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Calcium efflux and neurotransmitter release from rat hippocampal synaptosomes exposed to lead
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
92,35 1-357 (1988)
Calcium Eff lux and Neurotransmitter Release from Rat Hippocampal Synaptosomes Exposed to Lead’ DANIEL J. MINNEMA,’
I. A. MICHAELSON, AND G. P. COOPER
Department of Environmental Health, University of Cincinnati College of Medicine, 3223 Eden Avenue, Cincinnati. Ohio 45267-0056
Received May 12. 1987: accepted October 29. 198 7 Calcium E&IX and Neurotransmitter Release from Rat Hippocampal Synaptosomes Exposed to Lead. MINNEMA, D. J., MICHAELSON, I. A., AND COOPER G. P. (1988). Toxicol. Appl. Pharmacol. 92, 35 1-357. The results of several studies, employing various tissue preparations, have demonstrated that in vitro Pb exposure has similar effects on the release of several different transmitter substances. Pb has been observed to attenuate depolarization-evoked release and increase spontaneous (depolarization-independent) release. The current study confirms that Pb in vitro increases the spontaneous release of [‘Hlacetylcholine (ACh) from superfused synaptosomes prepared from rat hippocampus. Additionally, hippocampal synaptosomes, preloaded with 45Ca, were superfused under conditions similar to those used in the [3H]ACh-re1ease studies. Exposure to l-30 MM Pb produced a concentration-dependent increase in the efflux of %a that was quantitatively and temporally related to the Pb-induced release of [‘H]ACh from the hippocampal synaptosomes. Depolarization-evoked [‘HJACh release with high potassium did not produce a corresponding increase in 45Caefflux. It is concluded that the Pb-induced increase in spontaneous transmitter release is apparently due to either an increase in intraneuronal ionized calcium or the stimulation by Pb of Ca-activated molecules mediating transmitter ~&2Se.
0 1988 Academic
Press,
Inc.
Inorganic lead (Pb) is a recognized neurotoxin although its mechanisms of action are not well understood (Shellenberger, 1984; Cooper et al., 1984; Audesirk, 1985). Studies of the effects of in vitro Pb exposure on neuronal function have been performed in attempts to identify basic mechanisms of action which might be important in chronic in V&J exposures to Pb. Electrophysiological studies of peripheral synapses have demonstrated that acute exposure to relatively low concentrations of Pb in vitro has two basic effects on neurotransmitter release: (1) atten’ Supported by NIH ES-03399, ES-03992. and ES00159. A preliminary report was presented at the 16th Annual Meeting of the Society for Neuroscience, November 9- 14. 1986, Washington, DC. ’ To whom all correspondence and reprint requests should be addressed.
uation of depolarization-evoked release and (2) enhancement of spontaneous release (Manalis and Cooper, 1973). Both effects have recently been demonstrated for several mammalian CNS neurotransmitter systems (Suszkiw et al., 1984; Minnema et al., 1986; Minnema and Michaelson, 1986). Attenuation of depolarization-evoked release is due to inhibition of the neuronal influx of calcium (Ca) accompanying depolarization, apparently as the result of Pb competing with Ca for access to membrane Ca channels (Kostial and Vouk, 1957; Kober and Cooper, 1976; Suszkiw et al., 1984; Nachshen, 1984). The mechanisms underlying the enhanced spontaneous release remain unclear. The most popular hypothesis which has been advanced to account for the increased spontaneous neurotransmitter release produced by Pb and other polyvalent cations is that they
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Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
MINNEMA,
352
MICHAELSON,
interfere with intracellular Ca regulation, thereby increasing the level of ionized Ca within the nerve terminal (Heuser and Miledi, 197 1; deBassio et al., 197 1; Alnaes and Rahamimoff, 1974; Kajimoto and Kirpekar, 1972; Balnave and Gage, 1973). Since there has been no definitive test of this hypothesis, the current study was initiated to compare the effects of in vitro Pb exposure on synaptosomal transmitter release with its effects on 45Ca efflux. The efflux of 45Ca from cells preloaded with 45Ca has been used as an indirect indicator of changes in intracellular Ca concentration (Pounds, 1984; Reichardt and Kelly, 1983; Gilman et al., 1986; Brown et al., 1986). Presumably, any treatment that increases the intracellular free Ca concentration would activate the various cellular mechanisms involved in maintaining the cytosolic Ca concentration at approximately 1oe7M, including the Ca-ATPase pump (Lin and Way, 1984; Snelling and Nicholls, 1985). Therefore, if in vitro Pb exposure increases spontaneous transmitter release by increasing the intracellular Ca concentration, then Pb should cause an increase in neuronal 45Ca efflux that is related, both temporally and quantitatively, to transmitter release. METHODS Preparation ofsynaptosomes. Synaptosomes were prepared using a modification of the method of Gray and Whittaker (1962), as described elsewhere (Minnema and Michaelson, 1985). Adult male Long-Evans hooded rats (Charles River, Wilmington, MA) were terminated by decapitation. The brains were removed, rinsed in cold isotonic saline, and dissected on ice. The isolated hippocampal tissue from each brain (approximately 150 mg) was homogenized in 20 vol of cold 0.32 M sucrose buffered with 3.0 mM sodium phosphate, pH 7.4. The homogenate was centrifuged in a refrigerated RC2B Sorvall at 1OOOg(2°C) for 10 min. The resulting supemate (approximately 3 ml) was layered onto a precooked sucrose gradient consisting of 0.75 ml of 1.2 M sucrose and 1.5 ml of 0.8 M sucrose in 0.5 X 2-in. Beckman ultracentrifuge tubes. The tubes were centrifuged at 45,000 rpm for 25 min (inclusive of speed-up time) using a SW-50. I ultracentrifuge rotor resulting in approximately 223,OOOgat the 0.8-I .2 M sucrose interface. The isolated synaptosomes (approximately 0.5 ml) were transferred to 10 x 75-mm culture tubes, diluted to 1.5 ml with cold
AND COOPER
0.32 M sucrose, kept on ice, and used within a 4-hr period after animal termination. Synaptosomal protein was determined relative to a concurrently performed bovine serum albumin standard curve using the Coomassie blue method of Bradford (1976). An aliquot of synaptosomes equivalent to 240 rg protein (adjusted to 200 ~1 with cold 0.32 M sucrose) was added to 800 ~1 of N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes) buffer (at room temperature). In the 45Ca efflux studies, a Ca-free Hepes buffer was used. A 25 ~1 aliquot of either 45Ca (I r&i, approximately 10 mCi/mg) or [3H]choline (500 nCi, 80 Ci/mmol) was added and the sample was incubated for 30 min at 37’C under O#ZO2 (95/5%) in a Dubnoff metabolic shaker. Physiological buffer. The normal Hepes buffer consisted of I 18.5 mM NaCl, 4.75 mM KCI, 1.19 mM MgS04, 0.25 mM CaClr, 1 1.1 mM glucose, 1.14 mM ascorbic acid, and 17 mM Hepes, adjusted to pH 7.5 with 10 mM Tris base. For high-potassium buffer (used to examine depolarization-evoked release), the KC1 was adjusted to 6 1.75 mM, and the NaCl to 6 1.50 mM. Changes in CaC12, sodium acetate and, lead acetate concentrations were made with no other changes in buffer composition. Superfusion. Following incubation with either [rH]choline or %a, the loaded” synaptosomal suspensions were layered onto a 13-mm diameter, 0.65 pm pore-size cellulose acetate filter (NSI E06TF29325 Triton-Free. MicronSep, Honeoye Falls, NY). The layered synaptosomes, contained within the filter support chamber, were rinsed with two 0.5-ml aliquots of Hepes buffer (37°C). In the ‘%Ja efflux studies, these two aliquots of Hepes buffer contained 1 mM EGTA in order to facilitate the removal of extrasynaptosomal %a. The filter support chamber was then connected to a “push-pull” design superfusion