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Type A natriuretic peptides exhibit different bronchoprotective effects in rats
ejp ELSEVIER
European Journal of Pharmacology 271 (1994) 395-402
Type A natriuretic peptides exhibit different bronchoprotective effects in rats Thomas Fliige
a,b,c,*,
Heinz-Gerd Hoymann b, Jens Hohlfeld a, Uwe Heinrich b, Helmut Fabel Thomas O.F. Wagner a, Wolf-Georg Forssmann c
a,
a Department of Respiratory Medicine, Hannover Medical School, Hannover, Germany b Fraunhofer Institute of Toxicology and Aerosol Research, Hannover, Germany c Lower Saxony Institute for Peptide Research, Hannover, Germany Received 12 April 1994; revised MS received 8 September 1994; accepted 27 September 1994
Abstract
The protective effect of 11.4, 22.8 or 45.6 pmol/kg/min cardiodilatin/atrial natriuretic peptide (CDD/ANP-(99-126)), urodilatin (CDD/ANP-(95-126)) or vehicle intravenously against acetylcholine-induced bronchoconstriction was compared in spontaneously breathing, halothane-anesthetized Wistar rats. The inhalation of acetylcholine induced significant alterations of the spontaneous breathing parameters evaluated by whole-body plethysmography without significant differences between the treatment groups. Forced parameters detect airflow changes with a greater sensitivity and were measured in hyperventilation-induced temporary apnoea after the challenge. The forced expiratory volume in 0.1 s revealed a significant protective effect of 11.4 pmol/kg/min urodilatin compared to the controls whereas the parameters of the forced expiratory flow-volume curve were significantly preserved by 11.4 and 22.8 pmol/kg/min urodilatin (P < 0.05). Urodilatin showed protective effects against an acetylcholine challenge whereas CDD/ANP-(99-126) was without significant influence.
Keywords: Urodilatin; Natriuretic peptide, type A; Bronchoprotection; Acetylcholine; Lung function, rat
1. Introduction
Cardiodilatin/atrial natriuretic peptide ( C D D / ANP-(99-126)) is not only a diuretic and natriuretic peptide but also a specific vasodilator of renal, coronary and pulmonary vessels (Menard, 1991; Forssmann, 1994). Moreover it relaxes the bronchial smooth muscle in various animals (O'Donnell et al., 1985; Chou et al., 1986; Hamel and Ford-Hutchinson, 1986; Englebach et al., 1988; Watanabe et al., 1988; Ishii and Murad, 1989). The bronchodilating effect of C D D / ANP-(99-126) appears to be more prominent in the larger central airways than in the peripheral airways (Chou et al., 1986; Hamel and Ford-Hutchinson, 1986; Banerjee and Newman, 1990). In vitro studies with human airways revealed contradictory results (Labat et
* Corresponding author. Lower Saxony Institute for Peptide Research GmbH, Feodor-Lynen-Strasse 31, D-30625 Hannover, Germany. Tel. 49-511-855525, fax 49-511-855525. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00595-8
al., 1988; Candenas et al., 1991; Hulks et al., 1991; Fernandes et al., 1992). An increase of the forced expiratory volume in 1.0 s (FEVt. 0) was demonstrated in vivo during, and for a short period after the end of, an intravenous infusion in asthmatic subjects by different investigators (Hulks et al., 1989; Chanez et al., 1990). In addition, this peptide has been shown to inhibit the effects of methacholine, histamine, serotonin, leukotriene D4, and arachidonic acid on the guinea pig trachea in vitro (Hamel and Ford-Hutchinson, 1986; Potvin and Varma, 1989) and on the pulmonary inflation pressure in vivo (Potvin and Varma, 1989). The natriuretic peptide, urodilatin, was discovered by Schulz-Knappe et al. in 1988 (Schulz-Knappe et al., 1988). It consists of the entire molecule of human CDD/ANP-(99-126) plus a four-residue NH2-terminal extension: threonine-alanine-proline-arginine. Urodilatin, CCD/ANP-(95-126), is probably produced from the same gene code as type A natriuretic peptides in the heart but with different posttranslational pro-
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cessing to yield the 32-residue peptide (Schulz-Knappe et al., 1988). The first discovery of urodilatin in human urine, together with data from renal cell cultures, immunocytochemistry, radioimmunoassays, and other techniques, led to the conclusion that the distal tubules in the kidney synthesize a 126-amino acid prohormone identical to the one of CDD/ANP-(99-126) in cardiac atria (Goetz, 1991; Forssmann, 1994). Urodilatin shows an affinity for the same binding sites as CDD/ANP(99-126), and like CDD/ANP-(99-126) it activates guanylate cyclase in membranes from various tissues (Watanabe et al., 1988; Heim et al., 1989; Valentin et al., 1991). In vitro, urodilatin evokes a relaxation of vascular and bronchial smooth muscle (Schulz-Knappe et al., 1988; Duft et al., 1993). In vivo, the natriuretic and diuretic potency of urodilatin significantly exceeds that of CDD/ANP-(99-126) (Goetz, 1991; Villarreal et al., 1991; Abassi et al., 1992a; Hildebrandt et al., 1992; Bestle and Bie, 1993), but the effects on the systemic blood pressure are less pronounced (Saxenhofer et al., 1990). No immunoreactivity of urodilatin is found in human plasma (Goetz, 1991; Kentsch et al., 1992; Drummer et al., 1993; Forssmann, 1994). The aim of the present study was to investigate the protective effect of equimolar doses of intravenous CDD/ANP-(99-126), urodilatin or vehicle against bronchoconstriction induced by acetylcholine inhalation in anaesthetized Wistar rats.
2. Material and methods
2.1. Study design Forty-six 13-week-old female Wistar rats (Han: WlST, Zentralinstitut fiir Versuchstierzucht, Hannover, Germany) weighing 221.1 + 14.0 g (mean+ standard deviation) were studied. The internationally accepted principles in the care and use of experimental animals were strictly followed. The rats were anaesthetized with halothane/30% 0 2 and intubated orally with a tracheal cannula. An indwelling venous catheter in the tail vein was used for the intravenous infusion of 0.4 ml CDD/ANP-(99-126), urodilatin or vehicle (0.9% sodium chloride) at a constant rate for 12 min in a randomized order. CDD/ANP-(99-126) and urodilatin were dissolved in 0.9% sodium chloride and were given in doses of 11.4, 22.8 or 45.6 p m o l / k g / m i n (equivalent to 35.2, 70.3 or 140.5 n g / k g / m i n C D D / ANP-(99-126) and 40.0, 80.0 or 160.0 n g / k g / m i n urodilatin) using only one dosage per treatment group (n = 6). During the infusion period an acetylcholine challenge test was started after 5 min. Spontaneous pulmonary function parameters were evaluated under steady-state conditions prior to the infusion (baseline) and before and at the end of the acetylcholine inhala-
tion (pre/post-acetylcholine challenge). Forced parameters including volume-time and flow-volume relation were measured in hyperventilation-induced temporary apnoea immediately after the acetylcholine challenge. Heart rate was controlled by electrocardiography during the study. Vehicle-treated animals were used as controls on each study day. 2.2. Pulmonary function tests The techniques of quasi non-invasive mechanical lung function measurements (Heinrich and Wilhelm, 1984; Heinrich et al., 1989; Hoymann et al., 1994) used in this study are based on the methods reported by Likens and Mauderly (Likens and Mauderly, 1982; Tepper et al., 1987; Mauderly, 1989). The intubated animal was placed supine in a whole-body plethysmograph. The halothane concentration was adjusted individually between 1.5 and 3.0% (in 30% 0 2, gas flow 2.2 l/min) to get similar respiratory frequencies in all animals. This type of anaesthesia enables the performance of involuntary breathing manoeuvres and permits the measurement of lung compliance and resistance.
2.2.1. Spontaneous respiratory function parameters The parameters of spontaneous respiration including tidal volume (VT), dynamic lung compliance (Cdyn) and lung resistance (R L) were recorded continuously (analyzer model 6, Buxco, Sharon, USA) and evaluated at baseline, pre- and post-acetylcholine challenge. Respiratory volume changes were determined using a pneumotachograph (pressure transducer MP45, Validyne, Northridge, USA) at the rear wall of the chamber. Transpulmonary pressure changes (PAP) were analyzed by subtracting the oesophageal pressure, measured via a water-filled silicone tube in the position of maximal signal (tube diameter 1.6 mm; pressure transducer P23Db, Statham, Hato Ray, Puerto Rico), from the airway pressure determined in a side branch of the breathing port (air filled W101/RFT pressure transducer, MefAger~itewerkZw6nitz, Germany). Pap is necessary for calculating Cdyn and R L using the method of Amdur and Mead (Amdur and Mead, 1958).
2.2.2. Inhalation of acetylcholine The challenge test was performed using the concentration-dependent potency of an acetylcholine chloride aerosol to induce bronchospasm. The increase in lung resistance is a useful parameter to determine the degree of airway obstruction in the anaesthetized rat (Heinrich and Wilhelm, 1984; Pauluhn et al., 1987; Hohlfeld et al., 1992a; Hoymann et al., 1994). After 5 min of intravenous infusion a precisely defined acetylcholine aerosol (15 m g / m 3, mass median aerodynamic diameter (MMAD) 1.9 _+ 1.5/zm, dried) was added to the halothane and oxygen for 7 min. The aerosol was
ml ±S.D. ml/cm H20 _+S.D. cm H 2 0 / m l / s _+S.D.
VT e
% pre-Ach d +S.D. % pre-Ach d +S.D. % pre-Ach d +S.D.
