Control of Endocrine Activity The physiologic effects of hormones depend largely on their concentration in blood and extracellular fluid. Almost inevitably,

Control of Endocrine Activity

The physiologic effects of hormones depend largely on their concentration in blood and extracellular fluid. Almost inevitably, disease results when hormone concentrations are either too high or too low, and precise control over circulating concentrations of hormones is therefore crucial. The concentration of hormone as seen by target cells is determined by three factors:

1.Rate of production:

2.Rate of delivery

3.Rate of degradation and elimination:

Control of Endocrine Activity

[Hormone] as seen by target cells is determined by 3 factors:

Rate of production: Synthesis and secretion of hormones are the most highly regulated aspect of endocrine control. Such control is mediated by positive and negative feedback circuits.

Rate of delivery: An example of this effect is blood flow to a target organ or group of target cells - high blood flow delivers more hormone than low blood flow.

Rate of degradation and elimination: Hormones have characteristic rates of decay, and are metabolized and excreted from the body via several routes. Shutting off secretion of a hormone that has a very short halflife causes circulating hormone concentration to plummet, but if a hormone's biological halflife is long, effective concentrations persist for some time after secretion ceases.

Hormone Synthesis

Diversity of hormones structuresLots of interesting pathways of biosynthesis

Simplest of hormones-amino acidsGlycine and glutamate -act as NTs in brain

F and Y-are precursors of dopamine, NE and EpiWhich also function as NTs

Hormone Synthesis

Y also substrate for generation of thyroid hormones

W is precursor for serotonin, a CNS NTand melatonin, a pineal hormone

Hormone Synthesis

Steroid Hormones

Made within the SERSteroid secreting cells easily recognized

Complex multiple enzyme system for synthesis secretion

Hormone Synthesis

Thyroid HormonesMade on protienaceous substrates outside the cell

ThyroglobulinThen taken up via endocytosis into the thyroid

gland-released from carrier protein prior to secretion from thyroid.

UNIQUE PROCESS

Hormone Synthesis

ProhormonesResult from cleavage events after translation

Even have preprohormones

ExamplesRenin (enzyme from Kidney)

Acts on angiotensinogen (substrate from liver)Results in ANGIOTENSIN I which is converted

by another enzyme to Antgiotensin II

Hormone Synthesis

Prohormones

Angiotensin II and bradykins are examples of hormones that are released from liver cells as larger prohormones

Hormone Synthesis

NTsMade in axon end of neurons

Neuropeptides like oxytocin and vasopressin also made in neurons

Hormone Synthesis

Summary

Variety of processes and intracellular locations involved

SER, RER, Cholesterol from inside and outside the cell,

Secretory pathway involved in hormone modifications, particulary

glycosylation

Control of Hormone Secretion

Most hormones are made within cellsare packaged in secretory vesicles until

released

-Except ….


Internal and external effectorsExtrinsic-light, sounds, smell, temp,

Etc.

Stimulation of hormone secreting cells results in vesicle fusion with the PM and exocytosis of secretory granules



Hormones often stimulate secretion of hormones from other endocrine glands

Pit hormones TSH, FSH, LH and ACTH simulate target tissue cells of

thyroid, adrenal, gonads to secrete their own hormones

Hormones control other hormonesCascade effect


Neuroendocrine transduction

stimulation of hormone secretion by nerves


Hormone interaction with some membrane receptors results in

membrane depolarization-stimulates movement of Ca++into cells which

results in sec. vesicle exocytosis

Some chemical messenger inhibit secretion by resulting ……

Hormone Delivery

Several routes of delivery1.Endocrine

2. neurocrine- neuron contact target cell and releases hormone

3. neuroendocrine-neuron to blood4. Paracrine

5.lumonal-released into lumen of the gut

6. AutocrineSome delivered by all these routes

Hormone Circulation and metabolism

Peptide hormones have short half lives

Broken down by …

Most steroid hormones bound to plasma proteins. Steroid hormones

much more stable

Feedback Control of Hormone Production

Feedback circuits are at the root of most control

mechanisms in physiology, and are particularly prominent

in the endocrine system.

