IgG, IgM and IgA antibodies against the novel polyprotein in active tuberculosis.

BACKGROUND: The present study was aimed to evaluate whether IgG, IgM and IgA antibodies levels detected against a novel Mycobacterium tuberculosis polyprotein 38 F-64 F (with 38 F being the abbreviation for 38kD-ESAT6-CFP10 and 64 F for Mtb8.4-MPT64-TB16.3-Mtb8) are suitable for diagnosing active tuberculosis, and for monitoring the efficacy of chemotherapy on TB patients. METHODS: In this study, a total of 371 active TB patients without treatment were selected and categorized into S+/C+group (n=143), S-/C+group (n=106) or S-/C- group (n=122). A series of serum samples were collected from 82 active TB patients who had undergone anti-TB chemotherapy for 0-6 months at one month interval. Humoral responses (IgG, IgM and IgA) were determined for the novel Mycobacterium tuberculosis polyprotein using indirect ELISA methods in all of serum samples. RESULTS: For S+/C+, S-/C+and S-/C- active tuberculosis patients before anti-TB chemotherapy, the sensitivities of tests based on IgG were 65.7%, 46.2% and 52.5% respectively; the sensitivities based on IgM were 21.7%, 24.5% and 18.9%; and the sensitivities based on IgA were 25.2%, 17.9% and 23.8%. By combination of three isotypes, for all active tuberculosis patients, the test sensitivity increased to 70.4% with the specificity being 91.5%. After anti-TB chemotherapy, there were no significant differences between groups with different courses of anti-TB chemotherapy. CONCLUSIONS: The novel Mycobacterium tuberculosis polyprotein 38 F-64 F represents potential antigen suitable for measuring IgG, IgM and IgA antibodies. However, the serodiagnostic test based on the 38 F-64 F polyprotein appears unsuitable for monitoring the efficacy of chemotherapy.

Enhanced serodiagnostic utility of novel Mycobacterium tuberculosis polyproteins.

OBJECTIVES: The detection of Mycobacterium tuberculosis specific antibodies in human sera has been a rapid and important diagnostic aid for tuberculosis (TB) control and prevention. However, any single antigen is not enough to be used to cover the antibody profiles of all TB patients. METHODS: Seven single antigens (38 kDa, ESAT-6, CFP10, Mtb8.4, MPT64, TB16.3 and Mtb8) were evaluated serodiagnostically. Two novel M. tuberculosis polyproteins, 38kD-ESAT6-CFP10 (38F) and Mtb8.4-MPT64-TB16.3-Mtb8 (64F), were expressed and the novel 38F-64F indirect ELISA assay used to analyze antibody responses to polyproteins in serum samples. RESULTS: The sensitivity of the novel 38F-64F indirect ELISA alone was much higher than that of the sputum culture test (86.91% vs. 50.62%) and that of the sputum smear test (78.64% vs. 47.57%). The novel 38F-64F indirect ELISA had a sensitivity of 74.16% with sera from extrapulmonary TB patients and a sensitivity of 37.14% with sera from LTBI. The specificity of the novel 38F-64F indirect ELISA was 90.36% with the sera from healthy blood donors and 94.15% with the sera from non-TB patients. CONCLUSIONS: The novel 38F-64F indirect ELISA assay had effective diagnostic performance and would make meaningful contribution to the diagnosis of TB disease in developing countries.

The synergistic effects of insecticidal essential oils and piperonyl butoxide on biotransformational enzyme activities in Aedes aegypti (Diptera: Culicidae).

