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An attempt to differentiate the effects of angiotensin II, substance P and bradykinin on the field-stimulated guinea-pig vas deferens
European Journal of Pharmacology, 56 (1979) 21--29
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© Elsevier/North-Holland Biomedical Press
AN ATTEMPT TO DIFFERENTIATE THE EFFECTS OF ANGIOTENSIN II, SUBSTANCE P AND BRADYKININ ON THE FIELD-STIMULATED GUINEA-PIG VAS DEFERENS GERHARD ZETLER and ECKEHARD KAMPMANN
Abteilung fiir Pharmakologie der Medizinischen Hochschule Liibeck, Ratzeburger Allee 160, D-2400 Liibeck, Federal Republic of Germany Received 31 October 1978, revised MS received 4 January 1979, accepted 7 February 1979
G. ZETLER and E. KAMPMANN, An attempt to differentiate the effects of angiotensin II, substance P and bradykinin on the field-stimulated guinea-pig vas deferens, European J. Pharmacol. 56 (1979) 21--29. The ability of the angiotensin antagonists 1-Sar,8-Ala-angiotensin II (saralasin) and 1-Sar,8-Leu-angiotensin II (sarleusin) and the bradykinin-potentiating peptide B (BPP) to modify the twitch-enhancing effect of angiotensin II, bradykinin, and substance P, was studied in the isolated field-stimulated guinea-pig vas deferens. The twitchenhancing effect of angiotensin, bradykinin and substance P underwent tachyphylaxis which was strongest after angiotensin. Saralasin and sarleusin were without influence on the twitc h response and antagonized the effect of angiotensin, but not that of bradykinin or substance P, respectively. The features of the antagonism to angiotensin were compatible with the notion that saralasin is a competitive and sarleusin a dual antagonist. BPP did modify either the twitch response or the effects of angiotensin, bradykinin, and substance P. In the non-stimulated vas, the contracting effect of noradrenaline did not undergo tachyphylaxis and was not modified by angiotensin, bradykinin, or substance P. It is concluded that there exist in the guinea-pig vas specific peptide receptors, one of them having the properties of a "typical" angiotensin receptor. Bradykinin potentiator
Vas deferens
Angiotensin
1. Introduction In the isolated guinea-pig vas deferens the twitch response to a field stimulus is enhanced by substance P (SP), bradykinin and angiotensin II (Von Euler and Hedqvist, 1974a; SjSstrand and Swedin, 1974). A quantitative study of twelve peptides (Zetler, 1977) revealed differences in the twitch-enhancing effect: active were SP, eledoisin, bradykinin, angiotensin I and II, glucagon and carnosine; inactive were neurotensin, somatostatin, TRH, gastrin, pentagastrin, caerulein and cholecystokinin-octapeptide (results on the last two peptides are to be published). Furthermore, the active peptides differed as to potency (ECs0) and effectiveness (Ema~). The sum of these findings pointed to the possible existence of specific peptide receptors on the relevant structures of the guinea-pig vas. Muscarinic
Bradykinin
Substance P
Saralasin
receptors exist there since atropine antagonized the twitch-enhancing effect of carbachol in a competitive fashion with pA2 8.027, but left the SP effect intact (Zetler, 1977). From experiments using phenoxybenzamine, tetracaine and tetrodotoxin it was concluded that the peptides, acetylcholine, and carbachol may enhance the twitch by a prejunctional mechanism connected with the intramural neurons of the vas (Von Euler and Hedqvist, 1974b; Zetler, 1977). However, there are also good arguments for a postjunctional mechanism of action of these drugs except for angiotensin which may exert mainly a prejunctional action (SjSstrand and Swedin, 1974). Noradrenaline is perhaps the motor transmitter involved in the rapid twitch response of the field-stimulated guinea-pig vas deferens (Swedin 1971; Furness, 1974), although this was denied by Ambache and Zar
22
(1971), Ambache et al. (1972) and Von Euler and Hedqvist (1975). Complicated synaptic events also involving potassium and prostaglandins have been envisaged (Swedin, 1971; SjSstrand and Swedin, 1974; Stj~rne, 1976, 1977). The present experiments therefore aimed at the following restricted question: could the effects of SP, angiotensin II and bradykinin be differentiated with the aid of antagonists and a potentiator? To find o u t whether the angiotensin effects on the vas were "typical" we selected from the large series of specific angiotensin antagonists (cf. Regoli et al., 1974) the competitive antagonist 1-Sar,8-Ala-angiotensin II (Saralasin) and 1-Sar,8-Leu-angiotensin II (Sarleusin) which in isolated rat stomach and rabbit aortic strips acts as a dual (competitive and metactoid; Van den Brink and Lien, 1977) rather than competitive antagonist. It is already clear that saralasin is a competitive angiotensin antagonist for the presynaptic receptors of the rabbit pulmonary artery (Endo et al., 1977). A third pharmacological tool used in the present study was the bradykinin-potentiating peptide B (BPP; Pyr-Gly-Leu-Pro-Pro-Arg-ProLys-Ile-Pro-Pro). This compound strongly enhances the bradykinin effect on the isolated guinea-pig ileum (but only weakly that on the rat uterus) and inhibits the angiotensin I-converting enzyme and the plasma kininase (Kato and Suzuki, 1971; Ueda et al., 1971). BPP does not change the rat uterus contracting effect of angiotensin II (Ueda et al., 1971). 2. Materials and methods
The vasa deferentia of freshly killed guinea pigs weighing 250--400 g were carefully dissected free from their mesenteric connections and m o u n t e d between two vertical platinum electrodes in an organ bath containing 5 ml Tyrode solution of the following composition (mM): 137 NaC1, 2.7 KCI, 12 NaHCO3, 0.4 N a H 2 P O 4 - 1 H 2 0 , 1.8 CaC12-2 H20, 0.5 MgCl~ • 6 H20, 5.5 glucose • 2 H20. The bath fluid was kept at 37°C and gassed with car-
G. Z E T L E R , E. K A M P M A N N
bogen (95% 02 + 5% CO2). For the experiments with exogenous noradrenaline in the non-stimulated vas the bath solution contained Ca2+-EDTA, 10 -4 M. The load on the vas was 0.3 g and the contractions were recorded isotonically using a displacement transducer (Sachs/Freiburg) and a Servogor recorder (Metrawatt/Nfrnberg). Field stimulation was applied every minute for 3 sec and consisted of monophasic square pulses of 2--5 Hz, 2 msec in duration and 12 V in strenght (Stimulator II, Sachs/Freiburg). A single 30 Hz stimulus was applied several times during the course of an experiment to test the maximum twitch response of a given preparation. After an adaptation period lasting 30--90 min (with field stimulation) the first concent r a t i o n - r e s p o n s e curve to a given agonist drug (see fig. 1) was established and followed by a second curve after a recovery period lasting at least 30 min. The dosage was cumulative with concentrations increased immediately after every second twitch response since additional twitches did not reveal a further increase in the drug effect. This effect, i.e., the increase in twitch response, was expressed as a fraction of the difference between the response to 2--5 Hz and either the response to a 30 Hz stimulus (table 1) or the maximum effect (Emax) of a concentration--response curve. As suggested b y Waud (1968) the terms effectiveness (Emax) and p o t e n c y (pD2)will be used. pD2 is the negative logarithm of the molar concentration (ECs0) producing the half-maximum effect o f an agonist. The EC20, ECs0 and ECs0 were calculated from a log concentration--effect curve after linearization b y probit transformation and estimation of the regression function with the least squares method. The slope, i.e., than ~ of the linear part of a sigmoid log concentration-effect curve refers to a system of coordinates where Emax of the reference curve is 3.3 decades of length of the logarithmic abscissa. Slope = E m a x " 0.6 • 3.3/(log ECs0--1og EC20). The effects of the angiotensin antagonists and the bradykinin potentiator were evaluated b y means of a second concentration--response
PEPTIDE E F F E C T S ON T H E VAS D E F E R E N S
23
3. Results
curve, while the first curve served as control. As described in Results, intrinsic reductions in sensitivity occurred b e t w e e n two consecutive concentration-response curves and were allowed for b y correcting the controls with the aid of factors which will be mentioned. In experiments with antagonists the m e t h o d of Van den Brink and Lien (1977) was used to calculate the pA2 from the shift of the concentration-response curve, and the pD2' from the depression of its maximum. For statistical evaluation, P < 0.05 was set as threshold of probability. Student's t-test was applied to paired differences: t = k/s;. Multiple means were compared with Bartlett's test and the Scheff~ test. In the case of nonhomogeneous variances the Wilcoxon--Mann-Whitney ranking method was used. The drugs were angiotensin amide (Ciba), 1-Sar,8-Ala-angiotensin II (Serva), 1 -Sar,8-Leuangiotensin II (Serva), bradykinin triacetate (Serva), bradykinin-potentiating peptide B (Bachem), substance P (Bachem), and (--)noradrenaline (Hoechst). Angiotensin amide served as a valid though generally somewhat less active substitute for angiotensin II (Gross, 1971), and the term angiotensin will be used for the sake of shortness. A
i
A A
B
3.1. The agonists The agonists angiotensin, bradykinin, and SP produced concentration
1). The question of a possible direct smooth muscle-stimulating effect of the agonists was studied in preparations which reacted to a low agonist concentration with a normal enhanced twitch response. In the absence of field stimulation none of the three peptides induced a contraction of the vas at concentrations up to 300 pg/ml (2--3 • 10-4 M; neg log: 3.5--3.7).
