Hypoxia Impairs Vasodilation in the Lung

NORBERTF. VOELKEL, IVAN F. MCMURTRY,and JOHNT. REEVES,Cardiovascular Pulmonary Research Laboratory, University of Colorado HealthSciences Center, Denver, Colorado 80262

A B S T RA C T Alveolar hypoxia causes pulmonaryvasoconstriction; we investigated whether hypoxiacould also impair pulmonary vasodilation. Wefound inthe isolated perfused rat lung a delay in vasodilationfollowing agonist-induced vasoconstriction. The delaywas not due to erythrocyte or plasma factors, or toalterations in base-line lung perfusion pressure. Pre-treating lungs with arachidonic acid abolished hypoxicvasoconstriction, but did not influence the hypoxia-induced impairment of vasodilation after angiotensinII, bradykinin, or serotonin pressor responses. Progres-sive slowing of vasodilation followed angiotensin II-induced constriction as the lung oxygen tension fellprogressively below 60 Torr. KCI, which is not metab-olized by the lung, caused vasoconstriction; the subse-quent vasodilation time was delayed during hypoxia.However, catecholamine depletion in the lungsabolished this hypoxic vasodilation delay after KCI-induced vasoconstriction. In lungs from high altituderats, the hypoxia-induced vasodilation impairmentafter an angiotensin II pressor response was markedlyless than it was in lungs from low altitude rats. Weconclude from these studies that (a) hypoxia impairsvasodilation of rat lung vessels following constrictioninduced by angiotensin II, serotonin, bradykinin, orKCI, (b) hypoxia slows vasodilation following KCI-induced vasoconstriction probably by altering lunghandling of norepinephrine, (c) the effect of hypoxiaon vasodilation is not dependent on its constrictingeffect on lung vessels, (d) high altitude acclimationmoderates the effect of acute hypoxia on vasodilation,and (e) the hypoxic impairment of vasodilation ispossibly the result of an altered rate of dissociationof agonists from their membrane receptors on thevascular smooth muscle.

INTRODUCTION

Alveolar hypoxia causes pulmonary arterial constric-tion and alters responses to vasoactive substances (1).

Received for publication 28 November 197.9 and in revisedform 15 September 1.980.

238

It is not known, however, whether hypoxia affectspulmonary vasodilation. Recent work indicated thatlow oxygen impaired femoral artery relaxation afterconstriction that was induced by acetylcholine (2).Low oxygen also slowed isoproterenol-induced dila-tion of isolated arteries (3). Thus, hypoxia slowed therelaxation of isolated systemic vessels. Because theavailability of oxygen is a major controller of the pul-monary arterial bed, we wondered whether low oxygenwould also slow relaxation in the pulmonary circula-tion. Recently, it was suggested that the controllingeffect of oxygen on the pulmonary arterial bed is medi-ated by vasodilation (4). Our preliminary experimentssuggested that airway hypoxia in isolated rat lungsprolonged the time that was required for vasodilationafter angiotensin II-induced vasoconstriction (5). Weneeded, then, a systematic study of the effects of lowoxygen on the ability of lung vessels to relax.

We knew that because the lung arteries have theunique property of constricting with hypoxia, it wouldbe difficult to determine whether hypoxia had an inde-pendent action on the arterial relaxation. However,if we could abolish hypoxic vasoconstriction, but havea pulmonary arterial bed that would still constrict inresponse to a vasopressor agent, then we could studythe effect of hypoxia on the constriction and subsequentvasodilation in the absence of hypoxic vasoconstriction.Such experiments could provide new insight into theroles of hypoxia in vascular control in the lung.

METHODS

Adult male Sprague-Dawley rats with an average weight of350±30 g were used for the experiments. Following pento-barbital anesthesia (30 mg/kg i.p.) the lungs were isolated asdescribed (6, 7). The lungs were ventilated with 21% 02-5% C02-74% N2 by positive pressure and perfused atconstant flow (0.03 ml/g body wt) with heparinized bloodthat had been collected from three donor rats. After anequilibration period of 30 min at 38°C, each lung was chal-lenged with airway hypoxia (3% 02, 5% CO2, 92% N2) andangiotensin II (Sigma Chemical Co., St. Louis, Mo.) angio-tensin II was injected as a bolus into the pulmonary arteryline (all pulmonary arterial injections were of 0.1 ml vol) toassume normal vascular reactivity of the preparation. The

J. Clin. Invest. (D The American Society for Clinical Investigation, Inc. 0021-9738/81/01/0238/09 $1.00Volume 67 January 1981 238-246

pulmonary artery pressure was measured with a Stathamtransducer (Statham Instruments, Oxnard, Calif.) and recordedcontinuously on a Gilson recorder (Gilson Medical Electronics,Middleton, Wis.). For the angiotensin challenges, and chal-lenges with other vasoconstricting agents, we measured thevasodilation time as the time required for the pulmonary arterypressure to return from the peak of the pressor response to half-way to base line, t = APap/2. Whenever vasodilation time wasin excess of 240 s, we used this value for the statistical calecu-lations. Table I gives an overview of experiments.

