Effect of apoE genotype and vitamin E on biomarkers of oxidative stress in cultured neuronal cells and the brain of targeted replacement mice

The aetiology of apoE4 genotype-Alzheimer's disease (AD) association are complex. The current study emphasizes the impact of apoE genotype and potential beneficial effects of vitamin E (VE) in relation to oxidative stress. Agonist induced neuronal cell death was examined 1) in the presence of conditioned media containing equal amounts of apoE3 or apoE4 obtained from stably transfected macrophages, and 2) after pretreatment with alpha- and gamma-tocopherol, and -tocotrienol. ApoE3 and apoE4 transgenic mice were fed a diet poor or rich in VE to study the interplay of both apoE genotype and VE status, on membrane lipid peroxidation, antioxidative enzyme activity and glutathione levels in the brain. Cytotoxicity of hydrogen peroxide and glutamate was higher in neuronal cells cultured with apoE4 than apoE3 conditioned media. VE pre-treatment of neurons counteracted the cytotoxicity of a peroxide challenge but not of nitric oxide. No significant effects of apoE genotype or VE supplementation were observed on lipid peroxidation or antioxidative status in the brain of apoE3 and apoE4 mice. VE protects against oxidative insults in vitro, however, no differences in brain oxidative status were observed in mice. Unlike in cultured cells, apoE4 may not contribute to higher neuronal oxidative stress in the brain of young targeted replacement mice.

Peptide inhibitors of G protein-coupled receptor kinases

G protein-coupled receptor kinases (GRKs) are regulatory enzymes involved in the modulation of seven-transmembrane-helix receptors. In order to develop specific inhibitors for these kinases, we synthesized and investigated peptide inhibitors derived from the sequence of the first intracellular loop of the beta(2)-adrenergic receptor. Introduction of changes in the sequence and truncation of N- and C-terminal amino acids increased the inhibitory potency by a factor of 40. These inhibitors not only inhibited the prototypical GRK2 but also GRK3 and GRK5. In contrast there was no inhibition of protein kinase C and protein kinase A even at the highest concentration tested. The peptide with the sequence AKFERLQTVTNYFITSE inhibited GRK2 with an IC50 of 0.6 mu M, GRK3 with 2.6 mu M and GRK5 with 1.6 mu M. The peptide inhibitors were non-competitive for receptor and ATP. These findings demonstrate that specific peptides can inhibit GRKs in the submicromolar range and suggest that a further decrease in size is possible without losing the inhibitory potency. (c) 2005 Published by Elsevier Inc.

Cannabinoids inhibit human keratinocyte proliferation through a non-CB1/CB2 mechanism and have a potential therapeutic value in the treatment of psoriasis

Background: Cannabinoids from cannabis (Cannabis sativa) are anti-inflammatory and have inhibitory effects on the proliferation of a number of tumorigenic cell lines, some of which are mediated via cannabinoid receptors. Cannabinoid (CB) receptors are present in human skin and anandamide, an endogenous CB receptor ligand, inhibits epidermal keratinocyte differentiation. Psoriasis is an inflammatory disease also characterised in part by epidermal keratinocyte hyper-proliferation. Objective: We investigated the plant cannabinoids Delta-9 tetrahydrocannabinol, cannabidiol, cannabinol and cannabigerol for their ability to inhibit the proliferation of a hyper-proliferating human keratinocyte cell line and for any involvement of cannabinoid receptors. Methods: A keratinocyte proliferation assay was used to assess the effect of treatment with cannabinoids. Cell integrity and metabolic competence confirmed using lactate-dehydrogenase and adenosine tri-phosphate assays. To determine the involvement of the receptors, specific agonist and antagonist were used in conjunction with some phytocannabinoids. Western blot and RT-PCR analysis confirmed presence of CB1 and CB2 receptors. Results: The cannabinoids tested all inhibited keratinocyte proliferation in a concentration-dependent manner. The selective CB2 receptor agonists JWH015 and BML190 elicited only partial inhibition, the non-selective CB agonist HU210 produced a concentration-dependent response, the activity of theses agonists were not blocked by either C81 /C82 antagonists. Conclusion: The results indicate that while CB receptors may have a circumstantial role in keratinocyte proliferation, they do not contribute significantly to this process. Our results show that cannabinoids inhibit keratinocyte proliferation, and therefore support a potential role for cannabinoids in the treatment of psoriasis. (c) 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Antipsychotic drug action: antagonism, inverse agonism or partial agonism

