Nicotinic cholinergic receptor in brain detected by binding of α-[3H]bungarotoxin

346 Biochimica el Biophysica Acta, 3 6 2 ( 1 9 7 4 ) 3 4 6 - - 3 5 5 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA

27479

N I C O T I N I C C H O L I N E R G I C R E C E P T O R IN B R A I N D E T E C T E D BY B I N D I N G O F a - [ 3 H] B U N G A R O T O X I N

VESNA A. ETEROVIC and EDWARD L. BENNETT Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley, Calif. 94720 (U.S.A.) (Received February 21st, 1974)

Summary a - [ 3 H ] B u n g a r o t o x i n was p r e p a r e d b y c a t a l y t i c r e d u c t i o n of i o d i n a t e d a - b u n g a r o t o x i n w i t h t r i t i u m gas. Crude m i t o c h o n d r i a l f r a c t i o n f r o m r a t cerebral c o r t e x b o u n d 40 • 10 -1 s _ 6 0 • 10 -1 s m o l e s o f a-[ 3H] b u n g a r o t o x i n per m g of p r o t e i n . This binding was r e d u c e d b y 50% in t h e p r e s e n c e of a p p r o x . 10 -6 M d - t u b o c u r a r i n e or n i c o t i n e , 10 -s M a c e t y l c h o l i n e , 10 -4 M c a r b a m y l c h o l i n e or d e c a m e t h o n i u m or 10 -3 M a t r o p i n e . H e x a m e t h o n i u m a n d eserine were t h e least effective o f the drugs tested. Crude m i t o c h o n d r i a l f r a c t i o n was s e p a r a t e d into m y e l i n , nerve endings, and m i t o c h o n d r i a . T h e highest b i n d i n g o f t o x i n per m g o f p r o t e i n was f o u n d in nerve endings, as well as t h e greatest i n h i b i t i o n o f t o x i n binding b y d - t u b o c u r a r i n e . Binding of a-[ 3HI b u n g a r o t o x i n to m e m b r a n e s obtained b y o s m o t i c s h o c k o f t h e c r u d e m i t o c h o n d r i a l f r a c t i o n indicates t h a t t h e r e c e p t o r for t h e t o x i n is m e m b r a n e b o u n d . 12 Si.Labeled a - b u n g a r o t o x i n , prep a r e d w i t h Na I 2 s I and c h l o r a m i n e T, was highly specific f o r t h e a c e t y l c h o l i n e r e c e p t o r in d i a p h r a g m , h o w e v e r , it was less specific and less reliable t h a n a[ 3H] b u n g a r o t o x i n in brain. We c o n c l u d e t h a t a nicotinic cholinergic r e c e p t o r exists in brain, a n d t h a t c ~ - [ 3 H ] b u n g a r o t o x i n is a suitable p r o b e f o r this receptor.

Introduction A f t e r t h e p i o n e e r w o r k o f De R o b e r t i s [1] on t h e c h a r a c t e r i z a t i o n o f s y n a p t i c r e c e p t o r s in rat brain m a j o r progress in this area has b e e n a c h i e v e d using p e r i p h e r a l s y s t e m s . Cholinergic n i c o t i n i c r e c e p t o r s f r o m electric organs and m u s c l e have b e e n studied and purified e x t e n s i v e l y , a - T y p e * n e u r o t o x i n s f r o m cobras or B u n g a r u s m u l t i c i n c t u s v e n o m s have p r o v e d to b e v e r y specific Abbreviation:

fmole, femtomole

= 10 -15 m o l e .

* c~-Type t o x i n s f r o m s n a k e v e n o m s b l o c k i m p u l s e t r a n s m i s s i n n b y a c t i n g p o s t - s y n a p t i c a l l y , t h a t is, they bind to the receptors.

