Acta pharmacol. et toxicol. 1978, 42, 38 1-387
From the Department of Pharmacology, Linkoping University, Regionsjukhuset Linkoping, and the Allergy Clinic, Huddinge Hospital, Stockholm, Sweden
Inhibitory Effects of Imidazolines on Histamine Liberation from Human Leukocytes and on Tracheal Smooth Muscle Tone BY Rolf G . G . Andersson, Bjorn R. Lindgen and Helge Colldahl’)
(Received October 27, 1977; Accepted November 8, 1977)
Abstract: Antigen-induced IgE-mediated release of histamine from human leukocytes, an in vitro model of allergic reactions, was blocked by imidazole and imidazole-compoundssuch as oxymetazoline and clonidine.
The H-2-antihistaminesantagonized this effect of imidazolines. Alpha- and H-I-receptor blocking agents did not antagonize the effect. The contractile effects of the imidazolines were tested on tracheal preparations from the cow and guinea-pig.Imidazole was found to be a rather potent contracting agent, while oxymetazolineonly caused weak contractions. Clonidine relaxed the tracheal muscles, when used in the concentration range which were inhibitory in the leukocyteexperiments.The contractions caused by imidazolineswere non-competitively inhibited by clemastine, while the relaxing effects were blocked by a combination of propranolol, phentolamine and cimethidine. The results suggest that imidazolines which inhibit histamine release and relax bronchial smooth muscles may be of therapeutic importance in the treatment of human allergic disorders.
Key-words:
Imidazolines histamine release - tracheal contractility - man - guinea pig. ~
Bourne et al. (1972) have demonstrated that histamine-like P-adrenergic agonistsincrease the cyclic AMP level and inhibit the histamine release from human leukocytes. It was subsequently shown that these effects of exogenously applied histamine were blocked by H-2-receptor antagonists, but were unaffected by H- I-receptor antagonists (Lichtenstein & Gillespie 1975). No effect on either histamine release or cyclic A M P accumulation was observed from imidazole o r histidine compounds chemically related to histamine (Bourne et al. 1972). In an earlier paper, however, we demonstrated that high concentrations of imidazole inhibited the antigen-induced release of I)
Deceased on the 28th October, 1975.
histamine from the leukocytes of allergic donors in vitro (Andersson & Colldahl 1974). Other drugs (P-adrenergic agonists, prostaglandin E) which increase the cyclic A M P of leukocytes also inhibit histamine release (Lichtenstein & Margolis 1968). These drugs are also potent relaxing agents on most smooth muscle preparations (Andersson 1972). Alpha-adrenergic agonists on the other hand, increase the release of histamine from sensitized lung preparations (Kaliner et al. 1972). This type of drug is also able to contract human bronchial smooth muscles (Simonsson et al. 1972). In this work we have studied the inhibitory properties of imidazole and imidazole-derivates o n antigen-induced histamine release from human
382
R. G. G. ANDERSSON, B. R. LINDGREN AND H. COLLDAHL
leukocytes. We have also investigated the effects of these substances on tracheal smooth muscle tone in order to screen imidazole derivatives with properties which may be useful in therapy of asthma.
Materials and Methods Experimental procedure for human leukocytes Venous blood of normal or allergic human volunteers was subjected to sedimentation with 6% dextran containing 6 mM EDTA and the leukocytes were isolated as previously described (Lichtenstein & Osler 1964). The washed leukocytes were suspended in Tris-ACM and aliquots of the suspension were placed in tubes containing antigens and appropriate drugs. The histamine release was allowed to proceed for 45 min. at 37". At the end of the experiment the released amount of histamine was measured fluorometrically as described by Lichtenstein & Osler (1964), Colldahl (1973) and Lichtenstein & Gillespie (1975). The fluorescence readings for tubes containing leukocyte suspension in Tris ACM represent 'blank' values, which include the spontaneous release of histamine from the cells. Correction was made for the contribution of fluorescence from the reagents and the imidazole derivatives.
