Phormocology & Toxicology 1992, 71, 41-44.

In Vitro Tachyphylaxis to Isoprenaline in Guinea-Pig Trachea: Influence of Theophylline? A. Bergendall.*,A. Lindin', J. Lotvall', A. Ullman', B. E. Skoogh3 and C. C. Lofdahl'

'Department of Pharmacology, University of Goteborg, Box 3303 I , S-400 33 Goteborg, 'Department of Clinical Pharmacology, Sahlgrens Hospital, S-41345 Goteborg, and 'Department of Pulmonary Medicine, Renstroms Hospital, Box 17301, S-402 64 Goteborg, Sweden (Received September 25, 1991; Accepted January 7, 1992) Abstrucf: We used frontally opened tracheal rings, contracted with 10 pM of histamine to produce P-adrenoceptor tachyphylaxis by isoprenaline incubation and to evaluate whether theophylline can prevent this tachyphylaxis. Two concentration-response experiments with isoprenaline were performed in each ring. Between the concentration-response experiments the rings were incubated using four different treatments, isoprenaline (3 pM), theophylline (1 mM), theophylline + isoprenaline or vehicle for 60 min. Tachyphylaxis was evaluated as the difference in pD2 (-log ECw)during the first and second concentration-response experiment. Isoprenaline incubation produced a shift to the right of the concentrationresponse curve. The shift was small (approximately 0.2 log units) but significantly different from the control group. Theophylline failed to influence the process either alone or in combination with isoprenaline. In conclusion, P-adrenoceptor tachyphylaxis after isoprenaline incubation in isolated guinea-pig trachea proved to be a very small effect and we observed no influence by theophylline.

Considerable controversy exists about the occurrence and importance of P-adrenoceptor tachyphylaxis in the treatment of asthma (Conolly et al. 1987; Svedmyr & Lofdahl 1987). Different results have been obtained in various tissues and various species (Avner & Jenne 1981; Berti, et al. 1982; Davis & Conolly 1980; Douglas ef al. 1977; Fleisch & Titus 1972; Harvey & Tattersfield 1982; Holmberg et al. 1981; Larsson ei al. 1977; Lin et al. 1977; Spilker & Tyll 1976). In guinea-pig trachea, tachyphylaxis has been shown in several studies (Berti et al. 1982; Douglas et al. 1977; Elfellah & Turnbull 1978; Holmberg et al. 1981). Some bronchoactive drugs influence desensitization to PIadrenoceptor agonists. Corticosteroids counteract tachyphylaxis both in vitro and in vivo (Brodde et al. 1988; EllulMicallef & Fenech 1975; Hui et al. 1982; Svedmyr 1990) and ketotifen may also reverse tachyphylaxis (Bretz et al. 1983; Brodde et al. 1988; Svedmyr 1990). In a recent study it was shown that theophylline partly prevents the downregulation of P-receptors due to adrenergic treatment seen in polymorphonuclear leucocytes from asthmatic children (Otto et al. 1989). Furthermore, a study of rat aortic strips shows that aminophylline inhibits isoprenaline tachyphylaxis (Fleisch & Titus 1972). It is therefore also possible that theophylline decreases desensitization to P-agonists in tracheal smooth muscle. The aim of this study was to reproduce the tachyphylaxis phenomenon caused by isoprenaline incubation in guineapig trachea and t o determine whether theophylline prevents isoprenaline tachyphylaxis.

Materials and Methods The study was approved by the Animal Ethics Committee of the Medical Faculty of the University of Goteborg (DNo 157/90).

