Reversibility and Mode of Action of Black Widow Spider Venom on the Vertebrate Neuromuscular Junction A. G O R I O and A. M A U R O From The Rockefeller University, New York 10021. Dr. Gorio's present address is Fidia Research Laboratories, Abano Terme, Padova, Italy.

A B S T R AC T Black widow spider venom (BWSV) stimulates transmitter release and depletes synaptic vesicles from muscles bathed in a sodium free medium containing 1 mM EGTA. However, frog neuromuscular junctions treated with BWSV in glucosamine Ringer's and post-treated with antivenin recover normal function. This suggests that probably the p e r m a n e n t block of neuromuscular transmission is due to changes in permeability of the nerve e n d i n g plasma m e m b r a n e to cations such as Na +. When BWSV is applied in a medium lacking divalent cations and containing 1 mM EGTA, in most of the cases no effect is observed. We f o u n d that this inhibition can be overcome in three ways: (a) by adding divalent cations to the medium; (b) by increasing the tonicity of the medium with sucrose; (c) by raising the temperature of the medium. These results suggest that the lack of divalent cations influences the m e m b r a n e fluidity. Moreover, in view of the report by Yahara and Kakimoto-Sameshima (1977. Proc. Natl. Acad. Sci. U.S.A. 74:4511-4515) that hypertonic media induce capping of surface receptors in lymphocytes and thymocytes, we think that these data further support the hypothesis that BWSV stimulates release by a dual mode of action; namely, it increases the nerve e n d i n g permeability to cations and also stimulates release directly via a process of redistribution of m e m b r a n e components, a process which may also inhibit vesicle recycling. INTRODUCTION

When a crude homogenate of black widow spider venom (BWSV) glands is applied to a frog neuromuscular preparation, the miniature endplate potential (mepp) frequency rapidly increases several hundredfold. This rate of release is sustained for several minutes, after which it subsides and mepp's become rare, endplate potentials (epp's) cannot be evoked, and nerve endings are found to be depleted of synaptic vesicles (Clark et al.; 1970; Longenecker et al., 1970). While the mepp frequency is high, there is a corresponding increase in acetylcholine (ACh) release. Furthermore, after 1 h when mepp's are rare, neuromuscular transmission is blocked and vesicles are depleted; subsequent stimulation with a K+-rich solution does not affect the rate of mepp and of ACh release (Gorio et al., 1978 a). A protein, a-latrotoxin, of 130,000 mol wt, an isoelectric point ranging from pH 5.2 to 5.5 and without apparent lipolytic or proteolytic activity is responsible for all of these actions at the vertebrate neuromuscular junction J. GEN. PHYSIOL. 9 T h e Rockefeller University Press 9 0022-1295/79/02/0245-1951.00 V o l u m e 73

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(Frontali et al., 1976). T h e fact that a single protein was able to stimulate A C h release and block vesicle recycling was surprising and stimulated work to u n d e r s t a n d its m o d e o f action. Finkelstein et al. (1976) applied ct-latrotoxin to artificial black lipid m e m b r a n e s and observed a large increase in permeability to cations. T h e r e f o r e they suggested that the toxin, after binding to the nerve terminal plasma m e m b r a n e , increases the permeability to cations such as Ca ++ or Na +, leading to transmitter release. In addition, a provocative suggestion c o n c e r n i n g how a-latrotoxin stimulates release was made based on experiments which indicated that d u r i n g v e n o m action, a redistribution of the nerve terminal plasma m e m b r a n e c o m p o nents occur, which is similar to the patching and c a p p i n g of the surface receptors in lymphocytes when they are incubated with bivalent antibodies (Rubin et al., 1978). Later, it was shown that BWSV stimulates release and depletes synaptic vesicles in the absence o f both Na § and Ca ++, but it was also shown that the e n o r m o u s swelling o f the nerve endings, observed when large doses o f BWSV are applied, was absent if the m e d i u m contained glucosamine as a sodium substitute. This led to the suggestion that v e n o m has a double m o d e o f action: it increases the permeability of the nerve terminal plasma m e m b r a n e to cations, and it stimulates A C h release by a mechanism which may not be d e p e n d e n t on these permeability changes (Gorio et al., 1978 b). Using electrophysiological and morphological means, we have continued these studies to test the hypothesis o f BWSV dual m o d e o f action, and we believe that the experiments r e p o r t e d here reinforce this hypothesis. MATERIALS

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METHODS

Preparations All the experiments using pectoris nerve muscle preparations from small frogs, Rana pipiens, were performed at room temperature. Muscles were mounted in a lucite chamber, stretched over a lens and pinned down (Hurlbut and Ceccarelli, 1974). The nerve, dissected to the spinal cord, was drawn into two wells insulated with vaseline and stimulated with square pulses of 0.5 ms duration at an intensity three or four times the threshold for evoking muscle twitch. Muscles were usually mounted in Ringer's, and only preparations which twitched in response to nerve stimulation were used. Diaphragm neuromuscular preparations were quickly dissected from mice of 20 g weight, and mounted as described for the frog preparation. The solutions were changed by flushing the bath with the fluid contained in a suspended syringe.

Solutions The frog Ringer's solution consisted of 116 mM NaC1, 2.1 mM KCI, 1.8 mM CaCI2, and 6 mM Tris-HC1; the low-Ca++ Ringer's consisted of 112 mM NaC1, 2.1 mM KCI, 0.6 mM CaC12, 4 mM MgCI2, and 6 mM Tris-HC1, both at pH 7.2. D-Glucosamine was used as a sodium substitute in Na+-free Ringer's. Glucosamine was used as a sodium substitute in Na+-free Ringer's, inasmuch as glucosamine does not pass through venom-induced channels in artificial lipid membranes (Gorio et al., 1978 b). Approximately 7 mM KOH was added to the glucosamine solutions to obtain pH 6.5. At this pH glucosamine is about 90% ionized. For the recovery experiments, the Na+-free solutions consisted of 112 mM D-glucosamine-HCl, 4 mM MgCI2, and 6 mM Tris-HCl; the low-Ca ++ Ringer's was supplemented with 10 mM glucose and 100 p.M choline. BWSV antivenin was purchased

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from Merck Sharp & Dohme, West Point, Pa. The Ca++-free Ringer's aad the Ca++-free glucosamine Ringer's contained 1 mM EGTA and Mg ++ concentrations ranging from 10-s to 4 • l0 -3 M. EGTA was titrated at pH 7 with Tris-base. When K + was used as a Na substitute, the solutions consisted of 116 mM K-propionate, 4 mM MgCI~, 1 mM EGTA, and 6 mM Tris-HCl at pH 7.2. T h e Kreb's solutions for mouse diaphragms contained 142 mM NaCI, 8 mM NaHCO:~, 4 mM KHCO:), 1 mM NaH2PO4, 11 mM glucose, 1 mM Mg C12, and 2 mM CaClz. The divalent cations-free solution contained I mM EGTA. The glucosamine divalent cations-free Krebs' consisted of 150 mM glucosamine-HCl, 3 mM Tris-HC1, 10 mM glucose, and 1 mM EGTA at pH 6.5.

