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(Hypertension. 1995;25:1238-1244.)
© 1995 American Heart Association, Inc.

Articles

Association Between the Natriuretic Action of Angiotensin-(1-7) and Selective Stimulation of Renal Prostaglandin I₂ Release

Sean D. Hilchey; Caroline P. Bell-Quilley

From the Department of Pharmacology, New York Medical College, Valhalla.

Correspondence to Caroline P. Bell-Quilley, PhD, Safety Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd, PO Box 1539, King of Prussia, PA 19406-0939.

	Abstract

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Abstract We previously reported that angiotensin-(1-7) [Ang-(1-7)], a heptapeptide derived from the metabolism of either Ang I or Ang II, was biologically active in the rat isolated kidney, producing a marked diuresis and natriuresis that could be dissociated from the modest increase in glomerular filtration rate. The natriuretic response was accompanied by an increase in sodium concentration and concomitant decrease in urinary potassium concentration. Ang-(1-7) has also been shown to stimulate arachidonic acid release from isolated proximal tubules and elicit prostaglandin release from a number of tissues. Therefore, in the present study we tested the hypothesis that prostaglandins participate in the renal actions of Ang-(1-7). Rat isolated kidneys were perfused at 37°C with gassed (95% O₂/5% CO₂) Krebs-Henseleit buffer containing oncotic agents and amino acids for six 10-minute clearance periods at a constant pressure of 90 mm Hg. Ang-(1-7) was infused at a rate that achieved a final concentration of 3 pmol/mL in the presence and absence of 10 µmol/L indomethacin. Prostaglandin E₂ (PGE₂) and PGI₂ released into ureteral and venous effluents were measured by enzyme-linked immunoassay. During Ang-(1-7) infusion there was a selective increase in 6-keto-PGF₁, an index of PGI₂, appearing in both urine and perfusate; PGE₂ levels were unchanged. Inhibition of stimulated 6-keto-PGF₁ release with indomethacin halved the fourfold increase in urine flow and sevenfold increase in sodium excretion rate without altering the increase in urinary sodium concentration produced by Ang-(1-7). In contrast, the increased potassium excretion rate was unchanged, despite the reduction in urine flow, as indomethacin abolished the fall in urinary potassium concentration caused by Ang-(1-7) infusion alone. Thus, Ang-(1-7) is a specific stimulus for renal PGI₂ versus PGE₂ release. This effect may mediate Ang-(1-7)–induced natriuresis and diuresis and fall in urinary potassium concentration but does not appear to be involved in the doubling of urinary sodium concentration. It is possible that these observations have relevance to the link between prostaglandins and converting enzyme inhibitors in view of earlier reports that these antihypertensive agents substantially increase Ang-(1-7).

Key Words: angiotensins • natriuresis • indomethacin • prostaglandins • immunoenzyme techniques • kidney • rats

	Introduction

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It is becoming increasingly evident that metabolites of the renin-angiotensin system other than angiotensin II (Ang II) are biologically active. In particular, the spectrum of effects of des-[Phe⁸]Ang II, most commonly known as Ang-(1-7), has provoked special attention, as Ang-(1-7) is a major product of Ang I metabolism by several tissues,^{1 2} including the proximal tubule brush border.³ Relevant to the latter site of synthesis, we reported that Ang-(1-7) is a potent natriuretic agent in the rat isolated kidney in the absence of an effect on renal vascular resistance.⁴ This was in contrast to the potent vasoconstrictor action of Ang II over the same dose range. Moreover, the vascular response to Ang II was associated with a natriuresis only at the higher doses. A similar response of the anesthetized rat to renal arterial infusion of Ang-(1-7) has been reported.⁵

Additional properties of Ang-(1-7) include the ability to stimulate prostaglandin release from human astrocytes,⁶ glioma cells,⁷ and rabbit vas deferens⁸ with a potency similar to that of Ang II. On the other hand, Ang-(1-7) has been shown to be considerably more potent than Ang II in stimulating prostaglandin release from cultured endothelial cells.⁹ With respect to effects on renal eicosanoids, a recent study showed that Ang-(1-7) was as potent as Ang II as a stimulus for arachidonic acid release by isolated proximal tubule epithelial cells.¹⁰ However, no studies have addressed the effect of Ang-(1-7) on prostaglandin release by the intact kidney or have linked the changes in intrarenal prostaglandin release to the functional effects of Ang-(1-7).

