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Effect of paf-acether on isoprenaline-induced relaxation in isolated tracheal segments of rats and guinea pigs
European Journal of Pharmacology, 158 (1988) 135-137
135
Elsevier
EJP 20250
Short communication
Effect of paf-acether on isoprenaline-induced relaxation in isolated tracheal segments of rats and guinea pigs N a r e s h C h a n d *, W i l l i a m D i a m a n t i s a n d R. D u a n e Sofia Department of Pharmacology, Wallace Laboratories, Division of Carter- Wallace, lnc., Cranbury, NJ 08512, U.S.A.
Received 27 September 1988, accepted 11 October 1988 Platelet activating factor (paf-acether) was found to contract rat and guinea pig tracheal segments. It does not cause down regulation of fl-adrenoceptor in guinea pig trachea. In addition, paf-contracted rat tracheal segments lose their ability to relax in response to isoprenaline. Platelet activating factor (PAF, PAF-acether); Contractile responses; Isoprenaline; fl-Adrenoceptors; Smooth muscle (airway); (Rat, Guinea-pig) 1. Introduction
It has been suggested that platelet activating factor (paf-acether) plays an important role in the pathophysiology of inflammation, airway hyperreactivity and asthma (Benveniste, 1987; Barnes, 1988; Lagente et al., 1988; Page, 1988). In addition, paf-acether had also been reported to produce down regulation of fl-adrenoceptors in the h u m a n lung and guinea pig trachea (Agarwal et al., 1987; Agarwal and Townley, 1987). The data obtained in the present study showed that pafacether in threshold concentrations does not produce any functional fl-adrenoceptor defect in isolated tracheal segments of rats and guinea pigs.
2. Materials and methods
Two to four thoracic tracheal segments (3-5 m m in width) were dissected from CO2-euthanized adult male Hartley strain guinea pigs and Sprague-Dawley rats. The segments were mounted
in isolated tissue baths containing Krebs-Henseleit solution aerated with 5% CO 2 plus 95% 02 and maintained at 37 o C. The tissues were equilibrated for 2-3 h under an optimal resting load of 2 g for guinea pig trachea and 1 g for rat trachea. One segment of each pair was exposed to a threshold (0.1 /~M) concentration of paf-acether (C16: Bachem, Inc., Torrence, CA, USA) producing 100200 mg tension for a period of 30 or 120 min; another segment of each pair served as vehicletreated control and then both the segments were submaximally contracted with carbachol (see tables 1 and 2). In additional experiments one tissue of each pair was contracted with carbachol and another with paf-acether. After an 120-min stabilization period of the spasmogen-induced contraction(s), cumulative concentration-effect curves (relaxation) for isoprenaline were established isometrically and analyzed by linear regression analysis. The ICs0 of isoprenaline was determined from the linear portion of the concentration effect curves and compared by paired t-test (Bliss, 1945). 3. Results
* To whom all correspondence should be addressed: Department of Pharmacology,Wallace Laboratories, P.O. Box 1001, Cranbury, NJ 08512, U.S.A.
The exposure of guinea pig tracheal segments to a threshold concentration of paf-acether for a period of 30 or 120 min did not exert any effect
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
136 TABLE 1 R e l a x a t i o n r e s p o n s e s to c u m u l a t i v e a d d i t i o n of i s o p r e n a l i n e in c a r b a c h o l - a n d p a f - a c e t h e r - c o n t r a c t e d i s o l a t e d g u i n e a p i g t r a c h e a l s e g m e n t s . V a l u e s are m e a n s + S.E.M. Spasmogen(s)
Bath conc. (v.M)
Duration of exposure (min)
Spasmogen-induced tone (mg)
N
1365±103
5
Isoprenahne IC50 ( n M ) (95% c o n f i d e n c e limits)
Maximum relaxation (mg)
7.5 (5.8-9.6) slope = 70; r = 0.97
2113_+336
5.4 (3.8-7.7) slope = 96; r = 0.97
2 296 + 343
3.6 (2.4-5.3) slope = 101; r = 0.95
1 6 9 5 ± 191
3.2 (2.5-4.0) slope = 101; r = 0.95
2 169 ± 194
Carbachol
0.2±0.03
30
Paf-acether b + carbachol
0.1 0.1 ± 0.01
30 30
575 ± 224 855 ± 188
5
Carbachol
0.1 +__0.02
30
1204_+ 62
8
Paf-acether b + carbachol
0.1 0.1 ± 0.03
120 30
223 ± 13 1 117 _+ 132
Carbachol
0.3 ± 0.2
120
1 9 0 4 ± 376
4
9.4 (4.5-20) slope = 53; r = 0.96
2 1 2 6 ± 283
Paf-acether
1.5±1.4
120
1987±431
4
4.1 (2.9-5.8) ~ slope = 103; r = 1.0
2582+271
a p < 0.05, IC5o of i s o p r e n a l i n e a c h i e v e d in s e g m e n t s c o n t r a c t e d w i t h c a r b a c h o l a n d p a f - a c e t h e r (Bliss m e t h o d ) , b Tissues e x p o s e d to t h r e s h o l d c o n c e n t r a t i o n of p a f - a c e t h e r (0.1 t t M ) for a p e r i o d of 30 o r 120 r a i n b e f o r e c o n t r a c t e d w i t h c a r b a c h o l .