E lectrophysiology Electrophysiological studies were carried out using standard intracellular recording techniques with glass microelectrodes filled with 3 M KCI and with a resistance r a n g i n g from 10 to 30 M~. Records were displayed on a dual beam oscilloscope; one beam was AC-coupled at high gain to record mepp's and epp's and the other one was DC-coupled at low gain to record resting potentials.

Electron Microscopy Muscles were fixed in the recording chamber by draining the bath and filling it with fixative. Fixations were carried out with solutions consisting of 2% gluteraldehyde and 0.5% formaldehyde (freshly prepared from paraformaldehyde) in 0.1 M phosphate buffer pH 7.2 for 45 min. T h e n small pieces of muscle, suspected to be rich in neuromuscular junctions, were cut and put again into fresh fixative for 45 min. Successively the muscle bits were washed twice for 10 min each in phosphate buffer, and post-fixed in cold 1% osmium tetroxide in 0.1 M phosphate buffer for 90 min. T h e n the specimens were dehydrated in a graded series of alcohols and flat embedded in Epon 812 (Shell Chemical Co., New York). The pieces of muscles were oriented so that longitudinal or cross sections of nerve endings could be obtained. Semi-thin sections were cut with a glass knife, stained with toluidine blue and examined with a light microscope. When sections contained n u m e r o u s suspected neuromuscular junctions, serial thin sections were cut with a d i a m o n d knife, double-stained with uranyl acetate and lead citrate and examined in an electron microscope. Direct fixation with cold 2% osmium tetroxide, avoiding prefixation with aldehyde, was used in the experiments where K + was used as a Na substitute.

Black Widow Spider Venom Italian black widow spiders, Latrodectus mactans tredecimguttatus, collected outside of Rome, were decapitated and the cephalothoraxes kept frozen at -20~ When needed four cephalothoraxes were thawed, and the two glands from each of them were pulled out and homogenized in 1 ml of 120 mM Tris-HCl. T h e homogenate was stored in a cold room at 3~ Either 50 or 150/.LI of the crude homogenate was the standard dose used. When 150 gl was used, it will be indicated in the text. Since each venom gland contains about 2.5 #g of a-latrotoxin, the toxin concentration in the bath was 0.5 or 1.5 ~g/ml. Venom activity was routinely tested as described by Frontali et al. (1976). RESULTS

Effects of B W S V in Na +-Free, Ca ++-Free Medium I n the past we h a v e r e p o r t e d t h a t n e r v e t e r m i n a l s o f n e u r o m u s c u l a r p r e p a r a t i o n s s o a k e d i n a N a + - f r e e , Ca++-free m e d i u m f o r 75 m i n are well p r e s e r v e d a n d the vesicle p o p u l a t i o n is n o r m a l ( G o r i o et al., 1978 b). T h e a d d i t i o n o f 1 m M

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E G T A to this m e d i u m did not alter the result. Figs. 1 and 2 are electron m i c r o g r a p h s o f sections o f two nerve endings f r o m specimens which were bathed for 75 min in a glucosamine Ringer's containing 1 mM E G T A and 4 mM Mg ++. T h e terminals a p p e a r normal a n d contain a full c o m p l e m e n t o f synaptic

FIGURES 1 and 2. Electron micrographs of frog nerve endings (nt). The endings show a normal vesicle (v) distribution and clusters of mitochondria (mit). Where vesicles are absent cytoplasm is filled with glycogen. The preparation was fixed after a 75-rain incubation in a glucosamine-Ringer's containing 1 mM EGTA and 4 mM Mg++. (scn) Schwann cell nucleus; (m) myofibrils. Fig. 1: x 12,000. Fig. 2: x 16,500.

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FIGURES 3 and 4. Electron micrographs o f neuromuscular junctions from a preparation fixed after 60 minutes treatment with BWSV in glucosamine Ringer's containing 1 mM E G T A and 4 mM Mg ++. T h e endings (nt) are depleted o f synaptic vesicles. Many coated vesicles are present in Fig. 4. Arrows indicate smooth endoplasmic reticulum; (m) myofibrils. Fig. 3: x 8,000. Fig. 4: x 30,000.

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vesicles and mitochondria. T h u s incubation in Na+-free, Ca++-free m e d i u m containing 1 mM E G T A did not alter the structure o f the nerve endings. This is a sharp contrast to the observations made by Pumplin and Reese (1977) on preparations soaked for 120 rain in the same m e d i u m . T h e y observed a great loss of synaptic vesicles, which was n e v e r a p p a r e n t in o u r experiments. Inasm u c h as they used glucosamine Ringer's b u f f e r e d at p H 7.2, and inasmuch as at this p H a large a m o u n t o f glucosamine is not ionized, we favor the hypothesis that the u n c h a r g e d glucosamine could diffuse freely t h r o u g h the m e m b r a n e and create an osmotic imbalance which might have led to the observed loss o f vesicles. O u r solutions were at p H 6.5 (see Materials and Methods). Figs. 3 and 4 show sections o f nerve endings which were treated with BWSV when bathed in a glucosamine-Ringer's containing 1 mM E G T A and 4 mM Mg § Preparations were p e r f u s e d for 15-20 rain with this m e d i u m to eliminate most o f the Na + and then treated with BWSV. T h e nerve terminal is depleted o f synaptic vesicles, the mitochondria are fairly well preserved, and the swelling of the e n d i n g is limited, all these effects being characteristic o f the preparations treated with BWSV in Na+-free, Ca++-free m e d i u m (Gorio et al., 1978 b). This result is again in contrast with the observations made by Pumplin and Reese (1977), because when they applied brown widow spider v e n o m in Na+-free, Ca++-free m e d i u m , no depletion of synaptic vesicles was observed. T h e s e authors claim that brown widow and black widow spider venoms have a c o m m o n m o d e o f action, but perhaps this assumption deserves reexamination. T h e results were even m o r e striking when we looked at cross sections o f nerve endings f r o m control and treated preparations. We f o u n d that after soaking in glucosamine the nerve endings contained 94 +-- 31 vesicles per r a n d o m cross section, whereas v e n o m treated nerve endings contained only 4 + 3 vesicles.

