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Effects of captopril (SQ 14, 225) on the renin-angiotensin-aldosterone system in normal rats
European Journal of Pharmacology, 62 (1980) 269--275
269
© Elsevier/North-Holland Biomedical Press
EFFECTS OF CAPTOPRIL (SQ 14, 225) ON THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM IN NORMAL RATS TATSUO KOKUBU, EINOSUKE UEDA, MOTOTANE ONO, TADAO KAWABE, YUJI HAYASHI and TAKUYA KAN
The Second Department of Internal Medicine, Ehime University School of Medicine, Onsen-gun, Ehime 791-02, Japan Received 11 December 1979, accepted 8 January 1980
T. KOKUBU, E. UEDA, M. ONO, T. KAWABE, Y. HAYASHI and T. KAN, Effects ofcaptopril (SQ 14, 225) on the renin-angiotensin-aldosterone system in normal rats, European J. Pharmacol. 62 (1980) 269--275. High doses of captopril (SQ 14, 225) ( 1 2 0 - 1 6 0 mg/kg/day) were administered orally to normal rats, and the effects on the renin-angiotensin-aldosteronesystem were observed. Plasma angiotensin converting enzyme (ACE) activity was elevated significantly on the 3rd, 7th and 30th days of captopril administration. ACE activity in the lung and the kidney was significantly decreased on the 1st day then gradually increased, becoming significantly higher than that of controls by the 30th day. Plasma renin activity (PRA) was significantly elevated on the 1st day and remained at a high level until the 30th day. Renal renin content was found to be significantly lower on the 1st and 3rd days. Plasma aldosterone concentration was not affected by captopril treatment, whereas serum potassium concentration was found to be significantly lower on the 1st, 3rd and 30th days. It is suggested that besides its inhibitory action on ACE, captopril has a direct or indirect stimulating action on ACE production as well as on renin release. Angiotensin-convertingenzyme inhibition Tissue angiotensin-convertingenzyme
Serum angiotensin converting enzyme Renin-angiotensin
1. Introduction Captopril (D-3-mercapto-2-methylpropanoyl-L-proline, SQ 14, 225) is an orally active inhibitor of angiotensin converting enzyme (ACE) (EC 3.4.15.1) (Cushman et al., 1977). The administration of this compound produces various biochemical changes and lowers blood pressure in hypertensive rats (Muirhead et al., 1978; Laffan et al., 1978), in rabbits (Romero et al., 1974; Murthy et al., 1977; 1978), in dogs (McCaa et al., 1978; Watkins et al., 1978; Harris et al., 1978) and even in man (Gavras et al., 1974; 1978b: Cody et al., 1978). If the antihypertensive action of captopril is to be understood the effects of captopril on the renin-angiotensin system must be made clear.
Captopril
Although the effects of captopril on the renin-angiotensin system have been studied extensively, few investigators have reported a change in serum ACE activity (Gavras et al., 1978c). There have been no previous reports of changes in tissue ACE activity following captopril administration. The purpose of the present study was to examine changes in serum and tissue levels of ACE as well as other components of the renin-angiotensin system following oral administration of captopril to normal rats.
2. Material and methods Female Wistar rats weighing about 250 g were used. The rats were fed ordinary chow (Oriental Yeast Co., Tokyo, Japan) containing
270
130 mEq of Na/kg of chow. Each rat ate a b o u t 15 g of food per day, thus consuming 2 mEq of Na per day. Experiments were carried out as follows: (1) Control group: rats were given tap water ad lib, and (2) experimental group (captopril group): rats were given captopril-water in a concentration of 1 mg/ml ad libitum. Each rat drank 30~40 ml/day of water, that is 120~160 mg/kg/day of captopril. Captoprilwater was prepared freshly every day. On the 1st, 3rd, 7th and 30th days of captopril administration the animals were anesthetized with pentobarbital (40 mg/kg), the bilateral renal arteries and veins were ligated, and blood samples were taken from the inferior vena cava with heparinized plastic syringes. The blood was centrifuged at 700 × g for 15 min at 4°C and the separated plasma was divided among several small plastic tubes and stored in a freezer at --20 ° C for later use. The lungs and the kidneys were taken out immediately after bleeding and kept in a freezer until use. The following were assayed: plasma renin activity (PRA), plasma ACE activity, plasma aldosterone concentration (PAC), serum Na concentration, serum K concentration, plasma ACTH and plasma cortisol concentration. The ACE activity in pulmonary and renal tissues, and the renal renin content were also measured. The ACE activity was measured spectrophotometrically according to Cushman (Cushman and Cheung, 1971) with a slight modification. The incubation mixture contained 3.5 mM of Hip-His-Leu, 100 mM of borate buffer, pH 8.3, 300 mM of NaC1. Duplicate incubations were performed and the inter-assay error was within +- 10%. Biological assay was also performed using angiotensin I (ANGI) and bradykinin ( B K ) a s substrates. One ml of ANGI (200 ng/ml) was incubated with 0.1 ml of plasma and the ANGII thus formed was assayed using the uterus of a rat pretreated with hexestlol (1 mg/kg) 24 h prior to the experiment (Barret and Sambhi, 1969; Ueda et al., 1971). The
