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William D Atchison

Publications and source records attributed to William D Atchison.

At least 19 recordsLinked to original sources
PubMed2006

Acetylcholine release at neuromuscular junctions of adult tottering mice is controlled by N-(cav2.2) and R-type (cav2.3) but not L-type (cav1.2) Ca2+ channels.

The mutation in the alpha(1A) subunit gene of the P/Q-type (Ca(v)2.1) Ca(2+) channel present in tottering (tg) mice causes ataxia and motor seizures that resemble absence epilepsy in humans. P/Q-type Ca(2+)channels are primarily involved in acetylcholine (ACh) release at mammalian neuromuscular junctions. Unmasking of L-type (Ca(v)1.1-1.2) Ca(2+) channels occurs in cerebellar Purkinje cells of tg mice. However, whether L-type Ca(2+) channels are also up-regulated at neuromuscular junctions of tg mice is unknown. We characterized thoroughly the pharmacological sensitivity of the Ca(2+) channels, which control ACh release at adult tg neuromuscular junctions. Block of N- and R-type (Ca(v)2.2-2.3), but not L-type Ca(2+) channels, significantly reduced quantal content of end-plate potentials in tg preparations. Neither resting nor KCl-evoked miniature end-plate potential frequency differed significantly between tg and wild type (WT). Immunolabeling of Ca(2+) channel subunits alpha(1A), alpha(1B), alpha(1C), and alpha(1E) revealed an apparent increase of alpha(1B), and alpha(1E) staining, at tg but not WT neuromuscular junctions. This presumably compensates for the deficit of P/Q-type Ca(2+)channels, which localized presynaptically at WT neuromuscular junctions. No alpha(1C) subunits juxtaposed with pre- or postsynaptic markers at either WT or tg neuromuscular junctions. Thus, in adult tg mice, immunocytochemical and electrophysiological data indicate that N- and R-type channels both assume control of ACh release at motor nerve terminals. Recruitment of alternate subtypes of Ca(2+) channels to control transmitter release seems to represent a commonly occurring method of neuronal plasticity. However, it is unclear which conditions underlie recruitment of Ca(v)2 as opposed to Ca(v)1-type Ca(2+) channels.

Acetylcholine↗
PubMed2005

Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury.

The objective of the present study was to determine the relative effectiveness of methylmercury (MeHg) to alter divalent cation homeostasis and cause cell death in MeHg-resistant cerebellar Purkinje and MeHg-sensitive granule neurons. Application of 0.5-5 microM MeHg to Purkinje and granule cells grown in culture caused a concentration- and time-dependent biphasic increase in fura-2 fluorescence. At 0.5 and 1 microM MeHg, the elevations of fura-2 fluorescence induced by MeHg were biphasic in both cell types, but significantly delayed in Purkinje as compared to granule cells. Application of the heavy-metal chelator, TPEN, to Purkinje cells caused a precipitous decline in a proportion of the fura-2 fluorescence signal, indicating that MeHg causes release of Ca(2+) and non-Ca(2+) divalent cations. Purkinje cells were also more resistant than granule cells to the neurotoxic effects of MeHg. At 24.5 h after-application of 5 microM MeHg, 97.7% of Purkinje cells were viable. At 3 microM MeHg there was no detectable loss of Purkinje cell viability. In contrast, only 40.6% of cerebellar granule cells were alive 24.5 h after application of 3 microM MeHg. In conclusion, Purkinje neurons in primary cultures appear to be more resistant to MeHg-induced dysregulation of divalent cation homeostasis and subsequent cell death when compared to cerebellar granule cells. There is a significant component of non-Ca(2+) divalent cation released by MeHg in Purkinje neurons.

Air Pollutants↗
PubMed2005

Is chemical neurotransmission altered specifically during methylmercury-induced cerebellar dysfunction?

