Obligatory Metabolism of Angiotensin II to Angiotensin III for Zona Glomerulosa Cell–Mediated Relaxations of Bovine Adrenal Cortical Arteries

Obligatory Metabolism of Angiotensin II to Angiotensin III for Zona Glomerulosa Cell–Mediated... Abstract Hyperaldosteronism is associated with hypertension, cardiac hypertrophy, and congestive heart failure. Steroidogenic factors facilitate aldosterone secretion by increasing adrenal blood flow. Angiotensin (Ang) II decreases adrenal vascular tone through release of zona glomerulosa (ZG) cell–derived vasodilatory eicosanoids. However, ZG cell–mediated relaxation of bovine adrenal cortical arteries to Ang II is not altered by angiotensin type 1 or 2 receptor antagonists. Because traditional Ang II receptors do not mediate these vasorelaxations to Ang II, we investigated the role of Ang II metabolites. Ang III was identified by liquid chromatography–mass spectrometry as the primary ZG cell metabolite of Ang II. Ang III stimulated ZG cell–mediated relaxation of adrenal arteries with greater potency than did Ang II. Furthermore, ZG cell–mediated relaxations of adrenal arteries by Ang II were attenuated by aminopeptidase inhibition, and Ang III-stimulated relaxations persisted. Ang IV had little effect compared with Ang II. Moreover, ZG cell–mediated relaxations of adrenal arteries by Ang II were attenuated by an Ang III antagonist but not by an Ang (1-7) antagonist. In contrast, Ang II and Ang III were equipotent in stimulating aldosterone secretion from ZG cells and were unaffected by aminopeptidase inhibition. Additionally, aspartyl and leucyl aminopeptidases, which convert Ang II to Ang III, are the primary peptidase expressed in ZG cells. This was confirmed by enzyme activity. These data indicate that intra-adrenal metabolism of Ang II to Ang III is required for ZG cell–mediated relaxations of adrenal arteries but not aldosterone secretion. These studies have defined an important role of Ang III in the adrenal gland. The renin-angiotensin-aldosterone system (RAAS) is an important regulator of normal cardiovascular homeostasis (1). Dysregulation of this critical system leads to the pathogenesis of a variety of cardiovascular diseases, and pharmacologic intervention of the RAAS is therapeutically beneficial. Angiotensin (Ang) II is a potent vasoconstrictor and enhances the activity of the sympathetic nervous system (2). Inhibition of Ang II synthesis or Ang II receptor antagonism is beneficial in the treatment of hypertension (3). Ang II additionally stimulates the synthesis and secretion of aldosterone from the zona glomerulosa (ZG) region of the adrenal gland. Aldosterone is a mineralocorticoid that regulates extracellular fluid and electrolyte homeostasis (4). Elevated levels of aldosterone are associated with hypertension, cardiac fibrosis, left ventricular remodeling, endothelial dysfunction, vasculopathy, vascular remodeling, and renal injury (5). Mineralocorticoid receptor antagonists alleviate these deleterious effects. Thus, understanding the factors that regulate aldosterone release has important implications for cardiovascular health. Aldosterone secretagogues [Ang II, potassium, adrenocorticotropic hormone (ACTH)] might partially regulate aldosterone secretion by increasing adrenal blood flow (6–8). ACTH increases adrenal blood flow by 200% to 272% in vivo (9–12). ACTH also increases adrenal blood flow ex vivo in perfused adrenal glands (8, 13–16). Additionally, despite being a potent vasoconstrictor, Ang II did not decrease adrenal blood flow in sheep and increased adrenal blood flow in rats in vivo (17, 18). The effect of adrenal secretagogues on adrenal blood flow results from the unique vascular architecture of the adrenal gland. The subcapsular adrenal arteries are the only resistance vessels of the adrenal gland and control adrenal vascular resistance and blood flow (19). These vessels closely adhere to the ZG region, running parallel to or within the ZG region, before penetration into the gland and the formation of cortical and medullary capillary networks. The ZG cells produce vasorelaxing factors that contribute to the vascular effects of ACTH and Ang II. ACTH does not affect the vascular tone of the subcapsular adrenal arteries in vitro (20). However, in the presence of ZG cells, ACTH induces vascular relaxation by the release of ZG cell–derived epoxyeicosatrienoic acids (EETs) (21). EETs and their hydrolysis products, the dihydroxyeicosatrienoic acids (DHETs) relax adrenal arteries by activating calcium-activated potassium channels, causing membrane hyperpolarization. Although a potent vasoconstrictor of some arteries, Ang II directly relaxes adrenal cortical arteries through the release of endothelial nitric oxide (NO) (22). However, in the presence of ZG cells, this relaxation response to Ang II is augmented by ZG cell–derived EETs and DHETs (23). Another potential mechanism by which Ang II causes vasorelaxation of adrenal arteries is by metabolism of Ang II. In adrenal arteries, Ang II is metabolized primarily to Ang III and Ang (1-7) (24). These two metabolic pathways produce divergent effects. Aminopeptidase metabolism of Ang II to Ang III preserves the vasorelaxation response, and carboxypeptidase metabolism of Ang II to Ang (1-7) reduces the vasorelaxation response. The metabolism of Ang II to either metabolite removes the contraction response in adrenal arteries (24). With evidence supporting the importance of Ang II metabolism in the direct vasorelaxation response, we examined the role of Ang II metabolism in ZG cell–dependent vasorelaxation. The goals of these studies were to (1) pharmacologically characterize the receptor that mediates Ang II–stimulated, ZG cell–dependent vasorelaxation of adrenal arteries, (2) identify the ZG cell metabolites of Ang II, and (3) examine the role of the Ang II metabolites on ZG cell–dependent vasorelaxation of adrenal arteries. Our results have demonstrated that Ang III, not Ang II, mediates ZG cell–dependent vasorelaxation of adrenal arteries. Materials and Methods Reagents HEPES buffer and physiological saline solution ingredients, angiotensin peptides, nitro-l-arginine (L-NA), indomethacin, losartan, PD123,319, amastatin, and cell culture media were purchased from Sigma-Aldrich (St. Louis, MO). U46619 was purchased from Cayman Chemical (Ann Arbor, MI). D-Ala7-Ang (1-7) and Ile7-Ang III were purchased from Bachem Inc. (Torrance, CA). EC33, a glutamyl-aminopeptidase A inhibitor, was synthesized as previously described (25, 26). The 13C515N1-Ang IV internal standard was synthesized by the Medical College of Wisconsin Protein Nucleic Acid Facility (Milwaukee, WI). The 13C515N1-Ang III and 13C515N1-Ang (1-7) internal standards were synthesized by EZBiolab (Carmel, IN). All the solvents were high-performance liquid chromatography (LC) grade and purchased from Sigma-Aldrich. ZG cell isolation and culture Bovine adrenal ZG cells, bovine adrenal artery endothelial cells (BAAECs), and bovine adrenal artery smooth muscle cells (BAASMCs) were prepared by enzymatic dissociation of adrenal cortical slices, as previously described (22, 24, 27). For the vascular reactivity and mass spectrometry studies, freshly isolated ZG cells were used. For studies of aldosterone release, cultured ZG cells were used (28). Isometric tension recording Fresh bovine adrenal glands were acquired from a local slaughterhouse. Subcapsular cortical arteries closely adherent to the adrenal surface (200 to 300 μm) were dissected and cleaned of connective tissue in ice-cold HEPES buffer (150 mM NaCl, 10 mM HEPES, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 6 mM glucose; pH 7.4). Isolated arterial segments were threaded on two 40-μm stainless steel wires and mounted in a 610M 4-chamber wire myograph (Danish Myotechnologies, Aarhus, Denmark) containing physiological salt solution (PSS; 119 mM NaCl, 24 mM NaHCO3, 4.7 mM KCl, 2 mM CaCl2, 1.18 mM KH2PO4, 1.17 mM MgSO4, 0.026 mM EDTA, 5.5 mM glucose; pH 7.4), bubbled with 95% oxygen, 5% carbon dioxide at 37°C, as previously described (22, 29, 30). After 30 minutes of equilibration, the arteries were gradually stretched to a resting tension of 1 mN and stimulated with KCl (60 mM) and the thromboxane A2 mimetic U-46619 (100 nM) three times for 10 minutes at 10-minute intervals. The arteries were allowed to equilibrate for 30 minutes before the initiation of the experimental protocols. The arteries were precontracted with submaximal concentrations of U-46619 (10 to 30 nM) to 50% to 75% of their maximal KCl and U46619 stimulation. To examine the vasoactive factors released by ZG cells in response to Ang II stimulation, experiments were performed in the presence of ZG cells, as previously described (23). In brief, 5 to 10 × 105 cells were added to baths with intact arteries pretreated with the endothelial NO synthase inhibitor L-NA (30 μM) and the cyclooxygenase inhibitor indomethacin (10 μM). L-NA and indomethacin were added to block any direct relaxing effects of Ang peptides on adrenal arteries, such that the ZG cell–mediated responses only were studied (21, 23, 24). Cumulative concentration responses to Ang II, Ang III, or Ang IV (10 fM to 100 pM) were performed. The responses were repeated with arteries and ZG cells pretreated with the angiotensin type 1 (AT1) receptor antagonist losartan (10 μM), the angiotensin type 2 (AT2) receptor antagonist PD123,319 (10 μM), the Ang III antagonist Ile7-Ang III (10 μM), the Ang (1-7) antagonist D-Ala7-Ang (1-7), and the aminopeptidase inhibitors amastatin (10 and 100 μM) and EC33 (10 μM) (26, 31–35). Ang II metabolism The metabolism of Ang II by bovine adrenal arteries and ZG cells was examined under conditions replicating the isometric tension experiments. An adrenal artery ring or ZG cells (1 × 106), or the combination of the two, were incubated in 6 mL of PSS with 100 nM Ang II, bubbled with 95% oxygen, 5% carbon dioxide at 37°C for 10 minutes. PSS buffer alone containing 100 nM Ang II was used to control for spontaneous degradation. Buffer and cells/tissue were separated by centrifugation. Supernatant was removed and extracted the same day. Solid-phase extraction The internal standards [Sar1, Ile8-Ang II, 13C515N1-Ang III, 13C515N1-Ang IV, and 13C515N1-Ang (1-7); 30 ng] were added to the supernatant. The supernatant was prepared for solid-phase extraction by the addition of ethanol containing 1% trifluoroacetic acid (TFA) to a final volume of 15%, followed by adding 1 mL of water containing 1% TFA. The sample was applied to a preconditioned Sep Pak C18 SPE cartridge (Waters Corp., Milford, MA) and washed with 20 mL of water containing 1% TFA. Ang peptides were then eluted from the column using 6 mL of methanol containing 1% TFA and dried under a stream of nitrogen gas. For LC–mass spectrometry (LC-MS), the samples were then dissolved in 30 μL of 50% methanol/50% water containing 3% formic acid and 0.01% TFA and centrifuged, and the supernatant was analyzed. LC-MS analysis LC-MS and LC–tandem MS was performed using a modification of a previously described method (24, 29, 36). LC-tandem MS was used to identify the peptides, and LC/MS was used to quantify the Ang peptides. Analyses were performed using a Waters-Micromass Quattro micro-atmospheric pressure ionization electrospray triple quadrupole mass spectrometric system coupled with a Waters 2695 high-performance liquid chromatograph. The mass spectrometer is equipped with a Z-spray dual orthogonal ionization source and is controlled by MassLynx, version 4.1, software. The samples were separated using a reverse-phase C18 column (Jupiter 2.0 × 250 mm; Phenomenex) using water-methanol with 0.3% formic acid as a mobile phase at a flow rate of 0.2 mL/min. The