Transport properties of cell membranes

02.03.15 Transport Properties of Cell Membranes Conducted

by:

Park,Min

Chul,

Moss,Rebecca

Margaret,

Nairn,Thomas

Michael

Allen

and

Patchigalla,Hadassah (Class 14661: Group A5) Aims 1.

To develop an understanding of the theory behind data acquisition and signal processing.

2.

To become proficient in the use of two commonly used PowerLab programs; Scope and Chart.

3.

To reinforce knowledge of cell membrane transport properties using red blood cells (RBCs) as a model.

Introduction Most cells are freely permeable to water that diffuses through aquaporins (water channels) in the selectively permeable plasma membrane. This movement of water is termed osmosis and occurs in response to an osmotic pressure gradient across the cell membrane. The osmolality of a solution is the total concentration of particles of solute, measured by the number of osmotically active particles per kg-1 of water (Boron & Boulpaep, 2009). Osmolarity is the osmolar concentration expressed as osmoles per litre of solution rather than osmoles per kilogram of water. Despite the fact that it is osmolality which establishes osmotic pressure, for fluids in the human body deemed to be dilute the actual differences between osmolality and osmolarity in terms of numerical values the difference is less than 1% (Guyton & Hall. 2010). For two solutions of the same osmolarity, the net movement of water across the semi-permeable membrane separating them is zero, and the solutions are said to be isosmotic. A solution with a higher osmolarity is hyperosmotic, and one with lower osmolarity is described as being hypoosmotic. The tonicity of a solution describes the effect of that solution on the volume of the cells suspended in it, and is sometimes referred to as the effective osmolality (Boron &

Boulpaep, 2009). An isotonic solution does not alter the volume (or shape) of cells suspended in it because the osmolarity of the solution is the same as the cytosol of the cell. A hypertonic solution will cause the cells to decrease in volume, and a hypotonic solution will cause the cells to swell. The unique, biconcave, shape of RBCs in isotonic solutions enables them to change their volume over a much wider range of solution osmolarities than most other cells. They therefore simulate an osmometer in which the volume of the RBC is inversely related to the osmolarity of the solution. RBCs placed in a hypertonic solution will lose water, and shrink. This is termed crenation, where the shape of the RBC becomes shrivelled and spikey in appearance (Tortora and Derrickson,

2012). If the RBCs are placed in hypotonic solution, they swell until they are spherical in shape. Further swelling will cause rupture of the RBCs, and the loss of their contents (largely haemoglobin) into the surrounding solution (termed haemolysis). If RBCs are placed into solutions of penetrating solutes (such as urea), the time to haemolysis is an indication of the rate at which the solute is able to cross the cell membrane. As the solute enters the RBCs the osmotic gradient for water is increased, causing water to move more rapidly into the cells. Solutions of penetrating solutes are therefore hypotonic, regardless of their osmolarity. It is understood that small and water soluble molecules which have low affinity for lipids (phospholipid bilayer of cell membranes) may penetrate the membrane ( Johnson, L., 1998). Contrastingly, NaCl, the main solute used throughout the experiment is impermeable to RBCs. Hence, solutions of non-penetrating solutes do depend on their osmolarity. During the experiment Light Intensity Metre (LIM) transducer is used to measure the changes in red

blood cell (RBC) volume. The LIM transducer consists of a photo-sensor to detect the amount of light passing through test tubes containing different solutions and represents the amount of light detected as voltage output. The more light detected the higher the voltage and the less light detected the lower the voltage. In RBCs the molecule largely responsible for refraction of light is haemoglobin. If amount of RBCs thus haemoglobin increases then more light will be refracted and voltage output will be lower (MEDSCI 205 Laboratory Manual, 2015). Method Stock solutions of RBCs, washed and re-suspended in isotonic saline, were provided. These were used as osmometers for investigating the membrane transport properties of cells. Changes in cell volume were measured using a light intensity meter (LIM) that gave a voltage output in response to changes in cell volume. The voltage was then recorded using the Power Lab system and the Scope and Chart software. Three sets of experiments were carried out. First the LIM was calibrated using test tubes of isotonic solution with varying volumes of RBC suspension (ranging from 0-200 µL) and recording the voltage output using Chart software. A calibration curve was constructed and used to determine the concentration of RBCs in an unknown solution. A second set of experiments was then carried out in which 200 µL of RBCs was added to 10 mL of solutions of different concentrations. After mixing the test tubes were placed in the LIM and the Scope software used to show record the voltage output. The osmolarity of each solution was determined: Osmolarity = ф (osmotic coefficient) x n (number of ionic species) x c (concentration) The 0.075 M NaCl solution was made by using the equation c1v1 = c2v2. Using the given 0.154 M NaCl solution and knowing that 10mL of solution was required it was found that 4.87mL of 0.154 M NaCl solution and 5.13mL of water were required. Finally, solutions of different solute concentration were used to investigate solute permeability by recording the time to haemolysis using the LIM. Results: Part A Table 1 Test Tube Number

