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. Two alkaline degradation products of VX produced no cytotoxicity. Exposure for 24 h to 3 microM VX caused 36% and 94% irreversible loss of metabolism in hepatocytes and neuroblastoma cells, respectively. The insecticides parathion and chlorpyrifos stimulated hepatocyte metabolism but inhibited neuroblastoma cells. Their oxons were more active. Exposure of neuroblastoma cells for 4 h to VX, parathion, paraoxon, diisopropylfluorophosphate or chlorpyrifos gave an LC50 of 65, 775, 640, 340, or 672 microM, respectively, whereas 24 h gave an LC50 of 0.7, 3.7, 2.5, 29, and 31 microM, respectively. Preincubation of hepatocytes with phenobarbital enhanced their response to parathion and VX due to metabolic bioactivation. Atropine partially blocked the effects of VX and paraoxon on both cell types, which suggests the involvement of a muscarinic receptor as the target for cytotoxicity. There was no correlation between OP in vivo neurotoxicity and in vitro cytotoxicity. It is suggested that the former results from their cholinesterase inhibition, while the latter results from action on different targets and requires much higher concentrations.

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. The kinetics of the transient increase and subsequent decline increased with higher concentrations of SNTX. The biphasic and time-dependent response of hepatocytes to SNTX suggests that more than one mechanism may be involved in the toxicity of its different polypeptides. SNTX-induced cytotoxicity of hepatocytes was reduced by the presence of high titer antibodies against a heterologous jellyfish. Phenobarbital-induced cells became more vulnerable to SNTX, suggesting that some toxin component(s) require(s) bioactivation. Short-term exposure (1-2 h) to 10 microg/ml of the calcium ionophore calcimycin, or the non-selective monovalent cation ionophore gramicidin, had no effect on metabolic activity. However, 165 microg/ml gramicidin or 53 microg/ml calcimycin produced slight transient activation followed by steady decline in metabolic activity, while 20 h exposure to either ionophore produced total cell death. Exposure to even a 10-fold lower concentration of either ionophore killed 88% and 75%, respectively. This contrasts with the toxicity of SNTX which is detectable in minutes with as little as 3 microg/ml. Since pre-exposure to the organophosphate anticholinesterases VX and paraoxon, or the chemotherapeutic alkylating agents cyclophosphamide and mechlorethamine reduced the cytotoxic effects of SNTX, it suggests that phosphorylation or alkylation of cell protein(s) interferes with SNTX toxicity.

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 ... in establishing, modifying, leaving in effect, or revoking a tolerance for a pesticide chemical residue." This directive raises a number of scientific questions to be answered before the FQPA can be implemented. Among these questions is: What constitutes a common mechanism of toxicity? The ILSI Risk Science Institute (RSI) convened a group of experts to examine this and other scientific questions using the organophosphorus (OP) pesticides as the case study. OP pesticides share some characteristics attributed to compounds that act by a common mechanism, but produce a variety of clinical signs of toxicity not identical for all OP pesticides. The Working Group generated a testable hypothesis, anticholinesterase OP pesticides act by a common mechanism of toxicity, and generated alternative hypotheses that, if true, would cause rejection of the initial hypothesis and provide criteria for subgrouping OP compounds. Some of the alternative hypotheses were rejected outright and the rest were not supported by adequate data. The Working Group concluded that OP pesticides act by a common mechanism of toxicity if they inhibit acetylcholinesterase by phosphorylation and elicit any spectrum of cholinergic effects. An approach similar to that developed for OP pesticides could be used to determine if other classes or groups of pesticides that share structural and toxicological characteristics act by a common mechanism of toxicity or by distinct mechanisms.

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. Incubation of Torpedo membrane-bound nAChRs with the agonist carbamylcholine (Carb) stimulated the binding of [3H]thienyl-cyclohexylpiperidine ([3H]TCP), which binds to the receptor's noncompetitive antagonist binding site in its ionic channel, with high affinity (Kd of 196 nM). The agonist-stimulated binding of [3H]TCP (i.e., binding to activated nAChRs) was inhibited in a concentration-dependent manner by four organophosphate (OP) anticholinesterases, chlorpyrifos oxon (CPO), chlorpyrifos (CPS), parathion (PS), and paraoxon (PO) with IC50 (concentration that inhibits 50% of the effect) values of 5, 150, 200, and 300 microM, respectively. The binding of CPO was totally reversible. The OPs had no effect on equilibrium binding of [alpha-125I]bungarotoxin ([alpha-125I]BGT) to the receptor's acetylcholine (ACh)-binding site, but preincubation of the membranes with the OPs increased this site's affinity for Carb. In absence of agonist, 100 microM of the OPs increased the binding of [3H]TCP by two- to fivefold with the following order of decreasing potency: PS > CPO > CPS > PO. The data suggest that in addition to inhibition of acetylcholinesterase, these OPs bind to a site on the nAChR that is different from the sites that bind ACh or TCP and that this binding induces nAChR desensitization. The relevance of this direct action of OPs on nAChRs on their acute toxicities is discussed.

