Bronchopulmonary responses to prostaglandin F2α, histamine and acetylcholine in the dog

European Journal of Pharmacology, 32 (1975) 146--155

© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

B R O N C H O P U L M O N A R Y R E S P O N S E S T O P R O S T A G L A N D I N F2 ~, H I S T A M I N E AND A C E T Y L C H O L I N E IN T H E D O G MARTIN A. WASSERMAN Department of Hypersensitivity Diseases Research, The Upjohn Company, Kalamazoo, Michigan 49001, U.S.A.

Received 26 November 1974, revised MS received 4 February 1975, accepted 17 February 1975

M.A. WASSERMAN, Bronchopulmonary responses to prostaglandin F2 ~, histamine and acetylcholine in the dog, European J. Pharmacol. 32 (1975) 146--155. This investigation compared quantitatively the effects of prostaglandin F2a, histamine and acetylcholine on pulmonary airway resistance and dynamic lung compliance in the spontaneously breathing, anesthetized dog. Airway responses were evaluated by computer analysis before and after pharmacological blockade by either atropine or propranolol. In a dose range of 1.0--10.0 pg]kg i.v., prostaglandin F2a was the most potent bronchoconstrictor studied. At the highest dose, prostaglandin F2a increased airway resistance 153.3% and decreased lung compliance 55.4% from basal levels. Similar doses of either histamine or acetylcholine produced much less effect. Atropine significantly reduced the bronchopulmonary responses evoked by histamine, acetylcholine and prostaglandin F2~. Propranolol did not inhibit any of the respiratory effects of those bronchoconstrictors analyzed. The bronchopulmonary effects of prostaglandin F 2 (~ and histamine appear to be augmented by cholinergic stimuli. Pulmonary airway resistance Dynamic lung compliance

Prostaglandin F2 a Acetylcholine

1. Introduction Acute, reversible b r o n c h o s p a s m is a characteristic f e a t u r e o f t h e clinical p a t h o p h y s i o l o g y o f b r o n c h i a l a s t h m a (Beall et al., 1 9 7 3 ; Perm u t t , 1973). Widespread n a r r o w i n g o f the airways can be i n d u c e d p h a r m a c o l o g i c a l l y b y a wide variety o f agents including histamine, a c e t y l c h o l i n e , and prostagiandin F 2 ~ (PGF2 a ). Since the p o w e r f u l b r o n c h o c o n s t r i c t o r effects o f h i s t a m i n e were first observed by Dale and Laidlaw (1910), the physiological role o f this amine in allergic disease processes has b e e n e x a m i n e d b y n u m e r o u s investigators ( Schild et al., 1951; Brocklehurst, 1960; Austen, 1971). A f u n c t i o n a l p a r a s y m p a t h e t i c nervous syst e m is i m p o r t a n t in the regulation o f airway caliber in the dog ( W o o l c o c k et al., 1969; Cabezas et al., 1971). Reflex vagal-mediated

Histamine

Atropine

Propranolol

b r o n c h o c o n s t r i c t i o n m a y result f r o m stimulat i o n o f subepithelial 'irritant r e c e p t o r s ' b y various pharmacological, i m m u n o l o g i c a l and mechanical agents (Widdicombe, 1963; DeK o c k e t al., 1 9 6 6 ; Mills et al., 1969). Studies in t h e i n t a c t animal have s h o w n t h a t airway responses to the m e d i a t o r s o f a n a p h y l a c t i c bronc h o s p a s m are c o m p l e x and m a y involve b o t h a d i r e c t and i n d i r e c t e f f e c t on t r a c h e o b r o n c h i a l s m o o t h muscle ( D e K o c k et al., 1966). R e c e n t l y , certain prostaglandins have been studied as possible m e d i a t o r s o f bronchial asthma ( H o r t o n , 1969; C u t h b e r t , 1969). These acidic lipids are released during antigen--antib o d y i n t e r a c t i o n s f r o m guinea pig lung and p r o d u c e p o t e n t effects o n airway s m o o t h muscle {Piper and Vane, 1969; S w e a t m a n and Collier, 1968}. Since these prostaglandins are s y n t h e s i z e d and m e t a b o l i z e d in the lung {Piper

