SECONDARY MESSENGERs

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).

secondary MESSENGERS
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.

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

 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

 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
activity 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
special 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

 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.

Common mechanisms of secondary messenger systems

Types Of Second Messenger Molecules

Three basic types of secondary
messenger molecules:
Hydrophobic molecules: membraneassociated 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.

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.

PKA REGULATION by cAMP

The most common downstream
effector of cAMP isProtein 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.

 Protein synthesis-PKA directly

activate CREB, which bind the
cAMP response element(CRE)and
altering the transcription.

ANCHORAGE

How does each enzyme

find its appropriate set of
protein substrates ???

ANCHORAGE
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.

INACTIVATIO
N

feedback mechanism using phosphodiesterase.

Phosphodiesterase quickly converts cAMP to AMP, thus reducing
the amount of cAMP that can activate protein kinase A.

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-, IL-17, and IFN)

cAMP promote the release of anti-inflammatory mediators (eg, IL10) by immune cells.

cAMP activated PKA, which translocates into the nucleus and

activates transcription factor CREB (cAMP response element
binding protein).

 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-inflammatorymediator.
In contrast, cAMP elevation would inhibit expression of genes
driven by the transcription factor nuclear factor B (NF-B)
Decreasing intracellular cAMP, PDE4 could prevent PKA from
modulating pro- and anti-inflammatory mediators released by a
cell

Role of cAMP in Glycogen

breakdown

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).

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

2. INOSITOL TRIPHOSPHATE
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.

FUNCTION of IP3

Blocks Polyspermy in Sea Urchin

Fertilization -Fusion of eggs 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 sperms membrane to the eggs
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.

CGs release four main compounds: Proteases, Hyaluronic acid,

IP3 ROLE in PATHOPHYSIOLOGY

HUNTINGTONS 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.

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

3. DIACYLGLYCEROL
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.

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 (10100 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.

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.

Ligand gated
calcium channels
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

Stored-operated calcium channels :

Located in the plasma membrane of all nonexcitable 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.

Intracellular compartment:
Calcium is stored in higher
concentrations in endoplasmic
reticulum and 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-2) and slow skeletal
and fast skeletal form (calsequestrin1)

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 EFhand motifs .
Each EF-hand motif allows calmodulin
to sense intracellular calcium levels by
binding up to four Ca2+ ions.

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.

 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.

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.

 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.

FUNCTION

Skeletal

Smooth muscle contraction

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

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 relax 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 to various agents
In his earlier studies, Furchgott used strips of aorta that had
been dissected from the organ.

 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 cells 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

 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.

 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.

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.

 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

MECHANISM

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 /CalmodulinDependent 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.