Study of Membrane Permeability Using Alcohol-Induced Ciliary - Essay Example

STUDY OF MEMBRANE PERMEABILITY USING ALCOHOL-INDUCED CILIARY NARCOSIS OF Tetrahymena pyriformis INTRODUCTION Cilia, flagella and microvilli are cellular appendages with different characteristics and functions. Cilia are 6- to 10-µm cellular structures studding the membrane to allow movement. It is more than ten times smaller than the flagellum, which is 150 µm in length. In cross section, the characteristic 9 + 2 motif of microtubule arrangement can be appreciated. It has a characteristic oscillation motion in which they are relatively straight in forward stroke, but curved during the return stroke. This movement propels the cell perpendicular to the orientation of the cilia. In contrast, the longer flagellum, usually one or two, moves in an undulating manner, moving successive waves from the base to the tip in the process. As such, it propels the cell to move parallel to the flagellum’s orientation. On the other hand, microvilli are non-motile appendage with no internal structure supporting it (Reece, et al., 2011). The loss of activity of cilia, resulting to loss of cellular motility, is termed ciliate narcosis. This can be induced by different living conditions and substances to which it is exposed to, such as low temperature (Jackson, Goggin and Lucas, 2012), calcium (Nakanaka, Tanaka and Oosawa, 1984), magnesium and protein (Adshead, et al., 1975) concentrations, as well as the presence of certain substances, such as theophylline, bromhexine, ambroxol, terpin hydrate, mercaptoethanesulfonat-sodium, amrinon, salbutamol, tetracosactid-hexaacetate, histamine, phenol and nitric oxide (Graf, Graf and Wenz, 1999). In addition, a study using tracheal cells in vitro, showed that ciliary beat frequency was increased at low ethanol concentrations (0. 01 to 0. 1%), unchanged at (0. 5 to 1%), and decreased above 2% (Maurer and Liebman, 1988). Moreover, even if it does not penetrate the cellular membrane, the presence of glycerol in the culture can make cellular surroundings viscous, making it more difficult for cilia to move (Negus, 1949). Alcohols are amphiphatic molecules primarily composed of alkane (CH3) and hydroxyl (OH) moieties, such that the simplest alcohol is methane (CH3OH). When the hydrocarbon portion becomes longer, as in the case of ethanol (2 Cs), propranol (3 Cs) and butanol (4 Cs), the alcohol becomes effectively more hydrophobic. A compound similar to alcohols, propranolol in particular, is glycerol, which has hydroxyl group attached to each of the three carbon atoms (Masterton and Hurley, 2008). One of the well-studied organisms known to extensively use cilia for movement is the free-living Tetrahymena pyriformis, which belongs to the order Holotrichia, literally meaning cilia all over its membrane. The microorganism is a common fixture in human toxicity studies, because of its cilia’s similarity in terms of morphology and function to that of human epithelial cells (Graf, Graf and Wenz, 1999). Its cell membrane has the characteristic sandwich-like phospholipid bilayer, with the hydrophilic head facing the cytoplasm or the external surroundings, and the hydrophobic chain filling the middle. Given this structure, hydrophilic molecules can more easily pass the membrane, as compared to hydrophobic ones. However, the presence of transport mechanisms allows hydrophobic molecules to enter the cell (Reece, et al., 2011). In cellular permeability studies, an observable cellular change effected by previously identified substance must be utilized in order to clearly determine when and in what conditions the substance penetrated the cell membrane. In this case, the cellular change used was ciliate necrosis and the substance that causes it would be alcohol. This experiment was conducted in order to 1) observe narcosis of T. pyriformis ciliates by alcohol, and 2) discuss the results of ciliate narcosis in terms of membrane permeability. METHOD After preparing propranol and glycerol serial dilutions (propranol: 1.8 M, 1. 5M, 0. 4M, 0. 2M , 0M; glycerol: 2. 8M, 2M, 1. 7M, 1M, 0M) using distilled water and 5M stock solutions, a drop of each preparation was mixed with an equal volume of T. pyriformis culture on a watch glass. Each mixture was observed under x10 objective microscope lens. The observed percentage necrosis was then recorded. RESULTS Table 1. Percentage ciliary narcosis in serial dilutions of propranol and glycerol. solution concentration % narcosis Propranol (no. of carbons = 3) 1. 8M 1% 1. 5M 80% 0. 4M 50% 0. 2M 80% 0 M 0% Glycerol (no. of carbons = 3) 2. 8M 70% 2 M 60% 1. 7M 30% 1 M 90% 0 M 0% Table 1 shows percentage ciliary narcosis in serial dilutions of propranolol and glycerol. A graphical presentation of this data, showed non-linear curves, particularly of 4th order polynomial, representing the relation between concentration and % narcosis. 50% narcosis was observed from 0. 4M propranol (log 0. 4 = -0. 39794), and was estimated for 0. 075M glycerol (log 0. 075 = -1. 125). Since glycerol and propranolol have the same number of carbon atoms in their structures, they were compared based on the number of hydroxyl groups, wherein propranolol has one and glycerol has three. Plotting the data, there is an inverse relationship between number of hydroxyl groups and log concentration of 50% narcosis, such that the more hydroxyl molecules a substance has, the log concentration causing 50% narcosis decreases. The equation of the relation is log (M50%) = -0. 