BRONCHOPULMONARY RESPONSES IN THE DOG

147

et al., 1970), it is conceivable that they could contribute to the bronchoconstriction in bronchial asthma (Sweatman and Collier, 1968). The purpose of the present study was to compare the changes in pulmonary airway resistance (RA) and dynamic lung compliance (CD y N ) evoked by the intravenous (i.v.) injections of histamine, acetylcholine, and PGF~ in the spontaneously ventilated, pentobarbitalanesthetized dog. To ascertain whether observed responses also involved cholinergic activation, these same agents were tested in the present of atropine. The non-selective beta adrenergic receptor antagonist, propranolol, was administered to determine if elimination of sympathetic dilator tone would affect pharmacologically induced bronchoconstriction.

airflow (at the beginning and end of inspiration, the flow-resistive forces are zero) permits the calculation of CD y N ( L / c m water) according to the equation: CDy S = change in tidal volume/change in transpulmonary pressure.

2. Materials and methods

2.1. General considerations To effectively describe and evaluate airway and lung responses to various pharmacological agents, RA and CD y N were estimated by the technique of Diamond (1967). This method for the anesthetized dog is a modification of the procedure introduced by Amdur and Mead (1958) for the unanesthetized guinea pig. By simultaneously measuring transpulmonary pressure (cm water), airflow rate (L/sec) and tidal volume (L), and then analyzing the resulting waveforms, RA and CD y S may be calculated. At equal points of tidal volume during inspiration and expiration, the elastic tissue frictional forces (compliance) are the same; therefore, pressure changes measured between t w o such points would reflect only non-elastic flow-resistive forces. Graphical or computer-assisted measurements of the changes in transpulmonary pressure and airflow rate between these isovolumetric points permits the calculation of instantaneous RA (cm water/L/sec) according to the equation: RA = change in transpulmonary pressure/ change in airflow rate. Computing the pressure and volume change at points of zero

2.2. Experimental preparation Mongrel dogs of either sex (10--18 kg) were anesthetized with sodium pentobarbital (30 mg/kg) given i.v. Supplemental doses were administered as required to maintain a light level of anesthesia throughout the experiment. All test agents were administered through a cannulated femoral vein. To measure instantaneous airflow, a heated mesh screen pneumotachograph (0--40 liters/min; Electronics for Medicine, Inc.) was placed in series with a cuffed endotracheal tube of appropriate diameter inserted into the trachea. A differential pressure transducer (Statham PM5 + 0.15-350) indicated the pressure drop across the screen. Airflow rate was calibrated by passing compressed air through a Gilmont flowmeter (J-837) attached to the pneumotachograph. Tidal volume, the electrical integral of airflow rate, was calibrated by passing known volumes of air through the pneumotachograph. Transpulmonary pressure (alveolar pressure minus intrapleural pressure) was measured by connecting one port of a differential pressure transducer (Statham PM5 + 0.15--350) to an 18 gauge, 4 cm needle inserted through the 5th or 6th intercostal space to measure intrapleural pressure. The other port was joined to a small piece of rubber tubing placed between the endotracheal tube and pneumotachograph. Calibration of transpulmonary pressure was accomplished with a water manometer. O u t p u t signals from these transducers were amplified through SGA-2 amplifiers (Electronics for Medicine, Inc.) and then fed into an on-line analog Pulmonary Mechanics Computer (Buxco Electronics, Inc.). This device provided a continuous breath-by-breath analysis of the mechanical properties of the airways, i.e., RA and CD y S (Giles et al., 1973). The computer performed the necessary calculations at pre-

148 cisely the exact instant for measuring RA and CD Y N, thus facilitating the experiment. The computer o u t p u t was displayed on a directwriting Brush Model 220 recording device. Prior manual calculations of RA and C D y N were made according to the method of Amdur and Mead (1958). These values provided a calibration scale for the c o m p u t e r o u t p u t on the Brush recorder. Between five and ten consecutive breaths were analyzed and averaged before drug treatment (baseline control) and again at the height of the response after drug treatment.

