COMBINED CLINICAL AND BASIC SCIENCE SEMINAR Selected and edited by Richard T. Silver, M.D. and Alexander G. Bearn. M.D. Department of Medicine, The New York Hospital-Cornell Medical Center, New York, New York
Converting Enzyme, Angiotensin II and Hypertensive Disease
h4cderator: JOHN H. LARAGH. MD lecturers: RICHARD L. SOFFER. M.D.’ DAVID B. CASE, M.D. New York, New York
In today’s seminar we discuss some recent and highly complementary developments from both basic and clinical research which could have a tremendous impact on our understanding of and our ability to treat high blood pressure. First, Dr. Richard Soffer, who,has just recently joined our faculty at Cornell, will tell you about his research on the biochemistry and physiology of the converting enzyme. In a series of studies, Dr. Soffer and his group have prepared a highly purified form of this enzyme, identified the presence of zinc in its active center, prepared antibodies to the purified enzyme and then shown by tagging their antibody that the enzyme is strategically located throughout the vascular tree to play a key role in circulatory homeostasis. At the same time, Dr. David Case and his colleagues have been systematically evaluating the physiologic effects of blocking this converting enzyme in hypertensive patients by administering a nonapeptide inhibitor. This blockade prevents the formation of angiotensin II, normally generated by the activity of renin. Case’s studies suggest broad participation of angiotensio II in human essential hypertension since the blocking drug lowered the blood pressure in some 70 per cent of these patients and reduced it to normal in some 30 per cent. Thus, contrary to a popular view, renin excess does actively participate in the blood pressure support of most patients with common hypertension. Moreover, Case’s study shows that plasma renin activity measurements really do have specific physiologic meaning because the degree of the drop in blood pressure induced by the converting enzyme blockade was closely related to the control renin measurement. I think you will agree that these two lines of research are exciting and provocative, and that they are bringing us closer to exposing the primary causes of high blood pressure. HISTORIC ASPECTS OF THE RENIN-ANGIOTENSIN SYSTEM
From the Departments of Medicine and Biochemistry and the Cardiovascular Center, The New York HospitalCornell Medical Center, New York, New York. Work carried ouf in Dr. Soffer’s Laboratory was supported by Grant HL-17741 from the National Heart and Lung Institute. Requests for reprints should be &dressed to Dr. John H. Lara&, Tha New York Hospftaf-CornallMedical Center, 525 East 69th Street, New York, New York 10021. Manuscript accepted May 5, 1977. ’ Faculty Research Associate of the American Cancer Society.
Dr. Richard L. Soffer: Current understanding of the renin-angiotensin system is schematically summarized in Figure 1. A detailed review of the subject has appeared recently [l] and I will, therefore, briefly describe only selected points of historic interest. In 1898, Tigerstedt and Bergmann [2] found that the intravenous administration of extracts from kidney was associated with transient hypertension; they called the responsible agent “renin.” Their observation provided the first evidence that the kidney might play a direct role in the regulation of blood pressure, but it was largely disregarded until 1934 when Goldblatt et al. [3] reported that renal ischemia could cause permanent hypertension in dogs. Several years later, the concept was developed [4,5] that renin was an enzyme secreted into the bloodstream by the ischemic kidney where it acted on a plasma
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COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
Asp-Arq-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Va1-Tyr-Ser-R Anqiotensinogen
(Renin
Substrate)
Renin
$ Asp-Arq-Val-Tyr-
.Ile-His-Pro-Phe-His-Leu Anqiotensin
+ Leu-Val-Tyr-Ser-R
I
I I Convertinq
I
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
Enzyme
+ His-Leu
Anqiotensin
I
II
Receptor
i (Angiotensin
Effector
Figure 1.
January 1978
Response
The renin-angiotekin
protein substrate (angiotensinogen) to cleave off a vasopressor peptide (angiotensin). In 1954, Skeggs et al. [6] used partially purified preparations of porcine renin and equine angiotensinogen to generate this vasopressor peptide; they discovered that it could occur in two forms. Subsequently they established [ 71 that this was due to an enzymatic contaminant of their angiotensinogen preparation which catalyzed release of the dipeptide His-Leu from the COOH-terminus of angiotensin I to yield the octapeptide, angiotensin II. Angiotensin-converting enzyme was thus first detected as a component of horse plasma. Both angiotensins were found to be vasopressor when given intravenously; however, angiotensin II was implicated as the biologically active component of the renin-angiotensin system because only it increased the perfusion pressure of isolated rat kidneys [8] and caused contraction of aortic strips in vitro [9]. Until 1967, the pressor effect of angiotensin I was assumed to result from its conversion to angiotensin II catalyzed by the plasma enzyme which Skeggs et al. [6] had discovered. At that time, Ng and Vane [ 10,l l] found that this plasma activity was insufficient to account for the rapidity of conversion in vivo as manifested
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system.
by the vasopressor response to the decapeptide. They also found that angiotensin I was more potent when administered intravenously than intra-arterially and that it was almost quantitatively converted to angiotensin II during a single passage through the pulmonary circulation. Their results provided evidence that physiologic conversion is mediated by a tissue enzyme and that the lung plays a major role in this process. The rapidity of conversion in the pulmonary vascular bed suggested that the responsible enzyme might be located in close approximation to the circulating blood. In 1968, Bakhle [ 121 discovered that the conversion of angiotensin I catalyzed by canine pulmonary particles was inhibited by bradykinin-potentiating factor. The latter was a mixture of peptides from the venom of a South American pit viper, Bothrops jararaca, which Ferreira [ 131 had previously found to augment the action of bradykinin (Figure 2) by inhibiting its degradation. Bakhle’s observation was a seminal one. It provided the first clue that the same enzyme might catalyze conversion of angiotensin I as well as inactivation of bradykinin and it suggested that one or more of the components in bradykinin-potentiating factor might be a useful agent for selectively delineating the role of the
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CONVERTING ENZYME, ANGIOTENSIN II AND HYPERTENSIVEDISEASE
renin-angiotensin system. It has recently been established with pure preparations from rabbit [ 141 and hog [ 151 lung that a single enzyme can sequentially cleave Phe-Arg and Ser-Pro from the COOH-terminus of bradykinin and His-Leu from that of anigotensin I. Since bradykinin is a powerful vasodepressor agent, the action of converting enzyme on both these substrates may be regarded as physiologically vasopressor. Bakhle’s report stimulated great interest in the properties of the venom peptides. The structures of several were subsequently determined [ 16,171, and they were synthesized chemically [ 17,181. The pure compounds were found to inhibit the conversion of angiotensin I and the inactivation of bradykinin in vitro [ 191. A pentapeptide (Figure 2) was first used for studies in vivo [ 181 and shown to inhibit the vasopressor action of angiotensin I, potentiate the vasodepressor effect of bradykinin and to ameliorate experimental nephrogenic hypertension in an animal model [ 201. A nonapeptide (Figure 2) was found to exhibit comparable pharmacologic properties and to possess a longer duration of action [ 21-231. It has been established that this compound inhibits the conversion of angiotensin I in man [ 241. Dr. Case will describe its use as a probe for defining the contribution of the renin-angiotensin system to various forms of hypertension in man. Dr. Case will also discuss studies in which saralasin (Figure 2) has been used to delineate the role of angiotensin II in hypertension. Although this agent is an inhibitor of converting enzyme [ 251, it is thought to act pharmacologically as an antagonist to angiotensin II by competing with it for a binding site on a membranebound receptor [ 261. This receptor is the most recently recognized macromolecular component of the reninangiotensin system [ 271. It has thus far been characterized primarily by its aff inky for angiotensin II and has not yet been purified. Nor is it clear whether the same receptor molecule participates in the vascular response to angiotensin II and the increased elaboration of aldosterone [28] by cells of the adrenal zona glomerulosa which may at least partially be mediated by angiotensin Ill (Figure 2) [ 291. The mechanism whereby a cellular response is generated as a consequence of the interaction between a specific receptor and its angiotensin effector has not been established and represents an important area for future research. REGULATtON OF ANGIOTENSIN-CONVERTtNG ENZYME BY ANTtENtYME ANTIBODY
The biochemical properties of angiotensin-converting enzyme and its relationship to the metabolism of vasoactive peptides has recently been reviewed elsewhere [30]. This discussion will focus upon studies by my colleagues and me which are concerned with characterization of the enzyme in different rabbit organs
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Bradykinin
Pyr-Lys-Trp-Ala-Pro Bradykinin-Potentiating
Pentapeptide
Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro Bradykinin-Potentiating
Nonapeptide
Sar-Arg-Val-Tyr-Ile-His-Pro-Ala Saralasin
Arg-Val-Tyr-Ile-His-Pro-Phe Angiotensin
III
Figure 2. Structures of additional peptides discussed in this presentation.