T. K O K U B U ET AL.
uterus was suspended with 1 g tension in a 5 ml water bath containing modified Ringer solution, and the contraction of the uterus was measured by means of an isotonic transducer (Nihon Kohoden, TD-112 s). The sensitivity of the uterus was 1 ng/ml for ANGII and 30--40 ng/ml for ANGI. The assay was performed at concentrations lower than 5 ng/ml of ANGI, so that possible interference by ANGI could be ruled out. For measuring kininase activity, one ml of BK (5 pg/ml) was incubated with 0.1 ml of plasma and the remaining BK was assayed in the same way as ANGII. ACE activity in tissue was assayed as follows (Ueda et al., 1979): the tissue was homogenized in 25 mM borate buffer, pH 8.3 {50 vols. were added for pulmonary tissue and 10 vols. for renal tissue) with a Potter--Elvehjem homogenizer. An aliquot of the homogenized solution was transferred into a test tube (18 X 100 mm) and was homogenized again with Polytron ® (Kinematica, Lucerne, Switzerland) for 1 min. The solution was filtered through gauze, and centrifuged for 10 min at 700 X g. Cell debris were precipitated at this step, and no ACE activity was found in the precipitate. The supernatant which contained nuclear, lysozomal and soluble fractions was assayed for ACE. As a preliminary experiment, the ACE activity in various regions of the lung was compared, and a small variation in activity was found from one region to another. Because of this, one whole lung was used for the assay. A whole kidney was also used for the assay of enzyme activity in renal tissue. Renal renin content (RRC) was measured as follows (Gross et al., 1956): the kidney was homogenized in 100 mM phosphate buffer, pH 6.8 and centrifuged at 700 X g for 10 min. The supernatant, in a volume of 0.1 ml, was incubated with semipurified rat renin substrate (2000 ng ANGII/ml) (Sen et al., 1967) for 5 min in the presence of 8-hydroxyquinoline, EDTA-2Na and dimercaprol (BAL). The reaction was stopped by boiling the reaction mixture for 10 rain. The ANGI thus formed was measured radioimmunologically.
CAPTOPRIL ON RENIN-ANGIOTENSIN SYSTEM IN NORMAL RA T
Serum Na and K were measured with a flamephotometer (Beckman Flamephotometer, Klina). ACTH and cortisol were measured radioimmunologically. The pressor response to exogenous ANGI was measured in rats anesthetized with pentobarbital-Na. ANGI was injected through a cannula placed in the femoral vein and the blood pressure response was recorded through a cannula placed in the carotid artery.
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TABLE 1 Effect of oral administration of captopril on serum angiotensin-converting enzyme activity in rats. The assay was performed with various substrates to compare biological and spectrophotometric assays.
Angiotensin I Bradykinin Hip-His-Leu
Normal (nmol/ml/min)
Captopril (7th day) (nmol/ml/min)
0.084 1.45 42.2
0.374 4.32 116.0
3. Results There was no difference in b o d y weight between the control and the captopril group (265.8 + 3.1 g and 258.8 + 4.4 g respectively). Each rat drank a b o u t 30~40 ml/day captoprilwater that is 30~40 mg/day (120~160 mg/kg/ day) of captopril. Pressor responses to i.v. injection of ANGI (10 ng) ranged from 15 to 20% of the responses in the normal rats on the 3rd and 7th day, and were 25% on the 30th day of captopril administration. Changes of ACE activity in plasma, in the lung and in the kidney are shown in fig. 1. On the 1st day of captopril administration plasma ACE remained at the same level as in
PLASMA
nmollmllmin
J LUNG nmollgtissuelmin 1000
the controls, b u t it increased significantly on the 3rd, 7th and 30th day. In contrast to plasma ACE activity, ACE activity in the lung on the 1st day was significantly lower than that of the control group (P < 0.005). Pulmonary ACE activity returned to the control level on the 3rd and 7th days, and then increased significantly on the 30th day (P < 0.005}. ACE activity in the kidney changed in a way similar to that in the lung. ACE activity in the kidney decreased on the 1st day (P < 0.005) and increased significantly on the 30th day (P < 0.05). The plasma, which showed high ACE activ-
J KIDNEY nmollgtissuelmin 200.
100~ .>
10050 n-lO
n-lO
n-lO
Fig. 1. Effects of oral administration of captopril on the activity of angiotensin-converting enzyme in serum, lung and kidney in normal rats. Abscissa: days after captopril administration. ***P < 0.001, **P < 0.005, *P < 0.05.
272
T. K O K U B U ET AL.
nglmllh
i
PRA
RRC
PAC
pglg tissuelmin
S
nglml 20"
4
0.8"
, ~ ' < / ~
0.6"
?
i0"
0.4'
n-8
n-6 l
0.2.
n-lO
Fig. 2. E f f e c t s o f oral a d m i n i s t r a t i o n o f c a p t o p r i l o n plasma renin activity, renal renin c o n t e n t and plasma aldost e r o n e c o n c e n t r a t i o n in n o r m a l rats. Abscissa: days after c a p t o p r i l a d m i n i s t r a t i o n . * * * P < 0.001, **P < 0.005.
ity on spectrophotometric assay, was checked biologically as described in Methods. As shown in table 1, ACE activity measured with ANGI as the substrate was 4 times higher than in control plasma, 3 times higher when BK was used as the substrate and 3 times higher when Hip-His-Leu was used as the substrate. This plasma was dialyzed overnight in physiological saline to remove possible circulating captopril, and the ACE activity was compared with that of non-dialyzed plasma. There was no appreciable difference between them. Changes in PRA, RRC and PAC are shown in fig. 2. PRA increased significantly on the
1st day (P < 0.001) and remained at a high level until the 30th day. On the other hand, RRC decreased significantly on the 1st and on the 3rd day as compared with the control group (P < 0.005 and P < 0.001). PAC was not affected by captopril. Changes in serum electrolytes are shown in table 2. Though the serum Na concentration was not affected by captopril, the serum K level was significantlylowered on the 1st day (P < 0.001) and remained at a low level until the 30th day. As shown in table 3 plasma cortisol and plasma ACTH were not affected by captopril.