i
BB:
~
.: -
_-
Fig. 1. Twitch-enhancing effect of angiotensin (A and A A ; 4 Hz) and substance P (B and BB; 3 Hz), in the absence (A and B) and presence of 2 • 10-7 M sarleusin ( A A and BB). One field stimulus per minute. Sarleusin was added to the bath at the arrow. The vertical dashes indicate the steps of the cumulative dosage of a given agonist (/~g/ml): A, 0.01, 0.03, 0.1, 0.3, 1 and 3; AA, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 and 300; B and BB, 0.02, 0.1, 0.5, 2.5, 6.5 and 10.5. Ordinate scale: 20 m m between two thick horizontal lines.
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G. Z E T L E R , E. K A M P M A N N
TABLE 1 P o t e n c y (pD2), slope, and effectiveness (Emax) as o b t a i n e d f r o m c o n c e n t r a t i o n - - r e s p o n s e curves for angiotensin, b r a d y k i n i n and s u b s t a n c e P ( x -+ S~x). Emax refers to the t w i t c h r e s p o n s e as o b t a i n e d b y a field stimulus at 30 Hz (this r e s p o n s e was assigned a value o f 100).
Angiotensin Bradykinin Substance P
n
pD 2
Slope
Emax
8 8 8
7.28 -+ 0.151 6.67 ± 0.063 I 7.19 -+ 0.105
1.31 -+ 0.09 2.13 ± 0.19 1 1.16 ± 0.06
48 ± 7 69 -+ 4 1 89 -+ 3 2
Statistical significance exists for the d i f f e r e n c e s f r o m b o t h angiotensin and s u b s t a n c e P 1, and for t h a t f r o m angiotensin 2
3.2. C o n s e c u t i v e curves
c o n c e n tra tio n - - r e s p o n s e
According to the protocol of this study the effect of an antagonist was to be evaluated by means of the second of two consecutive doseresponse curves of a given agonist. Hence, the possibility of a desensitization had to be envisaged. The rapid development of specific tachyphylaxis is typical for angiotensin (review: Stewart, 1974) and was also ob-
i0010
served for the twitch-enhancing effect of this peptide in the hypogastricus-stimulated guinea-pig vas (Benelli et ah, 1964). In the present experiments reduced sensitivity to all three agonists was prominent during the second run of the concentration--response curve (fig. 2 and 3, table 2). This was concluded from the decrease in pD2, which meant that the ECs0 increased by a factor 3.63 (= antilog 0.56) in the case of angiotensin and by 1.45 (= antilog 0.16) for both bradykinin
I00-
/t
nglml
Fig. 2. T w i t c h - e n h a n c i n g e f f e c t o f b r a d y k i n i n (e, o) a n d s u b s t a n c e P (A, A). T w o successive c o n c e n t r a t i o n - r e s p o n s e curves each are s h o w n for each p e p t i d e (first curve, -= ~ and • A; s e c o n d curve, o . . . . . -o and A_ . . . . .A). T h e p o i n t s are m e a n s (x ± Sx, n = 8 for each curve). Abscissa (log): peptide c o n c e n t r a t i o n . Ordinate: Ema x • 100, referring t o t h e m a x i m u m e f f e c t o f t h e related first curve.
Fig. 3. M o d i f i c a t i o n o f t h e t w i t c h - e n h a n c i n g e f f e c t o f a n g i o t e n s i n b y saralasin a n d sarleusin. C o n c e n t r a t i o n - - r e s p o n s e curves are s h o w n for a n g i o t e n s i n (~±Sx): first c o n t r o l curve ( e ~, n f 8 ) ; s e c o n d c o n t r o l curve (o o, n = 8 ) ; second curves in p r e s e n c e o f 2.2 • 10 -7 M saralasin (A ~, n = 9), 5.2. 10 -8 M sarleusin (o . . . . . . o, n = 5), or 2.1" 10 -7 M sarleusin (o . . . . . . o, n = 6). Abscissa: c o n c e n t r a t i o n o f angiotensin. Ordinate: Ema x • 100, referring t o t h e m a x i m u m e f f e c t o f t h e first c o n t r o l curve.
P E P T I D E E F F E C T S ON T H E V A S D E F E R E N S
25
TABLE 2 M e a n d i f f e r e n c e s in p o t e n c y (PD2), s l o p e , a n d e f f e c t i v e n e s s (Emax) b e t w e e n t h e first a n d t h e s e c o n d c o n c e n t r a t i o n - r e s p o n s e c u r v e in t h e s a m e p r e p a r a t i o n . B o t h t h e p D a a n d t h e slope o f t h e first c u r v e are p r e s e n t e d in t a b l e 1. Emax refers to the r e l a t e d first c u r v e (Emax = 1.0).
Angiotensin Bradykinin Substance P
n
pD2
Slope
Emax " 1 0 0
8 8 8
--0.56 ± 0.13 1 --0.16 ± 0.06 1 --0.16 ± 0.06 I
+0.09 ± 0.17 - - 0 . 3 7 ± 0.11 1 --0.03 ± 0.05
--14 + 7 --11 + 2 1 --2 ± 2
I Statistically s i g n i f i c a n t d i f f e r e n c e f r o m zero.
and substance P. Emax remained constant for substance P and was somewhat reduced for angiotensin and bradykinin. There was no change in slope for either angiotensin or substance P, whereas the second curve was less steep for bradykinin.
3.3. The angiotensin antagonists In contrast to angiotensin, neither saralasin nor sarleusin (both at the concentration 2 • 10 -T M) produced any twitch-enhancing effect (fig. 1). Saralasin shifted the concentrat i o n - r e s p o n s e curve of angiotensin to the right but did not depress its maximum, whereas sarleusin also reduced the m a x i m u m (figs. 1 and 3). The saralasin effect was consistent with the notion that this analog is a competitive angiotensin antagonist. The effect of sarleusin points to a dual, i.e., competit, ive and metactoid (cf. Van den Brink and Lien, 1977) antagonist--receptor interaction as
already found for the isolated rat stomach strip (Regoli et al., 1973). According to the values of pA2 (table 3) the antagonistic power of sarleusin was greater than that of saralasin. Considering the pA2 and pD2, values, no statistically significant differences exist between the effects of the two concentrations of sarleusin. Both antagonists were practically inactive against bradykinin and substance P (fig. 1, table 4). Saralasin, it is true, reduced the potency of bradykinin by a factor 1.86 (antilog 0.27) but this effect was of minor importance since it was (a) not duplicated by sarleusin, and (b) very weak when compared with that against angiotensin (factor 6.6; sarleusin, factor 20.8). While the result that substance P was even somewhat more potent (factor 1.78) under the influence of sarleusin does not carry much weight it does stress the observation of a missing antagonism.