It is well established that many of the actions of Ang II involve an eicosanoid component, such as participation of thromboxane A₂ and products of the 12-lipoxygenase pathway in renal vasoconstriction.^{11 12 13 14} Importantly, the stimulation of arachidonic acid release from renal tubular epithelia has been linked to the inhibition of sodium reabsorption by high concentrations of Ang II.¹⁵

Based on these considerations we hypothesized that, as for Ang II, the ability of Ang-(1-7) to increase glomerular filtration rate (GFR) and water and electrolyte excretion rates might be mediated by vasodilator, natriuretic prostaglandins. Therefore, in the present study we examined the capacity of Ang-(1-7) to stimulate release of prostaglandin E₂ (PGE₂) and PGI₂ into venous and ureteral effluents of the rat isolated kidney and their possible role in the renal response to Ang-(1-7) through the use of indomethacin to inhibit prostaglandin synthesis. We used the isolated kidney preparation so that we could exclude systemic influences, eg, stimulation of vasopressin release by Ang-(1-7)¹⁶ or changes in aldosterone release as a response to changes in extracellular fluid volume. Additionally, we eliminated the possible contribution of prostaglandins arising from extrarenal sources.

Ang-(1-7) increased the release of 6-keto-PGF₁, an index of PGI₂ formation, without altering the release of PGE₂. As reported previously,⁴ Ang-(1-7) increased the loss of salt and water, in contrast to the stable excretion rates in the untreated time control group, in which initial sodium reabsorption exceeded 98.5%. Indomethacin inhibition of Ang-(1-7) stimulation of 6-keto-PGF₁ was accompanied by attenuation of the increase in water and sodium excretion rates, whereas indomethacin alone did not alter basal renal function.

	Methods

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Male Sprague-Dawley rats weighing 300 to 400 g were anesthetized with pentobarbital (65 mg/kg IP, Butler Co), and 0.4 mL mannitol (10% wt/vol) and 0.1 mL heparin (1000 U/mL, Elkins-Sinn Inc) were injected intravenously. A midline incision was made and the right kidney surgically isolated and perfused with a modified Krebs-Henseleit solution, exactly as described previously,¹³ via a catheter in the renal artery. Bovine serum albumin (Bovimar CRG7, Intergen Co) and Ficoll 70 (Pharmacia) were used as oncotic agents, and a mixture of 20 amino acids was included to improve the functional characteristics of the kidney. Vitamin B₁₂ was added at a concentration of 50 µg/mL for GFR determinations. Perfusate chloride concentration was adjusted to a normal concentration of 102 mmol/L by substitution of sodium acetate for NaCl. Concentrations of other electrolytes and organic constituents were as described previously.¹³ Urine was collected from a ureteral catheter, and the venous effluent, collected via a catheter placed in the lower abdominal vena cava, was recirculated through a humidified membrane lung and gassed with 95% O₂/5% CO₂. Perfusion pressure, monitored by a pressure transducer (Statham P23, Grass Instruments) placed proximal to the renal arterial catheter and recorded on a Grass polygraph, was maintained constant at 90 mm Hg by continual adjustments in perfusate flow rate. At the completion of surgery, rats were killed by a lethal intracardiac injection of KCl, and the kidneys were perfused in situ.

Experimental Protocol
After a 15-minute equilibration period, renal function was examined during six 10-minute clearance periods. The first was used as a pretreatment clearance period to establish the viability of the preparation. Initial sodium reabsorption of less than 98.5% served as a basis for termination of the experiment. Perfusate flow rate was determined from collection of the venous outflow over 30 seconds at the start and end of each period. Urine was collected over the entire 10-minute clearance period and the volume determined gravimetrically. Aliquots of perfusate and urine were taken for measurement of electrolytes and vitamin B₁₂ and assays of prostanoids.

