Catherine Pilon

Pharmacology of human dopamine D3 receptor expressed in a mammalian cell line : comparison with D2 receptor

Two cell lines were created by transfecting cDNAs of the human D2 receptor or the recently cloned human D3 receptor to CHO cells, and the properties of [125I]iodosulpride binding to membranes of these cells were compared. In cell lines expressing the D2 receptor subtype where the selectable marker, a phleomycin-resistance gene, was cotransfected in a different plasmid, a stable expression could be maintained for only few passages. In cell lines expressing the D3 receptor subtype, the selectable marker, a dihydrofolate reductase gene, was cotransfected in the same plasmid and a stable expression could be obtained. In addition, the D3 receptor gene could be amplified in these latter cell lines and a high expression level reached (up to 10(6) binding sites per cell). Sodium and, to a lesser extent, lithium similarly increased [125I]iodosulpride binding to D2 and D3 receptors. In the absence of guanylnucleotide, dopamine had a 24-fold higher apparent affinity at D3 than at D2 receptors. Gpp(NH)p induced rightward shift and steepening of dopamine competition curves at either subtype but the effects were more marked at D2 than at D3 receptors. Several agonists and antagonists, previously regarded as autoreceptor-selective, displayed higher affinities at D3 than at D2 receptors. Although most antagonists used as antipsychotics displayed high affinities at the D3 receptor, all were more potent at the D2 receptor. However, the ratio of Ki values varied over about 10-fold among these compounds, suggesting that they realize differential dopamine receptor subtype occupancy during treatments and that this might be reflected in their clinical profile.

Dopamine D3 Receptors Expressed by All Mesencephalic Dopamine Neurons

A polyclonal antibody was generated using synthetic peptides designed in a specific sequence of the rat D3 receptor (D3R). Using transfected cells expressing recombinant D3R, but not D2 receptor, this antibody labeled 45–80 kDa species in Western blot analysis, immunoprecipitated a soluble fraction of [125I]iodosulpride binding, and generated immunofluorescence, mainly in the cytoplasmic perinuclear region of the cells. In rat brain, the distribution of immunoreactivity matched that of D3R binding, revealed using [125I]R(+)trans-7-hydroxy-2-[N-propyl-N-(3′-iodo-2′-propenyl)amino] tetralin ([125I]7-trans-OH-PIPAT), with dense signals in the islands of Calleja and mammillary bodies, and moderate to low signals in the shell of nucleus accumbens (AccSh), frontoparietal cortex, substantia nigra (SN), ventral tegmental area (VTA) and lobules 9 and 10 of the cerebellum. Very low or no signals could be detected in other rat brain regions, including dorsal striatum, or in D3R-deficient mouse brain. Labeling of perikarya of AccSh and SN/VTA appeared with a characteristic punctuate distribution, mostly at the plasma membrane where it was not associated with synaptic boutons, as revealed by synaptophysin immunoreactivity. In SN/VTA, D3R immunoreactivity was found on afferent terminals, arising from AccSh, in which destruction of intrinsic neurons by kainate infusions produced a loss of D3R binding in both AccSh and SN/VTA. D3R-immunoreactivity was also found in all tyrosine hydroxylase (TH)-positive neurons observed in SN, VTA and A8 retrorubral fields, where it could represent D3autoreceptors controlling dopamine neuron activities, in agreement with the elevated dopamine extracellular levels in projection areas of these neurons found in D3R-deficient mice.

A functional test identifies dopamine agonists selective for D3 versus D2 receptors.

The functional potency of a series of dopamine agonists for increasing mitogenesis, measured by incorporation of [3H]thymidine, was established in transfected cell lines expressing human D2 or D3 receptors. The functional selectivity of agonists markedly differs from their binding selectivity. (+)7-OH-DPAT, pramipexole, quinerolane and PD 128,907, the most D3 receptor-selective compounds in binding studies, were 7, 15, 21 and 54 times more potent, respectively, at the D3 than at the D2 receptor in the functional test. Bromocriptine displayed a 10-fold functional selectivity toward the D2 receptor. The known behavioural actions of D3 selective agonists support a role for the D3 receptor in motor inhibitions, which should be taken into account for the treatment of motor dysfunctions by dopamine agonists.

