247,

J. Phy~iol. (1975), pp. 145-162 With 8 text -ft gure8 Printed in Great Britain

145

ON THE ASSOCIATION BETWEEN TRANSMITTER SECRETION AND THE RELEASE OF ADENINE NUCLEOTIDES FROM MAMMALIAN MOTOR NERVE TERMINALS

By EUGENE M. SILINSKY From the Department of Pharmacology, Northwestern University School of Medicine, 303 E. Chicago Avenue, Chicago, Illinois 60611, U.S.A.

(Received 6 August 1974) SUMMARY

1. Conventional electrophysiological techniques were used to record from isolated rat phrenic nerve-hemidiaphragm preparations. After periods of rest (20 min) or nerve stimulation (7/sec for 20 min) the bathing medium of the preparation was removed and assayed for adenosine triphosphate (ATP) and adenosine diphosphate (ADP) using a sensitive

modification of the firefly luciferase method (Silinsky, 1974). 2. In the presence of tubocurarine and normal (2 mm) calcium, fourteen periods of nerve stimulation (eight preparations) caused the appearance of ATP and/or ADP in amounts ranging from 28 to 641 p-mole. Experiments using carbachol (30 /Lm or 1 mm) suggested that this nucleotide efflux was not produced by a secondary action of released acetylcholine

(ACh). 3. Stimulation of isolated phrenic nerve trunks at 7/sec for 20 min did not cause the efflux of ATP or ADP. 4. In solutions of normal osmotic pressure and reduced calcium concentrations (0. 1 mm or 'calcium-free'), stimulation failed to release adenine

nucleotide from non-contracting preparations. 5. Diaphragms were bathed in normal calcium and indirectly stimulated at 11/sec for 80-90 min in the presence of 5 X 10-5 M hemicholinium-3. After all detectable signs of ACh release were eliminated, nerve stimulation failed to release ATP or ADP. 6. These results in conjunction with experiments on the hydrolysis of exogenous ATP suggest that ATP is released from the motor nerve ending and is subsequently degraded by enzymatic activity. It is also suggested that the released nucleotide may be derived from the cholinergic vesicle.

146

EUGENE M. SILINSKY INTRODUCTION

Recent evidence suggests that adenosine triphosphate (ATP) is present in peripheral cholinergic vesicles and may be released together with acetylcholine (ACh) from the motor nerve ending. In studies on nerve terminals of the electric organ of Torpedo, Whittaker and his collaborators (Whittaker, Dowdall & Boyne, 1972; Dowdall, Boyne & Whittaker, 1974) have found significant amounts of ATP associated with the synaptic vesicle fraction of tissue homogenates. As the nucleotide in this fraction was protected from the action of nucleotidases and did not exchange with exogenous ATP, these investigators concluded that ATP is contained withia the cholinergic synaptic vesicle and may be necessary for the binding of ACh. In a preliminary report, Silinsky & Hubbard (1973) have demonstrated the efflux of small quantities of ATP as a result of stimulating curarized mammalian nerve-muscle preparations through the nerve supply. Since neither the nerve trunk nor the post-synaptic membrane appeared to be the source of stimulus-specific nucleotide in these studies, it was suggested that the release of ATP accompanies the secretion of ACh from the motor nerve ending. It would thus be of interest to attempt to further localize the source of adenine nucleotide released by nerve impulses from the myoneural junction by determining if alterations in the size and number of ACh quanta released have any detectable effect on the efflux of nucleotide. This present work describes such an investigation made on the isolated rat phrenic nerve-hemidiaphragm preparation using conventional electrophysiological methods in conjunction with a modification of the firefly luciferase assay for ATP (Silinsky, 1974). METHODS

The left phrenic nerve-hemidiaphragm was dissected from 100 g SpragueDawley rats under ether anaesthesia and pinned in a Perspex chamber. The normal bathing medium contained (mM) NaCl, 137; CaCl2, 2; KCl, 5; MgC12, 0-01 or 1; NaH2PO4, 1; glucose, 11; NaHCO., 24 (Gage & Hubbard, 1966) and was bubbled with 95 % 02-5 % CO2 to a pH of 7-3 to 7-4. All preparations were washed with approximately 11. normal solution (flow rate 15-20 ml./min) before applying the test solutions. This was necessary as control experiments indicated that soluble ATP-degrading enzymes are present in the effluent of insufficiently washed diaphragms. In addition to vigorous oxygenation of the perfusion fluid in the feeding reservoir, solutions were oxygenated in two locations within the Perspex chamber in order to maintain a steady state with respect to the ionic composition of the diaphragm (Creese, 1954). All experiments were made at room temperature. Isolated stimulation pulses were delivered to the phrenic nerve by means of a polyethylene suction electrode. The stimulating current was 3-4 times the threshold value for muscle contraction and was monitored at regular intervals when preparations were bathed in high (15 /SM) concentrations of tubocurarine. Conventional

