Aida I. Sacaan

2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist.

In the present paper we describe 2-methyl-6-(phenylethynyl)-pyridine (MPEP) as a potent, selective and systemically active antagonist for the metabotropic glutamate receptor subtype 5 (mGlu5). At the human mGlu5a receptor expressed in recombinant cells, MPEP completely inhibited quisqualate-stimulated phosphoinositide (PI) hydrolysis with an IC50 value of 36 nM while having no agonist or antagonist activities at cells expressing the human mGlu1b receptor at concentrations up to 30 microM. When tested at group II and III receptors, MPEP did not show agonist or antagonist activity at 100 microM on human mGlu2, -3, -4a, -7b, and -8a receptors nor at 10 microM on the human mGlu6 receptor. Electrophysiological recordings in Xenopus laevis oocytes demonstrated no significant effect at 100 microM on human NMDA (NMDA1A/2A), rat AMPA (Glu3-(flop)) and human kainate (Glu6-(IYQ)) receptor subtypes nor at 10 microM on the human NMDA1A/2B receptor. In rat neonatal brain slices, MPEP inhibited DHPG-stimulated PI hydrolysis with a potency and selectivity similar to that observed on human mGlu receptors. Furthermore, in extracellular recordings in the CA1 area of the hippocampus in anesthetized rats, the microiontophoretic application of DHPG induced neuronal firing that was blocked when MPEP was administered by iontophoretic or intravenous routes. Excitations induced by microiontophoretic application of AMPA were not affected.

Molecular and functional characterization of recombinant human metabotropic glutamate receptor subtype 5.

We have isolated and characterized overlapping cDNAs that encode two isoforms of the human metabotropic glutamate receptor subtype 5 (hmGluR5). The deduced amino acid sequences of human and rat mGluR5a are 94.5% identical. However, a region in the putative cytoplasmic domain (SER926-ALA1121) displays significant sequence divergence. Genomic analysis of this region showed that the sequence divergence results from species-specific differences in the genomic sequences, not from alternative splicing. The distribution of mGluR5 mRNA in human brain was most strongly detected throughout the hippocampus, with moderate levels in the caudate-putamen, cerebral cortex, thalamus, and deep cerebellar nuclei, and at low levels in the cerebellar cortex. Activation of both hmGluR5a and hmGluR5b transiently expressed in Xenopus oocytes and HEK293 cells was coupled to inositol phosphate (InsP) formation and elevation of the intracellular free calcium ([Ca2+]i). The agonist rank order of potency for activating recombinant hmGluR5a receptors in either system was quisqualate > L-glutamate > 1S,3R-ACPD. Both the quisqualate stimulated InsP and [Ca2+]i were inhibited by (+)-MCPG. Recombinant human mGluR5a was also stably expressed in mouse fibroblast Ltk- cells, in which the efficacy and potency of quisqualate were unchanged for more than 30 cell passages.

Cloning and stable expression of the mGluR1b subtype of human metabotropic receptors and pharmacological comparison with the mGluR5a subtype

We isolated and characterized a cDNA encoding the human metabotropic glutamate receptor subtype 1b (hmGluR1b). In situ hybridization studies in human brain regions revealed a higher distribution of mGluR1 mRNA in the dentate gyrus of the hippocampus, the substantia nigra pars compacta and the Purkinje cell layer of the cerebellum compared to other regions studied. We established stable expression of recombinant hmGluR1b in L(tk-) mouse fibroblast and Chinese hamster ovary (CHO-dhfr-) cells. In both expression systems, agonist activation of hmGluR1b stimulated inositol phosphate (InsP) formation and elevation of the cytosolic free calcium ([Ca2+]i), and both responses were blocked by (S)-MCPG. The rank order of potency for agonists was quisqualate > glutamate > (1S,3R)-ACPD in both expression systems. Comparison of the agonist profiles of hmGluR1b and hmGluR5a, both stably expressed in L(tk-) cells, indicated the same rank order of potency (quisqualate > glutamate > or = (RS)-3,5-DHPG > or = (1S,3R)-ACPD), but each of the four agonists were more potent on hmGluR5a than on hmGluR1b. In antagonist studies, (S)-MCPG inhibited the agonist-induced InsP formation and elevation of [Ca2+]i in both hmGluR1b- and hmGluR5a-expressing cells. (S)-4CPG and (S)-4C3HPG both inhibited agonist responses only in hmGluR1b-expressing cells. However, in hmGluR5a-expressing cells the antagonist activity of (S)-4CPG and (S)-4C3HPG was dependent on the agonist used in the study, since they inhibited responses to glutamate but not to quisqualate. Stable cell lines expressing specific subtypes of human mGluRs represent valuable tools for the study of the mechanism of action of mGluRs at the molecular and cellular level and as screening targets for identification of subtype-selective agonists or antagonists.