system. A detailed description of the superfusion apparatus appears elsewhere (Minnema and Michaelson, 1985). The peristaltic pump was adjusted to provide a superfusion rate of 2.0 ml/min. The synaptosomes were superfused with Hepes buffer containing 0.25 mM Ca for 10 min prior to the collection of fractions. Fractions were then collected into liquid scintillation vials at I5-set intervals. Exposure of the synaptosomes to the various buffers (i.e., lead-free, lead, 6 1 mM KCI, etc.) was controlled by the manual switching of valves. Exposure to 6 1 mM KC1 was reproducibly limited to approximately 1 set by the use of an “injection-loop” valve arrangement. The temperature of the entire apparatus, with the exception of a fraction collector, was maintained at 37°C. At the end of the superfusion study the filter was removed, and the radioactivity remaining on the filter was determined. All radioactivity counting was performed using Aquasol- fluorophore and a Packard Model 460 liquid scintillation spectrometer with internal standardization, background correction, and automatically computed disintegrations per minute relative to an external quench curve. Acetycholine determination. Since the hippocampal synaptosomes were ‘*loaded” with the neurotransmitter
LEAD AND SYNAPTOSOMAL precursor, [3H]choline, it was necessary to demonstrate that the radiolabel released in the superfusate reflected mainly acetylcholine (ACh) rather than choline. To separate choline from ACh in the superfusate fractions, the choline-kinase ion-pair extraction method was used (Briggs and Cooper, 1981). A 200~~1 aliquot of each superfusate fraction was added to 50 ~1 of 50 nM glycylglycine buffer (pH 8.5) containing 10 mM ATP, 12.5 mM MgQ, and 0.01 units/ml choline kinase. After a 30-min incubation at 37”C, the sample was extracted with 300 r.d of 10 mg/ml sodium tetraphenylboron in butyronitrile. Following a 5-min centrifugation (lOOOg), 200~~1 aliquots of both the organic phase (reflecting ACh) and the aqueous phase (reflecting choline) were collected and quantified by liquid scintillation spectroscopy (as described above). Chemicals. [Methy/-3H]choline chloride (NET-109), %a chloride (NEZ-0 13), and Aquasol- were obtained from New England Nuclear (Boston, MA). Lead acetate and sodium acetate were obtained from Fisher Scientific (Pittsburgh, PA). Ultra-pure sucrose was obtained from Beckman (Palo Alto, CA). Choline kinase (C-7 138) adenosine-S-triphosphate (A-2383), tetraphenylboron. and glycylglycine were obtained from Sigma (St. Louis, MO). Butyronitrile was obtained from Kodak (Rochester, NY). All other chemicals were of reagent grade, and were obtained from available commercial sources. All solutions were prepared with double distilled water. Data handling and statistics. The amount of [3H]ACh or 45Ca released per fraction is expressed as a percentage of the total radioactivity: dpm released per fraction X 100 (total dpm released + dpm remaining on filter) Each point on the figures represents the mean f standard error of radiolabel efflux obtained from synaptosomal efflux studies conducted on different days. The number of trials(N) is indicated with each figure. The tissue from only one rat brain was used in preparing synaptosomes on any one day. Within the constraints imposed by both the amount of synaptosomal protein obtained and the duration of synaptosomal viability, only a maximum of hve to six trials could be performed per day. Therefore, on any given day, the synaptosomal preparation was exposed to all dosing conditions as indicated for the specific experiment. Analysis ofvariance (ANOVA) was used to statistically evaluate the ACh release and 45Caefflux data using a randomized-block factorial-pq design (Kirk, 1982). In all cases,the area under the curve (AUC) derived from the raw data was used in the analyses. Post hoc analyses were performed using the Newman-Keuls test at the 0.0 1 confidence level (Kirk, 1982).
RESULTS [‘HICholine and [“Hlacetylcholine determinations. Analysis of the superfusate frac-
4sCa EFFLUX
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i 2
4
FRACTION
6
8
NUMBER
10
12
(15
14
seconds
16
18
20
22
each)
FIG. 1. Relationship of Pb-induced [3H]ACh release to 45Ca efflux from rat hippocampal synaptosomes. Lead acetate ( 1, 3, 10, or 30 HIM) was added to the superfusing Hepes buffer during fractions 5-10. Sodium acetate (20 PM) was substituted for lead in control preparations. A 1-set pulse of 6 1 mM KCl-containing Hepes buffer was delivered at the beginning of fraction 19. The Hepes buffer contained 0.254 mM calcium. (A) Effect of Pb on [‘H]ACh release from synaptosomes preloaded with [3H]choline. (B) Effect of Pb on ?a efflux from synaptosomes preloaded 45Ca. N = 5 at each point.
tions by the choline-kinase ion-pair extraction method indicated that the radiolabel in the superfusate reflected greater than 90% ACh. The ratio of [3H]ACh to [3H]choline remained relatively constant during release, although a slight decrease in the ratio was observed following exposure to 10 and 30 PM Pb (Fig. 1, fractions 8- 17). Temporal characteristics ofconcentrationrelated Pb-induced ACh release (Fig. IA). There was a significant concentration-dependent (1 to 30 PM) Pb-induced increase in the release of [3H]ACh from rat hippocampal synaptosomes relative to [3H]ACh release in
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the presence of 20 FM sodium acetate. The onset of the increased [3H]ACh release was delayed by 15 to 30 set following Pb exposure. The release of [3H]ACh evoked by a lset exposure to 6 1 mrvi KC1 containing buffer at the beginning of fraction 19 had a rapid onset (< 15 set) and a total duration of less than 30 sec. The prior exposure to Pb did not alter the magnitude of the KCl-evoked [‘HIACh release. Temporal characteristics of concentrationrelated Pb-induced “‘Ca ejlux (Fig. 1B). There was a significant concentration-dependent (1 to 30 yM) Pb-induced increase in the efflux of 45Ca from rat hippocampal synaptosomes relative to 45Ca efflux in the presence of 20 PM sodium acetate. The onset of this increase in 45Ca efflux was delayed 30 to 45 set following Pb exposure, being slower than the [3H]ACh release (Fig. 1A) by approximately 15 sec. Except at the 30 PM Pb concentration, the peak increase in 45Ca efflux (fractions 11-12) followed the peak increase in [3H]ACh release (fractions 9-l 1). Only a very slight increase in 45Ca efflux was observed following membrane depolarization with 6 1 mM KC1 (fraction 19). Relationship of Pb-induced (spontaneous) transmitter release to 4’Ca eflux (Fig. 2). When plotted on a log-log scale the total amount of transmitter released by Pb (fractions 5- 18) was geometrically related to Pb concentration. This relationship was similar for three neurotransmitter systems (ACh, current study: dopamine, Minnema et al.. 1986; GABA, Minnema and Michaelson, 1986). A parallel relationship with respect to transmitter release was also observed for the efflux of 45Ca. DISCUSSION The dependence of depolarization-evoked transmitter release on the influx of extraneuronal Ca into the nerve terminal is well established (Augustine et al., 1987). The present results demonstrate minimal increases in 45Ca efflux accompanying depolarization-
AND COOPER
FIG. 2. Concentration-related effect of Pb on transmitter and ‘?a release from various rat synaptosomal prep arations. The Pb-induced release of [3H]dopamine from rat striatal synaptosomes (squares), [3H]GABA from rat cortical synaptosomes (diamonds), and [3H]ACh from rat hippocampal synaptosomes (triangles) exhibited similar Pb concentration-dependent effects on the magnitude of transmitter release. A similar (parallel) relationship was noted for the efflux of “%Ja from rat hippocampal synaptosomes (inverted triangles). The lines on the graph were determined using a least-squares analysis.