- 1 9 . 3 0 ++ 9.26 - 3 6 . 4 4 ++ 12.12 +61.84 ++ 45.00
- 1 1 . 8 0 ++ 6.01 - 3 2 . 6 5 ++ 11.37 +38.76 +4 18.73
- 2.04 2.26 - 6.32 ++ 3.39 + 5.14 ÷ + 2.90
0.15 0.135 0.011 0.289 0.030
0.23 0.172 0.047 0.272 0.046
- 1.07 2.90 - 4.54 q-+ 3.50 + 1.73 7.39
1.25
6
U R O 11.4 b
1.35
10
Controls ~
- 1 1 . 7 0 ++ 3.40 - 4 0 . 5 6 ++ 9.64 +65.46 ++ 30.39
- 0.79 5.16 - 3.53 5.47 + 1.97 6.44
0.10 0.159 0.018 0.290 0.037
1.39
6
U R O 22.8 ~
- 1 7 . 0 6 ++ 9.08 -37.69 + 17.62 +67.43 ++ 38.84
- 2.01 4.12 - 10.34 + 7.86 + 6.06 ÷ 5.77
0.21 0A86 0.047 0.281 0.081
1.46
6
U R O 45.6 b
- 1 9 . 7 3 ++ 7.69 - 4 3 . 6 0 4+ 11.24 +45.47 ++ 16.07
+ 3.98 ÷ 3.67 - 4.24 7.23 + 3.78 8.36
0.05 0.156 0.043 0.284 0.055
1.25
6
A N P 11.4 c
- 1 6 . 9 7 ++ 7.88 - 4 6 . 5 2 4+ 15.12 +72.33 ++ 41.32
- 1.27 2.41 + 4.61 24.55 - 1.77 11.47
0.08 0.184 0.037 0.308 0.095
1.41
6
A N P 22.8 c
- 1 7 . 1 1 ++ 9.27 - 4 6 . 4 6 ++ 8.79 +58.49 ++ 29.09
+ 1.27 2.64 + 2.25 8.63 -4.08 7.92
0.08 0.175 0.024 0.271 0.051
1.30
6
A N P 45.6 c
(paired t-test).
R at spontaneous pulmonary function par am et er s were evaluated unde r steady-state conditions (baseline) prior to the intravenous infusion of sodium chloride 0.9%, urodilatin or c a r d i o d i l a t i n / a t r i a l natriuretic peptide (99-126). During the infusion period an acetylcholine challenge test was started after 5 rain. All p a r a m e t e r s were measu r ed again before and at the en d of the acetylcholine inhalation, a Infusion of sodium chloride 0.9%. b T r e a t m e n t with urodilatin ( U R O ) 11.4, 22.8 or 45.6 p m o l / k g / m i m c T r e a t m e n t with c a r d i o d i l a t i n / a t r i a l natriuretic p e p t i d e (99-126) (ANP) 11.4, 22.8 or 45.6 p m o l / k g / m i n , d B e f o r e / a f t e r acetylcholine challenge (Ach). e Tidal volume, f Dynamic compliance, g Lung resistance. + P < 0.05 (paired t-test). + + P < 0.01
RL g
Cdy n f
Vx ~
Post-Ach d
RLg
Cdyn f
Pre-Ach d VT e
RLg
tr~ baseline +S.D. % baseline +S.D. % baseline +S.D.
n
Baseline
Cdy n f
Units
Parameter
Table 1 Spontaneous pulmonary function parameters at baseline, before and after acetylcholine challenge
ta.a
!
t~
g~
x..
T. Fliige et al. /European Journal of Pharmacology 271 (1994) 395-402
398
generated by a recently developed device including a computer-controlled system to select and continuously monitor the mass concentration and flow (Bronchy-H, Fraunhofer Institute of Toxicology and Aerosol Research, Hannover, Germany (Hohlfeld et al., 1992b)). In addition, the acetylcholine dose inhaled was calculated by the computer from the volume of minute ventilation and the concentration therein. The spontaneous pulmonary function values were evaluated at the time individual animals reached a total inhaled volume of 600 ml aerosol, corresponding to 9/~g acetylcholine (post-acetylcholine challenge).
2.2.3. Forced lung function parameters The forced lung function parameters were measured 1 min after the end of the acetylcholine inhalation in hyperventilation-induced temporary apnoea (Hoymann et al., 1994). Air reservoirs at a pressure of + 40 and - 5 0 cm H 2 0 were used for maximal inspiration (maximal PTP + 30 cm H20, flow 10 ml/s) and expiration to record forced expiratory volume-time relation and flow-volume curve. The evaluation included forced vital capacity (FVC (ml)) and forced expiratory volume in 0.1 s in relation to FVC (FEV0.1% (%FVC)) for the volume-time curve, and peak expiratory flow (PEF), maximum mid-expiratory flow (MMEF) and flow at 75, 50 and 25% FVC for the flow-volume curve (FEF75, FEFs0, FEF25, all flow values: ml/s). Forced parameters are aimed at detecting airflow obstruction with a greater sensitivity than measurements taken during tidal breathing (Mauderly, 1989) and were first incorporated into lung function tests with rats in 1977 (Diamond and O'Donnell, 1977). 2.3. Drugs Lyophilized, highly purified (> 99%) urodilatin acetate produced by the liquid phase peptide synthesis
method (32 amino acids, molecular weight 3506, batch number: A 08405) was purchased from Novabiochem, L~iufelfingen, Switzerland, and human CDD/ANP(99-126) (28 amino acids, molecular weight 3081, peptide purity 98.5%, batch number: 147D) from Saxon Biochemicals, Hannover, Germany. Both peptides were dissolved in physiological saline solution under sterile conditions immediately before infusion.
2.4. Statistics The data are presented as means and standard deviations ( + S.D.). The spontaneous pulmonary function parameters are reported as original values for the baseline. Changes in pre- and post-acetylcholine challenge were calculated as percent baseline and percent pre-acetylcholine challenge, respectively. The data for the forced expiratory manoeuvres are presented as originally measured. Analysis of variance (ANOVA) in combination with the Dunnett test were used to assess the significance of a difference between group mean values and the paired t-test for the determination of changes in a certain group pre- and post-acetylcholine challenge compared to baseline and pre-acetylcholine challenge, respectively. A value of P < 0.05 was considered significant.
3. Results
3.1. Baseline data Baseline measurements of the spontaneous pulmonary function parameters are shown in Table 1. VT, Cd~ and R L were in the normal range established in the laboratory and did not differ significantly between treatment groups. The respiratory frequency and the
Table 2 Heart rate at baseline, before and after acetylcholine challenge Parameter
Units
Controls a
U R O 11.4 b
U R O 22.8 b
U R O 45.6 b
ANP 11.4 c
ANP 22.8 c
ANP 45.6 c
Baseline
n
Heart rate
min - 1 + S.D.
10 427.00 30.46
6 439.26 29.84
6 424.00 6.78
6 445.76 60.10
6 447.68 20.22
6 420.06 10.91
6 394.96 24.07
% baseline + S.D.
+ 0.15 1.57
+ 0.04 1.00
- 2.29 0.46
+ 0.54 0.38
+ 0.90 3.04
+ 0.55 0.95
+ 0.04 3.42
% pre-Ach d + S.D.
-1.04 4.32
-1.13 2.25
-0.20 1.43
+0.27 2.21
+0.41 1.85
+0.53 3.42
-0.63 6.63
Pre-Ach d Heart rate
Post-Ach d Heart rate
The heart rate was evaluated under steady-state conditions (baseline) prior to the intravenous infusion of sodium chloride 0.9%, urodilatin or cardiodilatin/atrial natriuretic peptide (99-126). During the infusion period the course was monitored continuously by electrocardiography while an acetylcholine challenge test was started after 5 min. The heart rate was evaluated again before and at the end of the acetylcholine inhalation. a Infusion of sodium chloride 0.9%. b Treatment with urodilatin (URO) 11.4, 22.8 or 45.6 p m o l / k g / m i n , c Treatment with cardiodilatin/atrial natriuretic peptide (99-126) (ANP) 11.4, 22.8 or 45.6 p m o l / k g / m i n . ~ B e f o r e / a f t e r acetylcholine challenge (Ach).
T. Fliigeet al. / European Journal of Pharmacology 271 (1994) 395-402
399
Table 3 Parameters of the forced expiratory manoeuvres Parameter FEV0.1% d PEF e MMEF f FEF75 g FEFs0 g FEF25 g
Units
Controls a
n
10
% FVC _+S.D. ml/s + S.D. ml/s _+S.D. ml/s __S.D. ml/s ±S.D. ml/s _+S.D.
