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Diamide acts intracellularly to enhance transmitter release: The differential permeation of diamide, DIP, DIP + 1 and DIP + 2 across the nerve terminal membrane
Brain Research, 117 (1976) 27%285
277
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
DIAMIDE ACTS INTRACELLULARLY TO ENHANCE TRANSMITTER RELEASE: THE DIFFERENTIAL PERMEATION OF DIAMIDE, DIP, DIP + 1 AND DIP + 2 ACROSS THE NERVE TERMINAL MEMBRANE
P. L. CARLEN*, E. M. KOSOWER and R. WERMAN Neurobiology Unit, The Hebrew University, Jerusalem, and ( E.M.K.) Department o f Chemistry, Tel Aviv University, Ramat Aviv, Tel Aviv (Israel)
(Accepted March 29th, 1976)
SUMMARY The actions of the new potent thiol oxidizing agents, diazene dicarboxylic acid bis (N'-methyl piperazide) (DIP) and the N'-methyl iodide (DIP + 1) and the bisN'-methyl iodide (DIP + 2) salts of DIP, were tested at the frog neuromuscular junction. At 20 °C, DIP was as fast as the thiol oxidizing agent, diamide, in evoking transmitter release but was appreciably less effective at 6 °C. DIP + 1 and DIP + 2 did not increase transmitter release. Since the three agents are potent oxidizers of glutathione and since the effectiveness of the compounds appears to depend on their ability to exist, at least in part, in a neutral form at physiological pH, it is concluded that their action as promoters of transmitter release depends on their ability to permeate nerve terminal membranes. Thus, both diamide and DIP act to increase transmitter release by the intracellular oxidation of glutathione. The two charged agents, DIP + 1 and DIP + 2, are potent muscular depolarizing agents. It is probable that the quaternary nitrogen groups of these compounds render them cholinomimetics.
INTRODUCTION Diamide, an intracellular oxidizer of glutathione (GSH), has been shown to act presynaptically at the froga,S,la and locustx2 neuromuscular junctions. At the frog neuromuscular junction it increases acetylcholine (ACh) release, both in the form of an increased frequency of spontaneous miniature endplate potentials (MEPPS) and an increase in amplitude of endplate potentials (EPPs) evoked by neural stimuli3,1~. * Present address: Deaprtment of Medicine (Neurology), University of Toronto; address for rereprints: Addiction Research Foundation, 33 Russell Street, Toronto, Ontario M5S 2S1, Canada.
278 With low concentrations, all the evidence points to a specific intracellular presynaptic effect of the thiol oxidizing agent3,s, 13. There is additional physiological interest in that the action of diamide closely resembles that of prolonged repetitive stimulation of the presynaptic nerve`',3, 6. Diamide preferentially converts GSH to glutathione disulfide (GSSG) in comparison to its oxidizing action on other small dithiols found in nerve, and is appreciably less active against sulfhydryl groups in larger molecules 9. GSH is normally found in nerve tissue in appreciable concentrations (about 3 mM) 10 and its effective conversion to GSSG by diamide (rate constant 300 mole -1 • sec-1) s would increase free GSSG in nerve terminals. The presence of an appreciable excess of GSSG in nerve terminals would be expected to convert membrane dithiols into shorter disulfide bonds. It has been proposed that the membrane contractions that might result are responsible for vesicular release in the terminal a. Alternatively, it is possible that the major site of GSSG action could be at the sites of intracellular calcium (Ca `'+) sequestration and that diamide acts to increase intracellular Ca `'+ concentration 3. The possibility that the absence of GSH itself leads to release has not been ruled out 3. In order to ascertain whether the thiol groups accessible to an extracellular reagent are the site of diamide action, Kosower et al. v synthesized and examined a series of diamide-related compounds that were designed to vary in their ability to penetrate membranes. This was accomplished by adding a basic nitrogen to the diamide structure together with enough carbon atoms to make the compounds stable. It was anticipated that the highly charged quaternary salts of the basic charged nitrogen would penetrate membranes less well than potentially neutral compounds. The resulting compounds were diazene dicarboxylic acid bis (N'-methylpiperazide) (DIP), the N'-methyl iodide salt of DIP (DIP ÷ 1) and the bis-N'-methyl iodide salt of DIP (D1P + 2). The relevant structures are the following. (CH3)2NCON ~ CH3N
NCON(CH3) 2 Diam~de
NCON ~ NCON
/NCH 3
DIP CH3N
NCON =~ NCON\
N*(CH3) 2
DIP + I
f-~ (CH3)2*NN C O N ,
~ DIP +
7 \ NCON,~ JN+(CH3)2 2
It was demonstrated that the new compounds oxidized GSH in solution more rapidly than diamide, but penetrated the red blood cell membrane more slowly (DIP) or not at all (DIP q- 1, DIP q- 2) 7. All three compounds react in the order of hundreds to thousands times more rapidly than diamide with GSH. Thus DIP reacts a 100 times more rapidly than diamide with GSH when measured at 20 °C at neutral pH. DIP, however, was 2-4 times slower than diamide when intracellular GSH oxidation was measured at this temperature in red blood cells. The differential ability of the two compounds to act intraceUularly was accentuated at low temperatures, so that at 1 °C diamide was 10-15 times more rapid in action than DIPL
