Solve Organ Bath Problem with Guinea-Pig Ileum & Dog Mast Cells

[MiniJo]

Homework Statement

A botanist friend of yours has returned from a trip to the rain forest. She brings with her a package of dried leaves which contains what native healers believe to be an antidote to severe itching. You have access to an organ bath set up in which you can set up an isolated guinea-pig ileum to record responses to added agonists as well as a dog mastocytoma-derived cell line that responds to stimuli with the release of histamine. How would you use these preparations to explore the potential mechanism underlying the putative therapeutic effects of the leaves?

Homework Equations

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The Attempt at a Solution

Basically, this question is asking for the possible histamine-related effects of the leaves, if there are any. So I identified different stages to the experiment so far.

1. Control experiment.
The ileum has histamine receptors, therefore it will contract if it binds with histamine. Knowing this, I put the ileum into the organ bath and added histamine. Ideally this will produce a contraction.

2. Addition of leaves.
The leaves are believed to have antihistamine properties. So in another organ bath, I added the ileum, histamine and the leaves. I'm not sure what the best order for these additions is. Anyway. If the ileum stops contracting, then the leaves are inhibiting the reception of histamine or it's degrading the histamine. If the ileum continues to contract, then I'm not sure what's happening... maybe the leaves inhibit synthesis or release of histamine. We'll test this later.

3. To test for potential receptor blockade on the ileum or histamine degradation, prepare an organ bath, and add the ileum, a known histamine agonist, and the leaves. If the ileum contracts, we know that the leaves are responsible for degradation of histamine (since the agonist was able to be received). If there is no contraction, then the leaves are blocking the receptors. (Note that when I say "leaves" I actually mean some property of the leaves).

4. To test inhibition of release, set up another organ bath, and add the dog mast cells and the ileum. First as a control experiment, let the two things sit in the bath, making sure there's no contraction. Then stimulate the mast cells (UV, chemical agent...etc), this would cause degranulation, allowing the ileum to contract. In another organ bath, add the dog mast cells, the ileum and the leaves. Stimulate the mast cells to degranulate. If the ileum does not contract, then the leaves are interfering with degranulation (release of histamine). If the ileum does contract, then the leaves are ineffective against histamine.

5. To test for the inhibition of synthesis of histamine, we can add dog mast cells and ileum to an organ bath, stimulate the cells to degranulate and measure the level/amount of contraction in the ileum. Then, rinse the bath and add the leaves and mast cells immediately after. (These mast cells should have a lot less histamine compared to the beginning since they've just degranulated). Let the bath sit for a period of time. Add the ileum, then stimulate the mast cells again and measure the level of contraction in this second time around. If the level of contraction in the ileum is significantly less in the 2nd trial than in the first, then synthesis of histamine is inhibited by the leaves.

Does this seem like a proper solution?
If you see any flaws or have extra suggestions, please do tell me. I greatly appreciate any help. =)

 

[Moonbear]

You appear to be well along the right track here.

2. Addition of leaves.
The leaves are believed to have antihistamine properties. So in another organ bath, I added the ileum, histamine and the leaves. I'm not sure what the best order for these additions is. Anyway. If the ileum stops contracting, then the leaves are inhibiting the reception of histamine or it's degrading the histamine. If the ileum continues to contract, then I'm not sure what's happening... maybe the leaves inhibit synthesis or release of histamine. We'll test this later.

The one thing you have done pretty much in common for each experiment is to put everything in your organ bath at once. You do have a control that tests response to histamine alone, but how do you know the leaves aren't having an effect that is independent of histamine? I think there's an experimental control missing. (Your addition of histamine to the ileum bath is really a methodological control, to make sure your organ prep is working right, not an experimental control to help you directly test your hypothesis.)