Effect of Ions on the Efflux of Acetylcholine from Peripheral Nerve

WOLF-D. DETTBARN and PHILIP ROSENBERG From the Department of Neurology, College of Physicians and Surgeons of Columbia University, New York, and the Marine Biological Laboratory, Woods Hole, Massachusetts

ABSTRACT The nerves from the walking leg of lobster released acetylcholine (ACh) even when the ends were tied off, although this release was significantly increased when the nerve endings were not tied. The resting nerves were kept in sea water containing physostigmine. In absence of physostigmine no ACh was found in the surrounding fluid. Removal of Ca from the sea water reduced the release of ACh, while increased concentrations of Ca had no significant effect. Removal of Mg++ or increased Mg++ concentrations in the presence of normal Ca+ + concentrations increased the release of ACh. Increased K+ concentrations had a stimulating action on the efflux of ACh. Increased or reduced Na+ concentrations had only slight effects on the release of ACh in resting lobster nerve. During the 4 hr observation period the excised nerves were still able to synthe- size ACh. The choline acetylase activity was stimulated by increased concentrations of Mg++ and K+. The effects of ions on the release of ACh are similar to those reported at the junction.

INTRODUCTION Although it is known that peripheral nerve contains acetylcholine (ACh) (1-6), little information is available about its release from the nerve axon proper. This problem is of particular interest since Nachmansohn (6) has postulated that it is the reaction of ACh with a component within the membrane that controls the permeability changes during the conduction of an impulse. While ACh has a strong action on the neuromuscular junction, it is in- active even in high concentrations when applied to most peripheral nerve fibers. Evidence has accumulated that this is due to the presence of structural permeability barriers which prevent ACh from reaching the excitable membrane of peripheral nerve (7-9). These barriers will also make it difficult for internally released ACh to diffuse to the ouside. Only from freshly cut endings of the axon have significant amounts of ACh been reported to be released. This release can be increased by stimulation (1, 3, 4, 10-14). Recently it has been demonstrated that ACh has effects on the lobster walk-

447

The Journal of General Physiology THE JOURNAL OF GENERAL PHYSIOLOGY- VOLUME 50 · 966 ing leg nerves. It causes an initial increase in spike height, a prolongation of its duration, and depolarization leading to block of conduction (15-17). It therefore appeared that this preparation had a smaller permeability barrier to ACh than other peripheral nerve and might be a favorable material in which to reinvestigate the release of ACh from peripheral nerve. It also was of interest to study the effects of Ca ++, Mg++, K+, and Na + upon the resting release of ACh, as has been done previously on the nerve terminal (18).

METHODS The walking leg nerve of lobster (Homarus americanus) consists of over a thousand sensory fibers and a total of only 14 motor and inhibitory fibers (19). The nerve was removed from the first and second meropodite of either side, tied with nylon threads at both ends, blotted on filter paper, weighed, and was then placed into I ml of sea water. The sea water was replaced every hour for 4 consecutive hr. Then, each sample was made up to 6 ml with frog Ringer's solution and tested with the frog rectus bioassay for its ACh content (20). At the end of the 4 hr period, the nerves were weighed once more, homogenized in 8 ml of frog Ringer's solution, and tested for ACh. At the beginning of each experiment, two freshly excised nerves were homogenized immediately after weighing. Sea water of the following composition in mM/liter was used: KCl:10; NaC1:425; CaC12:10; MgCl2 :25; MgSO 4:25; NaHCO 3:2.4. Frog Ringer's solution had the following composition in mM/liter: KC1:2.5; NaCI: 115; CaCl2:1.8; NaHCO3 :2.4. Sea water and frog Ringer's contained 10-M physostigmine salicylate, unless the effect of cholinesterase on ACh content was being investi- gated. This concentration of physostigmine has little effect on electrical activity in 2 hr. The pH of the solutions was 7.6, and all experiments were carried out at room temperature of about 21°C. The results are presented as means - standard error (standard deviation of the mean). The usual controls were employed as to the identity of ACh (21, 22); however, since positive identification was not made, it is to be under- stood that the material released from the nerve is an ACh-like substance, although for the sake of convenience we shall refer to it as ACh. Each sample released from the lobster nerve was assayed between known concentrations of ACh which gave contractions of the frog muscle either slightly greater or slightly less than the unknown. The exact concentration of ACh-like material in the unknown sample was calculated by extrapolation. As seen in Fig. 1, 2 X 10 - 7 and 3 X 10- 7 M ACh give markedly different contractions so that one can arrive at the unknown concentration by extrapolation with considerable accuracy. Some of the tests employed for identification of ACh are also shown in Fig. 1. The resting potentials were recorded with the sucrose gap technique described by Staempfli (23).

