Br. J. Anaesth. (1982), 54,1217
MYONEURAL ACTIONS OF ORG NC 45
T. A. TORDA AND N. KlLOH
SUMMARY
In the toad sartorius preparation Org NC 45 reduced the mean quantal content of the end-plate potential both
m magnesium- and tubocuranne-treated preparations, as well as blocking post-juncaonal receptors. In a
concentration which permits recording of the miniature end-plate current no channel block was demonstrated.
Because of its short duration of action (Crul and
Booij, 1980; Marshall, Agoston et al., 1980), lack of
accumulation (Agoston et al., 1980; Buzello et al.,
1980) and minimal side-effects (Marshall, Agoston
et al., 1980; Marshall, McGrath et al., 1980; Agoston et al., 1980), OrgNC45 is likely to become
widely used in clinical anaesthesia. Its effects on the
chick biventer cervicis—reduction of the indirectly
elicited twitch without contracture; reduction of
response to acetylcholine—suggest a competitive,
"non-depolarizing" and post-junctional mode of action (Durant et al., 1979). We report a series of
electrophysiological studies to define more precisely
the sites of action of the drug at the myoneural
junction. Initial screening experiments (Torda and
Kiloh, 1982) demonstrated that, at concentrations
greater than that necessary to abolish indirectly
evoked contraction, OrgNC45 reduces the mean
quanta! content (m) of the end-plate potential
(EPP). Further studies were undertaken to demonstrate a presynaptic action at lesser concentrations,
also to confirm the postsynaptic component of the
neuromuscular block and to test for possible channel blocking action by examining the time course of
the miniature end-plate current (MEPC).
METHODS
The effect of Org NC 45 on quantal content of the
end-plate potential (EPP) was investigated in two
series of experiments on the toad (Bufo marinus)
sartorius nerve-muscle preparation. The methods
T. A. TORDA,* M.D., F F.A.R.A.CS , Department of Anaesthesia
and Resuscitation, School of Surgery, University of New South
Wales. N IQLOH, M.B , B.S., F F.A.R.A CS , School of Physiology
and Pharmacology, University of New South Wales.
Present address for correspondence: T. A. Torda, Department
of Anaesthesia and Intensive Care, Prince Henry Hospital, Sydney, N.S.W., 2036, Australia.
0007-0912/82/111217-05 $01 00
used are well known and have been described elsewhere in some detail (Torda, 1980). In the initial
series muscle contraction was prevented by decreasing evoked acetylcholine release by the use of high
magnesium solution containing: sodium chloride
115mmollitre~', magnesium chloride 15-18
mmol litre"1, potassium chloride 2.5mmol litre"1,
calcium chloride 1.8 mmol litre"1 and phosphate
buffer (pH7.2), 3 mmol litre"1. The muscle was
pinned out in a tissue bath and stimulated indirectly
by supramaximal square wave pulses of 50 us duration at 1 Hz frequency. The EPP were recorded
through microelectrodes of 10-20 Mil impedance
and displayed on an oscilloscope. The mean amplitude and variance of 80 to 120 EPP was determined
using a computer (PDP 8, Digital Equipment) and
m was calculated from the variance to mean ratio
(Del Castillo and Katz, 1954). In two studies, 40 or
more miniature end-plate potentials (MEPP) were
also recorded and m was also determined from the
ratio of EPP to MEPP amplitude. As EPP in magnesium-treated preparations are small, correction
for non-linear summation was not applied. After
three to six determinations of m, the perfusing
solution was changed to one containing Org NC 45
0.25 mg litre"1 (3.4xlO" 7 mol litre"1); after
20-3Omin three to six further determinations were
made. In three of the five cells, the preparation was
then returned to control solution for further determinations of m. Early experiments, not reported
here, using ampoule-packaged drug gave very variable results with inexplicable variations in quantal
size. When fresh drug, dissolved in saline adjusted
to pH4.0 with hydrochloric acid was used, the
results became reproducible. It is likely that the
ampoule preparation underwent some interaction in
vitro, which does not occur in vivo. Chelation with
* ion is a possibility (D. Savage, personal com© The Macmillan Press Ltd 1982
1218
BRITISH JOURNAL OF ANAESTHESIA
munication). The pH of the Ringer's solution was
not measurably affected by the small volume of acid
drug solution added.
