Tubocurarine chloride

The antibodies against pre-synaptic membrane receptor (PsmR) and acetylcholine receptor (AChR) in serum samples of myasthenia gravis (MG) patients and healthy donors were tested by enzyme-linked immunosorbent assays (ELISA). The serum samples of eight MC patients with different autoantibodies and those of six healthy donors without these two kinds of autoantibodies were collected to investigate their effects on the peri-neurially recorded membrane currents at mouse motor nerve terminals. After inhibition of both fast and Ca^sup 2+^-dependent K+ currents by tetra-ethylammonium (TEA), a positive wave was revealed, which was a balance of the slow K^sup +^(I^sub K,s^) and Ca^sup 2+^ currents (I^sub Ca^). Application of anti-PsmR antibody negative MC sera and healthy donor sera, whether anti-AChR antibody positive or negative, did not affect the positive wave. However, the positive wave shifted to prolonged Ca^sup 2+^-plateau when adding two of four anti-PsmR antibody positive serum samples from MC patients, indicating an inhibition of I^sub K,s^ by anti-PsmR antibody positive sera. Meanwhile, all serum samples derived from either patients or healthy donors did not affect I^sub Na^. [Neurol Res 2003; 25: 58-62]

Keywords: Mouse motor nerve terminals; peri-neurial recording; membrane currents; myasthenia gravis; potassium currents; pre-synaptic membrane receptor antibody

INTRODUCTION

Myasthenia gravis (MG) has been considered as an autoimmune disease that results from an autoantibody-induced damage of post-synaptic acetylcholine receptors (AChR)1,2. Examining anti-AChR antibody by radioimmunoassays or enzyme-linked immunosorbent assays (ELISA) was generally used to diagnose MG. However, it was found that some patients with severe MG symptoms may lack detectable anti-AChR antibody in their sera3 and an experimental allergic MG was duplicated by passive transfer of anti-AChR antibody negative sera4. These data imply that some factors in addition to anti-AChR antibodies may be involved in the pathogenesis of MG.

Lu ef al.5 reported that an antibody against a [beta]-bungarotoxin ([beta]-BuTx) binding protein was present in sera of some MG patients. They used [beta]-BuTx to capture its corresponding protein, tentatively named as presynaptic membrane receptor (PsmR), from preparation of crude human muscle. PsmR was then taken as antigen to test its antibodies in serum by ELISA. The anti-PsmR antibody was detected in 67 of 82 MG patients. It was presumed that MG could result from immune damage of not only AChR but also PsmR. It was also shown that the number of PsmR reactive T and B cells in the MG patients was much higher than that in the healthy persons6. The pathogenicity of autoantibody to PsmR in MG was established by passive transfer of sera from MG patients with anti-PsmR antibody7 or its IgG, as well as by immunization with [beta]-BuTx binding protein from calf diaphragm as antigen8. In the mice infected with antiPsmR antibody positive serum, the neurotransmitter release evoked by high-K^sup +^ solution was affected, i.e. the frequency of miniature endplate potential (mEPP) and the quantal content of end-plate potential decreased9.

As a pre-synaptic blocker, [beta]-BuTx blocks synaptic transmission by interfering with neurotransmitter release with an initial facilitation and a substantial inhibition10. Similar two-phase effect of the MG patient sera with anti-PsmR antibody on neurotransmitter release was observed11. [beta]-BuTx and dendrotoxin were shown to have a common binding site in nervous tissue12, and the binding site was demonstrated to be a kind of K+ channel13,14.

Peri-neurial recording allows to pick up the currents from the subendothelial space, which correspond to ion influx through ion channels within pre-terminal and terminal parts15-17. Although the recorded currents are not, strictly speaking, 'membrane currents', their size and duration reflect the conductance change originating from the pre-synaptic membrane to different ions. Therefore, the voltage changes attributable to Na+, K+ and Ca^sup 2+^ currents have been termed as Na+ (I^sub Na^), K+ (I^sub K^) and Ca^sup 2+^ currents (I^sub Ca^) respectively for convenience15,18,19. The peri-neurial recording has been widely used to elucidate the action of pre-terminal agents20-2.

