Pharmacological Research, Vol. 22, No . 1, 1990
49
BETA-ADRENOCEPTOR REACTIVITY AFTER EPITHELIUM REMOVAL IN GUINEA-PIG TRACHEA
IN VITRO LUISA DAFFONCHIO, ALICIA HERNANDEZ, ENRICA MARTINOTTI* and CLAUDIO OMINI* Institute of Pharmacological Sciences, University of Milan, Italy, *Institute of Biological Sciences, University of Pisa, Italy Received in final form 30 May 1989
SUMMARY In guinea-pig isolated tracheae precontracted with pilocarpine (2X10 -1 m) mechanical removal of the epithelium did not significantly modify the degree of relaxation induced by three different adrenergic agonists : epinephrine (adrenaline), isoproterenol (isoprenaline) and salbutamol . The failure of epithelium removal to modify isoproterenol relaxant activity was observed in both spontaneous tone and pilocarpine precontracted tracheae . In tracheae obtained from actively sensitized (ovalbumin) guinea-pigs, log-concentration response curves of epinephrine and salbutamol were unchanged by epithelium damage, whereas that of isoproterenol was slightly shifted to the left . In ovalbumin sensitized tracheae exposed to the antigen (ovalbumin, 50 ug/ml) epithelium removal enhanced the relaxant activity of the three adrenergic agonists used . Beta-adrenoceptor desensitization (isoproterenol, 10 - I m for 20 min twice) carried out in ovalbumin actively sensitized tracheae with or without epithelium, shifted the log-concentration response curves of isoproterenol to a similar rightward position . The present data suggest that the airway epithelium plays a minor role in the regulation of guinea-pig tracheal sensitivity to beta-adrenoceptor agonists, which is evident only in a classical model of experimental asthma . KEY
woaDs : epithelium, beta-adrenoceptors, trachea . INTRODUCTION
There is growing evidence that airway epithelium might generate substances able to modulate airway smooth muscle tone; therefore reduced release of such substances, due to different causes, has been suggested to contribute to the airway hyperreactivity observed in asthma [ 1-4] . In this regard, several investigators have already demonstrated that the mechanical removal of the epithelium in guinea-pig Correspondence to Dr . Luisa Daffonchio, Institute of Pharmacological Sciences, via Balzaretti 9, 20133 Milan, Italy. 1043-6618/90/010049-10/S03 .00/0
© 1990 The Italian Pharmacological Society
50
Pharmacological Research, Vol. 22, No . 1, 1990
trachea increases the responsiveness of the tissue to histamine, metacholine and particularly to the specific antigen in ovalbumin sensitized animals [5-7] . As far as beta-adrenoceptor reactivity is concerned, the available results on the possible influence of the epithelium are contradictory . In fact Farmer et al. [8], Hay et al. [5] and Holroyde [6] have shown that sensitivity to isoproterenol was increased in epithelium denuded guinea-pig trachea . On the other hand, Goldie et al. [7] and Flavahan et al. [9] did not find any difference or even a decrease in tissue sensitivity to isoproterenol in isolated tracheae without the epithelium . More recently, Lundblad and Persson [10] showed that epithelium removal shifted isoproterenol concentration response curves to the left, while terbutaline induced relaxation was reduced . Impaired beta-adrenoceptor function has been suggested to play an important role in asthma [11]. The above-mentioned studies have been performed using mainly isoproterenol as the beta-agonist . However, if the interaction between the epithelium and the adrenergic system is to have pathophysiological significance, it must operate with epinephrine, a physiological adrenergic agonist . Therefore, we investigated the changes in the relaxant activity of this compound after epithelium removal, in parallel with more selective beta-agonists . In particular, the influence of the epithelium has been evaluated in guinea-pig trachea in vitro under different experimental conditions, including the most used model of experimental asthma, i.e. the ovalbumin actively sensitized guinea-pig.
