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

INTRACELLULAR MAGNESIUM DOES NOT ANTAGONIZECALCIUM-DEPENDENT ACETYLCHOLINE SECRETION

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)

SUMMARY

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.

INTRODUCTION

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 04°C (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

256

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.

RESULTS

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.g Fig. 1 D).

It may be argued that the cytoplasmic concentration of ionized Mg is normallyquite high (2-3 mM; Baker, 1972) and thus that the lack of influence of additionalintraterminal Mg ions might be expected. The experiments in the next section suggestthat sufficient quantities of Mg were entering the cytoplasm to affect ACh secretion.

The effects of Ca +Mg liposomes on evoked ACh secretionIn the experiment shown in Fig. 2, upon addition ofCa liposomes, mff increased from

control levels (216-2 9) to a value of 8-5 (stable levels ofmn are always achieved within20 min after adding Ca liposomes.). Substitution with Ca+ Mg liposomes caused mnto increase further, to 11-9. The mf returned to control levels after superfusion with

PHy 314

257

E. D. KHARASCH, A. M. MELLOW AND E. M. SILINSKY

A B

Dia

DC

4mVL

2 msec

Fig. 1. Effects of 80 mM-Ca liposomes and 80 mM-Mg liposomes on evoked acetylcholine(ACh) release. Each end-plate potential (e.p.p.) is the computer-averaged response to 128stimuli presented at 0-5 Hz and reflects the mean number ofACh quanta release by a nerveimpulse (m). Control solution contains 04 mM-Ca, 1 mM-Mg. A, in control solution,mn = 30; B, Ca-liposomes, mn = 14-8; C, return to control, m = 3-1; D, Mg-liposomes,m = 2-9.

K.

0 0

.0** 0

Ca + Mg liposomes*0

.

0

0

00

50mm-Ca liposomesjI I I I

1 2 24 36 48 60 72 84 96Time (min)

Fig. 2. Enhancement of Ca-dependent ACh release by liposomal Mg. In this experimentthe control mn (= 2162-9: open circles) increased to a steady level of 8-5 after applicationof50-mM Ca liposomes (filled squares). After addition of 50 mM-Ca+ 30 mM-Mg liposomes(Ca +Mg liposomes: filled circles), ma increased further to 11 9 and then returned to thecontrol level after re-introduction of the control Ringer solution (open circles).

12

9

c

01m6

3

.205X

in

. -NNW

INTRACELLULAR Mg AND ACh RELEASE 259control Ringer solution. Similar qualitative results were observed in all fourpreparations investigated.The enhanced ACh release evoked by Ca+Mg liposomes was independent of

whether Ca+ Mg liposomes were presented to the neuromuscular junction before orafter Ca liposomes (e.g. Fig. 3). In the experiment shown in Fig. 3, the control m(1-3-14) was increased to /m = 8-6 within 8 min after superfusing with Ca+ Mg

10 0

0 08

Xc 6-_M X1~~~~~~

co |Ca liposomes_ ~~~~~~~04 0

022 L Ca +0k7Mg liposomes

1 2 24 36 48 60Time (min)

Fig. 3. Enhanced ACh secretion by intracellular Mg without prior treatment with Caliposomes. In this experiment m increased from control levels (mn = 1-3-1-4: open circles)to 8-6 in Ca+Mg liposomes (filled circles). As the e.p.p. in Ca+Mg liposomes wassuprathreshold for the muscle action potential, the preparation was superfused with Caliposomes prior to reaching a steady state in Ca + Mg liposomes. Note that m declines to6 1-6-3 in Ca liposomes (filled squares). Up to this time, 128 e.p.p.s were averaged tocalculate m (large symbols). Upon re-addition of Ca+Mg liposomes (small filled circles)only 32 e.p.p.s were averaged (smaller symbols), to shorten the time between averagesand enhance the probability of averaging e.p.p.s uncontaminated by action potentials;mn increased to 10 1 prior to producing suprathreshold action potentials. Returning to thecontrol Ringer solution restored mn to control levels (small open circles).

