Changes in secondary glutamate release underlie the developmental regulation of excitotoxic neuronal cell death.

Vulnerability to excitotoxicity increases during development in vivo and in vitro. To determine whether the mere presence of mature N-methyl-D-aspartate (NMDA) receptors coincides with the emergence of excitotoxicity or whether post-receptor signaling processes may also contribute, we examined the temporal relationship of NMDA receptor expression, function and toxicity using cortical cell cultures. Surface expression of all NMDA receptor subunits increased with time in culture. This correlated with NMDA receptor function, assessed both biochemically and electrophysiologically, but not with the appearance of excitotoxicity. Specifically, cells at day in vitro (DIV) 10 were less susceptible to NMDA receptor-induced neurotoxicity than those cultured for 14 days, even though receptor expression/function was identical. In addition, cell-attached single channel recordings revealed that NMDA receptor conductance, open probability, and frequency of channel openings were not significantly different between the two days. Intriguingly, depolarization-induced release of glutamate from cultures grown for 10 days was significantly lower than that released from cultures grown for 14 days. Further, exogenous addition of glutamate receptor agonists immediately after removal of NMDA rendered cultures at DIV 10 susceptible to excitotoxicity, while toxicity was significantly reduced by addition of an NMDA receptor antagonist immediately after exposure to NMDA at DIV 14. These data are the first to demonstrate that the subsequent, secondary release of glutamate plays an equal, if not more important, role than NMDA receptor development per se, in mediating the enhanced vulnerability of neurons to excitotoxicity that occurs with age.

SIN-1-induced cytotoxicity in mixed cortical cell culture: peroxynitrite-dependent and -independent induction of excitotoxic cell death.

3-Morpholinosyndnomine (SIN-1) has been reported to be a peroxynitrite (OONO(-)) donor because it produces both nitric oxide (NO) and superoxide (O(2)(-).) upon decomposition in aqueous solution. However, SIN-1 can decompose to primarily NO in the presence of electron acceptors, including those found in biological tissues, making it necessary to determine the release product(s) formed in any given biological system. In a mixed cortical cell culture system, SIN-1 caused a concentration-dependent increase in cortical cell injury with a parallel increase in the release of cellular proteins containing 3-nitrotyrosine into the culture medium. The increase in 3-nitrotyrosine immunoreactivity, a footprint of OONO(-) production, was specific for SIN-1 as exposure to neurotoxic concentrations of an NO donor (Z)-1-[2-aminoethyl)-N-(2-ammonioethyl) aminodiazen-1-ium-1,2-diolate (DETA/NO), or NMDA did not result in the nitration of protein tyrosine residues. Both SIN-1-induced injury and 3-nitrotyrosine staining were prevented by the addition of either 5,10,15,20-Tetrakis (4-sulfonatophenyl) prophyrinato iron (III) [FeTPPS], an OONO(-) decomposition catalyst, or uric acid, an OONO(-) scavenger. Removal of NO alone was sufficient to inhibit the formation of OONO(-) from SIN-1 as well as its cytotoxicity. Removal of O(2)(-). and the subsequently formed H(2)O(2) by superoxide dismutase (SOD) plus catalase likewise prevented the nitration of protein-bound tyrosine but actually enhanced the cytotoxicity of SIN-1, indicating that cortical cells can cope with the oxidative but not the nitrosative stress generated. Finally, neural injury induced by SIN-1 in unadulterated cortical cells was prevented by antagonism of AMPA/kainate receptors, while blockade of the NMDA receptor was without effect. In contrast, activation of both NMDA and non-NMDA receptors contributed to the SIN-1-mediated neurotoxicity when cultures were exposed in the presence of SOD plus catalase. Thus, whether SIN-1 initiates neural cell death in an OONO(-)-dependent or -independent manner is determined by the antioxidant status of the cells. Further, the mode of excitotoxicity by which injury progresses is determined by the NO-related species generated.

Differential modulation of prostaglandin H synthase-2 by nitric oxide-related species in intact cells.

