Lecture objectives • Explain the regulation and actions of hormones from the adrenal cortex • Describe how the adrenal gland is involved in acute and chronic stress. • Explain the unique way that thyroid hormones are synthesized and secreted, what regulates their levels in the blood, and how they act. Steroid hormones • Are made from cholesterol, are lipophilic & can enter target cell. – the adrenal cortical hormones, estrogens and androgens • Are immediately released from cell after synthesis • Interact with cytoplasmic or nuclear receptors – Activate DNA for protein synthesis • Are slower in action and have longer half-life than peptide hormones Adrenal corticol hormones • Synthesis is controlled by ACTH secretion. – ACTH acts through cAMP. – ACTH secretion is inhibited by cortisol. • There are 3 classes: i. Glucocorticoids ii. Mineralocorticoids iii. corticoids with both glucocorticoid and mineralocorticoid activities (corticosterone) NB: Glucocorticoids have weak mineralocorticoid activity; mineralocorticoids have weak glucocorticoid activity Enzymes involved in steroid hormone synthesis • Desmolases (or lyases): catalyse reactions which result in the removal of parts of the original cholesterol side-chain. – are located in the mitochondria and are linked to an electron transport system. • Hydroxylases: are membrane-bound and are present either in the mitochondrial or in the microsomal fraction of the cell. – require a cytochrome P-450, molecular oxygen and NADPH • Hydroxysteroid dehydrogenases: catalyse reversible reactions and depend either on NADP(H) or NAD(H). – are found both in the cell cytosol and in the microsomal fraction. • Aromatase: conversion of the A-ring to a phenolic structure (aromatization) – involves a complex sequence of hydroxylation reactions and loss of the angular C-19 methyl group. – Aromatase activity is mainly found in the ovary, the placenta and the brain. – Its substrate is either 4-androstenedione or testosterone. Steroid hormones: synthesis • Cholestryl ester in the adrenal cortex is cleaved by cholesterol esterase to release cholesterol • Cholesterol is transported to the mitochondria to synthesize pregenolone. • Cholesterol is cleaved by 20,22 desmolase to form pregnenolone. – It is the rate-determining step in steroidogenesis • Pregnenolone is the precursor molecule for all C18, C19 and C21 steroids – It is formed on the inner membrane of mitochondria then shuttled back to the smooth endoplasmic reticulum for further metabolism Steroid hormones Synthesis of corticoids • Pregnenolone forms progesterone by 3hydroxy steroid dehydrogenase (3HSD) • Progesterone is produced directly from pregnenolone – Can be secreted from the corpus luteum • Synthesis of cortisol from progesterone: – hydroxylations at C17, C21 and C11 • Synthesis of aldosterone from progesterone: – hydroxylations at C21, C11 and C18 – oxidation at carbon 18 Synthesis of steroid hormones Glucocorticoids: cortisol • Metabolic effects: – Promotes gluconeogenesis from amino acids. • it induces and maintains the activity of the glucogenic enzymes in the liver – Decreases amino acid uptake by muscle thereby decreasing protein synthesis – Decreases peripheral utilization of glucose Cushing‘s syndrome • Endocrine disorder caused by excessive secretion of cortisol into the blood. • Symptoms: – weight gain, particularly of the trunk and face. – lipodystrophy (around the shoulders) and a round face (moon face). • Other symptoms: – – – – loss of muscle fat (thinning of the skin) polyuria Polydipsia persistent hypertension and insulin resistance. Untreated Cushing's syndrome can lead to heart disease and increased mortality. Cortisol… • Stimulates catabolism of fatty acids and ketogenesis – induces and maintains the synthesis of hormone-sensitive lipase NB: Glucocorticoids have antiinflammatory and anti-allerginic action • Inhibit phospholipase A2 Cortisol vs synthetic corticoids Mineralocorticoids • Major hormone is aldosterone • Aldosterone acts on the kidney to increase blood pressure – through volume expansion by increasing sodium reabsorption and water retention – Its secretion from the adrenal cortex is induced by: • Na+/K+ ratio of the body and by angiotensin Aldosterone… – Stimulates hydrogen and potassium excretion, causing metabolic alkalosis with volume expansion • Causes hypertension and hypokalemia. • Angiontensin II, ACTH, elevated serum potassium, progesterone, and dopamine stimulate aldosterone synthesis Sex hormones • Testosterone: an