The clinical effects of treatment with ␤-adrenoceptor (␤-AR) agonists and antagonists in heart failure vary with duration of therapy, as do the effects of ␤-AR agonists in asthma. Therefore, we hypothesized that chronic effects of ''␤-blockers'' in asthma may differ from those observed acutely. We tested this hypothesis in an antigen (ovalbumin)-driven murine model of asthma. Airway resistance responses (R aw) to the muscarinic agonist methacholine were measured by using the forced oscillation technique. In comparison with nontreated asthmatic mice, we observed that: (i) The ␤-AR antagonists nadolol or carvedilol, given as a single i.v. injection (acute treatment) 15 min before methacholine, increased methacholine-elicited peak R aw values by 33.7% and 67.7% (P < 0.05), respectively; when either drug was administered for 28 days (chronic treatment), the peak R aw values were decreased by 43% (P < 0.05) and 22.9% (P < 0.05), respectively. (ii) Chronic treatment with nadolol or carvedilol significantly increased ␤-AR densities in lung membranes by 719% and 828%, respectively. (iii) Alprenolol, a ␤-blocker with partial agonist properties at ␤-ARs, behaved as a ␤-AR agonist, and acutely reduced peak Raw value by 75.7% (P < 0.05); chronically, it did not alter Raw. (iv) Salbutamol, a ␤-AR partial agonist, acutely decreased peak R aw by 41.1%; chronically, it did not alter Raw. (v) None of the ␤-blockers produced significant changes in eosinophil number recovered in bronchoalveolar lavage. These results suggest that ␤-AR agonists and ␤-blockers with inverse agonist properties may exert reciprocating effects on cellular signaling dependent on duration of administration.␤-blockers ͉ sympathomimetics ͉ airway resistance ͉ inverse agonist

Single-dose administration of beta-adrenoceptor agonists produces bronchodilation and inhibits airway hyperresponsiveness (AHR), and is the standard treatment for the acute relief of asthma. However, chronic repetitive administration of beta-adrenoceptor agonists may increase AHR, airway inflammation, and risk of death. Based upon the paradigm shift that occurred with the use of betablockers in congestive heart failure, we previously determined that chronic administration of beta-blockers decreased AHR in a murine model of asthma. To elucidate the mechanisms for the beneficial effects of beta-blockers, we examined the effects of chronic administration of several beta-adrenoceptor ligands in a murine model of allergic asthma. Administration of beta-blockers resulted in a reduction in total cell counts, eosinophils, and the cytokines IL-13, IL-10, IL-5, and TGF-b1 in bronchoalveolar lavage, and attenuated epithelial mucin content and morphologic changes. The differences in mucin content also occurred if the beta-blockers were administered only during the ovalbumin challenge phase, but administration of beta-blockers for 7 days was not as effective as administration for 28 days. These results indicate that in a murine model of asthma, chronic administration of beta-blockers reduces inflammation and mucous metaplasia, cardinal features of asthma that may contribute to airflow obstruction and AHR. Similar to heart failure, our results provide a second disease model in which beta-blockers producing an acutely detrimental effect may provide a therapeutically beneficial effect with chronic administration.

The human beta(2)-adrenergic receptor (betaAR) is rapidly desensitized in response to saturating concentrations of agonist by G protein-coupled receptor kinases (GRKs) and cAMP-dependent protein kinase A (PKA) phosphorylation of the betaAR, followed by beta-arrestin binding and receptor internalization. betaAR sites phosphorylated by GRK in vivo have not yet been identified. In this study, we examined the role of the carboxyl terminal serines, 355, 356, and 364, in the GRK-mediated desensitization of the betaAR. Substitution mutants of these serine residues were constructed in which either all three (S355,356,364A), two (S355,356A and S356, 364A), or one of the serines (S356A and S364A) were modified. These mutants were constructed in a betaAR in which the serines of the PKA consensus site were substituted with alanines (designated PKA(-)) to eliminate any PKA contribution to desensitization, and they were stably transfected into human embryonic kidney 293 cells. Treatment of the PKA(-) mutant with 10 microM epinephrine for 5 min caused a 3. 5-fold increase in the EC(50) value and a 42% decrease in the V(max) value for epinephrine stimulation of adenylyl cyclase. Substitution of all three serines completely inhibited the epinephrine-induced shift in the EC(50). Both double mutants, S355,356A and S356,364A, showed a nearly complete loss of the EC(50) shift, whereas the single substitutions, S356A and S364A, caused only a slight decrease in desensitization. None of the mutations altered the epinephrine-induced decrease in V(max,) which seems to be downstream of the receptor. The triple mutation caused a 45% decrease in epinephrine-induced internalization and a 90 to 95% reduction in phosphorylation of the betaAR relative to the PKA(-) (1.9+/- 0.2- and 16.6+/-3.8-fold phosphorylation over basal, respectively). The double mutants caused an intermediate reduction in internalization (20-21%) and phosphorylation (43-52%). None of the serine mutations altered the rate of betaAR recycling. Our data demonstrate that the cluster of serines within the 355 to 364 betaAR domain confer the rapid, GRK-mediated, receptor-level desensitization of the betaAR.