Endocrine Physiologylecture 2

Dale Buchanan Hales, PhD

Department of Physiology & Biophysics

Metabolic clearance rate (MCR)

• Defines the quantitative removal of hormone from plasma

• The bulk of hormone is cleared by liver and kidneys • Only a small fraction is removed by target tissue

– protein and amine hormones bind to receptors and are internalized and degraded

– Steroid and thyroid hormones are degraded after hormone-receptor complex binds to nuclear chromatin

• 99% of excreted hormone is degraded or conjugated by Phase I and Phase II enzyme systems

MCR of some hormones Hormone Half-life

Amines 2-3 min

Thyroid hormones: T4 T3

6.7 days0.75 days

Polypeptides 4-40 min

Proteins 15-170 min

Steroids 4-120 min

Hormone-Receptor interactions

• Definition: a protein that binds a ligand with high affinity and low capacity. This binding must be saturuable.

• A tissue becomes a target for a hormone by expressing a specific receptor for it. Hormones circulate in the blood stream but only cells with receptors for it are targets for its action.

Agonist vs. Antagonist

• Agonists are molecules that bind the receptor and induce all the post-receptor events that lead to a biologic effect. In other words, they act like the "normal" hormone, although perhaps more or less potently

• Antagonists are molecules that bind the receptor and block binding of the agonist, but fail to trigger intracellular signaling events

Hormone binding study

Hormone-receptor interactions

• Hormone--receptor interaction is defined by an equilibrium constant called the Kd, or dissociation constant.

• The interaction is reversible and how easily the hormone is displaced from the receptor is a quantitation of its affinity.

• Hormone receptor interactions are very specific and the Kd ranges from 10-9 to 10-12 Molar

Analysis of hormone interactions: Scatchard plots

Spare receptors

• In most systems the maximum biological response is achieved at concentrations of hormone lower than required to occupy all of the receptors on the cell.

• Examples: – insulin stimulates maximum glucose oxidation in

adipocytes with only 2-3% of receptors bound

– LH stimulates maximum testosterone production in Leydig cells when only 1% of receptors are bound

Spare Receptors

• Maximum response with 2-3% receptor occupancy

• 97% of receptors are “spare”• Maximum biological response is achieved when

all of the receptors are occupied on an average of <3% of the time

• The greater the proportion of spare receptors, the more sensitive the target cell to the hormone

• Lower concentration of hormone required to achieve half-maximal response

Binding vs. biological response

Spare receptors Amplification by 2nd messenger

Hormonal measurements

• Bioassay– an assay system (animal, organ, tissue, cell or enzyme

system) is standardized with know amounts of the hormone, a standard curve constructed, and the activity of the unknown determined by comparison

• example: testosterone stimulates growth of prostate gland of immature or castrate rat in a dose-dependent manner. Androgen content of unknown sample can be determined by comparison with testosterone.– disadvantage: cumbersome and difficult– advantage: measures substance with biological activity,

not just amount

Original bioassay systems defined the endocrine system

• Remove endocrine gland and observe what happened

• Prepare crude extract from gland, inject back into animal and observe what happened

• In isolated organ or cell systems, add extract or purified hormonal preparations and measure biological response

Hormonal measurements

• Chemical methods– chromatography– spectrophotometery

Radioimmunoassay• Radioactive ligand and unlabeled ligand compete for same antibody.

Competition is basis for quantitation– saturate binding sites with radioactively labeled hormone (ligand)– in parallel incubate complex with unknown and determine its

concentration by comparison– cold ligand (standard or unknown) competes with labeled ligand

for binding to antibody and displaces it in a dose-dependent way– amount of cold ligand is inversely proportional to amount of

radioactivity – (cold competes with hot so the more cold that binds antibody the

more hot is displaced resulting in fewer counts being associated with complex.

