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Budesonide inhibits plasma extravasation induced by capsaicin and by substance P in the rat nasal mucosa
Regulator), Peptides, 49 (1993) 159-166 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/$06.00
159
REGPEP 01598
Budesonide inhibits plasma extravasation induced by capsaicin and by substance P in the. rat nasal mucosa Elena Bacci a, Claude Bertrand a, Pierangelo Geppetti a, Jonathan Baker a, Jay A. Nadel b, Lauri A. Laitinen c and GOran Petersson a,, "Cardiovascular Research Institute and b Departments of Medicine and Physiology, University of California, San Francisco, CA 94143 (USA) and CDepartment of Pulmonary Medicine, University Central Hospital, Helsinki (Finland) (Received 30 July 1993; revised version received and accepted 24 September 1993)
Key words: Corticosteroid; Neurogenic inflammation; Vascular permeability; Trachea; Urinary bladder
Summary We studied the effect of the locally administered glucocorticoid budesonide on plasma extravasation induced by capsaicin and by substance P (SP) in the nasal mucosa of pathogen-free rats. Using Evans blue dye as a tracer, we measured plasma extravasation induced by capsaicin (150 #g kg- 1 i.v.) or SP (0.5 and 2.5 #g kg- 1 i.v.) in the rat naso- and maxilloturbinates after pretreatment with budesonide (0.1-50 #g twice/day for 2 days in the right nostril; 50/~g only for SP) or its vehicle. We found that budesonide inhibits plasma extravasation induced by capsaicin in a dose-dependent fashion in the nasal cavity. After the highest dose (50 #g) of budesonide, the values of Evans blue in the nasal mucosa were not different from the values observed after capsaicin vehicle alone. Budesonide also reduced plasma extravasation induced by capsaicin in the trachea and the urinary bladder of the rats in a dose-dependent fashion. Budesonide (50/~g) delivered to the nose inhibited the plasma extravasation caused by 0.5 but not by 2.5/~g SP kg- 1 in the nasal mucosa. We conclude that the postjunctional part of the neurogenic pathway is a target for glucocorticoid antiinflammatory action in the nasal mucosa, at least of the rat. Budesonide's effect on organs other than the nose can be explained by systemic absorption.
Introduction * Corresponding author. Present address: Department of Otorhino-laryngology, University of Lund, General Hospital, S-214 01 Malm6, Sweden. Fax: +46 40 336229.
Stimulation of sensory nerve endings by capsaicin releases neuropeptides (e.g., substance P), which cause neurogenic inflammation in rodent airways
160
[1,2]. An important aspect of neurogenic inflammation in the nasal mucosa is increased vascular permeability [2,3]. Increased vascular permeability with subsequent plasma extravasation is believed to account, at least in part, for symptoms (e.g., rhinorrhea) that occur in nasal inflammatory diseases. Glucocorticoids are commonly and effectively used in the therapy of many inflammatory diseases of the nose [4], but it is not known which of the many pathways involved in inflammation are affected by glucocorticoid treatment. It has been demonstrated that glucocorticoids inhibit plasma extravasation induced by neurogenic inflammation in the guinea pig and rat trachea [5,6]. In this study, we investigated whether locally administered glucocorticoids inhibit neurogenic plasma extravasation in the nasal mucosa of rats. We pretreated rats with budesonide for 2 days before giving an intravenous injection of capsaicin or substance P (SP), and we assessed changes in vascular permeability using Evans blue dye [7]. Our data show that budesonide inhibits plasma extravasation induced by capsaicin and by SP in the rat nasal mucosa. Materials and Methods
Animals We studied the effect of budesonide on capsaicinand SP-induced plasma extravasation in the nose of male, pathogen-free, F344 rats (200-250 g) from Simonsen Laboratories, Inc. (Gilroy, CA). The rats were housed in autoclaved plastic cages isolated by static filters (Microinsulator System, Lab Products, Maywood, N J) and placed inside a laminar flow rack enclosure (Stay-Clean, Lab Products) to prevent respiratory tract infection [ 6 ].
Pretreatment To determine the dose-response effect of budesonide on capsaicin-induced plasma extravasation, three groups of rats were treated for 2 days with budesonide (0.1 (n = 5), 1 (n = 5), 50 #g (n = 9) intranasally; ASTRA DRACO AB, Lund, Sweden). To
determine the response ofbudesonide on SP-induced plasma extravasation, two groups of rats were treated for 2 days with budesonide. After anesthesia with sodium methohexital (Brevital Sodium, Lilly, Indianapolis, IN), 60 mg kg 1 i.p., budesonide (25/d) was inoculated in the right nostril of each animal. The inoculation procedure was repeated four times, starting in the afternoon of the first day, then in the morning and in the afternoon of the second day, with the last inoculation being given in the morning of the third day, just before starting the experiment. A group of eight control rats received budesonide vehicle before capsaicin, and a group of four control rats received budesonide vehicle before SP, according to the administration protocol described above. The corticosteroids were given for 2 days prior to capsaicin or SP, because previous studies in the trachea suggested that this timing produces a maximal effect [6].
