The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site

This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.     

Identification of the 5-HT1A Receptor Binding Subunit in Rat Brain Membranes Using the Photoaffinity Probe [3H]8-Methoxy-2-[N-n-Propyl, N-3-(2-Nitro-4-Azidophenyl)Aminopropyl]Aminotetralin

The synthesis of a tritiated derivative of the 5-HT1A photoaffinity probe 8-methoxy-2-[N-n-propyl, N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin ([3H]8-methoxy-3'-NAP-amino-PAT) allowed the use of this probe for attempting the irreversible labeling of specific binding sites in rat brain membranes. Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of proteins solubilized from hippocampal microsomal membranes that had been incubated with 20 nM [3H]8-methoxy-3'-NAP-amino-PAT under UV light revealed a marked incorporation of 3H label into a 63-kilodalton protein termed PI. As expected of a possible correspondence between PI and 5-HT1A receptor binding sites, 3H labeling by the photoaffinity probe could be prevented by selective 5-HT1A ligands such as 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, buspirone, and gepirone and by N-ethylmaleimide, but not by the 5-HT2 antagonist ketanserin, noradrenaline- and dopamine-related drugs, monoamine oxidase inhibitors, and chlorimipramine. Furthermore, the regional and subcellular distributions of PI were identical to those of specific 5-HT1A binding sites. These results indicated that the binding subunit of the 5-HT1A receptor is a 63-kilodalton protein with a functionally important sulfhydryl group(s).     

Determination of 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiophen-2-yl]cyclopent-3-enecarboxylic acid (CP-191,166), an angiotensin II antagonist, in dog and rat plasma by high-performance liquid chromatography

A simple and precise high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of a novel angiotensin II antagonist, 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiopen-2-yl)cyclopent-3-enecarboxylic acid (CP-191,166, I), in dog and rat plasma. The internal standard (II, a saturated derivative of I) and analyte were extracted by liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Zorbax C₈ narrow-bore column with ultraviolet detection at 289 nm. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01–10.0 μg/ml (r²>0.99). In dog and rat plasma, intra- and inter-assay precision ranged from 0.00 to 3.36% and 0.00 to 4.95%, respectively. The average recoveries were similar (73%) for both I and II and the upper limit of quantification of I can be as high as 500 μg/ml. The method described has been successfully applied to the quantification of I in about 2000 dog and rat plasma samples over a nine-month period.     

[3H]N-[1-(2-Thienyl)cyclohexyl]-3,4-Piperidine ([3H]TCP) Binding in Human Frontal Cortex: Decreases in Alzheimer-Type Dementia

We studied [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine [( 3H]TCP) binding to human frontal cortex obtained at autopsy from 10 histologically normal controls and eight histopathologically verified cases with Alzheimer-type dementia (ATD). Extensively washed membrane preparations were used to minimize the effects of endogenous substances. In ATD frontal cortex, the total concentration (Bmax) of [3H]TCP binding sites was significantly reduced by 40-50%. The apparent dissociation constant (KD) values showed no significant change. The reduction in binding capacity was also apparent in Triton X-100-treated membrane preparations, and there was a linear correlation between the number of [3H]TCP binding sites and that of N-methyl-D-aspartate (NMDA)-sensitive [3H]glutamate binding sites. [3H]TCP binding sites spared in ATD brains retained the affinity for the ligand and the reactivity to NMDA, L-glutamate, and glycine. These results suggest that the primary change in NMDA receptor-ion channel complex in ATD brains is the reduction of its number, possibly reflecting the loss of neurons bearing these receptor complexes, and that the functional linkage within the receptor complexes spared in ATD brains remains normal.     

