Bioelectrochemistry: Ions, Surfaces, Membranes - American Chemical

26 Destruction of the Electrophysiological Potential of Excised Frog Skin by Surfactants

Downloaded by UNIV OF PITTSBURGH on May 15, 2016 | http://pubs.acs.org Publication Date: June 1, 1980 | doi: 10.1021/ba-1980-0188.ch026

R. D . K U L K A R N I and E . D . G O D D A R D Union Carbide Corporation, Tarrytown Technical Center, Tarrytown, N Y 10591

The effect of various surfactants on the electrophysiological potential of freshly excised frog abdominal skins bathed in Ringers solution was evaluated using sodium-linear alkyl benzene sulfonate (LAS, C ), sodium lauryl sulfate (SLS), sodium alkylethoxy sulfate (Neodol 25-3EOS, AES), cetyltrimethylammonium bromide (CTAB), and the nonionic Neodol 25-7EO. Surfactants can cause a rapid, irreversible decay of the frog skin potential, the nature and the rate of the potential decay being dependent on the type of surfactant used. In decreasing order of potential-destroying effectiveness, the grading of the surfactants was CTAB > SLS > LAS > AES > Neodol 25-7EO. A marked increase in potential decay rate was observed above their CMC for both CTAB and SLS. On the other hand, addition of Neodol 25-7EO caused a marked reduction in the decay rate produced by SLS, and this is ascribed in part to mixed micelle formation. 12

x x r h e n a freshly excised "still living" frog abdominal skin is bathed i n aerated Ringers solution, a steady-state potential is developed (1) under open circuit conditions. The magnitude of this potential, w i t h the innerside of the skin electrically positive i n relation to the outside, can be as high as 100-150 m V (1,2). The origin of such potential is thought to be metabolic sodium and potassium transport reactions associated w i t h active sodium transport or the "sodium p u m p " process (3,4,5), resulting in spontaneous transport of sodium ions from the outside to the inside of the skin. 1

Work carried out at Lever Brothers Research Center, Edgewater, New Jersey. 0-8412-0473-X/80/33-188-445$05.00/1 © 1980 American Chemical Society

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The existence of such potentials is vital to the functioning of animal systems (6) and is associated with a number of coupled transport processes ( 4 ) , control of cell volume ( 3 ) , and so on. Introduction of surfactant molecules, which are thought to interfere w i t h metabolic reactions responsible for the sodium pump, can destroy the generated potential. I n the case of skin, the rate of potential destruction is likely to be an indication of the aggressiveness of the surfactant towards the skin. This chapter deals w i t h the effect of various cationic, anionic, and nonionic surfactants on the decay of frog skin potential. Experimental Materials. The source of skin was the common American frog, Rana pipiens. The frogs, 3-5 i n . i n length, were obtained from Texas through The Connecticut Valley Biological Supply Company. They were stored in the refrigerator until use. The steady-state skin potential for these frogs was strongly dependent on their condition: sluggish frogs gave significantly lower potential than vigorous specimens. Just before use the frogs were taken out of the refrigerator, washed, and kept i n room-temperature water until they became active. They then were sacrificed and the abdominal skin was removed, washed, and stored i n normal Ringer's solution. The skins were used within 1 hr of their dissection. Sodium lauryl sulfate ( S L S ) was obtained from B D H Chemicals, L t d . and was > 99% pure. Cetyltrimethylammonium bromide ( C T A B ) was supplied by Fine Organics and was twice recrystallized before use. Sodium linear alkyl benzene ( L A S ) sulfonate with an average 12-carbon chain length was a Lever Brothers product purified by desalting i n hot 80% ethanol and extraction with petroleum ether. Nonionic surfactant Neodol 25-7EO, an ethoxylate based on a 12-15-carbon chain alcohol, was supplied by Shell, as was the Neodol 25-3EO sulfate ( A E S ) . Double-distilled water w i t h conductivity less than 1.5 X 10" mhos/ cm at 25 °C was used throughout. Apparatus. The apparatus used for the electrometric measurement is similar to that developed by Ussing (1,7) and is shown i n Figure 1. The cell consists of two conical compartments, each of 20-mL capacity, separated and sealed from each other by the membrane under investigation. These compartments are filled with sulfate Ringer's solution (111.2mM N a S 0 , 2.0mM K S 0 , L O m M C a S 0 , and 2.6mM N a H C 0 ) to an appropriate level. T h e solution i n each compartment is circulated and simultaneously aerated by the rising purified air bubbles i n the bubbling tubes as shown. The electrical potential measuring device consists of two calomel sleeve junction reference electrodes which are coupled to each of the half-cell compartments by agar bridges. These reference electrodes i n turn are connected to a Keithley 610 A electrometer coupled to a Honeywell Electronic 19 chart recorder. The experiments were conducted b y first mounting the membrane carefully i n between the two half-cell compartments, avoiding wrinkles, strains, and leakages. This was followed by the addition of 25-mL sulfate 6

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Apparatus for electrometric measurements

Ringers solution to each half cell and aeration of these cell compartments at 180 bubbles/min until a steady-state potential was recorded. Next, an aliquot of surfactant stock solution (generally 1-5 m L ) was added to the outer compartment to obtain the required surfactant concentration. T o avoid any hydrostatic disturbances across the membrane, the surfactant solution addition was accompanied by the simultaneous addition of an equal volume of sulfate Ringer's solution to the inner compartment. A l l the surfactant stock solutions were prepared i n the sulfate Ringer's solution. Throughout the experimentation care was taken to avoid any hydrostatic pressure head across the membrane. It was observed that even a small pressure difference not only altered the observed potential, but also changed the rate of its decay following surfactant insult. In general, application of positive pressure from outside to inside increased the skin potential and its decay after surfactant insult, while the reverse effect was observed with negative pressure. Accordingly, the hydrostatic head across the membrane was kept at minimum. This precaution was necessary to obtain reproducible results, especially when using low surfactant concentration and for certain mixed-surfactant systems. Figure 2 illustrates the reproducibility obtained when the above precautions were taken. Results and Discussion I n the present study, sulfate Ringer's solution was chosen over con-

