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Journal of Physiology (2002), 543.2, pp. 567-576
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.021048
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ABSTRACT |
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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.
(Received 21 March 2002; accepted after revision 25 June 2002)
Corresponding author W. D. Atchison: Department of Pharmacology and Toxicology, Michigan State University, B-331 Life Sciences Building, East Lansing, MI 48824-1317, USA. Email: atchiso1{at}msu.edu
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INTRODUCTION |
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Lambert-Eaton myasthenic syndrome (LEMS) is a paraneoplastic disorder in which the nerve-evoked, Ca2+-dependent release of acetylcholine (ACh) from the presynaptic nerve terminal is impaired (Elmqvist & Lambert, 1968; Lambert & Elmqvist, 1971). Patients with LEMS typically exhibit peripheral limb muscle weakness and fatigability, decreased reflexes, as well as various dysautonomias (Heath et al. 1988; Khurana et al. 1988; O'Neill et al. 1988). LEMS often is associated with small-cell lung carcinoma (Lennon et al. 1982). The underlying cause of LEMS is not fully understood, but the disease is thought to result from the generation of antibodies against the tumour. Passive transfer of LEMS to rodents by chronic injection of plasma, serum or IgGs faithfully duplicates the hallmark electrophysiological signs of LEMS, namely reduced quantal content and facilitation of end-plate potential (EPP) amplitudes during repetitive stimulation (Lang et al. 1983, 1987; Kim, 1985; Prior et al. 1985).
In LEMS, circulating autoantibodies are thought to target the voltage-gated Ca2+ channels involved in release of ACh from the nerve terminal (Lang et al. 1981, 1983; Lambert & Lennon, 1988). In several model systems, Ca2+ channel function is diminished after application of LEMS serum or plasma (Kim & Neher, 1988; Hewett & Atchison, 1991; Kim et al. 1993; Garcia & Beam, 1996; Garcia et al. 1996; Pinto et al. 1998). More directly, in passive transfer models of LEMS, the amplitude of Ca2+ currents recorded from nerve terminals of murine neuromuscular preparations is reduced (Smith et al. 1995; Xu et al. 1998). Furthermore, active zone particles, which presumably correspond to Ca2+ channels at the nerve terminal, appear to be disorganised and fewer in number at motor nerve terminals in LEMS patients (Fukunaga et al. 1982) or LEMS-treated mice compared with control groups (Fukunaga et al. 1983; Fukuoka et al. 1987). This reduction in the number of Ca2+ channels at the nerve terminal is likely to be responsible for the attenuated quantal content seen in LEMS.
Among the multiple types of voltage-gated Ca2+ channels that exist, several, including the L, N and P/Q types, have been shown to control neurotransmitter release; multiple subtypes often coexist at the same synapse to regulate transmitter release (Turner & Dunlap, 1995). At the mammalian neuromuscular junction it is primarily the P/Q-type Ca2+ channel that is involved in the release of ACh (Uchitel et al. 1992; Protti et al. 1996; Katz et al. 1996, 1997). L-type Ca2+ channels, which participate in the release of hormones (Lemos & Nowycky, 1989) and noradrenaline from chromaffin cells of the adrenal medulla (Owen et al. 1989), and N-type channels, which control ACh release at non-mammalian neuromuscular junctions (Sano et al. 1987), do not appear to be involved normally in the nerve-stimulated release of ACh from mammalian motor nerve terminals (Atchison & O'Leary, 1987; Atchison, 1989; Uchitel et al. 1992; Katz et al. 1996, 1997; Protti et al. 1996). Whereas N-type channels have been shown to be affected by LEMS serum (Peers et al. 1990; Suenaga et al. 1996), several recent studies suggest that following treatment with LEMS sera, the function of L-type Ca2+ channels is spared (Garcia & Beam, 1996) or unmasked (Smith et al. 1995; Xu et al. 1998). Specifically, treatment of mice for 30 days with plasma from LEMS patients attenuates the amplitude of the P/Q-type Ca2+ current and exposes a dihydropyridine (DHP)-sensitive L-type Ca2+ current. Inasmuch as induction of a DHP-sensitive Ca2+ current at murine motor nerve terminals might represent an adaptive response to the reduction of function of the normal complement of Ca2+ channels by LEMS autoimmune attack, the present study was undertaken to determine whether this DHP-sensitive component contributes to the release of ACh from motor nerve terminals in the LEMS passive transfer model and whether this component develops in tandem with the onset of LEMS.
