- Email: [email protected]
Interaction of bradykinin and angiotensin in the regulation of blood pressure in conscious rats
Gen. Pharmac. Vol. 22, No. 4, pp. 759-762, 1991 Printed in Great Britain. All rights reserved
0306-3623/91 $3.00 + 0.00 Copyright ~:~ 1991 Pergamon Press plc
I N T E R A C T I O N OF B R A D Y K I N I N A N D A N G I O T E N S I N THE R E G U L A T I O N OF B L O O D P R E S S U R E IN C O N S C I O U S RATS
IN
M. VAN DEN BUUSE* and J. KERKHOFF Rudolf Magnus Institute for Pharmacology, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands (Received 30 November 1990) Abstract---1. The interaction between bradykinin (BK) and the renin-angiotensin system was studied in conscious, catheterized rats. 2. Intravenous injection of BK induced dose-dependent decreases in blood pressure in normotensive Wistar and Wistar-Kyoto rats and spontaneously hypertensive rats. Pretreatment with the angiotensinconverting enzyme (ACE) inhibitor captopril markedly enhanced the effect of BK, such that the dose-response curve shifted significantly to the left in all three strains. 3. In a second series of experiments, captopril did not change basal blood pressure, but blocked the pressor response to angiotensin I (AI), but not angiotensin II (AII). 4. The partial agonist Sar~-Ala8-angiotensin II (SAR) increased blood pressure and blocked the pressor response to subsequent All treatment. 5. After pretreatment with BK (50 #g/kg), captopril evoked a decrease in blood pressure, while still blocking the effect of AI. 6. After pretreatment with BK, SAR decreased blood pressure, while still antagonizing the action of All. 7. These results suggest that ACE plays a role in the inactivation of circulating BK in normotensive and hypertensive rats. Conversely, BK can influence the activity of the renin-angiotensin system, probably by interacting with ACE.
between different forms of hypertension (Iimura and Shimamoto, 1989; Madeddu et al., 1987; Waeber et aL, 1986). In the present study we further investigated the interaction between the BK and the reninangiotensin system. We measured the effect of pretreatment with the ACE-inhibitor captopril on the blood pressure responses to bradykinin in normotensive and hypertensive rats. The results show that captopril differentially influences the hypotensive action of BK. In addition, we tested the effect of pretreatment with BK on the acute blood pressure response to captopril, AI, A l l or the All-antagonist saralasin (Sarl-AlaS-angiotensin II, SAR). A single i.v. injection of bradykinin was found to profoundly alter the responses to these compounds.
INTRODUCTION
Intravenous administration of the nonapeptide bradykinin (BK) results in a dose-dependent vasodilatation and decrease in blood pressure (Carretaro and Scicli, 1981; Pearson and Lang, 1967; Regoli and Baraba, 1980; Roche e Silva et al., 1949). This action is short-lasting, since BK is rapidly broken down by an aminopeptidase. This enzyme was shown to be identical to angiotensin-converting enzyme (ACE), which is responsible for the formation of angiotensin II (AII) from angiotensin I (AI) and angiotensinogen (Yang et al., 1970; Erd6s, 1979; Valloton, 1987). Thus, ACE-inhibitors have been shown to accentuate and prolong the blood pressure decrease produced by i.v. BK (Adamski and Grega, 1988; Ishida et al., 1989; Mullane and Moncada, 1980). Moreover, the antihypertensive effect of ACE-inhibitors may result from attenuated breakdown of the vasodilatory BK, in addition to inhibited formation of the vasoconstrictory A I I (Carbonell et al., 1988; Pontieri et al., 1990; Seino et al., 1989; Sharma, 1990). Other authors have questioned a possible role of BK in the antihypertensive action of ACE-inhibitors (see Filep et al., 1987, and references therein). It was also suggested that the role of kinins in the antihypertensive action of ACE-inhibitors could be different between normotensives and hypertensives and
MATERIALS AND METHODS Rats, operations and drugs Male rats were used, either normotensive Wistars or Wistar-Kyotos (WKY) or spontaneously hypertensive rats (SHR). The animals weighed 200-250 g and were housed 4-5 per cage until the preliminary operations. At least 24 hr before the experiments, the rats were operated under ether anesthesia. Catheters were implanted in the left femoral artery and the right jugular vein and exteriorized at the back of the neck. The rats were then housed individually. Mean arterial pressure (MAP) was recorded through the femoral artery cannula and a Statham P23D transducer on a Grass Model 7 polygraph. Drugs were dissolved in saline and injected through the jugular vein cannula in a volume of 1 ml/kg.
*To whom all correspondence should be sent at his present address: Marion Merrell Dow Research Institute, Strasbourg Research Center, 16, rue d'Ankara, 67009 Strasbourg Cedex, France. 759
M. VAN DEN BUUSEand J. KERKHOFF
760
Bradykinin was purchased from Janssen, Geel, Belgium. AI, AII and SAR were purchased from Bioproducts (UCB), Brussels, Belgium. Captopril (SQ14,255) was a gift from Squibb, Princeton, U.S.A.
Protocol Two types of experiments were performed. In Wistar rats, WKY and SHR we determined the dose response curve for BK before and after administration of 5 mg/kg captopril. In a separate group of Wistar rats, the effect of BK on the activity of the renin--angiotensin system was studied. Thus, first the effect of i.v. AII and AI was assessed. The dose of All (500 ng/kg) was chosen from preliminary dose-response experiments (see Results). The dose of AI was identical to that of AII. Subsequently, saline was injected or BK in a dose which elicited near-maximal depressor responses (50 pg/kg). In separate experiments BK was also tested in a threshold-dose and it was found that such a lower dose induced similar actions on the effect of captopril and SAR. After saline or BK, captopril (5 mg/kg) was injected to block ACE, followed by AII, and AI in doses identical to those described earlier. Then saline or BK were injected again. Finally, SAR was injected in a dose ten times that of AII. By a subsequent injection of AII the receptor-blocking effects of SAR were tested. There was always a 15min interval between injections, except for the last All injection, which was done 5 min after the administration of SAR. In this protocol we thus include drugs that have an intrinsic effect of the renin-angiotensin system (captopril, SAR), which was tested in the rats treated with saline instead of BK. In addition, the effect of BK on these drugs and on the responses to AII and AI were tested.
