PHA 316 Other Neurotransmitters: Nitric Oxide and Purines PDF

Other Peripheral Mediators Purine nucleosides and nucleotides Plus Nitric Oxide  Objectives Purines – List the different purine nucleoside an nucleotides that act as mediators – List the different purinergic receptors and their principal endogenous l...

Other Peripheral Mediators Purine nucleosides and nucleotides Plus Nitric Oxide  Objectives Purines – List the different purine nucleoside an nucleotides that act as mediators – List the different purinergic receptors and their principal endogenous ligands – Describe the role of adenosine, ADP and ATP as extracellular signaling molecules/ mediators and list some of the physiological processes mediated by them – Describe pharmacological interventions affecting purinergic signalling, key drug examples and their mechanisms of action and clinical use, and key interactions Nitric oxide  Purines Overview Recall that nitrogenous bases present in the DNA can be grouped into two categories: purines (Adenine (A) and Guanine (G)), and pyrimidine (Cytosine (C) and Thymine (T)) Purines are found in all of the body’s cells They are found in many foods with high purine foods are also high-protein foods and they include organ meats like kidney, fish like mackerel, herring, sardines and also yeas  Purine nucleoside and nucleotides Overview Recall purine nucleosides, especially adenosine, and nucleotides, especially ADP and ATP because of their crucial role in DNA/RNA synthesis and energy metabolism. But it is also important to note that they also produce a wide range of pharmacological effects that are unrelated to their role in energy metabolism. Focus here is on purine nucleoside and nucleotides as MEDIATORS where they function extracellularly as signalling molecules that produce a wide range of pharmacological effects.  Purine nucleoside and nucleotides as mediators - Overview Focus for this lesson is on purine nucleoside (adenosine) and purine nucleotides (ADP and ATP) as mediators 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. The physiological significance; and hence therapeutic relevance of the various receptor subtypes is still being unraveled  Nucleosides/Nucleotide as mediators – Site of action ATP,ADP and Adenosine work extracellularly as signalling molecules and produce diverse physiological/pharmacological effects. Fig 17.1  Nucleosides/Nucleotide as mediators – Physiological effects Purinergic signalling is involved in controlling a number of physiological processes Physiological control by purinergic signalling Regulation of Regulation of Regulation of Regulation of Neurotransmi coronary myocardial platelet immune tter in both blood flow function aggregation response CNS and PNS  Nucleosides/Nucleotide as mediators – Purinergic Receptors - Overview There are three main families of purine receptors each with several subtypes.(A, PY and PX) They utilize either cAMP or phospholipase C activation as their signalling system They respond to various adenine nucleoside and nucleotides, generally adenosine, ADP or ATP The subtypes in each family may be distinguished on the basis of their molecular structure as well as their agonist and antagonist selectivity. Refer to table Rang and Dale 17.1 for details: – Adenosine receptors (A1, A2A, A2B, A3): G-protein coupled – P2Y and subtypes: G-protein coupled – P2X and subtypes: receptor gated cation selective ion channels (ligand gated) EXERCISE: From table 17.1 what is the principal endogenous ligand for each of the above receptors?  Adenosine as mediator The simplest of the purines, adenosine is found in biological fluids throughout the body. Figure 17.1 Adenosine exists free in the cytosol of all cells and is transported in and out of cells mainly via a membrane transporter. Extracellular adenosine in tissues comes partly from this intracellular source and partly from extracellular hydrolysis of released ATP or ADP Adenosine can be inactivated to inosine by adenosine deaminase Virtually, all cells express one or more A-receptors and so adenosine produces many pharmacological effects, both in the periphery and in the CNS.  