TABLE 3 Saralasin a n d s a r l e u s i n as a n t a g o n i s t s o f a n g i o t e n s i n . T h e r e s u l t s (R- ± SR-x)were o b t a i n e d b y c o m p a r i n g t w o c o n c e n t r a t i o n - r e s p o n s e c u r v e s in e a c h e x p e r i m e n t , t h e first o n e a f t e r c o r r e c t i o n a l l o w i n g f o r t a c h y p h y l a x i s (see table 2 a n d t e x t ) . Emax b e l o n g s to t h e s e c o n d curve a n d refers t o t h e first o n e (Emax = 1). If n o t i n d i c a t e d b y n.s., d i f f e r e n c e s b e t w e e n m e a n s w i t h i n a c o l u m n are s t a t i s t i c a l l y s i g n i f i c a n t .
Saralasin 2.2 • 10 -7 M S a r l e u s i n 5.2 - 10 -8 M S a r l e u s i n 2.1 • 10 -7 M
n
PA2
9 5 6
7.34 ± 8.20 ± 7.98 ±
0.21 0.34 0.13 ~
n.s.
Emax " 1 0 0
pD~
102 ± 5 72 ± 4 51 ± 6
6 . 8 6 ± 0.11 ~ 6.66 ± 0.11
n.s
26
G. ZETLER, E. KAMPMANN
TABLE 4 Influence of saralasin (2.2 " 10 -7 M) and sarleusin (2.1 • 1 0 - 7 M) on the potency of bradykinin and substance P. The results ~-+ S~-x)were obtained by comparing two concentration--response curves in each experiment, the first used as reference curve after correction allowing for tachyphylaxis (see table 2 and text).
Bradykinin Substance P
Reference curve pD2
Saralasin (n = 8) change in PD2
6.17 -+ 0.10 6.32 ± 0.08
--0.27 -+ 0.05 1
6.83 ± 0.08 7.01 +- 0.16
+0.06 -+ 0.09
Sarleusin (n = 6) change in pD2 +0.03 ± 0.05 +0.25 + 0.04 I
i The difference from zero is statistically significant.
3.4. The bradykinin potentiating peptide B (SPP) BPP (1.5 • 10 -s M, given 5 min prior t o an agonist) was w i t h o u t e f f e c t o n t h e t w i t c h response and did n o t m o d i f y the p o t e n c y o f angiotensin, b r a d y k i n i n , and substance P (table 5). H o w e v e r , in t h e isolated guinea-pig ileum u n d e r t h e same e x p e r i m e n t a l c o n d i t i o n s b u t w i t h o u t field s t i m u l a t i o n , BPP ( 4 . 2 . 10 - 6 M and 1 . 7 " 1 0 - s M , given 5 m i n p r i o r t o b r a d y k i n i n ) increased t h e c o n t r a c t i n g e f f e c t o f 8 • 10 -9 M b r a d y k i n i n b y a f a c t o r o f 2.5 or 4, respectively.
TABLE 5 Potency (PD2) of angiotensin, bradykinin and substance P in the absence (reference curve as defined in table 4) and presence of the bradykinin potentiating peptide B (18 t~g/ml). n.s.: the difference from zero is not statistically significant. n
Reference
Change in pD2
curve
PD2 Angiotensin Bradykinin Substance P
6 8 6
7.03 +- 0.04 6.79 ± 0.11 7.08 -+ 0.16
--0.14 -+ 0.12 n.s. --0.05 +- 0.07 n.s. +0.17 -+ 0.09 n.s.
3.5. The non-stimulated vas A c c o r d i n g to Sj5strand ( 1 9 7 3 ) , V o n Euler and Hedqvist (1974a), SjSstrand and Swedin ( 1 9 7 4 ) and Blackwell et al. ( 1 9 7 8 ) s u b s t a n c e P b u t n o t angiotensin (SjSstrand and Swedin, 1 9 7 4 ) p o t e n t i a t e s t h e response o f t h e vas t o a field stimulus b y a partial d e p o l a r i z a t i o n o f t h e muscle cell m e m b r a n e w h i c h w o u l d facilit a t e and p r o m o t e a c t i o n potentials. A mechanism like this o n e can be e x p e c t e d also t o e n h a n c e t h e e f f e c t o f a s t i m u l a t o r y d r u g with a p o s t s y n a p t i c p o i n t o f a t t a c k at t h e s m o o t h muscle cell. T h e r e f o r e , e x p e r i m e n t s using p r e p a r a t i o n s w i t h o u t field s t i m u l a t i o n were d e v o t e d t o the q u e s t i o n o f w h e t h e r angiotensin and substance P w o u l d sensitize t h e vas t o e x o g e n o u s noradrenaline. When prepared as usual, t h e vas was o f e x t r e m e l y low sensitivity t o n o r a d r e n a l i n e and gave erratic results. The sensitivity c o u l d be increased b y o p e n i n g t h e vas and r e m o v i n g the m u c o s a b y scratching as suggested b y T h o a and Meangwyn-Davies (1968). A f t e r an a d a p t a t i o n period o f 3--4 h with n o r a d r e n a line doses every 5 min, these p r e p a r a t i o n s r e s p o n d e d t o n o r a d r e n a l i n e in a c o n c e n t r a t i o n ~ l e p e n d e n t fashion. The pD2 o f n o n c u m u l a t i v e doses o f n o r a d r e n a l i n e was 4.96 + 0.07 (1~+ s x ; n = 8 ) w h i c h indicates a very low p o t e n c y . N o desensitization o c c u r r e d w h e n t w o or m o r e c o n c e n t r a t i o n - - r e s p o n s e
PEPTIDE EFFECTS
ON THE VAS DEFERENS
TABLE 6 P o t e n c y ( p D 2 ) o f n o r a d r e n a l i n e in t h e n o n - s t i m u l a t e d v a s d e f e r e n s , in t h e a b s e n c e ( r e f e r e n c e c u r v e ) a n d p r e s e n c e o f a n g i o t e n s i n (2 p g / m l = 1 . 9 4 • 1 0 -6 M ) o r s u b s t a n c e P (2 p g / m l = 1 . 4 8 • 1 0 -6 M ) , r e s p e c t i v e l y . R e s u l t s are m e a n s : ~- +- S~x. n . s . : t h e d i f f e r e n c e f r o m z e r o is n o t s t a t i s t i c a l l y significant. n
Reference curve pD2
C h a n g e in p D 2
4 4
4 . 8 3 -+ 0 . 1 5 5 . 1 0 -+ 0 . 1 0
+ 0 . 0 3 -+ 0 . 0 5 n . s . - - 0 . 0 7 -+ 0 . 0 6 n . s .
Angiotensin Substance P
curves were established in the same preparation. Neither angiotensin nor substance P, when added at high concentrations to the organ bath 1 min prior to every single dose of noradrenaline, modified the concentration--effect curve of noradrenaline with regard to p o t e n c y (table 6). There was no change in Emax either.