liposomes. At this level of ACh release, many e.p.p.s were suprathreshold for themuscle action potential so the preparation was rapidly washed with Ca liposomes andmi declined to 6 1-6 3. Re-introducing Ca + Mg liposomes (filled circles) and averagingsmaller numbers of e.p.p.s (32 instead of 128) to enhance the probability of detectinge.p.p.s uncontaminated by action potentials, caused mn to increase to 101 beforesuprathreshold e.p.p.s appeared. Finally, superfusing with liposome-free Ringersolution caused m to return to the control level (open circles). It thus appears thatintraterminal Mg does indeed influence synchronous evoked transmitter secretion,but enhances rather than antagonizes the action of concurrently added Ca.

Studies on the effects of ion-containing liposomes on ACh release reported thus far have all beenmade at low control levels of -ir (mn < 10: see Figs. 1-3 and Rahamimoff et al. 1978). A fortuitousexcess of liposomes in a few experiments enabled us to investigate whether increases in ACh releasecould be evoked by ion-containing liposomes at higher Ca concentrations and thus more normallevels of ACh secretion. Fig. 4 illustrates an experiment made on the nerve-muscle preparation inFig. 3 using the same stock of Ca + Mg liposomes. In this fibre, however, the control Ringer solutionof 1-9 mM-Ca and 2 mM-Mg (4 mg 1.-1 tubocurarine chloride) produced m = 125. Superfusion with

9 2

E. D. KHARASCH, A. M. MELLOW AND E. M. SILINSKYCa + Mg liposomes in this Ringer solution caused wn to increase to suprathreshold levels (ia 330),an effect which was rapidly reversed upon re-introducing liposome-free Ringer solution. It thusappears that the ability of ion-containing liposomes to increase ACh secretion is not confined tolow levels of neurotransmitter secretion.

Future experiments where the effects of Ca liposomes are studied at different extracellular Caconcentrations should provide more information as to the levels of control m at which Ca liposomescannot increase M.

300

1 200

0

0000 O*

100 Ca + Mg liposomes

l l I l I I l I2 4 6 8 10 12 14 16 18

Time (min)

Fig. 4. Increases in ACh secretion produced by liposomes containing divalent cation atmore normal levels of HE. The control Ringer solution in this experiment contained1-9 mM-Ca, 2 mM-Mg and 4 mg l.-' tubocurarine chloride; mn in this solution was 125 (opencircles). After the addition of Ca+Mg liposomes m increased to 330 (filled circles), aneffect which was readily reversible. Values of mn were calculated from eqn. (1). Eachexperimental point reflects the averaged e.p.p. of 16 stimuli. The constancy of the control,n would be expected from its large size (see Rahamimoff, 1967). The flow rate in thisexperiment was 1 bath volume (2-3 ml.) per minute; thus Ca+Mg liposomes began toincrease m after only 3 bath volumes had been superfused over this fibre.

Observations on the effects of other Ca antagonists in liposomesMn ion, a Ca antagonist (Reuter, 1973) that is not a normal intracellular

constituent, failed to affect HE in all four fibres studied when applied to the nerve-endingcytoplasm in 10-12 5mM-MnCl2 liposomes.

Intracellular La (applied using 0 5 mM-LaCl3 liposomes) and intracellular Co(1 mM-CoCl2 liposomes), both potent antagonists of Ca transport (Baker, 1972;Reuter, 1973), produced small increases in evoked ACh release in several experimentswithout requiring the concomitant addition of liposomal Ca. It is noteworthy thatMn, Co and La are all high-affinity antagonists of Ca when applied in the bathingfluid of motor nerve endings (de Bassio, Schnitzler & Parsons, 1971; Meiri &Rahamimoff, 1972; Weakly, 1973; Crawford, 1974; Silinsky, 1978). Indeed, 1 /tM-LaCl3

260

INTRACELLULAR Mg AND ACh RELEASE 261produced a profound depression in m when added to the Ringer solution thatbathed the same fibres in which La liposomes produced increases in ACh release.