evoked transmitter release (Fig. 1, fraction 19). Any increase in intrasynaptosomal free Ca following membrane depolarization with high-potassium-containing buffer in the present experiment would reflect the influx of nonradioactive Ca from the superfusing buffer. Rapid accumulation by mitochondria is the major mechanism by which this increase in intracellular Ca is lowered (Nicholls, 1986). The plasma membrane Ca-ATPase, due to its restricted I’,,,,, , plays a lesser role in reestablishing normal intrasynaptosomal Ca concentrations following depolarizationevoked release (Nicholls, 1986). Even so, any of the Ca pumped out of the cell by the CaATPase would reflect mainly unlabeled free Ca that entered the nerve terminals following membrane depolarization. Since it is assumed that most of the 45Ca is bound (nonionized) within the synaptosomes, those treatments which do not mobilize the intraneuronal Ca stores will not alter 45Ca efflux. The observed lack of significant increase in 45Ca efflux accompanying (or following) depolarization-evoked transmitter release further supports the dependence of this phenomenon on extraneuronal Ca.
LEAD
AND
SYNAPTOSOMAL
The in vitro Pb-induced increase in spontaneous ACh release from rat hippocampal synaptosomes is similar to the effects of Pb observed for several other neurotransmitter systems (Minnema et al., 1986; Minnema and Michaelson, 1986). This similarity of in vitro Pb effects on release suggests that Pb acts through mechanisms common to all transmitter systems. The increased spontaneous release could be explained by several possible mechanisms, including (a) membrane depolarization, (b) inhibition of membrane Na,KATPase, (c) Ca-mimicking actions, and/or (d) increases in intracellular ionized Ca. The ability of Pb to increase spontaneous release in the presence of a Ca-free buffer argues against membrane depolarization as the mechanism mediating this Pb effect (Minnema et al., 1986; Minnema and Michaelson, 1986). Inhibition of membrane Na,K-ATPase increases transmitter release even in the absence of extraneuronal Ca (Baker and Crawford, 1975; Powis, 1983; Sweadner, 1985). This releasing action of known ATPase inhibitors (e.g., ouabain) is dependent on a transmembrane sodium gradient. Although some studies have indicated that Pb can inhibit membrane ATPase (Siegel and Fogt, 1977; Nechay and Saunders, 1978), the ability of Pb to induce release in the presence of low extraneuronal sodium suggests that Na,K-ATPase inhibition is not the mechanism mediating Pb’s action on spontaneous release (Minnema and Michaelson, 1986). Several studies suggest that Pb interferes with various aspects of intracellular Ca regulation (Pounds, 1984; Simons, 1986). While intracellular Ca regulation is an important aspect of a number of processes in a variety of cells, it is particularly critical in nerve cell terminals where Ca mediates transmitter release (Augustine et al., 1987). One possibility is that Pb may act directly as a Ca substitute at the intraneuronal sites mediating release. Although there is no direct support for this Camimetic action of Pb, several studies indicate that Pb can substitute for Ca to activate calmodulin-dependent phosphorylation (Cheung, 1984; Haberman et al., 1983;
4SCa EFFLUX
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Goldstein and At-, 1983). Although there exists a lack of conclusive evidence for a direct association of calmodulin with the transmitter release process (Augustine et al., 1987) Publicover ( 1985) has reviewed material suggesting that activation of calmodulin may be involved in asynchronous, spontaneous transmitter release. Activation of calmodulin has been demonstrated to stimulate the Caspecific ATPase pump (Kuo et al., 1979) which by itself could explain the observed increase in 45Ca efllux. The observed correlation between transmitter release and 45Ca efflux is consistent with a “Ca-mimetic” action of Pb at potential Ca-activated molecules (e.g., calmodulin, protein kinase C) which may simultaneously activate both transmitter release and the synaptic Ca pump. If Pb is indeed acting as a “Ca-mimetic,” it is interesting that the Pb-induced increase in 45Ca efflux was observed despite the assumed activation of calmodulin-dependent intrasynaptosomal Ca sequestration mechanisms (Ross and Carderas, 1983). Alternatively (or additionally), Pb could act to increase spontaneous transmitter release by increasing the intraneuronal ionized Ca concentration (Kolton and Yaari, 1982). One means by which the intraneuronal free Ca could be elevated is by inhibition of Ca extrusion; specifically, inhibition of the Mg2+-dependent Ca-ATPase. The extrusion of Ca by Ca-ATPase at the plasma membrane is the dominant means by which the intraneuronal Ca concentration is maintained during “resting” conditions (Snelling and Nicholls, 1985). Although Pb has been reported to be a weak inhibitor of this enzyme (Thompson and Nechay, 198 l), the Pb-induced increase in 45Ca efflux observed in the current study (Fig. 1B) would not be expected if Ca-ATPase inhibition is the mechanism by which Pb increases transmitter release. The similar concentration/release effects and temporal relationships between transmitter release and 45Ca efflux (Figs. 1 and 2) suggest that Pb may displace bound Ca from intraneuronal Ca sources. The slight temporal differences in onset and peak effects (i.e., the
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effect of Pb on transmitter release precedes its effect on 45Ca efflux) are consistent with the view that Pb increases the intraneuronal ionized Ca concentration, which would first interact at the intraneuronal site mediating transmitter release, and subsequently this Ca would be extruded from the nerve ending. The present study does not indicate the intraneuronal sites which serve as the source of the increased 45Ca efflux following Pb exposure. Studies using nonexcitable cells have shown that Pb increases whole cell Ca concentration, most of which is associated with the mitochondrial compartment (Pounds et al., 1982; Pounds and Rosen, 1986). It is difficult to explain the present results in terms of those studies in which Pb increases total cellular Ca, since the observed increase in 45Ca efflux from the nerve terminal would apparently be expected to lower intraneuronal Ca. One possibility is that the rate of “new” (unlabeled) Ca entering the cell following Pb exposure is greater than the efflux of Ca (as reflected by 45Ca). Alternatively, this apparent discrepency could reflect a biphasic Pb effect, either in a temporal and/or a dose-related fashion. For example, low Pb concentrations (< 10 PM) have been shown to inhibit microsomal and mitochondrial Ca uptake, whereas higher Pb concentrations (> 10 PM) increased Ca uptake (Suszkiw et al., 1984). Decreased mitochondrial and/or microsomal Ca uptake would be expected to increase intracellular ionized Ca. Clearly, the intracellular regulation of Ca is complex, and further studies will be necessary to explain the means by which Pb alters intraneuronal Ca homeostasis. In conclusion, the results of the present study confirm that Pb increases spontaneous neurotransmitter release in a similar fashion for several transmitter substances, and that this increase in release is correlated with a corresponding increase in 45Ca efflux. ACKNOWLEDGMENTS The authors thank Robert Greenland for his technical assistance, and Nancy Knapp for typing the manuscript.
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REFERENCES ALNAES, E., AND RAHAMIMOFF, R. (1974). Dual action of praseodymium (P?‘) on transmitter release at the frog neuromuscular junction. Nature (London) 247, 478-479. ALJDESIRK, G. (1985). Effects of lead exposure on the physiology of neurons. Prog. Neurobiol. 24, 199-23 1. AUGUSTINE, G., CHARLTON, M., AND SMITH, S. (1987). Calcium action in synaptic transmitter release. Annu. Rev. Neurosci.
10,633-693.
