Department of Pharmacology and Toxicology, Neuroscience Program and Institute of Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824-1317
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Xu, You-Fen, Sandra J. Hewett, and William D. Atchison. Passive transfer of Lambert-Eaton Myasthenic Syndrome induces dihydropyridine sensitivity of ICa in mouse motor nerve terminals. J. Neurophysiol. 80: 1056-1069, 1998. 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 ~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 µM 3,4-diaminopyridine and 1 mM tetraethylammonium were both reduced by LEMS plasma treatment. The fast component (ICa,f) was reduced by 35%, whereas the slow component (ICa,s) was reduced by 37%. -Agatoxin IVA (
-Aga-IVA; 0.15 µM) and
-conotoxin-MVIIC (
-CTx-MVIIC; 5 µM) 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
-Aga-IVA or
-CTx-MVIIC was blocked by 10 µM nifedipine and 10 µM Cd2+. Thus LEMS plasma appears to downregulate
-Aga-IVA-sensitive (P-type) and/or
-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]-
-conotoxin-GVIA (-
-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 ~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
-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.
Lambert-Eaton Myasthenic Syndrome (LEMS) is a presynaptic disorder of neuromuscular transmission, in which patients exhibit profound muscle weakness believed to be due to deficient Ca2+-dependent release of acetylcholine (ACh) in response to nerve stimuli (Lambert et al. 1956 Passive transfer of LEMS
Male ICR mice (20-50 g, Harlan Sprague-Dawley Laboratories, Madison, WI) were divided into three groups: a group receiving LEMS plasma, a control group receiving control plasma, and an untreated group that was kept for the same duration as the other two groups but that received no injections. Mice were injected intraperitoneally for 30 day with either 1.5 ml of control or LEMS plasma (Hewett and Atchison 1992b Neuromuscular preparation and solutions
After euthanasia, the mouse's rib cage was separated from the spinal column and bathed in an oxygenated solution of the following ionic composition (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 11 D-glucose, 12 NaHCO3, and 1 NaH2PO3. The solution was gassed with 95% O2-5% CO2 to buffer it at pH 7.4 at 25°C. The bath volume was 20 ml and was entirely replaced every 2-4 min during the dissection. The muscle, along with its branch of intercostal nerve, was dissected carefully to avoid damage to superficial fibers and their associated nerve terminals (McArdle et al. 1981 Perineurial recording
Electrophysiological recordings of voltage changes in the perineurial space induced by Ca2+ entry via voltage-gated Ca2+ channels were made from the isolated left triangularis sterni muscle preparations. Suprathreshold electrical stimuli were applied to the intercostal nerve trunk via a suction electrode and stimulator (S88, Grass Medical Instruments, Quincy, MA) with a stimulus isolation unit (SIU, Grass Medical Instruments). Stimulus frequencies ranged from 0.01 to 100 Hz. ICa was recorded using a glass microelectrode (1.0 mm ID, WP Instruments, Sarasota, FL) filled with 2 M NaCl and having a resistance of 2-5 M Preparation of synaptosomes and solutions
Synaptosomes were prepared from forebrains of male Sprague-Dawley rats (Harlan, 175-225 g) according to a modification of the method of Gray and Whittaker (1962) Binding experiments
Binding of [3H]-nitrendipine and [3H]-verapamil was initiated by addition of 40 µl of synaptosomal suspension (200-300 µg protein) to 400 µl of incubation buffer containing either 10-1,500 pmol of [3H]-nitrendipine or 0.2-12.8 nmol of [3H]-verapamil in ethanol for 90 min at 25°C in a room illuminated by a sodium lamp. The final concentration of ethanol was always <0.1% (vol/vol). In addition, the verapamil stock solutions and quench buffer contained 0.5 mg/ml bovine serum albumin to limit nonspecific binding of verapamil to test tubes and filters. Incubation was terminated by addition of 5 ml of cold quench solution followed by rapid filtration through glass fiber filters (Whatman GF) that had been presoaked in quench solution containing 0.1% (wt/vol) polyethylenimine. The filters were rinsed twice with 5 ml of cold quench solution. Scintillation cocktail was added 12 h before the radioactivity trapped on the filters was estimated in a liquid scintillation counter having an approximate efficiency of 45% for 3H. Nonspecific binding was measured in the presence of 5 µM unlabeled nifedipine or 25 µM unlabeled verapamil, respectively. Protein content was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. A 40-µl aliquot was taken from the stock solution to determine total ligand concentration. The average of triplicate values was used to determine the equilibrium dissociation constant (Kd) for binding and the maximum density of binding sites (Bmax) (Scatchard 1949 Immunoglobulin isolation
Control and LEMS sera and/or plasma were stored at Materials
Control human plasma donated by healthy volunteers was obtained from the American Red Cross (Lansing, MI). Plasma from three patients with typical clinical and electromyographic features of LEMS was generously provided by Dr. Andrew Massey (University of Kentucky Medical Center, Lexington, KY), Drs. Eva Feldman and James Albers (University of Michigan Medical Center, Ann Arbor, MI), and Dr. Shin Oh (University of Alabama Medical Center, Birmingham, AL). Statistical analysis
Data from perineurial recordings were analyzed using Student's unpaired t-test; significance was set at P < 0.01 for all experiments. Data from the equilibrium binding of [3H]-nitrendipine and [3H]-verapamil were analyzed by mixed design analysis of variance (ANOVA) followed by Student's t-test for paired samples. Data from Effect of K+ channel block on motor nerve terminal Ca2+ currents
ICa in mouse motor nerve terminals cannot be distinguished as a separate entity in standard Krebs solution, because its amplitude is obscured by voltage-gated K+ currents (IK) (Mallart 1986
Passive transfer of LEMS reduced Ca2+ currents
After perfusing the preparations with 300 µM 3,4-DAP and 10 mM TEA, 0.01-Hz stimulation elicited ICa with long duration from motor nerve terminals of the mice treated with control plasma as well as those treated with plasma from three patients clinically diagnosed with LEMS. However, the peak amplitude of ICa as well as the ratio of ICa:INa in LEMS plasma-treated motor nerve terminals were both smaller than those in the nerve terminals treated with control plasma (Fig. 2A and Table 1). The peak amplitude of ICa for LEMS plasma-treated mice was reduced by 36%: 39% for patient 1, 30% for patient 2, and 39% for patient 3. The ratio of ICa/INa was reduced by ~32%: 32% for patient 1; 32% for patient 2, and 30% for patient 3. In contrast, there was no significant difference in the value of peak amplitude of ICa or the ratio of ICa:INa between untreated preparations or those treated with control plasma (Table 1). INa in both the untreated motor nerve terminals and those treated with LEMS plasma from three patients was not significantly different from those treated with control plasma (Table 1).
