The effect of lactate on acetylcholine release evoked by various stimuli from torpedo synaptosomes

European Journal of Pharmacology, 129 (1986) 235-243

235

Elsevier EJP 00515

The effect of lactate on acetylcholine release evoked by various stimuli from Torpedo synaptosomes Y. M o r o t G a u d r y - T a l a r m a i n Laboratoire de Neurobiologie Cellulaire et Molbculaire, Dbpartementde Neurochimie, C.N.R.S., 91190 Gif sur Yvette, France

Received 25 February 1986, revised MS received 30 June 1986, accepted 8 July 1986

The effect of external lactate on acetylcholine (ACh) release was examined at the nerve electroplaque junction of Torpedo marmorata using cholinergic synaptosomes prepared from the electric organ. Lactate reduced the release of

ACh triggered by depolarization of synaptosomes with potassium. However, the release mechanism itself was not affected by lactate since, in its presence, the ACh release induced by different agents such as the calcium ionophore, A23187, or gramicidin D was equal to the release by control synaptosomes (without lactate). A possible site of action for lactate would be the voltage-dependent Ca2+ influx mediated by the natural calcium channel which is thought to couple depolarization and release and which would be bypassed during ionophore-induced Ca 2+ entry. An intracellular target was indicated because decreasing the extracellular pH made L( + )lactate a slightly more potent inhibitor. The involvement of a membrane transporter for lactate was suggested by the observation that the D ( - ) isomer of lactate was less potent than the natural L(+) isomer. The release of endogenous lactate by electric organ prisms was also determined and depolarization with high potassium strongly stimulated L( + )lactate release from prisms. These results suggest that lactate production by stimulated postsynaptic electroplaques may inhibit acetylcholine release from presynaptic nerve terminals, constituting an example of negative feedback. Acetylcholine release; Neuromuscular junction; Calcium channels; Calcium transport; Lactate metabolism in muscle; (Torpedo marmorata, Synaptosomes)

1. Introduction

Cholinergic nerve terminals are particularly abundant in the electric organ of Torpedo marmorata. They constitute a rich network which covers the ventral surface of the electroplaques, and thus c o m m a n d the synchronous electrical discharge of electroplaques. These nerve terminals have been isolated as a synaptosomal preparation (IsraEl et al., 1976) which holds an average membrane potential of - 5 0 mV (Meunier, 1984), has an active metabolism, i.e. synthesizes acetylcholine with uptake of the precursors, choline and acetate (Morel et al., 1977; O'Regan, 1984) and responds to depolarization by a Ca2+-dependent release of acetylcholine (IsraEl and Lesbats, 1981). These criteria show that isolated nerve endings are fully 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.

functional and very interesting for the study in vitro of mechanisms which regulate cholinergic neurotransmission. For instance, release of A T P from stimulated pieces of electric organ has been shown previously to have an inhibitory action on presynaptic activity (Meunier et al., 1975), so it seems that the products of electric organ metabolism can have feedback effects on the release of transmitter. Electroplaques result from differentiation of muscle tissue and during their electrical discharge, energy is supplied by creatine phosphate and A T P (Chmouliovsky-Moghissi and Dunant, 1979; Borroni, 1984). A sustained accumulation of lactic acid in electric organs accompanies the discharge (Caldwell and Keynes, 1963). This excess lactic acid results from the activity of lactate dehydro-

236 genase, an isoenzyme (LDH-5) of which has been shown to be present in the electric organ of Electrophorus electricus (Torres-Da Matta et al., 1975). During neuromuscular fatigue there is a correlation between the increase in lactate content of muscle and the decrease in contractile force that the muscle develops (Fitts and Holloszy, 1976; Tesch et al., 1983). For this reason, we wondered whether part of this inhibition might be due to a presynaptic effect of the production of an excess of lactate. We now investigated, (1) the effect of lactate on the release of ACh from synaptosomes prepared from the electric organ of Torpedo marmorata, ACh being detected by a chemiluminescent method described by IsraEl and Lesbats (1981), and (2) lactate extrusion from electroplaques, by measuring its release from resting and stimulated electrogenic prisms. The results show that depolarization of electroplaques considerably increases lactate release and that high external lactate causes an inhibition of the voltage-dependent efflux of endogenous ACh from a suspension of synaptosomes. This effect may be related to a reduction in the voltage-dependent calcium influx into the nerve terminal. Further studies showed that experimental procedures that were expected to change lactate entry into synaptosomes also modified the inhibitory action of lactate on ACh release, suggesting an intracellular target.