The positive, psychotic symptoms of schizophrenia can be treated by antipsychotic drugs and it has been assumed that these are antagonists at the D-2 and D-3 dopamine receptors in the brain. Recently, the D-2/D-3 partial agonist aripiprazole has been introduced as an antipsychotic drug. It has also been realized that, using in vitro assays, the other antipsychotic drugs are in fact inverse agonists at D-2/D-3 dopamine receptors. This raises questions about how these disparate drugs can achieve a similar clinical outcome. In this review, I shall consider the efficacies of these drugs in signalling assays and how these efficacies might affect treatment outcomes. It seems that the treatment outcome might depend on the overall level of cell stimulation, which is in turn dependent on the level of residual dopamine and the efficacy of the drug in signalling assays.

Signaling mechanisms of GPCR ligands

G protein-coupled receptors constitute one of the major classes of drug targets, so understanding the mechanisms of signaling through these receptors is of great importance. This review covers some of the recent advances in G protein-coupled receptor signaling. A high resolution structure of the beta(2)-adrenergic receptor has been reported, as well as several molecular switches involved in receptor activation. It has also been realised that receptors and G proteins and their subunits may not always separate upon receptor activation. The definition of the ability of these receptors to signal has been expanded considerably with the realisation that some signaling may occur independently of G proteins, that some signaling events may differ in their pharmacological profiles and that formation of heterodimers of these receptors may provide new avenues for both signaling and drug design.

G protein ss gamma subunits mediate presynaptic inhibition of transmitter release from rat superior cervical ganglion neurones in culture

The activation of presynaptic G protein-coupled receptors (GPCRs) is widely reported to inhibit transmitter release; however, the lack of accessibility of many presynaptic terminals has limited direct analysis of signalling mediators. We studied GPCR-mediated inhibition of fast cholinergic transmission between superior cervical ganglion neurones (SCGNs) in culture. The adrenoceptor agonist noradrenaline (NA) caused a dose-related reduction in evoked excitatory postsynaptic potentials (EPSPs). NA-induced EPSP decrease was accompanied by effects on the presynaptic action potential (AP), reducing AP duration and amplitude of the after-hyperpolarization (AHP), without affecting the pre- and postsynaptic membrane potential. All effects of NA were blocked by yohimbine and synaptic transmission was reduced by clonidine, consistent with an action at presynaptic alpha 2-adrenoceptors. NA-induced inhibition of transmission was sensitive to pre-incubation of SCGNs with pertussis toxin (PTX), implicating the involvement of G alpha(i)/(o)beta y subunits. Expression of G alpha transducin, an agent which sequesters G protein beta gamma (G beta y) subunits, in the presynaptic neurone caused a time-dependent attenuation of NA-induced inhibition. Injection of purified G beta gamma subunits into the presynaptic neurone inhibited transmission, and also reduced the AHP amplitude. Furthermore, NA-induced inhibition was occluded by pre-injection of G beta gamma subunits. The Ca2+ channel blocker Cd2+ mimicked NA effects on transmitter release. Cd2+, NA and G beta gamma subunits also inhibited somatic Ca2+ current. In contrast to effects on AP-evoked transmitter release, NA had no clear action on AP-independent EPSPs induced by hypertonic solutions. These results demonstrate that G beta gamma subunits functionally mediate inhibition of transmitter release by alpha 2-adrenoceptors and represent important regulators of synaptic transmission at mammalian presynaptic terminals.

