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Competitive antagonism of 8-ala-angiotensin II to angiotensins I and II on isolated rabbit aorta and rat ascending colon
EUROPEAN JOURNAL OF PHARMACOLOGY 15 (1971) 285-291. NORTH-HOLLAND PUBLISHING COMPANY
COMPETITIVE
ANTAGONISM
ANGIOTENSINS
OF 8-ALA-ANGIOTENSIN
II T O
I A N D II O N I S O L A T E D R A B B I T A O R T A
AND RAT ASCENDING COLON R.K. TURKER, M. YAMAMOTO, P.A. KHAIRALLAH and F.M. BUMPUS Research Division, Cleveland Clinic bbundation, Cleveland. Ohio, 44106, USA
Received 9 March 1971
Accepted 21 May 1971
R.K. TORKER, M. YAMAMOTO, P.A. KHAIRALLAH and F.M. BUMPUS, Competitive antagonism o f 8-ala-angiotensin H to angiotensins 1 and H on isolated rabbit aorta and rat ascending colon, European J. Pharmacol. 15 ( 1971 ) 285 -291. 8-Alanine angiotensin It has a specific competitive antagonistic effect against angiotensins 1 and II when assayed on the rabbit isolated aortic strip and rat colon; but by itself it has no myotropic action on these smooth muscle preparations even et very high concentrations. In pentobarbital anesthetized cats, 8-ala-angiotensin II does not antagonize the pressor and intestinal inhibitory effect of angiotensins 1 and 11. The possible mechanism of these effects of 8-ala-angiotensin II is discussed. 8-Ala-angiotensin II Competitive antagonism
t. INTRODUCTION Direct myotropic effects of angiotensin II have been reported in many tissues including intestinal and vascular smooth muscle (Page and Bumpus, 1961). From studies on the mechanism of tachyphylaxis to angiotensin, we have previously reported that this polypeptide has its own receptors on vascular smooth muscle (Khairallah et al., 1966). However, there is, as yet, no specific competitive antagonist for angiotensin and such a compound would support the presence of specific receptors. In 1966, Godfraind et al. reported that lidoflazine is a potent non-competitive inhibitor of the direct contractile response to angiotensin on the longitudinal muscle of guinea-pig ileum in vitro. However, lidoflazine did not antagonize responses to angiotensin on the rabbit isolated aorta
Angiotensin II Rabbit aorta
Angiotensin 1 Rat colon
(Ti.irker and Kayaalp, 1967a) and cat ileum in vivo (Tiirker et al., 1968). Another compound which nonspecifically blocks the pressor effect of angiotensin is guancydine (Cummings et al., 1969). Studying some analogs of angiotensin, Khairallah et al. (1970) reported that 8-ala-angiotensin II, given in concentrations over 500 ng/ml, effectively blocked the contractile response to angiotensin II without affecting the response to vasopressin and serotonin in the isolated guinea-pig ileum. These authors, however, did not study the mechanism of the inhibitory effect of 8-ala-angiotensin II. The data presented here demonstrate that 8-alaangiotensin II at very low concentrations competitively blocks the myotropic effect of angiotensins 1 and II on the rabbit isolated aortic strips and rat colon, but has no effect when given in vivo.
286
R.K. Tiirker et al., Competitive antagonism of angiotensin
2. MATERIALS AND METHODS Spirally cut rabbit aortic strips, 4 to 6 cm long, were prepared according to Furchgott and Bhadrakom (1953). They were mounted in a 10 ml isolated organ bath containing a physiologic salt solution (NaC1 112 mM, NaHCO3 25 mM, KC1 5 mM, NaH2PO4 1 mM, MgC12 0.5 mM, CaC12 2.5 mM, and glucose 11.5 mM) at 37°C and aerated with 95% O2, 5% CO2. Isometric contractions were recorded using a Grass force displacement transducer ( F T - 0 3 ) on a Grass polygraph (Model 7). The strips were placed under a 1 g passive stretch and allowed to equilibrate 2.5 to 4 hr. Rat ascending colons were prepared according to Regoli and Vane (1964) and mounted in a 10 ml isolated organ bath containing the same salt solution. Contractions were recorded isometrically. The preparations were subjected to 4 g passive stretch and allowed to equilibrate 0.5 to 1 hr. In a series of experiments rabbit aortic strips and rat colons were prepared for superfusion similar to the isolated blood-bathed organ technique (Vane, 1964). Strips were continuously superfused with warm, oxygenated physiologic salt solution at 7 ml/min. Angiotensin responses in both assay organs were measured before and after 8-ala-anglotensin II. In another series of experiments, similar tension changes of aortic strips were produced by angiotensin II, norepinephrine (NE) and serotonin. When the increased tension was established, 8-ala-angiotensin II was added to the bath and relaxation measured. In another 3 isolated rabbit aortic strip preparations the dose-response curve to norepinephrine was measured before and after the addition of 8-ala-angiotensin II. In other experiments, 4 cats weighing 2.5 to 4 kg were anesthetized by intravenous sodium pentobarbital. Arterial blood pressure was recorded from the right carotid artery. In situ motility and intraluminal pressure of the terminal ileum were recorded by using the method described by Tiirker and Kayaalp (1967b) and Tiirker (1969). Angiotensins I and II responses were measured before and after injections of 8-ala-angiotensin II. In order to evaluate the mechanism of antagonism, the criteria described by Schild (1947, 1949, 1957) were used. Calculated results were evaluated statistically using Student's t-test.
