TRANSPORTERS, CHANNELS & PUMPS

BODY FLUID COMPARTMENTS
Recall from the homeostasis lecture that the human body stripped of fat is ~60% water. Under normal conditions, 3/4 of the extracellular fluid (ECF) is interstitial water (IS) and 1/ 4 of ECF is blood plasma (IVS). Thus only 1/12 (i.e., 1/ 4 x 1/ 3) of the total body water is blood plasma.
   Total body water = ICF + ECF where ECF = IS + IVS

Figure 1. Fluid compartments of the body.

The fluid volumes of the body are measured by the isotope dilution method. In this procedure, a known quantity of a radioactive substance (marker) is administered, allowed to equilibrate within the body compartments and then its concentration is measured in a known volume of plasma. As the total amount administered is known, the volume of the diluted marker can be calculated from its final concentration in the plasma. This quantity is corrected for any of the substance excreted during equilibration and for the half-life (decay) of the radioactive isotope over time. To measure the intravascular space (plasma), radiolabeled albumin is infused; for extracellular markers, inulin is used. The whole body water volume is determined by infusing tritiated water.

SOLUTE & WATER FLOW
One of the key concepts that reoccur in physiology is the importance of gradients and the movement of solutes and water across barriers to maintain normal body function. Biological membranes are bilayers of lipid which restrict the movement of water soluble molecules, such as ions and glucose, from entering cells. In contrast, lipid soluble molecules cross membranes easily. 

DIFFUSION is the passive movement of materials down their concentration gradient. It takes place in an open system or across a permeable partition. There are two types of diffusion (Fig 1):

Simple diffusion - membrane permeable (lipid soluble) molecules cross membranes by simple diffusion.
Characteristics of simple diffusion include:
a. occurs from an area of higher to one of lower concentration i.e., down a concentration gradient.
b. requires no energy expenditure.
c. continues until equilibrium is reached.
d. occurs rapidly over short distances and slowly over long distances.
e. is directly related to temperature (molecules move faster at higher temperatures).
f. is inversely related to the size of the molecule (larger molecules move slower).

Facilitated diffusion- membrane impermeable (hydrophilic) molecules have restricted entry and may not enter cells at all.
Facilitated diffusion occurs with the aid of a transport protein but no input of energy is required. Transport proteins for facilitated diffusion move only one kind of molecule at a time. This process exhibits specificity, competition, and saturation. Glucose enters most cells in the body by facilitated diffusion.



Figure 1. Comparison of solute uptake by simple diffusion and by facilitated diffusion.

Impermeable molecules can enter or leave cells using transport proteins. Transport proteins are classified as either transporters or channels.

Transporter proteins bind to specific molecules (substrates) that they carry across the membrane by changing conformation (shape). They never form a direct connection between the ECF and ICF compartments. Small organic molecules such as glucose and amino acids cross membranes on transporters.

Some transporters bind a single substrate; others bind two or more and are called co-transporters (Fig 2).
Co-transporters are further classified into:
Symporter moves two (or more) substances in the same direction across a membrane.
Antiporter moves two (or more) substances in opposite directions across a membrane.




Figure 2. Co-Transporters include symporters and antiporters.
Channel proteins create a water filled passageway that connects the ECF and ICF compartments. Water and ions move through these channels. The open and closed state of the channels is determined by a part of the channel that acts as a “gate”. The gating of the channel is controlled by ligands (chemically gated), electrical state of the cell (voltage gated) and by tension (mechanically gated).

Solutes can also move across membranes by active transport.

ACTIVE TRANSPORT moves a molecule against its concentration gradient and requires the input of energy (ATP). Active transporters are called “pumps”. They exhibit specificity, competition, and saturation.

Active transport is involved in two types of solute movement:
Primary active transport uses ATP as energy. The transporters are enzymes with ATPase activity. The solute is “pumped” against its concentration gradient.

Secondary active transport couples two processes, primary active transport and facilitated transport. Active transport is used to generate an intracellular ion gradient, usually Na+. This ion gradient is then exploited to allow entry of a solute by either a co-transporter or a single channel. In secondary active transport solutes are moved across whole cells. The ATPase pump is located on the basal side of the cells (nearest to the blood) and the solute enters (or exits) the luminal side of the cell.

