ACETYLCHOLINE METABOLISM AT NERVE-ENDINGS R. I. Birks & F. C. Macintosh

ACETYLCHOLINE METABOLISM ATNERVE-ENDINGS

R. I. BIRKS M.Sc. Ph.D.

F. C. MacINTOSH M.A. Ph.D. F.R.S.

Department of PhysiologyMcCill University, Montreal

1 Synthesis of acetylcholine2 Storage of acetylcholine3 Release of acetylcholine4 Conclusions

References

The main peripheral cholinergic systems have now beenrecognized and their functional significance in most cases isfairly well understood; by contrast only fitful progress hasbeen made in the identification of central cholinergicmechanisms. In recent years the focus of attention hasshifted to the analysis of cholinergic transmission at thecellular level, i.e., to the mechanisms involved in the forma-tion, storage, release and action of acetylcholine at synapses(including neuro-effector junctions). The present article dealswith the first three of these, namely, synthesis, storage andrelease.

1. Synthesis of Acetylcholine

So far as we know, the acetylcholine of vertebrate tissues issynthesized entirely by a specific enzyme, choline acetylase(Nachmansohn & Machado, 1943), present in all cholinergicneurones (and in some other cells), and capable of trans-ferring acetyl groups from coenzyme A (CoA) to choline(Feldberg & Mann, 1946; Lipmann & Kaplan, 1946; Nach-mansohn & Berman, 1946). Theoretically acetylcholine canalso be formed by a reversed action of the true, specific oracetyl cholinesterase, whose normal role is the hydrolyticdestruction of acetylcholine, and whose cellular distributionis somewhat similar to that of choline acetylase, but it docsnot appear that conditions favouring this back-reaction arcever met with in vivo. Efficient synthesis of acetylcholinetherefore requires the presence in adequate quantity of(i) choline acetylase, (ii) free choline, (iii) CoA, and (iv) activeacetate for the formation of acetyl CoA. This last require-ment can be fulfilled in a variety of ways, since carbohydrates,amino acids and fatty acids can all serve as sources of acetylfor interaction with CoA (cf. Novelli, 1953). The chemicalenergy required for the acetylation of CoA is usually madeavailable in the form of adenosinetriphosphate (ATP); thecommon penultimate step may be the formation of adenylacetate (Berg, 1955).

Choline acetylase. This enzyme occurs in very unevenconcentration in different parts of the nervous system. Asmight be expected, its distribution closely parallels that of

acetylcholine (Feldberg & Vogt, 1948). The best recent data(Hebb, 1955; Hebb & Silver, 1956) re-emphasize the contrastsnoted by earlier workers, and strongly support the idea thatthe two substances do not occur in non-cholinergic neurones,but are present in substantial quantity throughout the axonsof cholinergic nerves, as well as at their endings. It is generallysupposed (cf. Feldberg & Vogt, 1948) that choline acetylase,like other neuronal enzymes, is manufactured in the cell bodyand is carried peripherally by movement of the axoplasm(Weiss, 1944). Support for this idea has recently beenprovided by Hebb & Waites (1956); they found that cholineacetylase accumulates in the proximal stump of a severedcholinergic nerve during the days immediately following thesection, when it is disappearing from the distal part of thenerve and from the nerve-endings. Further work by Hebb &Smallman (1956) has shown that most of the choline acetylaseof brain is sedimented with the mitochondrial fraction incentrifuged sucrose homogenates. These results do not con-clusively prove the mitochondrial location of the enzyme,since it is conceivable that in their experiments smallercellular components became adherent to the mitochondria asa result of the fractionation procedures; the fractions howeverappeared on histological examination to contain no otherstructures, and moreover a number of other anabolic systemshave been found to be predominantly localized in mito-chondria. Some sort of subcellular particle, at any rate, mustbe particularly rich in choline acetylase; and Hebb andSmallman's finding, that their fractions exhibited fullsynthesizing activity only after treatment with ether, suggeststhat the enzyme may be located inside the particle rather thanon its surface, and is for that reason relatively inaccessible toits substrates.

Whether acetylcholine can be stored in the cellular struc-tures that manufacture it has not been demonstrated. Someinsoluble cell constituent, at any rate, can bind acetylcholinequite firmly (Loewi & Hellauer, 1938; Trethewie, 1938).Recent developments in electron microscopy have focusedattention on particles much smaller than the mitochondria,the so-called synaptic vesicles, which may be involved in thecarriage of acetylcholine from the structures that synthesizeit to the presynaptic membrane where it is set free. Theevidence for this will be discussed below.