Frequency dependence of ICa
In motor nerve terminals treated with control plasma, ICa evoked in the presence of 10 mM TEA and 300 µM 3,4-DAP was reduced in both duration and amplitude by increasing the stimulation frequency from 0.01 to 10 Hz. For mice treated with LEMS plasma, 10-Hz frequency nerve stimulation decreased the duration of ICa but did not alter its amplitude (Fig. 3,A and C). The peak amplitude of ICa for control preparations was 2.3 ± 0.17 (SE) mV at 0.01 Hz and 1.2 ± 0.23 mV at 10 Hz. In LEMS-treated preparations, the peak amplitude of ICa was unchanged as stimulus frequency was increased (1.86 ± 0.24 at 0.01 Hz and 1.78 ± 0.23 mV at 10 Hz). In the presence of low concentrations of K+ channel blockers (1 mM TEA and 50 µM 3,4-DAP), increasing the stimulation frequency to 10 Hz had no effect on either amplitude or duration of ICa,f from either control or LEMS-treated mice. Conversely, at 10-Hz stimulation ICa,s for both groups was blocked almost completely (Fig. 3B).
Ca2+ dependence of ICa
In preparations treated with control plasma, increasing the extracellular Ca2+ concentration ([Ca2+]e) enhanced both the amplitude and duration of ICa. The peak amplitude of ICa saturated at 2 mM Ca2+, whereas the duration of ICa saturated at 4-6 mM. For LEMS plasma-treated preparations, a similar pattern was observed, however, the amplitude and duration of ICa were both increased further by raising the [Ca2+]e above that which was maximally effective for the controls. Saturation for the amplitude and duration of ICa occurred at 8-9 mM (Fig. 5, A-C). Therefore, in LEMS-treated mice, the [Ca2+]e had to increase by two to three times control levels to produce a maximal ICa.
To determine whether LEMS autoantibodies affect a particular type of Ca2+ channel in motor nerve terminals,
Effects of LEMS Igs on the binding of Ca2+ channel antagonists to cortical synaptosomes
As indicated above, in the chronic LEMS plasma-treated mouse motor nerve terminals, ICa was reduced, and this was accompanied by appearance of a DHP-sensitive ICa. In previous studies (Hewett and Atchison 1991 NITRENDIPINE BINDING.
When the binding of [3H]-nitrendipine (10-1,00 pmol) was measured after incubation in buffer alone, a single high-affinity binding site was observed. Nonspecific binding accounted for ~25% of total binding. Scatchard analysis of the data yielded a Kd value of 214 ± 84 pM and an apparent Bmax value of 62.0 ± 12.0 fmol/mg protein (Table 3). The Hill slope was approximately equal to one. These values are consistent with those reported by others for [3H]-nitrendipine binding in synaptosomes (Boles et al. 1984
VERAPAMIL BINDING.
Over a concentration range of 0.2-12.8 nmol, [3H]-verapamil bound a single high-affinity site in synaptosomes incubated with any Igs (Fig. 10). Nonspecific binding accounted for 40-50% of total binding. Scatchard analysis revealed that the Kd values for binding of [3H]-verapamil in the presence of control and LEMS Igs plus serum were similar: 17.1 ± 4.2 and 14.6 ± 9.1 nM, respectively. However, Bmax was reduced from 97.4 ± 15.0 to 37.3 ± 3.8 fmol/mg protein after incubation with LEMS Igs as compared with incubation with control Igs.
Passive transfer using plasma or sera from patients with LEMS produces the hallmark electrophysiological and ultrastructural characteristics of LEMS (Hewett and Atchison 1992b
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; Lambert and Elmqvist 1971
; Vincent et al. 1989
). The electrophysiological features of this disorder are reduced quantal content at low frequencies of nerve stimulation with marked facilitation of end-plate potential amplitudes at high frequencies of stimulation (Lambert and Elmqvist 1971
). LEMS is an autoimmune disease, which is associated frequently with small cell carcinoma of the lung (SCC) (Lang et al. 1981
; Lennon et al. 1982
). Mice injected with either plasma or immunoglobulins (Igs) derived from LEMS patients reproduce the electrophysiological and morphological features of the disease (Fukunaga et al. 1983
; Kim 1985
; Lang et al. 1983
).
, 1992a
,b
; Kim and Neher 1988
; Lang et al. 1987
; Roberts et al. 1985
). High-voltage-activated Ca2+ currents (ICa) from chromaffin cells and neuroblastoma-glioma cells were reduced in amplitude by LEMS antibodies (Kim and Neher 1988
; Peers et al. 1990
). Three ICa components (L, N, and T type) in human neuroblastoma (IMR 32) cells were markedly reduced in amplitude by LEMS Igs (Grassi et al. 1994
). Depolarization-dependent uptake of 45Ca2+ into isolated nerve terminals of rat forebrain was reduced by 40-50% by acute application of plasma and serum from patients with LEMS (Hewett and Atchison 1991
, 1992b
). In freeze-fracture micrographs of presynaptic membranes of mice injected with serum from LEMS patients, active zone particles were disorganized and reduced in number (Fukunaga et al. 1983
). These particles are thought to be voltage-dependent Ca2+ channels mediating ACh release. Thus the reduced neurotransmitter release by nerve impulses in LEMS could be related to a reduced ingress of Ca2+ into the nerve terminals.
