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Effect of converting enzyme inhibitors on tissue converting enzyme and angiotensin II: Therapeutic implications
Effect of Converting EnzymeInhibitors on Tissue Converting Enzymeand Angiotensin II: Therapeutic Implications THOMAS
UNGER,
MD,
DETLEV
GANTEN,
Local tissue renin-angiotensin systems have recently been discovered in various organs, and evidence is accumulating that inhibition of these local reninangiotensin systems may contribute to the actions of converting enzyme (CE) inhibitors. Measurements of CE activity and angiotensin II concentrations revealed that after oral administration of CE inhibitors, CE was inhibited not only in lung vascular endothelium and blood, but also in the heart, kidney, vascular wall, brain and other organs. The functional significance of tissue CE inhibition is suggested first by the antihypertensive effect of brain CE inhibition in spontaneously hypertensive rats,
A
lthough converting enzyme (CE) inhibitors have become established antihypertensive drugs and are currently under investigation for therapeutic use in such cardiac diseases as congestive heart failure and arrhythmias, their mechanisms of action are still not fully understood. Originally, a reduction of the vasoconstrictor peptide angiotensin II circulating in the blood, and possibly an accumulation of the vasodilator peptide bradykinin, were believed to be solely responsible for the actions of these drugs. However, doubts about this concept were raised early, based on various experimental and clinical findings.’ For instance, it was shown that blood pressure could be reduced in forms of hypertension that were not associated with a stimulated plasma renin-angiotensin system.2r3Further, the inhibition of the plasma renin-angiotensin system could often not be directly correlated with the antihypertensive effects of these drugs.3-5 Moreover, From the German Institute for High Blood Pressure Research and the Department of Pharmacology, University of Heidelberg, Heidelberg, Federal Republic of Germany. Address for reprints: Thomas Unger, MD, Department of Pharmacology, Im Neuenheimer Feld 366, 6900 Heidelberg, Federal Republic of Germany.
MD,
PhD,
and
RUDOLF
E. LANG,
MD
second by the concomitant persistence of blood pressure decrease and CE inhibition in vascular wall and kidney after long-term oral CE inhibitor treatment and third by ex vivo experiments demonstrating marked effects of oral CE inhibitor pretreatment on cardiac function in isolated rat hearts. Local inhibition of tissue renin-angiotensin systems may be an important factor involved in the beneficial effects of CE inhibitors in such cardiovascular diseases as arterial hypertension, congestive heart failure and cardiac arrhythmias. (Am J Cardiol 1987;59:18D-22D)
an increase in circulating endogenous bradykinin concentrations after CE inhibitor treatment has never been convincingly demonstrated. Recently, several laboratories have provided stringent evidence that the components of the renin-angiotensin system are generated locally in several organs involved in cardiovascular regulation, such as the kidney, heart, adrenal gland, vascular wall and central nervous system.6s7These findings have revolutionized our understanding of the renin-angiotensin system: The renin-angiotensin system can no longer be considered a hormonal system in the classical sense, because angiotensin II, the effector peptide of the system, can act asa circulating hormone as well as a locally generated modulator of organ function at the tissue level. In addition to the plasma renin-angiotensin system, local renin-angiotensin systems may therefore contribute to cardiovascular control. Concerning the actions of CE inhibitors, this new understanding of the renin-angiotensin system has given rise to the hypothesis that CE inhibitors exert their beneficial cardiovascular actions not only by reducing the activity of the plasma reninangiotensin system, but also by inhibiting tissue reninangiotensin systems in various target organs of cardiovascular control, such as brain, kidney, vascular wall
April 24, 1987
and hearts-l0 We report on some experimental findings in support of this hypothesis.
Inhibition of ConvertingEnzymein Tissue After Oral ConvertingEnzymeInhibitor Treatment Indirect evidence for tissue CE inhibition after CE inhibitor treatment was presented first by Asaad and Antonaccioll and by our laboratory? Long-term oral antihypertensive treatment of spontaneously hypertensive rats (SHRs and stroke-prone SHRs) with the CE inhibitor captopril increased renin concentrations in the vascular wall of the aorta, and this increase could be dissociated from the increase of plasma renin of renal origin. In analogy to the plasma renin-angiotensin system, where an increase in renin concentration is a consistent feature of CE inhibition (due to withdrawal of an angiotensin II-mediated negative feedback on renin), the increase of aortic renin was interpreted as being caused by a decrease in local angiotensin II generation in the vascular wall. Direct evidence for an inhibition of CE in various tissues after oral treatment with the CE inhibitors was subsequently provided by Cohen and Kurz,13 who showed that in SHRs a single oral dose of captopril or enalapril was able to reduce CE activity not only in blood serum and in lung, but also in aortic wall, heart, kidney and brain. In contrast to the blood serum, the reduction of CE activity in lung, aorta and kidney persisted for several days. A prolonged inhibition of CE activity in the aortic wall but not in the serum after repeated intraperitoneal administration of captopril was also observed by Velletri and Bean.14 In addition, these investigators demonstrated that CE was not confined to the endothelium, but was also localized in the tunica media of the vascular wall. These observations from acute studies were extended in our laboratory (Fig. 1). In strokeprone SHRs long-term oral antihypertensive treatment for several weeks with the CE inhibitors enalapril, ramipril and perindopril inhibited CE in most tissues investigated, including the aortic and mesenteric vascular wall, kidney, heart and brain.“-I7
THE AMERICAN
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OF CARDIOLOGY
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TABLE I Effect of a Single Oral Dose (10 mg/kg) of Ramiprii on Angiotensin ii Concentrations in Various Tissues of Nephrectomized Rabbits Control Hypothalamus Medulla oblongata Heart left ventricle left atrium right ventricle right atrium Aorta Adrenal gland
177.2 f 22.7 70.7 f 8.6 50.7 104.2 79.9 106.6 23.5 170.6
f f f f f f
7.5 7.6 7.3 23.1 1.4 13.0
Ramipril 959.8 f 112.2” 34.7 f 3.3‘ 57.5 63.2 68.9 47.4 15.8 98.0
f 9.9 41 10.8’ f 9.6 f 5.7’ f 0.6’ f 14.6’
Data are fmollg tissue and are mean f standard error of the mean. * p <0.05.
