Lifa Scieaces Vol . 18, pp . 1359-1366, 1976 . Priated in tha II.S .A .
Pargamon Press
ON THE EFFECT OF THE IONOPHORE X-537A ON NEUROMUSCULAR TRANSMISSION IN THE RAT S .-E . Janseon, E . Heinonen, V. Heinänen, J . Gripenberg, E .-M . Tolppanen and T . Salmi Departments of Anatomy and Physiology, University of Helsinki, Siltavuorenpenger, 00170 Helsinki 17, Finland . (Received in fiaal form January 19, 1976) Summary In the rat phrenic nerve-diaphragm muscle preparation, X-537A at 6x10-6 to 3x10 - 5 M (1~depolarized muscle fibre membranes, (2~caused an occasional transient increase in and ultimate block of spontaneous transmitter release, (3)did not increase the amplitude of the end-plate potential (epp~ but abruptl blocked stimulus-evoked transmitter release, and (4~produced an increase in the occurrence of "giant" miniature epp's (mepp's~ . The possibility ie discussed that the sporadically raised mepp frequency was due to an ionophore-induced depolarization of nerve terminals . The increased occurrence of "giant" mepp's apparently reflected a X-537A-induced spontaneous multiquantal release of acetylcholine . This was not dependent on extracellular calcium but appeared to be of presynaptic origin . The ionophoric antibiotic X-537A forma lipid-soluble complexes with monovalent and divalent canons allowing the passage of e .g, calcium through biological membranes (1~ . This makes the ionophore useful in the study of neural transmitter release mechanisms . In accordance with the Calcium Hypothesis (2~, X-537A increases spontaneous and stimulus-evoked release of acetylcholine at the frog neuromuscular junction (3) . The involvement of calcium in this ionophore-induced release is not clear by itself because the ion-binding properties of X-537~A are non-selective (4) . As Na+ is bound preferentially to Ca { (5), an increase in transmitter release could be explained by~a depolarization of nerve terminals due to an ionophore-induced intracellular accumulation of sodium ions (6) . Furthermore, X-537A induces transmitter release from peripheral adrenergic nerves, chromaffin vesicles, eynaptoeomea and adrenal glands by a calcium-independent mechanism (7, 8, 9, 10~, evidently by transporting the transmitter across cellular membranes . In this preliminary paper, the effects of X-537A on the release of acetylcholine were investigated using intracellular recording techniques in the rat phrenic nerve-diaphragm muscle preparation . 1359
1360
5-537A on Neuromuscular Transmission
Vol . 18, No . 12
Methode Adult male rata (180-220 g) of the Sprague-Daxley strain xere used . The phrenic nerve-diaphragm muscle preparation was mounted and euperfuaed with bathing solution, ae xill be described else where in detail (submitted for publication) . Intracellular recordings xere carried out xith glass microelectrodes drawn xith internal glass fibres (11) and filled with 3 M KC1-solution (DC-resistance 5-20 megaohms) . Muscle twitches in response to nerve stimulation xere abolished by cutting the muscle fibres (12) or by bathing the preparation in solutions contain high concentrations of Mgt+ (13) . End-plate potentials (app's and miniature app's (mepp's) were FM-recorded at a speed of 95 .3 cm s-1 . The amplitude and rise time of mepp's were measured from ink-jet paper recordings (Mingograf) from the foot-step at the baseline to the peak after FM-recorded material had been replayed at a speed 100 times aloxer than the recording speed. X-537A (Iioffmann-LaRoche) was dissolved in 95 9b ethanol . At the final concentration in the medium, ethanol had no effect on transmitter release . Results X-537A on restinbc membrane potential of muscle fibres . X-537A depolarized the muscle fibre membrane in a dose-dependent manner . The effect of X-537A at 1x10 - 5 and 3z10 - 5 M was studied in three preparations at each concentration . At 3x10 - 5 M, muscle fibres were depolarized by 10 mV during the first 15 minutes