androgen – male sex hormone synthesized in the testes – responsible for secondary male sex characteristics – produced from progesterone or pregnenolone through dehydroepiandrosterone • Estradiol: an estrogen – principal female sex hormone – produced in the ovary – responsible for secondary female sex characteristics Addison’s disease • Is a rare, chronic condition brought about by the failure of the adrenal glands • Both cortisol and aldosterone are not synthesized • Causes: autoimmune disease, TB • Symptoms: once advanced, can include severe fatigue and weakness, loss of weight, increased pigmentation of the skin, low BP, nausea, vomiting, salt cravings and painful muscles and joints Regulation of adrenal cortex Congenital adrenal hyperplasia (CAH) • Is a group of inherited disorders that result from loss-of-function mutations in one of several genes involved in adrenal steroid hormone synthesis • 21-hydroxylase deficiency: most common (>95% of CAH cases) • 11-hydroxylase deficiency • 3-β HSD deficiency • 17-hydroxylase deficiency CAH (cont’d) CYP 21 deficiency • ACTH levels are elevated, adrenal glands enlarge due to elevated plasma ACTH levels • Progesterone and 17-OH progesterone increase leading to masculinization (virilization) • Loss of Na+ in the urine leading to dehydration and hypotension which may lead to sudden death CAH CYP11 deficiency • Decrease in serum cortisol, aldosterone, and corticosterone • Increased production of deoxycorticosterone causes fluid retention and hypentension • Masculinization of the external genitalia CAH 3-β-HSD deficiency • No glucocorticoids, mineralocorticoids, androgens, or steroids. • Marked salt excretion in urine • Early death CYP 17 deficiency • Sex hormones and cortisol are not formed • Increased production of mineralocorticoids; sodium and water retention • Patient is phenotypically female but doesn’t mature Synthesis of androgens and estrogens • Begins with hydroxylation of progesterone (or of pregnenolone) to 17-OH progesterone • Enzyme 17, 20 lyase cleaves progesterone at C20 to yield androstenedione – It is the first adrenal androgen to be formed. Synthesis of androgens and estrogens • Reduction of the 17-keto group of androstenedione yields testosterone (from testis or gonads) – Androgens have 19 carbon atoms • Estrogens are synthesized from androgens by aromatization and oxidative removal of the methyl group at C19. Synthesis of Estrogens – An aromatic ring A is formed – Testosterone forms estradiol and dihydro testosterone (DHT) – Estrone is formed from androstenedione – In placenta, estradiol is hydroxylated to estriol. THE THYROID HORMONES • Thyroxine (3,5,3’,5’- tetraiodothyronine,T4) and 3,5,3’-triiodothyronine (T3), are tyrosine-based hormones produced by the thyroid gland. – L-3 monoiodotyrosine is the precursor of T3 and T4. – T3 can be formed from deiodination of T4 in tissues. • T4 is the major form of thyroid hormone in the blood but T3 is more biologically active than T4. Thyroid hormones Metabolic effects of thyroid hormones 1. Thyronines act on the body to increase the BMR 2. Increase the body's sensitivity to catecholamines 3. Lead to heat generation in humans, resulting from increased oxygen consumption and rates of ATP hydrolysis 4. Stimulate synthesis of proteins particularly enzymes Metabolic effects... 5. Increased thyroid hormone levels stimulate lipolysis by increasing the activity of hormone-sensitive lipase 6. Reduce plasma [cholesterol] by increasing its conversion to bile salts 7. Stimulate glycogenolysis in liver by potentiating the effect of adrenaline Mechanism of hormone action • Deals with how the hormone acts to change the physiologic state of its target cells. • What to consider: 1. structure and function of the receptor 2. how the bound receptor transduces a signal inside the cell and the end effectors of that signal. • Information is critical to understanding and treating diseases of the endocrine system, and in using hormones as drugs. How do hormones change their target cells? 