RESULTS The Release of ACh from Lobster Nerve in Sea Water Nerves tied at both ends and kept in sea water, continuously released ACh without being stimulated. Based on 38 experiments, the amount of ACh re- WOLF-D. DETrBARN AND PHILIP ROSENBERG Ions and Acetylcholine 449 leased was 10.0 ug/g nerve during the 4 hr observation period (Fig. 2). In the 1st hr, slightly less ACh was released than in the following 3 hr. At the end of the 4th hr, the nerves were homogenized and an average residue value was determined. The total concentration of ACh is the sum of these two values. Control nerves were homogenized immediately after dissection. Nerves with ends not tied released the same amount of ACh as those cut up into three or

C D

J60se 60 sec

FIGURE 1. Tracings of ACh bioassay with the frog's rectus abdominis muscle. A, contraction caused by 2 X 10- 7 MACh, an unknown sample, and 3 X 10-7 MACh. B shows the repetition of A. C, contraction caused by nerve homogenate incubated in sea water and 10- 4u physostigmine. No contraction is seen when the nerve is incubated in physostigmine free sea water. D, a contraction caused by 1.5 X 10-7 u ACh is inhibited by 5 X 10-a M atropine. E, a nerve homogenate boiled at pH 3 causes contraction, while boiling at pH 10 inhibits the contraction. F, contraction caused by 1.5 X 10-1 M ACh is inhibited by 5 X 0- 6 Mcurare. more samples (Fig. 3), although the time course of the release was altered, the greatest amount occurring in the st hr. When only the middle part of the nerves was submerged, 1.6 - 0.8 ,g/g/nerve was released during the 1st hr. When sea water without physostigmine was used, no ACh was detectable. ACh concentrations were identical irrespective of whether the homogenized suspensions were directly tested on the frog muscle or first acidified and boiled and then neutralized.

The Effect of Ca++ and Mg++ on the Release of ACh In the absence of Ca ++ and Mg++ the ACh efflux was only slightly, if at all, lower but the residue amount was considerably higher (Table I). A reduction 450 THE JOURNAL OF GENERAL PHYSIOLOGY · VOLUME 50 · 966 of the Ca++ concentration by only 50% had no significant effect on the release mechanism, whether in the presence or the absence of Mg++. When Ca++ was reduced to 0 and Mg++ remained at the normal concentration, the release of ACh into the outside fluid markedly decreased (Fig. 4). Removal of Mg++ without changing the Ca++ concentration caused a slight increase in release from the second to the fourth hr (Fig. 4). However, the

,Ip ACh/g 20

ig ACh/g

4 -. r r~~~~~ 10

3 -

2 12 9 .:I H_5

I _

1O 2 3 4 CON-I0IJ RESI- TOTAL TIME IN HOURS TROL DUE

FIGURE 2. Release of ACh from walking leg nerve of lobster as a function of time. The amounts of ACh released per hour are shown in the graph. The amount of ACh remaining in the nerves at the end of the 4th hr is given in an extra column ("residue"). The total concentration of ACh is the sum of ACh released during the 4 hr period plus the residue. The control column indicates the amount of ACh found in a nerve homogenized immediately after dissection from the lobster. The total value for ACh shows more than a 50% increase when compared with that of the control. Normal sea water was used at pH 7.8 and 21°C. nerves releasing the smallest amount of ACh, namely those kept in Ca++-free sea water, had the largest residue, while the residue of nerves kept in normal sea water or in Mg++-free solutions was smaller (Fig. 4). When the Mg++ concentration of the sea water was greatly increased to 200 mM, the amount of ACh released into the outside fluid was considerably increased, while under the same condition but in absence of Ca++ the release markedly decreased (Fig. 5). The Mg++-rich, Ca++-free nerves retained large amounts WOLF-D. DETTBARN AND PHILIP ROSENBERG Ions and Acetylcholine 451 of ACh, but even in the presence of Ca++, more ACh was retained than under control conditions of normal sea water. This relationship was also true for the total amounts of ACh (last three columns of Fig. 5). (See also Table I for a twofold increase in Mg++.) A fourfold increase of the Ca++ concentration from 10 to 40 mM had a slight, if any, stimulating effect on the outflow of ACh. The removal of Mg++ under these conditions had no significant effect (Table I).

