Evidence from guinea-pig trachealis that Uptake2 of isoprenaline is enhanced by hyperpolarization of the smooth muscle

Summary Previous studies (Bönisch et al. 1985; Trendelenburg 1986, 1987) have provided evidence that Uptake₂ of catecholamines is inhibited by depolarization of cells. The aim of this study was to further examine the relationship between Uptake₂ and membrane potential by testing the hypothesis that Uptake₂ is, conversely, stimulated by hyperpolarization of cells. The effects of -adrenoceptor agonists (isoprenaline and salbutamol) and -adrenoceptor antagonists (propranolol and ICI 118,551) on Uptake₂ of isoprenaline were examined in guinea-pig trachealis muscle, in which stimulation of -adrenoceptors mediates hyperpolarization of the smooth muscle cells (Allen et al. 1985), and in rat heart, in which -adrenoceptor agonists do not cause hyperpolarization.In guinea-pig trachealis muscle segments, propranolol and ICI 118,551 reduced Uptake₂ (as measured by the steady-state rate of corticosterone-sensitive formation of 3-O-methylisoprenaline normalized for the isoprenaline concentration) in tissues incubated in 2.5–250 nmol/l ³H-isoprenaline (in the range over which isoprenaline causes hyperpolarization of the muscle), but not in 1 nmol/l ³H-isoprenaline (which does not hyperpolarize the muscle). The normalized rates were greater in tissues incubated in 25 nmol/l than 1 nmol/l isoprenaline, and were enhanced by 2.5 gmol/l salbutamol in tissues incubated in 1 nmol/l isoprenaline. In rat hearts perfused with 1 or 25 nmol/l ³H-isoprenaline and U-0521 to inhibit catechol-O-methyltransferase, the rate of Uptake₂ of isoprenaline, normalized for the isoprenaline concentration, was unaffected by the isoprenaline concentration or the presence of propranolol, ICI 118,551 or salbutamol.The results of the study suggest that, in guinea-pig trachealis muscle, isoprenaline and salbutamol enhance Uptake₂ due to -adrenoceptor-mediated hyperpolarization of the muscle and that propranolol and ICI 118,551 decrease Uptake₂ of isoprenaline by preventing this hyperpolarization of the smooth muscle, and not by directly inhibiting the Uptake₂ transport process. Correspondence to: L. J. Bryan-Lluka at the above addressSome of the results of this study were presented to the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Vuocolo and Bryan-Lluka 1990) and the German Society for Pharmacology and Toxicology (Bryan-Lluka and Vuocolo 1991)     

Haemodynamic effects of adrenaline during treatment of hypertensive patients with propranolol and metoprolol

Summary A double blind cross-over trial of propranolol and metoprolol was carried out in eight hypertensive patients. At the end of each four-week period of medication, blood pressure and heart rate at rest were measured, and the haemodynamic effects of adrenaline infusion were studied. At rest, propranolol and metroprolol reduced the blood pressure and pulse rate to the same degree. Adrenaline infusion during propranolol medication caused a marked increase both in systolic and diastolic blood pressure, the blood flow in the forearm was unchanged, and the calculated vascular resistance showed a marked increase. Adrenaline infusion during metoprolol medication caused a less marked increase in systolic blood pressure and the diastolic pressure remained unchanged. Blood flow in the forearm increased and the vascular resistance in the forearm tended to decrease. Adrenaline infusion, therefore, caused different haemodynamic effects during non-selective -blockade with propranolol and during ₁-selective blockade with metoprolol. It seems probable that the adrenaline infusion test is comparable with adrenaline release during stress situations and the results may indicate that a ₁-selective blocker is to be preferred to a non-selective one as a therapeutic agent in the treatment of hypertension.     