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. If present, cocaine competed for the mAb and interfered with the binding of BE-FL, thereby reducing the fluorescence transmitted by the fiber. Calibration curves were prepared by measuring (over 30 s) the rates of fluorescence increase in the absence, or presence of cocaine. Ethanol or acid extracts of dry coca leaves were assayed by this fiber optic biosensor, gas chromatography and a fluorescent polarization immune assay. Biosensor values of cocaine content of leaves from five Erythroxylum species were not significantly different from gas chromatography values, but had higher variance. The biosensor assay was rapid and did not require cleanup of the crude leaf extracts. Cocaine in acid extracts was reduced significantly after 4 weeks at 23 degrees C and after 3 weeks at 37 degrees C. Fibers (mAb-coated), stored at 37 degrees C in phosphate-buffered solution (0.02% NaN3), gave stable responses for 14 days.

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. All drugs produced concentration-and time-dependent reduction in acidification rate following 24 hr exposure, which may be due to decreased number of viable cells and/or lowered metabolic rates of the live cells. The degree of irreversible inhibition of acidification rate was used as an index of cell death and the IC(50) values for the 10 drugs were comparable to those produced in the same cell line by a fluorescence assay using Calcein AM stain (r = 0.991), that fluoresces only in live cells, as well as the [(3)H]thymidine uptake assay (r = 0.976). There was also excellent correlation (r = 0.958) between IC(50) values of 24 hr exposure obtained from the Cytosensor with the 10 drugs and their published human lethal blood concentrations. An advantage of this new methodology over other in vitro assays is that it allows the determination of time points at which reversible change becomes irreversible.

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 = 7.6 nM). Cocaine or other test compounds competed with FL-BE for binding to the mAb in a concentration-dependent manner, thereby reducing the initial rate or steady-state fluorescence. Addition of cocaine to the flow buffer after reaching steady-state fluorescence enhanced the dissociation of bound FL-BE, and cocaine removal allowed fiber regeneration for multiple measurements. The detection limits for cocaine, cocaethylene, norcocaine, and BE were 5, 5, 29, and 30 ng/ml, respectively, but for ecgonine it was 4600 ng/ml and for methylecgonine it was 2000 ng/ml. Tropacocaine was detected at 10 ng/ml, but atropine was detected at 2900 ng/ml. The biosensor discriminated by 833-fold between cocaine and its stereoisomer pseudococaine. Structural features necessary for high-affinity recognition by this mAb are benzoate and 3 beta configuration, both of which are found in BE, cocaine, norcocaine, and cocaethylene.

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., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determination of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diisopropylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.

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. The data suggest that the receptor subtype down-regulations in the cortex and striatum are due to reductions in mRNA expression. Since the degrees of inhibition of acetylcholinesterase were similar in the 3 brain regions, it is suggested that the in situ concentrations of paraoxon were also similar. Accordingly, the absence of down-regulation of the m1 receptor in the hippocampus is not due to a lower concentration of paraoxon than in the cortex or striatum. It is possible that injections of higher parathion doses would produce down-regulation of mAChRs in the hippocampus, and that the hippocampus may have differences in the feed-back mechanisms for receptor regulation from those in the frontal cortex and the striatum.

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. Accordingly, data on radioligand binding to mAChRs in each mouse brain were correlated with brain AChE activity in the same mouse, and AChE inhibition served as a biomarker of exposure reflecting in situ paraoxon concentrations. Exposure to parathion for 14 d reduced maximal binding (Bmax) of [3H]quinuclidinyl benzilate ([3H]QNB), [3H]-N-methylscopolamine ([3H]NMS), and [3H]-4-diphenylacetoxy-N-methylpiperidine methiodide ([3H]-4-DAMP) by up to approximately 58% without affecting receptor affinities for these ligands. Maximal reduction in Bmax of [3H]QNB and [3H]-4-DAMP binding occurred in mice with highest AChE inhibition, while equivalent maximal reduction in Bmax of [3H]NMS occurred in mice with only approximately 10% AChE inhibition, without further change at higher parathion doses. This is believed to be due to the hydrophilicity of [3H]NMS, which limits its accessibility to internalized desensitized receptors. In submaxillary glands (mAChRs are predominantly m3 subtype), there were significant dose-dependent reductions in [3H]QNB binding and m3 mRNA levels in exposed mice, revealed by Northern blot analyses. The reduction in m3 receptors is suggested to result mostly from reduced synthesis at the transcription level, rather than from translational or posttranslational events. The data suggest that down-regulation of mAChRs occurs after dietary exposure for 14 d to sublethal concentrations of parathion in a field rodent species, and that significant though incomplete recovery in AChE and mAChRs occurs in 7 d following termination of exposure.

