Role of phosphoinositide metabolism in functional antagonism of airway smooth muscle contraction by β-adrenoceptor agonists

European Journal of Pharmacology - Molecular Pharmacology Section, 172 (1989) 175-183 Elsevier

175

EJM90018

Role of phosphoinositide metabolism in functional antagonism of airway smooth muscle contraction by fl-adrenoceptor agonists R o n a l d G . M . V a n A m s t e r d a m 1,,, H e r m a n M e u r s 1, F r a n s B r o u w e r t, J a n Bert P o s t e m a Adiet Timmermans 2 and Johan Zaagsma 1

1.2,

i Department of Pharmacology and Therapeutics, University of Groningen, A. Deusinglaan 2, 9713 A W Groningen, and 2 Department of Allergology, University Hospital, Oostersinge159, 9713 EZ Groningen, The Netherlands" Received 6 October 1988. revised MS received 6 February 1989, accepted 21 February 1989

Histamine and the muscarinic agonists, methacholine, oxotremorine, and McN-A-343, were used to contract guinea-pig tracheal smooth muscle preparations. Cumulative dose-relaxation curves with isoprenaline were performed subsequently. In addition, the concentration-dependent induction of phosphoinositide metabolism by the contractile agonists was measured in bovine tracheal smooth muscle. All agonists were found to induce a decrease of the apparent affinity of isoprenaline and a loss of relaxation, depending on the concentration and type of contractile agonist used. The differential effects of the contractile agonists, especially at higher and supramaximal concentrations, on these fl-adrenergic parameters could be explained by differences in phosphoinositide metabolism. Functional antagonism; Smooth muscle (airway); Muscarinic receptor agonists; Histamine; Phosphoinositide metabolism; fl-Adrenergic relaxation

1. Introduction

fl-Adrenoceptor agonists are known to become less effective in the treatment of asthma as the asthmatic episodes become more severe (Bouhuys, 1978; Barnes and Pride, 1983). This phenomenon could be explained from the observation that the potency and degree of the relaxation induced in various isolated airway preparations by fl-adrenoceptor agonists are gradually reduced in the presence of increasing concentrations of contractile agonists or mediators involved in the asthmatic reaction. Examples are muscarinic agonists (Van den Brink, 1973; Torphy et al., 1983; Raffestin et

* To whom all correspondence should be addressed: Dept. of Pharmacology and Therapeutics, University of Groningen, A. Deusinglaan 2, 9713 AW Groningen, The Netherlands.

al., 1985), histamine (Russell, 1984; Raffestin et al., 1985), serotonin (Russell, 1984) and leukotriene D 4 (Torphy, 1984). A functional antagonism between isoprenaline and methacholine, histamine or serotonin, respectively was recently also investigated in vivo using a canine model (Jenne et al., 1987). The data suggested that fl-adrenoceptor functioning is determined not only by the degree of contraction induced by the agonists but also by the nature of the agonist used. The loss of relaxation was most prominent for the muscarinic agonists, acetylcholine and methacholine, and was less for histamine, serotonin and leukotriene D 4 (Russell, 1984; Torphy, 1984). The biochemical mechanism of functional antagonism is still unclear. One site for the interaction between contractile and relaxant pathways in airway smooth muscle could be at the process of cyclic adenosine 3',5'-monophosphate (cAMP)