METHODS General
Male guinea-pigs (Dunkin Hartley, 350-400 g) were sacrificed by cervical dislocation and exsanguinated . The trachea was isolated, dissected free from connective tissues and divided in two equal segments randomly assigned to control (+E) or epithelium-denuded (-E) group respectively. The tracheal tubes were then opened longitudinally and cut into zig-zag strips [12] ; the epithelium was removed by gently rubbing the luminal surface with a cotton-tipped applicator . At the end of each experiment the tracheae were stained with Evans Blue in order to evaluate macroscopically the presence or absence of the epithelium . Some preparations were randomly submitted to histological examination as described below. Functional studies
Tissues were placed in 10 ml conventional organ bath containing Krebsbicarbonate solution oxygenated (0 2/CO,, 95/5%) and maintained at 37°C . A load of 1 . 2 g was applied to the tracheae and changes in the length of the preparations were measured by isotonic transducers (Basile 7006) connected to a Basile (Gemini 7070) pen recorder . After a 60 min equilibration period, tissues were maximally contracted with pilocarpine (2 X 10- 5 M) and when a stable plateau was reached (about 45 min) concentration response curves for epinephrine, isoproterenol and salbutamol were obtained by cumulative addition of the drugs . In
Pharmacological Research, Vol. 22, No. 1 , 1990
51
some experiments, isoproterenol concentration response curves were constructed in tracheae under spontaneous tone. The influence of epithelium removal on the relaxant action of the different betaagonists was also evaluated in ovalbumin sensitized guinea-pig tracheae contracted with pilocarpine . Animals were actively sensitized to the antigen by both intraperitoneal and subcutaneous injection of 100 mg/kg of ovalbumin and used 18-28 days later. In some experiments + E and - E tracheae taken from sensitized animals were challenged in vitro with the specific antigen (ovalbumin, 50 µg/ml) 1 h before exposure to pilocarpine . The effectiveness of ovalbumin sensitization was verified administering the antigen (ovalbumin, 50 ug/ml) to lung parenchymal strips taken from the same animals in the experiments where antigen challenge was not required directly on the trachea. Tissues whose tracheal or lung parenchymal preparation did not respond with a sustained contraction were eliminated from the study. The beta-adrenoceptor desensitization procedure was carried out in + E and - E tracheae taken from ovalbumin actively sensitized animals administering isoproterenol at 10 -6 M for 20 min twice . The relaxant activity of isoproterenol was subsequently evaluated when the pre-existing contraction was restored, as previously described [13] . Histological examination
Histological examination was carried out on + E and - E tracheal segments fixed with formalin. Transverse paraffin embedded sections were cut (4 µm) and stained with haemalum and eosin . Samples were then examined microscopically for the presence of the epithelium . Statistical analysis
Data were expressed as percentage of the maximal relaxation obtained with each agonist. Differences between log-concentration response curves were statistically evaluated by means of a computer aided program according to the variance analysis for comparison of regression lines [14]. This procedure implies the assessment of parallelism and therefore enables calculation of the dose ratio (DR) values and its 95% confidence limits (CL) . Drugs
The following drugs, all purchased from Sigma, were used : pilocarpine hydrochloride, (- )-isoproterenol hydrochloride, (-)-epinephrine (+ )-bitartrate, salbutamol hemisulfate, ovalbumin grade V .
RESULTS Rubbing the luminal surface of the trachea resulted in a complete removal of the epithelial layer without any sign of damage to the underlying tissues as shown in Fig . 1 . However, mechanical removal of guinea-pig tracheal epithelium did not
52
Pharmacological Research, Vol. 22, No. 1, 1990
Fig. 1 . Transverse paraffin embedded sections (4µm) of guinea-pig tracheal segments stained with haemalum and eosin . ( X 1 5). A, epithelium (E) intact ; B, epithelium rubbed.
modify the degree of relaxation of the beta-adrenergic agonists used (Fig. 2) . In fact, the log-concentration response curves for epinephrine, isoproterenol and salbutamol performed in -E, pharmacologically precontracted (pilocarpine 2 X 10-s M) guinea-pig tracheae were not significantly different from that obtained in + E tissues. In addition, the maximal relaxations obtained with the beta-agonists were not influenced by the removal of the epithelium (data not shown) . Since the pharmacologically elevated tone in the trachea might have modified the involvement of the epithelium in the relaxation induced by the beta-agonists, we examined the relaxant activity of isoproterenol in + E and - E guinea-pig tracheae under spontaneous tone . In this experimental condition, too, no difference was observed between the log-concentration response curves for isoproterenol in rubbed and control tissues (Fig . 3) . In preliminary experiments we measured the isoproterenol sensitivity of distal and proximal segments of the trachea . As previously described [15], no difference was found between the two preparations . Moreover, mechanical removal of the epithelium did not induce any change in isoproterenol sensitivity either in proximal or in distal segments. The DR values calculated between + E and - E tissues were 1 .01 (95% CL 0 . 77-1 . 34) and 1 . 11 (95% CL 0 . 86-1 .43) in proximal and distal preparations, respectively. In the tracheae obtained from actively sensitized guinea-pigs, mechanical damage of the epithelium did not modify the relaxant activity of epinephrine and
Pharmacological Research, Vol. 22, No. 1, 1990
53
M Control 0 Epithelium rubbed 100-
100-
9
8
7
100-
Fig. 2 . Log-concentration response curves for epinephrine, isoproterenol and salbutamol in control and epithelium rubbed tracheae taken from normal guinea-pigs . Each point represents the mean ± SEM from at least 5 replications. DR = dose ratio ; in parenthesis 95% confidence limits .