68.3 7.1 98.4 5.8 56.6 11.3 94.9 9.4 63.2 10.1 27.7 9.3
URO 11.4 b 6
77.6 4.2 113.6 9.2 77.3 11.4 112.2 8.6 83.2 11.2 42.4 10.2
* ** ** ** ** *
URO 22.8 b
URO 45.6 b
ANP 11.4 c
ANP 22.8 c
ANP 45.6 c
6
6
6
6
6
74.3 3.7 114.8 6.9 74.9 10.5 113.8 7.0 82.0 11.6 38.3 8.2
68.2 8.0 106.1 12.7 56.1 15.5 103.7 14.5 64.9 17.0 27.7 10.8
70.1 2.2 95.0 23.2 57.5 16.6 91.1 23.0 62.9 17.4 32.3 10.5
66.7 6.3 112.1 12.6 62.6 14.9 109.5 13.3 67.5 16.4 31.4 10.4
65.0 3.4 97.4 15.1 51.3 9.2 91.3 18.4 55.7 5.7 27.9 7.1
** ** ** ** *
During the intravenous infusion of sodium chloride 0.9%, urodilatin or cardiodilatin/atrial natriuretic peptide (99-126) an acetylcholine challenge test was started after 5 min. Forced parameters including volume-time and flow-volumerelation were measured in hyperventilation-induced temporary apnoea immediately after the end of the acetylcholine inhalation, a Infusion of sodium chloride 0.9%. b Treatment with urodilatin (URO) 11.4, 22.8 or 45.6 pmol/kg/min, c Treatment with cardiodilatin/atrial natriuretic peptide (99-126) (ANP) 11.4, 22.8 or 45.6 pmol/kg/min, d (forced expiratory volume in 0.1 s/forced vital capacity) × 100. e Peak expiratory flow. f Maximal mid-expiratory flow. g Forced expiratory flow at 75, 50 or 25% of forced vital capacity. * P < 0.05 (ANOVA + Dunnett test). * * P < 0.01 (ANOVA + Dunnett test).
inspiratory h a l o t h a n e c o n c e n t r a t i o n did n o t vary significantly (data n o t shown). H e a r t rate c o n t r o l l e d by e l e c t r o c a r d i o g r a p h y is d e m o n s t r a t e d in T a b l e 2. N o significant differences b e t w e e n the groups were f o u n d at baseline.
3.2. Infusion of urodilatin, CDD /ANP-(99-126) or vehicle preceding the acetylcholine challenge T h e i n f u s i o n of isotonic saline solution in the control a n i m a l s a n d of 11.4, 22.8 or 45.6 p m o l / k g / m i n u r o d i l a t i n or C D D / A N P - ( 9 9 - 1 2 6 ) resulted in n o significant differences b e t w e e n the t r e a t m e n t groups for the s p o n t a n e o u s respiratory f u n c t i o n p a r a m e t e r s after 5 m i n ( A N O V A + D u n n e t t test; T a b l e 1). C o m p a r i s o n with the individual b a s e l i n e values of each a n i m a l revealed a significant decrease of Cdyn in controls ( P < 0.01) a n d d u r i n g i n f u s i o n of 11.4 ( P < 0.01) a n d 45.6 p m o l / k g / m i n u r o d i l a t i n ( P < 0.05). I n addition, a significant increase of R L was d o c u m e n t e d after 5 m i n of 11.4 ( P < 0.01) or 45.6 p m o l / k g / m i n u r o d i l a t i n ( P < 0.05). Previous results showed n o significant b r o n c h o d i l a t i n g effect of the p e p t i d e s with respect to the p a r a m e t e r s of the forced expiratory m a n o e u v r e s at this time (data n o t shown). T h e h e a r t rate did n o t c h a n g e significantly in any g r o u p after a 5 - m i n i n f u s i o n ( T a b l e 2).
3.3. Effects of urodilatin, CDD /ANP-(99-126) or vehicle on the acetylcholine challenge T h e acetylcholine aerosol caused a b r o n c h o c o n s t r i c tion with a significant decrease in VT a n d Cdyn a n d a significant increase in R L in all groups ( P < 0.01). T h e r e were n o significant differences b e t w e e n the m e a n
values. C o m p a r i s o n of the groups t r e a t e d with 11.4 a n d 22.8 p m o l / k g / m i n u r o d i l a t i n or C D D / A N P - ( 9 9 - 1 2 6 ) evidenced smaller c h a n g e s in the a n i m a l s which received u r o d i l a t i n i n t r a v e n o u s l y ( T a b l e 1). As was expected, the respiratory f r e q u e n c y i n c r e a s e d significantly in all a n i m a l s d u r i n g the acetylcholine challenge ( + 5 6 . 1 + 8.1%, P < 0 . 0 1 ) with n o significant differences b e t w e e n the groups. T a b l e 3 s u m m a r i z e s the results of the forced expiratory m a n o e u v r e s . T h e FEW0.1% was significantly elevated in the a n i m a l s t r e a t e d with 11.4 p m o l / k g / m i n u r o d i l a t i n c o m p a r e d to the o t h e r groups ( P < 0.05). T h e flow-volume curve showed significantly higher values of P E F ( P < 0.01), M M E F ( P < 0.01), FEF75 ( P < 0.01), FEFs0 ( P < 0.01), a n d FEFz5 ( P < 0.05) in the rats with 11.4 a n d 22.8 p m o l / k g / m i n u r o d i l a t i n comp a r e d to those with e q u i m o l a r doses of C D D / A N P ( 9 9 - 1 2 6 ) a n d the controls. T h e F V C did n o t c h a n g e significantly in any t r e a t m e n t group. T h e r e were n o significant differences of these p a r a m e t e r s b e t w e e n the highest doses of u r o d i l a t i n a n d C D D / A N P - ( 9 9 - 1 2 6 ) or c o m p a r e d to the controls. T h e h e a r t rate r e m a i n e d u n c h a n g e d in all t r e a t m e n t groups (Table 2).
4. D i s c u s s i o n
A l t h o u g h n o significant differences of the s p o n t a n e o u s respiratory f u n c t i o n p a r a m e t e r s were f o u n d bet w e e n the t r e a t m e n t groups after 5 m i n of infusion, c o m p a r i s o n to the individual b a s e l i n e values showed a small significant decrease of Cdyn in the controls a n d d u r i n g 11.4 a n d 45.6 p m o l / k g / m i n urodilatin. This was c o m b i n e d with a small significant increase of R L in the two u r o d i l a t i n groups only.
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T. Fliige et al. / European Journal of Pharmacology 271 (1994) 395-402
In 1990 De Vries et al. showed that the clearance of human serum albumin infused in isotonic saline solution in rats was highest in the ileum and lung. High-dose CDD/ANP-(99-126) had no effect on pulmonary clearance but resulted in a significant increase in the ileum (De Vries et al., 1990). Zimmerman et al. (1990) reported, however, that pathophysiological levels of CDD/ANP-(99-126) provoke protein mobilization from arteries to the alveolar space, whereas pharmacological concentrations re-equilibrate the transwall gradient. Since there were no significant differences between the groups regarding the effects of urodilatin or CDD/ANP-(99-126) compared to the controls, it seems unlikely that the two peptides showed a relevant action on the pulmonary microcirculation in this study. The small changes of Cdyn and R L (Table 1) reported here may be the result of an increase in pulmonary plasma extravasation provoked by a relative volume overload. The infusion rate of 0.04 ml/min for 12 min was selected after review of the data of Hirata et al. (1985). They showed not only a significant decrease in mean arterial pressure and total peripheral vascular resistance, but also a reduction in cardiac output and central venous pressure after 10 min of infusion of comparable CDD/ANP-(99-126) doses at a rate of 0.02 ml/min in Wistar rats (Hirata et al., 1985). The haemodynamic changes were interpreted as the result of a direct relaxation of arterial smooth muscle and a venodilating effect of the peptide, reducing the cardiac pre- and afterload. Similar cardiovascular problems should be reduced in this study, by administration of a larger saline volume. General anaesthetics modify airway responsiveness by several mechanisms, including direct effects on airway smooth muscle and reductions in neural reflex activity. In dogs, halothane has been shown to reduce the response to cholinergic stimuli through both of these mechanisms (Hirshman et al., 1982; Warner et al., 1990). The inhalation of 9 /zg acetylcholine, however, produced a significant bronchoconstriction indicated by appropriate changes in R L and Cdy~ in all animal groups in this study while the inspiratory halothane concentration did not vary significantly between them. The more sensitive volume-time relation and flowvolume curve of the forced expiratory manoeuvres showed a significant protection of 11.4 and 22.8 pmol/kg/min urodilatin in the central (FEV0.1%, PEF, FEF75) and peripheral (FEFs0, FEF25) airways against the acetylcholine challenge (Diamond and O'Donnell, 1977; Tepper et al., 1987; Mauderly, 1989). Most forced parameters demonstrated a peak of the weaker CDD/ANP-(99-126) action at a dose of 22.8 p m o l / k g / m i n without reaching significance (Table 2). This contrasts with findings of the bronchodilating
