Instructor: Dr. Robert Harris
Office: 2530 Biological Sciences
Phone: 822-5709
Email: harris@zoology.ubc.ca

Texts:
Principles of Animal Physiology 2 nd ed.
Moyes and Schulte
Endocrinology 5 th ed.
Mac E. Hadley (recommended) or
Vertebrate Endocrinology 3 rd ed.
David O. Norris (acceptable)

Term Paper:
Midterm Exam:
Final Exam:
Course Total:
50%
30%
20%
100%


Endocrine signaling is probably the oldest control mechanism.

Many organisms get by quite nicely without any kind of nervous system.

Probably arose as mechanism for maintaining stable internal environments.


Endocrine signaling is probably the oldest control mechanism.

Many organisms get by quite nicely without any kind of nervous system.

Probably arose as mechanism for maintaining stable internal environments.


Broadly speaking, a hormone is a chemical that is produced by one specific cell type, secreted into the blood or interstitial fluid, and has specific effects on a different cell type(s).

Secreting cells are usually clumped together to form a discreet gland.

There are two types of glands:

Endocrine

Exocrine


Found in specialized cell types

These cells must synthesize the compound (or precursor) specifically for signaling.
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Cells must secrete the compound (or precursor) in response to a specific stimulii.


There must be a mechanism for clearance of the compound from the blood, or interstitial fluid.
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Drugs that affect the hormone will also affect the hormonal response in the target tissue.

Exogenously applied compound must be chemically identical to the hormone and must mimic the biological effects of the endogenous compound.


AUTOCRINE:

Hormone released feeds-back on the cell of origin, again without entering blood circulation.
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PARACRINE:
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Hormone released diffuses to its target cells through immediate extracellular space.
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Blood is not directly involved in the delivery.


ENDOCRINE:
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Most common (classical) mode, hormones delivered to target cells by blood.

NEUROENDOCRINE:

Hormone is produced and released by a neuron, delivered to target cells by blood.


Only target cells, or cells that have specific receptors, will respond to the hormone’s presence.

The strength of this response will depend on:

Blood levels of the hormone

The relative numbers of receptors for that hormone on or in the target cells
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The affinity (or strength of interactions) of the hormone and the receptor.


The affinity of hormones to their specific receptors is typically very high

The actual concentration of a circulating hormone in blood at any time reflects:

Its rate of release.

The speed of its inactivation and removal from the body.


The half-life is the time required for the hormone to loose half of its original effectiveness (or drop to half of its original concentration.

The time required for hormone effects to take place varies greatly, from almost immediate responses to hours or even days.

In addition, some hormones are produced in an inactive form and must be activated in the target cells before exerting cellular responses.
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In terms of the duration of hormone action, it ranges from about 20 minutes to several hours, depending on the hormone.


Tissue sensitivity may change in response to other hormones (priming).

Effects of photoperiod.
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Effects of temperature.


Extirpation/replacement (classical technique).
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Hormonal source is removed or ablated, to see if the response is abolished.

Once response has been abolished, source tissue or exogenous hormone is replaced in an attempt to restore response.

Injection of hormone
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Allograft
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Injection of live dissociated gland cells


Establish the physiological range of hormone concentrations.


Usually conducted in conjunction with extirpation experiments.
Endogenous source is removed and known concentrations or doses administered to determine the threshold and maximal doses for the hormone.

Other considerations:


Dose-response curve may be biphasic and not sigmoidal.
You must have 8-10 individual animals at each dosage to make results significant.


Radioimmunoassay (RIA):

Used to measure hormone concentration.

Uses antibodies specific to the hormone.
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Also uses radiolabelled purified hormone.
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Must create a standard curve (for competitive binding)

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Drawbacks:
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No guarantee that antibodies are recognizing the biologically active form of hormone.
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May be measuring inactive fragments.
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Often get cross-reactivity with other hormones.
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Closely related hormones have significant sequence homology.


Immunoradiometric assay (IMRA):

Also uses antibodies.

Uses two antibodies, one against C-terminus and one against N-terminus.

One AB is bound to a support matrix (usually avidin-coated beads).

Other AB is conjugated to an indicator of some sort.

Could be radiolabel or fluorescent label.

Measures virtually all the hormone in the sample.
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Only measures intact hormone.
 i.e. both end must be present.


High performance liquid chromatography (HPLC):






Can be used to separate out different hormones.
Like any biochemical purification protocol, it can be used to isolate a specific hormone.
Different hormones will have different migration rates on separation medium.
Can be used to track metabolites of a hormone.
Needs a great deal of characterization using purified hormone.
FPLC is a similar technique.