279 In this paper we compare the actions of these new thiol oxidizing agents with that of diamide at the frog neuromuscular junction. We demonstrate that of the three new compounds only DIP is active presynaptically. Furthermore the action of DIP is decreased more than that of diamide by cooling. METHODS The techniques were, in general, those reported in the preceding paper a. With only a few exceptions the experiments were carried out on the sternocutaneous nervemuscle preparation. This muscle is, in addition to other advantages, uniformly fiat and thin in small frogs. It is probable that this feature accounts for the more uniform temporal response of this preparation to diamide a. Control of temperature was accomplished by use of a Peltier cell. The three compounds, DIP, DIP + 1, DIP + 2, are easily soluble yellow solids and have a half-life for hydrolysis at pH 7.2 of 5.5 h, 1.5 h and 1.2 h respectively. Solutions of these compounds were therefore prepared immediately before use. These chemicals were either added from a point source or by flowing at least 10 bath-volumes of bathing solution containing the required concentration over the preparation. RESULTS Just as DIP was able to mimic the action of diamide in oxidizing intracellular GSH in red cells 7, DIP mimicked the action of diamide at the neuromuscular junction. In the experiment shown in Fig. 1, carried out at room temperature, 5 × 10-5 M DIP dramatically increased the rate of MEPPs which remained elevated throughout the 3.5 h observation period. In the same experiment the EPP amplitude increased with a corresponding time course but, after reaching a peak at 1 h after administration of DIP, slowly declined despite continued high MEPP rate. Although somewhat slow, the kinetics of the responses were within the range found for diamide at the same concentration and temperature. A series of experiments were carried out to assess the effect of temperature on the time of onset of increased MEPP frequency. In two sternocutaneous muscles fl om the same frog, 10-4 M diamide was added to one and l0 -4 M DIP was added to the paired muscle and both experiments were carried out at room temperature. In each case the MEPP frequency significantly increased within 1 min and rose to even higher frequencies. These experiments were then repeated at 6 °C, again using 10-4 diamide and DIP and a pair of sternocutaneous muscles from another frog. Fig. 2 shows the decrease of MEPP rate to less than one-fifth of control value as the preparation was cooled from room temperature (20 °C) to 6 °C, at which temperature the rest of the experiment was carried out. Diamide (10 -4 M) produced an increase in MEPP frequency that exceeded two standard deviations of the mean frequency at the low temperature within 3 min of its addition, and within 25 min a high MEPP frequency was observed. At that time 4 other endplates in the same preparation were examined and high MEPP frequencies were also found: 50, 55, 100 and 110 MEPPs/sec, respectively.
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Fig. 1. DIP increases EPP amplitude and MEPP frequency. EPP amplitudes from an average of 64 nerve-evoked responses at 1 Hz are plotted as shown by the smaller filled:circlesjointed by a continuous line. The MEPP frequency samples are shown by the larger filled circles joined by a dashed line. DIP was added from a point source to give a bath concentration of 5 × 10-5 M at the upward arrow. The MEPP frequencycontinued at a high rate until the end of the experiment, A sternocutaneous muscle was used and the bathing solution contained 3.2 mM Mg~+ and 0,3 mM Ca~+. In contrast, when the effect of 10-4 M DIP was examined at 6 °C in the paired sternocutaneous muscle, it took 12 min after DIP application before the increase in MEPP frequency was significant (Fig. 3). The rate of rise of MEPP frequency was quite slow and not until 70 min after addition of D I P was a relatively high MEPP rate obtained, and the frequency never exceeded 10/sec throughout the 3 h observation period. At that time 3 other endplates were examined in the same preparation and frequencies of 40, 33 and 12.6/sec, respectively, were found. Hence, at 6 °C, in this and similar experiments using paired sternocutaneous muscles from the same frog, a considerably larger delay in onset of MEPP rate increase was seen following DIP as compared with diamide. In addition, D I P action was characterized by a decrease in the rate of rise of the MEPP frequency and a lower final frequency than those that were obtained with the same dose of diamide. At this low temperature, the response to both thiol oxidizing agents was significantly delayed as compared to room temperature. The thiol oxidizing analogues of D I P made by adding one (DIP + l) or two (DIP + 2) positive charges to the D I P molecule were also tested for their effects on MEPP release. Fig. 4 shows an experiment where 10-4 M D I P + 1 was tested at room temperature. MEPP frequency was not increased and, in fact, showed a small b u t definite decrease, This decrease was associated with a profound depolarization of the muscle. The depolarization was readily reversible when the D I P + 1 was washed from the solution. Diamide was still effective in raising the MEPP frequency in this fiber (Fig. 4).
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Fig. 2. Cooling slows the diamide evoked increase in MEPP frequency slightly. The sternocutaneous muscle was cooled in a Peltier cell to 6 °C and this temperature was maintained until the end of the experiment. Bath temperatures until steady-state are plotted in the upper left part of the figure. MEPP frequency is plotted semilogarithmically against time. The mean MEPP frequency at 6 °C (0.83/sec) is indicated by the lower line and a second line indicates 2 standard deviations above the mean. At the vertical arrow, 10 -4 M diamide was added to the bath from a point source. The bathing solution contained 2 m M Mg ~÷ and 0.5 m M Ca 2÷.
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HOURS Fig. 3. Cooling significantly slows the DIP evoked increase in MEPP frequency. The other sternocutaneous muscle from the frog in the experiment of Fig. 2 was used. This muscle was also cooled down to 6 °C and this temperature was maintained until the end of the experiment (see temperature scale upper left). This graph was plotted as shown in Fig. 2. At the vertical arrow, 10 --4 M DIP was added to the bath from a point source. The bathing solution contained 2 m M Mg ~÷ and 0.5 m M Ca s÷.
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Fig. 4. DIP ÷ 1 produces depolarization of the muscle but fails to increase MEPP frequency. The lower part of the graph samples the MEPP frequency while the upper part of the graph is a plot of the resting potential. The initial horizontal line with small arrowheads indicates the time when 10-4 M DIP + 1 flowed continuously into the bath. The line with large arrowheads indicates when 10-4 M diamide flowed into the bath. All bathing solutions contained 0.3 mM Ca 2+ and 2 mM Mgs+. The experiment was carried out at room temperature.
D I P + 2, the double-quaternary salt of DIP, behavedin a fashion indistinguishable from D I P + 1 (Fig. 5), The application of this thiol oxidizing agent was associated with a profound, reversible depolerization and a small decrease in M E P P frequency. Again, in the experiment illustrated (Fig. 5), diamide was still able to elicit an increase in M E P P frequency. A concentration of 5 × 10 -5 M was used in this and similar experiments. CONCLUSIONS The present series of experiments represent an attempt to determine the site of action of diamide in evoking transmitter release3,8,tz, 13, by producing a series of thiol oxidizing molecules based on the structure of diamide with different abilities to permeate membranes. The resulting compounds, DIP, D I P ± 1 and D I P ÷ 2, are all much more active than diamide as oxidizers of G S H in free solution 7. The presence of two basic nitrogen atoms in the D I P molecule, however, produces a proportion of charged forms at neutral pH which may consequently hinder permeability through the lipid phase of membranes. In fact, approximately 50 % of D I P molecules are monoprotonated at p H 7.2 (ref. 7). D I P is only a slightly larger molecule than diamide and thus size considerations would not be expected to be important if permeation through the lipid phase of the membrane were a rate-limiting step. When the rate of oxidation of intraeellular G S H was examined in red blood cells at r o o m temperature, diamide took about 15 see to act whereas D I P took 30--60 see 7. This difference was accentuated by cooling and at 17 °C diamide acted in 30 see as
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MIN Fig. 5. DIP q- 2 produces depolarization of the muscle but fails to increase MEPP frequency. Plotted as shown in Fig. 4, the thinner initial horizontal line with small arrowheads indicates the time when 10 -5 M DIP + 2 continuously flowed into the bath. The line with the large arrowheads indicates when 10 -4 M diamide flowed into the bath. All bathing solutions contained 0.5 m M Ca ~+ and 2 m M Mg z+ until the flow of DIP q- 2 was finished. For the rest of the experiment the bathing solution contained zero Ca ~+ and 2 m M Mg 2+. The experiment was carried out at room temperature.
contrasted with 3 min for DIP. At 1 °C diamide required 2 min while DIP needed 20-30 rain to oxidize intracellular glutathione. Neither agent interfered with the ability of the red blood cells to regenerate GSH 7,". The differential abilities of DIP and diamide to oxidize GSH in free solution and intracellularly, accentuated by cooling, are presumably explained by differences in the ability of the two molecules to cross red blood cell membranes. By adding one or two positive charges to the DIP molecule (DIP q- 1 and DIP-I-2), molecules which were still much more reactive to free glutathione were obtained, but these molecules were totally inactive against GSH within red blood cells 7 and probably did not penetrate the red blood cell membrane. The new drugs showed a similar differential behavior when tested at the frog neuromuscular junction where diamide is known to act presynaptically to increase acetylcholine release a,13. The action of DIP was almost indistinguishable from that of diamide when both agents were examined at room temperature. At 6 °C, however, diamide action, although slowed, was appreciably more rapid and more extensive than that of DIP. The more highly charged DIP derivatives, DIP -[- 1 and DIP q- 2, did not produce any increase in transmitter release. It thus appears likely that the actions of diamide and DIP depend upon their ability to permeate the nerve terminal membrane. The depolarizing actions of DIP + 1 and DIP q- 2 bear remark. The depolarization was not accompanied by an increase in MEPP frequency, indicating that the presynaptic terminals were not depolarized by these agents. It is likely, therefore, that
