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Chapter 17. Nitric oxide. Intro. It is formed in an enzyme-catalysed reaction between molecular oxygen and Larginine.

NO is a key signaling molecule in the cardiovascular and nervous systems, and has a role in host defense. No is the endogenous activator of cGMP, an important second messenger in many cells. It has a particular high affinity for haem, and guanylate cyclase contains a heam group. Biosynthesis of nitric oxide and its control. Three known isoforms: an inducible form (iNOS or NOS-II; expressed in macrophages and kupffercells, and two so-called constitutive forms, present under physiological conditions in endothelium (eNOS or NOS-III) and in neurons (nNOS or NOS-I). Both nNOS and iNOS are soluble cytosolic enzymes. L-Argine is usually present in excess in endothelial cell cytoplasm, so the rate of production of NO is determined by the activity of the enzyme rather than by substrate availability. Very high doses of Largine can restore endothelial NO biosynthesis in some pathological states in which endothelial function is impaired. Possible explanations are: - Compartmentation: existence of a distinct pool which can become depleted despite apparently plentiful total cytoplasmic arginine concentrations; - Competition with endogenous inhibitors of NOS; - Reassembly/reactivation of enzyme. The activity of constitutive isoforms of NOS is controlled by intracellular calcium-calmodium: - Many endothelium-dependent agonist increase the cytoplasmic concentration of calcium ions; - Phosphorylation of specific residues on eNOS renders it more or less active at a given concentration of calciumcalmodulin; can alter NO synthesis in the absence of any change in Ca2+. Main physiological stimulus controlling endothelial NO synthesis in resistance vessels is probably shear stress. Is is transduced via a serine-threonine protein kinase called Akt or protein kinase B. Agonists that increase cAMP in endothelial cells also increase eNOS activity. Protein kinase C reduces eNOS activity by phosphorylating residues in the calmodulin-binding domain, thereby reducing the binding of calmodulin. Activity of iNOS is independent of Ca2+. This is because it has a very high affinity, so iNOS is activated even at low values of Ca2+ present under resting conditions. Haem has an affinity for NO > 10 000 times greater than for oxygen. In the presence of oxygen NO is converted to nitrate and the haem iron oxidized to methaemoglobin. Effects of nitric oxide. One of its most important biochemical effects is activation of soluble guanylate cyclase. Guanylate cyclase synthesis the second messenger cGMP. NO activates the enzyme by combining with its haem group. It contains another regulatory site, which is NO-independent. Effects of cGMP are terminated by phosphodiesterase enzymes. Sildenafil and Tadalafil are inhibitors of phosphodiesterase type V that are used to treat erectile dysfunction, because they potentiate NO actions in the corpora cavernosa of the penis by this mechanism.

Nitric oxide can activate guanylate cyclase in the same cells that producte it, giving rise to autocrine effects. The resulting increase in cGMP affects protein kinase G, cyclic nucleotide phosphodiesterases, ion channels and possibly other proteins. This inhibits Ca2+ induced smooth muscle contraction and platelet aggregation that occur in response to agonists. NO inhibits monocyte adhesion and migration, adhesion and aggregation of platelets and smooth muscle and fibroblast proliferation. - Vascular effects: mutant mice that lack the gene coding for eNOS are hypertensive, consistent with a role for NO biosynthesis in the physiological control of blood pressure. - Neuronal effects: It is implicated in the control of neuronal development and of synpaptic plasticity in the CNS. Mice carrying a mutation disrupting the gene coding nNOS have grossly distended stomachs similar to those seen in human hypertrophic pyloric stenosis. - Host defence: cytotoxic and/or cytostatic effects of NO are implicated in primitive non-specific host defence mechanisms against numerous pathogens. Therapeutic approaches. Inhalation of high concentrations of NO causes acute pulmonary oedema and methaemoglobineamia, but concentrations below 55 ppm are not toxic. NO inhibits bronchoconstriction, but the main action of inhaled NO is pulmonary vasodilatation. Therapeutically useful in respiratory distress syndrome. ADMA, approximately equipotent with l-NMMA, is present in human plasma and is excreted in urine. Its plasma concentration correlates with vascular mortality in patients receiving haemodialysis for chronic renal failure, and is increased in people with hypercholesterolaemia. Infusion of a low dose of L-NMMA into the brachial artery causes local vasoconstriction, owing to inhibition of the basal production of NO in resistance vessels of the infused arm, without influencing blood pressure or causing other systemic effects. Nitric oxide in pathophysiology. Nitric oxide is synthesized under physiogical and pathological circumstances. Either reduced or increased NO production can contribute to disease. Underproduction of neuronal NO (nNOS) is reported in babies with hypertrophic pyloric stenosis. Endothelial NO production is reduced in patients with hypercholesterolaemia and some other risk factos for atherosclerosis, and this may contribute to atherogenesis. Overproduction of NO may be important in neurodegenerative diseases and in septic shock. Chapter. 18. The heart. Cardiac rate and rhythm. Physiological sinus rhythm is characterized by impulses arising in the sinoarterial (SA) node and conducted in sequence through the atria, the AV node, bundle of his, purkinje fibres and ventricles. Electrophysiological