0922-4106/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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accumulation. This was suggested by a study of Torphy et al. (1985), who showed that methacholine concentrations causing a reduced fl-adrenergic relaxability of canine tracheal smooth muscle in response to isoprenaline also caused a reduced cAMP response to this agonist. The latter effect might be the consequence of interactions between the signal transduction mechanisms of contractile and fl-adrenergic stimuli. It has been demonstrated that contractile stimuli such as acetylcholine may cause inhibition of adenylate cyclase via an inhibitory guanine nucleotide-dependent regulatory protein (Gi) (Jones et al., 1987) which could antagonize the stimulation of adenylate cyclase by fl-adrenoceptor agonists, the latter process being mediated by the stimulatory regulatory protein (G,). Another transduction mechanism of contractile stimuli is phosphoinositide (PI) breakdown, which results in the generation of inositol phosphates and diacylglycerol (Grandordy et al., 1986, Takuwa et al., 1986). Inositol phosphates are involved in Ca 2+ movements, whereas diacylglycerol activates protein kinase C (Berridge, 1987). We have previously demonstrated that direct activation of protein kinase C in mononuclear leukocytes by the phorbol ester phorbol 12-myristate, 13-acetate (PMA) caused a reduced ~-adrenoceptor function, presumably by an alteration in the stimulatory regulatory protein (G,) (Meurs et al., 1986). Thus, apart from the activation of Gi, the latter mechanism may also be involved in the functional antagonism of /3-adrenoceptor agonist-induced cAMP generation and subsequent relaxation. We used for the present study three muscarinic agonists, methacholine (non-selective), oxotremorine (M 2 receptor-selective) and McN-A-343 (M 1 receptor-selective), known to have a different efficacy for stimulating muscarinic receptors in different systems (Brown et al., 1985; Leung et al., 1986) and, also histamine to contract guinea-pig tracheal smooth muscle. The functional antagonism of these contractions (induced by different concentrations of the agonists) by isoprenaline was studied. In addition, the dose-dependent generation of inositol phosphates by the agonists was determined in bovine tracheal smooth muscle. The aim of the study was to assess a

possible relationship between the relaxant behaviour of isoprenaline and the PI metabolism induced by the contractile agonists.

2. Materials and methods 2.1. Mechanical responses

Adult (400-600 g) outbred guinea-pigs of either sex were killed by a sharp blow on the head and were exsanguinated. The tracheas were rapidly removed and placed in Krebs-Henseleit (KH) solution (37°C) of the following composition (mM): NaC1 117.5, KC1 5.6, MgSO4 1.18, CaC12 2.5, NaH2PO 4 1.28, NaHCO 3 25.0 and glucose 5.5, gassed with 5% CO2 and 95% 02, pH 7.4. The tracheas were prepared free of serosal connective tissue and single ring preparations were mounted for isotonic recording in 20 ml water-jacketed organ baths (37 ° C) in KH solution using a 300 mg load. The tracheal smooth muscle was relaxed after equilibration for 30 min, using isoprenaline 0.1 /~M to establish the basal tone. The muscle was washed rapidly for 30 min and was contracted twice, using 10 #M and 10 + 90 #M methacholine, respectively, with a washing interval of 30 min. Following another hour of washing, the preparations were contracted with various concentrations of methacholine, oxotremorine, McN-A-343 or histamine and cumulative dose-relaxation curves were then made with isoprenaline. After the maximal response to the final concentration of isoprenaline had been obtained, the tissue was washed twice and 10/~M isoprenaline was added in order to again elicit maximal relaxation of the tissue. 2.2. Inositol phosphate measurement

Fresh bovine tracheas obtained from the slaughterhouse were transported to the laboratory within 30 min in ice-cold KH solution pregassed with 5% CO z and 95% 02. Tracheal muscle was dissected carefully and strips (12 × 3 mm) were prepared free of mucosa and serosal tissue in KH

