Journal of Ncurochrmistry. 1975. Vol. 25, pp. 483-496. Pergamon Press. Printed in Great Britain.

THE MECHANISM OF ACTION OF 0-BUNGAROTOXIN J. F. WERNICKE,’ A. D. VANKER’and B. D. HOWARD~ Department of Biological Chemistry, UCLA School of Medicine, and Molecular Biology Institute, University of California, Los Angeles, CA 90024, USA. (Received 5 Nonember 1974. Accepted 1 April 1975)

Abstract-B-Bungarotoxin, a presynaptically-acting polypeptide neurotoxin, caused an efflux from synaptosomes of previously accumulated y-aminobutyric acid and 2-deoxy-~-glucose.The toxin-induced efflux of y-aminobutyric acid occurred by a Na’-dependent process while that of 2-deoxyglucose was Na+-independent.These effects were also produced by treating synaptosomes with low molecular weight compounds, including fatty acids, that inhibit oxidative phosphorylation. After incubation with bbungarotoxin, synaptosomes exhibited increased production of l4COZ from [U-14C]glucoseand decreased ATP levels. b-Bungarotoxin treatment of various subcellular membrane fractions caused the production of a factor that uncoupled oxidative phosphorylation when added to mitochondria. Mitochondria from toxin-treated brain tissue exhibited a limitation in the maximal rate of substrate utilization. We conclude that b-bungarotoxin acts by inhibiting oxidative phosphorylation in the mitochondria of nerve terminals. This inhibition accounts for the observed P-bungarotoxin effects on synaptosomes and at neuromuscular junctions. We suggest that the effects on energy metabolism result from a phospholipase A activity found to be associated with the toxin.

P-BUNGAROTOXIN, which is a component of the venom of the snake Bungarus multicinctus, causes neuromuscular blockage by a presynaptic mode of action occurring in two stages (LEE& CHANG,1966; CHANCet al., 1973). An initial phase of increased rate of spontaneous acetylcholine release (increased miniature end-plate potential frequency) is followed by complete inhibition of nerve impulse-induced release of acetylcholine. The toxin has a mol. wt of 21,800 and consists of two subunits of mol. wt 8800 and 12,400 (KELLY& BROWN,1974). We have found that purified P-bungarotoxin also affects the storage of several putative neurotransmitters in mammalian brain in vitro (WERNICKE et al., 1974). The toxin is active when incubated with brain minces or synaptosomes and causes an efflux of GABA, norepinephrine and serotonin. The toxin also inhibits synaptosomal uptake of these compounds. Brain synapses are not disrupted by the toxin treatment. In this paper we give evidence that P-bungaroloxin acts by inhibiting oxidative phosphorylation in the mitochondria of nerve terminals. We suggest that this inhibition accounts for the observed P-bungarotoxin effects on synaptosomes and at neuromuscular junctions.

MATERIALS AND METHODS P-Bungurotoxin and enzymes

/I-Bungarotoxin and phospholipase A were purified by procedures reported previously (WERNICKEet al., 1974) from B. multicinctus venom, which was obtained from Miami Serpentarium (Miami, FL). Firefly luciferase, lysozyme, pancreatic ribonuclease, deoxyribonuclease and GIamylase were obtained from Worthington (Freehold, NJ). Chemicals

Radioactive chemicals were obtained from Amersham/ Searle (Arlington Heights, IL); tetrodotoxin from Calbiochem (La Jolla, CA); Ficoll, ouabain, sodium iodoacetate, egg yolk lecithin, bovine serum albumin, ADP and oleic and arachidonic acids from Sigma (St. Louis, MO); linoleic acid and sodium azide from Matheson, Coleman and Bell (Norwood, OH); 2,4-dinitrophenol and TMPD4 from Eastman (Rochester, NY); hyamine hydroxide from Packard (Downers Grove, IL) and Aquasol from New England Nuclear (Boston, MA). S-13 (5-chloro-3-t-butyl-2’-chloro-4 nitrosalicylanilide) was a gift from Dr. P. D. Boyer. Assay of /3-bungarotoxin toxicity

Toxin lethality was measured by injecting 2-fold serial dilutions of toxin into white mice intraperitoneally. For each dose, 4-8 mice were injected. The L D ~ , , is the dose that kills half the injected mice.

I Present address: Department of Neurology, UCLA, Preparation OJ synaptosomes Los Angeles, CA. Present address: Department of Biology, Georgia State Cerebral cortex from Sprague-Dawley rats (1 75-225 g) was homogenized in 0.32 M-sucrose and a crude synaptoUniversity, Atlanta, GA. To whom inquiries should be addressed. some-mitochondria1 fraction obtained as described (WERAhbreuiations used: TMPD, N,N,N’,N‘-trimethylphen- NICKE et a[., 1974). This fraction contained approx 3 W ylenediamine; EGTA, ethyleneglycol-bis-(P-aminoethyl 400 pg of protein per ml. For some experiments the synaptosomes were further purified by Ficoll density gradient ether) N,N’-tetraacetic acid. 483

484

J. F. WERNICKE, A. D. VANKERand B. D. HOWARD

centrifugation using conditions given (GOODKIN & HowARD,1974). Where indicated a quicker Ficoll gradient cen1972)was used. The Ficoll had trifugation method (VERITY, been deionized on AG 501-X8(D) resin (Biorad, Richmond, CA).

by addition of 5ml of 10% sucrose and filtration of the incubated sample on Millipore (AAWP) filters. The filters were washed, dried and counted as described in the preceding paragraph.

