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Induction of angiotensin I-converting enzyme in rat lung with captopril (SQ 14225)
European Journal of Pharmacology, 67 (1980) 473--475 © Elsevier/North-Holland Biomedical Press
473
Short communication INDUCTION OF ANGIOTENSIN I-CONVERTING ENZYME IN RAT LUNG WITH CAPTOPRIL (SQ 14225) F. FYHRQUIST, T. FORSLUND, I. TIKKANEN and CAROLA GRONHAGEN-RISKA Unit of Clinical Physiology, Minerva Institute for Medical Research, P.O. Box 819, SF-O0101 Helsinki 1 O, Finland Received 21 July 1980, revised MS received 8 September 1980, accepted 10 September 1980
F. FYHRQUIST, T. FORSLUND, I. TIKKANEN and C. GRONHAGEN-RISKA, Induction ofangiotensin I-converting enzyme in rat lung with captopril SQ 14225, European J. Pharmacol. 67 (1980) 473---475. Angiotensin I-converting enzyme (ACE, EC 3.4.15.1.) was measured in serum and in pulmonary plasma membranes of 40 spontaneously hypertensive rats (SHR, Okamoto Aoki strain), divided into 4 groups, and treated with SQ 14225 (Captopril®), 0.2 mg • ml -l in drinking water, for 0--24 weeks. Serum ACE activity increased 2.5--3 fold after 12--24 weeks of SQ 14225 treatment, parallelled by an increase of ACE concentration in purified pulmonary plasma membranes (25--52%), and in ACE concentration upon solubilization with Triton X-100 from such plasma membranes (96--120%). We conclude that the ACE inhibitor, SQ 14225, causes marked induction of pulmonary ACE biosynthesis. High serum ACE activity probably reflects increased total biosynthesis of the enzyme. Angiotensin I-converting enzyme inhibition Pulmonary angiotensin-converting enzyme
Serum angiotensin-converting enzyme Enzyme induction
1. Introduction Angiotensin I-converting enzyme (ACE, kininase II, EC 3.4.15.1.) converts angiotensin I to bioactive angiotensin II and also inactivates bradykinin. ACE is inhibited by D-methyl-3-mercapto-propanoyl-L-proline (SQ 14225, captopril), a new antihypertensive drug (Ondetti et al., 1977). The main locus of ACE is in the luminal membranes of pulmonary vascular endothelium (Ryan et al., 1975). The regulation of ACE biosynthesis is unkno~vn. Increased serum ACE activity in patients with essential hypertension during treatment with captopril (Larochelle et al., 1979, Fyhrquist and Forslund, unpublished observation) prompted us to study in spontaneously hypertensive rats (SHR) whether SQ 14225 affects the tissue concentration of ACE. Thus, the effect of SQ 14225 administered orally to SHR rats was studied in serum,
Captopril
in pulmonary plasma membranes, and after solubflization of ACE bound to pulmonary plasma membranes.
2. Materials and methods
2.1. Experimental procedure Forty female rats, age 4 weeks, of the Okamoto-Aoki strain (MSUegaard-Hansen, Ejby, Denmark), which develops spontaneous hypertension, were divided into four equal groups. Group 1 were controls, group 2 received SQ 14225 in the drinking water (0.2 m g . m1-1) during weeks 0--12, group 3 during weeks 12--24, and group 4 received the drug continuously during weeks 0--24. The animals were kept in 12 h dark and light cycles and were allowed standard rat food and drinking water ad libitum. Systolic blood pressure was recorded in conscious rats by a
474
tail cuff method (Harvard Apparatus Co., Massachusetts, U.S.A.), using a Doppler ultrasonic flowmeter (Parks Electronics Lab., Beawerton, U.S.A.). The lungs from rats of the various groups were rapidly excised during ether anesthesia after exhaustive aspiration of aortic blood. Pooled lungs were chilled on ice and homogenized at 0°C with an Ultra-Turrax in 20 mM Tris-HC1 buffer, pH 7.8, containing 5 mM MgC12, 30 mM KC1 and 0.25 M sucrose. The homogenate was filtered over double cheesecloth and the filtrate centrifuged at 700 g for 10 min. The supernatant was then centrifuged at 7 8 0 0 0 g for 90 min. The pellet containing plasma membranes was resuspended in a 10 mM Tris-HC1 buffer, pH 7.8, and was dialyzed overnight against the same buffer at 4 ° C. Solubilized ACE from plasma membranes was prepared by treating the membrane fraction with 0.1% Triton X-100 for 1 h at 0°C, followed by sonication for 20 sec. After a second ultracentrifugation at 78 000 g for 90 min, the supernatant contained solubilized enzyme.