OSMOSIS IS THE DIFFUSION OF WATER
Osmosis is the movement of water across membranes. Osmosis only refers to the movement of water and is facilitated diffusion. The water channel is called aquaporin. Water is never actively transported. Water flows from compartments with “dilute” solutions (where concentration of water is high) to “concentrated” solutions (where concentration of water is low) until the concentrations of water and solute are equal in both compartments. Note that the concentration of water is highest in pure water.

Osmotic pressure prevents the movement of water across a membrane. There is no osmotic pressure across a semi-permeable membrane when the water concentration on each side of the membrane is equal.

**** Non-penetrating solutes (e.g., Na+) determine the movement of water and consequently the size of the fluid compartment.

**** Penetrating solutes (e.g., steroid hormones, urea) diffuse freely across cell membranes so no net water movement will occur.

Osmolarity is the number of particles per liter of solution. To predict the osmotic movement of water we must know the concentration of the solutions. In chemistry, concentrations are usually expressed as molarity (M) or number of moles per liter of solution (mol/L). A mole is 6 x 1023 molecules. Molarity describes the number of molecules per liter of solution. Osmolarity (Osmol/L) deals with the number of particles in a liter of solution, not the number of molecules. Because some molecules dissociate into particles when they dissolve in a solution, the number of particles may not equal the number of molecules.

** Molarity (Mol/L) x (number of particles) = Osmolarity (Osmol/L).

For example in Gatorade, the molecule NaCl dissociates into two ions (Na+ and Cl-) but the glucose in Gatorade is only one molecule. NaCl and glucose are both non-penetrating molecules but NaCl contributes 2 osmotically active particles while glucose only one.

For glucose: 1 M glucose x (1 molecule) = 1 OsM glucose
For NaCl: 1M NaCl x (2 ions) = 2 OsM NaCl

Osmolality is an Osmol of solute per kg of water. Osmolarity and osmolality are often used interchangeably because physiological solutions are very dilute and very little of their weight comes from solute.

ISOSMOTIC SOLUTIONS: both compartments (A and B, see below) have the same number of solute particles (includes membrane penetrating and non-penetrating solutes).

HYPEROSMOTIC SOLUTIONS: when solution (A) has a higher number of solute particles than solution (B), solution (A) is hyperosmotic to (B).

HYPOSMOTIC SOLUTIONS: when (A) has a lower number of solute particles than solution (B), solution (A) is hyposmotic to (B). For example:
A = 100 mOsM                       A = 200 mOsM                        A = 100 mOsM
B = 100 mOsM                       B = 100 mOsM                         B = 200 mOsM
(A and B isosmotic)                 (A is hyperosmotic to B)            (A is hyposmotic to B)
Why are non-penetrating solutes important? Non-penetrating solutes are the bases for maintaining cell size at rest and for protection against cell shrinkage or swelling. To maintain homeostasis, the concentration of non-penetrating (osmotically active) particles in the ECF must be maintained within a narrow range (~300mOsM).

**** Osmolarity of the human body is ~300 mOsM.
TONICITY: is used to compare two fluid compartments (e.g., ICF and ECF). It refers to the concentration of non-penetrating solutes only and is always comparative. Tonicity can be estimated but not measured directly.

To determine the relative tonicity of a solution you must know the number of non-penetrating solutes in the solution and in the cell. If the cell has a higher number of non-penetrating solutes then the cell is “hypertonic” with respect to the solution, water will enter the cell, and the cell swells. In this example the solution is “hypotonic” relative to the cell.

For example, if a cell has intracellular tonicity of 100 mOsM. In which of the following solutions will it swell? Shrink?
Solution A = 100 mOsM
Solution B = 50 mOsM
Solution C = 200 mOsM

Answer: The cell will not change volume in solution A (isotonic to the cell). The cell will swell in solution B. The cell will shrink in solution C.

To illustrate the role of a solute in maintaining compartment size, consider what happens when water or solute is ingested (Figure 3).
               A                              B                             C











Figure 3. Effect of either water or solute addition on fluid compartment size. A.Basal state. B. After drinking 3L of water, the ICF increases by 2L and the ECF by 1L. C. After drinking 3L of isotonic NaCl, a solution with the same tonicity as the cells, all of the NaCl remains in the ECF compartment. Why?