and of antienzyme antibody developed in goats. Our interest in the enzyme was stimulated in late 1971 by which time evidence had accumulated that it was localized in apposition to the circulation and represented an important therapeutic target in nephrogenic hypertension. We reasoned that, under these circumstances, anticatalytic antibody introduced into the circulation would not be required to cross cellular permeability barriers in order to reach its site of action and that,
TABLE I
Properties of Rabbit Pulmonary Angiotensln-Converting Enzyme’
Molecularweight, native enzyme -
129,000 Molecular weight, reduced denatured enzyme - 129.000 NH2 - terminal residue = threonine COOKterminal residue = alanine Native heavy metal = zinc, one molar equivalent Amino acids - 837 residues/mol Tryptophan = 23 Slight hydrophobicity Carbohydrate - 176 residues/mole-26 Fucose = 4 Mannose = 43 Galactose = 57 N-Acetylglucosamine = 53 N-Acetylneuraminic acid = 19 * Data are from Das and Soffer
January 1978
The American
per cent by weight
[3 11.
Journal of Medlclne
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COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
I
I I
1
Seminal Z plasma I )
Serum ‘; . c.
DILUTED
L .. ... * _. Effect of antilung enzyme anrroooy on converting
ANTISERA . .. .
Pure enzyme
(pl 1 . , ..
,
Figure 3. enzyme acrrvlry In raoolt organ exrracts ana body fluids. From Das and Soffer (321. Residual activity was determined after incubating 3 munits of enzyme for 30 minutes with the indicated quantities
of antiserums.
therefore, it might exert its inhibitory effect in vivo. If this were the case it seemed to us that such antibody could be a useful biologically specific reagent for immunologically excising the renin-dependent component of blood pressure regulation. In order to examine these possibilities, a procedure was developed for isolating pure converting enzyme after solubilization from rabbit pulmonary membranes using a nonionic detergent [ 14,311. A purification factor of l,OOO-fold was required to obtain a homogeneous preparation from crude lung extracts indicating that the enzyme comprises about 0.1 per cent of total pulmonary protein. Some physiochemical characteristics of the enzyme are described in Table I. Such information on plasma membrane enzymes is relatively scarce since few have been obtained in pure water-soluble form. Therefore, converting enzyme may represent a useful tool for studying membrane-enzyme interaction and for investigating the general question of whether participation in an organized subcellular structure influences the catalytic properties of an enzyme. It contains a single large glycopolypeptide chain and a molar equivalent of tightly bound zinc which is required for catalytic activity. It is a moderately hydrophobic protein with a large number of tryptophanyl residues accounting for its high absorbance at 280 nm. Its paradoxically early elution during gel filtration through Sephadexo G-200 provided the first clue to its glycoprotein- nature [14,31].
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Antibodies against the rabbit glycoprotein were raised in goats and were found to be highly inhibitory in vitro [32]. Antiholoenzyme antibody was prepared by affinity chromatography using as ligand the pure enzyme fixed to an insoluble matrix. It was established that each antibody molecule could bind 2 molecules of enzyme and that at least 18 her cent of the antiholoenzyme antibody population recognized determinants which influence catalysis. Enzymatic hydrolysis of the physiologic substrates, angiotensin I and bradykinin, and of the model synthetic substrate, hippurylhistidyl-leucine (Hip-His-Leu), was inhibited similarly by antibody. Furthermore, inhibition dose-response curves obtained with the antilung enzyme antibody and a fixed amount of enzyme activity were identical regardless of whether the source of activity was the pure lung enzyme or crude fractions from various organs and body fluids (Figure 3). This result indicated that antibody prepared against the pulmonary glycoprotein could be expected to.inhibit angiotensin conversion throughout the body, provided it had access to the enzyme after introduction into the circulation. It also suggested that enzyme molecules in different organs possessed extensive homology with respect to those of their determinants which influence catalysis. We were interested in determining whether this immunologic homology also comprised those portions of the enzyme molecules which were unrelated to its catalytic action. To address this question [32] we
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CONVERTING ENZYME, ANGIOTENSIN II AND HYPERTENSIVE DISEASE
employed a competition radioimmune assay since this procedure encompasses determinants of the entire antigenic molecule. The assay measured displacement of radioiodinated pulmonary enzyme from an immune complex with antilung enzyme antibody by competing antigen in various crude fractions. Unlabelled pure lung enzyme was used as the reference antigen. Our rationale was that if enzyme molecules in a given extract were identical to the lung glycoprotein then the ratio of catalytic activity to competing antigen in that extract would correspond to the specific activity of the pure lung enzyme, i.e., 90 to 100 U/mg protein. It was established by this criterion that activities in kidney, brain and serum could be ascribed to an enzyme which was immunologically identical to that in the lung (Table II). Of special interest, however, was the finding that enzyme in seminal plasma was immunologically distinguished by this technic. Since its inhibition by antilung enzyme antibody was found to be identical to that observed with enzyme activities from the other sources, its lack of homology appears to reside in determinants unrelated to catalysis. The seminal plasma activity increases markedly during sexual maturation and may, therefore, play a special role in reproductive physiology ]331. Immunologic identity in the anticatalytic and competition radioimmune assays does not necessarily imply complete chemical identity. This was established by comparing pure preparations of rabbit lung and serum converting enzymes [34]. The latter constitutes a very minor component of crude serum and, in order to achieve the 60,000-fold enrichment required to obtain it in pure form, we exploited its immunologic homology with the pulmonary glycoprotein by employing antilung enzyme antibody as a ligand for affinity chromatography. As expected, most catalytic, structural and immunologic properties of the two purified enzymes were indistinguishable. However, the serum glycoprotein was found to contain three times more sialic acid on a molar basis than the lung enzyme. This was not completely unanticipated since a hepatic lectin is known to extract glycoproteins characterized by terminal nonreducing galactosyl residues from the circulation [35]. Our result suggests that the serum enzyme may be derived by sloughing or secretion of the tissue enzyme and subsequent modification of the microheterogenous glycoprotein population based on the specificity of this hepatic lectin. Under these circumstances, those molecules whose galactosyl residues are protected by sialylation would be expected to be spared and to selectively accumulate in the circulation. In order to assess the possibility that specific antibody might be an effective inhibitor of converting activity in vivo, it was necessary to determine whether most en-
TABLE II
Activity and lmmunoreactiviiy of Convertlng Enzyme from Various Rabbit Tissue Extracts and Fluids Catalytic
Catalytic Tissue Extract or Fluid
Lung nonidet P-40 extract Kidney nonidet P-40 extract Brain nonidet P-40 extract Serum Seminal plasma
COrlNing
Aclivily Anti (mUhng protein) (rg/mg protein)
545 59 4.6 1.8 219
Ulmg
5.49
93
0.617
95
0.049
94
0.0174 0.676
103 324
NOTE: Data are from Das and Soffer [32]. A unit of catalytic activity is the amount required to generate 1 .O ~mol of hippuric acid from
Hip-His-Leu per minute at 37%. Competing antigen was estimated using pure radioiodinated pulmonary enzyme as the displaceable antigen, pure unlabeled enzyme as the reference antigen and goat antibodies directed against the enzyme. The last column is the ratio of catalytic activity to competing antigen.
