Angiotensin-(1-7) can interact with the rat proximal tubule AT4 receptor system

Rajash K. Handa

Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-6520


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was undertaken to identify the non-AT1, non-AT2 angiotensin receptor that mediates the ANG-(1-7) inhibitory action on rat proximal tubule transport processes. ANG-(1-7) inhibited nystatin-stimulated, ouabain-suppressible O2 consumption (QO2) rates in freshly isolated rat proximal tubules (reflecting reduced basolateral Na+-K+-ATPase activity). Selective angiotensin-receptor subtype antagonists revealed that AT1 and AT4 receptors mediated the response of ANG-(1-7). Receptor autoradiography of the rat kidney demonstrated a high density of AT1 and AT4 receptors and no specific 125I-ANG(1-7) binding sites. Competition assays in rat kidney sections indicated that ANG-(1-7) competed predominantly for the AT1 receptor site, whereas its NH2-terminal-deleted metabolite, ANG-(3-7), competed primarily for the AT4-receptor site. Metabolism of 125I-ANG-(1-7) in rat proximal tubules generated peptide fragments that included ANG-(3-7), with the pentapeptide producing a concentration-dependent inhibition of nystatin-stimulated proximal tubule QO2 that was abolished by AT4-receptor blockade. These results suggest that the generation of ANG-(3-7) from the NH2-terminal metabolism of ANG-(1-7) caused the interaction of the parent peptide with the proximal tubule AT4 receptor, which elicited a decrease in energy-dependent solute transport.

angiotensin-(3-7); angiotensin IV; angiotensin receptor subtypes; metabolism; transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ANGIOTENSIN II [ANG II = ANG-(1-8)] is the major biologically active peptide of the renin-angiotensin system (RAS), having widespread effects on the internal environment. It is regarded as a hormone, neuromodulator, and neurotransmitter that plays a pivotal role in the regulation of cardiovascular, renal, and endocrine functions. Pharmacological and molecular biology approaches have characterized at least two subtypes of the ANG II receptor (AT1 and AT2) and further subdivisions within each subtype. Most ANG II actions in the brain and periphery have been ascribed to AT1-receptor activation with a counterregulatory role proposed for the AT2 receptor (25, 29).

Over the last decade it has become increasingly clear that there are a number of other angiotensin peptides that may contribute to the spectrum of activities of RAS, including ANG-(1-7) and ANG-(3-8) (= ANG IV). These peptides have biological actions that may be similar or more often opposite to those of ANG II, e.g., both peptides can oppose the vascular constrictor and growth-promoting effects of ANG II (4, 13). Although both ANG-(1-7) and ANG IV are capable of interacting with ANG II type AT1- and AT2-receptor subtypes to elicit a biological response, there is mounting evidence that each may also interact with its own unique receptor (4, 13). Radioligand binding studies have clearly demonstrated that ANG IV binds with high affinity and specificity to a novel non-AT1, non-AT2 membrane protein (termed the AT4 receptor) in a variety of tissues to influence cardiovascular function, growth, and memory (4, 27). The conclusion that ANG-(1-7) may also have its own unique receptor has largely been based on the observation that functional responses to ANG-(1-7) were minimally affected by AT1- or AT2-receptor blockade and yet markedly inhibited by either sarthran, a competitive nonselective angiotensin-receptor antagonist (5, 6, 15, 20), or by [D-Ala7]ANG(1-7), a putative ANG-(1-7)-receptor antagonist (23, 24). Activities associated with this novel ANG-(1-7) receptor have included systemic hypotension (5, 20, 23), coronary vessel dilatation (6), inhibition of vascular smooth muscle growth (15), and promotion of the concept that ANG-(1-7) acts as an antihypertensive peptide.

The kidney is a critical target organ for RAS in the long-term regulation of blood pressure, extracellular volume, and electrolyte composition. Recent studies have suggested that the kidney is not only an important source for ANG-(1-7) production (14) but also a target for the heptapeptide's biological activity (4, 13). In the rat kidney, ANG-(1-7) was reported to be natriuretic and to interact with an angiotensin non-AT1, non-AT2 receptor on proximal tubules to inhibit an ouabain-suppressible component of transepithelial Na+ transport (17). More recently, the angiotensin AT4 receptor has been localized to rat convoluted and straight proximal tubules (19). Activation of this receptor by ANG IV was also shown to inhibit a ouabain-suppressible component of transepithelial Na+ transport that was blocked by the specific AT4-receptor antagonist, divalinal-ANG IV (19). Consequently, the main focus of the present study was to determine whether the non-AT1, non-AT2 receptor activated by ANG-(1-7) to alter rat proximal tubule transepithelial Na+ transport was similar to or different from that of the AT4 receptor.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In Vitro Tissue O2 Studies

The receptor(s) involved in angiotensin peptide action on transcellular Na+ transport was pharmacologically characterized by measuring nystatin-stimulated tissue O2 consumption (QO2) rates in fresh suspensions of rat proximal tubules in the absence and presence of angiotensin-receptor antagonists. Nystatin-stimulated QO2 measurements can be used as a direct reflection of Na+-K+-ATPase activity and of Na+ transport because of the tight coupling between mitochondrial oxidative phosphorylation and Na+-K+-ATPase activity in the proximal tubule (21). In addition, ouabain-suppressible nystatin-stimulated QO2 provides an on-line, dynamic index of active Na+ transport (21).

