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The effects of captopril (SQ 14,225) on bradykinin-induced bronchoconstriction in the anesthetized guinea pig
287
European Journal of Pharmacology, 57 (1979) 287--294 © Elsevier/North-Holland Biomedical Press
THE EFFECTS OF CAPTOPRIL (SQ 14,225) ON BRADYKININ-INDUCED BRONCHOCONSTRICTION IN THE ANESTHETIZED GUINEA PIG ROLAND GREENBERG, GEORGE H. OSMAN, JR., EUGENE H. O ' K E E F E and MICHAEL J. ANTONACCIO
The Squibb Institute for Medical Research, P.O. Box 4000, Princeton, N.J. 08540, U.S.A. Received 15 December 1978, revised MS received 18 April 1979, accepted 7 May 1979
R. GREENBERG, G.H. OSMAN, JR., E.H. O'KEEFE and M.J. ANTONACCIO, The effects of captopril (SQ 14,225) on bradykinin-induced bronchoconstrictionin the anesthetized guinea pig, European J. Pharmacol. 57 (1979) 287--294. The effect of captopril'(SQ 14,225) a potent inhibitor of angiotensin converting enzyme (ACE: kininase II) on the bronchoconstrictor response to bradykinin was studied in the anesthetized guinea pig. The i.v. administration of captopril caused a profound long lasting hypotension without affecting pulmonary resistance or dynamic compliance. Similarly, the i.v. administration of bradykinin caused small increases in pulmonary resistance and decreases in dynamic compliance which were not altered by the administration of captopril. However, after /3-receptor blockade with propranolol, bradykinin-induced changes in resistance and compliance were enhanced; additional captopril administration further potentiated the bradykinin effects. The prostaglandin synthetase inhibitor indomethacin antagonized the bradykinin-induced bronchoconstriction in ~-blocked animals and its potentiation by captopril. In the isolated perfused guinea pig lung, bradykinin caused a dose dependent release of a prostaglandin-like substance which was significantly increased by captopril and antagonized by indomethacin. These results suggest that bradykinin causes a prostaglandin-mediated bronchoconstriction. Captopril, a potent inhibitor of ACE, prevents the degradation of bradykinin thus potentiating the bradykinin-induced bronchoconstriction, an effect observed in intact animals only in the absence of pulmonary ~-receptor activation. Bronchoconstriction Prostaglandin
Bradykinin
Guinea pig
1. Introduction The precise role of bradykinin in asthma is unknown. Bradykinin causes bronchoconstriction when administered by aerosol to asthmatics; however, it does not cause broncl~oconstriction in normal persons (Collier, 1970). There is little evidence available on the release of kinins during human anaphylaxis or asthma; however, Abe et al. (1967) reported elevated blood kinin concentrations in patients with severe bronchial asthma, indicating that kinins may be involved in human bronchial asthma. Bradykinin causes bronchoconstriction in both anesthetized and unanesthetized guinea pigs (Collier and Shorley, 1960; Drazen and
Propranolol
Captopril
Hypotension
Austen, 1974; 1975). This bronchoconstriction is inhibited by nonsteroidal anti-inflammatory drugs (Collier and Shorley, 1960; 1963). Bradykinin also causes the release of prostaglandins and thromboxane A: from isolated perfused guinea pig lungs. This release is inhibited by indomethacin and mepacrine (Palmer et al., 1973; Vargaftig and Dao Hai, 1972). It has been suggested that the bradykinin-induced bronchoconstriction is indirect and mediated through the generation of TXA2 from prostaglandins (Collier, 1969; Vane and Ferreira, 1974). Captopril (SQ14,225) has been recently reported to be an effective competitive inhibitor of angiotensin I-converting enzyme (ACE:kininaseII) (Ondetti et al., 1977).
288
Inhibition of ACE by captopril has been shown to potentiate both the hypotensive effect of exogenously administered bradykinin in the rat and rabbit and the contractile response to bradykinin in the isolated guinea pig ileum (Rubin et al., 1978a; Murthy et al., 1978). Experiments were therefore done to determine the effects of captopril on the bronchoconstrictor response to bradykinin in the anesthetized guinea pig. Additional experiments were done to determine the effect of captopril on bradykin-induced release of prostaglandins from the isolated perfused guinea pig lung.
2. Materials and methods
2.1. Pulmonary resistance and dynamic compliance Male guinea pigs weighing 480--520 g were anesthetized with urethane (1.5 g/kg, i.p.). Spontaneous respiration was arrested with succinylcholine chloride (1.5 mg/kg, i.p.). The guinea pigs were artificially ventilated with a Palmer respiration pump through a tracheal cannula at a rate of 72 strokes/min and a stroke volume of 3 ml. Pulmonary resistance and dynamic compliance were determined by the method described by Amdur and Mead (1958) as modified by Giles et al. (1973). Respiratory flow was measured with a Fleisch pneumotachograph (No. 000) and a Validyne (MP 45) differential pressure transducer. Transpulmonary pressure was determined by monitoring the difference between pressure in the external end of a tracheal cannula and the pressure in the pleural cavity by means of a Validyne MP 45 differential pressure transducer. The flow and pressure signals were fed into an on-line analog Pulmonary Mechanics Computer (Buxco Electronics, Inc.) which provided calculation of pulmonary resistance and dynamic compliance. The computer output of flow, pressure, volume, resistance and dynamic compliance were recorded on a
R. G R E E N B E R G ET AL.
Brush 260 recorder. Blood pressure was measured from a carotid artery with a Statham P23Db transducer and heart rate was derived from the pulse pressure. Both blood pressure and heart rate were recorded with the Buxco pulmonary mechanics computer. All parameters were digitalized by a Data Logger, Model DL-12 Buxco electronics and recorded on a Texas Instrument silent 700 ASR printer.