Possible implications of the dopamine D3 receptor in schizophrenia and in antipsychotic drug actions

The D(3) receptor may represent an important target for antipsychotic drugs which all bind with high affinity and do not induce upon repeated administration either tolerance or receptor upregulation. The D(3) receptor is localized in brain areas, namely the nucleus accumbens and cerebral cortex, implicated in neural circuits believed to display defective functioning in schizophrenia. Overexpression of the D(3) receptor, which accounts for the behavioral sensitization to levodopa in a rodent model of Parkinsons disease, might also be responsible for the sensitization to dopamine agonists observed in schizophrenia. The appearance of the D(3) receptor during brain development, early in proliferating neuroepithelia and later in neurons from limbic areas, suggests further studies to assess its participation in the neurodevelopmental disorders of schizophrenia. Finally, meta-analysis of approximately 30 studies comprising over 2500 patients indicate that a polymorphism in the coding sequence of the D(3) receptor is associated with a small but significant enhancement of vulnerability to the disease.

Functional implications of multiple dopamine receptor subtypes: the D1/D3 receptor coexistence

The D3 dopamine receptor, a D2-like receptor, is selectively expressed in the ventral striatum, particularly in the shell of nucleus accumbens and islands of Calleja, where it is found in medium sized substance P neurons. The latter co-express the D1 receptor whose interaction with the D3 receptor was studied by treating rats with selective agonists and antagonists. In agreement with the opposite cAMP response, they mediate in cultured neuroblastoma cells, the D1 and D3 receptors exerted opposite influences on c-fos expression in islands of Calleja. However, in agreement with the synergistic influence of cAMP on D3 receptor-mediated mitogenesis on the same cultured cells, D1 and D3 receptor stimulation in vivo synergistically enhanced preprotachykinin mRNA in the shell of accumbens. This indicates that the two receptor subtypes may affect neurons in either synergy or opposition according to the cell or signal generated. Levodopa-induced behavioral sensitization in hemiparkinsonian rats is another example of D1/D3 receptor interaction. Hence repeated levodopa administration induces the ectopic appearance of the D3 receptor in substance P/dynorphin, striatonigral neurons of the dorsal striatum. This induction is secondary to D1 receptor stimulation in neurons of the denervated side and fully accounts for the sensitization, i.e. the increased behavioral responsiveness to levodopa. During brain development, a similar process could operate to control the late appearance of the D3 receptor in D1-receptor bearing neurons of the ventral striatum at a time at which they start to be innervated by dopamine neurons. Finally, taking into account a variety of genetic, developmental, neuroimaging and pharmacological data, we postulate that imbalances between the levels of D1 and D3 receptors in the same neurons could be responsible for schizophrenic disorders.

Shorter variants of the D3 dopamine receptor produced through various patterns of alternative splicing

Using Polymerase Chain Reaction amplification of mRNAs from several areas of rat brain we have shown the occurrence of two shorter transcripts of the dopamine D3 receptor gene, in addition to that corresponding to the D3 receptor. Cloning and sequencing of these transcripts, together with the establishment of the exon-intron organization of the D3 receptor gene, shown these transcripts to result from different processes of alternative splicing. The first transcript encodes a 100 amino acid protein, being produced by splicing of an exon whose absence deletes the third transmembrane domain and gives rise downstream to a frameshift in the open reading frame. In the second transcript, an in frame 54 bp deletion is produced by splicing occurring at an internal acceptor site, suppressing half of the second extracellular loop and a small sequence in the fifth transmembrane domain. This transcript was stably expressed in CHO cells which, however, failed to reveal any dopaminergic ligand binding activity. The functional significance and possible role of these shorter variants of the dopamine D3 receptor in cell signalling remain to be established.

