Beta-adrenoceptor Desensitization Research Articles

P2624Association between renal dysfunction and cardiac sympathetic innervation in patients with heart failure

Abstract Introduction In heart failure (HF) a strict interaction exists between heart and kidney. Previous studies reported a significant impact of sympathetic overdrive, that causes beta-adrenoceptor desensitization, in both renal and heart failure progression. It can be hypothesized that renal failure might be associated with impaired sympathetic activity assessed directly at the myocardial level in patients affected by HF. Purpose Aim of the present observational study was to assess the relationship between renal dysfunction and cardiac sympathetic innervation in HF patients with mildly and severely reduced ejection fraction (HFrEF and HFmEF). Methods Two-hundred and sixty-three patients (84% males; 66±10.8 years) with mild-to-severe HF (EF 31±6.8%) underwent iodine-123 meta-iodobenzylguanidine (123I-MIBG) myocardial scintigraphy to assess sympathetic innervation, evaluating early and late heart to mediastinum (H/M) ratios and washout rate. All patients also underwent clinical evaluation and venous blood sample collection for the assessment of serum creatinine and consequently the estimation of glomerular filtration rate (eGFR) by EPI formula. Results A direct correlation was found between EPI-eGFR and late H/M (r=0,215; p<0.001) (figure 1), with an inverse correlation between NYHA class and late H/M (r=0,152; p=0.013), and a direct correlation between left ventricular EF and late H/M (r=0,348; p<0.001). These results were not confirmed for early H/M, nor washout rate. Dividing the population in reduced eGFR and normal eGFR (cut-off ≤60 ml/min/1,73m2), a statistical significant reduction of late H/M value was found in patients with reduced eGFR (late H/M = 1,49±0,21) compared with patients with preserved eGFR (late H/M = 1,56±0,26) (p=0.020). In a multivariate model, adjusting eGFR for NYHA class and left ventricular EF, reduced eGFR and left ventricular EF remained significant predictors of reduced late H/M (p=0.006 and p<0.001, respectively). Figure 1. linear regression curve Conclusions Patients with impaired renal function and HF show impaired cardiac sympathetic activity compared to HF patients with preserved renal function, and reduced eGFR estimated by EPI formula is a significant predictor of reduced late H/M evaluated by 123I-MIBG. Thus, in future studies, the combination of these two parameters might provide addictive prognostic information in HF patients.

Identification of proteasome subunit beta type 3 involved in the potential mechanism of corticosteroid protective effectiveness on beta-2 adrenoceptor desensitization by a proteomics approach.

Asthma is a chronic inflammatory disease characterized by airway inflammation with mucus hypersecretion and hyperresponsiveness to various nonspecific stimuli. Corticosteroids are usually used to prevent β2 adrenoceptor (β2AR) desensitization in clinical and experimental practice. But the exact mechanism of corticosteroid effectiveness on β2AR desensitization is unclear. To find the potential mechanisms related to the protective effects of corticosteroid on salbutamol induced β2AR desensitization by a proteomics approach. Thirty-two BALB/c (6-8 weeks old) mice were divided into four groups: group A, control group, phosphate buffered saline (PBS)-treated group; group B, asthmatic group, treated by ovalbumin (OVA); group C, β2AR desensitized asthmatic group, treated by OVA and salbutamol (SBT) and group D, corticosteroid-treated β2AR desensitized asthmatic group, treated with OVA, SBT and Dexamethasone (DEX). After administrated with those drugs, their serum total IgE, bronchoalveolar lavage fluid (BALF) cytokine concentration, airway resistance and membrane receptor number of β2AR were evaluated. After then, the mice of group C and D were sacrificed, their protein from lung tissue were extracted and then seperated by two-dimensional gel electrophoresis (2DE). Then, the isolated protein spots were analyzed by ImageMaster software and mass spectrometry. Bioinformatic tools were used to search these protein spots and find interesting protein spots associated with corticosteroid protective effect on β2AR desensitization. Finally, these protein spots were confirmed by Western blotting. With inflammatory cell count, cytokine concentration of BALF, pathological sections, total serum IgE, airway resistance, membrane receptor number and β2AR total amount changes, asthmatic mouse model and β2AR desensitization asthmatic mouse model were successfully established. Seventeen protein spots were found different expression between group C and group D, 4 protein spots were down-regulated and 13 protein spots were up-regulated compared to group C. Proteasome subunit beta type 3 was down-regulated. Increased proteasome subunit beta type 3 expression may be responsible for salbutamol-induced β2AR desensitization in asthmatic disease, and DEX possibly render the β2AR resensitization partially by decreasing the content of proteasome.

