Clinical and Experimental Pharmacology & Physiology (1979) 6,249-257.
DRUG INTERACTIONS IN CAT ISOLATED TRACHEAL SMOOTH MUSCLE
H.W.Mitchell and M. A. Denborough Department of Clinical Science, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia (Received 31 September 1977;revision received 20 December 1977) SUMMARY
1. The interactions between some drugs that contract and relax airways smooth muscle have been investigated in the cat isolated trachea. 2. Isoprenaline and theophylline inhibited serotonin-elicited contractions more than acetylcholine-mediated responses. This was observed both in terms of the degree of inhibition and the concentration of the relaxant drug producing this inhibition. 3. The acetylcholine- and serotonin-induced contractions were inhibited more by theophylline than by isoprenaline. Isoprenaline displaced acetylcholine and serotonin response curves to the right whereas theophylline caused a flattening of the curves. 4. Isoprenaline was more effective in inhibiting serotonin contractions than acetylcholine contractions when the tracheas were bathed in K+ depolarizing solution, suggesting that the difference in the susceptibility of serotonin and acetylcholine contractions to isoprenaline was not dependent on the electrical membrane potential. 5 . Isoprenaline inhibited the tonic component of acetylcholine contractions more than the phasic component. 6 . The differences in the pharmacological responses to the contractile and relaxant drugs in cat tracheal preparations provide further examples in smooth muscle of different mechanisms by which acetylcholine and serotonin induce contraction and isoprenaline and theophylline relaxation.
Key words: acetylcholine; contraction; dose-response relationship, drug; isoprenaline; muscle, smooth;relaxation; serotonin; theophylline; trachea. INTRODUCTION The mechanisms of action of drugs on airways smooth muscle have been investigated both in terms of mechanical responses to drug-contraction or relaxation-and cellular chemical Correspondence: H. W. Mitchell, Department of Clinical Science, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia 2601. 0305-1 870/79/0500-0249$02.00
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and electrical changes. In common with other types of muscle, contractions in airways smooth muscle can be brought about by drugs that produce membrane depolarization (Stephens & Kroeger, 1970; Kirkpatrick, 1975; Coburn & Yamaguchi, 1977). CaZ+is the link between the initial excitation and muscle contraction. The CaZ+may act to inhibit the relaxing proteins (troponin and tropomyosin) enabling actin and myosin to interact, producing contraction (Sparrow et al., 1970; Sparrow & Van Bockxmeer, 1972). In addition to Caz+,cyclic GMP may also modulate smooth muscle tone. An increase in the concentration of c-GMP has been reported in response to contractile stimuli in bovine trachea (Katsuki & Murad, 1977) but its exact role in muscle contractility is not known. In contrast, relaxation can be brought about by drugs that increase the concentration of cyclic AMP such as the 0-adrenoceptor agonists and phosphodiesterase inhibitors (Triner, Vulliemoz & Veroski, 1977; Katsuki & Murad, 1977). Part of the relaxant action of the c-AMP may be due to the activation of a Caz+ pump which would lead to a reduction in the amount of Ca2+available to the contractile machinery (Nilsson et al., 1977). In the case of drugs with actions on airways smooth muscle, although there may be common features in their intracellular actions, there is evidence that differing pathways may be involved. For example, in the bovine trachea,Kirkpatrick (1975) demonstrated that contractile responses to histamine were more dependent on the presence of CaZ+in the bathing medium than were responses to acetylcholine; this suggested that acetylcholine was more effective in mobilising intracellular CaZ+.In another study, serotonin, acetylcholine and carbachol were shown to have greatly differing quantitative effects on the accumulation of cGMP in bovine tracheas (Katsuki & Murad, 1977) suggesting that these agents may not be acting via identical pathways. In this paper, differences in the contractile response of cat tracheal smooth muscle to acetylcholine and serotonin and in the relaxant action of isoprenaline and theophylline are described. METHODS Prepmation of the isolated cat trachea Cats weighing more than 2 kg, were anaesthetised with 40 mglkg sodium pentabarbitone intraperitoneally, and then exsanguinated. A length of trachea was dissected from mid-way between the larynx and the bihrcation of the trachea and this was then placed in a physiological salt solution at 37°C. The composition of the solution was (mmol/l): NaCl 121, KCl 5.4, MgS04.7HzO 1.2, NaHZPO41.2, NaHCOJ 15, d-glucose 11.5 and CaCl2.2H2O 2.5. in K+ depolarizing solution the NaCl was replaced with KzSO4 (80 mmol/l). A tracheal ring was removed and cotton threads were attached to the cartilage at each end of the trachealis muscle. The remaining cartilage was then discarded. Two tracheal segments were suspended in an organ bath containing Ringers solution at 37°C and gassed with a 95% Oz/5% COz mixture. The upper threads were attached to force displacement transducers for measuring changes in isometric tension. The tissues were initially stretched to give a resting tension of about 0.5 g. A 60 min equilibration period was then allowed. Experimental procedure In most experiments concentration-response curves for the contractile drug (acetylcholine and serotonin) were obtained using a cumulative dosing regime. In some of the earlier experiments, in which the effects of isoprenaline on acetylcholineinduced responses
Drug interactions in cat isolated trachea