Effects of hypoxia on pressor responses to angiotensioniIL. Wemeasured the pressure rise and vasodilation time inresponse to 1 ,ug angiotensin II in seven blood-perfused ratlungs during normoxia (Pao2 133±3 Torr) and again 30 minlater during the 6th min of a continuing hypoxic pressorresponse (Pao2 38±3 Torr, experiment 1). Wethen used threedifferent maneuvers to determine whether base-line perfusionpressure had an effect on vasodilation time following angio-tensin II pressor responses. (a) In two rat lungs (experiment 2)we observed the effect of angiotensin II injection before andafter we raised the perfusion pressure by increasing the bloodflow from 9 to 18 ml/min. (b) In two other rat lungs (experiment3), we gave 1 ,ug angiotensin II at hourly intervals for 4 h, thustaking advantage of the slow increase in the base-line perfusionpressure with duration of perfusion. (c) In two experiments(experiment 4) we gave angiotensin II before and after the per-fusion pressure was raised by the addition of potassium chlorideto the blood reservoir.

In addition, in two rats (experiment 5) we gave increasingdoses of angiotensin II (0.5, 1.0, and 1.5 ,ug) to see whether themagnitude of the pressor response, which also seems todetermine the upstroke time, affected vasodilation time.

Wewished to abolish the pulmonary pressor response tohypoxia, but not to angiotensin II. From a preliminary study,we knew that 5 jxg/ml of sodium arachidonate (Nucheck,Eliason, Minn.), added to the blood reservoir, abolished thehypoxic pressor response (5, 8). To further characterize theeffect of arachidonic acid on lung vasoconstriction, we added5 ,ug/ml sodium arachidonate to the blood reservoir of sixrat lungs during normoxia. Bolus injection of angiotensin IIwas tested before and after arachidonate addition (experiment6). In seven rat lungs, 5 ,ug/ml sodium arachidonate was addedto the blood reservoir during the plateau phase of a hypoxicpressor response (3% FiO2, experiment 7). The pressor re-sponses to 3 and 0% Fio2 were measured within 60 min afterarachidonate addition.

Effects of varying degrees of hypoxia and changes in per-fusate on vasodilation time. In six blood-perfused rat lungs(experiment 8) we performed one hypoxic challenge (Fio2 3%)and one angiotensin II challenge. During the 6th min of the sec-ond hypoxic challenge, we gave the sodium arachidonate to theblood reservoir and observed the disappearance of the hypoxicpressor response. During subsequent hypoxic episodes, wegave angiotensin II 6 min after the ventilating gas waschanged in random order from one containing 21% oxygento a mixture containing either 7, 3, or 0% 02 in 5% Co2,

TABLE IOvervietw of Experiments

Pretreatment ofExperiment Animals Intervention Vasoconstnctor Arachidonate animals

1 (Fig. 1) 7 4 FIO2 to 3% Angiotensin II2 (Fig. 2) 2 T Blood flow Angiotensin II3 (Fig. 2) 2 t Base-line perfusion

pressure (time) Angiotensin II4 (Fig. 3) 2 t Base-line perfusion

pressure (KCI) Angiotensin II5 (Fig. 4) 2 1 Angiotensin II Angiotensin II6 (Fig. 4) 6 Arachidonate Angiotensin II *7 (Fig. 5) 7 4 FiO2 to 3%,0% Angiotensin I *8 (Fig. 6) 6 4 FiO2 7%,3%,0% Angiotensin II *9 (Fig. 7)* 2 4 F1o2 0% Angiotensin *

10 (Fig. 7)* 2 4 FiO2 0% Angiotensin *11 (Fig. 8) 4 4 FIO2 0% Serotonin *12 (Fig. 8) 4 4 FiO2 0% Bradykinin *13 (Fig. 9) 6 FIO2 21% KC1 *14 (Fig. 9) 6 4 FIO2 0% KC1 *15 Table 3 6 FiO2 21% tyramine KC1 * 6-OH-Dopamine