Desensitization of beta-adrenoceptors following repeated injections of 2-substituted 4-phenylquinolines.

The chronic effects of five 2-substituted 4-phenylquinoline derivatives on the sensitivity of the noradrenergic cyclic AMP-generating system in the rat brain cortex have been determined, and compared with those of the typical and atypical antidepressants imipramine and trazodone, respectively. Acute treatment (single i.p. dose of 20 mg kg-1) and sub-chronic treatment (20 mg kg-1 daily for 10 days) induced no significant desensitization of the beta-adrenoceptors. However, chronic treatment (20 mg kg-1 daily for 3 weeks) significantly decreased the isoprenaline-induced increase in cyclic AMP, suggesting desensitization. This effect, coupled with previous findings, points to a potential role of these compounds as antidepressants.

Alpha-adrenergic responsiveness in rat isolated perfused heart after abdominal aortic coarctation.

Chronic isoproterenol pre-treatment, a well-known model of compensatory hypertrophy associated with cardiac beta-adrenoceptor desensitization, enhances the inotropic response to phenylephrine in rat isolated perfused hearts, supporting the hypothesis that myocardial alpha-adrenoceptor stimulation contributes to the maintenance of myocardial performance in situations in which cardiac beta-adrenoceptor function is compromised. To further corroborate this hypothesis, the effects of abdominal aortic coarctation on cardiac alpha-adrenergic responsiveness were investigated in Langendorff heart preparations. Abdominal aortic coarctation causes cardiac hypertrophy (21%) as shown by a significant increase in the ratio of ventricular dry weight to bodyweight. In preparations from hypertrophied rats, both maximum increases in left ventricular systolic pressure and heart rate elicited by isoproterenol (10(-12) to 10(-4) M) were significantly reduced (the isoproterenol concentration producing 50% of the maximum positive inotropic and chronotropic responses was enhanced almost 21- and 2-fold, respectively). However, the positive inotropic response to phenylephrine (10(-12) to 10(-4) M) remained unaffected following abdominal aortic coarctation, when compared with sham-operated rats. In preparations from both groups, phenylephrine infusion did not induce significant changes in heart rate. These results show that although abdominal aortic stenosis induced desensitization of cardiac beta-adrenoceptors, it did not enhance cardiac alpha-adrenoceptor responsiveness. This suggests that such an enhancement depends on the experimental model used to induce cardiac hypertrophy associated with desensitization of cardiac beta-adrenoceptors.

Effects of long-term pretreatment with isoproterenol on inotropic responsiveness to alpha-adrenoceptor stimulation: study in isolated perfused rat hearts.

The effects of chronic pretreatment with isoproterenol (5 mg kg(-1)) daily for 10 days on cardiac alpha-adrenergic responsiveness in Langendorff heart preparations were investigated. Isoproterenol pretreatment caused cardiac hypertrophy (29%) as shown by a significant increase in the ratio of ventricular dry weight to body weight. In preparations from isoproterenol-pretreated rats, both maximum increases in left ventricular systolic pressure and heart rate elicited by isoproterenol (10(-12) to 10(-4) M) were significantly reduced (the isoproterenol concentration producing 50% of the maximum positive inotropic and chronotropic responses was enhanced almost 32- and 4-fold, respectively), while the positive inotropic response to phenylephrine (10(-12) to 10(-4) M) was significantly enhanced (the phenylephrine concentration producing 50% of the maximum positive inotropic effect was reduced almost 100-fold), compared with saline-pretreated rats. In preparations from both groups, phenylephrine infusion induced non-significant changes in heart rate and its positive inotropic response was reduced in the presence of propranolol (10(-7) M) in the perfusion medium. Even under beta-adrenoceptor blockade, the curve for the phenylephrine-induced positive inotropic effect remained shifted upward after isoproterenol pretreatment. Chronic isoproterenol pretreatment induces the expected cardiac beta-adrenoceptor desensitization while simultaneously enhancing the positive inotropic responsiveness to phenylephrine in Langendorff heart preparations. These findings support the hypothesis that cardiac alpha1-adrenoceptor stimulation may contribute to the maintenance of myocardial function under conditions in which beta-adrenoceptor function is compromised.