4. Discussion The peptides saralasin, sarleusin and BPP, when given alone at high concentrations, did n o t enhance the twitch response. They therefore add to the number of peptides devoid of this activity and mentioned in the Introduction. This result supports the notion that we are dealing with a specific pharmacological effect. This view is stressed by the powerful antagonism of saralasin and sarleusin against the twitch-enhancing effect of angiotensin. Obviously, these t w o specific angiotensin antagonists proved to have affinity to, b u t no activity on, the angiotensin-sensitive structure as was observed in other preparations (Regoli et al., 1974). The pattern of antagonism pointed to the competitive t y p e for saralasin and the dual t y p e (cf. Van den Brink and Lien, 1977) for sarleusin. This, too, is in accord with results obtained with these
27
antagonists in the isolated rat stomach and rabbit aortic strip (Regoli et al., 1973, 1974). Finally, both antagonists were w i t h o u t any clear-cut effect against bradykinin and substance P. The conclusion is that the typical specific angiotensin receptor exists in the structures which are essential for the twitchenhancing effect of peptides. This would explain w h y the well-known tachyphylaxis to angiotensin (Stewart, 1974; Paiva et al., 1977) also occurred in the present experiments. There also was desensitization, though to a lesser extent, to bradykinin and substance P. However, in spite of an equal change in potency (pD:), effectiveness (Emax) was reduced for bradykinin b u t n o t for substance P (table 2). This qualitative difference between the effects of the two peptides together with that in the steepness of the normal concentrat i o n - e f f e c t curves (fig. 2, table 1), points to u n k n o w n differences in the mechanisms leading to, or triggered by the activation of the receptors for bradykinin and substance P in the field-stimulated vas deferens. Transmitter exhaustion -- one mechanism suggested for the production of tachyphylaxis to angiotensin (Stewart, 1974) -- was probably n o t important in the present experiments since the desensitization to both bradykinin and substance P was less in spite of their effectiveness being superior to that of angiotensin (fig. 1, table 1). It must be stressed that in our experiments without field stimulation the vas produced no tachyphylaxis to noradrenaline. Hence, it is not likely that desensitization to the peptides involves the postsynaptic noradrenaline receptor. This receptor can also be excluded as the site of the mechanism for the twitchenhancing effect of the peptides. This must be concluded from the finding that even very high concentrations of both angiotensin and substance P did not modify the concentration--effect relationship in the non-stimulated vas (table 6). As far as angiotensin is concerned, our results agree with those of Benelli et al. (1964) w h o found that this peptide enhanced the twitch response to stimulation
28
of the hypogastric nerve b u t failed to potentiate the response of the vas to exogenous noradrenaline. However, there is also the view that substance P (and bradykinin) enhances the response of the guinea-pig vas to a field stimulus or to nerve stimulation by partial depolarization of the muscle membrane (SjSstrand, 1973; Von Euler and Hedqvist, 1974a; SjSstrand and Swedin, 1974). Furthermore, a purely postjunctional mechanism enhancing the responsiveness of the muscle cells has recently been postulated for the twitchenhancing effect of substance P in the mouse vas deferens (Blackwell et al., 1978). A muscle cell with an increased responsiveness to any depolarizing stimulus can be expected to show a facilitated response to a directly stimulating drug. However, in our experiments as in those of Benelli et al. (1964), the responsiveness to exogenous noradrenaline remained unchanged and was, in fact, b y no means regularly enhanced in the experiments of Blackwell et al. (1978). Hence, it is unlikely that the peptides influenced the postjunctional effector systems b e y o n d the noradrenaline receptor. Consequently, presynaptic receptors in the vas might be the site of action for the peptides active in our present experiments. This would explain the differences between the field-stimulated and the non-stimulated vas as regards the peptide effect in the former and its absence towards noradrenaline in the latter preparation. The receptors involved are specific since they are sensitive to a limited number of peptides. The use of BPP did not help to differentiate the peptide effects. The results obtained with the guinea-pig ileum proved that our sample of BPP, at the concentration used, was capable of potentiating the bradykinin effect in this preparation. The failure in the field-stimulated vas can have two causes: low or missing kininase activity of the vas tissue, or the absence of a receptor for BPP. We have no information about the kininase activity of the vas b u t draw attention to the view of Camargo
G. ZETLER, E. KAMPMANN
and Ferreira (1971) and Ufkes et al. (1977) that the bradykinin-potentiating effect of two pentapeptides in isolated smooth muscles is not due to kininase inhibition b u t to an increased affinity of the bradykinin receptor. The second possible cause, the absence of a BPP receptor in the vas, is reminiscent of differences in sensitivity to BPP of various bradykinin effects in isolated smooth muscle organs (Ufkes et al., 1976, 1977). Considering the suggestion that potentiating agents produce an allosteric transition of the bradykinin receptor (Camargo and Ferreira, 1971) one might think of differences between the bradykinin receptors of the guinea-pig ileum and those of the field-stimulated guinea-pig vas deferens.
Acknowledgement We are grateful to Mrs. Gisela Schmidt for skillful technical assistance.
References Ambache, N., L.P. Dunk, J. Verney and M.A. Zar, 1972, Inhibition of postganglionic motor transmission in vas deferens by indirectly acting sympathomimetic drugs, J. Physiol. (London) 227, 433. Ambache, N. and M.A. Zar, 1971, Evidence against adrenergic motor transmission in the guinea-pig vas deferens, J. Physiol. (London) 216, 359. Benelli, G., G. Della Bella and A. Gandini, 1964, Angiotensin and peripheral sympathetic nerve activity, Brit. J. Pharmacol. 22, 211. Blackwell, M., T.A. James and M.S. Start, 1978, Potentiation by substance P of contractions of the isolated vas deferens of the mouse elicited by electric field stimulation and by drugs, Brit. J. Pharmacot. 63, 323. Camargo, A. and S.H. Ferreira, 1971, Action of bradykinin potentiating factor (BPF) and dimercaprol (BAL) on the responses to bradykinin of isolated preparations of rat intestines, Brit. J. Pharmacol. 42, 305. Endo, T., K. Starke, A. Bangerter and H.D. Taube, 1977, Presynaptic receptor systems on the noradrenergic neurones of the rabbit pulmonary artery, Naunyn-Schmiedeb. Arch. Pharmacol. 296, 229.
PEPTIDE EFFECTS ON THE VAS DEFERENS Furness, J.B., 1974, Transmission to the longitudinal muscle of the guinea-pig vas deferens: the effect of pretreatment with guanethidine, Brit. J. Pharmacol. 50, 63. Gross, F., 1971, Angiotensin, in: Pharmacology of Naturally Occurring Polypeptides and LipidSoluble Acids, Vol. 1, International Encyclopedia of Pharmacology and Therapeutics, Sect. 72, ed. J.M. Walker (Pergamon Press Ltd., Oxford) p. 73. Kato, H. and T. Suzuki, 1971, Bradykinin-potentiating peptides from the venom of Agkistrodon halys blomhoffii. Isolation of five bradykinin potentiators and the amino acid sequences of two of them, potentiator B and C, Biochemistry 10, 972. Paiva, T.B., M.E. Miyamoto, L. Juliano and A.C.M. Paiva, 1977, Requirements for angiotensin tachyphylaxis in smooth muscles, J. Pharmacol. Exptl. Therap. 202, 294. Regoli, D., K. Park and F. Rioux, 1973, Pharmacology of angiotensin antagonists, Can. J. Physiol. Pharmacol. 51,114. Regoli, D., W.K. Park and F. Rioux, 1974, Pharmacology of angiotensin, Pharmacol. Rev. 26, 69. SjSstrand, N., 1973, Effects of acetylcholine and some other smooth muscle stimulants on the electrical and mechanicalresponses of the guineapig vas deferens to nerve stimulation, Acta Physiol. Scand. 89, 1. SjSstrand, N.O. and G. Swedin, 1974, On the mechanism of the enhancement by smooth muscle stimulants of the motor responses of the guineapig vas deferens to nerve stimulation, Acta Physiol. Scand. 9 0 , 5 1 3 . Stewart, J.M., 1974, Tachyphylaxis to angiotensin, in : Handbook of Experimental Pharmacology, Vol. X X X V I I : Angiotensin, eds. I.H. Page and F.M Bumpus (Springer, Berlin--Heidelberg--New Yorkl p. 170. Stj~/rne, L., 1976, The twitch contraction in guineapig isolated vas deferens -- result of cooperative interaction of potassium and noradrenaline released from adrenergic nerves in close junctions?, Acta Physiol. Scand. 9 8 , 5 1 2 . Stj~irne, L., 1977, Do potassium ions released from nerve modulate the sensitivity to transmitter in
29 "close" neuroeffector junctions of the vas deferens?, Neuroscience 2, 373. Swedin, G., 1971, Studies on neurotransmission mechanisms in the rat and guinea-pig vas deferens, Acta Physiol. Scand., Suppl. 369. Thoa, N.B. and G.D. Maengwyn-Davies, 1968, The guinea-pig isolated vas deferens: a method for increasing sensitivity to drugs, J. Pharm. Pharmacol. 20, 873. Ueda, E., T. Kokubu, H. Akutsu and Y. Yamamura, 1971, Inhibition of angiotensin I converting enzyme and kininase in rabbit plasma by bradykinin potentiating peptide B (pyr-gly-leu-pro-argpro-lys-ile-pro-pro), Experientia (Basel) 27, 1020. Ufkes, J.G.R., P.N. Aarsen and C. Van der Meer, 1976, The bradyk.inin potentiating activity of two pentapeptides on various isolated smooth muscle preparations, European J. Pharmacol. 40, 137. Ufkes, J.G.R., P.N. Aarsen and C. Van der Meet, 1977, The mechanism of action of two bradykinin-potentiating peptides on isolated smooth muscle, European J. Pharmacol. 44, 89. Van den Brink, F.G. and E.J. Lien, 1977, pD2-, pA2and pD2'-values of a series of compounds in a histaminic and a cholinergic system, European J. Pharmacol. 44, 251. Von Euler, U.S. and P. Hedqvist, 1974a, Effects of substance P on the response of guinea pig vas deferens to transmural nerve stimulation, Acta Physiol. Scand. 90, 651. Von Euler, U.S. and P. Hedqvist, 1974b, Phenoxybenzamine blockade of the enhancing effect of substance P on the twitch induced by transmural nerve stimulation in the guinea-pig vas deferens, Acta Physiol. Scand. 92, 283. Von Euler, U.S. and P. Hedqvist, 1975, Evidence for an a- and ~2-receptor mediated inhibition of the twitch response in guinea-pig vas deferens by noradrenaline, Acta Physiol. Scand. 93, 572. Waud, D.R., 1968, Pharmacological receptors, Pharmacol. Rev. 20, 49. Zetler, G., 1977, Effects of substance P and other peptides on the field-stimulated guinea pig vas deferens, in: Substance P, eds. Von Euler U.S. and B. Pernow (Raven Press, New York) p. 97.