Description of the experiments The rats were anesthetized with sodium methohexital, 70 mg kg i i.p., supplemented as needed. To assess vascular permeability, we injected Evans blue dye (3~o solution in 0.9~o NaC1, Polysciences, Inc., Warrington, PA). Evans blue has the property of binding to albumin and is therefore used to quantify plasma protein extravasation [7]. Immediately after the injection of the Evans blue (30 mg kg- 1 i.v. over 5 s), neurogenic plasma extravasation was induced by giving an intravenous injection of capsaicin (150/~g kg- 1 i.v. over 2 min; Sigma Chemical Co., St. Louis, MO), dissolved in a vehicle containing 0.75% ethanol, 0.375~o Tween 80, and 0.85~o NaCI. In a control group of rats (n = 5), the vehicle of capsaicin alone was given. Alternatively, plasma extravasation was produced by SP (0.5 (n = 5) or 2.5 (n = 4) #g kg- 1 i.v. over 20 s; Sigma), dissolved in water. In a control group of rats, water only was given. 5 min after the injection of capsaicin or SP, the chest was opened, a cannula was inserted into the ascending aorta through the left ventricle, and the circulation was perfused for 2 min with phosphate-
161 buffered saline (pH 3.5) at a pressure of 120 mmHg. The trachea was removed. The nose was dissected according to the procedure previously described [8]. Briefly, the head of the rat was cleared of external soft tissues, and the nasal cavities were opened with paramedian incisions through the dorsum and the floor of the nose. The naso- and maxilloturbinates were dissected free from the lateral wall of the nose. To test possible systemic and other possible effects of budesonide, the trachea and the urinary bladder was also removed. After blotting on bibulous paper, the trachea, the turbinates and the urinary bladder were weighed and placed in vials containing 3 ml of formamide (Fisher Scientific, Fair Lawn, N J) overnight at room temperature to extract Evans blue. Thereafter, the dye concentration in formamide was measured using a spectrofluorimeter (SLM 8000C, SLM Instruments Inc., Urbana, IL) [9]. Samples were excited through a monochromator at 620 nm (4 nm bandwidth). Emission light above 645 nm was filtered by a RG-645 cut-off filter (Schott, Duryea, PA) and detected by a photomultiplier (SLM). The signal was digitized and recorded by an interfaced IBM P C / A T computer. The signal to noise ratio was 6:1 for the weakest signal. The concentration of Evans blue was calculated from standard curves of Evans blue (0.005-10 #g m l - 1 in formamide) and expressed as ng of dye per mg of tissue. In most cases the septum had an opening that provides a communication between the two nasal cavities. To test whether the inoculated budesonide can move from the right to the left nasal cavity and to the trachea, we inoculated 25 #1 of Evans blue dissolved in budesonide vehicle into the right nostril of four rats. After 15 rain, we opened the chest and the nose to evaluate the distribution of the dye. The dye was present in the left nasal cavity, as well as in the esophagus of all rats. The dye was found in the upper third of the trachea in one rat.
Statistical analysis We have previously shown that capsaicin increases vascular permeability mainly in the naso- and max-
illoturbinates [8], so we pooled the values of Evans blue dye measured for the naso- and maxilloturbinates. Data are reported as mean + S.E.M. Possible differences between capsaicin (or SP) and capsaicin (or SP) vehicle-induced plasma extravasation was tested by Student's unpaired t-test. Mean values for different treatments were compared by one-way analysis of variance. Multiple comparisons between groups were tested by Dunnett's test. Differences between right and left turbinates were tested by Student's paired t-test. P-values < 0.05 were considered significant.
Results Capsaicin alone (150 #g k g - 1 i.v.) increased the concentration of Evans blue in the rat nasal mucosa from control values of 12.16 + 0.87 ng m g tissue (mean + SE) to 25.81 + 2.04 ng m g - 1 tissue ( P < 0.001). Pretreatment with budesonide inhibited Evans blue extravasation after capsaicin in a dosedependent fashion (Fig. 1). After 50 #g budesonide,
30
E v a n s blue
in turbinates (ng/mg of tissue)
15 ¸
0.1
1
50
I CAPSAIClN VEHICLE
BUDESONIDE VEHICLE then CAPSAICIN
I BUDESONIDE ~g) then CAPSAICIN
Fig. 1. Dose-dependentinhibitory effectof budesonide on Evans blue extravasationinduced by capsaicin(150 #g kg i i.v.)in nasoand maxilloturbinates of pathogen-free rats. Values are mean + S.E.M. Asterisks express a significant differencebetween treatment with budesonide and control (budesonide vehicle, *P < 0.05).
162 Rightturbln~ltes
30
I
60
Leftturblnates
1
[
I
Evans blue in trachea (ng/mg of tissue)
Evans blue 15 in turbinates (ng/mg of tissue)
i i,
30
15
0J 0
iol
1 5o I
BUDESONIDE BUDESONIDE VEHICLE (Fg)
Ol
1 50 I CAPSAICIN VEHICLE
BUDESONIDE BUDESONIDE VEHICLE (~g)
then
then
then
then
CAPSAICIN
CAPSAICIN
CAPSAICIN
CAPSAICIN
BUDESONIDE VEHICLE
BUDESONIDE ~g)
then
then
CAPSAICIN
CAPSAICIN
Fig. 2. Comparison of Evans blue cxtravasation induced by capsaicin (150 #g kg ~ i.v.) in right and left turbinates of pathogenfree rats after pretreatment with different doses of budesonide in the right nostril. No significant difference was observed between the right and left sides. Values are mean _+ S.E.M.
Fig. 3. Dose-dependent inhibitory effect of budesonidc on E~ans blue extravasation induced by capsaicin (150 /xg kg ' i.v.) in trachea of pathogen-free rats. Values are mean _+S.E.M. Asterisk expresses a significant difference between treatment with budesonide and control (*P<0.05).
the values were not different from the values observed after capsaicin vehicle alone (P= 0.96). There was no significant difference in the effect of budesonide on vascular extravasation between the right and left sides of the nose (Fig. 2). Budesonide
also reduced in a dose-dependent fashion the increase in Evans blue extravasation induced by capsaicin in the trachea (Fig. 3) and in the urinary bladder (Fig. 4) of the rats. SP alone increased the concentration of Evans
15
T 10
Evans blue in bladder (ng/mg of tissue)
5
0
D
0.1
1
50
I CAPSAICIN VEHICLE
BUDESONIDE VEHICLE then CAPSAICIN
I BUDESONIDE (~g) then CAPSAICIN
Fig. 4, Dose-dependent inhibitory effect of budesonide on Evans blue extravasation induced by capsaicin (150 pg kg ~ i.v.) in urinary bladder of pathogen-free rats. Values are mean + S.E.M. Asterisk expresses a significant difference between treatment with budesonide and control ( * P < 0.05).
163
90
60Evans blue (ng/mg of tissue} 30-
OTurbinates
Turbinates
Trachea
Bladder
Fig. 5. The effect ofbudesonide (50 #g twice/day, 2 days; hatched columns) or its vehicle (filled columns) on Evans blue extravasation caused by (A) SP (0.5 gg kg i in the naso- and maxilloturbinates, and by (B) SP (2.5 ~g kg 1) in the naso- and maxilloturbinates, the trachea and the urinary bladder of pathogen-free rats. Evans blue extravasation after SP-vehicle only is also shown (open columns). Values are mean _+S.E.M. Asterisks express a significant difference between treatment with budesonide and control (*P<0.05).
blue in the nasal mucosa in a dose-dependent fashion; from control values of 11.14+0.73 ng mg - 1 tissue to 29.40 + 3.37 ng mg- 1 tissue ( P < 0.05) after SP 0.5 #g kg - t i.v. and to 66.90 + 8.5 ng mg- 1 tissue ( P < 0.001) after 2.5/~g kg- 1 i.v. Budesonide (50/~g) reduced the extravasation caused by SP 0.5 ( P < 0.05) but not by SP 2.5 (P = 0.52) #g kg- 1 i.v. (Fig. 5). The SP evoked vascular extravasation after budesonide did not differ between sides of the nose (P = 0.54; data not shown). Budesonide inhibited the plasma extravasation after SP (2.5/~g kg- 1) in the urinary bladder and in the trachea (Fig. 5).