Influence of epithelium on beta-adrenoceptor desensitization of guinea pig tracheal smooth muscle

The effect of tissue incubation with a beta2-agonist of denuded and intact epithelium trachea on the responsiveness to isoprenaline and beta-receptor blocked by propranolol (CR-1) was examined in this study. We examined the effect of epithelium removal on the beta-adrenoceptor desensitization resulting from incubation of guinea pig trachea in the beta-adrenergic agonist isoprenaline (10 microM). Desensitization was measured as the change in EC50, the concentration of beta-agonist that produced 50% relaxation of tracheal rings contracted with methacholine. As a second measure of desensitization, we measured the shift in EC50 resulting from incubation of tracheal rings with the beta-adrenoceptor antagonist propranolol (20 nM), expressed as CR-1 ([post-propranolol EC50/baseline EC50]-1). Initially, we measured desensitization immediately after incubation in isoprenaline; subsequently, we repeated the protocol and allowed a 30 min rest between the end of incubation and the measurement. The sensitivity of denuded epithelium trachea to isoprenaline and (CR-1) was significantly higher than that of intact epithelium only in non-incubated preparations (p<0.05 to p<0.001). Incubation to isoprenaline caused a significant reduction in the tracheal response to isoprenaline in both the denuded groups (p<0.005 for both cases) and intact epithelium groups (p<0.05 for both cases). Incubation to isoprenaline also caused a significant reduction in (CR-1) value in both the denuded groups (p<0.005 for group 2 and p<0.001 for group 4) and intact epithelium only in group 1 (p<0.05). However, the changes in EC50 due to tissue incubation with isoprenaline were significantly greater in denuded than intact epithelium trachea (p<0.05 for all cases) and for CR-1 value only in groups 1 and 2 (p<0.05). These results indicate decrease in both tracheal response to beta-agonist (tolerance) and CR-1 (due to incubation of tissues with isoprenaline), which were greater in denuded epithelium groups.

Cysteinyl-leukotrienes in the regulation of β2-adrenoceptor function: an in vitro model of asthma

BackgroundThe response to β2-adrenoceptor agonists is reduced in asthmatic airways. This desensitization may be in part due to inflammatory mediators and may involve cysteinyl-leukotrienes (cysteinyl-LTs). Cysteinyl-LTs are pivotal inflammatory mediators that play important roles in the pathophysiology of asthma, allergic rhinitis, and other inflammatory conditions. We tested the hypothesis that leukotriene D4 (LTD4) and allergen challenge cause β2-adrenoceptor desensitization through the activation of protein kinase C (PKC).MethodsThe isoproterenol-induced cAMP accumulation was evaluated in human airway smooth muscle cell cultures challenged with exogenous LTD4 or the PKC activator phorbol-12-myristate-13-acetate with or without pretreatments with the PKC inhibitor GF109203X or the CysLT1R antagonist montelukast. The relaxant response to salbutamol was studied in passively sensitized human bronchial rings challenged with allergen in physiological salt solution (PSS) alone, or in the presence of either montelukast or GF109203X.ResultsIn cell cultures, both LTD4 and phorbol-12-myristate-13-acetate caused significant reductions of maximal isoproterenol-induced cAMP accumulation, which were fully prevented by montelukast and GF109203X, respectively. More importantly, GF109203X also prevented the attenuating effect of LTD4 on isoproterenol-induced cAMP accumulation. In bronchial rings, both montelukast and GF109203X prevented the rightward displacement of the concentration-response curves to salbutamol induced by allergen challenge.ConclusionLTD4 induces β2-adrenoceptor desensitization in human airway smooth muscle cells, which is mediated through the activation of PKC. Allergen exposure of sensitized human bronchi may also cause a β2-adrenoceptor desensitization through the involvement of the CysLT1R-PKC pathway.