features of cardiac muscle: - Pacemaker activity; - Absence of fast Na+ currend in SA and AV nodes, where slow inward Ca2+ current initiates action potentials; - Long action potential (plateau) and refractory peiod; - Influx of Ca2+ during the plateau. The heart contains intracellular calcium channels and voltage-dependent calcium channels in the plasma membrane. The main type of voltage dependent calcium channels is the L-type channel, which is also important in vascular smooth muscle. Regions that lack a fast inward current have a muchlonger refractory period than fastconducting regions. This is because recovery of the slow inward current following its inactivation during the action potential takes a considerable time, and the refractory period outlasts the action potential. Disturbances of cardiac rhythm. They are classified according to: - The site of origin- atrial, ventricular, junctional; - Whether the rate is increased or decreased. They may cause palpitations or symptoms from cerebral hypoperfusion. The commonest types of tachyarrhythmia are atrial fibrillation, where the heartbeat is completely irregular, and supraventricular tachycardia (SVT), where the heartbeat is rapid but regular. Bradyarrhytmias include various kinds of heart block and complete cessation of electrical activity. Four basic phenomena underlie disturbances of cardiac rhythm: - Delayed after-depolarisation; - Re-entry; - Etopic pacemaker activity; - Heart block. Main cause of delayed after-depolarisation is abnormally raised Ca2+, triggers inward current and hence a train of abnormal action potentials. After-depolarisation is known as the transient inward current. A rise in Ca2+ out of the cell in exchange for entry of three Na+, resulting in a net influx of one positive charge and hence membrane depolarization. Ca2+ opens non-selective cation channels in the plasma membrane, causing depolarization analogous to the endplate potential at the neuromuscular junction. Hypercalcaemia can delay repolarisation. Prolongation of the QT interval, hypokalaemia also prolongs the QT interval. Prolongation of the QT interval carries a risk of causing dangerous ventricular dysrhythmias. In normal cardiac rhythm, the conducted impulse dies out after it has activated the ventricles. Re-entry (zie plaatje)describes the situation in which the impulse re-excites regions of the myocardium after the refractory period has subsides, causing continuous circulation of action potentials. Ectopic pacemaker activity is encouraged by sympathetic activity and by partial depolarization during phase IV and can cause normally quiescent parts of the heart to take on spontaneous rhythm. Heart block results from fibrosis of, or ischaemic damage to, the conducting system. In complete heart block, the atria and ventricles beat independently of one another. Cardiac contraction. Stroke volume is determined by a combination of factors, including some intrinsic to the heart itself and other haemodynamic factors extrinsic to the heart. Intrisinc

factors regulate myocardial contractility via Ca2+ and ATP. Extrinsic circulatory factors include elasticity and contractile state of arteries and veins, and the volume and viscosity of the blood. Binding of Ca2+ to troponin C; changes the conformation of the troponin complex, permitting cross-bridging of myosin to actin and initiating contraction. Other factors that affect the force of contracting are the availability of oxygen and a source of metabolic energy such as free fatty acids. Ischaemic preconditioning; means an improved ability to withstand ischemia following previous ischaemic episodes. The frank-starling mechanism can be represented as a ventricular function curve: - Increased cardiac filling pressure (preload) increases ventricular end-diastolic volume. This increases stroke volume and hence cardiac output and mean arterial pressure. - Arterial and arteriolar vasoconstriction increases afterload. End-diastolic volume and hence stroke work are initially unchanged, but constant stroke work in the face of increased vascular resistance cause reduced stroke volume and hence increased end-diastolic volume. The starling mechanism plays little part in controlling cardiac output in healthy subjects, because changes in contractility, mainly as a result of changes in sympathetic activity, achieve the necessary regulation without any increase in ventricular filling pressure. Denervated heart patients relies on the starling mechanism to increased CO during exercise. Myocardial oxygen consumption and coronary blood flow. Main physiological factors that regulate coronary flow are: - Physical factors: coronary flow occurs during diastole. Diastole is shortened more than systole during tachycardia, reducing the period available for myocardial perfusion. The effective perfusion pressure is equal to the difference between the aortic and ventricular pressure. - Vascular control by metabolites/mediators: most important mechanism by which coronary flow is regulated. - Neural and humoral control: Coronary vessels have a dense sympathetic innervation, but sympathetic nerves exert only a small direct effect on the coronary circulation. Smaller vessels have B2-adrenoceptors that have a dilator effect. Autonomic transmitters. Sympathetic system. Main effects: - Increased force of contraction;

