Renal actions of the angiotensin AT2 receptor ligands CGP 42112 and PD 123319 after blockade of the renin-angiotensin system

ELSEVIER

European Journal of Pharmacology 259 (1994) 27-36

Renal actions of the angiotensin A T 2 receptor ligands CGP 42112 and PD 123319 after blockade of the renin-angiotensin system David Macari, Steven Whitebread, Frederic Cumin, Marc De Gasparo, Nigel Levens

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Cardiovascular Research, K 125 9.08, Ciba-Geigy Ltd., Basel, CH 4002, Switzerland (Received 30 December 1993; revised MS received 22 March 1994; accepted 29 March 1994)

Abstract

The purpose of this study was to investigate whether the selective angiotensin AT e receptor ligands, CGP 42112B (Nic-Tyr-(N~-benzoyloxycarbonyl-Arg)Lys-His-Pro-Ile-OH)and PD 123319 ((s)-l-[[4-(dimethylamino)-3-methyl-phenyl]methyl]5-(diphenylacetyl)-4,5,6,7-tetrahydro-lH-imidazo[4,5-c]-pyridine-6-carboxylic acid) are agonists at angiotensin receptors influencing blood pressure and renal function in the enalaprilat-treated anesthetized rat. The agonist angiotensin II significantly increased blood pressure and renal vascular resistance. Glomerular filtration rate was unchanged by angiotensin II. Effective renal blood flow decreased significantly in response to angiotensin II leading to a significant increase in filtration fraction. Angiotensin II did not induce significant change in urinary potassium excretion or free water formation but significantly increased both urine volume and urinary sodium excretion. At doses up to 3 orders of magnitude greater than angiotensin II, CGP 42112B also significantly increased blood pressure, filtration fraction, glomerular filtration rate, urine volume and urinary sodium excretion, but did not significantly affect effective renal blood flow or renal vascular resistance. The selective angiotensin AT 2 receptor ligand PD 123319 had no significant effects on blood pressure nor any measured parameter of renal function. The changes in blood pressure and renal function produced by angiotensin II and CGP 42112B could be completely blocked by the angiotensin AT 1 receptor antagonist losartan. The results therefore only support a role for angiotensin AT 1 receptors and not angiotensin AT 2 receptors in the control of renal function in the rat and demonstrate that at high doses the angiotensin AT z selective ligand CGP 42112B behaves as an agonist at angiotensin AT 1 receptors.

Key words: Angiotensin AT 1 receptor; Angiotensin AT 2 receptor; Angiotensin II; Kidney

1. Introduction

While the majority of studies would suggest that the renal actions of angiotensin II are mediated by angiotensin A T 1 receptors, there have been several recent reports suggesting that selective angiotensin A T 2 receptor ligands such as PD 123177 and PD 123319 can affect renal function. (Cogan et al., 1991; Wong et al., 1991; Loutzenhiser et al., 1991; Bovee et al., 1991; Keiser et al., 1992; Clark et al., 1993; Mento et al., 1992). PD 123177 and PD 123319 are selective for the angiotensin A T 2 receptor subtype but only within a certain range of concentrations (Whitebread et al., 1991; Chiu et al., 1989). Since, in the aforementioned studies, PD 123177 and PD 123319 were used at very

* Corresponding author. Tel. 0041 61 696 4046, fax 0041 61 696 5808. Elsevier Science B.V.

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high doses and, in the majority of cases, produced effects qualitatively similar to angiotensin A T x receptor antagonists, the possibility exists that PD 123177 and PD 123319 influence renal function by interacting with the angiotensin ATx receptor subtype. Thus, a role for the angiotensin A T z receptor in the control of renal function has not been convincingly demonstrated. The hypothesis that the angiotensin A T 2 receptor plays a role in the control of renal function in the rat has recently been reassessed by investigating the actions of the selective angiotensin A T 2 ligands C G P 42112B and PD 123319 in the sodium-depleted rat (Macari et al., 1993). In these studies, PD 123319 was inactive but C G P 42112B influenced many parameters of renal function. Since the renal actions of C G P 42112B were observed only at high doses and resembled those produced by the A T 1 receptor antagonist losartan it was concluded that C G P 42112B affected