Choline. This substance is a normal constituent of theextracellular fluid, where its concentration is of the order of10-' M (Bligh, 1952). Presumably, however, it is the intra-cellular level of free choline that is significant in relation toacetylcholine synthesis, and about this not much is known.It appears that choline does not generally penetrate veryreadily into cells (Lorente de N6, 1949; Bligh, 1953). Inexperiments on tissues containing intact nerve cells, forexample, sliced or minced brain (Quastel, Tennenbaum &Wheatley, 1936; Mann, Tennenbaum & Quastel, 1938) orsympathetic ganglia perfused with saline solutions (Brown &Feldberg, 1936), the extracellular choline concentrationappeared sometimes to be, and sometimes not to be, a limitingfactor for acetylcholine production. This is perhaps notsurprising, since the supply of active acetate in such prepara-tions may well be sub-optimal for acetylcholine synthesis, andthis rather than the intracellular choline level may be therate-controlling factor. Even in severe choline deficiency, areduction of tissue acetylcholine stores has not been clearlydemonstrated (Best, 1956). Probably, then, a sufficientquantity of choline is normally available at the intracellular

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sites of acetylation. Recent observations of our own stronglysuggest, however, that the intracellular stocks of choline arescanty, and must, when acetylcholine turnover is brisk, becontinually replenished from the extracellular fluid. Evidencefor this comes from experiments with a " hemicholinium "base HC3 (Schueler, 1955), which strongly depresses acetyl-choline synthesis by intact neurones (Macintosh, Birks &Sastry, 1956), almost certainly by competing with cholinefor a specific carrier located in the cell membrane (Birks,unpublished). The existence of such a choline-transportmechanism makes it easier to understand the observation(Birks, unpublished) that during prolonged activity a sym-pathetic ganglion can convert into acetylcholine—and release—as much as 30% of the choline carried to it by the blood.The presynaptic endings could hardly maintain such a rate ofcholine turnover unless they were equipped with some kindof special portal for the ingress of choline ions.

Coenzyme A and " active acetate ". CoA appears to be aubiquitous constituent of cells (Novelli, 1953), as would beexpected from its key position at the intersection of manymetabolic pathways. It is probably never a limiting factor foracetylcholine synthesis in vivo, except perhaps when its tissuelevels have been greatly reduced as a result of pantothenic aciddeficiency (Novelli, 1953; Bose, Gupta & De, 1954).

The " active acetate " required for the formation of acetylCoA can be supplied, as already noted, from a wide variety ofintracellular sources. A full discussion of the biochemicalmechanisms involved would be outside the scope of thisarticle. In cell-free systems either acetate or citrate canefficiently supply the two-carbon fragment (Balfour & Hebb,1952). Intact neurones, on the other hand, require an externalsource of glucose for the support of acetylcholine synthesis(Quastel et al. 1936; Kahlson & Macintosh, 1939; Crossland,Elliott & Pappius, 1955); lactate or pyruvate can replaceglucose, but acetate and citrate can not. For optimal syn-thesis, and for prolonged maintenance of the full acetylcholineoutput, the extracellular fluid must contain some other organicsubstance besides glucose and choline: this is an unidentifiedheat-labile factor present in plasma dialysates (Birks &Macintosh, unpublished). Glucose and the plasma factor areprobably significant in relation to the activation rather thanto the formation of the two-carbon fragment; it is doubtfulthat acetylcholine synthesis in vivo is ever limited by thesupply of acetate.

2. Storage of Acetylcholine

Brown & Feldberg (1936) found in their pioneer studies ofacetylcholine metabolism that the amount of acetylcholinethey could extract from a sympathetic ganglion appeared tohave little relation to the ganglion's immediate past history.For when a ganglion, perfused with Locke's solution con-taining glucose and eserine, was stimulated through its pre-ganglionic trunk for 30 min. or more, its output of acetyl-choline progressively declined, as already noted, to a lowsteady level and could be only partly restored by rest; yet sucha ganglion, when extracted, yielded about as much acetyl-choline as a ganglion that had never been stimulated.Synthesis, it seemed, had kept pace with release in thestimulated ganglion, but the newly-formed acetylcholine wasless easily discharged by nerve impulses. Perry (1953)explained these and other findings by postulating that theacetylcholine of the presynaptic endings exists in two forms,

one available for immediate release and one not so available;the unavailable form, he believed, can be made available at aslow steady rate which is independent of the parameters ofstimulation. He also suggested, on the basis of indirectevidence, that the presynaptic endings could take up most ofthe choline derived from the breakdown of the releasedtransmitter, and could use it for the synthesis of newavailable acetylcholine; extracellular acetylcholine protectedfrom hydrolysis by eserine could not be reclaimed in thisway.1

