Effects of (+)-tubocurarine on neuromuscular facilitation and depression in rat diaphragm

EF=FECTS OF ~+~-TU3OCURAR~NE ON NEU’R.OMUSCULAR FACILITATION AND DEPRESSION IN RAT DIAPWRAGM*t D. _I.HINMAN, byoh

R. S. JACOBS$.

Wniversity. Department of Pharmacology,

and A. Cr.

KARCZMAK

2160 South First Avenue, Naywood. IL 601.53, L!.S.A

Summary--E:Ffec& of f -I-i-tubucurarine on ~~~~~~~u~c~~~~~ facilitation and deprwion were studied in the rat phrenic nerve-diaphrqm. The paired end-pIate potential (EPP) method of anatysib was used. Tubocurarjne caused a significant decrease in the EPP,:EPP, ratio of EPP pairs elicited at a stimulus interval of 20 msec ifl the cut muscle preparation. potential of EPP, and EPP, ;\nd iontophoretic

of tubocurarine Furthermore.

Studies of tubacnrarine

with

respect to the equilibriunr

acetylchnline prrleMial pairs. indicated that the &I”ert on the EPP ratio was not associated with a postsynaptlc action of tubocururine.

tubocurarinc

did

not

alter

the amplitude

ratio

of the nerve

terminal

action

potcntialr.

The effects of tubocurarinc on the EPP,:EPP, ratio at varitrrrr; stimulus intervals were studied in the cut muscle preparation. lo the control condition, fxllittltion IEPP2:EPP, ratio > 1.0) occurred at 5 and

10 msec stimulus

intervals;

depression

(EPP2:EPP,

The postsynaptic actions of (+ )-tubocurarine on neuromuscular transmission are well established (LANGLEY, 1909; BROWN, DALE and FELDBERG. 1936; BUCHTHAL and LINDHARD, 1942; KUFFLER, 1943: DEL CASTELO and KATZ. 1957; TAKEUCHI and TAKEUCHI, f9&& The competitive ~~~~~~~~St~ between tuhocurarine and ~~~t~~~~~~~~~[ACt?) for ~ostsynapt~~ chotinergic receptors is commonfy accepted as one of the primary actions of t~~boc~rarin~ at the neuromuscular junction. LILLEHEILand NAES~(X961) suggested that tubocurarine exerted both presynaptic and postsynaptic actions at the neuromuscular jurrction. The presynaptic action of tubocurarine was manifested by a more rapid decline of end-plate potcrrtial (EPP) amplitude in a train of EPPs in the partially curarized as compared to the non-curarized preparation. The suggestion of a presynaptic action of tubocurarins was supported by the rest&s of HUBBARD, WZLSOWand M~AMOT@ iiS69i, fM?oas and BLABFR ($9711, BLABE% (197% HUBABD and WB.SBN it973). fn addition. tubocurarine has been shown to decrease the amount of ACh released by presynaptic stimulation J&AM.

ratio

< 1,s) occurred

BMNCHI Wd CEUDA, OLL and LOKG. 1971).

1964;

at longer

stimuiuR

GCRGIS, ~RMWEN.

SOK-

The purpose ofthis study was to further investigate the effects of tubocurarine on presynaptic mechsnisrns of neuromuscular transmission. The intcstigations cited above relating to the presynaptic actions of tubocurarine utilized the analysis of trains of EPPs or the rne~Isure~~~e~~t of 4Ch release by bioassay tcohniyues to anaf~ze ~~~~~~~~~~~~2~ I-cieaseprocesses. ItIthe present study, &e paired p&se method ol anatysis was employed to study the egects of tuboc~r~~rine on presynaptic mechanisms. Thr paired pulse method represents a simple model for evaluating depieiion of transmitter in the nerve terminal following the first * Preliminary reports of this work were prcxcnred in response (TAKE~MI, 195X; LILEY and NOKW, X053; Pllun~rclcoloqi.st16: 234 (1974) and Frdrl. Plw. I Ldll. 1111.ELMQWST:xtrd QUASTEL, 1965) and the post-activation .SOl~S C’.Y/J !3;0/.34: 751 (1975). increase in probability of release (HLIHBARU, 1463). P Supported by NIH Grants CM 77 and NS 06455. Also. tcchrriyues for direct measurement of par$ Present address: Department of Biological Sciences. University of California. Santa Barbara. California 93106. ameters of rrcurotnuscular transmission were utilized

428

D. J.

to differentiate presynaptic of tubocurarine.

