1.
SECOND MESSENGERS
Presenter : Dr.Anu Priya J

2.
SCHEME
• History
• Introduction
• Types
• cAMP Pathway
• cGMP Pathway
• IP3 / DAG Pathway
• Calcium as a second messenger
• Eicosanoids
• Applied aspects

3.
HISTORY
• Earl Wilbur Sutherland Jr. – 1971 Nobel Prize in Physiology
or Medicine
• Epinephrine – liver - glycogen to glucose – cAMP

4.
• Martin Rodbell & Alfred G. Gilman – 1994 Nobel Prize
HISTORY

5.
INTRODUCTION
• Cell to cell communication
• Chemical and physical messengers interact with receptors in
the plasma membrane , cytoplasm or nucleus.
• A series of signaling events that mediate the response to each
stimulus.
• Response – specific, amplified ,tightly regulated and
coordinated.

6.
Plasma membrane
EXTRACELLULAR
FLUID
CYTOPLASM
Reception Transduction Response
Receptor
Signaling
molecule
Activation
of cellular
response
Relay molecules in a signal transduction
pathway
321
Earl Sutherland – cell signaling- 3 processes

7.
• Signaling pathways are characterized by:
1. Multiple, hierarchical steps
2. Amplification of the hormone-receptor binding event which
magnifies the response
3. Activation of multiple pathways and regulation of multiple
cellular functions
4. Feedback mechanisms – tight regulatory control

8.
TYPES
• Hydrophobic molecules
-Membrane associated
-Phosphatidylinositol , Diacylglycerol
• Hydrophilic molecules
-Cytosolic
- cAMP, cGMP, inositol triphosphate,Ca2+

9.
• cAMP pathway
• cGMP pathway
• IP3 / DAG pathway
• Calcium as a second messenger
• Eicosanoids
Second Messengers

10.
11
Second Messengers
• General characteristics
– Low amounts in resting state
– Regulated synthesis
– Regulated destruction
– Act through other proteins

11.
Hormone
receptor
GDP
G protein
GTP
Adenylcyclase
cAMP
ATP
cAMP pathway

12.
cAMP pathway

13.
cAMP-dependent Protein Kinase A
C
R R
C
+ 4 cAMP
C
C R
R
cAMP
cAMP
cAMP
cAMP
+
The catalytic subunit is
now free to attack a
protein target.
cAMP pathway

14.
Active
phosphorylase
Kinase
Inactive
Phosphorylase
Kinase



 



P
P
Catalytic site
Calmodulin
2ATP 2ADP
cAPK
Kinase enzymes are the targets for the catalytic subunit of PKA
These target enzymes have some unique features.
Phosphorylase kinase, for example, is composed of 4 different subunits .
The delta subunit is calmodulin, a calcium binding protein, that regulates the activity.
The gamma subunit has the catalytic site . The enzyme is not active.
To activate phosphorylase kinase, the catalytic subunit of protein kinase A transfers 2
PO4s from 2 ATPs to the alpha and beta subunits. Phosphorylation at these sites renders
phosphorylase kinase active. Conversely, removing phosphate inactivates the kinase.

15.
P
The target kinases phosphorylate enzymes that control critical steps in a pathway.
For example, phosphorylase or glycogen synthase are targets of protein kinase.
For phosphorylase, the kinase enzyme is phosphorylase kinase.
The sequence of events is highlighted in the figure below
Adenylcyclase
C C
R R
cAMP dependent
protein kinase

 

Phosphorylase
kinase Phosphorylase
Thus hormones(external to the cell) have profound effect on glycogen degradation,
glycogen synthesis, and other processes by controlling the enzyme activities by the
presence or absence of phosphate groups.
P
cAMP pathway

16.
cAMP pathway

17.
• In addition to signaling in the cytoplasm, the catalytic subunit
of PKA can enter the nucleus of cells and phosphorylate and
activate the transcription factor cAMP response element
binding (CREB) protein.
• Phospho-CREB protein increases the transcription of many
genes
• Indirect effect of cAMP mediated by PKA
cAMP pathway