The following substances were used: Angiotensin II (Hypertensin, Ciba); 5-hydroxytryptamine creatinine sulfate (Sandoz); norepinephrine bitartrate (Parke Davis); 5-ile-angiotensin I, and 8-ala-angiotensin II. The latter peptide was synthesized in our laboratory (Park et al., 1967).
3. RESULTS 3.1. Isolated rabbit aortic strip
The maximum antagonism of 8-ala-angiotensin II was established within 2 min after addition of the peptide. After washing away the antagonist, responses of the strips to angiotensin II progressively increased and reached control levels within 45 min. The log dose-response curve for angiotensin II activity measured in the rabbit aortic strips was a straight line between 3.61 × 10 -9 M and 2.89 × 10-8 M. In the presence of either 1.55 × 10-8 M or 6.20 × 10-8 M 8-ala-angiotensin II curves were shifted to the right, but were still parallel (fig. 1). The dose ratios calculated from different molar concentrations of 8-ala-angiotensin II and the log k2 values were estimated. These results are summarized in table 1. The log k2 values for each concentration of antagonist were found to be similar and did not vary significantly. These values closely corresponded to the estimated pA2 value of 8-ala-angiotensin II against angiotensin II (table 2). In the same equilibration period, the pAlo value of 8-ala-angiotensin II against angiotensin II was found to be 7.41 + 0.07. The difference pA2 - pA 1o was 0.91. The dose-response curves to angiotensin I before and after addition of 8-ala-angiotensin II to the bath were also determined to be parallel (fig. 2). The dose ratios of angiotensin I in the presence of 8-ala-angiotensin II and log k2 values were calculated and summarized in table 1. The log k2 values were also found to be similar to the pA2 value (table 2). The difference between pA2 and pA l0 values was found to be 0.92. When equal tensions were induced by angiotensin II, NE and serotonin, the addition of 8-alaangiotensin II to the bath produced a relaxation within 2 min in those aortic strips contracted by angio-
R.K. Tiirker et al., Competitive antagonism o f angiotensin
287
Control
2.0-
A
& Responses in presence
of ( 8 - A l a ) A n g i o t e n s i n WI.S5xlO-SM
O
O Responses in presence
of (8-Ala) Angiotensin ]~ 6.2xlO-SM
1.6-
1.2-
0.8-
0.4-
I
3.61 xlO -9
I
7.23 xlO-9
I
I
1.44 xlO-8
2.89 xlO "8
I
5,78x10 -8
I
2.89x10 -7
M o l a r concentrations of angiotensin 1T
Fig. 1. Log dose--response curve of isolated rabbit aorta to angiotensin II before and after addition of 8-ala-angiotensin II. Each p o i n t represents the m e a n of 10 experiments + standard error o f the mean. Tension (g) indicates changes in tension.
Table 1 A n t a g o n i s m by 8-ala-angiotensin II o f angiotensins I and II responses on rabbit isolated aortic strips. Agonists
Angiotensin I
Bath concentration o f 8-ala angiotensin II (molar)
Dose ratios ( m e a n ± S.E.) a
log k2 (mean ± S.E.) a,b
7.75 X 10 -9
4.85 ± 0.386 (4) n (3.80-5.60) r
8.688 ± 0.852 (4) n (8.5577-8.7447) r
1.55 × 10 -a
8.20 ± 0.129 (4) (7:90-8.50)
8.666 ± 0.007 (4) (8.6480-8.6845)
X 10 -8
18.95 ± 0.523 (4) (17.90-20.20)
8.762 ± 0.0126 (4) (8.7365 - 8 . 7 9 2 0 )
7.75 X 10 .-9
3.26 ± 0.027 (4) (3.20-3.33)
8.464 ± 0.005 (4) (8.4531-8.4781)
1.55 × 10 - s
5.36 ± 0.165 (4) (5.00-5.75)
8.447 ± 0.016 (4) (8.4118-8.4824)
3.10 × 10- s
9.065 ± 0.134 (4) (8.75-9.40)
8.413 ± 0.007 (4) (8.3979-8.4329)
18.127 ± 0.497 (4) (17.21-19.30)
8.440 ± 0.012 (4) (8.4171-8.4701)
3.1 Angiotensin II
6.2
X 10 - s
a n = n u m b e r o f experiments; r = range. b log k 2 (dose ratio - 1) = log (antagonist concentration)
R.K. Tiirker et al., Competitive antagonism of angiotensin
288
5min I I
Table 2 PA2 and PAl0 values o f 8-ala-angiotensin II against angiotensins 1 and II on normal rabbit aortic strips. Agonists
pA 2 ± S.E.a
pAlo ± S.E.a
pA2_pA]o
Angiotensin II
8.32 ± 0.08 (10)
7.41 ± 0.07 (9)
0.91
8.60 ± 0.05
7.68 ± 0.04
0.92
(8)
(8)
Angiotensin I
a Number of experiments in parentheses.
