Rat Brain Membrane Preparations Research Articles

Isolation and Biological Activity of 8- Epitetrodotoxin and the Structure of a Possible Biosynthetic Shunt Product of Tetrodotoxin, Cep-226A, from the Newt Cynops ensicauda popei.

Tetrodotoxin (TTX, 1), a potent neurotoxin, has been found in various animal species in both marine and terrestrial environments. In this study, a new TTX analogue, 8- epiTTX (2), and a possible biosynthetic shunt compound of TTX, Cep-226A (3), were isolated from the newt Cynops ensicauda popei. The voltage-gated sodium ion channel (Nav) blocking activity of 2 and 6- epiTTX (4), a known analogue, were investigated by a colorimetric cell-based assay and compared with that of 1. The EC50 values for 2 and 4 were determined to be 110 ± 40 and 33 ± 11 nM, respectively, which were larger than that of 1 (1.9 ± 0.7 nM). The results indicated that the equatorial hydroxy group at C-8 in TTX significantly contributes to its Nav blocking activity, whereas the 6-epimer of TTX retains substantial activity, consistent with its previously reported toxicity in mice and binding affinity to rat brain membrane preparations. The presence of these epimers of TTX (2 and 4) and Cep-226A (3) in newts supports our hypothesis that TTX is derived from a monoterpene in terrestrial environments.

Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide.

Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology.

R-phenibut binds to the α2–δ subunit of voltage-dependent calcium channels and exerts gabapentin-like anti-nociceptive effects

Phenibut is clinically used anxiolytic, mood elevator and nootropic drug. R-phenibut is responsible for the pharmacological activity of racemic phenibut, and this activity correlates with its binding affinity for GABAB receptors. In contrast, S-phenibut does not bind to GABAB receptors. In this study, we assessed the binding affinities of R-phenibut, S-phenibut, baclofen and gabapentin (GBP) for the α2–δ subunit of the voltage-dependent calcium channel (VDCC) using a subunit-selective ligand, radiolabelled GBP. Binding experiments using rat brain membrane preparations revealed that the equilibrium dissociation constants (Kis) for R-phenibut, S-phenibut, baclofen and GBP were 23, 39, 156 and 0.05μM, respectively. In the pentylenetetrazole (PTZ)-induced seizure test, we found that at doses up to 100mg/kg, R-phenibut did not affect PTZ-induced seizures. The anti-nociceptive effects of R-phenibut were assessed using the formalin-induced paw-licking test and the chronic constriction injury (CCI) of the sciatic nerve model. Pre-treatment with R-phenibut dose-dependently decreased the nociceptive response during both phases of the test. The anti-nociceptive effects of R-phenibut in the formalin-induced paw-licking test were not blocked by the GABAB receptor-selective antagonist CGP35348. In addition, treatment with R- and S-phenibut alleviated the mechanical and thermal allodynia induced by CCI of the sciatic nerve. Our data suggest that the binding affinity of R-phenibut for the α2–δ subunit of the VDCC is 4 times higher than its affinity for the GABAB receptor. The anti-nociceptive effects of R-phenibut observed in the tests of formalin-induced paw licking and CCI of the sciatic nerve were associated with its effect on the α2–δ subunit of the VDCC rather than with its effects on GABAB receptors. In conclusion, our results provide experimental evidence for GBP-like, anti-nociceptive properties of R-phenibut, which might be used clinically to treat neuropathic pain disorders.