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were studied, the different concentrations of acetylcholine were added sequentially with washout between successive doses. The isoprenaline was then replaced after the washout. There were no significant differences (Students t-test, P > 0.6, 9 d.f.) in the control acetylcholine concentration-response curves using the two methods. Duplicate control curves to acetylcholine or serotonin were first obtained. The responses at each concentration were averaged and expressed as percentages of the maximal response obtained (%Emu). The relaxant drugs (0.001-10 /.unol/l isoprenaline or 0.1-10 mmol/l theophylline) were then added to the bath and responses to acetylcholine or serotonin reestablished. Contractions elicited by acetylcholine and serotonin in the presence of the relaxing drugs were expressed as a percentage of the maximal control response (%&A. A shift in the control curve was measured as the difference in the log concentrations of the contractile drug eliciting 5% Em,,. With isoprenaline a contact time of 2 min was allowed before administering the contractile drug, as earlier experiments had shown that the inhibitory effect of isoprenaline on acetylcholine and serotonin responses was constant from about 1 min to more than 10 min after its injection into the bath. Theophylline was administered 5 min prior to acetylcholine or serotonin. The relaxant drug was left in contact with the tissue while concentration-response curves to either acetylcholine or serotonin were established, and was then washed out. Statistical analyses were carried out using the paired t-test. Drugs The drugs used were: acetylcholine hydrochloride (Sigma), dl-isoprenaline hydrochloride (Sigma), 5-hydroxytryptamine creatinine sulphate (Serotonin, Sigma) and theophylline (Sigma). Concentrations refer to the free acid or base. Drug solutions were freshly prepared and were kept on ice during experiments. The stock solutions of isoprenaline were acidified with 1 mg/ml ascorbic acid to inhibit oxidation. RESULTS Effect o f isoprenaline on acetylcholine and serotonin contractions Isoprenaline (0.01 and 0.1 pmol/l) had no effect on acetylcholine-mediated contractions of the trachea. However at a higher concentration (1 pmol/l>, isoprenaline reduced the acetylcholine responses and the concentration-response curve was displaced to the right in a parallel fashion without change in the maximal response (Figs. 1 and 2a). No additional shift was produced by increasing the concentration of isoprenaline from 1 pmol/l to 10 /.unol/l. Isoprenaline was more effective in inhibiting serotonin- than acetylcholine-induced contractions (Fig. 1). Concentrations of 0.001 and 0.01 pmol/l isoprenaline reduced serotonin contractions and displaced the concentration-response curves to the right (Fig. 2b). Isoprenaline (0.1 pmol/l) caused a shift of about 500-fold in the concentration-response curve. In the presence of this concentration of isoprenaline, serotonin (100 pmol/l) produced a contractile response of approximately 60%Em, (Fig. 2b). Although greater responses could be obtained, the use of higher serotonin concentrations was precluded by the prolonged desensitisation which they produced.
Effect of theophylline on acetylcholine and serotonin contractions Theophylline (0.1 and 1 mmol/l) caused only slight reductions in the acetylcholine contractions whereas 10 mmol/l theophylline markedly inhibited the responses and caused a
0.5g /J H. W.Mitchell and M. A . Denborough
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0 P c
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s
0
I
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5-HT
Ach 5-HTAch lsoprenaline (mmol/l)
J-, Ach 5-HT Theophylline (mmol/l)
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Fig. 1. Contractions elicited in a cat tracheal preparation by acetylcholine (Ach, 10 rmol/l) and serotonin (5-HT, 0.1 pmol/l) in the absence and presence of isoprenaline (1 pmol/l) and theophylline (10 mmol/l).
Acetylcholine (pmol/l)
Serotonin (pmol/ll
Fig. 2. The effect of isoprenaline on mean concentration-response curves to (a) acetylcholine (Ach, n = 7), and (b) serotonin (SHT, n = 4) in cat isolated tracheal preparations. (a) Control curve for Ach ( 0 ) and then in the presence of 0.01 (o), 0.1 (a), 1 (A) and 10 (v) pmol/l isoprenaline. (b) Control curve to 5 HT ( 0 ) and then in the presence of 0.001 (a), 0.01 (o), and 0.1 (A) pmol/l isoprenaline.
Acetylcholine (pmol/l)
Serotonin (pmol/l)
Fig. 3. The effect of theophylline on mean concentration-responsecurves to (a) acetylcholine (Ach, n = 4), and (b) serotonin (5-HT, n = 4) in cat isolated tracheal preparations. Curves to Ach or 5-HT in the absence (0)and in the presence of 0.1 (a), 1.0 (A) and 10 (v) mmol/l theophylline.
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Table 1. The effect of isoprenaline (1 pmol/l) on acetylcholine (Ach)- and serotonin-induced contractions in normal and depolarized cat trachea*
K+depolarized
Normal solution plus isoprenaline mean (1 pmol/l) Ach (10 pmol/l) Ach (0.1 pmol/l) serotonin
5.5 43.3 0
s.e.m
solution plus isoprenaline
.
pt
mean
s.e.m.
27.6 47.0 13.5
8.7 (6) 7.1 (6) 8.1 (4)
2.0 (6) 10.1 (6) 0 (4)
0.7 (5 d.f.) >0.1
(3 d.f.1
*Responses expressed as percentage of original contractions obtained in normal solution. Number of preparations in parentheses. tcomparison between contractionselicited in the presence of isoprenaline in normal and K+ depolarizing solutions.
flattening of the concentration-response curve (Figs 1 and 3a). The maximum contraction obtained in the presence of 10 mmol/l theophylline was 24% Em,, . Theophylline inhibited serotonin-induced contractions to a greater extent than those to acetylcholine (Figs 1 and 3b). Theophylline (1 and 10 mmol/l) did not shift the concentration-response curves to the right, but reduced the slope of the curves. Theophylline (10 mmol/l) abolished serotonin-induced contractions.
Effect o f isoprenaline in K + depolarized preparations Experiments were carried out in tracheal preparations depolarized with 80 mmol/l K+ in order to eliminate the influence of the membrane potential on the interaction between isoprenaline and the contractile drug.
Ach 5-HT I
10
Ach‘5-HT I 10
K’
Ach I
10
5-HT
Ach 5 - H T I 10
Treatment of muscle Fig. 4. The effect of isoprenaline (ISOP,1 pmol/l) on acetylcholine (Ach, 1 and 10 pmol/l) and serotonin (5-HT, 0.1 pmol/l) induced contractions of cat isolated tracheal preparation in both normal and K + depolarizing solutions. Control responses were fust elicited t o Ach and 5-HT in normal solution. Isoprenaline was then added to the bath and 2 min later the responses t o Ach and 5-HT retested. Following several washouts the tissue was immersed in K+ depolarizing solution. When the tension had stabilised contractions to Ach and 5-HT were repeated and the effect of isoprenaline on these responses determined. In both bathing solutions isoprenaline inhibited 5-HT to a greater extent than Ach contractions.