Pharmacological Research, Vol. 22, No. 1, 1990
54 El Control
o
Epithelium rubbed
100-
-Log molar concentration
Fig. 3 . Log-concentration response curves for isoproterenol in control and epithelium rubbed tracheae under resting tone . Each point represents the mean ± SEM from 5 replications. DR dose ratio ; in parenthesis 95% confidence limits .
salbutamol (Fig . 4) . However, as far as isoproterenol was concerned, the loss of epithelium slightly but significantly shifted to the left its log-concentration response curves, with a calculated DR of 1 . 49 (95% CL 1 . 06-2 . 08; Fig . 4). On the other hand, after the anaphylactic challenge (ovalbumin, 50,ug/kg) in tracheae obtained from actively sensitized guinea-pigs, a statistically significant shift to the left of the log-concentration response curves for all three adrenergic compounds tested was observed in - E as compared to + E tissues . In particular, the DR values calculated for epinephrine, isoproterenol and salbutamol were : 1 . 75 (95% CL 1 . 28-2 . 39), 1 . 61 (95% CL 1 . 04-2 . 48) and 1 . 58 (95% CL 1 . 04-2 . 42) respectively (Fig. 5) . The beta-adrenoceptor desensitization procedure (isoproterenol, 10 -6 M for 20 min twice) significantly shifted to the right isoproterenol log-concentration response curves in both + E and - E tracheae . Moreover, results obtained indicated that epithelium removal did not modify the development of betaadrenoceptor desensitization . In fact, as shown in Fig . 6, isoproterenol logconcentration response curves constructed in desensitized tissues were comparable regardless of the presence or absence of the epithelium .
DISCUSSION The possible existence of an epithelium-derived relaxing factor which might modulate airway tone is suggestive, even if a conclusive demonstration has not yet been found [1, 10] . As far as the adrenergic activity is concerned, our data do not allow us to propose a major influence of the epithelium on the activity of the physiological adrenergic mediator . In fact, we could not detect any difference in the relaxant action of epinephrine in normal rubbed tracheae as compared to controls . The lack of influence of the epithelium on salbutamol induced relaxation is in
Pharmacological Research, Vol. 22, No . 1, 1990 M Control
O Control
O Epithelium rubbed
O Epithelium rubbed
100-
too 80 60 -
40 20 -
O• 7
8
6
100-
100-
8
7
8
7
100-
8
7
-Log molar concentration
- Log molar concentration
Fig . 4 (left). Log-concentration response curves for epinephrine, isoproterenol and salbutamol in control and epithelium rubbed tracheae taken from actively sensitized guinea-pigs . Each point represents the mean ±srs from 5 replications . DR = dose ratio : in parenthesis 95"4, confidence limits . ::"/'< 0 . 11 1, as compared to control . Fig . 5 (right) . Log-concentration response curves for epinephrine, isoproterenol and salbutamol in control and epithelium rubbed tracheae taken from actively sensitized guinea-pigs and challenged with the specific antigen . Each point represents the mean±sign from 5 replications . DR=dose ratio : in parenthesis 95% confidence limits . *P< 0 . 05, * *P< 0 . 01, as compared to control .
