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Ca2+-DEPENDENT CHANGES OF ACETYLCHOLINE RELEASE AND IP3 MASS IN TORPEDO CHOLINERGIC SYNAPTOSOMES
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Neurochem.Int. Vol.29,No, 6, pp. 637-643,1996 CopyrightQ 1996ElsevierScienceLtd Printedin Great Britain.All rightsreserved 01974186/96$15.00+0.00
PII: S0197-0186(96)00046-0
Q2’-DEPENDENT CHANGESOF ACETYLCHOLINE RELEASEAND IP3MASS IN TORPEDOCHOLINERGIC SYNAPTOSOMES MARIA ANGELICA CARRASCO,* YVETTE MOROT GAUDRY-TALARMAIN~ and JORDI MOLGO Laboratoire de Neurobiologie Cellulaire et Mo16culaire, Centre National de la Recherche Scientifique, 91198-Gif sur Yvette, Cedex, France (Received for publication 17 April 1996) Abstract—The aim of the present study was to investigate possible changes of inositol l,4,5-trisphosphate (IPJ mass in Torpedo cholinergic synaptosomes in conditions promoting stimulated acetylcholine (ACh) release. For this purpose, we used a radioreceptor IP3 mass assay and a chemihrminescent method for ACh detection. Torpedo cholinergic synaptosomes have consistent 1P, mass levels under resting conditions, The 1P, mass was neither modified by changes in external Ca2+ nor by a Ca Z+-freemedium containing EGTA. IP3mass and ACh release, measured in the same conditions and in parallel, were increased by depolarization with high K+ and by the ionophores A-23187 and gramicidin-D in a manner dependent on external Ca2+ emphasizing that Ca2+ entry, independently of the influx mechanism involved, leads to an IP3 increase. The phospholipase Cl inhibitors U-73122 and U-73343 reduced K+-stimulated IP3 levels while K+-evoked ACh release was almost completely blocked suggesting an additional effect of these drugs on depolarizationneurotransmitter secretion coupling. The effect reported showing an increase of IP3 by agents that stimulate ACh release may suggest a possible link between 1P, metabolism and the neurotransmitter release mechanism. However, such a link is probably not a direct one as implied by the results obtained with the
inhibitors of phospholipaseC. Copyright~ 1996ElsevierScienceLtd
The inositol phospholipid-dependent signal transduction pathway is now considered to play an important role in controlling a variety of cell functions in the nervous system (Henzi and MacDerrnott, 1992; Berridge, 1993). Inositol l,4,5-trisphosphate (IP~) is produced in cells by a number of agonists that interact with surface receptors coupled to phospholipase C. Activated phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5 -bisphosphate to produce diacylglycerol and IPj. This last product is a diffusible second messenger which mobilizes intracellular Ca2+ (Berridge, 1993). The aim of the present work was to determine whether IP~ mass levels could be detected in pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata which constitute a model
preparation of nerve endings from the peripheral nervous system retaining most of their metabolic and physiological properties (Morel et al., 1977; Israel and Lesbats, 1981). In addition, we have studied IP~ levels as a function of extracellular Ca2+ in the presence of various agents that increase acetylcholine (ACh) release. Furthermore, the effeets of a phospholipase C inhibitor were studied on both ACh release and IPq levels. Finally, the activity of IPJ 3-kinase, the enzyme which phosphorylates IP~ to form inositol 1,3,4,5tetrakisphosphate (IP1) was investigated. A preliminary account of this work has already been published in abstract form (Carrasco et al., 1995). EXPERIMENTALPROCEDURES Preparation of Torpedo cholinergicsynaptosomes
*Permanent address: Departamento de Fisiologia y Biofisica, Facultad de Medicina, Universidad de Chile, Casilla 70005, Correo 7, Santiago, Chile. tTo whom all correspondence should be addressed. Tel.: 33-1-69-82-36- 33; Fax: 33-l-69-82-94-66; E-mail: morot@)hermes.cnrs-gif. fr; molgofj)hermes.cnrs-gif. fr.
The method for preparing cholinergic synaptosomes from the electric organ of Torpedo marmorata have been described in detail previoudy (Morel et al., 1977; Israel and Lesbats, 1981). In brief, nerve endings were chopped up and diluted in an oxygenated physiological solution. Then, synaptosomes were collected from a discontinuous iso-osmotic NaCl and 637
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sucrose gradient devoided of Ca2+ and further concentrated (6,000 g, 20 rein) after dilution in a modified physiological solution of the following composition (mM): NaCl, 480; KC1, 3; MgCl,, 1.8; sodium phosphate, 1.2; NaHCO,, 5; glucose, 5.5; (pH =7,2). The syrmptosomal pellet was resuspended in the modified physiological solution to adjust the protein concentration to 7–8 mg/ml, All steps of the isolation procedure were carried out at 4“C.
A23187 and gramicidin-D from Boehringer (Mannheim, {l-[6-[[17beta-3-methoxyestraGermany). U-73122 I,3,5(10)trien-17-yl] amino]hexyl]-l H-pyrrole-2,5-dione} and U-73343 {1-[6-[[17beta-3-methoxyestra-1,3,5 (10) trien-l7yl]amino]hexyl]-2,5-pyrrolidine-dione} were from Biomol Research Laboratories (Plymouth Meeting, PA, USA), All other reagents were obtained from Sigma or Merck. RESULTS
Acetylcho[irrerelease determination Measureme,lts of the amount of ACh released from synaptosomal suspensions were performed at room temperature (2W22°C) using the choline oxidase-chemiluminescent method as previously described (Israel and Lesbats, 1981). Aliquots of the concentrated synaptosomal fraction (10 Al) were diluted into 250 PI ofa physiological medium containing (mM): NsrCl, 240; LiCl, 40; KC1, 3; MgCl,, 1.8; sucrose, 400; glucose, 5.5; Tris buffer, 50 (pH=8.6). In some experiments either various concentrations of Ca2+ (2–8 mM, as specified) or EGTA (1 mM) were added to the nominally Ca2+-free medium. ACh measurements were performed after 3 min incubation in the physiological medium containing the reagents for the chemiluminescent assay (Israd and Lesbats, 1981). Drugs were added directly to the medium and, at the concentrations used, had no effect on the ACh assay. Synaptosomal preparations contained sufficient amounts of acetylcholinesterase for the hydrolysis of released ACh, so that no exogenous enzyme was further added. The light emitted by the chemihrminescent reaction for ACh measurements was detected by a photomultiplier unit, recorded and calibrated by the addition of known amounts of standard ACh as previously described (Israel and Lesbats, 1981). Drugs used were added directly to the medium. 1P, mass determination IP3 mass determinations were performed in parallel experimental conditions, i.e. after 3 min of preincubation in the physiological medium, EGTA, calcium and stimulating agents were added directly to the medium. The IPXcontent of synaptosomes was measured using a radioreceptor method based on the displacement of bound 3[H]-IP, to 1P, receptors from rat cerebella (Bredt et al., 1989). For this purpose, after stopping the experiment with 30 @ of 60Y0HC104, followed bycentrifugation, the supernatants were neutralized with 170 @of 0.1 M MES (pH= 6.5), 20 mM EDTA and 2 M KOH (Stephens, 1990; Hidalgo et al., 1993). All 1P, determinations were made in triplicate. IP3 3-kinase activity measurement IP3 3-kinase activity was measured under standard conditions as described previously (Carrasco and Figueroa, 1995) with 50 nM [3H]-IP, as substrate. To measure this activity Torpedo synaptosomes were permeabilized by several successive freezing and thawing steps in liquid nitrogen. Chemicals 3[H]-Inositol l,4,5-trisphosphate (sp. act. 21 Ci mmol “) was purchased from New England Nuclear (Dupont NEN, France). rr-Inosito] l,4,5-trisphosphate was from Calbiochem (France). Acetylcholine, choline and horseradish peroxidase (EC1.1 1.1.7) type II were from Sigma (St Louis, MO, USA), Iuminol from Merck (Darmstadt, Germany), choline oxidase from Wako Co. (Japan), and the ionophore
Determination preparations
oj” IP3 levels in resting synaptosoma[
