Metal ion content of cholinergic synaptic vesicles isolated from the electric organ of Torpedo: Effect of stimulation-induced transmitter release

METAL ION CONTENT OF CHULINERGIC SYNAPTIC VESICLES ISOLATED FROM THE ELECTRIC ORGAN OF TORPEDO: EFFECT OF STIMULATION-INDUCED TRANSMITTER RELEASE R. SCHMIDT,’ H. ZIMMERMANNand V. P. WHITTAKER Abteilung fiir Neorochemie, Max-Pianck-lnstitut fiir Biophysikalische Chemie, Postfach 96% D-3400 GGttingen, Federal Republic of Germany synaptic vesicles were isolated from the Torpedo electric organ by a cornb~~at~~n of differential and density gradient ~ntrifugatjon. Iso-osmofar solutions of glycine or NaCf were used as homogenization and preparation media. The metal content of intact tissue and subcellular fractions were determined by atomic absorption spectroscopy. In the synaptic vesicle fraction ratios of metals to acetylcholine (g atom/mol) were: Na, 0.30; K, 0.10; Mg, 0.07; Ca, 0.28. Filtratian of isolated vesicles revealed that the strength of metal binding depends on the ionic potential of the metat cation. Thus alkali metat ions are bound to synaptic vesicles less tightly than alkaline earth metals. Incubation of vesicles with elevated levels of NaCl led to a partial exchange of Na with K but externaf concentrations of CaC1, in the physiological range were withaut effect on vesicle metal ion content. Stimulation of the electric organ in uiuo (5000 impulses, 5 Hz) caused a depletion of the acetylcholine and adenosine Y-triphsophate content of the vesicles whereas the levels of metal ions were increased. It is suggested that the release of acetylcholine from synaptic vesicfes exposes free negative charges to which extracellular metal cations can bind in ion exchange.

~~~~h~~n~r~~

Ike ~~~crrw organ of the ray Torpefio ~~~~#~~~~ has been used for the isolation of synaptic vesicles of high purity containing anly one type of transmitteracetylcholine (ACh) (SHERIDAN,WHITTAKER8~ ISRA&

amounts of phya~~~~~~il~ important metal ions in vesicles from tissue in different functional states could provide a further insight into the mechanism of stor-

age and release of the transmitter in these organelles. The analysis of the metal ion contents of synaptic vesicles does, however- involve a considerable number vesicles differ from synaptic vesicles isolated from of difficulties. ft is difficult to separate vesicles from brain tissue which are derived from a mixed populathe large excess of metal ions contained in tissue tion of terminals containing different types of transhomogenates and subcellular fractions derived from mitter and cannot be isolated under iso-osmolar conthem. Continued purification in ion-free media on the ditions ~W~~AKE~, MICH~E~N & KIRKLAND, other hand could result in loss of ions from vesicles 1964). Synaptic vesicles isolated from the Torpedo and also non-specific binding or redistribution of electric organ therefore lend themselves favourably to ions. Finally due to the low yield of vesicles, sensitive a stoichiametric analysis of the components they are methods of analysis have to be applied, such as composed of. atomic absorption spectroscopy, the one adopted They have been shown to contain adenosine S-trihere, phosphate (ATP) in addition to ACh (molar ratio We analysed the metaf ion content of electric tissue ACh: ATP = 5 : 1, DOWDALL, BOYNE & WHITTAKER, both qualitatively and quantitatively. The metal ion 1974). Stimulation of the electric organ causes a decontent of synaptic vesicles was determined in metal crease in the ACh and ATP content per vesicle (ZIM- ion-free and saline media after isolation from resting MERMANN & WHIT-FAKER, f974a; ~1~~~~~~~ & and stimulated tissue. Furthermore we investigated DENSTON, 1977) and these constituents are regained the possible influence of nonspecific cation binding during a subsequent recovery period (ZKMMERMANN & and exchange. Preliminary accounts of this work have WHITTAKER, 1974b; S~J~ZKIW & WHITSAKER, 1979). been given (SCHMIDT, ZIMMERMANN6% WHITTAKER, So far nothing is known about the metal ion content 1976; SCHMITT & Z~~M~~AN~~ 1977). of chofinergic synaptic vesicles. Dete~~nat~on of the 1955; ISRAEL, GAVTRON & L~BATS, 1970; WHITTAKER, ESSMAN& Dow, 1972). In this respect these

‘Present address: Ralph Lowell Laboratories, McLean Hospital, Harvard Medical School, Belmont, Massachusetts 02178, U.S.A. ~~~r~~a~~on~: ACh, acetylcholine; ATP, adenosine S-triphosphate; LDH, lactate dehydrogenase.

EXPERIMENTAL

PROCEDURES

Animals

Experiments were performed on Tf2rpeda ~~~~a~~ suppli& by air from the lnstitut de Biofogie Marine, Arcachon, France, and kept in tanks of circulating seawater at

R. SCHMIDT. H. Z~MMEKMANNand V. P. WtnrrAlits

626

16 C in the dark. The fish were anaesthetized by placing them in seawater containing 0.05% ethyl m-aminobenzoate methanesulphonate (Tricaine methane sulphonate. MS 222. Sandoz) until respiratory movements ol’ the spiracles ceased. The electric organs were removed and quickly frozen in liquid nitrogen. E/ec,trri,tr/ stimulution

und recording

Filtrate ot

Fish were stimulated with 5000 square wave pulses of 1.5 ms duration. IOV amplitude and a frequency of 5 Hz via platinum electrodes inserted through a hole in the skull. as described previously (ZIMMERMANN& WHITTAKER, 19740). To provide an unstimulated control, the nerves to one organ were cut at a point approximately I cm distal to that at which they emerge from the skull. The response of the innervated organ to electrical stimulation was recorded by means of a ‘peak-reading’ voltmeter and a pair of electrodes one placed on the dorsal skin and the other on the ventral skin above and below the organ. The response to the first pulse varied between 25 and 60V (mean: 41.2 k 1.8 V. s.I:.M.. )t = 33). After 15 min of repetitive stimulation electrical responses had fallen below I V.

Electric tissue frozen in liquid nitrogen was comminuted according to the method of WHITTAKER r’t ul. (1972). In order to avoid introduction of exogenous metal ions an iso-osmolar solution of glycine (0.8 M) insteah of NaCl (0.4 M) was used in some of the experiments for homogenifation and density gradients. The complete isolation procedure is given in Fig. I. Vesicles were first enriched by centrifugation of the supernatant fraction Slz and then washed in 0.8 M glycine. Final fractionation was by a step gradient modified from ISRAEL ef rrl. (1970)shown in Fig. 1. In some experiments the gradients contained additional layers of either 0.15 M sucrosee0.65 M glycine or 0.6 M sucrose 0.2 M glycine. This led to the recovery of an additional number of fractions from the gradient. For fnal evaluation of results the h-actions were combined in order to (it the original fractionation scheme. For comparison in one experiment synaptic vesicles were Isolated on ronal gradients according to WHITTAKER rt ul. (1972). In order IO obtain an amount of isolated vesicles suthcient for the analysis of metal ions 370g of electric tissue (equivalent to two fish) were frozen, comminuted and mixed with 560 ml of 0.8 M glycine. After filtration through cotton gauze, cell debris were sedimented by centrifugation at 1000 9 for 10 min. Less dense membrane fragments and mitochondria were removed from the supernatant Fraction by ccntrirugation at 17.000 R for 50 min. The synaptic vesicles contained in the supernatant fraction were then sedimented at 95.500 9 for 150 min using a Beckman Ti 35 rotor. The crude vesicle pellet was resuspended in 50 ml of 0.8 M glycinc to yield a fraction P, and loaded on a continuous Tonal density gradient. The gradient was prepared as described by WHITTAKER et ul. (1972) except that NaCl was replaced by iso-osmolar concentrations of glycine.

