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The Regulatory Role of Membrane Na+-K+-ATPase in Non-Quantal Release of Transmitter at the Neuromuscular Junction
The Regulatory Role of Membrane Na+-K+-ATPase in Non-Quanta1 Release of Transmitter at the Neuromuscular Junction FRANTISEKVYSKOEIL Institute of Physiology, Czechoslovak Academy of Sciences, 142 20 Prague (Czechoslovakia)
INTRODUCTION It was shown by Brooks in 1954 that acetylcholine (ACh) is released from the isolated diaphragm of the guinea-pig even in the absence of nerve stimulation. This fact was subsequently confirmed by several authors and spontaneous miniature end-plate potentials (MEPPs) were considered to be an electrophysiological correlate of the resting ACh output from skeletal muscle (see e.g. KrnjeviC and Mitchell, 1961). As early as in 1963, however, Mitchell and Silver’realized the serious discrepancy between the changes of biologically assayed ACh in .their experiments and electrophysiologically recorded MEPPs under different experimental conditions. Thus, for example, when the K’ concentration in the bathing fluid is increased from 5 to 30 mM, the MEPP frequency (representing the number of quanta released per unit time) is raised 200-300 times in the rat diaphragm (Liley, 1956) whereas the output of ACh increases only about 3-fold (Mitchell and Silver, 1963). A similar discrepancy appeared when the effect of temperature was studied. Mitchell and Silver therefore concluded that only a small fraction of the resting release derives from MEPPs and that, surprisingly, 97-99% of released ACh has a different origin. A similar conclusion was also drawn by Fletcher and Forrester (1975) who estimated that only 2% of overall ACh release from rat diaphragm at rest could represent quantal release and suggested that the remainder has a non-synaptic source. In our recent observation on the mouse diaphragm (Vizi and VyskoEil, 1979), where we compared the total and quantal release ACh before and during stimulation, we estimated that 1% of the total release represents quantal release. The non-quanta1 release of ACh was considered by some workers to be of “non-synaptic” origin and was attributed to the preterminal regions of motor axons (Brooks, 1954; Mitchell and Silver, 1963; Fletcher and Forrester, 1975; cf. Evans and Saunders, 1974), too far away from the postsynaptic membrane to evoke detectable depolarisation. Another possibility suggested by Katz and Miledi (1965) was that a steady and diffuse leakage may occur from the terminals all over the junction. This continuous leakage, because of hydrolysis by cholinesterase, can barely be recorded electrophysiologically. Nevertheless, Katz and Miledi (1 977) successfully tested this assumption on antiesterase
184 (diisopropylfluorophosphate, DFP) treated frog sartorius muscles where individual end-plates of superficial muscle fibres were subjected to a massive ionophoretic dose of (+)-tubocurarine (TC). During this local curarisation, various levels of hyperpolarisation wefe observed in different muscle fibres and this effect was attributed to the leakage of cytoplasmic ACh from nerve terminals causing a minute steady postsynaptic depolarisation, which is prevented by TC. The average depolarisation was only about 40 pV (at 18-24°C) in this preparation and such a change is so small that it does not provide a good opportunity for studying the characteristics of the steady leakage. We therefore decided to use the mouse diaphragm and to perform similar experiments on this preparation (VyskoEil and Ill&, 1977, 1978), where the electrical responses to ACh (MEPPs in particular) are more pronounced apparently because of the small diameter and high input impedance of the muscle fibres and compact junctional area (Salpeter and Eldefrawi, 1973). As will be demonstrated later, mouse end-plates were hyperpolarized by 1-2 mV during local curarization in the presence of anticholinesterases at 32°C. We checked the idea that the membrane Na*,K’-ATPase (Skou, 1965) of the nerve terminal membrane regulates nonquantal ACh liberation (Paton, Vizi and Zar, 1971 ; Vizi, 1973. 1975, 1977) in this preparation, and also in frog sartorius muscle.
