Inhibition of axonal morphogenesis by nonlethal, submicromolar concentrations of methylmercury.

We investigated the effects of sublethal concentrations of the neurotoxicant methylmercury (MeHg) on the developmental progression of cultured neurons to the stage of axonal morphogenesis. Chick (E8) forebrain neurons in vitro develop axons by a stereotyped developmental sequence nearly identical to that of widely used rat hippocampal neurons, but at much less cost and difficulty. In this chick forebrain system, 40% of neurons develop long axons after 2 days in culture, and 80% have axons after 4 days. A single, 2-h exposure to 0.5 or 0.25 microM MeHg reduced the number of neurons developing axons to approximately half that of controls without causing significant cell death for at least 2 days after treatment. Although MeHg caused an immediate depolymerization of neuronal microtubules, after 1 day of recovery the microtubule array of MeHg-treated neurons was indistinguishable by immunofluorescent assay from that of untreated cells at equivalent development stages. Thus, the inhibition of axonal development by submicromolar concentrations of MeHg did not appear to be the direct effect of microtubule disassembly. Chelation of Ca(2+) during MeHg exposure appeared to exert a small immediate protective effect, as previously reported, but was itself toxic within 1 day after chelation. We suggest that this inhibition of axonal morphogenesis by acute, sublethal concentrations of MeHg may play a role in the developmental syndrome caused by environmental exposure to MeHg.

Methylmercury affects multiple subtypes of calcium channels in rat cerebellar granule cells.

We tested the ability of methylmercury (MeHg) to block calcium channel current in cultures of neonatal cerebellar granule cells using whole-cell patch clamp techniques and Ba(2+) as charge carrier. Low micromolar concentrations of MeHg (0.25-1 microM) reduced the amplitude of whole cell Ba(2+) current in a concentration- and time-dependent fashion; however, this effect was not voltage-dependent and the current-voltage relationship was not altered. Increasing the stimulation frequency hastened the onset and increased the magnitude of block at both 0.25 and 0.5 microM MeHg but not at 1 microM. In the absence of stimulation, all concentrations of MeHg were able to decrease current amplitude. The ability of several Ca(2+) channel antagonists (omega-conotoxin GVIA, omega-conotoxin MVIIC, omega-agatoxin IVA, calcicludine, and nimodipine) to alter the MeHg-induced effect was tested in an effort to determine if MeHg targets a specific subtype of Ca(2+) channel. Each of the antagonists tested was able to decrease a portion of whole cell Ba(2+) current under control conditions. However, none were able to attenuate the MeHg-induced block of whole cell Ba(2+) current, suggesting either that the mechanism of MeHg-induced block involves sites other than those influenced specifically by Ca(2+) channel antagonists or that MeHg was able to "outcompete" these toxins for their binding sites. These results show that acute exposure to submicromolar concentrations of MeHg can block Ba(2+) currents carried through multiple Ca(2+) channel subtypes in primary cultures of cerebellar granule cells. However, it is unlikely that the presence of a specific Ca(2+) channel subtype is able to render granule cells more susceptible to the neurotoxicologic actions of MeHg.

Comparative effects of methylmercury on parallel-fiber and climbing-fiber responses of rat cerebellar slices.

The environmental neurotoxicant methylmercury (MeHg) causes profound disruption of cerebellar function. Previous studies have shown that acute exposure to MeHg impairs synaptic transmission in both the peripheral and central nervous systems. However, the effects of MeHg on cerebellar synaptic function have never been examined. In the present study, effects of acute exposure to MeHg on synaptic transmission between parallel fibers or climbing fibers and Purkinje cells were compared in 300- to 350-microm cerebellar slices by using extracellular and intracellular microelectrode-recording techniques. Field potentials of parallel-fiber volleys (PFVs) and the associated postsynaptic responses (PSRs) were recorded in the molecular layer by stimulating the parallel fibers in transverse cerebellar slices. The climbing-fiber responses were also recorded in the molecular layer by stimulating white matter in sagittal cerebellar slices. At 20, 100, and 500 microM, MeHg reduced the amplitude of both PFVs and the associated PSRs to complete block, however, it blocked PSRs more rapidly than PFVs. MeHg also decreased the amplitudes of climbing-fiber responses to complete block. For all responses, an initial increase in amplitude preceded MeHg-induced suppression. Intracellular recordings of excitatory postsynaptic potentials of Purkinje cells were compared before and after MeHg. At 100 microM and 20 microM, MeHg blocked the Na+-dependent, fast somatic spikes and Ca++-dependent, slow dendritic spike bursts. MeHg also hyperpolarized and then depolarized Purkinje cell membranes, suppressed current conduction from parallel fibers or climbing fibers to dendrites of Purkinje cells, and blocked synaptically activated local responses. MeHg switched the pattern of repetitive firing of Purkinje cells generated spontaneously or by depolarizing current injection at Purkinje cell soma from predominantly Na+-dependent, fast somatic spikes to predominantly Ca++-dependent, low amplitude, slow dendritic spike bursts. Thus, acute exposure to MeHg causes a complex pattern of effects on cerebellar synaptic transmission, with apparent actions on both neuronal excitability and chemical synaptic transmission.