Analysis Responses were measured as changes of baseline blood pressure and expressed as mean_+ standard error of the mean (SEM). The responses to AII, AI, BK, captopril or SAR were compared with an initial saline treatment. In some cases responses to AII and AI after treatment with captopril or SAR were compared to pretreatment responses. Also, responses to the peptides were compared between the saline group and the BK-treated group. Differences between groups were analyzed with analysis of variance and Student's t-test and considered to be significant when P < 0.05. RESULTS
A d m i n i s t r a t i o n o f B K (i.v.) induced a n immediate depressor response with a peak in the first 15 sec after administration. After a b o u t 2 m i n the effect of B K had largely disappeared. The acute pressor response to AI a n d AII was slower in onset a n d more prolonged. The peak of the pressor response was a r o u n d one min after i.v. injection. In a preliminary d o s e - r e s p o n s e experiment (n = 4), A I I was injected i.v. in the following doses: 0.7, 2, 6, 19, 56, 168, 500 a n d 1500 ng/kg. M a x i m u m pressor responses of these rats were 4_+ 1, 5 + 3 , 11 + 2 , 11 _+3, 20_+3, 34_+4, 39_+ 6 and 43_+ 5 m m H g respectively. F o r subsequent experiments the dose of 500 n g / k g was used (see below). In Fig. 1, the effect of different doses of B K o n M A P is s h o w n in Wistars, W K Y or S H R before a n d after t r e a t m e n t with captopril. In all three strains, B K p r o d u c e d dose-related falls in M A P , a l t h o u g h S H R showed a steeper d o s e - r e s p o n s e curve t h a n the n o r m o t e n s i v e rats. F o r instance, whereas 3.1 # g / k g B K did n o t significantly alter M A P in either strain, 5 0 p g / k g p r o d u c e d a M A P decrease of - 5 7 _ + 3 m m H g in Wistars, - 5 7 _ + 7 m m H g in W K Y a n d - 8 1 _+ 5 m m H g in SHR.
3O
SAL+BK E
0
e~'--~--6~6to \
~-0~o -30
\e....e
"~ .<3 -60
CAP+BK
n <
......
6,,:
W Ib l a b <
6~6\. "0--0%. 0~. o"--"~-oO 0e
-90 30 :x: E Q_
0 -30
SAL+BK
o_o~o~ 0
o\
%xk6~° WKY
o'%'o o...o\
< <"~ -60
CAP+BK
o--e--o\
\~)
~-.,?
-90 30 T
0
E 13._
• .
0"~0/"~/0~
~\T
•\._,\~+B~ o SHR
-30
< "~ -6o '\e -90
o.~
o.4
~.6
8.3
* oX 25
~oo
DOSE BK ( U g / k O
Fig. 1. The effect of i.v. injection of different doses of BK on blood pressure of Wistar, WKY and SHR, either without pretreatment (SAL + BK) or after pretreatment with captopril (CAP + BK). Data are mean + SEM of 5 7 rats per group. ANOVA indicated a significant overall differences between the two curves in either strain. Captopril at the present dose did not change basal b l o o d pressure. However, this drug greatly e n h a n c e d the response to subsequent B K treatment, as became evident from a parallel shift of the B K d o s e - r e s p o n s e curve to the left. As can be seen in Fig. 1, the threshold dose for a decrease in M A P by BK shifted from 3 - 6 # g / k g to a r o u n d 0.2 #g/kg. After captopril t r e a t m e n t the otherwise ineffective dose o f 6.3 # g / k g B K p r o d u c e d a M A P decrease of - 5 2 _+ 6 m m H g in Wistars, - 5 0 +_ 3 m m H g in W K Y a n d - 8 1 +_ 5 m m H g in SHR. These values are very similar to the responses o b t a i n e d after injection o f the 5 0 p g / k g dose in the first experiment. In the second series of experiments, W i s t a r rats were tested for the effect of B K o n the activity of the r e n i n - a n g i o t e n s i n system. As is s h o w n in Fig. 2a, saline injections produced little change in blood pressure. Subsequent injections of 5 0 0 n g / k g AII a n d AI produced pressor responses o f 47 +_ 6 and 44_+ 6 m m H g , respectively. Captopril t r e a t m e n t (5 m g / k g i . v . ) by itself induced no effect on b l o o d pressure, n o r did it significantly influence the pressor response to AII (56 +_ 7 mmHg). However, captopril virtually blocked the pressor response to AI (16 + 6 m m H g ) , illustrating t h a t the conversion o f AI to AII is essential for the pressor effect of this peptide. S A R ( S p g / k g i . v . ) induced a pressor response (34 +_ 7 m m H g ) , illustrating the partial agonistic activity of this peptide. S A R also inhibited the effect of a subsequent injection of AII (17 -+_4 m m H g ) , indicating the antagonistic properties of SAR.
Bradykinin and angiotensin interactions (a)
-I-
v
E E
DISCUSSION
70
35 0
13-
S All AI S
CAP AllAI S
SAR All
-35 -70
(b)
70
-1-
35
E E v
0
761
rl
-35 -70 Fig. 2. (a) (Top panel): maximal changes in pressure after i.v. injection of AII (500 ng/kg), AI (500 ng/kg), captopril (5 mg/kg) or SAR (5 #g/kg) in Wistar rats. AII and AI induce comparable increases in blood pressure. Captopril did not change MAP but blocks the actions of AI. SAR induces a pressor response and inhibits the response to All. (b) (Bottom panel): maximal changes in blood pressure in a separate group of rats after i.v. injection of All, AI, BK (50 pg/kg), captopril and SAR. All and AI induce similar changes in MAP as in the first group, whereas BK causes a fall in blood pressure. After BK, captopril induces a decrease in blood pressure, while blocking the action of AI. After BK, SAR induced a fall in blood pressure, while blocking the action of All. *P < 0.05 for difference with saline treated rats (first column).