347 reagents for the acetylcholine receptor in these systems [2]. However, information on a nicotinic acetylcholine receptor in brain remains scarce. Farrow and O'Brien [3] could not detect binding of nicotinic drugs to brain particles, but Moore and Loy [4] reported binding of an a-toxin to a sodium deoxycholate extract of brain particles. While the present work was in progress, a communication was published that describes the binding of ~2 s I-labeled a-bungarotoxin to brain membranes as a function of age of the rat [ 5 ]. The experiments reported here describe the pharmacological characteristics of a-[ 3 H] bungarotoxin binding to brain particles and the subcellular distribution of the toxin-binding component. 12 Si_Labeled a-bungarotoxin binding was studied as well, and the striking difference in binding between the two labeled derivatives of the same toxin is discussed. A summary report of these results has been presented at the Third Annual Meeting of the Society for Neuroscience, San Diego, California (U.S.A.), November 1973 [6]. Materials and Methods Crude venom from B. multicinctus was obtained from Miami Serpentarium. CM-Sephadex C-50 was from Pharmacia, CM-cellulose (CM-52) from Whatman. Nicotine hydrochloride, atropine and decamethonium iodide were obtained from K and K Laboratories, h e x a m e t h o n i u m chloride dihydrate from Mann Research Laboratories, carbamylcholine chloride from Sigma Chemical Co., and d-tubocurarine chloride from Calbiochem. Acetylcholine perchlorate was synthetized in our laboratory. BW-284c51 (1:5 bis (4-allyldimethyl-ammoniumphenyl) pentan-3-one diiodide) was a gift from Burroughs Wellcome and Co., and promethazine a gift from Wyeth. Acetylthiocholine was obtained from Calbiochem; and chloramine T from Matheson, Coleman and Bell. Na I 25 I carrier free and 12 s IC1 were purchased from New England Nuclear. Carrier free tritium was supplied by Lawrence Livermore Laboratory. a-Bungarotoxin was isolated from crude venom of B. multicinctus by ion-exchange chromatography on CM-Sephadex and CM-Cellulose as described by Mebs et al. [7]. It was distinguished from the other toxins of this venom by its amino acid composition, toxicity in mice and action on frog muscle, all of which compared favorably with the published values [7,8]. Upon electrophoresis in sodium dodecylsulfate-polyacrylamide gels, at least 90% of the material migrated in a single band, and the faint " c o n t a m i n a n t " band was most likely a dimer of a-bungarotoxin on the basis of its relative mobility. a-[ 3H] Bungarotoxin was prepared by catalytic reduction of a-bungarotoxin iodinated with 12 siC 1 of low specific activity, with carrier free tritium gas (details to be published). 3H-Labeled toxin was purified by gradient elution from a CM-cellulose column. Its toxicity in mice after subtaneous injection was similar to native toxin. The specific activity was 14.7 Ci/mmole, that is, 0.5 atom of tritium per molecule of toxin. For storage purposes, 1-ml'aliquots of a - [ 3 H ] b u n g a r o t o x i n (30pg/ml) in 0.2 M a m m o n i u m acetate, pH 5.8, were frozen in liquid nitrogen and kept at --10°C. After 6 months of storage, 6% of radioactivity was exchanged with the solvent, but the binding characteristics of the labeled toxin have not changed. 12 s I-Labeled a-bungarotoxin used in the binding studies was prepared

348 fr o m a-bungarotoxin and Na I 2 s I by oxidation with chloramine T as described by Berg et al. [ 9 ] . This was always used within a m o n t h after iodination. The initial specific activities were in the range of 3 - 1 0 4 - - 8 . 1 0 4 c p m / p m o l e . It should be n o ted t hat by this m e t h o d less than 10% of the a-bungarotoxin molecules are iodinated.

