Persitaltismo

7/29/2019 Persitaltismo

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De: http://www.biopac.com/bslprolessons/a05/a05.htm

BIOPAC Systems, Inc., 42 Aero Camino, Goleta, CA 93117Ph (805) 685-0066, Fax (805) 685-0067, Web: www.biopac.com

BSL PROLesson A052002 BIOPAC Systems, Inc. Updated 12-22-03

PHYSIOLOGICAL PROPERTIES OF VISCERAL SMOOTH MUSCLE

Overview

Dr. Katja HoehnMt. Royal CollegeChemical Biological and Environmental Sciences

Calgary, AB, CANADA

There are three types of muscle tissue in the human body, each specialized for certain tasksin maintaining homeostasis.

Skeletal muscle, generally under voluntary control, is so named because it is in mostcases attached to the skeleton. Contraction of skeletal muscle moves parts of thebody with respect to one another or moves the entire body.

Cardiac muscle is the muscle found only in the heart, where it functions to circulatethe blood in order to deliver nutrients to cells and remove wastes from cellsthroughout 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 andrespiratory system and in the walls of all but the smallest of blood vessels.Contraction of smooth muscle controls movement through these organs and bloodvessels as well as the pressure within them.

Smooth muscle is characterized histologically by small spindle-shaped cells with a centralnucleus. 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, whichmake 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 muscleand smooth muscle.

In both types of muscle, contraction is achieved through the interaction of actin and

myosin via the sliding filament mechanism.


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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 couplingin smooth muscle versus excitation contraction-coupling in skeletal muscle.

In smooth muscle the sarcoplasmic reticulum is poorly developed and there are no t-tubules.

On depolarization of the smooth muscle membrane, cytosolic Ca 2+ rises due to botha release of some Ca 2+ from the sarcoplasmic reticulum as well as the entry of Ca2+ into the cell from the extracellular fluid.

The cytosolic Ca 2+ does not bind troponin as it does in skeletal muscle (smoothmuscle does not contain troponin), but instead interacts with a regulatory molecule,calmodulin. The Ca 2+ calmodulin complex then binds to and activates a moleculecalled myosin kinase (or myosin light chain kinase), which catalyzes the transfer of aphosphate from ATP to myosin. This phosphorylated myosin can then interact withactin of the thin filaments and that initiates the cross-bridge cycling. Relaxation comesabout when the cytosolic Ca 2+ concentration returns to precontraction levels and the

myosin is dephosphorylated (the phosphate group is removed.) Ca 2+ in the cytosolis actively pumped back into the sarcoplasmic reticulum and back across the plasmamembrane.

Smooth muscle can be classified into two major categories: multi-unit smooth muscle andsingle-unit (or visceral) smooth muscle.

Mulit-unit smooth muscle is made up of individual muscle fibers, which can operateindependently, are not connected by gap junctions, and tend not to be spontaneouslyactive. Multi-unit smooth muscle is found only in a few locations in the body. Thisincludes the internal eye muscles that adjust the focus and the pupil size, the smoothmuscle of the large airways and large arteries, and the erector pili muscles attachedto 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 musclefibers that are structurally independent of each other, it consists of several motor unitsthat can contract independently, and its contraction is initiated by a stimulus from anerve (i.e., it is neurogenic, initiated by nerves). However, the nerves that stimulatecontraction in multi-unit smooth muscle are autonomic nerves (as they are in visceralsmooth 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 thetype of muscle we will be studying in todays lab and we will focus on this type ofsmooth muscle for the rest of this introduction. Visceral smooth muscle is made up ofmuscle cells interconnected by gap junctions, which provide electrical couplingbetween cells. Thus, an action potential generated in one muscle cell can easilyspread to adjacent cells, allowing the cells to contract as a single unit.

Some of the fibers in visceral smooth muscle can undergo spontaneous depolarization andtherefore act as pacemaker cells, which set the contractile pace for the unit. The pattern ofvery slow swings of depolarization and then hyperpolarization seen in visceral smooth muscleis known as the slow-wave potential or basic electrical rhythm. It is probably due to a slowleak of Na + into the cell, and the waxing and waning of the outwardly-directed Na + pump.

If the depolarizing swings are large enough to reach threshold, then a burst of actionpotentials are generated for the period of time during which the potential is above thethreshold level.

