NO and Purinergic Signalling (A*)

Question 1

List the types of purinergic receptors.

Are these receptors excitatory or inhibitory?

Which ligands do they respond to?

Are these LGICs or GPCRs?

Answer 1

• Classes of purinergic receptors include P1 (for adenosine) and P2 (for ATP and ADP).
• P1 receptors respond to adenosine.
• P1 receptors are all GPCRs.
• P1 receptor subtypes include A1, A2A, A2B and A3 receptors. These are either excitatory or inhibitory depending on subtype.
• P2 receptors respond to ATP and ADP.
• P2 receptors include P2X LGICs and P2Y GPCRs.
• P2X receptor subtypes include P2X1-7. These are all excitatory.
• P2Y receptor subtypes include P2Y1, 2, 3, 6, 11, 12, 13 and 14. These are either excitatory or inhibitory depending on the subtype.

Question 2

What is the structural difference between P2X and P2Y receptors?

Answer 2

P2X receptors are ligand-gated ion channels (permeable to Na+ and Ca2+) whereas P2Y receptors are GPCRs.

Question 3

Give an example of ATP acting as a cotransmitter.

What is the name given to this synergistic function of ATP with its cotransmitted neurotransmitter?

Answer 3

• In the neuromuscular junction, ATP is often coreleased with noradrenaline.
• Noradrenaline binds to alpha 1 receptors, which causes Ca2+ release from intracellular stores via the PKC pathway. This initiates muscle contraction.
• Simultaneously, ATP binds to P2X1 receptors (LGICs), causing Ca2+ influx.
• This Ca2+ influx A) contributes to the muscle contraction and B) resupplies the cell with intracellular Ca2+ stores.
• This synergistic function is known as cooperativity.
• ATP is usually released as a cotransmitter in other synapses as well, e.g. with ACh in the cortex, catecholamines in the hypothalamus, GABA in the dorsal horn, glutamate in the hippocampus and dopamine throughout the CNS.

Question 4

Give an example of presynaptic purinergic receptors.

Are these receptors excitatory or inhibitory?

Answer 4

• Most presynaptic purinergic receptors are P1 and P2Y receptors.
• These presynaptic receptors are usually (but not exclusively) inhibitory.

Question 5

Which enzymes synthesise adenosine?

Which molecule is the precursor to adenosine?

Answer 5

• Endonucleotidases produce adenosine.

- Adenosine is produced from ATP.

Question 6

What is the role of purinergic signalling at glial cells?

Answer 6

• Multiple P1 and P2 receptors are expressed on glial cells, but mostly P2X.
• In glia, purinergic signalling mediates:

1 - Short-term Ca2+ signalling.

2 - Proliferation.

3 - Differentiation.

4 - Cell death.

*See card 19.

Question 7

Which enzyme synthesises nitric oxide?

Form which molecule is nitric oxide synthesised?

List the isoforms of this enzyme.

Answer 7

• Nitric oxide synthase (NOS) synthesises nitric oxide.
• Nitric oxide is synthesised from L-arginine.

Isoforms include:

Constitutive:

1 - eNOS (e for endothelial).

2 - mtNOS (mt for mitochondrial).

3 - nNOS (n for neuronal).

Non-constitutive:

4 - iNOS (i for inducible (in astrocytes and microglia in response to immunological / inflammatory stimulation)).

Question 8

What stimulates nitric oxide release from neurones?

Answer 8

• For eNOS, mtNOS and nNOS, Ca2+ influx stimulates nitric oxide release from neurones.
• Nitric oxide cannot be stored, so nitric oxide release follows nitric oxide synthesis, which is also Ca2+ dependent.
• iNOS activity is regulated transcriptionally.

Question 9

Which neurones release nitric oxide as a neurotransmitter?

List 2 examples of nitric oxide release in the body.

Answer 9

• Most nitric oxide is released by parasympathetic postganglionic neurones. Examples include:

1 - Nitric oxide is released in the corpus cavernosum to produce an erection in the penis (nitric oxide causes vasodilation).

2 - Nitric oxide is released in endothelial cells to cause vasodilation.

Question 10

Give an overview of the nitric oxide signalling pathway.

Answer 10

1 - Nitric oxide increases the activity of guanylyl cyclase.

2 - Guanylyl cyclase converts GTP into cGMP.

3 - cGMP activates PKG (a kinase like PKA, PKC etc.), which has various cellular effects.

Question 11

Give an example of an enzyme that terminates the nitric oxide signalling pathway.

Give an example of a drug that inhibits this enzyme.