Subcellular fractionation of brain homogenates Male Sprague--Dawley rats (from Simonson, Gilroy, California, U.S.A.) of 2 0 0 -- 3 0 0 g were used. Subcellular fractionation was done following the conventional techniques of De Robertis et al. [10] and Whittaker and Barker [ 1 1 ] . A 10% h o m o g e n a t e of cerebral cortex in 0.32 M sucrose, pH 7.0 was centrifuged at 1000 × g f o r 10 min and the precipitate was discarded. T he supernatant was then centrifuged at 17 300 × g for 20 min (Sorvall RC-2B). This precipitate, called crude mitochondrial fraction, was fractionated into nerve endings, mitochondria and myelin by a discontinuous sucrose gradient [ 11]. The microsomal fraction was obtained by centrifuging at 100 000 × g (spinco L) for 90 min the supernatant from the crude mitochondrial fraction. The total particulate fraction was the precipitate after centrifuging the whole h o m o g e n a t e at 100 000 × g for 90 min. In some experiments the crude mitochondrial fraction was submitted to osmotic shock with 10 ml of distilled water per gram of original weight, and membranes pelleted at 17 000 × g after 30 min were used. Acetylcholinesterase (EC 3.1.1.7) was assayed by the m e t h o d of Ellman et al. [12] with 6 - 1 0 -4 M acetylthiocholine as substrate and 5" 10 -v M promethazine to inhibit cholinesterase (EC 3.1.1.8).

Binding assay To determine the binding of a-bungarotoxin, the precipitated particles, usually the crude mitochondrial fraction, were resuspended in Ringer's solution (115 mM NaC1, 5 mM KC1, 1.8 mM CaC12,1.3 mM M g S O 4 , 4 mM K H z P O 4 , 3 3 mM Tris--HC1 (pH 7.4), and 3.3 mM glucose). This suspension was incubated with a-[ 3H] b u n g a r o t o x i n in Ringer's solution at r o o m t em perat ure, in a final volume of 1.5--2 ml. The particle fraction contained 3--4.5 mg protein per ml and consisted o f the material obtained from 100 mg of original wet weight. When the effect of cholinergic drugs on the binding of a-bungarotoxin was studied, 1 ml of suspension was incubated with 0.5 ml of the drug in Ringer for 60 min. a-[ 3H] Bungarotoxin (0.5 ml) was then added and the incubation was continued for 15 min. U n b o u n d t oxi n was removed by centrifugation at 39 000 × g for 10 min. The precipitate was washed three times by resuspension in 10 ml Ringer's solution. For the drug com pet i t i on experiments, the washing solution contained 10 .3 M c om pet i t or . No radioactivity was detected in the supernatant after the third wash. The precipitate was oxidized to 3 H 2 0 and COz in a Packard 305 Sample Oxidizer and the p r o d u c t was c o u n t e d in a Packard Tri-Carb scintillation s pe c t r om e t e r with 30--34% efficiency. Protein c o n t e n t of each tube was determined by the m e t h o d of L ow ry et al. [13] in an aliquot taken f r o m the suspension after the second wash. The binding assay for ~z s I-labeled a- b ungar ot oxi n was as described for a-[ 3HI bungarot oxi n except t h a t the washed pellet was resuspended in 1 ml Ringer solution and c o u n t e d in a ~,-ray NaI well c o u n t e r f r om Nuclear Chicago with about 10% efficiency.

349 12 s I-Labeled a - b u n g a r o t o x i n standards were c o u n t e d simultaneously to correct for decay of 12 s I which has a half-life of 60 days. Binding to diaphragm was assayed as described by Berg et al. [ 9 ] , e x cept t hat the tissue was c o u n t e d w i t h o u t homogenization in the 7-ray counter. Ex cep t for the studies using diaphragm, the report ed values are means of duplicate determinations.