Immunocytochemistry:

Antibody based and uses two antibodies.

First AB recognizes the hormone.
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Second AB recognizes the first AB (must come from different species).

Second AB is labeled.

Can be used to localize cells secreting hormone.

Take tissue section, fix, permeabilize and label.

Note: All the problems associated with Abs apply.


Receptor binding assay (aka RRA):

Uses competitive binding between hot and cold hormone, usually on the membrane fraction of a cell homogenization.

Will provide the number of available binding sites on target tissue.

Will also provide the binding efficiency.


Molecular techniques:

Currently very “sexy”.

Usually transfect cell line with the gene for the hormone or receptor.

You can also measure mRNA levels (only for peptide hormones).

Must have at least a partial sequence.

Increased hormone production may not be associated with elevated mRNA levels.
 mRNA may code for a processing enzyme.

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The synthesis and secretion of most hormones are usually regulated by negative feedback systems.

Organized into “loops”.

As hormone levels rise, they stimulate target organ responses. These in turn, inhibit further hormone release.


The stimuli that induce endocrine glands to synthesize and release hormones belong to one of the following major types:

Humoral

Chemical changes in the blood (eg. Ca2+ or glucose).

Neural

Hormonal (as seen in feedback loops)


Peptide hormones: largest, most complex, and most common hormones. Examples include insulin and prolactin

Steroid hormones: lipid soluble molecules synthesized from cholesterol. Examples include gonadal steroids (e.g testosterone and estrogen) and adrenocortical steroids (e.g. cortisol and aldosterone).


Eicosanoids: small molecules synthesized from fatty acid substrates (e.g. arachidonic acid) located within cell membranes

Amines: small molecules derived from individual amino acids. Include catecholamines (e.g. epinephrine produced by the adrenal medulla), and thyroid hormones.

Figure 4.16

MAP kinases
• Activated RAS signals to
MAP-kinase-kinase-kinase
• MAPKKK signals to MAPkinase-kinase
• MAPKK signals to MAPkinase
• MAP-kinase phosphorylates a variety of target proteins
Figure 4.17

Transforming growth factor – b ( TGF -b) receptor
• An important serine-theronine kinase receptor enzyme
• Involved in regulating many cellular processes
• Malfunctions result in many diseases including Cancer, atherosclerosis etc.
Figure 4.18b


Ligand binds to transmembrane receptor

Receptor interacts with intracellular G-proteins

Named for their ability to bind guanosine nucleotides

Subunits of G-protein dissociate

Some subunits activate ion channels


Changes in membrane potential
Changes in intracellular ion concentrations

Some subunits activate amplifier enzymes

Formation of second messengers

• Large protein family (over 1000 members)
• Seven-transmembrane domain proteins
• Interact with a trimeric intracellular protein termed “G-protein”
• (guanine nucleotide binding protein)
G Protein Coupled Receptor
From: Boron & Boulpaep
Medical Physiology, 2003, Fig.4-3

Figure 3.25

Structure of heterotrimeric G proteins
Linked directly to the plasma membrane via a lipid anchor a
, b and g subunits
G-protein

There are many types of G proteins – 3 major families
Table 15-3. Three Major Families of Trimeric G Proteins*
FAMILY SOME FAMILY MEMBERS ACTION MEDIATED BY FUNCTIONS
I
II
III
G
G
G
G
G
G t i s olf o q
(transducin) a a a a bg bg and a a bg activates adenylyl cyclase; activates Ca 2+ channels activates adenylyl cyclase in olfactory sensory neurons inhibits adenylyl cyclase activates K + channels activates K + channels; inactivates Ca 2+ channels activates phospholipase Cb activates cyclic GMP phosphodiesterase in vertebrate rod photoreceptors activates phospholipase Cb
* Families are determined by amino acid sequence relatedness of the a subunits. Only selected examples are shown.
About 20 a subunits and at least 4 b subunits and 7 g subunits have been described in mammals.

Table 3.3

Figure 3.27

Important Tools for studying Gs/Gi
Cholera Toxin
– ADP-ribosylates Gs. This locks Gs in the GTPbound conformation, leading to constitutive activation of adenylyl cyclase, and accordingly, increased cAMP production.
Pertussis Toxin
– ADP-ribosylates Gi. This locks Gi in the inactive
GDP-bound conformation. Since activated Gi has an inhibitory effect on adenylyl cyclase, pertussis toxin potentiates cAMP accumulation.
These two toxins are used to determine which type of G protein a receptor is signaling through.

Figure 3.26

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Lecture Notes and synopsis are posted at: