The isolated ileum represents a valuable tool for evaluating the actions of pharmacological agents that affect the autonomic nervous system.  In this series of exercises, you will use the ILEUM computer simulation program to obtain additional data and determine the site(s) of action of an unknown agonist or antagonist.  Before proceeding, you should refer to your lecture notes and review the structure, function and pharmacology of the ANS.  If you wish to examine the experimental set up and procedures, see the ANS Pharmacology laboratory exercise later in the BSCI 441 section of this manual.

Using the ILEUM Program

     Turn on the computer and the printer.  Select the "Ileum" program from the Pharmacology menu and follow the instructions.  Please remember to bring paper for the printer.

     The program will first ask you to select an agonist or antagonist as your unknown.  Call your trusty TA, and he/she will make the selection for you!  No peeking, please!

     Return to the keyboard and press "1" for color and then select the number of lines between data traces by entering "2".  Make sure that the printer is loaded with paper and is "on line".  NOTE:  At one point, you will be asked if the trace appears correct.  Answer that question with a "N" for "NO" and your data printout will be easier to read.

     You are now ready to apply various drugs to an isolated ileum preparation.  A complete list of available drugs and a brief description of their effects may be found at the end of this section.

Please make careful note of the following instructions:
 

• To add a drug, type the abbreviation of the drug as it appears in the menu on the screen followed by pressing the Enter key.  If you key in an incorrect code, use the backspace key to erase your entry and retype the correct code.  If you use an unrecognized code, the program will abort and you may have to begin again.

• After selecting the drug, you will be asked for the dose.  Bath concentrations of drugs to be applied to the tissue are expressed in molar terms and entered in scientific notation using "E" for exponent.  Thus to add 2*10^-8M, enter "2E-8" and press the Enter key.

• After adding a drug, the resulting contraction will be printed.  The data are expressed as cm of contraction height.  Note that the program has experimental variability built into the results, so that repeated administration of the same dose of a drug will yield slightly different results.  You may want to consider this in the course of your experiments.

• If you apply an antagonist, it will remain in the bath until you remove it by pressing "W" for wash.  An agonist is added as one single dose which is automatically removed from the bath before the application of the next dose/drug.

• You will be able to record 4 or 5 drug additions on one side of a sheet of paper.  To conserve paper, turn the sheet 180^o and get two data sets on one side of a page.  Then turn the page over and use the other side.

Experiments

1.     Obtain and plot dose/response curves for the following agents:

• ACh
• ACh + eserine (10^-7M)
• ACh + hexamethonium (10^-7M)
• ACh + atropine (10^-7M)
• Carbachol + eserine (10^-7M)
• Methacholine + eserine (10^-7M)

From your data, what can you infer about the above agents?  Summarize your findings (briefly) and turn them in with your D/R curves (which may all be plotted on one page of semilog graph paper!).  Include these D/R plots with the one you obtained from the "real" ileum last week.

2.     Determine the site of action of DMPP.  Present your rationale, data,
        and your conclusions.

3.     Demonstrate the absence of sympathetic ganglia in the preparation.
        Present a summary of your rationale, data, and conclusions.

4.     Determine the site of action and identity (if possible) of your unknown
        agent.  It may NOT be a drug from the list above!  Present your rationale,
        data summary, and conclusions.  When you terminate the program
        (by pressing "Q"), the computer will print a code letter for your unknown
        on your data page.  Look for this letter and ask your TA to verify the identity
        of the unknown drug.

5.      Determination of the Site of Action of an Unknown Drug

    Design experiments to determine the site of action of an unknown pharmacologically active agent.  Be as specific as possible.  This task is accomplished by first constructing a flow chart of your experimental design.  For example, your unknown agent will elicit one of three possible responses from the ileum preparation:  contraction, relaxation, or no response.  What are the possible types of drugs used in the previous experiments above which could give you each of those responses?  How can you then distinguish among the possibilities?

    If you are able to construct and understand this flow chart, you have an excellent grasp of the pharmacology of the autonomic nervous system.