EXPERIMENTAL

NEUROLOGY

64,

428-444

(1979)

Effects of Antibodies to Torpedo Acetylcholine Receptor on the Acetylcholine Receptor-Ionic Channel Complex of Torpedo Electroplax and Rabbit Intercostal Muscle E. ELDEFRAWI, DAVID S. COPIO, C. SUE HUDSON, JOHN RASH, NABIL A. MANSOUR, AMIRA T. ELDEFRAWI, AND EDSON X. ALBUQUERQUE~

MOHYEE

Department

of Pharmacology School Received

and Experimental of Medicine, Baltimore,

August

3, 1978;

revision

Therapeutics, Maryland received

University 21201

October

of Maryland

17, I978

An electrophysiological, biochemical, and ultrastructural study of the acetylcholine (ACh) receptor-ionic channel complex was undertaken in electric organ membranes from the electric ray, Torpedo ocellata, and intercostal muscle of the rabbit. Rabbits immunized with receptor purified from Torpedo ocellata were paralyzed as is characteristic of experimental autoimmune myasthenia gravis. Incubation of Torpedo membranes with antisera from six paralyzed rabbits, but not control rabbits, resulted in inhibition of [3H]ACh and [rz51]rx-BGT binding by 19 to 95% and 43 to 86%, respectively, and did not significantly affect the binding of [3H]H,2-HTX. Anti-ACh-receptor antibodies bound to Torpedo microsacs were identified in electron micrographs by specific binding of ferritin-labeled goat anti-rabbit antibodies. Treatment of Torpedo microsacs with ACh-receptor antiserafrom rabbits inhibited their carbamylcholine-induced **Na efflux, which would suggest direct effect of the antibodies on Torpedo ACh-receptor function. HowAbbreviations: ACh-acetylcholine, ACh-esterase-acetylcholinesterase, a-BGTalpha-bungarotoxin, EAMG-experimental autoimmune myasthenia gravis, MEPPminiature end-plate potential, H,,-HTX-perhydrohistrionicotoxin, DFP-diisopropylfluorophosphate. 1 We are grateful to Drs. John Daly and Bernhard Witkop for providing the [3H]H,,HTX used in the present study. This study was supported in part by National Institutes of Health grant NS-12063 (to E.X.A.), National Science Foundation grant BNS76-21683 (to M.E.E.), and grants from the Muscular Dystrophy Association of America (to E.X.A., M.E.E., and J.E.R.) and the Pangborn Fund (to C.S.H.). Mr. Copio is now at Washington University, St. Louis and Dr. Mansour is at the University of Alexandria, Egypt. 428

0014-4886/79/050428-17$02.00/O Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

ACETYLCHOLINE

RECEPTOR

ANTIBODIES

429

ever, addition of the same anti-Torpedo-ACh-receptor antisera for 240 min to intercostal muscles of rabbits produced only a small reduction in the amplitude of the response to microiontophoretically applied ACh, miniature end-plate potentials, and end-plate potentials. It is suggested that the muscle weakness observed in rabbits immunized with Torpedo ACh-receptor protein may be due to time-requiring action on receptors such as their accelerated degradation, and the amount of antimuscle receptor in rabbit antisera against Torprdo receptor may be too small.

INTRODUCTION Acetylcholine (ACh)-receptors purified from the electric organs of the electric eel, Electrophorus electricus, or the electric ray, Torpedo sp., induce an immune response when injected into experimental animals. The immunized animals develop antibodies, which react with and precipitate the receptor protein from detergent extracts of ACh-receptor-containing membranes (26,30,34,37). In addition, the animals produce lymphocytes which yield positive skin tests when challenged intradermally with AChreceptor (30). This immune response, commonly designated “experimental autoimmune myasthenia gravis” (EAMG), is accompanied by muscular weakness and inability of the animal to stand unsupported. Several studies were initiated to pursue the underlying biochemical mechanisms of the effects of anti-ACh-receptors (10, 18, 26, 27, 30, 35 37). Most of these studies relied on the precipitation of an 1251-labeled (Ybungarotoxin-ACh-receptor antibody complex and led to the suggestion that almost all antibodies in the immunized animal’s serum were directed against determinants on the ACh-receptor other than the ACh or CYbungarotoxin (wBGT) binding sites (18, 26, 27, 30). In a few studies, the effect of antisera on the actual binding of ACh and/or a-BGT to the AChreceptor was studied, and the observations made ranged from the absence of any inhibition (IO) to partial (26) or total inhibition (35, 37). Immunization of animals with the purified ACh-receptor presumably results in the production of several populations of antibodies, directed at different determinants on the receptor molecule. Because inhibition of ACh binding by antisera might be due to precipitation of the detergentsoluble receptor, an action that is of no significance for receptor function in situ, a better index of direct in situ action is to study the effect of antisera on binding of ACh to the membrane-bound ACh-receptor and to compare the effect of the same sera on the intercostal muscle of a normal rabbit. Thus, the present study was initiated to determine the effect of anti-AChreceptor antibodies from paralyzed EAMG rabbits on the binding of [3H]ACh and 1251-labeled wBGT to membrane-bound Torpedo AChreceptors. Because a reduction in junctional ACh sensitivity and miniature