U18666A, a cholesterol-inhibition agent, modulates human neuronal nicotinic acetylcholine receptors heterologously expressed in SH-EP1 cell line

In this study, we evaluate the effects of (3β)‐3‐[2‐(diethylamino)ethoxy]androst‐5‐en‐17‐one dihydrochloride (U18666A), a cholesterol synthesis/transporter inhibitor, on selected human neuronal nicotinic acetylcholine receptors (nAChRs) heterologously expressed in the SH‐EP1 cell line using whole‐cell patch‐clamp recordings. The results indicate that with 2‐min pretreatment, U18666A inhibited different nAChR subtypes with a rank‐order of potency (IC₅₀ of whole‐cell peak current): α4β2 (8.0 ± 3.0 nM) > α3β2 (1.7 ± 0.4 μM) > α4β4 (26 ± 7.2 μM) > α7 (> 100 μM), suggesting this compound is more selective to α4β2‐nAChRs. Thus, the pharmacological profiles and mechanisms of U18666A acting on α4β2‐nAChRs were investigated in detail. U18666A suppresses both peak and steady state components of whole‐cell currents mediated by human α4β2‐nAChRs in response to nicotine. In nicotine‐induced concentration–response curves, U18666A reduces nicotine‐induced current at maximally effective agonist concentrations without influencing nicotine’s EC₅₀ value, suggesting a non‐competitive inhibition. U18666A‐induced inhibition of nAChR function is concentration‐, voltage‐, and use‐dependent, suggesting an open channel block. Taken into consideration of ～10 000‐fold enhancement of the potency of U18666A after 2‐min pre‐treatment, this compound also likely inhibits α4β2‐nAChRs through a close channel block. In addition, the U18666A‐induced inhibition in α4β2‐nAChRs is not mediated by either increased receptor endocytosis or altered cell cholesterol. These data indicate that U18666A is a potent antagonist of α4β2‐nAChRs and may be useful as a tool in the functional characterization and pharmacological profiling of nAChRs, as well as a potential candidate for smoking cessation.     

Density of NMDA-Coupled and Uncoupled 1-[1-(2-[3H]Thienyl)cyclohexyl]piperidine Recognition Sites in the Brain and Spinal Cord: Differential Effects of NMDA Agonists and Antagonists

Abstract: Binding of 1-[1-(2-[³H]thienyl)cyclohexyl]piperidine ([³H]TCP) to mouse brain and spinal cord membranes was studied using compounds selective for the NMDA-coupled 1-(1-phenylcyclohexyl)piperidine (PCP) and/or σ recognition sites. In both tissues, [³H]TCP labeled two populations of binding sites. Density of the low-affinity sites was approximately the same in both tissues, but the population of the high-affinity [³H]TCP sites was three times bigger in the brain than in the spinal cord. Self- and cross-displacement studies showed that the high-affinity [³H]TCP binding sites could be identical with NMDA receptor-coupled PCP sites, whereas the low-affinity [³H]TCP sites may be associated with σ binding sites in both tissues. The NMDA-coupled PCP sites labeled in the presence of 6.25 n M [³H]TCP constituted a much higher percentage of the total binding in the brain (75%) than in the spinal cord (44%). Consistent with this, reintroduction of glycine and glutamate significantly increased, but DA antagonists significantly inhibited [³H]TCP binding in the brain but not in the spinal cord. Together, these data suggest that a large component of [³H]TCP-labeled binding sites in the spinal cord may be associated with σ but not the NMDA receptor-coupled PCP sites.     

Biological activities and structure-activity relationship of substitution compounds of N-[2-(3-indolyl)ethyl]succinamic acid and N-[2-(1-naphthyl)ethyl]succinamic acid, derived from a new category of root-promoting substances, N-(phenethyl)succinamic acid analogs