Discussion
This study shows that budesonide inhibits plasma extravasation induced in the rat nasal mucosa by intravenous injection of capsaicin in a dose-dependent fashion and by SP. The highest close of budesonide completely abolished the extravasation induced by capsaicin and reduced the extravasation caused by a low dose but not by a high dose of SP in the nasal mucosa. The lack of difference in the effect of budesonide between the right and the left turbinates is apparently
due to the diffusion of the drug to the opposite nasal cavity, as we demonstrated by the inoculation of dye in the right nostril. The inhibitory effect of budesonide on plasma extravasation in the trachea may be due either to diffusion of the drug through the respiratory tract, as shown by nasal instillation of dye, or to systemic absorption. The fact that budesonide also reduced capsaicin-induced plasma extravasation in both the urinary bladder and in the trachea in a dose-dependent fashion is evidence for systemic absorption. Budesonide has been reported not to be metabolized in the nose or in the plasma [10]. Biotransformation of budesonide into low activity metabolites is reported to occur in the liver. The data reported in the literature about the systemic effects of locally administered steroids are controversial. Intranasally administered flunisolide, budesonide, or beclomethasone dipropionate significantly improved asthma and/or ocular symptoms [4,11-13]. On the other hand, Toogood and co-workers [14] showed that a dose of oral budesonide giving plasma levels similar to those achieved with a locally effective dose of budesonide had no significant effect on the occurrence of asthma symptoms when compared to placebo in humans. It has been reported that systemic availability of active budesonide in the rat after oral administration is 10% of the administered dose, with a half-life of 2.5 h [ 10]. Plasma extravasation in the nasal mucosa induced by capsaicin is considered to be specific and selectively mediated by sensory nerve stimulation because this effect of i.v. capsaicin is abolished in capsaicinpretreated animals [15]. In the trachea of rats, steroids inhibit plasma extravasation induced by capsaicin [6]. Various investigators have reported different effects of corticosteroids on neurogenic extravasation using different stimuli and using different corticosteroids and drug schedules. For example, methylprednisolone did not inhibit the tracheal extravasation caused by exposure to cigarette smoke and enhanced the extravasation caused by vagal nerve stimulation [ 16]. Likewise, pretreatment with betamethasone before gastric juice exposure
164
enhanced the tracheal extravasation [17]. The absence of corticosteroid inhibition in these studies could be due to differences in the dose or in the timing ofglucocorticoid administration or it could be due to differences among tissues. Corticosteroids may affect plasma extravasation associated with neurogenic inflammation in several ways and the effect might be different in different structures. The exact mechanism and level of the effect in the nasal mucosa is not clear. Because budesonide inhibited the plasma extravasation in the nasal mucosa after capsaicin and after a low dose of SP (equipotent stimuli), it appears that the inhibition of capsaicin-induced plasma extravasation is due to a post-junctional site of action in agreement with the effects of corticosteroids in the trachea [6]. One possible explanation for the observation that budesonide failed to inhibit the response after the high dose of SP in the nasal mucosa, but not in the trachea and in the bladder, is that the high dose of SP resulted in a 'supermaximar response. The effect of budesonide on the extravasation in the nasal mucosa might be due to an effect on neutral endopeptidase, because corticosteroids upregulates neutral endopeptidase [18], as demonstrated in the lower airways [19]. In the skin, corticosteroids are known to cause a 'blanching', an effect which is attributed to vasoconstriction [20]. In the nasal mucosa, the inhibition of the capsaicin-induced plasma extravasation is not likely to be due to a vasoconstriction effect by budesonide, because budesonide is not found to cause any vasoconstriction in the nasal mucosa in healthy subjects [21,22]. However, these findings do not exclude an effect on blood flow by corticosteroids in inflamed tissue but a reduction of blood flow in the nasal mucosa per se does not necessarily imply a subsequent reduction of the plasma extravasation [23]. The nose is the first structure that inhales air encounters on its way to the lungs and is, therefore, the first barrier to inhaled irritants and pathogens. Stimuli such as cigarette smoke [24], formaldehyde and ether [15] can induce neurogenic inflammation in the rodent nasal mucosa via a capsaicin-sensitive
mechanism. Glucocorticoids have been reported to inhibit virus-induced potentiation of plasma extravasation produced by capsaicin in the rat trachea [6]. In a controlled study in children with rhinitis, a higher incidence of sneezing was observed after inhalation of placebo as compared with inhalation of budesonide [4]. The authors attributed this result to the protective effect of budesonide on the irritation induced by the placebo inhalation. Some of the therapeutic effects of corticosteroids could be due to the inhibition of increased vascular permeability and the subsequent effects of plasma extravasation. However, it is not certain whether neurogenic inflammation plays a role in human airway disease. In contrast to rodent nasal airways, capsaicin is not found to cause plasma leakage as measured by increased albumin in nasal airway lavage of healthy subjects [25,26]. On the other hand, SP caused an increase of plasma exudation in patients with allergic rhinitis [27]. Furthermore, nasal resistance to airflow is increased by local instillation of SP in a dosedependent fashion in normal subjects and in patients with allergic rhinitis [28]. In asthmatic patients, the bronchial responses to inhaled tachykinin aerosols was recently reported to be inhibited by a neurokinin receptor antagonist [29]. So far, the relevance of neurogenic inflammation in upper airway disease is not tested by means of neurokinin receptor antagonists. In conclusion, our study demonstrates that budesonide inhibits plasma extravasation induced by capsaicin in a dose-dependent fashion and by SP, in the rat nasal mucosa, showing that the postjunctional part of the neurogenic pathway is a target on which glucocorticoids exert their antiinflammatory effect in the nasal mucosa.
