Increased pulmonary vascular permeability and oedema induced by intrathecally injected endothelins in rat

European Journal of Pharmacology 344 Ž1998. 251–259

Increased pulmonary vascular permeability and oedema induced by intrathecally injected endothelins in rat Philippe Poulat, Rejean Couture ´

)

Department of Physiology, Faculty of Medicine, UniÕersite´ de Montreal, ´ C.P. 6128, Succursale centre-Õille, Montreal, Que., Canada H3C 3J7 Received 23 June 1997; revised 25 November 1997; accepted 2 December 1997

Abstract The intrathecal Ži.t.. injection of endothelins to conscious rats was found to cause respiratory arrest. To gain some insights into this central phenomenon, peripheral vascular permeability and lung oedema were measured after i.t. and i.v. injections of these peptides. When injected at T-8 spinal cord level, endothelin-1 Ž65 and 650 pmol. and endothelin-3 Ž650 pmol. enhanced vascular permeability in the lungs by 22-fold and 7-fold, respectively, and caused sudden death at the highest dose. Less prominent increases Žbetween 1.4- and 2.2-fold. of vascular permeability were observed in other tissues Žtrachea, kidney, ears, skin of hind paws and back skin. with endothelin-1. Endothelin-1 Ž650 pmol. caused a similar increase Ž27-fold. in lung vascular permeability when injected at T-2, although the response was significantly less Ž P - 0.05. if injected at the L-4 Ž15-fold. spinal cord level. Only endothelin-1 produced lung oedema when injected at the T-2 or T-8 level. In contrast, intravenous injection of endothelins-1 and -3 Ž650 pmol. did not produce lung oedema and the lung vascular permeability was increased by only 1.4–1.6-fold and all rats survived. The prior i.t. injection of 6.5 nmol BQ-123 ŽcyclowD-Trp, D-Asp, L-Pro, D-Val, L-Leux., a selective endothelin ETA receptor antagonist, prevented the increases of lung vascular permeability and oedema and the mortality induced by i.t. endothelin-1 Ž650 pmol.. Whereas i.v. treatment with phentolamine Ž2 mgrkg. or pentolinium Ž25 mgrkgq 50 mgrkg per h = 15 min. abolished the lung vascular permeability changes evoked by endothelin-1 Ž650 pmol., atropine Ž1 mgrkg., N G-nitro-L-arginine Ž50 mgrkg. or indomethacin Ž5 mgrkg. had no effect. Moreover, the effects of endothelin-1 were attenuated in capsaicin pretreated rats Ž125 mgrkg, 10 days earlier. and almost abolished in rats subjected to sympathectomy with 6-hydroxydopamine Ž100 mgrkg, 24–48 h earlier.. All these treatments except atropine and N G-nitro-L-arginine prevented the endothelin-1-induced lung oedema and reduced the lethality by around 50%. These results suggest that the increases of pulmonary vascular permeability and oedema induced by i.t. endothelin-1 are due to an intense pulmonary vasoconstriction mediated by a-adrenoceptors following the release of catecholamines in response to the activation of endothelin ETA receptor in the spinal cord. This central phenomenon seems to be reflexogenic, including the involvement of primary afferent C-fibers and spinal cord ascending fibers to the brain. Thus, endothelin-1 could play a role in neurogenic pulmonary oedema through a central mechanism. q 1998 Elsevier Science B.V. Keywords: Endothelin; Spinal cord; Endothelin ETA receptor; Vascular permeability; Oedema; Lung

1. Introduction Endothelins belong to a family of structurally related 21 amino acid peptides including endothelin-1, endothelin-2, endothelin-3 and sarafotoxins. These peptides exert a variety of vascular and non-vascular effects through the activation of three guanine nucleotide-binding regulatory protein ŽG-protein.-coupled receptors containing seven putative transmembrane spanning domains ŽMasaki et al., 1994;

) Corresponding author. Tel.: q1-514-3437060; fax: q1-514-3432111; e-mail: [email protected]

0014-2999r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 1 4 - 2 9 9 9 Ž 9 7 . 0 1 5 6 9 - 0

Rubanyi and Polokoff, 1994; Rae et al., 1995.. The three receptors have been cloned: the endothelin ETA receptor, which has a higher affinity for endothelins-1 and -2 than for endothelin-3; endothelin ETB receptor, which shows similar affinities for the three peptides and the endothelin ETc receptor, which exhibits higher affinity for endothelin3 than for endothelin-1 ŽMasaki et al., 1994; Rubanyi and Polokoff, 1994.. Endothelins are produced by the endothelium and non-vascular endothelial cells of the respiratory, gastrointestinal, renal and urogenital tracts ŽRubanyi and Polokoff, 1994.. In addition, endothelin-1, endothelin-1 mRNA and endothelin-1 receptor binding sites were found in the brain, spinal cord and dorsal root ganglia ŽGiaid et

