Vasopressor and depressor effects of native angiotensins and inhibition of these effects in the Japanese quail

GENERAL AND COMPARATIVE ENDOCRINOLOGY 79, 12-22 (1990)

Vasopressor and Depressor Effects of Native Angiotensins and Inhibition of These Effects in the Japanese Quail

YOSHIO TAKEI AND YUKIO HASEGAWA*

Department of Physiology, Kitasato Universib School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228, and *Department of Clinical Chemistry, School of Pharmaceutical Sciences, Toho University, 2-2-l

Miyama, Funabashi, Chiba 274, Japan

Accepted July 25, 1989

The amino acid sequence of angiotensin I (ANG I) from the Japanese quail, Coturnix coturnixjaponica, obtained from incubation of homologous plasma and kidney extract was determined as H-Asp-Arg-Val-Tyr-Val-His-Pro-PheSer-Leu-OH. A bolus, intravenous injection of native ANG I or of ANG II induced an immediate vasodepressor response and a subsequent vasopressor response in quail which has been lightly anesthetized with urethane (0.75 gikg). The values for ED,, for the vasopressor and depressor effects of ANG II were 85 and 113 pmoVlO0 g body weight, respectively. The extent of the hypotension was dependent on the arterial pressure prior to injection. The effects of ANG I and II on heart rate were variable. Human [Asp’, Ile5, Hiss] ANG I and II were almost as potent as their quail counterparts with respect to the cardiovascular effects, but eel [Asn’, Val’, Gly? ANG I and II were less than half as potent. Human ANG III had little effect on arterial pressure in the quail. A bolus injection (108 l.&30 g) or infusion (1 ~&100 g/min) of [Sar’, Be*] ANG II almost abolished the cardiovascular effects of ANG I and II, but [Sar’, Alas] ANG II and [Sar’, Thrs] ANG II, which are effective inhibitors in mammals, had no inhibitory effects. The vasopressor and depressor effects of ANG I were abolished, while those of ANG II were slightly enhanced, after injection (100 l&O0 g) or infusion (1 l@OO g/min) of SQl4225, whereas des-Pro’ bradykinin and bradykinin potentiator B, which are effective inhibitors of ANG I converting enzyme in mammals, failed to inhibit the effect of ANG I. These results indicate that vascular ANG II receptors and ANG I converting enzyme in the quail may be different from those in mammals. o 1990 Academic press, I~C.

Angiotensin II (ANG II) given exoge- nously increases blood pressure in representative species of all classes of vertebrates (Nishimura, 1980). However, ANG II causes an immediate vasodepressor response prior to the vasopressor response in fowl (Moore er al., 1981a; Nishimura et al., 1982; Nakamura et al., 1982). Since only a vasopressor response has been observed after intravenous injection of ANG II in the duck (Wilson and Butler, 1983) and the pigeon (Evered and Fitzsimons 1981), it is not known whether or not the vasodepressor action of ANG II is a general phenomenon in avian species. In recent years, variants of ANG I from representative species of all classes of nonmammalian vertebrates have been sequenced and synthesized (Khosla et

al., 1983, 1985; Hasegawa et al., 1984), but, in the case of birds, only fowl ANG I has been sequenced. Thus, it appears to be of interest to determine the amino acid sequence and synthesize ANG I from another avian species, and, using the native angiotensins, to examine whether a vasodepressor response is a unique phenomenon in fowl or not.

A number of blockers of the renin- angiotensin system have been utilized in mammals to evaluate the physiological role of this system (Regoli er al., 1974; Peach, 1977). Those blockers that are frequently used include competitive inhibitors of ANG II and inhibitors of ANG I converting enzyme. These inhibitors have been used in nonmammalian vertebrates with the as-

12 0016~6480/90 $1.50 Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved

CARDIOVASCULAR EFFECTS OF ANGIOTENSINS 13

sumption that they are also effective in these animals. However, since ANG II receptors and ANG I converting enzyme of nonmammalian species may differ from those of mammals (Nishimura et al., 1978, 1982; Harper and Stephens, 1985; Takei, 1987; Takei et al., 1988), it remains to be determined whether or not these inhibitors which are active in mammals are actually effective in nonmammalian species.

