Brain Membrane Preparations Research Articles

Effects of 7,8-Dihydroxyflavone on Lipid Isoprenoid and Rho Protein Levels in Brains of Aged C57BL/6 Mice

Synaptic impairment may be the main cause of cognitive dysfunction in brain aging that is probably due to a reduction in synaptic contact between the axonal buttons and dendritic spines. Rho proteins including the small GTPase Rac1 have become key regulators of neuronal morphogenesis that supports synaptic plasticity. Small Rho- and Ras-GTPases are post-translationally modified by the isoprenoids geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), respectively. For all GTPases, anchoring in the plasma membrane is essential for their activation by guanine nucleotide exchange factors (GEFs). Rac1-specific GEFs include the protein T lymphoma invasion and metastasis 1 (Tiam1). Tiam1 interacts with the TrkB receptor to mediate the brain-derived neurotrophic factor (BDNF)-induced activation of Rac1, resulting in cytoskeletal rearrangement and changes in cellular morphology. The flavonoid 7,8-dihydroxyflavone (7,8-DHF) acts as a highly affine-selective TrkB receptor agonist and causes the dimerization and autophosphorylation of the TrkB receptor and thus the activation of downstream signaling pathways. In the current study, we investigated the effects of 7,8-DHF on cerebral lipid isoprenoid and Rho protein levels in male C57BL/6 mice aged 3 and 23 months. Aged mice were daily treated with 100 mg/kg b.w. 7,8-DHF by oral gavage for 21 days. FPP, GGPP, and cholesterol levels were determined in brain tissue. In the same tissue, the protein content of Tiam1 and TrkB in was measured. The cellular localization of the small Rho-GTPase Rac1 and small Rab-GTPase Rab3A was studied in total brain homogenates and membrane preparations. We report the novel finding that 7,8-DHF restored levels of the Rho proteins Rac1 and Rab3A in membrane preparations isolated from brains of treated aged mice. The selective TrkB agonist 7,8-DHF did not affect BDNF and TrkB levels, but restored Tiam1 levels that were found to be reduced in brains of aged mice. FPP, GGPP, and cholesterol levels were significantly elevated in brains of aged mice but not changed by 7,8-DHF treatment. Hence, 7,8-DHF may be useful as pharmacological tool to treat age-related cognitive dysfunction although the underlying mechanisms need to be elucidated in detail.

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.

Biocatalytic Detoxification of Paralytic Shellfish Toxins.

Small molecules that bind to voltage-gated sodium channels (VGSCs) are promising leads in the treatment of numerous neurodegenerative diseases and pain. Nature is a highly skilled medicinal chemist in this regard, designing potent VGSC ligands capable of binding to and blocking the channel, thereby offering compounds of potential therapeutic interest. Paralytic shellfish toxins (PSTs), produced by cyanobacteria and marine dinoflagellates, are examples of these naturally occurring small molecule VGSC blockers that can potentially be leveraged to solve human health concerns. Unfortunately, the remarkable potency of these natural products results in equally exceptional toxicity, presenting a significant challenge for the therapeutic application of these compounds. Identifying less potent analogs and convenient methods for accessing them therefore provides an attractive approach to developing molecules with enhanced therapeutic potential. Fortunately, Nature has evolved tools to modulate the toxicity of PSTs through selective hydroxylation, sulfation, and desulfation of the core scaffold. Here, we demonstrate the function of enzymes encoded in cyanobacterial PST biosynthetic gene clusters that have evolved specifically for the sulfation of highly functionalized PSTs, the substrate scope of these enzymes, and elucidate the biosynthetic route from saxitoxin to monosulfated gonyautoxins and disulfated C-toxins. Finally, the binding affinities of the nonsulfated, monosulfated, and disulfated products of these enzymatic reactions have been evaluated for VGSC binding affinity using mouse whole brain membrane preparations to provide an assessment of relative toxicity. These data demonstrate the unique detoxification effect of sulfotransferases in PST biosynthesis, providing a potential mechanism for the development of more attractive PST-derived therapeutic analogs.

