The interaction between salmeterol and beta 2-adrenoceptor agonists with higher efficacy on guinea-pig trachea and human bronchus in vitro.

1. In guinea-pig tracheal preparations precontracted with 1 mumol l-1 carbachol, formoterol, procaterol, fenoterol, salmefamol, salbutamol and terbutaline (in that order of potency) caused a concentration-dependent and almost complete, relaxation. However, under these conditions, the maximum relaxation by salmeterol was approximately 30% of the maximum attainable relaxation. 2. We have therefore explored the ability of salmeterol to inhibit the relaxant response to beta 2-adrenoceptor agonists of different chemical structure and relatively higher efficacy in smooth muscle preparations from guinea-pig trachea and human bronchus. 3. With 1 mumol l-1 salmeterol in the organ bath, the concentration-effect curves for the other agonists were shifted to the right in a variable way by 1.8-2.8 log units, fenoterol and salbutamol being the extremes. 4. When 20 mumol l-1 sulfonterol, another low efficacy beta 2-adrenoceptor agonist, was substituted for salmeterol, the difference in the magnitude of the rightward shift between fenoterol and salbutamol was eliminated. 5. In the human bronchus, formoterol and terbutaline had a higher apparent efficacy than salmeterol. With 1 mumol l-1 salmeterol in the organ bath, the concentration-effect curve for formoterol was shifted 2.7 log units to the right. 6. Salmeterol inhibits, competitively, relaxant responses to beta 2-adrenoceptor agonists with higher efficacy. The degree of inhibition seems to be dependent on the agonist used. This contrasts with results obtained with sulfonterol and suggests that salmeterol interacts with the beta 2-adrenoceptor in a complex way.

Desensitization by terbutaline of beta-adrenoceptors in the guinea-pig soleus muscle: biochemical alterations associated with functional changes.

1 The effects of adrenaline and terbutaline on cyclic adenosine 3',5'-monophosphate (cyclic AMP) content, 22Na-efflux, 42K-influx and subtetanic contractions have been assessed in soleus muscles isolated from guniea-pigs which had been maintained on food with or without terbutaline for 5 days. 2 Terbutaline and adrenaline increased cyclic AMP content and suppressed subtetanic contractions, and regression analysis indicates a statistically significant correlation between these two effects (P less than 0.01). 3 In muscles obtained from terbutaline-treated animals, the effects of terbutaline and adrenaline on cyclic AMP content, active Na-K-transport and subtetanic contractions were all considerably suppressed, but insulin stimulated 22Na-efflux and affected subtetanic contractions to the same extent as in the muscles obtained from the control group. 4 The results suggest that terbutaline treatment leads to a reduction in the number of beta 2-adrenoceptors in skeletal muscle or an impairment of their function. 5 The results provide further support for the idea that the effect of adrenaline or insulin on skeletal muscle contractions is the outcome of stimulation of active Na-K-transport.

Neuropeptides in guinea pig trachea: distribution and evidence for the release of CGRP into tracheal lumen.

The airways of the guinea pig are richly innervated by peptide-containing nerve fibers. Among the most abundant neuropeptides are calcitonin gene-related peptide (CGRP) and substance P (SP), which are stored in nerve fibers located predominantly within and beneath the epithelium, and vasoactive intestinal peptide (VIP), which is located in fibers running mainly among smooth muscle bundles and seromucous glands. Sensory denervation (capsaicin treatment) of adult guinea pigs caused an almost total disappearance of CGRP- and SP-containing nerve fibers, while the density of VIP-containing nerve fibers located in smooth muscle seemed to increase. In the isolated trachea, perfused luminally, CGRP was found to appear in the intraluminal fluid after exposure to capsaicin but not after electrical vagal stimulation. CGRP concentrations in the tracheal wall did not change significantly. Luminally applied CGRP did not affect smooth muscle tension, measured as intraluminal volume changes.

Neuropeptide Y: prejunctional inhibition of vagally induced contractions in the guinea pig trachea.

The effects of neuropeptide Y (NPY) on the contractile response to vagus nerve stimulation at different frequencies was studied in an isolated tracheal tube preparation from guinea pig. NPY had no effect on basal smooth muscle tension or on the contractile effect of carbachol, but inhibited vagally induced contractions in a concentration-dependent manner with a greater inhibition at low frequencies than at high. We suggest that the effect is exerted prejunctionally.

Comparison of the effects of forskolin and isoprenaline on tracheal, cardiac and skeletal muscles from guinea-pig.

