Physio Lab 3 Osmosis in Physioex

Physio Lab 3 Osmosis in PhysioEx Do activities 1-8

Activity 1-4, plays with 3 rats: One is normal One missing thyroid gland One missing pituitary gland (hypothalectomy)

Don’t let the hypothalectomy make you think they took out the hypothalamus (will die). It is the pituitary that is removed.

Activity 1, baseline rates Be able to tell which rats have higher or lower metabolic rate. Missing HPT, get low metabolic rate

Activity 2-3, hormone replacement therapy Add thyroxin, T4, the most abundant type of thyroid hormone. Exogenous means it is an outside source. Should have higher metabolism now. Should also rescue other rats, too. If normal rat given exogenous T4, what happens to its HP axis? Everything low.

Activity 3 Give TSH to normal rat, will lead to more stimulation of thyroid gland, increases metabolism. Give TSH to 2nd rat, no target organ, no effect. Nothing to bind to. TSH will affect the 3rd rat; binds to the thyroid gland, stimulates TH production.

Normal rat gets exogenous TSH, what happens with time to its endogenous HTP axis? All would go down. Normal TH though, just upsteam is high. Stop adding it, levels will go up.

Propythiouracil blocks iodination, follicular cells still make thyroglobulin, tries to release it, so there is no T3 or T4, like taking out the thyroid gland. Performing thyroidectomy, not doing anything to it. Equal metabolism compared to baseline. Give to third rat, metabolism will go down but not much. HTP axis is already low.

Hypothalactomy rat give TSH, now it’s back to normal. But give propothyrouracil, TSH can no longer rescue rat. In normal rat, will go down, like thyroidectomy.

Activity 5 Hormone replacement: calcitonin and estrogen Deplete sex hormones in rats, then try to rescue them. Give one rat daily injections of estrogen or calcitonin. Estrogen and calcitonin stimulates osteoclasts. Both rats should be helped, osteoporosis is not worsened, estrogen is better replacement therapy. There is a problem using estrogen because the women have higher incidence of heart attacks. Gonads and hormone replacement therapy: Birth Control Pills (BCP) Hypothalamus releases GTP-RH (gonadotropic releasing hormone) Pituitary releases FSH and LH Stimulates male gonads to make testosterone, and some estrogen and progesterone Stimulates females to release estrogen and progesterone, some testosterone As a female ages, her female levels go down, and the male hormones appear to get higher, but they stay the same, get more hair.

Male hormones lower with age too; their female hormone catch up, get more female characteristics. With age, she looks more dominate, he looks softer.

Hormone replacement therapy by using BCP If a female takes high levels of female hormones, negative feedback reduces endogenous levels, LH, FSH, estrogen, progesterone, go down, but her blood levels are high. BCP are high dosages, so endogenous levels go down. Sometimes it can take a year to get endogenous system back. Some women bounce back immediately: she takes the sugar pills for one week each month. If she forgets the first one-two pills the next week, her body is back to HPT axis, gets pregnant.

Be ready for questions on the HPT axis, endogenous reactions to shut down exogenous

Activity 6 Creating standard curve Plot your standard curve on the graph paper on the Lab manual.

X axis is glucose concentration Y axis absorbency

You should have some value points. Take a ruler and draw the best fit line. Make sure you start at the origin of the xy axis. When you work with a spectrophotometer, you always zero the machine. Do the standard curve to show that your curve obeys the Lambert-Beer law. Concentration and absorbency are linearly and directly related. If glucose doubles, absorbency doubles. This is a direct linear relationship. There are lab techs that do not create standard curves every week on their machine, may not know the machine is wrong. Need to calibrate machines frequently. There is a white light bulb in the machine that emits different light each day. They should make a standard curve every day. If your curve is off, your standards were made incorrectly, need to dump them out and make them again. There is no going ahead, it is not valid. Create a glucose standard curve to make sure you have a linear relationship with absorbency.

