Characterization of Novel Fluorescent Ligands with High Affinity for D₁ and D₂ Dopaminergic Receptors

Abstract

We have synthesized and characterized a series of novel fluorescently labeled ligands with high affinity and specificity for D1 and D2 dopamine receptors. D1-selective probes were synthesized using (R,S)-5-(4'-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl- [1H]-3-benzazepin-7-ol, the 4'-amino derivative of the high-affinity, D1-selective antagonist SCH-23390, whereas D2-selective probes were synthesized using the high-affinity, D2-selective antagonist N-(p-aminophenethyl)spiperone (NAPS). These ligands were coupled via spacer arms of various lengths to the fluorophores fluorescein and bodipy, which fluoresce in the yellow-green region, and to tetramethylrhodamine, which is a red fluorophore. The interaction of these fluorescent ligands with dopamine receptors was evaluated by examining their ability to compete for the binding of the radiolabeled antagonists [3H]SCH-23390 or [3H]methylspiperone to rat striatal D1 or D2 dopamine receptors, respectively. We report here that these novel fluorescent ligands exhibit very high affinity and specificity for either D1 or D2 dopamine receptors. The availability of various fluorescent ligands with different emission maxima and with high affinity and specificity for D1 and D2 dopamine receptors will now permit investigations involving the visualization and localization of these receptor subtypes at the single cell and intracellular levels in the CNS and on intact cells in culture.

2-[2-[4-[2-[2-[1,3-Dihydro-1,1-bis(4-hydroxyphenyl)-3-oxo-5-isobenzofuranthioureidyl]ethylaminocarbonyl]ethyl]phenyl] ethylamino]-5?-N-ethylcarboxamidoadenosine (FITC-APEC): A fluorescent ligand for A2a-adenosine receptors

“…Based on the moderately reduced potencies of other fluorescent ligands, the affinity of FITC-APEC was sufficiently high to permit validation studies of its potential as a novel fluorescent ligand. Other studies have shown that attachment of fluorescent substituents to high-affinity central benzodiazepine [3,4,6,24], peripheral benzodiazepine [5], and dopamine [7,9] receptor ligands results in a similar or more dramatic reduction in affinity of the parent molecule. One possibility is that coupling a large fluorescent moiety to a high-affinity molecule (Fig.…”

“…Recently, we reported that fluorescent ligands could be used to quantitate ligand-receptor interactions at central and peripheral benzodiazepine receptors in broken cell preparations [3][4][5]. Moreover, direct imaging of benzodiazepine [6] and dopamine [7][8][9][10][11] receptors using fluorescent ligands also has been described.…”

A Fluorescent Ligand-Binding Alternative Using Tag-lite® Technology

“…Briefly, the CCK(26-33) peptide was derivatized with our red d2 dye (red-CCK (26)(27)(28)(29)(30)(31)(32)(33)). This fluorescent ligand was expected to display a good affinity for both SNAP-tagfused CCK1 and CCK2 receptors.…”

In vivo labeling of L-type Ca2+ channels by fluorescent dihydropyridines: evidence for a functional, extracellular heparin-binding site.

“…On the other hand, for neuronal, w-conotoxin GVIA-sensitive Ca2' channels, distribution and lateral mobility were recently investigated with a fluorescent toxin derivative (14), but data on L-type Ca2+ channels are unavailable. For localization studies at the cellular or subcellular level or on living cells fluorescent probes exist for the following receptors: nicotinic acetylcholine (15), dopamine (16,17), f3-adrenergic (18,19), benzodiazepine (20), glycine (21), insulin (22), glucagon (23), and vasopressin (24,25). Fluorescent probes were also developed for voltage-dependent Na' channels (26,27 METHODS Synthesi of the (4,4-Dlfluoro-5,7-dmethyl4-bora-3a,4a-daz)-3-(s-indacene)proplonlc Acid (DMBodipy)-and (4,4-Dfhoro-7-stry-4-bora-3a,4a-diaza)-3-(s-Dee)propioc Acid (STBodipy)-Labeled DHP Enatoers.…”