Nitrogen monoxide (NO) has been reported to both activate and inhibit prostaglandin (PG) biosynthesis. This apparent paradox might be explained by the production/action of distinct NO-related species formed as a result of the prevailing redox states of different cellular systems. As such, the effect of NO donors with different redox characteristics on the modulation of prostaglandin H synthase-2 (PGHS-2) in primary mouse cortical astrocytes and COS-7 cells engineered to overexpress PGHS-2 was assessed. In general, compounds that released NO(*) or NO(-) enhanced, while a peroxynitrite (OONO(-)) generator inhibited, PGHS-2-dependent prostaglandin production. While the possibility of altered gene transcription was eliminated in the COS-7 system as PGHS-2 was maximally expressed, in primary astrocytes where PGHS-2 expression was induced by lipopolysaccharide (LPS), effects on protein expression were detected. Compounds that released NO(*) synergistically enhanced LPS-mediated PGHS-2 protein synthesis. None of these effects were mediated by cGMP. All donors lost their ability to modulate PGHS-2 expression and function when decayed. These results indicate that the ultimate effect of NO on PGHS-2 enzyme activity and expression is dictated by the prevalent NO-related species formed, suggesting that important interactions which may exist between NO and prostanoid pathways in vivo will be highly dependent on the inherent redox environment.

Mechanisms of the antioxidant effects of nitric oxide.

The Janus face of nitric oxide (NO) has prompted a debate as to whether NO plays a deleterious or protective role in tissue injury. There are a number of reactive nitrogen oxide species, such as N2O3 and ONOO-, that can alter critical cellular components under high local concentrations of NO. However, NO can also abate the oxidation chemistry mediated by reactive oxygen species such as H2O2 and O2- that occurs at physiological levels of NO. In addition to the antioxidant chemistry, NO protects against cell death mediated by H2O2, alkylhydroperoxides, and xanthine oxidase. The attenuation of metal/peroxide oxidative chemistry, as well as lipid peroxidation, appears to be the major chemical mechanisms by which NO may limit oxidative injury to mammalian cells. In addition to these chemical and biochemical properties, NO can modulate cellular and physiological processes to limit oxidative injury, limiting processes such as leukocyte adhesion. This review will address these aspects of the chemical biology of this multifaceted free radical and explore the beneficial effect of NO against oxidative stress.

A microtiter trypan blue absorbance assay for the quantitative determination of excitotoxic neuronal injury in cell culture.

An automated method for the determination of neuronal cell death using trypan blue is described. Following various excitotoxic insults, murine mixed cortical cell cultures are stained with trypan blue (0.05%; 15 min), followed by SDS (1%) lysis. The absorbance of the dye is measured spectrophotometrically at 590 nm using a microtiter plate reader. When compared to the biochemical lactate dehydrogenase assay, no statistical difference in the calculated levels of excitotoxic neuronal cell death was noted between the assays in any given paradigm. This method is fast and reliable. It eliminates the need for cell counting, thus allowing for high volume sample analysis with a minimum of sample error. Utility of this trypan blue absorbance spectrophotometric assay is likely to extend beyond the study of excitotoxic neuronal injury and should complement existing methods for measuring neuronal viability and cytotoxicity in cell culture.

Cyclooxygenase-2 contributes to N-methyl-D-aspartate-mediated neuronal cell death in primary cortical cell culture.

