Search

ABHD11 (α/β-hydrolase domain containing 11) is a non-annotated enzyme belonging to the family of metabolic serine hydrolases (mSHs). Its natural substrates and products are unknown. Using competitive activity-based protein profiling (ABPP) to identify novel inhibitors of human (h)ABHD11, three compounds from our chemical library exhibited low nanomolar potency towards hABHD11. Competitive ABPP of various proteomes revealed fatty acid amide hydrolase (FAAH) as the sole off-target among the mSHs. Our fluorescent activity assays designed for natural lipase substrates revealed no activity of hABHD11 towards mono- or diacylglycerols. A broader profiling using para-nitrophenyl (pNP)-linked substrates indicated no amidase/protease, phosphatase, sulfatase, phospholipase C or phosphodiesterase activity. Instead, hABHD11 readily utilized para-nitrophenyl butyrate (pNPC4), indicating lipase/esterase-type activity that could be exploited in inhibitor discovery. Additionally, a homology model was created based on the crystal structure of bacterial esterase YbfF. In contrast to YbfF, which reportedly hydrolyze long-chain acyl-CoA, hABHD11 did not utilize oleoyl-CoA or arachidonoyl-CoA. In conclusion, the present study reports the discovery of potent hABHD11 inhibitors with good selectivity among mSHs. We developed substrate-based activity assays for hABHD11 that could be further exploited in inhibitor discovery and created the first homology-based hABHD11 model, offering initial insights into the active site of this poorly characterized enzyme.

Monoacylglycerol lipase (MAGL) is a serine hydrolase that acts as a principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). In addition to terminating the signaling function of 2-AG, MAGL liberates arachidonic acid to be used as a primary source for neuroinflammatory prostaglandin synthesis in the brain. MAGL activity also contributes to cancer pathogenicity by producing precursors for tumor-promoting bioactive lipids. Pharmacological inhibitors of MAGL provide valuable tools for characterization of MAGL and 2-AG signaling pathways. They also hold great therapeutic potential to treat several pathophysiological conditions, such as pain, neurodegenerative disorders, and cancer. We have previously reported piperidine triazole urea, {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)methanone (JJKK-048), to be an ultrapotent and highly selective inhibitor of MAGL in vitro. Here, we characterize in vivo effects of JJKK-048. Acute in vivo administration of JJKK-048 induced a massive increase in mouse brain 2-AG levels without affecting brain anandamide levels. JJKK-048 appeared to be extremely potent in vivo. Activity-based protein profiling revealed that JJKK-048 maintains good selectivity toward MAGL over other serine hydrolases. Our results are also the first to show that JJKK-048 promoted significant analgesia in a writhing test with a low dose that did not cause cannabimimetic side effects. At a high dose, JJKK-048 induced analgesia both in the writhing test and in the tail-immersion test, as well as hypomotility and hyperthermia, but not catalepsy.

De novo synthesis of fatty acids is essential to maintain intensive proliferation of cancer cells. Unlike normal cells that utilize food-derived circulating lipids for their fuel, cancer cells rely on heightened lipogenesis irrespective of exogenous lipid availability. Overexpression and activity of the multidomain enzyme fatty acid synthase (FASN) is crucial in supplying palmitate for protumorigenic activity. Therefore, FASN has been proposed as an attractive target for drug development. As an effort to set up an effective toolkit to study FASN inhibitors in human and rodent tissues, we validated activity-based protein profiling (ABPP) as a viable approach to unveil inhibitors targeting FASN thioesterase domain (FASN-TE). ABPP was combined with multi-well plate-assays designed for classical substrate-based FASN activity analysis together with powerful monitoring of cancer cell proliferation using IncuCyte® Live Cell Analyzing System. FASN-TE inhibitors were identified by competitive ABPP using HEK293 cell lysates in a screen of in-house compounds (200+) designed to target serine hydrolase (SH) family. The identified compounds were tested for their inhibitor potencies in vitro using a substrate-based activity assay monitoring FASN-dependent NADPH consumption in LNCaP prostate cancer cell preparation, in parallel with selected reference inhibitors, including orlistat (THL), GSK2194069, GSK837149A, platensimycin and BI-99179. LNCaP lysate supernatant was validated as a reliable native preparation to monitor FASN-dependent NADPH consumption as opposed to human glioma GAMG cells, whereas FASN enrichment was a prerequisite for accurate assays. While inhibitor pharmacology was identical between human prostate and glioma cancer cell FASN preparations, notable differences were revealed between human and rodent FASN preparations, especially for inhibitors targeting FASN-TE. ABPP combined with substrate-based assays facilitated identification of pan thiol-reactive inhibitor scaffolds, exemplified by the 1,2,4-thiadiazole moiety. Finally, selected compounds were evaluated for their antiproliferative efficacy in situ using GAMG cells. These studies revealed that while the tested compounds acted as potent FASN inhibitors in vitro, only a few showed antiproliferative efficacy in situ. To conclude, we describe a versatile toolkit to study FASN inhibitors in vitro and in situ using human cancer cells and reveal dramatic pharmacological differences between human and rodent FASN preparations.

This article describes our systematic approach to exploring the utility of the 1,3,4-oxadiazol-2-one scaffold in the development of ABHD6 inhibitors. Compound 3-(3-aminobenzyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (JZP-169, 52) was identified as a potent inhibitor of hABHD6, with an IC₅₀ value of 216 nM. This compound at 10 μM concentration did not inhibit any other endocannabinoid hydrolases, such as FAAH, MAGL and ABHD12, or bind to the cannabinoid receptors (CB₁ and CB₂). Moreover, in competitive activity-based protein profiling (ABPP), compound 52 (JZP-169) at 10 μM selectively targeted ABHD6 of the serine hydrolases of mouse brain membrane proteome. Reversibility studies indicated that compound 52 inhibited hABHD6 in an irreversible manner. Finally, homology modelling and molecular docking studies were used to gain insights into the binding of compound 52 to the active site of hABHD6.

Inhibition of Autotaxin (ATX) is a potential treatment strategy for several diseases, including tumors with elevated ATX-lysophosphatidic acid (LPA) signaling. Combining structure-based virtual screening together with hen egg-white Autotaxin (ewATX) activity assays enabled the discovery of novel small-molecule ATX inhibitors with a 2,4-dihydropyrano[2,3-c]pyrazole scaffold. These compounds are suggested to bind to the lipophilic pocket, leaving the active site unrestrained. Our most potent compound, (S)-6-amino-4-(3,4-dichlorophenyl)-3-(4-[(4-fluorobenzyl)oxy]phenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile [(S)-25], inhibited human ATX (hATX) with an IC50-value of 134 nM. It also blocked ATX-evoked but not LPA-mediated A2058 melanoma cell migration. Noteworthy, molecular modeling correctly predicted the biologically active enantiomer of 2,4-dihydropyrano[2,3-c]pyrazoles, as verified by compound crystallization and activity assays. Our study established the ewATX activity assay as a valid and affordable tool in ATX inhibitor discovery. Overall, our study offers novel insights and approaches into design of novel ATX inhibitors targeting the hydrophobic pocket instead of the active site., final draft, peerReviewed

Endocannabinoids are the endogenous ligands of the G protein-coupled cannabinoid receptors. The principal brain endocannabinoid, 2-arachidonoylglycerol (2-AG), is enzymatically produced by postsynaptic neurons and then activates presynaptic CB1 receptors in a retrograde manner. The primary pathway for 2-AG generation is believed to be conversion from the diacylglycerols (DAGs) by two sn-1-specific lipases, DAGLα and DAGLβ. Previous studies with DAGL-deficient mice indicated that DAGLα is the major enzyme needed for retrograde synaptic 2-AG signalling. The current study investigated whether the CB1 receptor-mediated Gi/o protein activity is altered in brain cryosections of DAGL-deficient mice when compared to wild-type mice and whether the sn-1-specific DAGLs are able to generate 2-AG in brain cryosections. Functional autoradiography indicated that brain regional CB1 receptor-Gi/o-activity largely remained unaltered in DAGLα-knockout and DAGLβ-knockout mice when compared to wild-type littermates. Following comprehensive pharmacological blockade of 2-AG hydrolysis, brain sections generated sufficient amounts of 2-AG to activate CB1 receptors throughout the regions endowed with these receptors. As demonstrated by LC/MS/MS, this pool of 2-AG was generated via tetrahydrolipstatin-sensitive enzymatic pathways distinct from DAGLα or DAGLβ. We conclude that in addition to the sn-1-specific DAGLs, additional 2-AG generating enzymatic pathways are active in brain sections.