Effects of in vivo treatment with isoprenaline or prenalterol on beta-adrenoceptor mechanisms in the heart and soleus muscle of the cat

Summary The full agonist isoprenaline (5.3–6.6 nmol/kg min) and the partial -adrenoceptor agonist prenalterol (10.6–13.3 nmol/kg · min) were administered to cats continuously via osmotic minipumps (i.p.). After seven days the functional and adenylate cyclase responsiveness to the agonists, as well as the -adrenoceptor-binding characteristics, were studied in cardiac and soleus muscle preparations in vitro.After isoprenaline pretreatment, the papillary muscles and soleus muscle strips were 15–18 times less sensitive to isoprenaline compared with muscles from control cats. The stimulatory potency (pD₂) of prenalterol in the papillary muscle was not changed significantly. The affinity of the agonists to the -adrenoceptors was unaffected in both tissues by the pretreatment, but the densities of -adrenoceptors were significantly reduced, by 36% (myocardium) and 47% (soleus) respectively. In the cat papillary muscle the intrinsic sympathomimetic activity (ISA) of prenalterol on contractile parameters was reduced from 84 (T _max), 69 (dT/dt _max) and 71% (dT/dt _min) in control animals, to 33, 22 and 28%, respectively in the animals pretreated with isoprenaline.Prenalterol pretreatment did not induce any marked changes, either in the stimulatory potency or affinity of the agonists in the two tissuer or in the maximal response (ISA) of prenalterol in the papillary muscle.The marked reduction in the stimulatory potency of isoprenaline and the reduced ISA of prenalterol in the myocardium after isoprenaline pretreatment can not be explained by the reduction in -adreoceptor density alone. Since the affinity to the -adrenoceptors is unaffected, a reduced efficiency in the signal transmission must be the main cause. This alteration in signal transmission seems to be an event located distal to adenylate cyclase, since the relative decrease in the enzyme activity is even less than the loss of -adrenoceptors in both the myocardium and soleus muscle.The present results demonstrate that the effects on -adrenoceptors and functional responsiveness are different after prolonged treatment with a full -adrenoceptor agonist and a partial agonist, such as prenalterol.Some of these data were presented at the 66th Annual Meeting of the Federation of American Societies for Experimental Biology, April 15–23, 1982 New Orleans, USA     

Comparison of salmefamol with salbutamol aerosols in asthmatics

Summary In twelve asthmatic patients 200 µg of salmefamol and salbutamol given by metered aerosol produced a similar inital effect on FEV₁, FVC and PEFR without significant effect on heart rate or blood pressure. The duration of effect of salbutamol was approximately 4 hours; there was still an appreciable effect from salmefamol at 8 hours.     

A unique pressor response to isoprenaline in the pithed rat during triiodo-l-thyronine (T3)-induced hyperthyroidism

Summary Thyroid hormone appears to be involved in the regulation of -adrenoceptors affecting cardiovascular performance. In the present study, the influence of hyperthyroidism on -adrenoceptor-mediated response of the cardiovascular system was investigated in vivo using the pithed rat preparation. Hyperthyroidism was induced by triiodothyronine injections (500 g/kg, i.p.) for 6 days. A markedly accelerated basal heart rate and a wider pulse pressure with a significantly elevated systolic blood pressure were observed in hyperthyroid pithed rats. Although the basal and the maximal heart rates were increased in hyperthyroid rats, EC₅₀ of the heart rate response to isoprenaline did not significantly differ between euthyroid and hyperthyroid pithed animals. Markedly different responses of blood pressure to isoprenaline were obtained in the two groups; isoprenaline caused a dose-dependent decrease in diastolic pressure in euthyroid pithed rats, whereas it produced pressor response in hyperthyroid pithed rats. This unique pressor response to isoprenaline observed in hyperthyroid pithed rats was abolished by the ₁-adrenoceptor selective antagonist metoprolol but not by the -adrenoceptor antagonist phenoxybenzamine. The density of myocardial binding sites of the -type was markedly increased after T₃ treatment (65%), whereas that of the mesenteric artery was not altered. The results indicate that thyroid hormone exerts different effects on cardiac and vascular -adrenoceptors, and this different susceptibility to thyroid hormone may in part be responsible for the altered response of blood pressure to isoprenaline seen in hyperthyroid pithed rats.     

