R107474, 2-methyl-3-[2-(1,2,3,4-tetrahydrobenzo[4,5]furo[3,2-c]pyridin-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one, was investigated using in vitro and in vivo receptor assays and proved to be a potent and relatively selective alpha(2)-adrenoceptor antagonist. Performed assays in vitro were inhibition of binding to a large number of neurotransmitter receptor sites, drug receptor binding sites, ion channel binding sites, peptide receptor binding sites, and the monoamine transporters in membrane preparations of brain tissue or of cells expressing the cloned human receptors. The compound has subnanomolar affinity for halpha(2A)- and halpha(2C)-adrenoceptors (K(i) = 0.13 and 0.15 nM, respectively) and showed nanomolar affinity for the halpha(2B)-adrenoceptors and 5-hydroxytryptamine(7) (h5-HT(7)) receptors (K(i) = 1 and 5 nM, respectively). R107474 interacted weakly (K(i) values ranging between 81 and 920 nM) with dopamine-hD(2L), -hD(3) and -hD(4), h5-HT(1D)-, h5-HT(1F)-, h5-HT(2A)-, h5-HT(2C)-, and h5-HT(5A) receptors. The compound, tested up to 10 microM, interacted only at micromolar concentrations or not at all with any of the other receptor or transporter binding sites tested in this study. In vivo alpha(2A)- and alpha(2C)-adrenoceptor occupancy was measured by ex vivo autoradiography 1h after subcutaneous (sc) administration of R107474. It was found that R107474 occupies the alpha(2A)- and alpha(2C)-adrenoceptors with an ED(50) (95% confidence limits) of 0.014 mg/kg sc (0.009-0.019) and 0.026 mg/kg sc (0.022-0.030), respectively. Radiolabeled 2-methyl-3-[2-([1-(11)C]-1,2,3,4-tetrahydrobenzo[4,5]furo[3,2-c]pyridin-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one ([(11)C]R107474) was prepared and evaluated as a potential positron emission tomography (PET) ligand for studying central alpha(2)-adrenoceptors. [(11)C]R107474 was obtained via a Pictet-Spengler reaction with [(11)C]formaldehyde in 33 +/- 4% overall decay-corrected radiochemical yield. The total synthesis time was 55 min and the specific activity was 24-28 GBq/micromol. The biodistribution of [(11)C]R107474 in rats revealed that the uptake of [(11)C]R107474 after in vivo intravenous administration is very rapid; in most tissues (including the brain) it reaches maximum concentration at 5 min after tracer injection. In agreement with the known distribution of alpha(2)-adrenoceptors in the brain, highest uptake of radioactivity was observed in septum (3.54 +/- 0.52 ID/g, 5 min pi) and entorhinal cortex (1.57 +/- 0.10 ID/g, 5 min pi). Tissue/cerebellum concentration ratios for septum (5.38 +/- 0.45, 30 min pi) and entorhinal cortex (3.43+/-0.24, 30 min pi) increased with time due to rapid uptake followed by a slow washout. In vivo blocking experiments using the non-selective alpha(2)-adrenoceptor antagonist mirtazapine demonstrated specific inhibition of [(11)C]R107474 binding in selective brain areas. The receptor binding profile of mirtazapine is reported and the selectivity of inhibition of binding is discussed. These results suggest that [(11)C]R107474 deserves further investigation as a potential radioligand for studying alpha(2)-adrenoceptors using PET.