ATP-ADP RELEASE FROM NERVE ENDINGS

147

electrophysiological techniques were employed for both extracellular and intracellular recording. At the beginning of all experiments bipolar silver electrodes were placed on the diaphragm surface at end-plate regions (see Hadju & Knox, 1950), the nerve stimulated at a low rate and the resulting muscle action potentials (m.a.p.s) amplified and displayed on a Tektronix 502 cathode-ray oscilloscope (CRO). M.a.p.s. were monitored at 10-15 min intervals during the entire wash period. Test solutions (which contained, tubocurarine, reduced calcium concentrations, or other agents that depress neuromuscular transmission) were then washed through the bath. This caused a gradual elimination of the m.a.p. (and muscle contraction) leaving the compound end-plate potential (e.p.p.) as the detectable post-synaptic electrical event. At this point the bathing medium was replaced with approximately '2-5 ml. fresh solution. Periods of rest (20 min) were then alternated with periods of nerve stimulation (7/sec for 20 min, i.e. 8400 total stimuli) and the bathing medium removed and assayed within 30 see for ATP and ADP (see below). During removal and replacement of solutions, care was taken to avoid touching the preparation. At the conclusion of the second stimulation period (or in some instances, during a testing period) glass pipette micro-electrodes filled with 3 M-KCl and with resistances ranging from 10 to 20 MO were employed for intracellular recording of e.p.p.s and miniature end-plate potentials (m.e.p.p.s). The micro-electrode was connected to a high input impedance preamplifier which in turn was fed into the CRO. In some experiments micro-electrodes filled with 2 M sodium chloride (resistances from 1-3 MO) were used to record nerve terminal action potentials (n.t.p.s) simultaneously with focal e.p.p.s, the terminal first being located by recording extracellular m.e.p.p.s (Hubbard & Schmidt, 1963). Results were discarded if the m.a.p. did not return to the control level after returning the preparation to the normal bathing medium. For the experiments using hemicholinium-3, the above procedure was modified slightly (see Results). A series of control experiments were made on isolated phrenic nerve trunks using a polyethylene suction electrode for recording nerve action potentials (n.a.p.s) as well as for stimulation. The electrophysiological records presented in the text were photographed using a Polaroid camera. Firefly bioluminescence assay The light emitted from the mixing of a sample containing ATP with buffered firefly lantern extract (FLE) was used to determine nucleotide concentrations. The precise details of the assay system are described elsewhere (Silinsky, 1974). For the convenience of the reader, the main features will be outlined here. The light detection system consisted of an RCA 4516 photomultiplier tube housed in a black Perspex reaction chamber and driven by a stabilized d.c. power supply. The output of the photomultiplier was amplified and then recorded on a Brush-Gould pen recorder. Photographs of actual chart records are presented in the text. After reconstitution with distilled water, 0 43 ml. ice-cold FLE was pipetted in a reaction cuvette which was then placed into the tube housing directly above the photosensitive end-window of the photomultiplier. A syringe was then filled with 0-25 ml. of the sample or nucleotide standard and placed into a holder directly above the cuvette. As Fig. 1 A, s illustrates, upon opening the shutter of the reaction chamber, the base line of the chart record rises. This is because the crude luciferin-luciferase contains endogenous ATP and thus the emission record exhibits a resting luminescence (Fig. 1A, L). Upon injection of 4-2 p-mole ATP dissolved in physiological saline, a diminution of the resting luminescence was observed, followed by a burst of light emission peaking at approximately 1-5-2-0 see and then a decline in the intensity of luminescence. The level of the post-injection inhibition (due predominantly to the chloride content of the solution) marks the beginning of luminescence produced by saline solutions of

EUGENE M. SILINSKY

148

ATP and consequently was used as the base line for all measurements of light emission. Fig. 1 B illustrates light emission produced by the same concentration of ATP as in A, except that low-pass filtering was employed at the input of the recorder. Filtering was used in some instances but often made it difficult to distinguish the post-injection inhibition (and thus base line) from the level of the injection artifact. Confirmation of the precise base line was obtained by injecting either physiological saline into FLE (see legend for Fig. 5 C) or by the observation that 1O sec after ATP injection, the light emission pattern had declined to a level equidistant between the peak and the base line (see Fig. 1 A and B). Fig. 1 E illustrates a typical emission pattern produced by ADP, namely two rising phases of light emission without the presence of a sharp peak. Fig. 1 C and D shows that when ATP and ADP mixtures are assayed, the emission peak is less pronounced and declines much more slowly. The concentration of ATP and ADP in the bathing medium of the preparation was calculated using equations similar to the empirical mathematical expressions presented previously (Silinsky, 1974) and by comparison with standards such as those in Fig. 1. Light emission produced by other nucleoside triphosphates at physiological concentrations does not interfere with determinations of ATP and ADP concentrations. Adenosine monophosphate (AMP) is without effect in this system. L