mobile phase of 20% methanol in water linearly increased to 50% methanol over 30 minutes, followed by a linear increase to 60% methanol over 5 minutes. The positive ion electrospray ionization mass spectrometric conditions were as follows: capillary voltage, 3.0 kV; cone voltage, 20 V; desolvation temperature, 400°C; and source temperature, 100°C. LC-MS analysis was performed in positive electrospray mode in the single-ion recording mode. Aldosterone secretion Aldosterone secretion studies were performed as previously described (28). Cultured ZG cells were incubated with Ang II or Ang III (100 pM to 100 nM) for 2 hours before analysis of media for aldosterone. The responses were repeated with ZG cells pretreated with the aminopeptidase inhibitor amastatin (10 and 100 μM). Aldosterone production by cultured ZG cells was measured using enzyme-linked immunosorbent assay (27, 28). RNA sequencing Bovine adrenal ZG, endothelial, and smooth muscle cells (2 × 106 cells) were placed in RNAlater and shipped to Otogenetics for RNA extraction, polyA complementary DNA (cDNA) preparation, Illumina library preparation, and Illumina HiSeq2000 sequencing. Using DNAnexus, the reads were aligned and assembled to the bovine reference transcriptome. Once the reads were mapped, each transcript was quantified by calculating the reads per kilobase of transcript per 1 million mapped reads to normalize the data for transcript length and across samples of different coverage (37). This allowed a comparison of expression levels among transcripts and across the cell types. The reads per kilobase of transcript per 1 million mapped reads for the peptidases were plotted for the three cell types. The results for the specific peptidases were confirmed using reverse transcription polymerase chain reaction (RT-PCR). Table 1 lists the gene name, the common name used in our report, and Enzyme Commission number for the peptidases of interest. Table 1. Gene, Name, and Enzyme Commission Number of Peptidases Gene  Name  EC Number  ACE  Angiotensin converting enzyme  EC 3.4.15.1  ACE2/ACEH  Angiotensin converting enzyme-2  EC 3.4.17.23  ANPEP  Aminopeptidase N  EC 3.4.11.2  AQPEP  Aminopeptidase Q  EC 3.4.24.57  DNPEP  Aspartyl aminopeptidase A  EC 3.4.11.21  DPEP-1  Dipeptidase-1  EC 3.4.13.19  ENPEP  Glutamyl aminopeptidase A  EC 3.4.11.7  ERAP-1  ER leucine aminopeptidase  EC 3.4.11.3  ERAP-2  ER leucine aminopeptidase  EC 3.4.11.-  LAP-3  Leucine aminopeptidase  EC 3.4.11.5  METAP1  Methionine aminopeptidase-1  EC 3.4.11.18  METAP2  Methionine aminopeptidase-2  EC 3.4.11.18  MME  Neutral endopeptidase  EC 3.4.24.11  NPEPL1  Aminopeptidase-like 1  EC 3.4.11.1  NPEPPS  Alanyl aminopeptidase  EC 3.4.11.14  PEPD  X-proline dipeptidase  EC 3.4.13.9  PGPEP1  Pyroglutamyl peptidase  EC 3.4.31.9  PRCP  Prolyl carboxypeptidase  EC 3.4.16.2  PREP  Prolyl endopeptidase  EC 3.4.21.26  RNPEP  Arginyl aminopeptidase  EC 3.4.11.6  SCPEP-1  Serine carboxypeptidase-1  EC 3.4.16  XPNPEP-1  X-prolyl aminopeptidase-1  EC 3.4.11.9  XPNPEP-2  X-prolyl aminopeptidase-2  EC 3.4.11.9  XPNPEP-3  X-prolyl aminopeptidase-3  EC 3.4.11.9  Gene  Name  EC Number  ACE  Angiotensin converting enzyme  EC 3.4.15.1  ACE2/ACEH  Angiotensin converting enzyme-2  EC 3.4.17.23  ANPEP  Aminopeptidase N  EC 3.4.11.2  AQPEP  Aminopeptidase Q  EC 3.4.24.57  DNPEP  Aspartyl aminopeptidase A  EC 3.4.11.21  DPEP-1  Dipeptidase-1  EC 3.4.13.19  ENPEP  Glutamyl aminopeptidase A  EC 3.4.11.7  ERAP-1  ER leucine aminopeptidase  EC 3.4.11.3  ERAP-2  ER leucine aminopeptidase  EC 3.4.11.-  LAP-3  Leucine aminopeptidase  EC 3.4.11.5  METAP1  Methionine aminopeptidase-1  EC 3.4.11.18  METAP2  Methionine aminopeptidase-2  EC 3.4.11.18  MME  Neutral endopeptidase  EC 3.4.24.11  NPEPL1  Aminopeptidase-like 1  EC 3.4.11.1  NPEPPS  Alanyl aminopeptidase  EC 3.4.11.14  PEPD  X-proline dipeptidase  EC 3.4.13.9  PGPEP1  Pyroglutamyl peptidase  EC 3.4.31.9  PRCP  Prolyl carboxypeptidase  EC 3.4.16.2  PREP  Prolyl endopeptidase  EC 3.4.21.26  RNPEP  Arginyl aminopeptidase  EC 3.4.11.6  SCPEP-1  Serine carboxypeptidase-1  EC 3.4.16  XPNPEP-1  X-prolyl aminopeptidase-1  EC 3.4.11.9  XPNPEP-2  X-prolyl aminopeptidase-2  EC 3.4.11.9  XPNPEP-3  X-prolyl aminopeptidase-3  EC 3.4.11.9  Abbreviations: ANPEP, aminopeptidase N; DNPEP, aspartyl aminopeptidase; ENPEP, glutamyl aminopeptidase; PRCP, prolylcarboxypeptidase. View Large RNA isolation and reverse transcription polymerase chain reaction analyses Total RNA was isolated from ZG cells, BAAECs, and BAASMCs using a Qiagen RNeasy mini-kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). Total RNA was used for cDNA synthesis using the SuperScript first-strand synthesis system (Invitrogen, Carlsbad, CA). A standard 10 μL reaction contains 1 μg RNA, 50 ng of random hexamers, and 0.5 mM dNTPs. The mixture was incubated at 65°C for 5 minutes and on ice for 1 minute and then supplemented with 10× RT buffer (1× final concentration), 5 mM of MgCl2, 10 mM of dithiothreitol, ribonuclease inhibitor, and 50 U of SuperScript III RT. The reaction was incubated first at 25°C for 10 minutes, then at 50°C for 50 minutes, and, finally, at 85°C for 10 minutes, followed by incubation with 2 U of RNase H at 37°C for 30 minutes. For reverse transcription polymerase chain reaction (RT-PCR) analysis, 1 μL of the synthesized cDNA was used as the template in 25 μL reactions, each containing 0.2 mM dNTPS, 0.4 μM primers, 2 mM of MgCl2 and 1.25 U Taq DNA polymerase (Invitrogen) in 1× reaction buffer [20 mM Tris-HCl (pH 8.4), 50 mM KCl). The primers are indicated in Table 2. The reactions were incubated at 95°C for 3 minutes, followed by 37 amplification cycles, each including incubation at 95°C for 45 seconds, 60°C for 30 seconds, 72°C for 30 seconds, with an additional extension time at 72°C for 5 minutes. The RT-PCR products were then resolved in 1.5% agarose gels. Table 2. Primers Used in RT-PCR Gene Name  Accession Number  Forward  Reverse  DNPEP  NM001045952  5′-AGGTTGGTGTGGAGACCTAT-3′  5′-CTGGATGGATGTGGCAAGAA-3′  ENPEP  NM001038027  5′-CTATTGGACCCGAGAGCTAATG-3′  5′-CGTGTGGTTGACGGAAAGA-3′  ACE-2  NM001024502  5′-TGGGAGATGAAGCGAGAGATA-3′  5′-CTAAGGTCCAGGGTTCTGATTT-3′  PRCP  NM001038164  5′-GGGACATAGCTGAGGAAATGAA-3′  5′-CTCCAACCACCAAATGAGGATA-3′  ANPEP  NM001075144  5′-CCTACCTCACTCCCAACAATAAC-3′  5′-GTATGTCTTGCCTGCTTCCA-3′  RXFP3  XM005221625  5′-CTACCTGATGAAGAGCAAGCA-3′  5′-GCTCATGGAGGTGAGAAAGAA-3′  GAPDH  NM001034034  5′-ACGTGTCTGTTGTGGATCTG-3′  5′-CGTACCAGGAAATGAGCTTGA-3′  Gene Name  Accession Number  Forward  Reverse  DNPEP  NM001045952  5′-AGGTTGGTGTGGAGACCTAT-3′  5′-CTGGATGGATGTGGCAAGAA-3′  ENPEP  NM001038027  5′-CTATTGGACCCGAGAGCTAATG-3′  5′-CGTGTGGTTGACGGAAAGA-3′  ACE-2  NM001024502  5′-TGGGAGATGAAGCGAGAGATA-3′  5′-CTAAGGTCCAGGGTTCTGATTT-3′  PRCP  NM001038164  5′-GGGACATAGCTGAGGAAATGAA-3′  5′-CTCCAACCACCAAATGAGGATA-3′  ANPEP  NM001075144  5′-CCTACCTCACTCCCAACAATAAC-3′  5′-GTATGTCTTGCCTGCTTCCA-3′  RXFP3  XM005221625  5′-CTACCTGATGAAGAGCAAGCA-3′  5′-GCTCATGGAGGTGAGAAAGAA-3′  GAPDH  NM001034034  5′-ACGTGTCTGTTGTGGATCTG-3′  5′-CGTACCAGGAAATGAGCTTGA-3′  Abbreviations: ANPEP, aminopeptidase N; DNPEP, aspartyl aminopeptidase; ENPEP, glutamyl aminopeptidase; PRCP, prolylcarboxypeptidase. View Large Assay for aminopeptidase activity cleaving an N-terminal amino acid residue ZG cells were lysed in 50 mM Tris-HCl buffer (pH 7.4). Assays were performed in 96 well plates. ZG cell lysates (20 μL containing 100 µg protein) or buffer alone were incubated with 160 µL of a 1.25 mM solution of either the aspartyl-aminopeptidase A substrate (l-aspartyl-β-naphthylamide), glutamyl-aminopeptidase A substrate (l-glutamyl-β-naphthylamide) or leucyl-aminopeptidase substrate (l-leucyl-β-naphthylamide) in Tris-HCl buffer for various times. The reaction was stopped by adding 20 µL of 3M HCl. Hydrolysis of the amino acid results in the formation of the fluorescent β-naphthylamide, which was measured using a fluorescence plate reader (activation at 355 nm and emission at 460 nm). The results are expressed as relative fluorescence units. Statistical analysis Data are presented as the mean ± standard error of the mean. Statistically significant differences between the mean values were evaluated using analysis of variance, followed by the Student-Newman-Keuls multiple comparison test. A value of P < 0.05 was considered to indicate statistical significance. Results Ang II–stimulated, ZG cell–mediated vascular relaxation is not mediated by AT1 or AT2 receptors To determine which Ang receptor mediates ZG cell–dependent vascular relaxation in response to Ang II, vascular reactivity was performed on L-NA–treated adrenal arteries in the presence of Ang antagonists: losartan, an AT1 receptor antagonist; PD123,319, an AT2 receptor antagonist; D-Ala7-Ang (1-7), a Mas receptor antagonist; and Ile7-Ang III, an Ang III antagonist (31–34). L-NA was used to block any direct actions of Ang II on the adrenal arteries to allow the study of ZG cell–mediated relaxations only. Losartan, PD123,319, and D-Ala7-Ang (1-7) had no effect on ZG cell–mediated vascular relaxation of the adrenal arteries in response to Ang II [Fig. 1(a)–1(c)]. In contrast, the Ang III antagonist Ile7-Ang III significantly attenuated the ZG cell–mediated vascular relaxations to Ang II [Fig. 1(d)]. Reported studies have indicated that Ile7-Ang III inhibits Ang III binding and blocks the stimulation of aldosterone release by Ang III (32, 34, 38). Thus, these data suggest a potential role for Ang III in mediating the relaxations. Figure 1. View largeDownload slide ZG cell–mediated relaxations of adrenal arteries in response to Ang II in the presence of the (a) AT1 receptor antagonist losartan (10 μM), (b) AT2 receptor antagonist PD123,319 (10 μM), (c) Ang (1-7) antagonist d-Ala7-Ang (1-7) (10 μM), and (d) Ang III antagonist Ile7-Ang III (10 μM). ZG cells (5 to 10 × 105) were added to baths with intact arteries. Arteries were pretreated with the NO synthase inhibitor L-NA and cyclooxygenase inhibitor indomethacin to block direct relaxing effects of Ang II on adrenal arteries, allowing for measurement of ZG cell–mediated relaxations only. Each value represents the mean ± standard error of the mean (n = 6 to 10). *Statistically significantly different from the respective control, P < 0.05. Figure 1. View largeDownload slide ZG cell–mediated relaxations of adrenal arteries in response to Ang II in the presence of the (a) AT1 receptor antagonist losartan (10 μM), (b) AT2 receptor antagonist PD123,319 (10 μM), (c) Ang (1-7) antagonist d-Ala7-Ang (1-7) (10 μM), and (d) Ang III antagonist Ile7-Ang III (10 μM). ZG cells (5 to 10 × 105) were added to baths with intact arteries. Arteries were pretreated with the NO synthase inhibitor L-NA and cyclooxygenase inhibitor indomethacin to block direct relaxing effects of Ang II on adrenal arteries, allowing for measurement of ZG cell–mediated relaxations only. Each value represents the mean ± standard error of the mean (n = 6 to 10). *Statistically significantly different from the respective control, P < 0.05. Ang III is the primary Ang II metabolite of ZG cells The metabolism of Ang II was examined with a single bovine adrenal artery ring or with 1 × 106 ZG cells, or with the combination of the two, in conditions replicating the vascular reactivity experiments. No substantial Ang II metabolism was observed in the presence of an adrenal arterial ring (Fig. 2). In contrast, substantial Ang II metabolism was observed in the presence of ZG cells, with Ang III the primary metabolite (Fig. 2). Ang IV and Ang (1-7) were also produced by ZG cells, although to a lesser extent. The same Ang II metabolism profile was observed with the combination of an arterial ring and ZG cells. Figure 2. View largeDownload