In a previous study, it was demonstrated that N-(phenethyl)succinamic acid (PESA) derivatives form a new category of root-promoting substances which do not exhibit auxin-like activities, such as stem elongation and leaf epinasty (Soejima et al., 2000 [Plant Cell Physiol. 41s: 197]). In this study, N-[2-(3-indolyl)ethyl]succinamic acid (IESA) and N-[2-(1-naphthyl)ethyl]succinamic acid (NESA) were synthesized, and their biological activities were evaluated. In an adzuki root-promoting assay, IESA and NESA exhibited root-promoting activity equivalent to PESA. In adzuki stem elongation assays, elongation activity was not observed in the stem segments soaked in either an IESA or NESA aqueous solution, whereas the stem segments immersed in Indole-3-acetic acid (IAA) or 1-naphthylacetic acid (NAA) aqueous solution were clearly elongated. In an epinastic bending study, IAA and NAA exhibited leaf epinasty, whereas IESA and NESA did not, suggesting that the IESA and NESA derivatives belong to the same category of root-promoting substances as PESA derivatives and are different from auxin-like substances. In addition, eleven kinds of IESA derivatives and nineteen kinds of NESA derivatives were synthesized, and their root-promoting activities were measured. The activities of methyl ester derivatives were approximately three times higher than that of the acid compounds, with exceptions for some compounds. The partition coefficient (P) between 1-octanol and water for each IESA, NESA, and PESA derivative was measured in order to evaluate the hydrophobicity of their molecules and to determine their structure–activity relationship. The results indicate that the root-promoting activity of the acid compounds was significantly correlated with their hydrophobicity, whereas that of ester derivatives was not correlated.     

Enantioselective degradation of the herbicide mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid] by mixed and pure bacterial cultures

Abstract A consortium of three bacteria was isolated from top soil through their capacity to utilise the chlorinated, aromatic herbicide mecoprop as a single growth substrate. The consortium constituted a tight association of Alcaligenes denitrificans, Pseudomonas glycinea and Pseudomonas marginalis . The culture exclusively degraded the ( R )-(+)-isomer of the herbicide while the ( S )-(−)-enantiomer remained unaffected. The mecoprop-degrading community could also degrade 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid and racemic 2-phenoxypropionic acid. Initially, no single member of the consortium was able to degrade mecoprop as a pure culture but after prolonged incubation, A. denitrificans was able to grow on the herbicide as the sole source of carbon and energy.     

New synthesis of 6[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid and evaluation of the influence of adamantyl group on the DNA binding of a naphthoic retinoid

6[3-(1-Adamantyl)-4-methoxyphenyl]-2-naphthoic acid (Adapalene®), a synthetic aromatic retinoid specific for RARβ and RARγ receptors, has been prepared utilizing a Pd/C-mediated Suzuki coupling between 6-bromo-2-naphthoic acid and 4-methoxyphenyl boronic acid, followed by introduction of an adamantyl group in the position 3 of the formed 6-(4-methoxyphenyl)-2-naphthoic acid. The interaction of 6-(4-methoxyphenyl)-2-naphthoic acid/ethyl ester and the 3-adamantyl analogs with DNA was studied in aqueous solution at physiological conditions by UV–vis spectroscopy. The calculated binding constants K_ligand–DNA ranged between 1.1 × 10⁴ M⁻¹ and 1.1 × 10⁵ M⁻¹, the higher values corresponding to those of the adamantylated compounds. Molecular modeling studies have emphasized that the intercalative binding of adapalene and its derivatives to DNA is mainly stabilized by hydrophobic interactions related to the presence of the adamantyl group.     

Magnesium complexes of the antiviral 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) and of its 1-, 3-, and 7-deaza analogues in aqueous solution

 The stability constants of the 1 : 1 complexes formed between Mg²⁺ and the anions of the N1, N3, and N7 deaza derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PA^2–), i.e., of Mg(H;PA)⁺ and Mg(PA), were determined by potentiometric pH titration in aqueous solution (25  °C; I=0.1 M, NaNO₃) and compared with previous results [Sigel H, et al. (1992) Helv Chim Acta 75 : 2634–2656], obtained under the same conditions, for the corresponding complexes of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA^2–) and (phosphonomethoxy)ethane (PME^2–). Based on the analysis of a microconstant scheme it is concluded that in the monoprotonated complexes, Mg(H;PA)⁺, Mg²⁺ is coordinated to a significant part at the nucleobase, H⁺ being at the phosphonate group. By making use of log K ^Mg _Mg(R-PO3) versus pK ^H _H(R-PO3) straight-line plots (also obtained previously; see above) for simple phosphonates and phosphate monoesters, it is shown that all the Mg(PA) complexes, including those with PMEA^2– and PME^2–, are more stable than expected on the basis of the basicity of the ―PO^2– ₃ group. This proves that, to some extent, five-membered chelates, Mg(PA)_cl/O, involving the ether oxygen of the ―CH₂―O―CH₂―PO^2– ₃ chain are formed; their formation degree amounts to about 30–40% in equilibrium with the isomer having only a phosphonate-Mg²⁺ coordination. In the case of Mg(1-deaza-PMEA), probably a further isomer occurs in which also N3 of the nucleobase participates. The different properties between the Mg(PA) species and the Mg(AMP) complex are discussed. Received: 26 January 1998 / Accepted: 19 May 1998     

