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Introduction to
Endocrinology
Dr. Sai Sailesh Kumar G
Professor
Department of Physiology
NRIIMS
Email: dr.goothy@gmail.com
Measurement of Hormone Concentrations in the Blood
 Most hormones are present in the blood in extremely minute quantities; some
concentrations are as low as one billionth of a milligram (1 picogram) per milliliter.
 Therefore, it was difficult to measure these concentrations by the usual chemical means
 However, an extremely sensitive method that was developed about 50 years ago
 This method is called radioimmunoassay.
 More recently, additional methods, such as enzyme-linked immunosorbent assays,
have been developed for accurate, high-throughput measurements of hormones.
Radioimmunoassay
 First, an antibody that is highly specific for the hormone to be
measured is produced.
 Second, a small quantity of this antibody is
 (1) mixed with a quantity of fluid from the animal containing the
hormone to be measured
 (2) mixed simultaneously with an appropriate amount of purified
standard hormone that has been tagged with a radioactive isotope.
Radioimmunoassay
 One specific condition must be met:
 There must be too little antibody to bind completely both the
radioactively tagged hormone and the hormone in the fluid to be
assayed.
 Therefore, the natural hormone in the assay fluid and the radioactive
standard hormone compete for the binding sites of the antibody.
Radioimmunoassay
 Third, after binding has reached equilibrium,
 the antibody-hormone complex is separated from the remainder of the
solution,
 and the quantity of radioactive hormone bound in this complex is
measured by radioactive counting techniques.
Radioimmunoassay
 If a large amount of radioactive hormone has bound with the antibody,
 it is clear that there was only a small amount of natural hormone to
compete with the radioactive hormone,
 and therefore the concentration of the natural hormone in the assayed
fluid was small.
Radioimmunoassay
 Conversely, if only a small amount of radioactive hormone has bound,
 it is clear that there was a large amount of natural hormone to
compete for the binding sites
Radioimmunoassay
 Conversely, if only a small amount of radioactive hormone has bound,
 it is clear that there was a large amount of natural hormone to
compete for the binding sites
ELISA
 Enzyme-linked immunosorbent assays (ELISAs) can be used to
measure almost any protein, including hormones.
 Performed on plastic plates that each have 96 small wells
ELISA
 Enzyme-linked immunosorbent assay
 AB1 andAB2 are antibodies that recognize the hormone at
different binding sites
 AB3 is an antibody that recognizes AB2.
 E is an enzyme linked to AB3 that catalyzes the formation of a
colored fluorescent product (P) from a substrate (S).
 The amount of the product is measured using optical methods
and is proportional to the amount of hormone
ELISA
ELISA
 The ELISA method has become widely used in clinical and
research laboratories because
 (1) it does not use radioactive isotopes,
 (2) much of the assay can be automated using 96-well plates,
and
 (3) it has proved to be a cost-effective and accurate method for
assessing hormone levels.