Results

a-[3H] Bungarotoxin binding to brain subcellular particles High affinity binding of a - [ 3 H ] b u n g a r o t o x i n to brain crude mitochondrial fraction was observed with 1--40 nM t oxi n (Fig. 1). Maximum binding varied f r o m 40 • 10 -1 s to 60 • 10 -~ s moles (40--60 fmoles) per mg of protein for different preparations, depending probably on the purity of mitochondrial fractions. Half saturation was reached at 5.6 • 10 -9 molar toxin. At higher t oxi n concentrations, a lower affinity binding was observed. In a similar assay, the total particulate fraction f r om Saccharomyces cerevicae b o u n d only 8% of the value obtained for cortex. The binding of a - [ 3 H ] b u n g a r o t o x i n to brain particles was reduced by cholinergic drugs (Fig. 2). A nicotinic antagonist, d-tubocurarine, and an agonist, nicotine, p r o t e c t e d 50% of t oxi n binding sites at concentrations near 10 -6 M. Acetylcholine, carbamylcholine and d e c a m e t h o n i u m reached 50% prot ec tion between 10 -s and 10 -4 M. A muscarinic antagonist, atropine, had little effect until 10 -4 M, while concentrations of 10 -9 M affect muscarinic rece.ptors [ 1 4 ] . A ganglionic antagonist, h e x a m e t h o n i u m , and an acetylcholinesterase inhibitor, eserine, had little effect even at high concentrations. Native a-bungarotoxin at 10 -7 M inhibited a - [ 3 H ] b u n g a r o t o x i n binding by 80%. The remaining 20% was probably non-specific binding, since it was n o t affected by any of the drugs tested.

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F i g . 1 a - [ 3H] B u n g a r o t o x i n b i n d i n g t o c r u d e m i t o c h o n d r i a l f r a c t i o n . D e t a i l s a b o u t p r e p a r a t i o n o f t h e particles a n d the b i n d i n g assay are given in Materials a n d M e t h o d s . F i n a l particle c o n c e n t r a t i o n was 1 0 0 m g o r i g i n a l w e i g h t / m l , o r 4 . 3 m g p r o t e i n / m l . I n c u b a t i o n t i m e w a s 1 h. T h e i n s e r t is t h e d o u b l e r e c i p r o c a l p l o t o f o r i g i n a l d a t a , w h i c h g a v e a v a l u e f o r t h e h a l f s a t u r a t i o n o f 5 . 6 • 1 0 - 9 M.

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Fig. 2. I n h i b i t i o n o f a - [ 3H] B u n g a r o t o x i n b i n d i n g b y c h o l i n e r g i c d r u g s . C r u d e m i t o c h o n d r i a l f r a c t i o n ( 3 . 0 - - 4 . 5 m g p r o t e i n / m l ) w a s i n c u b a t e d w i t h t h e c o m p e t i t o r f o r 1 h. a - [ 3 H ] B u n g a r o t o x i n w a s t h e n a d d e d at a f i n a l c o n c e n t r a t i o n of 6.2 - 10 -9 M a n d t h e i n c u b a t i o n w a s c o n t i n u e d f o r 1 5 rain. T h e s a m p l e s w e r e t h e n p r o c e s s e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e d a t a r e l a t i v e t o a - b u n g a r o t o x i n , d - t u b o c u r a r i n e a n d c a r b a m y l c h o l i n e w e r e o b t a i n e d w i t h o n e m i t o c h o n d r i a l p r e p a r a t i o n ; t h o s e r e l a t i v e to nicotine, atropine and eserine with a second preparation, and those for d e c a m e t h o n i u m , h e x a m e t h o n i u m , a c e t y l c h o l i n e + B W - 2 8 4 c 5 1 , a n d B W - 2 8 4 c 5 1 w i t h a t h i r d . 1 0 0 % b i n d i n g w a s 1 8 , 20 a n d 2 5 f m o l e s / m g p r o t e i n , r e s p e c t i v e l y , f o r t h e t h r e e p r e p a r a t i o n s . +, ~ - b u n g a r o t o x i n ; A d - t u b o c u r a t i n e c h l o r i d e ; L~ n i c o t i n e h y d r o c h l o r i d e ; o, a c e t y l c h o l i n e p e r c h l o r a t e + 1.8 - 10 -5 M B W - 2 8 4 c 5 1 ; 0, c a r b a m y l c h o l i n e c h l o r i d e ; N d e c a m e t h o n i u m i o d i d e ; v a t r o p i n e ; m h e x a n l e t h o n i u m c h l o r i d e ; x, e s e r i n e s a l i e y l a t e ; ®, B W - 2 8 4 c 5 1 .

