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J Am Soc Nephrol 11:1377-1386, 2000
© 2000 American Society of Nephrology

Metabolism Alters the Selectivity of Angiotensin-(1-7) Receptor Ligands for Angiotensin Receptors

RAJASH K. HANDA

Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Washington State University, Pullman, Washington.

Correspondence to Dr. Rajash K. Handa, Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Washington State University, Pullman WA 99164-6520. Phone: 509-335-6624; Fax: 509-335-4650; E-mail: Handa{at}vetmed.wsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abstract. The present study examined whether metabolism of the putative angiotensin-(1-7) receptor agonist and antagonist [angiotensin-(1-7) and D-alanine7 angiotensin-(1-7), respectively] altered their ability to interact with angiotensin AT1, AT2, and AT4 receptor subtypes. Both angiotensin-(1-7) and D-alanine7 angiotensin-(1-7) competed with low affinity for 125I-sarcosine1, isoleucine8 angiotensin II binding to AT1 and AT2 receptors in rat liver and adrenal medulla membranes, respectively, and competed with low affinity for 125I-angiotensin IV binding to AT4 receptors in bovine kidney epithelial cell membranes. In vitro renal metabolism of the angiotensin-(1-7) receptor ligands (incubating peptides with rat cortical tissue homogenates) had minimal influence on low-affinity binding to AT1 and AT2 receptors, yet caused a significant and dramatic shift toward high-affinity binding for AT4 receptors. Low-affinity angiotensin II binding to the AT4 receptor was also shifted toward high-affinity binding following renal metabolism of the peptide. Conversely, angiotensins with high affinity for the AT4 receptor (e.g., angiotensin IV) were shifted toward low-affinity binding states following peptide metabolism. Incubation of 125I-angiotensin-(1-7) with rat cortical tissue generated the high-affinity AT4 receptor ligand 125I-angiotensin-(3-7), whereas the renal metabolism of 125I-angiotensin II generated both 125I-angiotensin-(3-7) and 125I-angiotensin IV. These results reveal that renal metabolism of angiotensin-(1-7) receptor ligands and angiotensin II yields products that have high affinity for the AT4 receptor and could potentially contribute to the biologic actions of the parent peptide in the kidney.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The renin-angiotensin system (RAS) plays a pivotal role in cardiovascular homeostasis, hydroelectrolyte balance, and cell function. Attesting to its importance in regulating the internal environment is the fact that overactivity of RAS [resulting in elevated angiotensin II (AngII) levels] can lead to arterial hypertension, congestive heart failure, and renal insufficiency (1). The widely held view that AngII is the only biologically active product of RAS has been challenged with findings suggesting that the diverse biologic actions of RAS in the body are likely due to the formation of AngII and several shorter AngII fragments acting on AngII type AT1, AT2, or atypical (non-AT1, non-AT2) angiotensin receptor subtypes (2,3,4,5). The carboxy-terminal and amino-terminal deleted fragments of AngII [Ang-(1-7) and Ang-(3-8), respectively] have been extensively examined and demonstrate numerous biologic activities that can be similar, or more often opposite, to those of AngII (2,3,4,5). This has led to the concept that peptides smaller than AngII may not only have unique biologic functions, but they may also serve to counter-regulate the actions of AngII.