Symposium overview: chemical modulation of neuroreceptors and channels via intracellular components.

Whereas the roles of G proteins and protein kinases in various neuroreceptors and ion channels have been studied extensively, their roles in the actions of drugs and toxicants on these receptors and channels remain to be elucidated. Almost all drugs and toxicants exert multiple actions on multiple target sites, and there is no reason to assume that a chemical modulates a receptor/channel via a single mechanism. In fact, experimental evidence is slowly but steadily being accumulated to indicate that certain drugs and toxicants modulate neuroreceptor/channel functions through interactions with intracellular components such as G proteins and protein kinases. Multiple actions of a toxicant on various receptors/channels may be explained on the basis of its interaction with the G protein/kinase system that is a common denominator of the target sites. This is a virgin field that promises a quantum leap in the coming years. Each presentation and discussion will focus on expected future developments and potential significance in the field of neurotoxicology.

Passive transfer of Lambert-Eaton myasthenic syndrome induces dihydropyridine sensitivity of ICa in mouse motor nerve terminals.

Mice were injected for 30 days with plasma from three patients with Lambert-Eaton Myasthenic Syndrome (LEMS). Recordings were made from the perineurial sheath of motor axon terminals of triangularis sterni muscle preparations. The objective was to characterize pharmacologically the identity of kinetically distinct, defined potential changes associated with motor nerve terminal Ca2+ currents (ICa) that were affected by LEMS autoantibodies. ICa elicited at 0.01 Hz were significantly reduced in amplitude by approximately 35% of control in LEMS-treated nerve terminals. During 10-Hz stimulation, ICa amplitude was unchanged in LEMS-treated motor nerve terminals, but was depressed in control. During 20- or 100-Hz trains, facilitation of ICa occurred in LEMS-treated nerve terminals whereas in control, no facilitation occurred during the trains at 20 Hz and marked depression occurred at 100 Hz. Saturation for amplitude and duration of ICa in control terminals occurred at 2 and 4-6 mM extracellular Ca2+, respectively; in LEMS-treated terminals, the extracellular Ca2+ concentration had to increase by two to three times of control to cause saturation. Amplitude of the two components of ICa observed when the preparation was exposed to 50 microM 3,4-diaminopyridine and 1 mM tetraethylammonium were both reduced by LEMS plasma treatment. The fast component (ICa,s) was reduced by 35%, whereas the slow component (ICa, s) was reduced by 37%. omega-Agatoxin IVA (omega-Aga-IVA; 0.15 microM) and omega-conotoxin-MVIIC (omega-CTx-MVIIC; 5 microM) completely blocked ICa in control motor nerve terminals. The same concentrations of toxins were 20-30% less effective in blocking ICa in LEMS-treated terminals. The residual ICa remaining after treatment with omega-Aga-IVA or omega-CTx-MVIIC was blocked by 10 microM nifedipine and 10 microM Cd2+. Thus LEMS plasma appears to downregulate omega-Aga-IVA-sensitive (P-type) and/or omega-CTx-MVIIC-sensitive (Q-type) Ca2+ channels in murine motor nerve terminals, whereas dihydropyridine (DHP)-sensitive (L-type) Ca2+ channels are unmasked in these terminals. Acute exposure (90 min) of rat forebrain synaptosomes to LEMS immunoglobulins (Igs; 4 mg/ml) did not alter the binding of [3H]-nitrendipine or [125I]-omega-conotoxin-GVIA (-omega-CgTx GVIA) when compared with synaptosomes incubated with an equivalent concentration of control Igs. Conversely, LEMS Igs significantly decreased the Bmax for [3H]-verapamil to approximately 45% of control. The apparent affinity of verapamil (KD) for the remaining receptors was not significantly altered. Thus acute exposure of isolated central nerve terminals to LEMS Igs does not increase DHP sensitivity, whereas it reduces the number of binding sites for verapamil but not for nitrendipine or omega-CgTx-GVIA. These results suggest that chronic but not acute exposure to LEMS Igs either upregulates or unmasks DHP-sensitive Ca2+ channels in motor nerve endings.

