Active Transport of Sodium
Paul R. Quinney and Howard A. Swartz' Butler University
Ions across Frog Skin
Indianapolis, Indiana
A demonstration experiment
I
he transport and transfer of materials across the cell membrane in living tissue is an important factor in cellular activity and function. The chemical reactions occurring within a cell are dependent upon a constant and regulated supply of reactants. The reactants must be transferred from the extracellular fluids to the intracellular fluids for utilization. Rut if the cell membrane were completely permeable to all materials, no regulation of transfer would be possible, and excessive quantities of reactants could enter into intracellular fluid. Thus the mechanisms by which a College of Pharmacy, Butler University; to whom correspondence should be directed.
Volume 41, Number 3, Morch 1964
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given material may pass through the cell membrane, either to enter or leave the intracellular fluid, assumes major importance. Any substance that may alter or effect this process will have a potent effect on cell biochemistry and physiology. The mechanisms by which materials may cross the cell membrane vary with the particle size of the material, pore size of the membrane, electrical charge of the material and the cell membrane electrical potential. Water may cross the cell membrane by osmosis, and some materials by diffusion; but the more important mechanism is active transport. This process involves enzymes and a carrier and allows for transfer across the cell membrane
Table 1.
Results of Sodium Ion Detection
Elapsed time (min) 5 15
1
Tube rontenta
+
1. 5 ml 0.9% NnCI 0% total activity (counts/min) N e t ion transoorted (millimoles) 2. 5 ml0.9% ~ a ~ i ( onj n o total activity (counts/min) Na+ ion transported (millimoles) 3. 5 ml0.9% NaCl + 0% KCN total activity (counts/min) N s + ion transported (millimoles) 4. 5 ml0.7%NaCI 0 2 total act~vlty(caunts/min) Ntl+ ion transoarted (millimoles) + 5. 5ml l . l % ~ a ~ I 'On total activity (counts/min) Na+ ion transported (millimolea)
30
600 0.026
4070 0.17
4850 0.21
4800 0.20
570 0.024
3715 0.16
4770 0.20
4900 0.21
+
+
in the presence of concentration gradiations. The process is illustrated below, where A represents the reactant material, C the carrier, and E the enzyme: Extracellular Fluid
Cell Membrane
Intramllular Fluid
Many materiak are actively transported across the cell membrane, including glucose and sodium ions. The sodium ions of the intracellular and extracellular fluid are associated with the cell membrane potential, and any change in the resting cell sodium ion distribution results in a detectable bioelectric current. Hence, movement of sodium ions across the cell membrane can be detected by electronic instr~ments,~ but the procedure is susceptible to wide fluctuations and error. The amount of ions involved are a t millimole levels; thus the determination by chemical methods is not feasible. A more specific and sensitive method, described in this paper, utilizes radioactive sodium ions and a suitable radiation detector and scaler. Frog s k i is employed as the membrane, as it is convenient and finds wide application in similar s t u d i e ~ . ~ The Experiment
One hundred milliliters of 0.9, 0.7, and 1.1% sodium chloride solutions were prepared. One microcurie of 22iValabeled sodium chloride was added to 60 ml of the 0.9% solution. Five samples of abdominal skin from double pithed frogs were tied securely to the ends of 1-in. plastic tubes. To three tubes, 5 ml of the non-labeled 0.9% NaCl was added. To one of these a few milligrams of KCN was added and agitated to dissolve. To the fourth tube, 5 ml of 0.7% solution *TUTTLE, W. W., AND SCEO'ITELIUS, B. A,, "Phy~iologyLaboratorv Msnusl." C. V. Mosbv Comoanv, . .. Saint Louis, Mo., 1963, p. 20. a L w , R. E., C H R I ~ ~ J. AN E.,, J . Am. Pharm. Assoe., Sci. Ed., 40,160(1951).
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was added; and to the fifth, 5 ml of 1.1% solution. To each of five 50-ml beakers, 10 ml of the labeled NaCl solution was added, and the tubes and their contents were placed in the beakers so that the fluid levels were equal (see Fig. 1). Oxygen a t 2-3 bubbles per second was introduced to both tubes and beakers (omitting in one of the 0.9% sodium chloride solutions). At intervals of 1, 5, 15, and 30 min, 1-ml samples were obtained from each tube, added to planchets and dried under an infrared lamp. The volumes were replaced with the correct nonlabeled NaCl solution. The activity was measured with a G-M detector and associated scaler. Constant geometry was maintained, and all samples were corrected for background. In the same way actiyity of a 1-ml sample of labeled NaCl solution was determined.
Frog Skin
Figure 1.
The activity in cpm/millimole of the IabeledNaCl solution was 23,469, calculated from the net coiTnts minute and millimole concentration/millilitei~o f the solution. The rate of transport of sodium ions in millimoles across the frog skm was calculated from the observed total volume activity in each tube (see Table 1). The results indicate that a lack of oxygen reduced the ability of the enzymes involved to catalyze the process (comparison: tubes 1 and 2). Potassium cyanide destroyed the enzymes (cytochromes), causing a near block of the transport process (comparison: tubes 1and 3). Concentration gradients did not alter the process (comparsion: tubes 1 , 4 and 5).
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