Thorax 1982;37 :88-96

Angiotensin converting enzyme activity and evolutionof pulmonary vascular disease in rats withmonocrotaline pulmonary hypertensionJM KAY, PM KEANE, KL SUYAMA, D GAUTHIER

From the Department ofLaboratory Medicine, St Joseph's Hospital and Department ofPathology,Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada

ABSTRACT We have investigated the role of angiotensin converting enzyme (ACE) in the develop-ment of pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular disease inrats given a single subcutaneous injection of the pyrrolizidine alkaloid monocrotaline. Thirty-sixyoung female Wistar rats were divided into a test group of 27 animals and a control group of nineanimals. Each test rat was given a single subcutaneous injection of monocrotaline (60 mg/kg bodyweight). On the first, third, fifth, seventh, tenth, twelfth, fourteenth, seventeenth, and twenty-seconddays after the injection of monocrotaline the mean right ventricular systolic blood pressure was

measured in one control and three test rats. The animals were then killed and we measured thespecific activity of ACE in serum and lung homogenate. We also evaluated muscularisation of pul-monary arterioles, medial hypertrophy of muscular pulmonary arteries, and right ventricular hyper-trophy. The sequence of changes was as follows: muscularisation of pulmonary arterioles and medialhypertrophy of muscular pulmonary arteries were apparent seven days after administration ofmono-crotaline; pulmonary hypertension and reduced lung ACE activity occurred after 10 days; rightventricular hypertrophy was detected after 12 days. Serum ACE activity was unchanged. If isconcluded that the reduction in lung ACE activity is a result rather than a cause of the pulmonaryhypertension. This reduction in lung ACE activity may be a protective mechanism designed to limitthe elevation of the pulmonary arterial pressure.

Monocrotaline is a pyrrolizidine alkaloid present inthe seeds and foliage of the leguminous plantCrotalaria spectabilis. The administration of thisalkaloid to rats produces pulmonary hypertension,hypertensive pulmonary vascular disease and rightventricular hypertrophy.1 The pulmonary vascularlesions comprise thickening of the pulmonary trunk,medial hypertrophy of muscular pulmonary arteries,and muscularisation of pulmonary arterioles. Inabout one-third of rats there is also an acute necro-tising pulmonary arteritis. The mechanism wherebymonocrotaline causes elevation of the pulmonaryarterial pressure is unknown. Recently we showedthat the activity of angiotensin converting enzyme(ACE) was significantly reduced in lung tissuehomogenates derived from rats with pulmonaryhypertension produced by subcutaneous injection of

Address for reprint requests: Dr JM Kay, Department ofLaboratory Medicine, St Joseph's Hospital, Hamilton,Ontario, Canada L8N 1Y4.

monocrotaline.2 Furthermore, the reduction in lungACE activity was inversely proportional to the sev-erity ofpulmonary hypertension and right ventricularhypertrophy. ACE is an exopeptidase that catalysesthe hydrolytic removal of carboxyterminal dipeptideresidues from polypeptide substrates.3 Two suchreactions ofACE may participate in the regulation ofblood pressure. First, it converts the inactivedecapeptide angiotensin I to the potent vaso-constrictive octapeptide angiotensin II. Secondly, itinactivates the vasodilator nonapeptide bradykinin.The role of ACE and angiotensin II in the humoralcontrol of the pulmonary circulation is not clear.Accordingly, we have studied serum and lung ACEactivity during the development of pulmonaryhypertension in rats given a single subcutaneousinjection of monocrotaline. We wished to determinethe temporal relationship between decreased lungACE activity on one hand, and pulmonary hyper-tension, right ventricular hypertrophy, medialhypertrophy of muscular pulmonary arteries, and

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muscularisation of pulmonary arterioles on the otherhand.