p-mole ATP

.p-mole ADP

L

4-2 -158

4-2

50

25 158

231

Fig. l. Light emission patterns produced by injecting physiological saline solutions of ATP (A, B), ADP (E), and ATP+ADP mixtures (C, D) into 0-43 ml. FLE. The number of p-mole of injected nucleotide in a volume of 0-25 ml. of solution is indicated beneath each emission record. Vertical calibration = 100 mV for A and B, 200 mV for C, D, and E. Horizontal calibration = 10 sec. In all Figures the moment of injection is indicated by a dot. Opening of the shutter of the reaction chamber is indicated by s. L, resting luminescence of the FLE. Further details in the text. Reagents FLE (types 50 and 250), ATP, ADP, carbachol, protease (Type VII), collagenase (Type I), apyrase (adenosine 5' triphosphatase and diphosphatase) were obtained from the Sigma Chemical Company, hemicholinium-3 from the Aldrich Chemical Co., tubocurarine chloride (TC) from the Nutritional Biochemicals Corporation, and acetylcholine chloride from Merck.

ATP-ADP RELEASE FROM NERVE ENDINGS

149

RESULTS

ATP and ADP efflux at normal levels of ACh release The purpose of this section is to determine whether of not ACh release is accompanied by the release of adenine nucleotide from the motor nerve ending at normal levels of transmitter output. As it has been demonstrated that prolonged depolarization of excitable membranes causes the outflow of adenine nucleotides (Abood, Koketsu & Miyamoto, 1962), it was

p-mole ATP p-mole ADP

Al

Control

0-85 73-5

0-4

57-5

A2 4.5 117.5

Fig. 2. Release of ATP and ADP by nerve impulses from a diaphragm bathed in normal (2 mM) calcium-high (15 /M) tubocurarine solutions. Al and A2 light emission produced by injecting bathing fluid from preparations stimulated at 7/sec for 20 min into FLE. Control-light emission produced by the bathing medium after 20 min of rest. The number of p-mole ATP and ADP in the injected volume (0.25 ml.) is indicated for each record. Resting luminescence for A, = 90 mY, horizontal calibration = 5 sec.

necessary to choose experimental conditions so that nucleotide efflux produced by depolarization would not obscure a possible association between the release of ACh and adenine nucleotide. Consequently, early experiments were made on preparations curarized to the level at which no e.p.p.s (and thus no post-synaptic membrane depolarization) could be detected. Fig. 2 illustrates that ATP and ADP were released by nerve impulses under these conditions. The control record was produced by the bathing fluid of the resting preparation and records A1 and A2 were made after stimulation periods. The amounts of ATP and ADP in 0-25 ml. of bathing medium are illustrated below each light emission record and the total amounts of ATP and ADP released by nerve impulses are presented in Table 1, Al and A2. Similar results were obtained from three other preparations bathed in 15 /tM tubocurarine, a concentration sufficient to eliminate all post-synaptic electrical activity (Table 1, preparations B-D). Experiments on four isolated phrenic nerves failed to reveal any stimulus-specific release of adenine nucleotide, after stimulation at 7/sec for

EUGENE M. SILINSKY 150 20 min. This suggests that the nerve trunk was not the source of nucleotide released by nerve impulses from the diaphragm preparation. Fig. 3 illustrates part of one such result, the amplitude of the compound nerve action potentials (Fig. 3, n.a.p.) remaining essentially unchanged for the duration of the experiment. In these studies on isolated nerve trunks, it was possible to approximate roughly the volume of intramuscular nerve TABLE 1. Release of ATP and ADP by nerve impulses from curarized preparations bathed in normal (2 mm) calcium solutions. Stimuli were presented at a frequency of 7/sec for 20 min (8400 total). Nucleotide increases are total p-mole/2-5 ml. of bathing solution. The tubocurarine concentration was 15/tM for periods A1-D and 1.5 /IM for E-H2. Applying the statistical test of Dixon (1953) to experiment C1 caused the nucleotide increase for this period to be rejected prior to calculating the mrrean ATP + ADP increase

Experimental* period

Al A2 B1 B2 Cl C2 D E F1 F2 F3

G,

ATP increase 4-5 41-0 2-5 1-8 - 2-2 1.9 -2*9

9'8 -0'3 16-3

3.3

G2

5-8

H1 H2

2*0 0-2

ADP increase 160 600 375 200 1040 440 200 485

350 250 325 257 22 435 124

ATP + ADP increase 165 641 378 202 1038 442 200 482 360 250 341 260 28 437 124 Mean 308 S.E. 42

* Letters refer to specific preparations and numbers to stimulation periods.