Methylmercury (MeHg) is an important environmental neurotoxicant that is present in seafood and affects the developing and mature nervous system. The neurotoxicity induced by MeHg is a concern, particularly for fish-eating populations and pregnant or nursing women. During MeHg-induced neurotoxicity, degeneration of the granule cell layer in the cerebellum occurs, which leads to deficits in motor function. I suggest that the action of MeHg on specific neurotransmitter receptors contributes to the selective vulnerability of granule cells. MeHg appears to stimulate M(3) muscarinic acetylcholine receptors and to inhibit GABA(A) receptor subtypes preferentially on cerebellar granule cells. This could lead to the loss of tonic inhibition of granule cells as a result of antagonism of GABA(A) receptors, and a M(3)-receptor-mediated increase in the intracellular concentration of Ca(2+) and block of a K(+)-dependent leak current. The net result would be increased spontaneous release of glutamate, which, coupled with a MeHg-induced impairment of glutamate uptake by astrocytes, could cause Ca(2+)-mediated cytotoxicity.

Acetylcholine↗
PubMed2005

Inwardly rectifying and voltage-gated outward potassium channels exhibit low sensitivity to methylmercury.

The concentration- and time-dependence of effects of methylmercury (MeHg) on voltage-gated outward K(+) (Kv) channels, inwardly rectifying K(+) (Kir) channels, voltage-gated Ca(2+) channels and GABA(A) receptor activated channels were compared in cerebellar granule cells in culture using whole cell patch clamp recording techniques. The objective was to determine if MeHg equally affects different types of ion channels. Under similar experimental conditions, these four ion channel types displayed markedly different sensitivity to MeHg. At 0.1-1 microM, MeHg caused apparent inhibition of Ca(2+)-channel and GABA(A) receptor-mediated currents, but did not cause any significant effect on Kv or Kir channels. Among the four channel types examined, GABA(A) receptors appeared to be the most sensitive to MeHg. The Kv channels, particularly the delayed rectifiers (DRs), appeared to be relatively resistant to MeHg compared with GABA(A) receptors and Ca(2+) channels. Kir channels were virtually unaffected by MeHg in the concentration range of 10-100 microM. The differential sensitivity of GABA(A) receptors and Kv channels to MeHg was also observed in granule and Purkinje cells in freshly isolated cerebellar slices of rat. The insensitivity of Kir channel to MeHg was also seen in Xenopus laevis oocytes expressing cloned Kir7.1 channels. Thus, these appear to be general properties of these channels as opposed to distinct effects associated with granule cells in culture. These results suggest that MeHg does preferentially affect certain types of ion channels. Hence, the effects of MeHg on membrane ion channels are not due simply to nonspecific actions on the membrane. Furthermore, at least certain types of Kir channels appear to be the most resistant type of ion channel reported to date to effects of MeHg.

Animals↗
PubMed2005

Fluid flow-induced increase in inward Ba2+ current expressed in HEK293 cells transiently transfected with human neuronal L-type Ca2+ channels.

Mechanical forces can alter the gating of several kinds of ion channels in many types of cells, but the mechanisms underlying the mechanosensitivity are not clearly understood. To date, there are very few reports on mechanosensitivity of Ca2+ channels, particularly neuronal Ca2+ channels. We examined the mechanical sensitivity of human recombinant L-type Ca2+ channels in response to fluid flow. Neuronal L-type Ca2+ channels (Ca(v) 1.2) were expressed transiently in HEK293 cells using expression cDNA clones of human alpha1C, alpha2delta, and beta subunits along with green fluorescent protein (GFP) as a reporter protein. Current (I(Ba)) through these heterologously-expressed channels was measured using whole cell recording technique with 20 mM Ba2+ as charge carrier. Transfected cells were exposed to a constant, increased fluid flow from a separate pipette during current recording. The L-type I(Ba) was found to be very sensitive to the flow-induced shear forces. Peak current amplitude increased by as much as approximately 50% during fluid flow as compared to that in the absence of fluid pressure. However, no change was observed in the amplitude of the average current during the final 5 ms of the 150-ms voltage step. Current amplitude promptly returned to normal control levels upon stopping fluid flow. The current-voltage relationship was not altered by fluid flow. The flow-induced increase in current amplitude exhibited an apparent shift in steady-state inactivation toward more negative potentials; inactivation was faster but was not voltage dependent. Activation was slightly faster under flow. Thus, increased mechanical tension associated with fluid flow can alter the fundamental properties of voltage-gated Ca2+ channels, even for channels which might not normally be exposed to fluid flow shear forces in their native environment.