28

D. Macari et al. /European Journal of Pharmacology 259 (1994) 27 36

renal function primarily by blockade of the angiotensin AT~ receptor. At the present time it is unclear whether CGP 42112B and PD 123319 are agonists or antagonists at the angiotensin AT 2 receptor (Bottari et al., 1993; Levens et al., 1992; Brechler et al., 1993; Kambayashi et al., 1993; Ernsberger et al., 1992; Zhou et al., 1993; Madhun et al., 1993). If CGP 42112B and PD 123319 exert agonistic properties, it is not surprising that changes in renal function attributable to angiotensin AT 2 receptor modulation were not observed in the sodium-depleted rat (Macari et al., 1993). In the following experiments, the renal actions of CGP 42112B in comparison to PD 123319 were assessed after blockade of the renin-angiotensin system with enalaprilat. The renin-angiotensin system was blocked in this study to increase the sensitivity of blood pressure and renal function to possible agonist effects of CGP 42112B and PD 123319. The renal actions of CGP 42112B and PD 123319 were studied within a wide range of infusion rates that produced plasma concentrations of either compound compatible with a selective interaction with angiotensin AT 2 receptors only or with both angiotensin AT l and angiotensin AT 2 receptors.

2. Materials and methods

2.1. Animals These experiments were conducted with male albino Sprague-Dawley rats weighing between 300 and 360 g (Tif:RAIf (SPF), Ciba-Geigy animal breeding colony, Sisseln, Switzerland). After arrival at our animal facilities, the rats were housed in groups of 5 in sawdustlittered, wire-topped plastic cages (35 x 18 x 57 cm). The cages were maintained in a room with a 12 h light/dark cycle (6 a.m.-6 p.m. light) at a temperature of 20-24°C and monitored humidity. The animals were allowed free access to a normal diet (Nafag, Gossau, Switzerland) and given tap water to drink. Before use in the following experiments, each animal was acclimatized to the above conditions for at least 7 days to ensure a standard state of sodium intake and hydration.

2.2. Anesthesia On the day of experiment, each animal was placed into a small plastic chamber (25 x 20 X 18 cm) the interior of which was continually ventilated with a mixture of 4% gaseous halothane (Hoechst-Pharma, Zurich, Switzerland) in 96% oxygen. Once immobile, the rats were given Inactin (Byk, Gulden, Constance, Germany) intraperitoneally (100 m g/ kg in a 50 m g/ m l

solution). Owing to the length of the experimental protocols, and the long half life of Inactin in the rat, this initial dose of anesthetic was not supplemented further. Surgical anesthesia was determined by the lack of both blink and pedal reflexes.

2.3. Operative procedures Following anesthesia with Inactin each animal was placed, in dorsal recumbency, upon a metal operating table and kept at a temperature of 36_+ 0.5°C. The trachea, right common carotid artery, jugular and femoral veins were cannulated with polyethylene tubing (PP 0.240 and PP 0.50 as appropriate, Portex, Hythe, Kent, UK) containing heparinized saline (10 units/ml; Liquemine, Hoffmann-La Roche, Basel, Switzerland). The carotid catheter was connected through an Isotec 6PL pressure transducer (Healthdyne, Marietta, GA, USA) to a Hellige Servomed pen recorder (Hellige, Freiburg, Germany) enabling a continuous record of the mean arterial blood pressure to be obtained throughout the experiment. The jugular and femoral venous catheters were connected to 5 ml and 20 ml plastic syringes housed in two separate Precidor infusion pumps (Infors, Basel, Switzerland) respectively, enabling drug administration to the animals. The bladder was located through a suprapubic incision. A small incision into the lumen was made through the head of the bladder, which was subsequently cannulated with 10 cm of polyethylene tubing (PP 0.19, Portex). The bladder catheter was bent at the center into a 90° angle, to facilitate the collection of urine. To prevent the catheter from slipping from the bladder during the experimental period, the end inserted in the lumen was flanged.

2. 4. Experimental procedures Following the operative methods described above, each animal received a priming solution of p-aminohippuric acid (2 mg/ml; Nephrotest; Biologische Arbeitsgemeinschaft, Lich/Hessen, Germany) and inulin (50 mg/ml; Inutest; Laevosan International, Zurich, Switzerland) dissolved in isotonic saline. 1 ml/kg of the priming solution was administered via the femoral vein. In some experiments the priming solution also contained losartan (1 or 3 mg/ml). Immediately after the priming injection, p-aminohippuric acid (60 m g / kg/h), inulin (500 m g / k g / h ) and enalaprilat (1.1 m g / kg/h) dissolved in isotonic saline were infused via the femoral vein at a rate of 20 ml/min for the duration of the experimental period. Following a 2 h equilibration period, two consecutive 30 min clearance periods were determined. At the end of the second clearance period, the experimental animals received bolus injections of either angiotensin