slide Ang II metabolism of adrenal arteries and ZG cells. A single bovine adrenal artery (BAA) ring (2 mm) or ZG cells (1 × 106 cells), or a combination of the two, were incubated with Ang II (100 nM) for 10 minutes. Supernatant was extracted and analyzed for Ang metabolites by LC-MS. Each value represents the mean ± standard error of the mean (n = 4). *Statistically significantly different from control, P < 0.05. Figure 2. View largeDownload slide Ang II metabolism of adrenal arteries and ZG cells. A single bovine adrenal artery (BAA) ring (2 mm) or ZG cells (1 × 106 cells), or a combination of the two, were incubated with Ang II (100 nM) for 10 minutes. Supernatant was extracted and analyzed for Ang metabolites by LC-MS. Each value represents the mean ± standard error of the mean (n = 4). *Statistically significantly different from control, P < 0.05. Ang III mediates ZG cell–dependent vascular relaxation of bovine adrenal arteries in response to Ang II Because (1) Ang III antagonism attenuated ZG cell–mediated vascular relaxation of adrenal arteries in response to Ang II and (2) Ang III is the primary Ang II metabolite of ZG cells, the ZG cell–dependent vascular response to Ang III was examined. Ang III stimulated a ZG cell–dependent relaxation response. The responses to Ang III were substantially greater than the responses to Ang II [10−10 M, Ang II, 35.8% ± 2.3%; Ang III, 48.7% ± 3.3%; Fig. 3(a)]. Figure 3. View largeDownload slide ZG cell–mediated relaxations of adrenal arteries in response to (a) Ang III and (b) Ang IV. ZG cells (5 to 10 × 105) were added to baths with intact arteries. Arteries were pretreated with the NO synthase inhibitor L-NA and cyclooxygenase inhibitor indomethacin to block the direct relaxing effects of Ang peptides on adrenal arteries, allowing for measurement of ZG cell–mediated relaxations only. Each value represents the mean ± standard error of the mean (n = 7 to 9). *Statistically significantly different from the respective Ang II concentration, P < 0.05. Figure 3. View largeDownload slide ZG cell–mediated relaxations of adrenal arteries in response to (a) Ang III and (b) Ang IV. ZG cells (5 to 10 × 105) were added to baths with intact arteries. Arteries were pretreated with the NO synthase inhibitor L-NA and cyclooxygenase inhibitor indomethacin to block the direct relaxing effects of Ang peptides on adrenal arteries, allowing for measurement of ZG cell–mediated relaxations only. Each value represents the mean ± standard error of the mean (n = 7 to 9). *Statistically significantly different from the respective Ang II concentration, P < 0.05. Because Ang IV is an aminopeptidase metabolite of Ang II and Ang III and a ZG cell metabolite of Ang II, the ZG cell–dependent vascular response to Ang IV was examined. In contrast to Ang III, the relaxation response to Ang IV was significantly less than that of Ang II [10−10 M, Ang II, 33.9% ± 2.0%; Ang IV, 15.3% ± 3.6%; Fig. 3(b)]. To examine the effect of metabolism of Ang II to Ang III on ZG cell–mediated vascular relaxation of adrenal arteries, amastatin (10 μM) and EC33 (10 μM), aminopeptidase inhibitors, were tested (26, 35). Ang II stimulated ZG cell–dependent relaxation responses that were significantly attenuated by amastatin [10−10 M, control, 35.8% ± 2.1%; amastatin, 15.3% ± 3.4%; Fig. 4(a)]. In contrast, amastatin had no effect on Ang III stimulated ZG cell–dependent relaxation responses in adrenal arteries [10−10 M, control, 50.0% ± 3.1%; amastatin, 41.9% ± 3.5%; Fig. 4(b)]. Additionally, Ang II stimulated ZG cell–dependent relaxation responses were significantly attenuated by EC33 [10−10 M, control, 30.4% ± 2.9%; EC33, 8.8% ±4.8%; Fig. 4(c)]. Figure 4. View largeDownload slide ZG cell–mediated relaxations of adrenal arteries in response to (a) Ang II in the presence of the aminopeptidase inhibitor amastatin (10 μM), (b) Ang III in the presence of the aminopeptidase inhibitor amastatin, and (c) Ang II in the presence of the aminopeptidase A inhibitor EC33 (10 μM). ZG cells (5 to 10 × 105) were added to baths with intact arteries. Arteries were pretreated with the NO synthase inhibitor L-NA and cyclooxygenase inhibitor indomethacin to block the direct relaxing effects of Ang II and Ang III on adrenal arteries, allowing for measurement of ZG cell–mediated relaxations only. Each value represents the mean ± standard error of the mean (n = 8 to 10). *Statistically significantly different from the respective control, P < 0.05. Figure 4. View largeDownload slide ZG cell–mediated relaxations of adrenal arteries in response to (a) Ang II in the presence of the aminopeptidase inhibitor amastatin (10 μM), (b) Ang III in the presence of the aminopeptidase inhibitor amastatin, and (c) Ang II in the presence of the aminopeptidase A inhibitor EC33 (10 μM). ZG cells (5 to 10 × 105) were added to baths with intact arteries. Arteries were pretreated with the NO synthase inhibitor L-NA and cyclooxygenase inhibitor indomethacin to block the direct relaxing effects of Ang II and Ang III on adrenal arteries, allowing for measurement of ZG cell–mediated relaxations only. Each value represents the mean ± standard error of the mean (n = 8 to 10). *Statistically significantly different from the respective control, P < 0.05. Metabolism of Ang II to Ang III does not affect aldosterone secretion Because the metabolism of Ang II to Ang III is necessary for ZG cell–dependent vascular relaxation of adrenal arteries, the effect of aminopeptidase metabolism of Ang II on aldosterone secretion was examined in bovine ZG cells. Ang II and Ang III were equipotent in stimulating aldosterone secretion [10−7 M, Ang II, 702 ± 122 pg/mL; Ang III, 744 ± 131 pg/mL; Fig. 5(a)]. In the presence of amastatin, the ability of Ang II and Ang III to stimulate aldosterone secretion remained unchanged [10−7 M, Ang II, 695 ± 123 pg/mL; Ang III, 762 ± 130 pg/mL; Fig. 5(b)]. Figure 5. View largeDownload slide Aldosterone secretion from cultured bovine ZG cells in response to Ang II and Ang III in the (a) absence or (b) presence of the aminopeptidase inhibitor amastatin (10 μM). Each value represents the mean ± standard error of the mean (n = 4). Figure 5. View largeDownload slide Aldosterone secretion from cultured bovine ZG cells in response to Ang II and Ang III in the (a) absence or (b) presence of the aminopeptidase inhibitor amastatin (10 μM). Each value represents the mean ± standard error of the mean (n = 4). Peptidases expressed in bovine ZG, endothelial, and smooth muscle cells RNA sequencing was performed on bovine ZG cells, BAAECs, and BAASMCs. Numerous aminopeptidases and peptidases that might metabolize Ang peptides were present in the transcriptome of these cells (Fig. 6). Table 1 lists the gene name, common name, and Enzyme Commission number for the peptidases. Both aspartyl aminopeptidase A (DNPEP) and glutamyl aminopeptidase A (ENPEP) metabolize Ang II to Ang III (26, 35, 39). DNPEP was highly expressed in all three cell types, especially in ZG cells (in relative expression, BAAEC, 6.13; BAASMC, 5.69; ZG, 23.64), and ENPEP was either not expressed or minimally expressed in the cells [Fig. 6(a)]. Angiotensin-converting enzyme 2 (ACE2) and prolylcarboxypeptidase (PRCP) metabolize Ang II to Ang (1-7) and Ang III to Ang (2-7) (3). ACE2 was not expressed in all three cell types, and PRCP was expressed in all three, albeit to a lesser extent than DNPEP. Aminopeptidase N (ANPEP), which metabolizes Ang III to Ang IV, was not expressed in the three types of cells. Of the leucyl-aminopeptidases, ERAP-1 and ERAP-2 metabolize Ang II to Ang III and Ang IV (40, 41); however, LAP-3 has not been tested for its ability to metabolize Ang II. Figure 6. View largeDownload slide RNA expression of peptidases in bovine adrenal arteries and ZG cells. RNA sequencing data for peptidases in (a) bovine ZG cells, (b) BAAECs, and (c) BAASMCs, expressed in reads per kilobase of transcript per 1 million mapped reads (RPKM). (d) Agarose gel electrophoresis of RT-PCR products of peptidases (upper) in bovine adrenal arteries and (lower) in bovine ZG cells. (1) DNPEP, 246 bp; (2) ENPEP, 262 bp; (3) ACE2, 274 bp; (4) PRCP, 276 bp; (5) ANPEP, 257 bp; (6) RNPEP, 272 bp; (7) NPEPPS, 255 bp; (8) LAP-3, 239 bp; and (9) bovine GAPDH, 228 bp. The gene names, common names, and Enzyme Commission numbers for the peptidases are listed in Table 1. Figure 6. View largeDownload slide RNA expression of peptidases in bovine adrenal arteries and ZG cells. RNA sequencing data for peptidases in (a) bovine ZG cells, (b) BAAECs, and (c) BAASMCs, expressed in reads per kilobase of transcript per 1 million mapped reads (RPKM). (d) Agarose gel electrophoresis of RT-PCR products of peptidases (upper) in bovine adrenal arteries and (lower) in bovine ZG cells. (1) DNPEP, 246 bp; (2) ENPEP, 262 bp; (3) ACE2, 274 bp; (4) PRCP, 276 bp; (5) ANPEP, 257 bp; (6) RNPEP, 272 bp; (7) NPEPPS, 255 bp; (8) LAP-3, 239 bp; and (9) bovine GAPDH, 228 bp. The gene names, common names, and Enzyme Commission numbers for the peptidases are listed in Table 1. To confirm the RNA sequencing results, RT-PCR analysis was performed using specific primers (Table 2). RNAs from bovine adrenal arteries and bovine ZG cells were examined. In bovine adrenal arteries, DNPEP and PRCP were highly expressed and ENPEP, LAP-3, and ANPEP were expressed to a lesser extent, and no ACE2 was detected [Fig. 6(d)]. In bovine ZG cells, DNPEP was highly expressed [Fig. 6(d)]. PRCP, LAP-3, and ANPEP were expressed to a lesser extent, but no ENPEP and ACE2 were detected in ZG cells. Aminopeptidase activity in bovine ZG cells Aspartyl-, glutamyl-, and leucyl-aminopeptidase enzymatic activities were examined in ZG cell lysates to confirm the RNA expression results. When ZG cell lysate was incubated with the aspartyl-aminopeptidase–specific substrate l-aspartyl-β-naphthylamide, hydrolysis of the aspartyl group and release of fluorescent β-naphthylamide occurred (Fig. 7). This aminopeptidase activity significantly increased with time. Similar lysate activity was obtained with the leucyl-aminopeptidase substrate l-leucyl-β-naphthylamide. In contrast, when ZG cell lysate was incubated with the glutamyl-aminopeptidase–specific substrate l-glutamyl-β-naphthylamide, no change was found in the formation of β-naphthylamide. When the amino acid β-naphthylamide substrates were incubated without lysates or lysates were incubated without substrates, no changes in fluorescence were observed over time. Figure 7. View largeDownload slide Aminopeptidase activity in bovine ZG cell lysates. Each value represents the mean ± standard error of the mean (n = 9). Asp-β-NA, l-aspartyl-β-naphthylamide (substrate for aspartyl-aminopeptidase A); Glu-β-NA, l-glutamyl-β-naphthylamide (substrate for glutamyl-aminopeptidase A); Leu-β-NA, l-leucyl-β-napthylamide (substrate for leucyl-aminopeptidase). Figure 7. View largeDownload slide Aminopeptidase activity in bovine ZG cell lysates. Each value represents the mean ± standard error of the mean (n = 9). Asp-β-NA, l-aspartyl-β-naphthylamide (substrate for aspartyl-aminopeptidase A); Glu-β-NA, l-glutamyl-β-naphthylamide (substrate for glutamyl-aminopeptidase A); Leu-β-NA, l-leucyl-β-napthylamide (substrate for leucyl-aminopeptidase). Discussion In previous studies, we found that Ang II relaxes adrenal arteries by acting directly on the adrenal vasculature and by a mechanism that involves ZG cells (Fig. 8). In the absence of ZG cell, Ang II causes endothelium-dependent relaxations of isolated adrenal arteries by activating AT2 receptors and promoting NO release (22, 24). In arteries without endothelium or treated with L-NA, Ang II does not cause relaxations. However, the presence of ZG cells restores the relaxations to