177

solution at room temperature. The tissue was stored overnight at 2 4 ° C in a 20 ml volume of K H solution continuously refreshed at a superfusion rate of 3.9 m l / m i n . Total inositol phosphates accumulation was measured essentially as described by Grandordy et al. (1986). Tracheal strips, weighing a total of about 4 g, were chopped with a Mcllwain tissue chopper, twice at a setting of 500 ffm followed by three times at a setting of 100 ffm. The tissue particles were washed four times in K H containing 5 mM LiC1 ( K H / L i C 1 ) and were then incubated with 50 /~Ci of [3H]inositol in a 15 ml volume of the same medium for 60 min at 37 ° C under continuous carbogenation and gentle shaking. LiC1 was added to enhance the accumulation of inositol phosphates by blocking the breakdown of inositol monophosphate to inositol (Berridge et al., 1982). After incubation, the tissue was washed twice with K H / L i C 1 and was finally suspended in a volume of 20 ml of this medium. Aliquots of 450 /~1 were incubated with 50 ffl of the agonist in K H / L i C 1 or vehicle for 25 min at 37 ° C in capped tubes pregassed with 5% CO 2 and 95% 02. Separate experiments had shown that inositol phosphates accumulation under these conditions is linear for at least 30 min. The incubations were terminated by adding 500 ttl of ice-cold 10% ( w / v ) trichloroacetic acid (TCA) to the samples, followed by vigorous shaking. After standing for 30 min on ice, the precipitated protein was removed by centrifugation and 800 /~1 of the supernatants was extracted three times with four volumes of water-saturated diethyl ether. The extracts were diluted to 10 ml with water and were applied to columns containing approximately 1 ml of Dowex A G 1X8 anion exchange resin in the formate form. The columns were washed with 10 ml water, followed b y the s e q u e n t i a l e l u t i o n of glycerophosphoinositol with 15 ml of 5 mM disodium tetraborate/30 m M sodium formate and total inositol phosphates with four times 2 ml of 0.1 M formic acid/1.0 M ammonium formate. The inositol phosphates samples were added to 15 ml of scintillation cocktail (Plasmasol ®, Packard, Groningen, The Netherlands) and were counted in a liquid scintillation spectrometer with an efficiency of about 35%.

2.3. Data analysis Contraction of tracheal muscle was expressed as a percentage of the response to 0.1 m M methacholine. The basal tone defined by the previous response to 0.1 ffM isoprenaline. The p D 2 ( - l o g ECs0 ) values of isoprenaline were evaluated from the dose-relaxation curves according to Van Rossum (1963). The maximal relaxing effect of isoprenaline (E .... ) was expressed as percentage of the full relaxation with 10 ktM isoprenaline determined after wash-out of the contractile agonist. Inositol phosphates accumulation was expressed as a percentage of the response to 1 m M methacholine. The data are expressed as means + S.E.M. The statistical significance of differences was estimated using the two-tailed Student's t-test.

2.4. Materials Methacholine-HC1, oxotremorine, histamineHC1 and (-)-isoprenaline-HC1 were obtained

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from Sigma Chemical Co. (St. Louis, MO). M c N A-343 was donated by M c N e i l Pharmaceuticals (Spring House, PA) and [3H]inositol (specific radioactivity 45-80 C i / m m o l ) was purchased from N e w England N u c l e a r (Boston, MA).

100

3. Results The contractile responses of the guinea-pig tracheal s m o o t h m u s c l e to the various agonists are depicted in fig. 1. M e t h a c h o l i n e and oxotremorine

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-log isoproterenol (M) Fig. 2. Relaxation of guinea-pig tracheal smooth muscle by isoprenaline after contraction caused by increasing concentrations of histamine (A), McN-A-343 (B), oxotremorine (C) and methacholine (D) (see inset). Isoprenaline pD2 values and percentage maximal relaxation are given (in the figure) as means + S.E.M. Contraction levels are expressed as percentages of the response to 0.1 mM methacholine in each experiment. The numbers of experiments are indicated in parentheses.