). Similarly, Garcia and Beam (1996)
demonstrated a reduction in whole cell high-voltage-activated ICa recorded from cultures of murine spinal motor neurons exposed semichronically to LEMS plasma. In each of these cases, currents sensitive to dihydropyridine (DHP)-type antagonists were either spared or unmasked after LEMS treatment.
-Agatoxin (Aga)-IVA and
-conotoxin (CTx)-MVIIC almost completely block ACh release and presynaptic ICa in mouse motor nerve terminals (Uchitel et al. 1992
; Xu and Atchison 1996
; Yan et al. 1994
). Although Hamilton and Smith (1992)
found evidence for the presence of some N-type Ca2+ channels in rat motor nerve terminals,
65% of the current could not be blocked by
-conotoxin-GVIA (
-CgTx-GVIA). Similar insensitivity to
-CgTx-GVIA as well as to DHPs has been reported in mouse motor nerve terminals (Anderson and Harvey 1987
; Penner and Dreyer 1986
). Thus P/Q-type Ca2+ channels may reflect the predominant type of Ca2+ channels in mammalian motor nerve terminals targeted by LEMS antibodies. The sensitivity of ICa in mammalian motor nerve terminals to LEMS Igs after passive transfer with serum from LEMS patients was reported by Smith et al. (1995)
. However, sensitivity to
-Aga-IVA or
-CTx-MVIIC was not examined.
; Xu and Atchison 1996
). Second, we sought to identify pharmacologically the types of high-voltage-activated ICa that were sensitive to LEMS Igs. Third, we sought to corroborate the striking observation that DHP-sensitive components of ICa were either unmasked (Smith et al. 1995
) or spared (Garcia and Beam 1996
) from block by LEMS Igs. These objectives were studied using perineurial measurements of ICa in triangularis sterni muscle from mice injected with plasma from LEMS patients. As reported by several groups (Penner and Dreyer 1986
; Shafer and Atchison 1992
; Xu and Atchison 1996
), it is possible to separate pharmacologically the current in the perineurial sheath into two kinetically and pharmacologically distinct components. We also performed binding studies for ligands reputed to interact with distinct populations of Ca2+ channels using rat cortical synaptosomes to assess if altered DHP sensitivity could be observed with acute application of LEMS Igs. This model was chosen because the pharmacological sensitivity of rat cortical synaptosomal Ca2+ channel function, faithfully mimics that at mammalian motor axon terminals (Anderson and Harvey 1987
; Atchison et al. 1988
; Nachshen and Blaustein 1979
; Suszkiw et al. 1986
; Uchitel et al. 1992
). In addition, acute exposure of this system to LEMS Igs causes disruption of Ca2+ channel function (Hewett and Atchison 1991
, 1992a
,b
) consistent with that seen during chronic passive transfer experiments.
-Aga-IVA and
-CTx-MVIIC. Conversely, acute treatment of central nerve terminals did not result in an alteration in DHP sensitivity, although the number of binding sites associated with the high-voltage-activated channel blocker verapamil was reduced by LEMS Igs.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
; Kim 1985
). To suppress the immune response to the injected plasma, the mice were first injected with cyclophosphamide (300 mg/kg ip) 1 day in advance. This treatment regimen reliably suppresses the full immune response (Lang et al. 1983
). Thirty-one days later, the left intercostal nerve with the triangularis sterni muscle was dissected from the mice for electrophysiological recording (McArdle et al. 1981
).
). A sufficient length of nerve trunk was dissected for external stimulation with a glass suction electrode. After dissection, the preparation was pinned out at its resting length in a small chamber of 5 ml. The solution circulated over the preparation at 5 ml/min. In all experiments, D-tubocurarine (20-50 µM) was used to abolish postsynaptic responses and procaine (100 µM) was used to prevent the repetitive firing of motor nerve terminals that occurs in the presence of K+ channel blockers.
-Aga-IVA and
-CTx-MVIIC were stored frozen at
70°C as stock solutions. Before experimentation, the stock solutions were thawed and diluted to the desired final concentration. Before applying
-Aga-IVA and
-CTx-MVIIC, bovine serum albumin (0.01% wt/vol) was added to the superfusion solution to prevent nonspecific binding of the toxins to the tube and chamber. Nifedipine and Bay K 8644 were prepared as 10 mM stock solutions in ethyl alcohol and stored at 4°C. The final concentration of ethyl alcohol was 0.1% (vol/vol). Before each experiment, nifedipine and Bay K 8644 were diluted with buffer to the desired final concentration. All experiments with nifedipine and Bay K 8644 were done in a darkened environment to prevent photo-oxidation. In the experiments using Cd2+, the bicarbonate buffering system was replaced with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; 10 mM) to avoid precipitation of CdCO3. For the experiments in which Ca2+ concentrations were decreased or increased, the osmolarity was maintained by adjusting the NaCl concentration.
; the electrode was placed inside the perineurial sheath of one of the branches of the intercostal nerve near the endplate region. The perineurium then was penetrated, and a steady negative 2-6 mV deflection denoted effective placement of the electrode for recording perineurial currents. Superficial nerve terminals were observed at a magnification of 400× using a microscope (Olympus BH-2, Olympus Optical, Tokyo, Japan) fitted with a water immersion objective and interference contrast (Nomarski) optics. Electrode movement during an experiment affects the amplitude of both the positive and negative components. If there was any evidence of electrode movement during the course of an experiment manifest as a change in the negative Na+ spike amplitude, records from that site were excluded from further analysis. Once a stable recording site was obtained, it could be held for as long as 80-90 min.