Design, synthesis and biological evaluation of bivalent ligands against A1–D1 receptor heteromers

To design and synthesize bivalent ligands for adenosine A1-dopamine D1 receptor heteromers (A1-D1R), and evaluate their pharmacological activities. Bivalent ligands and their corresponding A1R monovalent ligands were designed and synthesized. The affinities of the bivalent ligands for A1R and D1R in rat brain membrane preparation were examined using radiolabeled binding assays. To demonstrate the formation of A1-D1R, fluorescence resonance energy transfer (FRET) was conducted in HEK293 cells transfected with D1-CFP and A1-YFP. Molecular modeling was used to analyze the possible mode of protein-protein and protein-ligand interactions. Two bivalent ligands for A1R and D1R (20a, 20b), as well as the corresponding A1R monovalent ligands (21a, 21b) were synthesized. In radiolabeled binding assays, the bivalent ligands showed affinities for A1R 10-100 times higher than those of the corresponding monovalent ligands. In FRET experiments, the bivalent ligands significantly increased the heterodimerization of A1R and D1R compared with the corresponding monovalent ligands. A heterodimer model with the interface of helixes 3, 4, 5 of A1R and helixes 1, 6, 7 from D1R was established with molecular modeling. The distance between the two ligand binding sites in the heterodimer model was approximately 48.4 Å, which was shorter than the length of the bivalent ligands. This study demonstrates the existence of A1-D1R in situ and a simultaneous interaction of bivalent ligands with both the receptors.

Opiorphin highly improves the specific binding and affinity of MERF and MEGY to rat brain opioid receptors

Endogenously occurring opioid peptides are rapidly metabolized by different ectopeptidases. Human opiorphin is a recently discovered natural inhibitor of the enkephalin-inactivating neutral endopeptidase (NEP) and aminopeptidase-N (AP-N) (Wisner et al., 2006). To date, in vitro receptor binding experiments must be performed either in the presence of a mixture of peptidase inhibitors and/or at low temperatures, to block peptidase activity. Here we demonstrate that, compared to classic inhibitor cocktails, opiorphin dramatically increases the binding of [3H]MERF and [3H]MEGY ligands to rat brain membrane preparations. We found that at 0°C the increase in specific binding is as high as 40–60% and at 24°C this rise was even higher. In contrast, the binding of the control [3H]endomorphin-1, which is relatively slowly degraded in rat brain membrane preparations, was not enhanced by opiorphin compared to other inhibitors. In addition, in homologous binding displacement experiments, the IC50 affinity values measured at 24°C were also significantly improved using opiorphin compared to the inhibitor cocktail. In heterologous binding experiments the differences were less obvious, but still pronounced using [3H]MERF and MEGY compared to dynorphin1–11, or naloxone and DAGO competitor ligands.

Determination of Paralytic Shellfish Toxins in Shellfish by Receptor Binding Assay: Collaborative Study

A collaborative study was conducted on a microplate format receptor binding assay (RBA) for paralytic e shellfish toxins (PST). The assay quantifies the composite PST toxicity in shellfish samples based on the ability of sample extracts to compete with (3)H saxitoxin (STX) diHCl for binding to voltage-gated sodium channels in a rat brain membrane preparation. Quantification of binding can be carried out using either a microplate or traditional scintillation counter; both end points were included in this study. Nine laboratories from six countries completed the study. One laboratory analyzed the samples using the precolumn oxidation HPLC method (AOAC Method 2005.06) to determine the STX congener composition. Three laboratories performed the mouse bioassay (AOAC Method 959.08). The study focused on the ability of the assay to measure the PST toxicity of samples below, near, or slightly above the regulatory limit of 800 (microg STX diHCl equiv./kg). A total of 21 shellfish homogenates were extracted in 0.1 M HCl, and the extracts were analyzed by RBA in three assays on separate days. Samples included naturally contaminated shellfish samples of different species collected from several geographic regions, which contained varying STX congener profiles due to their exposure to different PST-producing dinoflagellate species or differences in toxin metabolism: blue mussel (Mytilus edulis) from the U.S. east and west coasts, California mussel (Mytilus californianus) from the U.S. west coast, chorito mussel (Mytilus chiliensis) from Chile, green mussel (Perna canaliculus) from New Zealand, Atlantic surf clam (Spisula solidissima) from the U.S. east coast, butter clam (Saxidomus gigantea) from the west coast of the United States, almeja clam (Venus antiqua) from Chile, and Atlantic sea scallop (Plactopecten magellanicus) from the U.S. east coast. All samples were provided as whole animal homogenates, except Atlantic sea scallop and green mussel, from which only the hepatopancreas was homogenized. Among the naturally contaminated samples, five were blind duplicates used for calculation of RSDr. The interlaboratory RSDR of the assay for 21 samples tested in nine laboratories was 33.1%, yielding a HorRat value of 2.0. Removal of results for one laboratory that reported systematically low values resulted in an average RSDR of 28.7% and average HorRat value of 1.8. Intralaboratory RSDr based on five blind duplicate samples tested in separate assays, was 25.1%. RSDr obtained by individual laboratories ranged from 11.8 to 34.9%. Laboratories that are routine users of the assay performed better than nonroutine users, with an average RSDr of 17.1%. Recovery of STX from spiked shellfish homogenates was 88.1-93.3%. Correlation with the mouse bioassay yielded a slope of 1.64 and correlation coefficient (r(2)) of 0.84, while correlation with the precolumn oxidation HPLC method yielded a slope of 1.20 and an r(2) of 0.92. When samples were sorted according to increasing toxin concentration (microg STX diHCl equiv./kg) as assessed by the mouse bioassay, the RBA returned no false negatives relative to the 800 microg STX diHCl equiv./kg regulatory limit for shellfish. Currently, no validated methods other than the mouse bioassay directly measure a composite toxic potency for PST in shellfish. The results of this interlaboratory study demonstrate that the RBA is suitable for the routine determination of PST in shellfish in appropriately equipped laboratories.

