Biol 456Biol 456Comparative Vertebrate Comparative Vertebrate

EndocrinologyEndocrinology

Instructor: Dr. Robert HarrisInstructor: Dr. Robert Harris

Office: 2530 Biological Office: 2530 Biological SciencesSciences

Phone: 822-5709Phone: 822-5709

Email: Email: harris@zoology.ubc.caharris@zoology.ubc.ca

Course requirements:Course requirements:Texts:Texts:Principles of Animal Physiology 2Principles of Animal Physiology 2ndnd ed. ed.

Moyes and SchulteMoyes and Schulte

Endocrinology 5Endocrinology 5thth ed. ed.

Mac E. Hadley (recommended)Mac E. Hadley (recommended)

oror

Vertebrate Endocrinology 3Vertebrate Endocrinology 3rdrd ed. ed.

David O. Norris (acceptable)David O. Norris (acceptable)

Mark BreakdownMark Breakdown

Term Paper: Term Paper: 50% 50%

Midterm Exam:Midterm Exam: 30% 30%

Final Exam:Final Exam: 20% 20%

Course Total: Course Total: 100%100%

Endocrine signaling is probably the Endocrine signaling is probably the oldest control mechanism.oldest control mechanism.

Many organisms get by quite nicely Many organisms get by quite nicely without any kind of nervous system.without any kind of nervous system.

Probably arose as mechanism for Probably arose as mechanism for maintaining stable internal maintaining stable internal environments.environments.

Endocrine signaling is probably the Endocrine signaling is probably the oldest control mechanism.oldest control mechanism.

Many organisms get by quite nicely Many organisms get by quite nicely without any kind of nervous system.without any kind of nervous system.

Probably arose as mechanism for Probably arose as mechanism for maintaining stable internal maintaining stable internal environments.environments.

Definitions:Definitions:

Broadly speaking, a hormone is a Broadly speaking, a hormone is a chemical that is produced by one chemical that is produced by one specific cell type, secreted into the blood specific cell type, secreted into the blood or interstitial fluid, and has specific or interstitial fluid, and has specific effects on a different cell type(s).effects on a different cell type(s).

Secreting cells are usually clumped Secreting cells are usually clumped together to form a discreet gland.together to form a discreet gland.

There are two types of glands:There are two types of glands: Endocrine Endocrine ExocrineExocrine

Features of Hormones:Features of Hormones:

Found in specialized cell typesFound in specialized cell types These cells must synthesize the These cells must synthesize the

compound (or precursor) specifically compound (or precursor) specifically for signaling.for signaling.

Cells must secrete the compound (or Cells must secrete the compound (or precursor) in response to a specific precursor) in response to a specific stimulii.stimulii.

Features of Hormones II:Features of Hormones II:

There must be a mechanism for There must be a mechanism for clearance of the compound from the clearance of the compound from the blood, or interstitial fluid.blood, or interstitial fluid.

Drugs that affect the hormone will also Drugs that affect the hormone will also affect the hormonal response in the affect the hormonal response in the target tissue.target tissue.

Exogenously applied compound must be Exogenously applied compound must be chemically identical to the hormone and chemically identical to the hormone and must mimic the biological effects of the must mimic the biological effects of the endogenous compound.endogenous compound.

MODES OF HORMONE MODES OF HORMONE DELIVERY I:DELIVERY I:

AUTOCRINE: AUTOCRINE: Hormone released feeds-back on the cell of Hormone released feeds-back on the cell of

origin, again without entering blood origin, again without entering blood circulation.circulation.

PARACRINE: PARACRINE: Hormone released diffuses to its target cells Hormone released diffuses to its target cells

through immediate extracellular space. through immediate extracellular space. Blood is not directly involved in the delivery.Blood is not directly involved in the delivery.

MODES OF HORMONE MODES OF HORMONE DELIVERY II:DELIVERY II:

ENDOCRINE: ENDOCRINE: Most common (classical) mode, Most common (classical) mode,

hormones delivered to target cells by hormones delivered to target cells by blood.blood.

NEUROENDOCRINE: NEUROENDOCRINE: Hormone is produced and released by a Hormone is produced and released by a

neuron, delivered to target cells by neuron, delivered to target cells by blood.blood.