284 the depolarization originated from the cholinergic chemosensitive membrane of the muscle. This is consistent with the presence of quaternary groups in the depolarizing molecules and the increased depolarizing ability of DIP + 2, which contains two such groups, compared to DIP ? 1 which contains only one. The quaternary nitrogen is characteristic of acetylcholine and other cholinomimetic agents. The apparent reduction in MEPP frequency that resulted from the use of DIP + 1 and DIP I- 2 may be the result of the reduction of the synaptic driving force and the shunting of the synaptic current that accompany depolarization. These actions would tend to reduce the size of MEPPs and possibly result in a shift of the smaller MEPPs into the noise, resulting in a lower apparent frequency. This possibility was not investigated in the present experiments. If the depolarization was indeed the result of a cholinergic action of the quaternary salts, receptor desensitization 1 and competition of these agents for receptor sites would also tend to reduce MEPP size, tending to produce an underestimation of frequency. Alternatively, acetylcholine has been reported to reduce MEPP releasO, 5 but the nicotinic agonist of acetytcholine, carbachol, has been reported to increase MEPP frequency 11. Unlike other cholinomimetics 1, DIP + 1 and DIP + 2 produced depolarizations which were not accompanied by rapid or obvious desensitization. These agents may therefore have some use in studying the cholinergic receptor 14. Diamide has been shown to increase transmitter release at glutaminergic synapses 1~. It is thus probable that DIP would also increase transmitter release at noncholinergic synapses. The strategy of molecular modification in order to understand and characterize the processes involved in depolarization-secretion coupling has not been taken advantage of hitherto. The experiments reported here may provide a first step in what may be an important new approach in understanding depolarizationsecretion coupling and the spontaneous release of quanta. It has been shown 8 that the conversion of glutathione into GSSG in nerve terminals by diamide replicates the manifestations of prolonged repetitive stimulation of the presynaptic axon2, 6. The manifestations of release of transmitter are clear evidence for a presynaptic site of action for diamide 3,13. Since the best native substrate for diamide action, GSH 9, is found in appreciable concentrations in nervO ° and is absent from the bathing solution, it was inferred that diamide operated intracellularly 3,s,13. The present experiments indicate that diamide does indeed act intracellularly. We have pointed out that there is one presynaptic action of calcium that is not replicated by diamide or other augmenters of transmitter release: some minimal, external concentration of Ca ~+ ions is necessary for the neurally evoked release of transmitter 3. The present study indicates that diamide and DIP enter the nerve terminal through the lipid membrane phase. The thiol oxidizing agents and the apparently impermeable GSH and GSSG would be excluded by size from a site available to calcium ions, a hydrophilic membrane channel. It is then possible that an essential step in neurally evoked depolarization-secretion coupling take place during the passage of Ca 2+ ions across the membrane.
285 ACKNOWLEDGEMENT Supported in p a r t by a
grant
from the C a n a d i a n Multiple Sclerosis Society.
REFERENCES 1 Axelsson, J. and Thesleff, S. W., The 'desensitizing' effect of acetylcholine on the mammalian motor end-plate, Actaphysiol. scand., 43 (1958) 15-26. 2 Braun, M., Schmidt, R. F. and Zimmerman, M., Facilitation at the frog neuromuscular junction during and after repetitive stimulation, Pfliigers Arch. ges. Physiol., 287 (1966) 41-55. 3 Carlen, P. L., Kosower, E. M. and Werman, R., The thiol-oxidizing agent diamide increases transmitter release by decreasing calcium requirements for neuromuscular transmission in the frog, Brain Research, 117 (1976) 257-276. 4 Ciani, S. and Edwards, C., The effect of acctylcholine on neuromuscular transmission in the frog, J. Pharmacol. exp. Ther., 142 (1963) 21-23. 5 Hubbard, J. I., Schmidt, R. F. and Yokota, T., The effect of acctylcholine upon mammalian motor nerve terminals, J. Physiol. (Lond.), 181 (1965) 810-829. 6 Hurlbut, W. P., Longenecker, H. B., Jr. and Mauro, A., Effects of calcium and magnesium on the frequency of miniature end-plate potentials during prolonged tetanization, J. Physiol. (Lond.), 219 (1971) 17-38. 7 Kosower, E. M., Kosower, N. S., Kenety-Londner, H. and Levy, L., Glutathione IX. New thioloxidizing agents: DIP, DIP + 1, DIP + 2, Biochem. biophys. Res. Commun., 59 (1974) 347-351. 8 Kosower, E. M. and Werman, R., New step in transmitter release at the myoneural junction, Nature New Biol., 233 (1971) 121-122. 9 Kosower, N. S., Kosower, E. M., Wertheim, B. and Correa, W., Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide, Biochem. biophys. Res. Commun., 37 (1969) 593-596. 10 McIlwain, H., Biochemistry and the Central Nervous System, 3rd ed., Churchill, London, 1966, p. 136. 11 Miyamoto, M. D. and Voile, R. L., Enhancement by carbachol of transmitter release from motor nerve terminals, Proc. nat. A cad. Sci. (Wash.), 71 (1974) 1489-1492. 12 Walther, C. and Rathmayer, W., The effect of Habrobracon venom on excitatory transmission in insects, J. comp. PhysioL, 89 (1974) 23-28. 13 Werman, R., Carlen, P. L., Kushnir, M. and Kosower, E. M., Effect of the thiol-oxidizing agent, diamide, on acetylcholine release at the frog endplate, Nature New BioL, 233 (1971) 120-121. 14 Ziskind, L. and Werman, R., Sodium ions are necessary for cholinergic desensitiziation in molluscan neurons, Brain Research, 88 (1975) 171-176.
