bruno (L12)

Question 1

explain purigenic signaling

Answer 1

intercellular signalling involving purine based molecules

purines: adenine, atp, adp, theobromine, caffeine, adenosine etc

Question 2

conversion from atp to adenosine

Answer 2

When you lose Phos from atp and you get conversion to adenosine → disadvantageous to the cell, turns off the energy demanding processes (adenosine does this)

Question 3

purigenic nerves

Answer 3

nerves that use purines as a neurotransmitter

Atp can be used as a neurotransmitter

Question 4

necessities to consider a molecule as a neurotransmitter

Answer 4

formation and storage of ATP
release of purine nucleotides
direct actions of purine nucleotides and nucleosides on smoot muscle
inactivation of ATP
antagonism and potentiation of responses to nerve stimulation and ATP

Question 5

purine production, release, metabolism and receptors

Answer 5

DIAGRAM IN L12 S5

Atp released from cells by endocytosis
Stored in presynaptic vesicles
Found in extracellular space, can activate receptors (like P2x and P2y), gets converted in extracellular space to give rise to different molecules (adp, amp or adenosine)

Interconversion of amp and adenosine (adenosine can be phosphorylated to give rise to AMP)

adenosine kinase by turning adenosine into AMP can create an inwards gradient for adenosine uptake
This produces a gradient for the atp to leak out of the cell
Equilibrative transporter pump, pumps atp down its concentration gradient

Question 6

why is purinergic signalling complex?

Answer 6

multiple receptors and signalling mechanisms

Atp activates P2X receptors
7 diff subunits in P2X (forms a polymer)
P2Y receptors are activated by many diff ligands
8 diff types of P2Y receptors (some are better activated by atp, some by adp, some by glucose)
Endogeneous ligands for P2Y receptors
Adenine receptor called P0
Adenosine receptor is P1 (4 diff types of P1)

Question 7

adenosine as a drug

Answer 7

purine receptors can be used as drug targets

Adenosine used to treat heart diseases

Question 8

ATP as a neurotransmitter in muscles

Answer 8

ATP is a neurotransmitter at AUTONOMIC NEUROEFFECTOR JUNCTIONS

Twitches in the muscle - excitatory junctions in the muscle
Alpha beta methylene ATP will desensitises the p2x receptors for reduction of size of these responses
Increase conc for more reduction in size
High conc you get an even greater reduction in response to nerve stimulation

Small peaks (bottom line) are spontaneous release of atp from synaptic vesicles
Disappear when you desensitise the receptors of atp (P2X)

EVIDENCE THAT ATP RELEASE IS USED AS A NEUROTRANSMITTER

Diagram on top right - varicosities with larger vesicles having neuropeptides and smaller vesicles having atp
Activate the receptors - ion channel gives rise to excitatory junction potential which can increase the influx of calcium and lead to smooth muscle contraction

Spontaneous twitching (excitatory)
Stimulation of the nerve stops activity - AP is occuring in the muscle, it is spontaneously twitching. If you stimulate the nerve, you get a hyperpolarization of the membrane and stop the twitching and contraction

Question 9

identification of P2X mediated synaptic transmission in the medial habenula of the brain

Answer 9

Recordings of synaptic transmissions at medial habenula
Apply diff compounds
When we block nicotinic receptors, they wont have any effect
Diff compound that affects the p2 receptors allows us to inhibit the actions of the agonists on these receptors
This suggests that the receptors that mediate this response are P2X and P2Y receptors

Purinergic transmitters are found in the hippocampus
Block glutamatergic transmission (synaptic transmission mediated by glutamate receptors) to discover the component of synaptics transmission that are sensitive to P2 receptor antagonists
so he showed that in the hippocampus and the cortex we find synaptic transmission mediated by the activation of p2x receptors

Question 10

ATP P2X receptors

Answer 10

ATP P2X RECEPTOR FAMILY
trimeric, so 3 subunits join to form an ion channel
both N and C terminals of the polypeptide chain is in the intracellular space

GLUTAMATE RECEPTOR FAMILY
GABA receptors are also part of this family, also form a receptor when its 4 subunits come together so is tetrameric
has the N terminal extracellular / C terminal intracellular

NICOTINIC RECEPTOR SUPERFAMILY
Nicotinic acetyl receptors - pentameric, 5 subunits join to form an ion channel
5HT3 receptors are ionotropic, also pentameric

—-»> In all these receptors, TM2 (transmembrane 2) actually creates the ion channel

Question 11

molecular architecture of trimeric ATP P2X and its ion access routes

Answer 11

Ion permeable pathway through the receptors to the pore of the hP2X4R

These ionotric receptors are like drain pipes through the membrane (from top to the bottom, orange in A)
Nicotinic receptors instead have a very ion dense area at the bottom so the ions would flow out through the sides

Fenestrations for the ions to flow in could be from the sides (orange in B)

Question 12

purinergic signalling to cause analgesia

Answer 12

ANALGESIA VIA P2X ANTAGONISTS OR A1 AGONISTS

purinergic signalling can take place in the peripheral and central pain pathways

Inject atp into someone’s skin will stimulate p2x receptors
If you use a1 agonists can induce analgesia
Atp activates the p2x receptors
Stimulate pain sensing fibres
Conversion of atp to adenosine to bind to a1 receptors

