D. Wray

AUTOIMMUNE AETIOLOGY FOR MYASTHENIC (EATON-LAMBERT) SYNDROME

The myasthenic (Eaton-Lambert) syndrome, associated with carcinoma of the bronchus in one patient and with immunological disorders in two others, improved after plasma exchange--observations supported by electromyographic evidence in two cases. Prednisolone and azathioprine treatment led to almost complete remission in one of the non-neoplastic cases and to improvement in the other. The IgG fraction of plasma from all three patients, injected daily (10 mg) into mice for 37-77 days, significantly reduced the initial compound muscle action potential and the quantal content of the end-plate potential measured in the diaphragm, when compared with control human IgG. These results indicate that an IgG autoantibody, binding to nerve terminal determinants, may be responsible for the disorder of neuromuscular transmission in the myasthenic syndrome, and that immunosuppressive drugs may be useful in treating the nonneoplastic form of the disease.

Antibodies to motor nerve terminals: an electrophysiological study of a human myasthenic syndrome transferred to mouse.

Immunoglobulin G(IgG) prepared from the plasma of patients with a presynaptic disorder of neuromuscular transmission (Lambert‐Eaton myasthenic syndrome, l.e.m.s.), or from normal pooled control human plasma, was injected into mice (10 mg daily) for up to 99 days. Micro‐electrodes were used to record end‐plate potentials from the diaphragm muscle bathed in normal Krebs solution containing tubocurarine (1.0‐4.6 microM). At 0.5 Hz nerve stimulation frequency, the quantal content was significantly reduced (P less than 0.01‐P less than 0.001) in mice treated with six l.e.m.s. patients’ IgG each compared with paired controls. The pooled quantal content was 55 +/‐ 3 (n = 110 end‐plates) for all test animals and 131 +/‐ 9 (n = 47) for all controls (P less than 0.001). During short trains at 20 or 40 Hz nerve stimulation, control muscles showed marked depression, while test muscles showed either facilitation or less marked depression. Quantal content throughout these trains remained lower than in controls. The results indicate that IgG antibody from l.e.m.s. patients can induce a similar physiologic disorder in injected mice, and they support the view that this antibody interferes with evoked release of transmitter in l.e.m.s. by binding to nerve terminal determinants.

The effect of myasthenic syndrome antibody on presynaptic calcium channels in the mouse.

1. The action of immunoglobulin G obtained from patients with Lambert‐Eaton myasthenic syndrome (LEMS IgG) was investigated by injecting mice, followed by intracellular recordings from the mouse diaphragm. 2. End‐plate potential quantal content was studied over a range of Ca2+ concentrations. Curves of log quantal content versus log Ca2+ concentration were shifted to the right by LEMS IgG. For low Ca2+ concentrations, release continued to follow Poisson statistics after LEMS IgG treatment. 3. Miniature end‐plate potential (m.e.p.p.) frequency was measured in solutions containing high K+ concentrations. LEMS IgG significantly reduced m.e.p.p. frequency at each K+ concentration studied. 4. M.e.p.p. frequency was measured at fixed high‐K+ concentration (15.9 mM) for a range of Ca2+ concentrations. The log‐log plot of m.e.p.p. frequency versus Ca2+ concentration was shifted downwards throughout by LEMS IgG. 5. M.e.p.p. frequency was not affected by LEMS IgG in Ca2+‐free solutions (K+ concentration 15.9 mM) or in solutions of low Ca2+ concentration (K+ concentration 5.9 mM). 6. At each Ca2+ concentration studied, m.e.p.p. amplitudes were not affected by LEMS IgG. 7. The data suggest that LEMS IgG acts on presynaptic voltage‐dependent Ca2+ channels to cause their loss of function, probably by down‐regulation.

Selective action of myasthenic syndrome antibodies on calcium channels in a rodent neuroblastoma x glioma cell line.