Methods

Thirty-six female Wistar rats (initial body weight113 g ± 11 SD) were divided into a test group of 27animals and a control group of nine animals. Eachtest rat was given a single subcutaneous injection ofa 2% aqueous solution of monocrotaline hydro-chloride. The dose of monocrotaline was 60 mg/kgbody weight. On the first, third, fifth, seventh, tenth,twelfth, fourteenth, seventeenth, and twenty-seconddays after the injection of monocrotaline the meanright ventricular systolic blood pressure (Prvs) wasmeasured in one control and three test rats. Underlight ether anaesthesia the right external jugular veinwas exposed and cannulated, using a modified21-gauge disposable needle measuring 0 7 mm inexternal diameter and 3A4 cm in length. The tip of thecannula was guided through the right atrium andtricuspid valve into the right ventricle. The pressurewas measured using a Hewlett Packard transducer(1280C) attached to a pressure coupler (8805C) andthermotip recorder (7702B). After measurement ofPrvs the abdomen was opened and a sample ofblood was obtained from the inferior vena cava. Theserum was separated and stored at - 20°C beforeACE assay using the method described below. Therats were then killed. The cervical and thoracicorgans were removed in one block. The left mainbronchus was clamped and ligated after which theleft lung was removed, washed in ice-cold physio-logical saline and stored at - 20°C to await ACEassay. The trachea was cannulated and the rightlung was distended by injecting 7 ml of 10% formolsaline which ensured that the pleural surfaces weresmooth. The trachea was then ligated, the cannulawithdrawn, and the remaining thoracic visceraimmersed in 10% formol saline until fixation wascomplete. The hearts were dissected and weighedusing a Sartorius semi-micro analytical balance.Right ventricular hypertrophy was evaluated byexpressing the weight of the free wall of the rightventricle (RV) as a percentage of the weight of theleft ventricle and interventricular septum (LV+S).A block of tissue was cut from each of the superior,middle, inferior, and median lobes of the right lungof every animal and was routinely processed forhistological examination. Sections were stained withhaematoxylin and eosin, by the martius-scarlet-bluemethod for fibrin4 and also by the Miller elastic-VanGieson method5 for distinguishing smooth muscle,collagen, and elastic tissue. Sections stained by thelatter method were used for the assessment of muscu-larisation of pulmonary arterioles and measurement

Fig 1 Normal pulmonary arteriole from a control rat.The wall is thin and devoid ofsmooth muscle. It consistsofa single elastic lamina lined by endothelial cells.Elastic- Van Gieson stain x 980.

of medial thickness of muscular pulmonary arteriesas described below.

MUSCULARISATION OF PULMONARYARTERIOLESThe pulmonary arteriole in a rat is an arterial vesselless than 20 ,um external diameter whose wall isnormally devoid of smooth muscle except adjacentto its origin from a muscular pulmonary artery. Itconsists of a single elastic lamina lined by endo-thelium (fig 1). Pulmonary venules are structurallyidentical to pulmonary arterioles, and these cannotbe distinguished from one another unless they can betraced to a larger vessel. In chronic pulmonaryhypertension the pulmonary arterioles becomemuscularised with the development of a thick medialcoat of circularly orientated smooth muscle boundedby internal and external elastic laminae (fig 2). Thusthey resemble muscular pulmonary arteries in struc-ture. We used the technique of Hunter et a16 to assessthe muscularisation of pulmonary arterioles. Allfour sections of the right lung from every test andcontrol rat were systematically examined using thex 40 objective in a microscope fitted with a calibratedeyepiece micrometer. All small blood vessels lessthan 50 ,um external diameter with a definite elasticcoat lying distal to respiratory bronchioles werecounted. These vessels lie adjacent to alveolar ductsor alveolar spaces. The number of such pulmonaryvessels with two elastic laminae (PVTEL) was

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Fig 2 Muscularised pulmonary arteriole from a ratwith pulmonary hypertension induced by monocrotaline.The wall is thick. A medial coat of circular smoothmuscle is bounded by internal and external elasticlamninae. Elastic- Van Gieson stain x 980.

expressed as a percentage of the total number ofvessels counted. A double elastic lamina was re-

corded when two distinct laminae separated by a

space were visible for at least half the diameter intransverse section, or at least half the length of thewall in longitudinal section. The counts of vesselsincluded occasional small venules which cannot bedistinguished from normal pulmonary arterioles.The sections were coded and examined withoutknowledge as to whether they were derived fromcontrol or test rats.

