Body Fluid Compartments and Cell Membranes Linda S. Costanzo, Ph.D.

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1 Body Fluid Compartments and Cell Membranes Linda S. Costanzo, Ph.D. OBJECTIVES: 1. Describe the major and minor body fluid compartments. 2. Compare the total cation and anion concentrations in meq/l in each compartment and comment on their relation to the law of macroscopic electroneutrality. 3. Compare the ion concentrations between the plasma water and the interstitial fluid in terms of the Gibbs-Donnan equilibrium. 4. Describe the cell membrane lipid bilayer structure and the role of the phosophlipids, cholesterol, and glycolipids in cell membranes. 5. Describe the concept of the fluid mosaic membrane model comparing integral and peripheral proteins. 6. Describe the role of the cytoskeleton in cell membrane protein localization and the unique role of glycoproteins. 7. Categorize types of membrane transport with respect to whether they are downhill or uphill and whether they are carrier-mediated. Suggested Reading: Physiology, edited by: R.M. Berne and M. N. Levy, Mosby, 6 th Ed. pp 5-7; Physiology, L.S. Costanzo, Saunders, pp 1-6, 8-12 I. VOLUME AND DISTRIBUTION OF BODY FLUIDS Water content (total body water, or TBW) comprises about 60% of body weight. The percentage varies between 50-70%, depending on gender and amount of adipose tissue. Males tend to have a higher percentage of water than females. Water content is inversely correlated with adipose tissue. Infants have up to 75% body weight as water, which is why severe diarrhea can be life-threatening. Water is distributed between two major compartments: intracellular fluid (ICF) and extracellular fluid (ECF), which are separated from each other by cell membranes. ICF is 2/3 of TBW and ECF is 1/3 of TBW. ECF is further sub-divided into two compartments, the interstitial fluid and plasma compartments, which are separated from each other by capillary walls. Interstitial fluid is 3/4 of ECF, and plasma water is 1/4 of ECF. Lymph, which is part of the ECF, is interstitial fluid that is collected in the lymphatic vessels and then returned to the plasma compartment. An additional minor compartment is the transcellular fluid, which is not part of ICF or ECF. Transcellular fluids are separated from the rest of the body fluids by a layer of cells, and they include gastrointestinal, peritoneal, pleural, and cerebrospinal fluids. Collectively, the volume of transcellular fluids is small, so they are ignored in the above summary numbers.

2 A simple tool is the rule. Approximately 60% of body weight is water (TBW), 40% of body weight is ICF, and 20% is ECF. (ICF is 2/3 of TBW, i.e., 40% of body weight; ECF is 1/3 of TBW, i.e., 20% of body weight.) II. COMPOSITION OF BODY FLUIDS A. Units A few tidbits on units. Please save for reference throughout the course! 1. Concentrations in body fluids are often expressed in molarity, such as mmol/l. 2. For electrolytes, we sometimes use equivalents, such as meq/l, which is the concentration in mmol/l x charge on the ion. Thus, for univalent ions, meq/l = mmol/l; for divalent ions, meq/l = 2 x mmol/l. For example, a Na + concentration of 1 mmol/l = 1 meq/l; a Ca 2+ concentration of 1 mmol/l = 2 meq/l.