Experimental procedure for iracheal smooth muscle Samples of tracheal smooth muscle were obtained either from bovine trachea or from guinea-pig trachea. Smooth muscle preparations free from cartilage tissue and mucosa were prepared from bovine trachea. From guinea-pig, chains of tracheal rings were prepared according to Akcasu (1959) ; these preparations consisted of both muscle and cartilage tissue. The preparations were mounted isometrically and tension was recorded by FT03 force transducer on a Grass polygraph. The preparations were equilibrated in Krebs-Henseleit buffer solution gassed with 95% 0, and 5% CO, at 37" for 1 hour before the drugs were tested. The bovine trachea was given an initial tension of 4 g, and the corresponding load of guinea-pig trachea 1 g. Cumulative dose response curves were constructed for the different drugs either on untreated muscles or on muscles contracted with histamine. The following drugs were used: Imidazole (Merck AG) Clonidine hydrochloride (Boehringer Ingelheim) Oxymetazoline chloride (Draco) Tolazoline hydrochloride (Ciba-Geigy AG) Metaoxedrin chloride (neosynephrine@)(Winthrop lab.) Metiamide (SK& F labs. Ltd.) Cimetidine (SK & F labs. Ltd.) Clemastine hydrogen fumarate (tavegyl@)(Sandoz).
bp 100
80
60
40
20
.-=c
-20
Fig. 1. Inhibition of antigen-induced histamine release from human leukocytes by some imidazolines; (0-0) repre) and (0-0)imidazole. Each curve is the mean of 5-7 experisents clonidine, (X-X)oxymethazoline, (Htolazoline ments.
383
HISTAMINE LIBERATION A N D TRACHEAL TONE
Results Inhibitory eflects of imidazole derivates on antigeninduced histamine liberationfrom human leukocytes High concentrations of imidazole 1.5-15 x lo-’ M inhibited the antigen-induced histamine release (fig. 1). This effect was observed independently of the kind of antigen used and confirms earlier results (Andersson & Colldahl 1974). Among the imidazole-derivatives tested in our study, clonidine was most active, a 20-70% inhibition occurred in the range 3.8 x 10-6-0.1 x lo-’ M with this drug (fig. 1). Oxymetazoline caused inhibition with essentially similar patterns, and almost complete inhibition was noted at 0.7 x lo-’ M (fig. 1). Dose response curves carried out in the presence of tolazoline showed a maximal inhibition of 40% at
25 x M (fig. 1). Then the degree of inhibition diminished. The antigen-induced histamine release increased instead at a tolazoline concentration of 25x lo-’ M. The capacity of H-2-receptor antagonists to block the ability of clonidine to inhibit histamine M) release is shown in fig. 2. Clonidine (2 x caused about a 50% inhibition of the histamine release. The inhibition was almost completely blocked by 4 x M cimetidine, while a similar concentration of metiamide antagonized the clonidine effect by about 60%. Cimetidine by itself caused no inhibition of the release of histamine from leukocytes challenged with an appropriate antigen. Tolazoline, used as an a-adrenergic antagonist or clemastine could not prevent the effect of clonidine. The inhibition of the histamine
100
80
60
40
20
Ag
Clon Met
Cim
+
+
+
+
-
+
+ +
+
c
-
-
L +
+
+)on antigen (Ag)-induced histamine release in the absence and Fig. 2. Inhibitory effect of clonidine (Clon; 2 x presenceofmetiamide(Met;4x 10-’)orcirnethidine(Cim;4x lo-”). MeanfS.E.(n=5-7).
384
R. G . G . ANDERSSON, B. R. LINDGREN A N D H. COLLDAHL
release by tolazoline (25 x M) or by oxymetazoline was, however, inhibited by cimetidine. Effects of imidazole-derivates on tracheal smooth muscle tone
The contractile effect of some substances containing an imidazole-ring was tested on tracheal preparations from the cow and the guinea-pig. When comparing the effects of imidazole, clonidine and oxymetazoline in the dose range lo-'lo-' M, the unsubstituted imidazole-molecule was the most powerful contracting agent on the tracheal muscles of both the cow (fig. 3) and the guinea-pig. The oxymetazoline contraction of bovine trachea was about 1/5 of that of imidazole (fig. 3). In guinea-pig trachea, low concentrations of this drug (< 1.5 x lo-' M)caused a slight contraction, while higher concentrations relaxed the muscle. High concentrations of clonidine (> 4 x M) and tolazoline increased the tension in bovine trachea (fig. 3). Clonidine was found to have the opposite effect on the guinea-pig trachea. The contractile effect of imidazolines was com-
pared with an a-adrenoceptor agonist, metoxedrine. The tension caused by this drug was only about 1/10 of the tension caused by imidazole, and half of that of oxymetazoline. The contractile effect of imidazole was found to be inhibited by clemastine in a noncompetitive manner (fig. 4). This H-1 -receptor-antagonist also blocked the contractions caused by oxymetazoline and clonidine in bovine trachea. In order to study the inhibitory properties of imidazolines on tracheal muscle, a constant tension was established in bovine trachea with 5 x lo-' M histamine. Imidazole further increased the tension but oxymetazoline and clonidine decreased the tension of this preparation (fig. 5). The inhibitory effects of the two latter drugs were only partially reduced in the presence of cimethidine. The inhibitory effect of clonidine was abolished in tracheal preparations which were preincubated with a combination of propranolol, phentolamine and cimethidine. After this treatment clonidine, instead contracted the tracheas (fig. 6 ) .
