Effect of thiols on beta 2-adrenoceptors in human mononuclear leucocytes

The effect of the disulfide reducing agent dithiothreitol (DTT) and other thiols on binding of the beta-adrenoceptor antagonist (-)-125iodocyanopindolol (125ICYP) to human mononuclear leucocytes (MNL) was investigated. Saturation experiments and dissociation kinetics revealed two classes of specific 125ICYP binding sites, one of high and the other of low affinity, respectively. In intact MNL DTT caused a decrease in specific binding. This was due almost selectively to a decrease in the affinity of high affinity binding sites, which decreased gradually in a concentration-dependent manner to the affinity of low affinity binding sites. In MNL membranes DTT decreased not only the affinity but also the number of high affinity binding sites. The DTT effect was completely reversible by simple reoxidation on air. The structural isomers (+/-)-DTT. (-)-DTT and dithioerythritol revealed identical effects on specific binding, whereas the monothiols mercaptoethanol and alpha-monothioglycerol, having a lower redox potential, were considerably less effective. In the same concentration range that influenced specific binding. DTT stimulated intracellular cAMP production. These results suggest functionally important disulfide bridges which regulate the affinity of beta-adrenoceptor binding sites in human MNL. They stabilize the receptor in a high affinity state; their reduction causes the conversion of the high affinity state into a low affinity state in a process associated with stimulation of adenylate cyclase. Available evidence indicates that a similar transformation is made by beta-adrenoceptor agonists. Consequently low affinity 125ICYP binding sites preexistent in untreated cells could represent a reduced receptor state resulting from agonist-receptor interaction in vivo

A partially automated radioligand binding assay system for use in clinical and pharmaceutical research

Using a Tecan robotic sample processor and IBM compatible PCs we have developed a flexible, partially automated radioligand binding assay system. It handles pipetting parameters of up to 16 saturation or competition experiments at a time with up to 24 radioligand- or competitor-concentrations in a range over 4 orders of magnitude per experiment. The system provides enough flexibility so that all pipetting parameters including different tube-, rack-sizes, sample volumina and pipetting sequences may be easily adapted to the large variety of experimental requirements in binding assays. It rationalizes and increases assay throughput (up to 70% spare of working time), improves reliance and reproducibility of results. Radioactive exposure is minimized to the time preparing the radioligand working solution and transferring the sample tubes to and from the sample processor. The system has proven effective in various investigations on binding interactions, as well as in clinical studies on receptor expression under physiologic, pathological and therapeutic conditions

Circannual variation in the expression of beta 2-adrenoceptors on human peripheral mononuclear leukocytes (MNLs)

Peripheral mononuclear leukocytes (MNLs) are widely used as a tissue model in studies of beta-adrenoceptor disturbances in hypertension and asthmatic diseases. The beta 2-adrenoceptor density (Bmax), however, depends not only on the gender of the person under study and on the time of day the blood specimens are obtained. Evidence is now reported for a circannual variation in the expression of beta 2-adrenoceptor sites on peripheral MNLs. In male volunteers the 24-h mean was found to be highest in the men studied in April/May (1135 +/- 10 sites/cell) and decreased to 891 +/- 16 sites/cell in August and to 712 +r90 sites/cell in December (means +/- SE, P less than 0.01 April/May compared to December). Concomitantly the circadian amplitude increased from 17.3% +/- 6.4% of 24-h mean in April/May to 28.2% +/- 1.4% of 24-h mean in August and to 34.2% +/- 4.2% of 24-h mean in December (means +/- SE, P less than 0.05, April/May compared to December). The circadian acrophase remained constant (190 degrees +/- 30 degrees equivalent to 12 h 40 min +/- 2 h 00 min, means +/- SE)