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P. Poulat, R. Couturer European Journal of Pharmacology 344 (1998) 251–259

al., 1989; Jones et al., 1989; Yoshizawa et al., 1990; Kar et al., 1991; Gulati and Rebello, 1992; Niwa et al., 1992., suggesting that endothelin-1 could also act as a putative neurotransmitter andror neuromodulator in the central nervous system ŽGulati and Srimal, 1992.. Recently, we reported that intrathecally injected endothelin-1 at T-9 spinal cord level produces dose-dependent and prolonged increases of mean arterial pressure concomitantly with dramatic decreases of heart rate in unanesthetized rats. The pressor response and bradycardia were ascribed to the activation of the sympatho-adrenal nervous system and to a vagal reflex mechanism, respectively ŽPoulat et al., 1994.. Both effects are mediated by the activation of endothelin ETA receptors in the spinal cord. This is consistent with the presence of endothelin ETA receptor binding sites in the rat spinal cord ŽGulati, 1991; Bertelsen et al., 1992.. Rats injected intrathecally with endothelin-1 Ž650 pmol. died by respiratory arrest within a few minutes. This toxic effect was prevented by the prior intrathecal injection of BQ-123 ŽcyclowD-Trp, D-Asp, L-Pro, D-Val, L-Leux., a selective endothelin ETA receptor antagonist ŽIhara et al., 1992.. Whereas this lethal effect of endothelin-1 was significantly reduced by the prior intrathecal injection of smaller doses of endothelin-1, the cardiovascular response to endothelin-1 was not subjected to desensitization. Moreover, the mortality was reduced in rats pretreated with either capsaicin, a neurotoxin for primary sensory C-afferents ŽBuck and Burks, 1986., or 6-hydroxydopamine, a neurotoxin for sympathetic fibers. The latter treatments had no effect against the cardiovascular response to endothelin-1. Thus, the lethal and cardiovascular responses to endothelin-1 appear to be mediated by distinct mechanisms following the activation of intraspinal endothelin ETA receptors ŽPoulat et al., 1994.. The purpose of the present study was threefold: first to compare the intrathecal and intravenous effects of endothelin-1 and endothelin-3 on increased vascular permeability in several peripheral organs and on pulmonary oedema in the conscious rat, second to ascertain the participation of intraspinal endothelin ETA receptors in the pulmonary hypermeability and oedema induced by endothelin-1 and third to examine the contribution of the sympathetic nervous system and other endogenous putative mediators in this central action of endothelin-1 on the lung.

2. Materials and methods 2.1. Animals Male Wistar rats Ž250–300 g. were purchased 3–5 days prior to experiments from Charles River, St. Constant, Quebec, Canada and housed four to five per cage under a 12 h light–dark cycle in a room with controlled temperature Ž208C. and humidity Ž53%. with food ŽCharles River Rodent. and water available ad libitum. All surgical and

chemical procedures conformed to the guiding principles for animal experimentation as enunciated by the Canadian Council on Animal Care and approved by the Animal Care Committee at the Universite´ de Montreal. ´ 2.2. Implantation of catheters Rats were anaesthetized with an intraperitoneal injection of 65 mgrkg sodium pentobarbitone ŽSomnotol w , M.T.C. Pharmaceuticals, Cambridge, Ontario. and two catheters were implanted. One was a polyethylene catheter ŽPE-50; Intramedic, Clay Adams, NJ. filled with physiological saline containing heparin Žsodium salt, 50 IUrml. and inserted into one jugular vein for i.v. pretreatment and Evans blue injection. The second catheter, used for intrathecal injections, consisted of a PE-10 tubing that was inserted into the spinal subarachnoid space through an incision made in the dura matter at the atlanto-occipital junction. This was pushed either to the level of the second or eight thoracic ŽT-2 or T-8. or fourth lumbar ŽL-4. spinal segment and fixed on the skull with cyanoacrylate glue. In an additional group of 7 rats, a third catheter ŽPE-50., filled with physiological saline containing 50 IUrml heparin sodium salt was inserted into the abdominal aorta through the femoral artery for direct blood pressure recording with a Statham pressure transducer ŽP231D. coupled to a Grass polygraph Žmodel 79; Grass Instruments Co., Quincy, MA.. These animals served to assess the efficacy of atropine and N G-nitro-L-arginine in blocking the hypotensive effects of carbachol and bradykinin, respectively. Each catheter was exteriorized at the back of the neck. Recovery from the anaesthetic was monitored closely under a warming lamp to maintain the body temperature and animals with obvious motor impairment andror abnormal behaviour were immediately humanely killed with an overdose of pentobarbital. Thereafter, rats were housed individually in polyethylene cage with a top grid and returned to their resident room. Experiments were conducted, at least 24 h after surgery, in conscious rats restrained in a contention plastic cage. 2.3. Measurement of Õascular permeability Vascular permeability was determined with the Evans blue dye method ŽJancso-Gabor et al., 1967.. Evans blue ´ ´ Ž35 mgrkg. was injected i.v. 10 min before intrathecal or i.v. injection of peptide. Intrathecal injections were made in a volume of 10 m l artificial cerebrospinal fluid ŽaCSF; composition in mM: 128.6 NaCl, 2.6 KCl, 2.0 MgCl 2 and 1.4 CaCl 2 ; pH adjusted to 7.2. followed by an additional 10 m l aCSF to flush the catheter Žvoid volume of the catheter.. I.v. injections were made in 0.1 ml saliner100 g body weight and 0.2 ml saline was used to flush the catheter. 5 min later, rats which survived to endothelin treatment were anaesthetized by inhalation of air saturated with metoxyflurane ŽMetofane w , CDMV, St. Hyacinthe,