1.5-
A v
:
:
~.
&
Control
I
Responses in presence of (8-Ala) Angiotensin ]~ 7.75x10-9M
I
I
I A~ 20ng/ml
A~ IOng/ml
A]I 20ng/ml
ATT 20ng/ml
8alo AIT Solo ATr IOng/ml 20ng/ml Fig. 4. Antagonistic effect of 8-ala-angiotensin II on the responses of angiotensin II on the isolated continuously superfused rabbit aorta and colOn. Upper tracing shows the tension changes of aorta. Lower tracing shows the tension changes of colon.
1.0-
.=
0.5-
2.~J5
519
111.8
I
23.6
Molar concentrations of angiotensin
Fig. 2. Log d o s e - r e s p o n s e curve of isolated rabbit aorta to angiotensin 1 before and after addition of 8-ala-angiotensin 1I. Each point represents the mean of 10 experiments + standard error of the mean. Tension (g) indicates changes in tension.
xIO-9 T
32 ng/ml
32 ng/ml
32 ng/ml
o
16 ng/ml
16r~/ml
50nglml
20nglml
L__J 5rain
Fig. 3. Tension changes in the isolated rabbit aorta to angiotensin I1 (A II), serotonin (S), and norepinephrine (NE) and the effect of 8-ala-angiotensin II.
R.K. Tiirker et al., Competitive antagonism of angiotensin
./"
289
5rain I I
j,
o
o
o
o
o
o
o
4
8
16
4
8
16
40
8ala A'n' 801aATr 3.3 7.7' I I x 10-9M
8ala Ang 1r
Angiotensin ] I (ng/ml)
1.5 x 10-SM
Fig. 5. Tension changes in the rat ascending colon to angiotensin II before and after addition of 8-ala-angiotensin II. 2.8H
Control
2.4-
0
0
Responses in presence of ( 8 - A l a
~.
A
Responses in presence of ( 8 - A l a ) A n g i o t e n s i n TT 155x10 8M
A n g i o t e n s i n ]I 31x10-SM 2.0m
o = s
16-
01 7.23x 10-9
I 1.446x I0 -8
I 2.892 xlO - 8
I 5.784 x I0 -B
I 2.89x~0 -7
M o l a r concentrations of angiotensin
2001
BPmmHg
Fig. 6. Log dose-response curve of isolated rat ascending colon to angiotensin II before and after addition of 8-alaangiotensin II. Each point represents the mean of 10 experiments + standard error of the mean. Tension (g) indicates changes in tension.
5 min I I
gL 120..J
0.25 pg/kg
0.5 pg/kg
0.25 pg/kg 8-olo ATT 5 pg/kg
0 25pg/kg DHE 2 mg/kg
Fig. 7. Blood pressure (upper tracing) and intraluminal pressure of terminal ileum (lower tracing) of the pentobarbital anesthetized cat. Effect of angiotensin II (A II), angiotensin I (A I), 8-ala-angiotensin II and dihydroergotamine (DHE).
290
R.K. Tiirker et al., Competitive antagonism of angiotensin
tensin II, while no relaxation was observed in the strips contracted by NE or serotonin (fig. 3). In the isolated continuously superfused rabbit aorta and rat colon, 8-ala-angiotensin II also antagonized the effect of angiotensin II in both assay organs (fig. 4), in a dose-dependent way. 3.2. Isolated rat ascending colon
8-Ala-angiotensin II antagonized the myotropic action of angiotensin II in this preparation (fig. 5). The dose-response relationship of angiotensin II before and after 8-ala-angiotensin II at molar concentrations of 1.55 × 10-8 to 3.1 × 10-8 showed parallel curves (fig. 6). Maximum response to angiotensins in both smooth muscle preparations can be obtained in the presence of 8-ala-angiotensin II. 3.3. In situ cat blood pressure and ileal motility Angiotensins I and II given intravenously in doses of 50 to 500 ng/kg produced pressor responses and decreased intraluminal pressure and motility of the ileum in a dose~iependent way. A single injection of 1 to 10 #g/kg of 8-ala-angiotensin II did not induce such a response and did not alter subsequent responses to angiotensins I and II (fig. 7). 4. DISCUSSION The antagonistic action of 8-ala-angiotensin II to angiotensin II in the isolated guinea-pig ileum has been reported by Khairallah et al. (1970). The present study clearly shows that 8-ala-angiotensin II is a competitive antagonist of angiotensins I and II. The following criteria which have been advocated by Schild (1957) appear to satisfy the concept of competitive antagonistic action of 8-ala-angiotensin II. First, the log dose-response curves of angiotensins I and II on the aorta are shifted progressively to the right and are parallel with each other. A similar parallel shift to the right in the rat ascending colon indicates that 8-ala-angiotensin II also competitively antagonizes the myotropic effect of angiotensin II in this preparation. Secondly, the calculated log (dose ratio - 1 ) against log antagonist concentration was linear and there was no change in the values of log k2 within the dose ranges of 8-ala-angiotensin II used. This also in-
