chronically hypoxic rats

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1 Thrax 1985;40: Lung angitensin cnverting enzyme activity in chrnically hypxic rats JM KAY, PM KEANE, KL SUYAMA, D GAUTHIER Frm the Department flabratry Medicine, St Jseph's Hspital, and Department fpathlgy, Faculty f Health Sciences, McMaster University, Hamiltn, Ontari, Canada ABSTRACT A study was carried ut t test the hypthesis that the reduced lung angitensin cnverting enzyme (ACE) activity which ccurs in chrnic hypxia is related t the develpment f pulmnary hypertensin rather than t hypxia per se. Right ventricular mean systlic pressure (Prvs, mm Hg) and ACE activity (nml/mg prtein/min) in lung tissue hmgenates were measured in seven grups f fur rats placed in a hypbaric chamber (380 mm Hg; 51 kpa) fr tw t 24 days. Identical measurements were made n 11 grups f fur rats, which were placed in the chamber fr 24 days and then allwed t recver in rm air fr ne t 153 days. After tw days f hypxia the mean Prvs (25.5 (SD 3-7)) and the mean lung ACE activity (56 (4.6)) did nt differ significantly frm cntrl values. Expsure t hypxia fr fur t 24 days caused a prgressive increase in mean Prvs t 44-4 (5*9) and a prgressive reductin in mean lung ACE activity t 34 (4.0). During recvery lung ACE activity increased and Prvs decreased, s that nrmal values were achieved by 15 and 56 days respectively. Decreased lung ACE activity may be related t haemdynamic factrs assciated with pulmnary hypertensin rather than t hypxia. Acute alvelar hypxia has been shwn in dgs t reduce the ability f the pulmnary vasculature t cnvert angitensin I t angitensin II' and t degrade bradykinin.? Chrnic alvelar hypxia in rats has been shwn t reduce the lung angitensin cnverting enzyme (ACE) activity.3'4 Recently there has been cntrversy5 abut whether the hypxia induced inhibitin f ACE activity is due t a direct effect f hypxia n the endthelial cell membrane6 r whether it is secndary t pulmnary haemdynamic changes.7-9 We have investigated the hypthesis that the reduced lung ACE activity which ccurs in chrnic hypxia is related t the develpment f pulmnary hypertensin rather than t hypxia per se. We measured the right ventricular mean systlic pressure (Prvs), right ventricular hypertrphy, and lung ACE activity in grups f rats expsed t chrnic hypxia fr tw t 24 days t determine whether the lung ACE activity diminished immediately with the nset f hypxia r whether it diminished gradually as pulmnary Address fr reprint requests: Dr JM Kay, Department f Labratry Medicine, St Jseph's Hspital, Hamiltn, Ontari L8N 4A6. Canada. Accepted 22 March 1985 hypertensin develped. We als measured Prvs, right ventricular hypertrphy, and lung ACE activity in grups f rats that were expsed t chrnic hypxia fr 24 days and then allwed t recver in rm air fr ne t 153 days. We wished t determine whether the lung ACE activity returned t nrmal immediately the rats were returned t rm air r whether it gradually increased as the pulmnary hypertensin slwly regressed. Methds We evaluated pulmnary hypertensin, right ventricular hypertrphy, serum ACE activity, and lung ACE activity in seven grups f fur yung female Wistar rats, which were placed in a hypbaric chamber (380 mm Hg; 51 kpa) fr tw, fur, eight, 11, 14, 18, and 24 days respectively. Identical measurements were made n 11 grups f fur similar rats placed in the hypbaric chamber4 fr 24 days and then allwed t recver in rm air fr ne, three, seven, 15, 24, 34, 56, 76, 104, 132, and 153 days respectively. Thirty eight yung female Wistar cntrl rats matched fr age were als studied during the curse f the experiment. The mean initial bdy weights f the grups f cntrl and test rats 587

