secondary messengers

1. SECONDARY MESSENGERs

2. secondary MESSENGERS
 Secondary messengers, intracellular signaling molecules released
by the cell to trigger physiological changes
 Amplifying components of intracellular signal transduction cascades.
 Examples of secondary messengers include cyclic AMP, cyclic
GMP, inositol trisphosphate, diacylglycerol, and calcium .
 Releases in response to exposure to extracellular signaling
molecules/ligands the first messengers, such as
neurotransmitters, hormones (epinephrine, growth hormone and
serotonin).

3. The first messengers such as peptide hormones,
neurotransmitters usually do not physically cross the
phospholipid bilayers.
First messengers need to be transduced into
secondary messengers, so that the extracellular signal
may be propagated intracellularly.
Secondary messengers greatly amplify the strength of
the signal.
Activate or inhibit the target enzymes of the pathway.
secondary MESSENGERS

4. History of secondary messengers
 Earl Wilbur Sutherland Jr. discovered
secondary messengers won the 1971 Nobel
Prize in Medicine
 He saw that epinephrine stimulate
glycogenolysis in liver cells, but
epinephrine alone would not convert
glycogen to glucose
 He found that epinephrine had to trigger a
secondary messenger, cyclic AMP for the
liver to convert glycogen to glucose.
Earl Wilbur Sutherland Jr.
1915-1974

5.  After receiving his M.D degree from Washington University Medical
School ,He started his research career in Biochemistry department of
Washington University of Medical School
 At that time he came in contact with Arthur Kornberg, Edwin Krebs,
C.DeDuve, Victor Najjar
 Sutherland collaborated with DeDuve on the origin and distribution
of a hyperglycemic-glycogenolytic factor present in commercial
insulin preparations
 And concluded that it came from the a-cells of the islets of
Langerhans, later renamed glucagon
 Sutherland than studied two parallel line of work-
 The enzyme phosphorylase which initiate the breakdown the
glycogen in liver and muscle
 How epinephrine and glucagon stimulated the release of glucose
from glycogen in the liver

6.  Liver slices take as a test system for the action of glucagon and
epinephrine, increased the glucose output when added in vitro
 In a control incubation the phosphorylase activity of the liver slices
showed a large drop
 When epinephrine and glucagon are added the phosphorylate
was restored
 At that time Krebs and Fisher studying the reactivation of inactive
rabbit muscle phosphorylase
 Shown that this occurred with ATP and Mg2+ or Mn2+ and a
enzyme, a kinase, was necessary for this reaction
 With this information they add hormones to inactive liver
phosphorylase in the presence of Mg2+ and ATP
 They observed activation of phosphorylase by epinephrine and
glucagon if they use relatively crude liver homogenate

7.  But if they centrifuged the extracts to remove the cellular debris, the
hormone action disappeared
 They were able to show that if the particulate fraction alone
incubated with hormones, a heat-stable factor was produced that
could in turn activate phosphorylase.
 The next step to isolate and identify the heat-stable factor
 This is a difficult task for isolate because it is rapidly destroyed by
phosphodiesterase
 This is the history of discovery adenosine-3',5'-phosphoric acid,
generally referred to as cyclic AMP.

8. Common mechanisms of secondary messenger systems

9. Types Of Second Messenger Molecules
 Three basic types of secondary
messenger molecules:
Hydrophobic molecules: membrane-
associated e.g. diacylglycerol,
phosphatidylinositol
Hydrophilic molecules: water-soluble
molecules, such as cAMP, cGMP, IP3,
and Ca2+, located within the cytosol.
Gases: nitric oxide (NO), carbon
monoxide (CO) and hydrogen sulfide
(H2S) which can diffuse both through
cytosol and across cellular membranes.

11. cAMP
 cAMP is a second messenger,
synthesized from ATP by
enzyme adenylyl cyclase.
 Adenylate cyclase is activated
by stimulatory G (Gs)-protein-
coupled receptors.
 Inhibited by adenylate cyclase
inhibitory G (Gi)-protein-
coupled receptors.

12. PKA REGULATION by cAMP
 The most common downstream effector of
cAMP is Protein kinase A(PKA).
 PKA is normally inactive as tetrameric
holoenzyme(two catalytic and two
regulatory units).
 The regulatory unit always block the
catalytic center of catalytic unit.
 Two cAMP molecules bind to each PKA
regulatory subunit.
 The regulatory subunit dissociate from the
catalytic subunit.
 The free catalytic subunits interact with
proteins to phosphorylate Ser or Thr
residues, either increases or decreases the
activity of the protein.

13.  Protein synthesis-PKA directly
activate CREB, which bind the cAMP
response element(CRE)and altering
the transcription.