As shown in Table 1, under basal conditions in Ca2+-free medium, the pelleted synaptosomal fraction contained an IP~ concentration of 471.3 +25.6 pmol mg-’ of protein. No significant difference was observed between the IPj level under this condition as compared with the values determined in Ca2+-containing medium (2–8 mM Ca2+) or containing 1 mM EGTA (Table 1). The IP~ mass remained unmodified during 1–5 min of incubation under the three control conditions. Changes in IP3 levels and ACh release during high K+induced depolarization Synaptosomal depolarization by high K+ induced an increase of IPq levels that depended on the presence of extracellular Ca2+. As shown in Fig. l(A), there was a significant increase in IPq mass in synaptosomes exposed for 1 min to extracellular K+ concentrations ranging from 6f&120 mM while no significant change was observed with 30 mM K+. When depolarization by K+ was induced in a nominally Caz+-free medium supplemented with EGTA, no significant change in IP3 mass was detected with the various K+ concentrations used (Fig. l(A)). Parallel ACh release experiments with increased extracellular K+, as expected (Israel and Lesbats, 1981; Meunier, 1984) caused a transient increase of Ca2+-dependent ACh release that depended on K+ concentration (Fig. l(B)). These results indicate that depolarization induced by high K+ (60 and 120 mM) causes an increase of both IP~ levels and ACh release. Table 1. IP3mass determinedin Torpedocholinergicsyrraptosomes under differentbasalconditions Experimentalcondition
IP3mass (pmOl/mgprotein)
Ca’+-freemedium Ca’+-freemedium + 1mM EGTA Ca’+-containingmedium(2–8mM)
471,3~ 25.6(tr=4) 484.2+ 44.5(n=3) 478.5~ 49.7(n=3)
Data are means ~ SEM of the number of differentTorpedopreparationsofsynaptosomesgivenin parentheses,performedin triplicate,
Ca2+-dependent changes of acetylcholine release
800
A
639
A
900
1
300J I,,,,,l,,,,l,,,,ri
o
60
,,,,,l,,,,rt,,,,,l
120
180 240
300
360
300
360
Time (s)
B
200 400 I
I
1
I
1
I
!
I
1
f
0 40 80120160
0 40 80 ~20180
[K+] (mM)
[K+] (mM)
Fig. 1. (A) 1P, levels (pmoles/mg protein) determined in Torpedo cholinergic synaptosomes depolarized by various K+ concentrationsduringl mininthepresenceofCa2+ (0) orinanominalfy Ca’+-free medium supplemented with 1 mMEGTA (~). (B) ACh release (nmoles/mg protein) from synaptosomes as a function of the K+ concentrations asin A. TheamountofACh released was measured after 1 min depolarization byhigh K+. Inane, data obtained from three to nine different synaptosomalpreparations were expressed asthemeantSEM.
0
● ✏✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌
o
60
120
f80
240
Time (s) ACh (150 pmol)
i
However, as shown in Fig. 1, while depolarization L“ IL with 30 mM K+ induced a significant increase of ACh I I I I 120 240 360 o release (Fig. l(B)), no significant change in IP3 levels was detected (Fig. l(A)). If ACh release occurs at Time (s) external K+ concentrations which do not increase IP3 Fig. 2. (A) Time course of the changes in IP3 mass (pmolevels and if ACh release is dependent on Ca2+ entry les/mg protein) of cholinergic synaptosomes depolarized by since no release was detected when the external Ca2+ 60 mM K+; in the presence of 4 mM Ca2+ (0) or in a Ca2+was reduced to low levels by EGTA, then small depo- free medium supplemented with 1 mM EGTA (A). Data are larizations open enough channels to produce intrathe mean~SEM of three to four different synaptosomal cellular Ca2+ concentration increases sufficient for preparations. (B) and (C) Time course of ACh release from ACh release. Stronger depolarization by K+ will lead cholinergic synaptosomes depolarized by 60 mM K+ in presence of 4 mM Ca2+. (B) The cumulative integral (area under to greater number of open Ca2+ channels and much the curve in (C)) expressing the amount and rate of the ACh higher levels of intracellular Ca2+. Under these con- release is presented as the function of time (Oto 6 rein) after ditions, it is likely that IP3 production may have a the depolarization by 60 mM K+ in presence of 4 mM Ca2+. (C) Time course of a typical chemiluminescent recording of higher Ca z+ threshold than the neurotransmitter ACh release (pmoles) using a known ACh (150 pmoles) for release mechanism which is known to require high calibration. Z+ microdomains (Llinas et ‘Z.> local intracellular Ca 1995). the presence of 60 mM K+ revealed the typical time When IP3 accumulation was studied in the presence of 60 mM K+, as a function of time (Fig. 2(A)), the course of transmitter release, as detected by the chemincrease was already detected at 30s, the shortest time iluminescent method (Fig. 2(B)). This time course is in accordance with previous results (Israel and Lesbats, assessed, and it was maintained during 5 min. Similar 1981; Dolezal et al., 1993) and with control measureresults were obtained with 120 mM K+ (data not shown). The continuous detection of ACh release in ments obtained by discontinuous ACh measurements
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(data not shown). It is worth noting that the maximum of the IP3 mass increase occurs in a similar time domain as the maximum rate of ACh release. Changes in IP3 levels and ACh release during the action of the Ca2+ionophore A-23 187 In order to bypass constitutive voltage-gated Ca2+ channels subtypes that mediate the Ca2+ influx during depolarization by high K+ (Moulian and Morot Gaudry-Talarmain, 1993) we used the Ca’+-ionophore A23187 to increase intrasynaptosomal Ca2+ levels. As shown in Table 2, the Ca2+-ionophore A-23187 (10 /M) was also effective in increasing IP3 levels (from 1–5 rein) provided the presence of extracellular Ca2+. Parallel ACh release measurements showed that A23187 increased ACh release (data not shown), as expected (Israel and Lesbats, 1981) only in the presence of extracellular Caz+. These results emphasize that Ca2+ entry is necessary to increase IP3 mass, independently of the Ca2+ influx mechanism involved. Changes in IP3 levels and ACh release during the action
of gramicidin-D Since depolarization induced by high K+ concentration also induces Na+ influx into synaptosomes and neurotoxins activating Na+ channels increase phosphoinositide hydrolysis (Gusovsky et al., 1987), experiments were performed with 1 PM gramicidinD. This antibiotic induces non-specific membrane channels for monovalent cations (Pressman, 1976) and depolarizes the synaptosomal membrane (Meunier, 1984). As shown in Table 2, gramicidin-D caused a marked increase in IP3 mass (assessed from 0.5–5 rein) only when Ca2+ was present in the physiological
medium. This indicates that monovalent influx of cations (Na+, Li+) is not the trigger of IP~ increase. AS previously shown (lsrael and Lesbats, 1981; Meunier, 1984) gramicidin-D caused a dramatic increase of ACh release (data not shown) owing to both depolarization-induced Caz+ influx from voltage-dependent Ca2+ channels and Ca2+ entry through the reversed operation of the Na+–Ca2+ exchange system (Morel and Meunier, 1981). Hence, the change in 1P, mass may be considered as depending on intracellular Ca2+ levels. Inhibition of PLC activity To further study IP3 metabolism in cholinergic synaptosomes we used inhibitors of PLC activity (Smith et al., 1990) and measured in parallel ACh release and IP3mass. As shown in Fig. 3, the membrane permeable aminosteroid U-73122 dose-dependently reduced Ca2+-dependent ACh release triggered by high K+ (120mM) medium with an EC50 of 0.79 t0.16 PM. The less active analog U-73343 required a higher drug concentration to reduce ACh release, the EC~~jwas 6.5 t 1.1 KM. The effects of U-73122 or U-73433 in 1P, mass levels were tested in synaptosomes in 120 mM K+ medium. At 2 PM neither compound prevented the 1P3mass increase obtained under 120 mM K+ while at 10 ,aM, both compounds limited the 1P, mass increase to 63.2 ~ 2.’7.0/. (n= 3) for U-73122 and 71.6. t6.1 % (n=3) for U-73343. Therefore, ACh release inhibition did not parallel the decrease in 1P3 mass with these drugs (Fig. 3). IP3 3-kinase activity Finally, the activity of IPj 3-kinase, enzyme that phosphorylates 1P, forming IP~,was measured. In two
