1. OTHER PERIPHERAL MEDIATORS

2. 5-HydroxyTryptamine (5-HT) & The Pharmacology of Migraine 25.01.2017

3. OVERVIEW
In this session we will discuss the role of 5-hydroxytryptamine (5-HT), which functions as:-
a neurotransmitter in the brain & periphery
A local hormone

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

5. 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.
Today, the terms ‘5-HT’ and ‘serotonin’ are used interchangeably

6. DISTRIBUTION, BIOSYNTHESIS AND DEGRADATION OF 5-HT

7. Distribution of 5-HT-1/4
5-HT occurs in the highest concentrations in three organs in the body-:
Wall of the intestine
Blood
CNS

8. Distribution of 5-HT..2/4 a) In the wall of the intestine:
Over 90% of the total amount in the body is present in the enterochromaffin cells in the gut (endocrine cells with distinctive staining properties).
These are cells derived from the neural crest, similar to those of the adrenal medulla
They are found mainly in the stomach and small intestine and are interspersed with mucosal cells.
Some 5-HT also occurs in the nerve cells of myenteric plexus, where it functions as an excitatory neurotransmitter.

9. Distribution of 5-HT..3/4 b) 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.
Refer to Haemostasis & Thrombosis

10. Distribution of 5-HT…4/4 c) In the CNS:
5-HT is a transmitter in the CNS and is found in high concentrations in localized regions of the midbrain.
Its functional role will be discussed later (Other transmitters and modulators)

11. Biosynthesis of 5-HT..1/4
Although 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 Noradrenaline (norepinephrine), except that the precursor amino acid is tryptophan instead of tyrosine.

12. Biosynthesis of 5-HT…2/4
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).

13. Biosynthesis of 5-HT…3/4
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.

14. Biosynthesis of 5-HT…4/4
Platelets (and neurons) 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

15. 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)

16. 5-HT Degradation
Degradation of 5-HT occurs mainly through oxidative deamination, catalyzed by monoamine oxidase A (MAO), followed by oxidation to 5-hydroxyindoleacetic acid (5-HIAA), the pathway being the same as that of NA catabolism.
5-HIAA 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

17. PHARMACOLOGICAL EFFECTS

18. 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.
The main sites of action are:-
GIT
Smooth muscle
Blood vessels
Platelets
Nerve endings
CNS

19. Pharmacological Effects-Sites of Action 1) 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).

20. Pharmacological Effects-Sites of Action 1) GIT…
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)
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

21. Pharmacological Effects-Sites of Action 2) 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

22. Pharmacological Effects-Sites of Action 3) 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, although the sensitivity varies greatly.

23. Pharmacological Effects-Sites of Action 3) 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.

24. Pharmacological Effects-Sites of Action 3) Blood Vessels…
5-HT also causes vasodilatation by acting on 5-HT1-receptors, partly by releasing nitric oxide from endothelial 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

25. Pharmacological Effects-Sites of Action 3) 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 first rises, because of constriction of large vessels, and then falls, because of arteriolar dilatation.
5-HT may play a role in the pathology of pulmonary hypertension

26. Pharmacological Effects-Sites of Action 4) 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; 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

27. Pharmacological Effects-Sites of Action 5) 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: when 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.

28. Pharmacological Effects-Sites of Action 5) Nerve endings…
Nettle stings contain 5-HT among other mediators
5-HT also inhibits transmitter release from adrenergic neurons in the periphery

29. Pharmacological Effects-Sites of Action 6) CNS:-
5-HT excites some neurons and inhibits others.
It may also act presynaptically to inhibit transmitter release from nerve terminals.
Different receptor subtypes and different membrane mechanisms mediate these effects
NB: role of 5-HT in the CNS will be discussed later

30. Summary Actions and Functions of 5-HT
1. Important actions are:
Increased GIT motility (direct excitation of smooth muscle and indirect action via enteric neurons).
Contraction of other smooth muscle (bronchi, uterus).
Mixture of vascular constriction (direct and via sympathetic innervation) and dilatation (endothelium dependent)

31. Summary Actions and Functions of 5-HT …
Platelet aggregation
Stimulation of peripheral nociceptive nerve endings
Excitation/inhibition of CNS neurons

32. Summary Actions and Functions of 5-HT…
2. Postulated Physiological and Pathophysiological Roles include:
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.