Towards Identifying the Binding Site for 125I‐Angiotensin 1‐7

Angiotensin 1–7 (Ang 1‐7) is a product of the renin‐angiotensin system (RAS) that exhibits central actions to reduce blood pressure and improve baroreflex sensitivity. For more than a decade Mas has been considered the receptor for Ang 1‐7, however, to date, no published studies have characterized Mas receptor radioligand binding in tissue membrane preparations. Our laboratory has demonstrated high affinity (low nanomolar KD) binding of a preparation of 125/127I‐Ang 1‐7 in liver membranes, however, this binding is not pharmacologically specific in that the IC50 of Ang 1‐7 is in the micromolar range and all angiotensin peptides compete for 125/127I‐Ang 1‐7 binding equivalently. 125/127I‐Ang 1‐7 binding to liver, kidney, brain and testes membrane preparations from both rats and mice has also failed to demonstrate a high affinity, pharmacologically specific binding site for 125/127I‐Ang 1‐7. Considering that rapid degradation of Ang 1‐7 could be adversely affecting the binding assay, we tested different peptidase inhibitors (JMV‐390, O‐phenanthroline and a proprietary combination of peptidase inhibitors Biotools®) to prevent degradation of the radioligand. Although we were able to reduce Ang 1‐7 degradation with these inhibitors (~80% preservation), we also reduced the total and specific binding of 125I‐Ang 1‐7. Indeed, competition binding assays for 125I‐Ang‐(1–7) with JMV‐390, O‐phenanthroline and Biotools® showed that 125I‐Ang‐(1–7) and all the inhibitors compete for the same binding site with JMV‐390 having a nanomolar IC50. It is important to note that the radioligand used for receptor binding studies contains an iodine125 on the tyrosine in position four of Ang 1‐7, and may have different characteristics for enzymatic metabolism. To assess this possibility, we determined the metabolism of 127I‐Ang 1‐7 (non‐radioactive iodine isotope) and indeed, the metabolism of Ang 1‐7 and 127I‐Ang 1‐7 in liver appear to differ, with 127I‐Ang 1‐7 being more resistant to metabolism. In addition, competition binding assays showed that 127I‐Ang 1‐7 is ~1000‐fold more potent than Ang 1‐7 in competing for 125I‐Ang 1‐7 binding. In conclusion, our results suggest that 125I‐Ang‐(1–7) may be binding to a peptidase rather than a receptor and the addition of the iodine molecule to the tyrosine in position four attenuates its degradation. Studies are underway to identify this peptidase and determine its potential therapeutic importance as a druggable target.Support or Funding InformationCardiovascular Neuroscience Research Fund 337667 Speth (PI) 03/31/15‐12/31/18 ‐ Nova Southeastern Univ.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of a Novel Radioligand for Characterization of the Mas Receptor for Angiotensin 1–7

It is well established that Mas (NP_002368) is the receptor for angiotensin 1–7 (Ang 1–7), however, to date, no published studies have characterized Mas receptor radioligand binding in tissue membrane preparations. Our laboratory has repeatedly failed to reliably demonstrate high affinity, saturable binding of 125I‐Ang 1–7 in liver, kidney, brain and testes membrane preparations from both rats and mice. While the presence of a single iodine‐125 molecule on the tyrosine of Ang II does not impair its binding to the AT1 and AT2 receptors, it is not known whether the presence of an iodine‐125 molecule on the tyrosine of Ang 1–7 is equally innocuous. To begin to address this question, we prepared a novel analog of Ang 1–7: Tyr0‐Ang 1–7, to provide an alternate site for radioiodination of Ang 1–7. Tyr0‐Ang 1–7 was radioiodinated using the chloramine T procedure of Hunter and Greenwood (1962) with a 7 times excess of peptide to iodine to minimize di‐iodination of the peptide. Two radioiodinated peaks, presumed to be mono125I‐Y0‐Ang 1–7 and mono125I‐Y4‐Ang 1–7, were resolved from the uniodinated peptide peak by HPLC (C18 reverse phase column, 14.5% acetonitrile: triethylamine phosphate, pH 3.0 isocratic mobile phase). Saturation and competition radioligand binding assays were run for each peak using rat liver membrane preparations in the presence and absence of 10 μM Ang 1–7. While the identity of the early and late 125I‐Y0‐ Ang 1–7 peaks has not yet been established, preliminary studies indicate that both 125I‐Y0‐Ang 1–7 peaks displayed high affinity, saturable binding to rat liver membranes: early peak: KD = 7.7±2.3 nM, BMAX = 3.3±0.72 fmol/mg wet weight; and late peak KD = 11±2, BMAX = 3.6±0.4 fmol/mg wet weight. Competition binding assays run using the late peak 125I‐Y0‐Ang 1–7 revealed IC50's of 32±15 nM for Ang 1–7 and 15 ± 9.6 nM for Y0‐Ang 1–7. Comparison of the ability of a variety of Ang peptides to compete for late peak 125I‐Y0‐Ang 1–7 binding at a 10 μM concentration gave a rank order of Ang 1–7>Ang 3–7>Ang IV>Ang III>Ang 2–7>Ang II>Ang I. These results suggest that addition of a tyrosine at the amino terminus of Ang 1–7 may increase its binding affinity for Mas and that the presence of an iodine‐125 on either Y0 or Y4 of Ang 1–7 does not preclude high affinity saturable binding to Mas.Support or Funding InformationThis work was supported by the Cardiovascular Neuroscience Fund, Nova Southeastern University and the Peptide Radioiodination Shared Resource, Georgetown University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Knockdown of interleukin-10 induces the redistribution of sigma1-receptor and increases the glutamate-dependent NADPH-oxidase activity in mouse brain neurons.