Instances of positive feedback certainly occur, but negative

feedback is much more common.

Feedback Control of Hormone Production

Negative feedback is seen when the output of a pathway inhibits

inputs to the pathway.

The heating system in your home is a simple negative feedback circuit.

Feedback loops are used extensively to regulate secretion of hormones

An important negative feedback loop is seen in control of thyroid hormone secretion. The

thyroid hormones thyroxine and triiodothyronine ("T4 and T3") are synthesized

and secreted by thyroid glands and affect metabolism throughout the body.

The basic mechanisms for control in this system (illustrated on next slide) are:

1.Neurons in the hypothalamus secrete thyroid releasing hormone (TRH), which stimulates cells in the anterior pituitary to secrete thyroid-stimulating hormone (TSH).

2. TSH binds to receptors on epithelial cells in the thyroid gland, stimulating synthesis and secretion of thyroid hormones, which affect probably all cells in the body.

3.When blood concentrations of thyroid hormones increase above a certain threshold, TRH-secreting neurons in the hypothalamus are inhibited and stop secreting TRH. This is an example of "negative feedback".

Inhibition of TRH secretion leads to shut-off of TSH secretion, which leads to shut-off of thyroid hormone secretion. As thyroid hormone levels decay below the threshold, negative feedback is relieved, TRH secretion starts again, leading to TSH secretion ...

+

+

-

-

Target cell response

TRH receptors only found in anterior pituitary

TSH receptors only found in thyroid gland

TH receptors found on every cell

Cascade effect

+

+

-

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Another type of feedback is seen in endocrine systems that regulate concentrations of blood components such as glucose.

Drink a glass of milk or eat a candy bar and the following (simplified) series of events will occur:

Glucose from the ingested lactose or sucrose is absorbed in the intestine and the level of glucose in blood rises.

Elevation of blood glucose concentration stimulates endocrine cells in the pancreas to release insulin.

Insulin has the major effect of facilitating entry of glucose into many cells of the body - as a result, blood glucose levels fall.

When the level of blood glucose falls sufficiently, the stimulus for insulin release disappears and insulin is no longer secreted.

Numerous other examples of specific endocrine feedback circuits will be presented in the sections on specific hormones or

endocrine organs.

Hormone Profiles: Concentrations Over Time

One important consequence of the feedback controls that govern hormone concentrations and the fact that hormones have a limited lifespan or half-life is that most hormones are secreted in "pulses". The following graph depicts concentrations of LH in the blood of a female dog over a period of 8 hours, with samples collected

every 15 minutes:

The pulsatile nature of LH secretion in this animal is evident.

LH is secreted from the anterior pituitary and critically involved in reproductive function; the frequency and amplitude of pulses are quite different at different stages of the reproductive cycle.

With reference to clinical endocrinology, examination of the graph should also demonstrate the caution necessary in interpreting endocrine data based on isolated samples.

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A pulsatile pattern of secretion is seen for virtually all hormones, with

variations in pulse characteristics that reflect specific physiologic states. In addition to the short-term pulses discussed here, longer-term temporal

oscillations or endocrine rhythms are also commonly observed and undoubtedly

important in both normal and pathologic states.

Mechanisms of Hormone Action

Immediately after discovery of a new hormone, a majority of effort is devoted to delineating its sites of synthesis and target cells, and in

characterizing the myriad of physiologic responses it invokes. An equally important area of study is to determine precisely how the hormone acts to change the physiologic

state of its target cells - its mechanism of action.

Mechanisms of Hormone Action

Understanding mechanism of action is itself a broad task, encompassing

structure and function of the receptor, how the bound receptor transduces a

signal inside the cell and the end effectors of that signal. This information is not only

of great interest to basic science, but critical to understanding and treating

diseases of the endocrine system, and in using hormones as drugs.