2 588 were 101 (SD 7.7) g and 104 (9.1) g respectively. Right ventricular mean systle pressure (Prvs) was measured by cannulating the right external jugular vein under light ether anaesthesia, a mdified 21 gauge dispsable needle being used.'0 The tip f the cannula was guided thrugh the right atrium and tricuspid valve int the right ventricle. The pressure was measured with a Hewlett-Packard transducer ( 1 280C) attached t a pressure cupler (8805C) and thermtip recrder (7702B). The pressure was measured in the chrnically hypxic rats frm 30 t 60 minutes after remval frm the hypbaric chamber. After measurement f Prvs the abdmen was pened and a sample f bld was btained frm the inferir vena cava. The serum was separated, frzen, and stred fr ne week t fur mnths t await ACE assay. The rats were then killed. The left lung was remved, washed in physilgical saline, frzen, and stred fr ne week t tw mnths t await ACE assay. ACE is stable fr at least ne year when stred at - 20 C. The lungs and sera were prepared fr assay by methds previusly described in detail.41' Briefly, the left lung was thawed, weighed with a Sartrius semimicr analytical balance, diced by hand, and then hmgenised in a Brinkman Plytrn PT The hmgenate was filtered and the filtrate was centrifuged at 1250 g fr 5 minutes. The prtein cncentratin was measured in the supernatant fluid that was then used fr enzyme assay. ACE activity was measured by means f a radienzymatic mdificatin f the assay described by Cushman and HYPOXIA E 60- (Ail EI 50 1 NORMOXIA Fig 1 Effect fchrnic hypxia and subsequent nrmxic recvery n mean right ventricular systlic pressure (Prvs). The mean value f the results derived frm the 38 cntrl rats is shwn as a cntinuus hrizntal line; each interrupted hrizntal line indicates I SD. The results frm the grups ffur test rats are given as mean and SD values. A clsed circle indicates that the value btained frm that grup ftest rats differs significantly frm the cntrl value (p < 0-05). Kay, Keane, Suyama, Gauthier Cheung.'2 This methd measures the rate f prductin f carbn 14 labelled hippuric acid frm hippuryl-l-histidyl-l-leucine. Lung ACE activity was expressed in nml/mg prtein/min. The heart was dissected after fixatin in 10% frml saline and weighed with a Sartrius semimicr analytical balance. Right ventricular hypertrphy was evaluated by expressing the weight f the free wall f the right ventricle (RV) as a percentage f the weight f the left ventricle and interventricular septum (LV+S). The results f the experiments, expressed as means with standard deviatins in parentheses, were evaluated with the t test fr unpaired data and linear regressin analysis. Differences in values were cnsidered t be significant when p < Results The Prvs in the 38 cntrl rats was ) mm Hg. After tw days f hypxia the mean Prvs (25.5 (3.7) mm Hg) was nrmal (fig 1). Expsure t hypxia fr fur t 24 days caused a prgressive increase in the mean Prvs t 44.4 (5p9) mm Hg. During nrmxic recvery the mean Prvs gradually decreased, until nrmal values were achieved 56 days after the animals' remval frm the hypbaric chamber (fig 1). In the 38 cntrl rats the rati f right t left ventricular weight (RV/(LV+S)) was 26&5% (2.7%). This rati was significantly increased t 32-5% (5-5%) in the grup f rats killed after being in the hypbaric chamber fr tw days (fig 2). Right ventricular hypertrphy prgressively increased s that after 24 days f hypxia the mean RV/(LV+S) 0- ak HYPOXIA 50 ] ~i I NORMOXIA 40] j 304..~~~ f ~ v-i-' 20O I I I l Fig 2 Effect fchrnic hypxia and subsequent nrmxic recvery n the rati f the weight fthe free wall f the right ventricle (RV) t the weight f the left ventricle and interventricular septum (LV+S). The mean value fthe results derived frm the 38 cntrl rats is shwn as a cntinuus hrizntal line; each interrupted hrizntal line indicates I SD. The results frm the grups ffur test rats are given as mean and SD values. A clsed circle indicates that the value btained frm that grup f test rats differs significantly frm the cntrl value (p < 0-05).