contained in the diaphragm by using the entire length of the phrenic nerve from its cervical origin to its intramuscular entrance into the diaphragm and by sucking only the very ends of the nerve into the stimulating and recording electrodes. The experiments conducted on the diaphragm preparation were made with a small length (7 mm or less) of free phrenic nerve and this was entirely sucked into the stimulating electrode. Subsequent experiments were performed on lightly curarized diaphragms (1 5 /tM tubocurarine) so that the electrical activity of surface end-plates could be monitored. It was assumed that even if very small amounts of ATP and ADP were released as a result of the transmitter-induced

ATP-ADP RELEASE FROM NERVE ENDINGS 151 depolarization, the localized voltage change at the end-plate would be an insignificant source of nucleotide as compared to the nerve ending. The results described below suggest this to be the case. B

A

S

Control

_

Fig. 3. Absence of stimulus-evoked nucleotide release from an isolated phrenic nerve trunk. A: light emission records, produced after stimulation (S) and after control periods. Resting luminescence = 90 mV, horizontal calibration = 10 sec. B: Superimposed n.a.p.s recorded during the stimulation period.

Fig. 4 illustrates a representative control and three stimulation periods (F1, F2, and F3) from one such preparation bathed in 1-5 /tM tubocurarine. The total stimulus evoked release of ATP and ADP for the three stimulation periods are presented in Table 1. Fig. 4B1 illustrates end-plate activity recorded at the beginning of 7/sec stimulation and Fig. 4B2 illustrates simultaneous intracellular (upper trace) and extracellular (lower trace) e.p.p.s recorded from another preparation at the termination of the last stimulation period. Table 1 E, C, and H presents results from three other diaphragms bathed in 1-5 /%M tubocurarine. As described previously (Silinsky & Hubbard, 1973), nerve stimulation in certain experiments produced an increase in the ADP concentration of the bathing medium without an accompanying increase in ATP (Table 1, periods C1, E and F2). Fig. 5 illustrates such an experiment in which stimulation (B) produces an increase of 482 p-mole ADP over control levels (A). Fig. 50 shows that the effluent from stimulated preparations incubated for 15 min with 1 mg/ml. of the enzyme apyrase (a 5'-nucleotidase which degrades ATP and ADP to AMP) exhibits a light emission record identical to that produced by injecting buffered physiological saline into FLE (see Silinsky, 1974, Fig. 4B). Several experiments made on the hydrolysis of exogenous ATP

EUGENE M. SILINSK Y

152

e A p-mole ATP p-mole ADP._'

2-60 120-0

B

I^I

Fig. 4. Adenine nucleotide release (A) and electrical recording (B) from preparations bathed in normal calcium and 1-5/zM tubocurarine. A: control and stimulation (F1, F2 and F3) periods as before. The number of p-mole ATP and ADP in 0-25 ml. bathing medium is indicated (see also Table 1). Resting luminescence = 100 mV, horizontal calibration = 3 sec. B: 1, extracellular end-plate activity at the beginning of F1. Vertical calibration = 01 mV. 2, simultaneous intracellular (upper) and extracellular (lower) e.p.p.s from another preparation near the end of the second stimulation period. Vertical calibration, upper trace = 0 2 mV, lower trace = 0.1 mV. Horizontel calibration = 2 msec for both 1 and 2. For all electrical recordings from end-plate regions, positivity is signalled upward.

A

p-mole'ATP p-mole ADP

095 58-5

B

C

0 65 107-0

Fig. 5. Stimulus-evoked release of ADP from a curarized diaphragm. A: control. B: stimulation period E of Table 1. C: light emission produced by the effluent in B after 15 min of incubation with apyrase, 1 mg/ml. This emission pattern is identical to those produced by nucleotide-free bathing medium (see Silinsky, 1974, Fig. 4B). The number of p-mole ATP and ADP in 0-25 ml. bathing fluid are indicated. Resting luminescence = 75 nmV, horizontal calibration = 5 sec.

