(Received for publication 3 February 1958)

Transcription

1 187 J. Physiol. (1958) I42, I87-I96 INTRACELLULAR DISTRIBUTIONS OF ACETYLCHOLINE AND CHOLINE ACETYLASE By CATHERINE 0. HEBB AND V. P. WHITTAKER From the A.R.C. Institute of Animal Physiology, Babraham, Cambridge (Received for publication 3 February 1958) There have been many suggestions that a large part of the acetylcholine (ACh) of nervous tissue is not in a freely diffusible form but instead is bound in some way to the tissue; and that the binding may involve some kind of subcellular particle such as the mitochondrion (Bodian, 1942; Brodkin & Elliott, 1953). From Feldberg's (1945) analysis of the problem it appears that the ester enters the bound state as it is produced: or, as he has phrased it, 'acetylcholine... is synthesized into this linkage'. It may be therefore that within the cell the ester is in close spatial association with the enzyme of synthesis, that is with choline acetylase. The intracellular distribution of this enzyme was studied by Hebb & Smallman (1956) who found that after differential centrifugation of rabbit brain homogenates, 50-70% of the choline acetylase present could be recovered in the granule fraction which included the bulk of the mitochondria; while most of the balance was dissolved enzyme. The present inquiry was directed toward obtaining more information about this distribution and about its relation to the distribution of ACh. METHODS Differential centrifugation. Homogenates of nerve tissue were prepared as described by Hebb & Smallman (1956) with the differences that the suspension medium used was 032M instead of 025M sucrose and contained eserine (5 x 10-5 M). In each centrifugation the gravitational effect is expressed in g. min; that is, as the product of the gravitational force g and the time of spinning in minutes. In all experiments a Servall bench centrifuge was used to prepare the first two particulate fractions, the nuclear fraction, P 1, and the mitochondrial fraction, P2. After collection of P2, in most but not all experiments, the microsomal fraction P3 was separated from the final supernatant S, by centrifugation in a Spinco Model L preparative ultracentrifuge. The Spinco was also used to refractionate P2 by the density gradient technique as described by Kuff & Schneider (1954) and by Blaschko, Hagen & Hagen (1957). To collect the subfractions of P2, a cutter was used similar to the one described by Randolph & Ryan (1950) and designed by Dr Schuster for Blaschko and his co-workers. We should like to record our thanks to the latter authors for demonstrating their technique to us.

2 188 CATHERINE 0. HEBB AND V. P. WHITTAKER Enzyme measurements. Choline acetylase activity was measured by the method described by Hebb & Smallman (1956). In this method the homogenate and particulate fractions are treated with ether in order to obtain full enzyme activity. An approximate estimate of succinic dehydrogenase activity was obtained by a colorimetric method similar to that recently described by Kuriaki & Nagano (1957). Estimation of ACCh. The ACh in the homogenate and its fractions was assayed on the frog rectus abdominis muscle preparation using the precautions described by Feldberg & Hebb (1947) to avoid errors of measurement from sensitization of the muscle. To determine the total ACh content of a sample, any 'bound' ester was first released by treating the sample with acid. Two procedures which gave identical results were used. In the first procedure, that used in most experiments, the sample was brought to ph 4 by addition of IN-HCl and heated in a boiling water-bath for 10 min. In the second procedure (cf. MacIntosh & Perry, 1950) trichloroacetic acid was added to the sample to give a final concentration of 10% (w/v) and the mixture was left in the cold for 90 min; it was then centrifuged, the precipitate resuspended in 10% (w/v) trichloroacetic acid and recentrifuged. The supernatants were combined, extracted with ether and then aerated to remove the ether. Free ACh was measured by assaying the untreated samples of the homogenate and its fractions as soon as possible after their preparation. Samples for assay of total ACh could be stored in the cold at ph 4 after acid treatment without change. The difference between the total and free ACh content represents the bound ACh. Histological observations. Microscopic examination of the homogenate and of the particulate fractions was done routinely. Fresh smears stained with Janus Green (0-02% in aqueous 0-32M sucrose and 0 002M-NaCl) were observed during a period of min, depending upon the rate of development and disappearance of the colour reaction. In all experiments smears were prepared in two ways: some were fixed in10 % neutral formol-saline and stained with haematoxylin and eosin; others were fixed in 2% osmic acid and stained with aniline acid fuchsin. In some experiments additional smears were fixed in 10% mercuric formaldehyde and stained with Sudan III and IV or with Sudan Black. Sudan Black in 50% diacetin was also tried on some smears fixed briefly in formol-saline. RESULTS The nuclear fraction P1, precipitated at ,500 g. min, was the least homogeneous fraction. Histological examination showed that it contained all the nuclei, a few mitochondria, and varying amounts of intact cells and tissue fragments, among which were neuroglia, pieces of blood vessels and connective tissue. The mitochondrial fraction P2, precipitated at 450,000 g. min, contained the bulk of the mitochondria. The microsomal fraction P3 was separated from the final supernatant,s, by centrifugation at 3,000,000 g. min. To obtain this fraction Hebb & Smallman (1956) had used a centrifugal force of only 1,500,000 g. min. We doubled it in order to be certain that the choline acetylase in the final supernatant was dissolved enzyme and not enzyme associated with particles that had not been precipitated by the final centrifugation. Our results confirmed their conclusion that the activity of the supernatant was due to dissolved enzyme. The histograms of Fig. 1 show the results of three experiments on rabbit brain, one on guinea-pig brain and one on the sheep caudate nucleus. There is a high degree of correlation between the distributions of enzyme and ester, a