Four experimental groups of six to seven kidneys each were studied as follows: time control; 10 µmol/L indomethacin; 3 pmol/mL Ang-(1-7); and Ang-(1-7) plus indomethacin given in combination as for the single treatment groups. Indomethacin was prepared as a concentrated solution of 10 mmol/L dissolved in NaHCO₃ (4.2% wt/vol) and added at the beginning of perfusion. Ang-(1-7) was infused from the beginning of the second clearance period. The peptide was dissolved in saline at a concentration of 1.5 mmol/L and administered at a rate of 3 µL/mL per minute according to the perfusate flow rate. NaHCO₃ and saline vehicles were administered as appropriate.

Prostaglandin Assays
Prostaglandins were measured in unextracted samples with the use of second-antibody, solid-phase, enzyme-linked immunoassay (ELISA) according to Pradelles et al.¹⁷ Microtiter plates (Immunoplate II, NUNC, Laboratory Disposable Products) were coated with goat anti-rabbit IgG (200 µL per well, Boehringer Mannheim Biochemicals) at a concentration of 10 µg/mL, diluted with 50 mmol/L potassium phosphate buffer, pH 7.4. After an overnight incubation at 4°C, plates were washed three times with successive 300-µL volumes of wash buffer composed of 0.05% Tween 20 in 10 mmol/L potassium phosphate buffer, pH 7.4. Plates were then filled with enzyme immunoassay buffer of the following composition: 0.1 mol/L potassium buffer, 0.4 mmol/L NaCl, 85 mmol/L Na₄EDTA, 0.01% wt/vol NaN₃, and 0.1% wt/vol bovine serum albumin. Plates were then covered with microtest plastic film (Fisher) and stored at 4°C for a minimum of 18 hours before their use in the competitive binding step. Coated plates could be stored in this manner for several weeks. After buffer removal, 50-µL aliquots of enzymatic tracer, antibody, and either prostaglandin standard (4 to 2000 pg/mL) or samples, diluted as appropriate with buffer, were placed in duplicate in the coated wells. Nonspecific binding was determined by replacement of antibody with buffer and maximal binding by equilibration in the absence of prostanoids. After overnight equilibration at 4°C, plates were washed three times with 300 µL enzyme immunoassay buffer. Wells were filled with 200 µL Ellman's reagent. The concentrated Ellman's stock solution [69.16 mmol/L acetylthiocholine iodide and 54.24 mmol/L 5,5'-dithiobis(2-nitrobenzoic acid) in phosphate buffer] was stored at -20°C and diluted 1:100 as required. After 1 to 2 hours of shaking, absorbance was read at 405 nm with automated subtraction of readings at 630 nm to correct for nonspecific absorbance (Microplate EL309, Biotek Instruments). Microtitration and washing steps were carried out with an automatic dispenser (Pro/Pette, Perkin-Elmer). Curve fitting was done with computerized mass action smoothed spline interpolation (Packard Instrument Co), and sample prostaglandin concentrations were automatically calculated by reference to the standard curve. The validity of application of ELISA for assay of perfusate and urine samples was established as described previously for radioimmunoassay of rat urine.¹⁸ Limits of sensitivity were 7.8 pg/mL for both prostanoids, this being the lowest standard for percent binding of maximum bound (B/Bo) less than 90%.

Determination of Electrolytes and Vitamin B₁₂ and Calculations
Perfusion pressure and perfusate flow rate for each period were calculated as the mean of measurements made at the beginning and end of each period. Renal vascular resistance was calculated as the ratio of perfusion pressure to perfusate flow rate. GFR was calculated from colorimetric measurements of the vitamin B₁₂ concentration in perfusate and urine.¹⁹ Vitamin B₁₂ absorbance of samples, diluted as appropriate into 96-microwell plates, was measured at 550 nm, with subtraction of the blank at 630 nm, with the use of the spectrophotometric plate reader. GFR was determined from the product of urine flow and vitamin B₁₂ concentration divided by the perfusate vitamin B₁₂ concentration. Perfusate and urinary electrolytes were measured with an automatic electrolyte analyzer (model 644, Ciba Corning). Electrolyte excretion rate was the product of urine flow and electrolyte concentration. Fractional excretion of water and electrolytes was calculated by dividing the absolute excretion by the filtered load. Urinary prostaglandin excretion rates were determined by multiplying prostaglandin concentration by urine flow.