Unexpectedly, the microsomal fraction bound a - [ 3 H ] b u n g a r o t o x i n as well (Table I). The bindings to mitochondrial and microsomal fractions were inhibited similarly by d-tubocurarine, suggesting that a similar kind of receptor is involved in both cases. Relative specific activity of acetylcholinesterase is also higher in microsomes (Table I, ref. 10). Contamination of the microsomal fraction with cholinergic nerve endings is possible but not necessary, as the presence of acetylcholinesterase in endoplasmic reticulum and cellular membrane has been demonstrated by histochemical methods [15]. Presence of TABLE I ~-[3H] BUNGAROTOXIN BINDING TO PRIMARY FRACTIONS FROM RAT CORTEX The preparation of the primary fractions, the binding assay and the enzyme assay were described in Materials and Methods. Final particle concentrations in mg protein/ml were: total particulate, 5.0; mitochondrial fraction, 4.9; microsomal fraction, 4.2. Final c~-[3H]bungarotoxin concentration was 6.3.10 -9 M. Final d-tubocuraxine concentration was 10-4 M. Relative specific activity for acetylcholinesterase is defined as specific activity in a given fraction divided by the specific activity of total particulate, which was 190 nmoles/min per mg protein.

Total particulate (fmoles bound/ mg protein)

Mitochondrial fraction (fmoles bound/ mg protein)

Microsomal fraction (fmoles bound/ mg protein)

Control + d-tubocurarine Percent inhibition

15 5 67%

15 6 60%

21 6 71%

Acetylcholinesterase

Relative specific activity 1.00

0.81

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Fig. 3. a - [ 3H] Bungarotoxin

binding to submitochondrial fractions. Submitochondrial fractions were prein M a t e r i a l s a n d M e t h o d s . a - [ 3 H ] B u n g a r o t o x i n final concentration was 6 . 2 • 1 0 -9 M. Protein concentrations in mg/ml were: myelin, 2.3; nerve endings, 3.2; mitochondria, 4.0. heavy hatching, control binding; light hatching, binding in the presence of 10 -4 M d-tubocurarine; no hatching, acetylcholinesterase relative specific activity.

p a r e d as d e s c r i b e d

acetylcholine receptor in axonal membrane of central neurons has been postulated [15] and an a-bungarotoxin-binding molecule has been described in the axonal membrane of the lobster walking leg [ 1 6 ] . Upon fractionation of the crude mitochondrial fraction into myelin, nerve endings and mitochondria, the highest specific activity for a-[ 3 HI bungarotoxin binding was found in nerve endings (Fig. 3). Some of the binding to myelin and mitochondria is probably due to cross-contamination with nerve endings, since it is partially inhibited by d-tubocurarine. Binding of a-[ 3H] bungarotoxin to membranes obtained by osmotic shock of the crude mitochondrial fraction is illustrated in Fig. 4. The concentration necessary for half-saturation of toxin binding sites was 3.2 • 10 .9 M. This value is very similar to the one found with crude mitochondria (5.6 • 10 -9 M) suggest-

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(3(-[3HI BUNGAROTOXIN (nM) F i g . 4. c~-[ 3 H ] B u ~ g a r o t o x i n b i n d i n g t o b r a i n m e m b r a n e s . C r u d e m i t o c h o n d r i a l f r a c t i o n w a s s u b m i t t e d to osmotic shock and tbe resulting membranes were incubated with ~-[ 3H] Bungazotoxin f o r 2 h. F i n a l membrane concentration w a s 4 m g p r o t e i n / m l . H a ] f - s a t u r a t i o n w a s r e a c h e d at 3 . 2 - 1 0 -9 M t o x i n c o n c e n tration.

