PNB Spring Lab Prep - Glucose Transport

What will be used from last week’s lab?

standard curve

What will you calculate in today’s lab?

unknown glucose concentration in the intestinal sacs you make today

What can happen to the intestine?

easily damaged; handle as little as possible, check for oxygenation

What prevents leakage of the sacs?

knots need to be tight (tie double knots)

What must be left to handle sacs?

5 cm of thread

What should be the size of the sac?

up to 2-3 cm

What sacs should be avoided?

if they have an air bubble inside

Where should intestines be disposed?

waste bag

What must you NOT do when cleaning up?

cut the feeder tube or dispose of small vials

What should not be mixed?

DNS and DNP

What is toxic?

DNS and DNP; wear gloves, dispose in separate waste containers

When must one predict the changes in glucose concentration?

if the intestine was NOT everted

What is the transport mechanism of fructose?

facilitated diffusion

What is the orientation of the intestine after sac eversion?

The mucosal side is now on the outside, and the serosal side is now on the inside of the sac

During the experiment, the initial concentration of glucose inside the sac is:

15 mM

What is the initial concentration of glucose outside the sac:

15 mM

In the control condition, after incubation, glucose in the sac will ______ in comparison to intitial concentrations of glucose. As a result, glucose concentrations in the sac will be _______ 15 mM?

increase; higher than

Are proteins digested by saliva?

false

What produces pancreatic juice?

pancreatic acini

What organs in the digestive system contain acini?

pancreas and salivary glands

Where should the tissue be kept?

in oxygenated Ringer’s at all times

What is the first step of the intestinal sac procedure?

place beaker 1 in the water bath at 37 C. Place beaker 2 in the ice bucket (if assigned cold ringers) or the 37 C water bath (if assigned DNP or low sodium)

What is the second step of the intestinal sac procedure?

Place an aerator into each beaker. Make sure each beaker is oxygenated (necessary for metabolism as well as stirring). Consult the TA if you encounter difficulties

What is the third step of the intestinal sac procedure?

You will be given a syringe with an attached plastic feeding tube. Fill the syringe with Ringer’s solution and 15mM glucose

What is the fourth step of the intestinal sac procedure?

Gently slip the plastic feeding tubing through the entire length of intestine. Note the dark line near the bottom of the feeding tube

What is the fifth step of the intestinal sac procedure?

Position the intestine so that the end of the intestine is about 5 mm below the dark line

What is the sixth step of the intestinal sac procedure?

securely tie the lower end of the intestine onto the tubing, at or slightly above the dark line, and 2 holes at the bottom of the feeding tube. Trim away any excess thread

What is the seventh step of the intestinal sac procedure?

securely tie a knot at the open end of the intestine

What is the eighth step of the intestinal sac procedure?

using the previously filled syringe (containing Ringer’s + glucose), exert light pressure on the syringe to fill the intestine with the solution. The filled intestine should resemble a sausage. Leakage and holes are common, therefore you must try to tie the sacs where there are no holes (no solution can be seen leaking out of the sac)

What is the ninth step of the intestinal sac procedure?

Securely tie off the intestine approximately 2 cm (3/4 inches) above the know that closed off the open end of the intestine (the end durthest from the feeding tube), to create one fluid filled sac about ½ - ¾ long. The sac should be firm and well filled. Leave about 5 cm (2 in.) of thread at each end of the sac to grab and drape over the edge of the incubation beakers. Tie another ligature about 0.5 cm above this knowt. This will be the first tie of the next sac

What is the tenth step of the intestinal sac procedure?

Cut the intestine between these two ligatures. You now have one prepared intestinal sac. Immediately place the sac into one of the test solution beakers (normal, cold, + DNP, or low Na+)

What is the eleventh step of the intestinal sac procedure?

repeat steps 10-11 to make enough sacs for the experiment (2 sacs in total). When done you should have 2 non-leaking sacs, each filled with Ringer’s + 15 mM glucose solution. Place 1 sac in each beaker. If you are able to make 1 or 2 extra sacs, please do and share with another group

How long should the sacs be incubated?

for 45 minutes

How every so often should the sacs be checked on?

every 5 minutes; make sure the sacs are not pressed against the walls of the beakers, and that they are oxygenated and covered with solution

What solution will be used to fill the sacs?

Mammalian Ringer’s + 15 mM glucose

What experimental conditions will be tested?

cold, DNP (lack of ATP), or low Na+

How does glucose transport occur?

from the mucosal to the serosal side

What is the expected result of the initial sample (15 mM glucose)?

15 mM

What is the expected result of Sac #1 (37 C) normal?

greater than 15 mM

What is optimally transported into the sac?

glucose

What is the expected result of Sac #2 (4 C)?

less than normal sac, but still greater than 15 mM

What is the result of colder temperatures?

slow down the chemical reactions required to transport glucose

What is the expected result of Sac #3 (37 C + DNP)?

less than normal sac, but still greater than 15 mM

What does DNP prevent?

production of ATP, leads to less primary active transport of glucose

What is the expected result of Sac #4 (37 C + low Na+)?

less than normal sac but still greater than 15 mM

What is the result of lower Na+?

less secondary active transport of glucose