TABLE 2 E f f e c t s o f oral a d m i n i s t r a t i o n o f captopril o n s e r u m e l e c t r o l y t e s in n o r m a l rats. Control
S e r u m Na (mEq/l)
139.5 + 0.63 (n = 8)
Serum K (mEq/1)
4.28 + 0.11 (n = 8)
Days o f oral a d m i n i s t r a t i o n o f c a p t o p r i l ( 1 2 0 - - 1 6 0 m g / k g / d a y ) 1st day
3rd day
7th day
3 0 t h day
140.0 -+ 0.73 (n = 6) ns 3.78 + 0.17 (n = 6)
138.7 -+ 1.52 (n = 7)
138.1 -+ 0.58 (n = 8)
139.8 + 0.65 (n = 8)
ns
ns
ns
3.56 -+ 1.36 (n = 7)
3.70 + 0.06 (n = 8)
3.68 + 0.08 (n = 6)
P < 0.001
P < 0.001
P < 0.001
P < 0.001
CAPTOPRIL ON RENIN-ANGIOTENSIN SYSTEM IN NORMAL RAT TABLE 3 Effect of oral administration of captopril on plasma ACTH and cortisol levels in normal rats.
Control (n = 6) 3rddayof captopril administration (n=8)
Plasma ACTH (pg/ml)
Plasma cortisol (ug/dl)
399.2 -+ 113.3 2 9 0 . 2 + 31.6
73.93 + 10.91 65.73-+ 9.42
4. Discussion The dose of captopril used in the present experiments was high enough to inhibit ACE activity in rats because the pressor response to exogenous ANGI was almost completely abolished. Serum ACE activity in captopril-treated rats was unexpectedly elevated. There might have been some problem in measuring the serum ACE without dialysis because circulating captopril might have inhibited the in vitro reaction of the enzyme. In assays done under such conditions the ACE activity found will be lower than the true value. However the enzyme activity was found to be higher than that in normal rats. Furthermore the activity was not increased by dialysis. In contrast to our results, Gavras et al. (1978c) reported that captopril reduced ACE activity in hypertensive patients to one tenth of its pre-administration value. We had previously found that the i.v. or i.p. injection of captopril lowered serum ACE activity, and also that a single oral administration lowered serum ACE activity (unpublished data}. So the effect of long-term administration of captopril in the drinking water might be different from that of a single administration. Possible mechanisms for the elevation of serum ACE activity in the present study are as follows; (1} elevated circulating ANGI stimulates the release of ACE from the lung or other organs, as an adaptation response to the increased circulating ANGI, (2) the blockade
273
of ACE on the surface of endothelial cells stimulates the production and release of ACE in those cells, (3) captopril inhibits the metabolic turnover of circulating ACE, and (4) the production of ACE is stimulated by captopril itself. Whatever the mechanism it is obvious that the increased circulating ACE is n o t effective to convert ANGI to ANGII because no pressor response to exogenous ANGI was observed. Changes of ACE activity in the lung and the kidney are difficult to explain in relation to the pressor response to ANGI. In the early period of captopril administration, tissue ACE activity and the pressor response to ANGI changed in parallel, b u t on the 30th day of administration, tissue ACE activity was significantly elevated whereas the pressor response to ANGI remained suppressed. The activity of the enzyme in tissue homogenates may not represent the activity in that organ in vivo. For example high angiotensinase activity is found in lung homogenate whereas no inactivation of ANGII occurs in the pulmonary circulation (Bakhle, 1968; Sander and Huggins, 1971}. We suspect that ACE on the surface of endothelium cells was mostly combined with captopril and that the ability to convert ANGI to ANGII was therefore reduced. In the period around the 30th day, ACE activity within the cell and not related to the conversion of circulating ANGI to ANGII, might have been somehow increased. Further experiments along the line of this speculation are in progress. Several investigators have reported that a marked elevation of PRA followed captopril administration (Gavras et al., 1974; 1978a, b, c; Ferguson et al., 1977; McCaa et al., 1978; Re et al., 1978; Watkins et al., 1978; Cody et al., 1978) and our results are consistent with theirs. The mechanism of elevation might not be simple as the phenomenon appeared soon after captopril administration. PRA was highest on the 1st day of administration, then decreased gradually, b u t was still higher than the normal level even on the 30th day of administration. Those results, in combination
274
with the finding of a decrease of RRC on the 1st and 3rd day, suggest that captopril might stimulate renin release from the kidney directly. PAC has been reported to be low following captopril administration in man (Gavras et al., 1978a) and in dogs (McCaa et al., 1978). Watkins et al. (1978) reported no change in PAC following captopril administration in one-kidney hypertensive dogs. In our experiments with normal rats, PAC did not change throughout the experiment. There might be some differences between species in the stimulatory mechanism of aldosterone secretion. A possible difference in the amount of sodium intake may also account for the discrepancy. We have considered an increase of ACTH or cortisol as a possible explanation for the lack of reduction in PAC. Captopril, however, did not increase the plasma level of either ACTH or cortisol. McCaa et al. (1978) reported that serum potassium concentration was not changed by captopril despite the increased sodium excretion. In our experiments, serum potassium concentration was significantly decreased on the 1st day, and stayed at a lower level throughout the experiment. The difference in findings could be ascribed to differences in the sodium intake or in the method of administration. In conclusion, the effects of captopril on the renin-angiotensin system may be more complicated than previously thought. In particular, the increase of ACE which occurred with long-term blockade of enzyme activity seems to deserve further study.
Acknowledgements SQ 14, 225 was kindly supplied by E.R. Squibb & Sons, Inc. and Sankyo Co., Ltd. The authors are grateful to Otsuka Assay Lab. for the ACTH and cortisol assays. This work was supported by Grant-in-Aid for Scientific Research (Japan) No. 337030 and by a Grant from Sankyo Co., Ltd.