Barium↗
PubMed2005

Methylmercury induces a spontaneous, transient slow inward chloride current in Purkinje cells of rat cerebellar slices.

Methylmercury (MeHg; 10-100 microM) induced a spontaneous, transient, slow inward current in Purkinje cells in rat cerebellar slices. Insensitivity of this current to tetrodotoxin suggests that its generation is not related to presynaptic firing. The present study was designed to attempt to identify the ionic origin of this current. Neither Gd(3+), a nonspecific cation channel blocker, nor tetrakis(2-pyridylmethyl)ethylethylenediamine, which chelates Zn(2+), could prevent this current. Following dialysis of cells with a low-[Cl(-)] pipette solution, the giant currents were inducible only when the cells were held at potentials more positive than 0 mV but not at potentials more negative than -60 mV. In addition, no giant currents were observed when cells were held at 0 mV under symmetrical [Cl(-)] conditions. Thus, this current seems to be mediated by Cl(-). However, it was insensitive to the glycine receptor antagonist strychnine. The anion channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or niflumic acid suppressed GABA(A) receptor-mediated spontaneous inhibitory postsynaptic currents. Niflumic acid also prevented appearance of this giant current; DIDS was only effective at more positive membrane potentials. Thus, this current seems to be carried by a voltage-dependent Cl(-) channel. Reducing extracellular Ca(2+) concentration and/or intracellular application of the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid seemed to be ineffective at preventing appearance of this current. Thus, these data do not seem to support the conclusion that this current is mediated by a Ca(2+)-activated Cl(-) channel. The role that this current plays in MeHg-induced neurotoxicity is unknown.

Animals↗
PubMed2004

Acute exposure to methylmercury causes Ca2+ dysregulation and neuronal death in rat cerebellar granule cells through an M3 muscarinic receptor-linked pathway.

Acute exposure to low concentrations of methylmercury (MeHg) causes a severe loss of intracellular calcium (Ca2+(i)) homeostasis, which apparently contributes to neuronal death of cerebellar granule cells in culture. We examined the role of muscarinic receptors in MeHg-induced Ca2+ dysregulation and cell death in rat cerebellar granule cells in vitro using fura-2 single-cell microfluorimetry and viability assays, respectively. The nonspecific muscarinic receptor antagonist atropine significantly delayed the onset of MeHg-induced Ca2+ elevations and reduced the amount of Ca2+ released into the cytosol. Depletion of the smooth endoplasmic reticulum (SER) Ca2+ pool with thapsigargin or down-regulation of muscarinic receptors and inositol-1,3,4-triphosphate (IP3) receptors with bethanechol (BCh) caused similar reductions in the amplitude of the MeHg-induced Ca2+ increase, suggesting that MeHg interacts with muscarinic receptors to cause Ca2+ release from the SER through activation of the IP3 receptors. To determine whether this Ca2+ release plays a role in MeHg-induced cell death, cells were exposed to MeHg in the presence of specific muscarinic receptor inhibitors. Acute exposure to increasing concentrations of MeHg (0.2-1.0 microM) caused a corresponding increase in cell death at 24.5 h post-exposure. Prior down-regulation of muscarinic and IP3receptors with BCh protected against cell death. Protection was ablated by atropine and the M3 receptor antagonist 4-diphenylacetoxyl-N-methylpiperidine methiodide (DAMP), but not by the neuronal nicotinic receptor antagonist dihydro-beta-erythroidine hydrobromide (DHE). Thus activation of M3 muscarinic receptors with subsequent generation of IP3 evidently contributes to elevated [Ca2+]i and subsequent cytotoxicity of cerebellar granule cells by MeHg.