Preparations. Eight female Dunkin-Hartley guinea-pigs (230-360 g) were killed by inhaling carbon dioxide for 3 min. in a closed box. The trachea was rapidly removed and put in an oxygenated dissection bath filled with Krebs-Ringer solution at room temperature. The solution had the following composition in mmol/l, NaCl 118; KCI 5.9; CaCI, 2.5; MgSO, 1.2; NaH2P0, 1.2; NaHCO, 25.5 and glucose 5.6. Ascorbic acid (0.3 mM) was added to the Krebs-Ringer as an antioxidant. The intermediate trachea was dissected free and mounted as four isolated and frontally-opened rings in 7 ml organ baths with temperated (38") and oxygenated (94% O2-6U/o COJ Krebs-Ringer solution. These baths could be flushed with fresh Krebs-Ringer, 1.3 ml/min. The isometric muscle tension was recorded continually using Grass force transducers (FT03) connected to a NB-MTO-I 6 analogue/digital converting board and a Macintosh I1 computer using the LabVIEW 11 signal-processing software (National Instruments, Austin, Texas, U.S.A.). This system permits real-time monitoring of the experiment and simultaneous and continuous saving of data for subsequent evaluation. Experimental design. The preparations were first pre-stretched to 49 mN and then readjusted to 29 mN repeatedly after 10, 20 and 30 min. After 30 rnin., theophylline (1 mM) was added to half the preparations and, 10 min. later, two wash-outs were made and flushing was continued for a further 30 min. After equilibration, flushing was stopped and the preparations were contracted by 10 pM of histamine (approximately EC," in preliminary studies). After 15 rnin., the contraction had stabilized and a first cumulative concentration-response curve was obtained by adding incremental concentrations of isoprenaline at 6-min. intervals (fig. I). After the concentration-response experiment, three wash-outs were performed. A 60-min. incubation period followed before a second concentration-response experiment for isoprenaline. During the incubation period, four different treatments were given. The first group was a control group and no drugs were added between the isoprenaline concentration-response curves. The second group received 1 mM of theophylline and the third group received 3 pM of isoprenaline during the incubation period. The fourth group was treated with 1 mM of theophylline and, after 5 min., 3 pM of isoprenaline was also added. After the incubation period the prepa-

42

A. BERGENDAL ET AL. Hist

1,o

-

lsopren conc. reap.

I

I

I

0

20

40

Hist

Incub

I

w

Theo

Isopren conc. rerp.

I

I

I 60

I

4a

10

0

SottHIst

I

80

Tlme (mln)

M and last time 3 x Fig. 1. Experimental design shown in an original recording. Abbreviations: Hist, histamine M); Isopren conc resp., isoprenaline concentration-response curve (3 x 10-9-10-6 M); WO, wash out performed 3 times; Incub, incubation (60 min.); Theo, theophylline (lo-' M); Sot, sotalol (lo-' M). 1 g=9.8 mN.

rations were washed three times and were then allowed to settle for 20 min. At this point, a new histamine contraction was induced at the same concentration and the second concentration-response experiment for isoprenaline was performed. The experiment ended with the addition of I mM of theophylline in order to ascertain the maximum relaxation and, after three wash-outs, sotalol(10 pM) and histamine (0.3 mM) were added to obtain maximum contraction. Data analysis. pD, values (-log EC,,) have been calculated by interpolation between the values achieved above and below 50% of the maximum effect on the concentration-response curves. Tachyphylaxis is expressed as the difference in pD, before and after incubation in each preparation. The data is presented as mean [S.E.M.]. A one way analysis of variation was used to evaluate any significant variation due to treatment group. If a significant variation was found, Fischer's least significant difference was used to evaluate differences between individual treatment groups. Any difference in the degree of contraction during the first and second histamine contraction was evaluated using Student's t-test (two tail) for paired data using 95% confidence intervals. A standard statistical package, Stat View 11. on Macintosh was utilized.