2.2. Analytical procedures ACE was measured according to Lieberman (1975), using L-hippuryl-L-histidyr-L-leucine as a substrate. Membrane-bound and solubilized enzyme fractions were assayed for ACE with adjustment of protein concentrations; membranes 0.6 mg. m1-1, solubilized enzyme 0.35 mg.m1-1. The results are expressed in pmol of hippuric acid released per min and liter. The identity of rat pulmonary ACE was tested with the following known inhibitors of the enzyme: Na2EDTA, SQ 20881, and SQ 14225, using the above-mentioned substrate.
2.3. Special reagents SQ 14225, captopril, and another highly specific inhibitor of ACE, SQ 20881 (teprotide), were generous gifts of E.R. Squibb and Sons, Princeton, N.J., U.S.A. L-hippuryl-Lhistidyl-L-leucine was purchased from Vega Biochemicals, Tuczon, Ariz., U.S.A.
F. FY H RQ U I S T ET AL.
3. Results
There was a marked increase of serum ACE activity of rats treated with SQ 14225 (fig. 1, groups 3 and 4) at sacrifice (24 weeks). This was paraUelled by increased ACE concentrations in pulmonary plasma membranes (25-52%), and in solubilized fractions of membrane enzyme (96--120%). An increase in solubilized ACE concentration was also detectable in the group which received SQ 14225 during weeks 0--12 only (fig. l, panel c), which implied that the effect of the tg
20O !
I
z-~ ec .-s
50
> z~ 0 to)
200
b
OE z7 m or)~ z '~_ tu 1 O0
;E z ,d
300 ".
200
100 1
2
3
GROUP
Fig. 1. ACE in serum (panel a), in pulmonary plasma membranes (b), and in a solubilized fraction of pulmonary plasma membranes (c) of spontaneously hypertensive rats. Treatment with SQ 14225 (captopril): group 1 = controls, group 2 = drug-treated during weeks 0--12, group 3 = drug-treated during weeks 12--24, group 4 = drug-treated during weeks 0--24. Mean + S.E.M. for serum ACE (a); mean values for pulmonary plasma membranes and solubilized protein (b, c) from lungs pooled within each group.
ANGIOTENSIN I-CONVERTING ENZYME IN RAT LUNG
475
Sg).
14225 (unpublished observation), would favour the idea that SQ 14225 caused an increased production of ACE, possibly by interfering with the production or effect of an adrenal factor that may modulate cellular ACE biosynthesis. Increased ACE in serum and pulmonary plasma membranes of SHR rats did not prevent the antihypertensive effect of the drug in these animals. In this respect, therefore, ACE induction with SQ 14225 may be of negligible importance. The increased tissue concentration of ACE may not be relevant for the biological activity of the enzyme. Also, the doses of SQ 14225 used may suffice to cover for even substantial increases of ACE in tissues and body fluid. Clearly, the mechanism of induction of ACE biosynthesis, with SQ 14225 is interesting, and merits further study.
4. Discussion
Acknowledgements
Our data indicate that the potent inhibitor of ACE, SQ 14225, or captopril, causes a marked increase of ACE concentration in pulmonary plasma membranes of SHR rats, presumably by induction of increased ACE biosynthesis. Kokubu et al. (1980) recently demonstrated and discussed the increase of serum ACE and lung tissue ACE in normal Wistar rats treated with SQ 14225 in doses several fold higher than those used by us. They measured ACE in a lung preparation containing nuclear, lysosomal, and soluble fractions. Thus, their data were not directly comparable to ours, obtained by studying ACE in purified pulmonary plasma membranes free of contaminating serum and solubflized enzyme. Kokubu et al. (1980) showed that ACE was increased in pulmonary tissue after treatment with SQ 14225. Our data agree with that report, and extend it by showing an increase of ACE concentration in rat pulmonary plasma membranes following SQ 14225 treatment. Preliminary observations from our laboratory that bilateral adrenalectomy prevented induction of ACE biosynthesis in lungs from Wistar rats treated with SQ
This study was supported by the Finnish Academy, by the Sigrid Juselius Foundation, and the Nordisk Insulinfond. We thank Dr. P.V. Pigott (Squibb, Europe) and Dr. Z.P. Horovitz (E.R. Squibb and Sons) for SQ 14225 and SQ 20881.