Table2. Effectof the ionophoresA-23187and gramicidin-Don IPj massdeterminedin T~rP~>dO cholinergicsynaptosomesin the presenceand absenceof Ca2+ Experimentalcondition control 10KMA-23187,
4
mM Ca’+
10ItM A-23187,I mM EGTA I PM gr&micidin-D, 4 rnM Caz+
1flM gramicidin-D,1 mM EGTA
Timeof’ stimulation(rein) 0.5
I 2 5 I 0.5 I 2 5 I 5
1P,mass(pmOl/mgprotein) 461.8+ 469.2+ 642.1~ 548.4~ 550.0i 438.9* 591.2~ 957.2t 812.9+ 766.7i 498.4+ 466.8~
14.9(n=4) 27.2(n=3) 116.0(n= 2)* 61.7(H=3)* 14.9(n= 3)* 31.7(?/=2) 52.9(n=3)* 212.3(n=2)’ 160.I (n= 3)* 178.0(n= 3)* 31.6(i4=3) 49.8(n=3)
The control valuecorrespondsto the pooled three basal conditionsshownin Table J. Data are memrsl SEM of the number of preparationsgivenin parenthesis,performedin triplicate. Statisticalsignificanceof eachconditionvs basalconditionwasassessedusingStudent’sr-test for unpairedobservations.*P< O.05.
Ca2+-dependent changes of acetylcholine release 120 I
0.01
1
0.1
f
10
100
Concentration (IJM) Fig. 3. Dose-response curves for the effect of the phospholipase C inhibitors U-73122 and U-73343 on ACh release triggered by high K+ (120 mM) in the presence of 8 mM Ca2+. Results are expressed as 0/0of control ACh release, each symbol represents the means f SEM of values obtained in 3&t different synaptosomal preparations,
different preparations of synaptosomes, incubated in Ca2+-containing medium, the enzyme presented an activity of 1.94 pmol 3[H]-IP1 rein-’ mg-’. This activity was reduced to 0.58 pmol 3[H]-IPt rein– mg– when the assay was performed in the presence of lmM EGTA indicating that 1P, 3-kinase is activated by Ca2+. DISCUSSION
Our results show that pure cholinergic synaptosomes isolated from Torpedo electric organ have consistent, measurable, IP3 mass levels under basal conditions. Furthermore, the IP3 content under resting conditions was neither modified by different Ca2+ concentrations nor by incubation with a Ca2+-free medium supplemented with EGTA. In this regard Torpedo synaptosomes differ from rat cerebral cortex slices where IP3 mass levels were found to be significantly reduced by omission or chelation of Ca2+ (Challiss and Nahorski, 1991). In addition, concentrated Torpedo synaptosomal preparations have about 20 times more IP3 content per mg of protein than rat cerebral cortex slices (Challiss and Nahorski, 1991;Tzigaret et al., 1993). Control experiments, similar to those performed by Hidalgo et al. (1993), strongly suggest that elevated IP3 mass levels in synaptosomes are not due to phosphatidylinositol 4,5-
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bisphosphate hydrolysis during the perchloric acid extraction procedure used. Therefore, it is likely that cholinergic nerve endings may have a high density of binding sites for IP3 at specialized regions where transmitter release occurs. IP3 levels were found, in the present study, to increase during depolarization induced by high K+ provided Ca2+ was present, in agreement with previous studies performed in rat cerebral cortex slices with a radioreceptor assay (Challiss et al., 1988; Challiss and Nahorski, 1991) and in rat cerebral cortex synaptosomes prelabeled with 3[H]-inositol (Audigier et al., 1988). Thus, our study confirms that depolarization induced by high K+, in the presence of Ca2+ increases IP3 mass levels in an homogeneous preparation of pure cholinergic synaptosomes using a specific radioreceptor method which allows the measurement of the active form of IP3. A similar Ca2+-dependent increase in 1P3mass was observed in synaptosomes exposed either to the Ca2+ionophore A-23187 or to .gramicidin-D. While the Ca’+-ionophore A-23187 requires Ca2+ to induce membrane depolarization, both high K+ and gramicidin-D induced depolarization independent of the presence of Ca2+ (Meunier, 1984). Therefore, depolarizationper se is not the stimulus responsible for the accumulation of IP3. Furthermore, Na+ entry does not seem to be involved in the increase of IP3 levels since gramicidin-D in the absence of Ca2+ did not increase IP3 mass. It is widely accepted that all PLC isoenzymes described so far are activated by Ca2+ (Rhee and Choi, 1992). Therefore, the increase of 1P, mass we obtained under various depolarizing challenges may be the consequence of Ca2+ entry into synaptosomes. There is increasing evidence suggesting that Ca2+ from intracellular stores might play a role in regulating Ca2+-dependent functions in neurones (Simpson et al., 1995). The question that our experiments raise is what is the functional role for the increase in IP3 that occurs in a similar time domain than ACh release. Are these changes just coincidental or do they obey to some regulation of the neurotransmitter release process? The results obtained using U-73122 and the less active analog U-73343, phospholipase Cfl inhibitors, revealed that these compounds markedly reduced ACh release, evoked by high K+, to a greater extent than IP~ mass. These results may indicate that the signaling pathway leading to an increase of IP3 during depolarization by high K+ involves a transducing mechanism in which PLCO is activated. The effects of the aminosteroid compounds appear to be consistent with the disruption of the G-protein/PLCP
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coupling and inhibition of IP3 synthesis (Smith et al., 1990). However, the interpretation of these results is complex since these drugs have been recently reported to have additional effects on phosphoinositide metabolism which can lead to changes in Ca2+ homeostasis (Grierson and Meldolesi, 1995). In the present study the aminosteroid compounds not only reduced IP3 mass levels but have additional undetermined effects that lead to an almost complete blockade of ACh release. One possibility that is worth considering for assigning a functional role for IP3 is that the increase in this diffusible messenger may contribute to promote the synthesis of IP~ and other highly phosphorylated inositol phosphates which have been shown to block synaptic transmission when injected into the presynaptic terminal of the squid giant synapse (Llinas et al., 1994). The specific binding of these inositol phosphates to the C2 region of synaptotagmin, an integral protein of synaptic vesicles, might account for this effect (Fukuda et al., 1994). In this respect, Ca2+dependent accumulation of IP3 and IP~ mass has been reported in rat cerebral cortex slices during depolarization by high K+ (Challis and Nahorski, 1991). Finally, in the present study we have shown that Torpedo synaptosomes are endowed with IP3 3-kinase, enzyme that phosphorylates IP3 forming IPJ, and that its activity is stimulated by Ca2+. In conclusion, as far as we know this is the first time that IP3 mass levels have been measured in pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata which constitute a model preparation of nerve endings from the peripheral nervous system. The effects reported may suggest a possible link between IP3 increase and ACh release. However, such a link is probably not a direct one as implied by the preliminary results obtained with the inhibitors of PLC8. Acknowledgements—This work was supported by grants from Direction des Recherches Etudes et Techniques (94/067 to J.M), International Cooperation Program of the European Community (CI1 CT 94-0129), FONDECYT (1940 538 to M. A. C.) and ECOS (Scientific Cooperation with Chile, B9304). We are greatful to M. Israel and R. Kado for helpful comments and critical reading of the manuscript and J. Stinnakre for suggestions and help with figures and data analysis. REFERENCES Audigier S. M. P., Wang J. K. T. and Greengard P. (1988) Membrane depolarization and carbamoylcholine stimulate phosphatidylinositol turnover in intact nerve terminals. Proc. Natl. Acad. Sci. USA 85, 2859–2863.