M.A. WASSERMAN 2. 4. Analysis o f data Statistical analyses were performed to compare quantitatively the differences in bronchopulmonary responses to the agonists alone and to compare responses after antagonist treatment. The paired t-test was used according to Snedecor (1956). A p value of 0.05 was selected as the level of significance. In addition, standard bioassay techniques were used to test the parallelism of the agonist dose--response curves (Finney, 1952). Data were expressed graphically as percent changes from initial control values. 2. 5. Drugs

2. 3. Drug dosage pro tocol After the initial preparation of the animal, a 30 min stabilization period followed. At this time, logarithmically graded doses of histamine or acetylcholine (1.0--10.0 pg/kg) were injected into the cannulated femoral vein. The cannula was then flushed with 2 ml of heparinized saline. Pre-injection control values and responses in the presence of drug were calculated. At least ten minutes elapsed between subsequent injections of each dose of drug tested in order to permit the return to basal levels. After completion of the histamine and acetylcholine treatments, responses to similar doses of PGF2 ~ were evaluated. 30 min after the last dose of PGF2 a, atropine sulfate was infused slowly at a rate of 0.033 mg/kg/min for 15 min (Harvard Constant Infusion Pump). Responses to the various doses of histamine, acetylcholine, and PGF2 ~ were repeated after atropine treatment. Each experiment was performed on 5 anesthetized dogs. A second series of experiments (4 dogs) was used to evaluate the effects of propranolol on the bronchopulmonary responses to single doses of histamine (10.0 pg/kg), acetylcholine (10.0 pg/kg), and PGF2~ (3.0 pg/kg). Initial control responses to the bronchoconstrictors were compared before and after a 15 min infusion of propranolol (0.02 mg/kg/min).

Histamine diphosphate and acetylcholine chloride (Sigma Chemical Company) were prepared as 1 mg/ml stock solutions in isotonic saline and kept refrigerated. Appropriate dilutions in isotonic saline were made fresh daily and refrigerated until the time of injection. PGF2 ~ (Upjohn) was prepared as a 1 mg/ml stock solution in a Tris--saline buffer solution (0.02 M) at pH 7 after initial solubilization in a small amount of absolute ethanol. The stock solution of PGF2a was frozen (--15°C) until the morning of the experiment. Daffy dilutions were prepared in Tris--saline buffer and kept on ice until ready for use. Doses of histamine and acetylcholine are expressed as the salts, while doses of PGF2 ~ are as the free acid.

3. Results 3.1. Effect o f histamine, acetylcholine and PGF2 ~ on airway mechanics An analog display from the Pulmonary Mechanics Computer illustrates the simplicity of the system (fig. 1). Each excursion of the writing pen represents the c o m p u t e d value for a single respiratory cycle. Routine calibrations of RA and CD v N were accomplished daily. From the trace on fig. 1, it is clearly evident that all three agonists tested: histamine (10 pg/kg),

BRONCHOPULMONARY RESPONSES IN THE DOG

.004 -1

149

HIST

ACH

PGFza

, 10 PO/kg IV

10 g / k g IV

3 pg/kg IV

19.2

,.

o

t

soc t

f

Fig. 1. Record from a Brush direct-writing recorder illustrating the analog output of the Pulmonary Mechanics Computer. Airway resistance (RA) and dynamic lung compliance (CDYN)responses to i.v. doses of histamine (HIST; 10 ug/kg), acetylcholine (ACH; 10 ug/kg) and prostaglandin F2~ (PGF2~; 3 ug/kg) are observed for the anesthetized dog. Each pen excursion represents the computed value for one ventilatory cycle. Paper speed = 1 mm/sec. Arrows indicate injection points.