ATP-ADP RELEASE FROM NERVE ENDINGS 153 suggested that part of the variation in ATP release might be accounted for by variations in the activity of hydrolytic enzymes. For example, two preparations where only ADP release was detectable (Table 1E and F) were immersed in a solution containing approximately 40 p-mole ATP for a 20 min period. After removing and subsequently assaying the ATP standard-bathing medium, none of the original 40 p-mole were detectable. Alternately, two other preparations hydrolysed much smaller amounts of exogenous ATP (20-30 % of the amount added remained unhydrolysed). Although the evidence presented thus far suggests that adenine nucleotide might be released from the nerve ending, other less apparent sources of nucleotide must be eliminated before any conclusions can be drawn. For example, a non-specific interaction between ACh and the basement membrane or the cell surface might be releasing ATP or ADP. In this regard it has recently been shown that digestion of the basement membrane by sequential treatment with collagenase and protease allows the amphibian nerve ending to be pulled free of the underlying muscle without any apparent damage to the nerve terminal (Betz & Sakmann, 1973). As preliminary experiments suggest that a similar effect occurs in rat diaphragm (Hall & Kelly, 1971), it was decided to investigate whether nucleotide is released by nerve impulses after enzymatic digestion of the basement membrane. A protocol similar to that used by Betz & Sakmann (1973) was employed (see legend for Fig. 6A) and after the elimination of post-synaptic electrical activity, 2 hr after enzyme treatment was begun, the preparation was returned to the normal bathing medium and stimulation begun at 7/sec. It was possible to detect a difference of 18-5 p-mole ATP between the first determination of the nucleotide concentration in the effluent from the stimulated preparation and the bathing medium of the resting diaphragm. However, as Fig. 6A illustrates, the ATP concentration in the effluent of the stimulated preparation was reduced by 18-5 pmole to the level of the control effluent in the 30 sec period between the first (1) and second (2) determinations. This suggests that ATP-degrading enzymes are present in the bathing medium after prolonged treatment with these proteolytic enzymes, thus complicating the interpretation of the experimental results. In two other preparations, it was observed that the beginning of enzymatic digestion of the basement membrane by collagenase (as detected by an increase in the size of the e.p.p. due to destruction of acetylcholinesterase) was again accompanied by the appearance of ATP-degrading enzymes in the effluent, leading to the termination of this line of investigation. Experiments made on carbachol-bathed diaphragms do, however, argue against the possibility that nucleotide efflux is a secondary consequence of ACh release. First, in preparation B of Table 1, two 20 min periods of

EUGENE M. SILINSKY 154 carbachol (1 mM) exposure failed to produce detectable increases in ATP or ADP in the effluent. In three other preparations bathed in 30 #cm carbachol (the concentration of tubocurarine ranging from 0 to 1.5 /M) no carbachol-induced nucleotide release was observed in a total of seven bathing periods. One such experiment is illustrated in Fig. 6B. In two other experiments, ACh (5 #m) failed to evoke nucleotide release from curarized preparations, thus precluding the remote possibility that ATP efflux is a consequence of ACh hydrolysis. B

A Ai|

1

M&- L.

2

Control

Carbachol

Fig. 6. Effects of protease-collagenase (A) and carbachol (B) on the release of ATP and ADP. A: the preparation was treated sequentially with collagenase, 1 mg/ml. for 1 hr followed by exposure to protease, 0-1 mg/ml. for an additional hr. The diaphragm was then returned to the normal physiological saline and stimulated at 7/sec for 20 min. (1) effluent from stimulated preparation. (2) second determination of light emission from the effluent in 1. It should be noted that approximately 18-5 p-mole ATP were hydrolysed in the 30 sec interval between determination 1 and 2. Record 2 is identical with the post-stimulation control. Resting luminescence = 100 mV, horizontal calibration = 5 sec. B: absence of detectable difference in nucleotide release between control and after 20 min of exposure to 30 IM carbachol. No tubocurarine was present in this experiment.

The results therefore suggest that adenine nucleotide is released from the motor nerve ending at normal levels of ACh output.

Effects of reduction in the number of ACh quanta released on the efflux of ATP and ADP It has been shown previously that reducing the ambient calcium concentration from 2 to 0-1 mm reduces the mean quantal content of the e.p.p. in rat diaphragm from approximately 200 to 1 (Hubbard, Jones & Landau, 1968; Hubbard, 1970). Consequently, preparations were bathed in low calcium solutions (either with or without 0-15 #M tubocararine), periods of rest alternated with periods of nerve stimulation as before and the bathing

ATP-ADP RELEASE FROM NERVE ENDINGS 155 medium assayed for adenine nucleotide. Fig. 7B illustrates the typical result with five such preparations bathed in low calcium, namely that nerve stimulation failed to release ATP or ADP in the absence of muscle contraction. No decrease in the hydrolysis of exogenous ATP was observed as a result of reducing the calcium concentration of the bathing medium from 2 to 0-1 mM in two experiments. Fig. 7 A shows representative extracellular (upper) and intracellular (lower) e.p.p.s and illustrates that B

A

= 01 S

Control

Fig. 7. Electrophysiological recording (A) and nucleotide release (B) from preparations bathed in low (0.1 mm) calcium solutions. A: upper trace, extracellular e.p.p. recorded during the last min of a 20min period of 7/sec stimulation. The tubocurarine concentration was 0 j15 #M. Calibration = 0-1 mV, 5 msec. Lower trace, intracellular e.p.p. recorded during 40/sec stimulation after the second period of 7/sec stimulation in another experiment. No tubocurarine was present in the bathing solution. Calibration = 1 mV, 20 msec. It is noteworthy that both intracellularly and extracellularly recorded e.p.p.s are well maintained in low calcium solutions. B: light emission patterns produced after stimulation (S) and control periods. Low-pass filtering was used for record S. Resting luminescence = 125 mV. Horizontal calibration = 5 sec.