HORMONE-TARGET CELL HORMONE-TARGET CELL SPECIFICITYSPECIFICITY

Only target cells, or cells that have Only target cells, or cells that have specific receptors, will respond to specific receptors, will respond to the hormone’s presence. the hormone’s presence. The strength of this response will The strength of this response will

depend on:depend on: Blood levels of the hormoneBlood levels of the hormone The relative numbers of receptors for that The relative numbers of receptors for that

hormone on or in the target cellshormone on or in the target cells The affinity (or strength of interactions) of The affinity (or strength of interactions) of

the hormone and the receptor.the hormone and the receptor.

Major endocrine glands in Major endocrine glands in the bodythe body

HALF-LIFE, ONSET, and HALF-LIFE, ONSET, and DURATION of HORMONE DURATION of HORMONE

ACTIVITYACTIVITY

The affinity of hormones to their The affinity of hormones to their specific receptors is typically very high specific receptors is typically very high

The actual concentration of a The actual concentration of a circulating hormone in blood at any circulating hormone in blood at any time reflects: time reflects: Its rate of release.Its rate of release. The speed of its inactivation and removal The speed of its inactivation and removal

from the body. from the body.

The half-life is the time required for the The half-life is the time required for the hormone to loose half of its original hormone to loose half of its original effectiveness (or drop to half of its original effectiveness (or drop to half of its original concentration.concentration.

The time required for hormone effects to The time required for hormone effects to take place varies greatly, from almost take place varies greatly, from almost immediate responses to hours or even days.immediate responses to hours or even days.

In addition, some hormones are produced in In addition, some hormones are produced in an inactive form and must be activated in an inactive form and must be activated in the target cells before exerting cellular the target cells before exerting cellular responses. responses.

In terms of the duration of hormone action, In terms of the duration of hormone action, it ranges from about 20 minutes to several it ranges from about 20 minutes to several hours, depending on the hormone. hours, depending on the hormone.

Considerations when Considerations when looking for hormonal looking for hormonal

responses:responses: Tissue sensitivity may change in Tissue sensitivity may change in

response to other hormones response to other hormones (priming).(priming).

Effects of photoperiod.Effects of photoperiod.

Effects of temperature.Effects of temperature.

Basic TechniquesBasic Techniques

Extirpation/replacement (classical Extirpation/replacement (classical technique).technique).

Hormonal source is removed or ablated, Hormonal source is removed or ablated, to see if the response is abolished.to see if the response is abolished.

Once response has been abolished, Once response has been abolished, source tissue or exogenous hormone is source tissue or exogenous hormone is replaced in an attempt to restore replaced in an attempt to restore response.response. Injection of hormoneInjection of hormone AllograftAllograft Injection of live dissociated gland cellsInjection of live dissociated gland cells

Establish the physiological range of Establish the physiological range of hormone concentrations.hormone concentrations. Usually conducted in conjunction with Usually conducted in conjunction with

extirpation experiments. extirpation experiments. Endogenous source is removed and known Endogenous source is removed and known

concentrations or doses administered to concentrations or doses administered to determine the threshold and maximal doses determine the threshold and maximal doses for the hormone.for the hormone.

Other considerations:Other considerations: Dose-response curve may be biphasic and not Dose-response curve may be biphasic and not

sigmoidal.sigmoidal. You must have 8-10 individual animals at each You must have 8-10 individual animals at each

dosage to make results significant.dosage to make results significant.

Radioimmunoassay (RIA):Radioimmunoassay (RIA):

Used to measure hormone concentration.Used to measure hormone concentration.

Uses antibodies specific to the hormone.Uses antibodies specific to the hormone.

Also uses radiolabelled purified hormone.Also uses radiolabelled purified hormone.

Must create a standard curve (for Must create a standard curve (for competitive binding)competitive binding)

Drawbacks:Drawbacks:

No guarantee that antibodies are recognizing No guarantee that antibodies are recognizing the biologically active form of hormone.the biologically active form of hormone.

May be measuring inactive fragments.May be measuring inactive fragments.

Often get cross-reactivity with other Often get cross-reactivity with other hormones.hormones.