Characterization of the antagonism with metoprolol,ICI 147,798, pindolol,mepindolol and bopindolol on the responses of the rat left atria to isoprenaline

Summary The aim of the study was to determine whether the antagonism with pindolol, mepindolol and bopindolol at the ₁-adrenoceptor of the rat left atria, a tissue with plenty of spare ₁-adrenoceptors for isopren aline maximum responses, was readily reversible or not. The effects of these drugs were compared to those of metoprolol, a readily reversible, and of ICI 147,798, an irreversible -adrenoceptor antagonist. Metoprolol at 10^–7 and 10^–6 M, ICI 147,798, pindolol, bopindolol (all at 10^–8 and 10^–7 M) and mepindolol at 10^–9 and 10^–8M inhibited the cardiac stimulation responses to a small extent, which is indicative of membrane stabilizing activity, and also caused surmountable antagonism of isoprenaline responses. The inhibitory effects on the isoprenaline responses of metoprolol and pindolol were readily reversible, that of mepindolol was slowly reversible and those of ICI 147,798 and bopindolol were not reversed in 3 h. The inhibitory effects on isoprenaline responses of metoprolol at 10^–6 M, pindolol and bopindolol at 10^–7 M and mepindolol at 10^–8 M were at equilibrium, which is indicative of reversible, whereas the inhibitory effects of ICI 147,798 were increased, which is indicative of irreversible antagonism, when the -blocker treatment time was increased from 1 to 2 h. We conclude that the antagonism with pindolol at the ₁-adrenocep-tors of the rat left atria is readily reversible, that of mepindolol is slowly reversible and that of bopindolol is very slowly reversible.Correspondence to S. A. Doggrell at the above address     

Failure of “antigastrin” (SC-15 396) to inhibit gastric acid and pepsin secretion in anaesthetized gastric fistula cats

Summary 1. The effect of antigastrin (SC-15 396) on gastric acid and pepsin secretion produced by the gastrin-analogue tetrapeptide amide Try. Met. Asp. Phe-NH₂ and by electrical stimulation of the vagus was investigated in anaesthetized gastric fistula cats.2. Antigastrin failed to inhibit both acid and pepsin response stimulated by either the tetrapeptide or vagus excitation.3. It was concluded that the ineffectiveness of antigastrin in cats is due to a species difference between rats and dogs on the one hand and cats on the other, and that antigastrin is not a specific gastrin antagonist.Supported by the Deutsche Forschungsgemeinschaft and by the Alfred Teufel-Stiftung.     

Lack of presynaptic modulation by isoprenaline of 3H-noradrenaline release from rabbit isolated ear artery

Summary The aim of the present investigation was to examine whether or not presynaptic facilitatory -adrenoceptors are detectable on the postganglionic nerves in the rabbit isolated ear artery. Strips of rabbit central ear artery were incubated with ³H-noradrenaline (10^–7 mol/l; 30 min or 10^–6 mol/l; 60 min). Subsequently, they were washed repeatedly with physiological salt solution. The strips were subjected to electrical-field stimulation (S₁–S₈) and the resultant ³H-overflow was determined.When the ear artery was stimulated with 150 pulses (0.5 ms; 3 Hz; 225 mA), isoprenaline (10^–9–10^–6 mol/l) either alone or in the presence of either rauwolscine (10^–6 mol/l) or phentolamine (10^–6 mol/l) did not alter the stimulation-evoked ³H-overflow. This was also the case in the presence of rauwolscine (10^–6 mol/l) plus either the selective phosphodiesterase inhibitor ICI 63 197 (3 × 10^–5 mol/l) or forskolin (10^–6 mol/l). When the ear artery was stimulated with 300 pulses (1 ms; 5 Hz; 225 mA), isoprenaline had no effect on the stimulation-evoked ³H-overflow. This was also the case when phentolamine (10^–6 mol/l) was present. Propranolol (10^–7–10^–5 mol/l) did not alter the stimulation-evoked ³H-overflow. In some experiments, the stimulation current was reduced to 175 mA in order to obtain similar reference release (S₃) values despite the presence of rauwolscine (150 pulses; 0.5 ms; 3 Hz). Even then, isoprenaline (10^–9–10^–6 mol/l) did not change stimulation-evoked ³H-overflow. The results suggest that postganglionic sympathetic nerves in rabbit central ear artery do not possess presynaptic facilitatory -adrenoceptors. Send offprint requests to J. Abrahamsen at the above address     

Role of brainstem and spinal noradrenergic and adrenergic neurons in the development and maintenance of hypertension in spontaneously hypertensive rats