T. KOKUBU ET AL.
References Bakhle, Y.S., 1968, Conversion of angiotensin I to angiotensin II by cell free extracts of dog lung, Nature 220,919. Barret, J.D. and Sambhi, M.R., 1969, Simultaneous assay of angiotensin I and II, and determination of converting enzyme activity, J. Pharmacol. Exp. Therap. 170,326. Cody, R.J.Jr., R.C. Tarazi, E.L. Bravo and F.M. Fouad, 1978, Haemodynamics of orally-active converting enzyme inhibitor (SQ 14, 225) in hypertensive patients, Clin. Sci. Mol. Med. 55, 453. Cushman, D.W. and H.S. Cheung, 1971, Spectrophotometric assay and properties of the angiotensinconverting enzyme of rabbit lung, Biochem. Pharmacol. 20, 1637. Cushman, D.W., H.S. Cheung, E.F. Sabo and M.A. Ondetti, 1977, Design of potent competitive inhibitor of angiotensin converting enzyme, Biochemistry 16, 5484. Ferguson, R.K., G.A. Turini, H.R. Brunner and H. Gavras, 1977, A specific orally active inhibitor of angiotensin-converting enzyme in man, Lancet 1, 775. Gavras, H., H.R. Brunner, J.H. Laragh, J.E. Sealey, I. Gavras and R. Vukovich, 1974, An angiotensin converting enzyme inhibitor to identify and treat vasoconstrictor and volume factors in hypertensive patients, New Engl. J. Med. 291,817. Gavras, H., H.R. Brunner, G.A. Turini, G.R. Kershaw, C.P. Tifft, S. Cuttelod, I. Gavras, R.A. Vukovich and D.N. McKinstry, 1978b, Antihypertensive effect of the oral angiotensin converting enzyme inhibitor SQ 14, 225 in man, New Engl. J. Med. 298,991. Gavras, H., I. Gavras, S. Textor, L. Volicer, H.R. Brunner and E.J. Ruchinska, 1978a, Effect of angiotensin converting enzyme inhibition of blood pressure, plasma renin activity and plasma aldosterone in essential hypertension, J. Clin. Endocrinol. Met. 4 6 , 2 2 0 . Gavras, H., C.S. Liang and H.R. Brunner, 1978c, Redistribution of regional blood flow after inhibition of the angiotensin converting enzyme, Circ. Res. 43 (Suppl. I), 1-59. Gross, F. and F. Sulser, 1956, Pressorische Substanzen in den Nieren experimental hypertonischer Ratten, Naunyn-Schmiedeb. Arch. Exp. Pathol. Pharmakol. 229,374. Harris, D.N., 1978, Effects of SQ 14, 225, an orally active inhibitor of angiotensin converting enzyme on blood pressure, heart rate and PRA of concious dogs, European J. Pharmacol. 51,345. Muirhead, E.E., R.L. Prewitt, Jr., B. Brooks and W.L. Brosius, Jr., 1978, Antihypertensive action of the
CAPTOPRIL ON RENIN-ANGIOTENSIN SYSTEM IN NORMAL RAT orally active converting enzyme inhibitor (SQ 14, 225) in spontaneously hypertensive rats, Circ. Res. 43 (Suppl. I), 1-53. Murthy, V.S., T.L. Waldron, M.E. Goldberg and R.R. Vollmer, 1977, Inhibition of angiotensin converting enzyme by SQ 14, 225 in conscious rabbits, European J. Pharmacol. 46, 207. 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, Circ. Res. 43 (Suppl. I), 1-40. Laffan, R.J., M.E., Goldberg, J.P. High, T.S. Schaeffer, M.H. Waugh and B. Rubin, 1978, Antihypertensive activity in rats of SQ 14, 225, an orally active inhibitor of angiotensin I-converting enzyme, J. Pharmacol. Exp. Therap. 204,281. McCaa, R.E., J.E. Hall and C.S. McCaa, 1978, The effects of angiotensin I-converting enzyme inhibitors on arterial blood pressure and urinary sodium excretion, Circ. Res. 43 (Suppl. I}, 1-32. Re, R., R. Novlline, M.T. Esconrron, C. Athanasouois, J. Burton and E. Haber, 1978, Inhibition of angiotensin converting enzyme for diagnosis of renal artery system, New Engl. J. Med. 298,582.
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Romero, J.C., S.W. Mak and S.W. Hoobler, 1974, Effect of blockade of angiotensin I-converting enzyme on the blood pressure of renal hypertensive rabbits, Cardiovasc. Res. 8, 681. Sander, G.E. and C.G. Huggins, 1971, Subcellular localization of angiotensin I converting enzyme in rabbit lung, Nature New Biol. 230, 27. Sen, S., R.R. Smeby and F.M. Bumpus, 1967, Isolation of a phospholipid renin inhibitor from kidney, Biochemistry 6, 1572. Ueda, E., H, Akutsu, T. Kokubu and Y. Yamamura, 1971, Partial purification and properties of angiotensin I converting enzyme from rabbit plasma, Jap. Circ. J. 35,801. Ueda, E., T. Joh, K. Nishimura, I. Kato, T. Ochi, Y. Hayashi, H. Kukita, T. Kawabe, N. Yoshida and T. Kokubu, 1979, Angiotensin I-converting enzyme activity in serum and in the lung of rabbits with experimental pneumonitis, Jap. J. Thorac. Dis. 17, 83. Watkins, B.E., J.O. Davis, R.H. Freeman, G.A. Stephens and J.M. DeForrest, 1978, Effects of the oral converting enzyme inhibitor (SQ 1 4 , 2 2 5 ) on onekidney hypertension in the dog, Proc. Soc. Exp. Biol. Med. 157,245.
