1. Chemical Mediators – Nitric Oxide Pharmacology January, 2012

2. 19/01/20162 Nitric Oxide Overview Nitric oxide (NO) is a ubiquitous mediator with diverse functions. It is generated from L-arginine by action of an enzyme, nitric oxide synthase (NOS), that occurs in endothelial, neuronal and inducible isoforms.

3. 19/01/20163 Nitric Oxide Overview… In this session we will focus on general aspects of NO, especially the following:-  Nature and biosynthesis  Degradation  Main effects  Mechanism of action  Therapeutic potential of drugs that act on the L-arginine/NO pathway.

4. 19/01/20164 Nitric Oxide – Nature and Source A free radical gas Formed in the atmosphere during lightning storms Also formed in a biochemical reaction in living systems (enzyme catalysed reaction between molecular oxygen and L-arginine) In general NO is a key signalling molecule in the cardiovascular and nervous systems and has a role in host defence

5. 19/01/20165 Nitric Oxide – Some Effects on Cells NO is an endogenous activator of soluble guanylate cyclase, leading to the formation of cyclic 3’5-guanosine monophosphate (cGMP), which functions as a ‘second messenger’ in many cells including: –Nerves –Smooth muscle –Monocytes –Platelets

6. 19/01/20166 Nitrogen and oxygen are neighbours in the periodic table, NO shares several properties with oxygen, in particular a high affinity for haem and other iron sulfur groups. This is important for activation of guanylate cyclase, which contains a haem group, and for the inactivation of NO by haemoglobin.

7. 19/01/20167 NO has a role in specific setting in: –Endothelium –Autonomic nervous system –Acts as a chemical transmitter and mediator of excitotoxicity in the CNS NO has a role in the innate mediator derived reactions of acute inflammation and the immune response

8. 19/01/20168 Nitric Oxide – Biosynthesis and Control Biosynthesis of NO is controlled by nitric oxide synthase enzymes (NOS) NOS enzymes exist in three isoforms: –An inducible form (iNOS or NOS-II) –Two so called ‘Constitutive’ forms –present in endothelium (eNOS or NOS-III), and in neurons (nNOS or NOS-I)

9. 19/01/20169 iNOS is expressed in various cells in response to pathological stimuli such as invading microorganisms Cells in which iNOS is expressed include macrophages and Kupffer cells, neutrophils, fibroblasts, vascular smooth muscle and endothelial cells Constitutive forms of NOS are present under physiological conditions in various tissues: endodhelium (eNOS), and in neurons (nNOS)

10. 19/01/201610 eNOS is not restricted to endothelium. It is also present in –Cardiac myocytes –Renal mesangial cells –Osteoblasts and osteoclasts –Airway epithelium –Platelets (in small amounts) Constitutive enzymes generate a small amounts of NO, whereas the activity of iNOS generates greater amounts due to its high activity and also present in larger amounts

11. 19/01/201611 The rate of production of NO in endothelial cells is determined by the activity of the enzyme rather than substrate availability (because L-arginine present in excess) However, very high doses of L-arginine can restore endothelial NO biosynthesis in pathological states associated with endothelial function impairment (e.g.hypercholesterolaemia)

12. 19/01/201612 The activity of constitutive isoforms of NOS is controlled by intracellular calcium- calmodulin and is controlled in two ways: –Increased cytosolic [Ca 2+ ] i mediated by endothelium-dependent agonists (e.g. Acetylcholine, bradykinin, substance P). This increases calcium-calmodulin, which activates eNOS or nNOS

13. 19/01/201613 –Phosphorylation of specific residues on eNOS rendering it more or less active at a given [calcium-calmodulin]. This can alter NO synthesis in the absence of any change in [Ca 2+ ] i In resistance vessels, shear stress is probably the main stimulus controlling endothelial NO synthesis

14. 19/01/201614 Shear stress is sensed by endothelial mechanoreceptors and transduced via protein kinase B (Akt), a serine-threonine protein kinase Agonists that increase endothelial cells cAMP (e.g. β 2 agonists) also increase eNOS activity, but via protein kinase A-mediated phosphorylation Protein kinase C reduces eNOS activity by phosphorylation of residues in the calmodulin binding domain, reducing calmodulin binding

15. 19/01/201615 Insulin increases eNOS activity via tyrosine kinase activation Although iNOS has a binding site for calcium-calmodulin (very high affinity), the activity of iNOS is independent of [Ca 2+ ] i

16. 19/01/201616 iNOS is induced by bacterial lipopolysaccharide and /or cytokines synthesised in response to lipopolysaccharide, particularly interferon-γ The antiviral effect of interferon-γ can be explained by this action Tumour necrosis factor-α and interleukin-1 do not alone induce iNOS rather each synergise with interferon-γ in this regard