BAKER, P., AND CRAWFORD, A. (1975). A note on the mechanism by which inhibitors of the sodium pump accelerate spontaneous release of transmitter from motor nerve terminals. J. Physiol. 247,209-226. BALNAVE, R., AND GAGE, P. (1973). The inhibitory effect of manganese on transmitter release at the neuromuscular junction of the toad. Brit. J. Pharmacol. 47,339-352. BRADFORD, M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
72,248-254.
BRICGS. C., AND COOPER, J. (1981). A synaptosomal preparation from the guinea pig ileum myenteric plexus. J. Neurochem. 36, 1097- 1108. BROWN, R., PRENDIVILLE, P., AND CAIN, C. (1986). Alpha- 1-adrenergic and H 1-histamine receptor control of intracellular Ca 2c in a muscle cell line: The influence of prior agonist exposure on receptor responsiveness. Mol. Pharmacol. 29,53 l-539. CHEUNG, W. (1984). Calmodulin: Its potential role in cell proliferation and heavy metal toxicity. Fed. Proc. 43,2995-2999. COOPER,G., SUSZKIW, J., AND MANALIS, R. (1984). Presynaptic effects of heavy metals. In Cellular and Molecular Neurotoxicology (T. Narahash, Ed.) pp. I-2 1. Raven Press, NY. DEBASSIO, W., SCHNITZLER, R., AND PARSONS, R. (t97 1). Influence of lanthanum on transmitter release at the neuromuscular junction. J. Neurobiof. 2, 263278. GILMAN, S., KUMAROO, K., AND HALLENBECK, J. ( 1986). Effects of pressure on uptake and release of calcium by brain synaptosomes. J. Appl. Physiol. 60, 1446-1450. GOLDSTEIN, G., AND AR, D. (1983). Lead activates calmodulin sensitive processes. LifeSci. 33, 1001-1006. GRAY, E.. AND WHI~AKER, V. (1962). The isolation of nerve endings from brain: An electron microscope study of cell fragments derived by homogenization and centrifugation. J. Anat. 96,79-87. HABERMANN, E., CROWELL, K., AND JANICKI, P. (1983). Lead and other metals can substitute for Ca” in calmodulin. Arch. Toxicol. 54,6 l-70. HEUSER, J., AND MILEDI, R. (1971). Effect oflanthanum ions on function and structure of frog neuromuscular junctions. Proc. R. Sot. London B. 179,247-260.
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KAJIMOTO,N.,ANDKIRPEKAR,
AND
SYNAPTOSOMAL
%(1972).Effectofmanganese and lanthanum on spontaneous release of acetylcholine at frog motor nerve terminals. Nature New Biol. 235,29-30. KIRK, R. (1982). Experimental Design: Procedures for the Behavioral Sciences, 2nd ed. Brooks/Cole, Belmont, CA. KOBER, T., AND COOPER,G. (1976). Lead competitively inhibits calcium dependent synaptic transmission in the bullfrog sympathetic ganglion. Nature (London) 262,704-705. KOLTON, L., AND YARRI, Y. (1982). Sites of action of lead on spontaneous transmitter release from motor nerve terminals. Isr. J. Med. Sci. 18, 165-170. KOSTIAL, K., AND VOUK. V. B. (1957). Lead ions and synaptic transmission in the superior cervical ganglion of the cat. Brit. J. Pharmacol. 12,2 19-222. Kuo. C., ICHIDA, S.. MATSUDA, T., KAKIUCHI, S., AND YOCHISDA, M. (1979). Regulation of ATP-dependent Ca-uptake of synaptic plasma membranes by Ca-dependent modulatory protein. Life Sci. 125,235-240. LIN, S.. AND WAY, E. (1984). Calcium transport in and out ofbrain nerve endings in vitro-The role of synaptosomal membrane Ca’+-ATPase in Ca*+-extrusion. Brain Res. 298,225-234. MANALIS, R., AND COOPER, G. (1973). Presynaptic and postsynaptic effects of lead at the frog neuromuscular junction. Nature (London) 243,354-356. MINNEMA, D., AND MICHAELSON. I. A. (1985). A superfusion apparatus for the examination of neurotransmitter release from synaptosomes. J. Neurosci. Methods 14,193-206. MINNEMA, D., AND MICHAELSON, I. A. (1986). Differential effects of inorganic lead and b-aminolevulinic acid in vitro on synaptosomal y-aminobutyric acid release. Toxicol. Appl. Pharmacol86,437-447. MINNEMA, D., GREENLAND, R., AND MICHAELSON, I. A. (1986). Effects of in vitro inorganic lead on dopamine release from superfused rat striatal synaptosomes. Toxicol. Appl. Pharmacol. 84,400-4 I 1. NACHSHEN, D. (1984). Selectivity of the Ca binding site in synaptosome Ca channels: Inhibition of Ca influx by multivalent metal cations. J. Gen. Physiol. 83,94 l967. NECHAY, B., AND SAUNDERS, J. (1978). Inhibitory characteristics of lead chloride in sodium- and potassiumdependent adenosinetriphosphatase preparations derived from kidney, brain and heart of several species. J. Toxicol. Environ. Health 4, 147-l 59.
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357
NICHOLLS, D. (1986). Intracellular calcium homeostasis. Brit. Med. Bull. 42,353-358. POUNDS, J. ( 1984). Effect of lead intoxication on calcium homeostasis and calcium-mediated cell function: A review. Neurotoxicology 5,295-332. POUNDS, J.. AND ROSEN, J. (1986). Cellular metabolism of lead: A kinetic analysis in cultured osteoclastic bone cells. Toxicol. Appl. Pharmacol. 83,53 l-545. POUNDS, J., WRIGHT, R., MORRISON, D., AND CASCIANO, D. (1982). Effect of lead on calcium homeostasis in the isolated rat hepatocyte. Toxicol. Appl. Pharmac-01.63,389-40 1. Powrs, D. (1983). Cardiac glycosides and autonomic neurotransmission. J. Auton. Pharmacol. 3, 127-l 54. PUBLICOVER, S. (1985). Calmodulin, synchronous and asynchronous release of neurotransmitter. Camp. Biothem. Physiol. 82A, 7- 11. REICHARDT. L., AND KELLY, R. (1983). A molecular description of nerve terminal function. Annu. Rev. Biothem. 52,87 1-926. Ross, D.. AND CARDERAS, H. (1983). Calmodulin stimulation of Ca-dependent ATP hydrolysis and ATP-dependent Ca transport in synaptic membranes. J. Neurochem. 41, 161-171. SHELLENBERGER, M. (1984). Effects of early lead exposure on neurotransmitter systems in the brain: A review with commentary. Neurotoxicology 5, 177-2 12. SIEGEL, G.. AND FOGT. S. (1977). Inhibition by lead ion of electrophorus electroplax (Na+-K+)-adenosine triphosphatase and K+-p-nitrophenylphosphatase. J. Biol. Chem. 252,5201-5205. SIMONS, T. (1986). Cellular interactions between lead and calcium. Brit. Med. Bull. 42,43 l-434. SNELLING, R., AND NICHOLLS, D. (1985). Calcium efflux and cycling across the synaptosomal plasma membrane. Biochem. J. 226,225-23 1. SUSZKIW. J., TOTH, G.. MURAWSKY, M., AND COOPER, G. (1984). Effects of Pb*+ and Cd*+ on acetylcholine release and Ca*+ movements in synaptosomes and subcellular fractions from rat brain and torpedo electric organ. Brain Res. 323,3 l-46. SWEADNER, K. (1985). Oubain-evoked norepinephrine release from intact rat sympathetic neurons: Evidence for carrier-mediated release. J. Neurosci. 5, 23972406. THOMPSON, J., AND NECHAY, B. (198 1). Inhibition by metals of canine renal calcium, magnesium-activated adenosinetriphosphatase. J. Toxicol. Environ. Health 7,901-908.