Subcellular fractions were fixed in buffered 1% glutaraldchyde and osmicated in buffered 1% 0~0, essentially as described by ZIMMERMANN & WHIT-~AKER (1974~). Thin sections (cut on an LKB-Ultratome III) were stained with lead citrate and uranyl acetate and observed in a JEM IOOB electron microscope.

‘centrifuged

13 300 g I

for

30 min

I

supernotontIS,: I centr’fuged at 200000a f0T 70 m,r

Fro. I. Isolation of synaptic vesicles from the electric organ of Torpedo marmoratu. The two schemes adopted for fractionation of the density gradient are given at the bottom of the Figure; g, average centrifugal force in gravity units.

Biochrmicrrl

mrthods

ACh (bioassay using the leech method), chohnesterases, fumarase (L-malate hydrolyase, EC 4.2.1.2) and lactate dehydrogenase (LDH) (L-lactate:NAD oxidoreductase, EC I. 1.1.27) were estimated in su bcellular fractions essentially as described by WHITTAKER & BARKER (1972). Units of fumarase activity is given as a unit change in extinction at 250nm/min measured in a 3 ml cuvette with a 1 cm light path. To measure free LDH in samples which contained osmotically sensitive structures. sucrose was added to the reaction medium to give a final osmolarity 0r 0.8 0s mol. To measure total LDH activity. occluded enzyme was released by adding Triton X-100 to samples before assay to give a final concentration of 0.1 y<%,.The difference between total and free LDH is referred to as occluded LDH. Cytochrome c oxidase (cytochrome c:O, oxidoreductase, EC 1.9.3.1) was measured according to the method of COOPERSTEIN & LAZAROW (1951). Cytochrome c was reduced by dithionite and separated from the reducing agent by passing the solution through a Sephadex G-25 column. The oxidation of cytochrome c was determined at room temperature by following the decrease in optical density at 550 nm in a reaction medium of 75 mM sodium phosphate buffer, pH 7.5. ATP was assayed by the firefly luciferinluciferase method (STANLEY& WILLIAMS, 1969) as modified

627

Metal ion content of cholinergic vesicles TABLE

1.

Metal Li Na K Mg Ca Zn

FOR TWEQUANTITATIVE DETERMINATION OF METAL ATOMICABSORPTION SPECTROPHOTOME’rRY

CONDITIONS

Wavelength (nm) 670.8 589.0 766.5 285.2 422.7 213.9

Additions to sample (in %) HCI La LiCl 0.85

1

0.85 0.85 0.85

1 1 1

0.5 0.5

IONS BY

Detection limit @g/l) 1.0 2.0 5.0 0.5 5.0 0.5

Magnesium and zinc were determined with compensation of nonspecific absorbance by a deuterium lamp. The detection limit is defined as the concentration giving a signal three times as high as the noise. by DOWDALLet al. (1974). Protein was measured using the method of SCHAFFNER& WEISSMANN (1973) with bovine serum albumin as a standard protein. Analysis of metal contents Solubilization of primary fractions. Primary fractions were solubilized by heating 0.5 g of sample with 0.5 ml of 65% HNOs at 185°C for 105min in a Teflon autoclave under pressure (KOTZ, KAISER,TSCH~PEL& T~LG, 1972). The Teflon cylinders were rinsed twice with 1 ml of water. Qualitative analysis. Electric tissue, solubilized as described above, was analysed qualitatively for metal cations by the general separation procedure (described, e.g. by JANDER& BLASIUS,1967), which is based on the different solubilities of the chlorides, sulfides and hydroxides of the different cations in acidic or alkaline solutions. Quantitative analysis. Quantitative analysi,; of metals was performed by atomic absorption spectrophotometry using the double-beam four-channel Beckman instrument, model 1248. Nonspecific absorbance due to absorption by molecules or radicals and to light scattering below 300 nm was compensated for by means of a deuterium lamp, which permitted the nonspecific absorbance to be measured at both sides of the resonance line and the reading to be subtracted electrically from the absorbance signal at the resonance line. In order to prevent clogging of the blade of the burner head by protein and sucrose contained in the samples, the blade was water-cooled. Samples were diluted 1: 10 with distilled water before determination. LiCl was used as an ionization-buffer for the determination of Na and K. The formation of chemical compounds (especially oxides, sulfides and spinels) was reduced by the addition of lanthanum chloride (Table 1). Correspondingly all standards contained the same additions. Since Ca is readily dissolved from glassware only polyethylene and polystyrene equipment was used. All equipment was freed from contamination by soaking in 0.5~ HCl overnight followed by several rinses with distilled water. Ca was removed from sucrose and glycine solutions by column chromatography with the cationexchanger Chelex 100 previously converted to its acidic form with 0.1 M HCI. The remaining contamination with metal ions was corrected for by running blank gradients with each preparation which were loaded with solutions of glycine or NaCl instead of fraction Pz. Addition of LiCl

The electric tissue does not contain measurable amounts of Li. In two experiments LiCl was added to the first super.

natant fraction S,2 (Fig. 1) in order to investigate the extent to which alkali metal ions could bind to particulate fractions, either by nonspecific adsorbance or redistribution on tissue homogenization. The concentration of LiCl was 10 mM which would correspond to approximately onetenth of the Na concentration in this fraction. Millipore filtration

In a further series of control experiments synaptic vesicles were filtered on Millipore filters (HAWP 02500, pore size 0.45 pm) with and without previous incubation in metal chloride solutions. Since filters were found to contain rather variable amounts of metals they were soaked before use for 10 h in 0.1 M HCI followed by several rinses with distilled water. Using this method the variation in metal ion content between individual filters was greatly reduced. Remaining contaminations were accounted for by determination of the metal ion contents of filter blanks. The acid washed filters retained 99.7% of the ATP contained in synaptic vesicles. After filtration of the vesicle suspension filters were washed twice with 5 ml of 0.8 M glycine dried in air and combusted in nitric acid as described for primary fractions. In a first series of experiments vesicles isolated in isoosmolar sucrose-glycine solutions were filtered directly after harvesting from the density gradient. In parallel experiments vesicles were shocked by incubation in a tenfold volume of distilled water in order to release their contents of soluble ions. After 1 h at 4°C suspensions were filtered as before. In a further series of experiments the synaptic vesicle fraction was incubated with various amounts of metal ions for various lengths of time at 4°C in order to investigate the possibility of nonspecific binding and (or) redistribution of metal ions on subcellular fractionation. Na+ was used as a representative of the alkali metals and Ca’+ of the alkaline earth metals. Subsequent to isolation synaptic vesicles were incubated in NaCl, CaCl, or glycine and filtered. Filters through which corresponding solutions of NaCl, CaCl, or glycine had been passed served as blanks. Chemicals

Chelex 100, 100-200 mesh, was from Bio-Rad Laboratories, Munich, F.R.G., Millipore filters were from Millipore GmbH, Neu-Isenberg, F.R.G. and cytochrome c (horse heart) from Boehringer, Mannheim, F.R.G. Acetylcholine perchlorate, acetylthiocholine iodide, AmidoschwarzlOB,2,2’-dinitro-5,5’-dithiobenzoicacid,HCl(Suprapur), HNOs (Suprapur), lanthanum-III-oxide (for atomic absorption spectroscopy), LiCl (Suprapur), NaCl (Suprapur) and sucrose (for density gradient centrifugation) were

62X

R. SCHMIDT.H. ZIMMERMANN and V. P.

obtained from Merck,

Darmstadt. F.R.G. Glycine was from Riedel-de Hain, Seelze-Hannover. F.R.G., Tricaine methane sulphonate (MS 222) From Sandoz, Basef, Switzerland, and bovine serum albumin (crystalline) from Serva, Heidelberg, F&G. NADH2. physostigmine sulphate, Tris and luciferin-luciferase mixture were obtained from Sigma,

Munich, F.R.G. Alt other chemicals were of the purest quality avaiiabte #.A. or Suprapur). RESULTS

~uaI~tative analysis of the electric tissue revealed the presence of Na, K, Mg, Ca and Zn, Traces of Fe were also found in the tissue and considered to be mainly derived from blood contained in tissue capil-

laries.