Equilibrium analysis of [3H]TCP binding: effects of glycine, magnesium and N-methyl-D-aspartate agonists

It has been reported that glutamate can increase the binding of [3H]TCP to phencyclidine (PCP) receptors by an action on receptors which are selective for N-methyl-D-aspartate (NMDA). Recently this laboratory has reported that glycine and magnesium can amplify this effect of NMDA agonists in well-washed, lysed cortical membranes. Here we report that maximally effective concentrations of glutamate (10 microM), NMDA (300 microM), MgCl2 (300 microM) and glycine (10 microM) increase the affinity of the PCP receptor for [3H]TCP by approximately 4-fold in the absence of any change in the density of PCP receptors. However, in combination with glutamate, magnesium had the further effect of increasing the Bmax by about 75%. Finally, a synaptosomal P2 preparation, which had not been washed to minimize the concentration of endogenous effectors had a Bmax value similar to the well-washed preparation, but had a KD value 8-fold lower. These data indicate that the primary effect of NMDA agonists, glycine, and low concentrations of magnesium ions is to convert the PCP receptor from a low-affinity to a high-affinity state. These data are discussed in relation to the functional regulation of the NMDA ionophore.

6-hydroxydopamine lesion of rat nigrostriatal dopaminergic neurons differentially affects nicotinic acetylcholine receptor subunit mRNA expression

AbstractNicotinic acetylcholine receptor (nAChR) subunit mRNA expression in the rat substantia nigra (SN) was assayed by semiquantitative RT-PCR following 6-hydroxydopamine (6-OHDA) lesion of nigrostriatal dopaminergic neurons. Six months after unilateral injection of 6-OHDA or saline into the SN, total RNA was isolated from ipsilateral and contralateral tissue samples. RT-PCR amplifications were performed with template titration using primers specific for sequences encoding1.nAChR α2–α7 and β2–β4 subunits2.Glutamic acid decarboxylase3.Glyceraldehyde 3-phosphate dehydrogenase for normalization of template mass. PCR products specific for α3, α4, α5, α6, α7, β2, β3, and glutamic acid decarboxylase were detected in the reactions containing SN RNA. This is the first evidence that α7 may be expressed in the SN. α2 and β4 PCR products were not detected in SN reactions, although they were observed in hippocampus and thalamus control reactions. A comparison of ipsilateral and contralateral SN RT-PCR reaction products showed substantial decreases in α5, α6, and β3 product yields following 6-OHDA, but not sham treatment. Neither the SN of sham-lesioned rats nor the thalamus of 6-OHDA-lesioned rats yielded similar results, indicating that the effects observed in 6-OHDA-treated SN were not caused by local mechanical damage or a nonspecific response, respectively. Effects of 6-OHDA treatment on α3, α4, α7, β2, or glutamic acid decarboxylase product yields from SN samples were small or undetectable. The results suggest that α5, β6, and β3 subunit-encoding mRNAs are expressed at substantially higher levels in dopaminergic than in nondopaminergic cell bodies in the SN.

Conformationally restricted analogues of nicotine and anabasine

A series of conformationally restricted analogues of nicotine has been synthesized and evaluated as agonists of neuronal acetylcholine receptors. Compound 2 (SIB-1663), which selectively activated human recombinant alpha 2 beta 4 and alpha 4 beta 4 nAChRs, was shown to be active in animal models of Parkinsons disease and pain.

Profiling of trans-azetidine-2,4-dicarboxylic acid at the human metabotropic glutamate receptors mGlu1b, -2, -4a and -5a.

