Design, synthesis and pharmacological profile of novel dopamine D2 receptor ligands.

The present study describes the synthesis and pharmacological profile of three novel heterocyclic compounds originally designed, on the basis of bioisosterism, as dopamine D2 receptor ligands: 1-[1-(4-chlorophenyl)-1H-pyrazol-4-ylmethyl]-4-phenyl-piperazine (LASSBio-579), 1-phenyl-4-(1-phenyl-1H-[1,2,3]triazol-4-ylmethyl)-piperazine (LASSBio-580) and 1-[1-(4-chlorophenyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-phenyl-piperazine (LASSBio-581). Binding studies performed on brain homogenate indicated that all three compounds bind selectively to D2 receptors. In addition, electrophysiological studies carried out in cultured hippocampal neurons suggested that LASSBio-579 and 581 act as D2 agonists, whereas LASSBio-580 acts as a D2 antagonist.

The N-methyl-D-aspartate neurotransmitter receptor is a mammalian brain target for the dinoflagellate Pfiesteria piscicida toxin.

Blooms of Pfiesteria piscicida, a dinoflagellate in eastern U.S. coastal rivers, are believed to secrete toxins that kill fish and produce short-term memory loss in humans.

Total synthesis of pyrrolizidines 223H', 239K', 265H', and 267H' found in Madagascan frogs (Mantella) and their affinities for nicotinic acetylcholine receptor.

The total synthesis of pyrrolizidines 223H', 239K', 265H', and 267H' has been achieved starting from 1,5-hexadiene via a common synthetic intermediate 5. The affinity of 1-4 for nicotinic acetylcholine receptor was evaluated.

Cytotoxicity of organophosphate anticholinesterases.

Organophosphate (OP) anticholinesterases were found to modulate metabolic activities of human neuroblastoma cells and hepatocytes, which was detectable by the Cytosensor microphysiometer. The nerve gas ethyl-S-2-diisopropylaminoethyl methylphosphorothiolate (VX), at 10 microM, produced significant reduction in cell metabolism within 2 min, as measured by changes in the acidification rate of the medium. The reduction was dose- and time-dependent and irreversible after 4 h of exposure.

Toxicity of sea nettle toxin to human hepatocytes and the protective effects of phosphorylating and alkylating agents.

The sea nettle jellyfish toxin (SNTX), which contains several polypeptides, was highly toxic to human hepatocytes. The Cytosensor microphysiometer was used continuously to monitor cell media acidification rate as an index of cellular metabolic activity. Cells exposed to > 1 microg SNTX protein/ml media exhibited a transient increase in metabolic activity, followed by a sharp decrease and cell death within minutes.

Common mechanism of toxicity: a case study of organophosphorus pesticides.

The Food Quality Protection Act of 1996 (FQPA) requires the EPA to consider "available information concerning the cumulative effects of such residues and other substances that have a common mechanism of toxicity ...

Chlorpyrifos, parathion, and their oxons bind to and desensitize a nicotinic acetylcholine receptor: relevance to their toxicities.

The nicotinic acetylcholine receptor (nAChR) of the electric organ of the electric ray. Torpedo sp., the richest source of nAChR, with similar structure and pharmacology to the mammalian skeletal muscle nAChR, carries several binding sites for different ligands.

Validation of the cytosensor for in vitro cytotoxicity studies.

The Cytosensor() microphysiometer was used to continuously monitor perturbations in metabolic rates of the human liver cell line ATCC-CCL-13 when exposed to each of 10 drugs. The effects of exposure to one concentration for 24 hr or to sequential increasing concentrations for 4 hr, and recovery after drug removal, were compared. Paracetamol (acetaminophen) and ethanol were used to establish the assay protocols and determine reversibility of drug effect.

Evaluation of a fiber optic immunosensor for quantitating cocaine in coca leaf extracts.

A fiber optic evanescent fluoroimmunosensor was used to rapidly detect and quantitate coca alkaloids as cocaine equivalents in leaf extracts of five Erythroxylum species. A monoclonal antibody (mAb) made against benzoylecgonine (BE), a major metabolite of cocaine, was immobilized covalently on quartz fibers and used as the biological sensing element in the portable fluorometer. Benzoylecgonine-fluorescein (BE-FL) was used as the optical signal generator when it bound to the fiber.

