TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

115, i l-20 ( 1992)

Inhibition of Mitochondrial Ca*+ Release Diminishes the Effectiveness Methyl Mercury to Release Acetylcholine from Synaptosomes’ PAUL C. LEVESQUE,~ MICHAEL F. HARE,~ AND WILLIAM

of

D. ATCHISON~‘~

Department of Pharmacology and Toxicology, Neuroscience Program, and Institute for Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824-1317 Received July 3 1, 199 I: accepted February 28, 1992

hibitor of mitochondrial Ca2+ transport, was tested for its ability to disrupt MeHg-induced release. RR alone increased [3H]ACh release by 8-10 and lo-13% at 20 and 60 PM, respectively. RRinduced release was attenuated only slightly in Ca’+-free solutions. Preincubation of [3H]choline-loaded synaptosomes with RR reduced the stimulatory effect of MeHg on release of [3H]ACh both in the presence and in the absence of Ca2+. The fluorescent potentiometric carbocyanine dye diS-C,(5) wasused to assessthe ability of RR to prevent MeHg-induced depolarization of intrasynaptosomal mitochondria. RR (20 PM) itself did not depolarize the mitochondrial membrane potential, nor did it prevent MeHg from depolarizing the mitochondria. The results indicate that extracellular Ca2+ contributes only partially to MeHg-induced spontaneous release of ACh. The results with RR suggest that MeHg interacts with mitochondria to induce release of bound intraterminal Ca*+ stores, resulting ultimately in stimulated release of ACh. The ability of RR to prevent release of mitochondrial Ca” and, subsequently, ACh is not due to prevention of access of MeHg to the mitochondria, nor to stabilization of the mitochondrial membrane. Finally, MeHg reduces choline uptake into nerve terminals. Thus, MeHg could interfere with cholinergic neurotransmission by affecting the regulatory step in ACh synthesis and by increasing the spontaneous release of transmitter. This combination of events would result in depletion of the readily releasable pool of ACh in axon terminals. 0 1992 Academic

Inhibition of Mitochondrial Ca2+ Release Diminishes the Effectiveness of Methyl Mercury to Release Acetylcholine from Synaptosomes. LEVESQUE, P. C., HARE, M. F., AND ATCHISON, W. D. (1992). Toxicol. Appl. Pharmucol. 115, 1 l-20. The interaction of methyl mercury (MeHg) with nerve-terminal mitochondria as a potential mechanism for its effects on the release of acetylcholine (ACh) was studied using rat brain synaptosomes. The primary goal was to assessthe relative contribution of extracellular Ca2+ and Ca2+ released from nerveterminal mitochondria to the previously described stimulatory effects of MeHg on spontaneous release of ACh. A secondary goal was to address possible mechanisms by which MeHg might interact with nerve-terminal mitochondria to elicit Ca’+ discharge and subsequent release of ACh. MeHg depressed the highaffinity uptake of [3H]choline into synaptosomes by approximately 25 and 45% when synaptosomes were incubated with [3H]choline in the presence of 10 and 100 MM MeHg, respectively. In Ca2+-containing solutions, 10 and 100 PM MeHg increased the release of [3H]ACh from [3H]choline-loaded synaptosomes by 10 and 30%, respectively; this effect was maximal at IO sec. Excluding Ca2+ from the reaction medium diminished the effectiveness of both 10 and 100 PM MeHg for inducing [3H]ACh release by about 30 and 25%, respectively, from that of Ca2+containing solutions; however, significant increases still occurred in nominally Ca2+-free solutions. Ruthenium red (RR), an in-

PRSS,

’ Supported by NIH Grant ES03299. The SPEX instrumentation was purchased with NIH Shared Instrumentation Grant ISlO-RR04927. This work was submitted by P.C.L. in partial fulfillment of the requirements for the Ph.D. degree in pharmacology and toxicology at Michigan State University. Preliminary results of parts of this work were presented at the 19th Annual Meeting of the Society for Neuroscience, October 29-November 3, 1989, Phoenix, Arizona and were published in abstract form in Society for Neuroscience Abstracts 15, 84.16, 1989. * Current address: Department of Physiology, University of Nevada College of Medicine, Anderson Medical Sciences Building, Reno, NV 89557-0046. 3 M.F.H. is the recipient of NIEHS Individual Postdoctoral Fellowship ES055 12. 4 W.D.A. is the recipient of Research Career Development Award SK04ES00178. ’ To whom correspondence and reprint requests should be addressed at Department of Pharmacology and Toxicology, B-33 1 Life Sciences Building, Michigan State University, East Lansing, Michigan 48824- 13 17. Fax: (5 17) 353-8915.

h.

Methyl mercury (MeHg)6 is a lipophilic mercurial that exerts prominent neurotoxic effects including sensory disturbances, cerebcllar ataxia, and generalized extremity weakness in exposed individuals after acute or chronic exposure (Hunter et al., 1940; Takeuchi et al., 1968; Bakir et al., 1973; Chang, 1977). The molecular and cellular mechanisms responsible for the effects of MeHg are as yet unclear, but could be due in part to biochemical and physiological effects of MeHg on the nerve, resulting in disruption of synaptic transmission with consequent sensory and motor defects. 6 Abbreviations used: MeHg, methyl mercury; ACh, acetylcholine; RR, ruthenium red; NMJ, neuromuscular junction; [Ca”],, intracellular concentration of calcium: Hepes, N-hydroxyethylpiperazine-M-2-ethanesulfonic acid, HPS, Hepes-buffered physiological saline; diS-C,(S), 3,3’-diethylthiadicarbocyanine iodide: DMSO, dimethyl sulfoxide: NaN3, sodium azide. 11 Copyright All rights of

0041-008x/92 $5.00 0 1992 by Academic Press, Inc. reproduction in any form reserved.

12

LEVESQUE,

MeHg affects transmitter

HARE, AND ATCHISON

release from central (Bondy et

creases [Ca2+]i. Moreover,

a portion of that Ca2+ increase

al., 1979; Komulainen and Tuomisto, 198 1, 1982; Saijoh et by MeHg may come from intrasynaptosomal mitochondria al., 1987; Minnema et al., 1989) and peripheral nerve endings (Komulainen and Bondy, 1987; Kauppinen et al., 1989). It (Barrett et al., 1974; Juang, 1976; Atchison and Narahashi, 1982; Atchison et al., 1984). The effects of MeHg on transmitter release have been characterized extensively by electrophysiological methods only at the neuromuscular junction (NMJ). One prominent effect of MeHg observed at this peripheral cholinergic synapse is a transient increase in asynchronous, spontaneous quanta1 release of acetylcholine (ACh) (Barrett et al., 1974; Juang, 1976; Atchison and Narahashi, 1982). Spontaneous release of ACh is not dependent on depolarization-induced influx of Ca*+ into the nerve-terminal or axonal impulse propagation. The frequency of spontaneous release, however, is directly proportional to the free Ca*+ concentration in the nerve terminal ([Ca*‘];) (Llinas and Nicholson, 1985). [Ca*+]i can be increased in the absence of external Ca2+ when Ca*+ is released from internal stores (Alnaes and Rahamimoff, 1975). MeHg increases the spontaneous release of ACh at the NMJ in the absence of extracellular Ca’+, suggesting a perturbation of mechanisms regulating [Ca*+]i (Atchison, 1986, 1987). This finding prompted subsequent electrophysiological (Levesque and Atchison, 1987, 1988) and neurochemical (Levesque and Atchison, 199 1) studies that implicated nerveterminal mitochondria as a source of Ca’+ for the increased spontaneous release of ACh induced by MeHg. Pretreatment of a neuromuscular preparation with ruthenium red (RR) or N,IV-bis(3,4-dimethoxyphenylethyl)-N-methylamineYS035, inhibitors of mitochondrial Ca2+ transport, completely prevented the stimulatory effects of MeHg on spontaneous release of ACh. Moreover, MeHg inhibited uptake of 45Ca2+ by isolated mitochondria and induced release of 45Ca2+ from preloaded mitochondria via a RR-sensitive mechanism. Taken together, these results suggest that MeHg is able to induce the release of Ca2+ from mitochondria and that release of this pool of Ca2+ by MeHg may contribute to the increased spontaneous release of ACh observed at the NMJ. Several neurochemical analyses have shown that MeHg increases spontaneous release of neurotransmitters from isolated nerve terminals (synaptosomes) (Bondy et al., 1979; Komulainen and Tuomisto, 198 1, 1982; Minnema et al., 1989) and brain slices (Saijoh et al., 1987). Spontaneous release of ACh, dopamine, GABA, glycine, and glutamate appear to be affected similarly by MeHg. Since the effects of MeHg on spontaneous release of transmitter from CNS preparations are not unique to a particular transmitter type, perhaps MeHg acts via a mechanism common to all transmitter systems. One mechanism that could cause increased spontaneous release of different neurotransmitters would be to increase [Ca2’]i. Using a fluorescent probe for Ca2’, Komulainen and Bondy (1987) demonstrated that MeHg significantly in-