If the depolarizing swings do not reach threshold, then the depolarizing andhyperpolarizing swings of action potential will occur without any action potentials andtherefore without any contractions. (See Fig. 8-32 and p. 274 in Sherwood 4th Editionfor further description).

Contraction in this type of smooth muscle does not depend on nerves for its initiation and itthus called myogenic (initiated by the muscle itself). Although nerves are not required to


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initiate contraction, nerves, (as well as hormones and a number of other factors which we willconsider below) can greatly modifythe rate and strength of contractions in this type ofmuscle.

For example, the strength of contractions can be altered by changing the startingpoint 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 somaticnervous system, which always excites skeletal muscle and always does so by releasing thesame neurotransmitter (acetylcholine), the autonomic nervous system can either excite orinhibit smooth muscle. The response depends on the type of neurotransmitter released by theautonomic nervous system and the subtypes of receptors that are present on the tissue. Ingeneral, the parasympathetic nervous system releases acetylcholine from postganglionicnerves onto the smooth muscle of the intestines.

Acetylcholine is an excitatory neurotransmitter in the gut, acting upon the muscarinic subtypeof receptors in the cell membrane. The sympathetic nervous system releases norepinephrinefrom postganglionic nerves onto the smooth muscle of the gut. Norepinephrine is an inhibitoryneurotransmitter in the gut. There are two adrenergic receptor subtypes that may respond tonorepinephrine, alpha receptors, and beta receptors. Both receptor subtypes are found invisceral smooth muscle and both have the same effect, namely the reduction of smoothmuscle rhythmicity and tonus.

Another difference between somatic nervous system innervation of skeletal muscle andautonomic nervous system innervation of visceral smooth muscle is that smooth muscle lacksthe highly structured one-to-one neuromuscular junctions found in skeletal muscle. Theterminal branches of autonomic fibers contain numerous swellings, called varicosities, andneurotransmitter is released from these varicosities into the interstitial fluid surrounding themuscle cells. The transmitter substance may have to diffuse a few micrometers to reach thenearest 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 celland each muscle cell may be influenced by more than one type of neurotransmitter. Usuallyonly the outermost layer of cells in innervated. The rest rely on conduction of electric currentfrom cell to cell via gap junctions.

Smooth muscle contraction has a number of special features. It is slow, sustained andresistant to fatigue:

It takes smooth muscle about 30 times as long to contract and relax as skeletalmuscle (up to 3000 msec (3 sec) for a smooth muscle contraction versus about 100msec 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 timeneeded for relaxation.

In spite of its slowness, smooth muscle can generate about the same tension ofcontraction per unit of cross-sectional area and can maintain that contraction at afraction of the energy cost of skeletal muscle.

o This energy efficiency is in part due to something called the latchphenomenon. It is thought that smooth muscle myofilaments may locktogether during prolonged contraction, partly as a result of the much slowercross-bridge cycling.

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

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


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volumes.

The strength of a smooth muscle contraction is much less sensitive to a change in the initiallength of the muscle than is the strength of a skeletal muscle contraction. The ability ofskeletal muscle to contract decreases markedly when it is stretched beyond its optimum initiallength. 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 overlappingarrangement of filaments. As a result, for example, the urinary bladder can still emptyefficiently 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 tothe inward flux of Ca 2+ through voltage-gated Ca 2+ channels. Thus, extracellular calciumplays a major role both in the development of the action potential and in the contractionprocess. Changing the Ca 2+ concentration of the extracellular fluid will have an effect oncontractions of visceral smooth muscle.

For comparative characteristics of muscle fibers--skeletal, singleunit smooth, multiunit smooth, and cardiac--see HumanPhysiology, 8e, Vander et al., p.331, Table 11-6.

About this lesson

In the following experiments, you will study some of the physiological and pharmacologicalproperties of visceral smooth muscle taken from the rabbit ileum. (The ileum is the last part ofthe small intestine). You will vary the Ca 2+ concentration, and the temperature and oxygencontent of the medium surrounding the muscle. In addition, you will investigate the effects ofacetylcholine, atropine and norepinephrine on the muscle tissue.

You will observe the recordings before and after the various treatments and look for andmeasure changes in the rhythmicity and in the tonus of the smooth muscle contractions. TheBiopac Student Lab PRO will be used to record the contractions.