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METHODS |
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Chronic treatment studies
Animal care, handling and experiments were performed in accordance with local university (Michigan State University All University Committee on Animal Use and Care) and national guidelines. Experiments were performed using male ICR mice (20-22 g, Harlan Sprague-Dawley Laboratories, Madison, WI, USA). Mice were injected once daily intraperitoneally (I.P.) for 30 days with 1.5 ml of plasma from patients clinically diagnosed with LEMS or plasma from healthy control patients. Prior to injection with the plasma, mice were first treated with 300 mg kg-1 I.P. of cyclophosphamide to suppress the immune response to exogenous proteins. After 30 days of plasma treatment, animals were killed by decapitation following anaesthesia with 80 % CO2 and 20 % O2. The diaphragm muscle with the attached phrenic nerve (Barstad & Lilleheil, 1968) was then removed and prepared for electrophysiological recording.
Acute treatment studies
Naive male ICR mice were killed as described above. After removal of the diaphragm muscle and the attached phrenic nerves, the tissue was incubated for 24 h at a room temperature of 23-25 °C in Dulbecco's minimum Essential medium with 20 % (v/v) of LEMS or control plasma. The medium was aerated continuously with 95 % O2 and 5 % CO2. The tissue was washed subsequently in buffered saline solution. Alternatively, the tissue was incubated for 2 h at room temperature in buffered saline solution containing 20 % (v/v) of the appropriate plasma under continual oxygenation (100 % O2). After the specified incubation time the tissue was prepared for electrophysiological recording.
Electrophysiological measurements
Diaphragm preparations were pinned out at resting tension in a Sylgard-coated chamber and perfused continuously with buffered saline solution at a rate of approximately 1-5 ml min-1. Tissues that were incubated for only 2 h were perfused at a similar rate but the perfusate contained buffered saline solution with 5 % (v/v) plasma. In order to prevent muscle contraction following stimulation of the phrenic nerve, the diaphragm muscle was cut approximately 4 mm on either side of the main intramuscular nerve branch (Glavinovic, 1979; Traxinger & Atchison, 1987). This technique does not produce significant changes in the muscle cable properties (Glavinovic, 1979; Lambert et al. 1981) and permits simultaneous recordings of EPPs and miniature EPPs (MEPPs) without the complicating effects of depressing transmitter release with high Mg2+-low Ca2+ solutions or inducing postjunctional receptor block with d-tubocurarine (Hubbard & Wilson, 1973). All recordings were made at a room temperature of 23-25 °C using conventional intracellular recording techniques. EPPs and MEPPs were recorded using borosilicate glass microelectrodes (o.d. 1.0 mm, WP Instruments, Sarasota, FL, USA) with a resistance of 5-25 M
when filled with 3 M KCl. The phrenic nerve was stimulated supramaximally using a suction electrode attached to a stimulus isolation unit (Grass SIU, Grass Instruments, Quincy, MA, USA) and stimulator (Grass S88). Recordings were made before and after addition of nimodipine. Experiments performed in the presence of nimodipine were done in the dark, in order to prevent photo-oxidation of this compound. Signals were amplified using either an Axoclamp-2 (Axon Instruments, Foster City, CA, USA) or WPI 721 (WP Instruments) amplifier and digitised into a PC computer for inspection using Axoscope 8.0 software (Axon Instruments), and then analysed using MiniAnalysis 4.0 software (Synaptosoft, Decatur, GA, USA). EPP and MEPP amplitudes were standardised to a membrane potential of -50 mV in order to correct for changes in membrane potential driving force (Katz & Thesleff, 1957). Recordings from each preparation were sampled and averaged from at least five different end-plates before and after addition of nimodipine, yielding an n value of 1. Quantal content (m) was calculated using the ratio of the mean amplitude of the corrected EPPs to the mean amplitude of the corrected MEPPs (Hubbard et al. 1969).
