Lack of involvement of [Ca2+]i in the external Ca2+-independent release of acetylcholine evoked by veratridine, ouabain and α-latrotoxin: Possible role of [Na+]i

43

J Physiology (1993) 87, 43-50 © Elsevier. Paris

Lack of involvement of [Ca2+]i in the external Ca2+-independent release of acetylcholine evoked by veratridine, ouabain and -latrotoxin: Possible role of [Na+]i V Adam-Vizi, Z Deri, P Bors, L T r e t t e r Department of Biochemisto' IL Semmelweis University of Medicine, Budapest H-1444, PO Box 262, Hungary

S u m m a r y - - Synaptosomes were challenged by veratridine, ouabain and et-latrotoxin, and the release of 14C-acetylcholine (ACb) was measured in the absence of external Ca 2+. We wished to test whether Ca 2÷ mobilized from internal stores triggered the ACh release that was independent of external Ca 2+. We found that none of the agents altered the [Ca2+]i in a Ca2+-free medium. Buffering the intracellular Ca 2+ concentration with BAPTA did not prevent the increase in release of 14C-ACh by veratridine or ouabain in the absence of Ca 2+, however, it greatly reduced the release evoked in a Ca2t-containing medium. In parallel samples the release of ACh and the change in the internal Na t concentration ([Nat]i) were measured. It was found that veratridine, ouabain and ot-latrotoxin all enhanced [Na÷]i in a concentration-dependent manner and a good quantitative relationship existed between the increase in [Na+]i and the release of ACh. c~-latrotoxin / ouabain / veratridine / ACh release / external Ca 2t

Introduction It has been reported by a number of investigators that certain depolarizing conditions evoke transmitter release not only in the ~resence but also in the absence of external Ca "÷. Veratridine, a compound acting on potential-dependent Na ÷ channels depolarizes nerve terminals, and in the presence of external Ca 2+ causes Ca 2+ influx (Biaustein, 1975; Akerman and Nicholis, 1981; Adam-Vizi and Ligeti, 1986 ; Satoh and Nakazato, 1991) and elevates [Ca2+]i (Adam-Vizi and Ashley, 1987) which correlates with the increase of the release of transmitters. In the absence of external Ca 2÷ there would be no Ca 2+ influx, yet increase in the release of transmitters was observed upon depolarization of the nerve terminals by veratridine in Ca2+-free solution (Haycock et al, 1978; Sandoval, 1980; Levi et al, 1980; Nelson and Blaustein, 1982; Schoffelmeer and Mulder, 1983; Adam-Vizi and Ligeti, 1984; Carroll and Benishin, 1984; Carvalho et al, 1986; McMahon et al, 1990). Ouabain, the inhibitor of the Na ÷, K+-ATPase which does not require the presence

of Ca 2+ in the external medium also has a transmitter-releasing effect (for review see Adam-Vizi, 1992). It stimulates the release of a number of neurotransmitters from the neuromuscular junction (Vizi and Vyskocil, 1979), cortex slices (Vizi, 1972 ; Gomez et al, 1975 ; Prado et al, 1990) and synaptosomes (Meyer and Cooper, 1981; Vyas and Marchbanks, 1981; Adam-Vizi and Ligeti, 1984; Santos et al, 1990; Satoh and Nakazato, 1992). Although the principal sites of action of veratridine and ouabain are different, both of them interfere with the Na + electrochemical gradient of the plasma membrane and are assumed to elevate the intracellular Na + concentration of the nerve terminals. Recently free [Na+]i of synaptosomes has been measured by a new fluorescent Na+-in dicator, sodium-binding benzofuran isophthalate (SBFI), (Deri and Adam-Vizi, 1993) and, indeed, increased [Na+]i was detected in the presence of these drugs. ~-Latrotoxin, the active compound of the black widow spider venom, was also shown to elevate the internal Na + concentration of synaptosomes (Deri and Adam-Vizi, 1993). This