Radiotracers, tritium labelling of neuropeptides

Tritium labelled endomorphins (Tyr-Pro-Trp-Phe-NH 2, EM1, Tyr-Pro-Phe-Phe-NH 2, EM2) and their analogs, Angiotensin-IV (Val- Tyr-Ile-His-Pro -Phe) and its analogs, Neuromedin N (LysIle-Pro-Tyr-Ile-Leu) and a new nociceptin analog (P he-Phe-Gly-pFPhe-Thr-Gly-Aib-Arg-LysSer-Ala-Arg-Lys-Arg-Lys-Asn-Gln-NH 2) and Substance P analogs (Arg-Pro-Lys-Pro-Gln-GlnPhe-Phe-Gly-Leu-Nle-NH 2, Arg-Pro-Lys-Pro-Gln-Gln-Phe) were synthesized by catalytic dehalogenation or saturation of the precursor pepti des with tritium gas. The specific radioactivity of the neuropeptides is high, ranging from 0.6 TBq/ mmol to 2.8 TBq/mmol. The labelled peptides were characterized by radioreceptor bindin g assays and used in mapping their metabolic pathway using rat brain membrane preparation.

Further Characterization of a Novel Tetrapeptide with an Analgesic Action in the Central and Peripheral Nervous System in Rats

The novel tetrapeptide FLPS has been previously shown to induce antinociception in a model of post-incisional pain in the rat. It has been demonstrated in membrane preparations of rat brain that 10 μM tetrapeptide did not compete with [3H]naloxone for its binding site on the opioid receptors which differentiates it from typical opioids. In the first set of experiments, the stimulation of [35S]GTPγS binding by the tetrapeptide to membrane preparations of recombinant cell lines expressing human μ, δ, and κ receptors has been investigated. The specific [35S]GTPγS binding did not change in the presence of 100 μM tetrapeptide and DAMGO, DPDPE, or U-69593 at submaximal and maximal concentrations, indicating that the tetrapeptide was not an agonist or antagonist of the opioid receptors and it did not stimulate or blocked the stimulation of G-proteins coupled to these receptors. Studies with naloxone and naloxone methiodide pretreatment suggested that the tetrapeptide was either directly or indirectly affected by a naloxone-binding site located primarily within the central nervous system and to a lesser extent in the periphery. Naloxone pretreatment had a dual effect on morphine antinociception based on the concentration used. At 2 mg/kg, naloxone competed reversibly with 25 mg/kg morphine on its binding site on the opioid receptors, while at 5.4 mg/kg it blocked antinociception induced by 10 mg/kg morphine, 2.7 mg/kg aspirin, or 75 mg/kg tetrapeptide. These data suggest that a new naloxone-binding site is involved in alleviation of pain by at least three classes of analsegics (opioids, cycloogenase 2 inhibitors, and the tetrapeptide). The biological activity of the peptide was discovered first and the mechanism of action is now being studied. In an attempt to identify the therapeutic target of the tetrapeptide, a total of 23 radioligand binding assays to targets within the CNS were conducted. The results of these assays were negative and they reinforce the notion that the tetrapeptide activates an unknown mechanism involved in pain perception after surgery.