3 INTRACELLULAR ACh AND CHOLINE ACETYLASE 189 fact which strongly suggests that the two have the same intracellular distribution. The correlation is particularly striking when the values for enzyme and ester in the supernatant fraction of the five experiments are compared. In one experiment, in which the optic lobes and cerebral hemispheres of six pigeons were analysed, there was again good agreement between the distributions of ACh and choline acetylase in the different fractions, but the results differed from the previous ones in that among the optic lobe fractions the supernatant contained the largest amount of enzyme and P2 had relatively less activity. Rabbit brain Rabbit brain Rabbit brain Guinea-pig Sheep caudate S brain nucleus _ (135%) (75%) (85%) (100%) (108%) v:.0 ;gtriid (134%) (70%) (79%O) (98>6%o) (99% ).50 tj P1P2P3 S Pl P2 P3 S PIP2P3 S P1P2P3 S Pl P2 P3 S Fig. 1. Histograms showing distribution expressed as percentage of total recovery in fractionated homogenate of ACh (upper row) and of choline acetylase (lower row). The figures in brackets show the total recoveries from the fractions, expressed as percentage of activities in the original homogenate. In all experiments there was a tendency for the sucrose-suspended homogenates to clump and sediment. Because of this, sampling of the whole homogenate for analysis was liable to error unless it was vigorously mixed immediately beforehand; and it was due to inadequate mixing in Expts. 1-3 of Fig. 1 that the values for total enzyme and ester recovered from the separate fractions did not correspond with those found for the original homogenate. Nevertheless, the correspondence between the recovered choline acetylase and recovered ACh in individual fractions was surprisingly good, when these were expressed as a percentage of the total recoveries. Histological examination of the mitochondrial fraction revealed the presence of particles which were variable in shape and size. All appeared to be sudanophilic, were stainable by aniline acid fuchsin and gave a colour reaction with Janus Green. None of the stains seemed to have any special affinity for one type of particle. Some particles appeared to be simple vesicles; others were more complex in structure. Both spherical and elongated particles were present. The largest were about 4-5 yl in length, the smallest too small to be visualized clearly with the conventional microscope. In the first two experiments in which P2 was refractionated this was done

4 CATHERINE 0. HEBB AND V. P. WHITTAKER over layers of 0x8, 1-2, 1x6 and 2-0M sucrose and the tubes were layered less than 1 hr before centrifuging. Since the 1-6 and 2-OM layers were virtually devoid of activity, the refractionation in subsequent experiments was done over layers of 0-8, 1-0, 1-2 and 1-6M sucrose. In these experiments the tubes were prepared 1-5 hr before centrifuging. Within these limits the time interval had no detectable effect on the results. As the material itself was suspended in 0-32M sucrose the gradient at the top of the tube was M. The results of the first experiment are given in Fig. 2. The three subfractions A, B, and C were collected by pipette from the layered tubes without using the Schuster cutter. A consisted of the 0-32M and 0-8M sucrose layers, I (100 UL. 50 r100 e= ~~~~(105%) o 0 C4> Subfractions A Sucrose concn. (M) c Fig. 2. Histograms showing distribution of ACh (upper row) and choline acetylase (lower row) in subfractions of P2 obtained by centrifugation over sucrose layers of graded densities. Ordinates are percentage of total activity recovered from the combined subfractions. The figures in brackets show the total recoveries from the subfractions expressed as percentage of activities in unfractionated P 2. B of the 1-2M layer and C of the remaining layers. There is good agreement between the distributions of choline acetylase and ACh in the three subfractions. Moreover, the activity in the combined subfractions represents full recovery of both enzyme and ester. In all other experiments in which P2 was refractionated the cutter was used to separate the subfractions. The results of two of these experiments, illustrated in Fig. 3, show that the highest concentrations of ACh and of choline acetylase are in the particles separating out within the density range M sucrose. However, the agreement between the ester and enzyme distributions is not so good as in previous experiments; further, whereas the enzyme is all recovered the recovery of the ester is only about 50 %. This low ester

