A novel, non-substrate-based series of glycine type 1 transporter inhibitors derived from high-throughput screening.

The synthesis and structure-activity relationships (SAR) of a series of indane and tetralin inhibitors of the type 1 glycine transporter, derived from a high-throughput screening (HTS) hit, are described. Key modifications that reduced the 5HT1B receptor affinity of the HTS hit and the P450 2D6 inhibition of subsequent analogues are delineated. While these modifications led to potent and selective GlyT1 inhibitors, HERG affinity and human microsomal clearance remain an issue for this series of compounds.

Inducible, brain region-specific expression of a dominant negative mutant of c-Jun in transgenic mice decreases sensitivity to cocaine.

Administration of cocaine induces the Fos family of transcription factors in the striatum, including the nucleus accumbens (NAc), a brain region important for the rewarding effects of addictive drugs. Several Fos proteins are induced acutely by cocaine, with stable isoforms of DeltaFosB predominating after chronic drug administration. However, it has been difficult to study the functional consequences of these Fos responses in vivo. Fos proteins heterodimerize with members of the Jun family to form active AP-1 transcription factor complexes. In the present study, we took advantage of this property and generated transgenic mice, using the tetracycline gene regulation system, that support the inducible, brain region-specific expression of a dominant negative mutant form of c-Jun (Deltac-Jun), which can antagonize the actions of Fos proteins. Expression of Deltac-Jun in the striatum and certain other brain regions of adult mice decreases their development of cocaine-induced conditioned place preference, suggesting reduced sensitivity to the rewarding effects of cocaine. In contrast, Deltac-Jun expression had no effect on cocaine-induced locomotor activity or sensitization. However, expression of Deltac-Jun in adult mice blocked the ability of chronic cocaine administration to induce three known targets for AP-1 in the NAc: the AMPA glutamate receptor subunit GluR2, the cyclin-dependent protein kinase Cdk5, and the transcription factor nuclear factor-kappaB (NFkappaB), without affecting several other proteins examined for comparison. Taken together, these results provide further support for an important role of AP-1-mediated transcription in some of the behavioral and molecular mechanisms underlying cocaine addiction.

Localization of splenic B cells activated for switch recombination by in situ hybridization with Igamma1 switch transcript and Rad51 probes.

B cells are activated for switch recombination by signals from Th cells, but the site at which this first occurs in vivo has yet to be identified. By in situ hybridization of splenic sections using riboprobes specific for the Igamma1 switch transcript and Rad51 mRNA, we have visualized B cells that are newly activated for switch recombination and characterized the spatial and temporal patterns of Igamma1 and Rad51 mRNA expression. Within 2 days after immunization with (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma-globulin, expression of Igamma1 switch transcripts and Rad51 mRNA was evident and was localized to B220+ B cells clustered within the T cell-rich periarteriolar lymphoid sheath (PALS) and surrounding follicles. By Ab staining, we have shown previously that cells switching from IgM to IgG expression can be visualized at 3 to 5 days postimmunization and colocalize to clusters of Rad51+ cells. Hybridization of adjacent sections with probes for Cmu and Cgamma1 mRNA now shows that switching from mu to gamma expression occurs within Rad51+ Igamma1+ regions of the PALS and peaks between days 3 and 5. Colocalized expression of Igamma1 and Rad51 transcripts was observed from days 2 through 12 of the immune response. Igamma1 and Rad51 transcripts were down-regulated but still detectable at 12 days postimmunization, when they were evident in peanut agglutinin-positive germinal center B cells. Taken together, these observations show that B cells are first activated for switch recombination in the T cell-rich PALS.

Rad51 expression and localization in B cells carrying out class switch recombination.

Rad51 is a highly conserved eukaryotic homolog of the prokaryotic recombination protein RecA, which has been shown to function in both recombinational repair of DNA damage and meiotic recombination in yeast. In primary murine B cells cultured with lipopolysaccharide (LPS) to stimulate heavy chain class switch recombination, Rad51 protein levels are dramatically induced. Immunofluorescent microscopy shows that anti-Rad51 antibodies stain foci that are localized within the nuclei of switching B cells. Immunohistochemical analysis of splenic sections shows that clusters of cells that stain brightly with anti-Rad51 antibodies are evident within several days after primary immunization and that Rad51 staining in vivo is confined to B cells that are switching from expression of IgM to IgG antibodies. Following switch recombination, B cells populate splenic germinal centers, where somatic hypermutation and clonal proliferation occur. Germinal center B cells are not stained by anti-Rad51 antibodies. Rad51 expression is therefore not coincident with somatic hypermutation, nor does Rad51 expression correlate simply with cell proliferation. These data suggest that Rad51, or a highly related member of the conserved RecA family, may function in class switch recombination.