RIA

radi

oact

ivit

y

Increasing amount of insulin

RIA

• advantages:– extremely sensitive due to use of radioisotope

– large numbers of samples can be processed simultaneously

– small changes in hormone concentrations can be reproducibly quantitated

– Easily automated for high-throughput analysis

• disadvantage:– can't determine if hormone measured has biological activity

– peptide hormones can be denatured and not active but still retain their antigenic character

Classes of hormones  

The hormones fall into two general classes based on their solubility in water. The water soluble hormones are the

catecholamines (epinephrine and norepinephrine) and peptide/protein hormones.

The lipid soluble hormones include thyroid hormone, steroid hormones and Vitamin D3

Types of receptors

Receptors for the water soluble hormones are found on the surface of the target cell, on the plasma membrane. These types of receptors are coupled to various second

messenger systems which mediate the action of the hormone in the target cell.

Receptors for the lipid soluble hormones reside in the nucleus (and sometimes the cytoplasm) of the target cell. Because these hormones can diffuse through the lipid

bilayer of the plasma membrane, their receptors are located on the interior of the target cell

Hormones and their receptorsHormone Class of

hormoneLocation

Amine (epinephrine)

Water-soluble Cell surface

Amine (thyroid hormone)

Lipid soluble Intracellular

Peptide/protein Water soluble Cell surface

Steroids and Vitamin D

Lipid Soluble Intracellular

Second messenger systems

Receptors for the water soluble hormones are found on the surface of the target cell, on the plasma membrane. These types of receptors are coupled to various second messenger systems which mediate the action of the hormone in the target cell

Second messengers for cell-surface receptors

Second messenger systems include: Adenylate cyclase which catalyzes the conversion of

ATP to cyclic AMP; Guanylate cyclase which catalyzes the conversion of

GMP to cyclic GMP (cyclic AMP and cyclic GMP are known collectively as cyclic nucleotides);

Calcium and calmodulin; phospholipase C which catalyzes phosphoinositide turnover producing inositol phosphates and diacyl glycerol.

Types of receptors

Second messenger systems

Each of these second messenger systems activates a specific protein kinase enzyme. These include cyclic nucleotide-dependent protein

kinases Calcium/calmodulin-dependent protein kinase, and

protein kinase C which depends on diacyl glycerol binding for activation. Protein kinase C activity is further increased by calcium which

is released by the action of inositol phosphates.

Second messenger systems

The generation of second messengers and activation of specific protein kinases results in changes in the activity of the target cell which characterizes the response that the hormone evokes.

Changes evoked by the actions of second messengers are usually rapid

Signal transduction mechanisms of hormones

Activation of adenylate

cyclase 

Inhibition of adenylate

cyclase 

Increased phospho-inositide turnover

Tyrosine kinase activation

 

-adrenergic 2-adrenergic 1-adgrenergic Insulin

LH, FSH, TSH, hCG

Opioid Angiotensin II Growth factors (PDGF, EGF, FGF, IGF-1

Glucagon Muscarinic cholinergic – M2

Muscarinic cholinergic – M3

Growth hormone

Vasopressin- V2   Vasopressin –V1 Prolactin

ACTH      

Cell surface receptor action

G-protein coupled receptorsAdenylate cyclase, cAMP and PKA

Amplification via 2nd

messenger

Transmembrane kinase-linked receptors

Certain receptors have intrinsic kinase activity. These include receptors for growth factors, insulin etc. Receptors for growth factors usually have intrinsic tyrosine kinase activity

Other tyrosine-kinase associated receptor, such as those for Growth Hormone, Prolactin and the cytokines, do not have intrinsic kinase activity, but activate soluble, intracellular kinases such as the Jak kinases.

In addition, a newly described class of receptors have intrinsic serine/threonine kinase activity—this class includes receptors for inhibin, activin, TGF, and Mullerian Inhibitory Factor (MIF).

Protein tyrosine kinase receptors

Receptors for lipid-soluble hormones reside within the cell Because these hormones can diffuse through the lipid

bilayer of the plasma membrane, their receptors are located on the interior of the target cell.