MEDIAL THICKNESS OF MUSCULAR

PULMONARY ARTERIES

The muscular pulmonary artery in a rat is definedas an arterial vessel with an external diameter lyingbetween 20 vm and 400 ,m. It has a tunica media ofcircularly orientated smooth muscle sandwichedbetween two elastic laminae (fig 3). A quantitativeestimation of medial hypertrophy was made bymeasuring the medial thickness and external diam-eter of muscular pulmonary arteries and muscu-

larised pulmonary arterioles. Histological sectionsstained by the elastic-Van Gieson method were

systematically examined in a microscope fitted with a

calibrated eyepiece micrometer. Only vessels that were

virtually circular in transverse section were measured.This reduced the number available for study, but

Fig 3 Normal muscular pulmonary artery from acontrol rat. The thin tunica media of circular smoothmuscle is bounded by internal and external elasticlaminae. Elastic- Van Gieson stain x 395.

it avoided error in the measurement of the diameter.The external diameter was taken as the mean of twomeasurements, at right angles to each other, of thedistance between diametrically opposite points onthe external elastic lamina. The medial thickness wasestimated as the mean of four measurements takenat approximately equally spaced points around thevessel wall. From these data medial thickness wasexpressed as a percentage of the external diameter ofthe vessel. A value for the average percentage medialthickness in each animal was obtained by totallingall the percentages of medial thickness, and dividingthe sum by the number of vessels examined. Thesections were coded and examined without know-ledge as to whether they were derived from controlor test animals.

ASSAY OF ACEThe specific activity of ACE was measured in serumand lung homogenate using a modification7 of thespectrophotometric assay described by Cushmanand Cheung.8 This method measures the rate ofproduction of hippuric acid from hippuryl-L-histidyl-L-leucine. Before assay the left lung wasthawed, weighed using a Sartorius semi-microanalytical balance, diced by hand, and then homo-genised in a Brinkman Polytron PT 10-35 at 4°C in12 volumes of assay buffer (500 m mol potassiumphosphate-750 m mol sodium chloride, pH 8 3). Thehomogenate was filtered through Whatman no 2

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paper and the filtrate was centrifuged at 5000 g for30 min. The protein concentration was measured9 inthe supernatant fluid which was then used for enzymeassay. Optimal assay conditions were obtained byusing 50 pl volumes of serum and 100 pi volumes oflung homogenate previously diluted with six timestheir volume of distilled water. These respectivealiquots of serum and diluted lung homogenate wereadded to 150 pl of 12 m mol hippuryl-L-histidyl-L-leucine in acid buffer. These conditions resulted inmaximal enzyme activity compatible with zero orderkinetics.'0The animals used in this experiment were cared for

in accordance with the regulations of the CanadianCouncil on Animal Care. They were allowed freeaccess to food and water, and were subjected to an

alternating sequence of 14 h of light and 10 h ofdarkness. The results of the experiment expressed asmeans with SD were evaluated using the t test forunpaired data. The respective mean values obtainedfrom the groups of test rats killed on the first, third,fifth, tenth, twelfth, fourteenth, seventeenth, andtwenty-second days after administration of mono-crotaline were compared with the mean valuesobtained from the group of nine control rats.Differences in mean values were considered to besignificant when p < 0-05.

