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Studies on the neuromuscular blocking action of MPTP in the mouse phrenic nerve-diaphragm
CopyrightQ
0028-3908j93 S&Q@$0.00 1993 Pergamon Press Ltd
STUDIES ON THE N~~R~M~S~~~AR ~~~CK~~ ACTION OF MPTP IN THE MOUSE PHRENIC NERVE-DIAPHRAGM K, S. Hsv,
W. M. Fu and S. Y. L~M-SHUU*
Institutes of Pb~~a~~o~ and Toxicology, Coliege of Medicine, Nation& Taiwan ~n~~~i~* No. t, 3en-Ai Road, tst Section, Taipei, Taiwan, R.O.C.
Z!hmawy--ne periphec3.l ~~~t~~t~ of l-methyl~-ph~yl-l,2,3,(i~tet~~~opyridins (MPTP] in the mouse phrenic n~ia~~a~ preparation was studied. The j~hib~tion of nerve-evoked twit&es was dependent on the ~~~tr~tions of MPTP, ranging from 1 to 2oQ@ M, which ordy slightly JUICES& the amplitude of the twitch evoked by direct stimulation of the mu&e. Pr&eatment with pargyline did not prevent this inhibitory action of MPTP. The inhibition of the twitch by MPTP was reversible and could be partially reversed by antioholinesterase agents such as neostigtnine and physostigmiae, as well as ecothiophatee. Pretreatment with either 0.7 FM d-tubocurarine or 2.2 PM succinylcholine, did not affect the twitches individually but markedly potentiated the effects of MPTP, by shifting the concentrationinhibition curve of MPTP to the left. MPTP not only directly inhibited acetylcholine (O.lmM)-induced contracture of the denervated moose Daphne but atso protected the muscle from the ~~bjto~ action oftr-bungarotoxin. Furtb~rmore, the specit?c binding of ~l~~-bu~~oto~~n to the mouse diaphragm wss also inhibited by MPTP. ~~troph~~oio~~~ stndies revealed that MPTP inhibited the frequency and amplitude of the m.e.p.p., as well as the amplitude of the e.p.p. All of these findings indioate that MPTP exerts a ~eur~u~ar b&king action trout a curare-like action, by binding to the nicotinic a~tyloboline receptors of the moose diap~a~. The inhibitory action of MPTP on the contraction of the dispel is considered to play a role in the respiratory failure and muscle paralysis of intoxication with MPTP_
Key word&-MP?T% mouse diaphragm, neuromuscuiar blockade, a~tyicholine
I-Methyl~pbenyl-1,2,3,6-tetmh~~r~py~~~ (MPTP) has been recognized as an agent in inducing Parkinson’s disease in an&m& which is similar to that in humans (Kopin, Burns, Cbiueh and Markey, 1986; Langston and Ballard, 1983). The cause of the degenerates of the dopamine (~A)~n~~~~ neurones in the nigrostriatal pathway of non-human primates by MPTP is responsible for the induction of Parkinson’s disease sopin and Markey? 1988). Studies on its mechanism of action indicated that MPTP has to be oxidized by monoamine oxidase (MAO) B in the glial cells of the brain to form MPP+ (Irwin and Langston, 1985). This process could be facilitated by the presence of certain metal ions, such as manganese, a~~~~~ copper, me~ury and zinc (Ro~to~ki~ Felix and Kalyanaraman, 1987; Wechsler, Cbeckoway, Franklin and Costa, 1991), MPP’ is then s~fi~ly t~~o~~ to dop~~e neurones thrQ~ the dopamine re-uptake system and destroys dopamine neurones by the inhibitor of mitochondrid NADH de~~dro3e~~ (I&pin and Markey, 1988). Pargy%ne, an irreversible MAO inhibitor, could thus prevent the toxicity of MPTP in viva (Langstan, Irwin, Langston and Forno, 1984).
*To WhDm& corre.sponaCneeshould be addmssed.
receptor.
All of these central effects of MFTP are well characterized but the peripheral ~eurotoxi~it~ of MPTP has awaited ~~a~~~on_ During the in~i~tio~ on the effects of paraquat and its synthetic by-product (2,~,2~-t~~y~~ne) on ~urarn~~lar trans~ssio~ in the mouse phrenic ne~~ap~~, it was found that 2,Z,r&ipyridine elicited a specific curare-like action (Lin-Shiau, Hsu and Fu, 1992). Because of the ~t~~t~~~ simiitity between pamquat aad Mm (all belong to pyridine derivatives), the effects of these three pyridine derivatives have been studied on the nerve-evoked twitches of the mouse diaphragm. The results obtained suggest that MPTP blocked Neromuscuiar transmission by binding to the nicotini~ a~~~bQ~ne rec+eptora This inhibitor action of MPTP on the cuntraetion of the diaphragm may contribute to the respiratory f&Sure in intoxication with MI’TP,
~~~
#&et& ~e~e~~~~~~
~re~r~r~~
Phrenic nerve-hemidiaphragm preparations were isolated from ICR strain mice (20-30g) according to the method of Biilbring (1946). The muscle preparation was suspended in IOml of modified Krebs’ solution (~rn~i~on in mM: NaCl 1313.6,KC1 4,8,
K. S. Hsu et al.
598
MgS04 1.2, NaHCO,, 12.5, CaCl, 2.5 and glucose Il. l), maintained at 37.0 f 05°C and oxygenated with 95% O2 and 5% CO,. For the chronically denervated studies, a section of about 1 cm of the left phrenic nerve was removed under pentobarbital (30 mg/kg) anaesthesia. The denervated diaphragm muscles and the contralateral innervated one were isolated lo-15 days later. Contractions of the diaphragm were elicited alternatively by stimulation of the phrenic nerve with supramaximal rectangular pulses of 0.05 msec duration and by direct stimulation of the muscle with pulses of 0.5 msec at 0.2 Hz. Tension was recorded by an isometric transducer (Grass FI.03) on a Grass Model 7 polygraph. Intracellular recording
Conventional microelectrode recording techniques were used (Fatt and Katz, 1951). Glass microelectrodes, filled with 3 M KCI, had resistances in the
range of 3-10 MR. The mouse diaphragm was placed in modified Krebs’ solution at 37.0 + 0.5”C and oxygenated with 95% O2 + 5% COZ. A high impedance amplifier (WPI) and Hitachi V-352 oscilloscope were used for the recordings. The amplitude and frequency of miniature endplate potentials (m.e.p.p.) were measured by conventional techniques and recorded with a Data 6100 waveform analyser. The amplitude of endplate potentials (e.p.p.), elicited at 1 Hz was determined in a modified Krebs’ solution, containing 0.5 mM CaZ+ and 2.0-2.4 mM M$+ Binding studies with [‘251Jx-bungarotoxin
The binding of [ 12’I]cz-bungarotoxin was studied in the mouse diaphragm preparations. The preparations were incubated with [ L251]o!-bungarotoxin at 37.0 + 0.5”C for 3.5 hr, in the presence or absence of various concentrations of MPTP. It was then washed repeatedly with 5 ml of modified Krebs’ solution for 5 min,
~10 min
MPTP 95 pM
10
20
min
30 Neo 0.3 pM
MPTP 95 pM
2il
4’0
io
102 pM Fig. 1. Reversible-inhibition by MPTP of the nerve-evoked twitches of mouse phrenic nerve-diaphragm. The nerve-muscle preparation was suspended in 10 ml of modified Krebs’ solution and stimulated indirectly and directly, alternately at 0.2 Hz. Note that the muscle contractions, evoked by direct stimulation of the muscle were slightly increased by MPTP. The inhibitory action of MPTP on the nerve-evoked twitches could be reversed by either washout (A) or 3 p M neostigmine (neo, B). Pretreatment with pargyline (C) did not prevent the neuromuscular blocking action of MPTP.