acetylcholine (10 gg/kg) and PGF2 ~ (3 pg/kg) alter airway and ventilatory function. Each drug produced an increase in RA and ventilation frequency, while concomitantly evoking a decrease in CD v N. The extremely potent and long lasting RA effects of PGF2~ are demonstrated in fig. 2. These duration--response curves were taken from a single representative experiment. Histamine and acetylcholine produced maximum increases in RA after approximately 30 sec and basal levels returned within 2--5 min. PGF2 ~ induced a maximum effect at 1 min and control values returned after 10 min (fig. 2). In a dose range of 1.0--10.0 pg/kg, histamine, acetylcholine and PGF2~ produced

lO

• Histamine 10 pg/kll

o i ¢' ~

u

s

i

dose-dependent elevations in RA and reductions in CDy N (figs. 3 and 4; table 1). Parallel line assays of the dose--response curves for the increase in RA induced by histamine and acetylcholine were performed. These curves were parallel and almost superimposable. The slope of the dose--response curve for PGF2 ~ on RA was significantly steeper than for histamine or acetylcholine; hence, single estimates of relative activities may not be justified. However, comparing various and arbitrary equiactive doses along each curve may be more appropriate (Daly et al., 1971; Wasserman and Levy, 1974a). Comparisons of activity for 30-40% increases in RA show that PGF2~ is approximately 3--5 times more potent than

•

~

O

pg/kg ProstaglendinFza 10 pg/kg

Acstylcholia 10

i

E

~

6

i,

i

o

I

"0 .....

I ½

1

1

L 2

I 5

I 10

TIME (MINUTES)

Fig. 2. Comparison of the onset and duration of airway resistance (cm water/L/sec) effects of histamine, acetylcholine and prostaglandin F2~ in the anesthetized dog. Drugs were administered as single bolus i.v. injections (10 pg/kg) at time zero.

150

M.A. WASSERMAN PGF2 ~ was again m o r e effective, especially t h e 3.0 a n d 10.0 p g / k g doses (p ~ 0.05; fig. 4; t a b l e 1). The slopes o f t h e d o s e - - r e s p o n s e curves f o r t h e decrease in CD y N p r o d u c e d b y h i s t a m i n e , a c e t y l c h o l i n e , and P G F : ~ were all parallel. T h e r e f o r e , m e a n i n g f u l c o m p a r i s o n s of relative p o t e n c i e s were m a d e . When a c e t y l c h o line was c o m p a r e d t o h i s t a m i n e , the p o t e n c y ratio w i t h c o n f i d e n c e limits was 1.02 ( 0 . 5 4 - 1.93). When P G F : ~ was c o m p a r e d to hista m i n e , t h e p o t e n c y r a t i o was 6.17 ( 3 . 1 1 - 16.14).

170 150

[] •

Response to egonist before ATROPINE (0.5 mg/kg) Response to agonist after ATROPINE (0.5 mg/kg)

100'

|

00

~

00

z

40

~ so 0

1 3 10 HISTAMINE (Po/kg)

1 3 10 ACETYLCHOLINE(pg/bo)

I

-

3 10 PGFza {pg/k9)

Fig. 3. Effect of atropine on the increase in airway resistance induced by various doses of histamine, acetylcholine and PGF2 a in the anesthetized dog. Each bar graph represents the mean + S.E. of observations in 5 animals. Atropine was infused at a rate of 0.033 mg/kg/min for 15 min. e i t h e r h i s t a m i n e or a c e t y l c h o l i n e . A c e t y l c h o line was e q u a l t o h i s t a m i n e in e v o k i n g elevat i o n s in RA in t h e d o s e range studied. The m e a n + S.E. f o r resting levels o f RA f o r all t h e s e e x p e r i m e n t s was 4.02 + 0.40 c m w a t e r / L / s e c . Histamine and acetylcholine decreased CD Y N equally at all doses e x a m i n e d , while 00

"~ 50

[ ] Response to agonist before ATROPINE {0.5 mg/kg) i l Response to ngonist after ATROPINE (0.5 mo/kg)

"[

2

~ 40

O

1 3 10 HISTAMINE (pg/kg)

1 3 10 ACETYLCHOLINE(pg/kg)

1

--3 10 PGF2a (pg/kg)

Fig. 4. Effect of atropine on the decrease in dynamic lung compliance induced by various doses of histamine, acetylcholine and PGF2 a in the anesthetized dog. Each bar graph represents the mean -+S.E. of observations in 5 animals. Atropine was infused at a rate of 0.033 mg/kg/min for 15 rain.