56
Pharmacological Research, Vol. 22, No. 1, 1990 o
Control
O Epithelium rubbed ∎ Desensitized
• Epithelium rubbed desensitized
- Log molar concentration
Fig . 6. Log-concentration response curves for isoproterenol in control and beta-adrenoceptor desensitized tracheae with and without the epithelium . Tissues were obtained from ovalbumin actively sensitized guinea-pigs . Each point represents the mean ± SEM from at least 3 replications . Dose ratio values (95% confidence limits) were: control versus epithelium rubbed : 1 . 51 (1 . 15-1 . 99), P
49
BETA-ADRENOCEPTOR REACTIVITY AFTER EPITHELIUM REMOVAL IN GUINEA-PIG TRACHEA
IN VITRO LUISA DAFFONCHIO, ALICIA HERNANDEZ, ENRICA MARTINOTTI* and CLAUDIO OMINI* Institute of Pharmacological Sciences, University of Milan, Italy, *Institute of Biological Sciences, University of Pisa, Italy Received in final form 30 May 1989
SUMMARY In guinea-pig isolated tracheae precontracted with pilocarpine (2X10 -1 m) mechanical removal of the epithelium did not significantly modify the degree of relaxation induced by three different adrenergic agonists : epinephrine (adrenaline), isoproterenol (isoprenaline) and salbutamol . The failure of epithelium removal to modify isoproterenol relaxant activity was observed in both spontaneous tone and pilocarpine precontracted tracheae . In tracheae obtained from actively sensitized (ovalbumin) guinea-pigs, log-concentration response curves of epinephrine and salbutamol were unchanged by epithelium damage, whereas that of isoproterenol was slightly shifted to the left . In ovalbumin sensitized tracheae exposed to the antigen (ovalbumin, 50 ug/ml) epithelium removal enhanced the relaxant activity of the three adrenergic agonists used . Beta-adrenoceptor desensitization (isoproterenol, 10 - I m for 20 min twice) carried out in ovalbumin actively sensitized tracheae with or without epithelium, shifted the log-concentration response curves of isoproterenol to a similar rightward position . The present data suggest that the airway epithelium plays a minor role in the regulation of guinea-pig tracheal sensitivity to beta-adrenoceptor agonists, which is evident only in a classical model of experimental asthma . KEY
woaDs : epithelium, beta-adrenoceptors, trachea . INTRODUCTION
There is growing evidence that airway epithelium might generate substances able to modulate airway smooth muscle tone; therefore reduced release of such substances, due to different causes, has been suggested to contribute to the airway hyperreactivity observed in asthma [ 1-4] . In this regard, several investigators have already demonstrated that the mechanical removal of the epithelium in guinea-pig Correspondence to Dr . Luisa Daffonchio, Institute of Pharmacological Sciences, via Balzaretti 9, 20133 Milan, Italy. 1043-6618/90/010049-10/S03 .00/0
© 1990 The Italian Pharmacological Society
50
Pharmacological Research, Vol. 22, No . 1, 1990
trachea increases the responsiveness of the tissue to histamine, metacholine and particularly to the specific antigen in ovalbumin sensitized animals [5-7] . As far as beta-adrenoceptor reactivity is concerned, the available results on the possible influence of the epithelium are contradictory . In fact Farmer et al. [8], Hay et al. [5] and Holroyde [6] have shown that sensitivity to isoproterenol was increased in epithelium denuded guinea-pig trachea . On the other hand, Goldie et al. [7] and Flavahan et al. [9] did not find any difference or even a decrease in tissue sensitivity to isoproterenol in isolated tracheae without the epithelium . More recently, Lundblad and Persson [10] showed that epithelium removal shifted isoproterenol concentration response curves to the left, while terbutaline induced relaxation was reduced . Impaired beta-adrenoceptor function has been suggested to play an important role in asthma [11]. The above-mentioned studies have been performed using mainly isoproterenol as the beta-agonist . However, if the interaction between the epithelium and the adrenergic system is to have pathophysiological significance, it must operate with epinephrine, a physiological adrenergic agonist . Therefore, we investigated the changes in the relaxant activity of this compound after epithelium removal, in parallel with more selective beta-agonists . In particular, the influence of the epithelium has been evaluated in guinea-pig trachea in vitro under different experimental conditions, including the most used model of experimental asthma, i.e. the ovalbumin actively sensitized guinea-pig.
