Transport Systems in Animals INTRODUCTION Multi-cellular organisms require some system to effectively transport gases, nutrients and wastes

from their cells to the outside environment. Consider a single cell in a fluid environment. Like all cells, its membrane is semi-permeable, permitting an exchange of some ions and small molecules (including water) with the external medium by the process of simple diffusion. Which ions and molecules may be transported in this way is determined by the specific chemical make-up of that cell's membrane. Thus, cells are not only semi-permeable, but they are selectively permeable - that is, limiting the passage of certain substances through the membrane (unless they are actively transported by a carrier molecule). An example of this "cellular individuality" may be found in the circulatory systems of animals. In this transport system, the endothelial cells which make up the capillary walls have relatively "leaky" membranes, while the tissues comprising veins and arteries are relatively impermeable. Capillaries, therefore, serve as the sites of "pick=up" and "delivery" of nutrients, gases and wastes and serve as the basic mechanism for the transport systems in animals. Larger vessels convey these materials from one capillary bed to another. The complexity of blood circulatory systems in all organisms can be viewed, therefore, from the evolutionary perspective of delivering the required materials from a source of high concentration (e.g. oxygen-rich air in the lungs) to an area of low concentration (e.g. oxygen-poor medium surrounding an active muscle cell). Different animals, depending primarily on their body size, have evolved different mechanisms for accomplishing this requirement. AIMS: 1. To demonstrate that, while simple diffusion is a rapid process of molecule exchange over small distances, larger multi-cellular organisms need specialized transport systems. 2. To demonstrate how animal transport systems combine bulk flow mechanisms with loading and unloading diffusion steps. 3. To examine the mammalian transport system and determine the tissues specialized for gas exchange and transport. 4. To examine some aspects of the human circulatory system and how they function. OBJECTIVES: 1. How do animal lungs maximize on the terms of Fick's Diffusion Equation? 2. Discuss your experimental conclusions to demonstrate simple diffusion across a semipermeable membrane. 3. State methods in which each of the following structures are adapted for oxygen transport in mammals: (i) capillaries, (ii) arteries and veins, (iii) the heart. 4. Describe the mechanisms for gas exchange and transport in two of the non-mammalian organisms on display and explain the benefits and limitations of those systems compared to mammalian ones. 5. Discuss the nature of haemoglobin and its function in the transport of oxygen in mammals.

6. Determine how certain aspects of the human circulatory system are related to sex, age and activity. FACTORS OF MOLECULAR SIZE AND STRUCTURE IN PERMEABILITY Purpose: To investigate the effects of molecular size and chemical structure on diffusion of various substances through a semipermeable membrane. Procedure: You will be given one of the following test solutions. a) Glucose - 0.01M solution b) Chloride - 0.9% NaCl c) 2% starch d) egg albumin (protein) Before setting up the experiment, test your solution in the appropriate way. a) Glucose Immerse 4 cm testape into the solution. Remove and wait 1 minute. A yellow colour indicates that it is free of glucose. A colour change to green indicates the presence of glucose. b) Chloride Acidify 5 drops of solution with 5 drops of 3M HNO 3. Add 1 drop of 0.5M AgNO 3 and allow to stand. A white precipitate indicates the presence of Cl. Add a few drops of IKI solution to several drops of the test solution. A blue colour indicates starch. To 2-3 mL of the test solution, add Biuret reagent one drop at a time with mixing. Violet colour indicates protein.

c) Starch d) Protein

Tie a knot in the end of a piece of soaked dialysis tubing. Using a pipette, half fill the bag with test solution. Tie another knot on the open end and immerse in a beaker of distilled water. Test the distilled water before immersion and every 15 mins. thereafter for 1 hour for the presence of your test solution. Report your results and those of the other groups on the bench who are doing different solutions. Questions 1. The molecular weight of glucose is 180. How fast does it penetrate the membrane as compared to chloride ion (MW=35)? 2. What chemical bonds can be found between glucose and water? 3. Does glucose enter the cell by simple diffusion?