properties of CDD/ANP-(99-126) in vitro and in vivo (Chou et al., 1986; Hamel and Ford-Hutchinson, 1986). A bolus injection of CDD/ANP-(99-126) in conscious sheep caused a significant reduction in the rise in R L induced by carbachol. However, it did not reverse the decrease in Cdyn after administration of histamine or carbachol, which was interpreted as a more prominent action on the larger central airways (Banerjee and Newman, 1990). On the other hand, in vitro urodilatin relaxes guinea pig tracheal tubes precontracted with methacholine significantly more potently than CDD/ANP-(99-126) (Duft et al., 1993). At present urodilatin is believed to act via the same receptors as CDD/ANP-(99-126) and the two peptides are equally potent in generating cyclic GMP at these sites (Heim et al., 1989; Rambotti and Spreca, 1991; Valentin et al., 1991). Despite a comparable plasma half-life of infused CDD/ANP-(99-126) or urodilatin of approximately 3 min (Yandle et al., 1986; Kentsch et al., 1990), differences were shown in the enzymes involved in their degradation. There is evidence that neutral endopeptidase (EC 3.4.24.11) plays an important role in the pulmonary metabolism of CDD/ANP-(99-126) (Perrella et al., 1991), but urodilatin is not affected by this enzyme (Gagelmann et al., 1988; Abassi et al., 1992a,b). This may cause pulmonary effects of urodilatin that are stronger a n d / o r last longer than those of CDD/ANP-(99-126). The results of this study are in accordance with this view. Another interesting finding is the absence of a significant baroreceptor-mediated reflex tachycardia despite the intravenous infusion of CDD/ANP-(99-126) doses that were shown to cause a reduction in arterial pressure (Hirata et al., 1985). This phenomenon was also described by other investigators and was interpreted as a result of the venodilating effects of CDD/ANP-(99-126) reducing the cardiac preload (Hirata et al., 1985). In the face of an increased infusion volume, such a mechanism is unlikely in this study, but there may be additional effects involved. The anaesthetic, halothane, is known to induce baroreceptor reflex attenuation via inhibition of sympathetic nerve activity in various animals. This might partially result from stereospecific activation of opioid receptors (Delle et al., 1989). In addition, Schultz et al. reported in 1988 that CDD/ANP-(99-126) evokes a decrease in sympathetic tone in rats. The authors demonstrated that this sympathetic inhibition was mediated by afferent vagal C-fibres modulated by CDD/ANP-(99-126) (Schultz et al., 1988). Neither CDD/ANP-(99-126) nor urodilatin caused a significant alteration in the heart rate in this study, possibly due to reflex inhibition of sympathetic tone. The significant protection 11.4 and 22.8 pmol/kg/min urodilatin but not equimolar CDD/ANP-(99-126) might indicate a stronger relaxing effect of the former
T. Fli~geet al. ~European Journal of Pharmacology 271 (1994) 395-402
peptide (Duft et al., 1993) compensating the acetylcholine-induced bronchoconstriction and the reduction in sympathetic tone. The highest doses of CDD/ANP(99-126) or urodilatin, 45.6 pmol/kg/min, did not show any protection against the acetylcholine challenge, which may be explained by bronchoconstrictor mechanisms exceeding the direct effects of both peptides on bronchial smooth muscle (Chou et al., 1986; Hamel and Ford-Hutchinson, 1986; Duft et al., 1993). This phenomenon should be due to a more pronounced vagal-mediated decrease in sympathetic tone related to the higher dose of the natriuretic peptides as the inspiratory halothane concentration and the acetylcholine dose did not differ between the treatment groups (Schultz et al., 1988). The effects of urodilatin or CDD/ANP-(99-126) on the pulmonary vasculature must also be considered (Kentsch et al., 1992). If the pulmonary artery pressure and thus the blood flow in the animals receiving the peptide infusion differed from those of the control group, the alterations in ventilation-perfusion relationship would have resulted in changes of the alveolar tension of oxygen and carbon dioxide. In turn, this would have affected bronchomotor tone. Under these circumstances, the stronger pulmonary vascular and bronchodilating properties of urodilatin (Hummel et al., 1992; Kentsch et al., 1992; Duft et al., 1993) might initiate overall effects significantly different from those of CDD/ANP-(99-126). To our knowledge, this is the first study comparing the protective effects of CDD/ANP-(99-126) and urodilatin on the bronchial system in vivo. Further investigations should elucidate whether the significantly higher effects of urodilatin shown in rats have relevance in the clinical situation of obstructive lung diseases in humans.
Acknowledgements We thank Mich61a M~ilzer and Edith Kaczmarek for administration of the substances and performing the calibrations and pulmonary function measurements. This study was conducted in the Lung Function Laboratory II of the Fraunhofer Institute of Toxicology and Aerosol Research, Hannover, Germany.
References Abassi, Z.A., J.R. Powell, E. Golomb and H.R. Keiser, 1992a, Renal and systemic effects of urodilatin in rats with high-output heart failure, Am. J. Physiol. 262, F615. Abassi, Z.A., J. Tate, S. Hunsberger, H. Klein, D. Trachewsky and H.R. Keiser, 1992b, Pharmacokinetics of ANF and urodilatin
401
during cANF receptor blockade and neutral endopeptidase inhibition, Am. J. Physiol. 263, E870. Amdur, M.O. and J. Mead, 1958, Mechanics of respiration in unanaesthetized guinea pigs, Am. J. Physiol. 192(2), 364. Banerjee, M.R. and J.H. Newman, 1990, Acute effects of atrial natriuretic peptide on lung mechanics and hemodynamics in awake sheep, J. Appl. Physiol. 69, 728. Bestle, M.H. and P. Bie, 1993, Renal effects of urodilatin and atrial natriuretic peptide in volume expanded conscious dogs, Acta Physiol. Scand. 149, 77. Candenas, M.L., E. Naline, L. Puybasset, P. Devillier and C. Advenier, 1991, Effect of atrial natriuretic peptide and of atriopeptins on the human isolated bronchus. Comparison with the reactivity of the guinea pig isolated trachea, Pulm. Pharmacol. 4, 120. Chanez, P., C. Mann, J. Bousquet, P.E. Chabrier, P. Godard, P. Braquet and F.B. Michel, 1990, Atrial natriuretic factor (ANF) is a potent bronchodilator in asthma, J. Allergy Clin. Immunol. 86 (3 PT 1), 321. Chou, J., E. Kubota, T. Sata and S.I. Said, 1986, Comparative relaxant activities of atrial natriuretic peptides (ANPs) and vasoactive intestinal peptide (VIP) on smooth muscle structures in lung, Fed. Proc. 45, 553 (Abstract). Delle, M., S.E. Ricksten and P. Thoren, 1989, The opiate antagonist naloxone counteracts the inhibition of sympathetic nerve activity caused by halothane anesthesia in rats, Anesthesiology 70, 309. De Vries, P.J., C.M. Tyssen, H.A. Struyker-Boudier and J.F. Smits, 1990, Atrial natriuretic factor increases albumin extravasation in conscious rats, Pfliig. Arch. 415, 507. Diamond, L. and M. O'Donnell, 1977, Pulmonary mechanics in normal rats, J. Appl. Physiol. 43, 942. Drummer, C., F. Fiedler, A. Bub, D. Kleefeld, E. Dimitriades, R. Gerzer and W.G. Forssmann, 1993, Development and application of a urodilatin (CDD/ANP-95-126)-specific radioimmunoassay, Pfliig. Arch. 423, 372. Duft, S., J.H. Wilkens, P. Marxen, M. Deeb, M. Kuhn, S. Piepenbrock, W.G. Forssmann, C. Fr61ich and J. Lichey, 1993, Urodilatin and atrial natriuretic factor relax isolated perfused guinea pig trachea and increase cyclic GMP, Am. Rev. Respir. Dis. 147(4), A847 (Abstract). Englebach, I.M., R.W. Lappe and J.M. Hand, 1988, Bronchoprotective and bronchodilator activity of Anaritide (human atrial natriuretic factor [102-126]) infusion in the anaesthetized guinea pig, Pulm. Pharmacol. 1, 119. Fernandes, L.B., J.M. Preuss and R.G. Goldie, 1992, Epithelial modulation of the relaxant activity of atriopeptides in rat and guinea-pig tracheal smooth muscle, Eur. J. Pharmacol. 212, 187. Forssmann, W.G., 1994, Urodilatin: from cardiac hormones to clinical trials, Exp. Nephrol. (in press). Gagelmann, M., D. Hock and W.G. Forssmann, 1988, Urodilatin (CDD/ANP-95-126) is not biologically inactivated by a peptidase from dog kidney cortex membranes in contrast to atrial natriuretic peptide/cardiodilatin (alpha-hANP/CDD-99-126), FEBS Lett. 233, 249. Goetz, K.L., 1991, Renal natriuretic peptide (urodilatin?) and atriopeptin: evolving concepts, Am. J. Physiol. 261, F921. Hamel, R. and A.W. Ford-Hutchinson, 1986, Relaxant profile of synthetic atrial natriuretic factor on guinea pig pulmonary tissues, Eur. J. Pharmacol. 121, 151. Heim, J.M., S. Kiefersauer, H.-J. Fiille and R. Gerzer, 1989, Urodilatin and beta-ANF. Binding properties and activation of particulate guanylate cyclase, Biochem. Biophys. Res. Commun. 163, 37. Heinrich, U. and A. Wilhelm, 1984, Lung function tests on hamsters and rats using the whole body plethysmograph, bga Schriften 5, 255. Heinrich, U., H. Muhle and H.G. Hoymann, 1989, Pulmonary function changes in rats after chronic and subchronic inhalation exposure to various particulate matter, Exp. Pathol. 37, 248.