Quantitative analysis of primary fractions was performed in order to investigate whether metal ions are lost or introduced during the preparation procedure and how the metal ions distributed during the various fractionation steps. The unfractionated tissue contained @mol/g wet wt): Ns, 209; K, 22.8; Mg, 4.3: Ca, 2.9; Zn, 0.06. Powdering of the frozen tissue caused no alterations in the ionic composition. Furthermore no cation was ~utr~u~ by the homogenization medium. About one-third of the cations stayed

WWITTAKEK

in the cell debris fraction (slightfy more alkaline earth than alkali metals). Fig. 2 presents the distribution of the various metal ions during the initial differential centrifugation steps (fractions P,l, P3_ S,) and compares them to the distributian of the vesicle marker ACh and cholinesterass activity. The majority of metal ions was obtaintd in the supernatant fraction Sj. In the crude vesick pellet fraction P3 a larger proportion of divalent than of monovalent metal ions was recovered. The percentages of the contents of intact electric tissue recovered in the crude vesicle fraction were: Na, 0.X: K, I 4: Mg,

6.6; Ca, 30.3; Zn, 23.7. The calcium content of the pellet fraction P,, was higher than that of all other metals. Fraction P,, is rich in the mitochondrial markers cytochrume c oxidase and fumarase (not presented) and ~holinesterase activity. The high Ca content of this fraction is mainly derived from its mitochondrial contents (SCHMIDT. ZIMMERMANN & Jo6, 1978). Thus on tissue homogenization alkali metab appeared to be rather freely

solubfe whereas Mg and particularly Ca and Zn were bound more tightly to sedimenting structures. The recoveries of the various metal ions (legend to Fig. 2) demonstrate that only small amounts of cations were lost or introduced during the fracfionation procedure. Onfy the recovery of Zn (IX>;) was rather high probably due to introduction of Zn from the caps covering the centrifuge tubes. The determination of Zn furthermore proved to be difficult as its

concentrations were extremely low (less than one 3000th of Nat. For these reasons Zn values derived from density gradient fractions have been omitted. Density grudient jiactions ~~ffc~e~~~u~ c~a~~~te~~~utju~. Crude synaptic

fractions [P,) were washed glycine and recentrifugatioa fraction (Pq) and then

vesicle by resuspension in Q.&M to yield a washed pellet

loaded on iso-osmolar sucrose-glydine density gradients as shown in Fig. 1. in Fig. 3 the biochemical characterization of the gradients is presented in the form of ue Duve plots. The vesicle markers ACh and ATP are enriched in frao tion 3 (vesicle fraction). The mitochondrial markers cytochrome c oxidase and fumarase sediment in the denser fractions of the gradient. The vesicle fraction Fir;. 2. Relative specific concentrations @SC) of metal ions, ACh and choiinesterase {ChE) in ti-te primary frac- contains only traces of either occluded or free cytoas can be deduced from the tions @lotted according to DE DUVE,PRESSMAN, GIANETTQ, plasmic constituents WATTIAUX& APFELMANS,1955). The areas of the columns

are proportional to the amounts of component and their heights to the relative enrichment of the component in the fraction. A larger fraction of the divafent than of the monovalent metal ions is obtained in the crude vesicle fraction P3. Mean absolute contents @nol.g wet wt. of tissue-‘) in the filtrate fraction (Fig. 1) were: Na, 149; K, 12.9; Mg, 2.5; Ca. 2.7; Zn, 0.05; AC4 0.25. The level of ChE was 343 I Li.g wet wt - I. On average the following percentages of component origina& contained in the E&rate were recovered in fractions P,,, P3 and S,: Na, 92; K, 98; Mg, 102; Ca. 1IO; Zn, 132; ACh, 71; ChE, 86. Values are means of seven experiments.

distribution of LDH activities. Most of the cholinesterase activity is recovered in fraction 5 (membrane fraction) but the vesicle fraction also contains choli-

nesterase activity, a phenomenon which is regularly observed also when vesicles are isolated by zonal centrifugation (TA~HIRO & STMX..ER, 1978). Qn average the vesicle fraction contained 1.35 _+0.10 (~s.E..M.) gmoI ACh/mg protein (n = 4) which is within the range of purity of synaptic vesicles obtained by zonal gradient centrifugation without additional purification steps (0.29-4.07 ~01 ACh/mg protein (see Table 2 of OHSAWA, DOWE, MORRIS & WHITTAKER. 1979)).

Metal ion content

ATP

I

0

50

1 100

iroctm

“umber

FIG. 3. Distribution of marker substances in the sucroseglycine gradient (Be Duue plots). Besides the vesicle markers ACh and ATP the mitochondrial mzrkers cyto-

chrome c oxidase and fumarase, the cytoplasmic marker lactate dehydrogenase (LDH) and cholinesterase activity (ChE) are given. Total LDH is represented by open columns and occluded LDH by cross-hatching. On average the following amounts (per g of wet wt.) were loaded on the gradient (recoveries in parentheses): ACh, 129 nmol (97); ATP, 26 nmol (101); cytochrome c oxidase, 0.007 IU (120); ChE, 29 IU (86); LDH, 0.35 IU (I I I); fumarase, 0.0023 extinction units. min-’ (105). Values are means of two experiments.