33. Summary Actions and Functions of 5-HT…
3. Clinical condition associated with disturbed 5-HT function includes:-
migraine,
carcinoid syndrome,
mood disorders and
anxiety

34. CLASSIFICATION OF 5-HT RECEPTORS

35. Classification of 5-HT Receptors
Currently, there are seven main receptor families (5-HT1-7), with further subtypes of 5HT1(A-F) and 5-HT2(A-C).
Many polymorphisms and splice variants have also be observed.
All are G- protein coupled receptors (GPCRs) except 5-HT3 which is a ligand-gated cation channel.
The six GPCR families are further subdivided into some 13 receptor types based on their sequence and pharmacology

36. Classification of 5-HT Receptors…
5HT1-receptor occur mainly in the CNS ( all subtypes i.e 5-HT1(A-D) and some blood vessels (5-HT1B/D subtype). Effects, some mediated through inhibition of adenylyl cyclase, include neural inhibition and vasoconstriction.
Specific agonist includes triptans e.g. sumatriptan (used in migraine therapy) and buspirone (used in anxiety).
Ergotamine is a partial agonist.
Specific antagonists include spiperone and methiothepin.

37. 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.

38. 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: pizotifen, kentanserin, methysergide and cyproheptadine.

39. Classification of 5-HT Receptors…
5-HT3 receptors occur in peripheral nervous system; especially nociceptive afferent neuron and enteric neurons, and in the 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.

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

41. Classification of 5-HT Receptors…
5-HT5 receptors (one subtype in humans) are located in the CNS.
5-HT6 receptors are located in the CNS and on leucocytes
5-HT7 receptors are located in the CNS and the gastrointestinal tract
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
NB: read Table 15.1 pg 197 (Rang & Dale’s Pharmacology)

42. DRUGS ACTING ON 5-HT RECEPTORS

43. Drugs Acting on 5-HT Receptors
Important drugs that act on 5-HT receptors in periphery include-:
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.
5-HT1B/D receptor agonist (e.g. sumatriptan) used for treating migraine.

44. Drugs Acting on 5-HT Receptors …
5-HT2-receptor antagonists (e.g. dihydroergotamine, methysergide, cyproheptadine, kentanserin, ketotifen, pizotifen).
These ‘classical’ 5-HT antagonists act mainly on the 5-HT2A-receptors.
They are, however non-selective, blocking other 5-HT receptors and act also on targets, such as alpha-adrenoceptors and histamine receptors.

45. 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.

46. Drugs Acting on 5-HT Receptors …
5-HT3-receptor antagonists (e.g. dolesatron, ondansetron, granisetron; tropisetron, palonosetron) are used as anti-emetic drugs particularly for controlling the severe nausea and vomiting that occurs with many forms of cancer chemotherapy.

47. Drugs Acting on 5-HT Receptors …
5-HT4-receptors agonists (e.g. metoclopramide; cisapride), which stimulate coordinated peristaltic activity (known as a ‘prokinetic action’) are used for treating gastrointestinal disorders.
Metoclopramide acts in this way, as well as by blocking dopamine receptors.
Similar but more selective drugs such as cisapride and tegaserod were introduced to treat irritable bowel syndrome, but withdrawn on account of cardiovascular side effects.

48. Drugs Acting on 5-HT Receptors … Ergot Alkaloids
Many of them act on 5-HT-receptors, but not selectively, and their actions are complex and diverse.
Are active substance produced by a fungus (Claviceps purpurea) that infects cereal crops, responsible for occasional poisoning incidents.

49. Ergot Alkaloids… The most important compounds are-:
Ergotamine, dihydroergotamine used in migraine prophylaxis.
Ergometrine, used in obstetrics to prevent postpartum haemorrhage (PPH).
Methysergide, used to treat carcinoid syndrome, and occasionally for migraine prophylaxis.
Bromocriptine, used in parkinsonism and endocrine disorders

50. Actions of Ergot Alkaloids
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

51. Actions of Ergot Alkaloids…
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, particularly in the CNS;
Methysergide is a potent antagonist at 5-HT2A-receptors

52. Clinical Use of Ergot Alkaloids
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 use is in treating the symptoms of carcinoid tumours
All these drugs can be used orally or by injection

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

54. Unwanted Effects of Ergot Alkaloids …
Methysergide also causes nausea and vomiting, but its most serious side effect, which probably restricts its clinical usefulness, is retroperitoneal and mediastinal fibrosis, which impairs the functioning of the gastrointestinal tract, kidneys, heart and lungs.
The mechanism of action of this is unknown.