2. Materials and methods

2.1. Preparation of Torpedo synaptosomes The synaptosomes were prepared from the electric organ of Torpedo marmorata by the method described by IsraEl et al. (1976) and Morel et al. (1977). Fishes were purchased from the Marine Station of Arcachon and were kept in artificial sea water. Nerve endings from 25-30 g of electric organ were collected from a discontinuous sucrose gradient in 40-50 ml of a synaptosomal fraction containing (mM): NaC1 280, KC1 3, MgCI 2 1.8, CaC12 3.4, sucrose 400, glucose 5.5, Na phosphate buffer 1.2; the p H was adjusted to pH 7.1 with N a H C O 3 5 mM.

2.2. Release of ACh from synaptosomes The release of ACh was followed continuously using the chemiluminescent method for the detection of ACh described by IsraEl and Lesbats (1981). In summary, ACh released from synaptosomes was immediately hydrolyzed by a specific acetylcholinesterase into choline and acetate, choline was then oxidized by choline oxidase (E.C.1.1.3.17) producing betaine and H202. The H202 formed was then detected by the bioluminescence created in the presence of luminol and peroxidase (E.C.I.ll.I.7). The composition of the assay medium was isoosmotic with elasmobranch physiological medium and was (mM): NaCI 280, KC1 3, MgC12 1.8, glucose 5.5 and, in routine conditions, the p H was maintained at 8.6 using 50 mM Tris base/Tris HC1 buffer. An aliquot (450485 /,1) of this medium plus 50-15 /~1 of the synaptosomal fraction was put in a glass tube continuously stirred by a small magnetic bar. Release experiments were done in the presence of the required concentration of calcium chloride (usually 3.5 mM) and purified acetylcholinesterase (E.C.3.1.1.7) (1 u n i t / 3 /,1). When desired, lactate and other compounds to be tested were added directly to the reaction mixture and the release was triggered by the addition of minute quantities of depolarizing agents such as KC1 3 M or gramicidin D, or by calcium ionophore A23187. The amplitude of each light response emitted by the chemiluminescent reaction corresponding to ACh release was calibrated by the addition of known amounts of ACh. The amplitude of the tracing a n d / o r the area under the curve were proportional to the external ACh. Total a n d / o r residual synaptosomal ACh contents were estimated at the end of the release period by lysis of the membrane with 0.5% Triton-X-100 (v/v) (IsraEl and Lesbats, 1981).

2.3. Lactate release experiments with electroplaques Small fragments containing one or two stacks of electrogenic prisms (about 50-100 mg each) were carefully dissected out and put in oxygenated (95% 02, 5% CO2) Torpedo physiological medium containing in mM: NaCI 280, KC1 7, CaCI 2 3.4,

237

MgC12 1.8, NaHCO 3 5, NaHPO 4 1.2, urea 300 and glucose 5.5, pH. 7.1. Two washing periods lasting about 60 min each (20-30 prisms in 300 ml) allowed the tissue to recover from the dissection. Groups of 15 prisms were preincubated at 4°C for 18h with drugs (curare, eserine) which were to be tested for their effect on lactate release. This prolonged incubation time allowed for the penetration by diffusion of the drugs into the tissue. Any excess of drugs was eliminated by rinsing the electrogenic prisms twice in 30 ml of the Torpedo medium. To measure lactate release, halves of electrogenic prisms (about 50 mg) were placed in histological crystal dishes containing 200 /tl of saline medium. The basal release of lactate was measured for 16 min, a 200 /~1 fraction was collected every 8 min and divided into two aliquots in order to measure the amount of lactate liberated. The fluorometric method (Leese and Bronk, 1972) was used for lactate estimation. Each aliquot (100 VI) was mixed with 450 V1 of glycine (333 mM) buffer containing an excess of the substrate NAD (2,5 mM). Exogenous lactate dehydrogenase (E.C.1.1.1.27) was then added to sample aliquots, while no enzyme was added in blank aliquots. The pyruvate generated by this reaction (50 min, 37 °C) was trapped at pH 9.5 with hydrazine (133 mM) while the amount of synthesized NADH was measured by its fluorescence at 360-465 nm.