Statistical Analyses
Differences between groups were assessed by one-way ANOVA conducted with the SAS/PC computer program (SAS Instruments). When the F value indicated overall significance, specific comparisons were made with the least significant difference test using an adjusted value of 0.01, as only half the possible comparisons were of interest. A value of P<.05 was considered statistically significant.²⁰

	Results

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Urinary and Venous Prostaglandin Release
Table 1 gives the time course of changes in 6-keto-PGF₁ and PGE₂ in urine. Compared with the time control, Ang-(1-7) infusion produced a considerably greater increase in 6-keto-PGF₁ excretion during the 60 minutes of perfusion that, by the last clearance period, was more than threefold the modest time-dependent increase in the control group (Table 1). Indomethacin was highly effective in inhibiting this Ang-(1-7)–stimulated 6-keto-PGF₁ excretion. However, the basal excretion of 6-keto-PGF₁ was resistant to indomethacin inhibition. PGE₂ excretion also increased as a function of time of perfusion. However, in contrast to the stimulatory effect on 6-keto-PGF₁, PGE₂ excretion in kidneys infused with Ang-(1-7) was identical to that in control, irrespective of the time point examined (Table 1). Indomethacin substantially inhibited PGE₂ excretion in both vehicle control and Ang-(1-7) groups.

View this table: [in this window] [in a new window]   Table 1. Effects of Angiotensin-(1-7) and Indomethacin on 6-Keto-PGF1 and PGE2 Released into Ureteral and Venous Effluents

In the venous compartment, 6-keto-PGF₁ levels were below the limits of detection before the 40-minute time point. Thereafter, 6-keto-PGF₁ concentrations were higher in the Ang-(1-7)–infused kidneys (Table 1) compared with control. Indomethacin reduced 6-keto-PGF₁ concentrations to below the limits of detection throughout. Once again, in contrast to the increase in 6-keto-PGF₁, the venous levels of PGE₂ (Table 1) were not affected by Ang-(1-7) but were inhibited by indomethacin. Thus, Ang-(1-7) produced a selective increase in PGI₂ in both vascular and tubular compartments that was sensitive to inhibition by indomethacin.

Renal Vascular Resistance, Perfusate Flow Rate, and GFR
There were no significant differences between the four treatment groups for renal vascular resistance and renal perfusate flow, which ranged between 2.3 and 2.8 mm Hg/(mL/min) and 30 and 39 mL/min, respectively, throughout the course of perfusion. However, Ang-(1-7) did cause a modest increase in GFR that was significantly different from the control group for the last two clearance periods and showed borderline significance (P<.08) for the other three Ang-(1-7) infusion clearance periods (Table 2). GFR also tended to be higher for the Ang-(1-7) plus indomethacin group compared with the respective indomethacin control group, but this effect did not reach statistical significance. Indomethacin alone at this normal chloride concentration did not influence GFR.

View this table: [in this window] [in a new window]   Table 2. Effects of Indomethacin on Changes in GFR of Rat Isolated Kidney With and Without Ang-(1-7) Infusion

Water and Electrolyte Excretion Rates
During the 60 minutes of perfusion in the control group, there was a modest time-dependent increase in sodium and water excretion rates, whereas potassium excretion declined slightly (Figure). Ang-(1-7) caused an immediate increase in water and sodium excretion rates that increased further to more than fourfold and sevenfold the pretreatment levels, respectively. The potassium excretion rate was less affected, as it increased only twofold. Although indomethacin alone did not affect basal water and electrolyte excretion rates, its ability to inhibit Ang-(1-7) stimulation of PGI₂ release was accompanied by a reversal of the increased excretion of water and sodium (Figure). In contrast, indomethacin did not change the Ang-(1-7) stimulation of potassium excretion rate (Figure).

View larger version (21K): [in this window] [in a new window]   Figure 1. Line graphs show changes in urine volume and sodium and potassium excretion rates of rat isolated kidney during angiotensin-(1-7) infusion at 3 pmol/mL in the presence and absence of indomethacin (10 µmol/L). Values are mean±SEM; n=6-7. *P<.05 vs respective control; P<.05 between angiotensin-(1-7) groups.