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ELDEFRAWI

ET AL.

end-plate potential (MEPP) may be due to a decrease in the total number of ACh-receptor-ionic channel complexes, or the number of active ones present, it was important to establish if the carbamylcholine-induced 22Na efflux from Torpedo microsacs was affected by preexposure in vitro to antiACh-receptor antisera. To examine if these antisera interfered with ionic flux by interacting directly with the ionic channel of the ACh-receptor, we studied their effects on the binding of [3H]perhydrohistrionicotoxin [3H]H12-HTX), a toxin that binds with high affinity to these ionic channels (12) [also called “ion conductance modulators” (1, 12) and “ionophores” (S)]. Our third objective was to study the effect of the sera of the diseased animals on the chemosensitivity of the neurally and iontophoretically evoked ACh potentials at the junctional region of the intercostal muscle of the rabbit. In addition, an electron microscopical study was carried out to determine whether or not receptor antibodies could be visualized and unambiguously identified as discrete structures bound to these ACh-receptor-enriched Torpedo membranes. MATERIALS

AND METHODS

Production of Antisera to PuriJed ACh-Receptors. ACh-Receptor was purified from Torpedo ocellata by affinity chromatography as previously described (14). Solutions (0.3 ml) containing 75 pg receptor protein were emulsified in equal volumes of Freund’s complete adjuvant (Difco Laboratories) and injected subcutaneously into paraspinal sites of New Zealand rabbits. At 41 days postinjection each rabbit was “boosted” with another 25 pg emulsified ACh-receptor. Within 7 days, all rabbits developed flaccid paralysis, at which time blood samples were collected by either arterial or cardiac puncture. Sera were isolated by centrifugation of clotted blood 10 min at 700 g and 4°C and were stored at -20°C until use. A micromethod for Ouchterlony double diffusion (33) was used to test for the presence of precipitating antibody in the sera. Control rabbits were injected and boosted with 5 mM Tris (pH 7.4) emulsified in Freund’s complete adjuvant. No weakness was noted in the controls. Preparation of Membrane Fragments (Microsacs) of Torpedo Electropfax. The frozen electric organ (50 g) was homogenized 2 min in 5 vol phosphate-buffered saline (90 mM KCI, 10 mM NaCI, 5 mM N%HP04, pH 7.2) in a Waring Blendor at maximum speed. It was then centrifuged 10 min at 5000 g and the supernatant fraction recentrifuged 60 min at 30,000 g . The resultant pellet was resuspended in Krebs original Ringer’s phosphate solution (107 mM NaCI, 5 mM KU, 0.65 mM CaCI,, 1.23 mM MgS04, 16 mM Na,HPO,), where the membrane formed microsacs. The final protein concentration, as determined by the method of Lowry et al. (31), was 1 to

ACETYLCHOLINE

IO

20

RECEPTOR

30

431

ANTIBODIES

60

90

TIME (mid

FIG. 1. The inhibition of 1*51-labeled a-BGT binding to microsacs by pretreatment with antiacetylcholine receptor antiserum from rabbit 2. Microsacs used: control (O), treated with control rabbit serum (A), treated with undiluted rabbit anti-ACh-receptor antiserum (m), preexposed 30 min to 50 pM d-tubocurarine (0). Vertical lines represent kstandard deviation of three experiments.