Rhythmicity refers to the pattern of the muscle contractions. In particular, you will belooking 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. Youwill observe and make note of changes in any of these aspects of rhythmicity. If theinterval between contractions is irregular or the amplitude of the contractions variesconsiderably, 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 refersto the amount of tension continuously generated by the muscle. Usually there are asmall percentage of muscle fibers that are in a constant state of contraction while themajority of the fibers undergo rhythmic contraction and relaxation. An increase in thepercentage of fibers in a state of continuous contraction results in increased muscletonus; a decrease results in a reduction of tonus. Shifts in the baseline position of therecording on the y-axis indicate changes in tonus.

IMPORTANT NOTE!

Wash your hands, the Petri dish and dissecting instruments, and rinse themuscle 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 pharmacologicalagents on the contraction of the visceral smooth muscle of the rabbit ileum.

2. In particular, the student should be able to:


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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 contrastit 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 ofthese characteristics in a chart recording.

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

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

g. describe how an increase in KCl in the extracellular fluid surrounding smooth musclecells causes depolarization.

h. describe the effects of norepinephrine, acetylcholine, and atropine on smooth musclecontractions.

Equipment

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:

o BIOPAC tissue bath (TISSUEBATH1)o BIOPAC water circulator (CIRCULATORA orCIRCULATORB)o Knife for cutting the ileum into 4 pieceso Forceps for handling the ileumo Petri dish for use during mounting of the intestinal segmento 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)o Fish hooko Needle clamp for holding the aeration tube in the muscle batho Beakers for changing solutionso Syringe (50 ml)o Solutions:

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


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le bath consists of a large glass tube surrounded by a jar, which serves as a water

connects to the bath and maintains the temperature at 37C.

ottom of the bath is a drain for the inner muscle bath.

into the bath and can be adjusted via the valve on the gas line.

ached at one end to a tissue bearer (which is inserted into the bath to anchor thee other end to the transducer via an S-hook at the 50-g ring.

le Prep

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

egment of intestine into a Petri dish filled with fresh Tyrode's solution.

the aeration tube from the smooth muscle bath. Attach one end of the intestine to theliding the S-shaped syringe needle through the wall of the intestine. Attach the other


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muscle to the fishhook suspended by thread from the transducer.

uscle bath with fresh warm Tyrode's solution from the large water bath. Carefullypreparation. Slide the upper end of the aeration tube into the clamp and attach the

ake sure the gas (95% O2 and 5% CO2) is bubbling by the tissue. Adjust the screwthat the flow of gas results in a small, steady stream of bubbles. Turn the heating

tension on the thread attaching the muscle to the transducer so there is no slack inm.

e temperature of the inner and outer baths. It should be between 34C and 37C.minutes in the warm Tyrode's, the gut should start to undergo spontaneous

ns. If the temperature begins to rise above this range, turn off the heating lamp. If ite the light closer and replace the outer bath with warmer Tyrode's solution.

e BSL PRO software on the host computer.he program should create a new "Untitled1" window.Smooth Muscle Template by choosing File > Open > choose Files of type: Graph(*GTL) > File name: "a05.gtl"

he template will establish the required settings.the desired file name.

generated by the rabbit ileum will be very small, we will use a small weight(about 5libration, putting it onto the transducer attachment labeled "50 g."

Cal1 using no weight at all (with only the s-hook hanging off the ring of ther).eight forCal2. (A rough estimate of the forces will suffice for this exercise, as we areprimarily in changes in rhythmicity and tonus in our recordings and not in absolute

e values for Cal1 and Cal2 below. This will help you in the unlikely event that yourhould crash. You would then not have to unhook the gut from the transducer to redo

ion, but could type in the calibration values that you recorded here.

Cal1, Input Value = _________mV Scale Value for Cal1 was 0l2, Input Value = _________mV Scale Value for Cal2 was ____grams


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Analysis

Hints for minimizing measurement error:

A. Always allow the muscle to recover to normal betweenexperimental protocols.

B. Record continuously! Stop your recording only to save yourdata.

C. Save after each manipulation.

D. Always record some normal contractions before and after eachexperimental manipulation before you stop recording momentarily tosave your file.

E. Insert a marker each time you begin a new experiment, changesolutions, add a drug or perform a manipulation (press the F9 key on aPC or the Esc key on a Mac) and write down what you have done in themarker label bar.

F. After recording the normal contractions, perform each of themanipulations listed below in the order given.

line

Baseline rhythmicity and tonus

muscle has stabilized, record several contractions showing the normal rhythmicity

.

have to adjust the position of the transducer to get a proper recording. Ask the

for help with this adjustment if necessary.