HINMAN. R. S. JAWRS and A. Cr. KAKLMAK

from postsynaptic

actions

METHODS All experiments were performed on the rat phrenic Holtzman female nerve-diaphragm preparation. albino rats were employed. Composition of the Krebs perfusing solution was: NaCl, 115 mM; KCI, 4.62 mM; KH,PO,, 1.15 mM: CaCI,, 2.46 mM; MgSO,, 1.12 mM; D-glucose, 0.88 mM; NaHC03, 26.8 mM. The perfusing solution was aerated with 95”,, O2 + 5”,, CO,. Bath temperature was 31-32 C. In the case of experiments with the cut muscle preparation, muscle contraction was prevented by cutting the muscle fibres transversely as described by BARSTAD and LILLEHEIL (1968). When transmission was blocked by low Ca’+/high Mg2+ perfusion soluin the Krebs tion, the Ca’+ and Mg” concentrations solution were 1.23 mM and 4.48-7.84 mM, respectively. Intracellular recording was carried out with conventional glass microelectrodes filled with 3 M KCI. End-plate foci were localized by selecting EPPs with maximum amplitude, and with rise time of one msec or less. The double intracellular microelectrode method of FATT and KATZ (1951) was used to determine the equilibrium potential of the end-plate membrane. A graph of membrane potential versus EPP amplitude was plotted by a computer program for linear regression. The membrane potential at which the EPP would be nullified was determined by extrapolation and this value was the equilibrium potential. The method for iontophoresis of acetylcholine was similar to that described by NASTUK (1951. 1953). Pairs of extracellular nerve terminal action potentials were recorded by the method described by HUBBARD and SCHMIDT (1963). In these experiments the interval between nerve action potentials was 20 msec. Facilitation and depression of neuromuscular transmission were determined by measuring the EPP,/EPP, ratio at stimulus intervals of 5 msec to 2 sec. The relationship between the EPP ratio and the stimulus interval is the cell recovery cycle (DESMEDT. 1966). Facilitation is defined as the condition in which EPP2/EPP, > 1.0 and depression is the condition in which EPP,/EPP, < 1.0.

Fig. I. Effect of tubocurarine on EPP pairs. Stlmuluy interval 20 msec. Calibration 5 mV: I msec.A: control. B: 1.34 x IO (’ hl tubocurarine added.

The EPP amplitudes were corrected for non-linear summation (MARTIN. 1955). The ratio of the amplitude of the first EPP to the amplitude of the second EPP (EPP2/EPP,) was calculated from the corrcctcd EPP amplitudes. The paired Student’s r-test was used for statistical analysis. Statistically significant difl‘erence was determined by P < 0.05 with the one-tailed t-test. (+ )-Tubocurarine chloride (Sigma) was used in all experiments.

RESC 1.X

The effects of tubocurarine on the EPPZ;EPP, ratio (EPP ratio) were determined in the cut muscle preparation using EPP pairs with a stimulus interval of 20 msec. The EPP ratio was determined in the absence and in the presence of 1.34 x 10mh M tubocurarine. The results are summarized in Table 1 and an example of the effect of tubocurarine is presented in Figure 1. In the control condition, the amplitudes of both EPPs were nearly equal. Addition 01 1.34 x 10mh M tubocurarine for a period of 15 min caused a significant reduction in the amplitude 01 both EPP, and EPP2. Furthermore. the EPP ratio

Table I. Effect of 1.34 x IO ’ M tubocurarinc on EPP pairs Resting potential (mV) Control Tubocurarine Wash

-33.8 f 3.0+ -33.0 _t 3.6 _ 34.x * 3.x

EPP, amplitude (mV) -. Y.YZ_+ ‘0’) 2.2 I + 0.0.1* 10.2x ? 1.03

EPPL EPP, O.Y8* 0.04 0.8’) * 0.(X* 0.04 & 0.03

Data expressed as mean k S.E.M. II = 4. * Statistically significant difference (P -c0.05)between control and tubocurarlnc treatment; Student’s t-test: one tailed test. t The low resting membrane potentials in these experiments arc characlerl?tlc ol’ the cut muscle preparation (see BARSTADand LILL.PHI.IL., lY6X).