compared to carbachol-contracted segments (P < 0.05) (table 1). The exposure of rat tracheal segments to threshold concentrations of paf-acether (0.1 ~M) for a period of 2 h was found to produce a
on isoprenaline concentration-effect curves in carbachol-contracted segments. However, the guinea pig tracheal segments contracted with pafacether alone exhibited a twofold leftward shift in the concentration-effect curve of isoprenaline as
TABLE 2 R e l a x a t i o n r e s p o n s e s to c u m u l a t i v e a d d i t i o n o f i s o p r e n a l i n e in c a r b a c h o l - a n d p a f - a c e t h e r - c o n t r a c t e d i s o l a t e d r a t t r a c h e a l s e g m e n t s . V a l u e s are m e a n s +_ S.E.M. Spasmogen(s)
Bath conc. (~M)
Duration of exposure (min)
Spasmogen-induced t o n e (mg)
N
1279+
74
7
Isoprenaline ICs0 ( n M ) (95% c o n f i d e n c e limits)
Maximum relaxation (mg)
12(7.7-19) slope = 33; r = 0.98
1139+144
4.7 (3.1-7.1) a s l o p e = 31; r = 0.98
1200 +
1 2 8 6 + 190
Carbachol
0.3+0.1
Paf-acether b + carbachol
0.1 0.2 + 0 . 3
120 30
159 + 108 1 148 + 68
7
Carbachol
0.1 + 0.01
120
1 283 _+ 166
5
6.9 (5.6-8.5) s l o p e = 43; r = 0.98
Paf-acether
5.1 + 2.2
120 .
1 0 7 6 + 91
5
N o effect
48 ±
71
27
a p < 0.05 i n d i c a t e s s i g n i f i c a n c e d e t e r m i n e d b y Bliss m e t h o d , b Tissues e x p o s e d to t h r e s h o l d c o n c e n t r a t i o n o f p a f - a c e t h e r (0.1 # M ) before contracted with carbachol.
137
twofold leftward shift in the concentration-effect (relaxation) curve of isoprenaline in carbacholcontracted tissues (table 2). However, the pafacether (5.1 ___2.2 #M, induced tone = 1076 + 91 mg; 2 h stabilization period)-contracted rat tracheal segments failed to relax in response to cumulative addition of isoprenaline. The second segment of each pair contracted with carbachol resulted in the expected isoprenaline concentration-effect curve (table 2).
4. Discussion In this study paf-acether produced strong contractile responses in guinea pig and rat tracheal segments. In sharp contrast to these findings, earlier Prancan et al. (1982) reported a concentration-dependent relaxation to paf-acether in guinea pig tracheal spirals. Although we do not have exact explanation for such a difference, factors such as the source, type or synthesis of paf-acether (C16 or C~8), solvent (ethanol/distilled water), s p i r a l / s e g m e n t could be responsible (Prancan et al., 1982, this study). Rat trachea once contracted with paf-acether did not relax even to repeated washings for 30-60 min. The data obtained in this study do not support the earlier reported fl-adrenoceptor functional defect induced by subthreshold/threshold concentrations of paf-acether in guinea pig trachea (Agarwal et al., 1987). The physiological significance of a two to threefold rightward (Agarwal et al., 1987) or leftward (this study) shift in concentration-effect curve of isoprenaline in the presence of paf-acether remains to be established. The rat tracheal segments pre-exposed to threshold concentrations of paf-acether and subsequently contracted with carbachol also exhibited a twofold leftward shift in the isoprenaline concentration-effect curve compared to the tissues which were contracted with carbachol alone. Interestingly, in contrast to guinea pig trachea, the rat tracheal segments which were contracted with
paf-acether alone failed to relax in response to isoprenaline. The species related differences in the ability of isoprenaline to relax paf-contracted tracheal segments may be explained by different mechanism(s) of Ca 2+ utilization and translocation in response to paf-acether in airway smooth muscles. In conclusion, the data obtained in this study showed that (i) paf-acether is capable of contracting isolated rat and guinea pig tracheal segments and (ii) the exposure of rat and guinea pig trachea to threshold concentrations of paf-acether does not produce any down regulation of fl-adrenoceptors.
