The effects of nifedipine and verapamil on antigen-induced bronchoconstriction in dogs

European Journal of Pharmacology, 92 (1983) 69- 75

69

Elsevier

T H E EFFECTS OF N I F E D I P I N E A N D VERAPAMIL O N A N T I G E N - I N D U C E D B R O N C H O C O N S T R I C T I O N IN D O G S PETER E. M A L O l,, M.A. W A S S E R M A N

i and R.L. GRIFFIN

Department of Hypersensitivity Diseases Research, The Upjohn Company, Kalamazoo, MI 49001, U.S.A. Received 5 April 1983, accepted 20 May 1983

P.E. MALO, M.A. WASSERMAN and R.L. GRIFFIN, The effects of nifedipine and verapamil on antigen-induced bronchoconstriction in dogs, European J. Pharmacol. 92 (1983) 69-75. The effects of calcium channel blockers, nifedipine and verapamil (i.v. and aerosol), were investigated in beagle dogs natively allergic to Ascaris suum antigen. Control exposures to an aerosol of Ascaris antigen provoked significant bronchopulmonary changes, i.e., increases in pulmonary resistance (RL) and decreases in dynamic lung compliance (CDvN). Pretreatment with either nifedipine or verapamil (200 t.tg/kg, i.v.) provided significant inhibition in the R L responses to Ascaris antigen (P < 0.015) while neither agent significantly affected CDVN changes. When administered as an aerosol, verapamil (1.0%; 10 breaths) significantly inhibited both the R L and CDvr~ responses to Ascaris antigen (P < 0.05), whereas a similar concentration of nifedipine was without effect. Resting basal levels of R L and Coy N were unaffected by either the i.v. route or by aerosols of either nifedipine or verapamil. These results suggest that calcium channel blockers may have beneficial effects against allergen-provoked bronchoconstriction; however, differences appear to exist in the choice of agent, route of administration and site of action. Ascaris sensitive dogs

Bronchospasm

Calcium antagonists

1. Introduction Nifedipine, a calcium slow channel blocking agent, has been shown to be clinically effective in the treatment of various cardiovascular disorders (Becket et al., 1976; Guazzi et al., 1977; Aoki et al., 1978; Kenmure and Scruton, 1979). The presumed mechanism of action of nifedipine is to specifically inhibit both the penetration of extracellular calcium through a cell membrane and the release of intracellular calcium (Guazzi et al., 1980). Because of the hypothesized role of calcium in smooth muscle contraction, recent studies have

Present address: Department of Pharmacology, Smith Kline and French Laboratories, Philadelphia, PA 19101, U.S.A. * Send all correspondence to: Peter Malo, F-129, Department of Pharmacology, Smith Kline and French Laboratories, Philadelphia, PA 19101, U.S.A. 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.

examined the action of nifedipine in the airways. In perfused canine tracheas, nifedipine produced tracheal relaxation, albeit not as effective as isoproterenol (Himori and Taira, 1980). Nifedipine can inhibit the Schultz-Dale reaction as well as histamine contractions in guinea pig tracheal preparations (Fanta et al., 1981; Weiss and Markowicz, 1981). In addition, nifedipine, while not modifying basal bronchial tone, did prevent exercise-induced bronchoconstriction in asthmatics (Denjean et al., 1980). Finally, we reported previously that nifedepine, by either the i.v. or aerosol route, inhibited pharmacologically induced bronchoconstriction in dogs (Malo et al., 1982a). Therefore, the purpose of the present investigation was to determine whether nifedipine (i.v. or aerosol) can inhibit an antigen-induced bronchospasm in the anesthetized dog. Also, another calcium channel blocker, verapamil, was studied under similar experimental conditions for comparison.

