Stepwise control of osteogenic differentiation by 5-HT(2B) receptor signaling: nitric oxide production and phospholipase A2 activation.

During development, antagonists of 5-HT(2) receptor subtypes cause morphological defects of mesodermal and neural crest derivatives including the craniofacial skeleton. We used an inducible mesoblastic cell line, C1, able to fully convert into osteocytes within 12 days, to assess the involvement of 5-HT(2) receptors during osteogenic differentiation. On day 5 of the osteogenic program, immediately before matrix mineralization, the cells selectively implement 5-HT(2B) receptors (5-HT(2B)R) which remain functional until terminal differentiation. In 5-HT-depleted medium, the receptor exhibits a constitutive activity leading to basal nitric oxide (NO) release and phospholipase A2 (PLA2)-dependent arachidonic acid (AA) production. Blockade of this intrinsic activity affects the efficiency of mineralization by decreasing calcium incorporation within the matrix by 40%. Optimal bone matrix mineralization involves both NO and PLA2 signaling pathways. Moreover, between day 5 and day 10, at the beginning of mineral deposition, the 5-HT(2B)R promotes prostaglandin E2 production through AA-dependent cyclooxygenase (COX) activation. From day 10 onwards, when C1 osteoblasts undergo conversion into osteocyte-like cells, COX activity is quenched. Altogether these observations indicate that the 5-HT(2B)R contributes in an autocrine manner to osteogenic differentiation and highlight a switch in the downstream targets of the receptor at the terminal stage of the program. Finally, in addition to its autocrine function, the 5-HT(2B)R responds to 5-HT by increasing NO production and AA release. These findings raise concern regarding the use of 5-HT(2B)R-related drugs that may interfere with bone metabolism in physiological or pathological situations.

Functional consequences of reduction in NMDA receptor glycine affinity in mice carrying targeted point mutations in the glycine binding site.

We have used site-directed mutagenesis in conjunction with homologous recombination to generate two mouse lines carrying point mutations in the glycine binding site of the NMDAR1 subunit (Grin1). Glycine concentration-response curves from acutely dissociated hippocampal neurons revealed a 5- and 86-fold reduction in receptor glycine affinity in mice carrying Grin1(D481N) and Grin1(K483Q) mutations, respectively, whereas receptor glutamate affinity remained unaffected. Homozygous mutant Grin1(D481N) animals are viable and fertile and appear to develop normally. However, homozygous mutant Grin1(K483Q) animals are significantly lighter at birth, do not feed, and die within a few days. No gross abnormalities in CNS anatomy were detected in either Grin1(D481N) or Grin1(K483Q) mice. Interestingly, in situ hybridization and Western blot analysis revealed changes in the expression levels of NMDA receptor subunits in Grin1(D481N) mice relative to wild type that may represent a compensatory response to the reduction in receptor glycine affinity. Grin1(D481N) mice exhibited deficits in hippocampal theta burst-induced long-term potentiation (LTP) and spatial learning and also a reduction in sensitivity to NMDA-induced seizures relative to wild-type controls, consistent with a reduced activation of NMDA receptors. Mutant mice exhibited normal prepulse inhibition but showed increased startle reactivity. Preliminary analysis indicated that the mice exhibit a decreased natural aversion to an exposed environment. The lethal phenotype of Grin1(K483Q) animals confirms the critical role of NMDA receptor activation in neonatal survival. A milder reduction in receptor glycine affinity results in an impairment of LTP and spatial learning and alterations in anxiety-related behavior, providing further evidence for the role of NMDA receptor activation in these processes.

The phosphoinositide pathway of lymphoid cells: labeling after permeabilization by alveolysin, a bacterial sulfhydryl-activated cytolysin.

