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Non-quantal acetylcholine release at mouse neuromuscular junction: Effects of elevated quantal release and aconitine
Neuroscience Letters, 117 (1990) 111-116 Elsevier Scientific Publishers Ireland Ltd.
111
NSL 07120
Non-quantal acetylcholine release at mouse neuromuscular junction: effects of elevated quantal release and aconitine Shan Ping Yu* and William Van der K l o o t Departments of Physiology and Biophysics, and of PharmacologicalSciences, Health Sciences Center SUNY, Stony Brook, NY 11794 (U.S.A.) (Received 26 March 1990; Revised version received 7 May 1990; Accepted 7 May 1990)
Keywords: Neuromuscular junction; Quanta; Acetylcholine; Aconitine; Ouabain; Voltage clamp The rate of non-quantal acetylcholine (ACh) release was estimated at the mouse neuromuscular junction by observing the effect of (+)-tubocurarine on endplate membrane potential or current in preparations pretreated with an irreversible anti-acetylcholinesterase (anti-AChE). Voltage clamping was an effective method for measuring nonquantal release. Non-quantal release was markedly inhibited by 10 gM aconitine. Non-quantal release was not significantly increased by 10 gM dihyroouabain (DHO). (It has been reported that ouabain increases the leak.) Non-quantal release was roughly doubled following exposure to hypertonic solution or to elevated K+-solution. This is in accord with the hypothesis that the leak is by way of ACh transporters incorporated into the terminal membrane following exocytosis, but other interpretations remain to be tested.
Our measurements of the non-quantal leak of acetylcholine (ACh) from mouse phrenic nerve terminals were undertaken as part of a larger project on the regulation of quantal size. For reasons that will become clear, the measurements could contribute little to our goal of understanding the mechanism for the quantal size increase. Nevertheless we thought the results of interest because they show the feasibility of studying the leak with the voltage clamp, reveal some unexpected drug effects and show that the previous treatment of the preparation with hypertonic solution or elevated K + produced subsequent changes in the leak. We are reporting these results because we will be unable to do further experiments together on this subject. The amount of ACh released by an isolated diaphragm treated with an anti-AChE far exceeded the amount that could be accounted for by the spontaneous MEPPs *Present address: Howard Hughes Medical Institute, Dept. of Neurobiology and Behavior, .SUNY, Stony Brook, NY 11794, U.S.A. Correspondence." W. Van der Kloot, Department of Physiology & Biophysics, Health Sciences Center, SUNY, Stony Brook, NY 11794, U.S.A. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
112 [7, 4]. Much of the spontaneous release is in the form of nonquantal leak, which was detected electrophysiologically by observing the effect of curare on the endplate. Katz and Miledi [5] in frog applied curare by ionophoresis, recording a hyperpolarization of about 50 I~V. Vysko6il and Ill6s [11, 12] recorded hyperpolarizations in the millivolt range in mouse diaphragm. The hyperpolarization is called the H-effect. Most of the subsequent studies on mammals were done by measuring the difference between the resting membrane potentials of the endplate zone and of the extrajunctional region [14]. We have been investigating treatments that increase quantal size at the mouse neuromuscular junction. Quantal size is roughly doubled after the diaphragm is soaked for 30 min in hypertonic solution. A variety of experiments suggest that size increased because additional ACh is loaded into the quanta shortly before release, and that this additional ACh comes from a recently synthesized, presumably cytoplasmic store [15, 17]. If the leak is proportional to [ACh] in the cytoplasm, we thought that measuring the H-effect might contribute to our understanding. When we undertook the measurements we did not know that the quantal size increase is blocked when acetylcholinesterase (ACHE) is inhibited, presumably because the supply of choline required for ACh synthesis is diminished [17]. Since the experiments on the H-effect must be done with AChE inhibited, they can tell us little about the