2.2. Isolated perfused guinea pig lung Guinea pig isolated lungs were perfused through the pulmonary artery and the effluent bioassayed for prostaglandins by a method similar to that described by Piper and Vane (1969). Male guinea pigs (350--400 g) were injected with heparin (500 U/kg, i.p.) and killed by a blow on the head. The pulmonary artery and trachea were cannulated and the lung removed and suspended in a heated glass chamber. The pulmonary artery was perfused with Krebs--Henseleit solution (mM: NaC1118.0, KC1 4.7, CaC122.5, KH2PO4 1.1, MgSO4 1.2, NaHCO3 25.0 and glucose 11.0) containing 3% dextran 70,000 mw with an Extracorporeal pump at a rate of 4 ml/min (Piper and Vane, 1969; Bhattacharya and Delaunois, 1955). The effluent from the lung superfused an assay tissue (chick rectum) which contracts in response to prostaglandins and is insensitive to bradykinin (Alabaster and Bakhle, 1972; Blumberg et al., 1977; Gilmore et al., 1968). In addition, the assay tissue was perfused at a rate of 1 ml/min with Krebs--Henseleit solution containing a mixture of antagonists which make it insensitive to histamine, acetylcholine, catecholamines and serotonin. The concentrations of the antagonists mepyramine, atropine and phenoxybenzamine were 0.1 pg/ ml, propranolol 2 pg/ml and methysergide 0.2 pg/ml (Piper and Vane, 1969). Indomethacin 10 pg/ml was also added to the solution perfusing the assay tissue to eliminate the possibility that the assay tissue might release endogenous prostaglandins (Eckenfels and
CAPTOPRIL AND BRADYKININ BRONCHOCONSTRICTION
Vane, 1972). Drugs were either injected into the cannulated pulmonary artery (through the lung} or directly over the assay tissue. Contractions of the chick rectum were measured with a Grass FT 03 force displacement transducer and recorded on a Beckman Dynograph recorder. 2.3. Materials and statistics The drugs used in this study were: succinylcholine C1 (Sigma), bradykinin triacetate (Sigma), captopril (D-3-mercapto-2-methylpropanoyl-L-proline) (Squibb), indomethacin (Sigma), mepyramine maleate (Ehemal0g), phenoxybenzamine HC1 (SKF), atropine SO4 (Sigma), D,L-propranolol HC1 (Sigma), prostaglandin E2 (Ono). Indomethacin was dissolved in a 2% sodium carbonate solution and the pH adjusted to 7.5 with HC1. A stock solution of PGE2 was prepared by dissolving 10 mg in 9 ml of a solution of 2% sodium carbonate and 1 ml of 95% ethanol. Drug concentrations are expressed as final concentrations of free base. The data were analyzed by Student's t-test for paired data. 3. Results
3.1. Respiratory and cardiovascular effects of captopril in the anesthetized guinea pig Captopril (0.1, 1 and 5 mg/kg, i.v.) was administered to four anesthetized guinea pigs for each dose before and after the administration of propranolol (1 mg/kg, i.v.). Captopril did not significantly alter either pulmonary resistance or dynamic compliance. However, these doses of captopril caused a severe long lasting hypotension both before and after propranolol (fig. 1). There were no significant changes in heart rate accompanying the hypotensive response of captopril. 3.2. The effect of captopril on the bronchoconstrictor response to bradykinin in the anesthetized guinea pig Bradykinin (1.0, 3.0 and 5.0 pg/kg, i.v.) was administered to groups of 5--7 guinea
289
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pigs for each dose 15 min before and 5 min after the administration of captopril (100 pg/ kg, i.v.). These doses of bradykinin caused small increases in pulmonary resistance and small decreases in dynamic compliance. The increase in pulmonary resistance in response to 5 pg of bradykinin was the only pulmonary response which was altered by captopril and then by only 10% (fig. 2). The maximum changes in resistance and dynamic compliance in response to bradykinin were obtained within 30 sec of administration and lasted for up to 3 min for resistance and 10 min for dynamic compliance. Captopril did not significantly alter the duration of these responses. The administration of captopril 15min after the administration of bradykinin (1.0 pg/ kg) in the above experiments had no effect on pulmonary resistance or dynamic compliance. However, the administration of captopril 15 min after bradykinin 3.0 and 5.0 pg/kg caused small increases in resistance and small
290
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decreases in dynamic compliance (fig. 3). In additional control experiments, bradykinin 1 and 3 #g/kg were administered twice at 15 min intervals to 4 and 5 guinea pigs respectively. There was no significant difference between the first and second bronchoconstrictor response to bradykinin. 3.3. The effect of captopril on the bronchoconstrictor response to bradykinin in the anesthetized guinea pig pretreated with propranolol The bronchoconstrictor response to bradykinin is enhanced by ~-adrenergic blockade with propranolol (Collier et al. 1965). Experiments were therefore done to evaluate the effect of captopril on the bronchoconstrictor response to bradykinin in guinea pigs which were pretreated with propranolol (1 mg/kg, i.v.). Bradykinin (0.1, 0.3 and 1.0 pg/kg, i.v.) was administered to groups of 5--7 propranolol-pretreated guinea pigs for each dose, 15 min before and 5 min after the administration of captopril (100#g/kg). These relatively small doses of bradykinin caused increases in resistance and decreases in dynamic compliance which were all significantly potentiated
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by captopril except the decrease in compliance in response to 0.1 pg bradykinin (fig. 2). A representative experiment is illustrated in fig. 4. The maximum changes in resistance and compliance were obtained within 30 sec of administration and lasted from 5 to 10 min. Captopril significantly prolonged the changes in both resistance and dynamic compliance after all doses of bradykinin. The administration of captopril 15 min after the administration of bradykinin (0.1 and 0.3 pg/kg) in the above experiments caused substantial increases in pulmonary resistance and decreases in dynamic compliance; however, only small changes were observed after bradykinin (1.0 #g/kg) (fig. 3). In additional control experiments bradykinin 1 #g/kg was administered twice at 15 min intervals to 4 guinea pigs pretreated with propranolol (1 mg/kg). There was no significant difference between the first and second bronchoconstrictor response to bradykinin. 3.4. The effect of indomethacin on the potentiation of bradykinin-induced bronchoconstriction by captopril in the anesthetized propanolol-treated guinea pig Bradykinin bronchoconstriction is believed to be due to the release of prostaglandins