Adenosine A1 receptor-mediated inhibition of cyclic AMP accumulation in type-2 but not type-1 rat astrocytes.

The effects of adenosine receptor-selective ligands on [3H]cyclic AMP accumulation have been investigated in type-1 and type-2 astrocyte-enriched cultures derived from neonatal rat forebrains. In type-1 astrocytes, 5'-N-ethylcarboxamidoadenosine (NECA) caused a concentration-dependent increase in [3H]cyclic AMP accumulation (EC50 = 1.2 microM) which was antagonised by pretreatment with either xanthine amine congener (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]-phenyl]- 1,3-dipropylxanthine, apparent Kd = 9 nM) or PD115,199 (N-[2-(dimethylamino)ethyl]-N-methyl-4-(1,3-dipropylxanthine) benzene sulphonamide, apparent Kd = 122 nM). In these cultures, N6-cyclopentyladenosine (CPA), did not affect forskolin- or isoprenaline-mediated elevations of [3H]cyclic AMP accumulation. These data indicate that type-1 astrocytes possess adenosine A2B but not adenosine A1 receptors coupled to adenylyl cyclase. In type-2 astrocyte-enriched cultures, 10 microM NECA caused significant elevations of [3H]cyclic AMP accumulation which were similarly inhibited by either 1 microM xanthine amine congener or 10 microM PD115,199 suggesting that they were primarily due to adenosine A2B receptor stimulation. However, CGS 21680 ((2-[[4-(2-carboxyethyl) phenethyl]-amino]adenosine-5'-N-ethylcarboxamide, 10 microM), also significantly increased [3H]cyclic AMP accumulation in type-2 astrocytes suggesting the additional presence of adenosine A2A receptors. Forskolin-mediated elevations of [3H]cyclic AMP accumulation in type-2 astrocytes were inhibited in a concentration-dependent manner by CPA. This effect was reversed by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microM), confirming the presence of adenosine A1 receptors negatively coupled to adenylyl cyclase in type-2 astrocytes.

Adenosine A1 receptor-mediated changes in basal and histamine-stimulated levels of intracellular calcium in primary rat astrocytes.

1. The effects of adenosine A1 receptor stimulation on basal and histamine-stimulated levels of intracellular free calcium ion concentration ([Ca2+]i) have been investigated in primary astrocyte cultures derived from neonatal rat forebrains. 2. Histamine (0.1 microM-1 mM) caused rapid, concentration-dependent increases in [Ca2+]i over basal levels in single type-2 astrocytes in the presence of extracellular calcium. A maximum mean increase of 1,468 +/- 94 nM over basal levels was recorded in 90% of type-2 cells treated with 1 mM histamine (n = 49). The percentage of type-2 cells exhibiting calcium increases in response to histamine appeared to vary in a concentration-dependent manner. However, the application of 1 mM histamine to type-1 astrocytes had less effect, eliciting a mean increase in [Ca2+]i of 805 +/- 197 nM over basal levels in only 30% of the cells observed (n = 24). 3. In the presence of extracellular calcium, the A1 receptor-selective agonist, N6-cyclopentyladenosine (CPA, 10 microM), caused a maximum mean increase in [Ca2+]i of 1,110 +/- 181 nM over basal levels in 30% of type-2 astrocytes observed (n = 53). The size of this response was concentration-dependent; however, the percentage of type-2 cells exhibiting calcium increases in response to CPA did not appear to vary in a concentration-dependent manner. A mean calcium increase of 605 +/- 89 nM over basal levels was also recorded in 23% of type-1 astrocytes treated with 10 microM CPA (n = 30). 4. In the absence of extracellular calcium, in medium containing 0.1 mM EGTA, a mean increase in [Ca2+]i of 504 +/- 67 nM over basal levels was recorded in 41% of type-2 astrocytes observed (n = 41) after stimulation with 1 microM CPA. However, in the presence of extracellular calcium, pretreatment with the A1 receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, for 5-10 min before stimulation with 1 microM CPA, completely antagonized the response in 100% of the cells observed. 5. In type-2 astrocytes, prestimulation with 10 nM CPA significantly increased the size of the calcium response produced by 0.1 microM histamine and the percentage of responding cells. Treatment with 0.1 microM histamine alone caused a mean calcium increase of 268 +/- 34 nM in 41% of the cells observed (n = 34). After treatment with 10 nM CPA, mean calcium increase of 543 +/- 97 nM was recorded in 100% of the cells observed (n = 33). 6. These data indicate that adenosine Al receptors couple to intracellular calcium mobilization and extracellular calcium influx in type-1 and type-2 astrocytes in primary culture. In addition, the simultaneous activation of adenosine Al receptors on type-2 astrocytes results in an augmentation of the calcium response to histamine H1 receptor stimulation.

