Fundamentals 11:00 - 12:00Scribe: David Davis

Thursday, September 17, 2009Proof: Molly Clark

Dr. WhikehartHormonesPage1 of 8

Hormones [S1]
Hormones have several different subdivisions.
The word hormone comes from a Greek word which means an impetus or stimulus to do something.

Outline [S2]
You might consider this class of compounds act as middle managers. They coordinate a number of activities in the body to make them operate as if they knew what they were doing.
We will talk about the classifications and their chemical types. We will talk about their characteristics and they certainly do have characteristics that are different from other biochemicals.
We will then move on to the synthesis and destruction of these, a little bit about assays and some things about general mechanisms.
The endocrine system itself, which was the original system in which hormones were studied.
Then, some miscellaneous details and we will sum up at the end.

Hormones [S3]
Hormones are essentially messengers or messenger molecules. They are meant to coordinate the operations of biological systems together.
This is something that you might think is done in higher organisms exclusively but that is not true. We know, for example, the insect and in the insect world there are hormones that are used for various things like attracting mates and things of that nature.
Hormones accomplish their job by increasing and decreasing the functions of a cell.
Here on this particular slide, you see an example of two related hormones known as Triiodothyronine (T3) and Thyroxine (T4). The only difference between those hormones is the inclusion of noninclusion of an extra iodine.
I think it should be obvious that this particular hormone is a derivative of tyrosine. One tyrosine is actually piggybacked on the other.
In addition to that, it has iodine added to it. These hormones are secreted by the thyroid gland. They affect general metabolic rates by increasing them.

The First Hormones to Be Discovered [S4]
The first hormone to be discovered was called antidiabetic factor.
That was way back in 1921 by three individuals, Banting, Best and Collip.
They worked in MacLeod’s lab in Toronto, Canada. We know about the first hormone due to Canadian research.
Just to give you an idea of how the discoveries are generated – the diabetic factor is an idea that came about from Oskar Minkoski and Josef von Mering in 1889.
This happened when they were doing experiments which involved creating a diabetic dog by removing its pancreas. Actually, they were looking at how fat digestion occurred.
To say that discoveries by chance is perfectly true and this is a perfect example of that.

Types (Classifications) of Hormones [S5]
We classify hormones in several different ways.
One is by a hormone class known as external cell – this means that these hormones come form some external position, usually the bloodstream and bind to the cell or go into the cell to produce its effect.
These external cell hormones are endocrine, paracrine or autocrine.
The endocrine which are the most well known will be secreted by something known as an endocrine gland into the bloodstream and you see over in that picture on the left side three different cells (three different types). They will then go to target cells and what they d is either bind to a receptor on the target cell or a receptor inside the target cell.
Paracrine and autocrine are hormones known more recently.
Paracrine hormone is one that is secreted for a group of cells in a tissue. Their work is very local. They are not secreted by an endocrine gland, but they are made by a nearby cell for that particular group of cells.
To look at it a bit more selfishly, we have cells that are called autocrine cells that make hormones just for themselves.

Endocrine Hormones….. [S6]
Endocrine cells are made and secreted by endocrine glands.
This can be either be from either the hypothalamic/pituitary system of the brain or by semi-independent systems.
They travel through the bloodstream to some target tissue or some target cell. These endocrine hormones were the original ones to be studied or to be known.
The paracine hormones are made by a local group of cells in a specified tissue,
The hormones are secreted into the interstitial fluid surrounding the cells. Interstitial fluid – the fluid around the cell. It never goes into the bloodstream.
An example, we see cholecystokini-8 which is a hormone of the gastrointestinal tract and you see if is simply a peptide. Hormones can be peptides, which is one type.
Autocrine hormones are hormones made by the cells that use them. They are self-modulating hormones
So, a cell gets stimulated to do something and it automatically puts out one of these autocrine hormones. The hormone binds to the outer surface of the cell – slows down the process. That is what self-modulation is.
Estradiol is a steroid hormone that supports the function of the cells in the womb.