3 3. Osmolarity is total solute concentration, expressed in units of mosmoles/liter. Osmolarity is concentration of solute particles, or concentration in mmol/l x number of particles that dissociate in solution (called g, the osmotic coefficient). For example, the osmolarity of 150 mmol/l NaCl = 150 mmol/l x 2 = 300 mosm/l (since NaCl dissociates into two particles in solution, i.e., g = 2). Osmolality is virtually the same thing as osmolarity, but expressed as mosmoles/kg H 2 0. Plasma osmolarity can be approximated as 2 x [Na + ]. 4. Substances like proteins are conventionally expressed in g/dl, where a dl (deciliter) is 100 ml and is also called %. 5. % can mean g per 100 ml. For example, 0.9% NaCl is 0.9 g NaCl/100 ml. It s weird, but that s what it means. Likewise, mg % can mean mg per 100 ml. For example, 5 mg% KCl means 5 mg KCl/100 ml. B. Composition The approximate ionic compositions of the plasma water, interstitial fluid, and intracellular compartments are shown in Table 1. Plasma water and interstitial fluid are ECF, while the muscle cell values represent ICF. Ion Interstitial fluid Plasma Water (meq/l) (meq/l) Muscle cell (meq/l) Cations Na K free Ca ~ 10-4 (rest bound) free Mg Total Cations Anions Cl HCO phosphates proteins other anions (ATP etc) Total Anions Table Note that the total ion concentration, in meq/l, for any compartment (e.g., plasma water) obeys the law of macroscopic electroneutrality,

4 i.e. the concentration of positive charges must always equal the concentration of negative charges. This law applies, and is always true, for any body fluid compartment. (Remember, macroscopic electroneutrality is not just a good idea it s the law!) 2. The individual ionic compositions of the ICF are very different from those of the ECF. For example, the Na + concentration is much lower in the ICF than in the ECF, while the K + concentration is much higher in the ICF than in the ECF. These differences in concentration across cell membranes are created and maintained by a cell membrane Na + /K + pump, that will be discussed in a subsequent lecture. The large Na + gradient across cell membranes that is created by the Na + /K + pump is, in turn, utilized by cells in many critical functions; for example, the Na + gradient is the basis of the upstroke of the action potential in nerve and muscle and is the energy source for the uphill transport of various other solutes (see later lectures). Also, the free Ca 2+ concentration is much lower in the ICF than in the ECF. Cell membrane Ca 2+ ATPase and Ca 2+ -Na + exchange help to keep intracellular free Ca 2+ in the submicromolar range. Also, a large fraction of the intracellular Ca 2+ is sequestered in cell organelles and is released only transiently in connection with important cell functions, such as muscle contraction, signal transduction, and release of hormones or neurotransmitter. 3. The compositions of the plasma and interstitial fluid (both part of the ECF) are very similar, but not identical. The major difference in the composition of plasma and interstitial fluid compartments is that the plasma contains large, negatively charged proteins, while the interstitial fluid is essentially protein-free. This difference, in turn, accounts for the small differences in concentration of small, diffuseable ions (e.g., Na +, K +, Cl - ) in these two compartments. The reason for these differences is that the small ions are free to diffuse between the plasma and the interstitial fluid across the capillary membranes, while the large protein anions are restricted to the plasma. Because the law of macroscopic electroneutrality must be obeyed in both plasma and interstitial fluid compartments, the plasma (with its negatively charged protein) will have a slightly higher concentration of diffuseable cations and a slightly lower concentration of diffuseable anions relative to the interstitial fluid. This special equilibrium, due to the presence of protein on one side of the membrane, is called the Gibbs- Donnan equilibrium, which is discussed below. III. GIBBS-DONNAN EQUILIBRIUM

5 A. Hypothetical example Figure 2. This simplified drawing explains what happens when a Gibbs-Donnan equilibrium is established. Solution 2 has protein (Pr-), and Solution 1 has no protein. In the initial system (before Gibbs-Donnan equilibrium is established), there is 9 meq/l of NaCl in Solution 1 and 9 meq/l of NaPr in Solution 2. The anion Pr - is assumed to be impermeable across the membrane that separates the two solutions, but Na + and Cl - are freely permeable across the membrane. Since Solution 2 initially contains no Cl -, Cl - will begin moving from Solution 1 to 2 down its concentration gradient. To preserve macroscopic electroneutrality (the law!), an equal number of Na + must move with the Cl -. As the Na + concentration in phase 2 increases, Na + backflux from Solution 2 to Solution 1 will eventually prevent further net Na + diffusion, and eventually also stop the further movement of Cl -. Because of the presence of the impermeant protein and because of the requirement for electroneutrality of both solutions, neither Na + nor Cl - can achieve equal concentrations on both sides; however, at equilibrium (the Gibbs- Donnan equilibrium), they achieve a compromise position. This equilibrium condition is expressed as an equality of ion products: where the subscripts 1 and 2 refer to the two solutions, and x is the number of meq/l of Cl - and Na + that have moved from Solution 1 to Solution 2. Solving the equation, for this example, we find that x = 3. At equilibrium, [Na + ] 1 = 6 meq/l, [Cl - ] 1 = 6 meq/l, [Na + ] 2 = 12 meq/l, and [Cl - ] 2 = 3 meq/l. [Pr - ] 2 remains at 9 meq/l. Note that at equilibrium, macroscopic electroneutrality still holds! In this case [Na + ] 1 = [Cl - ] 1, = 6 meq/l and [Na + ] 2 = [Cl - ] 2 + [Pr - ] 2 = 12 meq/l. The equilibrium condition also defines a constant ratio, r, called the Gibbs-Donnan ratio. This is:

6 r = [Na + ] 1 /[Na + ] 2 = [Cl - ] 2 /[Cl - ] 1 In this hypothetical example, r = 0.5. B. Real life example Extending to real life, we are interested in the Gibbs-Donnan ratio for plasma (p) and interstitial fluid (i), where r for the common ions is: Substituting actual values in plasma and interstitial fluid from Table 1, we find that r = Again, this redistribution of small ions across the capillary membrane is due to the presence of negatively charged protein in the plasma but not in the interstitial fluid. IV. CELL MEMBRANE STRUCTURE The membranes that separate the ICF and ECF serve as physical barriers and also contain a variety of proteins involved in transport of substances between the ICF and ECF. In addition, membrane proteins act as enzymes, receptors for ligands such as hormones and neurotransmitters, and as antigens. The basic structure of the cell membrane is a lipid bilayer, which consists of phospholipids, cholesterol, and sphingolipids. The phospholipids present in cell membranes are characterized as amphipathic, i.e. part of their structure is non-polar and hydrophobic, and part of their structure is polar and hydrophilic. Such amphipathic molecules are most stable when they are sitting at the interface between an aqueous phase (polar) and a lipid or oil phase (non-polar). For example at the interface between oil and water, phospholipids form a monolayer with the polar head of the molecule in the aqueous phase and the non-polar long-chain fatty acid tails in the non-polar oil phase. Phospholipids can also form a stable structure separating two aqueous solutions (e.g., ICF and ECF); in this arrangement, the non-polar tails point toward each other to form a bilayer, and the polar heads make contact with the aqueous solutions on either side. This lipid bilayer is the backbone of the cell membrane and accounts for the typical membrane thickness of about 10 nm. lipid monolayer

7 lipid bilayer Figure 3. A. Typical Lipid Profile of a Cell Membrane Lipid Percent by Weight Phosphatidylcholine 17.5 Sphingomyelin 16.0 Phosphatidylethanolamine 16.6 Phosphatidylserine 7.9 Phosphatidylinositol 1.2 Phosphatidic acid 0.6 Lysophosphatidylcholine 0.9 Cholesterol 26.0 Glycolipids 11.0 Table Phospholipids - The first seven lipids in Table 2 are phospholipids. The most abundant of these provide the structural barrier. We have already discussed their critical amphipathic properties. Phosphatidylinositol is present mainly on the cytoplasmic (ICF) side of the bilayer and is of special functional importance because it serves as the substrate for phosphatidylinositol 4,5-diphosphate (PIP 2 ). PIP 2 in turn can be broken down to inositol 1, 4, 5 trisphosphate (IP 3 ) and diacylglycerol (DAG) by the activation of the enzyme, phospholipase C (PLC) The activation of PLC normally occurs as part of a signal transduction cascade in response to the binding of a ligand (e.g. a hormone) to a membrane receptor protein. Both IP 3 and DAG serve as cell second messengers. 2. Cholesterol helps to buffer the fluidity of the non-polar fatty acids of the phospholipids. If present in the proper amount, cholesterol prevents the membrane from becoming either too rigid (wax-like) or