Neuromuscular blocking action of MPTP
599
with three changes of bathing solution. The treated diaphragms were trimmed, blotted with filter paper and then weighed. The radioactivity was counted by a y counter (LKB f282)+ Chemicals
~-Bungarot~xin
was isolated from the venam of described by Lee, Chang, Kau and Luh (1972). The homogeneity of the purified toxins was verified by disc gel electrophoresis (David, 1964). All of the chemicaf compounds listed above for the test experiments were purchased from Sigma Chemical Company, St Louis, Missouri, U.S.A. Hungary
m~ltici~ct~ by the method
Statistic analysis
The results are given as mean & SEM. Numbers of experiments are indicated by ~1.The significance of differences was evaluated by Student’s r-test. When more than one group was compared with one control, the significance was evaluated according to analysis of variance (ANOVA). Probability values (P) of less than 0.05 were considered significant.
I
:
1
10
&c6Ihration &,
106
Fig. 3. Facilitation by d-tubocurarine and succinylcholine of the neuromuscular blocking action of MPTP on the mouse diaphragm. The nerve-muscle preparation was suspended in 10 ml of modified Krebs’ solution and stimulated indirectly at 0.2Hz. Note that pretreatment with either 0.7&M d-tu~ura~ne (A, n =6-g) or 2.2pM s~nylcho~~ne (A, n 16-g) for 30 min .&i&xi the ~~tm~o~i~ibition curve for MPTP (0, n = 6-g) to the left. Twitch-inhibition by MPTP was estimated as the percentage of the respective control, prior to MPTP.
Inhibition of the twitch in the mouse diaphragm induced by MPTP
revealed IC, values @oncentrations for 50% inhibition) of 53, 0.7 and 13 PM, respectively (Fig. 2). Pretreatment with either 0.7pM d-tubocurarine or 2.2 PM succinylcholine potentiated the blocking action of MPTP and shifted the concentrationinhibition curve of MPTP to the left (Fig. 3).
As shown in Fig. l(A), MPTP inhibited the nerveevoked twitches of the mouse diaphragm but slightly increased the twitches evoked by direct stimulation of the muscle. Anticholinestera~ agents such as 0.3 yM neostigmine, 0.4 FM physosti~ine and 0.5 PM ecothiophate all partially reversed the inhibitory action of MFTP [Fig, I(B)]. By contrast, pretreatment with pargyline (a monoamine oxidase inhibitor) did not prevent the inhibitory action of MFTP [Fig. l(C)]. A comparison of the potencies on inhibition of the twitch by MPTP, d-tubocurarine and succinylcholine
As shown in Fig. 4, a~tylcho~~-indu~ contracture of the denervated mouse capes was i~bit~ by MPTP in a ~n~ntration~e~ndent manner. Similar to d-tubocurarine, MPTP protected the mouse diaphragm from the neuromuscular blocking action of u-bunprotoxin (Fig. 5 and Table 1). MPTP, either pretreatment or being applied after a-bungarotoxin restored the twitches after washout,
RESULTS
0.1
0.9
1
6
Concentration
10
66
I60
(rjc)
Pig. 2. Concentration-dependent inhibition of nerveevoked twitches of the mouse diaphragm by MPTP (O), ~-tu~urarine (A) and su~nylcholi~ (A). The nervemuscle preparation was suspended in !Oml of modified Krebs’ solution and stimufated indirectly. The amplitude of the twitch was measured 6Omin after d-tubocurarine and su~inylcholine and 9Omin after MPTP, in various concentrations. The ~n~bition of muscle contraction was calculated as the percentage of the control, prior to the application of drugs (n = 6-8).
Fig. 4. ~~~tionde~dent ~~~~ti~ by MPTP of a~ty~boiin~ind~ contractme is the denervated mouse diaphragm (IO-15 days after operation). MPTP, at various c&me&rations, was applied- to the- denervated mouse diaper for 30 min. followed bv the addition of ~tyl~ol~ne (6.1 mM). A~~l~ho~n~~d-~ contracture was then recorded and cakxdated as a percentage i~~tion of the contraction prior to the addition of MPTP (n = 6-g).
K. S. Hsu et al.
600
lg
I10 min
Ilimmmmr, t i0
B
h
a-BuTX
I-TC
4’0
6’
-770
b.
0.12 )rM
w
a-BuTX
0.7 uM
a-BuTX
MPTP 95 @I
a-BuTX 0.125 /LM
w
a-BuTX
0.125 PM
0.125 PM
W
W
MPTP 95 PM
W
W
Fig. 5. Reversal by d-tubocurarine and MF’TPof the neuromuscular blocking action of a-bungarotoxin in the mouse diaphragm. The phrenic nerve of the mouse was stimulated at 0.2 Hz and the twitches were recorded isometrically. a-Bungarotoxin (a-BuTX) alone irreversibly blocked the twitches (A), while pretreatment with either d-tubocurarine (d-TC, B) or MPTP (C and D) restored the twitches after washout (W). MPTP still reversed the twitches, even when applied 2Omin after a-BuTX (E).
either immediately or 30-40min after the blockade of the twitch (Table 1). In addition, MPTP inhibited the binding of [ 1251]cr-bungarotoxin to the mouse diaphragm (Fig. 6). The concentration of MPTP required for 50% inhibition of the specific binding of [‘*‘I]u-bungarotoxin was found to be 58 PM, which was comparable to that (53 PM) for 50% inhibition of nerve-evoked twitches (Fig. 6). Effects of MPTP e.p.p.
on tetanic contraction,
m.e.p.p. and
As shown in Fig. 7, MPTP had a greater inhibitory action on tetanic contraction than on single twitches; the higher the frequency of stimulation, the greater the inhibition induced by MPTP (Fig. 8). Moreover, MPTP inhibited the frequency and amplitude of miniature endplate potentials (m.e.p.p.s) (Fig. 9). The amplitude of endplate potentials (e.p.p.s) was also inhibited but the resting membrane potential remained unaffected (Table 2).