The mean molar ratio of ACh: ATP in the vesicle fractions was 4.55 f 0.41 (&SEAL, n = 4). Morphological characterization. Fine structural analyses of fraction 3 revealed that it contained the synaptic vesicles and only traces of contamination in the form of larger membrane fragments. Fraction 5 consisted of membrane fragments, fragments of nerve terminals, mitochondria and glycogen granules (Fig. 4). Quantitative analysis of metal ions. The distribution of metal ion contents over the density gradient is given in Table 2. It can be seen that all metal ions are enriched in the vesicle fraction (3) relative to neighbouring fractions. A satisfactory separation of K and Mg contents has been achieved from the contents of soluble ions in the lighter fractions as well as from the metal ion contents bound to particulate structures recovered in the membrane fraction (5). This is not so for Na and Ca. For Na the large contents of the supernatant fractions (la, lb, 2) are apparently de-

of cholinergic

629

vesicles

rived from the large excess of Na in the tissue homogenate. For Ca by far the greatest concentration occurs in the membrane fraction (5). That the subsidiary peak occurring in the vesicle fraction (3) is simply due to mitochondrial contamination can be excluded since the vesicle fraction does not contain a proportional amount of mitochondrial enzyme activity (Fig. 3). It is possible, however, that part of the Ca (which would have sedimented in fraction 5) is released during resuspension of the pellet fraction and/or the subsequent centrifugation step and thus contributes to the Ca content of fractions 1 to 4. If 0.4 M NaCl is substituted for 0.8 M glycine in the isolation of vesicles the Ca content of the gradient fractions is quite different. Using this preparation medium mitochondria lose 80% of its Ca (SCHMIDT et al., 1978). A similar effect can be observed for fraction 5 of the density gradient which is enriched in mitochondrial markers and where the Ca content falls by 82%. There is also a considerable reduction in Ca content of the less dense fractions of the gradient (not given). The content in the other cations is not significantly affected by homogenization of tissue in saline. In the synaptic vesicle fraction (compare last line of Table 2 with line 4) there is little change in K and Mg whereas the contents in Ca are markedly reduced. It is likely that the reduction in the Ca content of the denser fractions leads to a reduction in contamination of the less dense fractions and we therefore assume that the lower value of vesicular Ca content obtained by homogenization in saline approximates best the true vesicular Ca content. Stoichiometry of metal ions and acetylcholine in synaptic vesicles. The significance of the metal ion content of synaptic vesicles can best be appreciated by comparison with their transmitter content. In Table 3 the metal ion content per mol of ACh is given. For calculation of the ratio of Ca to ACh the value obtained from NaCl gradients was used. In the case of Na the vesicular value was corrected for contamination with soluble Na ions by subtracting the mean of the Na contents of the two neighbouring fractions (2 and 4a, Table 2) from the content of the vesicle fraction (3). Using this correction the ratio Na:ACh is reduced from 0.47 to 0.30. Table 3 also shows the ratios obtained from a single experiment where vesicles were isolated on a continuous sucrose-glycine gradient in a zonal rotor. The results are comparable to those obtained after separation of vesicles with a step gradient. They also indicate the large variance encountered due to the low yield of metal ions in the isolated synaptic vesicle fraction. Possible redistribution of metal ions during

subcellular

frac-

tionation

The results presented so far suggest that reproducible amounts of metal ions are isolated in combination with the various subcellular fractions. It is, however, not clear to what extent these values represent the in oiuo situation or

R. SCHMIDT, H. ZIMMERMAW and V. P. WHITTAWK

630

TARLt 2. METAL. ION CONTENT ot IXNSITY GRADIENT ~‘RAC’TIONS ~____~~

Fraction

Protein (/Ig.g wet wt of tissue ‘I

la lb

13.2 f 0.3 21.9 + 1.6

81.8 ) 42.5 f

2 3 (VF)

41.0 f 9.1 59.3 ) 3.4

29.2 49.5 (31.2 7.4 5.7 6.8 12.7 2.8

4a 4b 4c 5 (MF) 6

21.8 24.6 17.4 139.8 9.7

3 NaCl

* i ) ) *

2.8 3.3 2.6 16.2 3.3

Nii

K (ng-at0m.g

n.d.

13.2 10.4

8.3 i_ 1.8 4.4 & I.1

_+ 5.3 * 8.5 * 5.4) i 2.4 + 2.0 ) 2.6 f 2.3 ) 1.x

2.3 + 1.0 Il.8 * 7.3 I.3 0.7 0.6 0.7 0.2

n.d.

Ca

Mg wet wt of tissue- I)

+ 0.4 f 0.4 i 0.3 _I: 0.5 + 0. I

3.5 ) 0.6

57.9 f

3.6 i_ I.2 2.2 & 0.4 5.x * 1.6

37.3 * x.1 33.3 + 5.9 53.1 * 7.0

1.9 1.3 I.2 3.1 0.3

13.7 + 3.1

f + + i *

0.8 0.7 0.6 I.1 0.2

34.9 47.8 38.7 24X.6 l7.?

4.0 * 0.x

k & * i_ *

16.6

6.7 4.2 9.8 27.0 1.9

16.4 * 3.2

Density gradients were loaded with the resuspended and washed crude vesicle fraction Pi and centrifuged at 57,750g for I25 min. Each gradient was loaded with an equivalent of 30 g (wet wt) ol electric tissue. Na contents given in parentheses are corrected for soluble contamination from neighbouring fractions (see text). NaCl (column I) indicates that the vesicle fraction (VF) was prepared by substituting 0.4 M NaCl for 0.8 M gIycine in Fig. I. Values are means ) S.E.M.of four (glycine) or eight (NaCI) experiments; recoveries for glycine gradients (9; of parent fraction loaded) were: protein (94). Na (103). K (120) Mg (80). Ca (87); MF. membrane fraction; nd., not determined. are affected by redistribution of ions on tissue fractionation Millipore filtration-strength qf mrtul ion binding. In a first series of experiments to test this possibility the effect of Millipore filtration per se on intact and water shocked vesicles was investigated. Subjecting isolated synaptic vesicles to an additional filtration step permits the strength of metal ion binding to these subcellular organelles to be investigated. This is shown in Fig. 5. It demonstrates the binding capacity of the various metals to synaptic vesicles (‘x of ion retained on the filter) and relates it to the ionic potential (ratio of electrical charge to ionic radius) of the metal. Filtration leads to a loss parrticularly of alkali metals with smaller changes in divalent cations. The Figure suggests that the strength of ion binding to the vesicle is linearly related to the ionic potential of the metal. When synaptic vesicles were first shocked with distilled water and then subjected to filtration an additional loss of Na and K and particularly of Ca was observed. The proportion of Ca lost from shocked vesicles is higher than would be anticipated from its ionic potential. This would suggest that part of the vesicular Ca is not bound to membranes but also occurs in a soluble form in the synaptic vesicle core. The large variances are caused by the low ion contents of the vesicles and the difficulty of obtaining filters of reproducible purity (see Experimental Procedures). For appropriate blanks a solution was passed through Millipore filters, that had a similar ionic composition as the vesicle fraction but contained no biological material.

Addition of LiCI. Another way of obtaining information regarding the specificity of metal ion binding to synaptic vesicles is the addition of metal ions either to the homogenization medium or to the isolated vesicles, When LiCl (IOmM) was added to the first supernatant fraction S,, I”,, of the Li was recovered in the synaptic vesicle fraction, This is similar to the proportion of other alkali metal recovered from the same primary fraction (Na, 0.6”&,; K, 0.9’&) but differed from values obtained for alkaline earth metals (Mg, 2.7”,; Ca, 30.6aJ. Ion exchange of isolated vesicles. In a further series of experiments the effect of adding metal ions at varying concentrations to vesicles previously isolated in inorganic ion-free media was investigated. Na’ was chosen as representative for alkali and Ca *+ for alkaline earth metals. Vesicles were separated from the incubation medium by Millipore filtration. Figure 6 shows the time dependence of binding of Na and Ca when vesicles are incubated with excessive concentrations of NaCl (300 mM) and CaCI, (5 mM) respectively. These concentrations of metal ions are 2 to 3 times higher than those measured for the tissue homogenate (mM + s.E.M.: Na, 105 + 8; Ca, 2.4 + 0.1; ii = 13). The actual concents of freely soluble ions are presumably lower and approximated better by the assumption that the amounts recovered in the supernatant fraction S, (Fig. 1) are identical to the amounts of diffusible ions in the homogenate. These concentrations were calculated to be 60.4m~ for Na’ and 0.65 mM for Ca ‘+ As can be seen from Fig. 6 there is a time-dependent binding of both ions and chemical equilibrium is reached after 30 min.

TABLE 3. RATIOS OF CONCENTRATIONOF METAL IONS IN THE SYNAPTIC vt~su_u WAC‘TION TOTHATOF ACETYLCHOLINE

Type of gradient Step gradient Zonal gradient

Na 0.30 + 0.05 0.23

ratio (g-atom/mol) of K Mg 0.10 f 0.05 0.05

Methods as for Table 2. The zonal gradient details see Experimental Procedures).

