Quantitative imaging in live human cells reveals intracellular alpha(1)-adrenoceptor ligand-binding sites

Cellular distribution and binding characteristics of native alpha(1)-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 alpha(1)-AR-binding sites in native cells are intracellular. QAPB had high affinity and acted as a nonselective, competitive antagonist versus [(3)H]prazosin at cloned human alpha(1a)-, alpha(1b)-, and alpha(1d)-AR subtypes on membrane preparations and whole cells. RS100329 had 70-fold selectivity for alpha(1a)-ARs versus alpha(1b)- and alpha(1d)-ARs, validating its use to identify this subtype. In similar cells QAPB-associated fluorescence provided quantitative data analogous and comparable to [(3)H]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 alpha(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.