Recent reports show that striatal dopamine D1-type receptors from one side of the normal rat brain can control brain activity (as measured by c-fos induction) on both sides of the brain. However, this phenomenon has not yet been studied in the presence of sensitized dopamine D1-type receptors. Here we address this issue by investigating the extent to which dopamine D1-type receptors control brain activation in rats with unilaterally sensitized dopamine D1-type receptors. Gene induction assays were used to identify activated regions from midbrain to forebrain in unilaterally 6-hydroxydopamine lesioned (hemiparkinsonian) rats challenged with the full dopamine D1-type agonist SKF82958 (3 mg/kg, 0.5 and 2 h). The genes used are c-fos, the proven neuronal activity marker, and Regulator of G protein Signaling 2, a gene we propose as a marker of signaling homeostasis. SKF82958-mediated induction of both genes is greatly enhanced in hemiparkinsonian rats compared with shams, in both the lesioned and the intact hemisphere. For example, in the denervated caudate-putamen at 2 h postinjection, this enhancement is more than 80-fold for c-fos and up to 20-fold for Regulator of G protein Signaling 2; for the intact side this is 35-fold for c-fos and 27-fold for Regulator of G protein Signaling 2. Cortical induction of c-fos and Regulator of G protein Signaling 2 was generalized to most neocortical regions and was essentially equivalent in both the denervated and intact hemispheres. Interestingly, hippocampal structures also showed strong bilateral induction of both genes. This overall pattern of brain activation can be accounted for by the basal-ganglia thalamocortical and hippocampal circuits which both contain hemisphere-crossing connections and which can be initially activated in the lesioned hemisphere. Some regions, such as the intact striatum or the CA1 region, showed relatively low c-fos induction and relatively high Regulator of G protein Signaling 2 induction, possibly indicating that these regions are engaged in unusually strong signaling regulation activities. Our results show that, besides basal ganglia-thalamocortical circuits, dopamine D1-type-mediated brain activation in hemiparkinsonian rats also involves hippocampal circuits.

The present report examines the effects of acute NMDA antagonism on Regulator of G Protein Signaling 2 (RGS2) expression and adenylyl cyclase sensitivity in the rat striatum. MK-801 and phencyclidine rapidly down-regulate RGS2 mRNA. The down-regulation of RGS2 by MK-801 was dose dependent and transient. Because previous reports showed that RGS2 attenuates activity of adenylyl cyclase, RGS2 protein level and sensitivity of adenylyl cyclase to forskolin was tested 2 h after administration of MK-801 (1 mg/kg). In striatal membranes of these rats, RGS2 protein level was 17% lower and forskolin-stimulated cAMP production 38% higher than in controls. These findings reveal a cross-talk between NMDA receptors and adenylyl cyclase and suggest a general cross-talk mechanism by which RGS proteins transcriptionally regulated by ionotropic receptors can alter signaling properties of metabotropic receptors.

The diverse family of Regulators of G protein signaling (RGS) proteins are widely distributed proteins with multiple functions, including GAP activity for heterotrimeric G protein alpha subunits. Three members of the RGS family, RGS9-2, RGS4 and RGSz, have been shown to play an essential modulatory role in psychostimulant and opiate drug actions. Interestingly, these proteins show distinct structure, distribution pattern and cellular localization. In addition, each of these proteins is differentially regulated by drugs of abuse in particular brain networks and appears to modulate distinct signal transduction events. The striatal enriched RGS9 plays a prominent role in opiate and psychostimulant drug reward; RGS4 appears to modulate opiate dependence via actions in the locus coeruleus, whereas RGSz modulates analgesia via activation of the PKC pathway.

R214127 was shown to be a potent and noncompetitive metabotropic glutamate 1 (mGlu1) receptor-selective antagonist. The kinetics and pharmacology of [(3)H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone (R214127) binding to rat mGlu1a receptor Chinese hamster ovary (CHO)-dhfr(-) membranes was investigated, as well as the distribution of [(3)H]R214127 binding in rat brain tissue and sections. Specific binding to rat mGlu1a receptor CHO-dhfr(-) membranes was approximately 92% of total and was optimal at 4 degrees C. Full association was reached within 5 min, and [(3)H]R214127 bound to a single binding site with an apparent K(D) of 0.90 +/- 0.14 nM and a B(max) of 6512 +/- 1501 fmol/mg of protein. Inhibition experiments showed that [(3)H]R214127 binding was completely blocked by 2-quinoxaline-carboxamide-N-adamantan-1-yl (NPS 2390), (3aS,6aS)-6a-naphtalan-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan-1-on (BAY 36-7620), and 7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), but was not displaced by competitive mGlu1 receptor ligands such as glutamate and quisqualate, suggesting that R214127, NPS 2390, BAY 36-7620, and CPCCOEt bind to the same site or mutually exclusive sites. Experiments using rat cortex, striatum, hippocampus and cerebellum revealed that [(3)H]R214127 labeled a single high-affinity binding site (K(D) approximately 1 nM). B(max) values were highest in the cerebellum (4302 +/- 2042 fmol/mg of protein) and were 741 +/- 48, 688 +/- 125, and 471 +/- 68 fmol/mg of protein in the striatum, hippocampus, and cortex, respectively. The distribution of [(3)H]R214127 binding in rat brain was investigated in more detail by radioligand autoradiography. A high density of binding sites was detected in the molecular layer of the cerebellum. Moderate labeling was seen in the CA3 and dentate gyrus of the hippocampus, thalamus, olfactory tubercle, amygdala, and substantia nigra reticulata. The cerebral cortex, caudate putamen, ventral pallidum, and nucleus accumbens showed lower labeling. The high affinity and selectivity of [(3)H]R214127 for mGlu1 receptors renders this compound the ligand of choice to study the native mGlu1 receptor in brain.