In the central nervous system, three enzymes belonging to the serine hydrolase family are thought to regulate the life time of the endocannabinoid 2-arachidonoylglycerol (C20:4) (2-AG). From these, monoacylglycerol lipase (MAGL) is well characterized and, on a quantitative basis, is the main 2-AG hydrolase. The postgenomic proteins α/β-hydrolase domain containing (ABHD)6 and ABHD12 remain poorly characterized. By applying a sensitive fluorescent glycerol assay, we delineate the substrate preferences of human ABHD6 and ABHD12 in comparison with MAGL. We show that the three hydrolases are genuine MAG lipases; medium-chain saturated MAGs were the best substrates for hABHD6 and hMAGL, whereas hABHD12 preferred the 1 (3)- and 2-isomers of arachidonoylglycerol. Site-directed mutagenesis of the amino acid residues forming the postulated catalytic triad (ABHD6: S148-D278-H306, ABHD12: S246-D333-H372) abolished enzymatic activity as well as labeling with the active site serine-directed fluorophosphonate probe TAMRA-FP. However, the role of D278 and H306 as residues of the catalytic core of ABHD6 could not be verified because none of the mutants showed detectable expression. Inhibitor profiling revealed striking potency differences between hABHD6 and hABHD12, a finding that, when combined with the substrate profiling data, should facilitate further efforts toward the design of potent and selective inhibitors, especially those targeting hABHD12, which currently lacks such inhibitors.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is a lipid mediator involved in various physiological processes. In response to neural activity, 2-AG is synthesized post-synaptically, then activates pre-synaptic cannabinoid CB1 receptors (CB1Rs) in a retrograde manner, resulting in transient and long-lasting reduction of neurotransmitter release. The signalling competence of 2-AG is tightly regulated by the balanced action between 'on demand' biosynthesis and degradation. We review recent research on monoacylglycerol lipase (MAGL), ABHD6 and ABHD12, three serine hydrolases that together account for approx. 99% of brain 2-AG hydrolase activity. MAGL is responsible for approx. 85% of 2-AG hydrolysis and colocalizes with CB1R in axon terminals. It is therefore ideally positioned to terminate 2-AG-CB1R signalling regardless of the source of this endocannabinoid. Its acute pharmacological inhibition leads to 2-AG accumulation and CB1R-mediated behavioural responses. Chronic MAGL inactivation results in 2-AG overload, desensitization of CB1R signalling and behavioural tolerance. ABHD6 accounts for approx. 4% of brain 2-AG hydrolase activity but in neurones it rivals MAGL in efficacy. Neuronal ABHD6 resides post-synaptically, often juxtaposed with CB1Rs, and its acute inhibition leads to activity-dependent accumulation of 2-AG. In cortical slices, selective ABHD6 blockade facilitates CB1R-dependent long-term synaptic depression. ABHD6 is therefore positioned to guard intracellular pools of 2-AG at the site of generation. ABHD12 is highly expressed in microglia and accounts for approx. 9% of total brain 2-AG hydrolysis. Mutations in ABHD12 gene are causally linked to a neurodegenerative disease called PHARC. Whether ABHD12 qualifies as a bona fide member to the endocannabinoid system remains to be established.

[ 35 S]GTPγS autoradiography represents a powerful functional approach to detect receptor-dependent G i/o protein activity in anatomically defined brain structures. Inherent to this technique, however, is the notable basal signal evident in several brain regions in the absence of receptor stimulation by exogenously added agonist. In the rat brain, much of this basal labelling derives from tonic activation of adenosine A 1 and lysophosphatidic acid LPA 1 receptors in the gray and white matter regions, respectively. Despite the elimination of the two receptor activities, prominent basal [ 35 S]GTPγS labelling is still evident in discrete brain structures, possibly reflecting regional enrichment of G i/o and/or constitutive receptor activity or the presence of still unknown endogenous ligands activating their orphan receptors. Here, the anatomical distribution of the enhanced basal signal was systematically mapped in brain sections of 4-week-old male Wistar rats. Regions with prominent basal [ 35 S]GTPγS labelling represented neuroanatomically distinct structures, in particular various thalamic and hypothalamic nuclei. For instance, the paraventricular thalamic nucleus, the bed nucleus of the stria terminalis and the subfornical organ were highly labelled, as were the periaqueductal gray and the nucleus of the solitary tract. Pre-treatment with N -ethylmaleimide (NEM), an alkylating agent preventing all known receptor-driven G protein activity in cryostat sections markedly decreased the basal binding in all examined regions. In preliminary screening, selective antagonists for various brain-enriched G i/o -coupled receptors failed to suppress the basal signal in any of the studied regions.

Heterotrimeric G proteins play a key role in membrane-mediated cell-signalling and hormonal regulation. Our earlier studies gave evidence of G protein subunit Gai2 being under hormonal regulation in human in vivo. In this study, we used immortalized human oviduct epithelial cell line OE-E6/E7 as a model to study the hormonal regulation of Gai2. We aimed at clarifying whether estradiol or progesterone could individually regulate the expression of Gai2 and its potential signalling partners. Furthermore, we aimed to investigate which sex hormone receptors could potentially mediate the gene regulation in OE-E6/E7 cell line. OE-E6/E7 cells were cultured for 5 days with different concentrations of estradiol or progesterone. Quantitative real-time polymerase chain reaction (Q-PCR) was performed using cDNA of the hormone-treated cells to reveal any changes in gene expression. The presence of potential receptor targets in these cells was studied using PCR. Our data clearly showed that low concentrations of estradiol up-regulated the expression of Gai2 gene and down-regulated the expression of membrane progesterone receptor mPRa gene in OE-E6/E7 cell line. Progesterone had no significant effect on Gai2 gene expression, but it caused up-regulation of mPRa gene expression. In conclusion, it appears that sex hormones regulate the expression of Gai2 and mPRa genes in a reverse manner in OE-E6/E7 cells. Our results suggest that estrogen receptor ERb mediates the regulatory effects of estradiol in these cells.

Article, In mammalian brain, monoacylglycerol lipase (MAGL) is the primary enzyme responsible for terminating signaling function of the endocannabinoid 2-arachidonoylglycerol (2-AG). Previous in vivo studies with mice indicate that both genetic and chronic pharmacological inactivation of MAGL result in 8–30-fold increase of 2-AG concentration in the brain, causing desensitization and downregulation of cannabinoid CB1 receptor (CB1R) activity, leading to functional and behavioral tolerance. However, direct evidence for reduced CB1R activity in the brain is lacking. In this study, we used functional autoradiography to assess basal and agonist-stimulated CB1R-dependent Gi/o protein activity in multiple brain regions of MAGL-KO mice in comparison to their wild-type (WT) littermates. In addition, the role of endogenous cannabinoids in basal CB1R signaling was assessed after comprehensive pharmacological blockade of 2-AG hydrolysis by determining the contents of endocannabinoids (eCBs) in WT and MAGL-KO brain tissues by LC/MS/MS technology. To show whether lack of MAGL cause compensatory alterations in the serine hydrolase activity, we compared serine hydrolase pattern of WT and MAGL-KO using activity-based protein profiling. Consistent with studies using chronic pharmacological MAGL inactivation in vivo, we observed a statistically significant decrease of CB1R-Gi/o signaling in most of the studied brain regions. In MAGL-KO brain sections, elevated 2-AG levels were mirrored to heightened basal CB1R-dependent Gi/o-activity, as well as, dampened agonist-evoked responses in several brain regions. The non-selective serine hydrolase inhibitor methylarachidonoylfluorophosphonate (MAFP) was able to significantly elevate 2-AG levels in brain sections of MAGL-KO mice, indicating that additional serine hydrolases possess 2-AG hydrolytic activity in MAGL-KO brain sections., final draft, http://purl.org/eprint/status/PeerReviewed

In neuronal signalling mediated by the endocannabinoid 2-arachidonoylglycerol, both synthetic and inactivating enzymes operate within close proximity to the G(i/o)-coupled pre-synaptic CB(1) receptors, thus allowing for rapid onset and transient duration of this lipid modulator. In rat brain, 2-arachidonoylglycerol is inactivated mainly via hydrolysis by serine hydrolase inhibitor-sensitive monoacylglycerol lipase activity. We show in this study that comprehensive pharmacological elimination of this activity in brain cryosections by methyl arachidonylfluorophosphonate or hexadecylsulphonyl fluoride results in endocannabinoid-mediated CB(1) receptor activity, which can be visualized by functional autoradiography. URB597, a specific inhibitor of anandamide hydrolysis proved ineffective. TLC indicated that the bioactivity resided in 2-arachidonoylglycerol-containing fraction and gas chromatography-mass spectroscopy detected elevated levels of monoacylglycerols, including 2-arachidonoylglycerol in this fraction. Although two diacylglycerol lipase inhibitors, tetrahydrolipstatin (THL) and RHC80267, blocked the bulk of 2-arachidonoylglycerol accumulation in methyl arachidonylfluorophosphonate-treated sections, only THL reversed the endocannabinoid-dependent CB(1) receptor activity. Further studies indicated that at the used concentrations, THL rather specifically antagonized the CB(1) receptor. These findings confirm that in brain sections there is preservation of enzymatic pathways regulating the production of endogenous receptor ligands. Furthermore, the presently described methodology may serve as an elegant and intuitive approach to identify novel membrane-derived lipid modulators operating in the CNS.