D. Macari et al. / European Journal of Pharmacology 259 (1994) 27-36

II (0.7, 7.0, 20 or 70 ng/kg), CGP 42112B (1, 10, 100 or 1000/zg/kg), PD 123319 (1, 10, 100 or 1000 tzg/kg) or, in control animals, an equivalent volume of the isotonic saline vehicle (1 ml/kg) administered via the jugular vein. The bolus injection was followed in the case of the vehicle experiments by an infusion of isotonic saline (20/zl/min) administered via the jugular catheter and which continued for the duration of the experimental period. The injections of angiotensin II were immediately followed by infusions at (0.7, 7.0, 20 or 70 ng/kg/min). The injections of CGP 42112B and PD 123319 were immediately followed by infusion of either compound at the rate of 1, 10, 100 or 1000 p~g/kg/min. PD 123319 at 1000 ~ g / k g / m i n was dissolved in isotonic saline acidified with hydrochloric acid. There was an appropriate control group. Following an equilibration period of 15 min, there were two further consecutive 30 min clearance periods. In all experiments, urine was collected from the bladder catheter throughout each 30 min period into preweighed 5 ml polyethylene tubes. Urine volume was measured gravimetrically to an accuracy of +1 /zl. Blood samples (0.7 ml) were collected from the carotid catheter at the midpoint of each collection period. The volume removed was replaced with an equivalent quantity of blood obtained previously from donor animals. At the end of the experiment, 5 ml of blood was removed from the carotid catheter for the measure, ment of plasma losartan, CGP 42112B or PD 123319 concentrations (details below) and the animals were killed by opening the chest. Finally, the kidneys were removed, cleaned of adherent fat and weighed.

2.5. Experimental protocols The purpose of these experiments was to determine whether CGP 42112B and PD 123319 exert agonistic actions at angiotensin II receptors to influence blood pressure and renal function. To ensure that mainly agonistic effects were studied, the renin-angiotensin system was blocked by infusion of enalaprilat. In order to determine the subtype of receptor involved, the effects of angiotensin II and CGP 42112B on blood pressure and renal function were studied in the presence of the angiotensin AT 1 receptor antagonist losartan (1 and 3 mg/kg). Losartan was given as a single intravenous injection at the beginning of the experiment since it has a long biological half-life in the rat (Wong et al., 1990).

2.6. Chemical analysis p-Aminohippuric acid and inulin were measured by colorimetric methods (Brun, 1951; Schreiner, 1950). Urinary sodium and potassium concentrations were measured by flame photometry (AutoCal FP 743; In-

29

strumentation Laboratory, Milan, Italy) and urine and plasma osmolality by freezing point depression (Advanced Osmometer, Needham Heights, MA, USA).

2. 7. Measurement of the plasma levels of angiotensin H Animals were anesthetized and prepared with catheters in the jugular vein, carotid artery and bladder as described above. Enalaprilat (1.1 m g / k g / h ) was infused in isotonic saline via the jugular vein for 150 min. Control animals received isotonic saline at 20 /zl/min. At the end of the infusion period, a time corresponding to the end of the second clearance period, 5 ml of blood was drawn from the carotid artery into an ice-cold syringe containing 50 ~1 of an enzyme inhibitor solution used previously to prevent the production and breakdown of angiotensin II during blood collection (Wood et al., 1989). Following separation from cellular elements, the plasma was snap frozen in a mixture of dry ice and acetone and stored at -80°C until assay. Plasma angiotensin II concentrations were measured by radioimmunoassay as previously described, but without separation of angiotensins by high pressure liquid chromatography (Bunkenburg et al., 1991). The limit of detection of angiotensin II in the assay was 1 fmol/tube.

2.8. Measurement of the plasma levels of losartan, CGP 42112B and PD 123319 2.8.1. Blood collection In all experiments, 5 ml blood for the measurement of losartan, CGP 42112B or PD 123319 was withdrawn from the carotid catheter at the termination of each experiment. During blood collection, the animals continued to receive the infusion of CGP 42112B or PD 123319 or the isotonic saline vehicle. To prevent the breakdown of the peptidic structure of CGP 42112B, blood samples were collected into ice-cold tubes containing 50 tzl of the enzyme inhibitor solution referenced above. For consistency, plasma for the measurement of losartan and PD 123319 was also collected into this inhibitor solution. Following separation from cellular elements, the plasma was snap frozen in a mixture of dry ice and acetone and stored frozen at -80°C until assayed. 2.8.2. Measurement of losartan, CGP 42112B and PD 1233319 Plasma concentrations of losartan, CGP 42112B and PD 123319 were measured by radioreceptor assay as previously described, but with minor modifications (Whitebread et al., 1989). Losartan was measured using membranes of rat vascular smooth muscle cells which express angiotensin AT 1 receptors only. CGP 42112B and PD 123319 were measured using mem-