Isolation of rat proximal tubules. A suspension of cortical proximal tubules was obtained by a method that has been previously described (17). After a complete blood washout, the kidneys of pentobarbital sodium anesthetized male Wistar rats were briefly perfused with warm Krebs-Henseleit buffer (KHB) 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, supplemented with 1 mg/ml collagenase, 0.67 mg/ml hyaluronidase, and 0.67 mg/ml bovine serum albumin in situ. The kidneys were then excised and the cortex removed, minced, incubated for 20 min in enzyme-supplemented KHB at 37°C, and aerated with 95% O2-5% CO2. The tubule suspension was washed with ice-cold KHB followed by ice-cold Ca2+-free KHB (reduces clumping during the isolation step for proximal tubules). The tubule suspension was suspended in 45% isosmotic Percoll solution, and a band of proximal tubules was isolated by centrifugation at 4°C for 10 min at 19,430 g. The resultant fraction of proximal tubules was 95% pure and was washed to remove the Percoll and stored in ice-cold KHB until tissue QO2 was measured.

Determination of QO2. Suspensions of proximal tubules were incubated in KHB, in the absence or presence of angiotensin-receptor antagonists, for 10 min at 37°C in a shaker bath and aerated with 95% O2-5% CO2. A 0.1-ml aliquot of proximal tubules was placed in a thermoregulated chamber containing KHB, in the absence or presence of angiotensin-receptor antagonists at 37°C and sealed, and QO2 was measured polarographically with a Clarke O2 electrode. Other drugs such as 5 mM nystatin and angiotensin-peptide agonists were added in 25-µl boluses into the tubule-containing chamber via the injection port. The O2 tension in the closed chamber was linear and recorded as a function of time (1.5-2 min for each measurement), and the resulting slope indicated the QO2, which was calculated as a function of tubular protein content.

Radioligand Binding Studies

Cell culture. The Madin-Darby bovine kidney (MDBK) epithelial cell line was maintained in an atmosphere of 95% air-5% CO2 at 37°C in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (5%), calf bovine serum (5%), and the antibiotics penicillin (50 IU/ml), streptomycin (50 mcg/ml), and amphotericin B (5 µg/ml). Cultures were refed with fresh medium every 2 days. All the experiments presented in this study were performed at passage 10-16 on confluent cells that had been cultured for 5-7 days.

Cell membrane preparation. Confluent MDBK cells grown in 75-cm2 flasks were washed once with ice-cold PBS followed by the addition to the flask of 2 ml 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 EDTA, and 1.5 mmol/l 1,10-phenanthroline (divalent ion chelators), 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 ~10 s in 10 ml of isotonic buffer. The homogenate was centrifuged at 40,000 g for 30 min at 4°C. The supernatant was discarded, the pellet rehomogenized in 10 ml 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.

Radioreceptor assays. MDBK cell membranes (25 µg of protein) were incubated in a total volume of 250 µl isotonic buffer. Incubations were performed at 37°C for 60 min with 0.4 nmol/l 125I-ANG IV, and competition displacement curves were determined in the presence of unlabeled angiotensin peptides or angiotensin-receptor antagonists (10 pmol/l-30 µmol/l). Bound and free radioligands were separated by vacuum filtration in a cell harvester using No. 32 glass fiber filters and washed with 4 × 2 ml PBS containing (in mmol/l) 150 NaCl, 8.8 Na2HPO4, and 2 NaH2PO4, pH 7.2 at room temperature. Radioactivity retained by the protein-bound filters was measured by gamma counting. Competition data were analyzed by InPlot (GraphPad Software).

