General principles of endocrine functions Integration systems of the organism Physical; emotional; chemical; etc. • Hormonal system • Nervous system • Immune system Hormones Neurohormones Neurotransmitters Paracrine (autocrine) effectors CRH neurons Pituitary gland Adrenal gland Medulla • • • • Epinephrine and ©Norepinephrine • • • Cytokines such as IL-1 Viruses; bacteria; tumors How do cells communicate? • Intracrine • Autocrine • Paracrine • Neurocrine • Endocrine • Neuroendocrine source environment target eel 1. INTRACRINE 2. AUTOCRINE 3. PARACRINE TARGET CELLS SIGNALING CELL 5. ENDOCRINE BLOOD VESSEL ENDOCRINE CELLS TARGET CELLS 4. NEUROCRINE NEURONS 6. NEUROENDOCRINE NEUROSECRETORY NEURON BLOOD VESSEL ANTERIOR PITUITARY CELLS source synthesis/secretion no influence on specificity of effect - synthesis/secretion - main determinant of target eel (determined by localization) environment blood - universal environment - dilution and interactions r matrix/interstitial fluid J diffusion binding proteins proteases components of extracellular matrix - receptor = specificity - cell response - number of receptors - signaling pathways - other ligands - metabolisation of ligand/receptor - specificity and sensitivity - diffusion barrier - determinants of gradient - inhibition signaling pathways - effect of other ligands - binding proteins Hormones • Starling 1905 - secretin • Glandotropic hormones • Aglandotropic hormones • Target cells • Limited time of effect Hypothalamus Releasing hormones: GHRH, CRH, TRH, GnRH Inhibitory hormones: somatostatin, dopamine, vasopressin oxytocin Thyroid gland T3, T4, & calcitonin Adrenal glands Cortisol Aldosterone Adrenal androgens Epinephrine Norepinephrine Ovaries Estrogens Progesterone Pituitary gland Growth hormone, Prolactin. ACTH, MSH. TSH. FSH, & LH Parathyroid glands Parathyroid hormone Pancreas Insulin Glucagon Somatostatin Testes Testosterone Source: Molina PE: Endocrine Physiology, 4th Edition: wwyy.accessmedicine.com Copyright c The McGraw-Hill Companies, Inc. All rights reserved. Chemical nature of hormones DERIVED FROM AMINOACIDS -Adrenaline -Noradrenaline H3c~-o -Dopamine -Melatonine -T3/T4 STEROID -Cortisol -Aldosterone -Testosterone -Progesterone -Estradiol -Calcitriol HN.XH3 T o PEPTIDES AND PROTEINS -Hypothalamic hormones -Adenohypophyseal hormones -Insulin, glucagon, somatostatin -Gastrin, cholecystokinin, secretin -Natriuretic peptides -Erythropoietin, thrombopoietin -PTH, PHrP -etc h,n o ho ^ o nh n-,'' t' 0 hn \ /—NH ' o 0M\ y—nh gH /— i.....U0 o^nh " „hn nh f oh hn-v^nh h Nu OK((f Os NH f hn ^0 hn^^-oh hA>° nh2 nh; Chemical nature of hormones Hormone -characteristics Peptides -proteins Catecholamines Steroid hormones Thyroid hormones Ph-CH properties hydrophilic hydrophilic lipophilic lipophilic synthesis proteosynthesis Tyr modification CH precursors Tyr modifications storage secretory granules secretory granules not present colloid secretion controlled exocytosis controlled exocytosis diffusion diffusion transport free free/weakly bound bound bound elimination half-life short (4-40-170min) very short (2-3 min) moderate (up to 180 min) long (20 hours - 7 days) receptors membrane membrane cytosol nuclear effect short-term very short-term long-term long-term cell response quick very quick slow slow CHEMICAL STRUCTURE OF HORMONES DETERMINES THEIR BIOSYNTHESIS, STORAGE, RELEASE, TRANSPORTATION, ELIMINATION