The classification of dopamine receptors: relationship to radioligand binding. - Semantic Scholar
Radioligand-binding experiments have demonstrated that both D-1 (adenylate cyclase stimulatory) and D-2 (adenylate cyclase inhibitory) dopamine receptors. Annu Rev Neurosci. ; The classification of dopamine receptors: relationship to radioligand binding. Creese I, Sibley DR, Hamblin MW, Leff SE. Agonist interactions with dopamine receptors: Focus on radioligand-binding . The Classification of Dopamine Receptors: Relationship to Radioligand Binding.
Exposure of dopamine ligands to the cell: Initial concentration of the cells for the experiment was maintained at 1x cells mL-1 according to OECD guide lines. The rhodamine-labeled dopamine receptor agonists SKF was exposed to the Tetrahymena cells and incubated for two different times, 3 and 6 h.
Just prior to examination, the slides were covered with cover slips and a drop of immersion oil was added on the top. Two-dimensional photomicrographs were captured by an auto camera with 40xmagnification. Six hours incubation of ligands elicited the higher fluorescent activity.
The classification of dopamine receptors: relationship to radioligand binding.
The specific binding of the fluorescent probe to dopamine receptors was assessed with competition between agonist SKF and D1 specific antagonist SCH The cross binding of SKF to the receptors was faithfully checked by spiperone, a D2 antagonist. These two ligands were exposed to the cells at same concentrations as above mentioned and incubated for 6 h. Afterwards the binding of SKF to D1 receptor was scrutinized to determine competitive cross-reactivity of ligands binding in the cells.
Helium Neon Laser nm excitation and LP emission.
Identification of Dopamine Receptor in Tetrahymena thermophila by Fluorescent Ligands
The LSM capture photograph from a different plane; thus indicates the binding location of agonist in the cell. Slide preparation carried out as above mentioned. The investigation of D1 type receptor in Tetrahymena thermophila exerted photographic representation of ligands binding to the receptor. Figure 1 illustrates the photograph of control cells. They did not show fluorescent signals because they were not exposed with SKF In this experiment, the exposure of cells to fluorescent agonist SKF caused the emission of fluorescent signals.
The cells were treated with several concentrations of the agonist at two different incubation times.
Longer incubation causes saturation of receptor binding. But, more than 8 h exposure of ligand again caused the decrease of receptor binding as well as decrease of fluorescent signals.
Molecular And Pharmacological Studies Of Dopamine Receptors - David Sibley
The specific binding of the ligands was investigated with exposure to two ligands; one was agonist SKF and another was antagonist SCH Both ligands are specific to D1 receptors and were added together at the same concentration. The exposed cells were incubated for 6 h and viewed by employing fluorescence microscopy. Control Tetrahymena thermophila cell a 40xMagnification b xMagnification Fig. Fluorescence localization of D1 dopamine receptor binding of the D1 specific agonist fluorescent probe, rhodamine- SKF in Tetrahymena thermophila.
The entire photograph shows total binding of receptor after 3 h exposure of ligand. This time period causes saturation binding of ligands and reveals stronger signals. Binding of SKF Fig. SKF and D1 specific fluorescent probe antagonist SCH were applied together to the cells at equal five different nominal concentrations and incubated for 3 h. Cells did not show fluorescence signals of receptor binding for neither agonist nor antagonist.
Thus prove the specific binding of SKF Photographs are presented in the Fig. Interestingly the same result was found when, that the antagonist of one type dopamine receptor could SKF and SCH were used together in guinea-pig not prevent the binding of another type dopamine agonist Furukawa and Morishita, ; Zhu et al. Assay for the cross binding to D1 receptors. SKF and non-fluorescent D2 specific antagonist, spiperone were added together to the cells at the equal five different nominal concentrations and incubated for 3 h.
Spiperone competes only to binding to D2 tape receptors. Cells exhibited fluorescent signals of receptor binding. So, spiperone did not inhibit agonist binding. Ligand concentrations unit with each No. Binding of SKF It has also been previously reported that two types of dopamine receptors exists in dopaminergic cells. Therefore, the assessment of cross-affinity of receptor binding is also important.
That is why we have selected the D2 specific receptor antagonist spiperone which compete binding to D2 receptor due to its ligand properties Amenta et al. SKF and spiperone were added to the cells together with the five different concentrations of ligand again and incubated for 6 hours.
Abstract The D1 dopamine receptor D1R has been implicated in numerous neuropsychiatric disorders and various D1R-selective ligands have shown potential as therapeutic agents.
In an effort to identify novel selective allosteric modulators of the D1R we used a high throughput screening approach. Hit compounds were triaged through secondary functional and radioligand displacement binding assays to determine subtype selectivity and their allosteric versus orthosteric nature.
In addition we found approximately agonist and antagonist hits that failed to completely inhibit radioligand binding and thus have been classified as potential allosteric agonists and antagonists or negative allosteric modulators NAMs. These compounds are currently being characterized using additional assays to confirm their selectivity, activity, and classification. Four of the PAM hits have selected for chemical optimization, which is ongoing now. Unfortunately, truly specific drugs for this receptor have been difficult to obtain, primarily due to high conservation of the orthosteric binding site within dopamine receptor DAR subtypes and other G protein-coupled receptors GPCRs.
Hits were subjected to an extensive triage strategy to characterize DAR activity and selectivity. On the basis of these analyses, agonist and antagonist compounds were selected and evaluated using radioligand binding competition assays to identify the nature of their receptor interactions orthosteric or allosteric.
Compounds that are ineffective in competing for binding likely exert their functional activity via allosteric mechanisms. These experiments resulted in the identification of 47 agonists and 48 antagonists that had insignificant effects on radioligand binding when tested at concentrations up to 40 uM, despite exhibiting maximal functional effects at significantly lower concentrations.
These compounds would thus appear to be allosteric agonists and negative allosteric modulators of the D2R. Further characterization is ongoing. The D2R can activate a spectrum of signaling cascades primarily through G proteins and beta-arrestin recruitment, making it an attractive target for the development of signaling biased ligands. Unlike dopamine, which simultaneously activates G proteins and recruits beta-arrestins, a biased ligand affects only one pathway, and the development of such ligands can allow for a more fine-tuned study of receptor signaling.
Our lab has identified a compound ML that is a highly efficacious agonist at D2R-mediated G protein-linked signaling, but it does not recruit beta-arrestin. Rather, this compound is an antagonist of D2R-stimulated beta-arrestin-mediated signaling.