drug on pulmonary ACE may last for 12 weeks. The concentration of 5'-nucleotidase, a marker of plasma membranes, indicated a similar purity of plasma membranes in each group, and was unaltered by SQ 14225 treatment (range: 135--146 pmol • h -1 • 1-1). Solubilized pulmonary membrane ACE was completely inhibited by 5 mM Na2EDTA, by 0.1 mM SQ 14225, and by 0.1 mM SQ 20881. ICs0 of SQ 14225 with L-hippuryl-L-histidylL-leucine as a substrate was 2.9 × 10 -9 M. Spontaneous hypertension developed within 6 weeks in untreated rats. After 24 weeks control rats were hypertensive (195 + 6.3 mm Hg, mean -+ S.E.M.), like the rats of group 2 (181 + 3.8), while rats treated with SQ 14225 had a lower blood pressure (group 3 : 1 5 5 + 3.9, group 4 : 1 4 5 + 3.4 mm
References Kokubu, T., E. Ueda, M. Ono, T. Kawabe, Y. Hayashi and T. Kan, 1980, Effects of captopril (SQ 14225) on the renin-angiotensin-aldosterone system in normal rats, European J. Pharmacol. 62, 269. Larochelle, P., J. Genest, O. Kuchel, R. Boucher, Y. Gutkowska and D. KcKinstry, 1979, Effect of captopril (SQ 14225) on blood pressure, plasma renin activity and angiotensin I converting enzyme activity, Can. Med. Assoc. J. 121,309. Lieberman, J., 1975, Elevation of serum angiotensinconverting-enzyme (ACE) level in sarcoidosis, Am. J. Med. 59,365. Ondetti, M.A., B. Rubin and D.W. Cushman, 1977, Design of specific inhibitors of angiotensinconverting enzyme. New class of orally active antihypertensive agents, Science 196, 441. Ryan, J.W., U.S. Ryan, D.R. Schultz, C. Whitaker, A. Chung and F.E. Dorer, 1975, Subcellular localization of pulmonary angiotensin-converting enzyme (Kininase II), Biochem. J. 146,497.
473
Short communication INDUCTION OF ANGIOTENSIN I-CONVERTING ENZYME IN RAT LUNG WITH CAPTOPRIL (SQ 14225) F. FYHRQUIST, T. FORSLUND, I. TIKKANEN and CAROLA GRONHAGEN-RISKA Unit of Clinical Physiology, Minerva Institute for Medical Research, P.O. Box 819, SF-O0101 Helsinki 1 O, Finland Received 21 July 1980, revised MS received 8 September 1980, accepted 10 September 1980
F. FYHRQUIST, T. FORSLUND, I. TIKKANEN and C. GRONHAGEN-RISKA, Induction ofangiotensin I-converting enzyme in rat lung with captopril SQ 14225, European J. Pharmacol. 67 (1980) 473---475. Angiotensin I-converting enzyme (ACE, EC 3.4.15.1.) was measured in serum and in pulmonary plasma membranes of 40 spontaneously hypertensive rats (SHR, Okamoto Aoki strain), divided into 4 groups, and treated with SQ 14225 (Captopril®), 0.2 mg • ml -l in drinking water, for 0--24 weeks. Serum ACE activity increased 2.5--3 fold after 12--24 weeks of SQ 14225 treatment, parallelled by an increase of ACE concentration in purified pulmonary plasma membranes (25--52%), and in ACE concentration upon solubilization with Triton X-100 from such plasma membranes (96--120%). We conclude that the ACE inhibitor, SQ 14225, causes marked induction of pulmonary ACE biosynthesis. High serum ACE activity probably reflects increased total biosynthesis of the enzyme. Angiotensin I-converting enzyme inhibition Pulmonary angiotensin-converting enzyme
Serum angiotensin-converting enzyme Enzyme induction
1. Introduction Angiotensin I-converting enzyme (ACE, kininase II, EC 3.4.15.1.) converts angiotensin I to bioactive angiotensin II and also inactivates bradykinin. ACE is inhibited by D-methyl-3-mercapto-propanoyl-L-proline (SQ 14225, captopril), a new antihypertensive drug (Ondetti et al., 1977). The main locus of ACE is in the luminal membranes of pulmonary vascular endothelium (Ryan et al., 1975). The regulation of ACE biosynthesis is unkno~vn. Increased serum ACE activity in patients with essential hypertension during treatment with captopril (Larochelle et al., 1979, Fyhrquist and Forslund, unpublished observation) prompted us to study in spontaneously hypertensive rats (SHR) whether SQ 14225 affects the tissue concentration of ACE. Thus, the effect of SQ 14225 administered orally to SHR rats was studied in serum,
Captopril
in pulmonary plasma membranes, and after solubflization of ACE bound to pulmonary plasma membranes.