Salmeterol Does Not Alter Increased Bronchial Responsiveness Caused by Organic Dust Exposure

Exposure in a swine house induces airway inflammation and increases bronchial responsiveness to methacholine in healthy subjects. The aim was to investigate whether a long-acting beta2-agonist, salmeterol, alters the increased bronchial responsiveness induced in healthy subjects following exposure to organic dust in a swine barn. The study includes three separate parts. In the first part (part 1), healthy subjects inhaled salmeterol (50 microg bid, n = 8) or placebo (n = 8) over 2 weeks. In part 2, healthy subjects inhaled one single dose of salmeterol (100 microg, n = 6) or placebo (n = 6) 1 h prior to exposure in a swine barn, which was followed by a bronchial methacholine challenge. In part 3, eight healthy individuals inhaled placebo or salmeterol (100 microg), 2 h or 8 h prior to a bronchial methacholine provocation, without being exposed in the swine barn. Exposure caused an increase of bronchial responsiveness to methacholine by 3.2 doubling concentration steps (25 to 75th percentiles, 2.8 to 4.1) and 2.6 doubling concentration steps (25 to 75th percentiles, 1.4 to 3.7) in the placebo and salmeterol groups (2 weeks), respectively, with no significant differences between the groups (p = 0.3; part 1). Similar results were obtained when salmeterol was administered as a single dose (part 2) prior to exposure. However, salmeterol significantly attenuated the bronchial responsiveness to methacholine by 1.2 doubling concentration steps (0.8 to 1.7) 8 h after inhalation (part 3). Salmeterol inhalation did not protect against the increased bronchial responsiveness induced in healthy subjects following exposure to organic dust when administered for 2 weeks or as a single dose prior to exposure. This lack of protection cannot be explained by homologous beta2-adrenoceptor desensitization. We hypothesize that exposure to organic material may alter the airway response to beta2-agonists.

G-protein-coupled receptor kinase activity in human heart failure: Effects of β-adrenoceptor blockade

In human end-stage heart failure as well as in experimental animal models of heart failure, G-protein-coupled receptor kinase activity (GRK) is increased while beta-adrenoceptor responsiveness is diminished. In animal studies, beta-adrenoceptor blockers reverse the GRK-mediated desensitization and down-regulation of myocardial beta-adrenoceptors. The aim of this study was to investigate whether alterations in GRK activity are an early or late accompaniment of human heart failure and whether also in humans beta-adrenoceptor blocker treatment is able to influence myocardial GRK activity. We assessed in right atria, obtained from patients at different stages of heart failure, treated with or not treated with beta-adrenoceptor blockers, and in the four chambers of explanted hearts, obtained from patients with end-stage heart failure, beta-adrenoceptor density (by (-)-[(125)I]-iodocyanopindolol binding) and GRK activity (by an in vitro rhodopsin phosphorylation assay). With increasing severity of heart failure, plasma noradrenaline levels increased while myocardial beta-adrenoceptor density decreased with a maximum in GRK activity in end-stage heart failure. However, in relation to the progression of heart failure, we found that GRK activity transiently increased at an early stage of heart failure (NYHA I and II) but decreased back to control values in patients at NYHA III and IV. beta-Adrenoceptor blockers were able to reduce the early increase in GRK activity at NYHA I and II to control levels, whereas in those patients who did not have increased GRK activity (NYHA III and IV), they had only a marginal effect. According to our results, an increase in GRK activity is an early and transient event in the course of heart failure that can be prevented by beta-adrenoceptor blocker treatment.

Differential Effects of .BETA.2-Adrenoceptor Desensitization on the IgE-Dependent Release of Chemical Mediators from Cultured Human Mast Cells

In the present study, we examined the inhibitory effects of the beta2-adrenoceptor agonists isoproterenol, salbutamol, fenoterol, and clenbuterol, on the release of chemical mediators from cultured human mast cells after prolonged treatment with the agonists. Although preincubation of sensitized mast cells for 10 min with beta2-adrenoceptor agonists potently inhibited mediator release, prolongation of the preincubation period up to 240 min attenuated the inhibition. The attenuation of histamine release inhibition was potent when compared with that of prostaglandin D2 (PGD2) and cysteinyl leukotriene (LT) release inhibition. In contrast, forskolin inhibited mediator release and the inhibition increased gradually in proportion to the preincubation period. The reduced inhibition by the beta2-adrenoceptor agonists was compensated for by simultaneous treatment with cholera toxin. The beta2-adrenoceptor agonists elevated intracellular cAMP levels after 10-min incubation and the elevated levels were almost comparable to those after 240-min incubation. Forskolin elevated the intracellular cAMP levels more potently after incubation for 240 min than after 10 min. When mast cells were incubated for 3 d with the beta2-adrenoceptor agonists, similar attenuation of mediator release inhibition was observed. Elevation of intracellular cAMP levels was also attenuated, although beta2-adrenoceptor mRNA expression was potentiated. The present results collectively indicate that the attenuation of mediator release inhibition by beta2-adrenoceptor agonists under the present experimental conditions involves uncoupling between beta2-adrenoceptors and Gs proteins. Furthermore, the beta2-adrenoceptor desensitization causes differential attenuating effects on the inhibition of histamine, PGD2, and LT release, suggesting that downstream events involved in each inhibitory pathway have different sensitivity to receptor desensitization.

