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307 BISC Spring 2008 Frog Skin - A model for transport epithelia AIMS 1. To measure the voltage (potential difference) across a transport epithelium (the frog skin). 2. To investigate what causes this voltage and how it is maintained. 3. To investigate factors affecting active transport in this system. ZEROING THE VOLTMETER Insert the two calomel reference electrodes and the black Ag/AgCl electrode into a beaker of dilute Ringer's. (Under this circumstance, the voltage between the tips of the reference electrodes should be zero. ) Start the VI and when the voltmeter reading has stabilized (between 3 and -3 mV) click once on the voltmeter zero button. SETTING UP THE EXPERIMENTAL CHAMBER You will be provided with a 2 cm x 2 cm piece of skin taken from the abdomen (ventral side) of a frog. Gently place the skin between the two halves of the plexiglass chamber as illustrated in Fig. 1 and bind the two blocks together with one or more elastic bands. This is the equivalent of the ssing Chamber. Note which side of the skin is the serosal side (inside) and which side is the mucosal side (outside) - this is written on the blocks. Arrange the electrodes as in Fig. 1. Fill both chambers with normal Ringer's solution. While filling, try to keep the levels even on both sides. ADVICE: Keep the skin moist with Ringer's solution at all times and treat it very carefully. WARNING: Avoid excessive pressure when clamping the skin. This will likely damage the skin causing leakage. READING: Theory behind this lab is described in Eckert's Animal Physiology especially chapter 4 and pages 150151. A copy of a chapter from "Frog Neurobiology". Llinas and Precht eds is also available in the lab. WARNING: Only click the voltmeter zero button when both reference electrodes and the black current electrode are together in the same beaker. -1- BISC 307 Spring 2008 Aerate the Ringer's. screen): if you have a leak in the skin, you will not get a steady voltage. ADVICE: Keep the solution bathing the skin wellaerated using the bubbler. The rate of bubbling from the air tube only needs to be very slow. Check the voltage (on the computer Why do you have to aerate the frog skin preparation? Calomel electrode Bl ack Pin = reference Red Pi n = to current injection source Air in Calomel electrode (Red) = v oltage source Ag/AgCl electrode (Black) = current source Air in Mucosal Ski n Serosal Figure 1 -2- BISC 307 Spring 2008 MEASUREMENT OF SKIN POTENTIAL If the computer program is not already open (the screen should have knobs and dials marked voltage and current visible), then double click on the FROG SKIN icon located on the `desktop'. Something approximating Fig. 2 should appear on the computer screen. Plug the calomel electrodes into the black and red connectors of the amplifier/stimulator current injection module. THEORY: An electrically stable contact with the ionic solutions on either side of the skin can be obtained by using calomel reference electrodes. The electrodes can therefore be used to measure the potential difference between the two sides of the skin, with the voltage between electrodes (red vs black) being recorded in a number of ways. You will be using Macintosh computers and a program written especially for this exercise - to make things easy! Stop Watch 0.0 Current Control On Off sec Start Stop Reset Voltmeter Zero Figure 2 Immerse both red and black electrodes (one is attached to an Ag/AgCl electrode) in a beaker of Ringer's solution and note the voltage indicated by the "Voltmeter" on the computer screen. Use the mouse to click on the zero button of the voltmeter. This will compensate for differences in the electrodes and provide a zero reference to start. -200 mV Ammeter Zero -400 A THEORY: Since the ionic composition of the solution touching each of the electrodes is the same, the difference between electrodes should be zero. Any difference should only be a few mV or less and represents small differences in the composition of the two electrodes. This is `noise' in the experimental system. -3- BISC 307 Spring 2008 Transfer the electrodes to the ssing chamber, as shown in Fig. 1. ADVICE: Make sure the Ag/AgCl electrode attached to the calomel electrode is immersed in Ringer's but don't push it too far down (see Fig. 1). Given that the voltmeter displays the voltage of the electrode plugged into the red jack with respect to the reference jack (on either side of the frog skin), is the solution on the inside of the skin (serosal side) positive or negative with respect to the outside? Start the stopwatch and record the voltage at 1 min intervals for 10 min. If the voltage drifts, note the direction. After 10 min return the electrodes to the beaker of Ringer's and note the voltage and how much change there was, if any. After the electrodes have equilibrated, re-zero the voltmeter If the voltage across the skin drifted during the 10 min measurement period, was any of this drift due to changes in the electrode? MEASUREMENT OF IONIC CURRENT WITH VOLTAGE CLAMP Now, instead of measuring the voltage difference across the