Second messengers

What us cAMP?

• First messenger 

• Discovered in 1951 by Earle Sutherland 

What are the main features of cAMP?

• Present at low cons in resting calls by adenylate cyclase 

• Made rapidly when there's agonists 

• Binds to regulatory subunits PKA 

• Destroyed by phosphodiesterase - overproduction kills

What happens when protein kinase A binds to cAMP?

Activates catalytic subunits 

• Phosphorylates targets on serine/threonine residues

Residues = enzymes, receptors, ion channels, TFs

What happens to cAMP in the body?

How do you determine whether a drug acts via cAMP?

• Agent ↑ cAMP 

• Mimicked by dibutyryl cAMP and forskolin 

• Blocked by inhibitors of PKA 

• Potentate by phosphodiesterase inhibitors (IBMX)

What are the actions of cAMP?

• Inhibition of smooth muscle contraction 

• Stimulation of Ca²⁺ pump 

• Activate cardiac Ca²⁺ channels 

• Activation of potassium channels 

• Uncoupling of G-proteins from receptors 

• Stimulation of protein synthesis 

• Increased glycogen metabolism 

Glycogen metabolism cascade

What are phosphoinositides?

Membrane phospholipids based on inositol

• Metabolised to yield several 2^nd messengers and signalling molecules

• Stimulates Ca²⁺

Increases IP₃, protein kinase C (DAG) and tyrosine kinases (PIP₃)

• Diacylglycerol

Why do hormones need secondary messengers?

Hormones need a 2^nd messenger to release Ca²⁺ from cytoplasmic stores

• Inositol 1,4,5 trisphosphate (IP₃) made from phosphatidylinositol 45 bisphosphate hydrolysis (PIP₂)

IP₃ metabolism

Why is IP₄ made?

Stores that can release IP₃ have small amounts of Ca²⁺

• External Ca²⁺ is needed

• IP₄ binds to a Ca²⁺ channel to ↑ store

What are docking molecules?

They attract signalling proteins that have the right recognition (pH) domains to the plasma membrane

• PIP₂ and PIP₃

How is PIP₃ made?

PtdIns(4,5)P₂ can be phosphorylated by phosphatidylinositide-3-kinase (PI3-K) to give PtdIns(3,4,5)P₃, or PIP₃

• Stays membrane associated but can activate some kinases and scaffolding proteins

What are the roles of calcium ions?

• Activates ion channels, kinases, metabolic enzymes, structural proteins

• Membrane potential changes, secretion, shape changes, cell division, differentiation and intermediary metabolism apoptosis

Where can calcium ions be found?

• Excitable cells, via voltage gated channels

• From IP3 releasable Ca²⁺ stores

• From other internal stores (eg sarcoplasmic reticulum, cNADPH)

• Via store-operated channels (IP4)

• Via receptor-gated ion channels (NMDA receptors)

What is calmodulin?

Globular protein, intracellular Ca²⁺ receptor, 16Kda, binding 4 Ca²⁺ ions with low µM affinity

How does calmodulin work?

• Ca2+ binds

• Changes shape

• Effector binding site for other proteins

What is calmodulin kinase (CAMK) and specifically CAMKII?

• Activates Ca/calmodulin kinase I and II

CAMKII - regulation of receptor density in synapses (memory)

• Phosphorylation so it stays active after Ca levels go back to normal

1. Activation CAMKII

2. Nerves more closer

3. Stronger synapse

4. More communication

What activates protein phosphatase (PP2B) and what happens?

CAM activates it

• Dephosphorylation of TF NFAT

- Activates NFAT as there’s no P to stop it from going into nucleus to bind

• Cause cytokine production leading to immune response

Why is PP2B inhibition important?

Inhibited by cyclosporin

• Stops the body immune system from attacking transplant organs by binding to cyclophilin

Describe Ca²⁺ homeostasis

1) Ca²⁺ enters through voltage and ligand gated ion channels; from intracellular and extracellular stores.

2) It can be removed back into the stores, into mitochondria or out of the cell across the plasma membrane.

3) Removal is via Ca²⁺ATPase (Ca²⁺ pump) or Na+/Ca²⁺ exchange (ie sodium entry down its concentration ground provides the energy for Ca²⁺ removal against its concentration gradient.

4) Blockers of these processes increase intracellular calcium and can lead to inappropriate cell activation or cell death. Release of Ca²⁺ from mitochondria is a major means of cell death in apoptosis

Describe protein kinase C

• Multimember family

• Serine/threonine kinases usually activated by PtdIns(4,5)P₂ breakdown.

• All members of the family need phosphatidylserine (PS) and diacylglycerol (DAG) or a free fatty acid (eg arachadonic acid) for activity.

• Ca²⁺ needed by some members

• Can phosphorylate GPCRs

Protein kinase C activation

How does protein kinase C affect calcium ion homeostasis?

• Reduces receptor activity

• Negative feedback loop

What is nitric oxide?

Small, gaseous lipophilic signalling molecule

• Endothelial-derived relaxing factor in blood vessels

• Functions as a 2^nd messenger, NT and autocoid (locally released factor)

• Physiological and pathological processes

Biosynthesis of nitric oxide

Describe the properties of the 3 types of NOS isozymes

Describe nitric oxide biochemistry

• Reactions with iron

• Main target is guanylate cyclase (converts GTP to cGMP)

What does cGMP do?

• Activates ion channels, protein kinases, inhibits

• Stimulates cAMP phosphodiesterase

• May produce cADP-ribose (Ca²⁺ release)

Describe the toxicity of nitric oxide

• Free radical

  • Reacts with superoxide anion to give peroxoynitrite and hydroxyl radicals (reactive and toxic)

  • Bind to non-haem iron in proteins, leads to destruction and cell death (many are in mitochondria)

What are the roles of nitric oxide and its effect on the body?

• Muscle relaxant; cGMP activates protein kinases and ion channels causing decreased contractility and hyperpolarisation.

• Non-adrenergic, non-cholinergic (NANC) transmitter. In the CNS involved in long term potentiation and inhibition (LTP, LTD).

• From endothelial cells regulates blood pressure and tissue perfusion (cardiac perfusion, erectile tissue). Inhibits platelet aggregation and adhesion.

• Cell defence mechanisms. Production stimulated by bacterial endotoxins and cytokines

Describe the pharmacology of nitric oxide

1. NO donors–Nitro-vasodilators; Sodium nitroprusside

2. NOS inhibitors– L-NG-nitroarginine

3. GC inhibitors methylene blue

4. PDE inhibitors (Type V specific)–IBMX; Dipyridamole; Zaprinast; Sildenafyl (Viagra)