Slow receptor dissociation is not a key factor in the duration of action of inhaled long-acting ß2-adrenoceptor agonists.

BACKGROUND AND PURPOSE: ß(2) -Adrenoceptor agonists are important bronchodilators used for the treatment of chronic obstructive pulmonary disease and asthma. Clinical data on ß(2) -adrenoceptor agonists show a range of onset and duration of action. We have investigated whether the receptor binding kinetics of ß(2) -adrenoceptor agonists can explain their observed onset of action and duration of effect in the clinic. EXPERIMENTAL APPROACH: [(3) H]-DHA was used to label ß(2) -adrenoceptors expressed in CHO-cell membranes (K(d) of 0.084 nM). Competition kinetic experiments were performed in the presence of unlabelled ß(2) agonists at 37°C in HBSS containing GTP. To determine the kinetic parameters, three concentrations (10, 3 and 1 ×K(i) ) of the unlabelled compound were employed against a fixed concentration of [(3) H]-DHA (0.6 nM). KEY RESULTS: The clinically used ß(2) -adrenoceptor agonists exhibited a range of association and dissociation rates. The kinetic K(d) and the competition K(i) values of the eight ß(2) -adrenoceptor agonists examined were strongly correlated, suggesting that the method had produced accurate k(off) and k(on) rates. The kinetic on-rate was highly correlated with equilibrium binding affinity. CONCLUSIONS AND IMPLICATIONS: Although the ß(2) -adrenoceptor agonists displayed a range of kinetic rate parameters, simulations at relevant drug concentrations suggest that receptor kinetics do not play an important role in determining onset of action in the clinic. In addition, it is unlikely that receptor kinetics exert an important influence on the duration of action of these agonists, as indacaterol (once daily dosing) had a shorter residency time at the receptor than salmeterol (twice daily dosing).

Antipsychotic drugs influence transport of the beta-adrenergic antagonist [3H]-dihydroalprenolol into neuronal and blood cells.

The amine hypothesis suggests that the cause of schizophrenic or depressive psychosis is dysfunction of noradrenergic or serotonergic neurotransmission. We investigated pharmacological properties of [3H]-dihydroalprenolol (DHA) transport into C6, IMR32, native lymphocytes, B-lymphoblastoids and MOLT-3 cells. DHA transport was inhibited by a heterogeneous group of structurally related compounds exhibiting an amine group and various aromatic ring structures. It was verified on cells of neuronal/glial and blood cell origin but in detail on B-lymphoblastoids. The latter once showed strongest inhibition of DHA transport using tricyclic antidepressants (amitriptyline: IC50 = 2.86 microM, imipramine: IC50 = 3.33 microM) and haloperidol (IC50 = 3.98 microM) as a neuroleptic. Antipsychotics like clozapine (IC50 = 11 microM), olanzapine (IC50 = 15 microM), spiperone (IC50 = 66 microM) and EMD 49980 (ICso >> 100 microM) were less effective. In contrast to cells of blood origin, a stimulation of DHA transport by antipsychotics was not detectable using neuronal cells. As antipsychotics showed a distinct inhibition and, concerning cells of blood origin, a stimulation of transport after pre-incubation, further investigations seem to be of interest in respect to its involvement in the cellular uptake of drugs and therefore its impact on the quality of therapy of psychiatric patients.

Phosphorylation of beta-adrenergic receptor leads to its redistribution in rat heart during sepsis.

The role of receptor phosphorylation on the redistribution of beta-adrenergic receptors (beta-ARs) in rat hearts during different phases of sepsis was investigated. Sepsis was induced by cecal ligation and puncture (CLP). Changes in the distribution of beta-ARs in the sarcolemmal and light vesicle fractions were studied using (-)-[4,6-propyl-3H]dihydroalprenolol ([3H]DHA). Phosphorylation of beta-ARs was studied by perfusing hearts with [32P]H3PO4 followed by identification of the phosphorylated beta-ARs with immunoprecipitation using anti-beta 1-AR antibody. The results show that septic rat hearts exhibit an initial hypercardiodynamic (9 h after CLP; early sepsis) and a subsequent hypocardiodynamic (18 h after CLP; late sepsis) state. [3H]DHA binding studies show that, during early sepsis, the maximum binding capacity (Bmax) was increased by 26% in sarcolemma but was decreased by 30% in light vesicles, whereas, during late sepsis, the Bmax was decreased by 39% in sarcolemma but increased by 31% in light vesicles. These data indicate that beta-ARs in the rat heart were externalized from light vesicles to sarcolemma during early sepsis but were internalized from surface membranes to intracellular sites during late sepsis. The immunoprecipitation studies reveal that the externalization of beta-ARs during early sepsis was coupled with a concomitant decrease (-28.5 to -30.6%, P < 0.01) in the receptor phosphorylation, whereas the internalization of beta-ARs during late sepsis was accompanied by a simultaneous increase (30.3 to 33.8%, P < 0.01) in the receptor phosphorylation. Because the phosphorylation/dephosphorylation of beta 1-ARs regulate their functional coupling and may reflect their subcellular distribution, it is suggested that the increase in receptor phosphorylation seen in late sepsis leads to the receptor internalization observed in late sepsis; similarly, externalization of (dephosphorylated) receptors in early sepsis may give rise to the apparent decrease in sarcolemmal receptor phosphorylation observed during this interval.