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Control contractions were first elicited in normal physiological salt solution using acetylcholine (1 and 10 pmol/l) and serotonin (0.1 pmol/l). The serotonin contraction was intermediate in size between those obtained with the two concentrations of acetylcholine. The effect of 1 pmol/l isoprenaline on these contractions was then assessed. K+ depolarizing solution was then substituted for the normal salt solution; the responses to acetylcholine and serotonin were retested and the effect of isoprenaline on these contractile responses determined. In the normal and the depolarized preparations isoprenaline inhibited serotonin contractions to a greater extent than those to acetylcholine. The results are summarized in Table 1 and a representative trace is shown in Fig. 4. The percentage inhibitions of the 10 pmol/l acetylcholine and serotonin contractions were not significantly different in either bathing solutions. However, there was a significant reduction in the effect of isoprenaline of 1 pmol/l acetylcholine contractions in the K*depolarizing solution (P< 0.05). In the K+ bathing solution, control contractions in response to 1 pmol/l and 10 pmol/l acetylcholine and to the serotonin were 61.1% (s.e.m. = 3.0, n = 6), 61.7% (s.e.m. = 4.6, n = 6) and 72.5%(s.em. = 2.3, n = 4), respectively of the responses in normal salt solution. Effect of isoprenaline on phasic and tonic components of acetylcholine contractions During these experiments it was observed that isoprenaline inhibited sustained acetylcholine-induced tone more than phasic acetylcholine contractions (Fig. 5 ) . The results
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l I I I I !Oh ISOP(pmol/I) ISOP Ach Ach Ach Ach Ach Ach I00 I I 100 I loo Treatment of muscle I
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ISOP
Fig. 5. An example of an experiment showing the different action of isoprenaline on the phasic and tonic component of acetylcholine (Ach, 1 and 100 rmolll) induced contractions of the isolated cat trachea. The effect of isoprenaline (ISOP,1 rmol/l) on the Ach responses was f'iist established using the procedure described previously. Following washout, and recovery of the control contractions the effect of isoprenaline on the tonic component was measured. A contraction to Ach was elicited and the isoprenaline was added when the tension had stabilized. Isoprenaline was added cumulatively (at arrows) to give ten-fold increments in the concentration, until the maximum relaxation was obtained. The percentage relaxation to isoprenaline was greater on the tonic component of tone than on the phasic component.
obtained when a maximal concentration (1 pnolll) of isoprenaline was tested against the phasic and the tonic component of acetylcholine-induced contractions are shown in Table 2. Concentrations of acetylcholine that caused approximately 40% and 100% of Em, (1 and 100 pmol/l, respectively) were used.
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Drug interactions in cat isolated trachea
Table 2. The effect of isoprenaline (1 Mmol/l) on the phasic and tonic components of acetylcholine (Ach) contractions* Phasic
(1 pmol/l) Ach (100 Icmol/l) Ach
pt
Tonic
Mean
s.e.m.
Mean
s.e.rn.
17.1 65.1
2.6 (6) 9.8 (5)
2.4 40.6
2.4 (6) 8.6 (5)
< 0.01 (5 d.f.) < 0.01 (4 d.f.)
*Responses expressed as percentage of control contraction in the absence of isoprenaline. Number of preparations in parentheses. tcomparison between the phasic and tonic means.
DISCUSSION
In this paper it is shown that isoprenaline inhibits serotonin contractions more than responses to acetylcholine. A difference in the effect of isoprenaline on methacholine and histamine elicited contractions was found by Van den Brink (1973a,b) on the isolated calf trachea. Results reported by this author show that the maximum displacement of the control concentration-response curves caused by isoprenaline was more than ten times greater with histamine compared with rnethacholine. It was not possible to test the effect of histamine in the present experiments because the cat trachea is not contracted by this agent (Lulich, Mitchell & Sparrow, 1976). Contraction of tracheal smooth muscle cells elicited by drug such as acetylcholine, histamine and serotonin is associated with depolarization of the cell membrane (Stephens & Kroeger, 1970; Kirkpatrick, 1975; Coburn & Yamaguchi, 1977). However, there is a component of the contractile response that is not dependent on the membrane potential. The proportion of a contraction in response to acetylcholine and serotonin that is independent of the membrane potential has been estimated to range from about 70% to 50% respectively in the dog trachea (Coburn & Yamaguchi, 1977; Farley & Miles, 1977). Similar results were demonstrated in the present experiments since both acetylcholine and serotonin caused contractions when the trachea was bathed in K* depolarizing solution. In both normal and K+ depolarizing solutions, isoprenaline is more effective in counteracting serotonin than acetylcholine-induced contractions. This suggests that the difference in the functional antagonism between isoprenaline and the two contractile drugs is not dependent on the integrity of the membrane potential but on events subsequent to the initial excitation. There is evidence that different forms of contractile stimuli and drugs evoke mechanical responses of smooth muscle cells via different actions on the contractile machinery-for instance, by mobilization of different cellular Ca2+ fractions (Kirkpatrick, 1975; Sparrow & Simmonds, 1965). Our results with isoprenaline could reflect a difference in the intracellular of action of acetylcholine and serotonin on the tracheal smooth muscle Ca2+‘pools’. Both isoprenaline and theophylline inhibited serotonin and acetylcholine contractures, and this may be because both relaxant drugs increase the intracellular concentration of c-AMP in the trachea (Triner et ul., 1977; Katsuki & Murad, 1977). Nevertheless, there were differences between the actions of these two relaxant drugs on the cat trachea. Theophylline caused a quantitatively greater inhibition of contractile responses and flattened the concentration-response curves, whereas isoprenaline caused parallel shifts to the right
