Cross-Receptor Interactions between Dopamine D(2L) and Neurotensin NTS(1) Receptors Modulate Binding Affinities of Dopaminergics

Dopaminergic systems have been described to functionally interact with the neuromodulatory peptide neurotensin. Employing fluorescence detected coimmunoprecipitation and radioligand binding experiments, we herein demonstrate that coexpression of dopamine D(2L) receptor and the neurotensin receptor subtype NTS(1) leads to physical interaction and the formation of heteromers in transfected human embryonic kidney 293 cells. In this in vitro system, a trans-inhibitory effect on the agonist binding affinity of D(2) was observed in presence of neurotensin. To correlate between the functional properties of dopaminergic agents and the magnitude of neurotensin-induced modulation of D(2L) binding affinities in cells coexpressing D(2L) and NTS(1), a structurally diverse set of dopamine receptor agonists, partial agonists, and antagonists was tested. Ligand specific profiles indicating substantial bias between ligand efficacy and transmodulation were discovered, suggesting a heteromerization-based functional selectivity. In the presence of neurotensin, the novel D(2) agonist FAUC 326 displayed a 34-fold decrease of binding affinity in cells coexpressing D(2L) and NTS(1).     

Regulation of Dopamine D1 and D2 Receptors on Striatal Acetylcholine Release in Rats

The effects of dopamine (DA) D₁ and D₂ receptors on striatal acetylcholine (ACh) releases were investigated by in vivo microdialysis. All drugs were applied via dialysis membrane directly to the striatum. The levels of ACh release were increased by 10⁻⁴ M SKF38393, a D₁ receptor agonist. Although 10⁻⁴ M SCH23390, a D₁ receptor antagonist, exhibited an increase in the levels of ACh release, the agonist (10⁻⁴ M) induced-increase in the levels of ACh release was suppressed by coperfusion of the antagonist (10⁻⁴ M). In contrast, the levels of ACh release were decreased by the D₂ receptor agonist, N-434, in a dose-dependent manner (10⁻⁵ M to 10⁻⁷ M) and increased by the D₂ receptor antagonist, sulpiride, in a dose-dependent manner (10⁻⁵ M to 10⁻⁷ M). The agonist (10⁻⁵ M) induced-decrease in the levels of ACh release was suppressed by coperfusion of the antagonist (10⁻⁶ M). Coperfusion of D₁ (10⁻⁴ M) and D₂ (10⁻⁵ M) agonists blocked both effects of respective drug alone. In order to clarify the effect of endogenous DA, two drugs with different mechanisms for enhancing DA concentration in the synaptic cleft, the DA release-inducer methamphetamine, and the DA uptake inhibitor nomifensine were perfused separately. Both (10⁻⁴ M to 10⁻⁶ M) produced a dose- and a time-dependent decrease in the levels of ACh release. Significant higher levels of ACh release were observed in the striatum of the 6-hydroxydopamine (8

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)-treated rats with significant depletion of striatal DA content. These results suggest that in striatal DA-ACh interaction ACh release, as cholinergic interneuron's activity, is tonically inhibited via the D₂ receptor, mainly by dopaminergic input, and the D₁ receptor probably modifies the effect of the D₂ receptor indirectly.     

Computer-assisted image analysis of caveolin-1 involvement in the internalization process of adenosine A2A-dopamine D2 receptor heterodimers