zyme might be accessible to the circulation. Ryan et al. [36] had used antibody conjugated with microperoxidase to demonstrate that converting enzyme was localized on the luminal surface of pulmonary vascular endothelial cells. We [37] studied the distribution of extrapulmonary enzyme with fluorescein-labelled antilung enzyme antibody and found that it was restricted to vascular endothelial rather than parenchymal cells in most of the rabbit organs which we examined (Figure 4). The kidney provided an exception since both vascular endothelium and proximal tubular cells exhibited fluorescence. However, our results in conjunction with those of Ryan et al. [ 361 suggested that most converting enzyme molecules might be expected to interact with circulating antibodies. Our first experiments [38] on the effect of antienzyme antibody after intravenous administration were conducted with the homologous recipient animal, i.e., the rabbit. Initially, we infused sufficient antibody to completely inhibit the total enzyme content of a pair of rabbit lungs as extrapolated from the linear portion of an antibody inhibition dose-response curve performed with pure enzyme in vitro. This dose of antibody invariably resulted in an almost immediate immunespecific lethal reaction manifested by pulmonary edema. Animals which received smaller quantities of immune globulin survived for a limited period. The vasopressor response to angiotensin I was somewhat diminished and the vasodepressor effect of bradykinin was potentiated in these animals; however, it was not possible to administer sufficient antibody to anticipate major effects on the metabolism of these vasoactive peptides. The mechanism of the lethal response in the
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COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
Figure 4. Staining of rabbit organs by fluorescein-labeled antibody raised against pure rabbit pulmonar From Caldwell et al. [37]. Panels a, c, d, e and f illustrate lung, liver, adrenal cortex, pancreas and kidne b is a control specin rfen showing lung reacted with fluorescein-labeled preimmune globulin.
homologous recipient animal is not clear. It is probably unrelated to the anticatalytic action of the antibody preparations since venom peptides are extremely effective, although transient, inhibitors of the enzyme in vivo and yet are remarkably nontoxic. It is conceivable that the combination of antigen and antibody triggers a destructive immunologic process mediated by complement, which occurs most noticeably in the pulmonary vascular bed, because this is the first and one of the richest antigenic sites encountered by the antibody after intravenous infusion. If this is the case, then it may be possible to dissociate the lethal and anticatalytic actions of antibody by using Fab fragments which contain the antigen binding site of the holoantibody but lack that portion of it which interacts with complement. These fragments have been prepared and characterized as effective inhibitors of the enzyme in vitro, but their action in vivo has not yet been examined. An alternative recipient animal was sought with the aim of circumventing the immune lethal response.
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:on verTing en2yme. .especztivt ‘ly. t‘anet
Homology studies employing the competition radioimmune assay, double immunodiffusion and cross-anticatalytic action indicated that antirabbit enzyme antibody reacted with pulmonary converting enzymes from rat guinea pig and dog [39]. The effect of antibody in vivo was examined with the rat [39], and no immediate deleterious effects were observed as a consequence of its infusion into this heterologous recipient. Figure 5 shows typical data obtained with an animal which received three times the amount of antibody required to completely inhibit the converting activity of a pair of rat lungs as extrapolated from an antibody inhibition dose-response curve performed with crude rat pulmonary enzyme. Administration of antibody was invariably associated with a striking diminution in the vasopressor response to angiotensin I and with a dramatic augmentation of the vasodepressor action of bradykinin. Virtually identical data were generated from 1 hour to at least 48 hours after the animals received immune globulin; the half-life of anticatalytic antibody,
Volume 64
CONVERTING ENZYME, ANGIOTENSIN II AND HYPERTENSIVE DISEASE
3
50
00
500
ANGIOTENSIN I
;;
DO
IW
-z&p-
:s
;E
Antibody
i32 20w” ij
.. . . .
100
(ngramr)
,n/,.
0.05
0.1
i
0.5
1.0
BRADYKININ ( pgrams
_
50
ANGIOTENSINII
Y
ZE w-
&
lo
1000
(ngrams)
.
5.0
1
. .. Figure 5. Effect of anrlraoolt convernng enzyme antltwores on rat blood pressure response to vasoactlve peptides. From Conroy et al. [39]. Dose-response curves were obtained before and 1 hour affer the animal received 180 mg of immune globulin. Each point represents the mean of four determinations
...
COnhOl
..
and is bracketed by the values corresponding
measured in the serum of one of these, was several days. The pressor potency of norepinephrine was not altered during these experiments, and antibody-dependent changes in the metabolism of vasoactive peptides were completely immune-specific. The results with angiotensin I and bradykinin were those to be expected after successful immunologic blockade of converting enzyme. The small but definite immunespecific inhibition of the vasopressor response to angiotensin II was not anticipated. Conceivably, a receptor for angiotensin II may be located in appositon to converting enzyme on the vascular endothelial surface, and antienzyme antibody may block access of angiotensin II to its effector site by steric hindrance. The major points which I have attempted to develop may be summarized as follows. Angiotensin-converting enzyme is a COOH-terminal dipeptidyl peptidase, i.e., it catalyzes cleavage of dipeptide residues from the COOH-terminus of oligopeptide substrates. Its two most important currently recognized substrates are angiotensin I and bradykinin, and its action on each is physiologically vasopressor. The enzyme is a fairly well characterized molecule consisting of a single zinccontaining glycopolypeptide chain and, although it is a
to 1 standard devia-
plasma membrane component, it can be obtained fairly easily in pure water-soluble form. In most organs it is restricted to vascular endothelial cells; its active site is directly accessible to the circulation. The lung plays a major role in the physiologic conversion of angiotensin I because of its large vascular endothelial surface, because it receives the entire blood volume and because it does not extract angiotensin II. However, extrapulmonary angiotensin conversion does occur and the responsible enzyme throughout the body is immunologically similar to that in the lungs. Since angiotensin-converting enzyme catalyzes the generation of angiotensin II, it represents a therapeutic target for pathologic conditions resulting from an overactive renin-angiotensin system. It can be inhibited chemically by various peptides, and it is likely that orally effective nonpeptide inhibitors will be developed in the near future. Converting enzyme is accessible to the action of circulating macromolecules because of its anatomic localization in juxtaposition to the blood and it can be inhibited in vivo by anticatalytic antibody. Although immunologic studies with it are in their early stages, such antibody represents a biologically specific inhibitor which should ultimately complement the chemical
January 1978
The American Journal of Medicine
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COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
I .*
.*
VOLUME
- SODIUM
1 CARDIAC
$
VOLUME-
SODIUM
GAIN w-.