A fiber-optic cocaine biosensor.

A fiber-optic biosensor was developed for detection of cocaine, its metabolites, and other coca alkaloids, using a monoclonal antibody (mAb) against a derivatized benzoylecgonine (BE). The mAb was immobilized noncovalently on quartz fibers and a flow fluorometer was used to detect changes in evanescent wave fluorescence. A fluorescein (FL) conjugate of BE bound to the mAb specifically in a saturable manner and with high affinity (Kd = 7.

Choline derivatives and sodium fluoride protect acetylcholinesterase against irreversible inhibition and aging by DFP and paraoxon.

A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e.

Differential regulation of muscarinic receptor subtypes in rat brain regions by repeated injections of parathion.

Repeated injections with increasing moderate doses of parathion into adult male rats for 21 days resulted in 84-90% inhibition of acetylcholinesterase in the brain without overt signs of toxicity. Muscarinic acetylcholine receptor (mAChR) affinities for ligands were unaffected, but there was significant down-regulation of the m4 receptor subtype gene product, m1 mRNA and m3 mRNA in the frontal cortex as well as the m4 subtype and m4 mRNA in the striatum. However, in the hippocampus, there were no significant reductions in either the m1 receptor subtype nor its mRNA.

Down-regulation of muscarinic receptors and the m3 subtype in white-footed mice by dietary exposure to parathion.

The effect of ad libitum dietary exposure (as occurs in the field) to parathion for 14 d was investigated on the muscarinic acetylcholine receptor (mAChR) in brains and submaxillary glands of adults of a field species, the white-footed mouse Peromyscus leucopus. Immunoprecipitation using subtype selective antibodies revealed that the relative ratios of the m1-m5 mAChR subtypes in Peromyscus brain were similar to those in rat brain. There was little variability in acetylcholinesterase (AChE) activity in control mice brains but large variability in 39 exposed mice, resulting from differences in food ingestion and parathion metabolism.

Glutamate receptor inhibitors as potential insecticides.

Philanthotoxin (PhTX) is a neurotoxic constituent of the paralytic venom of the digger wasp, Philanthus triangulum. PhTX inhibits glutamate receptors of insect muscles mostly as a channel blocker, thereby producing muscle paralysis. Since glutamate receptor blockers may be of value as selective insect control agents, numerous derivatives of PhTX were synthesized and tested for their potencies as inhibitors of insect skeletal muscle glutamate receptors.

Differential effects of paraoxon on the M3 muscarinic receptor and its effector system in rat submaxillary gland cells.

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M3 subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [3H]quinuclidinyl benzilate ([3H]QNB) was most sensitive to atropine and the M3-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M2 antagonist AFDX116. This, and the binding characteristics of [3H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M3 subtype.

Synthesis and binding of [125I2]philanthotoxin-343, [125I2]philanthotoxin-343-lysine, and [125I2]philanthotoxin-343-arginine to rat brain membranes.

125I2-iodinated philanthotoxin-343 (PhTX-343), [125I2]PhTX-343-arginine, and [125I2]PhTX-343-lysine were synthesized and evaluated as probes for glutamate receptors in rat brain synaptic membranes. It was found that these probes were not specific for the glutamate receptors but may be useful for investigating the polyamine binding site. Filtration assays with Whatman GF/B fiber glass filters were unsuitable because the iodinated PhTX-343 analogues exhibited high nonspecific binding to the filters, thus hindering detection of specific binding to membranes.

Acetylcholinesterase fiber-optic biosensor for detection of anticholinesterases.

An optical sensor for anticholinesterases (AntiChEs) was constructed by immobilizing fluorescein isothiocyanate (FITC)-tagged eel electric organ acetylcholinesterase (AChE) on quartz fibers and monitoring enzyme activity. The pH-dependent fluorescent signal generated by FITC-AChE, present in the evanescent zone on the fiber surface, was quenched by the protons produced during acetylcholine (ACh) hydrolysis. Analysis of the fluorescence response showed Michaelis-Menten kinetics with a Kapp value of 420 microM for ACh hydrolysis.

Pharmacologic characterization of muscarinic receptors of insect brains.