2.4. Materials The materials used for the chemiluminescent method (choline oxidase, horseradish peroxidase, luminol and acetylcholinesterase) were prepared as described by IsraS1 and Lesbats (1981) for ACh release from synaptosomes. L( + )Lactate lithium salt, D( - )lactate lithium salt, iodoacetic acid, and LDH (L-lactate: NAD ÷ oxido-reductase E.C.1.1.1.27) from rabbit muscle (Type XI) were purchased from Sigma, St. Louis; NAD from Boehringer, FRG; glycine and hydrazine sulfate from Merck. The ionophores, gramicidin D and A23187, came from Boehringer, FRG. All ionophores were added at 1/3300 dilution of an ethanolic (gramicidin D) or 1/5000 dilution of an DMSO (A23187) stock solutions. D-Tubocurarine and eserine-salicylate were obtained respectively, from Serva-Heidelberg and Fluka AG, Buchs.

3. Results 3.1. Inhibitory effect of L( + )lactate on potassium depolarization-induced release of A Ch The release of ACh from synaptosomes was examined in the presence or absence of L( + )lactate in the assay medium as described in Materials and methods. The L( + )lactate effect was tested at a large range of concentrations. L( + )Lactate did not induce ACh leakage by itself (fig. 1). Figure 1 also shows that external %

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I-{+) LACTATE CONCENTRATION(raM) Fig. 1. Inhibition of potassium depolarization-evoked ACh release by L(+ )lactate. ACh release experiments were performed as described in Materials and methods. Synaptosomes were added to the assay medium (500 /~1) containing calcium (3.5 mM). L( + )Lactate was added to this suspension and left for 5 min. Release of ACh was induced first by addition of 60 mM potassium (e) and later by the addition of 3.5 vM A23187 (©). Results of 3 different experiments are shown on the graph (fit by eye) and individual data are expressed as percentages of their own controls (mean of 3-5 determinations per experiment). The tracings on the right illustrate one of these experiments in which ACh release from synaptosomes prepared from 10 mg original wet weight tissue was measured: (A) control sample, (B) lactate 9.1 mM, (C) lactate 22.2 mM. Arrows correspond to successive additions of lactate, KCI, external ACh standard, A23187 and Triton X-100. The addition of known amounts of acetylcholine chloride as external standard permits the calibration of ACh release by conversion of the amplitude of light emission into molar units using the corresponding amplitude of an ACh standard. Starred experiments were performed at 4 x higher amplification. The calibration bar for time indicates 1 min. At the end of the experiment, all the ACh still occluded inside the synaptosomes was liberated with Triton X-100 (0.5%, v/v); this remaining ACh was not significantly different in the control and in lactate-treated synaptosomes (compare the last light emissions of (A)(BXC) records). Note that lactate by itself did not induce any detectable release of acetylcholine at the moment of its addition.

238 L( + )lactate significantly r e d u c e d the evoked release of A C h triggered b y 60 m M KCI, an add i t i o n which d e p o l a r i z e d nerve endings b y coll a p s i n g t r a n s m e m b r a n e p o t a s s i u m g r a d i e n t s (IsraEl a n d Lesbats, 1981; Morel et al., 1977; Meunier, 1984). A m a x i m a l i n h i b i t i o n of 50% was o b t a i n e d at external lactate c o n c e n t r a t i o n s higher than 30 m M (fig. 1). D e p o l a r i z a t i o n of s y n a p t o s o m e s with K C I i n d u c e d a p a r t i a l release of c y t o p l a s m i c A C h o c c l u d e d in the nerve endings (IsraEl a n d Lesbats, 1981; Meunier, 1984). F u r t h e r a d d i t i o n of a c a l c i u m i o n o p h o r e , A23187 (3.5 /~M) to the assay m e d i u m c o n t a i n i n g 3.5 m M calcium caused a massive entry of calcium into the c y t o p l a s m o f the s y n a p t o s o m e s , which in turn triggered a new a n d m o r e substantial A C h release (the inset in fig. 1 a n d third l u m i n e s c e n t p e a k of the records). The s u m of the a m o u n t s of A C h released by the two agents equalled the total c y t o p l a s m i c A C h c o m p a r t m e n t as shown b y IsraEl a n d Lesbats (1981). If g r a m i c i d i n D was a d d e d as a third step, i n d u c ing a massive entry of sodium, no further release of A C h was o b s e r v e d (S. B i r m a n a n d F. M e u n i e r (1985) a n d results not shown). A f t e r these procedures, the s y n a p t o s o m a l m e m b r a n e s were finally lysed b y a d d i t i o n of T r i t o n - X 100, 0.5% ( v / v ) . T h e vesicular pool was thus l i b e r a t e d a n d no significant variations of this c o m p a r t m e n t of A C h were o b s e r v e d in the presence of lactate as c o m p a r e d to the control values (last l u m i n e s c e n t p e a k s in the records of fig. 1).