Synthesis of tritium‐labeled levetiracetam ((2S)‐2‐(2‐oxopyrrolidin‐1‐yl)butanamide) with high specific activity

A method for the preparation of [³H]levetiracetam with a high specific activity of 98 Ci/mmol (3.6 TBq/mmol) is described. The radioligand proved to be highly useful for the labeling of specific levetiracetam binding sites in rat brain membrane preparations. Copyright © 2011 John Wiley & Sons, Ltd.

The extracts of Fructus Akebiae, a preparation containing 90% of the active ingredient hederagenin: Serotonin, norepinephrine and dopamine reuptake inhibitor

Fructus Akebiae is a traditional Chinese herbal extract that has been used for the treatment of depressive disorders in China. Previous studies demonstrated that Fructus Akebiae extracts (FAE) displayed a potent antidepressant-like activity in animal behavior tests and found that the specific active ingredient from the extracts of Fructus Akebiae is hederagenin. However, the underlying mechanism is unknown. Here we provide evidences that FAE enhances the signaling of central monoamines via inhibition of the reuptake of the extracellular monoamines including serotonin (5-HT), norepinephrine (NE) and dopamine (DA). In rat brain membrane preparations and HEK293 cells transfected with human serotonin transporter (SERT), NE transporter (NET) and DA transporter (DAT), we found that FAE displayed marked affinity to rat and cloned human monoamine transporters in ex vivo and in vitro experiments, using competitive radio ligand binding assay. In uptake assays using rat synaptosomes and transfected cells, FAE was found to significantly inhibit all three monoamine transporters in a dose- and time-dependent manner, with a comparable or better potency to their corresponding specific inhibitors. In contrast, FAE (10μM), showed no significant affinity to a variety array of receptors tested from CNS. In support of our uptake data, in vivo microdialysis studies showed that administration of FAE (12.6, 25, 50mg/kg) significantly increased extracellular concentrations of 5-HT, NE and DA in frontal cortex of freely moving rats. Taken together, our current study showed for the first time that FAE is a novel triple inhibitor of monoamine transporters, which may be one the mechanisms of its antidepressant activity.

Novel selective cannabinoid CB1 receptor antagonist MJ08 with potent in vivo bioactivity and inverse agonistic effects