Ang-(3-8) (commonly acknowledged as AngIV) has high affinity and specificity for an atypical angiotensin receptor known as the AT4 receptor, which is widely distributed throughout the body (5), including the kidney (5,6). Investigators have also alluded to the possibility that many of the novel biologic actions of Ang-(1-7) in the brain and peripheral circulation may be mediated by an Ang-(1-7) receptor. This conclusion is largely based on the observation that Ang-(1-7) responses were minimally affected by AT1 and AT2 receptor blockade, and yet markedly inhibited by sarcosine derivatives of AngII (nonselective angiotensin receptor antagonists) and D-alanine7 Ang-(1-7) [putative Ang-(1-7) receptor antagonist] (3,4). Additional support for the existence of a unique Ang-(1-7) receptor has come from recent reports demonstrating that specific, high-affinity 125I-Ang-(1-7) binding sites are present in bovine aortic endothelial cells and canine coronary artery endothelium, and that both Ang-(1-7) and D-alanine7 Ang-(1-7) competed for the 125I-Ang-(1-7) binding site (3,7). There has also been speculation of an antihypertensive renal Ang-(1-7) receptor system based on reports that Ang-(1-7) is generated within the kidney (8,9) with increased levels after angiotensinconverting enzyme inhibition therapy (9), and that some of the renal actions of Ang-(1-7) on fluid and electrolyte excretion can be attenuated by D-alanine7 Ang-(1-7) (10,11,12). D-Alanine7 Ang-(1-7) has low affinity for AT1 and AT2 receptors in radioligand binding assays (13), and it selectively attenuated the biologic actions of Ang-(1-7) compared with AngII (13,14). These findings have led to the growing use of D-alanine7 Ang-(1-7) as a specific Ang-(1-7) receptor antagonist. Consequently, alterations in renal excretory function after administration of D-alanine7 Ang-(1-7) to normal, water-loaded, or spontaneously hypertensive rats have been interpreted as the result of a change in endogenous renal Ang-(1-7) receptor activity (10,11,12,15). A critical assumption of these and other studies is that metabolism of Ang-(1-7) receptor ligands does not alter their specificity for angiotensin receptors. We attempted to test the validity of this assumption by examining whether renal metabolism of Ang-(1-7) and D-alanine7 Ang-(1-7) altered the angiotensins' specificity and affinity for AT1, AT2, and AT4 receptors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
AT4 Receptor Binding Study
Cell Culture. Madin-Darby bovine kidney (MDBK) cells are an epithelial cell line that have biochemical characteristics of distal tubule/collecting duct cells (16,17). MDBK cells were maintained at 37°C in an atmosphere of 95% air/5% CO2, in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (5%), calf bovine serum (5%), and the antibiotics penicillin (50 IU/ml), streptomyocin (50 µg/ml), and amphotericin B (5 µg/ml). Cultures were re-fed with fresh media every 2 d. These cells were chosen because they are a renal epithelial cell line that contains abundant AT4 receptors with no detectable AT1 and AT2 receptors (18) or specific 125I-Ang-(1-7) binding sites (19). Frozen MDBK cells purchased from American Type Culture Collection (Manassas, VA) were of unknown passage. However, all of the experiments presented in this study were performed at passages 2 to 10 (after thawing frozen cells) on confluent cells that had been cultured for 5 to 7 d.

Cell Membrane Preparation. Confluent MDBK cells grown in 75-cm2 flasks were washed once with ice-cold phospate-buffered saline followed by the addition to the flask of 2 ml of ice-cold isotonic buffer [containing 150 mmol/L NaCl, 50 mmol/L Tris, 50 µmol/L Plummer's inhibitor (carboxypeptidase inhibitor), 20 µmol/L bestatin (aminopeptidase inhibitor), 5 mmol/L ethylenediaminetetra-acetic acid, 1.5 mmol/L 1,10 phenanthroline (divalent ion chelators), and 0.1% heat-treated bovine serum albumin, at pH 7.4]. The cells were dislodged by scraping with a rubber policeman, collected in a centrifuge tube, and homogenized for approximately 10 s in 10 ml of isotonic buffer. The homogenate was centrifuged at 40,000 x g for 30 min at 4°C. The supernatant was discarded, the pellet was rehomogenized in 10 ml of isotonic buffer, and the high-speed centrifugation was repeated. The final pellet was resuspended in isotonic buffer to a working concentration of 1 mg protein/ml.

AT1 and AT2 Receptor Binding Study
Cell Membrane Preparation. Liver and adrenal medulla tissue was obtained from decapitated adult male Sprague Dawley rats. The liver and adrenal medulla were chosen because they contain predominantly AT1 and AT2 receptors, respectively (20). The tissues were homogenized in 10 ml of hypotonic buffer (50 mM Tris, 1 mM ethylenediaminetetra-acetic acid, pH 7.4, at 4°C) for approximately 10 s. The homogenates were then centrifuged at 500 x g for 10 min at 4°C, the supernatant was saved on ice, and the pellet was resuspended in 10 ml of hypotonic buffer, rehomogenized, and recentrifuged. The supernatants were combined and centrifuged at 40,000 x g for 30 min at 4°C. The resulting pellet was then resuspended in 10 ml of isotonic buffer and homogenized, and the high-speed centrifugation was repeated. The final pellet was resuspended in isotonic buffer to a working concentration of 1 mg protein/ml.