MOUSE DIAPHRAGM AND NON-QUANTAL ACh RELEASE The whole diaphragm of a white mouse was dissected and divided into several strips, each of them pinned to a plastic plate and immersed in oxygenated perfusion saline (Liley, 1956). Intracellular recordings were made with 20-25 MS2 glass microelectrodes inserted into the muscle fibres close to the termination of small branches of the main intramuscular nerve trunk. In some cases, the bathM ‘neostigmine methylsulphate as an antiing medium contained 6 X cholinesterase (VyskoEil and IIIes, 1977), but experiments were also performed on diaphragm strips preincubated for 30 min in a solution containing an irreM ; VyskoEil and Ill&, 1978). versible cholinesterase inhibitor Soman ( Tetrodotoxin M) was present in the bath during experiments with anticholinesterases to eliminate spontaneous twitching. When superficial muscle fibres were successfully impaled with a microelectrode and large (1 -2 mV) MEPPs were observed on the oscilloscope screen, another pipette (50- 100 pm tip diameter) containing physiological saline (of M) the same composition as that in the bath) saturated with TC (about was immersed in the bath and rapidly placed close to the fibre. The effect of TC which diffused from the tip of the pipette was usually very rapid and MEPPs disappeared within seconds (Fig. 1A). In the normal solution, hyperpolarisation of the muscle fibre membrane (indicated in Fig. 1 as downward deflection) was routinely observed. It ranged in different fibres from 0.2 to 3.0 mV, the mean value (f S.E.M.) being 1.1 f 0.27 mV for a resting membrane potential (RMP) of -71 mV (20 fibres) in the presence of neostigmine. In experiments with Soman, similar results were obtained (mean hyperpolarization 0.9 f 0.30 mV at RMP -68 mV; VyskoEil and IIICs, 1978). The similar value of hyperpolarization obtained in the presence of neostigmine and after
185 PROST IGMINE
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0 Fig. 1. Mouse diaphragm. The effect of local curarisation on MEPPs and membrane potential M neostigmine (left) and after pretreatment of the diaphragm in the presence of 6 x with Soman (right). The muscle strips were incubated at 32OC in the normal solution (A), normal solution plus 2 x M ouabain (B) or a K* free solution (C) respectively. The bottom records (D) were obtained 10 min after readmission of K* into the K* free solution. Horizontal bars indicate the time of tubocuranine (TC) diffusion from pipette located in the end-plate area (VyskoEil and IllBs, 1978).
pretreatment with Soman makes it unlikely that the effect results from a direct postsynaptic depolarizing action of neostigmine, and is consistent with the interpretation (Katz and Miledi, 1977) that continuous ACh leakage is responsible. It has been found (Vizi and VyskoEil, 1979) that total ACh output is affected by activation or inhibition of M-ATPase, and we have investigated whether this can be detected electrophysiologically .
186 INHIBITION AND ACTIVATION OF Na*-K'-ACTIVATED MEMBRANE ATPase (M-ATPase) When strips of neostigmine-treated diaphragm were immersed in a solution containing 2 X M ouabain to block M-ATPase activity (Fig. 1B) the mean hyperpolarisation after TC increased t o 1.5 f 0.23 mV (mean RMP -65.5 mV). The suspension of muscles in a potassium-free solution (Fig. 1C) also led t o substantially higher TC-hyperpolarisation than in control experiments (mean 1.88 f 0.4 mV and 1.65 i 0.4 mV in prostigmine and after Soman treatment respectively). On the other hand, activation of M-ATPase by adding 5 mM K' after the muscles had been kept for 1-2 h in K-free solution abolished the hyperpolarisation when TC was applied 10-20 min after the readdition of K' ions (Fig. ID) i t . during the period of increased M-ATPase activity (Vizi and VyskoEil, 1979). It has recently been found (Vizi and VyskoEil, 1979) that block of M-ATPase by ouabain leads to an approximately two-fold increase in total resting release of ACh from the mouse diaphragm. This finding correlates with the present results showing the potentiation of TC hyperpolarisation by a factor of 1.51.7 during the blockade of membrane ATPase. Similarly, the activation of M-ATPase eliminated the electrophysiologically assayed non-quan tal leakage of ACh (Vyskocil and IlICs, 1977, 1978) and depressed the total release t o one tenth of the control value (Vizi and VyskoEil, 1979). FROG SARTORIUS AND NON-QUANTAL RELEASE DURING INHIBITION AND ACTIVATION O F M-ATPase It appeared of interest t o ascertain whether a similar dependence of nonquantal leakage on the activity of M-ATPase also exists in frog muscle. This preparation allows continuous microelectrode recordings from single muscle fibres for longer periods of time. In several preliminary experiments, sartorius muscles were continuously perfused with a standard oxygenated Ringer solution with 1 mM glucose at a temperature of 32-35"C, i.e. they were kept under conditions where one can expect a proper functioning of M-ATPase and higher output of ACh (Mitchell and Silver, 1963). To block