although ACh release is markedly reduced, the e.p.p. (and thus invasion of the nerve action potential into the terminal) is well maintained even during 40/sec stimulation (lower record). These results demonstrate that the large decline in the number of ACh quanta released by nerve impulses in low calcium is accompanied by a similar reduction in the release of adenine nucleotide. The release of ATP by nerve impulses from preparations bathed in low calciumhypertonic solutions was described previously (Silinsky & Hubbard, 1973). It is of

156

6EUGENE M. SILINSKY

interest that two preparations which failed to release ATP as a consequence of nerve stimulation when bathed in low calcium solutions of normal tonicity did release additional ATP when stimulated through the nerve supply in a bathing medium of increased tonicity. This suggests that perhaps the elevated osmotic pressure had reduced the activity of ATP-degrading enzymes. One preparation in which hypertonicity reversibly reduced the hydrolysis of exogenous ATP by approximately 50 p-mole in 20 min supports this contention. All experiments to this point were made on non-contracting preparations. In the early stages of washing one preparation in low calcium solution, before muscle contraction was abolished, nerve stimulation produced an increase of 709 p-mole adenine nucleotide over control levels. Although the exact source of this nucleotide is uncertain, this result is consistent with previous suggestions that ATP is released from contracting human muscle (Forrester & Lind, 1969; Forrester, 1972).

Bathing two preparations in solutions with no added calcium ('calciumfree') produced a detectable decrease in the hydrolysis of exogenous ATP. Consequently two diaphragms were studied in calcium-free media. Stimulation at 7/sec in these experiments resulted in a high percentage of e.p.p. failures as well as small increases in the frequencies of the m.e.p.p. However, stimulus-evoked release of adenine nucleotide was not observed under these conditions.

Effects of reduction in quantal size on the release of ATP and ADP Studies on rat diaphragm preparations have suggested that prolonged nerve stimulation in the presence of HC-3 produces large reductions in the size of the ACh quanta (Elmqvist & Quastel, 1965; Jones & Kwambumbumpen, 1970). It would thus be advantageous to investigate the effects of HC-3 on the release of ATP and ADP. After dissecting preparations in physiological saline containing 5 x 10-5 M-HC-3 and recording m.e.p.p.s intracellularly from different end-plate regions (Fig. 8 A1, insert) continuous nerve stimulation was began at a frequency of 11/sec and the compound m.a.p. recorded (Fig. 8 A1). When muscle contraction was eliminated, intracellular e.p.p.s (Fig. 8 A2, lower trace) were recorded simultaneously with the extracellular e.p.p. (Fig. 8 A2, upper trace). After 80-90 min of nerve stimulation at 11/sec, when all detectable evoked and spontaneous post-synaptic electrical activity was eliminated (Fig. 8 A3), stimulation was begun at 7/sec and the bathing medium removed and assayed for ATP and ADP. Fig. 8B, illustrates light emission records produced by injecting the bathing medium of the stimulated (S1) and resting (control) preparation. In all five preparations studied in the presence of HC-3, nerve impulses failed to release ATP or ADP after the electrophysiological signs of ACh release were eliminated. The brief rest afforded the preparation during the 20 min control period produced detectable post-synaptic electrical activity for only 1 see after stimulation was renewed. Fig. 8 B2 illustrates the first sec of a typical

157

ATP-ADP RELEASE FROM NERVE ENDINGS 1

3

2

rn-El.

T

S2 I

2

Fig. 8. Experiments made in 5 x 10-5 M-HC-3. A: 1, compound m.a.p. recorded from the end-plate region. Calibration = 0.5 mV, 2 msec. Insert spontaneous m.e.p.p.s recorded intracellularly at the beginning of an experiment. Calibration = 1 mV, 20 msec. 2, same experiment as 1 after 60 min of 11/sec stimulation in the presence of HC-3. intracellular (lower trace) and extracellular (upper trace) e.p.p.s recorded simultaneously with the extracellular record, the latter being from the same end-plate region as 1. Approximately 11 superimposed sweeps are shown. Calibration, upper trace = 0.1 mV, lower trace = 0 5mV, 2 msec. 3, absence of extracellular (upper) and intracellular (lower) e.p.p.s after 80min of 11/sec stimulation in HC-3. B: 1, nerve terminal action potential (n.t.p.) and focal e.p.p.s recorded after 70 (upper) and 80 (lower) min of 11/sec stimulation in HC-3. Note that in the lower trace, the focal e.p.p. was eliminated while the n.t.p. remained unchanged. Calibration, 0-2 mV, 1 msec. S1 and S21 light emission produced by the bathing media of the stimulated preparation (7/sec for 20 min). Control produced by bathing medium of resting preparation. Resting luminescence = 120 mV. 2, the first sec of extracellularly recorded post-synaptic electrical activity after resumption of stimulation following 20 min of rest. Calibration = 0-2 mV, 2 msec. See text for additional details.