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. Structure-activity relationship studies revealed that shortening the polyamine chain length reduced potency, and quaternarization of the nitrogen destroyed it. The potency was increased by a bulky anchoring group with moderate hydrophobicity at the end of the polyamine chain. The conversion of the tryosyl moiety to 3,5-diiodo-tyrosyl also increased potency and so did lengthening the butyryl chain from 4 to 10 carbons. Not only did PhTXs inhibit different subtypes of glutamate receptors, including the mammalian N-methyl-D-aspartate receptor, but also nicotinic receptors of insects and vertebrates. Because of this low selectively, and the hydrophilicity of the derivatives tested, which interferes with their penetration to the target receptor, these compounds cannot be used as insecticides. Nevertheless, the insect skeletal muscle glutamate receptor is a viable target for selective insecticides and major changes in PhTX structure may possibly produce derivatives that can be potential insecticides.

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. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 microM) reduced Bmax of [3H]4-DAMP binding from 27 +/- 4 to 13 +/- 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolin-induced cAMP formation in SMG cells with an EC50 of 200 nM, but paraoxon was greater than 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 microM atropine, that by paraoxon was unaffected by up to 100 microM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect beta-adrenoreceptor function in the preparation, since it did not affect the 10 microM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 microM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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. When binding was measured by a centrifugal assay, [125I2]PhTX-343-lysine bound with low affinity (KD = 11.4 +/- 2 microM) to a large number of sites (37.2 +/- 9.1 nmol/mg of protein). The binding of [125I2]PhTX-343-lysine was sensitive only to the polyamines spermine and spermidine, which displaced [125I2]PhTX-343-lysine with Ki values of (3.77 +/- 1.4) x 10(-5) M and (7.51 +/- 0.77) x 10(-5) M, respectively. The binding was insensitive to glutamate receptor agonists and antagonists. Binding results with [125I2]PhTX-343-arginine were similar to those of [125I2]-PhTX-343-lysine. Considering the high number of toxin binding sites (10000-fold more than glutamate) in these membranes and the insensitivity of the binding to almost all drugs that bind to glutamate receptors, it is evident that most of the binding observed is not to glutamate receptors. On the other hand, PhTX analogues with photoaffinity labels may be useful in the isolation/purification of various glutamate and nicotinic acetylcholine receptors; they could also be useful in structural studies of receptors and their binding sites.

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. The reversible inhibitor edrophonium (0.1 mM) inhibited AChE and consequently reduced fluorescence quenching. The biosensor response immediately recovered upon its removal. The carbamate neostigmine (0.1 mM) also inhibited the biosensor response but recovery was much slower. In the presence of ACh, the organophosphate (OP) diisopropylfluorophosphate (DFP) at 0.1 mM did not interfere with the ACh-dependent fluorescent signal quenching, but preexposure of the biosensor to DFP in absence of ACh inhibited totally and irreversibly the biosensor response. However, the DFP-treated AChE biosensor recovered fully after a 10-min perfusion with pralidoxime (2-PAM). Echothiophate, a quaternary ammonium OP, inhibited the ACh-induced fluorescence quenching in the presence of ACh and the phosphorylated biosensor was reactivated with 2-PAM. These effects reflected the mechanism of action of the inhibitors with AChE and the inhibition constants obtained were comparable to those from colorimetric methods. The biosensor detected concentrations of the carbamate insecticides bendiocarb and methomyl and the OPs echothiophate and paraoxon in the nanomolar to micromolar range. Malathion, parathion, and dicrotophos were not detected even at millimolar concentrations; however, longer exposure or prior modification of these compounds (i.e., to malaoxon, paraoxon) may increase the biosensor detection limits. This AChE biosensor is fast, sensitive, reusable, and relatively easy to operate. Since the instrument is portable and can be self-contained, it shows potential adaptability to field use.