Solutions and chemicals
The standard buffered saline solution contained (mM): NaCl 137.5, KCl 2.5, MgCl2 1, CaCl2 2, D-glucose 11, Hepes 14, pH adjusted to 7.4 at room temperature with NaOH and kept under continual oxygenation (100 % O2). In order to prevent the depolarisation-induced nerve conduction block that can occur when K+ is released from the cut muscle fibres, 2.5 mM KCl was used throughout the experiments (Hubbard & Wilson, 1973; Glavinovic, 1979; Traxinger & Atchison, 1987). Nimodipine was prepared as a 10 mM stock solution in 100 % ethanol, which was kept at 4 °C until use. The final working solution with nimodipine contained only 0.1 % ethanol (v/v). Plasma samples from patients clinically diagnosed as having LEMS were kindly provided by Dr Eva Feldman and Dr Jim Albers (University of Michigan Medical Center, Ann Arbor, MI, USA), Dr Andrew Massey (University of Kentucky Medical Center, Lexington, KY, USA) and Dr Shin Oh (University of Alabama Medical Center, Birmingham, AL, USA). Plasma was supplied with no identifiers and only patient age and gender were provided. Plasma was obtained during the course of routine plasma exchange therapy with the normal informed consent of the patient and in accordance with the approval of the Human Subject Committee of the respective institution. Control human plasma was donated by healthy volunteers and obtained as outdated blood donations from the American Red Cross (Lansing, MI, USA). Nimodipine, cyclophosphamide, and Hepes were purchased from Sigma (St Louis, MO, USA). All other reagents were of analytical grade or better.
Statistical analysis
Differences in m for both acute and chronic treatment studies in animals receiving LEMS or control plasma were analysed using a one-way analysis of variance followed by Tukey's test. P values were set to < 0.05 for all statistical tests.
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RESULTS |
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Effect of LEMS plasma on neuromuscular transmission at low-frequency stimulation
The duration of exposure to control plasma did not affect neuromuscular transmission from mouse hemidiaphragm preparations. Table 1 indicates that there was no significant difference between the resting membrane potentials, EPP amplitudes or MEPP amplitudes recorded from neuromuscular preparations of mice injected chronically with control plasma or from preparations obtained from naive mice and treated acutely with control plasma for 2 or 24 h (P > 0.05).
Conversely, Fig. 1A demonstrates that passive transfer of LEMS markedly reduced the evoked release of ACh. The m values of EPPs recorded following 0.5 Hz stimulation of the phrenic nerve from hemidiaphragm preparations obtained from mice injected for 30 days with LEMS plasma were reduced significantly in comparison to mice injected for the same duration with control plasma. Similar levels of reduction in m were observed in preparations from mice treated with plasma from each of three LEMS patients. The composite reduction, using values obtained from all patients, was 45.1 ± 3.1 % of control values (Fig. 1D).
