Autoradiographic localization of changes in pulmonary beta-adrenoceptors in an animal model of atopy

Vaccination of guinea pigs with Haemophilus influenzae leads to an impairment of beta-adrenoceptor function in lung. We have used an autoradiographic technique to study the distribution of changes in lung beta-adrenoceptor density. H. influenzae induced a decrease in beta-adrenoceptors in peripheral lung membranes of 22 +/- 5% (mean +/- S.E.M., n = 7), while the affinity of binding was unaffected. Tracheal beta-adrenoceptor binding was not influenced by H. influenzae. Autoradiography revealed a 27% reduction in beta-adrenergic binding sites on alveolar septa. Bronchial epithelial beta-adrenoceptors were decreased for 36%, and vascular smooth muscle and endothelial beta-adrenoceptors were also reduced. beta-Adrenoceptors on airway smooth muscle were unaffected. H. influenzae affected both the beta 1- and beta 2-subtypes of receptors. It is concluded that in this animal model of atopy beta-adrenoceptors may be decreased on several different cell types within the lungs, which may influence overall airway and vascular reactivity.     

Involvement of catecholamines in haemophilus influenzae induced decrease of β-adrenoceptor function

Summary The deeper airways of patients with asthmatic bronchitis are often infected with Haemophilus influenzae. Vaccination of guinea pigs with H. influenzae resulted in a significant impairment of the isoproterenol induced relaxation of isolated tracheal spirals by approximately 50% 4 days following vaccination. In the present study we further investigated the effects of some drugs affecting catecholamine release on the H. influenzae induced functional desensitization of tracheal spirals. Benserazide, an inhibitor of dopadecarboxylase, completely prevented the reduction in isoproterenol-induced relaxation after H. influenzae vaccination, while no effect on relaxation of tracheal spirals from control animals was detected. On the other hand, inhibiting the re-uptake of catecholamines with desipramine did not influence the relaxation in the H. influenzae vaccinated treacheal spirals. Treatment of control animals with desipramine however resulted in a decreased relaxation of the isolated spirals by 40%. One day following vaccination with H. influenzae the level of norepinephrine in lung tissue was significantly elevated by 71%, and in plasma by 77%, while after 4 days no significant effects were observed. The spontaneous release of norepinephrine, epinephrine and dopamine of tracheal incubates was increased at days 1 and 4 following vaccination. The release of catecholamines from minced lung incubates of H. influenzae pretreated guinea pigs did not differ from that of controls.On the basis of these results it may be suggested that catecholamine metabolism is changed in lungs from H. influenzae vaccinated animals. Catecholamines, accordingly may play a role in the desensitization of -adrenoceptors by H. influenzae.     

Involvement of the spleen in the endotoxin-induced deterioration of the respiratory airway and lymphocyte beta-adrenergic systems of the guinea pig

The beta-adrenergic binding sites on splenic lymphocyte membranes of the guinea pig were characterized with the radio-ligand [125I]cyanopindolol and showed a maximal number of binding sites (Bmax) of 125 fmol/mg protein and an affinity (Kd) of 170 pM. The potency of various beta-adrenoceptor antagonists to compete for [125I]cyanopindolol binding suggested that the receptor is of the beta 2 subtype. Endotoxin (1 mg/kg) induced a 35% decrease in the number of beta-adrenergic binding sites on lymphocytes, 4 days after i.p. administration. The reduction in the number of beta-adrenoceptors on the lymphocytes was accompanied by a 30% decrease in the relaxation of isolated guinea pig tracheal spirals to isoprenaline and a 20% reduction in the number of beta-adrenergic binding sites in peripheral lung tissue. The endotoxin-induced deterioration of the beta-adrenergic system in the respiratory airways was completely prevented by splenectomy. It is concluded that the spleen, and or cells or products derived from the spleen, are involved in the changes of the beta-adrenergic system in the respiratory airways and lymphocytes.     