, as described in detail by Atchison et al. (1988)
. After ultracentrifugation in a discontinuous sucrose gradient, the synaptosomal pellet was resuspended in physiological saline buffer to an approximate concentration of 15-25 mg/ml (Lowry et al. 1951
).
,b
). The proper volume of physiological saline buffer was added to maintain a constant incubation volume. Before the initiation of binding experiments, synaptosomes were allowed to equilibrate for 30 min at 37°C. Binding assays were carried out at room temperature.
-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 10 D-glucose, 10 HEPES, and 145 N-methyl-D-glucamine. Stock solutions of the radiolabels were made in incubation buffer.
).
-CgTx-GVIA binding was essentially identical to that described earlier except that synaptosomes were added to incubation buffer containing 30-480 pmol of [125I]-
-CgTx-GVIA and 5 mg/ml bovine serum albumin in deionized water. Nonspecific binding was measured in the presence of 1 µM unlabeled
-CgTx-GVIA. Radioactivity remaining on the filters was estimated using a gamma counter with an efficiency of 80% for 125I. Because binding of
-CgTx-GVIA is essentially irreversible, Scatchard analysis of the data could not be performed. Thus the average of duplicate values was used for estimation of Bmax by saturation analysis.
20°C until used. After thawing, fibrin was removed from the plasma samples by clotting, which was facilitated by the addition of 1 M CaCl2. The clot was removed by centrifugation at 2,500 × g for 15 min. Total immunoglobulins (Igs) were isolated by serial precipitations of serum in 30 and 50% saturated ammonium sulfate at 4°C, as described previously (Hewett and Atchison 1992a
).
). Igs from patients with SCC alone did not affect 45Ca uptake.
- Aga-IVA was generously provided by Dr. Nicholas Saccamano, Pfizer (Groton, CT) and Research Biochemicals (Natick, MA).
-CTx-MVIIC was obtained from Bachem (Torrance, CA) and the Peptide Institute (Osaka, Japan).
-CgTx-GVIA was purchased from Peninsula Laboratories (Belmont, CA). The radioisotopes [3H]-nitrendipine (74 Ci/mmol), [3H]-verapamil (66 Ci/mmol), and [125I]-
-CgTx (2200 Ci/mmol) were purchased from New England Nuclear (Boston, MA). Nifedipine, Bay K 8644, verapamil, tetraethylammonium chloride (TEA), 3,4-diaminopyridine (3,4-DAP), D-tubocurarine hydrochloride, bovine serum albumin, procaine hydrochloride and polyethylenimine were all obtained from Sigma Chemical (St. Louis, MO). All other reagents were of analytic grade or better.
-CgTx-GVIA binding experiments were analyzed using a randomized complete block ANOVA. Differences in binding study data were considered significant at P < 0.05.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
; Penner and Dreyer 1986
). It is necessary, therefore, to block IK using TEA and 3,4-DAP to reveal ICa. Shown in Fig. 1A are perineurial recordings in the absence and presence of 3,4-DAP and TEA. In the absence of K+ channel block, a double-notched downward voltage deflection is observed. The initial component is due to INa, and the subsequent component is due to IK. Addition of 300 µM DAP and 10 mM TEA causes a time-dependent reduction in the downward voltage trace, leaving the component associated with INa unaffected, and gradually unmasking an upward voltage response associated with ICa (Fig. 1A, d and e). Figure 1B is obtained from the continuous recording of Fig. 1A, but the time base has been adjusted to allow more precise analysis of the upward voltage changes. Tracings 1B, a-c, reflect additional time of the preparation in 300 µM DAP and 10 mM TEA and show a progressive increase of the Ca2+ voltage change plateau with nerve stimulation at 0.01 Hz. Maximal response to these K+ channel blockers was attained at 20 min (Fig. 1Bc). Addition of further DAP and TEA did not increase further the maximal amplitude of the sustained upward voltage change (ICa), although it did slow its rate of decay somewhat. Thus as reported previously by Penner and Dreyer (1986)
, 10 mM TEA and 300 µM 3,4-DAP display maximally effective block of IK.
View larger version (9K):
[in a new window]
FIG. 1.
Effect of tetraethylammonium (TEA) and 3,4-diaminopyridine (3,4-DAP) on the membrane currents of normal mouse motor nerve terminals. A: progressive suppression of IK in the presence of 300 µM 3,4-DAP and 10 mM TEA and its replacement by ICa. Superimposed recordings from the same site before (control) and 2 min (a), 4 min (b), 6 min (c), 8 min (d), and 10 min (e) after addition of 10 mM TEA and 300 µM 3,4-DAP. Each record is the average of 10 successive sweeps at 0.1 Hz. B: efficacy of TEA and 3,4-DAP at blocking IK. Progressive increase of ICa amplitude is a continuation of the recording of A. a: from the trace of Fig. 1Ae with a different scale. Superimposed recordings are from the same site in the presence of 300 µM 3,4-DAP and 10 mM TEA (a-c) and in the presence of 600 µM 3,4-DAP and 20 mM TEA (d). c: appears to be a maximal amplitude ICa at 20 min after addition of 10 mM TEA and 300 µM 3,4-DAP, whereas d appears to be the maximal effect of 20 mM TEA and 600 µM 3,4-DAP after 15 min of bath application. Each record in an average of 5-10 successive sweeps at 0.01 Hz
ICapacitative (Brigant and Mallart 1982
; Mallart 1985
). The negative component is associated with Na+ currents (INa) in the Nodes of Ranvier near the recording electrode, and the delayed positive waveform is related to a prolonged ICa at the nerve terminal. Perineurial signals result from the fact that the signal amplitude at any recording point increases with the number of endings that supply local circuit current flow to the recording site (see Mallart 1982
). Thus the magnitude of both positive and negative components should vary depending on the position of the microelectrode tip within the perineurial sheath (Brigant and Mallart 1982
; Mallart 1985
; Penner and Dreyer 1986
). Smith et al. (1995)
found a strong linear relationship between the peak amplitudes of ICa and ICapacitative of mouse motor nerve terminals using focal extracellular recordings, thus providing a convenient method to compare ICa amplitudes obtained from different terminals. In the present study, we also found a strong correlation between ICa and INa in control and LEMS-treated preparations (Fig. 2C). However, treatment with LEMS plasma caused a downward shift of the line from those of controls, indicating a reduced ICa in LEMS-treated terminals (Fig. 2C).