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Figure 1. Effect of treatment with plasma taken from Lambert-Eaton myasthenic syndrome (LEMS) patients on quantal content of mouse hemidiaphragm endplates A-C, hemidiaphragm preparations were taken from immunosuppressed mice treated for 30 days with 1.5 ml day-1 I.P. (A), or from naive mice and treated for 24 h (B), or 2 h (C) with plasma from one of three LEMS patients (Pt.). D, combined results from all three patients following 2 h, 24 h and 30 days of treatment with LEMS plasma. End-plate potential (EPP) and miniature EPP (MEPP) amplitudes were recorded using standard intracellular techniques. EPPs were elicited at a frequency of 0.5 Hz. Quantal content was determined from each neuromuscular junction preparation using the ratio of the average EPP amplitude to the average MEPP amplitude. Values are expressed as the percentage of quantal content from the LEMS-plasma-treated preparations to that of control-plasma-treated preparations. Each value represents the mean ± S.E.M. of at least five different preparations. The asterisk (*) indicates a value significantly different from control for panels A-C or from 30 days treatment for panel D (P < 0.05). | ||
Acute incubation of naive murine neuromuscular preparations with LEMS plasma also caused a significant reduction in m in comparison to preparations treated acutely with control plasma. In diaphragm preparations that were incubated for 2 h with LEMS plasma m was reduced to 61.4 ± 5.3 % of control levels for the composite of three patients (Fig. 1D). This composite reduction in m for all three patients was significantly different from that observed in neuromuscular preparations from mice treated chronically with LEMS plasma as well as from preparations treated acutely with control plasma (Fig. 1C and D). Plasma from only two of the individual LEMS patients significantly reduced m compared to preparations treated with control plasma when applied for 2 h (Fig. 1C). Further incubation of naive murine neuromuscular preparations with LEMS plasma (24 h) also reduced m to 56.3 ± 5.1 % of the value obtained following 24 h treatment with control plasma (Fig. 1D). Plasma from all three of the individual LEMS patients significantly reduced m compared to preparations treated with control plasma when applied for 24 h (Fig. 1B). However, the reduction in m observed following 24 h treatment with LEMS plasma was not significantly different from that of neuromuscular preparations from mice treated chronically with LEMS plasma or from naive murine neuromuscular preparations treated with LEMS plasma for 2 h (Fig. 1D).
None of the three LEMS treatment paradigms (30 days, 2 h and 24 h) significantly affected MEPP amplitudes or muscle resting membrane potentials (P > 0.05; results not shown).
Effect of LEMS plasma on neuromuscular transmission at high-frequency stimulation
In addition to a reduced m at low-frequency stimulation, another electrophysiological characteristic of LEMS is an increase in the amplitude of EPPs in comparison to that of the first EPP evoked during high-frequency stimulation of the nerve (Lang et al. 1983, 1987; Kim, 1985; Prior et al. 1985). Phrenic nerve stimulation at 40 Hz caused an overall reduction of EPP amplitudes from all three control treatment protocols (Fig. 2). On the other hand, LEMS plasma treatment in the 30 day injection, as well as the 2 h and 24 h treatment paradigms, each caused facilitation of the EPP amplitude in comparison to the initial EPP amplitude (Fig. 2).
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Figure 2. Effect of treatment with LEMS plasma on the facilitation of neuromuscular transmission EPP amplitudes were recorded using standard electrophysiological techniques from hemidiaphragm preparations taken from mice treated for 30 days with 1.5 ml day-1 I.P. of LEMS or control plasma (A) or naive mice and treated for 24 h (B) or 2 h(C) with LEMS or control plasma. EPPs were elicited by stimulation of the phrenic nerve at 40 Hz. | ||
The involvement of DHP-sensitive Ca2+ channels in neuromuscular transmission from motor nerve terminals treated with LEMS plasma
Normally, DHP-sensitive L-type Ca2+ channels are not involved in the nerve-stimulated release of ACh from adult mammalian motor nerve terminals (Atchison & O'Leary, 1987; Atchison, 1989; Uchitel et al. 1992; Katz et al. 1996; 1997; Protti et al. 1996). As shown in Fig. 3, the nerve-stimulated release of ACh recorded from diaphragm preparations of mice injected chronically with control plasma or from preparations treated acutely with control plasma was not sensitive to nimodipine. In addition, quantal content from hemidiaphragm preparations treated for 24 or 2 h with LEMS plasma was not altered in the presence of nimodipine (Figs 3B and C and 4B and C). However, as shown for sample records in Fig. 3A and for composite data from all preparations in Fig. 4A, nerve-stimulated release of ACh from motor nerve terminals of mice treated chronically with LEMS plasma was sensitive to nimodipine. As shown in Fig. 4A, m recorded from preparations obtained from mice treated chronically with LEMS plasma was reduced in the presence of nimodipine to 57.7 ± 3.3 % of their nimodipine-free ('control') value. Plasma from all three LEMS patients induced this nimodipine sensitivity of neuromuscular transmission.