may well be that the mechanism responsible for MeHg’s effects on spontaneous release of transmitter from synaptosomes, model CNS nerve endings, is similar to the mechanism proposed (Levesque and Atchison, 1987, 1988) to underlie the effect of MeHg at the NMJ, a peripheral cholinergic synapse. Thus, in the present study, we sought to link the various observations regarding the increased release of transmitter and the suspected origin of the increased [Ca*+]i. The primary objective was to determine whether MeHg increased spontaneous release of [3H]ACh from central nerve terminals subsequent to an interaction with mitochondria to release Ca2+. Effects of MeHg on release of [3H]ACh from rat brain synaptosomes loaded with [‘HIcholine were tested in the absence and presence of Ca”+. Attempts were made to alter the MeHg-induced release of [3H]ACh by pretreating labeled synaptosomes with RR to inhibit transport of Ca2+ by mitochondria in situ. The effect of MeHg on choline uptake, a regulatory step in ACh synthesis (Simon et al., 1976) was also measured. METHODS Chemicals andsolutions. Methyl mercury chloride was purchased from K + K Rare and Fine Chemicals (Plainview, NY). [acetyl-I-i4C]Choline chloride (10 mCi/mmol) and [mefhyl-‘HIcholine chloride (80 Ci/mmol) were purchased from ICN Radiochemicals (Irvine, CA) and from Research Products International Corp. (Mount Prospect, IL), respectively. Acetylcholinesterase (950 U/mg protein; electric eel Type III), choline phosphokinase (0.39 U/mg solid), ruthenium red, physostigmine sulfate, hemicholinium-3, Ficoll type 400-DL, 3-heptanone, gramicidin, valinomycin, and oligomycin were obtained from Sigma Chemical Co. (St. Louis, MO). Sodium azide (NaN,) was purchased from Mallinckrodt Chemical Works (St. Louis. MO). Tetraphenylboron sodium was obtained from Aldrich Chemical Co. (Milwaukee, WI). 3,3’-Diethyhhiadicarbocyanine iodide [diS-C,(S)] was obtained from Molecular Probes (Eugene, OR). N-HydroxyethylpiperazineW-2-ethanesulfonic acid (Hepes) was purchased from United States Biochemical Corp. (Cleveland. OH). Dimethyl sulfoxide (DMSO) was obtained from EM Science (Gibbstown. NJ). All other chemicals were of reagent grade or better. Distilled/deionized water was used in all experiments. For incubation of synaptosomes, physiologic Hepes solution (HPS) contained (mM): NaCI, 145; KCl, 5; MgCl,, 1; D-glucose, 10; Hepes, 10. Other special considerations with regard to solutions are given in the figure legends. All solutions were adjusted to pH 7.4 at room temperature by titrating with either Trizma Base, Hepes, or HCI as appropriate. Preporation of synuptosomes. Synaptosomes were isolated from the forebrains of male Sprague-Dawley rats (Harlan, 175-225 g) using methods identical to those detailed previously (Atchison et al., 1988; Shafer and Atchison, 1989; Levesque and Atchison, 199 1). For loading with [‘HIcholine, the synaptosomal pellets were combined and resuspended in 5 ml of physiological saline (HPS) at approximately IO mg protein/ml (Lowry et al., 195 I). All buffers were prepared daily from stock solutions and osmolarity was maintained at 320 -C 10 mOsm. Intrasynaptosomal ACh was labeled Labeling intrasynaptosomal ACh. via a modification of the method of Suszkiw and O’Leary (1983). The suspension of synaptosomes in 5 ml of HPS (approx. 10 mg protein/ml) was