An improved method allowing incorporation of [3H]myo-inositol into the phosphoinositide pool of human lymphoid cells is described. The procedure devised involves cell permeabilization with a thiol-activated membranolytic toxin, alveolysin, and optimization of the phosphoinositide labeling and extraction. In these conditions 4 to 10% of the added [3H]myo-inositol is found intracellularly and half of this amount (2-5%) is incorporated into the phosphoinositide pool in only 1 h as compared to the classical 0.2 to 0.3% incorporation obtained after 10 to 20 h. The integrity of coupling between receptors and phospholipase C was assessed by the inositol phosphate production after cell stimulation by various agonists.

Pharmacological characterization of metabotropic glutamate receptors linked to the inhibition of adenylate cyclase activity in rat striatal slices.

The pharmacological profile of mGlu receptors negatively linked to adenylyl cyclase was characterized in adult rat striatal slices. Among the mGlu agonists tested, (+)-2-aminobicyclo-[3.1.0]-hexane-2,6-di carboxylate (LY354740), was the most potent inhibitor of forskolin-stimulated cAMP formation (EC50 = 11 +/- 2 nM). Inhibition of forskolin stimulation by the group III agonist L-2-amino-4-phosphono-butanoate (L-AP4) was biphasic, the two parts of the concentration curve having EC50 values of 6 +/- 1 microM and 260 +/- 4 microM, suggesting a sequential recruitment of mGlu4/8 and mGlu7. The effects of several new phenylglycine derivative antagonists were tested on the inhibition of forskolin cAMP response by (2S,1'S,2'S)-2-(carboxy-cyclopropyl)-glycine (L-CCG I) and L-AP4. At 500 microM, (RS)-alpha-methyl-3-carboxy-methyl-pheny lglycine was unable to antagonize the effect of L-CCG I or L-AP4 but (S)-alpha-methyl-3-carboxy-phenylalanine inhibited the effect of L-AP4 with a low potency. Finally, (RS)-alpha-methyl-4-tetrazolylphenylglyc ine and particularly (RS)-alpha-methyl-4-phosphonophenylglyci ne, appeared to be the most potent and selective antagonists of L-AP4 induced inhibition of forskolin-stimulated cAMP production in adult rat striatal slices.

Pharmacological properties of native metabotropic glutamate receptors in freshly dissociated Golgi cells of the rat cerebellum.

We have examined the pharmacological properties of native metabotropic glutamate (mGlu) receptors in freshly isolated rat cerebellar Golgi cells using the whole-cell configuration of the patch-clamp technique. Group II mGlu receptor agonists inhibited voltage-gated Ca(2+) channels (VGCC) currents in a reversible and concentration-dependent manner with a rank order of potency being LY354740> DCG-IV > L-CCG-I > glutamate >>1S,3R-ACPD > NAAG. The maximum degree of inhibition obtained was similar for all drugs tested, saturating at about 33-41%, except for NAAG that had a non saturating effect of 50% at 1mM. Two novel group II mGlu receptor antagonists, LY341495 and Ro 65-3479, reversed VGCC current inhibition by LY354740 with pK(B) values of 7.0 and 6.3, respectively. In a subpopulation of Golgi cells, the antagonistic effect of LY341495 was only partial, suggesting a remaining effect of group I mGlu receptors. This was confirmed by experiments with S-DHPG, a selective group I mGlu receptor agonist. These experiments suggest that Golgi cells of the cerebellum express group II mGlu receptors that couple to the inhibition of VGCCs. Therefore, inhibition of VGCCs in cerebellar Golgi cells is a useful model system to evaluate novel group II mGlu receptor ligands.

Activity-dependent presynaptic autoinhibition by group II metabotropic glutamate receptors at the perforant path inputs to the dentate gyrus and CA1.