CAPTOPRIL AND BRADYKININ BRONCHOCONSTRICTION
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(Collier, 1969; Collier and Shorley, 1960). Experiments were therefore done to see if the 100 F
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prostaglandin synthetase inhibitor indomethacin would antagonize the bradykinin bronchoconstriction in the fl-blocked captopril-treated guinea pig. Bradykinin (1.0pg/kg) was administered i.v. to 8 guinea pigs pretreated with propranolol (1 mg/kg) before and after the administration of indomethacin (2.5 mg/kg) plus captopril (100 pg/kg). Bradykinin caused substantial increases in pulmonary resistance and decrease in dynamic compliance which were significantly reduced by the administration of indomethacin plus captopril, respectively (fig. 5). 3.5. The effect o f captopril on the bradykinininduced release o f a prostaglandin-like substance (PLS) from the isolated perfused guinea pig lung
Bradykinin has been shown to release prostaglandin and TXA2 from guinea pig isolated lungs (Palmer et al., 1973). Experi-
292
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ments were therefore done to see if captopril would increase bradykinin-induced release of prostaglandins. In 6 experiments bradykinin (50, 100 and 500 ng) was injected into the pulmonary artery at 15 min intervals prior to and during the infusion of captopril (10 /~g/ml). The effluent from the lung was bioassayed on the chick rectum after each dose of bradykinin. A typical experiment is illustrated in fig. 6. Captopril significantly increased the amount of prostaglandin-like substance (PLS) released by all doses of bradykinin {fig. 7). In four control experiments bradykinin (50, 100 and 500 ng) was injected into the pulmonary circulation twice at 15 min intervals. There was no significant difference between the amount of PLS released between the first and second administration of bradykinin. In four additional experiments, bradykinin (500 ng) was injected into the pulmonary artery prior to an during the infusion of captopril (10 pg/ml) plus indomethacin (10 pg/ml). Bradykinin caused the initial release of 10.9 + 3.1 ng of PLS which was significantly reduced to 1.0 + 1.0 ng/ml of PLS after the infusion of captopril and indomethacin P < 0.05. 4. Discussion
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Fig. 7. The effect of captopril on the bradykinininduced release of PLS from the isolated guinea pig lung. Increasing doses of bradykinin were injected into the pulmonary circulation at 15 rain intervals before and during the constant infusion of captopril (10 #g/ml). Each point on the dose--response curve is the mean of 5 experiments. The bars represent the standard error. Significance of the difference from controls. * P < 0.05, ** P < 0.01. Ordinate: PLS (ng). Abscissa: bradykinin (ng). o . . . . . . o control; o--------o, captopril (SQ 14,225) 10 pg/ml.
The results of this study show that relatively high doses of the potent ACE inhibitor captopril causes a profound long lasting hypotension in the anesthetized guinea pig without affecting pulmonary resistance or dynamic compliance. Captopril has been shown to lower the blood pressure in several hypertensive animal models, including renal and spontaneously hypertensive rats. However, captopril has very little hypotensive effect in normotensive animals, such as rat, rabbit, cat and monkey (Rubin et al., 1978b). In contrast, this study has shown that even moderate doses of captopril cause profound hypotension in the anesthetized guinea pig. This hypotension may be due to elevated
CAPTOPRIL AND BRADYKININ BRONCHOCONSTRICTION
plasma renin and consequently AI levels caused by the urethane anesthesia (Pettinger and Campbell, 1976), coupled with high levels of circulating plasma converting enzyme in the guinea pig (Cushman and Cheung, 1972). The results further show that bradykinin causes small increases in pulmonary resistance and small decreases in dynamic compliance which were not significantly altered by captopril. However, in guinea pigs pretreated with propranolol bradykinin caused large increases in pulmonary resistance and decreases in dynamic compliance which were potentiated by captopril. Bradykinin causes the release of catecholamines from the adrenal glands which can antagonize its bronchoconstrictor effect (Staszewska-Braczak and Vane, 1967; Piper et al., 1967). It has also been shown that bradykinin bronchoconstriction is enhanced by fl-blockade with propranolol (Collier et al., 1965; McCulloch et al., 1967 and Collier and James, 1967). The above results therefore suggest that inhibition of ACE with captopril increases the concentration of bradykinin causing an increase in both bronchoconstriction and release of catecholamines. The increased catecholamine secretion may be responsible for attenuating the bronchoconstriction. However, in the presence of propranolol where the bronchodilator effects of the released catecholamines are antagonized, captopril potentiates the bradykinin bronchoconstriction. These results are consistent with the findings of Rubin et al. (1978a) and Murthy et al. (1978) showing that captopril potentiates the contractile response to bradykinin in the isolated guinea pig ileum and the hypotensive responses to bradykinin in the rat and rabbit. The administration of captopril in normal or propranolol-treated guinea pigs did not cause any changes in pulmonary resistance or dynamic compliance in animals not receiving bradykinin. However, in guinea pigs receiving bradykinin prior to captopril, there was a significant increase in pulmonary resistance and decrease in dynamic compliance in response to captopril after the bronchocon-
293