The biochemical mechanisms underlying the increased toxicity of several plant essential oils (thymol, eugenol, pulegone, terpineol, and citronellal) against fourth instar of Aedes aegypti L. when exposed simultaneously with piperonyl butoxide (PBO) were examined. Whole body biotransformational enzyme activities including cytochrome P450-mediated oxidation (ethoxyresorufin O-dethylase [EROD]), glutathione S-transferase (GST), and beta-esterase activity were measured in control, essential oil-exposed only (single chemical), and essential oil + PBO (10 mg/liter) exposed larvae. At high concentrations, thymol, eugenol, pulegone, and citronellal alone reduced EROD activity by 5-25% 16 h postexposure. Terpineol at 10 mg/liter increased EROD activity by 5 +/- 1.8% over controls. The essential oils alone reduced GST activity by 3-20% but PBO exposure alone did not significantly affect the activity of any of the measured enzymes. All essential oils in combination with PBO reduced EROD activity by 58-76% and reduced GST activity by 3-85% at 16 h postexposure. This study indicates a synergistic interaction between essential oils and PBO in inhibiting the cytochrome P450 and GST detoxification enzymes in Ae. aegypti.

Plant terpenoids: acute toxicities and effects on flight motor activity and wing beat frequency in the blow fly Phaenicia sericata.

We evaluated the acute toxicities and the physiological effects of plant monoterpenoids (eugenol, pulegone, citronellal and alpha-terpineol) and neuroactive insecticides (malathion, dieldrin and RH3421) on flight muscle impulses (FMI) and wing beat signals (WBS) of the blow fly (Phaenicia sericata). Topically-applied eugenol, pulegone, citronellal, and alpha-terpineol produced neurotoxic symptoms, but were less toxic than malathion, dieldrin, or RH3421. Topical application of eugenol, pulegone, and citronellal reduced spike amplitude in one of the two banks of blow fly dorsolongitudinal flight muscles within 6-8 min, but with citronellal, the amplitude of FMIs reverted to a normal pattern within 1 hr. In contrast to pulegone and citronellal, where impulse frequency remained relatively constant, eugenol caused a gradual increase, then a decline in the frequency of spikes in each muscle bank. Wing beating was blocked permanently within 6-7 min of administering pulegone or citronellal and within 16 mins with eugenol. alpha-Terpineol-treated blow flies could not beat their wings despite normal FMI patterns. The actions of these monoterpenoids on blow fly flight motor patterns are discussed and compared with those of dieldrin, malathion, RH3421, and a variety of other neuroactive substances we have previously investigated in this system. Eugenol, pulegone and citronellal readily penetrate blow fly cuticle and interfere with flight muscle and/or central nervous function. Although there were differences in the effects of these compounds, they mainly depressed flight-associated responses, and acted similarly to compounds that block sodium channels and facilitate GABA action.

The G protein-coupled cannabinoid-1 (CB1) receptor of mammalian brain: inhibition by phthalate esters in vitro.

This research examines the in vitro interaction of phthalate diesters and monoesters with the G protein-coupled cannabinoid 1 (CB(1)) receptor, a presynaptic complex involved in the regulation of synaptic activity in mammalian brain. The diesters, n-butylbenzylphthalate (nBBP), di-n-hexylphthalate (DnHP), di-n-butylphthalate (DnBP), di-2-ethylhexylphthalate (DEHP), di-isooctylphthalate (DiOP) and di-n-octylphthalate (DnOP) inhibited the specific binding of the CB(1) receptor agonist [(3)H]CP-55940 to mouse whole brain membranes at micromolar concentrations (IC(50)s: nBBP 27.4 µM; DnHP 33.9 µM; DnBP 45.9 µM; DEHP 47.4 µM; DiOP 55.4 µM; DnOP 75.2 µM). DnHP, DnBP and nBBP achieved full (or close to full) blockade of [(3)H]CP-55940 binding, whereas DEHP, DiOP and DnOP produced partial (55-70%) inhibition. Binding experiments with phenylmethane-sulfonylfluoride (PMSF) indicated that the ester linkages of nBBP and DnBP remain intact during assay. The monoesters mono-2-ethylhexylphthalate (M2EHP) and mono-isohexylphthalate (MiHP) failed to reach IC(50) at 150 µM and mono-n-butylphthalate (MnBP) was inactive. Inhibitory potencies in the [(3)H]CP-55940 binding assay were positively correlated with inhibition of CB(1) receptor agonist-stimulated binding of [(35)S]GTPγS to the G protein, demonstrating that phthalates cause functional impairment of this complex. DnBP, nBBP and DEHP also inhibited binding of [(3)H]SR141716A, whereas inhibition with MiHP was comparatively weak and MnBP had no effect. Equilibrium binding experiments with [(3)H]SR141716A showed that phthalates reduce the B(max) of radioligand without changing its K(d). DnBP and nBBP also rapidly enhanced the dissociation of [(3)H]SR141716A. Our data are consistent with an allosteric mechanism for inhibition, with phthalates acting as relatively low affinity antagonists of CB(1) receptors and cannabinoid agonist-dependent activation of the G-protein. Further studies are warranted, since some phthalate esters may have potential to modify CB(1) receptor-dependent behavioral and physiological outcomes in the whole animal.