#g ACh/g 6

juP ACh/g 5 20

4 15

3 10

2i 1 5

I~~~~~~~·

A'I 0 1 2 3 4 CON- RESIDUE ".., .11 u- -p - 1l~

FIGURE 3. Release of ACh from tied and untied lobster nerves in sea water. Nerves which were not tied released more ACh than those with tied endings. No further increase of ACh was noticed when the untied nerves were cut up in three or more samples, although more ACh was released during the Ist hr. o tied, uncut; not tied, uncut; * not tied, cut. Sea water, pH 7.8 and 210C.

Total removal of Ca++ and Mg++ from sea water caused a depolarization of the resting potential by 10 0.8 my. The addition of Mg++ up to 200 mm to the Ca++-free solution decreased this depolarization slightly. Removal of Mg++ from sea water did not cause a depolarization.

The Effect of K+ on the Release of ACh

No significant changes in the outflow of ACh were found when the K+ concentration was reduced from 10 to 5 or 0 m. Increase in K+ concentration from 10 to 20 or 40 mM stimulated the outflow of ACh (Table II). Even the total amount of ACh increased according to the external K+ concentration. The effects of 70 m K+ presented in Fig. 6 were quite marked. The release, 452 THE JOURNAL OF GENERAL PHYSIOLOGY · VOLUME 50 · 966

residue, and total amount of ACh were all increased by this sevenfold increase in K+ concentration. Increase in the K+ concentration over that of the sea water caused a depolarization of the resting potential which in size was directly related to the external K+ concentration. An increase to 70 mM K+ depolarized the resting potential by 32 - 3 mv; a solution of 500 mM depolarized it by 69 7 myv.

TABLE I THE EFFECT OF CHANGES OF Ca ++ AND Mg++ CONCENTRATION IN SEA WATER ON THE RELEASE OF ACh FROM LOBSTER NERVE Normally sea water contained 10 mM Ca++ and 50 mM Mg++. Numbers of experiments are shown in parentheses. Results are expressed as mean standard deviation of the mean. The residue in nerve is the amount of ACh remaining after the 4 hr of release. Total ACh is the sum of the amount of ACh released during the 4 hr plus the residue. The column labeled Control represents the amount of ACh present in nerve, from the same lobster as used for the experimental studies, but which was homogenized and assayed immediately after dissection.

Amount of ACh found (g/g nrre)

Changes in the ionic Release of ACh Residue Total Control composition of sea water (S.W.) in mm/liter x hr 2 hr 3 hr 4 hr

S.W. (4) 0.44-0.2 1.2-0.5 2.0±0.6 1.940.5 6.642.3 12.1 20.1 0 Ca+++0 Mg++(12) 0.640.2 1.14-0.2 1.240.1 1.2-0.1 13.7-1.4 17.8

S.W. (2) 0.5-0.5 1.640.1 2.640.2 2.740.3 3.540.3 10.9 4.341.3 5 Ca+++50 Mg++(2) 040 1.640.1 1.840.2 2.340.2 5.9±0.3 11.6 5 Ca+++0 Mg++(2) 4-0 1.540.4 1.940.2 2.640.7 6.240.7 12.2

S.W. (2) 1.74-0.1 2.140.2 3.040.1 3.040.4 8.040.3 17.8 9.644.5 20 Ca+++50 Mg++(2) 1.64-0.2 2.1i0.1 2.94-0.2 2.740.2 7.141.2 16.4 20 Ca+++0 Mg++(2) 1.640.2 2.640.2 3.140.0 3.04-0.3 6.541.6 16.8