METHODS General
Male guinea-pigs (Dunkin Hartley, 350-400 g) were sacrificed by cervical dislocation and exsanguinated . The trachea was isolated, dissected free from connective tissues and divided in two equal segments randomly assigned to control (+E) or epithelium-denuded (-E) group respectively. The tracheal tubes were then opened longitudinally and cut into zig-zag strips [12] ; the epithelium was removed by gently rubbing the luminal surface with a cotton-tipped applicator . At the end of each experiment the tracheae were stained with Evans Blue in order to evaluate macroscopically the presence or absence of the epithelium . Some preparations were randomly submitted to histological examination as described below. Functional studies
Tissues were placed in 10 ml conventional organ bath containing Krebsbicarbonate solution oxygenated (0 2/CO,, 95/5%) and maintained at 37°C . A load of 1 . 2 g was applied to the tracheae and changes in the length of the preparations were measured by isotonic transducers (Basile 7006) connected to a Basile (Gemini 7070) pen recorder . After a 60 min equilibration period, tissues were maximally contracted with pilocarpine (2 X 10- 5 M) and when a stable plateau was reached (about 45 min) concentration response curves for epinephrine, isoproterenol and salbutamol were obtained by cumulative addition of the drugs . In
Pharmacological Research, Vol. 22, No. 1 , 1990
51
some experiments, isoproterenol concentration response curves were constructed in tracheae under spontaneous tone. The influence of epithelium removal on the relaxant action of the different betaagonists was also evaluated in ovalbumin sensitized guinea-pig tracheae contracted with pilocarpine . Animals were actively sensitized to the antigen by both intraperitoneal and subcutaneous injection of 100 mg/kg of ovalbumin and used 18-28 days later. In some experiments + E and - E tracheae taken from sensitized animals were challenged in vitro with the specific antigen (ovalbumin, 50 µg/ml) 1 h before exposure to pilocarpine . The effectiveness of ovalbumin sensitization was verified administering the antigen (ovalbumin, 50 ug/ml) to lung parenchymal strips taken from the same animals in the experiments where antigen challenge was not required directly on the trachea. Tissues whose tracheal or lung parenchymal preparation did not respond with a sustained contraction were eliminated from the study. The beta-adrenoceptor desensitization procedure was carried out in + E and - E tracheae taken from ovalbumin actively sensitized animals administering isoproterenol at 10 -6 M for 20 min twice . The relaxant activity of isoproterenol was subsequently evaluated when the pre-existing contraction was restored, as previously described [13] . Histological examination
Histological examination was carried out on + E and - E tracheal segments fixed with formalin. Transverse paraffin embedded sections were cut (4 µm) and stained with haemalum and eosin . Samples were then examined microscopically for the presence of the epithelium . Statistical analysis
Data were expressed as percentage of the maximal relaxation obtained with each agonist. Differences between log-concentration response curves were statistically evaluated by means of a computer aided program according to the variance analysis for comparison of regression lines [14]. This procedure implies the assessment of parallelism and therefore enables calculation of the dose ratio (DR) values and its 95% confidence limits (CL) . Drugs
The following drugs, all purchased from Sigma, were used : pilocarpine hydrochloride, (- )-isoproterenol hydrochloride, (-)-epinephrine (+ )-bitartrate, salbutamol hemisulfate, ovalbumin grade V .
RESULTS Rubbing the luminal surface of the trachea resulted in a complete removal of the epithelial layer without any sign of damage to the underlying tissues as shown in Fig . 1 . However, mechanical removal of guinea-pig tracheal epithelium did not
52
Pharmacological Research, Vol. 22, No. 1, 1990
Fig. 1 . Transverse paraffin embedded sections (4µm) of guinea-pig tracheal segments stained with haemalum and eosin . ( X 1 5). A, epithelium (E) intact ; B, epithelium rubbed.
modify the degree of relaxation of the beta-adrenergic agonists used (Fig. 2) . In fact, the log-concentration response curves for epinephrine, isoproterenol and salbutamol performed in -E, pharmacologically precontracted (pilocarpine 2 X 10-s M) guinea-pig tracheae were not significantly different from that obtained in + E tissues. In addition, the maximal relaxations obtained with the beta-agonists were not influenced by the removal of the epithelium (data not shown) . Since the pharmacologically elevated tone in the trachea might have modified the involvement of the epithelium in the relaxation induced by the beta-agonists, we examined the relaxant activity of isoproterenol in + E and - E guinea-pig tracheae under spontaneous tone . In this experimental condition, too, no difference was observed between the log-concentration response curves for isoproterenol in rubbed and control tissues (Fig . 3) . In preliminary experiments we measured the isoproterenol sensitivity of distal and proximal segments of the trachea . As previously described [15], no difference was found between the two preparations . Moreover, mechanical removal of the epithelium did not induce any change in isoproterenol sensitivity either in proximal or in distal segments. The DR values calculated between + E and - E tissues were 1 .01 (95% CL 0 . 77-1 . 34) and 1 . 11 (95% CL 0 . 86-1 .43) in proximal and distal preparations, respectively. In the tracheae obtained from actively sensitized guinea-pigs, mechanical damage of the epithelium did not modify the relaxant activity of epinephrine and
Pharmacological Research, Vol. 22, No. 1, 1990
53
M Control 0 Epithelium rubbed 100-
100-
9
8
7
100-
Fig. 2 . Log-concentration response curves for epinephrine, isoproterenol and salbutamol in control and epithelium rubbed tracheae taken from normal guinea-pigs . Each point represents the mean ± SEM from at least 5 replications. DR = dose ratio ; in parenthesis 95% confidence limits .