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suggesting that it was competing with the contractile drugs for some cellular process. However, the action of theophylline may not be confined to increasing c-AMP by inhibiting the enzyme phosphodiesterase (PDE) (Butcher & Sutherland, 1962). In rat uterus, Levy & Wilkenfeld (1 968) have reported a non-specific action of theophylline that was not apparently related to c-AMP. If theophylline has a direct effect on cell membranes or the contractile machinery in addition to increasing the c-AMP concentration, then this might explain why it has a greater effect than isoprenaline on contractions in the trachea. Our studies have shown that inhibition by isoprenaline of the tonic component of the response to acetylcholine is greater than that of the phasic component. The effectiveness of the isoprenaline on the two components of tone could not have been influenced by oxidation of the catecholamine in either of the experimental procedures. In preliminary tests it was found that the relaxant effect of the isoprenaline remained undiminished for at least 10 min, this was long enough for its effect on the acetylcholine contractions to be measured. Similar results have been reported with other smooth muscles using papaverine to inhibit drug elicited contractions (Ferrari, 1974) and using metabolic inhibitors on different phases of spontaneous activity in the gut (Boev, Golenhofen & Lukanow, 1976). Moreover, Mitchell & Sparrow (1977) reported that although the intrinsic tone of the isolated cat lung strip was reduced by 0-adrenoceptor agonists,histamine and prostaglandin Fza elicited contractions were highly resistant. These results may highhght differences in the physiological mechanisms involved in the maintenance of tone as opposed to the rapid changes associated with a phasic contraction. It was also noted that a maximal concentration of isoprenaline produced a smaller percentage inhibition of the contraction to 100 pmol/l than to 1 /.unol/l acetylcholine (Table 2). In this respect, our results are consistent with those reported by Buckner & Saini (1975) and Van den Brink (1973b). These authors used carbachol and methacholine on guinea-pig and calf tracheas respectively, and demonstrated that the percentage relaxation to isoprenaline became relatively smaller as the concentration of the contractile agent was increased. The principal findings reported in this paper are that isoprenaline and theophylline inhibit contractions elicited by serotonin more than those to acetylcholine, and that theophylline causes a greater degree of relaxation than isoprenaline in the cat trachea. ACKNOWLEDGMENTS
Howard W. Mitchell is a Wellcome (Australia) Research Fellow. The authors wish to acknowledge Dr M. P. Sparrow, Department of Pharmacology, University of Western Australia, for helpful discussion at the beginning of this study. REFERENCES Boev, K., Golenhofen, K. & Lukanow, J. (1976) Selective suppression of phasic and tonic activation mechanisms in stomach smooth muscle. Physiology of Smoorh Muscle (Eds E. Biilbring & M.F. Shuba), pp. 203-208. Raven Press, New York. Buckner, C.K. & Saini, R.K. (1975) On the use of functional antagonism to estimate dissociation constants for beta adrenergic receptor agonists in isolated guinea-pig trachea. Journal of Pharmacology and Experimental Therapeutics, 194,565 -5 74. Butcher, R.W. & Sutherland, E.W. (1962) Adenosine 33-phosphate in biological material. Journal of Biological Chemistry. 237,1244-1250. Cobum, RF. & Yamaguchi, T. (1977) Membrane potentialdependent and -independent tension in the canine tracheal muscle. Journal of Pharmacology and Experimental Therapeutics, 201, 276-284.
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Farley, J.M. & Miles, P.R (1977) Role of depolarisation in acetylcholine-induced contractions of dog trachealis muscle. Journal of Pharmacology and Experimental Therapeutics, 201,199-205. Ferrari, M. (1974) Effects of papaverine on smooth muscle and their mechanisms. Pharmacological Research Communications, 6,97-115. Katsuki, S. & Murad, F. (1977) Regulation ofadenosine cyclic 3',5'-rnonophosphate and guanosine cyclic 3',5'-monophosphate levels and contractility in bovine tracheal smooth muscle. Molecular Pharmacology, 13, 330-341. Kirkpatrick, C.T. (1975) Excitation and contraction in bovine tracheal smooth muscle. Journal ofphysiology, 244,263-281. Levy, B. & Wilkenfeld, B.E. (1968) The potentiation of rat uterine inhibitory responses to noradrenaline by theophylline and nitroglycerine. British Journal of Pharmacology, 34,604-61 2. Lulich, K.M., Mitchell, H.W. & Sparrow, M.P. (1976) The cat lung strip as an in vitro preparation of peripheral airways: a comparison of 0-adrenoceptor agonists, autacoids and anaphylactic challenge on the lung strip and trachea. British Journal ofPharmacology, 58, 71-79. Mitchell, H.W. & Sparrow, M.P. (1977) The effect of catecholamines on the in vivo and in vitro responses of the cat lung during anaphylaxis. British Journalof Pharmacology, 61,533-540. Nilsson, K.B., Andersson, G.G., Mohme-Lundholm, E. & Lundholm, L. (1977) Cyclic AMP and Cabinding in rnicrosomal fractions isolated from rabbit colon smooth muscle. Acta pharmacologica et toxicologica, 4 1 , 5 3 4 4 . Sparrow, M.P., Maxwell, L.C., Ruegg, J.C. & Bohr, D.F. (1970) Preparation and properties of a calcium ion-sensitive actomyosin from arteries. American Journal of Physiology, 219,1366-1 372. Sparrow, M.P. & Simmonds, W.J. (1965) The relationship of the calcium content of smooth muscle to its contractility in response to different modes of stimulation. Biochimica et Biophysica Acta, 109, 503-5 11. Sparrow, M.P. & Van Bockxmeer, F. (1972) Arterial tropomyosin and a relaxing protein fraction from vascular smooth muscle. Journalof Biochemistry, 72, 1075-1080. Stephens, N.L. & Kroeger, E. (1970) Effect of hypoxia on airway smooth muscle mechanisms and electrophysiology. Journal ofApplied Physiology, 28.630-635. Triner, L., Vulliemoz, Y. & Verosky, M. (1977) Cyclic 3'5'-adenosine monophosphate and bronchial tone. European Journal of Pharmacology, 41,37-46. Van den Brink, F.G. (1973a) The model of functional interaction, I. European Journal of Pharmacology, 22,270-278. Van den Brink, F.G. (1973b) The model of functional interaction 11. European Journal of Pharmacology, 22,279-286.