Question 13

chronic cough

Answer 13

Use of P2X3 antagonists to suppress the coughing effectively
Chronic cough condition - overstimulation of p2x3 receptors

Question 14

P2X7 receptor in pain and cell death

Answer 14

Shows improvement in abdominal pain and in general wellbeing
Useful in this one condition

P2X7R when activated under normal conditions doesn’t cause anything
Activated with very high concentrations of atp - this is when these pathological conditions take place
Sodium or potassium influx can stimulate cell death cascade
A property of p2x7r - properties of the receptor change with increased stimulation which gives rise to more ionic influx - coupled to another receptor through which more ions can flow

Not likely to dilate, conductance is quite high
Coupling to another protein can mediate additional ionic influx

Question 15

other functions of the P2X7 receptor

Answer 15

P2X7 receptors are unusual because they have a cytoplasmic ballast
this large protein entity on the c-terminal site of the receptor does not have a clear function

it is not an ion permeation pathway because it looks like the ions are getting through defenestrations on the side of the receptor
receptor is regulated for when it is is palmitoylated
so it does not desensitize in the presence of ATP
so when you remove the palmitoylations, then you get a desensitization receptor

Question 16

diversity of G protein coupled P2Y receptors

Answer 16

NATURAL LIGANDS AND SIGNALLING MECHANISMS

there are 8 p2y receptors which are bound by various agonists only some of which have ATP as the primary antagonist while the rest have a ADP or UTP or UDP or UDP-glucose
they are coupled to various g alpha subunit

Question 17

platelet aggregation

Answer 17

important in wound healing, haemostasis BUT could cause instead thrombosis in atherosclerosis

unnecessary and spontaneous platelets aggregation will cause blood clots which will increase blood pressure and cause heart diseases as well as strokes

compounds to treat platelets aggregation is called clopidogrel
this is a a different aside molecule which targets P2Y12 receptors

Question 18

all adenosine P1 receptors are GPCRs

Answer 18

TABLE IN L12 S22

Question 19

Adenosine as a cardiac “retaliatory metabolite”

Answer 19

adenosine has effects designed to detect the heart

Dilation of blood vessels by adenosine
Formation of new blood vessels around the heart
Affects the energy demands so reduces the heart rate, health has less demand on atp
Protection of the heart during a heart attack
Inhibits inflammation
Adaptation to a heart attack for any future heart attacks like increasing the number of blood vessels around the heart

Deflections (right) recorded from sinoatrial node - in 2 heart rate is delayed because of the use of adenosine

Question 20

adenosine A1 receptors in the control of cardiac dysrhythmias

Answer 20

Used in the clinical for supraventricular tachycardia
Spread through the heart to coordinate contractions of the ventricles and atria
But in supraventricular tachycardia, the increase of the heart rate is initiated not at the SAN but supraventricularly, so is ECTOPIC LOCUS OF RHYTHM GENERATION OF THE HEART (because NOT from atrial node)
Leads to very high and irregular heart rates

Black bar is adenosine injection - conversion from tachycardia to normal rhythm

Question 21

adenosine release - during seizure activity

Answer 21

Epileptic seizures releases more adenosine from the brain
Hippocampus is affected by epilepsy

before patients have a surgery to remove the hippocampus they inserted a fine tube into the hippocampus to sample the solution of extracellular fluid in the hippocampus
they detected a large increase of adenosine concentration during a seizure

Question 22

adenosine release - studied in a rodent’s brain tissue

Answer 22

They stimulated seizure activities in the tissue (red dotted bars)

you get a strong elevations of adenosine (measured by sensors)
so they tried to suppress the electrical activity of the brain
this study allows us to know that adenosine is being released as adenosine and not as ATP

ATP is being metabolized to adenosine and that is not being replaced quickly enough because of the intense activity
so adenosine is leaking out

Question 23

adenosine release - a natural anticonvulsant

Answer 23

Acting as a naturally occurring anticonvulsants
Control shows equal intervals between synaptic transmissions every 15 seconds

Adding an antagonists of these A1 receptors, being CTP, the blocking of these receptors will turn the non seizing tissues into tissues that’s a lot of seizure activity
we can make these very electrically evoked seizures much longer and more intense

this tells us that under normal conditions adenosine is acting as am anticonvulsant

Question 24

adenosine kinase - major regulator of extracellular adenosine concentration

Answer 24

adenosine kinase takes adenosine and turns it into AMP
this maintains an inward gradient for adenosine from outside the cell to the inside
we know this happens because if we poisoned the enzyme adenosine kinase with the compound iodotubercidin, we will see an increase in extracellular adenosine
so this prevented the uptake of adenosine into the cell so it is accumulating in the extracellular space
this will inhibit synaptic transmission so it is blocking the release of glutamate from presynaptic terminals

Question 25

astrogliosis and increased ADK as a response to seizures

Answer 25

in chronic epilepsy you get astrogliosis where you get a lot of these astrocytes
so there is a proliferation of these astrocytes in epileptic tissue
this is found in rodents and in humans hippocampus
hippocampus sclerosis is the scarring of the hippocampus tissue

Question 26

activity of adenosine kinase can influence seizures

Answer 26

this can regulate your response to seizure activity
if you inhibits adenosine kinase with iodotubercidin, it will elevate adenosine and act as an anticonvulsant to prevent a seizure activity
you can then show that this effect is due to adenosine A1 receptors