DISCUSSION

From these results it appears that Mg and other Ca antagonists do not depressCa-mediated release when applied to the inside of the nerve ending. These resultsconfirm suggestions made previously (Martin, 1977; Silinsky, 1977) that at cholinergicnerve endings, antagonism of depolarization-secretion coupling occurs at an externalsite, presumably at a region near the mouth of the Ca channel that controls alkalineearth cation entry. In this regard, Ca liposomes, whilst increasing mn, failed to affectthe asynchronous, neurally evoked discharge of m.e.p.p.s mediated by Ba and Srthrough the Ca channel (Silinsky, 1977, and unpublished data). This is in contrastto the pronounced inhibitory effects oflow ( < 0 25 mM) extracellular Ca concentrationson Ba (and Sr)-mediated evoked m.e.p.p.s (Silinsky, 1977, and unpublished data). Thisresult, in addition to confirming the suggestion that the high-affinity antagonism byCa occurs at the external face of the nerve ending (Silinsky, 1977), also providesfurther evidence that liposomal contents are being applied to an intraterminal site.If even small amounts of potent, reversible antagonists such as Ca, Mn or Co wereleaking to the extracellular fluid as a result of the local rupture of liposomes near theneuronal ending, then an antagonism of evoked m.e.p.p.s (by Ca) and m (by Co andMn) would be expected. As such antagonism was never observed, it appears thatliposomes in this study were delivering their entrapped material to the intracellularmilieu of the nerve ending.The mechanism by which intraterminal Mg enhances the effects of liposomal Ca

is not known. One plausible interpretation is that cytoplasmic Mg may be inhibitingCa extrusion or preventing Ca removal into storage sites (Baker, 1972). For example,if Mg binds to or even non-selectively screens negatively charged transport sites(Lehninger, 1970), the effective Ca concentration introduced by liposomes into nerveendings could be increased. The experimental observations with the more potentantagonists Co and La are in accord with this interpretation, as are the reported effectsof Na liposomes in increasing ACh secretion (Rahamimoff et al. 1978), behaviour thatmay be due to interference by Na with Ca transport (Reuter, 1973).

In the experiments described in this study, changes in m.e.p.p. frequency (m.e.p.p.f)produced by liposomal divalent cation, when observed, were quite small and generallyinsignificant compared with changes in m- (see also Rahamimoff et al. 1978). Suchresults are consistent with the premise that increases in m.e.p.p.q may be producedby a non-selective screening of negatively charged release sites by intracellular cation,whilst increases in m are mediated by selective binding to release sites (see Silinsky& Mellow, 1981; Silinsky. 1981). Thus, Ca at most concentrations used in liposomeswould produce large increases in m (by binding) but might not be in sufficiently highconcentration intracellularly to produce increases in m.e.p.p.f by a screeningmechanism. It is of interest that the trivalent cation La (in 0 5 mm-LaCl3 liposomes)produced dramatic, often unmeasurable increases in m.e.p.p.f. This effect, while notruling out other possible mechanisms, would be expected if La increased m.e.p.p.fby intracellular screening (see Hille, Woodhull & Shapiro, 1975).

E. D. KHARASCH, A. M. MELLOW AND E. M. SILINSKYThe experiments reported here suggest that the liposomes are indeed delivering

their entrapped contents into the nerve terminal. It is not known whether the eventrepresents lipid vesicles fusing with the plasma membrane and subsequentlydischarging their contents into the cytoplasm, whether simple diffusion occurs fromliposomes adhering to the nerve ending, or whether complete endocytosis of thevesicle with subsequent intracellular rupture occurs. Regardless of the precisemechanism ofthis transmembrane shuttle, however, the antagonism ofdepolarization-secretion coupling by extracellular Mg (and other divalent cations) appears to occuronly at the external surface of the cholinergic nerve ending.

This work was supported by an N.I.H. research grant, an N.I.H. predoctoral training grant (toA. M. M.) and a Northwestern University predoctoral fellowship (to E. D. K.). We thank DrA. F. Boyne for the loan of the ultrafiltration unit and Professor R. C. MacDonald for his commentson the manuscript.

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