Pharmacological implications of cellular localization of alpha1-adrenoceptors in native smooth muscle cells.

1. This study examines the cellular localization of alpha1-adrenoceptors and demonstrates that binding to intracellular receptive binding sites in native smooth muscle cells may influence the pharmacological profile of agonists or antagonists. The example tissue studied was rat basilar artery. 2. An alpha1-adrenoceptor antagonist and fluorescent analogue of prazosin, BODIPY-FL prazosin (QAPB) allowed visualization, with high resolution, of both plasma membrane and cytosolic binding sites on live native cells, as previously shown in cells harbouring recombinant receptors. QAPB-associated fluorescence binding was both time- and concentration-dependent in rat basilar smooth muscle cells and affinity for alpha1-adrenoceptors was calculated from specific binding curves as 1.1 nM. 3. Concentration-dependent binding of QAPB detected in smooth muscle cells dissociated from rat basilar arteries was composed of diffuse and clustered fluorescence. Visually the diffuse component of fluorescence was the more evident up to a concentration of 5 nM QAPB. Confocal visualization of an optical section through the cell showed that the clustered component was located mainly intracellularly. In rat basilar artery smooth muscle cells the intracellular binding sites were located in close proximity to the nuclear membrane. 4. 3D models of QAPB-associated fluorescence demonstrate that a high proportion of effective binding sites are intracellular, showing not only that a high proportion of receptors are located inside the cell but also that in this location they can bind ligands. This has implications for pharmacological analysis in relation to the consequences of intracellular binding per se and for differential effects upon the pharmacology of particular ligands according to whether they can enter the cell.