strictor response to bradykinin has disappeared. These results might be explained on the basis of a residual subthreshold amount of circulating or bound bradykinin remaining 15 min after the bronchoconstriction has disappeared. The administration of captopril then elevates this concentration of bradykinin possibly by displacing it from noncontractile tissues. Bradykinin is believed to cause bronchoconstriction in the guinea pig indirectly by releasing prostaglandin and TXA: (Collier, 1969). The bronchoconstrictor action of bradykinin is effectively antagonized by nonsteroidal anti-inflammatory drugs (Collier, 1970), which are potent inhibitors of prostaglandin synthesis (Vane, 1971). Bradykinin has also been shown to cause the release of prostaglandins and TXA2 from isolated guinea pig lungs. This release is inhibited by the prostaglandin synthetase inhibitor indomethacin or the phospholipase inhibitor mepacrine. It is further suggested that bradykinin activates a phospholipase causing the formation of arachidonic acid and the subsequent synthesis and release of prostaglandins and TXA2 (Vargaftig and Dao Hai, 1972; Palmer et al., 1973; Blackwell et al., 1978). The results of the present study have shown that the prostaglandin synthetase inhibitor indomethacin abolishes the bradykinin bronchoconstriction and its potentiation by captopril. In the isolated perfused lung bradykinin causes a dose dependent release of a PLS which is augmented by captopril and antagonized by indomethacin. These results are similar to those of Blumberg et al. (1977) who demonstrated the enhanced bradykinininduced release of PGEz from the perfused rabbit mesentary blood vessels by the ACE inhibitor SQ20,881. The present results are therefore consistent with a bradykinininduced prostaglandin mediated bronchoconstriction, which is potentiated by the reduction in the inactivation of bradykinin by captopril. References Abe, K., N. Watanabe, N. Kimagai, T. Mouri, T. Seki and K. Yoshinaga, 1967, Circulating plasma kinin
294 in patients with bronchial asthma, Experientia 23,626. Alabaster, V.A. and Y.S. Bakhle, 1972, The inactivation of bradykinin in the pulmonary circulation of isolated lungs, Brit. J. Pharmacol. 4 5 , 2 9 9 . Amdur, M.O. and J. Mead, 1958, Mechanics of respiration in unanesthetized guinea pigs, Amer. J. Physiol. 192, 364. Bhattacharya, B.K. and A.L. Delaunois, 1955, An improved method for the perfusion of isolated lung of guinea pig, Arch. Intern. Pharmacodyn. 101,495. Blackwell, G.J., R.J. Flower, F.P. Nijkamp and J.R. Vane, 1978, Phospholipase A2 activity of isolated perfused lungs: stimulation and inhibition by antiinflammatory steroids, Brit. J. Pharmacol. 62, 79. Blumberg, A.L., S.E. Denny, G.R. Marshall and P. Needleman, 1977, Blood vessel-hormone interactions: angiotensin, bradykinin and prostaglandin, Amer. J. Physiol. 232, H305. Collier, H.O.J., 1969, New light on how aspirin works, Nature 223, 35. Collier, H.O.J., 1970, Kinins and ventilation of the lungs in: Handbook of Experimental Pharmacology, Bradykinin and Kallidin and Kallikrein, Vol. XXV, ed. E.G. ErdSs (Springer, New York) p. 409. Collier, H.O.J. and G.W.L. James, 1967, Humoral factors affecting pulmonary inflation during anaphylaxis in the guinea pig in vivo, Brit. J. Pharmacol. 30,283. Collier, H.O.J., G.W.L. James and P.J. Piper, 1965, Intensification by adrenalectomy or by ~-adrenergic blockade of the bronchoconstrictor action of bradykinin in the guinea pig, J. Physiol. 180, 13P. Collier, H.O.J. and P.G. Shorley, 1960, Analgesic antipyretic drugs as antagonists of bradykinin, Brit. J. Pharmacol. 15,601. Collier, H.O.J. and P.G. Shorley, 1963, Antagonism by mefenamic and flufenamic acids of the bronchoconstrictor action of kinins in the guinea pig, Brit. J. Pharmacol. 20,345. Cushman, D.W. and H.S. Cheung, 1977, Studies in vitro of the angiotensin-converting enzyme of lung and other tissues, in:Hypertension, Eds. J. Geneset and E. Koiw (Springer-Verlag, New York) p. 532. Drazen, J.M. and K.F. Austen, 1974, Effects of intravenous administration of slow-reacting substance of anaphylaxis, histamine, bradykinin and prostaglandin F2~ on pulmonary mechanics in the guinea pig, J. Clin. Invest. 53, 1679. Drazen, J.M. and K.F. Austen, 1975, Atropine modification of the pulmonary effects of chemical mediators in the guinea pig, J. Appl. Physiol. 38, 834. Eckenfels, A. and J.R. Vane, 1972, Prostaglandin oxygen tension and smooth muscle tone, Brit. J. Pharmacol. 4 5 , 4 5 1 . Giles, R.E., J.C. William and M.P. Finkel, 1973, The bronchodilator and cardiac stimulant effects of Th 1165A, salbutamol and isoproterenol, J. Phar-
R. GREENBERG ET AL. macol. Exptl. Therap. 186,472. Gilmore, N., J.R. Vane and J.H. Wyllie, 1968, Prostaglandin released by the speen, Nature 218, 1135. McCulloch, M.W., C. Proctor and M.J. Rand, 1967, Evidence for an adrenergic homeostatic bronchodilator reflex mechanism, European J. Pharmacol. 2,214. Murthy, V.S., T.L. Waldron and M.E. Goldberg, 1978, The mechanism of bradykinin potentiation after inhibition of angiotensin converting enzyme by SQ 14,225 in conscious rabbits, Circulation Res. 43, Suppl. I, 41. Ondetti, M.A., D.W. Cushman and B. Rubin, 1977, Design of specific inhibitors of angiotensin-converting enzyme: a new class of orally-active antihypertensive agents, Science 196,441. Palmer, M.A., P.J. Piper and J.R. Vane, 1973, Release of rabbit aorta contracting substance (RCS) and prostaglandins induced by chemical or mechanical stimulation of guinea pig lungs, Brit. J. Pharmacol. 49,226. Pettinger, W.A. and W.B. Campbell, 1976, The rat as an animal model for study of the renin-angiotensinaldosterone-sodium (RAAS) axis, in: New Antihypertensive Drugs, eds. A. Scriabine and C.S. Sweet (Spectrum, New York) p. 179. Piper, P.J., H.O.J. Collier and J.R. Vane, 1967, Release of catecholamines in the guinea pig by substances involved in anaphylaxis, Nature 213,838. Piper, P.J. and J.R. Vane, 1969, Release of additional factors in anaphylaxis and its antagonism by antiinflammatory drugs, Nature 223, 29. Rubin, B., R.J. Laffan, D.G. Kotler, E.H. O'Keefe, D.A. DeMaio and M.E. Goldberg, 1978a, SQ 14,225 (D-3-mercapto-2-methylpropanoyl-L-proline), a noval orally active inhibitor of angiotensin I-converting enzyme, J. Pharmacol. Exptl. Therap. 204, 271. Rubin, B., M.J. Antonaccio and Z.P. Horovitz, 1978b, Captopril ( SQ 14,225) (D-3-mercapto-2-methylpropanoyl-L-proline), a novel orally active inhibitor of angiotensin converting enzyme and antihypertensive agent, Progr. Card. Dis. (in press). Staszewska-Barczak, J. and J.R. Vane, 1965, The release of catecholamines from adrenal medulla by peptides, J. Physiol 177, 57P. Vane, J.R., 1971, Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs, Nature 231,232. Vane, J.R. and S.H. Ferreira, 1974, Interactions between bradykinin and prostaglandins, in: Chemistry and Biology of the Kallikrein--Kinin System in Health and Disease, eds. J.J. Pisano and K.F. Austen, Fogarty International Center Proceedings No. 27, p. 255. Vargaftig, B.B. and N. Dao Hal, 1972, Selective inhibition by mepacrine of the release of "rabbit aorta contracting substance" evoked by the administration of bradykinin, J. Pharm. Pharmacol. 24, 159.