Radioreceptor Assays
MDBK, liver, and adrenal medulla cell membranes (25, 50, and 50 µg of protein, respectively) were incubated in a total volume of 250 µl of isotonic buffer. MDBK cell membrane incubations were performed at 37°C for 60 min with 0.6 nmol/L 125I-AngIV or for 90 min with 0.6 nmol/L 125I-divalinal-AngIV [both AngIV and divalinal-AngIV are selective AT4 receptor ligands (18)], and competition displacement curves were determined in the presence of unlabeled angiotensin peptides (0.1 nmol/L to 10 µmol/L). Binding to rat liver AT1 receptors or rat adrenal medulla AT2 receptors was examined by incubating rat tissue membranes with 0.6 nmol/L 125I-sarcosine1, isoleucine8 AngII (AT1/AT2 receptor ligand) for 2 h at 22°C in the presence of either 1 µmol/L PD 123319 (AT2 receptor antagonist) or 1 µmol/L losartan (AT1 receptor antagonist), respectively. Bound and free radioligands were separated by vacuum filtration in a cell harvester containing No. 32 glass fiber filters and radioligand-bound filters washed with 4 x 2 ml of phosphate-buffered saline (150 mmol/L NaCl, 8.8 mmol/L Na2HPO4, 2 mmol/L NaH2PO4, pH 7.2) at room temperature. Radioactivity retained by the protein-bound filters was measured by gamma counting.

Angiotensin Metabolism Studies
Male Sprague Dawley rats were anesthetized with an intraperitoneal injection of pentobarbital sodium, and the cortex of the kidney was carefully dissected free from medullary tissue, placed in ice-cold oxygenated Krebs—Henseleit buffer solution [containing (in mmol/L): 118 NaCl, 4 KCl, 1 KH2PO4, 27.2 NaHCO3, 1.25 CaCl2, 1.2 MgCl2, 3 glutamine, 1 sodium pyruvate, 1 L-lactic acid, 5 D-glucose, and 10 Hepes], and homogenized for approximately 10 s. A Lowry protein assay was then performed on the rat cortex homogenate, and aliquots of the cortical tissue (100 µg of protein) were incubated at 37°C with either 1 mmol/L Ang peptides or approximately 1.2 nmol/L 125I-Ang peptides in microcentrifuge tubes (in a total assay volume of 250 µl) for 0, 0.5, 1, 2, 5, 10, 20, 30, 40, and 80 min. The metabolism of Ang peptides was terminated by plunging the microcentrifuge tubes into ice-cold water and then immediately centrifuging the microcentrifuge tubes at 14,000 rpm for 5 min at 4°C. The supernatant was then stored at —90°C. Nonmetabolized angiotensins were either dissolved in distilled water or subjected to the same procedure as metabolized peptides, except that rat cortical tissue was not added during the incubation period. (The results of both control procedures were combined as they produced identical responses in competition curves.) Angiotensin samples were kept frozen for 2 to 5 d and then assayed for their binding to angiotensin receptors. The metabolism of 125I-Ang peptides was stopped by the addition of TCA (final concentration in reaction tube was 20%). Zero-time samples were generated by adding TCA to the tissue before the addition of 125I-Ang peptides. The microcentrifuge tubes were kept on ice, then centrifuged at 14,000 rpm for 5 min, and the supernatant was stored in tubes at —90°C. The next day, the tubes containing the supernatant were thawed, and we characterized the 125I-products by HPLC, using a reversed-phase C18 column linked to a radioactivity detector. The 125I-peptides were separated isocratically over 35 min with acetonitrile [generally 10.5% for 125I-Ang-(1-7) and 125I-D-alanine7 Ang-(1-7); 15% for 125I-Ang-(3-5); and 18% for 125I-AngII, 125I-AngIV, and 125I-divalinal-AngIV] and 83 mmol/L H3PO4 buffered at room temperature to pH 3.0 with triethylamine, at the measured flow rate of 1.75 ml/min. Angiotensin metabolites were identified by comparing their retention times with known 125I-angiotensin standards and by spiking samples with 125I-angiotensin markers.

Iodination of Angiotensin Peptides
All angiotensin peptides were monoiodinated using chloramine T, Na 125I, and sodium bisulfite, and separated from unlabeled and diiodinated peptide by HPLC with a reversed-phase C18 column. Radiolabeled peptide was eluted from the column with 83 mmol/L H3PO4 buffered to pH 3.0 with triethylamine and a linear acetonitrile gradient of 9 to 26% developed over 90 min. 125I-angiotensin peptides had a specific activity of approximately 2100 Ci/mmol.

Drugs
Losartan (AT1 receptor antagonist) was obtained from DuPont/Merck Pharmaceuticals, PD 123319 (AT2 receptor antagonist) was from Parke-Davis, and divalinal-AngIV [V{Psi}(CH2-NH2)YV{Psi}(CH2-NH2)HPF, putative AT4 receptor antagonist] was a kind gift from Dr. J. W. Harding (Washington State University). Ang-(3-5) was synthesized by the Washington State University peptide synthesis core facility. Ang-(1-7) and D-alanine7 Ang-(1-7) were purchased from Sigma and BioChem, respectively. The Madin-Darby bovine kidney cell line (MDBK) was obtained from American Type Culture Collection (no. CCL22).