We have tested the two enantiomers of trans-azetidine-2,4-dicarboxylic acid, (2S,4S)-azetidine-2,4-dicarboxylic acid ((2S,4S)-ADA) and (2R,4R)-azetidine-2,4-dicarboxylic acid ((2R,4R)-ADA) for activity at the human metabotropic glutamate receptors mGlu1b, mGlu2, mGlu4a and mGlu5a expressed in mammalian cells. In Chinese hamster ovary (CHO) cells expressing human mGlu2 receptors, 500 microM (2S,4S)-ADA inhibited forskolin-stimulated cAMP accumulation by 33 +/- 3% while 100 microM (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid induced an inhibition by 66 +/- 5%. The (2R,4R)-ADA enantiomer was inactive at human mGlu2 receptors. In CHO cells expressing human mGlu4a receptors, 10 microM L-AP4 inhibited forskolin-stimulated cAMP levels by 37 +/- 4% whereas both ADA enantiomers of trans-azetidine-2,4-dicarboxylic acid (500 microM) had no such effect. In CHO cells expressing human mGlu1b receptors and L cells expressing human mGlu5a receptors, both enantiomers, applied at 500 microM or 1 mM, were ineffective in stimulating inositolmonophosphate accumulation and did not affect quisqualate-stimulated inositolmonophosphate accumulation. We conclude that (2S,4S)-azetidine-2,4-dicarboxylic acid is a weak human mGlu2 receptor agonist and that (2R,4R)-azetidine-2,4-dicarboxylic acid is inactive at human mGlu2 receptors. Trans-azetidine-2,4-dicarboxylic acid has no significant agonistic effect on human mGlu4a receptors and neither agonistic nor antagonistic effects on human mGlu1b and mGlu5a receptors.

In vivo pharmacological characterization of (±)-4-[2-(1-methyl-2-pyrrolidinyl)ethyl]thiophenol hydrochloride (SIB-1553A), a novel cholinergic ligand: microdialysis studies

SIB-1553A ((+/-)-4-[2-(1-methyl-2-pyrrolidinyl)ethyl]thiophenol HCl) is a neuronal nicotinic acetylcholine receptor (nAChR) ligand which is active in rodent and primate models of cognition. In functional assays, SIB-1553A exhibits marked subtype selectivity for nAChRs as compared to nicotine. In addition SIB-1553A also exhibits affinities to histaminergic (H3) and serotonergic (5-HT1 and 5HT2) receptors and sigma binding sites. In the present investigation, we characterized SIB-1553A-induced neurotransmitter release in vivo. Following subcutaneous injection (s.c., 10 mg/kg), SIB-1553A rapidly entered the brain achieving concentration of approximately 20 microM 15 min post-injection and was eliminated from plasma with a terminal half-life of approximately 32 min. In freely moving rats, SIB-1553A (1-40 mg/kg, s.c.), markedly increased ACh release in the hippocampus and prefrontal cortex. In both regions, the magnitude of SIB-1553A-induced ACh release was greater than that seen with the prototypical nAChR agonist, nicotine (0.4 mg/kg, s.c.). Both isomers of SIB-1553A induced similar levels of increase in hippocampal ACh release. Increased hippocampal ACh release was also observed following oral administration of SIB-1553A (40 mg/kg) or after local perfusion into the hippocampus (1 mM). SIB-1553A-induced hippocampal ACh release was significantly attenuated by two nAChR antagonists, mecamylamine (MEC) and dihydro-beta-erythroidine (DHbetaE), and by the dopamine (DA) (D1) antagonist, SCH-23390, arguing that ACh release, in part, involves activation of nAChRs and a permissive DA synapse. In contrast to its robust effects on ACh release, SIB-1553A (40 mg/kg, s.c.) modestly increased striatal DA release (approximately 180% of baseline). Due to the proposed role of cholinergic pathways in learning and memory, the neurochemical profile of SIB-1553A suggests a potential for it to treat cognitive dysfunction.