at 37 oC and after 40 minutes the membrane potential xas -54 .2 t 11 .2 mY (mean ± S .D . of 12 fibres) as compared to more than -73 mY in the control . At the loxer concentration, the membrane depolarization levelled off at -68 to -65 mY . X-537A on mepp frequency. At moat junctions the mepp frequency remained unaffected by X-537A at 6x10 -6 to 3z10 - 5 M but in eight mepp experiments out of twenty, à transient increase in mepp frequency xas recorded from one or several junctions . When a raised mepp frequency was observed, it often gradually increased to ae high values as 500-600 mepp's e-1 . After this peak, the mepp frequency decreased until spontaneous transmitter release ceased at all junctions . In three experiments at 3x10 - 9 M, mepp's disappeared at 20, 7 and 10 minutes at 37 oC . A massive increase in mepp frequency which was subsequently followed by a blockage of neuromuscular transmission has been observed also in the frog (3) . The sporadic nature of the effect reported in the present paper may be due to species differenciea . XA on me am litude and rise time . In a control prepar ation, mepp amplitude was 0 . - 0 .29 mV mean ± S .D . of 2752 mepp's from 15 fibres in three preparation) . The mepp amplitude distribution in many cells xas positively skewed, but mepp's larger than 1 .5 mV xere rare and amounted to about 2 `,6 of all mepp'a . When such "giant" mepp'e occurred, they apparently reflected multiquantal release, i .e . release of either eupranormal or multiple packages of acetylcholine (14) . The most clear-cut effect of X-537A consisted of an increase in the number of such "giant" mepp'e . As Fig . 1 ahoxa, in
Vol . 18, No . 12
% 537A on Neuromuscular Traasmiesion
1361
addition to normal-ei$ed mepp's, a large number of mepp'e which were even larger than 2 mV were recorded . Thie .preliminary material proves little but .doea not exclude a grouping of amplitudes around multiples of the modal value . The effect was obtained after just a few minutes at 37 °C with X-537A at 3x10 -5 M. The "giant" mepp'e were usually 2-4 mY in amplitude and amounted to 10-30 ~ of~all mepp's . As during treatment with X-537A epontaneoua transmitter release ceased, the large amplitude mepp'e died away simultaneously with or even before normal mepp's .
1
f~ 2
~~~ r-~r~lnn 3 4
Mepp Ampitude, mV
FIG . 1 Mepp amplitude distribution after X-537A at 6x10-6 M . A total of 210 mepp'e were analysed from three celle (membrane potential -74, -69 and -60 mY~ in three preparations exposed to X-537A for 4-33 minutes at room temperature . In untreated preparations, the modal rise time of mepp'e that had an amplitude lees than arbitrarily 1 .1 mY was 0 .6 ma at room temperature while larger mepp's had a Blower rise time . The peak was at 0 .8 ma and in addition the distribution was skewed with many mepp'e in the range 2-3 .5 me (see 15) : The rising phase of such mepp'a was often distorted or S-shaped . In both mepp groups, X-537A increased the number of mepp's with particularly Blow time-to-peals values . Modal values were, however, altered little . In addition to mepp's with fast and straight rising phase (time-to-peak value lees than 1 ma), smoothly S-shaped mepp's and distorted potentials with foot-steps on rising and falling phases were seen (Fig . 2~ . The modal rise time was more than 1 ma and the range 0 .5 to 5 me . Seven experiments were carried out xith preparations bathed for at least one hour in eolutioae from which CaC1 2 waa omitted but which contained 0 .5-2 .5 mM EGTA . Despite the extremely low eztracellular calcium concentration during these conditions, X-537A induced the same skewed large-amplitude mepp'a ae seen in normal solutions containing calcium (Fig . 2~ . Aa increase in mepp frequency was not observed but the mepp's disappeared after treatment with X-537A in the same way ae in a solution which contained calcium. X-537A on the end-plate potential . Rega.rdlesely of how the muscle twitch was blocked, by cutting the preparation or by treat-