Results

QUALITATIVE MICROSCOPIC FINDINGS

The lungs of the nine control rats were normal andspecifically devoid of pneumonic changes. Thegroups of three test rats killed on the first, third,fifth, and seventh days after receiving monocrotalinehad histologically normal lungs. One of the threetest rats killed on the tenth day showed a mildproliferation of large intra-alveolar cells resembling

granular pneumocytes.11 One of the three test ratskilled on day 12 was unremarkable. The other twoshowed a mild proliferation of intra-alveolar cellsassociated with scanty foci of intra-alveolar fibrinousexudate. All three test rats killed on day 14 showed a

proliferation of large intra-alveolar cells. Foci ofintra-alveolar fibrinous exudate were also present inone of these rats. All three test rats killed on day 17showed a proliferation of large intra-alveolar cells.In addition, two of these rats showed foci of intra-alveolar fibrinous exudate. All three test rats killedon day 22 showed a proliferation and enlargement ofintra-alveolar cells together with capillary fibrinthrombi. In addition, two of these rats showedfoci of intra-alveolar fibrinous exudate. None of the27 test rats which received monocrotaline showedevidence of necrotising pulmonary arteritis.

QUANTITATIVE OBSERVATIONS

In the table are listed the results of the assessment ofmuscularisation of pulmonary arterioles (PVTEL),medial thickness of muscular pulmonary arteries,right ventricular hypertrophy (RV/(LV + S) %),Prvs, and the specific activity of ACE in lung tissuehomogenate and serum. The mean numbers of smallpulmonary blood vessels examined for evidence ofmuscularisation in the nine control and 27 test ratswere 396±65 and 410 ± 70, respectively. The meannumber of muscular pulmonary arteries measuredin the nine control rats was 15 ± 4. The mean numberof muscular pulmonary arteries and muscularisedpulmonary arterioles measured in the 27 test rats was34 ± 17. All the test rats examined 12 or more daysafter receiving monocrotaline had developed muscu-larisation of the pulmonary arterioles, medialthickening of the muscular pulmonary arteries (fig 4),right ventricular hypertrophy, pulmonary hyperten-sion, and a reduction in lung ACE activity. However,

Table Effect of monocrotaline on percentage of small pulmonary blood vessels with two elastic laminae, medial thickness ofmuscular pulmonary arteries, right ventricular weight, right ventricular mean systolic pressure, and lung and serum angiotensinconverting enzyme activity in rats

Control Test rats: days after inijection of monocrotaline(n = 9)

1 3 5 7 10 12 14 17 22

PVTEL (%) 4 9±1 2Medial thickness of 4-6±1t2MPA (%)

RV/(LV+S)% 30 9 ±3-2Prvs (mm Hg) 23e6±440

Lung ACE activity 86-9±3-8

5-0±0-4 7-0± 3-8 9 3 3-8 71 ± 0-8* 14 2± 2-3* 19 2± 3-1* 16 6± 3-1* 20-6± : 5* 19.5±4 5-1*40±09 5.3t± 2-0 5 3± 05 62± 1 0* 7-4± 2.1* 6-2± 07* 9-3± 1-5* 7-3± 1.1* 97± 0-1*

32-8±3-4 31 8± 2-1 29-7± 2-3 29-2± 0-2 32-7± 0 3 38-2± 2-1* 46-6± 9.5* 43-1± 7.5* 55-8±11-3*237±1t0 244± 3-8 223± 1-6 297± 5-8 298± 0.4* 43.8±15.4* 50-4± 9-8* 42-6± 7.4* 62.6±10.5*

(n = 2) (n = 2)87-1 ±2-7 79-7±13-9 79-6±12-7 75 4±12-2 5934- 9.8* 5221- 4.9* 44 7-15 4* 38-4±11t8* 35-9±11-8*

(nmol/mg protein/min)

Serum ACE (nmol/ 97-5±89 85-9±84 102 9± 5 5 114-3±16-1 73-2±20-1 88-9 -30-8 103 5± 3-4 96-24 9-2 84-1±13-1 85-3±23-1ml/mun)

PVTEL = small pulmonary blood vessels with two elastic laminae. MPA = muscular pulmonary arteries. RV = weight of free wall of right ventricle.LV+S = weight of left ventricle and interventricular septum. Prvs = right ventricular mean systolic blood pressure. ACE = angiotensin converting enzyme.* = test result significantly different from control p < 0 05. n= 3 except where otherwise stated.

