BHS 116.2- Physiology
Notetaker: Stephanie Cullen
Date: 11/14/12, 2nd Hour
Page: 1
Objective: Describe the mechanisms of action of the various types of hormones (adenylyl cyclasecAMP system, cell membrane phospholipid system, Ca++-Calmodulin system, and steroid and thyroid
hormone system).
Mechanisms of Hormone Action
- Hormone receptors
o All the hormones in any of the classes require a receptor in order to exert their effect on
the target cell
o Highly specific to each hormone
o 2,000-100,000 hormone receptors per cell in any of the 3 locations below
o Expression of the receptor is regulated by hormone
 If a lot of hormone is present, down regulate the receptor because they will be
activated
o Can be located in:
 Cell membrane (protein, peptide, or catecholamine)
 Water soluble hormones- have to bind receptor on the surface of the cell
 Cytoplasm (steroid hormones)- intracellular
 Lipid soluble hormones- can directly diffuse through the plasma
membrane of their target cell
 Do not need a cell surface receptor
 Nucleus (most steroid or thyroid hormone)- intracellular
 Lipid soluble hormones- can directly diffuse through the plasma
membrane of their target cell
 Do not need a cell surface receptor
- Intracellular signaling
o Hormone binds to its receptor whether it is in the cell
membrane, cytoplasm, or nucleus
 Elicits a number of different responses on target
cell:
o Change membrane permeability by opening or closing ion
channels (epinephrine)
 Get ion flux within the cell
o Activate intracellular enzymes
 Enzymes go on to activate other proteins in the cell
to bring about a cellular response of that hormone
 Insulin activates a kinase
o Activate genes to directly initiate transcription (produce
proteins that have a number of effects on that target cell)
 Thyroid hormone and steroid hormones
o Response is dependent on the hormone and which receptor it
is binding to but above are the 3 main responses
Second Messenger Systems
- Adenylyl cyclase-cAMP system
o Table 74-2 are some of the hormones that have receptors that are coupled to this system
- Cell membrane phospholipid system
o Table 74-3 are some of the target receptors involved in this system
BHS 116.2- Physiology
Notetaker: Stephanie Cullen
Date: 11/14/12, 2nd Hour
Page: 2
Adenylyl cyclase-cAMP System
- Hormone binds its receptor
- Receptor is coupled with cytoplasmic G-proteins
o G = guanosine nucleotide binding protein
- G-proteins (GTP binding)
o Comprised of α, β, and γ subunits
o At rest, α subunit is bound to GDP
- G-proteins
o Hormone binding to receptor triggers the release
of GDP and binding of GTP to α subunit
- There are 2 types of G proteins that influence the adenylyl
cyclase system
o Gs = stimulatory
o Gi = inhibitory
- Gs α subunit stimulates adenylyl cyclase to produce cAMP
from ATP (2nd messenger)
o 1st messenger: hormone
o 2nd messenger: cAMP
 Typically molecules that aren’t proteins or
steroids that have no other function other
than to activate something else
-
-
cAMP stimulates protein kinase A (PKA) which phosphorylates specific proteins
o Normally sitting around inactive until the cAMP triggers the phosphorylation
This phosphorylation will activate the previously inactive protein to phosphorylate other proteins
These phosphorylated proteins bring about the cellular response to the hormone.
o Opening of channels, stimulating gene production, stimulating release of another
hormone, etc.
o Whatever it is, it can have number of different effects on the cell
If receptor is coupled to Gi –protein, adenylyl cyclase is inhibited.