dicates competitive antagonism. Again, the difference pA2 - pAlo was found to be 0.91 for angiotensin II and 0.92 for angiotensin I which are very near to the theoretical value of 0.95 expected for competitive antagonism (Schild, 1957). The antagonistic action of 8-ala-angiotensin II was established within 2 min after exposure with the strips. However, this antagonism was easily washed out and complete recovery occurred within 45 min. The antagonistic potency of 8-ala-angiotensin II was found to be very high. The myotropic effect of 7.23 X 10-9 molar angiotensin II was completely antagonized by 7.75 X 10-9 molar 8-ala-angiotensin II. The effect of 8-ala-angiotensin II is specific for angiotensins I and II, since it did not inhibit the myotropic action of NE or serotonin even at very high concentrations. This finding also strongly indicates that angiotensin II has its own specific receptors in vascular smooth muscle cells. The question arises as to how 8-ala-angiotensin II interacts with angiotensins at the receptor site in aortic muscle. This may be due to the fact that the myotropic activity of angiotensin is due to a phenyl group in the position 8 and a free carboxyl terminus both of which interact with receptor (Khairallah et al., 1970). 8-ala-angiotensinII covers the receptor sites, preventing other molecules from interacting, but since it does not contain a phenyl side group, no contractile response is obtained. This study also clearly shows that angiotensins I and II act at the same receptor sites in the vascular wall. Recently Aiken hnd Vane (1970) reported that angiotensin I has a direct action on specific angiotensin I receptors, a direct but weak action on angiotensin II receptors and an indirect effect due to the variable conversion of decapeptide to angiotensin II. Since 8-ala-angiotensin II competitively antagonizes the myotropic effect of both polypeptides, it can be assumed that the same receptor system of the smooth muscle cell may be responsible for the effect of both polypeptides. It should be emphasized, however, that 8-alaangiotensin II does not antagonize the pressor and intestinal inhibitory effect of angiotensin when given intravenously to anesthetized cats. One possible explanation is that 8-ala-angiotensin II is rapidly metabolized and insufficient peptide reaches the receptor sites to block responses to angiotensins.
R.K.Tiirker et al., Competitive antagonism o f angiotensin ACKNOWLEDGEMENTS This investigation was supported by U.S. Public Health Service Research Grant HE--6835 from the National Institute of Heart and Lung Diseases.
REFERENCES Aiken, J.W. and J.R. Vane, 1970, The renin-angiotensin system: Inhibition of converting enzyme in the isolated tissues, Nature 288, 30. Cummings, J.R., A.N. Welter, J.L. Grace and W.D. Gray, 1969, Angiotensin blocking actions of guancydine, J. Pharmacol. Exptl. Therap. 170, 334. Furchgott, R.F. and S. Bhadrokom, 1953, Reaction of strips of rabbit aorta to epinephrine, isopropylarteranol, sodium nitrate and other drugs, J. Pharmacol. Exptl. Therap. 108, 129. Godfraind, T., A. Kaba and P. Polster, 1966, Specific antagonism to direct and indirect action of angiotensin on the isolated guinea-pig ileum. Brit. J. Pharmacol. 28, 93. Khairallah, P.A., I.H. Page, F.M. Bumpus and R.K. Tiirker, 1966, Angiotensin tachyphylaxis and its reversal, Circulation Res. 19,247. KhairaUah, P.A., A. Toth and F.M. Bumpus, 1970, Analogs of angiotensin II. Mechanism of receptor interaction, J. Med. Chem. 13,181.
291
Page, I.H. and F.M. Bumpus, 1961, Angiotensin, Physiol. Res. 41,331. Park, W.K., R.R. Smeby and F.M. Bumpus, 1967, Synthesis of [5-Isoleucine, 8-alanine] -angiotensin II by the solution method synthesis and the solid phase method synthesis, Biochim. 6, 3458. Regoli, D. and J.R. Vane, 1964, A sensitive method for the assay of angiotensin, Brit. J. Pharmacol. 23,351. Schild, H.O., 1947, pA, A new scale of the measurement of drug antagonism, Brit. J. Pharmacol. 2, 189. Schild, H.O., 1949, pA x and competitive drug antagonism, Brit. J. Pharmacol. 4,277. Schild, H.O., 1957, Drug antagonism and PAx, Pharmacol. Rev. 9,242. Tiirker, R.K. and S.A. Kayaalp, 1967a, Effect of lidoflazine on norepinephrine, angiotensin and serotonin responses in isolated smooth muscle preparations, Experientia 23,647. Tiirker, R.K. and S.O. Kayaalp, 1967b, Inhibitory effect of angiotensin on intestinal motility of the cat and its relation to sympathetic nervous system, Arch. Intern. Physiol. Biochim. 75,735. TiJrker, R.K., S.O. Kayaalp and A. Ozer, 1968, Further studies on lidoflazine (R 7904), Arzneimittel-Forsch. 18, 1209. Tiirker, R.K., 1969, Possible postganglionic adrenergic effect of angiotensin in the isolated perfused cat intestinal segment, Arch. Intern. Physiol. Biochim. 77,587. Vane, J.R., 1964, The use of isolated organs for detecting active substances in the circulating blood, Brit. J. Pharmacol. 23,360.