The lipid soluble hormone diffuses into the cell and binds to the receptor which undergoes a conformational change. The receptor-hormone complex is then binds to specific DNA sequences called response elements.

These DNA sequences are in the regulatory regions of genes.

The receptor-hormone complex binds to the regulatory region of the gene and changes the expression of that gene.

In most cases binding of receptor-hormone complex to the gene stimulating the transcription of messenger RNA.

The messenger RNA travels to the cytoplasm where it is translated into protein. The translated proteins that are produced participate in the response that is evoked by the hormone in the target cell

Responses evoked by lipid soluble hormones are usually SLOW, requiring transcription/translation to evoke physiological responses.

Receptors for lipid-soluble hormones reside within the cell

Mechanism of lipid soluble hormone

action

Receptor control mechanisms

• Hormonally induced negative regulation of receptors is referred to as homologous-desensitization

• This homeostatic mechanism protects from toxic effects of hormone excess.

• Heterologous desensitization occurs when exposure of the cell to one agonist reduces the responsiveness of the cell any other agonist that acts through a different receptor.

• This most commonly occurs through receptors that act through the adenylyl cyclase system.

• Heterologous desensitization results in a broad pattern of refractoriness with slower onset than homologous desensitization

I fought the law, but the law won…..

Mechanisms of endocrine disease

• Endocrine disorders result from hormone deficiency, hormone excess or hormone resistance

• Almost without exception, hormone deficiency causes disease – One notable exception is calcitonin deficiency

• Deficiency usually is due to destructive process occurring at gland in which hormone is produced—infection, infarction, physical compression by tumor growth, autoimmune attack

Mechanisms of endocrine disease

Type I Diabetes

• Deficiency can also arise from genetic defects in hormone production—gene deletion or mutation, failure to cleave precursor, specific enzymatic defect (steroid or thyroid hormones)

Mechanisms of endocrine disease

Congenital Adrenal Hyperplasia

• Inactivating mutations of receptors can cause hormone deficiency

Mechanisms of endocrine disease

Testicular Feminization Syndrome

• Hormone excess usually results in disease

• Hormone may be overproduced by gland that normally secretes it, or by a tissue that is not an endocrine organ.

• Endocrine gland tumors produce hormone in an unregulated manner.

Mechanisms of endocrine disease

Cushing’s Syndrome

• Exogenous ingestion of hormone is the cause of hormone excess—for example, glucocorticoid excess or anabolic steroid abuse

Mechanisms of endocrine disease

• Activating mutations of cell surface receptors cause aberrant stimulation of hormone production by endocrine gland.– McCune-Albright syndrome usually caused by

mosaicism for a mutation in a gene called GNAS1 (Guanine Nucleotide binding protein, Alpha Stimulating activity polypeptide 1).

– The activating mutations render the GNAS1 gene functionally constitutive, turning the gene irreversibly on, so it is constantly active. This occurs in a mosaic pattern, in some tissues and not others.

Mechanisms of endocrine disease

• Malignant transformation of non-endocrine tissue causes dedifferentiation and ectopic production of hormones

• Anti-receptor antibodies stimulate receptor instead of block it, as in the case of the common form of hyperthyrodism.

Mechanisms of endocrine disease

Grave’s Disease

• Alterations in receptor number and function result in endocrine disorders

• Most commonly, an aberrant increase in the level of a specific hormone will cause a decrease in available receptors

Mechanisms of endocrine disease

Type II diabetes

Hypothalamus and Pituitary

Hypothalamus and Pituitary

• The hypothalamus-pituitary unit is the most dominant portion of the entire endocrine system.

• The output of the hypothalamus-pituitary unit regulates the function of the thyroid, adrenal and reproductive glands and also controls somatic growth, lactation, milk secretion and water metabolism.

Hypothalamus and pituitary gland

Hypothalamus and pituitary gland

• Pituitary function depends on the hypothalamus and the anatomical organization of the hypothalamus-pituitary unit reflects this relationship.