MITOCHONDRIA,

MeHg. AND ACh RELEASE

13

concentration of 100 nM. Synaptosomes were incubated in the presence of incubated in the presence of 100 nM t3H]choline (85 Ci/mmol) for 30 min at 30°C under 02. After the loading incubation, synaptosomes were cen- [3H]choline for a total of 30 min in an oxygenated metabolic shaker at 30°C. The time course of [3H]choline uptake during the 30-min incubation was trifuged at 10,OOOgfor 10 min at 4°C. The loaded synaptosomes were then washed free of extrasynaptosomal radioactivity by two consecutive resus- determined by filtering IOO-~1aliquots of synaptosomes every 5 min through pensions in 5 ml cold HPS containing 20 NM hemicholinium, and repelleted 0.65pm Millipore filters. After the filters were rinsed twice with 2 ml of (1 O,OOOg,10 min. 4°C). Hemicholinium was used to block further uptake cold HPS, the filters were placed into glass vials and radioactivity was eluted of choline by the high-affinity uptake carrier. by adding 1.5 ml of a Triton X-lOO/HCI solubilizer. After IO min, 10 ml After washing, the final synaptosomal pellet was resuspended in oxygenated of aqueous scintillation cocktail was added and total tritium was estimated. HPS (approx. 10 mg protein/ml) plus 50 pM physostigmine and 20 pM Uptake of [3H]choline was determined in normal HPS, Na+-free HPS (subhemicholinium. Physostigmine was used to inhibit acetylcholinesterase and stituting N-methylglucamine for NaCI), or hemicholinium (20 PM)-COW prevent breakdown of ACh in subsequent release experiments. All HPS taining HPS to assessthe contribution of high-affinity uptake of choline to solutions used in [3H]ACh release experiments contained 20 pM hemichototal uptake. The effect of MeHg on [‘HIcholine uptake was determined by linium and 50 PM physostigmine. In experiments in which Ca’+-independent adding MeHg to the suspension of synaptosomes in HPS before adding release of [3H]ACh was measured, synaptosomes were loaded as described [3H]choline. above but were washed with and resuspended in Ca’+-free HPS, which was Fluorescence measurements. Experiments in which the potentiometric made by omitting CaCl,, increasing MgC& to IO mM. and reducing NaCI carbocyanine dye diS-Cz(5) was used to monitor alteration in membrane by 6 mM to maintain osmolarity. Previous studies in this lab have shown potential were conducted as described previously (Hare and Atchison. such solutions to contain between 6 and 12 pM CaZf as contaminant when 1992a,b). Briefly, a 50-~1 aliquot containing prewarmed synaptosomal protein analyzed using inductively coupled plasma emission spectroscopy (Atchison, (~0.5 mg) was added to 2 ml of HPS buffer containing 0.5 pM diS-C,(5) 1986, 1987) or fura- fluorescence (unpublished results). As wash steps were and incubated in a shaking water bath for 10 min at 37°C. Stock solutions performed at 4°C synaptosomal suspensions were incubated at 30°C for of diS-Cl(S) were dissolved in DMSO. Working solutions were made daily 10 min before their use in release experiments. by appropriate dilution of the stock solution with HPS. The final concenRelease ofACh. Release of [3H]ACh from prelabeled synaptosomes was tration of DMSO in the incubate was 0.05% (v/v). Determinations of diSinitiated by adding lOO+l aliquots (approx. 1 mg protein) of the labeled C2(5) fluorescence in synaptosomes were made using a SPEX fluorolog specsynaptosomes to 900 ~1 of HPS. After the solutions were allowed to mix for trofluorometer (SPEX. Edison, NJ) using polystyrene cuvettes and excitation and emission wavelengths of 649 and 682 nm. respectively (bandpass, 3.77 various intervals (ranging from 10 to 90 set), the synaptosomes were filtered rapidly through 0.65~pm Millipore filters under suction. The filters were nm each). A water-jacketed cuvette holder maintained the sample at a conwashed twice with 3 ml of ice-cold HPS. The filtrates were collected and stant 37°C and the sample was stirred continuously with a magnetic stirrer. assayed for [‘H]ACh. Ca*+-independent release was determined as above Experiments began when the baseline fluorescence was judged visually to except that labeled synaptosomes were washed and suspended in Ca’+-free be stable. HPS and all solutions used during release incubations were Ca’+-free. When Statistical analysis. ACh release data were analyzed statistically using MeHg and RR were tested for their effects on [3H]ACh release, aliquots of a randomized block analysis of variance (Steel and Torrie, 1960). Differences prelabeled synaptosomes were added to HPS buffers containing these agents. among treatment means were compared using Duncan’s and Dunnett’s tests. Both were tested for effects on release in the presence and absence of Ca2+. Fluorescence analysis of membrane potential and the choline uptake data When RR was tested for the ability to alter the effects of MeHg on release were analyzed for significance using Student’s paired t test. Differences were of [3H]ACh, labeled synaptosomes were incubated in the presence of this considered to be significant at p i 0.05. agent for 10 min before aliquots of the synaptosomes were added to normal or Ca2+-free HPS solutions containing MeHg. MeHg and the mitochondrial RESULTS inhibitor were tested only for their effectson spontaneous release of [3H]ACh. [3H]ACh released into the filtrates was asDetermination of [‘HvCI1. One drawback to using synaptosomes as model nerve tersayed by the method of Goldberg and McCaman (I 973) as modified by minals is that they are heterogeneous with respect to the Suszkiw and O’Leary (1983). Briefly, 250~~1 aliquots of the filtrates in scintransmitter released. In control experiments designed to detillation vials were mixed with 1 ml of a phosphorylation medium to yield termine whether our preparation of synaptosomes contained the following composition: 0.005 U choline kinase, 5 mM ATP, IO mM MgCIr. and 10 mM Na2HP04 buffer, pH 7.9. The mixture was incubated cholinergic nerve terminals, synaptosomes were analyzed for for 15 mm at 35°C to convert the free choline to phosphorylcholine. After the presence of the hemicholinium-sensitive (Yamamura and 15 min, 1.0 ml of tetraphenyl boron in heptanone (10 mg/ml) was added Snyder, 1973; Barker and Mittag, 1975), high-affinity choline and the vials were vortexed vigorously to extract [3H]ACh into the organic transporter (Haga and Noda, 1973; Yamamura and Snyder, phase. Phosphorylated [3H]choline is not extractable with tetraphenylboron and remains in the aqueous phase. After the aqueous and organic phases 1973) that is selectively localized to cholinergic nerve terhad separated, 10 ml of toluene-based scintillation fluor was added without minals (Kuhar et al., 1973; Yamamura and Snyder, 1973). disturbing the lower aqueous phase. Initially, internal [acety/-‘4C]choline Synaptosomes were incubated in the presence of a low con(approx. 1000 dpm) standards were added to all samples prior to the assays centration of [3H]choline (100 nM) to favor uptake via the and its recovery (>90%) was used to calculate the original content of [‘H]ACh high-affinity pathway and not by passive transport, which in the samples. To monitor the efficiency of the choline kinase-catalyzed have k, values for choline of 1-4 and 40-80 PM, respectively phosphorylation of choline and separation of free [3H]choIine from [3H]ACh, parallel control assayswere run with acetylcholinesterase (IO- I5 U/ml) added (Haga and Noda, 1973: Yamamura and Snyder, 1973). Reto hydrolyze all ACh in the samples. Radioactivity in these samples was sults of experiments in which [3H]choline uptake was deterclose to background (results not shown), indicating that the choline kinase mined are illustrated in Fig. 1. Total tritium in the synapassayprocedure was highly efficient for separating [‘HIcholine and [3H]ACh tosomes, as determined by filtration, increased rapidly and (>90%). All radioactivity was estimated using a Searle Model 6880 liquid saturated within lo- 15 min. After 15 min, the tritium conscintillation spectrometer. 13HjCholine uptake measurements. Choline uptake was initiated by tent of the synaptosomes remained relatively constant. This adding [‘HIcholine to synaptosomes in HPS (approx. 10 mg/ml) at a final may have been due to a balance between [3H]choline uptake

LEVESQUE.