European Journal of Pharmacology, 57 (1979) 287--294 © Elsevier/North-Holland Biomedical Press
THE EFFECTS OF CAPTOPRIL (SQ 14,225) ON BRADYKININ-INDUCED BRONCHOCONSTRICTION IN THE ANESTHETIZED GUINEA PIG ROLAND GREENBERG, GEORGE H. OSMAN, JR., EUGENE H. O ' K E E F E and MICHAEL J. ANTONACCIO
The Squibb Institute for Medical Research, P.O. Box 4000, Princeton, N.J. 08540, U.S.A. Received 15 December 1978, revised MS received 18 April 1979, accepted 7 May 1979
R. GREENBERG, G.H. OSMAN, JR., E.H. O'KEEFE and M.J. ANTONACCIO, The effects of captopril (SQ 14,225) on bradykinin-induced bronchoconstrictionin the anesthetized guinea pig, European J. Pharmacol. 57 (1979) 287--294. The effect of captopril'(SQ 14,225) a potent inhibitor of angiotensin converting enzyme (ACE: kininase II) on the bronchoconstrictor response to bradykinin was studied in the anesthetized guinea pig. The i.v. administration of captopril caused a profound long lasting hypotension without affecting pulmonary resistance or dynamic compliance. Similarly, the i.v. administration of bradykinin caused small increases in pulmonary resistance and decreases in dynamic compliance which were not altered by the administration of captopril. However, after /3-receptor blockade with propranolol, bradykinin-induced changes in resistance and compliance were enhanced; additional captopril administration further potentiated the bradykinin effects. The prostaglandin synthetase inhibitor indomethacin antagonized the bradykinin-induced bronchoconstriction in ~-blocked animals and its potentiation by captopril. In the isolated perfused guinea pig lung, bradykinin caused a dose dependent release of a prostaglandin-like substance which was significantly increased by captopril and antagonized by indomethacin. These results suggest that bradykinin causes a prostaglandin-mediated bronchoconstriction. Captopril, a potent inhibitor of ACE, prevents the degradation of bradykinin thus potentiating the bradykinin-induced bronchoconstriction, an effect observed in intact animals only in the absence of pulmonary ~-receptor activation. Bronchoconstriction Prostaglandin
Bradykinin
Guinea pig
1. Introduction The precise role of bradykinin in asthma is unknown. Bradykinin causes bronchoconstriction when administered by aerosol to asthmatics; however, it does not cause broncl~oconstriction in normal persons (Collier, 1970). There is little evidence available on the release of kinins during human anaphylaxis or asthma; however, Abe et al. (1967) reported elevated blood kinin concentrations in patients with severe bronchial asthma, indicating that kinins may be involved in human bronchial asthma. Bradykinin causes bronchoconstriction in both anesthetized and unanesthetized guinea pigs (Collier and Shorley, 1960; Drazen and
Propranolol
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Austen, 1974; 1975). This bronchoconstriction is inhibited by nonsteroidal anti-inflammatory drugs (Collier and Shorley, 1960; 1963). Bradykinin also causes the release of prostaglandins and thromboxane A: from isolated perfused guinea pig lungs. This release is inhibited by indomethacin and mepacrine (Palmer et al., 1973; Vargaftig and Dao Hai, 1972). It has been suggested that the bradykinin-induced bronchoconstriction is indirect and mediated through the generation of TXA2 from prostaglandins (Collier, 1969; Vane and Ferreira, 1974). Captopril (SQ14,225) has been recently reported to be an effective competitive inhibitor of angiotensin I-converting enzyme (ACE:kininaseII) (Ondetti et al., 1977).
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Inhibition of ACE by captopril has been shown to potentiate both the hypotensive effect of exogenously administered bradykinin in the rat and rabbit and the contractile response to bradykinin in the isolated guinea pig ileum (Rubin et al., 1978a; Murthy et al., 1978). Experiments were therefore done to determine the effects of captopril on the bronchoconstrictor response to bradykinin in the anesthetized guinea pig. Additional experiments were done to determine the effect of captopril on bradykin-induced release of prostaglandins from the isolated perfused guinea pig lung.
2. Materials and methods
2.1. Pulmonary resistance and dynamic compliance Male guinea pigs weighing 480--520 g were anesthetized with urethane (1.5 g/kg, i.p.). Spontaneous respiration was arrested with succinylcholine chloride (1.5 mg/kg, i.p.). The guinea pigs were artificially ventilated with a Palmer respiration pump through a tracheal cannula at a rate of 72 strokes/min and a stroke volume of 3 ml. Pulmonary resistance and dynamic compliance were determined by the method described by Amdur and Mead (1958) as modified by Giles et al. (1973). Respiratory flow was measured with a Fleisch pneumotachograph (No. 000) and a Validyne (MP 45) differential pressure transducer. Transpulmonary pressure was determined by monitoring the difference between pressure in the external end of a tracheal cannula and the pressure in the pleural cavity by means of a Validyne MP 45 differential pressure transducer. The flow and pressure signals were fed into an on-line analog Pulmonary Mechanics Computer (Buxco Electronics, Inc.) which provided calculation of pulmonary resistance and dynamic compliance. The computer output of flow, pressure, volume, resistance and dynamic compliance were recorded on a
R. G R E E N B E R G ET AL.
Brush 260 recorder. Blood pressure was measured from a carotid artery with a Statham P23Db transducer and heart rate was derived from the pulse pressure. Both blood pressure and heart rate were recorded with the Buxco pulmonary mechanics computer. All parameters were digitalized by a Data Logger, Model DL-12 Buxco electronics and recorded on a Texas Instrument silent 700 ASR printer.