Ang II in these arteries through the release of EETs and DHETs (23). The present study has demonstrated that ZG cell–dependent relaxations to Ang II are not mediated by traditional angiotensin AT1 or AT2 receptors or the Ang (1-7) Mas receptor. Only the Ang III antagonist Ile7-Ang III significantly attenuated these relaxations. The identity of the AT receptor that mediates these relaxations is not known (Fig. 8). A specific receptor for Ang III has yet to be identified, although evidence exists for a high affinity Ang III receptor. Specific binding of [3H]Ang II and [3H]Ang III in particulate fractions of rat adrenal glands demonstrated a high affinity binding site for [3H]Ang III with a Kd of 1.5 × 10−10 M and a binding capacity of 1.2 × 10−13 mol/mg of protein (34, 38, 42). Ang II did not suppress binding of [3H]Ang III to this high affinity site. [3H]Ang III binding was inhibited by the Ang III antagonist Ile7-Ang III, suggesting a specific receptor for Ang III. Our observation that Ile7-Ang III inhibits the ZG cell–mediated relaxations to Ang II provides further evidence that an unknown Ang III receptor might exist on ZG cells of the adrenal gland (Fig. 8). In contrast, in the absence of ZG cells, Ang III, like Ang II, directly relaxes adrenal arteries by activation of endothelial AT2 receptors and release of NO (24). Thus, the vasorelaxations to Ang III in the adrenal vasculature are complex. They involve distinct actions on ZG cells and the vascular endothelium and use different Ang receptors (Fig. 8). Figure 8. View largeDownload slide Summary of the regulation of adrenal vascular tone by Ang II and Ang III. Ang II–mediated vasorelaxation has both direct and indirect vascular action. (Left) In the direct action, relaxation of adrenal arteries by Ang II is mediated by activation of AT2 receptors on endothelial cells resulting in endothelial NO release, smooth muscle cell cyclic guanosine monophosphate (cGMP) formation, and relaxation. This direct action is inhibited by the AT2 antagonist PD123,319 and the NO synthase (NOS) inhibitor LNA. (Right) Ang II also has an indirect action via ZG cells. Ang II is metabolized to Ang III by an amastatin- and EC33-sensitive aminopeptidase (AP) on ZG cells. Ang III acts on an unknown AT receptor (AT?) to stimulate the synthesis and release of EETs and DHETs. The EETs act on an EET receptor (EET R), activating a calcium-activated, iberiotoxin (IBTX)-sensitive potassium (K) channel, allowing K efflux, membrane hyperpolarization, and relaxation. 14,15EEZE, 14,15-epoxyeicosa-5Z-enoic acid (EET antagonist); Arg, arginine; GTP, guanosine triphosphate; PL, phospholipid. Figure 8. View largeDownload slide Summary of the regulation of adrenal vascular tone by Ang II and Ang III. Ang II–mediated vasorelaxation has both direct and indirect vascular action. (Left) In the direct action, relaxation of adrenal arteries by Ang II is mediated by activation of AT2 receptors on endothelial cells resulting in endothelial NO release, smooth muscle cell cyclic guanosine monophosphate (cGMP) formation, and relaxation. This direct action is inhibited by the AT2 antagonist PD123,319 and the NO synthase (NOS) inhibitor LNA. (Right) Ang II also has an indirect action via ZG cells. Ang II is metabolized to Ang III by an amastatin- and EC33-sensitive aminopeptidase (AP) on ZG cells. Ang III acts on an unknown AT receptor (AT?) to stimulate the synthesis and release of EETs and DHETs. The EETs act on an EET receptor (EET R), activating a calcium-activated, iberiotoxin (IBTX)-sensitive potassium (K) channel, allowing K efflux, membrane hyperpolarization, and relaxation. 14,15EEZE, 14,15-epoxyeicosa-5Z-enoic acid (EET antagonist); Arg, arginine; GTP, guanosine triphosphate; PL, phospholipid. Additionally, Ang III is both more potent and efficacious than Ang II in stimulating ZG cell–mediated relaxation of adrenal arteries. These data support a role for Ang III in these relaxations. Although Ang II is considered the primary biologically active peptide of the RAAS, evidence has shown that Ang III is an active metabolite in the adrenal gland and central nervous system. In the central nervous system, Ang III plays a role in tonic blood pressure maintenance and hypertension (43). Inhibiting Ang II conversion to Ang III with aminopeptidase A inhibition lowers vasopressin release and blood pressure (26, 43). In the adrenal gland, Ang III is more potent than Ang II in stimulating aldosterone synthesis ex vivo in ZG cells of four species and equally potent in vivo (44–46). Moreover, evidence exists for intracellular Ang III in adrenal ZG cells and direct effects on isolated mitochondria (46). Most importantly, our data demonstrate that the endogenous production of Ang III mediates ZG cell–mediated relaxation of adrenal arteries in response to Ang II. The primary metabolite of Ang II in ZG cells is Ang III. The metabolism of Ang II by a single arterial ring was not substantial. However, in studies using isolated BAAECs or BASSMCs, Ang II was metabolized to Ang (1-7) and lesser amounts of Ang III and Ang IV (24). Thus, the vasculature is capable of Ang II metabolism to Ang III and other Ang peptides; however, it was not substantial compared with ZG cell–mediated metabolism under these experimental conditions. Ang II–stimulated, ZG cell–mediated relaxations of adrenal arteries was significantly attenuated by inhibition of aminopeptidases, the enzymes that metabolize Ang II to Ang III and Ang IV. Ang IV only minimally stimulates ZG cell–mediated relaxations of adrenal arteries, providing further evidence that Ang III, not a downstream aminopeptidase metabolite of Ang III, is the Ang peptide responsible for ZG cell–mediated relaxation (Fig. 8). The importance of Ang II metabolism to Ang III differs in the ZG cells according to whether aldosterone release or EET release are measured. The metabolism of Ang II to Ang III is essential for ZG cell–mediated relaxations of adrenal arteries; however, it is not necessary for, and has no effect on, stimulation of Ang II–induced aldosterone secretion. This is because Ang II and Ang III are equipotent in the stimulation of aldosterone secretion from bovine ZG cells. Thus, inhibition of aminopeptidase metabolism had no effect on Ang II–stimulated aldosterone production. This observation is in agreement with previous studies in primary rat ZG cells, in which Ang II and Ang III were equipotent in the stimulation of aldosterone secretion (45). In a human adrenocortical carcinoma cell line, Ang III was less potent than Ang II in stimulating aldosterone secretion (47). Thus, aminopeptidase inhibition blocks ZG cell–mediated relaxations to Ang II without affecting aldosterone release. The endogenous production of Ang III in ZG cells is further validated by the expression and activity of peptidases. RNA expression confirmed that the primary aminopeptidase present in ZG cells is aspartyl-aminopeptidase A. It is encoded by the DNPEP gene and metabolizes Ang II to Ang III (39). Leucyl-aminopeptidase-3, encoded by the LAP-3 gene, is also highly expressed; however, its ability to metabolize Ang II is not known. Glutamyl-aminopeptidase A, encoded by the ENPEP gene, converts Ang II to Ang III but is not detected in ZG cells (26). The activities of aspartyl-aminopeptidase A and leucyl-aminopeptidase, but not glutamyl-aminopeptidase A, are present in ZG cell lysates. These activity data are consistent with these RNA expression data. Identification of the aminopeptidases responsible for Ang III formation in ZG cells was beyond the scope of the present research. However, studies with aminopeptidase inhibitors and expression have provided some insights. Amastatin and EC33 inhibit the ability of Ang II to cause ZG cell–mediated relaxation of adrenal arteries. Thus, the aminopeptidase must be inhibited by amastatin and EC33. Aspartyl-aminopeptidase A (DNPEP) is not inhibited by either of the inhibitors (39). Thus, although aspartyl-aminopeptidase A is highly expressed, it is not responsible for Ang II conversion to Ang III in ZG cells. This might be because of its cytosolic localization. Similarly, aminopeptidase P1 (XPNPEP1), another cytosolic enzyme, is not inhibited by amastatin but is expressed in ZG cells (48). Glutamyl-aminopeptidase A (ENPEP) is inhibited by both amastatin and EC33 (26); however, it is not a candidate, because it is not expressed in ZG cells. This process of elimination leaves for consideration one of the leucyl-aminopeptidase family or another aminopeptidase not known to metabolize Ang II to Ang III. Three leucyl-aminopeptidases are expressed at different levels in ZG cells: leucyl-aminopeptidase-3 (LAP-3 gene), adipocyte leucyl-aminopeptidase (ERAP-1 gene), and endoplasmic reticulum leucyl-aminopeptidase (ERAP-2 gene). Leucyl-aminopeptidase-3 and adipocyte leucyl-aminopeptidase are inhibited by amastatin and, thus, represent possible candidates (40). The inhibitory activity of EC33 has not been determined for any of the three leucyl-aminopeptidases. Additionally, the sensitivity of endoplasmic reticulum leucyl-aminopeptidase-2 to amastatin inhibition is not known. It is also a possible participant. Adipocyte leucyl-aminopeptidase and endoplasmic reticulum leucyl-aminopeptidase-2 metabolize Ang II to Ang III (40, 41); however, this activity has not been documented for leucyl-aminopeptidase-3. This process of elimination leaves the leucyl-aminopeptidases as the leading candidates for the observed conversion of Ang II to Ang III in ZG cells (40, 41). Additionally, RNA for PRCP, the gene for prolylcarboxypeptidase or angiotensinase C, is expressed in ZG cells (49). However, ACE2 is not expressed. These findings suggest that prolylcarboxypeptidase, and not ACE2, is the enzyme responsible for metabolizing Ang II to Ang (1-7) in ZG cells. In conclusion, the regulation of adrenal blood flow plays an important role in the regulation of steroidogenesis. Ang II, a potent vasoconstrictor, causes vasorelaxation of adrenal cortical arteries by a direct endothelium-mediated mechanism and an indirect paracrine, ZG cell–mediated mechanism (Fig. 8). Ang II metabolism is important in both of these mechanisms. Ang II and its vascular endothelial metabolite Ang III increase adrenal blood flow and stimulate aldosterone production (24). Likewise, in the paracrine regulation of adrenal vascular tone by ZG cells, endogenous metabolism of Ang II to Ang III is an obligatory step in Ang II–stimulated, ZG cell–mediated relaxations of adrenal cortical arteries. Also, our study provides additional evidence for a unique Ang III receptor in the adrenal cortex. Thus, Ang III has important and distinct roles in the adrenal gland to regulate blood flow and steroidogenesis. Abbreviations: ACE2 angiotensin-converting enzyme 2 ACTH adrenocorticotropic hormone Ang angiotensin ANPEP aminopeptidase N AT1 angiotensin type 1 AT2 angiotensin type 2 BAAEC bovine adrenal artery endothelial cell BAASMC bovine adrenal artery smooth muscle cell cDNA complementary DNA DHET dihydroxyeicosatrienoic acid DNPEP aspartyl aminopeptidase EET epoxyeicosatrienoic acid ENPEP glutamyl aminopeptidase A LC liquid chromatography LC-MS liquid chromatography–mass spectrometry L-NA nitro-l-arginine MS mass spectrometry NO nitric oxide PRCP prolylcarboxypeptidase PSS physiological salt solution RAAS renin-angiotensin-aldosterone system RT-PCR reverse transcription polymerase chain reaction TFA trifluoroacetic acid ZG zona glomerulosa. Acknowledgments The authors thank Ms. Jessica Kelliher for technical assistance, Dr. Kasem Nithipatikom for assistance with the mass spectrometric analyses of angiotensin peptides, and Mrs. Gretchen Barg for secretarial assistance. 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Obligatory Metabolism of Angiotensin II to Angiotensin III for Zona Glomerulosa Cell–Mediated Relaxations of Bovine Adrenal Cortical Arteries