1. The effect of Lambert‐Eaton myasthenic syndrome (LEMS) immunoglobulin G (IgG) on Ca2+ channels in undifferentiated mouse neuroblastoma x rat glioma hybrid cells (NG 108 15) was studied using the whole‐cell patch clamp technique. 2. Sustained inward Ca2+ channel currents were evoked by depolarizing pulses from holding potentials of ‐80 and ‐40 mV, and were blocked by 5 microM‐nitrendipine (L‐type currents). Transient inward Ca2+ channel currents were activated from a holding potential of ‐80 mV by small depolarizing steps (T‐type currents). Noradrenaline (10 microM) was without effect on transient currents. 3. LEMS IgG selectively reduced sustained (L‐type) Ca2+ channel current amplitudes evoked from either holding potential used. In the presence of nitrendipine (5 microM), there was no significant effect of LEMS IgG on the remaining transient (T‐type) Ca2+ channel current amplitudes. 4. Studies of the potential for maximal inward current indicated that voltage sensitivities of both L‐ and T‐type Ca2+ channel current amplitudes were unaffected by LEMS IgG, whether recorded in the presence or absence of nitrendipine. LEMS IgG had no significant effect on the time‐to‐peak or decay of Ca2+ channel currents. 5. It is concluded that LEMS IgG acts selectively to cause functional loss of L‐type, but not T‐type, Ca2+ channels in NG 108 15 cells. Any effect of LEMS IgG on N‐type channels (not present in these undifferentiated cells) was not studied here. LEMS IgG also acts at motor nerve terminal Ca2+ channels leading to muscle weakness. Thus antigenic similarities must exist between L‐type channels in NG 108 15 cells and Ca2+ channels at motor nerve terminals.

Immunological Heterogeneity and Cellular Mechanisms in Myasthenia Gravis

The clinical and immunological heterogeneity that characterizes myasthenia gravis (MG) has implications for etiology, pathogenetic mechanisms, cellular interactions, and treatment. Moreover, it raises the important question of whether MG is a “single” disorder, attributable exclusively to autoantibodies directed against the postsynaptic nicotinic acetylcholine receptor (AChR). In this paper we present evidence that antibodies to determinants other than AChR may impair neuromuscular transmission in some MG patients, show data indicating a diversity of genetic factors influencing susceptibility to MG, and describe recent immunohistological and functional studies of the thymus and of thymic cells.

The effects of in vitro application of purified botulinum neurotoxin at mouse motor nerve terminals.

1. Purified botulinum neurotoxin type A (10 nM) was applied in vitro to mouse diaphragm muscles. Intracellular micro‐electrode recordings were made continuously in single fibres. 2. This treatment reduced end‐plate potential (e.p.p.) amplitudes with a time to half‐maximal effect of about 75 min at 22‐25 degrees C. E.p.p. rise‐times remained fast and unaffected by the toxin. 3. Miniature end‐plate potential (m.e.p.p.) frequency was reduced by the toxin to less than 5% of control frequency, and followed a similar time course to the block of e.p.p. amplitudes. The m.e.p.p. rise‐time and coefficient of variation (c.v.) of m.e.p.p. amplitude distributions both increased, but the time course of these increases lagged significantly behind the change in frequency. 4. A population of slow rise‐time m.e.p.p.s was present in controls at low frequency. This population was found to be unaffected by the toxin. 5. The above‐detailed in vitro changes could be explained by the toxin acting by a single common mechanism to inhibit the release process underlying both fast rise‐time m.e.p.p.s and e.p.p.s. A distinct release process, which leads to slow rise‐time m.e.p.p.s, was unaffected by the toxin.