Discussion This study demonstrated that P-adrenoceptor tachyphylaxis in isolated guinea-pig trachea evoked by isoprenaline incubation was a very small yet significant effect. We could not show that theophylline prevented tachyphylaxis. The isoprenaline concentration chosen to induce tachyphylaxis was based on earlier studies in which the concentrations varied from 0.1 pM to 10 pM during the incubation period (Avnet et al. 198I ; Bretz et al. 1983; Davis & Conolly 1980; Douglas et al. 1977; Lin et al. 1977). We used 3 pM of isoprenaline, as higher concentrations could not be washed out in spite of repeated wash-out procedures. Theophylline was used in a concentration of 1 mM, which is 10 times higher than the therapeutic plasma range in humans, and we therefore considered it of no interest to examine higher concentrations. Earlier studies in vitro have demonstrated tachyphylaxis

Drugs. Histamine dihydrochloride (Merck), ( +)-isoprenaline hemisulphate (Sigma), ( )-sotalol hydrochloride (gift from AB Hassle) and theophylline (gift from AB Draco) were all dissolved in distilled water. Control

lsoprenallne

Results There was no significant difference in the degree of contraction after the first and the second histamine contraction (95% confidence interval: -5.5 to 3.8%). The analysis of variance did not reveal any significant differences in pD2values during the first isoprenaline concentration-response curves depending on the treatment group. Isoprenaline incubation (3 pM) produced a rightward shift of the isoprenaline concentration-response curve (fig. 2). The mean shift was small, approximately 0.2 log units, but significantly different from the control group (table I). The presence of 1 mM of theophylline alone during incubation did not induce any significant shift of the isoprenaline concentration-response curve. Neither did theophylline inhibit the isoprenaline-induced shift of the concentration-response curve, even if there was a tendency towards a protective effect.

lsoprenallne t theophylllne

Theophylllne 100-

12 : ap

0. '-9 10

I

I-8

I-7

10 10 lsoprenallne (M)

'-6 10

ld9

los lo7 lsoprenallne (M)

lo6

Fig. 2. Mean [S.E.M.] isoprenaline concentration-response curves before (filled squares) and after (open circles) various treatments.

43

TACHYPHYLAXIS TO ISOPRENALINE IN ISOLATED TRACHEA Table 1. Isoprenaline pD,-values [S.E.M.] and % of maximum contraction before and after various treatments, * = P < 0.05. YUof maximum histamine contraction

Treatment Control n=6

Theophylline

7.59 (0.05)

n=6

Isoprenaline n=8 Isoprenaline

n=6

Difference in pD, before and after incubation -0.07 (0.02)

pD, during first isoprenaline curve 7.78 (0.08)

7.73 (0.05)

+ Theophylline

First histamine contraction 93 ( 5 )

Second histamine contraction 95 (10)

r

0.01 (0.06)

L

0.14 (0.03)

94 (2)

88 (3)

0.07 (0.04)

91 (2)

90 (3)

*

7.61 (0.05)

in isolated trachea from both rat and guinea-pig and from human bronchus (Avner & Jenne 1981; Davis & Conolly 1980; Douglas et al. 1977; Lin et al. 1977). We have used a similar protocol, but in earlier studies the magnitude of the shift was greater. Species variations and small differences in the design might explain the difference. In a study involving guinea-pig trachea (Douglas et al. 1977), the shift was somewhat larger than ours but still of the same order of magnitude. Human data revealed a more pronounced shift (Avner & Jenne 1981; Davis & Conolly 1980). The data from rat experiments is difficult to compare with our data as full concentration-response experiments were not performed in the study by Lin et al. (1977). Tachyphylaxis after in vivo treatment with P-agonists for 4-5 days has been shown in vitro in guinea-pig trachea (Holmberg et al. 1981). In this study, the shift of the isoprenaline concentration-response curve was also small, i.e. less than one log unit. The same study also demonstrated that tachyphylaxis is more pronounced in skeletal muscle than in bronchial smooth muscle. Clinical studies in man present more conflicting results. It is quite clear that non-bronchial P,-adrenoceptor responses (i.e. tremor, increase in heart rate, decrease in serum potassium) decrease with the prolonged use of P-agonists (Larsen & Hermansen 1977; Lipworth et al. 1989; Svedmyr et al. 1992). The development of tachyphylaxis to the bronchodilating effect in healthy individuals has been shown, but no tachyphylaxis in airways was seen in asthmatic patients under similar test conditions (Harvey et al. 1981; Holgate et al. 1977; Tashkin et al. 1982; van den Berg et al. 1982). The shift of the isoprenaline concentration-response curve in the present study was not significantly changed by theophylline even if there was a tendency towards a protective effect, and it was not possible to show any isoprenalineinduced tachyphylaxis in the presence of theophylline. The tachyphylaxis produced by isoprenaline was small and evaluating any changes might therefore prove difficult. However, the lack of any preventive effect by theophylline on tachyphylaxis contrasts with the effects by theophylline shown in rat aortic strips and in isolated leucocytes (Fleisch & Titus 1972, Otto et al. 1989). Our results indicate