. .
\
\
t ALDOSTERONE ”
LOSS
h
\
OUTPUT
+ + BLOODiRESSURE \ \
#CC
\
\
-
RENIN
RELEASE
1 \
+ ANGIO~ENSIN I I
____)
a
I’ \
1 ElLhD
Y# t VASOCONSTRICTION-
PRCSURE --w_
PERIPHERAL --W_
--
-____----
VASCULAR
RESISTANCE
Figure 6. Basic outline of the physiologic role of renin in maintaining blood pressure. Extracellular sodium loss lowers cardiac output and blood pressure, thereby threatening organ perfusion. However, renin is released in response to these events which in turn leads to angiotensin generation. Angiotensin raises blood pressure level acting as a direct vasoconstrictor. In addition, it stimulates aldosterone so that the kidneys will reabsorb more sodium chloride. These two actions reinforce each other in restoring
the system.
agents in delineating and modifying the contribution of the renin-angiotensin system to normal and pathologic processes. Dr. David B. Case: Dr. Soffer has presented a discussion of the biochemical components of the reninangiotensin system, culminating in a more detailed description of the converting enzyme or kininase II. At this point, I would like to extend this discussion to incorporate this biochemical system into a physiologic framework and into pathophysiologic hypotheses about the mechanisms involved in hypertension. Although there had been major advances in the understanding of the renal and adrenal relationships to salt balance and the regulation of the circulation, it was not until 55 years or so after the initial experiments of Tigerstedt and Bergman in 1898 that aldosterone was discovered [40], and then accurate methods for its measurement became available [41]. This set the stage for the crucial physiologic studies linking renin secretion to the control of aldosterone secretion which thereby linked blood pressure homeostasis to the simultaneous control of electrolyte balance. The breakthrough occurred when it was found that patients with malignant hypertension have massive oversecretion of aldosterone [42], a finding which led to the demonstration in human volunteer subjects of a highly specific action of infused angiotensin II to stimulate aldosterone secretion [43]. These studies exposed the renin-aldosterone hormonal connection and
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pointed out a particular derangement in it that is critically involved in causing malignant hypertension [44]. Subsequently, numerous studies in man and in various animals [45] have verified this renin-angiotensin-aldosterone axis and further characterized its fundamental role in blood pressure and in sodium and potassium homeostasis [46], and in the pathogenesis of particular forms of both experimental and clinical hypertension [47]. The more recent growth of all of this new knowledge has been aided by methodologic improvements and the increasing use of immunoassays instead of the more laborious pressor bioassays. Our present understanding of the renin axis is depicted in Figure 6. When sodium or extracellular volume is lost from the system, cardiac output declines as do the systemic and renal perfusion pressures. This decrease is one of several mechanisms by which the juxtaglomerular apparatus triggers secretion of renin. The release of renin is an early event in the compensatory response to the decrease in pressure. Increased renin levels lead to increased angiotensin II levels which work to restore pressure by producing vasoconstriction of peripheral resistance vessels, largely the arterioles. In addition, increased angiotensin II stimulates aldosterone secretion, leading to a restoration of extracellular volume by increased retrieval of distal tubular fluid sodium. Restoration of arterial pressure, effective volume and cardiac output in turn decrease renin secretion. Therefore, the system exhibits the character-
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CONVERTING ENZYME, ANGIOTENSIN II AND HYPERTENSIVE DISEASE
istic of a negative feedback loop. A series of baroreceptors and sodium chemoreceptors in the kidney and systemic circulation mediate the level of activity of this system and, therefore, provide the capability for quick responsiveness. In addition to this, angiotensin may have direct actions on the central nervous system which modify vascular tone. Altogether, we have a system responsive to subtle changes in extracellular sodium/ volume and perfusion pressure capable of initiation, and perpetuation of a compensatory response both by being able to influence sodium excretion and also by producing sustained vasoconstriction. Since the level of activity of this system should be inversely related to the state of sodium balance, clinical investigators sought ways to determine the normalcy or appropriateness of renin values by making simultaneous estimations of the state of sodium balance. One of the contexts chosen as a measure of sodium balance for renin estimations was the 24-hour urinary sodium excretion. Under steady-state conditions in normal subjects, the quantity of dietary sodium ingested is reflected in the urinary sodium content, excluding situations in which large amounts of insensible salt loss occur. Therefore, it was possible to describe a range of values of plasma renin activity corresponding to the span of values for 24-hour urinary sodium excretion [47] (see Figure 7). The same relationship could be derived from angiotensin II rather than plasma renin activity values, but the former are less accurate for clinical use and have not been widely used. As predicted, values for plasma renin activity were highest during dietary sodium deprivation and lowest when dietary sodium intake was greatest. A similar parallel band was derived for urinary aldosterone excretion in relation to urinary sodium excretion. The establishment of this nomogram relating plasma renin activity to its soduim balance thus provided a background with which to examine the major disorder of blood pressure regulation, hypertension. From careful analysis of several renin measurements during different salt diets on a metabolic ward, it was possible to characterize three different subgroups of hypertensive patients [47]. One group had suppressed renin levels during normal salt intake which rose within the same range as normal subjects during dietary sodium restriction. Since the range was the same as that in normal subjects, this subgroup was termed “normal-renin.” However, it was recognized that it might not be normal or appropriate to have “normal” levels of a pressor hormone in the presence of an increase in blood pressure. A second group had renin levels above the normal band which were most apparently high during periods of normal or high sodium intake, at a time when renin levels are normally suppressed. In this “high-renin” group, excess renin and angiotensin were
PLASMA 0
RENIN ACTIVITY
8
100
200 8
7
7
6 PRA
5
5
ng AI/
ml/hr 4
0
200
100
Urine
sodium
mEq/day
Vgure 7. Nomogram relating plasma renin activity ant the 24-hour urinary sodium excretion rate. The normal range is referenced against a measure of the state of sodium balance in normotensive healthy subjects. During dietary sodium deprivation, plasma renin levels rise.