Volume of Blood (µL)

Blood concentration (µL/mL)

Voltage (V)

1

0

0

0.000

2

50

5

-0.881

3

80

8

-1.485

4

110

11

-2.069

5

140

14

-2.822

6

170

17

-3.436

7

200

20

-4.035

Blood concentrations in the third column of the table were calculated by dividing the Volume of Blood by 10mL.

Figure 1a shows the decrease in Voltage outputs as Bovine Blood concentration increased. The Unknown bovine blood solution had the Voltage output of -2.350V.

The data from figure 1a was used to generate the LIM calibration curve. Here, Voltage outputs were plotted as a function of Bovine Blood concentration showing the negative linear relationship between Voltage outputs and Bovine Blood concentration. Using the calibration curve, the concentration of the Unknown bovine blood solution was found to be 12.0 µL/mL.

Part B - Table 2 Solutions

Osmotic Coefficient

Osmolarity (OsmL-1)

Tonicity

1. Distilled H20

0

0

Hypotonic

2. 0.075 M NaCl

0.93

0.140

Hypotonic

3. 0.154 M NaCl

0.93

0.286

Isotonic

4. 0.4 M NaCl

0.93

0.744

Hypertonic

5. 0.28 M Urea

1

0.28

Hypotonic

Osmolarity was calculated by using the equation Osmolarity = ф (osmotic coefficient) x n (number of ionic species) x c (concentration)

Figure 2 shows relative to isotonic 0.154 M NaCl solution, 3 solutions have positive Voltage output and 1 solution has negative Voltage output. The 3 solutions with positive Voltage outputs all have approximately the same Voltage output. The Distilled H20, 0.075 M NaCl and 0.28 M Urea solutions all lie above 0V showing its hypotonic property. Contrastingly, the 0.4 M NaCl solution lie well below 0V indicating its hypertonic property, Part C - Table 3 Solution

Osmolarity (Osm L-1)

Time to Haemolysis (s)

1. 10 mL 0.154 M NaCl

0.2864

Did

2. 2mL distilled H2O + 8mL 0.154 M NaCl

0.2292

within 20 secs.

3. 4mL distilled H2O + 6mL 0.154 M NaCl

0.1719

4. 6mL distilled H2O + 4mL 0.154 M NaCl

0.1146

16.2

5. 8mL distilled H2O + 2mL 0.154 M NaCl

0.0573

15.5

6. 10mL distilled H2O

0.0000

8.40

not

haemolyse

Osmolarity was calculated using the equation Osmolarity = ф (osmotic coefficient) x n (number of ionic species) x c (concentration)

The Time to Haemolysis in seconds for each solution increased from solution 6 of 8.4 seconds upto solution 4 of 16.2 seconds. The solutions 1, 2 and 3 did not haemolyse within 20 seconds. Data from solutions 4, 5 and 6 were used to generate the Figure below.

Here, the Time to Haemolysis in seconds was plotted as a function of Osmolarity in Osm/L. There is a positive linear relationship between the Time of Haemolysis and Osmolarity. The fastest recorded Time to Haemolysis was 8.4 seconds when the Osmolarity was 0 Osm/L. Discussion Part A As Red Blood Cell (RBC) concentration increased the LIM voltage output decreased linearly. Increase in RBC concentration means increase in the number of RBCs in a given volume of solution. As seen in the Introduction RBCs have the protein haemoglobin giving RBCs a different refractive index to the solution in which they are suspended and thus the ability to refract light. It follows that the increase in the number of RBCs would mean the increase in the number of haemoglobin in the given volume of solution. As concentration of haemoglobin increases, the degree that which light is refracted increases accordingly, hence the amount of light detected by the photo-sensor of LIM