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Figure 3. Effect of nimodipine on EPPs from LEMS- and control-plasma-treated neuromuscular junction preparations EPPs were recorded from neuromuscular junction preparations isolated from mice treated for 30 days with 1.5 ml day-1 I.P. of LEMS or control plasma (A) or naive mice and treated by incubation for 24 h (B) or 2 h (C) with LEMS or control plasma. Recordings from the same site are depicted before (-) and after (+) application of 10 µM nimodipine. Each tracing represents the average of at least 50 EPPs at a stimulation frequency of 0.5 Hz recorded from a single representative preparation. | ||
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Figure 4. Effect of nimodipine on the quantal content of mouse hemidiaphragm preparations treated with LEMS plasma Hemidiaphragm preparations were taken from mice treated for 30 days with 1.5 ml day-1 I.P. (A), or from naive mice and incubated continuously for 24 h (B) or 2 h (C) with plasma from one of three LEMS patients (Pt.). EPPs were elicited at a frequency of 0.5 Hz. Quantal content was determined from each preparation using the ratio of the average EPP amplitude to the average MEPP amplitude before and after the addition of 10 µM nimodipine. Values are the quantal content of preparations after the addition of nimodipine expressed as a percentage of the quantal content of the same preparation before nimodipine. Each value represents the mean ± S.E.M. of at least five different preparations. The asterisk (*) indicates a value significantly different from control (P < 0.05). | ||
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DISCUSSION |
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LEMS is associated with the loss of functional voltage-gated Ca2+ channels at motor nerve terminals and thus the reduction of the nerve-evoked release of ACh. Normally, P/Q-type, but not L- or N-type Ca2+ channels control the nerve-stimulated release of ACh from adult mammalian motor nerve terminals (Atchison & O'Leary, 1987; Atchison, 1989; Uchitel et al. 1992; Katz et al. 1996, 1997; Protti et al. 1996). Passive transfer of LEMS to mice by 30 days of plasma treatment attenuates the perineurial voltage changes associated with the amplitude of the P/Q-type Ca2+ current and induces the appearance of a DHP-sensitive L-type Ca2+ current at murine motor nerve terminals (Smith et al. 1995; Xu et al. 1998). The primary objective of the present study, therefore, was to determine if this novel DHP-sensitive L-type Ca2+ current becomes involved in synaptic transmission from adult mammalian motor nerve terminals in LEMS.
Nimodipine, a DHP-sensitive L-type Ca2+ channel blocker, had no effect on m from mice treated chronically for 30 days with control plasma, nor did it affect m in motor nerve terminals treated acutely for 2 or 24 h with either LEMS or control plasma. This finding is consistent with the observation of Xu et al. (1998) that the binding characteristics of [3H]-nitrendipine to rat synaptosomal nerve terminal preparations were not affected by acute application of LEMS IgG. Nimodipine did, however, attenuate m from motor nerve terminals isolated from mice treated for 30 days with LEMS plasma. Thus, following chronic treatment of mice with LEMS plasma, DHP-sensitive L-type Ca2+ channels become involved in the nerve-stimulated release of ACh from mammalian nerve terminals.
Although synaptic transmission from neuromuscular preparations isolated from mice and treated acutely with LEMS plasma for 2 and 24 h was not sensitive to nimodipine, m was reduced in comparison to hemidiaphragm preparations treated with control plasma. In addition, facilitation of EPP amplitudes occurred in neuromuscular preparations treated for 2 or 24 h with LEMS plasma. The observation that m is reduced after a brief exposure to LEMS plasma is consistent with the findings of Hewett & Atchison (1991), in which uptake of 45Ca2+ into rat forebrain synaptosomal preparations during KCl-induced depolarisation was reduced after acute exposure to LEMS IgG. However, this is the first study that we are aware of in which a consistent reduction in m is observed from neuromuscular preparations treated for such a short duration (2 h) with LEMS plasma. This finding may be due to the nature of our treatment paradigm, which consisted of a high concentration of plasma (20 %) during the acute incubation phase and the continual presence of a low concentration (0.5 %) of plasma during the recording phase.