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PHARMACOLOGY
92,35 1-357 (1988)
Calcium Eff lux and Neurotransmitter Release from Rat Hippocampal Synaptosomes Exposed to Lead’ DANIEL J. MINNEMA,’
I. A. MICHAELSON, AND G. P. COOPER
Department of Environmental Health, University of Cincinnati College of Medicine, 3223 Eden Avenue, Cincinnati. Ohio 45267-0056
Received May 12. 1987: accepted October 29. 198 7 Calcium E&IX and Neurotransmitter Release from Rat Hippocampal Synaptosomes Exposed to Lead. MINNEMA, D. J., MICHAELSON, I. A., AND COOPER G. P. (1988). Toxicol. Appl. Pharmacol. 92, 35 1-357. The results of several studies, employing various tissue preparations, have demonstrated that in vitro Pb exposure has similar effects on the release of several different transmitter substances. Pb has been observed to attenuate depolarization-evoked release and increase spontaneous (depolarization-independent) release. The current study confirms that Pb in vitro increases the spontaneous release of [‘Hlacetylcholine (ACh) from superfused synaptosomes prepared from rat hippocampus. Additionally, hippocampal synaptosomes, preloaded with 45Ca, were superfused under conditions similar to those used in the [3H]ACh-re1ease studies. Exposure to l-30 MM Pb produced a concentration-dependent increase in the efflux of %a that was quantitatively and temporally related to the Pb-induced release of [‘H]ACh from the hippocampal synaptosomes. Depolarization-evoked [‘HJACh release with high potassium did not produce a corresponding increase in 45Caefflux. It is concluded that the Pb-induced increase in spontaneous transmitter release is apparently due to either an increase in intraneuronal ionized calcium or the stimulation by Pb of Ca-activated molecules mediating transmitter ~&2Se.
0 1988 Academic
Press,
Inc.
Inorganic lead (Pb) is a recognized neurotoxin although its mechanisms of action are not well understood (Shellenberger, 1984; Cooper et al., 1984; Audesirk, 1985). Studies of the effects of in vitro Pb exposure on neuronal function have been performed in attempts to identify basic mechanisms of action which might be important in chronic in V&J exposures to Pb. Electrophysiological studies of peripheral synapses have demonstrated that acute exposure to relatively low concentrations of Pb in vitro has two basic effects on neurotransmitter release: (1) atten’ Supported by NIH ES-03399, ES-03992. and ES00159. A preliminary report was presented at the 16th Annual Meeting of the Society for Neuroscience, November 9- 14. 1986, Washington, DC. ’ To whom all correspondence and reprint requests should be addressed.
uation of depolarization-evoked release and (2) enhancement of spontaneous release (Manalis and Cooper, 1973). Both effects have recently been demonstrated for several mammalian CNS neurotransmitter systems (Suszkiw et al., 1984; Minnema et al., 1986; Minnema and Michaelson, 1986). Attenuation of depolarization-evoked release is due to inhibition of the neuronal influx of calcium (Ca) accompanying depolarization, apparently as the result of Pb competing with Ca for access to membrane Ca channels (Kostial and Vouk, 1957; Kober and Cooper, 1976; Suszkiw et al., 1984; Nachshen, 1984). The mechanisms underlying the enhanced spontaneous release remain unclear. The most popular hypothesis which has been advanced to account for the increased spontaneous neurotransmitter release produced by Pb and other polyvalent cations is that they
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interfere with intracellular Ca regulation, thereby increasing the level of ionized Ca within the nerve terminal (Heuser and Miledi, 197 1; deBassio et al., 197 1; Alnaes and Rahamimoff, 1974; Kajimoto and Kirpekar, 1972; Balnave and Gage, 1973). Since there has been no definitive test of this hypothesis, the current study was initiated to compare the effects of in vitro Pb exposure on synaptosomal transmitter release with its effects on 45Ca efflux. The efflux of 45Ca from cells preloaded with 45Ca has been used as an indirect indicator of changes in intracellular Ca concentration (Pounds, 1984; Reichardt and Kelly, 1983; Gilman et al., 1986; Brown et al., 1986). Presumably, any treatment that increases the intracellular free Ca concentration would activate the various cellular mechanisms involved in maintaining the cytosolic Ca concentration at approximately 1oe7M, including the Ca-ATPase pump (Lin and Way, 1984; Snelling and Nicholls, 1985). Therefore, if in vitro Pb exposure increases spontaneous transmitter release by increasing the intracellular Ca concentration, then Pb should cause an increase in neuronal 45Ca efflux that is related, both temporally and quantitatively, to transmitter release. METHODS Preparation ofsynaptosomes. Synaptosomes were prepared using a modification of the method of Gray and Whittaker (1962), as described elsewhere (Minnema and Michaelson, 1985). Adult male Long-Evans hooded rats (Charles River, Wilmington, MA) were terminated by decapitation. The brains were removed, rinsed in cold isotonic saline, and dissected on ice. The isolated hippocampal tissue from each brain (approximately 150 mg) was homogenized in 20 vol of cold 0.32 M sucrose buffered with 3.0 mM sodium phosphate, pH 7.4. The homogenate was centrifuged in a refrigerated RC2B Sorvall at 1OOOg(2°C) for 10 min. The resulting supemate (approximately 3 ml) was layered onto a precooked sucrose gradient consisting of 0.75 ml of 1.2 M sucrose and 1.5 ml of 0.8 M sucrose in 0.5 X 2-in. Beckman ultracentrifuge tubes. The tubes were centrifuged at 45,000 rpm for 25 min (inclusive of speed-up time) using a SW-50. I ultracentrifuge rotor resulting in approximately 223,OOOgat the 0.8-I .2 M sucrose interface. The isolated synaptosomes (approximately 0.5 ml) were transferred to 10 x 75-mm culture tubes, diluted to 1.5 ml with cold
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0.32 M sucrose, kept on ice, and used within a 4-hr period after animal termination. Synaptosomal protein was determined relative to a concurrently performed bovine serum albumin standard curve using the Coomassie blue method of Bradford (1976). An aliquot of synaptosomes equivalent to 240 rg protein (adjusted to 200 ~1 with cold 0.32 M sucrose) was added to 800 ~1 of N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes) buffer (at room temperature). In the 45Ca efflux studies, a Ca-free Hepes buffer was used. A 25 ~1 aliquot of either 45Ca (I r&i, approximately 10 mCi/mg) or [3H]choline (500 nCi, 80 Ci/mmol) was added and the sample was incubated for 30 min at 37’C under O#ZO2 (95/5%) in a Dubnoff metabolic shaker. Physiological buffer. The normal Hepes buffer consisted of I 18.5 mM NaCl, 4.75 mM KCI, 1.19 mM MgS04, 0.25 mM CaClr, 1 1.1 mM glucose, 1.14 mM ascorbic acid, and 17 mM Hepes, adjusted to pH 7.5 with 10 mM Tris base. For high-potassium buffer (used to examine depolarization-evoked release), the KC1 was adjusted to 6 1.75 mM, and the NaCl to 6 1.50 mM. Changes in CaC12, sodium acetate and, lead acetate concentrations were made with no other changes in buffer composition. Superfusion. Following incubation with either [rH]choline or %a, the loaded” synaptosomal suspensions were layered onto a 13-mm diameter, 0.65 pm pore-size cellulose acetate filter (NSI E06TF29325 Triton-Free. MicronSep, Honeoye Falls, NY). The layered synaptosomes, contained within the filter support chamber, were rinsed with two 0.5-ml aliquots of Hepes buffer (37°C). In the ‘%Ja efflux studies, these two aliquots of Hepes buffer contained 1 mM EGTA in order to facilitate the removal of extrasynaptosomal %a. The filter support chamber was then connected to a “push-pull” design superfusion system. A detailed description of the superfusion apparatus