endocannabinoids Flashcards
what are endocannabinoids
1)N-arachidonoyl ethanolamide
(anandamide or AEA) – partial agonist
2) 2-arachidonoyl glycerol
(2-AG) – full agonist
both agonists for CB1 and CB2 receptors
AEA is a bit more like THC in the sense that it is a partial agonsit whereas glycerol is a full agonist and more efficacious
both developed from arachodonic acid
endocannabinoids exist better in aqueuous environments both in and out of the cell better than THC or synthetic cannabis
Describe the biosynthesis of endocannabinoids
produced by our body
produced by enzymatic reactions
different enzymatic pathways involved
generally both derived from arachidonoyl acid
ADA particulary derived from a rare part of cell membrane- phosphatidylethanolamine
both activated by calcium
2 AG involves enzymes- DGL
both enzymes sensitive to calcium levels, which could be handy in controlling the levels of these 2 endocannabinoids in the body
some GPCRs that increase calcium can increase the production of ADA by increasing activitiy of relevant enzymes, increasing activation of CB1 receptors
Production of endocannabinoids
Stimuli for de novo synthesis
From phospholipid-derived precursors
Activation of GPCRs, PLC, [Ca2+]i, (depolarization)
Produced from
Neurons, cardiac myocytes, astrocytes, microglia, blood cells, vascular wall, gut etc
Target tissues
Neurons, cardiac myocytes, astrocytes, microglia, blood cells, vascular wall, gut etc
eCBS are not stored, they are synthesised de novo on demand
they are more of a local phenomenon, cells that produce encbs often act on themselves or nearby
collated in blood to some extent but not at high level and their are some enzymes that wouldbreak these down
therefore more local level used rather than in blood
Liquid chromatography mass spec
liquid chromatography
mass spec
measure the amount of a lipid
you have a target organ and are interested in endocannabinoids in that target organ
you can break down the cells, so anything in those cells is released eg. inorganic solvents,alcohol, methanol
the separated chemical groups will be funneled to mass spec
you bombard each chemical with high energy to break them up
detector at the end of this gives you a pic of the chemicals in the sample and the precise levels
you can see there’s a range of endocannabinoid concentrations in different tissue types
ideally you just want to measure ecb levels in a space in the synaptic cleft
not accurate but you can take solution through synaptic cleft and measure concentrations through same mass spec and chromatogrpahy technique
degradation of eCBs
whenever you manage eCB levels it will be balanced by any level of degradation that’s going on
when they are produced, they are taken up by cells
ADA in cells will be hydrolysed by FAAH back to arachidonic acid and ethanolaine
this can then be fed through COX system
so the downstream product could be prostaglandins
2-AG hydrolysed by GL
gives arachidonic acid and glycerol
the enzymes that degrade eCBS are usually inside cells
they might show dif expressions and are complementary locations to presynaptic cb1 receptors
how to enhance CB1 signalling through targeting degradation
experiment that supports how targetting degradation may be useful:
can target FAAH
degrades ADA
if you inhibit ADA you expect it to increase
if you inject in vivo a FAAH inhibitor, ADA increases
this increases CB1 receptor activation but only in areas with high ADA in first place
this helps you be selective over where you enhance CB1 signaling
Describe the anxiolytic role of ADA
can use Striatal slices from wild-type and FAAH KO mice
one area of interest is the anxiolytic role of ADAinhibiting degradation reduces anxiety and increased resistant to depression
use knockout model- replace gene of FAAH with something else
massive accumulation of ADA levels
then measure effects in terms of anxiety levels, using pain sensation
in the FAAH knockout model, mice were more tolerant to pain
showung behaviour of anxiolytic effect
Describe the endocannabinoid system
eCBs act on CB1 and CB2
these modulate tissue functions
you need a balance of production and degradation in both AdA and AG2
they can activate both cb1 and cb2 receptors
modulate tissue function
inhibiting one enzyme may not have the desired effect
you may have to inhibit a few enzymes before you see the desired effect
Describe the short term CB1 response
CB1 receptors act on pre synaptic channels, acting on potassium channels, reducing VGCCs, ultimately reducing calcium signal needed for neutrotransmitter release
Inhibitory effect on neurotransmission release
if this neurotransmitter is GABA, you inhbit an inhibitory signal leading to an overall excitatory effect
if neurotransmitteris excitatory then signal will be dampened
CB1 central and peripheral neurotransmission
central: Glutamate or GABA cortex, substantia nigra, caudate- putamen, hippocampus, amygdala, cerebellum Dopamine retina, caudate-putamen Acetylcholine cortex, hippocampus Noradrenaline retina
peripheral:
Noadrenaline arteries, heart, vas deferens, lung, bone
Acetylcholine intestine, urinary bladder
CGRP trachea, skin, dorsal root ganglia
What is the difference between cannabis and eCBs?
What is the difference between CB1 agonists and eCBs?
the difference is the signalling is more subtle with cannabis or synthetic compounds compared to ecbs
it’s more difficult to activate CB1 in one place compared to another
Describe retrograde signalling via eCBs
eCBs are not stored so not stored and released in the same sense from synaptic vesicles
they modulate levels of neurotransmitter release
generally produced by post synaptic neurones
phytocannabinoids generally go the opposite way
presynaptic neurone releases neurotransmitter, causes calcium increase in post synaptic neurone, stimulates lipid precursors to produce endocannabinoids and this acts on CB1 receptors in the pre synaptic neurone (even tho endocannabinoids are lipophilic some of it can still be released
negative feedback system
it’s a modulatory role with subtle effects
by targetting the inhibitors,
beauty of targeting degradation of ADA is that effect of those enzyme inhibitors would partly depend on the levels of endocannabinoids being used- so can be targetted because not all of neurociriculation has high levels of endocannabinoids all the time- depends on other neurotransimmiters and stimuli on those pre and post synaptic neurones
allows you to target endocannabinoid signalling in specific brain regions rather than blanket activation of CB1 receptors through synthetic cannabinoids
CB1 in the hippocampus
CB1 antagonists block depolarization-induced suppression of inhibition (DSI)
CB1 ↓ spontaneous IPSP of GABAergic interneurons
interneurones have a lot of CB1 receptors
when they are activated you get spikes (on graph)
the black block ont he graph shows excessive stimulation
if you stimulate pyramidal cells too much, you start to inhibit it
interneurones further inactivate pyramidal cells
this is called DSI
pyramidal cells show excitation
interneurones are inhibitory for glutamate release
causes inhibition of inhibitory neurones- mediated by endocannabinoids
endocannabinoids act on CB1 receptors on the interneurones (have a lot of CB receptors) activating GABA release
bottom graphs show that if you inhibit CB1 receptor on interneurones, whole phenmeon doesn’t work
Describe the eCBs system at synapses
Complementary localization of eCB components:
NAPE-PLD and FAAH in postsynaptic neurons
CB1 receptors in presynaptic terminals of interneurons
CB1 receptors located pre synaptically
if you used staining/probes/radioactively you would find CB1 presynaptically in those brain regions
also find enzymes that degrade ADA post synaptically
although CB1 is pre synaptic, the machinery controlling ADA levels like FAAH PLD etc (degradation) are all post synaptic
this retrograde signalling can happen not just for GABA but glutamate neurones as well
AEA vs 2 AG in synaptic plasticity
why do we have 2 or more
because they specialise in different neurotransmitters and synapses
ADA tends to be for glutaminergic transmission, 2AG might be more likely in GABAnergic neurones and synaptic plasticitiy
reason not clear
but in terms of empirical evidence looking through dif in vivo slices- that is the split between the function of both ADA and 2AG
but they both act in retrograde motion