We distinguished the functions of the different 5-hydroxytryptamine-2 (5-HT2) receptor (5-HT2R) subtypes in the tryptamine-induced 5-HT syndrome in rats using (1) the 5-HT2AR antagonist R93274 (N-[(3-p-fluorophenyl-1-propyl)-4-methyl-4-piperidinyl]-4-amino-5-iodo-2-methoxybenzamide), the 5-HT2A/CR antagonist R99647 (2-(dimethylaminomethyl)2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine), the 5-HT2B/CR antagonist SB-242084 (6-chloro-5-methyl-1-parallel to2-\(2-methyl-3-pyridyl)oxyl-5-pyridyl\carbamoyl\-indoline), and several 5-HT2R antagonists (ketanserin, risperidone, pipamperone and mianserin); and (2) chronic 5-HT2R activation by 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). In contrast to SB-242084, the selective 5-HT2AR antagonist R93274 as well as the non-selective 5-HT2AR antagonists (1199647, ketanserin, risperidone, pipamperone and mianserin) significantly inhibited tryptamine-induced forepaw treading and tremors, and reversed peripherally mediated cyanosis into hyperaemia; only the 5-HT2A/CR antagonists R99647 and mianserin inhibited the tryptamine-induced hunched back. Intermittent DOM administration (intravenously every 48 h for 12 days) did not change the centrally mediated tryptamine-induced forepaw treading, tremors and hunched back at 1, 4 or 7 days after the last DOM pretreatment. The DOM-induced head twitch response, measured immediately after every DOM injection, was not affected. In contrast, peripherally mediated cyanosis was reversed into hyperaemia in 75, 11 and 20% of all pretreated rats at 1, 4 and 7 days, respectively, after the last DOM administration. Taken together, these finding suggest that central 5-HT(2A)Rs mediate tryptamine-induced forepaw treading and tremors, that peripheral 5-HT(2A)Rs mediate tryptamine-induced cyanosis, and that 5-HT(2C)Rs mediate tryptamine-induced hunched back. Peripheral 5-HT(2A)Rs are more sensitive to desensitization after intermittent treatment with an agonist than central 5-HT(2A)Rs. (C) 2002 Lippincott Williams Wilkins.

Regulator of G protein signaling (RGS) proteins are a recently identified family of proteins which dampen G protein-coupled receptor-mediated signaling by accelerating the intrinsic GTPase activity of Galpha subunits of heterotrimeric G proteins. More than 20 different RGSs have been identified and at least 10 are expressed in the CNS. The present study describes in detail the localization in the rat brain of one member of this family, RGS2. The distribution of RGS2 mRNA and protein has been studied in parallel by performing in situ hybridization and immunoautoradiography on adjacent rat brain sections. Our localization study reveals that RGS2 mRNA and protein are widely expressed in the brain. Protein and mRNA are mostly colocalized such as in neocortex, piriform cortex, caudate-putamen, septum, hippocampus, locus coeruleus. Some mismatches were also observed such as presence of mRNA but not protein in the paraventricular nucleus, the substantia nigra pars compacta and the red nucleus, suggesting that RGS2 protein is present in neuronal projections. Previous reports describing an induction of RGS2 mRNA in the rat striatum after psychostimulants (amphetamine, cocaine) led us to focus on the distribution of RGS2 in the basal ganglia circuitry. The absence of RGS2 mRNA and protein in the globus pallidus suggests that RGS2 would play its regulatory role more in the direct (striatonigral) than in the indirect (striatopallidal) striatal output pathway. In addition, to delineate the implication of RGS2 in pre- and/or postsynaptic functions in the basal ganglia, we performed lesions of the nigrostriatal pathway by 6-hydroxydopamine (6-OHDA) and striatal quinolinic acid lesions. The 6-OHDA lesion did not modify RGS2 mRNA or protein levels in the caudate-putamen whereas the intrastriatal quinolinic acid infusion caused a marked reduction of RGS2 mRNA and protein in the lesioned zone. These data indicate that RGS2 is predominantly expressed in intrinsic striatal neurons. Moreover, the absence of detectable change in RGS2 expression after injections of 6-OHDA suggests also that RGS2 is not primarily involved in the hypersensitization of postsynaptic dopamine receptors observed after lesion of the nigrostriatal pathway.