I
-8
-6
-4
-2
log c o n c
(MI Fig. 3. Contractile effects of imidazolines (for symbols see fig. 1) and metaoxedrin (M - M) on bovine trachea. Mean k S.E.M.( n = 6 8 ) .
385
HISTAMINE LIBERATION AND TRACHEAL TONE Clem
1
clonidine A
R04
0,1
0,4
1,o
mM
A A
kist.
-5
-4
-3
0,04
0.4
i,o m M
-2
log conc
(MI
Fig. 4. Antagonistic effects of clemastine (Clem.) on imidazole-induced tracheal contraction. Concentrations of clemastine in (M) indicated in the figure.
Discussion Imidazole in high concentrations (> 1.5 x lo-’ M) was found to inhibit the antigen-induced histamine release from human leukocytes (fig. 1) (Andersson & Colldahl 1974). Bourne et al. (1972),
220
200
180
ae 180
dY
/
p
c
2
140
-
120
O
100
@ 0
80
m
c 0
a r V
10
40
Fig. 5. Influence of some imidazolines on tension development of bovine tracheal muscle contracted by histamine (5x lo-’ M). (Symbols are indicated in fig I). MeanfS.E.M. (n=5-7).
4
hist.
2
mln
Fig. 6. Effects of clonidine on tracheal smooth muscle tension. The preparations were contracted by histamine (5 x M) prior theclonidine addition. A, a test in the absence of antagonists. B, a test in the presence of proM), phentolamine (2 x M) and pranolol (3 x cimethidine (4 x M).
however, did not observe any effect of imidazole or imidazoleacetic acid in concentrations lower than lo-’ M. The potency of the inhibitory effect was increased in imidazolines containing a substituted benzyl-ring, viz, clonidine (2-2,6-dichloranilino)-2-imidazoline) and oxymetazoline (2- (4-tertbutyl-3-hydroxy-2,6-dimethylbenzyl)2fig. 1). Tolazoline (2-benzyl-2-imidazoline),classified as an a-adrenoceptor antagonist, on the other hand, showed an inhibitory effect only in low concentrations, while higher concentrations increased the histamine release. The effect of the imidazolines on the leukocytes was blocked by H-2-receptor antagonists but not by low concentrations of H-1- or a-receptor antagonists. Bourne et al. (1972) have previously reported that extracellular histamine inhibited the antigen-induced IgE-mediated release of histamine. This effect of histamine was also blocked by H-2-antagonists but not by H-l-antagonists (Lichtenstein & Gillespie 1975). In experiments on guinea-pig ileum it was observed that clonidine inhibited the release of acetylcholine in
386
R. G. G. ANDERSSON, B. R. LINDGREN AND H. COLLDAHL
cholinergic nerves; this effect was blocked by a-receptor antagonists while H-2-receptor antagonists had no effect on this preparation (Wikberg et al. 1975; Wikberg, personal communication). Our results favour the view of an H-Zreceptormediated effect of imidazolines in leukocytes. This assumption is further strengthened by the observation that an a-receptor agonist was almost ineffective in inhibiting histamine release from human leukocytes. The imidazole derivate, clonidine, has also been shown to be a potent agonist of H-2-receptors on slices from guinea-pig hippocampus (Audigier et al. 1976). These effects of imidazolines may be of value in the treatment of diseases in which histamine may play an important role in the pathogenesis, i.e. extrinsic asthma. Owing to this fact it also seemed interesting to study the effects of these drugs on bronchial smooth muscle tone. From our results imidazole was found to be a rather potent contracting agent both in the bovine and guinea-pig trachea, while clonidine and oxymetazoline mostly relaxed the tracheal muscles used (fig. 5). In low concentrations, however, oxymetazoline induced a contraction comparable to that of the a-adrenoceptor agonists, metaoxedrine, in bovine trachea. Clonidine had no contractile effects in the concentration range