Ang-(1-7) had opposite effects on urinary electrolyte concentrations, as sodium concentration doubled and potassium concentration was halved by the last clearance period (Table 3). Indomethacin did not alter this increase in sodium concentration. In contrast, indomethacin prevented the fall in potassium concentration produced by Ang-(1-7) (Table 3).

View this table: [in this window] [in a new window]   Table 3. Effects of Indomethacin and Ang-(1-7) on Urinary Sodium and Potassium Concentrations

As Ang-(1-7) had a positive effect on GFR, we also evaluated the data in terms of fractional excretion rates to discern those effects primarily attributable to a tubular site of action. Essentially the same results were obtained irrespective of whether the results were expressed in absolute or fractional terms. Ang-(1-7) infusion significantly increased fractional water and electrolyte excretion rates, and indomethacin attenuated these effects (Table 4). Thus, the diuretic and natriuretic effects of Ang-(1-7) and their modification by indomethacin appear to result primarily from a tubular site of action as opposed to an effect on filtered load.

View this table: [in this window] [in a new window]   Table 4. Effects of Indomethacin and Ang-(1-7) on Fractional Water and Electrolyte Excretion

	Discussion

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The renal functional responses of the rat isolated kidney to Ang-(1-7) were accompanied by increases in the release of 6-keto-PGF₁, and only 6-keto-PGF₁, an index of PGI₂, into both ureteral and venous compartments. Indomethacin inhibition of this enhanced PGI₂ release was associated with a diminution of the natriuretic and diuretic actions of the heptapeptide and prevention of the fall in urinary potassium concentration. Thus, the results of this study support our original hypothesis that the renal actions of Ang-(1-7) depend, at least in part, on prostaglandins. This is consistent with evidence that the depressor effect of Ang-(1-7) in the pithed rat also has a prostaglandin-dependent component.²¹ In addition, the indomethacin-sensitive effects of Ang-(1-7) in the kidney may be specifically mediated by a selective increase in the release of PGI₂. However, the absence of a significant effect of indomethacin on the ability of Ang-(1-7) to increase urinary sodium concentration or GFR indicates that these two effects are independent of an increase in cyclooxygenase-derived arachidonic acid metabolites.

With respect to the vascular compartment, Ang-(1-7) did not affect renal vascular resistance. However, infusion of the kidney with Ang-(1-7) was not entirely devoid of hemodynamic consequences, as GFR increased. Although this effect was modest, it was consistent with the small increases in GFR described earlier,⁴ which we suggested were indicative of an effect on microvascular resistances or mesangial cell contraction, as renal perfusate flow was not affected by Ang-(1-7). The ability of Ang-(1-7) to increase PGI₂ could explain this effect in view of its potency as an agent that causes relaxation of vascular smooth muscle and the mesangium.^{22 23 24} However, when the increased PGI₂ was blocked by indomethacin, the Ang-(1-7)–induced increase in GFR was not markedly affected, suggesting that PGI₂ may not be solely responsible for the ability of Ang-(1-7) to slightly increase GFR.

In contrast to its modest vascular action, Ang-(1-7) had substantial effects on water and electrolyte excretion rates. These diuretic and natriuretic responses of the rat isolated kidney were consistent with our earlier observations⁴ and with the recent report that Ang-(1-7) is also natriuretic when infused into the renal artery of the rat.⁵ On the other hand, intraperitoneal injection of Ang-(1-7) in water-loaded rats was shown to be antinatriuretic and antidiuretic.²⁵ Whether Ang-(1-7) administered by this route escapes metabolism before reaching the kidney is uncertain given the rapidity with which peptides are broken down. Moreover, although there is no direct evidence, it is possible that the reduction in excretion rates after intraperitoneal administration reflects direct effects of Ang-(1-7) on the gastrointestinal tract, as angiotensin receptors are abundant at this visceral site, and their stimulation affects electrolyte transport.²⁶

Indomethacin effectively reduced Ang-(1-7)–stimulated increases in PGI₂. However, basal PGI₂ excretion was not affected despite the fact that indomethacin inhibited basal levels of PGE₂. Previously, Terragno et al²⁷ showed that the susceptibility of PGE₂ to inhibition by indomethacin depended on the experimental conditions. The results of the present study provide an example of differences in the sensitivity of basal release of specific prostaglandins to indomethacin. It is well established that different tissues exhibit differential sensitivity to inhibitors of cyclooxygenase,²⁸ and recent studies have described cyclooxygenase isozymes that vary in their susceptibility to blockade by indomethacin.^{29 30} These differential sensitivities, coupled with the multiple cell types in the kidney and compartmentalization of synthesis of individual prostaglandins,³¹ provide a possible explanation for the resistance of basal PGI₂ release to indomethacin. Pharmacokinetic influences on the accessibility of indomethacin to discrete intrarenal sites of PGI₂ release³² may also be important.