3635(number of hydroxyl groups) – 0.0344. DISCUSSION Lipid solubility can be determined by partition coefficient, or the ratio of solubility in organic solvent (diethyl ether) to its solubility in water. The transport of molecules across lipid bilayer is affected by several factors. The size and molecular structure are characteristics of molecules that are important in determining their permeability to lipid membranes. Understandably, the size of a molecule increases with the number of atoms it is made up of. Other factors, such as the electronegativity of comprising atoms, also affect the size of the molecule. Lipid solubility, and in effect, partition coefficient, increases with the amount of hydrophobic moieties it contains. For example, methanol, ethanol and propanol contain one, two and three carbon atoms, and thus have increasing values of partition coefficient, 0. 14, 0. 26 and 1. 9, respectively. The same pattern is true for fatty acids. In contrast, certain moieties, such as hydroxyl group, make molecules more hydrophilic, or more likely to form hydrogen bonds with water. In effect, its lipid solubility decreases (Masterton and Hurley, 2008). Thus, while elongation of carbon chains increases lipid solubility, increasing the number of hydroxyl groups decreases them. Therefore, solutes that are too big cannot enter the cell without the aid of a transport system. Even then, big, hydrophobic molecules, such as steroids and vitamin A can pass by easily. On the other hand, certain small molecules, such as ions, do not readily permeate the lipid bilayer despite their size because of their charged state, which makes them hydrophilic. Large, polar molecules, such as glucose, cannot certainly pass through the cell, both because of its bulk and lipid insolubility, while small and hydrophobic molecules such as ethanol have a free pass (Reece, et al., 2011). In this experiment, the lipid solubility of propanol and glycerol were compared using the half maximal effective concentration in permeating the cell membrane and subsequently hindering ciliary movement, which was easily observed in highly ciliated T. pyriformis observed under the microscope. Propanol and glycerol both contain three carbons, and so the effect of their carbons on comparing lipid solubility was cancelled out. The difference thus lied in the number of hydroxyl group these compounds contain. Glycerol contains three, while propanol contains just one. The oxygen’s effect on the overall size of the molecules is insignificant, because of the small size of the atom. However, oxygen, together with nitrogen, form hydrogen bonds in the hydrogen portion of water. Thus, increasing the number of hydroxyl groups make the molecule more hydrophilic and less hydrophobic. In fact, the partition coefficient of propanol, 1. 9, is much greater than that of glycerol, which is estimated to be at 0. 00066 (Anon, n. d.). This is contrary to what was observed in this experiment, wherein it was shown that less concentration of glycerol was needed to cause half maximal effect than that of propanol. This is because comparison of half maximal effect is valid only if the effects of the two test substances are dose-dependent. As seen from figure 1, both curves did not correspond to dose response curves. However, it should have been in theory, since the entry of permeable solutes is also dependent on its concentration, such that the amount of solutes outside the cell must be greater than that in the cytoplasm, so that diffusion of these permeable molecules occur from outside to inside the cell (Reece, et al., 2011). This was not seen in the results, possibly because of the inaccuracy of the measurement of ciliate narcosis. It could have been better if a more objective cell count of ciliate narcosis was used instead of eyeballing the percentage of ciliate narcosis. CONCLUSION Observation of ciliate narcosis to study cellular permeability of substances proven to impede ciliary movement. Solute size, molecular structure and concentration are important factors that determine the molecule’s permeation through the cell membrane. REFERENCES Adshead, P. C., Martinez, J. R., Kilburn, K. H., and Hess, R. A., 1975. Ciliary inhibition and axonemal microtubule alterations in freshwater mussels. Annals New York Academy of Sciences, pp. 192-212. Jackson, C. L., Goggin, P. M. and Lucas, J. S., 2012. Ciliary Beat Pattern Analysis Below 37°C May Increase Risk of Primary Ciliary Dyskinesia Misdiagnosis. CHEST, 142(2), pp. 543-544. Masterton, W. L. and Hurley, C. N., 2008. Chemistry: Principles and Reactions. Connecticut: Brooks/Cole Maurer, D. R. and Liebman, J., 1988. Effects of ethanol on in vitro ciliary motility. Journal of Applied Physiology, 65(4), pp. 1617-1620 Nakaoka, Y., Tanaka, H. and Oosawa, F., 1984. Ca2+-dependent regulation of beat frequency of cilia in Paramecium. J. Cell Sci., 65, pp. 223-231 Nevus, V. E., 1949. Ciliary Action. Thorax, 4, 57-64. Reece, J.A., Urry, L.A., Cain, M.L., Wasserman, S.A., Minorsky, P.V. and Jackson, R.B., 2011 Campbell Biology. Pearson Education Inc., San Francisco. Anonymous, n. d. Membrane Permeability. Retrieved from: < http://faculty.weber.edu/jclark1/Cell%20Biology%20Labs/Mem%20Permeability.pdf>. [Accessed 13 January 2013] Read More