SummaryPrevious studies indicate that in brain tissue the endocannabinoid 2-AG is inactivated by monoglyceride lipase (MGL)-catalyzed hydrolysis, and a recent report has indicated that MGL activity could be specifically inhibited by URB754 [1]. In the present study, URB754 failed to inhibit 2-AG hydrolysis in rat brain preparations. In addition, brain cryosections were employed to assess whether URB754 could facilitate the detection of 2-AG-stimulated G protein activity. Nevertheless, whereas pretreatment with PMSF readily allowed detection of 2-AG-stimulated G protein activity, URB754 was ineffective. In contrast to previous claims, brain FAAH activity was also resistant to URB754. Thus, in our hands URB754 was not able to block the endocannabinoid-hydrolyzing enzymes and cannot serve as a lead structure for future development of MGL-specific inhibitors.

The basic secretagogues, such as compound 48/80 (c48/80) and mastoparans, are widely used histamine-releasing agents and their mechanism of action is commonly attributed to a direct, receptor-bypassing property to activate the Gi/o class of G proteins. We tested here whether c48/80 could directly stimulate [35S]guanosine-5′-[γ-thio]triphosphate ([35S]GTPγS) binding to rat brain sections in an attempt to visualize the entire signaling pool of Gi/o in its native neuroanatomical context. Instead of direct Gi/o activation, c48/80 (100 μg ml−1) from various suppliers stimulated brain phospholipase D (PLD) activity, leading to the generation of endogenous phospholipids capable of activating brain white matter-enriched, Gi/o-coupled lysophosphatidic acid (LPA) receptors. This response was sensitive to 1-butanol and was potently reversed by the LPA1/LPA3 receptor-selective antagonist Ki16425 (IC50 59±13 nM, mean±s.e.m.), and showed age-dependent decline, closely reflecting known developmental regulation of the PLD–LPA1 receptor axis in the CNS. In addition, c48/80 was found to modestly activate hippocampal 5-HT1A receptors in a pH-dependent and antagonist-sensitive manner. Consistent with the lack of direct Gi/o-activating properties in brain sections, c48/80 showed no activity in classical membrane [35S]GTPγS binding assays. Instead, c48/80 from one particular manufacturer elicited non-specific effect in these assays, therefore challenging the previous interpretations regarding the compound's ability to activate G proteins directly. We conclude that c48/80 is not a receptor-bypassing general G protein activator but rather activates PLD, leading to generation of endogenous LPA receptor-activating phospholipids. This property may also contribute to the compound's ability to release histamine from mast cells. British Journal of Pharmacology (2006) 147, 596–606. doi:10.1038/sj.bjp.0706671

All G protein-coupled melatonin receptors have two conserved cysteines in the interphase between transmembrane helix III and the second intracellular loop, in the region assumed important in receptor/G protein coupling. The cysteines are also potential targets of receptor S-nitrosylation. The effects of site-directed mutagenesis of these cysteines in the human MT1 melatonin receptor were investigated. The cysteines were mutated into serines either individually or as a pair and stable Chinese hamster ovary cell lines expressing the wild-type and mutant MT1 receptors were created. Receptor expression level, subcellular localization, ligand binding and G protein activation of the cell lines were analyzed. Serine substitution of C127 (Cys(3.52)) did not affect the ligand binding affinity and agonist potency but had an influence on receptor trafficking and G protein activation capacity. Serine substitution of C130 (Cys(3.55)) resulted in a decrease in the potency of melatonin to activate G proteins. When both cysteines were mutated into serines, normal MT1 receptor binding and activation were abolished. Computer modeling revealed that the mutations did not change the structure of the ligand binding pocket. Cysteine S-nitrosylation had no influence on G protein activation through MT1 receptors. Taken together, these data show that the two conserved cysteines in the end of transmembrane domain III of the MT1 melatonin receptor, especially C130 (Cys(3.55)), are needed for normal G protein activation and receptor trafficking.

2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid that binds to CB1 and CB2 cannabinoid receptors, inducing cannabimimetic effects. However, the cannabimimetic effects of 2-AG are weak in vivo due to its rapid enzymatic hydrolysis. The enzymatic hydrolysis of 2-AG has been proposed to mainly occur by monoglyceride lipase (monoacylglycerol lipase). Fatty acid amide hydrolase (FAAH), the enzyme responsible for the hydrolysis of N-arachidonoylethanolamide (AEA), is also able to hydrolyse 2-AG. In the present study, we investigated the hydrolysis of endocannabinoids in rat cerebellar membranes and observed that enzymatic activity towards 2-AG was 50-fold higher than that towards AEA. Furthermore, various inhibitors for 2-AG hydrolase activity were studied in rat cerebellar membranes. 2-AG hydrolysis was inhibited by methyl arachidonylfluorophosphonate, hexadecylsulphonyl fluoride and phenylmethylsulphonyl fluoride with ic 50 values of 2.2 nM, 241 nM and 155 μM, respectively. Potent FAAH inhibitors, such as OL-53 and URB597, did not inhibit the hydrolysis of 2-AG, suggesting that 2-AG is inactivated in rat cerebellar membranes by an enzyme distinct of FAAH. The observation that the hydrolysis of 1(3)-AG and 2-AG occurred at equal rates supports the role of MGL in 2-AG inactivation. This enzyme assay provides a useful method for future inhibition studies of 2-AG degrading enzyme(s) in brain membrane preparation having considerably higher MGL-like activity when compared to FAAH activity.

At nanomolar concentrations, SR141716 and AM251 act as specific and selective antagonists of the cannabinoid CB1 receptor. In the micromolar range, these compounds were shown to inhibit basal G-protein activity, and this is often interpreted to implicate constitutive activity of the CB1 receptors in native tissue. We show here, using [35S]GTPγS binding techniques, that micromolar concentrations of SR141716 and AM251 inhibit basal G-protein activity in rat cerebellar membranes, but only in conditions where tonic adenosine A1 receptor signaling is not eliminated. Unlike lipophilic A1 receptor antagonists (potency order DPCPX≫N-0840 ∼cirsimarin>caffeine), adenosine deaminase (ADA) was not fully capable in eliminating basal A1 receptor-dependent G-protein activity. Importantly, all antagonists reduced basal signal to the same extent (20%), and the response evoked by the inverse agonist DPCPX was not reversed by the neutral antagonist N-0840. These data indicate that rat brain A1 receptors are not constitutively active, but that an ADA-resistant adenosine pool is responsible for tonic A1 receptor activity in brain membranes. SR141716 and AM251, at concentrations fully effective in reversing CB1-mediated responses (10−6M), did not reduce basal G-protein activity, indicating that CB1 receptors are not constitutively active in these preparations. At higher concentrations (1–2.5 × 10−5M), both antagonists reduced basal G-protein activity in control and ADA-treated membranes, but had no effect when A1 receptor signaling was blocked with DPCPX. Moreover, the CB1 antagonists right-shifted A1 agonist dose–response curves without affecting maximal responses, suggesting competitive mode of antagonist action. The CB1 antagonists did not affect muscarinic acetylcholine or GABAB receptor signaling. When further optimizing G-protein activation assay for the labile endocannabinoid 2-arachidonoylglycerol (2-AG), we show, by using HPLC, that pretreatment of cerebellar membranes with methyl arachidonoyl fluorophosphonate (MAFP) fully prevented enzymatic degradation of 2-AG and concomitantly enhanced the potency of 2-AG. In contrast to previous claims, MAFP exhibited no antagonist activity at the CB1 receptor. The findings establish an optimized method with improved signal-to-noise ratio to assess endocannabinoid-dependent G-protein activity in brain membranes, under assay conditions where basal adenosinergic tone and enzymatic degradation of 2-AG are fully eliminated. British Journal of Pharmacology (2003) 140, 1451–1459. doi:10.1038/sj.bjp.0705577