3 Lung angitensin cnverting enzyme activity in chrnically hypxic rats. 80- i ' us~ ~~~~~DY '0 4'0 5'0 6' ,80 Fig 3 Effect fchrnic hypxia and subsequent nrmxic recvery n lung angitensin cnverting enzyme (ACE) activity. The mean value f the results derived frm the 38 cntrl rats is shwn as a cntinuus hrizntal line; each interrupted hrizntal line indicates 1 SD. The results frm the grups ffur test rats are given as mean and SD values. A clsed circle indicates that the value btained frm that grup ftest rats differs significantly frm the cntrl value (p < 0.05). was 51-8% (7-7%). During nrmxic recvery the mean RV/(LV+S) prgressively decreased until a nrmal mean value (29-3% (3.5%)) was achieved 104 days after remval frm the hypbaric chamber (fig 2). The mean specific activity f ACE in the lung tissue hmgenates derived frm the 38 cntrl rats was 62 (12-1) nmlmg prtein/min. After tw days f hypxia the mean lung ACE activity (56 (4.6) nml/mg prtein/min) did nt differ significantly frm the cntrl value. Expsure t hypxia fr fur t 24 days caused an uneven reductin in mean lung -r Fig 4 Effect fchrnic hypxia and subsequent nrmxic recvery n lung angitensin cnverting enzyme activity (LACE), mean right ventricular systlic pressure (Prvs), and rati fright t left ventricular weight (RVI(LV+S) %). Each pint represents the mean value frm a grup ffur test rats. E r- C0 E -ae uj z J v * TEST S " CONTROL 0 * ;;. 0 *.e *0 *.-*f 0 :.* SERUM ACE (nml/ml/min) Fig 5 Lung and serum ACE activity in cntrl rats, chrnically hypxic test rats, and test rats expsed t chrnic hypxia fllwed by nrmxic recvery. Values frm individual rats are shwn. ACE activity t 34 (4) nml/mg prtein/min (fig 3). During nrmxic recvery the mean lung ACE activity gradually increased until a nrmal value (54 (8.6) nml/mg prtein/min) was attained after 15 days. On the 34th day f nrmxic recvery the mean lung ACE was significantly raised t 80 (12-9) nml/mg prtein/min. This "rebund" was fllwed by nrmal mean values fr the recvery perid (fig 3). The relatin between Prvs, RV/(LV+S)%, and lung ACE activity during the hypxic and recvery phases f the experiment is shwn in figure 4. Thrughut the hypxic and recvery phases f the experiment, in the test rats the lung ACE activity had a significant negative crrelatin with the Prvs (r = -0 39) and RV/(LV+S)% (r = -0 71). In the 38 cntrl rats the mean serum ACE activity was 94 (21.7) nmlmlmin and there was a significant negative crrelatin between the serum ACE value and the duratin f the experiment in days (r = -0.54). Figure 5 shws that serum ACE activity des nt reflect lung ACE activity in female rats. In the test rats the serum ACE values did nt appear t differ frm the values in the cntrl rats during either the hypxic r the recvery phases f the experiment. Discussin Chrnic hypxic pulmnary hypertensin results frm a cmbinatin f vascnstrictin, rganic narrwing f muscularised pulmnary arteriles, and increased bld viscsity due t plycythaemia. If a high altitude dweller breathes pure xygen there is nly a slight reductin in pulmnary artery pressure, presumably due t relief f vascnstrictin.'3 Thus 589