Figure 2b shows similar treatments in a separate group of rats, only now i.v. saline had been replaced by 50 #g/kg BK. Thus, the initial AII and AI injections produced similar pressor responses as in the group described above (38 + 6 and 43 ___4 m m H g respectively). BK produced a marked fall in blood pressure ( - 5 1 + 5 mmHg), which had completely disappeared before the injection of captopril. In contrast to the saline-treated group, after BK captopril induced a marked fall in blood pressure ( - 5 1 _ 3 mmHg) which lasted 5-10 min. Nevertheless, the All-induced pressor response was still present (43 + 6 mmHg), as was the inhibitory effect of captopril on the response to AI (11 + 7 mmHg). As expected from the dose-response experiment (see Fig. 1), the second, (post-captopril) injection of BK induced a fall in blood pressure similar to the first BK treatment ( - 50 + 8 mmHg). Injection of SAR after BK treatment induced a decrease in blood pressure ( - 3 1 + 8 ) rather than the increase found in the saline-treated group. SAR also blocked the pressor effect of All. The response to All was reversed to a fall in blood pressure ( - 1 6 + 5mmHg), although this effect was not significantly different from the change in MAP induced by saline.
Bradykinin is a nonapeptide which is formed by the enzyme kallikrein from the precursor bradykininogen and cleaved by the proteolytic enzymes kininase I and II (McCaa, 1979; Regoli and Baraba, 1980; Valloton, 1987). It has been shown that kininase II is identical to ACE (Yang et al., 1970; Erd6s, 1979), indicating an overlap in the regulation of the renin-angiotensin and the kallikrein-bradykinin systems. Bradykinin may affect cardiovascular homeostasis also by interacting with other humoral systems, such as prostaglandines (Hui et al., 1986; Mullane and Moncada, 1980; Pontieri et al., 1990). A number of authors have suggested that elevated levels of kinins are partly or completely responsible for the hypotensive action of ACE-inhibitors (see Introduction). In our experiments, a nonhypotensive dose of captopril greatly enhanced the depressor responses to BK, most probably through inhibition of its breakdown. This effect was present in normotensive Wistar and WKY rats and in SHR. Although the dose-response curve for BK was steeper in SHR, the shift of the BK dose-response curve by captopril appeared to be similar in this strain as compared to the two normotensive strains. It needs to be elucidated whether the apparently steeper dose-response curve for BK in SHR is a result of a greater sensitivity of the vascular wall to BK in these rats, or merely a result of the higher blood pressure. In the present experiments we show that BK itself can also greatly influence the blood pressure responses to various experimental manipulations of the renin-angiotensin system. The most pronounced effects were the occurrence of an acute, marked fall in blood pressure after captopril injection, and the change of the acute pressor response to SAR into a depressor effect. The effects were found in the absence of changes in basal blood pressure, since the effect of the high dose of BK had already disappeared before captopril or SAR were injected. Moreover, in preliminary experiments we have observed that a lower, nonhypotensive dose of BK induced similar effects on the action of captopril and SAR (not shown). One mechanism by which we can explain the present result could be that after BK-treatment blood pressure in the animal is more strongly dependent on the constant active formation of All. Thus, captopril may block this formation and induce an otherwise absent fall in MAP. Also, SAR blocks endogenous All and induces a fall in MAP rather than an increase. It is possible that the SAR-induced decrease in MAP in BK-treated rats is a combination of the normally occurring pressor response (see Fig. 2a) and an even larger decrease in MAP caused by the antagonism of endogenous All. Perhaps the injection of BK triggers an increased activity of circulating or tissue ACE. This may be responsible for the rapid inactivation of BK (Adamski and Grega, 1988; Erd6s, 1979; McCaa, 1979), but may also enhance the formation of All. Other pressor mechanisms like the sympathetic nervous system may temporarily be shut off to prevent a large increase in basal blood pressure. Thus, 'normal' blood pressure is maintained, but in this situation the animal relies to a larger extent than normal on circulating or tissue angiotensin to
762
M. VAN DEN BUUSEand J. KERKHOFF
m a i n t a i n cardiovascular homeostasis a n d is therefore m o r e sensitive to any blockade o f the r e n i n angiotensin system. This hypothesis implies t h a t circulating or tissue A I I levels would be increased after injection of BK. A n alternative, a n d in fact opposite, e x p l a n a t i o n for our results in the second series o f experiments is suggested by recent results of Sasaguri et al. (1990). These a u t h o r s showed that B K m a y actually inhibit the activity o f ACE. In the in vivo situation in the n o r m o t e n s i v e rat this m a y not be sufficient to decrease b l o o d pressure for a longer period o f time after the vasodilatory effect o f B K waned. However, additional captopril treatment, which by itself does not block A C E sufficiently to decrease b l o o d pressure in these rats, after B K p r e t r e a t m e n t inhibits A C E activity to such an extent t h a t a ' t h r e s h o l d ' is reached and a fall in pressure results. Sasaguri et al. (1990) indeed showed additive effects of captopril a n d B K on A C E activity. S A R decreases blood