The actions of benzophenanthridine alkaloids, piperonyl butoxide and (S)-methoprene at the G-protein coupled cannabinoid CB1 receptor in vitro.

This investigation focused primarily on the interaction of two benzophenanthridine alkaloids (chelerythrine and sanguinarine), piperonyl butoxide and (S)-methoprene with G-protein-coupled cannabinoid CB(1) receptors of mouse brain in vitro. Chelerythrine and sanguinarine inhibited the binding of the CB(1) receptor agonist [(3)H]CP-55940 to mouse whole brain membranes at low micromolar concentrations (IC(50)s: chelerythrine 2.20 µM; sanguinarine 1.10 µM). The structurally related isoquinoline alkaloids (berberine and papaverine) and the phthalide isoquinoline ((-)-ß-hydrastine) were either inactive or considerably below IC(50) at 30 µM. Chelerythrine and sanguinarine antagonized CP-55940-stimulated binding of [(35)S] GTPγS to the G-protein (IC(50)s: chelerythrine 2.09 µM; sanguinarine 1.22 µM). In contrast to AM251, both compounds strongly inhibited basal binding of [(35)S]GTPγS (IC(50)s: chelerythrine 10.06 µM; sanguinarine 5.19µM). Piperonyl butoxide and S-methoprene inhibited the binding of [(3)H]CP-55940 (IC(50)s: piperonyl butoxide 8.2 µM; methoprene 16.4 µM), and also inhibited agonist-stimulated (but not basal) binding of [(35)S]GTPγS to brain membranes (IC(50)s: piperonyl butoxide 22.5 µM; (S)-methoprene 19.31 µM). PMSF did not modify the inhibitory effect of (S)-methoprene on [(3)H]CP-55940 binding. Our data suggest that chelerythrine and sanguinarine are efficacious antagonists of G-protein-coupled CB(1) receptors. They exhibit lower potencies compared to many conventional CB(1) receptor blockers but act differently to AM251. Reverse modulation of CB(1) receptor agonist binding resulting from benzophenanthridines engaging with the G-protein component may explain this difference. Piperonyl butoxide and (S)-methoprene are efficacious, low potency, neutral antagonists of CB(1) receptors. Certain of the study compounds may represent useful starting structures for development of novel/more potent G-protein-coupled CB(1) receptor blocking drugs.

Pinostrobin from Cajanus cajan (L.) Millsp. inhibits sodium channel-activated depolarization of mouse brain synaptoneurosomes.

This investigation focuses on the in vitro neuroactive properties of pinostrobin, a substituted flavanone from Cajanus cajan (L.) Millsp. of the Fabaceae family. We demonstrate that pinostrobin inhibits voltage-gated sodium channels of mammalian brain (IC(50)=23 µM) based on the ability of this substance to suppress the depolarizing effects of the sodium channel-selective activator veratridine in a synaptoneurosomal preparation from mouse brain. The resting membrane potential of synaptoneurosomes was unaffected by pinostrobin. The pharmacological profile of pinostrobin resembles that of depressant drugs that block sodium channels.

Effects of the essential oil constituent thymol and other neuroactive chemicals on flight motor activity and wing beat frequency in the blowfly Phaenicia sericata.