4 FIO2 3%, 0%16 Table 3 6 FIO2 21% KC1 * Reserpine

4 FIo2, tyramine17 Table 3 3 FIo2 21% tyramine *18 Table 3 4 FiO2 21% 0% Angiotensin II * 6-OH-Dopamine19 Table 3 2 FiO2 21% 0% Angiotensin II * Reserpine20 (Fig. 10) 6 FiO2 21% 4 3% 4 0% Angiotensin II *21 (Fig. 10) 4 FIO2 21% 4 3% 4 0% Angiotensin I * 3 d at altitude22 (Fig. 10) 4 FiO2 21% 3% 4 0% Angiotensin II * 5 wk at altitude

* Rat blood was used for all experiments except when plasma was used in experiment 9 and physiologicalsalt solution in experiment 10.

Hypoxic Vasodilation Impairment 239

balance N2. It is possible that erythrocyte or plasma angio-tensinases were responsible for the observed hypoxia-induceddelay in vasodilation. In two lungs (experiment 9) perfusedwith rat plasma in the presence of arachidonate, we observedthe responses to angiotensin II before and during hypoxia.Similar experiments were performed in two lungs (experiment10) perfused with a physiological salt solution composed as fol-lows (in mM): 119 NaCl, 4.7 KCl, 5.5 dextrose, 1.17 MgSO4,15 NaHCO3, 1.18 KHPO4, 50 sucrose, and 1.6 CaCl2; 5 vol% ofFicoll (Sigma Chemical Co.).

Vasodilation time following serotonin- and bradykinin-induced pulmonary vasoconstriction. To determinewhether the hypoxia-induced delay of vasodilation was specificfor angiotensin, we observed (in the presence of arachidonate)the effect of close arterial injections of serotonin (100 gg of5-hydroxytryptamine creatinine sulfate, Sigma Chemical Co.)in four blood-perfused rat lungs (experiment 11) during nor-moxia and hypoxia (0% 02), and of bradykinin (100 ,ug brady-kinin triacetate, Sigma Chemical Co.) in four rat lungs (experi-ment 12) during normoxia and hypoxia.

Vasodilation time following bolus KCl injection in normaland catecholamine-depleted lungs. To test whether thehypoxia-induced delay of vasodilation could be observedwith a pressor substance that is not metabolized duringpassage through the lung, we injected (in the presence ofarachidonate) 15 mg (in 0.1 ml vol) KCI into the pulmonaryarterial lines of six rat lungs (experiment 13) during normoxia.Because of KCl accumulation, each lung received only one in-jection of KCl. Thus, we gave KCl to six different rat lungs (ex-periment 14) during hypoxia (0% 02). Since potassium can re-lease norepinephrine from endogenous stores, any delay inrelaxation during hypoxia could be due to inhibition by hypoxiaof norepinephrine re-uptake or metabolism. To test the effectsof KCl apart from such metabolic influences, we evaluated theKCl response in lungs from rats treated to deplete catechol-amine stores. Wegave six rats (experiment 15) intraperitoneal6-hydroxydopaminehydrobromide (Regis Chemical Co., Mor-ton Grove, Ill.) 2 mg/kg on day 1, 5 mg/kg on day 2, and 10 mg/kgon day 3 -2 h before lung removal and perfusion. Weobservedthe effects of KCI injection (in the presence of arachidonate)in two of these lungs during normoxia, and in two lungsduring hypoxia. In the remaining two lungs we gave 2 mg oftyramine and observed no effect on pulmonary arterial pres-sure. Further, we gave six rats (experiment 16) intraperitonealreserpine (Ciba-Geigy Corporation, Summit, N. J.) 5 mg/kgdaily for 10 d (6). In four of the lungs (in the presence of arachi-donate) we observed the effects of the KCl injection duringhypoxia (0% 02) only. In the two remaining lungs the additionof 2 mgtyramine had no effect on pulmonary arterial pressure.By contrast, tyramine added to the reservoir of lungs fromthree untreated rats (experiment 17) increased the pulmonarvarterial pressure 5±1 mmHg.

The angiotensin II injection could also release norepi-nephrine from lung tissue (9, 10). We therefore performedexperiments in four lungs (experiment 18) from rats pretreatedwith 6-hydroxydopamine and in two lungs from rats pretreatedwith reserpine (experiment 19), in which angiotensin II ratherthan KCl was given during normoxia and hypoxia.