A functional aspect of horizontal molecular networks has been investigated experimentally, namely the heteromerization between adenosine A_2A and dopamine D₂ receptors and the possible role of caveolin-1 in the cotrafficking of these molecular complexes. This study has been carried out by means of computer-assisted image analysis procedure of laser images of membrane immunoreactivity of caveolin-1, A_2A, D₁, and D₂ receptors obtained in two clones of Chinese hamster ovary cells—one transfected with A_2A and dopamine D₁ receptors and the other one with A_2A and D₂ receptors. Cells were treated for 3 h with 10 μM D₁ receptor agonist SKF 38393, 50 μM D₂-D₃ receptor agonist quinpirole, and 200 nM A_2A receptor agonist CGS 21680. In A_2A-D₁-cotransfected cells, caveolin-1 was found to colocalize with both A_2A and D₁ receptors and treatment with SKF 38393 induced internalization of caveolin-1 and D₁ receptors, with a preferential internalization of D₁ receptors colocalized with caveolin-1. In A_2A-D₂-cotransfected cells, caveolin-1 was found to colocalize with both A_2A and D₂ receptors and either CGS 21680 or quinpirole treatment induced internalization of caveolin-1 and A_2A and D₂ receptors, with a preferential internalization of A_2A and D₂ receptors colocalized with caveolin-1. The results suggest that A_2A and D₂ receptors and caveolin-1 likely interact forming a macrocomplex that internalizes upon agonist treatment. These observations are discussed in the frame of receptor oligomerization and of the possible functional role of caveolin-1 in the process of co-internalization and, hence, in controlling the permanence of receptors at the plasma membrane level (prerequisite for receptor mosaic organization and plastic adjustments) and in the control of receptor desensitization. This paper is dedicated to Luciano Martini, Professor of Endocrinology in Milano and to Faustino Savoldi, Professor of Neurolgy in Pavia. Department of Human Anatomy and Physiology, Section of Antomy, University of Padova, 35121 Padova, Italy     

Identification of extrastriatal dopamine D2 receptors in post mortem human brain with [I]epidepride

The regional distribution of striatal and extrastriatal dopamine D₂ receptors in human brain was studied in vitro with(S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-[¹²⁵I]iodo-2,3-dimethoxybenzamide, [¹²⁵I]epidepride, using post mortem brain specimens from six subjects. Scatchard analysis of the saturation equilibrium binding in twenty-three regions of post mortem brain revealed highest levels of binding in the caudate (16.5 pmol/g tissue) and putamen (16.6 pmol/g tissue) with lower levels seen in the globus pallidus (7.0 pmol/g tissue), nucleus accumbens (7.2 pmol/g tissue), hypothalamus (1.8 pmol/g tissue), pituitary (1.3 pmol/g tissue), substantia innominata (1.0 pmol/g tissue), and amygdala (0.87 pmol/g tissue). Of note was the presence of dopamine D₂ receptors in the four thalamic nuclei studied, i.e. anterior nucleus (1.0 pmol/g tissue), dorsomedial nucleus (0.96 pmol/g tissue), ventral nuclei (0.72 pmol/g tissue), and pulvinar (0.86 pmol/g tissue), at levels comparable to the amygdala (0.87 pmol/g tissue) and considerably higher than levels seen in anterior cingulate (0.26 pmol/g tissue) or anterior hippocampus (0.36 pmol/g tissue). The frontal cortex had very low levels of dopamine D₂ receptors (0.17–0.20 pmol/g tissue) while the inferior and medial temporal cortex had relatively higher levels (0.31–0.46 pmol/g tissue). Inhibition of [¹²⁵I]epidepride binding by a variety of neurotransmitter ligands to striatal, ventral thalamic and inferior temporal cortical homogenates demonstrated that [¹²⁵I]epidepride binding was potently inhibited only by dopamine D₂ ligands. The present study demonstrates that dopamine D₂ receptors are present in basal ganglia, many limbic regions, cortex and in the thalamus. The density of thalamic D₂ receptors is comparable to many limbic regions and is considerably higher than in cortex. Very few frontal lobe D₂ receptors are present in man.     

Prostaglandin D2 and sleep/wake regulation

Prostaglandin (PG) D₂ is the most potent endogenous sleep-promoting substance. PGD₂ is produced by lipocalin-type PGD synthase localized in the leptomeninges, choroid plexus, and oligodendrocytes in the brain, and is secreted into the cerebrospinal fluid as a sleep hormone. PGD₂ stimulates DP₁ receptors localized in the leptomeninges under the basal forebrain and the hypothalamus. As a consequence, adenosine is released as a paracrine sleep-promoting molecule to activate adenosine A_2A receptor-expressing sleep-promoting neurons and to inhibit adenosine A₁ receptor-possessing arousal neurons. PGD₂ activates a center of non-rapid eye movement (NREM) sleep regulation in the ventrolateral preoptic area, probably mediated by adenosine signaling, which activation inhibits the histaminergic arousal center in the tuberomammillary nucleus via descending GABAergic and galaninergic projections. The administration of a lipocalin-type PGD synthase inhibitor (SeCl₄), DP₁ antagonist (ONO-4127Na) or adenosine A_2A receptor antagonist (caffeine) suppresses both NREM and rapid eye movement (REM) sleep, indicating that the PGD₂-adenosine system is crucial for the maintenance of physiological sleep.     