To characterize the biological profiles of MJ08, a novel selective CB(1) receptor antagonist. Radioligand binding assays were performed using rat brain and spleen membrane preparations. CB(1) and CB(2) receptor redistribution and intracellular Ca(2+) ([Ca(2+)](i)) assays were performed with IN CELL Analyzer. Inverse agonism was studied using intracellular cAMP assays, and in guinea-pig ileum and mouse vas deferens smooth muscle preparations. In vivo pharmacologic profile was assessed in diet-induced obesity (DIO) mice. In radioligand binding assay, MJ08 selectively antagonized CB(1) receptor (IC(50)=99.9 nmol/L). In EGFP-CB(1)_U2OS cells, its IC(50) value against CB(1) receptor activation was 30.23 nmol/L (SR141716A: 32.16 nmol/L). WIN 55,212-2 (1 μmol/L) increased [Ca(2+)](i) in the primary cultured hippocampal neuronal cells and decreased cAMP accumulation in CHO-hCB(1) cells. MJ08 (10 nmol/L-10 μmol/L) blocked both the WIN 55,212-2-induced effects. Furthermore, MJ08 reversed the inhibition of electrically evoked twitches of mouse vas deferens by WIN 55,212-2 (pA(2)=10.29±1.05). MJ08 and SR141716A both showed an inverse agonism activity by markedly promoting the contraction force and frequency of guinea pig ileum muscle. MJ08 significantly increased the cAMP level in CHO-hCB(1) cells with an EC(50) value of 78.6 nmol/L, which was lower than the EC(50) value for SR141716A (159.2 nmol/L). Besides the more potent pharmacological effects of cannabinoid CB(1) receptor antagonism in DIO mice, such as reducing food intake, decreasing body weight, and ameliorating dyslipidemia, MJ08 (10 mg/kg) unexpectedly raised the fasted blood glucose in vivo. MJ08 is a novel, potent and selective CB(1) receptor antagonist/inverse agonist with potent bioactive responses in vitro and in vivo that may be useful for disclosure the versatile nature of CB(1) receptors.

Alkamides and a neolignan from Echinacea purpurea roots and the interaction of alkamides with G-protein-coupled cannabinoid receptors

Multiple chromatographic separations of the CHCl 3-soluble extract of the roots of Echinacea purpurea led to the isolation of 19 compounds. Four natural products, three alkamides and nitidanin diisovalerianate, were identified, and five further compounds were detected for the first time in this species. Additionally, 10 known E. purpurea metabolites were isolated. The structures were determined by mass spectrometry and advanced 1D and 2D NMR techniques. The bioactivity of the isolated compounds was studied in [ 35S]GTPγS-binding experiments performed on rat brain membrane preparations. Both partial and inverse agonist compounds for cannabinoid (CB1) receptors were identified among the metabolites, characterized by weak to moderate interactions with the G-protein signaling mechanisms. The G-protein-modulating activities of the Echinacea compounds are rather far from the full agonist effects seen with the CB1 receptor agonist reference compound arachidonyl-2′-chloroethylamide (ACEA). However, upon coadministration with ACEA, a number of them proved capable of inhibiting the stimulation of the pure agonist, thereby demonstrating cannabinoid receptor antagonist properties.

JO 1784, a potent and selective ligand for rat and mouse brain σ-sites

JO 1784 ((+)-cinnamyl-1-phenyl-1-N-methyl-N-cyclopropylene) is a potent ligand for (+)-[3H]SKF 10,047 (2'-hydroxy-5,9-dimethyl-2-allyl-6,7-benzomorphan) binding sites in rat brain membrane preparations with an IC50 of 39 +/- 8 nM, which is comparable to that of haloperidol. The stereoisomer of JO 1784 is ten fold less potent. When administered to mice i.p. or p.o. JO 1784 displaced (+)-[3H]SKF 10,047 (5 muCi i.v.) from its sites in the brain with ID50 values of 1.2 and 3.5 mg kg-1, respectively. The high selectivity of JO 1784 for the sigma-binding site was assessed by its lack of significant affinity for more than 20 other sites including those for phencyclidine.

The Serine Protease Plasmin Cleaves the Amino-terminal Domain of the NR2A Subunit to Relieve Zinc Inhibition of the N-Methyl-d-aspartate Receptors