Answer 11

• Phosphodiesterases break down cGMP.
• Sildenafil (viagra) is a phosphodiesterase inhibitor (maintains NO signalling in corpus cavernosum - maintains vasodilation - maintains erection).
• There are also guanylyl cyclase inhibitors and inhibitors of nitric oxide synthase, but they’re not as clinically useful.

Question 12

Why is nitric oxide often involved in volume transmission?

Answer 12

Nitric oxide is often involved in volume transmission because it is a small molecule and is therefore able to diffuse long distances.

Question 13

What type of effect does nitric oxide have at neurotransmitter receptors?

Answer 13

• Nitric oxide has a neuromodulatory effect at neurotransmitter receptors.
• Therefore, nitric oxide is often cotransmitted with other neurotransmitters.

Question 14

Big boy A* Material:

Briefly describe a potential purinergic target for Alzheimer’s disease.

How can this target be used to treat Alzheimer’s disease?

Answer 14

• It was found that amyloid-beta, the primary pathological agent in Alzheimer’s, led to the formation of pores in the plasma membrane by a yet unknown process. This was found to result in leakage of ATP.
• This, in turn has been hypothesised to potentiate excitatory synaptic activity via P2X receptors, mostly through P2X7, but also some P2X4, leading to neurone death by excitotoxicity resulting from excessive influx of Na+ and Ca2+. This is confounded by upregulation of P2X7 receptors - a finding in postmortem studies of the brains of Alzheimer’s disease patients. Consistent with this theory, electrophysiological studies have identified increased excitatory activity in neurones following exposure to amyloid-beta.
• It has been found that administration of P2X antagonists restores physiological electrical activity, providing further evidence that the excitotoxicity seen in neurones exposed to amyloid-beta is, in part, mediated by overactivation of P2X receptors.
• Besides Alzheimer’s disease, P2X7 antagonists are also of interest for a number of neurodegenerative and neuroinflammatory diseases, such as ALS, Parkinson’s, Huntington’s and multiple sclerosis, due to their neuroprotective effects. However, nonspecific P2X antagonism, e.g. by suramin, is known to cause numerous side effects due to the ubiquity of the P2X receptor. Hence, a number of P2X7 antagonists with greater specificity for the P2X7 subtype and higher CNS penetrance have been developed over the past decade. To date, only one P2X7 antagonist has entered clinical trials, and was not pursued due to poor safety outcomes. No P2X7 antagonist has been tested in patients with neurodegenerative / neuroinflammatory disorders, therefore the efficacy of these drugs remains unknown. Further development of P2X7 antagonists is therefore warranted.
• Elucidation of the mechanisms underlying formation of the pores will enable the development of pharmacological therapies blocking the pore-forming process, which may lead to the development of more specific therapies with fewer adverse effects.
• Saez-Orellana et al., 2016.

Question 15

Big boy A* Material:

Briefly describe a potential purinergic target for Parkinson’s disease.

How can this target be used to treat Parkinson’s disease?

Answer 15

• A potential purinergic target for Parkinson’s disease is A2A receptors.
• A2A receptors are found abundantly in the basal ganglia, but particularly in striatal medium spiny neurones of the indirect pathway. This is a circuit in the basal ganglia that inhibits movement.
• In the striatum, A2A receptors are colocalized with D2 receptors, with which they form dimers. A2A receptors tonically inhibit D2 receptors - a class of dopamine receptors that inhibit the indirect pathway. Hence, A2A receptor activation is inhibitory to movement as it potentiates activity of the indirect pathway.
• Administration of istradefylline, an A2A receptor antagonist, is associated with a significant improvement in unified Parkinson’s disease ranking scale.
• This could be used in combination with dopamine replacement therapy to reduce the required dose of L-DOPA, which has numerous adverse effects, namely tardive dyskinesia. In clinical trials, istradefylline was well-tolerated and showed a good safety profile, ultimately leading to its approval as an adjuvant therapy for Parkinson’s disease in 2019. Although istradefylline is associated with involuntary motor activity in some patients, this likely offset by the advantage of reducing L-DOPA therapy, which itself poses a risk of L-DOPA-induced dyskinesia.

Question 16

Big boy A* Material:

Describe a potential purinergic target for schizophrenia.

How can this target be used to treat schizophrenia?