pressure after B K p r e t r e a t m e n t because its ability to antagonize the r e m a i n i n g e n d o g e n o u s angiotensin p r e d o m i n a t e s over its partial agonist activity. This hypothesis thus implies t h a t circulating or tissue AII levels would be decreased after injection o f BK. Recently, it was suggested t h a t e n d o g e n o u s B K plays a physiological role in b l o o d pressure regulation also by a t t e n u a t i n g the pressor effect o f AII, vasopressin and a l p h a - a d r e n o r e c e p t o r stimulation ( A u b e r t et al., 1988). A n otherwise inactive dose of BK a n t a g o n i s t induced pressor responses in rats pretreated with n o n p r e s s o r or pressor doses of AII, vasopressin or m e t h o x a m i n e . Also, the a t t e n u a t i n g effect of B K on the pressor action of AII ( A u b e r t et al., 1988) could play a role in the present experiments, such that the pressor (agonistic) action o f S A R is inhibited a n d the AII blocking effect (antagonism) remains. This indirect action of BK could also explain the absence of a n effect o f injected AII after b o t h B K a n d S A R injection (see Fig. 2b) as opposed to some residual pressor action of A I I after pretreatm e n t with S A R only (see Fig. 2a). F u r t h e r studies are needed to show w h e t h e r B K t r e a t m e n t increases or decreases circulating or tissue AII levels, as would be predicted by these two p r o p o s e d hypotheses. It is concluded t h a t B K m a y play an i m p o r t a n t role in cardiovascular regulation, not only t h r o u g h its direct vasodilatory effect, but also by its interaction with o t h e r vasoregulatory mechanisms, notably the r e n i n - a n g i o t e n s i n system. REFERENCES
Adamski S. W. and Grega G. J. (1988) Contribution of kininase II to the waning of vascular actions of bradykinin. Am. J. Physiol. 254, H1042-1050. Aubert J. F., Waeber B., Nussberger J., Vavrek R., Stewart J. M. and Brunner H. R. (1988) Influence of endogenous bradykinin on acute blood pressure response to vasopressors in normotensive rats assessed with a bradykinin antagonist. J. cardiot:asc. Pharmac. 11, 51~55. Carbonell L. F., Carretero O. A., Stewart J. M. and Scicli A. G. (1988) Effect of a kinin antagonist on the acute
antihypertensive activity of enalaprilat in severe hypertension. Hypertension 11, 239-243. Carretaro O. A. and Scicli A. G. (1981) Possible role of kinins in circulatory homeostasis. Hypertension 3 (Suppl. I),4 12. Erd6s E. G. (1979) Inhibitors of kininases. Fedn Proc./edn Am. Socs exp. Biol. 38, 2774 2777. Filep J., Rigter B. and F61des-Filep E. (1987) Antihypertensive and renal effects of captopril in spontaneously hypertensive rats: evidence against a role of the kallikrein kinin system. J. cardiot, asc. Pharmac. 10, 222 227. Hui S. C. G., Dai S. and Ogle C. W. (1986) Mechanisms of captopril-induced potentiation of the depressor responses to arachidonic acid in rats. Clin. exp. pharmac. Physiol. 13, 123 130. Iimura O. and Shimamoto K. (1989) Role of kallikrein kinin system in the hypotensive mechanisms of converting enzyme inhibitors in essential hypertension J. cardiorasc. Pharmac. 13, $63-$66. Ishida H., Scicli A. G. and Carretero O. A. (1989) Role of angiotensin converting enzyme and other peptidases in in z~ivo metabolism of kinins. Hypertension 14, 322-327. Madeddu P., Oppes M., Rubattu S., Dessi'-Fulgheri P., Glorioso N., Soro A. and Rappelli A. (1987) Role of renal kallikrein modulating the antihypertensive effect of a single dose ofcaptopril in normal- and low-renin essential hypertension. J. Hypert. 5, 645 648. McCaa R. E. (1979) Studies in vivo with angiotensin 1 converting enzyme (kininase II) inhibitors. Fedn. Proc. Fedn Am socs exp. Biol. 38, 2783 2787. Mullane K. M. and Moncada S. (1980) Prostacyclin mediates the potentiated hypotensive effect of bradykinin following captopril treatment. Eur. J. Pharmac. 66, 355 365. Pearson L. and Lang W. J. (1967) A comparison in conscious and anesthetized dogs of the effects on blood pressure of bradykinin, kallidin, eledoisin and kallikrein. Eur. J. Pharmac. 2, 83-87. Ponteiri V., Lopes O. U. and Ferreira S. H. (1990) Hypotensive effect of captopril. Role of bradykinin and prostaglandinlike substances. Hypertension 15 (Suppl. I), 155-158. Regoli D. and Baraba J. (1980) Pharmacology of bradykinin and related kinins. Pharmac. Rev. 32, 1 46. Roche e Silva M., Beraldo W. T. and Rosenfeld G. (1949) Bradykinin, a hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venoms and by trypsin. Am. J. Physiol. 156, 261 273. Sasaguri M., Ideishi M., Ikeda M. and Arakawa K. (1990) Inhibitory effects of kinins on angiotensin l conversion in the local circulation. Clin. exp. Hypert. AI2, 551 569. Seino M., Abe K., Nushiro N., Omata K. and Yoshinaga K. (1989) Role of endogenous bradykinin in the acute depressor effect of angiotensin converting enzyme inhibitor captopril assessed by a competitive antagonist of bradykinin. Clin. exp. Hypert. All, 35 43. Sharma J. N. (1990) Does kinin mediate the hypotensive action of angiotensin converting enzyme (ACE) inhibitors? Gen. Pharmac. 21, 451 457. Valloton M. B. (1987)The renin-angiotensin system. Trends pharmac. Sci. 8, 69-74. Waeber B., Aubert J. F., Vavrek R., Stewart J. M., Nussberger J. and Brunner H. R. (1986) Role of bradykinin in blood pressure regulation of conscious spontaneously hypertensive rats. J. Hypert. 4 (Suppl. 6), 597 598. Yang H. Y. T., Erd6s E. G. and Levin Y. (1970) A dipeptyl carboxypeptidase that converts angiotensin I and inactivates bradykinin. Biochim. biophys. Acta 214, 374 476.