Our previous study showed that a MG patient plasma which was anti-PsmR antibody positive and anti-AChR antibody negative selectively inhibited the peri-neurially recorded slow K+ current (I^sub K,s^) at mouse motor nerve terminals24, but it was unclear whether the anti-PsmR antibody negative sera from MG patients had no effect on I^sub K,s^, while the inhibition of I^sub K,s^ was a selective effect of anti-PsmR antibody positive sera. In the present study, the antibodies against PsmR and AChR in sera were tested by ELISA and the sera were collected to test their effects on I^sub K,s^ in mouse motor nerve terminals, respectively. The results indicated that only anti-PsmR antibody positive sera could cause inhibition of the current.

MATERIALS AND METHODS

Patients and sera

Sera used in this study were acquired with informed consent from six healthy donors and eight MG patients, none of whom was in association with thymoma or small-cell lung carcinoma, with an age varying between 2 to 61 years. All patients diagnosed by neurologists in the Department of Neurology based on clinical symptoms, responses of muscle to repetitive stimulation and neostigmine test had typical MG symptoms. Serum was separated from 3-5 ml of venous blood by centrifugation and stored at -30[degrees]C before use.

ELISA examination of antibody against PsmR and AChR

Both PsmR and AChR were purified separately from bovine diaphragm according to the methods described previously by Lu et al.5 and Lindstrom et al.25. Antibodies in sera were tested using the procedure of ELISA26. Briefly, Polyvinylchloride microtitre plates (Dynatech Lab., Chantilly, VA, USA) were respectively coated with 100 [mu]l of AChR (2 [mu]g ml^sup -1^), PsmR (2 [mu]g ml^sup -1^) and bovine serum album (10 [mu]g ml^sup -1^), all of which were diluted with buffered phosphate saline, then incubated at 4[degrees]C overnight. Unreactive sites were saturated with 150 [mu]l of 10% fetal calf serum (Gibco, Paisley, UK) in carbonate buffer (pH 9.6) at 37[degrees]C for 2 h. 100 [mu]l of 1/5 diluted sera from MG patients or healthy donors were added and incubated at 37[degrees]C for 1 h. Then, 100 [mu]l of 1/1000 diluted alkaline phosphatase conjugated anti-human IgG (Sigma Chemicals, St. Louis, MO, USA) was added to corresponding wells and incubated at room temperature for 2 h. The color was developed with p-nitrophenyl phosphate (Sigma Chemicals, St. Louis, MO, USA). Each sample was examined in duplicate, then the ratio of optical densities (OD) from the patient sample (P) and negative control (N) were read at 492 nm. On the basis of previous standard5,27, when P/N was more than 2.5 the serum sample was defined to be corresponding antibody positive, less than 2.0 negative and between 2.0 and 2.5 susceptible.

In the present study, eight MG serum samples tested antibodies by ELISA, two of them with antibodies negative both to AChR and PsmR, two only anti-AChR antibody, two only anti-PsmR antibody and two antibodies positive to both AChR and PsmR (Table 1), were chosen to study their effects on membrane currents of motor nerve terminals. Similar observations were performed with six serum samples from healthy donors, all of which were both anti-AChR and anti-PsmR antibody negative of course, as control.

Nerve-muscle preparation

The electrophysiological experiments were performed on intercostal nerve triangularis sterni preparations isolated from adult Kunming mice (18-22g, [male]). The preparations were prepared according to McArdle's method28 and continuously perfused with the modified Kreb's solution at a rate of 2-4 ml min^sup -1^. The solution contains the following (mM), NaCl 1 38, KCl 5, CaCl^sub 2^ 2, MgC^sub 2^ 1, NaH^sub 2^PO^sub 4^ 1, NaHCO^sub 3^ 12 and glucose 10, gassed with 95% O2 and 5% CO2 (pH 7.3). All electrophysiological experiments were performed at room temperature(18-22[degrees]C).

All experiments conformed to the guidelines of the NIH on the ethical use of animals and all experimental procedures were reviewed and approved by the Animal Care and Use Committee of Shanghai Institute of Physiology. All efforts were made to minimize the animal suffering and to reduce the number of animals used.