2.2. Isolated perfused guinea pig lung Guinea pig isolated lungs were perfused through the pulmonary artery and the effluent bioassayed for prostaglandins by a method similar to that described by Piper and Vane (1969). Male guinea pigs (350--400 g) were injected with heparin (500 U/kg, i.p.) and killed by a blow on the head. The pulmonary artery and trachea were cannulated and the lung removed and suspended in a heated glass chamber. The pulmonary artery was perfused with Krebs--Henseleit solution (mM: NaC1118.0, KC1 4.7, CaC122.5, KH2PO4 1.1, MgSO4 1.2, NaHCO3 25.0 and glucose 11.0) containing 3% dextran 70,000 mw with an Extracorporeal pump at a rate of 4 ml/min (Piper and Vane, 1969; Bhattacharya and Delaunois, 1955). The effluent from the lung superfused an assay tissue (chick rectum) which contracts in response to prostaglandins and is insensitive to bradykinin (Alabaster and Bakhle, 1972; Blumberg et al., 1977; Gilmore et al., 1968). In addition, the assay tissue was perfused at a rate of 1 ml/min with Krebs--Henseleit solution containing a mixture of antagonists which make it insensitive to histamine, acetylcholine, catecholamines and serotonin. The concentrations of the antagonists mepyramine, atropine and phenoxybenzamine were 0.1 pg/ ml, propranolol 2 pg/ml and methysergide 0.2 pg/ml (Piper and Vane, 1969). Indomethacin 10 pg/ml was also added to the solution perfusing the assay tissue to eliminate the possibility that the assay tissue might release endogenous prostaglandins (Eckenfels and
CAPTOPRIL AND BRADYKININ BRONCHOCONSTRICTION
Vane, 1972). Drugs were either injected into the cannulated pulmonary artery (through the lung} or directly over the assay tissue. Contractions of the chick rectum were measured with a Grass FT 03 force displacement transducer and recorded on a Beckman Dynograph recorder. 2.3. Materials and statistics The drugs used in this study were: succinylcholine C1 (Sigma), bradykinin triacetate (Sigma), captopril (D-3-mercapto-2-methylpropanoyl-L-proline) (Squibb), indomethacin (Sigma), mepyramine maleate (Ehemal0g), phenoxybenzamine HC1 (SKF), atropine SO4 (Sigma), D,L-propranolol HC1 (Sigma), prostaglandin E2 (Ono). Indomethacin was dissolved in a 2% sodium carbonate solution and the pH adjusted to 7.5 with HC1. A stock solution of PGE2 was prepared by dissolving 10 mg in 9 ml of a solution of 2% sodium carbonate and 1 ml of 95% ethanol. Drug concentrations are expressed as final concentrations of free base. The data were analyzed by Student's t-test for paired data. 3. Results
3.1. Respiratory and cardiovascular effects of captopril in the anesthetized guinea pig Captopril (0.1, 1 and 5 mg/kg, i.v.) was administered to four anesthetized guinea pigs for each dose before and after the administration of propranolol (1 mg/kg, i.v.). Captopril did not significantly alter either pulmonary resistance or dynamic compliance. However, these doses of captopril caused a severe long lasting hypotension both before and after propranolol (fig. 1). There were no significant changes in heart rate accompanying the hypotensive response of captopril. 3.2. The effect of captopril on the bronchoconstrictor response to bradykinin in the anesthetized guinea pig Bradykinin (1.0, 3.0 and 5.0 pg/kg, i.v.) was administered to groups of 5--7 guinea
289
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pigs for each dose 15 min before and 5 min after the administration of captopril (100 pg/ kg, i.v.). These doses of bradykinin caused small increases in pulmonary resistance and small decreases in dynamic compliance. The increase in pulmonary resistance in response to 5 pg of bradykinin was the only pulmonary response which was altered by captopril and then by only 10% (fig. 2). The maximum changes in resistance and dynamic compliance in response to bradykinin were obtained within 30 sec of administration and lasted for up to 3 min for resistance and 10 min for dynamic compliance. Captopril did not significantly alter the duration of these responses. The administration of captopril 15min after the administration of bradykinin (1.0 pg/ kg) in the above experiments had no effect on pulmonary resistance or dynamic compliance. However, the administration of captopril 15 min after bradykinin 3.0 and 5.0 pg/kg caused small increases in resistance and small
290
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decreases in dynamic compliance (fig. 3). In additional control experiments, bradykinin 1 and 3 #g/kg were administered twice at 15 min intervals to 4 and 5 guinea pigs respectively. There was no significant difference between the first and second bronchoconstrictor response to bradykinin. 3.3. The effect of captopril on the bronchoconstrictor response to bradykinin in the anesthetized guinea pig pretreated with propranolol The bronchoconstrictor response to bradykinin is enhanced by ~-adrenergic blockade with propranolol (Collier et al. 1965). Experiments were therefore done to evaluate the effect of captopril on the bronchoconstrictor response to bradykinin in guinea pigs which were pretreated with propranolol (1 mg/kg, i.v.). Bradykinin (0.1, 0.3 and 1.0 pg/kg, i.v.) was administered to groups of 5--7 propranolol-pretreated guinea pigs for each dose, 15 min before and 5 min after the administration of captopril (100#g/kg). These relatively small doses of bradykinin caused increases in resistance and decreases in dynamic compliance which were all significantly potentiated
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by captopril except the decrease in compliance in response to 0.1 pg bradykinin (fig. 2). A representative experiment is illustrated in fig. 4. The maximum changes in resistance and compliance were obtained within 30 sec of administration and lasted from 5 to 10 min. Captopril significantly prolonged the changes in both resistance and dynamic compliance after all doses of bradykinin. The administration of captopril 15 min after the administration of bradykinin (0.1 and 0.3 pg/kg) in the above experiments caused substantial increases in pulmonary resistance and decreases in dynamic compliance; however, only small changes were observed after bradykinin (1.0 #g/kg) (fig. 3). In additional control experiments bradykinin 1 #g/kg was administered twice at 15 min intervals to 4 guinea pigs pretreated with propranolol (1 mg/kg). There was no significant difference between the first and second bronchoconstrictor response to bradykinin. 3.4. The effect of indomethacin on the potentiation of bradykinin-induced bronchoconstriction by captopril in the anesthetized propanolol-treated guinea pig Bradykinin bronchoconstriction is believed to be due to the release of prostaglandins