DISCUSSION
In this study, it was demonstrated that MPTP, in concentrations ranging from 1 to 100 PM, markedly inhibited the nerve-evoked twitches of the mouse diaphragm and slightly increased the muscle-evoked twitches, indicating that the inhibitory action of MPTP was mediated by either inhibition of release of acetylcholine from the motor nerve terminals or antagonism of postsynaptic nicotinic acetylcholine receptors, rather than a direct inhibition of skeletal muscle fibres. Evidence obtained here indicated that MPTP exerts its effects on postsynaptic nicotinic acetylcholine receptors: the concentration-inhibition curves for MPTP, d-tubocurarine and succinylcholine were parallel to one another; MPTP directly blocked acetylcholine-induced contracture of the denervated mouse diaphragm. These findings suggest that MPTP mimicked d-tubocurarine (a well known competitive inhibitor of nicotinic acetylcholine
Neuromuscutar blocking action of MPTP
601
Table 1. Inhibition of the twitch by a?-bungarotoxinin mouse diaphragm treated with either MPTP or d-tubocurarine Time to Concentration
twitch block
a-Bungarotorin
Twitch restored
74.1 + 3.6
a-Bungarotoxin 4.8 48 95
MPTi
6 8 8
69.3 i 3.4 18.7 + 2.6’ 16.4 + 3.2*
16.3 & 2.1’ 87.3 f 3.2* 93.8 + 4.7*
0.125
KAhgarotoxin +
pargyiine a-Bungarotoxin + pargyline MPTG
152
0
0.125 102 76.8 rt 4.3’
48
a-Bungurotoxin + d-tubocurarine
I .o
16.3f3.7*
62.3 k 6.7+
Data are presented as mean f SEM. Twitches of the mouse diaphragm in modified Krebs’ solution were evoked by electrical stimulation of the phrenic nerve. Tbe restored twitch is defined as the recovery oFtwitches by washout of ~-bun~rotox~n. MFTP <48 @i, n = 8); pargyline ($52 PM, n = 4) and d-tubozurarine fl FM, R = 6), alone did not produce compiete neuromuscuSar blockade after 3 hr of treatment and the partial inbibition of the twitch was reversible after washout. *P < 0.05 as compared with control.
Fig. 6. Cancerttration-inhibition curve for MPTP (0, n = 6-8) and d-tubocurarine (II = 4-6) on the binding of [ 123~]cz-bungarotoxinto the mouse diaphragm. The mouse diaphragm was incubated with [ ~2s~a-bun~oto~in, in the presence or absence of various ~n~~tio~ of either MPTP or d-tubocurarine. The inhibition was cakulated as the percentage of the control specific binding of [ “SI]a-bungarotoxin alone. Data are presented as means k SEM (vertical bar).
or 2.2 FM su~~ylcho~~ne potentiated the inhibitory action of MPTP and aIso shifted the concentrationinhibition curve of MPTP to the left in a parallel manner. MPTP appeared to exert this inhibitory action directly, rather than through a metabolic receptors) (Van Maanen, 1950). Pretreatment with a conversion ta MPP+ since pretreatment with MAOs small ~ncent~at~on of either 0.7 yM d-tubocurari~a inhibitors, such as pargyline and ~~yl~prom~ne
(A)
Control
MPTP 95 @&. IO min
W
MPTP 95 @I, 20 min
20 min after washout
Fig. 7. Inhibition by MPTP (95 PM) of tetanic contraction of the mouse diaphragm. The phrenic nerve of the mouse diaphragm was train-stimulated (50 Hz, 1 set, 0.09 msec pulse duration), once every 30 xc in modified Krebs’ solution. The four successive panels showed the effect of MPTP on the tetanic responses at various time intervals (AA,B and C) and the recovery after washout (D).
K. S. Hsu et al.
602
A
1I mv 50 m*cc
0;s
i
s
io
so
loo
Frequency (Hz) Fig. 8. Frequency-dependent inhibition of amplitude of contraction of mouse diaphragm, induced by MPTP. MPTP inhibited the amplitude of contraction progressively as the stimulation frequency increased. The concentrations of MPTP were 4.8pM (C), 24pM (a), 48pM (A) and 95 PM (A). respectively (n = 4-6). (data not shown), did not prevent the inhibitory action of MPTP. a-Bungarotoxin has been known to be a specific and irreversible inhibitor of nicotinic acetylcholine receptors (Lee and Chang, 1966). In this investigation, it was shown that MPTP, given either as a pretreatment or applied after CI-bungarotoxin, was able to protect the diaphragm from the inhibitory action of a-bungarotoxin. Moreover, the specific binding of [‘*‘I]o!-bungarotoxin to the mouse diaphragm could be antagonized by MPTP,with an IC, value (58 PM) comparable to that (53 PM) for the neuromuscular blocking action of MPTP. All of these results support the contention that MPTP is not only a central neurotoxin but also a peripheral neurotoxin, by its specific binding to peripheral nicotinic acetylcholine receptors. Whether MPTP is capable of interacting with the central nicotinic acetylcholine receptors awaits clarification. MPTP exerted not only a neuromuscular blocking action but also tetanic fade (Wedensky inhibition). The neuromuscular blocking action was highly dependent on the stimulation frequency. Electrophysiological studies revealed that MPTP inhibited the amplitude of both the m.e.p.p. and the e.p.p. These effects of MPTP could be due to postsynaptic inhibition of acetylcholine receptors. The frequency of the m.e.p.p. was also significantly reduced by
B
MPTP 95 pM
-
-
Fig. 9. Effect of MPTP (95 PM) on spontaneous miniature endplate potentials (m.e.p.p.s) of the mouse phrenic nervediaphragm. (A) Control and (B) preparations treated with MPTP (95 PM) for 20 min. Note that MPTP reduced both the amplitude and frequency of m.e.p.p.s. MPTP and this effect may be due to a decrease of the release of transmitter from the nerve terminals. However, MPTP had no effect on the resting membrane potential nor on the action potential of the mouse diaphragm. Similarly, d-tubocurarine has been
Table 2. Depression of miniature endplate potentials (m.e.p.p.s) and endplate potentials (e.p.p.s) of the mouse diaphragm by MPTP or d-tubocurarine Treatment Control MPTP d-Tubocurarine
Concentration (W) 48 96 143 0.28
m.e.p.p. frequency (SW-‘)
1.24f 0.99 it 0.94 f 0.87 + 1.20 +
0.07 0.08. 0.03’ 0.08* 0.03
m.e.p.p. amplitude (mv) 1.32 k 0.08 1.21 * 0.07. 1.03f 0.07. 0.73 + 0.05. 0.84 f 0.07.
e.p.p. amplitude (mV) 3.32 k 0.68 2.46 f 0.32. 1.94 f 0.57* 0.96 + 0.23* 2.38 f 0.13’
RMP (-mV) 19.3 f 3.8 18.7 f 4.1 79.3 f 3.8 78.3 k 3.8 75.2 + 3.7
Data are presented as mean + SEM and obtained from 6 preparations, in each of which 6-8 mdplates were studied. MPTP was added cumulatively and the incubation time was more than 20min for each concentration. The endplate potentials (e.p.p.s) were evoked by nerve stimulation at 1Hz on the mouse diaphragm in 0.5 mM Ca’+ and 2.c2.4 mM M2+ modified Krebs’ solution. RMP: resting membrane potential. *P < 0.05 as compared with control.
