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Activation of 1 adrenergic receptors on atrial and ventricular myocytes increases the open probability of L-type Ca2+ channels allergy symptoms coughing night flonase 50 mcg buy lowest price, leading to a bigger plateau current allergy treatment children purchase genuine flonase line, Ca2+ store and trigger Ca2+ allergy testing questionnaire buy genuine flonase online, and thus a bigger Ca2+ transient and stronger contraction (inotropic effect) allergy medicine blood thinner cheap 50 mcg flonase otc. This produces an abrupt fall in heart rate allergy latex treatment 50 mcg flonase for sale, for example, on expiration or fainting. Hyperkalaemia depolarizes the myocytes, leading to conduction impairment and arrhythmias. Hypokalaemia has the opposite electrophysiological effect but is also arrhythmogenic, especially in the presence of a high sympathetic drive. Since this reduces myocardial O2 demand, these drugs are beneficial in stable angina. Molecular and cellular neurocardiology: development, cellular and molecular adaptations to heart disease. The mechanism and significance of the slow changes of ventricular action potential duration following a change of heart rate. Role of potassium in the regulation of systemic physiological function during exercise. The Journal of Physiology 1984; 353 (1), 1­50 66 5 Electrocardiography and arrhythmias 5. When a frog sciatic nerve/gastrocnemius muscle preparation accidentally fell onto an isolated frog heart such that the sciatic nerve touched the heart, both muscles contracted synchronously, suggesting that the heart generates electrical impulses. The currents generate small potential differences across the body surface, of around 1 mV, and these can be recorded by a sensitive voltmeter connected to metal electrodes placed on the skin surface. The potential difference is recorded on a strip of 67 Electrocardiography and arrhythmias 5. The lettering was allotted sequentially by Einthoven, reflecting a tradition in mathematics first used by Rene Descartes in the 17th century. Jimmie has his front and hind paws in pots of normal saline connected to a galvanometer (from the Illustrated London News, 22 May 1909). Mr Gladstone: "I understand the dog stood for some time in water to which sodium chloride had been added or in other words a little common salt. If my honourable friend has ever paddled in the sea he will understand the sensation. Had the experiment been painful the pain would no doubt have been immediately felt by those nearest the dog. Physiology; the Servant of Medicine: Chloroform in the Laboratory and in the Hospital. The magnitude of the skin potential difference depends on the size of the extracellular current, which in turn depends on the mass of myocardium that is activated. Subepicardial and apical myocytes (dashed trace) have shorter action potential durations than subendocardial and basal myocytes, so they repolarize first. Second-degree heart block is an intermittent failure of excitation to pass from the atria to the ventricles. In third-degree or complete heart block, the transmission fails completely, so the atria and ventricles beat independently, at different rates. The slow heart rate that results can cause breathlessness, particularly on exertion when heart rate should rise. Complete cessation of ventricular electrical activity is known as "asystole" and will cause a cardiac arrest and sudden cardiac death unless cardiopulmonary resuscitation is started promptly. The exact position makes little difference to the recording; the limb simply serves as a tube of conducting electrolyte solution (the extracellular fluid) connected to the torso. The Q wave is defined as the first downward spike (negative deflection), the R wave as the first upward spike (positive deflection) and the S wave as the second downward spike. There are three bipolar limb leads Pairs of limb electrodes, called the bipolar limb leads, can be connected across a voltmeter in three different combinations. When the left arm is connected to the positive terminal of the voltmeter and right arm to the negative terminal, this is called a Lead I recording. If the potential on the left side of the body is more positive than on the right during cardiac excitation, Lead I records a positive potential. Because each limb serves as an electrical conductor connected to the trunk, the limb electrodes in effect record from each shoulder and the pelvis. It coincides with the plateau of the ventricular action potential and rapid ventricular ejection. The trick is to feed the signal from two arm electrodes simultaneously into the negative terminal of the voltmeter, thereby producing an averaged signal located roughly in mid-chest. Red arrows show direction of biggest potential difference during ventricular