Biological assay guided fractionation of a dichloromethane extract of Synaptolepis kirkii led to the isolation of four new and five known daphnane-type diterpene orthoesters, whose structure was established by spectroscopic data. Full spectroscopic data of the new and known natural products are reported here for the first time. Pronounced neurotrophic and substantial antileukaemia activities of these compounds were found in in vitro assays.

This study documents differences in ligand binding and signal transduction properties between the human (h) 5-hydroxytryptamine (5-HT)4a and h5-HT4b receptor splice variants stably expressed in human embryonic kidney 293 cells. The fraction of the [3H]5-HT high-affinity site relative to the whole receptor population measured with [3H]GR113808 was higher for the h5-HT4a isoform (around 0.4) than for the 5-HT4b isoform (around 0.2) and was independent of the level of expression. The potency and efficacy of reference compounds tested for the cAMP response differed slightly but significantly between both variants. Most remarkably, 5-methoxytryptamine and prucalopride were found more potent on the 5-HT4b variant, whereas SDZ-HTF 919 and SB204070 were more potent on the 5-HT(4a) variant. Guanosine-5'-O-(3-[35S]thio)triphosphate binding on membranes and cAMP assays in whole cells revealed that only the h5-HT4b isoform coupled to Galphai/o-proteins in addition to its well-documented Galphas coupling. In contrast, the h5-HT4a receptor coupled only to Galphas-proteins, however, was able to trigger an increase in the intracellular calcium concentration ([Ca(2+)]i). The observed [Ca(2+)]i increase did not occur through inositol phosphate formation and was not sensitive to Bordetella pertussis toxin, forskolin, or 3-isobutyl-1-methylxanthine (pre)treatment but was due to Ca(2+) influx from the extracellular environment. Interestingly, the Ca(2+) pathway was dependent on high receptor expression levels and was compound-specific, because benzamide-like compounds triggered two to three times higher responses than indoleamines. Taken together, these data provide the first evidence for fine functional differences between C-terminal splice variants of the h5-HT4 receptor, which may contribute to a better understanding of the functional diversity of this receptor class.

The effect of antagonist pretreatment on the signaling properties of the human metabotropic glutamate 1a (hmGlu1a) receptor was examined in stably transfected L929sA cells. Pre-exposure of hmGlu1a receptor-expressing cells to the mGlu1 receptor antagonists (S)-4-carboxy-3-hydroxyphenylglycine and 7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester dramatically enhanced subsequent glutamate-induced phosphoinositide hydrolysis and intracellular [Ca(2+)] rise. We found clear indications that the antagonist-mediated enhancement of glutamate-evoked mGlu1a receptor signaling is caused by the development of mGlu1a receptor supersensitivity: the potency of glutamate was increased by 3-fold after 24 h antagonist pretreatment and the potency of the antagonists was significantly decreased in antagonist-pretreated cells. The kinetic profile of the antagonist-mediated enhancement showed that the maximal increase in intracellular [Ca(2+)] was already reached after 30-min pretreatment, suggesting that de novo receptor synthesis is not involved in the process of mGlu1a receptor supersensitization. Glutamate-mediated phosphoinositide hydrolysis increased up to 24 h after antagonist treatment. Although it seemed likely that the hmGlu1a receptor could desensitize after activation by endogenously present glutamate, removal of glutamate from the extracellular medium with GPT resulted in a much smaller enhancement of glutamate responsiveness. Moreover, the magnitude of antagonist-mediated receptor supersensitivity was much larger than the magnitude of agonist-induced receptor desensitization. These results suggest that antagonist-evoked mGlu1 receptor supersensitivity is not merely the result of a blockade of agonist-induced desensitization. Finally, we found that antagonist pretreatment doubled the amount of receptors at the cell surface. Our findings are the first lines of evidence that prolonged antagonist treatment can supersensitize the hmGlu1a receptor. In view of the potential therapeutic application of mGlu1 receptor antagonists, it will be important to know whether these phenomena occur in vivo.