14. ANCHORAGE
How does each enzyme find
its appropriate set of protein
substrates ???

15.  Cells maintain signaling specificity
 Protein scaffold complexes are key mechanism that
integrate cAMP signaling with other pathways
and signaling events.
 AKAPs act as scaffold proteins, they bind PKA and
physically tether these multi-protein complexes to
specific locations, such as the nucleus and other
compartments in cells.
ANCHORAGE

16. INACTIVATION
 feedback mechanism using phosphodiesterase.
 Phosphodiesterase quickly converts cAMP to AMP, thus reducing the
amount of cAMP that can activate protein kinase A.

17. PDE4 Promotes Inflammation by Degrading cAMP Within Immune
Cells
 Phosphodiesterase 4 (PDE4) is the predominant cAMP-degrading
enzyme expressed in inflammatory cells.
 cAMP helps regulate T cell function.
 cAMP helps maintain immune homeostasis by suppressing the
release of proinflammatory mediators (eg, TNF-α, IL-17, and IFN-
γ)
 cAMP promote the release of anti-inflammatory mediators (eg, IL-
10) by immune cells.
 cAMP activated PKA, which translocates into the nucleus and
activates transcription factor CREB (cAMP response element
binding protein).

18.  Decrease in PDE4 increases cAMP, leads to increased transcription of
genes that have CRE sites, including the gene for IL-10, which is an anti-
inflammatory mediator.
 In contrast, cAMP elevation would inhibit expression of genes driven by
the transcription factor nuclear factor κ B (NF-κB)
 Decreasing intracellular cAMP, PDE4 could prevent PKA from modulating
pro- and anti-inflammatory mediators released by a cell

19.  Glucagon stimulates liver cells to start
glycogenolysis through GPCR.
 Leading to the activation of adenylyl
cyclase and the formation of cAMP.
 The cAMP binds to protein kinase A
(PKA), activating it.
 PKA in turn phosphorylates other
downstream target proteins including
phosphorylase kinase (PhosK) and
glycogen synthase (GS).
Role of cAMP in Glycogen breakdown

20. 2. INOSITOL TRIPHOSPHATE
 Inositol triphosphate (IP3) is a
lipid-derived secondary
messenger.
 A product of the hydrolysis of the
phospholipid
phosphatidylinositol 4,5-
bisphosphate (PIP2) by the
enzyme phospholipase C

21.  Being water-soluble molecule
IP3 diffuses rapidly through the
cytosol.
 At endoplasmic reticulum(ER),
it binds to and opens IP3 -
gated Ca2+ channels in the ER
membrane.
 Ca2+ stored in the ER is released
through the open channels
resulting in increased
concentration of Ca2+ in the
cytosol.
2. INOSITOL TRIPHOSPHATE

22. FUNCTION of IP3
 Blocks Polyspermy in Sea Urchin
 Fertilization -Fusion of egg’s and sperm’ cellular membrane
 At this point egg is extremely susceptible to attack by other sperm attempting to
fertilize, so immediate action must be taken
 It can be achieved by two process- fast block and slow block
 In slow block, binding and fusion of the sperm’s membrane to the egg’s membrane
creates a cascade of events that enable the Gq Pathway.
 First Phospholipase C is activated.
 PLC cleaves Phosphatidylinositol 4,5 Bisphosphate to create two compounds: 1)
Inositol Triphosphate (IP3) and 2) Diacylglycerol (DAG).
 IP3 diffuses to the ER, where it opens Ca2+ channels.
 The release of Ca2+ ions stimulates the Cortical Granule Response.
 CG’s release four main compounds: Proteases, Hyaluronic acid, Peroxidases, Hyaline
Hyaline

24. IP3 ROLE in PATHOPHYSIOLOGY
HUNTINGTON’S DISEASE
 neurons in the brain degenerate.
 Affects medium spiny neurons (MSN) presents in stratium
 The cytosolic protein Huntingtin (Htt) has an additional 35 glutamine
residues added to its amino terminal region.
 This modified form of Htt is called Httexp.
 Httexp makes Type 1 IP3 receptors more sensitive to IP3, which leads
to the release of too much Ca2+ from the ER.
 The release of Ca2+ from the ER causes an increase in the cytosolic
and mitochondrial concentrations of Ca2+.
 This increase in Ca2+ is thought to be the cause of GABAergic MSN
degradation.