Answer 16

• Purinergic hypothesis of schizophrenia: dysfunction in purinergic signalling results in a decrease in adenosine transmission which underlies the imbalance between glutamatergic and dopaminergic transmission (Lara and Souza, 2000).
• The hypothesis posits that dysregulation of glutamatergic signalling arises from dysfunctional A2A receptor activity, which disrupts glutamate homeostasis through disturbed astrocyte-neurone signalling.
• In mice models of schizophrenia, antagonism of neuronal A2A receptors is beneficial for cognitive function but antagonism of astrocytic A2A receptors is detrimental to cognitive function (Matos et al., 2015).
• Purinergic miscommunication has been shown to lead to decreased presynaptic glutamate release, and downregulation of NMDA receptors.
• The purinergic hypothesis also suggests that other neurotransmitters, such as 5-HT, noradrenaline, dopamine and acetylcholine become dysregulated due to defective purinergic signalling. Collectively, these physiological changes are thought to underlie both positive, negative and motor symptoms of schizophrenia.
• Over the past decade, evidence has accumulated to suggest that stimulation of A2A receptors has potential to improve symptoms of schizophrenia. It is well-documented that A2A receptors form functional dimers with D2 receptors in the indirect striatal pathway of the basal ganglia. Here, A2A receptor activation antagonises D2 receptor function. Since schizophrenia is associated with dopaminergic hyperactivity, the therapeutic action of A2A stimulation in schizophrenia may be through attenuation of striatal dopaminergic transmission.
• Stimulation of A2A receptors would, in theory, be particularly useful for treating positive symptoms of schizophrenia. That is, the presence of abnormal thoughts and behaviours such as delusions and hallucinations. In schizophrenia, hyperactivity of dopaminergic transmission at the striatal indirect pathway leads to a disinhibition of thalamus, which receives inhibitory innervation from the basal ganglia in what is known as the cortico-basal ganglia-thalamo-cortical loop. This, in turn, results in a decrease in thalamic sensory gating (opening of filter), causing positive symptoms. The efficacy of A2A receptor stimulation for the treatment of schizophrenia has been investigated in clinical trials:
• Dipyridamole is an adenosine uptake inhibitor. Its primary function is as a vasodilator and anti-platelet drug, however is has also shown efficacy for the treatment of positive symptoms of schizophrenia as an adjuvant therapy (Wonodi et al., 2011). Although showing a good safety profile, dipyridamole is contraindicated in patients suffering from respiratory diseases since it is associated with increased risk of bronchospasm. Furthermore, the vasodilatory effect of dipyridamole effect makes it unsuitable for use in patients suffering from hypotension, and in geriatric patients, dipyridamole use is associated with increased risk of orthostatic hypotension. The ubiquity of A2A receptors makes diverse side effects an inevitable consequence of orthosteric agonism, hence the development of positive allosteric modulators (PAMs) of A2A receptors with high CNS penetrability will likely result in a preferable side effect profile. Various A2A PAMs have recently been discovered, however much of the research focus for these drugs has been directed towards inflammatory conditions.

Question 17

Big boy A* Material:

What is functional hyperaemia?

What is the role of NO in functional hyperaemia?

Briefly describe the epidemiology of stroke in the UK.

Describe a potential nitrergic target for stroke.

How can this target be used to treat stroke?

Answer 17

• Functional hyperaemia is the term used to describe the coupling of cerebral perfusion to neuronal activity.
• Functional hyperaemia is a result of the integration of neurones, glia and cerebral vasculature, known collectively as the neurovascular unit.
• Studies have found that functional hyperaemia is significantly attenuated in mice by both administration of the NOS antagonist, L-NAME, and genetic knockout of NOS. Therefore, NO plays an important role in functional hyperaemia. nNOS, the enzyme primarily responsible for functional hyperaemia, is upregulated in neurones by IP3 and Ca2+-dependent signalling mechanisms that are stimulated following increase neuronal activity.
• NO synthesised by upregulated nNOS is able to freely diffuse across astrocyte endfeet and into vascular smooth muscle cells. In conjunction with other signalling molecules, such as metabolites of arachidonic acid produced by astrocytes, this leads to vasodilation.
• Defective nitrergic signalling may play a role in numerous pathologies of the vasculature:
• Ischaemic stroke is the formation of an infarct following an obstruction to blood flow to the brain. It is the 4th most common cause of death in the UK, and is a leading cause of mental disability. The role of nitric oxide signalling in stroke is relatively well-documented, and the potential for NOS-targeting drugs for the treatment of stroke is under investigation.
• Following ischaemic stroke, NO is upregulated. NO secreted from the endothelium (eNOS), neurones (nNOS) and glia (iNOS) increase cerebral perfusion.
• However, although eNOS is useful for maintaining cerebral perfusion in the early stages, studies using nNOS and iNOS knockout mice have shown that these latter NOS isoforms worsen ischaemia, increasing the number and size of affected regions.
• This is thought to be related to nNOS and iNOS-induced upregulation of ROS and RNS, and the potential contribution to excitotoxicity that occurs as a result of stimulating glutamate release.
• Therefore, NOS inhibitors have been developed to protect against NO toxicity and neuronal necrosis caused by nNOS and iNOS.
• Among the first NOS inhibitors was L-NNA. Like many early NOS inhibitors, L-NNA has low specificity for isoforms of NOS. L-NNA has been used to demonstrate the theoretical potential of iNOS and nNOS inhibition. Administration of L-NNA has been shown to reduce stroke volume in eNOS knockout mice, but not in wildtype mice. This would indicate that inhibition of iNOS and nNOS produces a clinically useful effect, but that this effect is antagonised by inhibition of eNOS. This view is further supported by the finding that eNOS knockout mice exhibit hypertension, worsened ischaemia-reperfusion injury and impaired cerebral perfusion compared to control mice (Ito et al., 2010).
• Early studies voiced concerns that selective inhibition of nNOS will have deleterious effects on stroke, as nNOS is an important factor in the hyperaemic response to hypoxia, therefore inhibition of nNOS will worsen ischaemic damage. However, the opposite effect has so far been observed in animal studies. Another concern is that disruption to the role of neuronal and glial NO as a signalling molecule and as a mediator of synaptic plasticity may cause adverse effects. Indeed, nNOS knockout mice have been shown to display abnormal, aggressive behaviour, potentially due to the loss of other nitrergic signalling mechanisms.
• Another note on NO in blood supply to the brain: functional hyperaemia might be disturbed in Alzheimer’s disease because endothelial cells (eNOS), neurones (nNOS) and inflammatory glial cells (iNOS) are all dysfunctional in Alzheimer’s. Already said enough on this one so not going to write about it but perhaps worth mentioning - could be a good way of moving towards P2X antagonists.