Inhibitory effects of interferon-gamma on the heterologous desensitization of beta-adrenoceptors by transforming growth factor-beta 1 in tracheal smooth muscle.

Transforming growth factor-beta1 (TGF-beta 1) is generally considered to play an important role in the pathogenesis of chronic inflammation and fibrosis. This study was designed to determine mechanisms of reduced responsiveness of guinea-pig tracheal smooth muscle to beta-adrenoceptor agonists by TGF-beta 1, using isometric tension records and tissue cAMP measurement. Moreover, we examined the involvement of the signal transduction processes of TGF-beta superfamily in the desensitization of beta-adrenoceptors. After exposure to 0.2-2000 pm TGF-beta 1 for 4-8 h, the inhibitory effects of 1 microm isoprenaline (ISO) and 10 microm forskolin on 1 microm MCh-induced contraction were markedly reduced in a concentration-dependent fashion. The desensitization by TGF-beta 1 was greater against ISO than for forskolin. The values of EC75 for the curves for ISO after exposure to the normal bathing solution and TGF-beta 1 were 0.039 +/- 0.02 and 0.38 +/- 0.28 microm, respectively. The values of EC50 for the curves for forskolin under these conditions were 0.50 +/- 0.12 and 0.89 +/- 0.21 microm, respectively. On the other hand, the inhibitory effects of phosphodiesterase inhibitors such as theophylline and rolipram were not attenuated after exposure to TGF-beta 1. Concentration-inhibition curve for ISO was shifted to the right after exposure to 2000 pm TGF-beta 1 for 8 h more than that curve for forskolin. In contrast, the curve for theophylline was not shifted to the right by TGF-beta 1. When the tissues were incubated with TGF-beta 1 in the presence of IFN-gamma, an intracellular antagonist of TGF-beta signalling, IFN-gamma inhibited the reduced response to ISO and forskolin after exposure to TGF-beta 1 in a concentration-dependent fashion. After exposure to TGF-beta 1, the effects of cAMP accumulation of ISO was significantly reduced, however, neither forskolin-nor theophylline-induced cAMP accumulation was affected. IFN-gamma had no significant effect on cAMP accumulation either to ISO or forskolin. Impairment of the beta-adrenoceptors/adenylyl cyclase pathway are involved in heterologous desensitization of beta-adrenoceptors induced by TGF-beta 1 in airway smooth muscle. IFN-gamma functionally suppresses this phenomenon via cAMP-independent processes. Phosphodiesterase is still intact under this condition.

Blunted central bromocriptine-induced tachycardia in conscious, malnourished rats.

Bromocriptine-induced tachycardia, persisting after adrenalectomy, is mediated by central dopamine D2 receptor stimulation through activation of the sympathetic outflow to the heart. The present study investigated the effects of malnutrition during pregnancy on bromocriptine-induced tachycardia in adult conscious rats. Malnourished rats were obtained by feeding dams a multideficient diet (providing 8% protein) during mating and pregnancy. Birth weight was significantly reduced in malnourished rats when compared to control rats born to dams fed standard commercially diet (23% protein) during mating and pregnancy. Baseline mean aortic pressure and heart rate in malnourished rats were comparable to those of well-nourished rats. Tachycardia (33+/-9 beats/min.), but not the hypotensive response to intravenous bromocriptine (150 microg/kg) was significantly reduced in malnourished rats, compared with control rats (70+/-10 beats/min.). In malnourished rats, pretreatment with intravenous domperidone (500 microg/kg) blocked the bromocriptine-induced hypotension, without affecting the tachycardia. Neither cardiac vagal (40+/-6 beats/min.) nor sympathetic tone (76+/-6 beats/min.) was significantly altered by multideficient diet-induced malnutrition (51+/-6 and 67+/-10 beats/min., respectively). In isolated perfused heart preparations from malnourished rats, positive inotropic response to isoproterenol (10-8 to 10-4 M) was not significantly different compared to that in control rats. In summary, malnutrition during foetal life blunted the bromocriptine-induced tachycardia, an effect that could be related to central dopamine D2 receptor desensitization rather than to impairment of autonomic regulation of the heart or cardiac beta-adrenoceptor desensitization.