corresponding to that used for inhibiting histamine release. At higher concentrations, on the other hand, contractions could be demonstrated. The contractile effects of imidazole (fig. 4) and oxymetazoline were non-competitively antagonized by the H- 1-receptor antagonist clemastine. Whether the reactivity of human tracheal and bronchial tissues to imidazolines is quantitatively and qualitatively similar to those of cows and guinea-pigs remains to be studied. The treatment of hypertension in asthmatics with non-selective P-adrenoceptor antagonists may be hazardous because of the risk of severe bronchospasm (McNeil 1964). Selective P-adrenoceptor antagonists, however, have not been found to reduce the ventilatory capacity (Thiringer & Svedmyr 1976). In this study we have shown some effects of clonidine which may be favourable for asthmatics. Clonidine has been used as an antihypertensive drug for some years. It is therefore probable that clonidine is useful for the hyper-
tension treatment of asthmatics, and thus increases the therapeutic effect in this group of patients. In order to evaluate the usefulness of such a therapy, more clinical studies are needed. Acknowledgement Financial support has been provided by the Swedish Medical Research Council 0 4 x 4 3 8 .
References Akcasu, A. : The physiologic and pharmacologic characteristics of the tracheal muscle. Arch. int. Pharmacodyn 1959, 122, 201-207. Andersson, R. G. G. : Cyclic AMP and calcium ions in mechanical and metabolic responses of smooth muscle; Influence of some hormones and drugs. Acta physiol. scand. 1972, suppl. 382, 1-59. Andersson, R. G. G. & H. Colldahl: Relationship between antigen induced histamine release and cyclic AMP level in human leukocytes. Acta allergol. 1974, 29,248-255.
Audigier, Y., A. Virion & J. C. Schwartz: Stimulation of cerebral histamine H,-receptors by clonidine. Nature 1976,262,307-308.
Bourne, H. R., L. M. Lichtenstein & K. L. Melmon: Pharmacologic control of allergic histamine release in virro: Evidence for an inhibitory role of 3’5’ adenosine monophosphate in human leukocytes. J. Immunol. 1972,108,695-705.
Colldahl, H. :Changes in total histaminecontent of white blood cells in allergy patients. Acta allergol. 1973, 28, 3 16-332.
Kaliner, M., R. P. Orange & K. F. Austen: Immunological release of histamine and slow reacting substance of anaphylaxis from human lung. J. exp. Med. 1972,136,556-566.
Lichtenstein, L. M. & E. Gillespie: The effects of the HI and H2 antihistamines on allergic histamine release and its inhibition by histamine. J. Pharmacol. exp. Ther. 1975,192,441450.
Lichtenstein, L. M. & S. Margolis: Histamine release in virro : Inhibition by catecholamines and methylxanthines. Science 1968, 161,902-903. Lichtenstein, L. M. & A. G. Osler: Studies on the mechanism of hypersensitivity phenomena IX. Histamine release from human leukocytes by ragweed pollen antigen. J. exp. Med. 1964,120,507-530. McNeill, R. S.: Effect of a beta adrenergic blocking agent, propranolol on asthmatics. Lancet 1964, ii, 1101-1102.
Simonsson, B. G.,N. Svedmyr, B. E. Skoogh, R. Andersson & N. P. Bergh: In vivo and in vifrostudies on alphareceptors in human airways. Potentiation with bacterial endotoxin. Scand. J. r a p . Dis. 1972,53,227-236.
HISTAMINE LIBERATION A N D TRACHEAL TONE Thiringer, G. & N. Svedmyr: Interaction of orally administered metroprolol, practolol and propranolol with isoprenaline in asthmatics. Eur. J . d i n . Pharmacol. 1976, 10, 163-170.
387
Wikberg, J., R. Anderson & L. Lundholm: Differentiation of adrenergic a-receptors in guinea pig intestine and rabbit aorta. Blood Vessels 1975,12, 383.