0.07 * 0.02 0.03

represents

Ca 0.28 & 0.05 0.21

a single experiment

(for

Fig. 4. Electron micrographs of subcellular fractions from sucrose--glycine gradients. Upper micrograph: Vesicle fraction. It consists of synaptic vesicles with few contaminating membrane fragments. Lower micrograph: Membrane fraction. Besides Iarger membrane fragments it contains mitochondria (m) and numerous glycogcn particles (9). Bars indicate 0.5 pm. 631

Metal

100

.

% of control

0

d __--------k I_-------~-N0.30m.t *O”(L____

vesicles

Intact

shocked

633

ion content of cholinergic vesicles

a.

300

vesicles

/’

80 Ca

i

Ca.5lJf.t

/

/

100

/

No

--__

/

;I/

t

---__

i io

f

20 10n1c potentlal

30

-

LO

I p C/m I

tic vesicles isolated on sucrose-glycine gradients (4°C). After 30min chemical equilibrium is reached. Values represent means of four experiments with their standard errors (WM.).

FIG. 5. Relation between the metal ion content of the filtered vesicle fraction and the ionic potential. When synap-

tic vesicles isolated on sucrose-glycine gradients were filtered through Millipore filters metal ions were lost from vesicles. The percentage of original vesicular contents in ions which was retained on the filters is plotted against the ionic potential. The absolute values for metal ion contents of isolated vesicles are given in Table 2. Na values were corrected as described in Table 2 and Ca values derived from NaCl experiments were used. Filled circles and continuous line, filtration of intact vesicles (n = 6); open circles and dashed line, vesicles previous shocked with distilled water (n = 7). Vertical bars represent S.E.M. For calculation of ionic potentials (PC/m) (Na, 16.35; K, 12.05; Mg, 41.08; Ca, 30.23) ionic radii determined empirically by GoldSchmidt for a coordination number of 6 were used (cf. HILLER, 1952).

The effect of incubation of the synaptic vessicles in the presence of varying concentrations of NaCl is given in

Fig. 7. It will be noted that NaCl (up to 300 mM) is without effect on the alkaline earth metal content of the vesicles. The K content is, however, reduced by 40% even at Na concentrations which are in the range of those in the tissue homogenate (60 mM). By contrast the vesicular Na content increased by 20-30%. A further increase in the Na concentration does not lead to a further decrease in vesicular K content suggesting that the remaining K was not accessible to ion exchange with Na. Incubation of synaptic vesicles with CaCI, with Ca*+ concentrations in the physiological range (up to 2 mM) had no significant effect on vesicular metal ion contents. When synaptic vesicles are incubated with concentrations of CaCl, up to Lomb (4”C, 1 min) this is without effect on vesicular contents in both ACh and ATP. Effect of stimulation organs

(mm1

FIG. 6. Time course of binding of Na+ and Ca2+ to synapI

Electric

6’0

he

8’ 10

jo

tions of Ca containing structures to this fraction. Stimulation did not affect the protein content of the fraction and recovery of components was unchanged. Further separation of fraction Pi revealed that stimulation affects its various components in different ways (Table 5). The decrease in the Ca content of fraction Pi is mainly due to a fall in the Ca binding of the components sedimenting in fraction 5. The Ca content of this fraction decreased by 84”/, from 249 to 39 ng-atom, g wet wt- ’ (P < 0.01, t-test). On the contrary the synaptic vesicle fraction shows an increase particularly in its Ca content. In addition an increase in Mg contents could be observed. The Na content was unchanged whereas the K content was decreased. In view of the small absolute contents of K in this fraction this effect is, however, minute. Furthermore, stimulation caused a drastic depletion in vesicular

I ‘.-_A____ 5o 4

____________e

1’

1

I,

n

I

were stimulated

in vivo with 5000

pulses at 5 Hz via the electric lobe and subcellular fractions analysed for changes in ionic composition. Table 4 represents the changes in metal ion content which were observed in the washed crude synaptic vesicle fraction Pi. The most prominent change is the reduction in Ca contents. The large experimental error involved appears to be due to varying contribu-

30

100

ccnc

300 of

No

(mMI

FIG. 7. Influence of NaCl (1 h, 4°C) on metal ion contents of synaptic vesicles isolated on sucrose-glycine gradients. Part of the K is exchanged against Na whereas Ca and Mg are unaffected. Values represent means of four experiments with their standard errors (S.E.M.). Closed triangles, Na; open triangles, K; closed squares, Mg; open squares, Ca.

R. SCHMIDT, H.

634

TABLE,~.METAL ION CONTENT(NMOL.G WET WT-~)IN THE WASHED

CRUDE

1011

Na K Mg Ca Zn

VESICLEFRACTION, P;. STIMULATION

Control

Stimulated

166 & 45 25 & 6 123 * 30 658 + 179 9+2

192 _+61 17 +7 146 ) 57 374 f 160 13 _+3

AND

and V. P.

ZIMMERMANN

1’HANGEs

ON

guinea-pig

WWITTAUH

and rsrt forebrain; guinea-pig brain;

TAKER, 1966,

APREON, 1972, rabbit

brain

MAUGA>

HANK,

cortex);

b;:

WHYI-

TACHIKI &

indeed

K was

used as plasmic Most soluble

a marker for the distribution of soluble cytoconstituents. of the Mg is contained in the microsome and SLlpern~ttant fractions (BOROW~TZ F<.\PI'A & WEINER, 1965, bovine adrenal medulla; CLEMI-:NTL: &

Difference +26 -8 1-23 - 284 +4

MELIIOLESI,1975, guinea-pig exocrine pancreas). Ca is enriched in the mitochondrial fractions (BORUW~TZ or

Washed crude synaptic vesicle parent fractions P; were prepared according to Fig. 1, combusted in nitric acid and analysed for metal content. Values are means i_ S.E.M. from

seven experiments.

contents of ACR and ATP (both 94%) which is in a~eement with previous results (ZIM~RMANN & WHITTAKER, 197411).

al.,1965. bovine adrenal medulla; HANIG et trl.. 1972. rabbit brain cortex; CLEMENTI: & MEL~L~~SI. 1975_ guinea-pig exocrine pancreas; SC’HMIDTet al., 1978, Torpedo electric organ). The distribution of Zn roughly resembles that of Mg but it seems not to be specifically enriched in any fraction. A nonspecific distribution of Zn has been reported for the bovine adrenal medulla by BOROWlTz ~'tcrl. (1965).