3.2. Effects of atropine pretreatment on airway responses to histamine, acetylcholine and PGF2 A i r w a y r e s p o n s e s t o t h e t h r e e doses o f agonists t e s t e d were r e - e v a l u a t e d a f t e r a slow i n f u s i o n o f a t r o p i n e sulfate, 0.5 m g / k g {figs. 3 a n d 4). This cholinergic a n t a g o n i s t d i s p l a y e d intrinsic activity b y partially a t t e n u a t i n g t h e basal p a r a s y m p a t h e t i c t o n e p r e s e n t in t h e airw a y o f t h e dog. A t r o p i n e d e c r e a s e d resting I~A b y 17.3 + 4.1% a n d increased basal C D y N b y 1 0 . 6 + 3.2% (table 2). T h e s e values were s h o w n t o b e statistically significant (p < 0.05). T h e RA a n d CD v N e f f e c t s o f a c e t y l c h o l i n e were almost totally abolished after atropine p r e t r e a t m e n t . This w o u l d be e x p e c t e d since b o t h agonist and a n t a g o n i s t a c t on t h e cholinergic r e c e p t o r site. A f t e r a t r o p i n e , a c e t y l c h o line (10.0 p g / k g ) o n l y p r o d u c e d a 6.8 + 1.6% increase in RA and a 4.2 + 2.7% decrease in CDV N, w h e r e a s p r i o r to a t r o p i n e , t h e s e res p o n s e s were: 46.7 -+ 6.9% and 32.3 + 3.6%, respectively. The b r o n c h o p u l m o n a r y r e s p o n s e s to hista m i n e were also significantly r e d u c e d in t h e p r e s e n c e o f a t r o p i n e , y e t n o t to t h e s a m e e x t e n t as a c e t y l c h o l i n e . Only t h e CD y N res p o n s e to t h e largest d o s e o f h i s t a m i n e was spared significant m o d i f i c a t i o n b y a t r o p i n e . At this dose, h i s t a m i n e d e c r e a s e d CD y N 33.8 + 4.8% b e f o r e a t r o p i n e a n d 27.6 + 3.1% a f t e r a t r o p i n e (0.4 > p > 0.3). A p p a r e n t l y , hista m i n e is e i t h e r eliciting local reflex vagal activity or r e i n f o r c i n g intrinsic b r o n c h o m o t o r t o n e .

A C C U M U L A T I O N O F BASIC D R U G S IN 5HT O R G A N E L L E S

151

TABLE1 C o m p a r i s o n o f the b r o n c h o p u l m o n a r y e f f e c t s o f various i.v. doses o f h i s t a m i n e , a c e t y l c h o l i n e and PGF2 ~ in the dog. Data r e p r e s e n t t h e m e a n s + S.E. o f d e t e r m i n a t i o n s in 5 animals. C = c o n t r o l value b e f o r e drug; R = m a x i m u m r e s p o n s e after drug; A = a b s o l u t e change f r o m c o n t r o l value; %A = p e r c e n t change f r o m c o n t r o l value. Drug

Dose (ug/kg)