136 2
X-537A on Neuromuscular Transmission
Vol . 18, No . 12
ment with high concentrations of magnesium ions, X-537A did not increase the amplitude of the epp . Instead the epp was blocked in an all-or-nothing manner . Failures appeared abruptly and in no instance did the epp smoothly vanish in the noise of the baseline . This is shown in Fig. 3, in which the epp was suddenly blocked after 10 minutes of treatment with X-537A at 10- 5 M at 37 °C . At this stage of treatment, the extracellularly recorded action potential in the nerve trunk still persisted but in three ezperiment at 10 - 5 M, the action potential in the nerve trunk was blocked 10, 9 and 5 minutes after the epp failed . NOFiMgL NEp0.1M
CamFREE NED~1M
FIG. 2 Samples of X-537A-induced "giant" mepp's (6x10-6 M, room temperature) from preparations bathed either in normal solutions or in a calcium-free solution containing 2 .5 mM EGTA . Montage of ink-jet paper recordings (see Methods) . Normal-sized mepp's are indicated by arrows . Discussion The present results show that in the phrenic nerve-diaphragm muscle preparation, X-537A (1)depolarized muscle fibre membranes, (2)cauaed an occasional transient increase in and ultimate block of spontaneous transmitter release, (3)did not increase the epp amplitude but abruptly blocked stimulus-evoked transmitter releasey and (4)produced an increase in the occurrence of "giant" mepp's . The nature of the ionophore-induced membrane depolarization was not further investigated but at least in the frog, X-537A appears to depolarize muscle fibre membranes by inducing an intracellular accumulation of sodium ions (3) . It is not known if X-537A depolarized also the presynaptic nerve terminal . However,
Vol . 18, No . 12
R-537A on Neuromuscular Transmiseioa
1363
X-537A , 10b M 0 .8 min
28
FIG . 3 Computer (u-Linc) displays of epp aeries after exposure to 10-5 M X-537A at 37 oC . Each vertical column of points depicts the amplitude of an epp . The nerve was continoualy stimulated at 1 Hz and samples comprising 60 epp'e were taken at irregular intervals, Cut preparation, membrane potential -32 to -28 mv . this possibility would explain the increase in mepp frequency which was occasionally seen after treatment with X-537A as it remained uncertain ~rhether a facilitated calcium influx was involved in this effect, Firstly, X-537A did not increase the epp amplitude in a manner expected if the ionophore raised the intraterminal concentration of calcium . Secondly, as judged from preliminary experiments carried out is the present laboratory, 7C-537A was not partioularly effective in facilitating calcium uptake by nerve terminals : twenty times higher concentrations thaw employed in the present study were needed to obtain a clear-cut effect on the uptake of radiocalcium by nerve ending particles (eynaptosomes~ isolated from rat cerebral tissue . Thirdly, the massive increase in mepp frequency which was followed by a blockage of neuromuscular transmission resembles the presynaptic membrane depolarization which is produced by agents which increase membrane sodium conductance (16) . Another point which remained open was the mechanism by which X-537A blocked spontaneous and stimulus-evoked release of acetylcholine . A lowered acetylcholine-sensitivity of the postsynaptic membrane cannot be excluded but this possibility appears unlikely because of the abrupt nature of the epp block . Further, in contrast to results obtained by Kita and van der Kloot (3), we observed that X-537A blocked the eatracellularly recorded nerve action potential in the nerve trunk . Focal extracellular recordings were not carried out in the present study but it appears that the ionophore-induced epp block is due to an effect on the action potential generating mechanism in the presynaptic nerve fibre rather than to a poataynaptic action .
