Mass-Spectrometric Identification of a Novel Angiotensin Peptide in Human Plasma
Objective— Angiotensin peptides play a central role in cardiovascular physiology and pathology. Among these peptides, angiotensin II (Ang II) has been investigated most intensively. However, further angiotensin peptides such as Ang 1-7, Ang III, and Ang IV also contribute to vascular regulation, and may elicit additional, different, or even opposite effects to Ang II. Here, we describe a novel Ang II-related, strong vasoconstrictive substance in plasma from healthy humans and end-stage renal failure patients.
Methods and Results— Chromatographic purification and structural analysis by matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight (MALDI-TOF/TOF) revealed an angiotensin octapeptide with the sequence Ala-Arg-Val-Tyr-Ile-His-Pro-Phe, which differs from Ang II in Ala1 instead of Asp1. Des[Asp1]-[Ala1]-Ang II, in the following named Angiotensin A (Ang A), is most likely generated enzymatically. In the presence of mononuclear leukocytes, Ang II is converted to Ang A by decarboxylation of Asp1. Ang A has the same affinity to the AT1 receptor as Ang II, but a higher affinity to the AT2 receptor. In the isolated perfused rat kidney, Ang A revealed a smaller vasoconstrictive effect than Ang II, which was not modified in the presence of the AT2 receptor antagonist PD 123319, suggesting a lower intrinsic activity at the AT1 receptor. Ang II and Ang A concentrations in plasma of healthy subjects and end-stage renal failure patients were determined by matrix-assisted laser desorption/ionisation mass-analysis, because conventional enzyme immunoassay for Ang II quantification did not distinguish between Ang II and Ang A. In healthy subjects, Ang A concentrations were less than 20% of the Ang II concentrations, but the ratio Ang A / Ang II was higher in end-stage renal failure patients.
Conclusion— Ang A is a novel human strong vasoconstrictive angiotensin-derived peptide, most likely generated by enzymatic transformation through mononuclear leukocyte-derived aspartate decarboxylase. Plasma Ang A concentration is increased in end-stage renal failure. Because of its stronger agonism at the AT2 receptor, Ang A may modulate the harmful effects of Ang II.
The octapeptide, angiotensin II (Ang II) is generally accepted to play a central role in the physiology and pathology of vascular regulation. The secular therapeutic success of ACE inhibitors and AT1 blockers as cardiovascular protective drugs further underscored the role of Ang II in the pathophysiology of atherosclerosis and hypertension. Further peptides of the angiotensin family, including angiotensins III and IV and angiotensin 1-7 are less well characterized.1,2 These peptides have additional or antagonizing effects when compared with Ang II.3
Angiotensin 1-7 has been shown to be a vasodilator.4 Similarly, angiotensin IV is an agonist to the AT4 receptor mediating vasodilation.5 These various angiotensin peptides may gain growing importance as on ACE inhibition increased amounts of other angiotensin peptides are formed via ACE-independent pathways.6
To identify novel vasoconstrictive peptides, we screened chromatographic fractions obtained from human plasma for vasoconstrictive effects using the isolated perfused rat kidney as a bioassay.
Controls, Patients, and Isolation of Plasma
Details of the characteristics of patients and controls are given in the Table and in the supplemental Methods (available online at http://atvb.ahajournals.org). We obtained peripheral blood (10 mL) by catheterization of the cubital vein and collected the blood in tubes containing K2-EDTA (7.2 mg). The blood samples were centrifuged at 300g for 30 minutes at 4°C for isolation of plasma. The resulting plasma was fractionated by chromatography (see supplemental Methods).
For analytical purposes, samples were submitted to size-exclusion and reversed phase chromatography and mass-spectrometry (see supplemental Methods).
Measurements of Perfusion in the Isolated Perfused Rat Kidney
The effects of aliquots of lyophilized fractions of the analytic reversed-phase chromatography on vascular tone were evaluated in an isolated rat kidney perfused at a constant flow rate while perfusion pressure was continuously monitored. Details of the preparation and the perfusion procedure are described elsewhere7,8 and in the supplemental Methods. The underlying substance of the fraction with a vasoconstrictive effect was identified by matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight (MALDI-TOF/TOF) mass-spectrometry (see supplemental Methods).