In the first experiment of this study, we attempted to determine the amino acid sequence of quail ANG I. Since it was found that quail ANG I had the same amino acid sequence as fowl ANG I, the effects of quail (native), human, and eel ANG I and II on arterial pressure and heart rate were examined in the anesthetized quail. We used a small dose of urethane as an anesthetic, because this drug has been shown to exert a minimal influence on cardiovascular pa- rameters (Maggi and Meli, 1986). Finally we analyzed the effects of competitive inhibitors of ANG II and inhibitors of ANG I converting enzyme on the cardiovascular effects of ANG I and II. These experiments will help to characterize the ANG II receptors and converting enzyme of the quail, and they will provide information on effective inhibitors that will be useful for future experiments designed to evaluate the physiological role of the renin-angiotensin system in this bird.

MATERIALS AND METHODS

Animals

Male Japanese quail, Coturnix coturnix japonica, aged 4 weeks, were purchased from a local dealer (Su- zukei, Toyohashi). They were kept individually in small wire cages (21 X 12 x 17 cm3) under a 8L:16D photocycle at 25 + 1” for more than 2 weeks before use. Quail diet containing 150 meq/kg of Na (w/w) (Nippon Haigo Shiryo, Yokohama) and tap water were freely available until the day of experiment. The birds weighed 107 f 2mg (mean 2 SD, n = 42) at the time of experiment. In addition, more than 25 birds were used as a source of blood and kidney tissues for determination of the amino acid sequence of quail ANG I.

Drugs

The ANG I and II used were [Asp’, Va15, SeP] ANG I, and its ANG II, which have been shown to be native to the quail in this study, human [Asp’, Be’, Hiss] ANG I and its ANG II and III, and eel [Asn’, Va15, Gly? ANG I and its ANG II (Hasegawa ef al., 1983a; Khosla et al., 1985). Eel ANG I was synthesized and generously provided by Dr. Mahesh C. Khosla, De- partment of Brain and Vascular Research, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio. Competitive inhibitors of ANG II used were [Sar’, Ala*] ANG II, [Sari, Thrs] ANG II, and [Sar’, Ilea] ANG II. Inhibitors of ANG I converting enzyme used were des-Pro’ bradykinin (BK) (Naruse et al., 1981) and BK potentiator B, which is an undecapep tide obtained from the venom of Agkistrodon halys blomhofii (Kato and Suzuki, 1971), and SQ14225. SQ14225 (Captopril) was generously donated by San- kyo Pharmaceutical Co. Ltd., Tokyo. All peptides were purchased from the Peptide Institute Inc., Osaka, and other drugs were purchased from Wako Pure Chemicals, Tokyo. All peptides were dissolved in 0.9% NaCl, and stock solutions (100 nmol/ml for angiotensins and 2 mg/ml for inhibitors) were stored at - 25” until use.

Measurement of Blood Pressure

The birds were lightly anesthetized by an intramus- cular injection of urethane (0.75 g/kg) in the breast region and placed on their side on operating boards. The neck of each bird was lightly fuced between nails, and the body was wrapped gently with a cylindrical cloth. After tracheotomy, a polyethylene catheter (PElO, Clay Adams) was inserted into the right atrium through the right, external jugular vein for injections. Another polyethylene catheter was inserted into the right, common carotid artery for measurement of arterial pressure. Care was taken not to damage the va- gus nerve and other nerves that innervate the carotid sinus. Blood loss was slight during the surgery. Be- cause the anesthesia was so light, the birds moved occasionally during measurements. Thus, we utilized a system for measurement of blood pressure that allowed the birds to move a little without the cannula slipping out of the artery. The details of the apparatus for the measurements have been reported previously (Takei and Watanabe, 1990).

Determination of Amino Acid Sequence of Quail ANG Z

Collection of blood and kidney tissue. Two millili- ters of blood was withdrawn directly from the jugular vein of conscious quail into a chilled syringe which contained 20 pJ of 15% ammonium EDTA. Five days thereafter, 2 ml of blood was again withdrawn in the same way. Soon after the second withdrawal of blood, birds were decapitated and the kidneys were removed,

14 TAKE1 AND HASEGAWA

weighed, and stored frozen at -25” until use. Col- lected blood was centrifuged at 2700s for 15 min at 4”, and the plasma was stored at - 25” until use.