44 toxin increased the efflux o f a number o f transmitters in several preparations ( L o n g e n e c k e r et al, 1970; Tzeng and Siekevitz, 1978; Grasso and Senni, 1979; Meldolesi, 1982; Nicholls et al, 1982; for review see Rosenthal and Meldolesi, 1989), whether or not Ca ÷ is present in the medium (Tzeng et al, 1978; Grasso and Senni, 1979; Nicholls et al, 1982; Meldolesi et al, 1983). It was also shown that the toxin-induced release o f A C h (Deri et al, 1993) and glutamate ( M c M a h o n et al, 1990) was reduced in the absence o f external Na +, and it was suggested that a c o m p o n e n t o f the release of ACh was Na÷-de pendent (Deri et al, 1993). Experiments in the present work aimed to answer two q u e s t i o n s : 1) could there be a role for internal Ca 2÷ in the external Ca2÷-independent release o f acetylcholine evoked by veratridine or ouabain? It has been suggested that ouabain might release Ca 2÷ from intracellular pools by a Na +Ca 2+ exchange mechanism and hence trigger acetylcholine release from the neuromuscular junction (Baker and Crawford, 1975; Sandoval, 1980; C u n n i n g h a m and Neal, 1981). We have tested if there was any increase in [Ca2+]i in the presence o f ouabain or veratridine in a CaZ+-free medium. In addition, we have used B A P T A to buffer the intracellular [Ca 2+] and checked if that modified the external Ca2+-independent release o f acetylcholine evoked by these c o m p o u n d s ; and 2) is there any quantitative relationship between the increase o f [Na÷]i and A C h release? Synaptosomes were depolarized by veratridine, ouabain or oc-latrotoxin and the internal free Na ÷ concentration and the release o f 14C-ACh was measured in parallel samples. The results show that ACh release in the absence o f external Ca 2÷ occurs without a detectable change in [Ca2+]i; however, it can be quantitatively related to an elevated [Na+]i.

mC-acetylcholine release Synaptosomes were incubated in a standard medium (140 mM NaCI, 3 mM KCI, 2 mM MgC12, 2 mM CaCI2, 25 mM Hepes-Na, (pH 7.4) 10 mM glucose) containing 2p.M [14C]choline (0.1 ~tCi/ml; specific activity 55 mCi/mmol) for 30 min at 37°C. After sedimentation and washing, the pellet was resuspended in 0.32 M sucrose (20 mg/ml) and 50 lal aliquots were used for measuring the release of t4C-acetylcholine as detailed previously (Adam-Vizi and Ligeti, 1984). Separation of ~4C-acetylcholine from Jnc-choline released from synaptosomes was carried out as described by Rand and Johnson (1981).

[Ca2+]i Synaptosomes were loaded with fura-2 by incubation in the standard medium containing 5 ~tM fura-2-AM at 37°C for 20 min as described (Adam-Vizi and Ashley, 1987). The fluorescence of fura-2 was determined in a Perkin Elmer LS 50 fluorescence spectrometer. Excitation was at 340 nm, and emission at 500 nm.

[Na+]i Synaptosomes were loaded with SBFI (sodium-binding benzofuran isophthalate), a new fluorescent indicator (Minta and Tsien, 1989; Harootunian et al, 1989; Ali et al, 1989; Borin and Siffert, 1990) by incubation in the standard medium lacking sodium that contained 10 ~tM SBFI/AM for 60 min at 37°C as described elsewhere (Deri and Adam-Vizi, 1993). The 340/380-nm fluorescence intensity of synaptosomes were measured in a Perkin-Elmer LS 50 fluorescence spectrometer, and a calibration curve was used to quantify [Na+]i in mM.

Materials Standard laboratory chemicals were obtained from Calbiochem. [lac]choline was obtained from Amersham. ~Latrotoxin was a gift from AG Petrenko and Yu A Ushkaryov (Shemyakin Institute of Bioorganic Chemistry, Moscow, Russia).

Materials and methods Results Preparation of synaptosomes Synaptosomes were prepared from brain cortex of guinea pigs as described by Hajos (1975). Synaptosomes obtained from an 0.8 M sucrose gradient were diluted slowly with an equal volume of distilled water and centrifuged at 20000 g for 20 min. The pellet was suspended in 0.32 M sucrose (20 mg/ml of protein) and kept on ice for further manipulation.

[Ca2+]i of synaptosomes measured in a Ca2+-free medium. Lack of effect of veratridine and ouabain To investigate the potential involvement o f the intracellular Ca 2+ in the depolarization-evo~'ed ACh release in a Ca2+-free medium, [Ca2+]i was measured and synaptosomes were depolarized by either veratridine ( 5 - 4 0 ~tM) or o u a b a i n ( 2 0 -

45

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Fig 1. [Ca2+]i of synaptosomes measured in a Ca2÷-free medium. Veratridine (20 taM) or ouabain (100 llM) was applied as indicated and [Ca- ]i was momtored for 13 man.