Physiological roles of Hormones

Hormones control of activity of all cells in the body

Affect cellular synthesis and secretion of other hormones

After metabolic processes (catabolic and anabolic). Turnover of sugar,

proteins and fats

Affect Contraction, relaxation and metabolism of Muscle


Reproduction

Cell growth and proliferation

Excretion and reabsoroption of ions

Affect action of other hormones

Role in animal behavior


Some hormones only exist a few times in the life of an individual hCG

Sometimes still have hormone but not sensitive to it any longer

Sometimes no longer produce hormone-thyroid hormone, estrogen

General mechanisms of Hormone

action

ReceptorsSecond messengers

Phosphorylation involves STYKinases and phosphatases

Reminder about

General mechanisms of Hormone action

Steroid hormones have intracellular receptors.

So do Thyroid hormones

Endocrine pathophysiology

Failure of a gland to secrete enough hormone can lead to fatal consequences

No insulin-hyperglycemia-coma and death if untreated

General mechanisms of hormone action

Hormones regulate specific target tissuesNOT ALL CELLS IN the body

Determined by??Receptors-proteins bind hormones

Contribute to specificity of action

Can be PM or cytosolic or nuclear

Hormone response effected by Receptor Levels and hormone levels

Oxy and vasopressin AVP have similar structure and both hormones stimulate uterine smooth muscle contraction and

activate renal cAMPUterine receptors more sensitive to OXY

Renal receptor more sensitive to AVPNormal hormone conc. Each hormone only

activate appropriate cell type


When one hormone binds to the receptor of another hormone, this is called

CROSS TALK

Happens with lots of hormones.If hormone levels are high, will not only act

on its own receptor, but similar hormone receptors

Some hormones stimulate a number of tissues.

Insulin stimulates glucose uptake into skeletal muscle and Fat cells

But also talks to liver to shut down output of glucose from liver

High Insulin receptor levels on fat, muscle and liver, but low levels in

other tissues.

Insulin receptors at high levels in skeletal muscle

Fat cellsLIVER

Cells where INSULIN MODULATES glucose metabolism

Insulin receptors at low levels in all other tissues where this hormone

only has a modest effect on GROWTH

DOES NOT MODULATE GLUCOSE METABOLISM IN

THESE OTHER TISSUES

RECEPTORS FOR A PARTICULAR HORMONE ARE ONLY EXPRESSED IN CELLS

WHERE THE HORMONE ACTS.MORE ACTION-MORE

RECEPTORS

UNDERSTAND INSULIN EXAMPLE (IT IS AN

EASY ONE)


Have high levels of receptor in tissue that are primary responders

Hormones act via own receptors at normal concentrations

High hormone concentration can act on similar receptors

NE and EpiOxy and vaso

IGF-1 and insulin

In most cases, a maximum biological response to a

hormone is achieved when only a small % of the

receptors are occupied.

WHY?

There are 4 classes of membrane bound receptors

1. Those that are enzymes (have tyrosine kinase activity)

2. Ion channels3. Receptors coupled to G proteins

4. Receptor that don’t have enzymatic activity (utilize the JAK STAT

pathway)

Peptide hormones act via PM receptors





This means the receptor itself has enzymatic activity when the hormone

is bound.Usually KINASE activity

KINASE activity

X-OH X-OP

Phosphatase activity

Amino acid substratesS T Y


1. Those that are enzymesInsulin receptorEGF receptorNGF receptor

Usually induce cascade effect

2. Ion channelsLigand binding changes the conformation of

the receptor so that specific ions flow through it; the resultant ion movements alter the electric potential across the cell

membrane. The acetylcholine receptor at the nerve-

muscle junction


3. Receptors coupled to G proteins (7TMDS)

Odorant ReceptorsAdrenergic receptors

(epi and NE)

7 hydrophobic membrane spanning domainsInternal G protein interacting region

N terminal glycosylationC-term phosphorylation sites

Mediate signals for proteins, peptides, NTOdorants and photons

hydrophobic membrane spanning domains22 -28 hydrophobic AA

Many of the receptors for peptide hormones and NTs are linked to G

proteins

Most neuropeptide receptors



pathway)Examples

GH receptor, PRL receptorCardiotrophin 1 (CT1), CNTF receptor

Leptin receptor

The JAK/STAT Signaling Pathway

JAK STAT pathway Ligand binds receptor

Receptor DimerizesReceptor associate with JAK kinase

JAK phosphorylates receptorSTAT associates with phosphorylated

receptorJAK phosphorylated STAT

STAT forms dimer and translocates to the nucleus to regulate transcription

JAK STAT pathway

Used by Growth HormoneAnd prolactin

And EPOInterferons



2. Ion channels3. Receptors coupled to G proteins


pathway)