Internal Cell Hormones [S7]
Internal cell hormones are hormones that are made and function entirely within the confines of a cell.
They never go outside of a cell and they don’t function outside of a cell.
They are also known as second messengers. They have to be distinguished by some external hormones like steroids that enter a cell from the outside.
Be careful about that – so we have second messenger cells, they operate from JUST INSIDE THE CELL and we have the external cell hormones that come inside and usually bind someplace on the nucleus.
A very well known example is cyclic-AMP. This is a familiar molecule. We have seen this many times before. We know that AMP is used to make nucleic acids. We know that if you bind three phosphates to it, it becomes ATP, which is the energy compound for many reactions in cells.
Now, we have camp where the phosphate group is bound at the 3’-5’ end of the pentose sugar.
cAMP transfers a signal from the cell surface receptor to a series of molecules which often happen to be enzymes. It will amplify the original signal many fold.
This word amplify, the verb amplify, is the key to all hormone functions because it increase the original signal that was originally meant to be given. So, it isn’t internal cell hormones that do this as well.
This mechanism where we have an amplification is called a cascade mechanism. Cascade is a term that is borrowed by the electronics field. It produces the cascade effect by going from one enzyme to the next and every time is goes to a new enzyme, that new enzyme amplifies the original signal whatever that happened to be.
We have cyclic cGMP as well as camp and cGMP usually produces effects that are exactly the opposite of those of camp.
I did a tissue culture study several years ago looking at the cells of the cornea called the endothelium and we found that these cells were in growth phase – cAMP and cGMP ratios were at one point to one another and if the cells came together in a culture and they stopped growing and the ratio changed.
Calcium and Calmodulin work as a pair for another internal cell hormone system. Calcium usually binds to calmodulin making the calcium not available for whatever effect it happens to produce in a cell. So, that is a two way street – it binds and unbinds.
It is usually done with a series of enzymes.
Nitric Oxide is a gas that also happens to be another internal cell hormone. It does have real effects.

Hormone Characteristics [S8]
What are the characteristics of hormones?
Size (molecular weight) – typically, most hormones happen to be rather small in size.
Exceptions would include polypeptide hormones like growth hormone.
Here we see an example of oxytocin. This is a cyclic peptide that is used to stimulate contraction at birth.
Often, oxytocin, or an analog, will be used to stimulate the birth process – it is cyclic in the sense that there are two sulfur groups that connect two cysteines to the cyclic peptide.
Notice also the molecular weight, it is small. Most of the hormones that we know tend to be this small in size.

Duration of Existence…. [S9]
The duration of existence of hormones is generally that they have short half-lives.
You have to be careful about this term half live. There is a mathematical definition for it. You see that down there at the bottom of the slide.I don’t want you to worry about that because we aren’t going to do any calculations with that strict definition of half live
Because hormones are influenced by a lot of different things. There duration of existence is not strictly a half-life in the mathematical sense that we know.
Usually, if a particular hormone is made and it exists let us say that this is time and this is amount – all other things being equal, the concentration of the hormone would decay exponentially like something like this. (He drew a chart on the board with time as the X axis and Hormone amount on the Y axis.
All things are not equal because there are systems that manufacture their hormone while the hormone is being used up and also decaying. It usually has some optimal concentration that doesn’t’ vary much, but it does somewhat.
So, other factors that contribute to hormone availability to cause a cellular response would include the concept of bioavailability. You will run into that term in pharmacology as well. For example, is the hormone free or is it bound to a protein that prevents it from doing its thing. Sometimes it does its thing while being bound to the protein and sometimes it does not.
What is the rate of synthesis at a given time? Is it maximal or minimal and why does the rate change? It depends on what influences during synthesis.
What is the rate of degradation and what is the tissue contribution to that rate of degradation? So, to talk about the duration of existence of a hormone is a complicated thing. It isn’t simple.

More Information About Half Lives [S10]
Consider the hormone Thyroxine (T4). It has a half-life of seven days with a normal blood level of about 7.5 micrograms per 100 mL of blood. By the way, you will often see things indicated clinically that is chemicals in the bloodstream in terms of dL, which stand for deciliter. That is simply 100 mL usually of serum but, sometimes of plasma. The difference between serum and plasma is that one has the blood cells removed and the other has red blood cells AND clotting factors removed. That is the way that these concentrations are expressed clinically.
After 21 days, it is a time that would represent 3 half-lives. The amount would be reduced to about 0.94 micrograms per 100 mL except that the supply is constantly renewed by the thyroid gland.
Here are some typical half-lives of some various kinds of hormones. Notice how short they tend to be.
Thyroid releasing Hormone (releases the thyroid gland hormones) – 2 minutes.
Insulin – 6 minutes
Glucagon which operates exactly opposite to the effects of insulin – 7 minutes.
Growth hormone – 25 minutes.
Cortisol – 90 minutes.
Some hormones can actually exert their effects for days in terms of their half-lives.
If these processes are so complicated, we have to think about factors that involve synthesis of the hormone as well as factors that take away from the life of the hormone, why would we even consider the half-life of a hormone? The reason is very simple.
It gives us some comparison to how much control is necessary for the hormone to exert its particular influence. If you have a hormone with a short half-life, that is a fairly good indicator that the control of that hormone’s existence has to be very important and that is why, for example, things such as glucagon ad insulin have such short half-lives because we need that to be able to control things such as fatty acid synthesis and to be able to control the levels of uptake of glucose from the blood.