Pharmacological activation of metabotropic glutamate receptors (mGluRs) can inhibit synaptic transmission; however, relatively little evidence exists regarding the physiological conditions under which such autoreceptors are activated by synaptically released glutamate. Bath application of selective group II mGluR agonists profoundly inhibited field excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the perforant path inputs to both the mid-molecular layer of the dentate gyrus and the stratum lacunosum moleculare of the CA1. Application of the group II selective mGluR antagonist LY341495 resulted in an increase in the relative amplitude of a test fEPSP evoked 200 ms after a conditioning burst, but not after a single conditioning stimulus, in both pathways. Antagonist application also resulted in a marked increase in the relative amplitude of test population spikes evoked in the dentate gyrus following a conditioning burst. These observations are consistent with a presynaptic autoinhibitory action of group II metabotropic receptors that is revealed following burst stimulation of the pathway, consistent with their localisation in the preterminal zone. Activation of group II mGluRs during theta-gamma pattern discharge of projection neurones in the entorhinal cortex is likely to play an important role in the regulation of synaptic transmission and plasticity in the perforant pathway.

Characterization of [(3)H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using semliki forest virus vectors.

The binding properties of [(3)H]-LY354740 were characterized on rat metabotropic glutamate receptors mGlu2 and mGlu3 expressed in Chinese hamster ovary (CHO) cells using Semliki Forest virus vectors. The saturation isotherm gave K(D) values of 20+/-5 and 53+/-8 nM and B(max) values of 474+/-161 and 667+/-89 fmol/mg protein for mGlu2 and mGlu3 receptors, respectively. NMDA, CaCl(2), DHPG and kainate were inactive up to 1 mM, whereas LY341495, DCG IV and ibotenate inhibited [(3)H]-LY354740 binding with similar potencies on both receptors. L-CCG I, L-AP4, L-AP5, LY354740 and 1S,3R-ACPD were 2- to 4-fold more potent inhibitors of [(3)H]-LY354740 binding to mGlu2 than mGlu3 receptors. However, MPPG and L-AP3 had a 6-fold and DTT a 28-fold preference for mGlu2 over mGlu3. ZnCl(2), at 10 mM, inhibited more than 70% of [(3)H]-LY354740 binding to mGlu2 receptors. At the same concentration it did not affect significantly [(3)H]-LY354740 binding to mGlu3 receptors. On the contrary, glutamate, quisqualate, EGLU and NAAG showed a 3-, 5-, 7- and 12-fold preference for mGlu3 over mGlu2. Finally, GTPgammaS, which partially inhibited the binding on mGlu2 receptors, was inactive to inhibit [(3)H]-LY354740 binding on mGlu3 receptors.

mGluR-evoked augmentation of receptor-mediated cyclic AMP formation in neonatal and adult rat striatum.

1. The effects of selective agonists at group I, II and III metabotropic glutamate receptors (mGluRs) on adenosine A2 receptor-mediated cyclic AMP formation were compared in cross-chopped slices of adult and neonatal (8 days old) rat striatum, in the presence of 1 u ml(-1) adenosine deaminase. 2. The group II selective agonist, (2S,1R,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), elicited a potentiation of 5'-N-ethylcarboxamidoadenosine (NECA)-stimulated cyclic AMP production with similar potencies in adult (EC50 value 122 +/- 35 nM) and neonatal (EC50 value 285 +/-6 nM) brain. In contrast, the group I selective agonist (S)-dihydroxyphenylglycine ((S)-DHPG) augmented the NECA cyclic AMP response in neonatal striatum (EC50 value 9 +/- 1 microM), but at a concentration of 100 microM, (S)-DHPG failed to affect the NECA response in adult striatal slices. 3. The potentiation evoked by (S)-DHPG was specific for group I mGluRs as (2S,3S,4S,)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), a group II antagonist, was ineffective on the (S)-DHPG (100 microM) response at a concentration (500 microM) which reversed a similar augmentation elicited by DCG-IV (300 nM). Furthermore, a protein kinase C inhibitor (Ro 31-8220, 10 microM) markedly reversed the effect of (S)-DHPG without affecting the response to DCG-IV. 4. The mGluR agonist (2S,3S,4S,)-alpha-(carboxycyclopropyl)glycine (L-CCG-I), elicited a greater potentiation of NECA-stimulated cyclic AMP production in neonatal striatum in comparison with that observed in adult rat brain. Moreover, EC50 values obtained from adult and neonatal striatum were 2 +/-1 microM and 9 +/-1 microM, respectively. These differences in potency might reflect co-activation of both group I and group II mGluRs by L-CCG-I in neonatal striatum. 5. Distinct patterns of mGluR expression in various brain areas might account for previous conflicting data on the nature of the mGluR able to evoke such potentiated responses.