2 mg/ml. Maximum number of binding sites for [3H]ACh and r3H]H,,HTX was 0.7 and 0.5 nmol/mg proteins, respectively. Microsacs were mixed with an equal volume of sera and incubated 90 min at 21°C. Diisopropylfluorophosphate (DFP) was added to the mixture (final concentration of 0.1 mM) for 60 min to inhibit acetylcholinesterase (ACh-esterase) activity in the serum and Torpedo membranes. A loo-fold volume of Ringer’s solution was added and the mixture centrifuged 60 min at 30,000 g . This procedure was repeated and the pellet then resuspended in Ringer’s solution. Binding of Radiolabeled Ligands. Binding of [acetyk3H]ACh (49.5 Ci/ mol, New England Nuclear) to the microsacs was determined by equilibrium dialysis for 4 h at 21°C as previously detailed (12). ACh-Esterase in the microsac preparation was inhibited by incubation 1 h with 10F4 M DFP prior to dialysis. (w-BGT was isolated from the venom of Bungarus multicinctus (Miami Serpentarium) by ion-exchange chromatography on Sephadex CM 50 using ammonium acetate (pH 6.5) gradient and labeled with lz51 as previously described (16). The specific activity was 32 Wmmol.

432

ELDEFRAWI

PH]

ET AL.

ACh CONCENTRATION

(fit4

FIG. 2. Inhibition of [3H]ACh binding by pretreatment with anti-ACh-receptor antiserum from rabbit 2. Microsacs used: control(O), treated with control rabbit serum (A), treated with IO-fold diluted rabbit anti-ACh-receptor antiserum (II), treated with undiluted rabbit antiACh-receptor antiserum (m). Vertical lines as in Fig. 1.

The binding of 1251-labeled a-BGT was determined by a filter assay using Whatman GF/C glass fiber filters. To 1 ml tissue mixture, typically containing 0.014 mg protein/ml, 1251-labeled a-BGT was added to reach a final concentration of 50 nM. After exposure of microsacs to toxin for various lengths of time at 21°C unlabeled a-BGT (final concentration 1 PM) was added to stop the reaction. To reduce nonspecific lz51-labeled (r-BGT binding to the glass filters, each 200~~1 sample was mixed with an equal volume normal rabbit sera, the mixture filtered, and the filter washed with 20 ml cold Ringer’s solution. Filters were counted in a Packard 5230 auto gamma scintillation spectrometer. Each experiment was repeated three times using triplicate samples. [3H]H,,-HTX (sp act 4.8 CYmmol) was obtained by tritiation of isodihydrohistrionicotoxin, its activity tested on frog sartorius muscles as described (1 l), and its binding ( lOpa to 10e6 M) to the microsacs measured by equilibrium dialysis 4 h at 21°C (12). EfJEux of 22Nu. The method used for measuring receptor-mediated efflux of 22Na was that developed by Kasai and Changeux (24) and modified by Hess et al. (21) with a few more changes of our own (39). According to our procedure, carbamylcholine was added only after most of the nonspecific 22Na efflux ended and a steady efflux state was reached, thereby minimizing interference from nonspecific 22Na efflux. Electrophysiological Techniques. Conventional microelectrode tech-

ACETYLCHOLINE

RECEPTOR

ANTIBODIES

433

pl ANTISERA /ml MICROSAC PREPARATION

FIG. 3. Inhibition of [$H]ACh (0) and 12SI-labeled wBGT (m) binding by anti-ACh-receptor antiserum as a function of serum concentration from rabbit 2. The d-tubocurarine (dTC)insensitive (Y-BGT binding was measured in the presence of 50 pM dTC and subtracted from both control and experimental (i.e., antiserum-treated microsacs) values to determine the dTC-sensitive binding. Vertical lines as in Fig. 1.

niques were used for intracellular stimulation and recording from intercostal muscles (3). In the studies using microiontophoretic application of ACh, high-resistance pipets (250 to 450 Ma), filled with 2.5 M ACh, were used at the junctional and extrajunctional regions of the normal and seratreated animals. The duration of the cathodal current pulse varied from 0.5 to 1.O ms and this current pulse was superimposed on a low constant anodal braking current. When an increase in charge applied to the high-resistance pipets was required, precautions were taken to avoid capacitance leakage. The end-plate region of the normal and sera-treated muscles was localized visually and electrophysiologically by recording spontaneous MEPPs with fast rise times (less than 1.0 ms). Quanta1 content and size were determined from amplitudes of groups of 20 EPPs analyzed with the aid of a PDP-11 digital computer. Voltages were amplified by a Tektronix oscilloscope and sampled at a rate of 100 mslpoint using an analog-to-digital converter. EPP amplitudes were corrected for nonlinear summation of unit response (32). The average quanta1 content of the nerve-evoked EPPs was calculated by Poisson analysis of the last 180 EPP amplitudes of EPPs elicited by a 200impulse, l-Hz train delivered to the sciatic nerve (11, 22). The procedure used in this study was first to calculate the quanta1 content of EPPs in sequential groups of 20 and then to average those individual estimates. The effect of normal and EAMG sera on the end-plate was studied at 28 to 33°C. Electron Microscopic Analysis. To assess the relative proportion of ACh-receptor-containing microsacs, 5 ml serum from control or immunized rabbits was added to an equal volume of standard microsac preparation and incubated 3 h at 21°C. The membranes were then sepa-

434

ELDEFRAWI

ET AL.