Autoradiography Studies

Male rats were anesthetized with an intraperitoneal injection of pentobarbital sodium, and their kidneys were perfused in vivo with PBS containing (in mmol/l) 20 K2HPO4, 20 NaH2PO4, 125 NaCl, and 5 MgCl2, pH 7.4 at room temperature. The kidneys were then removed, frozen in isopentane at -25°C, and stored at -70°C until sectioned. Autoradiographic analysis of rat kidney binding was performed using 20-µm tissue sections mounted on gelatin-coated slides. Initially, sections were preincubated at room temperature for 30 min in isotonic buffer (same composition as that used above) and then incubated for 25 min in isotonic buffer containing 0.4 nmol/l 125I-peptide plus or minus 10 µmol/l displacers, rinsed, dried, and exposed to X-ray film (Kodak 5B5 in Wolf cassettes, stored at -70°C for 2-16 days, and then developed with Kodak D19). A computerized image-analysis system (MCID, Imaging Research) was used to quantitate film exposure based on a standard curve generated from known amounts of 125I.

Metabolism Studies

Rat proximal tubules were isolated as described in Isolation of rat proximal tubules, and suspended for 10 min in oxygenated KHB solution. These experiments were conducted at room temperature and with approximately one-fifth of the protein content per assay as was used in the QO2 study. This was because we found that 125I-ANG-(1-7) was completely metabolized within a 1-min exposure to proximal tubules at 37°C. Aliquots of the proximal tubule suspension were incubated with 125I-ANG-(1-7) (1.2 nmol/l in a total assay volume of 250 µl) in microcentrifuge tubes, and the metabolism of 125I-ANG-(1-7) was terminated by the addition of trichloroacetic acid (TCA, final concentration in reaction tube was 20%) at 0, 0.5, 4, 10, 20 and 40 min. Zero-time samples were generated by adding TCA to the tissue prior to the addition of 125I-ANG-(1-7). The microcentrifuge tubes were kept on ice and then centrifuged at 14,000 rpm for 5 min, and the supernatant was stored in tubes at -70°C. The next day the tubes containing the supernatant were thawed, and we characterized the 125I-products by HPLC, using a reverse-phase C18 column linked to a radioactivity detector. 125I-peptides were separated isocratically over 35 min with 89.5% solvent A-10.5% solvent B at the measured flow rate of 1.75 ml/min at room temperature (solvent A was 83 mmol/l H3PO4 buffered to pH 3.0 with triethylamine and solvent B was acetonitrile).

Iodination of Angiotensin Peptides

ANG-(1-7), ANG IV, and all other angiotensin-related peptides were monoiodinated using chloramine T, Na 125I, and sodium bisulfite, and separated from unlabeled and diiodinated peptide by HPLC utilizing a reverse-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 9-26% developed over 90 min. All 125I-angiotensin peptides had a specific activity of ~2,176 Ci/mmol.

Drugs

Losartan (AT1-receptor antagonist) was obtained from DuPont-Merck Pharmaceuticals, PD-123319 (AT2-receptor antagonist) from Parke-Davis, and divalinal-ANG IV (AT4-receptor antagonist) from Dr. Joseph W. Harding. Amino-terminal-deleted fragments of sarthran ([Sar1,Thr8]ANG II) and ANG-(1-7) were synthesized by the Washington State University peptide synthesis core facility. Angiotensin peptides were obtained from Sigma, Bachem, and Peninsula Laboratories. MDBK cells (CCL22) were a gift from Dr. William J. Wechter of Loma Linda University.

Statistics

All values represent means ± SE. Multiple groups were analyzed by one-way or two-way analysis of variance (ANOVA) with post hoc Sidak correction or Dunnett's test. Significance between two groups was analyzed by an unpaired Student t-test. Differences between means were taken to be significant at the 5% level. SAS and Sigmastat computer software programs were used to analyze the data.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