Elevations of intracellular Ca2+ as a probable contributor to decreased viability in cerebellar granule cells following acute exposure to methylmercury.

In these experiments we examined whether the elevations in intracellular Ca2+ concentration ([Ca2+]i) induced by methylmercury (MeHg)(described in our previous study) might contribute to cerebellar granule cell mortality following exposure to MeHg in vitro. Cells were exposed to 0.5 microM MeHg for 45 min or 1 microM MeHg for 38 min, conditions previously shown to induce elevations in [Ca2+]i in these cells. Control cells were exposed to buffer alone for 60 min. Viability was assessed using the Live/Dead viability/cytotoxicity kit. At 30 min post-MeHg exposure, there was no immediate increase in cell mortality; however, by 3.5 h after the onset of MeHg exposure, cell viability decreased to 74 and 54% of control values for 0.5 and 1.0 microM MeHg, respectively. At 24.5 h after MeHg exposure, cell viability declined to approximately 27%. Losses in cell viability at 3.5 h were prevented by pretreating the granule cells for 65 min with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA; 10 microM), then exposing the cells to MeHg in the continued presence of BAPTA; however, at 24.5 h, BAPTA no longer prevented MeHg-induced cell death. Exposure to the Ca2+ channel blockers omega-conotoxin MVIIC (1 microM) or nifedipine (1 microM), previously shown to delay elevations in [Ca2+]i with MeHg exposure in vitro, protected granule cells from MeHg-induced mortality at 3.5 h postexposure. These data suggest that at early time points, MeHg-induced increases in [Ca2+]i may contribute to granule cell mortality; however, the role of Ca2+ at later time points is unclear.

Action of methylmercury on GABA(A) receptor-mediated inhibitory synaptic transmission is primarily responsible for its early stimulatory effects on hippocampal CA1 excitatory synaptic transmission.

Bath application of methylmercury (MeHg) causes an early stimulation before block of synaptic transmission in the CA1 region of hippocampal slices. Effects of MeHg and Hg++ on inhibitory postsynaptic potentials (IPSPs) or currents (IPSCs) and excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) were compared to test whether or not early block by MeHg of GABA(A)-mediated inhibitory synaptic transmission and MeHg-induced alterations of the resting membrane potentials of CA1 neurons contribute to this initial enhancement of excitability. MeHg affected IPSPs and IPSCs similarly, and more rapidly than EPSPs and EPSCs. In contrast, although Hg++ blocked IPSPs more rapidly than EPSPs, times to block of IPSCs and EPSCs by Hg++ were virtually identical when CA1 neurons were voltage-clamped at their resting membrane potential levels. MeHg increased EPSC amplitudes before their subsequent decrease even when CA1 neuronal membranes were voltage-clamped at their resting potentials. This suggests that effects of MeHg on CA1 cell membrane potentials are not a major factor for MeHg-induced early stimulation of hippocampal synaptic transmission. Effects of MeHg and Hg++ on the reversal potentials for IPSCs also differed. Both metals blocked all outward and inward currents generated at different holding potentials. However, MeHg shifted the current-voltage (I/V) relationship to more positive potentials, although Hg++ shifted the I/V curve to more negative potentials. Hg++ was a less potent blocker of on IPSCs and EPSPs or EPSCs than was MeHg. To determine if the early increase in amplitude of population spikes or EPSPs is due to an action of MeHg at GABA(A) receptors, extracellular recordings of population spikes and intracellular recordings of EPSPs were compared with or without pretreatment of hippocampal slices with bicuculline. After preincubation of slices with 10 microM bicuculline for 30 to 60 min, MeHg only decreased the amplitudes of population spikes and EPSPs to block; no early increase of synaptic transmission occurred. Pretreatment of slices with strychnine, did not prevent MeHg-induced early increase in population spikes. MeHg also blocked responses evoked by bath application of muscimol, a GABA(A) agonist. Thus, block by MeHg of GABA(A) receptor-mediated inhibitory synaptic transmission may result in disinhibition of excitatory hippocampal synaptic transmission, and appears to be primarily responsible for the initial excitatory effect of MeHg on hippocampal synaptic transmission.