The neurokinin 3 (NK3) receptor antagonists represent a novel class of pharmacological agents, which are currently under evaluation for the treatment of psychiatric disorders. An efficient brain penetration is one of the main prerequisites to further evaluate compounds displaying high potency to bind the NK3 receptor. The present report describes a method for determining the in vivo occupancy of central NK3 receptors after peripheral administration of drugs. An ex vivo measurement of NK3 receptor occupancy by quantitative autoradiography employing [3H]senktide as the radioligand has been developed. The speed of the method, which is usually considered low due to the time dedicated to film exposure (from weeks to months), has been considerably increased by the use of the beta-imager. The high sensitivity of this new radioimager was used to visualize and quantitatively analyze the [3H]senktide binding sites in brain sections within hours. Using this method, we have demonstrated that the reference NK3 antagonist SR142801 dose dependently occupied the NK3 receptors in the gerbil brain after subcutaneous administration with an ED50 of 0.85 mg/kg. The less active enantiomer SR142806 occupied the NK3 receptors only by 25% at the highest used dose of 10 mg/kg. These values are in accordance with the reported behavioral effects of the compounds. Our results indicate that ex vivo receptor occupancy measurements can be dependently used to predict the central activity of NK3 antagonists. More generally, the combination of ex vivo receptor autoradiography with the beta-imager detection constitutes a new and fast method to evaluate the brain penetration of drug candidates.

The neurokinin 3 (NK3) receptor is predominantly expressed in the central nervous system (CNS). Species differences in neurokinin 3 (NK3) receptor pharmacology have led to the preferential use of guinea pigs and gerbils in the characterization of non-peptide NK3 antagonists. Little is known about the central localization of NK3 receptors in the CNS of these species. To study this, [(3)H]senktide and [(3)H]SR 142801 were used in autoradiography experiments to visualize the NK3 receptors in the guinea pig and gerbil brain and compared to with the distribution of [(3)H]senktide binding sites in the rat brain. In the three species, the NK3 receptor was similarly distributed within the cerebral cortex, the zona incerta, the medial habenula, the amygdaloid complex, the superior colliculus and the interpeduncular nucleus. Outside of these structures, our study has revealed that each species displayed a specific distribution pattern of central NK3 receptors. The rat was the only species where NK3 receptors could be visualized in the striatum, the supraoptic nucleus and the paraventricular nucleus of the hypothalamus. The guinea pig differed mainly from the two other species by the absence of detectable binding sites in the substantia nigra pars compacta and the ventral tegmental area. A specific localization of NK3 receptors in the anterodorsal and anteroventral thalamic nuclei characterized the gerbil. This last species is also unique by in the higher level of NK3 receptors in the dorsal and median raphe nuclei. All these differences suggest that the NK3 receptor mediates different functions in different species.