- Increased heart rate; - Increased automaticity; - Repolarisation and restoration of function following generalized cardiac depolarization; - Reduced cardiac efficiency. All result from activation of Beta1-adrenoceptors. All occur through increased intracellular cAMP. Increases the probability that the channels will open, increasing inward Ca2+ current and hence force of cardiac contraction. Also increases the Ca2+ sensitivity of contractile machinery. It facilitates Ca2+ capture by sarcoplasmic reticulum, thereby increasing amount of Ca2+ available for release. The increase in heart rate results from an increased slope of pacemaker potential. Parasympathetic system. Has little effect on contractility, main effects being on rate and rhythm: - cardiac slowing and reduced automaticity. - Inhibition of AV conduction. Result from occupation of muscarinic (m2) acetylcholine receptors. Receptors are negatively coupled to adenylate cyclase and thus reduce cAMP formation, acting to inhibit to slow Ca2+ current. They also open a potassium channel. The resulting increase in K+ permeability produces a hyperpolarising current that opposes the inward pacemaker current, slowing the heart and reducing automaticity. Coronary vessels lack cholinergic innervation. Cardiac natriuretic peptides. Atrial cells contain secretory granules, and store and release atrial natriuretic peptide (ANP). This has powerful effects on the kidney and vascular system. Release occurs during volume overload in response to stretching of the atria. Main effects are to increase Na+ and water excretion by the kidney; relax vascular smooth muscle; increase vascular permeability; and inhibit the release and/or actions of several hormones and mediators , including aldosterone, ang-II. Ischaemic heart disease. Angina. Occurs when the oxygen supply to the myocardium is insufficient for its needs. Chemical factors released by ischaemic muscle stimulate nociceptors. - Stable angina: produced by an increased demand on the heart and is caused by a fixed narrowing of the coronary vessels, almost always by atheroma. - Unstable angina: pain that occurs with less and less exertion, culminating in pain at rest. Myocardial infarction, namely platelet-fibrin thrombus associated with a ruptured atheromatous plaque, but without complete occlusion of the vessel. - Variant angina: uncommon. It occurs at rest and is caused by coronary artery spasm. Myocardial infarction. Cardiac myocytes rely on aerobic metabolism. If the supply of oxygen remains below a critical value, a sequence of events leading to cell death. Apoptosis may be an adaptive process in hypoperfused regions, sacrificing come jeopardised myocytes but thereby avoiding the disturbance of membrane function and risk of dysrhythmia inherent in necrosis. Prevention of irreversible ischaemic damage: - Thrombolytic and antiplatelet drugs to open the blocked artery and prevent their reocclusion; - Oxygen; - Opioids to prevent pain -> reduce sympathetic activity; - Beta-adrenoceptor antagonists. - Ace-inhibitors.

Drugs that affect cardiac function. Three groups. - Drugs that affect myocardial cells directly: * Autonomic neurotransmitters and related drugs * antidysrhythmic drugs * cardiac glycosides and other inotropic drugs * miscellaneous drugs and hormones - Drugs that affect cardiac function indirectly. Have actions elsewhere in the vascular system. - Calcium antagonists. Antidysrhythmic drugs. There are four classes: I. Drugs that block voltage-sensitive sodium channels; II. Beta-adrenoceptor antagonist; III. Drugs that substantially prolong cardiac action potential; IV. Calcium antagonists. Class I drugs: block sodium channels by binding to sites in the alfa subunit. This inhibits action potential , has been referred to as membrane-stabilizing activity. Their characteristic effect is to reduce the maximum rate of depolarization during phase 0. The central concept is of use-dependent channel block. Sodium channels exist in three distinct functional states: resting, open and refractory. Channels switch rapidly from resting to open in response to depolarization; known as activation. Maintained depolarization causes channels to change more slowly from open to refractory (inactivation), and the membrane must then be repolarized for a time to restore channel to the resting state before it can be activated again. Class I drugs bind when they are in either the open or the refractory state, less strongly to channels in resting state. Class II drugs: Comprise the B-adrenoceptor antagonists. Adrenaline can cause dysrhythmias. AV conduction depends critically on sympathetic activity; B-adrenoceptor antagonists increase the refractory period of the AV node and can therefore prevent recurrent attacks of SVT. Class III-drugs: originally based on the unusual behavior of a single drug, amiodarone. The special feature is that they substantially prolong the cardiac action potential. It involves blocking some of the potassium channels involved in cardiac repolarization, including the outward (delayed) rectifier. Action potential prolongation increases the refractory period. Prolonging of the cardiac action potential can paradoxically also have proarrhytmic effects -> torsade de pointes. Class IV-drugs: act by blocking voltage-sensitive calcium channels. Act on L-type channels. Slow conduction in the SA en AV nodes slowing the heart and terminating SVT by causing partial AV block. They shorten the plateau of the action potential and reduce the force of contraction. Clinical uses of class I antidysrhythmic drugs.