Statistical Analyses
All values quoted represent means ± SEM. Peptide metabolism and competition curves were analyzed by Inplot4 (GraphPad Software, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Renal Metabolism of Angiotensins
The metabolism profile and decay rates (t1/2 values) of several 125I-Ang peptides incubated with rat cortical tissue are shown in Figure 1. The rank order of metabolism decay rates was AngIV (AT4 receptor agonist) = Ang-(3-5) (AT4 receptor ligand) > AngII (AT1/AT2 receptor agonist) = D-alanine7 Ang-(1-7) [putative Ang-(1-7) receptor antagonist] > Ang-(1-7) [Ang-(1-7) receptor agonist] > sarcosine1, threonine8 AngII (sarthran, AT1/AT2 receptor antagonist) >> divalinal-AngIV (putative AT4 receptor antagonist). Based on these results, experiments examining whether metabolism of angiotensin peptides altered their affinity for angiotensin receptors initially used: (1) Ang-(1-7), D-alanine7 Ang-(1-7) and AngII that had been partially metabolized for 1 min in rat cortical tissue to achieve approximately 50% (range, 45 to 60%) metabolism of the peptide; (2) AngIV and Ang-(3-5) that had been incubated with rat cortical tissue for 0.5 min (the shortest reliable collectable time period) that resulted in >90% metabolism of the peptide; and (3) sarthran and divalinal-AngIV that had been incubated with rat cortical tissue for 1 and 10 min, respectively, which resulted in only 15 and 10% metabolism of the respective peptides.



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  		Figure 1. Metabolism of 125I-angiotensin peptides (approximately 1.2 nmol/L) incubated with 100 µg of rat cortical tissue at 37°C. Half-life decay rates (t1/2) were derived by fitting the metabolism curve to a single component exponential decay equation. The t1/2 value for divalinal-AngIV was calculated by linear regression analysis of the amount of peptide remaining at 0 to 80 min of tissue incubation. Data are means ± SEM from two to six experiments. For some data points, the standard error bar is less than the symbol size of the mean value.

 

Affinity for AT1 and AT2 Receptors
Preliminary studies demonstrated that neither distilled water nor oxygenated Krebs—Henseleit buffer (vehicle for rat cortical tissue) influenced 125I-ligand binding to angiotensin receptors (AT1, AT2, or AT4). As shown in Figure 2, A and B, D-alanine7 Ang-(1-7) had less affinity than Ang-(1-7) for both AT1 and AT2 receptors, with Ang-(1-7) having affinity for both receptor subtypes only at high micromolar concentrations. Metabolism of D-alanine7 Ang-(1-7) did not significantly alter its affinity for both AT1 and AT2 receptors. Similarly, metabolism of Ang-(1-7) did not significantly alter its affinity for the AT1 receptor. In contrast, only nonmetabolized Ang-(1-7) at a concentration of 10 µmol/L could compete for 30% of the AT2 receptor sites, suggesting that metabolism decreased the heptapeptide's ability to bind to the AT2 receptor. The presence of AT1 and AT2 receptors was confirmed by losartan (AT1 receptor antagonist) and PD 123319 (AT2 receptor antagonist) having relatively high affinity for their respective angiotensin receptor subtypes.



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  		Figure 2. Inhibition of 125I-sarcosine1, isoleucine8 AngII binding to rat liver AT1 receptors (A) and rat adrenal medulla AT2 receptors (B) by increasing concentrations of losartan (AT1 receptor antagonist), PD 123319 (AT2 receptor antagonist), and Ang-(1-7) receptor ligands (before and after renal metabolism). Data are means ± SEM from three experiments in duplicate. For some data points, the standard error bar is less than the symbol size of the mean value.

 

