Epinephrine-induced pulmonary oedema in rats is inhibited by corticotropin-releasing factor

Plru~n~ac~olo,~ic~ul Rr~srutrh.

Vol. 26, No. I, 1992

85

EPINEPHRINE-INDUCED PULMONARY OEDEMA IS INHIBITED BY CORTICOTROPIN-RELEASING

IN RATS FACTOR

S.M. SERDA and E.T. WEI* School

of’ Public

Health,

University

of California, USA

Berkeley,

California

947-70.

SUMMARY In various animal models of injury to skin, mucous membranes, muscle and brain, corticotropin-releasing factor (CRF) attenuated vascular leakage in the injured tissues. Here, the effects of CRF on a rat model of pulmonary oedema were examined. Male albino rats (220-290 g) received saline or CRF s.c., 30 min before pentobarbital anaesthesia, 60 mg/kg i.p., and 1 h before I-epinephrine bitartrate (Epi), 30 ,ug/kg i.v. Within 30 min after Epi all (n=27) saline-pretreated rats were dead from pulmonary oedema, but animals receiving human/rat CRF at doses of 7 to 57 ,ug/kg S.C. (n=25) were all alive. Body wt, wet and dry wt of lungs were used to calculate an oedema index. This index increased from 3.6fO.l to 9.6-t0.3 after Epi but was inhibited by 87% after CRF 28 ,ug/kg S.C. The ED50 of CRF for reducing pulmonary oedema was 3.2 (1.3-7.4) pug/kg S.C. Mean arterial pressure increased from 1191!14 to 167*2 mmHg after Epi 10 ,ug/kg i.v., but was not different (118+3 to 169+4 mmHg) after CRF pretreatment, 6 ,ug/kg s.c., a dose which reduced lung oedema. Pharmacokinetic estimates suggest that plasma levels of CRF sufficient to attenuate lung oedema in rats approximate those seen in pregnant women at delivery, raising the possibility that endogenous CRF may protect the maternal organism during parturition. KEY WORDS:corticotropin-releasing

factor, epineprhine,

pulmonary

oedema

INTRODUCTION Acute pulmonary oedema can be produced in rats by stimulated release of adrenomedullary catecholamines [l] or by intravenous injection of epinephrine (Epi) [2, 31. Meltzer, who first described Epi-induced pulmonary oedema in the rabbit in 1904 [4], explained this phenomenon as being due to the rapid redistribution of blood from the systemic into the pulmonary circulation. The increases in pulmonary microvascular pressure are transient, but thought sufficient to directly injure the endothelium of small blood vessels, resulting in increased *To whom correspondence should be addressed. I (M-66

I ~/Y2/OSOOSS-o7/$O~.oO/O

0 1992 The Italian Pharmacological

Soc~ci)

86

Pharmacolo~it~al

Research,

Vol. 26, No. 1, 1992

vascular permeability and haemorrhage [5]. This type of ‘pressure’ oedema [6-81 is inhibited by a-adrenoceptor antagonists, by proteolytic enzymes, by procedures such as pithing which lowers blood pressure and by ligation of the portal vein which reduces venous return [2, 3, 93. Previously, we reported that corticotropinreleasing factor (CRF), a 41-residue hypothalamic hormone [lo], reduced vascular leakage from the trachea of rats after exposure to formaldehyde vapours [ 11, 121. In the course of these studies, it was noticed that CRF appeared to protect the animals against the lethal and oedematogenic effects of formaldehyde. Here, the ability of CRF to inhibit Epi-induced pulmonary oedema in the rat was investigated.

MATERIALS

AND METHODS

Male Sprague-Dawley rats weighing 220-290 g (Simonsen Labs, G ilroy, CA) were used in all experiments. Animals received saline or CRF S.C.30 min before pentobarbital anaesthesia, 60 mg/kg i.p., and 1 h before 1-Epi bitartrate, 30 pg/kg i.v. (equivalent to 90 nmol/kg base, all doses refer to the bitartrate salt). This single dose of Epi is sufficient to produce pulmonary oedema in the pentobarbitalanaesthetized rat [2]. In some experiments, blood pressure was recorded from a polyethylene cannula placed in the left common carotid artery and connected to a transducer and polygraph. Mean arterial pressure (MAP) was calculated as MAP= diastolic blood pressure+l/3 (systolic blood pressure-diastolic blood pressure). For blood pressure experiments, the dose of Epi bitartrate was reduced to 10 pug/kg i.v. in order to avoid the lethal effects of Epi. To compare the efficacy of CRF by different routes of administration, saline or CRF, 5 pug/kg, was injected intra-arterially (via a cannulated common carotid artery) or intravenously (via a cannulated jugular vein) into the pentobarbitalanaesthetized rat and 45 min later, the animals were challenged with Epi. For the vagotomy experiments, saline or CRF was injected S.C.and 15 min later, the rats were anaesthetized with sodium pentobarbital. The vagus nerves were both ligated at the C3 level and the trachea cannulated. Epi bitartrate, 3O,~g/kg i.v., was then injected 1 h after saline or CRF. In other experiments, anaesthetizeh rats received a-helical-CRF(9-41) 92 pug/kgi.v. plus CRF, or dexamethasoneacetate, 2.5 mg/kg i.v. before Epi bitartrate, 30 pug/kgi.v. Respiratory rate was monitored with a thermistor placed near the nostrils and the time to cessation of breathing recorded. Animals that survived Epi injections were killed with concentrated pentobarbital 30 min after Epi and the lungs were dissected from all rats. The magnitude of lung oedema was estimated according to the procedures of Poulsen [ 131. Lungs were blotted on paper towels and weighed (wet wt), then placed in a 68 “C oven for 48 h and re-weighed again (dry wt). The ratio of 1000 (wet wt-dry wt)/body wt was termed the oedema index. Lungs (n=4 each from saline or CRF-treated groups) were also perfused intra-tracheally with 10% neutral-buffered formalin, dehydrated with a graded series of alcohol, cleared in xylene, infiltrated with liquid paraffin, cut into 5 p sections, and stained with hematoxylin and eosin for microscopic examination. The human/rat CRF and cr-helical-CRF(9-41) used here (Peninsula