From the Department of Pharmacology, Linkoping University, Regionsjukhuset Linkoping, and the Allergy Clinic, Huddinge Hospital, Stockholm, Sweden
Inhibitory Effects of Imidazolines on Histamine Liberation from Human Leukocytes and on Tracheal Smooth Muscle Tone BY Rolf G . G . Andersson, Bjorn R. Lindgen and Helge Colldahl’)
(Received October 27, 1977; Accepted November 8, 1977)
Abstract: Antigen-induced IgE-mediated release of histamine from human leukocytes, an in vitro model of allergic reactions, was blocked by imidazole and imidazole-compoundssuch as oxymetazoline and clonidine.
The H-2-antihistaminesantagonized this effect of imidazolines. Alpha- and H-I-receptor blocking agents did not antagonize the effect. The contractile effects of the imidazolines were tested on tracheal preparations from the cow and guinea-pig.Imidazole was found to be a rather potent contracting agent, while oxymetazolineonly caused weak contractions. Clonidine relaxed the tracheal muscles, when used in the concentration range which were inhibitory in the leukocyteexperiments.The contractions caused by imidazolineswere non-competitively inhibited by clemastine, while the relaxing effects were blocked by a combination of propranolol, phentolamine and cimethidine. The results suggest that imidazolines which inhibit histamine release and relax bronchial smooth muscles may be of therapeutic importance in the treatment of human allergic disorders.
Key-words:
Imidazolines histamine release - tracheal contractility - man - guinea pig. ~
Bourne et al. (1972) have demonstrated that histamine-like P-adrenergic agonistsincrease the cyclic AMP level and inhibit the histamine release from human leukocytes. It was subsequently shown that these effects of exogenously applied histamine were blocked by H-2-receptor antagonists, but were unaffected by H- I-receptor antagonists (Lichtenstein & Gillespie 1975). No effect on either histamine release or cyclic A M P accumulation was observed from imidazole o r histidine compounds chemically related to histamine (Bourne et al. 1972). In an earlier paper, however, we demonstrated that high concentrations of imidazole inhibited the antigen-induced release of I)
Deceased on the 28th October, 1975.
histamine from the leukocytes of allergic donors in vitro (Andersson & Colldahl 1974). Other drugs (P-adrenergic agonists, prostaglandin E) which increase the cyclic A M P of leukocytes also inhibit histamine release (Lichtenstein & Margolis 1968). These drugs are also potent relaxing agents on most smooth muscle preparations (Andersson 1972). Alpha-adrenergic agonists on the other hand, increase the release of histamine from sensitized lung preparations (Kaliner et al. 1972). This type of drug is also able to contract human bronchial smooth muscles (Simonsson et al. 1972). In this work we have studied the inhibitory properties of imidazole and imidazole-derivates o n antigen-induced histamine release from human
382
R. G. G. ANDERSSON, B. R. LINDGREN AND H. COLLDAHL
leukocytes. We have also investigated the effects of these substances on tracheal smooth muscle tone in order to screen imidazole derivatives with properties which may be useful in therapy of asthma.
Materials and Methods Experimental procedure for human leukocytes Venous blood of normal or allergic human volunteers was subjected to sedimentation with 6% dextran containing 6 mM EDTA and the leukocytes were isolated as previously described (Lichtenstein & Osler 1964). The washed leukocytes were suspended in Tris-ACM and aliquots of the suspension were placed in tubes containing antigens and appropriate drugs. The histamine release was allowed to proceed for 45 min. at 37". At the end of the experiment the released amount of histamine was measured fluorometrically as described by Lichtenstein & Osler (1964), Colldahl (1973) and Lichtenstein & Gillespie (1975). The fluorescence readings for tubes containing leukocyte suspension in Tris ACM represent 'blank' values, which include the spontaneous release of histamine from the cells. Correction was made for the contribution of fluorescence from the reagents and the imidazole derivatives.