Airway resistance (cm w a t e r / L / s e c ) C

R

A

%A

Histamine

1.0 3.0 10.0

3.45 ± 0.30 4 . 2 3 ± 0.67 3 . 6 4 ± 0.33

3.97 ± 0.31 5 . 2 6 + 0.65 5 . 5 3 ± 0.34

0.52 ± 0.08 1 . 0 3 + 0.13 1 . 8 9 ± 0.38

15.5 + 3.2 2 7 . 4 ± 6.2 5 6 . 0 ± 15.3

Acetylcholine

1.0 3.0 10.0

4 . 1 3 ± 0.27 3 . 9 7 ± 0.45 4 . 1 4 ± 0.43

4.76-+ 0.41 5 . 0 9 ± 0.54 5 . 9 9 ± 0.47

0 . 6 3 ± 0.12 1 . 1 2 ± 0.19 1.85 ± 0.15

16.2 ± 29.3± 46.7 ±

PGF 2

1.0 3.0 10.0

4.28 ± 0.47 4 . 1 5 ± 0.38 4.16 ± 0.29

5 . 6 3 ± 0.82 6 . 9 6 ± 0.77 10.37 + 0.99

1.35 ± 0.39 2,81+- 0.51 6.21 ± 0.98

Drug

Dose D y n a m i c lung c o m p l i a n c e ( L / c m water) (mg/kg) C R A

%A

Histamine

1.0 3.0 10.0

0.045 ± 0.00,7 0.043 ± 0.006 0.045 ± 0.005

0.040 ± 0.006 0.033 ± 0.004 0.029 ± 0.003

- - 0 . 0 0 5 ± 0.001 - 0 . 0 1 0 ± 0.002 - - 0 . 0 1 6 ± 0.003

--11.4 ± 2.0 --22.2 + 2.5 --33.8 ± 4.8

Acetylcholine

1.0 3.0 10.0

0 . 0 3 8 ± 0.005 0 . 0 3 9 ± 0.005 0.038 ± 0.004

0 . 0 3 3 ± 0.004 0 . 0 3 0 ± 0.004 0 . 0 2 6 ± 0.004

--0.005 ± 0.001 - - 0 . 0 0 9 ± 0.001 - - 0 . 0 1 2 ± 0.001

--13.5 ± 1.9 --22.1 ± 0.9 - - 3 2 . 3 ± 3.6

PGF2

1.0 3.0 10.0

0.039 -+ 0.005 0.039 + 0.005 0.038 + 0.005

0.030 ± 0.005 0.024± 0.004 0.017 + 0.003

--0.009 ± 0.001 --0.015-+ 0.001 --0.021± 0.003

--26.3 + 6.7 --41.1+ 5.0* --55.4+ 2.7*

4.4 5.2 6.9

3 0 . 1 ± 5.5 6 8 . 7 ± 11.5" 153.3 ± 2 5 . 8 * *

* p < 0.05; ** p < 0.01 c o m p a r i n g PGF2 ~ to h i s t a m i n e or a c e t y l c h o l i n e by the t-test for paired data. TABLE 2 Intrinsic airway e f f e c t s o f i.v. infusions o f a t r o p i n e and p r o p r a n o l o l in the s p o n t a n e o u s l y breathing, a n e s t h e t i z e d dog. D a t a r e p r e s e n t the m e a n s +- S.E. o f d e t e r m i n a t i o n s in 4 ( p r o p r a n o l o l ) or 5 ( a t r o p i n e ) animals. C = initial control values; R = r e s p o n s e s after 15 m i n i n f u s i o n o f a t r o p i n e o r p r o p r a n o l o l ; A = a b s o l u t e change from c o n t r o l values; %A = p e r c e n t change f r o m c o n t r o l values. Drug

Dose Airway resistance (cm w a t e r / L / s e c ) (mg/kg/ - min) C R A

%A

Atropine Propranolol

0.033 0.020

--17.3+- 4.1" -- 1.0 ± 7.4**

Drug

Dose D y n a m i c lung c o m p l i a n c e ( L / c m w a t e r ) (mg/kg/

Atropine Propranolol

4.99 + 0.52 3.89 + 0.40

4.16+-0.52 3.79 + 0.24

--0.83+ 0.15 --0.10 ± 0.31

rain)

C

R

A

%A

0.033 0.020

0.035 + 0.006 0.0383 + 0.003

0.038 +- 0.006 0.0382 + 0.003

+0.003 ± 0.001 --0.0001 + 0.003

+10.6 ± 3.2* -- 0.5 +- 8.3**

* T h e s e values w e r e statistically significant (p < 0.05). ** These values w e r e n o t statistically significant (p > 0.05).