Mitochondria1 respiration Preparation of other subcellular fractions A 0.2 ml sample of a crude synaptosome-mitochondria1 Synaptosomal plasma membranes (COTMAN& TAYLOR, fraction was added to 2.5 ml of respiration buffer (20 rnM1972) and human erythrocyte membranes (DODGE et al., Tris-HC1, pH 7.4, 100~ M - K C I5, mM-KH2P04, 1 mM1963) were obtained by published procedures. Rat erythro- EGTA). Oxygen uptake was measured at 37°C in a Rank cyte membranes were prepared from cells that had been oxygen electrode. The effect of various treated subcellular fractions on liver washed twice in lOm~-Tris-HCl, pH 7.4, 0.9% NaCl by centrifuging at 2000g for 1 min. The washed pellet was mitochondrial respiration was measured as follows. A osmotically shocked in 0 5 m~-Tris-HCl, pH 8.5, for 0 3 ml sample of a suspension of an appropriate subcellular 20 min and then washed twice by centrifuging at 49,000 g fraction was incubated at 37°C with 2.0 ml of 10 rnM-Tnsfor 60min in the same buffer. The final pellet was resus- HCl, pH 7.4, 200 mhi-sucrose, 20 mM-NaC1, 0.2 mM-CaC1, pended in 10mmTrisHC1, pH 74, 0.9% NaC1. Rat liver with and without /3-bungarotoxin or B. multicinctus phosmitochondria were prepared in 0.35-M-sucrose, 10 mhf- pholipase A. After incubation 0 1 ml of the sample was Tris-HC1, pH 7.4, 0.5m-EGTA. A 10% homogenate of mixed with 2.5 ml of respiration buffer, 0.1 ml of 0.4 Mliver was obtained with a Teflon-glass homogenizer (clear- potassium succinate and 0.05ml of a suspension of liver ance 0.16-0.2 mm) at 4°C. The homogenate was centrifuged mitochondria (13-3 mg of protein), and the uptake of at 2000g for 1 min. The pellet was resuspended in 30ml oxygen was then measured at 37" with an oxygen electrode. of medium and recentrifuged. The supernatant suspensions ADP was added as indicated under Results. were combined, and centrifuged at 23,000 g for 4 min. The pellet was washed and centrifuged 3 times with 20ml of Lipid analysis of treated erythrocyte membranes A suspension of human erythrocyte membranes (7mg medium. The final pellet was rehomogenized in 4ml of medium. Except for the last resuspension, all procedures of protein) in 7 m~-sodiumphosphate, pH 7-4, 1 m-CaC1, was incubated with and without 8-bungarotoxin or B. mulincluded 0.1% fatty acid free bovine serum albumin. ticinctus phospholipase A at 37°C for 60 min. The volume Measurement of storage processes in treated synaptosomes of incubation was 1 ml. After incubation membrane lipids Cerebral cortex from one rat was placed in 2ml of were extracted as described (UTES, 1972) and separated 20 mM-Tris-HC1 @H 7.4), 120mM-NaC1, 3 mM-KC1, 3 mM- by TLC (SKIPSKI et al., 1962) on silica gel G plates with MgSO,, 2mM-CaC12 and cut into pieces of approx 0.5- a solvent system of methanol-chloroform-acetic acid1 mm dia. The tissue was incubated with shaking for 5 min water (65:25:8:4, by vol). The lipid spots were detected by at 37°C. Then [14C]GABA (228 mCi/m-mol) and 2-deoxy- spraying with DragendorTs (WAGNER et al., 1961) and nin['H]glucose (8.5 Ci/m-mol) were added to 0.14 p~ and hydrin (SKIPSKIet al., 1962) reagents. The spots were ana0.29 p~ respectively, and the incubation continued for lyzed quantitatively as described (ROUSER et al., 1966). 10min. During incubation the samples were gassed with a mixture of 95% 0,, 5% COz. After incubation each Enzyme assays sample was mixed with 10 ml of 0.32 M-SUCrOSe and centriLactate dehydrogenase (OBERJAT & HOWARD, 1973) and fuged at lo00 g for 5 min to remove the salts of the incuba- phospholipase A (SALACHet al., 1971) activities were meation buffer. The pellet was washed by recentrifugation at sured by published procedures. Where indicated we also loo0 g for 5 min in 0.32 M-sucrose. The tissue was homo- used two other phospholipase A assays. One was based genized and a crude synaptosome+mitochondrial fraction on the ability of the reaction product, lysolecithin, to lyse et al., 1974). A 02ml erythrocytes. The erythrocytes were obtained by centrifugobtained as described (WERNICKE sample of this fraction was added to 1.8 ml of buffer con- ing oxalated rat blood at lOOOg for 10min at 4°C. The taining 8-bungarotoxin, metabolic inhibitor or no additive. plasma and Buffy layers were discarded and the erythroThe mixture was incubated with shaking at 37°C for cytes were washed twice by centrifuging in 10 vol of 0.15 M10 min. After incubation 5 ml of 1004 sucrose was added NaC1, 10 m-glucose, 10 m-sodium phosphate (pH 7.0). and the mixture filtered on a Millipore filter (AAWP), The final erythrocyte pellet was resuspended in 25 vol of which traps the synaptosomes and the previously accumu- this buffer. The material to be assayed was added to lated radioactivity retained within. The filter was washed lecithin, which had been purified on an aluminum oxide with 15ml of 10% sucrose, dried and counted by liquid column and dispersed at 1 mg/ml by sonication in the scintillation spectrometry (WERNICKE et al., 1974). Unless Tris-salts buffer described for radioactively labelling brain indicated otherwise the incubation buffer was the same tissue. The sample was incubated with shaking at 37°C Tris-salts buffer used for labelling the brain minces. For for 30 min. An equal volume of the rat erythrocyte suspeneach condition of incubation triplicate samples were used. sion was added and the incubation continued for 30min. In some experiments we measured the effect of 8-bungaro- The sample was centrifuged at lo00g for 20min. The toxin or metabolic inhibitor on the uptake of hemoglobin content of the supernatant was determined by [I4C]GABA into synaptosomes. A crude synaptosome- absorbance at 540nm. mitochondria1 fraction was obtained from a homogenate For another phospholipase assay, the substrate was bacof cerebral cortex and a 0.2 ml sample was added to 1-8ml terial membranes in which fatty acids of the phosphoglyof the Tris-salts buffer described above. The sample was cerides were radioactively labelled. The membranes were incubated with shaking at 37°C for lOmin, [I4C]GABA obtained from log phase Bacillus megaterium, which had (228 mCi/rn-mol) was added to 0054 pi and the incuba- been cultured at 37°Cin glucose-minimalmedium (SPIZIZEN, tion continued for Smin. GABA uptake was terminated 1958) containing 0.5% yeast extract and incubated with

Mechanism of action of B-bungarotoxin [1-'4C]palmitic acid (0.04 pCi/ml) for 90 min before being harvested by centrifugation. The cells were washed 3 times with 5% sucrose, resuspended in 10 mM-Tris-HCI (pH 7.6), ~ ~ M - E D Tand A , lysed with Iysozyme (05mg/ml). The membranes were washed by centrifuging twice at 46,000 g for 20min and resuspending in the same Tris-EDTA buffer. After treatment with deoxyribonuclease (50 pg/ml) at 37°C for 5 min, the membranes were washed again, and dialyzed against 2 changes of 5Ovol of the same buffer containing 2pg of palmitic acid per ml. The membranes were centrifuged, resuspended in 20 rnoshl-sodium phosphate (pH 7.4) and stored frozen. The material to be assayed was added to the membranes (300pg of protein, 0.01 pCi of I4C) in 20mOsM-sodium phosphate (pH 7.4) 0.15 mM-CaC1,. The reaction mixture (0.15 mi) was incubated at 37°C for 60min with shaking. Lipids were extracted as described (Kates, 1972) and separated by TLC (SKIPSKI et al., 1962) on silica gel G plates with a solvent system of hexane-diethyl ether-acetic acid (60:40:1). The fatty acid spot was scraped and the radioactivity measured by scintillation spectrometry using toluene scintillation fluid.

a)

485

A

Other determinations

PROTEIN (LOWRYet al., 1951) and membrane bound phosphorus (ROUSER, 1966) were measured spectrophotometrically. Synaptosomal ATP was extracted by the method of BRADFORD (1969) and measured with the firefly luciferase-luciferin system (STREHLER & TOTTER,1954) using a scintillation counter to measure emitted light (STANLEY & WILLIAMS, 1962).

RESULTS

Purity and assay of P-burzgarotoxin The P-bungarotoxin preparation used in the initial experiments on synaptosomes and mitochondria was purified to the same extent as that used in the studies of LEE& CHANG(1966) and CHANGet al. (1973) and produced the same pharmacological effects (EYERNICKE et al., 1974). The preparation gave one band on polyacrylamide gel electrophoresis (40 pg of protein applied; electrophoresis system number 9 of MAURER,1971). After denaturation with sodium dodecyl sulfate and mercaptoethanol, two major bands were detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis (WEBER & OSBORN,1969). By comparison with protein standards, the mol. wt of the proteins in these bands were estimated to be in the 9-13,000 range determined for the P-bungarotoxin subunits (KELLY & BROWN,1974). There was also a third band containing a trace amount of protein estimated to be approx 22,000 in mol. wt. This third band likely was undissociated P-bungarotoxin. However, as shown in Fig. 1 (tracing b), the preparation used in the initial experiments was not homogeneous, giving 2 bands on cellulose acetate electrophoresis. The major band contains 95% of the protein as determined from densitometer scans. Effects on storage of 2-deoxy-~-glucoseand GABA

The data in our original report on 8-bungarotoxin

FIG.1. Densitometer tracings of cellulose acetate electrophoresis of crude B. multicinctus venom (tracing a) and purified 8-bungarotoxin (tracings b and c). Electrophoresis of the toxin (IOpg of protein) and crude venom (25pg of protein) were as described (LEEet a/., 1972) except that 005 M-sodium phosphate buffer, pH 7.4 was used. The toxin preparation of tracing b was used for the experiments on synaptosomes and mitochondria.