Mechanism of hormone action
 Only cells with receptors for a specific hormone respond
 Those cells lacking the specific receptors are un affected
 Receptors on cell membrane – external receptors
 Receptors in cytoplasm – internal receptors
Mechanism of hormone action
 Peptide hormones bind to receptors on the cell membrane
 Steroid hormones bind with receptors in the cytoplasm
 Thyroid hormones bind with nuclear receptors
G proteins
 Peptide hormones binds to surface receptors
 Receptors are coupled with G proteins in the cell membrane
 Small and Large G proteins
 G protein coupled receptors are the membrane proteins that span the
membrane for 7 times
 Hence, they are called serpentine receptors (seven –helix receptors)
G proteins
 Hormone binds to receptor
 Hormone receptor complex
 HR complex binds to the G protein coupled receptors
 GDP exchanged for GTP
 Alpha sub-unit is separated
 Alpha sub-unit brings about physiological changes
G protein diseases
 Increased G protein activity
 Somatotroph tumor causing acromegaly
 Decrease in intrinsic GTPase activity
 G protein activity is prolonged
 More CAMP is produced
 Hyperplasia of somatotrophs of anterior pituitary- Acromegaly
G protein diseases
 Decreased G protein activity
 Type 1 pseudohypoparathyroidism
 Failure of G protein to respond to parathyroid hormone
 Features of hypoparathyroidism
 Without actual decrease in the parathyroid hormone
Transcription Translation effect
 Lipid soluble hormones easily enter the cell
 Steroid hormones
 1,25 DHCC
 Thyroid hormones
 Binds with receptor in cytoplasm or in nucleus
 Hormone receptor complex
 Stimulates transcription ( formation of mRNA)
Transcription Translation effect
 Stimulates transcription ( formation of mRNA)
 mRNA enters cytoplasm
 Activated ribosomes
 Synthesis of specific proteins
 Translation
 These proteins may be enzymes, structural proteins,
receptor proteins or secretory proteins
Transcription Translation effect
Second messengers
 Intracellular signal molecules that are formed by series of enzymatic
reactions
 Subsequent to the formation of HR complex
 Major second messengers are cAMP, DAG, IP3
 cAMP- cyclic AMP
 DAG- Diacylglycerol
 IP3- Inositol triphosphate
Second messengers
 Second messengers are formed by activation of four main
transduction systems
 Adenylyl cyclase –cyclic AMP system
 Membrane phospholipase – phospholipid system
 Guanylyl cyclase – cyclic GMP system
 Transcription of mRNAs
cAMP mediated hormone activity
 Proteins and poly peptide hormones
 Amino acid derivatives – catecholamines
 Some prostaglandlins
 Large in size so can not enter the cells
 Binds with receptors on cell surface
 Activates enzyme adynylil cyclase on inner surface of membrane
cAMP mediated hormone activity
 Activates enzyme adynylil cyclase on inner surface of membrane
 Increase in the formation of intra cellular cAMP
 cAMP acts as second messenger
 Converts inactive protein kinase to active form
 Protein kinase causes phosphorylation of proteins
 Brings different functions
cAMP mediated hormone activity
 Protein kinase causes phosphorylation of proteins
 Brings different functions
 Change in the membrane permeability
 Activation or inactivation of rate limiting enzymes
 Increase or decrease protein synthesis by action on
ribosomes
 Regulation of release of hormones
cAMP mediated hormone activity
Termination of cAMP actions
 cAMP is degraded to 5’AMP in the cell by the cytoplasmic enzyme
phosphodiesterase
 Note: Cholera toxin – stimulates cAMP
 Note: Pertussis toxin (Bordetella pertussis) (whooping cough) –
inhibits cAMP
Termination of cAMP actions
 Moderate intake of tea and coffee are good for heart
 Tea and coffee inhibits action of phosphodiesterase
 Increases cAMP
 Increase myocardial contractility
Membrane phospholipid-phospholipase system
 Hormone receptor complex
 Activates G alpha q protein
 Activates phospholipase C
 Hydrolysis of PIP2 (phosphatidyl inositol 4, 5 biphosphate)
 Formation of DAG and IP3
 IP3 release calcium from ER
 DAG stimulates intracellular enzyme protein kinase C
Enzyme-linked hormone receptors
 Some receptors, when activated, function directly as enzymes or are closely
associated with enzymes that they activate.
 Enzyme-linked receptors have their hormone-binding site on the outside of
the cell membrane and their catalytic or enzyme-binding site on the inside.
 When the hormone binds to the extracellular part of the receptor, an enzyme
immediately inside the cell membrane is activated (or occasionally
inactivated).
Enzyme-linked hormone receptors
 Leptin is a hormone secreted by fat cells and has many physiological
effects,
 but it is especially important in regulating appetite and energy balance
 leptin receptor, one of the signaling pathways occurs through a
tyrosine kinase of the Janus kinase (JAK) family, JAK2.