100 ~tM). In C a 2 + - c o n t a i n i n g m e d i u m v e r a t r i d i n e c a u s e s a slow, g r a d u a l rise in [Ca2+]i w h i c h is sust a i n e d as l o n g as v e r a t r i d i n e is p r e s e n t . F i g u r e 1 s h o w s that n e i t h e r o f these c o m p o u n d s c a u s e s a d e t e c t a b l e i n c r e a s e in [Ca2+]i w h e n a p p l i e d in a m e d i u m c o n t a i n i n g n o C a 2+ b u t 1 m M E G T A . As the m e t h o d o f m e a s u r i n g [Ca 2 + ]i in a s y n a p t o s o m a l s u s p e n s i o n m e a s u r e s an a v e r a g e Ca 2 + c o n c e n t r a t i o n , s m a l l local c h a n g e s o f [Ca 2 + ]i, w h i c h c o u l d b e s i g n i f i c a n t in the r e g u l a t i o n o f t r a n s m i t ter r e l e a s e , m a y not be d e t e c t e d .

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Fig 2. Synaptosomes were loaded with ]4C-choline and 14CACh release was measured as described in Materials and methods. BAPTA, where indicated, was present in 100 pM concentration from the loading throughout the experiment. Veratridine, ouabain or high K+ concentration were al~plied after 5 min preincubation in a Ca2÷-containing (a) or Ca-+-free medium containing 1 mM EGTA (b) and incubation was continued for 10 min. Data are the averages of four determinations made in triplicate (SEM).

The effect o f veratridine and ouabain on 14C-ACh r e l e a s e f r o m s y n a p t o s o m e s p r e t r e a t e d with BAPTA In o r d e r to i n v e s t i g a t e w h e t h e r C a 2+ w i t h i n the n e r v e t e r m i n a l , n o t r e v e a l e d b y the f l u o r e s c e n t [Ca 2+] m e a s u r e m e n t , is i n v o l v e d in t r i g g e r i n g A C h release, we p r e t r e a t e d s y n a p t o s o m a l s a m p l e s w i t h B A P T A ( 1 0 0 ~ M ) a n d s t u d i e d the r e l e a s e o f 14C-ACh. B A P T A is u s e d to b u f f e r the i n t r a c e l l u lar C a 2+ c o n c e n t r a t i o n a n d is s u p p o s e d to i n h i b i t c e l l u l a r f u n c t i o n s that are r e l a t e d to c h a n g e s o f

46 [Ca2+]i. In figure 2 14C-ACh release evoked by veratridine or ouabain in the presence of external Ca 2÷ (fig 2a) is compared to that observed in the absence of Ca 2+ (fig 2b) with and without treatment with BAPTA. It has been thoroughly documented that depolarization by a high K ÷ concentration in a Ca2+-containing medium opens voltage-dependent Ca 2÷ channels and Ca 2+ triggers the efflux of transmitters. It has also been shown that veratridine evokes Ca 2÷ influx, most likely via Na+-Ca 2+ exchange through the plasma membrane (Adam-Vizi and Ligeti, 1986). In accordance with these observations, 14C-ACh release evoked by high [K +] or veratridine was greatly reduced in a preparation where [Ca2+]i w a s buffered with 100 laM BAPTA (fig 2a). The effect of ouabain (100 !aM) was only slightly inhibited, in agreement with the finding that Ca 2÷ influx is not dramatically enhanced by ouabain during a 5rain incubation period (Adam-Vizi and Ligeti, 1986). Figure 2b shows that 14C-ACh release could also be elicited by ouabain and veratridine in a Ca2+-free medium, however, this release was not altered when the intracellular Ca 2÷ concentration was buffered with BAPTA. This result strongly suggests that no increase o f [Ca2+]i occurs when synaptosomes are depolarized by veratridine or ouabain in the absence of external Ca 2+, therefore external CaZ+-independent release of laC-ACh in response to these compounds appears to be unrelated to [Ca2+]i.

ratridine there appears to be a good quantitative relationship between the internal Na + concentration and the efflux of J4C-ACh. This appears to be also true when the Na ÷ concentration and the release of 14C-ACh were enhanced by 20 or 100 pM ouabain (table I). The rise of [Na+]i in response to ouabain is slower than that to veratridine (Deri and Adam-Vizi, 1993), however, values obtained after 10 min incubation (table I) show the same quantitative correlation between the two parameters. To investigate further whether ACh release could result from a rise in [Na+]i, the effect of 14C-ACh release (% of total labelled ACh)

