Chemical modification of the crayfish neuromuscular junction

Comp. gen. Pharmac., I973, 4, I ° 7 - I I6. [Scientechnica (Publishers) Ltd.]

Io7

CHEMICAL M O D I F I C A T I O N OF THE CRAYFISH NEUROMUSCULAR JUNCTION S. L. STUESSE* Department of Biological Sciences, State University of New York at Albany, Albany, New York I2222, U.S.A.

(Received 22 September, I972 ) ABSTRACT i. The effect of specific reagents on neuromuscular transmission in the crayfish claw abductor muscle and its excitatory nerve is reported. ~. Twenty mM I-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) causes a decrease in quantal content of the excitatory nerve. 3. Crayfish excitatory junctional potential heights and muscle membrane resistance are unaffected by pH 4.8 for up to 3o minutes. 4. EDC had no effect on membrane resistance or quantal size. 5. There is no evidence for a disulphide bond at or near the glutamate receptor. 6. Acid and alkaline phosphatase had no effect on neuromuscular transmission. 7. The effects of various other chemicals are reported. THE evidence is strong that the excitatory transmitter at the crayfish neuromuscular junction is glutamate (Robbins, 1958 , i959; van Harreveld, i959; Takeuchi and Takeuchi, I964; Ozeki and Sato, 197o); however, very little is known about the glutamate receptor site on the postsynaptic membrane or the role of various chemical groups on the nerve-terminal membrane in controlling the release of the transmitter. In the past glutamic acid and excitatory compounds with a similar structure have been used to determine what component groups and steric configurations of the transmitter are necessary for maximal activation of the postsynaptic membrane. Data from both the crayfish (Usherwood and Machili, i968 ) and the cat (Curtis and Watkins, i96o , 1963) have indicated that two carboxylate groups and one amino group, in the proper steric configuration, are necessary for maximum excitatory activity. The pKas of glutamic acid are 2"19, 4"52, and 9"67. At physiological p H both carboxyl groups and the amino * Present address : Physiology Department, Case Western Reserve University Medical School, Cleveland, Ohio 44Io6, U.S.A.

group would be almost completely ionized. Thus it seems reasonable to postulate that there might be ionic interactions between the excitatory transmitter and its receptor site. Experiments on the frog neuromuscular junction have shown that the release of acetylcholine can be affected by two compounds, which react with carboxylate groups or both phosphate and carboxylate groups. The former compound, a dihydroquinoline, initially potentiated and later inhibited transmitter release (Stuesse and Katz, 1973). The latter compound, a carbodiimide, in most cases inhibited transmitter release (Stuesse, I972 ). It is possible that these compounds reacted with cationic sites to which the Ca ~+, necessary for aeetylcholine release, normally binds, thus decreasing the probability of acetylcholine release. This explanation is especially attractive in the case of the dihydroquinoline because the inhibition of acetylcholine release was reversed by increasing the Ca z+ concentration in the bath (Stuesse and Katz, 1973). In addition to presynaptic actions, these compounds reduced acetylcholine sensitivity at the postsynaptic membrane (Edwards, Bunch,

