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PH2501- pharmacology > Lecture 3- secondary messengers > Flashcards

Lecture 3- secondary messengers Flashcards

1
Q

cAMP- discovery

A
  • First example of a second messenger
  • Discovered in 1957 by Earle Sunderland
  • Prototype of all signalling molecules
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2
Q

Main features of cAMP

A
  • Present at low concentrations in resting cells by adenylate cyclase
  • Produced rapidly in response to agonist
  • Binds to regulatory subunits of protein kinase A (PKA), activating the catalytic subunits. PKA phosphorylates many targets on serine/threonine residues (enzymes, receptors, ion channels, transcription factors)
  • cAMP destroyed by phosphodiesterase
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3
Q

Manipulation of cAMP

A

1) ATP (stimulated by forskolin) reacts with adenylate cyclase to form cAMP
2) cAMP binds to PKA (this has 4 subunits:2 regulatory; 2 catalytic) with the regulatory binding to the catalytic units to render them inactive. cAMP binds to regulatory sub-units and falls off. The catalytic subunits then change shape and activate. (dibutryl cAMP is an analogue that is lipid soluble which cAMP isn’t that mimics cAMP’s action)
- RpcAMPs is a competitive antagonist, stops cAMP binding site
- H89 Non-competitive antagonist
3) Phosphodiesterase is an enzyme which splits an ester bond at the phosphate section causing cAMP -> AMP
- Isobutyl methyl xanthine, papaverine, caffeine all inhibit the breakdown of cAMP can be used clinically as muscle relaxants e.g. asthma

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4
Q

Does a drug act via cAMP

A
  • The agent should increase cAMP
  • It should be mimicked by dibutyryl cAMP and forskolin (increases adenylate cyclase production of cAMP)
  • It should be blocked by inhibitory of PKA
  • It should be potentiated by inhibitors of phosphodiesterase (IBMX)
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5
Q

Actions of cAMP

A
  • 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
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6
Q

Glycogen metabolism

A

1) PKA (R2C2), cAMP then binds to the regulatory units–> 2R cAMP and 2C (active)
2) 2C phosphorylate, phosphorylase kinase (4 subunits alpha, beta, gamma, delta) –> the alpha and beta units get phosphorylated causing the enzyme to be active
3) phosphorylase kinase then phosphorylates glycogen phosphorylase causing this to become active
4) activated glycogen phosphorylase then turns glycogen –> glucose-1-phosphate
- This is a protein kinase cascade

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7
Q

Why are kinase cascades important

A
  • AMPLIFICATION: 1 molecule of phosphorylase kinase activates many glycogen phosphorylase molecules
  • CONTROL: Multiple messenger system, this means we can get very rapid production of glucose when we need it
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8
Q

Phosphoinositides

A
  • Membrane phospholipids base on inositol
  • Metabolised to yield several second messengers and signalling molecules
  • Stimulates Ca2+ increases (IP3), protein kinase C (DAG) and tyrosine kinase (PIP3)
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9
Q

Basic structure of phosphoinositol

A
  • Fatty acid
  • Glycerol
  • Phosphate
  • Phosphoinositol (2 phosphate groups)
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10
Q

Phosphatidylinositol-4,5-diphosphate (PIP2) hydrolysis

A
  • Many hormones act through Ca2+, but this is stored in cytoplasmic stores, not physically connected to the plasma membrane
  • Thus the hormones must produce a second messenger that releases Ca2+ from these stores
  • The messenger is inositol-1,4,5-trisphosphate (IP3), made from PIP2 hydrolysis
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11
Q

Hydrolysis of PIP2 to give IP3 and diacylglycerol (DAG)

A
  • PIP2 is cleaved by phospholipase C at the phosphate-glycerol
  • Fatty acids and glycerol is called DAG
  • AND IP3 (cyclic 6 C’s with 3 -OH and 3 P’s)
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12
Q

Metabolism of IP3

A

-Ins -> PI -> PIP -> PIP2-> Ins (1,4,5) P3 -> Ca2+ release
-PIP2 -> DAG -> PKC
-Ins (1,4,5) P3 -> Ins(1,3,4,5)P4 -> Ins(1,3,4)P3 ->InsP2 ->InsP -> Ins
+This is done to replenish Ca2+ stores, by making IP4 then creating IP3 (1,3,4) this IP3 is biologically inert so won’t cause release of Ca2+
-Last process can go straight from Ins(1,4,5)P3 ->InsP2 etc to form Ins.