believed to be playing a predominant role in maintaining the hypertensive state. Corroboration for this thesis came from studies showing the therapeutic benefit of treatments which lowered renin levels in high-renin patients: fi-adrenergic blockade with propranolol [48]; renal artery repair and nephrectomy in renovascular hypertension [49,50]. A third group of patients was identified who had low levels of plasma renin activity which did not rise into the normal range even during conditions of dietary sodium depletion. This “low-renin” subgroup was postulated to have a residual sodium or volume excess even after dietary sodium restriction which continued to suppress renal renin secretion. The sodium or volume factor appeared to be the predominant factor, as opposed to angiotensin, in sustaining the hypertension. Evidence to support this thesis came from studies showing that the administration of diuretics was more successful in lowering blood pressure in low-renin patients than in normal or high-renin patients [ 5 11. In addition, miner-
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155
LOW IO
RENIN
NORMAL RENIN n= IO
n=4
1
Average
HIGH
n=7
Average
RENIN
Average
n %DBP
I, T
15 J
Minutes
+
p
Converting Enzyme, Angiotensin II and Hypertensive Disease
h4cderator: JOHN H. LARAGH. MD lecturers: RICHARD L. SOFFER. M.D.’ DAVID B. CASE, M.D. New York, New York
In today’s seminar we discuss some recent and highly complementary developments from both basic and clinical research which could have a tremendous impact on our understanding of and our ability to treat high blood pressure. First, Dr. Richard Soffer, who,has just recently joined our faculty at Cornell, will tell you about his research on the biochemistry and physiology of the converting enzyme. In a series of studies, Dr. Soffer and his group have prepared a highly purified form of this enzyme, identified the presence of zinc in its active center, prepared antibodies to the purified enzyme and then shown by tagging their antibody that the enzyme is strategically located throughout the vascular tree to play a key role in circulatory homeostasis. At the same time, Dr. David Case and his colleagues have been systematically evaluating the physiologic effects of blocking this converting enzyme in hypertensive patients by administering a nonapeptide inhibitor. This blockade prevents the formation of angiotensin II, normally generated by the activity of renin. Case’s studies suggest broad participation of angiotensio II in human essential hypertension since the blocking drug lowered the blood pressure in some 70 per cent of these patients and reduced it to normal in some 30 per cent. Thus, contrary to a popular view, renin excess does actively participate in the blood pressure support of most patients with common hypertension. Moreover, Case’s study shows that plasma renin activity measurements really do have specific physiologic meaning because the degree of the drop in blood pressure induced by the converting enzyme blockade was closely related to the control renin measurement. I think you will agree that these two lines of research are exciting and provocative, and that they are bringing us closer to exposing the primary causes of high blood pressure. HISTORIC ASPECTS OF THE RENIN-ANGIOTENSIN SYSTEM
From the Departments of Medicine and Biochemistry and the Cardiovascular Center, The New York HospitalCornell Medical Center, New York, New York. Work carried ouf in Dr. Soffer’s Laboratory was supported by Grant HL-17741 from the National Heart and Lung Institute. Requests for reprints should be &dressed to Dr. John H. Lara&, Tha New York Hospftaf-CornallMedical Center, 525 East 69th Street, New York, New York 10021. Manuscript accepted May 5, 1977. ’ Faculty Research Associate of the American Cancer Society.
Dr. Richard L. Soffer: Current understanding of the renin-angiotensin system is schematically summarized in Figure 1. A detailed review of the subject has appeared recently [l] and I will, therefore, briefly describe only selected points of historic interest. In 1898, Tigerstedt and Bergmann [2] found that the intravenous administration of extracts from kidney was associated with transient hypertension; they called the responsible agent “renin.” Their observation provided the first evidence that the kidney might play a direct role in the regulation of blood pressure, but it was largely disregarded until 1934 when Goldblatt et al. [3] reported that renal ischemia could cause permanent hypertension in dogs. Several years later, the concept was developed [4,5] that renin was an enzyme secreted into the bloodstream by the ischemic kidney where it acted on a plasma
January 1978
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COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
Asp-Arq-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Va1-Tyr-Ser-R Anqiotensinogen
(Renin
Substrate)
Renin
$ Asp-Arq-Val-Tyr-
.Ile-His-Pro-Phe-His-Leu Anqiotensin
+ Leu-Val-Tyr-Ser-R
I
I I Convertinq
I
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
Enzyme
+ His-Leu
Anqiotensin
I
II
Receptor
i (Angiotensin
Effector
Figure 1.
January 1978
Response
The renin-angiotekin
protein substrate (angiotensinogen) to cleave off a vasopressor peptide (angiotensin). In 1954, Skeggs et al. [6] used partially purified preparations of porcine renin and equine angiotensinogen to generate this vasopressor peptide; they discovered that it could occur in two forms. Subsequently they established [ 71 that this was due to an enzymatic contaminant of their angiotensinogen preparation which catalyzed release of the dipeptide His-Leu from the COOH-terminus of angiotensin I to yield the octapeptide, angiotensin II. Angiotensin-converting enzyme was thus first detected as a component of horse plasma. Both angiotensins were found to be vasopressor when given intravenously; however, angiotensin II was implicated as the biologically active component of the renin-angiotensin system because only it increased the perfusion pressure of isolated rat kidneys [8] and caused contraction of aortic strips in vitro [9]. Until 1967, the pressor effect of angiotensin I was assumed to result from its conversion to angiotensin II catalyzed by the plasma enzyme which Skeggs et al. [6] had discovered. At that time, Ng and Vane [ 10,l l] found that this plasma activity was insufficient to account for the rapidity of conversion in vivo as manifested
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system.
by the vasopressor response to the decapeptide. They also found that angiotensin I was more potent when administered intravenously than intra-arterially and that it was almost quantitatively converted to angiotensin II during a single passage through the pulmonary circulation. Their results provided evidence that physiologic conversion is mediated by a tissue enzyme and that the lung plays a major role in this process. The rapidity of conversion in the pulmonary vascular bed suggested that the responsible enzyme might be located in close approximation to the circulating blood. In 1968, Bakhle [ 121 discovered that the conversion of angiotensin I catalyzed by canine pulmonary particles was inhibited by bradykinin-potentiating factor. The latter was a mixture of peptides from the venom of a South American pit viper, Bothrops jararaca, which Ferreira [ 131 had previously found to augment the action of bradykinin (Figure 2) by inhibiting its degradation. Bakhle’s observation was a seminal one. It provided the first clue that the same enzyme might catalyze conversion of angiotensin I as well as inactivation of bradykinin and it suggested that one or more of the components in bradykinin-potentiating factor might be a useful agent for selectively delineating the role of the
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CONVERTING ENZYME, ANGIOTENSIN II AND HYPERTENSIVEDISEASE
renin-angiotensin system. It has recently been established with pure preparations from rabbit [ 141 and hog [ 151 lung that a single enzyme can sequentially cleave Phe-Arg and Ser-Pro from the COOH-terminus of bradykinin and His-Leu from that of anigotensin I. Since bradykinin is a powerful vasodepressor agent, the action of converting enzyme on both these substrates may be regarded as physiologically vasopressor. Bakhle’s report stimulated great interest in the properties of the venom peptides. The structures of several were subsequently determined [ 16,171, and they were synthesized chemically [ 17,181. The pure compounds were found to inhibit the conversion of angiotensin I and the inactivation of bradykinin in vitro [ 191. A pentapeptide (Figure 2) was first used for studies in vivo [ 181 and shown to inhibit the vasopressor action of angiotensin I, potentiate the vasodepressor effect of bradykinin and to ameliorate experimental nephrogenic hypertension in an animal model [ 201. A nonapeptide (Figure 2) was found to exhibit comparable pharmacologic properties and to possess a longer duration of action [ 21-231. It has been established that this compound inhibits the conversion of angiotensin I in man [ 241. Dr. Case will describe its use as a probe for defining the contribution of the renin-angiotensin system to various forms of hypertension in man. Dr. Case will also discuss studies in which saralasin (Figure 2) has been used to delineate the role of angiotensin II in hypertension. Although this agent is an inhibitor of converting enzyme [ 251, it is thought to act pharmacologically as an antagonist to angiotensin II by competing with it for a binding site on a membranebound receptor [ 261. This receptor is the most recently recognized macromolecular component of the reninangiotensin system [ 271. It has thus far been characterized primarily by its aff inky for angiotensin II and has not yet been purified. Nor is it clear whether the same receptor molecule participates in the vascular response to angiotensin II and the increased elaboration of aldosterone [28] by cells of the adrenal zona glomerulosa which may at least partially be mediated by angiotensin Ill (Figure 2) [ 291. The mechanism whereby a cellular response is generated as a consequence of the interaction between a specific receptor and its angiotensin effector has not been established and represents an important area for future research. REGULATtON OF ANGIOTENSIN-CONVERTtNG ENZYME BY ANTtENtYME ANTIBODY
The biochemical properties of angiotensin-converting enzyme and its relationship to the metabolism of vasoactive peptides has recently been reviewed elsewhere [30]. This discussion will focus upon studies by my colleagues and me which are concerned with characterization of the enzyme in different rabbit organs
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Bradykinin
Pyr-Lys-Trp-Ala-Pro Bradykinin-Potentiating
Pentapeptide
Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro Bradykinin-Potentiating
Nonapeptide
Sar-Arg-Val-Tyr-Ile-His-Pro-Ala Saralasin
Arg-Val-Tyr-Ile-His-Pro-Phe Angiotensin
III
Figure 2. Structures of additional peptides discussed in this presentation.