Closely related hormones have significant Closely related hormones have significant sequence sequence

homology.homology.

Immunoradiometric assay (IMRA):Immunoradiometric assay (IMRA): Also uses antibodies.Also uses antibodies.

Uses two antibodies, one against C-terminus Uses two antibodies, one against C-terminus and one against N-terminus.and one against N-terminus.

One AB is bound to a support matrix One AB is bound to a support matrix (usually avidin-coated beads).(usually avidin-coated beads).

Other AB is conjugated to an indicator of Other AB is conjugated to an indicator of some sort.some sort.

Could be radiolabel or fluorescent label.Could be radiolabel or fluorescent label. Measures virtually all the hormone in the Measures virtually all the hormone in the

sample.sample. Only measures intact hormone.Only measures intact hormone.

i.e. both end must be present.i.e. both end must be present.

High performance liquid chromatography High performance liquid chromatography (HPLC):(HPLC):

Can be used to separate out different Can be used to separate out different hormones.hormones.

Like any biochemical purification protocol, it Like any biochemical purification protocol, it can be used to isolate a specific hormone.can be used to isolate a specific hormone.

Different hormones will have different Different hormones will have different migration rates on separation medium.migration rates on separation medium.

Can be used to track metabolites of a hormone.Can be used to track metabolites of a hormone. Needs a great deal of characterization using Needs a great deal of characterization using

purified hormone.purified hormone. FPLC is a similar technique.FPLC is a similar technique.

Immunocytochemistry:Immunocytochemistry:

Antibody based and uses two antibodies.Antibody based and uses two antibodies. First AB recognizes the hormone.First AB recognizes the hormone. Second AB recognizes the first AB (must Second AB recognizes the first AB (must

come from different species).come from different species). Second AB is labeled.Second AB is labeled. Can be used to localize cells secreting Can be used to localize cells secreting

hormone.hormone. Take tissue section, fix, permeabilize and label.Take tissue section, fix, permeabilize and label.

Note: Note: All All the problems associated with Abs the problems associated with Abs apply.apply.

Receptor binding assay (aka RRA):Receptor binding assay (aka RRA):

Uses competitive binding between Uses competitive binding between hot and cold hormone, usually on the hot and cold hormone, usually on the membrane fraction of a cell membrane fraction of a cell homogenization.homogenization.

Will provide the number of available Will provide the number of available binding sites on target tissue.binding sites on target tissue.

Will also provide the binding Will also provide the binding efficiency.efficiency.

Molecular techniques:Molecular techniques:

Currently very “sexy”.Currently very “sexy”. Usually transfect cell line with the gene Usually transfect cell line with the gene

for the hormone or receptor.for the hormone or receptor. You can also measure mRNA levels You can also measure mRNA levels

(only for peptide hormones). (only for peptide hormones). Must have at least a partial sequence.Must have at least a partial sequence. Increased hormone production may not Increased hormone production may not

be associated with elevated mRNA be associated with elevated mRNA levels.levels. mRNA may code for a processing enzyme.mRNA may code for a processing enzyme.

CONTROL OF HORMONE CONTROL OF HORMONE RELEASE:RELEASE:

The synthesis and secretion of most The synthesis and secretion of most hormones are usually regulated by hormones are usually regulated by negative feedback systemsnegative feedback systems.. Organized into “loops”.Organized into “loops”.

As hormone levels rise, they As hormone levels rise, they stimulate target organ responses. stimulate target organ responses. These in turn, inhibit further These in turn, inhibit further hormone release. hormone release.

The stimuli that induce endocrine The stimuli that induce endocrine glands to synthesize and release glands to synthesize and release hormones belong to one of the hormones belong to one of the following major types:following major types: HumoralHumoral

Chemical changes in the blood (eg. Ca2+ or Chemical changes in the blood (eg. Ca2+ or glucose).glucose).