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Figure 2. Potential decay pattern of the membrane in the presence of a representative alkyl benzene sulfonate surfactant. Reproducibility study.

have a lower permeability through the membrane, generate a higher membrane potential. Using sulfate also gave lower susceptibility to the hydrostatic pressure gradient across the membrane. The effect of different surfactants, all used at the same concentrations, on skin potential is shown i n Figure 3. In this figure, the normalized skin potential is plotted as a function of time following surfactant insult. Using normalized potential, i n which the starting potential is set at 100%, facilitates comparison of surfactant effects. Actual starting potentials varied appreciably from skin to skin, but good reproducibility for a particular treatment was obtained when normalized potentials were used (see Figure 2 ) . Figure 3 shows that after the first 10-20 min, the skin potential steadily decreases w i t h time with 0.005% surfactant i n the solution. The rate of potential decrease is higher for the cationic surfactant C T A B than for the anionic surfactants S L S and L A S . This observation is consistent w i t h the well-known fact that cationic surfactants such as C T A B are much more aggressive to biological membranes than anionic or nonionic surfactants. I n the absence of added surfactant, the skin potential maintained its value to within 10% even after 8 hr; addition of 0.005% C T A B completely destroyed the potential i n about 60 min. Once the potential is destroyed i n this way, repeated washing of the skin with



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Ringers solution w i l l not restore it. I n other words, the surfactants irreversibly impair the active metabolic sodium transport reactions w h i c h generate the potential. The distinct differences among various types of surfactant seen i n Figure 3 disappear at higher concentration levels. This is shown i n Figure 4 where normalized potential-time plots are given for 0.05% surfactant concentration. T h e effects for the different ionic surfactants are clearly much larger than for the nonionic Neodol 25-7EO. The above results clearly underline the importance of a charged head group on the surfactant molecule i n its attack on the membrane. Because of the wide disparity of decay rates caused b y ionic and nonionic surfactants, it was interesting to examine the effect of mixing such species. Figure 5 demonstrates that a 0.05% S L S solution causes a more rapid decay of skin potential than a similar solution to w h i c h 0.05% Neodol 25-7EO is added, i.e. an intermediate effect ( I n work to be published elsewhere ( 8 ) , it is shown that preapplication to frog skin of certain protectants such as polyoxethylenes or cationically substituted cellulose derivatives also mitigates the S L S effect.), rather than an additive effect, on the potential decay rate is obtained. The most likely cause of this is the formation of mixed micelles, which effectively decreases the concentration of free surfactant anion ( L S " ) monomer. However, reduction of the L S " monomer concentration does not provide a f u l l explanation for the observed effect. The critical micelle concentration (cmc) of

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Mitigating effect of Neodol 2S-7EO on SLS action


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SLS i n sulfate Ringers solution is 0.04% or 1.3 X 10" M. F r o m tabulated data of cmcs and salt effects (9,10) in the literature one can estimate the cmc of an equiweight mixture of SLS and Neodol 25-7EO to be at most one tenth of this concentration. Reference to Figures 5 and 6 indicates that the observed decay rate for the mixture clearly exceeds the additive effect of 0.05% Neodol 25-7EO and free L S " at a level of < 0.005%. One concludes either that the mixed micelles of SLS and Neodol 25-7EO have an effect greater than that of the nonionic micelles alone, or that the membrane activity of the free SLS (at its reduced level) is greater i n the presence of the nonionic surfactant. That the activity of the mixed micelles is less than that of SLS micelles is evident from results given later i n the chapter (see Figures 5, 10, and 11). It is also interesting that the potential destroying activity of A E S lies between that of an alkyl sulfate, for example, and a nonionic surfactant (see Figure 4). This might well result from shielding of the charge of the anionic head group by the neighboring ethoxy groups. Figures 6-11 demonstrate the effect of surfactant concentration on the decay of skin potential. Several features can be noted from these figures.


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1. A n increase i n the surfactant concentration increases the potential decay rate. Figures 10 and 11, discussed later, show that the decay rates observed w i t h SLS and C T A B show a pronounced increase above their respective cmcs. 2. W h i l e a continuous potential decay w i t h time is obtained most frequently, SLS, except at high concentration, shows an initial sharp rise i n potential followed by a steady decrease. The initial rise i n potential may be the result of physical adsorption of S L S onto the membrane and an increase of its charge, which w i l l influence its permeability to sulfate ions prior to the eventual potential destruction process. The higher the surfactant concentration, the lower the height of the maximum, and the quicker it is achieved. 3. A n exponential or nonlinear potential decay pattern is observed w i t h surfactant concentrations higher than 0.005%, and a linear potential decay is obtained at lower concentrations. Analysis of the D a t a . Surface-active agents, especially the ionic and, more especially, the cationic type, have pronounced biological activity. E a r l y experiments (11), for example, demonstrated the pronounced lytic activity of soaps and alkyl sulfates towards erythrocytes. Other experiments have demonstrated the interfacial activity of ionic surfactants towards various l i p i d materials such as cholesterol (12), and also towards protein (13). W h i l e such information is well known, knowledge of how surfactants impair cell structure, and, more particularly, interfere w i t h the metabolism of living cellular systems, is still


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Bioelectrochemistry: Ions, Surfaces, Membranes - American Chemical

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