Chronic passive transfer of LEMS to mice caused the typical clinical electrophysiological features seen in LEMS patients and reported in other studies: m was reduced in comparison to control groups and facilitation of the EPP amplitudes occurred at high-frequency stimulation (Lang et al. 1983, 1987; Kim, 1985; Prior et al. 1985). The reduction in m recorded from neuromuscular preparations obtained from mice treated chronically with LEMS plasma was significantly greater than that seen in neuromuscular preparations following 2 h LEMS treatment paradigms. This difference most likely reflects the time course required to induce LEMS after passive transfer to mice. As shown by Prior et al. (1985), passive transfer of LEMS to mice by daily injections of LEMS IgG does not induce a maximum reduction in m until approximately 10 days, and the half-maximal reduction in m occurs after 1.5 days of treatment.
The involvement of the DHP-sensitive L-type channels in synaptic transmission from mammalian motor nerve terminals following chronic LEMS treatment may reflect recruitment of the normally silent L-type channels that are already present at the terminal. Addition of the DHP agonist, Bay K 8644 to mammalian neuromuscular preparations increases m (Atchison & O'Leary, 1987; Atchison, 1989) and L-type channels have been shown to be involved in the spontaneous release of ACh from mammalian motor nerve terminals in the presence of physiological concentrations of extracellular KCl (Losavio & Muchnick, 1997). In addition, pre-treatment of mouse neuromuscular preparations with the intracellular Ca2+ buffer, DM-BAPTA-AM unmasks a nitrendipine-sensitive perineurial Ca2+ current (Urbano & Uchitel, 1999). However, this latter study did not find evidence for involvement of this unmasked L-type channel in ACh release. This discrepancy may be due to the spatial localisation of the L-type channels at the nerve terminal. If L-type channels are not in close proximity to the active zone release machinery (see Robitaille et al. 1990) then most likely the influx of Ca2+ through the L-type channels would have to occur in a more diffuse manner than that observed with Ca2+ channels clustered about the release sites, as is postulated to occur normally (Llinas et al. 1992). DM-BAPTA-AM could therefore buffer the Ca2+ entering through L-type channels before it reached the release apparatus.
One possible explanation for the unmasking of silent DHP-sensitive L-type channels seen after chronic LEMS treatment may be a reduction in the calcium-activated K+ (K(Ca)) current at the motor nerve terminal. K(Ca) channels are believed to be located in close proximity to the Ca2+ channels involved in transmitter release from motor nerve terminals (Robitaille & Charlton, 1992; Robitaille et al. 1993; Xu & Atchison, 1996; Protti & Uchitel, 1997). These K(Ca) channels respond to Ca2+ influx through Ca2+ channels localised at active zones (N-type in frog, and presumably P/Q-type in mice and rats) and may normally limit the duration and extent to which the nerve terminal remains depolarised in response to an action potential. Loss of Ca2+ channels due to LEMS plasma treatment may reduce activation of these K(Ca) channels, thus prolonging the duration and extent of nerve-terminal depolarisation. This enhanced nerve-terminal depolarisation could allow silent L-type channels that may be located at a site distant from the ACh release machinery to remain open long enough to become involved in synaptic transmission (Smith et al. 1995). The DHP sensitivity seen after chronic, but not acute LEMS treatment could reflect the difference in the extent to which m is reduced among the different treatment paradigms. Unlike 2 h LEMS treatment, chronic treatment of mice with LEMS plasma may reduce Ca2+ influx at the nerve terminal enough to decrease the K(Ca) current, which in turn could unmask silent L-type channels. Although there was no significant difference in the reduction of m between chronic treatment of mice with LEMS plasma and 24 h LEMS treatment, this finding may be misleading. It is likely that the predominant Ca2+ channel involved in transmitter release, presumably the P/Q-type in our preparations, may be lost to a greater extent in the chronically treated LEMS mice than following 24 h LEMS treatment, because m in the 30 day treatment regimen was reduced by ~40-45 % by nimodipine, whereas at 24 h, none of the quantal release was nimodipine sensitive. Thus, the apparent similar reduction in m may occur because L-type Ca2+ channels now comprise a significant portion of the nerve-evoked release of ACh in the chronically treated LEMS preparations. The possible distinct location of L-type Ca2+ channels from the normal release sites may preclude involvement of these Ca2+ channels in activation of K(Ca) channels. Thus, at the motor nerve terminals of mice treated chronically with LEMS plasma, activation of K(Ca) channels may be attenuated in comparison to motor nerve terminals exposed to LEMS plasma for only 24 h.