18.
cyclic AMP mediated response
Epinephrine (α2,β1,β2)
Acetylcholine(M2)
Norepinephrine
Glucagon
Somatostatin
ACTH
CRH
ADH
FSH
hCG
LH
MSH
PTH
TSH
Angiotensin II
Calcitonin

19.
cGMP Pathway
Ligand-receptor
Guanyl cyclase
GTP cGMP
Protein kinase G Active protein kinase G
Effects
PDE
GMP

20.
22
• smooth muscle relaxation
• vision
• ANP,NO
cGMP Pathway

21.
• cGMP Pathway in phototransduction
cGMP Pathway

22.
Nitric oxide (NO)
NO, a simple gas, is able to diffuse across the membrane, and alters the
activity of intracellular target enzymes. It’s extremely unstable, so its effects are
local. Ex. It signals the dilation of blood vessels.
Mechanism.
Acetylcholine is released from the terminus of nerve cell in the blood
vessel wall. The endothelial cells are stimulated to produce NO (from arginine),
which causes an increased synthesis of cGMP, a second messenger responsible for
blood vessel dilation.
Ach
Nerve cell endothelial cell
NO cGMP Vessel dilationAchR
cGMP Pathway

23.
IP3 /DAG
LIGAND RECEPTOR
↓
G protein
↓
Phospholipase C
↓
PIP2  IP3 + DAG
↓
Endoplasmic reticulum  opening of Ca channels
↓
Ca++
↓
Protein kinase C  Effects

24.
G protein
EXTRA-
CELLULAR
FLUID
Signaling molecule
(first messenger)
G protein-coupled
receptor
Phospholipase C
DAG
PIP2
IP3
(second messenger)
IP3-gated
calcium channel
Endoplasmic
reticulum (ER)
CYTOSOL
Ca2
GTP
Figure 11.14-1

25.
Figure 11.14-2
G protein
EXTRA-
CELLULAR
FLUID
Signaling molecule
(first messenger)
G protein-coupled
receptor
Phospholipase C
DAG
PIP2
IP3
(second messenger)
IP3-gated
calcium channel
Endoplasmic
reticulum (ER)
CYTOSOL
Ca2
(second
messenger)
Ca2
GTP

26.
Figure 11.14-3
G protein
EXTRA-
CELLULAR
FLUID
Signaling molecule
(first messenger)
G protein-coupled
receptor
Phospholipase C
DAG
PIP2
IP3
(second messenger)
IP3-gated
calcium channel
Endoplasmic
reticulum (ER)
CYTOSOL
Various
proteins
activated
Cellular
responses
Ca2
(second
messenger)
Ca2
GTP

27.
IP3 /DAG

28.
30
• Inositol tri-phosphate
• Hydrophilic
• Agonist for internal
calcium channel
• [Ca++]i rises
• Multiple effects through
Ca++-binding proteins
• Diacylglycerol
• Hydrophobic
• Targets PKC (a kinase)
• PKC requires Ca++ and
DAG
IP3 /DAG

29.
• Classical PKC family members (PKCα,PKCβ,PKCγ) require
both Ca2+ & DAG for activation.
• The novel PKCs (PKCδ,PKCε,PKCη) are independent of Ca2+
IP3 /DAG

30.
 Epinephrine (α1)
 Acetylcholine (M1,M3)
 Angiotensin
 GnRH
 GHRH
 Oxytocin
 TRH
 PDGF
IP3 /DAG

31.
• Calcium ions - once they enter the cytoplasm exert allosteric
regulatory effects on many enzymes and proteins.
• Calcium acts as a second messenger by indirect signal
transduction pathways such as via G protein-coupled
receptors.
Calcium as a 2nd Messenger