TRANSPORT SYSTEMS A model of a transport system is diagrammed in Figure 1. Note that, in living systems, the "sink" and "source" for one substance may be the "source" and "sink" for another. Thus simple diffusion of different substances is occurring in both directions across the permeable membranes. Transport systems are basically stirring mechanisms which allow rapid rates of transport in large organisms. Simple diffusion is involved in loading and unloading transport systems. Refer to Figure 1. The net efficiency of this operation is dependent on the following factors: (i) (ii) (iii) (iv) (v) the substance should be absorbed quickly as the carrier fluid (e.g., blood, in vertebrates) flows by the source area - i.e., diffusion into the systems must be rapid. the absorbed substance should remain in high concentration until it reaches the sink and not diffuse into inappropriate tissues. once the substance has reached the sink, it should diffuse rapidly out of the system. forward flow should be maintained within the system. carrier fluid pressure and volume must be held constant within the system.

Figure 1. Animal transport system

In mammals, the efficiency of the circulatory system has been optimized in a number of ways. Use your text and the following investigations in the lab to discover how each of the five efficiency criteria have been addressed in mammals. Specifically, you will study the transport of the gas, oxygen, within the body. Whereas many molecules (e.g., glucose) can be effectively delivered to cells in the body by simply dissolving it in the blood, oxygen must be concentrated by some secondary method (i.e. transport molecule) to reach the levels required by actively metabolizing cells. 1. Lungs - the oxygen source Examine the prepared slide labelled "Lung and bronchioli section". Make a carefully labelled diagram of the features which you see in the slide. Note the spherical spaces which are the alveoli. Determine the presence of capillaries and estimate the thickness of an alveolar and capillary wall. How does the design of the alveoli facilitate oxygen diffusion? Study Figures 2, noting the close association of each alveolus with a capillary network.

Figure 2. The Human Respiratory System

How lungs maximize gas exchange - Ficks Equation All adaptations that maximize respiratory gas exchange influence one or more components of the simple equation seen in Ficks law of diffusion. Ficks law applies to all gas exchange systems.

Q = DA( P1 P 2 ) L

Where Q is the rate at which a substance such as O2 diffuses between two locations D is the diffusion coefficient, which is a characteristic of the diffusing substance, the medium, and the temperature A is the cross-sectional area over which the substance is diffusing (note: internal surface of lungs has an enormous area of 750-1000 square feet) P1 and P2 are the partial pressures (concentrations) of the substance at two locations L is the distance between those locations Animals can maximize D for respiration by using air rather than water as their gas exchange medium. Why is this true? An important factor for gas transport is that the blood contains hemoglobin, a substance which reversibly combines with O2 and takes it out of solution. Therefore the internal O2 concentration is kept at almost zero. Important to exchange of gases at the alveoli is that the alveolar walls are extremely thin, so L in the equation is very small and that the blood capillaries into which the O2 is diffusing are closely associated with the alveolar walls. 2. Blood constituents - the transport molecules a) Human blood cells Examine the prepared slide of a blood smear and identify the different types of blood cells you see by using the chart on the next page and figure 3. The most abundant cells are red blood cells (erythrocytes) which are the sites of oxygen binding and transport. Scattered amongst these cells are a variety of white blood cells (leucocytes) containing darkly stained nuclei. While erythrocytes are responsible for gas exchange, the leucocytes function primarily in the body's defense and immune system. Think about the multi-functionality of the mammalian circulatory system and how one set of structures can be used for other purposes. Note the appearance of the erythrocytes. They should appear smoothly rounded. (Occasionally, the cells will appear "prickly" indicating improper fixing with the Wright's stain). Scan a known section of your slide and count the different cells and enter these data on the blackboard along with the other required information.

Figure 3. The composition of blood

Blood Cell Type Erythrocytes

Name

Abundance (# per cc) 5,000,000

Diameter

Function

Description

Drawing

R.B.C.

7.2 m

Gas transport

Cell resembles a bioconcave disc. Nucleus absent.