Affinity for AT4 Receptors
In a second series of experiments, we examined whether Ang-(1-7) receptor ligands (in the absence or presence of metabolism) had affinity for the AT4 receptor, and then extended these findings to other angiotensin peptides (Figures 3 and 4). To easily assess the magnitude of shift in the concentration—binding response curve following peptide metabolism, we show IC50 values for angiotensin peptide binding to the AT4 receptor in Table 1. The results indicate that Ang-(1-7) and D-alanine7 Ang-(1-7) have low affinity for the AT4 receptor. However, renal metabolism of the peptides produced significant and dramatic shifts of the concentration—response curve to the left by at least 10-fold (Figure 3, A and B). This indicated that renal metabolism of the peptides generates products that have higher affinity for the AT4 receptor than the native peptides. Likewise, AngII had low affinity for the AT4 receptor, and yet metabolism of the peptide substantially increased its affinity for the AT4 receptor (Figure 3C). Sarthran had low affinity for the AT4 receptor and remained 85% intact after a 1-min incubation with rat cortical tissue (Figure 1). There was no change in sarthran's affinity for the AT4 receptor after metabolism (Figure 3D). In converse experiments, we found that the concentration—response curve for high-affinity AT4 receptor ligands [AngIV and Ang-(3-5)] was shifted progressively toward low-affinity states with increasing exposure to metabolism (Figure 4, A and B). Divalinal-AngIV has high affinity for the AT4 receptor and was highly resistant to metabolism (Figure 1). We found no shift in the concentration—response curve for divalinal-AngIV after a 10-min incubation of the peptide with rat cortical tissue (Figure 4C).



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  		Figure 3. Inhibition of 125I-AT4 receptor ligand (125I-AngIV or 125I-divalinal-AngIV) binding to Madin-Darby bovine kidney (MDBK) AT4 receptors by increasing concentrations of Ang-(1-7) (A), D-alanine7 Ang-(1-7) (B), AngII (C), or sarthran (D). Angiotensins were subjected to either no metabolism or a 1-min period of metabolism in rat cortical tissue. Data are means ± SEM. For some data points, the standard error bar is less than the symbol size of the mean value. n = number of experiments performed in duplicate.

 


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  		Figure 4. Inhibition of 125I-AT4 receptor ligand (125I-AngIV or 125I-divalinal-AngIV) binding to MDBK AT4 receptors by increasing concentrations of AngIV (A), Ang-(3-5) (B), or divalinal-AngIV (C). Angiotensins were subjected to either no metabolism or a 0.5- to 10-min period of metabolism in rat cortical tissue. Data are means ± SEM. For some data points, the standard error bar is less than the symbol size of the mean value. n = number of experiments performed in duplicate.

 

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  		Table 1. Effect of peptide metabolism on the binding constants of angiotensin-related compounds for the AT4 receptora
 

Angiotensin Peptide Metabolites
Figure 5 depicts the generation of metabolites from 125I-angiotensins during the incubation period with rat cortical tissue. Renal metabolism of 125I-Ang-(1-7) yielded 125I-Ang-(2-7) and 125I-Ang-(3-7), which reached a peak at 0.5 and 1 min, respectively. Other metabolites generated from 125I-Ang-(1-7) proteolysis included 125I-tyrosine, 125-Ang-(1-6), and an undefined 125I-tyrosine-containing product (Figure 5A). The unidentified Ang-(1-7) metabolite was eluted from the HPLC column before the Ang-(1-6) peak, and therefore we can conclude (based on retention times) that the metabolite was not Ang-(4-5), Ang-(3-5) or Ang-(1-5). Of the remaining possible permutations of 125I-Ang-(1-7) degradation products, only the formation of Ang-(3-6) could contribute to the increased affinity of metabolized Ang-(1-7) for the AT4 receptor. This is due to the fact that Ang-(3-6) alone has both high affinity for the renal AT4 receptor (18) and an elution time that is less than Ang-(1-6). Both 125I-AngIV and 125I-Ang-(3-7) were generated from 125I-AngII metabolism, with peak concentrations achieved at 0.5 and 2 min, respectively (Figure 5B). Other metabolites included 125I-tyrosine and three unidentified products (A, B, and C), with the concentration of metabolite A > metabolite B > metabolite C. At this time, we can only exclude metabolite A as being 125I-Ang-(1-7), 125I-Ang-(1-5), or 125I-Ang-(3-5). As shown in Figure 5C, a major N-terminal deleted metabolite of 125I-AngIV metabolism was 125I-Ang-(3-7), which reached a maximum at 1 min. Other metabolites included 125I-tyrosine and two unidentified metabolites (A and B). We can conclude (based on retention times) that metabolite B is not 125I-Ang-(2-7), 125I-Ang-(3-6), 125I-Ang-(3-5), 125I-Ang-(3-4), or 125I-Ang-(4-5). Because metabolite B must contain a radiolabeled tyrosine moiety, we can deduce that it is a C-terminal deleted metabolite of 125I-AngIV that is either 125I-Ang-(4-8), 125I-Ang-(4-7), or 125I-Ang-(4-6). In the present study, we determined that both AngIV and Ang-(3-7) had high affinity for the MDBK AT4 receptor, with Ki values of 12.1 ± 0.1 and 10.8 ± 0.3 nmol/L, respectively (n = 3 each). Angiotensins require a free N-terminal valine and a minimal molecular sequence of valine-tyrosine-isoleucine to have high affinity for the AT4 receptor (5,18). On the basis of these ligand-structure AT4 receptor binding studies, identified metabolites generated during the incubation of angiotensins with rat cortical tissue bind to the AT4 receptor with a relative affinity order of AngIV >= Ang-(3-7) > Ang-(2-7) >> AngII > Ang-(1-7) >= Ang-(1-6) >> tyrosine.