32.
• 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+.
• Signaling occurs when the cell is stimulated to release calcium ions
(Ca2+) from intracellular stores, and/or when calcium enters the cell
through plasma membrane ion channels.
Calcium as a 2nd Messenger

33.
• sudden increase in the cytoplasmic Ca2+ level up to 500–1,000
nM by opening channels in the endoplasmic reticulum or
the plasma membrane.
• Phospholipase C pathway – IP3 & DAG
• Eicosanoids
Calcium as a 2nd Messenger

34.
• Many of Ca2+-mediated events occur when the released
Ca2+ binds to and activates the regulatory protein calmodulin.
• Calmodulin may activate calcium-calmodulin-
dependent protein kinases, or may act directly on other
effector proteins.
• Besides calmodulin, there are many other Ca2+-binding
proteins such as troponin C that mediate the biological effects
of Ca2+.
Calcium as a 2nd Messenger

35.
Calcium as a 2nd Messenger

36.
38
Calmodulin Targets
• Adenylate cyclase
• Phosphodiesterase
• Myosin light chain kinase
• Calmodulin-dependent kinases
• Calcineurin (a phosphatase)

37.
This class of lipids act as signaling molecules that bind to cell surface
molecules.
They include: PROSTAGLANDINS
PROSTACYCLIN
TROMBOXANES
LEUKOTRIENES.
The eicosanoids are rapidly broken down and therefore act in autocrine or
paracrine pathways. They stimulate a variety of responses in their target
cells, including blood platelet aggregation, inflammation, and smooth muscle
contraction.
EICOSANOIDS

38.
EICOSANOIDS

39.
Direct pathway
 Serotonin(5HT2)
 Glutamate(mGLUR1)
 Fibroblast growth factor β
 IFN α
 IFN γ
Indirect pathway
 Dopamine(D2)
 Adenosine(A1)
 Norepinephrine(α2)
 Serotonin(5HT1)
EICOSANOIDS

40.
• Caffeine and methylxanthines inhibit cAMP
phosphodiesterases
• Thus prolong cellular response mediated by cAMP and PKA
Applied

41.
• Cholera toxin – ADP ribosylation- Gαs
• Pertussis toxin – ADP ribosylation- Gαi
Applied

42.
• McCune-Albright syndrome
- A somatic mutation that constitutively activates the Gαs in a mosaic
pattern
- Excess cAMP
- Characteristic triad
i. Variable hyperfunction of multiple endocrine glands, including
precocious puberty in girls
ii. Bone lesions
iii. Pigmented skin lesions (café au lait spots)
Applied

43.
Huntington’s disease
• Disturbance of CREB protein in the brain can contribute to the
development and progression of Huntington’s disease.
• Autopsied brains of those who had Huntington's disease have
been found to have incredibly reduced amounts of CREB
protein
Applied

44.
Applied
Angina pectoris
Nitroglycerine
↓
Nitric oxide
↓
cGMP
↓
Protein kinase G
↓
Relax smooth muscle in coronary arteries

45.
Huntington’s disease
• Mutant Htt→ IP3 receptors more sensitive to IP3 → increased
release of Ca2+ from ER→increase in cytosolic and
mitochondrial concentration of calcium→ Ca2+ induced
degeneration of GABAergic medium spiny neurons
Alzheimer's Disease
• Familial – mutation of PS1,PS2,APP genes →increased IP3
mediated calcium release
Applied

46.
Eicosanoids are synthesized from arachidonic acid. The first enzyme involved
in their synthesis (cyclooxygenase, COX) is the target of ASPIRIN.
Aspirin actions:
-reduces inflammation and pain (inhibition of prostaglandins)
- reduces platelet aggregation and blood clotting (thromboxanes)
Applications:
- prevention of stroke
AA
COX aspirin
P
T
Applied

47.
THANK YOU

48.
References
• Berne & Levy - Physiology, 6th Edition
• Boron & Boulpaep - Medical Physiology, 2nd
Edition
• William’s textbook of Endocrinology,10th edition
• Internet references