Role of Src in hypersensitization to phosphodiesterase inhibitors in beta2-adrenoceptor-desensitized eosinophils.

When guinea pig eosinophils were incubated with fenoterol, a beta2-agonist, for 120 min, not only desensitization of beta2-adrenoceptors but also hyperresponsiveness to phosphodiesterase (PDE) inhibitors, such as theophylline and rolipram, was observed. The fenoterol-induced beta2-adrenoceptor desensitization was not affected by pretreatment with either genistein, a broad-spectrum tyrosine kinase inhibitor, or PP2, a specific Src family tyrosine kinase inhibitor. On the other hand, both genistein and PP2 abolished the hyperresponsiveness to PDE inhibitors in beta2-adrenoceptor-desensitized eosinophils. These findings suggested that Src family tyrosine kinases play important roles in the hypersensitization of PDE to PDE inhibitors in beta2-adrenoceptor-desensitized eosinophils.

Effects of angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade on beta-adrenoceptor signaling in heart failure produced by myocardial Infarction in rabbits: reversal of altered expression of beta-adrenoceptor kinase and G i alpha.

Both angiotensin-converting enzyme (ACE) inhibitors and angiotensin II type 1 (AT1) receptor blockers have been demonstrated to improve symptoms and prognosis in heart failure (HF). We compared the effects of ACE inhibition and AT1 receptor blockade on myocardial beta-adrenoceptor desensitization in rabbits with HF established 3 weeks after myocardial infarction (MI) with left circumflex coronary artery ligation. Rabbits with MI were randomized to no treatment, the ACE inhibitor temocapril (0.5 mg/kg/day) or AT1 receptor blocker valsartan (3 mg/kg/day). Echocardiographic examinations showed that, relative to rabbits with untreated MI, rabbits receiving temocapril or valsartan had a limitation of cardiac remodeling and prevention of the development of systolic dysfunction. Circulating plasma norepinephrine levels that were markedly elevated in MI animals were strongly inhibited by temocapril or valsartan therapy. beta-Adrenoceptor density, beta-adrenoceptor proportion showing high-affinity agonist binding, and basal and isoproterenol-stimulated adenylate cyclase activities were significantly reduced in MI rabbits. These defects were similarly reversed by temocapril or valsartan. Importantly, as found in human HF, myocardial protein levels of beta-adrenoceptor kinase 1 and G(i alpha) were significantly elevated in MI rabbits, suggesting that these molecules are contributing to the defects in myocardial beta-adrenoceptor signaling. The expression levels of these molecules were normalized equally by both treatments. The results suggest that pharmacologically different interventions in the renin-angiotensin system can equivalently improve the derangements in the beta-adrenoceptor signaling system in the failing heart. This may be important for the beneficial effects of these agents in HF.

β2-Adrenoceptor desensitization in human alveolar macrophages induced by inhaled terbutaline in vivo is not counteracted by budesonide