Animals↗
PubMed2004

Morphometric characterization of the neuromuscular junction of rodents intoxicated with 2,4-dithiobiuret: evidence that nerve terminal recycling processes contribute to muscle weakness.

2,4-Dithiobiuret (DTB) causes ascending motor weakness when given chronically to rodents. In muscles of animals with DTB-induced weakness, quantal release of acetylcholine (ACh) is impaired. We examined in detail the structural changes that occurred at neuromuscular junctions and their associated Schwann cells of extensor digitorum longus (EDL) muscles of male rats treated with DTB to the onset of muscle weakness, 5-8 days. Our objective was to assess the involvement of the Schwann cells and to determine the most likely primary targets of DTB. At the onset of muscle weakness, nerve terminals exhibited some enlarged regions, but did not sprout. Terminal Schwann cells became flatter and expanded to cover most of the endplate. The extent of invasion of the synaptic cleft by Schwann cell processes was not significantly different from controls; extension of Schwann cell sprouts away from the junction was not seen. Thus, the morphology of the Schwann cells, although clearly affected by DTB, does not suggest that they contribute directly to the physiological defects of DTB-treated terminals. Abnormal tubulovesicular structures or tangles of neurofilaments were clustered in the centers of about 25% of treated terminals. Fewer synaptic vesicles occupied the region opposite the postsynaptic folds. Vesicle volumes were variable and included some very large vesicles, corresponding with the variable MEPP amplitudes reported previously for terminals of DTB-treated rodents. The postsynaptic area stained by rhodamine-labeled alpha-bungarotoxin expanded with terminal swelling, apparently by unpleating of the postsynaptic folds. No loss of ACh receptors or spread of ACh receptors beyond terminal boundaries was detected. Morphometric data are consistent with the conclusion that DTB affects, either directly or indirectly, vesicular release of ACh and the subsequent vesicular recycling process.

Animals↗
PubMed2004

Disruption of intraneuronal divalent cation regulation by methylmercury: are specific targets involved in altered neuronal development and cytotoxicity in methylmercury poisoning?

Methylmercury is an environmental contaminant which causes relatively specific degeneration of the granular layer of the cerebellum, despite its ability to bind thiol groups in proteins of all cell types. The mechanisms underlying the specific targeting of cells during MeHg poisoning may depend on specific receptors and other targets related to divalent cation homeostasis, particularly intracellular calcium (Ca(2+)(i) signaling. MeHg disrupts Ca(2+)(i) homeostasis in a number of neuronal models, including cerebellar granule cells in primary culture, and contributes to MeHg-induced cell death, impaired synaptic function and disruption of neuronal development. Interestingly, the disruption of [Ca(2+)](i) regulation occurs through specific pathways which affect Ca(2+) regulation by organelles, particularly mitochondria and the smooth endoplasmic reticulum (SER). Cholinergic pathways which affect [Ca(2+)](i) signaling also appear to be critical targets, particularly muscarinic acetylcholine (ACh) receptors which are linked to Ca(2+) release through inositol-1,4,5-triphosphate (IP(3)) receptors. [Ca(2+)](i) dysregulation may also underlie observed alterations in cerebellar neuron development through interaction with specific target(s) in the developing axon. In this review, we examine the hypothesis that MeHg affects specific targets to cause disruption of neuronal development and cell death.

Animals↗
PubMed2003

Methylmercury differentially affects GABA(A) receptor-mediated spontaneous IPSCs in Purkinje and granule cells of rat cerebellar slices.