Experimental procedure for iracheal smooth muscle Samples of tracheal smooth muscle were obtained either from bovine trachea or from guinea-pig trachea. Smooth muscle preparations free from cartilage tissue and mucosa were prepared from bovine trachea. From guinea-pig, chains of tracheal rings were prepared according to Akcasu (1959) ; these preparations consisted of both muscle and cartilage tissue. The preparations were mounted isometrically and tension was recorded by FT03 force transducer on a Grass polygraph. The preparations were equilibrated in Krebs-Henseleit buffer solution gassed with 95% 0, and 5% CO, at 37" for 1 hour before the drugs were tested. The bovine trachea was given an initial tension of 4 g, and the corresponding load of guinea-pig trachea 1 g. Cumulative dose response curves were constructed for the different drugs either on untreated muscles or on muscles contracted with histamine. The following drugs were used: Imidazole (Merck AG) Clonidine hydrochloride (Boehringer Ingelheim) Oxymetazoline chloride (Draco) Tolazoline hydrochloride (Ciba-Geigy AG) Metaoxedrin chloride (neosynephrine@)(Winthrop lab.) Metiamide (SK& F labs. Ltd.) Cimetidine (SK & F labs. Ltd.) Clemastine hydrogen fumarate (tavegyl@)(Sandoz).
bp 100
80
60
40
20
.-=c
-20
Fig. 1. Inhibition of antigen-induced histamine release from human leukocytes by some imidazolines; (0-0) repre) and (0-0)imidazole. Each curve is the mean of 5-7 experisents clonidine, (X-X)oxymethazoline, (Htolazoline ments.
383
HISTAMINE LIBERATION A N D TRACHEAL TONE
Results Inhibitory eflects of imidazole derivates on antigeninduced histamine liberationfrom human leukocytes High concentrations of imidazole 1.5-15 x lo-’ M inhibited the antigen-induced histamine release (fig. 1). This effect was observed independently of the kind of antigen used and confirms earlier results (Andersson & Colldahl 1974). Among the imidazole-derivatives tested in our study, clonidine was most active, a 20-70% inhibition occurred in the range 3.8 x 10-6-0.1 x lo-’ M with this drug (fig. 1). Oxymetazoline caused inhibition with essentially similar patterns, and almost complete inhibition was noted at 0.7 x lo-’ M (fig. 1). Dose response curves carried out in the presence of tolazoline showed a maximal inhibition of 40% at
25 x M (fig. 1). Then the degree of inhibition diminished. The antigen-induced histamine release increased instead at a tolazoline concentration of 25x lo-’ M. The capacity of H-2-receptor antagonists to block the ability of clonidine to inhibit histamine M) release is shown in fig. 2. Clonidine (2 x caused about a 50% inhibition of the histamine release. The inhibition was almost completely blocked by 4 x M cimetidine, while a similar concentration of metiamide antagonized the clonidine effect by about 60%. Cimetidine by itself caused no inhibition of the release of histamine from leukocytes challenged with an appropriate antigen. Tolazoline, used as an a-adrenergic antagonist or clemastine could not prevent the effect of clonidine. The inhibition of the histamine
100
80
60
40
20
Ag
Clon Met
Cim
+
+
+
+
-
+
+ +
+
c
-
-
L +
+
+)on antigen (Ag)-induced histamine release in the absence and Fig. 2. Inhibitory effect of clonidine (Clon; 2 x presenceofmetiamide(Met;4x 10-’)orcirnethidine(Cim;4x lo-”). MeanfS.E.(n=5-7).
384
R. G . G . ANDERSSON, B. R. LINDGREN A N D H. COLLDAHL
release by tolazoline (25 x M) or by oxymetazoline was, however, inhibited by cimetidine. Effects of imidazole-derivates on tracheal smooth muscle tone
The contractile effect of some substances containing an imidazole-ring was tested on tracheal preparations from the cow and the guinea-pig. When comparing the effects of imidazole, clonidine and oxymetazoline in the dose range lo-'lo-' M, the unsubstituted imidazole-molecule was the most powerful contracting agent on the tracheal muscles of both the cow (fig. 3) and the guinea-pig. The oxymetazoline contraction of bovine trachea was about 1/5 of that of imidazole (fig. 3). In guinea-pig trachea, low concentrations of this drug (< 1.5 x lo-' M)caused a slight contraction, while higher concentrations relaxed the muscle. High concentrations of clonidine (> 4 x M) and tolazoline increased the tension in bovine trachea (fig. 3). Clonidine was found to have the opposite effect on the guinea-pig trachea. The contractile effect of imidazolines was com-
pared with an a-adrenoceptor agonist, metoxedrine. The tension caused by this drug was only about 1/10 of the tension caused by imidazole, and half of that of oxymetazoline. The contractile effect of imidazole was found to be inhibited by clemastine in a noncompetitive manner (fig. 4). This H-1 -receptor-antagonist also blocked the contractions caused by oxymetazoline and clonidine in bovine trachea. In order to study the inhibitory properties of imidazolines on tracheal muscle, a constant tension was established in bovine trachea with 5 x lo-' M histamine. Imidazole further increased the tension but oxymetazoline and clonidine decreased the tension of this preparation (fig. 5). The inhibitory effects of the two latter drugs were only partially reduced in the presence of cimethidine. The inhibitory effect of clonidine was abolished in tracheal preparations which were preincubated with a combination of propranolol, phentolamine and cimethidine. After this treatment clonidine, instead contracted the tracheas (fig. 6 ) .