Class Ia (e.g. disopyramide). - Ventricular dysrhythmias; - Prevention of recurrent paroxysmal atrial fibrillation triggered by vagal overactivity. Class Ib (e.g. lidocaine). - Treatment and prevention of ventricular tachycardia and fibrillation during and immediately after MI Class Ic - To prevent paroxysmal atrial fibrillation (flecainide). - Recurrent tachyarrhythimas associated with abnormal conducting pathways. Clinical uses of class II drugs (e.g. propranolol, timolol). - Reduce mortality following MI; - Prevent recurrence of tachyarrhythmias provoked by increased sympathetic activity. Clinical uses of class III drugs. - Amiodarone: tachycardia associated with the wolff-parkinson-white syndrome. It is also effective in many other supraventricular and ventricular tachyarrhythmias but has serious adverse effects. - Sotalol combines class III with class II actions. It is used in paroxysmal supraventricular dysrhythmias and supresses ventricular ectopic beats and short runs of ventricular tachycardia. Clinical uses of IV drugs. - Verapamil is the main drug that is used: Prevent recurrence of paroxysmal SupraVentricular tachycardia (SVT) and to reduce the ventricular rate in patients with atrial fibrillation; - Verapamil was previously given IV to terminate SVT; it is now seldom used for this because adenosine is safer. Details of individual drugs. Drugs that increase myocardial contraction. 1. Cardiac glycosides: main actions are on the heart but some adverse effects are extracardiac, including nausea , vomiting, diarrhoea and confusion. Cardiac effects are: - Cardiac slowing and reduced rate of conduction through AV node; - Increased force of contraction; - Disturbance of rhythm especially: block of av conduction and inceased ectopic pacemaker activity; Main mechanisms is through increased vagal activity and inhibition of the Na+/K+ pump. It binds to a site on the extracellular aspect of the alfa subunit of the Na+/K+ ATPase. Rate and rhythm: slow AV conduction by increasing vagal outflow via CNS activity. Beneficial in established rapid atrial fibrillation. If ventricular rate is excessively rapid, diastolic filling is inadequate. Increasing the refractory period of the AV node reduces ventricular rate. Larger doses of glycosides disturb sinus rhythm. Force of contraction: Cause of large increase in twitch tension in isolated preparation of cardiac muscle. They do not accelerate relaxation. The increased tension is caused by and increased Ca2+ transient. The action potential is only slightly affected and the slow inward current little changed.

Effects are increased if plasma K+ decreases, because of reduced competition at the K+-binding site on the Na+/K+ ATPase. 2. Other drugs that increase myocardial contractility. Certain B1-adrenoceptor agonists are used to treat acute but potentially reversible heart failure. On the basis of their positive inotropic action. Dobuatmine produces less tachycardia. Glucagon increases myocardial contractility by increasing synthesis of cAMP. Antianginal drugs. 1. Organic nitrates. Organic nitrates relax vascular and some other smooth muscles. In healthy subjects, this reduces stroke volume. Venous pooling occurs on standing and can cause postural hypotension and dizziness. There is a marked effect on larger muscular arteries. Reduces pulse wave reflection from arterial branches and consequently reduces central (aortic) pressure and cardiac afterload. - Myocardial oxygen consumption is reduces because of the reduction in both cardiac preload and afterload; - Redistribution of coronary flow towards ischaemic areas via collaterals; - Relief of coronary spasm. Nitrates are absorbed with release of NO. NO activates soluble guanylate cyclase , increasing formation of cGMP, which activates protein kinase G -> sequestration of intracellular Ca2+, with consequent relaxation. Repeated administration results in diminished relaxation, possibly because of depletion of free SH groups. It does not occur with ordinary formulations of shortacting drugs (e.g. glyceryl trinitrate), but does occur with longer-acting drugs (e.g. isosorbide mononitrate). Main adverse effects include postural hypotension and headache. Glyceryl trinitrate is rapidly inactivated by hepatic metabolism. Well abosorbed from the mouth and is taken as a tablet under the tongue, producting its effect within a few minutes. If swallowed, it is ineffective becuase of first-pass metabolism. Its effective duration is 30 minutes. Isosorbide is longer acting but has similar pharmacological actions. It is swallowed rather than taken sublingually 2. Beta-adrenoceptor antagonists. Prophylaxis of angina, and in treating patients with unstable agina. Reducing cardiac oxygen consumption. They reduce the risk of death following myocardial infarction. 3. Calcium antagonists. They bind to alfa1-subunit of the cardiac L-type calcium channel but at distinct sites. They prevent the channel from opening, thus reducing Ca2+ entry. Many show properties of use dependence. They also show voltage-dependent blocking actions, blocking more strongly when the membrane is depolarized ,causing calcium channel opening inactivation. - Cardiac actions: Can cause AV block and cardiac slowing by their actions on conducting tissues, but this is offset by a reflex increase in sympathetic activity secondary to their vasodilator action. They also have a negative inotropic effect, which results from the inhibition of the slow inward current during the action potential plateau. - Vascular smooth muscle: cause generalized arterial/arteriolar dilatation, thereby reducing blood pressure, but do not much affect the veins. They cause coronary

vasodilatation and are used in patients with coronary artery spasm. They are well absorbed from the gastrointestinal tract, and are given by mouth. Short-acting dihydropyridines cause flushing and headache because of their vasodilator action, and in chronic use dihydropyridines often cause ankle swelling related to arteriolar dilatation and increased permeability of postcapillary venules. Verapimil canse cause constipation probably because of effects on calcium channels in gastrointestinal nerves or smooth muscle.

Ch. 19. The vascular system.