3.2. Inefficacy of L( + )lactate to inhibit the release evoked by A23187 ionophore + calcium or gramicidin D ionophore In c o n t r a s t to the previous results, L( + )lactate a d d i t i o n d i d not affect A C h release if it was triggered b y the a d d i t i o n of the calcium channel f o r m e r A23187 (3.5 /zM) in the presence of 3.5 m M external calcium (fig. 2). Moreover, if s y n a p t o s o m e s were d e p o l a r i z e d b y c o l l a p s i n g the t r a n s m e m b r a n e s o d i u m g r a d i e n t using the cation i o n o p h o r e g r a m i c i d i n D (0.5/~M) (fig. 3) as shown b y IsraEl a n d L e s b a t s (1981) a n d M e u n i e r (1984) no n o t i c e a b l e difference in the r a t h e r large A C h release p r o d u c e d was o b s e r v e d in the presence of L( + ) l a c t a t e .

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Fig. 2. Absence of an effect of L( + )lactate on ACh release induced by calcium (3.5 mM) and ionophore A23187 (3.5 #M). Conditions of release experiments were the same as those presented in fig. 1. The lactate effect was tested in a concentration range of 4-36 mM. The synaptosomes were preincubated with lactate for 5 min and the release of ACh was then triggered by the addition of A23187 (3.5 /~M) in the presence of 3.5 mM external calcium chloride. The release of ACh was followed by the light emission obtained and calibrated with an ACh standard. Results of 2 different experiments are pooled on the graph and individual data (e) were calculated as percentages of their control values (mean of 2-4 control per experiment). The lower portion of the figures shows the ACh release taken from one experiment. Synaptosomes prepared from 10 mg original wet weight of tissue were used in the presence of: (A) control, (B) L( + )lactate 36.8 mM. The A23187-evoked ACh release (e) was constant whatever the external concentration of L( +)lactate. Arrows correspond to successive additions of saline medium or lactate (1'), A23187 (I"T), external ACh standard (TI"T), and Triton X-100 (? T 1"T) in the isoosmotic luminescent medium. Bound ACh liberated by disruption of the synaptosomal membrane by Triton X-100 (0.5%, v/v) (O) did not change in the presence or the absence of external lactate.

T h e b r e a k d o w n of m e m b r a n e s by a final add i t i o n of T r i t o n X-100 (0.5%) after A23187 (fig. 2) o r g r a m i c i d i n D (fig 3) a d d i t i o n , released equal quantities o f the o c c l u d e d A C h r e m a i n i n g in syna p t o s o m e s (last luminescent tracings of figs. 2 a n d 3) w h e t h e r or not they were treated with lactate.

3. 3. L( + )lactate inhibition of potassium depolarization-induced A Ch release: effects of L( + ) and D ( - ) stereoisomers of lactate and, effect of two pHs of release medium In o r d e r to test w h e t h e r lactate p e n e t r a t e s into nerve endings either as an a n i o n in a c a r r i e r - m e d i a t e d m e c h a n i s m o r b y diffusion as p r o t o n a t e d u n c h a r g e d lactic acid, we first l o o k e d at the stere-