I
-8
-6
-4
-2
log c o n c
(MI Fig. 3. Contractile effects of imidazolines (for symbols see fig. 1) and metaoxedrin (M - M) on bovine trachea. Mean k S.E.M.( n = 6 8 ) .
385
HISTAMINE LIBERATION AND TRACHEAL TONE Clem
1
clonidine A
R04
0,1
0,4
1,o
mM
A A
kist.
-5
-4
-3
0,04
0.4
i,o m M
-2
log conc
(MI
Fig. 4. Antagonistic effects of clemastine (Clem.) on imidazole-induced tracheal contraction. Concentrations of clemastine in (M) indicated in the figure.
Discussion Imidazole in high concentrations (> 1.5 x lo-’ M) was found to inhibit the antigen-induced histamine release from human leukocytes (fig. 1) (Andersson & Colldahl 1974). Bourne et al. (1972),
220
200
180
ae 180
dY
/
p
c
2
140
-
120
O
100
@ 0
80
m
c 0
a r V
10
40
Fig. 5. Influence of some imidazolines on tension development of bovine tracheal muscle contracted by histamine (5x lo-’ M). (Symbols are indicated in fig I). MeanfS.E.M. (n=5-7).
4
hist.
2
mln
Fig. 6. Effects of clonidine on tracheal smooth muscle tension. The preparations were contracted by histamine (5 x M) prior theclonidine addition. A, a test in the absence of antagonists. B, a test in the presence of proM), phentolamine (2 x M) and pranolol (3 x cimethidine (4 x M).
however, did not observe any effect of imidazole or imidazoleacetic acid in concentrations lower than lo-’ M. The potency of the inhibitory effect was increased in imidazolines containing a substituted benzyl-ring, viz, clonidine (2-2,6-dichloranilino)-2-imidazoline) and oxymetazoline (2- (4-tertbutyl-3-hydroxy-2,6-dimethylbenzyl)2fig. 1). Tolazoline (2-benzyl-2-imidazoline),classified as an a-adrenoceptor antagonist, on the other hand, showed an inhibitory effect only in low concentrations, while higher concentrations increased the histamine release. The effect of the imidazolines on the leukocytes was blocked by H-2-receptor antagonists but not by low concentrations of H-1- or a-receptor antagonists. Bourne et al. (1972) have previously reported that extracellular histamine inhibited the antigen-induced IgE-mediated release of histamine. This effect of histamine was also blocked by H-2-antagonists but not by H-l-antagonists (Lichtenstein & Gillespie 1975). In experiments on guinea-pig ileum it was observed that clonidine inhibited the release of acetylcholine in
386
R. G. G. ANDERSSON, B. R. LINDGREN AND H. COLLDAHL
cholinergic nerves; this effect was blocked by a-receptor antagonists while H-2-receptor antagonists had no effect on this preparation (Wikberg et al. 1975; Wikberg, personal communication). Our results favour the view of an H-Zreceptormediated effect of imidazolines in leukocytes. This assumption is further strengthened by the observation that an a-receptor agonist was almost ineffective in inhibiting histamine release from human leukocytes. The imidazole derivate, clonidine, has also been shown to be a potent agonist of H-2-receptors on slices from guinea-pig hippocampus (Audigier et al. 1976). These effects of imidazolines may be of value in the treatment of diseases in which histamine may play an important role in the pathogenesis, i.e. extrinsic asthma. Owing to this fact it also seemed interesting to study the effects of these drugs on bronchial smooth muscle tone. From our results imidazole was found to be a rather potent contracting agent both in the bovine and guinea-pig trachea, while clonidine and oxymetazoline mostly relaxed the tracheal muscles used (fig. 5). In low concentrations, however, oxymetazoline induced a contraction comparable to that of the a-adrenoceptor agonists, metaoxedrine, in bovine trachea. Clonidine had no contractile effects in the concentration range