25. 3. DIACYLGLYCEROL
 Diacylglycerol (DAG)
functions as a second
messenger signaling lipid
molecule.
 Product of the hydrolysis of
the phospholipid
phosphatidylinositol 4,5-
bisphosphate (PIP2) by the
enzyme phospholipase C

26.  Diacylglycerol remains within the plasma membrane, activate
serine/threonine protein kinase called protein kinase C (PKC), so
named because it is Ca2+ dependent.
 The initial rise in cytosolic Ca2+ induced by IP3 alters the PKC so that
it translocates from the cytosol to the cytoplasmic face of the plasma
membrane.
 There it is activated by the combination of Ca2+, diacylglycerol, and
the phospholipid phosphatidylserine
 Activated PKC phosphorylates target proteins like glucose
transporter, HMG-CoA reductase, cytochromeP450 etc.
3. DIACYLGLYCEROL

27. 4. CALCIUM IONS
 Once calcium enter the cytoplasm exert allosteric regulatory effects on
many enzymes and proteins (toxic in excess)
 Low cytoplasmic Ca++ at rest (10–100 nM).
 To maintain this low concentration, Ca2+ is actively pumped from the
cytosol to the extracellular space and into the endoplasmic
reticulum (ER)
 Certain proteins of the cytoplasm and organelles act as buffers by
binding Ca2+.
 Acts as a secondary messenger by signal transduction pathways such
as via G protein-coupled receptors.
 Signaling occurs when the cell is stimulated to release calcium ions
(Ca2+) from intracellular stores, or when calcium enters the cell
through plasma membrane ion channels.

28. Sources of Ca2+ :
 Extracellular compartment, nerve, cardiac
and smooth muscle cells
 Three types of plasma-membrane
localized calcium channels :
Voltage-dependent calcium channels :
 At physiological condition VDCCs are
closed (resting membrane potential)
 The concentration of calcium ions are
several times higher outside of the cell
than inside .
 Action potential depolarizes plasma
membrane, which results in the opening
of VDCCs and calcium ion rush into the
cell.

29.  transmembrane ion channels
 allow Ca2+ to pass through the
membrane in response to ligand
such as neurotransmitter like GABA,
acetyl choline
 e.g.–
> Nicotinic acetylcholine receptors
>glutamate/NMDA receptor
> ATP receptor
Ligand gated calcium
channels

30. Stored-operated calcium channels :
 Located in the plasma membrane of all non-
excitable cells (myocytes, endocrine cells etc.)
 They are major source of intracellular calcium
 Although it was initially considered to function only
in non excitable cells, growing evidence now points
towards a central role for in excitable cells too.

31. Intracellular compartment:
 Calcium is stored in higher concentrations
in endoplasmic reticulum and sarcoplasmic
sarcoplasmic reticulum
 In sarcoplasmic reticulum they are bound
with calsequestrin.
 Calsequestrin is highly acidic, containing up
to 50 Ca(2+)-binding sites, formed simply
by clustering of two or more acidic protein.
 Two forms of calsequestrin have been
identified i.e. Cardiac form (Calsequestrin-
and slow skeletal and fast skeletal form
(calsequestrin-1)

32. Ca2+ Sensors
Calmodulin
 Effects of Ca2+ are mediated through
ubiquitous Ca2+ sensing protein,
calmodulin(CaM).
 CaM is a 17 kDa Ca2+-binding protein
 Composed of, N- and C-terminal lobe
tethered by a loop, allows CaM to adopt a
variety of conformations
 Each lobe of CaM contains a pair of EF-
hand motifs .
 Each EF-hand motif allows calmodulin to
sense intracellular calcium levels by binding
up to four Ca2+ ions.

33. Calmodulin
 Activated by Ca2+ binding, it undergoes
conformational change that permits
Ca2+/calmodulin to bind various target
proteins
 The protein responds in an almost switch
like manner to increasing concentrations of
Ca2+.
 A tenfold increase in Ca2+ concentration
typically causes a fiftyfold increase in
calmodulin activation
 When an activated molecule of
Ca2+/calmodulin binds to its target,
calmodulin further changes its conformation.

34.  Whenever the concentration of Ca2+ in
the cytosol rises,Ca2+/calmodulin,
activates the plasma membrane Ca2+-
pump that uses ATP hydrolysis to pump
Ca2+ out of cells.

35. CaM-kinases
 Ca2+/calmodulin, plays a great role in protein phosphorylations,
catalyzed by a family of serine/threonine protein kinases called
Ca2+/calmodulin-dependent kinases (CaM-kinases).
 CaM-kinases phosphorylate gene regulatory proteins, such as the
CREB protein, and in this way activate or inhibit the transcription of
specific genes.
 One of the best-studied CaM-kinases is CaM-kinase II, found in most
animal cells, especially enriched in the nervous system.
 It is highly concentrated in synapses.