Preparation and Incubation of Mononuclear Leukocytes With Ang II and Angiotensinogen
Mononuclear leukocytes were obtained from healthy subjects according to established techniques9 (see supplemental Methods). To test whether Ang A is synthesized out of Ang II in the presence of mononuclear leukocytes 10−6 mmol/L AT-II and angiotensinogen, respectively, were added to the supernatant. After 0, 0.2, 0.5, 1, and 3 hours, aliquots of the supernatant were analyzed by MALDI mass spectrometry. The incubation with Ang II was repeated with thermically denatured mononuclear leukocytes. For this, mononuclear leukocytes were heated at 100°C for 10 minutes in a reaction vial.
Cell Culture, Transfection Method, and Membrane Preparation for Receptor Binding Studies
Details of the cell culture are given in the supplemental Methods. HEK 293 cells expressing either the AT1- or AT2-receptor protein were grown on 100-mm Petri dishes, washed twice with 5 mL of PBS (137 mmol/L, NaCl, 2.7 mmol/L KCl, 1.5 mmol/L KH2PO4, 8.0 mmol/L Na2HPO4, pH 7.4), harvested with a rubber policeman, and centrifuged at 400g for 10 minutes. For receptor binding studies, HEK 293 membranes were prepared, as described the supplemental Methods.
125I-Sar1,Ile8-Ang II Displacement Binding Analysis
Membranes (5 μg) were incubated in a final volume of 200 μL of Tris/BAME buffer containing 1 μmol/L 125I-Sar1,Ile8-Ang II alone or increasing concentrations of unlabeled ligand (1×10−12 to 1×10−6 mol/L) for 2 hours at 25°C at 300 rpm in a shaking water bath (supplemental Methods). The values were used for calculations of the Ki values of unlabeled ligands (see supplemental Methods).
Ca2+-Fluorescent Image Plate Reader Assay
Calcium was measured in VSMCs as previously published.10 The method is described in the supplemental Methods.
Quantification of Ang and Ang II
Ang A and Ang II was quantified by a MALDI-MS based method as recently described.11 The supernatants of mononuclear leukocytes after incubation with Ang II were directly quantified (see supplemental Methods).
Quantification of Ang II in Presence and Absence of Ang A by Enzyme Immunoassay
The “angiotensin II enzyme immunoassay kit” of SpiBio (Société de Pharmacologie et d’Immunologie, Massy, France) was used to test the cross-reactivity between Ang A and Ang II with a conventional enzymatic immunoassay used for Ang II-determinations (see supplemental Methods).
Impact of Ang A on Blood Pressure in Angiotensin II Receptor AT1A Knockout Mice
In these experiments, wild-type mice were compared with angiotensin II receptor AT1A KO mice, which had been bred as previously described by Ito et al12 (see supplemental Methods).
Human plasma was first fractionated by size-exclusion chromatography. A typical size-exclusion chromatogram is shown in Figure 1A. Each fraction obtained from the size-exclusion step was tested for vasoactivity in the isolated perfused rat kidney. One of the fractions showing a strong vasoconstrictive effect is labeled by an arrow in Figure 1A. This fraction was chromatographed by reversed-phase chromatography (Figure 1B). The reversed-phase chromatography allowed on the one hand to further fractionate the eluate, and on the other hand to desalt the eluate of the size-exclusion chromatography.
The fractions obtained from each reversed phase chromatography elution step were tested for vasoconstrictive properties in the isolated perfused rat kidney. The vasoconstrictive fraction detected using the isolated perfused kidney is again indicated by an arrow in Figure 1B. The insert of Figure 1B shows the vasoconstrictive effect of the labeled fraction.
Mass-spectrometric analysis of the underlying substance by MALDI-TOF revealed a mass peak at m/z 1002.54 (Figure 1C) corresponding to the peptide Ala-Arg-Val-Tyr-Ile-His-Pro-Phe, as confirmed by tandem MS (MALDI-TOF/TOF). In particular, the presence of alanine in the N-terminal position was obtained from the N-terminal b ions as shown in Figure 1D. The difference in the identified amino acid sequence compared with that of angiotensin II is given in Figure 1E in bold.
