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BSL PRO Lesson A05

2002 BIOPAC Systems, Inc. Updated 12-22-03

PHYSIOLOGICAL PROPERTIES OF VISCERAL SMOOTH MUSCLE

Overview
Dr. Katja Hoehn
Mt. Royal College Chemical Biological and Environmental Sciences Calgary, AB, CANADA There are three types of muscle tissue in the human body, each specialized for certain tasks in maintaining homeostasis.
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Skeletal muscle, generally under voluntary control, is so named because it is in most cases attached to the skeleton. Contraction of skeletal muscle moves parts of the body with respect to one another or moves the entire body. Cardiac muscle is the muscle found only in the heart, where it functions to circulate the blood in order to deliver nutrients to cells and remove wastes from cells throughout the body. Smooth muscle is generally not under voluntary control. It is usually organized in sheets found in the walls of hollow organs of the digestive, urinary, reproductive and respiratory system and in the walls of all but the smallest of blood vessels. Contraction of smooth muscle controls movement through these organs and blood vessels as well as the pressure within them.

Smooth muscle is characterized histologically by small spindle-shaped cells with a central nucleus. They do not exhibit the characteristic striations (stripes) seen in skeletal muscle. They lack myofibrils and do not have the clearly defined bands of actin and myosin, which make up the A-bands and the I-bands of the sarcomeres of skeletal muscle. In spite of their structural differences, a number of similarities exist between skeletal muscle and smooth muscle.
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In both types of muscle, contraction is achieved through the interaction of actin and myosin via the sliding filament mechanism. Both types of muscle use ATP to energize the sliding process.

In both types of muscle the final trigger for contraction is a rise in the cytosolic Ca 2+ concentration.

However, there are a number of important differences between excitation-contraction coupling in smooth muscle versus excitation contraction-coupling in skeletal muscle.
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In smooth muscle the sarcoplasmic reticulum is poorly developed and there are no ttubules. On depolarization of the smooth muscle membrane, cytosolic Ca 2+ rises due to both a release of some Ca 2+ from the sarcoplasmic reticulum as well as the entry of Ca 2+ into the cell from the extracellular fluid. The cytosolic Ca 2+ does not bind troponin as it does in skeletal muscle (smooth muscle does not contain troponin), but instead interacts with a regulatory molecule, calmodulin. The Ca 2+ calmodulin complex then binds to and activates a molecule called myosin kinase (or myosin light chain kinase), which catalyzes the transfer of a phosphate from ATP to myosin. This phosphorylated myosin can then interact with actin of the thin filaments and that initiates the cross-bridge cycling. Relaxation comes about when the cytosolic Ca 2+ concentration returns to precontraction levels and the myosin is dephosphorylated (the phosphate group is removed.) Ca 2+ in the cytosol is actively pumped back into the sarcoplasmic reticulum and back across the plasma membrane.

Smooth muscle can be classified into two major categories: multi-unit smooth muscle and singleunit (or visceral) smooth muscle.
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Mulit-unit smooth muscle is made up of individual muscle fibers, which can operate independently, are not connected by gap junctions, and tend not to be spontaneously active. Multi-unit smooth muscle is found only in a few locations in the body. This includes the internal eye muscles that adjust the focus and the pupil size, the smooth muscle of the large airways and large arteries, and the erector pili muscles attached to the hair follicles of the skin (which, when they contract, give you goose bumps). Its properties tend to be somewhat intermediate between those of skeletal muscle and single-unit smooth muscle. It is like skeletal muscle in that it consists of muscle fibers that are structurally independent of each other, it consists of several motor units that can contract independently, and its contraction is initiated by a stimulus from a nerve (i.e., it is neurogenic, initiated by nerves). However, the nerves that stimulate contraction in multi-unit smooth muscle are autonomic nerves (as they are in visceral smooth muscle) and not somatic nerves as for skeletal muscles. Visceral (or single-unit) is by far the most common type of smooth muscle. It is the type of muscle we will be studying in todays lab and we will focus on this type of smooth muscle for the rest of this introduction. Visceral smooth muscle is made up of muscle cells interconnected by gap junctions, which provide electrical coupling between cells. Thus, an action potential generated in one muscle cell can easily spread to adjacent cells, allowing the cells to contract as a single unit.