• The pituitary gland lies in a pocket of bone at the base of the brain, just below the hypothalamus to which it is connected by a stalk containing nerve fibers and blood vessels. The pituitary is composed to two lobes-- anterior and posterior

Hypothalamus and Pituitary

Posterior Pituitary: neurohypophysis

• Posterior pituitary: an outgrowth of the hypothalamus composed of neural tissue.

• Hypothalamic neurons pass through the neural stalk and end in the posterior pituitary.

• The upper portion of the neural stalk extends into the hypothalamus and is called the median eminence.

Hypothalamus and posterior pituitary

Midsagital view illustrates that magnocellular neurons paraventricular and supraoptic nuclei secrete oxytocin and vasopressin directly into capillaries in the posterior lobe

Anterior pituitary: adenohypophysis

• Anterior pituitary: connected to the hypothalamus by the superior hypophyseal artery.

• The antererior pituitary is an amalgam of hormone producing glandular cells.

• The anterior pituitary produces six peptide hormones: prolactin, growth hormone (GH), thyroid stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH).

Hypothalamus and anterior pituitary

Midsagital view illustrates parvicellular neurosecretory cells secrete releasing factors into capillaries of the pituitary portal system at the median eminence which are then transported to the anterior pituitary gland to regulate the secretion of pituitary hormones.

Anatomical and functional organization

Reituclar activating substance

Thalamus

neocortex

Limbic system

Optical system

Heat regulation (temperature)

Energy regulation (hunger,

BMI)

Autonomic regulation

(blood pressure etc)

Water balance (blood volume, intake--thirst, output—urine volume)

Metabolic rate, stress response, growth,

reproduction, lactation)

Sleep/wake

pain Emotion, fright, rage, smell vision

Anterior pituitary

hormonesposterior pituitary

hormones

Regulation of

Hypothalamus

Hypothalamus/Pituitary Axis

Hypothalamic releasing factors for anterior pituitary hormones

Travel to adenohypophysis via hypophyseal-portal circulation

Travel to specific cells in anterior pituitary to stimulate synthesis and secretion of trophic hormones

Hypothalamic releasing hormonesHypothalamic releasing hormone Effect on pituitary

Corticotropin releasing hormone (CRH)

Stimulates ACTH secretion

Thyrotropin releasing hormone (TRH)

Stimulates TSH and Prolactin secretion

Growth hormone releasing hormone (GHRH)

Stimulates GH secretion

Somatostatin Inhibits GH (and other hormone) secretion

Gonadotropin releasing hormone (GnRH) a.k.a LHRH

Stimulates LH and FSH secretion

Prolactin releasing hormone (PRH) Stimulates PRL secretion

Prolactin inhibiting hormone (dopamine)

Inhibits PRL secretion

Characteristics of hypothalamic releasing hormones

• Secretion in pulses• Act on specific membrane receptors• Transduce signals via second messengers• Stimulate release of stored pituitary hormones• Stimulate synthesis of pituitary hormones• Stimulates hyperplasia and hypertophy of target

cells• Regulates its own receptor

Hypothalamus and anterior

pituitary

Anterior pituitary

• Anterior pituitary: connected to the hypothalamus by hypothalmoanterior pituitary portal vessels.

• The anterior pituitary produces six peptide hormones: – prolactin, growth hormone (GH), – thyroid stimulating hormone (TSH), – adrenocorticotropic hormone (ACTH), – follicle-stimulating hormone (FSH), – luteinizing hormone (LH).

Anterior pituitary cells and hormones

Cell type Pituitary population

Product Target

Corticotroph 15-20% ACTH-lipotropin

Adrenal glandAdipocytesMelanocytes

Thyrotroph 3-5% TSH Thyroid gland

Gonadotroph 10-15% LH, FSH Gonads

Somatotroph 40-50% GH All tissues, liver

Lactotroph 10-15% PRL Breastsgonads

Anterior pituitary hormones