On the Reversibility of BWSV Effects (a) In Fig. 5 we have a series o f electrophysiological records showing that a p r e p a r a t i o n treated with BWSV is able to recover and show n o r m a l n e u r o m u s cular transmission. A muscle fiber is impaled in low-Ca ++ Ringer's and epp's, evoked by indirect stimulation, are r e c o r d e d (trace A). T h e n the p r e p a r a t i o n is p e r f u s e d with a Na+-free, Ca++-free m e d i u m and no electrical activity can be r e c o r d e d since glucosamine is i m p e r m e a n t to the post-synaptic m e m b r a n e o f the frog n e u r o m u s c u l a r junction (trace B). After soaking for 20 min in this m e d i u m , BWSV is a d d e d and left for 1 h. T h e v e n o m is then washed out with the same m e d i u m and the p r e p a r a t i o n is treated with 50 gl of antivenin for 15 rain to inactivate the v e n o m molecules b o u n d to the plasma m e m b r a n e o f the nerve endings. After this t r e a t m e n t the p r e p a r a t i o n is r e t u r n e d to low-Ca ++ Ringer's, but no mepp's could be r e c o r d e d n o r epp's evoked by nerve stimulation. T h e resting potential d u r i n g the soaking in glucosamine Ringer's was approximately constant a r o u n d - 7 0 mV, because the m e d i u m contained 2 7 mM K (see Materials and Methods); u p o n r e t u r n i n g to Na + Ringer's the resting potential recovered to - 8 5 mV. At this stage of the e x p e r i m e n t n e u r o m u s c u l a r transmission is blocked; however, since we treated the preparation with antivenin there should be no v e n o m molecules actively attached to the nerve endings. T r a c e C of Fig. 5 shows two small mepp's r e c o r d e d 30 min after we r e t u r n e d the

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muscle to Na + Ringer's. With time, m e p p ' s b e c a m e m u c h m o r e f r e q u e n t . 1 h a f t e r the r e a p p e a r a n c e o f small m e p p ' s , e p p ' s could be e v o k e d by stimulation o f the n e r v e (trace D). T h e long soaking p e r i o d in a Na+-free m e d i u m c a n n o t account for the late a p p e a r a n c e o f the e p p ' s , because, in control e x p e r i m e n t s a f t e r a r e t u r n to Na + Ringer's, m e p p ' s a n d e p p ' s are r e c o r d e d in 2-3 rain a f t e r 75 min in a Na+-free Ringer's (Rubin et al., 1978). T h e m o r p h o l o g i c a l a n d electrophysiological aspect o f this recovery process is now u n d e r detailed examination. T o obtain a r e p r o d u c i b l e time for recovery to take place, it is very i m p o r t a n t to follow strictly the described p r o c e d u r e . I f antivenin is a d d e d a f t e r the p r e p a r a t i o n has b e e n r e t u r n e d to Na + Ringer's, the recovery time is very erratic a n d it m a y be as long as 6 h. F u r t h e r m o r e , muscles when b a t h e d in r e g u l a r Na + Ringer's a n d treated with BWSV very rarely show any sign o f recovery w h e n

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FIGURE 5. Oscilloscope traces from electrophysiological recordings of experiments on the reversibility of BWSV effects. (A) The frog muscle was impaled in low-Ca ++ Ringer's and epp's were recorded. (B) When glucosamine Ringer's is the bathing fluid no electrical activity can be recorded; see text for details. BWSV is applied for 60 min in this medium, then the preparation is treated with antivenin and successively returned to low-Ca ++ Ringer's. (C) After 30 rain few mepp's are recorded, and (D) only after 90 minutes epp's could be evoked. Arrows indicate miniature endplate potentials. Recording was done with AC coupling.

treated with antivenin. T h e r e f o r e , it seems that the p r e s e n c e or the absence o f Na + in the b a t h i n g m e d i u m d u r i n g the application o f BWSV is related to the reversibility o f the toxin effect. Since, in the past we had r e p o r t e d evidence that an increased p e r m e a b i l i t y to Na + i n d u c e d by the toxin was p r o b a b l y responsible for the swelling o f the n e r v e endings (Gorio et al., 1978 b), it seems reasonable to postulate that p e r h a p s the s a m e m e c h a n i s m leads to swelling a n d to p e r m a n e n t block o f the n e u r o m u s c u l a r transmission. In one single case a p r e p a r a t i o n r e c o v e r e d s p o n t a n e o u s l y f r o m the v e n o m t r e a t m e n t , namely, a f t e r a recovery time o f 6 h high f r e q u e n c y bursts o f m e p p ' s a n d e p p ' s could be r e c o r d e d . T h i s muscle h a d the peculiarity that the n e r v e endings were resistant to new applications o f BWSV. Even very large doses could not increase the m e p p f r e q u e n c y a n d block n e u r o m u s c u l a r transmission. In contrast, the n e r v e endings o f p r e p a r a t i o n s which r e c o v e r e d a f t e r the

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p r o c e d u r e described previously utilizing antivenin, were again sensitive to BWSV. I n this case it is likely that antivenin pulls away v e n o m molecules f r o m a r e c e p t o r which is now again available, whereas in the spontaneously r e c o v e r i n g n e r v e endings either an inactive v e n o m molecule is still b o u n d to the r e c e p t o r or the r e c e p t o r m a y no longer be p r e s e n t . (b) We have recently r e p o r t e d that the addition o f v e n o m to p r e p a r a t i o n s b a t h e d in isotonic K2SO4 gave results which were difficult to i n t e r p r e t (Rubin et al., 1978). Because the p r e p a r a t i o n was totally depolarized d u r i n g the e x p o s u r e to K +, the e x p e r i m e n t could not be m o n i t o r e d electrophysiologically while the toxin was applied a n d only w h e n the muscle was r e t u r n e d to Na + Ringer's could electrical activity be r e c o r d e d . Control p r e p a r a t i o n s b a t h e d for 60 min in K + Ringer's recover e p p ' s after a 30-60-min flushing with n o r m a l Ringer's. Extensive washing is necessary p e r h a p s because K + is t r a p p e d inside the p e r i n e u r i u m a n d it takes a long time to wash out. O n the o t h e r h a n d , muscles b a t h e d in this m e d i u m and t r e a t e d with BWSV for 60 min show, d u r i n g the recovery time, high f r e q u e n c y bursts o f m e p p ' s a n d a f t e r ~ 1 h some o f the muscle fibers show e p p ' s (Rubin et al., 1978). We c o n f i r m e d these data also using K - p r o p i o n a t e instead of sulfate, a n d a d d i n g 1 m M E G T A to the m e d i u m (see Materials a n d Methods). Only electron microscopic analysis o f these e x p e r i m e n t s could have revealed w h e t h e r v e n o m was able to deplete the n e r v e endings of synaptic vesicles d u r i n g the 1 h o f e x p o s u r e to the toxin. Fig. 6 is an electron m i c r o g r a p h o f a n e r v e e n d i n g f r o m a p r e p a r a t i o n which was soaked for 75 min in K § isotonic Ringer's containing 4 m M Mg +§ a n d 1 m M E G T A . Synaptic vesicles are clustered n e a r the active zones, but the rest o f the cytoplasm is almost devoid ot t h e m . T h i s is the general f e a t u r e o f the end-plates examined. Figs. 7 and 8 are m i c r o g r a p h s o f two sections o f the same n e r v e e n d i n g f r o m a p r e p a r a t i o n that, a f t e r a 15-min soaking in isotonic K + Ringer's containing 4 m M Mg § a n d 1 m M E G T A , was t r e a t e d for 60 min with BWSV. T h e entire n e r v e terminal including the areas n e a r the active zones is completely devoid o f synaptic vesicles, which is as e x p e c t e d if BWSV was able to act in spite o f the substitution o f Na § with K +. Interestingly, in the control e x p e r i m e n t s , high-K + solutions were able to stimulate vesicle release a n d to obtain a partial depletion in spite o f the p r e s e n c e of 1 mM EGTA.