Perineurial signal recording

As described previously15,22,23, the peri-neurial signal was evoked by stimulating the nerve via a suction electrode and picked up by a 1 M NaCl-filled microelectrode inserted into the subendothelial space of the superficial nerve bundle near the endplate under visual control at 400x magnification using a water immersion 40 Zeiss objective. An Ag/AgCl wire in the bath served as the reference electrode. The signal magnified via a MEZ amplifier (8201 Nikon-Kohden, Japan) was displayed on a storage oscilloscope (Tektronix 5113A, Tektronix, Beaverton, OR, USA USA) and simultaneously digitized (Digidata 1200, Axon Instruments Inc., Foster City, CA, USA), then stored in a computer for further analysis.

A typical peri-neurial signal recorded in Kreb's solution is a predominant double negative peak, which corresponds to inward I^sub Na^ and outward fast K+ current (I^sub K^,^sub f^) in mouse nerve terminals17,29. Besides the I^sub K,f^, there are I^sub K,s^ and Ca^sup 2+^-activated K+ current (I^sub K,Ca^) in the terminals. When I^sub K,f^ and I^sub K,Ca^ are blocked by tetraethylammonium (TEA) chloride, a positive wave is revealed18,19,30. The positive wave is a balance of outward I^sub K,s^ and inward I^sub Ca^, and develops to a Ca^sup 2+^-plateau when I^sub K,s^ is blocked18,30, so we studied the effects on I^sub K,s^ of sera by observing the appearance of Ca^sup 2+^-plateau.

In all experiments, d-Tubocurarine (30-50 [mu]M) was used to abolish post-synaptic activity and procaine (100 [mu]M) to prevent repetitive firing of motor terminals. Neither d-Tubocurarine nor procaine had noticeable effect on membrane currents at the concentration used here22,31. TEA was purchased from Sigma Chemicals, d-Tubocurarine chloride from ICN Biomedicals (Costa Mesa, CA, USA), and procaine hydrochloride from Shanghai Second Pharmaceutical Factory. All other reagents were of analytical grade.

RESULTS

No effect of the anti-PsmR antibody negative sera on I^sub K,s^

As mentioned in Materials and Methods, after I^sub K,f^ and I^sub K,Ca^ were blocked by TEA (30 mM), a positive wave was revealed (Figure 1A). When the signal was stable, serum sample (16%, v/v) was added to the bath solution. No significant change in the positive wave could be found after application of sera from healthy donors or anti-PsmR negative sera from MG patients (see Figure 1B,C,D as examples). These observations showed that the sera without anti-PsmR antibody, whether anti-AChR anti-body positive or negative, did not influence I^sub K,s^ at mouse motor nerve terminals.

Inhibition of I^sub K,s^ by anti-PsmR positive sera

Similar experiments were performed with anti-PsmR antibody positive sera. As shown in Figure 2A, after adding the sera of No. 3984 sample, which was one of two serum samples with both anti-PsmR and anti-AChR antibodies, the duration of the positive wave increased progressively in 1-2 min up to a prolonged Ca^sup 2+^-plateau was developed in 4-5 min. The sera of No. 3702 sample, one of two serum samples with only anti-PsmR antibody, had the similar effect on the positive wave. Moreover, the effect of anti-PsmR antibody positive sera was related to their concentrations (comparing Figure 2Ba with 2Bb). The results show that only anti-PsmR antibody positive serum samples, no matter anti-AChR antibody positive or negative, inhibit I^sub K,s^. The other two anti-PsmR antibody positive serum samples did not cause obvious changes in waveform (data not shown).

In our previous work24, it was observed that a MG patient plasma with anti-PsmR antibody did not affect I^sub Na^. The same results were obtained with eight MG patients' sera mentioned above. Neither anti-PsmR antibody nor anti-AChR antibody positive sera blocked the invasion of action potential into nerve terminals. Thus, the inhibition of transmitter release in MG patient was not due to the failure of action potentials invading nerve terminals.

DISCUSSION

In a previous study, we reported that an anti-PsmR antibody positive MG plasma selectively inhibited the I^sub K,s^ but had no effect on I^sub Na^, I^sub Ca^ and other kinds of I^sub K^ at mouse motor nerve terminals24. In the present study, it was observed that all of 10 anti-PsmR antibody negative sera, in which four samples were from MG patients and others from healthy donors, had no effect on I^sub K,s^. However, two of four anti-PsmR antibody positive MG serum samples inhibited I^sub K,s^ obviously. The data indicate that some factors inhibiting I^sub K,s^ are exclusively involved in anti-PsmR antibody positive MG sera.