View larger version (17K):
[in a new window]
FIG. 2.
Comparison of ICa in motor nerve terminals of mice treated with control and LEMS patient plasma. A: ICa with long duration from a typical recording of control and Lambert-Eaton Myasthenic Syndrome (LEMS)-treated terminals in the presence of 10 mM TEA and 300 µM 3,4-DAP. Each record is the average of 5-6 successive sweeps at 0.01 Hz. B: moderate potassium channel block (50 µM 3,4-DAP and 1 mM TEA) induced clearly separated ICa,f and ICa,s, which are indicated by the arrows marked ICa,f and ICa,s, respectively. Each record is the average of 5-6 successive sweeps at 0.01 Hz. C: linear relationship of peak amplitude of ICa and INa. Recording was obtained at a stimulation frequency of 0.01 Hz in the presence of 300 µM 3,4-DAP and 10 mM TEA. Each point comes from a different branch of the nerve. Straight lines were fitted by least squares regression method. The correlation coefficient (r) is 0.62 for control and 0.59 for LEMS.
View this table:
TABLE 1.
Amplitude of ICa and INa in mouse motor nerve terminals untreated or treated with control and LEMS plasma
demonstrated the presence of two different ICa currents in mouse motor nerve terminals based on their differences in frequency dependence and sensitivity to extracellular Ca2+ concentration as well as inorganic and organic calcium-antagonists such as Cd2+, verapamil, and diltiazem. Two kinetically distinct components also show differential sensitivity to
-Aga-IVA, a P-type Ca2+ channel blocker, and
-CTx-MVIIC, a Q-type Ca2+ channel blocker. ICa,f appears to be more similar to a Q-type Ca2+ channel, and ICa,s appears more similar to a P-type Ca2+ channel (Xu and Atchison 1996
). The two ICa components can be separated clearly by use of lower concentrations of K+ channel blockers because they have different activation thresholds. Thus we could examine whether either current component was decreased preferentially by LEMS plasma treatment. As shown in Fig. 2B, 0.01-Hz nerve stimulation in the presence of 1 mM TEA and 50 µM 3,4-DAP elicited ICa,f and ICa,s, which both were reduced significantly in amplitude in the LEMS plasma-treated mice. Table 2 summarizes the results from nine control and seven LEMS-treated preparations; it shows that in the LEMS-treated group ICa,f and ICa,s were reduced by 35 and 37%, respectively, whereas the ratios of ICa,f:INa and ICa,s:INa were reduced by 29 and 47%, respectively, compared with control.
View this table:
TABLE 2.
Comparison of ICa,f and ICa,s in control and LEMS plasma-treated motor nerve terminals
View larger version (21K):
[in a new window]
FIG. 3.
Effect of nerve stimulation frequency on ICa in control and LEMS plasma-treated motor nerve terminals. A: current was recorded in the presence of 300 µM 3,4-DAP and 10 mM TEA. Superimposed recordings from the same site are depicted at stimulation frequencies of 0.01 Hz (1), 0.1 Hz (2), 1 Hz (3), and 10 Hz (4). Note that the amplitudes of ICa are decreased in the control but not in LEMS-treated group as the stimulation frequency is increased. B: current was recorded in the presence of 50 µM 3,4-DAP and 1 mM TEA. Superimposed recordings from the same site are depicted at a stimulation frequency of 0.01 Hz (1), 0.1 Hz (2), 1 Hz (3), and 10 Hz (4). Note that there is no difference between control and LEMS groups during the increased stimulation frequency, where the amplitude of ICa for both is not decreased. C: data were obtained in the presence of 300 µM 3,4-DAP and 10 mM TEA. Each value is the mean ± SE of 12 control preparations and 11 LEMS-treated preparations. INa was increased slightly as the stimulation frequency was increased in control and LEMS-treated preparations but there was no difference between the 2 groups.
View larger version (23K):
[in a new window]
FIG. 4.
Effects of train stimulation on ICa of motor nerve terminals in control and LEMS plasma-treated mice. A and B: current was recorded by giving train stimulation at 20 Hz for 100 ms and 100 Hz for 100 ms, respectively, in the presence of 300 µM 3,4-DAP and 10 mM TEA. Each record is the average of 15-20 successive sweeps at 0.1 Hz. Note that ICa amplitude in control is not facilitated at 20 Hz and is depressed at 100 Hz, whereas in the LEMS group, there is facilitation of ICa at both 20 and 100 Hz. C: recording was made by giving trains of stimuli at 100 Hz for 100 ms in the presence of 50 µM 3,4-DAP and 1 mM TEA. Note that responses from both the control and LEMS group appear to be facilitated. D: amplitudes of ICa during 20- and 100-Hz train stimulation were expressed as the percentage of the first ICa amplitude of the same train. Recording was made in the presence of 300 µM 3,4-DAP and 10 mM TEA. The values are the means ± SE of 5 control and 6 LEMS-treated preparations, respectively.
View larger version (22K):
[in a new window]
FIG. 5.
Effects of extracellular Ca2+ concentration on ICa of motor nerve terminals of mice treated with control and LEMS plasma. A and B: superimposed recordings were obtained from the same site at extracellular Ca2+ concentrations from 0.5 to 6 mM for control preparations and from 0.5 to 10 mM for LEMS-treated preparations. Recordings were made in the presence of 10 mM TEA and 300 µM 3,4-DAP and continuously at a single terminal for the various concentrations of Ca2+ administered sequentially at the time that the effect of the previous concentration appeared to be maximal. Each record is an average of 5-10 successive sweeps at 0.01 Hz.