1364
R-537A on Neuromuscular Transmission
Vol. 18, No . 12
The most interesting effects of X-537A showed out to be on the time course and amplitude of mepp's . These alterations appear to be of presynaptic origin : if X-537A affected exclusively the postsynaptic membrane one would expect uniformly enlarged and prolonged mepp's . In the present study, however, normal and altered mepp's were recorded from one and the name cell . This resulted in an amplitude and rise time distribution similar to that in a cell with an unusually high percentuage of "giant" mepp's occurring physiologically (14), It is unlikely that X-537A brought about these alterations by facilitating the passage of calcium through the presynaptic membrane because ionophore-induced mepp alterations were seen in virtually calcium-free solutions . If involved, calcium is probably mobilized from an intracellular store or activated at a membrane site . Preliminary experiments favour the second possibility because X-537A did not accelerate the efflux of radiocalcium from synaptosomes . X-537A might induce large-amplitude mepp's with skewed rising phase by at least six principally different mechanisms . 1~Because of the membrane depolarization after treatment with X-537Ar spontaneous abortive action potentials may occur and result in multiquantal release of acetylcholine, However, this mechanism would be exclusively dependent on eztracellular calcium. 2~X-537A might form lipid-soluble complexes with acetylcholine, just as with catecholamines (1~, thus facilitating the passage of acetylcholine through vesicle and preeynaptic membranes . 3)X-537A might bring single vesicles coalesce leading to the formation of multiquantal, releasable packages of acetylcholine . Preliminary electron microscopic studies did not, however, reveal enlarges synaptic vesicles in X-537A-treated synaptosomee, 4)X-537A might, perhaps by activating calcium at a critical membrane site, increase the drag éffect, i .e . the facilitation of transmitter release brought about by the release of a single quantum of acetylcholine (17, 18, 19i see also 20~, Mepp interval analysis xae not carried out on the present material but an interaction between single quanta would indeed explain the foot-steps on the rising and falling phases of the mepp's . S~Acetylcholine might be contained in multi-compartmental packages in the name way as has been suggested for noradrenaline in adrenergic nerves (21, 22~ . X-537A could then cause a fusion of the subcompartments to a multiquantal, releasable unit, 6~Finally, there is the possibility that X-537A releases large packages of acetylcholine from e .g . Schxann celle (23) . In conclusion, in the rat phrenic nerve-diaphragm preparation, the ionophore X-537A induces what appears spontaneous multiquantal release of acetylcholine . The underlaying this release remains unclear but it is not on eztracellular calcium .
muscle to be mechanism dependent
Vol . 18, No . 12
E-537A oa Neuromuscular Traaemiesion
1365
Acknowledgements This work was supported by a grant from the Finnish State Medical Research Council . jtef erenc e s 1 . H .C . PRESSMAN, Fed . Proc . ~, 1698-1703 (1973) " 2 . H . KATZ, The Release of Neural Transmitter Substances . Liverpool University Press~l9 9 3 . H . KITA and X . YAN,DER KLOOT, Nature ~, 658-660 (1974) . 4 . S . ESTRÂDè-O, H . CELIS, E . CALDERON, G . GALLO and M. MONTAL, J . Membr . Biol . 18, 201-218 (1974) . 5 . G. CORNELIUS, X . GXRTNER and D .H . HAYNES, Biochemistry 3052-3057 (1974) . 6 . D .I . DEYORE and W .L . NASTUK, Nature 2r , 644-646 (1975) " 7 . N .B . THOA, J .L . COSTA, J . MOSS and I .J . KOP]Id, Life Sçi . 14, 1705-1719 (1974) " 8 . R.G . JOHNSON and A . SCARPA, FERS Letters ~, 117-121 (1974) . 