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Changes of [Na+]i in response to veratridine, ouabain and o~-latrotoxin. Correlation between [Na+]i and the external Ca2+-independent release of 14C-ACh Internal free Na ÷ concentration of synaptosomes was followed by the new fluorescent Na ÷ indicator SBFI. As we have reported elsewhere (Deri and Adam-Vizi, 1993) an increased fluorescent signal was observed upon addition of veratridine to synaptosomes (fig 3, insert). In the present experiment synaptosomes were depolarized in the absence of external Ca 2÷ by veratridine applied in different concentrations and both the 340/380 fluorescence ratio and the release of 14C-ACh were followed in parallel samples. Veratridine enhanced [Na+]i from the resting value of 8.5 mM in a concentration-dependent manner (10-40 pM) (fig 3) using equilibrium values of [Na÷]i reached 3 min after addition of veratridine. Parallel to the [Na+]i changes the effiux of 14C-ACh release was also increased. In this concentration range of ve-

16

mn

40

(/~M)

Fig 3. [Na+]i and 14C-ACh release were measured in parallel samples after incubation of synaptosomes in the presence of different concentrations of veratridine for 10 min. The medium contained no Ca 2÷ but 1 mM EGTA. Insert, 340/380 nm fluorescense intensity ratio in synaptosomes loaded with SBFI. Veratridine was added as indicated by the arrow. [Na+]i in mM was calculated using a calibration curve (SEM values were less than 5% of the average values).

Table I. The effect of ouabain on [Na+]i and 14C-ACh release of synaptosomes.

[Na+]i (mM) Control Ouabain 20 pM 100 gM

ACh release (% of total)

8.5 + 1.2

16

_ 2.1

22 + 2.3 25 + 1.9

26.1 + 1.8 27.5 + 2.4

[Na+]i and 14C-ACh release were measured as for figure 3. Ouabain was added after a 5-min preincubation in a Ca2+-free medium and incubation was continued for 10 rain. Data are the averages of three determinations (SEM).

47 ct-latrotoxin was studied on both [Na+]i and 14EA C h release. Upon binding to its receptor the toxin augments the permeability of the plasma membrane to monovalent and divalent cations (Grasso et al, 1980; Nicholls et al, 1982; Meldolesi et al, 1984; Wanke et al, 1986; Scheer et al, 1986) and the latter were suggested to be the triggers of the toxin-elicited release of transmitters (Finkelstein et al, 1976; Hurlbut et al, 1971 ; Kharash et al, 1981 ; Mellow et al, 1982 ; Rosenthai et al, 1990; see also Rosenthal and Meldolesi, 1989). It has been reported that a component of the release of 14C-ACh due to the toxin requires the presence of Na ÷ in the medium (Deri et al, 1993). In synaptosomes, SBFI fluorescence was shown to be elevated by ct-latrotoxin (Deri and Adam-Vizi, 1993). The rise of [Na+]i as a function of ot-latrotoxin concentration is illustrated in figure 4 which shows that a toxin concentration of 1.5 x 10-1°-3.5 x 10-9M elevates [Na+]i from 8 mM to 48 mM. In this concentration range the efflux of IaC-ACh is also enhanced, and this is independent of the external Ca 2+. As with veratridine, the same curve represents the change of [Na+]i and 14C-ACh release, which suggests that intracellular Na ÷ may be an important factor in the regulation of external Ca2+-independent ACh release.

[No+] I (rnM)

t4C-ACh releose (~; of ~otol Iobelled ACh)

50

50

40 30

A ~

20

40

34o/380 3,~'

10

20

10 -1°

10 - °

1 0 -a

log [olpho--lotrotoxin] (M)

Fig4. [Na+]i and I4C-ACh release were measured as in figure 3. tx-Latrotoxin, in the concentrations indicated, was added after 5 min preincubation in a Ca2+-free medium and incubation was continued for 10 rain. Insert, 340/380nm fluorescence intensity ratio of synaptosomes loaded with SBFI. ~-Latrotoxin (10-9 M) was added as indicated by the arrow. Points represent the averages of three determinations (SEM values are less than 5% of the average values).