FIGURE 6 (apposite). Neuromuscular junctions from a frog preparation fixed after a 75-min incubation in isotonic K+-Ringer's containing 1 mM EGTA and 4 mM Mg ++. The synaptic vesicle (v) population is very reduced and only near the active zones of the nerve terminal (nt) there are two large clusters of vesicles, x 30,000. FIGURES 7 and 8 (opposite). Electron micrographs of two sections of the same neuromuscular junction from a frog muscle fixed after a 60-min treatment with BWSV in isotonic K+ Ringer's containing 1 mM EGTA and 4 mM Mg ++. The nerve ending (nt) is depleted of synaptic vesicles. The arrow points to a mitochondrion. Fig. 7: x 20,000. Fig. 8: x 16,000.

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Divalent Cation Dependency of B WS V Action

We have shown b e f o r e that BWSV depletes synaptic vesicles in the p r e s e n c e o f 1 m M E G T A even in a s o d i u m - f r e e m e d i u m , h o w e v e r a small a m o u n t o f divalent cation (e.g., Mg ++) must be p r e s e n t .

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I f BWSV is applied to a p r e p a r a t i o n soaked in a m e d i u m containing 1 mM E G T A , and if no Mg ++ is a d d e d , in most instances no obvious changes in m e p p frequency is observed (see also O r n b e r g , 1977). U n f o r t u n a t e l y , when a muscle is bathed in this m e d i u m , s p o n t a n e o u s fibrillations occur a n d it is very difficult to keep a stable impalement. T o o v e r c o m e this p r o b l e m the muscles were tightly stretched over the lens situated in the center of the r e c o r d i n g c h a m b e r , and this greatly i m p r o v e d the impalements. O t h e r investigators have used tetrodotoxin at a concentration o f 2 • 10-6 g/ml ( O r n b e r g , 1977) to block excitability. After a 15-20 min soaking in this m e d i u m , we incubated the p r e p a r a t i o n with 150 ~1 o f BWSV for a n o t h e r 15 min. We used this h i g h e r dose o f v e n o m to optimize the response at low concentration o f Mg ++. T h e n Ringer's containing 1 mM E G T A and different concentrations o f Mg ++ were p e r f u s e d into the c h a m b e r . Fig. 9

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FIGURE 9. Series of curves showing the effects of different Mg ++ concentrations on the increase of mepp frequency induced by BWSV. Frog preparations were incubated for 15-20 rain with BWSV in a Ringer's lacking divalent cations and containing 1 mM EGTA. Then solutions containing Mg ++ and 1 mM EGTA were perfused into the recording chamber; the observed increase of mepp frequency was dependent on Mg++ concentration. With 10-SM (11) and 10-4M (A) Mg +*, the mepp frequency increased up to 150/s in a few minutes but when Mg++ was at concentrations of 1 mM (9 or 4 mM (Q) the increase was very abrupt. With 4 mM Mg++ the frequency increased abruptly several hundredfold and lasted only 4-5 min. shows the effect o f Mg ++ concentrations r a n g i n g f r o m 10-~ to 4 x l0 -3 M on the v e n o m - i n d u c e d m e p p discharge. With 4 mM Mg ++ the m e p p f r e q u e n c y increased abruptly several h u n d r e d f o l d but subsided within a few minutes. With lower concentrations o f Mg ++, the increase o f m e p p frequency was slower but lasted longer. I n all cases, after 1 h o f t r e a t m e n t with BWSV, the m e p p rate was very low, but in the case o f 0.1 mM Mg ++, if Mg ++ was raised to 4 raM, a burst of m e p p ' s lasting 20-30 s was always observed. Each point o f Fig. 9 is an average o f two experiments which were p e r f o r m e d with the same v e n o m gland

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h o m o g e n a t e . With d i f f e r e n t h o m o g e n a t e s we obtained series of curves with a slightly slower increase in the f r e q u e n c y o f m e p p ' s but with the same c h a r a c t e r istics. (A single e x p e r i m e n t was p e r f o r m e d at the 10 -5 M Mg ++ c o n c e n t r a t i o n a n d it is shown in Fig. 9.) Probably this d i f f e r e n c e is d u e to the d i f f e r e n t a m o u n t o f active oz-latrotoxin contained in each gland. In one e x p e r i m e n t a f t e r addition o f BWSV to a p r e p a r a t i o n b a t h e d in Ringer's with E G T A a n d no Mg ++, the m e p p f r e q u e n c y increased to a b o u t 150/ s. T o check if this increase was caused by s o m e calcium c o n t a i n e d in the c r u d e h o m o g e n a t e we p e r f u s e d the c h a m b e r with a solution containing 10 -~ M Mg ++ a n d 1 m M E G T A . T h e m e p p f r e q u e n c y c o n t i n u e d u n c h a n g e d for 10 min t h e n slowly subsided to low levels. In a few e x p e r i m e n t s we have even used cobalt as a divalent cation. T h e p r e p a r a t i o n was i n c u b a t e d with 150 /zl o f v e n o m in the absence o f divalent cations, a n d was then flushed with solutions containing 1 or 4 m M Co ++ a n d 1 m M E G T A . With 1 m M Co ++ the m e p p f r e q u e n c y in 10 rain gradually r e a c h e d the f r e q u e n c y o f a b o u t 150/s, which was sustained for 12 min then gradually subsided to 5-6/s. I f solutions containing 4 mM Co ++ a n d 1 mM E G T A were used, the m e p p sharply increased to 400/s, similar to the effect observed w h e n 4 m M Mg ++ a n d 1 m M E G T A were used.