Adenosine as mediator: Cardiovascular System The main effects of adenosine in the Cardiovascular system: CV it exerts: Heart: a negative chronotropic effect by suppressing the automaticity of cardiac pacemakers, and a negative dromotropic effect through inhibition of AV-nodal conduction (antidysarrhythmic effect) Vasculature: Vasodilation and can cause hypotension (A2?) and cardiac depression (A1) Platelets: inhibition of platelet aggregation acting via A2A and A2B  Adenosine as mediator: Respiratory System The main effects of adenosine in Resp are: Respiratory system Adenosine receptors are found on all the cell types involved in asthma and the overall pharmacology is complex A1 receptors: promotion of mediator release from mast cells and hence Enhanced mucus secretion Bronchoconstriction Activation of leukotriens A2A receptors: Anti-inflammatory response  Adenosine related pharmacological agents: Clinical utility Areas of Clinical utility Cardiovascular system Asthma Inflammation CNS later Clinical uses of Adenosine and drugs that affect adenosine – CV system Cardio-Vascular system: Adenosine: – via intravenous bolus injection to terminate supraventricular tachycardia and convert to sinus rhythm. It is safer than Beta-blockers or verapamil, because of its short duration of action Dipyridamole: – Blocks adenosine cellular uptake. Used as antiplatelet drug with vasodilatory effects. Dipyridamole is used with other drugs to reduce the risk of blood clots after heart valve replacement. – Explain how inhibition of adenosine uptake contributes to activity of dipyridamole as antiplatelet agents? Clinical uses of Adenosine and drugs that affect adenosine CV – Drug Interactions Drug Interactions: – Adenosine is reported to interact with dipyridamole and concurrent administration requires reduction in dose of adenosine. – From your understanding of their respective mechanisms of action, explain the underlying mechanism for this interaction between adenosine and dipyridamole Clinical uses of Adenosine and drugs that affect adenosine – Respiratory Respiratory system: Methylxanthines e.g. theophylline Block adenosine receptors and is used in asthma Also said to inhibit phosphodiesterase – Explain how inhibition of adenosine receptors contributes to activity of theophylline as anti asthma agent? – Which adenosine receptors need to be blocked to produce the above effect in asthmatics? Clinical uses of Adenosine and drugs that affect adenosine - Summary Drug Interactions: methylxanthines e.g. theophylline or caffeine interaction with adenosine – Theophylline and caffeine interact with exogenously administered adenosine. What do you think would be the effect of this interaction between theophylline and adenosine?  Adenosine Adverse effects Caution in asthma? Explain the possible underlying mechanism Side effects include bradycardia, hypotension, dyspnoea  Adenosine diphosphate (ADP) as mediator ADP usually stored in vesicles and released by exocytosis – Exerts its activity thro P2Y receptors – When released, it exerts its biological effects predominantly through the P2Y family of receptors – Once released can be converted to adenosine by extonucletidases  ADP as mediator – Physiological Function Physiological function: ADP and platelets – ADP acts on platelets, causing aggregation How does this differ from effects of adenosine on platelets? – Released when platelets are activated - The secretory vesicles of blood platelets store both ATP and ADP in high concentrations, and release them when the platelets are activated – ADP acts on platelets, causing aggregation – The receptor involved is P2Y12. How does activation of P2Y12 receptors by ADP affect cAMP levels? Clinical uses of drugs that affect ADP - Summary Pharmacological agents – Clopidogrel, It is is a P2Y12 antagonists and exert their anti- aggregating effects through this mechanism. Used for prevention of arterial thromboembolic disorders usually in combination with aspirin Adenosine and related products  ATP as a mediator ATP as neurotransmitter ATP in nociception ATP in inflammation  ATP AS A MEDIATOR – Self study It is present in all cells in millimolar quantities and released when cells are damaged, Release is via number of mechanisms – Exocytosis – ATP transporter – Channels on membrane ATP exerts its action primarily through the P2X receptors primarily but also interact with P2Y receptors. 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. 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 Other peripheral mediators continued NITRIC OXIDE AS A MEDIATOR Nitric Oxide as a mediator Objectives Outline the nitric oxide biosynthesis pathway Describe the physiological role of nitric oxide as a mediator Describe the bio-synthesis and bio-signaling pathway for nitric oxide List key clinical conditions due to dysregulation of NO synthesis/signaling pathways Describe the pharmacological control of the above dysregulation  Nitric Oxide as a mediator Overview Nitric oxide has many physiological activities in various tissues It’s a key signalling molecule in the CV, NS and has role in host defence e.g – important vasodilator – Role in cell death and neurotransmission These can serve as potential sites for dysregulation and subsequent pharmacological intervention Will review biosynthesis and signalling and then potential sites for pharmacological intervention Endogenous Nitric Oxide Biosynthesis Endogenous NO