Effect of the Calcium‐Channel Agonist CGP 28392 on Transmitter Release at Mouse Neuromuscular Junctions

Transmitter release at the neuromuscular junction is dependent upon the entry of Ca’+ into the nerve terminal via voltage-dependent Ca2+ channels. At least three different types of neuronal Ca2+ channel have been classified,’ but those directly involved in the release of ACh at the mouse neuromuscular junction have not yet been clearly characterized. Organic Ca2+-channel antagonists (which act on L-type channels) are without effect on transmitter release at the skeletal neuromuscular junction.’.’ However, there are known to be Ca’+-antagonist-sensitive Ca2’ channels present at motor nerve terminals.’ Although not normally involved in transmitter release, L-type channels may play a role in release in the presence of Ca’+-channel agonists. To test this, we have investigated the effect of the Ca’+-channel agonist CGP 28392 on acetylcholine release at nerve terminals of mouse diaphragm muscles, comparing the effect with the antagonist nitrendipine.

The effect of Lambert-Eaton myasthenic syndrome antibody on slow action potentials in mouse cardiac ventricle

Immunoglobulin G (IgG) from Lambert-Eaton myasthenic syndrome (LEMS) patients acts at motor nerve terminal Ca2+ channels. It was injected into mice to investigate effects on cardiac Ca2+ channels. Intracellular recordings were made of slow action potentials in right ventricular muscle cells in the presence of high K+ concentrations and isoprenaline (1µM). Reduction in Ca2+ concentration reduced the rate of rise and amplitude, but not the duration, of slow action potentials whereas verapamil (1µM) blocked them. They were not blocked by tetrodotoxin (10 µM), and 4-aminopyridine (1mM) prolonged the decay phase without affecting the rate of rise and amplitude. The rate of rise, amplitude and duration of slow action potentials were not affected by LEMS IgG. These results show that LEMS IgG does not act on Ca2+ channel currents that underlie slow action potentials in mouse ventricles, suggesting antigenic differences between Ca2+ channels at motor nerve terminals and heart.

Actions of β-bungarotoxin on spontaneous release of transmitter at muscle end-plates treated with botulinum toxin

Rat leg muscles were injected subcutaneously with sublethal doses of type A botulinum neurotoxin, and the extensor digitorum longus muscle removed three days later. Intracellular microelectrode recordings were then made of miniature end-plate potentials (mepps). The mepp frequency was reduced by botulinum toxin, while mepp rise times were slowed. Mepp amplitude distributions became characteristically skew. beta-Bungarotoxin (140 nM) was applied to normal muscles in vitro and recordings were made 10-30 min later. The main effect was an increase in mepp frequency during this period. Mepp rise times were unaffected. When beta-bungarotoxin was applied in vitro to muscles treated with botulinum toxin there was also an increase in mepp frequency, although to a value less than in normal muscles. The mepp rise times were speeded up to normal values. The mepp amplitude and rise time distributions showed no obvious evidence for the addition of a second component to the distribution. The data appear to support the hypothesis that the sites for spontaneous release in botulinised muscle may be located at or near the usual release sites at the active zones.

Action of antibodies directed against the acetylcholine receptor on channel function at mouse and rat motor end-plates.

1. The acute effects of antibodies (both polyclonal and monoclonal) raised against the acetylcholine receptor were studied at mouse and rat end‐plates. Isolated muscles were incubated in solutions containing antibody for 2 1/4 to 3 1/2 h. Intracellular microelectrode techniques were then used to record miniature end‐plate potentials (MEPPs) and voltage noise. 2. Most antibody preparations investigated did not reduce MEPP amplitudes as compared with controls. One monoclonal (C7) and one polyclonal (J) preparation irreversibly reduced MEPP amplitudes. Both preparations caused reductions in acetylcholine‐induced depolarization and associated channel opening frequency (from voltage noise analysis). Single‐channel depolarization was not altered by these antibodies. 3. On the basis of these and previous results, four antibody binding regions on the receptor surface were distinguished according to whether channel function and/or alpha‐bungarotoxin binding were affected. Although most antibody preparations did not affect channel function, monoclonal antibody C7 appeared to alter function by acting on the channel itself so as to prevent channel opening.