P. Poulat, R. Couturer European Journal of Pharmacology 344 (1998) 251–259

Quebec. and perfused transcardially through aorta with 200–300 ml of saline solution after thoracotomy. At the end, the perfusion cannula was inserted into the pulmonary artery to wash out the remaining blood from the vascular bed of the lungs. Rats which did not survive up to 5 min, were also perfused the same way immediately after the respiratory arrest. The following tissues were removed: lungs, a liver lobe, one kidney, the two ears, a piece of 15 mm diameter of back skin, the skin of hind paws and the trachea. Tissues were weighed, dried for 24 h at 608C and weighed again. Thereafter, tissues were transferred in 4 ml of formamide for 36 h at 608C. After incubation, the Evans blue content in the samples was measured by spectrophotometry at 620 nm. The amount of Evans blue measured in tissues from rats injected with vehicle or endothelins was expressed in m g Evans bluerg of dry weight tissue. Positive values over control ŽaCSF or saline. in the differences between wet weight and dry weight tissue were taken as an index of oedema. Values in grams were converted directly into volume Žml. of oedema. 2.4. Experimental protocols 2.4.1. Spinal action of endothelins-1 and -3 The vascular permeability changes in the above mentioned tissues and lung oedema were measured following intrathecal injections, at the T-8 spinal cord level, of endothelin-1 at the dose of 6.5 pmol Žgroup 1 s 6 rats., 65 pmol Žgroup 2 s 8 rats., 650 pmol Žgroup 3 s 15 rats. as well as after intrathecal injection of 650 pmol endothelin-3 Žgroup 4 s 6 rats.. Two additional groups were used to assess the vascular permeability response in the same tissues and lung oedema after i.v. injection of 650 pmol endothelin-1 Žgroup 5 s 6 rats. and 650 pmol endothelin-3 Žgroup 6 s 6 rats.. Results were compared with those obtained after intrathecal injection of aCSF Ž n s 22 rats. or i.v. saline Ž n s 6 rats.. The vascular permeability changes and lung oedema induced by 650 pmol endothelin-1 were also measured when the peptide was injected at the T-2 Žgroup 7 s 7 rats. and L-4 Žgroup 8 s 6 rats. spinal cord levels. The injection of endothelin-1 Ž650 pmol. at the T-8 spinal level Žgroup 9 s 12 rats. served as a control group for the two intrathecal injection sites.

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2.4.2. Effect of BQ-123 Õersus endothelin-1 In 6 rats Žgroup 10., 6.5 nmol BQ-123 was injected intrathecally 15 min prior to 650 pmol endothelin-1 and the effects were measured on lung oedema and vascular permeability changes. The results were compared to those obtained in a control group which received only 650 pmol endothelin-1 Žgroup 9 s 12 rats.. 2.4.3. Effects of Õarious drugs Õersus endothelin-1 In 7 separate groups of 5–6 rats, the changes of lung vascular permeability and oedema induced by intrathecal injection of 650 pmol endothelin-1, at the T-8 spinal cord level, were measured after various treatments ŽTable 1.. The responses were compared to those measured in control rats which received 650 pmol endothelin-1 only Žgroup 9 s 12 rats and group 11 s 14 rats.. Under pentobarbitone anaesthesia Ž65 mgrkg, i.p.., rats received 6-hydroxydopamine Ž100 mgrkg, i.v.. 24–48 h prior to experiment in saline containing 1 mgrml ascorbic acid. The effect of the vehicle was assessed in 3 separate rats. Under slight anaesthesia with sodium pentobarbital Ž25 mgrkg, i.p.., rats received three s.c. injections of capsaicin Ž25, 40 and 60 mgrkg. over 3 consecutive days ŽBuck and Burks, 1986.. This 3 day treatment did not cause respiratory distress except in a few rats which were mechanically assisted for 1–2 min. Control rats Ž n s 6. which received the solvent only Ž25% ethanol, 50% dimethyl sulfoxide, 25% saline. under similar anaesthesia were pool with the other control rats Žgroup 11. as this vehicle did not affect the response to endothelin-1. The rats were used for experiments 10 days after the last injection of capsaicin. 2.5. Peptides and other compounds Endothelins-1 and -3 were purchased from Bachem Bioscience ŽKing of Prussia, PA.. BQ-123 ŽcyclowD-Trp, D-Asp, L-Pro, D-Val, L-Leux. was generously given by Banyu Pharmaceuticals ŽTsukuba, Japan.. Evans blue dye, phentolamine–HCl, pentolinium–HCl, atropine sulphate, indomethacin, N G -nitro-L-arginine, carbachol, heparin sodium salt Žgrade II. and capsaicin Ž8-methyl-N-vanillyl-