2. Materials and methods
2.1. Experimental procedure Forty female rats, age 4 weeks, of the Okamoto-Aoki strain (MSUegaard-Hansen, Ejby, Denmark), which develops spontaneous hypertension, were divided into four equal groups. Group 1 were controls, group 2 received SQ 14225 in the drinking water (0.2 m g . m1-1) during weeks 0--12, group 3 during weeks 12--24, and group 4 received the drug continuously during weeks 0--24. The animals were kept in 12 h dark and light cycles and were allowed standard rat food and drinking water ad libitum. Systolic blood pressure was recorded in conscious rats by a
474
tail cuff method (Harvard Apparatus Co., Massachusetts, U.S.A.), using a Doppler ultrasonic flowmeter (Parks Electronics Lab., Beawerton, U.S.A.). The lungs from rats of the various groups were rapidly excised during ether anesthesia after exhaustive aspiration of aortic blood. Pooled lungs were chilled on ice and homogenized at 0°C with an Ultra-Turrax in 20 mM Tris-HC1 buffer, pH 7.8, containing 5 mM MgC12, 30 mM KC1 and 0.25 M sucrose. The homogenate was filtered over double cheesecloth and the filtrate centrifuged at 700 g for 10 min. The supernatant was then centrifuged at 7 8 0 0 0 g for 90 min. The pellet containing plasma membranes was resuspended in a 10 mM Tris-HC1 buffer, pH 7.8, and was dialyzed overnight against the same buffer at 4 ° C. Solubilized ACE from plasma membranes was prepared by treating the membrane fraction with 0.1% Triton X-100 for 1 h at 0°C, followed by sonication for 20 sec. After a second ultracentrifugation at 78 000 g for 90 min, the supernatant contained solubilized enzyme.
2.2. Analytical procedures ACE was measured according to Lieberman (1975), using L-hippuryl-L-histidyr-L-leucine as a substrate. Membrane-bound and solubilized enzyme fractions were assayed for ACE with adjustment of protein concentrations; membranes 0.6 mg. m1-1, solubilized enzyme 0.35 mg.m1-1. The results are expressed in pmol of hippuric acid released per min and liter. The identity of rat pulmonary ACE was tested with the following known inhibitors of the enzyme: Na2EDTA, SQ 20881, and SQ 14225, using the above-mentioned substrate.
2.3. Special reagents SQ 14225, captopril, and another highly specific inhibitor of ACE, SQ 20881 (teprotide), were generous gifts of E.R. Squibb and Sons, Princeton, N.J., U.S.A. L-hippuryl-Lhistidyl-L-leucine was purchased from Vega Biochemicals, Tuczon, Ariz., U.S.A.
F. FY H RQ U I S T ET AL.
3. Results
There was a marked increase of serum ACE activity of rats treated with SQ 14225 (fig. 1, groups 3 and 4) at sacrifice (24 weeks). This was paraUelled by increased ACE concentrations in pulmonary plasma membranes (25-52%), and in solubilized fractions of membrane enzyme (96--120%). An increase in solubilized ACE concentration was also detectable in the group which received SQ 14225 during weeks 0--12 only (fig. l, panel c), which implied that the effect of the tg
20O !
I
z-~ ec .-s
50
> z~ 0 to)
200
b
OE z7 m or)~ z '~_ tu 1 O0
;E z ,d
300 ".
200
100 1
2
3
GROUP
Fig. 1. ACE in serum (panel a), in pulmonary plasma membranes (b), and in a solubilized fraction of pulmonary plasma membranes (c) of spontaneously hypertensive rats. Treatment with SQ 14225 (captopril): group 1 = controls, group 2 = drug-treated during weeks 0--12, group 3 = drug-treated during weeks 12--24, group 4 = drug-treated during weeks 0--24. Mean + S.E.M. for serum ACE (a); mean values for pulmonary plasma membranes and solubilized protein (b, c) from lungs pooled within each group.