synthesized from arginine by family of enzymes = Nitric oxide synthase (NOS) These intracellular enzymes are activated by calcium influx or by cytokines Differences between the isoforms of NOS enzymes and significance thereof – 2 constitutive (eNOS/NOS3 and nNOS/NOS1) – 1 Inducible (iNOS/NOS2) responsive to pathological stimuli. Found in macrophages and smooth muscles Activity of enzymes as determinants for rate of production of NO – Constitutive enzymes generate small amounts of NO. NOS2 produces much greater amounts, due to its (high activity and its abundance in pathological states) – When high dose L-Arginine can restore endothelial NO biosynthesis Effects of cGMP on dephosphorylation and inactivation of myosin light chain resulting in relaxation of muscle cells Endogenous Nitric Oxide Biosynthesis NO diffuses to sites of action in neighbouring cells. This is regulated by the redox state of haemoglobin alpha (i.e. Fe3+(signalling) versus Fe2+ (stop)) Nitric oxide is not stored in cells Nitric oxide (NO) synthase can be inhibited by arginine analogues e.g. L-NMMA, Inactivation of NO – Combination with haem on haemoglobin – by oxidation to nitrite and nitrate, which are excreted in urine; Drugs that cause release of NO do so by stimulating NOS and they include – Ach, muscarinic agonists, histamine Physiological Role of Nitric Oxide  Physiological Effects of Nitric Oxide See table 21.1 Gaseous, unstable, not Relaxation of vascular smooth muscles - stored in cells Half Life – Cardiovascular vasodilation less than 5-10 sec Platelet Aggregation inhibition Site of production versus site of action Host defense Virus, bacteria etc – Ability to diffuse through cell membranes Peripheral – GIT, penile Other substances that erection, upper resp etc stimulate endogenous Nervous System production of nitric oxide CNS - later Clinical conditions associated with derangement  Effects of Nitric oxide Smooth muscles – Vasodilator – Role in erectile tissue function in which smooth muscle relaxation is required to bring about the influx of blood that causes erection Cell adhesion – Reduced platelet aggregation – Reduced neutrophil adhesion Inflammation – Facilitates inflammation directly and through influence of prostaglandins  Nitric Oxide – Signaling Pathways Site of Production vs Site of Action NO as signaling mediator cGMP as second messenger Termination of activity Physiological effects  Common Pathological Condition Associated with Dysregulation of Nitric Oxide Over or under production – still area of immense research Over Activity – Hypotension – Neurodegenerative disorders – Septic shock Conditions associated with reduced NO activity – Atherosclerosis – Erectile dysfunction – Thrombosis – Hypercholesteremia  Exercise Table 21.1 What is the effect of NO on endothelium/vascular smooth muscle? – What physiological process is controlled by the above effect? – What is the effect of excess NO on the above physiological process? – What is the effect of excess NO on the above physiological process? Nitric Oxide – Signaling Pathways and dysregulation and related Pharmacological interventions High Level Pharmacological Intervention – Details later  Nitric Oxide related Pharmacological intervention – Clinical Use NO Pharm Interventions NO – inhalation of Inhibition of nitric low volumes for Nitric oxide NO replacement or oxide – mainly pulmonary donors/precurosors - potentiation research vasodilation Dietary supplement Respiratory distress Nitroprusside, with L-arginine or syndrome inorganic nitrates PDE type 5 Inhibitors Nitroglycerine - sildenafil  Exogenous Nitric Oxide donors Nitric oxide is released from several important drugs including – Nitroprusside Occurs spontaneously in the blood in the presence of oxygen – Nitrates Intracellular release and requires presence of thiol compounds. Tolerance may happen if endogenous thiol compounds are depleted – Nitrites Intracellular release and requires presence of thiol compounds. Tolerance may happen if endogenous thiol compounds are depleted Endogenous Nitric Oxide Biosynthesis Drugs that stimulate synthesis of NO by stimulating NOS. Such include – Acetylcholine – Other muscarinic agents – histamine High Level Pharmacological Intervention – Details later - Reflection Nitric oxide donors e.g. NTG – Why not NO direct? PDE Inhibitors e.g. Sildenafil – Why is concurrent use of nitrates e.g. nitroglycerine and sildenafil contraindicated? Potentiation of nitric oxide -Investigational – Selective donors replacement therapy – Dietary supplements Exercise: Investigate, use of L-Arginine supplement, Indications supported by literature, drug interactions that as a pharmacist you would need to be aware of – Antioxidants – Drugs that restore endothelial function in patients with metabolic risk factors