The hypothesis of Szentivanyi that bronchial asthma is associated with impaired 0-adrenoceptor function has been tested in several investigations in recent years. Most of these studies have been performed on leukocytes from peripheral blood, since these cells are easily accessible and since they are endowed with 0-adrenoceptors of the 02-subtype, as is human lung tissue. The interpretation of results from some of these studies is confounded by the findings that the (3-adrenoceptors of white blood cells are down-regulated by antiasthmatic therapy with β-adrenoceptor agonists. Such down-regulation of 0-adrenoceptors in white blood cells disappears within one week after discontinuation of treatment.
Some controversy still exists as to whether 0-adrenoceptors in white blood cells are altered or not in asthma. There are reports suggesting a reduced response to 0-adrenergic stimulation in untreated patients or a reduction of 0-adrenoceptor binding sites not ascribed to antiasthmatic therapy. However, most studies indicate that white blood cells from nonmedicated asthmatic patients have normal cyclic AMP (cAMP) responses to 0-adrenoceptor stimulation, normal adenylate cyclase activity, and normal amounts of 0-adrenoceptor binding sites.
Studies of various in vivo responses to 0-adrenergic stimulation in nonmedicated asthmatic patients have also yielded variable results. Shelhamer and co-work-ers showed reduced pulse pressure and plasma cAMP responses to intravenous infusions of isoprenaline both in asthmatic subjects and atopic subjects. Apold and Aksnes showed a diminished plasma cAMP response to adrenaline in untreated asthmatic children. However, Tattersfield and co-workers found no evidence in favor of reduced bronchodilator responses to salbutamol in patients with mild asthma. However, since asthmatic patients usually have at least some degree of (subclinical) bronchoconstriction, it is difficult to evaluate P-adrenoceptor sensitivity in the target organ by comparisons of bronchodilator responses to (3-agonists in asthmatic patients and healthy control subjects due to differences in baseline measurements. The healthy subjects will, of course, dilate their bronchi less readily due to the absence of bronchoconstriction. Other studies of P-adrenoceptor responsiveness in asthma have not clearly stated that the experiments were performed in a drug-free interval. To reduce the risk of asthma attacks you may if you onlineasthmainhalers.com.
All the above-mentioned studies have been carried out on patients with allergic asthma. We were interested also in studying the subgroup of patients with exercise-induced asthma (EIA), since exercise is a rather effective means of increasing circulating adrenaline, and EIA could be associated either with a blunted adrenaline response to exercise or a diminished bronchodilator response to adrenaline. We have previously reported that EIA patients have a normal plasma adrenaline response to exercise. The possibility that EIA is associated with blunted (mediated responses was therefore examined in the present study. The aim of the study was twofold: first, to examine P-adrenoceptor sensitivity both in lymphocytes in tntro and during isoprenaline infusions in vivo, since few in vivo studies have been performed, and second, to compare the responses of healthy control subjects to those of nonmedicated patients with EIA or nonexercise induced asthma (NEIA). Our in vivo studies with isoprenaline iniusions are performed with a technique, in which the various responses are related to the plasma concentrations of isoprenaline instead of the dose.
Material and Methods
The investigation was comprised of two studies. In study I, eight patients with a history of exercise-induced asthma (EIA), eight age-and sex-matched patients with asthma not induced by exercise (NEIA), and eight similarly matched healthy control subjects participated. Details of these subjects have been presented elsewhere. In study 2, eight patients with EIA, selected on the same grounds as the EIA patients in study 1, and seven age- and sex-matched healthy control subjects participated. All EIA patients but none of the control patients or NEIA patients participating in the present investigation had decreases of FEV, of >15 percent following a standardized pretrial exercise test described previously. At this pretrial examination, all subjects had basal pre-exercise FEVj values greater than 75 percent of their predicted values. In study 2, the EIA patients (seven males) were aged 16 to 35 years and the control subjects (six males), 15 to 36 years. Previous ventolin inhalers therapy in study 1 has been presented in detail. Previous therapy in the EIA group of study 2 was terbutaline aerosol when required (three patients), terbutaline aerosol and tablets when required (two), disodium cromoglycate (DSCG) when required (one), DSCG tid and salbutamol aerosol when required (one), and choline theophyllinate tablets when required (one patient). Thus, a total of 16 EIA patients, eight NEIA patients, and 15 healthy control patients participated in the investigation after a period of one week or more without any medication. Fourteen of the 16 EIA patients also had verified allergies. All patients in the present investigation had mild to moderate asthma, since they were required to be free from drug treatment during at least one week before the investigation. All subjects were nonsmokers and were asked to abstain from caffeine containing beverages on the day of the experiment. The subjects gave their informed consent before participating and the study protocol had been approved by the Ethics Committee of the Karolinska Institute.