5 INTRACELLULAR ACh AND CHOLINE ACETYLASE 191 recovery is due to the fact that before being assayed the samples were dialysed for hr against 032M sucrose in order to make them isotonic since in higher concentrations sucrose interfered somewhat with the assay of ACh. That the loss of ACh was due to dialysis was shown by control tests in which samples of unfractionated P2 were also dialysed for a similar period and lost approximately the same amount of ACh. In another experiment (Fig. 4) the fractionation procedure was modified. Three subfractions of P2, A, B and C, were first collected. Subfraction A contained all the 032M and half of the 08M sucrose layers; subfraction B 100 (50 %) (52%) V - <o -100 > (120%) (117 %) U 0 Hstogramst ~i o * Sucrose concn. (M)030812I Fig. 3. Hitgasshowing distribution of ACh (upper row) and choline acetylase (lower row) in subfractions of P2 obtained by centrifugation over sucrose layers of graded densities. Ordinates are percentage of total activity recovered from the combined subfiractions. The figures in brackets show the total recovreries from the subfractions exrpressed as percentage of activities in unfractioned P2. Assays for ACh were carried out on dialysed subfractions (see text). the remainder of the 0-8M, all the 1*OM and half of the 1-2M layers; while fraction C contained the remainder of the 1-2M and the whole of the 1-6M layers. Part of each of these subfractions was used to determine its choline acetylase activity; the remainder of each was made isotonic by the following procedure. The sample was first diluted with sufficient water to bring the concentration of sucrose to 0-32M; it was then centrifuged, the precipitate and supernatant collected separately and the precipitate resuspended in the original volume of 0-32M sucrose. The resuspended samples were called Ap, Bp and Cp to distinguish them from the untreated subfractions and were used for the assay of ACh in Fig. 4. Some of Bp was then refractionated over a narrower range of sucrose densities, 1-6M sucrose being omitted, and four further

6 192 CATHERINE 0. HEBB AND V. P. WHITTAKER subfractions collected. These subfractions were made isotonic by the dilution procedure just described and the resuspended precipitates, called a, b, c and d, were assayed for both ACh and choline acetylase activity. As shown by the histograms under (2) in Fig. 4, subfraction B contained most of the ester and enzyme activity. However, as in the two previous experiments, there was incomplete recovery (45 %) of ACh and this was again due to the treatment to make the subfractions isotonic, since in a control experiment it was found that with similar treatment a sample of unfractionated P 2 also lost about 50% of its ACh. While as shown in the figure there was full recovery of choline acetylase in the subfractions A, B and C, additional -~100 (1) (2) (3) O (84%) (45%) (114%) U ~ 0 0 r10 A(BP(C% 0 ~~(98%) 0n( 00%) (1 00%0/. o 1-50 V 4.._ 0 o -a1 0 I-L, P1 P2 P3 [ i Sucrose concn. (M) S ' Fig. 4. Histograms showing distribution of ACh (upper row) and choline acetylase (lower row). Ordinates are percentage of total activity recovered from the combined subfractions. The figures in brackets show the total recoveries from the subfractions expressed as percentage of activities in homogenate (1), in subfractions of P2 (2) and in.subfractions of Bp (3). measurements of choline acetylase made on samples of Ap, Bp and Cp showed that in addition to the loss of ACh they had also lost about 50% of the enzyme. That is, these samples were only about half as active as the untreated samples. However, in the case of Bp it was found that the loss was fully accounted for by enzyme present in the supernatant obtained from its preparation. It is probable, therefore, that the loss of ester and enzyme from the treated subfractions was due to diffusion into the suspending medium occurring as the result of dilution and centrifugation, or to incomplete sedimentation during the centrifugation. The results of the refractionation of Bp are shown in the histograms under (3) in Fig. 4. The particles containing the ester and enzyme are again spread over the M layers. Relatively few particles are as heavy as 1-2M sucrose,