-Aga-IVA- or
-CTx-MVIIC-sensitive Ca2+ channels as the target of LEMS autoantibodies
-Aga-IVA,
-CTx-MVIIC, and nifedipine were applied to identify pharmacologically, the types of ICa in control and LEMS plasma-treated mice. In control preparations, perfusing the preparation with 150 nM
-Aga-IVA caused block of ~90% of ICa within 15 min (Fig. 6A) The positive component remaining after treatment with
-Aga-IVA was not sensitive to Cd2+ even at 10 mM (results not shown). Thus the effect of
-Aga-IVA on ICa of control plasma-treated motor nerve terminals is similar to that of our previous observation in untreated motor nerve terminals (Xu and Atchison 1996
). However, 150 nM
-Aga-IVA was less effective in LEMS-treated preparations; it reduced ICa of LEMS-treated terminals 22% less than in control preparations. The ICa component that was resistant to
-Aga-IVA was sensitive to low concentrations of Cd2+ (10 µM), thus indicating the involvement of another type of Ca2+-sensitive channel in LEMS. The portion of ICa which remained after
-Aga-IVA was reduced further within 15 min of addition of 10 µM nifedipine (Figs. 6A and 8). Similar results were obtained using
-CTx-MVIIC. At 5 µM,
-CTx-MVIIC completely blocked ICa in the control plasma-treated terminals (Fig. 6B); the same concentration of
-CTx-MVIIC was 37% less effective at blocking ICa in LEMS plasma-treated terminals. The ICa that remained after
-CTx-MVIIC also was blocked by nifedipine (Figs. 6B and 8), and Cd2+ (results not shown).
View larger version (13K):
[in a new window]
FIG. 6.
Effect of -agatoxin-IVA (
-Aga-IVA) and
-conotoxin-MVIIC (
-CTx-MVIIC) on ICa in control and LEMS plasma-treated motor nerve terminals. A: ICa was recorded in the presence of 300 µM 3,4-DAP and 10 mM TEA. Superimposed recordings from the same site are depicted before and after application of
-Aga-IVA (0.15 µM) and nifedipine (10 µM) for control and LEMS groups. B: current was recorded in the presence of 300 µM 3,4-DAP and 10 mM TEA. Superimposed recordings from the same site are depicted before and after application of
-CTx-MVIIC (5 µM) and nifedipine (10 µM) for control and LEMS. Toxins were administered sequentially at the time that the effect of the previous toxin appeared maximal. Each record is an average of 6-7 successive sweeps at 0.01 Hz.
View larger version (32K):
[in a new window]
FIG. 8.
Summary of the effects of Ca2+ channel antagonists on ICa in control and LEMS plasma-treated motor nerve terminals. Values are the means ± SE of 4-6 control and LEMS-treated preparations.
-Aga-IVA and
-CTx-MVIIC on ICa in LEMS-treated terminals were additive with those of nifedipine, we examined the effect of nifedipine, verapamil, and Bay K 8644 on ICa in the absence of
-Aga-IVA and
-CTx-MVIIC. At 10 µM, nifedipine did not affect ICa in the control plasma-treated motor nerve terminals, but inhibited ICa by 25-35% in LEMS plasma-treated terminals (Fig. 7A). The current resistant to nifedipine in these animals could be abolished by 150 nM
-Aga-IVA just as in the controls (Fig. 7A). The same effect occurred when using
-CTx-MVIIC after nifedipine (results not shown). Thus the inhibitory effects of
-Aga-IVA or
-CTx-MVIIC on ICa were additive with those of nifedipine, suggesting the presence of
-Aga-IVA- and
-CTx-MVIIC-insensitive, nifedipine-sensitive L-type ICa in LEMS plasma-treated but not control motor nerve terminals. The nifedipine-sensitive components of current could be increased to a level similar to that measured in nifedipine-free solution by addition of 10 µM Bay K 8644. There was no significant difference in the percent inhibition of verapamil on ICa between control and LEMS-treated preparations (Figs. 7B and 8). At 10 µM, verapamil reduced ICa by 50% for control plasma-treated and 57% for LEMS plasma-treated preparations.
View larger version (15K):
[in a new window]
FIG. 7.
Effect of nifedipine and verapamil on ICa in control and LEMS plasma-treated motor nerve terminals. A: current was recorded in the presence of 50 µM 3,4-DAP and 1 mM TEA. Superimposed recordings from the same site are depicted before and after application of 10 µM nifedipine and 0.15 µM -Aga-IVA for control and LEMS groups. Toxins were administered sequentially at the time that the effect of the previous toxin appeared maximal. B: current was recorded in the presence of 50 µM 3,4-DAP and 1 mM TEA. Superimposed recordings from the same site are depicted before and after application of 10 µM verapamil for control and LEMS groups. Each record is an average of 6-7 successive sweeps at 0.01 Hz.
, 1992a
,b
), we found that plasma or Igs isolated from patients with LEMS could reduce the uptake of 45Ca2+ into synaptosomes during KCl-induced depolarization by 40-50%. We thus used this model to assess whether acute exposure to LEMS Igs would unmask a DHP-sensitive component in central nerve terminals. Specifically, we sought to determine whether in synaptosomes exposed to LEMS Igs, the pharmacologically distinct binding sites reputed to be associated with nerve terminal Ca2+ channels are altered either in number or in their affinity for antagonist.
; Suszkiw et al. 1986
; Turner and Goldin 1985
). Administration of LEMS Igs did not affect either the Kd or apparent Bmax (Table 3).
View this table:
TABLE 3.
Characteristics of binding of [3H]nitrendipine to rat brain synaptosomes in the absence and presence of LEMS Igs
-CONOTOXIN-GVIA BINDING.
The specific binding of [125I]-
-CgTx GVIA to synaptosomes in the presence of control and LEMS Igs also was examined (Fig. 9). Nonspecific binding accounted for <10% of total binding.