Berridge M. J. (1993) Inositol trisphosphate and calcium signaling. Nature 361, 314325. Bredt D. S., Mourey R. J., Snyder S. H. (1989) A simple, sensitive and specific radioreceptor assay forinositol-l,4,5, trisphosphate in biological tissues. Biochem. Biophys. Res. Commun. 159,976982. Carrasco M. A., Morot Gaudry-Talarmain Y., Molgo J. and Caviedes P. (1995) Inositol l,4,5-trisphosphate mass measurement in Torpedo cholinergic synaptosomes under conditions that release acetylcholine. Am. Soc. Neurosci. Abstr. 21, part 2, Abstr. 430.15. . Carrasco M. A., Figueroa S. (1995) Inositol l,4,5-trisphosphate 3-kinase activity in frog skeletal muscle. Comp. Biochem. Physiol. 11OB,747–753. Challiss R. A. J. and Nahorski S, R. (1991) Depolarization and agonist-stimulated changes in inositol-1,4,5, trisphosphate and inositol l,3,4,5-tetrakisphosphate mass accumulation in rat cerebral cortex. J. Neurochem. 57, 1042-1051. Challiss R. A. J., Batty I. H., Nahorski S. R. (1988) Mass measurements of inositol 1,4,5,-trisphosphate in rat cerebral cortex slices using a radioreceptor assay: effects of neurotransmitters and depolarization. Biochem. Biophys. Res. Commun. 157, 684691. Dolezal V., Sbia M., Diebler M. F., Varoqui H. and Morel N. (1993) Effect of N, N’-Dicyclohexylcarbodiimide on compartmentation and release of newly synthesized and preformed acetylcholine in Torpedo synaptosomes. J. Neurochem. 61, 1454-1460. Fukuda M., Aruga J., Niinobe M., Aimoto S. and Mikoshiba K. (1994) Inositol-1,3,4,5 -tetrakisphosphate binding to C2B domain of IP4 BP/Synaptotagmin H. Z BioL Chem. 269,29206-29211. Grierson J. P., Meldolesi J. (1995) Calcium homeostasis in mouse fibroblast cells: affected by U-73122, a putative phospholipase Cj blocker, via multiple mechanisms. Brit. J Pharmacol. 115, 11–14. Gusovsky F., McNeal E. T., Daly J. W. (1987) Stimulation of phosphoinositide breakdown in brain synaptoneurosomes by agents that activates sodium influx: Antagonism by tetrodotoxin, saxitoxin, and cadmium. Mol. Pharmacol. 32,479487. Henzi V. and MacDerrnott A. B. (1992) Characteristics and function of Ca2+ and inositol 1,4,5-trisphosphate-releasable stores of Ca2+ in neurons. Neuroscience 46, 251–273. Hidalgo C., Jorquera J., Tapia V. and Donoso P. (1993) Triads and transverse tubules isolated from skeletal muscle contain high levels of inositol l,4,5-trisphosphate. Y. Biol, Chem. 268, 15111–15117. Israel M. and Lesbats B. (1981) Continuous determination by a chemiluminescent method of acetylcholine release and compartmentation in Torpedo electric organ synaptosomes. J. Neurochem. 37, 1475–1483. Llinas R., Sugimori M. and Silver R. B. (1995) Time resolved calcium microdomains and synaptic transmission. J. Physiol. (Paris) 89, 77–81. Llin& R., Sugimori M., Lang E. J., Morita M., Fukuda M., Niinobe M. and Mikoshiba K. (1994) The inositol highpolyphosphate series blocks synaptic transmission by preventing vesicular fusion: A squid giant synapse study. Proc. Natl. Acad. Sci, USA 91, 1299G12993. Meunier F.-M. (1984) Relationship between presynaptic membrane potential and acetylcholine release in synaptosomes from Torpedo electric organ. J. PhysioL (London) 354, 121–137.
Ca2+-dependent changes of acetylcholine release Morel N. and Meunier F.-M. (1981) Simultaneous release of acetylcholine and ATP from stimulated cholinergic synaptosomes. J Neurochem. 36, 176G1773. Morel N., Israel M., Manaranche R. and Mastour-Frachon P. (1977) Isolation of pure cholinergic nerve endings from Torpedo electric organ. J, Cell. BioL 75, 43–55, Moulian N., Morot Gaudry-Talarmain Y. (1993) Agelenopsis aperta venom and FTX, a purified toxin, inhibit acetylcholine release in Torpedo synaptosomes. Neuroscience 54, 1035–1041. Pressman B. C, (1976) Biological applications of ionophores. Ann. Rev. Biochem. 45, 501–530. Rhee S. G., Choi K. O. (1992) Regulation of inositol-phospholipid-specific phospholipase C isozymes. J. Biol. Chem. 267, 12393-12396. Simpson P. B., Challiss R, A., Nahorski S. R. (1995) Neuronal Caz+-stores: activation and function. Trends
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Neurosci. 18,299-306. Smith R. J,, Sam L. M,, Justen J. M., Bundy G. A., Bala G. A., Bleasdale J. E. (1990) Receptor-coupled signal transduction in human polymorphonuclear neutrophik: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J. Pharmacoi, Exp. Ther. 253, 688%697, Stephens L.R. (1990) Preparation and separation of inositol tetrakisphosphates and inositol pentakisphosphates and the establishment of enantiomeric configurations by the use of L-iditol dehydrogenase. In Methods in Znositide Research (Irvine R. F., Ed.), pp. 9–30. Raven Press, New York. Tzigaret C., Mc Intosh T. K., Okiyama K. et al. (1993) Measurement of hippocampal levels of cellular second messengers following in situ freezing. J. Neurochem. 60, 827-834.