Effects of the paralytic agent 2,4-dithiobiuret on viability and morphology of rat pheochromocytoma (PC12) cells.

In previous studies, 2,4-dithiobiuret (DTB) caused a delayed onset neuromuscular weakness in rats which was associated with decreased quantal content, alterations in postsynaptic ion channel properties, and abnormalities in nerve terminal ultrastructure. The latter include features typical of degenerating or diseased motor endplates as well as a marked proliferation of smooth endoplasmic reticulum (SER), swelling of mitochondria and evidence for a decreased in intraterminal calcium concentrations at early stages of intoxication (Jones, 1989, Acta Neuropathol. 78:72). These in vivo studies do not allow us to distinguish between the initial effects of DTB on the nerve terminal and those evolving as a result of disuse or secondary to its action on the muscle fiber or Schwann cells. To begin to distinguish between primary and secondary effects of DTB, we examined DTB-treated rat PC12 cells for comparable changes. The direct effects of DTB on PC12 cells included signs of general toxicity. Cell death in sparsely- plated cultures increased from 8-9% in controls to 13.7% at 10 microM for 24 hr exposure, and continued to increase in a concentration-dependent fashion to 25% mortality at 25 microM. However, between 25 and 100 microM there was little additional increase in mortality. 10 to 40 microM DTB slightly decreased the ability of both differentiated and undifferentiated cells to adhere to a substrate. This effect was independent of cell mortality. In moderately-differ-entiated cells having processes up to 10 cell diameters and several varicosities, concentrations of DTB as high as those invoking increased cell mortality and comparable to those affecting the rat neuromuscular junction did not cause abnormalities in the structure of the SER. No masses of tubulovesicular profiles were seen with transmission electron microscopy, and large changes in the quantity or distribution were not detected at the light microscope with the fluorescent stains DiOC6 or rhodamine B. Other signs of neuronal degeneration (blebbing of the plasmalemma, large intracellular droplets, mitochondrial abnormalities) preceded or accompanied any evidence for abnormalities in the SER. Thus the effect of DTB on the SER at the rat motor nerve terminal may occur secondary to a more general toxic action on other cell types, or may be dependent on a level of neuronal activity not achieved in sparsely- plated cultures, or may require a greater degree of differentiation of the neuronal cells than provided by the PC12 cell model used in this study.

Pathways mediating Ca2+ entry in rat cerebellar granule cells following in vitro exposure to methyl mercury.

Cell imaging and the Ca(2+)-sensitive fluorophore fura-2 were used to examine methyl mercury's effect on Ca2+ homeostasis in rat cerebellar granule cells, a cell type preferentially targeted by methyl mercury. In vitro methyl mercury exposure (0.2-5.0 microM) induced a biphasic rise in fura-2 fluorescence ratio, consisting of a small first phase due to Ca2+ release from intracellular store(s) and a much larger second phase which required Ca2+ influx. The time-to-onset of these fura-2 fluorescence changes was inversely correlated with methyl mercury concentration. When examining various Ca2+ entry pathways as possible targets contributing to Ca2+ influx, we found that excitatory amino acid pathways were not directly involved. In contrast, the voltage-dependent Ca2+ channel blockers nifedipine and omega-conotoxin-MVIIC significantly delayed the time-to-onset of both phases, a response inconsistent with mere inhibition of Ca2+ entry. The nonselective voltage-dependent Ca2+ channel blocker Ni2+ had no effect on the methyl mercury response. Because methyl mercury alters cell membrane potentials, we hypothesized that voltage-dependent Na+ channels were activated initially; however, tetrodotoxin did not alter the methyl mercury-induced increases in fura-2 fluorescence ratio. Thus, methyl mercury alters Ca2+ homeostasis in cerebellar granule cells through nifedipine- and omega-conotoxin-MVIIC-sensitive pathways, suggesting that L-, N-, and/or Q-type Ca2+ channels may play a role in methyl mercury's mode of action or entry.

Effects of omega-agatoxin-IVA and omega-conotoxin-MVIIC on perineurial Ca++ and Ca(++)-activated K+ currents of mouse motor nerve terminals.