The Gi-linked platelet ADP receptor, now designated as P2Y12, accounts for ADP-induced inhibition of adenylyl cyclase in platelets and certain clonal rat cell lines. The pharmacology of this receptor is well characterized. Based on the functional approach of [35S]GTPγS autoradiography, we recently disclosed the widespread presence of Gi-linked ADP receptors in the rat nervous system. Based on initial pharmacological analysis, these receptors were strikingly similar with P2Y12. Here, we extend this analysis by comparing the potencies of six 2-alkylthio-substituted ATP analogues, including the adenosine-aspartate conjugate 2-hexylthio-AdoOC(O)Asp2 and five AR-C compounds (AR-C67085, AR-C69931, AR-C78511, AR-C69581, AR-C70300) with wide range of affinities towards P2Y12, in reversing 2-methylthio-ADP stimulated G protein activity in rat brain sections and human platelet membranes. Closely matching pIC50 values (r2=0.99) revealed pharmacological similarity between the two receptors with one exception: AR-C67085 more avidly recognized the platelet P2Y12. Further analysis of the rat brain pIC50 data against those available for three of the AR-C compounds in reversing P2Y12-mediated adenylyl cyclase inhibition in rat platelets (r2=0.96) and rat C6 glioma cells (r2=1.00) demonstrated that the three P2Y receptors are pharmacologically indistinguishable. We conclude that the rat brain Gi-linked ADP receptors, as revealed using [35S]GTPγS autoradiography, correspond to P2Y12.

The effects of low-level radiofrequency radiation (RFR) on ultraviolet (UV)-induced skin tumorigenesis were evaluated in ornithine decarboxylase (ODC) and non-transgenic mice.Transgenic female mice over-expressing the human ODC gene and their non-transgenic littermates (20 animals in the cage control group, and 45-49 animals in the other groups) were exposed for 52 weeks to UV radiation or a combination of UV radiation and pulsed RFR. The UV dose was 240 Jm(-2) (1.2 x human minimum erythemal dose) delivered three times a week. One group of animals was exposed to Digital Advanced Mobile Phone System (DAMPS)-type RFR, the other group to Global System for Mobile (GSM)-type RFR at a nominal average specific absorption rate of 0.5 W kg(-1), 1.5 h day(-1), for 5 days a week. The skin was carefully palpated weekly for macroscopic tumours. Histopathological analyses of all skin lesions and of a specified dorsal skin area were performed on all animals.UV exposure resulted in development of macroscopic skin tumours in 11.5 and 36.8% of non-transgenic and transgenic animals, respectively. The RFR exposures did not give a statistically significant effect on the development of skin tumours in either transgenic or non-transgenic animals, or in combined analysis, but tumour development appeared slightly accelerated especially in non-transgenic animals. No effects of RFR exposures were found on excretion of 6-hydroxymelatonin sulphate into urine or on polyamine levels in dorsal skin.RFR exposures did not significantly enhance skin tumourigenesis. However, the slightly accelerated tumour development may warrant further evaluation.

Histamine elicits its biological effects via three distinct G protein-coupled receptors, termed H1, H2, and H3. We have used guanosine 5'-(gamma-[35S]thio)triphosphate (GTPgamma[35S]) autoradiography to localize histamine receptor-dependent G protein activation in rat brain tissue sections. Initial studies revealed that in basal conditions, adenosine was present in tissue sections in sufficient concentrations to generate an adenosine A1 receptor-dependent GTPgamma[35S] signal in several brain regions. All further incubations therefore contained 8-cyclopentyl-1,3-dipropylxanthine (10 microM), a selective A1 receptor antagonist. Histamine elicited dose-dependent increments in GTPgamma[35S] binding to discrete anatomical structures, most notably the caudate putamen, cerebral cortex, and substantia nigra. The overall anatomical pattern of the histamine-evoked binding response closely reflects the known distribution of H3 binding sites and was faithfully mimicked by N(alpha)-methylhistamine, (R)-alpha-methylhistamine, and immepip, three H3-selective agonists. In all regions examined, the GTPgamma[35S] signal was reversed with thioperamide and clobenpropit, two potent H3-selective antagonists, whereas mepyramine, a specific H1 antagonist, and cimetidine, a prototypic H2 antagonist, proved ineffective. These data indicate that in rat brain tissue sections, GTPgamma[35S] autoradiography selectively detects H3 receptor-dependent signaling in response to histamine stimulation. As the existing evidence suggests that GTPgamma[35S] autoradiography preferentially reveals responses to G(i/o)-coupled receptors, our data indicate that most, if not all, central H3 binding sites represent functional receptors coupling to G(i/o), the inhibitory class of G proteins. Besides allowing more detailed studies on H3 receptor signaling within anatomically restricted regions of the CNS, GTPgamma[35S] autoradiography offers a novel approach for functional in vitro screening of H3 ligands.

The primary route of inactivation of the endocannabinoid 2-arachidonoylglycerol in the central nervous system is through enzymatic hydrolysis, mainly carried out by monoacylglycerol lipase (MAGL), along with a small contribution by the α/β-hydrolase domain (ABHD) proteins ABHD6 and ABHD12. Recent methodological progress allowing kinetic monitoring of glycerol liberation has facilitated substrate profiling of the human endocannabinoid hydrolases, and these studies have revealed that the three enzymes have distinct monoacylglycerol substrate and isomer preferences. Here, we have extended this substrate profiling to cover four prostaglandin glycerol esters, namely, 15-deoxy-Δ(12,14)-prostaglandin J2-2-glycerol (15d-PGJ2-G), PGD2-G, PGE2-G, and PGF2 α-G. We found that the three enzymes hydrolyzed the tested substrates, albeit with distinct rates and preferences. Although human ABHD12 (hABHD12) showed only marginal activity toward PGE2-G, hABHD6 preferentially hydrolyzed PGD2-G, and human MAGL (hMAGL) robustly hydrolyzed all four. This was particularly intriguing for MAGL activity toward 15d-PGJ2-G whose hydrolysis rate rivaled that of the best monoacylglycerol substrates. Molecular modeling studies combined with kinetic analysis supported favorable interaction with the hMAGL active site. Long and short MAGL isoforms shared a similar substrate profile, and hMAGL hydrolyzed 15d-PGJ2-G also in living cells. The ability of 15d-PGJ2-G to activate the canonical nuclear factor erythroid 2-related factor (Nrf2) signaling pathway used by 15d-PGJ2 was assessed, and these studies revealed for the first time that 15d-PGJ2 and 15d-PGJ2-G similarly activated Nrf2 signaling as well as transcription of target genes of this pathway. Our study challenges previous claims regarding the ability of MAGL to catalyze PG-G hydrolysis and extend the MAGL substrate profile beyond the classic monoacylglycerols.

Hyaluronan, a major matrix molecule in epidermis, is often increased by stimuli that enhance keratinocyte proliferation and migration. We found that small amounts of UDP-sugars were released from keratinocytes and that UDP-glucose (UDP-Glc) added into keratinocyte cultures induced a specific, rapid induction of hyaluronan synthase 2 (HAS2), and an increase of hyaluronan synthesis. The up-regulation of HAS2 was associated with JAK2 and ERK1/2 activation, and specific Tyr(705) phosphorylation of transcription factor STAT3. Inhibition of JAK2, STAT3, or Gi-coupled receptors blocked the induction of HAS2 expression by UDP-Glc, the latter inhibitor suggesting that the signaling was triggered by the UDP-sugar receptor P2Y14. Chromatin immunoprecipitations demonstrated increased promoter binding of Tyr(P)(705)-STAT3 at the time of HAS2 induction. Interestingly, at the same time Ser(P)(727)-STAT3 binding to its response element regions in the HAS2 promoter was unchanged or decreased. UDP-Glc also stimulated keratinocyte migration, proliferation, and IL-8 expression, supporting a notion that UDP-Glc signals for epidermal inflammation, enhanced hyaluronan synthesis as an integral part of it.