429

Presynaptic effects of (+)-tubocurarine Table 2. Effect of 1.34 x 10m6 M tubocurarine on equilibrium potential (EFpp) Resting potential (mv) Control Tubocurarine Wash

EPP, amplitude (mV)

-26.8 + 3.9t -27.7 k 3.7 -26.0 & 2.9

6.28 k 0.95 2.24 k 0.54 5.06 i 1.21

Data expressed as mean + S.E.M. n = 6. t The low resting membrane potentials in these experiments BARSTADand LILLE~EIL,1968). _

+0.22 k 6.78 -7.00 + 5.23 + 1.26 k 5.36

are characteristic

-0.92 + 6.34 -7.15 + 5.62 -0.25 & 5.36

of the cut muscle

preparation

(see

was significantly decreased in the presence of tubocurarine. These effects of tubocurarine were reversed when the perfusion was returned to normal Krebs solution.

junctions and the stimulus interval was 20 msec. As shown in Table 4 and Figure 3. 1.34 x IO-’ M tubocurarine did not alter nerve terminal action potential amplitude, duration, or NTAP,,INTAP, ratio.

Eyuilihrium

Fucilitrrtion

potenfial

The equilibrium potentials of EPP, and EPPz were determined in the cut muscle preparation in the presence and absence of 1.34 x lo-’ M tubocurarine. The stimulus interval in this set of experiments was 20 msec. The results from six experiments are summarized in Table 2. The equilibrium potential for EPP, was not significantly different from that for EPP2 either in the control or the tubocurarine-treated condition. Iontophoretic

ACh potentids

Pairs of iontophoretic ACh potentials were evoked at a stimulus interval of 50 msec in the normal nervediaphragm preparation. The time course of the ACh potential (approx. 40 msec) prevented investigation of ACh potentials elicited at shorter stimulus intervals. The effects of 1.34 x 10m6 M tubocurarine on iontophoretic ACh potential pairs are summarized in Table 3 and Figure 2 presents an individual experiment showing the actions of tubocurarine. The amplitudes of the two ACh potentials were nearly equal in both the control and the tubocurarine-treated conditions. Tubocurarine caused a significant reduction in the ACh potential amplitudes, but had no significant effect on the ACh potential amplitude ratio. Nerve

terminal

action

potentids

Pairs of nerve terminal action potentials (NTAP) were recorded extracellularly at single neuromuscular

Table

3. Effect of 1.34 x lo-’

Control Tubocurarine Wash

The cell recovery cycle was determined in the cut muscle preparation. The control cell recovery cycle is presented in Figure 4. Facilitation (EPP,/ EPP, > 1.0) predominated at stimulus intervals of 5 and 10 msec and depression (EPP2:EPP, C 1.0) predominated at stimulus intervals greater than 10 msec. Addition of 1.34 x 10. ’ M tubocurarine resulted in a significant decrease in the EPP ratio at stimulus intervals of 10, 20. 40. 80, 100, and 1000 msec. At intervals greater than 1000 msec there was no significant difference between the control EPP ratio and the tubocurarine-treated values. The action of tubocurarine on the cell recovery cycle was also studied in the cut muscle perfused with low Ca*‘/high Mg 2+ Krebs solution. The results of seven experiments are summarized in Figure 5. The magnitude and duration of facilitation in the low Ca’+/high Mg2+ Krebs solution were enhanced compared with facilitation in the normal Krebs solution The facilitation and depression was negligible. decayed in a linear fashion as the pulse interval was increased from 5 to l(XI msec; EPP, and EPP, were about equal at pulse interkals between 100 and 2000 msec. Addition of 1.34 x lo-’ M tubocurarine did not significantly suppress the facilitation in low Ca”, high Mg’+ Krebs solution. The EPP ratios in the presence and in the absence of tubocurarine were not statistically different at stimulus intervals of 5. 20. 40, 80, 100, 200, 400, 800. and 2000 msec. The EPP ratio

M tubocurarine

Resting potential (mV)

rrrul depressiorl

on iontophoretic

ACh potential, amplitude (mV)

-55.0 * 7.0 -53.8 + 7.1 - 57.5 * 17.5

4.19 + 0.75 0.93 f 0.28* 3.00 + 0.58

ACh potentials

Ratio of ACh potentials 1.04 + 0.04 1.05 f 0.05 1.09 * 0.15

Data expressed as mean i S.E.M. n = 4. * Statistically significant difference Student’s t-test: one tailed test.