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© Elsevier/North-Holland Biomedical Press
EFFECTS OF CAPTOPRIL (SQ 14, 225) ON THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM IN NORMAL RATS TATSUO KOKUBU, EINOSUKE UEDA, MOTOTANE ONO, TADAO KAWABE, YUJI HAYASHI and TAKUYA KAN
The Second Department of Internal Medicine, Ehime University School of Medicine, Onsen-gun, Ehime 791-02, Japan Received 11 December 1979, accepted 8 January 1980
T. KOKUBU, E. UEDA, M. ONO, T. KAWABE, Y. HAYASHI and T. KAN, Effects ofcaptopril (SQ 14, 225) on the renin-angiotensin-aldosterone system in normal rats, European J. Pharmacol. 62 (1980) 269--275. High doses of captopril (SQ 14, 225) ( 1 2 0 - 1 6 0 mg/kg/day) were administered orally to normal rats, and the effects on the renin-angiotensin-aldosteronesystem were observed. Plasma angiotensin converting enzyme (ACE) activity was elevated significantly on the 3rd, 7th and 30th days of captopril administration. ACE activity in the lung and the kidney was significantly decreased on the 1st day then gradually increased, becoming significantly higher than that of controls by the 30th day. Plasma renin activity (PRA) was significantly elevated on the 1st day and remained at a high level until the 30th day. Renal renin content was found to be significantly lower on the 1st and 3rd days. Plasma aldosterone concentration was not affected by captopril treatment, whereas serum potassium concentration was found to be significantly lower on the 1st, 3rd and 30th days. It is suggested that besides its inhibitory action on ACE, captopril has a direct or indirect stimulating action on ACE production as well as on renin release. Angiotensin-convertingenzyme inhibition Tissue angiotensin-convertingenzyme
Serum angiotensin converting enzyme Renin-angiotensin
1. Introduction Captopril (D-3-mercapto-2-methylpropanoyl-L-proline, SQ 14, 225) is an orally active inhibitor of angiotensin converting enzyme (ACE) (EC 3.4.15.1) (Cushman et al., 1977). The administration of this compound produces various biochemical changes and lowers blood pressure in hypertensive rats (Muirhead et al., 1978; Laffan et al., 1978), in rabbits (Romero et al., 1974; Murthy et al., 1977; 1978), in dogs (McCaa et al., 1978; Watkins et al., 1978; Harris et al., 1978) and even in man (Gavras et al., 1974; 1978b: Cody et al., 1978). If the antihypertensive action of captopril is to be understood the effects of captopril on the renin-angiotensin system must be made clear.
Captopril
Although the effects of captopril on the renin-angiotensin system have been studied extensively, few investigators have reported a change in serum ACE activity (Gavras et al., 1978c). There have been no previous reports of changes in tissue ACE activity following captopril administration. The purpose of the present study was to examine changes in serum and tissue levels of ACE as well as other components of the renin-angiotensin system following oral administration of captopril to normal rats.
2. Material and methods Female Wistar rats weighing about 250 g were used. The rats were fed ordinary chow (Oriental Yeast Co., Tokyo, Japan) containing
270
130 mEq of Na/kg of chow. Each rat ate a b o u t 15 g of food per day, thus consuming 2 mEq of Na per day. Experiments were carried out as follows: (1) Control group: rats were given tap water ad lib, and (2) experimental group (captopril group): rats were given captopril-water in a concentration of 1 mg/ml ad libitum. Each rat drank 30~40 ml/day of water, that is 120~160 mg/kg/day of captopril. Captoprilwater was prepared freshly every day. On the 1st, 3rd, 7th and 30th days of captopril administration the animals were anesthetized with pentobarbital (40 mg/kg), the bilateral renal arteries and veins were ligated, and blood samples were taken from the inferior vena cava with heparinized plastic syringes. The blood was centrifuged at 700 × g for 15 min at 4°C and the separated plasma was divided among several small plastic tubes and stored in a freezer at --20 ° C for later use. The lungs and the kidneys were taken out immediately after bleeding and kept in a freezer until use. The following were assayed: plasma renin activity (PRA), plasma ACE activity, plasma aldosterone concentration (PAC), serum Na concentration, serum K concentration, plasma ACTH and plasma cortisol concentration. The ACE activity in pulmonary and renal tissues, and the renal renin content were also measured. The ACE activity was measured spectrophotometrically according to Cushman (Cushman and Cheung, 1971) with a slight modification. The incubation mixture contained 3.5 mM of Hip-His-Leu, 100 mM of borate buffer, pH 8.3, 300 mM of NaC1. Duplicate incubations were performed and the inter-assay error was within +- 10%. Biological assay was also performed using angiotensin I (ANGI) and bradykinin ( B K ) a s substrates. One ml of ANGI (200 ng/ml) was incubated with 0.1 ml of plasma and the ANGII thus formed was assayed using the uterus of a rat pretreated with hexestlol (1 mg/kg) 24 h prior to the experiment (Barret and Sambhi, 1969; Ueda et al., 1971). The