Some of the fibers in visceral smooth muscle can undergo spontaneous depolarization and therefore act as pacemaker cells, which set the contractile pace for the unit. The pattern of very slow swings of depolarization and then hyperpolarization seen in visceral smooth muscle is known as the slow-wave potential or basic electrical rhythm. It is probably due to a slow leak of Na + into the cell, and the waxing and waning of the outwardly-directed Na + pump.
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If the depolarizing swings are large enough to reach threshold, then a burst of action potentials are generated for the period of time during which the potential is above the threshold level. If the depolarizing swings do not reach threshold, then the depolarizing and hyperpolarizing swings of action potential will occur without any action potentials and therefore without any contractions. (See Fig. 8-32 and p. 274 in Sherwood 4th Edition for further description).

Contraction in this type of smooth muscle does not depend on nerves for its initiation and it thus called myogenic (initiated by the muscle itself). Although nerves are not required to initiate contraction, nerves, (as well as hormones and a number of other factors which we will consider below) can greatly modify the rate and strength of contractions in this type of muscle.
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For example, the strength of contractions can be altered by changing the starting point of the membrane potential of pacemaker cells at the onset of the depolarizing swing.

The autonomic nervous system innervates visceral smooth muscle. In contrast to the somatic nervous system, which always excites skeletal muscle and always does so by releasing the same neurotransmitter (acetylcholine), the autonomic nervous system can either excite or inhibit smooth muscle. The response depends on the type of neurotransmitter released by the autonomic nervous system and the subtypes of receptors that are present on the tissue. In general, the parasympathetic nervous system releases acetylcholine from postganglionic nerves onto the smooth muscle of the intestines. Acetylcholine is an excitatory neurotransmitter in the gut, acting upon the muscarinic subtype of receptors in the cell membrane. The sympathetic nervous system releases norepinephrine from postganglionic nerves onto the smooth muscle of the gut. Norepinephrine is an inhibitory neurotransmitter in the gut. There are two adrenergic receptor subtypes that may respond to norepinephrine, alpha receptors, and beta receptors. Both receptor subtypes are found in visceral smooth muscle and both have the same effect, namely the reduction of smooth muscle rhythmicity and tonus. Another difference between somatic nervous system innervation of skeletal muscle and autonomic nervous system innervation of visceral smooth muscle is that smooth muscle lacks the highly structured one-to-one neuromuscular junctions found in skeletal muscle. The terminal branches of autonomic fibers contain numerous swellings, called varicosities, and neurotransmitter is released from these varicosities into the interstitial fluid surrounding the muscle cells. The transmitter substance may have to diffuse a few micrometers to reach the nearest cell, as opposed to a few nanometers in skeletal muscle. Receptor proteins on the muscle cell membrane are dispersed throughout its entire surface membrane. As a result, each autonomic terminal can influence more than one muscle cell and each muscle cell may be influenced by more than one type of neurotransmitter. Usually only the outermost layer of cells in innervated. The rest rely on conduction of electric current from cell to cell via gap junctions. Smooth muscle contraction has a number of special features. It is slow, sustained and resistant to fatigue:
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It takes smooth muscle about 30 times as long to contract and relax as skeletal muscle (up to 3000 msec (3 sec) for a smooth muscle contraction versus about 100 msec for skeletal muscle). The slow contraction is largely due to the slower rate of ATP splitting by myosin ATPase and the resultant slower cross-bridge cycling. A slower rate of Ca 2+ removal from the cytoplasm is responsible for the longer time needed for relaxation. In spite of its slowness, smooth muscle can generate about the same tension of contraction per unit of cross-sectional area and can maintain that contraction at a fraction of the energy cost of skeletal muscle.
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This energy efficiency is in part due to something called the latch phenomenon. It is thought that smooth muscle myofilaments may lock together during prolonged contraction, partly as a result of the much slower cross-bridge cycling.

In response to an increase in stretch, smooth muscle contracts. However, the increased tension brought about by stretch is only transient. Within a few minutes, the tension returns to normal.

This response is called the stress-relaxation response. It is important because it allows hollow organs (such as the bladder and uterus) to accommodate large volumes.