HARE, AND ATCHISON

jority of choline incorporated into synaptosomes, the large decrease in uptake produced by 100 PM MeHg may have been due, at least partly, to inhibition of high-affinity choline 8 transport. Exposure of [3H]choline-loaded synaptosomes to 10 or fiM MeHg caused a significant, concentration-dependent 100 4 increase in spontaneous release of [‘H]ACh (Fig. 3). The additional ACh release induced by MeHg over that occurring 0 in its absence is expressed as a percentage of control samples 0 6 12 18 24 30 incubated without MeHg. At 10 PM (Fig. 3) MeHg increased spontaneous release by 10% over control within 10 set of (mid TIME being introduced into the suspension of labeled synaptosomes. Incubation of the synaptosomes in the presence of Time course of [3H]choline uptake by synaptosomes incubated with 100 nM [3H]choline in control (A), Na+-free (0-Na; V), and hemi100 PM MeHg (Fig. 3) resulted in a 30% increase in sponcholinium-3 (20 PM)-containing (0) Hepes-buffered Kreb’s Ringer solutions taneous release of [3H]ACh. Under all conditions the effect (HPS). [3H]Choline was added to synaptosomal suspensions (10 mg/ml) at was already maximum at 10 set; measurements made after 0 min and aliquots were filtered every6 min to determine uptake. [3H]Choline 30 or 90 set of exposure to MeHg indicated no significant uptake refers to the net total tritium retained by filtered synaptosomes after additional elevation of release (p > 0.05). The increase in subtraction of filter blanks. Values are the mean ? SE of four different experiments. Values for each experiment are the averages of three replicates. spontaneous release of 13H]ACh induced by MeHg in the The asterisk indicates a significant reduction from control uptake (p < 0.05) absence of external Ca2+ was attenuated slightly over the by synaptosomes incubated in 0-Na+ and hemicholinium. release in the presence of Ca2+, but remained significant compared to that in control. RR, which blocks uptake and induces release of Ca” by mitochondria, was tested for effects on spontaneous release and release of tritium to the medium, as has been observed of transmitter. RR significantly enhanced release of [3H]ACh by others (Rowe11 and Duncan, 1981). Either inclusion of in a concentration-dependent manner (Fig. 4). The stimuhemicholinium or removal of Na+ inhibited the uptake of [3H]choline by over 65% throughout the incubation period. lator-y effect of RR on [3H]ACh release was reduced by only Thus at the low concentration of choline used, most of the lo-20% in Ca2+-free solutions from that in Ca2+-containing [3H]choline is incorporated into cholinergic synaptosomes possessing high-affinity transport for choline. High-affinity transport of choline is thought to be coupled directly to ACh synthesis (Barker and Mittag, 1975; Barker et al., 1975; Simon et at., 1976) and is believed to be the rate-limiting step for synthesis of this transmitter (Haga and Noda, 1973; Yamamura and Snyder, 1973). One possible mechanism by which MeHg could affect transmitter release would be to inhibit choline uptake and, in so doing, decrease nerve-terminal ACh levels. This could relate to the decrease in spontaneous release of ACh, which occurs after the MeHg-induced stimulation of miniature endplate potential frequency noted in electrophysiological experiments (Atchison and Narahashi 1982; Atchison, 1986). Results of experiments in which [3H]choline uptake was determined in the presence of MeHg are shown in Fig. 2. 10 100 Though not shown in the figure, uptake was examined for [MeHgl periods of up to 30 mitt; no time-dependent differences in (PM) the effect of MeHg were observed (p > 0.05). At 10 and 100 PM, MeHg reduced uptake by 16% (84 t- 15% of control; FIG. 2. Effects of 10 MM (dark bar) and 100 fiM (hatched bar) MeHg mean + SE) and 46% (54 ? 16% of control; mean -+ SE), on [‘HIcholine uptake in HPS. MeHg was added to the synaptosomes (IO respectively. At the higher concentration of MeHg, this de- mg/ml) just prior to adding [3H]choline. Aliquots of the synaptosomal suspensions were filtered after 5 min and total tritium retained on the filters crease was statistically significant (p < 0.05). On the basis was determined. Values are the mean ? SE of five different experiments. of this experiment, it cannot be determined whether the effect Values for each experiment are the average of three replicates. The asterisk of MeHg was specific for either high- or low-affinity uptake. indicates a significant reduction (p < 0.05) in [3H]choline uptake by MeHg However, since high-affinity uptake accounted for the ma- from that in MeHg-free controls.

MITOCHONDRIA,

10

15

MeHg, AND ACh RELEASE

100 [MeHgl (PM)

FIG. 3. Effects of 10 and 100 pM MeHg on release of [‘H]ACh from nondepolarized synaptosomes in the presence (dark bars) and absence (hatched bars) of 2.5 mM CaZ+. Aliquots of prelabeled synaptosomes were added to HP.7 (5 mM K+) containing MeHg ? Ca’+ and incubated for 10 sec. Results are expressed as the percentages of [3H]ACh released in response to MeHg, relative to [3H]ACh released during parallel incubations without MeHg. Values are the mean + SE of 10 different experiments. Values for each experiment are the average of three replicates. The asterisk indicates that the value for MeHg is significantly greater than its respective MeHgfree control values @ G 0.05).

solutions and was still significantly elevated over that in Ca*+free controls (p < 0.05). Efflux of [3H]ACh occurred rapidly and at 10 set was significantly and maximally elevated compared to control. No significant time-dependent effects of RR on t3H]-ACh release were observed (p > 0.05). Aliquots of [3H]choline-loaded synaptosomes were preincubated with the mitochondrial transport inhibitor for 10 min before adding MeHg. Preincubation of labeled synaptosomes with RR for 10 min significantly attenuated the stimulator-y effects of MeHg ( 100 PM) on spontaneous release of [3H]ACh in the presence and absence of Ca*+ (Fig. 5). Carbocyanine dyes have been useful in quantifying both the plasma membrane potential (I&,) and alterations in I& in synaptosomes (Blaustein and Goldring, 1975; Meunier, 1984; Hare and Atchison, 1992a). Moreover, carbocyanine dyes are concentrated in intrasynaptosomal mitochondria and their release upon depolarization of the mitochondria (Heinonen et al., 1984; Hare and Atchison, 1992a) can be used as a qualitative index of the mitochondrial membrane potential. MeHg depolarizes both synaptosomal plasma membranes and intrasynaptosomal mitochondrial membranes (Hare and Atchison, 1992b). The objective of this experiment was to determine if the protective action exerted by RR on MeHg+-induced ACh release is specific to the mitochondrial Ca” uniporter. Thus, we were interested in whether RR would also prevent or modulate the depolarization of & by

MeHg. If so, this might indicate that RR prevented access of MeHg to the Ca*+ release apparatus. RR (20 PM) added to the synaptosomal suspension decreased the fluorescence of diS-C,(5) by 39% after 5 min (Fig. 6A). In order to determine if RR also depolarized synaptosomal membranes, RR was added to synaptosomes in which & and $,.,, had been predepolarized with NaN3, oligomycin and gramicidin (Hare and Atchison, 1992a,b). In this case, RR decreased fluorescence by 58% (results not shown). Thus, RR produced a quench of fluorescence in predepolarized synaptosomes greater than that in synaptosomes with intact $, and &. This difference could be due to an increase in fluorescence associated with membrane depolarization concomitant with RR-induced quench of fluorescence. RR would also be expected to quench increases in fluorescence associated with membrane depolarization. To determine if this occurred, known amounts of diS-C,(S) were added to control and RR-treated synaptosomes and the subsequent increases in fluorescence compared. The quench by RR of the fluorescence response to various concentrations of diS-C,(5) added was a linear function of the amount of dye added (results not shown). Thus, it was possible to correct for treatment-induced increases in fluorescence in RR-pretreated synaptosomes. Depolarization of 9, with NaN3 and oligomycin resulted in an increase in synaptosomal fluorescence (Fig. 6B). RR reduced the fluorescence response to depolarization of 1c/, by 43%. Assuming that RR had no

2.5

zero

[Ca2+ (mM)

1

FIG. 4. Effects of ruthenium red (RR; 20 pM, dark bars, or 60 pM, hatched bars) on release of [‘H]ACh in the presence and absence (zero) of 2.5 mM Ca2+. Aliquots of prelabeled synaptosomes were added to HPS (5 mM K+) containing RR ? Ca2+ and incubated for 10 sec.Results are expressed as a percentage of [3H]ACh released in response to RR, relative to [3H]ACh released during parallel incubations without RR. Values are the mean f SE of five different experiments. Values for each experiment are the average of three replicates. The asterisk indicates a value significantly greater than that of its respective RR-free control (JI < 0.05).