Effect of the umami peptides on the ligand binding and function of rat mGlu4a receptor might implicate this receptor in the monosodium glutamate taste transduction.

1. The effect of several metabotropic ligands and di- or tripeptides were tested on the binding of [3H]-L(+)-2-amino-4-phosphonobutyric acid ([3H]-L-AP4) on rat mGlu4 receptor. For selected compounds, the functional activity was determined on this receptor using the guanosine-5'[gamma-35S]-thiotriphosphate [gamma-35S]-GTP binding assay. 2. Using the scintillation proximity assay, [3H]-L-AP4 saturation analysis gave binding parameters K(D) and Bmax values of 150 nM and 9.3 pmoles mg-1 protein, respectively. The specific binding was inhibited concentration-dependently by several mGlu receptor ligands, and their rank order of affinity was established. 3. Several peptides inhibited the [3H]-L-AP4 binding with the following rank order of potency: glutamate-glutamate>glutamate-glutamate-leucine=aspartate - glutamate>>glutamate - glutamate-aspartate>lactoyl-glutamate>>aspartate-aspartate. Aspartate-phenylalanine-methyl ester (aspartame) was inactive up to 1 mM and guanosine-5'-monophosphate and inosine-5'-monophosphate were inactive up to 100 micronM. 4. The [gamma-35S]-GTP binding functional assay was used to determine the agonist activities of the different compounds. For the rat mGlu4 agonists, L-AP4 and L-glutamate, the correlation between their occupancy and activation of the receptor was close to one. The peptides, Glu-Glu, Asp-Glu and Glu-Glu-Asp failed to stimulate the [gamma-35S]-GTP binding at receptor occupancy greater than 80% and Glu-Glu-Leu appeared to be a weak partial agonist. These peptides did not elicit a clear dose-dependent umami perception. However, Glu-lac showed a good correlation between its potency to stimulate the [gamma-35S]-GTP binding and its affinity for displacement of [3H]-L-AP4 binding. These data are in agreement with the peptide taste assessment in human subjects, which showed that the acid derivatives of glutamate had characteristics similar to umami.

Characterization of [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)glycine ([3H]-DCG IV) binding to metabotropic mGlu2 receptor-transfected cell membranes.

1. The binding of the new selective group II metabotropic glutamate receptor radioligand, [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine ([3H]-DCG IV), was characterized in rat mGlu2 receptor-transfected CHO cell membranes. 2. [3H]-DCG IV binding was pH-dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a Kd value of 160 nM and a Bmax value of 10 pmol mg(-1) protein. Binding was not sensitive to Na+-dependent glutamate uptake blockers or Cl-dependent glutamate binding inhibitors. Furthermore, up to concentrations of 1 mM, the glutamate ionotropic receptor agonists, N-methyl-D-aspartic acid (NMDA), (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate, did not affect [3H]-DCG IV binding. 3. Of the compounds observed to inhibit [3H]-DCG IV binding, the most potent were the recently described selective group II agonist, (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY 354740; Ki value 16 nM) and antagonist, 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl) propanoic acid (LY 341495; Ki value 19 nM). As expected, for a G-protein-coupled receptor, guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) inhibited [3H]-DCG IV binding in a concentration-dependent manner, with an IC50 value of 12 nNM. 4. A highly significant correlation was observed between the potencies of compounds able to inhibit [3H]-DCG IV binding and potencies obtained for agonist activity in a GTPgamma35S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu2 receptors, including L(+)-2-amino-3-phosphonopropionic acid (L-AP3), L(+)-2-amino-5-phosphonopentanoic acid (L-AP5), 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP), N-acetyl-L-aspartyl-L-glutamic acid (NAAG) and (RS)-alpha-methylserine-O-phosphate (MSOP).