(WERNICKE et al., 1974) were not sufficient to determine whether the toxin induced neurotransmitter efflux from brain tissue via the same process that operates to release neurotransmitter when a nerve impulse reaches a nerve terminal. We therefore investigated the effect of P-bungarotoxin on synaptosomal storage of the non-neurotransmitter compound, 2deoxyglucose, which can be accumulated within synaptosomes by a high affinity uptake process (DIAMOND & FISHMAN, 1973). After incubation with P-bungarotoxin, the ability of synaptosomes prepared from rat cerebral cortex to retain previously accumulated 2-deoxyglucose was reduced (Table 1). The storage of 2-deoxyglucose was altered by P-bungarotoxin to the same extent as the storage of the neurotransmitter GABA, each being reduced by approx 30%. The activity of /I-bungarotoxin depended on the ionic composition of the buffer in which the synaptosomes were treated with the toxin. The effect of Pbungarotoxin required was partially inhibited by 15 mM-Mg2+ and completely inhibited by 15 mMM n 2 + (Table 1). The effects of Mg2+ and M n 2 + were not due to irreversible inactivation of the toxin; when p-bungarotoxin was incubated with 15 mM-MgCl, or ISmM-MnCl, for 15min at 37°C and then diluted and tested on synaptosomes in a M n 2 + free buffer containing 3 mM-Mg2+, it had full biological activity. The effect of 8-bungarotoxin on 2-deoxyglucose storage did not require the presencc of N a + (Table 1); i n repeat experiments the storage of 2-deoxyglucose

J. F. WERNICKE, A. D. VANKERand B. D. HOWARD

486

from other components of the crude synaptosomemitochondria1 fraction because earlier Ficoll density gradient centrifugation studies demonstrated that Additions Retention almost all the accumulated [14C]GABA in this fracTreatment to buffer 2-Deoxyglucose GABA tion was located within synaptosomes (OBERJAT & HOWARD, 1973). We have performed similar studies look6 look2 Control Complete that demonstrate that the accumulated 2-deoxy71 f 1 p-Bungarotoxin Complete 67 f 3 [3H]glucose is also in synaptosomes. When tested EGTA, on Ficoll-purified synaptosomes suspended in the omit CaZ+ 104f4 104f3 standard incubation buffer, fl-bungarotoxin produced 102-t 2 Omit Na' 53 f 2 86 & 2 15 mM-Mg2+ 86 f 8 the same effects on GABA and 2-deoxyglucose stor15mM-Mn2+ 109 7 108 f 6 age as demonstrated with a crude synaptosome-mitochondrial fraction. Under the conditions used for the Measurements of synaptosomal retention of GABA and experiments reported in Table 1, most of the 14C 2-deoxy-~-@ucose were made as described under Materials retained in control synaptosomes or lost from toxinand Methods. B-Bungarotoxin was at 2.5 p g t d and EGTA at 0 . 2 m ~ When . NaCl was omitted it was replaced by treated synaptosomes is associated with GABA (WER& HOWARD,1973). 20.24 M-sucrose to maintain osmolarity. The results are NICE et al., 1974; OBERJAT expressed as a percentage of radioactivity retained in tissue Deoxyglucose is metabolized only to 2-deoxyglucose & FISHMAN, 1973; SOLS& incubated in the same buffer in the absence of B-bungaro- 6-phosphophate (DIAMOND toxin. The values are means f S.D. for triplicate incuba- CRANE, 1954). We did not determine what proportion tions of particles from the same preparation. Variation of of the 'H was associated with 2-deoxyglucose or with buffer composition (absence of Ca2+or Na2+and presence 2-deoxyglucose phosphate. Although we refer to of EGTA, 15mM Mg2+ or 1 5 m Mn2+), in itself, had effects on the storage of radioactive 2-deoxyglucose, negligible effect on retention of GABA and 2-deoxyglucose. other studies @IAMOND & FISHMAN, 1973) indicate The average amount of radioactivity retained in control that, under the conditions used, most of the comtissue corresponded to 36 pmol of 2-deoxyglucose/mg of pound is actually phosphorylated. protein and 51 pmol of GABA/mg of protein.

OF B-BUNGAROMXM TABLE1. DEPENDENCE

ACTIVITY ON

BUFFER CATION COMPOSITION

was consistently reduced to a greater extent by 6bungarotoxin treatment in Na+-free buffer than it was in buffer containing 120m-Na'. However, treatment with fl-bungarotoxin in Na+-free buffer did not affect the storage of GABA. This dependence on Na+ for the toxin's activity on GABA storage apparently does not reflect a requirement for an influx of Na+ into the synaptosomes because in standard incubation buffer 9 pM-tetrodotoxin, a blocker of Na' channels (NARAHASHI et al., 1964), did not inhibit the activity of fl-bungarotoxin. For these experiments we used a crude synaptosome-mitochondria1 fraction obtained from cerebral cortex. It was not necessary to separate synaptosomes TABLE 2. COMPARISON OF EFFECTS

Effects of metabolic inhibitors The experiments shown in Table 1 demonstrate that the fl-bungarotoxin effects were not specific for neurotransmitter storage processes and suggested that fl-bungarotoxin acted by interfering with energy metabolism. We therefore investigated whether the 6bungarotoxin effects could be mimicked by low mol. wt inhibitors of energy metabolism. As shown in Table 2, 2,4-dinitrophenol at 0.1 m~ and sodium azide at 3 m caused ~ a reduction of synaptosomal content of GABA and 2-deoxyglucose in 120 mM-Na+ buffer but only of 2-deoxyglucose in Na+-free buffer. Ouabain at 0.1 m~ reduced the storage of GABA in 1 2 0 m Na+ buffer but did not affect the storage of 2-deoxyglucose. Iodoacetate at I m did not reduce

B-BUNGAROTOXIN

OF AND METABOLIC INHIBITORS ON SYNAPTOSOMAL RETENTION OF PREVIOUSLY ACCUMULATED GABA AND 2-DEOXYGLUCOSE

Retention GABA Treatment Control B-Bungarotoxin 2,4-Dinitrophenol Sodium azide Sodium iodoacetate Ouabain

Complete buffer

Omit Na+

1OOf 7 66 6 45 2 44+3 96 f 4 60 f 4

105 f 1 91 f 3 105 f 7 96f 1 102 f 3 10055

*

2-Deoxyglucose Complete buffer Omit Na+ 1OOf 6 17 6 76 f 2 76 f 2 102 3 103 3

**

1OOf2 57 f 1 78 4 79 f 2 101 f 1 97 f 1

*

Conditions are as described in Table 1. Treatment consisted of incubation with 2.5 pg of B-bungarotoxin/ml, 0.1 m ~ 2,4-dinitrophenol, 3 m-sodium azide, 1 mM-sodium iodoacetate or 0.1 mM-ouabain. The results are expressed as a percentage of radioactivity retained in control preparations incubated in complete buffer. The values are means f S.D. for triplicate incubations. The average amount of radioactivity retained in the controls corresponded to 12 pmol of GABA/mg protein and 39 pmol of 2-deoxyglucose/mg protein.

Mechanism of action of P-bungarotoxin

TABLE 3. EFFECTOF

P-BUNGAROTOXW ON

SYNAPTOSOMAL

ATP Treatment Control 8-Bungarotoxin KC1 (40mM)

nmol ATP/mg protein 3.27 & 0.86 1.41 0.27 3.33 090

*

A 0 2 ml sample of synaptosomes, purified as described (GOODKIN & HOWARD, 1974) and containing 2.4mg prolein, was incubated with 2.0ml of normal or high potassium medium for 4Smin at 37°C with shaking under an atmosphere of 95% O,, 5% CO,. For control and toxintreated samples, the medium was 20 mM-Tris-HC1, pH 7.4, 83 mM-NaC1, 74 mM-sucrose, 3 mM-KCl, 3 mM-MgSO,, 2 w-CaCl,, 10 mM-glucose. For the high potassium sample the medium was the same except that sucrose was omitted and KCI was 40mM. When present, P-bungarotoxin was at 5 pg/ml. After incubation ATP was extracted and measured as described under Materials and Methods. The values are the means _+ S.D. for 3 experiments.

the storage of either compound. Thus, the effects of P-bungarotoxin on synaptosomal storage processes are very similar to those produced by two inhibitors of oxidative phosphorylation, 2,4-dinitrophenol and