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Fig. 3. Inefficacy of external lactate to modify ACh release induced by gramicidin D depolarization of synaptosomes.ACh release experiments were performed as described in Material and methods under the same conditions as those presented in fig. 1. Synaptosomes were added to 500 #1 of luminescent medium (isoosmotic with elasmobranch physiologicalmedium) containing 3.5 mM calcium. Various concentrations of L( + )lactate were added for 5 rain, then a fixed concentration of gramicidin D (0.5 #M) which depolarizes synaptosomes and triggered ACh release was added. Results of 2 experiments were used to draw the graph (e); individual data were expressed as percentages of their control values {2-4 values per experiment). After Triton-X-100 addition (0.5%, v/v), the remaining ACh was not significantlydifferent (©) in control and in lactate-treated synaptosomes. The lower part of the figure illustrates the ACh release from one experiment. This was obtained with synaptosomes (10 mg wet tissue): (A) control sample, (B) with lactate 27 mM. Arrows indicate the successive addition to the release medium of: saline medium or lactate (1'), gramicidin D (T $ ), external ACh standard (1" 1"1") and Triton-X-100 (1" 1"1"1").

ospecificity of the effect a n d second we compared the influence of different p H s of the assay m e d i u m o n the lactate effect. Figure 4 shows results from experiments in which A C h release was triggered by potassium depolarization in the presence of either of the isomers. We f o u n d that L( + )lactate inhibited A C h release more p o t e n t l y t h a n the D( - ) isomer. These effects were tested over a large range of concentrations (fig. 4). A way to increase the low percentage of lactic acid in the undissociated form as compared to the lactate anionic form which is present at physiological pH, was to acidify the p H of the assay m e d i u m a n d analyse the effect of lactate at different pHs. However, the l u m i n e s c e n t reaction mixture (choline oxidase, peroxidase, luminol) works better at an alkaline p H (IsraEl a n d Lesbats, 1985).

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Fig. 4. Comparison of L(+ )- and D ( - )lactate effect on ACh release evoked by potassium depolarization of synaptosomes. The conditions of release experiments were the same as described in Materials and methods and fig. 1. L(+)- or D( - )lactate (lithium salt) was added 5 min before the induction of ACh release with 60 mM potassium in the presence of 3.5 mM calcium. Results of 5 and 4 different experiments with respectively L( + )- and D( - )lactate were pooled. All the individual values were expressed as percentages of control ACh release (mean of 3-5 values per experiment). The data were then plotted and tested as a linear function of the lactate concentration. The 2 curves were drawn with equations determined using a program for linear regression analysis by the least squares method. The equations obtained were: with L( + )lactate: y = -1.73 x+102.3, n = 57, r = 0.43, P > 0.01 according to Fisher and Yates (1953): with D-lactate: y = -0.74 x+ 101.0, n = 47, r = 0.10, non significant probability.

F o r this reason, we could only explore the range of p H s from 8.0 to 8.6. The results o b t a i n e d (not shown) revealed that acidification of the p H of the assay m e d i u m slightly increased the i n h i b i t i t m of A C h release at the lower range (up to 15 m M ) of external L( + )lactate concentrations, b u t similar saturation curves were o b t a i n e d at values higher than 25 m M external lactate.

3.4. Comparison of the effect of L( + )lactate on potassium-induced A Ch release in sodium or lithium release media I n nerve terminals, p o t a s s i u m depolarization opens Ca 2÷ channels a n d also induces Ca 2÷ transport through exchange systems such as the N a + / C a 2÷ exchanger (see references in Discussion). T o d e t e r m i n e w h e t h e r a f u n c t i o n a l N a 2 + / C a 2÷ exchange is required for the inhibitory effect of lactate o n A C h release i n d u c e d by potassium depolarization we replaced all the external s o d i u m (280 m M ) in the release m e d i u m with

240 P R I S M S in TORPEDO MEDIUM

lithium (280 m M ) which then b e c o m e s the m a j o r cation and which is not a good substitute for the e x ch an g er ( B i rm a n and Meunier, 1985). Results presented in fig. 5 show that as powerful an inhibition of A C h release took place in the lithium m e d i u m as in the s o d i u m m e d i u m . Therefore, these results do not s u p p o r t an i m p o r t a n t role for the N a + / C a 2+ e x c h a n g e in this inhibitory effect of lactate.