Vascular smooth muscle is controlled by mediators secreted by sympathetic nerves and vascular endothelium, and by circulating hormones. Smooth muscle cell contraction is initiated by arise in Ca2+, which activates myosin ligh-chain kinase, causing phosphorylation of myosin, or by sensitisation of the myofilaments to Ca2+ by inhibition of myosin phopshatase. Agents cause vasoconstriction via one ore more mechanisms: - Release of intracellular Ca2+ via inositol triphosphate; - Depolarizing the membrane, opening voltage-gated calcium channels and causing Ca2+ entry; - Increasing sensitivity to Ca2+ via actions on myosin light-chain kinase and/or myosin phosphatase. Agents causing relaxation by: - Inhibiting Ca2+ entry through voltage-gated calcium channels either directly or indirectly by hyperpolarizing the membrane; - Increasing intracellular cAMP or cGMP; cAMP inactivates myosin light-chain kinase and facilitates Ca2+ efflux, cGMP opposes agonistinduced increases Ca2+. Endothelin. Stimuli to synthesis include many noxious vasoconstrictor mediators released by trauma or inflammation , including activated platelets, endotoxin etc. Inhibitors of ET synthesis include NO, natriuretic peptides, PGE2, PGI2, heparin and high shear stress. Endothelin receptor cause vasodilatation when infused directly into the brachial artery. ET-1 has an elimination half life of <5 minutes. There are two types of receptor, designated Eta and ETb, both of which are Gprotein-coupled. The predominant overall response is vasoconstriction. Endothelin-1 is a paracrine mediator rather than a circulating hormone. The role of endothelium in controlling vascular smooth muscle: Endothelial cells release vasoactive mediators including PGI2 and NO and endothelin. Many vasodilators act via endothelial NO production. The NO derives from arginine and is produced when Ca2+ increases in the endothelial cell, or sensitivity of endothelial NO synthase to Ca2+ is increased. NO relaxes smooth muscle by increasing cGMP formation. Endothelin is a potent and long-acting vasoconstrictor peptide released from endothelial cells by many chemical and physical factors. It is not confined to blood vessels.

The renin-angiotensin system. Synergises with the sympathetic nervous system. It stimulates aldosterone secretion and plays a central role in the control of Na+ excretion and fluid volume, as well as vascular tone. Renin is a proteolytic enzyme that is secreted by the juxtaglomerular apparatus. It is secreted in respons to various physiological stimuli, including a fall in Na+. The Na+ concentration in distale tubule is senses by the macula densa. Ang-II causes feedback inhibition. Angiotensin converting enzyme is a membrane bound enzyme on the surface of endothelial cells. The common isoform of ACE is also present in other vascular tissues, including the brain, heart, striated muscle and kidney. ACE inactivates bradykinin. The main actions of Ang-II are mediated via AT1 and/or AT2-receptors. Effects mediated by AT1-receptors include: - Generalised vasoconstriction; - Increased release of nor-adrenaline from sympathetic nerve terminals; - Stimulation of proximal tubular reabsorption of Na+; - Secretion of aldosterone; - Cell growth in the heart and arteries. Vasoactive drugs. Vasoconstrictor drugs. 1. Angiotensin II: is roughly 40 times as potent as noradrenaline in raising blood pressure. It constricts mainly cutaneous, splanchnic and renal vasculature, with less effect on blood flow to brain and skeletal muscle. No routine clinical uses. 2. Antidiuretic hormone. Is a posterior pituitary peptide hormone. A powerful vasoconstrictor in skin and some other vascular beds. Water retention is mediated through V2 receptors. ADH causes generalized vasoconstriction, including the coeliac, mesenteric and coronary vessels. 3. Endothelin. They have vasodilator and vasoconstrictor actions, but vasoconstriction predominates. Intravenous administration cause transient vasodilatation followed by profound and very long-lived vasoconstriction. Even more potent than ANG-II. Vasodilator drugs. Calcium antagonists: L-type calcium antagonists are discussed. Cause generalized arterial vasodilatation. Dihydropyridines act preferentially on vascular smooth muscle, whereas verapamil acts also on the heart. They act to : increase local tissue blood flow, reduce arterial pressure, reduce central venous pressure. Net effect is a reduction of cardiac preload, and afterload, hence reduction of cardiac work. Main uses are: - Antihypertensive therapy (e.g. At1 antagonists, calcium antagonists and alfa1 antagonists); - Treatment/prophylaxis angina (e.g. calcium antagonists, nitrates); - treatment of cardiac failure (e.g. angiotensin-converting enzyme inhibitors, AT1 antagonists). Hydralazine had unknown mechanism of action: acts mainly on arteries and