mechanism of quantal size increase. All experiments were performed on healthy Swiss Webster mice, weighting 1824 g. Both sexes were randomly used. The animals were sacrificed by cervical dislocation and the dissection of the diaphragm was finished within 5 min after decapitation. The right and left side of the diaphragm were separated into two preparations. Standard Tyrode solution contained (in mM): NaC1 135, KC1 5, CaC12 2, MgC12 1, NaHePO4 1, NaHCO3 15, glucose 11. A gas mixture of 95% O2 and 5% CO2 continuously bubbled through the solution, pH was 7.2 -7.4. Hypertonic solution contained 235 mM NaC1 with the other constituents unchanged. In 15 mM K ~-solution, KCI replaced an equivalent amount of NaC1. Preparations were incubated in 10 #M DFP (diisopropyl fluorophosphate; Sigma) for 30 min and then returned to Tyrode. The preparation was then pinned to the silicone rubber base of a plastic chamber containing about 10 ml of Tyrode solution. The solutions flowed into the experimental chamber at about 2 ml/min, temperature was 30°C (_+ I'~C). A 100 #M (+)-tubocurarine ((+)-TC) solution was in a micropipette with a tip diameter of 10-20/tm. At an appropriate time, the micropipette was lowered into the solution near the endplate region where the membrane potential or current was being monitored. ( + ) - T C diffused from the pipette tip onto the endplate region. Membrane potential or current was measured by intracellular recording or two microelectrode voltage clamping using a Degan or Axon clamp. High gain recording with a bandwidth from DC-1000 Hz was accomplished by using an Offset Subtracter (Axon) for subtracting the baseline. The H-effect we measured at normal mouse diaphragm neuromuscular junctions was a hyperpolarization of 1.30 +_0.17 mV (S.E.M., n = 39) (Fig. 1). In voltage clamp experiments, when the membrane potential of the end-plate region was clamped at
113
A
.
.
.
.
.
.
.
=.-----
2 .
.
.
.
.
.
1 (+)-TC
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
,
,]2mV
10 sec
Fig. 1. The effect of curare on the membrane potential at an endplate in the mouse diaphragm with and without AChE inhibition. A: without treatment with an anti-AChE. At the time of the arrow (+)-TC was applied to the endplate area as described in Methods. Note that the MEPPs disappeared after the application of (+)-TC but there was no shift in the base line, presumably because the ACh leaking from the terminal was promptly hydrolyzed by ACHE. B. The preparation was soaked for 30 min in 10/~M DFP and then washed with Tyrode. The application of (+)-TC caused the MEPPs to disappear and the endplate to hyperpolarize (the H-effect). When the ( + )-TC-containing pipette was moved away the membrane potential returned toward the resting level.
- 70 m V the ( + ) - T C reduced i n w a r d c u r r e n t b y 6.0 + 0.43 n A (n = 5) (Fig. 2). W h e n c l a m p e d at - 2 0 m V ( + ) - T C reduced i n w a r d c u r r e n t by 0.9 + 0.83 nA. P r e s u m a b l y the c h a n g e in c u r r e n t is less at - 2 0 m V because the m e m b r a n e p o t e n t i a l is closer to the reversal p o t e n t i a l a n d the A C h - g a t e d e n d p l a t e channels have a s h o r t e r m e a n o p e n time when the fiber is d e p o l a r i z e d . In mouse, increases in q u a n t a l size are b l o c k e d by t r e a t m e n t s t h a t should increase a x o p l a s m i c [Na +] [17]; similar results have been r e p o r t e d in frog [10]. In m o u s e there is evidence t h a t the rise in [Na +] acts by decreasing choline u p t a k e , which d e p e n d s on the N a + g r a d i e n t [16]. A t 10 l t M d i h y d r o o u a b a i n slightly increased the H-effect, b u t n o t significantly (Fig. 3). Vysko~il a n d Ill6s [11] r e p o r t e d t h a t the H-effect was significantly increased b y 20/~m o u a b a i n ; o u a b a i n is s u b s t a n t i a l l y less effective as an
114
10 nA L
los Fig. 2. The effect of ( + )-TC on the holding current at a voltage-clamped endplate in the mouse diaphragm. The endplate was clamped at - 70 inV. Application of ( + )-TC eliminated the miniature endplate currents (MEPCs) and reduced the inward current. The apparent increase in holding current following the removal of the ( + )-TC is unexplained but did not occur in all examples.