excitation (main dipole), in the frontal and transverse planes. There are six unipolar precordial leads In clinical practice, an additional six electrodes, V1­V6, are placed across the chest for connection to the positive terminal of the voltmeter. All three limb leads are connected to the negative terminal to create a mid-chest reference point. This is useful, because the wave of excitation travels in three dimensions, not just the frontal plane recorded through the limb leads. V5 and V6 are at the same level in the anterior axillary and mid-axillary line, respectively, and record the left ventricular activity best. The symbol for a vector is an arrow whose length represents the size of the vector and whose direction represents the angle of the vector. But this raises another question: in which direction does the cardiac dipole point For simplicity, we will consider only changes in the frontal plane here; however, since the ventricles are threedimensional bodies lying in an oblique, rotated position, the dipole size and direction change in the transverse plane too. Just as a diffuse mass can be represented by a centre of gravity, so a diffuse charge can be represented as a single charge at its electrical centre, or pole. Thus, during the spread of excitation the ventricles can be represented by one negative pole and one positive pole, that is, by an electrical dipole. The dipole magnitude is small, because only a small mass of muscle has been activated; and the dipole angle is ~120° to the horizontal, that is, pointing down and rightwards. Next, the remaining septum and most of the subendocardium depolarize, while the subepicardium is still polarized. Since the bulky left ventricle predominates, the dipole magnitude is large and the dipole angle is ~60°, that is, pointing down and leftwards. The last region to be excited by the advancing wave of depolarization is the base of the ventricles, close to the annulus fibrosus. The bulky left ventricle again predominates, creating a small dipole pointing headward. The sequence of ventricular activation thus causes the cardiac vector to rotate anticlockwise in the frontal plane, and to wax and wane in size as it rotates. You can get a sense of this effect by viewing a ruler face on, then slowly rotating it through 180°. The visible length changes from maximum to zero and back to maximum as the angle of view changes. Repolarization occurs in reverse, creating an upright T wave We now have the information needed to answer a puzzle raised earlier ­ why is the T wave upright This seems odd on first acquaintance, since repolarization is the electrical opposite of depolarization. The explanation is that the myocytes repolarize in reverse order to depolarization. That is, it possesses direction as well as magnitude, just like a mechanical force. Pale pink areas are myocytes at resting potential, so they carry a positive extracellular charge. Therefore, repolarization begins in the subepicardium and spreads towards the subendocardium, whereas depolarization does the reverse. The same can be said regarding action potential duration between the base and apex of the heart, such that depolarization spreads from base to apex, and repolarization from apex to base. Posterior base Epicardium Endocardium Apex Depolarization spreading outwards R wave 5. By 50 ms, the dipole has grown to its maximum size and swung anticlockwise to ~40°. Since myocytes repolarize in reverse order to depolarization, the dipoles are in the same direction. By 90 ms the dipole has dwindled in magnitude and, in the illustrated case, swung round to about -100°, creating a small, downward deflection in each limb lead, that is, an S wave. This indicates a more vertically orientated electrical axis, and brings us to the issue of the electrical axis of the heart. Predicting the temporal changes in body surface potentials based on the changes in electrical activity measured from the ventricles is known as the forward problem of electrocardiography. The dipole is biggest midway through excitation, because roughly half the wall is negative and half positive. The dipole waxes and wanes, and swings anticlockwise, as excitation sweeps through the ventricles. Several mathematical algorithms for this exist, although many more body surface electrodes are required to produce an accurate body surface map and knowing the position of the heart within the thorax is also crucial to their solutions. While the epicardial activation and repolarization sequences can be reasonably well determined, the absolute magnitude of the potential changes on the heart cannot. The axis also becomes more vertical during each inspiration because the descending diaphragm tugs on the pericardium and drags down the apex. The electrical axis depends also on the relative thickness of the right and left