Zinc is hypothesized to be co-released with glutamate at synapses of the central nervous system. Zinc binds to NR1/NR2A N-methyl-d-aspartate (NMDA) receptors with high affinity and inhibits NMDAR function in a voltage-independent manner. The serine protease plasmin can cleave a number of substrates, including protease-activated receptors, and may play an important role in several disorders of the central nervous system, including ischemia and spinal cord injury. Here, we demonstrate that plasmin can cleave the native NR2A amino-terminal domain (NR2A(ATD)), removing the functional high affinity Zn(2+) binding site. Plasmin also cleaves recombinant NR2A(ATD) at lysine 317 (Lys(317)), thereby producing a approximately 40-kDa fragment, consistent with plasmin-induced NR2A cleavage fragments observed in rat brain membrane preparations. A homology model of the NR2A(ATD) predicts that Lys(317) is near the surface of the protein and is accessible to plasmin. Recombinant expression of NR2A with an amino-terminal deletion at Lys(317) is functional and Zn(2+) insensitive. Whole cell voltage-clamp recordings show that Zn(2+) inhibition of agonist-evoked NMDA receptor currents of NR1/NR2A-transfected HEK 293 cells and cultured cortical neurons is significantly reduced by plasmin treatment. Mutating the plasmin cleavage site Lys(317) on NR2A to alanine blocks the effect of plasmin on Zn(2+) inhibition. The relief of Zn(2+) inhibition by plasmin occurs in PAR1(-/-) cortical neurons and thus is independent of interaction with protease-activated receptors. These results suggest that plasmin can directly interact with NMDA receptors, and plasmin may increase NMDA receptor responses through disruption or removal of the amino-terminal domain and relief of Zn(2+) inhibition.

Capsazepine reversible inhibition of opioid receptors by capsaicin

Event Abstract Back to Event Capsazepine reversible inhibition of opioid receptors by capsaicin Maria Wollemann1, Eniko Ioja1 and Sándor Benyhe1* 1 Institute of Biochemistry, Hungarian Academy of Sciences, Hungary Capsaicin inhibited the binding of endogenous opioid and opioid-like ligands (endomorphin-1, nociceptin and dynorphin-(1-17) in rat brain membrane preparations in vitro. The affinity decrease was most pregnant using endomorphin-1, followed by nociceptin and dynorphin. The inhibition of capsaicin on opioid binding was not observed using non endogenous peptides and synthetic ligands (DAMGO, morphine, naloxone). In the [3H]endomorphin binding assay capsazepine antagonized the inhibitory effect of capsaicin in rat brain membranes, which suggests that capsaicin acts on opioid receptors through the vanilloid receptor (TRPV1). In Chinese hamster ovary cells stable expressing -opioid receptors and lacking TRPV1 receptors, the inhibitory effect of capsaicin on the binding of [3H]endomorphin-1 was not present. It was concluded that the inhibitory effect of capsaicin on the affinity of opioid endogenous peptides in rat brain membrane preparations is not a direct effect, but indirectly other intermediate factors like protein kinases play an important role. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Pathophysiology and neurology - non-degenerative disorders Citation: Wollemann M, Ioja E and Benyhe S (2009). Capsazepine reversible inhibition of opioid receptors by capsaicin. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.120 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Mar 2009; Published Online: 04 Mar 2009. * Correspondence: Sándor Benyhe, Institute of Biochemistry, Hungarian Academy of Sciences, Szeged, Hungary, benyhe@brc.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Maria Wollemann Eniko Ioja Sándor Benyhe Google Maria Wollemann Eniko Ioja Sándor Benyhe Google Scholar Maria Wollemann Eniko Ioja Sándor Benyhe PubMed Maria Wollemann Eniko Ioja Sándor Benyhe Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Binding studies of novel, non-mammalian enkephalins, structures predicted from frog and lungfish brain cDNA sequences