What would happen if solid NaCl is ingested but no water? All of the NaCl remains within the ECF. This increases the osmolar concentration in the ECF compartment. To equalize the osmolarity inside and outside the cells, water is drawn out of the cells. The ECF will increase in volume and the cells will shrink. How does the NaCl distribute between the IS and IV spaces?

In later lectures we will see that the kidney’s primary functions are to (1) maintain fluid volumes of the body by regulating salt balance and (2) the osmolarity of the body by regulating water balance. Healthy people use two primary control systems to regulate water or sodium overload:
1. Osmolarity or ion concentration controls the elimination of water in the urine.
2. Changes in blood volume (or blood pressure) control sodium excretion-not osmolarity!

KEY CONCEPTS
1. Movement of solutes across membranes involves simple diffusion, facilitated diffusion, primary active transport and secondary active transport. Water always moves by facilitated diffusion called osmosis.
2. Movement of solute across a membrane is dependent on its size, charge, and lipid solubility.
3. Cellular volume is critically dependent on the steady state movement of solutes and water across membranes in exchange with the ECF. Cells swell in hypotonic conditions and shrink in hypertonic conditions.

THOUGHT PROBLEMS
1. Babla ate an anchovy pizza (very salty) during the super bowl but had nothing to drink. Did this meal alter the size of his ECF compartment? Why or why not?
2. Matin tried to pass his kidney stone by drinking 6 liters of water over 1 hour. His wife became concerned when he became confused and disoriented. At the ER, they were told that Matin had lowered (diluted) the sodium concentration of his IS compartment to 123 mOsM. Did the osmolarity of the IVS change? When the IS becomes hypotonic, does this affect the volume of brain cells (neurons)?
3. Josim ran a marathon. How did his perfuse sweating (loss of hyposmotic water) affect the osmolarity of the ECF (increase, decrease, remain unchanged)? Did the ECF volume increase, decrease, or remain unchanged? Was there a change in water distribution between ECF and ICF compartments?

ANSWERS
1. Yes, the ECF compartment increased. Due to an increase in osmolarity, water moved into the ECF from the ICF.
2. Yes, the sodium concentration in the IVS decreased to 123 mOsM.
If the IS becomes hypotonic to a cell, then water enters the cell causing the cell to swell.
Lowering the extracellular sodium concentration decreases the electrochemical gradient (i.e., makes more negative).
3. Osmolarity of ECF increases because the sweat lost is hyposmotic fluid.
ECF volume decreases because total body water decreases.
Yes, the ECF volume decreases which raises the osmolarity of the ECF. Water moves from the ICF to the ECF to “restore” osmotic equilibrium.

HOMEOSTASIS & BASIC MECHANISMS

TISSUES, ORGANS, SYSTEMS & FLUID COMPARTMENTS
Differentiated cells are cells specialized for a specific function.

Tissues are groups of cells which carry out related functions. The four tissue types include:
epithelium, muscle, nervous, and connective.

Organs are functional units formed by different tissues.

Organ systems include several organs that act in an integrated manner to perform a specific
function. They provide a means for exchange of materials between the external environment
surrounding the body and it’s interior. The ten organ systems of the human body include
cardiovascular, respiratory, digestive, endocrine, immune, integument, musculoskeletal, nervous,
reproductive, and urinary.

The body can be divided into two fluid compartments : ICF and ECF.
Intracellular fluid (ICF) is the cytoplasm within cell.
Extracellular fluid (ECF) surrounds the cells and serves as a buffer.



Figure 1. Fluid compartments of the body.

The ECF is divided into the interstitial fluid (ISF) that bathes the outside of the cells and the
intravascular fluid (IVF) (i.e., plasma, lymph, and cerebral spinal fluid) (Fig. 1).
In the adult 70 kg male, approximately 60% of body weight is water. Under normal conditions,
2/3 of this is ICF and 1/3 is ECF of which ¾ is interstitial fluid and ¼ intravascular fluid.

Because most capillaries that separate the ISF and IVF are leaky, the composition of these two
compartments is essentially identical. The main difference is that the IVF has higher protein
content. However, the composition of the ICF and ECF differ (Table 2, Fig. 2) due to the
hydrophobic nature of the cell membrane which prevents free exchange of ions and proteins.

ICF is a reducing environment that has a high concentration of K+, but low concentrations of
Na+ and free Ca++. Additionally, the concentrations of phosphates and proteins in the ICF are
greater than in the ECF (Table 1).

