D2L Receptors Research Articles

Interactions of the Novel Antipsychotic Aripiprazole (OPC-14597) with Dopamine and Serotonin Receptor Subtypes

OPC-14597 {aripiprazole; 7-(4-(4-(2,3-dichlorophenyl)-1-piperazinyl)butyloxy)-3,4-dihydro-2(1H)-quinolinone} is a novel candidate antipsychotic that has high affinity for striatal dopamine D2-like receptors, but causes few extrapyramidal effects. These studies characterized the molecular pharmacology of OPC-14597, DM-1451 (its major rodent metabolite), and the related quinolinone derivative OPC-4392 at each of the cloned dopamine receptors, and at serotonin 5HT6 and 5HT7 receptors. All three compounds exhibited highest affinity for D2L and D2S receptors relative to the other cloned receptors examined. Both OPC-4392 and OPC-14597 demonstrated dual agonist/antagonist actions at D2L receptors, although the metabolite DM-1451 behaved as a pure antagonist. These data suggest that clinical atypicality can occur with drugs that exhibit selectivity for D2L/D2S rather than D3 or D4 receptors, and raise the possibility that the unusual profile of OPC-14597 in vivo (presynaptic agonist and postsynaptic antagonist) may reflect different functional consequences of this compound interacting with a single dopamine receptor subtype (D2) in distinct cellular locales.

G Protein-Mediated Mitogen-Activated Protein Kinase Activation by Two Dopamine D2 Receptors

Two isoforms of dopamine D2 receptor, D2L (long) and D2S (short), differ by the insertion of 29 amino acids specific to D2L within the putative third intracellular loop of the receptor, which appears to be important in selectivity for G-protein coupling. We have generated D2L- and D2S-expressing Chinese hamster ovary (CHO) cells, and regulation of the mitogen-activated protein kinase (MAPK) pathway was examined in these cells. Both D2L and D2S mediated a rapid and transient activation of MAPK with dominant activation of p42-kDa MAPK. Pertussis toxin treatment completely abrogated stimulation of MAPK mediated by D2L and D2S, demonstrating that both receptors couple to pertussis toxin-sensitive G proteins in this signaling. Stimulation of MAPK mediated by both D2L and D2S receptor was markedly attenuated by coexpression of the C-terminus of β-adrenergic receptor kinase (βARKct), which selectively inhibits Gβγ-mediated signal transduction. Further analysis of D2L- and D2S-mediated MAPK activation demonstrated that D2L-mediated MAPK activation was not significantly affected by PKC depletion or partially affected by genistein. In contrast, D2S-mediated MAPK activation was potentially inhibited by PKC depletion and genistein was capable of completely inhibiting D2S-mediated MAPK activation. Together, these results suggest that D2L- and D2S-mediated MAPK activation is predominantly Gβγ subunit-mediated signaling and that protein kinase C and tyrosine phosphorylations are involved in these signaling pathways.

Sequestration of dopamine D2 receptors depends on coexpression of G-protein-coupled receptor kinases 2 or 5.

We examined the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [3H] sulpiride-binding activity from the cell surface and by transfer of [3H] spiperone-binding activity from the membrane fraction to the light vesicle fraction in sucrose-density gradients. In COS-7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (< 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10(-4) M dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway.

D2S, D2L, D3, and D4 dopamine receptors couple to a voltage‐dependent potassium current in N18TG2 × mesencephalon hybrid cell (MES‐23.5) via distinct G proteins