Enzyme-linked hormone receptors
 Leptin binds with the receptor
 conformational change of the receptor
 phosphorylation and activation of the intracellular associated JAK2
molecules
 phosphorylation of signal transducer and activator of transcription
(STAT) proteins
Enzyme-linked hormone receptors
 activates transcription by leptin target genes to initiate protein
synthesis. (Slow effects)
 also leads to the activation of other intracellular enzyme pathways
such as mitogen-activated protein kinases (MAPK) and
phosphatidylinositol 3-kinase (PI3K). (Rapid effects)
Permissive action
 one hormone must be present in adequate amounts to “permit” another hormone
to exert its full effect.
 The first hormone enhances a target cell’s responsiveness to the second hormone
by increasing the number of receptors for the second hormone.
 For example, thyroid hormone increases the number of receptors for epinephrine
in epinephrine’s target cells, increasing the effectiveness of epinephrine.
 In the absence of thyroid hormone, epinephrine is only marginally effective
Synergism
 occurs when the actions of several hormones are complementary and their
combined effect is greater than the sum of their separate effects.
 An example is the synergistic action of follicle-stimulating hormone and
testosterone,
 both of which are needed to maintain the normal rate of sperm production.
 Synergism results from each hormone’s influence on the number or affinity
(attraction) of receptors for the other hormone.
Antagonism
 occurs when one hormone causes the loss of another hormone’s receptors, reducing the
effectiveness of the second hormone.
 To illustrate, progesterone (a hormone secreted during pregnancy that decreases
contractions of the uterus)
 inhibits uterine responsiveness to estrogen (another hormone secreted during pregnancy
that increases uterine contractions).
 By causing loss of estrogen receptors on uterine smooth muscle, progesterone prevents
estrogen from exerting its excitatory effects during pregnancy and thus keeps the uterus
as a quiet (noncontracting) environment suitable for the developing fetus.
Down Regulation
 when the plasma concentration of hormones is chronically elevated,
 the total number of target-cell receptors for that hormone is gradually
reduced
 This phenomenon, known as downregulation, constitutes an important
locally acting negative feedback mechanism
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Introduction Endocrinology class -2.pptx

  • 1. Introduction to Endocrinology Dr. Sai Sailesh Kumar G Professor Department of Physiology NRIIMS Email: dr.goothy@gmail.com
  • 2.
  • 3.
  • 4. Measurement of Hormone Concentrations in the Blood  Most hormones are present in the blood in extremely minute quantities; some concentrations are as low as one billionth of a milligram (1 picogram) per milliliter.  Therefore, it was difficult to measure these concentrations by the usual chemical means  However, an extremely sensitive method that was developed about 50 years ago  This method is called radioimmunoassay.  More recently, additional methods, such as enzyme-linked immunosorbent assays, have been developed for accurate, high-throughput measurements of hormones.
  • 5. Radioimmunoassay  First, an antibody that is highly specific for the hormone to be measured is produced.  Second, a small quantity of this antibody is  (1) mixed with a quantity of fluid from the animal containing the hormone to be measured  (2) mixed simultaneously with an appropriate amount of purified standard hormone that has been tagged with a radioactive isotope.
  • 6. Radioimmunoassay  One specific condition must be met:  There must be too little antibody to bind completely both the radioactively tagged hormone and the hormone in the fluid to be assayed.  Therefore, the natural hormone in the assay fluid and the radioactive standard hormone compete for the binding sites of the antibody.
  • 7. Radioimmunoassay  Third, after binding has reached equilibrium,  the antibody-hormone complex is separated from the remainder of the solution,  and the quantity of radioactive hormone bound in this complex is measured by radioactive counting techniques.
  • 8. Radioimmunoassay  If a large amount of radioactive hormone has bound with the antibody,  it is clear that there was only a small amount of natural hormone to compete with the radioactive hormone,  and therefore the concentration of the natural hormone in the assayed fluid was small.