Difference of tolerances to isoprenaline between tracheal and vascular smooth muscles and cardiac conduction system in guinea pigs

Pretreatment to guinea pigs with sc isoprenaline (Iso) 10 micrograms/kg tid x 7 d reduced the effect of Iso on protecting histamine-induced asthma and decreased its pD2 values in relaxing isolated tracheal strip. This treatment did not change the asthmatic effect induced by histamine and the effect of Iso on positive chronotropic action, but elevated the blood pressure. These results suggest that it is easier to develop the tolerance of beta 2-adrenoceptors of respiratory smooth muscles than that of beta 1-adrenoceptors of heart. Radioligand binding assay showed that the treatment decreased the number of binding sites of beta-adrenoceptors on lungs of guinea pigs but did not change the binding affinity.     

Effect of respiratory tract viral infection on murine airway beta-adrenoceptor function, distribution and density.

1. The effects of a respiratory tract viral infection on beta-adrenoceptor density, distribution and function were investigated in murine airways. 2. Following intranasal inoculation of CBA/CaH mice with influenza A/PR-8/34 virus, the virus proliferated rapidly in trachea (peak titres 2 days post-inoculation) and lung (peak titres 4-6 days post-inoculation). Respiratory tract viral infection was associated with a significant increase in lung weight (88% higher than control mice at day 6 post-inoculation) that was related temporally to the development of peripheral lung inflammation and consolidation. 3. Analysis of specific binding of [125I]-cyanopindolol to beta-adrenoceptors revealed that on days 2, 4 and 8 post-inoculation with virus, mouse isolated tracheal sections contained, on average, 40% more beta-adrenoceptors than tracheal sections from time matched control mice. Subsequent quantitative autoradiographic studies demonstrated that this increase in total tracheal beta-adrenoceptors was due primarily to a 90% increase in the density of beta-adrenoceptors in the tracheal epithelium in virus-infected mice. 4. In contrast, virus-infection had no significant effect on the density of beta-adrenoceptors in tracheal airway smooth muscle, although within 2 days of inoculation with virus, mouse tracheal smooth muscle segments were approximately 2 fold less sensitive to the beta-adrenoceptor agonist, noradrenaline (mean pD2 = 6.57 +/- 0.04, n = 24) and to the adenylyl cyclase-activator forskolin (mean pD2 = 6.78 +/- 0.04, n = 12) compared to segments from control mice (mean pD2 = 6.84 +/- 0.06 for noradrenaline; mean pD2 = 7.03 +/- 0.07 for forskolin). Similar values were obtained 8 days post-inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of Haemophilus influenzae vaccination on anaphylactic mediator release and isoprenaline-induced inhibition of mediator release

The influence of Haemophilus influenzae on anaphylactic mediator release from ovalbumin-sensitized isolated guinea pig lungs was investigated. Lungs from H. influenzae-vaccinated animals released prostaglandins and thromboxanes following a smaller dose of ovalbumin than was effective in non-vaccinated animals. Histamine release was significantly increased in 4 day-vaccinated animals but not 1 or 10 days after vaccination, while broncho-constriction was potentiated in 1 and in 4 day-vaccinated animals. This increased histamine release was achieved following 2 micrograms ovalbumin. In contrast, doses of 10 micrograms and 1 mg ovalbumin respectively did not affect and decreased histamine release in the vaccinated group. The inhibition of anaphylactic mediator release by an infusion of 6 x 10(-9) M isoprenaline was significantly attenuated by H. influenzae vaccination. These results indicate an increased sensitivity to antigenic challenge and suggest that the functioning of beta-adrenoceptors was decreased as a result of H. influenzae vaccination.     

Adimolol, a long acting beta-adrenoceptor blocker in man.