BackgroundHuman lymphocyte antigen B-associated transcript 5 (BAT5, also known as ABHD16A) is a poorly characterized 63 kDa protein belonging to the α/β-hydrolase domain (ABHD) containing family of metabolic serine hydrolases. Its natural substrates and biochemical properties are unknown.Methodology/principal findingsAmino acid sequence comparison between seven mammalian BAT5 orthologs revealed that the overall primary structure was highly (≥95%) conserved. Activity-based protein profiling (ABPP) confirmed successful generation of catalytically active human (h) and mouse (m) BAT5 in HEK293 cells, enabling further biochemical characterization. A sensitive fluorescent glycerol assay reported hBAT5-mediated hydrolysis of medium-chain saturated (C14:0), long-chain unsaturated (C18:1, C18:2, C20:4) monoacylglycerols (MAGs) and 15-deoxy-Δ12,14-prostaglandin J2-2-glycerol ester (15d-PGJ2-G). In contrast, hBAT5 possessed only marginal diacylglycerol (DAG), triacylglycerol (TAG), or lysophospholipase activity. The best MAG substrates were 1-linoleylglycerol (1-LG) and 15d-PGJ2-G, both exhibiting low-micromolar Km values. BAT5 had a neutral pH optimum and showed preference for the 1(3)- vs. 2-isomers of MAGs C18:1, C18:2 and C20:4. Inhibitor profiling revealed that β-lactone-based lipase inhibitors were nanomolar inhibitors of hBAT5 activity (palmostatin B > tetrahydrolipstatin > ebelactone A). Moreover, the hormone-sensitive lipase inhibitor C7600 (5-methoxy-3-(4-phenoxyphenyl)-3H-[1], [3], [4]oxadiazol-2-one) was identified as a highly potent inhibitor (IC50 8.3 nM). Phenyl and benzyl substituted analogs of C7600 with increased BAT5 selectivity were synthesized and a preliminary SAR analysis was conducted to obtain initial insights into the active site dimensions.Conclusions/significanceThis study provides an initial characterization of BAT5 activity, unveiling the biochemical and pharmacological properties with in vitro substrate preferences and inhibitor profiles. Utilization of glycerolipid substrates and sensitivity to lipase inhibitors suggest that BAT5 is a genuine lipase with preference for long-chain unsaturated MAGs and could in this capacity regulate glycerolipid metabolism in vivo as well. This preliminary SAR data should pave the way towards increasingly potent and BAT5-selective inhibitors.

No G(i)-linked P2Y receptors have been cloned to date but the presence of such receptors is thought to be restricted to platelets and certain clonal cell lines. Using the functional approach of [(35)S]guanosine 5'-[gamma-thio]-triphosphate autoradiography, we uncovered the widespread presence of such receptors in the CNS. Under conditions in which the prominent signal due to tonic adenosine receptor activity is masked, ADP and ATP stimulated G-protein activity in multiple grey and white matter regions. Localization in the grey matter suggests inhibitory auto-/heteroreceptor function. In the white matter, activated G proteins appeared as 'hot spots' (presumed oligodendrocyte progenitors) with scattered distribution along the main fibre tracts. Responses to ATP were diminished under conditions that inhibited degradation, suggesting that prior conversion to ADP explained agonist action. Uracil nucleotides were ineffective but 2-methylthio-ADP activated G proteins approximately 500-fold more potently than ADP, although both were similarly degraded. Throughout the brain, ADP-dependent G-protein activity was reversed by 2-hexylthio-AdoOC(O)Asp(2), a non-phosphate ATP analogue, whereas selective P2Y(1) receptor antagonists proved ineffective. A similar receptor was also disclosed from the adrenal medulla. These data witness a hitherto unrecognized abundance of G(i/o)-linked ADP receptors in the nervous system. Biochemical and pharmacological behaviour suggests striking similarities to the elusive platelet P2Y(ADP) receptor.

Two endocannabinoids, arachidonoyl ethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) bind and activate G-protein-coupled cannabinoid receptors, but limited data exist on their relative ability to activate G-proteins. Here we assess agonist potency and efficacy of various cannabinoids, including 2-AG, HU-310 (2-arachidonoyl glyceryl ether, a third putative endocannabinoid), HU-313 (another ether analogue of 2-AG), AEA, R-methanandamide (an enzymatically stable analogue of AEA), and CP-55,940 at rat brain CB1 receptors using agonist-stimulated [35S]-GTPγS binding to cerebellar membranes and whole brain sections. Degradation of endocannabinoids under experimental conditions was monitored by HPLC. To enhance efficacy differences, agonist dose-response curves were generated using increasing GDP concentrations. At 10−6 M GDP, all compounds, except HU-313, produced full agonists responses ∼2.5 fold over basal. The superior efficacy of 2-AG over all other compounds became evident by increasing GDP (10−5 and 10−4 M). In membrane incubations, 2-AG was degraded by 85% whereas AEA and HU-310 were stable. Pretreatment of membranes with phenylmethylsulphonyl fluoride inhibited 2-AG degradation, resulting in 2 fold increase in agonist potency. Such pretreatment had no effect on AEA potency. Responses in brain sections were otherwise consistent with membrane binding data, but 2-AG evoked only a weak signal in brain sections, apparently due to more extensive degradation. These data establish that even under conditions of substantial degradation, 2-AG is a full efficacy agonist, clearly more potent than AEA, in mediating CB1 receptor-dependent G-protein activity in native membranes. British Journal of Pharmacology (2001) 134, 664–672; doi:10.1038/sj.bjp.0704297

Effects of 50-Hz sinusoidal magnetic fields (MFs) on embryo implantation, serum 17β-estradiol, progesterone, testosterone, and melatonin levels, and on estrogen receptor (ER) and progesterone receptor (PgR) densities in the uterus were studied during the preimplantation and implantation periods in rats. Pregnant Wistar rats were exposed to magnetic r.m.s. field strengths of 10 or 100 A/m (13 or 130 μT) or sham-exposed (controls) from day 0 of pregnancy for 24 h/day and killed during light and dark periods between 70 h and 176 h after ovulation. MFs did not influence the mean total number of implantations. The nocturnal mean serum melatonin concentration decreased by 34 and 38% at 10 and 100 A/m, respectively. At the same time, the first embryos, at an early developmental stage, arrived in the uterus in the MF-exposed groups. Serum estradiol and progesterone levels did not significantly change. Nuclear PgR and ER densities in the uterus decreased before implantation and there was an increased incidence of early stage embryos and fewer hatched embryos were found in the uterus at 100 A/m. During the early implantation period, the uterine cytosolic ER/PgR-ratio was increased at 100 A/m and no implants were concomitantly found in uterus. The nuclear ER/PgR-ratio decreased during implantation in both MF-groups due to decreased nuclear ER density. At the same time, 19% and 15% of the embryos (calculated from the corpora luteae) at 10 and 100 A/m, respectively, were yet morulae and not implanted. In summary, the results show that MFs do not impair implantation in rats although there may be some borderline changes in the transport and development of embryos and associated endocrinologic parameters.

Perfusion of hippocampal slices with an inhibitor of nitric oxide (NO) synthase-blocked induction of long-term potentiation (LTP) produced by a one-train tetanus and significantly reduced LTP by a two-train tetanus, but only slightly reduced LTP by a four-train tetanus. Inhibitors of heme oxygenase, the synthetic enzyme for carbon monoxide (CO), significantly reduced LTP by either a two-train or four-train tetanus. These results suggest that NO and CO are both involved in LTP but may play somewhat different roles. One possibility is that NO serves a phasic, signaling role, whereas CO provides tonic, background stimulation. Another possibility is that NO and CO are phasically activated under somewhat different circumstances, perhaps involving different receptors and second messengers. Because NO is known to be activated by stimulation of NMDA receptors during tetanus, we investigated the possibility that CO might be activated by stimulation of metabotropic glutamate receptors (mGluRs). Consistent with this idea, long-lasting potentiation by the mGluR agonist tACPD was blocked by inhibitors of heme oxygenase but not NO synthase. Potentiation by tACPD was also blocked by inhibitors of soluble guanylyl cyclase (a target of both NO and CO) or cGMP-dependent protein kinase, and guanylyl cyclase was activated by tACPD in hippocampal slices. However, biochemical assays indicate that whereas heme oxygenase is constitutively active in hippocampus, it does not appear to be stimulated by either tetanus or tACPD. These results are most consistent with the possibility that constitutive (tonic) rather than stimulated (phasic) heme oxygenase activity is necessary for potentiation by tetanus or tACPD, and suggest that mGluR activation stimulates guanylyl cyclase phasically through some other pathway.

A yeast functional colorimetric assay was employed to test the effects of site-directed point mutations on the function of the human Mel 1a melatonin receptor. Seven mutants were created in transmembrane domains III, V, and VII of the receptor to test the rhodopsin-based model of melatonin recognition. Two mutants in transmembrane domains III and VI were created to investigate the mechanisms of G protein activation in the melatonin receptor. Mutations in transmembrane domain V either potentiated agonist efficiencies (H195A) or totally abolished all responses to tested compounds (V192T+H195A). Mutation N124A in the conserved NRY motif in the end of transmembrane domain III seriously impaired receptor activation. Several mutants were found to have decreased ability to activate functional responses, reflecting the importance of these residues for receptor function. These data also suggest that activation of the receptor involves interaction of the 5-methoxy group of melatonin with the conserved histidine H195 in transmembrane domain V.