o Same process: GTP bound α binds to adenylyl cyclase but now inhibits the function of
adenylyl cyclase
 Prevents cAMP formation and the whole
cascade
o Many times the cells have a mix of Gi and Gs coupled
receptors
 Fine balance between whether these signaling
cascades are activated or inhibited
 Depends on which hormones are binding to
specific receptors and being secreted and
binding to the targets
BHS 116.2- Physiology
Notetaker: Stephanie Cullen
Date: 11/14/12, 2nd Hour
Page: 3
Cell Membrane Phospholipid System
- Hormone binds cell membrane receptor
- Receptor coupled to G-protein
o In this case, the protein is Gq
o Are other G proteins but these 3 tend to be prominent in a number of hormone-based
systems
- α binds GTP
- Gq α subunit activates Phospholipase C (effector protein)
o Enzymes whose job is to split phospholipids (phosphorylated lipids)
- Phospholipase C cleaves phosphotidylinositol biphosphate (PIP2) (membrane lipid) into
diacylglycerol (DAG) and inositol triphosphate (IP3)
o DAG and IP3 are second messengers
o Stimulate specific signaling cascades
- IP3 mobilizes Ca++ from the endoplasmic
reticulum resulting in activation of Calmodulin
o IP3 binds primarily to the rhianodine
receptor of the ER
o Triggers the opening for Ca++ to come
out of the ER into the cytoplasm
o Ca++ binds the calmodulin and
activates it
o Ca++ and IP3 are 2nd messengers
++
- Ca -Calmodulin System
o Changes in membrane potential or
hormones interacting with their
receptor can open Ca++ channels
o Upon entering a cell, Ca++ binds
calmodulin
o After Ca++ binds to calmodulin, calmodulin changes conformation and becomes an active
kinase so it now has enzymatic activity and can go on to phosphorylate other proteins
inside the cell
o Protein phosphorylation by Ca++/calmodulin leads to the cellular response to the hormone
 Remember, the Ca++-Calmodulin Complex in smooth muscle and how it leads to
contraction
 Myosin light chain kinase is activated by the active calmodulin
- DAG activates (phosphorylates) protein kinase C (PKC) which phosphorylates many proteins
leading to the cellular response to the hormone.
o Protein kinase C is different from phospholipase C
 Phospholipase C is an enzyme targeting lipids
 Protein kinase C is an enzyme targeting other proteins
o DAG is a 2nd messenger
Amplification of Hormone Signal
- A single hormone/receptor interaction (initial signal) can lead to an amplified signal throughout
the cell
o A little goes a long way
o One active receptor can activate a number of proteins: 10 fold
o Each G proteins α subunit can go on to activate a number of adenylyl cyclases to make a
number of cAMPs: another 100 fold amplification for a total of 1000
o Each cAMP can only activate a single protein kinase so still 1000
BHS 116.2- Physiology
Notetaker: Stephanie Cullen
o
o
Date: 11/14/12, 2nd Hour
Page: 4
Active kinases can phosphorylate/activate a large number of proteins another 100 fold:
100,000 fold total
Each protein can produce a number of end products: 10,000,000 fold from that single
hormone binding to a single receptor
Steroid Hormones Act Mainly on the Genetic Machinery of the Cell
- Steroid hormone enters cell by diffusion and binds to its receptor in either the cytoplasm (few) or
the nucleus (most)
- Cytosolic hormone-receptor complex diffuses into the nucleus through the nuclear pores and
binds to specific sites on DNA to stimulate gene transcription (transcription factor)
- If it binds to a nuclear receptor, then the hormone will diffuse into the nucleus and bind its
receptor in the nucleus
- Need the hormone/receptor complex (not just one or the other) to bind to the DNA
- Activation of transcription eventually resulting in new protein
Thyroid Hormone Increases Gene Transcription in Nucleus
- Thyroxine and triodothroxine bind receptors in nucleus
o Diffuses into the nucleus through the plasma membrane
o Hormone/receptor complex is a transcription factor
- Activate gene transcription for many proteins (especially metabolic enzymes)
- Continue to function for days to weeks (long periods of time)
Mechanism of Action of Thyroid Hormone and Most Steroid
Hormones
1. Hormone is released from plasma protein in the blood and
diffuses through the plasma and nuclear membranes and
binds its receptor in target tissue (steps 1 and 2 on the pic)
2. Hormone/Receptor complex binds to a hormone response
element on the DNA (3)
3. Transcription of specific genes (that have the hormone
response element) ensues (4 and 5)
4. Messenger RNA leaves the nucleus (6)
5. Protein synthesized in the cytoplasm (7)
6. New proteins exert the physiologic response (8 and 9)
o End protein can be channel, enzyme, another
transcription factor, receptor, etc.