Osmolarity and B W S V W h e n a n e u r o m u s c u l a r p r e p a r a t i o n is soaked in a h y p e r t o n i c m e d i u m the m e p p f r e q u e n c y increases by several fold and is sustained f o r several minutes, then it subsides a n d the n e r v e endings are depleted o f vesicles (Clark et al., 1970). I n T a b l e I we show the effects o f Con A a n d colchicine on the m e p p TABLE

I

EFFECTS OF CON A AND COLCHICINE

ON THE MEPP

FREQUENCY INDUCED BY 50% HYPERTONIC MEDIUM After application of Con A

Control

Dissolved in Ringer's

Dissolved in 1 M NaCI*

After application of colchicine and Con A

miniature endplate potentials per second

69---39 (14)

78-+20 (5)

5-+4 (5)

54-+30 (4)

Number in parentheses is number of trials. *The inhibitory action occurs only after Con A has first been dissolved in 1 M NaC|, a solution of relatively high ionic strength as compared with Ringer's. Undoubtedly, high ionic strength serves to enhance the solubility of Con A and, presumably, to minimize the tendency for Con A to aggregate. f r e q u e n c y i n d u c e d by h y p e r t o n i c Ringer's. I n these e x p e r i m e n t s sucrose was a d d e d to low-Ca ++ m e d i u m to increase the osrnolarity by 50%. W h e n a stable i m p a l e m e n t was o b t a i n e d a n d m e p p ' s r e c o r d e d , the h y p e r t o n i c Ringer's was then p e r f u s e d into the r e c o r d i n g c h a m b e r a n d 5 min later the m e p p ' s were r e c o r d e d . I n 14 e x p e r i m e n t s the rate was 69 -+ 39 m e p p ' s / s . T h e strategy o f the e x p e r i m e n t s was to r e a p p l y the h y p e r t o n i c m e d i u m a f t e r the p r e p a r a t i o n h a d

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been soaked for 1 h with 300/xg/ml o f C o n A. W h e n Con A was dissolved in 1 M NaCI a n d applied directly to the bath, the next increase in osmolarity failed to raise m e p p f r e q u e n c y to the previous level. H o w e v e r if Con A was dissolved in Ringer's and applied, no protection was observed (Table I). I n five experiments the m e p p frequency r e c o r d e d was 78 +- 20 per second. T h e application of Con A, even if dissolved in 1 M NaC1, does not affect the e p p amplitude and the K +induced release, as previously r e p o r t e d (see Rubin et al., 1978), W h e n preparations were treated, before application o f Con A dissolved in 1 M NaCI, with 10-4 M colchicine the response to the second application o f the hyperosmotic m e d i u m was almost normal, namely 54 -. 30 mepp's/s. It must be noticed also that BWSV action was p r e v e n t e d by Con A and that colchicine reversed the effect o f Con A (Rubin et al., 1978). Neither Con A nor colchicine had any striking effect on the resting m e p p frequency which was identical before and after treatment. In the previous sections we have described that when BWSV is applied to a p r e p a r a t i o n b a t h e d in divalent cation-free Ringer's containing 1 mM E G T A in most o f the cases no increase in m e p p frequency is observed. In addition, the rate o f m e p p s i n d u c e d by hypertonic m e d i u m lacking divalent cations decreased f r o m 69 - 39 to 18 +- 8/s (n = 4). However, if BWSV and the h y p e r t o n i c m e d i u m are applied together, a synergistic effect is observed (Misler and Hurlbut, 1978). In Fig. 10 we show three curves f r o m experiments p e r f o r m e d 450 4OO 350 3O0 250

E

2oo 150 lO0 5O

lO

20

30 40 Time in minutes

50

60

FIGURE 10. In this figure we show two examples of the effects of hypertonic medium on BWSV action. Frog preparations were bathed in Na+-Ringer's lacking divalent cations and containing 1 mM EGTA. When the same medium with the tonicity increased 50% (0) was flushed into the chamber, the mepp frequency rose to 18 +- 8 and was sustained for all the time of exposure. However, if BWSV was added to the soaking medium the perfusion with the hypertonic solutions gave a wide spectrum of mepp frequency increase, ranging from a slow rise to 100/s (&) to a very abrupt increase to - 400/s (m). See text for more details.

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with BWSV and h y p e r t o n i c m e d i u m . In these experiments preparations were bathed for 15-20 min in a m e d i u m lacking divalent cations containing 1 mM E G T A and then BWSV was a d d e d . After 15 rain the same Ringer's with osmolarity raised by 50% was flushed into the r e c o r d i n g c h a m b e r . We obtained a wide spectrum o f increase f r o m fiber to fiber e x t e n d i n g f r o m a slow rise in m e p p frequency to ]00/s which was sustained for - 10 rain, to a very a b r u p t increase to - 400/s which lasted a few minutes before subsiding. Temperature and B W S V

T h e mouse d i a p h r a g m was used to study the effect o f t e m p e r a t u r e on the action o f BWSV in the r a n g e o f 20-30 ~ C. T r e a t m e n t of m o u s e d i a p h r a g m s with BWSV leads to the same series o f events already described for f r o g n e u r o m u s cular junctions, namely, the m e p p f r e q u e n c y transiently increases and after 1 h the n e u r o m u s c u l a r transmission is blocked and the nerve endings are depleted o f vesicles (Gorio et al., 1978 a). T h e absence o f divalent cations f r o m the bathing m e d i u m inhibits v e n o m action at r o o m t e m p e r a t u r e 20-22 ~ C; however, at 36 ~ C n o r m a l v e n o m effect is observed. T o d e t e r m i n e the critical t e m p e r a t u r e , at which BWSV can trigger release in spite o f the absence o f divalent cations, we constantly p e r f u s e d the d i a p h r a g m for 20 min with a Krebs' lacking divalent cations and containing 1 mM E G T A . T h e n the flow was stopped and 50/zl o f BWSV a d d e d into the r e c o r d i n g c h a m b e r . After 15 min incubation the t e m p e r ature o f the bath was raised at a rate o f 0.33 ~ C per minute. In f o u r o f o u r f o u r experiments it was f o u n d that the critical t e m p e r a t u r e for the onset o f activity was between 30 and 31 ~ C. T h e increase in m e p p f r e q u e n c y could be relatively slow reaching a peak in 3-4 min or fast as shown in Fig. 11, where in 30 s the m e p p f r e q u e n c y rose f r o m < l / s to >400/s. I n one e x p e r i m e n t the t e m p e r a t u r e

S

0

2o

lmVi 0.5s

FIGURE I I. Oscilloscope traces from electrophysiological recordings of experiments on the effects of temperature and BWSV. Mouse diaphragm neuromuscular preparations were bathed at room temperature in a medium lacking divalent cations and containing 1 mM EGTA for 20 min. Then 50/zl of BWSV were added and after 15 rain the temperature rose. At 30.5 ~ C the mepp frequency increased in 30 s from a few seconds to >400/s. The slow fluctuation shown at time 0 s was probably due to muscle contractions induced by the lack of divalent cations. Recording was done with AC coupling.