Activation of spinal serotonin2A/2C receptors augments nociceptive responses in the rat

The role of spinal 5-HT_2A/2C receptors in the regulation of spinal nociceptive transmission was studied. The 5-HT_2A/2C agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and the antagonist ketanserin tartrate were administered intrathecally immediately before the formalin test. Activation of spinal 5-HT_2A/2C receptors increased the pain-like behavioural response in both the early and late phases. The findings support the hypothesis that spinal 5-HT_2A/2C receptors augment the spinal afferent nociceptive impulses induced by peripheral inflammation.     

Dopamine denervation leads to an increase in the intramembrane interaction between adenosine A2 and dopamine D2 receptors in the neostriatum

We have previously found, in striatal membrane preparations from young (2 months old) rats, that stimulation of adenosine A₂ receptors (with the selective adenosine A₂ agonist CGS 21680) increases the dissociation constants of high- (K_h) and low-affinity (K_l) dopamine D₂ binding sites (labelled with the selective dopamine D₂ antagonist [³H]raclopride) without changing the proportion of high affinity binding sites (R_h). In the present study in striatal preparations from adult (6 months old) rats, it was found that in addition to the increase in both K_h and K_l values, stimulation of adenosine A₂ receptors is associated with an increase in R_h. These result suggest that, in the adult rat, adenosine A₂ stimulation may inhibit the behavioural effects induced by dopamine D₂ stimulation both by decreasing the affinity and the transduction of dopamine D₂ receptors. We have also studied the intramembrane A₂-D₂ receptor interaction in an experimental model of Parkinson's disease, namely in rats with a unilateral 6-OH-dopamine-induced lesion of the nigro-striatal dopamine pathway. It was found that a unilateral dopamine denervation is associated with a higher density of striatal dopamine D₂ receptors in the order of 20%, without any change in their affinity compared with the unlesioned neostriatum. Furthermore, the density (B_max values) of dopamine D₂ receptors in the contralateral neostriatum was significantly higher (about 20%) than in the striatum from native animals. This finding suggests that an unilateral dopamine denervation also induces compensatory long-lasting changes of dopamine D₂ receptors in the contralateral neostriatum. In addition to the hightened sensitivity to dopamine agonists, it is known that the dopamine denervated striatum is more sensitive to adenosine antagonists like methylxanthines. If the adenosine A₂-dopamine D₂ interaction is the main mechanism of action mediating the central effects of methylxanthines, the dopamine denervation might also potentiate this interaction, i.e., dopamine D₂ receptors could be not only more sensitive to dopamine but also to adenosine A₂ receptor activation. Our results support this hypothesis, since membrane preparations from the denervated neostriatum are more sensitive to the effect of CGS 21680 on dopamine D₂ receptors. Thus a low dose of CGS 21680 (3 nM), which is not effective in membrane preparations from the neostriatum of naive animals, is still effective in membranes from the denervated neostriatum. These results underline the potential antiparkinsonian activity of adenosine A₂ antagonists.     

Synthesis and Evaluation of Methylated Arylazepine Compounds for PET Imaging of 5-HT2c Receptors

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PGE2 decreases reactivity of human platelets by activating EP2 and EP4

Introduction

Platelet hyperreactivity associates with cardiovascular events in humans. Studies in mice and humans suggest that prostaglandin E₂ (PGE₂) regulates platelet activation. In mice, activation of the PGE₂ receptor subtype 3 (EP3) promotes thrombosis, but the significance of EP3 in humans is less well understood.

Objectives

To characterize the regulation of thromboxane-dependent human platelet activation by PGE₂.

Patients/Methods

Platelets collected from nineteen healthy adults were studied using an agonist of the thromboxane receptor (U46,619), PGE₂, and selective agonists and/or antagonists of the EP receptor subtypes. Platelet activation was assayed by (1) optical aggregometry, (2) measurement of dense granule release, and (3) single-platelet counting.