(P < 0.05) between

control

and tubocurarine

treatment:

ntrol

Fig. 2. Effect of tuhocurarine on iontophoretic ACh tials. Stimulus intcrvnl 50 msec. Voltage calibration 5 msec. Current calibration I x 10 1.34 x IO ” ELItubocurarine

’ A. A:

poten2 mV:

control.

B:

added.

3. Effect of tubocurarinc on nerve terminal action Stimulus mlcr~al 30 mscc. <‘alihration SO0 /IV : 700 ~ux. C!pper truce: rcaponac to Iii-St stimulus. Lower trace: resp’onsc to second stimulus. A: control. B: 1.34 x 10 ” “rl tubocurarlne (( + )-TC) added. Fig.

polcnl~nls.

transmission. These results are in agreement with the conclusion of OSTUKA. ENVY and NWOM~RA ( 1962) that depression following repetitive stimulation is a presynaptic function. The release of ACh is depei7deIit on the sic.c of The results of the present study dcnlonstrate that the nerve terminal action potential (HAGWARA and tubocurarine caused a reversible decrease in the TASAKI, 1958: TAKELVHI and TAKEUCHI. 1962; KATZ EPP,;EPP, ratio. This decrease in the EPP ratio by and MILEIN, 1965). The decrease in EPP ratio in the tubocurarine was manifested at stimulus intervals of presence of tubocurdrine could result from a decrease IO--100 msec. Furthermore, the results demonstrate in NTAP,:NTAP, ratio but this possibility stems to that the effect of tubocurarine ~II the EPP ratio repbe excluded by the results of the present study. Previous reports have indicated that in the curarresents a presynaptic action of the drug. The responsiveness of the subsynaptic membrane ized preparation EPP, was smaller than EPP, at all stimulus intervals (Ecc-L~S. KATZ and Kut-t‘t,k,K. 1941; to ACh during EPP, and EPP, was determined by LILEY and MIRTH, 1953: LWDRCRG and Qun_lscti, measuring the EPP equilibrium potential and by recording the response to iontophoretically applied 1953a; THIES. t9h5). In contrast. the rcsuits of the AC%. The results indicate that the eqljilihriuni potenpresent study suggest that the cell recovery cycle of tial of EPP, was not si~ili~c~~~itly different from the the nolt-curari~ed nlarnrn~iliaii neuromuscular juncc~~~ilihriunl p~~tc~lti~llof EPP, under tither the contion exhibits both facilit~ltion and depression (Fig. 4). In the control condition, EPPz was larger than EPP, trol or the tubocurarinc trcatmcnt. Furthermore. the amplitudes of the lirst and the second AC% potentials at stimulus intervals of 5 and 10 msec and the EPP ratio was significantly greater than unity (P < 0.05) \+erc the same in the absence and presence of tuhoat the 5 msec stimulus interval. curarine. The results of these two sets of experiments Tubocurarine caused a decrease in the EPP ratio indicate that tubocurarinc causecl u decrease in the at stimulus intervals of 1OklOO msec (Fig. 4). This end-plate response to ACh. In addition. the results effect is consistent with either suppression of faciliindicate that the responsiveness of the subsynaptic cholinergic receptors to ACh is the same during EPP, tation or enhancement of depression by tubocurarinc. A differential effect of tubocurarine on facilitation or and EPP?. Thus. the EPP,,EPP, ratio is not dependent on postsynaptic proccsscs of ncLir(~n~Llsc~ll~lr depression GIII be determined by evaluating fkiliwas significantly decreased by tubocurarine two intervals, 10 and 1000 msec.

Action Amplitude (/iV)

Data

expressed

as mean i

potential, Duration (Icsec)

S EM.

II = 7.

only at

Action Amplitude (PVI

potential2 Duration

(px)

Actton potential amplitude ratio

431

Presynaptic effects of (+ )-tubocumrine content of the first EPP due to an increase

5

IO

20

40

100 zoo

400

STIMULUS INTERVAL

1000

(msect

Fig. 4. Effect of tubocur~r~tl~ on cell recovery cycle in normal Krebs solution. Open symbols represent control.

