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LAB 19 - Active Transport of Na by the Isolated Frog Skin


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Osmoregulation in animals requires the functioning of transport epithelia which can move ions by active transport against concentration and electrical gradients. Active transport requires the expenditure of energy to transport the molecule from one side of the membrane to the other, but active transport is the only type of transport that can actually take molecules up their concentration gradient as well as down. In amphibians, skin is a major osmoregulatory organ. It has to transport Na and Cl from fresh water where their concentrations are about 0.1 mM into the ECF of the frog where concentrations are about 110 mM. The purpose of this lab was to use an Ussing chamber to clamp frog skin between two solutions.

frog.gif (1551 bytes) Clamp your frog skin into the chamber and fill both sides with frog ECF (Ringer's) - 5 ml/side.

frog.gif (1551 bytes) Place the voltage sensing electrodes (pH reference electrodes) across the skin, and the current electrodes (skinny little silver wires) across the skin. With the current generator off, you measure the voltage which develops across the skin.

frog.gif (1551 bytes) Turn the short circuit current on and adjust it to make the trans skin voltageread 0 mV

frog.gif (1551 bytes) You then read the number of microamps of current required to do this (from the multimeter). It turns out that 1 microamp of current = 63 * 10 -11 moles of Na transported/min. This lets you get a firm count on just how much Na the skin can pick up for the frog from fresh water in a minute. All you have to know is the area of the skin clamped in your chamber and the total skin area of the frog.

frog.gif (1551 bytes) After doing this for identical solutions of the two sides of the skin, you repeat the measurement for the cases when the dilutions of frog saline are placed outside the skin. Michaelis/Menten kinetics are at work here, the idea is that the rate of Na pumping has to do with the density of Na pumps in the skin. If there is an excess of Na outside the skin, the pumps will be saturated and pumping will be at a maximum.

Cross section through a frog skin

Setup and Equipment

Ussing chamber was used to clamp frog skin between two solutions. One ,facing the inside, will be frog physiological saline ([NaCl] = 110 mM, [K] = 5 mM, pH 7.4). On the other side. the green side, we placed a series of solutions (50 % frog saline, 10 % frog saline, 1 % frog saline)