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SECOND MESSENGERS
Presenter : Dr.Anu Priya J
SCHEME
• History• Introduction• Types• cAMP Pathway• cGMP Pathway
• IP3 / DAG Pathway
• Calcium as a second messenger• Eicosanoids• Applied aspects
HISTORY
• Earl Wilbur Sutherland Jr. – 1971 Nobel Prize in Physiology or Medicine
• Epinephrine – liver - glycogen to glucose – cAMP
• Martin Rodbell & Alfred G. Gilman – 1994 Nobel Prize
HISTORY
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.
Plasma membrane
EXTRACELLULARFLUID
CYTOPLASM
Reception Transduction Response
Receptor
Signalingmolecule
Activationof cellularresponse
Relay molecules in a signal transductionpathway
321
Earl Sutherland – cell signaling- 3 processes
• 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
TYPES
• Hydrophobic molecules
-Membrane associated
-Phosphatidylinositol , Diacylglycerol
• Hydrophilic molecules
-Cytosolic
- cAMP, cGMP, inositol triphosphate,Ca2+
• cAMP pathway
• cGMP pathway
• IP3 / DAG pathway
• Calcium as a second messenger
• Eicosanoids
Second Messengers
11
Second Messengers
• General characteristics– Low amounts in resting state– Regulated synthesis– Regulated destruction– Act through other proteins
Hormone receptor
GDP
G protein
GTP
Adenylcyclase
cAMP
ATP
cAMP pathway
cAMP pathway
cAMP-dependent Protein Kinase A
C
R R
C
+ 4 cAMPC
C R
R
cAMP
cAMP
cAMP
cAMP
+ The catalytic subunit is now free to attack a protein target.
cAMP pathway
Active phosphorylaseKinase
Inactive PhosphorylaseKinase
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.
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 dependentprotein kinase
Phosphorylasekinase 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
cAMP pathway
• 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
cyclic AMP mediated response
Epinephrine (α2,β1,β2)
Acetylcholine(M2)
NorepinephrineGlucagonSomatostatin ACTHCRHADH
FSHhCGLHMSH PTHTSHAngiotensin IICalcitonin
cGMP Pathway
Ligand-receptor
Guanyl cyclase
GTP cGMP
Protein kinase G Active protein kinase G
Effects
PDE
GMP
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• smooth muscle relaxation• vision• ANP,NO
cGMP Pathway
• cGMP Pathway in phototransduction
cGMP Pathway
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
IP3 /DAG
LIGAND RECEPTOR
↓
G protein
↓
Phospholipase C
↓
PIP2 IP3 + DAG
↓
Endoplasmic reticulum opening of Ca channels
↓
Ca++
↓
Protein kinase C Effects
G protein
EXTRA-CELLULARFLUID
Signaling molecule(first messenger)
G protein-coupledreceptor
Phospholipase C
DAG
PIP2
IP3
(second messenger)
IP3-gatedcalcium channel
Endoplasmicreticulum (ER)
CYTOSOL
Ca2
GTP
Figure 11.14-1
Figure 11.14-2
G protein
EXTRA-CELLULARFLUID
Signaling molecule(first messenger)
G protein-coupledreceptor
Phospholipase C
DAG
PIP2
IP3
(second messenger)
IP3-gatedcalcium channel
Endoplasmicreticulum (ER)
CYTOSOL
Ca2
(secondmessenger)
Ca2
GTP
Figure 11.14-3
G protein
EXTRA-CELLULARFLUID
Signaling molecule(first messenger)
G protein-coupledreceptor
Phospholipase C
DAG
PIP2
IP3
(second messenger)
IP3-gatedcalcium channel
Endoplasmicreticulum (ER)
CYTOSOL
Variousproteinsactivated
Cellularresponses
Ca2
(secondmessenger)
Ca2
GTP
IP3 /DAG
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• 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
• 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
Epinephrine (α1) Acetylcholine (M1,M3) Angiotensin GnRH GHRH Oxytocin TRH PDGF
IP3 /DAG
• 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
• 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
• 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
• 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
Calcium as a 2nd Messenger
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Calmodulin Targets
• Adenylate cyclase
• Phosphodiesterase
• Myosin light chain kinase
• Calmodulin-dependent kinases
• Calcineurin (a phosphatase)
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 musclecontraction.
EICOSANOIDS
EICOSANOIDS
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
• Caffeine and methylxanthines inhibit cAMP phosphodiesterases
• Thus prolong cellular response mediated by cAMP and PKA
Applied
• Cholera toxin – ADP ribosylation- Gαs
• Pertussis toxin – ADP ribosylation- Gαi
Applied
• 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
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
Applied
Angina pectoris
Nitroglycerine
↓
Nitric oxide
↓
cGMP
↓
Protein kinase G
↓
Relax smooth muscle in coronary arteries
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
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
THANK YOU
References
• Berne & Levy - Physiology, 6th Edition
• Boron & Boulpaep - Medical Physiology, 2nd Edition
• William’s textbook of Endocrinology,10th edition
• Internet references