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Mechanisms of action of hormones and signaling molecules
- 1. MECHANISMS OF ACTION OF HORMONES AND SIGNALING MOLECULES DR.K.RAVI BABU MD BIOCHEMISTRY ASSISTANT PROFESSOR
- 4. Mechanisms of Action of Hormones and Signaling Molecules The nervous system and endocrine system are the major control mechanisms that integrate the functions of the tissues in the body. The nervous system transmits electrochemical signals between the brain and peripheral tissues for coordinating the diverse body functions. The endocrine system releases chemical mediators or hormones into the circulation. Neural regulation of endocrine glands is effected.
- 5. Mechanisms of Action of Hormones and Signaling Molecules : continue…. Neurotransmitters have several features in common with hormones. EH Starling in 1905 introduced the term “hormone”. Signal molecules are of different types and the process of transferring the signal into the cell is called signal transduction. There are two types of cells in signal transduction—the sender cell where the signal originates and the target cell that receives the signal. The signal alters or modulates the activity/function of the cell.
- 6. Mechanisms of Action of Hormones and Signaling Molecules continue….. Autocrine signaling occurs when same cell acts as sender and recipient, e.g. growth, differentiation, immune and inflammatory response. Paracrine signaling is effected by local mediators which have their effect near the site of secretion without entering the circulation (rapid & transient ). Juxtacrine signaling :gap junctions or through protein molecules on the surface of the two cells. Endocrine signaling is between cells which are located at a distance from each other and the signal may be hormones or chemical messengers secreted into circulation. target cell, they bind to specific target cell receptors with high affinity. Plasma carrier proteins exist for all classes of endocrine hormones. Carrier proteins for peptide hormones prevent hormone destruction by plasma proteases. Carriers for steroid and thyroid hormones allow these hydrophobic hormones to be present in the plasma.
- 7. CLASSIFICATION OF HORMONES Hormones can be classified in different ways : 1.Based on chemical nature a.Protein or peptide hormones : e.g.,Insulin, Growth hormone,Vasopressin and Oxytocin b.Steroid hormones :Mineralocorticoids,Glucocorticoids and Sex hormones c.Derivatives of amino acids : e.g.,Thyroid hormones,adrenal medullary hormones 2.Based on solubility properties : a.Lipophilic (Steroids and Thyroids ) b.Hydrophilic (Insulin,GH and ADH ) 3.Based on mechanism of action : Most widely used,based on the location of the receptors and the nature of the signal used to mediate hormone action.
- 8. Mechanisms of Action of Hormones and Signaling Molecules Carriers for small, hydrophilic amino acid-derived hormones prevent their filtration through the renal glomerulus, greatly prolonging their circulating half-life. The classical definition of a hormone is “substances released from ductless or endocrine glands directly to the blood”. it is synthesized by one type of cells and transported through blood to act on another type of cells. Based on mechanism of action, the hormones may be classified into two : I. Hormones with cell surface receptors II. Hormones with intracellular receptors.
- 11. Hormones Acting through Cyclic AMP Cyclic AMP (cAMP) was first discovered by Earl Sutherland in 1961, who was awarded Nobel prize in 1971. Signal transduction pathways are like a river flowing in one direction only; components closer to the receptor are called “upstream” and closer to the response are called “downstream”.
- 14. Fig. Action of hormones through G-protein
- 16. Hormones Acting through Cyclic AMP Signal Transduction through G-Protein Action is through G-protein coupled receptors (GPCRs). Binding of different types of signal molecules to G-protein coupled receptors is a general mechanism of signal transduction. The GPCRs are transmembrane proteins with 7 helical segments spanning the membrane. When any ligand binds, the GPCRs activate heterotrimeric GTP binding regulatory proteins (G-proteins). The G-protein in turn will interact with effector proteins which may be enzymes or ion channel proteins, which result in the desired effect. Different types of G-proteins are present in the cells that are coupled with different receptors and activating different effector proteins. The extracellular messenger, the hormone (H) combines with the specific receptor (R) on the plasma membrane .