152

M.A. WASSERMAN

Post-atropine examination of the airway responses to PGF2 ~ demonstrates that this prostaglandin indeed possesses some cholinergicmediated c o m p o n e n t in its activity. The very large increases in RA evoked by the 3.0 and 10.0 pg/kg doses of PGF2~ were more than halved in the presence of atropine, while the CD Y N responses to the same doses were significantly reduced, but to a lesser degree (figs. 3 and 4). The 10.0 pg/kg dose of PGF2~ produced an increase in RA of 153.3 + 25.8% before atropine and 66.8 + 9.1% after atropine, whereas the CDy S response decreased from 55.4 + 2.7% to 42.7 + 3.8%. The profuse salivation following all doses of PGF2 ~ was abolished after atropine.

is shown on fig. 5. Data plotted are the mean +S.E. from a series of 4 dogs. The infusion of propranolol did not significantly alter the resting tone of the airways (p > 0.05; table 2). In some animals, RA increased and CDV N decreased after propranolol, while the opposite was true in other animals. This wide variability is reflected in the rather large standard errors which were calculated (table 2). The responses to the agonists tested after propranolol were not significantly different from those before propranolol. Therefore, propranolol (0.3 mg/ kg) appears to have no effect on the airway responses to various classes of bronchoconstrictors in the normal, healthy, anesthetized dog.

3.3. Effects o f propranolol on the airway responses to histamine, acetylcholine and PGF 2

4. Discussion

The effects of a slow infusion of propranolol (total dose: 0.3 mg/kg) on the increase in RA and decrease in CD y S evoked by single, representative doses of histamine (10.0 pg/kg), acetylcholine (10.0 pg/kg) and PGF2, (3.0 pg/kg) C~ •

Responseto ogonistbefore PROPRANOLOL(0.3 mg/kg) Responseto agonistafter PROPRANOLOL(0.3 mg/kg)

240 f

60

200 160 1

40.

/

°[ ~ 80

20

~ 4o

0

HIST ACt4 PGF2Q (10 pg/kg) (10 pg/kg) (3 pg/kg)

0

HIST ACH PGF2a (10 pg/kg) (10 pg/kg) (3 pg/kg)

Fig. 5. Effect of propranolol on the increase in airway resistance and the decrease in dynamic lung compliance induced by single doses of histamine (HIST; 10 ug/kg), acetylcholine (ACH; 10 tLg/kg) and prostaglandin F2a (PGF2a; 3 u g / k g ) in the anesthetized dog. Each bar graph represents the mean -+ S.E. of observations in 4 animals. Propranolol was infused at a rate of 0.02 mg/kg/min for 15 min.

Intense bronchoconstriction is a major pathological feature of human bronchial asthma. Airway narrowing may be produced by several agents released endogenously either as a primary result of antigen--antibody reactions or secondarily through secretory and reflex mechanisms (Schild et al., 1951; Collier and James, 1967; Austen, 1973). Histamine, acetylcholine and PGF2 ~ are among the most widely studied agents which can induce or contribute to the bronchospasm during asthma. These naturally occurring substances can function as mediators of the hyperreactive pathological state. They all can cause the characteristic tissue changes of mucosal edema and hypersecretion in addition to smooth muscle contraction. An on-line analog Pulmonary Mechanics Computer facilitated the quantitative evaluation of non-elastic (RA) and elastic (CDy N ) forces in spontaneously breathing, anesthetized dogs. Spontaneously breathing animals more faithfully reflect lung mechanics in the intact animal than do artificially ventilated animals (Mills and Widdicombe, 1970). The computerized device permitted an objective analysis of a number of chemically unrelated pharmacological mediators. The relative contribution of cholinergic and sympathetic impulses