transducer decreases. Therefore the LIM voltage output decreases. As for the Unknown blood solution, it makes sense that its concentration is 12.0µL/mL. The voltage output of -2.350V was lower than Test tube number 4 of -2.069V with concentration of 11.0µL/mL. Explanation of why the LIM calibration curve was linear is as follows. Starting from test tube 2, the volume of blood added to each test tube increases by 30 µL. Therefore, the amount of RBCs and thus protein molecules (mainly haemoglobin) added to each test tube increases constantly (i.e. concentration increases constantly). Since the haemoglobin molecules, which refract light, increases constantly by each test tube, it follows that the amount of light refracted and thus detected by the photo-sensor decreases correspondingly. Consequently, the relationship between blood concentration and voltage output is linear. Note: the linear relationship described and seen above will no longer hold true once it reaches a point when the test tube is completely saturated with blood at which point it will give the lowest possible voltage output reading. Part B Diagram 1: Relationship between osmolarity and tonicity (Jones, M., Fosbery, R., Taylor, D. & Gregory, J. 2011). 0.4 M NaCl solution was hyperosmotic and hypertonic. The solution was hyperosmotic since the osmolarity of the solution (0.744 Osm/L) was higher than the osmolarity of RBCs (0.28 Osm/L). Referring to Figure 2, the voltage output of 0.4 M NaCl solution was -3.376V. The large negative value indicates that the amount of light refracted is high and therefore amount of light detected by the LIM transducer photo-sensor was small. This means that the protein concentration (largely haemoglobin) of RBCs has increased refracting more light. It follows that the mechanism in which protein concentration would have increased is cell crenation from the solution being hypertonic. 0.154 M NaCl solution was isosmotic and isotonic. The solution was isosmotic because its osmolarity was the same as RBC’s osmolarity. Data from Figure 2 indicates there is almost no change in RBC volume which is shown by the voltage output being 0.013V (close to 0). Solution was isotonic since there was no net water movement (hence no change in RBC volume) as the osmolarity of the solution and RBC’s osmolarity were equal. Both distilled water and 0.075 M NaCl solutions were hypoosmotic and hypotonic. The solutions both had lower osmolarity than RBCs and therefore it was hypoosmotic. From Figure 2 and by using LabChart, the voltage output of distilled water solution and 0.075 M NaCl solution were 4.311V and 4.322V respectively. The positive voltage output indicates a lot of light being detected by the photosensor and the amount of light refracted is low. Referring back to 0.4 M NaCl solution which had protein concentration increase, these 2 solutions had protein concentration decrease (net water movement into cells). Consequently, the decrease in protein concentration in RBCs was caused by the solutions being hypotonic. The 0.28 M Urea solution was a rather unique solution since in terms of osmolarity it was isosmotic but in terms of tonicity was hypotonic. The solution was isosmotic because it had the same

osmolarity as the RBC osmolarity. But, looking at Figure 2, the voltage output of the urea solution is 4.410V. This meant the urea solution was a hypotonic solution as the distilled water and 0.075 M NaCl solutions. The reason why the urea solution was hypotonic despite being isosmotic lies in urea being freely permeable to the selectively permeable plasma membrane of RBC. As seen previously, all the solutions in this part of the experiment dealt with NaCl (excluding water) which was impermeable to the plasma membrane. The impermeability of NaCl and its difference in osmolarity with respective to RBCs establishes the initial osmotic pressure gradient. However, urea being freely permeable to plasma membrane indicates it is independent of the osmolarity. As seen from the introduction, solutions of penetrating solutes are hypotonic, regardless of their osmolarity. Overall, from the isosmotic urea and isosmotic NaCl solutions experiments, the difference between osmolarity and tonicity becomes clear. Osmolarity refers to the concentration of solutes and tonicity refers to the effect that a particular concentration of solutes have on the changes of cell volume. Part C Comparison (using Figure 3a) of the Voltage output graph of each solution to the 10mL distilled water solution graph would show which solutions underwent complete haemolysis and which did not. 10mL of distilled water with osmolarity of 0 showed the lowest time to haemolysis. The selectively permeable membrane of RBC is freely permeable to water through water channels called aquaporins. As seen earlier in the introduction, water moves by osmosis and with the very steep osmotic pressure gradient (because the solution is absent from any solutes) it follows that pure distilled water takes the least amount of time to move into RBCs and cause haemolysis. From solutions 5 to 1 the concentration of solute (NaCl) increases. Referring back to Table 3, it can be deduced as the solution gradually becomes more and more concentrated, the osmotic pressure gradient will become less and less steep. This continues until it reaches a point where the solution’s osmolarity matches RBC’s osmolarity of 0.28 Osm/L which in this case it would be solution 1 with 0.2864 Osm/L. Solution 5 with 8mL distilled H2O + 2mL 0.154 M NaCl has osmolarity of 0.0573 Osm/L. This is still considerably lower than the RBC’s osmolarity of 0.28 Osm/L but higher than 0 Osm/L of distilled water. Thus time to haemolysis was longer than distilled water solution since the osmotic pressure gradient became less steep. Solution 4 with 6mL distilled H2O + 4mL 0.154 M NaCl has osmolarity of 0.1146 Osm/L. Following from solution 5 this solution had higher osmolarity than solution 5 and 6. Hence, the time to taken to Haemolysis was the longest out of the 3 measuring 16.2 seconds. Once again the reason for the increase in Time to Haemolysis is attributed to the increase in solute concentration and therefore the decrease in osmotic pressure gradient. The group concluded that for solutions 1, 2 and 3, the Time to complete Haemolysis exceeded the experimental range of 20 seconds. The solutions 1 and 2 have osmolarity values very close to RBC osmolarity value of 0.28 Osm/L. It follows that the solutions are isotonic in that it does not cause any volume changes to the RBCs and hence haemolysis. However, it is odd that solution 3 did not haemolyse within 20 seconds. Its osmolarity of 0.1719 Osm/L was nowhere near to be able to be considered as similar to RBC osmolarity. A plausible explanation to this would be that some of the RBCs in solution 3 haemolysed and