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Neurochem.Int. Vol.29,No, 6, pp. 637-643,1996 CopyrightQ 1996ElsevierScienceLtd Printedin Great Britain.All rightsreserved 01974186/96$15.00+0.00
PII: S0197-0186(96)00046-0
Q2’-DEPENDENT CHANGESOF ACETYLCHOLINE RELEASEAND IP3MASS IN TORPEDOCHOLINERGIC SYNAPTOSOMES MARIA ANGELICA CARRASCO,* YVETTE MOROT GAUDRY-TALARMAIN~ and JORDI MOLGO Laboratoire de Neurobiologie Cellulaire et Mo16culaire, Centre National de la Recherche Scientifique, 91198-Gif sur Yvette, Cedex, France (Received for publication 17 April 1996) Abstract—The aim of the present study was to investigate possible changes of inositol l,4,5-trisphosphate (IPJ mass in Torpedo cholinergic synaptosomes in conditions promoting stimulated acetylcholine (ACh) release. For this purpose, we used a radioreceptor IP3 mass assay and a chemihrminescent method for ACh detection. Torpedo cholinergic synaptosomes have consistent 1P, mass levels under resting conditions, The 1P, mass was neither modified by changes in external Ca2+ nor by a Ca Z+-freemedium containing EGTA. IP3mass and ACh release, measured in the same conditions and in parallel, were increased by depolarization with high K+ and by the ionophores A-23187 and gramicidin-D in a manner dependent on external Ca2+ emphasizing that Ca2+ entry, independently of the influx mechanism involved, leads to an IP3 increase. The phospholipase Cl inhibitors U-73122 and U-73343 reduced K+-stimulated IP3 levels while K+-evoked ACh release was almost completely blocked suggesting an additional effect of these drugs on depolarizationneurotransmitter secretion coupling. The effect reported showing an increase of IP3 by agents that stimulate ACh release may suggest a possible link between 1P, metabolism and the neurotransmitter release mechanism. However, such a link is probably not a direct one as implied by the results obtained with the
inhibitors of phospholipaseC. Copyright~ 1996ElsevierScienceLtd
The inositol phospholipid-dependent signal transduction pathway is now considered to play an important role in controlling a variety of cell functions in the nervous system (Henzi and MacDerrnott, 1992; Berridge, 1993). Inositol l,4,5-trisphosphate (IP~) is produced in cells by a number of agonists that interact with surface receptors coupled to phospholipase C. Activated phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5 -bisphosphate to produce diacylglycerol and IPj. This last product is a diffusible second messenger which mobilizes intracellular Ca2+ (Berridge, 1993). The aim of the present work was to determine whether IP~ mass levels could be detected in pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata which constitute a model
preparation of nerve endings from the peripheral nervous system retaining most of their metabolic and physiological properties (Morel et al., 1977; Israel and Lesbats, 1981). In addition, we have studied IP~ levels as a function of extracellular Ca2+ in the presence of various agents that increase acetylcholine (ACh) release. Furthermore, the effeets of a phospholipase C inhibitor were studied on both ACh release and IPq levels. Finally, the activity of IPJ 3-kinase, the enzyme which phosphorylates IP~ to form inositol 1,3,4,5tetrakisphosphate (IP1) was investigated. A preliminary account of this work has already been published in abstract form (Carrasco et al., 1995). EXPERIMENTALPROCEDURES Preparation of Torpedo cholinergicsynaptosomes
*Permanent address: Departamento de Fisiologia y Biofisica, Facultad de Medicina, Universidad de Chile, Casilla 70005, Correo 7, Santiago, Chile. tTo whom all correspondence should be addressed. Tel.: 33-1-69-82-36- 33; Fax: 33-l-69-82-94-66; E-mail: morot@)hermes.cnrs-gif. fr; molgofj)hermes.cnrs-gif. fr.
The method for preparing cholinergic synaptosomes from the electric organ of Torpedo marmorata have been described in detail previoudy (Morel et al., 1977; Israel and Lesbats, 1981). In brief, nerve endings were chopped up and diluted in an oxygenated physiological solution. Then, synaptosomes were collected from a discontinuous iso-osmotic NaCl and 637
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sucrose gradient devoided of Ca2+ and further concentrated (6,000 g, 20 rein) after dilution in a modified physiological solution of the following composition (mM): NaCl, 480; KC1, 3; MgCl,, 1.8; sodium phosphate, 1.2; NaHCO,, 5; glucose, 5.5; (pH =7,2). The syrmptosomal pellet was resuspended in the modified physiological solution to adjust the protein concentration to 7–8 mg/ml, All steps of the isolation procedure were carried out at 4“C.
A23187 and gramicidin-D from Boehringer (Mannheim, {l-[6-[[17beta-3-methoxyestraGermany). U-73122 I,3,5(10)trien-17-yl] amino]hexyl]-l H-pyrrole-2,5-dione} and U-73343 {1-[6-[[17beta-3-methoxyestra-1,3,5 (10) trien-l7yl]amino]hexyl]-2,5-pyrrolidine-dione} were from Biomol Research Laboratories (Plymouth Meeting, PA, USA), All other reagents were obtained from Sigma or Merck. RESULTS
Acetylcho[irrerelease determination Measureme,lts of the amount of ACh released from synaptosomal suspensions were performed at room temperature (2W22°C) using the choline oxidase-chemiluminescent method as previously described (Israel and Lesbats, 1981). Aliquots of the concentrated synaptosomal fraction (10 Al) were diluted into 250 PI ofa physiological medium containing (mM): NsrCl, 240; LiCl, 40; KC1, 3; MgCl,, 1.8; sucrose, 400; glucose, 5.5; Tris buffer, 50 (pH=8.6). In some experiments either various concentrations of Ca2+ (2–8 mM, as specified) or EGTA (1 mM) were added to the nominally Ca2+-free medium. ACh measurements were performed after 3 min incubation in the physiological medium containing the reagents for the chemiluminescent assay (Israd and Lesbats, 1981). Drugs were added directly to the medium and, at the concentrations used, had no effect on the ACh assay. Synaptosomal preparations contained sufficient amounts of acetylcholinesterase for the hydrolysis of released ACh, so that no exogenous enzyme was further added. The light emitted by the chemihrminescent reaction for ACh measurements was detected by a photomultiplier unit, recorded and calibrated by the addition of known amounts of standard ACh as previously described (Israel and Lesbats, 1981). Drugs used were added directly to the medium. 1P, mass determination IP3 mass determinations were performed in parallel experimental conditions, i.e. after 3 min of preincubation in the physiological medium, EGTA, calcium and stimulating agents were added directly to the medium. The IPXcontent of synaptosomes was measured using a radioreceptor method based on the displacement of bound 3[H]-IP, to 1P, receptors from rat cerebella (Bredt et al., 1989). For this purpose, after stopping the experiment with 30 @ of 60Y0HC104, followed bycentrifugation, the supernatants were neutralized with 170 @of 0.1 M MES (pH= 6.5), 20 mM EDTA and 2 M KOH (Stephens, 1990; Hidalgo et al., 1993). All 1P, determinations were made in triplicate. IP3 3-kinase activity measurement IP3 3-kinase activity was measured under standard conditions as described previously (Carrasco and Figueroa, 1995) with 50 nM [3H]-IP, as substrate. To measure this activity Torpedo synaptosomes were permeabilized by several successive freezing and thawing steps in liquid nitrogen. Chemicals 3[H]-Inositol l,4,5-trisphosphate (sp. act. 21 Ci mmol “) was purchased from New England Nuclear (Dupont NEN, France). rr-Inosito] l,4,5-trisphosphate was from Calbiochem (France). Acetylcholine, choline and horseradish peroxidase (EC1.1 1.1.7) type II were from Sigma (St Louis, MO, USA), Iuminol from Merck (Darmstadt, Germany), choline oxidase from Wako Co. (Japan), and the ionophore
Determination preparations
oj” IP3 levels in resting synaptosoma[