TABLE

1

Inhibition of Binding of Labeled Acetylcholine ([3H]ACh) (10m6 M) and Bungarotoxin (1251-labeled (Y-BGT) (lo-@ M) to Torpedo Microsacs by Preincubation with Sera from Rabbits with Experimental Antoimmune Myasthenia Gravis Compared to Control Sera. Inhibition Rabbit No.

[3H]ACh

(%) of binding 1251-labeled a-BGT

Control sera 5 9 15 16

-4.gn 2.6 -0.5 -5.7

f + + k

9.1 3.1 5.0 2.9

5.5 11.0 0.9 4.7

-c k 2 r

13.2 5.5 6.8 6.4

Immunized sera 1 2 3 8 11 13

18.8 95.5 62.2 40.0 65.0 60.7

? f f 2 2 f

1.9 1.6 2.0 6.9 3.1 2.7

42.5 86.2 86.1 57.2 51.9 64.4

f k + f -c +

6.9 1.9 0.9 2.6 3.8 1.4

a A negative value reflects an increase in binding.

rated by centrifugation 30 min at 30,000 g. Each pellet was homogenized in 5 ml Ringer’s solution and divided into two aliquots. One control and one experimental sample were rinsed and centrifuged three times at 30,000 g for 30 min. The other control and experimental samples were incubated 8 h at 4°C with a 1500 dilution of ferritin-conjugated goat-anti-rabbit IgG (Cappel Laboratories, Downingtown, Pennsylvania). These preparations were pelleted, washed twice by suspension in Ringer’s, and repelleted three times by centrifugation 30 min at 30,000 g. The four pellets were fixed in cold 2.5% glutaraldehyde buffered in Ringer’s, postfixed in buffered 1% OsO1, stained in 0.5% aqueous uranyl acetate, dehydrated in an ethanol series, and embedded in plastic mixture (10% Epon 812, 20% Araldite 6005, 70% dodecanyl succinic anhydride plus 1.5% DMP-30 as catalyst). Sections were cut on a Porter-Blum MT II ultramicrotome using a diamond knife, picked up on 200-mesh grids, and viewed on a Siemens Elmiskop 101 using either a standard specimen holder or a &24”C double-tilt device. RESULTS Binding Studies. ACh-Receptor-containing Torpedo membranes incubated with lz51-labeled a-BGT revealed a linear relationship between 1251-labeled a-BGT binding and protein concentration. The specificity of

ACETYLCHOLINE

RECEPTOR

20

Microrocr

ANTIBODIES

435

+

8 Conlrol SW0 0 Control sero + ‘Garb 0.1mM x Control MO + dTC 0.1 mH + Cmb 0.1mY 0.1 mu 4 ACh-receptor antiwa+Corb

Carb

TIME

in minute8

FIG. 4. Effect of rabbit anti-ACh-receptor antiserum on the efflux of *2Na from Torpedo microsacs. Zero time represents the time at which microsacs that had been incubated with ZZNaCl were diluted 200-fold with buffer. Samples were taken at various times afterward, filtered on HAWP 0.45~pm Millipore filter, and rinsed twice, and their radioactivity on the filters was counted. Vertical bars represent *standard deviation of three experiments. Carb-carbamylcholine, dTC-d-tubocurarine.

toxin binding was confirmed by inhibition with d-tubocurarine (Fig. 1). Antibodies to ACh-receptor, as detected by a single sharp precipitation arc with the Ouchterlony double diffusion technique, were present in the sera of all rabbits immunized with the purified ACh-receptor but were never observed in control sera. The titer of antibodies against Torpedo receptor was between 10 and 30 Fmol cu-BGT binding sites precipitated per liter of serum quantitated as described (30). In measuring the effect of immune sera on the binding of lz51-labeled a-BGT or [3H]ACh to microsacs, 20- to 40-fold excess antibodies to receptor sites were used. Binding of 1251-labeled a-BGT to microsacs was inhibited by pretreatment with immunized rabbit serum, but pretreatment with control serum had no measurable effect (Fig. 1). Microsacs preexposed to immunized rabbit sera greatly inhibited the binding of [3H]ACh to Torpedo microsacs (Fig. 2). As the amount of serum added to the microsac preparation was increased, an increase in

ELDEFRAWI

436

ET AL.