603
Neuromuscular blocking action of MFTP reported to act on both postsynaptic and presynaptic acetylcholine receptors (Wilson, 1982). MPTP has been shown to be a selective compound, capable of inducing Parkinsonism-like symptoms in mice as well as in primates (Heikkila, 1984a, b). The selective destruction of dopamine neurones by MPTP depends on the metabolic activation of MPTP to the formation of MPP+ by the mitochond~al MAO, of glial cells and then MPP+ is transported to dopaminergic nerve terminals by dopamine re-uptake system (Irwin and Langston, 1985). MPP+ is concentrated in the mitochondria by the mitochondrial transport system where MPP+ inhibits mitochondrial respiration through an antagonism of NADH dehydrogenase (Sihha, Singh and Krishna, 1986). An inhibitor of MAO,, such as pargyline has been shown to be able to protect the dopamine neurones from the inhibitory action of MPTP (Heikkila, 1984a). By contrast, pargyline was shown in this study to be inactive in attenuating the inhibitory action of MPTP [Fig. l(C) and Table 11. This fact, together with the rapid and reversible effect of MPTP, suggests a direct action of MPTP on the nicotinic acetylchohne receptor. Since MPTP is a pyridine derivative with a tertiary nitrogen moiety, it is considered that MPTP might bind to the nicotinic acetylcholine receptor by interacting with the nitrogen moiety of the pyridine ring with the anionic active site of the receptors. It has been reported that the chemical structure required for binding to nicotinic acetylcholine receptors are ciaimed to be a mono-quaternary or bistertiary amine and the former had less stable bonds with nicotinic acetylcholine receptors of skeletal muscle and electrical organs than the latter compounds (Kharkevich and Skoldinov, 1985; Kitz, Karis and Ginsburg, 1969). In this respect, the bonds between MPTP and nicotinic acetylcholine receptors are less stable than for compounds with one or two quaternary nitrogen atoms (e.g. d-tubocurarine, succinylcholine). In fact, IC5,, values for the neuromuscular blocking actions of d-tubocurarine, succinylcholine and MPTP, obtained in this study were 0.7, 13 and 58 PM, respectively, which correlates with their relative affinity for nicotinic acetylcholine receptors. In conclusion, it was demonstrate in this study that MPTP by itself was a reversible antagonist of postsynaptic nicotinic acetylcholine receptors of the mouse phrenic nervediaphragm. This neuromuscular blocking action of MPTP on the contraction of the diaphragm is considered to be involved in the respiratory failure associated with intoxication with MPTP. Acknowledgements-This work was supported by research grants from the National Science Council, Republic of China (NSC79-0412-B002-122) and (NSC80-O412-B002-08~). REFERENCES
Billbring E. (1946) Obviations on the isolated phrenic nerve diaphragm prep~ation of the rat. Br, J. Phurmac. 1: 38-61.
David B. J. (1964) Disc electrophoresis II. Method and application to human serum proteins. Am. N.Y. Acad. Sci. 121: 404. Fatt P. and Katz B. (1951) An analysis of the endplate potential recorded with an intracellular electrode. j. Physiol., Land. 115: 320-370. Heikkila R. E. (1984a) Dooaminerzic neurotoxicitv of 1-me~yl-~-phe~yl-l,2,~,6-te~rahydr~py~dine in mice. Science 224: 1451-1453. Heikkila R. E. (1984b) Protection against the do~minergic neurotoxicity of 1-methyl4phenyl-1,2,3,~tetrahydropyridine by monoamine oxidase inhibitors. Nature 311: 467-469. Irwin I. and Langston J. W. (1985) Selective accumulation of MPP+ in the substantia nigra: A key to neurotoxicity? Life Sci. 36: 213-218. Kharkevich D. A. and Skoldinov A. P. (1985) On some principles of interaction of curare-like agents with acetylcholine receptors of skeletal muscle. J. Pharm. Pharmac. 32: 733-739. Kitz R. J., Karis J. H. and Ginsburg S. (1969) A study in vitro of new short-acting nondepolarizing neuromu~u~r blocking- agents. Eiochem. Phurmac. 18: 871881. Kopin I. J. and Markey S. P. (1988) MPTP Toxicity: Implications for research in Parkinson’s disease. A. Rev. Nebosci. 11: 81-96. Kopin I. J., Burns S. R., Chiueh C. C. and Markey S. P. (19861MPTP-induced narkinsonian svndromes in humans and animals. In: Alzhkimer’s and Parkinson’s Diseases. (Fisher A., Hanin 1. and Lachman C., Eds), pp. 519-530. Plenum Publishing, New York. Korytowski W., Felix C. and Kalvanaraman B. (1987) Mechanism of oxidation of I-methyi4phenyl-2,3didydroovridinium (MPTP? B&hem. Bionhvs. Res. Commun. * I
ii4: 692-698.
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I
Langston J. W. and Ballard P. A. (1983) Parkinson’s disease in a chemist working with l-methyls-~enyl-1,2,5,6tetrahydropy~dine (MPTP). New Engi. 1. Med. 309: 310. Langston J. W., Irwin I., Langston E. B. and Fomo L. S. (1984) Pargyline prevents MPTP-induced parkinsonism in primates. Science 225: 1480-1482. Lee C. Y. and Chang C. C. (1966) Modes of actions of purified toxins from elapid venoms on neuromuscular transmission. Mem. Inst. Butantan. Simp. 33: 355-372. Lee C. Y., Chang S. L., Kau S. T. and Luh S. Y. (1972) Chromatographic separation of the venom of Bungarus multicinctus and characterization of its components. J. Chromar. 72: 71-81. Lin-Shiau S. Y., Hsu K. S. and Fu W. M. (1992) Studies on curare-like action of 2,2’,2”-tripyridine in the mouse phrenic nerve diaphragm. Br. $. Pharmuc. 156: 55-60. Sihha B. K., Singh Y. and Krishna G. (1986) Formation of superoxide and hydroxyl radicals from l-methyly-phenylpyridium ion MPP+: Reductive activation by NADPH cytochrome P-450 reductase. Biochem. Rionhys. Res. Corn&t. 135: 283-288. Van Maanen E. F. (1950) The antagonism between acetylcholine and the curare alkaloids o-tubocurarine, C-curarine-I, C-toxiferine-II and Berythroidine in the rectus abdominis of the frog. J. Pharmac. Exp. Ther. 99: 255-264. Wechsler H., Checkoway G. M., Franklin G. M. and Costa L. G. (1991) A pilot studv of occuoational environmental risk factors’for-Parkinson% disease. Neurotoxicofogy 12: 387-392. Wilson D. F. (1982) Influence of presynaptic receptors on neuromu~ular tran~ssion in rat. Am. J. Physiol. 242~ C366-372.