277
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
DIAMIDE ACTS INTRACELLULARLY TO ENHANCE TRANSMITTER RELEASE: THE DIFFERENTIAL PERMEATION OF DIAMIDE, DIP, DIP + 1 AND DIP + 2 ACROSS THE NERVE TERMINAL MEMBRANE
P. L. CARLEN*, E. M. KOSOWER and R. WERMAN Neurobiology Unit, The Hebrew University, Jerusalem, and ( E.M.K.) Department o f Chemistry, Tel Aviv University, Ramat Aviv, Tel Aviv (Israel)
(Accepted March 29th, 1976)
SUMMARY The actions of the new potent thiol oxidizing agents, diazene dicarboxylic acid bis (N'-methyl piperazide) (DIP) and the N'-methyl iodide (DIP + 1) and the bisN'-methyl iodide (DIP + 2) salts of DIP, were tested at the frog neuromuscular junction. At 20 °C, DIP was as fast as the thiol oxidizing agent, diamide, in evoking transmitter release but was appreciably less effective at 6 °C. DIP + 1 and DIP + 2 did not increase transmitter release. Since the three agents are potent oxidizers of glutathione and since the effectiveness of the compounds appears to depend on their ability to exist, at least in part, in a neutral form at physiological pH, it is concluded that their action as promoters of transmitter release depends on their ability to permeate nerve terminal membranes. Thus, both diamide and DIP act to increase transmitter release by the intracellular oxidation of glutathione. The two charged agents, DIP + 1 and DIP + 2, are potent muscular depolarizing agents. It is probable that the quaternary nitrogen groups of these compounds render them cholinomimetics.
INTRODUCTION Diamide, an intracellular oxidizer of glutathione (GSH), has been shown to act presynaptically at the froga,S,la and locustx2 neuromuscular junctions. At the frog neuromuscular junction it increases acetylcholine (ACh) release, both in the form of an increased frequency of spontaneous miniature endplate potentials (MEPPS) and an increase in amplitude of endplate potentials (EPPs) evoked by neural stimuli3,1~. * Present address: Deaprtment of Medicine (Neurology), University of Toronto; address for rereprints: Addiction Research Foundation, 33 Russell Street, Toronto, Ontario M5S 2S1, Canada.
278 With low concentrations, all the evidence points to a specific intracellular presynaptic effect of the thiol oxidizing agent3,s, 13. There is additional physiological interest in that the action of diamide closely resembles that of prolonged repetitive stimulation of the presynaptic nerve`',3, 6. Diamide preferentially converts GSH to glutathione disulfide (GSSG) in comparison to its oxidizing action on other small dithiols found in nerve, and is appreciably less active against sulfhydryl groups in larger molecules 9. GSH is normally found in nerve tissue in appreciable concentrations (about 3 mM) 10 and its effective conversion to GSSG by diamide (rate constant 300 mole -1 • sec-1) s would increase free GSSG in nerve terminals. The presence of an appreciable excess of GSSG in nerve terminals would be expected to convert membrane dithiols into shorter disulfide bonds. It has been proposed that the membrane contractions that might result are responsible for vesicular release in the terminal a. Alternatively, it is possible that the major site of GSSG action could be at the sites of intracellular calcium (Ca `'+) sequestration and that diamide acts to increase intracellular Ca `'+ concentration 3. The possibility that the absence of GSH itself leads to release has not been ruled out 3. In order to ascertain whether the thiol groups accessible to an extracellular reagent are the site of diamide action, Kosower et al. v synthesized and examined a series of diamide-related compounds that were designed to vary in their ability to penetrate membranes. This was accomplished by adding a basic nitrogen to the diamide structure together with enough carbon atoms to make the compounds stable. It was anticipated that the highly charged quaternary salts of the basic charged nitrogen would penetrate membranes less well than potentially neutral compounds. The resulting compounds were diazene dicarboxylic acid bis (N'-methylpiperazide) (DIP), the N'-methyl iodide salt of DIP (DIP ÷ 1) and the bis-N'-methyl iodide salt of DIP (D1P + 2). The relevant structures are the following. (CH3)2NCON ~ CH3N
NCON(CH3) 2 Diam~de
NCON ~ NCON
/NCH 3
DIP CH3N
NCON =~ NCON\
N*(CH3) 2
DIP + I
f-~ (CH3)2*NN C O N ,
~ DIP +
7 \ NCON,~ JN+(CH3)2 2
It was demonstrated that the new compounds oxidized GSH in solution more rapidly than diamide, but penetrated the red blood cell membrane more slowly (DIP) or not at all (DIP q- 1, DIP q- 2) 7. All three compounds react in the order of hundreds to thousands times more rapidly than diamide with GSH. Thus DIP reacts a 100 times more rapidly than diamide with GSH when measured at 20 °C at neutral pH. DIP, however, was 2-4 times slower than diamide when intracellular GSH oxidation was measured at this temperature in red blood cells. The differential ability of the two compounds to act intraceUularly was accentuated at low temperatures, so that at 1 °C diamide was 10-15 times more rapid in action than DIPL