179 were full m u s c a r i n i c agonists, whereas M e N - A - 3 4 3 p r o d u c e d a b o u t 80% a n d h i s t a m i n e 90% of the maximal methacholine-induced contraction. The p o t e n c y ( p D 2) sequence was o x o t r e m o r i n e (7.22 + 0.04) > m e t h a c h o l i n e (6.30 _+ 0.12) > h i s t a m i n e (5.47 + 0.09) > M c N - A - 3 4 3 (4.83 + 0.17). T h e p D 2 a n d the m a x i m a l effect of the isopren a l i n e - i n d u c e d r e l a x a t i o n d e p e n d e d strongly on the c o n c e n t r a t i o n a n d n a t u r e of the contractile a g o n i s t used, as is illustrated in fig. 2. F o r example, with 0.3 ffM histamine, 1 ffM M e N - A - 3 4 3 or 0.03 ffM m e t h a c h o l i n e that i n d u c e d c o m p a r a b l y low c o n t r a c t i o n levels of a b o u t 25-30%, strikingly similar p D 2 values were o b t a i n e d for i s o p r e n a l i n e (8.85 + 0.14, 8.83 + 0.22 or 8.72 + 0.17, respectively) while the c o n t r a c t i o n s were fully reversed. A t i n t e r m e d i a t e c o n t r a c t i o n levels of a b o u t 65%, the p D 2 values for i s o p r e n a l i n e varied from 7.68 + 0.09 with 1 ~tM m e t h a c h o l i n e to 8.21 + 0.27 with 10 ffM M e N - A - 3 4 3 (P < 0.01). W h e n high conc e n t r a t i o n s of m e t h a c h o l i n e a n d o x o t r e m o r i n e were used, the p D 2 values for i s o p r e n a l i n e were f o u n d to be decreased further to 6.77 + 0.07 for 0.1 m M m e t h a c h o l i n e a n d 7.16 + 0.11 for 0.1 m M o x o t r e m o r i n e . A t such ( s u p r a ) m a x i m a l c o n c e n t r a tions, not o n l y were the p D 2 values d e c r e a s e d

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Fig. 4. Relationship between (A) pECso values for isoprenaline relaxation of guinea-pig tracheal smooth muscle and the logarithm of inositol phosphates accumulation of bovine trachea and (B) maximal isoprenaline relaxation of guinea-pig tracheal muscle and inositol phosphates accumulation in bovine trachea as obtained in the presence of different concentrations of methacholine (e), oxotremorine (D), MeN-A-343 (o) and histamine (0)- The values were derived from the data as presented in figs. 2 and 3 and were correlated separately for histamine and the muscarinic agonists in panel A (r = correlation coefficient). The slopes of the regression lines are given in the text.

180 of isoprenaline were found in the sequence McNA-343 < histamine < oxotremorine < methacholine. The amount of inositol phosphates accumulation induced in bovine trachea by the four contractile agonists used (fig. 3) differed from the contractile response to these agonists. In contrast to the contractile response, the inositol phosphates production by histamine and oxotremorine reached less than 50% of the methacholine-induced effect and only a 6.5% effect was obtained with McN-A-343. The inositol phosphates response to different concentrations of the contractile agonists were compared with the pD 2 and Ema x values of isoprenaline. The pD 2 values of isoprenaline at all concentrations of the different agonists showed a highly significant inverse correlation with inositol phosphates accumulation (fig. 4A). However, the slopes of the regression lines for the muscarinic agonists ( - 1.03) and for histamine ( - 0.59) were different. Consequently, with concentrations of oxotremorine (1/~M) and histamine (10/~M) that were equieffective on IP accumulation different pD 2 values were found for isoprenaline, 7.22 + 0.10 and 8.00 + 0.16, respectively (P < 0.001). Also the maximal relaxation caused by isoprenaline was inversely correlated with inositol phosphates accumulation (fig. 4B). The latter relationship was similar for histamine and the muscarinic agonists. Thus, the increase in phosphoinositide metabolism caused by histamine and by the muscarinic agonists resulted in a similar decrease of the maximal relaxation induced by the/3-agonist. From the data presented in fig. 4B it can be calculated that, with contractile agonist concentrations inducing less than 14% of the maximal inositol phosphates production, the contractions were fully antagonized by isoprenaline. This is illustrated by the fact that the fully reversible contractions induced by all the concentrations of McN-A-343 were accompanied by an inositol phosphates response of 6.5% maximally (fig. 3). Increasing the histamine concentration from 10 to 100/~M induced a reduction of the Ema x values of isoprenaline whereas the apparent affinity remained the same. This confirms previous data (Torphy et al., 1985). However, increases in the concentrations of oxotremorine and methacholine

further reduced the pD 2 and Ema x values of isoprenaline until the maximal inositol phosphates accumulation was reached (cf. fig. 3).

