Download Lab #9: Smooth muscle properties (rat)

LabSmooth muscle properties (rat)
Introduction
Smooth muscle is very different structurally and functionally from skeletal and cardiac muscle studied previously.
Skeletal and cardiac muscle contain striations, composed of repeating actin and myosin fibers arranged so that they line up
(producing "bands" when under light microscopy). Smooth muscle is composed of essentially the same proteins as the
other muscle types, but they arranged in no systematic manner- the proteins are arranged more-or-less at random in the
cytoplasm. This more loose arrangement (and greater range of movement) is thought to be the primary reason why smooth
muscle is able to produce contractions over a greater range of changes in initial length.
There are other differences. First, receptors for neurotransmitters are scattered over the surface of the muscle, not
just in neuromuscular junctions. Second, Ca++ is stored in vesicles near the surface of the cell, not in the sarcoplasmic
reticulum. Third, smooth muscle contracts more slowly. As a result of both of these latter features, smooth muscle can
obtain Ca++ from the extracellular environment. (What does this suggest about its rate of fatigue?) Fourth, smooth muscle
fibers can produce graded contractions. Fifth, some kinds of smooth muscle contract spontaneously (single-unit; e.g. gut,
uterus, blood vessels). (Others (multi-unit) need nervous or hormonal stimulation to contract (e.g. iris, sphincters of the
gut and urinary tracts).)
The spontaneous contraction of single-unit smooth muscle is due to leakiness of Ca++; resulting in "pacemaker"
activity. In the gut, the depolarization is 10-15mV with a periodicity of 3-20s. These "slow waves" may or may not initiate
action potentials. An action potential stimulates conduction to neighboring muscle cells (through "gap junctions"),
resulting in a localized "twitch" or twitches. Slow waves produce sufficient numbers of action potentials to maintain a
steady-state contraction rate termed muscle "tone". This tone can be varied by hormonal, neurohormonal and nervous
(enteric and autonomic) control.
The autonomous nervous system provides "dual" innervation to practically all glands and organs by its
sympathetic and parasympathetic divisions. In the gut and urogenital tracts, parasympathetic input (neurotransmitter ACh)
is stimulatory while sympathetic input (neurotransmitter NE) in inhibitory. EPI, produced by the adrenal medulla in
response to sympathetic input, acts as a NE agonist. Thus, gut motility and urogenital function are regulated by
neurotransmitters and neurohormones. Like in the heart, atropine is a parasympathetic (ACh) antagonist.
In this exercise you will use segments of the mid-portion of the small intestine (jejunum; or a portion of the uterus)
of the rat. The preparation will be mounted in a muscle bath. This serves many functions- primarily it maintains the tissue
in the narrow dissolved gas, temperature, pH and ionic environment range required for normal functioning. The
preparation is kept in Tyrode's solution (Ringer's solution with glucose and Ca++) at 37oC in the presence of added O2.
Contraction force and contraction frequency and muscle tone will be monitored and recorded using the strain-gauge force
transducer.
Materials and Methods:
The organ bath
1. Make sure the circulating constant water bath is set at 37+ 1oC.
2. Make sure that all solutions to be used (Tyrode's, Ca ++-free Tyrode's, ACh, EPI, and Atropine) are at 37 oC before
use.
3. Note the organ bath- it functions to keep the preparation aerated and at the necessary temperature for effective
muscle contraction.
i) The circulating constant temperature bath circulates water through a jacket that surrounds the chamber into
which the preparation is lowered.
ii) The chamber itself has a capacity of 25ml.
iii) The stopcock located at the bottom drains the chamber. Also, the stopcock allows for solutions to be
introduced to the preparation to first pass through a coiled series of tubes within the out jacket. This allows
for this solution to be warmed prior to contacting the preparation.
The preparation
1. A rat will be euthanized and 2 cm. segments of the jejunum isolated and maintained in warmed, aerated Tyrode's.
2. The tissue is mounted between the force transducer and the hook in the muscle chamber.
3. Keep the preparation well aerated using an air pump and air stone built into the base of the organ chamber. Adjust
the airflow so the small air stone at the bottom produces a small stream of air bubbles.
4. Monitor the temperature and wait for the reestablishment of spontaneous contractions (5- 20 min).
Application of external solutions
Introduce the solutions in the order given. For each introduction:
1. Add Tyrode's solution.
2. Allow the preparation several minutes to establish (or reestablish) baseline contractions.
3. Add the drug.
4.
5.
6.
7.
8.
Give the preparation several minutes to respond.
Record and annotate.
Turn the red stopcock at the bottom to slowly drain the solution.
Gently pour in fresh 37oC Tyrode's solution (or Ca+ free Tyrode’s when noted) and drain to rinse the preparation.
Refill with Tyrode's (or Ca+-free Tyrode's).
Solutions and treatments:
i) Tyrode's + EPI
ii) Tyrode's + ACh. (Watch carefully (and record) as you add the drug- you are looking for a particular change
in the response of the tissue to the initial introduction of the drug. Also, be prepared to drain the solution
quickly and rinse with plain Tyrode’s.)
iii) Ca+-free Tyrode's
iv) Ca+-free Tyrode's + ACh (WATCH the muscle carefully- again, note any responses)
v) Tyrode's + Atropine
vi) Tyrode's + Atropine + ACh
Starling's Law of the heart
Does smooth muscle respond as cardiac muscle? Refill the apparatus with Tyrode's and test this experimentally
using the micropositioner to change the tension on the suspending string.
Resultsa) Determine the tone, contraction rate and contraction strength of the tissue in response to each treatment.
Determine the mean +SD of several contractions. Present the data in Table form with interesting Chart traces as
figures when appropriate.
b) Plot change in baseline (mV) vs. contraction intensity (mV). (It may be helpful to consult with those students who
did the same analysis on the frog heart preparation. However, there is no need to calibrate and convert to grams as
was done previously in the cardiac muscle exercise; mV will work fine for our purposes.)
Discussiona) External application of each chemical mimics the effects of various nervous and neurohormone stimuli. Interpret
changes tone, rate and/or strength as appropriate.
b) Why do you suppose temperature is so much more important to the functioning of smooth muscle as opposed to
skeletal muscle?
c) Differentiate between the source of Ca++ that stimulates contraction in skeletal vs. smooth muscle. Interpret the
Ca++-free Tyrode's data in this light.
d) When you added ACh to the Ca++-free Tyrode's, you might have seen a brief spasm. Where did the Ca++
responsible for this come from?
e) Does Starling's Law of the Heart apply to smooth muscle? Is so, what functional significance could this serve for
the gut and uterus?
f) Atropine is derived from the belladonna (meaning "beautiful woman") plant. This drug was used to dilate the
pupils of woman's eyes- giving them a "come-hither" look. Research the autonomic control of pupil diameter and
compare Atropine's effects on the pupil, the gut and on cardiac muscle. Interpret.