BACKGROUND: The effects were evaluated of the plant terpenoid thymol and eight other neuroactive compounds on flight muscle impulses (FMIs) and wing beat frequency (WBF) of tethered blowflies (Phaenicia sericata Meig.). RESULTS: The electrical activity of the dorsolongitudinal flight muscles was closely linked to the WBF of control insects. Topically applied thymol inhibited WBF within 15-30 min and reduced FMI frequency. Octopamine and chlordimeform caused a similar, early-onset bursting pattern that decreased in amplitude with time. Desmethylchlordimeform blocked wing beating within 60 min and generated a profile of continuous but lower-frequency FMIs. Fipronil suppressed wing beating and induced a pattern of continuous, variable-frequency spiking that diminished gradually over 6 h. Cypermethrin- and rotenone-treated flies had initial strong FMIs that declined with time. In flies injected with GABA, the FMIs were generally unidirectional and frequency was reduced, as was seen with thymol. CONCLUSIONS: Thymol readily penetrates the cuticle and interferes with flight muscle and central nervous function in the blowfly. The similarity of the action of thymol and GABA suggests that this terpenoid acts centrally in blowflies by mimicking or facilitating GABA action.

The cannabinoid receptor agonist CP-55,940 and ethyl arachidonate interfere with [(3)H]batrachotoxinin A 20 alpha-benzoate binding to sodium channels and inhibit sodium channel function.

Recent investigations in our laboratory showed that voltage-gated sodium channels (VGSCs) in brain are sensitive to inhibition by various synthetic cannabinoids and endocannabinoids. The present experiments examined the effects of the cannabinoid-1 (CB1) receptor agonist CP-55,940 and ethyl arachidonate on [(3)H]batrachotoxinin A 20 alpha-benzoate ([(3)H]BTX-B]) binding and VGSC-dependent depolarization of the nerve membrane in synaptoneurosomes isolated from mouse whole brain. CP-55,940 acted as a full inhibitor of [(3)H]BTX-B binding and its IC(50) was established at 22.3 microM. At its maximum effect concentration, ethyl arachidonate achieved partial (approximately 70%) inhibition and was less effective than CP-55,940 as an inhibitor of binding (IC(50)=262.7 microM). The potent CB1 receptor antagonist AM251 (2 microM) had no significant effect on the displacement of [(3)H]BTX-B by either compound (P>0.05). Scatchard analyses showed that CP-55,940 and ethyl arachidonate reduce the binding of [(3)H]BTX-B by lowering its B(max) but ethyl arachidonate also increased the K(d) of radioligand binding. In kinetic experiments, CP-55,940 and ethyl arachidonate were found to boost the dissociation of [(3)H]BTX-B from VGSCs to rates that exceed the maximum velocity achievable by veratridine, indicating they operate as allosteric inhibitors of [(3)H]BTX-B binding. Neither compound was effective at changing the initial rate of association of [(3)H]BTX-B with sodium channels. CP-55,940 and ethyl arachidonate inhibited veratridine-dependent (TTX-suppressible) depolarization of the plasma membrane of synaptoneurosomes with IC(50)s of 3.2 and 50.1 microM respectively. These inhibitory effects were again not influenced by 2 microM AM251. Our data demonstrate that the potent cannabinoid receptor agonist CP-55,940 and the ethyl ester of arachidonic acid have the ability to associate with VGSCs and inhibit their function independently of effects on CB1 receptors. Binding data comparisons using mouse brain preparations indicate CP-55,940 is approximately 10,000 times more potent as a CB1 receptor ligand than a sodium channel ligand while ethyl arachidonate shows a much smaller differential. Ethyl arachidonate has been shown previously to be the principal metabolite of ethanol in the brains of intoxicated individuals and effects of this ester on VGSCs and CB1 receptors may contribute to the depressant effects of alcohol.

20(S)-protopanaxadiol and the ginsenoside Rh2 inhibit Na+ channel-activated depolarization and Na+ channel-dependent amino acid neurotransmitter release in synaptic fractions isolated from mammalian brain.

