Cell Physiology and Body Fluids-Virtual Experiments
Online Virtual Experiments:
For Virtual Experiment Visit this link. Click on "Exercise 1: Cell Transport Mechanisms and Permeability". Click on one of the following titles:
- Simple Diffusion,
- Facilitated Diffusion,
- Osmosis, Filtration,
- Active Transport.
1. Simple Diffusion: In this activity
we will be simulating the process of diffusion across the plasma membrane.
Notice the two glass beakers at the top of the screen (see figure with labels).
You will be filling each beaker with fluid. Imagine that the right beaker
represents the inside of a cell, while the left beaker represents the
extracellular (interstitial) fluid. Between the two beakers is a membrane
holder into which you will place one of four dialysis membranes found on the
right side of the screen. Each of these membranes has a different “MWCO,” which
stands for “molecular weight cut off.” Molecules with a molecular weight of
less than this value may pass through the membrane, while molecules with higher
molecular weight values cannot. Now, let’s set up
a mock dialysis machine experiment (see this video). These machines are used on
patients who have lost kidney function. Urea, a breakdown product of amino
acids, must be removed from the patient’s blood or it will become toxic to the
body and cause death. Dialysis machines take a patient’s blood and pass it
through a selectively permeable membrane in order to remove urea from the
blood. On one side of the membrane is the patient’s blood; on the other side
are fluids carefully selected to mimic the concentrations found in the body of
substances such as Na, K, Ca and HCO3. To simulate this process: One beaker
will represent the dialysis patient’s blood and the other beaker will contain
no urea. Normally, dialysis machines are set to run so that the blood is
subjected to diffusion twice, and urea is reduced by 75% rather than 50%. In
addition, excess water is drawn from the patient, who has no other way to
dispose of excess fluid. Dialysis patients need to have routine lab tests done
to ensure that ion concentrations are maintained at normal levels. Questions: Which materials diffused from the left beaker to the right beaker?
Which did not?
Why?
2. Facilitated Diffusion: Simple diffusion accounts for the transmembrane transport of some
ions, but not all of them. Some molecules that are too
polar to diffuse still manage to
get through the plasma membrane’s lipid bilayer. Similarly, some
molecules that
are too large to
pass through protein channels still manage to cross the membrane. How?
The passage of
such molecules and the non-diffusional movement of ions through a
membrane is mediated by
integral proteins known as transporters. Transporters are embedded
within the plasma membrane and work
by undergoing a conformational
change that allows transport to occur. A molecule first binds to a
receptor site on a
transporter. When bound, the transporter changes shape so that the
binding site moves from one side
of the membrane to the other side. The molecule then dissociates from
the transporter
and is released on the other side
of the membrane. This type of transport is called facilitated diffusion.
It is considered a form of passive transport because no cellular energy
is expended
in the process. In facilitated diffusion, molecules are still moving
from one location to another along a concentration gradient, but it is
transport proteins that result in this movement—not random thermal
motion.
Among the most important facilitated-diffusion systems in the body are
those
that move glucose across the membrane. Without transporters, the
relatively
large, polar glucose molecule would never be able to pass into a cell.
However,
the number of transport proteins in a given cell membrane is finite, so
only a
certain amount of glucose can be transported per unit of time. Transport
of
glucose into the cell is especially interesting in that the glucose is
converted to glucose- 6-phosphate as soon as it enters the cell, so that
there
is always a low concentration of glucose inside the cell, which favors
transport into the cell. The following figure illustrate the main parts of this
virtual experiment and the whole experiment is shown in the following video.
The results of this experiment are shown in the following table. The graph
that depict the relationship between the number of transporters and the transport
rate is shown in this figure. .Questions: 1. Does the diffusion rate of Na/Cl change with the number of receptors? 2. What is the mechanism of the Na/Cl transport? 3. If you put the same amount of glucose in the right beaker as in the left, would you be able to observe any diffusion?
4. Can you give explanation why the diffusion rate for NaCl does not change with the change of number of transporters? 5. Ca you give explanation why the slops of the two concentrations of glucose seen in the graph
that depict the relationship between the number of transporters and the transport
rate are different?, being more steeper for the higher glucose concentration than the slop for low glucose concentration.