~o8

STUESSE

M a r f e y , M a r o i s , a n d v a n M e t e r , 197o; Stuesse, 1972; Stuesse a n d K a t z , I973). T h e studies o n crayfish r e p o r t e d in this p a p e r a r e s i m i l a r to those p r e v i o u s l y c a r r i e d o u t o n t h e frog. C h e m i c a l s w h i c h r e a c t specifically w i t h v a r i o u s c h e m i c a l g r o u p s w e r e p e r f u s e d o n to a crayfish n e r v e - m u s c l e p r e p a r a t i o n a n d t h e results w e r e a n a l y s e d . MATERIALS AND METHODS DISSECTION The abductor (opener) muscle of the crayfish (Procambarus clarkff) cheliped and its excitatory nerve bundle were used. Crayfish claws were excised by applying pressure to the joint closest to the body until the crayfish autonomized the claw. The adductor (closer) muscle of the cheliped was cut away leaving the opener muscle exposed and intact. T h e n the three nerve bundles in the meropodite were exposed and all but the bundle containing the excitatory nerves to the abductor muscle were removed. Most of the dissection was performed in a large volume of cold van Harreveld's (I966) solution. After dissection, the claw was kept overnight in a refrigerator (2-4 ° C.) in van Harreveld's solution. INTRAGELLULAR RECORDINGS Excitatory junctional potentials (Rips) were recorded intracellularly from the abductor muscle of the claw using 5-20 M f l , 3 M KCl-filled micro-electrodes and a conventional electrophysiological set-up (preamplifier, amplifier, oscilloscope). The claw was placed in a chamber o f io ml. volume and held in place by embedding the middle section of the preparation in Valap (I vaseline : i lanoline : I paraffin). Solutions were changed in one of two ways: 5 ° ml. of the new solution were perfused through the chamber with a dual syringe infusion-withdrawal p u m p , or the solution was changed by pipette. Care was taken to make sure that the solution level in the chamber remained unchanged. Platinum electrodes were used to stimulate the excitatory nerve. Large voltages (approximately IO V.) were needed to stimulate the nerves, since the nerve bundles were in a large volume of van Harreveld's solution. To avoid muscle contraction, low stimulation rates were used, 5-2o per second, depending on the muscle). Ejps were recorded on film and the amplitudes of a m i n i m u m of 5-1o ejps were averaged. EXTRACELLULAR RECORDINGS To record from a single junctional area in crayfish, extracellular recording may be used (Dudel and Kuffler, i96i ). The surfaces of the muscle were searched carefully with a low-resistance ( I - 3 M ~ ) 3 M NaCl-filled micro-electrode, while the excitatory nerve was stimulated at a

Comp. gen. Pharmac.

low rate (for example, 2 per second). At certain localized regions on the membrane, presumably end-plates (Takeuehi and Takeuchi, 1964) , an extracellular end-plate potential was recorded (eepp). (The noise level of the set-up was 5o laV. so miniature end-plate potentials could not be recorded.) I f the stimulus frequency is carefully chosen, some stimuli will not produce an eepp, i.e., there will be some failures of synaptic transmission. I f the quantal content, the mean number of packets of transmitter released per impulse, is kept low, then the release of packets o f transmitter can be approximated by a Poisson distribution (del Castillo and Katz, 1954; Dudel and Kuffler, I96I ). The values for quantal content (rn) and quantal size, the average size of the response to one packet of transmitter released (q), can be computed from the following formulae:

m=ln(N/no) or m=I/(C.V.) 2 and q=~/m, where c.v. is the coefficient of variation, N, is the total number of stimuli delivered, n o is the number of failures of synaptic transmission, and Y is the mean response size in mV. to one packet of transmitter released. A change in m would indicate that the chemical had a presynaptic effect, while a change in q a postsynaptic effect (seethe review of Martin (1966) for further details). MEMBRANE RESISTANCE Current was injected across the muscle-fibre membrane through a single 3 M KCl-filled microelectrode (2-IO M f l ) , and the potential change across the m e m b r a n e was recorded with the same micro-electrode. The injection technique does not alter the input resistance or voltage gain of the amplifier (WPI Model M 4A). After treatment of the muscle-fibres with 20 m M l-ethyl- 3 (3dimethylaminopropyl) carbodiimide HCI-(EDC) and 30 m M glycine methyl ester (GME) for up to 3 ° minutes, the m e m b r a n e characteristics were again observed. The effect of acidic p H on membrane resistance was also investigated. Since Tris does not buffer well at acidic pH, the solution acidity was checked both before and after an experiment. Spot checks were also m a d e using p H paper during the course of the experiments. The compound N-ethoxycarbonyl-2-ethoxy-i, 2-dihydroquinoline ( E E D O ) is not water-soluble, and so it was dissolved in dimethylsulphoxide (DMSO) and then added to van Harreveld's. The final concentration of E E D Q w a s usually ~ × IO-4 M in o'o 4 per cent D M S O - v a n Harreveld, and o.o 4 per cent D M S O - v a n Harreveld without E E D O was the control solution for these experiments. Dilute hydrochloric acid or Tris base was added to solutions to bring them topH 7'2- 7"4 or, in the case of some E D C experiments, p H 4.8. All solutions containing reactive chemicals were made fresh daily. This was especially important in the case o f E D C (Mann Research Laboratories),