A

jl

5oc

400 is 0 2 2 w

3oc

Q 0” 8 g

2oc

2

100

AMPLITUDE

(mV)

FIG. 5. Distribution of amplitudes of the spontaneous miniature end-plate potentials recorded from the intercostal muscle of the rabbit, in control condition (A) and after exposure 120 to 200 min to sera (IgG) from immunized rabbits (B). Temperature 28 to 33°C.

the inhibitory effect on both a-BGT and ACh binding was observed (Fig. 3), giving 50% inhibition with 20-fold diluted antiserum. All antisera tested inhibited significantly binding of ACh and (Y-BGT, whereas control sera did not (Table 1). Binding of [3H]H,,-HTX (40 nM) to microsacs treated with anti-AChreceptor antiserum was not significantly different from its binding to microsacs treated with control serum, suggesting that the anti-ACh-receptor antibody binding site(s) is (are) different from the HI,-HTX binding sites. 22Na EfjYux Studies. The carbamylcholine-induced efflux of 22Na from Torpedo microsacs has been established as a biochemical correlate of in siru electrophysiologic events (15,21,24,39). The 0.1 mM carbamylcholineinduced 22Na efflux from Torpedo microsacs was unaltered by pretreatment with sera from control rabbits. This receptor-activated 22Na efflux was inhibited 30 -+ 7% (N = 3) by the presence of 0.1 mM d-tubocurarine and 60 k 11% (N = 3) by pretreatment of the microsacs with sera from the moribund rabbits (Fig. 4). Effect of Sera (IgG) from the Immunized

Rabbits on the Chemosensi-

ACETYLCHOLINE

RECEPTOR TABLE

437

ANTIBODIES

2

Effect of Myasthenic Rabbit Serum on Membrane Potential, Acetylcholine (ACh) Sensitivity, and Miniature End-Plate Potentials (MEPPs) of the Normal Intercostal Muscle of the Rabbit Time 0 60 120 180 240 The amplitude no significant n Numbers * Numbers

Resting membrane potential -78 -83 -74 -80 -78

t 2 k t rt

2.3 (6) 1.6 (7) 3.1 (15) 2.0 (12) 2.0 (13)

ACh sensitivity (mV/nC) 3173 3297 3011 2315 1976

f c t + +

633 (4) 511 (3) 430 (8) 611 (7) 827 (8)

MEPP frequency 0.97 0.83 0.91 0.86 0.60

k 2 t f +

0.02 0.30 0.06 0.05 0.10

(400)b (400) (730) (810) (905)

of the ACh potential was not adjusted for loss of membrane potential because depolarization was recorded. in parentheses represent numbers ofjunctions assessed and potentials recorded. in parentheses represent numbers of single potentials recorded.

tive Properties of the Innervated Middle-Layer Intercostal Muscle of the Rabbit. Serum (25 ml) from moribund rabbits, diluted with Ringer’s solu-

tion (15 ml), was applied to the intercostal muscle. After exposure to the serum for periods varying from 60 to 120 min the amplitude, frequency, and time course of the spontaneous MEPPs remained unaltered (Fig. 5 and Table 2). Examination of the histogram in Fig. 5 shows a marked similarity between control (A) and 120 min serum-treated (B) muscles. The medians for both A and B records shown in Fig. 5 were identical, but after prolonged exposure (about 240 min) to the sera a slight shift to the left was observed, indicating a decrease in MEPP amplitude. In addition a slight (10%) increase in amplitude and frequency of the MEPPs was observed in some cells after 60 min of exposure to the serum. Exposure to the serum for 60 to 120 min did not affect the amplitude of the EPP. When treatment with serum was extended to 240 min, EPP amplitude was reduced 10% in 47 of 52 single end-plate recordings. After 180 to 240 min incubation with EAMG serum, a slight decrease in junctional ACh sensitivity was noted. It should, however, be noted that this estimate was not sufficiently low to really indicate that the serum had an inhibitory effect on AChreceptors. The decrease seen both in MEPP amplitude and in ACh sensitivity after more than 240-min exposure to the serum may reflect an effect of the serum on the junctional region, because using control serum alone produced an effect similar to that seen with EAMG serum in two of five cases. Electron Microscopic Studies. Microsacs incubated with control sera exhibited smooth membrane surfaces devoid of attached material (Fig. 6a), whereas approximately 5 to 20% of the microsacs incubated with antisera exhibited a “fuzzy coat” of 30 x 70 A particles presumably attached to

438

ELDEFRAWI

ET AL.