Using whole-cell recording techniques we compared effects of the environmental cerebellar neurotoxicant methylmercury (MeHg) on spontaneous IPSCs (sIPSCs) of both Purkinje and granule cells in cerebellar slices of the rat. In Purkinje cells, bath application of 10, 20 or 100 microM MeHg initially increased then suppressed the frequency of sIPSCs to zero. In granule cells, the initial increase in frequency was not observed in approximately 50% of cells examined, but suppression of sIPSCs by MeHg occurred in every cell tested. For both cells, time to onset of effects of MeHg was inversely related to the concentration; moreover, the pattern of changes in mIPSCs induced by MeHg in the presence of tetrodotoxin was similar to that in sIPSCs. For ea ch concentration of MeHg, it took 2-3 times longer to block sIPSCs in Purkinje cells than it did in granule cells. MeHg also initially increased then decreased amplitudes of sIPSCs to block in both cells; again the response was more variable in granule cells. In most Purkinje and some granule cells, MeHg induced a giant, slow inward current during the late stages of exposure. Appearance of this current appeared to be MeHg concentration dependent, and the direction of current flow was reversed by changing the holding potentials. Reduction of the [Cl-] in the internal solution caused inwardly directed, but not outwardly directed giant currents to disappear, suggesting that this current is a Cl(-)-mediated response. However, bicuculline and picrotoxin failed to block it. MeHg apparently acts at both presynaptic and postsynaptic sites to alter GABA(A) receptor-mediated inhibitory synaptic transmission. GABA(A) receptors in granule cells appear to be more sensitive to block by MeHg than are those in Purkinje cells, although the general patterns of effects on the two cells are similar.

Animals↗
PubMed2003

Comparative effects of methylmercury and Hg(2+) on human neuronal N- and R-type high-voltage activated calcium channels transiently expressed in human embryonic kidney 293 cells.

Expression cDNA clones of alpha1B-1 or alpha1E-3 subunits coding for human neuronal N-(Cav2.2) or R-subtype (Cav2.3) Ca2+ channels, respectively, was combined with alpha2-bdelta and beta3-a Ca2+ channel subunits, and transfected into human embryonic kidney cells for transient expression to determine whether specific types of neuronal voltage-sensitive Ca2+ channels are affected differentially by methylmercury (MeHg) and Hg2+. For both Ca2+ channel subtypes, MeHg (0.125-5.0 microM) or Hg2+ (0.1-5 microM) caused a time- and concentration-dependent reduction of current. MeHg caused an initial, rapid component and a subsequent more gradual component of inhibition. The rapid component of block was completed between 100 and 150 s after beginning treatment. At 0.125 to 1.25 microM, MeHg caused a more gradual decline in current. Apparent IC50 values were 1.3 and 1.1 microM for MeHg, and 2.2 and 0.7 microM for Hg2+ on N- and R-types, respectively. For N-type current, effects of Hg2+ were initially greater on the peak current than on the sustained current remaining at the end of a test pulse; subsequently, Hg2+ blocked both components of current. For R-type current, Hg2+ affected peak and sustained current approximately equally. Kinetics of inactivation also seemed to be affected by Hg2+ in cells expressing N-type but not R-type current. Washing with MeHg-free solution could not reverse effects of MeHg on either type of current. The effect of Hg2+ on N- but not R-type current was partially reversed by Hg2+-free wash solution. Therefore, different types of Ca2+ channels have differential susceptibility to neurotoxic mercurials even when expressed in the same cell type.

Calcium↗
PubMed2003

Iberiotoxin-induced block of Ca2+-activated K+ channels induces dihydropyridine sensitivity of ACh release from mammalian motor nerve terminals.