Vasodilation time followving angiotensin lI-induced con-striction in lungs from lows and high altitude rats. Pressorresponses to 1 ,tg angiotenin were measured (in the presenceof arachidonate) during normoxia (21% 02) and hypoxia(3% and 0%02) in lungs from three groups of rats: rats kept atthe laboratory altitude of 1,600 m (experiment 20, n = 6), ratsexposed for 3 d to a simulated altitude of 4,250 m(experiment21, n = 4), and rats exposed for 5 wk to a simulated altitude of4,250 m (experiment 22, n = 4).

Statistical analysis. Data are displayed as mean±+SD. TheStudent's t test was used to evaluate significant differencesbetween groups. When we wished multiple comparison ofgroups, we used a two-way analysis of variance and theStudent-Newman-Keuls test. Differences were considered tobe significant if P < 0.05.

RESULTS

Effects of hypoxia on pressor responses to angio-tensin II. The pressor responses to bolus injection of1 ug angiotensin II were comparable during normoxia(21±1 mmHg) and during hypoxia (18+2 mmHg)but the vasodilation time was longer during hypoxiathan during normoxia (65±5 vs. 22±3 s) (Fig. 1). How-ever, because of hypoxic vasoconstriction, the angio-tensin II response during hypoxia began from a higherbase line and reached a higher peak pressure thanduring normoxia. Increasing the base-line perfusionpressure, either by prolonging the perfusion time or byraising the flow, prolonged vasodilation time (Fig. 2),but the prolongation was less than that caused byhypoxia. Raising the perfusion pressure by the additionof KCI did not prolong the vasodilation time in responseto angiotensin II during normoxia (Fig. 3). Increasinglyhigher doses of angiotensin II raised the peak pressureand seemed to shorten the vasodilation time (Fig. 4).

Effects of sodium arachidonate on angiotensin IIvasoconstriction during normoxia and hypoxia.During normoxia, addition of arachidonate to the reser-

40*

Pap(mm Hg)

30*

20-

10*

0-

tAlI

6 40 80 120 160TIME (seconds)

FIGURE I Vasopressor response to 1 ,mg angiotensin II (All)given as a bolus into the pulmonary artery (arrow) in perfusedrat lungs during normoxia (filled circles) and hypoxia (opencircles). The pulmonary arterial pressures are shown as themean+SDeach 10 s. The blood-oxygen tensions in the lungeffluent are shown (Pao2). Because of vasoconstriction duringhypoxia, the pressor responses to angiotensin II started from ahigher base-line perfusion pressure. The notation P < 0.05is to signify that the relaxation phase (as measured by vaso-dilation time) was slowed during hypoxia.

240 N. F. Voelkel, I. F. McMuirtri, andj. T. Reeves

t (Pop)30o(2 )

(s) 26

22-

18.

14

0

0

* 0

0 0

00

0

t(sO) 0.(s) 30.

* 0 000 20

10,

10 12 14 16 18 20BASE-LINE PERFUSION PRESSURE

(mm Hg)

FiGuRE 2 The relationship between base-line perfusion pres-sure and vasodilation time, t = (Pap/2), following injection of1 ,ug angiotensin II in four isolated blood-perfused rat lungs.The open circles indicate increased perfusion pressure withlong lung-perfusion times in two lungs. The filled circlesindicate that base-line perfusion pressure was increased intwo lungs by increasing the blood flow to the lung, step-wise, from 9 to 18 mllmin.

voir raised the pulmonary arterial pressure 7-12 mmHg (n = 6). In every instance the pressure rise wastransient, pulmonary arterial pressure returned to baseline within 10 min after arachidonate addition. Thepressor response to angiotensin II after arachidonatewas 15+2% less than before arachidonate (n = 6),vasodilation time was the same before and afterarachidonate (Table II, experiment 6).

In seven rat lungs the hypoxic pressor responseranged from 12 to 19 mmHg. As hypoxia continued,addition of arachidonate caused a transient pressurerise (7+2 mmHg) and then a fall to prehypoxic base-line levels of pulmonary arterial pressure. Subsequentchallenges with hypoxia (FiO2 3 or 0%) elicited no vaso-constriction for -60 min after arachidonate addition.Thus, we elected to use arachidonate to abolishhypoxic vasoconstriction and permit study of the re-maining angiotensin pressor response.