Laboratories, Belmont, CA) had been synthesized by solid phase methods and was purified to ~95% by high performance liquid chromatography. 1-Epi bitartrate and dexamethasone acetate were obtained from Sigma Chemical Co., St Louis, MO. Chemicals were dissolved in either distilled water or saline and administered at 0.05 ml/100 g body weight. All data are presented as mean+st+t. Statistical significance was assessed by analysis of variance (ANOVA) with the Studentized range-test or the Student’s f-test [14]. The median effective dose (ED50) of CRF for inhibiting pulmonary oedema was calculated according to Litchfield and Wilcoxon [ 151.

RESULTS Injection of Epi bitartrate, 30 pug/kg i.v., into anaesthetized rats produced laboured breathing and within the observation period of 30 min all saline-pretreated animals (n=27) were dead with a mean survival time of 3.5kO.6 min. In these animals, redstained froth was present in the upper respiratory tract and fluids oozed freely from the trachea when it was cut. The oedema index for untreated rats was 3.6fO. 1 (n=6). This index in Epi-treated rats was 9.6f0.3 (n=27) and, relative to normal animals of similar body weight, corresponded to a 2.14-fold increase in lung weights. CRF administered S.C. 1 h before Epi inhibited the development of pulmonary oedema in a dose-dependent relationship as shown in Fig. 1. The ED50 (95% CL) of CRF for inhibiting pulmonary oedema relative to saline-treated rats was 3.2 (1.3, 7.4) ,ug/kg S.C. Animals receiving the higher CRF doses of 7 to 57 pug/kg S.C.(n=25) were all alive at the end of the 30 min observation period (P< 0.001 Student’s t-test, relative to saline controls). The enlarged lung surfaces of Epi-treated rats were spotted with many haemorrhagic patches. Microscopically, numerous aggregates of erythrocytes could be seen in the perivascular, peribronchiolar and perialveolar spaces, and in

ED50 = 3.2 (1.3-7.4)

&kg

S.C.

I : :::: 0.5

1 1.0

10.0

CRF &kg

dose-response Fig. 1. Log-probit 6-7 per dose level ).

SC.

relationship

Injected

1 hr before

Epinephrine

of CRF for inhibiting

pulmonary

o&ma

(H=

88

200

T

Ii0

O-0

Saline

0-0

CRF

6 &kg

Research,

S.C. 1 hr before

epinephrine

b

-‘5 Min ofter

Pharmacological

epinephrine

bitartrote

5 10 W/kg

Vol. 26, No, I, 1992

lb i.v.

Fig. 2. Mean arterial pressure response to Epi bitartrate IO pug/kg i.v. in pentobarbitalanaesthetized rats with or without CRF pretreatment (6 pg/kg S.C. 30 min before Epi).

the alveolar lumen. Extensive interstitial oedema, ruptured alveolar membranes and emphysematous air pockets were observed. If a colloidal dye such as Monastral blue (30 mg/kg i.v. at a volume of 0.2 ml/100 g) was administered before Epi, the dye could be subsequently detected in transudates within the alveoli, in scattered deposits on the alveolar wall, and as particles engulfed by alveolar macrophages. By contrast, the lungs of CRF-treated rats after Epi retained their normal pink colour and haemorrhagic patches were not observed. The lung sections from these rats revealed some interstitial alveolar oedema and thrombosis in the alveolar capillaries but no displacement of erythrocytes into the perivascular, peribronchiolar, or perialveolar spaces. Monastral blue administered to CRF-treated rats was retained within the vascular compartment after Epi. In saline and CRF-treated animals, the MAP, averaged over 10 min before Epi injection were 119&4 mmHg (n=8) and 118f3 mmHg (n=8), respectively (Fig. 2). After a sublethal dose of 10 ,ug/kg i.v. Epi, the MAP of saline-treated rats increased to a peak of 167f2 mmHg, a level similar to those found in rats pretreated with CRF 6 ,ug/kg S.C. 1 h before Epi (169*4 mmHg). In these animals, pretreatment with CRF at 6 ,ug/kg S.C. inhibited lung oedema produced by the lower dose of Epi (oedema index for saline-treated rats was 5.2kO.5 vs 3.9kO.2 for CRF-treated rats, PcO.025, Student’s r-test). As shown in Table I, a-helical-CRF(9-41), a synthetic CRF receptor antagonist [16], prevented the inhibitory effects of CRF on Epi-induced pulmonary oedema. CRF was more effective when injected by the intravenous jugular route than when given by the intra-arterial carotid route, suggesting that its sites of action were peripherally and not centrally mediated. Bilateral vagotomy slightly increased the oedema index after Epi injection, but it had no effect on the prevention of pulmonary oedema by CRF. Dexamethasone acetate (2.5 mg/kg i.v.), a potent synthetic corticosteroid, did not affect Epi-induced pulmonary oedema.