We utilized the approach of stably expressing different dopamine (DA) receptors into identified cell lines in an attempt to better understand the coupling of these receptors to membrane ion channels via second messenger systems. Recently, we examined the N18TG2 × mesencephalon (MES-23.5) cell line that is phenotypically similar to mesencephalic dopamine-containing neurons. Whole-cell voltage-clamp methods were used to investigate a voltage-dependent K+ current present in these cells. Untransfected MES-23.5 cells displayed a voltage-dependent slow-onset, slowly inactivating outward current which was not altered by bath application of either the D2 DA receptor agonist quinpirole (QUIN; 10–100 μM) or the D1 DA receptor agonist SKF38393, indicating that these cells were devoid of DA receptors. The K+ current studied was activated upon depolarization from a holding potential of −60 mV to a level more positive than −20 mV and was observed to be sensitive to bath application of tetraethylammonium. When MES-23.5 cells were transfected to stably express the D2S, D2L, D3, and D4 receptors, the same current was observed. In cells expressing D2L, D2S, and D3 receptors, application of the DA receptor agonists QUIN (1–80 μM), 7-hydroxy-dipropylaminoteralin (7-OH-DPAT, 1–80 μM), and dopamine (DA, 1–80 μM), increased the peak outward current by 35–40%. In marked contrast, cells stably expressing the D4 receptor demonstrated a significant DA agonist-induced reduction of the peak K+ current by 40%. For all four receptor subtypes, the D2-like receptor antagonist sulpiride (SUL 5 μM), when coapplied with QUIN (10 μM), totally abolished the change in K+ current normally observed, while coapplication of the D1-like receptor antagonist SCH23390 was without effect. The modulation of K+ current by D2L, D3, and D4 receptor stimulation was prevented by pretreatment of the cells with pertussis toxin (PTX, 500 ng/ml for 4 h). In addition, the intracellular application of a polyclonal antibody which specifically recognizes GOα completely blocked the ability of D2L, D3, and D4 receptors to modulate outward K+ currents. In contrast, the intracellular application of an antibody directed against Goα was without effect, whereas intracellular application of an antibody recognizing Gsα abolished the ability of the D2S receptor to enhance K+ current. These findings demonstrate that different members of the D2 DA receptor family may couple in a given cell to a common effector in dramatically different ways. Synapse 31:108–118, 1999. © 1999 Wiley-Liss, Inc.

Rapid measurements of intracellular calcium using a fluorescence plate reader.

Intracellular calcium is a universal second messenger that can serve as a broad-based measure of receptor activity. Recent developments in multi-well plate fluorescence readers facilitate measurement of intracellular free-calcium levels and reduce reliance on slower, more cumbersome or expensive data collection methods. In this report, we describe a rapid and sensitive method to assay intracellular calcium ions in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells from multi-well plates using a fluorometer equipped with on-line injectors. We examine the compatibility of visible-light excitable dyes Calcium Green-1 and Oregon Green 488 BAPTA-1. Using this assay, we were able to detect and quantify activity from muscarinic and beta-adrenergic receptors endogenous to HEK293 cells and detect calcium signals generated by activation of Gi-coupled recombinant mu-opioid and dopamine D2L receptors, and the Gs-coupled melanocortin subtype 4 (MC4) receptor. Fluorescence signals, stable in HEK293 cells, required the use of Oregon Green 488 BAPTA-1 and an inhibitor of organic anion transport in CHO cells. Under appropriate conditions, both cell types can be used to collect complete concentration-response data for a variety of receptors (including a recombinant muscarinic M1 receptor expressed in CHO cells) from a single plate of dye-loaded cells.

Adenylyl cyclase interaction with the D2 dopamine receptor family; differential coupling to Gi, Gz, and Gs.

1. The D2-type dopamine receptors are thought to inhibit adenylyl cyclase (AC), via coupling to pertussis toxin (PTX)-sensitive G proteins of the Gi family. We examined whether and to what extent the various D2 receptors (D2S, D2L, D3S, D3L, and D4) couple to the PTX-insensitive G protein Gz, to produce inhibition of AC activity. 2. COS-7 cells were transiently transfected with the individual murine dopamine receptors alone, as well as together with the alpha subunit of Gz. PTX treatment was employed to inactivate endogenous alpha i, and coupling to Gi and Gz was estimated by measuring the inhibition of cAMP accumulation induced by quinpirole, in forskolin-stimulated cells. 3. D2S or D2L receptors can couple to the same extent to Gi and to Gz. The D4 dopamine receptor couples preferably to Gz, resulting in about 60% quinpirole-induced inhibition of cAMP accumulation. The D3S and D3L receptor isoforms couple slightly to Gz and result in 15 and 30% inhibition of cAMP accumulation, respectively. 4. We have demonstrated for the first time that the two D3 receptor isoforms, and not any of the other D2 receptor subtypes, also couple to Gs in both COS-7 and CHO transfected cells, in the presence of PTX. 5. Thus, the differential coupling of the D2 dopamine receptor subtypes to various G proteins may add another aspect to the diversity of dopamine receptor function.

