INHIBITORY EFFECTS OF MYASTHENIC SERUM ON THE SYNTHESIS OF ACETYLCHOLINE IN THE RAT BRAIN

Folia Psychiatrica et Neurologica Japonica, Vol. 22, No. 1, 1968

INHIBITORY EFFECTS OF MYASTHENIC SERUM ON THE SYNTHESIS OF ACETYLCHOLINE

IN THE RAT BRAIN

BY

Takashi MATSUYAMA, M.D., Kazuyasu NAKAO, M.D., Shizuo YOSHIDA, M.D., Toru UOZUMI, M.D. and Kenzo MATSUOKA, M.D. The First Department of Surgery, Osaka University,

School of Medicine, Osaka, Japan

INTRODUCTION

In 193416), Nevin reported the so called “myasthenic substance” which is contained in the serum of a patient with myasthenia gravis and disturbs the transmission of nerve impulses in the neuromuscular junction. This substance has since been investigated by Walkerze), Wilsonz’), Gross-Brockhoffll) and many other investigatorszo.zz.~3.24.2s). Although several systematic works have been made on the functional mechanism of this substance, no definite conclusion has been reached as to the problem whether this substance will interfere with the neuromuscular transmission by affecting the presynaptic site, by affecting the postsynaptic site and acting like curare which antagonizes acetylcholine(Ach), or by some other functional mechanism.

While Struppler22.23), Wilson27) and Tsukiyama60) regarded the so-called “myasthenic substance” as a curare-like one which affects postsynaptic transmission, Windsorzs) suggested, in his preliminary report, that this substance has the effect of a depolarizing block on the neuromuscular junction and not a curare-like effect. Minot,ls), Desmedt4-7) et a12.3,9J3J4-17) suggested an inhibitory effect on the synthesis and the release of Ach in the presynaptic site on the basis of electrophysiological experiments but the evidence seems to be insufficient.

We attempted to confirm whether the so-called “myasthenic substance” in serum has inhibitory effects on the synthesis of acetylcholine and affects the neuromuscular transmission at a presynaptic site.

However, as it is impossible to examine the possible inhibitory effect of the myasthenic patient’s serum directly on the human neuromuscular junction, we conducted an experimental study using cerebral cortex slices obtained from rats in the attempt to elucidate whether the myasthenic patient’s serum may have an inhibitory action on the Ach synthesis induced after Quastel’s method’s).

MATERIALS AND METHODS

The present experiment was carried out on 23 patients with myasthenia gravis; 15 cases of the generalized and 8 cases of the ocular type. As controls, the serum from 24

Received for publication March 9, 1968

56 T. Matsuyama, K. Nakao, S. Yoshida, T. Uozumi and K. Matsuoka

normal subjects was examined. The serum examined was obtained early in the morning from the cubital vein,

incubated at 37°C for 30 minutes, and centrifuged at 2,00Or/min. for 10 minutes. Then, the resulting supernatant fluid was separated.

For evaluation of the effect of muscle exercise, a tourniquet was applied to the upper arm after collecting 10 ml. of blood from the cubital vein and the patient then was made to grasp a hand-dynamometer repeatedly 100 times with a pressure slightly higher than the systolic given on the tourniquet and blood was immediately collected and serum separated. 1) Method of synthesizing Ach10J*,21),

The brain of male rats (Donryu strain) was removed and the dura mater and the visible vessels cleaned away. The brain was washed with cooled Krebs-Ringer phosphate saline (KRPS) containing 0.1 mg/ml eserine sulfate and 10 mM of glucose. Slices of the cerebral cortex were prepared on a cooled glass plate. Fifty to 70 mg. of the slice was incubated for 120 minutes in 3 ml. of KRPS under an aerobic condition at 37°C while shaking 80 times per minute. The solution was separated from the slices by centrifuga- tion. The Ach content of the supernatant fluid was evaluated as the free Ach content.