(S)-4-carboxy-3-hydroxyphenylglycine activates phosphatidyl inositol linked metabotropic glutamate receptors in different brain regions of the neonatal rat

In the present investigation, effects of several agonists and antagonists of metabotropic glutamate receptors (mGluRs) which are coupled to phosphatidyl inositol (PI) hydrolysis were evaluated in slices of neonatal rat hippocampus, striatum, cortex and cerebellum. The rank order of potency of agonists in the PI hydrolysis assay was identical in all brain regions: quisqualic acid (Quis) > (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG) > 1S, 3R-aminocyclopentane dicarboxylic acid (1S,3R-ACPD) >> L-glutamate (Glu). All agonists were equiefficacious in the four brain regions tested. The responses to 3,5-DHPG, a highly selective Class I mGluR agonist, were attenuated by (S)-4-carboxyphenylglycine ((S)-4CPG), (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG) and 1-aminoindan-1,5-dicarboxylic acid (UPF-523) with a rank order of potency of (+)-MCPG > or = (S)-4CPG > or = UPF-523 in the different brain regions. These results suggest little selectivity among these putative mGluR antagonists in the different brain regions studied. Interestingly, (S)-4-carboxy-3-hydroxyphenylglycine ((S)-4C3HPG), a compound reported to act as antagonist at Class I mGluRs, produced concentration-dependent increases in PI hydrolysis in all four brain regions suggesting that (S)-4C3HPG acts as an agonist. In striatum, hippocampus and cortex, (S)-4C3HPG was equiefficacious to Quis, 3,5-DHPG, 1S,3R-ACPD and Glu. However, in the cerebellum, (S)-4C3HPG displayed weak agonist activity (37% of that of a maximally effective concentration of Quis). The effects of (S)-4C3HPG in the PI hydrolysis assay appeared to be mediated by the activation of an mGluR subtype since it was significantly blocked by (S)-4CPG, an mGluR antagonist. In addition, the agonistic effects of (S)-4C3HPG appear to be unrelated to inhibition of [3H]-Glu uptake into rat hippocampal or cerebellar synaptosomes. These results demonstrate a unique pharmacological profile of (S)-4C3HPG which can be interpreted as (S)-4C3HPG being a highly selective mGluR5 agonist or alternatively, that the effects of (S)-4C3HPG may be mediated through a novel Class I mGluR subtype(s), yet to be identified.

Pharmacological characterization of SIB-1663, a conformationally rigid analog of nicotine

SIB-1663 ([+/-]-7-methoxy-2,3,3a,4,5,6,9b-hexahydro-1H-pyrrolo-[3,2h]-isoquinoline) is a conformationally restricted analog of nicotine (NIC). SIB-1663 exhibited modest affinities to cholinergic receptors (K(i) values displacing the binding of [(3)H]-nicotine (NIC) and [(3)H]-quinuclinidylbenzilate (QNB) binding were 1.0+/-0.3 and 2.6+/-0.3 microM, respectively) with no appreciable affinity to nearly 40 other receptors. SIB-1663 selectively activated alpha2beta4 and alpha4beta4 human recombinant neuronal nicotinic acetylcholine receptors (nAChRs) with no appreciable activation of alpha4beta2 nAChRs, the presumed high-affinity nAChRs in rodent brain. These properties led us to examine profile of SIB-1663 in native preparations. SIB-1663 increased DA release from the rat striatum (STR) and olfactory tubercles and NE release from hippocampus, thalamus and prefrontal cortex (PFC). SIB-1663 was equiefficacious to NIC in STR-DA and PFC-NE release assays and less efficacious than NIC in other release assays. SIB-1663 appeared to be partial agonist in the hippocampal NE release assay. SIB-1663-induced neurotransmitter release in vitro was relatively insensitive to the nAChR antagonists, mecamylamine (MEC) or dihydro-beta-erythroidine (DHbetaE) providing equivocal evidence for nAChR activity. SIB-1663 (3-30 mg/kg, s.c.) increased locomotor activity in naive rats in a novel environment, increased ipsilateral turning in rats with unilateral 6-OHDA nigrostriatal lesion and increased withdrawal latencies in the tail-flick assay. The in vivo effects of SIB-1663 in these assays showed varying degrees of sensitivity to nAChR antagonists in that the locomotor activity and turning behavior of SIB-1663 were partially sensitive to MEC, whereas the antinociceptive activity was completely sensitive to MEC. In addition, SIB-1663 (s.c. or i.c.v.) attenuated antinociceptive activity NIC given by the same route suggesting a partial agonist activity. SIB-1663 also increased the retention of avoidance learning in normal rats when administered immediately after the acquisition session. These data indicate that SIB-1663, a conformationally restricted analog of NIC, with distinct nAChR subtype selectivity from NIC exhibits contrasting pharmacology with some of its in vivo actions involving nAChRs.