The absence of any functional effect of indomethacin given alone is consistent with our recent observation that the renal response to indomethacin depends on chloride concentration³³ such that opposite effects are observed at high and low concentrations of the anion. For example, during perfusion with high chloride, a marked natriuresis is observed, whereas at low chloride concentrations, indomethacin is antinatriuretic. At the normal concentration of chloride used in the present study, the opposing responses to indomethacin would be expected to result in no overall change in renal function.

The magnitude of the increases in excretion rates suggests that multiple sites are involved in the action of Ang-(1-7). The large increases in PGI₂ that accompanied the increases in sodium and water excretion rates support an involvement of this prostanoid in this tubular response, a possibility that is further supported by the observation that PGI₂ inhibition was associated with a considerable reduction in salt and water excretion without significant changes in GFR, pointing to a tubular site of action. Notwithstanding the evidence for the participation of PGI₂, the incomplete reversal of the effect of Ang-(1-7) by indomethacin, despite the return of PGI₂ to basal levels, lends support to the involvement of additional mechanisms. Moreover, the observation that indomethacin reversed the fall in potassium concentration, without affecting Ang-(1-7)–induced increases in urinary sodium concentration, is further evidence for multiple sites of action involving multiple mechanisms. These may include Ang-(1-7) stimulation of endothelium-derived relaxing factor,³⁴ which is natriuretic in the isolated kidney,³⁵ or enhanced release of cytochrome P-450–dependent metabolites of arachidonic acid in the proximal tubule. Ang-(1-7) is equipotent to Ang II as a stimulus for arachidonic acid release by isolated proximal epithelial cells, and Ang II inhibition of sodium transport at this site has been associated with stimulation of epoxide metabolites.¹⁰

The association between the stimulation of excretion and an increase in PGI₂ is consistent with earlier reports demonstrating that PGI₂ infusion into the renal artery of the dog, at a concentration that was devoid of an effect on GFR, was natriuretic and diuretic.³⁶ On the other hand, PGE₂ had a greater natriuretic potency than PGI₂ at concentrations producing a renal vasodilator response.^{22 24} It has been suggested that increases in prostaglandin excretion simply reflect a flow-dependent stimulation of prostaglandin release.^{37 38 39} However, this seems an unlikely explanation for the increase in PGI₂ for two reasons. First, the effect of Ang-(1-7) was selective for PGI₂. Second, the tubular actions of Ang-(1-7) were blunted by cyclooxygenase inhibition, supporting the idea that increased release of PGI₂ is a prerequisite for the enhanced water and electrolyte excretion rates. On the other hand, the mechanism for the increased urinary sodium concentration can be separated from an increase in PGI₂, as indomethacin was without effect. In this regard, a mineralocorticoid effect cannot account for the reciprocal changes in urinary concentrations of potassium and sodium produced by Ang-(1-7), as aldosterone is not present in the isolated kidney preparation. In fact, this study clearly demonstrates that the fall in potassium concentration and increase in sodium concentration are dissociated events, the former being linked to a cyclooxygenase-dependent mechanism.

The lack of effect of Ang-(1-7) on PGE₂ was unexpected in view of both the diuretic and natriuretic activities of PGE₂^{22 37} and the frequent association between increased flow rate and enhanced PGE₂ excretion.^{38 39} However, a number of studies do not support a relationship between urine flow rate and renal PGE₂ production.^{40 41} The results of the present study provide yet another example.