Phur-mur~olqic~url Research, Vol. 26, No. I, 1992

X‘)

Table I Factors affecting CRF modulation of Epi-induced pulmonary edema Treatments

Time’ (min)

Oedema

Index

Saline+Saline CRF s.c.+Saline Saline+Epi CRF s.c.+Epi

28 28

60 60 60 60

3.6+0. I 3.9fO. I 9.9kO.8” 4.4kO.4

Saline+CRF s.c.+Epi a-CRF(9-4 I )+CRF s.c.+Epi

28 28

30 30

4.1 f0.3 9.4+2.2**

5 5 5 5

45 45 45 45

10.5+0.8 4.8+0.2** I I .6fO.S 8.3fO.S*”

Vagotomy+Saline+Epi Vagotomy+CRF s.c.+Epi

28

60 60

10.910.4 4.0fO. I **

Saline+Epi Dexamethasone

-

30 30

9.5kO.7 10.9+0.2

Saline i.v.+Epi CRF i.v.+Epi Saline i.a.+Epi CRF i.a.+Epi

i.v.+Epi

‘Time between last drug administration and injection of saline or I -epinephrine bitartrate 30 pg/kg i.v. a-helical-CRF(9-41) 92 pg/kg was injected 5 min before CRF. The dose of dexamethasone acetate was 2.5 mg/kg (n=6-8 per group). ‘“P
DISCUSSION The results here show that CRF can prevent the massive, rapidly fatal, pulmonary oedema that develops in rats after intravenous injection of Epi. The potency and efficacy of CRF on this endpoint, as shown by an ED50 of 3.2 ,uglkg and histological evidence of the virtual absence of haemorrhage and protein exudation into the alveolar air spaces, exceeded activities found in other tests of CRFs antiintlammatory effect [ 121. For example, CRF inhibits vascular leakage in skin and muscle after strong heat or mechanical injuries, but with EDSOs of 29 ,ug/kg and 24 ,ug/kg, respectively, and a maximum inhibition efficacy of 60 to 65% [ 12, 17 1. The potency and selectivity of CRF for the lung may have physiological significance since pharmacokinetic data (18, 191 suggest that plasma levels of CRF producing anti-oedematogenic actions on the lung approach the high levels of CRF seen in the final stages of human pregnancy [20,21]. Athough CRF may be inactivated by binding to plasma protein during human pregnancy [22], it is possible that endogenous CRF-like substances may exert protective antiinflammatory effects on the maternal organism during parturition. increased venous return and elevated peripheral resistance are important elements of Epi-induced pulmonary oedema because a-adrenoceptor antagonists can protect both against the lethal effects of Epi and the oedema [2. 31. CRF.

90

Pharmacological Research, Vol. 26, NO. I, 1992

injected intravenously, lowers blood pressure in several species, including the rat, because of a direct vasodilatory action on certain vascular beds [lo, 23,241. Urotensin I, a CRF superfamily peptide found in the urophysis of fish, is also hypotensive and attenuates adrenergic vasoconstriction in vascular smooth muscle preparations [25]. The systemic hypotensive effect of CRF, however, did not appear to play a role in the prevention of Epi-induced pulmonary oedema because CRF prevented oedema when MAP was not lowered by subcutaneous CRF, and because CRF did not affect the surge in blood pressure produced by Epi. The mechanism by which CRF prevents distension and rupture of lung alveolar membranes is still unclear. Parasympathetic innervation of the lung does not play a part because vagotomy did not alter the CRF effect. Also, release of adrenal steroids is unlikely to contribute to protection because dexamethasone did not reduce oedema. The results are compatible with a direct hormonal action on cell-cell or cell-substratum adhesion mechanisms, but experimental evidence on this possibility is still lacking. Recently, it has been reported that vasoactive intestinal peptide [26] and atria1 natriuretic peptide can prevent oedema in the isolated perfused lung [27, 281. Thus, these peptides, like CRF, exhibit unusual protective effects on lung permeability. It would appear that further studies of these peptides and CRF may provide clues on how pulmonary oedema can be modified.

ACKNOWLEDGEMENTS We thank Alan P. H. Lin and Gordon G. Gao for assistance. Supported by grants DA-00091, ES-04505 from the USPHS, the Chevron Risk Assessment Research Program, the Northern California Occupational Health Center and the University of California Toxic Substances Teaching and Research Program.

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