1. OTHER PERIPHERAL MEDIATORS: PURINES
26th Jan, 2017

2. Purine
A double ringed, crystalline organic base, C5H4N4, from which is derived the nitrogen bases adenine and guanine, as well as uric acid as a metabolic end product.
Purines are found in all of the body’s cells, and in virtually all foods.
The high purine foods are also high-protein foods and they include organ meats like kidney, fish like mackerel, herring, sardines and also yeast

3. Overview In this session we will describe:
the role of purine nucleosides and nucleotides as chemical mediators sub-serving a wide range of functions
The mechanisms responsible for their synthesis and release
The various receptors on which they act
Drugs that affect purinergic signalling

4. Overview…
There is an increasing interest in purine pharmacology and the potential role of purinergic agents in the treatment of pain and a variety of disorders, particularly of thrombotic and respiratory origin
The full complexity of purinergic control systems, and their importance in many pathophysiological mechanisms, is only now emerging.

5. Overview… In comparison:
5-HT has a longer pharmacological history than purines, and numerous drugs in current use act wholly or partly on 5-HT receptors, of which no fewer than 14 subtypes have been identified
Purine pharmacology is much sparser

6. Overview…
In both cases, the physiological significance; and hence therapeutic relevance of the various receptor subtypes is still being unravelled
(NB: to unravel=to explain something that is difficult to undestand)
Focus will be on the more secure hypotheses, recognizing that the picture is far from complete

7. Learning Objectives To understand and be able to describe:-
Purine receptors
Adenosine as a mediator
ADP as a mediator
ATP as a mediator 7

8. Introduction
Nucleosides, especially adenosine, and nucleotides, especially ADP (adenosine diphosphate) and ATP ( adenosine triphosphate), will be familiar to you because of their crucial role in DNA/RNA synthesis and energy metabolism,
but it may come as a surprise to learn that they also produce a wide range of pharmacological effects that are unrelated to their role in energy metabolism. 8

9. Nucleoside does not contain phosphate groups
Nucleotide=nitrogen base (purine or pyrimidine) + phosphate group + pentose sugar (ribose or deoxyribose). They are units of DNA
Nucleoside=nitrogen base (purine or pyrimidine) +pentose sugar (ribose or deoxyribose). They are units of RNA
Nucleoside does not contain phosphate groups
Nucleotidases break down nucleotides (such as thymine nucleotide) into nucleosides (such as thymidine) and phosphate groups. 9

10. 1. Purine Receptors
There are three main families of purine receptors each with several subtypes.
The subtypes in each family may be distinguished on the basis of their molecular structure as well as their agonist and antagonist selectivity 10

11. 1. Purine Receptors… The three main types of purine receptor are:-
Adenosine receptors
P2Y metabotropic receptors
P2X ionotropic receptors 11

12. 1. Purine Receptors…
Adenosine receptors (subtypes A1, A2A, A2B and A3), formerly known as P1-receptors;
These respond to adenosine, and are G-protein-coupled receptors (GPCRs) that regulate cAMP
Linked to stimulation or inhibition of adenylate cyclase
They are present in many different tissues

13. 1. Purine Receptors… 2. P2Y metabotropic receptors (P2Y1-14),
which are G-protein-coupled receptors that utilize either cAMP or phospholipase C activation as their signalling system (refer to How Drugs Act: Molecular Aspects)
they respond to various adenine nucleotides, generally preferring ATP over ADP or AMP

14. 1. Purine Receptors… P2X ionotropic receptors (P2X1-7)
are multimeric ATP-gated cation channels
Refer table 16.1 pg 206 of Pharmacology by Rang and Dale

15. Drugs Acting on Purine Receptors
Methylxanthines, especially analogues of theophylline, are A1/A2-receptor antagonists: however they also increase the cAMP (cyclic 3’5’-adenosine monophosphate) by inhibiting phosphodiestrase, which contributes to their pharmacological actions independently of adenosine receptor antagonism.
P2-receptors are blocked by Suramin 15

16. 2. Adenosine as a Mediator
The simplest of the purines, adenosine is found in biological fluids throughout the body.
Adenosine differs from ATP in that it is not stored by and released from secretory vesicles.
Rather, it exists free in the cytosol of all cells and is transported in and out of cells mainly via a membrane transporter.
Little is known about the way in which this is controlled but the extracellular concentrations are usually quite low compared with intracellular levels 16

17. 2. Adenosine as a Mediator
Adenosine in tissues comes partly from this intracellular source and partly from extracellular hydrolysis of released ATP or ADP
Virtually, all cells express one or more A-receptors and so adenosine produces many pharmacological effects, both in the periphery and in the CNS.

18. 2. Adenosine as a Mediator…
Based on its ability to inhibit cell function and thus minimize the metabolic requirements of cells, one of its functions may be as an ‘acute’ protective agent that is released immediately when tissue integrity is threatened (e.g. by coronary or cerebral ischaemia)
Under less extreme conditions, variations in adenosine release may play a role in controlling blood flow and (through effects on the carotid bodies) respiration, matching them to the metabolic needs of the tissues 18

19. 2. Adenosine as a Mediator …
Adenosine affects many cells and tissues, including smooth muscle and nerve cells. It is not a conventional transmitter but may be important as local hormone and ‘homeostatic modulator’. 19

20. 2. Adenosine as a Mediator …
Important sites of action include the heart and the lung
Adenosine acts through A1-, A2- and A3-G-protein receptors, coupled to inhibition or stimulation of adenylate cyclase.
A1- and A2-receptors are blocked by xanthines, such as theophylline