Question 18

Big boy A* Material:

List 2 metabolic pathways involving NO other than the guanylyl cyclase pathway.

Answer 18

• Normally, NO signals through multiple pathways. Guanylyl cyclase is one example (explained in the lecture), but NO is also involved in posttranslational modification:

1 - Nitrosylation (addition of NO-), e.g. of cysteine residues, which are produced in inflammatory conditions.

2 - Nitration (addition of NO2), e.g. of tyrosine.

• Products of tyrosine nitration such as 3-nitrotyrosine are sometimes used as a marker of so-called ‘nitrosative stress’, e.g. in diabetes.
• The relative contribution of NO to each of these pathways depends on the microenvironment.

Question 19

From novel neuronal signalling mechanisms A* cards:

What is the importance of glial ATP receptors?

What is the function of these receptors in glia?

Answer 19

• Glial P2X receptors (an LGIC that responds to ATP) are important because, in the extracellular space, ATP is a damage signal (since it is released by perforated cells).
• In glia, ATP is detected by P2X receptors, and this triggers two defensive signalling mechanisms:

1 - Induces a Ca2+ wave from intracellular stores by triggering a downstream signalling pathway mediated by inositol (1,4,5) triphosphate.

• Both the Ca2+ and inositol (1,4,5) triphosphate can enter adjacent glia through gap junctions, spreading the signal.

2 - Stimulates the cell to release more ATP into the extracellular space in order to spread the signal.

Question 20

A*:

Describe the discovery of purinergic signalling.

Answer 20

• In 1964, Burnstock et al. (1964) published findings that pulsatile electrical stimulation of the vagus led to relaxation in gastrointestinal (GIT) smooth muscle which persisted in the presence of atropine, a muscarinic antagonist, and following degeneration of the adrenergic innervation. This sparked international debate as, until this time, GIT motility was thought to be influenced exclusively by excitatory vagal innervation and inhibitory splanchnic innervation. Subsequent studies found that the smooth muscle relaxation found by Burnstock et al. was abolished by tetrodotoxin, a neurotoxin used to block action potential conduction, indicating that the smooth muscle relaxation was in fact mediated by non-adrenergic-non-cholinergic (NANC) neurotransmission in vagal efferent fibres. The neurotransmitter responsible for the smooth muscle relaxation was later demonstrated in 1970 by Burnstock et al. to be ATP, leading to the now widely accepted view that excitatory cholinergic neurones and inhibitory NANC neurones form two distinct and functionally antagonistic pathways that together comprise the vagal innervation of the GIT.
• This discovery was the first description of purinergic signalling. Today, numerous purinergic signalling molecules are known to be a coreleased with various neurotransmitters including noradrenaline, neuropeptide Y, acetylcholine, NO, dopamine, GABA and glutamate in both peripheral and central nerves where they…
• Here, you could say ‘mediate a neuromodulatory function via numerous receptor subtypes’ - then list purine receptor subtypes and THEN give examples of receptor functions.