Summary In young and adult spontaneously hypertensive rats (SHR), dopamine -hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) activities in discrete areas of the brainstem and spinal cord were measured as indices of noradrenergic and adrenergic neuronal activities. In young SHR, the DBH activities were elevated in the locus coeruleus (LC), A2 cell area and thoracic intermediolateral cell area (IML). The elevation disappeared at adult SHR. In young SHR, no significant change of PNMT activity was observed in the A1, A2, nucleus tractus solitarii (NTS), LC and IML areas, while, in adult SHR, the PNMT activity in the A1 cell area and DBH activity in the NTS were elevated. Lowering of blood pressure by hydralazine decreased the PNMT activity elevated in the A1 cell area and elevated it in the NTS.Plasma levels of norepinephrine and epinephrine, as measured in blood samples collected via aortic cannula at resting state, were much lower than many reported values in blood collected from the decapitated trunk. In young SHR, a significant elevation of plasma norepinephrine and DBH levels was confirmed as signs of peripheral sympathetic nervous activation. The elevation disappeared at adult SHR. Plasma epinephrine levels raised under restraint stress were much higher in SHR at all ages than in normotensive controls.In young SHR, the selective activation of noradrenergic neurons of the IML, A2 and LC areas, accompanied by activation of the peripheral sympathetic nervous system, initiates the hypertension. In adult SHR, the activation of adrenergic neurons in the A1 cell area including the nucleus reticularis lateralis may not be involved in the maintenance of hypertension but may be the results of hypertension.     

Comparative pharmacology of human β-adrenergic receptor subtypes—characterization of stably transfected receptors in CHO cells

Although many ₁-receptor antagonists and ₂-receptor agonists have been used in pharmacotherapy for many years their pharmacological properties at all three known subtypes of -adrenergic receptors are not always well characterized. The aim of this study was, therefore, to provide comparative binding characteristics of agonists (epinephrine, norepinephrine, isoproterenol, fenoterol, salbutamol, salmeterol, terbutalin, formoterol, broxaterol) and antagonists (propranolol, alprenolol, atenolol, metoprolol, bisoprolol, carvedilol, pindolol, BRL 37344, CGP 20712, SR 59230A, CGP 12177, ICI 118551) at all three subtypes of human -adrenergic receptors in an identical cellular background. We generated Chinese hamster ovary (CHO) cells stably expressing the three -adrenergic receptor subtypes at comparable levels. We characterized these receptor subtypes and analyzed the affinity of routinely used drugs as well as experimental compounds in competition binding studies, using the non-selective antagonist ¹²⁵I-cyanopindolol as a radioligand. Furthermore, we analyzed the -receptor-mediated adenylyl cyclase activity in isolated membranes from these cell lines. The results from our experiments show that all compounds exhibit distinct patterns of selectivity and activity at the three -receptor subtypes. In particular, a number of ₂- or ₃-receptor agonists that are inverse agonists at the other subtypes were identified. In addition, ₁-receptor antagonists with agonistic activity at ₂- and ₃-receptors were found. These specific mixtures of agonism, antagonism, and inverse agonism at different subtypes may have important implications for the therapeutic use of the respective compounds.     

Characteristics of pentobarbital discrimination in the gerbil: Transfer and antagonism

Experiment 1. Gerbils were trained in a T-shaped maze to discriminate the effects produced by pentobarbital (P-barb. 15 mg/kg, i.p.) and the effects of saline. The response, a left or right turn in the maze, was thus contingent upon the prevailing training condition (P-barb. or saline). The criterion of performing 8 correct first trial choices in 10 consecutive sessions was reached within 20 training sessions. Tests with descending doses of P-barb. yielded an ED₅₀ of 9 mg/kg. Tests with phenobarbital (40 mg/kg) or diazepam (2 and 4 mg/kg) solely maintained the drug response. P-barb. discrimination was reversed by megimide (ED₅₀: 8.5–9.6 mg/kg) and metrazol (ED₅₀: 24.9–27.9 mg/kg). Thus megimide was approximately 3 times more effective than metrazol. Metrazol (40 and 80 mg/kg) also counteracted the phenobarbital and diazepam response. Picrotoxin (2.5 and 5 mg/kg) was less effective whereas caffeine (100 mg/kg) and piracetam (100–1000 mg/kg) did not upset P-barb. discrimination. Experiment 2. Naive gerbils had to discriminate mixtures of P-barb. (15 mg/kg) plus either 40 or 80 mg/kg of metrazol from saline already at the start of the discriminative training. The drug combinations produced discriminable effects since most gerbils reached the acquisition criterion (8/10), although more slowly than gerbils trained with P-barb. solely. Gerbils trained without a drug stimulus (saline vs. saline) never attained the criterion during 60 consecutive sessions. In conclusion, reversal of established discrimination (Expt. 1) does not necessarily mean that the same drug combination lacks discriminable effects as demonstrated in Experiment 2.     