- 17. Hormones Acting through Cyclic AMP continue…… The H-R complex activates the regulatory component of the protein designated as G-protein or nucleotide regulatory protein. G-proteins are so named, because they can bind GTP and GDP. GDP-GTP exchange is mediated by the GEF (Guanine nucleotide exchange factor). The G- protein is a trimeric membrane protein consisting of alpha, beta and gamma subunits . Alfred Gilman and Martin Rodbell : G-protein. Robert Lefkowitz and Brian Kobilka :G-protein-coupled receptors (GPCRs). Most of the hormone/ligand signals are transducted through GPCR. Around 50% of medicines are acting through GPCR
- 18. G-protein Activates Adenyl Cyclase When the hormone receptor complex is formed, the activated receptor stimulates the G-protein, which carries the excitation signal to adenylate cyclase . The hormone is not passed through the membrane; but only the signal is passed; hence this mechanism is called signal transduction. The adenyl cyclase is embedded in the plasma membrane .
- 20. Subunit Activation of G-protein The inactive G-protein is a trimer with alpha, beta and gamma subunits. When activated, GTP binds and the beta-gamma subunits dissociate from the alpha subunit. Adenylate cyclase is activated by G-alpha-GTP . The binding of hormone to the receptor triggers a configurational change in the G-protein which induces the release of bound GDP and allows GTP to bind. The hormone has an amplified response, since several molecules of G-alpha-GTP are formed.
- 25. CAMP response element binding protein Signal transducers and activators of transcription Mitpgen activated protein kinase Janus kinase CREB- binding protein
- 30. Hormones Acting through Cyclic AMP : Inactivation The active G-alpha-GTP is immediately inactivated by GTPase. The G-alpha-GDP form is inactive . The activation is switched off when the GTP is hydrolyzed to GDP by the GTPase activity of the alpha subunit . This is a built-in mechanism for deactivation. Thus GTPase acts as a molecular switch. The alpha subunit, which is bound to GDP, can re-associate with beta and gamma subunits. The GTP-GDP exchange rate decides the activity of adenyl cyclase.
- 31. Cyclic AMP Adenyl cyclase or adenylate cyclase converts ATP to cAMP (3’,5’-cyclic AMP), and phosphodiesterase hydrolyzes cAMP to 5’ AMP . Cyclic AMP is a second messenger produced in the cell in response to activation of adenylate cyclase by active G-protein. During hormonal stimulation, cyclic AMP level in the cell increases several times. The level of cyclic AMP in the cell is regulated by its rate of production by adenylate cyclase (AC) and hydrolysis by phosphodiesterase (PDE). The action of PDE is also regulated by hormones and drugs. Therefore, cellular level of cyclic AMP can be increased by inhibition of PDE. e.g., Insulin activates PDE, decreasing the cellular level of cAMP while caffeine and theophylline inhibit PDEs increasing cAMP levels.
- 32. Cyclic AMP continue…… Second Messenger Activates PKA The cAMP (second messenger), in turn, activates the enzyme, PKA (Cyclic AMP dependent protein kinase). Cyclic AMP binds to the regulatory subunits of PKA so that the catalytic subunits having kinase activity can phosphorylate proteins. The cascade amplification effect is seen in this series of activation reactions. This PKA is a tetrameric molecule having two regulatory (R) and two catalytic (C) subunits (R2 C2) . This complex has no activity. But cAMP binds to the regulatory subunit and dissociates the tetramer into regulatory and catalytic subunits. The catalytic subunit is now free to act.
- 33. KINASE PHOSPHORYLATES THE ENZYMES The catalytic subunit then transfers a phosphate group from ATP to different enzyme proteins . Phosphorylation usually takes place on the OH groups of serine, threonine or tyrosine residues of the substrates. Hence, these kinases are called Ser/Thr kinases. The enzymes may be activated or inactivated by this phosphorylation. This is an example of covalent modification. Glycogen phosphorylase and hormone sensitive lipase are controlled by cyclic AMP.
- 37. There are Many G-proteins About 30 different G-proteins are identified, each being used for different signal transduction pathways. The G-protein, which stimulates adenyl cyclase, is called Gs (G- stimulatory) and the opposite group is called Gi (G-inhibitory). An example of inhibitory G-protein is the inhibition of adenylate kinase. The alpha subunit of the Gs and Gi are different, but beta and gamma are the same . G-proteins are also involved in toxic manifestations of cholera and pertussis. Mutations in gene encoding the alpha subunit of Gs-protein or abnormalities in G-protein signaling have been found to result in the action of toxins .