16

LEVESQUE,

24 -_

e

145r

HARE, AND ATCHISON

I

zero

2.5

[Ca2+l (mM)

FIG. 5. Effects of MeHg (100 pM) on release of [‘H]ACh from nondepolarized synaptosomes in the absence (open bar) or after preincubation with RR (20 pM, dark bars, or 60 pM, hatched bars) in the presence and absence (zero) of 2.5 mM Ca2f . Aliquots of prelabeled synaptosomes incubated with RR for 10 min were added to HPS (5 mM K+) containing MeHg f Ca2+ and incubated for 10 sec. Results are expressed as the percentage of [‘H]ACh released in the absence of MeHg and RR with or without Ca2+. Values are the mean f SE of seven different experiments. Values for each experiment are the average of three replicates. Asterisks indicate values significantly different from those obtained in the absence of MeHg-induced release of [‘H]ACh from synaptosomes treated with RR 0, < 0.05).

effects on fluorescence other than quenching, the predicted decrease in the fluorescence response by RR due to depolarization of $,,, would be 42%. Thus, the RR-induced decrease in the fluorescence response to depolarization of 1c/, could be completely accounted for by quenching of the fluorescence signal, and it is unlikely, then, that RR depolarized &,, . The fluorescence response of RR-treated synaptosomes to 10 PM MeHg was 78% of that of controls (Fig. 7). If the only effect of RR was to quench the MeHg-induced fluorescence increase, the expected increase in fluorescence should have been 57% of controls. Thus, RR may have exacerbated the depolarizing action of MeHg.

bation of synaptosomes with an inhibitor of mitochondrial Ca2+ transport reduced the effectiveness of MeHg for increasing spontaneous release of [3H]ACh. Thus, the effects of MeHg on ACh release from central neurons, as measured neurochemically, are consistent with those obtained previously in electrophysiological studies at the NMJ. The preparation of rat forebrain synaptosomes utilized in this study contained functional cholinergic nerve terminals since most of the [3H]choline added was taken up via Na+dependent, hemicholinium-sensitive transport, which is exclusively localized in choline& nerve terminals (Kuhar et al., 1973; Yamamura and Snyder, 1973). Uptake of [3H]choline via high-affinity transport implies that intrasynaptosomal ACh synthesis occurred normally since these two events are coupled (Barker et al., 1975; Simon et al., 1976). Choline uptake by synaptosomes was reduced significantly by MeHg at 100 PM. Whether MeHg specifically inhibits high-affinity choline uptake or affects uptake of choline indirectly was not determined. High-affinity choline transport

3 g

2

s Q) ‘0 I IL

arldeiollgo 0

0

2

4

6

6

I 10

Time (mid

DISCUSSION Since MeHg has been shown previously to affect peripheral cholinergic neurotransmission and is a potent CNS neurotoxicant, we studied its effects on uptake of choline and release of ACh in the CNS in vitro using rat brain synaptosomes. MeHg partially inhibited uptake of [3H]choline and induced spontaneous release of [3H]ACh from [3H]cholineloaded synaptosomes. Enhanced release of t3H]ACh was attenuated slightly in Ca 2+-free solutions but was still significantly greater than that from MeHg-free controls. Preincu-

con

RR

prod

FIG. 6. Effect of ruthenium red (RR) on the fluorescence of diS-C2(5) in synaptosomes. (A) RR (20 pM) was added at 0.5 min. At 5.5 min. 10 mM NaN3 (azide) and 4 pM oligomycin (oligo) were added to RR-treated (solid line) or control (dashed line) synaptosomes. Lines represent the mean of duplicate tracings from three animals. (B) Increases in fluorescence resulting from exposure to NaNl and oligomycin. Values are the means f SE taken 5 min after NaN3 and oligomycin treatment. Con, control; RR, RRtreated synaptosomes; and pred, predicted increase in fluorescence based only on the fluorescence quenching properties of RR (see text).

MITOCHONDRIA,

MeHg, AND ACh RELEASE

86.

YeHg

4.y If ___________----------control -I ,.-2 - 1---______-----__-----_--m--* 4’ 0

2

4 Time

6

8

10

8

(mid

FIG. 7. Effects of MeHg on diS-C*(5) fluorescence in the presence and absence of RR. At 0.5 min 20 pM RR was added (dashed line) followed at 5.5 min by 10 pM MeHg. Lines represent the mean of duplicate recordings from three separate experiments.

is reduced by agents that depolarize the cell. Concentrations of MeHg >, 10 PM cause a time-dependent depolarization of synaptosomal membrane potentials (Hare and Atchison, 1992b). Thus at 100 PM MeHg, inhibition of choline uptake might occur, in part, by membrane depolarization. This could account for the much greater reduction in choline uptake induced by 100 PM than that induced by 10 PM MeHg. Whatever the mechanism, inhibition of this regulatory step in the synthesis of ACh by MeHg could affect neurotransmission at central cholinergic synapses. MeHg stimulates spontaneous release of ACh from synaptosomes (Minnema et al., 1989, present study), brain slices (Saijoh et al., 1987), and the NMJ (Atchison, 1986, 1987) in the absence of external Ca2+. MeHg may stimulate spontaneous release by releasing Ca2+ from bound intracellular stores, such as intraterminal mitochondria, which can sequester and store large quantities of Ca2’ (Scott et al., 1980; Nicholls and Akerman, 198 1; Nicholls, 1986). MeHg inhibits mitochondrial respiration (Verity et al., 1975; Sone et al., 1977; O’Kusky, 1983; Cheung and Verity, 198 1) and ATP production (Sone et al., 1977; Kauppinen et al., 1989), disruption of which would result in increased [Ca2+]i (Ashley et al., 1982; Heinonen et al., 1984). MeHg also induces an immediate efflux of 45Ca2+ from isolated mitochondria of the CNS (Minnema et al., 1989; Levesque and Atchison, 1991). If MeHg produced this efflux of Ca2’ from mitochondria in situ, [Ca2+]i would likely increase. MeHg significantly increases [Ca2’]i in synaptosomes and NG108 cells incubated in Ca2’-free solutions (Komulainen and Bondy, 1987; Hare et al., 1991; Hare and Atchison, 1992~); however, the source of this Ca2+ has not been examined in detail. The MeHg-induced increase in [Ca2’]i may originate from mitochondria. Depolarization (and, hence, inactivation of mitochondrial uptake of Ca2+) of mitochondria attenuated, but did not block, the increase in [Ca2+] by MeHg+ (Komulainen and Bondy, 1987). Moreover, MeHg was less effective at

17

inducing release of Ca 2+ from isolated brain mitochondria that were pretreated with RR to inhibit uptake and induce release of Ca2+ (Levesque and Atchison, 199 1). The present results with RR provide important evidence for a link between release of mitochondrial Ca2+ and stimulation by MeHg of spontaneous release of ACh from nerve terminals. RR inhibits Ca2’ transport into or out of mitochondria via the Ca2+ uniport protein located on the inner mitochondrial membrane (Moore, 197 1; Luthra and Olson, 1977; Jurkowitz et al., 1983). RR may have reduced the effectiveness of MeHg for inducing ACh release from synaptosomes by preventing release of Ca2+ from mitochondria by MeHg. Pretreatment of 45Ca2+-loaded mitochondria with RR blocks the release of 45Ca2t induced by MeHg, which occurs in the absence of RR (Levesque and Atchison, 199 1). The fact that RR was equally effective at altering MeHginduced release of [3H]ACh in the presence and absence of external Ca2+ suggests that RR blocked the stimulatory effect of MeHg on ACh release by preventing release of mitochondrial Ca2+ by MeHg and not by altering plasma membrane Ca2+ transport. RR could also reduce the effect of MeHg on transmitter release by inducing the efflux of a pool of Ca2+ from mitochondria (Carafoli, 1982) upon which MeHg could otherwise act. Specific mitochondrial Ca2+ efflux pathways, not associated with the uniporter, are insensitive to RR. Thus RR induces a net efflux of Ca2+ from mitochondria (Moore, 197 I; Vasington et al., 1972). RR-induced release of Ca2+ from mitochondria has been suggested to underlie its stimulation of spontaneous release of transmitter observed at the NMJ (Alnaes and Rahamimoff, 1975; Bemath and Vizi, 1987; Levesque and Atchison, 1987) and from brain slices (Gomez and Farrell, 1985). RR probably induced spontaneous release of [3H]ACh from synaptosomes in the present study by this mechanism since it stimulated ACh release even in the absence of external Ca2*. RR releases Ca2+ from isolated rat brain mitochondria without adversely affecting I& (Nicholls, 1978). This is believed to be a specific action of RR on the mitochondrial Ca2+ uniporter (Moore, 1971). Depolarization of \I/,,, by MeHg was not prevented by RR, whereas Ca2+ efflux from isolated mitochondria (Levesque and Atchison, 1991) and transmitter release from motor axon terminals (Levesque and Atchison, 1987) and synaptosomes (Levesque and Atchison, 199 1) were prevented. This suggests that the mechanism of RR block is not likely due to RR preventing access of MeHg to mitochondrial binding sites nor is it likely that RR complexed with MeHg. Moreover, prior mitochondrial depolarization does not block MeHg-induced transmitter release from the neuromuscular junction (Levesque and Atchison, 1987) and does not completely block MeHg-induced elevations in [Ca’+]i in synaptosomes (Komulainen and Bondy, 1987). Therefore, MeHg-induced depolarization of I/+,, and the subsequent release of mitochondrial Ca2+ into the cytosol are not sufficient to cause transmitter release at