30

D. Macariet al. / European Journal of Pharmacology 259 (1994) 27-36

brane preparations of PC12W cells which express angiotensin AT 2 receptors only. [125I][Sarl,IleS]Angiotensin lI (2200 Ci/mmol; Anawa, Wangen, Switzerland) was used in the assay as the displaced tracer. Standard curves were constructed by incubating 175/zl of a membrane preparation (50-100 /zg protein/ml) of PC12W cells or cultured rat aortic smooth muscle cells with 25 /zl of the radio-iodinated ligand (0.175 nM in the final assay), various concentrations of an appropriate standard (CGP 42112B, PD 123319 in the angiotensin AT 2 receptor assay and losartan in the angiotensin AT 1 receptor assay - 25 ~l) and 25 tzl of control plasma. The standards were dissolved in buffer (Tris-HC1 50 mM pH 7.4, NaC1 125 mM, MgC12 6.5 mM, Na2-EDTA 1 raM) containing 10% dimethyl sulfoxide. Plasma samples were diluted appropriately (1 : 10, 1 : 100 or 1 : 1000) in buffer (TrisHCI 50 mM, pH 7.4, NaC1 125 mM, MgC12 6.5 mM, Naz-EDTA 1 mM). Samples were measured in the same way, except that the standards were replaced with the same volume of diluted plasma and 25/xl of buffer containing 10% dimethyl sulfoxide. The incubation, 2 h at 25°C, was terminated by the addition of 2 ml ice-cold phosphate buffered saline (pH 7.4). Bound radioactivity was immediately separated from free by passing through Whatman G F / F (Whatman, Maidstone, UK) filters coated with bovine serum albumin (2 mg/ml). The filters were rinsed twice with 4 ml ice-cold phosphate buffered saline (pH 7.4) and trapped radioactivity measured in a gamma counter (1277 Gammamaster, Wallac, Finland). The quantities of losartan, CGP 42112B or PD 123319 present in the plasma samples were determined from the appropriate standard curve made at the same plasma dilution. The detection limits of the assay, as defined by the quantity of standard required to reduce binding of the radioiodinated ligand by 10%, were 1.0 nM for CGP 42112B, 10.0 nM for PD 123319 and 100 nM for losartan.

saline. All solutions of PD 123319 were prepared in isotonic saline, except for the highest dose administered which was acidified with hydrochloric acid. Enalaprilat was obtained form Merck, Sharp & Dohme Research Laboratories, West Point, PA, USA. 2.10. Expression o f data

Blood pressure was taken as the average of this parameter over each clearance period. Effective renal blood flow and glomerular filtration rate were determined as the clearance of p-aminohippuric acid and inulin, respectively. Filtration fraction, renal vascular resistance, urinary sodium and urinary potassium excretion and free water formation were calculated using standard formulae. In all experiments, the first two and last two clearance periods were averaged together to give two single values. The second average clearance value was subtracted from the first average clearance value to give, in each case, the change produced by drug or vehicle. These values are shown in the figures and were used for data analysis. Data from several experiments are grouped together and expressed as the mean and S.E. Control experiments for the angiotensin II, CGP 42112B and PD 123319 experiments were performed in the same way but without administration of the drug. 2.11. Statistical analysis

Statistical analysis was by one-way analysis of variance. Significant values of each performed analaysis of variance were followed by Scheffe's test or Tukey's honest significant difference test for unequal sample groups to determine the significance of the change produced by each dose of drug to control values (Armitage and Berry, 1987). In all analyses P < 0.05 was regarded as being statistically significant.

2. 9. Drugs used

The angiotensin AT 1 receptor antagonist losartan (2-butyl-4-chloro-l-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]4-yl]methyl]-lH-imidazole-5-methanol) and the angiotensin AT e receptor ligand PD 123319 ((s)-l-[[4-(dimethylamino)-3-methyl-phenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-lH-imidazo[4,5-c]-pyridine-6carboxylic acid) were synthesized by Ciba-Geigy. The angiotensin AT 2 receptor ligand CGP 42112B (NicTyr-(N"-benzoyloxycarbonyl-Arg)Lys-His-Pro-Ile-OH) is the trifluoroacetate salt of CGP 42112 and was synthesized for Ciba-Geigy by Neosystem, Strasbourg, France. Angiotensin II amide (Hypertensin) was synthesised by Ciba-Geigy. All solutions of angiotensin II, losartan, and CGP 42112B were prepared in isotonic