FIG. 6. Electron micrographs of a--Torpedo electroplax membranes treated with control rabbit serum appear free of any attached material. b--Torpedo electroplax membranes, reacted with experimental autoimmune myasthenia gravis rabbit anti-ACh receptor immunoglobulin G (IgG), exhibit a layer of attached material (arrows). c-To determine the degree of nonspecific ferritin binding, membranes were reacted with control rabbit serum and then with ferritin-conjugated goat IgG directed against rabbit IgG. Some ferritin is observed between microsacs, but no ferritin label is apparent on the membranes. d-Microsacs were reacted with rabbit anti-ACh-receptor IgG and then “sandwich labeled” with the ferritinconjugated goat anti-rabbit IgG. Five to twenty percent of the microsacs exhibited ferritin granules (arrows) bound to the layer of attached material on the membranes. Thus, the number of labeled microsacs approximates the amount of ACh-receptor-containing membrane in the preparation. All magnifications as in d.

ACh-receptor-containing membranes (Fig. 6b). Microsacs treated with control sera (lacking antireceptor IgG) followed by ferritin-conjugated goat anti-rabbit immunoglobulin were not labeled with ferritin. Because unattached ferritin was present in areas between microsacs (Fig. 6c), the

ACETYLCHOLINE

RECEPTOR

ANTIBODIES

439

demonstrated lack of ferritin labeling of control microsacs was not due to penetration failure or to excessive washing. Furthermore, the extremely low level of nonspecific ferritin “trapping” by the very sparse basal lamina in control preparations confirms the effectiveness of the rinse procedure. However, antisera-treated microsacs incubated with ferritin-conjugated goat anti-rabbit immunoglobulin exhibited numerous attached ferritin granules (Fig. 6d), thereby clearly establishing that the “fuzzy coat” was composed of a layer of attached rabbit IgG and that the labeled microsacs contained ACh-receptors. For more detailed information on labeling of ACh-receptor-containing membranes from myasthenic humans, EAMG rabbits, and Torpedo electroplax see (36). DISCUSSION Two main observation are made in the present study. The first is that sera from rabbits paralyzed as a result of immunization with Torpedo AChreceptor contain antibodies that inhibit to variable degrees ACh and oBGT binding to its Torpedo ACh-receptor. The second is that these EAMG sera inhibit the receptor function in Torpedo membranes (i.e., carbamylcholine-induced 22Na efflux from Torpedo microsacs), yet they cause little or no inhibition of neuromuscular transmission when applied to normal rabbit intercostal muscle at 28 to 33°C. On the other hand, we found that the moribund rabbit had reduced EPP and junctional ACh sensitivity (unpublished results). Our finding of inhibition of a-BGT binding to the membrane-bound ACh-receptor by sera from several EAMG rabbits contrasts with the finding of no or little (12%) inhibition observed in a rabbit immunized with Torpedo ACh-receptor or with Electrophorus ACh-receptor, respectively (23). However, the inhibition of toxin binding observed agrees with that reported on sera from goats immunized with Electrophorus ACh-receptot-, which inhibited 51% of lz51-labeled a-BGT binding to membranebound ACh-receptors in Electrophorus electroplax (27). These differences may be due to one or more variations between individual rabbits, state of the disease, antigens, or method of testing. It is anticipated that of the many antibodies produced against the purified ACh-receptor antigen, only a few may be directed against the active sites. The ratio of total precipitating antibodies to active-site-inhibiting antibodies was estimated at 80: 1 in one case (18) and between 7: 1 and 20: 1 in another (25). This ratio appears to change in the same animal as Zum and Fulpius (41) showed that in one rabbit the ratio of antibodies directed against the toxin-binding site increased markedly just before the appearance of paralysis. The active-site-inhibiting antibodies, which are detected by their inhibition of [3H]ACh binding to membrane-bound ACh-receptors, vary in the degree of their inhibition from 18.8 to 95.5% (Table 1) in

