Published September 1, 1978
A NEW METHOD FOR EXCITATION-CONTRACTION
UNCOUPLING IN FROG SKELETAL MUSCLE
J. DEL CASTILLO and GLADYS ESCALONA DE M O T F A
From the Laboratory of Neurobiology and the Department of Pharmacology, University of Puerto
Rico, School of Medicine, Medical Sciences Campus, San Juan, Puerto Rico 00901. Dr. Escalona de
Motta's present address is the Department of Biology, University of Puerto Rico, Rio Piedras
Campus, Rio Piedras, Puerto Rico 00931.
ABSTRACT
KEY WORDS formamide 9 excitation-contraction
uncoupling
frog skeletal muscle
neuromuscular
transmission
The mechanical activity initiated by membrane
depolarization is a serious obstacle to intracellular
electrophysiological work in skeletal muscle, as
the twitch associated with the action-potential
soon dislodges or breaks the intracellular microelectrodes and damages the muscle fiber. To avoid
this, two main techniques have been used.
The first technique is to work in hypertonic
solutions with 2.5 x the normal osmolarity. Fenn
(6) and Hodgkin and Horowicz (7) showed that
these solutions abolish contraction but leave the
action-potential unaffected, an observation confirmed since then by many authors. However,
hypertonic solutions of nonpermeant compounds
such as sugars or NaC1 block neuromuscular transmission (5).
The second technique depends upon an osmotic
shock which disrupts or disorganizes the transverse tubular system. The muscle is first immersed
in a hypertonic solution of a solute that penetrates
782
the cell membrane, such as glycerol. If the muscle
is left in this solution, the tension developed
decreases temporarily and the fibers recover their
initial volume and twitch tension (1). However, if
the muscle is rapidly transferred from the hypertonic solution to an isotonic medium, the fibers
swell and lose their power to generate tension.
This appears to be due to the breakage of the
transverse tubular system (8, 3), which becomes
vesiculated and loses continuity with the extracellular space, as shown by the observation that
horseradish peroxidase does not penetrate into its
interior any longer. These changes are accompanied by a decrease in membrane capacity (4).
This method has been used successfully by
several authors, but it requires relatively long
periods of immersion, up to 2 h, in the hypertonic
glycerol solution, it works only on small, thin
muscles, and many fibers are damaged and have
low resting potentials. In addition, for reasons
which are not clear, this method does not always
work. A new version of this technique using ethylene glycol instead of glycerol was introduced by
Sevcik and Narahashi (9). Muscles were im-
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The mechanical activity of frog sartorius muscle fibers can be uncoupled from the
electrical activity o f their surface m e m b r a n e s by immersing the preparation in
Ringer solution containing either 1.5 or 2.0 M of f o r m a m i d e for 1 5 - 3 0 min. This
uncoupling is not reversed when the muscle is transferred to normal frog Ringer
solution. F o r m a m i d e does not affect the electrical activity of the sciatic nerve
branch, and both endplate potentials and miniature endplate potentials m a y be
recorded from the uncoupled muscles. Prolonged exposure to f o r m a m i d e , b e y o n d
the time n e e d e d to paralyze, causes neuromuscular block.
Published September 1, 1978
platinum electrodes. Both the FMD concentration
and the time of exposure necessary to achieve uncoupling seem to depend upon the condition of
the frogs. Muscles obtained from one batch of
frogs stopped twitching after from 5 to 15 min in a
2.0 M FMD solution. However, this same concentration caused damage to muscles taken from frogs
of other shipments. These, in turn, were best uncoupled, in about 15-30 min, with a 1.5 M FMD
solution. It is, therefore, advisable to determine,
by trial and error, the FMD concentrations and
times of exposure needed to uncouple the muscles
from any particular batch of frogs to avoid causing
damage to the fibers.
As soon as contractions stopped in any of the
above mentioned conditions, the muscles were
transferred to normal Ringer's solution. These
muscles remained uncoupled during the rest of the
experiment, usually 6-8 h, with resting potentials
ranging from 80 to 90 mV. In a few preparations,
exposed to 2.0 M FMD for periods over 20 min,
the resting potentials decreased to values lower
than 60 mV, but this decrease was reversed if the
muscles were left overnight at 5~ in normal
Ringer's solution. Next morning, their resting
potentials were again normal, and the muscles
were still paralyzed.