Table 1 List of treatments given to rats injected with intrathecal endothelin-1 Ž650 pmol. Group

Treatment

Dose

Route

Time prior to endothelin-1

n

10 12 13 14 15 16 17 18

BQ-123 Žendothelin ETA receptor antagonist. 6-hydroxydopaminea phentolamine Ž a-adrenoceptor antagonist. pentolinium Žganglionic blocker. capsaicina indomethacin Žcyclooxygenase inhibitor. atropine Žmuscarinic acetylcholine receptor antagonist. N G -nitro-L-arginine ŽNO synthase inhibitor.

6.5 nmol 100 mgrkg 2 mgrkg 25 mgrkgq 50 mgrkg per h 125 mgrkg 5 mgrkg 1 mgrkg 50 mgrkg

i.t. i.v. i.v. i.v. s.c. i.v. i.v. i.v.

15 min 24–48 h 15 min 15 min 10 days 60 min 15 min 30 min

6 6 5 6 6 6 5 6

a

See Section 2 for protocols.

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P. Poulat, R. Couturer European Journal of Pharmacology 344 (1998) 251–259

6-nonenamide. were all purchased from Sigma Chemicals ŽSt. Louis, MO. and 6-hydroxydopamine was obtained from Labkemi AB ŽStockholm, Sweden.. Formamide and ascorbic acid were purchased from American Chemicals ŽMontreal, Canada. and Baker ŽPhillipsburg, NJ., respectively. Stock solutions Ž1 mgrml. of endothelins and BQ-123 were dissolved in aCSF and divided into aliquots of 100 m l each and stored at y208C for a maximum of two months. Indomethacin was dissolved in Trizma base Ž0.2 M; Sigma.. N G -nitro-L-arginine was dissolved in saline with strong agitation at 50–608C. This solution was allowed to cool to normal body temperature before injection. All other compounds injected i.v. were prepared in saline. Doses of peptides are expressed in mol of the salt. 2.6. Statistical analysis of data Results were expressed as means " S.E.M. Statistical differences were evaluated with a one-way analysis of variance ŽANOVA. followed by a post-hoc Dunnett’s test for multiple comparisons with one control. Only probability values Ž P . less than 0.05 were considered to be statistically significant. 3. Results 3.1. Effects of endothelins The effects of intrathecal injection of endothelins-1 and -3 on the vascular permeability changes in various tissues are shown in Fig. 1. Intrathecal injection of endothelin-1 at

the T-8 spinal cord level Ž65 and 650 pmol. increased by 22-fold vascular permeability in the lungs when compared to aCSF values. The vascular permeability was also significantly increased in the trachea, the ears, the skin of the back and of hind paws, but to a smaller extent Žbetween 1.4- and 2.2-fold. than in the lungs. A non-dose-dependent increase Ž1.2–1.7-fold. of vascular permeability was seen in the kidney from 6.5 to 650 pmol endothelin-1. In contrast, endothelin-1 failed to modify vascular permeability in the liver at any doses. At the dose of 650 pmol, endothelin-3 increased vascular permeability in the lungs Ž7-fold., the trachea Ž1.6-fold. and the back skin Ž1.5-fold.. These effects of endothelin-3 were therefore less pronounced than those evoked by the same dose of endothelin-1, particularly in the lungs ŽFig. 1.. A significant lung oedema was also induced by endothelin-1 at 65 and 650 pmol. Conversely, the highest dose of endothelin-3 failed to induce lung oedema ŽTable 2.. All rats injected with 650 pmol endothelin-1 Ž n s 15. or 650 pmol endothelin-3 Ž n s 6. died within 3–5 min after injection and one out of 8 rats after 65 pmol endothelin-1. The symptoms before death were reduction of the respiratory rate followed by respiratory arrest and abundant foam was dripping from the mouth. The lungs were swollen but the heart was still beating vigorously in those rats when the transcardial perfusion of saline was initiated. Intravenous injection of 650 pmol endothelins-1 and -3 Žwhich corresponds to 2.1–2.6 nmolrkg. enhanced vascular permeability in the lungs by 1.4 and 1.6 fold, respectively ŽFig. 2.. Thus, these effects were relatively small when compared to those elicited by similar intrathecal

Fig. 1. Evans blue exudation in several tissues after intrathecal injection at the T-8 spinal cord level of aCSF Ž n s 22., endothelin-1 at 6.5 pmol Ž n s 6., 65 pmol Ž n s 8., 650 pmol Ž n s 15. or endothelin-3 at 650 pmol Ž n s 6. in conscious rats. Values of Evans blue Ž m grg of dry weight tissue. represent the mean " S.E.M. of n rats. Statistical significance of differences between endothelins and aCSF values is indicated by ) P - 0.05, ) ) P - 0.01; ))) P - 0.001.