Forskolin, a potent activator of adenylate cyclase, and isoprenaline, an unselective beta-adrenoceptor agonist, were studied in vitro on tissues from guinea-pig with respect to relaxation of the carbachol-contracted trachea, increase in the force of contraction of the papillary muscle and depression of subtetanic contractions of the soleus muscle, three well-characterized beta-adrenoceptor-mediated effects. Forskolin and isoprenaline relaxed the trachea and increased the force of contraction of the papillary muscle. Isoprenaline but not forskolin caused a depression of the subtetanic contraction of the soleus muscle. Forskolin did not seem to potentiate the effects of isoprenaline on the tissues studied; the combined effects appeared to be a mere addition. Forskolin did not increase the efficacy of the partial agonist prenalterol either. It is concluded that there is no simple relation between c-AMP generation and the functional response to beta-adrenoceptor agonists. Forskolin should not be used uncritically to probe beta-adrenoceptor-mediated effects.

Bronchodilating properties of the VIP receptor agonist Ro 25-1553 compared to those of formoterol on the guinea-pig isolated trachea.

Ro 25-1553 is a 31-amino acid analogue of vasoactive intestinal peptide (VIP) and has recently been shown to be highly selective for the VPAC(2)-receptor. The bronchodilating property of this compound was evaluated in vitro on preparations of guinea-pig trachea, with the long-acting beta(2)-adrenoceptor selective agonist, formoterol, as a reference. In strip-preparations precontracted with carbachol, Ro 25-1553 caused a concentration-dependent and complete relaxation of the tracheal smooth muscle. Ro 25-1553 was 3-7 times less potent than formoterol on a molar basis, but the efficacy was comparable with that of formoterol. Both compounds showed a rapid onset of action and a similar durability of effect. Ro 25-1553 appeared to interact with formoterol as well as with salmeterol in an additive way. In vagus nerve-trachea tube preparations, when added to the external medium, Ro 25-1553 concentration-dependently and completely inhibited nerve-induced contractions. This occurred in the same concentration range as needed for relaxation of precontracted strips. Ro 25-1553 was active also when administered into the tracheal lumen albeit the concentration had to be increased. The present study supports and extends previous results suggesting that Ro 25-1553 may be a powerful alternative to the beta(2)-adrenoceptor agonists which prevail today.

Formoterol and salmeterol are both long acting compared to terbutaline in the isolated perfused and ventilated guinea-pig lung.

An isolated, perfused and ventilated guinea-pig lung was used to compare the duration of effect of the bronchodilating beta 2-adrenoceptor agonists formoterol, salmeterol and terbutaline. Lung conductance was measured real time with the aid of a computer. Bronchoconstriction was induced in the preparation every 10 min by bolus injections of acetylcholine into the pulmonary artery. Lung conductance was reduced by about 70% after acetylcholine. The test compounds or the vehicle was administered for 1 min as aerosols generated from solutions: formoterol (10 mumol/l), salmeterol (100 mumol/l) and terbutaline (1000 mumol/l). This treatment inhibited the response to acetylcholine by 50-60% within the first 10 min for all three test compounds. The onset of action appeared to be slower for salmeterol than for formoterol and terbutaline. The inhibitory effect of terbutaline disappeared completely during the next 20 min of continuous perfusion (single pass), while both formoterol and salmeterol displayed a significant inhibitory effect 40 min after their administration. Formoterol, when inhaled at a higher dose (100 mumol/l), caused a 90% inhibition of the response to acetylcholine. This effect was completely reversed by 0.1 mumol/l propranolol in the perfusion medium. There were in general no major changes in the basal conductance measured between the acetylcholine provocations.

Analysis of the beta-receptor mediated effect on fast-contracting skeletal muscle in vitro.

Subtetanic contractions of the isolated extensor digitorum longus (EDL) of the guinea-pig, a fast-contracting muscle, were evoked by transmural field stimulation. Isoprenaline, adrenaline, terbutaline, and noradrenaline each caused a dose-dependent increase in the force of contraction, their potencies decreasing in that order. Tyramine was without effect in this respect. Curare depressed the contractions of EDL by about 20% but did not appreciably change the response to the beta-adrenoceptor agonists. The effects of isoprenaline and noradrenaline were blocked by propranolol (unselective) and H 35/25 (1-(p-tolyl)-2-isopropylamino-1-propanol, beta2-selective) but not by practolol (beta1-selective). Moreover, the increase in the force of subtetanic contractions of EDL produced by noradrenaline was unaffected by phentolamine. It is concluded that the adrenoceptor mediating the increase in the force of contraction of the isolated EDL is of the beta2-type and that the site of action is direct on the muscle. Its similarity to the receptor mediating the inverse effect on the slow-contracting soleus-muscle is pointed out.

Uptake and disappearance of 4-methyl-alpha-ethylmetatyramine in relation to its releasing action on 5-hydroxytryptamine in the brain.