N a + - K + p u m p i n h i b i t o r t h a n d i h y d r o o u a b a i n . W e h a v e n o e x p l a n a t i o n for these a p p a r e n t differences b e t w e e n the t w o sets o f e x p e r i m e n t s . A c o n i t i n e ( 1 0 / i M ) , w h i c h o p e n s s o d i u m c h a n n e l s [9], d e c r e a s e d t h e H - e f f e c t by 80% (Fig. 3). In the p r e s e n c e o f a c o n i t i n e m i n i a t u r e e n d p l a t e p o t e n t i a l ( M E P P ) a m p l i t u d e s w e r e n o r m a l , so it d o e s n o t a p p e a r to h a v e a p o s t j u n c t i o n a l effect. Since i n h i b i t i o n o f the N a + - K + e x c h a n g e p u m p did n o t i n h i b i t t h e H-effect, it is u n l i k e l y t h a t the effect o f a c o n i t i n e is d u e to a rise in i n t r a c e l l u l a r s o d i u m . P e r h a p s a c o n i t i n e
E;~ Hyperpolorizetion (mV) 0
1
2
3
4-
I
I
I
I
5 - -
°=39
e). Control
,
b). Hypertonie
n=31
:~
c). DHO n=lO
d). Aconitine
,k n=24
e). Control
n=26
f). 15 mM KCl
o
1'o
l's
& Membrene current (nA) Fig. 3. A summary of the effects of the treatments tested on the H-response (striped bars) and the endplate current (filled bars) during ( + )-TC application. The bars show the mean and the error bars show the SEM. *Mean is significantly different from the control. All preparations were pretreated with DFP. a: the membrane hyperpolarization produced by (+)-TC application. This is the control for the potential measurements, b: the response in preparations pretreated for 30 min in hypertonic solution. The recordings were made in Tyrode. c: in Tyrode containing 10 pM DHO (dihydroouabain). d: in Tyrode containing 10 pM aconitine, e: the decrease in inward holding current caused by ( + )-TC application (controls for the current measurements), f: the change in current in preparations in 15 mM K ÷-solution.
115
directly blocks the ACh transport system responsible for the H-effect. Aconitine should be tested as a possible inhibitor of vesicular ACh uptake and its effects on the chemically measured leak should also be determined. Hypertonic treatment (30 min in 235 mM NaC1 solution) roughly doubled the Heffect (Fig. 3). After 30 min in 15 mM potassium solution and return to Tyrode the H-response was again about doubled (Fig. 3). The major conclusion is that pretreatment with either hypertonic solution or elevated K+-solution can alter the subsequent H-response. Both treatments markedly increase the rate of spontaneous quantal release. Edwards et al. [3] and VyskoOl et al. [14] suggested that the ACh leak might be through vesicular membrane, inserted into the nerve terminal when synaptic vesicles fuse during exocytosis, because non-quantal release is blocked by vesamicol, an inhibitor of ACh transport into isolated synaptic vesicles [1, 2]. Our results are compatible with this hypothesis, but other interpretations are possible and have not been ruled out. Meriney et al. [6] studied the leak at frog neuromuscular junctions by using outside-out patches of AChR-rich membrane as a detector. They did not detect an increase in non-quantal release following stimulation, and believe that the spontaneous release detected by their patch near the nerve terminal was far too low to account for the leak measured biochemically. Such experiments should be repeated at mouse neuromuscular junction, where the leak is many times greater. It is also worth noting that Meriney et al. [6] inactivated AChE by exposure to collagenase. This treatment should be studied to see whether the H-effect is detectable; there is some evidence that the leak is increased following inhibition of intracellular AChE [8], which may favor the accumulation of ACh in the cytoplasm. Supported by Grant 10320 from the NINDS. 