17. 19/01/201617 iNOS is induced in macrophages and other cells Induction of iNOS is inhibited by: –Glucocorticoids –Several cytokines, including transforming growth factor-β

18. 19/01/201618 Nitric Oxide – Degradation and Carriage Nitric oxide is degraded through a series of chemical reactions –Nitric oxide reacts with oxygen to form Dinitrogen tetroxide or nitrogen tetroxide (N 2 O 4) –N 2 O 4 combines with water to produce a mixture of nitric and nitrous acids –Nitrite ions are oxidised to nitrate by oxyhaemoglobin;- these reactions summarised as follows:-

19. 19/01/201619 2NO + O 2 N 2 O 4 N 2 O 4 + H2ONO 3 - + NO 2 - + 2H + NO 2 - + HbONO 3 - + Hb

20. 19/01/201620 Low NO concentrations are relatively stable in air because the reaction between NO and oxygen is a second -order reaction For this reason, small amounts of NO produced in the lung escape degradation and can be detected in exhaled air NO reacts very rapidly with even low concentrations of superoxide anion (O 2 - ) to produce peroxynitrite anion (ONOO - ), which is responsible for some of its toxic effects.

21. 19/01/201621 Endothelium-derived NO acts locally on underlying vascular smooth muscle or on adherent monocytes or platelets A strong, but still controversial, case has been made that NO can also act at a distance in the mammalian circulation via reversible interaction with haemoglobin Haem of haemoglobin has an affinity for NO greater than for oxygen

22. 19/01/201622 In the absence of oxygen, NO bound to haem is relatively stable In the presence of oxygen, NO is converted to nitrate and the haem iron oxidised to methaemoglobin Therefore, NO is inactivated by combination with the haem of haemoglobin or by oxidation to nitrite and nitrate which are excreted in urine

23. 19/01/201623 Alternatively, under physiological conditions, a specific cystine residue in globin combines reversibly with NO to produce S-nitrocylated haemoglobin, which acts as an intravascular NO storage protein S-nitrocylated haemoglobin is believed to be involved in various NO-related activities such as control of vascular resistance, blood pressure and respiration

24. 19/01/201624 Proposed mechanisms through which S- nitrosylated haemoglobin may be involved in NO-related activities include: –Nitrocylation of haemoglobin is reversible –Hb takes up NO in the lungs (depending on oxygenation) and releases it in tissues, including the respiratory centre in the brain, in concert with release of oxygen

25. 19/01/201625 –Haemoglobin acts as an oxygen sensor and could regulate vascular tone (and hence tissue perfusion) in response to local partial pressure of oxygen-PO 2 by releasing NO (this mechanism impaired in sickle cell disease)

26. 19/01/201626 –NO from S-nitrocylated haemoglobin is not released into the cytoplasm of erythrocytes (would be promptly inactivated by haem), but is transported out of red cells via cystine residues in the haemoglobin binding domain of an anion exchanger called AE1 –AE1 is the most abundant protein in red cell membranes, and is responsible for exchange of chloride and bicarbonate ions across the cell membrane

27. 19/01/201627 Nitric Oxide – Biochemical and Cellular Effects Nitric oxide reacts with various metals, thiols and oxygen species, thereby modifying proteins, DNA and lipids Through its haem group, activation of soluble guanylate cyclase, heterodimer present as distinct isoenzymes in vascular and nervous tissue (most important biochemical effects)

28. 19/01/201628 Activated guanylate cyclase synthesises the second messenger cGMP and many physiological effects of low concentrations of NO are mediated by cGMP Effects of NO are terminated by phosphodiesterase enzymes NO diffuses from site of synthesis and activates guanylate cyclase in neighbouring cells where the resulting increase in cGMP affects kinase G, cyclic nucleotide phosphodiesterases, ion channels and possibly other proteins

29. 19/01/201629 The effect of NO on neighbouring cells ends in the inhibition of the [Ca 2+ ] i -induced smooth muscle contraction and platelet aggregation that would occur in response to agonists NO also hyperpolarises vascular smooth muscles through potassium channel activation NO inhibits monocyte adhesion and migration; and proliferation of smooth muscle cells and fibroblasts, cellular effects probably underlying antiatheroscelerotic action of NO

30. 19/01/201630 Large amounts of NO (released following NOS induction or excess stimulation of N- methyl-D-aspartic acid [NMDA] receptors in the brain) cause cytotoxic effects These cytotoxic effects may result from direct effect of NO or effect of NO via peroxynitrite anions and contribute to host defence, but also to neural damage associated with overstimulation of NMDA receptors by glutamate Paradoxically, NO is also cytoprotective under some circumstances

31. 19/01/201631 Nitric Oxide – Vascular Effects The endothelial L-arginine/NO pathway is tonically active in resistance vessels reducing vascular resistance, and hence systemic blood pressure Mutant mice that lack the gene coding for eNOS are hypertensive suggesting a role for NO biosynthesis in the physiological control of blood pressure