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Abstract

Abstract Hyperaldosteronism is associated with hypertension, cardiac hypertrophy, and congestive heart failure. Steroidogenic factors facilitate aldosterone secretion by increasing adrenal blood flow. Angiotensin (Ang) II decreases adrenal vascular tone through release of zona glomerulosa (ZG) cell–derived vasodilatory eicosanoids. However, ZG cell–mediated relaxation of bovine adrenal cortical arteries to Ang II is not altered by angiotensin type 1 or 2 receptor antagonists. Because traditional Ang II receptors do not mediate these vasorelaxations to Ang II, we investigated the role of Ang II metabolites. Ang III was identified by liquid chromatography–mass spectrometry as the primary ZG cell metabolite of Ang II. Ang III stimulated ZG cell–mediated relaxation of adrenal arteries with greater potency than did Ang II. Furthermore, ZG cell–mediated relaxations of adrenal arteries by Ang II were attenuated by aminopeptidase inhibition, and Ang III-stimulated relaxations persisted. Ang IV had little effect compared with Ang II. Moreover, ZG cell–mediated relaxations of adrenal arteries by Ang II were attenuated by an Ang III antagonist but not by an Ang (1-7) antagonist. In contrast, Ang II and Ang III were equipotent in stimulating aldosterone secretion from ZG cells and were unaffected by aminopeptidase inhibition. Additionally, aspartyl and leucyl aminopeptidases, which convert Ang II to Ang III, are the primary peptidase expressed in ZG cells. This was confirmed by enzyme activity. These data indicate that intra-adrenal metabolism of Ang II to Ang III is required for ZG cell–mediated relaxations of adrenal arteries but not aldosterone secretion. These studies have defined an important role of Ang III in the adrenal gland. The renin-angiotensin-aldosterone system (RAAS) is an important regulator of normal cardiovascular homeostasis (1). Dysregulation of this critical system leads to the pathogenesis of a variety of cardiovascular diseases, and pharmacologic intervention of the RAAS is therapeutically beneficial. Angiotensin (Ang) II is a potent vasoconstrictor and enhances the activity of the sympathetic nervous system (2). Inhibition of Ang II synthesis or Ang II receptor antagonism is beneficial in the treatment of hypertension (3). Ang II additionally stimulates the synthesis and secretion of aldosterone from the zona glomerulosa (ZG) region of the adrenal gland. Aldosterone is a mineralocorticoid that regulates extracellular fluid and electrolyte homeostasis (4). Elevated levels of aldosterone are associated with hypertension, cardiac fibrosis, left ventricular remodeling, endothelial dysfunction, vasculopathy, vascular remodeling, and renal injury (5). Mineralocorticoid receptor antagonists alleviate these deleterious effects. Thus, understanding the factors that regulate aldosterone release has important implications for cardiovascular health. Aldosterone secretagogues [Ang II, potassium, adrenocorticotropic hormone (ACTH)] might partially regulate aldosterone secretion by increasing adrenal blood flow (6–8). ACTH increases adrenal blood flow by 200% to 272% in vivo (9–12). ACTH also increases adrenal blood flow ex vivo in perfused adrenal glands (8, 13–16). Additionally, despite being a potent vasoconstrictor, Ang II did not decrease adrenal blood flow in sheep and increased adrenal blood flow in rats in vivo (17, 18). The effect of adrenal secretagogues on adrenal blood flow results from the unique vascular architecture of the adrenal gland. The subcapsular adrenal arteries are the only resistance vessels of the adrenal gland and control adrenal vascular resistance and blood flow (19). These vessels closely adhere to the ZG region, running parallel to or within the ZG region, before penetration into the gland and the formation of cortical and medullary capillary networks. The ZG cells produce vasorelaxing factors that contribute to the vascular effects of ACTH and Ang II. ACTH does not affect the vascular tone of the subcapsular adrenal arteries in vitro (20). However, in the presence of ZG cells, ACTH induces vascular relaxation by the release of ZG cell–derived epoxyeicosatrienoic acids (EETs) (21). EETs and their hydrolysis products, the dihydroxyeicosatrienoic acids (DHETs) relax adrenal arteries by activating calcium-activated potassium channels, causing membrane hyperpolarization. Although a potent vasoconstrictor of some arteries, Ang II directly relaxes adrenal cortical arteries through the release of endothelial nitric oxide (NO) (22). However, in the presence of ZG cells, this relaxation response to Ang II is augmented by ZG cell–derived EETs and DHETs (23). Another potential mechanism by which Ang II causes vasorelaxation of adrenal arteries is by metabolism of Ang II. In adrenal arteries, Ang II is metabolized primarily to Ang III and Ang (1-7) (24). These two metabolic pathways produce divergent effects. Aminopeptidase metabolism of Ang II to Ang III preserves the vasorelaxation response, and carboxypeptidase metabolism of Ang II to Ang (1-7) reduces the vasorelaxation response. The metabolism of Ang II to either metabolite removes the contraction response in adrenal arteries (24). With evidence supporting the importance of Ang II metabolism in the direct vasorelaxation response, we examined the role of Ang II metabolism in ZG cell–dependent vasorelaxation. The goals of these studies were to (1) pharmacologically characterize the receptor that mediates Ang II–stimulated, ZG cell–dependent vasorelaxation of adrenal arteries, (2) identify the ZG cell metabolites of Ang II, and (3) examine the role of the Ang II metabolites on ZG cell–dependent vasorelaxation of adrenal arteries. Our results have demonstrated that Ang III, not Ang II, mediates ZG cell–dependent vasorelaxation of adrenal arteries. Materials and Methods Reagents HEPES buffer and physiological saline solution ingredients, angiotensin peptides, nitro-l-arginine (L-NA), indomethacin, losartan, PD123,319, amastatin, and cell culture media were purchased from Sigma-Aldrich (St. Louis, MO). U46619 was purchased from Cayman Chemical (Ann Arbor, MI). D-Ala7-Ang (1-7) and Ile7-Ang III were purchased from Bachem Inc. (Torrance, CA). EC33, a glutamyl-aminopeptidase A inhibitor, was synthesized as previously described (25, 26). The 13C515N1-Ang IV internal standard was synthesized by the Medical College of Wisconsin Protein Nucleic Acid Facility (Milwaukee, WI). The 13C515N1-Ang III and 13C515N1-Ang (1-7) internal standards were synthesized by EZBiolab (Carmel, IN). All the solvents were high-performance liquid chromatography (LC) grade and purchased from Sigma-Aldrich. ZG cell isolation and culture Bovine adrenal ZG cells, bovine adrenal artery endothelial cells (BAAECs), and bovine adrenal artery smooth muscle cells (BAASMCs) were prepared by enzymatic dissociation of adrenal cortical slices, as previously described (22, 24, 27). For the vascular reactivity and mass spectrometry studies, freshly isolated ZG cells were used. For studies of aldosterone release, cultured ZG cells were used (28). Isometric tension recording Fresh bovine adrenal glands were acquired from a local slaughterhouse. Subcapsular cortical arteries closely adherent to the adrenal surface (200 to 300 μm) were dissected and cleaned of connective tissue in ice-cold HEPES buffer (150 mM NaCl, 10 mM HEPES, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 6 mM glucose; pH 7.4). Isolated arterial segments were threaded on two 40-μm stainless steel wires and mounted in a 610M 4-chamber wire myograph (Danish Myotechnologies, Aarhus, Denmark) containing physiological salt solution (PSS; 119 mM NaCl, 24 mM NaHCO3, 4.7 mM KCl, 2 mM CaCl2, 1.18 mM KH2PO4, 1.17 mM MgSO4, 0.026 mM EDTA, 5.5 mM glucose; pH 7.4), bubbled with 95% oxygen, 5% carbon dioxide at 37°C, as previously described (22, 29, 30). After 30 minutes of equilibration, the arteries were gradually stretched to a resting tension of 1 mN and stimulated with KCl (60 mM) and the thromboxane A2 mimetic U-46619 (100 nM) three times for 10 minutes at 10-minute intervals. The arteries were allowed to equilibrate for 30 minutes before the initiation of the experimental protocols. The arteries were precontracted with submaximal concentrations of U-46619 (10 to 30 nM) to 50% to 75% of their maximal KCl and U46619 stimulation. To examine the vasoactive factors released by ZG cells in response to Ang II stimulation, experiments were performed in the presence of ZG cells, as previously described (23). In brief, 5 to 10 × 105 cells were added to baths with intact arteries pretreated with the endothelial NO synthase inhibitor L-NA (30 μM) and the cyclooxygenase inhibitor indomethacin (10 μM). L-NA and indomethacin were added to block any direct relaxing effects of Ang peptides on adrenal arteries, such that the ZG cell–mediated responses only were studied (21, 23, 24). Cumulative concentration responses to Ang II, Ang III, or Ang IV (10 fM to 100 pM) were performed. The responses were repeated with arteries and ZG cells pretreated with the angiotensin type 1 (AT1) receptor antagonist losartan (10 μM), the angiotensin type 2 (AT2) receptor antagonist PD123,319 (10 μM), the Ang III antagonist Ile7-Ang III (10 μM), the Ang (1-7) antagonist D-Ala7-Ang (1-7), and the aminopeptidase inhibitors amastatin (10 and 100 μM) and EC33 (10 μM) (26, 31–35). Ang II metabolism The metabolism of Ang II by bovine adrenal arteries and ZG cells was examined under conditions replicating the isometric tension experiments. An adrenal artery ring or ZG cells (1 × 106), or the combination of the two, were incubated in 6 mL of PSS with 100 nM Ang II, bubbled with 95% oxygen, 5% carbon dioxide at 37°C for 10 minutes. PSS buffer alone containing 100 nM Ang II was used to control for spontaneous degradation. Buffer and cells/tissue were separated by centrifugation. Supernatant was removed and extracted the same day. Solid-phase extraction The internal standards [Sar1, Ile8-Ang II, 13C515N1-Ang III, 13C515N1-Ang IV, and 13C515N1-Ang (1-7); 30 ng] were added to the supernatant. The supernatant was prepared for solid-phase extraction by the addition of ethanol containing 1% trifluoroacetic acid (TFA) to a final volume of 15%, followed by adding 1 mL of water containing 1% TFA. The sample was applied to a preconditioned Sep Pak C18 SPE cartridge (Waters Corp., Milford, MA) and washed with 20 mL of water containing 1% TFA. Ang peptides were then eluted from the column using 6 mL of methanol containing 1% TFA and dried under a stream of nitrogen gas. For LC–mass spectrometry (LC-MS), the samples were then dissolved in 30 μL of 50% methanol/50% water containing 3% formic acid and 0.01% TFA and centrifuged, and the supernatant was analyzed. LC-MS analysis LC-MS and LC–tandem MS was performed using a modification of a previously described method (24, 29, 36). LC-tandem MS was used to identify the peptides, and LC/MS was used to quantify the Ang peptides. Analyses were performed using a Waters-Micromass Quattro micro-atmospheric pressure ionization electrospray triple quadrupole mass spectrometric system coupled with a Waters 2695 high-performance liquid chromatograph. The mass spectrometer is equipped with a Z-spray dual orthogonal ionization source and is controlled by MassLynx, version 4.1, software. The samples were separated using a reverse-phase C18 column (Jupiter 2.0 × 250 mm; Phenomenex) using water-methanol with 0.3% formic acid as a mobile phase at a flow rate of 0.2 mL/min. The mobile phase of 