A rapid release of NO was observed after the addition of 10-8 M AngII in HBSS. The increase in current is proportional to the NO concentration. Addition of HBSS alone produced no detectable response.
Representative amperogram for 10-8 M angiotensin II (AngII)-mediated NO release from porcine pulmonary arterial endothelial (PPAE) cells in HBSS containing 1 mM L-Arg. Vehicle is HBSS containing 1 mM L-Arg.
The average peak concentrations of NO released from PPAE cells after stimulation with 10-4 (n = 3), 10-6 (n = 12), 10-7 (n = 4), 10-8 (n = 13), 10-10 (n = 4), and 10-12 (n = 4) M AngII are illustrated in Figure 4. The average maximal NO releases were 112.7 ± 4.6, 75.6 ± 13, 61.5 ± 5.3, 55.5 ± 13.6, 56.5 ± 5.7, and 29.5 ± 6.4 nM NO, respectively. The overall ANOVA P value is <0.001. The NO response to 10-4 M AngII was significantly higher than all other concentrations (P < 0.05), and NO release from 10-12 M AngII was significantly lower than all higher concentrations tested (P < 0.05). NO release elicited from 10-6 M AngII was significantly different from NO release from 10-8 M AngII (P < 0.05).
NO release from PPAE cells in response to different concentrations of AngII. ***P < 0.05, greater than all other concentrations; *P < 0.05, less than all other concentrations; **P < 0.05, significantly different from 10-8 M AngII.
After incubation with 1 mM L-NAME (in maintenance media) for 1 h before the assay, PPAE cells did not release NO when stimulated with 10-8 M AngII in HBSS with L-NAME and L-Arg (Figure 5A). When the same PPAE cells were reincubated in L-NAME-free maintenance media for 1 h and assayed in fresh HBSS + 1 mM L-Arg, there was release of NO with a peak NO concentration of 64 ± 4 nM (n = 4) in response to 10-8 M AngII (Figure 5A).
(A) In the presence of 1 mM NG-nitro-L-arginine methyl ester (L-NAME), PPAE cells do not release NO in response to AngII (1). The removal of L-NAME restores the AngII-stimulated NO release in the same cells (2). (B) In the absence of L-Arg, PPAE cells do not release NO in response to AngII (1). The replacement of L-Arg restores the AngII-stimulated NO release in the same cells (2).
Figure 5B represents a typical amperogram of NO measurements from cells in L-Arg-free HBSS. NO release due to 10-8 M AngII was not detectable in the absence of L-Arg. When 1 mM L-Arg was added, 78 ± 12 nM NO (n = 5) was released from the same cell population.
Effects of AngII Receptor Antagonists on AngII-Mediated NO Release. To identify the AngII receptor subtype that mediates NO release, experiments were performed in the presence of losartan, an AT1 receptor antagonist, or PD123319, an AT2 receptor antagonist. Figure 6A represents a typical amperogram of the AngII (10-8 M) response of PPAE cells in the presence of losartan (10-7 M) and Figure 6B in the presence of PD123319 (10-7 M). NO release was not inhibited by either the AT1 or the AT2 receptor inhibitor (60 ± 8 nM [n = 8] and 57 ± 8 nM [n = 5], respectively).
(A) Representative amperogram for AngII-mediated NO release from PPAE cells in HBSS containing 1 mM L-Arg and 10-7 M losartan. AngII-stimulated NO release was not affected by the presence of the AT1 receptor antagonist (60 ± 8 nM NO, n = 8 versus 55.5 ± 13.6, n = 13). (B) Representative amperogram for AngII-mediated NO release from PPAE cells in HBSS containing 1 mM L-Arg and 10-7 M PD123319. AngII-stimulated NO release was not affected by the presence of the AT2 receptor antagonist (57 ± 8 nM NO, n = 5 versus 55.5 ± 13.6, n = 13).