18

LEVESQUE,

HARE,

the neuromuscular junction or in synaptosomes. This suggest that a RR-sensitive Ca2+ pool, presumably mitochondrial, may mediate the MeHg-induced increases in spontaneous transmitter release. Thus, MeHg acts at many sites associated with mitochondrial functioning. One in particular, the RRsensitive Ca*+ uniporter, may be linked to MeHg-induced increases in the spontaneous release of transmitter. RR may have exacerbated the depolarizing action of MeHg. This may have been an effect on &, since in synaptosomes in which the $, had been depolarized, MeHg produced similar depolarizations in control and RR-treated synaptosomes (results not shown). It is possible that RR may have activated a K+ conductance in the mitochondria since nanomolar concentrations of RR have been reported to increase K+ permeability in beef heart mitochondria (Jung and Brierley, 1984). The effectiveness of RR at blocking MeHg-induced increases in spontaneous release of transmitter observed in the present study differed from that in previous experiments at the NMJ (Levesque and Atchison, 1987, 1988) in which RR completely blocked MeHg-induced increases in spontaneous quanta1 release of ACh. Perhaps this previous inhibition was due in part to postsynaptic effects. RR is thought to affect postsynaptic responses to several neurotransmitters (Robertson and Wann, 1987). Measurements of synaptosomal ACh release are direct measurements of presynaptic terminal function, whereas the electrophysiological measurements at the NMJ are indirect, being made from the postsynaptic response to ACh release. Thus, results of neurochemical analysis of ACh release would be independent of any postsynaptic actions of RR. Alternatively, if MeHg increases nonquantal release of ACh in addition to stimulating quanta1 release, an inhibitory action of RR on quanta1 release might be masked somewhat. Considering its somewhat specific effects on Ca2+ transport, RR may inhibit only quanta1 release induced by MeHg since nonquantal release is less sensitive than quanta1 release to changes in [Ca”]i (Vyskocil et al., 1989). ACh released in a nonquantal manner would be detected by the neurochemical methods utilized in the present experiments but not by the electrophysiological studies at the NMJ, which measured only the effects of MeHg on spontaneous quanta1 release of ACh. MeHg-induced release of [3H]ACh from synaptosomes was somewhat attenuated in Ca’+-free solutions. An analogous effect of MeHg was observed at the NMJ (Atchison, 1986, 1987). Although MeHg significantly increases synaptosomal free-Ca*+ levels in the absence of external Ca2’, the maximum increase in [Ca’+]i is less than that observed in synaptosomes (Komulainen and Bondy, 1987; Hare et al., 199 1) or NG 108- 15 cells (Hare and Atchison, 1992~) in Ca2+containing media, suggesting that MeHg caused Ca2+ entry into the nerve. Taken together, these results imply that external Ca2+ contributes at least in part to the elevated [Ca’+]i for the stimulatory effect of MeHg on spontaneous release

AND

ATCHISON

of transmitter. The mechanism by which extracellular Ca” apparently contributes to the MeHg-induced rise in [Ca”+]i is unclear and it is not known whether this occurred in the present experiments. It is doubtful that MeHg elicits Ca’+ influx through voltage-sensitive Ca*+ channels since MeHg has been shown to block depolarization-induced flux of 45Ca’f into synaptosomes during time intervals similar to those during which MeHg stimulated [3H]ACh release in the present study (Atchison et al., 1986; Shafer and Atchison, 1989; Hewett and Atchison, 1992) and to block rapidly currents carried by Ba*+ through putative Ca” channels in pheochromocytoma cells (Shafer and Atchison, 199 1). It has been suggested that 100 pM MeHg increases the permeability of synaptosomal plasma membranes to Ca2’ (Komulainen and Bondy, 1987), but these effects on membrane permeability were not observed with 30 pM MeHg or lower concentrations. This would not explain the apparent partial dependence on external Ca2’ of the effect of 10 pM MeHg on [3H]ACh release observed in the present study. Recently, Arakawa et al. ( 199 1) demonstrated an increase in an apparently nonspecific cation current in dorsal root ganglion cells with mercurials, so perhaps channel types other than Ca2+ channels contribute to this action. There is no conclusive evidence that MeHg caused Ca” influx through the plasma membrane in the present study and the contribution of external Ca2+ to the stimulatory effects of MeHg on spontaneous release of ACh remains to be determined. An alternate explanation for the reduced effect of MeHg on spontaneous release of [3H]ACh in Ca*+-free solutions is that there is less intrasynaptosomal Ca’+. Incubating synaptosomes in Ca’+-free solutions reduces intramitochondrial Ca2+ content (Scott et al., 1980). In the present study, [3H]choline-loaded synaptosomes were washed twice and resuspended in Cazf-free medium before they were used in Ca’+-free release experiments. If there is less Ca2+ within the nerve terminal for MeHg to release, the maximum increase in [Ca2’]i would be less than that if mitochondria contained a larger quantity of Ca’+. Since spontaneous quanta1 release of ACh is strongly dependent on [Ca2+]i, less ACh would be released by MeHg under these conditions. The effect of RR on [3H]ACh release was also reduced in Ca’+-free solutions. Rather than increase Ca2+ influx into synaptosomes, RR may block entry of Ca *+ through voltage-gated channels (Taipale et al.. 1989). This suggests that the reduced effect of RR on [3H]ACh release in Ca2+-free solutions may not be due to the absence of external Ca2+ but is probably related to effects of RR on mitochondrial Ca’+. Reduced intraterminal bound Cal+ levels may also explain the attenuated effect of MeHg in Ca2+-free media at the NMJ since bathing the neuromuscular preparation in Ca2+-free solutions markedly reduced the Ca2+ content of the tissue (Atchison, 1986). In summary, the results indicate that MeHg may interfere with cholinergic neurotransmission in the CNS by reducing ACh synthesis and by stimulating spontaneous release of

MITOCHONDRIA,

MeHg, AND ACh RELEASE

ACh. Since newly formed ACh is released preferentially (Collier and Macintosh, 1969) and since high-affinity choline uptake is coupled kinetically to ACh synthesis, the combination of reducing ACh synthesis and stimulating spontaneous release could result in transmitter depletion. This might explain the paradoxical cessation of ACh release which occurs secondary to the stimulation by MeHg of miniature end-plate potential frequency at the NMJ. The effects of MeHg on spontaneous release of transmitter from CNS nerve endings are similar to those observed previously at the NMJ. MeHg may stimulate spontaneous release of transmitter subsequent to disrupting intraterminal Ca2+ homeostasis and elevating [Ca2’]i. The results indicate specifically that MeHg may induce release of Ca2+ from nerve-terminal mitochondria, potentially leading to elevated [Ca2+]i. Perturbation of [Ca2’]i by MeHg may underlie its effects on other transmitter systems in both the peripheral and the central nervous systems. Moreover, release of neurotransmitters is only one example of a Ca2+-dependent process. If MeHg indeed alters cellular Ca2+ regulation by interfering with transmembrane Ca2+ fluxes or Ca2+ buffering by intracellular organelles, one could predict effects of MeHg on other Ca2+-dependent cellular functions in neuronal as well as in nonneuronal cells. ACKNOWLEDGMENTS The authors acknowledge the secretarial assistanceof Gretchen Humphries and Kimberly Isaacson.