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Fig. 6. Stimulation of lactate release from prisms of electric organ by depolarization with high potassium. A detailed account of this experiment is presented in Materials and methods. In brief, halved electrogenic prisms were preincubated in physiological medium (A), supplemented with eserine 10 -4 M (B), or curare 5.10 3 M (C). After washing, they were placed in a histological dish and each tissue was incubated for 8 rain with 200 /~1 of fresh saline medium. Lactate release by the tissue during this time interval was measured fluorometrically as described in Materials and methods. The lower part of the diagram shows lactate release stimulated after potassium depolarization of the tissue. After 16 min in normal saline medium, 100 mM KCI was added to the superfusate and lactate release was monitored during several further 8 rain periods. In the upper panel, similar experiments were done but the production of lactic acid was inhibited at 16 min by bathing the prisms with saline medium containing 5 mM iodoacetic acid.

lactate efflux was then increased when m i n u t e quantities of KCI 3 M were ad d ed to the b a t h i n g m e d i u m . In fig. 6C,B, prisms were p r e i n c u b a t e d (as described in Materials and m e t h o d s ) with saline m e d i u m c o n t a i n i n g the A C h antagonist, D - t u b o curarine, or eserine in o r d er to inhibit respectively the activation of the nicotinic A C h receptor and the acetylcholinesterase activity present in the n er v e e l e c t r o p l a q u e space. U n d e r these two conditions (columns B and C), the basal release of lactate was not different from the c o n t r o l ( c o l u m n A) but a greater st i m u l at i o n of lactate release was o b s e r v e d after d e p o l a r i z a t i o n of the tissue with KCl. The basal release of lactate was also measured (upper panels of fig. 6) after a d d i t i o n of

241 iodoacetic acid (5 mM), a known inhibitor of glycolysis. As expected, a decrease in the resting levels began to appear 15 min after the addition of the drug to normal prisms or to prisms preincubated with curare or eserine.

4. Discussion Our results show that the presence of lactate outside cholinergic synaptosomes inhibited the ACh release induced by depolarization of the membrane with high potassium. In contrast with these results, no effect of lactate was seen when ACh release was triggered by the direct entry of calcium into nerve endings through the calcium ionophore A23187 in the presence of normal external calcium. Hence, it appears that the mechanism by which lactate inhibits ACh release is related to natural calcium entry into the Torpedo nerve endings. Furthermore, this result shows that the ACh release mechanism itself was unimpaired by lactate. A voltage sensitive Ca 2+ current and an alternate process, the N a + / C a z+ exchange, have been described in nerve terminals and both of these are thought to contribute to depolarization-stimulated Ca 2+ uptake by synaptosomes (Blaustein and Ector, 1976). Several authors (Marsal et al., 1980; Dunant et al., 1980; Birman and Meunier, 1985) show evidence for the presence of these mechanisms in Torpedo electric organ. A way to inhibit the N a + / C a 2+ exchange is to replace the external sodium with lithium (Birman and Meunier, 1985). Results of experiments on ACh release obtained with lithium release medium show that lactate inhibition was at least as potent in the presence of lithium as in normal sodium medium. Moreover, ACh release was not modulated by external lactate when a very large release of ACh was promoted by collapsing the sodium gradient of synaptosomes in the presence of gramicidin D. Gramicidin D induces a passive entry of sodium which subsequently activates the N a + / C a 2+ exchange (Birman and Meunier, 1985). These results reinforce the idea that the inhibitory action of lactate is on the voltage-sensitive calcium current through the Ca 2+ channels present in the membrane of

Torpedo nerve terminals and that the N a + / C a z+ exchange does not participate in the induction of the lactate effect. It has often been shown in experiments on the regulation of cytoplasmic pH in muscles and other cells (Roos, 1975; Mainwood and Worsley-Brown, 1975, De Hemptinne et al., 1983; and also reviews of Roos and Boron, 1981; Moody, 1984; Thomas, 1984) that, at a physiological pH, weak organic acids pass through the cell membrane by two mechanisms: first, a carrier-mediated mechanism is necessary for charged molecules to cross the membrane; a second passive mechanism permits the acid to enter the cell in its protonated form. For lactic acid, which has a pK of 3.7, less than 0.5% is in the undissociated form near the physiological pH (see Mainwood and Worsley-Brown, 1975). Results obtained with the stereoisomers L ( + ) - and D( - )lactate show that the natural L ( + ) isomer was a more powerful inhibitor of neurotransmitter release. These data favor a mechanism which should be carrier-mediated, and resemble the data presented by De Hemptinne et al. (1983) on Purkinje cells showing that after 10 min 20 mM external lactate induced a higher acidification with the natural L ( + ) than with the D ( - ) isomer. These authors and others (Mason and Thomas, 1985) have also shown that a decrease in internal pH was not accompanied by significant depolarization of the membrane. This is probably true for Torpedo synaptosomes as well, since no leakage of ACh occurred during application of L( + )lactate (fig. 1) or D ( - )lactate (not shown). A second possibility would be that lactic acid also penetrates as the acid molecule. With a pK = 3.7, more undissociated lactic acid molecules are present at pH 8.0 than at pH 8.6 and could be responsible for the faster inhibition of ACh release. These data are in accord with the work of Roos (1975), Mainwood and Worsley-Brown (1975) and De Hemptinne et al. (1983) showing that, though in minor quantities, permeation of acid molecules takes place very efficiently. In both cases, the final consequence should be an increase of lactate ions in the cytoplasm, which may lead to intracellular acidification with many possible effects: calcium concentration, enzyme activities, channels properties and rate of