Second Messengers of Hormone Action

Cyclic NucleotidesGenerated by Nucleotide cyclyzing

enzymes-located on inner surface of PM

ATP cAMPAdenylate cyclase

GTP cGMPguanylate cyclase

cAMP and cGMP Combine with cyclic dependent protein

kinases cAMP associates and activates

cyclic dependent protein kinase A (PKA)2 Regulatory and 2 Catalytic subunitscAMP binds R subunits which frees C

which has enzymatic activity

Genomic actions of cAMP Many second messengers, result in

immediate responseOthers have actions which are blocked

by actino. D or Cyclohex.For example, PKA activates CREB

cAMP response element binding proteinCREB binds gene promoters are CRE

elements (camp responsive elements) to modulate transcription

Enzyme phos. Leads to a cascade effectResults in amplification

So little hormone or second messenger have a large effect

X-OH X-OP

y-OH y-OP

z-OH z-OP

cAMP and cGMP rapidly metabolized

Action of kinasesReversed by phosphatases

When initiate a response, also initiate a means to inhibit response

cAMP and cGMP getInactivated by phosphodiesterases (PDE)

To 5’AMP or 5’ GMP cyclase

Activity of PDEs inhibited by methylxanthines

Caffeine, theophylline, and theobromineIncreased cAMP/signaling

Many hormones use adenylate cyclase to cause a physiological response.

Some activate this enzymeOthers can inhibit

Receptor G-protein interactions mediate different signal transduction pathways

PLCAdenylate cyclase

PLA2Ion Channel*

*G proteins don’t always induce Second Messenger

Some Second Messengers

Multiple Membrane Messengers

PI (phosphoinosotides) are phospholipids in PM of all eukaryotes

Breakdown to form second messengers• AA• IP3

• DAG

Protein Kinase C (PKC)Multifunctional enzyme

Many typesCa++ dependent and independent

Inactive until associates with PM then activated by DAG

Active enzyme is membrane boundPKC-many roles including growth and

proliferation. Activated by most peptide mitogenic hormones

Eicosanoids and Hormone Action

Eicosanoids are produced by cells in response to some hormones

Intracellular second messengers1982 Nobel Prize

They are rapidly degraded, so they are not transported to distal sites within the body.

Eicosanoids and Hormone Action

Common EicosanoidPGE2PGF2a

Prostacylin (PGI2)

PGI2 and PGE2 can activate adenylate cyclase

Eicosanoids are produced exclusively

within the PMPrimarily derived from AA that is release from phospholipids via action

of PLA2

AA can also produce leukotrienesAA----5HPETE by

5’lipoxgenase

Prostacyclin (PGI2)

Produced by blood vessel wallMost potent natural inhibitor of blood

platelet aggregationActivates AC which increase cAMP Which inhibits platelet aggregations

ThromboxaneA2 specific product of the platelets

2 types of prostaglandins thromboxane (vasoconstrictor) and PGI2

(vasodilator) Opposite effects

A vasoconstrictor is any substance that acts to constrict blood vessels

Vasoconstrictors are also used clinically to increase blood pressure or to reduce local blood flow.

LeukotrienesMade by leukocytes

Important in vascular contraction and permeability

Lots of diseases associated with increased levels

AsthmaChronic bronchitis, CF, septic shock

Psoriasis, Inflamm Bowel Dis

Many prostaglandins act as local mediators -paracrine and autocrine signaling

Destroyed near the site of their synthesis. Modulate the responses of other hormones and can have

profound effects on many cellular processes.