0028-3908j93 S&Q@$0.00 1993 Pergamon Press Ltd
STUDIES ON THE N~~R~M~S~~~AR ~~~CK~~ ACTION OF MPTP IN THE MOUSE PHRENIC NERVE-DIAPHRAGM K, S. Hsv,
W. M. Fu and S. Y. L~M-SHUU*
Institutes of Pb~~a~~o~ and Toxicology, Coliege of Medicine, Nation& Taiwan ~n~~~i~* No. t, 3en-Ai Road, tst Section, Taipei, Taiwan, R.O.C.
Z!hmawy--ne periphec3.l ~~~t~~t~ of l-methyl~-ph~yl-l,2,3,(i~tet~~~opyridins (MPTP] in the mouse phrenic n~ia~~a~ preparation was studied. The j~hib~tion of nerve-evoked twit&es was dependent on the ~~~tr~tions of MPTP, ranging from 1 to 2oQ@ M, which ordy slightly JUICES& the amplitude of the twitch evoked by direct stimulation of the mu&e. Pr&eatment with pargyline did not prevent this inhibitory action of MPTP. The inhibition of the twitch by MPTP was reversible and could be partially reversed by antioholinesterase agents such as neostigtnine and physostigmiae, as well as ecothiophatee. Pretreatment with either 0.7 FM d-tubocurarine or 2.2 PM succinylcholine, did not affect the twitches individually but markedly potentiated the effects of MPTP, by shifting the concentrationinhibition curve of MPTP to the left. MPTP not only directly inhibited acetylcholine (O.lmM)-induced contracture of the denervated moose Daphne but atso protected the muscle from the ~~bjto~ action oftr-bungarotoxin. Furtb~rmore, the specit?c binding of ~l~~-bu~~oto~~n to the mouse diaphragm wss also inhibited by MPTP. ~~troph~~oio~~~ stndies revealed that MPTP inhibited the frequency and amplitude of the m.e.p.p., as well as the amplitude of the e.p.p. All of these findings indioate that MPTP exerts a ~eur~u~ar b&king action trout a curare-like action, by binding to the nicotinic a~tyloboline receptors of the moose diap~a~. The inhibitory action of MPTP on the contraction of the dispel is considered to play a role in the respiratory failure and muscle paralysis of intoxication with MPTP_
Key word&-MP?T% mouse diaphragm, neuromuscuiar blockade, a~tyicholine
I-Methyl~pbenyl-1,2,3,6-tetmh~~r~py~~~ (MPTP) has been recognized as an agent in inducing Parkinson’s disease in an&m& which is similar to that in humans (Kopin, Burns, Cbiueh and Markey, 1986; Langston and Ballard, 1983). The cause of the degenerates of the dopamine (~A)~n~~~~ neurones in the nigrostriatal pathway of non-human primates by MPTP is responsible for the induction of Parkinson’s disease sopin and Markey? 1988). Studies on its mechanism of action indicated that MPTP has to be oxidized by monoamine oxidase (MAO) B in the glial cells of the brain to form MPP+ (Irwin and Langston, 1985). This process could be facilitated by the presence of certain metal ions, such as manganese, a~~~~~ copper, me~ury and zinc (Ro~to~ki~ Felix and Kalyanaraman, 1987; Wechsler, Cbeckoway, Franklin and Costa, 1991), MPP’ is then s~fi~ly t~~o~~ to dop~~e neurones thrQ~ the dopamine re-uptake system and destroys dopamine neurones by the inhibitor of mitochondrid NADH de~~dro3e~~ (I&pin and Markey, 1988). Pargy%ne, an irreversible MAO inhibitor, could thus prevent the toxicity of MPTP in viva (Langstan, Irwin, Langston and Forno, 1984).
*To WhDm& corre.sponaCneeshould be addmssed.
receptor.
All of these central effects of MFTP are well characterized but the peripheral ~eurotoxi~it~ of MPTP has awaited ~~a~~~on_ During the in~i~tio~ on the effects of paraquat and its synthetic by-product (2,~,2~-t~~y~~ne) on ~urarn~~lar trans~ssio~ in the mouse phrenic ne~~ap~~, it was found that 2,Z,r&ipyridine elicited a specific curare-like action (Lin-Shiau, Hsu and Fu, 1992). Because of the ~t~~t~~~ simiitity between pamquat aad Mm (all belong to pyridine derivatives), the effects of these three pyridine derivatives have been studied on the nerve-evoked twitches of the mouse diaphragm. The results obtained suggest that MPTP blocked Neromuscuiar transmission by binding to the nicotini~ a~~~bQ~ne rec+eptora This inhibitor action of MPTP on the cuntraetion of the diaphragm may contribute to the respiratory f&Sure in intoxication with MI’TP,
~~~
#&et& ~e~e~~~~~~
~re~r~r~~
Phrenic nerve-hemidiaphragm preparations were isolated from ICR strain mice (20-30g) according to the method of Biilbring (1946). The muscle preparation was suspended in IOml of modified Krebs’ solution (~rn~i~on in mM: NaCl 1313.6,KC1 4,8,
K. S. Hsu et al.
598
MgS04 1.2, NaHCO,, 12.5, CaCl, 2.5 and glucose Il. l), maintained at 37.0 f 05°C and oxygenated with 95% O2 and 5% CO,. For the chronically denervated studies, a section of about 1 cm of the left phrenic nerve was removed under pentobarbital (30 mg/kg) anaesthesia. The denervated diaphragm muscles and the contralateral innervated one were isolated lo-15 days later. Contractions of the diaphragm were elicited alternatively by stimulation of the phrenic nerve with supramaximal rectangular pulses of 0.05 msec duration and by direct stimulation of the muscle with pulses of 0.5 msec at 0.2 Hz. Tension was recorded by an isometric transducer (Grass FI.03) on a Grass Model 7 polygraph. Intracellular recording
Conventional microelectrode recording techniques were used (Fatt and Katz, 1951). Glass microelectrodes, filled with 3 M KCI, had resistances in the
range of 3-10 MR. The mouse diaphragm was placed in modified Krebs’ solution at 37.0 + 0.5”C and oxygenated with 95% O2 + 5% COZ. A high impedance amplifier (WPI) and Hitachi V-352 oscilloscope were used for the recordings. The amplitude and frequency of miniature endplate potentials (m.e.p.p.) were measured by conventional techniques and recorded with a Data 6100 waveform analyser. The amplitude of endplate potentials (e.p.p.), elicited at 1 Hz was determined in a modified Krebs’ solution, containing 0.5 mM CaZ+ and 2.0-2.4 mM M$+ Binding studies with [‘251Jx-bungarotoxin
The binding of [ 12’I]cz-bungarotoxin was studied in the mouse diaphragm preparations. The preparations were incubated with [ L251]o!-bungarotoxin at 37.0 + 0.5”C for 3.5 hr, in the presence or absence of various concentrations of MPTP. It was then washed repeatedly with 5 ml of modified Krebs’ solution for 5 min,
~10 min
MPTP 95 pM
10
20
min
30 Neo 0.3 pM
MPTP 95 pM
2il
4’0
io
102 pM Fig. 1. Reversible-inhibition by MPTP of the nerve-evoked twitches of mouse phrenic nerve-diaphragm. The nerve-muscle preparation was suspended in 10 ml of modified Krebs’ solution and stimulated indirectly and directly, alternately at 0.2 Hz. Note that the muscle contractions, evoked by direct stimulation of the muscle were slightly increased by MPTP. The inhibitory action of MPTP on the nerve-evoked twitches could be reversed by either washout (A) or 3 p M neostigmine (neo, B). Pretreatment with pargyline (C) did not prevent the neuromuscular blocking action of MPTP.
