Abstract

Cellular distribution and binding characteristics of native α₁-adrenoceptors (ARs) were determined in a live, single, human smooth muscle cell (SMC) with confocal laser scanning microscopy and a fluorescent ligand, BODIPY-FL prazosin (QAPB). This allowed single-cell competitive ligand binding and showed that 40% of α₁-AR-binding sites in native cells are intracellular. QAPB had high affinity and acted as a nonselective, competitive antagonist versus [³H]prazosin at cloned human α_1a-, α_1b-, and α_1d-AR subtypes on membrane preparations and whole cells. RS100329 had 70-fold selectivity for α_1a-ARs versus α_1b- and α_1d-ARs, validating its use to identify this subtype. In similar cells QAPB-associated fluorescence provided quantitative data analogous and comparable to [³H]prazosin binding in whole cells. In human, dissociated, prostatic smooth muscle cells QAPB-associated fluorescence binding exhibited specific high-affinity binding properties (FK_D = 0.63 ± 0.02 nM), which was 3- to 4-fold higher compared with recombinant cells (FK_D = 2.1–2.3 nM). Internal consistency in the data showed that affinity is greater, in general, in membrane preparations than in cells but also greater in the native prostatic tissues or cells than in equivalent recombinant receptors. Fluorescence revealed binding sites both on the plasmalemmal membrane and on intracellular compartments: at all locations RS100329 inhibited QAPB binding identifying the sites as α_1A-ARs. Quantitative three-dimensional mapping of QAPB-associated fluorescence binding in native human cells showed that 40% of high-affinity-binding sites was in intracellular compartments. This provides a potential new site for physiological agonism and makes intracellular access a potential differentiator of drug action.