279 In this paper we compare the actions of these new thiol oxidizing agents with that of diamide at the frog neuromuscular junction. We demonstrate that of the three new compounds only DIP is active presynaptically. Furthermore the action of DIP is decreased more than that of diamide by cooling. METHODS The techniques were, in general, those reported in the preceding paper a. With only a few exceptions the experiments were carried out on the sternocutaneous nervemuscle preparation. This muscle is, in addition to other advantages, uniformly fiat and thin in small frogs. It is probable that this feature accounts for the more uniform temporal response of this preparation to diamide a. Control of temperature was accomplished by use of a Peltier cell. The three compounds, DIP, DIP + 1, DIP + 2, are easily soluble yellow solids and have a half-life for hydrolysis at pH 7.2 of 5.5 h, 1.5 h and 1.2 h respectively. Solutions of these compounds were therefore prepared immediately before use. These chemicals were either added from a point source or by flowing at least 10 bath-volumes of bathing solution containing the required concentration over the preparation. RESULTS Just as DIP was able to mimic the action of diamide in oxidizing intracellular GSH in red cells 7, DIP mimicked the action of diamide at the neuromuscular junction. In the experiment shown in Fig. 1, carried out at room temperature, 5 × 10-5 M DIP dramatically increased the rate of MEPPs which remained elevated throughout the 3.5 h observation period. In the same experiment the EPP amplitude increased with a corresponding time course but, after reaching a peak at 1 h after administration of DIP, slowly declined despite continued high MEPP rate. Although somewhat slow, the kinetics of the responses were within the range found for diamide at the same concentration and temperature. A series of experiments were carried out to assess the effect of temperature on the time of onset of increased MEPP frequency. In two sternocutaneous muscles fl om the same frog, 10-4 M diamide was added to one and l0 -4 M DIP was added to the paired muscle and both experiments were carried out at room temperature. In each case the MEPP frequency significantly increased within 1 min and rose to even higher frequencies. These experiments were then repeated at 6 °C, again using 10-4 diamide and DIP and a pair of sternocutaneous muscles from another frog. Fig. 2 shows the decrease of MEPP rate to less than one-fifth of control value as the preparation was cooled from room temperature (20 °C) to 6 °C, at which temperature the rest of the experiment was carried out. Diamide (10 -4 M) produced an increase in MEPP frequency that exceeded two standard deviations of the mean frequency at the low temperature within 3 min of its addition, and within 25 min a high MEPP frequency was observed. At that time 4 other endplates in the same preparation were examined and high MEPP frequencies were also found: 50, 55, 100 and 110 MEPPs/sec, respectively.
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Fig. 1. DIP increases EPP amplitude and MEPP frequency. EPP amplitudes from an average of 64 nerve-evoked responses at 1 Hz are plotted as shown by the smaller filled:circlesjointed by a continuous line. The MEPP frequency samples are shown by the larger filled circles joined by a dashed line. DIP was added from a point source to give a bath concentration of 5 × 10-5 M at the upward arrow. The MEPP frequencycontinued at a high rate until the end of the experiment, A sternocutaneous muscle was used and the bathing solution contained 3.2 mM Mg~+ and 0,3 mM Ca~+. In contrast, when the effect of 10-4 M DIP was examined at 6 °C in the paired sternocutaneous muscle, it took 12 min after DIP application before the increase in MEPP frequency was significant (Fig. 3). The rate of rise of MEPP frequency was quite slow and not until 70 min after addition of D I P was a relatively high MEPP rate obtained, and the frequency never exceeded 10/sec throughout the 3 h observation period. At that time 3 other endplates were examined in the same preparation and frequencies of 40, 33 and 12.6/sec, respectively, were found. Hence, at 6 °C, in this and similar experiments using paired sternocutaneous muscles from the same frog, a considerably larger delay in onset of MEPP rate increase was seen following DIP as compared with diamide. In addition, D I P action was characterized by a decrease in the rate of rise of the MEPP frequency and a lower final frequency than those that were obtained with the same dose of diamide. At this low temperature, the response to both thiol oxidizing agents was significantly delayed as compared to room temperature. The thiol oxidizing analogues of D I P made by adding one (DIP + l) or two (DIP + 2) positive charges to the D I P molecule were also tested for their effects on MEPP release. Fig. 4 shows an experiment where 10-4 M D I P + 1 was tested at room temperature. MEPP frequency was not increased and, in fact, showed a small b u t definite decrease, This decrease was associated with a profound depolarization of the muscle. The depolarization was readily reversible when the D I P + 1 was washed from the solution. Diamide was still effective in raising the MEPP frequency in this fiber (Fig. 4).