As shown in Table 1, under basal conditions in Ca2+-free medium, the pelleted synaptosomal fraction contained an IP~ concentration of 471.3 +25.6 pmol mg-’ of protein. No significant difference was observed between the IPj level under this condition as compared with the values determined in Ca2+-containing medium (2–8 mM Ca2+) or containing 1 mM EGTA (Table 1). The IP~ mass remained unmodified during 1–5 min of incubation under the three control conditions. Changes in IP3 levels and ACh release during high K+induced depolarization Synaptosomal depolarization by high K+ induced an increase of IPq levels that depended on the presence of extracellular Ca2+. As shown in Fig. l(A), there was a significant increase in IPq mass in synaptosomes exposed for 1 min to extracellular K+ concentrations ranging from 6f&120 mM while no significant change was observed with 30 mM K+. When depolarization by K+ was induced in a nominally Caz+-free medium supplemented with EGTA, no significant change in IP3 mass was detected with the various K+ concentrations used (Fig. l(A)). Parallel ACh release experiments with increased extracellular K+, as expected (Israel and Lesbats, 1981; Meunier, 1984) caused a transient increase of Ca2+-dependent ACh release that depended on K+ concentration (Fig. l(B)). These results indicate that depolarization induced by high K+ (60 and 120 mM) causes an increase of both IP~ levels and ACh release. Table 1. IP3mass determinedin Torpedocholinergicsyrraptosomes under differentbasalconditions Experimentalcondition
IP3mass (pmOl/mgprotein)
Ca’+-freemedium Ca’+-freemedium + 1mM EGTA Ca’+-containingmedium(2–8mM)
471,3~ 25.6(tr=4) 484.2+ 44.5(n=3) 478.5~ 49.7(n=3)
Data are means ~ SEM of the number of differentTorpedopreparationsofsynaptosomesgivenin parentheses,performedin triplicate,
Ca2+-dependent changes of acetylcholine release
800
A
639
A
900
1
300J I,,,,,l,,,,l,,,,ri
o
60
,,,,,l,,,,rt,,,,,l
120
180 240
300
360
300
360
Time (s)
B
200 400 I
I
1
I
1
I
!
I
1
f
0 40 80120160
0 40 80 ~20180
[K+] (mM)
[K+] (mM)
Fig. 1. (A) 1P, levels (pmoles/mg protein) determined in Torpedo cholinergic synaptosomes depolarized by various K+ concentrationsduringl mininthepresenceofCa2+ (0) orinanominalfy Ca’+-free medium supplemented with 1 mMEGTA (~). (B) ACh release (nmoles/mg protein) from synaptosomes as a function of the K+ concentrations asin A. TheamountofACh released was measured after 1 min depolarization byhigh K+. Inane, data obtained from three to nine different synaptosomalpreparations were expressed asthemeantSEM.
0
● ✏✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌✌
o
60
120
f80
240
Time (s) ACh (150 pmol)
i
However, as shown in Fig. 1, while depolarization L“ IL with 30 mM K+ induced a significant increase of ACh I I I I 120 240 360 o release (Fig. l(B)), no significant change in IP3 levels was detected (Fig. l(A)). If ACh release occurs at Time (s) external K+ concentrations which do not increase IP3 Fig. 2. (A) Time course of the changes in IP3 mass (pmolevels and if ACh release is dependent on Ca2+ entry les/mg protein) of cholinergic synaptosomes depolarized by since no release was detected when the external Ca2+ 60 mM K+; in the presence of 4 mM Ca2+ (0) or in a Ca2+was reduced to low levels by EGTA, then small depo- free medium supplemented with 1 mM EGTA (A). Data are larizations open enough channels to produce intrathe mean~SEM of three to four different synaptosomal cellular Ca2+ concentration increases sufficient for preparations. (B) and (C) Time course of ACh release from ACh release. Stronger depolarization by K+ will lead cholinergic synaptosomes depolarized by 60 mM K+ in presence of 4 mM Ca2+. (B) The cumulative integral (area under to greater number of open Ca2+ channels and much the curve in (C)) expressing the amount and rate of the ACh higher levels of intracellular Ca2+. Under these con- release is presented as the function of time (Oto 6 rein) after ditions, it is likely that IP3 production may have a the depolarization by 60 mM K+ in presence of 4 mM Ca2+. (C) Time course of a typical chemiluminescent recording of higher Ca z+ threshold than the neurotransmitter ACh release (pmoles) using a known ACh (150 pmoles) for release mechanism which is known to require high calibration. Z+ microdomains (Llinas et ‘Z.> local intracellular Ca 1995). the presence of 60 mM K+ revealed the typical time When IP3 accumulation was studied in the presence of 60 mM K+, as a function of time (Fig. 2(A)), the course of transmitter release, as detected by the chemincrease was already detected at 30s, the shortest time iluminescent method (Fig. 2(B)). This time course is in accordance with previous results (Israel and Lesbats, assessed, and it was maintained during 5 min. Similar 1981; Dolezal et al., 1993) and with control measureresults were obtained with 120 mM K+ (data not shown). The continuous detection of ACh release in ments obtained by discontinuous ACh measurements
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(data not shown). It is worth noting that the maximum of the IP3 mass increase occurs in a similar time domain as the maximum rate of ACh release. Changes in IP3 levels and ACh release during the action of the Ca2+ionophore A-23 187 In order to bypass constitutive voltage-gated Ca2+ channels subtypes that mediate the Ca2+ influx during depolarization by high K+ (Moulian and Morot Gaudry-Talarmain, 1993) we used the Ca’+-ionophore A23187 to increase intrasynaptosomal Ca2+ levels. As shown in Table 2, the Ca2+-ionophore A-23187 (10 /M) was also effective in increasing IP3 levels (from 1–5 rein) provided the presence of extracellular Ca2+. Parallel ACh release measurements showed that A23187 increased ACh release (data not shown), as expected (Israel and Lesbats, 1981) only in the presence of extracellular Caz+. These results emphasize that Ca2+ entry is necessary to increase IP3 mass, independently of the Ca2+ influx mechanism involved. Changes in IP3 levels and ACh release during the action
of gramicidin-D Since depolarization induced by high K+ concentration also induces Na+ influx into synaptosomes and neurotoxins activating Na+ channels increase phosphoinositide hydrolysis (Gusovsky et al., 1987), experiments were performed with 1 PM gramicidinD. This antibiotic induces non-specific membrane channels for monovalent cations (Pressman, 1976) and depolarizes the synaptosomal membrane (Meunier, 1984). As shown in Table 2, gramicidin-D caused a marked increase in IP3 mass (assessed from 0.5–5 rein) only when Ca2+ was present in the physiological
medium. This indicates that monovalent influx of cations (Na+, Li+) is not the trigger of IP~ increase. AS previously shown (lsrael and Lesbats, 1981; Meunier, 1984) gramicidin-D caused a dramatic increase of ACh release (data not shown) owing to both depolarization-induced Caz+ influx from voltage-dependent Ca2+ channels and Ca2+ entry through the reversed operation of the Na+–Ca2+ exchange system (Morel and Meunier, 1981). Hence, the change in 1P, mass may be considered as depending on intracellular Ca2+ levels. Inhibition of PLC activity To further study IP3 metabolism in cholinergic synaptosomes we used inhibitors of PLC activity (Smith et al., 1990) and measured in parallel ACh release and IP3mass. As shown in Fig. 3, the membrane permeable aminosteroid U-73122 dose-dependently reduced Ca2+-dependent ACh release triggered by high K+ (120mM) medium with an EC50 of 0.79 t0.16 PM. The less active analog U-73343 required a higher drug concentration to reduce ACh release, the EC~~jwas 6.5 t 1.1 KM. The effects of U-73122 or U-73433 in 1P, mass levels were tested in synaptosomes in 120 mM K+ medium. At 2 PM neither compound prevented the 1P3mass increase obtained under 120 mM K+ while at 10 ,aM, both compounds limited the 1P, mass increase to 63.2 ~ 2.’7.0/. (n= 3) for U-73122 and 71.6. t6.1 % (n=3) for U-73343. Therefore, ACh release inhibition did not parallel the decrease in 1P3 mass with these drugs (Fig. 3). IP3 3-kinase activity Finally, the activity of IPj 3-kinase, enzyme that phosphorylates 1P, forming IP~,was measured. In two