7 INTRACELLULAR ACh AND CHOLINE ACETYLASE 193 but this layer contains a significant proportion of the total activity recovered; it appears that at least 80% of the particles carrying ACh and choline acetylase have a density equivalent varying from 0-8 to 1-2M sucrose and that their average density is nearer to the lower of these values. It may also be seen that the recovery of the ester and enzyme in the four subfractions, a, b, c, and d was complete. Since these had been treated in the same manner as Bp to make them isotonic, it appears that of the activity originally present in the particulate about half was due to ester and enzyme which was more firmly attached and less labile than the remaining half, which was lost from the particulate during the preparation of Bp from subfraction B. 032 A :_*a' _ Clear White z Oz8 _=_ B White - -- White EE b C Yellow o - Brown 1-2 V) D Grey Brown 1.6 E Clear Dark ppt. Fig. 5. Diagram showing distribution of pigmented material in P2 obtained from rabbit brain after centrifugation over layers of sucrose of graded densities. Sucrose concentrations of the layers shown on the left; the arrows on the right indicate levels at which the tubes were cut; the letters A-E designate the subfractions so collected. Their choline acetylase and succinic dehydrogenase activities are given in Table 1. Refractionation over sucrose effected some degree of separation of pigmented from non-pigmented material in the mitochondrial fraction. The appearance of the layered tubes after centrifuging is shown diagrammatically for a typical experiment in Fig. 5. In this experiment the tubes were cut so as to get all the strongly pigmented material which was in the 1 2M sucrose layer into one subfraction, D; and so that all the subfractions corresponded as nearly as possible to the original sucrose layers. In all five subfractions not only choline acetylase was estimated but also succinic dehydrogenase, which is known to be a mitochondrial enzyme. The results are given in Table 1. While choline acetylase was almost exclusively associated with the lighter and non-pigmented layers, the highest concentration of succinic dehydrogenase was in the fraction which contained most of the pigmented material. Presumably then the bulk of the mitochondria was in this fraction; and the inference could be drawn that choline acetylase was associated with some particles other than the mitochondria.

8 194 (7ATHERINE 0. HEBB AND V. P. WHITTAKER Bound and free states of ACCh and choline acetylase In Fig. 6, constructed from the averaged results of three experiments on rabbit brain, the distribution is shown of free and bound ACh in the homogenate and its fractions. There is some free ACh in the homogenate which is wholly accounted for by the free ACh in the supernatant; this fraction also TABLE 1. Choline acetylase and succinic dehydrogenase activity of separate subfractions A-E expressed as percentage of total recovered activity. Values refer to experiment illustrated in Fig 5. Choline Succinic acetylase dehydrogenase Fraction activity (%) activity (%) A 12 1 B 33 2 C D 6 75 E (4- U_ 0 i, 50 so 0 40 (4 C Homogenate P 1 P 2 P 3 S Fig. 6. Distribution of free 0, and bound * ACh in the homogenate and fractions prepared from rabbit brain. Averaged values of three experiments. contained some bound ACh, but it is not certain that the amount found is significant. If it were, it would indicate that ACh 'binding' cannot be explained simply in terms of a vesicular hypothesis, as for example its sequestration within the tissue organelles. As was to be expected no free ACh was found in the fractions P1-P 3. The bound ACh associated with them was inactive until released by experimental treatments which included acidification with HCI or trichloroacetic acid, shaking with ether, or freezing and thawing. We have also to distinguish between the bound and free states of choline acetylase. In agreement with earlier work (Hebb & Smallman, 1956; Gardiner, 1957) we found that the choline acetylase of the supernatant was active on