No DNA sequence code for des[Asp1]-[Ala1]-Ang II, which in the following will be named Angiotensin A (where A stands for alanine) or Ang A, was found in data-bases covering the human genome. Therefore, we addressed the question, whether Ang A may arise from Ang II by decarboxylation of Asp. Although a number of tissues may be likely candidates for this transformation, we first tested human mononuclear leukocytes. During the incubation of lipopolysaccharide (LPS)-activated mononuclear leukocytes in the presence of Ang II a significant increase in Ang A levels in the supernatant was detected. The amount of Ang A and Ang II in the supernatant is quantified in Figure 1F (relative to internal standard corticotropin [ACTH]). The synthesis and release of Ang II by mononuclear leukocytes was recently described.11 LPS-stimulation enhanced the Ang II conversion, but mononuclear leukocytes also converted Ang II without LPS-stimulation (rel. mass-signal intensity [AU] 0.03±0.01 versus 0.007±0.009; n=3). No increase in Ang A levels were detected with intact mononuclear leukocytes in the absence of Ang II nor when a denatured mononuclear leukocyte preparation was used (data not shown). During incubation of LPS-activated mononuclear leukocytes in the presence of renin substrate or angiotensinogen, significant increases in Ang A, Ang II, and Ang I levels in the supernatant, but no mass-signal of a modified des[Asp1]-[Ala1]-Ang I were detected (data not shown). Therefore, a modified angiotensinogen is to be excluded as a source of Ang A.
These experiments suggest that Ang A is not derived from a precursor protein like angiotensinogen nor modified angiotensinogen, but is generated from Ang II by enzymatic decarboxylation involving mononuclear leukocytes. A nonenzymatic decarboxylation was excluded by the finding that after heat-denaturing Ang A synthesis is abolished.
After isolation of Ang A from human plasma, we analyzed the vasoconstrictive action of this novel peptide in the isolated perfused rat kidney model. In this model, Ang A caused a dose-dependent vasoconstriction, which was 90% of the maximal effect induced by Ang II (Figure 2A). The effect induced by Ang A was abolished in the presence of the angiotensin-receptor antagonist AT1 EXP 3174. The EC50 value (mol/L) of Ang A induced vasoconstriction ([4.43±1.95]×10−7) was an order of magnitude lower than that of Ang II-mediated vasoconstriction ([5.20±2.52]×10−8). The AT2 receptor antagonist PD123319 had no significant effect on the vasoconstriction induced by Ang A.
Thus, Ang A revealed a lower vasoconstrictive potency and efficacy than Ang II, suggesting that it acts only as a partial agonist. Accordingly, Ang A was less active than Ang II in an in vivo assay (Figure 2B). Increasing concentrations of the peptides were injected into the femoral vein of wild-type and Ang II AT1A receptor–deficient mice, and the blood pressure changes were monitored by a femoral artery catheter. Both peptides induced a strong hypertensive response, but Ang A only at ≈10 times higher concentrations than Ang II. Both peptides signal via the AT1A receptor, because the blood pressure response was blunted in knockout animals (Figure 2B). The kinetics of response to Ang A and Ang II application were comparable in this set-up (Figure 2C).
To elucidate whether the lower potency of the vasoconstriction induced by Ang A could be attributed to a lower affinity of Ang A to the AT1 receptor than that of Ang II, and/or to a higher affinity to the AT2 receptor, binding of Ang A and Ang II to AT1 and AT2 receptors expressed in human embryonic kidney (HEK) 293 cells was studied. We characterized the receptor binding of both peptides by displacement of the radioligand [125I]-Sar,1 Ile8 angiotensin II by increasing concentrations of unlabeled Ang II and Ang A (Figure 3A and 3B). IC50-values of the rat AT1-receptor (Figure 3A) and the rat AT2-receptor (Figure 3B) were [-log IC50] 9.54±0.04 and 9.92±0.04 for Ang A, and [-log IC50] 9.49±0.06 and 9.59±0.04 for Ang II. Whereas no significant difference in the affinity of AT1 receptors for Ang A and Ang II was observed (Figure 3A), the affinity of AT2 receptors for Ang A was significantly higher than for Ang II (Figure 3B). VSMCs stimulated with Ang II or Ang A showed an dose-dependent increase in cytosolic calcium with a significant difference within their EC50 values ([-log mol/L] Ang II: 7.7±0.1 and Ang A: 7.0±0.1, P<0.05, n=5 independent experiments; Figure 3C). In the presence of EXP3174 (1 μmol/L) cytosolic calcium increase by half-maximal agonistic concentration of Ang II (10 nmol/L) or Ang A (100 nmol/L) was completely inhibited (Figure 3D). These data suggest that the lower potency of Ang A to induce vasoconstriction is caused neither by a lower affinity to AT1 receptors nor by higher activity at the AT2 receptor, but due to a lower intrinsic activity at the AT1 receptor.
