Endocrine Physiology lecture 2

Dale Buchanan Hales, PhD Department of Physiology & Biophysics

 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

Metabolic clearance rate (MCR)

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

MCR of some hormones

Hormone Amines Thyroid hormones: T4 T3 Polypeptides Proteins Steroids Half-life 2-3 min 6.7 days 0.75 days 4-40 min 15-170 min 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

radioactivity 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 receptors

Hormone Amine (epinephrine) Amine (thyroid hormone) Peptide/protein Steroids and Vitamin D Class of hormone Water-soluble Lipid soluble Water soluble Lipid Soluble Location Cell surface Intracellular Cell surface 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 cellsurface 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 b-adrenergic LH, FSH, TSH, hCG Inhibition of adenylate cyclase a2-adrenergic Opioid Increased phosphoinositide turnover a1-adgrenergic Angiotensin II Tyrosine kinase activation

Insulin Growth factors (PDGF, EGF, FGF, IGF-1

Glucagon
Vasopressin- V2 ACTH

Muscarinic cholinergic M2

Muscarinic cholinergic M3
Vasopressin V1

Growth hormone
Prolactin

Cell surface receptor action

G-protein coupled receptors

Adenylate 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 activitythis class includes receptors for inhibin, activin, TGFb, 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.

Receptors for lipid-soluble hormones reside within the cell

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.

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

Mechanisms of endocrine disease

Deficiency usually is due to destructive process occurring at gland in which hormone is producedinfection, infarction, physical compression by tumor growth, autoimmune attack Type I Diabetes

Mechanisms of endocrine disease

Deficiency can also arise from genetic defects in hormone productiongene deletion or mutation, failure to cleave precursor, specific enzymatic defect (steroid or thyroid hormones) Congenital Adrenal Hyperplasia

Mechanisms of endocrine disease

Inactivating mutations of receptors can cause hormone deficiency

Testicular Feminization Syndrome

Mechanisms of endocrine disease

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.
Cushings Syndrome

Mechanisms of endocrine disease

Exogenous ingestion of hormone is the cause of hormone excessfor example, glucocorticoid excess or anabolic steroid abuse

 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. Graves Disease

Mechanisms of endocrine disease

Mechanisms of endocrine 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
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

Hypothalamus and Pituitary

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

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), folliclestimulating 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

neocortex Reituclar activating substance

Thalamus

Limbic system Emotion, fright, rage, smell

Optical system vision

Sleep/ wake
Heat regulation (temperature)

pain

Energy regulation (hunger, BMI)

Autonomic regulation (blood pressure etc)

Water balance (blood volume, intake--thirst, outputurine volume)

Metabolic rate, stress response, growth, reproduction, lactation)

Regulation of Hypothalamus

posterior pituitary hormones

Anterior pituitary hormones

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 hormones

Hypothalamic releasing hormone Effect on pituitary

Corticotropin releasing hormone (CRH) Thyrotropin releasing hormone (TRH) Growth hormone releasing hormone (GHRH) Somatostatin
Gonadotropin releasing hormone (GnRH) a.k.a LHRH Prolactin releasing hormone (PRH) Prolactin inhibiting hormone (dopamine)

Stimulates ACTH secretion

Stimulates TSH and Prolactin secretion Stimulates GH secretion Inhibits GH (and other hormone) secretion Stimulates LH and FSH secretion Stimulates PRL secretion 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 Corticotroph Pituitary Product population 15-20% Target

Thyrotroph

3-5%

Adrenal gland ACTH b-lipotropin Adipocytes Melanocytes TSH Thyroid gland

Gonadotroph
Somatotroph

10-15%
40-50%

LH, FSH
GH

Gonads
All tissues, liver Breasts gonads

Lactotroph

10-15%

PRL

Anterior pituitary hormones