Preparation of crude angiotensin. Crude angiotensin was prepared by the modiied version of Boucher’s procedure, as reported previously (Hasegawa ef al., 1983b). Kidney tissue (13.7 g) was homogenized with 14 ml of 5.8 mM 2Na-EDTA and centrifuged at 18,OOOg for 15 min at 2-4”. The supematant was dialyzed against 5.8 mM 2Na-EDTA at 4” for 24 hr. The dialysate was then acidified to pH 3.0 with 1 M HCl, and kept at 4” for 30 min. After neutralization, the kidney extract was centrifuged at 18,OOOg as above. The supematant (14 ml), plasma dialyzed against 5.8 mM 2Na-EDTA (55 ml), and 2.5% phenylmethyl sul- fonyl fluoride (0.5 ml) were incubated together with Dowex 5OW-X2 resin (PH 7.4, NH,-form) at 20” for 20 hr and loaded onto a column that contained Dowex 5OW-X2 resin (PH 6.0, NH,-form). The column was washed with 0.2 M ammonium acetate-acetic acid (pH 6.0), 10% acetic acid, and water. The active principle was eluted with 0.1 M diethylamine and 0.5 M ammonium hydroxide. The eluate was evaporated to dry- ness, and the activity was determined by the vasopressor bioassay in the quail with synthetic [Asp’, Val’, SeP] ANG I as a standard.

Purification and analysis of quail angiotensin. Crude angiotensin was suspended in 0.1% formic acid and passed through a Sep-Pak C,, cartridge (Waters Associates) for desalting. The active principle was eluted with 5% methanol, dried, and bioassayed as above to assess recovery. The dried residue was then dissolved in 0.05 M ammonium fonnate-formic acid (pH 3.0) and loaded onto a column of SP-Sephadex C-25 (26.5 x 0.9 cm). The active principle was eluted with a linear gradient from 0.05 M ammonium for- mate-formic acid (pH 3.0) to 0.5 M ammonium for- mate @H 6.5), and each fraction was bioassayed as above. The fraction with vasoactivity was desalted on a Sep-Pak C,s cartridge and eluted with 8% methanol that contained 0.05% tritluoroacetic acid (TFA). The dried material was further purified by high-performance liquid chromatography (HPLC) on a column of TSKgel ODS 80 TM (4.6 mm i.d. x 30 cm, Tosoh, Tokyo) using an eluent composed of methanol, water, and TFA (430:570:1) at a flow rate of 0.5 ml/mitt, and each fraction was bioassayed. The active principle gave the same retention time (52.8 mitt) as that of synthetic [Asp’, Val’, SeP] ANG I and was found to be homogeneous by high-performance thin-layer chromatography on a plate of silica gel 60 (Merck). Finally, the amino acid sequence of the quail angiotensin was analyzed by stepwise Edman degradation using a gas- phase automated protein sequencer (Applied Biosys- terns, Model 470A) coupled to an HPLC system for identification of the phenylthiohydantoin-derivatized amino acids produced (Applied Biosystems, Model 120 PTH analyzer).

Experimental Protocols for Physiological Studies

Relative pofency of angiotensins. [Asp’, Val’] ANG II, which is native to the quail, was used as the standard angiotensin. In order to examine their relative potencies, quail, human, and eel ANG I and II and human ANG III were injected into birds over 10 set at doses of 0, 5, 10, 20, 50, 100, 200, and 500 pmol/lOO g in a volume of 50 pl. Each injection was followed by flushing of the cannula (dead space: 10 pl) with 30 ~1 of 0.9% NaCl. Each bird received different doses of angiotensins once, and the order of injection of angiotensins and doses was determined by the Latin squares method. The injections were repeated after the arterial pressure returned to the level recorded prior to the injections, because repeated injections of 100 pmol/lOO g of quail ANG II in this way generated reproducible effects. BK was also injected after the last injection of angiotensin at a dose of 500 pmoYlO0 g. At the begin- ning and end of injections, 100 pmoYlO0 g of quail ANG II was injected to confii the reproducibility of the response. The average decrease in the response at the end was 4.5 2 2.1% (n = 9).

Effect of inhibitors. The values of ED, of quail ANG I and II determined in the above experiment were used to examine the effects of inhibitors. The injection volume of angiotensin and inhibitors was 50 ~1. The preliminary experiment indicated that [Sari, Iles] ANG II and SQ14225 appear to be the only effective inhibitors of the vasopressor and depressor effects of ANG II and ANG I, respectively. Therefore, in the first experiment, quail ANG II was injected 5 min before and 5 min after injection of 0.9% NaCl or 100 cLg/lOO g of the inhibitors in the following order: [Sari, Ala*] ANG II, [Sar’, Thrs] ANG II, [Sar’, Ile’] ANG II, and SQ14225, in six quail. After injection of [Sar’, Ile’] ANG II, injection of quail ANG II was repeated every 5 min until its effect was fully restored. In the next experiment, quail ANG I was injected 5 min before and 5 min after injection of 0.9% NaCl or 100 pg/lOO g of [Sari, Ile’] ANG II, des-Pro* BK, BK potentiator B, and SQ14225, in that order, in another six quail. des-Pro* BK was injected 30 min after injection of [Sar’, Ile’] ANG II, and the injection of ANG I was given every 30 min for up to 2 hr after injection of SQ14225.