70 2. Materials and methods

Pulmonary resistance (RE) and dynamic lung compliance (CDYN) were estimated by the technique of Diamond (1972). 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/s) and tidal volume (1), and then analyzing the resulting waveforms, R E and CDyN 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 two such points reflect only non-elastic flow-resistance forces. Graphical or computer-assisted measurements of the changes in transpulmonary isovolumetric points permit the calculation of instantaneous R L (cm w a t e r / l / s ) according to the equation: R L = change in transpulmonary pressure/ change in airflow rate. Computing the pressure and volume change at points of zero airflow (at the beginning and end of inspiration, the flow-resistive forces are zero) permits the calculation of C Dy s (1/cm water) according to the equation: C Dy N = change in tidal volume/change in transpulmonary pressure. Beagle 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 jugular vein. To measure instantaneous airflow, a heated mesh screen pneumotachograph (0-40 l/rain; 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 (J837) attached to the pneumotachograph. Tidal volume, the electrical integral of airflow rate, was calibrated by pasing 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. Output 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., R L and C DYN- The computer performed the necessary calculations at precisely the exact instant for measuring R L and CDy N, thus facilitating the experiment. The output was displayed on a direct writing Grass Model 7B Polygraph. Between five and ten consecutive breaths were analyzed and averaged before drug treatment and again at the height of the response after treatment. In addition to pulmonary functions, mean systemic arterial blood pressure (MAP) and heart rate (HR) were monitored continuously. MAP was measured via a cannulated femoral artery with a Statham P23Db Transducer. The arterial cannula was fitted with a K75 Three-Way Stopcock (Pharmaseal) for blood samples to be collected for blood gas analysis. Blood gases were monitored with the use of an Instrumentation Laboratory Model 813 p H / B l o o d Gas Analyzer. H R was counted electronically from lead II of an electrocardiograph ( E K G / T a c h o g r a p h Preamplifier, Grass Model 7P4D). Tracings from all cardiovascular and pulmonary systems were recorded on the Grass Polygraph. All beagle dogs used in this study had been screened previously for Ascaris antigen sensitivity and had a history of positive reactions to an intrabronchial challenge of Ascaris suum antigen extract (Greer Laboratories, Lenoir, NC). The extract, a 1:10 (w/v) concentrate was diluted to various concentrations with physiological saline. Only those animals with a consistent reaction to

71 1 : 10000 or 1 : 1000 a n t i g e n d i l u t i o n were chosen for this study. A positive reaction for each challenge was considered to be an increase in R L > 80% a n d a decrease in CDv N > 40%. 5 beagle dogs were used in this study. N i f e d i p i n e a n d verapamil pret r e a t m e n t s were used by i.v. (200 ~ g / k g ) a n d by the aerosol routes (1.0%; 10 breaths). Aerosols of Ascaris a n t i g e n a n d test drugs were generated b y a M a r k VII Bird ~ Respirator with an in-line micronebulizer. The flowrate, sensitivity a n d pressure settings o n the respirator were adjusted to keep c o n s t a n t the v o l u m e of drug nebulized with each breath. N o a t t e m p t was m a d e to calculate exactly how m u c h drug was actually inhaled but rather by k n o w i n g the c o n c e n t r a t i o n of each drug used (1.0%) a n d the average volume nebulized per b r e a t h (0.007 ml), the total a m o u n t of the drug delivered over a u n i f o r m n u m b e r of breaths (10) could be a p p r o x i m a t e d , i.e. 700 ~g. After the initial p r e p a r a t i o n of the animals, a 30-min stabilization period followed. At this time, a pre-selected c o n c e n t r a t i o n of Ascaris a n t i g e n was a d m i n i s t e r e d i n t r a b r o n c h i a l l y as a n aerosol a n d the response m o n i t o r e d . 3 to 4 weeks later, a n i m a l s were treated with either n i f e d i p i n e or verapamil 15 m i n prior to rechallenge with the antigen. N i f e d i p i n e a n d verapamil were both administered as a n i.v. i n f u s i o n over 10 min, while as aerosols, both were nebulized as a 1.0% solution for 10 tidal breaths. Statistical evaluations to c o m p a r e results of these studies were performed using the t-test for u n p a i r e d data (Snedecor, 1956). A value for significance was chosen at the P < 0.05 level. D a t a were expressed graphically as well as in a t a b u l a r fashion as the m e a n + S.E. a n d represent the percent change of the particular e n d p o i n t unless otherwise stated. N i f e d i p i n e was synthesized at the U p j o h n Company, E x p e r i m e n t a l Chemistry U n i t , K a l a m a z o o , MI, a n d was kept frozen in complete darkness to p r e v e n t p h o t o - d e g r a d a t i o n . The c o m p o u n d was solubilized with some difficulty a n d required w a r m i n g in a small a m o u n t of absolute ethanol prior to the a p p r o p r i a t e d i l u t i o n for either the i.v. (200 /~g/kg solution) studies or the aerosol (10 m g / m l solution) studies. Verapamil was a generous gift from K n o l l Pharmaceuticals ( W h i p p a n y ,

N J) a n d was solubilized in physiological saline to the appropriate d i l u t i o n (200 /~g/kg i.v. or 10 m g / m l for aerosol investigation).