Cannabinoids stimulate fibroblastic colony formation by bone marrow cells indirectly via CB2 receptors

Recently, the cannabinoid receptors CB1 and CB2 were shown to modulate bone formation and resorption in vivo, although little is known of the mechanisms underlying this. The effects of cannabinoids on mesenchymal stem cell (MSC) recruitment in whole bone marrow were investigated using either the fibroblastic colony-forming unit (CFU-f) assay or high-density cultures of whole bone marrow. Levels of the CB1 and CB2 receptors were assessed by flow cytometry. Treatment of CFU-f cultures with the endocannabinoid 2-arachidonylglycerol (2-AG) dose-dependently increased fibroblastic and differentiated colony formation along with colony size. The nonspecific agonists CP 55,940 and WIN 55,212 both increased colony numbers, as did the CB2 agonists BML190 and JWH015. The CB1-specific agonist ACEA had no effect, whereas the CB2 antagonist AM630 blocked the effect of the natural cannabinoid tetrahydrocannabivarin, confirming mediation via the CB2 receptor. Treatment of primary bone marrow cultures with 2-AG stimulated proliferation and collagen accumulation, whereas treatment of subcultures of MSC had no effect, suggesting that the target cell is not the MSC but an accessory cell present in bone marrow. Subcultures of MSCs were negative for CB1 and CB2 receptors as shown by flow cytometry, whereas whole bone marrow contained a small population of cells positive for both receptors. These data suggest that cannabinoids may stimulate the recruitment of MSCs from the bone marrow indirectly via an accessory cell and mediated via the CB2 receptor. This recruitment may be one mechanism responsible for the increased bone formation seen after cannabinoid treatment in vivo.

Modelling the interdependence between the stoichiometry of receptor oligomerization and ligand binding for a coexisting dimer/tetramer receptor system

Many G protein-coupled receptors have been shown to exist as oligomers, but the oligomerization state and the effects of this on receptor function are unclear. For some G protein-coupled receptors, in ligand binding assays, different radioligands provide different maximal binding capacities. Here we have developed mathematical models for co-expressed dimeric and tetrameric species of receptors. We have considered models where the dimers and tetramers are in equilibrium and where they do not interconvert and we have also considered the potential influence of the ligands on the degree of oligomerization. By analogy with agonist efficacy, we have considered ligands that promote, inhibit or have no effect on oligomerization. Cell surface receptor expression and the intrinsic capacity of receptors to oligomerize are quantitative parameters of the equations. The models can account for differences in the maximal binding capacities of radioligands in different preparations of receptors and provide a conceptual framework for simulation and data fitting in complex oligomeric receptor situations.

An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro

G-protein-coupled receptors are desensitized by a two-step process. In a first step, G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-activated receptors that subsequently bind to a second class of proteins, the arrestins. GRKs can be classified into three subfamilies, which have been implicated in various diseases. The physiological role(s) of GRKs have been difficult to study as selective inhibitors are not available. We have used SELEX (systematic evolution of ligands by exponential enrichment) to develop RNA aptamers that potently and selectively inhibit GRK2. This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation with an IC50 of 4.1 nM. Phosphorylation of rhodopsin catalyzed by GRK5 was also inhibited, albeit with 20-fold lower potency (IC50 of 79 nM). Furthermore, C13 reveals significant specificity, since almost no inhibitory activity was detectable testing it against a panel of 14 other kinases. The aptamer is two orders of magnitude more potent than the best GRK2 inhibitors described previously and shows high selectivity for the GRK family of protein kinases.

The phytocannabinoid Δ9-tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum

Background and purpose: The phytocannabinoid Delta(9)-tetrahydrocannabivarin (Delta(9)-THCV) has been reported to exhibit a diverse pharmacology; here, we investigate functional effects of Delta(9)-THCV, extracted from Cannabis sativa, using electrophysiological techniques to define its mechanism of action in the CNS. Experimental approach: Effects of Delta(9)-THCV and synthetic cannabinoid agents on inhibitory neurotransmission at interneurone-Purkinje cell (IN-PC) synapses were correlated with effects on spontaneous PC output using single-cell and multi-electrode array (MEA) electrophysiological recordings respectively, in mouse cerebellar brain slices in vitro. Key results: The cannabinoid receptor agonist WIN 55,212-2 (WIN55) decreased miniature inhibitory postsynaptic current (mIPSC) frequency at IN-PC synapses. WIN55-induced inhibition was reversed by Delta(9)-THCV, and also by the CB1 receptor antagonist AM251; Delta(9)-THCV or AM251 acted to increase mIPSC frequency beyond basal values. When applied alone, Delta(9)-THCV, AM251 or rimonabant increased mIPSC frequency. Pre-incubation with Delta(9)-THCV blocked WIN55-induced inhibition. In MEA recordings, WIN55 increased PC spike firing rate; Delta(9)-THCV and AM251 acted in the opposite direction to decrease spike firing. The effects of Delta(9)-THCV and WIN55 were attenuated by the GABA(A) receptor antagonist bicuculline methiodide. Conclusions and implications: We show for the first time that Delta(9)-THCV acts as a functional CB1 receptor antagonist in the CNS to modulate inhibitory neurotransmission at IN-PC synapses and spontaneous PC output. Delta(9)-THCV- and AM251-induced increases in mIPSC frequency beyond basal levels were consistent with basal CB1 receptor activity. WIN55-induced increases in PC spike firing rate were consistent with synaptic disinhibition; whilst Delta(9)-THCV-and AM251-induced decreases in spike firing suggest a mechanism of PC inhibition.