and of antienzyme antibody developed in goats. Our interest in the enzyme was stimulated in late 1971 by which time evidence had accumulated that it was localized in apposition to the circulation and represented an important therapeutic target in nephrogenic hypertension. We reasoned that, under these circumstances, anticatalytic antibody introduced into the circulation would not be required to cross cellular permeability barriers in order to reach its site of action and that,
TABLE I
Properties of Rabbit Pulmonary Angiotensln-Converting Enzyme’
Molecularweight, native enzyme -
129,000 Molecular weight, reduced denatured enzyme - 129.000 NH2 - terminal residue = threonine COOKterminal residue = alanine Native heavy metal = zinc, one molar equivalent Amino acids - 837 residues/mol Tryptophan = 23 Slight hydrophobicity Carbohydrate - 176 residues/mole-26 Fucose = 4 Mannose = 43 Galactose = 57 N-Acetylglucosamine = 53 N-Acetylneuraminic acid = 19 * Data are from Das and Soffer
January 1978
The American
per cent by weight
[3 11.
Journal of Medlclne
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COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
I
I I
1
Seminal Z plasma I )
Serum ‘; . c.
DILUTED
L .. ... * _. Effect of antilung enzyme anrroooy on converting
ANTISERA . .. .
Pure enzyme
(pl 1 . , ..
,
Figure 3. enzyme acrrvlry In raoolt organ exrracts ana body fluids. From Das and Soffer (321. Residual activity was determined after incubating 3 munits of enzyme for 30 minutes with the indicated quantities
of antiserums.
therefore, it might exert its inhibitory effect in vivo. If this were the case it seemed to us that such antibody could be a useful biologically specific reagent for immunologically excising the renin-dependent component of blood pressure regulation. In order to examine these possibilities, a procedure was developed for isolating pure converting enzyme after solubilization from rabbit pulmonary membranes using a nonionic detergent [ 14,311. A purification factor of l,OOO-fold was required to obtain a homogeneous preparation from crude lung extracts indicating that the enzyme comprises about 0.1 per cent of total pulmonary protein. Some physiochemical characteristics of the enzyme are described in Table I. Such information on plasma membrane enzymes is relatively scarce since few have been obtained in pure water-soluble form. Therefore, converting enzyme may represent a useful tool for studying membrane-enzyme interaction and for investigating the general question of whether participation in an organized subcellular structure influences the catalytic properties of an enzyme. It contains a single large glycopolypeptide chain and a molar equivalent of tightly bound zinc which is required for catalytic activity. It is a moderately hydrophobic protein with a large number of tryptophanyl residues accounting for its high absorbance at 280 nm. Its paradoxically early elution during gel filtration through Sephadexo G-200 provided the first clue to its glycoprotein- nature [14,31].
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Antibodies against the rabbit glycoprotein were raised in goats and were found to be highly inhibitory in vitro [32]. Antiholoenzyme antibody was prepared by affinity chromatography using as ligand the pure enzyme fixed to an insoluble matrix. It was established that each antibody molecule could bind 2 molecules of enzyme and that at least 18 her cent of the antiholoenzyme antibody population recognized determinants which influence catalysis. Enzymatic hydrolysis of the physiologic substrates, angiotensin I and bradykinin, and of the model synthetic substrate, hippurylhistidyl-leucine (Hip-His-Leu), was inhibited similarly by antibody. Furthermore, inhibition dose-response curves obtained with the antilung enzyme antibody and a fixed amount of enzyme activity were identical regardless of whether the source of activity was the pure lung enzyme or crude fractions from various organs and body fluids (Figure 3). This result indicated that antibody prepared against the pulmonary glycoprotein could be expected to.inhibit angiotensin conversion throughout the body, provided it had access to the enzyme after introduction into the circulation. It also suggested that enzyme molecules in different organs possessed extensive homology with respect to those of their determinants which influence catalysis. We were interested in determining whether this immunologic homology also comprised those portions of the enzyme molecules which were unrelated to its catalytic action. To address this question [32] we
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CONVERTING ENZYME, ANGIOTENSIN II AND HYPERTENSIVE DISEASE
employed a competition radioimmune assay since this procedure encompasses determinants of the entire antigenic molecule. The assay measured displacement of radioiodinated pulmonary enzyme from an immune complex with antilung enzyme antibody by competing antigen in various crude fractions. Unlabelled pure lung enzyme was used as the reference antigen. Our rationale was that if enzyme molecules in a given extract were identical to the lung glycoprotein then the ratio of catalytic activity to competing antigen in that extract would correspond to the specific activity of the pure lung enzyme, i.e., 90 to 100 U/mg protein. It was established by this criterion that activities in kidney, brain and serum could be ascribed to an enzyme which was immunologically identical to that in the lung (Table II). Of special interest, however, was the finding that enzyme in seminal plasma was immunologically distinguished by this technic. Since its inhibition by antilung enzyme antibody was found to be identical to that observed with enzyme activities from the other sources, its lack of homology appears to reside in determinants unrelated to catalysis. The seminal plasma activity increases markedly during sexual maturation and may, therefore, play a special role in reproductive physiology ]331. Immunologic identity in the anticatalytic and competition radioimmune assays does not necessarily imply complete chemical identity. This was established by comparing pure preparations of rabbit lung and serum converting enzymes [34]. The latter constitutes a very minor component of crude serum and, in order to achieve the 60,000-fold enrichment required to obtain it in pure form, we exploited its immunologic homology with the pulmonary glycoprotein by employing antilung enzyme antibody as a ligand for affinity chromatography. As expected, most catalytic, structural and immunologic properties of the two purified enzymes were indistinguishable. However, the serum glycoprotein was found to contain three times more sialic acid on a molar basis than the lung enzyme. This was not completely unanticipated since a hepatic lectin is known to extract glycoproteins characterized by terminal nonreducing galactosyl residues from the circulation [35]. Our result suggests that the serum enzyme may be derived by sloughing or secretion of the tissue enzyme and subsequent modification of the microheterogenous glycoprotein population based on the specificity of this hepatic lectin. Under these circumstances, those molecules whose galactosyl residues are protected by sialylation would be expected to be spared and to selectively accumulate in the circulation. In order to assess the possibility that specific antibody might be an effective inhibitor of converting activity in vivo, it was necessary to determine whether most en-
TABLE II
Activity and lmmunoreactiviiy of Convertlng Enzyme from Various Rabbit Tissue Extracts and Fluids Catalytic
Catalytic Tissue Extract or Fluid
Lung nonidet P-40 extract Kidney nonidet P-40 extract Brain nonidet P-40 extract Serum Seminal plasma
COrlNing
Aclivily Anti (mUhng protein) (rg/mg protein)
545 59 4.6 1.8 219
Ulmg
5.49
93
0.617
95
0.049
94
0.0174 0.676
103 324
NOTE: Data are from Das and Soffer [32]. A unit of catalytic activity is the amount required to generate 1 .O ~mol of hippuric acid from
Hip-His-Leu per minute at 37%. Competing antigen was estimated using pure radioiodinated pulmonary enzyme as the displaceable antigen, pure unlabeled enzyme as the reference antigen and goat antibodies directed against the enzyme. The last column is the ratio of catalytic activity to competing antigen.