3. Results

3.1. Effect of enalaprilat on the plasma levels of angiotensin H

Plasma angiotensin II concentrations, at a time corresponding to the end of the second clearance period, averaged 40.3 + 17.4 fmol/ml (n = 6). Following 150 min of enalaprilat infusion (1.1 m g / k g / h ) plasma angiotensin II concentrations were reduced to the detection limits of the assay (1 fmol/tube; n = 6). This small quantity of angiotensin II immunoreactivity remaining after enalapril treatment probably represents background since the same low levels of angiotensin II

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C G P 42112B d i d n o t significantly a l t e r effective r e n a l b l o o d flow o r r e n a l v a s c u l a r r e s i s t a n c e b u t significantly i n c r e a s e d f i l t r a t i o n f r a c t i o n at infusion r a t e s o f 100 a n d 1 0 0 0 / ~ g / k g / m i n . A t a n infusion r a t e of 1000 /zg/kg/min, C G P 42112B significantly i n c r e a s e d g l o m e r u l a r filtration rate. I n c o n t r a s t to t h e effects o f a n g i o t e n s i n II a n d C G P 42112B, P D 123319 h a d no significant effects on any m e a s u r e d p a r a m e t e r of r e n a l h e m o d y n a m i c s .

10-4 10-3 102 10-1 100 101 102 103 log infusion rate (llg/kg/min)

Fig. 1. Effect of angiotensin II, CGP 42112B and PD 123319 on blood pressure after blockade of the renin-angiotensin system. Results are expressed as mean + S.E. Angiotensin II (open squares); CGP 42112B (black squares); PD 123319 (open diamonds). Horizontal broken line and accompanying shading are the mean + S.E. of the control group of experiments (n = 8). The number of animals in each treatment group is shown in parentheses. Statistics by analysis of variance followed by Scheffe's test. Significance values (* P < 0.05) are relative to controls. In some cases the S.E. bars are smaller than the accompanying symbols.

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3.2. Effect of angiotensin II, CGP 42112B and PD 123319 on blood pressure after blockade of the reninangiotensin system (Fig. 1) Angiotensin II produced a dose-dependent increase in b l o o d p r e s s u r e which was significantly d i f f e r e n t f r o m c o n t r o l v a l u e s with infusion r a t e s o f 20 a n d 70 n g / k g / min. A l t h o u g h c o n s i d e r a b l y less p o t e n t t h a n ang i o t e n s i n II, C G P 42112B also p r o d u c e d a d o s e - d e p e n d e n t i n c r e a s e in b l o o d p r e s s u r e which was significantly d i f f e r e n t f r o m c o n t r o l v a l u e s with infusion r a t e s o f 100 a n d 1 0 0 0 / z g / k g / m i n . In c o n t r a s t to a n g i o t e n s i n II a n d C G P 42112B, P D 123319 d i d n o t significantly affect b l o o d p r e s s u r e .

3.3. Effect of angiotensin II, CGP 42112B and PD 123319 on renal hemodynamics after blockade of the renin-angiotensin system (Fig. 2)

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A n g i o t e n s i n II d i d n o t significantly affect g l o m e r u lar f i l t r a t i o n rate, b u t significantly d e c r e a s e d effective r e n a l b l o o d flow at an infusion r a t e o f 70 n g / k g / m i n . B o t h r e n a l v a s c u l a r r e s i s t a n c e a n d filtration f r a c t i o n i n c r e a s e d d o s e - d e p e n d e n t l y . T h e i n c r e a s e in r e n a l vasc u l a r r e s i s t a n c e p r o d u c e d by a n g i o t e n s i n II was significantly d i f f e r e n t f r o m c o n t r o l v a l u e s at infusion r a t e s o f 20 a n d 70 n g / k g / m i n , while f i l t r a t i o n f r a c t i o n r e a c h e d statistical significance f r o m c o n t r o l v a l u e s at an infusion r a t e of 70 n g / k g / m i n .

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Fig. 2. Effect of angiotensin II, CGP 42112B and PD 123319 on renal hemodynamics after blockade of the renin-angiotensin system. Resuits are expressed as mean+S.E. Angiotensin II (open squares); CGP 42112B (black squares); PD 123319 (open diamonds). Horizontal broken line and accompanying shading are the mean + S.E. of the control group of experiments. The number of animals in each treatment group is the same as in Fig. 1. Statistics by analysis of variance followed by Scheffe's test, Significance values (* P < 0.05) are relative to controls.