CRAYFISH NEUROMUSCULAR JUNCTION

I973, 4

since it decomposes in water. Experiments were performed at room temperature (approximately 20 ° C.). RESULTS EFFECT OF E D C ON THE CRAYFISH EXCITATORY JUNCTIONAL POTENTIAL

Investigations w i t h n u m e r o u s c o m p o u n d s on the crayfish n e u r o m u s c u l a r j u n c t i o n yielded only a few substances w h i c h c h a n g e d the

in ejp a m p l i t u d e was reversible u p o n w a s h i n g for a p p r o x i m a t e l y 2o m i n u t e s in control solution (van H a r r e v e l d ' s solution at p H 7"4 or p H 4.8). N o differences in r e c o v e r y t i m e could be seen a t these two p H s , a l t h o u g h a faster recovery time at p H 4.8 m i g h t be e x p e c t e d u n d e r c e r t a i n circumstances (see Discussion). T h i s lack of difference m a y h a v e been d u e p a r t i a l l y to the i n a c c u r a c y of the

0.5mv 1 5m sec

A

C

xo9

.

Fza. i.--Effect of carbodiimide on crayfish excitatory junctional potentials. A, Control ejps in van Harreveld's solution. B, zo minutes post 20 m M E D C + 3 o m M glycine methyl ester. C, 20 minutes post 20 m M E D C + 3 o m M glyeine methyl ester. D, 5 minutes post van Harreveld's solution. E, zo minutes post van Harreveld's solution. F, 3° minutes post van Harreveld's solution. e x c i t a t o r y j u n c t i o n a l p o t e n t i a l size. O n e o f these was E D C . T w e n t y m M E D C a t p H 4.8 w i t h 3 ° m M glyeine m e t h y l ester p r o d u p e d a decrease in ejp height (Fig. x). This decrease was usually 5 ° p e r cent or m o r e in 15-2o minutes t i m e ; the effect was also seen w i t h o u t the presence of G M E . T h e decrease

ejp measurements. G l y c i n e m e t h y l ester b y itself was w i t h o u t effect on the ejp a m p l i t u d e . EFFECT OF E D C ON M E M B R A N E RESISTANCE AND M E M B R A N E POTENTIAL DIFFERENCE T h e ejp decrease in the presence of E D C could have been due to a decrease in muscle

110

Comp. gen. Pharmac.

STUESSE

in m e m b r a n e resistance in 5 experiments (Fig. 2), or m e m b r a n e potential difference i n more t h a n 3 ° experiments, was found. However, the conditions u n d e r which the m e m b r a n e potential was m o n i t o r e d were such that a change of less t h a n 5 inV. would n o t have been noticed. Glycine methyl ester by itself also had no effect.

A

f

\

EFFECT OF E D C QUANTAL SIZE

B

I

S

20 inV. IOm seconds

\

FIO. 2.--Effect of 20 m M EDC on membrane resistance in crayfish abductor muscle. A, Control in van Harreveld's solution. B, 9o minutes post EDC.

ON

QUANTAL CONTENT AND

T o d e t e r m i n e w h e t h e r the decrease in ejp size seen with E D C was due to a pre- or postsynaptic action of EDC, extracellular recordings from a single j u n c t i o n a l area were made. I n this way, both q u a n t a l content a n d q u a n t a l size could be measured. Analysis of a b o u t 2o0 eepps per e x p e r i m e n t i n d i c a t e d that w i t h i n 20 m i n u t e s E D C reduced the average n u m b e r of packets of t r a n s m i t t e r released per impulse (m) to as little as a q u a r t e r of the original a m o u n t .

Table L--EFFECT OF E D C + G M E ON CRAYFISHNEUROMUSCULARTRANSMISSION EXPERIMENTNo.

TREATMENT

O UANTAL CONTENT

(~UANTAL SIZE

(m= I/(C.V.)2

(x/m; mY.)