Neuromuscular blocking action of MPTP
599
with three changes of bathing solution. The treated diaphragms were trimmed, blotted with filter paper and then weighed. The radioactivity was counted by a y counter (LKB f282)+ Chemicals
~-Bungarot~xin
was isolated from the venam of described by Lee, Chang, Kau and Luh (1972). The homogeneity of the purified toxins was verified by disc gel electrophoresis (David, 1964). All of the chemicaf compounds listed above for the test experiments were purchased from Sigma Chemical Company, St Louis, Missouri, U.S.A. Hungary
m~ltici~ct~ by the method
Statistic analysis
The results are given as mean & SEM. Numbers of experiments are indicated by ~1.The significance of differences was evaluated by Student’s r-test. When more than one group was compared with one control, the significance was evaluated according to analysis of variance (ANOVA). Probability values (P) of less than 0.05 were considered significant.
I
:
1
10
&c6Ihration &,
106
Fig. 3. Facilitation by d-tubocurarine and succinylcholine of the neuromuscular blocking action of MPTP on the mouse diaphragm. The nerve-muscle preparation was suspended in 10 ml of modified Krebs’ solution and stimulated indirectly at 0.2Hz. Note that pretreatment with either 0.7&M d-tu~ura~ne (A, n =6-g) or 2.2pM s~nylcho~~ne (A, n 16-g) for 30 min .&i&xi the ~~tm~o~i~ibition curve for MPTP (0, n = 6-g) to the left. Twitch-inhibition by MPTP was estimated as the percentage of the respective control, prior to MPTP.
Inhibition of the twitch in the mouse diaphragm induced by MPTP
revealed IC, values @oncentrations for 50% inhibition) of 53, 0.7 and 13 PM, respectively (Fig. 2). Pretreatment with either 0.7pM d-tubocurarine or 2.2 PM succinylcholine potentiated the blocking action of MPTP and shifted the concentrationinhibition curve of MPTP to the left (Fig. 3).
As shown in Fig. l(A), MPTP inhibited the nerveevoked twitches of the mouse diaphragm but slightly increased the twitches evoked by direct stimulation of the muscle. Anticholinestera~ agents such as 0.3 yM neostigmine, 0.4 FM physosti~ine and 0.5 PM ecothiophate all partially reversed the inhibitory action of MFTP [Fig, I(B)]. By contrast, pretreatment with pargyline (a monoamine oxidase inhibitor) did not prevent the inhibitory action of MFTP [Fig. l(C)]. A comparison of the potencies on inhibition of the twitch by MPTP, d-tubocurarine and succinylcholine
As shown in Fig. 4, a~tylcho~~-indu~ contracture of the denervated mouse capes was i~bit~ by MPTP in a ~n~ntration~e~ndent manner. Similar to d-tubocurarine, MPTP protected the mouse diaphragm from the neuromuscular blocking action of u-bunprotoxin (Fig. 5 and Table 1). MPTP, either pretreatment or being applied after a-bungarotoxin restored the twitches after washout,
RESULTS
0.1
0.9
1
6
Concentration
10
66
I60
(rjc)
Pig. 2. Concentration-dependent inhibition of nerveevoked twitches of the mouse diaphragm by MPTP (O), ~-tu~urarine (A) and su~nylcholi~ (A). The nervemuscle preparation was suspended in !Oml of modified Krebs’ solution and stimufated indirectly. The amplitude of the twitch was measured 6Omin after d-tubocurarine and su~inylcholine and 9Omin after MPTP, in various concentrations. The ~n~bition of muscle contraction was calculated as the percentage of the control, prior to the application of drugs (n = 6-8).
Fig. 4. ~~~tionde~dent ~~~~ti~ by MPTP of a~ty~boiin~ind~ contractme is the denervated mouse diaphragm (IO-15 days after operation). MPTP, at various c&me&rations, was applied- to the- denervated mouse diaper for 30 min. followed bv the addition of ~tyl~ol~ne (6.1 mM). A~~l~ho~n~~d-~ contracture was then recorded and cakxdated as a percentage i~~tion of the contraction prior to the addition of MPTP (n = 6-g).
K. S. Hsu et al.
600
lg
I10 min
Ilimmmmr, t i0
B
h
a-BuTX
I-TC
4’0
6’
-770
b.
0.12 )rM
w
a-BuTX
0.7 uM
a-BuTX
MPTP 95 @I
a-BuTX 0.125 /LM
w
a-BuTX
0.125 PM
0.125 PM
W
W
MPTP 95 PM
W
W
Fig. 5. Reversal by d-tubocurarine and MF’TPof the neuromuscular blocking action of a-bungarotoxin in the mouse diaphragm. The phrenic nerve of the mouse was stimulated at 0.2 Hz and the twitches were recorded isometrically. a-Bungarotoxin (a-BuTX) alone irreversibly blocked the twitches (A), while pretreatment with either d-tubocurarine (d-TC, B) or MPTP (C and D) restored the twitches after washout (W). MPTP still reversed the twitches, even when applied 2Omin after a-BuTX (E).
either immediately or 30-40min after the blockade of the twitch (Table 1). In addition, MPTP inhibited the binding of [ 1251]cr-bungarotoxin to the mouse diaphragm (Fig. 6). The concentration of MPTP required for 50% inhibition of the specific binding of [‘*‘I]u-bungarotoxin was found to be 58 PM, which was comparable to that (53 PM) for 50% inhibition of nerve-evoked twitches (Fig. 6). Effects of MPTP e.p.p.
on tetanic contraction,
m.e.p.p. and
As shown in Fig. 7, MPTP had a greater inhibitory action on tetanic contraction than on single twitches; the higher the frequency of stimulation, the greater the inhibition induced by MPTP (Fig. 8). Moreover, MPTP inhibited the frequency and amplitude of miniature endplate potentials (m.e.p.p.s) (Fig. 9). The amplitude of endplate potentials (e.p.p.s) was also inhibited but the resting membrane potential remained unaffected (Table 2).