The ginsenoside Rh(2) and its aglycone 20(S)-protopanaxadiol are known to inhibit the binding of [(3)H]batrachotoxinin 20alpha-benzoate to site 2 on voltage-gated sodium channels and electrophysiological investigations conducted by others have shown that ginsenosides cause voltage-dependent inhibition of reconstituted forms of the sodium channel. Here we describe the actions of Rh(2) and 20(S)-protopanaxadiol on sodium channel function and release of neurotransmitters resulting from activation of native sodium channels in synaptic preparations isolated from whole mouse brain. Rh(2) and 20(S)-protopanaxadiol inhibited veratridine-dependent (tetrodotoxin-suppressible) depolarization of synaptoneurosomes as determined using the rhodamine 6G method although 20(S)-protopanaxadiol was more potent as an inhibitor than Rh(2). Veratridine- (sodium channel-) dependent release of the neurotransmitters L-glutamate and GABA was almost fully inhibited by 20(S)-protopanaxadiol, however, less complete inhibition was observed with Rh(2). At its maximum inhibitory concentration, Rh(2) also produced release of l-glutamate and GABA from synaptosomes, in contrast to 20(S)-protopanaxadiol. We conclude that low to moderate micromolar concentrations of Rh(2) and 20(S)-protopanaxadiol inhibit sodium channel function and sodium channel-activated release of neurotransmitters. Apparently the ginsenoside Rh(2) cannot achieve complete inhibition of sodium channel-activated transmitter release because at high concentrations it also stimulates release.

Inhibition of [3H]batrachotoxinin A-20alpha-benzoate binding to sodium channels and sodium channel function by endocannabinoids.

A number of putative endocannabinoids were found to modify the binding of [(3)H]batrachotoxinin A-20alpha-benzoate ([(3)H]BTX-B) to site 2 on voltage-gated sodium channels of mouse brain and achieve functional inhibition of sodium channels in vitro. 2-Arachidonoyl-glycerol (2-AG), arachidonoyl glycerol ether (AGE), N-arachidonoyl-dopamine (NADA) gave almost complete inhibition of [(3)H]BTX-B binding with IC(50) values of 90.4, 51.2 and 20.7 microM, respectively. The CB1 receptor antagonist AM251 (2 microM) had no effect on the displacement of radioligand by these endocanabinoids. Arachidonoyl-glycine (A-Gly) and arachidonoyl-GABA (A-GABA) were apparently less effective inhibitors of [(3)H]BTX-B binding giving 14.8+/-2.2 and 23.9+/-4.8% inhibition at 100 microM. Phenylmethanesulphonylfluoride (PMSF) did not alter the inhibitory effects of 2-AG, AGE, NADA and A-Gly on binding, but the efficacy of 100 microM A-GABA was increased by 60.3+/-6.3% (P<0.05). Scatchard analyses showed that 2-AG, AGE and NADA reduce the binding of [(3)H]BTX-B by lowering B(max) although increases in K(D) were also evident for AGE and NADA. Our kinetic experiments found that 2-AG, AGE and NADA increase the dissociation velocity of radioligand from site 2 on sodium channels demonstrating that these endocannabinoids operate as allosteric inhibitors of [(3)H]BTX-B binding. 2-AG, AGE and NADA inhibited veratridine-dependent (TTX-suppressible) depolarization of the plasma membrane of synaptoneurosomes at low micromolar concentrations and again the capacities of A-Gly and A-GABA to inhibit this response were less pronounced. The three most effective endocannabinoids (2-AG, AGE and NADA) were then examined in a synaptosomal transmitter release assay where they were observed to inhibit sodium channel- (veratridine-dependent) release of l-glutamate and GABA in the low micromolar range. These effects also occurred through a mechanism that was not influenced by 2 microM AM251. It is concluded that direct inhibition of sodium channel function leading to reduced neuronal excitation and depression of presynaptic release of amino acid transmitters is a property shared by several endocannabinoids.

Ethanolamine and related amino alcohols increase basal and evoked release of [3H]-D-aspartic acid from synaptosomes by enhancing the filling of synaptic vesicles.