P. Poulat, R. Couturer European Journal of Pharmacology 344 (1998) 251–259 Table 2 Effects of endothelins and various treatments on lung oedema in conscious rat Treatment a

Oedema Žml.

n

Control ŽaCSF, T-8.

1.60"0.09

22

Endothelin-1 Ž6.5 pmol, T-8. Endothelin-1 Ž65 pmol, T-8. Endothelin-1 Ž650 pmol, T-8. Endothelin-3 Ž650 pmol, T-8.

2.09"0.07 2.24"0.11b 2.15"0.16 b 1.57"0.03

6 8 15 6

1 2 3 4

Control Žsaline, i.v.. Endothelin-1 Ž650 pmol, i.v.. Endothelin-3 Ž650 pmol, i.v..

1.88"0.03 1.94"0.03 1.72"0.03

6 6 6

5 6

Endothelin-1 Ž650 pmol,T-8. Endothelin-1 Ž650 pmol, T-2. Endothelin-1 Ž650 pmol, L-4.

2.36"0.12 b 2.26"0.11b 2.13"0.38

12 7 6

9 7 8

Endothelin-1 Ž650 pmol, T-8. BQ-123qendothelin-1 6-hydroxydopamineqendothelin-1 Phentolamineqendothelin-1 Pentoliniumqendothelin-1 Endothelin-1 Ž650 pmol, T-8. Capsaicinqendothelin-1 Indomethacinqendothelin-1 Atropineqendothelin-1 N G -nitro-L-arginineqendothelin-1

2.36"0.12 c 1.74"0.03 d 1.65"0.02 e 0.98"0.05 b,f 0.74"0.07 b,f 2.11"0.12 a 1.65"0.02 d 1.62"0.22 d 2.67"0.14 c 2.40"0.20 b

12 6 6 5 6 14 6 6 5 6

9 10 12 13 14 11 15 16 17 18

Group

Values of lung oedema represent the mean"S.E.M. of n rats. They were calculated as the difference between the wet weight Žg. and dry weight Žg. and converted into volume Žml.. Statistical comparison to control Ž a,b,c . or endothelin-1 Ž650 pmol, T-8. without any pretreatment Ž d,e,f . is indicated by a,d P - 0.05; b,e P - 0.01 and c,f P - 0.001. a See Table 1 and Section 2 for protocols.

doses of either peptide and all rats survived. When given i.v. only endothelin-1 increased vascular permeability in the other organs Žkidney, 1.9-fold; ears, 1.7-fold; back

255

skin, 1.4-fold and hind paw skin, 2.5-fold. ŽFig. 2.. No lung oedema was produced by either peptide injected i.v. ŽTable 2.. Increases of vascular permeability in the lungs were not significantly different when endothelin-1 Ž650 pmol. was injected at the T-2 Ž27-fold. or T-8 Ž20-fold. spinal cord level while it was significantly lower Ž15-fold, P - 0.05. when the peptide was injected at the L-4 level ŽFig. 3.. The effects of endothelin-1 on lung oedema were significant when injected at T-2 and T-8 but not at L-4 spinal levels ŽTable 2..

3.2. Effect of BQ-123 Õersus the response to endothelin-1 in the lungs BQ-123 Ž6.5 nmol i.t.; 15 min earlier., a selective antagonist at endothelin ETA receptor, reduced markedly the effect of 650 pmol endothelin-1 on the vascular permeability and oedema in the lungs ŽFig. 4 and Table 2.. Furthermore, the 6 rats receiving the dose of 650 pmol endothelin-1 survived after pretreated with 6.5 nmol of the antagonist.

3.3. Effects of Õarious pretreatments on the response to endothelin-1 in the lungs The increase of vascular permeability induced by 650 pmol endothelin-1 in the lungs was abolished Ž P - 0.001. in rats pretreated with phentolamine Ž2 mgrkg, i.v., 15 min. or pentolinium Ž25 mgrkg followed by 50 mgrkg

Fig. 2. Evans blue exudation in several tissues after intravenous injection of saline Ž n s 6., endothelin-1 at 650 pmol Ž n s 6. or endothelin-3 at 650 pmol Ž n s 6. in conscious rats. Values of Evans blue Ž m grg of dry weight tissue. represent the mean" S.E.M. of n rats. Statistical significance of differences between endothelins and saline values is indicated by ) ) P - 0.01, ) ) ) P - 0.001.