We also examined the effects of slow infusion of the inhibitors at a rate of 1 ug/lOO g/l0 pl/min following a priming injection of 5 pg/lOO g in 50 ~1. The injection of quail ANG II was made more than 15 min after the start of infusion of 0.9% NaCl, [Sari, Ala’] ANG II, [Sar’, Th?] ANG II, [Sar’, Ile’] ANG II, or SQ14225 in six birds, and the injection of quail ANG I was made after infusion of 0.9% NaCl, l&r’, Ile’] ANG II, des- Pro* BK, BK potentiator B, or SQ14225 in another six birds. If no inhibition was observed following the in-


fusion of an inhibitor, the next infusion was initiated 15 min after the termination of the previous infusion, but in the case of [Sari, Iles] ANG II, 1 hr was allowed to pass before the start of the next infusion.

Analyses of Responses

The ED, of each angiotensin was calculated from a logistic-log transformation based on the changes in arterial pressure of all birds at doses between 5 and 500 pmol. The fitting to the logistic curve was executed using the Newton-Raphson algorithm for estimates of maximum likelihood. Since the maximal change, which is necessary for calculation of the logistic-log transformation, did not appear to be attained even at 500 pmol, it was estimated as follows: the change in arterial pressure after 500 pmol of quail ANG II was increased by l-10 mm Hg, and using each value as a maximal change, curve fitting was executed. Then, the value that produced the best fit, judged by the x2 test, was selected as the maximal change of a bird. The ratio of potency of each angiotensin to the standard quail ANG II was calculated from the ratio of values of ED, after the parallelism of the logistic curves was confirmed. In the case of human ANG III, the potency ratio was calculated from the ratio of doses of quail ANG II and human ANG III that produced the same change. The relationship between two variables was examined by linear regression analysis. All other statistical comparisons were made by the paired t test. Statistical significance was determined at P < 0.05. All results are expressed as means f SE of the mean. However, 95% confidence intervals are attached to the

ED, because the mean and the variance are given as logarithms. All computations were carried out on an IBM 4341 computer using an APL interpreter.

RESULTS

Amino Acid Sequence of Quail Angiotensin

The total angiotensin-like vasoactivity after the incubation of kidney extract and plasma was equivalent to 24 nmol of [Asp’, Val’, SeP] ANG I. Thus, the yield was 436 pmol/ml plasma. After the Sep-Pak treat- ment of the crude extract, SP-Sephadex chromatography and HPLC, the total level fell to 16, 14, and 10 nmol, respectively. The analysis by sequential Edman degradation of 2 nmol of the final product showed that the amino acid sequence of quail ANG I is H-Asp-Arg-Val-Tyr-Val-His-Pro- Phe-Ser-Leu-OH.

Vasopressor and Depressor Actions of Angiotensins

A bolus injection of quail ANG II caused

PA

Dose (picomoles/lOO g) FIG. 1. Changes in arterial pressure (PA), mean arterial pressure (MA), and heart rate (HR) after a

bolus injection of native [Asp’, Val’] ANG II in a quail which has been lightly anesthetized with urethane (0.75 g/kg). A vasodepressor response and a subsequent vasopressor response were always observed after injection but the response of the heart rate was not consistent.


an immediate, transitory decrease and a arterial pressure, even at high doses. BK subsequent increase in arterial pressure in caused only a small effect on arterial pres- the quail (Fig. 1). The extent of these sure, even at high doses. BK caused only a changes was dose dependent. The effect of small increase in blood pressure (2.5 + 2.1 ANG II on heart rate was variable and de- mm Hg, n = 6) at 500 pmoYlO0 g. Human pended on dosage and other conditions angiotensins were as potent as their quail (Fig. 1). However, there was an tendency counterparts in terms of both vasopressor for tachycardia to be induced in birds and depressor effects, but eel angiotensins whose normal heart rate was low, while were much less potent (Fig. 2, Table 1). bradycardia occurred in birds whose heart ANG II was generally more potent than rate was high. All variants of ANG I and II ANG I when compared on a molar basis. tested induced both decreases and in- The potency of human ANG III relative to creases in arterial pressure in a dose- quail ANG II was only 1.8 + 0.6% (n = 9). dependent manner (Fig. 2). However, hu- The extent of the hypotension immediately man ANG III had only a small effect on after injection was significantly correlated