20% methanol in water linearly increased to 50% methanol over 30 minutes, followed by a linear increase to 60% methanol over 5 minutes. The positive ion electrospray ionization mass spectrometric conditions were as follows: capillary voltage, 3.0 kV; cone voltage, 20 V; desolvation temperature, 400°C; and source temperature, 100°C. LC-MS analysis was performed in positive electrospray mode in the single-ion recording mode. Aldosterone secretion Aldosterone secretion studies were performed as previously described (28). Cultured ZG cells were incubated with Ang II or Ang III (100 pM to 100 nM) for 2 hours before analysis of media for aldosterone. The responses were repeated with ZG cells pretreated with the aminopeptidase inhibitor amastatin (10 and 100 μM). Aldosterone production by cultured ZG cells was measured using enzyme-linked immunosorbent assay (27, 28). RNA sequencing Bovine adrenal ZG, endothelial, and smooth muscle cells (2 × 106 cells) were placed in RNAlater and shipped to Otogenetics for RNA extraction, polyA complementary DNA (cDNA) preparation, Illumina library preparation, and Illumina HiSeq2000 sequencing. Using DNAnexus, the reads were aligned and assembled to the bovine reference transcriptome. Once the reads were mapped, each transcript was quantified by calculating the reads per kilobase of transcript per 1 million mapped reads to normalize the data for transcript length and across samples of different coverage (37). This allowed a comparison of expression levels among transcripts and across the cell types. The reads per kilobase of transcript per 1 million mapped reads for the peptidases were plotted for the three cell types. The results for the specific peptidases were confirmed using reverse transcription polymerase chain reaction (RT-PCR). Table 1 lists the gene name, the common name used in our report, and Enzyme Commission number for the peptidases of interest. Table 1. Gene, Name, and Enzyme Commission Number of Peptidases Gene  Name  EC Number  ACE  Angiotensin converting enzyme  EC 3.4.15.1  ACE2/ACEH  Angiotensin converting enzyme-2  EC 3.4.17.23  ANPEP  Aminopeptidase N  EC 3.4.11.2  AQPEP  Aminopeptidase Q  EC 3.4.24.57  DNPEP  Aspartyl aminopeptidase A  EC 3.4.11.21  DPEP-1  Dipeptidase-1  EC 3.4.13.19  ENPEP  Glutamyl aminopeptidase A  EC 3.4.11.7  ERAP-1  ER leucine aminopeptidase  EC 3.4.11.3  ERAP-2  ER leucine aminopeptidase  EC 3.4.11.-  LAP-3  Leucine aminopeptidase  EC 3.4.11.5  METAP1  Methionine aminopeptidase-1  EC 3.4.11.18  METAP2  Methionine aminopeptidase-2  EC 3.4.11.18  MME  Neutral endopeptidase  EC 3.4.24.11  NPEPL1  Aminopeptidase-like 1  EC 3.4.11.1  NPEPPS  Alanyl aminopeptidase  EC 3.4.11.14  PEPD  X-proline dipeptidase  EC 3.4.13.9  PGPEP1  Pyroglutamyl peptidase  EC 3.4.31.9  PRCP  Prolyl carboxypeptidase  EC 3.4.16.2  PREP  Prolyl endopeptidase  EC 3.4.21.26  RNPEP  Arginyl aminopeptidase  EC 3.4.11.6  SCPEP-1  Serine carboxypeptidase-1  EC 3.4.16  XPNPEP-1  X-prolyl aminopeptidase-1  EC 3.4.11.9  XPNPEP-2  X-prolyl aminopeptidase-2  EC 3.4.11.9  XPNPEP-3  X-prolyl aminopeptidase-3  EC 3.4.11.9  Gene  Name  EC Number  ACE  Angiotensin converting enzyme  EC 3.4.15.1  ACE2/ACEH  Angiotensin converting enzyme-2  EC 3.4.17.23  ANPEP  Aminopeptidase N  EC 3.4.11.2  AQPEP  Aminopeptidase Q  EC 3.4.24.57  DNPEP  Aspartyl aminopeptidase A  EC 3.4.11.21  DPEP-1  Dipeptidase-1  EC 3.4.13.19  ENPEP  Glutamyl aminopeptidase A  EC 3.4.11.7  ERAP-1  ER leucine aminopeptidase  EC 3.4.11.3  ERAP-2  ER leucine aminopeptidase  EC 3.4.11.-  LAP-3  Leucine aminopeptidase  EC 3.4.11.5  METAP1  Methionine aminopeptidase-1  EC 3.4.11.18  METAP2  Methionine aminopeptidase-2  EC 3.4.11.18  MME  Neutral endopeptidase  EC 3.4.24.11  NPEPL1  Aminopeptidase-like 1  EC 3.4.11.1  NPEPPS  Alanyl aminopeptidase  EC 3.4.11.14  PEPD  X-proline dipeptidase  EC 3.4.13.9  PGPEP1  Pyroglutamyl peptidase  EC 3.4.31.9  PRCP  Prolyl carboxypeptidase  EC 3.4.16.2  PREP  Prolyl endopeptidase  EC 3.4.21.26  RNPEP  Arginyl aminopeptidase  EC 3.4.11.6  SCPEP-1  Serine carboxypeptidase-1  EC 3.4.16  XPNPEP-1  X-prolyl aminopeptidase-1  EC 3.4.11.9  XPNPEP-2  X-prolyl aminopeptidase-2  EC 3.4.11.9  XPNPEP-3  X-prolyl aminopeptidase-3  EC 3.4.11.9  Abbreviations: ANPEP, aminopeptidase N; DNPEP, aspartyl aminopeptidase; ENPEP, glutamyl aminopeptidase; PRCP, prolylcarboxypeptidase. View Large RNA isolation and reverse transcription polymerase chain reaction analyses Total RNA was isolated from ZG cells, BAAECs, and BAASMCs using a Qiagen RNeasy mini-kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). Total RNA was used for cDNA synthesis using the SuperScript first-strand synthesis system (Invitrogen, Carlsbad, CA). A standard 10 μL reaction contains 1 μg RNA, 50 ng of random hexamers, and 0.5 mM dNTPs. The mixture was incubated at 65°C for 5 minutes and on ice for 1 minute and then supplemented with 10× RT buffer (1× final concentration), 5 mM of MgCl2, 10 mM of dithiothreitol, ribonuclease inhibitor, and 50 U of SuperScript III RT. The reaction was incubated first at 25°C for 10 minutes, then at 50°C for 50 minutes, and, finally, at 85°C for 10 minutes, followed by incubation with 2 U of RNase H at 37°C for 30 minutes. For reverse transcription polymerase chain reaction (RT-PCR) analysis, 1 μL of the synthesized cDNA was used as the template in 25 μL reactions, each containing 0.2 mM dNTPS, 0.4 μM primers, 2 mM of MgCl2 and 1.25 U Taq DNA polymerase (Invitrogen) in 1× reaction buffer [20 mM Tris-HCl (pH 8.4), 50 mM KCl). The primers are indicated in Table 2. The reactions were incubated at 95°C for 3 minutes, followed by 37 amplification cycles, each including incubation at 95°C for 45 seconds, 60°C for 30 seconds, 72°C for 30 seconds, with an additional extension time at 72°C for 5 minutes. The RT-PCR products were then resolved in 1.5% agarose gels. Table 2. Primers Used in RT-PCR Gene Name  Accession Number  Forward  Reverse  DNPEP  NM001045952  5′-AGGTTGGTGTGGAGACCTAT-3′  5′-CTGGATGGATGTGGCAAGAA-3′  ENPEP  NM001038027  5′-CTATTGGACCCGAGAGCTAATG-3′  5′-CGTGTGGTTGACGGAAAGA-3′  ACE-2  NM001024502  5′-TGGGAGATGAAGCGAGAGATA-3′  5′-CTAAGGTCCAGGGTTCTGATTT-3′  PRCP  NM001038164  5′-GGGACATAGCTGAGGAAATGAA-3′  5′-CTCCAACCACCAAATGAGGATA-3′  ANPEP  NM001075144  5′-CCTACCTCACTCCCAACAATAAC-3′  5′-GTATGTCTTGCCTGCTTCCA-3′  RXFP3  XM005221625  5′-CTACCTGATGAAGAGCAAGCA-3′  5′-GCTCATGGAGGTGAGAAAGAA-3′  GAPDH  NM001034034  5′-ACGTGTCTGTTGTGGATCTG-3′  5′-CGTACCAGGAAATGAGCTTGA-3′  Gene Name  Accession Number  Forward  Reverse  DNPEP  NM001045952  5′-AGGTTGGTGTGGAGACCTAT-3′  5′-CTGGATGGATGTGGCAAGAA-3′  ENPEP  NM001038027  5′-CTATTGGACCCGAGAGCTAATG-3′  5′-CGTGTGGTTGACGGAAAGA-3′  ACE-2  NM001024502  5′-TGGGAGATGAAGCGAGAGATA-3′  5′-CTAAGGTCCAGGGTTCTGATTT-3′  PRCP  NM001038164  5′-GGGACATAGCTGAGGAAATGAA-3′  5′-CTCCAACCACCAAATGAGGATA-3′  ANPEP  NM001075144  5′-CCTACCTCACTCCCAACAATAAC-3′  5′-GTATGTCTTGCCTGCTTCCA-3′  RXFP3  XM005221625  5′-CTACCTGATGAAGAGCAAGCA-3′  5′-GCTCATGGAGGTGAGAAAGAA-3′  GAPDH  NM001034034  5′-ACGTGTCTGTTGTGGATCTG-3′  5′-CGTACCAGGAAATGAGCTTGA-3′  Abbreviations: ANPEP, aminopeptidase N; DNPEP, aspartyl aminopeptidase; ENPEP, glutamyl aminopeptidase; PRCP, prolylcarboxypeptidase. View Large Assay for aminopeptidase activity cleaving an N-terminal amino acid residue ZG cells were lysed in 50 mM Tris-HCl buffer (pH 7.4). Assays were performed in 96 well plates. ZG cell lysates (20 μL containing 100 µg protein) or buffer alone were incubated with 160 µL of a 1.25 mM solution of either the aspartyl-aminopeptidase A substrate (l-aspartyl-β-naphthylamide), glutamyl-aminopeptidase A substrate (l-glutamyl-β-naphthylamide) or leucyl-aminopeptidase substrate (l-leucyl-β-naphthylamide) in Tris-HCl buffer for various times. The reaction was stopped by adding 20 µL of 3M HCl. Hydrolysis of the amino acid results in the formation of the fluorescent β-naphthylamide, which was measured using a fluorescence plate reader (activation at 355 nm and emission at 460 nm). The results are expressed as relative fluorescence units. Statistical analysis Data are presented as the mean ± standard error of the mean. Statistically significant differences between the mean values were evaluated using analysis of variance, followed by the Student-Newman-Keuls multiple comparison test. A value of P < 0.05 was considered to indicate statistical significance. Results Ang II–stimulated, ZG cell–mediated vascular relaxation is not mediated by AT1 or AT2 receptors To determine which Ang receptor mediates ZG cell–dependent vascular relaxation in response to Ang II, vascular reactivity was performed on L-NA–treated adrenal arteries in the presence of Ang antagonists: losartan, an AT1 receptor antagonist; PD123,319, an AT2 receptor antagonist; D-Ala7-Ang (1-7), a Mas receptor antagonist; and Ile7-Ang III, an Ang III antagonist (31–34). L-NA was used to block any direct actions of Ang II on the adrenal arteries to allow the study of ZG cell–mediated relaxations only. Losartan, PD123,319, and D-Ala7-Ang (1-7) had no effect on ZG cell–mediated vascular relaxation of the adrenal arteries in response to Ang II [Fig. 1(a)–1(c)]. In contrast, the Ang III antagonist Ile7-Ang III significantly attenuated the ZG cell–mediated vascular relaxations to Ang II [Fig. 1(d)]. Reported studies have indicated that Ile7-Ang III inhibits Ang III binding and blocks the stimulation of aldosterone release by Ang III (32, 34, 38). Thus, these data suggest a potential role for Ang III in mediating the relaxations. Figure 1. View largeDownload slide ZG cell–mediated relaxations of adrenal arteries in response to Ang II in the presence of the (a) AT1 receptor antagonist losartan (10 μM), (b) AT2 receptor antagonist PD123,319 (10 μM), (c) Ang (1-7) antagonist d-Ala7-Ang (1-7) (10 μM), and (d) Ang III antagonist Ile7-Ang III (10 μM). ZG cells (5 to 10 × 105) were added to baths with intact arteries. Arteries were pretreated with the NO synthase inhibitor L-NA and cyclooxygenase inhibitor indomethacin to block direct relaxing effects of Ang II on adrenal arteries, allowing for measurement of ZG cell–mediated relaxations only. Each value represents the mean ± standard error of the mean (n = 6 to 10). *Statistically significantly different from the respective control, P < 0.05. Figure 1. View largeDownload slide ZG cell–mediated relaxations of adrenal arteries in response to Ang II in the presence of the (a) AT1 receptor antagonist losartan (10 μM), (b) AT2 receptor antagonist PD123,319 (10 μM), (c) Ang (1-7) antagonist d-Ala7-Ang (1-7) (10 μM), and (d) Ang III antagonist Ile7-Ang III (10 μM). ZG cells (5 to 10 × 105) were added to baths with intact arteries. Arteries were pretreated with the NO synthase inhibitor L-NA and cyclooxygenase inhibitor indomethacin to block direct relaxing effects of Ang II on adrenal arteries, allowing for measurement of ZG cell–mediated relaxations only. Each value represents the mean ± standard error of the mean (n = 6 to 10). *Statistically significantly different from the respective control, P < 0.05. Ang III is the primary Ang II metabolite of ZG cells The metabolism of Ang II was examined with a single bovine adrenal artery ring or with 1 × 106 ZG cells, or with the combination of the two, in conditions replicating the vascular reactivity experiments. No substantial Ang II metabolism was observed in the presence of an adrenal arterial ring (Fig. 2). In contrast, substantial Ang II metabolism was observed in the presence of ZG cells, with Ang III the primary metabolite (Fig. 2). Ang IV and Ang (1-7) were also produced by ZG cells, although to a lesser extent. The same Ang II metabolism profile was observed with the combination of an arterial ring and ZG cells. Figure 2. View largeDownload slide Ang II