BackgroundIn the central nervous system, interleukin-10 (IL-10) provides trophic and survival effects directly on neurons, modulates neurite plasticity, and has a pivotal importance in the neuronal regeneration in neurodegenerative and neuroinflammatory conditions. This cytokine is primarily produced by glial cells and has beneficial effects on the neuronal viability. However, the mechanisms of IL-10-elicited neuroprotection are not clear.ResultsMembrane preparations, isolated from wild-type (Wt) and IL-10 knockout (KO) mice brain were used in this study. It has been shown that compared to wild-type mice, in IL-10 KO mice brain, the amount of immunoglobulin binding protein (BiP) is greatly increased, whereas the content of sigma receptor-1 (SigR1) is not changed significantly. Co-immunoprecipitation experiments have shown that the association of SigR1 with small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1), NR2B subunit of NMDA-receptor (NMDAR) and inositol-3-phosphate receptor (IP3R) is higher in the IL-10 KO mice brain than in the Wt mice brain. Besides, we have found that either glutamate or sigma ligands, separately or together, do not change glutamate-induced NADPH-oxidase (NOX) activity in Wt-type mice brain membrane preparations, whereas in IL-10 KO mice high concentration of glutamate markedly increases the NOX-dependent production of reactive oxygen species (ROS). Glutamate-dependent ROS production was decreased to the normal levels by the action of sigma-agonists.ConclusionsIt has been concluded that IL-10 deprivation, at least in part, can lead to the induction of ER-stress, which causes BiP expression and SigR1 redistribution between components of endoplasmic reticulum (ER) and plasma membrane. Moreover, IL-10 deficiency can change the specific organization of NMDAR, increasing the surface expression of SigR1-sensitive NR2B-containing NMDAR. In these conditions, glutamate-dependent ROS production is greatly increased leading to the initiation of apoptosis. In this circumstances, sigma-ligands could play a preventive role against NMDA receptor-mediated excitotoxicity.

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.

Inhibition of PMCA activity by tau as a function of aging and Alzheimer's neuropathology

Ca2+-ATPases are plasma membrane and intracellular membrane transporters that use the energy of ATP hydrolysis to pump cytosolic Ca2+ out of the cell (PMCA) or into internal stores. These pumps are the main high-affinity Ca2+ systems involved in the maintenance of intracellular free Ca2+ at the properly low level in eukaryotic cells. The failure of neurons to keep optimal intracellular Ca2+ concentrations is a common feature of neurodegeneration by aging and aging-linked neuropathologies, such as Alzheimer's disease (AD). This disease is characterized by the accumulation of β-amyloid senile plaques and neurofibrillary tangles of tau, a protein that plays a key role in axonal transport. Here we show a novel inhibition of PMCA activity by tau which is concentration-dependent. The extent of inhibition significantly decreases with aging in mice and control human brain membranes, but inhibition profiles were similar in AD-affected brain membrane preparations, independently of age. No significant changes in PMCA expression and localization with aging or neuropathology were found. These results point out a link between Ca2+-transporters, aging and neurodegeneration mediated by tau protein.

Tritiated Opioid Receptor Ligands as Radiotracers

Tritiated opioid ligands are essential tools for the identification of opioid receptors. This review deals with the syntheses of tritiated opioid peptide derivatives, including enkephalins, dynorphins, dermorphins, deltorphins and endomorphins, and also discusses tritium-labeled nonpeptide opioids. It additionally focuses on the relevance of tritium-labeled opioid compounds as research tools for investigating opioid receptor pharmacology. Agonists and antagonists are used for the characterization of new opioid ligands by means of radioreceptor binding assays. Further topics covered in this review are the distribution of the endogenous peptides in the central nervous system and peripheral tissues, and degradation studies of opioids in brain membrane preparations and the blood.

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.