Activity 7 Use the curve to measure glucose plasma levels on 5 patients, only one is normal patient. Some are diabetic, some borderline. What if they have impaired glucose receptors? Treatment is diet and exercise because they have type 2 diabetes. They make insulin, but receptors are down-regulated. What if woman was pregnant and had high glucose? That is gestational diabetes. She can make insulin, but the pregnancy hormones download the receptors, as if she had type 2 diabetes. This is a high risk pregnancy, which will be a lot of work for her. She has to come to the lab for a blood glucose test every hour for four hours. If she fails that, goes to nutritionist, taught what foods she can eat; can’t have milk in the morning, although it is okay after lunch time. Milk has a high glycemic index in the morning. No chocolate and carbs. She has to have smaller portions of carbs. One serving of wheat thins is 8 crackers. Sent home with glucose meter, puncture finger one hour after every meal, bring the print out to all doctor appointments. Keep a journal of what she ate. “When you ate this, it caused this spike”. One The baby of this woman gains weight in the thoracic region. The thoracic region gets stuck, may need C- section. Cannot go even one day over due date. During the last few weeks of pregnancy, doctor will keep checking the size to see if they need to induce labor. After delivery, mom will be ok. Baby could have problems maintaining glucose levels, is watched for a few days. There is a 70% chance that later on the mom and the baby will get diabetes. Stress can cause gestational diabetes. One piece of bread is 1 ½ portions…you would have to cut ¼ of the bread slice, fold it in half to make a sandwich. If not compliant, need insulin shots.

Activity 8 Cortisol and ACTH Determine Cushing’s disease (pituitary), or syndrome (excess ACTH from source other than pituitary or high cortisol for some reason). Five different patients are evaluated.

Low cortisol, low ACTH 2 adrenal insufficient, give ACTH, will go to normal

High Cortisol low ACTH is Cushing’s syndrome, tumor or exogenous cortisol

High Cortisol high ACTH is Cushing’s disease. Pit is oversecreting

Low Cortisol high ACTH is primary adrenal insuff (Addison’s).

High Cortisol, low ACTH, Cushing’s syndrome

REVIEW SHEET 8. Animals metabolism will increase, more oxygen used. Patient 2 treated with prednisone. It is exogenous, so is Cushing’s syndrome. PhysioEx 4: Endocrine System Physiology

(Complete all 8 activities and review sheet)

The endocrine system regulates the activity of every cell, tissue, and organ within the body. While each endocrine gland releases a specific hormone(s) with a specific activity, the overall action of the endocrine system is to maintain homeostasis within the body, i.e., maintain a stable internal environment.

Endocrine glands release hormones directly into the blood stream, without use of an epithelial- lined duct. Hormones are chemical signals and these molecules are either hydrophilic or hydrophobic. Those that are hydrophilic are amino acid derived and travel easily in the blood plasma, but may be cleared from the body more readily by the kidneys. These hormones must also bind to a receptor on the 2+ surface of the cell membrane and then activate a second messenger system (e.g., cAMP, IP 3/Ca , or tyrosine kinase). Your instructor will discuss during lecture some example hydrophilic hormone mechanisms and second messenger systems. In contrast, hydrophobic hormones are lipid-derived and can not travel in the aqueous blood plasma very easily. Instead, they require a transport protein. By traveling with a transport protein they are protected from the aqueous environment, but more importantly, the half-life of this type of hormone within the body is much longer because these hormones may distribute into body fat compartments, which can act as a reservoir for the hormone. Additionally, these hormones are not cleared from the body by the kidney as easily. Hydrophobic hormones are able to cross the lipid bilayer of a cell and bind to a receptor within the cell’s cytoplasm or within the nucleus itself. Again, your instructor will highlight in lecture some example hydrophobic hormones and their modes of action. The exception to the above categories is thyroid hormone (TH): this is an amino-acid derived hormone that requires a protein carrier in the blood plasma and can cross the cell membrane to bind to its receptor within the nucleus. In other words, this protein behaves as if it were a hydrophobic hormone.

Regardless of the hormone’s mechanism for binding to a cell’s receptor, ALL HORMONES ALTER THE PROTEIN ACTIVITY WITHIN A TARGET CELL. How they do this can be variable, but ultimately the cell’s activity level has changed (increased or decreased) due to a change in protein activity (again, either increased or decreased). Protein activity can be changed by the following:

Alter gene expression (turn on or turn off the expression of genes)

Phosphorylation of a protein to activate it.