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Hildebrandt, D.A., H.L. Mizelle, M.W. Brands and J.E. Hall, 1992, Comparison of renal actions of urodilatin and atrial natriuretic peptide, Am. J. Physiol. 262, R395. Hirata, Y., M. Ishii, T. Sugimoto, H. Matsuoka, K. Kangawa and H. Matsuo, 1985, The effects of human atrial 28-amino acid peptide on systemic and renal hemodynamics in anesthetized rats, Circ. Res. 57, 634. Hirshman, C.A., G. Edelstein, S. Peetz, R. Wayne and H. Downes, 1982, Mechanism of action of inhalational anesthesia on airways, Anesthesiology 56, 107. Hohlfeld, J., H.G. Hoymann, W. Koch, R. Klingebiel and U. Heinrich, 1992a, Standardization of aerosol delivery with a new aerosol delivery with a new aerosol generating device for use in animal physiology, in: Drittes Grafschafter Kolloquium: Aerosole und Lunge (SchmaUenberg-Grafschaft). Hohlfeld, J., R. White, W. Koch and U. Heinrich, 1992b, A new aerosol generating device for use in animal physiology, Eur. Respir. J. 5 (Suppl.15), 531S. Hoymann, H.G., U. Heinrich, R. Beume and U. Kilian, 1994, Comparative investigation of the effects of zardaverine and theophylline on pulmonary function in rats, Exp. Lung Res. 20(3), 235. Hulks, G., A. Jardine, J.M.C. Connell and N.C. Thomson, 1989, Bronchodilator effect of atrial natriuretic peptide in asthma, Br. Med. J. 299(6707), 1081. Hulks, G., K.G. Crabb, J.C. McGrath and N.C. Thomson, 1991, In vitro effects of atrial natriuretic factor and sodium nitroprusside on bronchomotor tone in human bronchial smooth muscle, Am. Rev. Respir. Dis. 143, A344 (Abstract). Hummel, M., M. Kuhn, A. Bub, H. Bittner, D. Kleefeld, P. Marxen, B. Schneider, R. Hetzer and W.G. Forssmann, 1992, Urodilatin: a new peptide with beneficial effects in the postoperative therapy of cardiac transplant recipients, Clin. Invest. 70, 674. Ishii, K. and F. Murad, 1989, ANP relaxes bovine tracheal smooth muscle and increases c-GMP, Am. J. Physiol. 256 (Cell. Physiol. 25), C495. Kentsch, M., D. Ludwig, B. Hentschel, R. Gerzer and G. MiillerEsch, 1990, Effects of urodilatin (95-126 ANF) in healthy volunteers, Eur. J. Clin. Invest. 20 (Suppl.), 13 (Abstract). Kentsch, M., D. Ludwig, C. Drummer, R: Gerzer and G. Muller-Esch, 1992, Haemodynamic and renal effects of urodilatin in healthy volunteers, Eur. J. Clin. Invest. 22, 319. Labat, C., X. Norel, J. Benveniste and C. Brink, 1988, Vasorelaxant effects of atrial peptide II on isolated human pulmonary muscle preparations, Eur. J. Pharmacol. 150, 397. Likens, S.A. and J.L. Mauderly, 1982, Effect of elastase or histamine on single-breath N 2 washouts in the rat, J. Appl. Physiol. (Respir. Environ. Exercise Physiol.) 52(1), 141. Mauderly, J.L., 1989, Effect of inhaled toxicants on pulmonary function, in: Concepts in Inhalation Toxicology, eds. R.O. McClellan and R.F. Henderson (Hemispere Publishing Corporation, New York) p. 347. Menard, O., 1991, Atrial natriuretic factor and the lung, Rev. Mal. Respir. 8, 153. O'Donnell, M., R. Garippa and A.F. Welton, 1985, Relaxant effect
of atriopeptins in isolated guinea pig airway and vascular smooth muscle, Peptides 6, 597. Pauluhn, J., L. Machemer and G. Kimmerle, 1987, Effects of inhaled cholesterinesterase inhibitors on bronchial tonus and on plasma and erythrocyte acetylcholine esterase activity in rats, Toxicology 46, 177. Perrella, M.A., K.B. Margulies, C.M. Wei, L.L. Aarhus, D.M. Heublein and J.C.J. Burnett, 1991, Pulmonary and urinary clearance of atrial natriuretic factor in acute congestive heart failure in dogs, J. Clin. Invest. 87, 1649. Potvin, W. and D.R. Varma, 1989, Bronchodilator activity of atrial natriuretic peptide in guinea pigs, Can. J. Physiol. Pharmacol. 67(10), 1213. Rambotti, M.G. and A. Spreca, 1991, Ultrastructural demonstration of guanylate cyclase in rat lung after activation by ANF, Cell Mol. Biol. 37, 455. Saxenhofer, H., A. RaseUi, P. Weidmann, W.G. Forssmann, A. Bub, P. Ferrari and S.G. Shaw, 1990, Urodilatin, a natriuretic factor from kidneys, can modify renal and cardiovascular function in men, Am. J. Physiol. 259, F832. Schultz, H.D., D.G. Gardner, C.F. Deschapper, H.M. Coleridge and J.C.G. Coleridge, 1988, Vagal C-fiber blockage abolishes sympathetic inhibition by atrial natriuretic factor, Am. J. Physiol. 255, R6. Schuiz-Knappe, P., K. Forssmann, F. Herbst, D. Hock, R. Pipkorn and W.G. Forssmann, 1988, Isolation and structural analysis of 'urodilatin', a new peptide of the cardiodilatin-(ANP)-family, extracted from human urine, Kiln. Wochenschr. 66, 752. Tepper, J.S., M.J. Wiester, D.L. Costa, W.P. Watkinson and M.F. Weber, 1987, Cardiopulmonary effects in awake rats four and six months after exposure to methyl isocyanate, Environ. Health Perspect. 72, 95. Valentin, J.P., L.A. Sechi, C. Qiu, M. Schambelan and M.H. Humphreys, 1991, Urodilatin displaces labeled atrial natriuretic peptide from renal binding sites and is equally potent in generating cyclic GMP, Hypertension 18, 3939 (Abstract). Villarreal, D., R.H. Freeman and R.A. Johnson, 1991, Renal effects of ANF (95-126), a new atrial peptide analogue, in dogs with experimental heart failure, Am. J. Hypertens. 4, 508. Warner, D.O., J. Vettermann, J.F. Brichant and K. Rehder, 1990, Direct and neurally mediated effects of halothane on pulmonary resistance in vivo, Anesthesiology 72, 1057. Watanabe, H., H. Furui, K. Yamaki, R. Suzuki, K. Takagi and T. Satake, 1988, Atrial natriuretic polypeptide causes dose-dependent relaxant effect of guinea pig tracheal smooth muscle, Am. Rev. Respir. Dis. 137, 102 (Abstract). Yandle, T.G., A.M. Richards, M.G. Nicholls, R.C. Cuneo, E.A. Espiner and J.H. Livesey, 1986, Metabolic clearance rate and plasma half life of alpha-human atrial natriuretic peptide in man, Life Sci. 38, 1827. Zimmermann, R.S., N.C. Trippodo, A.A. MacPhee, A.J. Mattinez and R.W. Barbee, 1990, High-dose atrial natriuretic factor enhances albumin escape from systemic but not the pulmonary circulation, Circ. Res. 67, 461.
European Journal of Pharmacology 271 (1994) 395-402
Type A natriuretic peptides exhibit different bronchoprotective effects in rats Thomas Fliige
a,b,c,*,
Heinz-Gerd Hoymann b, Jens Hohlfeld a, Uwe Heinrich b, Helmut Fabel Thomas O.F. Wagner a, Wolf-Georg Forssmann c
a,
a Department of Respiratory Medicine, Hannover Medical School, Hannover, Germany b Fraunhofer Institute of Toxicology and Aerosol Research, Hannover, Germany c Lower Saxony Institute for Peptide Research, Hannover, Germany Received 12 April 1994; revised MS received 8 September 1994; accepted 27 September 1994
Abstract
The protective effect of 11.4, 22.8 or 45.6 pmol/kg/min cardiodilatin/atrial natriuretic peptide (CDD/ANP-(99-126)), urodilatin (CDD/ANP-(95-126)) or vehicle intravenously against acetylcholine-induced bronchoconstriction was compared in spontaneously breathing, halothane-anesthetized Wistar rats. The inhalation of acetylcholine induced significant alterations of the spontaneous breathing parameters evaluated by whole-body plethysmography without significant differences between the treatment groups. Forced parameters detect airflow changes with a greater sensitivity and were measured in hyperventilation-induced temporary apnoea after the challenge. The forced expiratory volume in 0.1 s revealed a significant protective effect of 11.4 pmol/kg/min urodilatin compared to the controls whereas the parameters of the forced expiratory flow-volume curve were significantly preserved by 11.4 and 22.8 pmol/kg/min urodilatin (P < 0.05). Urodilatin showed protective effects against an acetylcholine challenge whereas CDD/ANP-(99-126) was without significant influence.