242 e x c h a n g e s w i t h a n t i p o r t e r s such as N a + / H +, CI-/HCOf .... T o see w h e t h e r the p h y s i o l o g i c a l release of l a c t a t e w o u l d be i n c r e a s e d by s t i m u l a t i o n o f elect r o p l a q u e s as was s e e n a f t e r i n t e n s i v e w o r k w i t h o t h e r tissues; skeletal m u s c l e ( M a i n w o o d a n d W o r s l e y - B r o w n , 1975; M a s o n a n d T h o m a s , 1985, R e n n i e a n d W a t t , 1985); fish m u s c l e ( W o k o m a a n d J o h n s t o n , 1981) a f t e r s u s t a i n e d c r u i s i n g speeds, a n d g u i n e a - p i g b r a i n (Barberis, 1978). R e suits o f e x p e r i m e n t s o n l a c t a t e e f f l u x f r o m p r i s m s of e l e c t r o p l a q u e s s h o w that lactic a c i d was p r o d u c e d t h r o u g h m e t a b o l i s m a n d that high levels o f lactic a c i d w e r e a c c u m u l a t e d a f t e r p o t a s s i u m dep o l a r i z a t i o n a n d w e r e t h e n e x t r u d e d f r o m the tissue. T h e efflux r a t e o f l a c t a t e ( a b o u t 5 p m o l / g p e r m i n ) f r o m electric o r g a n p r i s m s was in the s a m e r a n g e as t h a t p r e v i o u s l y o b t a i n e d w i t h skeletal m u s c l e ( M a i n w o o d a n d W o r s l e y - B r o w n , 1975). T h e s e d a t a w e r e o b t a i n e d in Torpedo saline m e d i u m w h i c h d o e s n o t p r e s e n t all the c o n d i t i o n s o f p H a n d salt c o m p o s i t i o n w h i c h h a v e b e e n d e m o n s t r a t e d to i n c r e a s e c o n s i d e r a b l y the e f f l u x o f l a c t a t e f r o m m u s c l e cells ( M a i n w o o d a n d W o r s l e y - B r o w n , 1975). I n c o n c l u s i o n , the p r e s e n t results o b t a i n e d o n the n e r v e e l e c t r i c o r g a n j u n c t i o n s h o w t h a t the a c c u m u l a t i o n of e x t e r n a l l a c t a t e r e d u c e s the A C h release t r i g g e r e d b y d e p o l a r i z a t i o n o f n e r v e end i n g s w i t h p o t a s s i u m . T h e n e r v e e l e c t r o p l a q u e of Torpedo is a m o d i f i e d n e u r o m u s c u l a r f u n c t i o n a n d the p r e s e n t results i n d i c a t e t h a t a m a j o r m e t a b o l i c p r o d u c t of i n t e n s i v e work, lactate, c a n i n f l u e n c e A C h release b y p r e s y n a p t i c c h o l i n e r g i c nerve endings through a negative feedback process.

Acknowledgements This work was supported by the 'Centre National de la Recherche Scientifique'. I am grateful to Dr. M. Israel for his encouragement and advice at all stages of this work, and to Drs. F.M. Meunier, N. Morel, S. O'Regan and S. Birman for stimulating, helpful discussions and aid with English. I thank J.P. Bouillot for the photography and D. Chaslard for typing the manuscript.

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