A comparative study in eight healthy normotensive males of the effects on blood pressure, heart rate and beta-adrenoceptor function following single oral doses of adimolol (600 mg), propranolol (240 mg) and placebo. Both active treatments produced small but significant reductions in blood pressure and heart rate, supine and erect. These effects persisted for up to 7 days after adimolol. The heart rate increases following both dynamic exercise and intravenous isoprenaline were attenuated by both propranolol and adimolol. With adimolol evidence of functional beta-adrenoceptor antagonism was sustained for up to 7 days. Lymphocyte beta-adrenoceptor binding studies showed that both adimolol and propranolol significantly reduced affinity for beta-adrenoceptors. In addition, adimolol significantly reduced receptor number and even by 3 days after dosing Bmax had only returned to half the control value. In a small sub-group of subjects there was no evidence to suggest that adimolol had additional alpha-adrenoceptor antagonist properties. Adimolol was detected in plasma for up to 3 days after dosing. The mean terminal elimination half-life was 14 h, compared to 3 h for propranolol. This study confirms that adimolol has prolonged beta-adrenoceptor antagonist activity with effects persisting for up to 7 days after a single dose. The reduction in beta-adrenoceptor number following adimolol suggests that this prolonged effect may not be solely due to competitive antagonism but may additionally depend upon non-competitive antagonism at beta-adrenoceptors.     

Deficiency of beta-adrenoceptor-mediated relaxation in suncus trachea

Characteristics of beta-adrenoceptor in the tracheal smooth muscle of Suncus murinus (suncus) were studied in comparison with those of rats and guinea pigs. Isoproterenol induced concentration-dependent relaxation of suncus trachea. However, the maximal relaxation was very small, and about 1000 times higher concentration was necessary compared to the case of the guinea pig. The order of the sensitivity to isoproterenol and the maximal relaxation was guinea pig much greater than rat greater than suncus. Tracheae from younger suncus were more sensitive to isoproterenol, but the maximal relaxation was not significantly different from the results using adult animals. Forskolin, a direct activator of adenylate cyclase, relaxed tracheae to similar extents in the three species. Though the affinity of specific [3H]dihydroalprenolol binding was not significantly different, the maximal number of binding sites was in the order of rat greater than guinea pig greater than suncus. However, the differences in density of the binding among the three species were not as great as differences in isoproterenol-induced relaxation. These results suggest that 1) beta-adrenoceptor-mediated relaxation of tracheal smooth muscle is not well-developed in the suncus and 2) the deficient relaxation is probably caused by insufficient coupling between adrenoceptors and adenylate cyclase.     

Adrenaline-induced tracheal relaxation ex vivo is depressed following aerosol antigen challenge of sensitized guinea-pigs.

We investigated the changes in beta-adrenoceptor responses induced in guinea-pig tracheal and cardiac tissues by the anaphylactic reaction. Antigen aerosol challenge in sensitized guinea-pigs resulted in a marked reduction in adrenaline relaxation in isolated trachea ex vivo. The isoprenaline effect was also slightly decreased by antigen exposure, suggesting a possible impairment of tracheal beta-adrenoceptor function. On the other hand, the chronotropic and inotropic activity of adrenaline in isolated atria was not modified by the anaphylactic shock, suggesting a specific involvement of lung beta-adrenoceptors in the allergic reaction.     

Impaired activation of adenylyl cyclase in lung of the Basenji-greyhound model of airway hyperresponsiveness: decreased numbers of high affinity beta-adrenoceptors.

1. To evaluate mechanisms involved in the impaired beta-adrenoceptor stimulation of adenylyl cyclase in tissues from the Basenji-greyhound (BG) dog model of airway hyperresponsiveness, we compared agonist and antagonist binding affinity of beta-adrenoceptors, beta-adrenoceptor subtypes, percentage of beta-adrenoceptors sequestered, and coupling of the beta-adrenoceptor to Gs alpha in lung membranes from BG and control mongrel dogs. We found that lung membranes from the BG dog had higher total numbers of beta-adrenoceptors with a greater percentage of receptors of the beta 2 subtype as compared to mongrel lung membranes. 2. Agonist and antagonist binding affinity and the percentage of beta-adrenoceptors sequestered were not different in BG and mongrel dog lung membranes. However, the percentage of beta-adrenoceptors in the high affinity state for agonist was decreased in BG lung membranes suggesting an uncoupling of the receptor from Gs alpha. 3. Impaired coupling between the beta-adrenoceptor and G protein documented by the decreased numbers of beta-adrenoceptors in the high affinity state in BG lung membranes, is a plausible explanation for the reduced stimulation of adenylyl cyclase and the resultant reduction in airway smooth muscle relaxation in this model.     

