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Bio 306 Molecular and Cellular Biology

By Kim Sims,2014-06-22 12:32
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Bio 306 Molecular and Cellular Biology

Bio 306: Molecular and Cellular Biology

    CELL SIGNALING WITH cAMP

I. Introduction

     Signaling between cells often involves the secretion of a ligand molecule from one cell that interacts with receptor molecules on the surface of a different cell. When that happens, a signaling pathway is activated within the responding cell that results in a change in cellular function. One important signaling pathway involves the generation of a “second messenger” molecule called cyclic adenosine monophosphate (cAMP). This was the first cellular signaling pathway to be described at the molecular level (Sutherland, Nobel Prize 1971)

     cAMP is synthesized by the action of an intramembrane enzyme called adenylyl cyclase. When certain ligands (such as epinephrine) bind to their receptors they activate a G-protein that subsequently activates adenylyl cyclase. cAMP concentrations increase which then functions to activate other cellular enzymes. As long as cAMP concentrations are elevated cell function will continue to be affected. Another cellular enzyme, phosphodiesterase, catalyzes the degradation of cAMP to AMP. This reaction lowers the level of cytoplasmic cAMP and reduces the response of the cell. The level of cell response depends on the balance of synthesis and degradation of cAMP.

     In addition to ligands that stimulate the production of cAMP, other ligands will cause a decrease in the production of cAMP. This could be due to an inhibition of adenylyl cyclase or stimulation of phosphodiesterase. A cellular response will depend on the interaction of stimulatory and inhibitory ligands.

     Chinese hamster ovary (CHO) cells are ovarian cells that have been isolated and cultured outside of the body (in-vitro). This is a common cell-line used in numerous scientific studies. We will be using this cell-line to study the dynamics of cAMP cell signaling. Acetylcholine is a ligand that will act on these cells causing a reduction in cAMP levels by activating an inhibitory G-protein thereby inhibiting adenylyl cyclase activity. By treating CHO-m2 cells with acetylcholine, atropine (an acetylcholine receptor antagonist), forskolin (an adenylyl cyclase activator) and/or IBMX (a phosphodiesterase inhibitor), we can look at cell signaling and the role cAMP plays in that process.

     We will measure cAMP levels using a technique called ELISA (enzyme-linked

    immunosorbent assay). The ELISA we will use is a competitive immunoassay. The wells of a

    microtiter plate are coated with antibodies to rabbit antibodies (goat anti-rabbit IgG). The wells are filled with cell sample containing cAMP, a cAMP molecule that has alkaline phosphatase covalently linked to it (cAMP-AP conjugate) and a rabbit antibody to cAMP (rabbit anti-cAMP). The rabbit anti-cAMP antibody will bind, in a competitive manner, with either the cAMP in the sample or the cAMP-AP conjugate. The goat anti-rabbit IgG on the well will bind to the rabbit antibody, holding it and its bound cAMP/cAMP-AP to the well. After incubation the excess reagents are washed away and substrate to alkaline phosphatase is added. The alkaline phosphatase reaction produces a yellow color. The yellow color generated is read on a microplate reader at 405 nm. Note that more cAMP present in the sample will cause less cAMP-AP to bind to the rabbit antibody (and, thus, to the well). This will result in less alkaline phosphatase reaction product. Therefore, the intensity of the yellow color is inversely proportional to the concentration of cAMP in the samples.

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    II. Cell Treatments

    A. Aspirate the media out of the cell culture wells using a micropipette.

    B. Add 200 l treatment media to the cells as described below and let incubate for 10

    minutes @RT. (ACh=acetylcholine, FK=forskolin, AT=atropine, IBMX=

    isomethylbutylxanthine)

    1. 1 mM ACh, 250 M FK

    2. 1 mM ACh, 250 M FK, 50 M IBMX

    3. 0.1 mM ACh, 250 M FK

    4. 0.1 mM ACh, 250 M FK, 50 M IBMX

    5. 0.01 mM ACh, 250 M FK

    6. 0.01 mM ACh, 250 M FK, 50 M IBMX

    7. 0.001 mM ACh, 250 M FK

    8. 0.001 mM ACh, 250 M FK, 50 M IBMX

    9. 250 M FK

    10. 250 M FK, 50 M IBMX

    11. media only

    12. media, 50 M IBMX

    13. 1 mM ACh, 250 M FK, 10 M AT

    14. 1 mM ACh, 250 M FK, 50 M IBMX, 10 M AT

    C. Aspirate the media and lyse the cells in 0.5 ml of 0.1M HCl for 10 minutes.

    D. Transfer the cell lysate to microcentrifuge tubes and centrifuge for 5 minutes. oC.) (Samples may be stored @-20

    III. ELISA

    A. Prepare cAMP standards

    1. Label 5 glass tubes S1-S5

    2. Pipet 900 l of 0.1M HCl into tube #S1

    3. Pipet 750 l of 0.1M HCl into tubes #S2-S5.

    dd 100 l of the 2000 pmol/ml cAMP standard to tube #S1. Vortex. 4. A

    5. Add 250 l of tube #S1 to tube #S2 and vortex thoroughly.

    6. Continue this for tubes #S3-S5. B. Plate Set Up: The following wells are needed. All wells are in duplicate. Use the

    plate layout sheet as a reference.