D2S, D2L, D3, and D4 dopamine receptors couple to a voltage-dependent potassium current in N18TG2 x mesencephalon hybrid cell (MES-23.5) via distinct G proteins.

We utilized the approach of stably expressing different dopamine (DA) receptors into identified cell lines in an attempt to better understand the coupling of these receptors to membrane ion channels via second messenger systems. Recently, we examined the N18TG2 x mesencephalon (MES-23.5) cell line that is phenotypically similar to mesencephalic dopamine-containing neurons. Whole-cell voltage-clamp methods were used to investigate a voltage-dependent K+ current present in these cells. Untransfected MES-23.5 cells displayed a voltage-dependent slow-onset, slowly inactivating outward current which was not altered by bath application of either the D2 DA receptor agonist quinpirole (QUIN; 10-100 microM) or the D1 DA receptor agonist SKF38393, indicating that these cells were devoid of DA receptors. The K+ current studied was activated upon depolarization from a holding potential of -60 mV to a level more positive than -20 mV and was observed to be sensitive to bath application of tetraethylammonium. When MES-23.5 cells were transfected to stably express the D2S, D2L, D3, and D4 receptors, the same current was observed. In cells expressing D2L, D2S, and D3 receptors, application of the DA receptor agonists QUIN (1-80 microM), 7-hydroxy-dipropylaminoteralin (7-OH-DPAT, 1-80 microM), and dopamine (DA, 1-80 microM), increased the peak outward current by 35-40%. In marked contrast, cells stably expressing the D4 receptor demonstrated a significant DA agonist-induced reduction of the peak K+ current by 40%. For all four receptor subtypes, the D2-like receptor antagonist sulpiride (SUL 5 microM), when coapplied with QUIN (10 microM), totally abolished the change in K+ current normally observed, while coapplication of the D1-like receptor antagonist SCH23390 was without effect. The modulation of K+ current by D2L, D3, and D4 receptor stimulation was prevented by pretreatment of the cells with pertussis toxin (PTX, 500 ng/ml for 4 h). In addition, the intracellular application of a polyclonal antibody which specifically recognizes Goalpha completely blocked the ability of D2L, D3, and D4 receptors to modulate outward K+ currents. In contrast, the intracellular application of an antibody directed against Goalpha was without effect, whereas intracellular application of an antibody recognizing Gsalpha abolished the ability of the D2S receptor to enhance K+ current. These findings demonstrate that different members of the D2 DA receptor family may couple in a given cell to a common effector in dramatically different ways.

Human Dopamine D3 and D2L Receptors Couple to Inward Rectifier Potassium Channels in Mammalian Cell Lines

The molecular mechanisms coupling the D3 dopamine receptor to downstream effectors have neither been well defined nor well characterized. Here we examine the coupling of the human D3 receptor to G-protein coupled inward rectifier potassium channels (GIRKs) in mammalian cells. Human D3 receptors couple strongly to homomeric human GIRK2 channels coexpressed in Chinese hamster ovary (CHO) cells, with a coupling efficiency comparable to that of D2L receptors. The coupling between D3 receptors and native GIRK channels was examined in an AtT-20 mouse pituitary cell line stably expressing the human D3 receptor. AtT-20 cells endogenously express somatostatin and muscarinic receptors coupled to GIRK channels. RT-PCR and Western blot analyses revealed that AtT-20 cells natively express Kir3.1 and Kir3.2 channel isoforms, but not D2 or D3 dopamine receptors. In D3 receptor expressing AtT-20 cells, application of the D2/D3 receptor agonist, quinpirole, induces pertussis toxin-sensitive inward rectifying K+currents that are blocked by barium. Activation of D3 receptors leads to both homologous desensitization of this receptor and an unusual unidirectional heterologous desensitization of somatostatin receptors. AtT-20 cells may be a good model to examine the functional role of D3 dopamine receptors in regulating neurotransmitter secretion.

D2 dopamine receptors stimulate mitogenesis through pertussis toxin-sensitive G proteins and Ras-involved ERK and SAP/JNK pathways in rat C6-D2L glioma cells.