Control 3° minutes post 2o m M EDC + 30 m M GME

2'46

0'o8

I'I2

O'IO

0"22

O'2I

0.09

O'14

Control 3° minutes post EDC+GME

O'8I 0"22

o.I 7 o-I 7

4*

Control 20 minutes post 2o m M EDC + 3° m M GME

0"5I o'19

5*

Control 15 minutes post 20 m M E D C + 3° m M GME

0"20

Control 3° minutes post EDC + GME

o-o5

* Ouantal content calculated from m = l n (W/no) only. m e m b r a n e resistance or m e m b r a n e potential difference. However, no m e a s u r a b l e change

T h e q u a n t a l size was u n c h a n g e d in two experiments a n d decreased in a third (TableI).

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CRAYFISH NEUROMUSCULAR JUNCTION

1973, 4

experiments showed that this was actually due to the a m m o n i u m sulphate with which the enzyme had been precipitated during EFFECT OF U R E A O N EJP SIZE preparation. T h e decrase in ejp amplitude EDC is gradually decomposed in an aque- was seen even after the enzyme had been ous solution yielding urea as a by-product. It denatured by placing it in a boiling-water was possible that the decrease seen with EDC bath for 2o minutes, and application of a might be due to urea present in the solution. solution of (NH4),SO 4 mimicked the action However, i o - 2 o m M urea added to van of the alkaline phosphatase solution. It Harreveld solution was without effect on the was thought that any action the alkaline phosphatase might have would be masked ejp amplitude (3 experiments). by the ( N H , ) s S O * present. Therefore, EFFECT OF ACIDIC p H ON EJP SIZE, O__UANTAL another source of alkaline phosphatase (Cal CONTENT, AND MEMBRANE RESISTANCE Biochemical Corp.) with no (NH4)sSO 4 In most experiments with EDC the solu- present was tested on the crayfish preparations were at p H 4-8. Therefore control tion. This enzyme, in concentrations up to experiments were performed at this p H to 5.o units per ml., had no effect on the crayAcid phosphatase see if acidic p H affected the parameters fish ejp amplitude.

Thus, in the crayfish EDC acts primarily on the presynaptic (nerve) terminal.

Table II.--EFFECT OF ACIDIC pH ON O UANTAL CONTENT OF THE EXCITATORY NERVE TO THE ABDUCTOR MUSCLE IN CRAYFISH

TIME IN pH 4'8 (minutes) EXPERIMENT

NO.

CONTROL

IO

o'i6 o'39 o'74 o.5i o.38

O'8I

20

30

O'II

o'I6

0.36 --

o'76

--

0-42

6o

0-65 0.48

Quantal content calculated from the formula m = In (W/n0). Based on a minimum of 200 stimuli. measured. There was no affect on membrane resistance (4 experiments), on ejp size (6 experiments), or on the quantal content (Table II). EFFECTS OF OTHER CHEMICALS ON CRAYFISH EXCITATORY JUNCTIONAL POTENTIALS

Since the carbodiimide used could react with either phosphate or carboxylate groups (Khorana, i953, Hoare and Koshland, 1967) , an attempt was made to distinguish between these possibilities. T h e conditions of reversibility had suggested that phosphate groups were involved (see Discussion). When 1.8 units per ml. alkaline phosphatase (Worthington Biochemical Corp.), p H 8-o, were applied to the crayfish preparation, a decrease in ejp size was seen. However, control

(Worthington Biochemical Corp.) also did not change ejp amplitude. Since, by using phosphatases, we were unable to confirm that the presynaptic action of EDC was due to its reaction with phosphate grOiaps, reagents which react with carboxylate groups, 5 x io -8 M W-ethoxycarbonyl2-ethoxy- 1,2-dihydroquinoline (EEDQ) (Belleau and Malek, 1968 ) and Woodward's Reagent K (up to IO m M ) , were applied to the crayfish preparation. Neither of these chemicals affected ejp size or rise time. T h e response of the eel electroplax to ACh is diminished by treatment with a strong reducing agent, dithiothreitol (Karlin and Bartels, I966 ). Presumably this is due to the reduction of disulphide bonds. In crayfish the presumed glutamate receptor was unaffected

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STUESSE

Comp. gen. Pharmac.

Table IlL--EFFECT OF VARIOUS TREATMENTS ON CRAYFISH EXCITATORY JUNCTIONAL POTENTIALS (EJPS) TREATMENT OR CHEMICAL AND SOURCE

REACTING GROUP

CONCENTRATION AND TIME

EFFECT ON EJP

van Harreveld's, pH 9.0

30-60 minutes

None

van Harreveld's, pH 4"7

30-60 minutes

None

DithiothreitolCleland's reagent (Cal Biochemical)

S-S groups

raM, ~o minutes I o m m , I o minutes

None None

Woodward's Reagent K (Pilot Chemicals Inc.)

