Essay: Dopamine receptors

Abstract
Dopamine receptors are found within the brains of both humans and rats. Of these receptors there are two subtypes D1 and D2, within these there are also 5 further subunits (D1, D2, D3, D4 and D5) (Rang et al., 2016). Drugs that act upon dopamine receptors demonstrate selectivity for these subtypes. In this study the goal was to understand which subtypes different drugs are selective for based on their ability to displace a known D2 receptor antagonist Spiperone. A radiolabelled ligand of tritiated Spiperone was used in a competitive ligand binding assay to assess the binding of three dopamine antagonistic drugs, Haloperidol, Sulpiride and SKF38393. The assays were carried out using rat brain membrane and the three drugs at a range of concentrations from 1×10-11 to 1×10-6M. The bound radioactivity of the samples was then counted the following day. The drug Haloperidol and Sulpiride were able to displace Spiperone to 0% and 16.4% binding. This demonstrates their affinity to the D2 receptor subtype similarly to Spiperone. SKF38393 however had little effect on the percentage binding of Spiperone and even at its highest concentration of 1×10-6M Spiperone remained 78% bound. This suggests that SKF38393 may have antagonistic on the D1 subtype receptors, for which Spiperone has less affinity than D2, as it was still able to cause some reduction in binding. We can therefore state that Haloperidol and Sulpiride are D2 receptor antagonists.
Introduction
Dopamine receptors are often targets of neuropharmocological treatments for disorders of the brain such as Schizophrenia and Parkinson’s disease (Seeman et al., 1994). Dopamine receptors are G-protein coupled receptors, of which there are two subtypes (D1 and D2) and a further 5 subunits (D1, D2, D3, D4 and D5). D1 and D5 subunits belong to the D1 subtype, whilst D2, D3 and D4 are subunits of the D2 subtype (Rang et al., 2016). All five have been identified in both human and rat brains (Mengod et al., 1992). Drugs often have selectivity towards a particular subtype or subunit of dopamine receptor. The aim of the present study was to assess the various receptor subunit selectivities of three drugs – Haloperidol, Sulpiride and SKF38393, these are the cold ligands. This was assessed based on their effects on the binding of radioactive Spiperone, the hot ligand, to dopamine receptors of Rat brain membrane in a competitive ligand binding assay. Spiperone is a D2 receptor antagonist (van Wieringen et al., 2013). If the drugs, “cold ligands”, are able to completely displace the radioactive Spiperone ligands it is acting as a competitive antagonist of Spiperone, binding to the same receptors i.e. D2 subtype receptors. If there is little effect on the binding of Spiperone this means the cold ligand binds to different dopamine receptors the D1 subtype to which Spiperone has less selectivity for. In order to use these drugs for treatment it is important to understand which dopamine receptors they act on, in order to understand which dopaminergic pathways they will effect.
Methods
To examine the selectivity of the three drugs to different subunits of dopamine receptors their effects on the binding of Spiperone were assessed. To do this competitive ligand binding assays were carried out for all three drugs at a range of concentrations. Serial dilutions were made of the cold ligands (drugs) to obtain 10 stocks at 6 concentrations. These concentrations ranged from 10-5 M to 10-10M, and were diluted using Tris buffer. 6 pairs of Eppendorf tubes were used, 2 for each of the six cold ligand concentrations. To each of the tubes the following were added 350μM of ice cold Tris buffer, 50μM of rat brain membrane (1mg protein/mL) and 50μM of cold ligand (from the 6 concentrations). It was important that these be added in that specific order. The 50μM of tritiated Spiperone (hot ligand) was added to each tube. The 12 tubes were then vortexed and placed on ice for 30 minutes. Following the incubation the samples were filtrated by vacuum filtration in a Millipore filtration device. A glass fibre filter was placed into each well so that the sample solutions were pulled through the filters and the filter washed using 2 x 4mL of ice cold Tris buffer. The samples were then filtered. Once filtration had occurred the filters were removed from the well and each placed into a scintillation vial. 4mL scintillation fluid was then added to each vial. The tubes were incubated for several hours before the bound radioactivity was counted the following day. This incubation allowed for any remaining water in the filters to be absorbed so that it did not affect the counts. A Scintillation counter was used for 4 minutes to count the bound radioactivity of each filter, obtaining the activity in disintegrations per minute (dpm).
Results
From the graph we can see that Haloperidol and Sulpiride follow similar patterns. A decline is seen from maximum percentage binding of 96.68% with Haloperidol and 100% with Sulpiride at their lowest concentrations where Spiperone was not displaced. To 0% in the case of Haloperidol where all of the hot ligand was displaced, and 16.4% for Sulpiride where most of it was displaced. The curve for Sulpiride is shifted to the right suggesting that Sulpiride is less potent than Haloperidol. However the effect of SKF38393 does not follow a similar path at these concentrations, the percentage binding remains above 90% until 1×10-6 M where it drops to 78% binding of Spiperone. Table 1 shows the inhibitor constant, Ki, values calculated using the Cheng-Prusoff equation. For Haloperidol and Sulpiride these were 0.30nM and 3.15nM respectively, however it is not possible to calculate these for SKF38393 because IC50 cannot read from the graph as binding of Spiperone was not reduced by 50%.
Ki (nM)
Haloperidol 0.30
Sulpiride 3.15
SKF38393 ND
Discussion
From these results we can infer that Haloperidol and Sulpiride are antagonists to the same dopamine receptors as Spiperone, the D2 subtype. This can be inferred as percentage binding of Spiperone was greatly decreased by both drugs. For Sulpiride percentage binding of the hot ligand was reduced from 100% at 1×10-11 M to 16.4% at 1×10-6 M. Whilst for Haloperidol the highest percentage binding of Spiperone was 96.68% at 1×10-11M which was then reduced to 0% at 1×10-6M concentration. We can also infer that Haloperidol is more potent at displacing Spiperone. This is because Haloperidol was able to displace Spiperone more effectively at lower concentrations than Sulpiride. For example at 1×10-9M of both drugs Haloperidol displaced Spiperone to 48.8% binding, whereas Sulpiride was only able to displace Spiperone to 87.1% binding. This can also be seen in the Ki values for Haloperidol and Sulpiride, for Haloperidol Ki=0.30 whereas for Sulpiride Ki=3.15. A lower Ki suggests higher potency as less concentration is required to produce half maximum inhibition. These results demonstrate the ability of the two drugs to displace Spiperone from the dopamine receptors and reducing percentage binding in rat brain membrane. Since Spiperone was displaced when using these drugs we can infer that they bind to the same dopamine receptor the D2 subtype, including subunits D2, D3 and D4. However SKF38393 had little effect on the binding of Spiperone, only decreasing to from 99% binding at 1×10-11M to 78% at 1×10-6M. From this we can suggest that SKF38393 binds to D1 subtype dopamine receptors due to its small reducing effect on Spiperone. This is because Spiperone only shows weak affinity for D1 and D5 subunits. These results have great implications for treatments of diseases related to dopamine transmission within the brain, such as Schizophrenia and Parkinson’s disease. Dopamine D2 receptors have been found to be increased in the brains of Schizophrenics (Seeman et al., 1987). The “dopamine hypothesis of schizophrenia” states that it is the excessive transmission at D2 dopamine receptors in particular brain regions that causes many of the positive symptoms of schizophrenia (Brisch et al., 2014). These symptoms include hallucinations, delusions and distortion of real life (NHS., 2016). A dopamine receptor antagonist such as Haloperidol and Sulpiride could be effective in reducing these symptoms as the drugs would act as inhibitors at the dopamine D2 receptor, controlling its hyperactivity.