4. Discussion

The present study demonstrated that the apparent affinity and the maximal relaxant effect of isoprenaline in the functional antagonism of tracheal smooth muscle contraction by histamine and acetylcholine receptor agonists depend strongly on the nature and the concentration of the contractile agonist and not on the contraction level per se. This was particularly true for high contraction levels as also found by Torphy (1984). We have recently demonstrated that the muscarinic agonists methacholine, oxotremorine and McN-A-343, have a different efficacy to induce PI metabolism in bovine tracheal smooth muscle. A significant linear correlation was found between the contractile response (pECs0 value) and PI metabolism (pEC3. 5 value), suggesting a direct relationship between the two parameters (Meurs et al., 1988). In addition to having an effect on the contractile potencies of the various agonists, PI metabolism could also be involved in the functional antagonism of the contraction by fl-adrenoceptor stimulation. Since the potency (pEC3. 5) of the contractile agonists to induce PI metabolism in bovine trachea (fig. 3) was also found to correlate linearly ( r = 0.948) with their contractile potencies (pECs0) in guinea-pig trachea (fig. 1), we considered the possibility of a relationship between contractile agonist-induced PI breakdown and pD 2 and Ema x for isoprenaline-induced relaxation. We found that the increase of inositol phosphates accumulation caused by the contractile agonists, rather than the contraction per se, is directly related to the reduction of pD 2 and Ema x values of isoprenaline. Differences between the agonists became clear at high concentrations of the contractile agonists, when increases of PI metabolism were no longer paralleled by increases of tracheal smooth muscle contraction. Full relaxation in response to isoprenaline was still obtained with a guinea-pig trachea contracted with a concentration of McN-A-343 that induced about

181

80% contraction but a maximal inositol phosphates response of only 6.5% (cf. fig. 3). However, only partial relaxation was obtained with the agonists that induced similar (sub)maximal contraction levels but higher (> 14%) inositol phosphates accumulation. Our findings clearly suggest a functional (perhaps causal) relationship between the levels of PI metabolism and of/3-adrenoceptor function in the airway smooth muscle. Two possible explanations could account for this: (1) competition of the two transduction mechanisms involving adenylate cyclase activation and PI breakdown for the functional response and (2) interference of the inositol phosphates response with the /~-adrenoceptormediated activation of adenylate cyclase. The second possibility is supported by the observation of Torphy et al. (1985) that the reduction in isoprenaline relaxability of tracheal smooth muscle was parallelled by a reduced cAMP accumulation. The report of Meurs et al. (1986) that direct activation of protein kinase C with PMA caused a reduced fl-adrenoceptor function also supports this possibility. Although the involvement of phosphoinositide metabolism in the functional antagonism of fladrenoceptor agonists could apply to contractile agonists in general, the behavior of histamine deviated somewhat from that of the muscarinic agonists. The decrease in the relaxation in response to isoprenaline (Emax) a t increasing concentrations of histamine showed the same linear relationship with PI metabolism (fig. 4B). The apparent affinity (pD2) of isoprenaline was, however reduced less than could have been expected from the accumulation of inositol phosphates as found with the muscarinic agonists. The mechanism that underlies the relative maintenance of isoprenaline potency with increasing histamine concentrations is still unclear. An obvious explanation could be that species differences between guinea-pig and calf lead to an overestimation of the inositol phosphates-accumulating potency of histamine in the guinea-pig. Thus the 37% level found for bovine airway smooth muscle with 10 - 4 M histamine may not have been reached in the guinea-pig. However, such an overestimation is unlikely because the loss of maximal