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ET AL.

sera from moribund animals. This suggests that these active-site-inhibiting antibodies may not be important for the development of paralysis, although they may contribute to it. It supports the earlier proposals that pathogenesis of EAMG is due mainly to the speeding of ACh-receptor metabolism caused by binding of antibodies (19). Direct evidence that inhibition of ACh binding to the ACh-receptor is unnecessary for pathogenesis of EAMG is that immunization of rats with any of the four immunologically distinct Torpedo ACh-receptor subunits, even the ones that do not carry the ACh binding site, induces voluntary muscle paralysis (29). It is not surprising to find that the rabbit antisera did not inhibit [3H]H,2HTX binding to the ionic channel of the ACh-receptor, because the antigen used (i.e., Torpedo ACh-receptor) did not bind any [3H]H,,-HTX and might be void in the ionic channel component. In addition, the binding sites for [3H]H,,-HTX in Torpedo membranes may not be accessible to antibody action. Most antisera show similar inhibition of ACh and (Y-BGT binding (Fig. 3), whereas some (from rabbits 1 and 3) show distinct differences in their abilities to inhibit the two ligands (Table I). This variability occurs despite the fact that all rabbits were inoculated with the same antigen and schedule, and the sera were collected when the animals were paralyzed. The disparity may result in part from differences in the molecular weight of ACh and a-BGT. For example, it was found that antibodies to an enzyme with various substrates had greater inhibitory effect on reactions with substrates of higher molecular weight (7). An alternative explanation is that among the antibody populations, one affects the ACh binding site, and another affects the a-BGT binding site, which are then assumed to be partially or totally separate sites (6, 13, 17). Using sera from animals which showed high titers of anti-ACh-receptor antibodies, we were able to verify previous studies using human myasthenic sera on normal human muscle, i.e., no alterations in the electrophysiologic properties of neuromuscular transmission of rabbit (Fig. 5) and human (2) intercostal muscles were seen when Torpedo receptor antibodies and myasthenic sera were added to the muscles, respectively. Nevertheless, intercostal muscle end-plates of myasthenic patients (4) and EAMG rabbits (Albuquerque, unpublished) were found to have low junctional ACh sensitivity. In addition, preliminary studies of the spectral analysis of the noise produced by iontophoretic application of the ACh disclose no change in the elementary single channel conductance and life time (Albuquerque et al., unpublished). This is similar to the finding in human myasthenic muscle where no change in the characteristics of the single channel were noted even though ACh sensitivity was reduced (9). However, it is different from the effect of antibodies against eel receptor

ACETYLCHOLINE

RECEPTOR

ANTIBODIES

441

when applied to cultures of myoblasts (19). The number of ACh-receptors was reduced, but the remaining ones exhibited reduced mean channel conductance and open time by 15% and 23%, respectively. There is an apparent discrepancy between the biochemical and the electrophysiologic data. Torpedo receptor antibodies, when added to rabbit intercostal muscle, do not inhibit neuromuscular transmission (Table 2 and Fig. 5), yet these same antibodies inhibit [3H]ACh binding to Torpedo ACh-receptor (Table 1 and Figs. 2, 3), bind to these receptors (Fig. 6), and inhibit their carbamylcholine-induced 22Na efflux (Fig. 4). There is a difference between studying the muscle of the immunized animal and studying the muscle from a control animal after antisera from another animal are applied to it in vitro. In the first case, the muscle may have less receptors present because of accelerated breakdown of receptors caused by the circulating antibodies. This is a time-requiring process that takes possibly one or more days. In the second case, only if homologous antibodies reach the ACh-receptors in the end plate and directly inhibit binding to the receptor’s active site or the receptor’s function will any effect be observed. This process takes one or more hours. Because AChreceptors in the end plate are accessible to antibodies (40), there are two factors, one or both of which may explain this apparent discrepancy. One may be related to the homology between antibodies and receptor. Rabbit antibodies against Electrophorus (23, 35, 38), but not Torpedo AChreceptors (24), were found to inhibit the carbamylcholine-induced depolarization in Electrophorus electroplax. It has also been estimated that the titer of antibodies against extrajunctional ACh-receptors in EAMG rats (induced by Electrophorus ACh-receptor) could be as low as only l/1,000 that of antibody to Electrophorus ACh-receptor (27). Although we did not test our immune sera for antibodies against muscle AChreceptors, it has been reported that sera from rabbits immunized with Torpedo receptor contain antibodies that react 100% with Torpedo receptor and only 0.22 to 0.5% with mammalian muscle receptors (28). The second factor is the local concentration of ACh to which the Ach-receptor-antibody complex is exposed. In the binding study, [“H]ACh is used at 1 pM, and iontophoretically or neurally evoked ACh may reach much higher concentrations in the synaptic gap. Such a high concentration might compete with some antibodies for the ACh binding site on the receptor and displace these active-site-inhibiting antibodies, thereby restoring neuromuscular transmission to normal. High concentrations of a specific ligand have been utilized to separate antibodies that are bound to antigenic determinants at the active sites of phosphorylcholine-binding mouse myeloma protein (8). Other antibodies that are not bound to the active sites may then cause acceleration of receptor degradation and other changes that end in paralysis of the immunized animal (20).