DRUG INTERACTIONS IN CAT ISOLATED TRACHEAL SMOOTH MUSCLE
H.W.Mitchell and M. A. Denborough Department of Clinical Science, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia (Received 31 September 1977;revision received 20 December 1977) SUMMARY
1. The interactions between some drugs that contract and relax airways smooth muscle have been investigated in the cat isolated trachea. 2. Isoprenaline and theophylline inhibited serotonin-elicited contractions more than acetylcholine-mediated responses. This was observed both in terms of the degree of inhibition and the concentration of the relaxant drug producing this inhibition. 3. The acetylcholine- and serotonin-induced contractions were inhibited more by theophylline than by isoprenaline. Isoprenaline displaced acetylcholine and serotonin response curves to the right whereas theophylline caused a flattening of the curves. 4. Isoprenaline was more effective in inhibiting serotonin contractions than acetylcholine contractions when the tracheas were bathed in K+ depolarizing solution, suggesting that the difference in the susceptibility of serotonin and acetylcholine contractions to isoprenaline was not dependent on the electrical membrane potential. 5 . Isoprenaline inhibited the tonic component of acetylcholine contractions more than the phasic component. 6 . The differences in the pharmacological responses to the contractile and relaxant drugs in cat tracheal preparations provide further examples in smooth muscle of different mechanisms by which acetylcholine and serotonin induce contraction and isoprenaline and theophylline relaxation.
Key words: acetylcholine; contraction; dose-response relationship, drug; isoprenaline; muscle, smooth;relaxation; serotonin; theophylline; trachea. INTRODUCTION The mechanisms of action of drugs on airways smooth muscle have been investigated both in terms of mechanical responses to drug-contraction or relaxation-and cellular chemical Correspondence: H. W. Mitchell, Department of Clinical Science, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia 2601. 0305-1 870/79/0500-0249$02.00
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and electrical changes. In common with other types of muscle, contractions in airways smooth muscle can be brought about by drugs that produce membrane depolarization (Stephens & Kroeger, 1970; Kirkpatrick, 1975; Coburn & Yamaguchi, 1977). CaZ+is the link between the initial excitation and muscle contraction. The CaZ+may act to inhibit the relaxing proteins (troponin and tropomyosin) enabling actin and myosin to interact, producing contraction (Sparrow et al., 1970; Sparrow & Van Bockxmeer, 1972). In addition to Caz+,cyclic GMP may also modulate smooth muscle tone. An increase in the concentration of c-GMP has been reported in response to contractile stimuli in bovine trachea (Katsuki & Murad, 1977) but its exact role in muscle contractility is not known. In contrast, relaxation can be brought about by drugs that increase the concentration of cyclic AMP such as the 0-adrenoceptor agonists and phosphodiesterase inhibitors (Triner, Vulliemoz & Veroski, 1977; Katsuki & Murad, 1977). Part of the relaxant action of the c-AMP may be due to the activation of a Caz+ pump which would lead to a reduction in the amount of Ca2+available to the contractile machinery (Nilsson et al., 1977). In the case of drugs with actions on airways smooth muscle, although there may be common features in their intracellular actions, there is evidence that differing pathways may be involved. For example, in the bovine trachea,Kirkpatrick (1975) demonstrated that contractile responses to histamine were more dependent on the presence of CaZ+in the bathing medium than were responses to acetylcholine; this suggested that acetylcholine was more effective in mobilising intracellular CaZ+.In another study, serotonin, acetylcholine and carbachol were shown to have greatly differing quantitative effects on the accumulation of cGMP in bovine tracheas (Katsuki & Murad, 1977) suggesting that these agents may not be acting via identical pathways. In this paper, differences in the contractile response of cat tracheal smooth muscle to acetylcholine and serotonin and in the relaxant action of isoprenaline and theophylline are described. METHODS Prepmation of the isolated cat trachea Cats weighing more than 2 kg, were anaesthetised with 40 mglkg sodium pentabarbitone intraperitoneally, and then exsanguinated. A length of trachea was dissected from mid-way between the larynx and the bihrcation of the trachea and this was then placed in a physiological salt solution at 37°C. The composition of the solution was (mmol/l): NaCl 121, KCl 5.4, MgS04.7HzO 1.2, NaHZPO41.2, NaHCOJ 15, d-glucose 11.5 and CaCl2.2H2O 2.5. in K+ depolarizing solution the NaCl was replaced with KzSO4 (80 mmol/l). A tracheal ring was removed and cotton threads were attached to the cartilage at each end of the trachealis muscle. The remaining cartilage was then discarded. Two tracheal segments were suspended in an organ bath containing Ringers solution at 37°C and gassed with a 95% Oz/5% COz mixture. The upper threads were attached to force displacement transducers for measuring changes in isometric tension. The tissues were initially stretched to give a resting tension of about 0.5 g. A 60 min equilibration period was then allowed. Experimental procedure In most experiments concentration-response curves for the contractile drug (acetylcholine and serotonin) were obtained using a cumulative dosing regime. In some of the earlier experiments, in which the effects of isoprenaline on acetylcholineinduced responses
Drug interactions in cat isolated trachea