3. Filtration: At the same time that diffusion is allowing cells to take in oxygen and nutrients while expelling carbon dioxide and metabolic wastes, another process is also taking place. This process occurs mainly in capillaries of the body (such as those in the kidneys) where fluid pressure of the blood—called hydrostatic pressure—forces materials across a capillary wall. Both blood and interstitial fluid contain dissolved solutes. Usually, the osmotic pressure of the interstitial fluid is not as great as the hydrostatic pressure of the blood, so there is a net movement of fluid and/or solutes out of capillaries—a process called filtration. What is filtered out depends solely on the molecular size of the solute and the size of the “pores” in the membrane. Filtration is considered a passive process, since it occurs without the expenditure of metabolic energy. The setup of this virtual experiment is shown in the figure. This experiment is demonstrated in the following video. The results of this experiment is shown in the figure. Questions: 1. Does the membrane MWCO affect filtration rate? 2. Does the amount of pressure applied affect the filtration rate? 3. Did all solutes pass through all the membranes? If not, which one(s) did not? Why? 4. How can the body selectively increase the filtration rate of a given organ or organ system?
4. Active transport: Active transport differs from passive transport in
that energy derived from metabolism is used to move solutes across the
membrane. It also differs in that solutes are moved from an area of low concentration to an area of
high concentration—the opposite of facilitated diffusion. As with facilitated
diffusion, binding of a substance to a
transporter is required. Since the
bound substance is moving “uphill” to an area of higher concentration, the transporters are
often spoken of as pumps. The net movement from lower to higher
concentration and the maintenance of a higher steady-state concentration on one
side of a membrane can be achieved only by the continuous input of energy into the
active-transport mechanism. The energy input can alter the affinity of the
binding site on the transporter so that
there is a higher affinity when facing one
direction over the other, or the energy may alter the rates at which the transporter moves the
binding site from one side of a membrane to the
other. As with facilitated diffusion, the number of transport molecules per
cell is finite. Energy is derived
from hydrolysis of ATP by a transporter which is an ATPase that catalyzes the
breakdown of ATP and phosphorylates itself. This phosphorylation of the
transporter will either alter the affinity of the binding site or the rate of
conformational change.
- Sodium-potassium ATPase, responsible for the outward flow of sodium and inward flow of potassium. Sodium is the primary ion found in the extracellular fluid, while potassium is the ion found, for the most part, inside cells.
- Calcium transport,
- Hydrogen transport, and
- Hydrogen-potassium transport.
NOTES: Referring to the figure of results: A: In experiment “1” the NaCl is present in L beaker and nothing on the R beaker in the presence of 1 mM ATP. B: In experiment “2” the NaCl is present in L beaker and KCl on the R beaker in the presence of 1 mM ATP. C. In experiment “3” the NaCl is present in L beaker and KCl on the R beaker in the presence of 4 mM ATP instead of 1 mM ATP. D. In experiment “4” the same experimental condition as in experiment 3 EXCEPT the number of transporters were increase to 900.
Virtual Experiment on Osmosis & Diffusion & Osmotic fragility test:
In biological systems a solution consists of water, which is called the 'solvent' and the molecules (or ‘solutes') dissolved in the water. All molecules are in constant motion and collide with one another and the side of a container. If molecules of a solute are concentrated in one area, their movement allows them to become evenly distributed throughout the solution. This movement of molecules from a higher concentration to an area where there is a lower concentration is called diffusion. In biological systems, concentration gradients are usually established across a membrane. When a cell uses oxygen, the intracellular concentration of oxygen decreases, and when a cell produces carbon dioxide, the level of carbon dioxide inside the cell increases. Thus, there will be two concentration gradients across the same cell membrane: oxygen has a higher concentration outside the cell, and carbon dioxide has a higher concentration inside the cell. These two concentration gradients, coupled with the fact that these molecules readily move across a lipid bilayer, allow oxygen and carbon dioxide to diffuse across the cell membrane. Polar, or charged, molecules diffuse very poorly through the lipid bilayer, and their movement across cell membranes is facilitated by membrane proteins, which forms channels and transporter molecules. Channels are water-filled pores that allow small charged molecules to pass across a membrane, with a net movement or ‘flux' down their concentration gradients. Channels are usually very specific and allow only one or a few types of molecules {or ions} to pass through. For example, potassium channels allow potassium ions and water to pass through but do not allow other ions to cross the cell membrane. Solutions in the body have a variety of solute molecules and the combined concentration of all molecules in a solution is called its ‘osmolarity’; this term describes the number of molecules in a liter. If two