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\_'{.. .................... sEe r...,. .... _s__,...0S t- X F. .................... .............. ..... ...|O s _ _z.. . Ps

F ____ _ v sr _ wzaxr: _-_ :.A......... , _ _ s _K .................. 4i_ s _ tC; ............. ,,11w. | !,

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*e * l9.A \z..:_ . .g. .,s | :::t.j.\. _ i: B.f:884 * .................. ^ | .^.b ................ '.'

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Fig 4 Muscular pulmonary artery from a rat withpulmonary hypertension induced by monocrotaline. Thetunica media is thickened. Elastic- Van Gieson stainx 250.

there was no reduction in the serum ACE activity. Infig 5 the data have been arranged so as to illustratethe time sequence in the development of muscular-isation of pulmonary arterioles, medial hypertrophyof muscular pulmonary arteries, right ventricularhypertrophy, pulmonary hypertension, and reduc-tion in lungACE activity. In the case of each param-eter, the respective mean values obtained from thegroups of three test rats killed on the first, third,fifth, seventh, tenth, twelfth, fourteenth, seventeenth,and twenty-second days after monocrotaline ad-ministration have been expressed as a percentage ofthe mean value obtained from the group of ninecontrol rats. The sequence of changes was as follows:muscularisation of pulmonary arterioles and medialhypertrophy of muscular pulmonary arteries wereapparent seven days after administration of mono-crotaline; pulmonary hypertension and reduced lungACE activity occurred 10 days after monocrotalineadministration; right ventricular hypertrophy wasdetected after 12 days.

Discussion

This study has shown that muscularisation ofpulmonary arterioles and medial hypertrophy ofmuscular pulmonary arteries occur seven days afterthe atministration of a single subcutaneous in-

do

C7

:3 5 7 10 12 14 17Days atter monocrotaline injection

Fig 5 The time sequence in the development ofmuscularisation ofpulmonary arterioles, medial hyper-trophy of muscular pulmonary arteries (MPA), rightventricular hypertrophy, pulmonary hypertension,and reduction in lung angiotensin converting enzyme(ACE) activity after a single subcutaneous injection ofmonocrotaline. With each parameter the respective meanvalues obtainedfrom each group of three test ratskilled on a particular day have been expressed as apercentage of the mean value obtainedfrom the group ofnine control rats. An open circle indicates p > 0 05. Aclosed circle denotes p < 0 05.

jection of monocrotaline. At this time the lung ACEactivity is normal. Pulmonary hypertension is first

22

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apparent 10 days after injection and this coincideswith a reduction in lung ACE activity. Rightventricular hypertrophy occurs after 12 days.Presumably the vascular lesions lead to the develop-ment of pulmonary hypertension which in turn isfollowed by right ventricular hypertrophy. It seemsthat the reduction in lung ACE activity is a resultrather than a cause of the pulmonary hypertension.ACE has been found in association with the micro-vascular beds of many tissues including the spleen,pancreas, adrenal, liver, and kidney.12 However,it has been calculated that most of the ACE activityin the body occurs in the lungs13 where the enzymehas been localised on the luminal surface of pul-monary endothelial cells,'4 particularly in relationto flask-shaped indentations called caveolae intra-cellulares (fig 6). The caveolae represent a specialised

C

A

Fig 6 Alveolar capillary wall of rat lung fixed byperfusion of the pulmonary artery with glutaraldehyde.The capillary (C) is lined by an endothelial cell (E)the surface of which shows indentations named caveolaeintracellulares (arrows). The alveolar space (A) is linedby a membranous pneumocyte (M). This is separatedfrom the endothelial cell by a thin amorphous zone (1)comprising the fused basement membranes of the twocells. Electron micrograph. Uranyl acetate and leadcitrate stain x 102 000.