arterioles, causing a fall in blood pressure accompanied by reflex tachycardia and an increased cardiac output. It interferes with the action of inositol triphosphate on Ca2+ release from the sarcoplasmic reticulum. It can cause an immune disorder resembling systemic lupus erythematosus. Ethanol: dilates cutaneous vessels, causing the familiar drunkards flush. Indirectly acting vasodilator drugs. Two main groups: - Sympathetic vasoconstriction; - The renin-angiotensin system. 1. Renin inhibitors: reduce plasma renin activity, but their effects on blood pressure in patients with hypertension are dissapointing. 2. Angiotensin-converting enzyme inhibitors ACE: ACE cleaves the C-terminal pair of amino acids from peptide substrates. It active site contains a zinc atom. Captopril contains a sulfhydryl group appropriately placed to bind the zinc atom, coupled to a proline residue that binds the site on the enzyme that normally accommodates the terminal leucine of AG-I. ACE inhibitors affect capacitance and resistance vessels, and reduce cardiac load as well as arterial pressure. They do not effect cardiac contractility, so cardiac output normally increases. Unwanted effects in large doses are rashes, taste disturbance, neutropenia and heavy proteinura. ACE inhibitors that do not posses a sulfhydryl group do not cause these unwanted effects. A dry cough (bradykinin build up) -> commonest adverse effect. Clinical uses: - Hypertension; - Cardiac failure; - People at high risk of ischaemic heart disease; - Diabetic nephropathy 3. ANG-II receptor subtype 1 antagonists (sartans): Are non-peptide , orally active AT1 receptor antagonists. It is not known whether any of the beneficial effects of ace inhibitors are bradykinin/NOmediated, so it is unwise to assume that AT1 receptor antagonists will necessarily share all the therapeutic properties of ACE inhibitors. Clinical uses of vasoactive drugs. 1. Hypertension. Essential hypertension: increased cardiac output may be an early feature, but by the time it is diagnosed there is usually increased peripheral resistance and the cardiac output is normal. The main systems include the sympathetic nervous system, the renin-angiotensin-aldosterone system, and tonically active endothelium-derived

autocoids. Start treatment with wither an ACE inhibitor or an AT1 antagonist. Betaadrenoceptor antagonists are less well tolerated than ACE inhibitors or AT1 antagonists. They are useful for hypertensive patients with some additional indication for beta-blockade, such as angina or heart failure. Addition of a third or fourth drug is often needed, and a long-acting alfa1 antagonist is one option in this setting. Spironolactone (a competitive antagonist of aldosterone) needs careful monitoring of plasma K+ concentration because it inhibits urinary K+ excretion as well as causing oestrogen-related adverse effects. 2. Cardiac failure. The underlying abnormality is a cardiac output that is inadequate to meet the metaboic demands of the body during excercise. When CO is insufficient, an increase in fluid volume occurs, partly because increased venous pressure causes increased formation of tissue fluid, and partly because reduced renal blood flow activates the RAAS, causing Na+ and water retention. Drugs used for treatment do the following: - Increases natriuresis: increasing salt and water excretion; - Inhibit the RAAS: this is inappropriately activated in patients with cardiac failure. Betaadrenoceptor antagonists inhibit renin secretion. ACE-inhibitors and AT1 antagonists block the formation of ANG-II and inhibit its action, thereby reducing vascular resistance improving tissue perfusion and reducing cardiac afterload. - Antgonise Beta-adrenoceptor: heart failure is accompanied by potentially harmful activation of the sympathetic nervous system. Proving a rationale for using beta blockers for this disorder. - Inhibit ADH: vasopressin is released inappropriately in heart failure and may contribute to the hyponatraemia. - Relax vascular smooth muscle: Glyceryl trinitrate, its venodilator effect reduces venous pressure, and its effects on arterial compliance and wave reflection reduce cardiac work. - Increase the contractility: cardiac glycosides are used either in patients with heart failure who also have chronic rapid atrial fibrillation, or in patients who remain symptomatic despite treatment with diuretic and ACE inhibitor. 3. Shock and hypotensive state. Is medical emergency by inadequate perfusion of vital organs, usually because of a very

low blood pressure. Leads to anaerobic metabolism and hence to increased lactate production. Mediators promoting vasodilatation in shock converge on two main mechanisms: - Activation on ATP-sensitive potassium channels by reduced cytoplasmic ATP and increased lactate and protons. - Increased synthesis of NO, which activates myosin light-chain phosphate and activates Kca-channels. Patients with shock are not homogenous population making it hard to perform valid clinical trial.

4. Peripheral vascular disease. Commonest symptom is pain in the legs on walking (claudication), followed by pain at rest and in severe cases gangrene of the feet or legs. Treatment is often surgical or by angioplasty. Drug treatment includes antiplatelet drugs, a statin and an ACE inhibitor. 5. Raynauds disease. Inappropriate vasoconstriction of small arteries and arterioles gives rise to raynauds phenomenon. Treatment involves stopping smoking and avoiding cold. 5. Pulmonary hypertension. Systolic pulmonary pressure in adults is normally 20mmHg. Pulmonary artery pressure is difficult to measure and requires cardiac catheterization. If pulmonary pressure is raised, it usually causes some leak of the tricuspid valve, allowing regurgitation of blood from the right ventricle to the right atrium. Increased pulmonary pressure can result form an increased cardiac output.

Ch. 20 Atherosclerosis and lipoprotein metabolism.