487

sodium azide, but not to those produced by ouabain (an inhibitor of Na', K+-ATPase) or iodoacetate (an inhibitor of glycolysis). This finding prompted an investigation of energy metabolism in toxin-treated synaptosomes. Effects on energy metabolism When synaptosomes were incubated with P-bungarotoxin, there was a substantial decline in synaptosoma1 ATP levels. In the experiments reported in Table 3, the amount of ATP in toxin-treated synaptosomes was 43% of that present in control synaptosomes, which had been incubated without P-bungarotoxin. In addition, as shown in Fig. 2, P-bungarotoxin caused a 150% increase in the rate of I4CO2 production by synaptosomes incubated with [U-'4C]glucose. Incubation of synaptosomes in 40 mM-KC1 medium, which causes synaptosomal depolarization and neurotransmitter release (DEBELLEROCHE & BRADFORD, 1972), did not alter the level of ATP and this treatment increased 14C02 production only slightly. Thus, the toxin-induced metabolic changes are not simply due to stimulation of the potassiuminducible neurotransmitter release process. It is known that uncoupled mitochondria exhibt increased substrate oxidation while ATP production is decreased. The possibility that the toxin-induced metabolic changes were due to uncoupling of oxidative phosphorylation led us to an examination of the effects of P-bungarotoxin on mitochondria1 respiration. EfSects on mitochondria

It is difficult to assess the respiratory activity of mitochondria treated directly with 8-bungarotoxin because Ca2+, which in itself causes uncoupling of oxidative phosphorylation (LEHNINGER et a/., 1967), is required for /I-bungarotoxin activity (CHANG et a/., 1973; WERNICKE et al., 1974). Consequently, incubation of brain or liver mitochondria with P-bungarotoxin in a buffer lacking Caz+ and containing EGTA, I I I I 15 30 45 60 a Ca2+ chelating agent, caused no alteration of mitoT I M E (rntn) chondrial respiration, and as expected, incubation in FIG.2. Effect of P-bungarotoxin on production of 14C02 the presence of Ca2+ caused uncoupling of oxidative from [U-'4C]glucose by synaptosomes. A 0.2 ml sample phosphorylation even without fi-bungarotoxin. of Ficoll-purified synaptosomes (GOODKIN & HOWARD, We thus examined the effects of P-bungarotoxin on 1974), containing 1.4 mg protein, was incubated with 1.8 ml of 3 mM-KC1 medium (for control and toxin-treated sam- mitochondria .by indirect procedures. As shown in ples) or 40mM-KC1. The compositions of the media are Fig. 3, P-bungarotoxin reacted with synaptosomes to given in Table 3. P-Bungarotoxin when present was at produce a factor that partially uncoupled oxidative 5 pg/ml. Each sample also contained 0.8 p~-[U-'~C]glu- phosphorylation when added to mitochondria. For cose (255 mCi/mmol). The samples were incubated at 37°C this experiment the synaptosomes were incubated in with shaking in 25 ml Erlenmeyer flasks fitted with serum a Ca2+-containing buffer with or without P-bungarocaps and plastic collection cups (Kontes) that contained toxin. Samples of the incubated synaptosomes were filter paper wetted with 0 2 ml of hyamine hydroxide. After added to a large excess of Ca2+ free, EGTA-containincubation, 0-2ml of ice-cold 1 M-trichloroacetic acid was ing medium in which mitochondria were suspended. injected into the mixture and the flask allowed to stand After addition of substrate (succinate), oxygen uptake in ice for 60 min. The plastic cups were cut off into 10 ml of Aquasol and the radioactivity measured by liquid scin- was measured with an oxygen electrode. Prior studies Control; -0. /%bun-. had shown that treatment of synaptosomes with 8tillation spectrometry. 0-0, 40 mM KC1. The data points are the bungarotoxin produced a factor that partially ungarotoxin; A-A, coupled oxidative phosphorylation in mitochondria means 2 S.D. for triplicate incubations.

L,

J. F. WERNICKE, A. D. VANKERand B. D. HOWARD

488

somes themselves was not detectable under the conditions used. The oxygen consumption of liver mitochondria incubated with toxin-treated synaptosomes in ADP-deficient medium was 85% greater than that of mitochondria incubated with control synaptosomes. Addition of ADP further increased the rate of respiration. The final rate was approximately the same as that exhibited by control mitochondria after ADP addition. Thus, addition of toxin-treated synaptosomes to liver mitochondria caused a partial loss of respiratory control. For the experiments described in Fig. 3 the respiratory control index (respiratory rate after addition of ADP divided by the respiratory I I I I rate before ADP addition) of mitochondria incubated 1 2 3 4 TIME (min) with toxin-treated synaptosomes was 2.8 while that of mitochondria incubated with control synaptosomes FIG. 3. Succinate-dependent oxygen uptake by liver mitochondria mixed with toxin-treated or control synapto- was 44. As shown in Fig. 4a the extent of uncoupling somes. Ficoll-purified (GOODKIN & HOWARD, 1974) synap- depended on the duration of incubation of synaptotosomes (0.5 mg of protein) were incubated without or with somes with toxin. The results illustrated in Fig. 4b demonstrate that 5 fig of j-bungarotoxin/ml for 90 min and were then added to liver mitochondria. The succinate-dependent oxygen liver mitochondria treated with B-bungarotoxin in the uptake was measured as described under Materials and presence of Ca2+uncoupled the oxidative phosphorylMethods. The arrow marks the addition of ADP (at ation of other liver mitochondria examined in the 1 . 7 5 ~ 1 ~to) the mixture. (a) control synaptosomes; (b) absence of Ca2+.As expected from the work of others toxin-treated synaptosomes. (WOJTZCAK & LEHNINGER, 19611, liver mitochondria incubated with Caz+ produced an uncoupling factor isolated from brain. It was easier to quantify this effect even in the absence of P-bungarotoxin but signifiwhen the test mitochondria were prepared from liver cantly less than that produced when the toxin was rather than brain because the liver mitochondria present. In each case illustrated in Fig. 4 the respiraremained more tightly coupled during the isolation tory control index (given in the parentheses next to procedure. The respiratory activity of the synapto- the data points) of the liver mitochondria decreased after the addition of toxin-treated membranes. The uncoupling activity of each toxin-treated preparation shown in Fig. 4 was eliminated by the presence of 0.1% bovine serum albumin in the respiration I buffer when oxygen uptake was measured. This find(4.6) a 0 " ing suggests that the toxin-induced uncoupling factor ( 4 41 I 1561 is a fatty acid. Fatty acids uncouple oxidative phosI I I I phorylation and bovine serum albumin protects ( 2.3 I against the uncoupling effect of fatty acids (WOJTZCAK & LEHNINGER, 1961), presumably because of its ability to bind fatty acids tightly ( E ~ Y F ~etRal., 1947). (7.0) 1531 b) Toxin

\

.

;

liGE.zz

1 '

(70)

1531 17

A second inhibitory effect

0

20

40

60

80

I00

120

In addition to producing a factor that uncouples oxidative phosphorylation, treatment with fi-bungaroFIG.4. Time-dependent production by 8-bungarotoxin of toxin causes another effect on mitochondria. This an uncouphng factor from (a) synaptosomes and (b) mito- second effect was detected by measuring the respirchondria. Ficoll-purified (VERITY, 1972) synaptosomes ation of a synaptosome-mitochondria1 fraction iso(0.5 mg of protein) or liver mitochondria (1.1 mg of protein) lated from brain minces that had been treated with were incubated without or with 5 jig of P-bungarotoxin/ml P-bungarotoxin. As shown in Fig. 5b the respiratory and were added to test liver mitochondria. The succinate- rate of a synaptosome-mitochondria1 fraction predependent oxygen uptake was measured as described un- pared from toxin-treated tissue increased after addider Materials and Methods. The rate of oxygen uptake tion of substrate (succinate) but did not further inby liver mitochondria is illustrated after addition of membrane preparations that had incubated with b-bungaro- crease after subsequent addition of ADP. Particles toxin M () and without b-bungarotoxin (M for ) from control tissue (Fig. 5a) responded to ADP with varying times. The values in parentheses next to the data a respiratory control index of 1.7.This toxin-induced points are the respiratory control indices of the liver mito- effectis not due to uncoupling of oxidative phosphorylchondria after addition of the toxin-treated or control ation because before ADP addition the substrate membranes. rate of oxidation in particles from toxin-treated tissue MEMBRANE INCUBATION TIME lrnml