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P. Poulat, R. Couturer European Journal of Pharmacology 344 (1998) 251–259

Fig. 3. Evans blue exudation in the lungs after intrathecal injection of either aCSF Ž ns 22. or 650 pmol endothelin-1 at T-8 Ž ns12., T-2 Ž ns 7. or L-4 Ž ns6. spinal cord level in conscious rats. Values of Evans blue Ž m grg of dry weight tissue. represent the mean"S.E.M. of n rats. Statistical significance of differences between endothelin-1 and aCSF is indicated by ) ) ) P - 0.001 while comparison for endothelin-1 values between the T-2 and L-4 spinal cord levels is indicated by † P - 0.05.

per h, i.v., 15 min. and markedly reduced Ž P - 0.001. in rats treated 24–48 h earlier with 6-hydroxydopamine Ž100 mgrkg, i.v.. ŽFig. 4.. Likewise, the effect of 650 pmol endothelin-1 was significantly reduced in rats pretreated with capsaicin Ž945 " 19 m grg of dry weight tissue; n s 6, P - 0.01. when compared to control rats Ž1589 " 104 m grg of dry weight tissue; n s 14.. In contrast, the endothelin-1-induced vascular permeability in the lungs was not significantly modified after pretreatment with indomethacin Ž5 mgrkg, i.v., 60 min., N G -nitro-L-arginine Ž50 mgrkg, i.v., 30 min. or atropine Ž1 mgrkg, i.v., 15 min. with respective values of 1468 " 147 Ž n s 6., 1496 " 127 Ž n s 6. and 1459 " 95 Ž n s 5. m grg of dry weight tissue.

Fig. 4. Evans blue exudation in the lungs after intrathecal injection of aCSF Ž ns6. or 650 pmol endothelin-1 in control rats Ž ns12. or in rats pretreated with BQ-123 Ž ns6., 6-hydroxydopamine Ž ns6., phentolamine Ž ns 5. or pentolinium Ž ns6.. Values of Evans blue Ž m grg of dry weight tissue. represent the mean"S.E.M. of n rats. Statistical significance of differences between control ŽET-1. and treated rats is indicated by †††P - 0.001 while statistical comparison to aCSF is indicated by ) ) P - 0.01, ) ) ) P - 0.001.

Moreover, the vehicles for 6-hydroxydopamine Žsaline containing 1 mgrml ascorbic acid. and indomethacin ŽTrizma base 0.2 M. did not affect the response to endothelin-1 on lung vascular permeability Žvalues were 1410 " 110 Ž n s 3. and 1484 " 130 Ž n s 4. m grg of dry weight tissue, respectively.. Endothelin-1-induced lung oedema was absent in rats pretreated with either capsaicin, 6-hydroxydopamine, indomethacin, phentolamine or pentolinium ŽTable 2. and about 50% of the rats subjected to each treatment were still alive at the time of the perfusion. In the case of the two latter treatments, lung oedema was significantly lower than control values which may be due to a decrease of peripheral vascular resistance and a corresponding reduction of hydrostatic blood pressure in lung capillaries. On the other hand, atropine and N G-nitro-L-arginine affected neither lung oedema ŽTable 2. nor the mortality produced by endothelin-1. These two latter treatments were found to block significantly the hypotensive effects induced by carbachol Ž2.7 = 10y8 molrkg, i.v.; n s 3. and bradykinin Ž1.6 = 10y7 molrkg, i.v.; n s 4., respectively Žresults not shown.. The vehicles for 6-hydroxydopamine and indomethacin had no significant effect on the response to endothelin-1 on lung oedema Žvalues were 2.05 " 0.16 Ž n s 3. and 2.20 " 0.10 Ž n s 4. ml, respectively..

4. Discussion In conscious rats, intrathecal injections of endothelin-1 augmented markedly pulmonary vascular permeability and oedema. This central action of endothelin is likely mediated primarily by endothelin ETA receptors in the spinal cord on the basis of the following considerations: Ž1. endothelin-3 produced smaller increases of vascular permeability than endothelin-1 and failed to produce pulmonary oedema and Ž2. BQ-123, a selective antagonist of endothelin ETA receptors, inhibited the pulmonary vascular permeability and oedema changes induced by endothelin-1 and reduced the mortality to 0%. This is in agreement with the presence of endothelin ETA receptor binding sites in the rat spinal cord ŽGulati, 1991; Bertelsen et al., 1992. and with previous studies supporting a role for endothelin ETA receptors in cardiovascular changes induced by intrathecal injection of endothelin-1 in conscious rat ŽPoulat et al., 1994. and in endothelin-induced inositol phosphate production in rat spinal cord slices ŽPoulat et al., 1996a.. A minor contribution of endothelin ETB receptors in the spinal action of endothelins on vascular permeability cannot be excluded at this time because a residual response to endothelin-1 persisted after treatment with BQ-123. This remains to be addressed with the use of selective endothelin ETB receptor agonists and antagonists. It is unlikely that intrathecal effects of endothelins-1 and -3 are due to leakage of the peptides into the pulmonary or systemic