AngIV-Mediated NO Release in PPAE Cells. To determine whether a metabolic product of AngII causes NO release, the effect of AngIV was examined. After addition of 10-6 (Figure 7) or 10-8 M AngIV, NO release was 144 ± 13 nM (n = 7) and 74.3 ± 9.6 nM (n = 3), respectively, which is statistically higher (P < 0.05) than that of 10-6 M AngII (75.6 ± 13, n = 12) and 10-8 M AngII (55.5 ± 13.6, n = 13).
Representative amperogram for AngIV-mediated NO release from PPAE cells in HBSS containing 1 mM L-Arg. AngIV (10-6 M) elicited a much greater NO release than AngII (144 ± 13 nM NO, n = 7 versus 75.6 ± 13, n = 12).
Furthermore, the partial nonpeptide antagonist of the AngIV receptor, divalinal AngIV (1 μM), blocked both 10-8 M AngII- (n = 3) and 10-8 M AngIV- (n = 4) stimulated NO release, but not 10 μM A23187-mediated NO release (Figure 8), strongly suggesting that AngII- as well as AngIV-stimulated NO release is mediated by the AngIV receptor.
In the presence of 1 μM divalinal AngIV, PPAE cells release NO in response to 10 μM A23187 but not 10-8 M AngII or 10-8 M AngIV.
NO Release from PAE Cells. To determine if there are differences in AngII-evoked responses between porcine pulmonary artery and aortic endothelial cells, the response of PAE cells to AngII was also studied. There was no detectable release of NO by PAE cells when 10-8 M or 10-6 M AngII (n = 10) was added (Figure 9). However, in response to 10-8 or 10-6 M Bk, 52 ± 17 nM (n = 13) and 88 ± 8 nM (n = 3) NO, respectively, was released by the aortic endothelial cell culture (Figure 9). In contrast, 10-8, 10-6, and 10-5 M Bk (n = 10) elicited no detectable response from PPAE cells (not shown).
Representative amperogram for NO release from porcine aortic endothelial (PAE) cells in HBSS containing 1 mM L-Arg. AngII (10-6 M) did not stimulate NO release. Bradykinin (BK; 10-8 M) elicited 52 ± 17 nM NO (n = 13).
As a positive control, cells were also exposed to a calcium ionophore, a receptor-independent stimulator of ecNOS. A23187 (10 μM) stimulated NO release from both PPAE and PAE cell cultures: 122 ± 29 nM NO (n = 14) and 54 ± 16 nM NO (n = 7), respectively.
Measurements of ecNOS Activity by the L-Arg/L-Citrulline Conversion Assay
AngII and AngIV Activation of ecNOS in PPAE Cells. As shown in Table 1, AngII and AngIV significantly increased ecNOS activity in the total membrane fraction of PPAE cells (P < 0.05). In cytosol fractions, the ecNOS activities of AngII-and AngIV-stimulated cells were comparable to controls and were <5% of total membrane fraction activity (not shown). Similar to the electrochemical NO measurements, AngIV-stimulated activation of ecNOS was greater compared to activation by the same concentration of AngII.
Effect of AngII and AngIV on ecNOS Activity in PPAE cellsa
Effects of AngII and AngIV Receptor Antagonists on AngII and AngIV Activation of ecNOS in PPAE Cells. To determine whether AngII- or AngIV-mediated activation of ecNOS is mediated through AngII receptors and/or AngIV receptors, the effects of [Sar1 Ile8]-AngII (a nonspecific AngII receptor antagonist), losartan, PD123177 (a specific AT2 receptor antagonist), and divalinal AngIV were examined. AngII- and AngIV-stimulated increases in ecNOS activity were not blocked by [Sar1 Ile8]-AngII, losartan, or PD123177 (Figure 10). However, ecNOS activation by AngII and AngIV were both inhibited by the AngIV receptor blocker (Figure 11).