QO2 Studies

We have previously shown that ANG-(1-7) and ANG IV can concentration dependently inhibit QO2 in both control and nystatin-stimulated rat proximal tubules (17, 19). The present studies were performed on a suspension of freshly isolated rat proximal tubules to which nystatin, a polyene antibiotic used to increase the plasma membrane permeability to Na+, was added. The employed concentrations of nystatin and extracellular Na+ were such that the intracellular Na+ concentration was raised to saturating levels, resulting in maximal activation of basolateral Na+-K+-ATPase activity. Under these conditions, any confounding action of angiotensin peptides on apical Na+ entry was bypassed, and consequently the peptide effect on the basolateral pump can be tested. Basal QO2 and nystatin-stimulated QO2 values were similar in control compared with receptor antagonist-incubated groups and averaged 26.0 and 41.2 nmol O2 · min-1 · mg protein-1, respectively (Table 1). The results depicted in Fig. 1 demonstrate the effect of ANG-(1-7) and ANG IV (each 1 pmol/l) on nystatin-stimulated proximal tubule QO2 and their modification following incubation with angiotensin-receptor antagonists (each 1 µmol/l). ANG-(1-7) and ANG IV inhibited proximal tubule QO2 to a similar degree (~22%). ANG-(1-7) caused a 9.62 ± 1.00 nmol O2 · min-1 · mg protein-1 decrease in QO2 values in vehicle-treated tubules that was significantly attenuated by the AT4-receptor antagonist divalinal-ANG IV (QO2 fall of only 3.83 ± 1.05 nmol O2 · min-1 · mg protein-1) and abolished by either sarthran (a nonselective angiotensin-receptor antagonist) or a combination of divalinal-ANG IV and losartan (AT1-receptor antagonist). The significantly lower QO2 value in the control group of the sarthran-treated tubules was most likely due to random sampling variability and was not responsible for the inability of ANG-(1-7) to decrease QO2 because 1) Table 1 shows the pooled data of control groups prior to the application of ANG-(1-7) or ANG IV and shows no statistical difference between vehicle and receptor antagonist-treated groups and 2) the nystatin-stimulated QO2 value for PD-123319 (AT2-receptor antagonist)-treated tubules was similar to that of the sarthran-treated group and decreased ~8 nmol O2 · min-1 · mg protein-1 following the addition of ANG-(1-7) (not shown). The response of ANG IV was abolished by either divalinal-ANG IV or sarthran treatment. These results suggest that ANG-(1-7) can inhibit proximal tubule transcellular Na+ transport by acting on tubular AT1 and AT4 receptors, whereas ANG IV acts exclusively via the AT4 receptor. In addition to known receptor blockade actions of sarthran at the AT1 and AT2 receptor, the present study indicates that sarthran (or its metabolites) can also block the AT4 receptor. We have previously reported that ANG-(1-7) and ANG IV do not alter the increased QO2 induced by carbonyl cyanide p-tri(fluoromethoxy)phenylhydrazone (mitochondrial oxidative phosphorylation uncoupler) nor alter QO2 of tubules treated with nystatin and ouabain (Na+-K+-ATPase inhibitor) (17, 19). This indicates that the angiotensin responses were not due to an alteration in mitochondrial respiratory activity but most likely due to an inhibition of the Na+-K+-ATPase exit step of transcellular Na+ transport.

                              
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Table 1.   QO2 values in control and angiotensin-receptor antagonist-incubated groups



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Fig. 1.   Nystatin-stimulated proximal tubule (PT) O2 consumption (QO2) values in absence and presence of 1 pmol/l ANG-(1-7) (top) or 1 pmol/l ANG IV (bottom) and their modification following angiotensin-receptor blockade (each 1 µmol/l); n = 6-13 measurements for each treatment group. dival, Divalinal-ANG IV; los, losartan. * P < 0.05 from respective control response. dagger  P < 0.001 from angiotensin-induced QO2 change observed in vehicle-treated group, using 2-way ANOVA with repeated measures 1-way and post hoc Sidak correction.

Radioligand Binding Studies

Because the results from the QO2 studies indicated that ANG-(1-7) and sarthran interacted with the proximal tubule AT4 receptor, the following experiments were conducted to determine their relative affinity for the renal AT4 receptor. These experiments could not be performed in rat proximal tubule membranes because we were unable to prevent substantial metabolism of 125I-ANG IV by tubule proteases. Consequently, studies were conducted in the MDBK cell line because 125I-ANG IV remains intact during the incubation period and these epithelial cells appear to lack AT1 and AT2 receptors and yet express high levels of the AT4 receptor (18). The results shown in Fig. 2A indicate that ANG IV and divalinal-ANG IV have high affinity for the AT4 receptor, whereas losartan and PD-123319 demonstrate extremely poor affinity for the AT4 receptor. Although sarthran had low affinity for the AT4 receptor, successive NH2-terminal deletions of sarthran substantially increased the affinity of the peptide for the AT4 receptor. Similarly, ANG-(1-7) had poor affinity for the AT4 receptor and yet NH2-terminal deletions of the heptapeptide increased its affinity for the AT4 receptor (Fig. 2B). Therefore it was most likely that [des-Sar1, Arg2]sarthran and ANG-(3-7) generated from the proteolytic cleavage of sarthran and ANG-(1-7), respectively, were responsible for the effect of the parent peptide at the AT4 receptor site in the proximal tubule QO2 studies.


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Fig. 2.   Inhibition of 125I-ANG IV binding in Madin-Darby bovine kidney (MDBK) cell membranes by increasing concentrations of angiotensin-receptor antagonists and amino-terminal-deleted fragments of sarthran (A) and by ANG-(1-7) and its amino-terminal-deleted fragments (B). Data are means ± SE from 3 experiments in duplicate.