21
© Elsevier/North-Holland Biomedical Press
AN ATTEMPT TO DIFFERENTIATE THE EFFECTS OF ANGIOTENSIN II, SUBSTANCE P AND BRADYKININ ON THE FIELD-STIMULATED GUINEA-PIG VAS DEFERENS GERHARD ZETLER and ECKEHARD KAMPMANN
Abteilung fiir Pharmakologie der Medizinischen Hochschule Liibeck, Ratzeburger Allee 160, D-2400 Liibeck, Federal Republic of Germany Received 31 October 1978, revised MS received 4 January 1979, accepted 7 February 1979
G. ZETLER and E. KAMPMANN, An attempt to differentiate the effects of angiotensin II, substance P and bradykinin on the field-stimulated guinea-pig vas deferens, European J. Pharmacol. 56 (1979) 21--29. The ability of the angiotensin antagonists 1-Sar,8-Ala-angiotensin II (saralasin) and 1-Sar,8-Leu-angiotensin II (sarleusin) and the bradykinin-potentiating peptide B (BPP) to modify the twitch-enhancing effect of angiotensin II, bradykinin, and substance P, was studied in the isolated field-stimulated guinea-pig vas deferens. The twitchenhancing effect of angiotensin, bradykinin and substance P underwent tachyphylaxis which was strongest after angiotensin. Saralasin and sarleusin were without influence on the twitc h response and antagonized the effect of angiotensin, but not that of bradykinin or substance P, respectively. The features of the antagonism to angiotensin were compatible with the notion that saralasin is a competitive and sarleusin a dual antagonist. BPP did modify either the twitch response or the effects of angiotensin, bradykinin, and substance P. In the non-stimulated vas, the contracting effect of noradrenaline did not undergo tachyphylaxis and was not modified by angiotensin, bradykinin, or substance P. It is concluded that there exist in the guinea-pig vas specific peptide receptors, one of them having the properties of a "typical" angiotensin receptor. Bradykinin potentiator
Vas deferens
Angiotensin
1. Introduction In the isolated guinea-pig vas deferens the twitch response to a field stimulus is enhanced by substance P (SP), bradykinin and angiotensin II (Von Euler and Hedqvist, 1974a; SjSstrand and Swedin, 1974). A quantitative study of twelve peptides (Zetler, 1977) revealed differences in the twitch-enhancing effect: active were SP, eledoisin, bradykinin, angiotensin I and II, glucagon and carnosine; inactive were neurotensin, somatostatin, TRH, gastrin, pentagastrin, caerulein and cholecystokinin-octapeptide (results on the last two peptides are to be published). Furthermore, the active peptides differed as to potency (ECs0) and effectiveness (Ema~). The sum of these findings pointed to the possible existence of specific peptide receptors on the relevant structures of the guinea-pig vas. Muscarinic
Bradykinin
Substance P
Saralasin
receptors exist there since atropine antagonized the twitch-enhancing effect of carbachol in a competitive fashion with pA2 8.027, but left the SP effect intact (Zetler, 1977). From experiments using phenoxybenzamine, tetracaine and tetrodotoxin it was concluded that the peptides, acetylcholine, and carbachol may enhance the twitch by a prejunctional mechanism connected with the intramural neurons of the vas (Von Euler and Hedqvist, 1974b; Zetler, 1977). However, there are also good arguments for a postjunctional mechanism of action of these drugs except for angiotensin which may exert mainly a prejunctional action (SjSstrand and Swedin, 1974). Noradrenaline is perhaps the motor transmitter involved in the rapid twitch response of the field-stimulated guinea-pig vas deferens (Swedin 1971; Furness, 1974), although this was denied by Ambache and Zar
22
(1971), Ambache et al. (1972) and Von Euler and Hedqvist (1975). Complicated synaptic events also involving potassium and prostaglandins have been envisaged (Swedin, 1971; SjSstrand and Swedin, 1974; Stj~rne, 1976, 1977). The present experiments therefore aimed at the following restricted question: could the effects of SP, angiotensin II and bradykinin be differentiated with the aid of antagonists and a potentiator? To find o u t whether the angiotensin effects on the vas were "typical" we selected from the large series of specific angiotensin antagonists (cf. Regoli et al., 1974) the competitive antagonist 1-Sar,8-Ala-angiotensin II (Saralasin) and 1-Sar,8-Leu-angiotensin II (Sarleusin) which in isolated rat stomach and rabbit aortic strips acts as a dual (competitive and metactoid; Van den Brink and Lien, 1977) rather than competitive antagonist. It is already clear that saralasin is a competitive angiotensin antagonist for the presynaptic receptors of the rabbit pulmonary artery (Endo et al., 1977). A third pharmacological tool used in the present study was the bradykinin-potentiating peptide B (BPP; Pyr-Gly-Leu-Pro-Pro-Arg-ProLys-Ile-Pro-Pro). This compound strongly enhances the bradykinin effect on the isolated guinea-pig ileum (but only weakly that on the rat uterus) and inhibits the angiotensin I-converting enzyme and the plasma kininase (Kato and Suzuki, 1971; Ueda et al., 1971). BPP does not change the rat uterus contracting effect of angiotensin II (Ueda et al., 1971). 2. Materials and methods
The vasa deferentia of freshly killed guinea pigs weighing 250--400 g were carefully dissected free from their mesenteric connections and m o u n t e d between two vertical platinum electrodes in an organ bath containing 5 ml Tyrode solution of the following composition (mM): 137 NaC1, 2.7 KCI, 12 NaHCO3, 0.4 N a H 2 P O 4 - 1 H 2 0 , 1.8 CaC12-2 H20, 0.5 MgCl~ • 6 H20, 5.5 glucose • 2 H20. The bath fluid was kept at 37°C and gassed with car-
G. Z E T L E R , E. K A M P M A N N
bogen (95% 02 + 5% CO2). For the experiments with exogenous noradrenaline in the non-stimulated vas the bath solution contained Ca2+-EDTA, 10 -4 M. The load on the vas was 0.3 g and the contractions were recorded isotonically using a displacement transducer (Sachs/Freiburg) and a Servogor recorder (Metrawatt/Nfrnberg). Field stimulation was applied every minute for 3 sec and consisted of monophasic square pulses of 2--5 Hz, 2 msec in duration and 12 V in strenght (Stimulator II, Sachs/Freiburg). A single 30 Hz stimulus was applied several times during the course of an experiment to test the maximum twitch response of a given preparation. After an adaptation period lasting 30--90 min (with field stimulation) the first concent r a t i o n - r e s p o n s e curve to a given agonist drug (see fig. 1) was established and followed by a second curve after a recovery period lasting at least 30 min. The dosage was cumulative with concentrations increased immediately after every second twitch response since additional twitches did not reveal a further increase in the drug effect. This