adaptation of the pulmonary endothelial surfacewhich greatly increases the amount of ACE incontact with the blood. It may be that during pul-monary hypertension a reduction in the size ornumber of caveolae may occur. This would decreasethe surface area and permit streaming of substratepast the enzyme. The reduction in lung ACE activityin pulmonary hypertension may be a protectivemechanism designed to limit the elevation of thepulmonary arterial pressure. Decreased enzymeactivity would lead to a reduction in the amount ofthe vasoconstrictor agent angiotensin II and anincrease in the amount of the vasodilator substancebradykinin. Rats with spontaneous systemic hyper-tension have been found to have reduced ACEactivity in the serum, kidney, and anterior pituitarywith normal values in lung, testis, epididymis,posterior pituitary, and hypothalamus.15 It has beensuggested that regulation of ACE activity mayoccur independently in different organs. Perhaps thisregulation is brought about by morphologicalchanges in the endothelial surface in response toalterations in intravascular pressure.

In 1976 Oparil and her co-workers studied serumACE activity in patients with sarcoidosis, chronicobstructive pulmonary disease, and shock lung.16The patients with sarcoidosis had increased ACEactivity while those with chronic obstructive pul-monary disease and shock lung had decreasedactivity in the serum. They suggested that serumACE activity is a reflection of pulmonary ACEactivity. Our results do not support this concept.Our rats with pulmonary hypertension induced bymonocrotaline had decreased lung ACE activity withnormal levels in the serum. In a previous paper weshowed that rats with pulmonary hypertensiondue to chronic hypobaric hypoxia also had decreasedlung ACE activity with normal levels in the serum.2In experimental acute lung injury caused by thio-urea'7 and paraquat'8 there is an elevation of serumACE activity which corresponds to a reduction inlung ACE activity. However, these changes aretransient and last between one and two hours.Presumably acute injury to the pulmonary capillaryendothelial cells causes ACE to be released from thelung into the blood. It has been reported that asingle subcutaneous injection of monocrotaline(60 mg/kg body weight) caused ultrastructuralevidence of interstitial alveolar oedema and swellingof alveolar capillary endothelial cells within 24 h inyoung male Sprague-Dawley rats.19 If such changesdid occur in our animals then they were not ac-companied by changes in lung or serum ACE levels.We did not examine the lungs of our rats with theelectron microscope but no histological abnormalitywas detected until the tenth day after monocrotaline

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administration when one animal showed a mildproliferation of large intra-alveolar cells resemblinggranular pneumocytes.11 This non-specific reactionof the lung to injury was noted three days aftermuscularisation of pulmonary arterioles and medialhypertrophy of muscular pulmonary arteries hadfirst been observed.The work reported in this paper appears to be the

first systematic study of the time course of thedevelopment of pulmonary hypertension and associ-ated pulmonary vascular disease after administrationof a single dose of monocrotaline. We found thatmuscularisation of pulmonary arterioles and medialhypertrophy ofmuscular pulmonary arteries occurredat seven days followed by pulmonary hypertension at10 days. Right ventricular hypertrophy was detectedafter 12 days. Meyrick and her co-workers20 haverecently described the development of pulmonaryhypertension and associated pulmonary vasculardisease in Sprague-Dawley rats fed on a diet con-taining powdered Crotalaria spectabilis seeds at aconcentration of 1 g powdered seed per kg rat food.They found that muscularisation of the smallpulmonary arterial vessels and pulmonary hyper-tension were evident on day 14. Increased medialthickness of muscular pulmonary arteries up to 200/tm external diameter was detected on day 21 whileright ventricular hypertrophy was not apparent untilday 28. There are at least three reasons why theseresults differ from our own. First, Meyrick and herco-workers used male Sprague-Dawley rats weighingbetween 220 and 240 g whereas we used femaleWistar rats weighing 113 g. Secondly, while we gavea precisely measured dose of monocrotaline to ourrats, Meyrick and her co-workers merely fed theirrats on powdered Crotalaria spectabilis seeds.Accordingly, it is not clear what dose was given toindividual rats, and there must have been con-siderable variation between animals. It has beencalculated that a diet containing 0-l % of Crotalariaspectabilis seeds is equivalent to a daily dose of about10 mg/kg body weight of monocrotaline since theseeds contain about 3% of the pure alkaloid.21 Webelieve that in order to study the evolution of thepulmonary vascular disease it is important to give adefinite known dose of monocrotaline at a specifictime. Thirdly, Meyrick and her co-workers used adifferent method to prepare the pulmonary vascula-ture for examination. This involved deep freezingthe lungs for an unstated time followed by thawingand injection of warm micropaque and gelatin underpressure. Huxtable and his co-workers22 placedmonocrotaline in the drinking water of rats at a