DISCUSSION
In this study, it was demonstrated that MPTP, in concentrations ranging from 1 to 100 PM, markedly inhibited the nerve-evoked twitches of the mouse diaphragm and slightly increased the muscle-evoked twitches, indicating that the inhibitory action of MPTP was mediated by either inhibition of release of acetylcholine from the motor nerve terminals or antagonism of postsynaptic nicotinic acetylcholine receptors, rather than a direct inhibition of skeletal muscle fibres. Evidence obtained here indicated that MPTP exerts its effects on postsynaptic nicotinic acetylcholine receptors: the concentration-inhibition curves for MPTP, d-tubocurarine and succinylcholine were parallel to one another; MPTP directly blocked acetylcholine-induced contracture of the denervated mouse diaphragm. These findings suggest that MPTP mimicked d-tubocurarine (a well known competitive inhibitor of nicotinic acetylcholine
Neuromuscutar blocking action of MPTP
601
Table 1. Inhibition of the twitch by a?-bungarotoxinin mouse diaphragm treated with either MPTP or d-tubocurarine Time to Concentration
twitch block
a-Bungarotorin
Twitch restored
74.1 + 3.6
a-Bungarotoxin 4.8 48 95
MPTi
6 8 8
69.3 i 3.4 18.7 + 2.6’ 16.4 + 3.2*
16.3 & 2.1’ 87.3 f 3.2* 93.8 + 4.7*
0.125
KAhgarotoxin +
pargyiine a-Bungarotoxin + pargyline MPTG
152
0
0.125 102 76.8 rt 4.3’
48
a-Bungurotoxin + d-tubocurarine
I .o
16.3f3.7*
62.3 k 6.7+
Data are presented as mean f SEM. Twitches of the mouse diaphragm in modified Krebs’ solution were evoked by electrical stimulation of the phrenic nerve. Tbe restored twitch is defined as the recovery oFtwitches by washout of ~-bun~rotox~n. MFTP <48 @i, n = 8); pargyline ($52 PM, n = 4) and d-tubozurarine fl FM, R = 6), alone did not produce compiete neuromuscuSar blockade after 3 hr of treatment and the partial inbibition of the twitch was reversible after washout. *P < 0.05 as compared with control.
Fig. 6. Cancerttration-inhibition curve for MPTP (0, n = 6-8) and d-tubocurarine (II = 4-6) on the binding of [ 123~]cz-bungarotoxinto the mouse diaphragm. The mouse diaphragm was incubated with [ ~2s~a-bun~oto~in, in the presence or absence of various ~n~~tio~ of either MPTP or d-tubocurarine. The inhibition was cakulated as the percentage of the control specific binding of [ “SI]a-bungarotoxin alone. Data are presented as means k SEM (vertical bar).
or 2.2 FM su~~ylcho~~ne potentiated the inhibitory action of MPTP and aIso shifted the concentrationinhibition curve of MPTP to the left in a parallel manner. MPTP appeared to exert this inhibitory action directly, rather than through a metabolic receptors) (Van Maanen, 1950). Pretreatment with a conversion ta MPP+ since pretreatment with MAOs small ~ncent~at~on of either 0.7 yM d-tubocurari~a inhibitors, such as pargyline and ~~yl~prom~ne
(A)
Control
MPTP 95 @&. IO min
W
MPTP 95 @I, 20 min
20 min after washout
Fig. 7. Inhibition by MPTP (95 PM) of tetanic contraction of the mouse diaphragm. The phrenic nerve of the mouse diaphragm was train-stimulated (50 Hz, 1 set, 0.09 msec pulse duration), once every 30 xc in modified Krebs’ solution. The four successive panels showed the effect of MPTP on the tetanic responses at various time intervals (AA,B and C) and the recovery after washout (D).
K. S. Hsu et al.
602
A
1I mv 50 m*cc
0;s
i
s
io
so
loo
Frequency (Hz) Fig. 8. Frequency-dependent inhibition of amplitude of contraction of mouse diaphragm, induced by MPTP. MPTP inhibited the amplitude of contraction progressively as the stimulation frequency increased. The concentrations of MPTP were 4.8pM (C), 24pM (a), 48pM (A) and 95 PM (A). respectively (n = 4-6). (data not shown), did not prevent the inhibitory action of MPTP. a-Bungarotoxin has been known to be a specific and irreversible inhibitor of nicotinic acetylcholine receptors (Lee and Chang, 1966). In this investigation, it was shown that MPTP, given either as a pretreatment or applied after CI-bungarotoxin, was able to protect the diaphragm from the inhibitory action of a-bungarotoxin. Moreover, the specific binding of [‘*‘I]o!-bungarotoxin to the mouse diaphragm could be antagonized by MPTP,with an IC, value (58 PM) comparable to that (53 PM) for the neuromuscular blocking action of MPTP. All of these results support the contention that MPTP is not only a central neurotoxin but also a peripheral neurotoxin, by its specific binding to peripheral nicotinic acetylcholine receptors. Whether MPTP is capable of interacting with the central nicotinic acetylcholine receptors awaits clarification. MPTP exerted not only a neuromuscular blocking action but also tetanic fade (Wedensky inhibition). The neuromuscular blocking action was highly dependent on the stimulation frequency. Electrophysiological studies revealed that MPTP inhibited the amplitude of both the m.e.p.p. and the e.p.p. These effects of MPTP could be due to postsynaptic inhibition of acetylcholine receptors. The frequency of the m.e.p.p. was also significantly reduced by
B
MPTP 95 pM
-
-
Fig. 9. Effect of MPTP (95 PM) on spontaneous miniature endplate potentials (m.e.p.p.s) of the mouse phrenic nervediaphragm. (A) Control and (B) preparations treated with MPTP (95 PM) for 20 min. Note that MPTP reduced both the amplitude and frequency of m.e.p.p.s. MPTP and this effect may be due to a decrease of the release of transmitter from the nerve terminals. However, MPTP had no effect on the resting membrane potential nor on the action potential of the mouse diaphragm. Similarly, d-tubocurarine has been
Table 2. Depression of miniature endplate potentials (m.e.p.p.s) and endplate potentials (e.p.p.s) of the mouse diaphragm by MPTP or d-tubocurarine Treatment Control MPTP d-Tubocurarine
Concentration (W) 48 96 143 0.28
m.e.p.p. frequency (SW-‘)
1.24f 0.99 it 0.94 f 0.87 + 1.20 +
0.07 0.08. 0.03’ 0.08* 0.03
m.e.p.p. amplitude (mv) 1.32 k 0.08 1.21 * 0.07. 1.03f 0.07. 0.73 + 0.05. 0.84 f 0.07.
e.p.p. amplitude (mV) 3.32 k 0.68 2.46 f 0.32. 1.94 f 0.57* 0.96 + 0.23* 2.38 f 0.13’
RMP (-mV) 19.3 f 3.8 18.7 f 4.1 79.3 f 3.8 78.3 k 3.8 75.2 + 3.7
Data are presented as mean + SEM and obtained from 6 preparations, in each of which 6-8 mdplates were studied. MPTP was added cumulatively and the incubation time was more than 20min for each concentration. The endplate potentials (e.p.p.s) were evoked by nerve stimulation at 1Hz on the mouse diaphragm in 0.5 mM Ca’+ and 2.c2.4 mM M2+ modified Krebs’ solution. RMP: resting membrane potential. *P < 0.05 as compared with control.
