Different mechanisms of action of beta2-adrenergic receptor agonists: a comparison of reproterol, fenoterol and salbutamol on monocyte cyclic-AMP and leukotriene B4 production in vitro.

BACKGROUND: Beta2-adrenergic receptor agonists have several effects on airway function, most of which are mediated in a variety of cell types resulting in increased c-AMP-production and inhibition of inflammatory mediator production. However, their stimulating effects on cAMP-production became known to be inversed by increasing phosphodiesterase (PDE) activity and degradation of cAMP. Therefore, in this study we have evaluated the efficacy of reproterol, a dual acting beta2-adrenoceptor agonist and PDE-inhibitor, as compared to salbutamol and fenoterol with respect to production of cAMP and LTB4 in cultured monocytes. METHODS: Isolated human monocytes (10(5)/ml) were incubated (n = 9) in suspension with beta2-adrenoceptor agonists (10(-10) -10(-4) M) for 30 minutes with and without IBMX. Then, cAMP production was determined following treatment with Triton-X100. Production of LTB4 was measured following incubation of beta2-adrenoceptor agonists for 4 hrs in the presence of LPS (10 mg/ml). cAMP and LTB subset 4 were measured in culture supernatants by enzyme immunoassay. RESULTS: At 10(-5) M, production of cAMP was significantly stimulated by reproterol > fenoterol > salbutamol in a dose-dependent manner to an extent of *128%, *65%, 13% (*p<0.04) respectively. In contrast, LTB4-production was inhibited significantly to a similar degree by salbutamol and reproterol in a dose-dependent manner by 59% and 49% (10(-5) M, p<0.03), respectively, with decreasing inhibition (15%) after fenoterol. Following co-incubation with IBMX, cAMP production only increased significantly (p<0.002) after fenoterol (+110%) compared to salbutamol (+29%) and reproterol (+50%) (ANOVA, p<0.001). CONCLUSION: These data suggest effects of the theophylline constituent of reproterol to inhibit adenylyl cyclase induced phosphodiesterase activity. The advantageous synergistic effects of reproterol on cAMP-production need to be further explored in trials.

Inhibition by reproterol of cAMP PDE in intact mastocytoma P-815 cells.

In vitro studies in rat mastocytes and human monocytes suggested that reproterol (a selective beta(2)-adrenoceptor agonist with a theophylline moiety) exerts anti-inflammatory actions through inhibition of cyclic AMP (cAMP) PDE activity. Thus, reproterol was tested for its ability to inhibit cAMP PDE in cultured mouse mastocytoma P-815 cells. cAMP PDE activity was measured in intact cells by spectrofluorometry using the fluorescent substrate 2'-O-anthraniloyl cAMP. Reproterol was more potent than theophylline to inhibit cAMP PDE (pIC(50)=4.28+/-0.25 vs. 3.16+/-0.05). This contrasted with disrupted cells, where the PDE inhibitory potency of reproterol was low (pIC(50)=2.85+/-0.03) and similar to that of theophylline (pIC(50)=2.66+/-0.19). No cAMP PDE inhibition was found with other beta(2)-agonists tested (fenoterol, salbutamol, salmeterol and formoterol). Finally, the selective PDE inhibitors calmidazolium (100 nM), milrinone (5 microM) and rolipram (50 microM) inhibited cAMP PDE activity by approximately 20, 30 and 25% respectively. In conclusion, reproterol potently and non-specifically inhibited intracellular cAMP phosphodiesterases in intact mastocytoma cells. This can explain the previously reported beta(2)-adrenoceptor-independent anti-inflammatory actions of reproterol in vitro. Further studies are required to define the anti-inflammatory potential of reproterol in asthma.

beta-agonistic bronchodilators: comparison of dose/response in working rat hearts.

STUDY OBJECTIVES: Different beta-agonists are compared with regard to their cardiodepressive side effects. DESIGN: The metaphenolic bronchodilators reproterol, salbutamol, fenoterol, and terbutaline were introduced at a dosage of 0.0005 micromol to a maximum of 10 micromol per gram of heart tissue into the isolated working rat heart under hypoxic conditions, and the response was observed during subsequent reoxygenation. As an index of external heart work, aortic flow was measured. Heart rate, coronary flow, and developed pressure were recorded. At the end of heart perfusion, mitochondria were isolated and analyzed for adenosine triphosphatase activity, adenosine triphosphate (ATP) synthesis, and membrane fluidity. Moreover, intact mitochondria and lipid peroxidation were investigated using a model system. MEASUREMENTS AND RESULTS: Compared to controls, reproterol gave the most favorable results, with an increase of 25 to 30% of aortic flow during reoxygenation at a concentration of 10 micromol/g heart tissue. In contrast, both fenoterol and salbutamol at a concentration of 1 micromol/g heart tissue decreased aortic flow during reoxygenation, whereas terbutaline had a negative influence on aortic flow at 0.01 to 0.1 micromol/g heart tissue. Mitochondria of these hearts were isolated at the end of the experiment. Mitochondrial ATP synthesis was increased above controls at nearly all concentrations of reproterol. ATP synthesis was decreased at 1 micromol and 10 micromol fenoterol. As little as 0.0005 micromol terbutaline decreased ATP synthesis by 50%. In intact mitochondria, adenosine diphosphate (ADP) to oxygen ratios were found to be increased with terbutaline and fenoterol, indicating ADP consumption by myokinase activation. Lipid peroxidation was increased in a model system between concentrations of 0.002 micromol/mg and 0.04 micromol/mg phosphatidylcholine by fenoterol and terbutaline, whereas a decrease was noted with reproterol. Membrane fluidity was found increased after addition of reproterol, which supports the evidence of efficient ATP synthesis by this compound. CONCLUSIONS: Cardiodepressive side effects and greater toxicity of fenoterol and terbutaline were found under the conditions of our experiment. Salbutamol and, in particular, reproterol appear much better tolerated. In addition to partial beta-adrenergic agonism, reproterol may exert an inhibitory influence on adenosine receptor sites and phosphodiesterase, which could result in membrane stabilization by saving cyclic adenosine monophosphate or ATP.