32. 19/01/201632 Increased NO generation may contribute to the generalised vasodilation occuring during pregnancy

33. 19/01/201633 Nitric Oxide – Neuronal Effects NO is a non-adrenergic and non-cholinergic (NANC) neurotransmitter in many tissues, neurotransmitter other than acetylcholine and noradrenaline NO as a NANC is important in upper airways, GIT, and in the control of penile erection NO is implicated in the control of neuronal development and of synaptic adaptability in the CNS

34. 19/01/201634 Mice carrying a mutation disrupting the gene coding nNOS have grossly distended stomachs similar to those seen in human hypertrophic pyloric stenosis ( a disorder characterized by pyloric hypertrophy causing gastric outflow obstruction, which occurs in approximately 1 to 150 male infants and is corrected surgically).

35. 19/01/201635 nNOS knock out mice resist stroke damage caused by middle cerebral artery ligation but are aggressive and oversexed (Characteristics that may not be unambiguously disadvantageous, at least in the context of natural selection)

36. 19/01/201636 Nitric Oxide – Host Defence Cytotoxic and/or cytostatic effects of NO are implicated in primitive non-specific host defence mechanisms against numerous pathogens including viruses, bacteria, fungi, protozoa and parasites as well as against tumour cells

37. 19/01/201637 NO damages invading pathogens through nitrosylation of nucleic acids, and combination with haem-containing enzymes, such as Mitochondrial enzymes involved in cell respiration

38. 19/01/201638 Nitric Oxide – Therapeutic Approaches Use of NO itself –Pulmonary vasodilatation is the main action of inhaled NO –Inspired NO acts preferencially on ventilated alveoli, and could be therapeutically useful in respiratory distress –Inhaled NO dilates blood vessels in ventilated alveoli (exposed to inspired gas) thus reducing shunting

39. 19/01/201639 –Therefore NO used in ICUs to reduce pulmonary hypertension and to improve oxygen delivery in patients with respiratory distress syndrome, but not known whether improve long-term survival

40. 19/01/201640 Use of NO Donors –Nitric oxide donors (e.g. Nitroprusside and organic nitrovasodilators, glyceryl trinitrate (GTN) are well established agents for angina and acute heart failure syndromes

41. 19/01/201641 Inhibition of nitric oxide synthesis –There is therapeutic interest in selective inhibitors of different isoforms of NOS –Drugs for long-term treatment should not inhibit eNOS to avoid adverse cardiovascular effects –Selective inhibitors of iNOS versus the two constitutive forms have been described (e.g. N- iminoethyl-L-lysine) and have potential for the treatment of inflammatory and other conditions in which iNOS has been implicated (e.g. asthma)

42. 19/01/201642 –Inhibition of NO biosynthesis (e.g. By NG- monomethyl-L-arginine ( L-NMMA) is being investigated in disorders where there is overproduction of NO such as inflammation and neurodegenerative disease Potentiation of NO –Several means through which the L-arginine/NO pathway could be enhanced are under investigation –The hope is that potentiating NO will prevent atherosclerosis or its thrombotic complications or have beneficial effects attributable to NO

43. 19/01/201643 Some established possibilities for potentiation of NO include –Drugs that restore endothelial function in patients with metabolic risk factors for vascular disease (e.g. Angiotensin converting enzyme - ACE inhibitors, statins, insulin, oestrogens) –β 2 -adrenoceptor agonists and related drugs (e.g. Nebivolol, a β 1 -adrenoceptor antagonist that is metabolised to an active metabolite that activates the L-arginine/NO pathway)

44. 19/01/201644 –Type V phosphodiesterase inhibitors (e.g. Sildenafil, tadalafil) potentiate NO actions in the corpora cavernosa of the penis and are used: To treat erectile dysfunction Other possible uses (pulmonary hypertension, gastric stasis) are being investigated

45. 19/01/201645 Clinical Conditions in which Nitric Oxide May Play a Part Sepsis: can cause multiple organ failure. Whereas NO benefits host defence by killing invading organisms, excessive NO causes harmful hypotension However, disappointingly, L-NMMA (N- monomethyl-L-arginine) worsened survival in one controlled clinical trial Systemic vasodialtation ( due to excess NO?) is typical in patients with hepatic cirrhosis and chronic low grade endotoxaemia

46. 19/01/201646 Nitrosative stress and nitration of proteins in airway epithelium may contribute to steroid resistance in asthma, and the ineffectiveness of glucocorticoids in chronic obstructive pulmonary disease Nitric Oxide biosynthesis is reduced in patients with hypercholesterolaemia and some other disorders that predispose to athreromatous vascular disease including cigarette smoking and diabetes mellitus

47. 19/01/201647 Further reading: Pharmacology by Rang & Dale-(7 th Edition Chapter 20)