system on the results, Twenty hours later, ramipril was given orally at a dose of 10 mg/kg to 30 animals. This dose had previously been shown to normalize blood pressure in stroke-prone SHRs.15J6Four hours after treatment, all animals were killed, and tissue angiotensin II concentrations were measured in various organs including heart, aorta, adrenal gland and brain. The CE inhibitor-induced changes in tissue angiotensin II concentrations are shown in Table I. With the exception of the heart ventricles and the hypothalamus, tissue angiotensin II was significantly lowered by oral ramipril treatment. Because the animals had been nephrectomized before treatment, these changes occurred independently of the plasma renin-angiotensin system. The fact that CE inhibitor treatment did not change angiotensin II levels in the heart ventricles and even increased angiotensin II in the hypothalamus, while reducing angiotensin II in the other organs, suggested a tissue-specific regulation of angiotensin II.
&WEEKS ORAL TREATMENT
SHR
Reductionof AngiotensinII in TissueAfter Oral ConvertingEnzymeInhibitor Treatment Reduced tissue CE activity after systemic CE inhibitor treatment suggested an inhibition of angiotensin II generation in tissue. However, alternative pathways for a local angiotensin II biosynthesis independent of CE18Jgcould not be excluded at this point. To demonstrate that CE inhibitor treatment really reduced tissue angiotensin II concentrations, direct measurements of the peptide were thought to be necessary. With the help of a recently developed high performance liquid chromatography-controlled radioimmunoassay for angiotensin peptides in tissue,z0we were able to show a reduction of tissue angiotensin II concentrations in several organs after oral pretreatment with the CE inhibitor ramipril.21 We nephrectomized 60 rabbits bilaterally to exclude an influence of the plasma renin-angiotensin
IS
9490-3
aMK
01
q lHoe L98
1 10 rnglkgld
FIGURE 1. Percent of inhibition of converting enzyme in various tissues after long-term oral treatment of spontaneously hypertensive rats (SHR) with the converting enzyme inhibitors perindoprii (S 9490-3), ramiprii (HOE 498) and enaiaprii (MK 421). For details see references 16 and 17.
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Implications of Converting EnzymeInhibition in Tissue After Oral Converting EnzymeInhibitor Treatment The data just reported demonstrate that oral treatment with CE inhibitors inhibited tissue CE and reduced angiotensin II levels in important target organs for cardiovascular regulation. Thus, they provided a solid foundation for the hypothesis that tissue CE inhibition may contribute to the cardiovascular actions of CE inhibitors. However, it remained to be established if the observed tissue renin-angiotensin system inhibition was only a phenomenon associated with CE inhibitor treatment, or if tissue CE inhibition was causally linked to the actions of these drugs. Attempts to address this question met with a number of experimental difficulties. First, systemic CE inhibitor treatment invariably antagonizes the plasma renin-angiotensin system together with tissue renin angiotensin systems. It is well known that the activity of the plasma renin-angiotensin system and its pathophysiologic importance vary greatly with the disease and the conditions studied, e.g., high renin or low renin hypertension, high or low salt intake, congestive heart failure or ischemic heart disease. Presumably, the relative contribution of tissue renin-angiotensin systems to cardiovascular dysfunction may vary as well, and, in addition, may depend on the degree of stimulation of the plasma renin-angiotensin system. Therefore, it has been extremely difficult to design in vivo studies on the action of CE inhibitors, in which their effects on the tissue renin-angiotensin system could be clearly separated from those on the plasma renin-angiotensin system. Second, the importance of tissue renin-angiotensin system inhibition in various target organs, such as kidney, vascular wall, heart or brain, may be different depending on the relative contribution of the organ to cardiovascular control under the conditions studied. For instance, in stroke-prone SHRs, brain angiotensin appears to contribute to the maintenance of hypertension6 whereas this may not be the case in other types of hypertension. Third, local and systemic effects of CE inhibitors other than angiotensin II reduction complicate the issue. These effects may be related to CE inhibition (e.g., bradykinin accumulation) or may not. Despite these difficulties in analyzing the functional role of tissue renin-angiotensin systems and separating tissue CE inhibition from other effects of CE inhibitors, a number of experimental findings suggest that tissue CE inhibition indeed constitutes an important factor within the cardiovascular actions of CE inhibitors. The examples given next include the actions of CE inhibitors on tissue renin-angiotensin system in brain, kidney, vascular wall and heart. Blood pressure-lowering effects of brain converting enzyme inhibition in spontaneously hypertensive rats: Several investigators have demonstrated that in SHRs administration of CE inhibitors captopril, enalapril and ramipril into the brain ventricles lowered blood pressure to a greater extent than the intravenous administration of the same doses.g-22-24 In addition, the
ENZYME
INHIBITOR