Dopamine D2 receptors are members of the G protein-coupled receptor superfamily and are expressed on both neurons and astrocytes. Using rat C6 glioma cells stably expressing the rat D2L receptor, we show here that dopamine (DA) can activate both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) pathways through a mechanism involving D2 receptor-G protein complexes and the Ras GTP-binding protein. Agonist binding to D2 receptors rapidly activated both kinases within 5 min, reached a maximum between 10 and 15 min, and then gradually decreased by 60 min. Maximal activation of both kinases occurred with 100 nM DA, which produced a ninefold enhancement of ERK activity and a threefold enhancement of JNK activity. DA-induced kinase activation was prevented by either (+)-butaclamol, a selective D2 receptor antagonist, or pertussis toxin, an uncoupler of G proteins from receptors, but not by (-)-butaclamol, the inactive isomer of (+)-butaclamol. Cotransfection of RasN17, a dominant negative Ras mutant, prevented DA-induced activation of both ERK and JNK. PD098059, a specific MEK1 inhibitor, also blocked ERK activation by DA. Transfection of SEK1 (K --> R) vector, a dominant negative SEK1 mutant, specifically prevented DA-induced JNK activation and subsequent c-Jun phosphorylation without effect on ERK activation. Furthermore, stimulation of D2 receptors promoted [3H]thymidine incorporation with a pattern similar to that for kinase activation. DA mitogenesis was tightly linked to Ras-dependent mitogen-activated protein kinase (MAPK) and JNK pathways. Transfection with RasN17 and application of PD098059 blocked DA-induced DNA synthesis. Transfection with Flag delta169, a dominant negative c-Jun mutant, also prevented stimulation of [3H]thymidine incorporation by DA. The demonstration of D2 receptor-stimulated MAPK pathways may help to understand dopaminergic physiological functions in the CNS.

Differential innervation of individual melanotropes suggests a role for nonsynaptic inhibitory regulation of the developing and adult rat pituitary intermediate lobe

Dopamine and GABA were detected in intermediate lobe axons around birth, and early axons were closely apposed to glial cells and processes, possibly using them for guidance. In the adult, axons containing colocalized dopamine and GABA were distributed in a distinct pattern within the lobe, with plexuses located dorsally and ventrally. Axons preferentially followed glial processes in interlobular septa, yet were also interspersed between melanotropes. Individual melanotropes were contacted by varying numbers of axon terminals, with some devoid of contacts. Boutons contained both small clear vesicles and large dense-cored vesicles; membrane specializations were not well-developed. From these findings we concluded that in addition to direct synaptic inhibition, dopamine and GABA could stimulate their receptors by mechanisms similar to “parasynaptic” [Schmitt (1984) Neuroscience, 13:991–1001] or “volume” [Agnati et al. (1995) Neuroscience, 69:711–726] transmission as proposed for the CNS. Humoral agents passing into the intermediate lobe from portal vessels, thus acting as classical hormones, further regulate the melanotropes. Moreover, approximately 50% of the axonal elements were closely apposed to glia, suggesting that glia could have regulatory roles. Previous studies from our laboratory [Chronwall et al. (1987) Endocrinology, 120:1201–1211; Chronwall et al. (1988) Endocrinology, 123:1992:1202] demonstrated heterogeneity in proopiomelanocortin (POMC) biosynthesis among individual melanotropes, prompting the hypothesis that the degree of innervation could govern the expression of certain molecules. We combined immunohistochemistry and in situ hybridization histochemistry to evaluate whether melanotrope molecular heterogenity is spatially correlated with axons and terminals. Tentatively, melanotropes expressing low levels of POMC and α1A subunit P/Q type Ca2+ channel mRNAs often were apposed to axons, whereas those with low levels of D2L receptor mRNA rarely were contacted by axons, suggesting that innervation could be one of the factors inducing and maintaining heterogeneity. Synapse 28:227–243, 1998. © 1998 Wiley-Liss, Inc.

Differential innervation of individual melanotropes suggests a role for nonsynaptic inhibitory regulation of the developing and adult rat pituitary intermediate lobe.