Effects of omega-agatoxin-IVA (omega-Aga-IVA) and omega-conotoxin-MVIIC (omega-CTx-MVIIC) on mouse motor nerve terminal Ca+2 currents and Ca(+2)-activated K+ currents (IK,Ca) were compared using the triangularis sterni preparation and perineurial recording techniques. omega-Aga-IVA caused concentration- and time-dependent block of both the fast (ICa-f) and slow (ICa-s) components of Ca+2 current. Low concentrations (10 nM) caused preferential block of ICa-s. Higher concentrations (100-150 nM) of omega-Aga-IVA blocked ICa-f effectively. omega-CTx-MVIIC blocked both ICa-s and ICa-f with equal sensitivity; however, higher concentrations and longer exposure times than those required for omega-Aga-IVA were needed. omega-CTx-MVIIC could block the residual ICa-f that remained after pretreatment with Cd+2 or omega-Aga-IVA. Increasing the extracellular Ca+2 concentration partially antagonized the effects of both omega-Aga-IVA and omega-CTx-MVIIC on ICa-s and ICa-f. Washing the preparation with toxin-free solution only slightly antagonized the effect of omega-Aga-IVA and was ineffective in omega-CTx-MVIIC-treated preparations. Low concentrations of omega-Aga-IVA and omega-CTx-MVIIC increased the duration of IK,Ca whereas higher concentrations reduced the amplitude of IK,Ca. Thus, at mouse motor nerve terminals, both omega-Aga-IVA- and omega-CTx-MVIIC-sensitive Ca+2 currents exist. omega-Aga-IVA appears to be more selective in blocking nerve terminal Ca+2 current than does omega-CTx-MVIIC. Paradoxically, block of ICa-s alone by omega-Aga-IVA and, to a lesser extent, omega-CTx-MVIIC was associated with increased duration of IK,Ca whereas block of ICa-s and ICa-f by omega-Aga-IVA and omega-CTx-MVIIC was associated with reduced amplitude of IK,Ca.

Tetrandrine blocks voltage-dependent calcium entry and inhibits the bradykinin-induced elevation of intracellular calcium in NG108-15 cells.

Tetrandrine, a plant alkaloid used in Chinese traditional medicine blocks voltage-dependent Ca2+ entry in NG108-15 cells as assessed using fura-2 microfluorimetry. Cells were depolarized with 50 mM KCI for one min resulting in a transient increase in the 340/380 nm ratio of fura 2 fluorescence, indicative of an increase in [Ca2+]i. Treatment of the same cell with 100 microM tetrandrine for seven min followed by an identical K+ depolarization blocked the increase in 340/380 nm fluorescence ratio. Washing with tetrandrine-free solutions for 20 min partially reversed this effect. Bradykinin (Bk) induces transient and repetitive increases in [Ca2+] due to release of Ca2+ from intracellular stores via activation of the inositol trisphosphate (IP3) second messenger system. After pre-treatment with 100 microM tetrandrine for seven min, the Bk (1 microM, 1 min) response was significantly reduced. Likewise, the effect of angiotensin II (AT-II), which also causes an IP3 dependent release of Ca2+ from intracellular stores, was ablated by tetrandrine. Thus, in addition to block of voltage-dependent Ca2+ channels, tetrandrine also caused disturbances in intracellular Ca2+ signalling.

Mercurial-induced alterations in neuronal divalent cation homeostasis.

Mercurials such as Hg2+ and methylmercury (MeHg) are environmental contaminants. Both are neurotoxic upon chronic and acute exposure, however, these toxic manifestations are distinct. The mechanisms underlying this cytotoxicity remain unknown, but may be related to a disruption in divalent cation homeostasis because both disrupt Ca(2+)-dependent processes in several model systems. These effects include a block in nerve-evoked neurotransmitter release as well as an increase in spontaneous transmitter release. This suggests that mercurials simultaneously decrease Ca2+ influx following nerve stimulation, and increase intracellular Ca2+ concentration ([Ca2+]i) in the nerve terminal. Although these effects appear to be at odds, they can be justified mechanistically. Both Hg2+ and MeHg block voltage-activated Ca2+ channels in the nerve terminal. The mechanism of block by these mercurials is different, since Hg2+ and MeHg are competitive and noncompetitive inhibitors of Ca2+ influx, respectively. The functional consequence in both instances remains decreased Ca2+ influx into the nerve terminal following the invasion of an action potential leading to decreased nerve-evoked release of neurotransmitter. The effects of mercurials on voltage-activated Ca2+ channels are distinct from those which mediate the increases in spontaneous transmitter release. Reducing extracellular Ca2+ concentration ([Ca2+]e) decreased, but did not prevent, the mercurial-induced increases in spontaneous transmitter release, suggesting that both intra- and extracellular sources of Ca2+ contribute to mercurial-induced elevations in [Ca2+]i in a nerve terminals. The effects of MeHg on divalent cation homeostasis have been studied using isolated nerve terminals from the rat brain (synaptosomes) and cells in culture (NG108-15 and isolated cerebellar granule cells) loaded with the Ca(2+)-selective fluorescent indicator fura-2. In synaptosomes, MeHg caused an Ca(2+)e-independent elevation in intrasynaptosomal Zn2+ concentration ([Zn2+]i) as well as an Ca(2+)e-dependent elevation in [Ca2+]i. The elevations in [Zn2+]i and [Ca2+]i were mediated by release of Zn2+ from soluble synaptosomal proteins and increased plasma membrane permeability, respectively. In NG108-15 cells, the effects of MeHg on divalent cation concentrations were more complex. First, MeHg mobilized Ca2+ from an intracellular store sensitive to inositol-1,4,5-tris-phosphate (IP3) which was independent of IP3 generation. Second, MeHg increased the intracellular concentration of an endogenous polyvalent cation, possibly Zn2+. Finally, MeHg caused an increase in the plasma membrane permeability to Ca2+ which was attenuated by high concentrations of the voltage-activated Ca2+ channel blocker nifedipine or by the voltage-activated Na+ channel blocker tetrodotoxin (TTX). While these studies demonstrate mercurials interfere with divalent cation regulation in neuronal systems, the consequences of these effects are not yet known.

