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  • J. Physiol. (1981), 314, pp. 255-263 255With 4 text-figure8Printed in Great Britain


    BY E. D. KHARASCH, A. M. MELLOW AND E. M. SILINSKY*From the Laboratory of Presynaptic Happenings, Department of Pharmacology,

    Northwestern University Medical School, 303 East Chicago Avenue,Chicago, Illinois 60611, U.S.A.

    (Received 30 July 1980)


    1. The effects ofintracellular application ofCa and Mg ions on evoked acetylcholinesecretion at frog motor nerve terminals were studied. Ca and Mg were applied to thenerve-ending cytoplasm using liposomes as a vehicle.

    2. Under conditions in which intracellular application of Ca produced many-foldincreases in evoked acetylcholine secretion, the addition of Mg intracellularly failedto affect evoked acetylcholine release.

    3. When Mg was applied to the nerve-ending cytoplasm concurrently with Ca,acetylcholine release was further increased above the level produced by introducingCa alone.

    4. The results suggest that intracellular Mg does not antagonize depolarization-secretion coupling and that antagonism of transmitter release by extracellular Mgoccurs only at the external surface of the nerve ending.


    The secretion ofneurotransmitter substances is thought to be coupled to membranedepolarization by the movement of Ca ions from the extracellular fluid throughspecific aqueous channels in the nerve-terminal membrane (Katz, 1969). Mg ions,which antagonize Ca currents in an apparently competitive fashion when present inthe fluid bathing the external surface of most Ca channels (Katz & Miledi, 1969a;Reuter, 1973), are presumed to act as competitive inhibitors of depolarization-secre-tion coupling (Jenkinson, 1957) by occluding the site of Ca entry into the nerveending (Martin, 1977; Silinsky, 1977). At variance with such an interpretation, whichassumes a single extracellular locus for the action of Mg, are the observations thatMg under some conditions enters nerve fibres (Baker & Crawford, 1971) and mayantagonize the excitatory actions ofCa at an intraterminal site involved in transmittersecretion (Katz & Miledi, 1969b; Miledi, 1973). For example, in studies on squid giantsynapse it has been shown that asynchronous transmitter release induced by theionophoretic application of Ca into the nerve ending is somewhat reduced by theintraterminal ionophoresis of Mg or Mn (Miledi, 1973). Other studies on the effectsof extracellular Mg have suggested even more complex inhibitory and excitatory

    * Please address all correspondence to Dr Silinsky.

    0022-3751/81/4450-1123 807.50 C) 1981 The Physiological Society

  • E. D. KHARASCH, A. M. MELLOW AND E. M. SILINSKYinteractions between intraterminal Mg and Ca (e.g. Cooke, Okamoto & Quastel, 1973;Hurlbut, Longnecker & Mauro, 1973). In view of these diverse impressions of thesites and actions of Mg, it appears of interest to study directly the effects ofintraterminal Mg on the Ca-dependent secretion of acetycholine (ACh) at the skeletalneuromuscular junction, a synapse which favours accurate electrophysiologicalmeasurements of quantal transmitter secretion. This paper describes such a studyusing ion-containing lipid vesicles (liposomes) as a vehicle for delivering ions into thecytoplasm of the nerve ending (Papahadjopoulos, 1970; Pagano & Weinstein, 1978;Theoharides & Douglas, 1978; Rahamimoff, Meiri, Erulkar & Barenholz, 1978;Gutman, Lichtenberg, Cohen & Boonyaviroj, 1979). A brief report of some of theseresults has been published (Mellow, Silinsky & Boyne, 1979).

    METHODSPreparation of liposomes

    Ion-containing liposomes were prepared using a modification of published methods (see referencesin Introduction). Egg phosphatidylcholine in hexane (Sigma Chemical Co) was evaporated todryness under vacuum and flushed with N2 gas. Ten millilitres of the solution to be entrapped withinthe liposomes were added to the dry lipid and mechanically shaken (Vortex) for 10 min. The milkylipid dispersion was then transferred to a pre-cooled plastic vial and sonicated (Branson) at highpower for 20 min under a continuous stream of N2. Local heating of the lipid suspension wasprevented by an ice-water cooling jacket and by intermittent sonication (20 sec min-'). At the endof 20 min, lipid suspensions generally had a clear, amber colour and further sonication had no effecton their appearance. This is indicative of the formation of small, unilamellar vesicles (liposomes)of20-50 nm diameter (e.g. Papahadjopoulos, 1970). After sonication the liposomes were centrifugedat 5000 g for 30 min at 04C (Sorvall RC 2-B refrigerated centrifuge) to remove Ti fragments fromthe sonifier probe. The supernatant was diluted 1:7 with the control Ringer solution and filteredwith an Amicon MMC unit under He gas at.440 for appropriate numbers (> 5) of sample volumes(see below) using Diaflo XM100 membranes. The liposomes were then gently warmed to roomtemperature and applied to the neuromuscular junction with a roller pump.The integrity of liposomes was evaluated by adding 45CaCl2 to the lipid suspension prior to

    sonication and then filtering the sonicated liposomes. It was found that after approximately fivesample volumes had been filtered, radioactivity declined to a constant level, suggesting thatliposomes were intact and not leaking their 45CaC12 contents and that this method was efficientlyremoving extraliposomal divalent cation. The removal of extraliposomal Ca was assayed directlyin some experiments. This confirmed that five sample volumes were sufficient to remove extra-liposomal Ca.