Acknowledgements We thank Donald M. McDonald, M.D., Ph.D. for useful discussions, Isao Yamawaki, M.D., Brendan Chart, Amy Haskell and Simona Ikeda for technical
165
assistance, Thomas Hartmann, Ph.D., for assisting with spectrofluorometry, Natalie Holt for typing the manuscript. This study was supported in part by NIH PPG-HL24136. Elena Bacci was supported in part by grants from the Italian Ministry of Education and from NIH (USA/Italy Exchange Program). Claude Bertrand was supported in part by a grant from the Fondation pour la Recherche M6dicale. G6ran Petersson was supported in part by grants from Tore Nilsons Fund for Medical Research, the Medical Faculty, University of Lund, Sweden, the Swedish Society of Medicine and the Swedish Medical Research Council (Including No. 8686). References 1 Lundblad, L., Lundberg, J.M., Brodin, E. and A.nggfird, A., Origin and distribution of capsaicin-sensitive substance P-immunoreactive nerves in the nasal mucosa, Acta Otolaryngol., 96 (1983) 485-493. 2 Lundblad, L., Saria, A., Lundberg, J.M. and Anggfird, A., Increased vascular permeability in rat nasal mucosa induced by substance P and stimulation of capsaicin-sensitive trigeminal neurons, Acta Otolaryngol., 96 (1983) 479-484. 3 Saria, A., Lundberg, J.M., Skofitsch, G. and Lembeck, F., Vascular protein leakage in various tissues induced by substance P, capsaicin, bradykinin, serotonin, histamine and by antigen challenge, Naunyn-Schmiedeberg's Arch. Pharmacol., 324 (1983) 212-218. 4 Henriksen, J.M. and Wenzel, A., Effect ofintranasally administered corticosteroid (budesonide) on nasal obstruction, mouth breathing, and asthma, Am. Rev. Respir. Dis., 30 (1984) 1014-1018. 5 ErjefNt, 1. and Persson, C.G.A., Anti-asthma drugs attenuate inflammatory leakage of plasma into airway lumen, Acta Physiol. Scand., 128 (1986) 653-654. 6 Piedimonte, G., McDonald, D.M. and Nadel, J.A., Glucocorticoids inhibit neurogenic plasma extravasation and prevent virus-potentiated extravasation in the rat trachea, J. Clin. Invest., 86 (1990) 1409-1415. 7 Jancsd-Gfibor, A,. Szolcs/myi, J. and Jancsd, N., A simple method for measuring the amount of azovan blue exuded into the skin in response to an inflammatory stimulus, J. Pharm. Pharmacol., 19 (1967) 486-487. 8 Petersson, G., Bacci, E., McDonald, D.M. and Nadel, J.A., Neurogenic plasma extravasation in the rat nasal mucosa is potentiated by peptidase inhibitors, J. Pharm. Exp. Ther., 264 (1993) 509-514.
9 Saria, A. and Lundberg, J.M., Evans blue fluorescence: quantitative and morphological evaluation of vascular permeability in animal tissues, J. Neurosci. Methods, 8 (1983) 4149. 10 Ryrfeldt, A., Andersson, P., Edsb~icker, S., T~Snnesson, M.A., Davies, D. and Pauwels, R., Pharrnacokinetics and metabolism of budesonide, a selective glucocorticoid, Eur. J. Respir. Dis. Suppl., 63 (1982) 86-95. 11 Bjerrum, P. and Ilium, P., Treatment of seasonal allergic rhinitis with budesonide and disodium cromoglycate, Allergy, 40 (1985) 65-69. 12 Orgel, H.A., Meltzer, E.O., Kemp, J.P. and Welch, M.J., Clinical, rhinomanometric, and cytologic evaluation of seasonal allergic rhinitis treated with beclomethasone dipropionate as aqueous nasal spray or pressurized aerosol, J. Allergy Clin. Immunol., 77 (1986) 858-864. 13 Reed, C.E., Marcoux J.P. and Welsh, P.W., Effects of topical nasal treatment on asthma symptoms, J. Allergy Clin. lmmunol., 81 (1988) 1042-1047. 14 Toogood, J.H,. Frankish, C.W., Jennings, B.H., Baskerville, J.C., Borga, O., Lefcoe, N.M. and Johansson, S.-A., A study of the mechanism of the antiasthmatic action of inhaled budesonide, J. Allergy Clin. Immunol., 85 (1990) 872-880. 15 Lundblad, L., Lundberg, J.M. and Angg~rd, A., Local and systemic capsaicin pretreatment inhibits sneezing and the increase in nasal vascular permeability induced by certain chemical irritants, Naunyn-Schmiedeberg's Arch. Pharmacol., 326 (1984) 254-261. 16 Lundberg, J.M., Martling, C.R., Saria, A., Fotkers, K. and Rosell, S., Cigarette smoke-induced airway oedema due to activation of capsaicin-sensitive vagal afferents and substance P release, Neuroscience, 10 (1983) 1361-1368 17 Martling, C.-R. and Lundberg, J.M., Capsaicin sensitive afferents contribute to acute airway edema following tracheal instillation of hydrochloric acid or gastric juice in the rat, Anesthesiology, 68 (1988) 350-356. 18 Borson, D.B. and Gruenert, D.C., Glucocorticoids induce neutral endopeptidase in transformed human tracheal epithelial cells, Am. J. Physiol., 260 (1991) L83-L89. 19 Piedimonte, G., McDonald, D.M. and Nadel, J.A., Neutral endopeptidase and kininase I1 mediate glucocorticoid inhibition of neurogenic inflammation, J. Clin. Invest., 68 (1991) 754-760. 20 McKenzie, A.W., Percutaneous absorption of steroids, Arch. Dermatol., 86 (1962) 611. 21 Bende, M., Lindqvist, N. and Pipkorn, U., Effect of a topical glucocorticoid, budesonide, on nasal mucosal blood flow as measured with 133Xe wash-out technique, Allergy, 38 (1983) 461-464. 22 Lindqvist, N., Holmberg, K. and Pipkorn, U., Intranasally administered budesonide, a glucocorticoid, does not exert its
166 clinical effect through vasoconstriction, Clin. Otolaryngol., 14 (1989) 519-523. 23 Svensson, C., Pipkorn, U., Alkner, U., Baumgarten, C.R. and Persson, C.G.A., Topical vasoconstrictor (oxymetazoline) does not affect histamine-induced mucosal exudation of plasma in human nasal airways, Clin. Exp. AII.~ 22 (1992) 411-416. 24 Lundberg, J.M., Lundblad, L., Saria, A. and AnggOxd, A., Inhibition of cigarette smoke-induced oedema in the nasal mucosa by capsaicin pretreatment and a substance P antagonist, Naunyn-Schmiedeberg's Arch. Pharmacol., 326 (1984) 181-185. 25 Bascom, R., Kagey-Sobotka, A. and Proud D., Effect of intranasal capsaicin on symptoms and mediator release, J. Pharm. Exp. Ther., 259 (1991) 1323-1327.