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was raised at the rate o f 1~ C p e r m i n u t e until 32 ~ C was r e a c h e d , w h e r e u p o n the m e p p f r e q u e n c y j u m p e d f r o m a few to several h u n d r e d s p e r second without an i n t e r m e d i a t e increase. This b e h a v i o r suggested that all the release sites were activated simultaneously. T o exclude the possibility that an increase in permeability to Na + could be responsible for the v e n o m activity at h i g h e r t e m p e r a t u r e , some e x p e r i m e n t s were p e r f o r m e d using glucosamine as a s o d i u m substitute (see Materials a n d Methods). We p e r f u s e d the p r e p a r a t i o n for 20 rain at 36 ~ C with the glucosamine-containing a n d divalent cation-free Krebs', then we a d d e d v e n o m a n d allowed the p r e p a r a t i o n to cool down to r o o m t e m p e r a t u r e o v e r a p e r i o d o f 45 min. (In this p r o c e d u r e , v e n o m is in contact with the p r e p a r a t i o n at t e m p e r a tures above 30~ C for 25-30 min.) T h e n cold divalent cation-free, N a + Krebs' was p e r f u s e d into the c h a m b e r , w h e r e u p o n only few m e p p s were r e c o r d e d in t h r e e e x p e r i m e n t s ; m o r e o v e r , addition o f Ca ++ failed to increase m e p p frequency in three e x p e r i m e n t s . I n the control e x p e r i m e n t s BWSV was not a d d e d . I m m e d i a t e l y after the r e a p p e a r a n c e o f m e p p ' s in the divalent cation-free, Na + Krebs', n o r m a l Krebs' was r e i n t r o d u c e d into the r e c o r d i n g c h a m b e r a n d a few minutes later the BWSV was applied. A n o r m a l v e n o m effect was observed. T h e s e e x p e r i m e n t s showed that BWSV can stimulate release even in the absence o f Na + a n d divalent ions at h i g h e r t e m p e r a t u r e s . T h e s e observations were c o r r o b o r a t e d by a m o r p h o l o g i c a l e x a m i n a t i o n of the control and v e n o m - t r e a t e d p r e p a r a t i o n s , which, after attaining r o o m t e m p e r a ture were fixed a n d processed for electron microscopy. Fig. 12 is an electron m i c r o g r a p h o f a cross section of a n e r v e e n d i n g f r o m a control muscle which contains a good c o m p l e m e n t o f vesicles, whereas the v e n o m p r e p a r a t i o n shown in Fig. 13 is devoid of t h e m . DISCUSSION

F u r t h e r c o n f i r m a t i o n has b e e n o b t a i n e d that ct-latrotoxin, the c o m p o n e n t o f BWSV active at the v e r t e b r a t e n e u r o m u s c u l a r j u n c t i o n , depletes synaptic vesicles a n d blocks n e u r o m u s c u l a r transmission by a m e c h a n i s m which m a y not involve induced permeability changes. M o r e o v e r , there are strong suggestions that v e n o m m a y act by inducing a redistribution o f the m e m b r a n e c o m p o n e n t s , which results in a c h a n g e of the relationship between p l a s m a m e m b r a n e a n d cytoplasmic structures leading to vesicle fusion. BWSV can stimulate release even in N a +- a n d Ca++-free m e d i u m . As a Na + substitute we used o-glucosamine, which is i m p e r m e a n t to the postsynaptic m e m b r a n e a n d to the v e n o m - i n d u c e d channels in black lipid m e m b r a n e s . T h e reduction in size a n d d i s a p p e a r a n c e o f any electrical activity d u r i n g the introduction of glucosamine Ringer's into the r e c o r d i n g c h a m b e r can be used as a p a r a m e t e r to m o n i t o r the time-course o f the substitution o f Na + with glucosamine. I f Na + Ringer's is substituted with one which consists of 50% NaC1 a n d 50% glucosamine-HC1, the a m p l i t u d e o f m e p p ' s decreases to - 50% (Misler, 1976). I n o u r e x p e r i m e n t s electrical activity is abolished f r o m superficial fibers in < 1 rain o f p e r f u s i o n with Na+-free solution, indicating r a p i d elimination o f Na + f r o m the synaptic cleft. T h e r e f o r e , it seems clear that p e r f u s i o n for 15-20 rain is sufficient to eliminate most o f the Na + f r o m the

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e x t r a c e l l u l a r s p a c e o r at least to r e d u c e it to a c o n c e n t r a t i o n c o m p a r a b l e to t h a t within the cytoplasm. Because of the presence of EGTA and the very reduced c o n c e n t r a t i o n o f N a +, we c a n c o n c l u d e t h a t in this c o n d i t i o n v e n o m d o e s n o t s t i m u l a t e r e l e a s e b y i n c r e a s i n g C a ++ o r N a + e n t r y .

FIGURE 12. Section o f nerve ending from mouse d i a p h r a g m s fixed after having been incubated in glucosamine Krebs' lacking divalent cations. (See Results for details). T h e nerve terminal is well preserved and contains a good n u m b e r o f vesicles (v) and mitochondria (mit). x 17,500. FIGURE 13. Section of endplate from mouse d i a p h r a g m fixed after treatment with BWSV while incubated in glucosamine Krebs' lacking divalent cations (see Results for details). T h e nerve e n d i n g (nt) is depleted of vesicles and contains mitochondria (mit). • 10,000.