Slices were homogenated after addition of 3 ml. of Frog Ringer solution, containing eserin but no postassium ions. The homogenated material was adjusted to pH4.0 by adding an acetic acid buffer solution and heated on the boiling water bath for 2 minutes. The material was then cooled immediately. The amount of Ach detected in this material was evaluated as the bound Ach content.

The summation of the free Ach and the bound Ach contents was evaluated as the total Ach content. 2) Preparation of test solutions

were prepared for the purpose of examining the effect on the synthesis of Ach. 3 ) Determination of Ach

Ach was biologically assayed using the frog rectus abdominis muscle. (1)

Test solutions containing 2 ml. of KRPS and 1 ml. of various sera to be examined

Preparation of the standard Ach solution: Standard Ach solutions containing 0.01, 0.02, 0.04, and 0.06 pg/ml, respectively of Ach-chloride were prepared from the Frog Ringer solution.

The rectus abdominis muscle was suspended for 1 hour in Frog Ringer solution in a Magnus tube, and then for another 15 minutes in Frog Ringer solution containing 20 &ml of eserine sulfate. The frog muscle which showed contraction in the standard Ach solution of 0.01 pg/ml was used for experiment.

Preparation of the standard curve of the frog muscle contraction in the standard Ach solution: After the sensitivity to Ach was intensified by suspending in Frog Ringer solution in a Magnus tube for 5 minutes and then in the eserine-added Frog Ringer solution for another 5 minutes; the contraction of the frog muscle in the standard Ach solution was recorded on a kymogram for 90 seconds. The standard curve, i.e. the curve showing the relation between the concentration of the standard Ach solution from 0.01 to 0.06pg/ml and the degree of contraction of the frog muscle was recorded.

The contraction of the frog muscle in the test solutions was measured in the same manner as in the preparation of the standard curve. The Ach content of the test solutions was then

(2) Experimental animals:

( 3 )

(4) Determination of the Ach concentration in the test solution:

Inhibitory Effects of Myasthenic Serum on Synthesis of Acetylcholine 57

calculated from the above standard curves. The amount of Ach production was calculated as the Ach-chloride per gram cerebral cortex (wet weight).

(5) Evaluation of the inhibitory effect of myasthenic serum on the Ach-induced contraction of the frog rectus abdominis muscle:

The sera from normal subjects and myasthenic patients after exercise were diluted 6-fold with Frog Ringer solution. An Ach solution of 0.03 pg/ml was prepared from each of the serum dilutions and the contraction of the frog rectus abdominis muscle measured in order to examine the inhibitory action of the myasthenic serum on the contraction of the frog rectus abdominis muscle induced by Ach in comparison with that of the normal subject.

RE s u L T s

1) Individual differences of Ach synthesis in KRPS: The free and bound Ach from the rat weighing 100 to 119 gm. was 3.242

0.08 pg/g and 2.70k0.15 pg/g (mean and standard error), weighing 120-139 gm. was 3.2620.05 pg/g and 2.7920.07 rg/g, weighing 140-159 gm. was 3.2720.08 pg/g and 2.81a0.05 pg/g, weighing 160-179 gm. was 3.3420.07 pg/g and 2.8020.07 pg/g respectively. Each group consisted of ten animals.

The amount of Ach slightly increased according to an increase in the body weight, but there was no difference between the right and left cerebral cortex. Then, following experiments were performed using both right and left cerebral cortex. One side was to estimate of the amount of Ach in KRPS as the control and the other side the amount in the test solution. In order to minimize individual difference, the following correction method was employed. As the total average of the amount of free Ach was 3.282 0.07 pg/g and of bound Ach was 2.78k0.09 pg/g in KRPS, each value obtained from KRPS was always corrected to 3.28 Fg/g of free Ach and to 2.78 pg/g of bound Ach. The value obtained from the test solution was corrected according to the rate of correction of the value from KRPS. 2) Effect of normal and myasthenic sera on the contraction of the frog rectus

abdominis muscle by Ach: The contraction of frog rectus abdominis muscle was measured in the two solu-

tions containing 0.03 pg/ml of Ach, one prepared with six times diluted normal human serum and the other with diluted myasthenic serum. The results are shown in Figure 1, which shows the degree of muscle contraction compared with the standard Ach solution. There was no significant difference in contraction between the normal and myasthenic sera. 3) Amount of Ach in the slice of the cerebral cortex before incubation:

The amount of free Ach in the slice of cerebral cortex before incubation was scanty and it was impossible to estimate this with the method employed.