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were studied, the different concentrations of acetylcholine were added sequentially with washout between successive doses. The isoprenaline was then replaced after the washout. There were no significant differences (Students t-test, P > 0.6, 9 d.f.) in the control acetylcholine concentration-response curves using the two methods. Duplicate control curves to acetylcholine or serotonin were first obtained. The responses at each concentration were averaged and expressed as percentages of the maximal response obtained (%Emu). The relaxant drugs (0.001-10 /.unol/l isoprenaline or 0.1-10 mmol/l theophylline) were then added to the bath and responses to acetylcholine or serotonin reestablished. Contractions elicited by acetylcholine and serotonin in the presence of the relaxing drugs were expressed as a percentage of the maximal control response (%&A. A shift in the control curve was measured as the difference in the log concentrations of the contractile drug eliciting 5% Em,,. With isoprenaline a contact time of 2 min was allowed before administering the contractile drug, as earlier experiments had shown that the inhibitory effect of isoprenaline on acetylcholine and serotonin responses was constant from about 1 min to more than 10 min after its injection into the bath. Theophylline was administered 5 min prior to acetylcholine or serotonin. The relaxant drug was left in contact with the tissue while concentration-response curves to either acetylcholine or serotonin were established, and was then washed out. Statistical analyses were carried out using the paired t-test. Drugs The drugs used were: acetylcholine hydrochloride (Sigma), dl-isoprenaline hydrochloride (Sigma), 5-hydroxytryptamine creatinine sulphate (Serotonin, Sigma) and theophylline (Sigma). Concentrations refer to the free acid or base. Drug solutions were freshly prepared and were kept on ice during experiments. The stock solutions of isoprenaline were acidified with 1 mg/ml ascorbic acid to inhibit oxidation. RESULTS Effect o f isoprenaline on acetylcholine and serotonin contractions Isoprenaline (0.01 and 0.1 pmol/l) had no effect on acetylcholine-mediated contractions of the trachea. However at a higher concentration (1 pmol/l>, isoprenaline reduced the acetylcholine responses and the concentration-response curve was displaced to the right in a parallel fashion without change in the maximal response (Figs. 1 and 2a). No additional shift was produced by increasing the concentration of isoprenaline from 1 pmol/l to 10 /.unol/l. Isoprenaline was more effective in inhibiting serotonin- than acetylcholine-induced contractions (Fig. 1). Concentrations of 0.001 and 0.01 pmol/l isoprenaline reduced serotonin contractions and displaced the concentration-response curves to the right (Fig. 2b). Isoprenaline (0.1 pmol/l) caused a shift of about 500-fold in the concentration-response curve. In the presence of this concentration of isoprenaline, serotonin (100 pmol/l) produced a contractile response of approximately 60%Em, (Fig. 2b). Although greater responses could be obtained, the use of higher serotonin concentrations was precluded by the prolonged desensitisation which they produced.
Effect of theophylline on acetylcholine and serotonin contractions Theophylline (0.1 and 1 mmol/l) caused only slight reductions in the acetylcholine contractions whereas 10 mmol/l theophylline markedly inhibited the responses and caused a
0.5g /J H. W.Mitchell and M. A . Denborough
252
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Fig. 1. Contractions elicited in a cat tracheal preparation by acetylcholine (Ach, 10 rmol/l) and serotonin (5-HT, 0.1 pmol/l) in the absence and presence of isoprenaline (1 pmol/l) and theophylline (10 mmol/l).
Acetylcholine (pmol/l)
Serotonin (pmol/ll
Fig. 2. The effect of isoprenaline on mean concentration-response curves to (a) acetylcholine (Ach, n = 7), and (b) serotonin (SHT, n = 4) in cat isolated tracheal preparations. (a) Control curve for Ach ( 0 ) and then in the presence of 0.01 (o), 0.1 (a), 1 (A) and 10 (v) pmol/l isoprenaline. (b) Control curve to 5 HT ( 0 ) and then in the presence of 0.001 (a), 0.01 (o), and 0.1 (A) pmol/l isoprenaline.
Acetylcholine (pmol/l)
Serotonin (pmol/l)
Fig. 3. The effect of theophylline on mean concentration-responsecurves to (a) acetylcholine (Ach, n = 4), and (b) serotonin (5-HT, n = 4) in cat isolated tracheal preparations. Curves to Ach or 5-HT in the absence (0)and in the presence of 0.1 (a), 1.0 (A) and 10 (v) mmol/l theophylline.
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Drug interactions in cat isolated trachea
Table 1. The effect of isoprenaline (1 pmol/l) on acetylcholine (Ach)- and serotonin-induced contractions in normal and depolarized cat trachea*
K+depolarized
Normal solution plus isoprenaline mean (1 pmol/l) Ach (10 pmol/l) Ach (0.1 pmol/l) serotonin
5.5 43.3 0
s.e.m
solution plus isoprenaline
.
pt
mean
s.e.m.
27.6 47.0 13.5
8.7 (6) 7.1 (6) 8.1 (4)
2.0 (6) 10.1 (6) 0 (4)
0.7 (5 d.f.) >0.1
(3 d.f.1
*Responses expressed as percentage of original contractions obtained in normal solution. Number of preparations in parentheses. tcomparison between contractionselicited in the presence of isoprenaline in normal and K+ depolarizing solutions.
flattening of the concentration-response curve (Figs 1 and 3a). The maximum contraction obtained in the presence of 10 mmol/l theophylline was 24% Em,, . Theophylline inhibited serotonin-induced contractions to a greater extent than those to acetylcholine (Figs 1 and 3b). Theophylline (1 and 10 mmol/l) did not shift the concentration-response curves to the right, but reduced the slope of the curves. Theophylline (10 mmol/l) abolished serotonin-induced contractions.
Effect o f isoprenaline in K + depolarized preparations Experiments were carried out in tracheal preparations depolarized with 80 mmol/l K+ in order to eliminate the influence of the membrane potential on the interaction between isoprenaline and the contractile drug.
Ach 5-HT I
10
Ach‘5-HT I 10
K’
Ach I
10
5-HT
Ach 5 - H T I 10
Treatment of muscle Fig. 4. The effect of isoprenaline (ISOP,1 pmol/l) on acetylcholine (Ach, 1 and 10 pmol/l) and serotonin (5-HT, 0.1 pmol/l) induced contractions of cat isolated tracheal preparation in both normal and K + depolarizing solutions. Control responses were fust elicited t o Ach and 5-HT in normal solution. Isoprenaline was then added to the bath and 2 min later the responses t o Ach and 5-HT retested. Following several washouts the tissue was immersed in K+ depolarizing solution. When the tension had stabilised contractions to Ach and 5-HT were repeated and the effect of isoprenaline on these responses determined. In both bathing solutions isoprenaline inhibited 5-HT to a greater extent than Ach contractions.