CAPTOPRIL AND BRADYKININ BRONCHOCONSTRICTION
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(Collier, 1969; Collier and Shorley, 1960). Experiments were therefore done to see if the 100 F
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prostaglandin synthetase inhibitor indomethacin would antagonize the bradykinin bronchoconstriction in the fl-blocked captopril-treated guinea pig. Bradykinin (1.0pg/kg) was administered i.v. to 8 guinea pigs pretreated with propranolol (1 mg/kg) before and after the administration of indomethacin (2.5 mg/kg) plus captopril (100 pg/kg). Bradykinin caused substantial increases in pulmonary resistance and decrease in dynamic compliance which were significantly reduced by the administration of indomethacin plus captopril, respectively (fig. 5). 3.5. The effect o f captopril on the bradykinininduced release o f a prostaglandin-like substance (PLS) from the isolated perfused guinea pig lung
Bradykinin has been shown to release prostaglandin and TXA2 from guinea pig isolated lungs (Palmer et al., 1973). Experi-
292
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ments were therefore done to see if captopril would increase bradykinin-induced release of prostaglandins. In 6 experiments bradykinin (50, 100 and 500 ng) was injected into the pulmonary artery at 15 min intervals prior to and during the infusion of captopril (10 /~g/ml). The effluent from the lung was bioassayed on the chick rectum after each dose of bradykinin. A typical experiment is illustrated in fig. 6. Captopril significantly increased the amount of prostaglandin-like substance (PLS) released by all doses of bradykinin {fig. 7). In four control experiments bradykinin (50, 100 and 500 ng) was injected into the pulmonary circulation twice at 15 min intervals. There was no significant difference between the amount of PLS released between the first and second administration of bradykinin. In four additional experiments, bradykinin (500 ng) was injected into the pulmonary artery prior to an during the infusion of captopril (10 pg/ml) plus indomethacin (10 pg/ml). Bradykinin caused the initial release of 10.9 + 3.1 ng of PLS which was significantly reduced to 1.0 + 1.0 ng/ml of PLS after the infusion of captopril and indomethacin P < 0.05. 4. Discussion
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Fig. 7. The effect of captopril on the bradykinininduced release of PLS from the isolated guinea pig lung. Increasing doses of bradykinin were injected into the pulmonary circulation at 15 rain intervals before and during the constant infusion of captopril (10 #g/ml). Each point on the dose--response curve is the mean of 5 experiments. The bars represent the standard error. Significance of the difference from controls. * P < 0.05, ** P < 0.01. Ordinate: PLS (ng). Abscissa: bradykinin (ng). o . . . . . . o control; o--------o, captopril (SQ 14,225) 10 pg/ml.
The results of this study show that relatively high doses of the potent ACE inhibitor captopril causes a profound long lasting hypotension in the anesthetized guinea pig without affecting pulmonary resistance or dynamic compliance. Captopril has been shown to lower the blood pressure in several hypertensive animal models, including renal and spontaneously hypertensive rats. However, captopril has very little hypotensive effect in normotensive animals, such as rat, rabbit, cat and monkey (Rubin et al., 1978b). In contrast, this study has shown that even moderate doses of captopril cause profound hypotension in the anesthetized guinea pig. This hypotension may be due to elevated
CAPTOPRIL AND BRADYKININ BRONCHOCONSTRICTION
plasma renin and consequently AI levels caused by the urethane anesthesia (Pettinger and Campbell, 1976), coupled with high levels of circulating plasma converting enzyme in the guinea pig (Cushman and Cheung, 1972). The results further show that bradykinin causes small increases in pulmonary resistance and small decreases in dynamic compliance which were not significantly altered by captopril. However, in guinea pigs pretreated with propranolol bradykinin caused large increases in pulmonary resistance and decreases in dynamic compliance which were potentiated by captopril. Bradykinin causes the release of catecholamines from the adrenal glands which can antagonize its bronchoconstrictor effect (Staszewska-Braczak and Vane, 1967; Piper et al., 1967). It has also been shown that bradykinin bronchoconstriction is enhanced by fl-blockade with propranolol (Collier et al., 1965; McCulloch et al., 1967 and Collier and James, 1967). The above results therefore suggest that inhibition of ACE with captopril increases the concentration of bradykinin causing an increase in both bronchoconstriction and release of catecholamines. The increased catecholamine secretion may be responsible for attenuating the bronchoconstriction. However, in the presence of propranolol where the bronchodilator effects of the released catecholamines are antagonized, captopril potentiates the bradykinin bronchoconstriction. These results are consistent with the findings of Rubin et al. (1978a) and Murthy et al. (1978) showing that captopril potentiates the contractile response to bradykinin in the isolated guinea pig ileum and the hypotensive responses to bradykinin in the rat and rabbit. The administration of captopril in normal or propranolol-treated guinea pigs did not cause any changes in pulmonary resistance or dynamic compliance in animals not receiving bradykinin. However, in guinea pigs receiving bradykinin prior to captopril, there was a significant increase in pulmonary resistance and decrease in dynamic compliance in response to captopril after the bronchocon-
293