Increase

0 IAsp: Val’l ANG ,I . [ASP: 118’1 ANG I,

g) 20-

5 Decrease

5 10 ,

0 [ASP: Val: Ser’l A& ,

. iAS!3: Ile: HIS’, ANG ,

A [Asn: Val: Gly’l ANG ,

5 10 20 50 100 200 500

Dose (picomoles/ 100 g) FIG. 2. Dose-response relationships for the vasopressor (upper) and depressor (lower) effects of

angiotensins in the urethane-anesthetized quail (n = 9). Changes in arterial pressure are corrected for the changes in controls injected with 0.9% NaCl. The values of ED, calculated from the logistic-log transformation are given in Table 1. The resting arterial pressure was 93.0 -’ 10.3 mm Hg (n = 9) before the start of injections and 86.9 k 8.3 mm Hg (n = 9) after the last injection. The vertical bars show SE of the mean. *Significantly (P < 0.05) diierent from the control level.


TABLE 1 THE RELATIVE POTENCIES AND VALUES OF EDH, FOR THE VASOPRESSOR AND DEPRESSOR EFFECTS OF

ANGIOTENSIN I AND II (ANG I AND II) IN THE QUAIL (n = 9)

Potency on molar basis (%) EDso (pmol/lOO g)

Angiotensins Pressor Depressor

[Asp’,Val’] ANG II 100 100 85.4 (84.4-86.4) 112.7 (110.7-114.6)

[Asp1,11e5] ANG II 86.6 + 7.4 120.9 f 9.1 104.9 (103.5-106.3) 100.7 (98.6-102.8)

[Asn’,Val’] ANG II 22.3 k 4.3* 37.2 k 5.7* 374.1 (369.6-378.7) 382.9 (375.7-390.2)

[Asp’,Val’,SeP] ANG I 68.9 2 13.3 99.4 2 26.3 121.5 (120.4-122.7) 240.5 (236.2-244.9)

[Asp1,11e5,His~ ANG I 55.5 +- 11.5* 86.0 k 23.9 154.7 (153.2-156.3) 194.3 (191.2-197.5)

[Asnl,ValS,GlyT ANG I 17.4 2 3.5* 39.4 k 9.3* 488.3 (482.M94.2) 407.8 (402.2-413.4)

Note. The relative potency is expressed as a mean k SE of the mean. Since the ED, is given as a logarithm after logistic-log transformation, the variation of the mean is expressed in terms of 95% confidence intervals. *P < 0.05 compared with 100%.

with the arterial pressure prior to injection (Fig. 3).

Effects of Inhibitors of the Renin-Angiotensin System

A bolus injection of competitive inhibitors of ANG II (100 pg/lOO g) caused a transitory increase in arterial pressure: 6.4 + 1.1 mm Hg for [Sat-‘, Ala*] ANG II, 8.4 + 1.5 mm Hg for [Sar’, Thr8] ANG II, and 16.9 k 2.3 mm Hg for [Sar’, Ile8] ANG II (n = 6 in each case). The increase disap- peared within 2 min. Injection of the same dose of des-Pro’ BK or of BK potentiator B caused immediate decreases (33.3 + 3.7 and 11.0 k 2.6 mm Hg, respectively) and subsequent increases (13.0 k 4.5 and 42.0 + 4.4 mm Hg, respectively) in arterial pressure, whereas SQ14225 caused only a small increase (4.9 + 1.2 mm Hg) in arterial pressure (n = 6 in each case). The arterial pressure returned to the control level within 5 min after injection of these inhibitors. [Sar’, Ile8] ANG II increased the arterial pressure by 10.7 + 2.4 mm Hg (n = 6) during infusion at the rate of 1 pg/lOO g/mm, while all

other inhibitors tested had no effects on arterial pressure during infusion.

