Jarmo T. Laitinen

The serine hydrolases MAGL, ABHD6 and ABHD12 as guardians of 2-arachidonoylglycerol signalling through cannabinoid receptors

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.

Despite substantial degradation, 2‐arachidonoylglycerol is a potent full efficacy agonist mediating CB1 receptor‐dependent G‐protein activation in rat cerebellar membranes

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.

An optimized approach to study endocannabinoid signaling: evidence against constitutive activity of rat brain adenosine A1 and cannabinoid CB1 receptors

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 (10−6 M), did not reduce basal G‐protein activity, indicating that CB1 receptors are not constitutively active in these preparations. At higher concentrations (1–2.5 × 10−5 M), 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.

Biochemical and pharmacological characterization of human α/β-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12).

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.

Different patterns of light exposure in relation to melatonin and cortisol rhythms and sleep of night workers

Koller M, Harma M, Laitinen JT, Kundi M, Piegler B, Haider M. Different patterns of light exposure in relation to melatonin and Cortisol rhythms and sleep of night workers. J. Pineal Res. 1994: 16: 127–135.

Guanosine 5′-(γ-[35S]Thio)triphosphate Autoradiography Allows Selective Detection of Histamine H3 Receptor-Dependent G Protein Activation in Rat Brain Tissue Sections†

Abstract: Histamine elicits its biological effects via three distinct G protein‐coupled receptors, termed H1, H2, and H3. We have used guanosine 5′‐(γ‐[35S]thio)triphosphate (GTPγ[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 GTPγ[35S] signal in several brain regions. All further incubations therefore contained 8‐cyclopentyl‐1,3‐dipropylxanthine (10 µM), a selective A1 receptor antagonist. Histamine elicited dose‐dependent increments in GTPγ[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 Nα‐methylhistamine, (R)‐α‐methylhistamine, and immepip, three H3‐selective agonists. In all regions examined, the GTPγ[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, GTPγ[35S] autoradiography selectively detects H3 receptor‐dependent signaling in response to histamine stimulation. As the existing evidence suggests that GTPγ[35S] autoradiography preferentially reveals responses to Gi/o‐coupled receptors, our data indicate that most, if not all, central H3 binding sites represent functional receptors coupling to Gi/o, the inhibitory class of G proteins. Besides allowing more detailed studies on H3 receptor signaling within anatomically restricted regions of the CNS, GTPγ[35S] autoradiography offers a novel approach for functional in vitro screening of H3 ligands.

Characterization of Melatonin Receptors in the Rat Area postrema: Modulation of Affinity with Cations and Guanine Nucleotides

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).

A rhodopsin-based model for melatonin recognition at its G protein-coupled receptor

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.

Effects of mobile phone radiation on UV-induced skin tumourigenesis in ornithine decarboxylase transgenic and non-transgenic mice.

Purpose: The effects of low-level radiofrequency radiation (RFR) on ultraviolet (UV)-induced skin tumorigenesis were evaluated in ornithine decarboxylase (ODC) and non-transgenic mice. Materials and methods: 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 ×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. Results: 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. Conclusion: RFR exposures did not significantly enhance skin tumourigenesis. However, the slightly accelerated tumour development may warrant further evaluation.

The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D–lysophosphatidic acid receptor axis and 5‐HT1A receptors in rat brain sections

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 compounds 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 compounds ability to release histamine from mast cells.