4-Methyl-alpha-ethylmetatyramine (H75/12) was given intraperitoneally to mice and rats. At various time intervals the animals were killed and the concentration of H75/12 and 5-hydroxytryptamine (5-HT) in the brain and of H75/12 in the heart and, in some cases, in plasma was determined. H75/12 was rapidly taken up by the heart and then disappeared bi- or multiphasically. A maximum concentration of about 6 mug/g was obtained in the brain 30--60 min after the administration of 25 mg/kg. H75/12 disappeared from the brain with a half-life of 1.0 and 1.5 hrs in the mouse and rat, respectively. In the mouse the relative uptake of H75/12 increased with the dose given, i.e. the dose-uptake curve had an upward bending. Significant release of 5-HT in the brain was obtained after a single injection of 25 and 100 mg/kg in the rat and mouse, respectively. The 5-HT release was prevented by tricyclic, antidepressive agents, particularly by chlorimipramine. However, these agents had no certain effect of the uptake of H75/12 in the brain. Moreover, it was not possible to demonstrate a decreased uptake of H75/12 in the rat brain after a chronic, transverse cerebral hemisection, although the monoamine-containing neurones had degenerated as indicated by a complete disappearance of 5-HT. It is concluded that only a minor part of the H75/12 measured is located in monoaminergic neurones. The uptake of H75/12 in the rat brain appeared to be increased in starving animals, possibly indicating decreased metabolism of the drug.

Studies on the synthesis and subcellular distribution of dopamine in the rat adrenal medulla.

Rats received intravenous injections of 3H-tyrosine and were killed at various time intervals thereafter. 3H-dopamine (DA) in the adrenals reached a maximum within 1.5 min after the administration of 3h=tyrosine. From the 15th min it disappeared with an apparent half life of 90 min. 3H-Adrenaline (A) plus 3H-noradrenaline(NA) increased much more slowly and reached a plateau 120-240 min after the injection. The approximate synthesis rate of adrenal A plus NA, calculated from the specific activity curves, ranged from 0.3 to 2.2 nmoles/h per kg b.w. The highest value was noted the first few minutes, the lowest 1-2 hrs after the administration of 3H-tyrosine. In some experiments subcellular fractionation of the adrenals was performed. In untreated animals the amount of DA and A plus NA recovered from the supernatant fraction was about 10 and 8 per cent, respectively, of the total amount recovered from the supernatant and particulate fractions. In the adrenals of animals receiving 3H-tyrosine 3.75 or 60 min beforehand these figures were significantly elevated whereas the DA and A plus NA of the particulate fraction did not deviate significantly from control values. The specific activities of 3H-DA were the same in the supernatant and particulate fractions within 3.75 min after the injection of 3H-tyrosine.

The effect of the enantiomers of formoterol on inherent and induced tone in guinea-pig trachea and human bronchus.

The long-acting beta2-adrenoceptor agonist formoterol is, like all other members of this class of drugs, used as a racemate in the clinic. While the effects of the individual enantiomers have been studied on airway smooth muscle from guinea pig, comparable data on human bronchial smooth muscle are scanty or absent. Therefore, we compared the effects of the enantiomers of formoterol on inherent and induced tone in isolated human bronchi with that on guinea-pig trachea in vitro. The human bronchi either were studied under resting tension conditions or were precontracted with 10 microM carbachol or 0.1 mM histamine. The guinea-pig trachea was precontracted with 0.01, 0.1 or 1 microM carbachol. The racemate and (R,R)-formoterol caused a concentration-dependent relaxation of all preparations with an EC50 of about 1 nM. In the guinea-pig trachea, the concentration-effect curve for formoterol was moved to the right in response to an increased concentration of carbachol. In both human bronchus and guinea-pig trachea, (S,S)-formoterol was more than 1,000 times less potent than (R,R)-formoterol. Thus the relaxing effect of formoterol in human airways as well as in guinea-pig trachea was shown to lie with the (R,R)-enantiomer. Notably, (S,S)-formoterol did not exert any contractile effects within the tested concentration range in either airway preparation. Therefore, we conclude that with regard to relaxant effects the pure (R,R)-enantiomer of formoterol does not offer a benefit over the racemate.

The effect of the vasoactive intestinal polypeptide agonist Ro 25-1553 on induced tone in isolated human airways and pulmonary artery.