26 Rajakulasingam, K., Polosa, R., Lau, L.C., Church, M.K.. Holgate, S.T. and Howarth, P.H., Nasal effects of bradykinin and capsaicin: influence on plasma protein leakage and role of sensory nerves, J. Appl. Physiol., 72 (1992) 1418-1424. 27 Braunstein, G., Fajac, I., Lacronique, J. and Frossard. N., Clinical and inflammatory responses to exogenous tachykinins in allergic rhinitis, Am. Rev. Resp. Dis., 144 (1991) 630-635. 28 Lurie, A., Nadel, J.A., Roisman, G., Siney, H. and Dusser, D.J., Role of neutral endopeptidase and kininase lI on substance P-induced increase in nasal obstruction in patients with allergic rhinitis, Am. Rev. Resp. Dis., in press. 29 Ichinose, M., Nakajima, N., Takahashi, T., Yamauchi, H.. Inoue, H., and Takishima, T., Protection against bradykinininduced bronchoconstriction in asthmatic patients by neurokinin receptor antagonist, Lancet, 340 (1992) 1248.
159
REGPEP 01598
Budesonide inhibits plasma extravasation induced by capsaicin and by substance P in the. rat nasal mucosa Elena Bacci a, Claude Bertrand a, Pierangelo Geppetti a, Jonathan Baker a, Jay A. Nadel b, Lauri A. Laitinen c and GOran Petersson a,, "Cardiovascular Research Institute and b Departments of Medicine and Physiology, University of California, San Francisco, CA 94143 (USA) and CDepartment of Pulmonary Medicine, University Central Hospital, Helsinki (Finland) (Received 30 July 1993; revised version received and accepted 24 September 1993)
Key words: Corticosteroid; Neurogenic inflammation; Vascular permeability; Trachea; Urinary bladder
Summary We studied the effect of the locally administered glucocorticoid budesonide on plasma extravasation induced by capsaicin and by substance P (SP) in the nasal mucosa of pathogen-free rats. Using Evans blue dye as a tracer, we measured plasma extravasation induced by capsaicin (150 #g kg- 1 i.v.) or SP (0.5 and 2.5 #g kg- 1 i.v.) in the rat naso- and maxilloturbinates after pretreatment with budesonide (0.1-50 #g twice/day for 2 days in the right nostril; 50/~g only for SP) or its vehicle. We found that budesonide inhibits plasma extravasation induced by capsaicin in a dose-dependent fashion in the nasal cavity. After the highest dose (50 #g) of budesonide, the values of Evans blue in the nasal mucosa were not different from the values observed after capsaicin vehicle alone. Budesonide also reduced plasma extravasation induced by capsaicin in the trachea and the urinary bladder of the rats in a dose-dependent fashion. Budesonide (50/~g) delivered to the nose inhibited the plasma extravasation caused by 0.5 but not by 2.5/~g SP kg- 1 in the nasal mucosa. We conclude that the postjunctional part of the neurogenic pathway is a target for glucocorticoid antiinflammatory action in the nasal mucosa, at least of the rat. Budesonide's effect on organs other than the nose can be explained by systemic absorption.
Introduction * Corresponding author. Present address: Department of Otorhino-laryngology, University of Lund, General Hospital, S-214 01 Malm6, Sweden. Fax: +46 40 336229.
Stimulation of sensory nerve endings by capsaicin releases neuropeptides (e.g., substance P), which cause neurogenic inflammation in rodent airways
160
[1,2]. An important aspect of neurogenic inflammation in the nasal mucosa is increased vascular permeability [2,3]. Increased vascular permeability with subsequent plasma extravasation is believed to account, at least in part, for symptoms (e.g., rhinorrhea) that occur in nasal inflammatory diseases. Glucocorticoids are commonly and effectively used in the therapy of many inflammatory diseases of the nose [4], but it is not known which of the many pathways involved in inflammation are affected by glucocorticoid treatment. It has been demonstrated that glucocorticoids inhibit plasma extravasation induced by neurogenic inflammation in the guinea pig and rat trachea [5,6]. In this study, we investigated whether locally administered glucocorticoids inhibit neurogenic plasma extravasation in the nasal mucosa of rats. We pretreated rats with budesonide for 2 days before giving an intravenous injection of capsaicin or substance P (SP), and we assessed changes in vascular permeability using Evans blue dye [7]. Our data show that budesonide inhibits plasma extravasation induced by capsaicin and by SP in the rat nasal mucosa. Materials and Methods
Animals We studied the effect of budesonide on capsaicinand SP-induced plasma extravasation in the nose of male, pathogen-free, F344 rats (200-250 g) from Simonsen Laboratories, Inc. (Gilroy, CA). The rats were housed in autoclaved plastic cages isolated by static filters (Microinsulator System, Lab Products, Maywood, N J) and placed inside a laminar flow rack enclosure (Stay-Clean, Lab Products) to prevent respiratory tract infection [ 6 ].
Pretreatment To determine the dose-response effect of budesonide on capsaicin-induced plasma extravasation, three groups of rats were treated for 2 days with budesonide (0.1 (n = 5), 1 (n = 5), 50 #g (n = 9) intranasally; ASTRA DRACO AB, Lund, Sweden). To
determine the response ofbudesonide on SP-induced plasma extravasation, two groups of rats were treated for 2 days with budesonide. After anesthesia with sodium methohexital (Brevital Sodium, Lilly, Indianapolis, IN), 60 mg kg 1 i.p., budesonide (25/d) was inoculated in the right nostril of each animal. The inoculation procedure was repeated four times, starting in the afternoon of the first day, then in the morning and in the afternoon of the second day, with the last inoculation being given in the morning of the third day, just before starting the experiment. A group of eight control rats received budesonide vehicle before capsaicin, and a group of four control rats received budesonide vehicle before SP, according to the administration protocol described above. The corticosteroids were given for 2 days prior to capsaicin or SP, because previous studies in the trachea suggested that this timing produces a maximal effect [6].