Dopamine and GABA were detected in intermediate lobe axons around birth, and early axons were closely apposed to glial cells and processes, possibly using them for guidance. In the adult, axons containing colocalized dopamine and GABA were distributed in a distinct pattern within the lobe, with plexuses located dorsally and ventrally. Axons preferentially followed glial processes in interlobular septa, yet were also interspersed between melanotropes. Individual melanotropes were contacted by varying numbers of axon terminals, with some devoid of contacts. Boutons contained both small clear vesicles and large dense-cored vesicles; membrane specializations were not well-developed. From these findings we concluded that in addition to direct synaptic inhibition, dopamine and GABA could stimulate their receptors by mechanisms similar to "parasynaptic" [Schmitt (1984) Neuroscience, 13:991-1001] or "volume" [Agnati et al. (1995) Neuroscience, 69:711-726] transmission as proposed for the CNS. Humoral agents passing into the intermediate lobe from portal vessels, thus acting as classical hormones, further regulate the melanotropes. Moreover, approximately 50% of the axonal elements were closely apposed to glia, suggesting that glia could have regulatory roles. Previous studies from our laboratory [Chronwall et al. (1987) Endocrinology, 120:1201-1211; Chronwall et al. (1988) Endocrinology, 123:1992:1202] demonstrated heterogeneity in proopiomelanocortin (POMC) biosynthesis among individual melanotropes, prompting the hypothesis that the degree of innervation could govern the expression of certain molecules. We combined immunohistochemistry and in situ hybridization histochemistry to evaluate whether melanotrope molecular heterogenity is spatially correlated with axons and terminals. Tentatively, melanotropes expressing low levels of POMC and alpha1A subunit P/Q type Ca2+ channel mRNAs often were apposed to axons, whereas those with low levels of D2L receptor mRNA rarely were contacted by axons, suggesting that innervation could be one of the factors inducing and maintaining heterogeneity.

Resistance of the dopamine D2L receptor to desensitization accompanies the up-regulation of receptors on to the surface of Sf9 cells.

Dopamine D2 receptor agonists are commonly used in the control of PRL-secreting adenomas, and the sensitivity of dopamine agonists during long term therapy is exquisite. However, the molecular mechanisms responsible for the maintenance of this cellular sensitivity to dopamine agonists remain poorly understood. In the present study, we examined the agonist-induced regulation of the human D2L receptor expressed to a specific activity of approximately 1 pmol receptor/mg protein in Sf9 insect cells. Treatment of D2L receptor-expressing cells with dopamine for up to 3 h resulted in no detectable change in the ligand-binding properties of the receptor and a approximately 120-fold reduction in the potency, but not the efficacy, of D2L receptors to mediate dopamine inhibition of forskolin-stimulated adenylyl cyclase activity. This resistance of the D2L receptor to agonist-induced desensitization was accompanied by a approximately 28% translocation of intracellular D2L receptors to the cell surface, as quantified by cellular fractionation and radioligand binding and visualized by whole cell immunocytochemical staining and confocal microscopy. Immunoblot analysis of the P2 membrane fraction revealed that surface D2L receptors comprised monomers and dimers. Treatment of D2L receptor-expressing cells with the protein synthesis inhibitor cycloheximide significantly reduced the basal expression level of receptors, but did not block the agonist-induced up-regulation of receptors. Longer periods of dopamine exposure for 24 h brought about a small increase in surface receptor density. However, when these studies were conducted in the presence of cycloheximide, receptor density was marginally reduced, suggesting that receptor synthesis accounts for the maintenance of cellular receptor density under these conditions. We conclude that the resistance of the D2L receptor-coupled adenylyl cyclase system to agonist-induced desensitization is attributed to the up-regulation of surface receptors after the translocation of existing intracellular receptors and de novo receptor synthesis.

Resistance of the Dopamine D2L Receptor to Desensitization Accompanies the Up-Regulation of Receptors on to the Surface of Sf9 Cells

Resistance of the Dopamine D2L Receptor to Desensitization Accompanies the Up-Regulation of Receptors on to the Surface of Sf9 Cells

Activation of type II adenylate cyclase by D2 and D4 but not D3 dopamine receptors.