NeuralNeural Hormonal (as seen in feedback loops)Hormonal (as seen in feedback loops)

CHEMISTRY OF CHEMISTRY OF HORMONESHORMONES

Peptide hormonesPeptide hormones: largest, most : largest, most complex, and most common hormones. complex, and most common hormones. Examples include insulin and prolactinExamples include insulin and prolactin

Steroid hormonesSteroid hormones: lipid soluble : lipid soluble molecules synthesized from cholesterol. molecules synthesized from cholesterol. Examples include gonadal steroids (e.g Examples include gonadal steroids (e.g testosterone and estrogen) and testosterone and estrogen) and adrenocortical steroids (e.g. cortisol adrenocortical steroids (e.g. cortisol and aldosterone).and aldosterone).

EicosanoidsEicosanoids: small molecules : small molecules synthesized from fatty acid synthesized from fatty acid substrates (e.g. arachidonic acid) substrates (e.g. arachidonic acid) located within cell membraneslocated within cell membranes

AminesAmines: small molecules derived : small molecules derived from individual amino acids. Include from individual amino acids. Include catecholamines (e.g. epinephrine catecholamines (e.g. epinephrine produced by the adrenal medulla), produced by the adrenal medulla), and thyroid hormones.and thyroid hormones.

Receptor Tyrosine Receptor Tyrosine KinasesKinases

Figure 4.16

MAP kinasesMAP kinases

Figure 4.17

• Activated RAS signals to MAP-kinase-kinase-kinase

• MAPKKK signals to MAP-kinase-kinase

• MAPKK signals to MAP-kinase

• MAP-kinase phosphorylates a variety of target proteins

Transforming growth factor – Transforming growth factor – TGFTGFreceptorreceptor

Figure 4.18b

• An important serine-theronine kinase receptor enzyme

• Involved in regulating many cellular processes

• Malfunctions result in many diseases including Cancer, atherosclerosis etc.

G-Protein-Coupled G-Protein-Coupled ReceptorsReceptors Ligand binds to transmembrane receptor Ligand binds to transmembrane receptor

Receptor interacts with intracellular Receptor interacts with intracellular G-G-proteinsproteins Named for their ability to bind Named for their ability to bind guanosineguanosine

nucleotidesnucleotides Subunits of G-protein dissociateSubunits of G-protein dissociate

Some subunits activate ion channelsSome subunits activate ion channels Changes in membrane potentialChanges in membrane potential Changes in intracellular ion concentrationsChanges in intracellular ion concentrations

Some subunits activate amplifier enzymesSome subunits activate amplifier enzymes Formation of Formation of second messengerssecond messengers

G-proteins coupled receptors G-proteins coupled receptors (GPCRs)(GPCRs)

• 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

G-Protein-Coupled G-Protein-Coupled ReceptorsReceptors

G-proteinsG-proteins

Linked directly to the plasma membrane via a lipid anchor

Structure of heterotrimeric G proteins

, and subunits

G-protein

There are many types of G proteins – 3 There are many types of G proteins – 3 major familiesmajor families

Table 15-3. Three Major Families of Trimeric G Proteins*

* Families are determined by amino acid sequence relatedness of the subunits. Only selected examples are shown. About 20 subunits and at least 4 subunits and 7 subunits have been described in mammals.

activates phospholipase C-

GqIII

activates cyclic GMP phosphodiesterase in vertebrate rod photoreceptors

Gt (transducin)

activates phospholipase C-

and

activates K+ channels; inactivates Ca2+ channels

Go

activates K+ channels

inhibits adenylyl cyclaseGiII

activates adenylyl cyclasein olfactory sensory neurons

Golf

activates adenylyl cyclase; activates Ca2+ channels

GsI

FUNCTIONSACTION MEDIATED BYSOME FAMILY MEMBERSFAMILY

Table 3.3

Second MessengersSecond Messengers

Figure 3.27

Cyclic-AMP SignalingCyclic-AMP Signaling

Important Tools for studying Gs/Gi

Cholera Toxin – ADP-ribosylates Gs. This locks Gs in the GTP-bound conformation, leading to constitutive activation of adenylylcyclase, 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 areceptor is signaling through.

Figure 3.26

Inositol-Phospholipid Inositol-Phospholipid SignalingSignaling

Lecture Notes and synopsis are Lecture Notes and synopsis are posted at: posted at:

http://www.zoology.ubc.ca/http://www.zoology.ubc.ca/~harris/~harris/