- 39. There are Many Protein Kinases More than thousand protein kinases are now known. Some important hormone responsive protein kinases are, cAMP-dependent kinases, epidermal growth factor-dependent tyrosine kinase, insulin-dependent tyrosine kinase. All the known effects of cAMP in eukaryotic cells result from activation of protein kinases, which are serine/threonine kinases .
- 40. Glycogen Phosphorylase is a Typical Example Glycogen phosphorylase and hormone sensitive lipase are activated by cAMP mediated cascade . The termination of the effect of the hormonal action by phosphorylation is effected by the action of protein phosphatases. e.g.,glycogen phosphorylase becomes itself is inhibited by phosphorylation of its regulatory subunit. When cyclic AMP level falls, the regulatory subunit is dephosphorylated and protein phosphatase becomes active, which in turn hydrolyzes phosphate group from the enzyme. Protein kinases as well as protein phosphatases are involved in the action of different hormones.
- 41. The actions of cAMP in eukaryotic cells is multifaceted and these include:continue …… A. Activation of protein kinase and phosphorylation of effector proteins like enzymes and ion channels. These enzymes may directly phosphorylate enzymes or secondary kinases that phosphorylate other enzyme. E.g., i. PKA phosphorylates hormone sensitive lipase thus activating it. ii. Phosphorylase kinase that phosphorylates glycogen phosphorylase. iii. When ion channel proteins or transporters are phosphorylated, the membrane potential is modified, thus regulating the influx of calcium.
- 42. The actions of cAMP in eukaryotic cells is multifaceted and these include: B. cAMP also has a long lasting effect on gene expression. The translocation of the active PKA subunits to the nucleus induces phosphorylation of cAMP regulated gene regulatory proteins or CREBs (CAMP response element binding protein ) These proteins will bind to cAMP sensitive regulatory elements (CRE) on genes, thus controlling their expression. C. G-protein mediated signal transduction also requires scaffold and adapter proteins that increase the fidelity and speed of a signaling cascade. Anchoring proteins localize and concentrate the signaling proteins at their site of action. The interaction of these proteins involve specific domains within the protein like SH2 (Src homology type 2), PTB (phosphor tyrosine binding), etc.
- 43. Calcium-based Signal Transduction Intracellular regulator of cell function like contraction of muscles, secretion of hormones and neurotransmitters, cell division and regulation of gene regulation. Rapid but transient increase in cytosolic calcium result from either opening of calcium channels in the plasma membrane or calcium channels in the ER. The released calcium can be rapidly taken-up by ER to terminate the response.
- 44. Calcium-based Signal Transduction continue……. The intracellular calcium concentration is low (10-7) where as extracellular calcium concentration is very high (10-3), maintaining a 10,000 fold calcium gradient across the membrane. The inside has a negative potential therefore influx of calcium is rapid. Even small increase in cytosolic free calcium can have maximal effect on calcium regulated cellular functions. There are mainly 3 types of calcium transport systems: a. Voltage gated calcium channels b. Sodium/calcium antiport transporter c. Calcium transporting ATPase.
- 45. Calcium-based Signal Transduction continue……. The calcium transporting ATPase transporter accumulates calcium within the lumen of ER (sarcoplasmic reticulum) in muscle. These calcium ions can be released into the cytoplasm by an inositol triphosphate (IP3) gated calcium channel or by a ligand gated calcium release channel (ryanodine receptor). When cytosolic calcium increases, binding regulatory proteins, activation of several calcium binding regulatory proteins occurs. Calmodulin is expressed in various tissues and mediates the regulatory actions of calcium ions. Calcium binding causes conformational change in calmodulin resulting in interaction with kinases, phosphatases, NOS etc. Some of these CAM kinases can phosphorylate a wide range of proteins that alter cellular functions. When bound to calmodulin, CAM kinase II also autophosphorylates, so that its activity is sustained. Intracellular calcium acts as a mediator of hormone action either independently or in conjunction with cAMP.