352

ing that the same receptor is involved in both cases, and that it is a membranebound molecule. 5 2 s I - L a b e l e d c ~ - b u n g a r o t o x i n b i n d i n g to brain a n d d i a p h r a g m

Results obtained with two different preparations of 52 Si_labele d abungarotoxin are presented. One of these preparations (Prepn 3) showed saturable binding at 3--4 pmoles bound per gram of original weight (approx. 70 fmoles/mg protein). This a m o u n t is similar to the value obtained with a[ 3HI bungarotoxin (40--60 fmoles/mg protein), but only 50% of this binding was inhibited by preincubation with 10 -3 M d-tubocurarine (Fig. 5a). At saturating toxin concentrations maximum binding was reached in 10 min. The a m o u n t and the rate of 12 Si_labele d ~-bungarotoxin binding showed little change with temperature between 0 and 37°C (results not shown). When the same toxin was assayed for specific binding to rat diaphragm, more than 80% of bound radioactivity was found in the area containing end plates (Fig. 6a). These results confirm previous findings [9] that ~2 SI_labeled ~-bungarotoxin is highly specific for the acetylcholine receptor in diaphragm. It is also shown that " o l d " ~2 Si_labele d ~-bungarotoxin differs from freshly iodinated toxin mainly by markedly decreased total binding due primarily to very large loss in the specific binding component. On the contrary, when " o l d " 52 Si_labele d ~-bungarotoxin was incubated with brain particles non-specific binding was greatly increased. A second preparation of ~2 Si.labele d ~-bungarotoxin (Prepn 4) showed only the characteristics of non-specific binding to brain even when freshly prepared (Fig. 5b). This binding was not saturable; it was not decreased in the presence of d-tubocurarine, or carbamylcholine, and the a m o u n t bound was increased about five times over previous results (Fig. 5b). However, the same toxin preparation showed highly specific binding to diaphragm. The saturation (a) (b)

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F i g . 5. 1 2 5 i _ l a b e l e d a - b u n g a r o t o x i n b i n d i n g t o c r u d e m i t o e h o n d r i a l f r a c t i o n . P a r t i c l e s a t a f i n a l c o n c e n t r a t i o n o f 1 0 0 m g o r i g i n a l w e i g h t / m l w e r e i n c u b a t e d w i t h d - t u b o e u r a r i n e o r R i n g e r ' s s o l u t i o n f o r 1 h. U p o n a d d i t i o n o f 1 2 5 i . l a b e l e d a - b u n g a r o t o x i n t h e i n c u b a t i o n w a s c o n t i n u e d f o r 1 0 r a i n . (a) D a t a o b t a i n e d w i t h 125 I - l a b e l e d ~ - b u n g ~ a r o t o x i n f r o m P r e p n 3. B i n d i n g i n t h e p r e s e n c e o f 2 . 5 • 1 0 - 4 M d - t u b o c u r a r i n e is a l s o shown (...... ). ( b ) A s i m i l a r e x p e r i m e n t p e r f o r m e d w i t h 1 2 $ 1 . 1 a b e l e d a - b u n g a r o t o x i n f r o m P r e p n 4 (zx). ©, b i n d i n g i n t h e p r e s e n c e o f 3 . 3 • 1 0 -4 M d - t u b o c u r a r i n e ; e , b i n d i n g i n t h e p r e s e n c e o f 3 . 3 • 1 0 - 4 M carbamycholine.

353 curve was very similar to the one shown on Fig 6a. Preincubation with dtubocurarine or carbamylcholine inhibited most of the binding to the area containing end plates. About half of the binding to the area w i t h o u t end plates was inhibited as well, suggesting that some acetylcholine receptor may exist in this area (Fig. 6b) (see also ref. 9). In conclusion, although 12 Si.labele d ~-bungarotoxin seems to be specific for the acetylcholine receptor from diaphragm, different toxin preparations gave inconsistent results with brain particles. Discussion It is believed that bungarotoxins do not act on central nervous system in vivo, due to their failure to cross the blood--brain barrier, but instead act peripherally [17]. Thus, the failure to act centrally does not argue for or against the presence of nicotinic synapses in the central nervous system. The presence of such synapses in the spinal cord (Renshaw cells) has been clearly demonstrated [18]. The binding of a - [ 3 H ] b u n g a r o t o x i n to brain cortex as described here strongly suggests the presence of a mcotinic acetylcholine receptor in this tissue. The component(s) binding a-[ 3H]bungarotoxin is present in low concentration, in the order of pmoles/g. This result agrees with those reported in two previous communications [4,5]; the value of 17.5 nmoles/g reported elsewhere [19] seems increasingly improbable. 100