The role which Ca(2+)-activated K(+) (K(Ca)) channels play in regulating acetylcholine (ACh) release was examined at mouse motor nerve terminals. In particular, the ability of the antagonist iberiotoxin to recruit normally silent L-type Ca(2+) channels to participate in nerve-evoked release was examined using conventional intracellular electrophysiological techniques. Incubation of cut hemidiaphragm preparations with 10 microM nimodipine, a dihydropyridine L-type Ca(2+) channel antagonist, had no significant effect on quantal content of end-plate potentials. Nevertheless, 1 microM S-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K 8644) enhanced quantal content to 134.7 +/- 3.5% of control. Iberiotoxin (150 nM) increased quantal content to 177.5 +/- 9.9% of control, whereas iberiotoxin plus nimodipine increased quantal content to only 145.7 +/- 10.4% of control. Coapplication of 1 microM Bay K 8644 with iberiotoxin did not significantly increase quantal content further than did treatment with iberiotoxin alone. The effects of iberiotoxin and nimodipine alone or in combination on the miniature end-plate potential (MEPP) frequency following KCl-induced depolarization were examined using uncut hemidiaphragm preparations. Nimodipine alone had no effect on MEPP frequency from preparations incubated in physiological saline containing 5 to 20 mM KCl. Moreover, iberiotoxin alone or combined with nimodipine also had no effect on MEPP frequency in physiological salines containing 5 to 15 mM KCl. At 20 mM KCl, however, iberiotoxin significantly increased MEPP frequency to 125.6% of iberiotoxin-free values; combined treatment with nimodipine and iberiotoxin prevented this increase in MEPP frequency. Thus, loss of functional K(Ca) channels unmasks normally silent L-type Ca(2+) channels to participate in ACh release from motor nerve terminals, particularly under conditions of intense nerve terminal depolarization.

Acetylcholine↗
PubMed2003

Effects of toxic environmental contaminants on voltage-gated calcium channel function: from past to present.

Voltage-gated Ca2+ channels are targets of the number of naturally occurring toxins, therapeutic agents as well as environmental toxicants. Because of similarities of their chemical structure to Ca2+ in terms of hydrated ionic radius, electron orbital configuration, or other chemical properties, polyvalent cations from aluminum to zinc variously interact with multiple types of voltage-gated Ca2+ channels. These nonphysiological metals have been used to study the structure and function of the Ca2+ channel, especially its permeability characteristics. Two nonphysiological cations, Pb2+ and Hg2+, as well as their organic derivatives, are environmental neurotoxicants which are highly potent Ca2+ channel blockers. These metals also apparently gain intracellular access in part by permeating through Ca2+ channels. In this review the history of Ca2+ channel block produced by Pb2+ and Hg2+ as well as other nonphysiological cations is traced. In particular the characteristics of Ca2+ channel block induced by these environmental neurotoxic metals and the consequences of this action for neuronal function are discussed.

Animals↗
PubMed2003

Ca2+ channels as targets of neurological disease: Lambert-Eaton Syndrome and other Ca2+ channelopathies.