effect, i.e., the increase in twitch response, was expressed as a fraction of the difference between the response to 2--5 Hz and either the response to a 30 Hz stimulus (table 1) or the maximum effect (Emax) of a concentration--response curve. As suggested b y Waud (1968) the terms effectiveness (Emax) and p o t e n c y (pD2)will be used. pD2 is the negative logarithm of the molar concentration (ECs0) producing the half-maximum effect o f an agonist. The EC20, ECs0 and ECs0 were calculated from a log concentration--effect curve after linearization b y probit transformation and estimation of the regression function with the least squares method. The slope, i.e., than ~ of the linear part of a sigmoid log concentration-effect curve refers to a system of coordinates where Emax of the reference curve is 3.3 decades of length of the logarithmic abscissa. Slope = E m a x " 0.6 • 3.3/(log ECs0--1og EC20). The effects of the angiotensin antagonists and the bradykinin potentiator were evaluated b y means of a second concentration--response
PEPTIDE E F F E C T S ON T H E VAS D E F E R E N S
23
3. Results
curve, while the first curve served as control. As described in Results, intrinsic reductions in sensitivity occurred b e t w e e n two consecutive concentration-response curves and were allowed for b y correcting the controls with the aid of factors which will be mentioned. In experiments with antagonists the m e t h o d of Van den Brink and Lien (1977) was used to calculate the pA2 from the shift of the concentration-response curve, and the pD2' from the depression of its maximum. For statistical evaluation, P < 0.05 was set as threshold of probability. Student's t-test was applied to paired differences: t = k/s;. Multiple means were compared with Bartlett's test and the Scheff~ test. In the case of nonhomogeneous variances the Wilcoxon--Mann-Whitney ranking method was used. The drugs were angiotensin amide (Ciba), 1-Sar,8-Ala-angiotensin II (Serva), 1 -Sar,8-Leuangiotensin II (Serva), bradykinin triacetate (Serva), bradykinin-potentiating peptide B (Bachem), substance P (Bachem), and (--)noradrenaline (Hoechst). Angiotensin amide served as a valid though generally somewhat less active substitute for angiotensin II (Gross, 1971), and the term angiotensin will be used for the sake of shortness. A
i
A A
B
3.1. The agonists The agonists angiotensin, bradykinin, and SP produced concentration
1). The question of a possible direct smooth muscle-stimulating effect of the agonists was studied in preparations which reacted to a low agonist concentration with a normal enhanced twitch response. In the absence of field stimulation none of the three peptides induced a contraction of the vas at concentrations up to 300 pg/ml (2--3 • 10-4 M; neg log: 3.5--3.7).
i
BB:
~
.: -
_-
Fig. 1. Twitch-enhancing effect of angiotensin (A and A A ; 4 Hz) and substance P (B and BB; 3 Hz), in the absence (A and B) and presence of 2 • 10-7 M sarleusin ( A A and BB). One field stimulus per minute. Sarleusin was added to the bath at the arrow. The vertical dashes indicate the steps of the cumulative dosage of a given agonist (/~g/ml): A, 0.01, 0.03, 0.1, 0.3, 1 and 3; AA, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 and 300; B and BB, 0.02, 0.1, 0.5, 2.5, 6.5 and 10.5. Ordinate scale: 20 m m between two thick horizontal lines.
24
G. Z E T L E R , E. K A M P M A N N
TABLE 1 P o t e n c y (pD2), slope, and effectiveness (Emax) as o b t a i n e d f r o m c o n c e n t r a t i o n - - r e s p o n s e curves for angiotensin, b r a d y k i n i n and s u b s t a n c e P ( x -+ S~x). Emax refers to the t w i t c h r e s p o n s e as o b t a i n e d b y a field stimulus at 30 Hz (this r e s p o n s e was assigned a value o f 100).
Angiotensin Bradykinin Substance P
n
pD 2
Slope
Emax
8 8 8
7.28 -+ 0.151 6.67 ± 0.063 I 7.19 -+ 0.105
1.31 -+ 0.09 2.13 ± 0.19 1 1.16 ± 0.06
48 ± 7 69 -+ 4 1 89 -+ 3 2
Statistical significance exists for the d i f f e r e n c e s f r o m b o t h angiotensin and s u b s t a n c e P 1, and for t h a t f r o m angiotensin 2
3.2. C o n s e c u t i v e curves
c o n c e n tra tio n - - r e s p o n s e
According to the protocol of this study the effect of an antagonist was to be evaluated by means of the second of two consecutive doseresponse curves of a given agonist. Hence, the possibility of a desensitization had to be envisaged. The rapid development of specific tachyphylaxis is typical for angiotensin (review: Stewart, 1974) and was also ob-
i0010
served for the twitch-enhancing effect of this peptide in the hypogastricus-stimulated guinea-pig vas (Benelli et ah, 1964). In the present experiments reduced sensitivity to all three agonists was prominent during the second run of the concentration--response curve (fig. 2 and 3, table 2). This was concluded from the decrease in pD2, which meant that the ECs0 increased by a factor 3.63 (= antilog 0.56) in the case of angiotensin and by 1.45 (= antilog 0.16) for both bradykinin
I00-
/t
nglml
Fig. 2. T w i t c h - e n h a n c i n g e f f e c t o f b r a d y k i n i n (e, o) a n d s u b s t a n c e P (A, A). T w o successive c o n c e n t r a t i o n - r e s p o n s e curves each are s h o w n for each p e p t i d e (first curve, -= ~ and • A; s e c o n d curve, o . . . . . -o and A_ . . . . .A). T h e p o i n t s are m e a n s (x ± Sx, n = 8 for each curve). Abscissa (log): peptide c o n c e n t r a t i o n . Ordinate: Ema x • 100, referring t o t h e m a x i m u m e f f e c t o f t h e related first curve.
Fig. 3. M o d i f i c a t i o n o f t h e t w i t c h - e n h a n c i n g e f f e c t o f a n g i o t e n s i n b y saralasin a n d sarleusin. C o n c e n t r a t i o n - - r e s p o n s e curves are s h o w n for a n g i o t e n s i n (~±Sx): first c o n t r o l curve ( e ~, n f 8 ) ; s e c o n d c o n t r o l curve (o o, n = 8 ) ; second curves in p r e s e n c e o f 2.2 • 10 -7 M saralasin (A ~, n = 9), 5.2. 10 -8 M sarleusin (o . . . . . . o, n = 5), or 2.1" 10 -7 M sarleusin (o . . . . . . o, n = 6). Abscissa: c o n c e n t r a t i o n o f angiotensin. Ordinate: Ema x • 100, referring t o t h e m a x i m u m e f f e c t o f t h e first c o n t r o l curve.
P E P T I D E E F F E C T S ON T H E V A S D E F E R E N S
25
TABLE 2 M e a n d i f f e r e n c e s in p o t e n c y (PD2), s l o p e , a n d e f f e c t i v e n e s s (Emax) b e t w e e n t h e first a n d t h e s e c o n d c o n c e n t r a t i o n - r e s p o n s e c u r v e in t h e s a m e p r e p a r a t i o n . B o t h t h e p D a a n d t h e slope o f t h e first c u r v e are p r e s e n t e d in t a b l e 1. Emax refers to the r e l a t e d first c u r v e (Emax = 1.0).