Functional activity of the cannabinoid 1 receptor is not affected by opioid antagonists in the rat brain

BackgroundWIN55212-2 is a synthetic cannabinoid agonist and selective to cannabinoid 1 (CB1) receptors, which are distributed mainly in the central nervous system. Opioid receptors and CB1 receptors have several similarities in terms of their intracellular signal transduction mechanisms, distributions, and pharmacological action. Several studies have therefore sought to describe the functional interactions between opioids and cannabinoids at the cellular and behavioral levels. The present study investigated agonist-stimulated [35S]GTPγS binding by WIN55212-2 in rat brain membranes and determined the antagonism by selective opioid antagonists at the level of receptor-ligand interaction and intracellular signal transduction.MethodsSprague-Dawley rats (male, n = 20) were euthanized for the preparation of brain membranes. In agonist-stimulated [35S]GTPγS binding by WIN55212-2, the values of EC50 and maximum stimulation (% over basal) were determined in the absence or presence of the µ, κ and δ opioid receptor antagonists naloxone (20 nM), norbinaltorphimine (3 nM), and naltrindole (3 nM), respectively. Ke values for opioid antagonist inhibition in the absence or presence of each opioid receptor antagonist were calculated using the following equation: [nanomolar antagonist] / (dose ratio of EC50 - 1).ResultsIn WIN55212-2-stimulated [35S]GTPγS binding in the rat brain membranes, the values of EC50 and maximum stimulation (% over basal) were 154 ± 39.5 nM and 27.6 ± 5.3% over basal, respectively. Addition of selective opioid antagonists did not produce a significant rightward shift in the WIN55212-2 concentration-response curve, and Ke values were not applicable.ConclusionsOur results suggest that the functional activity of WIN55212-2-stimulated [35S]GTPγS binding was not affected by opioid antagonists in the rat brain membranes. Although the exact mechanism remains unclear, our results may partially elucidate their actions.

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.

Pharmacological Assessment of <i>γ</i>-Secretase Activity from Rodent and Human Brain

γ-Secretase is involved in the final processing of the amyloid precursor protein into a heterogeneous pool of β-amyloid (Aβ) peptides. Current Alzheimer’s disease drug discovery efforts include targeting γ-secretase activity in brain to attenuate production of the neurotoxic Aβ species. The resulting pharmacology may be affected by species-specific differences in the γ-secretase core complex or its associated proteins. Therefore, we utilized partially purified γ-secretase membranes derived from the brains of different species, including human cortex, to quantitatively assess the de novo production of both Aβ42 and Aβ40 following treatment with known γ-secretase inhibitors and modulators. We determined that the inhibitory activity of a Notch-1 sparing γ-secretase inhibitor and the modulatory activity of two classes of γ-secretase modulators were equipotent at affecting the production of Aβ across rodent and human brain membrane preparations. Additionally, the observed modulator-specific Aβ profile in isolated brain membranes across species was similar to that observed in HeLa cell membranes, and the brain and CSF of guinea pigs following oral administration. By utilizing rapidly purified γ-secretase, we were able to probe and compare the complex pharmacology of γ-secretase in the brain across common rodent species and human cortex.

Characterization of Ligand Binding to the σ1Receptor in a Human Tumor Cell Line (RPMI 8226) and Establishment of a Competitive Receptor Binding Assay

The standard assay for the determination of σ(1) receptor affinities of novel compounds is a competitive binding assay using [(3)H]-(+)-pentazocine as radioligand and membrane preparations from guinea pig brain. Herein, a novel competitive binding assay was developed employing the hematopoietic cell line of human multiple myeloma (RPMI 8226), which expresses a large amount of the human σ(1) receptor. Membrane fragments of RPMI 8226 cells were prepared and characterized. A Western blot analysis confirmed the high density of σ(1) receptors in this cell line. Assay conditions were carefully optimized leading to an incubation period of 120 min, an incubation temperature of 37°C, and receptor material for each well was prepared from 300,000 cells. It was shown that a large excess (10 μM) of (+)-pentazocine, haloperidol, and di-o-tolylguanidine provided the same results during determination of the nonspecific binding. Saturation experiments with the radioligand [(3)H]-(+)-pentazocine led to a K(d)-value of 36±0.3 nM and a B(max)-value of 477±7 fmol/mg protein. These data resulted in approximately 122,000 σ(1) binding sites per cell. The assay was validated by using six known σ(1) ligands and eight σ(1) ligands prepared in our lab. The K(i)-values determined with RPMI 8226-derived receptor material are in good accordance with the K(i)-values obtained with guinea pig brain membrane preparations. Compared with guinea pig brain preparations, the RPMI 8226-derived receptor material represents a better standardized receptor material with a high density of human σ(1) receptors.

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.