Effects of mercurials on ligand- and voltage-gated ion channels: a review.

Both organic and inorganic mercurials are neurotoxic, an action attributable to their prominent reactivity with numerous biological ligands. While many sites within the central nervous system can be potentially affected by mercurials, ligand-and voltage-gated ion channels represent a plausible early target. There are several reasons for this. First, ion channels are located on the plasma membrane in large numbers, thus increasing the likelihood of mercurial-channel interaction. Second, ion channels may allow the passage of mercurials of similar size and charge as those ions which normally pass through the channel, a process which can hinder physiologic ion transport and also lead to disruption of intracellular events. Third, all mercurials have a high affinity for sulfhydryl groups on cysteines which may comprise critical regions of an ion channel. Consistent with an ability of neurotoxic metals to disrupt ion channel function, other heavy metals such as Cd2+, Pb2+, Co2+ and Zn2+ inhibit agonist binding to ligand-gated ion channels and inhibit ion flux through both ligand- and voltage-gated ion channels. Ion channels play a crucial role in cellular homeostasis. Changes in the intracellular concentrations of ions, necessary to initiate and sustain processes such as neurotransmitter release, growth cone elongation and gene expression, arise at least in part via flux through voltage- and ligand-gated ion channels. Since such a large battery of events are mediated by ion channels, it follows that their disruption by mercurials could lead to potentially deleterious consequences for the cell. This review will focus on the possible role that alteration in ion channel function may play in the pathological events seen following exposure either in vivo or in vitro to mercurials, and in particular methylmercury (MeHg).

Methylmercury acts at multiple sites to block hippocampal synaptic transmission.

To explore the mechanisms by which methylmercury (MeHg) blocks central synaptic transmission, intracellular recordings of action potentials and resting membrane potentials were made in CA1 neurons of rat hippocampal slices. At 4 to 100 microM, MeHg blocked action potentials in a concentration- and time-dependent manner. MeHg also depolarized Ca1 neuronal membranes. However, this effect occurred more slowly than block of action potentials because the resting membrane potentials remained unchanged when threshold stimulation-evoked action potentials were blocked. Thus, MeHg may initially alter the threshold level of neuronal membrane excitability and subsequently depolarize the membrane leading to block of synaptic transmission. To identify potential sites of action of MeHg, effects of MeHg on the responses of CA1 neurons to orthodromic stimulation of Schaffer collaterals, antidromic stimulation of the alveus, direct injection of current at cell soma and iontophoretic application of glutamate were compared. At 20 and 100 microM, MeHg blocked action potentials evoked by stimulation of Schaffer collaterals and by current injection at the cell soma at similar times. In contrast, action potentials evoked by stimulation of the alveus were blocked more rapidly by 100 microM MeHg than were action potentials evoked by current injection at CA1 neuronal soma. MeHg also blocked the responses of CA1 neurons to iontophoresis of glutamate, but time to block of these responses was slower than block of the corresponding orthodromically-evoked responses by stimulation of Schaffer collaterals. Compared to excitatory postsynaptic potentials, inhibitory postsynaptic potentials appeared to be more sensitive to MeHg, because block of inhibitory postsynaptic potentials occurred before block of excitatory postsynaptic potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of 2,4-dithiobiuret on the synthesis and release of acetylcholine and dopamine from rat pheochromocytoma (PC12) cells.