Table2. Effectof the ionophoresA-23187and gramicidin-Don IPj massdeterminedin T~rP~>dO cholinergicsynaptosomesin the presenceand absenceof Ca2+ Experimentalcondition control 10KMA-23187,
4
mM Ca’+
10ItM A-23187,I mM EGTA I PM gr&micidin-D, 4 rnM Caz+
1flM gramicidin-D,1 mM EGTA
Timeof’ stimulation(rein) 0.5
I 2 5 I 0.5 I 2 5 I 5
1P,mass(pmOl/mgprotein) 461.8+ 469.2+ 642.1~ 548.4~ 550.0i 438.9* 591.2~ 957.2t 812.9+ 766.7i 498.4+ 466.8~
14.9(n=4) 27.2(n=3) 116.0(n= 2)* 61.7(H=3)* 14.9(n= 3)* 31.7(?/=2) 52.9(n=3)* 212.3(n=2)’ 160.I (n= 3)* 178.0(n= 3)* 31.6(i4=3) 49.8(n=3)
The control valuecorrespondsto the pooled three basal conditionsshownin Table J. Data are memrsl SEM of the number of preparationsgivenin parenthesis,performedin triplicate. Statisticalsignificanceof eachconditionvs basalconditionwasassessedusingStudent’sr-test for unpairedobservations.*P< O.05.
Ca2+-dependent changes of acetylcholine release 120 I
0.01
1
0.1
f
10
100
Concentration (IJM) Fig. 3. Dose-response curves for the effect of the phospholipase C inhibitors U-73122 and U-73343 on ACh release triggered by high K+ (120 mM) in the presence of 8 mM Ca2+. Results are expressed as 0/0of control ACh release, each symbol represents the means f SEM of values obtained in 3&t different synaptosomal preparations,
different preparations of synaptosomes, incubated in Ca2+-containing medium, the enzyme presented an activity of 1.94 pmol 3[H]-IP1 rein-’ mg-’. This activity was reduced to 0.58 pmol 3[H]-IPt rein– mg– when the assay was performed in the presence of lmM EGTA indicating that 1P, 3-kinase is activated by Ca2+. DISCUSSION
Our results show that pure cholinergic synaptosomes isolated from Torpedo electric organ have consistent, measurable, IP3 mass levels under basal conditions. Furthermore, the IP3 content under resting conditions was neither modified by different Ca2+ concentrations nor by incubation with a Ca2+-free medium supplemented with EGTA. In this regard Torpedo synaptosomes differ from rat cerebral cortex slices where IP3 mass levels were found to be significantly reduced by omission or chelation of Ca2+ (Challiss and Nahorski, 1991). In addition, concentrated Torpedo synaptosomal preparations have about 20 times more IP3 content per mg of protein than rat cerebral cortex slices (Challiss and Nahorski, 1991;Tzigaret et al., 1993). Control experiments, similar to those performed by Hidalgo et al. (1993), strongly suggest that elevated IP3 mass levels in synaptosomes are not due to phosphatidylinositol 4,5-
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bisphosphate hydrolysis during the perchloric acid extraction procedure used. Therefore, it is likely that cholinergic nerve endings may have a high density of binding sites for IP3 at specialized regions where transmitter release occurs. IP3 levels were found, in the present study, to increase during depolarization induced by high K+ provided Ca2+ was present, in agreement with previous studies performed in rat cerebral cortex slices with a radioreceptor assay (Challiss et al., 1988; Challiss and Nahorski, 1991) and in rat cerebral cortex synaptosomes prelabeled with 3[H]-inositol (Audigier et al., 1988). Thus, our study confirms that depolarization induced by high K+, in the presence of Ca2+ increases IP3 mass levels in an homogeneous preparation of pure cholinergic synaptosomes using a specific radioreceptor method which allows the measurement of the active form of IP3. A similar Ca2+-dependent increase in 1P3mass was observed in synaptosomes exposed either to the Ca2+ionophore A-23187 or to .gramicidin-D. While the Ca’+-ionophore A-23187 requires Ca2+ to induce membrane depolarization, both high K+ and gramicidin-D induced depolarization independent of the presence of Ca2+ (Meunier, 1984). Therefore, depolarizationper se is not the stimulus responsible for the accumulation of IP3. Furthermore, Na+ entry does not seem to be involved in the increase of IP3 levels since gramicidin-D in the absence of Ca2+ did not increase IP3 mass. It is widely accepted that all PLC isoenzymes described so far are activated by Ca2+ (Rhee and Choi, 1992). Therefore, the increase of 1P, mass we obtained under various depolarizing challenges may be the consequence of Ca2+ entry into synaptosomes. There is increasing evidence suggesting that Ca2+ from intracellular stores might play a role in regulating Ca2+-dependent functions in neurones (Simpson et al., 1995). The question that our experiments raise is what is the functional role for the increase in IP3 that occurs in a similar time domain than ACh release. Are these changes just coincidental or do they obey to some regulation of the neurotransmitter release process? The results obtained using U-73122 and the less active analog U-73343, phospholipase Cfl inhibitors, revealed that these compounds markedly reduced ACh release, evoked by high K+, to a greater extent than IP~ mass. These results may indicate that the signaling pathway leading to an increase of IP3 during depolarization by high K+ involves a transducing mechanism in which PLCO is activated. The effects of the aminosteroid compounds appear to be consistent with the disruption of the G-protein/PLCP
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coupling and inhibition of IP3 synthesis (Smith et al., 1990). However, the interpretation of these results is complex since these drugs have been recently reported to have additional effects on phosphoinositide metabolism which can lead to changes in Ca2+ homeostasis (Grierson and Meldolesi, 1995). In the present study the aminosteroid compounds not only reduced IP3 mass levels but have additional undetermined effects that lead to an almost complete blockade of ACh release. One possibility that is worth considering for assigning a functional role for IP3 is that the increase in this diffusible messenger may contribute to promote the synthesis of IP~ and other highly phosphorylated inositol phosphates which have been shown to block synaptic transmission when injected into the presynaptic terminal of the squid giant synapse (Llinas et al., 1994). The specific binding of these inositol phosphates to the C2 region of synaptotagmin, an integral protein of synaptic vesicles, might account for this effect (Fukuda et al., 1994). In this respect, Ca2+dependent accumulation of IP3 and IP~ mass has been reported in rat cerebral cortex slices during depolarization by high K+ (Challis and Nahorski, 1991). Finally, in the present study we have shown that Torpedo synaptosomes are endowed with IP3 3-kinase, enzyme that phosphorylates IP3 forming IPJ, and that its activity is stimulated by Ca2+. In conclusion, as far as we know this is the first time that IP3 mass levels have been measured in pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata which constitute a model preparation of nerve endings from the peripheral nervous system. The effects reported may suggest a possible link between IP3 increase and ACh release. However, such a link is probably not a direct one as implied by the preliminary results obtained with the inhibitors of PLC8. Acknowledgements—This work was supported by grants from Direction des Recherches Etudes et Techniques (94/067 to J.M), International Cooperation Program of the European Community (CI1 CT 94-0129), FONDECYT (1940 538 to M. A. C.) and ECOS (Scientific Cooperation with Chile, B9304). We are greatful to M. Israel and R. Kado for helpful comments and critical reading of the manuscript and J. Stinnakre for suggestions and help with figures and data analysis. REFERENCES Audigier S. M. P., Wang J. K. T. and Greengard P. (1988) Membrane depolarization and carbamoylcholine stimulate phosphatidylinositol turnover in intact nerve terminals. Proc. Natl. Acad. Sci. USA 85, 2859–2863.