0306-3623/91 $3.00 + 0.00 Copyright ~:~ 1991 Pergamon Press plc
I N T E R A C T I O N OF B R A D Y K I N I N A N D A N G I O T E N S I N THE R E G U L A T I O N OF B L O O D P R E S S U R E IN C O N S C I O U S RATS
IN
M. VAN DEN BUUSE* and J. KERKHOFF Rudolf Magnus Institute for Pharmacology, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands (Received 30 November 1990) Abstract---1. The interaction between bradykinin (BK) and the renin-angiotensin system was studied in conscious, catheterized rats. 2. Intravenous injection of BK induced dose-dependent decreases in blood pressure in normotensive Wistar and Wistar-Kyoto rats and spontaneously hypertensive rats. Pretreatment with the angiotensinconverting enzyme (ACE) inhibitor captopril markedly enhanced the effect of BK, such that the dose-response curve shifted significantly to the left in all three strains. 3. In a second series of experiments, captopril did not change basal blood pressure, but blocked the pressor response to angiotensin I (AI), but not angiotensin II (AII). 4. The partial agonist Sar~-Ala8-angiotensin II (SAR) increased blood pressure and blocked the pressor response to subsequent All treatment. 5. After pretreatment with BK (50 #g/kg), captopril evoked a decrease in blood pressure, while still blocking the effect of AI. 6. After pretreatment with BK, SAR decreased blood pressure, while still antagonizing the action of All. 7. These results suggest that ACE plays a role in the inactivation of circulating BK in normotensive and hypertensive rats. Conversely, BK can influence the activity of the renin-angiotensin system, probably by interacting with ACE.
between different forms of hypertension (Iimura and Shimamoto, 1989; Madeddu et al., 1987; Waeber et aL, 1986). In the present study we further investigated the interaction between the BK and the reninangiotensin system. We measured the effect of pretreatment with the ACE-inhibitor captopril on the blood pressure responses to bradykinin in normotensive and hypertensive rats. The results show that captopril differentially influences the hypotensive action of BK. In addition, we tested the effect of pretreatment with BK on the acute blood pressure response to captopril, AI, A l l or the All-antagonist saralasin (Sarl-AlaS-angiotensin II, SAR). A single i.v. injection of bradykinin was found to profoundly alter the responses to these compounds.
INTRODUCTION
Intravenous administration of the nonapeptide bradykinin (BK) results in a dose-dependent vasodilatation and decrease in blood pressure (Carretaro and Scicli, 1981; Pearson and Lang, 1967; Regoli and Baraba, 1980; Roche e Silva et al., 1949). This action is short-lasting, since BK is rapidly broken down by an aminopeptidase. This enzyme was shown to be identical to angiotensin-converting enzyme (ACE), which is responsible for the formation of angiotensin II (AII) from angiotensin I (AI) and angiotensinogen (Yang et al., 1970; Erd6s, 1979; Valloton, 1987). Thus, ACE-inhibitors have been shown to accentuate and prolong the blood pressure decrease produced by i.v. BK (Adamski and Grega, 1988; Ishida et al., 1989; Mullane and Moncada, 1980). Moreover, the antihypertensive effect of ACE-inhibitors may result from attenuated breakdown of the vasodilatory BK, in addition to inhibited formation of the vasoconstrictory A I I (Carbonell et al., 1988; Pontieri et al., 1990; Seino et al., 1989; Sharma, 1990). Other authors have questioned a possible role of BK in the antihypertensive action of ACE-inhibitors (see Filep et al., 1987, and references therein). It was also suggested that the role of kinins in the antihypertensive action of ACE-inhibitors could be different between normotensives and hypertensives and
MATERIALS AND METHODS Rats, operations and drugs Male rats were used, either normotensive Wistars or Wistar-Kyotos (WKY) or spontaneously hypertensive rats (SHR). The animals weighed 200-250 g and were housed 4-5 per cage until the preliminary operations. At least 24 hr before the experiments, the rats were operated under ether anesthesia. Catheters were implanted in the left femoral artery and the right jugular vein and exteriorized at the back of the neck. The rats were then housed individually. Mean arterial pressure (MAP) was recorded through the femoral artery cannula and a Statham P23D transducer on a Grass Model 7 polygraph. Drugs were dissolved in saline and injected through the jugular vein cannula in a volume of 1 ml/kg.
*To whom all correspondence should be sent at his present address: Marion Merrell Dow Research Institute, Strasbourg Research Center, 16, rue d'Ankara, 67009 Strasbourg Cedex, France. 759
M. VAN DEN BUUSEand J. KERKHOFF
760
Bradykinin was purchased from Janssen, Geel, Belgium. AI, AII and SAR were purchased from Bioproducts (UCB), Brussels, Belgium. Captopril (SQ14,255) was a gift from Squibb, Princeton, U.S.A.
Protocol Two types of experiments were performed. In Wistar rats, WKY and SHR we determined the dose response curve for BK before and after administration of 5 mg/kg captopril. In a separate group of Wistar rats, the effect of BK on the activity of the renin--angiotensin system was studied. Thus, first the effect of i.v. AII and AI was assessed. The dose of All (500 ng/kg) was chosen from preliminary dose-response experiments (see Results). The dose of AI was identical to that of AII. Subsequently, saline was injected or BK in a dose which elicited near-maximal depressor responses (50 pg/kg). In separate experiments BK was also tested in a threshold-dose and it was found that such a lower dose induced similar actions on the effect of captopril and SAR. After saline or BK, captopril (5 mg/kg) was injected to block ACE, followed by AII, and AI in doses identical to those described earlier. Then saline or BK were injected again. Finally, SAR was injected in a dose ten times that of AII. By a subsequent injection of AII the receptor-blocking effects of SAR were tested. There was always a 15min interval between injections, except for the last All injection, which was done 5 min after the administration of SAR. In this protocol we thus include drugs that have an intrinsic effect of the renin-angiotensin system (captopril, SAR), which was tested in the rats treated with saline instead of BK. In addition, the effect of BK on these drugs and on the responses to AII and AI were tested.