cholinesterase the muscles were preincubated, as described above, in a solution containing either M). When Soman o r another irreversible inhibitor Armin (U.S.S.R.) (1 X the baseline had become sufficiently stable after impalement, a pipette (1 00 pm diameter) containing Ringer solution and 10-2M ouabain was placed close t o the junction for several minutes. As indicated in Fig. 2 a slight depolarisation of about 1 mV developed during this period. It was almost completely eliminated by local curarisation via a TC-containing pipette, which suggests that this depolarisation might be caused by ouabain-stimulated nonquantal leakage of ACh. After removal of both pipettes, the RMP and MEPPs usually recovered and in several cases the whole procedure was repeated (Fig. 2, bottom record). If sartorius muscles were bathed in a K'-free solution for 10-15 min local
187
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Fig. 2. The effect of ouabain on the neuromuscular junction of a frog sartorius muscle pretreated with Soman. Intracellular records from one muscle cell (starting R M P = -82 mV) before and after local application of ouabain (OU)and tubocurarine (TC). Inset illustrates the experimental situation and localisation of electrode and OU- and TC-filled pipettes. For other details see text. Fig. 3. TC-induced hyperpolarisation of postsynaptic membrane of one frog m. sartorius fibre pretreated with Soman in a potassium free (K+-free) solution (RMP = 97 mV) and 7 min after readmission of 5 mM potassium (+K+)into the Ringer solution (RP = 100 mV) at 35OC.
curarisation caused a hyperpolarisation similar to that seen in the mouse diaphragm ranging from 0.25-1 mV in four experiments (Fig. 3, top record). The reactivation of M-ATPase after 1 h by addition of 5 mM K' led to the disappearance of the hyperpolarising effect of TC (Fig. 3, bottom record). It thus seems very likely that in the frog muscle ACh leakage is also dependent on the activity of M-ATPase.
FINAL REMARKS There is no longer any doubt that the largest part of the ACh released at the resting neuromuscular junction, is in nonquantal form, probably originating from cytoplasmic stores in nerve terminals (cf. Tauc et al., 1974). The question arises about the physiological meaning of this spontaneous output of ACh. Since it is released in relatively large amounts (Vizi and VyskoEil, 1979) the
188 possibility exists that it has a trophic influence on target muscle cells. Katz and Miledi ( 1 977) suggested that it may induce or regulate junctional cholinesterase, but it may even be involved in more general “trophic” regulations (Thesleff, this volume). Whatever role other than impulse transmission is played by ACh it appears that the non-quanta1 release is controlled t o a large extent by the activity of M-ATPase. The mechanism by which this membrane enzyme controls nonquantal liberation of transmitter is not clear yet and should be studied in more detail. SUMMARY The subsynaptic area of mouse diaphragm fibres was hyperpolarised by 1-2 niV during local curarisation of the junctional zone in the presence of neostigmine or after treatment of the muscle with organophosphate cholinesterase inhibitors. In a solution containing 5 mM K’ the mean hyperpolarisation was 1 .I f 0.27 mV. After adding 2 X lo-’ M ouabain the hyperpolarisation increased t o 1.5 f 0.25 mV. In a K+-free bathing medium (i.e. blockade of membrane ATPase) the curare induced hyperpolarisation was also increased, to 1.8 f 0.4 mV. Reactivation of membrane ATPase by addition of K’ after a period in a K’-free medium reduced the hyperpolarisation t o zero. Similar results were also obtained in the frog sartorius muscle. The spontaneous non-quanta1 leakage of acetylcholine at rest, manifested as hyperpolarisation during local curarisation, appears t o be regulated by the activity of Na+-K+-ATPase of the nerve terminals directly, whereas the spontaneous quantal release (miniature end-plate potentials) is affected in the mouse preparation by the Na+-K+pump only indirectly as a result of the slow changes in ionic distribution (Vizi and VyskoEil, 1979). ACKNOWLEDGEMENT We wish to thank Dr. Pave1 Hnik for his help in the preparation of manuscript. REFERENCES Brooks, V.B. (1954) The action of botulinum toxin on motor nerve filaments. J. Physiol. (Lond.), 123, 501-515. Evans, C.A.N. and Saunders, N.R. (1974) An outflow of acetylcholine from normal and regenerating ventral roots of the cat. J. Physiol. (Lond.), 240, 15-32. Fletcher, P. and Forrester, T. (1975) The effect of curare on the release of acetylcholine from mammalian nerve terminals and an estimate of quantum content. J. Physiol. (Lond.), 251, 131-144. Katz, B. and Miledi, R. ( 1 965) The quantal release of transmitter substances. In Studies in Physiology, Springer, Berlin, pp. 118-125. Katz, B. and Miledi, R. (1977) Transmitter leakage from motor nerve endings. Proc. R . Soc. Lond. B, 196,59-72. KrnjeviC, K. and Mitchell, J.F. (1961) The release of acetylcholine in the isolated rat diaphragm. J. Physiol. (Lond.), 155,246-262.
