Berthold Endocrine Experiment

Remove- replacement-injection

1. Chemical classes of hormones

2. Biosynthesis of a particular hormone

3.Transport of the hormones

4.Recognition & signaling of the hormone

5.Functions of the hormones.

6.Degradation of the hormone.

Objectives

Endocrine Methods• Remove-replacement-injection• Purification and cloning• Synthesis and production of hormone• Test biological activity with pure hormones• Development of antibodies• Localization by immunocytochemistry• Establish assays (RIA)• Microarray,deep sequencing, proteomics• Knock-out/Knock down/mutants

Hormone Types/Functions

Three structural divisions:

1) Amines--H2O sol. (small--AA) catecholamines and thyroid hormones

2) Steroids--lipid sol. cyclic hydrocarbon derivatives from cholesterol

3) Peptide/protein -- H2O sol. largest, complex

Amines

• Hormones derived from tyrosine and tryptophan.

• Include hormones secreted by adrenal medulla, thyroid, and pineal glands.

Thyroid Hormones

• Tyrosine derivatives bound together.

• Contain 4 iodine atoms (T4).

• Contain 3 iodine atoms (T3).

• Small, non-polar molecules.– Soluble in plasma

membranes.

Steroids• Lipids derived from

cholesterol.• Are lipophilic

hormones.– Testosterone– Estradiol– Cortisol– Progesterone

                      

Peptides/Proteins

• Chains of amino acids (< 100 amino acids in length).– ADH – Insulin

• Long polypeptides (>100) bound to one or more carbohydrate groups.– FSH– LH

Biosynthesis of Peptides and Protein Hormones

DNA (The gene)

RNA (Primary transcript)

mRNA

Pre-(Pro)-hormone

Pro-hormone

Hormone

RNA processing

translation

Proteolysis via signal peptide cleavage

Proteolysis via second modificationGlycosylationphosphorylation

5’ 3’Proopiomelanocortin (POMC) gene

5’ 3’ mRNA

1 2 3 4 5 6 7N C

Signal peptide

-MSH

ACTH

-MSH CLIP

-lipotropin

4 6 7

6 8

-lipotropin -endorphin

-MSH enkephalin

Products in corticotrophic cell of the anterior pituitary

Products in the intermdeiary gland

Classification of chemical communication systems

1) Autocrine--secretion that affects the same cell which the secretion originated

Ex: Adrenergic nerve endings

2) Paracrine--secretion that affects neighboring cells

Ex: Inflammatory response

3) Endocrine--a secretion of a chemical substance that is released into the blood and affects a distant target

4) Exocrine--secretion of a substance that is released onto surface of animal--including internal structures

Exocrine Glands/tissues:

--possess ducts

--salivary glands, intestinal epithelium, secretory cells in stomach, and secretory cells of the liver and pancreas

Endocrine Glands/tissues:

--lack a definite duct

--Adrenal gland, GI tract, heart, kidney, ovary, pancreas, thyroid, pituitary, placenta, testes, and thymus

Fig. 7-8, p.265

Hypothalamus

Connecting stalk(infundibulum)

Hypothalamus

Opticchiasm

Posteriorlobe ofpituitary

Anteriorpituitary

(b)

(a)Posteriorpituitary

Anteriorlobe ofpituitary

Bone

Anterior and posterior pituitary glands.

• Also called the neurohypophysis.

• Formed by down growth of the brain during fetal development.

• Is in contact with the infundibulum.

• Nerve fibers extend through the infundibulum.

Posterior Pituitary

• Master gland (also called adenohypophysis).• Derived from a pouch of epithelial tissue

that migrates upward from the mouth.• Consists of 2 parts:• Pars distalis: anterior pituitary.• Pars tuberalis: thin extension in contact with

the infundibulum.

Anterior Pituitary

Hypothalamic Control of Posterior Pituitary

• Hypothalamus produces:– ADH: supraoptic nuclei.

– Oxytocin: paraventricular nuclei.

• Hormones transported along the hypothalamo-hypophyseal tract.

• Stored in posterior pituitary.

• Release controlled by neuroendocrine reflexes.

Posterior Pituitary(neurohypophysis)--releases

neurohormones

1) antidiuretic hormone (vasopressin)

2) oxcytocin

Larhammar et al, Ann. N.Y. Acad. Sci. 1163: 201–208 (2009)

• Hormonal control rather than neural.

• Hypothalamus synthesizes releasing hormones and inhibiting hormones.

• Hormones are transported to axon endings of median eminence.– Delivers blood and hormones to anterior

pituitary via portal system.

Hypothalamic Control of the Anterior Pituitary

Hypothalamic Control of the Anterior Pituitary

• Hormones secreted into the hypothalamo-hypophyseal portal system regulate the secretions of the anterior pituitary.

• Anterior pituitary and hypothalamic secretions are controlled by the target organs they regulate.

• Negative feedback inhibition by target gland hormones.