A bolus injection or infusion of the vehicle (0.9% NaCl) did not affect vasopressor and depressor effects of any angiotensin injected. Among three competitive inhibitors of ANG II, only [Sar’, Ile81 ANG II inhibited vasopressor and depressor effects of ANG II after both bolus injection and infusion (Table 2). After a bolus injection, the vasopressor response to quail ANG II was decreased from 44.9 + 4.6 to 13.9 + 2.9 mm Hg and the vasodepressor response from 10.1 k 2.5 to 1.3 2 1.3 mmHg (n = 6 in each case). [Sar’, Ile81 ANG II was also the sole inhibitor capable of blocking the cardiovascular effects of quail ANG I after a bolus injection or an infusion (Table 2). Af- ter a bolus injection of [Sari, Ile8] ANG II, the vasopressor response was decreased from 42.6 + 4.6 to 10.9 + 2.1 mm Hg, and the vasodepressor response from 9.8 + 2.1 to 0 mm Hg (n = 6 in each case). The inhibitory effect of [Sar’, Ile8] ANG II con- tinued for 15-20 min after a bolus injection of 100 &OO g (Fig. 4).

Among three inhibitors of converting en-

18 TAKEI AND HASEGAWA

4 Y 0.43X - 23 n 10.

.E

8

E 8

0

Resting blood pressure (mmHg)

FIG. 3. The relationship between the extent of the vasodepressor response to 100 pmol/lOO g of native [Asp’, Va15] ANG II or human [Asp’, Be’] ANG II, and the arterial pressure prior to injection in nine quail anesthetized with urethane (0.75 g/kg). In some birds, 0.25 glkg of urethane was added to stabilize the resting level. The regression line and corresponding equation for each ANG II are given in the figure. The correla- tions are signiticant for both quail ANG II (r = 0.927) and human ANG II (r = 0.835).

zyme examined, SQ14225 was the only inhibitor that effectively inhibited the vasopressor and depressor effects of ANG I after both bolus injection and infusion (Table 2). After a bolus injection of SQ14225 the vasopressor and depressor effects of quail ANG I were inhibited, with decreases of changes in arterial pressure from 42.6 2 4.6 to 8.3 2 2.1 mm Hg and from 9.8 2 2.1 to 0 mm Hg, respectively (n = 6 in each case). The inhibitory effect of SQ14225 appears to continue for more than 2 hr after a bolus injection of 100 p&100 g (Fig. 5). However, the cardiovascular effects of quail ANG II were slightly augmented after administra- tion of SQ14225 (Table 2).

DISCUSSION

Amino Acid Sequence of Quail Angiotensin

Although comparative studies have been performed on the chemical structure of angiotensins from vertebrates, information for nonmammalian species is still scarce

TABLE 2 THE EFFECT OF A SLOW INFUSION (1 p&O0 g/min)

OF COMPETITIVE INHIBITOIS OF ANGIOTENSIN II (ANG II) OR OF INHIBITORS OF ANG I CONVERTING ENZYME ON THE VASOPRESSOR (UPPER Rows) AND

DEPRESSOR (LOWER Rows) EFFECTS OF NATIVE ANG I AND II IN THE QUAIL (n = 6)

Pressor and depressor effects (mm W

Inhibitors ANG II ANG I

Vehicle 39.2 + 5.1 38.7 + 5.9 3.0 k 1.3 4.5 + 1.8

[Sar’,Ala*] ANG II 38.7 f 5.4 -

3.0 k 1.3 -

[Sar’,Thrs] ANG II 35.0 f. 5.3 -

2.7 -t 0.9 -

[Sar1,11e8] ANG II 8.7 f 1.74 10.5 k 2.4* 0* 0*

des-Pro’ BK - 35.1 2 6.0 - 21.2 f 14.8

BK potentiator B - 33.2 2 5.8 - 20.6 2 12.2

SQ14225 44.9 _' 5.3 2.3 -t- 0.2* 1.7 2 1.7 0*

Note. The doses of ANG I and II injected are equivalent to the ED,. Values are means -C SE of the mean. Mean, resting arterial pressure was 80.8 + 3.7 mm Hg before the start of infusion and 86.5 + 4.2 mm Hg after the last infusion. *P < 0.05 compared with the value after infusion of the vehicle. BK, bradykinin.

B 50-

i 40- ?