4 590 we cnsider that a slight reductin in Prvs may have ccurred during the minutes between remval f the hypxic rats frm the hypbaric chamber and cardiac catheterisatin in rm air. We d nt think that this affects ur cnclusins since the lung ACE levels were decreased in pulmnary hypertensive rats catheterised under nrmxic cnditins. One wuld have expected the lung ACE t increase rapidly t nrmal if its activity had been inhibited by hypxia. The mean Prvs was nrmal in the grup f rats examined after tw days f hypxia but the mean rati f right t left ventricular weight (32.5% (5.5%) was significantly greater than the cntrl value f 26-5% (2-7%). This apparent discrepancy culd have resulted frm a raised Prvs reverting t nrmal during the minutes between remval frm the hypbaric chamber and cardiac catheterisatin in rm air. We believe, hwever, that the right ventricular hypertrphy in this grup f rats is spurius and represents a statistical artefact related t the unusually lw mean rati f right t left ventricular weight in the cntrl rats f this experiment. In all ur previus studies the mean rati has been abut 30%.4" '4 In favur f this explanatin, the pulmnary arterial vessels were nrmal in the rats killed after tw days f hypxia, whereas there was muscularisatin f the pulmnary arteriles in the rats killed after fur days, when the mean Prvs was 36 mm Hg and the RV/(LV+S) rati was 39-9%. This experiment has shwn that after tw days f expsure t chrnic hypbaric hypxia rats have nrmal lung ACE activity and n pulmnary hypertensin. Expsure t hypxia fr fur t 24 days caused a prgressive develpment f pulmnary hypertensin assciated with an uneven reductin in lung ACE activity. During nrmxic recvery the lung ACE activity gradually increased and the pulmnary hypertensin gradually decreased ver perids f 15 and 56 days. We made direct measurements f ACE activity in lung tissue hmgenates. Our findings cnfirm the recent bservatins f Caldwell and Blatteis,3 wh expsed rats t hypbaric hypxia fr tw weeks and then injected graded dses f angitensin I and angitensin II int the right atrium. Dse-arterial pressr respnse curves were cnstructed. Ttal vascular ACE activity was estimated by determining the mean prprtin f the magnitudes f the angitensin I systemic pressr respnses and cmparing these with equimlar dses f angitensin II. They fund that ttal vascular angitensin I cnversin was 55% in the chrnically hypxic rats and 84% in cntrl rats. Furthermre, the ttal vascular cnversin f angitensin I t angitensin II was reduced in the rats expsed t chrnic hypxia even ne t three Kay, Keane, Suyama, Gauthier days after they had been remved frm the hypbaric chamber. Our results and thse f Caldwell and Blatteis3 suggest that the decreased lung ACE activity may be related t haemdynamic factrs assciated with pulmnary hypertensin, rather than t hypxia per se. This prpsal is in keeping with ur bservatin that thrughut the hypxic and recvery phases f the experiment in the test rats the lung ACE activity had a significant negative crrelatin with the severity f pulmnary hypertensin and degree f right ventricular hypertrphy. ACE is lcated n the luminal surface f pulmnary endthelial cells. Meyrick and Reid'5 have described structural changes in the pulmnary endthelial cells f rats expsed t chrnic hypbaric hypxia. Frm the third day f expsure the endthelial cells f newly muscularised arteries appeared thickened with flcculent cytplasm cntaining scanty small and elngated mitchndria, ribsmes, rugh endplasmic reticulum, a small Glgi apparatus, and pincyttic vesicles. The pulmnary capillary endthelial cells were up t fur times thicker than nrmal and had abnrmally electrn lucent cytplasm. Perhaps these structural changes are related t decreased synthesis f ACE. In 1976 Oparil and her cwrkers'6 suggested that