Chronic administration of 2,4-dithiobiuret (DTB), causes delayed-onset neuromuscular weakness in rats. This effect results from inhibition of quantal release of acetylcholine (ACh) from motor nerve terminals. The effects of noncholinergic neurotransmission are unknown. The purpose of the present study was to examine the presynaptic mechanisms involved in DTB-induced inhibition of ACh release, particularly, the specificity of action of DTB for cholinergic secretion. Differentiated pheochromocytoma (PC12) cells were used to compare the effects of DTB on the content and release of ACh and dopamine (DA) using neurochemical techniques. At concentrations of 50 to 1000 microM, DTB had little or no effect on [3H]choline uptake or on the spontaneous release of endogenous or [3H]ACh, but caused a significant decrease in release of endogenous or [3H]ACh elicited by depolarization with elevated extracellular [K+]. DTB reduced evoked release of ACh without altering cellular levels of ACh or choline, suggesting that DTB acts directly on mechanisms involved in ACh release. These alterations occurred without prominent alterations in [Ca2+]i as measured by fluorescence microscopy of individual PC12 cells loaded with fura-2. Moreover, DTB did not affect the increase of [Ca2+]i of PC12 cells in response to KCl-induced depolarization. alpha-Latrotoxin-stimulated release of ACh was not inhibited by DTB. DTB-induced suppression of depolarization-evoked release of [3H]ACh was associated with an increased level of [3H]ACh in the vesicular pool although the cytosolic pool was unaffected. High concentrations of DTB also reduced depolarization-evoked release of DA and inhibited DA synthesis resulting in a decrease in the readily releasable pool of DA. These effects occurred at higher concentrations and after longer exposures to DTB than were necessary to alter ACh release. Inasmuch as DA synthesis in the PC12 cell has been shown to be modulated by ACh release, this effect on DA release may reflect a consequence of the diminished release of ACh. These results suggest that DTB alters the release of ACh by interrupting either the mobilization and/or release of the vesicular pool of ACh.

Nifedipine and tetrodotoxin delay the onset of methylmercury-induced increase in [Ca2+]i in NG108-15 cells.

Methylmercury (MeHg) causes a multiphasic disruption of intraneuronal cation regulation. Release of Ca2+ from internal stores and entry of extracellular Ca2+ (Ca2+e) contribute to the temporally distinct early (first Ca2+ phase) and late (second Ca2+ phase) components of increased intracellular Ca2+ concentration ([Ca2+]i). The present study was designed to explore the mechanisms mediating the second Ca2+ phase. Fluorescence intensity was monitored from single NG108-15 cells loaded with fura-2 before and during acute application of 2 microM MeHg. Nifedipine (1 or 10 microM but not 0.1 microM) significantly delayed the time-to-onset of the second Ca2+ phase. Nifedipine (1 microM but not 0.1 microM) also caused a concentration-dependent delay in the onset of both the first Ca2+ phase which is independent of Ca2+e and the elevation of non-Ca2+ cation (non-Ca2+ phase). The L-type dihydropyridine (DHP) Ca2+ channel agonist Bay K-8644 (10 nM) had no effect on the time-to-onset of the second Ca2+ phase. Neither the N-type Ca2+ channel blocker omega-conotoxin GVIA (up to 1 microM) nor the nonselective Ca2+ channel blocker Ni2+ (1 mM) altered the time-to-onset of the second Ca2+ phase. Removal of Na+e or addition of the voltage-dependent Na+ channel antagonist tetrodotoxin (TTX, 1 microM) significantly delayed the onset of the second Ca2+ phase. In a manner similar to that for 1 microM nifedipine, TTX also delayed the onset of the other phases. Thus, we hypothesize that MeHg depolarizes the plasma membrane leading to an increase in the activation of voltage-dependent Na+ and Ca2+ channels which promotes, directly or indirectly, the influx of Ca2+ during the second Ca2+ phase.

Decreased calcium currents in motor nerve terminals of mice with Lambert-Eaton myasthenic syndrome.