Autoradiography Studies

Additional studies addressed whether a novel high-affinity ANG-(1-7) receptor could be detected in the rat kidney and to confirm that NH2-terminal-deleted products of ANG-(1-7) and sarthran metabolism could account for the ligand-AT4 receptor interaction in the rat kidney. Specific binding sites for 125I-ANG IV were localized to the cortex and outer strip of the outer medulla and were displaced by both ANG IV and divalinal-ANG IV, indicative of an AT4 receptor site (Figs. 3 and 4). Specific 125I-ANG II binding sites were found in the glomeruli, cortex, and inner medulla and displaced by either ANG II or losartan, implying that these receptors were largely of the AT1-receptor subtype (Figs. 3 and 4). Receptor autoradiography did not detect high-affinity ANG-(1-7) receptors in the rat kidney as both the density of total and nonspecific 125I-ANG-(1-7) binding (Fig. 3, E and F) or 125I-[D-Ala7]ANG-(1-7) binding (not shown) were similar. The absence of specific 125I-ANG-(1-7) binding in the rat kidney was not due to any detectable degradation of the ligand in the incubation buffer. Furthermore, if limited metabolism of 125I-ANG-(1-7) had occurred at the kidney surface, we would expect to detect some specific binding because 125I-ANG-(3-7) is a major product of 125I-ANG-(1-7) metabolism (see Metabolism Studies, below) and demonstrates specific binding in the rat kidney (Fig. 3, G and H). Similar to the findings in the MDBK cell radioligand binding study, we found ANG-(1-7) and sarthran relatively ineffective in competing with 125I-ANG IV for its binding site in the rat kidney. On the other hand, their NH2-terminal-deleted metabolites, ANG-(3-7) and [des-Sar1,Arg2]sarthran, respectively, were able to compete with 125I-ANG IV for the renal AT4 receptor (Fig. 4). In contrast, ANG-(1-7) and sarthran competed with 125I-ANG II for AT1 receptors in the kidney, whereas their NH2-terminal-deleted metabolites were unable to displace 125I-ANG II from its receptor (Fig. 4).


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Fig. 3.   Total and nonspecific binding of 125I-ANG IV (A and B, respectively; 2-day exposure), 125I-ANG II (C and D, respectively; 16-day exposure), 125I-ANG-(1-7) (E and F, respectively; 16-day exposure), and 125I-ANG-(3-7) (G and H, respectively; 2-day exposure) in the rat kidney. Specific 125I-ANG IV binding was localized to convoluted and straight proximal tubules in cortex and outer stripe of outer medulla. Specific 125I-ANG II binding was localized to cortex and inner medulla with especially high density of binding overlying glomeruli and renomedullary interstitial cells near vasa recta bundles. No specific high-affinity 125I-ANG-(1-7) binding was observed. Distribution of specific 125I-ANG-(3-7) binding was similar to that of 125I-ANG IV binding and most likely reflected binding to AT4 receptor as 125I-ANG(3-7) binding could be displaced by ANG IV (not shown).



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Fig. 4.   Specific 125I-ANG II and 125I-ANG IV binding in the rat kidney and its displacement by various agonists and antagonists. Densitometric measurements were taken from 6 different tissue sections in absence or presence of 10 µmol/l competitor. Specific binding was calculated from serial rat kidney sections by subtracting nonspecific binding from total binding. Displacement by competitors was then expressed as a percentage of specific binding. * P < 0.05 from 100% specific binding of corresponding 125I-peptide using 1-way ANOVA and Dunnett's test.

Metabolism Studies

This study was conducted to identify products of ANG-(1-7) metabolism that could potentially interact with the AT4 receptor in rat proximal tubules. The metabolites were identified by both comparing their retention times with known 125I-angiotensin standards and by spiking samples with 125I-angiotensin markers. The retention times for 125I-ANG-(1-4) and 125I-ANG(3-6) standards were identical and could not be differentiated by altering the acetonitrile concentration in the mobile phase. Consequently, the chromatogram peak with a similar retention time to 125I-ANG-(1-4) and 125I-ANG-(3-6) may represent either one or both peptides. Incubation of 125I-ANG-(1-7) with rat proximal tubules resulted in a time-dependent metabolism of the radioactive probe, yielding several 125I-tyrosine-containing peptides (Fig. 5). 125I and 125I-tyrosine were generated almost immediately and increased over time. 125I-ANG-(2-7), 125I-ANG-(1-6), 125I-ANG-(1-4), and/or 125I-ANG-(3-6) were generated after 30 s of exposure, whereas 125I-ANG-(3-7) and 125I-ANG-(3-4) were observed at 4 min, and 125I-ANG-(4-5) was observed at 20 min. MDBK cell radioligand binding studies indicated that peptides generated during the metabolism of 125I-ANG-(1-7) in rat proximal tubules bound to the AT4 receptor with a relative binding affinity order of ANG IV = ANG-(3-7) > ANG-(2-7) = ANG-(3-6) >> ANG-(1-7) = ANG-(1-6) > ANG-(1-4) = ANG-(3-4) = ANG-(4-5) = tyrosine (not shown). Consequently, ANG-(3-7) is a major product of ANG-(1-7) metabolism in rat proximal tubules and has the highest affinity for the renal AT4 receptor.