In the second series of experiments movement of
the preparation was prevented by the addition of
tubocurarine 1.2mglitre~' (1.5xlO~ 6 mol litre"1))
instead of magnesium ion. Similar determinations of
m were made in five cells with and without
OrgNC45, 0.2 5 ing litre-1; three cells were held
long enough to complete wash-out of the drug to
return to control conditions for further determinations.
Post-junctional block was demonstrated by recording the effect of OrgNC 45 (0.25 mg litre"1) on
the amplitude of the miniature end-plate potential in
four cells.
The effect of Org NC 45 in concentrations up to
0.5 mg litre"1 on the rise-time and time constant of
decay (TD) of the MEPC was investigated using focal
extracellular recording of the MEPC (Torda and
Gage, 1977) in the presence of neostigmine,
lmglitre" 1 (3.0 x 10"*mollitre"1). At each drug
concentration MEPC were recorded on FM tape,
which was later played back through a Neurograph
N3 (Transidyne Corp.) transient recorder. For each
estimate of TD, 20 to 40 MEPC were "captured" and
averaged by computer.
When tubocurarine was used to prevent movement of the fibres, the quantal content of the EPP
was greater than in the magnesium-treated preparation (table II). The effect of OrgNC45 was very
similar, however—average of 48% reduction (range
39 to 72%)—which was reversible. The membrane
potential was not altered by the drug in either the
magnesium- or the tubocurarine-treated preparations.
The amplitude of MEPP was reduced by an average of 55% (range 43-61%) by OrgNC45
0.25 mg litre"1 in the four preparations studied
(table III).
Finally, there was no significant effect on either
the 20% to 80% rise-time or the time constant of
decay of the MEPC by Org NC 45 in the concentrations tested, although the MEPC amplitude was
reduced by up to 58% (table IV).
TABLEII. The effect of OrgNC 450.25 mglitre ' on quantal content
of the EPP in curarized preparations. The percent column expresses
the treated value as percentage of the mean of the control and washout
values
Quantal content
Cell
RESULTS
The results from the five magnesium treated preparations are summarized in table I. OrgNC45
O^Smglitre"1 reduced mean quantal content of the
EPP by an average of 50% (range 34-67%). The
effect was readily reversible on wash-out.
Control
OrgNC45
1
2
3
4
5
28.9
21.6
38.2
41.5
14.8
8.2
11.2
23.2
20.4
10 0
Mean
29.0±11.2
14.6±6.7
Washout
27.6
44.4
18.2
Percent
28
46
61
47
61
30.1±13.3 48.5±13 3
±SD
TABLEI. The effect of OrgNC450.25mg litre'1 on quantal content
of the EPP of magnesium-treated preparations. The values obtained
from EPP/MEPP rano are not included in the means. The percent
column expresses the treated value as percent of the mean of the control
and washout values. * Value derived from EPP/MEPP ratio
TABLE m . The effect of OrgNC45 0.25mg litre-' on MEPP
amplitude (mV). The percentages are based on the mean of the control
Quantal content
and washout values
Cell
Control
OrgNC45
Washout
Percent
MEPP amplitude
9.91
5.03
9.16
53
Org NC 45
Washout
Percent
Cell
Control
3.79
1.24
4.11
31
1.97
0.65
33
40
1.14
1.15
1
0.45
3.09
2.20
4.00
62
45
1.17
1.26
2
0.55
2.68*
1.35*
4.20*
39*
39
1.32
1.60
3
0.57
3.80
2.06
54
57
1.34
1.05
4
068
4.54*
2.89*
64*
Mean
±SD
4.5113.10
2.24±1.68
5.76±2.95 46.6±13.8
Mean
±SD
1.24±0.10
0.56±0.09
1.27±0.23 45.3 + 8.3
ACTIONS OF ORG NC 45
1219
TABLE IV. The tfftct of OrgNC4S on the amplitude, growth time
(20% to 80%) and time constant of decay (TD) of MEPC in the
neosngmine-treated toad sartorius
Amplitude
(* rrmrth
(ng litre"1)
Control
5
10
20
50
100
500
MV
%
(ms)
(ms)
208 ±42
193 ±30
194±50
146 ±42
145±45
138±36
87 ±16
100
93
93
70
70
66
42
0.56
0.54
0.54
0.55
0.55
0.53
0.54
3 55
3.30
3.92
3.32
3.92
3.70
3.20
DISCUSSION
All the initial clinical studies to which reference has
been made in the introductory paragraph confirm
the clinical value of Org NC 45 as a relatively shortacting neuromuscular blocking drug with few sideeffects. Its actions, as distinct from its effects, have
been the subject of only one study to date (Durant et
al., 1979). Screening experiments suggested that
OrgNC45 in concentrations greater than that required to immobilize the indirectly-stimulated preparation, reduced the quantal content of the EPP
(Torda and Kiloh, 1982).