Description of the experiments The rats were anesthetized with sodium methohexital, 70 mg kg i i.p., supplemented as needed. To assess vascular permeability, we injected Evans blue dye (3~o solution in 0.9~o NaC1, Polysciences, Inc., Warrington, PA). Evans blue has the property of binding to albumin and is therefore used to quantify plasma protein extravasation [7]. Immediately after the injection of the Evans blue (30 mg kg- 1 i.v. over 5 s), neurogenic plasma extravasation was induced by giving an intravenous injection of capsaicin (150/~g kg- 1 i.v. over 2 min; Sigma Chemical Co., St. Louis, MO), dissolved in a vehicle containing 0.75% ethanol, 0.375~o Tween 80, and 0.85~o NaCI. In a control group of rats (n = 5), the vehicle of capsaicin alone was given. Alternatively, plasma extravasation was produced by SP (0.5 (n = 5) or 2.5 (n = 4) #g kg- 1 i.v. over 20 s; Sigma), dissolved in water. In a control group of rats, water only was given. 5 min after the injection of capsaicin or SP, the chest was opened, a cannula was inserted into the ascending aorta through the left ventricle, and the circulation was perfused for 2 min with phosphate-
161 buffered saline (pH 3.5) at a pressure of 120 mmHg. The trachea was removed. The nose was dissected according to the procedure previously described [8]. Briefly, the head of the rat was cleared of external soft tissues, and the nasal cavities were opened with paramedian incisions through the dorsum and the floor of the nose. The naso- and maxilloturbinates were dissected free from the lateral wall of the nose. To test possible systemic and other possible effects of budesonide, the trachea and the urinary bladder was also removed. After blotting on bibulous paper, the trachea, the turbinates and the urinary bladder were weighed and placed in vials containing 3 ml of formamide (Fisher Scientific, Fair Lawn, N J) overnight at room temperature to extract Evans blue. Thereafter, the dye concentration in formamide was measured using a spectrofluorimeter (SLM 8000C, SLM Instruments Inc., Urbana, IL) [9]. Samples were excited through a monochromator at 620 nm (4 nm bandwidth). Emission light above 645 nm was filtered by a RG-645 cut-off filter (Schott, Duryea, PA) and detected by a photomultiplier (SLM). The signal was digitized and recorded by an interfaced IBM P C / A T computer. The signal to noise ratio was 6:1 for the weakest signal. The concentration of Evans blue was calculated from standard curves of Evans blue (0.005-10 #g m l - 1 in formamide) and expressed as ng of dye per mg of tissue. In most cases the septum had an opening that provides a communication between the two nasal cavities. To test whether the inoculated budesonide can move from the right to the left nasal cavity and to the trachea, we inoculated 25 #1 of Evans blue dissolved in budesonide vehicle into the right nostril of four rats. After 15 rain, we opened the chest and the nose to evaluate the distribution of the dye. The dye was present in the left nasal cavity, as well as in the esophagus of all rats. The dye was found in the upper third of the trachea in one rat.
Statistical analysis We have previously shown that capsaicin increases vascular permeability mainly in the naso- and max-
illoturbinates [8], so we pooled the values of Evans blue dye measured for the naso- and maxilloturbinates. Data are reported as mean + S.E.M. Possible differences between capsaicin (or SP) and capsaicin (or SP) vehicle-induced plasma extravasation was tested by Student's unpaired t-test. Mean values for different treatments were compared by one-way analysis of variance. Multiple comparisons between groups were tested by Dunnett's test. Differences between right and left turbinates were tested by Student's paired t-test. P-values < 0.05 were considered significant.
Results Capsaicin alone (150 #g k g - 1 i.v.) increased the concentration of Evans blue in the rat nasal mucosa from control values of 12.16 + 0.87 ng m g tissue (mean + SE) to 25.81 + 2.04 ng m g - 1 tissue ( P < 0.001). Pretreatment with budesonide inhibited Evans blue extravasation after capsaicin in a dosedependent fashion (Fig. 1). After 50 #g budesonide,
30
E v a n s blue
in turbinates (ng/mg of tissue)
15 ¸
0.1
1
50
I CAPSAIClN VEHICLE
BUDESONIDE VEHICLE then CAPSAICIN
I BUDESONIDE ~g) then CAPSAICIN
Fig. 1. Dose-dependentinhibitory effectof budesonide on Evans blue extravasationinduced by capsaicin(150 #g kg i i.v.)in nasoand maxilloturbinates of pathogen-free rats. Values are mean + S.E.M. Asterisks express a significant differencebetween treatment with budesonide and control (budesonide vehicle, *P < 0.05).
162 Rightturbln~ltes
30
I
60
Leftturblnates
1
[
I
Evans blue in trachea (ng/mg of tissue)
Evans blue 15 in turbinates (ng/mg of tissue)
i i,
30
15
0J 0
iol
1 5o I
BUDESONIDE BUDESONIDE VEHICLE (Fg)
Ol
1 50 I CAPSAICIN VEHICLE
BUDESONIDE BUDESONIDE VEHICLE (~g)
then
then
then
then
CAPSAICIN
CAPSAICIN
CAPSAICIN
CAPSAICIN
BUDESONIDE VEHICLE
BUDESONIDE ~g)
then
then
CAPSAICIN
CAPSAICIN
Fig. 2. Comparison of Evans blue cxtravasation induced by capsaicin (150 #g kg ~ i.v.) in right and left turbinates of pathogenfree rats after pretreatment with different doses of budesonide in the right nostril. No significant difference was observed between the right and left sides. Values are mean _+ S.E.M.