Results

Healthy volunteers demonstrated significant interindividual variation in platelet response to PGE₂. PGE₂ completely inhibited U46,619-induced platelet aggregation and ATP release in 26% of subjects; the remaining 74% had partial or no response to PGE₂. Antagonism of EP4 abolished the inhibitory effect of PGE₂. In all volunteers, a selective EP2 agonist inhibited U46,619-induced aggregation. Furthermore, the selective EP3 antagonist DG-041 converted all PGE₂ nonresponders to full responders.

Conclusions

There is significant interindividual variation of platelet response to PGE₂ in humans. The balance between EP2, EP3, and EP4 activation determines its net effect. PGE₂ can prevent thromboxane-induced platelet aggregation in an EP4-dependent manner. EP3 antagonism converts platelets of nonresponders to a PGE₂-responsive phenotype. These data suggest that therapeutic targeting of EP pathways may have cardiovascular benefit by decreasing platelet reactivity.     

Specific bindings of prostaglandin D2, E2 and F2α in postmortem human brain

Binding activities specific for each of [³H]prostaglandin (PG) D₂, E₂ and F_2α were detected in the P₂ fraction of the human brain homogenates. The bindings were time-dependent, saturable and of high affinity;K_dvalues were 30 nM for all the PG bindings. Regional distribution of these binding activities was determined by measuring specific bindings with 10 nM [³H]PG-D₂, [³H]PG-E₂ and [³H]PG-F_2α in the P₂ fractions from 17 brain regions. The PG-D₂ binding activity was high in the hypothalamus, amygdala and hippocampus followed by cerebellar nuclei, thalamus, nucleus accumbens and cerebral cortex. The PG-E₂ binding sites were similarly concentrated in the hypothalamus and the limbic system, but, unlike the PG-D₂ binding, no significant binding of [³H]PG-3₂ was observed in cerebellar nuclei, cerebellar cortex and putamen. Compared with these two PG bindings, PG-F_2α binding activity was low in many areas, but significant binding was detected in the amygdala, cingulate cortex, cerebellar medulla, hippocampus, nucleus accumbens, midbrain and hypothalamus. These results suggest the presence and specific distribution of three distinct types of PG binding activities, i.e. specific binding of PG-D₂, PG-E₂ and PG-F_2α, in the human brain.     

Serotonin2 (5-HT2) receptor binding in the frontal cortex of schizophrenic patients

Summary Serotonin₂ (5-HT₂) receptor binding was studied, using³H-spiperone as the ligand, in post-mortem brain specimens obtained from schizophrenic patients (N=11) and non-psychiatric controls (N=11). The maximum number of binding sites (B_max) was significantly decreased in schizophrenic patients as compared to normal controls. This difference did not appear to be due to neuroleptic treatment. No difference in K_d (an inverse measure of the affinity of³H-spiperone to its binding sites) was observed between the two groups. However, studies with unmedicated schizophrenic patients are needed to draw any definite conclusion. The role of serotonergic processes in the psychobiology of schizophrenia is discussed.     

Transformation of Pro-Leu-Gly-NH(2) Peptidomimetic Positive Allosteric Modulators of the Dopamine D(2) Receptor into Negative Modulators

The synthesis of dimethyl derivatives of 5.6.5 spiro bicyclic lactam Pro-Leu-Gly-NH(2) peptidomimetics was carried out to test the hypothesis that by placing methyl groups on the β-methylene carbon of the thiazolidine ring steric bulk would be introduced into the topological space that the β-methylene carbon is believed to occupy in the negative allosteric modulators of the dopamine D(2) receptor. With such a modification, a positive allosteric modulator would be converted into a negative allosteric modulator. This hypothesis was shown to be correct as 3a and 4a where found to be negative allosteric modulators, whereas their unmethylated derivatives were positive allosteric modulators of the dopamine D(2) receptor.     