Mechanism of action of p-bungarotoxin TABLE 4. RESPIRATORY RATE OF A SYNAPTOSOME-MITOCHONDRIAL FRACTION OBTAINED FROM TOXIN-TREATED AND CONTROL CEREBRAL CORTEX

Substrate 15 mM-Succinate

Treatment

Control Toxin 15 mM-Pyruvate Control Toxin 15 m~-Glutamate Control Toxin 15 mM-Ascorhate, Control 0.12 mM-TMPD Toxin

Respiratory rate* Substrate Substrate plus ADP 1.78 1.38 1-36 1.10 0.97 0.53 2.93 2.83

4.73 1.97 3.77 1.83 4.99 1.04 3.86 3.08

Rat cerebral cortex was incubated with or without pbungarotoxin, and a synaptosome-mitochondria1 fraction was prepared as described in the legend to Fig. 5. The respiratory rate of this fraction was measured as described under Materials and Methods. * pmol O,/min per 100 mg of protein.

was not greater than that of control particles. Thus, in this case the toxin treatment appears primarily to cause an alteration of the respiratory chain so as to decrease the maximal rate of substrate utilization. Consistent with this hypothesis is the finding that in the presence of succinate the uncoupling agents 2,4dinitrophenol at 0.02 mM and S-13 (5-chloro-3-tbutyl-2-chloro-4-nitrosalicylanilide)at 0-05PM did not increase the substrate-dependent respiratory rate of the particles from toxin-treated tissue, while the respiratory rate of control particles increased 2-fold after treatment with these agents. We have not located the respiratory chain site of this apparent toxin-induced limitation in the rate of substrate utilization. However, as shown in Table 4, we have also observed the effect when respiration was measured with pyruvate and glutamate, which are NADHlinked substrates, and with ascorbate using TMPD (JACOBS, 1960) as an electron transfer agent between ascorbate and the cytochrome-c region of the respiratory chain. In some experiments, before addition of ADP the respiratory rate of particles from toxintreated tissue was lower than that of control particles. The oxygen uptake observed with the crude mitochondrial fraction used for the experiments of Fig.

489

5 results from respiration by both the synaptosomes and the free mitochondria present in this fraction. We separated the synaptosomes and mitochondria by Ficoll density centrifugation and found that the maximal respiratory rate of each is decreased by toxin treatment in the manner described in Fig. 5 for the crude synaptosome-mitochondria1fraction. The majority of the mitochondria present in the preparation used for the experiment described in Fig. 5 come from neuronal and glial cell bodies rather than nerve terminals. We do not know whether treatment of brain minces with b-bungarotoxin alters essentially all the mitochondria in the brain minces before the tissue is homogenized. Alternatively, the toxin treatment may produce an inhibitory agent that alters most of the mitochondria during homogenization and subsequent handling of the tissue. Evidence for the latter mechanism comes from the finding that the crude mitochondria1 fraction from a homogenate of toxin-treated brain tissue eliminates the ADP response of control mitochondria when the two preparations are incubated together (Fig. 5c).

Phospholipase A assays We reported earlier (WERNICKE et ai., 1974) that our P-bungarotoxin preparation contained less than 3% of the specific phospholipase A activity of Vipera russelli phospholipase A. We also gave evidence that the effects of P-bungarotoxin were not due to contamination with the phospholipase A previously established to be present in B. rnulticinctus venom. Nevertheless, because of the results presented in Fig. 4 and the fact that the uncoupling could be eliminated by bovine serum albumin, we have reinvestigated, by more sensitive assays for the enzyme, the possibility that the effects of P-bungarotoxin were due to contaminating phospholipase A. The results presented in Table 5 demonstrate that with dispersed egg yolk lecithin as a substrate, the purified P-bungarotoxin contains less than 0.1% of the specific enzyme activity of the B. rnulticinctus venom phospholipase A. The activity of a mixture of P-bungarotoxin and phospholipase A was additive, so the P-bungarotoxin preparation does not contain a phospholipase inhibitor.

5. PHOSPHOLIPASE ACTIVITIES OF 8-BUNGAROTOXIN AND PHOSPHOLIPASE-A TABLE Assay 1

Protein None

Phospholipase A p-Bungarotoxin

Concentration (ng/ml) -

17 8

4 6700

Activity* 0.47 2.68 1.60 068 0.47

Concentration (ng/ml) -

250

100 50 100,000

Assay 2

Activity? 0.170

+ 0.01 1

0.860 f 0.160 0.458 f 0.031

0.198 0002 0.240 f 0.012

Assay 1 was as reported (SALACH et al., 1971).Assay 2 was as described under Materials and Methods. * ApH unit x 10' x min-'. t A,,, of incubated erythrocyte supernatant; values are means f mcan deviation for duplicate determinations.

J. F. WERNICKE, A. D. VANKERand B. D. HOWARD

490 c) Control +Toxin

7 TIME (rnin)

FIG. 7. Production by j-bungarotoxin and phospholipase A of uncoupling factor from erythrocyte and synaptosoma1 plasma membranes. Rat erythrocyte membranes and 2 4 6 8 1 0 TIME ( m i d synaptosomal plasma membranes each containing 37 pg of FIG.5. Oxygen uptake by a crude synaptosome-mitochon- phosphorus were incubated without or with 1 pg of 1-bundrial fraction prepared from toxin-treated and control cere- garotoxin/ml or 0.01 fig of phospholipase A/ml. The membral cortex. One-half of a rat cerebral cortex was minced brane preparations were then added to liver mitochondria in 2 0 ml of the TrisHCl buffer described in Table 3. The and the succinate-dependentoxygen uptake was measured as described under Materials and Methods. The rate of mince preparations were incubated without or with 5 pg mitochondria1 oxygen uptake is illustrated after addition of p-bungarotoxin/ml for 60 min with shaking at 37°C under an atmosphere of 95% 02,5% COz. After incubation of membrane preparations that had incubated with and without 1-bungarotoxin or phospholipase A for varying the tissue was washed by centrifugation, was homogenized, times. Erythrocyte membranes incubated with phospholiand a crude synaptosome-mitochondria1 fraction was prepase A (R-O), with 1-bungarotoxin (A-A), with no pared in the buffer and by methods described (VERITY, additive (M Synaptosomal ). plasma membranes incu1972). The respiration of this fraction was measured as bated with phospholipase A (m-m), with 1-bungarodescribed under Materials and Methods. The control with no additive (M). toxin (A-A), sample (tracing a ) contained 2.0 mg of protein, the toxintreated sample (tracing b) contained 2.2 mg of protein and the 1 :1 mixture of control and toxin-treated sample (tracing c) contained 2.1 mg of protein. The arrows mark the lipase A. Thus the P-bungarotoxin activity on synaptosomes cannot be attributed to phospholipase A addition of succinate at 14 mM or ADP at 1.75 mM. contamination. Nor can the production of a n uncoupling factor by P-bungarotoxin be attributed to such contamination. As shown in Fig. 7, both P-bungarotoxin and the phospholipase A produce a n uncoupling factor when incubated with erythrocyte and 100synaptosomal plasma membranes. Whereas the activity of the phospholipase A on erythrocyte mem80branes is, if anything, slightly greater than on synap-e tosomal plama membranes, /I-bungarotoxin has c c " greater activity on synaptosomal plasma membranes 's 60than o n erythrocyte membranes. Furthermore, as ac X W shown in Table 6, the activity of P-bungarotoxin on 40synaptosomal plasma membranes is approx 1% that 3 4 of phospholipase A. This is more than 10-fold greater m than the relative specific activities with dispersed 3 20lecithin as a substrate, The ability of P-bungarotoxin to produce a n uncoupling factor is a specific property of the toxin; neither pancreatic ribonuclease a t 5 pgJ ml nor pancreatic a-amylase a t 10 pg/ml produced an uncoupling agent when incubated with these membranes. Our B-bungarotoxin preparation is not wntaminated with the established venom phospholipase A, but it does have phospholipase A-activity. This activity was detected by analysis of the lipids extracted from human erythrocyte membranes that had been incubated with the toxin. As shown in Fig. 8 6-bungaro1 0