Berridge M. J. (1993) Inositol trisphosphate and calcium signaling. Nature 361, 314325. Bredt D. S., Mourey R. J., Snyder S. H. (1989) A simple, sensitive and specific radioreceptor assay forinositol-l,4,5, trisphosphate in biological tissues. Biochem. Biophys. Res. Commun. 159,976982. Carrasco M. A., Morot Gaudry-Talarmain Y., Molgo J. and Caviedes P. (1995) Inositol l,4,5-trisphosphate mass measurement in Torpedo cholinergic synaptosomes under conditions that release acetylcholine. Am. Soc. Neurosci. Abstr. 21, part 2, Abstr. 430.15. . Carrasco M. A., Figueroa S. (1995) Inositol l,4,5-trisphosphate 3-kinase activity in frog skeletal muscle. Comp. Biochem. Physiol. 11OB,747–753. Challiss R. A. J. and Nahorski S, R. (1991) Depolarization and agonist-stimulated changes in inositol-1,4,5, trisphosphate and inositol l,3,4,5-tetrakisphosphate mass accumulation in rat cerebral cortex. J. Neurochem. 57, 1042-1051. Challiss R. A. J., Batty I. H., Nahorski S. R. (1988) Mass measurements of inositol 1,4,5,-trisphosphate in rat cerebral cortex slices using a radioreceptor assay: effects of neurotransmitters and depolarization. Biochem. Biophys. Res. Commun. 157, 684691. Dolezal V., Sbia M., Diebler M. F., Varoqui H. and Morel N. (1993) Effect of N, N’-Dicyclohexylcarbodiimide on compartmentation and release of newly synthesized and preformed acetylcholine in Torpedo synaptosomes. J. Neurochem. 61, 1454-1460. Fukuda M., Aruga J., Niinobe M., Aimoto S. and Mikoshiba K. (1994) Inositol-1,3,4,5 -tetrakisphosphate binding to C2B domain of IP4 BP/Synaptotagmin H. Z BioL Chem. 269,29206-29211. Grierson J. P., Meldolesi J. (1995) Calcium homeostasis in mouse fibroblast cells: affected by U-73122, a putative phospholipase Cj blocker, via multiple mechanisms. Brit. J Pharmacol. 115, 11–14. Gusovsky F., McNeal E. T., Daly J. W. (1987) Stimulation of phosphoinositide breakdown in brain synaptoneurosomes by agents that activates sodium influx: Antagonism by tetrodotoxin, saxitoxin, and cadmium. Mol. Pharmacol. 32,479487. Henzi V. and MacDerrnott A. B. (1992) Characteristics and function of Ca2+ and inositol 1,4,5-trisphosphate-releasable stores of Ca2+ in neurons. Neuroscience 46, 251–273. Hidalgo C., Jorquera J., Tapia V. and Donoso P. (1993) Triads and transverse tubules isolated from skeletal muscle contain high levels of inositol l,4,5-trisphosphate. Y. Biol, Chem. 268, 15111–15117. Israel M. and Lesbats B. (1981) Continuous determination by a chemiluminescent method of acetylcholine release and compartmentation in Torpedo electric organ synaptosomes. J. Neurochem. 37, 1475–1483. Llinas R., Sugimori M. and Silver R. B. (1995) Time resolved calcium microdomains and synaptic transmission. J. Physiol. (Paris) 89, 77–81. Llin& R., Sugimori M., Lang E. J., Morita M., Fukuda M., Niinobe M. and Mikoshiba K. (1994) The inositol highpolyphosphate series blocks synaptic transmission by preventing vesicular fusion: A squid giant synapse study. Proc. Natl. Acad. Sci, USA 91, 1299G12993. Meunier F.-M. (1984) Relationship between presynaptic membrane potential and acetylcholine release in synaptosomes from Torpedo electric organ. J. PhysioL (London) 354, 121–137.
Ca2+-dependent changes of acetylcholine release Morel N. and Meunier F.-M. (1981) Simultaneous release of acetylcholine and ATP from stimulated cholinergic synaptosomes. J Neurochem. 36, 176G1773. Morel N., Israel M., Manaranche R. and Mastour-Frachon P. (1977) Isolation of pure cholinergic nerve endings from Torpedo electric organ. J, Cell. BioL 75, 43–55, Moulian N., Morot Gaudry-Talarmain Y. (1993) Agelenopsis aperta venom and FTX, a purified toxin, inhibit acetylcholine release in Torpedo synaptosomes. Neuroscience 54, 1035–1041. Pressman B. C, (1976) Biological applications of ionophores. Ann. Rev. Biochem. 45, 501–530. Rhee S. G., Choi K. O. (1992) Regulation of inositol-phospholipid-specific phospholipase C isozymes. J. Biol. Chem. 267, 12393-12396. Simpson P. B., Challiss R, A., Nahorski S. R. (1995) Neuronal Caz+-stores: activation and function. Trends
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Neurosci. 18,299-306. Smith R. J,, Sam L. M,, Justen J. M., Bundy G. A., Bala G. A., Bleasdale J. E. (1990) Receptor-coupled signal transduction in human polymorphonuclear neutrophik: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J. Pharmacoi, Exp. Ther. 253, 688%697, Stephens L.R. (1990) Preparation and separation of inositol tetrakisphosphates and inositol pentakisphosphates and the establishment of enantiomeric configurations by the use of L-iditol dehydrogenase. In Methods in Znositide Research (Irvine R. F., Ed.), pp. 9–30. Raven Press, New York. Tzigaret C., Mc Intosh T. K., Okiyama K. et al. (1993) Measurement of hippocampal levels of cellular second messengers following in situ freezing. J. Neurochem. 60, 827-834.