 ECF is an oxidizing environment that has low concentration of K+ but high concentrations of
Na+ and free Ca++ (Table 1).

TABLE 1. ELECTROLYTES (mM) IN HUMAN CELLS

Ion                                    ECF (Plasma)                                  ICF (Cytosol)
Na+                                        140.0                                                 15.0
K+                                             4.4                                                140.0
Ca++                                         1.2*                                                  0.0005
Cl-                                         105.0                                                    7.0
* Plasma contains bound as well as free Ca++

In most cells, there is a passive leak of K+ across the plasma membrane allowing K+ ions to move
from the inside of cells to the outside. This leak is matched by pumping K+ back into the cell via
the Na+ -K+ ATPase, an integral membrane protein. The movement (pumping) of K+ back into the
cells requires energy (ATP). During each cycle of the ATPase, two K+ are exchanged for 3 Na+
and one molecule of ATP is hydrolyzed to ADP.

When K+ is pumped into cells, Na+ is pumped out. This generates an unequal distribution of Na+
and K+ across the plasma membrane which is called a chemical gradient. The unequal
distribution of ions also establishes a charge (electro) gradient with the inside of the cell more
negative relative to the outside of the cell. The “electrochemical” gradient represents a storehouse
of energy (called the electrochemical potential). Sodium ions can enter cells through special
protein channels. When Na+ enters, it moves passively down its electrochemical gradient. Its
entry is matched by the rate of its removal via the Na+ -K+ ATPase so that the intracellular
concentration of Na remains low and constant. The actions of the Na+-K+ ATPase pump balance
the amounts of Na+ and K+ entering and leaving the cell per unit time; however, their intracellular
and extracellular concentrations are NOT equal. This is called a steady state. Metabolic energy
(ATP) is expended to maintain a steady state.

EQUILIBRIUM, STEADY STATE & HOMEOSTASIS
The keys to maintaining stability of the ECF are self-regulatory mechanisms which allow us to
adapt to a changing environment. To understand these adaptations, we need to consider the
concepts of equilibrium and steady state.

Equilibrium is a condition in which the opposing forces are balanced. There is no net transfer of
a substance (or of energy) from one compartment to another. An equilibrium state will occur if
there is sufficient time for exchange and if there is no barrier to movement from one compartment
to the other. No energy expenditure is required to maintain an equilibrium state.

 Steady state, is a condition in which the amount (or concentration) of a substance is constant
within a compartment and does not change with time. There is no net gain or net loss of a
substance in a compartment because the input and output are equal. A steady state is not
necessarily an equilibrium state. Energy expenditure may be needed to maintain a steady
state.

Homeostasis is the maintenance of the ECF as a steady state. When conditions outside of the
body change (e.g., temperature), these changes are reflected in the composition of the ECF which
surrounds the individual cells of the body. The ECF is the site of exchange where nutrients are
delivered and cellular wastes removed. Therefore the composition of the ECF dynamically changes with time, but certain factors must be kept within a narrow range for optimal functioning
of cells, tissues, and organs. These specific factors include oxygen (O2) and carbon dioxide
(CO2), glucose and other metabolites, osmotic pressure, concentrations of H+, Ca++, K+, Mg++,
and temperature. Uncorrected deviations can lead to disease and/or death.

HOMEOSTATIC REGULATION
To maintain homeostasis, the functions of various organ systems must be integrated. Both
homeostasis and integration require that the cells of the body (~ 75 trillion!) communicate with
each other in a rapid and efficient manner. There are two basic types of extrinsic physiological
control paths: local and reflex.

Local control involves paracrine (between neighbors) and/or autocrine (self-to-self) responses.
Proteins called cytokines mediate local control.

Reflex control involves the nervous and endocrine systems. Reflex control responds to changes
that are more widespread or systemic in nature. In a reflex control pathway (or loop), the decision
to respond is made at a distance from the target cell or tissue. Reflex control has three basic
components (Fig 2): an input stimulus, integrator of the stimulus, and a response (effector).


Figure 2. Components of a reflex loop.

The integrating center evaluates the incoming signal, compares it with a set point (desired value),
and decides on an appropriate response. The effector carries out the appropriate response to bring
the situation back to within normal limits. Reflex pathways are closed loops.

Mass balance in the body refers to a steady state in which the total amount of a substance
equals its intake plus its production minus its output.
                     Total body content of X = intake of X + production of X – output of X.