  • 9. Radioimmunoassay  Conversely, if only a small amount of radioactive hormone has bound,  it is clear that there was a large amount of natural hormone to compete for the binding sites
  • 10. Radioimmunoassay  Conversely, if only a small amount of radioactive hormone has bound,  it is clear that there was a large amount of natural hormone to compete for the binding sites
  • 11. ELISA  Enzyme-linked immunosorbent assays (ELISAs) can be used to measure almost any protein, including hormones.  Performed on plastic plates that each have 96 small wells
  • 12. ELISA  Enzyme-linked immunosorbent assay  AB1 andAB2 are antibodies that recognize the hormone at different binding sites  AB3 is an antibody that recognizes AB2.  E is an enzyme linked to AB3 that catalyzes the formation of a colored fluorescent product (P) from a substrate (S).  The amount of the product is measured using optical methods and is proportional to the amount of hormone
  • 13. ELISA
  • 14. ELISA  The ELISA method has become widely used in clinical and research laboratories because  (1) it does not use radioactive isotopes,  (2) much of the assay can be automated using 96-well plates, and  (3) it has proved to be a cost-effective and accurate method for assessing hormone levels.
  • 15. Mechanism of hormone action  Only cells with receptors for a specific hormone respond  Those cells lacking the specific receptors are un affected  Receptors on cell membrane – external receptors  Receptors in cytoplasm – internal receptors
  • 16. Mechanism of hormone action  Peptide hormones bind to receptors on the cell membrane  Steroid hormones bind with receptors in the cytoplasm  Thyroid hormones bind with nuclear receptors
  • 17.
  • 18. G proteins  Peptide hormones binds to surface receptors  Receptors are coupled with G proteins in the cell membrane  Small and Large G proteins  G protein coupled receptors are the membrane proteins that span the membrane for 7 times  Hence, they are called serpentine receptors (seven –helix receptors)
  • 19. G proteins  Hormone binds to receptor  Hormone receptor complex  HR complex binds to the G protein coupled receptors  GDP exchanged for GTP  Alpha sub-unit is separated  Alpha sub-unit brings about physiological changes
  • 20.
  • 21. G protein diseases  Increased G protein activity  Somatotroph tumor causing acromegaly  Decrease in intrinsic GTPase activity  G protein activity is prolonged  More CAMP is produced  Hyperplasia of somatotrophs of anterior pituitary- Acromegaly
  • 22. G protein diseases  Decreased G protein activity  Type 1 pseudohypoparathyroidism  Failure of G protein to respond to parathyroid hormone  Features of hypoparathyroidism  Without actual decrease in the parathyroid hormone
  • 23. Transcription Translation effect  Lipid soluble hormones easily enter the cell  Steroid hormones  1,25 DHCC  Thyroid hormones  Binds with receptor in cytoplasm or in nucleus  Hormone receptor complex  Stimulates transcription ( formation of mRNA)
  • 24. Transcription Translation effect  Stimulates transcription ( formation of mRNA)  mRNA enters cytoplasm  Activated ribosomes  Synthesis of specific proteins  Translation  These proteins may be enzymes, structural proteins, receptor proteins or secretory proteins
  • 26. Second messengers  Intracellular signal molecules that are formed by series of enzymatic reactions  Subsequent to the formation of HR complex  Major second messengers are cAMP, DAG, IP3  cAMP- cyclic AMP  DAG- Diacylglycerol  IP3- Inositol triphosphate
  • 27. Second messengers  Second messengers are formed by activation of four main transduction systems  Adenylyl cyclase –cyclic AMP system  Membrane phospholipase – phospholipid system  Guanylyl cyclase – cyclic GMP system  Transcription of mRNAs
  • 28. cAMP mediated hormone activity  Proteins and poly peptide hormones  Amino acid derivatives – catecholamines  Some prostaglandlins  Large in size so can not enter the cells  Binds with receptors on cell surface  Activates enzyme adynylil cyclase on inner surface of membrane
  • 29. cAMP mediated hormone activity  Activates enzyme adynylil cyclase on inner surface of membrane  Increase in the formation of intra cellular cAMP  cAMP acts as second messenger  Converts inactive protein kinase to active form  Protein kinase causes phosphorylation of proteins  Brings different functions