Certain prostaglandins cause blood platelets to aggregate and adhere to the walls of blood vessels. Because

platelets play a key role in clotting blood and plugging leaks in blood vessels, these prostaglandins can affect

the course of vascular disease and wound healing; aspirin inhibits their synthesis by acetylating

prostaglandin H2 synthase.

Not all hormones work via cell surface receptors

Steroid and Thyroid Hormone receptors

Receptors are present in cytosol and/or nucleusEstrogen and estrogen receptor (ER)

Testosterone and androgen receptor (AR)Thyroid hormones and Thyroid hormone

receptors (TR)Cortisol and Glucocorticoid receptor (GR)

Hormones enter cell by diffusion (hydrophobic)

Usually bind receptor in cytosol (displace a binding protein)

Translocates to the nucleusBinds promoter at specific elements

Regulates gene expression

Hormone/receptor complex translocates to the nucleus and binds promoters at

hormone response elementEREs (estrogen response elements)

AREsTREsGREs

The hormone and receptor complex directly bind response elements with the DNA

Can allCommunicate

with the nucleus

Hormones are complexLots of things to be studied regarding

hormones (see next slide)

Methods are used to performENDOCRINOLOGY EXPERIMENTS?

General considerations

1. Source2. Structure determination * (inject or oral)

3. Biosynthesis4. Control of secretion

5. Cellular mechanism of secretion6. Circulation and metabolism7. Biological actions/functions

8. Mechanisms of action

Histological and cytological studies

Hypertrophic-enlarged This means bigger cells

Contain more ER and GolgiOpposite of atrophic

Hyperplasia or HyperplasticAn increase in number

Hypertrophic-enlarged This means bigger cells

Contain more ER and GolgiOpposite of atrophic

Hyperplasia or HyperplasticAn increase in number

Can have Hypertrophy or Hyperplasia

or both depending on condition

Immunocytochemistry

This is method to examine peptide or peptide hormone in a tissue.

Must have an antibody against that protein.

Antibody bind hormone (protein).Use Fluorescent dye to bind antibody to

visualize location of protein.

Immunocytochemistry to show marker of Hodgkins lymphoma

Immunocytochemistry

This is method CAN ALSO BE used to determine what tissue produces a

hormone and/or where in the cell it is localized

Surgical Methods

Endocrine organs can be transplanted to a new location

Ectopic-abnormal site

Hypophysectomy-removal of pituitaryPituitary target organs become atrophic

Ectopic-abnormal

site

Removal of both members of paired (bilateral roans) such as adrenals or gonads usually leads to COMPLETE

loss of dependent tissue/organFUNCTION.

Only unilateral (one) removalHave compensatory hypertrophy

To account for ablated organ

ParabiosisAnimals are sutured together and

share vascular systems

Remove endocrine gland of one mouse, the organs of other animal

will hypertrophy. Chemical communication between

animals

Obese gene-genetic defect in this gene causes obesity and type II diabetes

The obesity gene codes for a hormone called leptin that is made exclusively in FAT

diabetes gene-genetic defect in this gene causes obesity and type II diabetes

The diabetes gene codes for the leptin receptor which is primarily expressed in the

hypothalamus

Ob/ob mice-no leptin db/db-no leptin receptor

2 commonly used rodent models of type II diabetes

Parabiosis of ob/ob and db/db miceOb/ob mice-no leptin

db/db-no leptin receptor

Gray mouse is wild type

Ob mouse and wild type-get leaner ob mouse. Sharing Hormone

db mouse and wild type-db does not get leaner because of defective

receptor, not a problem with the hormone.

db mouse and ob mouse-ob mouse gets better as it gets circulating

hormone from db mouse. Db mouse does not improve because of defective

receptor

This pivotal parabiosis experiment showed that ob gene coded for

circulating factor and that db did not.

Positional cloning is method to identify and clone the gene that

creates a phenotype. So-finding the genotype

Positional cloning is method to identify and clone the gene that

creates a phenotype. So-finding the genotype

Obese mice-defect in obese geneTook over 10 years to find gene

Same with diabetes gene

Obese mice-defect in obese geneFound was fat

specific

Chemicals

Alloxan or streptozoticn destroy islets which produce insulin-induce Type I

diabetes in an animal

Cobalt chloride destroy glucagon secreting cells

Induce diabetes chemically or surgically

Hormone Replacement Therapy (HRT)

Reverse the undesirable effects of hormone loss following surgery or

disease state or age.