9 INTRACELLULAR ACh AND CHOLINE ACETYLASE 195 incubation without further treatment, but most of the enzyme in the particulate was inactive unless first treated with ether. There is thus some parallel with the difference in the state of free and bound ester. Moreover, it was found that treatments which activated or freed the enzyme had an analogous effect on the release of bound ACh. Thus ether treatment, which was 100 % effective in activating the enzyme, also released all the ACh. On the other hand, freezing and thawing, a procedure which is known to disrupt mitochondria, produced only a partial release of enzyme and similarly only' a partial release of ACh. For instance, in one experiment freezing and thawing 20 times caused a 50% activation of the enzyme in P2 and the release of 51 % ACh. Repeating the freezing and thawing more often had little more effect. This degree of activation corresponds to the loss which occurred in the amount of enzyme and ester when dialysing P2 or its subfractions, and is probably also due to liberation of a more labile fraction of ACh and choline acetylase. A discussion of the state of the bound ACh is given elsewhere (Whittaker, 1958). DISCUSSION The evidence we have cited here all tends to support the conclusion that ACh and choline acetylase reside in the same subcellular particle of nervous tissue. A similar conclusion was reached by Bellamy (personal communication) who found both the ACh and the choline acetylase, in the mitochrondrial fraction obtained from rat brain homogenates by differential centrifugation. Our evidence is cumulative and circumstantial. There is the close correlation between the intracellular distribution of choline acetylase and ACh which extends to the refractionated mitochondrial preparations; and there is the additional evidence that release of enzyme and ester from the particulate to the suspending medium occurs in the same proportions with such different treatments as freezing and thawing, dilution, and mixing with ether. The finding that about 50% of the particulate ACh and choline acetylase is released not only with the relatively mild procedures necessary to change the suspending medium from hypertonic to isotonic sucrose, but with the more drastic procedure of freezing and thawing as well, can plausibly be interpreted to mean that some of the constituent particles are more fragile than others, perhaps because they represent an ageing part of the total population. Another interpretation is that within the particle both enzyme and ester are partly free and partly bound to some part of its structure. Disruption of the particle then would lead to the discharge of only a portion of the active materials; the rest would remain attached until the structure was further decomposed. Whichever of these interpretations proves to be right the evidence is consistent with the view that ACh and the enzyme are held within the same subcellular particles; and that the ACh is *bound' and the enzyme inactive

10 196 CATHERINE 0. HEBB AND V. P. WHITTAKER because of a membrane barrier preventing exchange of substrates and reaction product between the inside of the particle and the surrounding fluid. It appears that the particles which contain ACh and its synthesizing enzyme are different from mitochondria. We have found that the particulate containing the highest concentration of succinic dehydrogenase, and presumably therefore most of the mitochondria, can be separated from the particulate containing most of the choline acetylase. Similar results have since been obtained by one of us (V.P.W.) for the distribution of ACh and succinic dehydrogenase. Although the particles which contain ACh and choline acetylase do not seem to be mitochondria their true nature has yet to be established. To accomplish that it will clearly be necessary to separate them more completely from the other particles which make up the so-called mitochondrial fraction. SUMMARY 1. Experiments on fractionated homogenates of brain tissue have shown that the intracellular distribution of acetylcholine is similar to that of choline acetylase. 2. The evidence indicates that both enzyme and ester reside in the same subcellular particle which, though found in the same fraction as the mitochondria, is thought to be another type of organelle. We wish to acknowledge valuable technical assistance given by Miss Jean Gilson and Miss Miriam Leonard. REFERENCES BLASCHKO, H., HAGEN, J. M. & HAGEN, P. (1957). Mitochondrial enzymes and chromaffin granules. J. Phy8iol. 139, BODIAN, D. (1942). Cytological aspects of synaptic function. Physiol. Rev. 22, BRODKIN, E. & ELLIOTT, K. A. C. (1953). Binding of acetylcholine..4mer. J. Physiol. 173, FELDBERG, W. (1945). Present views on the mode of action of acetylcholine in the central nervous system. Physiol. Rev. 25, FELDBERG, W. & HEBR, C. 0. (1947). The effect of magnesium ions and of creatine phosphate on the synthesis of acetylcholine. J. Physiol. 106, GARDINER, J. (1957). Experiments on the site of action of a choline acetylase inhibitor. J. Physiol. 138, 13P. HEBB, C. 0. & SMALLMAN, B. N. (1956). Intracellular distribution of choline acetylase. J. Physiol. 134, KUFF, E. L. & SCHNEIDER, W. C. (1954). Intracellular distribution of enzymes. XII. Biochemical heterogeneity of mitochondria. J. biol. Chem. 206, KURIARI, K. & NAGANO, H. (1957). Susceptibility of certain enzymes of the central nervous system to tetrodotoxin. Brit. J. Pharmacol. 12, MACINTOSH, F. C. & PERRY, W. M. L. (1950). Biological estimation of acetylcholine. Meth. med. Re8. 3, RANDOLPH, M. L. & RYAN, R. R. (1950). A slicer for sampling liquids. Science, 112, 528. WHITTAKER, V. P. (1958). The nature of bound acetylcholine. Biochem. J. 68, 21 P.