Discussion One of the conclusions drawn from the present experiments is that intracellular Ca 2+ is unlikely to be involved in the external Ca2+-independent acetylcholine release evoked by ouabain or veratridine. The suggestion that Ca 2÷ released from the intracellular stores triggers the Ca2÷-inde pendent transmitter release was put forward by Baker and Crawford (1975) to explain the effect of ouabain on the neuromuscular junction. This explanation was also adopted for the stimulatory effect of veratridine on GABA release observed in a Ca2÷-free medium (Sandovai, 1980; Cunningham and Neal, 1981) and the Ca2÷-independent release of ACh (Adam-Vizi and Ligeti, 1984) and noradrenaline (Torok and Magyar, 1986; Torok et al, 1987) in response to ouabain. This seemed to be a plausible explanation since both veratridine and ouabain alter the Na ÷ electrochemical potential, and Na ÷ had been shown to induce Ca 2÷ efflux from the mitochondria (Crompton et al, 1976; Silbergeld, 1977; Nicholls and Crompton, 1980). However, inconsistent with this hypothesis were reports that Ca 2÷ release from the mitochondria by protonophores (Sanchez-Prieto et al, 1987) or by anoxia (Kauppinen et al, 1988) failed to cause glutamate release, furthermore, that veratridine did not stop the release of GABA from synaptosomes depleted of Ca 2÷ (Sihra et al, 1984). In the present experiments no evidence was found for the role of intracellular Ca 2÷ in the stimulated release of 14C-ACh by ouabain or veratridine. No increase of [Ca2+]i by either of these compounds could be detected. Although this cannot be regarded as conclusive, because an overall Ca 2+ concentration is measured, experiments with BAPTA also argue against the involvement of intracellular Ca 2÷. If Ca 2÷ were involved in the Ca2+-independent effect of ouabain or veratridine, BAPTA, by buffering [Ca2+]i, should influence the release of ACh. This indeed occurred in the presence of Ca 2+, however, in Ca2+-free medium both ouabain and veratridine were still able to stimulate ACh release. An alternative explanation for the Ca2÷-inde pendent release of amino acid neurotransmitters is that carriers present in the plasma membrane work in a reverse mode due to an alteration of the Na + electrochemical gradient (for review see Nicholls, 1989). This latter is obviously an important factor in the effect of ouabain and veratridine. However, the explanation cannot easily be applied to the release of acetylcholine, as no

48 Na+-dependent ACh carrier has yet been described. In spite of this we found a quantitative correlation between the intracellular Na ÷ concentration and ACh release which seems to be consistent in the case of veratridine, ouabain and c~-latrotoxin. In the interpretation of this phenomenon some possibilities can be envisaged : 1) alteration of [Na+]i is only 'an epiphenomenon' and the factor that is more directly involved in the ACh release process is secondary to a change in [Na+]i. This could be intracellular Ca 2+ mobilized from the stores, however, as discussed above, this appears unlikely. Another parameter to be considered is the intracellular pH which may be shifted by a rise of [Na÷]i. In preliminary experiments with the intracellular pH indicator, BCECF we have not detected marked change in pH using small concentrations of veratridine and ouabain where ACh release Was already stimulated. To control for the possible role of pH, Nordmann and Stuenkel (1991) who applied intracellular Na ÷ pulses and measured vasopressin secretion from the neurohypopbysis, induced changes in intracellular pH with propionic acid, but this failed to produce the same secretory response as Na+; 2) cholinergic nerve terminals may possess a Na ÷dependent carrier that mediates the efflux of ACh when the Na ÷ electrochemical gradient favors an outward transport. It would be worthwhile to investigate if the high-affinity choline uptake could work in the reverse direction and could carry ACh when the [Na ÷] within the nerve terminals is enhanced. It has been suggested, as an alternative to vesicular exocytosis, that ACh is transported through the plasma membrane by a channel or a carrier even in the presence of Ca 2+ (Tauc, 1982 ; Israel et al, 1984; Birman et al, 1986; Dunant, 1986; see also Van der Kloot, 1988). There is as yet no information as to whether this process is sensitive to changes in [Na+]i; 3) it is also possible that intracellular Na ÷ is directly involved in the regulation of ACh release. Nordmann and Stuenkel (1991) made a strong case for a role of Na ÷, suggesting that it may be an intrinsic regulator of neurosecretion. They demonstrated a marked increase in the release of vasopressin on increasing [Na+]i without a concomitant change in [Ca2+]i. These experiments may throw new light upon earlier experiments in which injection of Na ÷ into the squid giant synapse (Charlton and Atwood, 1977), addition of Na+-containing liposomes to the frog neuromuscular junction (Rahamimoff et al, 1978) and Na÷-ionophores (Sandoval, 1980; Meiri et al, 1981; Sitges,

1989a,b) were able to stimulate neurotransmission. Whatever is the role of [Na+]i in the Ca2+-independent release of transmitters it appears to be a significant factor in the release of acetylcholine.

Acknowledgments This work was supported by ETT, OTKA and USHungarian Joint Fund to VA-V Thanks are expressed to AG Petrenko and Yu A Ushkariov (currently in the Department of Molecular Genetics, Howard Hughes Medical Institute, Texas) for the kind gift of c~latrotoxin.

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