189 Liley, A.W. (1956) The effect of presynaptic polarisation on the spontaneous activity at the mammalian neuromuscular junction. J. Physiol. (Lond.), 134,427-443. Mitchell, J.F. and Silver, A. (1963) The spontaneous release of acetylcholine from the denervated hemidiaphragm of the rat. J. Physiol. (Lond.); 165, 117-1 29. Paton, W.D.M., Vizi, E.S. and Zar, M.A. (1971) The mechanism of acetylcholine release from parasympathetic nerves. J. Physiol. (Lond.), 21 5,819-848. Salpeter, M.M. and Eldefrawi, M.E. (1 973) Sizes of end-plate compartments, densities of acetylcholine receptor and other quantitative aspects of neuromuscular transmission. J. Histochem. Cytochem,, 21,769-778. Skou, J.C. (1965) Enzymatic basis for active transport of Na' and K' across cell membrane. Physiol. Rev., 45,596-617. T a w , L., Hoffmann, A., Tsuji, S., Hinzen, D.H. and Faille, L. (1974) Transmission abolished on a cholinergic synapse after injection of acetylcholinesterase into the presynaptic neurone. Nature, 250,496-498. Vizi, E.S. (1973) Does stimulation of Na*-K'-Mg2+-activated ATP-ase inhibit acetylcholine release from nerve terminals? Brit. J. Pharmacol., 48, 346-347. Vizi, E.S. (1 975) Release mechanisms of acetylcholine and the role of Na*-K'-activated ATP-ase. In Cholinergic Mechanisms, P.G. Waser (Ed.), New York, Raven Press, pp. 199-21 1. Vizi, E.S. ( 1 977) Termination of transmitter release by stimulation of sodium-potassium activated ATPase. J. Physiol. (Lond.), 267, 261 -280. Vizi, E.S. and VyskoEil, F. (1979) Changes in total and quantal release of acetylcholine in the mouse diaphragm during activation and inhibition of membrane ATPase. J. Physiol, (Lond.), 286, 1-14. VyskoEil, F. and Illts, P. (1977) Nonquantal release of transmitter at mouse neuromuscular junction and its dependence on the activity of Na'-K'-ATPase. Pfliigers Arch. ges. Physiol., 370,295-297. VyskoEil, F. and Illts, P. (1978) Electrophysiological examination of transmitter release in nonquantal form in the mouse diaphragm and the activity of membrane ATPase. Physiol. bo hem oslo v , 2 7 , 4 4 9-4 5 5,
INTRODUCTION It was shown by Brooks in 1954 that acetylcholine (ACh) is released from the isolated diaphragm of the guinea-pig even in the absence of nerve stimulation. This fact was subsequently confirmed by several authors and spontaneous miniature end-plate potentials (MEPPs) were considered to be an electrophysiological correlate of the resting ACh output from skeletal muscle (see e.g. KrnjeviC and Mitchell, 1961). As early as in 1963, however, Mitchell and Silver’realized the serious discrepancy between the changes of biologically assayed ACh in .their experiments and electrophysiologically recorded MEPPs under different experimental conditions. Thus, for example, when the K’ concentration in the bathing fluid is increased from 5 to 30 mM, the MEPP frequency (representing the number of quanta released per unit time) is raised 200-300 times in the rat diaphragm (Liley, 1956) whereas the output of ACh increases only about 3-fold (Mitchell and Silver, 1963). A similar discrepancy appeared when the effect of temperature was studied. Mitchell and Silver therefore concluded that only a small fraction of the resting release derives from MEPPs and that, surprisingly, 97-99% of released ACh has a different origin. A similar conclusion was also drawn by Fletcher and Forrester (1975) who estimated that only 2% of overall ACh release from rat diaphragm at rest could represent quantal release and suggested that the remainder has a non-synaptic source. In our recent observation on the mouse diaphragm (Vizi and VyskoEil, 1979), where we compared the total and quantal release ACh before and during stimulation, we estimated that 1% of the total release represents quantal release. The non-quanta1 release of ACh was considered by some workers to be of “non-synaptic” origin and was attributed to the preterminal regions of motor axons (Brooks, 1954; Mitchell and Silver, 1963; Fletcher and Forrester, 1975; cf. Evans and Saunders, 1974), too far away from the postsynaptic membrane to evoke detectable depolarisation. Another possibility suggested by Katz and Miledi (1965) was that a steady and diffuse leakage may occur from the terminals all over the junction. This continuous leakage, because of hydrolysis by cholinesterase, can barely be recorded electrophysiologically. Nevertheless, Katz and Miledi (1 977) successfully tested this assumption on antiesterase