Cyclooxygenase isozymes (COX-1 and COX-2) are found to be constitutively expressed in brain, with neuronal expression of COX-2 being rapidly induced after numerous insults, including cerebral ischemia. Because overactivation of N-methyl-D-aspartate (NMDA) receptors has been implicated in the cell loss associated with ischemia, we characterized the expression of the COX isozymes in murine mixed cortical cell cultures and used isozyme-selective inhibitors to determine their relative contribution to NMDA receptor-stimulated prostaglandin (PG) production and excitotoxic neuronal cell death. Immunocytochemical analysis of mixed cortical cell cultures revealed that COX-2 expression was restricted to neurons, whereas COX-1 was expressed in both neurons and astrocytes. Brief exposure to NMDA (5 min; 100 microM) elicited a time-dependent accumulation of PGs in the culture medium that preceded neuronal cell death and correlated with the induction of COX-2 mRNA. COX-1 expression remained unchanged. Flurbiprofen, a nonselective COX-1/COX-2 inhibitor, blocked NMDA-stimulated PG production and attenuated neuronal death in a concentration-dependent manner. Similar results were obtained with the specific COX-2 inhibitor NS-398 (10-30 microM) but not with the selective COX-1 inhibitor valeryl salicylate (10-300 microM). Inhibition of total constitutive COX activity with aspirin (100 microM, 1.5 h) before NMDA exposure did not prevent subsequent NMDA-mediated neuronal cell death. However, neuronal injury in aspirin-pretreated cultures was attenuated by flurbiprofen administration after NMDA exposure. Finally, the protection afforded by COX-2 inhibition was specific for NMDA because neither flurbiprofen nor NS-398 protected neurons against kainate-mediated neurotoxicity. Together, these results support the conclusion that newly synthesized COX-2 protein contributes to NMDA-induced neuronal injury.

Hypoxia modulates nitric oxide-induced regulation of NMDA receptor currents and neuronal cell death.

Nitric oxide (NO) released from a new chemical class of donors enhances N-methyl-D-aspartate (NMDA) channel activity. Using whole cell and single-channel patch-clamp techniques, we have shown that (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-NO (PAPA-NO) and diethylamine NO, commonly termed NONOates, potentiate the glutamate-mediated response of recombinant rat NMDA receptors (NR1/NR2A) expressed in HEK-293 cells. The overall effect is an increase in both peak and steady-state whole cell currents induced by glutamate. Single-channel studies demonstrate a significant increase in open probability but no change in the mean single-channel open time or mean channel conductance. Reduction in oxygen levels increased and prolonged the PAPA-NO-induced change in both peak and steady-state glutamate currents in transfected HEK cells. PAPA-NO also enhanced cell death in primary cultures of rodent cortical neurons deprived of oxygen and glucose. This potentiation of neuronal injury was blocked by MK-801, indicating a critical involvement of NMDA receptor activation. The NO-induced increase in NMDA channel activity as well as NMDA receptor-mediated cell death provide firm evidence that NO modulates the NMDA channel in a manner consistent with both a physiological role under normoxic conditions and a pathophysiological role under hypoxic conditions.

Inducible nitric oxide synthase expression in cultures enriched for mature oligodendrocytes is due to microglia.

Expression of inducible nitric oxide (NO) synthase (NOS-2) occurs during inflammation in the central nervous system (CNS) and has been linked to demyelination accompanying certain CNS inflammatory diseases. Although astrocytes and microglia are thought to be the major sources of NOS-2 expression in the CNS in vivo, recent evidence suggested that the myelin-producing oligodendrocytes (OLs) themselves can express NOS-2 in culture. Given the potentially important pathological implications of this finding, the purpose of this study was to examine further the expression of NOS-2 by OLs in vitro. After exposure to lipopolysaccharide (LPS) and interferon-gamma (IFNgamma), primary cultures enriched for mature OLs released NO in a time-dependent manner, although the amount varied considerably between different culture preparations. Increased NO production was accompanied by expression of NOS-2 mRNA and protein, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Immunofluorescence analysis revealed that the cell-type expressing NOS-2 in these cultures was galactocerebroside (Gal C)-negative but CD11b-positive. Further, NO production could be attenuated in cultures treated with the microglial/macrophage toxin, leucine methyl ester, prior to LPS/IFNgamma stimulation. Thus, microglia were the source of NOS-2 catalytic activity in these cultures. The present results indicate that LPS and IFNgamma are not effective stimuli for induction of NOS-2 in OLs in primary cell culture.

Interferon-gamma reduces cyclooxygenase-2-mediated prostaglandin E2 production from primary mouse astrocytes independent of nitric oxide formation.

