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).

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

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: 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.

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 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.

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 ???

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

INACTIVATION

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-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).

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

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

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

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

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

IP3 ROLE in PATHOPHYSIOLOGYHUNTINGTON’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.

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

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

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.

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.

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

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.

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 (calsequestrin-1)

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.

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 OXIDEHISTORY

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 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

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 /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.