Phosphorylation of a protein to deactivate it. Note that second messengers, used by most hydrophilic hormones, can cause all three events to occur, while hydrophobic hormones traditionally alter gene expression.

Hormones are released from an endocrine gland in one of three ways: hormonal, neural, or humoral. Hormonal means there is a hormone released by another endocrine gland that causes a subsequent endocrine gland to release its hormone. The best example of this type of release mechanism would be the endocrine glands of the Hypothalamus-Pituitary-X axis (where X- stands for another endocrine gland such as the thyroid, gonads, or adrenal glands). An example of a neural mechanism would be the release of epinephrine from the adrenal medulla: A preganglionic neuron of the sympathetic nervous system directly synapses to the Chromaffin cells located in the adrenal medulla. The posterior pituitary also releases its stored hormones via direct synapse of hypothalamic neurons stimulating release of Oxytocin or ADH. Lastly, humoral mechanisms detect a decline or increase in a particular blood substance and adjust hormonal release. For example, when blood sugar levels are high, insulin is released. Conversely, when blood sugar levels are low, glucagon is released. The parathyroid gland also releases a hormone in a humoral fashion. If blood calcium levels are too low, parathyroid hormone is released to stimulate the kidneys to reabsorb more calcium, the intestines absorb more calcium from ingested materials, and osteoclast activity is increased—ultimately leading to an increase in blood calcium levels.

The regulation of most hormones is under the control of negative feedback mechanisms. This basically means that when a hormone’s presence in the blood is high, the hormone will feedback to an endocrine gland that will ultimately slow or stop the hormone’s release. Conversely, if blood levels of a hormone are too low, the decrease in feedback will result in the endocrine gland releasing more of that hormone.

Today’s lab will help you understand the negative feedback mechanisms used by the endocrine system. Additionally, your instructor will cover this in depth during lecture.

Insulin and Diabetes background for PhysioEx:

Insulin is a hormone released after a meal. This hormone signals insulin-dependent cells (e.g., skeletal muscle) to produce more glucose carrier molecules in their cell membranes. These integral proteins allow glucose to enter the cell and therefore, glucose levels in the blood will decrease. A person who does not make insulin or does not have a properly working insulin receptor, will fail to make glucose carriers and therefore, glucose will remain in the blood stream. This condition is known as diabetes mellitus Type 1 and Type 2, respectively.

Before beginning the next activity, you will first determine a glucose standard curve using a machine called a spectrophotometer. Your instructor will describe this machine to you and how it is used. You will have another opportunity, later in the course, to physically use a spectrophotometer. So, today’s experiment is simply to introduce you to the concept behind the spectrophotometer. Basically, the principle is this: the more solute in a solution the more concentrated it is (think of making sun tea- how does the carafe look after ½ hour in the sun vs. 4 hours in the sun?). With increased solute concentration, more light will be absorbed by the solution (the solution appears darker.) Or, another way of saying this, the more concentrated the solution the less light that will pass through the solution because the solute particles impede the light from going through the solution. The spectrophotometer measures how much or how little light is able to pass through a solution. It doesn’t tell you on its readout screen how much got through, rather it tells you how much was trapped, or absorbed by the solution. Hence, the “absorbance” reading. (Note: the term is “absorbance,” not absorption.)

A standard curve is important because it allows you to generate a linear relationship between known concentrations of solutes and their respective absorbance readings. Generating a standard curve of “knowns” allows you to take an “unknown” solution and determine its concentration using your graph—if the standard curve is linear and obeys the Lambert-Beer Law. If the standard curve is not linear, then it can not be used to give an accurate determination of your “unknown” solution. On the next page be sure to generate your standard curve.

 Before you begin, answer this: Why would a person with diabetes mellitus have a higher absorbance reading of their plasma vs. a person without diabetes?  Below, manually graph your standard curve that you generate during the PhysioEx exercise six. Label the axes, and include the appropriate units of measurement. Be sure each axis is linear. Do not attach the printed graph from the PhysioEx exercise. The idea here is to give you some practice with generating graphs.