zyme might be accessible to the circulation. Ryan et al. [36] had used antibody conjugated with microperoxidase to demonstrate that converting enzyme was localized on the luminal surface of pulmonary vascular endothelial cells. We [37] studied the distribution of extrapulmonary enzyme with fluorescein-labelled antilung enzyme antibody and found that it was restricted to vascular endothelial rather than parenchymal cells in most of the rabbit organs which we examined (Figure 4). The kidney provided an exception since both vascular endothelium and proximal tubular cells exhibited fluorescence. However, our results in conjunction with those of Ryan et al. [ 361 suggested that most converting enzyme molecules might be expected to interact with circulating antibodies. Our first experiments [38] on the effect of antienzyme antibody after intravenous administration were conducted with the homologous recipient animal, i.e., the rabbit. Initially, we infused sufficient antibody to completely inhibit the total enzyme content of a pair of rabbit lungs as extrapolated from the linear portion of an antibody inhibition dose-response curve performed with pure enzyme in vitro. This dose of antibody invariably resulted in an almost immediate immunespecific lethal reaction manifested by pulmonary edema. Animals which received smaller quantities of immune globulin survived for a limited period. The vasopressor response to angiotensin I was somewhat diminished and the vasodepressor effect of bradykinin was potentiated in these animals; however, it was not possible to administer sufficient antibody to anticipate major effects on the metabolism of these vasoactive peptides. The mechanism of the lethal response in the
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COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
Figure 4. Staining of rabbit organs by fluorescein-labeled antibody raised against pure rabbit pulmonar From Caldwell et al. [37]. Panels a, c, d, e and f illustrate lung, liver, adrenal cortex, pancreas and kidne b is a control specin rfen showing lung reacted with fluorescein-labeled preimmune globulin.
homologous recipient animal is not clear. It is probably unrelated to the anticatalytic action of the antibody preparations since venom peptides are extremely effective, although transient, inhibitors of the enzyme in vivo and yet are remarkably nontoxic. It is conceivable that the combination of antigen and antibody triggers a destructive immunologic process mediated by complement, which occurs most noticeably in the pulmonary vascular bed, because this is the first and one of the richest antigenic sites encountered by the antibody after intravenous infusion. If this is the case, then it may be possible to dissociate the lethal and anticatalytic actions of antibody by using Fab fragments which contain the antigen binding site of the holoantibody but lack that portion of it which interacts with complement. These fragments have been prepared and characterized as effective inhibitors of the enzyme in vitro, but their action in vivo has not yet been examined. An alternative recipient animal was sought with the aim of circumventing the immune lethal response.
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:on verTing en2yme. .especztivt ‘ly. t‘anet
Homology studies employing the competition radioimmune assay, double immunodiffusion and cross-anticatalytic action indicated that antirabbit enzyme antibody reacted with pulmonary converting enzymes from rat guinea pig and dog [39]. The effect of antibody in vivo was examined with the rat [39], and no immediate deleterious effects were observed as a consequence of its infusion into this heterologous recipient. Figure 5 shows typical data obtained with an animal which received three times the amount of antibody required to completely inhibit the converting activity of a pair of rat lungs as extrapolated from an antibody inhibition dose-response curve performed with crude rat pulmonary enzyme. Administration of antibody was invariably associated with a striking diminution in the vasopressor response to angiotensin I and with a dramatic augmentation of the vasodepressor action of bradykinin. Virtually identical data were generated from 1 hour to at least 48 hours after the animals received immune globulin; the half-life of anticatalytic antibody,
Volume 64
CONVERTING ENZYME, ANGIOTENSIN II AND HYPERTENSIVE DISEASE
3
50
00
500
ANGIOTENSIN I
;;
DO
IW
-z&p-
:s
;E
Antibody
i32 20w” ij
.. . . .
100
(ngramr)
,n/,.
0.05
0.1
i
0.5
1.0
BRADYKININ ( pgrams
_
50
ANGIOTENSINII
Y
ZE w-
&
lo
1000
(ngrams)
.
5.0
1
. .. Figure 5. Effect of anrlraoolt convernng enzyme antltwores on rat blood pressure response to vasoactlve peptides. From Conroy et al. [39]. Dose-response curves were obtained before and 1 hour affer the animal received 180 mg of immune globulin. Each point represents the mean of four determinations
...
COnhOl
..
and is bracketed by the values corresponding
measured in the serum of one of these, was several days. The pressor potency of norepinephrine was not altered during these experiments, and antibody-dependent changes in the metabolism of vasoactive peptides were completely immune-specific. The results with angiotensin I and bradykinin were those to be expected after successful immunologic blockade of converting enzyme. The small but definite immunespecific inhibition of the vasopressor response to angiotensin II was not anticipated. Conceivably, a receptor for angiotensin II may be located in appositon to converting enzyme on the vascular endothelial surface, and antienzyme antibody may block access of angiotensin II to its effector site by steric hindrance. The major points which I have attempted to develop may be summarized as follows. Angiotensin-converting enzyme is a COOH-terminal dipeptidyl peptidase, i.e., it catalyzes cleavage of dipeptide residues from the COOH-terminus of oligopeptide substrates. Its two most important currently recognized substrates are angiotensin I and bradykinin, and its action on each is physiologically vasopressor. The enzyme is a fairly well characterized molecule consisting of a single zinccontaining glycopolypeptide chain and, although it is a
to 1 standard devia-
plasma membrane component, it can be obtained fairly easily in pure water-soluble form. In most organs it is restricted to vascular endothelial cells; its active site is directly accessible to the circulation. The lung plays a major role in the physiologic conversion of angiotensin I because of its large vascular endothelial surface, because it receives the entire blood volume and because it does not extract angiotensin II. However, extrapulmonary angiotensin conversion does occur and the responsible enzyme throughout the body is immunologically similar to that in the lungs. Since angiotensin-converting enzyme catalyzes the generation of angiotensin II, it represents a therapeutic target for pathologic conditions resulting from an overactive renin-angiotensin system. It can be inhibited chemically by various peptides, and it is likely that orally effective nonpeptide inhibitors will be developed in the near future. Converting enzyme is accessible to the action of circulating macromolecules because of its anatomic localization in juxtaposition to the blood and it can be inhibited in vivo by anticatalytic antibody. Although immunologic studies with it are in their early stages, such antibody represents a biologically specific inhibitor which should ultimately complement the chemical
January 1978
The American Journal of Medicine
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COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
I .*
.*
VOLUME
- SODIUM
1 CARDIAC
$
VOLUME-
SODIUM
GAIN w-.