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, 0.17 nM in houseflies and 0.13 nM in the cockroach). However, the receptor concentration was low (108, 64.7, and 108 fmol/mg protein for the three species, respectively). The association and dissociation rates of [3H]QNB binding to honey bee brain membranes, sensitivity of [3H]QNB binding to muscarinic agonists, and high affinity for atropine were also features generally similar to muscarinic receptors of mammalian brains. In order to further characterize the three insect brain muscarinic receptors, the displacement of [3H]QNB binding by subtype-selective antagonists was studied. The rank order of potency of pirenzepine (PZ), the M1 selective antagonist, 11-[2-[dimethylamino)-methyl)1-piperidinyl)acetyl)-5,11- dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepin-6 one (AF-DX 116), the M2-selective antagonist, and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) the M3-selective antagonist, was also the same as that of mammalian brains, i.e., 4-DAMP greater than PZ greater than AF-DX 116. The three insect brain receptors had 27-50-fold lower affinity for PZ (Ki 484-900 nM) than did the mammalian brain receptor (Ki 16 nM), but similar to that reported for the muscarinic receptor subtype cloned from Drosophila. Also, the affinity of insect receptors for 4-DAMP (Ki 18.9-56.6 nM) was much lower than that of the M3 receptor, which predominates in rat submaxillary gland (Ki of 0.37 nM on [3H]QNB binding). These drug specificities of muscarinic receptors of brains from three insect species suggest that insect brains may be predominantly of a unique subtype that is close to, though significantly different from, the mammalian M3 subtype.

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 2.74 nM; Bmax of 82 fmol/mg protein) was detected and [3H]CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with [3H]CD and considered to be of the M2 subtype. Up to 70% of the [3H]CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. [3H]CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of [3H]CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM [3H]CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting [3H]CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration. Nevertheless, the high affinities that cardiac muscarinic receptors have for these toxicants point to their extra vulnerability. It is suggested that the success of iv administration of the muscarinic receptor inhibitor atropine in initial therapy of poisoning by OP anticholinesterases may be related in part to the extra sensitivity of M2 receptors to certain OPs.

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. Other nucleotides, particularly the nonhydrolyzable ATP analogs beta,gamma-imidoadenosine-5'-triphosphate (AMP-PNP) and beta,gamma-methyladenosine-5'-triphosphate (AMP-PCP) also potentiated [3H]DA and [3H]NE uptake. Several endogenous 5'-nucleotide triphosphates (e.g. GTP, UTP and CTP) potentiated [3H]CA uptake, but were less effective than ATP. Among the ATP metabolites, only ADP potentiated uptake whereas AMP and adenosine did not. [3H]Dopamine uptake measured in Krebs bicarbonate buffer had a Km of 2.1 microM and a Vmax of 163.9 pmol/mg prot./min. In presence of ATP, [3H]DA uptake had much higher affinity (Km = 0.56 microM) and larger capacity (Vmax = 333 pmol/mg prot./min) than uptake in absence of added ATP. Furthermore, [3H]DA uptake in presence of ATP had faster rate of uptake, and was independent of temperature while in absence of added ATP it was temperature-dependent. This ATP-dependent [3H]DA uptake was retained by synaptosomal ghosts that were obtained after lysing the striatal synaptosomes and removing their contents of synaptic vesicles and mitochondria. It is proposed that, in addition to the carrier-mediated (neuronal) uptake of CA, there is neuronal uptake that is regulated by ATP and inhibited by cocaine, which may be more relevant for terminating the synaptic action of CA because of its faster rate of uptake and larger capacity.

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. The incorporation of a butyl side-group in the polyamine also enhanced potency. These results are explained on the basis of the known non-competitive antagonistic blockage by philanthotoxin-433 of the channel gated by postjunctional glutamate receptors when the channel is open.

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. Recovery from PhTX block differed among the three amino acids. NMDA responses recovered completely within a few minutes whereas responses to kainate and quisqualate recovered more slowly. PhTX had no effect on equilibrium binding of [3H]glutamate to rat brain cortical membranes studied in buffer treated to eliminate microorganisms. Based on the drug specificity of this [3H]glutamate binding, it is suggested to be mostly to the NMDA receptor. Low concentrations of PhTX (1-10 microM) potentiated binding of [3H] MK-801, a specific noncompetitive inhibitor of the NMDA receptor. However, higher PhTX concentrations inhibited this binding with an IC50 of 20 microM, similar to its inhibition of the oocyte-expressed NMDA receptor. Inhibition of [3H]MK-801 binding by PhTX was noncompetitive. It is suggested that PhTX, like the more potent MK-801, binds to an allosteric site on the NMDA receptor and inhibits its function but its binding site is not identical with the MK-801 binding site.