S.W. (2) 1.2-0.3 2.440.7 3.04-0.6 2.5±0.4 6.840.1 15.9 7.640.2 40 Ca+++50 Mg++(2) 1.04-0.0 2.940.3 3.4-0.9 2.440.1 4.740.7 14.4 40 Ca+++O Mg++(2) 1.04-0.1 2.9-0.1 2.940.5 2.0±0.2 4.6-0.6 13.4

S.W. (4) 1.440.2 2.3i0.3 2.5±0.2 2.440.5 5.840.9 14.4 11.8±1.2 0 Ca-++100 Mg++(4) 0.90.1 1.14-0.1 1.240.1 1.240.1 16.31.7 20.7 10 Ca++l00 Mg++(4) 1.440.3 4.0-1.1 4.040.8 2.9±0.5 8.4±0.6 20.7

The Effect of Na+ on the Release of ACh The removal of the entire Na+ (425 mM) from sea water caused a slightly increased outflow of ACh. A further slight increase was noticed when Na+ was replaced with isotonic sucrose solution. In both Na+-free solutions the total amount of ACh was greater than that in the normal sea water (Table II). An increase in the Na+ concentration by 30% caused a somewhat greater outflow, while lowering the Na+ by the same amount had no effect. Doubling the Na + WOLF-D. DETTBARN AND PHILIP ROSENBERG Ions and Acetylcholine 453 concentration to 850 mM caused an increased release, while replacement of 425 mM of Na+ with sucrose caused a slight decrease in comparison to the control (Table II). Na+-free sea water, replaced with an equiosmotic amount of sucrose, caused a hyperpolarization of 15 4 3 my after an initial small de-

FIeREi 4. Release of ACh from lobster nerve in Ca+-- or Mg++-free sea water. Re- moval of Ca ++ markedly reduces the release of ACh, while removal of Mg++ without changing the Ca++ concentration causes a slight increase. The nerves kept in Ca++-free sea water show a large amount of ACh remaining inside after the 4 hr period, which corresponds to the reduced release. Total values of ACh are higher in the 4 hr nerves than in the control nerve. pH 7.8, 21°C. o Control, A 0 Cat+ , · OMg . polarization. Smaller changes in Na+ concentration had no effect on the resting potential. DISCUSSION The theory assuming the action of ACh to be essential for the control of the ionic permeability of excitable membranes during electrical activity in axons 454 THE JOURNAL OF GENERAL PHYSIOLOGY · VOLUME 50 - 966 as well as at junctions was partly based on the evidence that potent competi- tive and reversible inhibitors of either ACh-esterase or ACh receptoI reversi- bly block electrical activity of excitable membranes. This evidence seemed, however, not satisfactory to some investigators, who felt that it was essential to show in addition a direct action of ACh on axonal membranes. Direct

jug ACh/g

A& a!un ACh/n 50 7

6 E 40

30 4 ·.·.·· 3 20

2 ····· 10 'a ······ 1 n . ,_ . I 1.'. v - _11 2 3 4 CON- RESIDUE TOTAL TIME IN HOURS TROL FIGURE 5. The effect of Mg on the release and synthesis of ACh in lobster nerve fibers. A fourfold increase of Mg+ in sea water to 200 mM causes a steady rise in the release of ACh over that of the control during the 4 hr period. In spite of the higher release rate, the amounts found remaining are still higher than those in control nerves. Removal of Ca ++ in the presence of high Mg++ causes a reduced release of ACh below that of the control. These nerves show also the highest concentration of ACh residue. The total ACh is highest in the nerves kept in the Mg++-rich sea water. pH 7.8, 21 C. 0 control, 0 Ca, 200 mM Mg, A 200 mM Mg. effects of ACh have, however, now been demonstrated on peripheral nerves (15-17, 23 a). The release of ACh, i.e. the appearance of the ester in extracellular perfusion fluid, was originally also considered to be confined to the junction. How- ever, ACh release under resting conditions from dog sciatic and vagus nerve but not from toad sciatic nerve was reported, but unfortunately it was not stated whether the nerve endings were tied off (1). Several other authors re- WOLF-D. DETrBARN AND PHILIP ROSENBERG Ions and Acetylcholine 455

ported ACh release from freshly cut vertebrate nerve endings during electrical activity (1, 3, 10-14); Lissak (4) reported release of ACh from intact nerve during electrical activity, while double the amount was released under similar conditions from the cut nerve endings. A more thorough evaluation of the ACh release from axons and the effects of ionic alterations on this release appeared desirable.