Keywords: Urodilatin; Natriuretic peptide, type A; Bronchoprotection; Acetylcholine; Lung function, rat
1. Introduction
Cardiodilatin/atrial natriuretic peptide ( C D D / ANP-(99-126)) is not only a diuretic and natriuretic peptide but also a specific vasodilator of renal, coronary and pulmonary vessels (Menard, 1991; Forssmann, 1994). Moreover it relaxes the bronchial smooth muscle in various animals (O'Donnell et al., 1985; Chou et al., 1986; Hamel and Ford-Hutchinson, 1986; Englebach et al., 1988; Watanabe et al., 1988; Ishii and Murad, 1989). The bronchodilating effect of C D D / ANP-(99-126) appears to be more prominent in the larger central airways than in the peripheral airways (Chou et al., 1986; Hamel and Ford-Hutchinson, 1986; Banerjee and Newman, 1990). In vitro studies with human airways revealed contradictory results (Labat et
* Corresponding author. Lower Saxony Institute for Peptide Research GmbH, Feodor-Lynen-Strasse 31, D-30625 Hannover, Germany. Tel. 49-511-855525, fax 49-511-855525. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00595-8
al., 1988; Candenas et al., 1991; Hulks et al., 1991; Fernandes et al., 1992). An increase of the forced expiratory volume in 1.0 s (FEVt. 0) was demonstrated in vivo during, and for a short period after the end of, an intravenous infusion in asthmatic subjects by different investigators (Hulks et al., 1989; Chanez et al., 1990). In addition, this peptide has been shown to inhibit the effects of methacholine, histamine, serotonin, leukotriene D4, and arachidonic acid on the guinea pig trachea in vitro (Hamel and Ford-Hutchinson, 1986; Potvin and Varma, 1989) and on the pulmonary inflation pressure in vivo (Potvin and Varma, 1989). The natriuretic peptide, urodilatin, was discovered by Schulz-Knappe et al. in 1988 (Schulz-Knappe et al., 1988). It consists of the entire molecule of human CDD/ANP-(99-126) plus a four-residue NH2-terminal extension: threonine-alanine-proline-arginine. Urodilatin, CCD/ANP-(95-126), is probably produced from the same gene code as type A natriuretic peptides in the heart but with different posttranslational pro-
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cessing to yield the 32-residue peptide (Schulz-Knappe et al., 1988). The first discovery of urodilatin in human urine, together with data from renal cell cultures, immunocytochemistry, radioimmunoassays, and other techniques, led to the conclusion that the distal tubules in the kidney synthesize a 126-amino acid prohormone identical to the one of CDD/ANP-(99-126) in cardiac atria (Goetz, 1991; Forssmann, 1994). Urodilatin shows an affinity for the same binding sites as CDD/ANP(99-126), and like CDD/ANP-(99-126) it activates guanylate cyclase in membranes from various tissues (Watanabe et al., 1988; Heim et al., 1989; Valentin et al., 1991). In vitro, urodilatin evokes a relaxation of vascular and bronchial smooth muscle (Schulz-Knappe et al., 1988; Duft et al., 1993). In vivo, the natriuretic and diuretic potency of urodilatin significantly exceeds that of CDD/ANP-(99-126) (Goetz, 1991; Villarreal et al., 1991; Abassi et al., 1992a; Hildebrandt et al., 1992; Bestle and Bie, 1993), but the effects on the systemic blood pressure are less pronounced (Saxenhofer et al., 1990). No immunoreactivity of urodilatin is found in human plasma (Goetz, 1991; Kentsch et al., 1992; Drummer et al., 1993; Forssmann, 1994). The aim of the present study was to investigate the protective effect of equimolar doses of intravenous CDD/ANP-(99-126), urodilatin or vehicle against bronchoconstriction induced by acetylcholine inhalation in anaesthetized Wistar rats.
2. Material and methods
2.1. Study design Forty-six 13-week-old female Wistar rats (Han: WlST, Zentralinstitut fiir Versuchstierzucht, Hannover, Germany) weighing 221.1 + 14.0 g (mean+ standard deviation) were studied. The internationally accepted principles in the care and use of experimental animals were strictly followed. The rats were anaesthetized with halothane/30% 0 2 and intubated orally with a tracheal cannula. An indwelling venous catheter in the tail vein was used for the intravenous infusion of 0.4 ml CDD/ANP-(99-126), urodilatin or vehicle (0.9% sodium chloride) at a constant rate for 12 min in a randomized order. CDD/ANP-(99-126) and urodilatin were dissolved in 0.9% sodium chloride and were given in doses of 11.4, 22.8 or 45.6 p m o l / k g / m i n (equivalent to 35.2, 70.3 or 140.5 n g / k g / m i n C D D / ANP-(99-126) and 40.0, 80.0 or 160.0 n g / k g / m i n urodilatin) using only one dosage per treatment group (n = 6). During the infusion period an acetylcholine challenge test was started after 5 min. Spontaneous pulmonary function parameters were evaluated under steady-state conditions prior to the infusion (baseline) and before and at the end of the acetylcholine inhala-
tion (pre/post-acetylcholine challenge). Forced parameters including volume-time and flow-volume relation were measured in hyperventilation-induced temporary apnoea immediately after the acetylcholine challenge. Heart rate was controlled by electrocardiography during the study. Vehicle-treated animals were used as controls on each study day. 2.2. Pulmonary function tests The techniques of quasi non-invasive mechanical lung function measurements (Heinrich and Wilhelm, 1984; Heinrich et al., 1989; Hoymann et al., 1994) used in this study are based on the methods reported by Likens and Mauderly (Likens and Mauderly, 1982; Tepper et al., 1987; Mauderly, 1989). The intubated animal was placed supine in a whole-body plethysmograph. The halothane concentration was adjusted individually between 1.5 and 3.0% (in 30% 0 2, gas flow 2.2 l/min) to get similar respiratory frequencies in all animals. This type of anaesthesia enables the performance of involuntary breathing manoeuvres and permits the measurement of lung compliance and resistance.
2.2.1. Spontaneous respiratory function parameters The parameters of spontaneous respiration including tidal volume (VT), dynamic lung compliance (Cdyn) and lung resistance (R L) were recorded continuously (analyzer model 6, Buxco, Sharon, USA) and evaluated at baseline, pre- and post-acetylcholine challenge. Respiratory volume changes were determined using a pneumotachograph (pressure transducer MP45, Validyne, Northridge, USA) at the rear wall of the chamber. Transpulmonary pressure changes (PAP) were analyzed by subtracting the oesophageal pressure, measured via a water-filled silicone tube in the position of maximal signal (tube diameter 1.6 mm; pressure transducer P23Db, Statham, Hato Ray, Puerto Rico), from the airway pressure determined in a side branch of the breathing port (air filled W101/RFT pressure transducer, MefAger~itewerkZw6nitz, Germany). Pap is necessary for calculating Cdyn and R L using the method of Amdur and Mead (Amdur and Mead, 1958).
2.2.2. Inhalation of acetylcholine The challenge test was performed using the concentration-dependent potency of an acetylcholine chloride aerosol to induce bronchospasm. The increase in lung resistance is a useful parameter to determine the degree of airway obstruction in the anaesthetized rat (Heinrich and Wilhelm, 1984; Pauluhn et al., 1987; Hohlfeld et al., 1992a; Hoymann et al., 1994). After 5 min of intravenous infusion a precisely defined acetylcholine aerosol (15 m g / m 3, mass median aerodynamic diameter (MMAD) 1.9 _+ 1.5/zm, dried) was added to the halothane and oxygen for 7 min. The aerosol was
ml ±S.D. ml/cm H20 _+S.D. cm H 2 0 / m l / s _+S.D.
VT e
% pre-Ach d +S.D. % pre-Ach d +S.D. % pre-Ach d +S.D.
- 1 9 . 3 0 ++ 9.26 - 3 6 . 4 4 ++ 12.12 +61.84 ++ 45.00
- 1 1 . 8 0 ++ 6.01 - 3 2 . 6 5 ++ 11.37 +38.76 +4 18.73
- 2.04 2.26 - 6.32 ++ 3.39 + 5.14 ÷ + 2.90
0.15 0.135 0.011 0.289 0.030
0.23 0.172 0.047 0.272 0.046
- 1.07 2.90 - 4.54 q-+ 3.50 + 1.73 7.39
1.25
6
U R O 11.4 b
1.35
10
Controls ~
- 1 1 . 7 0 ++ 3.40 - 4 0 . 5 6 ++ 9.64 +65.46 ++ 30.39
- 0.79 5.16 - 3.53 5.47 + 1.97 6.44
0.10 0.159 0.018 0.290 0.037
1.39
6
U R O 22.8 ~
- 1 7 . 0 6 ++ 9.08 -37.69 + 17.62 +67.43 ++ 38.84
- 2.01 4.12 - 10.34 + 7.86 + 6.06 ÷ 5.77
0.21 0A86 0.047 0.281 0.081
1.46
6
U R O 45.6 b
- 1 9 . 7 3 ++ 7.69 - 4 3 . 6 0 4+ 11.24 +45.47 ++ 16.07
+ 3.98 ÷ 3.67 - 4.24 7.23 + 3.78 8.36
0.05 0.156 0.043 0.284 0.055
1.25
6
A N P 11.4 c
- 1 6 . 9 7 ++ 7.88 - 4 6 . 5 2 4+ 15.12 +72.33 ++ 41.32
- 1.27 2.41 + 4.61 24.55 - 1.77 11.47
0.08 0.184 0.037 0.308 0.095
1.41
6
A N P 22.8 c
- 1 7 . 1 1 ++ 9.27 - 4 6 . 4 6 ++ 8.79 +58.49 ++ 29.09
+ 1.27 2.64 + 2.25 8.63 -4.08 7.92
0.08 0.175 0.024 0.271 0.051
1.30
6
A N P 45.6 c
(paired t-test).
R at spontaneous pulmonary function par am et er s were evaluated unde r steady-state conditions (baseline) prior to the intravenous infusion of sodium chloride 0.9%, urodilatin or c a r d i o d i l a t i n / a t r i a l natriuretic peptide (99-126). During the infusion period an acetylcholine challenge test was started after 5 rain. All p a r a m e t e r s were measu r ed again before and at the en d of the acetylcholine inhalation, a Infusion of sodium chloride 0.9%. b T r e a t m e n t with urodilatin ( U R O ) 11.4, 22.8 or 45.6 p m o l / k g / m i m c T r e a t m e n t with c a r d i o d i l a t i n / a t r i a l natriuretic p e p t i d e (99-126) (ANP) 11.4, 22.8 or 45.6 p m o l / k g / m i n , d B e f o r e / a f t e r acetylcholine challenge (Ach). e Tidal volume, f Dynamic compliance, g Lung resistance. + P < 0.05 (paired t-test). + + P < 0.01
RL g
Cdy n f
Vx ~
Post-Ach d
RLg
Cdyn f
Pre-Ach d VT e
RLg
tr~ baseline +S.D. % baseline +S.D. % baseline +S.D.
n
Baseline
Cdy n f
Units
Parameter
Table 1 Spontaneous pulmonary function parameters at baseline, before and after acetylcholine challenge
ta.a
!
t~
g~
x..