3. R e s u l t s

Those b r o n c h o p u l m o n a r y changes in R L and CDy y following an aerosol exposure to Ascaris a n t i g e n (12 tidal breaths) are plotted in fig. 1. Prior to Ascaris challenge, an aerosol exposure to an equal n u m b e r breaths of s a l i n e / e t h a n o l vehicle alone produced small decreases in R E ( - 1 0 . 7 + TABLE 1 Historical aerosol challenges with Ascaris suum extract in antigen-sensitivebeagle dogs. Number of Animal challengesb Identification 1 2 3 4 5

II87

RL a

CDYN

Cc

%Ad C

t~A

6.0 1.1 4.3 3.5 4.8

441 363 334 445 371

0.035 0.028 0.034 0.073 0.020

-60 - 78 - 71 - 64 - 73

1 2 3 4

964

5.0 4.6 7.3 4.8

86 125 200 150

0.032 0.039 0.019 0.025

- 43 -63 - 71 - 60

1 2 3 4

9232

5.6 6.7 4.8 3.9

543 570 485 550

0.019 0.023 0.030 0.028

-

1 2 3 4

9127

4.2 3.8 4.8 5.0

89 155 125 100

0.044 0.038 0.039 0.050

- 32 - 48 -45 - 47

1 2 3 4

156

3.0 3.8 4.2 4.7

125 150 160 130

0.020 0.025 0.021 0.030

-

0.032 ±0.003

- 60 ±3

Mean ± S.E.

4.5 271 ±0.28 ±38

74 63 64 68

60 62 58 54

pulmonary resistance (cm water/l/s); C D y N = dynamic lung compliance (1/cm water). ~' A representative portion of the historical record kept on these animals in which each challenge is separated usually by 3-4 weeks of recovery time. C = resting control values in absolute units. a %A = percent change from resting control. a RL =

72

B

A 70 z

%

60

rr

o~ 500-

50

"~ 400.

40' o

rr

o

300-

=, 200

30" 20"

.E 100

10E3

o

Fig. I. Effect of nifedipine (i.v, and aerosol) pretreatment vs. Ascaris antigen-induced bronchospasm in anesthetized dogs. Each bar represents the mean percent change +- S.E.M. in airway resistance (R L) or dynamic lung compliance (C DYy ) for 5 animals. * P < 0.05 when value compared to Ascaris response alone. [] Ascaris antigen alone; • Nifedipine (200 / t g / k g , i.v.)+Ascaris antigen 15 min later; ~ Nifedipine aerosol (1.0%; 10 breaths)+Ascaris antigen 15 min later.

from previous Ascaris responses alone (R e: 271 + 38, CDvN: - 6 0 + 3; table 1). Pretreatment with nifedipine (200 btg/kg, i.v.) had virtually no effect on resting levels of R L, C DYN or H R (table 2), but did significantly reduce MAP by 57 +_ 3% from 106 + 6 mmHg to 45 + 3 mmHg (P < 0.01; table 2). When nifedipine-treated

5.0) and larger, highly variable increases in C D y N ( + 30% _+ 31%). With Ascaris challenge in sensitized beagles, R L increased 300 + 50% and CDy N decreased 61 + 4% from resting baseline values of 4.17 + 0.74 cm w a t e r / 1 / s and 0.033 +_ 0.005 l / c m water, respectively. This Ascaris reaction with a vehicle pretreatment was not significantly different

TABLE2 E f f e c t o f c a l c i u m antag•nists•nc•ntr••br•nch•pu•monaryandcardi•vascu•arfuncti•nsintheanesthetizedd•g. Dr ug

Route

Dose

Saline/ ethanol Nifedipine

Aerosol

Verapamil

Aerosol

Nifedipine

i.v.

10 m g / m l ; 10 breaths 10 m g / m l ; 10 breaths 200/lg/kg

Verapamil

i.v.