In the present study, two of four anti-PsmR positive serum samples showed no effect on I^sub K,s^, which may be concerned with different antigen determinants of PsmR. Although anti-PsmR contained in the two serum samples did not affect the current, its binding to pre-synaptic slow K+ channel may affect neurotransmission presynapticly by other unknown path ways, implying that the structure or functional changes of presynaptic slow K+ channel are involved in the pathogenesis of MG.

For the pathogenesis of MG, increasing evidence has been obtained to support the hypothesis reported by Lu ef al.5 that MG is not only due to the post-synaptic damage produced by anti-AchR autoantibody but also presynaptic damage by anti-PsmR autoantibody. Our finding seems to be consistent with this hypothesis. The results of previous24 and present studies that anti-PsmR antibody positive sera selectively inhibited slow K+ channel indicate that anti-PsmR antibody has presynaptic effects on synaptic transmission, supporting the theory that the presynaptic damage produced by anti-PsmR autoantibody is another pathogenesis of MG besides the damage of AChR.

Moreover, the present results provide an explanation for why some MG patient sera increase frequency of mEPP before decreasing it11. As we know that [beta]-BuTx, a kind of pre-junctional blocker extracted from snake venom, inhibits ACh release from nerve terminals and the inhibition is preceded by a facilitory phase of ACh release10. On the other hand, peri-neurial recording has further showed that [beta]-BuTx inhibited I^sub K,s^ in mouse motor nerve terminals21. PsmR is a protein captured by using [beta]-BuTx and is confirmed to belong to a subclass of voltage gated K+ channel13,14. An inhibition of I^sub K,s^ by anti-PsmR antibody will induce depolarization of membrane and increase frequency of mEPP. The action of anti-PsmR antibody in MG may cause the similar effect as that of blocking ACh release by [beta]-BuTx.

In the past years, we also observed some clinical differences among MG patients with different autoantibodies in their sera in Hua-Shan Hospital27. For instance, the patients with only anti-PsmR antibody often had a shorter course of MG. While the patients with only anti-AChR antibody had a longer one and their MG symptoms tended to recur after steroid therapy. In addition, the curative effects in patients with both kinds of autoantibodies were not as good as in patients with only one. Moreover, it has been observed that some MG patients had anti-PsmR antibody alone in the sera in initial stage of MG, but several months later the anti-AChR antibody was also detected, in final stage only anti-AChR antibody remained. These data indicated that the appearance of autoantibodies, both anti-PsmR and anti-AChR antibodies, were not only involved in the pathogenesis of MG but also corresponding to various stages of MG. We believe that to further analyze the alternation between these two kinds of autoantibodies and the relationship between the alternation and clinical symptoms during the course of MG may advance our knowledge of MG pathogenesis and throw a new light on the therapeutic field.

CONCLUSION

Anti-PsmR antibody existed in some MG patients sera while some MG patients lacked detectable anti-AChR antibody in their sera. Anti-PsmR antibody positive MG sera rather than negative sera could inhibit the slow K+ current at mouse motor nerve terminals. It seemed that through acting on the slow K+ channels anti-PsmR antibodies produced pre-synaptic damage. So besides anti-AchR antibodies which caused postsynaptic damage, anti-PsmR antibodies may be involved in the pathogenesis of MG.

ACKNOWLEDGEMENTS

This work was supported by the National Basic Research program of China (G1999054000) and National Natural Science Foundation of China (39870249, 30170302).

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Tong-Hui Xu, Jun Ding, Yu-Liang Shi, Mu-Feng Li, Chuan-Zhen Lu* and Jian Qiao*

Key Laboratory of Neurobiology, Institute of Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, * Department of Neurology, Hua-Shan Hospital, Fudan University, Shanghai, P.R. China

Correspondence and reprint requests to: Yu-Liang Shi, Shanghai Institute of Physiology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, P.R. China, [ylshi@server.shcnc.ac.cn] Accepted for publication September 2002.

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