In vitro studies suggest that glucocorticoids may counteract beta-agonist-induced desensitization of beta-adrenoceptors by actions at the transcriptional level, but the clinical relevance of such findings is not clear. Oral terbutaline treatment decreases beta-adrenoceptor sensitivity in alveolar macrophages in vivo. This effect is not counteracted by inhaled or orally taken steroids. We therefore examined whether inhaled terbutaline elicited a similar effect on beta(2)-adrenoceptor sensitivity in alveolar macrophages, and if co-treatment with an inhaled steroid, budesonide, would prevent such down-regulation. Bronchoalveolar lavage (BAL) and lung function tests, including bronchodilator responses to inhaled terbutaline, were performed before and after 2 weeks of regular inhalation of terbutaline, 0.5 mg three times daily, and budesonide, 400 microg twice daily, or placebo, in 24 healthy volunteers. Four untreated subjects served as controls. A marked, approx. 90%, decrease in isoprenaline-induced cAMP accumulation in alveolar macrophages was found in both treatment groups after 2 weeks, with no difference between placebo and budesonide (P = 0.45). In the untreated control group, cAMP responses to both isoprenaline and prostaglandin E(1) tended to be lower on the second occasion. A limited, non-specific desensitization of adenylate cyclase activity thus contributed to the marked desensitization elicited by terbutaline inhalations. The bronchodilator response to inhaled terbutaline did not change after treatment in any of the three groups (F = 0.9, P = 0.50). In conclusion, inhalation of a beta-agonist induced marked down-regulation of beta(2)-adrenoceptor sensitivity in alveolar macrophages in vivo without influencing the bronchodilator response to a beta(2)-agonist in healthy subjects. Co-treatment with an inhaled steroid failed to counteract the desensitization of alveolar macrophage beta(2)-adrenoceptors.

Beta2-adrenoceptors: mechanisms of action of beta2-agonists

The human beta2-adrenoceptor is a member of the 7 transmembrane family of receptors. It is encoded by a gene on chromosome 5 and is widely distributed in the respiratory tract. Following beta2-adrenoceptor activation, intracellular signalling is mainly produced by inducing cyclic AMP. This produces airway relaxation through phosphorylation of muscle regulatory proteins and modification of cellular Ca2+concentrations. Beta2-agonists have been characterised into those which directly activate the receptor (salbutamol/terbutaline), those which are taken up into a membrane depot (formoterol) and those which interact with a receptor-specific, auxiliary binding site (salmeterol). These differences in mechanism of action are reflected in the kinetics of airway smooth muscle relaxation and bronchodilation in asthmatic patients. Beta-adrenoceptor desensitisation is associated with beta2-agonist activation and differs depending on the cell type. It is reflected in the different profiles of clinical tolerance to chronic beta2-agonist therapy. A number of polymorphisms of the beta2-receptor have been described which appear to alter the behaviour of the receptor, including the degree of downregulation and response to beta2-agonists.

Beta2-agonist exerts differential effects on the development of cord blood T cells but not on peripheral blood T cells.

Asthma is a chronic inflammatory disease characterized by reversible airway obstruction caused by edematous airway lining, thickened mucosal secretions, and smooth muscle constriction. Beta2-adrenoceptor agonists are widely used in the treatment of bronchial asthma because of their ability to induce relaxation of airway smooth muscle. Evidence indicates that desensitization and down-regulation of beta-adrenoceptors occurs in long-term beta2-agonist therapy, and these medications were thought to cause increased severity of, and mortality in, asthma. The purpose of this study was to delineate further the potential adverse effects of beta2-agonists on the development of T lymphocytes. T cells isolated from umbilical cord blood and adult peripheral blood were cultured in the presence of salbutamol. Intracellular staining with fluorescence-labeled antibodies was used to differentiate the frequency of type 1 T-helper (Th1) and type 2 T-helper (Th2) cells. The results showed a statistically significant inverse relationship between the concentration of salbutamol and the ratio of Th1 over Th2 on cord blood T cells. However, this trend was not observed in adult peripheral blood T cells. The data revealed another potential adverse effect in which chronic beta2-agonist exposure predisposed differentiation of T lymphocytes towards Th2 while that of Th1 was relatively suppressed, especially in cord blood T cells. Hence, beta2-agonists, despite their effect in symptomatic rescue in asthma, should not be used indiscriminately as long-term therapeutic agents.

Impaired G(s)alpha and adenylyl cyclase cause beta-adrenoceptor desensitization in chronically hypoxic rat hearts.

The effects of beta-adrenoceptor stimulation with isoproterenol on electrically induced contraction and intracellular calcium ([Ca(2+)](i)) transient, and cAMP in myocytes from both hypertrophied right and nonhypertrophied left ventricles of rats exposed to 10% oxygen for 4 wk, were significantly attenuated. The increased [Ca(2+)](i) transient in response to cholera toxin was abolished, whereas increased cAMP after NaF significantly attenuated. The biologically active isoform, G(s)alpha-small (45 kDa), was reduced while the biologically inactive isoform, G(s)alpha-large (52 kDa), increased. The increased electrically induced [Ca(2+)](i) transient and cAMP with 10-100 microM forskolin were significantly attenuated in chronically hypoxic rats. The content of G(i)alpha(2), the predominant isoform of G(i) protein in the heart, was unchanged. Results indicate that impaired functions of G(s) protein and adenylyl cyclase cause beta-adrenoceptor desensitization. The impaired function of the G(s) protein may be due to reduced G(s)alpha-small and/or increased G(s)alpha-large, which does not result from changes in G(i) protein. Responses to all treatments were the same for right and left ventricles, indicating that the impaired cardiac functions are not secondary to cardiac hypertrophy.