concentration of 20 mg/I for three weeks. It was

calculated that the daily dose was 3 6 mg/kg. Theystated that right ventricular hypertrophy was present

KaYv, Keane, Suyama, Gauthier

after two weeks of treatment and the right ventricularblood pressure was significantly elevated after 22days. Medial hypertrophy of pulmonary arteries wasdetectable by 14 days and was marked by 21 days.However, the pulmonary vasculature was notevaluated quantitatively. They measured lung ACEactivity by perfusing the lung and studying the rateof metabolism of hippuryl-histidyl-leucine. When therate of metabolism was expressed per dry weight oflung tissue there was apparently a significantdecrease in lung ACE activity. However, when themetabolism was expressed as rate per lung then therewas no significant reduction. Accordingly, theseworkers concluded that monocrotaline administra-tion did not impair the activity of ACE in the lung.The mechanism whereby monocrotaline induces

pulmonary hypertension is unknown. Monocrotalineitself is a stable and relatively inactive substance butthere is evidence that it is metabolised in the liver toproduce a highly reactive unstable pyrrole deriva-tive.23 Intravenous injection of the pyrrole derivativeleads to the development of pulmonary hyper-tension, right ventricular hypertrophy, and pulmon-ary vascular disease.24 It has been suggested thatmonocrotaline pyrrole damages the pulmonarycapillary endothelium leading to cytoplasmic swell-ing followed by cellular disruption and subsequentthrombosis with fibrin deposition.24 It was furtherpostulated that the resulting occlusion of alveolarcapillaries impedes blood flow leading to pulmonaryhypertension accompanied by medial hypertrophyof muscular pulmonary arteries and right ventricularhypertrophy. In our study of the evolution of pul-monary vascular disease, muscularisation of pul-monary arterioles and medial hypertrophy of mus-cular pulmonary arteries were apparent after sevendays and pulmonary hypertension was detectableafter 12 days. Fibrin thrombi, however, were notdetected in the alveolar capillaries until 22 days aftermonocrotaline administration. Furthermore, thestated hypothesis does not account for the prolifera-tion of smooth muscle which has been described inthe pulmonary veins of rats given Crotalariaspectabilis seeds25 and the pyrrolizidine alkaloidfulvine.26 Alveolar hypoxia, which is known to beassociated with pulmonary vasoconstriction, hasbeen invoked as the mechanism responsible for thepulmonary hypertension induced by monocrotaline.27Alveolar hypoxia also leads to a reduction in lungACE activity.2 28 However, the histological type ofhypertensive pulmonary vascular disease encoun-tered in monocrotaline intoxication is quite differentfrom that which occurs in chronic hypoxic pulmonaryhypertension.29 Furthermore, although we did notmeasure systemic arterial oxygen tension, this wasdone by Meyrick and her co-workers who found no