Acknowledgements The authors thank Mr. Thomas P. Mahoney for technical assistance and Mrs. Janet Rittman for secretarial assistance.
References Agarwal, K. and R.G. Townley, 1987, Effect of platelet-activating factor on beta-adrenoceptors in human lung, Biochem. Biophys. Res. Commun. 143, 1. Agarwal, D.K., P. Byorth and R.G. Townley, 1987, Effect of platelet-activating factor (PAF) on isoproterenol-induced relaxation in the airways, J. Allergy Clin. Immunol. 79, 171. Barnes, P.J., 1988, Platelet-activating factor and asthma, J. Allergy Clin. Immunol. 81, 152. Benveniste, J., 1987, A role of paf-acether (platelet-activating factor) in bronchial hyperreactivity?, Am. Rev. Resp. Dis. 135, $2. Bliss, C.I., 1945, Confidence limits for biological Assays, Biometrics 1, 57. Lagenty, V., M. Cirino, S. Desquand, J. Lefort and B.B. Vargaftig, 1988, Involvement of paf-acether in anaphylactic bronchoconstriction induced in guinea pigs by aerosolized antigen, Int. Arch. Allergy Appl. Immunol. 85, 14. Page, C.P., 1988, The role of platelet-activating factor in asthma, J. Allergy Clin. Immunol. 81, 144. Prancan, A., J. Lefort, M. Barton and B.B. Vargaftig, 1982, Relaxation of the guinea pig trachea induced by plateletactivating factor and by serotonin, European J. Pharmacol. 80, 29.
135
Elsevier
EJP 20250
Short communication
Effect of paf-acether on isoprenaline-induced relaxation in isolated tracheal segments of rats and guinea pigs N a r e s h C h a n d *, W i l l i a m D i a m a n t i s a n d R. D u a n e Sofia Department of Pharmacology, Wallace Laboratories, Division of Carter- Wallace, lnc., Cranbury, NJ 08512, U.S.A.
Received 27 September 1988, accepted 11 October 1988 Platelet activating factor (paf-acether) was found to contract rat and guinea pig tracheal segments. It does not cause down regulation of fl-adrenoceptor in guinea pig trachea. In addition, paf-contracted rat tracheal segments lose their ability to relax in response to isoprenaline. Platelet activating factor (PAF, PAF-acether); Contractile responses; Isoprenaline; fl-Adrenoceptors; Smooth muscle (airway); (Rat, Guinea-pig) 1. Introduction
It has been suggested that platelet activating factor (paf-acether) plays an important role in the pathophysiology of inflammation, airway hyperreactivity and asthma (Benveniste, 1987; Barnes, 1988; Lagente et al., 1988; Page, 1988). In addition, paf-acether had also been reported to produce down regulation of fl-adrenoceptors in the h u m a n lung and guinea pig trachea (Agarwal et al., 1987; Agarwal and Townley, 1987). The data obtained in the present study showed that pafacether in threshold concentrations does not produce any functional fl-adrenoceptor defect in isolated tracheal segments of rats and guinea pigs.