HALF-LIFE, WAY OF ELIMINATION AND THE MECHANISM OF EFFECT ON TARGET CELLS Hormones • Pleiotropic effects Multiplicity • Permissive effect Arterioles- a2 receptors Endocrine organs * specialised cells - specialised organs („endocrine") * „secretory" cells - organs with endocrine function * cells without specialised secretory function * cells converting hormone precursors adipose tissue Adipokines Clinical aspects • Production of hormones by tumors - PARANEOPLASTIC SYNDROMES Lung tumors Liver and kidney tumors GIT tumors - ADH (hyponatremia) - erythropoietin I - ACTH (Cushing syndrome) - ACTH (Cushing syndrome) (polycythemia) - PTHrP (hypercalcaemia) Secretion of hormones and its regulation • Neuronal control • hypothalamus • sympathetic/parasympathetic nervous system • Hormonal control • Regulation od secretion by ions or substrates (Glu, AA) INSULIN Hormone secretion is controlled by feedback system biologická odpověď hypotalamus 1© adenohypofýza 1 © *roPni hormon I hormon cílové žlázy I biologický účinek endokrinní buňka I hormon Feedback negative X positive simple X complex Taken from Kittnar et al. Lékařská fyziologie. 1st edition. Grada 2011. kalcemie Cyclic changes in hormone secretion External 24 h light-dark cycle Endogenous circadian rhythm SCN (Master clock) #2f Entrainment Photic Zeitgeber Synchronization Non photic Zeitgeber ■ Sleep-wake cycle ■ Physical activity ■ Social time ■ Meals oscillators Cellular oscillators SCN: - Afferent - retina - Efferent - hypothalamic nucleus - Melatonin - GHRH/GHIH - ADH - ACTH - Cortisol - Insulin - Ghrelin - Adiponectin - Leptin Neuronal/hormona = SNC-dependent Satiety/fasting Body temperature ft) t3 o o o o 77 O Lepttn (% change from 0BO0 h) 00 Plasma Cortisol (nmol/L) > Normalized CSF IR-hCRH (SD) Tl Peripheral LH cane. (ng/mL) „ o no a en oo K g ST g f B Portal Qn-RHcgng (pg/mL) m Serum TSH (mU>L) ( ) i: -» -* rj l\3 lj ftg ooiouiotnotn 1 _i_L Plasma melatonin (pmol/L) •»■'■ & CA .-it g g § § < o 5 3 2. o Hormone transport Chemical properties of hormone Transport protein(s) bond and its significance • Albumin • Globulins • Specific proteins - TBG, SHBG, CBG Bond strength ^Alternative'' binding - TBG versus transthyretin •Protection •Reservoir •Ubiquitous distribution •Transport across plasmatic membrane (SHBG-megalin) DYNAMIC BALANCE BETWEEN HORMONE AND TRANSPORT PROTEIN Hormone elimination Different length of time in circulation Metabolisation by • Target cells • Enzymatic systems in blood • Organs - mainly liver Elimination • Liver • Kidneys PHASE I Hydroxylation, decarboxylation Oxidation, reduction PHASE II Glucuronidation Sulphatation Methylation Conjugation with glutathione \7 Vascular system \7 \7 bile urine Hormones and cell response Target cells Specificity High affinity Selectivity hormone • oo O°°o??0 MECHANISMS Conformation changes Phosphorylation/dephosphorylation + protein recruitment GTP binding (G proteins) cAMP binding (efector proteins) Precursor molecule generation in PM Non-covalent Ca2+ bond SIGNALING PATHWAYS CELL RESPONSE Receptor binding Signal amplification and transduction efector molecules % of occupied receptors conformation change synergy antagonism possible loss of sensitivity feedback-loop regulation CELL RESPONSE IS MEDIATED BY RELEVANT RECEPTORS Receptor level of cell response regulation Active receptor Desensitized receptor • Downregulation • Upregulation • Homologous desensitization • Heterologous desensitization Phosphorylation (specific kinases) Dephosphorylation (specific phosphatases) Modification by proteins of inhibited signaling pathway Receptor protein Agonist TOOtlJ J'. .Y< .'