The amount of bound Ach before incubation was 1.5420.12 pg/g. Therefore, the amount of bound Ach in the cerebral cortex after incubation was calculated by subtrac- tion of 1.54 from the value actually measured. 4) Effect of the potassium ion concentration:

The potassium ion concentration of the test solution was in the range of 5.33 to 6.00mEq/L. The free Ach synthetized in the solution containing 5.33, 5.52, 5.67, 5.74 and 6.00 mEq/L potassium ion was 3.28k0.04 pg/g, 3.29r0.07 pg/g, 3.28+


Free Ach

Normal subjects 3.60* 0.07 .~

Myasthenia gravis 2.95 *O .08 generalized 2.9 1 f 0.08 ocular 3.02 k0.06

90

70

60

Bound Ach

1.63 +O .06

1.27 +0.07 1.23 f0.07 1.34f0.06

Normal Myasthenia Fig. 1. Effect of normal and myathenic sera on the con-

traction of the frog muscle by acetylcholine compared with the standard 0.03 p/g of acetylcholine solution.

0.06 pg/g, 3.2820.04 pg/g, and 3.2820.06 pg/g respectively. The bound Ach values were 2.78kO. 18 pg/g, 2.76.tO. 12 pg/g, 2.8220.16 pg/g, 2.82kO. 14 pg/g and 2.782 0.16 pg/g, respectively. Each value was the average of five samples. There was no significant difference in regard to the amount of Ach with potassium ion ranging from 5.33 to 6.00 mEq/L. 5 ) Eflects of normal and myasthenic serum on the Ach synthesis in the rat brain slice: (1) Normal subjects

The amount of free Ach produced in the solution containing normal serum was 3.60k0.07 pg/g and that of the bound Ach 1.6320.06 Fg/g.

The relationship of age to Ach production was examined and it was found that the free Ach produced was 3.53k0.19 pg/g in the 10 to 19 years age group, 3.62t0.26 pg/g in the 20 to 29 years group, 3.5920.09 pg/g in the 30 to 39 years group, 3.6320.63 pg/g in the 40 to 49 years group, and 3.60+0.32pg/g in the 50 to 69 years group; and the bound Ach produced was 1.62A0.15 pg/g, respectively. The free and bound Ach produced, therefore, did not significantly differ according to the age.

By sex distinction, the free Ach produced was 3.61t0.01 pg/g with the male serum, and 3.5820.01 pg/g with the female; and the bound ACh produced was 1.63kO.09 pg/g with the male serum and 1.64kO.08 pg/g with the female. In short, there was no significant difference according to sex.

Table 1. Ach synthesis in the solution containing various sera in all cases.


generalized type (7 cases)

ocular type (3 cases)

Myasthenia gravis

(2) Myasthenic serum The free Ach produced in the solution after addition of the serum from the patients

with myasthenia gravis was 2.9520.08 pg/g and the bound Ach was 1.27&0.07 pg/g. When compared with the normal human serum, the free and bound Ach produced

were decreased by about 18% and about 22%, respectively in the solution containing myasthenic serum. The difference is highly signficant (PCO.0 1) .

As for the effect of the type of disease, the free and bound Ach produced are shown in Table 1. In short, there was evidence of any influence of the disease type on the Ach synthesis. 6) Effects of normal human serum and tnyasthenic serum on the Ach synthesis in the

rat brain slice before and after exercise. ( 1 ) Normal human serum

In the normal healthy person, the Ach produced before exercise was 3.67+0.14 pg/g for the free and 1.64ZkO.09 pg/g for the bound form.