Alloimmunization-induced changes in beta-adrenoceptor expression and cAMP on B lymphocytes

In this report we have examined the effect of alloimmunization on beta-adrenergic expression in lymphocytes. We have observed a variation in the number of beta-adrenoceptors (Bmax) according to the degree of immunization without modifications in their affinity (Kd). This phenomenon was accompanied with parallel variations of intracellular cAMP levels. A decrease in Bmax values was observed during the first and second immunizations. Then the Bmax began to increase, exceeding control values up to the fourth and fifth immunizations, and remaining constant at the sixth immunizations. Only B cell-enriched populations showed variation in Bmax values of beta-adrenoceptors with alloimmunization. In contrast, the Bmax values of T cell-enriched populations did not change. Kd values were similar in all cell types tested. The number of binding sites was not dependent on the animal's age. Modifications in cAMP levels of B cell-enriched populations were correlated with changes in beta-adrenoceptor expression. These results suggest that beta-adrenoceptor expression and cAMP intracellular levels in B cell-enriched populations vary with the number of alloimmunizations. In addition, the antibody synthesis induced by allogenic stimulus was inversely proportional to the number of beta-adrenoceptors expressed on B cells. Perhaps these findings are evidence of a control mechanism that regulates antibody synthesis during the immune response.     

Concomitant glucocorticoid treatment prevents the development of beta-adrenoceptor desensitization in the guinea pig lung

The aim of the present study was to investigate, whether concomitant administration of the synthetic glucocorticoid betamethasone (BM), theophylline (THEO), or the muscarinic antagonist ipratropium bromide (IPRA) could influence the desensitization-associated decrease of beta-adrenoceptors in the guinea pig lung during prolonged in vivo treatment with the beta 2-agonist terbutaline (TER). The animals were sacrificed 20 hrs after the last drug dosage and the lung membrane homogenates were prepared for 3H-dihydroalprenolol (3H-DHA) binding in vitro. Treatment with TER 200 micrograms/kg subcutaneously twice a day for five days decreased by 22% the maximum number of binding sites (Bmax) at saturation in comparison with the saline-treated controls. Concomitant administration of BM 2 mg/kg intraperitoneally abolished this effect of TER, whereas THEO 20 mg/kg or IPRA 5 micrograms/kg failed to modify it. None of the in vivo treatments affected the binding affinity of 3H-DHA. In vitro, TER inhibited in a concentration-dependent manner 3H-DHA binding to the lung membranes of untreated guinea pigs. At high concentrations IPRA, but not THEO or BM, showed some binding to the beta-receptors as well. Thus, it is concluded that glucocorticoids may prevent beta-adrenoceptor desensitization in the lungs via an indirect mechanism, e.g. inhibition of phospholipase A2 enzyme.     

1-Isopropylamino-3-(4-indanoxy)-2-propanol HCl: A potent β-adrenoceptor antagonist

1. The beta-adrenoceptor blocking activity of 1-isopropylamino-3-(4-indanoxy)-2-propanol HCl (USVC 6524) was determined in the anaesthetized dog, the isolated rat uterus and the isolated guinea-pig tracheal strip.2. USVC 6524 inhibited the positive inotropic, positive chronotropic and vasodilator responses to isoprenaline in the dog in a dose of 10 mug/kg and higher. On the basis of comparative pA(2) values, USVC 6524 is approximately 10 times more potent as a beta-adrenoceptor antagonist than propranolol.3. Cardiac depressant effects produced by USVC 6524 were relatively mild and occurred only after the onset of a strong beta-adrenoceptor blockade.4. USVC 6524 also blocked beta-adrenoceptors in the isolated rat uterus and guinea-pig tracheal spiral strip.     