2. Materials and methods
Two to four thoracic tracheal segments (3-5 m m in width) were dissected from CO2-euthanized adult male Hartley strain guinea pigs and Sprague-Dawley rats. The segments were mounted
in isolated tissue baths containing Krebs-Henseleit solution aerated with 5% CO 2 plus 95% 02 and maintained at 37 o C. The tissues were equilibrated for 2-3 h under an optimal resting load of 2 g for guinea pig trachea and 1 g for rat trachea. One segment of each pair was exposed to a threshold (0.1 /~M) concentration of paf-acether (C16: Bachem, Inc., Torrence, CA, USA) producing 100200 mg tension for a period of 30 or 120 min; another segment of each pair served as vehicletreated control and then both the segments were submaximally contracted with carbachol (see tables 1 and 2). In additional experiments one tissue of each pair was contracted with carbachol and another with paf-acether. After an 120-min stabilization period of the spasmogen-induced contraction(s), cumulative concentration-effect curves (relaxation) for isoprenaline were established isometrically and analyzed by linear regression analysis. The ICs0 of isoprenaline was determined from the linear portion of the concentration effect curves and compared by paired t-test (Bliss, 1945). 3. Results
* To whom all correspondence should be addressed: Department of Pharmacology,Wallace Laboratories, P.O. Box 1001, Cranbury, NJ 08512, U.S.A.
The exposure of guinea pig tracheal segments to a threshold concentration of paf-acether for a period of 30 or 120 min did not exert any effect
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
136 TABLE 1 R e l a x a t i o n r e s p o n s e s to c u m u l a t i v e a d d i t i o n of i s o p r e n a l i n e in c a r b a c h o l - a n d p a f - a c e t h e r - c o n t r a c t e d i s o l a t e d g u i n e a p i g t r a c h e a l s e g m e n t s . V a l u e s are m e a n s + S.E.M. Spasmogen(s)
Bath conc. (v.M)
Duration of exposure (min)
Spasmogen-induced tone (mg)
N
1365±103
5
Isoprenahne IC50 ( n M ) (95% c o n f i d e n c e limits)
Maximum relaxation (mg)
7.5 (5.8-9.6) slope = 70; r = 0.97
2113_+336
5.4 (3.8-7.7) slope = 96; r = 0.97
2 296 + 343
3.6 (2.4-5.3) slope = 101; r = 0.95
1 6 9 5 ± 191
3.2 (2.5-4.0) slope = 101; r = 0.95
2 169 ± 194
Carbachol
0.2±0.03
30
Paf-acether b + carbachol
0.1 0.1 ± 0.01
30 30
575 ± 224 855 ± 188
5
Carbachol
0.1 +__0.02
30
1204_+ 62
8
Paf-acether b + carbachol
0.1 0.1 ± 0.03
120 30
223 ± 13 1 117 _+ 132
Carbachol
0.3 ± 0.2
120
1 9 0 4 ± 376
4
9.4 (4.5-20) slope = 53; r = 0.96
2 1 2 6 ± 283
Paf-acether
1.5±1.4
120
1987±431
4
4.1 (2.9-5.8) ~ slope = 103; r = 1.0
2582+271
a p < 0.05, IC5o of i s o p r e n a l i n e a c h i e v e d in s e g m e n t s c o n t r a c t e d w i t h c a r b a c h o l a n d p a f - a c e t h e r (Bliss m e t h o d ) , b Tissues e x p o s e d to t h r e s h o l d c o n c e n t r a t i o n of p a f - a c e t h e r (0.1 t t M ) for a p e r i o d of 30 o r 120 r a i n b e f o r e c o n t r a c t e d w i t h c a r b a c h o l .
compared to carbachol-contracted segments (P < 0.05) (table 1). The exposure of rat tracheal segments to threshold concentrations of paf-acether (0.1 ~M) for a period of 2 h was found to produce a
on isoprenaline concentration-effect curves in carbachol-contracted segments. However, the guinea pig tracheal segments contracted with pafacether alone exhibited a twofold leftward shift in the concentration-effect curve of isoprenaline as
TABLE 2 R e l a x a t i o n r e s p o n s e s to c u m u l a t i v e a d d i t i o n o f i s o p r e n a l i n e in c a r b a c h o l - a n d p a f - a c e t h e r - c o n t r a c t e d i s o l a t e d r a t t r a c h e a l s e g m e n t s . V a l u e s are m e a n s +_ S.E.M. Spasmogen(s)
Bath conc. (~M)
Duration of exposure (min)
Spasmogen-induced t o n e (mg)
N
1279+
74
7
Isoprenaline ICs0 ( n M ) (95% c o n f i d e n c e limits)
Maximum relaxation (mg)
12(7.7-19) slope = 33; r = 0.98
1139+144
4.7 (3.1-7.1) a s l o p e = 31; r = 0.98
1200 +
1 2 8 6 + 190
Carbachol
0.3+0.1
Paf-acether b + carbachol
0.1 0.2 + 0 . 3
120 30
159 + 108 1 148 + 68
7
Carbachol
0.1 + 0.01
120
1 283 _+ 166
5
6.9 (5.6-8.5) s l o p e = 43; r = 0.98
Paf-acether
5.1 + 2.2
120 .