36.  CaM-kinase II function as a molecular memory device, switching to an
active state when exposed to Ca2+/calmodulin and remain active even
after the Ca2+ signal has decayed.
 This is because the kinase phosphorylates itself (autophosphorylation).
 In its autophosphorylated state, the enzyme remains active even in the
absence of Ca2+
 The enzyme maintains this activity until serine/threonine protein
phosphatases inhibit the autophosphorylation and shut the kinase off.
 CaM-kinase II activation serve as a memory trace and seems to have a role
in some types of memory and learning in the vertebrate nervous system.
 Mutant mice that lack a brain-specific form of the enzyme have specific
defects in their ability to remember where things are.

38. FUNCTION
 Skeletal

39. Smooth musclecontraction

40. NFAT(NUCLEAR FACTOR OF ACTIVATED T CELLS)
ACTIVATION
 In unstimulated cells, phosphorylated
NFAT is located in the cytosol.
 Ca2+/calmodulin complex binds to and
activates calcineurin, a protein-serine
phosphatase.
 Activated calcineurin then
dephosphorylates phosphate residues
on cytosolic NFAT
 NFAT, exposing a nuclear localization
sequence that allows NFAT activity and
expression of gene essential for T cell
activation

41. 5. NITRIC OXIDE
HISTORY
 NO functions as a messenger molecule began with an accidental
observation
 It had been known for many years that acetylcholine acts in the body to
the smooth muscle cells of blood vessels, but the response could not be
duplicated in vitro
 When portions of a major blood vessel such as the aorta were incubated in
physiologic concentrations of acetylcholine in vitro, the preparation usually
showed little or no response
 In the late 1970s, Robert Furchgott, a pharmacologist at New York State
medical center, was studying the in vitro response of pieces of rabbit aorta
various agents
 In his earlier studies, Furchgott used strips of aorta that had been dissected
from the organ.

42.  Furchgott switched from strips of aortic tissue to aortic rings and aortic
rings responded to acetylcholine by undergoing relaxation
 The strips had failed to display the relaxation response because the
endothelial layer that lines the aorta had been rubbed away during the
dissection
 This surprising finding suggested that the endothelial cells were somehow
involved in the response by the adjacent muscle cells.
 Acetylcholine binds to receptors on the surface of endothelial cells,
leading to the production and release of an agent that diffuses through
the cell’s plasma membrane and causes the muscle cells to relax.
 The diffusible agent was identified in 1986 as nitric oxide by Louis
Ignarro and Salvador Moncada

43.  Nitric oxide (NO) is a gas,
diffuse through the plasma
membrane and affect nearby
cells.
 Synthesized from arginine and
oxygen by the NO synthase.
 NO then activate soluble guanylyl
cyclase, to produce cGMP.

44.  The function of NO is the dilation
of blood vessels.
 The acetylcholine
(neurotransmitter) acts on
endothelial cells to stimulate NO
synthesis.
 NO, diffuses to neighboring
smooth muscle cells where it
interacts with the guanylyl
cyclase.
 This increase enzymatic activity
resulting in the synthesis of cGMP.
 The cGMP then induces muscle
relaxation and blood vessel
dilation.

45. NITROGLYCERINE ACT AS VASODILATER
A BRIEF HISTORY
 Nitroglycerin is an oily liquid that may explode
when subjected to heat, shock or flame.
 Alfred Nobel developed the use of nitroglycerin
as a blasting explosive by mixing the
nitroglycerin with inert absorbents such as
diatomaceous earth
 Named them as dynamite and patented it in
1867.
 Dr. William Murrell experimented with the use of
nitroglycerin to relieve angina pectoris and to
reduce the blood pressure.

46.  A few months before his death in 1896, Alfred Nobel was prescribed
nitroglycerine for this heart condition
 He said to his friend that "Isn't it the irony of fate that I have been
prescribed nitro-glycerin, to be taken internally !
 They call it Trinitrin, so as not to scare the chemist and the public, so
that it also called as glyceryl trinitrin

47. MECHANISM

48. REFERANCE
 Kaestner, Lars. "Calcium Signalling." (2013): n. pag. Web.
<http://dx.doi.org/10.1016/j.cell.2007.11.028>.
 Fujisawa, H. "Regulation of the Activities of Multifunctional Ca2 /Calmodulin-Dependent Protein
Kinases." Journal of Biochemistry 129.2 (2001): 193-99. Web. <10.1007/s00018-008-8086-2>.
 Carnegie, Graeme K., Christopher K. Means, and John D. Scott. "A-kinase Anchoring Proteins:
From Protein Complexes to Physiology and Disease." IUBMB Life 61.4 (2009): 394-406. Web.
<10.1002/iub.168>.
 Alberts, Bruce. Molecular Biology of the Cell, 5th Edition. New York: Garland Science, 2008. Print.