Fig. 3. Dose-dependent inhibitory effect of budesonidc on E~ans blue extravasation induced by capsaicin (150 /xg kg ' i.v.) in trachea of pathogen-free rats. Values are mean _+S.E.M. Asterisk expresses a significant difference between treatment with budesonide and control (*P<0.05).
the values were not different from the values observed after capsaicin vehicle alone (P= 0.96). There was no significant difference in the effect of budesonide on vascular extravasation between the right and left sides of the nose (Fig. 2). Budesonide
also reduced in a dose-dependent fashion the increase in Evans blue extravasation induced by capsaicin in the trachea (Fig. 3) and in the urinary bladder (Fig. 4) of the rats. SP alone increased the concentration of Evans
15
T 10
Evans blue in bladder (ng/mg of tissue)
5
0
D
0.1
1
50
I CAPSAICIN VEHICLE
BUDESONIDE VEHICLE then CAPSAICIN
I BUDESONIDE (~g) then CAPSAICIN
Fig. 4, Dose-dependent inhibitory effect of budesonide on Evans blue extravasation induced by capsaicin (150 pg kg ~ i.v.) in urinary bladder of pathogen-free rats. Values are mean + S.E.M. Asterisk expresses a significant difference between treatment with budesonide and control ( * P < 0.05).
163
90
60Evans blue (ng/mg of tissue} 30-
OTurbinates
Turbinates
Trachea
Bladder
Fig. 5. The effect ofbudesonide (50 #g twice/day, 2 days; hatched columns) or its vehicle (filled columns) on Evans blue extravasation caused by (A) SP (0.5 gg kg i in the naso- and maxilloturbinates, and by (B) SP (2.5 ~g kg 1) in the naso- and maxilloturbinates, the trachea and the urinary bladder of pathogen-free rats. Evans blue extravasation after SP-vehicle only is also shown (open columns). Values are mean _+S.E.M. Asterisks express a significant difference between treatment with budesonide and control (*P<0.05).
blue in the nasal mucosa in a dose-dependent fashion; from control values of 11.14+0.73 ng mg - 1 tissue to 29.40 + 3.37 ng mg- 1 tissue ( P < 0.05) after SP 0.5 #g kg - t i.v. and to 66.90 + 8.5 ng mg- 1 tissue ( P < 0.001) after 2.5/~g kg- 1 i.v. Budesonide (50/~g) reduced the extravasation caused by SP 0.5 ( P < 0.05) but not by SP 2.5 (P = 0.52) #g kg- 1 i.v. (Fig. 5). The SP evoked vascular extravasation after budesonide did not differ between sides of the nose (P = 0.54; data not shown). Budesonide inhibited the plasma extravasation after SP (2.5/~g kg- 1) in the urinary bladder and in the trachea (Fig. 5).
Discussion
This study shows that budesonide inhibits plasma extravasation induced in the rat nasal mucosa by intravenous injection of capsaicin in a dose-dependent fashion and by SP. The highest close of budesonide completely abolished the extravasation induced by capsaicin and reduced the extravasation caused by a low dose but not by a high dose of SP in the nasal mucosa. The lack of difference in the effect of budesonide between the right and the left turbinates is apparently
due to the diffusion of the drug to the opposite nasal cavity, as we demonstrated by the inoculation of dye in the right nostril. The inhibitory effect of budesonide on plasma extravasation in the trachea may be due either to diffusion of the drug through the respiratory tract, as shown by nasal instillation of dye, or to systemic absorption. The fact that budesonide also reduced capsaicin-induced plasma extravasation in both the urinary bladder and in the trachea in a dose-dependent fashion is evidence for systemic absorption. Budesonide has been reported not to be metabolized in the nose or in the plasma [10]. Biotransformation of budesonide into low activity metabolites is reported to occur in the liver. The data reported in the literature about the systemic effects of locally administered steroids are controversial. Intranasally administered flunisolide, budesonide, or beclomethasone dipropionate significantly improved asthma and/or ocular symptoms [4,11-13]. On the other hand, Toogood and co-workers [14] showed that a dose of oral budesonide giving plasma levels similar to those achieved with a locally effective dose of budesonide had no significant effect on the occurrence of asthma symptoms when compared to placebo in humans. It has been reported that systemic availability of active budesonide in the rat after oral administration is 10% of the administered dose, with a half-life of 2.5 h [ 10]. Plasma extravasation in the nasal mucosa induced by capsaicin is considered to be specific and selectively mediated by sensory nerve stimulation because this effect of i.v. capsaicin is abolished in capsaicinpretreated animals [15]. In the trachea of rats, steroids inhibit plasma extravasation induced by capsaicin [6]. Various investigators have reported different effects of corticosteroids on neurogenic extravasation using different stimuli and using different corticosteroids and drug schedules. For example, methylprednisolone did not inhibit the tracheal extravasation caused by exposure to cigarette smoke and enhanced the extravasation caused by vagal nerve stimulation [ 16]. Likewise, pretreatment with betamethasone before gastric juice exposure
164
enhanced the tracheal extravasation [17]. The absence of corticosteroid inhibition in these studies could be due to differences in the dose or in the timing ofglucocorticoid administration or it could be due to differences among tissues. Corticosteroids may affect plasma extravasation associated with neurogenic inflammation in several ways and the effect might be different in different structures. The exact mechanism and level of the effect in the nasal mucosa is not clear. Because budesonide inhibited the plasma extravasation in the nasal mucosa after capsaicin and after a low dose of SP (equipotent stimuli), it appears that the inhibition of capsaicin-induced plasma extravasation is due to a post-junctional site of action in agreement with the effects of corticosteroids in the trachea [6]. One possible explanation for the observation that budesonide failed to inhibit the response after the high dose of SP in the nasal mucosa, but not in the trachea and in the bladder, is that the high dose of SP resulted in a 'supermaximar response. The effect of budesonide on the extravasation in the nasal mucosa might be due to an effect on neutral endopeptidase, because corticosteroids upregulates neutral endopeptidase [18], as demonstrated in the lower airways [19]. In the skin, corticosteroids are known to cause a 'blanching', an effect which is attributed to vasoconstriction [20]. In the nasal mucosa, the inhibition of the capsaicin-induced plasma extravasation is not likely to be due to a vasoconstriction effect by budesonide, because budesonide is not found to cause any vasoconstriction in the nasal mucosa in healthy subjects [21,22]. However, these findings do not exclude an effect on blood flow by corticosteroids in inflamed tissue but a reduction of blood flow in the nasal mucosa per se does not necessarily imply a subsequent reduction of the plasma extravasation [23]. The nose is the first structure that inhales air encounters on its way to the lungs and is, therefore, the first barrier to inhaled irritants and pathogens. Stimuli such as cigarette smoke [24], formaldehyde and ether [15] can induce neurogenic inflammation in the rodent nasal mucosa via a capsaicin-sensitive
mechanism. Glucocorticoids have been reported to inhibit virus-induced potentiation of plasma extravasation produced by capsaicin in the rat trachea [6]. In a controlled study in children with rhinitis, a higher incidence of sneezing was observed after inhalation of placebo as compared with inhalation of budesonide [4]. The authors attributed this result to the protective effect of budesonide on the irritation induced by the placebo inhalation. Some of the therapeutic effects of corticosteroids could be due to the inhibition of increased vascular permeability and the subsequent effects of plasma extravasation. However, it is not certain whether neurogenic inflammation plays a role in human airway disease. In contrast to rodent nasal airways, capsaicin is not found to cause plasma leakage as measured by increased albumin in nasal airway lavage of healthy subjects [25,26]. On the other hand, SP caused an increase of plasma exudation in patients with allergic rhinitis [27]. Furthermore, nasal resistance to airflow is increased by local instillation of SP in a dosedependent fashion in normal subjects and in patients with allergic rhinitis [28]. In asthmatic patients, the bronchial responses to inhaled tachykinin aerosols was recently reported to be inhibited by a neurokinin receptor antagonist [29]. So far, the relevance of neurogenic inflammation in upper airway disease is not tested by means of neurokinin receptor antagonists. In conclusion, our study demonstrates that budesonide inhibits plasma extravasation induced by capsaicin in a dose-dependent fashion and by SP, in the rat nasal mucosa, showing that the postjunctional part of the neurogenic pathway is a target on which glucocorticoids exert their antiinflammatory effect in the nasal mucosa.