-CgTx GVIA bound to synaptosomes with a half-saturation of between 0.1 and 0.2 nM. Because binding of
-CgTx GVIA is essentially irreversible, the maximum density of binding sites was estimated by extrapolation of the saturated component to the y axis. The apparent Bmax value for
-CgTx binding in synaptosomes treated with control or LEMS Igs did not differ. The control value (260.6 ± 6.7) was consistent with those reported by others (Cruz and Olivera 1986
; Marqueze et al. 1988
; Wagner et al. 1988
).
View larger version (10K):
[in a new window]
FIG. 9.
Comparative effects of control (- -) and LEMS Igs (-
-) on
-conotoxin-GVIA (
-CgTx-GVIA) binding to rat forebrain synaptosomes. Equal aliquots of synaptosomes were incubated with control or LEMS Igs (4 mg/ml) in the presence of 10% (vol/vol) human serum 90 min before initiation of binding. Specific binding of [125I]-CgTx was determined as described in METHODS. Values shown are the means ± SE of 3 experiments. When SE bars are not shown the SE is smaller than the size of the symbol.
View larger version (21K):
[in a new window]
FIG. 10.
Comparative effects of control and LEMS immunoglobulins (Igs) on verapamil binding to synaptosomes. Equal aliquots of synaptosomes were incubated with control or LEMS Igs (4 mg/ml) in the absence (A) or presence of 10% (vol/vol) human serum (B) 90 min before initiation of binding. Specific binding of [3H]-verapamil was determined as described in METHODS. Values shown are the means ± SE of 6 experiments. When SE bars are not shown, the SE is smaller than the size of the symbol.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
; Kim 1985
; Kim and Neher 1988
; Lambert and Elmqvist 1971
; Smith et al. 1995
). These include depletion of active zone particles, reduction of quantal content, and facilitation of release at high frequencies of stimulation. In the present study, passive transfer of LEMS to mice reduced the amplitude of presynaptic ICa, caused frequency-dependent facilitation of ICa, and induced sensitivity to DHP-type antagonists in mouse triangularis sterni muscles, corroborating our previous results (Smith et al. 1995
). However, unlike our previous study, in which we were unable to resolve kinetically distinct components of ICa, the present study shows that both fast and slow components of ICa were reduced roughly in parallel by LEMS autoantibodies. Moreover, the pharmacological identity of the current components affected by LEMS passive transfer was determined. Finally, acute exposure of synaptosomes to LEMS plasma was shown not to alter the binding of the DHP antagonist nitrendipine, whereas it did reduce the maximum binding of the phenylalkylamine verapamil.
, 1992b
) and that protein components, the sizes of which are consistent with those of the Ca2+ channel complex, are recognized by Igs of 14 patients with LEMS (Hajela and Atchison 1995
).
). The perineurial recording method used in the present study lacks the unequivocal nature of voltage clamp for resolving electrically distinct events, however, it does provide a reproducible, reliable estimation of events occurring at the mammalian motor axon terminal, a structure not amenable to voltage-clamp methodology. We have shown previously that the two components of ICa recorded at motor axon terminals of triangularis sterni are primarily sensitive to
-Aga-IVA and
-CTx-MVIIC (Xu and Atchison 1996
). In the present study,
-Aga-IVA and
-CTx-MVIIC inhibit ICa from motor nerve terminals of mice treated with control plasma by 90-95%, confirming that channels sensitive to these peptide toxins (putative P- and/or Q-type Ca2+ channels) are primarily responsible for the Ca2+ influx in mammalian motor nerve terminals. In LEMS plasma-treated mouse motor nerve terminals, whereas ICa is reduced by 30-40% compared with control,
-Aga-IVA and
-CTx-MVIIC again block the majority of the remaining ICa, except for the presence of 20-30% toxin-insensitive but DHP-sensitive L-type ICa. These results demonstrate a preferential effect of LEMS antibodies on P- or Q-type Ca2+ channels in mammalian motor nerve terminals to cause their loss of function. This is consistent with the recent report that LEMS sera preferentially affects P- or Q-type Ca2+ currents, but not L-type currents, of mouse motoneuron cell bodies (Garcia and Beam 1996
).
; Atchison and O'Leary 1987
), which suggest that DHP-sensitive Ca2+ channels do not normally contribute to ACh release at murine neuromuscular junctions. However, apparently L-type Ca2+ channels do contribute to the Ca2+ influx in LEMS plasma-treated mouse motor nerve terminals. Similar findings have been reported by Smith et al. (1995)
, who showed that 43% of ICa in mouse phrenic motor nerve terminals exposed to LEMS antibodies became sensitive to DHPs but insensitive to
-CgTx-GVIA. They proposed that activation of L-type Ca2+ channels in LEMS-treated preparations was due to depolarization of the terminal membrane resulting from inactivation of an IK,Ca because intracellular Ca2+ concentrations were decreased by LEMS antibodies. The question arises whether this apparent L-type Ca2+ channel activation also would occur after acute exposure to LEMS Igs.
,b
) and the apparent Bmax for verapamil. No effect of LEMS plasma was seen on the apparent affinity of the remaining binding sites for ligand, suggesting that reduction of available verapamil binding sites occurs without effects on the functional characteristics of the remaining sites. The same concentration of Igs did not affect the binding characteristics of [3H]-nitrendipine. Thus acutely LEMS Igs appears to spare DHP-sensitive binding sites. Superficially, this result agrees with the observations of Garcia and Beam (1996)
that DHP-sensitive components of ICa were spared by LEMS Igs. The question then is whether induction of DHP sensitivity to LEMS Igs represents an acute or adaptive response. Perhaps DHP-sensitive channels are simply unmasked because they are spared clustering by the antibodies and subsequent lysis, or they may be actively upregulated teleologically to help the terminal cope with reduced ingress of Ca2+ caused by loss of P/Q types of channels normally present. Evidently another mechanism is involved in the activation of L-type Ca2+ channels during chronic treatment of mouse motor nerve terminals with LEMS serum or plasma, an activation that does not occur during acute treatment. For instance, the appearance of DHP-sensitive, L-type Ca2+ currents in the LEMS-treated motor nerve terminals may be due to new protein and mRNA synthesis for L-type Ca2+ channels or covalent modifications of preexisting proteins for L-type Ca2+ channels to compensate for conductance lost from P- or Q-type Ca2+ channels due to LEMS antibody attack. The DHP-sensitive, L-type Ca2+ currents do not appear in the acutely treated preparations because feedback regulation requires time for accumulation of new proteins synthesized for L-type Ca2+ channels, and synaptosomes lack the necessary biosynthetic machinery needed to transcribe and translate new L channels.