In lungs treated in this fashion with arachidonateacid, hypoxia was associated with slowed relaxationafter angiotensin II injection (Fig. 5, Table II, experi-

Pap(mm Hg) Po02 134 Torr

t lug AZ t KCI

I min TIME

FIGURE 3 Vasopressor responses to angiotensin II (All)during normoxia before and after increasing the base-lineperfusion pressure by addition of 15 meq KCI to the bloodreservoir in an isolated perfused rat lung. t(Pap/2) = 16 and18 s before KC1 addition, and 17 and 18 s after base line hadbeen raised by KC1 addition.

ANGIOTENSINIO.5ug

011.0ug 1.5ug

0

5 15A Pap (mmH)

0

25 35

FIGURE 4 Relationship between vasodilation time, t(Pap/2)and the maximum increase in pulmonary arterial pressure(APap) with angiotensin II injection in two isolated blood-perfused lungs. The boluses of angiotensin II in the dosesshown were given into the pulmonary artery at 20-minintervals.

ment 7). Vasodilation time increased when the Po2 ofpulmonary venous blood fell to 60 Torr and increasedsixfold at a Po2 of 25 Torr (Fig. 6). Angiotensin II re-sponses during normoxia in this series of experimentsshowed no change of vasodilation time in comparisonwith the data obtained in these lungs before arachi-donate had been added. Vasodilation time was not af-fected by removing cells and plasma (Fig. 7, Table II,experiments 9 and 10).

Vasodilation time following serotonin or bradykinin-induced vasoconstriction. Vasodilation time was pro-longed following serotonin or bradykinin-induced vaso-constriction during hypoxia (Fig. 8, Table II, experi-ments 11 and 12). Valuies for vasodilation time duringhypoxia were not calculated since complete vasodila-tion did not occur while hypoxia lasted. However,t = A Pap/2 was >240 s.

Vasodilation time followving bolus KCl injections innormal and catecholamine-depleted lungs. Theboluses of KCI induced pressor responses that weresimilar in magnitude to those of angiotensin II. Hypoxiaslowed vasodilation after the KC1 induced constriction(Fig. 9) and the magnitude of the slowing was com-parable to that observed after angiotensin II was ad-ministered. After catecholamine stores had beendepleted by pretreating the rats with either 6-hydroxy-dopamine or reserpine, hypoxia no longer slowed re-laxation after KCI induced constriction (Table III).

In catecholamine-depleted lungs, the pressor re-sponse to angiotensin II was normal during normoxiaand hypoxia. The subsequent vasodilation was pro-longed by hypoxia (Table I). Thus, the catecholaminedepletion did not affect the contraction or relaxationwhen angiotensin II was given during normoxia orhypoxia.

Vasodilation time following angiotensin II-inducedconstriction in lungs from low; and high altitude rats.The pressor responses to angiotensin II were slightly

Hypoxic Vasodilation Impairment 241

TABLE IISummary of Vasodilation Time

APapExperiment 2 2

6 FIo2 21% 26+5*FiO2 21% 28±44

7 (Fig. 5) F1o2 21% 28±3*3% 72+4*0% 180±6*

FIO2 21% 30±343% 76±440% 190±5t

9 (Fig. 7) FIo2 21% 22, 200% 175, 180

10 (Fig. 7) FIO2 21% 24, 200% 140, 135

11 (Fig. 8) FIo2 21% 63±40% >240

12 (Fig. 8) FIO2 21% 42±40% >240

20 (Fig. 10) FIO2 21% 27±33% 68±40% 195±5

21 (Fig. 10) FIo2 21% 26±33% 48+40% 74±4

22 (Fig. 10) FIO2 21% 24+23% 28±30% 38±3

* Before arachidonate.4 After arachidonate.

greater in the rats maintained for 5 wk at high al-titude, than in the low altitude rats, or those thathad been at high altitude for only 3 d. Vascular hyper-trophy that resulted from long high altitude residence,presumably permitted a rather large response to angio-tensin II (Table IV) (11). However, the vasodilationtime during hypoxia decreased with increasing durationof stay at high altitude (Fig. 10, Table II, experiments20-22). The vasodilation times during normoxia andhypoxia in these low altitude rat lungs pretreated witharachidonate (Table IV) were similar to the values ob-served in the low altitude rat lungs not pretreated witharachidonate (Fig. 1). The values of Po2 were similar inthe two groups.

DISCUSSION

In the present study we found that angiotensin IIgiven to isolated perfused rat lungs during hypoxia

30.

