1. OTHER PERIPHERAL MEDIATORS: 5-HYDROXYTRYPTAMINE (5-HT) & PURINE PEPTIDES
OTHER CHEMICAL MEDIATORS
5HT
PEPTIDES & PROTEINS
METHYLXANTHINES
ATRIAL NATRIURETIC PEPTIDES
NITRATES
NITRIC OXIDE

2. OVERVIEW
In this session we will discuss two types of mediators, both of which play a role as neurotransmitter in the brain and periphery and also probably function as local hormones.
These mediators are:
5-HydroxyTryptamine (5-HT)
Purines (nucleosides and nucleotides)

3. Overview…
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

4. 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 understand)
Focus will be on the more secure hypotheses, recognizing that the picture is far from complete.

5. 5-HYDROXYTRYPTAMINE
(5-HT)

6. Learning Objectives At the end of the session students must be
able to understand and describe:-
Distribution, biosynthesis, and degradation
Pharmacological effects
Classification of 5-HT receptors
Drugs acting on 5-HT receptors
Clinical conditions in which 5-HT plays a role

7. 5-HYDROXYTRYPTAMINE
Serotonin was the name given in the last century to an unknown vasoconstrictor substance found in the serum after blood has clotted.
It was identified chemically as 5-hydroxytryptamine (5-HT) in 1948, and shown to originate from platelets.
It was subsequently found in the GIT and the CNS, and shown to function both as a neurotransmitter and as a local hormone in the peripheral vascular system.

8. DISTRIBUTION, BIOSYNTHESIS AND DEGRADATION OF 5-HT

9. Distribution of 5-HT
5-HT occurs in the highest concentrations in three organs in the body-:
Wall of the intestine
Blood
CNS

10. Distribution of 5-HT…
In the wall of the intestine: Over 90% of the total amount in the body is present in enterochromaffin cells, which are cells derived from the neural crest, similar to those of the adrenal medulla, that are interspersed with mucosal cells, mainly in the stomach and small intestine.

11. Distribution of 5-HT…
Some 5-HT also occurs in the nerve cells of myenteric plexus, where it functions as an excitatory neurotransmitter.
In blood: 5-HT is present in high concentration in platelets, which accumulate it from the plasma by an active transport system, and release it when they aggregate at the site of tissue damage.

12. Distribution of 5-HT (cont.)
In the CNS: 5-HT is a transmitter in the CNS and is found in high concentrations in localized regions of the midbrain.

13. THE BIOSYNTHESIS OF 5-HT
Though 5-HT is present in the diet, most of this is metabolized before entering the blood stream.
Endogenous 5-HT arises by biosynthesis, which follows a pathway similar to that of NA, except that the precursor amino acid is tryptophan instead of tyrosine.
FIND OUT: Which foods in the diet have serotonin in them?

14. Biosynthesis of 5-HT…
Is formed from amino-acid tryptophan which is present in the diet, but most is metabolized before entering the blood stream.
Tryptophan is converted to 5-hydroxytryptophan ( in chromaffin cells and neurons, but not in platelets) by the action of tryptophan hydroxylase ( an enzyme confined to 5-HT producing cells).
Tryptophan is converted to 5-hydroxytryptophan ( in chromaffin cells and neurons, but not in platelets)

15. Biosynthesis of 5-HT (cont.)
The 5-hydroxytryptophan is then decarboxylated to 5-HT, by the non-specific amino acid decarboxylase that also participates in the synthesis of catecholamines and histamine.
5-HT is transported into 5-HT-containing cells by a specific transport system.

16. Biosynthesis of 5-HT (cont)
Platelets (and neurones) possess a high affinity 5-HT uptake mechanism, and platelets become loaded with 5-HT as they pass through the intestinal circulation, where the local concentration is relatively high.
The mechanisms of synthesis, storage, release and re-uptake of 5-HT are very similar to those of NA, and many drugs affect both processes indiscriminately

17. 5-HT is often stored in neurons and chromaffin cells as a co-transmitter together with various peptide hormones, such as somatostatin, substance P or vasoactive intestinal polypeptide (VIP)

18. 5-HT Degradation
Degradation of 5-HT occurs mainly through oxidative deamination, catalysed by monoamine oxidase (MAO), followed by oxidation to 5-hydroxyindoleacetic acid (5-HIAA), which is excreted in the urine, and serves as an indicator of 5-HT production in the body.
This is used, for example in the diagnosis of carcinoid syndrome

19. Pharmacological Effects
The actions of 5-HT are numerous and complex, and there is considerable species variation.
This complexity reflects a profusion of 5-HT receptor subtypes, which has been revealed in recent years.

20. Pharmacological Effects-Sites of Action
The main sites of action are:
GIT:-
5-HT stimulates gastrointestinal motility, this being partly through a direct effect on the smooth muscles (5-HT2-receptors) and partly as a result of an indirect excitatory effect on enteric neurons (5-HT3 and 5-HT4-receptors).

