1. Presented by ROHIT BISHT (M. pharma Pharmacology I.S. F. College of pharmacy , Moga,Punjab

2. ENDOTHELIUM 3. ENDOTHELIUM The endothelium is the thin layer of cells that lines the interior surface of Blood vessels and lymphatic vessels forming an interface between circulating blood and lymph in the lumen and the rest of the vessel wall. The cells that form the endothelium are called endothelial cells. Endothelial cells in direct contact with blood are called vascular endothelial cells where as those in direct contact with lymph are known as lymphatic endothelial cells. 4. Endothelial cells release substances acting directly on vascular smooth muscle cells, causing either contraction or relaxation. 5. In 1980 Furchgott & Zawadzki first described endothelium- dependent relaxation of the blood vessels by acetylcholine. Further studies in 1984 revealed that other factors such as bradykinin, histamine and 5-hydroxytryptamine release endothelium derived relaxing factor (EDRF), which can modulate vessel tone. In 1987 Furchgott proposed that EDRF might be nitric oxide (NO) based on a study of the transient relaxations of endothelium-denuded rings of rabbit aorta to acidified inorganic nitrite (NO-) solutions and the observations that superoxide dismutase (SOD, which removes O2 -) protected EDRF. ENDOTHELIUM DERIVED RELAXING FACTOR 6. In 1988 Palmer et al could detect NO production both biologically and chemically by chemiluminescence. The following year in 1989 the enzyme responsible for NO production, NO synthase, was discovered and NO pathway was proposed. Neuronal and humoral mediators, e.g. Ach, adrenaline , noradrenaline , histamine ,5-HT, ATP, adenosine, substance P , arginine vasopressin (AT/VP), bradykinin, thrombin and Ca++ - ionophore A 23187 , VEGF , insulin, angiotensin , TNF-, IL-6 , arginase , asymmetric dimethylarginine (ADMA), dimethylarginine dimethylaminohydrolase (DDAH), etc. acting in their corresponding receptors or cellular structures can affect production and release of NO. 7. NITRIC OXIDE A chemical compound with formula NO is a free radical gas. It is first identified as endothelial derived releasing factor(E D R F ). At high concentration , fight against infectious organism and cancer cell. At lower concentration helps in regulating the circulatory and central nervous system. Nitric oxide differs from other neurotransmitter and hormones in a way that it is not regulated by storage, release , or targeted degradation. No does not require receptor for its action when synthesized immediately utilized. Ca++ clamudulin complex is necessary for nitric oxide synthesis. 8. The structure and nature of Nitric Oxide Nitric oxide is a di atomic free radical consisting of one atom of nitrogen and one atom of oxygen. Lipid soluble and very small for easy passage between cell membranes. Short lived, usually degraded or reacted within a few seconds. The natural form is a gas. N O 9. Synthesis of Nitric Oxide Nitric oxide is synthesized from L-arginine. This reaction is catalyzed by nitric oxide synthase, a 1,2,9,4 amino acid enzyme. COO- C (CH2)3 NH C H2N H NH2+ +H3N Arginine NOS NADPH + O2 NAD+ COO- C (CH2)3 NH C H+H3N N + H2N H OH N-w-Hydroxyarginine COO- C (CH2)3 NH H+H3N + NO NOS C O NH2 Citrulline 10. Release & m/a of No- 11. Intracellular mechanism: When nitric oxide forms in large parts because superoxide anion has a height affinity for No. Superoxide anion reduces No bioavailability. Nitric oxide also binds to the heme moiety of hemoglobin and heme moiety of enzyme gunayl cyclase , which is found in smooth muscle cell and most other cells of body. When NO formed by vascular endothelium it rapidly diffuses into the blood where it binds to hemoglobin & subsequently broken down. 12. It also diffuses into vascular smooth muscle cells adjacent to the endothelium where it binds to & activate gunyl cyclase . This enzyme catalyse the dephosprylation of GTP to cGMP which serve as a second messenger for many important cellular function , particular for signaling smooth muscle contraction. cGMP induces smooth muscle relaxation by multiple mechanism including- increased intracellular cGMP which inhibit ca++ entry into the cell and decrease intracellular ca++ concentration. activates k+ channel which leads to hyper polarization & relaxation . Stimulates a cGMP dependent protein kinase that activates myosin light chain phosphate (MLCK) the enzyme that dephosphorylate myosin light chain leads to smooth muscle relaxation 13. Types of NOS NOS I or n NOS Central and peripheral neuronal cells, brain, spinal cord, platelets. Ca++ dependent, used for neuronal communication Constitutive NOS II or I NOS Most nucleated cells, particularly macrophages Independent of intracellular Ca++ and its regulation depend upon de novo synthesis. Inducible in presence of inflammatory cytokines, bacterial liposaccharides. NOS III or e NOS Present on Vascular endothelial cells and neuronal cells Ca+2 dependent Vascular regulation NOS Constitutive Inducible 14. Nitric Oxide in the human body has many uses which are best summarized under five categories. NO in the nervous system NO in the circulatory system NO in the muscular system NO in the immune system NO in the digestive system No in the reproductive system NO in the gene toxicity No in the apoptosis 14 Role of nitric oxide 15. NO is a signaling molecule, but not necessarily a neurotransmitter. NO signals inhibition of smooth muscle contraction, adaptive relaxation, and localized vasodilation. n nos action in C N S have been associated with pain perception in spinal cord level. NO diffuses out of the cells making it vescular storage in vesicles and release by exocytosis NO does not bind to surface receptors, but instead exits cytoplasm, enters the target cell, and binds with intracellular guanyl cyclase Present in presynaptic terminal 15 Nitric oxide in the Nervous system NO serves in the body as a neurotransmitter, but there are definite differences. 