TABLE II THE EFFECT OF CHANGES OF K+ AND Na + CONCENTRATION IN SEA WATER ON THE RELEASE OF ACh FROM LOBSTER NERVE Number of experiments are shown in parentheses. Results are expressed as mean 4 standard deviation of the mean. Explanation of terms same as in Table I.

Amount of ACh found (g/g nerve) Changes in the ionic Release of ACh Residue Total Control composition of sea water (S.W.) in m/liter i hr 2 br 3 hr 4 hr

S.W. (4) 2.440.7 2.740.6 3.840.6 4.041.2 6.640.4 19.5 13.840.9 0 KC1 (4) 3.00.5 4.240.7 5.64-1.5 3.6±1.1 5.140.3 21.5 5 KCI (4) 2.340.2 3.240.2 3.940.7 3.841.2 5.640.4 18.8

S.W. (4) 3.140.4 4.340.8 4.440.5 2.540.1 5.3+0.7 19.6 21.2±2.6 20 KCI (4) 4.340.7 6.041.0 4.740.2 2.640.1 4.7+0.5 22.3 40 KCI (4) 4.040.6 5.740.3 5.440.6 2.940.3 6.8-1.2 24.8

S.W. (6) 2.5-0.3 4.740.4 4.740.4 3.340.6 8.041.2 23.2 19.140.9 0 Na + (6) 4.440.9 5.640.8 4.840.8 2.6±0.1 8.3±0.2 25.7 0 Na++Sucrose* (6) 3.340.5 6.340.8 6.340.8 4.5±0.5 9.241.7 29.6

S.W. (6) 2.140.4 3.440.9 3.140.5 3.240.7 4.6-0.3 16.4 12.042.4 500 NaCl (6) 3.440.6 4.940.7 4.240.6 2.940.9 3.840.3 19.2 300 NaCl (6) 2.440.5 3.14-0.3 4.240.4 3:040.4 6.440.5 19.1

S.W. (4) 1.840.4 3.140.2 4.2±0.6 3.640.4 7.642.1 20.3 15.644.0 850 NaCI (6) 3.940.6 5.0±1.0 4.0±0.7 2.440.4 3.140.5 18.4 S.W.+Sucrose* (6) 1.740.6 3.24-0.8 3.540.7 3.540.7 7.241.7 19.1

* An amount of sucrose was added which was isotonic with 425 mrr NaCI.

The data presented in this paper unequivocally demonstrate a significant continuous release of ACh from the axon of the lobster walking leg. The increase in release with time might be due to an increased permeability caused by damage, lack of oxygen, and other factors. This release takes place only in presence of physostigmine, but this is a condition prevailing also at the junction. In fact, it was emphasized by Dale and his associates that ACh appears in the perfusion fluid of junctions only in presence of physostigmine (24). Since physostigmine is a powerful inhibitor of ACh-esterase, the necessity of block- ing the extremely rapid physiological removal mechanism in order to obtain 456 THE JOURNAL OF GENERAL PHYSIOLOGY VOLUME 50 · 1966 an extracellular appearance raises the question whether this appearance takes place physiologically, i.e., with the enzyme at full activity. The total amount of ACh in nerves used for the leakage experiments was in many cases close to twofold that of the control nerve, in which ACh was determined at the beginning of the experiment. This increase of total amount must apparently be attributed to a continuous ACh formation by choline acetylase. When the fibers were tied and not cut, the release was smaller than that observed from cut and untied fibers, but only in the cut fibers was an effect on the total amount formed noticeable. The increased release from open