21. 1. Site of action-GIT (cont.)
5-HT also stimulates fluid secretion and elicits nausea and vomiting by stimulating smooth muscle and sensory nerves in the stomach (5-HT3- and 5-HT4-receptors)

22. Site of action-GIT (cont.)
The peristaltic reflex, evoked by increasing the pressure within a segment of intestine, is mediated, partly at least, by the release of 5-HT from chromaffin cells in response to the mechanical stimulus.
Chromaffin cells also respond to vagal stimulation by releasing 5-HT

23. 2. Site of action- Smooth Muscle
Elsewhere in the body (e.g. uterus and bronchial tree) smooth muscle is also contracted by 5-HT in many species, but only to a minor extent in humans

24. 3. Site of Action-Blood vessels
The effect of 5-HT on blood vessels depends on various factors, including the size of the vessel, the species and the prevailing sympathetic activity.
Large vessels, both arteries and veins are usually constricted by 5-HT, though the sensitivity varies greatly.

25. Site of Action-Blood vessels…
This is a direct action on vascular smooth muscle cells, mediated through 5-HT2A-receptors
Activation of 5-HT1 receptors causes constriction of large intracranial vessels, dilatation of which contributes to headache.

26. Site of Action-Blood vessels…
5-HT also causes vasodilatation by acting on 5-HT1-receptors, partly by releasing nitric oxide from endothelia cells, and partly by inhibiting noradrenaline release from sympathetic nerve terminals.
Thus 5-HT2A receptors predominantly give rise to vasoconstriction, whereas 5-HT1 receptors produce dilatation
5-HT2A-receptors – VASOCINSTRICTION
5-HT1 receptors - VASODILATATION

27. Site of Action-Blood vessels…
When 5-HT2A receptors are blocked by ketanserin the vasodilator effect is revealed.
If 5-HT is injected intravenously, the blood pressure usually rises, because of constriction of large vessels, then falls, because of arteriolar dilatation.

28. 4. Site of Action-Platelets
5-HT causes platelet aggregation, via 5-HT2A-receptors, and the platelets that collect in the vessel release more 5-HT.
If the endothelium is intact, 5-HT release from adherent platelets causes vasodilatation, which helps to sustain blood flow;

29. Site of Action-Platelets…
if it is damaged (e.g. by atherosclerosis),
5 -HT causes constriction and impairs blood flow further
These effects of platelet-derived 5-HT are thought to be important in vascular disease

30. 5. Site of Action-Nerve endings
5-HT stimulates nociceptive ( pain-mediating) sensory nerve endings, an effect mediated mainly by 5-HT3-receptors.
If injected into the skin, 5-HT causes pain: given systemically, it elicits a variety of autonomic reflexes through stimulation of afferent fibres in the heart and lungs, which further complicate the cardiovascular response.

31. 6. Site of Action-CNS 5-HT excites some neurons and inhibits others.
It also acts presynaptically to inhibit transmitter release from nerve terminals.

32. SUMMARY:ACTIONS AND FUNCTIONS OF 5-HT
Important actions are:
Increased GIT motility (direct excitation of smooth muscle and indirect action via enteric neurons).
Contraction of smooth muscle (bronchi, uterus).

33. SUMMARY:ACTIONS AND FUNCTIONS OF 5-HT…
Mixture of vascular constriction (direct and via sympathetic innervation) and dilatation (endothelium dependent)
Platelet aggregation
Stimulation of peripheral nociceptive nerve endings
Excitation/inhibition of CNS neurons

34. POSTULATED PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL ROLES
In periphery: peristalsis, vomiting, platelet aggregation and haemostasis, inflammatory mediator, sensitization of nociceptors and microvascular control.
In CNS: many postulated functions; include control of appetite; sleep; mood; hallucinations, stereotyped behaviour; pain perception and vomiting.

35. Clinical condition associated with disturbed 5-HT function includes migraine, carcinoid syndrome, mood disorders and anxiety

36. …TO CONTINUE…….

37. Classification of 5-HT Receptors
Currently, there are seven main receptor types:-
5-HT1-7, with further subtypes (A-D)
of 5HT1 and 5-HT2.
All are G- protein coupled receptors except 5-HT3 which is ligand-gated cation channel.

38. Classification of 5-HT Receptors…
5HT1-receptor occur mainly in the CNS ( all subtypes i.e 5-HT1A-D) and some blood vessels (5-HT1D subtype). Effects are neural inhibition and vasoconstriction. Act by inhibiting adenylate cyclase. Specific agonist includes sumatriptan (used in migraine therapy) and buspirone (used in anxiety). Ergotamine is a partial agonist. Specific antagonists include spiperone and methiothepin.