CO2- groups

I o mM, 55 minutes pH 8.0

None

Alkaline phosphatase, pH 8.0 (Worthington Biochemical Corp.)

PO4-

i .8 units per ml, io-3o minutes

Decrease in ejp---due to (NH4)2SO4 in the solution. Seen even after heating enzyme in boiling water for ~o minutes

Acid phosphatase, pH 4"9 (Worthington Biochemical Corp.)

PO(

I'o unit per ml. 9° minutes

None

Alkaline phosphatase, pH 8.0 (Cal Biochemical Corp.)

PO4-

2"8 units per ml. 5.0 units per ml. 3° minutes

None

EEDQ

COc

5"Io- ZM, 30 minutes

None None

NaBH4

Non-specific reducing agent

I0 mM, I hour

Formaldehyde

Amino, guanidyl, amide, SH

5 mM, I5 minutes ~o mM, IO minutes

None Reduced resting potential from --80 mV. to -- IO niT.

SH

W-Ethylmaleimide (Aldrich Chemical Co.)

SH

5× Io-4M 5-8 minutes

Spontaneous repetitive ejps, muscle contraction

Iodoacetamide (Aldrich Chemical Co.)

SH

5 x IO- aM, 25 minutes

None, in one experiment. Spontaneous repetitive ejps leading to muscle contraction in two experiments

I mM, 9° minutes

None Comment: had buffering problems with 5 m M solution--was very acidic

~-Nitro-4-carboxyphenyl sulphenyl chloride

o.o5-I m M

Almost immediate contraction None

Azobenzene-2-sulphenyl bromide, pH5"l (Nutritional BiochemInc.)

0.005-o.02 m M I hour

Tryptophancontaining peptides

Decrease

(NH,) ~SO,

20 mM, Io-2o minutes

NH,Br

4° mM, 2o minutes

None

Na,SO4

2o mM, 2o minutes

None

I973, 4

CRAYFISH NEUROMUSCULAR JUNCTION

by either of two strong reducing agents: dithiothreitol (up to I O m M ) and sodium borohydride (up to I o m M ) . Another series of compounds applied to the crayfish neuromuscular junction reacted with sulphydryl groups: azobenzene-~-sulphenyl bromide (Fontana, Veronese, and Scoffone, I968 ) N-ethylmaleimide, and iodoacetamide (see Webb, I966 ). These compounds m a y have had multiple reaction sites because they quickly led to repetitive ejps and muscle contraction. Table III summarizes the results obtained with these and other chemicals when applied to the crayfish preparation. Each experiment was repeated at least three times and the table shows the ranges of concentration used and the duration of application of the solution. DISCUSSION Experiments on frog neuromuscular junctions have shown that EDC treatment results in a decrease in both the average n u m b e r of packets of transmitter released per impulse (m) and quantal size (q) with little or no effect on m e m b r a n e resistance (Edwards and others, I97O; Stuesse, I972 ). T h e effect of EDC on crayfish neuromuscular transmission was similar to that in frog; a decrease in quantal content, a possible decrease in quantal size, and no noticeable change in m e m b r a n e resistance. T h e big difference, however, was that in crayfish the decrease in quantal release due to EDC in the presence of a nucleophile was reversible, while in frogs it is not. I n the reaction scheme outlined by Edwards and others (I97O), it was speculated that if the reaction of EDC in the presence of glycine methyl ester was irreversible, one could possibly attribute any changes to the reaction of EDC with carboxylate groups, while if it were reversible, phosphate groups would be implicated. I f EDC is actually reacting with PO t - groups to form a phosphoramidate compound which is acid labile, the time course of reversibility should be faster at p H 4"8 than at p H 7.o. No differences in reversal times were seen; however, with the technique used, differences of less than 15-2o per cent would not have