metabolism of adrenal arteries and ZG cells. A single bovine adrenal artery (BAA) ring (2 mm) or ZG cells (1 × 106 cells), or a combination of the two, were incubated with Ang II (100 nM) for 10 minutes. Supernatant was extracted and analyzed for Ang metabolites by LC-MS. Each value represents the mean ± standard error of the mean (n = 4). *Statistically significantly different from control, P < 0.05. Figure 2. View largeDownload slide Ang II metabolism of adrenal arteries and ZG cells. A single bovine adrenal artery (BAA) ring (2 mm) or ZG cells (1 × 106 cells), or a combination of the two, were incubated with Ang II (100 nM) for 10 minutes. Supernatant was extracted and analyzed for Ang metabolites by LC-MS. Each value represents the mean ± standard error of the mean (n = 4). *Statistically significantly different from control, P < 0.05. Ang III mediates ZG cell–dependent vascular relaxation of bovine adrenal arteries in response to Ang II Because (1) Ang III antagonism attenuated ZG cell–mediated vascular relaxation of adrenal arteries in response to Ang II and (2) Ang III is the primary Ang II metabolite of ZG cells, the ZG cell–dependent vascular response to Ang III was examined. Ang III stimulated a ZG cell–dependent relaxation response. The responses to Ang III were substantially greater than the responses to Ang II [10−10 M, Ang II, 35.8% ± 2.3%; Ang III, 48.7% ± 3.3%; Fig. 3(a)]. Figure 3. View largeDownload slide ZG cell–mediated relaxations of adrenal arteries in response to (a) Ang III and (b) Ang IV. ZG cells (5 to 10 × 105) were added to baths with intact arteries. Arteries were pretreated with the NO synthase inhibitor L-NA and cyclooxygenase inhibitor indomethacin to block the direct relaxing effects of Ang peptides on adrenal arteries, allowing for measurement of ZG cell–mediated relaxations only. Each value represents the mean ± standard error of the mean (n = 7 to 9). *Statistically significantly different from the respective Ang II concentration, P < 0.05. Figure 3. View largeDownload slide ZG cell–mediated relaxations of adrenal arteries in response to (a) Ang III and (b) Ang IV. ZG cells (5 to 10 × 105) were added to baths with intact arteries. Arteries were pretreated with the NO synthase inhibitor L-NA and cyclooxygenase inhibitor indomethacin to block the direct relaxing effects of Ang peptides on adrenal arteries, allowing for measurement of ZG cell–mediated relaxations only. Each value represents the mean ± standard error of the mean (n = 7 to 9). *Statistically significantly different from the respective Ang II concentration, P < 0.05. Because Ang IV is an aminopeptidase metabolite of Ang II and Ang III and a ZG cell metabolite of Ang II, the ZG cell–dependent vascular response to Ang IV was examined. In contrast to Ang III, the relaxation response to Ang IV was significantly less than that of Ang II [10−10 M, Ang II, 33.9% ± 2.0%; Ang IV, 15.3% ± 3.6%; Fig. 3(b)]. To examine the effect of metabolism of Ang II to Ang III on ZG cell–mediated vascular relaxation of adrenal arteries, amastatin (10 μM) and EC33 (10 μM), aminopeptidase inhibitors, were tested (26, 35). Ang II stimulated ZG cell–dependent relaxation responses that were significantly attenuated by amastatin [10−10 M, control, 35.8% ± 2.1%; amastatin, 15.3% ± 3.4%; Fig. 4(a)]. In contrast, amastatin had no effect on Ang III stimulated ZG cell–dependent relaxation responses in adrenal arteries [10−10 M, control, 50.0% ± 3.1%; amastatin, 41.9% ± 3.5%; Fig. 4(b)]. Additionally, Ang II stimulated ZG cell–dependent relaxation responses were significantly attenuated by EC33 [10−10 M, control, 30.4% ± 2.9%; EC33, 8.8% ±4.8%; Fig. 4(c)]. Figure 4. View largeDownload slide ZG cell–mediated relaxations of adrenal arteries in response to (a) Ang II in the presence of the aminopeptidase inhibitor amastatin (10 μM), (b) Ang III in the presence of the aminopeptidase inhibitor amastatin, and (c) Ang II in the presence of the aminopeptidase A inhibitor EC33 (10 μM). ZG cells (5 to 10 × 105) were added to baths with intact arteries. Arteries were pretreated with the NO synthase inhibitor L-NA and cyclooxygenase inhibitor indomethacin to block the direct relaxing effects of Ang II and Ang III on adrenal arteries, allowing for measurement of ZG cell–mediated relaxations only. Each value represents the mean ± standard error of the mean (n = 8 to 10). *Statistically significantly different from the respective control, P < 0.05. Figure 4. View largeDownload slide ZG cell–mediated relaxations of adrenal arteries in response to (a) Ang II in the presence of the aminopeptidase inhibitor amastatin (10 μM), (b) Ang III in the presence of the aminopeptidase inhibitor amastatin, and (c) Ang II in the presence of the aminopeptidase A inhibitor EC33 (10 μM). ZG cells (5 to 10 × 105) were added to baths with intact arteries. Arteries were pretreated with the NO synthase inhibitor L-NA and cyclooxygenase inhibitor indomethacin to block the direct relaxing effects of Ang II and Ang III on adrenal arteries, allowing for measurement of ZG cell–mediated relaxations only. Each value represents the mean ± standard error of the mean (n = 8 to 10). *Statistically significantly different from the respective control, P < 0.05. Metabolism of Ang II to Ang III does not affect aldosterone secretion Because the metabolism of Ang II to Ang III is necessary for ZG cell–dependent vascular relaxation of adrenal arteries, the effect of aminopeptidase metabolism of Ang II on aldosterone secretion was examined in bovine ZG cells. Ang II and Ang III were equipotent in stimulating aldosterone secretion [10−7 M, Ang II, 702 ± 122 pg/mL; Ang III, 744 ± 131 pg/mL; Fig. 5(a)]. In the presence of amastatin, the ability of Ang II and Ang III to stimulate aldosterone secretion remained unchanged [10−7 M, Ang II, 695 ± 123 pg/mL; Ang III, 762 ± 130 pg/mL; Fig. 5(b)]. Figure 5. View largeDownload slide Aldosterone secretion from cultured bovine ZG cells in response to Ang II and Ang III in the (a) absence or (b) presence of the aminopeptidase inhibitor amastatin (10 μM). Each value represents the mean ± standard error of the mean (n = 4). Figure 5. View largeDownload slide Aldosterone secretion from cultured bovine ZG cells in response to Ang II and Ang III in the (a) absence or (b) presence of the aminopeptidase inhibitor amastatin (10 μM). Each value represents the mean ± standard error of the mean (n = 4). Peptidases expressed in bovine ZG, endothelial, and smooth muscle cells RNA sequencing was performed on bovine ZG cells, BAAECs, and BAASMCs. Numerous aminopeptidases and peptidases that might metabolize Ang peptides were present in the transcriptome of these cells (Fig. 6). Table 1 lists the gene name, common name, and Enzyme Commission number for the peptidases. Both aspartyl aminopeptidase A (DNPEP) and glutamyl aminopeptidase A (ENPEP) metabolize Ang II to Ang III (26, 35, 39). DNPEP was highly expressed in all three cell types, especially in ZG cells (in relative expression, BAAEC, 6.13; BAASMC, 5.69; ZG, 23.64), and ENPEP was either not expressed or minimally expressed in the cells [Fig. 6(a)]. Angiotensin-converting enzyme 2 (ACE2) and prolylcarboxypeptidase (PRCP) metabolize Ang II to Ang (1-7) and Ang III to Ang (2-7) (3). ACE2 was not expressed in all three cell types, and PRCP was expressed in all three, albeit to a lesser extent than DNPEP. Aminopeptidase N (ANPEP), which metabolizes Ang III to Ang IV, was not expressed in the three types of cells. Of the leucyl-aminopeptidases, ERAP-1 and ERAP-2 metabolize Ang II to Ang III and Ang IV (40, 41); however, LAP-3 has not been tested for its ability to metabolize Ang II. Figure 6. View largeDownload slide RNA expression of peptidases in bovine adrenal arteries and ZG cells. RNA sequencing data for peptidases in (a) bovine ZG cells, (b) BAAECs, and (c) BAASMCs, expressed in reads per kilobase of transcript per 1 million mapped reads (RPKM). (d) Agarose gel electrophoresis of RT-PCR products of peptidases (upper) in bovine adrenal arteries and (lower) in bovine ZG cells. (1) DNPEP, 246 bp; (2) ENPEP, 262 bp; (3) ACE2, 274 bp; (4) PRCP, 276 bp; (5) ANPEP, 257 bp; (6) RNPEP, 272 bp; (7) NPEPPS, 255 bp; (8) LAP-3, 239 bp; and (9) bovine GAPDH, 228 bp. The gene names, common names, and Enzyme Commission numbers for the peptidases are listed in Table 1. Figure 6. View largeDownload slide RNA expression of peptidases in bovine adrenal arteries and ZG cells. RNA sequencing data for peptidases in (a) bovine ZG cells, (b) BAAECs, and (c) BAASMCs, expressed in reads per kilobase of transcript per 1 million mapped reads (RPKM). (d) Agarose gel electrophoresis of RT-PCR products of peptidases (upper) in bovine adrenal arteries and (lower) in bovine ZG cells. (1) DNPEP, 246 bp; (2) ENPEP, 262 bp; (3) ACE2, 274 bp; (4) PRCP, 276 bp; (5) ANPEP, 257 bp; (6) RNPEP, 272 bp; (7) NPEPPS, 255 bp; (8) LAP-3, 239 bp; and (9) bovine GAPDH, 228 bp. The gene names, common names, and Enzyme Commission numbers for the peptidases are listed in Table 1. To confirm the RNA sequencing results, RT-PCR analysis was performed using specific primers (Table 2). RNAs from bovine adrenal arteries and bovine ZG cells were examined. In bovine adrenal arteries, DNPEP and PRCP were highly expressed and ENPEP, LAP-3, and ANPEP were expressed to a lesser extent, and no ACE2 was detected [Fig. 6(d)]. In bovine ZG cells, DNPEP was highly expressed [Fig. 6(d)]. PRCP, LAP-3, and ANPEP were expressed to a lesser extent, but no ENPEP and ACE2 were detected in ZG cells. Aminopeptidase activity in bovine ZG cells Aspartyl-, glutamyl-, and leucyl-aminopeptidase enzymatic activities were examined in ZG cell lysates to confirm the RNA expression results. When ZG cell lysate was incubated with the aspartyl-aminopeptidase–specific substrate l-aspartyl-β-naphthylamide, hydrolysis of the aspartyl group and release of fluorescent β-naphthylamide occurred (Fig. 7). This aminopeptidase activity significantly increased with time. Similar lysate activity was obtained with the leucyl-aminopeptidase substrate l-leucyl-β-naphthylamide. In contrast, when ZG cell lysate was incubated with the glutamyl-aminopeptidase–specific substrate l-glutamyl-β-naphthylamide, no change was found in the formation of β-naphthylamide. When the amino acid β-naphthylamide substrates were incubated without lysates or lysates were incubated without substrates, no changes in fluorescence were observed over time. Figure 7. View largeDownload slide Aminopeptidase activity in bovine ZG cell lysates. Each value represents the mean ± standard error of the mean (n = 9). Asp-β-NA, l-aspartyl-β-naphthylamide (substrate for aspartyl-aminopeptidase A); Glu-β-NA, l-glutamyl-β-naphthylamide (substrate for glutamyl-aminopeptidase A); Leu-β-NA, l-leucyl-β-napthylamide (substrate for leucyl-aminopeptidase). Figure 7. View largeDownload slide Aminopeptidase activity in bovine ZG cell lysates. Each value represents the mean ± standard error of the mean (n = 9). Asp-β-NA, l-aspartyl-β-naphthylamide (substrate for aspartyl-aminopeptidase A); Glu-β-NA, l-glutamyl-β-naphthylamide (substrate for glutamyl-aminopeptidase A); Leu-β-NA, l-leucyl-β-napthylamide (substrate for leucyl-aminopeptidase). Discussion In previous studies, we found that Ang II relaxes adrenal arteries by acting directly on the adrenal vasculature and by a mechanism that involves ZG cells (Fig. 8). In the absence of ZG cell, Ang II causes endothelium-dependent relaxations of isolated adrenal arteries by activating AT2 receptors and promoting NO release (22, 24). In arteries without endothelium or treated with L-NA, Ang II does not cause relaxations. However, the presence of ZG cells restores the relaxations to Ang II in these