8 too fluid (olive oil-like). 3. Glycolipids These lipids are linked to carbohydrate moieties that extend out from the bilayer into the ECF. They often act as membrane antigens (such as blood group antigens A and B) or, in some cases, as membrane receptors. B. Membrane Proteins Fluid Mosaic Model In this concept of membrane structure, proteins are thought to be either imbedded in or adsorbed onto the surface of the lipid bilayer (see figure below). They are not rigidly held in place (although there are exceptions, see below), but may diffuse in the plane of the bilayer. There are two main types of membrane protein: 1. Integral or intrinsic proteins these proteins usually span the bilayer and have hydrophobic amino acids that make contact with the fatty acid chains of the bilayer and hydrophilic amino acids that contact the ECF and the ICF. It requires detergent action to remove integral proteins from the lipid. Functionally, integral proteins may be receptors, enzymes, ion channels, carrier proteins etc. 2. Peripheral or extrinsic proteins these proteins are bound to either the interior or exterior membrane surface, and are associated with the membrane through hydrophilic interactions. Figure Localization of membrane proteins Some cell types require membrane proteins to be confined to specific regions of the membrane (for example, in epithelial cells, the Na + /K + - ATPase (Na + /K + -pump). Epithelial cells, such as those lining the intestinal mucosa, are polarized cells that sit between two different extracellular fluids; their apical membranes contact the aqueous fluid in the lumen of the intestine, while their basolateral membranes contact the interstitial fluid. For functional reasons that will be described later, the Na + /K + - ATPase is confined exclusively to the basolateral membrane. This can

9 occur because the Na + /K + - ATPase is linked to the membrane cytoskeleton via linking proteins, ankyrin and fodrin (see figure below). The linkage prevents the ATPase from diffusing from the basolateral to the apical cell membrane. Figure Membrane Glycoproteins Carbohydrate moieties can link to membrane proteins especially at the ECF side of the membrane. Growth, differentiation, and maintenance of many cell types depend on interactions between anchoring glycoproteins in the extracellular matrix, such as fibronectin, and specific glycoproteins on the cell surface, called integrins. The interaction sets off intracellular transduction events that, during early development, guide differentiation, and, at cell maturity, regulate cell division and programmed cell death (apoptosis). V. OVERVIEW OF MEMBRANE TRANSPORT The types of membrane transport are: Simple diffusion Facilitated diffusion Primary active transport Secondary active transport (cotransport and countertransport) Osmosis (of water) A. Energetics 1. Downhill transport means a substance is transported down an electrochemical gradient and requires no consumption of metabolic energy. Simple and facilitated diffusion illustrate downhill transport. 2. Uphill transport means a substance is transported against an electrochemical gradient and requires consumption of metabolic energy (either directly in the form of ATP or indirectly in the form of

10 an ion gradient). Primary and secondary active transport illustrate uphill transport. B. Carrier- or non-carrier mediated Transport is further characterized by whether it is carrier-mediated or not. Carrier-mediated transport includes all types except simple diffusion. The features of carrier-mediated transport include: saturation, stereospecificity, and competition. Saturation. Because carrier proteins have a limited number of binding sites for the transported solute, saturation of transport occurs. The point at which all binding sites are occupied by solute is called the transport maximum or T m. (T m is analogous to Vmax in enzyme kinetics.) Stereospecificity. Binding of solute to transporters is highly stereospecific. For example, in the small intestine, the transporter for glucose absorption recognizes the natural isomer, D-glucose,