9 . R .W . HOLTZ, Biochim. Biophys . Acts ~, 13 -152 (1975) " 10 . A . RICCI, Jr ., K .M . SANDERS, J . PORTMORE and W .G . VAN DER KLOOT, Life 5-1 . 16, 177-184 (1975) " 11 . K . TASAKI, Y . TSUKÂKAVA, S . ITO, M .J . üAYNER and X .J . YU, Physiol . Hehav. 3, 1009-1010 (1968) . 12 . J .A .B . BARSTAD, Experientia 18, 579-580 (1962) . 13 . J . 1)EL CASTILLO and L . ENGBAEK, J . Physiol . (Lond .) 124, 370-384 (1954) " 14 . A .K . LILEY, J . Physiol . (Lond .) 1~6, 595-605 (1957) " 15 . R .L .E . MENRATH and J .G . HLACHMAN, Proc . Univ . Otago Med . Sçh. 48, 72-73 (1970) . 16 . S .-E . JANSSON, E .X . ALBUQUERQUE and J . DALY, J . Pharmacol . Ezp . Ther . 182,, 525-537 (1974) . 17 " A .R . MARTIN and G. PILAR, J . Physiol . (Lond .) ~, 1-16 (1964) . 18 . J .C . BORNSTEIN, Nature 248, 529-531 (1974) " 19 . E .F . BARRETT J .N . HARRETT, A .R . MARTIN and R . RAHAMIMOFF, J . Physiol : ~Lond.) ~, 453-463 (1974) . 20 . J.I . HUHHARD and S .F . JONES, J . Physiol . (Lond .) ~2, 1-21 (1973) " 21 . B. FOLKOÜ and J . HÄGGENDAHL, in New Aspects of Storage and Release Mechanisms of Catecholamines . Haver Symposium II . Eda . H .J . Schümaan and G . Kroneberg, pp . 91-97 . Springer Verlag~ Berlin, Heidelberg, New York (1970) . 22 . L. STJARNE, in Handbook of Ps cho harmacolo . Eds . L .L . Iversen, S .D . Iversen andS .H . Snyder . Yol . . Plenum Press, London (1975) . 23 . R . MILEDI and C .R . SLATER, Proc . RoY. Soc . H. 162, 289-306 (1968) .
Pargamon Press
ON THE EFFECT OF THE IONOPHORE X-537A ON NEUROMUSCULAR TRANSMISSION IN THE RAT S .-E . Janseon, E . Heinonen, V. Heinänen, J . Gripenberg, E .-M . Tolppanen and T . Salmi Departments of Anatomy and Physiology, University of Helsinki, Siltavuorenpenger, 00170 Helsinki 17, Finland . (Received in fiaal form January 19, 1976) Summary In the rat phrenic nerve-diaphragm muscle preparation, X-537A at 6x10-6 to 3x10 - 5 M (1~depolarized muscle fibre membranes, (2~caused an occasional transient increase in and ultimate block of spontaneous transmitter release, (3)did not increase the amplitude of the end-plate potential (epp~ but abruptl blocked stimulus-evoked transmitter release, and (4~produced an increase in the occurrence of "giant" miniature epp's (mepp's~ . The possibility ie discussed that the sporadically raised mepp frequency was due to an ionophore-induced depolarization of nerve terminals . The increased occurrence of "giant" mepp's apparently reflected a X-537A-induced spontaneous multiquantal release of acetylcholine . This was not dependent on extracellular calcium but appeared to be of presynaptic origin . The ionophoric antibiotic X-537A forma lipid-soluble complexes with monovalent and divalent canons allowing the passage of e .g, calcium through biological membranes (1~ . This makes the ionophore useful in the study of neural transmitter release mechanisms . In accordance with the Calcium Hypothesis (2~, X-537A increases spontaneous and stimulus-evoked release of acetylcholine at the frog neuromuscular junction (3) . The involvement of calcium in this ionophore-induced release is not clear by itself because the ion-binding properties of X-537~A are non-selective (4) . As Na+ is bound preferentially to Ca { (5), an increase in transmitter release could be explained by~a depolarization of nerve terminals due to an ionophore-induced intracellular accumulation of sodium ions (6) . Furthermore, X-537A induces transmitter release from peripheral adrenergic nerves, chromaffin vesicles, eynaptoeomea and adrenal glands by a calcium-independent mechanism (7, 8, 9, 10~, evidently by transporting the transmitter across cellular membranes . In this preliminary paper, the effects of X-537A on the release of acetylcholine were investigated using intracellular recording techniques in the rat phrenic nerve-diaphragm muscle preparation . 1359