The GTP-Insensitive Component of High-Affinity [3H]8-Hydroxy-2-(Di-n-Propylamino)tetralin Binding in the Rat Hippocampus Corresponds to an Oxidized State of the 5-Hydroxytryptamine1A Receptor

Previous studies on central 5-hydroxytryptamine1A (5-HT1A) receptors have consistently shown the existence of a GTP-insensitive component of agonist binding, i.e., binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) that persists in the presence of 0.1 mM GTP or guanylylimidodiphosphate (GppNHp). The molecular basis for this apparent heterogeneity was investigated pharmacologically and biochemically in the present study. The GppNHp-insensitive component of [3H]8-OH-DPAT binding increased spontaneously by exposure of rat hippocampal membranes or their 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate-soluble extracts to air; it was reduced by preincubation of solubilized 5-HT1A binding sites in the presence of dithiothreitol and, in contrast, reversibly increased by preincubation in the presence of various oxidizing reagents like sodium tetrathionate or hydrogen peroxide. In addition, exposure of hippocampal soluble extracts to short-cross-linking reagents specific for thiols produced an irreversible increase in the proportion of GppNHp-insensitive over total [3H]8-OH-DPAT binding. The pharmacological properties of this GppNHp-insensitive component of [3H]8-OH-DPAT binding were similar to those of 5-HT1A sites in the absence of nucleotide. Sucrose gradient sedimentation of solubilized 5-HT1A binding sites treated by dithiothreitol or sodium tetrathionate showed that oxidation prevented the dissociation by GTP of the complex formed by the 5-HT1A receptor binding subunit (R[5-HT1A]) and a guanine nucleotide-binding protein (G protein). Moreover, the oxidation of -SH groups by sodium tetrathionate did not prevent the inactivation of [3H]8-OH-DPAT specific binding by N-ethylmaleimide, in contrast to that expected from an interaction of both reagents with the same -SH groups on the R[5-HT1A]-G protein complex. These data suggest that the appearance of GTP-insensitive [3H]8-OH-DPAT specific binding occurs as a result of the (spontaneous) oxidation of essential -SH groups (different from those preferentially inactivated by N-ethylmaleimide) on the R[5-HT1A]-G protein complex.     

[3H]8-Hydroxy-2-(Di-n-Propylamino)Tetralin Binding to Pre- and Postsynaptic 5-Hydroxytryptamine Sites in Various Regions of the Rat Brain

The specific binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([ 3H]8-OH-DPAT) to 5-hydroxytryptamine (5-HT)-related sites was investigated in several regions of the rat brain. Marked differences were observed in the characteristics of binding to membranes from hippocampus, striatum, and cerebral cortex. Hippocampal sites exhibited the highest affinity (KD approximately 2 nM) followed by the cerebral cortex (KD approximately 6 nM) and the striatum (KD approximately 10 nM). Ascorbic acid inhibited specific [3H]8-OH-DPAT binding in all three regions but millimolar concentrations of Ca2+, Mg2+, and Mn2+ enhanced specific binding to hippocampal membranes, whereas only Mn2+ increased it in the cerebral cortex and all three cations inhibited specific binding to striatal membranes. Guanine nucleotides (0.1 mM GDP, GTP) inhibited binding to hippocampal and cortical membranes only. As intracerebral 5,7-dihydroxytryptamine markedly decreased [3H]8-OH-DPAT binding sites in the striatum, but not in the hippocampus, the striatal sites appear to be on serotoninergic afferent fibers. In contrast, in the hippocampus the sites appear to be on postsynaptic 5-HT target cells, as local injection of kainic acid decreased their density. Both types of sites appear to be present in the cerebral cortex. The postsynaptic hippocampal [3H]8-OH-DPAT binding sites are probably identical to the 5-HT1A subsites, but the relationship between the presynaptic binding sites and the presynaptic autoreceptors controlling 5-HT release deserves further investigation.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

