Pheromones

Other Cellular Regulators-Act like hormones

Neurotransmitters

Other Cellular Regulators

Besides well recognized kinds of hormone, other substances play important roles as chemical messengers

Ca++

Glucose-specific stimuli for insulin secretion from the b cells of the pancreas.

Amino acids

None of these effectors are not TRADITIONAL hormones, but act like hormones.

Besides classics

NTsPheromones

GlucoseCalcium

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

Peptide Hormones -translated on secretory pathway (ER..golgi. Sec ves)

Made in RER

Can have continuous or regulated secretion

Hormone Synthesis

Steroid Hormones

Made within the SERSteroid secreting cells easily recognized by large

amounts of SER

Complex multiple enzyme system for synthesis secretion

Hormone Synthesis

Thyroid Hormones

Made on protienaceous substrates outside the cellThyroglobulin

Then 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 and converted to active hormone in the

blood.

Hormone Synthesis

NTs

Made 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 thyroid and steroid hormonesWhich are not in secretory vesicles

Control of Hormone Secretion

Internal and external effectors

Extrinsic-light, sounds, smell, temp, Etc.

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

Control of Hormone Secretion

Glycos.In Cis

Sorting in

Trans Golgi

Control of Hormone Secretion

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

Control of Hormone Secretion

Neuroendocrine transduction

stimulation of hormone secretion by nerves

Control of Hormone Secretion

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 hyper polarization

Hormone Delivery-several routes

Endocrine, Para, auto

neurocrine- neuron contact target cell and releases hormone

neuroendocrine-neuron to blood

lumonal-released into lumen of the gut

Some delivered by all multiple routes

Hormone Circulation and metabolism

Peptide hormones have short half lives

Exopeptidases and endopeptidases

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.

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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+

-

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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 luteinizing hormone 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, 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

What do hormones do???

Physiological roles of Hormones

Hormones control 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

Physiological roles of Hormones

Reproduction

Cell growth and proliferation

Excretion and reabsoroption of ions

Affect action of other hormones

Role in animal behavior

Physiological roles of Hormones

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

Receptors

Second messengers

Phosphorylation involves STY

Kinases 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

Hormone response effected by Receptor Levels and hormone levels

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)

Hormone response effected by Receptor Levels and hormone levels

Have high levels of receptor in tissue that are primary responders

Hormones act via own receptors at normal concentrations

At high hormone concentrations, hormones 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?

Agonists and Antagonists Exocrine

Endocrine, Paracrine, AutocrineSecretinHumoral

Development origin of hormoneHomeostasis pH= ___ Temp=___CIf you secrete one, then you secrete

many…..

Endocrine Activity-rate of

production, delivery, and degradation

Steroid hormones, made in SERThyroid hormones-outside of cell

Not Secreted in vesicles like peptide hormones

Feedback circuits

Contribute to complexity of Hormone Action

Pulsatile secretion

General mechanisms of Hormone

action

Receptors

Second messengers

Kinases and phosphatases

CROSS talk

Hormones are complexLots of things to be studied regarding

hormones

Methods are used to performENDOCRINOLOGY EXPERIMENTS?

General considerations

1. Source2. Structure determination

3. Biosynthesis4. Control of secretion

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

8. Mechanisms of action

Scientific Method – observation + experiments

formulate hypotheses

Must be testable via observation or experimentation

Lot of Data Theory

Theory accepted Law of Principle or DOGMA

Always need a control vehicle if using solution

Sham operation if doing surgery

Always limit variables

Show specificity

Effects Usually Time and Dose Dependent

Old principle of Logic

Occam’s razor of several reasonable

explanations….

The simplest is most probable.

Types of Experiments in Endocrinology

Chemical ID

10 -amino acid sequence

20 –secondary 30 – R group interactions

40 –association with other proteins

Modifications like glycosylation, phosphorylation and sulfation

Methods of Endocrine Analysis

Microscopy(light, EM to whole-body scanning

techniques – (CAT, PET, MRI)

Imaging studies are important component of endocrinology studies

Imaging also important component of diagnosis and treatment

The picture shows a tumor cell

disintegrating after an attack by a T cell. 

Two additional, intact tumor cells are shown

in. The successful cytotoxic T

lymphocyte may now make these cells its

targets.

Bioassays

Different approaches to examine hormone activity

Based on activity (enzymatic)Or association with another molecule

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

Site directed mutagenesis

Histological and cytological studies

Hypertrophic-enlarged This means bigger cells

Contain more ER and GolgiOpposite of atrophic

Hyperplasia or HyperplasticAn increase in number

Enlarged spleen cells

Enlarged fat cells at top

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 to two proteins

Immunocytochemistry

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

hormone and/or where in the cell it is localized

Radioisotope Studies

I125 take up by thyroid

Radioactive Ca measured

P to perform phosphorylation studiesHalf life studiesKinase studies

Radioisotope Studies

I125 take up by thyroidthe amount of iodide the thyroid absorbs is a reliable indicator

of how much hormone the gland is producing

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

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

RIA

At first was only useful for petptides. Now possible to trick antibody

producing cells to make specific anitbodies against all type of

chemical substancesCan measure Steroid and Thyroid

hormones now with this assay

Electrophysiology membrane potential

Electrophysiology The cell-attached patch clamp uses a

micropipette attached to the cell membrane to

allow recording from a single ion channel.

"Current Clamp" is a common technique in electrophysiology. This is a whole-cell

current clamp recording of a neuron firing due to it being depolarized by current

injection

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

Pharmacological experiments

Actinomycin D-inhibits transcriptionCycloheximide-inhibits translation

Colchicine-disrupt microtubulesCytochalasin B-disrupts microfilament

Pharmacological experiments

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

Pharmacological experiments

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

Disadvantages 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

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