antihypertensive effect of a low dose of captopril (15 pg/kg) injected into brain ventricles was associated with a marked inhibition of CE in the brain, but not in the peripheryeg CE inhibitor-induced accumulation of bradykinin in the brain could be ruled out as the cause for the blood pressure decrease, because bradykinin, in contrast to its peripheral vasodilatory effects, causes blood pressure increases when acting on receptors in the central nervous system.25Therefore, it was concluded from these experiments that inhibition of the stimulated brain renin-angiotensin system in SHRs lowered blood pressure independently of the plasma renin-angiotensin system. Persistent blood pressure reduction and tissue converting enzyme inhibition after long-term oral converting enzyme inhibitor treatment: Stroke-prone SHRs were treated orally for several weeks with the CE inhibitors enalapril(30 mg/kg/day) and ramipril(3 mg/kg/day). Blood pressure was normalized and plasma and tissue CE in various organs was inhibited during treatment. Upon withdrawal of the drugs, the reduced pressor responses to intravenous angiotensin I and CE activity in the blood plasma were restored to normal within 1 day, while blood pressure remained decreased for an additional 2 weeks, and the CE inhibition persisted in the kidney and the aortic and mesenteric vascular wa11.15J6These findings demonstrated that the prolonged antihypertensive action of the CE inhibitors was unrelated to CE inhibition in the plasma and lung vascular endothelium, but was associated with a persistent tissue CE inhibition in the kidney and the vascular wall. Effects of converting enzyme inhibition in the isolated heart: Rats were treated orally with the CE inhibitors enalapril (30 mg/kg) or ramipril (1 mg/kg]. One hour later the animals were killed, and the hearts were isolated and perfused according to the technique of Langendorff. The activity of CE in these hearts was inhibited for up to 24 hours. The effects of angiotensin I given to the perfusate [decrease in coronary flow, increase in force of contraction) were reduced and those of bradykinin (increase in coronary flow, decrease in force of contraction] were potentiated. In addition, the effects of an electrical stimulation of cardiac sympathetic nerves on coronary flow and heart rate were attenuated.27 Further studies using a similar isolated heart technique showed that captopril and ramiprilat given in the perfusate drastically reduced the reperfusion-induced ventricular fibrillation after ischemia.28~2g Oral pretreatment with ramipril 1 hour before sacrifice had the same effect.2g Thus, in the absence of the plasma renin-angiotensin system, inhibition of CE in the heart reduced the local renin-angiotensin system activity including the angiotensin II-mediated facilitation of neurotransmission, and, in addition, exerted a beneficial action on postischemic arrhythmias. These 3 examples may suffice to demonstrate that the inhibition of tissue CE in target organs can produce considerable cardiovascular actions independently of the plasma renin-angiotensin system. Whether the observed effects of local CE inhibition are entirely due to
April 24, 1987
a reduction of local angiotensin II generation, or whether interference with other peptide systems plays an additional role, cannot be answered as yet. Although much remains to be learned about the functional role of local renin-angiotensin system in various organs, the data we have reported strengthen the hypothesis that tissue CE inhibition contributes to the beneficial actions of CE inhibitors in cardiovascular disease.
References 1. Unger Th, Canten D, Lang RE. Pharmacology of converting enzyme inhibitors: new aspects. Clin Exp Hypertens 1983;A5:1333-1354, 2. Gavras H, Brunner HR. Turini GA, Kershaw GR, Tifft CP, Cutteelod S. Gavras I. Vukowich RA, McKinstry DN. Antihypertensive effect of the oral angiotensin-converting enzyme inhibitor SQl4.225 in man. N Engl J Med 1978:298:991-995. 3. Antonaccio MJ, Rubin V. Horovitz ZP. Effects of captopril in animal models of hypertension. Clin Exp Hypertens 1980;2:613-637. 4. Brunner HR. Gavras H, Waeber B, Kershaw GR, Turini GA, Vulkovich RA, McKinstry DN. Oral angiotensin-converting enzyme inhibitor in long-term treatment of hypertensive patients. Ann Intern Med 1979;90:19-23. 5. Bravo EL, Tarazi RC. Converting enzyme inhibition with an orally active compound in hypertensive man. Hypertension 1979;1:39-46. 6. Ganten D, Lang RE, Lehmann E, Unger Th. Brain angiotensin: on the way to becoming a well-studied neuropeptide system. Biochem Pharmacol 1984;
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converting enzyme. I Cardiovasc Pharmacol 1982;4:315-325. 15. Unger Th. Fleck Th, Ganten D. Long RE, Rettig R. 2-(N-(s)-l-EthoxycarbonyI-3-phenylpropyl-L-alonyl)-(~S,3S.5S]-2-azabicyclo (3.3.8)octane-3-carboxylic acid [HOE 498): antihypertensive action and persistent inhibition of tissue converting enzyme activity in spontaneously hypertensive rats. Arzneimittelforschung 1984;34:1426-1430, 16. Unger Th, Ganten D, Lang RE, Schijlkens BA. Is tissue convering enzyme inhibition a determinant of the antihypertensive efficacy of converting enzyme inhibitors? Studies with the two different compounds, HOE 498 and MK 421, in spontaneously hypertensive rats. J Cardiovasc PharmacoJ1984;6: 872-880.
17. Unger Th, Moursi M. Ganten D, Hermann K, Lang RE. Antihypertensive action of the converting enzyme inhibitor perindopril(S9490-31 in spontaneously hypertensive rats: Comparison with enalapril (MK 421) and ramipril (HOE 498). J Cardiovasc Pharmacol 1986;8:276-285. 18. Okunishi H, Miyazaki M. Toda N. Evidence for a putatively new angiotensin II-generating enzyme in the vascular wall. J Hypertens 1984;2:277-284. 19. Lanzillo JJ,Dasarathy Y, Stevens J.Fanburg BL. Conversion of angiotensin I to angiotensin II by a latent endothelial cell peptidyl dipeptidase that is not angiotensin-converting enzyme. Biochem Biophys Res Commun 1986J34.2: 770-776.
20. Hermann K, Lang RE. Unger Th, Bayer C, Ganten D. Combined highperformance liquid chromatography-radioimmunoassay for the characterization and quantitative measurement of neuropeptides. J Chromatogr 1984; 312:273-284.
467-478.