1 0 7 6 + 91
5
N o effect
48 ±
71
27
a p < 0.05 i n d i c a t e s s i g n i f i c a n c e d e t e r m i n e d b y Bliss m e t h o d , b Tissues e x p o s e d to t h r e s h o l d c o n c e n t r a t i o n o f p a f - a c e t h e r (0.1 # M ) before contracted with carbachol.
137
twofold leftward shift in the concentration-effect (relaxation) curve of isoprenaline in carbacholcontracted tissues (table 2). However, the pafacether (5.1 ___2.2 #M, induced tone = 1076 + 91 mg; 2 h stabilization period)-contracted rat tracheal segments failed to relax in response to cumulative addition of isoprenaline. The second segment of each pair contracted with carbachol resulted in the expected isoprenaline concentration-effect curve (table 2).
4. Discussion In this study paf-acether produced strong contractile responses in guinea pig and rat tracheal segments. In sharp contrast to these findings, earlier Prancan et al. (1982) reported a concentration-dependent relaxation to paf-acether in guinea pig tracheal spirals. Although we do not have exact explanation for such a difference, factors such as the source, type or synthesis of paf-acether (C16 or C~8), solvent (ethanol/distilled water), s p i r a l / s e g m e n t could be responsible (Prancan et al., 1982, this study). Rat trachea once contracted with paf-acether did not relax even to repeated washings for 30-60 min. The data obtained in this study do not support the earlier reported fl-adrenoceptor functional defect induced by subthreshold/threshold concentrations of paf-acether in guinea pig trachea (Agarwal et al., 1987). The physiological significance of a two to threefold rightward (Agarwal et al., 1987) or leftward (this study) shift in concentration-effect curve of isoprenaline in the presence of paf-acether remains to be established. The rat tracheal segments pre-exposed to threshold concentrations of paf-acether and subsequently contracted with carbachol also exhibited a twofold leftward shift in the isoprenaline concentration-effect curve compared to the tissues which were contracted with carbachol alone. Interestingly, in contrast to guinea pig trachea, the rat tracheal segments which were contracted with
paf-acether alone failed to relax in response to isoprenaline. The species related differences in the ability of isoprenaline to relax paf-contracted tracheal segments may be explained by different mechanism(s) of Ca 2+ utilization and translocation in response to paf-acether in airway smooth muscles. In conclusion, the data obtained in this study showed that (i) paf-acether is capable of contracting isolated rat and guinea pig tracheal segments and (ii) the exposure of rat and guinea pig trachea to threshold concentrations of paf-acether does not produce any down regulation of fl-adrenoceptors.
Acknowledgements The authors thank Mr. Thomas P. Mahoney for technical assistance and Mrs. Janet Rittman for secretarial assistance.
References Agarwal, K. and R.G. Townley, 1987, Effect of platelet-activating factor on beta-adrenoceptors in human lung, Biochem. Biophys. Res. Commun. 143, 1. Agarwal, D.K., P. Byorth and R.G. Townley, 1987, Effect of platelet-activating factor (PAF) on isoproterenol-induced relaxation in the airways, J. Allergy Clin. Immunol. 79, 171. Barnes, P.J., 1988, Platelet-activating factor and asthma, J. Allergy Clin. Immunol. 81, 152. Benveniste, J., 1987, A role of paf-acether (platelet-activating factor) in bronchial hyperreactivity?, Am. Rev. Resp. Dis. 135, $2. Bliss, C.I., 1945, Confidence limits for biological Assays, Biometrics 1, 57. Lagenty, V., M. Cirino, S. Desquand, J. Lefort and B.B. Vargaftig, 1988, Involvement of paf-acether in anaphylactic bronchoconstriction induced in guinea pigs by aerosolized antigen, Int. Arch. Allergy Appl. Immunol. 85, 14. Page, C.P., 1988, The role of platelet-activating factor in asthma, J. Allergy Clin. Immunol. 81, 144. Prancan, A., J. Lefort, M. Barton and B.B. Vargaftig, 1982, Relaxation of the guinea pig trachea induced by plateletactivating factor and by serotonin, European J. Pharmacol. 80, 29.