REFERENCES Alnaes, E., and Rahamimoff, R. (1975). On the role of mitochondria in transmitter release from motor nerve terminals. J. Physiol. (London) 248, 285-306. Arakawa, O., Nakahiro, M., and Narahashi, T. (199 1). Mercury modulation of GABA-activated chloride channels and non-specific channels in rat dorsal root ganglion neurons. Brain Res. 551, 58-63. Ashley, R. H., Brammer, M. J., and Marchbanks, R. (1982). Measurement of intrasynaptosomal free calcium by using the fluorescent indicator quin2. Biochem. J. 219, 149-158. Atchison, W. D. (1986). Extracellular calcium-dependent and -independent effects of methylmercury on spontaneous and potassium-evoked release of acetylcholine at the neuromuscular junction. J. Pharmacol. Exp. Ther. 237,672-680. Atchison, W. D. (1987). Effects of activation of sodium and calcium entry on spontaneous release of acetylcholine induced by methylmercury. J. Pharmacol. Exp. Ther. 241, 13 1- 139. Atchison, W. D., Adgate, L., and Beaman, C. M. (1988). Effectsof antibiotics on uptake of calcium into isolated nerve terminals. J. Pharmacol. Exp. Ther. 245,394-40 1. Atchison, W. D., Clark, A. W., and Narahashi, T. (1984). Presynaptic effects of methylmercury at the mammalian neuromuscular junction. In Cellular and Molecular Neurotoxicology (T. Narahashi, Ed.), pp. 23-43. Raven Press, New York. Atchison, W. D., Joshi, U., and Thornburg, J. E. (1986). Irreversible suppression of calcium entry into nerve terminals by methylmercury. J. Pharmacol. Exp. Ther. 238,6 18-624.

19

Atchison, W. D., and Narahashi, T. (1982). Methyl mercury-induced depression of neuromuscular transmission in the rat. Neurotoxicology 3, 37-50. Bakir, F., Damluji, S., Amin-Zaki, L., Murthada, M., Khalidi, A., Al-Rawi, N. Y., Tikriti, S., Dhahir, H. L., Clarkson, T. W., Smith, J. C., and Doherty, R. A. (1973). Methylmercury poisoning in Iraq. Science 181, 230-240. Barker, L. A., Downdall, M. J., and Mittag, T. W. (1975). Comparative studies on synaptosomes: High-affinity uptake and acetylation of N-[Me‘HIcholine and N[Me-‘HJN-hydroxyethyl pyrrolidinium. Brain Res. 86, 343-348. Barker, L. A., and Mittag, T. W. (1975). Comparative studies of substrates and inhibitors of choline transport and choline acetyltransferase. J. Pharmacol. Exp. Ther. 192, 86-94. Barrett, J., Botz, D., and Chang, D. B. (1974). Block of neuromuscular transmission by methylmercury. In Behavioral Toxicology, Ear/y Detection of Occupational Hazards (C. Xintaras, B. L. Johnson, and I. de Groot. Eds.). U.S. Department of HEW Document, Washington, DC. Bemath, S.,and Vizi, E. S. (1987). Inhibitory effectof ionized free intracellular calcium enhanced by ruthenium red and m-chlorocarbonylcyanide phenyl hydrazone on the evoked release of acetylcholine. Biochem. Pharmacol. 36,3683-3687. Blaustein, M. P., and Goldring, J. M. (1975). Membrane potentials in pinched-off presynaptic nerve terminals monitored with a fluorescent probe: Evidence that synaptosomes have potassium diffusion potentials. J. Physiol. (London) 247, 589-6 15. Bondy, S. C.. Anderson, C. L., Hanington, M. E., and Prasad, K. N. (1979). The effectsof organic and inorganic lead and mercury on neurotransmitter high-affinity transport and release mechanisms. Environ. Res. 19, 102Ill. Carafoli, E. (1982). The transport of calcium across the inner membrane of mitochondria. In Membrane Transport of Calcium (E. Carafoli, Ed.), pp. IO9- 140. Academic Press, London. Chang, L. W. (1977). Neurotoxic effects of mercury-A review. Environ. Res. 14, 329-373. Cheung, M., and Verity, M. A. (198 1). Methyl mercury inhibition of synaptosome protein synthesis: The role of mitochondrial dysfunction. Environ. Res. 24, 286-298. Collier, B., and Macintosh, F. D. (1969). The source of choline for acetylcholine synthesis in a sympathetic ganglion. Can. J. Physiol. Pharmacol. 46, 127-135. Goldberg, A. M., and McCaman, R. E. (1973). The determination of picomole amounts of acetylcholine in mammalian brain. J. Neurochem. 20, l-8. Gomez, M. V., and Farrell, N. (1985). The effect oftityustoxin and ruthenium red on the release of acetylcholine from slices of cortex of rat brain. Nezcropharmacology 24, 1103-l 107. Haga, T., and Noda, H. (1973). Choline uptake systems of rat brain synaptosomes. Biochim. Biophys. Acta 291, 564-575. Hare, M. F., and Atchison, W. D. (1992a). Differentiation between alterations in plasma and mitochondrial membrane potentials in svnaptosomes using a carbocyanine dye. J. Neurochem. 58, 132 1- 1329. Hare, M. F., and Atchison, W. D. (1992b). Comparative action of methyl mercury and divalent inorganic mercury on nerve terminal and intraterminal mitochondrial membrane potentials. J. Pharmacol. Exp. Ther. 261, 166-172. Hare, M. F., and Atchison, W. D. (1992~). Methyl mercury-induced increases in [Ca’+& in NG 108- 15 cells arise from extra- and intracellular sources. The Toxicologist 12, 3 13. Hare. M. F., Denny, M. F., and Atchison, W. D. (1991). Organic and inorganic mercury alter synaptosomal calcium concentrations. Neurosci. Abstr. 17, 1463.

20

LEVESQUE.