Effect of AngII (Panel A) and AngIV (Panel B) on endothelial-type constitutive NO synthase (ecNOS) activity in PPAE cells. Cells were incubated with or without [Sar1Ile8] AngII (10 μM), losartan (10 μM), or PD123177 (10 μM) for 15 min followed by a 2-h incubation in the presence of AngII (1 μM) or AngIV (1 μM) at 37°C. After incubation, ecNOS activity was measured in the total membrane fraction. 1, control; 2, AngII (Panel A) or AngIV (Panel B); 3, AngII (Panel A) or AngIV (Panel B) + [Sar1Ile8] AngII; 4, AngII (Panel A) or AngIV (Panel B) + losartan; 5, AngII (Panel A) or AngIV (Panel B) + PD123177. ecNOS activity in cells incubated with [Sar1Ile8] AngII (10 μM), losartan (10 μM), or PD123177 (10 μM) alone was comparable to control (not shown). Data are ± SEM (n = 8). *P < 0.05 versus control.
Effect of divalinal AngIV on AngII (Panel A) and AngIV (Panel B) stimulation of ecNOS activity in PPAE cells. Cells were incubated with or without divalinal AngIV (5 μM) for 15 min followed by a 2-h incubation in the presence of AngII (1 μM) or AngIV (1 μM) at 37°C. After incubation, ecNOS activity was measured in the total membrane fraction. 1, control; 2, AngII (Panel A) or AngIV (Panel B); 3, AngII (Panel A) or AngIV (Panel B) + divalinal AngIV. ecNOS activity in cells incubated with divalinal AngIV (5 μM) alone was comparable to control (not shown). Data are ± SEM (n = 8). *P < 0.05 versus control.
Discussion
The results of this study demonstrate that acute administration of physiologic concentrations of AngII, a potent vasoconstrictor, is associated with the release of NO, a known vasodilator, from cultured PPAE cells. The AngII-mediated NO release occurred rapidly and was short-lived. It required extracellular L-Arg, the substrate for NOS, and was inhibited by L-NAME, a competitive NOS inhibitor, thereby indicating that AngII-mediated NO release occurred via activation of NOS, which was supported by the results of the L-Arg/L-citrulline conversion assay. AngII-stimulated NO synthesis and ecNOS activation were not mediated by either AT1 or AT2 receptors, suggesting the involvement of a different receptor and/or a metabolic product of AngII. AngIV was found to stimulate NO release and increase ecNOS activity from PPAE cell cultures. Furthermore, both AngII- and AngIV-mediated NO release and ecNOS stimulation were inhibited by divalinal AngIV, an AngIV receptor antagonist, thereby indicating that under our experimental conditions the AngIV receptor is involved in AngII- and AngIV-stimulated NO synthesis and activation of ecNOS. Additionally, Bk failed to cause NO release from PPAE cells but stimulated NO production from PAE cells. In contrast, AngII did not stimulate NO release from PAE cells.
AngII has been observed by others to induce endothelium-dependent vasodilation in some tissues, such as canine renal and cerebral arteries (16), fowl aorta (17), and rat and rabbit cerebral arteries (27,28), an effect that was suggested to be mediated, in part, by EDRF (NO). AngII has also been reported to produce a biphasic vascular response (contraction followed by a relaxation) in conscious rabbits (29) and in anesthetized rats (18), the depressor effect postulated to be partially mediated by NO. By using an electrochemical sensor for direct measurements of NO, our study demonstrates that AngII can stimulate the release of NO, a potent vasodilator (30). Our results are in agreement with observations by Thorup et al. (23), who recently demonstrated, using a porphyrinically modified carbon fiber electrode, that AngII also stimulates NO release in isolated and perfused renal resistance arteries of the rat.
It has been suggested that stimulation of AT2 receptors may mediate, at least in part, the depressor response of AngII (18). However, in the current study, neither the AT1 nor the AT2 receptor antagonist inhibited AngII-mediated NO release by PPAE cells. This suggests the involvement of a different receptor and/or an AngII metabolic product. In addition, in the isolated and perfused renal resistance arteries of the rat, losartan and candesartan, another selective AT1 inhibitor, failed to completely block (only 77 and 63%, respectively) NO release generated from 10-8 M AngII (23), indicating that, at least in part, some receptor other than the AT1 receptor is involved. Our hypothesis is further supported by reports that AngII-mediated increases in cGMP or nitrite (indirect methods of NO detection) from a human proximal tubule cell line (31) and a neuroblastoma neuro 2a cell line (32) were not inhibited by losartan or PD123319 (AT1 and AT2 receptor blockers, respectively).