solutions with different osmolarity are separated by a membrane that is permeable only to water, then water molecules will move across the membrane to equalize the ‘osmotic gradient’; that is, water will move from the solution with a lower concentration of solutes to the one with a higher concentration of solutes. This special type of diffusion, where water moves down its concentration gradient, is called osmosis. Biological membranes are selectively permeable because they have channels and transporter molecules that allow certain solutes to pass but not others. Problems may arise when cells encounter molecules that cannot pass through the cell membrane. Sodium is a good example of a non-penetrating solute. Most cell membranes have very few sodium channels. Some sodium may enter the cell but it is quickly pumped out. So sodium may be considered to be non-penetrating. When cells are placed in sodium chloride solutions, depending on the concentration, water may flux across the membrane and makes the cell swell or shrink. The term ‘tonicity of a solution ‘is used to describe what happens to the cell's volume when it is placed into the solution. If there is no net flux of water, the solution is 'isotonic.' If water enters the cell and it swells, the solution is said to be hypotonic, but if water leaves the cell and it shrinks, the solution is hypertonic. This lab examines the tonicity of sodium chloride solutions in relation to erythrocytes (red blood cells}. You will incubate blood samples in different concentrations of sodium chloride and measure the color of the solutions with a spectrophotometer at 510 nm. In normal conditions, this wavelength of light is reflected by the cell membranes. If the cells are placed in a hypotonic solution, water will move into the cells by osmosis and they will burst, or ‘hemolyze.' Less light will be reflected and the amount of light transmitted through the solution (i.e., the transmittance value from the spectrophotometer) will increase. On the other hand, if the cells are placed in a hypertonic solution and they shrink, the transmittance will decrease. This technique, therefore, allows you to monitor the size and integrity of the red blood cells incubated in salt solutions.
Procedure (see video): In this lab, red blood cells are incubated in a series of salt solutions from 0 mM to 240 mM, or more commonly and conveniently is to use 0.9, 0.75, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.20, and 1.0 g/dl concentration of NaCl solution. A spectrophotometer uses light with a wavelength of 510 nm and the transmittance is used as an indication of the red blood cells. If a salt solution makes the cells swell, or even burst (hemolyze), the transmittance will be high. If, on the other hand, a solution makes the cells shrink, the transmittance will be low. In this way, light transmittance is used to examine whether a salt solution at a particular concentration makes the cells shrink or swell. Defibrinated blood is diluted 1:20 with physiological saline to make a blood solution. Deionized water and a stock solution of 1M or 1% NaCl solution are used to make up solutions between 0 and 240 mM NaCl or 0.9, 0.75, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.20, and 1.0 g/dl of NaCl solution. The appropriate volumes of water and stock solution are measured using a pipetter. The wheel on the side is rotated to draw a fluid up the attached pipette. The fluids are mixed and 5 mL are placed in a tube and mixed with 1 mL of diluted blood. The tubes are allowed to stand for 15 minutes. During this time, the spectrophotometer, set at a wavelength of 510 nm, is calibrated using the tube containing deionized water and the blood solution as a ‘blank. Normally, hemolysis onset at: 0.45-0.50 % NaCl and hemolysis complete at: 0.30-0.33 % NaCl (see results).
Medical
application: Conditions
in which the osmotic fragility test is affected are of two types:
- Low osmotic fragility (increased resistance to hemolysis) is characteristic of: Thalassemia, Iron deficiency anemia, Sickle cell anemia, other red cell disorders in which codocytes (target cells) and leptocytes are found, and after splenectomy.
- High osmotic fragility (increased tendency to hemolysis) occurs in: Hereditary spherocytosis, In spherocytosis associated with autoinunune hemolytic anemia, Severe burns, or chemical poisoning, or in Hemolytic disease of the newborn (erythroblastosis fetalis), Venous blood cells are more fragile than arterial blood cells, and stored blood.
QUESTIONS: 1. Would you say the fragility is increased or decreased in the following situations? a. Hemolysis begins at 0.55% NaCl and is complete at 0.45% NaCl. b. Hemolysis beings at 0.3% NaCl and is completed at 0.25% NaCl. 2. Name one condition where fragility is increased and say why? 3. Name one condition where fragility is decreased. 4. Name one other etiology where fragility is increased in the RBC. 5. What is the difference between isotonic and iso-osmotic solutions? 6. Define the terms hemolysis and crenation. When do they occur? 7. Name some hemolytic agents. 8. What is mechanical fragility and how is it tested? 9. Do all the normal RBCs present in the given sample of blood have the same osmotic fragility to hypotonic solutions? 10. What happens to the osmotic fragility of RBCs in an individual after splenectomy? 11. Define the terms osmosis and osmotic pressure. How much osmotic pressure is exerted by the blood and what is its importance in the body? 12. Explain the mechanism of hemolysis of RBCs in the body