arteries through the release of EETs and DHETs (23). The present study has demonstrated that ZG cell–dependent relaxations to Ang II are not mediated by traditional angiotensin AT1 or AT2 receptors or the Ang (1-7) Mas receptor. Only the Ang III antagonist Ile7-Ang III significantly attenuated these relaxations. The identity of the AT receptor that mediates these relaxations is not known (Fig. 8). A specific receptor for Ang III has yet to be identified, although evidence exists for a high affinity Ang III receptor. Specific binding of [3H]Ang II and [3H]Ang III in particulate fractions of rat adrenal glands demonstrated a high affinity binding site for [3H]Ang III with a Kd of 1.5 × 10−10 M and a binding capacity of 1.2 × 10−13 mol/mg of protein (34, 38, 42). Ang II did not suppress binding of [3H]Ang III to this high affinity site. [3H]Ang III binding was inhibited by the Ang III antagonist Ile7-Ang III, suggesting a specific receptor for Ang III. Our observation that Ile7-Ang III inhibits the ZG cell–mediated relaxations to Ang II provides further evidence that an unknown Ang III receptor might exist on ZG cells of the adrenal gland (Fig. 8). In contrast, in the absence of ZG cells, Ang III, like Ang II, directly relaxes adrenal arteries by activation of endothelial AT2 receptors and release of NO (24). Thus, the vasorelaxations to Ang III in the adrenal vasculature are complex. They involve distinct actions on ZG cells and the vascular endothelium and use different Ang receptors (Fig. 8). Figure 8. View largeDownload slide Summary of the regulation of adrenal vascular tone by Ang II and Ang III. Ang II–mediated vasorelaxation has both direct and indirect vascular action. (Left) In the direct action, relaxation of adrenal arteries by Ang II is mediated by activation of AT2 receptors on endothelial cells resulting in endothelial NO release, smooth muscle cell cyclic guanosine monophosphate (cGMP) formation, and relaxation. This direct action is inhibited by the AT2 antagonist PD123,319 and the NO synthase (NOS) inhibitor LNA. (Right) Ang II also has an indirect action via ZG cells. Ang II is metabolized to Ang III by an amastatin- and EC33-sensitive aminopeptidase (AP) on ZG cells. Ang III acts on an unknown AT receptor (AT?) to stimulate the synthesis and release of EETs and DHETs. The EETs act on an EET receptor (EET R), activating a calcium-activated, iberiotoxin (IBTX)-sensitive potassium (K) channel, allowing K efflux, membrane hyperpolarization, and relaxation. 14,15EEZE, 14,15-epoxyeicosa-5Z-enoic acid (EET antagonist); Arg, arginine; GTP, guanosine triphosphate; PL, phospholipid. Figure 8. View largeDownload slide Summary of the regulation of adrenal vascular tone by Ang II and Ang III. Ang II–mediated vasorelaxation has both direct and indirect vascular action. (Left) In the direct action, relaxation of adrenal arteries by Ang II is mediated by activation of AT2 receptors on endothelial cells resulting in endothelial NO release, smooth muscle cell cyclic guanosine monophosphate (cGMP) formation, and relaxation. This direct action is inhibited by the AT2 antagonist PD123,319 and the NO synthase (NOS) inhibitor LNA. (Right) Ang II also has an indirect action via ZG cells. Ang II is metabolized to Ang III by an amastatin- and EC33-sensitive aminopeptidase (AP) on ZG cells. Ang III acts on an unknown AT receptor (AT?) to stimulate the synthesis and release of EETs and DHETs. The EETs act on an EET receptor (EET R), activating a calcium-activated, iberiotoxin (IBTX)-sensitive potassium (K) channel, allowing K efflux, membrane hyperpolarization, and relaxation. 14,15EEZE, 14,15-epoxyeicosa-5Z-enoic acid (EET antagonist); Arg, arginine; GTP, guanosine triphosphate; PL, phospholipid. Additionally, Ang III is both more potent and efficacious than Ang II in stimulating ZG cell–mediated relaxation of adrenal arteries. These data support a role for Ang III in these relaxations. Although Ang II is considered the primary biologically active peptide of the RAAS, evidence has shown that Ang III is an active metabolite in the adrenal gland and central nervous system. In the central nervous system, Ang III plays a role in tonic blood pressure maintenance and hypertension (43). Inhibiting Ang II conversion to Ang III with aminopeptidase A inhibition lowers vasopressin release and blood pressure (26, 43). In the adrenal gland, Ang III is more potent than Ang II in stimulating aldosterone synthesis ex vivo in ZG cells of four species and equally potent in vivo (44–46). Moreover, evidence exists for intracellular Ang III in adrenal ZG cells and direct effects on isolated mitochondria (46). Most importantly, our data demonstrate that the endogenous production of Ang III mediates ZG cell–mediated relaxation of adrenal arteries in response to Ang II. The primary metabolite of Ang II in ZG cells is Ang III. The metabolism of Ang II by a single arterial ring was not substantial. However, in studies using isolated BAAECs or BASSMCs, Ang II was metabolized to Ang (1-7) and lesser amounts of Ang III and Ang IV (24). Thus, the vasculature is capable of Ang II metabolism to Ang III and other Ang peptides; however, it was not substantial compared with ZG cell–mediated metabolism under these experimental conditions. Ang II–stimulated, ZG cell–mediated relaxations of adrenal arteries was significantly attenuated by inhibition of aminopeptidases, the enzymes that metabolize Ang II to Ang III and Ang IV. Ang IV only minimally stimulates ZG cell–mediated relaxations of adrenal arteries, providing further evidence that Ang III, not a downstream aminopeptidase metabolite of Ang III, is the Ang peptide responsible for ZG cell–mediated relaxation (Fig. 8). The importance of Ang II metabolism to Ang III differs in the ZG cells according to whether aldosterone release or EET release are measured. The metabolism of Ang II to Ang III is essential for ZG cell–mediated relaxations of adrenal arteries; however, it is not necessary for, and has no effect on, stimulation of Ang II–induced aldosterone secretion. This is because Ang II and Ang III are equipotent in the stimulation of aldosterone secretion from bovine ZG cells. Thus, inhibition of aminopeptidase metabolism had no effect on Ang II–stimulated aldosterone production. This observation is in agreement with previous studies in primary rat ZG cells, in which Ang II and Ang III were equipotent in the stimulation of aldosterone secretion (45). In a human adrenocortical carcinoma cell line, Ang III was less potent than Ang II in stimulating aldosterone secretion (47). Thus, aminopeptidase inhibition blocks ZG cell–mediated relaxations to Ang II without affecting aldosterone release. The endogenous production of Ang III in ZG cells is further validated by the expression and activity of peptidases. RNA expression confirmed that the primary aminopeptidase present in ZG cells is aspartyl-aminopeptidase A. It is encoded by the DNPEP gene and metabolizes Ang II to Ang III (39). Leucyl-aminopeptidase-3, encoded by the LAP-3 gene, is also highly expressed; however, its ability to metabolize Ang II is not known. Glutamyl-aminopeptidase A, encoded by the ENPEP gene, converts Ang II to Ang III but is not detected in ZG cells (26). The activities of aspartyl-aminopeptidase A and leucyl-aminopeptidase, but not glutamyl-aminopeptidase A, are present in ZG cell lysates. These activity data are consistent with these RNA expression data. Identification of the aminopeptidases responsible for Ang III formation in ZG cells was beyond the scope of the present research. However, studies with aminopeptidase inhibitors and expression have provided some insights. Amastatin and EC33 inhibit the ability of Ang II to cause ZG cell–mediated relaxation of adrenal arteries. Thus, the aminopeptidase must be inhibited by amastatin and EC33. Aspartyl-aminopeptidase A (DNPEP) is not inhibited by either of the inhibitors (39). Thus, although aspartyl-aminopeptidase A is highly expressed, it is not responsible for Ang II conversion to Ang III in ZG cells. This might be because of its cytosolic localization. Similarly, aminopeptidase P1 (XPNPEP1), another cytosolic enzyme, is not inhibited by amastatin but is expressed in ZG cells (48). Glutamyl-aminopeptidase A (ENPEP) is inhibited by both amastatin and EC33 (26); however, it is not a candidate, because it is not expressed in ZG cells. This process of elimination leaves for consideration one of the leucyl-aminopeptidase family or another aminopeptidase not known to metabolize Ang II to Ang III. Three leucyl-aminopeptidases are expressed at different levels in ZG cells: leucyl-aminopeptidase-3 (LAP-3 gene), adipocyte leucyl-aminopeptidase (ERAP-1 gene), and endoplasmic reticulum leucyl-aminopeptidase (ERAP-2 gene). Leucyl-aminopeptidase-3 and adipocyte leucyl-aminopeptidase are inhibited by amastatin and, thus, represent possible candidates (40). The inhibitory activity of EC33 has not been determined for any of the three leucyl-aminopeptidases. Additionally, the sensitivity of endoplasmic reticulum leucyl-aminopeptidase-2 to amastatin inhibition is not known. It is also a possible participant. Adipocyte leucyl-aminopeptidase and endoplasmic reticulum leucyl-aminopeptidase-2 metabolize Ang II to Ang III (40, 41); however, this activity has not been documented for leucyl-aminopeptidase-3. This process of elimination leaves the leucyl-aminopeptidases as the leading candidates for the observed conversion of Ang II to Ang III in ZG cells (40, 41). Additionally, RNA for PRCP, the gene for prolylcarboxypeptidase or angiotensinase C, is expressed in ZG cells (49). However, ACE2 is not expressed. These findings suggest that prolylcarboxypeptidase, and not ACE2, is the enzyme responsible for metabolizing Ang II to Ang (1-7) in ZG cells. In conclusion, the regulation of adrenal blood flow plays an important role in the regulation of steroidogenesis. Ang II, a potent vasoconstrictor, causes vasorelaxation of adrenal cortical arteries by a direct endothelium-mediated mechanism and an indirect paracrine, ZG cell–mediated mechanism (Fig. 8). Ang II metabolism is important in both of these mechanisms. Ang II and its vascular endothelial metabolite Ang III increase adrenal blood flow and stimulate aldosterone production (24). Likewise, in the paracrine regulation of adrenal vascular tone by ZG cells, endogenous metabolism of Ang II to Ang III is an obligatory step in Ang II–stimulated, ZG cell–mediated relaxations of adrenal cortical arteries. Also, our study provides additional evidence for a unique Ang III receptor in the adrenal cortex. Thus, Ang III has important and distinct roles in the adrenal gland to regulate blood flow and steroidogenesis. Abbreviations: ACE2 angiotensin-converting enzyme 2 ACTH adrenocorticotropic hormone Ang angiotensin ANPEP aminopeptidase N AT1 angiotensin type 1 AT2 angiotensin type 2 BAAEC bovine adrenal artery endothelial cell BAASMC bovine adrenal artery smooth muscle cell cDNA complementary DNA DHET dihydroxyeicosatrienoic acid DNPEP aspartyl aminopeptidase EET epoxyeicosatrienoic acid ENPEP glutamyl aminopeptidase A LC liquid chromatography LC-MS liquid chromatography–mass spectrometry L-NA nitro-l-arginine MS mass spectrometry NO nitric oxide PRCP prolylcarboxypeptidase PSS physiological salt solution RAAS renin-angiotensin-aldosterone system RT-PCR reverse transcription polymerase chain reaction TFA trifluoroacetic acid ZG zona glomerulosa. Acknowledgments The authors thank Ms. Jessica Kelliher for technical assistance, Dr. Kasem Nithipatikom for assistance with the mass spectrometric analyses of angiotensin peptides, and Mrs. Gretchen Barg for secretarial assistance. 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