1 Anderson, D.C., King, S.C. and Parsons, S.M., Inhibition of [3H]acetylcholine active transport by tetraphenylborate and other anions, Mol. Pharmacol., 24 (1983) 55-59. 2 Anderson, D.C., King, S.C. and Parsons, S.M., Pharmacological characterization of the acetylcholine transport system in purified Torpedoelectric organ synaptic vesicles, Mol. Pharmacol., 24 0983) 48-54. 3 Edwards, C., Dole~al, V., Tu~ek, S., Zemkovfi, H. and Vysko~il, F., Is an acetylcholine transport system responsible for nonquantal release at the rodent myoneural junction?, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 3514-3518. 4 Fletcher, P. and Forrester, T., The effect of curare on the release of acetylcholine from mammalian motor nerve terminals and an estimate of quantum content, J. Physiol., 251 (1975) 131-144. 5 Katz, B. and Miledi, R., Transmitter leakage from motor nerve endings, Proc. R. Soc. Lond. Ser. B., 196 (1977) 59-72. 6 Meriney, S.D., Young, S.H. and Grinnell, A.D., Constraints on the interpretation of nonquantal acetylcholine release from frog neuromuscular junctions, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 20982102. 7 Mitchell, J.F. and Silver, A., The spontaneous release of acetylcholine from the denervated hemidiaphragm of the rat, J. Physiol., 165 (1963) 117-129. 8 Molenaar, P.C. and Polak, R.L., Is the non-quantal resting release of ACh from the isolated rat diaphragm an artifact due to cholinesterase inhibition? In M.J. Dowdall and J.N. Hawthorne (Eds.), Cellular and Molecular Basis of Cholinergic Function, Ellis Horweed, U.K., 1987, pp. 221-225.
116 9 Schmidt, H. and Schmitt, O., Effect of aconitine on the sodium penetration of the node of Ranvier, Pflfigers Arch. Eur. J. Physiol., 349 (1974) 133 148. 10 Van der Kloot, W., The packing of acetylcholine into quanta at the frog neuromuscular junction is inhibited by increases in intracellular sodium, Pfliigers Arch. Eur. J. Physiol., 412 (1988), 258 263. 1 I Vysko6il, F. and ll16s, P., Non-quantal release of transmitter at mouse neuromuscular junction and its dependence on the activity of Na ~-K ~ ATPase, Pflfigers Arch. Eur. J. Physiol., 370 (1977), 295 297. 12 Vysko6il, F. and Ill6s, P., Electrophysiological examination of transmitter release in non-quantal form in the mouse diaphragm and the activity of membrane ATPase, Physiol. Bohemoslov.. 27 (1978), 449 455. 13 Vysko6il, F., Nikolsky, E. and Edwards, C., An analysis of the mechanisms underlying the non-quantal release of acetylcholine at the mouse neuromuscular junction, Neuroscience, 9 (1983) 429 435. 14 Vysko~il, F., Zemkov~., H. and Edwards, C., Non-quantal acetylcholine release. In LC. Sellin, R. Libelius and S. Thesleff(Eds), Neuromuscular Junction, Elsevier, Amsterdam, 1989, pp. 197 205. 15 Weiler, M., Roed, I.S. and Whittaker, V.P., The kinetics of acetylcholine turnover in a resting cholinergic nerve terminal and the magnitude of the cytoplasmic compartment, J. Neurochem., 38 (1982), 1187 1191. 16 Yamamura, H.I. and Snyder, S.H., High-affinity transport of choline into synaptosomes of rat brain, J. Neurochem., 21 (1973), 1355-1374. 17 Yu, S.P., The Regulation of Quantal Size at the Mouse Neuromuscular Junction, Ph.D. Thesis, State University of New York at Stony Brook, 1990.