Assays for enhanced activity of low efficacy partial agonists at the D-2 dopamine receptor

Background and purpose: Low efficacy partial agonists at the D-2 dopamine receptor may be useful for treating schizophrenia. In this report we describe a method for assessing the efficacy of these compounds based on stimulation of [S-35]GTP gamma S binding. Experimental approach: Agonist efficacy was assessed from [S-35]GTP gamma S binding to membranes of CHO cells expressing D2 dopamine receptors in buffers with and without Na+. Effects of Na+ on receptor/G protein coupling were assessed using agonist/[H-3] spiperone competition binding assays. Key results: When [S-35]GTP gamma S binding assays were performed in buffers containing Na+, some agonists (aripiprazole, AJ-76, UH-232) exhibited very low efficacy whereas other agonists exhibited measurable efficacy. When Na+ was substituted by N-methyl D-glucamine, the efficacy of all agonists increased (relative to that of dopamine) but particularly for aripiprazole, aplindore, AJ-76, (-)-3-PPP and UH-232. In ligand binding assays, substitution of Na+ by N-methyl D-glucamine increased receptor/G protein coupling for some agonists -. aplindore, dopamine and (-)-3-PPP-but for aripiprazole, AJ-76 and UH-232 there was little effect on receptor/G protein coupling. Conclusions and implications: Substitution of Na+ by NMDG increases sensitivity in [S-35] GTPgS binding assays so that very low efficacy agonists were detected clearly. For some agonists the effect seems to be mediated via enhanced receptor/G protein coupling whereas for others the effect is mediated at another point in the G protein activation cycle. AJ-76, aripiprazole and UH-232 seem particularly sensitive to this change in assay conditions. This work provides a new method to discover these very low efficacy agonists.

Allosteric effects of antagonists on signalling by the chemokine receptor CCR5

Antagonists of the chemokine receptor, CCRS, may provide important new drugs for the treatment of HIV-1. In this study we have examined the mechanism of action of two functional antagonists of the chemokine receptor CCRS (UK-396,794, UK-438,235) in signalling and internalisation assays using CHO cells expressing CCR5. Both compounds were potent inverse agonists versus agonist-independent [S-3]GTP gamma S binding to membranes of CHO cells expressing CCR5. Both compounds also acted as allosteric inhibitors of CCL5 (RANTES) and CCL8 (MCP-2) -stimulated [S-35]GTP gamma S binding to CHO-CCR5 membranes, reducing the potency and maximal effects of the two chemokines. The data are consistent with effects of the allosteric inhibitors on both the binding and signalling of the chemokines. Both compounds inhibited CCR5 internalisation triggered by chemokines. When CHO-CCR5 cells were treated with either of the two compounds for prolonged periods of time (24 h) an increase (similar to 15%) in cell surface CCRS was detected. (C) 2007 Elsevier Inc. All rights reserved

Evidence that GABA rho subunits contribute to functional ionotropic GABA receptors in mouse cerebellar Purkinje cells