Pharmacological Research, Vol. 22, No. 1, 1990
54 El Control
o
Epithelium rubbed
100-
-Log molar concentration
Fig. 3 . Log-concentration response curves for isoproterenol in control and epithelium rubbed tracheae under resting tone . Each point represents the mean ± SEM from 5 replications. DR dose ratio ; in parenthesis 95% confidence limits .
salbutamol (Fig . 4) . However, as far as isoproterenol was concerned, the loss of epithelium slightly but significantly shifted to the left its log-concentration response curves, with a calculated DR of 1 . 49 (95% CL 1 . 06-2 . 08; Fig . 4). On the other hand, after the anaphylactic challenge (ovalbumin, 50,ug/kg) in tracheae obtained from actively sensitized guinea-pigs, a statistically significant shift to the left of the log-concentration response curves for all three adrenergic compounds tested was observed in - E as compared to + E tissues . In particular, the DR values calculated for epinephrine, isoproterenol and salbutamol were : 1 . 75 (95% CL 1 . 28-2 . 39), 1 . 61 (95% CL 1 . 04-2 . 48) and 1 . 58 (95% CL 1 . 04-2 . 42) respectively (Fig. 5) . The beta-adrenoceptor desensitization procedure (isoproterenol, 10 -6 M for 20 min twice) significantly shifted to the right isoproterenol log-concentration response curves in both + E and - E tracheae . Moreover, results obtained indicated that epithelium removal did not modify the development of betaadrenoceptor desensitization . In fact, as shown in Fig . 6, isoproterenol logconcentration response curves constructed in desensitized tissues were comparable regardless of the presence or absence of the epithelium .
DISCUSSION The possible existence of an epithelium-derived relaxing factor which might modulate airway tone is suggestive, even if a conclusive demonstration has not yet been found [1, 10] . As far as the adrenergic activity is concerned, our data do not allow us to propose a major influence of the epithelium on the activity of the physiological adrenergic mediator . In fact, we could not detect any difference in the relaxant action of epinephrine in normal rubbed tracheae as compared to controls . The lack of influence of the epithelium on salbutamol induced relaxation is in
Pharmacological Research, Vol. 22, No . 1, 1990 M Control
O Control
O Epithelium rubbed
O Epithelium rubbed
100-
too 80 60 -
40 20 -
O• 7
8
6
100-
100-
8
7
8
7
100-
8
7
-Log molar concentration
- Log molar concentration
Fig . 4 (left). Log-concentration response curves for epinephrine, isoproterenol and salbutamol in control and epithelium rubbed tracheae taken from actively sensitized guinea-pigs . Each point represents the mean ±srs from 5 replications . DR = dose ratio : in parenthesis 95"4, confidence limits . ::"/'< 0 . 11 1, as compared to control . Fig . 5 (right) . Log-concentration response curves for epinephrine, isoproterenol and salbutamol in control and epithelium rubbed tracheae taken from actively sensitized guinea-pigs and challenged with the specific antigen . Each point represents the mean±sign from 5 replications . DR=dose ratio : in parenthesis 95% confidence limits . *P< 0 . 05, * *P< 0 . 01, as compared to control .
56
Pharmacological Research, Vol. 22, No. 1, 1990 o
Control
O Epithelium rubbed ∎ Desensitized
• Epithelium rubbed desensitized
- Log molar concentration
Fig . 6. Log-concentration response curves for isoproterenol in control and beta-adrenoceptor desensitized tracheae with and without the epithelium . Tissues were obtained from ovalbumin actively sensitized guinea-pigs . Each point represents the mean ± SEM from at least 3 replications . Dose ratio values (95% confidence limits) were: control versus epithelium rubbed : 1 . 51 (1 . 15-1 . 99), P