1360
5-537A on Neuromuscular Transmission
Vol . 18, No . 12
Methode Adult male rata (180-220 g) of the Sprague-Daxley strain xere used . The phrenic nerve-diaphragm muscle preparation was mounted and euperfuaed with bathing solution, ae xill be described else where in detail (submitted for publication) . Intracellular recordings xere carried out xith glass microelectrodes drawn xith internal glass fibres (11) and filled with 3 M KC1-solution (DC-resistance 5-20 megaohms) . Muscle twitches in response to nerve stimulation xere abolished by cutting the muscle fibres (12) or by bathing the preparation in solutions contain high concentrations of Mgt+ (13) . End-plate potentials (app's and miniature app's (mepp's) were FM-recorded at a speed of 95 .3 cm s-1 . The amplitude and rise time of mepp's were measured from ink-jet paper recordings (Mingograf) from the foot-step at the baseline to the peak after FM-recorded material had been replayed at a speed 100 times aloxer than the recording speed. X-537A (Iioffmann-LaRoche) was dissolved in 95 9b ethanol . At the final concentration in the medium, ethanol had no effect on transmitter release . Results X-537A on restinbc membrane potential of muscle fibres . X-537A depolarized the muscle fibre membrane in a dose-dependent manner . The effect of X-537A at 1x10 - 5 and 3z10 - 5 M was studied in three preparations at each concentration . At 3x10 - 5 M, muscle fibres were depolarized by 10 mV during the first 15 minutes at 37 oC and after 40 minutes the membrane potential xas -54 .2 t 11 .2 mY (mean ± S .D . of 12 fibres) as compared to more than -73 mY in the control . At the loxer concentration, the membrane depolarization levelled off at -68 to -65 mY . X-537A on mepp frequency. At moat junctions the mepp frequency remained unaffected by X-537A at 6x10 -6 to 3z10 - 5 M but in eight mepp experiments out of twenty, à transient increase in mepp frequency xas recorded from one or several junctions . When a raised mepp frequency was observed, it often gradually increased to ae high values as 500-600 mepp's e-1 . After this peak, the mepp frequency decreased until spontaneous transmitter release ceased at all junctions . In three experiments at 3x10 - 9 M, mepp's disappeared at 20, 7 and 10 minutes at 37 oC . A massive increase in mepp frequency which was subsequently followed by a blockage of neuromuscular transmission has been observed also in the frog (3) . The sporadic nature of the effect reported in the present paper may be due to species differenciea . XA on me am litude and rise time . In a control prepar ation, mepp amplitude was 0 . - 0 .29 mV mean ± S .D . of 2752 mepp's from 15 fibres in three preparation) . The mepp amplitude distribution in many cells xas positively skewed, but mepp's larger than 1 .5 mV xere rare and amounted to about 2 `,6 of all mepp'a . When such "giant" mepp'e occurred, they apparently reflected multiquantal release, i .e . release of either eupranormal or multiple packages of acetylcholine (14) . The most clear-cut effect of X-537A consisted of an increase in the number of such "giant" mepp'e . As Fig . 1 ahoxa, in
Vol . 18, No . 12
% 537A on Neuromuscular Traasmiesion
1361
addition to normal-ei$ed mepp's, a large number of mepp'e which were even larger than 2 mV were recorded . Thie .preliminary material proves little but .doea not exclude a grouping of amplitudes around multiples of the modal value . The effect was obtained after just a few minutes at 37 °C with X-537A at 3x10 -5 M. The "giant" mepp'e were usually 2-4 mY in amplitude and amounted to 10-30 ~ of~all mepp's . As during treatment with X-537A epontaneoua transmitter release ceased, the large amplitude mepp'e died away simultaneously with or even before normal mepp's .