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  		Figure 5. Metabolites generated by the renal metabolism of 125I-Ang-(1-7) (Panel A, n = 4), 125I-AngII (Panel B, n = 2), and 125I-AngIV (Panel C, n = 3). Data are means ± SEM. For some data points, the standard error bar is less than the symbol size of the mean value.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A growing number of studies have used both Ang-(1-7) and D-alanine7 Ang-(1-7) as an Ang-(1-7) receptor agonist and antagonist, respectively, to elucidate the role of the Ang-(1-7) receptor system in biologic systems. The results of the present study indicate that Ang-(1-7) and D-alanine7 Ang-(1-7) have low affinity for known AT1, AT2, and AT4 receptors and are in agreement with the notion that these ligands may be specific for the proposed Ang-(1-7) receptor. However, renal metabolized Ang-(1-7) receptor ligands demonstrated relatively high affinity and selectivity for the AT4 receptor. Consequently, metabolites of Ang-(1-7) receptor ligands could potentially act as agonists and/or antagonists at the AT4 receptor, and thus contribute to the overall biologic activity of the parent peptide. In support of this contention, picomolar concentrations of Ang-(1-7) and its major NH2-terminal deleted metabolite, Ang-(3-7), can decrease energy-dependent solute transport in rat proximal tubules by interacting with the AT4 receptor (21). Also, subnanomolar concentrations of Ang-(1-7), Ang-(3-7), D-alanine7 Ang-(1-7), and AngIV can inhibit phosphorylation of the extracellular signal-regulated kinase-2 (Erk-2) protein by 25 to 60% in MDBK cells (19); a bovine renal epithelial cell line containing abundant AT4 receptors without any detectable expression of AT1 and AT2 receptors (18) and specific 125I-Ang-(1-7) binding sites (19). These results are in agreement with earlier work suggesting that Ang-(3-7) is a biologic active peptide (22). Despite the fact that both Ang-(1-7) and AngIV can elicit vasodilative (23,24,25,26) and/or vasoconstrictive (23,24,27,28) activity, there are no reports suggesting an interaction of Ang-(1-7) with the AT4 receptor system in the regulation of vascular contractile function. Whether this reflects differences in tissue receptor profile, ligand degradation products, and/or AT4 receptor isoforms in vascular versus renal epithelial tissue is unknown. Nevertheless, our results suggest that metabolism of peptides such as Ang-(1-7) and D-alanine7 Ang-(1-7) allows them to potentially interact with the AT4 receptor, at least in kidney epithelial tissue.

Because our knowledge of the renal trafficking and processing of angiotensin peptides such as Ang-(1-7) and AngIV is rudimentary, we used the proteolytic activity of rat cortical kidney homogenates to metabolize peptides to ensure the participation of all extracellular, membrane-bound, and intracellular enzymes that could potentially be involved in kidney angiotensin peptide metabolism. Consequently, renal enzymes from multiple origins (e.g., ectoenzymes and cytosolic, lysosomal, and microsomal enzymes) may have participated in the breakdown of angiotensin peptides. Although the general consensus is that ectoenzymes (those present at the cell surface with their catalytic moiety on the outside of the cell membrane) are responsible for metabolizing angiotensins present in the circulation and presumably renal interstitial fluid, recent findings have revealed that angiotensin proteolytic enzymes (endopeptidases and exopeptidases) can be secreted into the rat tubule fluid (29). Furthermore, angiotensins such as AngII can be transported into kidney cells (30), where they could be exposed to some type of intracellular enzymatic regulation such as that described for internalized AngII in bovine adrenal medullary cells (31). However, the HPLC profile of the 125I-Ang-(1-7) metabolites generated by enzymes present in cortical tissue homogenates suggested that carboxypeptidases and aminopeptidases were largely responsible for Ang-(1-7) metabolism in the rat cortex. We have previously reported similar findings for Ang-(1-7) metabolism in rat proximal tubules (21). In addition, others have shown relatively high levels of Ang-(3-7) and Ang-(2-7) in rat urine (9), which are presumably of renal origin and derived from Ang-(1-7) metabolism.