    1. Blank

    2. TA: total enzyme activity

    3. NSB: non-specific binding

    4. Bo: total binding

    5. cAMP standards

    a) S1=200 pmol/ml

    b) S2=50 pmol/ml

    c) S3=12.5 pmol/ml

    d) S4=3.125 pmol/ml

    e) S5=0.781 pmol/ml

    6. Cell culture samples 1-14

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    C. Procedure

    1. Pipet 50;;l of the pink Neutralizing Reagent into each well EXCEPT the TA

    and Blank wells.

    2. Pipet 100 l of 0.1M HCl into the NSB and the Bo (0pmol/mL standard)

    wells.

    3. Pipet 100 l standards #1-#5 into the appropriate wells.

    4. Pipet 100 l samples into the appropriate wells.

    5. Pipet 50;;l of 0.1M HCl into the NSB wells.

    6. Pipet 50;;l of blue Conjugate into each well EXCEPT the TA and Blank

    wells.

    7. Pipet 50 l of yellow antibody into each well EXCEPT the Blank, TA and

    NSB wells.

    NOTE: Every well used should be brown in color except the NSB wells which should be purple.

    The Blank and TA wells are empty at this point and have no color.

8. Incubate the plate at room temperature for 1 hours in the orbital shaker at

    300 rpm. Cover the wells with parafilm or the plate sealer sheet. 9. Empty the contents of the wells by inverting the plate and wash by adding 200

    l of wash solution to every well. Repeat the wash 2 more times for a total

    of 3 washes.

    10. After the final wash, empty or aspirate the wells, and firmly tap the plate on a

    kimwipe to remove any remaining wash buffer.

    11. Add 5 l of blue conjugate to the TA wells.

    12. Add 200 l of the p-Npp substrate solution to every well.

    13. Incubate at room temperature for 1 hour without shaking.

    14. Add 50;;l of stop solution to every well. This stops the reaction and the plate

    should be read immediately.

    15. Reading the plate:

    a) Turn on the plate reader 15 minutes before the reaction is finished.

    b) Place the 405 nm cube in the light path.

    c) “Blank” the plate reader on air. (The data will be corrected for the

    blank wells later during the calculations.)

    d) Push the plate holder to the left until it resists slightly.

    e) Hit “blank” on the keypad.

    f) The readout should be all zeros if this is successful. The plate should

    be ejected automatically when finished. Otherwise, press stop.

    g) Place your 96-well plate in the reader, push to the left to engage the

    reader, hit “Start” on the keypad.

     for each well. If you need 2 copies you h) The printout will report A405

    can read the plate again OR Xerox the printout.

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D. Calculations

    1. Create an Excel table including all raw data and calculations using the format

    below. Include TA, NSB, Bo, S1-S5 and cell culture samples.

    Average Specific Percent Concentration Sample ODs measured OD OD bound cAMP (pmol/ml)

    2. Calculate the average OD value for every duplicate value.

    3. Calculate the corrected NSB value: subtract the average blank OD value

    from the average NSB OD value. Report this on the table as a specific OD

    (even though it is not).

    4. Calculate the specific OD for Bo, S1-S5 and culture samples: Subtract the

    corrected NSB from the average OD for each.

    5. Calculate the percent bound using the following formula:

    [Specific OD/Specific Bo OD] x 100 = % Bound

    6. Plot % bound (y-axis) vs. cAMP concentration (x-axis) for the standards using

    Excel. Fit a line to this plot (should be logarithmic).

    7. Calculate cAMP concentrations in cell culture samples: Use the formula for

    the line to determine the concentration of cAMP in the cell culture samples.

    IV. Lab Report

    A. Include the data table and graph used to calculate cAMP concentrations. B. Create two bar graphs, one with samples 1 - 14 and one using the data from

    treatments 1, 3, 5, 7 and 13.

    C. What is the effect of forskolin on cAMP concentrations? Support your answer with

    experimental results. Explain its mechanism of action. Why was forskolin included

    in virtually all samples?

    D. What was the effect of acetylcholine on cAMP concentrations? Was there a

    concentration effect? Support with data. Explain its mechanism of action. E. What was the effect of IBMX on cAMP concentrations? Support with data. Explain

    its mechanism of action.

    F. What was the effect of atropine on cAMP concentrations? Support with data.

    Explain its mechanism of action.

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