Ionotropic gamma-amino butyric acid (GABA) receptors composed of heterogeneous molecular subunits are major mediators of inhibitory responses in the adult CNS. Here, we describe a novel ionotropic GABA receptor in mouse cerebellar Purkinje cells (PCs) using agents reported to have increased affinity for rho subunit-containing GABA(C) over other GABA receptors. Exogenous application of the GABA(C)-preferring agonist cis-4-aminocrotonic acid (CACA) evoked whole-cell currents in PCs, whilst equimolar concentrations of GABA evoked larger currents. CACA-evoked currents had a greater sensitivity to the selective GABA(C) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) than GABA-evoked currents. Focal application of agonists produced a differential response profile; CACA-evoked currents displayed a much more pronounced attenuation with increasing distance from the PC soma, displayed a slower time-to-peak and exhibited less desensitization than GABA-evoked currents. However, CACA-evoked currents were also completely blocked by bicuculline, a selective agent for GABA(A) receptors. Thus, we describe a population of ionotropic GABA receptors with a mixed GABA(A)/GABA(C) pharmacology. TPMPA reduced inhibitory synaptic transmission at interneurone-Purkinje cell (IN-PC) synapses, causing clear reductions in miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency. Combined application of NO-711 (a selective GABA transporter subtype 1 (GAT-1) antagonist) and SNAP-5114 (a GAT-(2)/3/4 antagonist) induced a tonic GABA conductance in PCs; however, TPMPA had no effect on this current. Immunohistochemical studies suggest that rho subunits are expressed predominantly in PC soma and proximal dendritic compartments with a lower level of expression in more distal dendrites; this selective immunoreactivity contrasted with a more uniform distribution of GABA(A) alpha 1 subunits in PCs. Finally, co-immunoprecipitation studies suggest that rho subunits can form complexes with GABA(A) receptor alpha 1 subunits in the cerebellar cortex. Overall, these data suggest that rho subunits contribute to functional ionotropic receptors that mediate a component of phasic inhibitory GABAergic transmission at IN-PC synapses in the cerebellum.

Cannabinoid influences on palatability: microstructural analysis of sucrose drinking after Delta(9)-tetrahydrocannabinol, anandamide, 2-arachidonoyl glycerol and SR141716

Rationale: Central cannabinoid systems have been implicated in appetite control through the respective hyperphagic and anorectic actions of CB1 agonists and antagonists. The motivational changes underlying these actions remain to be determined, but may involve alterations to food palatability. Objectives: The mode of action of cannabinoids on ingestion was investigated by examining the effects of exogenous and endogenous agonists, and a selective CB1 receptor antagonist, on licking microstructure in rats ingesting a palatable sucrose solution. Methods: Microstructural analyses of licking for a 10% sucrose solution was performed over a range of agonist and antagonist doses administered to non-deprived, male Lister hooded rats. Results: Delta(9)-tetrahydrocannabinol (0.5, 1 and 3 mg/kg) and anandamide (1 mg/kg and 3 mg/kg) significantly increased total number of licks. This was primarily due to an increase in bout duration rather than bout number. There was a nonsignificant increase in total licks following administration of 2-arachidonoyl glycerol (0.2, 1.0 and 2.0 mg/kg), whereas administration of the CB1 antagonist SR141716 (1 mg/kg and 3 mg/kg) significantly decreased total licks. All drugs, with the exception of anandamide, significantly decreased the intra-bout lick rate. An exponential function fitted to the cumulative lick rate curves for each drug revealed that all compounds altered the asymptote of this function without having any marked effects on the exponent. Conclusions: These data are consistent with endocannabinoid involvement in the mediation of food palatability.

The effects of phorbol esters with different activities on protein kinase C

The sapintoxins are a series of naturally occurring fluorescent phorbol esters with a range of selective biological activities (e.g. pro-inflammatory but non-tumour promoting). Their ability to activate protein kinase C (PKC) in vitro has been studied. Both tumour promoting and non-promoting phorbol derivatives activate the enzyme in vitro at low concentrations. 12-deoxyphorbol-13-phenylacetate-20 acetate (DOPPA) acts as a partial agonist in the activation of protein kinase C. Structurally distinct phorbol esters may therefore preferentially activate different forms of protein kinase C. -sapinine, a biologically inactive compound, binds to protein kinase C without stimulating the enzyme and prevents subsequent activation by phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (TPA).