1
f~ 2
~~~ r-~r~lnn 3 4
Mepp Ampitude, mV
FIG . 1 Mepp amplitude distribution after X-537A at 6x10-6 M . A total of 210 mepp'e were analysed from three celle (membrane potential -74, -69 and -60 mY~ in three preparations exposed to X-537A for 4-33 minutes at room temperature . In untreated preparations, the modal rise time of mepp'e that had an amplitude lees than arbitrarily 1 .1 mY was 0 .6 ma at room temperature while larger mepp's had a Blower rise time . The peak was at 0 .8 ma and in addition the distribution was skewed with many mepp'e in the range 2-3 .5 me (see 15) : The rising phase of such mepp'a was often distorted or S-shaped . In both mepp groups, X-537A increased the number of mepp's with particularly Blow time-to-peals values . Modal values were, however, altered little . In addition to mepp's with fast and straight rising phase (time-to-peak value lees than 1 ma), smoothly S-shaped mepp's and distorted potentials with foot-steps on rising and falling phases were seen (Fig . 2~ . The modal rise time was more than 1 ma and the range 0 .5 to 5 me . Seven experiments were carried out xith preparations bathed for at least one hour in eolutioae from which CaC1 2 waa omitted but which contained 0 .5-2 .5 mM EGTA . Despite the extremely low eztracellular calcium concentration during these conditions, X-537A induced the same skewed large-amplitude mepp'a ae seen in normal solutions containing calcium (Fig . 2~ . Aa increase in mepp frequency was not observed but the mepp's disappeared after treatment with X-537A in the same way ae in a solution which contained calcium. X-537A on the end-plate potential . Rega.rdlesely of how the muscle twitch was blocked, by cutting the preparation or by treat-
136 2
X-537A on Neuromuscular Transmission
Vol . 18, No . 12
ment with high concentrations of magnesium ions, X-537A did not increase the amplitude of the epp . Instead the epp was blocked in an all-or-nothing manner . Failures appeared abruptly and in no instance did the epp smoothly vanish in the noise of the baseline . This is shown in Fig. 3, in which the epp was suddenly blocked after 10 minutes of treatment with X-537A at 10- 5 M at 37 °C . At this stage of treatment, the extracellularly recorded action potential in the nerve trunk still persisted but in three ezperiment at 10 - 5 M, the action potential in the nerve trunk was blocked 10, 9 and 5 minutes after the epp failed . NOFiMgL NEp0.1M
CamFREE NED~1M
FIG. 2 Samples of X-537A-induced "giant" mepp's (6x10-6 M, room temperature) from preparations bathed either in normal solutions or in a calcium-free solution containing 2 .5 mM EGTA . Montage of ink-jet paper recordings (see Methods) . Normal-sized mepp's are indicated by arrows . Discussion The present results show that in the phrenic nerve-diaphragm muscle preparation, X-537A (1)depolarized muscle fibre membranes, (2)cauaed an occasional transient increase in and ultimate block of spontaneous transmitter release, (3)did not increase the epp amplitude but abruptly blocked stimulus-evoked transmitter releasey and (4)produced an increase in the occurrence of "giant" mepp's . The nature of the ionophore-induced membrane depolarization was not further investigated but at least in the frog, X-537A appears to depolarize muscle fibre membranes by inducing an intracellular accumulation of sodium ions (3) . It is not known if X-537A depolarized also the presynaptic nerve terminal . However,
Vol . 18, No . 12
R-537A on Neuromuscular Transmiseioa
1363
X-537A , 10b M 0 .8 min
28
FIG . 3 Computer (u-Linc) displays of epp aeries after exposure to 10-5 M X-537A at 37 oC . Each vertical column of points depicts the amplitude of an epp . The nerve was continoualy stimulated at 1 Hz and samples comprising 60 epp'e were taken at irregular intervals, Cut preparation, membrane potential -32 to -28 mv . this possibility would explain the increase in mepp frequency which was occasionally seen after treatment with X-537A as it remained uncertain ~rhether a facilitated calcium influx was involved in this effect, Firstly, X-537A did not increase the epp amplitude in a manner expected if the ionophore raised the intraterminal concentration of calcium . Secondly, as judged from preliminary experiments carried out is the present laboratory, 7C-537A was not partioularly