Angiotensin Bradykinin Substance P
n
pD2
Slope
Emax " 1 0 0
8 8 8
--0.56 ± 0.13 1 --0.16 ± 0.06 1 --0.16 ± 0.06 I
+0.09 ± 0.17 - - 0 . 3 7 ± 0.11 1 --0.03 ± 0.05
--14 + 7 --11 + 2 1 --2 ± 2
I Statistically s i g n i f i c a n t d i f f e r e n c e f r o m zero.
and substance P. Emax remained constant for substance P and was somewhat reduced for angiotensin and bradykinin. There was no change in slope for either angiotensin or substance P, whereas the second curve was less steep for bradykinin.
3.3. The angiotensin antagonists In contrast to angiotensin, neither saralasin nor sarleusin (both at the concentration 2 • 10 -T M) produced any twitch-enhancing effect (fig. 1). Saralasin shifted the concentrat i o n - r e s p o n s e curve of angiotensin to the right but did not depress its maximum, whereas sarleusin also reduced the m a x i m u m (figs. 1 and 3). The saralasin effect was consistent with the notion that this analog is a competitive angiotensin antagonist. The effect of sarleusin points to a dual, i.e., competit, ive and metactoid (cf. Van den Brink and Lien, 1977) antagonist--receptor interaction as
already found for the isolated rat stomach strip (Regoli et al., 1973). According to the values of pA2 (table 3) the antagonistic power of sarleusin was greater than that of saralasin. Considering the pA2 and pD2, values, no statistically significant differences exist between the effects of the two concentrations of sarleusin. Both antagonists were practically inactive against bradykinin and substance P (fig. 1, table 4). Saralasin, it is true, reduced the potency of bradykinin by a factor 1.86 (antilog 0.27) but this effect was of minor importance since it was (a) not duplicated by sarleusin, and (b) very weak when compared with that against angiotensin (factor 6.6; sarleusin, factor 20.8). While the result that substance P was even somewhat more potent (factor 1.78) under the influence of sarleusin does not carry much weight it does stress the observation of a missing antagonism.
TABLE 3 Saralasin a n d s a r l e u s i n as a n t a g o n i s t s o f a n g i o t e n s i n . T h e r e s u l t s (R- ± SR-x)were o b t a i n e d b y c o m p a r i n g t w o c o n c e n t r a t i o n - r e s p o n s e c u r v e s in e a c h e x p e r i m e n t , t h e first o n e a f t e r c o r r e c t i o n a l l o w i n g f o r t a c h y p h y l a x i s (see table 2 a n d t e x t ) . Emax b e l o n g s to t h e s e c o n d curve a n d refers t o t h e first o n e (Emax = 1). If n o t i n d i c a t e d b y n.s., d i f f e r e n c e s b e t w e e n m e a n s w i t h i n a c o l u m n are s t a t i s t i c a l l y s i g n i f i c a n t .
Saralasin 2.2 • 10 -7 M S a r l e u s i n 5.2 - 10 -8 M S a r l e u s i n 2.1 • 10 -7 M
n
PA2
9 5 6
7.34 ± 8.20 ± 7.98 ±
0.21 0.34 0.13 ~
n.s.
Emax " 1 0 0
pD~
102 ± 5 72 ± 4 51 ± 6
6 . 8 6 ± 0.11 ~ 6.66 ± 0.11
n.s
26
G. ZETLER, E. KAMPMANN
TABLE 4 Influence of saralasin (2.2 " 10 -7 M) and sarleusin (2.1 • 1 0 - 7 M) on the potency of bradykinin and substance P. The results ~-+ S~-x)were obtained by comparing two concentration--response curves in each experiment, the first used as reference curve after correction allowing for tachyphylaxis (see table 2 and text).
Bradykinin Substance P
Reference curve pD2
Saralasin (n = 8) change in PD2
6.17 -+ 0.10 6.32 ± 0.08
--0.27 -+ 0.05 1
6.83 ± 0.08 7.01 +- 0.16
+0.06 -+ 0.09
Sarleusin (n = 6) change in pD2 +0.03 ± 0.05 +0.25 + 0.04 I
i The difference from zero is statistically significant.
3.4. The bradykinin potentiating peptide B (SPP) BPP (1.5 • 10 -s M, given 5 min prior t o an agonist) was w i t h o u t e f f e c t o n t h e t w i t c h response and did n o t m o d i f y the p o t e n c y o f angiotensin, b r a d y k i n i n , and substance P (table 5). H o w e v e r , in t h e isolated guinea-pig ileum u n d e r t h e same e x p e r i m e n t a l c o n d i t i o n s b u t w i t h o u t field s t i m u l a t i o n , BPP ( 4 . 2 . 10 - 6 M and 1 . 7 " 1 0 - s M , given 5 m i n p r i o r t o b r a d y k i n i n ) increased t h e c o n t r a c t i n g e f f e c t o f 8 • 10 -9 M b r a d y k i n i n b y a f a c t o r o f 2.5 or 4, respectively.
TABLE 5 Potency (PD2) of angiotensin, bradykinin and substance P in the absence (reference curve as defined in table 4) and presence of the bradykinin potentiating peptide B (18 t~g/ml). n.s.: the difference from zero is not statistically significant. n
Reference
Change in pD2
curve
PD2 Angiotensin Bradykinin Substance P
6 8 6
7.03 +- 0.04 6.79 ± 0.11 7.08 -+ 0.16
--0.14 -+ 0.12 n.s. --0.05 +- 0.07 n.s. +0.17 -+ 0.09 n.s.
3.5. The non-stimulated vas A c c o r d i n g to Sj5strand ( 1 9 7 3 ) , V o n Euler and Hedqvist (1974a), SjSstrand and Swedin ( 1 9 7 4 ) and Blackwell et al. ( 1 9 7 8 ) s u b s t a n c e P b u t n o t angiotensin (SjSstrand and Swedin, 1 9 7 4 ) p o t e n t i a t e s t h e response o f t h e vas t o a field stimulus b y a partial d e p o l a r i z a t i o n o f t h e muscle cell m e m b r a n e w h i c h w o u l d facilit a t e and p r o m o t e a c t i o n potentials. A mechanism like this o n e can be e x p e c t e d also t o e n h a n c e t h e e f f e c t o f a s t i m u l a t o r y d r u g with a p o s t s y n a p t i c p o i n t o f a t t a c k at t h e s m o o t h muscle cell. T h e r e f o r e , e x p e r i m e n t s using p r e p a r a t i o n s w i t h o u t field s t i m u l a t i o n were d e v o t e d t o the q u e s t i o n o f w h e t h e r angiotensin and substance P w o u l d sensitize t h e vas t o e x o g e n o u s noradrenaline. When prepared as usual, t h e vas was o f e x t r e m e l y low sensitivity t o n o r a d r e n a l i n e and gave erratic results. The sensitivity c o u l d be increased b y o p e n i n g t h e vas and r e m o v i n g the m u c o s a b y scratching as suggested b y T h o a and Meangwyn-Davies (1968). A f t e r an a d a p t a t i o n period o f 3--4 h with n o r a d r e n a line doses every 5 min, these p r e p a r a t i o n s r e s p o n d e d t o n o r a d r e n a l i n e in a c o n c e n t r a t i o n ~ l e p e n d e n t fashion. The pD2 o f n o n c u m u l a t i v e doses o f n o r a d r e n a l i n e was 4.96 + 0.07 (1~+ s x ; n = 8 ) w h i c h indicates a very low p o t e n c y . N o desensitization o c c u r r e d w h e n t w o or m o r e c o n c e n t r a t i o n - - r e s p o n s e
PEPTIDE EFFECTS
ON THE VAS DEFERENS
TABLE 6 P o t e n c y ( p D 2 ) o f n o r a d r e n a l i n e in t h e n o n - s t i m u l a t e d v a s d e f e r e n s , in t h e a b s e n c e ( r e f e r e n c e c u r v e ) a n d p r e s e n c e o f a n g i o t e n s i n (2 p g / m l = 1 . 9 4 • 1 0 -6 M ) o r s u b s t a n c e P (2 p g / m l = 1 . 4 8 • 1 0 -6 M ) , r e s p e c t i v e l y . R e s u l t s are m e a n s : ~- +- S~x. n . s . : t h e d i f f e r e n c e f r o m z e r o is n o t s t a t i s t i c a l l y significant. n
Reference curve pD2
C h a n g e in p D 2
4 4
4 . 8 3 -+ 0 . 1 5 5 . 1 0 -+ 0 . 1 0
+ 0 . 0 3 -+ 0 . 0 5 n . s . - - 0 . 0 7 -+ 0 . 0 6 n . s .