P. Poulat, R. Couturer European Journal of Pharmacology 344 (1998) 251–259

circulation since the same dose of peptides administered intravenously had only a slight effect on vascular permeability in the lungs and did not produce lung oedema. Moreover, all rats survived to i.v. injection of both endothelins. In agreement with our data, increased plasma protein extravasation was shown after i.v. administration of endothelin-1 Ž1.0 nmolrkg. in selected vascular beds, including the gastrointestinal and respiratory tracts, spleen and kidney of conscious rats ŽSirois et al., 1992; Filep et al., 1993.. Cyclooxygenase inhibition by indomethacin did not affect the vascular permeability in upper and lower bronchi after i.v. injection of endothelin-1 ŽFilep et al., 1993.. This is in line with the present study which also excludes the involvement of cyclooxygenase derived eicosanoids in pulmonary vascular permeability induced by intrathecal endothelin-1. Nevertheless, prostaglandins appear to play a role in the spinal action of endothelin-1 in pulmonary oedema. In contrast, nitric oxide and muscarinic acetylcholine receptors are unlikely involved in the spinal action of endothelin-1 on increases of pulmonary vascular permeability and oedema since treatment with either N G-nitro-L-arginine or atropine was without consequence. In the present study, lung oedema was not induced by i.t. endothelin-3 or by i.v. injection of endothelins-1 and -3 which led to increases of vascular permeability. Lung oedema was observed only after i.t. endothelin-1 which also caused marked vascular permeability changes. This indicates that both vascular phenomena are controlled by distinct mechanisms which may be as follows: any small changes of vascular permeability may occur in the postcapillary and collecting venules without significant increases of hydrostatic pressure in the capillaries when locally released mediators are able to cause the formation of pores following the contraction of endothelial cells. The presence of a greater colloid osmotic pressure in the capillaries would therefore prevent the production of oedema under this condition. Thus, oedema would not occur unless a high hydrostatic pressure in the capillaries is achieved. A greater increase of vascular permeability and oedema could be seen simultaneously if both vascular mechanisms operate under optimal condition. According to our results, indomethacin would exert an inhibitory effect only against the increased intracapillary hydrostatic pressure which is congruent with the ability of this drug to prevent swelling induced by pro-inflammatory agents and with that of prostaglandins E 1 and E 2 to cause oedema without vascular permeability changes ŽInsel, 1990.. Increased pulmonary vascular permeability is a classical feature of the adult respiratory distress syndrome described in human and experimental models ŽGosling et al., 1994.. In that situation, immunoreactive endothelin-1 was found elevated in the plasma and in the broncho-alveolar lavage fluid, suggesting the involvement of systemic andror pulmonary endothelin-1 and an abnormal pulmonary metabolism of endothelin-1 ŽPritze et al., 1992; Simmet et

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al., 1992; Druml et al., 1993; Langleben et al., 1993.. Moreover, plasma endothelin-1 levels in both systemic and pulmonary arteries were higher than at recovery in a case of high altitude pulmonary oedema with pulmonary hypertension ŽDroma et al., 1996.. Evidence also suggests a role for endothelin-1 in hypoxia-induced pulmonary hypertension ŽTjen-A-Looi et al., 1996.. These studies are consistent with the potent pulmonary vasoconstrictor activity of this peptide ŽRae et al., 1995. and its ability to increase capillary permeability in rat airways ŽSirois et al., 1992; Filep et al., 1993.. Despite one cannot rule out completely the possibility that intrathecal injection of endothelin-1 could induce the release of endogenous endothelin-1 into the systemic andror pulmonary circulation, the peripheral sympathetic nervous system appear to be essential in the spinal action of endothelin in the present study. Indeed, among the numerous pretreatments used to prevent the endothelin-1-induced vascular permeability and oedema, 6-hydroxydopamine, phentolamine and pentolinium, appeared to be the most effective. This evidence is a solid argument against a peripheral andror pulmonary action of endothelins. Similarly to the spinal effects of endothelins, the occurrence of the adult respiratory distress syndrome can be correlated with the activation of the sympathetic nervous system. For instance, the severity of the adult respiratory distress syndrome obtained after lesion of the anterior hypothalamic nuclei in rats was reduced by alpha adrenergic blockade and peripheral catecholamine depletion ŽDe Oliveira and de Oliveira, 1988.. Also, severe pulmonary edema and hypertension occurred within minutes after massive sympathetic nervous system activation with intracisternal veratrine administration in chloralose anaesthetized dogs ŽMaron et al., 1994.. Finally, the contribution of sympathetic nerves in increasing lung vascular permeability was demonstrated in an isolated rat sympathetic nerve–lung preparation ŽSakakibara et al., 1992.. In this in vitro study, interaction was found between neurotransmitters released from capsaicin-sensitive nerves and norepinephrine released from the sympathetic nerve terminals in enhancing lung vascular permeability upon electrical nerve stimulation. Thus, intrathecal endothelin-1 could increase vascular permeability and oedema in the lungs by massive stimulation of the sympathetic nervous system following the activation of endothelin ETA receptor in the spinal cord. Sympathetic neurons supplying the lung in rodents were located in stellate ganglia and in thoracic sympathetic chain ganglia T-2 to T-4. The majority of the sympathetic neurons also contain neuropeptide Y. Within the airways, their axons were found in the smooth muscle layer, around blood vessels including the pulmonary artery and vein and to a lesser extent in the lamina propria ŽKummer et al., 1992.. Nevertheless, increased lung vascular permeability and sudden death are not necessarily coupled phenomenon because both effects could be separated. For instance, a