Next we quantified the Ang A/Ang II ratio in human plasma by MALDI mass spectrometry. As shown in Figure 3E, the Ang A/Ang II ratio was higher in end-stage renal failure patients than in healthy subjects. Measurements in seven healthy subjects revealed an Ang A concentration of 6.7±4.7 pg/mL, which is 9.7±3.3% (calculated for each patient; min.: 2.1%; max.: 25.2%) of the Ang II concentration found in the same subjects (88.0±12.3 pg/mL). The Ang A concentration was increased in plasma of end-stage renal failure patients (n=7) to 28.4±11.0 pg/mL; this corresponds to 30.9±9.1% (calculated for each patient; min.: 5.1%; max.: 73.6%; P<0.05) of the Ang II concentration found in the same patients (127.5±29.4 pg/mL).
A conventional enzyme immunoassay for Ang II quantification did not distinguish between Ang II and Ang A (Figure 3F).
Ang A is a novel angiotensin peptide, which is present in human plasma; it is derived from Ang II by enzymatic decarboxylation, emphasizing the physiological importance of Ang A. A yet unknown decarboxylase present in human mononuclear cells is one potential cellular source of Ang A. At present, the nature of this decarboxylase, its substrate specificity, and its tissue distribution are unknown. It remains to be shown whether other cells or tissues may also contribute to Ang A formation.
The Ang A/Ang II ratio in plasma of healthy subjects was below 0.2 in all, but this ratio increased to up to 0.7 in patients with stage 5 chronic kidney disease. The increased Ang A/Ang II ratio in patients with stage 5 chronic kidney disease may either indicate an increased activity of decarboxylase in human mononuclear cells from patients with chronic kidney disease stage 5, a reduced enzymatic degradation of Ang A in these patients, or renal excretion. An increase of the Ang A half-life is not unlikely, because modifications of half life of low molecular proteins or peptides in chronic renal failure patients are described frequently (eg,13,14). Presently it is unknown whether the increased Ang A/Ang II ratio in plasma may be related to well-known uremic complications in patients with chronic kidney disease stage 5. Conventional enzyme immunoassays do not distinguish between Ang II and Ang A; these assays quantify the sum of Ang II and Ang A.
Interestingly, the affinity of Ang A to the AT1 receptor is nearly equal to that of Ang II. However, the potency to induce vasoconstriction is lower than that of Ang II. Thus, Ang A is a less potent, and only partial agonist at the AT1 receptor. This potential partial agonism will be the more effective in subjects with an increased Ang A/Ang II ratio. It is notable that Ang III, which lacks the amino-terminal aspartate residue, has a similar affinity to the AT1 receptor as Ang A. In contrast to Ang A, for Ang III the same biological activity as for Ang II has been demonstrated.15 In addition, Ang II and Ang III cause a similar vascular effect when injected intracerebroventricularly or directly into certain brain regions.16,17 In the case of Ang II the increase in blood pressure can be inhibited by a selective inhibitor of aminopeptidase A (APA), which mediates the generation of Ang III by the cleavage of the N-terminal aspartate residue of Ang II.18 These data suggest that the vasoconstrictive effect of Ang II is mediated by Ang III. Whether Ang III provokes an increase in blood pressure and release of plasma arginine-vasopressin (arginine vasopressin) through the activation of AT1 and/or AT2 receptors or a yet unidentified effector molecule is still not clear.19 In the case of Ang A, the processing to Ang III could be impaired, because the APA has a preference for N-terminal acidic residues such as aspartate or glutamate. Therefore, it is intriguing to speculate that decarboxylation of aspartate in Ang II may prevent or delay Ang III formation.
Interestingly, the affinity of Ang A to the AT2 receptor is higher than that of Ang II. Whether the intrinsic activity of Ang A at the AT2 receptor also translates into an increase in intrinsic activity requires a suitable model to study AT2-mediated signaling events. For example, the mechanisms by which the AT2 receptor mediates vasodilation and which signaling pathways are involved remain obscure. Several authors favor an indirect mechanism, such as stimulation of the B2 bradykinin receptor, possibly via an intracellular acidification and resulting in an increased NO and cGMP production.20,21 Furthermore, there are reports suggesting that phosphotyrosine activity is increased after AT2 receptor stimulation.22–24 Further elucidation of AT2 receptor signaling potentially will help to study the downstream effects of Ang A binding to AT2 receptor. Our data demonstrate that Ang-A represents a new metabolite of Ang II system, which is the first one acting as a partial agonist. Further studies will be required to analyze whether this new peptide reveals altered metabolism to Ang III and Ang IV peptides, thereby adding to the complexity of the regulation of the cardiovascular system.
We thank Brigitte Egbers for technical assistance.
Original received August 7, 2006; final version accepted November 16, 2006.
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