ventricle walls. Hypertrophy of the right ventricle (often due to pulmonary disease) produces right axis deviation. This creates a potential difference between the ischaemic myocytes and surrounding healthy ones, caused at least in part through opening of adenosine triphosphatesensitive K+ channels and accumulation of extracellular K+. This is usually caused by an atherosclerotic lesion producing a narrowing in a coronary artery that only limits oxygen delivery to the myocardium when demand is increased as heart rate rises during exercise. This can be treated by emergency revascularization using a catheter and injection of dye (angiography) to identify the blockage, and passing a wire through the blockage and inflating a small balloon on the wire (angioplasty) surrounded by an expandable metal frame known as a stent. This has been shown to improve morbidity and mortality in randomized clinical trials. Measurements of plasma troponin levels released from damaged myocytes may be required to diagnose or exclude a myocardial infarction. The answer is complex, but a simple way of looking at it is that a pathological arrhythmia usually requires a trigger to generate an extra-stimulus within the heart. This extra-stimulus needs to have the right properties and be perfectly timed within a vulnerable window, and there needs to be a suitable substrate within the heart to maintain the propagation of the extra-stimulus. The substrate can be structural and/or electrophysiological, as well as static and/or dynamic. The shorter the duration of an ectopic beat action potential, and therefore refractory period, the more likely it is to be able to produce a re-entrant circuit (see the following section). The relationship between action potential duration and the preceding diastolic interval is known as electrical restitution. This concept was first demonstrated by George Ralph Mines, the pioneering English cardiac electrophysiologist. Myocytes emerging from their refractory period find themselves re-excited prematurely by the return of the local excitation wave (re-entry). A slow conduction velocity of wave propagation favours re-entry, as does a short refractory period, because it allows time for the myocytes to regain excitability. That way, both waves of excitation Triggers and a vulnerable window Arrhythmic triggers include afterdepolarization and abnormal automaticity. They may also be driven by local injury currents from ischaemic regions depolarizing neighbouring border zones, which may account some ventricular tachycardia seen during ischaemia/reperfusion known as an accelerated idioventricular rhythm. Triggered activity may result from afterdepolarizations as described in Section 3. This is exacerbated by cardiac ischaemia and adrenergic receptor stimulation by catecholamines. This is coupled to an increase in Na+ conductance due to activation of the Na+/Ca 2+ exchanger as the cell attempts to defend against the high levels of intracellular Ca 2+. Afterdepolarizations do not always result in the generation of an action potential and ectopic beat. They must be of sufficient magnitude to reach the threshold and follow the refractory period of the previous beat. Atrial or ventricular ectopic beats are common in completely normal, healthy hearts where they rarely lead to arrhythmias. Limbs A and B represent two pathways with different electrophysiological properties surrounding an area of central anatomical or functional conduction block. Limb B has a region of slow conduction velocity and unidirectional conduction block (if the cells are refractory at that point in time at point 1 in limb B). Therefore, the initial impulse conducts anterogradely only through limb A and is blocked in limb B (labelled 1). The impulse then continues to conduct retrogradely into limb B (labelled 2) and through the zone of slow conduction velocity (labelled 3). As the wavefront returns, if limb A is no longer refractory, then the wavefront can propagate down limb A again and a re-entry circuit is formed. Cardiac Electrophysiology and catheter ablation [Oxford Specialist Handbooks in Cardiology]. In ischaemic and dilated cardiomyopathy, there is scarring and fibrosis as well as remodelling of ion channels and sympathetic innervation, particularly in scar border zones.