The strength of a smooth muscle contraction is much less sensitive to a change in the initial length of the muscle than is the strength of a skeletal muscle contraction. The ability of skeletal muscle to contract decreases markedly when it is stretched beyond its optimum initial length. Smooth muscle, in contrast, can generate considerable force even when stretched to more than twice of the resting length. This is due to the irregular, highly overlapping arrangement of filaments. As a result, for example, the urinary bladder can still empty efficiently even when very full and yet does not become flabby when empty. Action potentials in smooth muscle cells, unlike those in skeletal muscle, are primarily due to the inward flux of Ca 2+ through voltage-gated Ca 2+ channels. Thus, extracellular calcium plays a major role both in the development of the action potential and in the contraction process. Changing the Ca 2+ concentration of the extracellular fluid will have an effect on contractions of visceral smooth muscle. For comparative characteristics of muscle fibers--skeletal, single unit smooth, multiunit smooth, and cardiac--see Human Physiology, 8e, Vander et al., p.331, Table 11-6. About this lesson In the following experiments, you will study some of the physiological and pharmacological properties of visceral smooth muscle taken from the rabbit ileum. (The ileum is the last part of the small intestine). You will vary the Ca 2+ concentration, and the temperature and oxygen content of the medium surrounding the muscle. In addition, you will investigate the effects of acetylcholine, atropine and norepinephrine on the muscle tissue. You will observe the recordings before and after the various treatments and look for and measure changes in the rhythmicity and in the tonus of the smooth muscle contractions. The Biopac Student Lab PRO will be used to record the contractions.
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Rhythmicity refers to the pattern of the muscle contractions. In particular, you will be looking at three aspects of rhythmicity: (1) the rate (frequency) of the contractions, (2) the amplitude (size) of the contractions and the (3) regularity of the contractions. You will observe and make note of changes in any of these aspects of rhythmicity. If the interval between contractions is irregular or the amplitude of the contractions varies considerably, then the contractions are arrhythmic. The smooth muscle in the walls of the digestive tract (and many other locations) maintains a constant low level of contraction, known as tone or tonus. Tonus refers to the amount of tension continuously generated by the muscle. Usually there are a small percentage of muscle fibers that are in a constant state of contraction while the majority of the fibers undergo rhythmic contraction and relaxation. An increase in the percentage of fibers in a state of continuous contraction results in increased muscle tonus; a decrease results in a reduction of tonus. Shifts in the baseline position of the recording on the y-axis indicate changes in tonus. IMPORTANT NOTE! Wash your hands, the Petri dish and dissecting instruments, and rinse the muscle bath before handling or mounting the intestinal segment. Contaminants from your hands or tools could kill the tissue.

Objectives

1. To study the effects of media ionic composition, temperature, and various pharmacological agents on the contraction of
the visceral smooth muscle of the rabbit ileum. 2. In particular, the student should be able to:

a. outline some of the differences between skeletal, cardiac and smooth muscle b. distinguish structurally and functionally between two types of smooth muscle, visceral (single-unit) and multi-unit
smooth muscle. State where you would expect to find each.

c. describe excitation-contraction coupling in smooth muscle and compare and contrast it with excitation-contraction
coupling in skeletal muscle.

d. describe the following properties of visceral smooth muscle contraction: tonus, rhythmicity, rate, regularity, and
amplitude. Describe how you would identify each of these characteristics in a chart recording.

e. discuss the importance of calcium in smooth muscle contraction. Describe the effect of removing calcium from the
medium on smooth muscle contraction.

f. describe the effects of oxygen depletion, sustained depolarization and temperature changes on smooth muscle
contractions.

g. describe how an increase in KCl in the extracellular fluid surrounding smooth muscle cells causes depolarization. h. describe the effects of norepinephrine, acetylcholine, and atropine on smooth muscle contractions.

Equipment
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BIOPAC Acquisition Unit (MP30) BIOPAC variable force transducer with S-hook (SS12LA) A piece of rabbit ileum Filter paperfor placing the muscle on during cutting 95% O2, 5%CO2 gasto bubble the physiological solution 2 oxygen tanks Each lab group should have:
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BIOPAC tissue bath (TISSUEBATH1) BIOPAC water circulator (CIRCULATORA or CIRCULATORB) Knife for cutting the ileum into 4 pieces Forceps for handling the ileum Petri dish for use during mounting of the intestinal segment Ring weight (between 3 and 7 grams) that has been pre-weighed and has its weight recorded on it (for calibration of the force transducer) Fish hook Needle clamp for holding the aeration tube in the muscle bath Beakers for changing solutions Syringe (50 ml) Solutions:
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Tyrode's solution Calcium-free Tyrode's solution 1 M KCl 10 -4 M norepinephrine 10 -4 M acetylcholine 10 -3 M atropine 2% CaCl2

Setup
Hardware

The smooth muscle bath consists of a large glass tube surrounded by a jar, which serves as a water jacket. A water circulator connects to the bath and maintains the temperature at 37C. The tube at the bottom of the bath is a drain for the inner muscle bath. O2 gas is bubbled into the bath and can be adjusted via the valve on the gas line. The muscle is attached at one end to a tissue bearer (which is inserted into the bath to anchor the muscle) and at the other end to the transducer via an S-hook at the 50-g ring.