-

-

E

I

I

I

I

Mechanism of action of P-bungarotoxin

TABLE6. PRODUCTION

OF UNCOUPLING FACTOR FROM BY VARYING CONCENTRATIONS OF /~-BUNGAROTOXIN AND PHOSPHOLIPASE A

SYNAPTOSOMAL PLASMA MEMBRANES

Treatment

Dose (pg/ml)

Control

P-Bungarotoxin

10 2

Phospholipase A

0.1

0.02

0.01

Incubation time (min)

Respiratory rate*

0 60

3.81 3.99 4.61 12.6 13.7 9.8 10.7 12.4 12.9 5.6 8.3 4.98 7.5

100 60

100 60 100 60 100 60 100 60 100

As described under Materials and Methods, synaptosoma1 plasma membranes were incubated with j3-bungarotoxin, phospholipase A or neither for the times indicated. The membranes were added to liver mitochondria and the succinate-dependent oxygen uptake measured. * pmol OJmin per 100mg of mitochondria1 protein.

toxin catalyzes the production of lysophosphoglycerides under these conditions. Incubation as described for Fig. 8 resulted in the formation of 0.22p-101 of lysophosphatidylcholine and 0.21 pmol of lysophosphatidylserine plus lysophosphatidylethanolamineper mg of membrane protein. Lysophosphoglycerides were formed after incubation of human erythrocyte membranes with as little as 0.1 pg of fi-bungarotoxin per ml. The lysophosphoglycerides were not formed when Ca2+was omitted from the buffer during incubation with P-bungarotoxin or when the membranes were incubated with 10 pg of pancreatic ribonuclease per ml as a control. Further purijication of fi-bungarotoxin We further purified the toxin to determine which of the two protein fractions in our P-bungarotoxin preparation (Fig. lb) contained the neurotoxic and phospholipase activities. The purification involved the chromatographic procedures described (WERNICKE et al., 1974) except that the third column (CM-cellulose) was eluted with a 600 ml linear gradient of 0.3-0.7 Mammonium acetate (PH 5.G6.5). Under these conditions the elution curves of the major and minor protein component overlapped, but the minor component eluted slightly ahead of the major component. One fraction from the initial one-third of the material eluted from this column was kept and designated preparation A. Fractions containing the last one-third of the material eluted from this column were pooled, dialyzed against 0.05 M-ammonium acetate (PH 5.0) and rechromatographed on CM-cellulose as above. The last one-third of the material eluted was concentrated by lyophilization and desalted on a Sephadex

49 1

G-25 column. This more purified material was designated preparation B. The purified toxin gave a single symmetrical peak on Sephadex G-75 chromatography and one band on polyacrylamide gel electrophoresis; however, as shown in Fig. 1 (tracing c) 2 bands were detected on cellulose acetate electrophoresis. Densitometer scans revealed that the minor band contained 1% of the total protein. Preparation A contained the same proteins but the minor component was 17% of the total. We then compared the specific activities of preparations A and B with respect to toxicity (mouse lethality) and phospholipase A activity. If one or both of these activities resided only in the minor protein fraction, the respective specific activity of preparation A would be 17 times greater than that of preparation B. Toxicity studies revealed that the ~ 1 of) prep~ ~ arations A and B was 0 1 pg and 005 pg/g mouse, respectively. This difference is not significant. As shown in Table 7 the specific phospholipase-A activity of preparation B is slightly greater than that of preparation A. These studies demonstrate that both the phospholipase-A and toxin activities reside in the major protein fraction of preparations A and B. The contaminating protein fraction might also have fibungarotoxin activity since B. rnulticinctus venom apparently contains several P-bungarotoxin isozymes (LEEe f al., 1972).

SF

-

z

PE +

@

LPSand L P E 4

LPCn "

Q1D

ID

(ID

abed

FIG. 8. Thin layer chromatography tracing of lipids extracted from human erythrocyte membranes treated with P-bungarotoxin or phospholipase A. The membranes were incubated with (a) no additive, (b) /I-bungarotoxin (10 pg/ ml), (c) phospholipase A (10 pg/ml) or (d) phospholipase A (0.1 pg/ml). The lipids were extracted and chromatographed as described under Materials and Methods. The spots are: PE, phosphatidyl ethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine: LPS, lysophosphatidylserine; LPE, lysophosphatidylethanolamine; SPN, sphingomyelin; LPC, lysophosphatidylcholine. SF and 0 refer to solvent front and origin respectively.

J. F. WERNICKE, A. D. VANKERand B. D. HOWARD

492 7. PHOSPHOLIPASE A TABLE

ACTIVITIES OF p-BUNGAROTOXIN

TABLE9. RELEASEOF

LACTATE DEHYDROGENASE FROM SYNAPTOSOMES

PREPARATIONS CONTAINING VARYING AMOUNTS OF CONTA-

MINATING PROTEIN

Dose (pg/ml)

p-Bungarotoxin None Preparation A (17% contamination) Preparation B (1% contamination)

Phospholipase A activity*

-

600f 100

4 10 25

6100 f 650 9300 f 500 13,200 f 800

4 10 25

9500 f 800 12,800 f 600 14,100 f 300

As described under Materials and Methods, 8-bungarotoxin was incubated with bacterial membranes in which the phosphoglycerides were radioactively labelled. After incubation the free fatty acids were isolated and the radioactivity measured. The values are the means f mean deviation for samples incubated in duplicate. * Radioactive fatty acids formed (d.p.m./sample). Effects of fatty acids To determine whether the fatty acids liberated by /3-bungarotoxin treatment could reduce synaptosomal GABA storage we incubated a crude synaptosome-mitochondrial fraction obtained from a homogenate of cerebral cortex with various fatty acids and then measured the ability of the treated particles to accumulate GABA. As shown in Table 8, linoleic, arachidonic, , linolenic and oleic acids, each at 0.03 m ~significantly inhibited the accumulation of GABA by synaptosomes. The effect was more striking when the concentration of each fatty acid was 01 m ~ We . have

TABLE 8. EFFECT OF F

Treatment Control Oleate

Linoleate Linolenate Arachidonate

A ~ ACIDS Y ON SYNAPTOSOMES

GABA

UPTAKE INTO

GABA Uptake CalciumConcentration Complete free + 0.2 (mM) buffer mM-EGTA 003 0.1 0.03 0.1 003 0.1 0.03 01

100+3 39f3 20+3 34,l 9+2 34 f 7 3+8 42f 1 6f06

102f2 47+5 17k2 33k7 12+3 -

-

A crude synaptosome-mitochondria1 fraction was obtained and GABA uptake measured as described under Materials and Methods. The results are expressed as a percentage of GABA uptake by control tissue, which accumulated an average of 280pmol of GABA/mg protein. The values are means f S.D. for triplicate incubations of the same preparation.