COMPETITIVE
ANTAGONISM
ANGIOTENSINS
OF 8-ALA-ANGIOTENSIN
II T O
I A N D II O N I S O L A T E D R A B B I T A O R T A
AND RAT ASCENDING COLON R.K. TURKER, M. YAMAMOTO, P.A. KHAIRALLAH and F.M. BUMPUS Research Division, Cleveland Clinic bbundation, Cleveland. Ohio, 44106, USA
Received 9 March 1971
Accepted 21 May 1971
R.K. TORKER, M. YAMAMOTO, P.A. KHAIRALLAH and F.M. BUMPUS, Competitive antagonism o f 8-ala-angiotensin H to angiotensins 1 and H on isolated rabbit aorta and rat ascending colon, European J. Pharmacol. 15 ( 1971 ) 285 -291. 8-Alanine angiotensin It has a specific competitive antagonistic effect against angiotensins 1 and II when assayed on the rabbit isolated aortic strip and rat colon; but by itself it has no myotropic action on these smooth muscle preparations even et very high concentrations. In pentobarbital anesthetized cats, 8-ala-angiotensin II does not antagonize the pressor and intestinal inhibitory effect of angiotensins 1 and 11. The possible mechanism of these effects of 8-ala-angiotensin II is discussed. 8-Ala-angiotensin II Competitive antagonism
t. INTRODUCTION Direct myotropic effects of angiotensin II have been reported in many tissues including intestinal and vascular smooth muscle (Page and Bumpus, 1961). From studies on the mechanism of tachyphylaxis to angiotensin, we have previously reported that this polypeptide has its own receptors on vascular smooth muscle (Khairallah et al., 1966). However, there is, as yet, no specific competitive antagonist for angiotensin and such a compound would support the presence of specific receptors. In 1966, Godfraind et al. reported that lidoflazine is a potent non-competitive inhibitor of the direct contractile response to angiotensin on the longitudinal muscle of guinea-pig ileum in vitro. However, lidoflazine did not antagonize responses to angiotensin on the rabbit isolated aorta
Angiotensin II Rabbit aorta
Angiotensin 1 Rat colon
(Ti.irker and Kayaalp, 1967a) and cat ileum in vivo (Tiirker et al., 1968). Another compound which nonspecifically blocks the pressor effect of angiotensin is guancydine (Cummings et al., 1969). Studying some analogs of angiotensin, Khairallah et al. (1970) reported that 8-ala-angiotensin II, given in concentrations over 500 ng/ml, effectively blocked the contractile response to angiotensin II without affecting the response to vasopressin and serotonin in the isolated guinea-pig ileum. These authors, however, did not study the mechanism of the inhibitory effect of 8-ala-angiotensin II. The data presented here demonstrate that 8-alaangiotensin II at very low concentrations competitively blocks the myotropic effect of angiotensins 1 and II on the rabbit isolated aortic strips and rat colon, but has no effect when given in vivo.
286
R.K. Tiirker et al., Competitive antagonism of angiotensin
2. MATERIALS AND METHODS Spirally cut rabbit aortic strips, 4 to 6 cm long, were prepared according to Furchgott and Bhadrakom (1953). They were mounted in a 10 ml isolated organ bath containing a physiologic salt solution (NaC1 112 mM, NaHCO3 25 mM, KC1 5 mM, NaH2PO4 1 mM, MgC12 0.5 mM, CaC12 2.5 mM, and glucose 11.5 mM) at 37°C and aerated with 95% O2, 5% CO2. Isometric contractions were recorded using a Grass force displacement transducer ( F T - 0 3 ) on a Grass polygraph (Model 7). The strips were placed under a 1 g passive stretch and allowed to equilibrate 2.5 to 4 hr. Rat ascending colons were prepared according to Regoli and Vane (1964) and mounted in a 10 ml isolated organ bath containing the same salt solution. Contractions were recorded isometrically. The preparations were subjected to 4 g passive stretch and allowed to equilibrate 0.5 to 1 hr. In a series of experiments rabbit aortic strips and rat colons were prepared for superfusion similar to the isolated blood-bathed organ technique (Vane, 1964). Strips were continuously superfused with warm, oxygenated physiologic salt solution at 7 ml/min. Angiotensin responses in both assay organs were measured before and after 8-ala-anglotensin II. In another series of experiments, similar tension changes of aortic strips were produced by angiotensin II, norepinephrine (NE) and serotonin. When the increased tension was established, 8-ala-angiotensin II was added to the bath and relaxation measured. In another 3 isolated rabbit aortic strip preparations the dose-response curve to norepinephrine was measured before and after the addition of 8-ala-angiotensin II. In other experiments, 4 cats weighing 2.5 to 4 kg were anesthetized by intravenous sodium pentobarbital. Arterial blood pressure was recorded from the right carotid artery. In situ motility and intraluminal pressure of the terminal ileum were recorded by using the method described by Tiirker and Kayaalp (1967b) and Tiirker (1969). Angiotensins I and II responses were measured before and after injections of 8-ala-angiotensin II. In order to evaluate the mechanism of antagonism, the criteria described by Schild (1947, 1949, 1957) were used. Calculated results were evaluated statistically using Student's t-test.