.VI, .'rXiei»^ÄW..M'. WW .'.'< r IV. nmxu».'w>jnm\t/),i,M t Uncoupling Signaling from signaling cascade cascade Endosome (a) Receptor inactivation (b) Receptor internalization Lysosome (c) Receptor down-regulation Figure 13.10. Major mechanisms for the termination of receptor-dependent signal transduction. Textbook of Biochemistry With Clinical Correlations. Sixth Edition, Edilcd by Thomas M. Devlin. Copyrighl © 2006 John Wiley & Sons, Inc. Hormones - proteins and peptids Hormones Paracrine/autocrine peptides Signal (26) □ Prepraopiomelanocortin (265) (146) ft-Lipotropin (91) N-terminal peptide (76) I ACTH (39) r-Lipolropin (58) I □ (13) CLIP Nucleus ß -Endorphin (31) [ ) Peptide length in amino acids MSH sequences Koeppen & Stanton: Berne and Levy Physiology, 6th Edition, Copyright © 2006 by Mosby, an imprint of Elsevier, Inc. All rights reserved Capillary lumen Secretory vesicles , O ° ° °° o O £ Immature secretory granules Mature secretory granules Plasma membrane Lysosome preprohormone - prohormone - hormone (+ fragments) G protein-coupled receptors (GPCR) Gs - Gs, G0|f - activation of AC inhibition of AC •G0 (2, brain) •Gt (2, photorec. - cAMP-PDE) •Gz (inhibition of K+channels) Gq/11 - activation of PLCß G12/13" inhibition and activation of RhoGEF Peptide and protein: Glucagon, Angiotensin, GnRH, SS, GHRH, FSH, LH, TSH, ACTH Amino acid derived: Epinephrine, norepinephrine Ion channels, PI3Ky, PLC-ß, adenylate cyclases Biological responses Adenylate cyclase I CAMP PLC-ß DAG Ca++ PKC Adenylate cyclase t cAMP Gene expression regulation factors Nucleus Receptor tyrosinkinases i EGF receptor PDGF receptor 11 4 B T T Insulin receptor Cys-rich domain Ig-like domain } Tyrosine kinase domain • 58 RTKs/20 subfamilies • Usually dimerisation after ligand binding • ATP as a source of P for phosphorylation of intracellular domains/associated proteins • Insulin • IGF-1/2 Receptors associated with cytosolic TK • GH, prolactin, leptin, erythropoietin • Dimeric receptor without TK activity • Association with JAK kinase • After ligand binding -dimerisation, transphosphorylation, activation signal transducers and activators of transcription Receptor serin/threonin kinases Anti-Müllerian hormone, inhibitin Form of dissociated heterodimer SMAD = „latent transcription factors" TGF-ß-related hormones RII/RI dimer ^Active smad) Ccq-smaqI) Nucleus Regulation of specilic gene expression gamma-activated sequence-like elements (GLEs, promotor region of some genes) Receptor guanylate cyklases Signal transduction - system of second messengers HORMONE = FIRST MESSENGER INTRACELLULAR SIGNALING MOLECULE GENERATED AFTER HORMONE-RECEPTOR BONDING = SECOND MESSENGER CAMP • TSH, glucagon, ACTH, hypothalamic hormones, ADH etc. • Proteinkinase A Modulation of signaling pathways by compartmentalization (A-kinase anchoring proteins (AKAPs)) cGMP • ANP, BNP, CNP • NO as a signaling molecule • Proteinkinase G • DAG and IP • PIP2 - phospholipase C system • Ca2+/Ca2+-calmodulin AC-cAMP system PKA CREB (cAMP-responslve element-binding protein) Epac (E) as an another effector molecule (exchange protein activated by cAMP) cyclic nucleotide gated (CNG) channels