1. Activation of enzymes and other dynamic molecules 2. Modulation of gene expression: Stimulating transcription of a group of genes can alter a cell's phenotype by leading to synthesis of new proteins. Inhibiting transcription shuts off the corresponding proteins NB: Location of a hormone receptor determines its mode of action Hormone receptors 1. Cell surface receptors • For protein and peptide hormones, catecholamines and eicosanoids Mechanism of action Formation of second messengers which alter the activity of other molecules - usually enzymes within the cell 2. Intracellular receptors (cytoplasm and/or nucleus) • For steroid and thyroid hormones Mechanism of action Hormones with cell surface receptors • Act through formation of a second messenger within the cell • Second messenger is formed after formation of a hormone-receptor complex – Triggers a series of molecular interactions that alter the physiologic state of the cell (signal transduction) Cell surface receptors • Have 3 domains 1. Extracellular (ligand-binding domain) 2. Transmembrane domain 3. Cytoplasmic or intracellular domains • Tails or loops of the receptor that are within the cytoplasm react to hormone binding – Are the effector regions of the molecule Examples of cell surface receptors: 1.Simple, single-pass polypeptide (protein) e.g. growth factor receptors Cell surface receptors… 2. More than one subunit. e.g. insulin 3. Transmembrane- 7 loops. E.g. beta-adrenergic receptor: Beta-3 adrenergic receptor Second messenger systems 1. Cyclic AMP (Adenylate cyclase system) Used by catecholamines, glucagon, LH, FSH, calcitonin, ADH, parathyroid hormone, TSH etc 2. Tyrosine kinase activity Insulin, human growth hormone, many growth factors 3. Calcium and/or phosphoinositides Epinephrine, norepinephrine, ADH, GnRH, TRH 4. cGMP: e.g. nitric oxide Activation of adenylate cyclase yiels cAMP Hormone receptor is coupled to a G protein Activation of adenylate cyclase • Interaction between the receptor and adenylate cyclase (AC) is mediated by a G protein • G-proteins are heterotrimeric – Have 3 subunits: α, ß, and γ. • There are different kinds of G proteins – Stimulatory G-protein (Gs) • Activates AC using the alpha subunit Gsα to form c-AMP • Gs is activated by receptors for hormones e.g. epinephrine and glucagon. G proteins… – Inhibitory G-protein (Gi) • AC is inhibited by the alpha subunit of Gi . cAMP isn’t formed Adrenergic receptors • Are G protein-coupled receptors for adrenaline and noradrenaline • 2 main groups of adrenergic receptors: – α and β; they have several subtypes (α1, α2) . – β receptors have subtypes β1, β2, and β3. • All are linked to Gs proteins – NB: β2 also couples to Gi • α1 couples to a G protein known as Gq – it results in increased intracellular Ca2+ resulting in smooth muscle contraction. • α2 couples to Gi – it causes a decrease of cAMP activity, resulting in e.g. smooth muscle contraction. • β receptors couple to Gs; increase intracellular cAMP activity – results in heart muscle contraction – smooth muscle relaxation and, – glycogenolysis Summary: Depending on the type of receptor, one hormone may affect different cells differently Adrenergic receptors β-receptor types • Respond to epinephrine and to blocking agents (antagonists) like propanolol • Β1- adrenergic receptors are located mainly in the heart • Β2- adrenergic receptors are located in the lungs, GIT, liver, uterus, vascular smooth muscle, skeletal muscle • Β3- adrenergic receptors are in fat cells Clinical utility of drugs which affect adrenergic nervous system a. Agonists of β2- adrenergic receptors are used in treatment of asthma (relaxation of the smooth muscles of the bronchi) b. Antagonists of β1- adrenergic receptors are used in treatment of hypertension and angina (slow heart and reduced force of contraction) c. Antagonists of the α1- adrenergic receptors lower BP (relaxation of smooth muscle and dilation of blood vessels GPCR Also known as seven-transmembrane domain receptor, 7TM receptor, heptahelical receptor, serpentine receptor, and G proteinlinked receptor (GPLR) G protein activation ATP Activation of adenylate cyclase • Adenylate cyclase (AC) is a transmembrane protein • The β-adrenergic receptor is the G-protein coupled receptor (GPCR) for epinephrine – in muscle and adipose. • In absence of hormone, the G-protein α subunit has bound GDP. • With bound hormone, α subunit of a Gprotein (Gα) binds GTP Activation of AC • The α, β, and γ subunits are complexed together. • Hormone binding causes: – a conformational change in the receptor that is transmitted to a G-protein – The nucleotide-binding site on Gα releases GDP and binds GTP (GDP-GTP exchange) Activation of AC: Stimulatory Gprotein • Gα -GTP dissociates from the inhibitory βγ subunit complex, binds to AC and activates it • Activated AC catalyzes synthesis of cAMP. – cAMP is degraded by phosphodiesterase Protein Kinase A • (cAMP-dependent protein Kinase) – phosphorylates serine or threonine residues of various cellular proteins, altering their activity. – Catalytic subunit of PKA are active when released from the regulatory subunits Turning off of the hormonal signal 1. Gα hydrolyzes GTP to GDP + Pi (GTPase). – GDP-Gα binds the inhibitory βγ complex. – Adenylate cyclase is inactivated. 