GABA transporters mediate glycine release from cerebellum nerve endings: roles of Ca(2+)channels, mitochondrial Na(+)/Ca(2+) exchangers, vesicular GABA/glycine transporters and anion channels

GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [(3)H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [(3)H]glycine release were characterized. GABA stimulated [(3)H]glycine release in a concentration-dependent manner (EC(50) = 8.26 μM). The GABA-evoked release was insensitive to GABA(A) and GABA(B) receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca(2+) entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca(2+)-independent release involved mitochondrial Ca(2+), as it was prevented by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca(2+) did not trigger exocytotic release because the [(3)H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [(3)H]glycine so that less [(3)H]glycine can become cytosolic when GABA taken up exchanges with [(3)H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [(3)H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca(2+)-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition.     

Some characteristics of two azoreductase systems in rat liver. Relevance to the activity of 2-[4'-di(2"-bromopropyl)-aminophenylazo]benzoic acid (CB10-252), a compound possessing latent cytotoxic activity.

The system involved in the reduction of 2-[4'-di(2'-bromopropyl) aminophenylazolbenzoic acid (CB10-252), an agent designed for treating primary liver cell cancer, has been demonstrated to be localised mainly in the 108 000 X g supernatant fraction of rat liver homogenate. It is also present in other organs particularly in the spleen. DAB-azoreductase as shown previously is present almost entirely in the microsomal fraction and is found in high concentration only in liver. The pH maximum for CB10-252-azoreductase implying the importance of the 2'-carboxyl group in determining substrate specificity. The use of enzyme inhibitors and other additives showed that CB10-252 WAS NOT AXANTHINE OXIDASE OR DIHYDROFOLATE REDUCTASE. Its activity was not affected by carbon monoxide, phenobarbitone (PB), or 3-methylcholanthrene (MC) pretreatment. Enhancement of the activity by ferrous ions and FAD indicated that at least part of the reduction system could involve a flavoprotein with FAD as the prosthetic group. The activity of CB10-252-azoreductase and methylred-azoreductase was reduced by menadione (vitamin K3), cyanide and propylgallate. A diaphorase preparation from pig heart reduced both CB10-252 and methylred with both NADPH- and NADH-generating systems.     

Identification of novel thiazolo[5,4-d]pyrimidine derivatives as human A1 and A2A adenosine receptor antagonists/inverse agonists

In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a free amino group at position-7. The new compounds were tested for their affinity and potency at human (h) A₁, A_2A, A_2B and A₃ adenosine receptors expressed in CHO cells. The results reveal that the higher affinity of these new set of thiazolopyrimidines is toward the hA₁ and hA_2A adenosine receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual A₁/A_2A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and a phenyl moiety at position 2, displayed the highest affinity (hA₁ K_i?=?10.2?nM; hA_2A K_i?=?4.72?nM) and behaved as a potent A₁/A_2A antagonist/inverse agonist (hA₁ IC₅₀?=?13.4?nM; hA_2A IC₅₀?=?5.34?nM).     

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.     