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Fig. 5.   Time course for metabolism of 125I-ANG-(1-7) in the rat proximal tubules and subsequent generation of 125I-products. Points represent percentage of total radioactivity detected following HPLC, and each value is the mean of 2 experiments. 50% ACN wash, residual radioactivity detected during 50% acetonitrile wash of C18 column.

ANG-(3-7) Studies

Results from the radioligand binding, autoradiographic, and metabolism studies suggest that ANG(1-7) is metabolized to generate ANG-(3-7), which could be the high-affinity ligand for the renal AT4 receptor. Consequently, it was necessary to determine whether ANG-(3-7) was biologically active in the rat proximal tubule preparation and whether the effect of the peptide was mediated by the AT4 receptor. As shown in Fig. 6, ANG-(3-7) produced a concentration-dependent inhibition of nystatin-stimulated QO2 that was to the right of the concentration-response curve generated by ANG-(1-7). The inhibitory effect of ANG-(3-7) was absent in ouabain-treated tubules (not shown) and abolished by divalinal-ANG IV treatment, indicating that the ouabain-suppressible response was mediated by the AT4 receptor (Fig. 6, inset).


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Fig. 6.   Concentration-dependent inhibitory response curve of ANG-(1-7) and ANG-(3-7) on nystatin-stimulated rat proximal tubule QO2. Inset, tissue treatment with divalinal-ANG IV abolished ANG-(3-7) inhibitory response. * P < 0.05 using unpaired Student's t-test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study identified the renal non-AT1, non-AT2 receptor that was responsible for the inhibitory action of ANG-(1-7) on proximal tubular Na+ transport as the AT4 receptor. Furthermore, ANG-(3-7) generated from ANG-(1-7) metabolism appears to be the ligand responsible for the activation of the proximal tubule AT4-receptor system. These conclusions were based on the facts that 1) receptor autoradiography did not detect high-affinity 125I-ANG-(1-7) binding sites in the rat kidney, 2) the inhibitory action of ANG-(1-7) on stimulated rat proximal Na+ transport was attenuated by the specific AT4-receptor antagonist divalinal-ANG IV and abolished by additional AT1-receptor blockade, 3) ANG-(1-7) had extremely low affinity for the renal AT4 receptor, whereas ANG-(3-7) demonstrated high affinity for the renal AT4 receptor, 4) 125I-ANG-(3-7) was a major product of 125I-ANG-(1-7) metabolism in rat proximal tubules, and 5) ANG-(3-7) inhibited proximal tubule Na+ transport that was abolished by AT4-receptor blockade. Consequently, metabolism of ANG-(1-7) to generate ANG-(3-7) can activate the AT4 receptor to elicit an inhibitory response on rat proximal Na+ transport. The results also suggested that the competitive nonselective angiotensin-receptor antagonist sarthran was an inhibitor of the AT4 receptor. This was most likely due to its metabolism to generate [des-Sar1,Arg2]sarthran, which was also shown to have high affinity for the renal AT4 receptor.