Nitric oxide (NO) and prostaglandins (PGs) modulate inflammatory and immune responses in the central nervous system (CNS). Both NO and PG synthesis have been described in appropriately stimulated astrocytes. In other systems, both positive and negative modulation of cyclooxygenase (COX) activity, hence PG synthesis, have been described by NO. Since interferon (IFN)-gamma is known to upregulate the production of NO from astrocytes, the present study was designed to investigate the effect of IFNgamma on PG production from activated astrocytes and to determine whether this effect is mediated by NO. Astrocytic PG production was induced by exposure of murine cortical cultures to lipopolysaccharide (LPS). This induction was time- and concentration-dependent, and prevented by inhibitors of transcription and translation, as well as the selective COX-2 inhibitor, NS-398. LPS-induced expression of COX-2 mRNA and protein was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Exposure of LPS-treated astrocytes to IFNgamma resulted in a concentration-dependent decrease in PGE2 accumulation which was accompanied by a striking parallel increase in NO formation. However, the NOS inhibitors, N(G)-nitro-L-arginine or N6-(1-iminoethyl)-lysine, failed to reverse the IFNgamma-mediated diminution of LPS-induced PGE2 production, indicating that the IFN-gamma-mediated reduction in COX-2-dependent PGE2 production occurred independent of NO formation. Additional experiments demonstrated that IFN-gamma acted mainly by downregulating the expression of COX-2 protein. Present results indicate that PG and NO synthesis in mouse cortical astrocytes in vitro are under the direct reciprocal control of IFNgamma.

Analysis of the neuroprotective effects of various nitric oxide donor compounds in murine mixed cortical cell culture.

Nitric oxide (NO) has been implicated in both the pathogenesis of and protection from NMDA receptor-mediated neuronal injury. This apparent paradox has been attributed to alternate redox states of nitrogen monoxide, whereby, depending on the redox milieu, nitrogen monoxide can be neuroprotective via nitrosation chemistry or react with superoxide to form secondary toxic species. In our murine mixed cortical cell culture system, the NONOate-type NO donors diethylamine/NO complex sodium (Dea/NO), (Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium++ +-1,2-diolate (Papa/NO), and spermine/NO complex sodium (Sper/NO), as well as the S-nitrosothiols S-nitroso-L-glutathione (GSNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) (NO+ equivalents), decreased NMDA-induced neuronal injury in a concentration-dependent manner. 8-Bromo-cyclic GMP did not mimic the inhibitory effects of the donors, suggesting that the neuroprotection was not the result of NO-stimulated neuronal cyclic GMP production. Furthermore, neuronal injury induced by exposure of cultures to H2O2 was not altered by the presence of Dea/NO, indicating the absence of a direct antioxidant effect. NONOates did, however, reduce NMDA-stimulated uptake of 45Ca2+, whereas high potassium-induced 45Ca2+ accumulation, a measurement of entry via voltage-gated calcium channels, was unaffected. The parallel reduction of 45Ca2+ accumulation and NMDA neurotoxicity by NONOates mimicked that seen with an NMDA receptor antagonist. Electrochemical measurements of NO via an NO-sensitive electrode demonstrated that neuroprotective concentrations of all donors produced appreciable amounts of NO over the 5-min time frame. Determination of the formation of NO+ equivalents, as assessed by N-nitrosation of 2,3-diaminonaphthylene, revealed little or no observable N-nitrosation by Sper/NO, GSNO, and SNAP with significant N-nitrosation observed by Papa/NO and Dea/NO. However, addition of ascorbate (400 microM) effectively prevented the nitrosation of 2,3-diaminonaphthylene produced by Dea/NO and Papa/NO without altering their neuroprotective properties or their effects on 45Ca2+ accumulation. Present results indicate that the intrinsic NO/NO+ characteristics of NO donor compounds may not be a good predictor of their ability to inhibit NMDA receptor-mediated neurotoxicity at the cellular level.

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.

Potentiation of oxygen-glucose deprivation-induced neuronal death after induction of iNOS.