skin you will measure the current flowing across the skin when the skin voltage is 0. In the literature, this current is known as the "short circuit current" or SCC. Replace the calomel electrodes in the ssing Chamber so that they are again positioned across the frog skin. Ensure that the On/Off button of the Current Control is set to "OFF" and the "KNOB" is twisted all the way to the left (use the mouse to adjust the knob, click on the button to change from ON to OFF. ADVICE: For the following exercises you will want to have a record of the time it takes for solution changes as well as the time you spend recording the skin current. Just keep the clock running continuously for the rest of the lab period and record the actual elapsed time from the beginning of your current measurements. ADVICE: If the voltage increases, the Ag/AgCl current injection, your electrodes must be reversed. Switch them if necessary. -4- BISC 307 Spring 2008 Insert the Ag/AgCl electrodes into the chamber as shown in Fig. 1. Click the Current control to "ON". Slowly increase the current (using the mouse) and ensure that the mV reading of the Voltmeter approaches zero as the current is increased. Once you achieve 0 mV manually, switch on the software voltage clamp with its clamp voltage set to 0.0. The program will continually adjust current to maintain the clamp voltage. Record the clamp current for 5 min and log your recording. Turn off the clamp and check that the voltage difference between the calomel electrodes is still zero (or close to it) by placing them in the beaker of Ringer's. Remove the saline on each side of the ssing Chamber and replace it as follows: First, take out the current (Ag/AgCl) electrodes to provide access for the rinsing wash bottle and syringe. Gently draw off the Ringer with the syringe taking care not to touch the skin itself. Next, add a few ml of fresh normal Ringer to the mucosal side and Na-free ringer to the serosal side. Immediately draw this off and replace it. Repeat this rinse procedure 3 times before eventually filling each side. (Note In this case, you should wind up with Na-free Ringer on the serosal side and normal Ringer on the mucosal side. ) Measure the skin voltage briefly, with no external current, and then measure short circuit current as before. It will likely drift for a while. Give it a few minutes to stabilize and try to get 5 minutes at a stable level. -5- THEORY - VOLTAGE CLAMP: Since solutions are identical on both sides of the skin, any net current across the skin when the voltage across the skin is held at zero be must due to active transport by the skin epidermal cells. We don't measure this current directly. Instead, we apply an opposing current from a suitable "current generator" and measure the applied current necessary to clamp the skin voltage at zero. When the applied current = current generated by the skin, then the net current is zero. The Ag/AgCl electrode adds negative charge in the form of Cl- to the serosal side and removes it on the mucosal side. At the point where Clions added = Na+ ions transported, the skin voltage = 0 and the applied current (which you can measure with the Ammeter on the computer screen) equals the current flowing across the skin. (see Eckert pg. 140). WARNING: Be very careful when changing solutions in the ssing Chamber. Do not touch the skin with the syringe. ADVICE: A small amount of fluid remains in the chamber each time you empty it, so you need to rinse well and at least three times quickly otherwise there will still be some of the previous solution in the chamber. Estimate when the first measurement is made after rinsing is complete so as to be able to compare the time course of the effect of these solutions to the effect of metabolic inhibitors. BISC 307 Spring 2008 When the current seems reasonably stable quickly change to normal Ringer on both sides. You will need to rinse as before. Again get a skin voltage and a set of representative SCC measurements. Now try it the other way around with Nafree Ringer on the mucosal side and normal Ringer on the serosal side. Finally try it with Na-free Ringer on both sides. Go back to normal Ringer on both sides and obtain some baseline measurements. Let the skin acclimate for 5 to 10 minutes before continuing with the next step. QUESTIONS: On which side does the drop to Nafree Ringer have the greatest impact on the skin current? On the skin voltage? Why? ADVICE: Always ensure that the air is bubbling at approximately the same rate as it was before changing solutions. Don't have the bubbles going so fast that they cause the skin to flap around. WARNING: Don't leave the skin sitting in zero Na for too long. EFFECT OF ADH: Obtain baseline data by recording SCC vs time for 10 minutes with normal Ringer on both sides. Add 50 l of 0.1 mg/mL [Arg8]-vasotocin (AVT) to the serosal side of the skin. ADVICE: Unfortunately, there is usually a significant time lag before the added ADH takes effect and then the skin may take considerable time to stabilize. You can't really do much about this except wait for the current to stabilize. Record the SCC