II3

been detected. Another source of variability was that it was difficult to measure the reversibility under both conditions in one preparation. Single measurements for each condition were made on a n u m b e r of preparations and the variations among preparations m a y have obscured any differences between reversal times. Woodward's Reagent K, W R K (Io m M , p H 8"0) and E E D Q (5 × IO-3 M ) , two reagents which are known to react with carboxylate groups (Belleau and Malek, I968), had no effect on the size of ejps in the crayfish. However, in frog 5 × I ° - 4 M E E D Q affects quantal content and decreases quantal size (Stuesse and Katz, i973). Since EDC reacts with both PO 4- and C O , - groups and it decreased ejp size, perhaps it is the modification of PO4- groups which caused the reduction. I f this is so, then alkaline phosphatase or acid phosphatase might also reduce the ejp size, since they hydrolyse available phosphate groups from compounds. Alkaline phosphatase, from two different sources, and acid phosphatase where used, but none affected the ejp. However, the enzymes would not react with all the phosphate groups on the m e m b r a n e surface. Either the phosphatase did not hydrolyse key phosphate groups at or near the receptor, or the hydrolysis of these groups does not affect the ejp amplitude. Albuquerque, Sokoll, Sonesson, and Thesleff (i968) had similar problems using enzymes which hydrolysed proteins. In their work sulphydryl blockers reduced ACh sensitivity of the muscle m e m b r a n e indicating that a protein was involved, but not one of five enzymes which hydrolyse protein affected ACh sensitivity. Since enzymes are large molecules which are only partially purified, and one has a good deal of non-specific protein contamination in the solution when using them, accessibility of the enzyme to the m e m b r a n e surface could be blocked. It is also possible that the effect of EDC is not due to its modification of phosphate groups at the receptor site but to those at nearby sites. T h e n perhaps the carbodiimide molecule attached to the phosphate sterically inhibits the ACh receptor and decreases the ACh sensitivity.

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STUESSE

The ejp size was found to be unchanged by p H changes from 4.8 to 9.0. This is in agreement with work by Ozeki and Sato (I97O), who showed that the crayfish response to iontophoretically applied glutamate was unchanged between p H 3"4 and p H 9"3- This helps to eliminate certain groups which have their pIf, values in this range, such as the a-amino groups of lysine or hydroxylysine, the imidazole groups of histidine, and possibly the suphydryl group of cysteine, all of which have pK~,s in the range of p H used. If glutamate reacted with the undissociated form of a molecule and it became dissociated, or vice versa, one would expect a change in glutamate sensitivity. It is interesting that acidic p H did not affect membrane resistance or quantal content. This is in contrast to results reported at the frog neuromuscular junction, where acetylcholine is the excitatory transmitter. Acidic pH in frog led to an increased membrane resistance and decreased quantal content (Del Castillo, Nelson, and Sanchez, I962). The effect of modifying sulphydryl groups in the crayfish neuromuscular preparation was investigated using three different reagents specific for sulphydryl groups, azobenzene-2-sulphenyl bromide (ASB) (Fontana, and others, I968), N-ethylmaleimide (NEM), and iodoacetamide (seeWebb, 1966 ). All of these reagents behaved similarly. They either had no effect (at low concentrations) or they caused repetitive, spontaneous ejps (not seen with ASB) which quickly led to muscle contraction (Chang, Lu, Wang, and Chang, 197o); Chang attributed this primarily to a direct action of the SH reagents on the sarcotubular system. However, there was also a gradual block of nerve conduction. Thus, as might be expected, the reagents had multiple sites of action. This seems to be the case with crayfish muscle; if there is any effect on the glutamate receptor itself, which is doubtful, it is obscured by the action of the sulphydryl reagents on the nerve and interior of the muscle. The response of ACh receptors to ACh and carbamylcholine can be inhibited by

Comp. gen. Pharmac.