The human 5-hydroxytryptamine5A (h5-ht5A) receptor was expressed in Escherichia coli (h5-ht5A-E. coli) to verify its pharmacological profile in the absence of G proteins. In addition, the ability of the h5-ht5A receptor to interact with mammalian Gi/o and Gs proteins was investigated by a new reconstitution approach. Agonists displayed lower affinities for h5-ht5A-E. coli than for stably transfected h5-ht5A-HEK 293 cells, due to the absence of G protein coupling in E. coli. Lysergic acid diethylamide behaved as a neutral antagonist, showing equal affinities for the G protein-coupled and the uncoupled receptor. To analyze the G protein coupling behavior of the h5-ht5A receptor, h5-ht5A-E. coli membranes or h5-ht5A-Sf9 insect cell membranes were fused by vortexing to membranes from baculovirus-infected Sf9 cells expressing mammalian G proteins. The ability of the h5-ht5A receptor to differentiate between Gi/Go/Gz and Gs proteins was explored by investigation of agonist binding affinities and agonist-induced stimulation of [35S]GTP gamma S binding. The h5-ht5A receptor failed to interact with Gz and Gs proteins and coupled equally well to Gj and Go proteins to form a complex with high affinity for agonists. Under the applied conditions, however, Gi proteins were found to be better activated than Go proteins in the [35S]GTP gamma S binding assay.

Establishing a stable cell line that expresses a particular protein of interest is often a laborious and time-consuming experience. With constitutive expression systems, a gradual loss of the highly expressing clones over a given time span and/or a severe counter-selection due to toxicity of the expressed protein for the host cell line are major drawbacks. In both cases, inducible expression systems offer a valuable alternative. Over the years, many regulated expression systems have been developed and evaluated. In the present study, we compare the efficiency, the advantages and the drawbacks of a tetracycline- and an ecdysone-inducible system for expression of the reporter protein chloramphenicol acetyltransferase and of different G-protein-coupled serotonin (5-HT) receptors. A high level of expression of different 5-HT receptors was obtained with the tetracycline-inducible system. In the cell line L929, which stably expresses the tetracycline-responsive transactivator, a maximum ligand binding of 20,000 and 9500 fmol/mg protein was measured for the h5-HT(1B) and h5-ht(1F) receptors, respectively. In the HEK293rtTA cell line, levels of 15,700, 3000, and 9100 fmol bound ligand/mg protein were obtained for the h5-HT(1B), h5-ht(1F) and h5-HT(4b) receptors, respectively. These high expression levels remained stable for several months of continuous culture. Although the ecdysone-inducible expression system was useful for tightly regulated expression, the levels were far lower than those obtained with the tetracycline system (e.g. 640 fmol bound ligand/mg protein for the h5-ht(1F) receptor in HEK293EcR).

The 5-HT2A and 5-HT2C receptors belong to the same subtype of the G-protein coupled receptor family and have several agonist and antagonist ligands in common. To gain more insight into the differences in the regulation of the two receptors, we studied the effect of agonist and antagonist pre-treatment on radioligand receptor binding and 5-HT-induced inositol phosphate formation on rat 5-HT2A and rat 5-HT2C receptors stable expressed in NIH 3T3 cells. We compared short (15 min) and prolonged (48 h) pre-treatment of the cells with the natural agonist, 5-HT and with the antagonist pipamperone, which can be readily washed out. The rat 5-HT2C receptor showed an agonist-induced down-regulation (decrease in Bmax of labelled agonist and antagonist binding) and desensitisation (decrease in 5-HT-induced inositol phosphate formation and potency of 5-HT). Antagonist pre-treatment induced an increase in rat 5-HT2C receptor-mediated inositol phosphate formation as well as increased agonist and antagonist radioligand binding. These findings are consistent with the classical model of G-protein coupled receptor regulation. In contrast, the rat 5-HT2A receptor expressed in the same host cell behaved differently, unlike the classical model. Pre-treatment with 5-HT for 15 min and 48 h did not change receptor levels measured by radioligand binding, but the signal transduction response (inositol phosphate formation) was significantly reduced. Pre-treatment with the antagonist pipamperone for 15 min and 48 h caused an increase in antagonist radioligand binding but a reduction in agonist radioligand binding and a decrease in inositol phosphate formation and potency of 5-HT. Hence, the rat 5-HT2A receptor apparently undergoes agonist desensitisation without down-regulation of the total receptor number. Antagonist pre-treatment causes a paradoxical desensitisation, possibly by uncoupling of the receptor from G-proteins. The uncoupled receptor does not bind 5-HT in the nanomolar range but retains its antagonist binding properties. Paradoxical antagonist-induced desensitisation of rat 5-HT2A receptors has also been observed in vivo.