o Sometimes the response takes longer because need to
transcribe a new gene
BHS 116.2- Physiology
Notetaker: Stephanie Cullen
Date: 11/14/12, 2nd Hour
Page: 5
Objective: Describe what a radioimmunoassay and ELISA are and how they work.
How Do We Measure the Plasma Levels of Hormones?
- Radioimmunoassay (RIA)
o Typically isn’t used much anymore (was prominent 20-30
years ago)
o Start with a known amount of radioactive (radio labeled)
hormone used to compete with plasma hormone then add a
cold amount of nonlabeled hormone
 Set up a standard curve
 All based on amount of radiolabeled hormone bound to
antibody vs the amount that is free
o Antibody is added and binds to the hormone
o Radioactivity is measured and the amount of bound/free
hormone is determined
 No cold/non-labeled hormone present, have large
amount of bound and a low amount of free
 The more cold (unlabeled) horomone added, the competition for binding site
increases
 Can infer by the bound to free ratio where on the standard curve the patient’s
hormone would be
o Plasma hormone level is inversely proportional to the amount of radioactive hormone
bound
 More hormone in patient’s blood = less radioactive bound
- Enzyme-Linked Immunosorbent Assay (ELISA)
o Color-o-metric diagnostic
o Plasma containing hormone added to assay wells and it binds to
antibody coating the wells (antibodies against insulin coat the
bottom of each well of the plate)
o 2nd antibody is added (from the patient’s plasma) and binds to the
hormone (insulin)
 Wash out the plasma
 Hormone stays bound to its antibody
 Add another antibody that also binds the insulin
 Create a sandwich (sandwich ELISAs)
o 3rd antibody is linked to an enzyme binds to 2nd antibody
 Has an enzyme coupled to the 3rd antibody
 Wash out the excess
o Substrate is added and is converted to a different color by the
enzyme
 Substrate is sensitive to the enzyme that is coupled to the
3rd antibody which causes the color change
o Color change is measured and compared to a standard curve to
estimate hormone levels (proportionate to degree of color change)
 More insulin bound = more enzyme we have in the end
converting more substrate into the active substrate (darker
color is produced by these reactions)
 Directly proportional binding type of assay (not inversely proportional like RIA)
 Darker wells in top picture have more binding
o Can do on small microchips and is very automated to use small amounts
Objective: Describe the general causes of endocrine disorders.
BHS 116.2- Physiology
Notetaker: Stephanie Cullen
Date: 11/14/12, 2nd Hour
Page: 6
General Causes of Endocrine Disorders
- Hyposecretion
o Genetic- Lack of an enzyme involved in synthesis
 Don’t produce hormone so secrete low amounts (if any)
o Dietary- Lack of iodine for thyroid hormone production
o Chemical or toxic- Insecticides
o Immunologic- Autoimmunity
o Other disease processes- Cancer
o Iatrogenic- Physician-induced, surgical removal
 Thyroid removal also removes the parathyroid
 Causes hyposecretion of parathyroid hormone too
o Idiopathic- Unknown cause
- Hypersecretion
o Tumors that ignore the normal regulation (feedback pathway)
o Immunologic factors that mimic hormones and stimulating increased production of that
hormone
- Abnormal Target Cell Responsiveness
o Lack of hormone receptor
o Lack of enzyme in the signaling pathway so can’t get the normal response that the
hormone would normally produce in that cell
Clicker Question: A hormone binds to which of the following to initiate the target cell response?
a. G protein
b. PKC
c. Adenylyl cyclase
d. Receptor
o Binds the receptor which activates the G protein