Molecular probes for the A2A adenosine receptor based on a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine scaffold

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high affinity and selectivity as antagonists for the human A_2A adenosine receptor (AR). We extended ether-linked chain substituents at the p-position of the phenyl group using optimized O-alkylation. The conjugates included an ester, carboxylic acid and amines (for amide condensation), an alkyne (for click chemistry), a fluoropropyl group (for ¹⁸F incorporation), and fluorophore reporter groups (e.g., BODIPY conjugate 14, K_i 15 nM). The potent and A_2AAR-selective N-aminoethylacetamide 7 and N-[2-(2-aminoethyl)-aminoethyl]acetamide 8 congeners were coupled to polyamidoamine (PAMAM) G3.5 dendrimers, and the multivalent conjugates displayed high A_2AAR affinity. Theoretical docking of an AlexaFluor conjugate to the receptor X-ray structure highlighted the key interactions between the heterocyclic core and the binding pocket of the A_2AAR as well as the distal anchoring of the fluorophore. In conclusion, we have synthesized a family of high affinity functionalized congeners as pharmacological probes for studying the A_2AAR.     

Different mechanisms involved in apoptosis following exposure to benzo[a]pyrene in F258 and Hepa1c1c7 cells

The present study compares and elucidates possible mechanisms why B[a]P induces different cell signals and triggers apparently different apoptotic pathways in two rather similar cell lines (hepatic epithelial cells of rodents). The rate and maximal capacity of metabolic activation, as measured by the formation of B[a]P-tetrols and B[a]P-DNA adducts, was much higher in mouse hepatoma Hepa1c1c7 cells than in rat liver epithelial F258 cells due to a higher induced level of cyp1a1. B[a]P increased intracellular pH in both cell lines, but this change modulated the apoptotic process only in F258 cells. In Hepa1c1c7 cells reactive oxygen species (ROS) production appeared to be a consequence of toxicity, unlike F258 cells in which it was an initial event. The increased mitochondrial membrane potential found in F258 cells was not observed in Hepa1c1c7 cells. Surprisingly, F258 cells cultured at low cell density were somewhat more sensitive to low (50nM) B[a]P concentrations than Hepa1c1c7 cells. This could be explained partly by metabolic differences at low B[a]P concentrations. In contrast to the Hepa1c1c7 model, no activation of cell survival signals including p-Akt, p-ERK1/2 and no clear inactivation of pro-apoptotic Bad was observed in the F258 model following exposure to B[a]P. Another important difference between the two cell lines was related to the role of Bax and cytochrome c. In Hepa1c1c7 cells, B[a]P exposure resulted in a "classical" translocation of Bax to the mitochondria and release of cytochrome c, whereas in F258 cells no intracellular translocation of these two proteins was seen. These results suggest that the rate of metabolism of B[a]P and type of reactive metabolites formed influence the resulting balance of pro-apoptotic and anti-apoptotic cell signaling, and hence the mechanisms involved in cell death and the chances of more permanent genetic damage.     

Differential effects of cholesterol and 7-dehydrocholesterol on ligand binding of solubilized hippocampal serotonin1A receptors: implications in SLOS

The serotonin1A receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive, behavioral, and developmental functions. Solubilization of the hippocampal serotonin1A receptor by 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) is accompanied by loss of membrane cholesterol which results in a reduction in specific agonist binding activity. Replenishment of cholesterol to solubilized membranes restores the cholesterol content of the membrane and significantly enhances specific agonist binding activity. In order to test the stringency of the requirement of cholesterol in this process, we solubilized native hippocampal membranes followed by replenishment with 7-dehydrocholesterol (7-DHC). 7-DHC is an immediate biosynthetic precursor of cholesterol differing only in a double bond at the 7th position in its sterol ring. Our results show, for the first time, that replenishment of solubilized hippocampal membranes with 7-DHC does not restore ligand binding activity of the serotonin1A receptor, in spite of recovery of the overall membrane order. This observation shows that the requirement for restoration of ligand binding activity is more stringent than the requirement for the recovery of overall membrane order. These novel results have potential implications in understanding the interaction of membrane sterols with this important neuronal receptor under pathogenic conditions such as the Smith-Lemli-Opitz syndrome.