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low incidence of mortality was found in rats which received 65 pmol endothelin-1 while this dose was equipotent to 650 pmol in increasing lung vascular permeability. Moreover, the increased vascular permeability and lung oedema were abolished by treatments Žphentolamine, pentolinium. which did not prevent completely the mortality but reduced it by about 50% only. Endothelins might be unique in increasing pulmonary vascular permeability through a spinal mechanism. The intrathecal injection of substance P or bradykinin at the T-9 spinal cord level failed to mimic this effect of endothelins although they enhanced vascular permeability in cutaneous tissues of the back, the hind paws and the ears. These spinal effects of substance P and bradykinin were ascribed to a spinobulbar pathway involving primary afferent C-fibers and a cholinergic vagal mechanism ŽKerouac ´ et al., 1987; Jacques and Couture, 1990.. Similarly to endothelins ŽPoulat et al., 1994., substance P, neuropeptide K and neuropeptide gamma injected intrathecally activate the sympatho-adrenal system and the peripheral release of catecholamines that lead to increases of arterial blood pressure and heart rate ŽHassessian et al., 1990; Pham et ´ al., 1993; Couture et al., 1995; Poulat et al., 1996b.. Despite the autonomic effects of these neuropeptides are apparently similar to those evoked by endothelins, none of them produced respiratory distress. Thus, the distinct action of endothelins on pulmonary vascular permeability could result from pulmonary vascular hypertension following a more selective and greater activation of the sympathetic nervous activity in this territory. The observation that endothelin-1 administered at the L-4 spinal cord level enhanced lung vascular permeability, although with lesser efficacy than at the T-2 and T-8 levels, suggests the activation of ascending fibers in the spinal cord. The data with capsaicin also points to a sensory component involving primary afferent C-fibers. In that respect, endothelin-1 was found to induce ventral root depolarization through the release of substance P in a newborn rat spinal cord preparation in vitro ŽYoshizawa et al., 1989.. Although the exact significance of this in the endothelin-induced pulmonary vascular permeability and oedema remains to be clarified, the input in the spinal dorsal horn of sensory information coming from lung primary afferents may influence the sympathetic reflex induced by endothelins. Thus, the desensitization or neurodegeneration of these primary afferents by capsaicin ŽBuck and Burks, 1986. would explain the protective effect of this treatment against the spinal action of endothelins. In keeping with these data, immunoreactive endothelin was enhanced in rat injured spinal cord ŽMcKenzie et al., 1995.. Because intrathecal endothelin-1 mimics neurogenic pulmonary oedema, this peptide can thus be suggested as a potential central mediator responsible of the adult respiratory distress syndrome following head trauma or spinal cord injury which is invariably associated with pulmonary vascular permeability changes and respiratory complica-

tions ŽGosling et al., 1994; Jackson and Groomes, 1994; Slack and Shucart, 1994.. In conclusion, intrathecal injection of endothelin-1 induced a pronounced increase of vascular permeability and oedema in the lungs. This effect appears to be due to an intense pulmonary vasoconstriction mediated by a-adrenoceptors following the release of catecholamines in the systemic and most likely in the pulmonary circulation in response to the activation of endothelin ETA receptor in the spinal cord. Whereas nitric oxide and muscarinic acetylcholine receptors do not seem to take part to this central action of endothelin, a capsaicin-sensitive sensory component was revealed. In addition, prostaglandins would be involved in the oedema response. The data with capsaicin together with the finding that endothelin-1 is effective when injected at various segments of the spinal cord point to the possibility that the peripheral sympathetic nervous system is activated through a reflex mechanism involving primary afferent C-fibers and spinal cord ascending fibers projecting to supraspinal nervous centers. Hence, endothelin-1 might be of pathophysiological significance in the development of adult respiratory distress syndrome caused by spinal cord injury and head trauma.

Acknowledgements This work was supported by the Grant-in-Aid MT-8925 from the Medical Research Council of Canada ŽMRCC.. P.P. was a postdoctoral fellow from the MRCC.

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