D. Macariet al. / European Journal of Pharmacology 259 (1994) 27-36

32

3.4. Effect o f angiotensin II, CGP 42112B and PD 123319 on the urinary excretion of water and electrolytes after blockade o f the renin-angiotensin system (Fig. 3)

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Fig. 3. Effect of angiotensin II, CGP 42112B and PD 123319 on urine volume and urinary electrolyte excretion after blockade of the reninangiotensin system. Results are expressed as mean+S.E. Angiotensin II (open squares); CGP 42112B (black squares); PD 123319 (open diamonds). Horizontal broken line and accompanying shading are the mean +S.E. of the control group of experiments. The number of animals in each treatment group is the same as in Fig. 1. Statistics by analysis of variance followed by Scheffe's test. Significance values (* P < 0.05) are relative to controls.

Like a n g i o t e n s i n II, C G P 42112B also p r o d u c e d a d o s e - d e p e n d e n t increase in b o t h u r i n e v o l u m e a n d u r i n a r y s o d i u m excretion. T h e increase in u r i n e v o l u m e p r o d u c e d by C G P 42112B was significantly different from control values at i n f u s i o n rates of 100 a n d 1000 / x g / k g / m i n while the increase in s o d i u m excretion p r o d u c e d by C G P 42112B was significantly different from control values at infusion rates b e t w e e n 10 a n d 1 0 0 0 / z g / k g / m i n . C G P 42112B, again like a n g i o t e n s i n

D. Macari et al. / European Journal of Pharmacology 259 (1994) 27-36

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II, did not significantly affect the renal excretion of potassium or the formation of free water. When infused at doses between 1 and 1 0 0 0 / z g / k g / min, PD 123319 had no significant effect on any measured parameter of urinary excretion.

3.5. Effect of losartan on changes in blood pressure and renal function produced by angiotensin H and CGP 42112B following blockade of the renin-angiotensin system (Fig. 4) In these studies the ability of the angiotensin AT 1 receptor antagonist losartan to inhibit the effects of angiotensin II and CGP 42112B on blood pressure and renal function were assessed. The pressor and renal

actions of the highest doses of angiotensin II (70 n g / k g / m i n ) and CGP 42112B (1000 m g / k g / m i n ) studied are presented in Fig. 4. Doses of 1 and 3 mg/kg losartan progressively attenuated the pressor and renal actions of angiotensin II. All of the effects of angiotensin II were completely blocked by 3 mg/kg losartan while all of the effects of CGP 42112B were completely blocked by 1 mg/kg losartan.

3.6. Relationship between administered dose and the plasma concentrations of losartan, CGP 42112B and PD 123319 (Fig. 5) The results illustrate that a linear relationship exists between the injected dose of losartan or the infusion

Table 1 Baseline parameters of renal function after blockade of the renin-angiotensin system

No losartan 1 mg/kg iosartan 3 mg/kg losartan P

Blood Effective renal pressure blood flow (mmHg) ( m l / m i n / g kidney)

Renal vascular resistance (mm H g / m l / m i n / g kidney)

Glomerular Filtration Urine filtration rate fraction volume (ml/min/g (%) (/~l/min kidney) 100 g)

96 + 14 97 + 13 94 + 13 N.S.

2.0 + 0.8 2.1 + 0.7 1.8 5- 0.8 N.S.

1.1 + 0.3 1.1 + 0.3 1.0 + 0.2 N.S.

8.0 5- 1.8 8.0 5- 1.8 7.2 5- 1.8 N.S.

Urinary sodium excretion (~,mol/min/ 100 g)

27.8 + 7.1 11.6 + 5.9 1.7 + 1.0 27.6 5- 4.1 8.0 5- 5.8 1.1 5- 1.0 27.5 + 4.6 11.9 + 6.1 1.2 + 1.1 N.S. N.S. N.S.

Urinary potassium extretion (/~mol/min/ 100 g)

Free water formation (~,lmin)

0.87 + 0.21 0.81 + 0.11 0.84 5- 0.17 N.S.

62.2 + 20.3 58.8 + 14.1 63.9 5- 16.5 N.S.

For each experiment, the first two clearance periods were averaged together to form a single value. For each major series of experiments (no losartan, 1 mg/kg and 3 mg/kg losartan) all of the single values are collected together in the table and expressed as mean 5- the standard deviation. By ANOVA followed by the Tukey honest significant difference test for unequal sample groups, there were no significant differences between the three series of experiments. Furthermore, using the same statistical procedures there were no significant differences between the groups that comprise the three major experimental series.