EUGENE M. SILINSKY stimulation period made after 20 min of rest. During the first three stimuli, the compound m.a.p. is reduced to an e.p.p. which is maintained only for three subsequent stimuli before being eliminated. Thus the majority of the 8400 stimuli would not be releasing detectable amounts of ACh and, as illustrated in Fig. 8B, S2, failed to release additional amounts of ATP or ADP as well. It has been suggested that HC-3 may depress nerve conduction in isolated frog nerves (Frazier, 1968). Although the concentrations of HC-3 and stimulation frequencies used in the amphibian studies are not directly applicable to the mammalian preparation, it was decided to investigate the possibility that nerve conduction was impaired in the present experiments. Consequently in three preparations, nerve terminals were located extracellularly (see Methods) and the n.t.p. and focal e.p.p. monitored during the entire period of stimulation in HC-3. In all three experiments, the n.t.p. remained essentially at the control level at a time when the e.p.p. was eliminated. One such experiment is illustrated in Fig. 8 B1, the upper photograph taken at 80 min and the lower at 90 min after stimulation was begun at 11/sec. Furthermore, in one of these preparations it was possible to record action potentials from eight different fine intramuscular nerve branches, presumably at or near the nerve terminal after all postsynaptic electric activity was eliminated. These results suggest that HC-3 is blocking transmission from nerve to muscle rather than disturbing propagation of the action potential into the nerve ending. In one preparation, the stimulation rate was reduced from 11/sec to 7/sec approximately 60 min after the beginning of nerve stimulation, a time when moderate sized e.p.p.s were still detectable. In this instance, nerve stimulation for 20 min produced an increase of 510 p-mole ATP + ADP over control levels. At a later time in this same experiment, when e.p.p.s and m.e.p.p.s were no longer observed, nerve stimulation failed to produce nucleotide release. The results presented here suggest, therefore, that large reductions in the number and size of the released ACh quantum eliminate the release of detectable amounts of ATP and ADP. 158

DISCUSSION

It is evident from these experiments that stimulation of the curarized phrenic nerve-hemidiaphragm through the nerve supply consistently causes the release of adenine nucleotide when preparations are bathed in normal calcium solutions. Under the present conditions, neither the nerve trunk, preterminal intramuscular nerve, post-synaptic membrane nor a secondary action of released ACh are responsible for the stimulus-specific release of

ATP-ADP RELEASE FROM NERVE ENDINGS 159 ATP and ADP. These results in conjunction with control experiments estimating the degree of hydrolysis of exogenous ATP suggest that ATP is released from the motor nerve ending and is subsequently being degraded into ADP by enzymatic activity. In low calcium solutions the number of ACh quanta released by nerve impulses is markedly reduced without an accompanying reduction in the hydrolysis of exogenous ATP. The absence of any detectable stimulusevoked release of ATP or ADP at such low levels of ACh secretion would therefore be quite expected if ACh and ATP are released together in stoichiometric amounts. Although other interpretations are possible, the experiments made in the presence of HC-3 suggest that in the diaphragm preparation, the release of ATP is intimately associated with the quantum of ACh. Is it thus possible that ATP is released together with ACh from the synaptic vesicle? Indeed, the ATP concentration in peripheral cholinergic vesicles is quite high (0.1 M, A. F. Boyne, personal communication) and therefore the vesicle would be a likely compartment from which ATP secretion might occur. Zimmermann & Whittaker (1974) have recently demonstrated that prolonged stimulation of Torpedo nerve endings produced a parallel fall in vesicular ACh and vesicular ATP. It would thus be of interest to compare the ratio of ACh/ATP released in the present studies on mammalian tissue to the ACh/ATP ratios detected in the cholinergic vesicles of Torpedo. The mean ATP +ADP release per phrenic nerve impulse in 2 mm calcium was approximately 3'7 x 10-14 mole (Table 1). Using Krnjevi6 & Mitchell's (1961) estimate for the number of end-plates per hemidiaphragm (104) produces an average of 3-7 x 10-18 mole or 22 x 105 molecules of ATP + ADP released by a nerve impulse per end-plate. Estimates of the number of ACh molecules released by a nerve impulse per end-plate in this preparation range from 14-4 x 105 to 90 x 105 (see review by Hubbard, 1970). Remarkably, the mean ratio of ACh/ATP detected in the cholinergic synaptic vesicles of stimulated Torpedo nerve endings was approximately 5/1 (Dowdall et at. 1974). The simplest explanation for these results is that ATP is packaged within the cholinergic synaptic vesicles in the mammalian motor nerve terminal and is released together with ACh by the exocytotic discharge of the vesicular contents. Whether ATP release necessarily accompanies the secretion of ACh from motor nerve endings in all circumstances or whether the association between ACh and nucleotide is a general property of cholinergic systems is by no means certain. Abood et al. (1962) have suggested that ATP is released from stimulated frog nerve. The large quantities of nervous tissue employed in these amphibian studies as well as the 50/sec stimulation rate continued for extended periods are by no means comparable to the 1-2 mg rat phrenic nerve stimulated at 7/sec. 6 P HY 247