Neuromuscular transmission can be interrupted
in numerous ways. Interference with neurosecretdon
(reduction of quantal content of the EPP) is typical
of the magnesium ion (Del Castillo and Katz, 1954)
or the aminoglycoside antibiotics (Elmquist and
Josefsson, 1962). The post-junctional receptors can
be blocked competitively, as by tubocurarine (Jenkinson, 1960), or irreversibly as with a bungarotoxin
(Katz and Miledi, 1973a). Decamethonium- and
suxamethonium-induced block, usually called depolarization block, is also post-junctional in nature,
although the exact mechanism must be considered
uncertain or variable (Galindo, 1971a; Galindo and
Kennedy, 1974; Adams and Sakman, 1978; Bowman, 1980). Additionally, at least three other possible mechanisms of transmission block exist. Desensitization by prolonged exposure to agonists (Rang
and Ritter, 1970a; Bowman, 1980) and metaphilic
antagonism, which is an interaction between a desensitizing drug and antagonist (Rang and Ritter,
1970b; Ryall, 1979). The final possible mechanism
involves block of the open ionic channel. This is the
type of action which local anaesthetics exert at the
neuromuscular junction (Adams, 1976). Decamethonium in clinical concentrations appears to
act as a channel blocker as well as whatever other
actions it may have (Adams and Sakman, 1978) and
tubocurarine has also been noted to affect the time
constant of decay of MEPC in neostigmine-treated
muscle (Katz and Miledi, 1973b) and under conditions of hyperpolarization, suggesting open channel
block (Katz and Miledi, 1978; Colquhoun, Dryer
and Sheridan, 1979). Gallamine and pancuronium
may also have such action when the end-plate is
hyperpolarized (Katz and Miledi, 1978). It was with
these considerations in mind that the present series
of experiments was planned. The mean open channel life-time was measured from the time constant of
decay of the MEPC, which does not allow for investigation of the hyperpolarized state (Anderson and
Stevens, 1973).
The effect of Org NC 45 on the MEPP confirmed
a potent post-synaptic action, reversible and similar
to the principal action of all the clinically used
"non-depolarizing" neuromuscular blocking drugs.
In a concentration of O^Smglitre"1, OrgNC45
reduced the MEPP by 55%.
The experiments estimating quantal content confirmed that the MEPP is already reduced by concentrations of Org NC 45 less than that necessary to
prevent muscle contraction. This is similar to the
action of tubocurarine found by most workers
(Galindo, 1971b; Hubbard and Wilson, 1973). A
similar effect for pancuronium has also been described (Galindo, 1972a). It is likely that such prejunctdonal effect is of some significance, but its
contribution to the clinical effect of these drugs is
still open to speculation. Some believe that it is of
considerable importance at physiological rates of
nerve firing (Galindo, 1972b).