Muscarinic receptors in brain membranes from honey bees, houseflies, and the American cockroach were identified by their specific binding of the non-selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate ([3H]QNB) and the displacement of this binding by agonists as well as subtype-selective antagonists, using filtration assays. The binding parameters, obtained from Scatchard analysis, indicated that insect muscarinic receptors, like those of mammalian brains, had high affinities for [3H]QNB (KD = 0.47 nM in honey bees, 0.

Structure-activity relationships of philanthotoxin analogs and polyamines on N-methyl-D-aspartate and nicotinic acetylcholine receptors.

The effects of varying the structure of philanthotoxin (PhTX) were investigated on binding of the channel blockers: [3H]perhydrohistrionicotoxin (H12-HTX) to the nicotinic acetylcholine receptor (nACh-R) of Torpedo electric organ and [3H]MK-801 [( 3H]-5-methyl-10,11-dihydro-5H-dibenzocyclo-hepten-5,10-imine maleate) to the N-methyl-D-aspartate receptor (NMDA-R) of rat brain cortex. The four moieties of PhTX (tyrosine, butyrate, spermine and the terminal amino group) were modified or conjugated resulting in 36 compounds. Although the potencies of the PhTX analogs on both receptors were higher with increasing lipophilicity and the polyamine chain length, there was considerable divergence between the two receptors' channels in the structural activity requirements for blockade by PhTX analogs.

Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases.

The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-[3H]cis-methyldioxolane ([3H]CD), which has been used to label a high affinity population of M2 receptors. A single population of sites (KD 2.

Structure-activity relationships of analogues of the wasp toxin philanthotoxin: non-competitive antagonists of quisqualate receptors.

Fifty-two analogues of the wasp toxin, philanthotoxin-433, have been synthesized and tested on a glutamatergic, nerve-muscle preparation from locust leg. Reduction in amplitude of the neurally-evoked muscle twitch was used to construct dose-inhibition relationships from which IC50S were estimated. The most active analogues were characterized by one or more of the following: increased hydrophobicity of aromatic and tyrosyl regions; an increased number of protonated groups in the polyamine region; a guanidinium instead of a spermine terminal amino moiety.

ATP-regulated neuronal catecholamine uptake: a new mechanism.

Uptake of the catecholamines (CA), dopamine (DA) and norepinephrine (NE) into synaptosomes prepared from rat and bovine brains was potentiated by ATP (from 0.1 to 5.0 mM) in a dose-dependent manner.

Inhibition of rat brain glutamate receptors by philanthotoxin.

The actions of philanthotoxin (PhTX) were studied on the function of glutamate receptors expressed in Xenopus oocytes injected with rat brain mRNA and on binding of radioligands to rat brain glutamate receptors. PhTX reversibly inhibited the oocyte responses to quisqualate, N-methyl-D-aspartate (NMDA) and kainate in a dose-dependent manner. The NMDA receptor was the most sensitive to PhTX action (10-fold more than the kainate receptor) and the least sensitive was the smooth current component of the quisqualate response.

Actions of the insecticide 2(nitromethylene)tetrahydro-1,3-thiazine on insect and vertebrate nicotinic acetylcholine receptors.

The nitromethylene heterocyclic compound 2(nitromethylene)tetrahydro)1,3-thiazine (NMTHT) inhibits the binding of [125I]alpha-bungarotoxin to membranes prepared from cockroach (Periplaneta americana) nerve cord and fish (Torpedo californica) electric organ. Electrophysiological studies on the cockroach fast coxal depressor motorneuron (Df) reveal a dose-dependent depolarization in response to bath-applied NMTHT. Responses to ionophoretic application of NMTHT onto the cell-body membrane of motorneuron Df are suppressed by bath-applied mecamylamine (1.

Allosteric inhibition of nicotinic acetylcholine receptors of vertebrates and insects by philanthotoxin.

The effects of pure philanthotoxin (PhTX), a component of the venom of the wasp Philanthus triangulum, were studied on nicotinic acetylcholine receptors (nAChRs) of vertebrates and insects so as to compare their sensitivities and the mechanism of action of PhTX. Electrophysiological techniques were used on frog muscles and cockroach thoracic ganglia and biochemical techniques were applied to membranes from Torpedo electric organ and honeybee brain. PhTX (1-20 microM) inhibited reversibly the indirectly elicited muscle twitch and reduced the endplate current peak amplitude and its decay time constant in a concentration-dependent manner.