Radiolabelling of the human 5-HT2A receptor with an agonist, a partial agonist and an antagonist: effects on apparent agonist affinities.

Previous work has shown that 5-hydroxytryptamine (5-HT)2A receptors can be radiolabelled with various radioligands, including partial agonists, such as [125I]-DOI and [3H]-DOB, and antagonists, such as [3H]-ketanserin and [3H]-spiperone. Because 5-HT has high affinity for the 5-HT2A receptor when displacing [3H]-DOB, the purpose of the present study was to determine whether or not the receptor could be labelled with [3H]-5-HT and what would be the effect of labelling the receptor with various radioligands having differing efficacies at the receptor. Consequently, the human 5-HT2A receptor stably expressed in NIH 3T3 cells was radiolabelled with the endogenous agonist [3H]-5-HT, the partial agonist [3H]-DOB, and the antagonist [3H]-ketanserin. The receptor could be radiolabelled with [3H]-5-HT with a Kd value of 1.3 +/- 0.1 nM and a Bmax value of 3461 +/- 186 fmoles/mg protein and the radiolabelling was sensitive to the stable guanosine 5'-triphosphate (GTP) analogue guanylyl-imidodiphosphate (GMP-PNP). Ketanserin labeled significantly more receptors (Kd = 1.1 +/- 0.1 nM: Bmax = 27,684 +/- 1500 fmoles/mg protein) than [3H]-DOB (Kd = 0.8 +/- 0.08 nM: Bmax = 8332 +/- 16 fmoles/mg protein) which, in turn, labelled significantly more receptors than [3H]-5-HT. The apparent affinity of antagonists did not change when the receptor was radiolabelled with either [3H]-agonists or [3H]-antagonists; however, agonists had a higher apparent affinity for [3H]-agonist-labeled receptors than for [3H]-antagonist-labeled receptors. Therefore, the apparent affinity of agonists for the 5-HT2A receptor estimated from displacement experiments depends on the intrinsic efficacy of the radioligand used.

Cloning and functional expression of alternative spliced variants of the human metabotropic glutamate receptor 8.

Two new spliced variants of the human metabotropic glutamate receptor 8 (HmGluR8), designated HmGluR8b and HmGluR8c, were identified in a human fetal brain cDNA library. The HmGluR8b and c differ from previously reported HmGluR8a by the out-of-frame insertions of 55-bp and 74-bp, respectively. The 55-bp insertion which contains a stop codon resulted in substitution of the last 16 amino acids in the C-terminus of HmGluR8a with 16 different amino acids in HmGluR8b. The 74-bp insertion introduces a frame shift in the predicted translation resulting in termination of the polypeptide before the putative seven transmembrane domains. Thus, the predicted HmGluR8c protein is 501 amino acids long and could represent a secreted isoform of the receptor. The pattern of mRNA expression of mGluR8 variants in human brain were analyzed by RT-PCR, Northern blot and in situ hybridization. Both HmGluR8a and b are expressed with similar abundance in fetal and adult brains. The in situ hybridization results indicate a predominantly glial cell expression of HmGluR8c in human brain. The three isoforms were transiently expressed in CHO cells from Semliki Forest Virus vectors. [3H]l-AP4 binding was performed on the cell membranes and the saturation curves showed the presence of a binding site with KD values of 249 and 182 nM and Bmax values of 13.6 and 10.5 pmoles/mg protein for HmGluR8a and b, respectively. For the six mGluR ligands studied, a similar rank order of potency was observed on both HmGluRa and b: l-AP4>l-SOP=l-CCG I>l-glutamate>DCG IV>LY 354740.

Reducing conditions differentially affect the functional and structural properties of group-I and -II metabotropic glutamate receptors.