BACKGROUND AND PURPOSE: Previous studies have shown that brain ischemia and other insults can induce a marked increase in inducible nitric oxide synthase (iNOS) expression in astrocytes and some immune cells, but the biological significance of this phenomenon has not been elucidated. The purpose of the present study was to determine whether this induction of astrocyte iNOS alters neuronal vulnerability to severe hypoxic insults. METHODS: Astrocytic iNOS was induced by exposure of murine cortical cultures to interferon gamma in combination with either interleukin-1 beta or lipopolysaccharide. Cultures were exposed to combined oxygen-glucose deprivation. The extracellular concentration of glutamate was measured by high-performance liquid chromatography. N-Methyl-D-aspartate (NMDA) receptor activity was assessed by measurement of 45Ca2+ influx: neuronal death was assessed by morphological examination and quantitated by measurement of lactate dehydrogenase efflux to the bathing medium. RESULTS: In murine neocortical cell cultures containing neurons and astrocytes, neuronal injury induced by combined oxygen-glucose deprivation was not reduced by the addition of the nitric oxide synthase inhibitors NG-nitro-L-arginine or LG-nitro-arginine methyl ester. However, after induction of astrocyte iNOS activity with interferon gamma plus lipopolysaccharide or interleukin-1 beta, oxygen-glucose deprivation-induced neuronal injury was markedly enhanced and nitric oxide synthase inhibitors became protective. This iNOS-mediated potentiation was associated with a large increase in both extracellular glutamate accumulation and 45Ca2+ influx into neurons. The potentiation could be blocked by MK-801 but not CNQX, suggesting critical involvement of NMDA receptor activation. CONCLUSIONS: These results support the idea that nitric oxide production mediated by induced astrocytic iNOS can potentiate NMDA receptor-mediated neuronal death consequent to hypoxic-ischemic insults.

Murine encephalitogenic lymphoid cells induce nitric oxide synthase in primary astrocytes.

A cytokine-inducible form of nitric oxide synthase (iNOS), capable of producing large quantities of nitric oxide (NO), can be induced in many cell types. We demonstrate that conditioned medium from encephalitogenic myelin basic protein-sensitized lymphoid cells (MBP-CM) induces the expression of iNOS in primary cultures of murine astrocytes in a time- and concentration-dependent manner. iNOS mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) as early as 3 h post-exposure. Accumulation of nitrite into the astrocyte culture medium, an indirect measure of NO, was measurable 3 h post-exposure, plateaued at 24 h, and was prevented by the simultaneous administration of the NOS inhibitors, L-N(G)-nitroarginine methyl ester, N(G)-nitro-L-arginine or aminoguanidine. Astrocyte expression of iNOS protein, detected by immunohistochemistry and immunoprecipitation/Western blot, was prevented by inhibitors of RNA or protein metabolism, consistent with its dependence on de novo protein synthesis.

Expression of the neurofibromatosis 1 (NF1) gene in reactive astrocytes in vitro.

Neurofibromin, the product of the neurofibromatosis 1 (NF1) gene, is an important tumor suppressor protein expressed most abundantly in the nervous system. Within the central nervous system, neurofibromin has been localized to neurons and oligodendrocytes but not astrocytes. As individuals with NF1 are at an increased risk for optic pathway gliomas and astrocytomas, we chose to re-evaluate the level of neurofibromin expression in primary cultures of murine cortical astrocytes under control and reactive conditions. Astrocytes under control conditions expressed low levels of NF1 mRNA and protein. Following stimulation with dibutyryl-cyclic AMP or interferon-gamma in combination with either lipopolysaccharide or interleukin-1 beta, there was a marked increase in NF1 mRNA and protein expression. These results suggest that neurofibromin may be involved in the process of reactive astrocytosis seen in response to CNS injury.

Selective potentiation of NMDA-induced neuronal injury following induction of astrocytic iNOS.

Nitric oxide (NO) produced by the constitutive NO synthase (cNOS) in neurons has been implicated in mediating excitotoxic neuronal death. In our murine cortical cell culture system, NMDA neurotoxicity was not blocked by addition of the NOS inhibitors, NG-nitro-L-arginine or aminoguanidine. However, following activation of inducible NOS in astrocytes by interleukin-1 beta plus interferon-gamma, NMDA but not kainate neurotoxicity was markedly potentiated. This selective potentiation of NMDA neurotoxicity was blocked by NOS inhibition or antioxidants (superoxide dismutase/catalase or Tempol) and could be mimicked by NO generators (SIN-1 or SNAP) or the oxygen radical generator, pyragallol. These results raise the possibility that NO production by astrocytes may contribute to NMDA receptor-mediated neuronal death, perhaps through interaction with oxygen radicals.