2. Phosphodiesterases catalyze hydrolysis of cAMP to 5’ AMP. Methylxanthines including caffeine, theophylline inhibit the enzyme) 3. Receptor desensitization varies with the hormone. – E.g. the activated receptor is phosphorylated via a G-protein receptor - The phosphorylated receptor may bind to a protein β-arrestin. - β-Arrestin promotes removal of the receptor from the membrane by clathrinmediated endocytosis. - β-Arrestin may also bind a cytosolic phosphodiesterase, bringing the enzyme close to where cAMP is being produced, and turns off the signal. 4. Protein Phosphatases remove the phosphates attached to proteins via protein Kinase A. Stimulatory receptor ... • In presence of NAD+, the cholera toxin produced by Vibrio cholerae catalyzes ADP-ribosylation of an Arg residue in Gsα – The GTPase activity of the G-protein is inhibited – GTP is not hydrolyzed to GDP – AC remains activated – Elevated cAMP causes an efflux of Cl- ions leading to secretion of H2O, Na+, K+ and HCO3- into the gut resulting in severe diarrhea ADP-ribosylation of αs Inhibitory receptors • The active Gα-GTP of Gi proteins inhibit adenylate cyclase • Mediation of inhibition of AC is by Gi • Examples of hormones whose actions are mediated by Gi: epinephrine at α2adrenergic receptor, opiates, somatostatin, TRH and GRH • The βγ subunits of Gs are similar to those of Gi; the α subunits are different. • Events are the same as for Gs receptors – Gαi –GTP may directly inhibit AC or, – the released Gi βγ subunit may bind the αsubunit of Gs reversing the activation of AC Effect of Pertussis toxin (produced by the bacterium Bordetella pertussis) on Gi – causes whooping cough – catalyzes ADP-ribosylation of a cys residue in the α-subunit of Gi. – This prevents Gi from exchanging GDP for GTP – Gi is locked in the GDP form and AC cannot be inhibited, leading to increased cellular [cAMP]. • Elevated intracellular cAMP affects normal biological signaling. • The toxin causes several systemic effects, e.g. increased release of insulin causing hypoglycaemia • α2-adrenergic receptors: found in platelets, nerve termini, pancreatic islets Inhibitory receptor Activation of PKA and gene transcription CBP co-activates activated CREB CREB: cAMPResponse element Binding protein Phosphoinositide cascade • Some hormones e.g. norepinephrine at α1adrenergic receptor, and serotonin, vasopressin, GnRH, activate a signal cascade based on the membrane lipid phosphatidylinositol (PI) • PI is phosphorylated twice using ATP – Phosphatidylinositol 4,5-BP (PIP2) is formed Phoshoinositide cascade… • PIP2 is a membrane lipid • PIP2 is cleaved by Phospholipase C (PLC). • Different isoforms of PLC respond to different signals • A G-protein designated Gq activates one form of PLC. Phosphoinositide cascade Phosphoinositide cascade ... • When a G protein receptor GPLC is activated, GTP exchanges for GDP. • Gqα-GTP activates PLC. • Activated PLC cleaves PIP2 yielding two second messengers: – inositol-1,4,5-trisphosphate (IP3) – diacylglycerol (DAG) Phosphoinositide… • IP3 activates Ca2+ release channels in endoplasmic reticulum (ER) membranes. • Ca++ stored in the ER is released to the cytosol – it may bind to calmodulin, or – may help to activate PKC. • Diacylglycerol acts by increasing the affinity of protein kinase C (PKC) for Ca2+ Phosphoinositide… • PKC catalyzes phosphorylation of several cellular proteins & enzymes, altering their activity. • IP3 has a short half life. – It is rapidly dephosphorylated to inositol 1,4BP and to inositol 1-P which are inactive Effects mediated by phosphoinositide cascade • • • • • Glycogenolysis in liver cells Serotonin release by platelets Insulin secretion Smooth muscle contraction Histamine secretion by mast cells Calcium • Most abundant mineral element in the body • Plays essential roles in: – – – – cell signaling muscle contraction neurotransmission fertilization and cell division • When released into cells, it can bind calcium sensing proteins (e.g. calmodulin) and trigger different biological effects Calcium… • Examples of biological effects: – muscle contraction – insulin release from the pancreas – blocking the entry of additional sperm cells once an egg has been