-Aga-IVA-insensitive, but nifedipine-sensitive, L-type Ca2+ currents involved in ACh release processes have been found at newly formed (Sugiura and Ko 1997
) and reinnervating mammalian neuromuscular junctions (Katz et al. 1995
). However, the pattern of effect of DHP antagonists at these channels is to increase ACh release rather than to decrease it. Nevertheless, it may be possible that the apparent activation of L-type Ca2+ channels in LEMS-treated motor nerve terminals results from regeneration of the nerve terminals after LEMS antibody-induced degradation and necrosis. However, morphological studies have not suggested that nerve terminal degeneration and/or sprouting occurs. Instead, it appears that LEMS antibodies only reduce the density and number of Ca2+ channels with the remaining Ca2+ channels aggregating into clusters; no change of other membrane structures was reported (Fukunaga 1983). Furthermore, in synaptosomes incubated with LEMS Igs, lactate dehydrogenase release was not increased, whereas depolarization-dependent uptake of 45Ca2+ was decreased (Hewett and Atchison 1992a
,b
), providing further support that the reduction in Ca2+ channel function by LEMS antibodies was not simply a result of disruption of nerve terminal membrane integrity. Thus we think it unlikely that motor nerve terminals degraded, necrosed and regenerated after injection of LEMS plasma.
; Glossmann and Striessnig 1988
; Miller 1987
). However, recent evidence suggests that verapamil may not be as selective for L-type channels as was previously thought. In cerebellar granule cells (Carboni and Wojcik 1988
) and neocortical neurons (Mangano et al. 1991
) in primary culture, Ca2+ influx resulting from cell depolarization is only partially inhibited by nitrendipine. In contrast, verapamil completely blocked the depolarization-induced influx, suggesting that verapamil either blocks all subtypes of voltage-sensitive Ca2+ channels (Carboni and Wojcik 1988
) or blocks the activity of a separate, perhaps novel type Ca2+ channel (Mangano et al. 1991
). A verapamil-sensitive ICa current also exists at mouse motor nerve terminals (Anderson and Harvey 1987
; Penner and Dryer 1986; Smith et al. 1995
), and nerve-evoked release of ACh at mammalian neuromuscular junctions is sensitive to verapamil as well (Nachshen and Blaustein 1979
). We found that the ICa remaining after treatment with
-Aga-IVA and
-CTx-MVIIC could be blocked by 10 µM verapamil. Thus the best pharmacological correlation between the functional effectiveness of LEMS Igs on motor axon terminals and inhibition of ligand binding on nerve terminals exists for verapamil. Our results show that a portion of P- or Q- type ICa in motor nerve terminals is sensitive to verapamil as well; this is consistent with the results of Ishibashi et al. (1995)
, who showed that verapamil blocked current carried through P-type Ca2+ channels in rat dissociated Purkinje neurons. Thus the total effect of inhibition of verapamil-sensitive P- or Q-type ICa by LEMS antibodies and appearance of DHP-sensitive ICa cause no greater percent reduction in the verapamil-sensitive currents in the LEMS-treated motor nerve terminals than those of control plasma-treated motor nerve terminals.
). Therefore in LEMS plasma-treated motor nerve terminals, the loss of frequency-dependent depression for ICa during high frequencies of stimulation may well reflect the low [Ca2+]i, which is insufficient to trigger Ca2+-induced inactivation. However, in the presence of low concentrations of K+ channel blockers, there was no difference in ICa between control and LEMS plasma-treated preparations during high-frequency stimulation or train stimulation; facilitation of ICa amplitude occurred in both control and LEMS-treated groups. A more likely explanation is that delayed rectifier K+ channels, spared in the presence of low concentrations of K+ channels blockers, repolarized the membrane promptly to prevent Ca2+ increase (Mallart 1985
). Therefore these results demonstrate that caution is required when using perineurial recording technique to assess and compare the frequency-dependent effects on Ca2+ channel function between control and LEMS-treated motor nerve terminals.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank Drs. Margarita Contreras and Ravindra Hajela for helpful discussions. We acknowledge the generous donations of sera/plasma from Dr. Mark Glasberg, Henry Ford Hospital, Detroit, MI; Drs. Eva Feldman and Jim Albers, Department of Neurology, University of Michigan; and Dr. Shin Oh, Department of Neurology, University of Alabama, Birmingham. Similarly, the generous gift of -Agatoxin-IVA by Dr. Nicholas Saccamano, Medicinal Chemistry Research, Pfizer, is gratefully acknowledged.
This work was supported by National Institute of Environmental Health Sciences Grant ES-05822. S. J. Hewett was supported by a Viets Fellowship from the Myasthenia Gravis Foundation.
![]() |
FOOTNOTES |
---|
Present address of S. J. Hewett: Dept. of Pharmacology, School of Medicine, University of Connecticut Health Center, Farmington, CT 06030.
Address for reprint requests: W. D. Atchison, Dept. of Pharmacology and Toxicology, Michigan State University, B-331 Life Sciences Bldg., East Lansing, MI 48824-1317.
Received 12 November 1997; accepted in final form 21 May 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|