Chromosomal localization of the human D3 dopamine receptor gene

SummaryA novel dopamine D3 receptor gene that may be involved in psychiatric diseases has recently been characterized. It has been assigned to chromosome 3 by hybridization with a D3 receptor probe to human sorted chromosomes, and localized to band 3q 13.3 by in situ hybridization.

Two Intracellular Signaling Pathways for the Dopamine D3 Receptor: Opposite and Synergistic Interactions with Cyclic AMP

Abstract: As cerebral neurons express the dopamine D1 receptor positively coupled with adenylyl cyclase, together with the D3 receptor, we have investigated in a heterologous cell expression system the relationships of cyclic AMP with D3 receptor signaling pathways. In NG108‐15 cells transfected with the human D3 receptor cDNA, dopamine, quinpirole, and other dopamine receptor agonists inhibited cyclic AMP accumulation induced by forskolin. Quinpirole also increased mitogenesis, assessed by measuring [3H]thymidine incorporation. This effect was blocked partially by genistein, a tyrosine kinase inhibitor. Forskolin enhanced by 50–75% the quinpirole‐induced [3H]thymidine incorporation. This effect was maximal with 100 nM forskolin, occurred after 6–16 h, was reproduced by cyclic AMP‐permeable analogues, and was blocked by a protein kinase A inhibitor. Forskolin increased D3 receptor expression up to 135%, but only after 16 h and at concentrations of >1 µM. Thus, in this cell line, the D3 receptor uses two distinct signaling pathways: it efficiently inhibits adenylyl cyclase and induces mitogenesis, an effect possibly involving tyrosine phosphorylation. Activation of the cyclic AMP cascade potentiates the D3 receptor‐mediated mitogenic response, through phosphorylation by a cyclic AMP‐dependent kinase of a yet unidentified component. Hence, transduction of the D3 receptor can involve both opposite and synergistic interactions with cyclic AMP.

Antipsychotics with inverse agonist activity at the dopamine D3 receptor.

SummaryIn NG 108-15 cells expressing the recombinant human D3 receptor, dopamine agonists enhance [3H]thymidine incorporation and decrease cAMP accumulation. In these cells, but not in wild type cells, haloperidol, fluphenazine, and various other antipsychotics inhibited basal [3H]thymidine incorporation in a concentration-dependent manner. In contrast, other dopamine antagonists such as nafadotride or (+)AJ 76, two D3-preferring antagonists, were without effect. The concentration-response curve of haloperidol was shifted to the right in presence of nafadotride, with a potency compatible with its nanomolar apparent affinity as neutral antagonist. Pertussis toxin treatment abolished or markedly reduced the responses to haloperidol or fluphenazine. In contrast, no significant enhancement of cAMP accumulation could be observed, under the influence of haloperidol or eticlopride. These data indicate that some dopamine antagonists behave as inverse agonists, and thus appear to inhibit an agonist-independent activity of the D3 receptor on [3H]thymidine incorporation pathway, but not on the cAMP pathway.

Role of dopamine D3 receptors in thermoregulation : a reappraisal

Dopamine agonist-induced hypothermia has been proposed to be mediated by the D3 receptor (D3R), as it is elicited by (+)7-OH-DPAT and antagonized by S 14297, two putative D3R-preferential ligands. Here we show, however, that S 14297 is a full and partial agonist at D3R and D2R, respectively. Hypothermia was induced in rats by agonists with potencies correlated with their D3R and D2R functional potencies, and was reversed by antagonists, with a rank order of potency typical of the D2R. Moreover, BP 897, a highly potent and selective but partial D3R agonist was inactive in producing hypothermia or reversing (+)7-OH-DPAT-induced hypothermia. (+)7-OH-DPAT was as potent and efficient in inducing hypothermia in wild-type as in D3R-deficient mice. Hence, our results suggest that hypothermia does not result from a selective stimulation of the D3R.