Analysis Responses were measured as changes of baseline blood pressure and expressed as mean_+ standard error of the mean (SEM). The responses to AII, AI, BK, captopril or SAR were compared with an initial saline treatment. In some cases responses to AII and AI after treatment with captopril or SAR were compared to pretreatment responses. Also, responses to the peptides were compared between the saline group and the BK-treated group. Differences between groups were analyzed with analysis of variance and Student's t-test and considered to be significant when P < 0.05. RESULTS
A d m i n i s t r a t i o n o f B K (i.v.) induced a n immediate depressor response with a peak in the first 15 sec after administration. After a b o u t 2 m i n the effect of B K had largely disappeared. The acute pressor response to AI a n d AII was slower in onset a n d more prolonged. The peak of the pressor response was a r o u n d one min after i.v. injection. In a preliminary d o s e - r e s p o n s e experiment (n = 4), A I I was injected i.v. in the following doses: 0.7, 2, 6, 19, 56, 168, 500 a n d 1500 ng/kg. M a x i m u m pressor responses of these rats were 4_+ 1, 5 + 3 , 11 + 2 , 11 _+3, 20_+3, 34_+4, 39_+ 6 and 43_+ 5 m m H g respectively. F o r subsequent experiments the dose of 500 n g / k g was used (see below). In Fig. 1, the effect of different doses of B K o n M A P is s h o w n in Wistars, W K Y or S H R before a n d after t r e a t m e n t with captopril. In all three strains, B K p r o d u c e d dose-related falls in M A P , a l t h o u g h S H R showed a steeper d o s e - r e s p o n s e curve t h a n the n o r m o t e n s i v e rats. F o r instance, whereas 3.1 # g / k g B K did n o t significantly alter M A P in either strain, 5 0 p g / k g p r o d u c e d a M A P decrease of - 5 7 _ + 3 m m H g in Wistars, - 5 7 _ + 7 m m H g in W K Y a n d - 8 1 _+ 5 m m H g in SHR.
3O
SAL+BK E
0
e~'--~--6~6to \
~-0~o -30
\e....e
"~ .<3 -60
CAP+BK
n <
......
6,,:
W Ib l a b <
6~6\. "0--0%. 0~. o"--"~-oO 0e
-90 30 :x: E Q_
0 -30
SAL+BK
o_o~o~ 0
o\
%xk6~° WKY
o'%'o o...o\
< <"~ -60
CAP+BK
o--e--o\
\~)
~-.,?
-90 30 T
0
E 13._
• .
0"~0/"~/0~
~\T
•\._,\~+B~ o SHR
-30
< "~ -6o '\e -90
o.~
o.4
~.6
8.3
* oX 25
~oo
DOSE BK ( U g / k O
Fig. 1. The effect of i.v. injection of different doses of BK on blood pressure of Wistar, WKY and SHR, either without pretreatment (SAL + BK) or after pretreatment with captopril (CAP + BK). Data are mean + SEM of 5 7 rats per group. ANOVA indicated a significant overall differences between the two curves in either strain. Captopril at the present dose did not change basal b l o o d pressure. However, this drug greatly e n h a n c e d the response to subsequent B K treatment, as became evident from a parallel shift of the B K d o s e - r e s p o n s e curve to the left. As can be seen in Fig. 1, the threshold dose for a decrease in M A P by BK shifted from 3 - 6 # g / k g to a r o u n d 0.2 #g/kg. After captopril t r e a t m e n t the otherwise ineffective dose o f 6.3 # g / k g B K p r o d u c e d a M A P decrease of - 5 2 _+ 6 m m H g in Wistars, - 5 0 +_ 3 m m H g in W K Y a n d - 8 1 +_ 5 m m H g in SHR. These values are very similar to the responses o b t a i n e d after injection o f the 5 0 p g / k g dose in the first experiment. In the second series of experiments, W i s t a r rats were tested for the effect of B K o n the activity of the r e n i n - a n g i o t e n s i n system. As is s h o w n in Fig. 2a, saline injections produced little change in blood pressure. Subsequent injections of 5 0 0 n g / k g AII a n d AI produced pressor responses o f 47 +_ 6 and 44_+ 6 m m H g , respectively. Captopril t r e a t m e n t (5 m g / k g i . v . ) by itself induced no effect on b l o o d pressure, n o r did it significantly influence the pressor response to AII (56 +_ 7 mmHg). However, captopril virtually blocked the pressor response to AI (16 + 6 m m H g ) , illustrating t h a t the conversion o f AI to AII is essential for the pressor effect of this peptide. S A R ( S p g / k g i . v . ) induced a pressor response (34 +_ 7 m m H g ) , illustrating the partial agonistic activity of this peptide. S A R also inhibited the effect of a subsequent injection of AII (17 -+_4 m m H g ) , indicating the antagonistic properties of SAR.
Bradykinin and angiotensin interactions (a)
-I-
v
E E
DISCUSSION
70
35 0
13-
S All AI S
CAP AllAI S
SAR All
-35 -70
(b)
70
-1-
35
E E v
0
761
rl
-35 -70 Fig. 2. (a) (Top panel): maximal changes in pressure after i.v. injection of AII (500 ng/kg), AI (500 ng/kg), captopril (5 mg/kg) or SAR (5 #g/kg) in Wistar rats. AII and AI induce comparable increases in blood pressure. Captopril did not change MAP but blocks the actions of AI. SAR induces a pressor response and inhibits the response to All. (b) (Bottom panel): maximal changes in blood pressure in a separate group of rats after i.v. injection of All, AI, BK (50 pg/kg), captopril and SAR. All and AI induce similar changes in MAP as in the first group, whereas BK causes a fall in blood pressure. After BK, captopril induces a decrease in blood pressure, while blocking the action of AI. After BK, SAR induced a fall in blood pressure, while blocking the action of All. *P < 0.05 for difference with saline treated rats (first column).