The theoretical significance of these findings is
interesting. The work of Standaert (1964) and Standaert and Riker (1967) demonstrated that cholinergic drugs can act prejunctionally and that this action
is blocked by tubocurarine. This work led to postulation of the existence of pre-junctional cholinergic
receptors (Blaber and Karczmar, 1967; Hubbard,
Wilson and Miyamoto, 1969) which may play a part
in modulation of acetylcholine release (Bowman and
Webb, 1976; Miyamoto, 1978). Block of these "R2"
receptors may result in reduced acetylcholine mobilization during tetanus and account for, or be a factor
in, tetanic fade.
Finally, it has been suggested that open channel
block as demonstrated by Katz and Miledi may also
contribute to tetanic fade (Bowman, 1980). Our
rather crude experiments failed to demonstrate such
BRITISH JOURNAL OF ANAESTHESIA
1220
an effect for OrgNC45. In this context it is worth
noting that shortening of open channel life-time by
tubocurarine, pancuronium and gallaminp has only
been demonstrated in the hyperpolarized preparation and was undetectable at physiological membrane potential.
In summary, therefore, Org NC 45 reduces quantal content of the EPP, as well as blocking receptors
competitively in the toad sartorius. Open channel
block at normal membrane potential has not been
demonstrated. Although these findings will not influence clinical practice, they have implications for a
better understanding of neuromuscular transmission and of the drugs which block it.
ACKNOWLEDGEMENTS
We are deeply indebted to Professor P. W. Gage of the School of
Physiology and Pharmacology of the University of New South
Wales for his help, encouragement and the use of his laboratory,
to Ms J. Allen for much assistance and to Dr D. S. Savage of
Organon Technics for supplies of NC 45 and Organon (Australia)
for generous financial assistance; finally to our ever patient Mi
Schubert for innumerable typescripts.
other pancuronium analogues in the cat. / . Pharm. Pharmacol.,
31,831.
Elmquist, D., and Josefsson, J. O. (1962). The nature of
neuromuscular block produced by neomycine Acta Physiol.
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(1971b). Prejuncnonal effect of curare: Its relative importance. / . Nturophysiol., 34,289.
(1972a). Curare and pancuronium compared: effects on
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(1972b). The role of prejunctional effects in myoneural
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Kennedy, R. (1974). Further observations on depolarizing
neuromuscular block The so-called phase II block. Br. J.
Anaesth., 46,405.
Hubbard, J. I., and Wilson, D. F. (1973). Neuromuscular
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blocking drugs and the effect of d-tubocurarine. / . Phystol.
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and substances which depolarize the motor end-plate. / .
Phystol. (Lond.), 152, 309.
Katz, B., and Miledi,R. (1973a). The effect of a bungarotoxin on
acetylcholine receptors. Br. J. Pharmacol., 49, 138.
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ACTIONS OF ORG NC 45
ACTIONS NEUROMUSCULAIRES DE
L'ORG NC 45
RESUME
Dans la preparation du muscle couturier de crapeau, l'Org NC 45
diminue le contenu Hem en take moyen du potentiel postjonctionneJ dans les preparations traitees au magnesium et aussi
dans celles traitees a la tubocuranne, et il bloque les recepteurs
post-jonctionnels. A une concentration qui permette 1'enregistrement du ties faible courante post-jonctionnel, on n'a pas pu
retrouver de bloc de canal.
1221
sowohl in mit Magnesium und Tubokurann behandelten
Praparaten, als auch in den Wockiercnden postsynaptischen Rezeptoren herab. In einer Konzentraoon, die die Messung des
Mimatur-Endplattenpotential8 erlaubt, zeigte sicfa kein KanalHock.
A(XIONES MIONEURALES DEL ORG NC 45
SUMARIO
DIE MYONEURALE WIRKUNG VON ORG NC45
ZUSAMMENFASSUNG
An einem Praparat aus dem M. sanorius der RrOte seme Org
NC45 den mittleren Quanteninhalt des Endplattenpotentials
En la preparacion del sartorio de zapo, el Org NC 45 redujo el
tenor cu£ntico medio del potencial de la piaca-terminal tanto en la
preparacion tratada por magnesio como en la tratada por
tubocurarina, y tambien bloqueo los receptorcs post-pincionalrs.
En una concentration que permita el registro de la corriente
miniatura de la placa-terminal, no fue demostrado ning^in bk>queo del canal.