97 ( 6 )

that there are either species differences o r organ differences relating to the theophylline effect on P-adrenoceptors and the effects mediated by them. In conclusion, our in vitro data in guinea-pig supports the theory that P,-adrenoceptor tachyphylaxis is a problem of minor importance in the P-adrenoceptor therapy, and we were unable to demonstrate any effect by theophylline on this very small tachyphylaxis. Acknowledgements Many thanks to Lena Bernsten for teaching the in vitro technique and for assistance during the experiments. The study was supported by grants from the Swedish Medical Research Council and from the Swedish Heart-Lung Foundation.

References Avner, B. P. & J. W. Jenne: Desensitization of isolated human bronchial smooth muscle to beta-receptor agonists. J. Allergy Clin. Immunol. 1981, 68, 51-57. Berti, F., L. Daffonchio, G . C. Folco, C. Omini & T. Vigano: Desensitization of beta-adrenoceptors in guinea-pig trachea: a prostaglandin mediated phenomenon. J. Auton. Pharmacol. 1982, 2, 247-253. Bretz, U., U. Martin, L. Mazzoni & U. M. Ney: Beta-adrenergic tachyphylaxisin the rat and its reversal and prevention by ketotifen. Eur. J. Pharmacol. 1983, 86, 321-328. Brodde, 0.-E., U. Howe, S. Egerszegi, N. Konietzko & M. C. Michel: Effect of prednisolone and ketotifen on beta2-adrenoceptors in asthmatic patients receiving beta2-bronchodilators. Eur. J. Clin. Pharmacol. 1988, 34, 145-150. Conolly, M. E., K. K. Hui, S. E. Borst & J. W. Jenne: Betaadrenergic tachyphylaxis (densensitization) and functional antagonism. In: Drug therapy for asthma. Research and clinical practice. Eds.: J. W. Jenne and S . Murphy. New York, Basel, Marcel Dekker 1987, 259-296. Davis, C. & M. E. Conolly: Tachyphylaxis to beta-adrenoceptor agonists in human bronchial smooth muscle: studies in vitro. Brit. J. Clin. Pharmacol. 1980, 10,417423. Douglas, J. S., A. J. Lewis, P. Ridgway, C. Brink & A. Bouhuys: Tachyphylaxis to beta-adrenoceptoragonists in guinea pig airway smooth muscle in vivo and in vitro. Eur. J. Pharmacol. 1977, 42, 195-205. Elfellah, M. S. & M. J. Turnbull: Effect of pretreatment with bronchodilator drugs on in vitro responsiveness of guinea pig lung adenylate cyclase. Eur. J. Pharmacol. 1978, 51, 211-217.