184 (diisopropylfluorophosphate, DFP) treated frog sartorius muscles where individual end-plates of superficial muscle fibres were subjected to a massive ionophoretic dose of (+)-tubocurarine (TC). During this local curarisation, various levels of hyperpolarisation wefe observed in different muscle fibres and this effect was attributed to the leakage of cytoplasmic ACh from nerve terminals causing a minute steady postsynaptic depolarisation, which is prevented by TC. The average depolarisation was only about 40 pV (at 18-24°C) in this preparation and such a change is so small that it does not provide a good opportunity for studying the characteristics of the steady leakage. We therefore decided to use the mouse diaphragm and to perform similar experiments on this preparation (VyskoEil and Ill&, 1977, 1978), where the electrical responses to ACh (MEPPs in particular) are more pronounced apparently because of the small diameter and high input impedance of the muscle fibres and compact junctional area (Salpeter and Eldefrawi, 1973). As will be demonstrated later, mouse end-plates were hyperpolarized by 1-2 mV during local curarization in the presence of anticholinesterases at 32°C. We checked the idea that the membrane Na*,K’-ATPase (Skou, 1965) of the nerve terminal membrane regulates nonquantal ACh liberation (Paton, Vizi and Zar, 1971 ; Vizi, 1973. 1975, 1977) in this preparation, and also in frog sartorius muscle.
MOUSE DIAPHRAGM AND NON-QUANTAL ACh RELEASE The whole diaphragm of a white mouse was dissected and divided into several strips, each of them pinned to a plastic plate and immersed in oxygenated perfusion saline (Liley, 1956). Intracellular recordings were made with 20-25 MS2 glass microelectrodes inserted into the muscle fibres close to the termination of small branches of the main intramuscular nerve trunk. In some cases, the bathM ‘neostigmine methylsulphate as an antiing medium contained 6 X cholinesterase (VyskoEil and IIIes, 1977), but experiments were also performed on diaphragm strips preincubated for 30 min in a solution containing an irreM ; VyskoEil and Ill&, 1978). versible cholinesterase inhibitor Soman ( Tetrodotoxin M) was present in the bath during experiments with anticholinesterases to eliminate spontaneous twitching. When superficial muscle fibres were successfully impaled with a microelectrode and large (1 -2 mV) MEPPs were observed on the oscilloscope screen, another pipette (50- 100 pm tip diameter) containing physiological saline (of M) the same composition as that in the bath) saturated with TC (about was immersed in the bath and rapidly placed close to the fibre. The effect of TC which diffused from the tip of the pipette was usually very rapid and MEPPs disappeared within seconds (Fig. 1A). In the normal solution, hyperpolarisation of the muscle fibre membrane (indicated in Fig. 1 as downward deflection) was routinely observed. It ranged in different fibres from 0.2 to 3.0 mV, the mean value (f S.E.M.) being 1.1 f 0.27 mV for a resting membrane potential (RMP) of -71 mV (20 fibres) in the presence of neostigmine. In experiments with Soman, similar results were obtained (mean hyperpolarization 0.9 f 0.30 mV at RMP -68 mV; VyskoEil and IIICs, 1978). The similar value of hyperpolarization obtained in the presence of neostigmine and after
185 PROST IGMINE
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0 Fig. 1. Mouse diaphragm. The effect of local curarisation on MEPPs and membrane potential M neostigmine (left) and after pretreatment of the diaphragm in the presence of 6 x with Soman (right). The muscle strips were incubated at 32OC in the normal solution (A), normal solution plus 2 x M ouabain (B) or a K* free solution (C) respectively. The bottom records (D) were obtained 10 min after readmission of K* into the K* free solution. Horizontal bars indicate the time of tubocuranine (TC) diffusion from pipette located in the end-plate area (VyskoEil and IllBs, 1978).