21. Ganten D, Balz W. Hense H. Jung W, Rohde A, Bayer C. Angiotensin (ANGJ peptides in tissue of rabbits: characterization and regulation after nephrectomy and converting enzyme inhibitor (CEI] treatment (abstr]. Naunyn Schmiedebergs Arch Pharmacol 1985;329:R63. 22. Hutchinson JS, Mendelsohn FAO, Doyle AE. Blood pressure responses normotensive and spontaneously hypertensive rats to intracerebroventricular and peripheral administration of captopril. Hypertension 1980;2:546-550. 23. Stamler JF, Brody MJ. Phillips MI. The central and peripheral effects of captopril [SQ14225)on the arterial pressure of the spontaneously hypertensive rat. Brain Res 1980;186:499-503. 24. Phillips MI, Kimura B. Converting enzyme inhibitors and brain angiotensin. J Cardiovasc Pharmacol 1986;8:suppl 10:582-590. 25. Unger Th, Rockhold RW, Yukimura T, Rettig R, Rascher W, Ganten D. Role of kinins and substance P in blood pressure regulation of normotensive and spontaneously hypertensive rats. In: Buckley JP, Ferrario CM, eds. Central Nervous System Mechanisms in Hypertension. New York: Raven Press,
10. Velletri P, Bean BL. The effects of captopril on rat aortic angiotensinconverting enzyme. J Cardiovasc Pharmacol 1982;4:315-325. 11. Asaad MM, Antonaccio MJ. Vascular wall renin in spontaneously hypertensive rats. Potential relevance to hypertension. Maintenance and antihypertensive effect of captopril. Hypertension 1982;4:487-493. 12. Unger Th, Hiibner D, Schiill 8. Lang RE, Rascher W, Rettig R, Ganten D. Effect of chronic oral coptopril treatment on tissue renin concentration and converting enzyme activity in stroke-prone spontaneously hypertensive rats. In: Rascher W, Clough D, Ganten D, eds. Hypertensive Mechanisms, Stuttgart: Schattauer Verlag, 1982:768-773. 13. Cohen ML, Kurz K. Angiotensin converting enzyme inhibition in tissue from spontaneously hypertensive rats after treatment with captopril or MK 421. J Pharmacol Exp Ther 1982;220:63-69. 14. Velletri P. Bean 8. The effects of captopril on rat aortic angiotensin-
26. Unger Th, Ganten D. Lang RE, Schiilkens BA. Persistent tissue converting enzyme inhibition following chronic treatment with HOE 498 and MK 421 in spontaneously hypertensive rats. J Cardiovasc Pharmacol 1985;7:36-41. 27. Xiang K, Linz W. Becker H, Ganten D. Lang RE. Schalkens BA, Unger Th. Effects of converting enzyme inhibitors: ramipril and enalapril on peptide action and sympathetic neurotransmission in the isolated heart. Eur J Pharmacol 1985;113:215-223. 28. van Gilst WH, de Graeff AP, Kingma JH, Wesseling H. de Langen CDJ. Captopril reduces purine loss and reperfusion arrhythmias in the rat heart after coronary artery occlusion. Eur J Pharmacol 1984;100:113-117. 29. Linz P. Schiilkens BA, Yi-Fan H. Beneficial effects of converting enzyme inhibitors in ischemic rat heart. J Cardiovasc Pharmacol 1986:8:suppll&591599.
33:3523-3528.
7. Campbell DJ. The site of angiotensin production. J Hypertens 1985;3: 199-207. 8. Antonaccio MJ. Kerwin L. Pre and post-junctional inhibition of vascular 1981;3:1:1-54-Isympathetic function by captopril in SHR. Hypertension fi2.
9. Unger Th. Kaufmann-Biihler I. Schijlkens B. Ganten D. Brain converting enzyme inhibition: a possible mechanism for the antihypertensive action of captopril in spontaneously hypertensive rats. Eur J Pharmacol 1981a;70:
1981:115-127.
Discussion Dr. Hansson (Giiteborg, Sweden): I thought your data were very interesting and I do not dispute your explanations at the end. I just want to remind you that a number of other drugs that are not considered to lower blood pressure, mainly through effects on the renin-angiotensin system, will also show this rather slow return of blood pressure. Beta blockers do the same thing and so do diuretics. Dr. Unger (Heidelberg, Germany): I agree with you and this is why I tried to throw in a word of caution. Our observation does not really prove the hypothesis, but I think the hypothesis that tissue inhibition is important is worth pursuing further.
Dr. Amery (Leuven, Belgium): You show that angiotensin I did not change, which is quite interesting. Did you look at all the factors in the system, all the renins and local renins? Dr. Unger: Yes, we did in previous studies and found that renin was increased in various organs after converting enzyme inhibitor treatment. Dr. Brunner (Lausanne, Switzerland): I notice that after ramipril you have a decrease in angiotensin II, but the level is still very high. I am a little bit concerned about this. The second question is whether you measured angiotensin II in the vascular wall after 7 days of converting enzyme inhibi-
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NEW ANGIOTENSIN
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tion? Measurement of converting enzyme activity is very nice, but if you really wanted to make a point, I think you have to be sure that angiotensin II is still reduced. Dr. Unger: You would like to see a greater reduction? Dr. Brunner: If you block the system, it should be low. We have not measured angiotensin II in tissue, but at least in plasma when we use our method combining high performance liquid chromatography with radioimmunoassay, we find almost no angiotensin II in the plasma. Dr. Unger: This is in agreement with our findings on plasma angiotensin II when we use extraction and high performance liquid chromatography. Dr. Brunner: Levels were still higher than 50. I do not know what the units were-pg/ml or fmol/ml? Dr. Unger: They were in fmol/ml. You are referring to the organs, because I did not show plasma angiotensin II values. Yes, about 50% of angiotensin II remains in tissue, but if you consider that local renin-angiotensin systems in various organs are inhibited to a certain degree and each may contribute to some extent to the general inhibition of the system, I wonder whether one has to really postulate that the system has to be blocked 100% everywhere. In your own published data the inhibition or the reduction of angiotensin II after an oral dose of enalapril was also limited in time and did not correspond to the time of blood pressure reduction.
ENZYME
INHIBITOR
Did you not have normalization of angiotensin II plasma levels 24 hours after the drug while blood pressure was still reduced? Dr. Brunner: No, angiotensin II has not gone up to normal levels. We know we still have a methodologic problem, because we get in vitro angiotensin II generation and if angiotensin I is high, we still get angiotensin II due to in vitro conversion and the high performance liquid chromatography will, of course, not avoid this. We are now developing methods to avoid in vitro conversion. I think there remain problems with the measurement of angiotensin II. This is why I am worried about your whole construction, because I think we have to be very sure about the methodology used before we can make any statements. Dr. Ball (Glasgow, Scotland): The presence of a brain renin-angiotensin system is obviously of great interest. Do you know in which cell type the renin-angiotensin system is actually present in the brain? Dr. Unger: Well, published data point to neuronal cells and it has indeed been shown that the renin-angiotensin system components are present in neuronal cells in the brain. Most recently, some data have been presented suggesting that some of the renin-angiotensin system components may also occur in glial cells, but this finding needs further confirmation.