16. Role in Neurodegenerative disease Implicated in :- Alzheimer disease Parkinson disease Huntington disease Amyotrophic leteral sclerosis All are related to the excessive release of NO & glutamate both. But in Parkinson's disease Glial cells produce excessive levels of nitric oxide, which may be neurotoxic for a sub population of dopaminergic neurons, especially those not expressing NADPH- diaphorase activity. The presence of glial cells expressing nitric oxide synthase in the substantia nigra of patients with Parkinson's disease represents a consequence of dopaminergic neuronal loss. 17. Play a role in long term memory As a retrograde messenger that facilitates long term potentiation of neurons (memory) Synthesis mechanism involve Ca2+/Calmodulin activates NOS-I activates Guanyl cyclase cycle of nerve action potentials catalyzes cGMP production 18. Nitric oxide in the circulatory system NO serves as a vasodilator Released in response to high blood flow rate and signaling molecules (Ach and bradykinin) Highly localized and effects are brief If NO synthesis is inhibited, blood pressure increases NO aids in gas exchange between hemoglobin and cells Hemoglobin is a vasoconstrictor, Fe scavenges NO NO is protected by cysteine group when O2 binds to hemoglobin. During O2 delivery, NO locally dilates blood vessels to aid in gas exchange 19. Nitric oxide in the Muscular system NO was originally called EDRF (endothelium derived relaxation factor) NO signals inhibition of smooth muscle contraction Ca 2+ is released from the vascular lumen activating NOS NO is synthesized from NOS III in vascular endothelial cells This causes guanyl cyclase to produce cGMP A rise in cGMP causes Ca2+ pumps to be activated, thus reducing Ca2+ concentration in the cell This causes muscle relaxation 20. Role in Blood vessels 21. Role in the Immune system NOS II catalyzes synthesis of NO used in host defense reactions Activation of NOS II is independent of Ca2+ in the cell Synthesis of NO happens in most nucleated cells, particularly macrophages. NO is a potent inhibitor of viral replication. NO is a bactericidal agent NO is created from the nitrates extracted from food near the gums. This kills bacteria in the mouth that may be harmful to the body. 22. Role In the Digestive system NO is used in adaptive relaxation NO promotes the stretching of the stomach in response to filling. When the stomach gets full, stretch receptors trigger smooth muscle relaxation through NO releasing neurons. Role In the Reproductive system: Nos localized in pelvic nerve neuron innervating the corpora cavrinosa and the neuronal plexuses of the adventitial layer of the penile arteries proven most effective for erectyl dysfunction. 23. No and its derivatives produced in inflamed tissue contribute to the carcinogenesis process due to direct or indirect DNA damage. Direct DNA damage: DNA deamination , peroxynitrite induced adult formation single strand break in the DNA Indirect DNA damage: interaction of NO reactive species with other molecule like amines , thioles , lipids. -NO after reaction with O2/superoxide forms genotoxicity . Role In Genotoxicity: 24. Role in wound healing & tissue repair No is powerful stimulator of cell division maturation and differentiation . Necessary mediator of neuro vascularization i.e. angiogenesis and lymph ducts to nourish the healing of tissue. Role in Apoptosis Nitric oxide and its reaction products either promotes or prevent apoptosis . Pro-apoptosis effect of NO- induction of apoptosis by NO resulting in the accumulation of tumor suppressor protein p-53. Anti-apoptotic effect of NO- some studies suggest that endogenous I Nos expression or exposure to low dose of NO donors inhibits apoptosis. 25. Role in Inflammation NO has shown to act as a mediator of inflammatory processes. This process has enhanced the effect of cyclooxygenases and stimulates the production of pro- inflammatory eicosanoids. 26. PROSTACYCLINE Prostacyclin (or PGI2) is a prostaglandin member of the family of lipid molecules known as eicosanoids. It inhibits platelet activation and is also an effective vasodilator. In 1960s, ,Professor John Vane, began to explore the role of prostaglandins in anaphylaxis and respiratory diseases. Sir John discovered that aspirin and other oral anti-inflammatory drugs work by inhibiting the synthesis of prostaglandins. Sir John and a team had identified a lipid mediator they called PG-X, which inhibits platelet aggregation. PG-X, which later would become known as prostacyclin, is 30 times more potent than any other then-known anti-aggregatory agent. 