1. The effects of immunoglobulin G (IgG) from patients with Lambert-Eaton myasthenic syndrome on Ca2+ currents in mammalian motor nerve terminals are unknown. Therefore, we recorded these currents in phrenic nerves of mice injected with serum from either LEMS patients, myasthenia gravis patients, or healthy control individuals. 2. In control preparations, the endplate currents induced by repetitive stimulation at > or = 20 Hz were depressed as expected. However, in the LEMS animals quantal content decreased and either depression did not occur or synaptic facilitation occurred. 3. Ca2+ currents were smaller in LEMS animals. At 0.5 Hz stimulation frequency, normalized Ca2+ currents in LEMS animals were 57 +/- 14% of those in control. At higher frequencies, Ca2+ currents become smaller in control but not in LEMS animals. 4. Ca2+ currents in controls were unaffected by addition of nifedipine but were reduced by 37% upon addition of omega-conotoxin GVIA. In LEMS animals, however, the currents were depressed by 43% by nifedipine but were unaffected by omega-conotoxin GVIA. Thus, LEMS is associated with reduced Ca2+ currents and a shift to dihydropyridine sensitivity.

The proteins synaptotagmin and syntaxin are not general targets of Lambert-Eaton myasthenic syndrome autoantibody.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune neuromuscular disease in which impairment of Ca2+ entry into the nerve ending and consequent impaired release of acetylcholine (ACh) results in muscle weakness. The identity of the primary antigenic target molecule(s) of the autoantibodies is uncertain. Electrophysiological studies and 45Ca2+ uptake studies implicate a direct effect on the Ca2+ channel complex at the motor nerve terminal. Some recent studies, however, suggest a more indirect interference caused by binding of autoantibodies to synaptotagmin or syntaxin, molecules presumed to be involved in docking and/or coupling the synaptic vesicles to the Ca2+ channels in the active zone for vesicle exocytosis and transmitter release. Western blot analyses of rat and human brain membrane proteins and pure recombinant synaptotagmin and syntaxin were used to examine directly the targets of LEMS autoantibodies and determine specifically whether or not synaptotagmin and/or syntaxin were general targets in LEMS. IgG from 14 patients with LEMS was used to probe western blots of gels containing synaptotagmin, syntaxin, rat synaptosomal proteins, and human brain membrane proteins. Several similar immunoreactive bands were observed using both rat and human brain membranes. These include high-molecular-weight protein bands whose size would be consistent with being components of Ca2+ channels. No reactive component was observed against either syntaxin or synaptotagmin in IgG of the 14 LEMS patients. However, both human and rat brain membranes contain proteins recognized by antibodies directed against synaptotagmin or syntaxin, indicating their immunologic relatedness and evolutionary conservation. These results suggest that large-molecular-weight proteins consistent with being Ca2+ channel subunits rather than syntaxin and synaptotagmin are general targets of LEMS autoantibodies.

Methylmercury mobilizes Ca++ from intracellular stores sensitive to inositol 1,4,5-trisphosphate in NG108-15 cells.

Fluorescence intensity was monitored from individual NG108-15 cells loaded with the Ca(++)-selective probe fura-2, and exposed to 2 microM methylmercury (MeHg). The initial effect of 2 microM MeHg was an elevation in intracellular Ca++ concentration ([Ca++]i), which was not blocked by lowering extracellular Ca++ (Ca++e), nifedipine (0.1 microM) or by Ni++ (1 mM). Addition of 100 microM Mn++ to Ca(++)-containing medium did not alter fluorescence intensity at either the Ca(++)-insensitive excitation wavelength of 360 nm or the Ca(++)-sensitive wavelength of 380 nm. Depolarization with K+ decreased the intensity at both wavelengths, indicating Mn++ entry. In the presence of Mn++, MeHg decreased the 380 nm, but not the 360 nm signal. Bradykinin (Bk) caused a transient increase in the fluorescence ratio, which was blocked by the endoplasmic reticulum Ca(++)-adenosine triphosphatase inhibitor thapsigargin. Pretreatment with Bk and thapsigargin reduced significantly the increase in ratio induced by MeHg from 21.9 +/- 3.4 to 6.9 +/- 1.8% of base line. Bk had no effect when applied after MeHg. Caffeine reduced the Bk-induced increase in [Ca++]i and the MeHg-induced increase in ratio from 21.9 +/- 3.4 to 9.0 +/- 2.1%. Thus, Bk, caffeine and MeHg all appear to release a common pool of intracellular calcium (Ca2+i). When applied after MeHg, Bk increased inositol 1,4,5-trisphosphate (IP3) by 305 +/- 27% compared to 270 +/- 29% in controls. Thus, MeHg did not induce Ca++ release by IP3 generation, nor did it block the effects of Bk by interfering with IP3 synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)