plasma ACE activity may be a reflectin f pulmnary cnversin and that it can be altered by pulmnary disease. Our results d nt supprt this idea. As we have shwn befre, rats with pulmnary hypertensin and decreased lung ACE activity have nrmal levels in the serum.4 " In this experiment ur female cntrl rats shwed a significant negative crrelatin between the serum ACE value and the duratin f the experiment. This finding cnfirms the bservatins f ther wrkers that the serum ACE level is related t age bth in human subjects" 18 and in rats.'7 This wrk is supprted by a grant frm the Medical Research Cuncil f Canada. The hypbaric chamber was designed and cnstructed with the aid f a grant frm St Jseph' s Hspital Fundatin. We thank Jennifer Hines fr assisting in the preparatin f the manuscript. References 1 Leuenberger PJ, Stalcup SA, Mellins RB, Greenbaum LM, Turin GM. Decrease in angitensin I cnversin by acute hypxia in dgs. Prc Sc Exp Bil Med 1978;15S8: Stalcup SA, Lipset JS, Legant PM, Leuenberger PJ, Mellins RB. Inhibitin f cnverting enzyme activity by acute hypxia in dgs. J Appl Physil 1979;46: CaldweIl RW, Blatteis CM. Effect f chrnic hypxia n angitensin-induced pulmnary vascnstrictin and cnverting enzyme activity in the rat. Prc Sc Exp Bil Med 1983; 172:

5 Lung angitensin cnverting enzyme activity in chrnically hypxic rats 4 Keane PM, Kay JM, Suyama KL, Gauthier D, Andrew K. Lung angitensin cnverting enzyme activity in rats with pulmnary hypertensin. Thrax 1982;37: Stalcup SA, Davidsn D. Systemic circulatry adjustments t acute hypxia and rexygenatin in unanesthetized sheep. J Clin Invest 1984;73: Stalcup SA, Lipset JS, Wan J-M, Leuenberger P, Mellins RB. Inhibitin f angitensin cnverting enzyme activity in cultured endthelial cells by hypxia. J Clin Invest 1979;63: Pitt BR, Lister G. Pulmnary metablic functin in the awake lamb: effect f develpment and hypxia. J Appl Physil 1983;55: Szidn P, Bairey N, Oparil S. Effect f acute hypxia n the pulmnary cnversin n angitensin I t angitensin II in dgs. Circ Res 1980;46: Szidn P, Oparil S, Osikwicz G, Byse FM. Effect f hypxia n the cnversin f angitensin I t II in cultured prcine pulmnary endthelial cells. Bichem Pharmacl 1983;32: Kay JM, Heath D. Crtalaria spectabilis: the pulmnary hypertensin plant. Springfield: Charles C Thmas, 1969: Kay JM, Keane PM, Suyama KL, Gauthier D. Angitensin 591 cnverting enzyme activity and evlutin f pulmnary vascular disease in rats with mncrtaline pulmnary hypertensin. Thrax 1982;37: Cushman DW, Cheung HS. Spectrphtmetric assay and prperties f the angitensin cnverting enzyme f rabbit lung. Bichem Pharmacl 1971;20: Penalza D, Sime F, Bancher N, Gamba R. Pulmnary hypertensin in healthy man brn and living at high altitudes. Medicina Thracalis 1962; 19: Kay JM. Effect f intermittent nrmxia n chrnic hypxic pulmnary hypertensin, right ventricular hypertrphy, and plycythemia in rats. Am Rev Respir Dis 1980; 121: Meyrick B, Reid L. The effect f cntinued hypxia n rat pulmnary arterial circulatin. Lab Invest 1978;38: Oparil S, Lw J, Kerner TJ. Altered angitensin I cnversin in pulmnary disease. Clin Sci Ml Med 1976;51: Beutler E, West C. Sex- and age-related differences in activity f angitensin-cnverting enzyme in serum. Clin Chem 1983;29: 1570 (letter). 18 Lieberman J. Elevatin f serum angitensin-cnvertingenzyme (ACE) level in sarcidsis. Am J Med 1975;59: Thrax: first published as /thx n 1 August Dwnladed frm n 27 September 2018 by guest. Prtected by