    Composition ofsolutionThe control Ringer solution used for liposome filtration and for bathing frog neuromuscular

    junctions contained (mM): CaCl2, 0 3-04; MgCl2, 1-2, NaCl, 115; KCl, 2; HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid), 2 (pH 7 1); and neostigmine bromide (1 mgl7-). Thevarious ionic solutions encapsulated within liposomes were as follows: 80 mM-CaCl2 (80 mM-Caliposomes); 80mM-MgCl2 (80 mM-Mg liposomes); 50 mM-CaCl2 + 45 mM-KCI (Ca liposomes); 50 mM-CaCI2 + 30mM-MgCl2 (Ca + Mg liposomes). All solutions were buffered with 2 mM-HEPES, adjustedto pH 7-1 using KOH. As KCl liposomes do not affect evoked ACh secretion (Rahamimoffet at. 1978),KCI was used when necessary to maintain the liposomes isotonic to the Ringer solution. Forliposomes made from multivalent cations other than Ca or Mg (e.g. Mn, Co or La), lowerconcentrations of the cation were employed because of the higher affinity and lower solubility ofthese agents (see references in Results).

    Electrophy8iological detailsCutaneous pectoris nerve-muscle preparations of frog (Rana pipiens) were dissected and

    superfused with flowing Ringer solution. Preparations were transilluminated by a fibre opticssystem similar to that described by Dreyer & Peper (1974). Such illumination, in conjunction with


  • INTRACELLULAR Mg AND ACh RELEASEa stereomicroscope ( x 100 magnification) enabled nerve terminals to be located visually (cf. Dreyer& Peper, 1974). Stimulation pulses were delivered to the nerve through a suction electrode.Electrical potential changes were recorded intracellularly at end-plate regions with glass micro-electrodes filled with 3M-KCI, the reference electrode being a silver-silver chloride pellet. Electrodeswere filled by the fibreglass method of Tasaki, Tsukuhara, Ito, Wayner & Yu (1968) and hadresistances ranging from 10 to 20 MC. Signals from the micro-electrode were fed into a conventionalpreamplifier, the output of which was delivered in parallel into an oscilloscope, a pen recorder, anda computer of average transients. The output of the computer was displayed on anotheroscilloscope. Amplitudes of miniature end-plate potentials (m.e.p.p.) were determined fromoscilloscope traces and muscle resting potentials were measured from pen records.Under conditions in which m.e.p.p.s could be recorded directly, the mean number ofACh quanta

    released synchronously by a nerve impulse (mn) was determined from the ratio of the meancomputer-averaged, end-plate potential amplitude (e:p-p:) to the mean m.e.p.p. amplitude(m:e:p-p:), (del Castillo & Katz, 1954) using suitable numbers of e.p.p.s to reduce the coefficientof variation to < 5 % (see Rahamimoff, 1967). Corrections for non-linear summation were madewhen necessary (Martin, 1955; Stevens, 1976).When tubocurarine chloride (TC; Sigma Chemical Co.) was used to paralyse neuromuscular

    transmission (e.g. Fig. 4), N was calculated using the following equation (Ceccarelli & Hurlbut,1975; Silinsky, 1981):

    (1+4[TC]), (1)

    where U.pIpT. is the evp-.. in curare corrected for non-linear summation, m.e.p.p. is for thenon-curarized preparation, [TC] is in mg 1.-i, and 4 represents the affinity constant (1. mg-') of TCfor the ACh receptor. All electrophysiological experiments were carried out at room temperature.


    Effects of Ca liposomes and Mg liposomes on evoked ACh secretionFig. 1 illustrates an experiment in which the effects of 80 mM-Ca liposomes and

    80 mM-Mg liposomes were studied in the same fibre. As the mre~p1 was unaffectedby the presence of either type of liposome, each e.p.p. is a direct reflection of m, theelectrophysiological correlate of the depolarization-secretion process. In thisexperiment, 10 min in 80 mM-Ca liposomes produced a stable, five-fold increase in m(Fig. 1 B) over the control level (Fig. 1 A), an effect which was completely reversed15 min after returning to liposome-free Ringer solution (Fig. 1 C). In six otherexperiments using 50-80 mM-Ca liposomes, m ranged from 3 to 9 times the controlvalue. In contrast to these results, addition of 80 mM-Mg liposomes failed to affectIn in all six preparations studied (e

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