Melatonin is produced rhythmically by the pineal gland and the retina with increased synthesis during darkness. Pineal melatonin serves as the 'chemical expression of darkness' conveying information on the ambient light-dark cycle into rhythmic bodily functions. On-going debate on modes and sites of action ranges from views of melatonin affecting each and every cell ('cure-all') to those of melatonin having restricted actions through specific high-affinity receptors. The present review deals with the latter view. The use of 2-[125I]-iodomelatonin has allowed the exact localization and characterization of high-affinity melatonin receptors that signal through the G(i/o) class of G proteins. Molecular cloning of melatonin receptor genes has confirmed that most, if not all, high-affinity melatonin-binding sites represent the G-protein-coupled melatonin receptors. Based on sequence dissimilarities, melatonin receptors are classified into three subtypes, Mel1a, Mel1b and Mel1c. A distribution wider than originally thought of melatonin receptors in the human brain and peripheral sites has brought these receptors into focus of several drug companies, promising exciting times for research on melatonin and new therapeutic possibilities.

Haem oxygenase-1 (HO-1) is a stress protein and a rate-limiting enzyme in haem degradation, generating ferrous iron, carbon monoxide and bile pigments. HO-1 has been suggested to be protective against oxidative stress. In the normal rodent brain the enzyme is localized in selected neuron populations, but heat shock, glutathione depletion in vivo and oxidative stress in vitro induce HO-1 predominantly in glial cells. We studied HO-1 expression in the brain following transient occlusion of the middle cerebral artery, and found increased mRNA levels in the ischaemic region from 4 h to 7 days after 90 min of ischaemia. The mRNA levels peaked at 12 h, and were localized perifocally. HO-1-immunoreactive astrocytes and microglial cells were seen in the perifocal area, in the ipsilateral and occasionally in the contralateral hippocampus. Some perifocal neurons were also HO-1-immunoreactive. In the infarcted area HO-1-positive microglia/macrophages were detected in double-labelling experiments. A microassay measuring the conversion of [14C]haem to [14C]bilirubin showed a two-fold increase in haem oxygenase activity in the infarcted core. These observations show a long-term induction of HO-1 protein and its activity following ischaemia-reperfusion brain injury, and indicate increased capacity for haem degradation and the generation of biologically active bile products, carbon monoxide and iron in astrocytes and some microglia/macrophages during focal brain ischaemia.

The recent elucidation of the primary structures of different melatonin receptors as well as the deduction of the secondary structure of rhodopsin has allowed us to construct a model for melatonin recognition at its G protein-coupled receptor. To achieve this, we have used the quantum mechanics method Austin model 1 to fully optimize the structures of melatonin and several analogs. We also synthesized three compounds and used the three-dimensional analysis comparative molecular field analysis (CoMFA) to generate a model for the structure-activity relationships of melatonin and 27 melatonin-like compounds. This model predicted with good accuracy the affinities of the synthesized compounds for the melatonin receptor. We propose that recognition of the functional moieties of melatonin occurs through specific interaction of these moieties with fully conserved amino acid residues present in transmembrane helices V, VI and VII of the melatonin receptor. These residues are not found in other members of the G protein-coupled receptor family. The rhodopsin-based model can explain the importance of some structural features of melatonin and related active compounds.

Heme oxygenase activity is the sole known physiological source for the production of carbon monoxide (CO), a gaseous messenger candidate. A sensitive radioenzymatic microassay was validated to study regional distribution of heme oxygenase activity within the rat brain. The assay utilized a 14,000 × g supernatant of brain homogenate and [14C]heme as the substrate. Thin layer chromatography revealed that incubation of cerebellar supernatant with [14C]heme yielded a single reaction product, indistinguishable from bilirubin, that was selectively extracted into toluene. Radioactivity in toluene increased linearly in respect to time and added protein, was totally dependent on NADPH and was not detected with boiled homogenate. The reaction was dose-dependently inhibited by Zn-protoporphyrin IX (IC50 0.3 μM) and by an antibody generated against rat NADPH-cytchrome P450 reductase indicating specific involvement of heme oxygenase. As little as 36 fmol [14C]bilirubin/min could be readily detected requiring only microgram-quantities of cerebellar homogenate. Heme oxygenase activity measurements from discrete brain regions revealed for the first time marked differences in enzyme activity with the increasing order: frontal cortex < cerebellum = caudate-putamen < hippocampus = hypothalamus = colliculi ⪡ trapezoid body. This activity pattern closely reflects the distribution of immunoreactivity and mRNA for heme oxygenase. The present microassay should offer a valuable tool for studies directly assessing a possible role for CO in neural signaling.

1. Innervation of the mammalian pineal gland is mainly sympathetic. Pineal synthesis of melatonin and its levels in the circulation are thought to be under strict adrenergic control of serotonin N-acetyltransferase (NAT). In addition, several putative pineal neurotransmitters modulate melatonin synthesis and secretion. 2. In this review, we summarize what is currently known on the pineal cholinergic system. Cholinergic signaling in the rat pineal gland is suggested based on the localization of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), as well as muscarinic and nicotinic ACh binding sites in the gland. 3. A functional role of ACh may be regulation of pineal synaptic ribbon numbers and modulation of melatonin secretion, events possibly mediated by phosphoinositide (PI) hydrolysis and activation of protein kinase C via muscarinic ACh receptors (mAChRs). 4. We also present previously unpublished data obtained using primary cultures of rat pinealocytes in an attempt to get more direct information on the effects of cholinergic stimulus on pinealocyte melatonin secretion. These studies revealed that the cholinergic effects on melatonin release are restricted mainly to intact pineal glands since they were not readily detected in primary pinealocyte cultures.

Background Lysophosphatidic acid (LPA) is a signalling phospholipid with multiple biological functions, mainly mediated through specific G protein-coupled receptors. Aberrant LPA signalling is being increasingly implicated in the pathology of common human diseases, such as arteriosclerosis and cancer. The lifetime of the signalling pool of LPA is controlled by the equilibrium between synthesizing and degradative enzymatic activity. In the current study, we have characterized these enzymatic pathways in rat brain by pharmacologically manipulating the enzymatic machinery required for LPA degradation. Results In rat brain cryosections, the lifetime of bioactive LPA was found to be controlled by Mg2+-independent, N-ethylmaleimide-insensitive phosphatase activity, attributed to lipid phosphate phosphatases (LPPs). Pharmacological inhibition of this LPP activity amplified LPA1 receptor signalling, as revealed using functional autoradiography. Although two LPP inhibitors, sodium orthovanadate and propranolol, locally amplified receptor responses, they did not affect global brain LPA phosphatase activity (also attributed to Mg2+-independent, N-ethylmaleimide-insensitive phosphatases), as confirmed by Pi determination and by LC/MS/MS. Interestingly, the phosphate analog, aluminium fluoride (AlFx-) not only irreversibly inhibited LPP activity thereby potentiating LPA1 receptor responses, but also totally prevented LPA degradation, however this latter effect was not essential in order to observe AlFx--dependent potentiation of receptor signalling. Conclusions We conclude that vanadate- and propranolol-sensitive LPP activity locally guards the signalling pool of LPA whereas the majority of brain LPA phosphatase activity is attributed to LPP-like enzymatic activity which, like LPP activity, is sensitive to AlFx- but resistant to the LPP inhibitors, vanadate and propranolol.

A microdialysis method combined with a sensitive radioimmunoassay was used to monitor extracellular cyclic GMP (cGMP) levels in the frontal cortex and the cerebellum of anesthetized rats in vivo. Basal cGMP release remained constant throughout the perfusion period and was approximately 2 fmol/30 min in the frontal cortex and approximately 4 fmol/30 min in the cerebellum. The nitric oxide (NO) donor sodium nitroprusside (SNP) stimulated cGMP release transiently in both regions. However, the maximal response was 3-fold in the frontal cortex (obtained with 5 microM SNP) but 90-fold in the cerebellum (obtained with 1 mM SNP). Perfusion with the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) suppressed cerebellar cGMP release by 74% indicating that NO is the major regulator of basal cGMP levels in the cerebellum. Quite opposite, L-NAME exhibited no potency in the frontal cortex suggesting that other activators of guanylyl cyclase may regulate basal cortical cGMP levels in vivo.