- 46. Phosphorylase Kinase Activation The active protein kinase can now convert the phosphorylase kinase to an active phosphorylated form, which converts phosphorylase-b to phosphorylase-a. Phosphorylase kinase itself is a tetrameric enzyme (alpha, beta, gamma, delta). Of these the gamma subunit has the catalytic site and the other 3 subunits have regulatory effects. Phosphorylase kinase is activated by Ca++ and phosphorylation of alpha and beta subunits by PKA. Phosphorylation of alpha and beta subunits relieves autoinhibition of catalytic activity of gamma subunit. Binding of Ca++ to the delta subunit which is identical to calmodulin (CaM) is also necessary for full activity of delta subunit since it also has a role in dysregulating the gamma subunit. Calcium triggers muscle contraction as well as glycogen breakdown through the action of phosphorylase kinase.
- 48. Phosphorylase Kinase Activation The rate of glycogenolysis is linked to rate of muscle contraction. The dephosphorylation of the active form by protein phosphatase 1 (PP1) involves removal of phosphate group from phosphorylase a and alpha and beta subunits of phosphorylase kinase. The activity of PP1 is controlled differently in liver and muscle. The catalytic subunit of PP1 in muscle is active only when it is bound to glycogen through the glycogen binding GM subunit. The phosphorylation of PP1 by an insulin stimulated protein kinase (site1) activates the enzyme where as phosphorylation at site 2 by PKA makes its action ineffective. When cAMP level is high, PP1 is inhibited by inhibitor1 which is activated by phosphorylation by PKA. The effect of cyclic AMP is not only by increasing the phosphorylation of enzymes, but also by decreasing dephosphorylation.
- 49. Hormones can increase the cytosolic calcium level by Hormones can increase the cytosolic calcium level by the following mechanisms: A. By altering the permeability of the membrane. B. The action of Ca-H+ -ATPase pump which extrudes calcium in exchange for H+ . C. By releasing the intracellular calcium stores. D. Calmodulin, the calcium dependent regulatory protein within the cell has four calcium binding sites. When calcium binds there is a conformational change to the calmodulin, which has a role in regulating various kinases. Calmodulin is a 17 kDa protein which has structural and functional similarity with the muscle protein troponin C. Examples of enzymes or functional proteins regulated by calmodulin are: Adenyl cyclase, calcium-dependent protein kinases, calcium-magnesium-ATPase, cyclic nucleotide phosphodiesterase, nitric oxide synthase and phosphorylase kinase.
- 51. Abnormal G-protein signaling Cholera toxin is encoded by a bacteriophage present inside the bacteria Vibrio cholerae. The enterotoxin contains two A subunits and 5 B subunits. The B subunit binds to a ganglioside GM1 on the surface of intestinal mucosal cell. The A subunit then enters into the inner part of the membrane, which leads to ribosylation of the alpha subunit of Gs protein. This results in the inhibition of the inherent GTPase activity and irreversible activation of G protein. Adenyl cyclase remains continuously active and keeps cyclic AMP levels high. This prevents absorption of salts from intestine leading to watery diarrhea and loss of water from body. In the large intestine, chronic elevation of cAMP results in a sustained PKA mediated phosphorylation of chloride channels (CFTRs) that normally regulate salt and water transport. Hyperactivity of these channels will result in loss of sodium chloride with watery diarrhea (liquid stools), that may have fatal results. The patient may lose as much as 1 L of water per hour.
- 52. Abnormal G-protein signaling continue…… Pertussis toxin ribosylates the alpha subunit of Gi-protein and prevents the Gi- GDP complex from interacting with the activated receptor. Effects of bacterial toxins from Clostridium tetani are exerted through proteases, that attack proteins involved in synaptic vesicle and plasma membrane fusion. The toxin has two polypeptides, one of which binds to cholinergic motor neurons and facilitates the entry of the second polypeptide. Failure to release the neurotransmitter leads to fatal paralysis of the chest muscles. Mutations in gene encoding the alpha subunit of Gs-protein (gsp gene) has been found to result in decreased GTPase activity of the alpha subunit, leading to continued activation of Gs alpha and adenyl cyclase. The resultant increase in cAMP has been found to lead to PKA-dependent phosphorylation of cyclic AMP sensitive gene regulatory proteins. Over-expression of cAMP inducible genes has been found to produce growth hormone/ ACTH secreting tumors of the pituitary
- 54. Second messengers They are produced in response to binding of signal molecules (hormones) to receptors. They bring about activation of enzyme cascades or changes in plasma membrane potential. They may be water soluble or lipid soluble. Water soluble messengers are cAMP, cGMP, Inositol triphosphate (ITP). Lipid soluble messengers are Diacyl glycerol (DAG) and Phosphatidyl inositol triphosphate (PIP3).