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It has b e e n suggested that an increase in cytoplasmic Na + concentration can displace Ca ++ f r o m intracellular stores leading to increased vesicle release (Lowe et al., 1976; Alnaes a n d R a h a m i m o f f , 1975). A n o t h e r suggested way to displace intracellular Ca ++ is to decrease intracellular K + (Baker a n d C r a w f o r d , 1975), which could be i n d u c e d by toxin channels in the presence o f N a substitutes. But we have shown that BWSV is able to deplete the endings o f synaptic vesicles even w h e n K + is substituting for Na + t h e r e f o r e , even this hypothesis should be discarded. O n the o t h e r h a n d , o u r evidence indicates that indeed an increase in n e r v e terminal permeability does occur d u r i n g BWSV t r e a t m e n t . N e r v e endings treated with large doses of v e n o m swell e n o r m o u s l y , but if Na + was substituted with glucosamine no swelling was o b s e r v e d (Gorio et al., 1978 b). F u r t h e r m o r e , it seems that the p r e s e n c e o f Na + in the bathing m e d i u m is strongly related to the irreversibility o f the v e n o m effect. All these data suggest that the swelling a n d irreversible block o f the n e u r o m u s c u l a r transmission are d u e to the s a m e m e c h a n i s m , n a m e l y , an increase o f the N a + permeability i n d u c e d by the toxin. T h e fact that m i t o c h o n d r i a o f n e r v e endings f r o m p r e p a r a t i o n s b a t h e d in a Ca++-containing m e d i u m a n d treated with BWSV are e n o r m o u s l y swollen also suggests that v e n o m increases the terminal Ca ++ permeability (Smith et al., 1977). Soaking in Ca++-containing Ringer's without t r e a t m e n t with the toxin does not change m i t o c h o n d r i a l s h a p e (Gorio et al., 1978 b), a n d m i t o c h o n d r i a are k n o w n to swell as a consequence o f Ca ++ uptake. A n o t h e r interesting aspect o f BWSV's m o d e o f action is the r e q u i r e m e n t for divalent cations in the b a t h i n g m e d i u m . I f v e n o m is a d d e d to a p r e p a r a t i o n b a t h e d in a m e d i u m lacking divalent cations a n d containing 1 m M E G T A , only on a few occasions was an effect observed. H o w e v e r , the addition to this m e d i u m o f 0.1 m M Mg ++, which c o r r e s p o n d s to a concentration o f a b o u t 5 • 10 -7 M free Mg ++ (Schmid a n d Reilly, 1957), consistently led to increased m e p p frequencies. I f the Mg +§ concentration in the bath is increased t h e r e is a parallel increase in m e p p frequency; with 4 m M Mg ++ the discharge is very vigorous a n d lasts only a few minutes. T h i s m u c h s t r o n g e r stimulation o b t a i n e d with 4 m M Mg § m i g h t be e x p l a i n e d by an influx o f Mg ++ into the n e r v e e n d i n g , which was suggested by H u r l b u t et al, (1971) to explain the Mg § d e p e n d e n c e o f the increase in m e p p f r e q u e n c y i n d u c e d by tetanic stimulation of the n e r v e in E G T A - c o n t a i n i n g m e d i u m . In a study recently c o m p l e t e d , Misler a n d H u r l b u t 1 obtained convincing evidence for the correlation of m e p p f r e q u e n c y with the electrochemical potential d i f f e r e n c e o f Mg ++ across the f r o g n e r v e terminal m e m b r a n e in p r e p a r a t i o n s treated with BWSV. T h u s , the results p r e s e n t e d h e r e a n d those o f O r n b e r g (1977) a n d o f Misler a n d H u r l b u t 1 s u p p o r t the hypothesis that o n e o f the m o d e s o f action of BWSV is to increase permeability o f the n e r v e t e r m i n a l to Mg ++ a n d also to o t h e r divalent cations. I n a previous r e p o r t we showed that if p r e p a r a t i o n s are b a t h e d in a m e d i u m containing low concentrations o f concanavalin A (Con A) T h e BWSV action is 1 Misler, S., and W. P. Hurlbut. Action of black widow spider venom on quantitized release of acetylcholine at the frog neuromuscular junction: dependence upon external Mg2+. Proc. Natl. Acad. Sci. U. S. A. In press.

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inhibited; however, if the preparation was preincubated with colchicine the full effect was observed (Rubin et al., 1978). This led to the suggestion that perhaps venom stimulates release and inhibits recycling of vesicles by a mechanism involving a redistribution of the membrane components similar to patching and capping of lymphocyte surface receptors exposed to multivalent antibodies. Yahara and Kakimoto-Sameshima (1977) have reported that surface receptors o f mouse lymphocytes or thymocytes redistribute into caps if exposed to a hypertonic medium o f - 600 raM. This effect is strongly inhibited by Con A, and the inhibition is reversed by colchicine. In Fig. 10 we show that BWSV inhibition caused by the absence of divalent cations can be reversed if the osmolarity of the medium is raised by 50%. These observations further suggest the involvement of a rearrangement of surface molecules in BWSV action. We feel that this correlation is significant. In this regard, we note that Con A is able to inhibit not only BWSV but also the increase of mepp frequency induced by a hyperosmotic medium which is also reversed by colchicine. I f divalent ions are absent both stimulatory effects are reduced, particularly, that due to BWSV. However, if applied together they have a synergistic effect. This suggests that perhaps in both cases the same basic mechanism is involved in stimulating release. A strong indication that the lack of divalent cations may interfere with the mobility of the membrane components comes from the experiments p e r f o r m e d with mouse diaphragms. In these experiments a higher temperature (30-31 ~ C) overcomes the block o f venom action induced by the lack of divalent ions at room temperature perhaps by increasing the membrane fluidity. T h e possibility, that the observed effect may be due to an increase o f Na + permeability is excluded by the electrophysiological and morphological experiments with glucosamine. T h e lack of Na § and divalent cations again suggest that the venom stimulatory effects may be mediated by a rearrangement o f membrane components. Singer (1976) has suggested that the clustering of receptor molecules induces a stress in the membrane which, through a critical series of changes, may activate an actomyosin-like system attached to the cytoplasmic face of the membrane. Following this contractile process an endocytosis will occur. Indeed it was reported that in lymphocytes the formation of patches and of caps always leads to the formation of subpatches or subcaps containing actin and myosin (Burguignon and Singer, 1977). Moreover, it was shown that during BWSV action vesicles fuse near the active zone (Ceccarelli et al., 1976) and that, from our preliminary autoradiographic studies, a-latrotoxin binds to the nerve endings at the active zones, z T h e r e f o r e we would propose that because of the nature of the active zones, which appear to be in a fixed position, contraction of actomyosin filaments could pull vesicles towards the plasma membrane, leading to exocytosis. CONCLUSION

Venom from Italian black widow spiders, Latrodectus mactans tredecigmuttatus, is able to stimulate release, to inhibit vesicle recycling and to block permanently 2 Gorio, A., M. C. Tzeng, and P. Siekevitz. Unpublished observations.