603
Neuromuscular blocking action of MFTP reported to act on both postsynaptic and presynaptic acetylcholine receptors (Wilson, 1982). MPTP has been shown to be a selective compound, capable of inducing Parkinsonism-like symptoms in mice as well as in primates (Heikkila, 1984a, b). The selective destruction of dopamine neurones by MPTP depends on the metabolic activation of MPTP to the formation of MPP+ by the mitochond~al MAO, of glial cells and then MPP+ is transported to dopaminergic nerve terminals by dopamine re-uptake system (Irwin and Langston, 1985). MPP+ is concentrated in the mitochondria by the mitochondrial transport system where MPP+ inhibits mitochondrial respiration through an antagonism of NADH dehydrogenase (Sihha, Singh and Krishna, 1986). An inhibitor of MAO,, such as pargyline has been shown to be able to protect the dopamine neurones from the inhibitory action of MPTP (Heikkila, 1984a). By contrast, pargyline was shown in this study to be inactive in attenuating the inhibitory action of MPTP [Fig. l(C) and Table 11. This fact, together with the rapid and reversible effect of MPTP, suggests a direct action of MPTP on the nicotinic acetylchohne receptor. Since MPTP is a pyridine derivative with a tertiary nitrogen moiety, it is considered that MPTP might bind to the nicotinic acetylcholine receptor by interacting with the nitrogen moiety of the pyridine ring with the anionic active site of the receptors. It has been reported that the chemical structure required for binding to nicotinic acetylcholine receptors are ciaimed to be a mono-quaternary or bistertiary amine and the former had less stable bonds with nicotinic acetylcholine receptors of skeletal muscle and electrical organs than the latter compounds (Kharkevich and Skoldinov, 1985; Kitz, Karis and Ginsburg, 1969). In this respect, the bonds between MPTP and nicotinic acetylcholine receptors are less stable than for compounds with one or two quaternary nitrogen atoms (e.g. d-tubocurarine, succinylcholine). In fact, IC5,, values for the neuromuscular blocking actions of d-tubocurarine, succinylcholine and MPTP, obtained in this study were 0.7, 13 and 58 PM, respectively, which correlates with their relative affinity for nicotinic acetylcholine receptors. In conclusion, it was demonstrate in this study that MPTP by itself was a reversible antagonist of postsynaptic nicotinic acetylcholine receptors of the mouse phrenic nervediaphragm. This neuromuscular blocking action of MPTP on the contraction of the diaphragm is considered to be involved in the respiratory failure associated with intoxication with MPTP. Acknowledgements-This work was supported by research grants from the National Science Council, Republic of China (NSC79-0412-B002-122) and (NSC80-O412-B002-08~). REFERENCES
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David B. J. (1964) Disc electrophoresis II. Method and application to human serum proteins. Am. N.Y. Acad. Sci. 121: 404. Fatt P. and Katz B. (1951) An analysis of the endplate potential recorded with an intracellular electrode. j. Physiol., Land. 115: 320-370. Heikkila R. E. (1984a) Dooaminerzic neurotoxicitv of 1-me~yl-~-phe~yl-l,2,~,6-te~rahydr~py~dine in mice. Science 224: 1451-1453. Heikkila R. E. (1984b) Protection against the do~minergic neurotoxicity of 1-methyl4phenyl-1,2,3,~tetrahydropyridine by monoamine oxidase inhibitors. Nature 311: 467-469. Irwin I. and Langston J. W. (1985) Selective accumulation of MPP+ in the substantia nigra: A key to neurotoxicity? Life Sci. 36: 213-218. Kharkevich D. A. and Skoldinov A. P. (1985) On some principles of interaction of curare-like agents with acetylcholine receptors of skeletal muscle. J. Pharm. Pharmac. 32: 733-739. Kitz R. J., Karis J. H. and Ginsburg S. (1969) A study in vitro of new short-acting nondepolarizing neuromu~u~r blocking- agents. Eiochem. Phurmac. 18: 871881. Kopin I. J. and Markey S. P. (1988) MPTP Toxicity: Implications for research in Parkinson’s disease. A. Rev. Nebosci. 11: 81-96. Kopin I. J., Burns S. R., Chiueh C. C. and Markey S. P. (19861MPTP-induced narkinsonian svndromes in humans and animals. In: Alzhkimer’s and Parkinson’s Diseases. (Fisher A., Hanin 1. and Lachman C., Eds), pp. 519-530. Plenum Publishing, New York. Korytowski W., Felix C. and Kalvanaraman B. (1987) Mechanism of oxidation of I-methyi4phenyl-2,3didydroovridinium (MPTP? B&hem. Bionhvs. Res. Commun. * I
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Langston J. W. and Ballard P. A. (1983) Parkinson’s disease in a chemist working with l-methyls-~enyl-1,2,5,6tetrahydropy~dine (MPTP). New Engi. 1. Med. 309: 310. Langston J. W., Irwin I., Langston E. B. and Fomo L. S. (1984) Pargyline prevents MPTP-induced parkinsonism in primates. Science 225: 1480-1482. Lee C. Y. and Chang C. C. (1966) Modes of actions of purified toxins from elapid venoms on neuromuscular transmission. Mem. Inst. Butantan. Simp. 33: 355-372. Lee C. Y., Chang S. L., Kau S. T. and Luh S. Y. (1972) Chromatographic separation of the venom of Bungarus multicinctus and characterization of its components. J. Chromar. 72: 71-81. Lin-Shiau S. Y., Hsu K. S. and Fu W. M. (1992) Studies on curare-like action of 2,2’,2”-tripyridine in the mouse phrenic nerve diaphragm. Br. $. Pharmuc. 156: 55-60. Sihha B. K., Singh Y. and Krishna G. (1986) Formation of superoxide and hydroxyl radicals from l-methyly-phenylpyridium ion MPP+: Reductive activation by NADPH cytochrome P-450 reductase. Biochem. Rionhys. Res. Corn&t. 135: 283-288. Van Maanen E. F. (1950) The antagonism between acetylcholine and the curare alkaloids o-tubocurarine, C-curarine-I, C-toxiferine-II and Berythroidine in the rectus abdominis of the frog. J. Pharmac. Exp. Ther. 99: 255-264. Wechsler H., Checkoway G. M., Franklin G. M. and Costa L. G. (1991) A pilot studv of occuoational environmental risk factors’for-Parkinson% disease. Neurotoxicofogy 12: 387-392. Wilson D. F. (1982) Influence of presynaptic receptors on neuromu~ular tran~ssion in rat. Am. J. Physiol. 242~ C366-372.