This research examines the effects of ethanolamine and other amino alcohols on the dynamics of acridine orange (AO), oxonol V, and [3H]-D-aspartic acid in synaptic preparations isolated from mammalian brain. Ethanolamine concentration-dependently enhanced AO release from synaptosomes. Similar effects were observed with methylethanolamine and dimethylethanolamine, but not choline. The enhancement of AO efflux by ethanolamine was independent of extrasynaptosomal calcium (in contrast to KCl-induced AO efflux), was unaffected by tetrodotoxin and did not involve depolarization of the synaptosomal plasma membrane. KCl was unable to release AO from synaptosomes following exposure to ethanolamine, however ethanolamine and other amino alcohols were found to enhance both basal and KCl-evoked release of [3H]-D-aspartic acid from synaptosomes. Using isolated synaptic vesicles we demonstrate that amino alcohols are able to 1) abolish the ATP-dependent intravesicular proton concentration (i.e. stimulate efflux of AO) in a similar way to carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2) increase the ATP-supported transvesicular membrane potential (i.e. quench oxonol V fluorescence) in contrast to CCCP and 3) enhance intravesicular uptake of [3H]-D-aspartic acid. These results suggest that positively charged, membrane impermeant amino alcohol species are generated within synaptic vesicles as they sequester protons. Cationic forms of these amino alcohols boost the transvesicular electrical potential which increases transmitter uptake into synaptic vesicles and facilitates enhancement of basal and evoked release of transmitter. Our data suggest a potential role for ethanolamine and related amino alcohols in the regulation of synaptic vesicle filling. These findings may also have relevance to neuropathophysiological states involving altered production of ethanolamine.

Vanilloid (subtype 1) receptor-modulatory drugs inhibit [3H]batrachotoxinin-A 20-alpha-benzoate binding to Na+ channels.

This investigation was conducted to provide further insight into the effects of vanilloid (subtype 1) receptor (VR1) drugs at voltage-gated sodium channels and examine the potential of this interaction to influence release of neurotransmitters from synaptosomes prepared from mammalian brain. The VR1 modulatory drugs capsaicin, olvanil and capsazepine inhibited the binding of batrachotoxinin-A 20-alpha-benzoate ([(3)H]BTX-B) to receptor site 2 of voltage-gated sodium channels. All drugs reduced the affinity of radioligand for sodium channels, and capsazepine also decreased the number of [(3)H]BTX-B binding sites. In kinetic experiments, no reduction in radioligand association rate was found, but capsaicin, olvanil and capsazepine all enhanced the dissociation rate of [(3)H]BTX-B. All drugs inhibited veratridine-evoked release of L-glutamic acid, gamma-amino butyric acid and L-aspartic acid from synaptosomes; however, their inhibitory effects on transmitter release were much weaker when 35 mM potassium chloride was used to depolarize synaptosomes. The study compounds, in common with other central nervous system depressants, interact with a region on the voltage-gated sodium channel that permits negative allosteric coupling with receptor site 2 and this mechanism likely accounts for blockade of sodium channel-activated transmitter release.

Depolarization-induced release of ethanolamine from brain synaptic preparations in vitro.

The release of ethanolamine from mouse brain synaptosomes and synaptoneurosomes has been investigated. The depolarizing agents veratridine (50 microM), KCl (35 mM) and 4-aminopyridine (2 mM) enhanced the release of [3H]ethanolamine from preloaded synaptosomes under superfusion conditions. Tetrodotoxin (2 microM) strongly inhibited veratridine- and 4-aminopyridine-stimulated release of [3H]ethanolamine but had no effect on KCl-evoked or resting release. In the absence of calcium, a reduction in the resting release of [3H]ethanolamine occurred and release evoked by veratridine, and KCl was markedly reduced. Exposure of preloaded synaptosomes to 5 mM ethanolamine (but not 5 mM serine or 5 mM choline) calcium-dependently increased the efflux of [3H]ethanolamine, however, this was not accompanied by membrane depolarization. When these experiments were performed using synaptoneurosomes, qualitatively similar results were obtained. The resting and evoked release of [3H]ethanolamine was however approximately 2.5-fold higher compared to synaptosomes on a brain equivalent basis, suggesting that uptake and release occur at sites in addition to the nerve ending. Our data are consistent with the idea that a significant amount of ethanolamine accumulates presynaptically and undergoes calcium-dependent release upon depolarization possibly via classical exocytosis. In contrast, ethanolamine-induced release of [3H]ethanolamine likely involves mostly diffusional exchange across the neuronal membrane rather than base exchange. The present results add support to the concept that ethanolamine may play a role as a synaptic signaling molecule in mammalian brain.