HARE, AND ATCHISON

Heinonen, E., Akerman, K. E. O., and Kaila, K. (1984). Depolarization of the mitochondrial membrane potential increases free cytosolic calcium in synaptosomes. Neurosci. Lett. 49, 33-37. Hewett, S. J., and Atchison, W. D. (1992). Effectsof charge and lipophilicity on mercurial-induced reduction of %a uptake into isolated nerve terminals of the rat. Toxicol. Appl. Pharmacol. 113,267-273. Hunter, D., Bomford, R., and Russell, D. S. (1940). Poisoning by methyl mercury compounds. Q. .r. Med. 9, 193-241. Juang, M. S. (1976). An electrophysiological study of the action of methylmercuric chloride and mercuric chloride on the sciatic nerve-sartorius muscle preparation of the frog. Toxicol. Appl. Pharmacol. 37, 339-348. Jung, D. W., and Brierley, G. P. (1984). The permeability of uncoupled heart mitochondria to potassium ion. J. Biol. Sci. 259, 6904-69 I 1. Jurkowitz, M. S., Geisbuhler, T., Jung, D. W., and Brierley, G. P. (1983). Ruthenium red-sensitive and -insensitive release of Ca2+ from uncoupled heart mitochondria. Arch. Biochem. Biophys. 223, 120- 128. Kauppinen, R. A., Komulainen, H., and Taipale, H. (1989). Cellular mechanisms underlying the increase in cytosolic free calcium concentration induced by methylmercury in cerebrocortical synaptosomes from guinea pig. J. Pharmacol. Exp. Ther. 248, 1248-1254. Komulainen, H., and Bondy, S. C. (1987). Increased free intrasynaptosomal Ca’+ by neurotoxic organometals: Distinctive mechanisms. Toxicol. Appl. Pharmacol.

88,77-86.

Komulainen, H., and Tuomisto, J. (198 1). Interference of methylmercury with monoamine uptake and release in rat brain synaptosomes. Acta Pharmacol.

Toxicol.

48,2 14-222.

Komulainen, H., and Tuomisto, J. (1982). Effects of heavy metals on monoamine uptake and release in brain synaptosomes and blood platelets. Neurobehav. Toxicol. Teratol. 4, 647-649. Kuhar, M. J., Sethy, V. H., Roth, R. V., and Aghajanian, G. C. (1973). Choline: Selective accumulation by central cholinergic neurons. J. Neurochem.

20,581-593.

Levesque, P. C., and Atchison, W. D. (1987). Interactions of mitochondrial inhibitors with methylmercury on spontaneous quanta1 release of acetylcholine. Toxicol. Appl. Pharmacol. 87, 315-324. Levesque, P. C., and Atchison, W. D. (1988). Effect of alteration of nerve terminal Ca2+ regulation on increased spontaneous quanta1 release ofacetylcholine by methylmercury. Toxicol. Appl. Pharmacol. 94, 55-65. Levesque, P. C., and Atchison, W. D. (1991). Disruption of brain mitochondrial calcium sequestration by methylmercury. J. Pharmacol. Exp. Ther. 256,236-242.

Llinas, R., and Nicholson, C. (1975). Calcium role in depolarization-secretion coupling: An aequorin study in squid giant synapse. Proc. Nat1 Acad. Sci. (USA)

72, I87-

190.

Lowry, 0. H.. Rosebrough, N. J., Fan-, A. L., and Randall, R. J. (1951). Protein measurements with the folin phenol reagent. J. Biol. Chem. 193, 265-275. Luthra, R., and Olson, M. S. (1977). The inhibition of calcium uptake and release by rat liver mitochondria by ruthenium red. PEBS Lett. 81, 142146. Meunier, F. (1984). Relationship between presynaptic membrane potential and acetylcholine release in synaptosomes from Torpedo electric organ. J. Physiol. (London) 354, 12 1- 137. Minnema, D. J., Cooper, G. P., and Greenland, R. D. (1989). Effects of methylmercury on neurotransmitter release from rat brain synaptosomes. Toxicol. Appl. Pharmacol. 99,5 IO-52 1. Moore, C. L. (197 1). Specific inhibition of mitochondrial Ca2+ transport by ruthenium red. Biochem. Biophys. Res. Commun. 42, 298-305.

Nicholls, D. G. (1978). Calcium transport and proton electrochemical potential gradient in mitochondria from guinea-pig cerebral cortex and rat heart. Biochem. J. 170, 51 l-522. Nicholls, D. G. (1986). Intracellular calcium homeostasis. Br. Med. Bull. 42,353-358. Nicholls, D. G., and Akerman, K. E. 0. (I 98 1). Biochemical approaches to the study of cytosolic calcium regulation in nerve endings. Philos. Trans. R. Sot. London Ser. B. 296, 115-122. O’Kusky, J. (1983). Methylmercury poisoning of the developing nervous system: Morphological changes in neuronal mitochondria. Acta Neuropathol. (Berlin) 61, 1I6- 122. Robertson, B., and Wann, K. T. (1987). On the action of ruthenium red and neuraminidase at the frog neuromuscular junction. J. Phvsiol. (London) 382,411-423.

Rowell, P. P., and Duncan, G. E. (1981). The subsynaptosomal distribution and release of [‘Hlacetylcholine synthesized by rat cerebral cortical synaptosomes. Neurochem. Res. 6, 1265- 128 1. Saijoh, K., Inoue, Y., and Sumino, K. (1987). Stimulating effect of methyl mercury chloride on [‘Hlacetylcholine release from guinea-pig striatal slices. Toxicol. In Vitro 1, 233-237. Scott, I. D., Akerman. K. E. O., and Nicholls, D. G. (1980). Calcium-ion transport by intact synaptosomes. Intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential. Biochem. J. 192, 873-880. Shafer, T. J., and Atchison, W. D. (1989). Block of ‘%a uptake into synaptosomes by methylmercury: Car+- and Na+-dependence. J. Pharmacol. Exp.

Ther

248, 696-702.

Shafer, T. J., and Atchison, W. D. (1991). Methylmercury blocks N- and L-type Ca2+ channels in nerve growth factor-differentiated pheochromocytoma (PC12) cells. J. Pharmacol. Exp. Ther. 258, 697-702. Simon, J. R., Atweh, S., and Kuhar, M. J. (1976). Sodium-dependent high affinity choline uptake: A regulatory step in the synthesis of acetylcholine. J. Neurochem.

26,909-922.

Sone, N., Larsstuvold, M. K., and Kagawa, Y. (1977). Effect of methylmercury on phosphorylation, transport, and oxidation in mammalian mitochondria. Biochem. J. 82,859-868. Steel, R. D. G., and Torrie. J. H. (1960). Principles and Procedures of Statistics. McGraw-Hill, New York. Suszkiw, J. B., and O’Leary, M. E. (1983). Temporal characteristics of potassium-stimulated acetylcholine release and inactivation of calcium influx in rat brain synaptosomes. J. Neurochem. 41, 868-873. Taipale, H. T., Kauppinen, R. A., and Komulainen, H. (1989). Ruthenium red inhibits the voltage-dependent increase in cytosolic free calcium in cortical synaptosomes from guinea-pig. Biochem. Pharmacol. 38, 11091113. Takeuchi, T., Matsumoto, H., Sasaki, M., Kambara, T., Shin&hi. Y., Hirata, Y., Nobukiro, M., and Satoh, H. (1968). Pathology of Minamata disease. Kumamoto

Med.

J. 34,52

1-524.

Vasington, F. D.. Gazzotti, P., Tiozzo, R., and Carafoli, E. (1972). The effect of ruthenium red on Car+ transport and respiration in rat liver mitochondria. Biochim. Biophys. Acta 256,43-54. Verity, M. A., Brown, W. J., and Cheung, M. (1975). Organic mercurial encephalopathy: In vivo and in vitro effectsof methylmercury on synaptosomal respiration. J. Neurochem. 25, 759-766. Vyskocil, F., Zemkova, H., and Edwards, C. (1989). Non-quanta1 acetylcholine release. In Neuromuscular Junction (L. Sellin, R. Libelius, and Thesleff, Eds.), pp. 197-205. Elsevier, Amsterdam. Yamamura, H. I., and Snyder, S. H. (1973). Choline: High-affinity uptake by rat brain synaptosomes. Science 178, 626-628.