Children lacking GH are given this hormone to avoid stunted growth

Immunological Neutralization of

Hormone activity Antibodies against a hormone injected.

Bind hormone and inhibit its actionMostly used as Experimental rather

than treatment approach to understand the actions of specific hormones

Inject anti-NGF antibodiesno growth and dev’t of SNS

Bioassays

Different approaches to examine hormone activity

Based on activity (enzymatic)Or association with another molecule

Bioassays


Structure-Activity StudiesSite directed mutagenesis

Bioassays


Structure-Activity StudiesSite directed mutagenesis

Structure-Activity Studies

Mutate part of the gene or one base of the gene to determine if that part is important in hormone activity and

function

Site directed mutagenesis

Radioisotope Studies

I125 take up by thyroid

Radioactive Ca measured

P to perform phosphorylation studiesHalf life studiesKinase studies

Radioisotope Studies

I125 take up by thyroid

Chemical ID

10 -amino acid sequence

20 –secondary 30 – 40 –association with other proteins

Modifications like glycosylation, phosphorylation and sulfation

RIADetection of hormones at

minute concentrations.

Need an antibody

RIADetection of hormones at minute concentrations. Need an antibody

RIA

Nobel Prize in Medicine (1977) to Rosalyn Yalow

Electrophysiology membrane potential

Pharmacological experiments

Actinomycin D-inhibits transcriptionCycloheximide-inhibits translation

Colchicine-disrupt microtubulesCytochalasin B-disrupts microfilament


Actinomycin D and Cycloheximide

Can be used to determine if an action of a hormone is genomic

Specific Example

To determine if effect of a hormone is dependent on new proteins synthesis, treat target cells with CH then look at

hormone action.

If action is blocked, know the effect is genomic


Colchicine and Cytochalasin can be used to tell if signaling or secretion is

dependent on cytoskeleton

Tissue Extracts and purification

Type I diabetics need daily injections of insulin

Used to come from pigs, cattle, horse.Slaughterhouse blood

Contaminants from animalsSpecificity issues

Insulin now made recombinant

Sheep melatonin Bovine GH

Tissue Extracts and purificationDisadvantage of using hormones

purified from animals or Slaughterhouse blood

-Contaminants from animals-Specificity issues

-Cost, much cheaper to make recombinantly

Sheep melatonin Bovine GH

Recombinant DNA methods

Way in which we make insulin

Genetic engineering in various speciesFish, mice, rats.

Transgenic Animals

introduce gene in animal-Usually replace wild type with a mutant

-Or express gene from a different promoter.

Transgenic Mice over expressing TropomodulinHave enlarged right atrium and ventricle and are larger

Labeled for two different proteins which are normally present in myofibrils. The alternating bands of tropomodulin (green) and alpha-

actinin (red) show the dense packing of myofibril throughout the interior of the cell.

The normal alternating pattern of tropomodulin and alpha-actinin immunoreactivity has been disturbed. The yellow color indicates

colocalization of both red and green labels (an abnormal distribution). Transgenic mice with this level of tropomodulin overexpression suffer

from cardiomyopathy

transgenic mice that

overexpress TGFß1 in the CNS

animals developed severe hydrocephalus

transgenic colony serves as a model of congenital

hydrocephalus

GH receptor

knockout

GFP mice

overexpress neurotrophin-3 (NT-3) in skeletal muscle

When lifted by the tail, wildtype extend their hindlimbs and digits. In contrast, all transgenic NT-3 mice retract their hindlimbs to the body and clench their paws in a

"clasping phenotype"

Transgenic mice has different

coat color

Transgenic mice extremely useful in studying diseases

Test is Feb 10STUDY your NOTES

and the study guide

Documents

Control of Endocrine Activity The physiologic effects of hormones depend largely on their concentration in blood and extracellular fluid. Almost inevitably,