Characterization of beta-adrenoreceptors of rainbow trout (Oncorhynchus mykiss) erythrocytes.

Although many studies have characterized these receptors according to pharmacological criteria, this work represents only the second direct characterization of the rainbow trout beta-adrenergic receptors. Radioligand binding assays using (+/-)-4-(3-t-butylamino-2-hydroxy-propoxy)-[5,7-3H]benzimidazol-2- one ([3H]CGP 12177) and 1-[4,6-propyl 3H]dihydroalprenolol ([3H]DHA) were conducted to determine equilibrium binding times, ligand-receptor dissociation constants (KD) and binding capacities (Bmax). Furthermore, we assessed the influence of erythrocyte handling, suspension medium and endogenous catecholamines on Bmax and KD. Maximal binding was obtained when erythrocytes were handled minimally and maintained suspended in plasma rather than physiological saline. Washing and resuspending the erythrocytes, as well as the transfer of the erythrocytes into saline, significantly impaired apparent radioligand affinity and receptor density. Endogenous catecholamines, at levels considered normal for non-stressed animals, did not interfere with the radioligand binding assays, and thus eliminated the need to wash and resuspend erythrocytes. Based on the binding characteristics after intentional lysis of erythrocytes, it is shown that the total receptor population of trout erythrocytes can be estimated by propranolol-displaceable DHA binding, the density of high-affinity surface receptors can best be determined by isoproterenol-displaceable CGP binding, and the number of receptors located within the erythrocytes can be calculated from the difference between the total receptor density and the number of isoproterenol-displacable DHA binding sites. Each of these components must be considered when performing radioreceptor assays using these radioligands and this has significant implications for the interpretation of erythrocyte beta-adrenorecptor localization and mobilization.

Interactions of cryptosin with mammalian cardiac beta-adrenoceptors.

Cryptosin - a new cardenolide from the leaves of Cryptolepis buchanani R & S was found to be a potent positive inotropic agent. In experiments with dog heart ex vivo, the rise in the cardiac rate associated with an increase in dP/dtmax and left ventricular pressure (LVP) correlated with changes in the beta-adrenoceptor densities as measured by the binding of 3H-Dihydroalprenolol (DHA). A significant change in the beta-adrenoceptor densities was observed when cryptosin was incubated with guinea pig and dog heart sarcolemmal membranes in vitro. Analysis of the binding of 3H-DHA in post-cryptosin treated membranes indicated a non-specific type of interaction of cryptosin with mammalian cardiac beta-adrenoceptors.

Binding of beta-adrenergic antagonists 3H-DHA and 3H-CGP 12177 to intact rainbow trout (Salmo gairdneri) and carp (Cyprinus carpio) red blood cells.

beta-Adrenergic ligand binding to trout (Salmo gairdneri) and carp (Cyprinus carpio) red cells using a hydrophobic (3H-DHA) and a hydrophilic (3H-CGP) ligand was studied. Conditions which lead to the appearance (in vivo and in vitro hypoxia in carp cells) or disappearance ("winter" acclimation history in trout red cells) of beta-adrenergic responses were associated with either an appearance or marked increase of isoproterenol-displaceable binding (carp), or a disappearance of isoproterenol-displaceable binding (trout), respectively. The apparent number of binding sites was markedly greater for the hydrophobic than for the hydrophilic ligand, suggesting that binding of the hydrophobic ligand to internalized receptors and to nonreceptor sites may affect the results obtained with intact cells.