Double-blind cross-over comparison of clenbuterol and salbutamol tablets in asthmatic out-patients

Summary A double-blind cross-over comparison of a new ₂-sympathomimetic bronchodilator, clenbuterol, with salbutamol and placebo has been made during a 24 day period of out-patient treatment of 19 adults with moderately severe asthma. Oral clenbuterol (10 µg 3 times a day) and salbutamol (4 mg 3 times a day) were equally and significantly (p<0.001) more effective than placebo, when daily records of peak expiratory flow or use of isoprenaline inhalations were the criteria of activity. Daily records of symptoms according to a questionnaire also suggested relief of the subjective effects of asthma during treatment with both active drugs (p<0.01).     

Metabolic effects of oral salbutamol in late pregnancy

Summary Ten women in late pregnancy were given an oral dose of salbutamol 4 mg. Heart-rate and blood-pressure were recorded and blood-samples for measurement of cyclic AMP, C-peptide, glucose, lactate, glycerol, non-esterified fatty acids (NEFA) and -hydroxybutyrate (3-HB) were collected every 30 min for 120 min. In seven of the women the same experiment was performed without the salbutamol. After salbutamol maternal heart-rate was significantly increased at 30 and 90 min, and diastolic blood-pressure was significantly decreased between 30 and 120 min; systolic blood-pressure was unaltered. Plasma cyclic AMP was significantly increased by salbutamol at 30 and 120 min and C-peptide at 60 min. Plasma glucose was significantly elevated 60 min after salbutamol and glycerol after 90 min. Plasma NEFA and 3-HB increased both with and without the drug, with a tendency towards higher levels in the group that received salbutamol. Lactate levels were unchanged after salbutamol and fell when the drug was not given, but the difference was not significant. The results show clear circulatory and metabolic effects of a single clinical dose of salbutamol.     

Coronary vascular responses to nicotine in the anaesthetized dog

Summary The effect of the intra-coronary (i.c.) injection of nicotine on large coronary artery diameter and coronary blood flow was examined in anaesthetized dogs. In sixteen untreated dogs nicotine (20 g i.c.) had a biphasic effect on arterial pressure (initial increase, 7 ± 2 mmHg; secondary decrease, –8 ± 3 mmHg) which was accompanied by small and variable effects on heart rate and an increase in LV dP/dt. Nicotine increased large coronary artery diameter by 5.8 ± 0.8% but had a biphasic effect on coronary blood flow (initial increase, 41 ± 7 ml/min; secondary decrease, –10 ± 2 ml/min). Bilateral vagotomy or muscarinic receptor blockade with atropine (0.1 mg/kg i. v.) did not significantly affect the nicotine-induced changes in coronary artery diameter or coronary blood flow. The additional antagonism of -adrenoceptors with propranolol (1 mg/kg i. v.) abolished the effect of nicotine in coronary artery diameter ( CD = 0.2 ± 0.2%) and the initial increase in coronary blood flow ( CBF = 1 ± 1 ml/min) but enhanced the secondary decrease in flow ( CBF = –25 ± 3 ml/min). The nicotine-induced decrease in coronary blood flow observed after muscarinic and -adrenoceptor blockade was attenuated by antagonism of ₁-adrenoceptors with prazosin (10 g/kg i. c., CBF = –15 ± 3 ml/min) and abolished after additional antagonism of ₂-adrenoceptors with idazoxan (50 g/kg i. c., CBF = –2 ± 1 ml/min). These results indicate that in the anaesthetized dog intra-coronary injection of nicotine results in -adrenoceptor mediated dilatation of both large and small coronary arteries. In the coronary resistance vessels, but not in the large coronary artery, the dilatation is opposed by ₁-and ₂-adrenoceptor mediated vasoconstriction. Send offprint requests to O. L. Woodman at the above address     

