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Vol. 294, Issue 2, 434-443, August 2000
1-Adrenoceptor Ligand-Binding Sites1
Autonomic Physiology Unit, Division of Neuroscience and Biomedical
Systems, Institute of Biomedical & Life Sciences, University of
Glasgow, Glasgow, United Kingdom
Cellular distribution and binding characteristics of native
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 1-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
1a-, 1b-, and 1d-AR
subtypes on membrane preparations and whole cells. RS100329 had 70-fold
selectivity for 1a-ARs versus 1b- and
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 (FKD = 0.63 ± 0.02 nM), which was 3- to 4-fold higher compared with recombinant cells (FKD = 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 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.
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