Figure 2b shows similar treatments in a separate group of rats, only now i.v. saline had been replaced by 50 #g/kg BK. Thus, the initial AII and AI injections produced similar pressor responses as in the group described above (38 + 6 and 43 ___4 m m H g respectively). BK produced a marked fall in blood pressure ( - 5 1 + 5 mmHg), which had completely disappeared before the injection of captopril. In contrast to the saline-treated group, after BK captopril induced a marked fall in blood pressure ( - 5 1 _ 3 mmHg) which lasted 5-10 min. Nevertheless, the All-induced pressor response was still present (43 + 6 mmHg), as was the inhibitory effect of captopril on the response to AI (11 + 7 mmHg). As expected from the dose-response experiment (see Fig. 1), the second, (post-captopril) injection of BK induced a fall in blood pressure similar to the first BK treatment ( - 50 + 8 mmHg). Injection of SAR after BK treatment induced a decrease in blood pressure ( - 3 1 + 8 ) rather than the increase found in the saline-treated group. SAR also blocked the pressor effect of All. The response to All was reversed to a fall in blood pressure ( - 1 6 + 5mmHg), although this effect was not significantly different from the change in MAP induced by saline.
Bradykinin is a nonapeptide which is formed by the enzyme kallikrein from the precursor bradykininogen and cleaved by the proteolytic enzymes kininase I and II (McCaa, 1979; Regoli and Baraba, 1980; Valloton, 1987). It has been shown that kininase II is identical to ACE (Yang et al., 1970; Erd6s, 1979), indicating an overlap in the regulation of the renin-angiotensin and the kallikrein-bradykinin systems. Bradykinin may affect cardiovascular homeostasis also by interacting with other humoral systems, such as prostaglandines (Hui et al., 1986; Mullane and Moncada, 1980; Pontieri et al., 1990). A number of authors have suggested that elevated levels of kinins are partly or completely responsible for the hypotensive action of ACE-inhibitors (see Introduction). In our experiments, a nonhypotensive dose of captopril greatly enhanced the depressor responses to BK, most probably through inhibition of its breakdown. This effect was present in normotensive Wistar and WKY rats and in SHR. Although the dose-response curve for BK was steeper in SHR, the shift of the BK dose-response curve by captopril appeared to be similar in this strain as compared to the two normotensive strains. It needs to be elucidated whether the apparently steeper dose-response curve for BK in SHR is a result of a greater sensitivity of the vascular wall to BK in these rats, or merely a result of the higher blood pressure. In the present experiments we show that BK itself can also greatly influence the blood pressure responses to various experimental manipulations of the renin-angiotensin system. The most pronounced effects were the occurrence of an acute, marked fall in blood pressure after captopril injection, and the change of the acute pressor response to SAR into a depressor effect. The effects were found in the absence of changes in basal blood pressure, since the effect of the high dose of BK had already disappeared before captopril or SAR were injected. Moreover, in preliminary experiments we have observed that a lower, nonhypotensive dose of BK induced similar effects on the action of captopril and SAR (not shown). One mechanism by which we can explain the present result could be that after BK-treatment blood pressure in the animal is more strongly dependent on the constant active formation of All. Thus, captopril may block this formation and induce an otherwise absent fall in MAP. Also, SAR blocks endogenous All and induces a fall in MAP rather than an increase. It is possible that the SAR-induced decrease in MAP in BK-treated rats is a combination of the normally occurring pressor response (see Fig. 2a) and an even larger decrease in MAP caused by the antagonism of endogenous All. Perhaps the injection of BK triggers an increased activity of circulating or tissue ACE. This may be responsible for the rapid inactivation of BK (Adamski and Grega, 1988; Erd6s, 1979; McCaa, 1979), but may also enhance the formation of All. Other pressor mechanisms like the sympathetic nervous system may temporarily be shut off to prevent a large increase in basal blood pressure. Thus, 'normal' blood pressure is maintained, but in this situation the animal relies to a larger extent than normal on circulating or tissue angiotensin to
762
M. VAN DEN BUUSEand J. KERKHOFF
m a i n t a i n cardiovascular homeostasis a n d is therefore m o r e sensitive to any blockade o f the r e n i n angiotensin system. This hypothesis implies t h a t circulating or tissue A I I levels would be increased after injection of BK. A n alternative, a n d in fact opposite, e x p l a n a t i o n for our results in the second series o f experiments is suggested by recent results of Sasaguri et al. (1990). These a u t h o r s showed that B K m a y actually inhibit the activity o f ACE. In the in vivo situation in the n o r m o t e n s i v e rat this m a y not be sufficient to decrease b l o o d pressure for a longer period o f time after the vasodilatory effect o f B K waned. However, additional captopril treatment, which by itself does not block A C E sufficiently to decrease b l o o d pressure in these rats, after B K p r e t r e a t m e n t inhibits A C E activity to such an extent t h a t a ' t h r e s h o l d ' is reached and a fall in pressure results. Sasaguri et al. (1990) indeed showed additive effects of captopril a n d B K on A C E activity. S A R decreases blood