appears elsewhere (Minnema and Michaelson, 1985). The peristaltic pump was adjusted to provide a superfusion rate of 2.0 ml/min. The synaptosomes were superfused with Hepes buffer containing 0.25 mM Ca for 10 min prior to the collection of fractions. Fractions were then collected into liquid scintillation vials at I5-set intervals. Exposure of the synaptosomes to the various buffers (i.e., lead-free, lead, 6 1 mM KCI, etc.) was controlled by the manual switching of valves. Exposure to 6 1 mM KC1 was reproducibly limited to approximately 1 set by the use of an “injection-loop” valve arrangement. The temperature of the entire apparatus, with the exception of a fraction collector, was maintained at 37°C. At the end of the superfusion study the filter was removed, and the radioactivity remaining on the filter was determined. All radioactivity counting was performed using Aquasol- fluorophore and a Packard Model 460 liquid scintillation spectrometer with internal standardization, background correction, and automatically computed disintegrations per minute relative to an external quench curve. Acetycholine determination. Since the hippocampal synaptosomes were ‘*loaded” with the neurotransmitter
LEAD AND SYNAPTOSOMAL precursor, [3H]choline, it was necessary to demonstrate that the radiolabel released in the superfusate reflected mainly acetylcholine (ACh) rather than choline. To separate choline from ACh in the superfusate fractions, the choline-kinase ion-pair extraction method was used (Briggs and Cooper, 1981). A 200~~1 aliquot of each superfusate fraction was added to 50 ~1 of 50 nM glycylglycine buffer (pH 8.5) containing 10 mM ATP, 12.5 mM MgQ, and 0.01 units/ml choline kinase. After a 30-min incubation at 37”C, the sample was extracted with 300 r.d of 10 mg/ml sodium tetraphenylboron in butyronitrile. Following a 5-min centrifugation (lOOOg), 200~~1 aliquots of both the organic phase (reflecting ACh) and the aqueous phase (reflecting choline) were collected and quantified by liquid scintillation spectroscopy (as described above). Chemicals. [Methy/-3H]choline chloride (NET-109), %a chloride (NEZ-0 13), and Aquasol- were obtained from New England Nuclear (Boston, MA). Lead acetate and sodium acetate were obtained from Fisher Scientific (Pittsburgh, PA). Ultra-pure sucrose was obtained from Beckman (Palo Alto, CA). Choline kinase (C-7 138) adenosine-S-triphosphate (A-2383), tetraphenylboron. and glycylglycine were obtained from Sigma (St. Louis, MO). Butyronitrile was obtained from Kodak (Rochester, NY). All other chemicals were of reagent grade, and were obtained from available commercial sources. All solutions were prepared with double distilled water. Data handling and statistics. The amount of [3H]ACh or 45Ca released per fraction is expressed as a percentage of the total radioactivity: dpm released per fraction X 100 (total dpm released + dpm remaining on filter) Each point on the figures represents the mean f standard error of radiolabel efflux obtained from synaptosomal efflux studies conducted on different days. The number of trials(N) is indicated with each figure. The tissue from only one rat brain was used in preparing synaptosomes on any one day. Within the constraints imposed by both the amount of synaptosomal protein obtained and the duration of synaptosomal viability, only a maximum of hve to six trials could be performed per day. Therefore, on any given day, the synaptosomal preparation was exposed to all dosing conditions as indicated for the specific experiment. Analysis ofvariance (ANOVA) was used to statistically evaluate the ACh release and 45Caefflux data using a randomized-block factorial-pq design (Kirk, 1982). In all cases,the area under the curve (AUC) derived from the raw data was used in the analyses. Post hoc analyses were performed using the Newman-Keuls test at the 0.0 1 confidence level (Kirk, 1982).
RESULTS [‘HICholine and [“Hlacetylcholine determinations. Analysis of the superfusate frac-
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FIG. 1. Relationship of Pb-induced [3H]ACh release to 45Ca efflux from rat hippocampal synaptosomes. Lead acetate ( 1, 3, 10, or 30 HIM) was added to the superfusing Hepes buffer during fractions 5-10. Sodium acetate (20 PM) was substituted for lead in control preparations. A 1-set pulse of 6 1 mM KCl-containing Hepes buffer was delivered at the beginning of fraction 19. The Hepes buffer contained 0.254 mM calcium. (A) Effect of Pb on [‘H]ACh release from synaptosomes preloaded with [3H]choline. (B) Effect of Pb on ?a efflux from synaptosomes preloaded 45Ca. N = 5 at each point.
tions by the choline-kinase ion-pair extraction method indicated that the radiolabel in the superfusate reflected greater than 90% ACh. The ratio of [3H]ACh to [3H]choline remained relatively constant during release, although a slight decrease in the ratio was observed following exposure to 10 and 30 PM Pb (Fig. 1, fractions 8- 17). Temporal characteristics ofconcentrationrelated Pb-induced ACh release (Fig. IA). There was a significant concentration-dependent (1 to 30 PM) Pb-induced increase in the release of [3H]ACh from rat hippocampal synaptosomes relative to [3H]ACh release in
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the presence of 20 FM sodium acetate. The onset of the increased [3H]ACh release was delayed by 15 to 30 set following Pb exposure. The release of [3H]ACh evoked by a lset exposure to 6 1 mrvi KC1 containing buffer at the beginning of fraction 19 had a rapid onset (< 15 set) and a total duration of less than 30 sec. The prior exposure to Pb did not alter the magnitude of the KCl-evoked [‘HIACh release. Temporal characteristics of concentrationrelated Pb-induced “‘Ca ejlux (Fig. 1B). There was a significant concentration-dependent (1 to 30 yM) Pb-induced increase in the efflux of 45Ca from rat hippocampal synaptosomes relative to 45Ca efflux in the presence of 20 PM sodium acetate. The onset of this increase in 45Ca efflux was delayed 30 to 45 set following Pb exposure, being slower than the [3H]ACh release (Fig. 1A) by approximately 15 sec. Except at the 30 PM Pb concentration, the peak increase in 45Ca efflux (fractions 11-12) followed the peak increase in [3H]ACh release (fractions 9-l 1). Only a very slight increase in 45Ca efflux was observed following membrane depolarization with 6 1 mM KC1 (fraction 19). Relationship of Pb-induced (spontaneous) transmitter release to 4’Ca eflux (Fig. 2). When plotted on a log-log scale the total amount of transmitter released by Pb (fractions 5- 18) was geometrically related to Pb concentration. This relationship was similar for three neurotransmitter systems (ACh, current study: dopamine, Minnema et al.. 1986; GABA, Minnema and Michaelson, 1986). A parallel relationship with respect to transmitter release was also observed for the efflux of 45Ca. DISCUSSION The dependence of depolarization-evoked transmitter release on the influx of extraneuronal Ca into the nerve terminal is well established (Augustine et al., 1987). The present results demonstrate minimal increases in 45Ca efflux accompanying depolarization-
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FIG. 2. Concentration-related effect of Pb on transmitter and ‘?a release from various rat synaptosomal prep arations. The Pb-induced release of [3H]dopamine from rat striatal synaptosomes (squares), [3H]GABA from rat cortical synaptosomes (diamonds), and [3H]ACh from rat hippocampal synaptosomes (triangles) exhibited similar Pb concentration-dependent effects on the magnitude of transmitter release. A similar (parallel) relationship was noted for the efflux of “%Ja from rat hippocampal synaptosomes (inverted triangles). The lines on the graph were determined using a least-squares analysis.