20

10-

pop(mm Hg) AA

t lgAE t lgAH- HYPOPi OX L\\\\, _P__X_I_A_

I min TIME

FIGURE 5 Vasopressor responses to angiotensin II (AII)during hypoxia (3% FiO2) in an isolated blood-perfused ratlung. Left: Hypoxia caused an 8-mm Hg increase in perfusionpressure; vasodilation was delayed following angiotensin IIinjection. Right: 15 min after the addition of 5 i.g/ml sodiumarachidonate acid (AA) to the blood reservoir, hypoxia nolonger caused vasoconstriction but the vasodilation afterangiotensin II-induced vasoconstriction was delayed.

Pap30 (mm Hg)

10]

Fi02%

21

301

10 7

30q J 3

30i*1 ~~~~~~~0

101 t iugAIEI min ' TIME

200.

120~

40.

OF

TIME FOR 50%VASILATION(s)

,4," nee* P<0.05

7 3 0 FiO2 %21120 60

PaO2 Torr20

FIGURE 6 Vasopressor responses with varying degrees ofhypoxia to 1 ,ug angiotensin II injections (arrow) in oneexperiment with an isolated blood-perfused rat lung. Sodiumarachidonate had been added to the blood reservoir 15 minbefore the first angiotensin II challenge. Hypoxic vasocon-striction but not pressor responses to angiotensin II wereabolished. Shown at the right of each curve is the oxygentension (Pao2) of the pulmonary effluent blood when the lungwas ventilated with a mixture containing (from top to bottom)21, 7, 3, or 0% oxygen. Bottom: Relationship between vaso-dilation time and the oxygen tension in the pulmonary effluentblood after injection of 1 ,ug angiotensin II during hypoxiaof varying degrees of severity. Vasodilation time increasedwhen the Po2 in the pulmonary venous blood fell to 60 Torror less. Asterisk (*) indicates that vasodilation time duringhypoxia exceeded that during normoxia.

242 N. F. Voelkel, I. F. McMurtry, and J. T. Reeves

30.

10*

30-

10

Pop(mm Hg)

PLASMAPERFUSEDLUNG

PaO2 132 Torr PaO2 27 Torr

tilugAlE-

min

Pap(mm Hg)

PHYSIOL SALT SOLUTION

PaO2 130 Torr P002 31 Torr

lug AIE luggAI

I-min YP....

FIGuRE 7 Pressor responses to angiotensin II (All) injection(arrows) during normoxia and during hypoxia (Fio2 0%) inone lung perfused with rat plasma (top) and in one lung per-

fused with physiological salt solution (bottom).

caused the expected pressor response, but that subse-quent vasodilation was delayed when compared withnormoxic lungs. We then attempted to separate theconstricting effect of hypoxia from a hypoxic impair-ment of vasodilation. During normoxia neither raisingthe perfusion pressure by increasing flow before ad-ministering angiotension II, nor varying the dose ofangiotensin II to yield varying peak pressures pro-

duced the delayed vasodilation observed duringhypoxia. These findings suggested that there was an

effect of hypoxia that was independent of base-lineor peak pressure. We then abolished the hypoxicpressor response by administering sodium arachidon-

ate to the perfusate of the preparation (5, 8). Arachi-donate, a prostaglandin precursor, appears to stimulatethe synthesis of vasodilator prostaglandins, probablyprostacyclin, PGI2 (8), a potent pulmonary vasodilator(12-14). In the dose of arachidonate used, a pressorresponse to angiotensin II persisted. Arachidonateitself did not affect the relaxation of the lung vesselsafter angiotensin II constriction during normoxia or

hypoxia. In the presence of arachidonate, hypoxia didnot alter perfusion pressure, but still delayed vasodila-tion after angiotensin 1I-induced constriction. Thus, we

considered that hypoxia, per se, slowed relaxation fol-lowing an angiotensin II-induced constriction. Theeffect was independent of hypoxic vasoconstriction, andtherefore, independent of capillary recruitment whichmight have altered the transit time of lung perfusateor volume of distribution of the vasoactive substances.