- 58. Hormones Acting through PIP2 Cascade The major player in this type of signal transduction is phospholipase C that hydrolyses phosphatidyl inositol in membrane lipids to 1,4,5-Inositol triphosphate (IP3) and Diacyl Glycerol (DAG) that act as second messengers. PIP3 (Phosphatidyl Inositol 3,4,5- phosphate) is another second messenger produced by the action of a phosphoinositide kinase. The phospholipase C may be activated either by G- proteins or calcium ions. DAG can also be generated by the action of phospholipase D that produces phosphatidic acid which is hydrolyzed to DAG.
- 59. Hormones Acting through PIP2 Cascade The binding of hormones like serotonin to cell surface receptor triggers the activation of the enzyme phospholipase-C which hydrolyzes the phosphatidyl inositol to diacylglycerol. IP3 can release Ca++ from intracellular stores, such as from endoplasmic reticulum and from sarcoplasmic reticulum . The elevated intracellular calcium then triggers processes like smooth muscle contraction, glycogen breakdown and exocytosis
- 61. Hormones Acting through PIP2 Cascade PIP3 can be formed by the action of PI3-kinases that are activated through growth factors and cytokine mediated receptor tyrosine kinases. PIP3 which is a lipid second messenger has a role in regulation of cell motility, membrane trafficking and cell survival signaling pathways. The major mediator of PIP3 action is PKB (Protein kinase B) which has a role in glucose transport, glycogen metabolism and cell death signaling pathways. Active PKB/Akt is the major mediator of PIP3 action. It represses the activity of cell death signaling pathways. The PDK (Phosphatidyl inositol dependent kinase) and IP3 kinase are also involved in glucose transport and glycogen metabolism. There is “cross talk” between the various signal transduction pathways that are coordinately regulated.
- 62. Diacylglycerol Pathway Diacylglycerol (DAG) The messenger formed by the hydrolysis of PIP2 activates protein kinase C (PKC) which in turn would phosphorylate other target proteins. PKC activates several serine threonine kinases that phosphorylate several substrates including transcription factors, ion channels and transporters. Most effects of IP3 and DAG are found to be synergistic. DAG also increases the affinity of protein kinase-C for calcium. The enzymes are thus activated, even at physiological levels of calcium within the cell.
- 63. Role of Cyclic GMP Cyclic GMP (cGMP) is another important second messenger involved in contractile function of smooth muscles, visual signal transduction and maintenance of blood volume. Cyclic GMP degradation is catalyzed by membrane bound PDEs. i. It is formed from GTP by the action of guanyl cyclase. ii. Drugs like nitroprusside, nitroglycerin, sodium nitrite and atriopeptides (a group of peptides produced by atrial cardiac tissue). All these compounds act as potent vasodilators, by inhibiting the phosphodiesterase.
- 64. Role of Cyclic GMP continue….. iii. Cyclic GMP activates cGMP-dependent protein kinase G (PKG), which phosphorylates important effector proteins that can regulate calcium dependent contraction or motility by modulating calcium influx. e.g., smooth muscle myosin, leading to relaxation and vasodilatation. iv. Cyclic GMP is also involved in the rhodopsin cycle. v. NO (Nitric oxide) is the major activator of guanylate cyclase. NO in turn is produced by the action of NOS (Nitric oxide synthase) in tissues like vascular endothelial cells. NO can easily diffuse through the membrane and activate guanylate cyclase. Increased level of cyclic GMP in smooth muscle triggers rapid and
- 65. Role of Cyclic GMP continue….. The vasodilatation resulting from NO induced increase in cGMP . The drugs that act via NO release are nitroprusside, nitrites (used in angina as coronary vasodilators) and sildenaphil citrate (Viagra). Even though nitroglycerin was used to relieve angina.