Perry's hypothesis of two forms of acetylcholine remains anattractive one, but needs some modification in the light ofnewer observations (Birks & Macintosh, unpublished experi-ments). In ganglia that arc perfused with eserinized plasmarather than with eserinized Locke's solution containingglucose, and can thus synthesize acetylcholine much moreefficiently, the extractable acetylcholine rises progressivelyduring an hour to about double the normal level, whether ornot the presynaptic fibres are being stimulated. If the per-fusion is then continued with eserine-free plasma, this surplusacetylcholine quickly disappears. The surplus acetylcholinemust therefore have accumulated in spaces which in theabsence of eserine contain active cholinesterase, and whichare not external to the presynaptic terminals; for, in theabsence of stimulation, no detectable acetylcholine leaks intothe eserinized effluent. The " surplus " is apparently not" available " in Perry's sense, since, as already mentioned, theoutput of acetylcholine from a ganglion perfused witheserinized plasma does not rise along with the acetylcholinecontent, but is maintained indefinitely at a steady level. Theacetylcholine contained in ganglia that have been perfusedwith eserinized Locke's solution and stimulated for a longtime is probably for the most part surplus acetylcholine whichcan be neither released, nor de-esterified so that it can be usedto make new available acetylcholine.

The simplest way to account for all these findings is tosuppose that the surplus acetylcholine is free in the pre-synaptic axoplasm and that the available acetylcholine is heldwithin subcellular particles of some sort. The idea that pre-synaptic acetylcholine is released from a particulate store hasalready been reached, on quite different grounds, by otherinvestigators (for references see Castillo & Katz, 1956 andEccles, 1957), most of whom have identified the acetylcholine-containing particles with the minute vesicles, of diameterabout 40 mix., which appear from electron micrographs toabound in the presynaptic axoplasm of all junctions so farexamined (Palade & Palay, 1954; de Robertis & Bennett,1955; Robertson, 1956). Katz and his co-workers (Fatt &Katz, 1952; Castillo & Katz, 1956), whose beautiful studieshave done most to elucidate this problem, have shown thatthe end-plates of resting amphibian and mammalian musclenormally exhibit small fluctuations of membrane potentialwhich can be due only to the spontaneous release of" packets " or " quanta " of acetylcholine, random in timebut roughly equal in size, and each containing hundreds orthousands of acetylcholine molecules (Fatt & Katz, 1952).The normal end-plate potential, which triggers the propagatedresponse of a muscle fibre, is undoubtedly set up by thenearly simultaneous release of a great number of suchquanta (Castillo & Katz, 1954b; Martin, 1955). Thesequantal phenomena have so far been seen only at theneuromuscular junction, but it seems highly probable that

1 See «ho Perry, p. 220 ofthli number of the Bulletin.—ED.

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they occur at all cholinergic endings, and indeed at synapsesgenerally.

The idea just discussed, that the extractable acetylcholineof nervous tissue is located mainly in the vesicular elementsthat characterize the presynaptic axoplasm, introduces certaindifficulties. Nerve axons, for example, seem to be free of theseelements (Robertson, 1956). Yet cholinergic fibres may con-tain about as much acetylcholine, weight for weight, as ganglia(Macintosh, 1941), and under certain circumstances mayrelease a little of it into an external medium in response tostimulation (Lissak, 1939). These findings might appear torule out the synaptic vesicles as carriers of acetylcholine.Since, however, the synaptic endings account for most of aganglion's acetylcholine but probably for no more than 1 % ofits volume, they must contain acetylcholine (and acetylcholine-carrying particles) in far higher concentration than the axonsfrom which they are derived. The acetylcholine of the axonsmay perhaps be held in vesicles so sparsely scattered that theyhave been overlooked by electron micrographers. Anotherpossibility is that the acetylcholine of the axons is all retainedwithin the mitochondria or other structures that manufactureit, and that it is only in the synaptic terminals that these loadtheir product into the smaller vesicles which deliver it to thepresynaptic membrane.

On balance, then, the evidence, while far from conclusive,suggests that two kinds of particle are involved in the supplyof acetylcholine to the releasing mechanism: one, perhapsidentical with the mitochondrion, whose function is mainlyrelated to synthesis of acetylcholine, and another, which maybe the synaptic vesicle, concerned with its storage and release.How these two are linked can only be guessed: possibly thevesicles originate from the endoplasmic reticulum (Palade,1956; Hodge, McLean & Mercer, 1956) which is in turnformed as a result of mitochondrial activity (Bernhard &Rouiller, 1956). Once formed, the vesicles can presumablyreach the presynaptic membrane as a result of their randomthermal movement; no special transport mechanism wouldseem to be required.