Ro 25-1553 is a metabolically stable analogue of endogenous vasoactive intestinal polypeptide (VIP). This compound is a potent bronchodilator in vitro as well as in vivo. Moreover, Ro 25-1553 has been shown to be highly selective of the VPAC2 receptor. We assessed the effect of Ro 25-1553 on isolated human bronchi and pulmonary arteries in vitro. Macroscopically normal human airways and pulmonary arteries were obtained from patients undergoing surgery for lung cancer. The relaxing capability of Ro 25-1553 on bronchial and pulmonary artery tone was measured using standard techniques. Bronchial rings were pre-contracted with 0.1 mM histamine, and tone in pulmonary artery rings was induced with 10 microM PGF2alpha. Increasing concentrations of Ro 25-1553 within a range of 1 pM to 10 microM were added and isometric tension changes were recorded. Ro 25-1553 caused a concentration-dependent relaxation of airway and pulmonary artery preparations, with an EC50 of approximately 10 nM and a maximal relaxation of 70%-75% of the induced tone. The presence of VPAC2 receptors in the two tissues, though low in density, was confirmed by in situ hybridization, immunocytochemistry and ligand binding. These findings indicate that the VIP analogue Ro 25-1553 may be useful in the treatment of asthma and/or chronic obstructive pulmonary diseases.

An in vitro method for the study of beta-receptor mediated effects on slow contracting skeletal muscle.

The soleus muscle of a guinea-pig was dissected out under pentobarbitone anaesthesia and mounted on a holder in an organ bath containing Krebs solution. The tendon was attached to a force transducer and subtetanic contractions were evoked by electrical field stimulation (0.5 ms pulses at 10-12 Hz for 1.5 or 3 s every 22 s). The experiments were performed at 37 degrees. Terbutaline, a selective agonist at beta2-adrenoceptors, reduced the force of subtetanic contractions in a dose-dependent manner, the EC50 being 0.2 muM. The reduction was due to a lessened degree of fusion. The results conform to previous in vivo studies.

Analysis of the beta-receptor mediated effect on slow-contracting skeletal muscle in vitro.

Subtetanic contractions of the guinea-pig isolated soleus, a slow-contracting skeletal muscle, were evoked by transmural field-stimulation. Isoprenaline caused a dose-dependent depression of the contractions. This effect was inhibited by propranolol and H 35/25 (1-(p-tolyl-2-isopropylamino-1-propanol) but not by practolol. Similar results were obtained for terbutaline. Tazolol and H 80/62 (1-isopropylamino-3-(p-hydroxyphenoxy)-2-propanol (HCl), selective beta1-agonists, had no effect per se but inhibited the effect of terbutaline. Adrenaline, noradrenaline, and dopamine all caused a dose-dependent decrease in the force of the soleus contractions, their potencies being in that order. Tyramine did not appreciably affect the contractions nor did it inhibit the effect of terbutaline. Pretreatment with reserpine, if anything, increased the response to terbutaline. It is concluded, in conformity with previous in vivo studies, that the adrenergic receptor mediating the effect on the soleus muscle contractions is of the beta2-type. Indirect sympathomimetic effects do not contribute to the responses observed on the isolated soleus muscle.

Pentobarbitone and skeletal muscle contractions: on the interaction with the effect elicited by the beta-adrenoceptor agonist, terbutaline.

The soleus, a slow-contracting muscle, and the extensor digitorum longus (EDL), a fast-contracting muscle from guinea-pig were prepared for isometric recording in vitro. Subtetanic contractions were evoked by transmural field-stimulation. Pentobarbitone increased the force of contraction in both muscles. In the soleus it shifted the stimulation frequency-response curve to the left. Terbutaline caused a decrease in the force of subtetanic contractions of the soleus, an effect which was dependent on the stimulation frequency. In the presence of pentobarbitone, the stimulation frequency had to be lowered by about 2 HZ in order to maintain the optimum response to terbutaline. The EDL responded to terbutaline with an increased force of contraction. In this case the stimulation frequency was less critical and the effects were the same in the presence and in the absence of pentobarbitone. Experiments with alpha-chloralose yielded results similar to those obtained with pentobarbitone.

beta 1-Adrenoceptors mediating relaxation of the guinea-pig trachea: experiments with prenalterol, a beta 1-selective adrenoceptor agonist.

In the presence of 17 beta-oestradiol, prenalterol, a beta-selective adrenoceptor agonist, caused a dose-dependent relaxation of the isolated, pilocarpine-contracted guinea-pig trachea. This effect was blocked by the antagonists propranolol (non-selective) and practolol (beta 1-selective) but not by IPS 339 [(t-butylamino-3-ol-2-propyl)oximino-9-fluorene HCl] (beta 2-selective). The relaxing effect of terbutaline, a beta 2-selective adrenoceptor agonist, was more efficiently blocked by IPS 339 than by practolol. These data support the hypothesis that the guinea-pig trachea contains both beta 1- and beta 2-adrenoceptors mediating relaxation and that the beta 1-adrenoceptors are selectively stimulated by prenalterol. The efficacy of prenalterol was less than that of terbutaline, thus confirming its partial agonistic activity. In the absence of 17 beta-oestradiol, the ability of prenalterol to relax the pilocarpine-contracted trachea was lost. It is suggested that 17 beta-oestradiol may act as a functional antagonist to pilocarpine as it caused a partial relaxation itself.