Atherogenesis. Atheroma is a focal disease of the intima of large and medium-sized arteries. Fatty streaks are earliest structurally apparent lesion and progress to fibrous and/or fatty plaques. Symptoms depend on the vascular bed and occur only when blood flow through the vessel is reduced below that needed to meet the metabolic demands of tissues downstream from obstruction. 1. Endothelial dysfunction with altered PG and NO; 2. Injury of dysfunctional endothelium leads to expression of adhesion molecules. Encourages monocyte attachment and migration of monocytes from the lumen into the intima. 3. LDL particles are transported into the vessel wall and generate free radicals that oxidase LDL (oxLDL) resulting in lipid peroxidation; 4. oxLDL is taken up by macrophages; 5. Subendothelial collections of foam cells and T lymphocytes form fatty streaks; 6. Cholesterol can be mobilised from the artery wall and transported in plasma in the form of HDL-c; 7. Activated platelets, macrophages, causing proliferation of smooth muscle and deposition of connective tissue components. 8. A plaque can rupture, forming a substrate for thrombosis. Lipoprotein transport in blood. There are four main classes, differin gin the relative proportion of the core lipids, and in the type of apoprotein. They also differ in size and density:

- HDL-c: particles adsorb cholesterol derived from cell breakdown in tissues and transfer it to VLDL and LDL particles. - LDL-c: particles with large component of cholesterol; some LDL cholesterol is taken up by the tissues and some by the liver, by endocytosis via specific LDL receptors. - VLDL: which transport cholesterol and newly synthesized triglycerides to the tissues, where triglycerides are removed as before leaving LDL particles. - Chylomicrons: transport triglycerides adn cholesterol from gastrointestinal tract to the tissues, where tiglyceride is split by lipoprotein lipase, releasing free fatty acids and glycerol. These are taken up in muscle and adipose tissue. Chylomicron remnants are taken up in the liver, where cholesterol is stored, secreted in bile, oxidized to bile acids or converted into VLDL.

Lipid-lowering drugs. 1. Satins: HMG-CoA reductase inhibitors. Main biochemical effect of statins is to reduce plasma LDL-c. Short-acting statins are given by mouth at night to reduce peak cholesterol synthesis in the early morning. Mild unwanted effects include myalgia, gastrointestinal disturbance, raised concentrations of liver enzymes in plasma, insomnia and rash. 2. Fibrates. Cause a marked reduction in circulating VLDL. The mechanism of action is complex. They are agonists for PPARs; in humans , the main effects are to increase transcription of the genes for lipoprotein lipase, apoA1 and apoA5. They increase hepatic LDL-c uptake. Fibrates reduce plasma-c reactive protein and fibrinogen, improve glucose tolerance, and inhibit vascular smooth muscle inflammation. 3. Drugs that inhibit cholesterol absorption.

Their effect is modest and they are bulky, unpalatable and cause diarrhoea. Agents that interfere with cholesterol absorption usually as an adjunct to diet plus statin: - Ezetimibe; - Stanol-enriched foods; - Bile acid-binding resins. 4. Fish oil derivatives. Omega-3 marine triglyceride reduce plasma triglyceride concentrations but increase cholesterol. Mechanism of action of fish oil on plasma triglyceride is unknown.

Ch. 21. Haemostasis and thrombosis.


Haemostasis is arrest of blood loss from damaged blood vessels. A wound causes vasoconstriction accompanied by: - Adhesion and activation of platelets; - Fibrin formation. Thrombosis is the pathological formation of a haemostatic plug within the vasculature in the absence of bleeding. Virchows triad. This comprises injury to the vessel wall; altered blood flow and abnormal coagulability of the blood. A thrombus , which forms in vivo, should be distinguished from a clot, which forms in static blood in vitro. Clots are amorphous, consisting of a diffuse fibrin meshwork in which red and white blood cells are trapped indiscriminately. An arterial thrombus -> white thrombus consisting mainly of platelets and leucocytes in a fibrin mesh, associated with atherosclerosis. Venous thrombus -> red thrombus and consists of a small white head and a large jelly-like red tail. Drugs affecting heamostasis and thrombosis in three distinct ways: - Affecting blood coagulation; - affecting patelet function; - affecting fibrin removal (fibrinolysis). Blood coagulation. Coagulation cascade. Means the conversion of fluid blood to a solid gel or clot. Main event is the conversion by thrombin of soluble fibrinogen to insoluble strands fibrin. Important inhibitor is antithrombin III, which neutralizes all the serine proteases in the cascade. Vascular endothelium also actively limits thrombus extension. Thrombin cleaves fibrinogen, producing fragments that polymerise to form fibrin. It also activates factor XIII, a fibrino ligase, which strengthens fibrin-to-fibrin links, thereby stabilizing the coagulum. Thrombin also causes platelet aggregation, stimulates cell proliferation and modulates smooth muscle contraction. Vascular endothelium in haemostasis and thrombosis. Normally, it provides a non-thrombogenic surface by virtue of surface heparan sulfate, a cofactor for antithrombin III. It also plays an active part in haemostasis, synthesising and storing several key haemostatic components; VWF, TF and

plasminogen activator inhibitor (PAI-1).