UNGER,
MD,
DETLEV
GANTEN,
Local tissue renin-angiotensin systems have recently been discovered in various organs, and evidence is accumulating that inhibition of these local reninangiotensin systems may contribute to the actions of converting enzyme (CE) inhibitors. Measurements of CE activity and angiotensin II concentrations revealed that after oral administration of CE inhibitors, CE was inhibited not only in lung vascular endothelium and blood, but also in the heart, kidney, vascular wall, brain and other organs. The functional significance of tissue CE inhibition is suggested first by the antihypertensive effect of brain CE inhibition in spontaneously hypertensive rats,
A
lthough converting enzyme (CE) inhibitors have become established antihypertensive drugs and are currently under investigation for therapeutic use in such cardiac diseases as congestive heart failure and arrhythmias, their mechanisms of action are still not fully understood. Originally, a reduction of the vasoconstrictor peptide angiotensin II circulating in the blood, and possibly an accumulation of the vasodilator peptide bradykinin, were believed to be solely responsible for the actions of these drugs. However, doubts about this concept were raised early, based on various experimental and clinical findings.’ For instance, it was shown that blood pressure could be reduced in forms of hypertension that were not associated with a stimulated plasma renin-angiotensin system.2r3Further, the inhibition of the plasma renin-angiotensin system could often not be directly correlated with the antihypertensive effects of these drugs.3-5 Moreover, From the German Institute for High Blood Pressure Research and the Department of Pharmacology, University of Heidelberg, Heidelberg, Federal Republic of Germany. Address for reprints: Thomas Unger, MD, Department of Pharmacology, Im Neuenheimer Feld 366, 6900 Heidelberg, Federal Republic of Germany.
MD,
PhD,
and
RUDOLF
E. LANG,
MD
second by the concomitant persistence of blood pressure decrease and CE inhibition in vascular wall and kidney after long-term oral CE inhibitor treatment and third by ex vivo experiments demonstrating marked effects of oral CE inhibitor pretreatment on cardiac function in isolated rat hearts. Local inhibition of tissue renin-angiotensin systems may be an important factor involved in the beneficial effects of CE inhibitors in such cardiovascular diseases as arterial hypertension, congestive heart failure and cardiac arrhythmias. (Am J Cardiol 1987;59:18D-22D)
an increase in circulating endogenous bradykinin concentrations after CE inhibitor treatment has never been convincingly demonstrated. Recently, several laboratories have provided stringent evidence that the components of the renin-angiotensin system are generated locally in several organs involved in cardiovascular regulation, such as the kidney, heart, adrenal gland, vascular wall and central nervous system.6s7These findings have revolutionized our understanding of the renin-angiotensin system: The renin-angiotensin system can no longer be considered a hormonal system in the classical sense, because angiotensin II, the effector peptide of the system, can act asa circulating hormone as well as a locally generated modulator of organ function at the tissue level. In addition to the plasma renin-angiotensin system, local renin-angiotensin systems may therefore contribute to cardiovascular control. Concerning the actions of CE inhibitors, this new understanding of the renin-angiotensin system has given rise to the hypothesis that CE inhibitors exert their beneficial cardiovascular actions not only by reducing the activity of the plasma reninangiotensin system, but also by inhibiting tissue reninangiotensin systems in various target organs of cardiovascular control, such as brain, kidney, vascular wall
April 24, 1987
and hearts-l0 We report on some experimental findings in support of this hypothesis.
Inhibition of ConvertingEnzymein Tissue After Oral ConvertingEnzymeInhibitor Treatment Indirect evidence for tissue CE inhibition after CE inhibitor treatment was presented first by Asaad and Antonaccioll and by our laboratory? Long-term oral antihypertensive treatment of spontaneously hypertensive rats (SHRs and stroke-prone SHRs) with the CE inhibitor captopril increased renin concentrations in the vascular wall of the aorta, and this increase could be dissociated from the increase of plasma renin of renal origin. In analogy to the plasma renin-angiotensin system, where an increase in renin concentration is a consistent feature of CE inhibition (due to withdrawal of an angiotensin II-mediated negative feedback on renin), the increase of aortic renin was interpreted as being caused by a decrease in local angiotensin II generation in the vascular wall. Direct evidence for an inhibition of CE in various tissues after oral treatment with the CE inhibitors was subsequently provided by Cohen and Kurz,13 who showed that in SHRs a single oral dose of captopril or enalapril was able to reduce CE activity not only in blood serum and in lung, but also in aortic wall, heart, kidney and brain. In contrast to the blood serum, the reduction of CE activity in lung, aorta and kidney persisted for several days. A prolonged inhibition of CE activity in the aortic wall but not in the serum after repeated intraperitoneal administration of captopril was also observed by Velletri and Bean.14 In addition, these investigators demonstrated that CE was not confined to the endothelium, but was also localized in the tunica media of the vascular wall. These observations from acute studies were extended in our laboratory (Fig. 1). In strokeprone SHRs long-term oral antihypertensive treatment for several weeks with the CE inhibitors enalapril, ramipril and perindopril inhibited CE in most tissues investigated, including the aortic and mesenteric vascular wall, kidney, heart and brain.“-I7
THE AMERICAN
JOURNAL
OF CARDIOLOGY
Volume 59
19D
TABLE I Effect of a Single Oral Dose (10 mg/kg) of Ramiprii on Angiotensin ii Concentrations in Various Tissues of Nephrectomized Rabbits Control Hypothalamus Medulla oblongata Heart left ventricle left atrium right ventricle right atrium Aorta Adrenal gland
177.2 f 22.7 70.7 f 8.6 50.7 104.2 79.9 106.6 23.5 170.6
f f f f f f
7.5 7.6 7.3 23.1 1.4 13.0
Ramipril 959.8 f 112.2” 34.7 f 3.3‘ 57.5 63.2 68.9 47.4 15.8 98.0
f 9.9 41 10.8’ f 9.6 f 5.7’ f 0.6’ f 14.6’
Data are fmollg tissue and are mean f standard error of the mean. * p <0.05.