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evidence of systemic arterial hypoxemia.20 A pro-liferation of mast cells occurs in a proportion ofrats treated with monocrotaline.30 Rat mast cellscontain serotonin which is known to constrict smallpulmonary blood vessels in some animals. It waspostulated that monocrotaline or one of its metab-olites might stimulate the proliferation of pulmonarymast cells and that the release of serotonin from themmight lead to constriction of small pulmonary bloodvessels. This hypothesis was especially attractivesince it would account for the latent period whichelapses between the administration of monocro-taline and the development of pulmonary hyper-tension. It was found p-chlorophenylalanine whichinhibits serotonin synthesis reduced the severity ofpulmonary hypertension in rats treated with mono-crotaline but did not prevent it completely.3'However, in a more recent study, cinnarizine whichinhibits vasoconstriction induced by serotonindid not prolong the survival or reduce the incidenceof right ventricular hypertrophy and hypertensivepulmonary vascular disease in rats given a singleinjection of monocrotaline.32 It has been suggestedthat the lung mast cell hyperplasia which occurs inrats treated with monocrotaline is not concerned inthe genesis of the pulmonary hypertension but is asecondary phenomenon related to the developmentof exudative lesions in the lung parenchyma.33

In conclusion, the present study has revealed thatthe sequence of changes occurring in rats given asingle injection of monocrotaline is muscularisationof pulmonary arterioles and medial hypertrophy ofmuscular pulmonary arteries followed by pulmonaryhypertension followed by right ventricular hyper-trophy. The depression in lung ACE activity occursafter the development of the pulmonary vasculardisease and appears at the same time as the pul-monary hypertension. Accordingly, it is consideredthat decreased lung ACE activity is an effect ratherthan a cause of the pulmonary hypertension. Thisreduction in lung ACE activity in pulmonary hyper-tension may be a protective mechanism designed tolimit the elevation of the pulmonary arterial pressure.

We thank Jennifer Hines for assisting in the prep-aration of the manuscript. This research is supportedby a grant from the Medical Research Council ofCanada.

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2 Keane PM, Kay JM, Suyama, KL, Gauthier D, Andrew K.Lung angiotensin converting enzyme activity in ratswith pulmonary hypertension. Thorax 1982; in press.

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I Lendrum AC, Fraser DS, Slidders W, Henderson R.Studies on the character and staining of fibrin. J ClinPathol 1962;15:401-13.

5 Miller PJ. An elastin stain. Med Lab Technol 1971;28:148-9.

6 Hunter C. Barer GR, Shaw JW, Clegg EJ. Growth of theheart and lungs in hypoxic rodents: a model of humanhypoxic disease. Clin Sci Mol Med 1974;46:375-91.

7Le Treut A, Couliou H, Delbary M, Larzul J-J, DeLabarthe B, Le Gall J-Y. Le dosage de l'enzyme deconversion de l'angiotensine I par methode spectro-photometrique. Clin Chim Acta 1979;98:1-4.

8 Cushman DW, Cheung HS. Spectrophotometric assay andproperties of the angiotensin converting enzyme ofrabbit lung. Biochem Pharmacol 1971;20:1637-48.

9 Meijers CA, Rutten JCJM. A simple rapid method forthe determination of total nitrogen in urine. Clin ChimActa 1969;24:308-10.

10 Lieberman J. Elevation of serum angiotensin-converting-enzyme (ACE) level in sarcoidosis. Am J Med 1975;59:365-72.

"Kay JM, Smith P, Heath D. Electron microscopy ofCrotalaria pulmonary hypertension. Thorax 1969;24:511-26.

12 Caldwell PRB, Seegal BC, Hsu KC. Angiotensin-con-verting-enzyme: vascular endothelial localisation. Science1976;191 :1050-1.

1 Ng KKF, Vane JR. Conversion of angiotensin I to angio-tensin 11. Nature 1967;216:762-6.

14 Ryan JW, Ryan US, Shultz DR, Whitaker C, Chung A.Subcellular localisation of pulmonary angiotensin-converting enzyme (kininase lIj. Biochem J 1975;146:497-9.

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