11 but does not recognize or transport the L-isomer. (Simple diffusion, in contrast, does not distinguish between the natural and unnatural isomers.) Competition. Although transporters exhibit a high degree of stereospecificity, still they can be tricked into transporting chemically similar solutes. Thus, D-galactose can compete for the D-glucose transporter and, by occupying some of the glucose binding sites, inhibit D-glucose transport; this is an example of competitive inhibition. There is also a phenomenon of noncompetitive inhibition, in which compounds that are structurally unrelated to the transported compound bind to the transporter and prevent it from functioning. In the case of D-glucose transport, phloretin is a noncompetitive inhibitor. VI. PRACTICE PROBLEMS 1. A body fluid compartment has a Na + concentration of 15 mm; this compartment is most likely the: A. interstitial fluid compartment B. ICF C. plasma D. small intestinal lumen E. cerebrospinal fluid 2. A solution contains 0.5 mm MgCl 2. The concentrations of Mg 2+ and Cl - in meq/l are respectively: A. 0.5, 0.5 B. 1, 2 C. 2, 1 D. 1, 1 E. 0.5, 1 3. The Donnan ratio for diffusable ions between two solutions (Solution 1 relative to Solution 2) is found to be Accordingly, which of the following describes the Gibbs-

12 Donnan equilibrium condition for these two solutions? A. [Na + ] 1 = 140 mm, [Na + ] 2 = 155 mm B. [Cl - ] 1 = 106 mm, [Cl - ] 2 = mm C. [K + ] 1 = 4 mm, [K + ] 2 = 4.7 mm D. [Ca 2+ ] 1 = 2 mm, [Ca 2+ ] 2 = 2.5 mm E. [Mg 2+ ] 1 = 1 mm, [Mg 2+ ] 2 = 1.25 mm 4. The plasma contains anionic proteins that cannot cross the capillary membranes. When the plasma is in equilibrium with the interstitial fluid: A. the concentration of permeable anions is higher in the plasma than in the interstitial fluid. B. the concentration of permeable cations is higher in the interstitial fluid than in the plasma. C. the concentration of permeable anions will be the same in each fluid phase. D. the concentration of permeable cations will be the same in each fluid phase. E. the concentration of permeable anions is higher in the interstitial fluid than in the plasma. 5. An impermeable anion is in phase 1 which is in Gibbs-Donnan equilibrium with phase 2 containing only permeable ions. The following data are obtained: [K + ] 1 = 6 mm, [K + ] 2 = 3.6 mm, and [Cl - ] 1 = 100 mm. [Cl - ] 2 is then: A.100 mm B.110 mm C. 90 mm D. 167 mm E. 3.6 mm

13 6. In problem 5, at least one other permeable cation must also be present because: A. there must always be 2 different cations present. B. [K + ] 2 < [Cl - ] 2 which appears to violate macroscopic electroneutrality. C. [Cl - ] 1 must equal [Cl - ] 2. D. the permeable anion concentrations in phase 1 must always equal the permeable cation concentrations in phase 1. E. [Cl - ] 1 < [Cl - ] The main permeability barrier in the cell plasma membrane resides in the: A. peripheral proteins B. integral proteins C. lipid bilayer D. lipid monolayer E. phosphatidalinositol layer ANSWERS 1. B (a Na + concentration in this range can only be in the ICF given the choices) 2. D ( [Mg 2+ ] = (2 meq/mmole)(0.5 mmole/l) = 1 meq/l, [Cl - ] = (1 meq/mmole)(1 mmole/l) = 1 meq/l) 3. B (this is the only choice that expresses the correct Donnan ratio, viz. [Cl - ] 2 /[Cl - ] 1 = 100.7/106 = 0.95) 4. E (Since the interstitial fluid has no protein anions, electroneutrality must be satisfied by the permeable anions, which means that the latter will be higher in the interstitial fluid compartment). 5. D (Since: [K + ] 1 /[K + ] 2 = [Cl - ] 2 /[Cl - ] 1, then [Cl - ] 2 = (6)(100)/3.6 = 167 mm) 6. B (There must be a cation, M + (assuming a monovalent cation for simplicity) whose concentration in phase 2 is mm, so that [K + ] 2 + [M + ] 2 = [Cl - ] 2. This means that [M + ] 1 = mm. The impermeable anion concentration must then have been mm). 7. C