released its contents to the solution. As discussed earlier, RBCs contain protein haemoglobin and when haemolysed, the protein released to the solution medium will increase its osmolarity until such a point the osmolarity of the solution matches that of the RBCs. At this point no more haemolysis will occur since there is no osmotic gradient. Conclusion Overall, the theory behind data acquisition and signal processing was understood by conducting all parts of the experiment. Each part required the knowledge of the relationship between voltage output and changes in RBC volumes. For example, in Part A, the group had to realise the decrease in voltage output was caused by the increase in light refraction, hence increase in blood concentration. By participating in all aspects of the experiment, the lab group has also become proficient is the use of 2 commonly used PowerLab programs mainly Scope and Chart. All parts of the experiment were done using the 2 programs and analysis and discussion of data above required the extensive and almost exclusive use of both programs. Lastly, the knowledge of cell membrane transport properties using RBCs was revised and emphsised. From Part B, the group learnt that the selectively permeable plasma membrane of RBCs was freely permeable to urea but not permeable to NaCl. This allowed distinguishing between the concepts osmolarity and tonicity possible. From Part C, the lab group learnt osmolarity and time to haemolysis are directly proportional. As the concentration of solution increased so did the time since osmotic pressure gradient decreased. Looking back at the 3 parts of the experiment, an obvious negative linear relationship between Bovine blood concentration and voltage output was observed (Part A). Next, the association between osmolarity and tonicity was observed by using solutions of different osmolarity and the LIM transducer. Plus, the consequence of urea being permeable to plasma membrane in terms of its tonicity was observed (Part B). Lastly, the positive linear association between Osmolarity and Time to Haemolysis was observed in Part C of the experiment. Experimental Errors / Limitations For Part C, there were inconsistencies in starting Scope at the same time as adding blood to each test tube. Plus, reading the time taken to Haemolysis using Figure 3a was difficult since there were 3 graphs which overlapped with one another. It was hard to choose a point of the graphs on the figure to use to determine the time since there were miniscule bumps even when the graph showed the plateau area. References Boron, W., & Boulpaep, E. (2007). Medical Physiology (2nd ed). Philadelphia: Saunders MEDSCI 205 Laboratory Manual (2010). Laboratory 1: Introduction to Experimental Measurement and Membrane Transport. Tortora, G., & Derrickson, B. (2012). Principles of Anatomy and Physiology. United States of America: John Wiley & Sons. Johnson, L. (1998). Essential Medical Physiology. United States of America: Lippincott-Raven. Guyton & Hall. (2010). Textbook of Medical Physiology (12th ed). Philadelphia: Saunders Jones, M., Fosbery, R., Taylor, D. & Gregory, J. (2011). AS Level and A Level Biology. United Kingdom: Cambridge University Press.