ANGIOTENSIN I-CONVERTING ENZYME IN RAT LUNG
475
Sg).
14225 (unpublished observation), would favour the idea that SQ 14225 caused an increased production of ACE, possibly by interfering with the production or effect of an adrenal factor that may modulate cellular ACE biosynthesis. Increased ACE in serum and pulmonary plasma membranes of SHR rats did not prevent the antihypertensive effect of the drug in these animals. In this respect, therefore, ACE induction with SQ 14225 may be of negligible importance. The increased tissue concentration of ACE may not be relevant for the biological activity of the enzyme. Also, the doses of SQ 14225 used may suffice to cover for even substantial increases of ACE in tissues and body fluid. Clearly, the mechanism of induction of ACE biosynthesis, with SQ 14225 is interesting, and merits further study.
4. Discussion
Acknowledgements
Our data indicate that the potent inhibitor of ACE, SQ 14225, or captopril, causes a marked increase of ACE concentration in pulmonary plasma membranes of SHR rats, presumably by induction of increased ACE biosynthesis. Kokubu et al. (1980) recently demonstrated and discussed the increase of serum ACE and lung tissue ACE in normal Wistar rats treated with SQ 14225 in doses several fold higher than those used by us. They measured ACE in a lung preparation containing nuclear, lysosomal, and soluble fractions. Thus, their data were not directly comparable to ours, obtained by studying ACE in purified pulmonary plasma membranes free of contaminating serum and solubflized enzyme. Kokubu et al. (1980) showed that ACE was increased in pulmonary tissue after treatment with SQ 14225. Our data agree with that report, and extend it by showing an increase of ACE concentration in rat pulmonary plasma membranes following SQ 14225 treatment. Preliminary observations from our laboratory that bilateral adrenalectomy prevented induction of ACE biosynthesis in lungs from Wistar rats treated with SQ
This study was supported by the Finnish Academy, by the Sigrid Juselius Foundation, and the Nordisk Insulinfond. We thank Dr. P.V. Pigott (Squibb, Europe) and Dr. Z.P. Horovitz (E.R. Squibb and Sons) for SQ 14225 and SQ 20881.
drug on pulmonary ACE may last for 12 weeks. The concentration of 5'-nucleotidase, a marker of plasma membranes, indicated a similar purity of plasma membranes in each group, and was unaltered by SQ 14225 treatment (range: 135--146 pmol • h -1 • 1-1). Solubilized pulmonary membrane ACE was completely inhibited by 5 mM Na2EDTA, by 0.1 mM SQ 14225, and by 0.1 mM SQ 20881. ICs0 of SQ 14225 with L-hippuryl-L-histidylL-leucine as a substrate was 2.9 × 10 -9 M. Spontaneous hypertension developed within 6 weeks in untreated rats. After 24 weeks control rats were hypertensive (195 + 6.3 mm Hg, mean -+ S.E.M.), like the rats of group 2 (181 + 3.8), while rats treated with SQ 14225 had a lower blood pressure (group 3 : 1 5 5 + 3.9, group 4 : 1 4 5 + 3.4 mm
References Kokubu, T., E. Ueda, M. Ono, T. Kawabe, Y. Hayashi and T. Kan, 1980, Effects of captopril (SQ 14225) on the renin-angiotensin-aldosterone system in normal rats, European J. Pharmacol. 62, 269. Larochelle, P., J. Genest, O. Kuchel, R. Boucher, Y. Gutkowska and D. KcKinstry, 1979, Effect of captopril (SQ 14225) on blood pressure, plasma renin activity and angiotensin I converting enzyme activity, Can. Med. Assoc. J. 121,309. Lieberman, J., 1975, Elevation of serum angiotensinconverting-enzyme (ACE) level in sarcoidosis, Am. J. Med. 59,365. Ondetti, M.A., B. Rubin and D.W. Cushman, 1977, Design of specific inhibitors of angiotensinconverting enzyme. New class of orally active antihypertensive agents, Science 196, 441. Ryan, J.W., U.S. Ryan, D.R. Schultz, C. Whitaker, A. Chung and F.E. Dorer, 1975, Subcellular localization of pulmonary angiotensin-converting enzyme (Kininase II), Biochem. J. 146,497.