34

D. Macariet al. / European Journal of Pharmacology 259 (1994) 27-36

rates of both CGP 42112B and PD 123319 and the plasma levels of each compound. 3. 7. Basal blood pressure and parameters of renal function in enalaprilat treated animals after blockade of the renin-angiotensin system (Table 1) The average of the first two control periods for each measured parameter obtained in animals either untreated or treated with losartan is shown in Table 1. Each parameter lies within the normal range obtained in our laboratory. Neither 1 m g / k g nor 3 m g / k g losartan significantly affected baseline values of renal function.

4. Discussion

The purpose of this study was to determine whether the selective angiotensin A T 2 receptor ligands CGP 42112B and PD 123319 have agonistic properties at angiotensin II receptors that influence blood pressure and renal function in the anesthetized rat. Previous studies have shown that angiotensin II receptors exist as two major subtypes, termed A T 1 and A T 2 (Whitebread et al., 1989; Chiu et al., 1989; Levens et al., 1992; Bottari et al., 1993). The selective angiotensin AT1 receptor antagonist losartan displays a high affinity for the angiotensin A T 1 receptor ( K i ~ 16 nM) but a low affinity ( K i > 100 /.~M) for the angiotensin A T 2 receptor. In contrast, both the peptide ligand CGP 42112B and the non-peptide ligand PD 123319 have a high affinity ( K i ~ 0.1 nM and 12 nM respectively) for the angiotensin A T E receptor but a low affinity for the angiotensin A T 1 receptor subtype ( K i ~ 2 /zM and > 1 0 0 / z M respectively). Angiotensin II has both high and equal affinity for the angiotensin A T l ( K d 0.5 nM) and angiotensin A T 2 (Kd 0.5 nM) receptor subtypes. In order to effectively study the agonistic properties of CGP 42112B and PD 123319, the endogenous renin-angiotensin system was blocked with enalaprilat. Enalaprilat resulted in an essentially complete blockade of the renin-angiotensin system, since plasma angiotensin II levels were reduced to the detection limits of the assay and the subsequent administration of doses of losartan up to 3 m g / k g did not significantly affect baseline blood pressure or parameters of renal function. Initial experiments defined the pressor and renal effects of angiotensin II and the angiotensin A T 2 selective ligands CGP 42112B and PD 123319. Angiotensin II served as a reference agonist against which the actions of CGP 42112B and PD 123319 were compared. Angiotensin II significantly increased blood pressure and renal vascular resistance in a dose-de-

pendent manner while tending to decrease effective renal blood flow. When threshold doses are compared, infusion rates of CGP 42112B approximately 3 orders of magnitude higher than angiotensin II were needed to significantly increase blood pressure. Although renal vascular resistance tended to increase in response to the highest infusion rates of CGP 42112B, neither this parameter nor effective renal blood flow changed significantly. CGP 42112B is thus a considerably less potent systemic and renal vasoconstrictor than angiotensin II. Glomerular filtration rate was unaltered in response to angiotensin II while filtration fraction increased. CGP 42112B increased filtration fraction and, at 1000 / x g / k g / m i n , also increased glomerular filtration rate. These findings may suggest that both angiotensin II and CGP 42112B change renal hemodynamics by preferentially constricting the efferent arteriole (Blantz and Gabbi, 1987). The reason why CGP 42112B and angiotensin differentially affect glomerular filtration rate is unknown at the present time. Within the range of infusion rates studied, angiotensin II increased urinary volume and urinary sodium excretion in the absence of a change in glomerular filtration rate. The increase in sodium excretion and urine volume was observed only at infusion rates of angiotensin II which increased blood pressure, suggesting the involvement of pressure natriuresis and diuresis mechanisms (Khraibi et al., 1989; Mizelle et al., 1992). Inhibition of tubular sodium transport may also be a contributing factor (Harris and Navar, 1985). CGP 42112B also increased urine volume and urinary sodium excretion at pressor doses, which in the case of the highest dose of CGP 42112B infused, could also be explained by an increase in glomerular filtration rate. CGP 42112B when infused at 1 0 / z g / k g / m i n increased sodium excretion without significant changes in glomerular filtration rate or blood pressure. This observation could be explained by inhibition of tubular transport although an influence on extrarenal mechanisms cannot be ruled out. Although CGP 42112B is highly selective, it also binds to angiotensin A T 1 receptors at concentrations above 1/xM. Thus at high infusion rates, a progressive interaction of CGP 42112B with angiotensin A T 1 receptors would be expected. Considering the relative affinity of CGP 42112B for angiotensin A T 1 and angiotensin A T 2 receptors and assuming that plasma levels after 90 min of infusion are equal to the concentration of this peptide at angiotensin II receptors, then infusion of all doses of CGP 42112B would be compatible with saturation of angiotensin A T 2 receptors (Whitebread et aI., 1989; Chiu et al., 1989). However, at 1 0 0 0 / z g / k g / m i n corresponding to a plasma concentration of 7 / x M , CGP 42112B would be expected to saturate more than 80% of the angiotensin A T 1 recep-

