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Determination of the transcriptional activity of the β-adrenoceptor (β-AR) gene in human mononuclear leucocytes (MNL) may provide important information concerning variations in receptor expression in clinical studies. We used a new method with highly fluorochrome-labeled gene. probes for in situ hybridization which allows a rapid detection of mRNA at the cellular level. MNL from venous blood of healthy volunteers were permeabilized, fixed and attached t o adhesive glass slides. After incubation with fluorochrome-labeled cloned hamster OAR-DNA probes the amount of DNA-mRNA hybrids formed in the cytoplasm of individual cells was analyzed by rnicrofluorimetry of specifically bound fluorochrorne. In nine of ten normal subjects studied so far a fraction of OAR-mRNA positive MNL could be detected. The proportion of positive cells as well as their signal intensity revealed a high interindividual variance.

Peripheral mononuclear leukocytes (pMNL) bear a population of β2-adcenoceptors. Radioreceptor assays with (-)-125Iodocyanopindolol (125I-CYP) are often used to determine the expression of these hormone receptors under physiological and pathological conditions. Doubts on the occurence of just one class of binding sites as well as the availability of new laboratory equipment prompted us to revise the procedure employed for investigation of these receptors. pMNL were harvested from venous human blood by density centrifugation with LymphopaqueR LymphoprepR, or FicollR yielding immunologically distinct pMNL fractions. Receptor binding assays were performed semi-automatically with 125I-CYP in the range from 0.6-600 pmol/1. Analysis of the data (modified affinity spectra, Scatchard plot) revealed two classes of binding sites (high-and low affinity binding). The binding isotherms were sigmoidal in the concentration range from 0.6-3.0 pmol/1. Parameters estimated for the high affinity binding site may vary by a factor of 10, depending on the mathematical model employed.

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.

The second messenger 3':5'-cyclic adenosine monophosphate (cAMP) inhibits the proliferation of human B lymphocytes. In lymphoid malignancies, cAMP levels or the number of beta2-adrenergic receptors seem to be decreased. In order to explore this phenomenon further, the function of the beta2-adrenergic receptor complex was examined in mononuclear leucocytes (MNLs) from patients with B-cell chronic lymphocytic leukaemia (CLL). Peripheral blood MNLs from 25 CLL patients (16 male, nine female; aged 62 ± 9 years) and 10 healthy volunteers (seven male, three female; aged 47 ± 19 years) were used. The binding characteristics of beta2-adrenergic receptors (beta2-AR) on MNLs were determined by radioligand binding assays with [125I]-cyanopindolol ([125I]-CYP). The number of high-affinity binding sites for [125I]-CYP was significantly lower in CLL patients (313 ± 300 sites per cell; mean ± SD) than in control subjects (1479 ± 1268 sites per cell). Moreover, the density of beta2-AR decreased with disease progression, from Binet stage A (371 ± 236, n = 13) to B (236 ± 136, n = 7) and C (141 ± 59, n = 5) (P < 0.05; Kruskal-Wallis analysis). Functional analyses of the beta2-AR complex were performed by measuring the cellular cAMP content of MNLs in response to different stimulators. The cAMP production of MNLs upon isoprenaline stimulation (ISO; 10 min, 10-4 mol L-1) was slightly lower in CLL patients (12.5 ± 7.04 pmol 10-6 cells) than in control subjects (15.91 ± 10.08 pmol 10-6 cells), and decreased with CLL progression (stage A 14 ± 7; stage B 13.66 ± 3.91; stage C 3.07 ± 0.79 pmol 10-6 cells). In contrast, cAMP accumulation in response to cholera toxin (CHO; 10-4 g ml-1, 120 min) was not different in control subjects (70.07 ± 31.30 pmol 10-6 cells) and CLL patients (stage A 95.24 ± 123.07 , stage B 70.76 ± 57.37, stage C 33.21 ± 33.73 pmol 10-6 cells). When stimulated with forskolin (100 mumol L-1, 15 min), control MNLs produced about ten-fold more cAMP than CLL MNLs (188.56 ± 92.53 vs. 17.88 ± 10.32 pmol 10-6 cells); this response was not stage dependent. Taken together, the results show that the beta2-AR transmembrane signalling is impaired in CLL patients. The correlation of some beta2-AR signalling defects with disease progression suggests that they may contribute to the disease progression of CLL patients.

We have developed a partially automated method for the performance of equilibrium radioligand binding studies which is applied by our group in investigations on circadian variations and stimulation studies on β2 adrenoceptor sites in human peripheral mononuclear leucocytes (pMNL). Using a Tecan Robotic Sample Processor, binding assays with 12 concentrations of ' iodocyanopindolol (1-150 pmol/1, total binding in triplicates, unspecific binding in the presence of 10-5 mol/l timolol in duplicates) are prepared automatically with all titer tubes per experiment arranged in a microtiterplate-sized rack. After incubation in a waterbath for 2hr at 37o C, the whole rack is centrifuged at 500% and transferred back to the lab robot. Bound radioactivity is separated from the unbound ligand by removing the supernatant by the machine. The radioactive counts are evaluated using personal computers. The lab robot enhances reproducibility of experimental results and frees lab workers from time-consuming pipetting jobs. Radioactive exposure is minimized to the time preparing the radioligand working solution and transferring the sample tubes from the robot to the waterbath, to the centrifuge and back to the robot. The variability of our software allows easy adaptation to other binding studies with intact cells.

Summary Peripheral mononuclear leukocytes (MNLs) are widely used as a tissue model in studies of ß-adrenoceptor disturbances in hypertension and asthmatic diseases. The ß2-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 ß2-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±90 sites/cell in December (±SE,P<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 (±SE,P<0.05, April/May compared to December). The circadian acrophase remained constant (190°±30° equivalent to 12 h 40 min±2 h 00 min, ±SE).