Recent studies have established that AT4 receptors are expressed in the kidney: rat mesangial cells (3), apical and basolateral membranes of the rat and rabbit proximal convoluted tubule (10, 19), opossum proximal convoluted tubule (11), rat straight proximal tubule (19), bovine distal/collecting tubule (18), and the human collecting duct (8). Functions associated with this novel renal ANG IV-AT4-receptor system have included increased cortical renal blood flow (7), counteraction of ANG II-mediated constriction of mesangial cells (3), and inhibition of proximal tubule Na+ transport (19). Although [based on reports that ANG-(1-7) is generated within the kidney and can alter renal excretory function] there is also speculation of a renal ANG-(1-7)-receptor system (13), disagreement exists as to the exact nature of the biological effect and receptor subtype involved. ANG-(1-7) was shown to elicit a dose-dependent natriuretic and diuretic response when infused into the renal artery of the anesthetized rat or into the isolated rat kidney. This response was independent of renal innervation, renal vascular resistance, or increases in glomerular filtration rate, suggesting a tubular site of action (9, 17). Consistent with this notion, the heptapeptide inhibited apical-to-basolateral Na+ flux across cultured rabbit proximal epithelial cells (2). In contrast, intraperitoneal or subcutaneous administration of micromolar concentrations of ANG(1-7) to water-loaded, conscious rats resulted in an antidiuresis that could be blocked by a putative ANG-(1-7)-receptor antagonist (24). In this latter study, the heptapeptide also caused a large fall in the filtered load of Na+ into the nephron with minimal alteration in urinary Na+ excretion. Consequently, a greater fraction of the filtered load of Na+ was being excreted, which would be consistent with an overall inhibitory action of ANG-(1-7) on tubular Na+ reabsorption (24). A renal micropuncture study in anesthetized rats reported that luminal perfusion of the convoluted proximal tubule with 1 pmol/l to 10 nmol/l ANG-(1-7) did not alter net fluid reabsorption, and that luminal perfusion of the loop of Henle (including straight proximal tubule) with only the highest concentration of ANG-(1-7) elicited an AT1-receptor-mediated increase in fluid, K+, or Na+ reabsorption (30). However, ANG-(1-7) had a biphasic effect on fluid and bicarbonate reabsorption when applied basolaterally to the microperfused isolated rat proximal straight tubule with 1 pmol/l and 10 nmol/l concentrations producing an AT1-receptor-mediated stimulation and inhibition of transport, respectively (16). In contrast, we have demonstrated that simultaneous exposure of apical and basolateral surfaces of isolated rat proximal tubules (containing both convoluted and straight tubules) to 0.1 pmol/l to 10 nmol/l ANG-(1-7) elicited only a concentration-dependent inhibition of transepithelial Na+ transport (17), and assuming no offsetting action downstream of the proximal tubule could account for the natriuresis observed in vivo. The decrease in transepithelial Na+ transport that was achieved with 1 pmol/l ANG-(1-7) was largely mediated by a sarthran inhibitable, non-AT1, non-AT2 receptor with a smaller contribution by a sarthran inhibitable, losartan-sensitive receptor (17). The results of the present study indicate that the AT4 receptor was responsible for the non-AT1, non-AT2 receptor component of the inhibitory action of ANG-(1-7) because it was abolished by divalinal-ANG IV, a specific AT4-receptor antagonist (19). Although the evidence to date suggests that the integrated kidney response to ANG-(1-7) is a natriuresis, the actions of ANG-(1-7) on nephron transport processes appear complex and may be related to ANG-(1-7) and its metabolites being able to act on different angiotensin-receptor subtypes distributed throughout the nephron.

ANG-(1-7) can be generated from the enzymatic processing of ANG I in the renal vasculature (1) and tubules (26) and may account for the high levels of ANG-(1-7) found in the urine compared with plasma (14). However, the subsequent metabolic fate of the heptapeptide and whether biologically active ANG(1-7) fragments are generated in the kidney are unknown. Proximal tubules are rich in ectopeptidases and endopeptidases that can hydrolyze angiotensins at both NH2-terminal and COOH-terminal ends, as well as internal bonds (4). Metabolism of 125I-ANG-(1-7) in rat proximal tubules revealed that ANG-(3-7) and ANG-(1-6) were the major NH2-terminal and COOH-terminal deleted fragments of ANG-(1-7) degradation, respectively. Subsequent radioligand binding results indicated that of all the generated metabolic products, ANG-(3-7) had the highest affinity for the AT4 receptor. In addition, ANG-(3-7) produced a concentration-dependent inhibition of proximal tubule Na+ transport that could be abolished by divalinal-ANG IV, further implicating ANG-(3-7) as a biologically active product of ANG-(1-7) metabolism responsible for the inhibitory action of ANG-(1-7) at the proximal tubule AT4-receptor site. Despite the high affinity of ANG-(3-7) for the AT4 receptor, it was less potent than ANG-(1-7) in inhibiting energy-dependent proximal tubule transport. This was in part due to ANG-(1-7) acting on an additional losartan-sensitive receptor site to inhibit transport, the contribution of which may increase at higher peptide concentrations (16, 30). The assertion that ANG-(3-7) is a biologically active peptide of RAS is supported by studies that have shown that the pentapeptide can improve recognitive function, increase blood pressure, and dilate coronary vessels (4-6), albeit at high concentrations.