Overall [S11]
Overall, I can tell you reasonably that most hormones, once they are made, only remain minutes before inactivation.
Removal by tissues like liver, kidney, and lungs is a source of having them take place.
How, for example, you wanted to do an experiment to find out the half-life of a particular hormone. You would carry that experiment out. You would have to poison your organism from making any of the new hormone. You would start out at a given level measuring that hormone and after a period of time, say 1-4 minutes, and see how much hormone remains in the circulation.
Both, the synthesis and release of hormones are equally important.
Endocrine hormones are released in the blood rapidly once signaling takes place.
As part of the system of checks and balances, some hormones have counterparts that produced opposing effects.
Insulin promotes glucose uptake while glucagon promotes glucose release from the liver.

Example of Varying Levels of Hormones During the Menstrual Cycle [S12]
To show you how much hormone concentrations can vary, here are some hormone levels during the menstrual cycle.
We have estradiol, which is indicated by the black line and progesterone which is indicated by the red curves
If we take a typical menstrual cycle of about 25 days, how much of a variation is there with these particular hormones?
Because, they are going to be involved in preparing the womb for impregnation.

How Fast Can Hormones Cause Effects? [S13]
How fast can hormones cause effects?
A good way to look at that, if you didn’t want to do the animal experiment that I talked about before, is to run a glucose tolerance test
This gives you some idea of how much glucose remains in the blood plasma or the blood serum after an individual takes in a meal or a test dose of glucose. An individual that has a glucose tolerance test comes to the laboratory and is usually given a bottle filled with glucose – it is super sweet and some people can’t tolerate it very well.
You would ingest this and you look to see what happens to the level of glucose in a given period of time.
You see up here some typical results that would take place. If you look at the normal level (described as the gold/yellow line), you will see that the level goes up pretty rapidly for 60 minutes then starts to level down to a normal level which is between 90 and 100 mg per dL.
If you have a serious diabetic, you are going to get a curve that looks like something on top (the red line), that goes up and it tends to stay up for a good period of time. What that indicates there is that there is going to be an insulin insufficiency for that person to uptake the glucose into their cells. It could be more tan that, but that is the simple explanation.
We might have someone that is hypoglycemic and those individuals can give varying results for one of those glucose tolerance tests. A perfectly good example is shown here where they tend to run, in a normal situation, a little bit below normal and they take the glucose water and there isn’t much of a change at all. This indicates that there was another problem. It could indicate that there is a tumor that causes excess output of insulin, so the cells are immediately flooded on the inside with glucose – more than what they need. In fact, there is a condition in which glucose can be toxic.
I think you can see here some examples of how hormones can cause effects just by loading up an individual with a glucose sample.
I like to ask questions about things like this, so be sure to study this.

The principle of Radioimmunoassays [S14]
How do we test hormones? The most common method used today is called a radioimmunoassay.
As a matter of fact, this test became so reliable that it is now automated.
When I was doing research, we used to be able to get one of these kits to test a particular hormone. They cost between 500 and 600 hundred dollars and it gave you maybe 50 or 60 assays.
It was easy to do.
Let me give you an idea of the principle behind it. We can refer to the little examples that you see down below.
What we have are a radioactive and a nonradioactive compound. The compound, in this case, being the hormone that we are interested in.
The radioactive and the nonradioactive hormone will compete with one another for a reaction with an antibody complex.
So, there are two components to this test – one is the radioactive substance that we are looking for and the other is the antibody that will bind to the radioactive or nonradioactive substance that we are looking for.
In running the test, you are going to mix all of these components together and both the radioactive, which is the reference sample, and the substance that you are looking for, which is nonradioactive, will compete for the antibody. If we have a sample, for example, that is rather low in concentration, then the radioactive reference sample is going to win out and you will have a very high radioactivity – that is what you see in the mixture on the left hand side on the bottom.
If we go to the opposite extreme on the other side, where the sample is very high in concentration that is not very radioactive, then the radioactive substance is going to lose out in the relative competition in the radioactive and nonradioactive samples and there will be very low radioactivity when you get your results.
If you were to run a series of controls, which you always must do. (He drew another grid on the board with radioactivity on the Y-axis and concentration on the X-axis.)
As you run this test with a given number of samples, you are going to end up generating a line that looks something like this and then, you will run your unknown and your unknown will be somewhere along this line and it will give you the concentration.