. .
\
\
t ALDOSTERONE ”
LOSS
h
\
OUTPUT
+ + BLOODiRESSURE \ \
#CC
\
\
-
RENIN
RELEASE
1 \
+ ANGIO~ENSIN I I
____)
a
I’ \
1 ElLhD
Y# t VASOCONSTRICTION-
PRCSURE --w_
PERIPHERAL --W_
--
-____----
VASCULAR
RESISTANCE
Figure 6. Basic outline of the physiologic role of renin in maintaining blood pressure. Extracellular sodium loss lowers cardiac output and blood pressure, thereby threatening organ perfusion. However, renin is released in response to these events which in turn leads to angiotensin generation. Angiotensin raises blood pressure level acting as a direct vasoconstrictor. In addition, it stimulates aldosterone so that the kidneys will reabsorb more sodium chloride. These two actions reinforce each other in restoring
the system.
agents in delineating and modifying the contribution of the renin-angiotensin system to normal and pathologic processes. Dr. David B. Case: Dr. Soffer has presented a discussion of the biochemical components of the reninangiotensin system, culminating in a more detailed description of the converting enzyme or kininase II. At this point, I would like to extend this discussion to incorporate this biochemical system into a physiologic framework and into pathophysiologic hypotheses about the mechanisms involved in hypertension. Although there had been major advances in the understanding of the renal and adrenal relationships to salt balance and the regulation of the circulation, it was not until 55 years or so after the initial experiments of Tigerstedt and Bergman in 1898 that aldosterone was discovered [40], and then accurate methods for its measurement became available [41]. This set the stage for the crucial physiologic studies linking renin secretion to the control of aldosterone secretion which thereby linked blood pressure homeostasis to the simultaneous control of electrolyte balance. The breakthrough occurred when it was found that patients with malignant hypertension have massive oversecretion of aldosterone [42], a finding which led to the demonstration in human volunteer subjects of a highly specific action of infused angiotensin II to stimulate aldosterone secretion [43]. These studies exposed the renin-aldosterone hormonal connection and
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pointed out a particular derangement in it that is critically involved in causing malignant hypertension [44]. Subsequently, numerous studies in man and in various animals [45] have verified this renin-angiotensin-aldosterone axis and further characterized its fundamental role in blood pressure and in sodium and potassium homeostasis [46], and in the pathogenesis of particular forms of both experimental and clinical hypertension [47]. The more recent growth of all of this new knowledge has been aided by methodologic improvements and the increasing use of immunoassays instead of the more laborious pressor bioassays. Our present understanding of the renin axis is depicted in Figure 6. When sodium or extracellular volume is lost from the system, cardiac output declines as do the systemic and renal perfusion pressures. This decrease is one of several mechanisms by which the juxtaglomerular apparatus triggers secretion of renin. The release of renin is an early event in the compensatory response to the decrease in pressure. Increased renin levels lead to increased angiotensin II levels which work to restore pressure by producing vasoconstriction of peripheral resistance vessels, largely the arterioles. In addition, increased angiotensin II stimulates aldosterone secretion, leading to a restoration of extracellular volume by increased retrieval of distal tubular fluid sodium. Restoration of arterial pressure, effective volume and cardiac output in turn decrease renin secretion. Therefore, the system exhibits the character-
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CONVERTING ENZYME, ANGIOTENSIN II AND HYPERTENSIVE DISEASE
istic of a negative feedback loop. A series of baroreceptors and sodium chemoreceptors in the kidney and systemic circulation mediate the level of activity of this system and, therefore, provide the capability for quick responsiveness. In addition to this, angiotensin may have direct actions on the central nervous system which modify vascular tone. Altogether, we have a system responsive to subtle changes in extracellular sodium/ volume and perfusion pressure capable of initiation, and perpetuation of a compensatory response both by being able to influence sodium excretion and also by producing sustained vasoconstriction. Since the level of activity of this system should be inversely related to the state of sodium balance, clinical investigators sought ways to determine the normalcy or appropriateness of renin values by making simultaneous estimations of the state of sodium balance. One of the contexts chosen as a measure of sodium balance for renin estimations was the 24-hour urinary sodium excretion. Under steady-state conditions in normal subjects, the quantity of dietary sodium ingested is reflected in the urinary sodium content, excluding situations in which large amounts of insensible salt loss occur. Therefore, it was possible to describe a range of values of plasma renin activity corresponding to the span of values for 24-hour urinary sodium excretion [47] (see Figure 7). The same relationship could be derived from angiotensin II rather than plasma renin activity values, but the former are less accurate for clinical use and have not been widely used. As predicted, values for plasma renin activity were highest during dietary sodium deprivation and lowest when dietary sodium intake was greatest. A similar parallel band was derived for urinary aldosterone excretion in relation to urinary sodium excretion. The establishment of this nomogram relating plasma renin activity to its soduim balance thus provided a background with which to examine the major disorder of blood pressure regulation, hypertension. From careful analysis of several renin measurements during different salt diets on a metabolic ward, it was possible to characterize three different subgroups of hypertensive patients [47]. One group had suppressed renin levels during normal salt intake which rose within the same range as normal subjects during dietary sodium restriction. Since the range was the same as that in normal subjects, this subgroup was termed “normal-renin.” However, it was recognized that it might not be normal or appropriate to have “normal” levels of a pressor hormone in the presence of an increase in blood pressure. A second group had renin levels above the normal band which were most apparently high during periods of normal or high sodium intake, at a time when renin levels are normally suppressed. In this “high-renin” group, excess renin and angiotensin were
PLASMA 0
RENIN ACTIVITY
8
100
200 8
7
7
6 PRA
5
5
ng AI/
ml/hr 4
0
200
100
Urine
sodium
mEq/day
Vgure 7. Nomogram relating plasma renin activity ant the 24-hour urinary sodium excretion rate. The normal range is referenced against a measure of the state of sodium balance in normotensive healthy subjects. During dietary sodium deprivation, plasma renin levels rise.
believed to be playing a predominant role in maintaining the hypertensive state. Corroboration for this thesis came from studies showing the therapeutic benefit of treatments which lowered renin levels in high-renin patients: fi-adrenergic blockade with propranolol [48]; renal artery repair and nephrectomy in renovascular hypertension [49,50]. A third group of patients was identified who had low levels of plasma renin activity which did not rise into the normal range even during conditions of dietary sodium depletion. This “low-renin” subgroup was postulated to have a residual sodium or volume excess even after dietary sodium restriction which continued to suppress renal renin secretion. The sodium or volume factor appeared to be the predominant factor, as opposed to angiotensin, in sustaining the hypertension. Evidence to support this thesis came from studies showing that the administration of diuretics was more successful in lowering blood pressure in low-renin patients than in normal or high-renin patients [ 5 11. In addition, miner-
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155
LOW IO
RENIN
NORMAL RENIN n= IO
n=4
1
Average
HIGH
n=7
Average
RENIN
Average
n %DBP
I, T
15 J
Minutes
+
p