We have examined the influence of reducing conditions on the activity of group-I or -II metabotropic glutamate receptors. In cultured cerebellar granule cells or in hippocampal slices, the reducing agent dithiothreitol (DTT) inhibited the stimulation of polyphosphoinositide (PPI) hydrolysis elicited by group-I mGlu receptor agonists without affecting responses to norepinephrine or carbamylcholine. Similarly, DTT reduced the increase in intracellular free Ca(2+) induced by glutamate in HEK-293 cells expressing mGlu5 receptors. In adult hippocampal slices, the selective group-II mGlu receptor agonist, (2S,1'R,2'R,3'R)-2-(2, 3-dicarboxycyclopropyl)glycine (DCG-IV) had no effect per se on PPI hydrolysis, but potentiated the response to quisqualate. Although DTT substantially attenuated the action of quisqualate, it did not affect the potentiation by DCG-IV, suggesting that group-II mGlu receptors are resistant to extracellular reduction. Accordingly, DTT did not affect the inhibition of forskolin-stimulated cAMP formation induced by maximally effective concentrations of group-II mGlu receptor agonists in hippocampal slices or in CHO cells expressing mGlu2 receptors. At structural level, DTT differentially affected the aggregation state of mGlu1a, -2/3 or -5 receptors. In immunoblots performed under non-reducing conditions, mGlu1a, -2/3 or -5 antibodies labeled exclusively a high-molecular weight band, corresponding to receptor dimers. Under reducing conditions, mGlu1a or -5 receptors were detected as monomers, whereas a large proportion of mGlu2/3 receptors was still present in a dimeric form. We conclude that reducing conditions differentially influence the aggregation state of group-I and -II mGlu receptors and suggest that dimerization affects the functional activity of native mGlu receptors.

Effect of metabotropic glutamate receptor activation on receptor-mediated cyclic AMP responses in primary cultures of rat striatal neurones.

Co-activation of group I metabotropic glutamate (mGlu) receptors and adenosine receptors resulted in an augmented cyclic AMP response in primary cultures of rat striatal neurones. L-glutamate and the selective group I agonist, (S)-dihydroxyphenylglycine (S-DHPG) evoked concentration-dependent potentiations of cyclic AMP accumulation stimulated by the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), with EC50 values of 3.41+/-0. 39 and 5.69+/-1.64 microM, respectively, and maximal augmentations of approximately 350% at concentrations of 100 microM. The S-DHPG potentiation was inhibited by group I mGlu receptor antagonists and a protein kinase C inhibitor, Ro 31-8220, implicating products of PI hydrolysis in this effect. Furthermore, L-glutamate and S-DHPG stimulated PI hydrolysis in striatal neuronal cultures with similar EC50 values to those observed for the augmentation of NECA cyclic AMP responses (5.19+/-1.18 and 3.78+/-1.42 microM, respectively). In situ hybridization and immunofluorescence techniques indicate that group I mGlu receptor-evoked potentiations are likely to be mediated via mGlu5 receptors, which are expressed at high levels in these cultures. In contrast to cross-chopped slices of neonatal rat striatum, of equivalent age, the group II mGlu receptor agonist, (2S, 2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) was without effect on NECA- or forskolin-stimulated cyclic AMP responses in primary striatal neuronal cultures. This lack of effect might be due to a low level of expression of group II mGlu receptors in cultured striatal neurones.

Multiple pathways for regulation of the KCl-induced [3H]-GABA release by metabotropic glutamate receptors, in primary rat cortical cultures.