Pretreatment of rat proximal tubule suspensions with the competitive nonselective angiotensin-receptor antagonist sarthran was able to abolish the inhibitory transport effects of ANG-(1-7). The efficacy of sarthran to abolish the biological actions of ANG-(1-7), while AT1- and AT2-receptor blockade have minimal effects, has also been reported in conscious spontaneously hypertensive rats, pithed rats, coronary vessels, vascular smooth muscle cells, and proximal tubules, and has suggested the presence of a novel ANG-(1-7) receptor (5, 6, 15, 17). The results of the present study indicate that in addition to the known blocking actions of sarthran at the ANG II type AT1- and AT2-receptor subtypes, sarthran metabolites can recognize the AT4 receptor. Radioligand binding and autoradiographic studies revealed that although sarthran has poor affinity for the AT4 receptor, its NH2-terminal metabolite [des-Sar1,Ala2]sarthran has high affinity for the AT4 receptor. Therefore sarthran was most likely NH2 terminally degraded in the proteolytic milieu of the proximal tubule suspension, and a sarthran metabolite was responsible for blocking the biological response of ANG-(1-7) and ANG IV at the AT4-receptor site. Similarly, removal of the first two NH2-terminal amino acids from an alternative nonspecific angiotensin-receptor antagonist [Sar1,Ile8]ANG II also conferred high affinity for the renal AT4 receptor (unpublished results). Although there is evidence for the existence of a novel ANG-(1-7) receptor based in part on the use of [D-Ala7]ANG-(1-7), a putative ANG-(1-7)-receptor antagonist (6, 22-24, 28), it is conceivable that NH2-terminal degradation of the ANG-(1-7)-receptor antagonist may also increase its affinity for the AT4 receptor in a manner similar to that observed with ANG-(1-7), sarthran, and [Sar1, Ile8]ANG II. These results highlight the fact that metabolic products of ANG-(1-7) and angiotensin peptide-receptor antagonists may influence the AT4-receptor system in tissues with NH2-terminal-directed proteolytic activity.

The QO2 results together with those reported by Garcia and Garvin (16) and ourselves (17) indicate that in addition to the AT4 receptor, low picomoles per liter concentrations of ANG-(1-7) can alter proximal tubule transport by interacting with a sarthran-inhibitable, losartan-sensitive, PD-123319-insensitive receptor, presumed to reflect an ANG-(1-7) interaction with proximal tubule AT1 receptors. The autoradiography results clearly demonstrate that ANG-(1-7), and not a metabolite such as ANG-(3-7), was responsible for the heptapeptide's interaction with the kidney AT1 receptor. However, there was a complete absence of high-affinity 125I-ANG-(1-7) binding sites in the rat kidney, including no high-affinity binding to AT1 receptors. This is in keeping with reports that the apparent dissociation constant of ANG-(1-7) for the losartan-sensitive, PD-123319-insensitive renal AT1A receptor (mediates the renal actions of ANG II) is in the micromolar range (12). We have not examined whether the interaction of ANG-(1-7) with the losartan-sensitive, non-AT4-receptor site can be blocked by [D-Ala7]ANG-(1-7) because the putative ANG-(1-7)-receptor antagonist is rapidly metabolized in the proximal tubule preparation (half-life 35 s, unpublished results), and would be completely degraded prior to the addition of ANG-(1-7) to the tissue-containing chamber. Thus the ability of picomole per liter concentrations of ANG-(1-7) to interact with a losartan-sensitive-receptor site to alter proximal tubule transport requires further investigation because the heptapeptide lacks a carboxy terminal phenylalanine moiety that is essential for high-affinity binding to known AT1 receptors in the kidney (12), and losartan has poor affinity for the renal AT4 receptor (3, 8, 11, 18) and for the recently described high- and low-affinity ANG-(1-7)-receptor sites in vascular endothelial cells (28).

It should be emphasized that although autoradiography demonstrated the absence of a high density of high-affinity ANG-(1-7) binding sites in the rat kidney, these results cannot exclude the possibility of the presence of low-affinity ANG-(1-7) binding sites or low density of high-affinity binding sites that were below the level of autoradiographic detection. With regard to the latter, ANG-(1-7) receptors could not be detected by the more sensitive method of emulsion autoradiography (radioactive kidney sections coated with emulsion for 8 wk at -70°C, not shown). However, only isolating and sequencing the cDNA encoding the ANG-(1-7) receptor will provide the necessary tools to unequivocally determine whether ANG-(1-7) receptors exist in the kidney.

In summary, we propose that the inhibition of rat proximal tubule Na+ transport activity by ANG-(1-7) is due in part to metabolism of the heptapeptide to generate ANG-(3-7), which then activates the proximal tubule AT4-receptor system. Thus ANG-(1-7) and its metabolites can interact with a number of angiotensin-receptor subtypes, including AT1, AT2, AT4, and perhaps a novel ANG-(1-7) receptor, that may account for the diverse biological actions of the heptapeptide on the internal environment.


    ACKNOWLEDGEMENTS

I am indebted to Drs. Joseph W. Harding (Washington State University) for providing divalinal-ANG IV and access to the HPLC system, to Robert C. Speth for providing access to the MCID image analysis system, and to Steve M. Simasko who provided access to cell culturing facilities.


    FOOTNOTES

This work was supported by a grant-in-aid from the Washington Affiliate of the American Heart Association.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. K. Handa, Dept. of VCAPP, College of Veterinary Medicine, Washington State Univ., Pullman, WA 99164-6520.

Received 20 July 1998; accepted in final form 24 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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