  • 30. cAMP mediated hormone activity  Protein kinase causes phosphorylation of proteins  Brings different functions  Change in the membrane permeability  Activation or inactivation of rate limiting enzymes  Increase or decrease protein synthesis by action on ribosomes  Regulation of release of hormones
  • 32. Termination of cAMP actions  cAMP is degraded to 5’AMP in the cell by the cytoplasmic enzyme phosphodiesterase  Note: Cholera toxin – stimulates cAMP  Note: Pertussis toxin (Bordetella pertussis) (whooping cough) – inhibits cAMP
  • 33. Termination of cAMP actions  Moderate intake of tea and coffee are good for heart  Tea and coffee inhibits action of phosphodiesterase  Increases cAMP  Increase myocardial contractility
  • 34. Membrane phospholipid-phospholipase system  Hormone receptor complex  Activates G alpha q protein  Activates phospholipase C  Hydrolysis of PIP2 (phosphatidyl inositol 4, 5 biphosphate)  Formation of DAG and IP3  IP3 release calcium from ER  DAG stimulates intracellular enzyme protein kinase C
  • 35.
  • 36. Enzyme-linked hormone receptors  Some receptors, when activated, function directly as enzymes or are closely associated with enzymes that they activate.  Enzyme-linked receptors have their hormone-binding site on the outside of the cell membrane and their catalytic or enzyme-binding site on the inside.  When the hormone binds to the extracellular part of the receptor, an enzyme immediately inside the cell membrane is activated (or occasionally inactivated).
  • 37. Enzyme-linked hormone receptors  Leptin is a hormone secreted by fat cells and has many physiological effects,  but it is especially important in regulating appetite and energy balance  leptin receptor, one of the signaling pathways occurs through a tyrosine kinase of the Janus kinase (JAK) family, JAK2. 
  • 38. Enzyme-linked hormone receptors  Leptin binds with the receptor  conformational change of the receptor  phosphorylation and activation of the intracellular associated JAK2 molecules  phosphorylation of signal transducer and activator of transcription (STAT) proteins
  • 39. Enzyme-linked hormone receptors  activates transcription by leptin target genes to initiate protein synthesis. (Slow effects)  also leads to the activation of other intracellular enzyme pathways such as mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K). (Rapid effects)
  • 40.
  • 41. Permissive action  one hormone must be present in adequate amounts to “permit” another hormone to exert its full effect.  The first hormone enhances a target cell’s responsiveness to the second hormone by increasing the number of receptors for the second hormone.  For example, thyroid hormone increases the number of receptors for epinephrine in epinephrine’s target cells, increasing the effectiveness of epinephrine.  In the absence of thyroid hormone, epinephrine is only marginally effective
  • 42. Synergism  occurs when the actions of several hormones are complementary and their combined effect is greater than the sum of their separate effects.  An example is the synergistic action of follicle-stimulating hormone and testosterone,  both of which are needed to maintain the normal rate of sperm production.  Synergism results from each hormone’s influence on the number or affinity (attraction) of receptors for the other hormone.
  • 43. Antagonism  occurs when one hormone causes the loss of another hormone’s receptors, reducing the effectiveness of the second hormone.  To illustrate, progesterone (a hormone secreted during pregnancy that decreases contractions of the uterus)  inhibits uterine responsiveness to estrogen (another hormone secreted during pregnancy that increases uterine contractions).  By causing loss of estrogen receptors on uterine smooth muscle, progesterone prevents estrogen from exerting its excitatory effects during pregnancy and thus keeps the uterus as a quiet (noncontracting) environment suitable for the developing fetus.
  • 44. Down Regulation  when the plasma concentration of hormones is chronically elevated,  the total number of target-cell receptors for that hormone is gradually reduced  This phenomenon, known as downregulation, constitutes an important locally acting negative feedback mechanism