Smooth Muscle Prep

IMPORTANT: Wash your hands, the Petri dish and dissecting instruments, and rinse the muscle bath before handling or mounting the intestinal segment. Contaminants from your hands or tools could kill the tissue.

1. Place a segment of intestine into a Petri dish filled with fresh Tyrode's solution. 2. Remove the aeration tube from the smooth muscle bath. Attach one end of the intestine to the tube by sliding the Sshaped syringe needle through the wall of the intestine. Attach the other end of the muscle to the fishhook suspended

by thread from the transducer. 3. Fill the muscle bath with fresh warm Tyrode's solution from the large water bath. Carefully insert the preparation. Slide the upper end of the aeration tube into the clamp and attach the airline. Make sure the gas (95% O2 and 5% CO2) is bubbling by the tissue. Adjust the screw clamp so that the flow of gas results in a small, steady stream of bubbles. Turn the heating lamp on. 4. Adjust the tension on the thread attaching the muscle to the transducer so there is no slack in the system. 5. Check the temperature of the inner and outer baths. It should be between 34C and 37C. After 5-10 minutes in the warm Tyrode's, the gut should start to undergo spontaneous contractions. If the temperature begins to rise above this range, turn off the heating lamp. If it falls, move the light closer and replace the outer bath with warmer Tyrode's solution.

Software 1. Launch the BSL PRO software on the host computer.

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The program should create a new "Untitled1" window.

2. Open the Smooth Muscle Template by choosing File > Open > choose Files of type: Graph Template (*GTL) > File
name: "a05.gtl" The template will establish the required settings. 3. Save As the desired file name.
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Calibration
Because the force generated by the rabbit ileum will be very small, we will use a small weight (about 5 grams) for our calibration, putting it onto the transducer attachment labeled "50 g."

1. Calibrate Cal1 using no weight at all (with only the s-hook hanging off the ring of the transducer). 2. Use the weight for Cal2. (A rough estimate of the forces will suffice for this exercise, as we are interested primarily in
changes in rhythmicity and tonus in our recordings and not in absolute values). 3. Record the values for Cal1 and Cal2 below. This will help you in the unlikely event that your system should crash. You would then not have to unhook the gut from the transducer to redo a calibration, but could type in the calibration values that you recorded here. Cal1, Input Value = _________mV Scale Value for Cal1 was 0 Cal2, Input Value = _________mV Scale Value for Cal2 was ____grams

Recording & Analysis

Hints for minimizing measurement error: A. Always allow the muscle to recover to normal between experimental protocols. B. Record continuously! Stop your recording only to save your data. C. Save after each manipulation. D. Always record some normal contractions before and after each experimental manipulation
before you stop recording momentarily to save your file. E. Insert a marker each time you begin a new experiment, change solutions, add a drug or perform a manipulation (press the F9 key on a PC or the Esc key on a Mac) and write down what you have done in the marker label bar. F. After recording the normal contractions, perform each of the manipulations listed below in the order given.

Segment 1: Baseline

Baseline rhythmicity and tonus

1. Once the muscle has stabilized, record several contractions showing the normal rhythmicity and tonus.
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You may have to adjust the position of the transducer to get a proper recording. Ask the instructor for help with this adjustment if necessary.

Segment 2: Effects of Extracellular Calcium

Effect of Calcium-free Tyrode's Solution

Effect of 2% Calcium chloride drops Given what you know about the importance of the extracellular calcium concentration to smooth muscle contractions, what hypothesis would you make about the effect, on the tonus and rhythmicity of contraction, of replacing the regular Tyrode's solution in the extracellular fluid with calcium-free Tyrode's?

1. Replace the regular Tyrode's solution with Ca 2+ -free Tyrode's solution. Record the response of the muscle over the
next few minutes. 2. Add a few drops of 2% CaCl2. Note the effect.

Do the results confirm your hypothesis?

3. Replace the Tyrode's solution in the bath with fresh normal Tyrode's solution and allow the contractions to stabilize
before proceeding to part B. Segment 3: Effects of Chemical Depolarization

Effects of KCI Normally, the membrane potential oscillates above and below threshold, thus generating the pattern of muscle contraction. Sustained depolarization can be induced by adding KCl to the bath.