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13
Q

PIP3

A
  • PtdIns(4,5)P2 can be phosphorylated by phosphatidylinositide-3-kinase (PI3-K) to give PIP3
  • This remains membrane-associated but can activate a number of kinases and scaffolding proteins (act like glue, help to build complexes)
  • Both PIP2 and PIP3 can act as docking molecules, attracting signalling proteins that have the appropriate recognition domains (e.g. pH domains) to the plasma membranes
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14
Q

Ca2+

A
  • Activates ion channels, kinases, metabolic enzymes structural proteins etc
  • Involved in membrane potential changes, secretion (neurotransmitters, enzymes, fluids, etc), intermediary metabolism, apoptosis, shape changes, cell division and differentiation
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15
Q

Calmodulin

A
  • Intracellular Ca2+ receptors, 16Kda, binding 4 Ca2+ ions with a low microM affinity
    -When a cell is activated Ca2+ increases by about 1mcM (any more leads to toxicity), causing Ca2+ to bind
    -When Ca2+ binds it straightens out (2 Ca2+ sites at each end and a long rod/ helix in the middle)
    -
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16
Q

Calmodulin Kinase

A
  • Activates Ca/calmodulin kinase I and II
  • CAMKII is involved in regulation of receptor density in synapses (memory)
  • CAMKII undergoes autophosphorylation so that it remains active even after the Ca2+ concentration has returned to basal levels
17
Q

Calcineurin

A
  • Protein phosphates (PP2B) activated by CAM (Calmodulin)
  • Dephosphorylates the transcription factor NFAT
  • NFAT causes cytokine production, leading to an immune response
  • Cyclosporin binds to the protein cyclophilin to inhibit calcineurin
18
Q

Protein Kinase C

A
  • Multimember family
  • Serine/threonine kinase usually activated by PtdIns (4,5)P2 breakdown
  • All members of the family need phosphatidylserine (PS) and diacylglycerol (DAG) or a free fatty acid (arachidonic acid) for activity
  • All activated in the plasma membrane
  • Ca2+ needed by some members
19
Q

Activation of PKC

NB LOOK OVER AGAIN

A
  • Removal of Ca2+ and DAG; Downregulation by proteolysis ->
  • PKCcytosolic –(Ca2+ via PIP2 breakdown)–>
  • PKC.Ca2+ mem –>
  • PKC.Ca2+.DAG mem (active)
20
Q

Properties of NOS isozymes

A
  • TYPE I: Cerebellum (neuronal); nNOS; Constitutive; Regulates
  • TYPE II: Immunologically activated (macrophages);iNOS; inducible; No effect
  • TYPE III: Vascular endothelial cells; eNOS; Constitutive; Regulates
  • All forms of NOS need calmodulin to bind to become active, this will only happen if there is an increase in calcium
  • However, if iNOS it has a v.high affinity for calmodulin, therefore, it doesn’t matter if there is Ca2+ present, NO will be produced with iNOS
21
Q

Biochemistry of NO

A
  • The biological activity primarily due to reaction with (haem) Fe.
  • The most important target is guanylate cyclase which converts to GTP to cGMP
  • cGMP activates ion channels, protein kinases, inhibits or stimulates cAMP phosphodiesterase and may produce cADP-ribose (Ca2+ release)
22
Q

Physiology of NO

A
  • 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 BP and tissue perfusion (cardiac perfusion, erectile tissue). Inhibit platelet aggregation and adhesion
  • Cell defence mechanism. Production stimulated by bacterial endotoxins and cytokines
23
Q

Ca2+ elevation

A
  • Excitable cells, via voltage gated ion channels
  • From IP3-releasable Ca2+ stores
  • From other internal stores (e.g. sarcoplasmic reticulum, cNADPH)
  • Via store-operated channels (IP4)
  • Via receptor gated ion channels (NMDA receptors)
24
Q

Ca2+ homeostasis

A

1) Ca2+ enters through voltage and ligand gated ion channel;s; 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 (i.e. Na entry down its conc gradient provides energy for Ca2+ removal against conc gradient)
4) Blockers of these processes increase intracellular Ca2+ and can lead to inappropriate cell activation or cell death. Release of Ca2+ from mitochondria is a major means of cell death in apoptosis

25
Q

Actions of PKC on Ca2+ homeostasis

A
  • PKC is activated by Ca2+
  • PKC can reduce Ca2+ release into cells
  • Receptor couples with Gq protein (G-protein which activates phospholipase C)
  • PKC when active can stick phosphate groups on receptor causing it be desensitised, and so can no longer recognise G-protein so no production of phospholipase C
  • PKC also stimulates phosphatase which act to turn IP3 into IP2 which will reduce Ca2+ release
  • IP3 binds to channels on endoplasmic reticulum to release Ca2+, PKC can phosphorylate these channels meaning that IP3 is less able to release Ca2+
  • PKC phosphorylates Ca/ATPase and Na,Ca2+pump causing an increase in activity
26
Q

Nitric oxide

A
  • NO is small, gaseous lipophilic signalling molecule, first identified as a component of endothelial-derived relaxing factor (EDRF) in blood vessels
  • Can function as second messenger a neurotransmitter and an autocoid
  • Involved in physiological and pathological processes
27
Q

Biosynthesis of no

A
  • Nitric oxide synthase (NOS) has 2 blocks: the enzyme part which takes O2 + arginine= NO + Citruline
  • The activity of NOS is modulated by calmodulin (Ca2+ for eNOS and nNOS)
28
Q

Toxicity of NO

A
  • NO is a free radical; it can react with the superoxide anion to give peroxoynitrite and then hydroxyl radicals. The latter are very reactive and toxic
  • it can also bind to non-harm Fe in proteins irreversible leading to destruction of the proteins. Many of these are in the mitochondria, leading to cell death

PH2501- pharmacology (47 decks)

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