Lymphocyte

2,000

8-12 m

Produce antibodies

Cytoplasm agranular. Spherical nucleus occupies most of cell. Cytoplasm agranular. Nucleus fairly large and horseshoeshaped. Cytoplasm granular. Nucleus has two lobes.

Monocyte

200

15 m

Phagocytosis

Leukocytes

Eosinophil

200

15 m

Phagocytosis

Basophil

50

10 m

Phagocytosis

Cytoplasm granular. Nucleus has two lobes.

Neutrophil

5,500

10 m

Phagocytosis

Cytoplasm granular. Nucleus with 1-5 lobes, depending on age of cell.

b) Determination of haemoglobin - the hematocrit In this section you will determine the hematocrit of bovine whole blood. Blood spun in a centrifuge separates into two distinct portions, the packed cells (red and white) and the plasma. If the blood is placed in a small tube of constant bore and then spun in the centrifuge, the height of the column of packed cells expressed as a percentage of the height of the cells and plasma combined is known as the hematocrit and is an indirect measure of the haemoglobin content of the blood. Procedure: 1. 2. Obtain a heparinized capillary tube from the demonstrator. Place the end of the capillary tube into the bovine blood and allow the blood to fill the tube by capillary action until it is at least 3/4 full. Place a finger over one end of the tube to prevent the sample running out and seal the other end by pressing it into the sealing compound with a slight twisting motion. Tilt the tube about 45 degrees to release the tube and withdraw. This should prevent the plug from being sucked out. Tap the tube, plugged end down, once or twice on a smooth surface to strengthen the seal. Place the sealed tube into one of the numbered slots of the hematocrit centrifuge and remember the number of your slot. Once the centrifuge is full the demonstrator will spin the samples for approximately 5 minutes. It is advisable to prepare duplicate samples in case one of the seals leak. Once the samples have been spun, remove your capillary tube and determine the hematocrit of your blood. Record the data on the appropriate chart on the blackboard.

3.

4. 5. 6.

7.

c) Amphibian blood (frog) Examine the prepared slides of frogs blood. The majority of cells present in this vertebrates blood consist of erythrocytes. How do these cells differ from your own? Speculate on whether mammalian erythrocytes are more efficient at transporting oxygen and how the frog differs in its gas exchange requirements. Compare the relative surface area to volume ratio for the two types of erythrocytes. d) Insect haemolymph (grasshopper) Examine the prepared slide of insect haemolymph and note the extremely reduced cell concentration for these organisms. Insects do not use their haemolymph for transportation of oxygen, employing instead a form or direct cell contact with the environment. This has left the insects with little need for extensive transport molecule packages (i.e., red blood cells). Draws the cells that you see and speculate on the possible functions of this transport system.

3.Haemoglobin Haemoglobin (Hb) is a respiratory pigment found in many different animals. Its structure varies slightly in molecular weight and oxygen-combining properties according to the species in which it is found. All types of Hb possess a haeme group with an iron (Fe) atom at its centre (Figure 4). When O2 binds to this iron atom, the pigment is said to be oxygenated and is called oxyhaemoglobin (Hb0). This complex renders the blood a bright red colour. When deoxygenated, Hb gives the blood a darker, purplish-red colour. In the Vertebrata, Hb is carried by erythrocytes and is a complex protein molecule consisting of four intricately folded polypeptide chains. Each Hb molecule has an affinity for up to four oxygen molecules (Figure 4).
CH3 C C N : : N ....... Fe CH CH2