- 66. Endothelial NOS: NOS-3 or eNOS or endothelial NOS is seen in endothelial cells, platelets, endocardium and myocardium. the NO˙ is constantly produced and released, so as to have arterial relaxation. It is localized in the plasma membrane. It is activated by calcium. The gene for endothelial NOS is on chromosome 7.
- 67. Mechanism of Action of Nitric Oxide : NO diffuses to the adjacent smooth muscle and activates guanylate cyclase. Increased level of cyclic GMP activates protein kinase in smooth muscles, which causes dephosphorylation of myosin light chains, leading to relaxation of muscles. Thus NO is a vasodilator.
- 69. Physiological Actions of Nitric Oxide Blood vessels: NO˙ is a potent vasodilator. The normal blood pressure is maintained by the NO˙ liberated by endothelial NOS (NOSe). NO˙ causes cerebral, coronary, renal and muscle arteries to dilate. A deficiency of NO˙ is associated with hypertension. Excessive production of NO˙ results in refractory hypotension, which may be seen in patients with septicemic shock.
- 70. Hormones with Intracellular Receptors i. The hormones in this group include the steroid hormones and thyroid hormones. They diffuse through the plasma membrane and bind to the receptors in the cytoplasm . ii. The hormone receptor (HR) complex is formed in the cytoplasm. The complex is then translocated to the nucleus. Steroid hormone receptor proteins have a molecular weight of about 80–100 kD. Each monomer binds to a single steroid molecule at a hydrophobic site, but on binding to genes they dimerize iii. In the nucleus, the HR binds to the hormone response elements (HRE) or steroid response elements (SRE) .
- 71. Hormones with Intracellular Receptors The SRE acts as an enhancer element and when stimulated by the hormone, would increase the transcriptional activity . The newly formed mRNA is translated to specific protein, which brings about the metabolic effects. Binding to the SRE sequence leads to dimerization of the receptor. Steroid hormones influence gene expression, so that the rate of transcription is increased. The stability of mRNA is also increased. This would lead to induction of protein synthesis. Steroid receptors have been found to enhance initiation of transcription by formation of complexes at promoters . iv. Best examples of the effect of hormones on genes are: a. The induction of synthesis of amino transferases by glucocorticoids. b. Synthesis of calcium binding protein by calcitriol.
- 77. Insulin Signaling Pathway Insulin acts by binding to a plasma membrane receptor on the target cells. It has 2 alpha and 2 beta subunits. Insulin binds with the alpha subunits. This binding activates the tyrosine kinase activity of the beta subunit, leading to autophosphorylation of the beta subunit . This event, in turn, phosphorylates insulin receptor substrates (IRS). There are different IRS molecules, named as IRS 1 to 4. Activation of IRS2 results in activation of the PI-3 kinase, which eventually activates various protein kinases, PKB, PKC, SGK (serum glucocorticoid regulated kinase), etc.
- 78. Insulin Signaling Pathway This leads to transcription of specific genes for key enzymes of glycolysis, such as glucokinase. There are more than 100 enzymes influenced by insulin. An alternate pathway involves activation of IRS1. The message is later transmitted into a series of serine/threonine kinases, such as IRS2 → GRB2→ mSOS→ Ras → Raf → MEK→ MAPK, etc. which causes cell growth and new DNA synthesis. GRB = growth factor receptor binding protein; mSOS = mammalian son of sevenless; MAPK = mitogen activated protein kinase. A third pathway is IRS3→ mTOR → p70S6K. ( mTOR= mammalian target of rapamycin; p70S6K = p70 ribosomal protein S6 kinase). This pathway leads to increased synthesis of glucose transporters, insulin receptors, etc.
- 82. mTOR Mammalian target of rapamycin (mTOR) also known as FK506 binding protein 12-rapamycin associated protein 1 (FRAP1) is a protein which in humans is encoded by the FRAP1 gene. The mTOR is a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. The mTOR integrates the input from upstream pathways, including insulin, growth factors (such as IGF-1 and IGF-2), and mitogens. It also senses cellular nutrient and energy levels and redox status. The mTOR pathway is dysregulated in certain cancers. Rapamycin is a bacterial product that can inhibit mTOR by associating with its intracellular receptor FKBP12. The FKBP12-rapamycin complex binds directly to the FKBP12-Rapamycin Binding (FRB) domain of mTOR.