200 # g / k g

Aerosol

Reb

C Dv N

Cc

%A

C

5.33 _ 2.6 5.29 _ 1.03 4.71 + 2.24 3.58 + 0.54 3.33 + 0.44

- 10.7

0.020 +- 0.009 0.059 -+ 0.034 0.021 +_0.007 0.033 + 0.005 0.033 + 0.007

- 1.0% 7.4% 2.0% 10.5%

MAP %3

C~

HR %~

Cc

%A

30%

130_+ 9

4%

213--_22

- 1.2

32%

128--,23

-41.2

178_+ 3

14%

0

67 +- 7

4.0

210 +- 19

- 4.3

0

106-+ 6

- 57.3 **

210 ± 16

- 3.0

- 3 9 **

210--, 6

- 1.0

-6.1%

93-+ 5

Values represent the mean ± S.E. from 5 animals. R L = pulmonary resistance (cm w a t e r / l / s ) ; CDy y = dynamic lung compliance ( l / c m water); MAP = mean systemic arterial blood pressure (mmHg); H R = heart rate (beats/min). c C = resting control values in absolute units; %A = percent change. ** P < 0.01. b

73

!1

A z

~- 60' J

500

so

,~ 400

.~- 40

rr" 300

E o 0 30

.__. 200

,<

.c

c_.

2o

too-

Fig. 2. Effect of verapamil (i.v. and aerosol) pretreatment vs. Ascaris antigen-induced bronchospasm in anesthetized dogs. Each bar represents the mean percent change+ S.E.M. in airway resistance (RE) or dynamic lung compliance (CDvN) for 5 animals. * P < 0.05 when value compared to Ascaris response alone. [] Ascaris antigen alone; • Verapamil (200/.t g/kg, i.v.)+ Ascaris antigen 15 min later; [] Verapamil aerosol (1.0%; 10 breath)+Ascaris antigen 15 min later.

dogs were now exposed to Ascaris antigen aerosol, the R E response was significantly inhibited (P < 0.05), while the CDV N response was unaffected (fig. 1). When nifedipine was administered as an aerosol (1.0%, 10 tidal breaths), basal levels of R E, CDYN, HR and MAP were affected minimally (table 2). Nifedipine aerosol had no effect against an Ascaris antigen challenge (fig. 1). When verapamil was tested under the conditions described above, the following results ensued. By the i.v. route, verapamil (200/~g/kg) did not significantly affect resting R E, CDy N or HR, but did decrease MAP by 3 9 + 2 % from 9 3 + 5 mmHg to 57 + 2 mmHg (P < 0.01; table 2). Ascaris antigen challenges were significantly inhibited for R L , but not C o y N by i.v. veraparnil (fig. 2). Aerosolized verapamil had essentially no effect on resting values of R E , CDYN, MAP or HR (table 2), but did significantly reduce the Ascaris antigen-induced increases in R E and decreases in C o y N (fig. 2). Finally, neither verapamil or nifedipine produced any significant effect on the blood gas profiles.

4.Discussion

The ability of nifedipine (i.v. or aerosol) to inhibit pharmacologically induced broncho-

constriction in normal, anesthetized beagle dogs has been previously reported by our laboratory (Malo et al., 1982a). A logical extension of these studies was to repeat them against an antigen-induced bronchospasm. Therefore, the effect of nifedipine and verapamil by i.v. and aerosol administration in a group of Ascaris antigen-sensitive beagle dogs was tested. A single dose of 200 /zg/kg, i.v. and 10 m g / m l aerosol concentration were chosen from previous work utilizing dose response curves against pharmacologically-induced bronchospasm (Malo et al., 1982a). In the present study, we show that i.v. nifedipine can provide substantial protection in large airway constriction (RL) due to an antigen-provoked bronchospasm; this agrees with in vitro work performed by Weiss and Markowicz (198 I). In spite of this effect in the larger airways, it was surprising to observe that nifedipine failed to provide any protection against Ascaris antigen effects in the smaller airways of the lung as measured by C DVN. In addition, aerosolized nifedipine, while providing some protection against pharmacologically induced bronchoconstriction, failed to produce any inhibition against Ascaris antigen exposure. This was somewhat disquieting, based on the assumption that the aerosolized calcium antagonist had direct access to the airway and should have provided adequate protection. Patel (1981a) suggested that nifedipine, by its blocking