Application of Supercritical Carbon Dioxide for the Preparation of a Piroxicam-β-Cyclodextrin Inclusion Compound

Purpose. Piroxicam is a poorly soluble NSAID, whose solubility is enhanced when included into -cyclodextrin. The preparation of a piroxicam--cyclodextrin inclusion compound using supercritical CO₂ was investigated. Methods. The solubility and the stability of piroxicam in supercritical CO₂ were determined. Then, the influence of the temperature, the pressure and the time of exposure on the inclusion rate was studied. Results. The solubility of piroxicam varied over a wide range depending on the temperature and pressure (from 0.006 to 1.500 mg/g of CO₂). The temperature and the time of exposure had a great influence on the inclusion yield, while pressure did not and a complete inclusion was achieved by keeping a physical mixture of piroxicam and -cyclodextrin (1:2.5 mol/mol) for 6 hours at 150°C and 15 MPa of CO₂. This complex was characterized by Differential Scanning Calorimetry, differential solubility and Fourier Transform Infrared Spectrometry. Conclusions. Supercritical carbon dioxide may prove to be a novel useful complexation method of drugs into -cyclodextrin.     

A comparison of the β1 and β2 effects of subcutaneous isoprenaline,sulbutamol and terbutaline in adult man

Summary The selectivity of salbutamol 250 and 500 µg and terbutaline 250 and 500 µg compared with isoprenaline when administered subcutaneously has been investigated in 6 human volunteers who showed consistant bronchospasm when challenged with histamine by nebulizer. Changes in heart rate, forearm blood flow, tremor and F.E. V.₁ were recorded and subjected to analysis of variance. Measurements were made at a fixed time interval after administration of active drugs or placebo and compared with pre-drug baseline recordings. The study was performed double-blind. A statistically significant difference was observed when all active drugs were compared with placebo, in the parameters of heart rate, F.E.V.₁ and tremor. However, when the active drugs were compared with each other, no statistically significant difference was observed.     

Effect of dibutyryl cyclic-AMP on levels of dopamine-β-hydroxylase in isolated superior cervical ganglia

Summary When rat superior cervical ganglia were incubated with dibutyryl cyclic-AMP their content of dopamine--hydroxylase increased approximately two-fold over a period of six hours. This effect was blocked by cycloheximide. The possibility is discussed that cyclic AMP may mediate the rise in ganglion dopamine--hydroxylase which occurs during increased sympathetic activity.     

Effects of single oral doses of bisoprolol and atenolol on airway function in nonasthmatic chronic obstructive lung disease and angina pectoris

Summary A randomized, placebo-controlled, double-blind crossover investigation in 12 patients with non-asthmatic chronic obstructive lung disease and co-existing stable angina pectoris was done to compare two ₁-selective adrenoceptor blocking agents, atenolol 100 mg and bisoprolol 20 mg. Systolic and diastolic blood pressures (SBP, DBP), heart rate (HR) as well as airway resistance (AWR, and less frequently forced expiratory volume in 1 s (FEV₁) and intrathoracic gas volume (ITGV) were measured in the sitting position before and at various times up to 24 h after drug intake.During the first 4 h both beta-blockers produced a significant reduction in HR in comparison to placebo (p<0.01). Atenolol 100 mg significantly increased AWR relative to placebo and bisoprolol (p<0.05). After 24 h, a significant reduction in HR (p<0.01) could only be demonstrated after bisoprolol, whereas atenolol alone led to a significant elevation in AWR relative to placebo and bisoprolol (p<0.05) at that time.It is concluded that bisoprolol appears to have a high degree of beta₁-selectivity, thus providing a wide split between beta₁- and beta₂-adrenoceptor blockade. Bisoprolol in its therapeutic dose range is expected to be relatively safe as regards bronchoconstriction in patients suffering both from hypertension and/or angina pectoris and chronic obstructive lung disease.