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EUGENE M. SILINSKY

Electrical stimulation of the phrenic nerve trunk causes action potentials to invade sensory nerve endings antidromically. As such endings are generously supplied with mitochondria (Katz, 1961), it might be argued that depolarization of the sensory nerve ending liberates adenine nucleotide. It would be difficult to justify such an argument merely on quantitative grounds as sensory nerve endings are quite rare in this preparation (see Yellin, 1974, Fig. 1). Moreover, in the experiments conducted in low calcium and in those made in the presence of HC-3, action potentials were propagating down the nerve trunk, through the intramuscular nerves and into the fine terminal branches yet neither ATP nor ADP were released under these conditions. It thus appears that in this present study nucleotide release by nerve impulses is associated with the process of transmitter release. Kato, Katz & Collier (1974) in experiments on cat superior cervical ganglion have concluded that ATP is not released together with ACh from the preganglionic nerve endings. These studies do not, however, exclude the possibility that ATP might be released from the preganglionic nerve ending and subsequently degraded before entering the vasculature. Perfusing known quantities of ATP through the blood vessels and assaying the perfusion medium for degradation of the nucleotide may not be an adequate hydrolysis control for, if ATP is released from the nerve ending into an extracellular compartment (the synaptic cleft), it must then cross capillary endothelium to enter the vascular system. As endothelia have large concentrations of enzymes that degrade ATP to adenosine (for references see Burnstock, 1972) which would not be detectable by the luciferase assay used by Kato et al. the failure to observe nucleotide release may be a technical problem. In discussing their results, Kato et al. have concluded that the experiments of Silinsky & Hubbard (1973) may have demonstrated the release of ATP from contracting muscle. Such an interpretation is indeed puzzling since all post-synaptic electrical activity was eliminated in the latter study on the diaphragm.

Discussions of the many possible functions for released ATP and other phosphates at the neuromuscular junction (e.g. as a 'trophic' factor, as a means of increasing blood flow to active muscle, etc.) have been presented previously and will not be repeated here (see Drachman, 1971 and Forrester, 1972 for references). Of more direct interest, however, is the possibility that ATP may be involved in controlling the release of ACh from the motor nerve ending. Ribeiro & Walker (1973) have recently demonstrated that ATP in concentrations as small as 0-1 mm depressed the release of ACh by approximately 50 %, an effect independent of the calcium chelator action of ATP. Interpreting their results in conjunction with the results of Silinsky & Hubbard (1973), Ribeiro & Walker have suggested that ATP may be involved in a negative feed-back mechanism whereby, under conditions of repetitive stimulation, sufficient ATP is released from the motor nerve ending to act back on the terminal and depress the release of transmitter by subsequent stimuli. From the results presented here, if a nerve impulse releases 3-7 x 10-18 mole ATP into a synaptic cleft of 450/1m3 (Salpeter & Eldefrawi, 1973) the transient concentration of ATP would be 0-08 mm. It is thus possible that the ATP concentration in the synaptic cleft after brief repetitive stimulation could be as high as 0-1 mm. Of interest in this regard are the experiments

ATP-ADP RELEASE FROM NERVE ENDINGS 161 of Abood & Matsubara (1968) suggesting that nerve endings in rat brain contain an ATP-binding protein which is not dependent upon divalent cations nor is involved in ATP-linked enzymatic activity. It is quite conceivable, therefore, that small molecules such as ATP after being released from the nerve ending affect a specific protein component in the nerve ending perhaps in an allosteric manner, and depress the subsequent release of ACh. Although one cannot conclude that ATP regulates the release of ACh, such a mechanism could indeed explain some of the paradoxical results that have arisen from attempts to reconcile quantitative determinations of neuromuscular depression with the simple vesicle hypothesis (see Ginsborg, 1970 for review). I wish to thank J. R. Musick and Drs R. E. Ten Eick, D. M. J. Quastel, R., Spehlmann, and D. H. Singer for the loan of equipment and discussion regarding the

manuscript. REFERENCES

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On the association between transmitter secretion and the release of adenine nucleotides from mammalian motor nerve terminals.

247, J. Phy~iol. (1975), pp. 145-162 With 8 text -ft gure8 Printed in Great Britain 145 ON THE ASSOCIATION BETWEEN TRANSMITTER SECRETION AND THE RE...
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