Leu- and Met-enkephalin were the first endogenous opioid peptides identified in different mammalian species including the human. Comparative biochemical and bioinformatic evidence indicates that enkephalins are not limited to mammals. Various prodynorphin (PDYN) sequences in lower vertebrates revealed the presence of other enkephalin fingerprints in these precursor polypeptides. Among the novel enkephalins Ile-enkephalin (Tyr-Gly-Gly-Phe-Ile) was primarily observed in the African clawed frog (Xenopus laevis) PDYNs, while the structure of Phe-enkephalin (Tyr-Gly-Gly-Phe-Phe) was predicted by analyzing brain cDNA sequences encoding a PDYN of the African lungfish (Protopterus annectens). Ile-enkephalin can also be found in the PDYNs of four other fish species including the eel, bichir, zebrafish and tilapia, but no further occurrence for the Phe-enkephalin motif is available as yet. Based on sequencing data, the biological relevance of Phe- and Ile-enkephalin is suggested, because both of them can arise by regular posttranslational enzymatic processing of the respective neuropeptide precursors. In various receptor binding assays performed on rat brain membrane preparations both of the new peptides turned out to be moderate affinity opioids with a weak preference for the δ-opioid receptor (DOP) sites. Phe-enkephalin of the lungfish displayed rather unexpectedly low affinities toward the μ-opioid receptor (MOP) and DOP, while exhibiting moderate affinity toward the κ-opioid receptor (KOP). In receptor-mediated G-protein activation assays measured by the stimulation of [35S]GTPγS binding, Met-enkephalin produced the highest stimulation followed by Leu-enkephalin, Ile-enkephalin and Phe-enkephalin, whereas the least efficacious among these endogenous peptides was still more effective than the prototype opiate agonist morphine in these functional tests.

Capsaicin inhibits the in vitro binding of peptides selective for μ- and κ-opioid, and nociceptin-receptors

Capsaicin inhibited the equilibrium specific binding of endogenous opioid-like peptide ligands such as endomorphin-1, nociceptin, and dynorphin (1–17) in rat brain membrane preparations. We studied the in vitro effect of capsaicin (1–10 μM) on homologous and heterologous competitive binding of opioid ligands, using unlabeled synthetic peptides and the following tritiated compounds: [ 3H]endomorphin-1, [ 3H]endomorphin-2, [ 3H]nociceptin (1–17) and [ 3H]dynorphin (1–17). Capsaicin-dependent inhibition was also observed in [ 35S]GTPγS stimulation assays in the presence of certain opioid peptides. The inhibition of opioid binding was further investigated using other synthetic and natural μ-opioid ligands such as [ d-Ala 2, NMePhe 4,Gly 5-ol]enkephalin (DAMGO), morphine and naloxone. The decrease in opioid ligand affinity upon capsaicin treatments was most apparent with endomorphin-1, followed by nociceptin and dynorphin. The binding of other investigated opioids were not affected in the presence of capsaicin. In [ 3H]endomorphin-1 binding assays, capsazepine antagonized the inhibitory effect of capsaicin in rat brain membranes suggesting the involvement of TRPV1 receptors. In Chinese hamster ovary (CHO) cells stably expressing μ-opioid receptors, but lacking vanilloid receptors, the inhibition by capsaicin on the binding of [ 3H]endomorphin-1 was not present. It is concluded that the inhibitory effect of capsaicin on the receptor binding affinity of endogenous opioid peptides in brain membrane preparations seems not to be a direct effect, it is rather a negative feedback interaction with opioid receptors.

Intrinsic potency of synthetically prepared brevetoxin cysteine metabolites BTX-B2 and desoxyBTX-B2

In mammals and shellfish, brevetoxins produced by the dinoflagellate Karenia brevis are rapidly metabolized to cysteine conjugates. These metabolites identified by mass spectrometry are produced in abundance in mammals and are potentially major bioactive products for intoxication. They are also abundant metabolites in shellfish where they are, in contrast to mammals, retained for prolonged periods, posing a potential threat to shellfish consumers. In this work, we analyze the intrinsic potency of the semi-synthetic cysteine brevetoxin sulfoxide (BTX-B2) and the cysteine brevetoxin (desoxyBTX-B2), each confirmed for purity by LC–MS and NMR techniques, on receptor site 5 of the voltage-gated sodium channels (VGSCs) in brain, heart and skeletal muscle. We show that both brevetoxin conjugates compete with the tritiated reduced parent brevetoxin ([ 3H]PbTx-3) in rat brain membrane preparations and in HEK cells expressing skeletal muscle or cardiac VGSC, albeit, with 8–16-fold lower affinity than the PbTx-3. On neuroblastoma cell assays we show a 3-fold reduction in cytotoxic potency for BTX-B2 relative to PbTx-3, and an 8-fold reduction for desoxyBTX-B2. In conclusion, the major transformation product of brevetoxin observed in diverse species through cysteine adduction and oxidation leads to metabolites with reduced potency on brain, skeletal muscle and heart cells.

