Endocrine Physiology lecture 2

1. Endocrine Physiologylecture 2 Dale Buchanan Hales, PhD Department of Physiology & Biophysics

2. 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

3. Hormone Half-life Amines 2-3 min Thyroid hormones: T4 T3 6.7 days 0.75 days Polypeptides 4-40 min Proteins 15-170 min Steroids 4-120 min MCR of some hormones

4. 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.

5. 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

6. Hormone binding study

7. 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

8. Analysis of hormone interactions: Scatchard plots

9. 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

10. 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

11. Binding vs. biological response Spare receptors Amplification by 2nd messenger

12. 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

13. 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

14. Hormonal measurements • Chemical methods • chromatography • spectrophotometery

15. 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.

16. RIA radioactivity Increasing amount of insulin

17. 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

18. 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

19. 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

20. Hormone Class of hormone Location 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 Hormones and their receptors

21. 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

22. 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.

23. Types of receptors

24. 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.

25. 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

26. Activation of adenylate cyclase Inhibition of adenylate cyclase Increased phospho-inositide turnover Tyrosine kinase activation b-adrenergic a2-adrenergic a1-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 Signal transduction mechanisms of hormones

27. Cell surface receptor action

28. G-protein coupled receptors Adenylate cyclase, cAMP and PKA

29. Amplification via 2nd messenger

30. 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, TGFb, and Mullerian Inhibitory Factor (MIF).

31. Protein tyrosine kinase receptors

32. 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.

33. 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.

34. Mechanism of lipid soluble hormone action

35. 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

36. I fought the law, but the law won…..

37. 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

38. Mechanisms of endocrine disease • Deficiency usually is due to destructive process occurring at gland in which hormone is produced—infection, infarction, physical compression by tumor growth, autoimmune attack Type I Diabetes

39. Mechanisms of endocrine disease • 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) Congenital Adrenal Hyperplasia

40. Mechanisms of endocrine disease • Inactivating mutations of receptors can cause hormone deficiency Testicular Feminization Syndrome

41. 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. Cushing’s Syndrome

42. Mechanisms of endocrine disease • Exogenous ingestion of hormone is the cause of hormone excess—for example, glucocorticoid excess or anabolic steroid abuse

43. 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.

44. 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. Grave’s Disease

45. 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

46. Hypothalamus and Pituitary

47. 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.

48. Hypothalamus and pituitary gland

49. Hypothalamus and pituitary gland

50. 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