D. Macari et al. / European Journal of Pharmacology 259 (1994) 27-36

tor population. Thus, C G P 42112B may produce changes in blood pressure and renal function by interacting with angiotensin AT~ receptors. In this regard, the pressor and renal effects of C G P 42112B could be completely blocked by a dose of 1 m g / k g losartan (plasma concentration 1 ~M). Considering the relative affinity of losartan for angiotensin A T t and angiotensin A T 2 receptors and again assuming that plasma levels are equal to the concentration of the compound at angiotensin II receptors, then at 1 / z M , losartan would totally saturate angiotensin A T 1 receptors (Whitebread et al., 1989; Chiu et al., 1989) but would not bind to angiotensin A T 2 receptors. Thus it appears that the increase in blood pressure and changes in renal function produced by C G P 42112B are due entirely to angiotensin A T 1 receptor stimulation. The pressor and renal actions of angiotensin II in this study were also blocked by a dose of 3 m g / k g losartan which produced a plasma concentration of 10 /~M. At this plasma concentration, losartan would not be expected to bind to more than 5% of the angiotensin A T 2 receptor population. Thus the pressor and renal actions of angiotensin II must be primarily due to angiotensin A T t receptor stimulation, a conclusion shared by others (Cogan et al., 1991; Wong et al., 1991; Loutzenhiser et al., 1991; Bovee et al., 1991; Clark et al., 1992). The results of the present studies therefore demonstrate that the pressor and renal effects of angiotensin II and C G P 42112B are mediated by angiotensin AT~ receptors. The reason why neither PD 123319 nor C G P 42112B produced effects on blood pressure and renal function attributable to an interaction with angiotensin A T 2 receptors in the present studies could have two explanations. Either C G P 42112B and PD 123319 are not agonists at angiotensin A T 2 receptors or angiotensin A T 2 receptors do not play an important role in modulating blood pressure and renal function. Previous studies conducted by our group shed light on these two possibilities. In the anesthetized sodium-depleted rat neither C G P 42112B nor PD 123319 had effect on blood pressure and renal function at doses compatible with a selective interaction with angiotensin A T 2 receptors (Macari et al., 1993). Thus, when studied under conditions compatible with the expression of either agonistic or antagonistic properties at angiotensin A T 2 receptors, neither C G P 42112B nor PD 123319 affected blood pressure or renal function. These observations would suggest that the angiotensin A T 2 receptor does not play an important role in the control of blood pressure or renal function, at least in the anesthetized rat. This conclusion is also supported by observations in the present studies where the pressor and renal effects of angiotensin II were blocked by a selective dose of losartan. U n d e r these conditions, angiotensin

35

A T 2 receptors would" be open to stimulation by angiotensin II but no changes in blood pressure and renal function were observed. Two important observations emerge from these studies. Firstly, when taken in aggregate with previous studies from our group (Macari et al., 1993) the present observations are compatible with the conclusion that angiotensin A T 2 receptors do not exert a controlling influence on blood pressure and renal function in the anesthetized rat. Secondly, C G P 42112B is increasingly used in studies to investigate the physiological role of angiotensin A T 2 receptors. However, it is apparent that C G P 42112B interacts significantly with the angiotensin A T 1 receptor and acts as an agonist at the angiotensin A T t receptor when the renin-angiotensin system is blocked. Conversely, when the renin-angiotensin system is activated, C G P 42112B acts as an antagonist of the angiotensin A T t receptor (Macari et al., 1993). Since C G P 42112B has the properties of a partial agonist at angiotensin A T t receptors this may also be the case at angiotensin A T 2 receptors. Considerable care must therefore be taken in the interpretation of studies with this compound where an interaction with the angiotensin A T 1 receptor has not been rigorously excluded and the possibility that this compound may be a partial agonist at angiotensin A T 2 receptors considered.

Acknowledgments The authors are indebted to Mr. Rene Gasser, Mr. Karl Pfeiffer, Ms. Monique Burgunder, Ms. Dominique Monna and Ms. Michele Mele for their excellent technical assistance during the performance of these studies.

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