Considering the possible intrarenal sites at which increased PGI₂ mediates the renal responses to the heptapeptide, it has been shown that Ang-(1-7) inhibits sodium transport and is a potent stimulus for arachidonic acid release by proximal tubule epithelial cells via activation of phospholipase A₂.¹⁰ The magnitude of the natriuretic effect of Ang-(1-7) also points to a proximal site of action. However, an examination of the distribution of cyclooxygenase along the nephron with the use of immunohistochemical localization techniques has shown that the enzyme levels in proximal tissue are extremely low.^{42 43} Accordingly, the capacity of this nephron segment to generate prostaglandins is minimal. It therefore seems unlikely that the increase in PGI₂ or the indomethacin-sensitive natriuresis results from an effect of Ang-(1-7) at a proximal tubular site. On the other hand, the collecting duct has a high prostaglandin synthesizing capacity, and PGI₂ inhibits sodium reabsorption at this site.^{44 45} In this instance, this effect cannot be attributed to antagonism of antidiuretic hormone by PGI₂,⁴⁴ which is absent in the isolated kidney, and may be due to a direct effect of PGI₂, as has been shown in studies with isolated collecting tubule preparations.⁴⁵ However, in proportion to the entire nephron, the contribution of the collecting tubule to salt and water reabsorption is small. It is likely, therefore, that additional cyclooxygenase-mediated effects of intrarenally generated PGI₂ are involved, such as redistribution of intrarenal blood flow and associated changes in peritubular pressures leading to decreases in reabsorption.

This is the first evidence that Ang-(1-7) affects renal prostaglandin release, in contrast to an earlier study with cultured rabbit renomedullary interstitial cells in which Ang-(1-7) had no effect.⁴⁶ This increased PGI₂ release is consistent with previous work showing that Ang-(1-7) elicits prostaglandin release from a variety of other cell types, including vascular smooth muscle⁴⁷ and endothelial⁹ cells. However, PGI₂ stimulation alone is peculiar to the whole kidney, as these previous studies demonstrated that Ang-(1-7) also increased PGE₂. This selective increase in PGI₂ also distinguishes the effect of Ang-(1-7) on renal prostaglandin synthesis from the reported effect of Ang II. Thus, Ang II is a potent stimulus of PGE₂ and thromboxane B₂ as well as 6-keto-PGF₁ in both in vitro and in vivo kidney preparations from several species.^{11 12 14} Possible mechanisms for the selective action of Ang-(1-7) include stimulation of renal cells that preferentially generate PGI₂; redirection of endoperoxide to PGI₂ synthesis as a result of diminished release of 12-hydroperoxyeicosatetraenoic acid, which has been shown to inhibit prostacyclin synthetase⁴⁸ ; or activation of an angiotensin receptor specifically linked to prostacyclin synthase. With regard to the latter, Ang-(1-7) stimulation of prostaglandin release from endothelial cells shows differential sensitivity to angiotensin type 1 and type 2 receptor antagonists.^{9 47} Within the kidney, there is evidence for angiotensin receptor heterogeneity linked to different intracellular messengers and differences in Ang II versus Ang-(1-7) binding.⁴⁹ This ability of Ang-(1-7) to selectively affect PGI₂ release provides a unique example of specific regulation of renal PGI₂ release as well as a paradigm in which the responses to increases in PGI₂ intrarenally are manifest, as opposed to those experimental designs in which PGI₂ is infused into the renal artery, often with attendant renal vasodilator effects.

In conclusion, the potent natriuretic action of Ang-(1-7) can be linked to selective stimulation of renal PGI₂ release into both vascular and tubular compartments. As this heptapeptide metabolite of Ang I and Ang II is substantially increased by inhibitors of converting enzyme,⁵⁰ it is possible that a selective increase in PGI₂ as a result of increased Ang-(1-7) formation may have importance for the mechanisms underlying the action of these antihypertensive drugs.

	Acknowledgments

 
This work was supported by American Heart Association grants 91-014G and 94-318 and National Institutes of Health research grant RO1-HL-25394. We thank Dr M. Khosla for generous supplies of Arg-(1-7) and Dr J.C. McGiff for his helpful comments on the manuscript.

	Footnotes

 
This work was presented in preliminary form at the 66th Scientific Sessions of the American Heart Association, Atlanta, Ga, November 8-11, 1993.

Received September 23, 1994; first decision November 9, 1994; accepted February 16, 1995.

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