evoked transmitter release (Fig. 1, fraction 19). Any increase in intrasynaptosomal free Ca following membrane depolarization with high-potassium-containing buffer in the present experiment would reflect the influx of nonradioactive Ca from the superfusing buffer. Rapid accumulation by mitochondria is the major mechanism by which this increase in intracellular Ca is lowered (Nicholls, 1986). The plasma membrane Ca-ATPase, due to its restricted I’,,,,, , plays a lesser role in reestablishing normal intrasynaptosomal Ca concentrations following depolarizationevoked release (Nicholls, 1986). Even so, any of the Ca pumped out of the cell by the CaATPase would reflect mainly unlabeled free Ca that entered the nerve terminals following membrane depolarization. Since it is assumed that most of the 45Ca is bound (nonionized) within the synaptosomes, those treatments which do not mobilize the intraneuronal Ca stores will not alter 45Ca efflux. The observed lack of significant increase in 45Ca efflux accompanying (or following) depolarization-evoked transmitter release further supports the dependence of this phenomenon on extraneuronal Ca.
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The in vitro Pb-induced increase in spontaneous ACh release from rat hippocampal synaptosomes is similar to the effects of Pb observed for several other neurotransmitter systems (Minnema et al., 1986; Minnema and Michaelson, 1986). This similarity of in vitro Pb effects on release suggests that Pb acts through mechanisms common to all transmitter systems. The increased spontaneous release could be explained by several possible mechanisms, including (a) membrane depolarization, (b) inhibition of membrane Na,KATPase, (c) Ca-mimicking actions, and/or (d) increases in intracellular ionized Ca. The ability of Pb to increase spontaneous release in the presence of a Ca-free buffer argues against membrane depolarization as the mechanism mediating this Pb effect (Minnema et al., 1986; Minnema and Michaelson, 1986). Inhibition of membrane Na,K-ATPase increases transmitter release even in the absence of extraneuronal Ca (Baker and Crawford, 1975; Powis, 1983; Sweadner, 1985). This releasing action of known ATPase inhibitors (e.g., ouabain) is dependent on a transmembrane sodium gradient. Although some studies have indicated that Pb can inhibit membrane ATPase (Siegel and Fogt, 1977; Nechay and Saunders, 1978), the ability of Pb to induce release in the presence of low extraneuronal sodium suggests that Na,K-ATPase inhibition is not the mechanism mediating Pb’s action on spontaneous release (Minnema and Michaelson, 1986). Several studies suggest that Pb interferes with various aspects of intracellular Ca regulation (Pounds, 1984; Simons, 1986). While intracellular Ca regulation is an important aspect of a number of processes in a variety of cells, it is particularly critical in nerve cell terminals where Ca mediates transmitter release (Augustine et al., 1987). One possibility is that Pb may act directly as a Ca substitute at the intraneuronal sites mediating release. Although there is no direct support for this Camimetic action of Pb, several studies indicate that Pb can substitute for Ca to activate calmodulin-dependent phosphorylation (Cheung, 1984; Haberman et al., 1983;
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Goldstein and At-, 1983). Although there exists a lack of conclusive evidence for a direct association of calmodulin with the transmitter release process (Augustine et al., 1987) Publicover ( 1985) has reviewed material suggesting that activation of calmodulin may be involved in asynchronous, spontaneous transmitter release. Activation of calmodulin has been demonstrated to stimulate the Caspecific ATPase pump (Kuo et al., 1979) which by itself could explain the observed increase in 45Ca efllux. The observed correlation between transmitter release and 45Ca efflux is consistent with a “Ca-mimetic” action of Pb at potential Ca-activated molecules (e.g., calmodulin, protein kinase C) which may simultaneously activate both transmitter release and the synaptic Ca pump. If Pb is indeed acting as a “Ca-mimetic,” it is interesting that the Pb-induced increase in 45Ca efflux was observed despite the assumed activation of calmodulin-dependent intrasynaptosomal Ca sequestration mechanisms (Ross and Carderas, 1983). Alternatively (or additionally), Pb could act to increase spontaneous transmitter release by increasing the intraneuronal ionized Ca concentration (Kolton and Yaari, 1982). One means by which the intraneuronal free Ca could be elevated is by inhibition of Ca extrusion; specifically, inhibition of the Mg2+-dependent Ca-ATPase. The extrusion of Ca by Ca-ATPase at the plasma membrane is the dominant means by which the intraneuronal Ca concentration is maintained during “resting” conditions (Snelling and Nicholls, 1985). Although Pb has been reported to be a weak inhibitor of this enzyme (Thompson and Nechay, 198 l), the Pb-induced increase in 45Ca efflux observed in the current study (Fig. 1B) would not be expected if Ca-ATPase inhibition is the mechanism by which Pb increases transmitter release. The similar concentration/release effects and temporal relationships between transmitter release and 45Ca efflux (Figs. 1 and 2) suggest that Pb may displace bound Ca from intraneuronal Ca sources. The slight temporal differences in onset and peak effects (i.e., the
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effect of Pb on transmitter release precedes its effect on 45Ca efflux) are consistent with the view that Pb increases the intraneuronal ionized Ca concentration, which would first interact at the intraneuronal site mediating transmitter release, and subsequently this Ca would be extruded from the nerve ending. The present study does not indicate the intraneuronal sites which serve as the source of the increased 45Ca efflux following Pb exposure. Studies using nonexcitable cells have shown that Pb increases whole cell Ca concentration, most of which is associated with the mitochondrial compartment (Pounds et al., 1982; Pounds and Rosen, 1986). It is difficult to explain the present results in terms of those studies in which Pb increases total cellular Ca, since the observed increase in 45Ca efflux from the nerve terminal would apparently be expected to lower intraneuronal Ca. One possibility is that the rate of “new” (unlabeled) Ca entering the cell following Pb exposure is greater than the efflux of Ca (as reflected by 45Ca). Alternatively, this apparent discrepency could reflect a biphasic Pb effect, either in a temporal and/or a dose-related fashion. For example, low Pb concentrations (< 10 PM) have been shown to inhibit microsomal and mitochondrial Ca uptake, whereas higher Pb concentrations (> 10 PM) increased Ca uptake (Suszkiw et al., 1984). Decreased mitochondrial and/or microsomal Ca uptake would be expected to increase intracellular ionized Ca. Clearly, the intracellular regulation of Ca is complex, and further studies will be necessary to explain the means by which Pb alters intraneuronal Ca homeostasis. In conclusion, the results of the present study confirm that Pb increases spontaneous neurotransmitter release in a similar fashion for several transmitter substances, and that this increase in release is correlated with a corresponding increase in 45Ca efflux. ACKNOWLEDGMENTS The authors thank Robert Greenland for his technical assistance, and Nancy Knapp for typing the manuscript.
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REFERENCES ALNAES, E., AND RAHAMIMOFF, R. (1974). Dual action of praseodymium (P?‘) on transmitter release at the frog neuromuscular junction. Nature (London) 247, 478-479. ALJDESIRK, G. (1985). Effects of lead exposure on the physiology of neurons. Prog. Neurobiol. 24, 199-23 1. AUGUSTINE, G., CHARLTON, M., AND SMITH, S. (1987). Calcium action in synaptic transmitter release. Annu. Rev. Neurosci.
10,633-693.
BAKER, P., AND CRAWFORD, A. (1975). A note on the mechanism by which inhibitors of the sodium pump accelerate spontaneous release of transmitter from motor nerve terminals. J. Physiol. 247,209-226. BALNAVE, R., AND GAGE, P. (1973). The inhibitory effect of manganese on transmitter release at the neuromuscular junction of the toad. Brit. J. Pharmacol. 47,339-352. BRADFORD, M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
72,248-254.