DOI, a 5-HT2A/2C receptor agonist, attenuates clozapine-induced cortical dopamine release

(±)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI, 1.25, 2.5 and 5 mg/kg), a serotonin (5-HT)_2A/2C agonist, produced an inverted U-shaped increase in DA release in rat medial prefrontal cortex (mPFC) with a significant effect only at 2.5 mg/kg. This effect was completely abolished by M100907 (0.1 mg/kg), a 5-HT_2A antagonist, and WAY100635 (0.2 mg/kg), a 5-HT_1A antagonist, neither of which when given alone affected dopamine release. DOI (2.5 mg/kg), but not the 5-HT_2C agonist Ro 60-0175 (3 mg/kg), attenuated clozapine (20 mg/kg)-induced mPFC dopamine release. These results suggest that 5-HT_2A receptor stimulation increases basal cortical dopamine release via 5-HT_1A receptor stimulation, and inhibits clozapine-induced cortical dopamine release by diminishing 5-HT_2A receptor blockade.     

Single nucleotide polymorphisms and mRNA expression for melatonin MT(2) receptor in depression

Polymorphisms (rs 4753426 and rs 794837) and expression of the melatonin MT₂ receptor gene were evaluated in 181 patients with recurrent depressive disorder (rDD) and 149 healthy subjects of Polish origin. We found an increased risk for rDD in patients with the C allele and a decreased risk in patients with the T allele (rs4753426). Patients with the AT heterozygote (rs794837) had an increased mRNA level. The significance of the MT₂ receptor gene and the risk of rDD are suggested.     

[H]Spiperone binds selectively to rat striatal D2 dopamine receptors in vivo: a kinetic and pharmacological analysis

We have determined the kinetic, equilibrium saturation, and pharmacological characteristics of [³H]spiperone ([³H]SPIP) binding to rat brain regional particulate fractions following i.v. injections of [³H]SPIP and compared these parameters to those determined in vitro with traditional ligand-homogenate binding assays. [³H]SPIP binding to rat striatum in vivo and in vitro occurs to a single class of non-interacting binding sites which possess the pharmacological properties of a D₂ dopamine (DA) receptor. The potencies of neuroleptic drugs in inhibiting DA receptor-mediated behaviors correlate with their potencies at displacing striatal [³H]SPIP binding in vivo. While striatum possesses a similar density of [³H]SPIP binding sites in vivo (34 pmol/g) and in vitro (31 pmol/g), binding affinity in vivo is about 200 times lower than in vitro. This difference in binding affinities appears to arise from alterations of [³H]SPIP association and dissociation rate constants brought about by tissue homogenization. The implications of our findings for external imaging of DA receptors and studies of DA receptor function in human brain homogenates are discussed.     

Postnatal lead exposure induces supersensitivity to the stimulus properties of a D2-D3 agonist

To examine the impact of lead (Pb) exposure during the ontogeny of dopaminergic (DA) systems on resultant DA function, rats were exposed postnatally (0–21 days of age) via the lactating dam to 0, 100 or 350 ppm Pb acetate in drinking water. At 2 months of age, the postnatally Pb-exposed rats were trained to discriminate the stimulus properties of either the D₁ receptor agonist SKF38393 (6.0 mg/kg) or the D₂-D₃ receptor family subtype agonist quinpirole (0.05 mg/kg) from saline using a standard two-lever operant food-reinforced drug discrimination paradigm. In each training group, dose-effect curves describing drug lever responding to lower doses of the training drug and to preadministration of selective DA antagonists were obtained to examine Pb-induced changes in DA sensitivity. Doses of other DA agonists were substituted for the training drug to determine the generality of any changes in DA sensitivity, and doses of non-DA compounds were substituted to determine the specificity of any changes in DA sensitivity. In the D₁/saline training condition, Pb exposure was not associated with any specific or consistent changes in DA sensitivity. In contrast, exposure to Pb was associated with D₂-D₃ receptor subtype supersensitivity as was indicated by significantly elevated levels of drug lever responding in the presence of quinpirole and haloperidol and to at least one dose of apomorphine. No differences in the dose-effect curves for either (+)-amphetamine or NMDA were observed in the D₂-D₃-trained control and Pb-exposed groups, but an increase in drug lever responding in the presence of pentobarbital was noted in the Pb-exposed group relative to control. Taken together, these findings are consistent with a Pb-induced functional D₂-D₃ supersensitivity possibly mediated via autoreceptors. Moreover, this functional D₂-D₃ supersensitivity necessarily represents a permanent effect of postnatal Pb exposure since both blood and brain Pb levels were negligible at the time drug discrimination training began.