Inhibition of fatty acid amide hydrolase, a key endocannabinoid metabolizing enzyme, by analogues of ibuprofen and indomethacin

There is evidence in the literature that the nonsteroidal anti-inflammatory drugs indomethacin and ibuprofen can interact with the cannabinoid system both in vitro and in vivo. In the present study, a series of analogues of ibuprofen and indomethacin have been investigated with respect to their ability to inhibit fatty acid amide hydrolase, the enzyme responsible for the hydrolysis of the endogenous cannabinoid anandamide. Of the fourteen compounds tested, the 6-methyl-pyridin-2-yl analogue of ibuprofen (“ibu-am5”) was selected for further study. This compound inhibited rat brain anandamide hydrolysis in a non-competitive manner, with IC 50 values of 4.7 and 2.5 μM being found at pH 6 and 8, respectively. By comparison, the IC 50 values for ibuprofen were 130 and 750 μM at pH 6 and 8, respectively. There was no measurable N-acylethanolamine hydrolyzing acid amidase activity in rat brain membrane preparations. In intact C6 glioma cells, ibu-am5 inhibited the hydrolysis of anandamide with an IC 50 value of 1.2 μM. There was little difference in the potencies of ibu-am5 and ibuprofen towards cyclooxygenase-1 and -2 enzymes, and neither compound inhibited the activity of monoacylglycerol lipase. Ibu-am5 inhibited the binding of [ 3H]-CP55,940 to rat brain CB 1 and human CB 2 cannabinoid receptors more potently than ibuprofen, but the increase in potency was less than the corresponding increase in potency seen for inhibition of FAAH activity. It is concluded that ibu-am5 is an analogue of ibuprofen with a greater potency towards fatty acid amide hydrolase but with a similar cyclooxygenase inhibitory profile, and may be useful for the study of the therapeutic potential of combined fatty acid amide hydrolase–cyclooxygenase inhibitors.

The β-Adrenergic Receptor Antagonist Metipranolol Blunts Zinc-Induced Photoreceptor and RPE Apoptosis

To determine the effect of zinc on retinal cells at concentrations at which it is known to cause oxidative stress. Furthermore, the effects of metipranolol, known to prevent retinal damage, and of other antiglaucoma drugs were determined on zinc-injured retinal cells. Lipid peroxidation assays were conducted on rat brain and bovine retina-retinal pigment epithelial (RPE) membrane preparations. Immunohistochemistry, immunoblot analysis and the terminal-deoxynucleotidyl transferase dUTP-linked nick-end labeling (TUNEL) procedure determined the effects of zinc with or without trolox or metipranolol on photoreceptor death in situ. The effect of treatments on cultured RPE cells was analyzed using cell viability assays, immunoblot analysis, and the TUNEL procedure. Zinc-induced lipid peroxidation of rat brain and bovine retina-RPE membranes, although the effect of the latter was of a (twofold) greater magnitude. Both effects, however, were similarly attenuated by metipranolol, desacetylmetipranolol, and trolox. Antiglaucoma drugs other than metipranolol had no effect. Intraocular injection of 150 microM zinc and treatment of cultured RPE cells with zinc led to mainly photoreceptor apoptosis and apoptotic death of RPE cells (50% death at 18 microM rising to 10% at 50 microM), respectively. Zinc-induced apoptosis of cultured RPE cells and photoreceptors were attenuated only by metipranolol and trolox. The combined data suggest that oxidative injury to RPE cells and photoreceptors may be caused by elevated levels of zinc in diseases such as age-related macular degeneration (AMD) and that metipranolol may act as an efficacious antioxidant to blunt this process.