Progressively impaired vasodilation after angio-tensin II-induced constriction, progressively de-creasing Po2, and a hypoxia-induced delay in vasodila-tion after constriction by serotonin and bradykininraised the possibility that hypoxia could have alteredlung vascular endothelial function. Specifically,hypoxia could have decreased uptake or metabolismof each of these substances. Because impaired vaso-

dilation during hypoxia was also observed in lungsperfused with physiological salt solution, inactivationby hypoxia of the angiotensinases in the red cells or

plasma (15, 16) could not be implicated. However,hypoxia could have slowed the normally limited angio-tensin breakdown within the lung tissue (17-19).Likewise, hypoxia could inhibit degradation of brady-kinin or serotonin (20, 21) and account for the impairedvasodilation. Our findings that hypoxia slowed vasodila-tion followed KCl-induced constriction only in lungswith intact catecholamine stores were compatible with

Pap mmHg)

30 Pao2 134 Torr

10 t 100ug BRADYKININ

30] Pa2 32 Torra

10 t 100ug BRADYKININ I

min TIME

POp (mm HO)Pc

I min TIME

FIGURE 8 Pressure response to bolus injection (arrows) of bradykinin (left) and serotonin(right) in blood-perfused lungs pretreated with sodium arachidonate. Shown are responsesduring normoxia (top) and during hypoxia (Fio2 0%) (bottom).

Hypoxic Vasodilation Impairment 243

302 125 Torr

t 100ug SEROTONIN

Pao2 30 Torr

t 100 ug SEROTONIN

/ / HYPOXIA4 X/X71Z

//X/, HYPOXA ~/,

TABLE IIIVasodilatiotn Time durinig Normoxia and Hypoxia in

Blood-perfused Lungs from Catecholam ine-depletedand Nondepleted Rats

I = _____

Pressor substance Po2 APap 2 i

Torr Torr s

No catecholaimine depletion

0 30 50 70 90TIME (s)

110 130

FIGURE 9 Change in pulmonary artery perfusion pressureexpressed as percent of peak response (pressure minus base-line pressure x 100, divided by peak pressure rise withKCI) after bolus injection (arrow) of KCl into the pulmonaryartery of 12 blood-perfused rat lungs pretreated with sodiumarachidonate. In six rat lungs KCI was administered duringnormoxia, in six different rat lungs KCI was given duringhypoxia. Mean+SDare shown at 10-s intervals. The P < 0.05indicates that vasodilation time during hypoxia exceeded thatof normoxia.

KCIKCIAII*AII

135±342±3

133±233+3

23±220±216±116±1

70±5164±1030±2

184+7

Catecholaminie depletion by reserpine pretreatment

KCIAllAll

29±3133±230±3

20±117±217±1

40±5t28±3

180±5

Catecholamine depletion by 6-OH-dopamine pretreatment

the concept that KCI released norepinephrine (22, 23)from the lung, and that hypoxia impaired subsequentnorepinephrine catabolism or re-uptake. It is possible,therefore, that hypoxia had acted on the metabolismof a variety of vasoconstrictor substances to prolongtheir effect. However, since pulmonary removal ofserotonin (24, 25) and norepinephrine (26, 27) hasbeen shown to be overwhelmed by microgram doses ofthese substances, the transport systems were mostlikely exhausted by the doses necessary to achievemarked pulmonary vasoconstriction. Furthermore,ouabain (1 mM)and cooling of the lungs, both of whichhave been shown to decrease pulmonary serotoninand norepinephrine removal (28-30), did not mimicthe effect of hypoxia. Both substantially raised the mag-

nitude of serotonin and angiotensin II pressor re-

sponses and slowed vasodilation time to a lesser ex-

tent than did hypoxia (data not shown). Finally, hypoxicimpairment of the active endothelial cell transport sys-

tem does not explain our findings, because the pul-monary arterioles, not the capillaries with their largeendothelial surface, are probably the major site ofagonist-induced vasoconstriction (31). Therefore, we

reason that some property of the vascular smoothmuscle cell is affected at relatively high oxygen

tensions leading to impaired vasodilation.Angiotensin, serotonin, bradykinin, and norepi-

nephrine probably initiate vasoconstriction by in-creasing the free cytoplasmic calcium concentrationin smooth-muscle cells. We had considered thathypoxia could prolong the effect of these substancesby delaying sequestration and/or extrusion of intra-cellular calcium. The injection of a bolus of concen-

KCIKCIKCIAllAll

130±340±330±2

131±230±2

27±322±217±216±115±1

50±550+5153+±331±3

165±4

22244

Showni are the pressor substances given as a bolus by closearterial injection, the Po2 of the venous blood draining thelung, the maximal increase in pulmonary arterial pressure

(APap) following the pressor injection, and the vasodilationtime (t = APap/2), which is the time for the pulmonary arterialpressure to fall back halfway to base-line values. All lungs

had been pretreated vith arachidonic acid to abolish thehypoxic pulmonary pressor response.