Beta-adrenoceptor dysfunction after inhibition of NO synthesis.

G(s) protein-coupled beta-adrenoceptors rapidly desensitize on exposure to agonists in reconstituted membrane preparations, whereas rapid tachyphylaxis to beta-adrenoceptor-mediated vasodilation does not readily occur in vivo. This study examined the possibility that endothelium-derived nitrosyl factors prevent the rapid desensitization of beta-adrenoceptors in the vascular smooth muscle of resistance arteries in pentobarbital-anesthetized rats. The fall in mean arterial blood pressure and in hindquarter vascular resistance produced by the beta-adrenoceptor agonist isoproterenol (ISO, 0.1 to 10 microg/kg IV) was slightly but significantly smaller in rats treated with the NO synthase inhibitor N:(G)-nitro-L-arginine methyl ester (L-NAME, 100 micromol/kg IV) than in saline-treated rats. The ISO-induced fall in mesenteric resistance was similar in L-NAME-treated and in saline-treated rats. The fall in hindquarter vascular resistance and in mesenteric resistance produced by ISO (8 x 10 microg/kg IV) was subject to tachyphylaxis on repeated injection in rats treated with L-NAME (100 micromol/kg IV) but not in rats treated with saline. Injections of L-S:-nitrosocysteine (1200 nmol/kg IV), a lipophobic S:-nitrosothiol, before each injection of ISO (10 microg/kg IV) prevented tachyphylaxis to ISO in L-NAME-treated rats. The vasodilator effects of ISO (0.1 to 10 microg/kg IV) in L-NAME-treated rats that received 8 injections of ISO (10 microg/kg IV) were markedly smaller than in L-NAME-treated rats that received 8 injections of saline. These results indicate that (1) the vasodilator actions of ISO in pentobarbital-anesthetized rats only minimally involve the release of endothelium-derived nitrosyl factors, (2) the effects of ISO are subject to development of tachyphylaxis in L-NAME-treated rats, and (3) tachyphylaxis to ISO is prevented by L-S:-nitrosocysteine. These findings suggest that endothelium-derived nitrosyl factors may prevent desensitization of beta-adrenoceptors in vivo.

Pathophysiological basis of heart failure

The progression of heart failure is related to local and systemic neuroendocrine activation. On the level of the myocardium, neuroendocrine activation (angiotensin II, endothelin, aldosterone, norepinephrine) as well as mediators of inflammation and free oxygen radicals contribute to hypertrophy, dilation and remodeling of the ventricles. In addition, vascular endothelial dysfunction and alterations of skeletal muscle contribute to clinical symptoms of heart failure patients. Changes in ventricular geometry during the progression of cardiac disease are associated with specific subcellular alterations on the level of the myocytes. Especially, disturbed intracellular Ca2+ handling resulting in altered excitation contraction coupling may lead to impaired systolic and diastolic function. Disturbed Ca2+ homeostasis has been associated with reduced re-uptake capacity of the sarcoplasmic reticulum for Ca2+ and an enhanced activity of the sarcolemmal Na+/Ca(2+)-exchanger. In consequence, alterations in force-frequency behavior were attributed to a decline in intracellular Ca2+ transients at higher stimulation rates. The reduced expression and desensitization of myocardial beta-adrenoceptors and alterations on the level of the G-proteins result in a reduced basal and catecholamine-stimulated activity of adenylate cyclase and a reduction in intracellular cAMP content. In consequence, reduced phosphorylation of intracellular functional proteins in the failing human heart contributes to altered Ca2+ handling. The Frank-Starling mechanism seems to be unaltered in isolated human myocardium from failing hearts. Endothelin and angiotensin may contribute to the regulation of myocardial contractility in the human heart, but their functional relevance in the regulation of myocardial contractility under clinical conditions remains to be evaluated.