C C CH C N C N C CH CH2 C CH3

HC CH3 C C

CH2 CH2 COOH

C HC C C CH

C C CH3

CH

CH2 COOH

Figure 4. The Haeme Molecule As the erythrocytes file through the lung capillaries, the Hb is placed into an oxygen-rich environment (partial pressure, (pO 2) = 100 mm Hg) and becomes almost 100% saturated with oxygen. The extent to which the HbO dissociates is determined primarily by the pO 2 in the fluid around the erythrocyte. A relatively small drop in the pO 2 in the tissues causes a large dissociation of O2 from HbO (the steep part of curve in Figure 5). Dissociation is also influenced by pH and is more rapid in an acid environment. During exercise, when more O2 is used by the muscle cells, more CO2 is formed and picked up by the blood where it reacts with H2O to form carbonic acid. This lowered pH causes the HbO to release more O2 to the tissues where the need is greatest (lower curve in Figure 5). This pH

dependent dissociation is called the Bohr effect. On returning to the lungs, release of CO2 by the blood reduces its acidity and facilitates the formation of HbO. Due to conformational changes in the Hb molecule when it binds to its first O2 molecule, it has greater affinity for O2 which facilitates binding of the other three O2 molecules. This phenomenon is called co-operative binding.

Figure 5. Human dissociation curves at pH 7.2, 7.4 and pH 7.6

BLOOD CHARACTERISTICS OF SOME ANIMALS Haemoglobin content in gm/100 mL 15 ------11.0 ------------------5.0 4.7 6.5-9.9 ---------------0.0 ---7.8 8.5 10.5 ---------8.5 8.4 -------

Animal mammals man sheep rabbit dog vicuna llama platypus birds ostrich fish (Marine) Scomber scombrus (mackrel) Atlantic salmon Notothenia neglecta Notothenia rossii Onchorhyncus kisutch Lophius piscatorius Opsanus tau Spheroides maculatus Stenotomus chrysops Prionotus carolinus Chaenocephalus aceratus fish (freshwater) Atlantic salmon Ictiobius cyprinellus Itiobius bubalus Cyprinus carpio Carassius auratus Rutilus rutilus Esox lucius Salvelinus fontinalus Salvelinus fontinalus Salmo gairdneri Marine worms Urechis caupo

Hematocrit % 46.0 35.3 35.0 34.6 29.8 38.6 50.0 33.8 37.1 39.4 25.2 24.0 32.5-52.5 15.5 19.5 17.5 32.6 24.0 0.0 24.8 24.6 32.9 31.3 36.0 46.0 29.1 27.2 37.0 26.0 18-40

O2 Cap in vol % 21.0 15.9 15.6 ---17.0 23.5 23.5 13.9 15.8 12.3 ---------5.07 6.21 6.75 7.30 7.66 0.65 8.8 ---------11.3 13.4 8.96 ------9-10 2.66-7.22

Certain other animals (e.g. some Arthropoda and Mollusca) have a similar respiratory pigment in their haemolymph containing copper in place of iron. This pigment is called haemocyanin. Speculate on how this molecule might have evolved and why certain arthropods require a form of gas transport system whereas others do not. 4. Blood Vessels Obtain a prepared slide labelled "Artery, vein and nerve". Scan the slide to locate the artery and vein. Compare their structures. Notice that the vein will be somewhat collapsed, due to the thinner walls while the artery retains its shape by virtue of thick elastic walls. This elasticity of arteries responds to the high blood pressure originating with the heartbeat, and serves to transmit the pulses along to adjacent sections of the vessel. Veins, on the other hand, do not experience this high blood pressure since much pressure is lost through the walls of the capillary beds. How does the structure of an artery provide for the efficient transport of blood? If blood pressure is very low in the veins, how is blood returned to the heart? Make a labelled diagram of a vein and artery under the low power of the microscope.

Figure 6. The walls of arteries and veins

5. Mammalian Heart In groups of four, examine the sheep heart and the model hearts on display. Identify the four chambers, major vessels and valves of the heart. A model human heart has been placed near the sheep dissection for a comparison of these two mammals. Arrange the two hearts in the same orientation and use the key booklet to locate the structures. Compare the relative sizes of the chambers in the two hearts and speculate as to the evolutionary pressures which have given rise to these differences. What important roles are played by the heart and its associated valves and vessels in the efficient transport of oxygen throughout the body? Refer to the five criteria listed earlier.

Figure 7. The human heart and circulation