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Fig. 2. Cooling slows the diamide evoked increase in MEPP frequency slightly. The sternocutaneous muscle was cooled in a Peltier cell to 6 °C and this temperature was maintained until the end of the experiment. Bath temperatures until steady-state are plotted in the upper left part of the figure. MEPP frequency is plotted semilogarithmically against time. The mean MEPP frequency at 6 °C (0.83/sec) is indicated by the lower line and a second line indicates 2 standard deviations above the mean. At the vertical arrow, 10 -4 M diamide was added to the bath from a point source. The bathing solution contained 2 m M Mg ~÷ and 0.5 m M Ca 2÷.
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HOURS Fig. 3. Cooling significantly slows the DIP evoked increase in MEPP frequency. The other sternocutaneous muscle from the frog in the experiment of Fig. 2 was used. This muscle was also cooled down to 6 °C and this temperature was maintained until the end of the experiment (see temperature scale upper left). This graph was plotted as shown in Fig. 2. At the vertical arrow, 10 --4 M DIP was added to the bath from a point source. The bathing solution contained 2 m M Mg ~÷ and 0.5 m M Ca s÷.
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Fig. 4. DIP ÷ 1 produces depolarization of the muscle but fails to increase MEPP frequency. The lower part of the graph samples the MEPP frequency while the upper part of the graph is a plot of the resting potential. The initial horizontal line with small arrowheads indicates the time when 10-4 M DIP + 1 flowed continuously into the bath. The line with large arrowheads indicates when 10-4 M diamide flowed into the bath. All bathing solutions contained 0.3 mM Ca 2+ and 2 mM Mgs+. The experiment was carried out at room temperature.
D I P + 2, the double-quaternary salt of DIP, behavedin a fashion indistinguishable from D I P + 1 (Fig. 5), The application of this thiol oxidizing agent was associated with a profound, reversible depolerization and a small decrease in M E P P frequency. Again, in the experiment illustrated (Fig. 5), diamide was still able to elicit an increase in M E P P frequency. A concentration of 5 × 10 -5 M was used in this and similar experiments. CONCLUSIONS The present series of experiments represent an attempt to determine the site of action of diamide in evoking transmitter release3,8,tz, 13, by producing a series of thiol oxidizing molecules based on the structure of diamide with different abilities to permeate membranes. The resulting compounds, DIP, D I P ± 1 and D I P ÷ 2, are all much more active than diamide as oxidizers of G S H in free solution 7. The presence of two basic nitrogen atoms in the D I P molecule, however, produces a proportion of charged forms at neutral pH which may consequently hinder permeability through the lipid phase of membranes. In fact, approximately 50 % of D I P molecules are monoprotonated at p H 7.2 (ref. 7). D I P is only a slightly larger molecule than diamide and thus size considerations would not be expected to be important if permeation through the lipid phase of the membrane were a rate-limiting step. When the rate of oxidation of intraeellular G S H was examined in red blood cells at r o o m temperature, diamide took about 15 see to act whereas D I P took 30--60 see 7. This difference was accentuated by cooling and at 17 °C diamide acted in 30 see as
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contrasted with 3 min for DIP. At 1 °C diamide required 2 min while DIP needed 20-30 rain to oxidize intracellular glutathione. Neither agent interfered with the ability of the red blood cells to regenerate GSH 7,". The differential abilities of DIP and diamide to oxidize GSH in free solution and intracellularly, accentuated by cooling, are presumably explained by differences in the ability of the two molecules to cross red blood cell membranes. By adding one or two positive charges to the DIP molecule (DIP q- 1 and DIP-I-2), molecules which were still much more reactive to free glutathione were obtained, but these molecules were totally inactive against GSH within red blood cells 7 and probably did not penetrate the red blood cell membrane. The new drugs showed a similar differential behavior when tested at the frog neuromuscular junction where diamide is known to act presynaptically to increase acetylcholine release a,13. The action of DIP was almost indistinguishable from that of diamide when both agents were examined at room temperature. At 6 °C, however, diamide action, although slowed, was appreciably more rapid and more extensive than that of DIP. The more highly charged DIP derivatives, DIP -[- 1 and DIP q- 2, did not produce any increase in transmitter release. It thus appears likely that the actions of diamide and DIP depend upon their ability to permeate the nerve terminal membrane. The depolarizing actions of DIP + 1 and DIP q- 2 bear remark. The depolarization was not accompanied by an increase in MEPP frequency, indicating that the presynaptic terminals were not depolarized by these agents. It is likely, therefore, that