In the nervous system, voltage-gated Ca2+ channels regulate numerous processes critical to neuronal function including secretion of neurotransmitters, initiation of action potentials in dendritic regions of some neurons, growth cone elongation, and gene expression. Because of the critical role which Ca2+ channels play in signaling processes within the nervous system, disruption of their function will lead to profound disturbances in neuronal function. Voltage-gated Ca2+ channels are the targets of several relatively rare neurological or neuromuscular diseases resulting from spontaneously-occurring mutations in genes encoding for parts of the channel proteins, or from autoimmune attack on the channel protein responses. Mutations in CACNAIA, which encodes for the alpha1A subunit of P/Q-type Ca2+ channels, lead to symptoms seen in familial hemiplegic migraine, episodic ataxia type 2, and spinocerebellar ataxia type 6. Conversely, autoimmune attack on Ca2+ channels at motor axon terminals causes peripheral cholinergic nerve dysfunction observed in Lambert-Eaton Myasthenic Syndrome (LEMS), the best studied of the disorders targeting voltage-gated Ca2+ channels. LEMS is characterized by decreased evoked quantal release of acetylcholine (ACh) and disruption of the presynaptic active zones, the sites at which ACh is thought to be released. LEMS is generally believed to be due to circulating antibodies directed specifically at the Ca2+ channels located at or near the active zone of motor nerve terminals (P/Q-type) and hence involved in the release of ACh. However, other presynaptic proteins have also been postulated to be targets of the autoantibodies. LEMS has a high degree of coincidence (approximately 60%) with small cell lung cancer; the remaining 40% of patients with LEMS have no detectable tumor. Diagnosis of LEMS relies on characteristic patterns of electromyographic changes; these changes are observable at neuromuscular junctions of muscle biopsies from patients with LEMS. In the majority of LEMS patients, those having detectable tumor, the disease is thought to occur as a result of immune response directed initially against voltage-gated Ca2+ channels found on the lung tumor cells. In these patients, effective treatment of the underlying tumor generally causes marked improvement of the symptoms of LEMS as well. Animal models of LEMS can be generated by chronic administration of plasma, serum or immunoglobulin G to mice. These models have helped dramatically in our understanding of the pathogenesis of LEMS. This "passive transfer" model mimics the electrophysiological and ultrastructural findings seen in muscle biopsies of patients with LEMS. In this model, we have shown that the reduction in amplitude of Ca2+ currents through P/Q-type channels is followed by "unmasking" of an L-type Ca2+ current not normally found at the motor nerve terminal which participates in release of ACh from terminals of mice treated with plasma from patients with LEMS. It is unclear what mechanisms underlie the development of this novel L-type Ca2+ current involved in release of ACh at motor nerve terminals during passive transfer of LEMS.

Animals↗
PubMed2003

Evidence for interactions between intracellular calcium stores during methylmercury-induced intracellular calcium dysregulation in rat cerebellar granule neurons.

Acute exposure to methylmercury (MeHg) causes severe disruption of intracellular Ca(2+) ([Ca(2+)](i)) regulation, which apparently contributes to neuronal death. Activation of the mitochondrial permeability transition pore (MTP) evidently contributes to this effect. We examined in more detail the contribution of mitochondrial Ca(2+) ([Ca(2+)](m)) to elevations of [Ca(2+)](i) caused by acute exposure to a low concentration of MeHg in primary cultures of rat cerebellar granule neurons. In particular, we sought to determine whether interactions occurred between Ca(2+)(i) pools in response to MeHg. Prior depletion of Ca(2+)(m) using carbonyl cyanide m-chlorophenylhydrazone (CCCP) and oligomycin significantly decreased the amplitude of [Ca(2+)](i) release from intracellular stores, and delayed the onset of whole-cell [Ca(2+)](i) elevations, caused by 0.5 microM MeHg. CCCP alone hastened the MeHg-induced release of Ca(2+) within the cell, whereas oligomycin alone delayed the MeHg-induced influx of extracellular Ca(2+). In granule cells loaded with rhod-2 acetoxymethylester to measure changes in [Ca(2+)](m), MeHg exposure caused a biphasic increase in fluorescence. The initial increase in fluorescence occurred in the absence of extracellular Ca(2+) and was abolished by mitochondrial depolarization. The secondary increase was associated with spreading of the dye from punctate staining to whole-cell distribution, and was delayed significantly by the MTP inhibitor cyclosporin A and the smooth endoplasmic reticulum Ca(2+) ATPase inhibitor thapsigargin. We conclude that MeHg causes release of Ca(2+) from the mitochondria through opening of the MTP, which contributes the bulk of the elevated [Ca(2+)](i) observed during MeHg neurotoxicity. Additionally, the Ca(2+) that enters the mitochondria seems to originate in the smooth endoplasmic reticulum, providing a mechanism for the observed mitochondrial Ca(2+) overload.