Lactate dehydrogenase

Synaptosomes disrupted

Treatment

activity released*

(%I

Triton X-100 Linoleate Oleate Control

135 f 11 10.8 f 0.3 109 f 1.1 11.4 f 1.1

100 8.0 8.1 8.4

After purification on a Ficoll density-gradient (GOODKIN & HOWARD, 1974), synaptosomes were incubated as described under Materials and Methods for GABA uptake. The buffer contained 0.1% Triton X-100,0.1 mM-linoleate, 0 1 m-oleate or no additive. The samples were incubated at 37°C for 15min, chilled, centrifuged at 12,OOOg for 15min at 4°C and the supernatants assayed for lactate dehydrogenase activity. The values are the means f mean deviation for samples incubated in duplicate. * nmol Of substrate reacted/min per ml of supernatant solution. determined that the fatty acids do not inhibit GABA accumulation by disrupting the synaptosomes. This conclusion was reached by measuring the release of lactate dehydrogenase from synaptosomes treated with linoleic acid or oleic acid. Lactate dehydrogenase is a marker for cytoplasm occluded within synapto1968). The amount of lactate dehydsomes (FONNUM, rogenase released by Triton X-100 was taken to represent 100%synaptosome disruption since Triton X-100 destroys synaptosome plasma membranes under the conditions used (FISZER & DEROBERTIS, 1967). The results given in Table 9 indicate that neither 0.1 mlinoleic acid nor 0 1 m-oleic acid caused more disruption of synaptosomes than incubation without fatty acids. Incubation of synaptosomes with fatty acids also reduced the storage of newly accumulated 2-deoxyglucose. The ability of fatty acids to cause an efflux of GABA and 2-deoxyglucose was dependent on the ionic composition of the buffer in which the synaptosomes were treated (Table 10). In Na+-free buffer the fatty acid-induced efflux of GABA was reduced while that of 2-deoxyglucose was increased. Mn2' at 15 m~ inhibited the effects of the fatty acids. Thus the effects of Na' and Mn2+ on fatty acid-induced reduction of synaptosomal storage of GABA and 2-deoxyglucose were similar to the effects of these ions on /3bungarotoxin-induced reduction of GABA and 2deoxyglucose storage. However, unlike j-bungarotoxin, oleic and linoleic acids did not require Ca2+ to reduce synaptosomal storage of GABA (Table 8). The results shown in Tables 8 and 10 indicate that fatty acids had a greater effect on uptake of GABA than on storage of previously accumulated GABA.

Calcium accumulation The results of Tables 8 and 10 are consistent with the hypothesis that b-bungarotoxin acts by liberating

Mechanism of action of P-bungarotoxin OF TABLE10 EFFECT

FATTY ACIDS ON ABILITY OF SYNAPTO-TABLE 11. SOMES TO RETAlN PREVIOUSLY ACCUMULATEI) GABA AND

493

EFFECTOF

fi-BUNCAROTOXIN

ON

CALCIUM

UPTAKE BY SYNAPTOSOMES

2-L)EOXY-I)-QLUCOSE Treatment Control Oleate Linoleate

Additions to buffer Complete Complete Omit Na' 15 mM-Mn2+ Complete Omit Na' 15mM-Mn2+

Incubation time (min)

Retention GABA 2-Deoxyglucose 100 i 7 71 f 2 95 f 4 86 k 3

k7 95 f 6

68

79

14

Calcium uptake* Treatment Control P-Bungarotoxin

1Dof4 80 If: 3

2

14.3 f 1.0

5

60 6 98 f 1 7013

10 60

18.0 1.4 19.6 f 0.7 14.6 k 2.2

51 & 8

97 f 3

+

13.9 f 1.6 16-8 f 1.5 19.4 1.3 12.4 & 0.4

Synaptosomes were purified on a Ficoll density gradient (GOODKIN & HOWARD, 1974) and Ca" uptake measured as described under Materials and Methods for GABA

Conditions are as described in Table 1. Treatment con- uptake except that the buffer contained 0.18 pCi/ml sisted of incubation with 0.1 mwoleate or 0.1 mwlinoleate. 4 5 c a 2 f and the Ca2+ concn was 0.1 mM. The synaptoThe results are expressed as a percentage of radioactivity somes were incubated in this buffer for 2-60min in the retained in control tissue incubated in the same buffer. presence and absence of 2.5 pg/ml 8-bungarotoxin. The The values are means f S.D. for triplicate incubations values are the means S.D. for triplicate incubations. using tissue from the same preparation. The a v amount of * nm Of Caz+/mg of protein. radioactivity retained by control tissue corresponded to 63 pmol of GABA/mg of protein and 47 pmol of 2-deoxyglucose/mg of protein. 1965). The fact that, in Ca2' free, EGTA-containing buffer, P-bungarotoxin is slightly activated by 0.1 m ~ Sr2+ but not a t all by 2 m ~ - B a ~indicates + that Sr" fatty acids from some nerve-ending component and does not act simply by displacing endogenous Ca2+ that the Ca2+ requirement for P-bungarotoxin acti- from its complex with EGTA. The stability constants vity reflects this fatty acid-liberating activity. A Ca2+ for the 1:1complexes of EGTA with the cations mearequirement is a common characteristic of phospholi- sured at 2 0 T , are as follows (SCHWARZENBACH e t al., pase enzymes from various sources (GATT& BAREN- 1957): Ca2+ (log K = 10.97); Sr2+ (log K = 8.50); HOLZ, 1973). However, Ca2' could have a function Ba2+ (log K = 8.41). Therefore, Ba2+ at 2mM would in /%bungarotoxin activity in addition to its require- be expected to displace more Ca2+ from its complex ment for the liberation of fatty acids. P-Bungarotoxin- with EGTA than would Sr2+ at 0.1 m ~ However, . induced alteration of synaptosomal plasma mem- Ba2+ does not substitute for Ca2+while 0.1 mM-Sr2+ branes could cause an influx of Ca2', which then does. is sequestered by the mitochondria of the nerve terminals (KELLY& BROWN,1974). According to this hypothesis, the Ca2+, rather than the fatty acids, TABLE12. EFFECTOF THE SUBSTITUTION OF BARIUM OR STRONTIUM FOR CALCIUM ON THE ACTIVITY OF would be the main inhibitors of oxidative phosphorylP-BUNGAROTOXIN ation. Mitochondria have the ability to accumulate Ca2+ and oxidative phosphorylation becomes unTreatment Buffer GABA uptake et al., 1967). We coupled in the process (LEHNINGER have performed two experiments that indicate that p- Control Complete 100 f 5 bungarotoxin does not act by a mechanism involv- fl-Bungarotoxin Complete 32 f 2 ing CaZ+ influx. As shown in Table 11 there was Ca2+ free 103 f 3 no observable difference between the uptake of 45Ca2+ + 2 m Ba2+ ~ 102 f 4 +0.2mM Sr2+ 91 3 by control synaptosomes and by those treated with + 1 mM Sr2+ 81 f 5 P-bungarotoxin. Secondly and more importantly we 77 f 2 + 2 mM Sr" examined the ability of Baz+ and Sr2+ to substitute for Ca" in the toxin-induced reduction of GABA A crude synaptosome-mitochondria1 fraction was storage in synaptosomes. As shown in Table 12, Sr2+ partially substituted for Ca2+ but Ba2+ did not. If obtained and GABA uptake measured as described under p-bungarotoxin acts by causing mitochondria1 Ca2+ Materials and Methods. The Ca2+ free buffer contained accumulation, under the conditions used SrZ+ should 0.2 mM-EGTA. P-Bungarotoxin was at 2.5 pg/ml. The inhibit P-bungarotoxin, not activate it. Liver mito- results are expressed as a percentage of uptake by control tissue in complete medium. Variation in the buffer (omischondria can accumulate Sr2+ in addition to Ca2+, sion of Ca2+ or addition Of EGTA, BaZ+or Sr") in itself but after Sr2+ accumulation the mitochondria retain had negligible effect on GABA uptake. The values are normal respiratory control and oxidative phosphoryl- means f S.D. for triplicate incubations using tissue from ation remains coupled (LEHNINGER et a!., 1967). Fur- the same preparation. Control tissue in complete medium thermore, Sr2+ stabilizes the mitochondria against accumulated an average of 225 pmol of GABA/mg of prothe uncoupling effects of Ca2+ (CAPLAN & CARAFOLI,tein. ~~~~