Bacterial cell wall components decrease the number of guinea-pig lung beta-adrenoceptors

Infections of the deeper respiratory airways can contribute to the progression of chronic asthmatic bronchitis. In the present report a number of microorganisms affecting the number of beta-adrenoceptors in guinea-pig lung homogenates are described. Haemophilus influenzae, Streptococcus pneumoniae, Bordetella pertussis and Escherichia coli O111B4 induced a significant decrease of the number of beta-adrenoceptors (by approximately 20%). Staphylococcus aureus, influenza A virus and Escherichia coli J5 were not active. These data point to a common factor shared by gram-negative bacilli; i.e. endotoxin. Purified endotoxin of E. coli O111B4 also decreased the number of beta-adrenoceptors, while E. coli J5-LPS did not. This suggests that neutral polysaccharides of bacterial cell walls, especially those in the 'O'-antigenic side chain of gram-negative endotoxins may be responsible for the decrease of beta-adrenoceptor number and therefore contribute to the pathogenesis of chronic asthmatic bronchitis. Intact endotoxin seems to be necessary since neither the isolated lipid nor the polysaccharide part of E. coli O111B4 LPS affected the number of beta-adrenoceptors in the lung.     

Decreased number of beta-adrenoceptors in cerebral cortex of hypothyroid rats

The influence of thyroid hormone deficiency on beta-adrenoceptors in the rat cerebral cortex was investigated using the 3H-dihydroalprenolol binding assay. The maximal number of binding sites (Bmax) and the dissociation constants (KD) were determined in the brain tissue from euthyroid animals and from rats made hypothyroid by feeding 6-propyl-2-thiouracil. Hypothyroidism resulted in a 37% decrease in beta-adrenoceptor density (Bmax). The dissociation constants (KD) of both groups were not significantly different.     

曲尼司特对豚鼠肺组织β肾上腺素受体向下调节的影响

用放射配体结合分析法分别测定了正常组、特布他林组、特布他林十曲尼司特组豚鼠的肺组织β受体最大结合力和解离常数,结果显示:给特布他林后豚鼠肺组织β受体发生明显的向下调节.曲尼司特可预防此向下调节的发生.     

Kappa-opioid receptor agonist inhibits the cholera toxin-sensitive G protein in the heart

To explore the signaling mechanisms of the negative modulation of beta-adrenoceptors by kappa-Opioid receptors (kappa-OR) in the heart, the possibility of the interaction at the level of G protein and receptor was determined. Cholera toxin, an activator of the stimulatory G protein (Gs), elevated electrically induced intracellular Ca2+ ([Ca2+]i) transients and induced ribosylation of the alpha-subunit of Gs (Gsalpha) in rat ventricular myocytes. The effects were significantly attenuated by U50,488H, a specific agonist of kappa-OR, and were abolished by nor-binaltorphimine, a selective kappa-OR antagonist. The content of Gsalpha, however, was not affected by U50,488H. Receptor binding experiments showed that neither Bmax nor Kd of the binding of [3H]CGP-12177, a beta-adrenoceptor antagonist, was affected by U50,488H. The current study provides the first evidence that kappa-OR stimulation inhibits the ribosylation of the alpha-subunit of the Gs protein, thus inhibiting the action of cholera toxin on the protein.     

Pharmacological and biochemical characterization of the beta-adrenergic signal transduction pathway in different segments of the respiratory tract