Animals↗
PubMed2002

Passive transfer of Lambert-Eaton syndrome to mice induces dihydropyridine sensitivity of neuromuscular transmission.

Lambert-Eaton myasthenic syndrome (LEMS) is a paraneoplastic disorder in which autoantibodies apparently target the voltage-gated Ca2+ channels that regulate acetylcholine (ACh) release at motor nerve terminals. P/Q-type Ca2+ channels are primarily involved in ACh release at mammalian neuromuscular junctions. Passive transfer of LEMS to mice by repeated administration of plasma from LEMS patients reduces the amplitude of the perineurial P/Q-type current, and unmasks a dihydropyridine (DHP)-sensitive L-type Ca2+ current at the motor nerve terminal. The present study sought to determine if this DHP-sensitive component contributes to ACh release. Mice were treated for 30 days with plasma from healthy human controls or patients with LEMS. For some studies, diaphragms from naive mice were incubated with LEMS or control human plasma for 2 or 24 h. End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from neuromuscular junctions in the hemidiaphragm. Treatment of mice with LEMS plasma evoked the characteristic electrophysiological signs of LEMS: reduced quantal content and facilitation of EPP amplitudes at high-frequency stimulation. Quantal content was also reduced in muscles incubated acutely with LEMS plasma. Nimodipine, a DHP-type blocker of L-type Ca2+ channels, did not significantly affect the quantal content of muscles treated for 2 or 24 h with either control or LEMS plasma, or following chronic treatment with control plasma. However, following 30 days treatment with LEMS plasma, nimodipine significantly reduced the remaining quantal content to 57.7 +/- 3.3 % of pre-nimodipine control levels. Thus, DHP-sensitive Ca2+ channels become involved in synaptic transmission at the mouse neuromuscular junction after chronic, but not acute treatment with LEMS plasma. However, reductions in quantal release of ACh occur even after very short periods of exposure to LEMS plasma. As such, development of the L-type Ca2+ channel contribution to ACh release during passive transfer of LEMS appears to occur only after quantal release is significantly impaired for an extended duration, suggesting that an adaptive response of the ACh release apparatus occurs in LEMS.

Acetylcholine↗
PubMed2002

Acute exposure to methylmercury opens the mitochondrial permeability transition pore in rat cerebellar granule cells.

Cerebellar granule cells are preferentially targeted during methylmercury (MeHg) poisoning. Following acute MeHg exposure, granule cells in culture undergo an increase in intracellular Ca2+ concentration ([Ca2+]i) that apparently contributes to cell death. This effect consists of several temporally and kinetically distinct phases. The initial elevation arises from release of Ca2+(i) stores; the second phase results from entry of Ca2+(e). In these experiments, we tested the hypothesis that release of mitochondrial Ca2+ through the mitochondrial permeability transition pore (MTP) contributes to the initial release of Ca2+(i). Neonatal rat cerebellar granule cells in culture and single cell microfluorimetry were used to examine MeHg-induced changes in [Ca2+]i and mitochondrial membrane potential (Psi(m)). Pretreatment with the MTP inhibitor cyclosporin A (CsA, 5 microM) delayed the initial phase of increased [Ca2+]i induced by 0.2 and 0.5 microM MeHg, but not 1.0 microM MeHg. CsA (5 microM) also delayed the irreversible loss of Psi(m) induced by 0.5 microM MeHg. Ca2+(e) was not required for either effect, because the effect of CsA on the first phase increase in [Ca2+]i and loss of Psi(m) was not altered in nominally Ca2+-free buffer. Increasing concentrations of MeHg (0.2-2.0 microM) caused increasing incidence of cell death at 24 h postexposure. Treatment with CsA provided protection against cytotoxicity at lower MeHg concentrations (0.2-0.5 microM), but not at higher MeHg concentrations (1.0-2.0 microM). Thus, the MTP appears to play a significant role in the cellular effects following acute exposure of cerebellar granule neurons to MeHg.

Animals↗