*

494

J. F. WERNICKE, A. D. VANKER and B. D. HOWARD

treatment of mitochondria with Bungarus fasciatus DISCUSSION We have shown that P-bungarotoxin acts on synap- venom and has been attributed to the release of fatty et al., 1965). The tosomes to cause an efflux of newly accumulated acids from triglycerides (ZIEGLER GABA and 2-deoxyglucose. This activity of the toxin effect might also be caused by release of lysophosis Ca*+-dependent. The efflux of GABA also requires phoglycerides since lysolecithin has been reported to Na+, but the efflux of 2-deoxyglucose does not. These depress mitochondrial oxidation of succinate and 8et al., 1957). effects are also produced by low mol. wt inhibitors hydroxybutyrate (WITTER Because the metabolism of neuromuscular junction of oxidative phosphorylation indicating that B-bungarotoxin acts by interfering with synaptosomal nerve terminals cannot be studied apart from other energy metabolism. This hypothesis is supported by accompanying tissue, e.g. muscle, the types of the finding that the toxin caused a decrease in synap- measurements reported here could not be made at tosomal ATP levels and inhibition of mitochondrial neuromuscular junctions. Nevertheless, we believe oxidative phosphorylation. Incubation of fl-bungaro- that the ability of P-bungarotoxin to inhibit oxidative toxin with synaptosomal or erythrocyte membranes phosphorylation accounts for all of the known pharproduced a factor that partially uncoupled oxidative macological effect of the toxin at neuromuscular juncphosphorylation when added to mitochondria. A tions. Our hypothesis on the mechanism of action second effect on mitochondria could also be observed, of 8-bungarotoxin is made more attractive by the i.e. a toxin-induced limitation in the maximal rate of finding that other low mol. wt uncoupling agents, e.g. 2,4dinitrophenol, cause effects at neuromuscular electron transport. Analysis of lipids extracted from toxin-treated junctions very similar to those produced by 8-bungarohuman erythrocyte membranes demonstrated that the toxin, i.e. increased miniature end-plate potential a-bungarotoxin preparation has phospholipase A frequency and subsequent blockade (KRAArz & et activity. The present studies suggest but do not defini- TRAUTWEIN,1957; BEANIet al., 1966; GLAGOLEVA tively establish that the neurotoxic and phospholi- al., 1970). One consequence of the action of uncouppase activities reside on the same protein; however, ling agents on mitochondria is an efflux of Ca2+ et al., 1965). Thus, the simplest explanation of the inhibition of oxidative from the mitochondria (DRAHOTZ phosphorylation by fl-bungarotoxin is that it results by uncoupling mitochondria in nerve terminals, flfrom this phospholipase A activity. The phospholi- bungarotoxin and the other agents would increase pase likely liberates fatty acids from some membrane cytosol Caz+ levels, which could account for the inof the synaptosomes. The fatty acids in turn cause creased spontaneous rate of quanta1 acetylcholine inhibition of oxidative phosphorylation. Consistent release observed before blockade. Calcium ions are with this interpretation is the finding that fatty acids required for the neurotransmitter release process react with synaptosomes to produce the same effects (RUBIN, 1961) and may activate it. The transient inon GABA and 2-deoxyglucose storage as those pro- creased rate of acetylcholine release may also be duced by treatment with 8-bungarotoxin. caused by partial depolarization of nerve terminals We believe that the fatty acids liberated by P-bun- as a result of a direct effect of the toxin-induced ungarotoxin treatment were sufficient to account for the coupling factor on the presynaptic plasma membrane. uncoupling effects observed in Fig. 3 and 4. While 2,4Dinitrophenol appears to decrease the resting we have not directly measured the amount of fatty potential of excitable membranes by directly altering acid liberated, this value was calcdlated from the the membranes ( m n& WUTWEIN, 1957; ABOOD amount of lysophosphoglycerides formed by treat- et aE., 1961). The second stage of the P-bungarotoxin ment of erythrocyte membranes with 6-bungarotoxin activity, neuromuscular blockade, would be caused by (Fig. 8). Addition to liver mitochondria of erythrocyte a depletion of energy stores, a consequence of the membranes that had been treated with b-bungaro- uncoupling activity. The other effect of the toxin on toxin as described for Fig. 8 caused the mitochondria mitochondria, limitation of electron transport, would to become partially uncoupled. The lipids extracted also contribute to decreased energy stores. The from the treated membranes contained a total of latency for toxin-induced neuromuscular blockade is 2.9 p o l of lysophosphoglycerides; thus 2.9 pnol of shortened if the nerve to the muscle is repeatedly fatty acids were liberated. This results in a fatty acid stimulated during toxin treatment (CHANGet al., concentration of 4 p or ~ 7pnol/g of mitochondrial 1973). Such repeated stimulation might simply acceprotein in the oxygen electrode when mitochondrial lerate the rate at which nerve terminal energy stores respiration was measured. Oleic acid at 0.4 ,m causes are depleted. mitochondrial swelling (WOJTZCAK & LEHNINGER, We do not know whether, in vivo, 8-bungarotoxin 1961) and stimulation of mitochondrial ATPase can acts at the external surface of the nerve terminal be detected after incubation with 1 5 p o l of oleic plasma membrane or enters the nerve terminal and acid/g of mitochondrial protin (J~ORST et al., 1962). acts on mitochondria directly. Entrance could be The phospholipase A activity in the P-bugarotoxin accomplished by the pinocytosis process that has preparation likely also produces the toxin-induced been postulated to occur at nerve terminals during limitation of the maximal rate of mitochondrial res- synaptic transmission (HUBBARD, 1973). One other piration. A similar effect has been observed after foreign protein (horseradish peroxidase) is believed to

Mechanism of action of P-bungarotoxin

enter nerve terminals by this means (ZACKS & SAITO, 1969; HEUSER& REESE, 1973). Such a mechanism would offer another explanation for the decreased latency of toxin-induced neuromuscular blockade when the nerve to the muscle is stimulated repeatedly. With repeated stimulation more P-bungarotoxin would enter the terminal thereby increasing the rate at which mitochondria became inhibited. In addition, entrance of P-bungarotoxin into nerve terminals by a pinocytosis process linked to neurotransmitter release would explain the toxin’s apparent in vivo specificity for nerve terminal mitochondria. The toxin would not have access to the mitochondria of other tissues that do not undergo pinocytosis in this manner. Pharmacologically, the consequences of neuromuscular blockade would predominate and mask any effects caused by occasional pinocytosis by other types of cells. The difficulty with which the phospholipase activity associated with P-bungarotoxin is measured may simply reflect our ignorance of what is the proper membrane substrate. We do not know on what membrane of nerve-endings the phospholipase acts. Our purified synaptosomal plasma membrane preparation may not contain a sufficient amount of the proper membrane substrate for optimal activity; furthermore, maximal activity may only be obtained with membranes from neuromuscular junction nerve-endings. The specificity of this phospholipase for membranes and the apparent preference for neuronal membranes (Fig. 7) merit additional study. It should be noted that this property of the phospholipase is not unique to this enzyme; other phospholipases are known to exhibit differing specifcities for various classes and physical states of phospholipids (GATT & BARENHOLZ, 1973). Botulinum toxin and apparently tetanus toxin act presynaptically to inhibit neurotransmitter release (BROOKS, 1956; CURTIS& DEGROAT, 1968). The mechanism of action of these toxins may also involve depletion of energy stores in nerve endings. Acknowledgeiizents-We thank RlCHAKU L. NORMAN for preparing synaptosomal plasma membranes and Drs. PAULD. BOER and GEORGE J. POPJAKfor useful discussions. Research supported by National Institutes of Health Grant MH 21875. Note added in proof: We recently have been able to measure the phospholipase A activity of the P-bungarotoxin preparation using a pH stat and dispersed lecithin as a substrate. REFERENCES ABOODL. G., KOKETSU K. & NOUAK. (1961) Am. J . Pharmac. 20, 431436. BEANIL., BIANCHI C. & LEDDAF. (1966) Br. J . Pharmac. Chemothrr. 27, 299-312. BRADFORD H. F. (1969) J . Neurochem. 16, 675684. BROOKS V. B. (1956) J. Physiol. 134, 264277. BORSTP., Loos J. A,, CHRISTE. J. & SLATERE. C. (1962) Eiochim. biophys. Acta 62, 509-518. BOYERP. D., BALLOU C. A. & LUCK1. M. (1947) J . biol. Chent. 167, 407-424.

495

CAPLAN A. I. & CARAFOLI E. (1965) Biochinz. biophys. Acta 104, 317-329. CHANGC. C., CHENT. F. & LEEC. Y. (1973) J. Pharmac. exp. Ther. 184, 339-345. GANG C. C., HUANGM. C. & LEEC. Y. (1973) Nature, Lond. 243, 166-167. COTMANC. W . & TAYLOR D. (1972) J. Cell Bid. 55, 696711.

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