111
NSL 07120
Non-quantal acetylcholine release at mouse neuromuscular junction: effects of elevated quantal release and aconitine Shan Ping Yu* and William Van der K l o o t Departments of Physiology and Biophysics, and of PharmacologicalSciences, Health Sciences Center SUNY, Stony Brook, NY 11794 (U.S.A.) (Received 26 March 1990; Revised version received 7 May 1990; Accepted 7 May 1990)
Keywords: Neuromuscular junction; Quanta; Acetylcholine; Aconitine; Ouabain; Voltage clamp The rate of non-quantal acetylcholine (ACh) release was estimated at the mouse neuromuscular junction by observing the effect of (+)-tubocurarine on endplate membrane potential or current in preparations pretreated with an irreversible anti-acetylcholinesterase (anti-AChE). Voltage clamping was an effective method for measuring nonquantal release. Non-quantal release was markedly inhibited by 10 gM aconitine. Non-quantal release was not significantly increased by 10 gM dihyroouabain (DHO). (It has been reported that ouabain increases the leak.) Non-quantal release was roughly doubled following exposure to hypertonic solution or to elevated K+-solution. This is in accord with the hypothesis that the leak is by way of ACh transporters incorporated into the terminal membrane following exocytosis, but other interpretations remain to be tested.
Our measurements of the non-quantal leak of acetylcholine (ACh) from mouse phrenic nerve terminals were undertaken as part of a larger project on the regulation of quantal size. For reasons that will become clear, the measurements could contribute little to our goal of understanding the mechanism for the quantal size increase. Nevertheless we thought the results of interest because they show the feasibility of studying the leak with the voltage clamp, reveal some unexpected drug effects and show that the previous treatment of the preparation with hypertonic solution or elevated K + produced subsequent changes in the leak. We are reporting these results because we will be unable to do further experiments together on this subject. The amount of ACh released by an isolated diaphragm treated with an anti-AChE far exceeded the amount that could be accounted for by the spontaneous MEPPs *Present address: Howard Hughes Medical Institute, Dept. of Neurobiology and Behavior, .SUNY, Stony Brook, NY 11794, U.S.A. Correspondence." W. Van der Kloot, Department of Physiology & Biophysics, Health Sciences Center, SUNY, Stony Brook, NY 11794, U.S.A. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
112 [7, 4]. Much of the spontaneous release is in the form of nonquantal leak, which was detected electrophysiologically by observing the effect of curare on the endplate. Katz and Miledi [5] in frog applied curare by ionophoresis, recording a hyperpolarization of about 50 I~V. Vysko6il and Ill6s [11, 12] recorded hyperpolarizations in the millivolt range in mouse diaphragm. The hyperpolarization is called the H-effect. Most of the subsequent studies on mammals were done by measuring the difference between the resting membrane potentials of the endplate zone and of the extrajunctional region [14]. We have been investigating treatments that increase quantal size at the mouse neuromuscular junction. Quantal size is roughly doubled after the diaphragm is soaked for 30 min in hypertonic solution. A variety of experiments suggest that size increased because additional ACh is loaded into the quanta shortly before release, and that this additional ACh comes from a recently synthesized, presumably cytoplasmic store [15, 17]. If the leak is proportional to [ACh] in the cytoplasm, we thought that measuring the H-effect might contribute to our understanding. When we undertook the measurements we did not know that the quantal size increase is blocked when acetylcholinesterase (ACHE) is inhibited, presumably because the supply of choline required for ACh synthesis is diminished [17]. Since the experiments on the H-effect must be done with AChE inhibited, they can tell us little about the mechanism of quantal size increase. All experiments were performed on healthy Swiss Webster mice, weighting 1824 g. Both sexes were