Investigators have recently begun to examine metabolites of AngII, including AngII(1-7) and AngIV, neither of which binds substantially to AT1 or AT2 receptors (26,33). AngIV has been shown to have a binding site pharmacologically distinct from the classic AngII receptors (26). AngIV binding sites have been found in a variety of tissues (e.g., brain, heart, liver, kidney, uterus, aorta, and lung) and in many species (e.g., bovine, equine, canine, feline, rabbit, guinea-pig, and porcine) (26). Importantly, it has been demonstrated that AngIV receptors are highly concentrated on endothelial cells (26). Our recent results (34) have demonstrated that PPAE cells express high levels of AngIV receptors, and neither AngII nor any of the AngII blockers interfere with AngIV binding in PPAE cells.
An AngIV-mediated vasodilatory response has been well documented. In recent studies we have found that AngIV leads to relaxation of freshly isolated porcine pulmonary resistance arteries (34). In addition, AngIV has been demonstrated to increase blood flow in the cortex of the rat kidney, an effect preserved during AT1 and AT2 receptor blockade (26). Finally, Haberl et al. suggested that AngII, AngIII, and AngIV may participate in the regulation of cerebral blood flow in the rabbit by an EDRF-dependent mechanism because coadministration with L-Arg synergistically produced potent dilation (35).
By using a sensor uniquely designed for direct measurements of NO release from cell cultures, we have demonstrated that stimulation of the AngIV receptor leads to NO release from PPAE cells. However, whether the intact AngII itself, its metabolite AngIV, or a combination of AngII and its metabolic products is responsible for NO release in these cells remains to be elucidated. AngII is converted to AngIV by aminopeptidases that cleave 2 amino acids from the peptide (33,36). Haberl et al. observed that AngII- but not AngIV- mediated endothelium-dependent vasodilation in rabbit brain arterioles was completely blocked in the presence of amastatin, an aminopeptidase inhibitor, suggesting that AngII-mediated vasodilation in rabbit arterioles is due to conversion of AngII to AngIV (35). Whether this is the case in PPAE cells remains to be established. However, we have recently reported that aminopeptidases A and M are present in PPAE cells, and inhibitors of these enzymes block AngII conversion to AngIV in these cells (34).
Porsti et al. (37) and Brosnihan et al. (38) observed that AngII(1-7)-mediated vasorelaxation of porcine and canine coronary arteries occurred via a B2 bradykinin receptor and a non-AT1 non-AT2 receptor, suggesting that Bk is an intermediate. AngII was a relatively weak vasodilator, whereas AngIII and AngIV had no vasorelaxation effect in porcine coronary arterial rings (37). In contrast, in the current studies both AngII and AngIV caused NO release, whereas Bk was incapable of stimulating NOS in PPAE cell cultures. Therefore, Bk does not appear to be an intermediate signaling mechanism in AngII- or AngIV-mediated NO release in PPAE cells.
This study also indicates that significant differences in response to AngII and Bk exist depending on the source of cultured endothelial cells. Calcium ionophore A23187, a non-receptor-mediated stimulator of NOS (39), elicited a rapid NO release from both PPAE and PAE cell cultures, thereby demonstrating the ability of each culture type to enzymatically synthesize NO. Bk stimulated NO production from PAE cells, which has also been reported by others (40), but failed to stimulate NO-release from PPAE cell cultures (passages 3 to 7). However, it has been reported that Bk receptors are sensitive to trypsinization and decrease with increasing passage (41); therefore, the inability of Bk to cause NO release in PPAE cell cultures should be interpreted with caution. Nonetheless, Bk, as well as A23187, served to demonstrate the presence of constitutive NOS activity in PAE cell cultures. In contrast, AngII did not stimulate NO release from PAE cells. Considering that previous studies have demonstrated AngII-mediated NO release from a bovine aortic endothelial cell culture (22), the observed species and tissue-related heterogeneity of endothelial responses to various hormones is interesting.