Mass flow is mass balance over time, such that:
   Mass flow (amt/min) = [concentration (amt/vol)] X [volume flow (vol/min)]
For example, infusion of 4g of glucose in 10 ml at a rate of 2 ml/min gives a mass flow of:
                  (4g /10 ml) X (2ml/min) = 0.8 g/min
There are several different types of reflex pathways within the body. These include negative
feedback, positive feedback, feed forward, tonic control, antagonistic control and circadian
rhythms.
In Negative feedback loops, the response removes the stimulus (Fig 3). A critical consequence of
negative feedback control is that it allows the system to resist deviation of a given parameter from
a preset range (or set point). Negative feedback is the most common form of homeostatic control
in biological systems.

Figure 3. Negative feedback control systems responds to external change that lowers body
temperature.
In physiological systems, we encounter two types of negative feedback systems (Fig.4): simple
(A) and complex (B). The complex negative feedback system permits finer control.


Figure 4. Simple and complex negative feedback loops. (A) Simple negative feedback
involves two cellular compartments. (B) Complex negative feedback involves more than two
cellular components. Typically the feed back signal inhibits secretion at all previous levels.
In positive feedback loops, the response reinforces the stimulus rather than decreasing or
removing it (Fig. 5) and is therefore an unstable condition. The consequence of positive feedback is not to maintain homeostasis but to elicit a change. Positive feedback loops are found during
development or maturation. They are finite loops; often negative feedback will reduce or
terminate these responses.


Figure 5. Positive feedback loops. A positive feedback occurs when a hormone
signal increases its stimulation rather than decreasing it.

Feed-forward Control enables the body to anticipate a change and start a reflex loop. For
example, the sight, smell, or even the thought of food starts our mouths to water. The saliva
lubricates the food particles during chewing.

Tonic Control permits the activity of the organ system to be modulated (either up or down). This
is like the volume control on a radio which enables you to make the sound louder or softer by
turning a single knob. For example, the diameter of a blood vessel is set by the activity of the
sympathetic nervous system (Fig. 6). A moderate rate of signaling from the nerve results in a
blood vessel of intermediate diameter. An increase in the rate of signaling by the nerve results in
constriction of the vessel; a decrease in signaling leads to dilation.



Figure 6. Tonic control. Physiological parameters that are under tonic control are regulated
by modulation (up-down) rather than by on-off switches. Tonic control is an important
regulator of blood flow to the organs.

Antagonistic Control modulates the activity of an organ system by two separate regulators
which act in opposition. For example (Fig. 7), chemical signals (neurotransmitter) from a
sympathetic neuron increase heart rate, whereas neurotransmitters from a parasympathetic
neuron decrease it.


Figure 7. Antagonistic control of heart rate.

Circadian Rhythms allow control systems to fluctuate in a predictable, timed manner over a 24
hour cycle as their set points change. Circadian rhythms govern many biological functions,
including blood pressure, body temperature, and metabolic processes. Circadian rhythms arise
from special group of cells in the brain (hypothalamus) which are programmed by either the lightdark,
day-night cycle by input from the retina or our sleep (rest) -activity periods. When the
circadian clock is altered (e.g., jet lag), temperature rhythms and the secretion of various
hormones are also altered.

KEY CONCEPT
The human body is an inter-dependent set of self-regulating systems whose primary function is to
maintain an internal environment compatible with living cells and tissues (homeostasis).

Important Generalizations of Homeostatic Control Systems
• Stability of internal variables is achieved by balancing inputs and outputs to the body and
among organ systems.
• In negative feedback systems, a change in a variable is corrected by bringing the body back to
the initial set point. Note that set points can be “reset” at a higher or lower physiological
value.
• Not always possible to maintain everything relatively constant by homeostatic control
mechanisms in response to change. There is a hierarchy of importance in the maintenance of
life.

Problem:
Identify the components of the reflex loop in the following scenario.
You have finished the marathon in just under three hours. You are tired, sweating profusely, and
start to drink Cocacola. After a few minutes you are still tired but no longer sweating or thirsty.

Answer:
Sweating = loss of ECF water (stimulus); the stimulus is recognized by the hypothalamus
(integrator); the thirst response (effector) is triggered; the individual drinks Cocacola; this
removes the stimulus; i.e., no longer thirsty; this is classic negative feedback