pretreatment with Soman makes it unlikely that the effect results from a direct postsynaptic depolarizing action of neostigmine, and is consistent with the interpretation (Katz and Miledi, 1977) that continuous ACh leakage is responsible. It has been found (Vizi and VyskoEil, 1979) that total ACh output is affected by activation or inhibition of M-ATPase, and we have investigated whether this can be detected electrophysiologically .
186 INHIBITION AND ACTIVATION OF Na*-K'-ACTIVATED MEMBRANE ATPase (M-ATPase) When strips of neostigmine-treated diaphragm were immersed in a solution containing 2 X M ouabain to block M-ATPase activity (Fig. 1B) the mean hyperpolarisation after TC increased t o 1.5 f 0.23 mV (mean RMP -65.5 mV). The suspension of muscles in a potassium-free solution (Fig. 1C) also led t o substantially higher TC-hyperpolarisation than in control experiments (mean 1.88 f 0.4 mV and 1.65 i 0.4 mV in prostigmine and after Soman treatment respectively). On the other hand, activation of M-ATPase by adding 5 mM K' after the muscles had been kept for 1-2 h in K-free solution abolished the hyperpolarisation when TC was applied 10-20 min after the readdition of K' ions (Fig. ID) i t . during the period of increased M-ATPase activity (Vizi and VyskoEil, 1979). It has recently been found (Vizi and VyskoEil, 1979) that block of M-ATPase by ouabain leads to an approximately two-fold increase in total resting release of ACh from the mouse diaphragm. This finding correlates with the present results showing the potentiation of TC hyperpolarisation by a factor of 1.51.7 during the blockade of membrane ATPase. Similarly, the activation of M-ATPase eliminated the electrophysiologically assayed non-quan tal leakage of ACh (Vyskocil and IlICs, 1977, 1978) and depressed the total release t o one tenth of the control value (Vizi and VyskoEil, 1979). FROG SARTORIUS AND NON-QUANTAL RELEASE DURING INHIBITION AND ACTIVATION O F M-ATPase It appeared of interest t o ascertain whether a similar dependence of nonquantal leakage on the activity of M-ATPase also exists in frog muscle. This preparation allows continuous microelectrode recordings from single muscle fibres for longer periods of time. In several preliminary experiments, sartorius muscles were continuously perfused with a standard oxygenated Ringer solution with 1 mM glucose at a temperature of 32-35"C, i.e. they were kept under conditions where one can expect a proper functioning of M-ATPase and higher output of ACh (Mitchell and Silver, 1963). To block cholinesterase the muscles were preincubated, as described above, in a solution containing either M). When Soman o r another irreversible inhibitor Armin (U.S.S.R.) (1 X the baseline had become sufficiently stable after impalement, a pipette (1 00 pm diameter) containing Ringer solution and 10-2M ouabain was placed close t o the junction for several minutes. As indicated in Fig. 2 a slight depolarisation of about 1 mV developed during this period. It was almost completely eliminated by local curarisation via a TC-containing pipette, which suggests that this depolarisation might be caused by ouabain-stimulated nonquantal leakage of ACh. After removal of both pipettes, the RMP and MEPPs usually recovered and in several cases the whole procedure was repeated (Fig. 2, bottom record). If sartorius muscles were bathed in a K'-free solution for 10-15 min local
187
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. L. I dL ry I
K- free
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Fig. 2. The effect of ouabain on the neuromuscular junction of a frog sartorius muscle pretreated with Soman. Intracellular records from one muscle cell (starting R M P = -82 mV) before and after local application of ouabain (OU)and tubocurarine (TC). Inset illustrates the experimental situation and localisation of electrode and OU- and TC-filled pipettes. For other details see text. Fig. 3. TC-induced hyperpolarisation of postsynaptic membrane of one frog m. sartorius fibre pretreated with Soman in a potassium free (K+-free) solution (RMP = 97 mV) and 7 min after readmission of 5 mM potassium (+K+)into the Ringer solution (RP = 100 mV) at 35OC.