prior exposure to dithiothreitol, a reducing agent which reduces disulphide bonds (Karlin and Bartels, (I966) in the electroplax, and Collins and Katz (I972) in the frog). In contrast to the ACh receptor, the glutamate receptor does not seem to have a disulphide bond at or near its active site which is accessible. Two potent reducing agents, dithiothreitol and sodium borohydride, had no effect on crayfish ejps. Formaldehyde is a very reactive relatively unspecific chemical which reacts primarily with amino and sulphydryl groups but can also form methylene cross-bridges with amide, guanidyl, heterocyclic, and phdiaolic groups. In crayfish, low concentrations of formaldehyde (5 mM) did not effect the ejp size. High concentrations of formaldehyde (2o raM) caused a rapid fall in muscle membrane resting potential (from --8o mV. to -- io inV. in IO minutes). This was the only reagent used which modifies amino groups and, although it had no effect at low concentrations, one cannot rule out the presence of amino, guanidyl, or some other positively charged group on the glutamate receptor. 2-Nitro-4-carboxyphenyl sulphenyl chloride, a reagent specific for trytophan residues in peptides (Veronese, Boccu, and Fontana, I968), had no effect on crayfish ejps. Therefore, it is unlikely that tryptophan would be found at the glutamate receptor. SUMMARY The results with EDC lead one to speculate that, because of its reversibility, the reaction of EDC with phosphate groups leads to the decreased quantal content in crayfish. This is substantiated by the fact that neither E E D Q or W R K , carboxylate-modifying reagents, affected crayfish neuromuscular transmission. However, in view of the alkaline and acid phosphatase data, one can not rule out the possibility that EDC is reacting with carboxylate groups. By contrast, in frog skeletal muscle E E D Q leads to an increase and then decrease in quantal content and a decrease in quantal size (Stuesse and Katz, I973). Also, EDC causes a decreased quantal content in frog, but the reversibility differs from that in

I973, 4

CRAYFISH NEUROMUSCULARJUNCTION

crayfish, i m p l i c a t i n g carboxylate groups in the reaction. Both release of g l u t a m a t e a n d m e m b r a n e resistance are unaffected by pHs in crayfish. A g a i n , this does not seem to be true in the frog (del Castillo a n d others, 1962 ). T h e evidence presented here o n disulphide inhibitors is consistent with the i n t e r p r e t a t i o n that a n accessible disulphide b o n d does not play a n i m p o r t a n t role either in release of g l u t a m a t e or at or n e a r the g l u t a m a t e receptor s t r u c t u r e ; again, this is not true for the A C h receptor ( K a r l i n a n d Bartels, i966 ). I n general, the chemical groups controlling g l u t a m a t e release a n d its postsynaptic receptor i n crayfish seem to be less accessible or more resistant to chemical modification t h a n those of acetylcholine in frog skeletal muscle. However, it is hoped that a more extensive use of chemicals will yield more v a l u a b l e i n f o r m a t i o n i n the future.

ACKNOWLEDGEMENTS The authors wish to express gratitude to Dr. Charles Edwards and Dr. Peter Marfey for helpful discussions during the course of this work. This work was supported by grants numbers GMo~oI 4 and NSo768I from USPHS and preliminary results were reported in an abstract (Stuesse, S., and Edwards, C. (I97I), Fed Proc. Fedn Am. Socs exp. Biol., 3o: 6I 7 (Abstr. ~358). Dr. Stuesse was an NIH predoctoral trainee, grant GMo2oI4, during the time that these studies were done. REFERENCES ALBUQUERQUE, E. X., SOKOLL, M. D., SONESSON, B., THESLEFF, S. (1968), ' Studies on the nature of the cholinergic receptor ', Eur. 07. Pharmac., 4 4o-4 6. BELLEAU, B., and MALEK, G. (1968), ' A new convenient reagent for peptide synthesis ', 07. Am. chem. Soc., 9o~ x65i-i652. DEL CASTILLO,J., and KATZ, B. (I954) , ' Q.uantal components of the endplate potential ', 07.Physiol., xa4~ 56o-573 • DEL CASTILLO,J., NELSON, Z. E., and SANCHEZ,V. (I962), 'Mechanism of the increased acetylcholine sensitivity of the skeletal muscle in low pH solutions ', 07. cell. comp. Physiol., 599 35-4. CHANG, C. C., Lu, S. S., WANG, P. N., and CHANG, S. T. (I97O), ' A comparison of the effects of various sulfhydryl reagents on neuromuscular transmission ', Eur. 07. Pharmac, n~ i95-2o 3. COLLINS, E., and KATZ, N. L. (I972), personal communication.

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Inhibitions of the Central Nervous System and Aminobutyric Acid (ed. Roberts), pp. 424-444- Oxford: Pergamon.

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