After exercise, the free and bound Ach produced were 3.6720.15pg/g and 1.64a 0.09 pg/g, respectively.

In other words, no significant difference was observed in Ach between before and after exercise of the upper arm in the normal healthy person.

before 2.91f0.15 1.23f0.06 after 2.67 f0 . 22 1.01f0.19

before 3.02 f0.09 1.34f0.09 after 2.85fO. 32 1.22f0.32

Table 2. Ach synthesis in the solutions containing various sera before and after exercise (pg/g)

Free Ach Bound Ach

Normal subjects before 3.67f0.14 1.64f0.09 (10 cases) after 3.67fO. 15 1.64f0.09

(2) Myasthenic serum In the patients with myasthenia gravis, the free and bound Ach synthesized before

exercise was 2.93a0.11 p g / g and 1.27Zk0.06 pg/g, respectively. With the serum after exercise, the free and bound Ach produced were 2.73+

0.16 p g / g and 1.0520.15 &g, respectively. In short, the synthesis of Ach was further decreased after exercise as compared

with before exercise. The difference is significant (P<O.O5). With regard to the influence of the disease type, the synthesis of Ach in the general-

ized type and the ocular type are shown in Table 2. The synthesis of Ach after exercise was therefore lowered by about 11% in the

generalized type and by about 6% in the ocular type as compared with the values before exercise.


DISCUSSION

1) On the method of Ach determination: In employment of a bioassay method for the determination of Ach, two problems

must be considered: i.e. whether the Ach determined by this method will actually repre- sent the accurate amount of Ach itself, and whether the concentration of electrolytes in the test solutions for the Ach determination is kept constant.

Most experiments for the determination and detection of Ach employ the bioassay method utilizing the pharmacological action of Ach. This bioassay approach is quite sensitive and it is possible to determine Ach of measurable concentration by 0.01- 0.02 rg/ml by the frog rectus abdominis muscle method. However, on the other hand, it is impossible for this method to exclude the interference of analogous substances such as acetylcholine esters and betaine esters12) because of its poor specificity for Ach. More- over, there is no satisfactory method available for the separate assay of such analogous choline esters and betain esters. Therefore, the investigations hitherto introduced in this line have reported the total of the substance which have an Ach-like muscle contracting action including the acetylcholine esters and others in addition to Ach in the strictest sense as Ach. We, too, evaluated the total substances showing Ach-like action in the cortex slice suspension after completion of Ach synthesis, as the free Ach content and the total substances having the same action in the cerebral cortex slice as the bound Ach content.

In the bioassay using the frog rectus abdominis muscle, the determination will be less accurate because of the nonspecific contraction of the muscle unless the ion com- position in the sample is kept constant. In the present experiments, although there was a slight variation in the K+ and Ca++ ion concentration of the serum to be examined, synthesis of Ach was carried on in suspensions containing 5.33 to 6.00mEq/l of K+ ion and 4.93 to 5.60mEq/l of Ca++ ion; and the resultant Ach synthesized was determined by bioassay. The lack of difference observed in the Ach determined signifies that the determination was unaffected within the range of K” and Ca++ ion concentration. Accordingly, it is believed that the serum electrolyte concentrations in the samples examined in this investigation need not be considered. 2) On the mechanism of the blocking of the neuromuscular transmission in the patients

with myasthenia gravis. Since Walker26) reported in 1935 that prostigmin was effective in the treatment of

myasthenia gravis, it has come to be considered that as this substance has an anti- cholinestrase action and the cause for this disease might be a blocking of neuromuscular transmission. On the other hand, the paralysis following administration of d-tubo- curarine chloride in normal subjects closely resembles the paralysis appearing in the patients with this disease, who are more sensitive to curare than the normal subjects1.8). From these findings, it was suggested that curare-like substances might cause a blocking of the neuromuscular transmission in this disease. Lately, many investigations have been reported on curare-like substances in the serum and tissues of the patients with this disease.