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cts of Extracellular Calcium

Effect of Calcium-free Tyrode's Solution

Effect of 2% Calcium chloride drops

now about the importance of the extracellular calcium concentration to smoothns, what hypothesis would you make about the effect, on the tonus and rhythmicityreplacing the regular Tyrode's solution in the extracellular fluid with calcium-free


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he regular Tyrode's solution with Ca 2+ -free Tyrode's solution. Record the responsescle over the next few minutes.

drops of 2% CaCl2. Note the effect.

nfirm your hypothesis?

he Tyrode's solution in the bath with fresh normal Tyrode's solution and allow thens to stabilize before proceeding to part B.

cts of Chemical Depolarization

Effects of KCI

mbrane potential oscillates above and below threshold, thus generating the pattern ofn. Sustained depolarization can be induced by adding KCl to the bath.

of 1 M KCl to the bath.

KCl to remain in the bath for about 1 minute, then replace with regular Tyrode's

ct did this have on rhythmicity? How does excess K + in the extracellular fluid causeation? What event is triggered at the cell membrane, which results in initiation of then process?

preparation to recover, and then replace the regular Tyrode's with calcium-free

he contraction to diminish so that the preparation appears to be dormant, then addml if necessary) of 1M KCl solution and observe the response.The muscle cells areed by the addition of KCl, just as they were in steps 1 and 2, but does theation result in contraction? Explain.

he bath with regular Tyrode's and allow the rhythmicity and contraction amplitude tonormal before proceeding to Segment 4C .

cts of Change in Temperature


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Effects of Change in Temperature

tem is affected by changes in temperature, not only due to the effects of temperatureon, but also because of the effect of temperature on enzyme activity. Formulate aing the effect of cooling on muscle rhythmicity and tonus.

he warm Tyrode's with room-temperature (21C) Tyrode's.

he "Heater" switch on the water circulator and note the effect as the temperaturer the next few minutes.

he "Heater" switch on the water circulator, replace the bath solution with Tyrode's,until the preparation has stabilized at 37C before proceeding.

cts of Norepinephrine and Acetylcholine


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Effects of Norepinephrine

Effects of Acetylcholine

epinephrine and acetylcholine will be tested in this part of the experiment. What doill be the effects of these two drugs?

ml of 10 -4 M norepinephrine to the muscle bath (bath concentration will be roughly). Observe the response over the next minute. Drain and refill the bath with fresh


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contractions are stabilized, add 1.0 ml of 10 -4 M acetylcholine to the muscle bathcentration will be approximately 2 x 10 -6 ).

refill the bath. Allow the preparation to stabilize before proceeding.

cts of Atropine

Effects of Atropine

on two subtypes of acetylcholine receptors called muscarinic and nicotinic receptorsable 7-4 in Sherwood text, 4th Edition). Nicotinic receptors are found in autonomicl muscle and in the central nervous system. Muscarinic receptors are found on

mooth muscle and glands, as well as in the CNS. Atropine blocks the muscarinicholine agent. When applied to the bath, it should block the muscarinic receptor sites

scle and therefore inhibit the action of acetylcholine.

the bath filled with regular Tyrode's solution. Add 1 ml of 10 -3 M atropine. Waitately 1 minute. What happens to the contractions?

draining the bath, add 1 ml of 10 -4 M acetylcholine. Is there a response? How doesnse compare to the results obtained in part D?

cts of Oxygen Depletion

alian tissue needs oxygen for normal metabolism. Formulate a hypothesis for theepletion on muscle rhythmicity and tonus.

he oxygen supply to the smooth muscle preparation. Note the effect over the nexttes.

e stream of bubbles and allow the preparation to stabilize before proceeding.

nfirm your hypothesis?

data.


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Notes

To save recorded data, choose File menu > Save As > file type: BSL PROfiles

(*.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 putyour entire data file on screen.

b. Print your data on 4 pages with 3 graphs per page for optimal results.

If you are using an inkjet printer, you will have to set printer preferences.

Clean-up1. 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. Rinse the muscle bath twice with distilled water.

3. Turn off and rinse the aeration tube.

4. Empty and rinse all beakers or flasks.

5. Turn off all electrical equipment.

APPENDIXGRAPH TEMPLATE SETTINGS

Click here to open a PDF of the graph template file settings.

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