442

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In summary, different antibodies appear to be produced in rabbits against Torpedo ACh-receptor, some of which inhibit its binding of ACh and/or a-BGT to different degrees. This inhibition varies among sera from moribund rabbits. These antibodies may inhibit receptor function in Torpedo microsacs, as exemplified by carbamylcholine-activated 22Na efflux, though they have no apparent effect on neuromuscular transniission when applied to normal rabbit muscle, a discrepancy that may be explainable. We believe that paralysis in moribund EAMG rabbits (which is an acute phase) and myasthenia gravis (which is mostly a chronic human disease) is due to inhibition of neuromuscular transmission mainly because of reduced numbers of ACh-receptors, although inhibition of ACh binding may contribute to the paralysis. It is necessary to study in more detail the molecular interaction of EAMG serum with its targets, to identify the principal units which are active in producing the inhibition of ACh and a-BGT binding, and to understand more about the factors involved in degeneration of the synaptic folds of immunized rabbits. REFERENCES 1. ALBUQUERQUE, E. X., E. A. BARNARD, T. H. CHIU, A. J. LAPA, J. 0. DOLLY, S. JANSSON, I. DALY, AND B. WITKOP. 1973. Acetylcholine receptor and ion conductance modulator sites at the murine neuromuscular junction: Evidence from specific toxin reactions. Proc. Natl. Acad. Sci. U.S.A. 70: 949-953. 2. ALBUQUERQUE, E. X., F. J. LEBEDA, S. H. APPEL, R. ALMON, F. C. KAUFFMAN, R. F. MAYER, T. NARAHASHI, AND I. Z. YEH. 1976. Effects of normal and myasthenic serum factors on innervated and chronically denervated mammalian muscles. Ann. N.Y. Acad. Sci. 274: 475-492. 3. ALBUQUERQUE, E. X., AND R. J. MCISAAC. 1970. Fast and slow mammalian muscles after denervation. Exp. Neurol. 26: 183-202. 4. ALBUQUERQUE, E. X., J. E. RASH, R. F. MAYER, AND J. R. SATTERFIELD. 1976. An electrophysiological and morphological study of the neuromuscular junction in patients with myasthenia gravis. Exp. Neurol. 51: 536-563. 5. BON, C., AND J.-P. CHANGEUX. 1977. Cernleotoxin: a possible marker of the cholinergic ionophore. Eur. J. Biochem. 74: 43-51. 6. BULGER, J. E., J.-J. L. Fu, E. F. HINDY, R. L. SILBERSTEIN, AND G. P. HESS. 1977. Allosteric interactions between the membrane-bound acetylcholine receptor and chemical mediators. Kinetic studies. Biochemistry 16: 684-692. 7. CINADER, B. 1%7. Antibodies to Biologically Active Molecules. Oxford, Pergamon Press. 8. CLAFLIN, J. L., AND J. M. DAVIE. 1975. Specific isolation and characterization of antibody directed to binding site antigenic determinants. J. Zmmunol. 114: 70-75. 9. CULL-CANDY, S. G., R. MILEDI, AND A. TRAUTMAN. 1978. Acetylcholine-induced channels and transmitter release at human endplates. Nature (London) 271: 14-15. 10. DAMLE, V., S. HAMILTON, R. VALDERRAMA, AND A. KARLIN. 1976. Binding properties of acetylcholine receptor in membrane from Torpedo electric tissue. The Pharmacologist IS: 146. 11. DEL CASTILLO, J., AND B. KATZ. 1954. Quanta1 components of the end plate potential. J. Physiol. (London) 124: 560-573.

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