Bryan Hummel
Vert.
Physiology 99
October
5, 1999.
Introduction: An
American bullfrog was used to extracellularly stimulate and measure the
contraction of skeletal muscle. The
muscle is stimulated by action potentials from the attached nerves. When the muscle membranes depolarize, they
become permeable to Ca2+ ions which flow into the cell and cause
muscle contraction. The muscle can also
be stimulated directly by an electrode.
The force of the contractions was measured under different stimuli to
determine the effects of acetylcholine and curare on the latent period,
contraction period and relaxation period.
Materials and Methods: An
American bullfrog was dissected to expose one of the sciatic nerves between the
spine and the “knee” joint. At the hip
joint, a suture was tied around the nerve and the nerve was moistened with
Ringer’s solution. Next the gastrocnemius
muscle was detached from the surrounding tissues, cut at the Achilles’ tendon,
and tied to a force transducer. A
stimulator was touched to the nerve in order to stimulate it into producing an
action potential or directly to the muscle in order to get a contraction. A computer was used to both record the
magnitude of the force put on the force transducer, and the time it took for
the muscle to go through its phases of contraction.
Results: When
the stimulating electrode was in contact with the sciatic nerve, the threshold
voltage was 0.2 Volts. The maximal
contraction was recorded at 0.6 Volts.
When the muscle was directly stimulated, the threshold voltage was 0.8
Volts, and the maximal contraction was recorded at 1.7 Volts. The average latent period was .02 seconds,
the average contraction lasted .05 seconds, and the average relaxation period
was .085 seconds. After the injection
of acetylcholine, the muscle showed no response, but with the addition of the
curare (an acetylcholine receptor antagonist), there were a few muscle twitches
without any electrical stimulation. The
length of time it took to cause tetanus at 15 pulses per second was 44.4
seconds.
Discussion: The
direct stimulation of the muscle required a higher voltage in order to get a
contraction. This most likely occurs
for two reasons. First is the fact that
once a nerve reaches its threshold voltage, it perpetuates the action potential
down the nerve to the muscle. This
brings us to the second reason, the nerve branches out and reaches many
thousand parts of the muscle, which all get depolarized. When stimulating directly, only the cells
surrounding the electrode will be depolarized to the point of releasing
calcium, thus the contraction is on a part of the muscle rather than the whole
thing. With nerve stimulation, even
after the nerve reached its threshold, increased voltage would still increase
contraction to a point. This was due to
the electrode being able to depolarize more of the individual nerves in the
compound sciatic nerve until the whole compound nerve was depolarized. As the wave of depolarization moved down the
sciatic nerve towards the muscle, a latent period was observed. During this latent period, the muscle
membranes are becoming depolarized and allowing calcium to flow into the cell
from the lumen of the sarcoplasmic reticulum.
The calcium causes the troponin to change conformation, which in turn
removes tropomyosin and allows for the formation of cross-bridges. The next stage was actual muscle
contraction, which involves interaction between actin (thin) and myosin (thick)
filaments. The myosin extends
cross-bridges that attach to actin and pull the two filaments past one another
by the movement of the myosin head. The
myosin head rotates and pulls on the actin filaments which result in an overall
shortening of the sarcomere. This happens
in a cyclic fashion so that there are always cross bridges attached to maintain
muscle tension. Using ATP as an energy source, the myosin head detaches from
the actin and undergoes a conformational change in the myosin fiber until the
next open binding site. During the period of relaxation, the intracellular
calcium is actively transported to the sarcoplasmic reticulum; one ATP
transports two calcium molecules. In
the S.R. a large protein (calsequestrin) binds to calcium and essentially takes
it out of the fluid in the S. R., which allows a smaller concentration gradient
for active transport to act against.
When the intracellular calcium levels get low enough, the calcium that
was bound to troponin dissociates and flows down the concentration gradient
into the intracellular fluid where it can be transported to the sarcoplasmic
reticulum. Upon dissociation, troponin
returns to its initial shape and the tropomyosin returns to the actin binding
site and will no longer allow for the myosin to establish cross-bridges. Because acetylcholine is the
neurotransmitter that stimulates muscle cell depolarization, and curare is an
acetylcholine receptor antagonist, one would expect the addition of curare to
prevent the depolarization, calcium influx and muscle contraction. This was
unfortunately not what happened in lab, the muscle showed no response upon the
addition of the acetylcholine, and was still able to be stimulated after the
addition of curare. This was probably
due to the fact that the syringe only allows the chemical to be inserted in a
localized region, and the rest of the muscle remains unaffected. There is mechanical summation in the muscle
cells due to the arrangement of the sarcomeres. Although each sarcomere doesn’t shorten a large amount, the
series of contracting sarcomeres can shorten the muscle considerably. The stimulated skeletal muscle cannot stay
stimulated forever. A state of tetanus
occurs after the muscle is re-stimulated before the muscle had time to deplete
its internal calcium levels, thus the active state of the muscle contraction is
prolonged until the muscle can not shorten any further. A graph of tetanus
would look similar to Figure 1. Some
muscles such as cardiac muscles will not undergo tetanus.
Figure
1. A graph of stimulation frequency
versus muscle contraction.
Tetanus is different than
fatigue. Tetanus can and often does
cause fatigue, but fatigue cannot cause tetanus. Fatigue is caused by the muscle running out of energy sources. This happened in lab much more quickly than
it would happen in vivo. This is
because the frog would have kept a constant supply of the precursors to ATP
flowing to the muscle, and in lab only ringer’s solution was provided. Also the frog would not stimulate its muscle
as forcefully or as often as we could accomplish in lab. Because the frog muscle was allowed to pull
on the force transducer, its contractions would be considered isotonic. If the muscle were attached between two
unmoving objects, it would be considered isometric. My final conclusion is that I am finished writing this lab
report, and our frog is finished as well.
Bryan Hummel
Vert. Physiology 99
October 5, 1999.