The D2-like dopamine receptors couple to a variety of signal transduction pathways, including inhibition of adenylate cyclase, mitogenesis, and activation of potassium channels. Although these effects are mediated via pertussis toxin-sensitive G proteins, G(i/o), it is likely that some of these effects are influenced by the release of G protein betagamma subunits. Type II adenylate cyclase (ACII) is highly regulated by multiple biochemical stimuli, including protein kinase C, forskolin, G protein alpha subunits, and G protein betagamma subunits. The ability of betagamma subunits to activate this enzyme in the presence of activated alpha(s) has been particularly well characterized. Although stimulation by betagamma subunits has been described as conditional on the presence of activated alpha(s), betagamma subunits also potentiate ACII activity after activation of protein kinase C. We created stable cell lines expressing ACII and the D2L receptor, the D3 receptor, or the D4.4 receptor. Activation of D2L or D4.4 receptors, but not D3 receptors, potentiated beta-adrenergic receptor/Gs-stimulated activity of ACII, as measured by the intracellular accumulation of cAMP. Similarly, stimulation of D2L or D4.4 receptors potentiated phorbol ester-stimulated ACII activity in the absence of activated alpha(s), whereas stimulation of D3 receptors did not. The effect of D2-like receptor stimulation was blocked by pretreatment with pertussis toxin and by inhibition of protein kinase C. We propose that activation of both D2L and D4.4 dopamine receptors potentiated phorbol-12-myristate-13-acetate-stimulated ACII activity through the release of betagamma subunits from pertussis toxin-sensitive G proteins. In contrast, the lack of D3 receptor-mediated effects suggests that stimulation of D3 receptors does not result in an appreciable release of betagamma subunits.

Differential Regulation of Type I and Type VIII Ca2+-stimulated Adenylyl Cyclases by Gi-coupled Receptors in Vivo

Coupling of intracellular Ca2+ to cAMP increases may be important for some forms of synaptic plasticity. The type I adenylyl cyclase (I-AC) is a neural-specific, Ca2+-stimulated enzyme that couples intracellular Ca2+ to cAMP increases. Since optimal cAMP levels may be crucial for some types of synaptic plasticity, mechanisms for inhibition of Ca2+-stimulated adenylyl cyclases may also be important for neuroplasticity. Here we report that Ca2+ stimulation of I-AC is inhibited by activation of Gi-coupled somatostatin and dopamine D2L receptors. This inhibition is due primarily to Gialpha and not betagamma subunits since coexpression of betagamma-binding proteins with I-AC did not affect somatostatin inhibition. However, betagamma released from Gs did inhibit I-AC, indicating that the enzyme can be inhibited by betagamma in vivo. Interestingly, type VIII adenylyl cyclase (VIII-AC), another Ca2+-stimulated adenylyl cyclase, was not inhibited by Gi-coupled receptors. These data indicate that I-AC and VIII-AC are differentially regulated by Gi-coupled receptors and provide distinct mechanisms for interactions between the Ca2+ and cAMP signal transduction systems. We propose that I-AC may be particularly important for synaptic plasticity that depends upon rapid and transient cAMP increases, whereas VIII-AC may contribute to transcriptional-dependent synaptic plasticity that is dependent upon prolonged, Ca2+-stimulated cAMP increases.

NMDA and dopamine D2L receptor interaction in human neuroblastoma SH-SY5Y cells involves tyrosine kinase and phosphatase.

Blockade of N-methyl-D-aspartate (NMDA) receptors by the specific antagonists dizocilpine and (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid on human neuroblastoma SH-SY5Y cells expressing human D21 receptors resulted in a significant increase in the density of D2L receptors. In order to understand the mechanism of dopamine D2L receptor induction following NMDA receptor blockade we used specific protein tyrosine kinase and phosphatase inhibitors to demonstrate their involvement in this interaction. The induction of dopamine D2L receptor was measured by radioreceptor binding assay. The density of the dopamine D2L receptor was increased to 109% by the inhibition of protein tyrosine kinase and prevented by the inhibition of phosphatase 1 or 2A. Inactivation of NMDA receptors might effect the phosphorylation-dephosphorylation states of the regulatory proteins and lead to the induction of the D2L receptor gene.