Whatever may be the form of the particle in which acetyl-choline is carried, the total amount of acetylcholine that canbe stored within a neurone is no doubt limited by the capacityof the carrier particles rather than by the neurone's ability tosynthesize acetylcholine. Resting nerve-endings, which con-tain their full complement of acetylcholine, continue tomanufacture and release it, but far more slowly than they dowhen they are active. Presumably, when activity ceases, theformation of acetylcholine and the formation of carriermaterial are both sharply reduced as soon as the depletedtransmitter stores have been replenished. It is hard to guesshow this braking action is applied to the transmitter " produc-tion line ": it can hardly be due either to exhaustion of rawmaterial, or to a mass-action effect resulting from accumula-tion of the finished product (unless all the available space inthe axoplasm is filled with it). A similar difficulty exists inconnexion with other specific cell products, such as thesecretory granules of exocrine gland cells.

3. Release of AcetylcholineFor any acetylcholine-containing particle within a pre-

synaptic ending, the probability that it will discharge itstransmitter store within the next few milliseconds is extremelysmall. The probability is greatly increased, though still smallOn a sympathetic ganglion it is about 10-*), when the ending

during that time is invaded by a nerve impulse. So far thefacts are well established. It is still far from clear, however,just what change in the particle's environment, consequent onthe arrival of the impulse, makes it more likely that the particlewill be mobilized and will unload its acetylcholine into theextracellular space. Presumably the particle must fuse at leasttemporarily with the membrane: it will not suffice that theparticle should merely rupture at the membrane's innersurface; for in that case, the acetylcholine that accumulates ineserinized nerve-endings should be available for release by thenerve impulse, but it is not. The local potential gradientsassociated with depolarization can hardly be steep enough tomove the particles, supposing that these are charged, morethan a small fraction of their diameter in the time available. Itis easier to imagine that the fusion of particles and membranemay be facilitated by a local diminution of axoplasmicviscosity that would permit increased Brownian movement ofparticles, or by some modification of the surface properties ofeither particle or membrane that would lead to diminution ofshort-range repulsive forces between them. Such changes asthese might result from a modification of the ionic composi-tion of the axoplasm close to the membrane, such as is knownto occur in axons when they become depolarized, or to analteration in the membrane itself.

If it is an ionic change in the axoplasm that triggers therelease of acetylcholine, this change can hardly be theentry of sodium ions, since the amount of acetylcholineliberated from a stimulated ganglion is independent of theexternal sodium level over a wide range (Hutter & Kostial,1955); moreover both the spontaneous quantal release ofacetylcholine, and the more copious outpouring that occurswhen the endings are depolarized by a rise of external potas-sium, can occur in the complete absence of sodium (Castillo &Katz, 1955; Hutter & Kostial, 1955). It is even less likely thatmovements of potassium ions arc responsible: the release ofacetylcholine by application of potassium is clearly second-ary to the depolarization this produces. A more attractivehypothesis would be that it is the inward movement of calciumions (Flflckiger & Keynes, 1955) that mobilizes the acetyl-choline units. The presence of calcium in the extracellularfluid is indispensable for the release of acetylcholine both bypresynaptic impulses and by potassium (Harvey & Mac-intosh, 1940); when the calcium concentration is changed,there is a corresponding and striking change in both the totalamount of acetylcholine discharged by each impulse (Hutter& Kostial, 1954), and in the number of quanta contributingto the discharge at each junction (Fatt & Katz, 1952; Castillo& Katz, 1954b). In an equally remarkable way, magnesiumdepresses acetylcholine release and antagonizes the junctionaleffects of calcium (Castillo & Engbaek, 1954; Castillo & Katz,1954a; Hutter & Kostial, 1954). One difficulty aboutsupposing that the entry of calcium is directly responsible formobilizing the acetylcholine units is that the spontaneousrelease of acetylcholine at frog motor-nerve endings is littleaffected by changes in external calcium or magnesium (Fatt &Katz, 1952). Quite possibly, however, it is the intracellular levelof calcium that matters; this is apparently always low (Keynes&Lewis, 1956),anditmaybenearlyindependentoftheexternallevel except where a temporary depolarization facilitates entryof these ions. Calcium ions that enter an axon are extrudedonly very slowly (FlOckiger & Keynes, 1955), and if the sameis true for nerve-endings, a rise of intracellular calcium mightaccount for the increased acetylcholine release, both resting