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153

to airway smooth muscle was also estimated from such data. The results of the present investigation indicate that PGF2~ is clearly the most potent bronchoactive agent studied. In a dose range of 1.0--10.0 pg/kg i.v., PGF2~ increased RA approximately 3 to 5 times more than did similar doses of either histamine or acetylcholine. PGF2 ~ was also significantly more effective in reducing CD y N than either of the other two agents. Histamine and acetylcholine were equip o t e n t for both RA and C D y N responses. These data would indicate a relative order of potency for canine bronchopulmonary responses to be: PGF2~ > histamine = acetylcholine. When peripheral airways constrict, CDy N decreases markedly, thereby restricting lung distension and expelling gas from the lungs (Colebatch et al., 1966). RA increases only slightly, since these peripheral airways (respiratory bronchioles and alveolar ducts) contribute only a small fraction of the total resistance to airflow (Colebatch et al., 1966). Variations in RA may be reflected in alterations of the larger, more central, conducting airways (trachea to terminal bronchioles), where the greatest resistance to airflow is observed (Colebatch et al., 1966). Therefore, each of the bronchoconstrictors in the present study appears to act at all levels of the tracheobronchial tree. After demonstrating that potent effects on airway dynamics are evoked by histamine, acetylcholine and PGF2~ the effect of autonomic blockade on pharmacologically induced bronchoconstriction was tested. A clarification of the mechanism of bronchoconstriction is invaluable to the fundamental understanding of the control of smooth muscle tone. Pretreatment with the classical, cholinergic end organ antagonist, atropine (0.5 mg/kg), nearly abolished the RA and CD~N responses to the cholinergic agonist, acetylcholine. The airway responses induced by histamine were significantly reduced after atropine. Mills and Widdic o m b e (1970) observed that, in spontaneously breathing guinea pigs, vagotomy reduced by 50% or more the decrease in total lung conduc-

tance and compliance evoked by histamine. They suggested that a vagal reflex arc was activated by stimulation of so-called 'lung irritant receptors' in the bronchial epithelium. Efferent (atropine) or afferent (cooling) vagal blockade can diminish the response of airway smooth muscle to histamine (Gold et al., 1972). Therefore, previous findings and present results may be explained by postulating the occurrence of a cholinergic bronchoconstrictor reflex in addition to the direct effect of histamine on smooth muscle. Alternatively, SjSstrand and Swedin (1968) have demonstrated in vitro that smooth muscle stimulating agents may act synergistically with released neurotransmitters to produce enhanced responses. Therefore, if atropine inhibits the effect of the normal cholinergic nervous discharge to the airways, then reduced histamine responses would be observed. Douglas et al. (1973) concur with this hypothesis in which complex reflex pathways need not be involved. The powerful canine airway effects of P G F 2 , were also significantly reduced in the presence of atropine. In addition, the abundant salivation produced concomitantly by PGF2 was abolished after atropine. The salivary response may be caused by the endogenous liberation of acetylcholine by PGF2 ~ (Hahn and Patil, 1974). Baum and Shropshire (1971) have shown that some prostaglandins could influence not only adrenergic nerve transmission, b u t also could enhance the responses of the guinea pig ileum to either cholinergic nerve stimulation or exogenous acetylcholine. Therefore, the effects of PGF2 ~ on airway smooth muscle appear to possess some cholinergic component, possibly reflexive or augmentative, in addition to a direct constricting effect. Propranolol (0.3 mg/kg), a ~-adrenergic receptor antagonist, had no intrinsic effect on airway smooth muscle and did not make the airways hyperreactive to any of the constricting agents. In a previous study, this dose of propranolol was shown to inhibit the effect of isoproterenol against histamine-induced bronchospasm by more than 50% (Wasserman and Levy, 1974b). Also, these investigators have

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shown that the pulmonary resistance response to histamine was not significantly changed after propranolol 1.0 mg/kg (Wasserman and Levy, 1974b). Sympathetic nerve innervation of the airways may be minimal in the dog and may only play a minor role in the regulation of airway caliber. In histamine-sensitive guinea pigs, Dennis and Douglas (1970) have shown that propranolol did not increase the effect of histamine. They concluded that sympathetic tone must indeed be small. Cabezas et al. (1971) demonstrated in dogs that thoracic sympathetic nerve stimulation had no effect on airway diameter after vagotomy, but, with intact vagi, supramaximal sympathetic stimulation could partially reduce airway tone; hence, vagal constrictor impulses to the airways predominate. It appears that beside uptake, distribution, solubility in physiological fluids, and drug metabolism, a relative preponderance of vagal or sympathetic tone can contribute to the magnitude of airway smooth muscle contraction induced by various pharmacological agents.

Acknowledgment The author wishes to gratefully acknowledge the excellent technical (electronic) assistance of Mr. Richard M. Eustice.

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