Variations in vascular response to angiotensins between species as well as between tissues of the same species have been well documented. Major differences have been shown to exist both in the proportion and the distribution of AT1 and AT2 receptors between the same tissues in different species (33,42). Santos et al. observed that human and bovine, but not porcine, aortic endothelial cell cultures converted AngI into AngII(1-7) (43). Swanson et al. demonstrated significant variation in 125I-AngIV binding sites in adrenal glands of different species (26). In addition, the proportion of AT1 to AT2 receptors has been demonstrated to vary significantly in different tissues within the same species (42,44). Also, the AngIV binding density varied markedly between tissues of one species, as shown in guinea pig (26).
Differences in mechanisms of Bk-mediated vasodilation have also been demonstrated, both between species as well as between various vascular beds of the same species. Bk relaxes, for example, the feline superior mesenteric artery in a cyclooxygenase-dependent and oxygen-radical-mediated process, whereas it dilates the canine superior mesenteric artery in an endothelium-dependent mode which does not rely on free radical formation (45,46,47). Furthermore, Bk relaxes canine coronary artery via NO release, but dilates canine mesenteric veins in a prostaglandin-mediated process (48). The observed heterogeneity of response of individual vascular beds to vasoactive agents is not at all surprising and may bear considerable physiologic and pathologic significance.
Although our results do not give a complete picture of vascular physiology, it is tempting to speculate on their potential importance in vivo, especially in view of several established observations. For instance, circulating levels of AngII are elevated in renin-dependent systemic hypertension; however, pulmonary artery pressures have been found to be within the normal range (49,50). Therefore, AngII- and AngIV-mediated NO release may provide a protective mechanism for the pulmonary circulation in renin-dependent systemic hypertension. One mechanism of such protection is provided by Olson et al., who observed that in bovine pulmonary artery endothelial cells, but not bovine coronary endothelial cells, ecNOS mRNA expression was increased after 6 h of AngII exposure and protein increased after 4 and 8 h of AngII exposure, effects that were inhibited by saralasin (51). Nitrite (an NO metabolite) was increased after 18 h of AngII exposure (51). Our data suggest a more “acute” mechanism in which AngII (and/or AngIV) directly stimulates ecNOS activity and NO release.
In conclusion, we have demonstrated for the first time, by direct measurements using an NO sensor uniquely designed for cell cultures, that AngII and AngIV stimulate NO release from cultured porcine pulmonary artery endothelial cells. This NO release appears to be mediated by the AngIV receptor. Whether AngII itself stimulates NO release or is converted into an angiotensin metabolite, which can function as an NOS agonist, remains to be determined. These studies further suggest the presence of tissue-related differences in angiotensin- and Bk-mediated NO release.
Acknowledgments
Acknowledgments
This work was supported in part by Grant DK 28330 from the U.S. Public Health Service (to Dr. Tisher) and by U.S. Public Health Service Training Grant DK 07518 (to Drs. Nathalie Hill-Kapturczak and Matthias H. Kapturczak). The research was also supported in part by National Heart, Lung, and Blood Institute Grant HL-52136 (to Dr. Block) and the Medical Research Service of the Veterans Administration. We are indebted to Humberto Herrera for the preparation of tissue cultures. Losartan was a gift from Dr. Ronald D. Smith (Du Pont Merck, Wilmington, DE). PD123319 was a gift from Dr. Joan A. Keiser (Parke-Davis, Ann Arbor, MI). Divalinal AngIV was kindly provided by Hedral Therapeutics (Portland, OR).
Footnotes
American Society of Nephrology
This work was presented in part at the 30th annual meeting of the American Society of Nephrology, November 2 to 5, 1997, San Antonio, Texas, and has appeared in abstract from (J Am Soc Nephrol 8: A1522, 1997 and J Am Soc Nephrol 8: A1528, 1997).
Dr. Thomas Coffman served as Guest Editor and supervised the review and final disposition of this manuscript.
- © 1999 American Society of Nephrology
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