In rat cortical primary cultures, group II- and III-metabotropic glutamate receptor-selective agonists concentration-dependently reduced KCl-induced [3H]GABA release, with IC50 values of 11 nM for LY354740, 80 nM for L(+)-2-amino-4-phosphonobutyric acid (L-AP4), 180 nM for DCG-IV, and 330 nM for L-SOP. The group II antagonists, LY341495 and EGLU, reversed the effect of LY354740, and the group III antagonist MTPG reversed the effect of L-AP4. In the presence of omega-conotoxin GVIA, LY354740 inhibited the remaining [3H]GABA release, whereas L-AP4 was inactive. In contrast, in the presence of nifedipine, L-AP4 inhibited the remaining [3H]GABA release, but LY354740 was no longer active. The PKA inhibitor, H89, blocked the effects of both L-AP4 and LY354740, whereas the PKC inhibitor Ro 31-8220 blocked only the effect of LY354740. Both Ro 31-8220 and H89 reduced the [3H]GABA release to 60% of control. In whole-cell, voltage-clamp experiments, LY354740 and L-AP4 inhibited voltage-gated calcium channel currents with IC50 values of 28 nM and 22 microM, respectively. The results suggest that, in these cells, KCl-induced [3H]GABA release is modulated by two different mechanisms, one involving group II receptors and a direct control of the Ca2+ channel activity, and the other mediated by group III receptors and possibly involving a regulation located downstream of the Ca2+ channel activation.

Evidence for a role of 5-HT1C receptors in the antiserotonergic properties of some antidepressant drugs.

A variety of antidepressants of different chemical classes were tested for their in vivo and in vitro activity at 5-HT1C receptors in the brain. Conventional tricyclic antidepressants (imipramine, desipramine, maprotiline, clomipramine, trimipramine, amitriptyline, nortriptyline, doxepin, amoxapine) as well as mianserin and trazodone were found to display high to low nanomolar affinity for 5-HT1C receptors. Antidepressants of other chemical classes and with other mechanisms of action (affecting amine uptake systems: fluoxetine, citalopram, sertraline, fluvoxamine, nomifensine, amineptine; or monoamine oxidase inhibitors: moclobemide, iproniazid) had negligible affinities (micromolar range) for 5-HT1C receptors, except fluoxetine. When tested in an in vivo rat model thought to reveal functional agonistic or antagonistic properties at 5-HT1C receptors, all antidepressants displaying high affinity for this receptor type (except clomipramine and trimipramine) were antagonists at 5-HT1C receptors. Antidepressants with a lower affinity for 5-HT1C receptors (except nomifensine) were inactive in this functional in vivo model. Taken together, these results suggest that antagonism at brain 5-HT1C receptors is a component of the antiserotonergic properties of a number of established antidepressants. In addition, the study confirmed that 5-HT1A receptors functionally interact with 5-HT1C receptors, which suggests that some degree of activity at 5-HT1A receptors may also be an important property for antidepressant activity.

Involvement of a cyclic-AMP pathway in group I metabotropic glutamate receptor responses in neonatal rat cortex.

3,5-Dihydroxyphenylglycine (DHPG), (S)-3-hydroxyphenylglycine and (S)-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG) stimulated phosphoinositide hydrolysis in neonatal rat cortical slices, but with lower maximal effect, in comparison with 2S,1'S,2'S-2-(2'-carboxycyclopropyl)glycine (L-CCG I) or (1S,3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid (1S,3R-ACPD). DHPG, 1S,3R-ACPD, and S-4C3HPG also evoked a rapidly desensitizing increase in [Ca2+]i in cortical layers of neonatal brain slices. (R,S)-alpha-methyl-4-tetrazolyl-phenylglycine (MTPG), and (R,S)-alpha-methyl-4-phosphono-phenylglycine (MPPG) inhibited the increase of phosphoinositide hydrolysis elicited by 1S,3R-ACPD but not that by R,S-DHPG. In contrast, the selective group II receptor agonist (1S,2S,5R,6S)-2-amino-bicyclo-[3.1.0]-hexane-2,6-dicarboxylate (LY 354740) potentiated the response of R,S-DHPG. Finally, 8-(4-chlorophenylthio)-cAMP, a membrane permeant analogue of cAMP, reversed the stimulatory effect of 1S,3R-ACPD and S-4C3HPG on phosphoinositide hydrolysis and [Ca2+]i mobilization, without affecting the response induced by R,S-DHPG. These data suggest that, in neonatal rat cortex, the activation of group II metabotropic glutamate receptors potentiates the phosphoinositide hydrolysis and [Ca2+]i responses mediated by group I metabotropic glutamate receptors.