system on the results, Twenty hours later, ramipril was given orally at a dose of 10 mg/kg to 30 animals. This dose had previously been shown to normalize blood pressure in stroke-prone SHRs.15J6Four hours after treatment, all animals were killed, and tissue angiotensin II concentrations were measured in various organs including heart, aorta, adrenal gland and brain. The CE inhibitor-induced changes in tissue angiotensin II concentrations are shown in Table I. With the exception of the heart ventricles and the hypothalamus, tissue angiotensin II was significantly lowered by oral ramipril treatment. Because the animals had been nephrectomized before treatment, these changes occurred independently of the plasma renin-angiotensin system. The fact that CE inhibitor treatment did not change angiotensin II levels in the heart ventricles and even increased angiotensin II in the hypothalamus, while reducing angiotensin II in the other organs, suggested a tissue-specific regulation of angiotensin II.
&WEEKS ORAL TREATMENT
SHR
Reductionof AngiotensinII in TissueAfter Oral ConvertingEnzymeInhibitor Treatment Reduced tissue CE activity after systemic CE inhibitor treatment suggested an inhibition of angiotensin II generation in tissue. However, alternative pathways for a local angiotensin II biosynthesis independent of CE18Jgcould not be excluded at this point. To demonstrate that CE inhibitor treatment really reduced tissue angiotensin II concentrations, direct measurements of the peptide were thought to be necessary. With the help of a recently developed high performance liquid chromatography-controlled radioimmunoassay for angiotensin peptides in tissue,z0we were able to show a reduction of tissue angiotensin II concentrations in several organs after oral pretreatment with the CE inhibitor ramipril.21 We nephrectomized 60 rabbits bilaterally to exclude an influence of the plasma renin-angiotensin
IS
9490-3
aMK
01
q lHoe L98
1 10 rnglkgld
FIGURE 1. Percent of inhibition of converting enzyme in various tissues after long-term oral treatment of spontaneously hypertensive rats (SHR) with the converting enzyme inhibitors perindoprii (S 9490-3), ramiprii (HOE 498) and enaiaprii (MK 421). For details see references 16 and 17.
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Implications of Converting EnzymeInhibition in Tissue After Oral Converting EnzymeInhibitor Treatment The data just reported demonstrate that oral treatment with CE inhibitors inhibited tissue CE and reduced angiotensin II levels in important target organs for cardiovascular regulation. Thus, they provided a solid foundation for the hypothesis that tissue CE inhibition may contribute to the cardiovascular actions of CE inhibitors. However, it remained to be established if the observed tissue renin-angiotensin system inhibition was only a phenomenon associated with CE inhibitor treatment, or if tissue CE inhibition was causally linked to the actions of these drugs. Attempts to address this question met with a number of experimental difficulties. First, systemic CE inhibitor treatment invariably antagonizes the plasma renin-angiotensin system together with tissue renin angiotensin systems. It is well known that the activity of the plasma renin-angiotensin system and its pathophysiologic importance vary greatly with the disease and the conditions studied, e.g., high renin or low renin hypertension, high or low salt intake, congestive heart failure or ischemic heart disease. Presumably, the relative contribution of tissue renin-angiotensin systems to cardiovascular dysfunction may vary as well, and, in addition, may depend on the degree of stimulation of the plasma renin-angiotensin system. Therefore, it has been extremely difficult to design in vivo studies on the action of CE inhibitors, in which their effects on the tissue renin-angiotensin system could be clearly separated from those on the plasma renin-angiotensin system. Second, the importance of tissue renin-angiotensin system inhibition in various target organs, such as kidney, vascular wall, heart or brain, may be different depending on the relative contribution of the organ to cardiovascular control under the conditions studied. For instance, in stroke-prone SHRs, brain angiotensin appears to contribute to the maintenance of hypertension6 whereas this may not be the case in other types of hypertension. Third, local and systemic effects of CE inhibitors other than angiotensin II reduction complicate the issue. These effects may be related to CE inhibition (e.g., bradykinin accumulation) or may not. Despite these difficulties in analyzing the functional role of tissue renin-angiotensin systems and separating tissue CE inhibition from other effects of CE inhibitors, a number of experimental findings suggest that tissue CE inhibition indeed constitutes an important factor within the cardiovascular actions of CE inhibitors. The examples given next include the actions of CE inhibitors on tissue renin-angiotensin system in brain, kidney, vascular wall and heart. Blood pressure-lowering effects of brain converting enzyme inhibition in spontaneously hypertensive rats: Several investigators have demonstrated that in SHRs administration of CE inhibitors captopril, enalapril and ramipril into the brain ventricles lowered blood pressure to a greater extent than the intravenous administration of the same doses.g-22-24 In addition, the