From the results reported by various investigators, it is quite evident that the “myasthenic substance”, which causes a block of the neuromuscular transmission, may be present in the serum of myasthenic patients. Concerning the blocking mechanism of this substance, most investigators believe that this effect may come directly from the


“myasthenic substance” or may be a result of the curare-like action of a combination product of the “myasthenic substance” and a decomposition substance of Ach. How- ever, none of their investigations give full evidence for the curare-like effect of the substance, but only a presumption. Windsor28) and others3-7,9,13,14) oppose these assump- tions and suggest an inhibitory effect of this substance on the synthesis of Ach. It is still unknown whether the substance may affect the synthesis of Ach presynaptically or have a postsynaptic curare-like action.

In the present experiment, the diluted serum obtained from myasthenic patients after loading by exercise did not inhibit the Ach-induced contraction of the frog muscle. However, the myasthenic patient’s diluted serum, obtained after loading by exercise, inhibited the muscular contraction in the frog neuromuscular speciments in the experiments carried out by Wilson27), and Windsor2”. Our experimental results seem to be contradictory to those obtained by Wilson and Windsor. This discrepancy may come from a difference in the experimental method. In the present experiment, the effect of the myasthenic patient’s serum on the contraction of the frog muscle was investigated after Ach had been exogenously given, whereas in the report of Wilson and Windsor, the effect was studied on the muscle contraction occurring after the nerve stimulation, in which nerve-impulse was transmitted by Ach which had been released from the nerve end-plates as a result of electrical stimulation of the nerve. If neuromuscular transmission is blocked by a curare-like action on the muscle end-plates by a “myasthenic substance” in the myasthenic patient’s serum, a similar blocking of the neuromuscular transmission by “myasthenic substance”, followed by a lowering of the muscle contraction as observed by Wilson and Windsor may be expected in our experiment. Actually, no reduction of the muscle contraction occurred. This seems to indicate that the “myasthenic substance” may not act as a curare-like substance. As another possibility, it may be considered that the presynaptic action of the substance may inhibit the synthesis or isolation of Ach and then lead to the blocking of the neuromuscular transmission.

Therefore, in order to further investigate whether the myasthenic serum might have an inhibiting action on the synthesis of Ach, we experimentally produced Ach synthesis in slices of the rat cerebral cortex and tested the effect of myasthenic patient’s serum on this. As a result, reduction of the free and bound Ach synthesis was demonstrated (Table 1 ). In addition, the myasthenic serum obtained after loading by exercise showed a greater inhibitory effect on the Ach synthesis as compared with the serum obtained before the loading exercise (Table 2 ) . This finding has something in common with Tsukiyama’s24) report that the serum obtained after the loading exercise caused electro- myographically, a more potent blocking of the neuromuscular transmission as compared with the pre-load serum.

We have demonstrated that the serum obtained from myasthenic patients inhibits the process of synthesis of Ach in the experimental system utilizing the cerebral cortex which is regarded as a tissue synthesizing the largest quantity of Ach and bioassay which is the most sensitive for Ach. Although some question may be raised as to the bioassay method employed, since a minute amount of Ach-like substances other than Ach itself may be present in the cerebral cortex slices and these substance may possibly react to the bioassay, we believe that the results of our present experiments have provided an important clue for the presence of a certain factor inhibiting the Ach synthesis in the serum of patients with myasthenia gravis.


CONCLUSION

The possible inhibiting effects of serum obtained from patients with myasthenia gravis on the synthesis of Ach was investigated using slices of rat cerebral cortex and the following findings were obtained:

The myasthenic serum inhibited the synthesis of free and bound Ach in the rat cerebral cortex slices.

The myasthenic serum obtained after loading by exercise showed a further potent inhibitory effect.

( 1 )

(2)

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INHIBITORY EFFECTS OF MYASTHENIC SERUM ON THE SYNTHESIS OF ACETYLCHOLINE IN THE RAT BRAIN