2 I 30-

h 2 20-

$ 5 lo-

t .G

01 _$ ~~~ * % Before

z 5 10 15 20

injection

i

0 Decrease

0 Increase 1 * * *

m

5 Minutes after injection of tSa/,ii& ANG II

FIG. 4. The time course of changes in the inhibitory effect of 100 pg/lOO g of [Sar’, Be81 ANG II on the vasopressor and depressor effects of 85 pmol/lOD g (ED& of native [Asp’, Val’] ANG II in the urethane- anesthetized quail (n = 6). Injection of the vehicle did not affect the ANG II effect. The vertical bars show SE of the mean. *The inhibition was significant (P < 0.05).


c3 Decrease 0 Increase * * * * * All &kNl * * *

Before 5 30 60 90 120 Injection

Minutes after injection of XI14225

FIG. 5. The time course of changes in the inhibitory effect of 100 pg/lOO g of SQ14225 on the vasopressor and depressor effects of 113 pmoYlO0 g (ED& of native [Asp’, Val’, Ser9] ANG I in the urethane- anesthetized quail (n = 6). Injection of the vehicle did not affect the ANG I effect. The vertical bars repre- sent SE of the mean. *The inhibition was significant (P < 0.05).

compared to that for mammalian species (Khosla et al., 1983). The paucity of information is especially apparent in the case of birds since only fowl ANG I has been sequenced (Nakayama et al., 1973). Compar- ative studies of ANG I show that variations in amino acids occur at positions 1,5, and 9 (Nishimura, 1980). The nature of the amino acids at these positions may affect the affinity of ANG I for the converting enzyme and the binding affinity of ANG II for its receptors in each species. Thus, it is impor- tant to sequence, synthesize, and use native angiotensins in physiological studies of the renin-angiotensin system in nonmammalian vertebrates. We have identified quail ANG I as [Asp’, Val’, Se?] ANG I in the present study and, thus, quail ANG I is identical to fowl ANG I. Since quail and fowl belong to the same order of classitica- tion (Galliformes), the biochemical nature also appears to reflect taxonomical proxim- ity which is based on the anatomy.

Vasodepressor Eflect of Angiotensin

Moore et al. (1981a) reported that the vasodepressor effect of human ANG II which

was observed in the conscious fowl disap- peared after urethane anesthesia. It has been shown that urethane anesthesia is characterized by the preservation of a number of cardiovascular reflexes, but high doses often produce adverse reactions such as hypotension (Maggi and Meli, 1986). Moore et al. (1981a) injected fowl with twice the amount of urethane per body weight that we used for the quail and their dose decreased the blood pressure of the fowl. Thus, the difference in the vasodepressor response between the quail and fowl may be due to the difference in the levels of anesthesia.

The vasodepressor effect of angiotensin was observed in the fowl (Moore et al., 1981a; Nishimura et al., 1982) and in the quail in this study, but it was not observed in the pigeon (Evered and Fitzsimons, 1981) or the duck (Wilson and Butler, 1983). Because the extent of the vasodepressor response was significantly correlated with the level of resting arterial pressure, as observed in this study, it is possible that the difference in the responses of these avian species is due to the difference in the resting level of arterial pressure. The mean, resting arterial pressure before injection was 115 + 5 mm Hg (n = 6) in the conscious fowl (Nakamura et al., 1982); 93 + 10 mm Hg (n = 9) in the anesthetized quail in this study; 131 + 6 mm Hg (n = 8) in the conscious pigeon (Evered and Fitzsimons, 1981); and approximately 158 mm Hg in the conscious duck (Wilson and Butler, 1983). Thus, the absence of a vasodepressor response in the pigeon and duck is not due to the low resting blood pressure. Since quail and fowl belong to the order Galliformes, while pigeons (Columbiformes) and ducks (Anseriformes) belong to different orders, it is possible that the vasodepressor response to angiotensin is unique to the gallinacean birds.

Relative Potency of Angiotensins

The present study has shown that eel ANG I and II are much less potent than


their quail and human counterparts in terms of their vasopressor and depressor effects in the quail. Human ANG III had only 2% of the activity of quail ANG II. These results are quite similar to those obtained in the fowl (Nishimura et al., 1982; Table 3). However, the relative potency of angiotensins in the rat is different from those found with quail and fowl: eel ANG II and human (or rat) ANG III have greater activities in the rat than in these two avian species (Ta- ble 3). Only Watanabe et al. (1977) observed a comparably low vasopressor activity of eel ANG II in the rat. Recently, we obtained preliminary results showing that eel ANG I and II are more effective than human and quail counterparts in terms of vasopressor effect in the eel (Takei, 1987). These results show that native ANG II is most effective in each class of vertebrates, and further suggest that ANG II receptors have evolved in parallel with the evolution of their ligand molecules.