39. Classification of 5-HT Receptors
5-HT2-receptors occur in CNS and many peripheral sites (especially blood vessels; platelets; autonomic neurons). Neuronal and smooth muscle effects are excitatory. Some blood vessels dilated as a result of nitric oxide release from endothelial cells.

40. Classification Of 5-HT Receptors
5-HT2-receptors act through phospholipase C/ inositol pathway. Specific ligands include LSD (lysergic acid diethylamide: agonist in CNS, antagonist in periphery). Specific antagonists: kentanserin, methysergide and cyproheptadine.

41. Classification of 5-HT Receptors
5-HT3 receptors occur in peripheral nervous system; especially nociceptive afferent neuron and enteric neurons, and in CNS. Effects are excitatory, mediated via direct receptors-coupled ion channels. Specific agonist: 2-methyl-5-HT. Specific antagonist: ondansentron; tropisetron. Antagonists are used mainly as antiemetic drugs, but may also be anxiolytic.

42. Classification of 5-HT Receptors
5-HT4 receptors occur mainly in enteric nervous system (also in CNS). Effects are excitatory, causing increased gastrointestinal motility. Act by stimulating adenylate cyclase. Specific agonist include metoclopramide (used to stimulate gastric emptying).

43. Classification of 5-HT Receptors
Little is known so far about the function and pharmacology of 5-HT5-7 receptors.
Many new receptor-selective agonists and antagonists are being developed

44. DRUGS ACTING ON 5-HT RECEPTORS
Important drugs that act on 5-HT receptors in periphery include-:
(1) 5-HT1D receptor agonist (e.g. sumatriptan) used for treating migraine. Selective 5-HT1A agonists, such as 8-OH-DPAT( 8-hydroxy-2-(di-n-propylamino) tetralin; are potent hypotensive agents, acting by central mechanism, but are not used clinically.

45. DRUGS ACTING ON 5-HT RECEPTORS
(2) 5-HT3-receptor antagonists (e.g. ondansetron, granisetron; tropisetron) used as anti-emetic drugs particularly for controlling the severe nausea and vomiting that occurs with many forms of cancer chemotherapy.

46. DRUGS ACTING ON 5-HT RECEPTORS
(3) 5-HT2-receptor antagonists (e.g. dihydroergotamine, methysergide, cyproheptadine, kentanserin, ketotifen, pizotifen). These ‘classical’ 5-HT antagonists act mainly on the 5-HT2-receptors. They are, however non-selective, and act also on targets, such as alpha-adrenoceptors and histamine receptors.

47. DRUGS ACTING ON 5-HT RECEPTORS
Dihydrergotamine and methysergide belong to the ergot family and are used mainly for migraine prophylaxis. Ketotifen is sometimes used to treat asthma but the role of 5-HT receptors in this condition is unclear. Other 5-HT2 antagonists are used to control the symptoms of carcinoid tumours.

48. DRUGS ACTING ON 5-HT RECEPTORS
(4) 5-HT4-receptors agonists (e.g. metoclopramide; cisapride), which stimulate coordinated peristaltic activity (known as a ‘prokinetic action’) are used for treating gastrointestinal disorders

49. ERGOT ALKALOIDS
Many of them act on 5-HT-receptors, but not selectively, and their actions are complex and diverse.
Active substance produced by fungus (Claviceps purpurea) infecting cereal crops, responsible for occasional poisoning incidents.
The most important compounds are-:

50. ERGOT ALKALOIDS Ergotamine, dihydroergotamine used in migraine.
Ergometrine, used in obstetrics to prevent postpartum haemorrhage.
Methysergide, used to treat carcinoid syndrome, and occasionally for migraine prophylaxis.
Bromocriptine, used in parkinsonism and endocrine disorders

51. Actions
Main sites of action are 5-HT receptors, dopamine receptors and adrenoceptors (mixed agonist, antagonist and partial agonist effects).
They all cause stimulation of smooth muscle, some being relatively selective for vascular smooth muscle and others acting mainly on the uterus

52. Ergotamine and dihydroergotamine are, respectively, a partial agonist and an antagonist at alpha adrenoceptors; They act selectively on 5-HT1-receptors.
bromocriptine is an agonist on dopamine receptors, particulary in the CNS;
methysergide is a potent antagonist at 5-HT2-receptors

53. Clinical Use
The only use of ergotamine is in the treatment of attacks of migraine unresponsive to simple analgesics
Methysergide is occasionally used for migraine prophylaxis, but its main us is in treating the symptoms of carcinoid tumours
All these drugs can be used orally or by injection

54. Unwanted Effects
Include nausea and vomiting, vasoconstriction (ergot alkaloids are contraindicated in patients with peripheral vascular disease because of its vasoconstrictor action)

55. Clinical Conditions in Which 5-HT Plays a Role
There are two situations in which the peripheral actions of 5-HT are believed to be important, namely:
-migraine
-carcinoid syndrome

56. Migraine
Migraine is a common and debilitating condition, affecting 10-15% of people, the causation of which is not well understood.
The classical pattern of events in a migraine attack consists of an initial visual disturbance (the aura), in which a flickering pattern, followed by a blind spot, progresses gradually across an area of the visual field.