74 action of the slow calcium channel, may prevent mediator release from the mast cells in response to exercise. Therefore, nifedipine by this mode of action should prevent histamine release due to antigen-induced bronchospasm. It may be possible that the concentration used was not sufficient to alleviate the bronchoconstriction. Based on the average volume nebulized per breath (0.007 ml) with the Mark VII Bird respirator/micronebulizer, using a 1.0% solution of nifedipine, 10 breaths should provide an estimated delivered dose of 700 /~g directly to the airway. Also, the delivered aerosol dose appeared to be absorbed systemically by alveolar capillary beds because of hypotension observed after administration. Therefore, although this estimated dose was higher than that used i.v., it could be possible that the concentration was still insufficient to prevent the direct and indirect reflex mechanisms associated with an antigen-induced bronchospasm. Solubility problems with the compound which prevented the use of higher concentrations of nifedipine, may have also prevented upon aerosolization, sufficiently high tissue concentrations capable of blocking mediator release. This may be possible even in light of the hypotension observed upon aerosol delivery of the drug. Nifedipine may be capable of exerting effects on vascular smooth muscle at one concentration, while exerting action on respiratory smooth muscle at a higher concentration (D.J. Triggle, personal communication). Therefore, the higher activity concentration may not have been achieved due to a solubility problem incurred inside the animal. Nifedipine may exert some selectivity in terms of mediator blockade. This is probably related to different usage of calcium pools by different mediators. In the guinea pig, nifedipine provided protection against histamine-induced bronchoconstriction (Fanta et al., 1982); however, both nifedipine and verapamil were ineffective against bronchospasm evoked by LTD 4 (R.R. Osborn, personal communication). In addition, Weichman et al. (1983) reported that the intracellular calcium antagonist, TMB-8, was effective in blocking LTD4-induced tracheal contractions. Therefore, nifedipine, at the dosage employed, may be effective against only certain mediators employing a flux of calcium into the

cell. Also, since the dog is an animal with a predominant cholinergic system, the inhibitory action of nifedipine may have been overwhelmed by a possible recruitment of a cholinergic reflex pathway by the plethora of mediators released during antigen-induced bronchospasm. A component of the response to histamine may involve an indirect vagal-mediated reflex as well as possible direct mediator effects on bronchial smooth muscle (Malo et al., 1982b). As a result, nifedipine may be blocking only the direct mediator activity due to calcium regulation, but may fail to establish any major cholinergic action. Patel (1981b) reported in a single blind trial with exercise-induced asthmatics that inhaled verapamil was equally effective as sodium cromoglycate in preventing decreases in FEV1 0. These reported actions would tend to agree with the presently reported ability of verapamil to provide substantial protection to the larger airways by either mode of administration. However, it is interesting to note that verapamil, like nifedipine, failed to provide any protection on the C Dr N response when given by i.v. injection. The inability of either calcium channel blocker to effectively alter the response of the smaller airways to Ascaris antigen may be due to pathophysiology not sensitive to these calcium channel blocking drugs. The decreases in CDv N could have been the result of atelectasis secondary to severe bronchoconstriction and airway closure; however, neither functional residual capacity nor total lung capacity were recorded so this is not conclusively established. In addition, the decreases in CDvN may have been due to a reflex stimulation of the vagus nerves (Woolcock et al., 1969) in which case, both nifedipine and verapamil may have been ineffective. Finally, extensive collateral ventilation in the dog, may mask any action by nifedipine or verapamil in the sublobar segment of the lung. While Smith et al. (1979) reported that collateral channels and small airways have similar responses to hypocapnia and methacholine, it is not known to what extent collateral ventilation affects the response to antigen-induced bronchospasm nor to what degree this system is influenced by calcium channel blockers. Although there was some variability in the con-

75 t r o l r e s p o n s e to r e p e a t e d a n t i g e n c h a l l e n g e , the r e s p o n s e s o b s e r v e d to p e r i o d i c a n t i g e n c h a l l e n g e o v e r a n u m b e r o f m o n t h s was fairly c o n s i s t e n t w i t h the a n i m a l s u s e d in the p r e s e n t study. I n addition, whereas successive antigenic challenges m a y p r o d u c e d e c l i n i n g r e s p o n s e s as m e d i a t o r s bec o m e d e p l e t e d , this c o n d i t i o n a p p a r e n t l y was n o t m a n i f e s t e d in this g r o u p of a n i m a l s , b a s e d u p o n t h e h i s t o r i c a l r e c o r d o f s u c c e s s i v e m o n t h l y challenges. A s a result, the a n i m a l s s e r v e d as their o w n c o n t r o l s in w h i c h we c o m p a r e d the a c t i o n of the calcium channel blockers on Ascaris responses. In conclusion, our study demonstrated that n i f e d i p i n e a n d v e r a p a m i l are c a p a b l e o f p r o v i d i n g p r o t e c t i o n in an a n t i g e n - i n d u c e d b r o n c h o c o n s t r i c tion, d e p e n d i n g u p o n the r o u t e o f a d m i n i s t r a t i o n . In addition, there may be varying degrees of prot e c t i o n p r o v i d e d b y the c a l c i u m c h a n n e l b l o c k e r s d u e to s t r u c t u r a l d i f f e r e n c e s , as seen in the g r e a t e r a b i l i t y o f v e r a p a m i l to i n h i b i t a n t i g e n - i n d u c e d bronchospasm than nifedipine. Therefore, because o f the r e g u l a t o r y n a t u r e of c a l c i u m in a i r w a y s m o o t h m u s c l e a n d m a s t cell f u n c t i o n , c a l c i u m c h a n n e l a n t a g o n i s t s m a y h a v e a v a r i e d a n d useful r o l e in the t r e a t m e n t o f c e r t a i n t y p e s of a i r w a y diseases.