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antihypertensive effect of a low dose of captopril (15 pg/kg) injected into brain ventricles was associated with a marked inhibition of CE in the brain, but not in the peripheryeg CE inhibitor-induced accumulation of bradykinin in the brain could be ruled out as the cause for the blood pressure decrease, because bradykinin, in contrast to its peripheral vasodilatory effects, causes blood pressure increases when acting on receptors in the central nervous system.25Therefore, it was concluded from these experiments that inhibition of the stimulated brain renin-angiotensin system in SHRs lowered blood pressure independently of the plasma renin-angiotensin system. Persistent blood pressure reduction and tissue converting enzyme inhibition after long-term oral converting enzyme inhibitor treatment: Stroke-prone SHRs were treated orally for several weeks with the CE inhibitors enalapril(30 mg/kg/day) and ramipril(3 mg/kg/day). Blood pressure was normalized and plasma and tissue CE in various organs was inhibited during treatment. Upon withdrawal of the drugs, the reduced pressor responses to intravenous angiotensin I and CE activity in the blood plasma were restored to normal within 1 day, while blood pressure remained decreased for an additional 2 weeks, and the CE inhibition persisted in the kidney and the aortic and mesenteric vascular wa11.15J6These findings demonstrated that the prolonged antihypertensive action of the CE inhibitors was unrelated to CE inhibition in the plasma and lung vascular endothelium, but was associated with a persistent tissue CE inhibition in the kidney and the vascular wall. Effects of converting enzyme inhibition in the isolated heart: Rats were treated orally with the CE inhibitors enalapril (30 mg/kg) or ramipril (1 mg/kg]. One hour later the animals were killed, and the hearts were isolated and perfused according to the technique of Langendorff. The activity of CE in these hearts was inhibited for up to 24 hours. The effects of angiotensin I given to the perfusate [decrease in coronary flow, increase in force of contraction) were reduced and those of bradykinin (increase in coronary flow, decrease in force of contraction] were potentiated. In addition, the effects of an electrical stimulation of cardiac sympathetic nerves on coronary flow and heart rate were attenuated.27 Further studies using a similar isolated heart technique showed that captopril and ramiprilat given in the perfusate drastically reduced the reperfusion-induced ventricular fibrillation after ischemia.28~2g Oral pretreatment with ramipril 1 hour before sacrifice had the same effect.2g Thus, in the absence of the plasma renin-angiotensin system, inhibition of CE in the heart reduced the local renin-angiotensin system activity including the angiotensin II-mediated facilitation of neurotransmission, and, in addition, exerted a beneficial action on postischemic arrhythmias. These 3 examples may suffice to demonstrate that the inhibition of tissue CE in target organs can produce considerable cardiovascular actions independently of the plasma renin-angiotensin system. Whether the observed effects of local CE inhibition are entirely due to
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a reduction of local angiotensin II generation, or whether interference with other peptide systems plays an additional role, cannot be answered as yet. Although much remains to be learned about the functional role of local renin-angiotensin system in various organs, the data we have reported strengthen the hypothesis that tissue CE inhibition contributes to the beneficial actions of CE inhibitors in cardiovascular disease.
References 1. Unger Th, Canten D, Lang RE. Pharmacology of converting enzyme inhibitors: new aspects. Clin Exp Hypertens 1983;A5:1333-1354, 2. Gavras H, Brunner HR. Turini GA, Kershaw GR, Tifft CP, Cutteelod S. Gavras I. Vukowich RA, McKinstry DN. Antihypertensive effect of the oral angiotensin-converting enzyme inhibitor SQl4.225 in man. N Engl J Med 1978:298:991-995. 3. Antonaccio MJ, Rubin V. Horovitz ZP. Effects of captopril in animal models of hypertension. Clin Exp Hypertens 1980;2:613-637. 4. Brunner HR. Gavras H, Waeber B, Kershaw GR, Turini GA, Vulkovich RA, McKinstry DN. Oral angiotensin-converting enzyme inhibitor in long-term treatment of hypertensive patients. Ann Intern Med 1979;90:19-23. 5. Bravo EL, Tarazi RC. Converting enzyme inhibition with an orally active compound in hypertensive man. Hypertension 1979;1:39-46. 6. Ganten D, Lang RE, Lehmann E, Unger Th. Brain angiotensin: on the way to becoming a well-studied neuropeptide system. Biochem Pharmacol 1984;
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converting enzyme. I Cardiovasc Pharmacol 1982;4:315-325. 15. Unger Th. Fleck Th, Ganten D. Long RE, Rettig R. 2-(N-(s)-l-EthoxycarbonyI-3-phenylpropyl-L-alonyl)-(~S,3S.5S]-2-azabicyclo (3.3.8)octane-3-carboxylic acid [HOE 498): antihypertensive action and persistent inhibition of tissue converting enzyme activity in spontaneously hypertensive rats. Arzneimittelforschung 1984;34:1426-1430, 16. Unger Th, Ganten D, Lang RE, Schijlkens BA. Is tissue convering enzyme inhibition a determinant of the antihypertensive efficacy of converting enzyme inhibitors? Studies with the two different compounds, HOE 498 and MK 421, in spontaneously hypertensive rats. J Cardiovasc PharmacoJ1984;6: 872-880.
17. Unger Th, Moursi M. Ganten D, Hermann K, Lang RE. Antihypertensive action of the converting enzyme inhibitor perindopril(S9490-31 in spontaneously hypertensive rats: Comparison with enalapril (MK 421) and ramipril (HOE 498). J Cardiovasc Pharmacol 1986;8:276-285. 18. Okunishi H, Miyazaki M. Toda N. Evidence for a putatively new angiotensin II-generating enzyme in the vascular wall. J Hypertens 1984;2:277-284. 19. Lanzillo JJ,Dasarathy Y, Stevens J.Fanburg BL. Conversion of angiotensin I to angiotensin II by a latent endothelial cell peptidyl dipeptidase that is not angiotensin-converting enzyme. Biochem Biophys Res Commun 1986J34.2: 770-776.
20. Hermann K, Lang RE. Unger Th, Bayer C, Ganten D. Combined highperformance liquid chromatography-radioimmunoassay for the characterization and quantitative measurement of neuropeptides. J Chromatogr 1984; 312:273-284.