DISCUSSION

Distrihufion

Many analyses of metal ion contents in sub~llu~ar fractions have been performed using isotope techniques. This approach has, however, two main shortcomings: the uptake per se of a certain isotope into a tissue or a subcellular fraction does not allow conclusions about its specificity of binding and the uptake of radiolabel is determined not only by the capacity of the binding sites but also by the rate of net uptake and isotope exchange. Analysis by atomic absorption spectrophotometry was therefore selected as a more direct approach. Distribution

of metal

ions in primary

fractions

oimetul

ions in densit_y gradients

Three main fractions were obtained in our studies. The soluble fraction was rich in free metal ions partly derived from the cytoplasmic pool and partly by washout effects from subcellular particles. The membrane fraction was heterogeneous in composition. It contained sealed structures derived from presynaptic nerve terminals, fragments of pre- and postsynaptic membranes and mitochondria. Presumably due to the presence of mitochondria (SCHMIDT et al., 1978) it had a particularly high content of Ca. Synaptic vesicles were concentrated between the 0.2 M and O.4M sucrose sokition; the fraction was enriched in the vesicle markers ACh and ATP and contained traces of membrane fragments as the only

Most of Na and K was recovered in the supernacontamination detectable by electron microscopy. All tant fraction (S3) which is rich in soluble cytoplasmic components whereas‘ Ca was more enriched in the metal ions analysed were detected in the vesicle fraction. Part of the Na content could be attributed to particulate fractions; the distribution of Mg is intermediate. Although a direct comparison with other contamination by soluble Na salts and part of the Ca fractionation studies is difficult because of differences content was most likely derived from the membrane in procedures good agreement exists on the following fraction. Even after correction for these contaminations vesicuiar Na and Ca were particularly high points: reaching in g-atom units almost one third of the vesiK is mainly recovered in the soluble supernatant cular ACh. fractions (HOLLAND & AUDITORE, 1955, brain, liver, Table 6 compares the specific concentration of kidney of guinea-pigs and rats; RYALL, 1962; 1964, TABLE 5. EFFECT OF STIM[ILATIONON CONTENTS IN METAL~ONS

Condition Control Stimulated as y0 of control

ANU

A~~T~L~~oL~N~ OF THE SYNAPTIC VESICLL: FUACTIOP*’

Content of inorganic cations (ng-at0m.g wet wt ‘) or ACh (nmo1.g wet wt-‘) Ca ACh K Na Mg 49.5 li: 8.5 (31.2 + 5.4) 44.7 rf: 10.7 (30.8 * 7.4)

Total cation content ng equiv.g fly at0m.g wet wt -. ’ wet wt-’

f 1.8& 7.3

5.8 i 1.6

16.4 + 3.2

7x.9 * 6.1

144.1

-_I 166.3

6.3 _t 1.3

8.9 + 1.4

41.8 * 7.7

4.6 + 1.2

92.4

143.1

$3

154

255

6

64

X6

Methods as for Table 2. Results for stimulation experiments are derived from nine experiments. Na concentrations given in parentheses are corrected as for Tabie 2. For determination of total metal ion contents the corrected Na values and the Ca values derived from NaCl experiments were used. Ony the changes in the contents in Ca and ACh were significant (P < 0.01; t-test).

635

Metal ion content of choiinergic vesicles TABLE

6.

SPECIFIC CONCENTRATIONS

OFMETAL IONS (NO ATOM/MGPROTEIN) IN VESICLE FRACTIONS ISOLATEU FROMVARIOUS TISSUES

Na

K

Mg

Ca

214 -

48 -

44 -

35 60

526

200

9-l

217 70.5

-.

-_

-

125

Author

Source

HANIGet al. (1972)

Rabbit brain cortex

Mouse brain cortex

Ross (1977)

Torpedo electric organ Chromaffin granules

Present investigation BOROWITZ (1967)

bovine adrenalmedulla Chromaffingranules bovine adrenal medulla

SERCK-HANSSEN & CHRISTIANSEN (1973)

metal ions of the synaptic

vesicle fraction obtained from electric tissue to those obtained by other authors from brain synaptic vesicles and chromaffin granules. For all metals investigated the values are highest for Torpedo vesicles. This could be due to the higher purity of cholinergic vesicles but also to the larger diameter (twice that of brain vesicles) and the comparatively low protein content of cholinergic vesicles

potentials alkaf metals are at least in part exchangeable during tissue fra~ionation. As long as the number of negative electrical charges of the vesicles is not changed one would, however, expect constancy of the total positive charge contributed by vesicular metal ions.

(OHSAWA et al., 1979).

Isolated synaptic vesicles contain in addition to ACh and ATP the metal ions Na, K, Mg and Ca. The molar (atomic) ratio of the other constituents relative to ACh is 0.22,0.30,0.10,0,07 and 0.28 respectively or 0.97 together. At present we do not know to what extent these components are osmotically active and to what extent the positive charges of ACh and metal cations are counterbalanced by the negative charges of ATP, chloride or vesiculin (STADLER & WHITTAKER,1978). For the interior of chohnergic synaptic vesicles isolated from the Torpedo electric organ an ACh concentration of 0.55 M has been calculated

Specificity

of metal ion content of synaptic vesicles

The ionic composition of the isolated synaptic vesicle fraction was reproducible and different from other subcellular fractions-the homogenate, the unfractionated tissue and the solutions used for preparation of vesicles. On the other hand when Li’ ions which are not contained in electric tissue were added to the parent fraction Srz proportions of Li comparable to other alkali metals were recovered in the synaptic vesicle fraction. The addition of elevated Na+ concentrations (comparable to those in the cell homogenate) to isolated vesicles led to a loss of K and to an increase in the Na content of the vesicles whilst their Mg and Ca contents were not affected. On the other hand the addition of physiological concentrations of Ca2’ to the vesicle fraction did not influence the vesicular metal ion content. Thus it appears likely that the transition on tissue homogenization of synaptic vesicles from a high intracellular concentration of K+ to an elevated Na+ concentration could lead to an exchange of K’ versus Na+ whereas the alkaline earth metals are not likely to be affected to a significant extent. The loss of up to two-thirds of the alkali metal ion contents of the vesicles on Millipore filtration might be a consequence of a nonspecific uptake of these ions to vesicles isolated into a medium of low ionic strength. A large proportion of these ions could be bound rather loosely to the vesicle surface and could become lost on filtration. The loss of Ca and Mg under these experimental conditions is considerably smaller. In fact the correlation between ionic potential and strength of metal ion binding to intact vesicles suggests that the exchangeability of vesicular metal ions is largely dependent upon their ionic potentials. Thus alkaline earth metals and Mg in particular appear to be rather tightly bound constituents of synaptic vesicles, whereas due to their lower ionic

Signr~cance of metal ion content of synaptic vesicles

{BREER,MORRIS& WHITTAKER, 1978; ONSAWA et al.,

1979). If one assumes that all components were free in solution inside the vesicle they would add up to an osmolarity of 1.08 osmol. This is comparable with estimates of the osmolarity reported for body fluids of sharks and rays (0.8 to 1.1 osmol, BURGER, 1967; HOLMES& DONALDSON, 1969). It has been shown that Ca and Mg ions increase the mean apparent molecular weight of aggregates of ATP with biogenic amines (BERNEIS,PLETSCHER & DA PRADA,1969) and also that complexes of these metal ions with ATP or guanosine 5’-triphosphate incorporate biogenic amines (BERNEIS, DA PRADA & PLET~CHER, 1971). Such a binding mechanism in addition to the active catecholamine uptake (KIRSHNER, 1962) and the carrier mediated ATP uptake (KosTRON,WINKER, &ER & KOENIG, 19776) might help to maintain high concentrations of the secretory product in the storage granule (chromatlin granule) (see also PLETSCHER, DA PRADA, STEFFEN,L~TOLD AZ BEIINEIS, 1973; DA PRADA & PLEISCHER,1974). Since cholinergic vesicles from Torpedo also contain ATP part of the vesicular metal ion content could be involved in complex binding with the nucleotide (such as MgATP). However complexes with ATP and alkaline earth metal ions as described for the catecholamines do not seem to exist for ACh (KOBOS & RECHNITZ, 1976).