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CONCENTRATION OF COMPETITOR (M)

Fig. 6. (a) 125i.Labele d c~-bungarotoxin binding to rat diaphragm. Binding to diaphragm was assayed by the m e t h o d of Berg et al. [9] . 1251_Labele d &-bungarotoxin from Prepn 3 was used. Incubation time was 1 0 5 min. T h e c p m b o u n d u p o n incubation with "old" toxin were corrected for radioactive decay. 4, binding of 12SIdabeled a-bungarotoxin i day after iodination to the area containing end plates; ~, binding of 125i.labeled cz-bungarotoxin I day after iodination to the area without end plates; e, binding of 125i.labele d ~-bungarotoxin 80 days after iodination to the area containing end plates; o binding of 125 I-labeled c~-bungarotoxin 80 days after iodination to the area without end plates. (b) Inhibition of the binding of 12 Si_labeled c~-bungarotoxin to diaphragm b y d-tubocurarine and carbamylcholine. Pieces of diaphragm were incubated with or without the competitors in Ringer's solution for 1 h, 125i_Labele d ~-bungarotoxin (Prepn 4) w a s then added at a final concentration of 8.75 • 10 -8 M and the incubation was continued for 1 0 5 rain. Further procedure was as described b y Berg et al. [9]. e, inhibition of binding to the area containing end plates by d-tubocurarine; o, inhibition of binding to the area without end plates by d-tubocurarine; 4 inhibition of binding to the area containing end plates b y carbamylcholine; A inhibition of binding to the area without end plates b y carbamylcholine.

354 Table II shows the striking similarities between pharmacological profiles of the acetylcholine r e c e p t o r f r om ganglionic neurones in tissue culture [20] and the a-[ 3H] b u n g a r o t o x i n binding to cerebral cortex report ed here. Nicotinic receptors f r o m muscle and electroplax were more sensitive to decamethonium, as was expected, but otherwise t h e y were n o t grossly different from the neuronal receptors. Results obtained with a - [ 3 H ] b u n g a r o t o x i n and ,2 Si.labele d a-bungarot o x i n invite some comparisons. While a - [ 3 H ] b u n g a r o t o x i n was a reliable reagent for brain acetylcholine r e c e pt or and its binding characteristics did not noticeably change after 6 months of storage, 12s I-labeled a-bungarotoxin showed a marked t e n d e n c y to progressively higher and higher non-specific binding in brain a p p r o x i m a t e l y a m o n t h after iodination. This behavior was clearly different f r om t hat exhibited with diaphragm where " o l d " toxin showed decreased binding. Moreover, some ~ 2 s I-labeled a-bungarotoxin preparations did n o t show saturable binding to brain particles or c o m p e t i t i o n with cholinergic drugs, even when the usual specific binding was seen for diaphragm. Thus conditions for specific binding of a-bungarotoxin to brain acetylcholine re c ep to r seem to be more stringent than those required for diaphragm. Brain and muscle receptors are probably not identical as suggested by their sensitivities to drugs (Table II), but the difference in 12 s I-labeled a-bungarotoxin nonspecific binding is more likely related to dissimilar biochemical composition of b o t h organs. Salvaterra and Moore [5] r e p o r t e d specific binding of 125Ilabeled a- b u n g ar ot oxi n to brain particles. Their toxin was iodinated with 12 siC1 instead of Na 1251 and chloramine T used by us. It is possible that failure of freshly prepared 12 Si_labeled a-bungarotoxin to bind specifically to brain acetylcholine r e c e pt or is due to some damage of the protein by the strong o x i d an t chloramine T. The loss of specificity with time is probably due to increasing radiation damage from the high specific activity iodine. TABLE n DRUG AFFINITIES FOR ACETYLCHOLINE RECEPTORS FROM SEVERAL SOURCES In the f o u r initial c o l u m n s , p r o t e c t i o n against ~ - b u n g a r o t o x i n b i n d i n g by the r e s p e c t i v e d r u g w a s m e a s u r e d . Figttres i n d i c a t e the c o n c e n t r a t i o n n e c e s s a r y to d e c r e a s e t o x i n b i n d i n g to 50%. C o l u m n 1 s h o w s d a t a f r o m Fig. 2. C o l u m n s 2, 3 a n d 4 w e r e o b t a i n e d w i t h 125I-labeled ~ - b u n g a r o t o x i n (refs. 20, 21 a n d 9, r e s p e c t i v e l y ) . D a t a s h o w n in C o l u m n 5 w e r e o b t a i n e d b y m e a s u r i n g c h a n g e s in ionic fluxes o f " m i c r o s a c s " f r o m electric o r g a n s u p o n a p p l i c a t i o n o f the d r u g (ref. 22); t h e figures are c o n c e n t r a t i o n s n e c e s s a r y for half o f t h e m a x i m a l r e s p o n s e .