T. K O K U B U ET AL.
uterus was suspended with 1 g tension in a 5 ml water bath containing modified Ringer solution, and the contraction of the uterus was measured by means of an isotonic transducer (Nihon Kohoden, TD-112 s). The sensitivity of the uterus was 1 ng/ml for ANGII and 30--40 ng/ml for ANGI. The assay was performed at concentrations lower than 5 ng/ml of ANGI, so that possible interference by ANGI could be ruled out. For measuring kininase activity, one ml of BK (5 pg/ml) was incubated with 0.1 ml of plasma and the remaining BK was assayed in the same way as ANGII. ACE activity in tissue was assayed as follows (Ueda et al., 1979): the tissue was homogenized in 25 mM borate buffer, pH 8.3 {50 vols. were added for pulmonary tissue and 10 vols. for renal tissue) with a Potter--Elvehjem homogenizer. An aliquot of the homogenized solution was transferred into a test tube (18 X 100 mm) and was homogenized again with Polytron ® (Kinematica, Lucerne, Switzerland) for 1 min. The solution was filtered through gauze, and centrifuged for 10 min at 700 X g. Cell debris were precipitated at this step, and no ACE activity was found in the precipitate. The supernatant which contained nuclear, lysozomal and soluble fractions was assayed for ACE. As a preliminary experiment, the ACE activity in various regions of the lung was compared, and a small variation in activity was found from one region to another. Because of this, one whole lung was used for the assay. A whole kidney was also used for the assay of enzyme activity in renal tissue. Renal renin content (RRC) was measured as follows (Gross et al., 1956): the kidney was homogenized in 100 mM phosphate buffer, pH 6.8 and centrifuged at 700 X g for 10 min. The supernatant, in a volume of 0.1 ml, was incubated with semipurified rat renin substrate (2000 ng ANGII/ml) (Sen et al., 1967) for 5 min in the presence of 8-hydroxyquinoline, EDTA-2Na and dimercaprol (BAL). The reaction was stopped by boiling the reaction mixture for 10 rain. The ANGI thus formed was measured radioimmunologically.
CAPTOPRIL ON RENIN-ANGIOTENSIN SYSTEM IN NORMAL RA T
Serum Na and K were measured with a flamephotometer (Beckman Flamephotometer, Klina). ACTH and cortisol were measured radioimmunologically. The pressor response to exogenous ANGI was measured in rats anesthetized with pentobarbital-Na. ANGI was injected through a cannula placed in the femoral vein and the blood pressure response was recorded through a cannula placed in the carotid artery.
271
TABLE 1 Effect of oral administration of captopril on serum angiotensin-converting enzyme activity in rats. The assay was performed with various substrates to compare biological and spectrophotometric assays.
Angiotensin I Bradykinin Hip-His-Leu
Normal (nmol/ml/min)
Captopril (7th day) (nmol/ml/min)
0.084 1.45 42.2
0.374 4.32 116.0
3. Results There was no difference in b o d y weight between the control and the captopril group (265.8 + 3.1 g and 258.8 + 4.4 g respectively). Each rat drank a b o u t 30~40 ml/day captoprilwater that is 30~40 mg/day (120~160 mg/kg/ day) of captopril. Pressor responses to i.v. injection of ANGI (10 ng) ranged from 15 to 20% of the responses in the normal rats on the 3rd and 7th day, and were 25% on the 30th day of captopril administration. Changes of ACE activity in plasma, in the lung and in the kidney are shown in fig. 1. On the 1st day of captopril administration plasma ACE remained at the same level as in
PLASMA
nmollmllmin
J LUNG nmollgtissuelmin 1000
the controls, b u t it increased significantly on the 3rd, 7th and 30th day. In contrast to plasma ACE activity, ACE activity in the lung on the 1st day was significantly lower than that of the control group (P < 0.005). Pulmonary ACE activity returned to the control level on the 3rd and 7th days, and then increased significantly on the 30th day (P < 0.005}. ACE activity in the kidney changed in a way similar to that in the lung. ACE activity in the kidney decreased on the 1st day (P < 0.005) and increased significantly on the 30th day (P < 0.05). The plasma, which showed high ACE activ-
J KIDNEY nmollgtissuelmin 200.
100~ .>
10050 n-lO
n-lO
n-lO
Fig. 1. Effects of oral administration of captopril on the activity of angiotensin-converting enzyme in serum, lung and kidney in normal rats. Abscissa: days after captopril administration. ***P < 0.001, **P < 0.005, *P < 0.05.
272
T. K O K U B U ET AL.
nglmllh
i
PRA
RRC
PAC
pglg tissuelmin
S
nglml 20"
4
0.8"
, ~ ' < / ~
0.6"
?
i0"
0.4'
n-8
n-6 l
0.2.
n-lO
Fig. 2. E f f e c t s o f oral a d m i n i s t r a t i o n o f c a p t o p r i l o n plasma renin activity, renal renin c o n t e n t and plasma aldost e r o n e c o n c e n t r a t i o n in n o r m a l rats. Abscissa: days after c a p t o p r i l a d m i n i s t r a t i o n . * * * P < 0.001, **P < 0.005.
ity on spectrophotometric assay, was checked biologically as described in Methods. As shown in table 1, ACE activity measured with ANGI as the substrate was 4 times higher than in control plasma, 3 times higher when BK was used as the substrate and 3 times higher when Hip-His-Leu was used as the substrate. This plasma was dialyzed overnight in physiological saline to remove possible circulating captopril, and the ACE activity was compared with that of non-dialyzed plasma. There was no appreciable difference between them. Changes in PRA, RRC and PAC are shown in fig. 2. PRA increased significantly on the
1st day (P < 0.001) and remained at a high level until the 30th day. On the other hand, RRC decreased significantly on the 1st and on the 3rd day as compared with the control group (P < 0.005 and P < 0.001). PAC was not affected by captopril. Changes in serum electrolytes are shown in table 2. Though the serum Na concentration was not affected by captopril, the serum K level was significantlylowered on the 1st day (P < 0.001) and remained at a low level until the 30th day. As shown in table 3 plasma cortisol and plasma ACTH were not affected by captopril.