Feedback Control of the Anterior Pituitary

Feedback Control of the Anterior Pituitary

• Negative feedback at 2 levels:– The target gland hormone can act on the

hypothalamus and inhibit releasing hormones.– The target gland hormone can act on the

anterior pituitary and inhibit response to the releasing hormone.

Fig. 7-11, p.269

Fig. 7-12, p.270

Water vs. Lipid

Water soluble Lipid soluble

hydrophilic hydrophobic

external (2nd mess.) internal

external receptors cytoplasmic rec.

short half-life long half-life

intermediary resp. Long-term resp.

protein activation gene activation

Mechanisms of Hormone Action

• Hormones of same chemical class have similar mechanisms of action.– Location of cellular receptor proteins.

• Target cell must have specific receptors for that hormone (specificity).

• Hormones bind to receptors with high bond strength (affinity).

• Low capacity of receptors (saturation).

Hormones That Bind to Nuclear Receptor Proteins

• Lipophilic steroid and thyroid hormones bound to plasma carrier proteins.

• Hormones dissociate from carrier proteins to pass through lipid component of the target cell membrane.

• Receptors for the lipophilic hormones are known as nuclear hormone receptors.

Nuclear Hormone Receptors• Function within cell to activate genetic

transcription.• mRNA directs synthesis of specific enzyme

proteins that change metabolism.• Receptor must be activated by binding to hormone

before binding to specific region of DNA called HRE (hormone responsive element).– Located adjacent to gene that will be transcribed.

Mechanisms of Steroid Hormone Action

• Steroid receptors located in cytoplasm.

• Bind to steroid hormone.

• Translocates to nucleus.

• DNA-binding domain binds to specific HRE of the DNA.

• Dimerization occurs.

• Stimulates transcription.

Mechanism of Thyroid Hormone Action

• Receptor proteins located in nucleus.

• T3 binds to ligand-binding domain.

• DNA-binding domain can then bind to the half-site of the HRE.

• Other half-site is vitamin A derivative 9-cis-retinoic acid.

• Two partners can bind to the DNA to activate HRE.

Hormones That Use 2nd Messengers

• Cannot pass through plasma membrane.• Catecholamines, polypeptides, and

glycoproteins bind to receptor proteins on the target cell membrane.

• Actions are mediated by 2nd messengers (signal-transduction mechanisms).– Extracellular hormones are transduced into

intracellular second messengers.

Hormones That Use 2nd Messengers

2nd messenger systems:• Adenylate cyclase• Phospholipase C• Tyrosine kinase• NO• various kinases

• Hormone binds to receptor protein.

• Dissociation of a subunit of G-protein.

• G-protein binds and activates adenylate cyclase.

• ATP cAMP + PPi

• cAMP attaches to inhibitory subunit of protein kinase.

Adenylate Cyclase-cAMP

• Activates protein kinase.• Phosphorylates enzymes within the cell to produce

hormone’s effects.• Modulates activity of enzymes present in the cell.• Alters metabolism of the cell.• cAMP inactivated by phosphodiesterase.

– Hydrolyzes cAMP to inactive fragments.

Adenylate Cyclase-cAMP

CatecholaminesACTHFSHLHTSHGlucagonPTHCalcitonin

• Binding of epinephrine to alpha-adrenergic receptor activates a G-protein, (phospholipase C).

• Phospholipase C splits phospholipid into inositol triphosphate (IP3) and diacylglycerol (DAG).

• Both derivatives serve as second messengers.

Phospholipase-C-Ca++

• IP3 diffuses through cytoplasm to ER.

• Binding of IP3 to receptor protein in ER causes Ca++ channels to open.

• Ca++ diffuses into the cytoplasm.• Ca++ binds to calmodulin.• Calmodulin activates specific protein kinase enzymes.• Alters the metabolism of the cell, producing the

hormone’s effects.

Phospholipase-C-Ca++

CatecholaminesTRHLHRHOxytocinADH

1)By release of Ca2+ from intracellular Calcium storages

2)By influx of Calcium from cell exterior Ca2+ channel

Two ways to increase cytosol free Ca 2+

• Receptor protein on cell membrane is tyrosine kinase.• Insulin receptor consists of 2 units that dimerize when

they bind with insulin.• Insulin binds to ligand–binding site, activating

enzymatic site.• Autophosphorylation occurs, increasing tyrosine kinase.• Activates signaling molecules, altering the metabolism

of the cell.

Tyrosine Kinase

PRL

Stat

GH/PRL signaling mechanism

PI3K Src

P

D2D1

Box1

YYYY

P

P

D2D1

Box1

YYYY

P

PP

PRL

JAK2JAK2

Stat 1

P

Stat 3

Stat 5

P

Extra-cellular

Intra-cellular

Fyn MAPK

BiologicalResponse

AKT ? AKT ERK

PRLR alpha

PRL

PRLPRL

PRL

PRL

Circulation

Kinase

Signal Molecule

Known

Not Sure

Legend

OVERVIEW

Physiological Effects of Hormones

1.Reproduction (Gonads)

2.Stress & Steroids (Adrenal)

3.Metabolism (Pancreas and Thyroid)

4.Electrolyte/Water Balance

Gonads and Placenta• Gonads (testes and ovaries):

– Secrete sex hormones.• Testosterone.