Ang-(3-7) was generated by the renal metabolism of Ang-(1-7) and is known to be an agonist with high affinity for the renal epithelial AT4 receptor (21). The multiple products of D-alanine7 Ang-(1-7) metabolism were not identified. However, assuming that proteolytic cleavage of D-alanine7 Ang-(1-7) by renal proteases resembles that described for Ang-(1-7) (21), then the increased affinity of the peptide for AT4 receptors would most likely be due to the formation of NH2-terminal deleted fragments [e.g., D-alanine7 Ang-(2-7) and D-alanine7 Ang-(3-7)]. Similar to findings with Ang-(1-7) receptor ligands, we found that AngII had low affinity for the AT4 receptor and that there was a dramatic shift toward high affinity for the AT4 receptor following renal metabolism of the peptide. This was likely due to the observed formation of the highaffinity AT4 receptor ligands AngIV and Ang-(3-7). This raises the intriguing possibility that AngII could influence renal AT4 receptor activity by generating metabolites that are AT4 receptor agonists. We have recently reported that picomolar concentrations of AngII and AngIV inhibit oxygen consumption of nystatin-treated rat proximal tubules, which likely reflects a decrease in Na+ -K+ -ATPase activity (6). The effect of AngIV was mediated by the AT4 receptor, whereas the effect of AngII was mediated by a sarthran-inhibitable, non-AT4 receptor (6), which was presumably due to an action on AT1 and/or AT2 receptors. It is probable that AngIV and Ang-(3-7) were generated from the metabolism of AngII in the rat proximal tubule preparation, but were at too low a concentration (subpicomolar) for us to detect an AT4 receptor-mediated response. Interestingly, Gesualdo and colleagues have shown that nanomolar concentrations of AngII and AngIV can stimulate plasminogen activator inhibitor type 1 (PAI-1) expression in human proximal tubule epithelial (HK-2) cells, and that AngII's effect was mediated exclusively by the formation of AngIV acting on a non-AT1, non-AT2 receptor (32). Others have also demonstrated in nonrenal tissues that the biologic activity of AngII on the pulmonary vasculature (26,33) and on cardiac endothelial PAI-1 expression (34) was largely dependent on the generation of AngIV acting on AT4 receptors. Taken together, these results support the contention that metabolism of angiotensins, such as Ang-(1-7) and AngII, could potentially influence AT4 receptor-mediated functions in the kidney.

To confirm that the affinity of metabolized angiotensin peptides for the AT4 receptor could be shifted in either direction, we were able to demonstrate that high-affinity AT4 ligands moved toward low-affinity states after incubation with rat cortical tissue. It was initially puzzling that a 0.5- or 2-min period of AngIV metabolism did not cause a dramatic loss in AngIV's affinity for the AT4 receptor, despite near complete degradation of the peptide. This was likely due in part to the concomitant formation of Ang-(3-7) from the metabolic breakdown of AngIV. Also, we cannot exclude the possibility that the renal metabolism rates of angiotensins may be slower than their radioactive iodinated counterparts. We also examined the ability of metabolized Ang-(3-5) to bind the AT4 receptor, because Ang-(3-5) is the minimal molecular angiotensin sequence that can demonstrate high affinity for the AT4 receptor (5,18). Consequently, N-terminal or C-terminal deletions of the tripeptide should result in a more rapid loss in its ability to bind the AT4 receptor. Our results demonstrated a near complete loss in the tripeptide's ability to bind the AT4 receptor following a 10-min incubation with rat cortical tissue. This finding provides some assurance that the experimental conditions did not mask a greater loss of metabolized AngIV's ability to bind to the AT4 receptor. An argument could be made that the results obtained are not related to metabolism of angiotensin products, but to other factors present in the incubate. We believe that this is most unlikely because: (1) oxygenated Krebs—Henseleit buffer (vehicle for rat cortical tissue) does not influence 125I-ligand binding to the AT4 receptor; (2) in contrast to Ang-(1-7), D-alanine7 Ang-(1-7), and AngII, sarthran's low affinity for the AT4 receptor was unchanged following a 1-min period of incubation with rat cortical tissue, which was likely due to the fact that the peptide remained 85% intact; and (3) in contrast to AngIV and Ang-(3-5), divalinal-AngIV was remarkably resistant (90% intact) to metabolism following a 10-min incubation with rat cortical tissue and was associated with no change in its high affinity for the AT4 receptor. Thus, the findings of the present study suggest that proteolysis can either degrade or promote the interaction of angiotensins with the AT4 receptor.