Although in the respiratory system there is great therapeutic interest in manipulating and understanding the beta-adrenoceptor-G-protein-adenylate cyclase (AC) signal transduction pathway, little is known on segmental differences among lung, bronchus, and trachea with regard to the receptor concentration and interaction to G-proteins and coupling to AC. In this study, patterns of distribution and absolute quantities of beta-adrenoceptor subtypes beta(1) and beta(2) were determined in membranes of equine lung parenchyma, bronchial and tracheal epithelium with the underlying smooth muscle by saturation and competition binding assays using the radioligand (-)-[125I]-iodocyanopindolol (ICYP). Additionally, the functional coupling of beta-adrenoceptors to G-proteins (assessed by beta-agonist competition binding in the presence and absence of GTP) as well as the coupling efficiency and biochemical activities of AC was investigated in each region. The specific ICYP binding was rapid, reversible, saturable with time and of high affinity. The radioligand binding identified more total beta-adrenoceptors in the lung than in bronchus or trachea (428+/-19, 162.4+/-4.8, 75.6+/-1.2 fmol/mg protein, respectively) with about 40% of receptors in the high affinity state. The beta(2)-adrenoceptor subtype predominated in all segments (approximately 74-80%), as the highly selective beta(2)-adrenoceptor antagonist ICI 118,551 was about 10,000 times more potent in inhibiting ICYP binding than was the beta(1)-selective adrenoceptor antagonist CGP 20712A, and beta-adrenoceptor agonists inhibited ICYP binding with an order of potency: (-)-isoprenaline>(-)-adrenaline>(-)-noradrenaline. The dissociation constant (K(d)) was higher in the trachea than in bronchus or lung (13.0+/-0.9 pM vs. 20.0+/-2.3 pM vs. 30.8+/-4.4 pM, P<0.05, respectively). The beta(2)-adrenoceptor-mediated AC response was tissue-dependent; stimulants acting on beta-adrenoceptor (isoproterenol), G-protein (GTP, NaF) and AC (forskolin, Mn(2+)) enhanced AC responses in all three regions, but the AC activity was higher in tracheal crude membranes than in bronchus or lung (trachea>bronchus>lung), hence, the number of beta(2)-adrenoceptors correlated inversely with the amount of AC. We conclude that (1) the stoichiometry of components within the pulmonary beta-adrenoceptor-G-protein complex is segment-dependent, and (2) the receptor number or AC activity is possibly the rate-limiting factor in the beta-adrenoceptor-G-protein-AC-mediated physiological responses. Thus, it is speculated that this could have important therapeutic consequences in beta-adrenoceptor agonist-induced receptor regulation in bronchial asthma.     

Increase of Bmax-values of beta-adrenoceptor sites after chronic treatment with reserpine?

After treatment of rats with 5 x 0.5 mg/kg/d reserpine in membrane preparations from the parotid gland no increase in the total number of beta-adrenoceptors (Bmax) was observed using the antagonist ligand (-)3H-dihydroalprenolol. There occurred, however, a pronounced increase of the high affinity agonist binding component. Thus, it appears that not the absolute number of beta-adrenoceptor sites but the relative amount of high affinity sites is most sensitive against the sensitization process. The results are in contrast to those of other authors. We suspect that increases of Bmax-values after reserpine can be simulated by the loss of noradrenaline whereas in control membranes noradrenaline is still bound to the adrenoceptors thus preventing the radioactive ligand from access to these sites. We highly recommend to use preincubation methods prior to ligand binding studies in order to remove endogenous ligands still bound to the high affinity site of the beta-adrenoceptor.     

Relationship between the (--)-[3H]-dihydroalprenolol binding to beta-adrenoceptors and transmembrane 86Rb efflux of the BC3H1 nonfusing muscle cell line

1 We have studied the binding properties of the beta-adrenoceptor antagonist [3H]-dihydroalprenolol ([3H]-DHA) on a membrane preparation of the non-fusing muscle cells BC3H1. 2 [3H]-DHA appears to bind to two classes of sites. The first site has a high affinity (KD = 0.53 nM) and a low capcity (Bmax = 58 fmol/mg of protein). The second site has a low affinity (KD = 110 nM) and a high capacity (Bmax = 1100 fmol/mg of protein). 3 The pharmacological properties of the high affinity low capacity site correspond to the known properties of the beta 2-adrenoceptors since the agonists inhibit [3H]-DHA binding following the series isoprenaline greater than adrenaline greater than noradrenaline greater than phenylephrine and the antagonists following the series alprenolol congruent to propranolol greater than butoxamine greater than practolol greater than phentolamine. 4 The binding properties of the beta-adrenoceptors were correlated with the effect of beta-adrenoceptor agonists and antagonists on 86Rb efflux rate from BC3H1 Cells. 5 There is very good correlation between the dissociation constants obtained by inhibition of [3H]-DHA binding by the antagonists alprenolol and propranolol, and the inhibition constants calculated from their antagonism of the 86Rb efflux rate stimulation by adrenaline. The ratio of the dissociation constants obtained by inhibition of [3H]-DHA binding by agonists and their EC50, calculated from 86Rb efflux curves, is higher than 1. This high KD/EC50 ratio indicates a high coupling efficiency between receptor occupancy by agonists and the biological effect measured.