effective in facilitating calcium uptake by nerve terminals : twenty times higher concentrations thaw employed in the present study were needed to obtain a clear-cut effect on the uptake of radiocalcium by nerve ending particles (eynaptosomes~ isolated from rat cerebral tissue . Thirdly, the massive increase in mepp frequency which was followed by a blockage of neuromuscular transmission resembles the presynaptic membrane depolarization which is produced by agents which increase membrane sodium conductance (16) . Another point which remained open was the mechanism by which X-537A blocked spontaneous and stimulus-evoked release of acetylcholine . A lowered acetylcholine-sensitivity of the postsynaptic membrane cannot be excluded but this possibility appears unlikely because of the abrupt nature of the epp block . Further, in contrast to results obtained by Kita and van der Kloot (3), we observed that X-537A blocked the eatracellularly recorded nerve action potential in the nerve trunk . Focal extracellular recordings were not carried out in the present study but it appears that the ionophore-induced epp block is due to an effect on the action potential generating mechanism in the presynaptic nerve fibre rather than to a poataynaptic action .
1364
R-537A on Neuromuscular Transmission
Vol. 18, No . 12
The most interesting effects of X-537A showed out to be on the time course and amplitude of mepp's . These alterations appear to be of presynaptic origin : if X-537A affected exclusively the postsynaptic membrane one would expect uniformly enlarged and prolonged mepp's . In the present study, however, normal and altered mepp's were recorded from one and the name cell . This resulted in an amplitude and rise time distribution similar to that in a cell with an unusually high percentuage of "giant" mepp's occurring physiologically (14), It is unlikely that X-537A brought about these alterations by facilitating the passage of calcium through the presynaptic membrane because ionophore-induced mepp alterations were seen in virtually calcium-free solutions . If involved, calcium is probably mobilized from an intracellular store or activated at a membrane site . Preliminary experiments favour the second possibility because X-537A did not accelerate the efflux of radiocalcium from synaptosomes . X-537A might induce large-amplitude mepp's with skewed rising phase by at least six principally different mechanisms . 1~Because of the membrane depolarization after treatment with X-537Ar spontaneous abortive action potentials may occur and result in multiquantal release of acetylcholine, However, this mechanism would be exclusively dependent on eztracellular calcium. 2~X-537A might form lipid-soluble complexes with acetylcholine, just as with catecholamines (1~, thus facilitating the passage of acetylcholine through vesicle and preeynaptic membranes . 3)X-537A might bring single vesicles coalesce leading to the formation of multiquantal, releasable packages of acetylcholine . Preliminary electron microscopic studies did not, however, reveal enlarges synaptic vesicles in X-537A-treated synaptosomee, 4)X-537A might, perhaps by activating calcium at a critical membrane site, increase the drag éffect, i .e . the facilitation of transmitter release brought about by the release of a single quantum of acetylcholine (17, 18, 19i see also 20~, Mepp interval analysis xae not carried out on the present material but an interaction between single quanta would indeed explain the foot-steps on the rising and falling phases of the mepp's . S~Acetylcholine might be contained in multi-compartmental packages in the name way as has been suggested for noradrenaline in adrenergic nerves (21, 22~ . X-537A could then cause a fusion of the subcompartments to a multiquantal, releasable unit, 6~Finally, there is the possibility that X-537A releases large packages of acetylcholine from e .g . Schxann celle (23) . In conclusion, in the rat phrenic nerve-diaphragm preparation, the ionophore X-537A induces what appears spontaneous multiquantal release of acetylcholine . The underlaying this release remains unclear but it is not on eztracellular calcium .
muscle to be mechanism dependent
Vol . 18, No . 12
E-537A oa Neuromuscular Traaemiesion
1365
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