corresponding to that used for inhibiting histamine release. At higher concentrations, on the other hand, contractions could be demonstrated. The contractile effects of imidazole (fig. 4) and oxymetazoline were non-competitively antagonized by the H- 1-receptor antagonist clemastine. Whether the reactivity of human tracheal and bronchial tissues to imidazolines is quantitatively and qualitatively similar to those of cows and guinea-pigs remains to be studied. The treatment of hypertension in asthmatics with non-selective P-adrenoceptor antagonists may be hazardous because of the risk of severe bronchospasm (McNeil 1964). Selective P-adrenoceptor antagonists, however, have not been found to reduce the ventilatory capacity (Thiringer & Svedmyr 1976). In this study we have shown some effects of clonidine which may be favourable for asthmatics. Clonidine has been used as an antihypertensive drug for some years. It is therefore probable that clonidine is useful for the hyper-
tension treatment of asthmatics, and thus increases the therapeutic effect in this group of patients. In order to evaluate the usefulness of such a therapy, more clinical studies are needed. Acknowledgement Financial support has been provided by the Swedish Medical Research Council 0 4 x 4 3 8 .
References Akcasu, A. : The physiologic and pharmacologic characteristics of the tracheal muscle. Arch. int. Pharmacodyn 1959, 122, 201-207. Andersson, R. G. G. : Cyclic AMP and calcium ions in mechanical and metabolic responses of smooth muscle; Influence of some hormones and drugs. Acta physiol. scand. 1972, suppl. 382, 1-59. Andersson, R. G. G. & H. Colldahl: Relationship between antigen induced histamine release and cyclic AMP level in human leukocytes. Acta allergol. 1974, 29,248-255.
Audigier, Y., A. Virion & J. C. Schwartz: Stimulation of cerebral histamine H,-receptors by clonidine. Nature 1976,262,307-308.
Bourne, H. R., L. M. Lichtenstein & K. L. Melmon: Pharmacologic control of allergic histamine release in virro: Evidence for an inhibitory role of 3’5’ adenosine monophosphate in human leukocytes. J. Immunol. 1972,108,695-705.
Colldahl, H. :Changes in total histaminecontent of white blood cells in allergy patients. Acta allergol. 1973, 28, 3 16-332.
Kaliner, M., R. P. Orange & K. F. Austen: Immunological release of histamine and slow reacting substance of anaphylaxis from human lung. J. exp. Med. 1972,136,556-566.
Lichtenstein, L. M. & E. Gillespie: The effects of the HI and H2 antihistamines on allergic histamine release and its inhibition by histamine. J. Pharmacol. exp. Ther. 1975,192,441450.
Lichtenstein, L. M. & S. Margolis: Histamine release in virro : Inhibition by catecholamines and methylxanthines. Science 1968, 161,902-903. Lichtenstein, L. M. & A. G. Osler: Studies on the mechanism of hypersensitivity phenomena IX. Histamine release from human leukocytes by ragweed pollen antigen. J. exp. Med. 1964,120,507-530. McNeill, R. S.: Effect of a beta adrenergic blocking agent, propranolol on asthmatics. Lancet 1964, ii, 1101-1102.
Simonsson, B. G.,N. Svedmyr, B. E. Skoogh, R. Andersson & N. P. Bergh: In vivo and in vifrostudies on alphareceptors in human airways. Potentiation with bacterial endotoxin. Scand. J. r a p . Dis. 1972,53,227-236.
HISTAMINE LIBERATION A N D TRACHEAL TONE Thiringer, G. & N. Svedmyr: Interaction of orally administered metroprolol, practolol and propranolol with isoprenaline in asthmatics. Eur. J . d i n . Pharmacol. 1976, 10, 163-170.
387
Wikberg, J., R. Anderson & L. Lundholm: Differentiation of adrenergic a-receptors in guinea pig intestine and rabbit aorta. Blood Vessels 1975,12, 383.