Antifungal properties of surangin B, a coumarin from Mammea longifolia.

The natural product electron transport inhibitor surangin B was examined for its ability to inhibit in vitro mycelial growth and spore germination in several species of fungi. As an inhibitor of mycelial growth, surangin B showed strongest activity against Rhizoctonia solani (IC50 = 3.8 microM) and Botrytis cinerea (IC50 = 11.2 microM). Inhibitory effects were less pronounced in Alternaria dauci, Fusarium oxysporum and Penicillium sp. (IC50 values > 30 microM) and absent in Trichoderma harzianum. Surangin B reduced the level of spore germination in Fusarium oxysporum (IC50 = 2.3 microM) and Botrytis cinerea (IC50 = 1.4 microM), although Alternaria dauci was considerably more tolerant of this coumarin (IC50 = 500 microM). Our results indicate that surangin B may have potential as an antifungal agent.

Quantitation of paralytic shellfish toxins using mouse brain synaptoneurosomes.

A membrane potential assay based on synaptoneurosomes prepared from mouse brain was evaluated further for its utility in estimating saxitoxin and related bioactives. Saxitoxin concentrations quantitated in mussel extracts by the synaptoneurosomal technique correlated well with spiked concentrations in these samples (r2 = 0.995; slope=1.048). Other experiments found that the synaptoneurosomal assay can detect saxitoxin-like bioactives in zooplankton samples and the concentrations measured were consistent with preliminary estimations of saxitoxin equivalents using the [3H] saxitoxin receptor binding technique. Veratrine, a mixture of alkaloids that activate sodium channels, had similar potential as a substitute for veratridine in the synaptoneurosomal assay. The results provide additional evidence that the mouse brain synaptoneurosomal membrane potential assay has excellent capability for quantitation of saxitoxin-like activity in shellfish tissues and may also be applied to zooplankton samples.

Inhibition of voltage-sensitive sodium channels by the cannabinoid 1 receptor antagonist AM 251 in mammalian brain.

The cannabinoid 1 receptor antagonist AM 251 is known to block the inhibitory effects of endocannabinoids and synthetic cannabinoid agonists on transmitter release through an action at presynaptic cannabinoid 1 receptors in brain. We examined the ability of AM 251 to inhibit sodium channel-dependent functions and the binding of [3H]batrachotoxinin A 20-alpha-benzoate to sodium channels in mouse brain synaptic preparations. Depolarization of synaptoneurosomes by the sodium channel site 2-specific neurotoxin veratridine, which is abolished by tetrodotoxin, was found to be inhibited in a concentration-dependent fashion by AM 251 (IC50=8.9 microM). Veratridine-dependent (tetrodotoxin suppressible) release, of L-glutamic acid and GABA from synaptosomes was also reduced by AM 251 [IC50s=8.5 microM (L-glutamic acid), 9.2 microM (GABA)]. The binding of the radioligand [3H]batrachotoxinin A 20-alpha-benzoate to site 2 on sodium channels was displaced by AM 251 (IC50=11.2 microM). Scatchard analysis of binding showed that at its IC50, AM 251 increased (by 2.3 times) the KD of radioligand without altering Bmax, suggesting a competitive mechanism of inhibition by AM 251. Kinetic experiments indicated that AM 251 inhibits equilibrium binding by allosterically accelerating the dissociation of the [3H]-batrachotoxinin A 20-alpha-benzoate:sodium channel complex. Our data suggest that micromolar concentrations of AM 251 are capable of reducing neuronal excitability and inhibiting release of excitatory and inhibitory transmitters through blockade of voltage-sensitive sodium channels in brain.

Mechanisms for resin acid effects on membrane currents and GABA(A) receptors in mammalian CNS.