Acknowledgements We thank Donald M. McDonald, M.D., Ph.D. for useful discussions, Isao Yamawaki, M.D., Brendan Chart, Amy Haskell and Simona Ikeda for technical
165
assistance, Thomas Hartmann, Ph.D., for assisting with spectrofluorometry, Natalie Holt for typing the manuscript. This study was supported in part by NIH PPG-HL24136. Elena Bacci was supported in part by grants from the Italian Ministry of Education and from NIH (USA/Italy Exchange Program). Claude Bertrand was supported in part by a grant from the Fondation pour la Recherche M6dicale. G6ran Petersson was supported in part by grants from Tore Nilsons Fund for Medical Research, the Medical Faculty, University of Lund, Sweden, the Swedish Society of Medicine and the Swedish Medical Research Council (Including No. 8686). References 1 Lundblad, L., Lundberg, J.M., Brodin, E. and A.nggfird, A., Origin and distribution of capsaicin-sensitive substance P-immunoreactive nerves in the nasal mucosa, Acta Otolaryngol., 96 (1983) 485-493. 2 Lundblad, L., Saria, A., Lundberg, J.M. and Anggfird, A., Increased vascular permeability in rat nasal mucosa induced by substance P and stimulation of capsaicin-sensitive trigeminal neurons, Acta Otolaryngol., 96 (1983) 479-484. 3 Saria, A., Lundberg, J.M., Skofitsch, G. and Lembeck, F., Vascular protein leakage in various tissues induced by substance P, capsaicin, bradykinin, serotonin, histamine and by antigen challenge, Naunyn-Schmiedeberg's Arch. Pharmacol., 324 (1983) 212-218. 4 Henriksen, J.M. and Wenzel, A., Effect ofintranasally administered corticosteroid (budesonide) on nasal obstruction, mouth breathing, and asthma, Am. Rev. Respir. Dis., 30 (1984) 1014-1018. 5 ErjefNt, 1. and Persson, C.G.A., Anti-asthma drugs attenuate inflammatory leakage of plasma into airway lumen, Acta Physiol. Scand., 128 (1986) 653-654. 6 Piedimonte, G., McDonald, D.M. and Nadel, J.A., Glucocorticoids inhibit neurogenic plasma extravasation and prevent virus-potentiated extravasation in the rat trachea, J. Clin. Invest., 86 (1990) 1409-1415. 7 Jancsd-Gfibor, A,. Szolcs/myi, J. and Jancsd, N., A simple method for measuring the amount of azovan blue exuded into the skin in response to an inflammatory stimulus, J. Pharm. Pharmacol., 19 (1967) 486-487. 8 Petersson, G., Bacci, E., McDonald, D.M. and Nadel, J.A., Neurogenic plasma extravasation in the rat nasal mucosa is potentiated by peptidase inhibitors, J. Pharm. Exp. Ther., 264 (1993) 509-514.
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166 clinical effect through vasoconstriction, Clin. Otolaryngol., 14 (1989) 519-523. 23 Svensson, C., Pipkorn, U., Alkner, U., Baumgarten, C.R. and Persson, C.G.A., Topical vasoconstrictor (oxymetazoline) does not affect histamine-induced mucosal exudation of plasma in human nasal airways, Clin. Exp. AII.~ 22 (1992) 411-416. 24 Lundberg, J.M., Lundblad, L., Saria, A. and AnggOxd, A., Inhibition of cigarette smoke-induced oedema in the nasal mucosa by capsaicin pretreatment and a substance P antagonist, Naunyn-Schmiedeberg's Arch. Pharmacol., 326 (1984) 181-185. 25 Bascom, R., Kagey-Sobotka, A. and Proud D., Effect of intranasal capsaicin on symptoms and mediator release, J. Pharm. Exp. Ther., 259 (1991) 1323-1327.
26 Rajakulasingam, K., Polosa, R., Lau, L.C., Church, M.K.. Holgate, S.T. and Howarth, P.H., Nasal effects of bradykinin and capsaicin: influence on plasma protein leakage and role of sensory nerves, J. Appl. Physiol., 72 (1992) 1418-1424. 27 Braunstein, G., Fajac, I., Lacronique, J. and Frossard. N., Clinical and inflammatory responses to exogenous tachykinins in allergic rhinitis, Am. Rev. Resp. Dis., 144 (1991) 630-635. 28 Lurie, A., Nadel, J.A., Roisman, G., Siney, H. and Dusser, D.J., Role of neutral endopeptidase and kininase lI on substance P-induced increase in nasal obstruction in patients with allergic rhinitis, Am. Rev. Resp. Dis., in press. 29 Ichinose, M., Nakajima, N., Takahashi, T., Yamauchi, H.. Inoue, H., and Takishima, T., Protection against bradykinininduced bronchoconstriction in asthmatic patients by neurokinin receptor antagonist, Lancet, 340 (1992) 1248.