21. Functional Aspects-Adenosine Receptors
The main effects of adenosine are:
-hypotension (A2) and cardiac depression (A1)
-inhibition of atrioventricular conduction (antidysrhythmic effect, A1)
-inhibition of platelet aggregation (A2)
-bonchoconstriction (probably secondary to mast cell activation, A3)
-presynaptic inhibition in CNS (responsible for neuroprotective effect, A1) 21

22. Adenosine is very short acting and sometimes used for its antidysrhythmic effect
New adenosine agonists and antagonists are in development, mainly for treatment of ischaemic heart disease and stroke 22

23. Uses of Adenosine
Because of its inhibitory effects on cardiac conduction, adenosine may be used as an intravenous bolus injection to terminate supraventricular tachycardia
It is safer than Beta-adrenoceptor antagonists or verapamil, because of its short duration of action
Selective adenosine receptor antagonists could also have advantages over theophylline in the treatment of asthma 23

24. Adenosine and the CVS
Inhibits cardiac conduction and it is likely that all four of the adenosine receptors are involved in this effect
Because of this, adenosine itself may be used as a drug, being given as an intravenous bolus injection to terminate supraventricular tachycardia
It is safer than Beta-adrenoceptor antagonists or verapamil, because of its short duration of action

25. Adenosine and the CVS…
Longer lasting analogues have been discovered that also show greater receptor selectivity
Adenosine uptake is blocked (thus its action prolonged) by dipyridamole, a vasodilator and antiplatelet drug

26. Adenosine and Asthma
Adenosine receptors are found on all the cell types involved in asthma and the overall pharmacology is complex
However, by acting through its A1 receptor, adenosine promotes mediator release from mast cells, and causes enhanced mucus secretion, bronchoconstriction and leukocyte activation

27. Adenosine and Asthma…
Methylxanthines, especially analogues of theophylline, are adenosine receptor antagonists.
Theophylline has been used for the treatment of asthma

28. Adenosine in the CNS Act through A1 and A2A receptors
Has an inhibitory effect on many CNS neurons and
The stimulation experienced after consumption of methylxanthines such as caffeine occurs partly as a result of block of these receptors

29. ADP AS A MEDIATOR 29

30. ADP as a Mediator ADP is usually stored in vesicles in cells.
When released, it exerts its biological effects predominantly through the P2Y family of receptors. 30

31. ADP and Platelets
The secretory vesicles of blood platelets store both ATP and ADP in high concentrations, and release them when the platelets are activated.
One of the many effects of ADP is to promote platelet aggregation, so this system provides positive feedback-an important mechanism for controlling this process. 31

32. ADP and Platelets… The receptor involved is P2Y12.
Clopidogrel, prasugrel and the earlier agent, ticlopidine, are P2Y12 antagonists and exert their anti-aggregating effects through this mechanism

33. ADP and Platelets… ADP acts on platelets, causing aggregation.
This is important in thrombosis.
It also acts on vascular and other types of smooth muscle, as well as having effects on CNS 33

34. ATP AS A MEDIATOR 34

35. ATP as a Mediator
ATP exerts its action primarily through the P2X receptors.
The extracellular domain of these multimeric receptors can bind three molecules of ATP.
When activated, the receptor gates the cation-selective ion channels that trigger ongoing intracellular signalling
The other actions of ATP in mammals are mediated through the P2Y receptors. 35

36. ATP as a Mediator…
Suramin (a drug originally developed to treat trypanosome infections) and an experimental compound PPADS antagonize ATP and have broad-spectrum inhibitory activity at most P2X and P2Y-receptors
Cytoplasmic ATP may be released, independently of exocytosis, when the cells are damaged (e.g. by ischaemia)

37. ATP as a Mediator…
ATP released from cells is rapidly dephosphorylated by a range of tissue-specific nucleotidases, producing ADP and adenosine, both of which produce a wide variety of receptor mediated effects.
The role of intracellular ATP in controlling membrane potassium channels, which is important in the control of vascular smooth muscle and of insulin secretion is quite distinct from its transmitter function

38. ATP as a Neurotransmitter
ATP functions as a neurotransmitter (or co-transmitter) at peripheral neuroeffector junctions and central synapses.
ATP is stored in vesicles and released by exocytosis.
Cytoplasmic ATP may be released when cells are damaged.
It also functions as an intracellular mediator, inhibiting the opening of membrane potassium channels 38

39. ATP as a Neurotransmitter…
ATP acts on two types of purinoceptors (P2), one of which is (P2x) is a ligand-gated ion channel responsible for fast synaptic responses.
P2X2, P2X4, P2X6, are the predominant receptor subtypes expressed in neurons
P2X1 predominates in smooth muscles
The other (P2Y) is coupled to various second messengers. 39

40. ATP as a Neurotransmitter…
Suramin blocks the P2x-receptor.
Released ATP is rapidly converted to ADP and adenosine that may act on other purinergic receptors

41. ATP in Nociception
ATP causes pain when injected, as a result of activation of P2X2 and/or P2X3 receptors in afferent neurons involved in the transduction of norciception
Oddly, perhaps, the same receptors seem to be involved in taste perception on the tongue.
Elsewhere in the CNS, P2X4 receptors on microglia may be important in the development of neuropathic pain 41

42. ATP in Inflammation
The P2X7 receptor is widely distributed on cells of the immune system, and ATP, apparently acting through this receptor, causes the release from macrophages and mast cells of cytokines and other mediators of the inflammatory response. 42

43. Future Prospects
While it is true that few currently available drugs act through purinergic receptors when compared, e.g. with 5-HT receptors, the area as a whole holds promise for future therapeutic exploitation, particularly in the treatment of asthma, pain and gastrointestinal disorders, provided compounds with sufficient receptor selectivity can be found. 43

44. Further Reading on Purines:
Rang & Dale’s Pharmacology (7th Edition-Chapter 16) 44

45. THANK YOU! 45