Resin acids from bleached wood pulp are toxic to fish. 12,14-Dichlorodehydroabietic acid (12,14-Cl(2)DHA) raises cytoplasmic Ca(2+) in synaptosomes and blocks neural GABA(A) receptors; however, the underlying mechanism remains unclear in these earlier rodent studies. 12,14-Cl(2)DHA (50µM) almost completely blocked native GABA(A) currents (rat cortical cultures) but had no significant effect on picrotoxin-sensitive recombinant human receptors in oocytes (α1, ß2 and γ2L: the most prevalent isoforms in mammalian brain). In oocytes, 12,14-Cl(2)DHA failed to produce a calcium-activated chloride current, in contrast to the calcium ionophore ionomycin (10µM). However, in cultured cortical pyramidal cells, both ionomycin and 12,14-Cl(2)DHA produced chloride-selective currents of similar magnitude (presumably secondary to Ca(2+) release). 12,14-Cl(2)DHA was unable to stimulate phosphate labelling of [(3)H ]-inositol in mouse synaptosomes, indicating that the study compound does not cause Ca(2+) release via an IP(3) mechanism. Calcium pump ATPase inhibition also seems unlikely since thapsigargin did not elevate free calcium in synaptosomes. 12,14-Cl(2)DHA clearly blocks GABA(A) currents indirectly: we infer that its toxicity may be secondary to the elevations in cytoplasmic Ca(2+) via an unidentified recognition site (or receptor) found in neuronal cells.

Stimulation by surangin B of endogenous amino acid release from synaptosomes.

The effect of surangin B, an insecticidal natural product coumarin, on presynaptic release of endogenous amino acids was investigated using a purified synaptosomal fraction isolated from mouse brain. Surangin B stimulated the release of glutamic acid (GLU), gamma-aminobutyric acid (GABA), serine, alanine and the aminosulfonic acid taurine from synaptosomes at micromolar concentrations. In all cases, these responses were reduced by removing calcium from the saline and surangin B-evoked release of GLU, GABA, aspartic acid (ASP) and alanine was significantly inhibited by the sodium channel blocker tetrodotoxin. Rotenone (a complex I inhibitor) and carbonyl cyanide chlorophenylhydrazone (CCCP; an uncoupler), were more potent releasers of amino acids from synaptosomes than surangin B, however, carboxin (a complex II-selective inhibitor), was extremely weak to ineffective in this regard. The stimulatory effect of surangin B and complex III-selective inhibitors on release of GLU, GABA, ASP and alanine by synaptosomes was significantly reduced by N,N,N',N'-tetramethyl-p-phenylenediamine, suggesting that blockade of complex III in intraterminal mitochondria is an important effect of this coumarin. Our results demonstrate that surangin B, in common with CCCP and inhibitors of complex I and III, cause release of both neurotransmitter and non-neurotransmitter amino acids from nerve endings in vitro. However, in contrast to most classical agents which interfere selectively with mitochondrial function, the release of endogenous amino acids from synaptosomes by surangin B also involves a moderate extracellular calcium ion-dependent component and relies partially on sodium ion entry into the nerve ending.

A rapid assay for the brevetoxin group of sodium channel activators based on fluorescence monitoring of synaptoneurosomal membrane potential.

A functional pharmacologically-based assay for the brevetoxin group of sodium channel activators was developed using synaptoneurosomes isolated from the brains of CD1 mice. The assay can detect the depolarizing effect of brevetoxin congeners PbTx-2 and PbTx-3 as enhancements of the veratridine-dependent increase in fluorescence of the voltage-sensitive fluorescent probe rhodamine 6G. The assay is relatively rapid and can detect brevetoxin activity in the nanomolar range. The synaptoneurosomal assay has been used to analyse mussel tissue extracts spiked with PbTx-2, and composite toxicity, expressed as PbTx-3 equivalents in extracts of oysters naturally exposed to brevetoxins. In this latter context, the synaptoneurosomal technique was shown to compare favorably with the cytotoxicity assay, the receptor binding assay and HPLC/MS. Our results support the concept that this membrane potential assay detects brevetoxins based on their interaction with sodium channels.