To gain some understanding of the factors regulating high affinity melatonin (MT) receptors in the rat, we conducted a series of studies using quantitative autoradiography of [2-125I]iodo-MT binding in vitro with validated assay conditions. MT receptor status and the relative protein content of the autoradiographic sections were assessed in the anterior pituitary gland, the area postrema, the caudal (tail) artery (CA), the anterior cerebral artery (ACA), and the suprachiasmatic nuclei (SCN) of 9-, 96-, and 306-day-old Wistar rats. When age-associated changes in protein content were taken into account, MT receptor expression in the area postrema and the SCN remained relatively constant between 9-306 days of age. On the contrary, a dramatic loss of MT receptors was observed in the arteries of 306-day-old rats (98% and 89% loss compared to the 9-day-old rats in the ACA and CA, respectively). In the anterior pituitary gland, MT receptors were expressed only in the 9-day-old rats. The above changes reflected major changes in binding capacity and minor changes in binding affinity. Neither removal of endogenous circulating MT (acute light exposure for 24 h, pinealectomy, or superior cervical ganglionectomy) nor MT injections (1 mg/kg for 10 days 6 h after lights on) affected MT receptor status in the ACA, CA, area postrema, or SCN. Our data suggest that MT receptor expression is differentially regulated during development and that permanent alterations in MT levels do not affect rat MT receptor status.

Ependymal cells line the brain ventricles and separate the CSF from the underlying neuronal tissue. The function of ependymal cilia is largely unclear however they are reported to be involved in the regulation of CSF homeostasis and host defence against pathogens. Here we present data that implicates a role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the inhibition of ependymal ciliary function, and also that the PACAP effects are not entirely dependent on adenylyl cyclase activation. Primary ependymal cultures were treated with increasing doses of PACAP27 or adenylyl cyclase toxin (ACT), and ciliary beating was recorded using high-speed digital video imaging. Ciliary beat frequency (CBF) and amplitude were determined from the videos. Ependymal CBF and ciliary amplitude were attenuated by PACAP27 in a concentration- and time-dependent manner. The peptide antagonist PACAP6-27 blocked PACAP27-induced decreases in amplitude and CBF. Treatment with ACT caused a decrease in amplitude but had no effect on CBF, this suggests that the inhibition of CBF and amplitude seen with PACAP27 may not be completely explained by G(s)-AC-cAMP pathway. We present here the first observational study to show that activation of PAC1 receptors with PACAP27 has an important role to play in the regulation of ependymal ciliary function.

Melatonin binding sites were localized and characterized in the vasculature of the rat by using the melatonin analogue 2-[125I]iodomelatonin (125I-melatonin) and quantitative in vitro autoradiography. The expression of these sites was restricted to the caudal artery and to the arteries that form the circle of Willis at the base of the brain. The arterial 125I-melatonin binding was stable, saturable, and reversible. Saturation studies revealed that the binding represented a single class of high-affinity binding sites with a dissociation constant (Kd) of 3.4 x 10(-11) M in the anterior cerebral artery and 1.05 x 10(-10) M in the caudal artery. The binding capacities (Bmax) in these arteries were 19 and 15 fmol/mg of protein, respectively. The relative order of potency of indoles for inhibition of 125I-melatonin binding at these sites was typical of a melatonin receptor: 2-iodomelatonin greater than melatonin greater than N-acetylserotonin much much greater than 5-hydroxytryptamine. Norepinephrine-induced contraction of the caudal artery in vitro was significantly prolonged and potentiated by melatonin in a concentration-dependent manner, suggesting that these arterial binding sites are functional melatonin receptors. Neither primary steps in smooth muscle contraction (inositol phospholipid hydrolysis) nor relaxation (adenylate cyclase activation) were affected by melatonin. Melatonin, through its action on the tone of these arteries, may cause circulatory adjustments in these arteries, which are believed to be involved in thermoregulation.

We have localized and characterized the binding of the melatonin agonist, 2-[125I]iodomelatonin, in the rat area postrema (AP), by using quantitative autoradiography in vitro. At equilibrium conditions, Scatchard analysis revealed saturable high-affinity binding to a single class of sites (Kd 45.9 +/- (SE) 6.0 pM and Bmax 30.8 +/- 4.6 fmol/mg protein, n = 4 experiments with a total of 18 rats). Melatonin and 6-hydroxymelatonin were potent displacers of 2-[125I]iodomelatonin binding in the AP (IC50 20 and 500 pM, respectively) while N-acetylserotonin exhibited only a modest potency (IC50 25 nM). Micromolar concentrations of guanine nucleotides dose-dependently and specifically inhibited agonist binding at 22 degrees C. Saturation studies revealed that this was due to a decrease in binding affinity. Divalent cations (4 mM CaCl2 or 2 mM MgCl2) had no detectable effect on the affinity of the binding site, whereas physiological concentrations of NaCl significantly decreased the binding affinity. These results demonstrate specific high-affinity binding sites for 2-[125I]iodomelatonin in the rat AP and suggest coupling of these putative receptors to guanosine nucleotide binding regulatory protein(s).

Melatonin binds and activates G protein-coupled melatonin receptors. The density and affinity of the endogenous melatonin receptors change throughout the 24-hr day, and the exposure of recombinant melatonin receptors to melatonin often results in desensitization of the receptors. Receptor density, G protein activation and expression level were analyzed in CHO cell lines stably expressing the human MT1 receptors after 1 or 72 hr of exposure to melatonin (agonist, 10 nm) and luzindole (antagonist/inverse agonist, 10 microm). The 72-hr exposure to luzindole significantly increased the apparent receptor density in cell lines with both high and low MT1 receptor expression levels (MT1(high) and MT1(low) cells, respectively). In the constitutively active MT1(high) cells, luzindole pretreatment also stimulated the functional response to melatonin in [(35)S]GTPgammaS binding assays, whereas melatonin pretreatment attenuated the functional response at both time points. Receptor ELISA was used to analyze the cell membrane and total expression level of the MT1 receptor in intact and permeabilized cells, respectively. Luzindole pretreatment decreased the total cellular level of MT1 receptor in the MT1(high) cells at both time points but increased the cell surface expression of MT1 receptor at 72 hr. Melatonin significantly decreased MT1 receptor cell surface expression only in MT1(high) cells after a 1-hr treatment. These results indicate that melatonin treatment desensitizes MT1 receptors, whereas luzindole increases ligand binding and G-protein activation. Luzindole also stimulates downregulation of the MT1 receptor protein, interfering with the synthesis and/or degradation of the receptor.

BACKGROUND: Heterotrimeric G proteins take part in membrane-mediated cell signalling and have a role in hormonal regulation. This study clarifies the expression and localization of the G protein subunit G alpha(i2) in the human endometrium and Fallopian tube and changes in G alpha(i2) expression in human endometrium during the menstrual cycle. METHODS: The expression of G alpha(i2) was identified by Polymerase chain reaction (PCR), and localization confirmed by immunostaining. Cyclic changes in G alpha(i2) expression during the menstrual cycle were evaluated by quantitative real-time PCR. RESULTS: We found G alpha(i2) to be expressed in human endometrium, Fallopian tube tissue and in primary cultures of Fallopian tube epithelial cells. Our studies revealed enriched localization of G alpha(i2) in Fallopian tube cilia and in endometrial glands. We showed that G alpha(i2) expression in human endometrium changes significantly during the menstrual cycle, with a higher level in the secretory versus proliferative and menstrual phases (P < 0.05). CONCLUSIONS: G alpha(i2) is specifically localized in human Fallopian tube epithelial cells, particularly in the cilia, and is likely to have a cilia-specific role in reproduction. Significantly variable expression of G alpha(i2) during the menstrual cycle suggests G alpha(i2) might be under hormonal regulation in the female reproductive tract in vivo.

To identify novel selective CB2 lead compounds, a comparative model of the CB2 receptor was constructed using the high-resolution bovine rhodopsin X-ray structure as a template. The CB2 model was utilized both in building the database queries and in filtering the hit compounds by a docking and scoring method. In G-protein activation assays, 1-isoquinolyl[3-(trifluoromethyl)phenyl]methanone (40, NRB 04079) was found to act as a selective agonist at the human CB2 receptor.

The effect of magnetic field (MF) exposure on melatonin production was studied in female CD(2)F(1)(BALB/c x DBA/2) mice. The mice were exposed to a 50 Hz MF at 100 microT for 52 days and nocturnal urine was collected 1, 3, 7, 14, 16 and 23 days after the beginning of MF exposure. The animal room was illuminated for 12 h daily at 200 lux. To study the circadian rhythm of melatonin production, night and day samples of urine were collected once, at about 40 days after the beginning of MF exposure. Urinary 6-hydroxy melatonin sulfate (6-OHMS) was determined to assess melatonin production. The pineal glands were analyzed for melatonin content at the middle of the dark period. No statistically significant peak of melatonin was observed in either group. The light-regulated natural melatonin rhythm was absent in sham-exposed mice. The MF exposure caused a significant day-night difference in the 6-OHMS levels, but did not affect the total excretion of 6-OHMS during the 24-hour period. A possible interpretation of the findings is that MF exposure increases the sensitivity of the pineal gland to light in this strain normally insensitive to the circadian light variations. Further studies on interaction of light and MF exposure might help in understanding the inconsistencies of earlier research on MFs and melatonin.