strictor response to bradykinin has disappeared. These results might be explained on the basis of a residual subthreshold amount of circulating or bound bradykinin remaining 15 min after the bronchoconstriction has disappeared. The administration of captopril then elevates this concentration of bradykinin possibly by displacing it from noncontractile tissues. Bradykinin is believed to cause bronchoconstriction in the guinea pig indirectly by releasing prostaglandin and TXA: (Collier, 1969). The bronchoconstrictor action of bradykinin is effectively antagonized by nonsteroidal anti-inflammatory drugs (Collier, 1970), which are potent inhibitors of prostaglandin synthesis (Vane, 1971). Bradykinin has also been shown to cause the release of prostaglandins and TXA2 from isolated guinea pig lungs. This release is inhibited by the prostaglandin synthetase inhibitor indomethacin or the phospholipase inhibitor mepacrine. It is further suggested that bradykinin activates a phospholipase causing the formation of arachidonic acid and the subsequent synthesis and release of prostaglandins and TXA2 (Vargaftig and Dao Hai, 1972; Palmer et al., 1973; Blackwell et al., 1978). The results of the present study have shown that the prostaglandin synthetase inhibitor indomethacin abolishes the bradykinin bronchoconstriction and its potentiation by captopril. In the isolated perfused lung bradykinin causes a dose dependent release of a PLS which is augmented by captopril and antagonized by indomethacin. These results are similar to those of Blumberg et al. (1977) who demonstrated the enhanced bradykinininduced release of PGEz from the perfused rabbit mesentary blood vessels by the ACE inhibitor SQ20,881. The present results are therefore consistent with a bradykinininduced prostaglandin mediated bronchoconstriction, which is potentiated by the reduction in the inactivation of bradykinin by captopril. References Abe, K., N. Watanabe, N. Kimagai, T. Mouri, T. Seki and K. Yoshinaga, 1967, Circulating plasma kinin
294 in patients with bronchial asthma, Experientia 23,626. Alabaster, V.A. and Y.S. Bakhle, 1972, The inactivation of bradykinin in the pulmonary circulation of isolated lungs, Brit. J. Pharmacol. 4 5 , 2 9 9 . Amdur, M.O. and J. Mead, 1958, Mechanics of respiration in unanesthetized guinea pigs, Amer. J. Physiol. 192, 364. Bhattacharya, B.K. and A.L. Delaunois, 1955, An improved method for the perfusion of isolated lung of guinea pig, Arch. Intern. Pharmacodyn. 101,495. Blackwell, G.J., R.J. Flower, F.P. Nijkamp and J.R. Vane, 1978, Phospholipase A2 activity of isolated perfused lungs: stimulation and inhibition by antiinflammatory steroids, Brit. J. Pharmacol. 62, 79. Blumberg, A.L., S.E. Denny, G.R. Marshall and P. Needleman, 1977, Blood vessel-hormone interactions: angiotensin, bradykinin and prostaglandin, Amer. J. Physiol. 232, H305. Collier, H.O.J., 1969, New light on how aspirin works, Nature 223, 35. Collier, H.O.J., 1970, Kinins and ventilation of the lungs in: Handbook of Experimental Pharmacology, Bradykinin and Kallidin and Kallikrein, Vol. XXV, ed. E.G. ErdSs (Springer, New York) p. 409. Collier, H.O.J. and G.W.L. James, 1967, Humoral factors affecting pulmonary inflation during anaphylaxis in the guinea pig in vivo, Brit. J. Pharmacol. 30,283. Collier, H.O.J., G.W.L. James and P.J. Piper, 1965, Intensification by adrenalectomy or by ~-adrenergic blockade of the bronchoconstrictor action of bradykinin in the guinea pig, J. Physiol. 180, 13P. Collier, H.O.J. and P.G. Shorley, 1960, Analgesic antipyretic drugs as antagonists of bradykinin, Brit. J. Pharmacol. 15,601. Collier, H.O.J. and P.G. Shorley, 1963, Antagonism by mefenamic and flufenamic acids of the bronchoconstrictor action of kinins in the guinea pig, Brit. J. Pharmacol. 20,345. Cushman, D.W. and H.S. Cheung, 1977, Studies in vitro of the angiotensin-converting enzyme of lung and other tissues, in:Hypertension, Eds. J. Geneset and E. Koiw (Springer-Verlag, New York) p. 532. Drazen, J.M. and K.F. Austen, 1974, Effects of intravenous administration of slow-reacting substance of anaphylaxis, histamine, bradykinin and prostaglandin F2~ on pulmonary mechanics in the guinea pig, J. Clin. Invest. 53, 1679. Drazen, J.M. and K.F. Austen, 1975, Atropine modification of the pulmonary effects of chemical mediators in the guinea pig, J. Appl. Physiol. 38, 834. Eckenfels, A. and J.R. Vane, 1972, Prostaglandin oxygen tension and smooth muscle tone, Brit. J. Pharmacol. 4 5 , 4 5 1 . Giles, R.E., J.C. William and M.P. Finkel, 1973, The bronchodilator and cardiac stimulant effects of Th 1165A, salbutamol and isoproterenol, J. Phar-
R. GREENBERG ET AL. macol. Exptl. Therap. 186,472. Gilmore, N., J.R. Vane and J.H. Wyllie, 1968, Prostaglandin released by the speen, Nature 218, 1135. McCulloch, M.W., C. Proctor and M.J. Rand, 1967, Evidence for an adrenergic homeostatic bronchodilator reflex mechanism, European J. Pharmacol. 2,214. Murthy, V.S., T.L. Waldron and M.E. Goldberg, 1978, The mechanism of bradykinin potentiation after inhibition of angiotensin converting enzyme by SQ 14,225 in conscious rabbits, Circulation Res. 43, Suppl. I, 41. Ondetti, M.A., D.W. Cushman and B. Rubin, 1977, Design of specific inhibitors of angiotensin-converting enzyme: a new class of orally-active antihypertensive agents, Science 196,441. Palmer, M.A., P.J. Piper and J.R. Vane, 1973, Release of rabbit aorta contracting substance (RCS) and prostaglandins induced by chemical or mechanical stimulation of guinea pig lungs, Brit. J. Pharmacol. 49,226. Pettinger, W.A. and W.B. Campbell, 1976, The rat as an animal model for study of the renin-angiotensinaldosterone-sodium (RAAS) axis, in: New Antihypertensive Drugs, eds. A. Scriabine and C.S. Sweet (Spectrum, New York) p. 179. Piper, P.J., H.O.J. Collier and J.R. Vane, 1967, Release of catecholamines in the guinea pig by substances involved in anaphylaxis, Nature 213,838. Piper, P.J. and J.R. Vane, 1969, Release of additional factors in anaphylaxis and its antagonism by antiinflammatory drugs, Nature 223, 29. Rubin, B., R.J. Laffan, D.G. Kotler, E.H. O'Keefe, D.A. DeMaio and M.E. Goldberg, 1978a, SQ 14,225 (D-3-mercapto-2-methylpropanoyl-L-proline), a noval orally active inhibitor of angiotensin I-converting enzyme, J. Pharmacol. Exptl. Therap. 204, 271. Rubin, B., M.J. Antonaccio and Z.P. Horovitz, 1978b, Captopril ( SQ 14,225) (D-3-mercapto-2-methylpropanoyl-L-proline), a novel orally active inhibitor of angiotensin converting enzyme and antihypertensive agent, Progr. Card. Dis. (in press). Staszewska-Barczak, J. and J.R. Vane, 1965, The release of catecholamines from adrenal medulla by peptides, J. Physiol 177, 57P. Vane, J.R., 1971, Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs, Nature 231,232. Vane, J.R. and S.H. Ferreira, 1974, Interactions between bradykinin and prostaglandins, in: Chemistry and Biology of the Kallikrein--Kinin System in Health and Disease, eds. J.J. Pisano and K.F. Austen, Fogarty International Center Proceedings No. 27, p. 255. Vargaftig, B.B. and N. Dao Hal, 1972, Selective inhibition by mepacrine of the release of "rabbit aorta contracting substance" evoked by the administration of bradykinin, J. Pharm. Pharmacol. 24, 159.