jg ACh/g 28

20 pg ACh/ ri 4 XI 16 3 iIW 12 2 a MIi~1 a

4. leil.I n 1 2 3 4 CON- RESIDUE TOTAL TIME IN HOURS TROL FIGURE 6. The effect of K+ on the release and synthesis of ACh in lobster nerve fibers. A sevenfold increase of K+ over that of sea water causes an increased release of ACh during the 4 hr period. The residue is increased and so is the total amount of ACh in nerves kept in K+-rich (70 m) sea water. pH 7.8, 21°C. o control, A 70 rn K+. nerve endings is not surprising since even in the lobster nerve fibers permeability barriers still surround the axonal membrane. In the absence of Ca++ the outflow of ACh into the sea water was markedly lower than that of Ca++-containing solutions. However, the formation of ACh was not affected. The residue was much higher than in the presence of Ca++. Thus, it seems that not the formation of ACh, but its outflow into the surrounding fluid, is impaired. This effect becomes even more striking when Mg++ are increased. Their presence apparently leads to a strongly increased ACh synthesis, as has been also reported for choline acetylase in vitro (25-27); never- theless, the outflow into the surrounding medium is impaired in the absence of Ca++. The importance of Ca++ and Mg++ in synaptic, and in particular in neuromuscular, transmission has been recognized for a long time (18, 28-30). It is WOLF-D. DETTBARN AND PHILIP ROSENBERG Ions and Acetylholine 457 known that the release of ACh during the nerve impulse fails when Ca is withdrawn from the external solution. The addition of Mg++ to a Ca+ + - deficient nerve terminal will further decrease the release of ACh (31-33). The interrelationship of Ca concentration and release of ACh in our experiments is in fairly good agreement with the dependency of the frequency of the miniature end plate potentials (MEPP) on Ca++ concentration. Reduction of Ca++ causes a decrease in the number of these potentials, while additional increase over the normal concentration shows no further stimulating effect (34). The effect of Mg++ on the release mechanism of ACh on peripheral nerve corresponds also to its action on the frequency of the MEPP at the junction. The mechanism of ACh-release from nerve terminals has been discussed (18, 35). The present experiments support the view that Ca++ facilitates the outflow of ACh and that Ca+ + and Mg++ have a naturally antagonistic effect on the outflow. The mechanism of the Ca++ action in the process of the ACh outflow is as yet unknown. As in other preparations (25, 36, 37), increased K+ concentrations stimulated the outflow of ACh from the peripheral nerve of lobster. K+ in increased concentrations also had a stimulating effect on ACh synthesis. An action of K+ in an isolated choline acetylase system has been described (38). Alteration of the Na+ concentration in sea water caused only small changes in ACh outflow. It appeared that any change in the Na+ concentration slightly increased the outflow of ACh. It is difficult to decide from our experiments whether the release of ACh from the resting peripheral nerve is increased by depolarization. As we have seen, an increase of K+ over the normal concentration of 10 mM will cause a proportional depolarization with an increased efflux of ACh simultaneously. This certainly would correspond to observations made on the nerve terminal (18). We have also seen that increased K+ has a stimulating effect on the synthesis of ACh. It is, therefore, difficult to determine whether the increased release in presence of high K+ is due to the depolarization, to increased synthesis, or, as might well be, to both effects. Depolarization is not the only factor leading to an increased release of ACh, as can be seen from the results with Ca++ and Mg++. While removal of Ca++ from sea water will cause a depolarization, the outflow of ACh drops significantly. The absence of Mg++ from sea water increases the outflow of ACh without a depolarization. Our experiments indicate that without being stimulated the peripheral nerve of lobster continuously releases ACh. It is also of special interest that Ca++-, Mg++-, or K+-induced changes in the release of ACh are essentially the same as those described for the neuromuscular junction. The effects of electrical stimulation on the release of ACh are now being studied.

We are grateful to Dr. David Nachmansohn for his continuing advice and interest in these studies. Mr. Gordon Kirschberg provided able assistance in the early phases of the study. 458 THE JOURNAL OF GENERAL PHYSIOLOGY · VOLUME 50 · 1966

This investigation was supported by a Public Health Service Research Grant NB-03304-05 and by the National Science Foundation Grant NSF-GB-1913. Dr. Dettbarn was supported by Research Career Development Award 1-K3-NB-25, 133-01. Dr. Rosenberg was supported by Research Career Development Award 5-K3-NB-21, 862-02. Received for publication 27 January 1966.

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