Filled symbols

represent

1.34 x 10mh M tubocurarine

added. Asterisks (*) indicate stntlstically sigmficant encc (P < 0.05) between control and tubocurarine ment; paired Student’s r-test. Bars indicate S.E.M.

differtreat,I = 6.

in probability of release. Tubocurarine has been shown by analysis of trains of EPPs to decrease releasable stores and increase probability of release (HUBBARD CT I(/.. 1969: GAI.IYIX). 1971 : Hr HIIAKI) and WILSOV. 1973). The effects of tubocurarine on the EPP ratio described in the present study are consistent with its effects on readily releasable stores and probability of release. Altogether. the present data support the notion of a presynaptic action of tubocurarine. These data. as well as the results of other investigators. suggest that tub~~curarine af%ects storage of ACh in the nerve terminal, It is of interest in this context that tubocurarine inhibited uptake of choline in the phrenic nervediaphragm preparation ((‘HANG and LIX. 1970) and inhibited ACh synthesis of brain tissue ( BIIATNAC~AR and MAC‘INTOSH, 1967). IHowe\er.

these two effects

were manifested only at high concentrations

of tubo-

curarine.

tation and depression as two independent phenomena. Relatively pure facilitation can be measured by reducing the quanta1 content by low Ca’+,!high Mg” Krebs solution (BALNAW and GAGE, 1970). The control cell recovery cycle in the low Ca”ihigh Mg” Krebs solution (Fig. 5) revealed marked facilitation and minimal depression and this is consistent with results reported by LL!NDHER(; and QWLIS~H (1953b).

DEL CASTILLO and KATZ (1953,

1954). HIJR-

RAKII (1963) and THIES (1963). Under these conditions, tubocurarine did not significantly decrease EPP ratio (Fig. 5). These results indicate that tubocurarine does not suppress facilitation and that it may enhance depression preferentially. Neuromuscular depression probably reflects depletion of the readily available store by the quanta of ACh released by the first nerve terminal action potential (T~~~~~J~,HI,1958; O’I-SL:KAtit (I/.. 1962; ELhlQwsT and QGASTFL. 1965.). Thus, depression is dependent upon the size of the readily available store and upon the quanta1 content of the first EPP. Enhancement of depression may result from a decrease in readily releasable stores a&or an increase in the quanta1

5

IO 20 40

loo 200 400 Too0

STIMULUS INTERVAL tmsec) Figure 5. Effect of tubocurarine on cell recovery cycle in low Ca’+ :high Mg’+ Krebs solution. Symbols as in Figure 4. 11= 7,

A~IFRRAUI. A. and BFTI.. W. (lY71). Does curare a&t tr~itlsnlilt~r relc~se’? J. f’li~xir~i.. twtf. 213: h9i- 705. BALYWVC, R. J. and GAGI, P. W. ( 1970). Temperature scnsitivity of the time course of f~lcilit~ttiofl of tr~lnsmitter release. Rruir~ Rex 21: 37~300. BAKSI’AII. J. A. and LILLFHEIL, 6. (IOU). Transversaly cut diaphragm preparatio.1 from rat. An ad.juv:tnt tool in the study of the physiology and pharmacology of the myoneural ,junction. 3~11.5 1111.Phrrr/,~tr~o[l!,,~. 7‘ht;r. 175: 373 90. BEANI. L.. BIANC‘HI. C. and LI:OIX F. (lY63). The etrect of tubocurarine on ACh release from nerve terminals. J. Phrsi0l.. 1..0!,~/. 174: 172 I x3. Bt~Al\;r.l~.R. and Vusrwix. F. ( 1967). The action of tubocumrinc and atropinc on the normal and denervatcd rilt di;lphragm. .1. Phi .sicd.. Lad. 188: 53~-66. BHATNAGAK, S. P. and Wn(-Ir;xctst~. F. C. (1967~. Elkcrs of quaternary bxes ~tnd i~~or~~~~~ic c;ltion.s on atetylcholine synthesis in nervous t&sue. C~tt. J. ~~~~..si(~~. ~~I~~~~?~~/~. 45: ‘749 3%. BLABEK. 1.. C. (1973). The prcjunctional action oB’ some non-depolarizmg blocking druss. Br. J. P/I(o.H~~. C‘/w Uloriw. 47: IO’) I I6 Bourn. J. M. and MFKKY. E. H. (IYhY). Intlucnce of (Itubocurarlnc. dccnmethonium nnd succlnylcholine on repetitively evoked end-plate potentials. J. Phrrrwc~. cvp. Thcr. 167: 334.-343.

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