curarisation caused a hyperpolarisation similar to that seen in the mouse diaphragm ranging from 0.25-1 mV in four experiments (Fig. 3, top record). The reactivation of M-ATPase after 1 h by addition of 5 mM K' led to the disappearance of the hyperpolarising effect of TC (Fig. 3, bottom record). It thus seems very likely that in the frog muscle ACh leakage is also dependent on the activity of M-ATPase.
FINAL REMARKS There is no longer any doubt that the largest part of the ACh released at the resting neuromuscular junction, is in nonquantal form, probably originating from cytoplasmic stores in nerve terminals (cf. Tauc et al., 1974). The question arises about the physiological meaning of this spontaneous output of ACh. Since it is released in relatively large amounts (Vizi and VyskoEil, 1979) the
188 possibility exists that it has a trophic influence on target muscle cells. Katz and Miledi ( 1 977) suggested that it may induce or regulate junctional cholinesterase, but it may even be involved in more general “trophic” regulations (Thesleff, this volume). Whatever role other than impulse transmission is played by ACh it appears that the non-quanta1 release is controlled t o a large extent by the activity of M-ATPase. The mechanism by which this membrane enzyme controls nonquantal liberation of transmitter is not clear yet and should be studied in more detail. SUMMARY The subsynaptic area of mouse diaphragm fibres was hyperpolarised by 1-2 niV during local curarisation of the junctional zone in the presence of neostigmine or after treatment of the muscle with organophosphate cholinesterase inhibitors. In a solution containing 5 mM K’ the mean hyperpolarisation was 1 .I f 0.27 mV. After adding 2 X lo-’ M ouabain the hyperpolarisation increased t o 1.5 f 0.25 mV. In a K+-free bathing medium (i.e. blockade of membrane ATPase) the curare induced hyperpolarisation was also increased, to 1.8 f 0.4 mV. Reactivation of membrane ATPase by addition of K’ after a period in a K’-free medium reduced the hyperpolarisation t o zero. Similar results were also obtained in the frog sartorius muscle. The spontaneous non-quanta1 leakage of acetylcholine at rest, manifested as hyperpolarisation during local curarisation, appears t o be regulated by the activity of Na+-K+-ATPase of the nerve terminals directly, whereas the spontaneous quantal release (miniature end-plate potentials) is affected in the mouse preparation by the Na+-K+pump only indirectly as a result of the slow changes in ionic distribution (Vizi and VyskoEil, 1979). ACKNOWLEDGEMENT We wish to thank Dr. Pave1 Hnik for his help in the preparation of manuscript. REFERENCES Brooks, V.B. (1954) The action of botulinum toxin on motor nerve filaments. J. Physiol. (Lond.), 123, 501-515. Evans, C.A.N. and Saunders, N.R. (1974) An outflow of acetylcholine from normal and regenerating ventral roots of the cat. J. Physiol. (Lond.), 240, 15-32. Fletcher, P. and Forrester, T. (1975) The effect of curare on the release of acetylcholine from mammalian nerve terminals and an estimate of quantum content. J. Physiol. (Lond.), 251, 131-144. Katz, B. and Miledi, R. ( 1 965) The quantal release of transmitter substances. In Studies in Physiology, Springer, Berlin, pp. 118-125. Katz, B. and Miledi, R. (1977) Transmitter leakage from motor nerve endings. Proc. R . Soc. Lond. B, 196,59-72. KrnjeviC, K. and Mitchell, J.F. (1961) The release of acetylcholine in the isolated rat diaphragm. J. Physiol. (Lond.), 155,246-262.
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