• Estradiol.

• Progesterone.

• Placenta:– Secretes large amounts of estrogen and progesterone.

Sertoli cells: synthesis androgen-binding protein and inhibin.

Leydig cells: produce and secrete testosterone

Testosterone: key hormone for produce sperm, develop male sex characters, protein synthesis and general growth

ss

Cell type

Ploidy/Chromosomes #N Process

Time of completion

Oogonium diploid/46 2NOocytogenesis (mitosis) third trimester

primary Oocyte diploid/46 4N

Meiosis I (Folliculogenesis)

Dictyate in prophase I until ovulation

secondary Oocyte haploid/23 2N Meiosis II

Halted in metaphase II until fertilization

Ovum haploid/23 1N

1. hypothalamic GnRH control of FSH / LH release,

2. ovarian follicular development to ovulation and subsequent corpus luteum formation

3. the feedback control of FSH / LH secretion by ovarian hormones.

•the corpus luteum regresses,

•there is a rapid fall in the secretion of oestrogen and progesterone,

•the endometrium undergoes shrinkage due to extracellular fluid loss,

•the spiral arteries constrict,

•the endometrial blood flow decreases with cell death and destruction of blood vessels `

Adrenal Glands

• Paired organs that cap the kidneys.• Each gland consists of an outer cortex and inner

medulla.• Adrenal medulla:

– Derived from embryonic neural crest ectoderm (sympathetic ganglia).

– Synthesizes and secretes:• Catecholamines (mainly epinephrine but some

norepinephrine).

Adrenal Medulla

• Innervated by sympathetic nerve fibers.– Increase respiratory rate. – Increase heart rate, cardiac output; and

vasoconstrict blood vessels, thus increasing venous return.

– Stimulate glycogenolysis.– Stimulate lipolysis.

Adrenal Glands

• Adrenal cortex:– Does not receive neural innervation.– Must be stimulated hormonally.– Consists of 3 zones:

• Zona glomerulosa: – Aldosterone: regulate Na+ and K+ balance.

• Zona fasciculata: – Cortisol: regulate glucose metabolism.

• Zona reticularis:– Androstenedione and DHEA: supplement sex steroids.

Pancreas• Endocrine portion consists of islets of Langerhans.• Alpha cells secrete glucagon.

– Stimulus is decrease in plasma glucose concentrations.– Stimulates lipolysis.

• Beta cells secrete insulin.– Stimulus is increase in plasma glucose concentrations.– Promotes entry of glucose into cells.

Regulating Blood Glucose

Two major hormones:

1) insulin

2) glucagon

Origin: cells, cells-- islet of Langerhans

Control: BG high--insulin, BG low--glucagon

Diabetes MellitusTwo types:

1) Type I (IDDM)

--severe, insulin dependant, juvenile onset,

--loss of beta cell mass

2) Type II (NIDDM)

--less severe, non-insulin-dependant, adult onset, more common

--defective insulin receptors

untreatedfat metabolism(ketones) ketoacidosis ketoacidosis

Thyroid Hormones• Thyroid gland located just below the larynx.

• Thyroid is the largest of the pure endocrine glands.

• Follicular cells secrete thyroxine.

• Parafollicular cells secrete calcitonin.

Production of Thyroid Hormones

• I- (iodide) actively transported into the follicle and secreted into the colloid.

• Oxidized to (Io) iodine.• Iodine attached to tyrosine.

– Attachment of 1 iodine produces monoiodotyrosine (MIT).

– Attachment of 2 iodines produces diiodotyrosine (DIT).

• MIT and DIT or 2 DIT molecules coupled.

Production of Thyroid Hormones

• T3 and T4 produced.

• TSH stimulates pinocytosis into the follicular cell.– Enzymes hydrolyze to T3 and T4 from

thyroglobulin.

• Attached to thyroid-binding protein and released into blood.

T3 Effects • Stimulates cellular respiration by:

– Production of uncoupling proteins.– Stimulate active transport Na+/ K+ pumps.– Lower cellular [ATP].

• Increases metabolic heat.• Increases metabolic rate.

– Stimulates increased consumption of glucose, fatty acids and other molecules.

Hormonal Regulation: H2O, Electrolytes

Major organs: kidney, intestine, and bone

Antidiuretic hormone (ADH): also vasopressin, regulates H2O turnover in kidney

-- premeability of H2O in duct ( urine)

Aldosterone: Na+ reabsorption, blood osmolarity

Atrial natriuretic peptide (ANP): Na+ , ( urine)