current until it stabilizes. EFFECT OF INHIBITORS: Repeat voltage clamp (as described above) in normal saline; this is very important as you require some baseline data before you add an inhibitor. Group 1 When you have a stable baseline in normal Ringer's, add 200 l of 0.15 M KCN (potassium cyanide) to the mucosal side and continue recording. After about 5 min, add a further 200 l of 0.15 M KCN to the serosal side. Record SCC until a reasonably stable result is observed. ADVICE: Each lab station will use a different inhibitor. You will need to know what the other groups did so you can include their data in your report. All groups will then share their data with each other. Do not leave the lab without getting data from the other groups. -6- BISC 307 Spring 2008 Group 2 When you have stable baseline, add 0.5 ml of 10 mM ouabain to the mucosal side of the Ussing Chamber and continue recording SCC for about 5 min. Now add 0.5 ml of 10 mM ouabain to the serosal side and continue recording until a stable level is achieved. Group 3 When you have a stable baseline, add 200 l of 0.10 mM amiloride to the serosal side of the ssing Chamber. Record SCC for 5 min and then add 200 l of 0.10 mM amiloride to the mucosal side. Record until a stable response is achieved. ALTERNATE EXERCISES The ion transport and water uptake capabilities of the skin are under hormonal control. Hormones circulating in the blood, as well as neuromodulators released by nerve endings within the skin, can increase or decrease the Na permeability, pump activity and water permeability. In this way the frog skin is a model for many other transport epithelia such as gut or bladder. Instead of treating the skin with a metabolic inhibitor, you may have the opportunity to observe the effect of cholinergic (acetylcholine), or adrenergic agonists (isoproterenol), or the amphibian equivalent of hormones such as ADH (antidiuretic hormone), aldosterone or progesterone. Your TA will inform you of your options at the beginning of the lab exercise. ANALYSIS AND DISCUSSION Start your report (the Introduction) with a brief description of the structure and various functions of frog skin. Compare it briefly to other transport epithelia (give a "textbook" level of treatment of this material, not a primary literature review. Use some drawings). Answer the following question and use the references below to get started towards writing a 'good' report. Most of the literature below is fairly old. If the effects of hormones were investigated, you should search the literature for more recent studies that are relevant to your observations. ADVICE: For your current measurements calculate (using the computer) the flow of ions across the skin in each solution from the equation: current ( A) ion flow ( mol / sec) = F where, F = Faraday's constant (96,490 coulombs/mole). -7- BISC 307 Spring 2008 Some Discussion Questions: 1. How does ion transport lead to a relatively stable potential across the skin when you have the same saline formulation on both sides? 2. What is the advantage of measuring current with no net voltage across the skin? How does this current provide a measure of net ion flux? 3. What is the main ion transported across the skin and which direction is it transported? Justify your choice from your experimental observations. What is the ion flux per cm2 of skin (given that the diameter of the circular patch of skin is 1.8 cm). 4. Why is such transport important to the frog? Can you suggest other systems in vertebrates that play the same role? 5. 6. 7. What powers the transport system? What does the response to KCN tell you about the transport mechanism? Plot current vs time after adding the KCN, ouabain and amiloride. What accounts for the differences in response time between amiloride and ouabain? Plot the time course of the effect of ADH. Considering the role of ADH in the vertebrate kidney, is its effect on frog skin that which you would expect? -8- BISC 307 Spring 2008 SOME APPROPRIATE REFERENCES Koefed-Johnsen, V. and ssing, H.H. (1958). The nature of the frog skin potential. Acta Physiol. Scand. 42: 298-308. Ussing, H.H. (1989) Epithelial Transport: Frog Skin as a Model System. In: Membrane Transport. People and Ideas. D.C. Tosteson editor. Waverly Press, Baltimore. pp 337-362. Nielsen, R. (1982). A coupled Na+-K+ pump for mediating transepithelial sodium transport in frog skin. In: Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.) 16: 87-108. Nielsen, R. (1982) Effect of Amiloride, Ouabain and Ba++ on the Nonsteady-State Na-K Pump Flux and Short Circuit Current in Isolated Frog Skin Epithelia. J. Membrane Biol. 65:227-234. Weber, W.M., Blank, U. and Clauss, W. (1995). Regulation of electrogenic Na+ transport across leech skin. American Journal of Physioliology 37R: 605-613. ssing, H.H. and Zerahn, K. (1951). Active transport of Na+ in frog skin. Acta physiol. Scand. 23: 110-127. Lindemann B and Voute C 1976 Structure and Function of the Epidermis. Neurobiology. R Ll nas and W Precht eds. Springer-Verlag New York pp 169-210 In: Frog -9-

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