T. Fliige et al. /European Journal of Pharmacology 271 (1994) 395-402
398
generated by a recently developed device including a computer-controlled system to select and continuously monitor the mass concentration and flow (Bronchy-H, Fraunhofer Institute of Toxicology and Aerosol Research, Hannover, Germany (Hohlfeld et al., 1992b)). In addition, the acetylcholine dose inhaled was calculated by the computer from the volume of minute ventilation and the concentration therein. The spontaneous pulmonary function values were evaluated at the time individual animals reached a total inhaled volume of 600 ml aerosol, corresponding to 9/~g acetylcholine (post-acetylcholine challenge).
2.2.3. Forced lung function parameters The forced lung function parameters were measured 1 min after the end of the acetylcholine inhalation in hyperventilation-induced temporary apnoea (Hoymann et al., 1994). Air reservoirs at a pressure of + 40 and - 5 0 cm H 2 0 were used for maximal inspiration (maximal PTP + 30 cm H20, flow 10 ml/s) and expiration to record forced expiratory volume-time relation and flow-volume curve. The evaluation included forced vital capacity (FVC (ml)) and forced expiratory volume in 0.1 s in relation to FVC (FEV0.1% (%FVC)) for the volume-time curve, and peak expiratory flow (PEF), maximum mid-expiratory flow (MMEF) and flow at 75, 50 and 25% FVC for the flow-volume curve (FEF75, FEFs0, FEF25, all flow values: ml/s). Forced parameters are aimed at detecting airflow obstruction with a greater sensitivity than measurements taken during tidal breathing (Mauderly, 1989) and were first incorporated into lung function tests with rats in 1977 (Diamond and O'Donnell, 1977). 2.3. Drugs Lyophilized, highly purified (> 99%) urodilatin acetate produced by the liquid phase peptide synthesis
method (32 amino acids, molecular weight 3506, batch number: A 08405) was purchased from Novabiochem, L~iufelfingen, Switzerland, and human CDD/ANP(99-126) (28 amino acids, molecular weight 3081, peptide purity 98.5%, batch number: 147D) from Saxon Biochemicals, Hannover, Germany. Both peptides were dissolved in physiological saline solution under sterile conditions immediately before infusion.
2.4. Statistics The data are presented as means and standard deviations ( + S.D.). The spontaneous pulmonary function parameters are reported as original values for the baseline. Changes in pre- and post-acetylcholine challenge were calculated as percent baseline and percent pre-acetylcholine challenge, respectively. The data for the forced expiratory manoeuvres are presented as originally measured. Analysis of variance (ANOVA) in combination with the Dunnett test were used to assess the significance of a difference between group mean values and the paired t-test for the determination of changes in a certain group pre- and post-acetylcholine challenge compared to baseline and pre-acetylcholine challenge, respectively. A value of P < 0.05 was considered significant.
3. Results
3.1. Baseline data Baseline measurements of the spontaneous pulmonary function parameters are shown in Table 1. VT, Cd~ and R L were in the normal range established in the laboratory and did not differ significantly between treatment groups. The respiratory frequency and the
Table 2 Heart rate at baseline, before and after acetylcholine challenge Parameter
Units
Controls a
U R O 11.4 b
U R O 22.8 b
U R O 45.6 b
ANP 11.4 c
ANP 22.8 c
ANP 45.6 c
Baseline
n
Heart rate
min - 1 + S.D.
10 427.00 30.46
6 439.26 29.84
6 424.00 6.78
6 445.76 60.10
6 447.68 20.22
6 420.06 10.91
6 394.96 24.07
% baseline + S.D.
+ 0.15 1.57
+ 0.04 1.00
- 2.29 0.46
+ 0.54 0.38
+ 0.90 3.04
+ 0.55 0.95
+ 0.04 3.42
% pre-Ach d + S.D.
-1.04 4.32
-1.13 2.25
-0.20 1.43
+0.27 2.21
+0.41 1.85
+0.53 3.42
-0.63 6.63
Pre-Ach d Heart rate
Post-Ach d Heart rate
The heart rate was evaluated under steady-state conditions (baseline) prior to the intravenous infusion of sodium chloride 0.9%, urodilatin or cardiodilatin/atrial natriuretic peptide (99-126). During the infusion period the course was monitored continuously by electrocardiography while an acetylcholine challenge test was started after 5 min. The heart rate was evaluated again before and at the end of the acetylcholine inhalation. a Infusion of sodium chloride 0.9%. b Treatment with urodilatin (URO) 11.4, 22.8 or 45.6 p m o l / k g / m i n , c Treatment with cardiodilatin/atrial natriuretic peptide (99-126) (ANP) 11.4, 22.8 or 45.6 p m o l / k g / m i n . ~ B e f o r e / a f t e r acetylcholine challenge (Ach).
T. Fliigeet al. / European Journal of Pharmacology 271 (1994) 395-402
399
Table 3 Parameters of the forced expiratory manoeuvres Parameter FEV0.1% d PEF e MMEF f FEF75 g FEFs0 g FEF25 g
Units
Controls a
n
10
% FVC _+S.D. ml/s + S.D. ml/s _+S.D. ml/s __S.D. ml/s ±S.D. ml/s _+S.D.
68.3 7.1 98.4 5.8 56.6 11.3 94.9 9.4 63.2 10.1 27.7 9.3
URO 11.4 b 6
77.6 4.2 113.6 9.2 77.3 11.4 112.2 8.6 83.2 11.2 42.4 10.2
* ** ** ** ** *
URO 22.8 b
URO 45.6 b
ANP 11.4 c
ANP 22.8 c
ANP 45.6 c
6
6
6
6
6
74.3 3.7 114.8 6.9 74.9 10.5 113.8 7.0 82.0 11.6 38.3 8.2
68.2 8.0 106.1 12.7 56.1 15.5 103.7 14.5 64.9 17.0 27.7 10.8
70.1 2.2 95.0 23.2 57.5 16.6 91.1 23.0 62.9 17.4 32.3 10.5
66.7 6.3 112.1 12.6 62.6 14.9 109.5 13.3 67.5 16.4 31.4 10.4
65.0 3.4 97.4 15.1 51.3 9.2 91.3 18.4 55.7 5.7 27.9 7.1
** ** ** ** *
During the intravenous infusion of sodium chloride 0.9%, urodilatin or cardiodilatin/atrial natriuretic peptide (99-126) an acetylcholine challenge test was started after 5 min. Forced parameters including volume-time and flow-volumerelation were measured in hyperventilation-induced temporary apnoea immediately after the end of the acetylcholine inhalation, a Infusion of sodium chloride 0.9%. b Treatment with urodilatin (URO) 11.4, 22.8 or 45.6 pmol/kg/min, c Treatment with cardiodilatin/atrial natriuretic peptide (99-126) (ANP) 11.4, 22.8 or 45.6 pmol/kg/min, d (forced expiratory volume in 0.1 s/forced vital capacity) × 100. e Peak expiratory flow. f Maximal mid-expiratory flow. g Forced expiratory flow at 75, 50 or 25% of forced vital capacity. * P < 0.05 (ANOVA + Dunnett test). * * P < 0.01 (ANOVA + Dunnett test).
inspiratory h a l o t h a n e c o n c e n t r a t i o n did n o t vary significantly (data n o t shown). H e a r t rate c o n t r o l l e d by e l e c t r o c a r d i o g r a p h y is d e m o n s t r a t e d in T a b l e 2. N o significant differences b e t w e e n the groups were f o u n d at baseline.
3.2. Infusion of urodilatin, CDD /ANP-(99-126) or vehicle preceding the acetylcholine challenge T h e i n f u s i o n of isotonic saline solution in the control a n i m a l s a n d of 11.4, 22.8 or 45.6 p m o l / k g / m i n u r o d i l a t i n or C D D / A N P - ( 9 9 - 1 2 6 ) resulted in n o significant differences b e t w e e n the t r e a t m e n t groups for the s p o n t a n e o u s respiratory f u n c t i o n p a r a m e t e r s after 5 m i n ( A N O V A + D u n n e t t test; T a b l e 1). C o m p a r i s o n with the individual b a s e l i n e values of each a n i m a l revealed a significant decrease of Cdyn in controls ( P < 0.01) a n d d u r i n g i n f u s i o n of 11.4 ( P < 0.01) a n d 45.6 p m o l / k g / m i n u r o d i l a t i n ( P < 0.05). I n addition, a significant increase of R L was d o c u m e n t e d after 5 m i n of 11.4 ( P < 0.01) or 45.6 p m o l / k g / m i n u r o d i l a t i n ( P < 0.05). Previous results showed n o significant b r o n c h o d i l a t i n g effect of the p e p t i d e s with respect to the p a r a m e t e r s of the forced expiratory m a n o e u v r e s at this time (data n o t shown). T h e h e a r t rate did n o t c h a n g e significantly in any g r o u p after a 5 - m i n i n f u s i o n ( T a b l e 2).