D2L, D2S, and D3 dopamine receptors stably transfected into NG108‐15 cells couple to a voltage‐dependent potassium current via distinct G protein mechanisms

D2L, D2S, and D3 dopamine receptors stably transfected into NG108‐15 cells couple to a voltage‐dependent potassium current via distinct G protein mechanisms

P-9-10 - BTS 73 947, a novel D1/D5 antagonist with predicted atypical antipsychotic activity

Aripiprazole is the first dopamine D2/D3 receptor partial agonist successfully developed and ultimately approved for treatment of a broad spectrum of psychiatric and neurological disorders. Aripiprazole's dopamine D2 and serotonin 5-HT1A receptor partial agonist activities have been postulated to confer clinical efficacy without marked sedation, and a relatively favorable overall side-effect profile. Using aripiprazole's unique profile as a benchmark for new dopamine partial agonist development may facilitate discovery of new antipsychotics. We conducted an in vitro comparative analysis between aripiprazole, and its human metabolite OPC-14857 (7-(4-[4-(2,3-dichlorophenyl)-1-piperazinyl)butoxy)-2(1H)-quinolinone)); RGH-188 (trans-1-[4-[2-[4-(2,3-dichlorophenyl)piperazine-1-yl]ethyl]cyclohexyl]-3,3-dimethylurea), and its metabolite didesmethyl-RGH-188 (DDM-RGH-188); as well as bifeprunox, sarizotan, N-desmethylclozapine (NDMC; clozapine metabolite), and SDZ 208-912 (N-[(8α)-2-chloro-6-methylergolin-8-yl]-2,2-dimethylpropanamide). In vitro pharmacological assessment included inhibition of forskolin-stimulated cAMP accumulation and the reversal of dopamine-induced inhibition in clonal Chinese hamster ovary cell lines expressing D2S, D2L, D3 Ser-9 and D3 Gly-9 for human dopamine receptors. All test compounds behaved as dopamine D2/D3 receptor partial agonists. Aripiprazole's intrinsic activity at dopamine D2S and D2L receptors was similar to that of OPC-14857 and RGH-188; lower than that of dopamine and bifeprunox; and higher than that of DDM-RGH-188, SDZ 208-912, sarizotan, and NDMC. Aripiprazole's intrinsic activity at dopamine D3 Ser-9 and D3 Gly-9 receptors was similar to that of OPC-14857 and sarizotan; lower than that of dopamine, bifeprunox, RGH-188 and DDM-RGH-188; and higher than that of SDZ 208-912 and NDMC. A consolidated assessment of these findings may help defining the most appropriate magnitude of intrinsic activity at dopamine D2/D3 receptors for clinical efficacy and safety.

Interaction of NMDA and dopamine D2L receptors in human neuroblastoma SH-SY5Y cells.

To understand the mechanism of interaction of the dopamine D2L receptors with NMDA receptors, we have developed a model by transfecting human neuroblastoma SH-SY5Y cells with the human dopamine D2L receptor gene. In vitro blockade of NMDA receptors by the specific antagonists MK-801 and (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) on human neuroblastoma SH-SY5Y cells expressing human dopamine D2L receptors resulted in a significant increase in the density of D2L receptors without a significant change in receptor affinity. Moreover, the dopamine receptor mRNA level increased by approximately 50% by the blockade of NMDA with MK-801. These results suggest a possible interaction of NMDA and dopamine D2L receptors in neuroblastoma SH-SY5Y cells. This system would serve as an excellent model to study the molecular mechanisms involved in the interaction of these two receptors.

Melanotrope dopamine D2 receptor isoform expression in the developing rat pituitary

This study measured melanotrope mRNA and protein expression for the dopamine D2 receptor, and its long isoform, in relation to the appearance of dopamine in axons of the postnatal rat pituitary intermediate lobe. At postnatal day 2, prior to the onset of dopaminergic innervation, D2 receptor (D2T) mRNA was expressed heterogeneously in a subpopulation of melanotropes which also expressed the long isoform (DL). The D2L mRNA appeared to be predominant during early postnatal development, since the D2T probe, which did not discriminate between the isoforms, and the D2L probe hybridized generally to the same cells, as demonstrated in serial sections. Immunohistochemical methods, using two different antisera for the D2T receptor, however, indicated a low level of protein in most melanotropes. Localization of D2L protein corresponded well to D2L receptor mRNA distribution. At day 10, representing a time when dopamine is present in axons throughout the lobe, both D2T receptor mRNA and protein were detected in a significantly larger population of melanotropes than those expressing D2L mRNA and protein. This suggests the appearance of detectable short isoform (D2S) mRNA in virtually all melanotropes and implicates dopamine as a possible signal for increasing D2S isoform mRNA expression.