Interferon-gamma and interleukin-1 beta induce nitric oxide formation from primary mouse astrocytes.

An inducible form of nitric oxide synthase (iNOS) capable of producing large quantities of nitric oxide (NO) exists in some cell types. We demonstrate by immunoprecipitation and nitrite formation that interleukin-1 beta (IL1 beta) plus interferon-gamma (INF gamma) induce the expression of nitric oxide synthase in primary cultures of murine cortical astrocytes. This induction is time and dose dependent, and inhibited by the NOS inhibitor NG-nitro-L-arginine and the protein synthesis inhibitor cycloheximide.

Inhibition of nitric oxide formation does not protect murine cortical cell cultures from N-methyl-D-aspartate neurotoxicity.

We examined the role of nitric oxide in N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity in rat and mouse primary cortical cell cultures. In rat and mouse cultures, the NO synthase inhibitor, NG-Nitro-L-arginine, blocked cGMP formation but not neuronal cell death following a 5-10 min exposure to 300-500 microM NMDA. NG-Monomethyl-L-arginine was also unable to prevent neuronal death. In contrast, the non-competitive NMDA receptor antagonist, dextrorphan, prevented both cGMP formation and cell death. While other data suggest that the synthesis of nitric oxide can mediate NMDA receptor-mediated neurotoxicity, present results suggest that such synthesis is not necessarily required.

Disruption of synaptosomal calcium channel function by Lambert-Eaton myasthenic immunoglobulin is serum-dependent.

An autoantibody to nerve terminal Ca2+ channels has been suggested to mediate the pathogenesis of the neuromuscular disorder Lambert-Eaton Myasthenic Syndrome (LEMS). We demonstrated previously that in the presence of control human serum, immunoglobulins isolated from a patient with LEMS reduced flux of Ca2+ into isolated nerve terminals during depolarization. The objective of the present study was to determine the role of serum in reducing uptake of 45Ca2+ into rat brain synaptosomes by LEMS IgG. Depolarization-dependent uptake of 45Ca2+ through voltage-gated Ca2+ channels was determined using synaptosomes incubated with control (disease-free) and LEMS IgG with or without control human serum. In the absence of human serum, LEMS IgG did not reduce uptake of 45Ca2+ into synaptosomes. However, in the presence of control human serum (10% of total incubation volume), 45Ca2+ uptake was reduced significantly by LEMS IgG (2 and 4 mg/ml), but not by IgG from disease-free patients or by 10% (v/v) control human serum alone. This concentration of serum was found to be optimal; higher concentrations produced significant reductions in Ca2+ uptake, whereas at lower concentrations the serum/IgG combination was ineffective. The depressant effect of high concentrations of serum alone on 45Ca2+ uptake was mimicked by equal concentrations of bovine serum albumin suggesting that deficits in 45Ca2+ uptake produced by high concentrations of serum were due to increased protein binding of the radiolabel. Heat-inactivating the serum abolished its ability to interact with the LEMS immunoglobulins to depress 45Ca2+ uptake. This suggested a role for complement in this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Specificity of Lambert-Eaton myasthenic syndrome immunoglobulin for nerve terminal calcium channels.