57. Migraine
This visual disturbance is followed, about 30 minutes later, by a severe throbbing headache, starting unilaterally, often with photophobia, nausea, vomiting and prostration, which lasts for several hours.
Sometimes attacks are precipitated by particular foods or by visual stimuli, but more often they occur without obvious cause.

58. Antimigraine Drugs
It is important to distinguish between drugs used to treat acute attacks of migraine and drugs used for prophylaxis
NB: Prophylactic= done or used in order to prevent a disease
e.g. Prophylactic treatment is the treatment that prevents a disease ( it can be a medicine, device or course of action)

59. Antimigraine Drugs Acute Attack
Simple analgesics (e.g aspirin, paracetamol) can be given with metoclopramide to speed up absorption.
Ergotamine (5-HT1D-receptor partial agonist)
Sumatriptan (5-HT1D agonist) is effective but short acting (half life of about 2 hours)

60. Antimigraine Drugs
Newer compounds (e.g. zolmitriptan)are claimed to be faster acting and not to cause chest pain
Prophylaxis (considered for patients with more than one severe attack per month)
Beta-adrenoceptor antagonists (e.g propranolol, metoprolol)

61. Antimigraine Drugs
Pizotifen (5-HT2-receptor antagonist). Adverse effects include weight gain, antimuscarinic effects.
Other 5-HT2-receptor antagonists
-cyproheptadine: also has antihistamine and calcium antagonist actions
-methysergide: rarely used because of risk of retroperitoneal fibrosis and renal failure

62. Antimigraine Drugs
Tricyclic antidepressants (e.g amitryptyline) may be effective even though patients are not depressed
Clonidime, an alpha 2-adrenoceptor agonist has been used, but efficacy is doubtful.
Calcium antagonists (e.g dihydropyridines, verapamil): headache is a side effect of these drugs, paradoxically, may reduce frequency of migraine attacks. There mechanism of action is unknown.

63. Carcinoid Syndrome
A rare disorder associated with malignant tumuors of enterochromaffin cells, usually arising in the small intestine and metastasizing to the liver. These tumours secrete a variety of hormones. 5-HT is the most important, but neuropeptides, such as substance P, and other agents, such as prostaglandins and bradykinin are also produced.

64. The release of these substances into the bloodstream results in various unpleasant symptoms, including flushing, diarrhea and bronchoconstriction, as well as hypotension, which may cause dizziness or fainting.

65. PURINES

66. Learning Objectives ATP as a neurotransmitter ADP and platelets
Adenosine as a mediator
Purine receptors

67. Purines
Nucleosides, especially adenosine, and nucleotide, especially ADP (adenosine diphosphate) and ATP ( adenosine triphosphate), produce a wide range of pharmacological effects that are unrelated to their role in energy metabolism.

68. 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.

69. 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

70. 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. The other (P2Y) is coupled to various second messengers. Suramin blocks the P2x-receptor.
Released ATP is rapidly converted to ADP and adenosine

71. 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, though one of many, for controlling this process.

72. Adenosine as a Mediator
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

73. Adenosine as a Mediator (cont.)
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

74. Adenosine as a Mediator (cont.)
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’.

75. Adenosine as a Mediator (cont.)
Adenosine acts through A1-, A2- and A3-receptors, coupled to inhibition or stimulation of adenylate cyclase. A1- and A2-receptors are blocked by xanthines, such as theophylline

76. Purine Receptors There are two main types:
P1-receptors (subtypes A1, A2, and A3); these respond to adenosine, and are G-protein-coupled receptors (GPCRs). Linked to stimulation or inhibition of adenylate cyclase; they are present in many different tissues

77. P2-receptors (subtypes P2x and P2Y, each with several further subdivisions): these respond to ATP and/or ADP.

78. 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)

79. 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

80. 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

81. Selective adenosine receptor antagonists could also have advantages over theophylline in the treatment of asthma

82. 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

83. Exercise
Discuss the distribution, biosynthesis, storage, release, re-uptake and degradation of NA and 5-HT respectively, showing the similarities and differences in the mechanisms.
What are the actions and functions of
5-HT?

84. Classify the main 5-HT receptor subtypes; in each state the location, main effects, agonists and antagonists
What are the important drugs that act on 5-HT receptors in the periphery?

85. Further Reading:
Rang & Dale’s Pharmacology (7th Edition-Chapter 15-16)

86. THANK YOU!