hyperpolarization-activated cyclic nucleotide modulated (HCN) channels phosphodiesterases ATP cAMP IL Protein phosphorylation (membrane, cytosolic, & nuclear proteins) Ionic current (e.g., K+) RAP GTP Activation of effector proteins Cellular response PLC - DAG and IP3 system Ca2+ - calmodulin system O Calmodulin binds Ca2+ four calcium ions Calmodulin Calcium- calmodulin complex Q Calmodulin change conformation, resulting in an active complex Target protein Q The two globular hands" of the complex wrap around a binding site on a target protein Calmodulin-binding site (a) Structure of Ca2^-calmodulin complex (b) Function of Ca2^-calmodulin complex Copyright © 2009 Pearson Education, inc. O 3 o Q. C 8- •a 3 Q. n> 3 ,3 £U in fl> Extracellular fluid Channel closed Change in electrical properties of cell Calcium enters cell through open channel As second messenger Muscle Secretion Calmodulin contraction Ca O -O O 1 lm< Ca-calmodulin Activates enzymes Protein- P \ Response in cell (muscle contraction, altered metabolism, altered transport) Extracellular signals (hormones, neurotransmitters) NO as a signalling molecule - cGMP °2\ Arqinine / eNOsY M NO Citrulline Cellular response (\ smooth muscle tone) NOS Protein Modification Gene Expression Mitochondrial Respiration Cytoprotection cGMP signalling y signalling f \ ONOOJ In situ NO T le NO ^ J Oxidation Reduction (Normoxia) {Hypoxia) i J > NO,' NO/ROS 8-nitro-cGMP 1 S-Guartylation Donald JA, Forgan LG, Cameron MS: The evolution of nitric oxide signalling in vertebrate blood vessels. J Comp Physiol B-Biochem Syst Environ Physiol 2015, 185(2):153-171. Clinical aspects Syndromes of resistance to hormones (i.e. IR, IGF-1, TR(3) Syndromes caused by CPCRs and G proteins mutations • ADH - nephrogenic diabetes insipidus • ACTH - familiar ACTH resistance • GnRH - hypogonadotrophic hypogonadism • FSH - hypergonadotrophic ovarial dysgenesis • LH - male pseudohermaphroditism • Melanocortin 4 - obesity • PTH/PTHrP - Blomstrand lethal chondrodysplasia Hormones acting through nuclear receptors HORMONES -Thyroid hormones - TRot/ß <^ i heterodimers -Estrogens - ERct/ß -Testosterone - AR -Progesterone - PR homodimers -Aldosterone - MR -Cortisol - GR VITAMINS -1,25-[0H]2D3 - VDR -All-trons-retinoic acid - RA receptors a, ß, y -9-c/s-retinoic acid - retinoid X receptor RXR a, ß, y PRODUCTS OF METABOLISM AND XENOBIOTICS -Fatty acids- PPAR a, (3, y -Oxysterols - liver X receptor LXR a, p -Bile acids - BAR -Hem - RevErb a, p -Phospholipids - homologue of liver receptor LRH-1, SF-1 -Xenobiotics - pregnane X receptor PXR - constitutive androstane receptor CAR Orphan receptors Variable receptors Explanation of some effects and pathologies General mechanism of effect of hormones acting through nuclear receptors Prohormone (ligand precursor) Hormone (ligand) -High affinity of ligand bond = due to R structure -Recognition of specific promotor region -Dimerisation of receptors (homodimers, heterodimers) -Remodelation of chromatin for gene expression (HDAC) -Gene expression at the end decreased or increased WHY ONLY NUCLEAR RECEPTORS? -Synthesis in cytoplasm -Stay until ligand binding or until transport to nucleus -Regulation mechanism - modification, count of receptors -Important parameter - selectivity of target cells -Tissue-specific factors, coactivators and corepressors Nuclear receptors ATD DBD LBD (amino terminus domain) (DNA binding