The following substances were used: Angiotensin II (Hypertensin, Ciba); 5-hydroxytryptamine creatinine sulfate (Sandoz); norepinephrine bitartrate (Parke Davis); 5-ile-angiotensin I, and 8-ala-angiotensin II. The latter peptide was synthesized in our laboratory (Park et al., 1967).
3. RESULTS 3.1. Isolated rabbit aortic strip
The maximum antagonism of 8-ala-angiotensin II was established within 2 min after addition of the peptide. After washing away the antagonist, responses of the strips to angiotensin II progressively increased and reached control levels within 45 min. The log dose-response curve for angiotensin II activity measured in the rabbit aortic strips was a straight line between 3.61 × 10 -9 M and 2.89 × 10-8 M. In the presence of either 1.55 × 10-8 M or 6.20 × 10-8 M 8-ala-angiotensin II curves were shifted to the right, but were still parallel (fig. 1). The dose ratios calculated from different molar concentrations of 8-ala-angiotensin II and the log k2 values were estimated. These results are summarized in table 1. The log k2 values for each concentration of antagonist were found to be similar and did not vary significantly. These values closely corresponded to the estimated pA2 value of 8-ala-angiotensin II against angiotensin II (table 2). In the same equilibration period, the pAlo value of 8-ala-angiotensin II against angiotensin II was found to be 7.41 + 0.07. The difference pA2 - pA 1o was 0.91. The dose-response curves to angiotensin I before and after addition of 8-ala-angiotensin II to the bath were also determined to be parallel (fig. 2). The dose ratios of angiotensin I in the presence of 8-ala-angiotensin II and log k2 values were calculated and summarized in table 1. The log k2 values were also found to be similar to the pA2 value (table 2). The difference between pA2 and pA l0 values was found to be 0.92. When equal tensions were induced by angiotensin II, NE and serotonin, the addition of 8-alaangiotensin II to the bath produced a relaxation within 2 min in those aortic strips contracted by angio-
R.K. Tiirker et al., Competitive antagonism o f angiotensin
287
Control
2.0-
A
& Responses in presence
of ( 8 - A l a ) A n g i o t e n s i n WI.S5xlO-SM
O
O Responses in presence
of (8-Ala) Angiotensin ]~ 6.2xlO-SM
1.6-
1.2-
0.8-
0.4-
I
3.61 xlO -9
I
7.23 xlO-9
I
I
1.44 xlO-8
2.89 xlO "8
I
5,78x10 -8
I
2.89x10 -7
M o l a r concentrations of angiotensin 1T
Fig. 1. Log dose--response curve of isolated rabbit aorta to angiotensin II before and after addition of 8-ala-angiotensin II. Each p o i n t represents the m e a n of 10 experiments + standard error o f the mean. Tension (g) indicates changes in tension.
Table 1 A n t a g o n i s m by 8-ala-angiotensin II o f angiotensins I and II responses on rabbit isolated aortic strips. Agonists
Angiotensin I
Bath concentration o f 8-ala angiotensin II (molar)
Dose ratios ( m e a n ± S.E.) a
log k2 (mean ± S.E.) a,b
7.75 X 10 -9
4.85 ± 0.386 (4) n (3.80-5.60) r
8.688 ± 0.852 (4) n (8.5577-8.7447) r
1.55 × 10 -a
8.20 ± 0.129 (4) (7:90-8.50)
8.666 ± 0.007 (4) (8.6480-8.6845)
X 10 -8
18.95 ± 0.523 (4) (17.90-20.20)
8.762 ± 0.0126 (4) (8.7365 - 8 . 7 9 2 0 )
7.75 X 10 .-9
3.26 ± 0.027 (4) (3.20-3.33)
8.464 ± 0.005 (4) (8.4531-8.4781)
1.55 × 10 - s
5.36 ± 0.165 (4) (5.00-5.75)
8.447 ± 0.016 (4) (8.4118-8.4824)
3.10 × 10- s
9.065 ± 0.134 (4) (8.75-9.40)
8.413 ± 0.007 (4) (8.3979-8.4329)
18.127 ± 0.497 (4) (17.21-19.30)
8.440 ± 0.012 (4) (8.4171-8.4701)
3.1 Angiotensin II
6.2
X 10 - s
a n = n u m b e r o f experiments; r = range. b log k 2 (dose ratio - 1) = log (antagonist concentration)
R.K. Tiirker et al., Competitive antagonism of angiotensin
288
5min I I
Table 2 PA2 and PAl0 values o f 8-ala-angiotensin II against angiotensins 1 and II on normal rabbit aortic strips. Agonists
pA 2 ± S.E.a
pAlo ± S.E.a
pA2_pA]o
Angiotensin II
8.32 ± 0.08 (10)
7.41 ± 0.07 (9)
0.91
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a Number of experiments in parentheses.