isoprenaline relaxability could indeed be predicted from the inositol phosphates accumulation as induced by the muscarinic agonists. Relaxant prostanoids, e.g. prostaglandin E 2 and prostacyclin, generated in response to histamine have been shown to suppress the maximal contractile effect of histamine (Orehek et al., 1975; Shore et al., 1985). In separate experiments we used 8.4 /~M of the cyclooxygenase inhibitor indomethacin, given 30 min prior to a cumulative histamine dose-response curve and the subsequent relaxation with isoprenaline. The maximal histamine-induced contraction (91.6 _+ 2.3) was significantly increased, to 102.3 _+ 3.7 (P < 0.02), in the presence of indomethacin, indeed indicating a contribution of relaxant prostanoids. However, in agreement with earlier findings (Russell, 1984), indomethacin did not significantly reduce the potency of isoprenaline (data not shown). An additive action of cAMP-generating prostaglandins can thus be excluded as explanation for the lower sensitivity to histamine of the isoprenaline pD 2 values. A third possibility may be the H 2 receptorstimulating property of histamine that results in stimulation of adenylate cyclase in lung parenchyma by as little as 1/~M (Foreman et al., 1985; 1986). This could have potentiated the isoprenaline-induced relaxation. However, 10/~M of the H 2 antagonist cimetidine, added 30 min prior to 1 mM histamine, was found not to influence the subsequent isoprenaline-induced relaxation (data not shown). However, in studying the effects of/~-agonists on PI hydrolysis in bovine tracheal smooth muscle, Hall and Hill (1988) reported that histamine-induced inositol phosphates accumulation was more strongly inhibited than the accumulation induced by carbachol. Similar effects were also observed by Madison and Brown (1988) on canine tracheas. Such a differential inhibition by isoprenaline of PI metabolism induced by histamine and methacholine during our functional experiments would imply a relative overestimation of the inositol phosphates response to histamine determined in the absence of the B-adrenoceptor agonist. It has been demonstrated that, following allergen provocation of allergic asthmatics, B-adren-

182 oceptor f u n c t i o n was reduced in the lymphocytes of these patients. This was m a i n l y due to a n alteration in the G s protein, causing u n c o u p l i n g of the receptor from adenylate cyclase (Meurs et al., 1987). A p p l i c a t i o n of the p h o r b o l ester P M A to n o n - c h a l l e n g e d lymphocytes resulted in a similar decrease (Meurs et al., 1986; 1987). It was concluded that activation of p r o t e i n kinase C is involved in the u n c o u p l i n g of the r - r e c e p t o r a n d adenylate cyclase, resulting in a loss of c A M P generation. T h a t a similar m e c h a n i s m could also apply to airway s m o o t h muscle was indicated b y a study of Dale a n d O b i a n i m e (1985) who f o u n d that P M A - c o n t r a c t e d guinea-pig lung p a r e n c h y m a was less sensitive to the relaxing effect of isoprenaline. Acetylcholine, the release of which m a y be caused by vagal reflexes d u r i n g asthmatic attacks (Cropp, 1975), could reach local c o n c e n t r a t i o n s that could well a c c o u n t for the PI m e t a b o l i s m needed to reduce fl-adrenoceptor function. Similarly, local c o n c e n t r a t i o n s of histamine as high as 1 m M are released d u r i n g allergic asthmatic attacks ( A d a m s a n d Lichtenstein, 1979). I n conclusion, b o t h the a p p a r e n t affinity a n d the m a x i m a l effect of fl-adrenoceptor agonists that i n d u c e airway smooth muscle relaxation, appear to be d e t e r m i n e d by the degree of PI t u r n o v e r i n d u c e d b y contractile agonists. The possibility however, r e m a i n s that the effect of PI m e t a b o l i s m on fl-adrenoceptor f u n c t i o n is parallelled b y a p r o p o r t i o n a l effect of G i - m e d i a t e d adenylate cyclase inhibition.

Acknowledgement This work was supported by the Netherlands Asthma Foundation.

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