Lymphocytes: Sixty ml of peripheral venous blood was diluted in an equal volume of balanced salt solution containing 5000 IU heparin. The diluted blood was carefully layered on a solution of Ficoll-Paque (Pharmacia) with a density of1.077 ± 0.001 g/cm. After centrifugation (30 minutes at 400 x g and 18°C), the lymphocyte layer was carefully removed and diluted in the balanced salt solution. The lymphocyte suspension was centrifuged at 250 x g for 10 minutes at 18°C. The supernatant was discarded, and the pellet resuspended and washed using the same procedure. The final pellet was suspended in Dulbeccos phosphate buffered saline (PBS) containing 5.5 mM glucose and kept cold until incubated. The cells were counted in a Burker chamber and diluted with PBS to give a final concentration in the incubation step of 2-3 X106 cells/ml. Only plastic materials or siliconized glassware was used. The procedure yielded a cell preparation consisting of more than 90 percent mononuclear cells of which more than 90 percent were lymphocytes. Viability, as measured by trypan blue dye exclusion, was >95 percent.
To study P-adrenoceptor mediated cyclic AMP (cAMP) accumulation, the cells were incubated with 0 (blank), or 10~-10-4 M isoprenaline. Ascorbic acid (110 μ.M) was present in all incubates to prevent catecholamine oxidation. In study 1, concentration-effect curves for isoprenaline were also performed in the absence and presence of 30 nM (-)-propranolol and 50 jiM atenolol. The shifts of the dose-response curves were used to calculate pA^values for the two p-adrenoceptor antagonists. In these experiments, the isoprenaline response was amplified by the addition of the phosphodiesterase inhibitor 3-isobutyl-l-methyl-xanthine (IBMX, 0.5 mM).
The incubation was started by adding 40 |xl of lymphocyte suspension to 160 c.1 of ice-cold PBS containing the above-mentioned drugs. The cells were then incubated for 20 minutes at 37°C, after which the incubation was stopped by heating to 85 to 90°C for three minutes. The samples were subsequently cooled and stored at — 20°C until analyzed for cAMP contents by the protein binding method of Brown and co-workers. Incubations were performed in duplicate or triplicate, and cAMP was subsequently determined in duplicate, using 50 \l\ aliquots from each incubate.
Plasma Assays: Venous blood samples were collected in ice-cold centrifuge tubes containing EDTA (10 mM final concentration). Following centrifugation at + 4°C, the plasma was removed and stored at — 80°C until analyzed. Plasma catecholamines were determined by high performance cation exchange liquid chromatography with electrochemical detection. In our laboratory, the sensitivity is better than 0.05 nM (7 to 9 pg/ml) for noradrenaline, adrenaline, and dopamine, and approximately 0.1 nM for isoprenaline in plasma. For the endogenous catecholamines the interassay and intraassay coefficients of variation have been estimated to be 9 to 12 percent at 0.1 to 0.2 nM and 2 to 3 percent above 1 to 2 nM; for isoprenaline these coefficients are 3 to 9 percent at 0.5 nM and 1.4 to 5 percent at 3 nM. Plasma cAMP was determined by the protein binding method of Brown et аl, and glycerol by an enzymatic micromethod.
Venous cannulae were inserted into antecubital veins on both arms (one for blood sampling and one for infusions). After five to ten minutes of rest, 60 ml of blood was taken for the lymphocyte studies. After a further 30 minute rest period, the subjects in study 1 performed an exercise test, the results of which have been reported. Ibirty minutes after this exercise test, an isoprenaline infusion test was performed using two dose levels of isoprenaline (0.02 and 0.1 jig x kg’ x min). During isoprenaline infusions, heart rate was monitored continuously by ECC. Blood pressure was determined with a conventional cuff technique at rest and after six and eight minutes on each isoprenaline dose level. After the eight minute measurements, a 10 ml blood sample was drawn and the isoprenaline infusion interrupted for lung function measurements in a whole-body plethysmograph, followed by spirometric measurements of FEVt and vital capacity. The plethysmographic measurements included lung volumes and airway resistance, from which specific airway conductance (Sgaw) was calculated. Due to the preceding exercise test in study 1, results from the isoprenaline infusion test in that study were only used for exploratory purposes. In study 2, a similar procedure was used, but the subjects were not subjected to the exercise test and three dose levels of isoprenaline (0.02, 0.05 and 0.1 jigxkg_1xmin 4, each step lasting eight minutes) were used.
Results are presented as mean values ± SEM. Plasma concentration-response curves for isoprenaline in the in vivo infusion tests and dose-response curves in the in vitro experiments were compared by analysis of covariance. When appropriate, Students f-test for paired or unpaired variates was used. A value of p<0.05 was considered significant.