Acknowledgements We are grateful to Dr. W. Wierenga and Mr. B. Evans, Experimental Chemistry Research II, The Upjohn Company, for synthesizing the nifedipine usd in this study.

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asthma by a calcium antagonist (nifedipine), Eur. J. Clin. Invest. 10, 9. Diamond, L.0 1972. Potentiation of bronchomotor responses by beta adrenergic antagonists, J. Pharm. Exp. Ther. 181,434. Fanta, C.H., C.S. Venugopalan and J.M. Drazen, 1981, Nifedipine inhibits constriction of guinea pig tracheobronchial smooth muscle in vitro, Am. Rev. Respir. Dis. 123, 180. Fanta, C.H., C.S. Venugopalan, P.G. Lacouture and J.M. Drazen, 1982, Inhibition of bronchoconstriction in the guinea pig by a calcium channel blocker, nifedipine, Am. Rev. Respir. Dis. 125, 61. Guazzi, M., M.T. Olivari, A. Polese, C. Fiorentini, F. Magrini and P. Moruzzi, 1971, Nifedipine: a new antihypertensive with rapid action, Clin. Pharm. Ther. 22, 528. Guazzi, M., C. Fiorentini, M. Olivari, A. Bartorelli, G. Necchi and A. Polese, 1980, Short and long term efficacy of a calcium antagonistic agent (nifedipine) combined with methyldopa in the treatment of severe hypertension, Circulation 61, 913. Himori, N. and Taira, 1980, Differential effects of the calcium antagonistic vasodilators, nifedipine and verapamil on tracheal musculature and vasculature of the dog, Brit. J. Pharm. 68, 595. Kenmure, A.C.F. and J.H. Scruton, 1979, A double-blind controlled trial of the anti-anginal efficacy of nifedipine compared with propranolol, Brit. J. Clin. Prac. 33, 49. Malo, P.E., M.A. Wasserman and R.L. Griffin, 1982a, Effects of intravenous nifedipine on PGF2,, and histamine-induced bronchoconstriction in anesthetized dogs, J. Pharm. Exp. Ther. 221,410. Malo, P.E., M.A. Wasserman and R.L. Griffin, 1982b, The effects of lidocaine and hexamethonium on prostaglandin F2,~- and histamine-induced bronchoconstriction in normal and Ascaris sensitive dogs, Drug Dev. Res. 2, 567. Patel, K.R. 1981a, Calcium antagonists in exercise-induced asthma, Brit. Med. J. 282, 932. Patel, K.R., 1981b, The effect of calcium antagonist, nifedipine in exercise-induced asthma, Clin. Allergy 11,429. Smith, L.J., C.R. Inners, P.B. Terry, H.A. Menkes and R.J. Traystman. 1979, Effects of methacholine and hypocapnia on airways and collateral ventilation in dogs, J. Appl. Physiol. Respir. Environ. Exercise Physiol. 46, 966. Snedecor, G.W., 1956, Statistical Methods (Iowa State College Press, Ames) p. 122. Weichman, B.M., R.M. Muccitelli, S.S. Tucker and M.A. Wasserman, 1983, Effect of calcium antagonists on leukotriene D4-induced contractions of the guinea pig trachea and lung parenchyma, J. Pharmacol. Exp. Ther., in press. Weiss, E.B. and J. Markowicz, 1981, Inhibition of anaphylaxis in airways smooth muscle by the calcium channel drugs verapamil and nifedipine, Am. Rev. Respir. Dis. 123, 42. Woolcock, A.J., P.T. Macklem, J.C. Hogg, N.J. Wilson, T.A. Nadel, N.R. Frank and J. Brain, 1969, Effect of vagal stimulation on central and peripheral airways in dogs, J. Appl. Physiol. 269, 806.