Adenosine accumulates in the basal forebrain during prolonged wakefulness and induces sleep. There is abundant evidence showing that the sleep-inducing effects are mediated locally in the basal forebrain through the adenosine A1 receptor. In previous studies an increase in the mRNA expression but no apparent change in the ligand binding of the A1 receptors have been found. In the present study we used [(35)S]GTPgammaS autoradiography to assess regional A1 receptor dependent G-protein activity in rat brain during prolonged wakefulness and recovery sleep. We found that the G-protein activity was increased in the cortex but not in the basal forebrain during the first hours of sleep deprivation, suggesting different A1 receptor mediated responses to increasing adenosine concentrations in different brain areas.

1. Several G-protein-coupled receptors (GPCRs) have been localized to various layers of the vertebrate retina, using autoradiographic and immunohistochemical techniques, but the functional data concerning G protein activation are limited. Here, we establish optimized assay conditions to detect receptor-dependent G protein activity in membranes and tissue sections of the rat retina. 2. Agonist-stimulated [35S]GTPγS-binding responses were characterized for the Gi/o-linked adenosine A1, cannabinoid CB1, m2/m4 muscarinic acetylcholine, and GABAB receptors. Initial assumption was that G protein activity under “basal conditions” is high due to enrichment and activity of rhodopsin and transducin in this tissue. 3. We found that pretreatment of retina membranes with hydroxylamine (10 mM), a rhodopsin-inactivating drug, substantially (up to 60%) reduced basal G protein activity, thereby improving signal-to-noise ratio to detect agonist-stimulated G protein activation for all studied receptors. [35S]GTPγS autoradiography revealed that hydroxylamine specifically reduced basal binding in the transducin-enriched photoreceptor layer. In contrast, hydroxylamine did not affect GPCR signaling in brain membranes, indicating specific action on retinal transducin. 4. For all studied receptors, [35S]GTPγS autoradiography allowed localization of G protein activity to different retinal layers, with the bulk of signal detected in the ganglion cell layer. Strongest responses were observed for adenosine and muscarinic receptor agonists. Additional G protein activity was detected in the inner plexiform layer. 5. Responses to all tested agonists were reversed in the presence of appropriate receptor-selective antagonists, indicating receptor-mediated G protein activation. 6. We suggest that [35S]GTPγS-binding assays conducted under the established conditions should facilitate further studies on functional GPCRs in the mammalian retina.

Models of G protein-coupled melatonin receptor structure suggest that ligand recognition occurs in a binding pocket formed by transmembrane helices III, V and VII. Constitutively active mutations in G protein-coupled receptors have revealed that transmembrane helix III/intracellular loop 2 interface and transmembrane domain VI are critical regions in receptor activation. In this study, nine site-directed mutants of the human MT1 melatonin receptor were created to test the importance of specific amino acids in these regions in ligand recognition and receptor activation events. We analyzed ligand binding, G protein activation and subcellular localization of MT1 receptors transiently expressed in COS-7 cells. Receptor ELISA was employed to study expression levels of N-terminally HA epitope tagged wild-type and mutant MT1 receptors. Mutations in histidine H195 (His5.46) in transmembrane domain V reduced receptor affinity for 2-[ 125 I ]iodomelatonin. Several other mutants had diminished expression on the plasma membrane. Amino acids M107 (Met3.32) in transmembrane domain III and S280 (Ser7.46) in transmembrane domain VII were found not to participate in ligand recognition in human MT1 receptor. Constitutive activity was not obtained with mutations in N124 (Asn3.49) or P253 (Pro6.50). These mutants failed to bind 2-[ 125 I ]iodomelatonin and had reduced expression levels. The need to upgrade current melatonin receptor models has become evident. Several important amino acids for the human MT1 melatonin receptor function were revealed in the current study, with effects of mutations ranging from slightly reduced affinity or efficacy to complete loss of function.

Increased expression of heme oxygenase-1 (HO-1) is a common feature in a number of neurodegenerative diseases. Interestingly, the spatial distribution of HO-1 expression in diseased brain is essentially identical to that of pathological expression of tau. In this study, we explored the relationship between HO-1 and tau, using neuroblastoma cells stably transfected with sense and antisense HO-1 constructs as well as with the vector alone. In transfected cells overexpressing HO-1, the activity of heme oxygenase was increased, and conversely, the level of tau protein was dramatically decreased when compared with antisense HO-1 or CEP transfected cells. The suppression of tau protein expression was almost completely reversed by zinc-deuteroporphyrin, a specific inhibitor of heme oxygenase activity. The activated forms of ERKs (extracellular signal-regulated kinases) were also decreased in cells overexpressing HO-1 although no changes in the expression of total ERK-1/2 proteins were observed. These data are in agreement with the finding that the expression of tau is regulated through signal cascades including the ERKs, whose activities are modulated by oxidative stresses. The expression of tau and HO-1 may be regulated by oxidative stresses in a coordinated manner and play a pivotal role in the cytoprotection of neuronal cells.

A recently introduced technique of [gamma-35S]GTP autoradiography was used to localize and characterize muscarinic receptor-dependent activation of G-proteins in tissue sections of the chick optic tectum, a brain region with relatively high expression of G-protein-coupled receptors for the neurotransmitter acetylcholine. Within the highly stratified tectal structure, the bulk of muscarinic receptor-mediated [gamma-35S]GTP signal was localized to the stratum griseum et fibrosum superficiale with considerably lower binding responses in other tectal layers. Quantitative comparison of [gamma-35S]GTP binding responses in tectal sections and membranes revealed a close match between the two tissue preparations for the response elicited by the cholinergic agonist carbachol, its dose-dependent reversal with the non-selective muscarinic antagonist atropine, its approximately 100-fold sensitivity towards blockade with M1-type (pirenzepine) over M2-type (gallamine) muscarinic antagonists, as well as absolute requirement for micromolar concentrations of GDP (EC50 approximately 10 microM) for the receptor-mediated [gamma-35S]GTP response. The pharmacological profile is consistent with that of cm4, a recently cloned chicken homolog of the mammalian m4 muscarinic acetylcholine receptor. Moreover, the strict GDP-dependence of the binding response suggest activation of Gi/o, the inhibitory class of G-proteins. These data provide the first functional characterization of the chick tectal muscarinic receptors. A close match between [gamma-35S]GTP responses in membranes and tissue sections strongly suggest that [gamma-35S]GTP autoradiography offers great potential for studies on G-protein-mediated signaling, with particular use within anatomically restricted regions that are not readily approached with more conventional techniques. It is anticipated that [gamma-35S]GTP autoradiography should greatly facilitate studies on signaling capacity of an individual receptor subtype whilst in its native cellular environment.

A microdialysis method combined with a sensitive radioimmunoassay was used to monitor cGMP release in the frontal cortex of the anesthetized rats in vivo. We assessed the relative contribution of endogenous nitric oxide (NO), and effects of exogenous carbon monoxide (CO) and phosphodiesterase activity, as possible regulators of cortical CGMP levels. Perfusion with CO-saturated aCSF (approximately 1 mM CO) failed to significantly stimulate cortical cGMP levels. For comparison, cerebellar cGMP levels increased by 2-fold during CO stimulation, followed by a prolonged response that was fully reversible with the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Cortical perfusion with zinc protopophyrin-IX (100 microM), a widely used inhibitor of the CO-generating enzyme heme oxygenase, suppressed cGMP levels by 50%, a response that spontaneously recovered in spite of the continuous presence of the metalloporphyrin. Perfusion with isobutylmethyl xanthine IBMX (1 mM) resulted in 5-fold increase in cortical cGMP levels, as compared to basal levels without IBMX. In the presence of IBMX, L-NAME suppressed basal cortical cGMP levels by 70% indicating that NO synthase activity generates the bulk of cGMP in this brain region, as previously shown for basal cGMP production in the hippocampus and the cerebellum. These data also emphasize a crucial role for phosphodiesterase activity in the maintenance of cGMP levels in vivo in the frontal cortex. The relatively weak responses to exogenous CO lend little support for a role of this gas in regulating basal cortical cGMP levels in vivo.