3.3. Effects of urodilatin, CDD /ANP-(99-126) or vehicle on the acetylcholine challenge T h e acetylcholine aerosol caused a b r o n c h o c o n s t r i c tion with a significant decrease in VT a n d Cdyn a n d a significant increase in R L in all groups ( P < 0.01). T h e r e were n o significant differences b e t w e e n the m e a n
values. C o m p a r i s o n of the groups t r e a t e d with 11.4 a n d 22.8 p m o l / k g / m i n u r o d i l a t i n or C D D / A N P - ( 9 9 - 1 2 6 ) evidenced smaller c h a n g e s in the a n i m a l s which received u r o d i l a t i n i n t r a v e n o u s l y ( T a b l e 1). As was expected, the respiratory f r e q u e n c y i n c r e a s e d significantly in all a n i m a l s d u r i n g the acetylcholine challenge ( + 5 6 . 1 + 8.1%, P < 0 . 0 1 ) with n o significant differences b e t w e e n the groups. T a b l e 3 s u m m a r i z e s the results of the forced expiratory m a n o e u v r e s . T h e FEW0.1% was significantly elevated in the a n i m a l s t r e a t e d with 11.4 p m o l / k g / m i n u r o d i l a t i n c o m p a r e d to the o t h e r groups ( P < 0.05). T h e flow-volume curve showed significantly higher values of P E F ( P < 0.01), M M E F ( P < 0.01), FEF75 ( P < 0.01), FEFs0 ( P < 0.01), a n d FEFz5 ( P < 0.05) in the rats with 11.4 a n d 22.8 p m o l / k g / m i n u r o d i l a t i n comp a r e d to those with e q u i m o l a r doses of C D D / A N P ( 9 9 - 1 2 6 ) a n d the controls. T h e F V C did n o t c h a n g e significantly in any t r e a t m e n t group. T h e r e were n o significant differences of these p a r a m e t e r s b e t w e e n the highest doses of u r o d i l a t i n a n d C D D / A N P - ( 9 9 - 1 2 6 ) or c o m p a r e d to the controls. T h e h e a r t rate r e m a i n e d u n c h a n g e d in all t r e a t m e n t groups (Table 2).
4. D i s c u s s i o n
A l t h o u g h n o significant differences of the s p o n t a n e o u s respiratory f u n c t i o n p a r a m e t e r s were f o u n d bet w e e n the t r e a t m e n t groups after 5 m i n of infusion, c o m p a r i s o n to the individual b a s e l i n e values showed a small significant decrease of Cdyn in the controls a n d d u r i n g 11.4 a n d 45.6 p m o l / k g / m i n urodilatin. This was c o m b i n e d with a small significant increase of R L in the two u r o d i l a t i n groups only.
400
T. Fliige et al. / European Journal of Pharmacology 271 (1994) 395-402
In 1990 De Vries et al. showed that the clearance of human serum albumin infused in isotonic saline solution in rats was highest in the ileum and lung. High-dose CDD/ANP-(99-126) had no effect on pulmonary clearance but resulted in a significant increase in the ileum (De Vries et al., 1990). Zimmerman et al. (1990) reported, however, that pathophysiological levels of CDD/ANP-(99-126) provoke protein mobilization from arteries to the alveolar space, whereas pharmacological concentrations re-equilibrate the transwall gradient. Since there were no significant differences between the groups regarding the effects of urodilatin or CDD/ANP-(99-126) compared to the controls, it seems unlikely that the two peptides showed a relevant action on the pulmonary microcirculation in this study. The small changes of Cdyn and R L (Table 1) reported here may be the result of an increase in pulmonary plasma extravasation provoked by a relative volume overload. The infusion rate of 0.04 ml/min for 12 min was selected after review of the data of Hirata et al. (1985). They showed not only a significant decrease in mean arterial pressure and total peripheral vascular resistance, but also a reduction in cardiac output and central venous pressure after 10 min of infusion of comparable CDD/ANP-(99-126) doses at a rate of 0.02 ml/min in Wistar rats (Hirata et al., 1985). The haemodynamic changes were interpreted as the result of a direct relaxation of arterial smooth muscle and a venodilating effect of the peptide, reducing the cardiac pre- and afterload. Similar cardiovascular problems should be reduced in this study, by administration of a larger saline volume. General anaesthetics modify airway responsiveness by several mechanisms, including direct effects on airway smooth muscle and reductions in neural reflex activity. In dogs, halothane has been shown to reduce the response to cholinergic stimuli through both of these mechanisms (Hirshman et al., 1982; Warner et al., 1990). The inhalation of 9 /zg acetylcholine, however, produced a significant bronchoconstriction indicated by appropriate changes in R L and Cdy~ in all animal groups in this study while the inspiratory halothane concentration did not vary significantly between them. The more sensitive volume-time relation and flowvolume curve of the forced expiratory manoeuvres showed a significant protection of 11.4 and 22.8 pmol/kg/min urodilatin in the central (FEV0.1%, PEF, FEF75) and peripheral (FEFs0, FEF25) airways against the acetylcholine challenge (Diamond and O'Donnell, 1977; Tepper et al., 1987; Mauderly, 1989). Most forced parameters demonstrated a peak of the weaker CDD/ANP-(99-126) action at a dose of 22.8 p m o l / k g / m i n without reaching significance (Table 2). This contrasts with findings of the bronchodilating
properties of CDD/ANP-(99-126) in vitro and in vivo (Chou et al., 1986; Hamel and Ford-Hutchinson, 1986). A bolus injection of CDD/ANP-(99-126) in conscious sheep caused a significant reduction in the rise in R L induced by carbachol. However, it did not reverse the decrease in Cdyn after administration of histamine or carbachol, which was interpreted as a more prominent action on the larger central airways (Banerjee and Newman, 1990). On the other hand, in vitro urodilatin relaxes guinea pig tracheal tubes precontracted with methacholine significantly more potently than CDD/ANP-(99-126) (Duft et al., 1993). At present urodilatin is believed to act via the same receptors as CDD/ANP-(99-126) and the two peptides are equally potent in generating cyclic GMP at these sites (Heim et al., 1989; Rambotti and Spreca, 1991; Valentin et al., 1991). Despite a comparable plasma half-life of infused CDD/ANP-(99-126) or urodilatin of approximately 3 min (Yandle et al., 1986; Kentsch et al., 1990), differences were shown in the enzymes involved in their degradation. There is evidence that neutral endopeptidase (EC 3.4.24.11) plays an important role in the pulmonary metabolism of CDD/ANP-(99-126) (Perrella et al., 1991), but urodilatin is not affected by this enzyme (Gagelmann et al., 1988; Abassi et al., 1992a,b). This may cause pulmonary effects of urodilatin that are stronger a n d / o r last longer than those of CDD/ANP-(99-126). The results of this study are in accordance with this view. Another interesting finding is the absence of a significant baroreceptor-mediated reflex tachycardia despite the intravenous infusion of CDD/ANP-(99-126) doses that were shown to cause a reduction in arterial pressure (Hirata et al., 1985). This phenomenon was also described by other investigators and was interpreted as a result of the venodilating effects of CDD/ANP-(99-126) reducing the cardiac preload (Hirata et al., 1985). In the face of an increased infusion volume, such a mechanism is unlikely in this study, but there may be additional effects involved. The anaesthetic, halothane, is known to induce baroreceptor reflex attenuation via inhibition of sympathetic nerve activity in various animals. This might partially result from stereospecific activation of opioid receptors (Delle et al., 1989). In addition, Schultz et al. reported in 1988 that CDD/ANP-(99-126) evokes a decrease in sympathetic tone in rats. The authors demonstrated that this sympathetic inhibition was mediated by afferent vagal C-fibres modulated by CDD/ANP-(99-126) (Schultz et al., 1988). Neither CDD/ANP-(99-126) nor urodilatin caused a significant alteration in the heart rate in this study, possibly due to reflex inhibition of sympathetic tone. The significant protection 11.4 and 22.8 pmol/kg/min urodilatin but not equimolar CDD/ANP-(99-126) might indicate a stronger relaxing effect of the former
T. Fli~geet al. ~European Journal of Pharmacology 271 (1994) 395-402
peptide (Duft et al., 1993) compensating the acetylcholine-induced bronchoconstriction and the reduction in sympathetic tone. The highest doses of CDD/ANP(99-126) or urodilatin, 45.6 pmol/kg/min, did not show any protection against the acetylcholine challenge, which may be explained by bronchoconstrictor mechanisms exceeding the direct effects of both peptides on bronchial smooth muscle (Chou et al., 1986; Hamel and Ford-Hutchinson, 1986; Duft et al., 1993). This phenomenon should be due to a more pronounced vagal-mediated decrease in sympathetic tone related to the higher dose of the natriuretic peptides as the inspiratory halothane concentration and the acetylcholine dose did not differ between the treatment groups (Schultz et al., 1988). The effects of urodilatin or CDD/ANP-(99-126) on the pulmonary vasculature must also be considered (Kentsch et al., 1992). If the pulmonary artery pressure and thus the blood flow in the animals receiving the peptide infusion differed from those of the control group, the alterations in ventilation-perfusion relationship would have resulted in changes of the alveolar tension of oxygen and carbon dioxide. In turn, this would have affected bronchomotor tone. Under these circumstances, the stronger pulmonary vascular and bronchodilating properties of urodilatin (Hummel et al., 1992; Kentsch et al., 1992; Duft et al., 1993) might initiate overall effects significantly different from those of CDD/ANP-(99-126). To our knowledge, this is the first study comparing the protective effects of CDD/ANP-(99-126) and urodilatin on the bronchial system in vivo. Further investigations should elucidate whether the significantly higher effects of urodilatin shown in rats have relevance in the clinical situation of obstructive lung diseases in humans.
Acknowledgements We thank Mich61a M~ilzer and Edith Kaczmarek for administration of the substances and performing the calibrations and pulmonary function measurements. This study was conducted in the Lung Function Laboratory II of the Fraunhofer Institute of Toxicology and Aerosol Research, Hannover, Germany.
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