Based on the work of Hans Ussing (Danish) and Earnst Huf (German.) These
experiments rely upon the fact that frogs which live in freshwater have
a permeable skin through which many small molecules will easily pass, such
as respiratory gases, and water. Since their bodily fluids are hypertonic
to the surrounding water, water will tend to enter the animal by osmosis.
To avoid overfilling and subsequent bursting, the frog must produce large
amounts of a very dillute urine using the kidney. Even an efficient kidney
will not be able to recover all of the salts used in the production of the
urine so some salt will be lost to the environment as the water is excreted.
So, in order to replace this salt, the frog skin is equiped to pump sodium
chloride from the freshwater into the animal. It is this active transport
that we will examine.
Why are we interested in this at this stage of the course? It gives us a
way of modelling the processes involved in establishing resting potentials
in nerves.
Procedure
Double pith a frog. There are several ways to do this. Firmly grasping the
frog in one hand, quickly push a sharp metal probe through the skin from
the back of the neck up into the brain. move the pointer tip around in the
brain to destroy it. This is then a single pithed frog, one in which all
higher brain function has been destroyed. Then, you can take the pointer
and insert it into the spinal column and push it down until the legs extend
and then relax. This is then a double pithed frog, one in which both higher
brain function and reflex function is destroyed. The pointer should be long
enough to reach the end of the spinal column, and should be easy to insert.
If it is not easy, then the probe is probably outside the column.
Remove a large section of skin from the abdominal surface of the frog and
rinse it in normal frog Ringers solution. The skin section will not be large
enough to form a complete partition between the two halves of the Ussing
chamber. So, cut two pieces of parafilm to fit the chamber and cut holes
in the parafilm to expose the frog skin when sandwiched in the middle. Ensure
that there is enough overlap between the skin and the parafilm to allow
for a generous amount of vaseline to effect the seal. Coat the ends of the
Ussing chamber with a layer of vaseline, and clamp the two halves together,
either side of the layer of skin.. Clamp the two halves together with bulldog
clips which will effect a good seal. Put fresh normal frog Ringers at room
temperature in both halves of the chamber, and allow the system to equilibrate.
When filling the chambers, be very careful to fill each side at the same
rate. The same goes for emptying the chambers.
Put the two calomel (pH) electrodes into the same beaker of normal Ringers
solution, turn on the meter and zero the millivolt reading on the oscilloscope.
This is a dual beam instrument so you can use one beam to fix zero on the
screen, and the other to measure the deflection. You can adjust the sensitivity
of the Y-axis later once you know how much potential is present. Then push
the Calomel electrodes through the holes in the top of the Ussing chamber.
Keep the electrodes in Ringers at all times. Connect up the platinum conductivity
electrodes to the chamber (hold in place with a clamp stand) but do not
connect them to the ammeter just yet.
Part 1. Voltage
When the system has calmed down, note the voltage (potential) across the
skin. measure this for a period of several minutes. Does it increase? Adjust
the voltage sensitivity to be a good deflection of the trace. If there is
no potential, or very low, there may be a leak either through, or around
the skin. Check this to see that it is OK. If necessary, start again with
a fresh piece of skin. It may be possible to seal any small punctures in
the skin with some vaseline. Try this first before using another frog. Which
side of the the frog skin is positive?
Part 2. External circuit
Connect up the short circuiting platinum electrodes to the ammeter (the
electrode on the inside surface of the animal should be conected to the
negative terminal of the ammeter, which I think is the one marked R). Slowly
turn up the current being put into the circuit (using the potentiometer
knob). If you have the leads reversed, the millivolt meter reading will
increase as you increase the input. Swap the leads if this occurs. Adjust
the input until the oscilloscope (millivolt meter) reads zero, this may
be a bit tricky. The reading on the ammeter is a measure of the amount of
current (in microamperes) required to completely counter the work of the
frog skin, and therefore a measure of the amount of charge moved by the
skin. The external current also has the effect of reducing the potential
across the skin to zero. This will negate the effect of other ions such
as chloride and potassium, and allow a more accurate determination of the
movement of sodium by the pumps.
Turn the external current off for a few minutes, and then read the current
again. Repeat this several times until you get an average value.
Calculation of sodium flux
The sodium flux across the skin (in moles) = It/F
where I is the current in amperes (=coulombs per second), t is the time
in seconds, and F is the Faraday number (96500 coulombs.) So, as an example,
if the ammeter reads 50mAmp, then the flux of sodium is 5.18 x 10-10
M per second. Convert this to flux per unit area of skin.
What is the average sodium flux that you measure? Compare this to the other
group.
Part 3: The effect of changing the ionic concentration.
1. Repeat the experiments with chloride-free Ringers. What effect does this
have upon the voltage generated by the skin ( as determined in part 1.)
What does this tell us about the movement of chloride ions across the skin?
2. Put potassium-free Ringers on the outside of the skin, and normal Ringers
on the inside. What effect does this have? What about the reverse experiment
(K-free on inside)? What does this suggest about the role of potassium in
the pumping of sodium, and about the asymmetry of the skin?
3. Add Ouabain to the outside solution, and note the effect on the voltage.
Then add it to the inside, and note the effect. Where are the sodium pumps
located? Be very careful with the ouabain, wear gloves and avoid contact.
As this will block the action of sodium pumps, it will easily disrupt heart
pacemaker function in humans.
4. Repeat the above experiments using sodium-free Ringers. What effect does
the sodium concentration have upon the current needed to negate the potential?
Do this one last!
Material and methods for Lab one. FROG SKIN CONDUCTIVITY
Frogs (jumbo frogs from Carolina Biological)
Ringers solution
Low sodium Ringers solution
Sodium-free Ringers solution
Potassium-free Ringers solution
Chloride-free Ringers solution
Ouabain solution (Concentration?)
0.01M KCN
Batteries in the variable current apparatus. Three type 411 Eveready, 15V?
Calomel Electrods
Platinum electrodes (for shorting circuit)
Voltmeter/potentiometer apparatus