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t h e t r a n s m i s s i o n at f r o g n e u r o m u s c u l a r j u n c t i o n s by a d u a l m e c h a n i s m . B W S V i n c r e a s e s t h e p e r m e a b i l i t y o f t h e n e r v e e n d i n g p l a s m a m e m b r a n e to c a t i o n s ( e . g . , N a +, Ca++), b u t also m a y i n d u c e a r e d i s t r i b u t i o n o f t h e m e m b r a n e c o m p o n e n t s , w h i c h r e s u l t s in a c h a n g e o f t h e r e l a t i o n s h i p b e t w e e n p l a s m a m e m b r a n e a n d c y t o p l a s m i c s t r u c t u r e s l e a d i n g to vesicle f u s i o n . H o w e v e r , t h e s e two p h e n o m e n a o c c u r s i m u l t a n e o u s l y a n d t h e r e f o r e in s t a n d a r d c o n d i t i o n s ( n a m e l y , i f Ca ++ is p r e s e n t ) , s t i m u l a t i o n o f r e l e a s e m a y be e v o k e d by b o t h m e c h a n i s m s . I f a single v e n o m m o l e c u l e is a c t i n g s i m u l t a n e o u s l y in b o t h ways o r if t h e r e a r e two b i n d i n g sites, o n e n e a r t h e active z o n e to d i r e c t l y s t i m u l a t e r e l e a s e a n d t h e o t h e r o n e in l i p i d p a t c h e s o f t h e e n d i n g p l a s m a m e m b r a n e a c t i n g as a n i o n o p h o r e , m u s t still be d e t e r m i n e d . I t is h o p e d t h a t a u t o r a d i o g r a p h i c e x p e r i m e n t s in p r o g r e s s will give a n a n s w e r to t h e s e q u e s t i o n s . We gratefully acknowledge the assistance of Mrs. Lois Lynch, Mrs. Asneth Kloesman, and Mr. Roland Blischke with the electron microscopy. Dr. AlfYedo Gorio is a fellow of Muscular Dystrophy Associations of America. Received for publication 1 June 1978. REFERENCES

ALNAES, E., and R. RAHAMIMOFF. 1975. On the role of mitochondria in transmitter release from motor nerve terminals. J. Physiol. (Lond.). 245:285-306. BAKER, P. F., and A. C. CRAWVORD. 1972. Mobility and transport o f magnesium in squid giant axons.J. Physiol. 227:855-874. BOURGUIGNON, L. Y. W., and J. SINGER. 1977. On the mechanism of capping. J. Cell Biol. 75:(2,pt. 2)214 a. CZCCARELLI, B., D. PELUCHETTI, F. GROHOVAZ, and N. |EZZI. 1976. Freeze-fracture studies on changes at neuromuscular junctions induced by black widow spider venom. In Proceedings o f the 6th European Congress on Electron Microscopy. Biological Sciences. 2:376-378. CLARK, A. W., A. MAURO, H. E. LONGENECKER, JR., and W. P. HURLBUT. 1970. Effects of black widow spider venom on the frog neuromuscular junction. Nature (Lond.). 225: 703-705. FtNKELSTEIN, A., L. L. RtmIy, and M. C. TZENG. 1976. Black widow spider venom: effect of purified toxin on lipid bilayer membranes. Science (Wash. D.C.). 195:10091011. FRONTALI, N., B. CECCARELLI, A. GORIO, A. MAURO, P. S1EKEVITZ, M. C. TZENG, and W. P. HURLBUT. 1976. Purification from black widow spider venom of a protein factor causing the depletion of synaptic vesicles at neuromuscular junctions. J. Cell Biol. 68: 462-479. GORIO, A., W. P. I-[URLBUT, and B. CECCARELLI. 1978 a. Acetylcholine compartments in mouse diaphragm: a comparison of the effects of black widow spider venom, electrical stimulation and high concentration of potassium.J. Cell Biol. 78:716-733. GORIO, A., L. L. RUBIr,I, and A. MAURO. 1978 b. Double mode of action of black widow spider venom on frog neuromuscular j u n c t i o n . J . Neurocytol. 7:193-205. HURLBUT, W. P., and B. CECCARELLI. 1974. T r a n s m i t t e r release and recycling of synaptic vesicle m e m b r a n e at the neuromuscular junction. Adv. Cytopharmacol. 2:141-154. HURLBUT, W. P., ]-I. E. LONGENECKER, JR., and A. MAURO. 1971. Effects of calcium and

GORIO AND MAURO Dual Mode of Action of Black Widow Spider Venom

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magnesium on the fi-equency o f miniature end-plate potentials d u r i n g prolonged depolarization. J. Physiol. (Lond.). 219:17-38. LONGENECI(ER, H. E., JR., W. P. HURLUUT, A. MAURO, and A. W. CLARK. 1970. Effects of black widow spider venom on the frog neuromuscular junction. Nature (Lond.). 225: 701-703. LowE, D. A., B. P. RICHARDSON, P. TAYLOR, and P. DONATSCH. 1976. Increasing intracellular sodium triggers calcium release from bound pools. Nature (Lond.). 260: 337-338, MISLER, S. 1976. Cationic inhibition of the endplate potential: tests o f an electrostatic screening hypothesis. Ph.D. Thesis. New York University Medical Center, New York. MISLER, S., and W. P. HURLBUT. 1978. Effects of Mg ++ and osmotic pressure on black widow spider venom action. Biophys. J. 21:178 a. (Abstr.) ORNBERG, R. L. 1977. T h e divalent cation d e p e n d e n c e of spontaneous quantal release. Soc. Neurosci. 3:375. (Abstr.) PUMPLIN, D. W., and T. S. REESE. 1977. Action of brown widow spider venom and botulinum toxin on the frog neuromuscular junction examined with the freezefracture technique.J. Physiol. (Lond.). 273:443-457. RUBIN, L. L., A. GORIO, and A. MAURO. 1978. Effect o f concanavalin A on black widow spider venom activity at the neuromuscular junction: implications for mechanism of venom action. Brain Res. 143" 107-124. SCHMII), R. W., and G. N. REILLEY. 1957. New complexon for titration of calcium in the presence o f magnesium. Anal. Chem. 29:264-268. SINGER, S, J. 1976. T h e fluid mosaic model of m e m b r a n e structure: some applications to ligand-receptor and cell-cell interactions. In Surface Membrane Receptors. R. A. Bradshaw, W. A. Frazier, R. C. Merrel, D. I. Gottlieb and R. A. Hogue-Angeletti, editors. Plenum Publishing Corporation, New York. 1-24. SMIT~, J. E., A. W. CLARK, and T. A. KUSTER. 1977. Suppression by elevated calcium of black widow spider venom activity at frog neuromuscular junctions. J. Neurocytol. 6: 519-539. YAHARA, I., and F. Kal~IMOTO-SaMESHXMA. 1977. L i g a n d - i n d e p e n d e n t cap formation: redistribution of surface receptors on mouse lymphocytes and thymocytes in hypertonic m e d i u m . Proc. Natl. Acad. Sci. U.S.A. 74"4511-4515.