636

R. SCHMIDT. H. ZIMMERMANN

It has been proposed for chromaffin granules that thegranular uptake and subsequent exocytosis of Ca2 + might help to bwer the cytoplasmic Ca” concentration after stimulation of the chromaffin cell (SER~K-HANSSEN & CHRISTIANSEN. 1973; BOROWITZ, LESLIE& BAUGH, 1975; WINKLER,PEER, KOSTRON& GEXSSLER. 1975). A possible role for the metal ion content in transmitter release will be discussed in the subsequent section. Changes

in metal

ion content

on stimulation

Stimulatjon leads to an increase in vesicular contents of Mg and particularly Ca. There is a small decrease in both K and Na which is, however, of little significance due to the small alkali metal contribution in absolute amounts of positive charge. The large increase in the Ca content of the vesicle fraction is particularly significant as the parent fraction Pi shows a stimulation-induced decrease in Ca content. The loss of Ca from the latter fraction can mainly be assigned to gradient fraction 5 which was found to contain larger membrane fragments and also mitochondria. Thus the vesicle fraction is unlikely to be contaminated by particles normally present in fraction 5. These results suggest that the release of ACh from synaptic vesicles makes available negative electrical charge which was masked in vesicles from unstimulated controls. If the binding of metal ions to the negative charges corresponds to their ionic potential Mg would be most tightly bound. If a balance sheet is drawn of total positive charges contributed by ACh and metal ions (Table 5) it can be seen that the amount of charge in ng equivalents after stimulation was about 85% of the control value. Since stimulation of 5 Hz will also cause a reduction in synaptic vesicle number (up to 50%, ZIMM~RMA~~ & WH~TAKER, 1974~) the total amount of positive charge per vesicle may even be slightly increased. It has been suggested previously that the release of ACh (WHITTAKER.1973) or other transmitter substances which carry a positive charge (UVNAS.1973)

and V. P. WHI~TAKER

occurs in ion exchange against metal cations on cxocytosis (Z~MME~MA~N, 1979). Our results are in broad agreement with such a model. Since Ca’ + ,XJ~,~t+does not cause release from intact synaptic vesicles iI appears possible that the ion exchange of vesicular ACh t’s metal ions takes place during the process of exe- and endocytosis when the lumen of the vesicle is open to the extracellutar space, This ion exchange could be the actual driving force for release of transmitter from the fusing vesicle. A possible candidate for the carrier of negative charge inside the vesicle would be the vesicular gly~osaminogiy~dn (vesiculin) (STADLER& WHITTAKER,197X). Reloading of synaptic vesicles could be a reversal of this cation exchange. CARPENTER & PARSONS (1978) report that synaptic vesicles isolated on NaCl. sucrose density gradients from -f. c~~~~~ur~~icu lose their Na content in Na-free media on addition of an ionophor. The hyperpolarization of the vesicle membrane caused by the release of Na ions is subsequently partially reversed by the spontaneous influx of choline and ACh. Changes storayr

in the metal ion cmtwt

of other

swr’c~tor~~

vrsicles

Analysis of the ionic contents of secretory storage vesicles and their dynamic changes has mainly been restricted to alkaline earth metals. A stimulationdependent increase in the Ca content of secretory granules has been reported for neutrophile leucocytes (w00DfN & WIENEKE,1964) and later for c~orna~n granules isolated from the perfused adrenal gland (BOROWITZ,1969; SERCK-HANSSEN & CHRISTIANSEN, 1973). BOROWITZ (1969) furthermore observed an increase in the Mg contents of a less dense (adrenaline-rich) fraction but a decrease in a denser (noradrenaline-rich fraction). According to KOSTRON, WINKLER,GEISSLER& KOENIG (1977a) chromaffin granules possess a specific Ca” + uptake system. Whether this also applies to cholinergic vesicles needs to be elucidated.

REFERENCES BERNEIS K. H., DA PRADA M. & PLETSCHER A. (1971) A possible mechanism for uptake of biogenic amines by storage organefles: incorporation into nucleotide-metal aggregates. Exprrientia 27, 917-918. BERNEIS K. H., PLETSCHER A. & DA PRADA M. (1969) Metal-dependent

their storage and release.

Nature,

Land.

aggregation

of biogenic

amines:

a hypothesis

for

244, 281-283.

B~ROWITZ J. L. (1967) Calcium binding by subceilular fractions of bovine adrenal medulla. J. CrlI Physiol. 69, 305-310. BOROWJTZ J. L. (1969) Effect of a~tylchoiine on the subcellutar distribution of 45Ca in bovine adrenal medulla. Bi~c~em. Pharmac.

l&715-723.

BORO~ITZ J. L.. FUWA K. & WEJNER N. (1965) Distribution of metals and catecholamines in bovine adrenal medulla subcellular fractions. Nature, Lond. 205, 42-43. BOROWITZ J L., LESLIE S. W. & BAUGH L. (1975) Adrenal catecholamine release: possible termination mechanisms. in Calcium Transport in Confrnction nnd Secretion (eds CARAF~LJ E., CLEMENT! F., DRABIKOWSKI W. & MARGRET~I A.). pp. 227-234. North-Holland, Amsterdam. BREER H., MORRJS S. J. & WHJ’RAKER V. P. (1978) A structural model of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata deduced from density measurements at different osmotic pressures. Eur. J. Eiochrm. 87, 453.-458.

637

Metal ion content of cholinergic vesicles

BURGERJ. W. (1967) Problems in the electrolyte economy of the spiny dogfish, Squnlus acanthias. In Sharks,Skatesand ~~~~(edsGILBERT P. W., MATHEWSON R. F. & RALL D. P.), pp. 177-185. The John Hopkins Press, Baltimore. CARPENTER R. S. & PARSONS S. M. (1978) Electrogenic behaviour of synaptic vesicles from Torpedo californica. J. biol. Chem. 253, 326329. CLEMENTE F. & MELD~LESIJ. (1975) Calcium and pancreatic secretion. J. Cell Biol. 65, 88-102. C~C~PERSTEIN S J. & LAZAROWA. (1951) A microspectrophotometric method for the determination of cytochrome oxidase. J. biol. Chem. 189,, 6655670. DA PRADA M. & PLETSCHER A. (1974) Mechanisms of 5hydroxytryptamine storage in subcellular organelles of blood platelets. Adu. Biochem. Psychopharmacol 10, 31 I-320. DE DUVE C., PRE~~MANB. C., GIANETTO R., WATTIAUX R. & APPELMANSF. (1955) Tissue fractionation studies-6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem. J. 60, 604617. DOWDALLM. J., BOYNEA. F. & WHITTAKERV. P. (1974) Adenosine triphosphate, a constituent of cholincrgic synaptic vesicles. Biochem. M. 140, I-12. HANIG R. C.,TACHIKIK. H. & APRISONM. H. (1972) Subcellular distribution of potassium, sodium, magnesium, calcium and chloride in cerebral cortex. J. Neurochem. 19, 1501-1507. HILLERJ.-E. (1952) Grundrg der Kristallchemie. Walter de Gruyter & Co., Berlin. HOLLANDW. C. & AUDITOREG. V. (1955) Distribution of potassium in liver, kidney and brain of the rat and guinea-pig. Am. J. Physiol. 183, 309-3 13.