d-Tubocurarine Nicotine Acetylcholine + inhibitor* Carbamylcholine Decamethonium Hexamethonium Atropine Eserine

Brain c r u d e mitochondrial fraction

Chick sympathetic neurones

Chick muscle cells

Rat diaphragm

"Microsacs" f r o m Electrophorus electricus

7.4" 1 0 - 7 1 . 3 - 1 0 -6 2 . 5 " 1 0 -5 1.4" 10-4 2 . 2 - 1 0 -4 ~ 1 0 -3 5.6 "10-4 ~ 10 -3

2.5" 10- 7 5 . 0 . 1 0 -7 1 . 6 . 1 0 -5

7 . 4 . 1 0 -7

10-5

1.5.10-7

2.0-10-4 6.0.10-4 ~ 10 -3

* A c e t y l c h o l i n e plus an i n h i b i t o r of a c e t y l c h o l i n e s t e r a s e .

~1o-5 3 . 1 . 1 0 -7 ~10-5 2 . 3 - 1 0 -7 >~ 10 -5

10-4

10-4 )10- 3 3 . 0 . 1 0 -5

4 . 0 . 1 0 -6 3.3.10-5 1 . 2 . 1 0 -6 6.2.10-5

355 The widespread existence of acetylcholine, cholineacetylase and acetylcholinesterase in central nervous system has prompted the search for central cholinergic pathways. Krnjevid [23] indicates that an ascending cholinergic system may mediate cortical arousal. However, the responses of most central neurones to iontophoretically applied acetylcholine were different from peripheral nicotinic synapses. Alternative mechanisms for acetylcholine central action have been proposed. According to Koelle's model [15], in some instances acetylcholine reacts with axonal acetylcholine receptors to facilitate the liberation of more acetylcholine or other neurotransmitters. Since microsomal fraction contains mainly non-synaptic membranes [24], specific binding of ~[ 3HI bungarotoxin to this fraction could be an indication of the existence of extrasynaptic acetylcholine receptors.

Acknowledgements This work was supported, in part, by the U.S. Atomic Energy Commission. One author (V.A.E.) had a postdoctoral fellowship of Consejo Nacional de Investigaciones Cientificas y Tecnicas of Argentina. Thanks are given to Hiromi Morimoto for performing the assays for acetylcholinesterase and to Marie Hebert for assistance in the preparation of purified a-bungarotoxin. Professor P. Fromageot and Dr J.P. Changeux provided details in addition to the original description [25] of the catalytic exchange of tritium for iodine in snake toxins, for which the authors are indebted.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

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