Inhibitors of the Renin-Angiotensin System

In the quail, [Sar’, Ile*] ANG II is an effective inhibitor which antagonizes the vascular effects of ANG II, while [Sar’, Ala*] ANG II and [Sar’, Thr*] ANG II, which are potent inhibitors in mammals (Khosla et al., 1974), are ineffective. These results indicate that vascular ANG II receptors in the quail may differ from those in mam-

mals, as has already been suggested to be the case in the fowl (Nishimura et al., 1982). In the fowl, however, [Sar’, Ala81 ANG II and [Sar’, Thr*] ANG II appear to be weak but effective inhibitors: [Sar’, Ala81 ANG II infused at a rate of 12.5 pg/kg/min inhibits the vasodepressor action of human ANG II (Moore et al., 198Ib); and [Sar’, The*] ANG II infused at a rate of 5 pg/kg/min decreases by 50% the vasopressor response to native ANG II (Ni- shimura et al., 1982). The inhibitory effects of [Sar’, Ala’] ANG II and of [Sar’, Thr*] ANG II are, respectively, 10% and less than 4% of that of [Sar’, Ile’] ANG II in the fowl. In this study, however, both inhibitors failed to inhibit the vasopressor and depressor effects of native ANG II when infused at a rate of 10 p,g/kg/min after a priming injection of 50 &kg. It is possible that vascular ANG II receptors in the quail also differ from those in.the fowl.

The effects of ANG II competitive inhibitors have been examined in other nonmammalian vertebrates, and the results appear to vary among species. Both [Sar’, Ile’] ANG II and [Sar’, Ala81 ANG II are effective inhibitors in the turtle, Pseudemys scriptu (Stephens, 1981), [Sar’, Ileg] ANG II is effective but [Sar’, Ala*] ANG II and [Sar’, Thr’] ANG II are ineffective in the bullfrog (Harper and Stephens, 1985), and neither [Sar’, Ile*] ANG II nor [Sar’, Th$] ANG II appears to be effective in the eel (Nishimura et al., 1978). Together with the

TABLE 3 THE RELATIVE POTENCIES OF ANGIOTENSINS IN THE RAT, FOWL, AND QUAIL WITH RESPECT TO THE

VASOPRESSOR EFFECT

Animals

Angiotensins Rat” Ratb Rat’ Fowld Qd [Asp’,Val’] ANG II 100 100 100 100 100 [Asp’,Ile5] ANG II 100 74 50 117 87 [Asn’,Val’] ANG II 93 - 25 27 22 [Asp’,Va15,SeP] ANG I - 85 69 56 69 [Asp’,Ile’,Hisg] ANG I 50 54 38 - 56 [Ile’] ANG III 38 - - 2 2

D Regoli et al. (1974); bKhosla et al. (1977); ‘Watanabe et al. (1977); dNisbimura et nl. (1982).


results of varying pressor potencies of angiotensins in different classes of vertebrates, it is suggested that vascular ANG II receptors have evolved during vertebrate phylogeny.

The present study also showed that SQ14225 is a potent inhibitor of the cardiovascular actions of ANG I, but des-Pro2 BK and BK potentiator B, which are effective inhibitors in mammals, are ineffective. Since the effect of ANG II is not inhibited by SQ14225, ANG I may be active after its conversion to ANG II. These results indicate that the converting enzyme of the quail differs from that of mammals. Since amino acid of ANG I at position 9 differs in all classes of vertebrates ever examined (Ni- shimura, 1980), it is possible that converting enzyme, which cleaves ANG II between the eighth and ninth position, is also different among these animals. However, the enzyme may retain common character- istics throughout the vertebrate phylogeny because SQ14225 is an effective inhibitor in all mammalian and nonmammalian vertebrates so far examined (fowl, Nishimura et al., 1982; rat snake, Ho et al., 1984; and bullfrog, Harper and Stephens, 1985).

ACKNOWLEDGMENTS

The authors are very grateful to Dr. Mahesh C. Khosla, Department of Brain and Vascular Research, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, for his generous gift of eel angiotensin I, and to Sankyo Pharmaceutical Co. Ltd. for pro- viding SQ14225. They also thank Dr. Haruo Suzuki and Ms. Etsuko B. Mukouyama, Department of Bio- physical Chemistry, Kitasato University School of Medicine, for their help in operating a protein sequencer. This investigation was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan (62748449).

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Vasopressor and depressor effects of native angiotensins and inhibition of these effects in the Japanese quail