H. W. Mitchell and M . A. Denborough
254
Control contractions were first elicited in normal physiological salt solution using acetylcholine (1 and 10 pmol/l) and serotonin (0.1 pmol/l). The serotonin contraction was intermediate in size between those obtained with the two concentrations of acetylcholine. The effect of 1 pmol/l isoprenaline on these contractions was then assessed. K+ depolarizing solution was then substituted for the normal salt solution; the responses to acetylcholine and serotonin were retested and the effect of isoprenaline on these contractile responses determined. In the normal and the depolarized preparations isoprenaline inhibited serotonin contractions to a greater extent than those to acetylcholine. The results are summarized in Table 1 and a representative trace is shown in Fig. 4. The percentage inhibitions of the 10 pmol/l acetylcholine and serotonin contractions were not significantly different in either bathing solutions. However, there was a significant reduction in the effect of isoprenaline of 1 pmol/l acetylcholine contractions in the K*depolarizing solution (P< 0.05). In the K+ bathing solution, control contractions in response to 1 pmol/l and 10 pmol/l acetylcholine and to the serotonin were 61.1% (s.e.m. = 3.0, n = 6), 61.7% (s.e.m. = 4.6, n = 6) and 72.5%(s.em. = 2.3, n = 4), respectively of the responses in normal salt solution. Effect of isoprenaline on phasic and tonic components of acetylcholine contractions During these experiments it was observed that isoprenaline inhibited sustained acetylcholine-induced tone more than phasic acetylcholine contractions (Fig. 5 ) . The results
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Fig. 5. An example of an experiment showing the different action of isoprenaline on the phasic and tonic component of acetylcholine (Ach, 1 and 100 rmolll) induced contractions of the isolated cat trachea. The effect of isoprenaline (ISOP,1 rmol/l) on the Ach responses was f'iist established using the procedure described previously. Following washout, and recovery of the control contractions the effect of isoprenaline on the tonic component was measured. A contraction to Ach was elicited and the isoprenaline was added when the tension had stabilized. Isoprenaline was added cumulatively (at arrows) to give ten-fold increments in the concentration, until the maximum relaxation was obtained. The percentage relaxation to isoprenaline was greater on the tonic component of tone than on the phasic component.
obtained when a maximal concentration (1 pnolll) of isoprenaline was tested against the phasic and the tonic component of acetylcholine-induced contractions are shown in Table 2. Concentrations of acetylcholine that caused approximately 40% and 100% of Em, (1 and 100 pmol/l, respectively) were used.
25 5
Drug interactions in cat isolated trachea
Table 2. The effect of isoprenaline (1 Mmol/l) on the phasic and tonic components of acetylcholine (Ach) contractions* Phasic
(1 pmol/l) Ach (100 Icmol/l) Ach
pt
Tonic
Mean
s.e.m.
Mean
s.e.rn.
17.1 65.1
2.6 (6) 9.8 (5)
2.4 40.6
2.4 (6) 8.6 (5)
< 0.01 (5 d.f.) < 0.01 (4 d.f.)
*Responses expressed as percentage of control contraction in the absence of isoprenaline. Number of preparations in parentheses. tcomparison between the phasic and tonic means.
DISCUSSION
In this paper it is shown that isoprenaline inhibits serotonin contractions more than responses to acetylcholine. A difference in the effect of isoprenaline on methacholine and histamine elicited contractions was found by Van den Brink (1973a,b) on the isolated calf trachea. Results reported by this author show that the maximum displacement of the control concentration-response curves caused by isoprenaline was more than ten times greater with histamine compared with rnethacholine. It was not possible to test the effect of histamine in the present experiments because the cat trachea is not contracted by this agent (Lulich, Mitchell & Sparrow, 1976). Contraction of tracheal smooth muscle cells elicited by drug such as acetylcholine, histamine and serotonin is associated with depolarization of the cell membrane (Stephens & Kroeger, 1970; Kirkpatrick, 1975; Coburn & Yamaguchi, 1977). However, there is a component of the contractile response that is not dependent on the membrane potential. The proportion of a contraction in response to acetylcholine and serotonin that is independent of the membrane potential has been estimated to range from about 70% to 50% respectively in the dog trachea (Coburn & Yamaguchi, 1977; Farley & Miles, 1977). Similar results were demonstrated in the present experiments since both acetylcholine and serotonin caused contractions when the trachea was bathed in K* depolarizing solution. In both normal and K+ depolarizing solutions, isoprenaline is more effective in counteracting serotonin than acetylcholine-induced contractions. This suggests that the difference in the functional antagonism between isoprenaline and the two contractile drugs is not dependent on the integrity of the membrane potential but on events subsequent to the initial excitation. There is evidence that different forms of contractile stimuli and drugs evoke mechanical responses of smooth muscle cells via different actions on the contractile machinery-for instance, by mobilization of different cellular Ca2+ fractions (Kirkpatrick, 1975; Sparrow & Simmonds, 1965). Our results with isoprenaline could reflect a difference in the intracellular of action of acetylcholine and serotonin on the tracheal smooth muscle Ca2+‘pools’. Both isoprenaline and theophylline inhibited serotonin and acetylcholine contractures, and this may be because both relaxant drugs increase the intracellular concentration of c-AMP in the trachea (Triner et ul., 1977; Katsuki & Murad, 1977). Nevertheless, there were differences between the actions of these two relaxant drugs on the cat trachea. Theophylline caused a quantitatively greater inhibition of contractile responses and flattened the concentration-response curves, whereas isoprenaline caused parallel shifts to the right
256
H. W.Mitchell and M.A . Denborough