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21. Ganten D, Balz W. Hense H. Jung W, Rohde A, Bayer C. Angiotensin (ANGJ peptides in tissue of rabbits: characterization and regulation after nephrectomy and converting enzyme inhibitor (CEI] treatment (abstr]. Naunyn Schmiedebergs Arch Pharmacol 1985;329:R63. 22. Hutchinson JS, Mendelsohn FAO, Doyle AE. Blood pressure responses normotensive and spontaneously hypertensive rats to intracerebroventricular and peripheral administration of captopril. Hypertension 1980;2:546-550. 23. Stamler JF, Brody MJ. Phillips MI. The central and peripheral effects of captopril [SQ14225)on the arterial pressure of the spontaneously hypertensive rat. Brain Res 1980;186:499-503. 24. Phillips MI, Kimura B. Converting enzyme inhibitors and brain angiotensin. J Cardiovasc Pharmacol 1986;8:suppl 10:582-590. 25. Unger Th, Rockhold RW, Yukimura T, Rettig R, Rascher W, Ganten D. Role of kinins and substance P in blood pressure regulation of normotensive and spontaneously hypertensive rats. In: Buckley JP, Ferrario CM, eds. Central Nervous System Mechanisms in Hypertension. New York: Raven Press,
10. Velletri P, Bean BL. The effects of captopril on rat aortic angiotensinconverting enzyme. J Cardiovasc Pharmacol 1982;4:315-325. 11. Asaad MM, Antonaccio MJ. Vascular wall renin in spontaneously hypertensive rats. Potential relevance to hypertension. Maintenance and antihypertensive effect of captopril. Hypertension 1982;4:487-493. 12. Unger Th, Hiibner D, Schiill 8. Lang RE, Rascher W, Rettig R, Ganten D. Effect of chronic oral coptopril treatment on tissue renin concentration and converting enzyme activity in stroke-prone spontaneously hypertensive rats. In: Rascher W, Clough D, Ganten D, eds. Hypertensive Mechanisms, Stuttgart: Schattauer Verlag, 1982:768-773. 13. Cohen ML, Kurz K. Angiotensin converting enzyme inhibition in tissue from spontaneously hypertensive rats after treatment with captopril or MK 421. J Pharmacol Exp Ther 1982;220:63-69. 14. Velletri P. Bean 8. The effects of captopril on rat aortic angiotensin-
26. Unger Th, Ganten D. Lang RE, Schiilkens BA. Persistent tissue converting enzyme inhibition following chronic treatment with HOE 498 and MK 421 in spontaneously hypertensive rats. J Cardiovasc Pharmacol 1985;7:36-41. 27. Xiang K, Linz W. Becker H, Ganten D. Lang RE. Schalkens BA, Unger Th. Effects of converting enzyme inhibitors: ramipril and enalapril on peptide action and sympathetic neurotransmission in the isolated heart. Eur J Pharmacol 1985;113:215-223. 28. van Gilst WH, de Graeff AP, Kingma JH, Wesseling H. de Langen CDJ. Captopril reduces purine loss and reperfusion arrhythmias in the rat heart after coronary artery occlusion. Eur J Pharmacol 1984;100:113-117. 29. Linz P. Schiilkens BA, Yi-Fan H. Beneficial effects of converting enzyme inhibitors in ischemic rat heart. J Cardiovasc Pharmacol 1986:8:suppll&591599.
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7. Campbell DJ. The site of angiotensin production. J Hypertens 1985;3: 199-207. 8. Antonaccio MJ. Kerwin L. Pre and post-junctional inhibition of vascular 1981;3:1:1-54-Isympathetic function by captopril in SHR. Hypertension fi2.
9. Unger Th. Kaufmann-Biihler I. Schijlkens B. Ganten D. Brain converting enzyme inhibition: a possible mechanism for the antihypertensive action of captopril in spontaneously hypertensive rats. Eur J Pharmacol 1981a;70:
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Discussion Dr. Hansson (Giiteborg, Sweden): I thought your data were very interesting and I do not dispute your explanations at the end. I just want to remind you that a number of other drugs that are not considered to lower blood pressure, mainly through effects on the renin-angiotensin system, will also show this rather slow return of blood pressure. Beta blockers do the same thing and so do diuretics. Dr. Unger (Heidelberg, Germany): I agree with you and this is why I tried to throw in a word of caution. Our observation does not really prove the hypothesis, but I think the hypothesis that tissue inhibition is important is worth pursuing further.
Dr. Amery (Leuven, Belgium): You show that angiotensin I did not change, which is quite interesting. Did you look at all the factors in the system, all the renins and local renins? Dr. Unger: Yes, we did in previous studies and found that renin was increased in various organs after converting enzyme inhibitor treatment. Dr. Brunner (Lausanne, Switzerland): I notice that after ramipril you have a decrease in angiotensin II, but the level is still very high. I am a little bit concerned about this. The second question is whether you measured angiotensin II in the vascular wall after 7 days of converting enzyme inhibi-
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tion? Measurement of converting enzyme activity is very nice, but if you really wanted to make a point, I think you have to be sure that angiotensin II is still reduced. Dr. Unger: You would like to see a greater reduction? Dr. Brunner: If you block the system, it should be low. We have not measured angiotensin II in tissue, but at least in plasma when we use our method combining high performance liquid chromatography with radioimmunoassay, we find almost no angiotensin II in the plasma. Dr. Unger: This is in agreement with our findings on plasma angiotensin II when we use extraction and high performance liquid chromatography. Dr. Brunner: Levels were still higher than 50. I do not know what the units were-pg/ml or fmol/ml? Dr. Unger: They were in fmol/ml. You are referring to the organs, because I did not show plasma angiotensin II values. Yes, about 50% of angiotensin II remains in tissue, but if you consider that local renin-angiotensin systems in various organs are inhibited to a certain degree and each may contribute to some extent to the general inhibition of the system, I wonder whether one has to really postulate that the system has to be blocked 100% everywhere. In your own published data the inhibition or the reduction of angiotensin II after an oral dose of enalapril was also limited in time and did not correspond to the time of blood pressure reduction.
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Did you not have normalization of angiotensin II plasma levels 24 hours after the drug while blood pressure was still reduced? Dr. Brunner: No, angiotensin II has not gone up to normal levels. We know we still have a methodologic problem, because we get in vitro angiotensin II generation and if angiotensin I is high, we still get angiotensin II due to in vitro conversion and the high performance liquid chromatography will, of course, not avoid this. We are now developing methods to avoid in vitro conversion. I think there remain problems with the measurement of angiotensin II. This is why I am worried about your whole construction, because I think we have to be very sure about the methodology used before we can make any statements. Dr. Ball (Glasgow, Scotland): The presence of a brain renin-angiotensin system is obviously of great interest. Do you know in which cell type the renin-angiotensin system is actually present in the brain? Dr. Unger: Well, published data point to neuronal cells and it has indeed been shown that the renin-angiotensin system components are present in neuronal cells in the brain. Most recently, some data have been presented suggesting that some of the renin-angiotensin system components may also occur in glial cells, but this finding needs further confirmation.