Biological response is determined by

Assessment of endocrine function hormonal assays

Concentration of hormone Transport of hormone to the effector tissue Receptor interaction Number of receptors Affinity of interaction Post-receptor effects (intracellular signaling mechanisms)

Assessment of endocrine function

Early studies of endocrine system Anatomical

Measurements of hormone levels in plasma and urine Measurements of hormone levels over time Assessment of diurnal and nocturnal cycle Measurement of both arms of hormone feedback system (also points to the origin of dysfunction) Dynamic tests stimulation (entry at or below the level of hormone action and test peripheral parts of signaling cascade) suppression of hormone production and secretion (test the negative feedback)

Size of organ Goiter enlarged thyroid Changes in cytological characteristics Hypertrophy enlarged, hyperactive Hyperplasia increased cell number Atrophy reduced in size, hypoactive

Physiological studies of endocrine system

Bioassays - in vivo

Removal and replacement Injectem and inspectem Modern version Gene knock-out and overexpression

Based on response of target tissue Employs animals or tissues to asses presence of active substance based on known physiological effects Lack sensitivity, precision, specificity No need for purified molecules In experiments pharmacological vs. physiological doses http://history.nih.gov/exhibits/thinblueline/timeline.html

Assays for plasma hormone levels next step

RIA - radioimmunoassays

In vitro

In vitro assays
Immunometric Competitive binding of radiolabelled and natural hormone to antibodies Anti-hormone antibody can serve as high affinity binding for a hormone Reduced binding of radioligand in the presence of hormone Readout radiation

Radioimmunoassays Nonisotopic immunoassays

RIA radioimmunoassays

Spin-offs of RIA

Need for purified hormone and antibodies Information about concentration of hormone, no information about the receptor

ELISA Binding of hormone to AB reported by color rather than radiation AB can be replaced by natural binding proteins such as recombinant receptors

ELISA

Advantages

Enzyme-Linked ImmunoSorbent Assay Biochemical reaction to identify the presence of antigen or antibodies in a sample Direct: Antigen detection Indirect: Antibodies detection

Non-radioactive High specificity Color change or florescence allows to use photometroscopy Amplification of minute levels allows for high sensitivity Qualitative and Quantitative Used to find blood serum antigens

Direct ELISA: Antigen detection

Indirect ELISA: Antibodies detection

Enzyme linked Antibodies to the antibodyantigen complex Antibodies applied to surface

Enzyme linked Antibodies to the antibodyantigen complex Antigens in sample Antibodies in sample (HIV Antibodies, for example) Known Antigens applied to surface

Activity assays

Radioreceptor assays

Receptor assays new in vitro bioassays

Similar to RIA except intact cells or membranes are used as antigen Tests for receptors on cells or cellular components Provide the information about binding to the receptor of choice (in case of multiple receptors for the same hormone) Assess total activity (binding power) No information about biological activity (measures only the first step in the cascade, no information about second messengers)

Radioreceptor assays

Interpreting results of receptor binding studies

Scatchard plot Determines association constant for the receptor

Receptor affinity (binding strength) Defined in terms of equilibrium dissociation constant in single class, noncooperative system - concentration of hormone that is required for binding 50% of sites Receptor specificity (selectivity) Defined by structural features of hormones and analogs

Receptor specificity

Receptor cooperativity
How binding of the hormone to the receptor affects the subsequent binding Noncooperative Positively cooperative Negatively cooperative

How much hormone do we really need amplification of hormone signaling

Amplification in signaling cascades

Progressive steps in receptor signaling

Hormones levels are very low and signal needs to be amplified Maximal biological response occurs below the saturation level Spare receptors

Spare receptors

No spare receptors

Maximum biological response is achieved with only a fraction of receptors occupied Spare receptors - receptors present in numbers that exceed those required for maximal response Role Augment sensitivity to low hormone concentrations by increasing probability of binding Assure continuous availability of receptors during internalization

Spare receptors

Increase of biological response for low concentration of hormone when spare receptors are present

No spare receptors Signal amplified by spare receptors

Regulation of receptor number

Desensitization

Number of receptors in the membrane can be regulated dependent on the stage of development or differentiation Upregulation and downregulation Homospecific and heterospecific regulation

Homologous Requires receptor occupancy Usually involves internalization, phosphorylation Heterologous Can affect function of unoccupied receptors Mediated by different enzymes Mostly occurs with receptors acting through the adenylyl cyclase

Desensitization of adrenergic receptor

Desensitization

Second messengers produced by activated receptor activate ARK ARK phosphorylates the receptor Phosphorylated receptor is less active and binds arrestin (a protein that prevents interaction of receptor with G protein)

Dysfunction due to changes in receptor number and function

Dysfunction caused by anti-receptor antibodies

Homologous down-regulation of insulin receptors by insulin in chronic hyperinsulinemia in obesity Caused by clustering Internalization in clathrin coated vesicles Degradation in lysosomes Heterologous upregulation of adrenergic receptors in hyperthyroidism

Anti-receptor antibodies Graves disease - TSH receptor Insulin resistance Myastenia gravis - nicotinic acetylcholine receptors

Dysfunction caused by postreceptor defects

Effects of Plasma-Binding Proteins

Defects in G protein signaling Activation of Gs by cholera toxin resulting in elevated levels of cAMP McCune-Albright Syndrom (polyostotic fibrous dysplasia) mutation of Gs Vitamin D resistance - inherited deficiency in enzymes in metabolic pathways for vitamin D conversion Androgen resistance

Polypeptide hormones circulate unbound Steroid hormones bound to albumin and specific binding proteins Less than 1-3% in free form Amount of free hormone can be determined through equilibrium dialysis

New in vitro bioassays

Assessment of endocrine function

Incubation of plasma with endocrine tissues, membrane preps, or cultured cells Very sensitive Enables measurements of bioactive hormone when immunoreactive hormone can not be detected

Measurements of hormone levels in plasma and urine Measurements of hormone levels over time Assessment of diurnal and nocturnal cycle Measurement of both arms of hormone feedback system (also points to the origin of dysfunction) Dynamic tests stimulation (entry at or below the level of hormone action and test peripheral parts of signaling cascade) suppression of hormone production and secretion (test the negative feedback)

Alterations in trophic and target hormone pairs

Scientific vs. diagnostic methods

Surgery Histology and immunocytochemistry Immunological neutralization of hormone activity Mutagenesis of hormone of receptors Hybridization Isotope studies Radioimmunoassays Autoradiography Pharmacology Electrophysiology

Therapeutic methods

Surgery Hormone replacement therapy Fetal tissue transplants Neutralizing antibodies Pharmacology Gene therapy