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Ellul-Micdllef,R. & F. F. Fenech: Effect of intravenous prednisolone in asthmatics with diminished adrenergic responsiveness. Lancet 1975, 27, 1269-1271. Fleisch, J. H. & E. Titus: The prevention of isoproterenol desensitization and isoproterenol reversal. J. Pharmacol. Exp. Therap. 1972, 181,425433. Harvey, J. & A. Tattersfield: Airway response to salbutamol: effect of regular salbutamol inhalations in normal, atopic, and asthmatic subjects. Thorax 1982, 37,280-287. Harvey, J. E., C. J. Baldwin, P. J. Wood, K. G. M. M. Alberti & A. E. Tattersfield: Airway and metabolic responsiveness to intravenous salbutamol in asthma: effect of regular inhaled salbutam01. Clin. Sci. 1981, 60, 579-585. Holgate, S. T., C. J. Baldwin & A. E. Tattersfield: Beta-adrenergic agonist resistance in normal human airways. Lancet 1977, 2,375. Holmberg, E.. A. B. Jeppsson & B. Waldeck: Selective development of tolerance to beta-adrenoceptor agonists in skeletal muscle as compared with airway smooth muscle from the guinea-pig. Clin. Exp. Pharmacol. Physiol. 1981, 8, 49-56. Hui, K. K. P., M. E. Conolly & D. P. Tashkin: Reversal of human lymphocyte beta-adrenoceptor desensitization by glucocorticoids. Clin. Pharmacol. Therap. 1982, 32, 566-571. Larsen, J. J. & K. Hermansen: Pharmacodynamics of a new selective beta2-adrenergic bronchodilator 3-(4-methoxygenzylamino)-4hydroxy-alpha-(tert. butylaminomethy1)-benzyl-alcohol, HCI (QH25). Acta pharmacol. et toxicol. 1977, 40, 42-64. Larsson, S., N. Svedmyr & G. Thiringer: Lack of bronchial beta adrenoceptor resistance in asthmatics during long-term treatment with terbutaline. J. Allergy Clin. Immunol. 1977, 59, 93-100.

Lin, C.-S., L. Hurwitz, J. Jenne & B. P. Avner: Mechanism of isoproterenol-induced desensitization of tracheal smooth muscle. J. Pharmacol. Exp. Therap. 1977, 203, 12-22. Lipworth, B. J., A. D. Struthers & D. G. McDevitt: Tachyphylaxis to systemic but not to airway responses during prolonged therapy with high dose inhaled salbutamol in asthmatics. Amer. Rev. Respir. Dis. 1989, 140, 586-592. Otto, J., S . Giinther & R. Urbanek: The effects of theophylline on beta2-adrenoceptors on polymorphonuclear leukocytes of asthmatic children and juveniles. Eur. J. Pediatr. 1989, 149, 661-664. Spilker, B. & J. Tyll: On the question of tachyplaxis to isoproterenol in guinea pigs. Eur. J. Pharmacol. 1976, 36, 283-288. Svedmyr, N.: Action of corticosteroids on beta-adrenergic receptors. Amer. Rev. Respir. Dis. 1990. 141, S31LS38. Svedmyr, N. & C.-G. Lofdahl: Physiology and pharmacodynamics of beta-adrenergic agonists. In: Drug therapyfor asthma. Research and clinicalpractice. Eds.: J. W. Jenne and S. Murphy. New York, Marcel Dekker Basel, 1987, 177-21 1, Svedmyr, N. & C.-G. Lofdahl: Intravenous sympathomimetic drugs in acute severe asthma. In: Bronchial asthma, third edition. E. Weiss a. coworkers Littlepress 1992, in press. Tashkin, D. P., M. E. Conolly, R. 1. Deutsch, K. K. Hui, M. Littner, P. Scarpace & I . Abrass: Subsensitization of beta-adrenoceptors in airways and lymphocytes of healthy and asthmatic subjects. Amer. Rev. Respir. Dis. 1982, 125, 185-193. van den Berg, W., J. G . Leferink, J. K. Fokkens, J. Kreukniet, R. A. A. Maes & P. L. B. Bruynzeel: Clinical implications of druginduced desensitization of the beta receptor after continuous oral use of terbutaline. J . Allergy Clin. Immunol. 1982, 69, 410-417.

In vitro tachyphylaxis to isoprenaline in guinea-pig trachea: influence of theophylline?

We used frontally opened tracheal rings, contracted with 10 microM of histamine to produce beta-adrenoceptor tachyphylaxis by isoprenaline incubation ...
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