About 1% of the population suffers from schizophrenia and antipsychotic drugs/neuroleptics are often very effective in treating hallucinations and delusions (i.e. psychotic periods) ocurring during this mental disease. The clinical efficacy of these drugs is related to their affinity for D2 dopamine receptors and, particularly to their ability to block the mesolimbic dopaminergic system. However excessive blockade of striatal D2receptors by such drugs will cause extrapyramidal symptoms similar to Parkinson's disease. Whereas these side-effects were especially observed with the first generation of antipsychotics, they are less of a concern with the newer generation of "atypical antipsychotics". This is probably related to their fast dissociation from the D2 receptors, thereby allowing the receptors to remain responsive to fast fluctuations in dopamine concentration in the striatum. In vitro studies dealing with antipsychotic drug- D2 receptor interactions often rely on radioligand binding experiments on more or less pure cell membrane preparations. On the other hand, functional assays to monitor drugs for antagonism, partial and inverse agonism are carried out in intact cells. Both assays can be carried out under similar experimental conditions when performed in intact cells. Using this strategy we significantly improved our knowledge about the complex nature of drug-angiotensin AT1-receptor interaction and allowed us the unravel the molecular mechansim of "partial insurmountable antagonism". Such a 'full intact cell approach' has only been sparely used in the dopamine receptor field. This prompts us to make advantage of the already acquired technical skills to perform a thorough comparison of the interaction between various antipsychotics and the D2S and D2L receptor isoforms in intact cells. Preliminary research during the last year provided evidence for the feasibility of this approach and already yielded a number of original results. Experiments will initially be carried out on recombinant CHO cells permanently expressing human D2S (already available) and D2L receptors (will be produced by transfection and cloning). Binding studies will be performed with the hydrophobic antagonist [3H]-spiperone and the hydrophilic antagonist [3H]-raclopride. A decreased cAMP production constitutes the primary functional response in these cells (as well as in endogenous D2 receptor-expressing cells). To get a more convenient and faster read-out system, we will accomodate the receptors to trigger calcium transients by making use of CHO-AEQ cells that stably express G?16 and apoaequorin (obtained from Euroscreen). In this respect, we have already produced human D2L receptorexpressing CHO-AEQ cells and we plan to produce those expressing the human D2S receptors. D2-agonist dose response curves will yield information about the intrinsic activity of each ligand (relative to dopamine as control). For antagonists, these assays will provide information about their competitive vs. noncompetitive behavior (co-incubation experiments) and their degree of insurmountability (i.e. their ability to decrease the maximal response of the agonist following antagonist preincubation). Functional wash-out experiments, in which cells are pre-incubated with antagonists, washed and treated with a large volume of fresh medium for different lengths of time, will provide information about the antagonist's dissociation rate. These data can be compared with those from related radioligand binding experiments (either direct information by dissociation of radiolabelled antagonists or indirect information by monitoring the delay of radioligand association after pretreatment of the cells with unlabelled antagonist and washing). Attention will also be devoted to the potential influence of the D2 receptor "environment" on its interaction with antipsychotic drugs. In this respect, it has been found that D2 receptors not only interact with G proteins (such as Gi/o in native CHO cells and G16 in CHO-AEQ cells) but also with other cytoplasmic proteins such as fillamin en H-FABP. The influence of these latter proteins will be investigated by co-transfection experiments. Crucial experiments with the most interesting antipsychotics will finally be performed on endogenous D2receptor expressing neuronal cell lines.
|Effective start/end date||1/01/08 → 31/12/10|
Flemish discipline codes
- Biological sciences