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8alo AIT Solo ATr IOng/ml 20ng/ml Fig. 4. Antagonistic effect of 8-ala-angiotensin II on the responses of angiotensin II on the isolated continuously superfused rabbit aorta and colOn. Upper tracing shows the tension changes of aorta. Lower tracing shows the tension changes of colon.
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Fig. 2. Log d o s e - r e s p o n s e curve of isolated rabbit aorta to angiotensin 1 before and after addition of 8-ala-angiotensin 1I. Each point represents the mean of 10 experiments + standard error of the mean. Tension (g) indicates changes in tension.
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32 ng/ml
32 ng/ml
32 ng/ml
o
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Fig. 3. Tension changes in the isolated rabbit aorta to angiotensin I1 (A II), serotonin (S), and norepinephrine (NE) and the effect of 8-ala-angiotensin II.
R.K. Tiirker et al., Competitive antagonism of angiotensin
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289
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Fig. 5. Tension changes in the rat ascending colon to angiotensin II before and after addition of 8-ala-angiotensin II. 2.8H
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Responses in presence of ( 8 - A l a ) A n g i o t e n s i n TT 155x10 8M
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Fig. 6. Log dose-response curve of isolated rat ascending colon to angiotensin II before and after addition of 8-alaangiotensin II. Each point represents the mean of 10 experiments + standard error of the mean. Tension (g) indicates changes in tension.
5 min I I
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0.25 pg/kg
0.5 pg/kg
0.25 pg/kg 8-olo ATT 5 pg/kg
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Fig. 7. Blood pressure (upper tracing) and intraluminal pressure of terminal ileum (lower tracing) of the pentobarbital anesthetized cat. Effect of angiotensin II (A II), angiotensin I (A I), 8-ala-angiotensin II and dihydroergotamine (DHE).
290
R.K. Tiirker et al., Competitive antagonism of angiotensin
tensin II, while no relaxation was observed in the strips contracted by NE or serotonin (fig. 3). In the isolated continuously superfused rabbit aorta and rat colon, 8-ala-angiotensin II also antagonized the effect of angiotensin II in both assay organs (fig. 4), in a dose-dependent way. 3.2. Isolated rat ascending colon
8-Ala-angiotensin II antagonized the myotropic action of angiotensin II in this preparation (fig. 5). The dose-response relationship of angiotensin II before and after 8-ala-angiotensin II at molar concentrations of 1.55 × 10-8 to 3.1 × 10-8 showed parallel curves (fig. 6). Maximum response to angiotensins in both smooth muscle preparations can be obtained in the presence of 8-ala-angiotensin II. 3.3. In situ cat blood pressure and ileal motility Angiotensins I and II given intravenously in doses of 50 to 500 ng/kg produced pressor responses and decreased intraluminal pressure and motility of the ileum in a dose~iependent way. A single injection of 1 to 10 #g/kg of 8-ala-angiotensin II did not induce such a response and did not alter subsequent responses to angiotensins I and II (fig. 7). 4. DISCUSSION The antagonistic action of 8-ala-angiotensin II to angiotensin II in the isolated guinea-pig ileum has been reported by Khairallah et al. (1970). The present study clearly shows that 8-ala-angiotensin II is a competitive antagonist of angiotensins I and II. The following criteria which have been advocated by Schild (1957) appear to satisfy the concept of competitive antagonistic action of 8-ala-angiotensin II. First, the log dose-response curves of angiotensins I and II on the aorta are shifted progressively to the right and are parallel with each other. A similar parallel shift to the right in the rat ascending colon indicates that 8-ala-angiotensin II also competitively antagonizes the myotropic effect of angiotensin II in this preparation. Secondly, the calculated log (dose ratio - 1 ) against log antagonist concentration was linear and there was no change in the values of log k2 within the dose ranges of 8-ala-angiotensin II used. This also in-