HOLMESW. N. & DONALDSONE. M. (1969) The body compartments and the distribution of electrocytes. In Fish Physiology, Excretion, ionic Regulation, and Metabolism (eds HOARW. S. & RANDALLD. J.), Vol. 1, pp. I-89. Academic Press, New York. ISRAELM., GAUTRONJ. & LESBATS B. (1970) Fractionnement de I’organe electrique de la Torpille: Locahsation subcellulaire de I’acetylcholine. J. Neurochem. 17, 1441-1450. JANDERG. & BLASIUSB. (1967) Lehrbuch der analytischen und priiparatiuen anorganischen Chemie. Hirzel Verlag, Stuttgart. KIRSHNERN. (1962) Uptake of catecholamines by a particulate fraction of the adrenal medulla. J. biol. Chem. 237, 2311-2317. KOBOS R. K. & RECHNITZG.

A. (1976) AcetylcholineeATP

binding by direct membrane electrode measurement. Biochem.

biophys. Res. Commun. 71, 762-767. KOSTRON H., WINKLER H..

GEISSLERD. &

KSNIG P.

(1977a) Uptake of calcium by chromaffin granules in vitro. J.

Neurochem. 28, 487493. KOSTRON H., WINKLER H., PEER

L. J. & KBNIG P. (1977b) Uptake of adenosine triphosphate by isolated adrenal chromaffin granules: A carrier-mediated transport. Neuroscience 2, 159-166. KOTZ L., KAISERG., TSCH~PELP. & T~LG G. (1972) Aufschlub biologischer Matrices fur die Bestimmung sehr niedriger Spurenelementgehalte bei begrenzter Einwaage mit Salpeterslure unter Druck in einem TeflongefLB. Z. Anal. Chem. 260, 207-209. MANGANJ. L. & WHITTAKER V. P. (1966) The distribution of free amino acids in subcellular fractions of guinea-pig brain, Biochem. J. 98, 128137. OHSAWAK.. DOWE G. H. C., MORRISS. J. & WHITTAKERV. P. (1979) The lipid and protein content of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata purified to constant composition. Implications for vesicle structure. Brain Res. 161, 447457. PLETSCHER A., DA PRADAM., STEFFENH., L~TOLDB. & BERNEISK. H. (1973) Mechanisms of catecholamine accumulation in adrenal chromaffin granules. Brain Res. 62, 317-326. ROSS D. H. (1977) Calcium content and binding in synaptosomal subfractions during chronic morphine treatment, Neurochem. Res. 2, 581-593. RYALLR. W. (1962) Sub-cellular distribution of pharmacologically active substances in guinea-pig brain. Nature, Lond. 1%,680-681. RYALLR. W. (1964) The subcellular distributions of acetylcholine, substance P, 5hydroxytryptamine. y-aminobutyric acid and glutamic acid in brain homogenates. J. Neurochem. 11, 131-145. SCHAFFNER W. & WEISSMANN C. (1973) A rapid, sensitive and specific method for the determination of protein in dilute solution. Analyt. Biochem. 56, 502-514. SCHMIDTR. & ZIMMERMANN H. (1977) The effect of stimulation on the distribution of Ca2+ and other metal ions in the cholinergic synaptic vesicle and nerve terminal sac fraction derived from the electric organ of Torpedo marmorma. In Synapses (eds COTTRELL G. A. & USHERW~~DP. N. R.), pp. 362-363. Blackie, Glasgow. SCHMIDTR., ZrMMERMANN H. & Jo6 F. (1978) Release of calcium from mitochondria in the electric organ of Torpedo marmomtaduring nerve activity. Proc. Europ. Sot. Neurochem. (ed. NEUHOFFV.), Vol. 1, p, 595. Verlag Chemie, Weinheim. SCHM!DTR., ZIMMERMANNH. & WHITTAKERV. P. (1976) Quantitative analysis of the metal ion content of chohnergic sYnaptic vesicles by atomic absorption spectrophotometry and changes induced by electrical stimulation. Expl. Brain Res. 24, 19-20. grack-HANSEN G. & CHRISTIANSEN E. N. (1973) Uptake stimulated in vitro. Biochim. biophys. Acta 307, 404414.

of calcium

in chromaffin

SHERIDANM. N., WHIlTAKER V. P. & ISRAELM. (1966) The subcellular fractionation Zellhorsch. mikrosk. Anat. 74, 291-307.

granules

of bovine

adrenal

medulla

of the electric organ of Torpedo2.

STADLERH. & WHITTAKERV. P. (1978) Identification of vesiculin as a glycosaminoglycan. Brain Res. 153, 408-413. STANLEY P. E. & WILLIAMS S. G. (1969) Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal. Biochem. 29, 381-392.

63X

R. SCHMIDT. H. ZIMMERMANNand V. P. WHITIAKI II

SUSZKIW J. B. & WHITTAKER V. P. (1979) Role of vesicle recycling in vesicular storage and rclcase of acctylcholme 111 Torpedo electroplaque synapses. Prog. Bruin Res. 49, in press. TASHIRO T. & &ADLER H. (1978) Chemical composition of cholinergic synaptic vesicles from Torprdn MW~OI’LII~hascd on improved purification. Eur. J. Biochrm. 90, 479487. C!vrI& B. (1973) An attempt to explain nervous transmitter release as due to nerve impulse-induced cation cxchangc 4<.ro pl~j~iol. xmd. 87, 168-l 75. WHIT~‘AK~KV. P. (1973) The storage of acetylcholine in presynaptic nerve terminals. Sci. Bus. 121ed/c. nn,l. RCI. 17 31. WHITTAKI:R V. P. & BARK~.K L. A. (1972) The subcellular fractionation of brain tissue Gth special reference 10 the preparation of synaptosomes and their component organelles. In Meth. Neloochc,m. 2. I 51. WHITTAUR V. P., ESSMANW. B. & Dowc G. H. C. (1972) The isolation of pure synaptic vcs~clcs from the electric organs of elasmobranch fish of the family Torpedinidae. Biochmfil. J. 128, 833-846. WHII-IAK~R V. P., MICHAELSON I. A. & KIRKLAND R. J. A. (1964) The separation of synaptic vesicles from ncrvc-ending particles (‘synaptosomes’). Biochcm J. 90, 293 303. WINKUR H.. PEER L.. KOSTRON H. & Gmst.tx D. (1975) Calcium and the catecholaminc-storing vesicles of adrenal medulla. In C&km Trap7sporr it! Contraction and Srcrrfiorl (eds CAKAFOLI E.. Ct.tMt:iv’rI F.. DRADIKOWSKI W. & MARGRETH A.). pp. 243 251. North-Holland, Amsterdam. W~KHNN A. M. & WII-:NI:KI.A. A. (1964) The participation of calcium, adenosine triphosphate and adenosine triphosphatase in the extrusion of the granule protems from the polymorphonuclear leucocyte. Biochern. J. 90. 49X-509. ZIMMERMANNH. (1979) Vesicle recycling and transmitter release. Neuroscience 4, 1773 1803. ZIMMI:RMANNH. & D~:NSTONC. R. (1977) Separation of synaptic vesicles of different functional slates from the chohnerglc synapses of the Torpedo electric organ. Neuroscirrw 2. 715 730. ZIMM~RMAXN H. & WHITTAK~~RV. P. (1974~) Effect of electrical stimulation on the yield and composition of synapttc vesicles from the cholinergic synapses of the electric organ of Torpedo.. A combined biochemical. electrophysiological and morphological study. J. Neurochcm. 22, 435- 450. ZIMMIRMAKN H. & WHITTAKI:R V. P. (1974h) Different recovery rates of the electrophysiological. biochemical and morphological parameters in the cholinergic synapses of the Torpedo electric organ after stimulation J. Ncuroc~hcvx 22. 1109 1114.

(Acceptrd 9 October

1979)