randomly used. The animals were sacrificed by cervical dislocation and the dissection of the diaphragm was finished within 5 min after decapitation. The right and left side of the diaphragm were separated into two preparations. Standard Tyrode solution contained (in mM): NaC1 135, KC1 5, CaC12 2, MgC12 1, NaHePO4 1, NaHCO3 15, glucose 11. A gas mixture of 95% O2 and 5% CO2 continuously bubbled through the solution, pH was 7.2 -7.4. Hypertonic solution contained 235 mM NaC1 with the other constituents unchanged. In 15 mM K ~-solution, KCI replaced an equivalent amount of NaC1. Preparations were incubated in 10 #M DFP (diisopropyl fluorophosphate; Sigma) for 30 min and then returned to Tyrode. The preparation was then pinned to the silicone rubber base of a plastic chamber containing about 10 ml of Tyrode solution. The solutions flowed into the experimental chamber at about 2 ml/min, temperature was 30°C (_+ I'~C). A 100 #M (+)-tubocurarine ((+)-TC) solution was in a micropipette with a tip diameter of 10-20/tm. At an appropriate time, the micropipette was lowered into the solution near the endplate region where the membrane potential or current was being monitored. ( + ) - T C diffused from the pipette tip onto the endplate region. Membrane potential or current was measured by intracellular recording or two microelectrode voltage clamping using a Degan or Axon clamp. High gain recording with a bandwidth from DC-1000 Hz was accomplished by using an Offset Subtracter (Axon) for subtracting the baseline. The H-effect we measured at normal mouse diaphragm neuromuscular junctions was a hyperpolarization of 1.30 +_0.17 mV (S.E.M., n = 39) (Fig. 1). In voltage clamp experiments, when the membrane potential of the end-plate region was clamped at
113
A
.
.
.
.
.
.
.
=.-----
2 .
.
.
.
.
.
1 (+)-TC
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
,
,]2mV
10 sec
Fig. 1. The effect of curare on the membrane potential at an endplate in the mouse diaphragm with and without AChE inhibition. A: without treatment with an anti-AChE. At the time of the arrow (+)-TC was applied to the endplate area as described in Methods. Note that the MEPPs disappeared after the application of (+)-TC but there was no shift in the base line, presumably because the ACh leaking from the terminal was promptly hydrolyzed by ACHE. B. The preparation was soaked for 30 min in 10/~M DFP and then washed with Tyrode. The application of (+)-TC caused the MEPPs to disappear and the endplate to hyperpolarize (the H-effect). When the ( + )-TC-containing pipette was moved away the membrane potential returned toward the resting level.
- 70 m V the ( + ) - T C reduced i n w a r d c u r r e n t b y 6.0 + 0.43 n A (n = 5) (Fig. 2). W h e n c l a m p e d at - 2 0 m V ( + ) - T C reduced i n w a r d c u r r e n t by 0.9 + 0.83 nA. P r e s u m a b l y the c h a n g e in c u r r e n t is less at - 2 0 m V because the m e m b r a n e p o t e n t i a l is closer to the reversal p o t e n t i a l a n d the A C h - g a t e d e n d p l a t e channels have a s h o r t e r m e a n o p e n time when the fiber is d e p o l a r i z e d . In mouse, increases in q u a n t a l size are b l o c k e d by t r e a t m e n t s t h a t should increase a x o p l a s m i c [Na +] [17]; similar results have been r e p o r t e d in frog [10]. In m o u s e there is evidence t h a t the rise in [Na +] acts by decreasing choline u p t a k e , which d e p e n d s on the N a + g r a d i e n t [16]. A t 10 l t M d i h y d r o o u a b a i n slightly increased the H-effect, b u t n o t significantly (Fig. 3). Vysko~il a n d Ill6s [11] r e p o r t e d t h a t the H-effect was significantly increased b y 20/~m o u a b a i n ; o u a b a i n is s u b s t a n t i a l l y less effective as an
114
10 nA L
los Fig. 2. The effect of ( + )-TC on the holding current at a voltage-clamped endplate in the mouse diaphragm. The endplate was clamped at - 70 inV. Application of ( + )-TC eliminated the miniature endplate currents (MEPCs) and reduced the inward current. The apparent increase in holding current following the removal of the ( + )-TC is unexplained but did not occur in all examples.