TABLE 2 E f f e c t s o f oral a d m i n i s t r a t i o n o f captopril o n s e r u m e l e c t r o l y t e s in n o r m a l rats. Control
S e r u m Na (mEq/l)
139.5 + 0.63 (n = 8)
Serum K (mEq/1)
4.28 + 0.11 (n = 8)
Days o f oral a d m i n i s t r a t i o n o f c a p t o p r i l ( 1 2 0 - - 1 6 0 m g / k g / d a y ) 1st day
3rd day
7th day
3 0 t h day
140.0 -+ 0.73 (n = 6) ns 3.78 + 0.17 (n = 6)
138.7 -+ 1.52 (n = 7)
138.1 -+ 0.58 (n = 8)
139.8 + 0.65 (n = 8)
ns
ns
ns
3.56 -+ 1.36 (n = 7)
3.70 + 0.06 (n = 8)
3.68 + 0.08 (n = 6)
P < 0.001
P < 0.001
P < 0.001
P < 0.001
CAPTOPRIL ON RENIN-ANGIOTENSIN SYSTEM IN NORMAL RAT TABLE 3 Effect of oral administration of captopril on plasma ACTH and cortisol levels in normal rats.
Control (n = 6) 3rddayof captopril administration (n=8)
Plasma ACTH (pg/ml)
Plasma cortisol (ug/dl)
399.2 -+ 113.3 2 9 0 . 2 + 31.6
73.93 + 10.91 65.73-+ 9.42
4. Discussion The dose of captopril used in the present experiments was high enough to inhibit ACE activity in rats because the pressor response to exogenous ANGI was almost completely abolished. Serum ACE activity in captopril-treated rats was unexpectedly elevated. There might have been some problem in measuring the serum ACE without dialysis because circulating captopril might have inhibited the in vitro reaction of the enzyme. In assays done under such conditions the ACE activity found will be lower than the true value. However the enzyme activity was found to be higher than that in normal rats. Furthermore the activity was not increased by dialysis. In contrast to our results, Gavras et al. (1978c) reported that captopril reduced ACE activity in hypertensive patients to one tenth of its pre-administration value. We had previously found that the i.v. or i.p. injection of captopril lowered serum ACE activity, and also that a single oral administration lowered serum ACE activity (unpublished data}. So the effect of long-term administration of captopril in the drinking water might be different from that of a single administration. Possible mechanisms for the elevation of serum ACE activity in the present study are as follows; (1} elevated circulating ANGI stimulates the release of ACE from the lung or other organs, as an adaptation response to the increased circulating ANGI, (2) the blockade
273
of ACE on the surface of endothelial cells stimulates the production and release of ACE in those cells, (3) captopril inhibits the metabolic turnover of circulating ACE, and (4) the production of ACE is stimulated by captopril itself. Whatever the mechanism it is obvious that the increased circulating ACE is n o t effective to convert ANGI to ANGII because no pressor response to exogenous ANGI was observed. Changes of ACE activity in the lung and the kidney are difficult to explain in relation to the pressor response to ANGI. In the early period of captopril administration, tissue ACE activity and the pressor response to ANGI changed in parallel, b u t on the 30th day of administration, tissue ACE activity was significantly elevated whereas the pressor response to ANGI remained suppressed. The activity of the enzyme in tissue homogenates may not represent the activity in that organ in vivo. For example high angiotensinase activity is found in lung homogenate whereas no inactivation of ANGII occurs in the pulmonary circulation (Bakhle, 1968; Sander and Huggins, 1971}. We suspect that ACE on the surface of endothelium cells was mostly combined with captopril and that the ability to convert ANGI to ANGII was therefore reduced. In the period around the 30th day, ACE activity within the cell and not related to the conversion of circulating ANGI to ANGII, might have been somehow increased. Further experiments along the line of this speculation are in progress. Several investigators have reported that a marked elevation of PRA followed captopril administration (Gavras et al., 1974; 1978a, b, c; Ferguson et al., 1977; McCaa et al., 1978; Re et al., 1978; Watkins et al., 1978; Cody et al., 1978) and our results are consistent with theirs. The mechanism of elevation might not be simple as the phenomenon appeared soon after captopril administration. PRA was highest on the 1st day of administration, then decreased gradually, b u t was still higher than the normal level even on the 30th day of administration. Those results, in combination
274
with the finding of a decrease of RRC on the 1st and 3rd day, suggest that captopril might stimulate renin release from the kidney directly. PAC has been reported to be low following captopril administration in man (Gavras et al., 1978a) and in dogs (McCaa et al., 1978). Watkins et al. (1978) reported no change in PAC following captopril administration in one-kidney hypertensive dogs. In our experiments with normal rats, PAC did not change throughout the experiment. There might be some differences between species in the stimulatory mechanism of aldosterone secretion. A possible difference in the amount of sodium intake may also account for the discrepancy. We have considered an increase of ACTH or cortisol as a possible explanation for the lack of reduction in PAC. Captopril, however, did not increase the plasma level of either ACTH or cortisol. McCaa et al. (1978) reported that serum potassium concentration was not changed by captopril despite the increased sodium excretion. In our experiments, serum potassium concentration was significantly decreased on the 1st day, and stayed at a lower level throughout the experiment. The difference in findings could be ascribed to differences in the sodium intake or in the method of administration. In conclusion, the effects of captopril on the renin-angiotensin system may be more complicated than previously thought. In particular, the increase of ACE which occurred with long-term blockade of enzyme activity seems to deserve further study.
Acknowledgements SQ 14, 225 was kindly supplied by E.R. Squibb & Sons, Inc. and Sankyo Co., Ltd. The authors are grateful to Otsuka Assay Lab. for the ACTH and cortisol assays. This work was supported by Grant-in-Aid for Scientific Research (Japan) No. 337030 and by a Grant from Sankyo Co., Ltd.