Kidney structures that constitutively or inducibly express AT4 receptors include glomerular mesangial cells (35), proximal tubules (6,32,36,37), and more distal segments of the nephron (18,38). Functions associated with activation of the nephron AT4 receptor system encompass an attenuation of AngII-stimulated mesangial cell contractility (39), inhibition of energy-dependent solute transport in the proximal tubule (6), and stimulation of proximal tubule PAI-1 expression (32). There is the expectation that Ang-(1-7) receptors are also present in the rat kidney based on functional studies demonstrating that Ang-(1-7) can alter renal fluid and electrolyte reabsorptive function (10,11,12,27,40) and that urinary Ang-(1-7) levels are severalfold greater than those in plasma (8,9). Conventional x-ray film and emulsion autoradiographic techniques have failed to detect Ang-(1-7) receptors in the rat kidney (21), but this could simply be related to small populations of the receptor being present in the kidney. However, there are also conflicting views regarding the exact nature of the biologic effect of Ang-(1-7) in the kidney, as well as the receptor subtype involved. Several in vitro studies have demonstrated that Ang-(1-7) can have a biphasic action on kidney transport processes with picomolar and nanomolar concentrations of Ang-(1-7), resulting in the stimulation and inhibition, respectively, of proximal tubule fluid and bicarbonate reabsorption (40), and renal membrane Na+ -K+ -ATPase activity (41). However, most investigators have failed to observe an antinatriuretic/antidiuretic response to Ang-(1-7) infused intravascularly into the anesthetized or conscious rat or isolated rat kidney, and instead have consistently observed a natriuresis and diuresis (12,27,42,43). On the other hand, intraperitoneal or subcutaneous administration of Ang-(1-7) to water-loaded conscious rats resulted in a selective antidiuresis (10,11); however, it remains unclear whether this reflects a direct action of circulating Ang-(1-7) at the level of the kidney and/or is due to an action at an extrarenal site(s). Others have also reported that Ang-(1-7) has no effect (44), stimulates (40,41,44), or inhibits (27,40,41) tubular reabsorptive function in early segments of the rat nephron. A number of receptor subtypes have been reported to mediate the renal actions of Ang-(1-7), including losartan-sensitive AT1 or Ang-(1-7) receptors (11,21,40,44), losartan-insensitive Ang-(1-7) receptors (12), and AT4 receptors (19,21). In an effort to determine the tonic effect of endogenous Ang-(1-7) on kidney function, recent studies have infused the putative Ang-(1-7) receptor antagonist D-alanine7 Ang-(1-7) into anesthetized or conscious rats (normotensive, water-loaded, or spontaneously hypertensive) and have observed increases in Na+ and water excretion (10,11,12,15). These results have been interpreted to suggest that either D-alanine7 Ang-(1-7) binds to an Ang-(1-7) receptor to block the tonic stimulatory action of endogenous Ang-(1-7) on renal Na+ and water reabsorption, or that D-alanine7 Ang-(1-7)'s interaction with the receptor site results in increased Na+ and water excretion. The results of the present study add an additional level of complexity to the interpretation of Ang-(1-7) and D-alanine7 Ang-(1-7)'s action in the kidney, because metabolites of Ang-(1-7) receptor ligands can potentially act on renal AT4 receptors to influence kidney function (19,21).

Naturally occurring ligands that specifically interact with the AT4 receptor include AngIV (45) and hemorphins (46,47). The specificity and high affinity of AngIV for the AT4 receptor are encoded by the ligand's first three N-terminal residues (valinetyrosine-isoleucine) (5,18). Substitution of residues in each of these three positions in the AngIV molecule reveals a large number of amino acid sequences that allow the peptide to bind with high affinity to the AT4 receptor (48,49). With few exceptions, these structural data suggest that high-affinity AT4 receptor ligands require a free N-terminal amine, hydrophobic, aromatic, and hydrophobic amino acids in positions 1 through 3, respectively, and an undefined C-terminal extension (48,49). We and others have also shown that metabolites of Ang-(1-7), AngII, and their amino acid-substituted analogs can functionally interact with the AT4 receptor (21,26,32,33,34). Taken together, these findings suggest that proteolysis of angiotensins [such as Ang-(1-7) and AngII receptor ligands], and likely other endogenous nonangiotensin peptides, may expose internal amino acid sequences that allow them to bind with high affinity to the AT4 receptor and alter cellular biologic activity.


   Acknowledgments
 
This work was supported by a grant from the Washington Affiliate of the American Heart Association. The author thanks Dr. Joseph W. Harding for providing divalinal-AngIV and access to the HPLC system and Dr. Steve M. Simasko, who provided access to cell culturing facilities.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication September 28, 1999. Accepted for publication December 1, 1999.




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