AUTONOMIC REGULATION OF INTESTINAL SMOOTH MUSCLE
BACKGROUND
In this experiment, you will:
Observe the spontaneous contraction of intestinal smooth muscle in the absence of input from the central
nervous or endocrine systems.
Demonstrate the effects of inputs from the two autonomic branches
Demonstrate the roles of external calcium and internal calcium stores in control of contraction
Demonstrate that the cholinergic receptors of the gut are muscarinic
The abdominal portions of the intestine are innervated by branches of the vagus nerves for its
parasympathetic inputs and by postganglionic fibers originating in the superior mesenteric ganglion for its
sympathetic inputs. The intestine has numerous postganglionic parasympathetic neurons and branches of
postganglionic sympathetic neurons arranged in two plexi, the submucosal plexus which overlies the
muscularis mucosae and the myenteric plexus which lies intermediate to the longitudinal and circular
muscle layers. The gut plexi are not responsible for driving or sustaining the spontaneous activity of the gut
muscle, but they are necessary for coordination of the normal modes of gut behavior, such as peristalsis.
As with the heart and blood vessels, the cholinergic receptors of gut muscle cells are of the muscarinic
type. The adrenergic receptors are mainly of the alpha-adrenergic type. As a muscarinic agonist you will
be using Mecholyl, a more stable congener of acetylcholine. As an adrenergic agonist, you will be using
epinephrine.
Although in this exercise we will look at only the classical autonomic neurotransmitters, you should be
aware that the enteric neurons also release a large variety of peptide neuromodulators. You should also
recall that enteroendocrine cells release substances that may have effects on muscle activity.
Intestinal smooth muscle cells are of the single-unit type. Spontaneously active cells are common in this
tissue. These cells show slow rhythmic depolarizations called slow waves that result in episodes of
contraction in the cells. Depending on conditions, a slow wave may or may not result in a burst of action
potentials. If action potentials occur, the resulting contraction will be more intense. Since all cells are
connected to their neighbors by gap junctions, a spontaneously-active cell can serve as a pacemaker driving
simultaneous activity in the population of cells surrounding it.
In smooth muscle generally, the free Ca ++ that appears in the cytoplasm during excitation comes from two
sources - Ca++ that enters through L Ca++ channels during depolarization and Ca++ that is released from
calciosomes. You will be able to demonstrate that the muscle depends on extracellular Ca++ for slowwave activity but has an internal store of Ca++ that is specifically released by cholinergic
neurotransmission.
PROCEDURE
You will be using an isolated gut preparation that has seen very widespread use in pharmacology. At one
time, a similar preparation using the clam heart was the most sensitive assay for acetylcholine, with a
sensitivity threshold of about 10-9M. In recent years, the mammalian intestinal preparation has been a very
valuable model for study of opiate receptors, which are abundant in the gut.
Run the GUT setup program.
Set up for repeat recording by pressing
File
Repeat recording
4 ENTs. Enter the name you have selected for your set of files.
Set the amplifier gain to 1500 or 5K. Center the trace using the OFFSET knob. If the trace goes off the
screen during the course of the experiment, bring it back using the OFFSET. During a contraction, the
trace will move upward.
The computer will save screens of about 15 sec each. For periods in which you do not want to be
collecting data, you can pause the collection by pressing ESC. Remember to keep a log of the files that
you do collect.
You will be provided with a strip of intestine, usually from the jejunum. Keep it moist with Locke's
solution at all times and minimize its contact with the atmosphere. Fill the inner chamber of the muscle
bath with pre-warmed Locke's solution. Tie a piece of wire to both ends of the strip. Attach one end of the
strip to the L-shaped arm of the muscle holder. Bring the other wire through the flat hole in the muscle
holder. Lower the muscle holder into the bath and clamp it into position. Slide all but one of the leaves of
the transducer to one side. Tie the upper wire to the sensor leaf of the transducer; leave some slack in the
wire Start a trace on the monitor. Raise the transducer by using the knob on its clamp until there is a
corresponding rise in the trace. Make sure that the wire is not rubbing against the muscle holder. Insert the
aerator needle and adjust the flow so that there is a tiny stream of bubbles that does not disturb the tissue or
its connections. Make sure that the bath temperature remains at 36-39oC - gut activity diminishes
temporarily as the temperature falls more than a degree or two below this value, and it stops permanently if
the temperature rises more than a degree or two above this value. Bath solutions that you add should be
pre-warmed.
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The gut should begin to contract and relax spontaneously. Notice the pattern of the activity: rate,
amplitude, etc.
At the beginning of a new data screen, add a few drops of Mecholyl to the muscle bath. The response
will differ somewhat depending on the dose and the piece of gut - if the effect is not dramatic, add
some more. Collect at least one screen of data showing this response.
After the response to Mecholyl has waned somewhat, add a few drops of epinephrine. Follow this
response for at least one screen.
Pause the recording. Drain the muscle bath and refill it with pre-warmed Ca++-free Locke's solution.
Wait 5 minutes and drain again. Repeat this washing procedure a total of 3 times.
Start the recording again. Is spontaneous activity present? If so, wash again with Ca ++-free Locke's.
Add the same amount of Mecholyl that you used to produce activity in Step 2. If you get a response,
wait for the response to be complete, and then add a second dose of Mecholyl.
Recover spontaneous activity by returning the gut to standard Locke's solution, with several washes to
remove residual Mecholyl.
Add two droppers of atropine, a muscarinic blocker. Is there any effect on the spontaneous activity?
Now add the dose of Mecholyl that you found adequate to cause a substantial activation of the gut in
step 2.