suggesting that it was competing with the contractile drugs for some cellular process. However, the action of theophylline may not be confined to increasing c-AMP by inhibiting the enzyme phosphodiesterase (PDE) (Butcher & Sutherland, 1962). In rat uterus, Levy & Wilkenfeld (1 968) have reported a non-specific action of theophylline that was not apparently related to c-AMP. If theophylline has a direct effect on cell membranes or the contractile machinery in addition to increasing the c-AMP concentration, then this might explain why it has a greater effect than isoprenaline on contractions in the trachea. Our studies have shown that inhibition by isoprenaline of the tonic component of the response to acetylcholine is greater than that of the phasic component. The effectiveness of the isoprenaline on the two components of tone could not have been influenced by oxidation of the catecholamine in either of the experimental procedures. In preliminary tests it was found that the relaxant effect of the isoprenaline remained undiminished for at least 10 min, this was long enough for its effect on the acetylcholine contractions to be measured. Similar results have been reported with other smooth muscles using papaverine to inhibit drug elicited contractions (Ferrari, 1974) and using metabolic inhibitors on different phases of spontaneous activity in the gut (Boev, Golenhofen & Lukanow, 1976). Moreover, Mitchell & Sparrow (1977) reported that although the intrinsic tone of the isolated cat lung strip was reduced by 0-adrenoceptor agonists,histamine and prostaglandin Fza elicited contractions were highly resistant. These results may highhght differences in the physiological mechanisms involved in the maintenance of tone as opposed to the rapid changes associated with a phasic contraction. It was also noted that a maximal concentration of isoprenaline produced a smaller percentage inhibition of the contraction to 100 pmol/l than to 1 /.unol/l acetylcholine (Table 2). In this respect, our results are consistent with those reported by Buckner & Saini (1975) and Van den Brink (1973b). These authors used carbachol and methacholine on guinea-pig and calf tracheas respectively, and demonstrated that the percentage relaxation to isoprenaline became relatively smaller as the concentration of the contractile agent was increased. The principal findings reported in this paper are that isoprenaline and theophylline inhibit contractions elicited by serotonin more than those to acetylcholine, and that theophylline causes a greater degree of relaxation than isoprenaline in the cat trachea. ACKNOWLEDGMENTS
Howard W. Mitchell is a Wellcome (Australia) Research Fellow. The authors wish to acknowledge Dr M. P. Sparrow, Department of Pharmacology, University of Western Australia, for helpful discussion at the beginning of this study. REFERENCES Boev, K., Golenhofen, K. & Lukanow, J. (1976) Selective suppression of phasic and tonic activation mechanisms in stomach smooth muscle. Physiology of Smoorh Muscle (Eds E. Biilbring & M.F. Shuba), pp. 203-208. Raven Press, New York. Buckner, C.K. & Saini, R.K. (1975) On the use of functional antagonism to estimate dissociation constants for beta adrenergic receptor agonists in isolated guinea-pig trachea. Journal of Pharmacology and Experimental Therapeutics, 194,565 -5 74. Butcher, R.W. & Sutherland, E.W. (1962) Adenosine 33-phosphate in biological material. Journal of Biological Chemistry. 237,1244-1250. Cobum, RF. & Yamaguchi, T. (1977) Membrane potentialdependent and -independent tension in the canine tracheal muscle. Journal of Pharmacology and Experimental Therapeutics, 201, 276-284.
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Farley, J.M. & Miles, P.R (1977) Role of depolarisation in acetylcholine-induced contractions of dog trachealis muscle. Journal of Pharmacology and Experimental Therapeutics, 201,199-205. Ferrari, M. (1974) Effects of papaverine on smooth muscle and their mechanisms. Pharmacological Research Communications, 6,97-115. Katsuki, S. & Murad, F. (1977) Regulation ofadenosine cyclic 3',5'-rnonophosphate and guanosine cyclic 3',5'-monophosphate levels and contractility in bovine tracheal smooth muscle. Molecular Pharmacology, 13, 330-341. Kirkpatrick, C.T. (1975) Excitation and contraction in bovine tracheal smooth muscle. Journal ofphysiology, 244,263-281. Levy, B. & Wilkenfeld, B.E. (1968) The potentiation of rat uterine inhibitory responses to noradrenaline by theophylline and nitroglycerine. British Journal of Pharmacology, 34,604-61 2. Lulich, K.M., Mitchell, H.W. & Sparrow, M.P. (1976) The cat lung strip as an in vitro preparation of peripheral airways: a comparison of 0-adrenoceptor agonists, autacoids and anaphylactic challenge on the lung strip and trachea. British Journal ofPharmacology, 58, 71-79. Mitchell, H.W. & Sparrow, M.P. (1977) The effect of catecholamines on the in vivo and in vitro responses of the cat lung during anaphylaxis. British Journalof Pharmacology, 61,533-540. Nilsson, K.B., Andersson, G.G., Mohme-Lundholm, E. & Lundholm, L. (1977) Cyclic AMP and Cabinding in rnicrosomal fractions isolated from rabbit colon smooth muscle. Acta pharmacologica et toxicologica, 4 1 , 5 3 4 4 . Sparrow, M.P., Maxwell, L.C., Ruegg, J.C. & Bohr, D.F. (1970) Preparation and properties of a calcium ion-sensitive actomyosin from arteries. American Journal of Physiology, 219,1366-1 372. Sparrow, M.P. & Simmonds, W.J. (1965) The relationship of the calcium content of smooth muscle to its contractility in response to different modes of stimulation. Biochimica et Biophysica Acta, 109, 503-5 11. Sparrow, M.P. & Van Bockxmeer, F. (1972) Arterial tropomyosin and a relaxing protein fraction from vascular smooth muscle. Journalof Biochemistry, 72, 1075-1080. Stephens, N.L. & Kroeger, E. (1970) Effect of hypoxia on airway smooth muscle mechanisms and electrophysiology. Journal ofApplied Physiology, 28.630-635. Triner, L., Vulliemoz, Y. & Verosky, M. (1977) Cyclic 3'5'-adenosine monophosphate and bronchial tone. European Journal of Pharmacology, 41,37-46. Van den Brink, F.G. (1973a) The model of functional interaction, I. European Journal of Pharmacology, 22,270-278. Van den Brink, F.G. (1973b) The model of functional interaction 11. European Journal of Pharmacology, 22,279-286.