T. KOKUBU ET AL.
References Bakhle, Y.S., 1968, Conversion of angiotensin I to angiotensin II by cell free extracts of dog lung, Nature 220,919. Barret, J.D. and Sambhi, M.R., 1969, Simultaneous assay of angiotensin I and II, and determination of converting enzyme activity, J. Pharmacol. Exp. Therap. 170,326. Cody, R.J.Jr., R.C. Tarazi, E.L. Bravo and F.M. Fouad, 1978, Haemodynamics of orally-active converting enzyme inhibitor (SQ 14, 225) in hypertensive patients, Clin. Sci. Mol. Med. 55, 453. Cushman, D.W. and H.S. Cheung, 1971, Spectrophotometric assay and properties of the angiotensinconverting enzyme of rabbit lung, Biochem. Pharmacol. 20, 1637. Cushman, D.W., H.S. Cheung, E.F. Sabo and M.A. Ondetti, 1977, Design of potent competitive inhibitor of angiotensin converting enzyme, Biochemistry 16, 5484. Ferguson, R.K., G.A. Turini, H.R. Brunner and H. Gavras, 1977, A specific orally active inhibitor of angiotensin-converting enzyme in man, Lancet 1, 775. Gavras, H., H.R. Brunner, J.H. Laragh, J.E. Sealey, I. Gavras and R. Vukovich, 1974, An angiotensin converting enzyme inhibitor to identify and treat vasoconstrictor and volume factors in hypertensive patients, New Engl. J. Med. 291,817. Gavras, H., H.R. Brunner, G.A. Turini, G.R. Kershaw, C.P. Tifft, S. Cuttelod, I. Gavras, R.A. Vukovich and D.N. McKinstry, 1978b, Antihypertensive effect of the oral angiotensin converting enzyme inhibitor SQ 14, 225 in man, New Engl. J. Med. 298,991. Gavras, H., I. Gavras, S. Textor, L. Volicer, H.R. Brunner and E.J. Ruchinska, 1978a, Effect of angiotensin converting enzyme inhibition of blood pressure, plasma renin activity and plasma aldosterone in essential hypertension, J. Clin. Endocrinol. Met. 4 6 , 2 2 0 . Gavras, H., C.S. Liang and H.R. Brunner, 1978c, Redistribution of regional blood flow after inhibition of the angiotensin converting enzyme, Circ. Res. 43 (Suppl. I), 1-59. Gross, F. and F. Sulser, 1956, Pressorische Substanzen in den Nieren experimental hypertonischer Ratten, Naunyn-Schmiedeb. Arch. Exp. Pathol. Pharmakol. 229,374. Harris, D.N., 1978, Effects of SQ 14, 225, an orally active inhibitor of angiotensin converting enzyme on blood pressure, heart rate and PRA of concious dogs, European J. Pharmacol. 51,345. Muirhead, E.E., R.L. Prewitt, Jr., B. Brooks and W.L. Brosius, Jr., 1978, Antihypertensive action of the
CAPTOPRIL ON RENIN-ANGIOTENSIN SYSTEM IN NORMAL RAT orally active converting enzyme inhibitor (SQ 14, 225) in spontaneously hypertensive rats, Circ. Res. 43 (Suppl. I), 1-53. Murthy, V.S., T.L. Waldron, M.E. Goldberg and R.R. Vollmer, 1977, Inhibition of angiotensin converting enzyme by SQ 14, 225 in conscious rabbits, European J. Pharmacol. 46, 207. 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, Circ. Res. 43 (Suppl. I), 1-40. Laffan, R.J., M.E., Goldberg, J.P. High, T.S. Schaeffer, M.H. Waugh and B. Rubin, 1978, Antihypertensive activity in rats of SQ 14, 225, an orally active inhibitor of angiotensin I-converting enzyme, J. Pharmacol. Exp. Therap. 204,281. McCaa, R.E., J.E. Hall and C.S. McCaa, 1978, The effects of angiotensin I-converting enzyme inhibitors on arterial blood pressure and urinary sodium excretion, Circ. Res. 43 (Suppl. I}, 1-32. Re, R., R. Novlline, M.T. Esconrron, C. Athanasouois, J. Burton and E. Haber, 1978, Inhibition of angiotensin converting enzyme for diagnosis of renal artery system, New Engl. J. Med. 298,582.
275
Romero, J.C., S.W. Mak and S.W. Hoobler, 1974, Effect of blockade of angiotensin I-converting enzyme on the blood pressure of renal hypertensive rabbits, Cardiovasc. Res. 8, 681. Sander, G.E. and C.G. Huggins, 1971, Subcellular localization of angiotensin I converting enzyme in rabbit lung, Nature New Biol. 230, 27. Sen, S., R.R. Smeby and F.M. Bumpus, 1967, Isolation of a phospholipid renin inhibitor from kidney, Biochemistry 6, 1572. Ueda, E., H, Akutsu, T. Kokubu and Y. Yamamura, 1971, Partial purification and properties of angiotensin I converting enzyme from rabbit plasma, Jap. Circ. J. 35,801. Ueda, E., T. Joh, K. Nishimura, I. Kato, T. Ochi, Y. Hayashi, H. Kukita, T. Kawabe, N. Yoshida and T. Kokubu, 1979, Angiotensin I-converting enzyme activity in serum and in the lung of rabbits with experimental pneumonitis, Jap. J. Thorac. Dis. 17, 83. Watkins, B.E., J.O. Davis, R.H. Freeman, G.A. Stephens and J.M. DeForrest, 1978, Effects of the oral converting enzyme inhibitor (SQ 1 4 , 2 2 5 ) on onekidney hypertension in the dog, Proc. Soc. Exp. Biol. Med. 157,245.