N a + - K + p u m p i n h i b i t o r t h a n d i h y d r o o u a b a i n . W e h a v e n o e x p l a n a t i o n for these a p p a r e n t differences b e t w e e n the t w o sets o f e x p e r i m e n t s . A c o n i t i n e ( 1 0 / i M ) , w h i c h o p e n s s o d i u m c h a n n e l s [9], d e c r e a s e d t h e H - e f f e c t by 80% (Fig. 3). In the p r e s e n c e o f a c o n i t i n e m i n i a t u r e e n d p l a t e p o t e n t i a l ( M E P P ) a m p l i t u d e s w e r e n o r m a l , so it d o e s n o t a p p e a r to h a v e a p o s t j u n c t i o n a l effect. Since i n h i b i t i o n o f the N a + - K + e x c h a n g e p u m p did n o t i n h i b i t t h e H-effect, it is u n l i k e l y t h a t the effect o f a c o n i t i n e is d u e to a rise in i n t r a c e l l u l a r s o d i u m . P e r h a p s a c o n i t i n e
E;~ Hyperpolorizetion (mV) 0
1
2
3
4-
I
I
I
I
5 - -
°=39
e). Control
,
b). Hypertonie
n=31
:~
c). DHO n=lO
d). Aconitine
,k n=24
e). Control
n=26
f). 15 mM KCl
o
1'o
l's
& Membrene current (nA) Fig. 3. A summary of the effects of the treatments tested on the H-response (striped bars) and the endplate current (filled bars) during ( + )-TC application. The bars show the mean and the error bars show the SEM. *Mean is significantly different from the control. All preparations were pretreated with DFP. a: the membrane hyperpolarization produced by (+)-TC application. This is the control for the potential measurements, b: the response in preparations pretreated for 30 min in hypertonic solution. The recordings were made in Tyrode. c: in Tyrode containing 10 pM DHO (dihydroouabain). d: in Tyrode containing 10 pM aconitine, e: the decrease in inward holding current caused by ( + )-TC application (controls for the current measurements), f: the change in current in preparations in 15 mM K ÷-solution.
115
directly blocks the ACh transport system responsible for the H-effect. Aconitine should be tested as a possible inhibitor of vesicular ACh uptake and its effects on the chemically measured leak should also be determined. Hypertonic treatment (30 min in 235 mM NaC1 solution) roughly doubled the Heffect (Fig. 3). After 30 min in 15 mM potassium solution and return to Tyrode the H-response was again about doubled (Fig. 3). The major conclusion is that pretreatment with either hypertonic solution or elevated K+-solution can alter the subsequent H-response. Both treatments markedly increase the rate of spontaneous quantal release. Edwards et al. [3] and VyskoOl et al. [14] suggested that the ACh leak might be through vesicular membrane, inserted into the nerve terminal when synaptic vesicles fuse during exocytosis, because non-quantal release is blocked by vesamicol, an inhibitor of ACh transport into isolated synaptic vesicles [1, 2]. Our results are compatible with this hypothesis, but other interpretations are possible and have not been ruled out. Meriney et al. [6] studied the leak at frog neuromuscular junctions by using outside-out patches of AChR-rich membrane as a detector. They did not detect an increase in non-quantal release following stimulation, and believe that the spontaneous release detected by their patch near the nerve terminal was far too low to account for the leak measured biochemically. Such experiments should be repeated at mouse neuromuscular junction, where the leak is many times greater. It is also worth noting that Meriney et al. [6] inactivated AChE by exposure to collagenase. This treatment should be studied to see whether the H-effect is detectable; there is some evidence that the leak is increased following inhibition of intracellular AChE [8], which may favor the accumulation of ACh in the cytoplasm. Supported by Grant 10320 from the NINDS. 