Generally, uncoupling did not impair the electrical activity of the nerve, and both endplate
potentials and miniature endplate potentials could
be recorded. However, prolonged exposure to
FMD would also block neuromuscular transmission, though still not affecting the electrical activity of the nerve. For this reason, to do work on
neuromuscular transmission the preparation in the
FMD solution should be stimulated every few
seconds and changed to normal Ringer's solution
while nerve stimulation still elicits weak twitching
in a few fibers. Occasionally, the mechanical
responses to stimulation may increase too much
upon returning the muscles to normal Ringer's
solution; but, if this happens, a second and brief
exposure to FMD is usually sufficient to produce
uncoupling while neuromuscular transmission remains unaffected. Muscles treated in this manner
have remained paralyzed for periods of up to 3
days, at 5~ with normal resting potentials. FMD
is, indeed, a very effective uncoupling agent for
both teaching and research purposes.
The mechanism of action of FMD is unclear.
Unpublished observations of C6rdoba, GarciaSalazar, and del Castillo showed that FMD even
at 1 M concentration failed to remove water from
DEL CASTILLOAND ESCALONADE MoTrA New Method for Excitation-Contraction Uncoupling
783
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mersed in ethylene glycol (876 or 1095 mM) for
periods of 2-4 h at 5~ and then retumed suddenly to normal Ringer's solution. Twitches
occurred spontaneously, and the contraction
elicited by nerve stimulation gradually decreased.
Muscles uncoupled with this technique are said to
be in better condition than those treated with
glycerol; the average resting potential of the
muscle fibers decreases only by 10-13 mV and remains constant for as long as 2 h. Unfortunately,
and also for obscure reasons, the ethylene glycol
method does not always work.
Recently, we needed to record endplate currents from the frog sartorius with a double microelectrode voltage clamp technique, and neither
the glycerol nor the ethylene glycol method
worked on our preparations. We decided to
attempt uncoupling the mechanical activity with
formamide (FMD), a compound known to exert,
at a concentration of 1.75 M, a relaxing action on
glycerinated muscle fibers (10) and at concentrations lower than 1 M to block completely and
reversibly the mechanical activity of mammalian
smooth muscle (2).
We found that immersion of sciatic-sartorius
preparations of the frog Rana pipiens for 15-30
rain in a 1.5-2.0 M solution of FMD in frog
Ringer's solution produces a complete and longlasting uncoupling of the mechanical activity of
the muscles. These muscles cease to contract in
response to either direct or indirect electrical
stimulation while they are in the FMD solution.
Therefore, in spite of the high concentrations of
FMD used, the uncoupling caused by this compound does not depend upon an osmotic shock
in the usual sense. When uncoupled muscles are
transferred back into normal Ringer's solution, no
spontaneous twitching occurs, no gross swelling is
observed, and the muscles retain their normal
transparency while remaining paralyzed. The
possibility that returning the muscles to normal,
isotonic Ringer's solution may cause further
changes in their function cannot be excluded, but
there is no indication that these changes, if any,
contribute to the uncoupling observed.
Using the following procedure, we have obtained the best uncoupling results. The sciaticsartorius preparation, pinned to a layer of Sylgard
plastic (Dow Coming Corp., Midland, Mich.) in
the bottom of a petri dish, was immersed in the
FMD solution and the muscle twitch was tested
every 10 or 20 s with both direct and indirect
stimuli (1 ms duration) applied by means of two
Published September 1, 1978
muscle cells and to inhibit the activity of actomyosin-like ATPases of the guinea pig ileum.
However, F M D exerts striking changes in the
optical density of suspensions of freshly prepared
skeletal muscle microsomes ("relaxing factor")
which suggest that the vesicles formed by membranes of the sarcoplasmic reticulum swell under
the influence of that compound. It has been
suggested by the above mentioned authors that
F M D may act by preventing the m o v e m e n t or
storage of calcium ions, a mechanism that could
also account for the observed block in neuromuscular transmission.
This work was supported by United States Public
Health Service grants NS07464, NS14938, and
GM05567, contribution no. 76 of the Laboratory of
Neurobiology.
Received for publication 22 December 1977, and in
revised form 16 March 1978.
1. CAetrro, C. 1968. Volume and twitch tension
changes in single muscle fibers in hypertonic solutions. J. Gen. Physiol. $2:793-809.
2. C6P.DOSA, F., S. SCHOOF, S. V~LEZ, and J. DEE
CASTILLO. 1968. Inhibitory action of formamide on
smooth muscle contraction. Life Sci. 7:897-903.
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