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Muscarinic receptor and beta-adrenergic receptor binding were measured simultaneously in a membrane fraction of bovine tracheal smooth muscle using [3H]-L-quinuclidinyl benzilate and [125I]-(-) iodocyanopindolol. The binding characteristics, affinity and receptor density, obtained in the double receptor assay and in the control experiments were the same within experimental error. Moreover, there appears to be neither a significant influence of an excess of d,1-propranolol on [3H]-L-quinuclidinyl benzilate binding nor a significant influence of an excess of 1-quinuclidinyl benzilate on [125I]-(-)iodocyanopindolol binding. The method is advantageous where both receptors have to be assayed and where limited amounts of biological material, like in biopsy specimen, are available.

In association experiments of (-)125Iodocyanopindolol (125ICYP) with human mononuclear cells (MNC) at 70 pM and a temperature of 37 degrees C equilibrium was reached within 30 min. However, when the same experiments were performed at a concentration of 4 pM 125ICYP, equilibrium was only reached after 3 hours. The consequences of incomplete equilibrium for the interpretation of binding experiments under the incorrect assumption that equilibrium has been reached, was investigated at equilibration times of one, two and three hours. The dissociation constant, Kd, decreased from 7.4 +/- 0.2 pM after one hour to 2.5 +/- 0.4 pM after three hours of incubation while the receptor density, RO, decreased from 970 +/- 170 to 713 +/- 58 sites/cell. Analysis of computer simulated binding curves confirmed the decrease in Kd and RO at prolonged incubations. We conclude that in 125ICYP binding in intact MNC one hour of incubation is not sufficient to obtain equilibrium at the lower concentrations. This leads to an overestimation of Kd- and to a lesser extent of RO-values. Extending the incubation time to three hours on the other hand may lead to a loss of cells and therefore to an underestimation of RO.

The distribution of beta-adrenergic receptors in rat nasal glands has been investigated with the use of an in vitro autoradiographic technique. Radioligand binding studies indicated that [125I]cyanopindolol binds specifically to beta-adrenergic receptors in cryostat sections of these glands. Autoradiograms generated after incubation with 0.02 nM [125I]cyanopindolol and dipping in nuclear K2 emulsion, showed specific labelling of the striated excretory ducts. These in vitro observations suggest a sympathetic control of the ion and water content of the glandular secretion.

(-)125I-Iodocyanopindolol (125ICYP) binding to intact and ultrasonically treated human mononuclear cells (MNC) was studied. Specific binding of 125ICYP defined as the difference in binding in the presence and absence of 2 microM (+/-)-propranolol displayed in intact cells a dissociation constant (Kd) of 11.8 +/- 2.7 pM and a beta-adrenoceptor number (Rt) of 2371 +/- 154 sites/cell. This specific binding, however, still had a complex character. In broken cells a homogeneous class of binding sites with a Kd-value of 6.7 +/- 1.0 pM and a Rt-value of 883 +/- 89 sites/cell was found. When in intact cells nonspecific binding was determined with CGP-12177, a hydrophilic beta-adrenoceptor antagonist, a homogeneous class of binding sites was found with a Kd of 7.0 +/- 0.3 pM and a Rt of 1645 +/- 95 sites/cell. Inhibition curves with (+)-, (-)-, (+/-)-propranolol, timolol and CGP-12177, obtained with ultrasonically treated cells were monophasic. In intact cells propranolol and timolol not only displaced 125ICYP from its specific sites, but in an almost monophasic way also from nonspecific binding sites. CGP-12177, however, showed a clear plateau. It is concluded that in broken MNC a loss of binding sites may occur, whereas in intact cells additional binding interferes with the correct determination of specific 125ICYP binding. The latter can be reduced to a minimum by using hydrophylic ligands such as CGP-12177 to measure nonspecific binding.