Endogenous expression of histamine H1 receptors functionally coupled to phosphoinositide hydrolysis in C6 glioma cells: regulation by cyclic AMP.

1. The effects of histamine receptor agonists and antagonists on phospholipid hydrolysis in rat-derived C6 glioma cells have been investigated. 2. Histamine H1 receptor-stimulation caused a concentration-dependent increase in the accumulation of total [3H]-inositol phosphates in cells prelabelled with [3H]-myo-inositol. The rank order of agonist potencies was histamine (EC50 = 24 microM) > N alpha-methylhistamine (EC50 = 31 microM) > 2-thiazolylethylamine (EC50 = 91 microM). 3. The response to 0.1 mM histamine was antagonized in a concentration-dependent manner by the H1-antagonists, mepyramine (apparent Kd = 1 nM) and (+)-chlorpheniramine (apparent Kd = 4 nM). In addition, (-)-chlorpheniramine was more than two orders of magnitude less potent than its (+)-stereoisomer. 4. Elevation of intracellular cyclic AMP accumulation with forskolin (10 microM, EC50 = 0.3 microM), isoprenaline (1 microM, EC50 = 4 nM) or rolipram (0.5 mM), significantly reduced the histamine-mediated (0.1 mM) inositol phosphate response by 37%, 43% and 26% respectively. In contrast, 1,9-dideoxyforskolin did not increase cyclic AMP accumulation and had no effect on the phosphoinositide response to histamine. 5. These data indicate the presence of functionally coupled, endogenous histamine H1 receptors in C6 glioma cells. Furthermore, the results also indicate that H1 receptor-mediated phospholipid hydrolysis is inhibited by the elevation of cyclic AMP levels in these cells.

Adenosine A2B-receptor-mediated cyclic AMP accumulation in primary rat astrocytes.

1. The effects of adenosine receptor agonists and antagonists on the accumulation of cyclic AMP have been investigated in primary cultures of rat astrocytes. 2. Adenosine A2-receptor stimulation caused a concentration-dependent increase in the accumulation of [3H]-cyclic AMP in cells prelabelled with [3H]-adenine. The rank order of agonist potencies was 5'-N-ethylcarboxamidoadenosine (NECA; EC50 = 1 microM) > adenosine (EC50 = 5 microM) > 2-chloroadenosine (EC50 = 20 microM) >> CGS 21680 (EC50 > 10 microM). The presence of 0.5 microM dipyridamole, an adenosine uptake blocker, had no effect on the potency of adenosine. 3. The response to 10 microM NECA was antagonized in a concentration-dependent manner by the non-selective adenosine receptor antagonists, xanthine amine congener (apparent KD = 12 nM), PD 115,199 (apparent KD = 134 nM) and 8-phenyltheophylline (apparent KD = 126 nM). However, the A1-receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, had no significant effect on the responses to NECA or 2-chloroadenosine at concentrations up to 1 microM. 4. Stimulation of A1-receptors with the selective agonist, N6-cyclopentyladenosine, did not alter the basal accumulation of [3H]-cyclic AMP but inhibited a forskolin-mediated elevation of [3H]-cyclic AMP accumulation by a maximal value of 42%. This inhibition was fully reversed in the presence of 0.1 microM, 8-cyclopentyl-1,3-dipropylxanthine. 5. The time course for NECA-mediated [3H]-cyclic AMP accumulation was investigated. The results suggest that there is a substantial efflux of cyclic AMP from the cells in addition to the rapid and sustained elevation of intracellular cyclic AMP (5 fold over basal) which was also observed. 6. These data indicate that rat astrocytes in primary culture express an A2B-adenosine receptor coupled positively to adenylyl cyclase. Furthermore, the presence of A1-receptors negatively coupled to adenylyl cyclase appears to have no significant effect on the A2B-receptor-mediated cyclic AMP responses to NECA and 2-chloroadenosine.