Pharmacological characterization of interactions of RO 25-6981 with the NR2B (epsilon2) subunit.

We used ligand binding to ascertain whether the pharmacological actions of RO 25-6981 [(R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidinepropanol] match those of other NR2B (epsilon2) subunit specific agents. RO 25-6981 inhibited binding of 125I-MK801 [iodo-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine maleate] to receptors made from NR1a/epsilon2 but not NR1a/epsilon1. Increasing the concentration of spermidine did not change the efficacy of RO 25-6981 and minimally changed the IC(50) value. Chimeric epsilon1/epsilon2 receptors demonstrated that the structural determinants for high affinity actions of RO 25-6981 were contained completely within the first 464 amino acids, but no receptor retained wildtype features when the size of the epsilon2 component was decreased further. Epsilon1Q336R receptors were more inhibited by ifenprodil and RO 25-9681 than wildtype epsilon1 receptors in ligand binding assays but not in functional assays. Selected mutations of epsilon2E200 and epsilon2E201 also decreased the sensitivity of receptors to ifenprodil and RO 25-6981. These results suggest that RO 25-6981 shares structural determinants with ifenprodil and other modulators in the NR2B subunit.

L-AP4 inhibition of depolarization-evoked cGMP formation in rat cerebellum.

The effects of the group III mGluR agonist, L-2-amino-4-phosphonobutyrate (L-AP4), on depolarization-stimulated cGMP levels in adult rat cerebellar slices were determined. L-AP4 elicited a concentration-dependent, complete inhibition of cGMP formation stimulated by 4-aminopyridine (4-AP; 1 mM), yielding an IC50 value of 4.2 +/- 1.6 microM (n = 3). The 4-AP response was also reduced by the P-type Ca2+ channel toxins omega-conotoxin MVIIC (3 microM; 39 +/- 7% inhibition) and omega-Agatoxin IVA (30 nM; 53 +/- 4%), and was abolished in the absence of Ca2+ or in the presence of Co2+. The inhibitions of the 4-AP cGMP response by 10 microM L-AP4 and 30 nM omega-Agatoxin IVA were not additive, indicating that part of the actions of L-AP4 in the cerebellum involves the modulation of P-type Ca2+ channels.

Brain 5-HT1C receptors and antidepressants.

A variety of antidepressants of different chemical classes were tested for their in vivo and in vitro activity at 5-HT1C receptors in the brain. Conventional tricyclic antidepressants (imipramine, desipramine, maprotiline, clomipramine, trimipramine, amitriptyline, nortriptyline, doxepin, amoxapine, oxaprotiline) and two atypical antidepressants (mianserin and trazodone) were found to display affinity for 5-HT1C receptors in the nanomolar range. Antidepressants of other chemical classes and mechanisms of action (serotonin uptake inhibitors: fluoxetine, citalopram, sertraline, fluvoxamine; noradrenaline-dopamine uptake inhibitors: nomifensine, bupropion, amineptine; or monoamine oxidase inhibitors: moclobemide, iproniazid) had affinities in the micromolar range for 5-HT1C receptors, except fluoxetine. When tested in an in vivo functional model revealing agonistic or antagonistic properties at 5-HT1C receptors, all antidepressants displaying high affinity for this receptor type (except fluoxetine, clomipramine, trimipramine and oxaprotiline) were antagonists at 5-HT1C receptors. Antidepressants with lower 5-HT1C receptor affinity (except nomifensine) were inactive in this functional in vivo model. Antagonism at brain 5-HT1C receptors is a component of the antiserotonergic properties of a number of established antidepressants, especially of the tricyclic class.