BRICGS. C., AND COOPER, J. (1981). A synaptosomal preparation from the guinea pig ileum myenteric plexus. J. Neurochem. 36, 1097- 1108. BROWN, R., PRENDIVILLE, P., AND CAIN, C. (1986). Alpha- 1-adrenergic and H 1-histamine receptor control of intracellular Ca 2c in a muscle cell line: The influence of prior agonist exposure on receptor responsiveness. Mol. Pharmacol. 29,53 l-539. CHEUNG, W. (1984). Calmodulin: Its potential role in cell proliferation and heavy metal toxicity. Fed. Proc. 43,2995-2999. COOPER,G., SUSZKIW, J., AND MANALIS, R. (1984). Presynaptic effects of heavy metals. In Cellular and Molecular Neurotoxicology (T. Narahash, Ed.) pp. I-2 1. Raven Press, NY. DEBASSIO, W., SCHNITZLER, R., AND PARSONS, R. (t97 1). Influence of lanthanum on transmitter release at the neuromuscular junction. J. Neurobiof. 2, 263278. GILMAN, S., KUMAROO, K., AND HALLENBECK, J. ( 1986). Effects of pressure on uptake and release of calcium by brain synaptosomes. J. Appl. Physiol. 60, 1446-1450. GOLDSTEIN, G., AND AR, D. (1983). Lead activates calmodulin sensitive processes. LifeSci. 33, 1001-1006. GRAY, E.. AND WHI~AKER, V. (1962). The isolation of nerve endings from brain: An electron microscope study of cell fragments derived by homogenization and centrifugation. J. Anat. 96,79-87. HABERMANN, E., CROWELL, K., AND JANICKI, P. (1983). Lead and other metals can substitute for Ca” in calmodulin. Arch. Toxicol. 54,6 l-70. HEUSER, J., AND MILEDI, R. (1971). Effect oflanthanum ions on function and structure of frog neuromuscular junctions. Proc. R. Sot. London B. 179,247-260.
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KAJIMOTO,N.,ANDKIRPEKAR,
AND
SYNAPTOSOMAL
%(1972).Effectofmanganese and lanthanum on spontaneous release of acetylcholine at frog motor nerve terminals. Nature New Biol. 235,29-30. KIRK, R. (1982). Experimental Design: Procedures for the Behavioral Sciences, 2nd ed. Brooks/Cole, Belmont, CA. KOBER, T., AND COOPER,G. (1976). Lead competitively inhibits calcium dependent synaptic transmission in the bullfrog sympathetic ganglion. Nature (London) 262,704-705. KOLTON, L., AND YARRI, Y. (1982). Sites of action of lead on spontaneous transmitter release from motor nerve terminals. Isr. J. Med. Sci. 18, 165-170. KOSTIAL, K., AND VOUK. V. B. (1957). Lead ions and synaptic transmission in the superior cervical ganglion of the cat. Brit. J. Pharmacol. 12,2 19-222. Kuo. C., ICHIDA, S.. MATSUDA, T., KAKIUCHI, S., AND YOCHISDA, M. (1979). Regulation of ATP-dependent Ca-uptake of synaptic plasma membranes by Ca-dependent modulatory protein. Life Sci. 125,235-240. LIN, S.. AND WAY, E. (1984). Calcium transport in and out ofbrain nerve endings in vitro-The role of synaptosomal membrane Ca’+-ATPase in Ca*+-extrusion. Brain Res. 298,225-234. MANALIS, R., AND COOPER, G. (1973). Presynaptic and postsynaptic effects of lead at the frog neuromuscular junction. Nature (London) 243,354-356. MINNEMA, D., AND MICHAELSON. I. A. (1985). A superfusion apparatus for the examination of neurotransmitter release from synaptosomes. J. Neurosci. Methods 14,193-206. MINNEMA, D., AND MICHAELSON, I. A. (1986). Differential effects of inorganic lead and b-aminolevulinic acid in vitro on synaptosomal y-aminobutyric acid release. Toxicol. Appl. Pharmacol86,437-447. MINNEMA, D., GREENLAND, R., AND MICHAELSON, I. A. (1986). Effects of in vitro inorganic lead on dopamine release from superfused rat striatal synaptosomes. Toxicol. Appl. Pharmacol. 84,400-4 I 1. NACHSHEN, D. (1984). Selectivity of the Ca binding site in synaptosome Ca channels: Inhibition of Ca influx by multivalent metal cations. J. Gen. Physiol. 83,94 l967. NECHAY, B., AND SAUNDERS, J. (1978). Inhibitory characteristics of lead chloride in sodium- and potassiumdependent adenosinetriphosphatase preparations derived from kidney, brain and heart of several species. J. Toxicol. Environ. Health 4, 147-l 59.
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NICHOLLS, D. (1986). Intracellular calcium homeostasis. Brit. Med. Bull. 42,353-358. POUNDS, J. ( 1984). Effect of lead intoxication on calcium homeostasis and calcium-mediated cell function: A review. Neurotoxicology 5,295-332. POUNDS, J.. AND ROSEN, J. (1986). Cellular metabolism of lead: A kinetic analysis in cultured osteoclastic bone cells. Toxicol. Appl. Pharmacol. 83,53 l-545. POUNDS, J., WRIGHT, R., MORRISON, D., AND CASCIANO, D. (1982). Effect of lead on calcium homeostasis in the isolated rat hepatocyte. Toxicol. Appl. Pharmac-01.63,389-40 1. Powrs, D. (1983). Cardiac glycosides and autonomic neurotransmission. J. Auton. Pharmacol. 3, 127-l 54. PUBLICOVER, S. (1985). Calmodulin, synchronous and asynchronous release of neurotransmitter. Camp. Biothem. Physiol. 82A, 7- 11. REICHARDT. L., AND KELLY, R. (1983). A molecular description of nerve terminal function. Annu. Rev. Biothem. 52,87 1-926. Ross, D.. AND CARDERAS, H. (1983). Calmodulin stimulation of Ca-dependent ATP hydrolysis and ATP-dependent Ca transport in synaptic membranes. J. Neurochem. 41, 161-171. SHELLENBERGER, M. (1984). Effects of early lead exposure on neurotransmitter systems in the brain: A review with commentary. Neurotoxicology 5, 177-2 12. SIEGEL, G.. AND FOGT. S. (1977). Inhibition by lead ion of electrophorus electroplax (Na+-K+)-adenosine triphosphatase and K+-p-nitrophenylphosphatase. J. Biol. Chem. 252,5201-5205. SIMONS, T. (1986). Cellular interactions between lead and calcium. Brit. Med. Bull. 42,43 l-434. SNELLING, R., AND NICHOLLS, D. (1985). Calcium efflux and cycling across the synaptosomal plasma membrane. Biochem. J. 226,225-23 1. SUSZKIW. J., TOTH, G.. MURAWSKY, M., AND COOPER, G. (1984). Effects of Pb*+ and Cd*+ on acetylcholine release and Ca*+ movements in synaptosomes and subcellular fractions from rat brain and torpedo electric organ. Brain Res. 323,3 l-46. SWEADNER, K. (1985). Oubain-evoked norepinephrine release from intact rat sympathetic neurons: Evidence for carrier-mediated release. J. Neurosci. 5, 23972406. THOMPSON, J., AND NECHAY, B. (198 1). Inhibition by metals of canine renal calcium, magnesium-activated adenosinetriphosphatase. J. Toxicol. Environ. Health 7,901-908.