1. Add 1 ml of 1 M KCl to the bath. 2. Allow the KCl to remain in the bath for about 1 minute, then replace with regular Tyrode's solution. 3. What effect did this have on rhythmicity? How does excess K + in the extracellular fluid cause depolarization? What
event is triggered at the cell membrane, which results in initiation of the contraction process? 4. Allow the preparation to recover, and then replace the regular Tyrode's with calcium-free Tyrode's. 5. Wait for the contraction to diminish so that the preparation appears to be dormant, then add 1ml (or 2 ml if necessary) of 1M KCl solution and observe the response. The muscle cells are depolarized by the addition of KCl, just as they were in steps 1 and 2, but does the depolarization result in contraction? Explain. 6. Replace the bath with regular Tyrode's and allow the rhythmicity and contraction amplitude to return to normal before proceeding to Segment 4C . Segment 4: Effects of Change in Temperature

Effects of Change in Temperature Any biological system is affected by changes in temperature, not only due to the effects of temperature on Brownian motion, but also because of the effect of temperature on enzyme activity. Formulate a hypothesis regarding the effect of cooling on muscle rhythmicity and tonus.

1. Replace the warm Tyrode's with room-temperature (21C) Tyrode's. 2. Turn off the "Heater" switch on the water circulator and note the effect as the temperature drops over the next few
minutes. 3. Turn on the "Heater" switch on the water circulator, replace the bath solution with Tyrode's, and wait until the preparation has stabilized at 37C before proceeding. Segment 5: Effects of Norepinephrine and Acetylcholine

Effects of Norepinephrine

Effects of Acetylcholine The effects of norepinephrine and acetylcholine will be tested in this part of the experiment. What do you hypothesize will be the effects of these two drugs?

1. Apply 1.0 ml of 10 -4 M norepinephrine to the muscle bath (bath concentration will be roughly 2 x 10 -6 ). Observe the
response over the next minute. Drain and refill the bath with fresh Tyrode's. 2. Once the contractions are stabilized, add 1.0 ml of 10 -4 M acetylcholine to the muscle bath (bath concentration will be

approximately 2 x 10 -6 ). 3. Drain and refill the bath. Allow the preparation to stabilize before proceeding. Segment 6: Effects of Atropine

Effects of Atropine Acetylcholine acts on two subtypes of acetylcholine receptors called muscarinic and nicotinic receptors (see p. 227 and Table 7-4 in Sherwood text, 4th Edition). Nicotinic receptors are found in autonomic ganglia, in skeletal muscle and in the central nervous system. Muscarinic receptors are found on cardiac muscle, smooth muscle and glands, as well as in the CNS. Atropine blocks the muscarinic subtype of acetylcholine agent. When applied to the bath, it should block the muscarinic receptor sites in the smooth muscle and therefore inhibit the action of acetylcholine.

1. Start with the bath filled with regular Tyrode's solution. Add 1 ml of 10 -3 M atropine. Wait approximately 1 minute. What
happens to the contractions? 2. Without draining the bath, add 1 ml of 10 -4 M acetylcholine. Is there a response? How does the response compare to the results obtained in part D? Segment 7: Effects of Oxygen Depletion Any living mammalian tissue needs oxygen for normal metabolism. Formulate a hypothesis for the effect of oxygen depletion on muscle rhythmicity and tonus.

1. Turn off the oxygen supply to the smooth muscle preparation. Note the effect over the next few minutes. 2. Restart the stream of bubbles and allow the preparation to stabilize before proceeding.
Do the results confirm your hypothesis?

3. Save the data.

Notes
To save recorded data, choose File menu > Save As > file type: BSL PRO files (*.ACQ) File name: (Enter Name) > Save button To erase all recorded data (make sure you have saved it first), and begin from Time 0, choose: MP30 menu > Setup Acquisition > Click on "Reset"

Printing your data

1. To print out the data for each group member: a. Click on the "horizontal autoscale" and then on "vertical autoscale" icons to put your entire data file on screen. b. Print your data on 4 pages with 3 graphs per page for optimal results.
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If you are using an inkjet printer, you will have to set printer preferences.

Clean-up
1. Remove the piece of intestine and place it in a plastic disposal bag. Do not cut the thread when removing the S-hook.
Save the S-hook and fish hook!

2. 3. 4. 5.

Rinse the muscle bath twice with distilled water. Turn off and rinse the aeration tube. Empty and rinse all beakers or flasks. Turn off all electrical equipment.

APPENDIX
GRAPH TEMPLATE SETTINGS Click here to open a PDF of the graph template file settings.

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