Lambert-Eaton Myasthenic Syndrome (LEMS) is a presynaptic, neuromuscular disorder characterized by impaired nerve-evoked release of ACh. Repetitive nerve stimulation, which increases the probability of quantal release, improves the transmission defect. An autoantibody to Ca2+ channels of presynaptic motor nerve terminals is thought to mediate the pathogenesis of this disease. The goal of the present study was to examine the specificity of LEMS autoantibodies for nerve terminal Ca2+ channels as compared to other voltage-sensitive ion channels in nerve terminals, and to determine if non-specific membrane damage contributed to the pathogenesis of LEMS. The ion channel specificity of LEMS autoantibody was assessed by comparing the ability of acute application of IgG isolated from the plasma of a patient with LEMS to reduce depolarization-dependent uptake of 45Ca2+ and 22Na+ into or efflux of 86Rb+ from rat forebrain synaptosomes. The clinical diagnosis of LEMS was confirmed electrophysiologically by treatment of mice for 30 days with plasma (1.5 ml/day) taken from this patient. Characteristic reduction of quantal content elicited at 1 Hz and facilitation at 20 Hz was observed in mice treated with LEMS plasma compared to those treated with control plasma. One s, K(+)-stimulated 45Ca2+ uptake was inhibited 36.5 +/- 14.5% and 44.5 +/- 9.8% by acute application of 2 and 4 mg/ml LEMS IgG, respectively; IgG from patients with small cell carcinoma of the lung (SCC) had no effect on 45Ca2+ entry. The same concentrations of LEMS IgG affected neither voltage-dependent uptake of 22Na+ into veratridine-depolarized synaptosomes nor 86Rb+ efflux from K(+)-depolarized synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of charge and lipophilicity on mercurial-induced reduction of 45Ca2+ uptake in isolated nerve terminals of the rat.

The goal of this study was to compare the ability of neurotoxic mercurials which differ in ionic charge and/or lipophilicity to block nerve-terminal calcium channels. To do so, we examined the acute effects of methyl mercury (MeHg+), ethyl mercury (EtHg+), inorganic mercury (Hg2+), dimethyl mercury (Me2Hg), p-chloromercuribenzoate (PCMB), and p-chloromercuriphenyl-sulfonate (PCMBS-) (10-1000 microM) on 45Ca2+ flux into rat forebrain synaptosomes at rest and during depolarization. Basal (depolarization-independent) entry of 45Ca2+ was measured during 10-sec exposure to mercurials in solutions containing 5 mM KCl. Concentrations of 50, 100, 250, 500, and 1000 microM of Hg2+, MeHg+, and EtHg+ reduced basal influx of 45Ca2+. PCMB reduced basal influx at concentrations of 10, 50, and 100 microM, but increased influx at 1000 microM. PCMBS- and (Me)2Hg had no effect on basal flux at any concentration tested. Uptake of 45Ca2+ was measured after 1 sec of K(+)-induced depolarization (41.25 mM) to determine influx through voltage-dependent Ca2+ channels ("fast" phase) or during the last 10 sec of a 20-sec period of depolarization for uptake associated with a reversed Na+/Ca2+ exchanger and a residual noninactivating Ca2+ channel component ("slow" phase). Fast and slow components of 45Ca2+ uptake into synaptosomes were blocked in a concentration-dependent manner by MeHg+, EtHg+, and Hg2+. For block of the fast component, the calculated IC50's and confidence intervals were (microM) EtHg+, 92 (82, 102); Hg2+, 155 (149, 161); and MeHg+, 196 (120, 272). IC50's and the confidence intervals for the slow component of uptake were (microM) Hg2+, 49 (43, 55); MeHg+, 72 (67, 77); and EtHg+, 147 (142, 152). In contrast, Me2Hg, PCMB, and PCMBS- (10-1000 microM) caused no appreciable reduction in either phase of 45Ca2+ uptake. Increasing [Ca2+]e was unable to overcome the block induced by MeHg+ and EtHg+ (100 microM) on either phase of 45Ca2+ uptake into synaptosomes. Likewise, increasing [Ca2+]e failed to overcome block of the slow component by Hg2+ (100 microM). Increasing [Ca2+]e was able to overcome, in part, block of the fast phase induced by Hg2+ (100 microM) although the percentage of reversal was not statistically significant. The magnitude of block of 45Ca2+ uptake increased as a function of increasing [K+]e for MeHg+ and EtHg+, suggesting the block to be voltage-dependent. Thus, mercurials of dissimilar charge and lipophilicity affect synaptosomal Ca2+ uptake differentially.(ABSTRACT TRUNCATED AT 400 WORDS)