Drugs that act on the coagulation cascade. Vitamin K. It is essential for the formation of clotting factors II, VII, IX, X. These are all glycoproteins with several gamma-carboxyglutamic acid (Gla). Gamma-carboxylation occurs after the syntehsis of the chain. Carboxylase enzyme requires vit K as a cofactor. Ca2+ binding does not occur in the absence of gamma-carboxylation. Protein C and S are also vitamin K dependent. Thrombosis. Main drugs used to prevent or treat red thrombus are: - Injectable anticoagulants (heparin); - Oral anticoagulants (warfin). Heparins act immediately. Consequently patients with venous thrombosis are treated immediately with an injectable anticoagulant. Injectable anticoagulants. Heparin: doses are specified in units of activity rather than of mass. Heparin fragments or a synthetic pentasaccharide referred to as low-molecular-weight heparins (LMWHs) are used increasingly in place of unfractionated heparin. Heparin inhibits coagulation by activating antithrombin III. Antithrombin III inhibits thrombin. Thrombin is considerably more sensitive to inhibitory effect of heparin than is factor X. To inhibit heparin, it is necessary for heparin to bind to the enzyme ad well as to antithrombine III; to inhibit factor X, it is necessary only for heparin to bind to antithrombin III. The LMWHS increase the action of antithrombin III on factor Xa but not on thrombin, because the molecules are too small to bind both enzymes. Heparin acts immediately following IV injection. LMWHS are given subcutaneously, they have longer elimination half-life than unfractionated heparin, and this is independent of dose -> do not prolongs aPTT. Unwanted effects

are: - Haemorrhage: treated by stopping therapy, giving protramine sulfate -> heparin antagonist which forms an inactive complex with heparin. - Thrombosis: uncommon but serious adverse effect of heparin -> heparin-inducedthrombocytopenia (HIT). More serious thrombocytopenia occuring 2-14 days after the start of therapy and is caused by IgM or IgG antibodies against complexes of heparin and platelet factor IV. Circulating immune complexes bind to Fc receptors on circulating platelets, therby activation them and releasing more platelet factor IV and causing thrombocytopenia. LMWHs are less liable than standard heparin to activate platelets to release platlet factor IV, and they bind less avidly to platelet factor IV. Vitamin K antagonists: warfin. Is the most important oral anticoagulant; warfin and other vit K antagonists require frequent blood tests to individualize dose. They interfere with the posttranslational gamma-carboxylation of glutamix acid residues in clotting factors II, VII, IX, X. Their effect takes several days to develop because of the time taken for degradation of performed carboxylated clotting factors. The peak concentration in the blood occurs within an hour of ingestion, but because of the mechanism of action this does not coincide with the peak pharmacological effect, which occurs about 48hours later. Warfin crosses the placenta and is not given in the first months of pregnancy because it is teratogenic, nor in the later stages because it can cause intracranial haemorrhage in the baby during delivery. It appears in milk during lactation. Factors that potentiate oral anticoagulants: - Liver disease: interferes with synthesis of clotting factors; - Agents that inhibit hepatic drug metabolism (co-trimoxazole); - Drugs that inhibit platelet function (e.g. aspirin); - Drugs that displace warfarin from binding sites on plasma albumin (result in a transient increased in the concentration of free warfarin in plasma; - Drugs that inhibit reduction of vitamin K; - Drugs that decrease the availability of vitamin K. Platelet adhesion and activation. A low platelet count results in thrombocytopenic purpura. When platelets are activated, they undergo a sequence of reactions: - Adhesion following vascular damage; - Shape change; - Secretion of the granule contents (including agonists, ADP and 5hydroxytrypatamine); - Biosynthesis of labile mediator such as platelet-activating factor and thromboxane (TX) A2; - Aggregation , promoted by various agonists, including collagen, thrombin, ADP, 5hydroxytrypatamine and TXA2, acting on specific receptors on the platelet surface; activation by agonists leads to expression of GPIIB/IIIa receptors that bind fibrinogen; - Exposure of acidic phospholipid on the platelet surface, promoting thrombin formation. Antiplatelet drugs. Aspirin. Alters the balance between TXA2, which promotes aggregation, and PGI2, which inhibits it. It inactivates COX by irreversibly acetylating a serine residue in its active site. This reduces both TXA2 synthesis in platelets and PGI2 synthesis in endothelium. Vascular endothelial cells can synthesise new enzyme via regeneration of COX-1 and via COX-2, whereas platelets cannot. Platelets are replaced in 7-10 days. Adverse effects of aspirin are mainly on gastrointestinal tract -> clearly dose-

related. Dipyridamole. A phophodiestarase inhibitor. A modified release of dipyridamole reduced the risk of stroke and death in such patients by around 15%- a similar effect to that of aspirin. Headache was the commonest adverse effect -> cause no excess risk of bleeding. Thienopyridine derivatives. Ticlopidine inhibits ADP-dependent aggregation. Its efficacy is reducing stroke is similar to that of aspirin, but idosyncratic unwanted effects, including sever dyscrasias. Clopidogrel is structurally related to ticlopidine and also inhibits ADP-induced aggregation through an active metabolite. It can cause rash or diarrhoea, but neutropenia is no more common than with aspirin. Fibrinolysis. Fibrinolytic drugs. Drugs are used clinically principally to repon the occluded coronary artery in patients with acute myocardial infarction, less commonly in patients with life-threatening venous thrombosis or pulmonary embolism. Streptokinase is a protein extracted form cultures of streptococci. It activates plasminogen. Alteplase and duteplase are single and double-chain recombinant tPA. They are more active on fibrin-bound plasminogen than on plasma plasminogen. Unwanted effects. Main hazard is bleeding, including gastrointestinal haemorrhage and stroke. Streptokinase can cause allergic reactions and low-grade fever.

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