* Angiotensin II.Values in the catecholamine depleted lungs which differ

(P < 0.05) from nondepleted lungs.

trated KCI into the pulmonary artery should havecaused a transient depolarization of, and calcium entryinto, the pulmonary arterial smooth-muscle cells. Inthe catecholamine-depleted lungs, in which norepi-nephrine release presumably did not complicate theinterpretation, calcium entry should have been themajor, if not sole, stimulus for vasoconstriction; Ca++sequestration and extrusion should have been the onlyprocess mediating relaxation. In this case, hypoxiacaused no slowing of the subsequent vasodilation.Although calcium flux could not be measured in theseexperiments, these findings suggest that under circum-stances where receptor interactions can be excluded,the hypoxia-induced delay of vasodilation was notbecause of impaired calcium extrusion or sequestra-tion. Hypoxia could possibly alter the affinity of re-

ceptor binding sites on the vascular cell membranes,

244 N. F. Voelkel, I. F. McMurtry, and J. T. Reeves

100

i) 80z0U,LwL 60Cry

L 400-

0--R 20

6666

422

TABLE IVHypoxic Vasodilation Time after Angiotensin II

Injection in Rat Lungs from Rats with andtvithout Exposure to High Altitude

Base-line t = _PapPo, pressor APap 2

Torr Torr Torr s

Rat lungs from low altitude (controls) (n = 6)

135+439±4

12+112±1

+15+1+ 15±2

Lungs from high altitude rats (acute exposure 3d)at 4,500 m (n = 4)

132+438±4

13±113.5+ 1

+ 14±* 1+14+1

Lungs from high altitude rats (chronic exposure 5 wkat 4,500 m) (n = 4)

134±240+2

13.5+ 114.5+ 1

+ 19±1+20±2

Shown are the Po2 of the venous blood draining the lung,the pulmonary arterial perfusion pressure just before injectionof angiotensin II (base-line pressure), the maximal increasein pressure with angiotensin II injection (APap) and the vaso-dilation time (t = APap/2). All lungs had been pretreatedwith arachidonic acid to abolish the hypoxic pressor response.n is the number of experiments.* Values differ (P < 0.05) from low altitude control values.

LOW ALTITUDEI

l1

3 DAYS AT HIGH ALTIT E

t ~~~~~~:t

or any intracellular response subsequent to the agonist-receptor interaction, or both. Weare intrigued by thepossibility that hypoxia could alter agonist-receptorinteractions because prolonged vasodilation duringhypoxia was seen only with agonists that bind tospecialized receptors; when KCl, which presumablycauses cell calcium influx without binding or activa-tion of a hormone receptor, was used, vasodilationin catecholamine-depleted lungs was not delayedduring hypoxia.

In recent studies, we proposed that metabolic adapta-tions to hypoxia can occur with chronic exposure tohigh altitude. Such adaptation might account for theblunting of hypoxic vasoconstriction in lungs fromhigh altitude acclimated rats (11, 32). We expectedand found that in lungs from rats exposed for severalweeks to high altitude, hypoxia would also be lesseffective in prolonging vasodilation time after angio-tensin-induced vasoconstriction. It is possible thatsome adaptation of cell membrane function or cellmetabolism during high altitude residence allows arelatively rapid vasodilation of pulmonary arterialsmooth muscle during hypoxia.

Our results indicate that moderate degrees of alveolarhypoxia prolong the action of some pharmacologicvasoconstrictor suibstances on the pulmonary circula-tion in the absence of hypoxic vasoconstriction. Weconclude that alveolar hypoxia probably impairsvasodilation because of interference with vascularmuscle properties that adapt during high altituderesidence and not because of impairment of the meta-bolic degradation of the vasoconstrictor agonists.Such hypoxia-induced delay of relaxation following avasoconstriction has not previously been shown forlung vessels and might be considered as an actionof hypoxia on the lung circulation apart from its knownconstrictor effect.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Healthgrants HL-14985 and HL-20429.

HYPOXIA 0% 02

TIME

FIGURE 10 Pressor responses to bolus injection of 1 Ag angio-tensin II (arrows) in blood-perfused rat lungs pretreated withsodium arachidonate. Left: Responses during normoxia inlungs (from above downward) from a low altitude rat (1,600m), a rat that had lived for 3 d at a simulated altitude of 4,250m, and a rat that had lived for 5 wk at a simulated altitude of4,250 m. Right: Responses to angtiotensin II in each of therats when the lungs were ventilated with 0%oxygen.

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