- 83. Jak/STAT Pathway Some hormones (GH, prolactin, erythropoietin, cytokines) when complexed with the receptor, activate cytoplasmic tyrosine kinases, such as Tyk, Jak, etc. (Jak means Janus kinase ). Janus is a greek mythological figure with two heads; the name is given because Jak dimerises. The name for the month of January is also derived from this Janus; Jak in turn activates STAT (signal transducers and activators of transcription). The phosphorylated STAT dimerizes and translocates into the nucleus, where it binds to a specific DNA element and activates transcription.
- 84. NFkB and Glucocorticoids NFkB is the abbreviation for nuclear factor kappa-light-chain-enhancer of activated B-cells. It is a protein complex that controls the transcription of DNA. NFkB is found in almost all animal cell types and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens. NFkB plays a key role in regulating the immune response to infection. Incorrect regulation of NFkB has been linked to cancer ,inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development.
- 85. NFkB and Glucocorticoids NFkB has also been implicated in processes of synaptic plasticity and memory. Active NFkB turns on the expression of genes that keep the cell proliferating and protect the cell from conditions that would otherwise cause it to die via apoptosis. NFkB is a transcription factor. It is a heterodimeric complex; it has two subunits, p50 and p65. Normally NFkB is in cytoplasm, and is complexed with inhibitors, and so is inactive. Stimuli such as cytokines, ROS, mitogens will activate IKK (= IkB kinase), which phosphorylates IkB (=inhibitor of NFkB).
- 86. NFkB and Glucocorticoids Phosphorylated IkB is attached with ubiquitin and is degraded by proteasomes. NFkB is now free, which translocates to nucleus. It binds to various gene promoters and activates transcription of proteins involved in inflammatory response. Glucocorticoids are used widely in clinical practice as anti- inflammatory agents. Glucocorticoids increase IkB; the glucocorticoid receptors bind to the p65 subunit of NFkB; in both ways glucocorticoids inhibit the NFkB activity, and so reduce the inflammation.
- 87. NUCLEAR RECEPTORS Their middle region contains the DNA binding domain (DBD) through which they bind the specific region of DNA, termed hormone response elements . The receptors also have a ligand binding domain (LBD) at the carboxy terminal half. A hinge region separates the DBD and LBD regions. Nuclear receptors were identified for all the hormones.
- 91. Clinical Case Study A 25-year-old woman working in a rural school suddenly began to pass profuse watery stools almost continuously. Immediately she started to vomit and her general condition deteriorated. She was rushed to the hospital and on admission, she was cyanotic, skin turgor was poor, BP was 70/50 mm Hg and pulse was rapid and weak. The stool sample was taken for culture and treatment was started immediately. What is the diagnosis? What is the basis of clinical findings?
- 92. Clinical Case Study Answer The patient is suffering from cholera, an acute diarrheal disease that can result in rapidly progressive dehydration and death within a few hours. It is caused by the bacteria, vibrio cholera. People are infected when they consume contaminated food or water. Patients present with watery diarrhea and vomiting, with no fever. Diagnosis is confirmed by identifying bacteria is stool sample. Treatment includes replacing lost fluids and giving antibiotics.
- 93. Clinical Case Study Answer continue……. Certain toxins produced by the bacteria (choleragen) leads to activation of the enzyme, adenyl cyclase, converting cAMP to ATP. The activated protein leads to continuous activation of protein kinase A which opens a chloride channel (CFTR channel) and inhibits Na+ - H+ exchanger. The net effect is inhibition of absorptive sodium transport system in intestinal villus cells and activationof secretory chloride channel transport in crypt cells. This leads to accumulation of sodium chloride in lumen; water moves passively to maintain osmolality and watery diarrhea results. Unless replaced, this leads to shock (due to fluid depletion) and acidosis (due to loss of bicarbonate). The cholera toxin can also enhance intestinal secretion via prostaglandins and neural histamine receptors.