27. E D H F : In blood vessels Endothelium-Derived Hyperpolarizing Factor or EDHF is proposed to be a substance or electrical signal that is generated or synthesized in and released from the endothelium; its action is to hyperpolarise vascular smooth muscle cells, causing these cells to relax, thus allowing the blood vessel to expand in diameter. Both a vascular endothelial cytochrome P450 (CYP450) product of arachidonic acid metabolism and the potassium ion (K+) have been identified as endothelium-derived hyperpolarizing factors (EDHFs) in animal vascular tissues. 28. Pathways Of EDHF:- There are two general pathways that explain EDHF:- 1. Diffusible factors are endothelium-derived substances that are able to pass through internal elastic layer (IEL), reach underlying vascular smooth muscle cells at a concentration sufficient to activate ion channels , and initiate smooth muscle hyperpolarisation and relaxation. 2. Contact-mediated mechanisms bestow endothelial hyperpolarisation that passively spreads to the smooth muscle through inter cellular coupling and therefore EDHF is considered as a solely electrical event. 29. Although the phenomenon of EDHF has been observed and reported in scientific literature, to date the chemical identity of the factor(s) has not been determined. 1) In some cases, members of a class of arachidonic acid derivatives, the epoxyeicosatrienoic acids (EETs), have been found to mediate the vasodilatation. These compounds are formed by epoxidation of any one of four double bonds of the arachidonic acid carbon backbone by cytochrome p450 epoxygenase enzymes. Structure of Arachidonic acid 30. 2) In addition, in some cases hydrogen peroxide has been suggested to function as an EDHF in some vascular beds; although the validity of this observation is debated. because the H2O2 candidacy is questioned by the fact that it may have an inhibitory action on K+ channels, at least, in some vascular beds. 3) It Has been suggested that EDHF is Potassium Ions (K+) as the activation of endothelial K-Ca+ channels causes an efflux of K+ from endothelial cells towards the extracellular space. 31. An increase in extracellular K+ has been shown to activate an ouabain -sensitive electrogenic Na+K+- ATPase followed by hyperpolarization and smooth muscle cell relaxation. However the involvement of K+ ions into EDHF- mediated relaxation does not necessarily involve the activation Na+K+-ATPase channels. It is more likely that K+ ions and gap junctions can be involved in EDHF-mediated relaxation simultaneously, and may also act synergistically. 32. 4. An alternative explanation for the EDHF phenomenon is that direct intercellular communication via gap junctions allows passive spread of agonist-induced endothelial hyperpolarization through the vessel wall. In some arteries, eicosanoids and K+ ions may themselves initiate a conducted endothelial hyperpolarization, thus suggesting that electro tonic signalling may represent a general mechanism through which the endothelium participates in the regulation of vascular tone. 33. INTERACTION B/W NO,EDHF AND PROSTACYCLIN:- The three main mediators of endothelial vasodilator function, NO, prostacyclin, and EDHF appear not to be mutually exclusive and act synergistically in a complex manner to maintain the health of the vasculature . In arteries, NO is the predominant endothelium-derived vasodilator but has relatively less prominent contribution in the resistance vessels of the microcirculation where EDHF appears to predominate. NO may inhibit EDHF responses as some studies could only demonstrate. EDHF responses once NO production had been inhibited. 34. Pharmacological inhibitors Targets Comments Apamin S KCa + Highly specific Charybdotoxin IKCa +-BKCa + Can inhibit some Kv channels Iberiotoxin B KCa + Highly specific Tetraethylammonium SKCa +-IKCa +-BKCa + Inhibit other K+channels at >102)m Tetraethybutylammonium S KCa +-I KCa +-B KCa + Inhibit other K+channels at >102m BaCl2 KIR + Ouabain Na+/K+ ATPase Can affect gap junction activity at >104m KCL K+ currents Dilates at >102m through KIR + and Na+/K+ ATPase activation 18 -glycyrrhetic acid Gap junctions Possesses nonjunctional effects on membrane currents Connexin mimetic peptides Gap junctions Highly specific Catalase Hydrogen peroxide 35. EDHF IN DISEASES Experimental evidence indicates that a shift away from NO- mediated endothelium-dependent relaxation toward EDHF dependent relaxation occurs in disease states. Alteration of EDHF-mediated responses has been reported with aging, hypertension, atherosclerosis, hypercholesterolemia, heart failure, angioplasty, eclampsia, diabetes. EDHF in Hypertension Evidence suggests that CYP expression and EET generation are increased in hypertension and in hypercholesterolemia. Polymorphisms within CYP epoxygenases being associated with an enhanced risk of developing coronary artery disease and hypertension. 36. EDHF in diabetes EDHF-mediated responses are depressed in some models of type I and type II diabetes with the exception of murine models. EDHF in hypercholesterolemia In hypercholesterolemia, it has been observed that there is significant contribution of KCa+ channel activation and a lower Nitric Oxide release with acetylcholine. 37. Demonstrated level of E D R Fs-