pressure after B K p r e t r e a t m e n t because its ability to antagonize the r e m a i n i n g e n d o g e n o u s angiotensin p r e d o m i n a t e s over its partial agonist activity. This hypothesis thus implies t h a t circulating or tissue AII levels would be decreased after injection o f BK. Recently, it was suggested t h a t e n d o g e n o u s B K plays a physiological role in b l o o d pressure regulation also by a t t e n u a t i n g the pressor effect o f AII, vasopressin and a l p h a - a d r e n o r e c e p t o r stimulation ( A u b e r t et al., 1988). A n otherwise inactive dose of BK a n t a g o n i s t induced pressor responses in rats pretreated with n o n p r e s s o r or pressor doses of AII, vasopressin or m e t h o x a m i n e . Also, the a t t e n u a t i n g effect of B K on the pressor action of AII ( A u b e r t et al., 1988) could play a role in the present experiments, such that the pressor (agonistic) action o f S A R is inhibited a n d the AII blocking effect (antagonism) remains. This indirect action of BK could also explain the absence of a n effect o f injected AII after b o t h B K a n d S A R injection (see Fig. 2b) as opposed to some residual pressor action of A I I after pretreatm e n t with S A R only (see Fig. 2a). F u r t h e r studies are needed to show w h e t h e r B K t r e a t m e n t increases or decreases circulating or tissue AII levels, as would be predicted by these two p r o p o s e d hypotheses. It is concluded t h a t B K m a y play an i m p o r t a n t role in cardiovascular regulation, not only t h r o u g h its direct vasodilatory effect, but also by its interaction with o t h e r vasoregulatory mechanisms, notably the r e n i n - a n g i o t e n s i n system. REFERENCES
Adamski S. W. and Grega G. J. (1988) Contribution of kininase II to the waning of vascular actions of bradykinin. Am. J. Physiol. 254, H1042-1050. Aubert J. F., Waeber B., Nussberger J., Vavrek R., Stewart J. M. and Brunner H. R. (1988) Influence of endogenous bradykinin on acute blood pressure response to vasopressors in normotensive rats assessed with a bradykinin antagonist. J. cardiot:asc. Pharmac. 11, 51~55. Carbonell L. F., Carretero O. A., Stewart J. M. and Scicli A. G. (1988) Effect of a kinin antagonist on the acute
antihypertensive activity of enalaprilat in severe hypertension. Hypertension 11, 239-243. Carretaro O. A. and Scicli A. G. (1981) Possible role of kinins in circulatory homeostasis. Hypertension 3 (Suppl. I),4 12. Erd6s E. G. (1979) Inhibitors of kininases. Fedn Proc./edn Am. Socs exp. Biol. 38, 2774 2777. Filep J., Rigter B. and F61des-Filep E. (1987) Antihypertensive and renal effects of captopril in spontaneously hypertensive rats: evidence against a role of the kallikrein kinin system. J. cardiot, asc. Pharmac. 10, 222 227. Hui S. C. G., Dai S. and Ogle C. W. (1986) Mechanisms of captopril-induced potentiation of the depressor responses to arachidonic acid in rats. Clin. exp. pharmac. Physiol. 13, 123 130. Iimura O. and Shimamoto K. (1989) Role of kallikrein kinin system in the hypotensive mechanisms of converting enzyme inhibitors in essential hypertension J. cardiorasc. Pharmac. 13, $63-$66. Ishida H., Scicli A. G. and Carretero O. A. (1989) Role of angiotensin converting enzyme and other peptidases in in z~ivo metabolism of kinins. Hypertension 14, 322-327. Madeddu P., Oppes M., Rubattu S., Dessi'-Fulgheri P., Glorioso N., Soro A. and Rappelli A. (1987) Role of renal kallikrein modulating the antihypertensive effect of a single dose ofcaptopril in normal- and low-renin essential hypertension. J. Hypert. 5, 645 648. McCaa R. E. (1979) Studies in vivo with angiotensin 1 converting enzyme (kininase II) inhibitors. Fedn. Proc. Fedn Am socs exp. Biol. 38, 2783 2787. Mullane K. M. and Moncada S. (1980) Prostacyclin mediates the potentiated hypotensive effect of bradykinin following captopril treatment. Eur. J. Pharmac. 66, 355 365. Pearson L. and Lang W. J. (1967) A comparison in conscious and anesthetized dogs of the effects on blood pressure of bradykinin, kallidin, eledoisin and kallikrein. Eur. J. Pharmac. 2, 83-87. Ponteiri V., Lopes O. U. and Ferreira S. H. (1990) Hypotensive effect of captopril. Role of bradykinin and prostaglandinlike substances. Hypertension 15 (Suppl. I), 155-158. Regoli D. and Baraba J. (1980) Pharmacology of bradykinin and related kinins. Pharmac. Rev. 32, 1 46. Roche e Silva M., Beraldo W. T. and Rosenfeld G. (1949) Bradykinin, a hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venoms and by trypsin. Am. J. Physiol. 156, 261 273. Sasaguri M., Ideishi M., Ikeda M. and Arakawa K. (1990) Inhibitory effects of kinins on angiotensin l conversion in the local circulation. Clin. exp. Hypert. AI2, 551 569. Seino M., Abe K., Nushiro N., Omata K. and Yoshinaga K. (1989) Role of endogenous bradykinin in the acute depressor effect of angiotensin converting enzyme inhibitor captopril assessed by a competitive antagonist of bradykinin. Clin. exp. Hypert. All, 35 43. Sharma J. N. (1990) Does kinin mediate the hypotensive action of angiotensin converting enzyme (ACE) inhibitors? Gen. Pharmac. 21, 451 457. Valloton M. B. (1987)The renin-angiotensin system. Trends pharmac. Sci. 8, 69-74. Waeber B., Aubert J. F., Vavrek R., Stewart J. M., Nussberger J. and Brunner H. R. (1986) Role of bradykinin in blood pressure regulation of conscious spontaneously hypertensive rats. J. Hypert. 4 (Suppl. 6), 597 598. Yang H. Y. T., Erd6s E. G. and Levin Y. (1970) A dipeptyl carboxypeptidase that converts angiotensin I and inactivates bradykinin. Biochim. biophys. Acta 214, 374 476.