55. CLINICAL CONDITIONS IN WHICH 5-HT PLAYS A ROLE

56. Clinical Conditions in Which 5-HT Plays a Role
There are three situations in which the peripheral actions of 5-HT are believed to be important, namely:
1. migraine
2. carcinoid syndrome
3. pulmonary hypertension

57. 1. Migraine
Migraine is a common and debilitating condition, affecting 10-15% of people.
Although the causes are not well understood, both genetic and environmental factors seems to be important
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 (a ‘scontillating scotoma’) progresses gradually across an area of the visual field.

58. 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.

59. Migraine…
In women, migraine may be linked to the menstrual cycle or other reproductive events.
It appears that rapidly falling oestrogen levels can precipitate attacks in susceptible subjects.

60. Pathophysiology of Migraine
Read

61. 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)

62. Antimigraine Drugs… Acute Attack
Simple analgesics (e.g aspirin, paracetamol) can be given with or without 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)
Newer compounds (e.g. zolmitriptan)are claimed to be faster acting and not to cause chest pain

63. Antimigraine Drugs… Prophylaxis
(considered for patients with more than one severe attack per month)
Beta-adrenoceptor antagonists (e.g propranolol, metoprolol)
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

64. Antimigraine Drugs Prophylaxis …
Tricyclic antidepressants (e.g amitryptyline) may be effective even though patients are not depressed
Clonidine, 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. Their mechanism of action is unknown.

65. 2. Carcinoid Syndrome
A rare disorder associated with malignant tumuors of enterochromaffin cells, usually arising in the small intestine and metastasize 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.

66. 2. Carcinoid Syndrome…
The release of these substances into the bloodstream results in various unpleasant symptoms, including:
flushing,
diarrhoea and
bronchoconstriction,
as well as hypotension, which may cause dizziness or fainting.

67. 2. Carcinoid Syndrome…
Fibrotic stenosis of heart valves, which can result in cardiac failure, also occurs.
The syndrome is readily diagnosed by measuring the urinary excretion of the main metabolite of 5-HT, 5-HIAA
Excretion in the disease may increase 20-fold and is raised even during periods when the tumour is asymptomatic.

68. 2. Carcinoid Syndrome…
5-HT2 antagonists, such as cyproheptadine, are effective in controlling some of the symptoms of carcinoid syndrome
A complementary therapeutic approach is to use octreotide (a long-acting analogue of somatostatin), which supresses hormone secretion from neuroendocrine, including carcinoid, cells

69. 3. Pulmonary Hypertension
It is an extremely serious disease characterized by the progressive remodeling of the pulmonary vascular tree.
This leads to an inexorable rise in pulmonary arterial pressure which, if untreated (and treatment is difficult), inevitably leads to right heart failure and death.
The role of 5-HT in this pathology was suggested by the fact that at least one form of the condition was precipitated by the use of appetite suppressants (e.g. dexfenfluramine) that were at one time widely prescribed as ‘weight loss’ or ‘slimming’ aids

70. 3. Pulmonary Hypertension…
Pulmonary hypertension is still considered to be a disease in which 5-HT plays an important role and which therefore may become a target for novel drug development.

71. Evaluation:-
Have you understood ? and can now describe the following?:-
Distribution, Biosynthesis, and Degradation of 5-HT
Pharmacological effects of 5-HT
Classification of 5-HT receptors
Drugs acting on 5-HT receptors
Clinical conditions in which 5-HT plays a role

72. Assignment
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?

73. Assignment…
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?
Write short notes on migraine

74. Further Reading on 5-HT and the Pharmacology of Migraine:
Rang & Dale’s Pharmacology (7th Edition-Chapter 15)

75. THANK YOU FOR BEING ATTENTIVE!