Beta-adrenergic receptors of human leukocytes. Studies with intact mononuclear and polymorphonuclear cells and membranes comparing two radioligands in the presence and absence of chloroquine.

Owing to the large differences in reported values for beta-adrenergic receptor numbers and binding affinity in normal leukocytes, we undertook a systematic re-examination of the binding of two widely used beta antagonists, (-)-[3H]dihydroalprenolol (DHA) and (+/-)-[125I]iodohydroxybenzylpindolol (HYP), to intact normal mononuclear (MN) leukocytes and polymorphonuclear (PMN) leukocytes and membrane preparations. Assays were conducted in the presence and absence of chloroquine, which has been proposed recently to eliminate ligand uptake into a non-receptor cell compartment such as lysosomes. The binding curves relating radioligand concentration to specific sitesper intact cell were biphasic. At high (10-24 nM) (-)-DHA ligand concentration in the absence of chloroquine, a large number (20,000-60,000 sites/cell) of low affinity (Kd 12-15 nM) stereospecific binding sites were detected in both cell types. This class of binding sites was eliminated by 10 microM chloroquine not only in PMN cells but also in the lysome-poor MN cells (greater than or equal to 90% lymphocytes), leaving 2000-3000 specific high affinity (-)-DHA sites/cell. In the absence of chloroquine, comparably low numbers of specific high affinity binding sites/cell were also obtained by the use of appropriately low concentrations of (-)-DHA or (+/-)-HYP (800 pM or less). However, even at these low radioligand concentrations chosen to measure high affinity specific binding, the addition of 10 microM chloroquine produced a moderate reduction in the number of sites/cell, without a detectable change in the apparent Kd. Mean (+/- S.E.M.) site numbers obtained in the presence of chloroquine were: 1331 +/- 100 sites/MN cell and 1135 +/- 129 sites/PMN cell (Kd 143-153 pM) using (-)-DHA; and 1487 +/- 210 sites/MN cell and 1065 +/- 69 sites/PMN cell [avg. Kd(+/-) 224-274 pM] using (+/-)-HYP. Chloroquine had no effect on agonist-stimulated cAMP production but produced an apparent increase in the effectiveness of (-)-propranolol as an inhibitor of DHA binding. Competition studies on the binding of DHA and HYP with zinterol and practolol confirmed that the receptor was of the beta 2-subtype for both MN and PMN cells. The detection of a moderately larger number of high affinity binding sites at saturation (Scatchard analysis) by (+/-)-HYP than by (-)-DHA was a consistent finding with either intact cells or membranes, with or without chloroquine. The possible overestimation of receptor numbers by a racemic ligand such as (+/-)-HYP is discussed and leads us to favor the use of a pure stereoisomer such as (-)-DHA. A system employing 800 pM (-)-[3H]DHA, 1 microM (-)-propranolol and 10 microM chloroquine with intact MN and PMN cells yielded reproducible and plausible results. Our values for beta-adrenergic receptor numbers of intact MN and PMN cells and membranes are compared to others in the literature.