domain) (ligand binding domain) -Coregulatory proteins binding (independent on ligand) - Phosphorylation sites -DNA binding (zinc fingers) -Dimerisation -ERE, PRE, GRE, MRE, ARE -Ligand binding (agonist, antagonist) -Coregulatory proteins binding (dependent on ligand) -Dimerisation -Nuclear translocation -Chaperone association (HSP) Example - steroid hormones X thyroid hormones Pathway 1 (Steroid hormones) (-) Hormone — HRE Pathway 2 (Thyroid hormones, vitamin D. PPARs) {-) Hormone Blocking general transcription laclor HRE Gene Chromatin structure Ě Gene {+) Hormone Repressed transcription Dissociation ot co-repressors stimulated transcription Stimulated transcription Termination of hormone action Receptor-mediated endocytosis and subsequent lysosome degradation Phosphorylation/ dephosphorylation of receptor or proteins of signaling pathway Ubiquitination and proteosomal degradation Binding of regulatory factor on corresponding protein (enzyme) Inner enzymatic activity and its regulation Clinical aspects Hormone overproduction Hormone underproduction Changes in sensitivity of target tissues and/or change in cell response Higher rate of inactivation or degradation of hormones Insufficient production or higher degradation of transport proteins Changes of transport hormones production during physiological conditions (pregnancy) Clinical aspects A. Decreased hormone responsiveness B. Decreased hormone sensitivity Source: Molina PE: Endocrine Physiology, 4th Edition; www.actessmediclrje.tonn Source: Molina pe: Endocrine Physiology, 4th Edition: wnw^accessmedicHie.corn Copyright & Tl>e McGraw-Hill Conn panic*, inc. All rights reserved. Copyright & T Its McGraw-Hill Companies, I no. All rights reserved. Decreased number of receptors Decreased concentration of hormone-activating enzyme(s) Increased concentration of non-competitive inhibitor Decreased number of target cells Decreased affinity of hormone to receptor Decreased number of receptors Increased rate of hormone degradation Increased concentration of antagonists/competitive inhibitors Determination of hormone levels in blood -HIGH SENSITIVITY DEMANDS -WIDE CONCENTRATION RANGE Antigen-antibody interaction-based methods -Anibody requirements (poly- X monoclonal) -Monoclonal antibodies = specific epitopes -Radioactive labeled antibodies -Necessity of quantification! -RIA, ELISA Methods based on HPLC-MS Nucleic acid-based methods -hybridization techniques -restriction fragmentation, electrophoresis, sequencing Separation techniques - free X bound hormones dialysis Cortisol Vitamin D-2S-OH - Progesterone - DHEA - T3 - Testosterone—mala - Norepinephrine - !- SH Prolactin Testosterone—female Vitamin D 1.25-OH Estradiol—lemale Estradiol—male -LH -Aldosterone TSH FT* -fnsuiin - FT3 -Epinephrine Growth hormone —I- 100 10,000 r 1,000,000 Concerttralion in picomolesA. EXTREMELY LOW LEVELS OF HORMONES IN BLOOD RIA = radioimmunoassay Antibody affinity = K1/K_1 = [AbAg]/[Ab][Ag] 125l-Analyte i 8 .i, — ft Competitive binding 6 Ab - Ag* 10 Ab' 8 Ab + 16 Ag* + 4Ag°^± 2Ab-Ag° 2Ag° -< -< **** :-<*-<0i [i # 0 j -< < + **** + 00 + j * * * i -< -< **** oo I * * * i < •< **** l-O -<*j J* 0 *i Bound 1 IV T Calibration of standards Ab + Ag* + Aga Ab • Ag*+Ab< Ag° + Ag* t Ag° 8 16 0 i 8 0 ; 4 16 2 ! a 0 6 16 10 2 8 16 12 i 4 4 i 12 8 8 16 36 2 6 | 14 30 Constant Vanabte Bound Free □ "I- T 12 16 20 24 28 32 36 Concern ration }NSB