dicates competitive antagonism. Again, the difference pA2 - pAlo was found to be 0.91 for angiotensin II and 0.92 for angiotensin I which are very near to the theoretical value of 0.95 expected for competitive antagonism (Schild, 1957). The antagonistic action of 8-ala-angiotensin II was established within 2 min after exposure with the strips. However, this antagonism was easily washed out and complete recovery occurred within 45 min. The antagonistic potency of 8-ala-angiotensin II was found to be very high. The myotropic effect of 7.23 X 10-9 molar angiotensin II was completely antagonized by 7.75 X 10-9 molar 8-ala-angiotensin II. The effect of 8-ala-angiotensin II is specific for angiotensins I and II, since it did not inhibit the myotropic action of NE or serotonin even at very high concentrations. This finding also strongly indicates that angiotensin II has its own specific receptors in vascular smooth muscle cells. The question arises as to how 8-ala-angiotensin II interacts with angiotensins at the receptor site in aortic muscle. This may be due to the fact that the myotropic activity of angiotensin is due to a phenyl group in the position 8 and a free carboxyl terminus both of which interact with receptor (Khairallah et al., 1970). 8-ala-angiotensinII covers the receptor sites, preventing other molecules from interacting, but since it does not contain a phenyl side group, no contractile response is obtained. This study also clearly shows that angiotensins I and II act at the same receptor sites in the vascular wall. Recently Aiken hnd Vane (1970) reported that angiotensin I has a direct action on specific angiotensin I receptors, a direct but weak action on angiotensin II receptors and an indirect effect due to the variable conversion of decapeptide to angiotensin II. Since 8-ala-angiotensin II competitively antagonizes the myotropic effect of both polypeptides, it can be assumed that the same receptor system of the smooth muscle cell may be responsible for the effect of both polypeptides. It should be emphasized, however, that 8-alaangiotensin II does not antagonize the pressor and intestinal inhibitory effect of angiotensin when given intravenously to anesthetized cats. One possible explanation is that 8-ala-angiotensin II is rapidly metabolized and insufficient peptide reaches the receptor sites to block responses to angiotensins.
R.K.Tiirker et al., Competitive antagonism o f angiotensin ACKNOWLEDGEMENTS This investigation was supported by U.S. Public Health Service Research Grant HE--6835 from the National Institute of Heart and Lung Diseases.
REFERENCES Aiken, J.W. and J.R. Vane, 1970, The renin-angiotensin system: Inhibition of converting enzyme in the isolated tissues, Nature 288, 30. Cummings, J.R., A.N. Welter, J.L. Grace and W.D. Gray, 1969, Angiotensin blocking actions of guancydine, J. Pharmacol. Exptl. Therap. 170, 334. Furchgott, R.F. and S. Bhadrokom, 1953, Reaction of strips of rabbit aorta to epinephrine, isopropylarteranol, sodium nitrate and other drugs, J. Pharmacol. Exptl. Therap. 108, 129. Godfraind, T., A. Kaba and P. Polster, 1966, Specific antagonism to direct and indirect action of angiotensin on the isolated guinea-pig ileum. Brit. J. Pharmacol. 28, 93. Khairallah, P.A., I.H. Page, F.M. Bumpus and R.K. Tiirker, 1966, Angiotensin tachyphylaxis and its reversal, Circulation Res. 19,247. KhairaUah, P.A., A. Toth and F.M. Bumpus, 1970, Analogs of angiotensin II. Mechanism of receptor interaction, J. Med. Chem. 13,181.
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Page, I.H. and F.M. Bumpus, 1961, Angiotensin, Physiol. Res. 41,331. Park, W.K., R.R. Smeby and F.M. Bumpus, 1967, Synthesis of [5-Isoleucine, 8-alanine] -angiotensin II by the solution method synthesis and the solid phase method synthesis, Biochim. 6, 3458. Regoli, D. and J.R. Vane, 1964, A sensitive method for the assay of angiotensin, Brit. J. Pharmacol. 23,351. Schild, H.O., 1947, pA, A new scale of the measurement of drug antagonism, Brit. J. Pharmacol. 2, 189. Schild, H.O., 1949, pA x and competitive drug antagonism, Brit. J. Pharmacol. 4,277. Schild, H.O., 1957, Drug antagonism and PAx, Pharmacol. Rev. 9,242. Tiirker, R.K. and S.A. Kayaalp, 1967a, Effect of lidoflazine on norepinephrine, angiotensin and serotonin responses in isolated smooth muscle preparations, Experientia 23,647. Tiirker, R.K. and S.O. Kayaalp, 1967b, Inhibitory effect of angiotensin on intestinal motility of the cat and its relation to sympathetic nervous system, Arch. Intern. Physiol. Biochim. 75,735. TiJrker, R.K., S.O. Kayaalp and A. Ozer, 1968, Further studies on lidoflazine (R 7904), Arzneimittel-Forsch. 18, 1209. Tiirker, R.K., 1969, Possible postganglionic adrenergic effect of angiotensin in the isolated perfused cat intestinal segment, Arch. Intern. Physiol. Biochim. 77,587. Vane, J.R., 1964, The use of isolated organs for detecting active substances in the circulating blood, Brit. J. Pharmacol. 23,360.