Effects of ATP and Ions on Rabbit Psoas Muscle

• Qualitative -- students observe contractile tissue, cause it to react
• Quantitative -- students observe and measure contraction under various circumstances
• Hands-on -- students prepare and interact with the organism (or at least a part of it)
• Feasible -- Carolina Biological Supply kit #20-3525

•  Contraction is a biochemical process common to all animals
•  Contraction is needed for posture, locomotion, peristalsis, circulation, ventilation, and generation of heat
•  Contraction is a rapid, chained, cyclical process

1. 1-2 cc of glycerol solution is decanted into a Petri dish
2. 1-2 cm of psoas are cut and added to the dish
3. Glass teasing needles (easily made) are used to tease the psoas into strands of a few muscle cells
4.  Length of each strand is measured
5. Contraction and % contraction are measured/calculated, using Mineral Solution, ATP Solution, and ATP + Mineral Solution
6. Students write about outcome; interpret/explain

• Alter/adjust pH, temperature
• Alter balance/concentration of various chemicals

(Dr. Joseph Becker suggests that EDTA will precipitate the Ca⁺⁺ from solution)
• Solicit student modifications

The Effects of ATP and Ions on Rabbit Psoas Muscle Cells

Background:

1. In response to a chemical command from a nerve fiber, the muscle cell admits sodium (Na⁺) from the bloodstream, then releases Calcium (CA⁺⁺) and other ions from internal storage vesicles.
2. In the presence of these ions, an enzyme breaks down an energy storage chemical called ATP (adenosine triphosphate -- a phosphorilated nucleotide.)
3. When broken down, the ATP is used by a protein in the muscle cell (myosin).  The myosin shortens, pulling against another protein (actin), therefore shortening the entire muscle cell.  This shortening produces motion and releases heat.
4. The internally-released ions are pumped back into storage, more ATP is assembled in the mitochondria, and Na⁺  leaves the cell, permitting a repetition of the entire process.

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Objectives:

•  Students will prepare single strands of muscle cell, and examine muscle cells microscopically, identifying bands of darker and lighter proteins (actin and myosin);
• Using isolated muscle fibers, students will predict, observe, describe, and quantify the effects of ions by themselves, ATP by itself, and ions + ATP (in combination.)

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Materials/Supplies:

You will need the following:

• Petri Dish (1 per lab group)
• Microscope slides (at least 6 per lab group)
• glass teasing needles (2 per lab group)
• Dissecting microscopes (1 per lab group)
• Effects of ATP and Ions Kit
• (available from Carolina Biological Supply Company) (1 per 4-5 lab groups)
• Compound Microscopes (1 per lab group)

 

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Students' Procedures:

1. The rabbit psoas comes in a test tube filled with glycerol; it is tied to a wooden dowel. From the test tube, you or your instructor will pour off 1 or 2-CC of glycerol into each Petri dish.
2. Using scissors, you or your teacher will cut about 1-2 cm of muscle tissue free from the dowel for each lab group, then place this length into the glycerol in each of the Petri dishes.
3. Students then transfer their Petri dish to a dissecting microscope. Using this microscope, they should then CAREFULLY tease the muscle fibers apart with the glass needles until they have isolated at least 6 strands, only a few cells thick.
4. The muscle strands should be transferred to 6 flat microscope slides, then straightened to the best of the students’ ability. The length of each strand should then be measured and its length recorded on the provided chart.
5. Students then visualize the muscle fibers at 400x, drawing what they see, and identifying and labeling dark and light bands and all other characteristics possible.
6. In the space provided on the data chart, students should predict the results when ions, ATP, and the combination are added to each respective slide.  Then they actually add the chemicals while observing under low-dry magnification.  After this, they record their results, including change in each fiber length, average change in fiber length,  and average percent change in fiber length for each provided solution.
 

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Notes to the Instructor:

There are 2 potential problems with the materials provided in the Cabisco kit:

• Only 1 set of 3 dropper bottles is provided with each length of rabbit psoas. You can compensate for this by subdividing the chemicals into your own dropper bottles, one set at each lab station.
• Materials are shipped cold, and must be kept cold until the lab exercise is conducted.  Alert the necessary individuals that you must be notified promptly when materials arrive, and have some means at hand to keep them cold.

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Data / Basic Comprehension Questions:

Part A: Predict the effects of each solution on muscle cells.

ATP solution:
 

Ion Solution:
 

ATP + Ion Solution:
 

Part B:  Complete the charts.
 

	Strands 1 &2  (To be treated with Ions only)	Strands 3 & 4  (To be treated with ATP only)	Strands 5 & 6  (To be treated with ATP + Ions)
Length before adding chemical	|	|	|
Average Length
Observations

 
 
 

Length After Adding Chemical	|	|	|
Average Length After Adding Chemical
Average Change (Before Versus After)

 
Average Percent Change:
 

Other Observations:(For example, were your predictions accurate?)
 
 

 
 Questions:

1. Why were the teasing needles in this lab exercise made of glass rather than steel?

2. Which solution was most effective in causing the muscle cells to contract?  Why?

3. Explain why you would need calcium and other minerals in your diet, even if your skeleton contained no minerals at all.

4. Explain why each muscle cell contains many mitochondria.

5. Why does your upper arm become thicker when you contract your biceps muscle?

6. In this lab exercise, we can only cause the muscle cells to contract once.  After that, they will not respond to the chemicals any more.  Why is this true?

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