Angiotensin Substance P
curves were established in the same preparation. Neither angiotensin nor substance P, when added at high concentrations to the organ bath 1 min prior to every single dose of noradrenaline, modified the concentration--effect curve of noradrenaline with regard to p o t e n c y (table 6). There was no change in Emax either.
4. Discussion The peptides saralasin, sarleusin and BPP, when given alone at high concentrations, did n o t enhance the twitch response. They therefore add to the number of peptides devoid of this activity and mentioned in the Introduction. This result supports the notion that we are dealing with a specific pharmacological effect. This view is stressed by the powerful antagonism of saralasin and sarleusin against the twitch-enhancing effect of angiotensin. Obviously, these t w o specific angiotensin antagonists proved to have affinity to, b u t no activity on, the angiotensin-sensitive structure as was observed in other preparations (Regoli et al., 1974). The pattern of antagonism pointed to the competitive t y p e for saralasin and the dual t y p e (cf. Van den Brink and Lien, 1977) for sarleusin. This, too, is in accord with results obtained with these
27
antagonists in the isolated rat stomach and rabbit aortic strip (Regoli et al., 1973, 1974). Finally, both antagonists were w i t h o u t any clear-cut effect against bradykinin and substance P. The conclusion is that the typical specific angiotensin receptor exists in the structures which are essential for the twitchenhancing effect of peptides. This would explain w h y the well-known tachyphylaxis to angiotensin (Stewart, 1974; Paiva et al., 1977) also occurred in the present experiments. There also was desensitization, though to a lesser extent, to bradykinin and substance P. However, in spite of an equal change in potency (pD:), effectiveness (Emax) was reduced for bradykinin b u t n o t for substance P (table 2). This qualitative difference between the effects of the two peptides together with that in the steepness of the normal concentrat i o n - e f f e c t curves (fig. 2, table 1), points to u n k n o w n differences in the mechanisms leading to, or triggered by the activation of the receptors for bradykinin and substance P in the field-stimulated vas deferens. Transmitter exhaustion -- one mechanism suggested for the production of tachyphylaxis to angiotensin (Stewart, 1974) -- was probably n o t important in the present experiments since the desensitization to both bradykinin and substance P was less in spite of their effectiveness being superior to that of angiotensin (fig. 1, table 1). It must be stressed that in our experiments without field stimulation the vas produced no tachyphylaxis to noradrenaline. Hence, it is not likely that desensitization to the peptides involves the postsynaptic noradrenaline receptor. This receptor can also be excluded as the site of the mechanism for the twitchenhancing effect of the peptides. This must be concluded from the finding that even very high concentrations of both angiotensin and substance P did not modify the concentration--effect relationship in the non-stimulated vas (table 6). As far as angiotensin is concerned, our results agree with those of Benelli et al. (1964) w h o found that this peptide enhanced the twitch response to stimulation
28
of the hypogastric nerve b u t failed to potentiate the response of the vas to exogenous noradrenaline. However, there is also the view that substance P (and bradykinin) enhances the response of the guinea-pig vas to a field stimulus or to nerve stimulation by partial depolarization of the muscle membrane (SjSstrand, 1973; Von Euler and Hedqvist, 1974a; SjSstrand and Swedin, 1974). Furthermore, a purely postjunctional mechanism enhancing the responsiveness of the muscle cells has recently been postulated for the twitchenhancing effect of substance P in the mouse vas deferens (Blackwell et al., 1978). A muscle cell with an increased responsiveness to any depolarizing stimulus can be expected to show a facilitated response to a directly stimulating drug. However, in our experiments as in those of Benelli et al. (1964), the responsiveness to exogenous noradrenaline remained unchanged and was, in fact, b y no means regularly enhanced in the experiments of Blackwell et al. (1978). Hence, it is unlikely that the peptides influenced the postjunctional effector systems b e y o n d the noradrenaline receptor. Consequently, presynaptic receptors in the vas might be the site of action for the peptides active in our present experiments. This would explain the differences between the field-stimulated and the non-stimulated vas as regards the peptide effect in the former and its absence towards noradrenaline in the latter preparation. The receptors involved are specific since they are sensitive to a limited number of peptides. The use of BPP did not help to differentiate the peptide effects. The results obtained with the guinea-pig ileum proved that our sample of BPP, at the concentration used, was capable of potentiating the bradykinin effect in this preparation. The failure in the field-stimulated vas can have two causes: low or missing kininase activity of the vas tissue, or the absence of a receptor for BPP. We have no information about the kininase activity of the vas b u t draw attention to the view of Camargo
G. ZETLER, E. KAMPMANN
and Ferreira (1971) and Ufkes et al. (1977) that the bradykinin-potentiating effect of two pentapeptides in isolated smooth muscles is not due to kininase inhibition b u t to an increased affinity of the bradykinin receptor. The second possible cause, the absence of a BPP receptor in the vas, is reminiscent of differences in sensitivity to BPP of various bradykinin effects in isolated smooth muscle organs (Ufkes et al., 1976, 1977). Considering the suggestion that potentiating agents produce an allosteric transition of the bradykinin receptor (Camargo and Ferreira, 1971) one might think of differences between the bradykinin receptors of the guinea-pig ileum and those of the field-stimulated guinea-pig vas deferens.
Acknowledgement We are grateful to Mrs. Gisela Schmidt for skillful technical assistance.
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29 "close" neuroeffector junctions of the vas deferens?, Neuroscience 2, 373. Swedin, G., 1971, Studies on neurotransmission mechanisms in the rat and guinea-pig vas deferens, Acta Physiol. Scand., Suppl. 369. Thoa, N.B. and G.D. Maengwyn-Davies, 1968, The guinea-pig isolated vas deferens: a method for increasing sensitivity to drugs, J. Pharm. Pharmacol. 20, 873. Ueda, E., T. Kokubu, H. Akutsu and Y. Yamamura, 1971, Inhibition of angiotensin I converting enzyme and kininase in rabbit plasma by bradykinin potentiating peptide B (pyr-gly-leu-pro-argpro-lys-ile-pro-pro), Experientia (Basel) 27, 1020. Ufkes, J.G.R., P.N. Aarsen and C. Van der Meer, 1976, The bradyk.inin potentiating activity of two pentapeptides on various isolated smooth muscle preparations, European J. Pharmacol. 40, 137. Ufkes, J.G.R., P.N. Aarsen and C. Van der Meet, 1977, The mechanism of action of two bradykinin-potentiating peptides on isolated smooth muscle, European J. Pharmacol. 44, 89. Van den Brink, F.G. and E.J. Lien, 1977, pD2-, pA2and pD2'-values of a series of compounds in a histaminic and a cholinergic system, European J. Pharmacol. 44, 251. Von Euler, U.S. and P. Hedqvist, 1974a, Effects of substance P on the response of guinea pig vas deferens to transmural nerve stimulation, Acta Physiol. Scand. 90, 651. Von Euler, U.S. and P. Hedqvist, 1974b, Phenoxybenzamine blockade of the enhancing effect of substance P on the twitch induced by transmural nerve stimulation in the guinea-pig vas deferens, Acta Physiol. Scand. 92, 283. Von Euler, U.S. and P. Hedqvist, 1975, Evidence for an a- and ~2-receptor mediated inhibition of the twitch response in guinea-pig vas deferens by noradrenaline, Acta Physiol. Scand. 93, 572. Waud, D.R., 1968, Pharmacological receptors, Pharmacol. Rev. 20, 49. Zetler, G., 1977, Effects of substance P and other peptides on the field-stimulated guinea pig vas deferens, in: Substance P, eds. Von Euler U.S. and B. Pernow (Raven Press, New York) p. 97.