1 Anderson, D.C., King, S.C. and Parsons, S.M., Inhibition of [3H]acetylcholine active transport by tetraphenylborate and other anions, Mol. Pharmacol., 24 (1983) 55-59. 2 Anderson, D.C., King, S.C. and Parsons, S.M., Pharmacological characterization of the acetylcholine transport system in purified Torpedoelectric organ synaptic vesicles, Mol. Pharmacol., 24 0983) 48-54. 3 Edwards, C., Dole~al, V., Tu~ek, S., Zemkovfi, H. and Vysko~il, F., Is an acetylcholine transport system responsible for nonquantal release at the rodent myoneural junction?, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 3514-3518. 4 Fletcher, P. and Forrester, T., The effect of curare on the release of acetylcholine from mammalian motor nerve terminals and an estimate of quantum content, J. Physiol., 251 (1975) 131-144. 5 Katz, B. and Miledi, R., Transmitter leakage from motor nerve endings, Proc. R. Soc. Lond. Ser. B., 196 (1977) 59-72. 6 Meriney, S.D., Young, S.H. and Grinnell, A.D., Constraints on the interpretation of nonquantal acetylcholine release from frog neuromuscular junctions, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 20982102. 7 Mitchell, J.F. and Silver, A., The spontaneous release of acetylcholine from the denervated hemidiaphragm of the rat, J. Physiol., 165 (1963) 117-129. 8 Molenaar, P.C. and Polak, R.L., Is the non-quantal resting release of ACh from the isolated rat diaphragm an artifact due to cholinesterase inhibition? In M.J. Dowdall and J.N. Hawthorne (Eds.), Cellular and Molecular Basis of Cholinergic Function, Ellis Horweed, U.K., 1987, pp. 221-225.
116 9 Schmidt, H. and Schmitt, O., Effect of aconitine on the sodium penetration of the node of Ranvier, Pflfigers Arch. Eur. J. Physiol., 349 (1974) 133 148. 10 Van der Kloot, W., The packing of acetylcholine into quanta at the frog neuromuscular junction is inhibited by increases in intracellular sodium, Pfliigers Arch. Eur. J. Physiol., 412 (1988), 258 263. 1 I Vysko6il, F. and ll16s, P., Non-quantal release of transmitter at mouse neuromuscular junction and its dependence on the activity of Na ~-K ~ ATPase, Pflfigers Arch. Eur. J. Physiol., 370 (1977), 295 297. 12 Vysko6il, F. and Ill6s, P., Electrophysiological examination of transmitter release in non-quantal form in the mouse diaphragm and the activity of membrane ATPase, Physiol. Bohemoslov.. 27 (1978), 449 455. 13 Vysko6il, F., Nikolsky, E. and Edwards, C., An analysis of the mechanisms underlying the non-quantal release of acetylcholine at the mouse neuromuscular junction, Neuroscience, 9 (1983) 429 435. 14 Vysko~il, F., Zemkov~., H. and Edwards, C., Non-quantal acetylcholine release. In LC. Sellin, R. Libelius and S. Thesleff(Eds), Neuromuscular Junction, Elsevier, Amsterdam, 1989, pp. 197 205. 15 Weiler, M., Roed, I.S. and Whittaker, V.P., The kinetics of acetylcholine turnover in a resting cholinergic nerve terminal and the magnitude of the cytoplasmic compartment, J. Neurochem., 38 (1982), 1187 1191. 16 Yamamura, H.I. and Snyder, S.H., High-affinity transport of choline into synaptosomes of rat brain, J. Neurochem., 21 (1973), 1355-1374. 17 Yu, S.P., The Regulation of Quantal Size at the Mouse Neuromuscular Junction, Ph.D. Thesis, State University of New York at Stony Brook, 1990.