284 the depolarization originated from the cholinergic chemosensitive membrane of the muscle. This is consistent with the presence of quaternary groups in the depolarizing molecules and the increased depolarizing ability of DIP + 2, which contains two such groups, compared to DIP ? 1 which contains only one. The quaternary nitrogen is characteristic of acetylcholine and other cholinomimetic agents. The apparent reduction in MEPP frequency that resulted from the use of DIP + 1 and DIP I- 2 may be the result of the reduction of the synaptic driving force and the shunting of the synaptic current that accompany depolarization. These actions would tend to reduce the size of MEPPs and possibly result in a shift of the smaller MEPPs into the noise, resulting in a lower apparent frequency. This possibility was not investigated in the present experiments. If the depolarization was indeed the result of a cholinergic action of the quaternary salts, receptor desensitization 1 and competition of these agents for receptor sites would also tend to reduce MEPP size, tending to produce an underestimation of frequency. Alternatively, acetylcholine has been reported to reduce MEPP releasO, 5 but the nicotinic agonist of acetytcholine, carbachol, has been reported to increase MEPP frequency 11. Unlike other cholinomimetics 1, DIP + 1 and DIP + 2 produced depolarizations which were not accompanied by rapid or obvious desensitization. These agents may therefore have some use in studying the cholinergic receptor 14. Diamide has been shown to increase transmitter release at glutaminergic synapses 1~. It is thus probable that DIP would also increase transmitter release at noncholinergic synapses. The strategy of molecular modification in order to understand and characterize the processes involved in depolarization-secretion coupling has not been taken advantage of hitherto. The experiments reported here may provide a first step in what may be an important new approach in understanding depolarizationsecretion coupling and the spontaneous release of quanta. It has been shown 8 that the conversion of glutathione into GSSG in nerve terminals by diamide replicates the manifestations of prolonged repetitive stimulation of the presynaptic axon2, 6. The manifestations of release of transmitter are clear evidence for a presynaptic site of action for diamide 3,13. Since the best native substrate for diamide action, GSH 9, is found in appreciable concentrations in nervO ° and is absent from the bathing solution, it was inferred that diamide operated intracellularly 3,s,13. The present experiments indicate that diamide does indeed act intracellularly. We have pointed out that there is one presynaptic action of calcium that is not replicated by diamide or other augmenters of transmitter release: some minimal, external concentration of Ca ~+ ions is necessary for the neurally evoked release of transmitter 3. The present study indicates that diamide and DIP enter the nerve terminal through the lipid membrane phase. The thiol oxidizing agents and the apparently impermeable GSH and GSSG would be excluded by size from a site available to calcium ions, a hydrophilic membrane channel. It is then possible that an essential step in neurally evoked depolarization-secretion coupling take place during the passage of Ca 2+ ions across the membrane.
285 ACKNOWLEDGEMENT Supported in p a r t by a
grant
from the C a n a d i a n Multiple Sclerosis Society.
REFERENCES 1 Axelsson, J. and Thesleff, S. W., The 'desensitizing' effect of acetylcholine on the mammalian motor end-plate, Actaphysiol. scand., 43 (1958) 15-26. 2 Braun, M., Schmidt, R. F. and Zimmerman, M., Facilitation at the frog neuromuscular junction during and after repetitive stimulation, Pfliigers Arch. ges. Physiol., 287 (1966) 41-55. 3 Carlen, P. L., Kosower, E. M. and Werman, R., The thiol-oxidizing agent diamide increases transmitter release by decreasing calcium requirements for neuromuscular transmission in the frog, Brain Research, 117 (1976) 257-276. 4 Ciani, S. and Edwards, C., The effect of acctylcholine on neuromuscular transmission in the frog, J. Pharmacol. exp. Ther., 142 (1963) 21-23. 5 Hubbard, J. I., Schmidt, R. F. and Yokota, T., The effect of acctylcholine upon mammalian motor nerve terminals, J. Physiol. (Lond.), 181 (1965) 810-829. 6 Hurlbut, W. P., Longenecker, H. B., Jr. and Mauro, A., Effects of calcium and magnesium on the frequency of miniature end-plate potentials during prolonged tetanization, J. Physiol. (Lond.), 219 (1971) 17-38. 7 Kosower, E. M., Kosower, N. S., Kenety-Londner, H. and Levy, L., Glutathione IX. New thioloxidizing agents: DIP, DIP + 1, DIP + 2, Biochem. biophys. Res. Commun., 59 (1974) 347-351. 8 Kosower, E. M. and Werman, R., New step in transmitter release at the myoneural junction, Nature New Biol., 233 (1971) 121-122. 9 Kosower, N. S., Kosower, E. M., Wertheim, B. and Correa, W., Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide, Biochem. biophys. Res. Commun., 37 (1969) 593-596. 10 McIlwain, H., Biochemistry and the Central Nervous System, 3rd ed., Churchill, London, 1966, p. 136. 11 Miyamoto, M. D. and Voile, R. L., Enhancement by carbachol of transmitter release from motor nerve terminals, Proc. nat. A cad. Sci. (Wash.), 71 (1974) 1489-1492. 12 Walther, C. and Rathmayer, W., The effect of Habrobracon venom on excitatory transmission in insects, J. comp. PhysioL, 89 (1974) 23-28. 13 Werman, R., Carlen, P. L., Kushnir, M. and Kosower, E. M., Effect of the thiol-oxidizing agent, diamide, on acetylcholine release at the frog endplate, Nature New BioL, 233 (1971) 120-121. 14 Ziskind, L. and Werman, R., Sodium ions are necessary for cholinergic desensitiziation in molluscan neurons, Brain Research, 88 (1975) 171-176.