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and triggered by excitation, that follows FIG. 1.repetitive activation of the endings (Iiley& North, 1953; Castillo & Katz, 1954c;Liley, 1956a, 1956b). At present the biggestobstacle to any theory relating the lability ofthe acetylcholine stores to the internal cal-cium level would be the finding (Fluckiger& Keynes, 1955) that in Loligo axons theactivated calcium influx appears to be onlyabout six times the resting influx: to meetthis objection some adjuvant hypothesiswould have to be constructed. Furtherinformation about the factors controllingcalcium (and magnesium) fluxes at nerve-endings may clarify the situation.

Various other procedures are known toaffect acetylcholine liberation, both spon-taneous and induced by excitation, but theireffects have not as yet helped very muchin elucidating the mechanisms of release.Among these may be mentioned raising thetemperature (Fatt & Katz, 1952; Brown,1954; Kostial & Vouk, 1956), which canfacilitate both lands of release; applicationof botulinum toxin, which stops both kinds(Brooks, 1956); moderate anodal polariza-tion of the presynaptic endings, which in-creases the nerve-induced release withoutmuch effect on the spontaneous release(Castillo & Katz, 1954d); and raising theosmotic pressure, which greatly increasesthe frequency of the spontaneous discharge(Fatt & Katz, 1952; Boyd & Martin, 1956;Furshpan, 1956).

In addition repetitive presynaptic excita-tion may, as we have seen, lead to reducedacetylcholine output, with accompanying" fatigue " of transmission when the extracellular mediumis deficient in the materials required for optimal synthesis.When these materials are supplied, as in a ganglion perfusedwith plasma, we find (Birks, unpublished) that the acetyl-choline output per preganglionic volley is independent of thestimulation frequency over the range 2-20/sec.; at higherfrequencies than these it is the output per minute that isconstant, while the output per volley varies inversely with thefrequency and is poorly synchronized with the stimuli (cf.Castillo & Katz, 1954c); only at very low frequencies, 1/sec.or below, is the volley output higher than over the middlerange of frequencies. Such results as these suggest that atfrequencies below 20/sec. the time between impulses is undernormal circumstances sufficient for restitution of the availableacetylcholine; at higher frequencies the release is limited bythe rate at which the stores can be made available, thislimiting rate being, perhaps, the rate at which synapticvesicles move up to the inner surface of the membrane. Itseems improbable that junctional transmission is everimpaired, under physiological conditions, because there is ashortage of available acetylcholine at the nerve-endings.Prolonged high-frequency excitation tends to cause excessiverather than deficient post-synaptic action; if block occurs, itis because the transmitter is released asynchronously and thepost-synaptic membrane has no opportunity to recover fromits effects.

SCHEMATIC REPRESENTATION OF THE PROBABLE COURSE]]OF ACETYLCHOLINE METABOLISM AT NERVE-ENDINGS

INTRAC6U.ULAR EXTRAC«U.UL««

Synchronoustjtction _trtogtrtdby actionaattmlal

Processes involved in acetylcholine synthesis are shown In upper part of diagram;processes involved In acetylcholine storage and release are shown In lower part.Broken lines represent phase boundaries.

4. Conclusions

The findings discussed above permit the construction of atentative scheme (fig. 1) that would be compatible with whatwe know about acetylcholine metabolism in cholinergicneurones. In brief, mitochondria manufactured in theperikaryon are carried by axoplasmic flow to the nerve-endings. They contain choline acetylase, and contain or cansynthesize CoA, which becomes acetylated most efficientlywhen carbohydrate is being aerobically utilized. Cholineenters the nerve-endings by a special transport mechanism,and acetyl groups are transferred to it from CoA by themitochondrial choline acetylase; the acetylcholine thusformed is loaded by mechanisms unknown into small carrier-particles, the synaptic vesicles, which reach the presynapticmembrane by Brownian movement. A unit (some hundredsor thousands of molecules) of acetylcholine is released, anda unit of post-synaptic action occurs, whenever a vesiclemakes an effective collision with the presynaptic membrane.Such effective collisions occur singly at random intervalswhen the membrane is at rest, more frequently when it hasrecently been excited, and synchronously in large numberswhen it is depolarized by a nerve impulse. The problem ofwhat determines an effective collision is unsolved: the calciumconcentration of the presynaptic axoplasm may be animportant factor.

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