fertilized. Calmodulin • Is a CALcium MODULated proteIN. • Senses calcium levels and relays signals to various calcium-sensitive enzymes, ion channels and other proteins. • Binds 4 molecules of Ca2+ to form activated Ca2+-calmodulin complex – Leads to activation of enzymes like: • adenylate cyclase • phosphorylase kinase • calmodulin-dependent protein kinase Tyrosinase activity TT Tyrosine kinase: insulin receptor • The Insulin Receptor Tyrosine Kinase (IRTK) consists of 2α and 2β subunits linked by disulfide bonds. – The α chains are extracellular and have insulin binding domains – The linked β chains penetrate through the plasma membrane (transmembrane). • The α chains are regulatory and the β chains are catalytic • The insulin receptor (IR) is a tyrosine kinase Insulin receptor ... • Binding of insulin induces a conformational change in the receptor – the intracellular tyrosine domains on the β chains autophosphorylate • Phosphorylated tyrosine residues turn on the catalytic activity of the receptor (kinase activity) – The tyrosine kinase phosphorylates a number of intracellular proteins e.g. insulin receptor substrates (IRS, 1, 2, 3, 4) Insulin receptor… • Insulin-receptor substrates (IRS) are proteins that are attracted to the phosphorylated sites on the activated insulin receptor. • Activated IRS act as adaptor proteins – they bring a kinase (e.g. glucokinase, PFK1) and its substrate together rather than activating a kinase, • The main activity of activation of the IR is inducing glucose uptake. – Insulin “insensitivity” or a decrease in IR signaling leads to Type II DM. Insulin receptor (cont’d) • Phosphorylation of IRSs is on serine or threonine residues – Activated IRSs are coupled to several additional protein kinase signal systems: 1. Activation of phosphatidylinositol-3 kinase pathway • forms PI-3,4,5-triphosphate (PIP3). • PIP3 activates PKB (Akt) & PKC • Membrane trafficking of Glut 4; growth, apoptosis 2. Mitogen-activated protein kinases (MAP kinases). Insulin receptor • Activated protein kinases can activate protein phosphatases leading to the biological effects of insulin. – E.g. insulin-activation of pyruvate dehydrogenase • crucial in insulin's stimulation of hepatic lipid synthesis – Dephosphorylation of glycogen synthase NB: The binding of the growth hormone to its receptor also leads to activation of cytoplasmic tyrosine kinases cGMP Atrial natriuretic factor • Is released from the heart in response to elevated blood pressure • Binds on a membrane receptor with an intracellular guanylate cyclase domain. • Guanylate cyclases are not coupled to G proteins – Guanylate cyclase forms cGMP from GTP • cGMP activates protein kinase G important in regulation of smooth muscle relaxation, platelet function, cell division etc Nitric oxide • Is a vasodilator • Stimulates a soluble guanylate cyclase (not membrane bound). It is cytoplasmic – NO is synthesized in endothelial cells from Arg by nitric oxide synthase (NOS). – NOS is a calcium-calmodulin activated enzyme. • Vascular smooth muscle is contracted by agents that raise calcium levels in smooth muscle cells and relaxed by agents that raise calcium levels in endothelial cells. Transcriptional enhancer mechanism • Used by steroid and thyroid hormones, vit D & vit A (retinoic acid). • Steroid hormones receptors are: – ligand-activated proteins that regulate transcription of selected genes. – function as ligand-dependent transcription factors. – found in the cytosol and the nucleus. – belong to the steroid and thyroid hormone receptor super-family of proteins Transcriptional enhancer ... • Binding of the hormone on the, receptors causes conformational change • The receptors are activated – they recognize and bind to specific nucleotide sequences in the DNA known as hormoneresponse elements (HREs). • Transcription is altered. • The response can either activate or repress gene expression. Transcription enhancer… • mRNA is synthesized in the nucleus and enters the cytoplasm and promotes protein synthesis for: – Enzymes as catalysts – Tissue growth and repair – Regulate enzyme function Hormone receptor regulation • Is an important part of endocrine function • It occurs through: – up or down-regulation of the number of receptors – desensitization of the receptors. by increasing or decreasing receptor synthesis by internalization of membrane receptors after ligand binding (endocytosis through coated pits) by uncoupling of the receptor from its signal transduction pathway (desensitization).