Alternatively, the ability of chronic LEMS treatment to induce the involvement of L-type channels in synaptic transmission may reflect more than the simple unmasking of channels already present. This is supported by the inability of acute LEMS plasma treatment to induce DHP-sensitive transmitter release despite reducing m. For example, processes that take a longer time, such as synthesis of new mRNA and then new channels, may be required. Along these lines, L-type channels become involved in transmitter release at newly forming or regenerating mammalian neuromuscular preparations (Katz et al. 1996; Sugiura & Ko, 1997; Rosato Siri & Uchitel, 1999; Santafe et al. 2000, 2001). However, morphological data do not support the notion that LEMS treatment induces degeneration or sprouting of new nerve terminals (Fukunaga et al. 1982, 1983; Fukuoka et al. 1987).
Finally, various studies support the idea that the fast nature of the transmitter release process at motor nerve terminals necessitates a close association between voltage-gated Ca2+ channels and transmitter release sites (Robitaille et al. 1990; Sugiura et al. 1995; Seagar & Takahashi, 1998). Perhaps relocalisation of L-type channels to active zones may be needed before they become involved in transmitter release (Polo-Parada et al. 2001).
The involvement of L-type Ca2+ channels in transmitter release from adult mammalian motor nerve terminals may act as a compensatory mechanism in LEMS to overcome the chronic loss of functional P/Q-type Ca2+ channels. Indirect evidence suggests that a similar phenomenon occurs in humans with LEMS. An association has been demonstrated between respiratory failure and the administration of verapamil in a patient with LEMS (Krendel & Hopkins, 1986). In addition, the signs of LEMS developed in a patient with ischaemic heart disease who received the L-type antagonist, diltiazem, and disappeared as the patient's serum levels of diltiazem decreased (Ueno & Hara, 1992). Thus, it is possible that in patients with LEMS, L-type Ca2+ channels also become involved in synaptic transmission at the neuromuscular junction. As such, these patients might benefit from therapies directed specifically at facilitating current flow through L-type Ca2+ channels.
In conclusion, following passive transfer of LEMS by chronic injection, the ACh secretory process assumes a considerable component of L-type Ca2+ channel sensitivity. While the mechanism underlying this adaptive change is unclear, it may reflect a compensatory process by which transmission is sustained in the face of an autoimmune attack at the nerve terminal.
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Acknowledgements
This work was supported by NIEHS grant ES0-5822 to W.D.A. and by a Viets Fellowship to M.T.F. from the Myasthenia Gravis Association. LEMS plasma was generously provided by Dr Eva Feldman and Dr Jim Albers (University of Michigan Medical Center, Ann Arbor, MI, USA), Dr Andrew Massey (University of Kentucky Medical Center, Lexington, KY, USA), and Dr Shin Oh (University of Alabama Medical Center, Birmingham, AL, USA).
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LEMS-plasma-treated group ; 
, control-plasma-treated group. Values are expressed as the mean ratio ± S.E.M. of the amplitude of EPPn (where n represents the stimulus number) to that of the first EPP elicited in the train (EPP1). The asterisk (*) indicates a value significantly different from that of the first EPP (P < 0.05).