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A relatively small quantity of ions is exchanged per action potential the numerous ionic currents described in this chapter are all small allergy medicine injection 50 mcg flonase order with mastercard, even the Na+ current allergy partners wilmington nc buy cheap flonase 50 mcg line. Consequently allergy medicine kid flonase 50 mcg visa, the change in intracellular ion concentration caused by a single action potential is tiny allergy testing raleigh nc buy cheap flonase on-line. Since a myocyte contains about 200 000 million Na+ ions allergy medicine addiction order flonase 50 mcg otc, Na+ concentration rises by a mere 0. The Na+-K+ and Na+-Ca2+ transporters can therefore restore the chemical composition of the sarcoplasm without excessive expenditure of metabolic energy. The numerous subtypes of channel are summarized in more detail, for reference purposes, in Table 4. In K+ channels, four individual proteins, called subunits, make up the ring; in Na+ and Ca 2+ channels, four -like domains of a single protein make up the ring. The simplest cardiac channel is the inwardly rectifying channel (K ir), where each subunit has two hydrophobic transmembrane helices. This probably evolved into the voltage-sensitive K+ channels, with six transmembrane helices per subunit. The myocyte was loaded with a fluorescent dye to measure free sarcoplasmic Ca2+ concentration. The resting potential approaches the Nernst equilibrium potential for K+, modified by a small, inward background leak of Na+, because the membrane is far more permeable to K+ than it is to Na+ at rest. Voltage-gated Na+ channels admit a brief influx of positive ions into the cell at the start of an action potential, depolarizing the cell. The overshoot of the action potential (+30 mV) approaches a Nernst equilibrium potential for Na+, modified by a small outward background current of K+. This nearly counterbalances the outward K+ current and generates a long plateau at around 0 potential, lasting 200­400 ms. The plateau is terminated by an increasing efflux of K+ ions through slowly activating, delayed rectifier K+ channels. However, depolarization reduces K ir conductance (inward rectification) because intracellular Mg2+ ions and polyamines impede the inner mouth of the channel when current is flowing outwards. Voltage-gated potassium channel (Kv family) Two members of this family are particularly important for cardiac function. Mutation studies show that positively charged arginine and lysine residues in the S4 loops act as a voltage sensor, making this a voltage-activated channel. Inactivation is brought about by a ball-on-chain (the last 20 amino acids of the intracellular N-terminal), which slowly blocks the intracellular opening following depolarization. The inward rectifier channel (Kir), with two transmembrane helices per subunit, probably evolved into voltage-sensitive K+ channels, with six transmembrane helices, which evolved into Na+ and Ca2+ channels. Phosphorylation of sites on intracellular loops by protein kinases can regulate channel activity. Ca2+ channel phosphorylation by cyclic adenosine monophosphate-activated protein kinase A promotes the open state, following adrenergic stimulation. Positively charged arginine and lysine residues in S4 again act as the voltage sensor, and amino acids in the pore-lining P loop confer Na+ selectivity. The antiarrhythmic drugs lidocaine, procaine and quinidine inhibit Na+ channels by plugging the pore. The m or activation gate (probably the S6 helices) is closed at resting potentials, blocking the pore. A second intracellular gate, the h or inactivation gate, is open at a resting potential. When the membrane is depolarized to the threshold potential, an outward shift in the positively charged, voltage-sensitive S4 helices open the activation gate, allowing Na+ passage. Because the inactivation gate moves more slowly than the activation gate, there is a millisecond or so when the channel is open, before the inactivation gate closes. Voltage-gated, L-type calcium channel the cardiac L-type Ca 2+ channel comprises a single poreforming protein, voltage-gated calcium channel subunit alpha Cav1. Outward displacement of the charged S4 helices of each domain by depolarization opens the activation gate (S6 helices). As long as the inactivation gate is closed, the myocyte is incapable of re-excitation (refractory). Closure of the inactivation gate is enhanced by intracellular Ca 2+ via a binding protein, calmodulin (Ca 2+-dependent inactivation). Consequently, the rise in intracellular Ca 2+ during the action potential helps to terminate opening. Adrenergic stimulation leads to the phosphorylation of the C-terminus domain by protein kinase A, increasing the duration of the open state. L-type Ca 2+ channel blockers, such as verapamil and nifedipine, have the opposite effect; they attenuate iCa and thus weaken the heartbeat. This may be helpful, in that it reduces the work of the Ca 2+ pumps/exchangers, and thus reduces O2 demand during hypoxia. The action potential can also be regulated physiologically, and can be altered by disease, as described next. Catecholamines boost the plateau current and contractile force the contractile force of a cardiac myocyte is proportional to the size of the plateau current iCa, which can be increased by circulating adrenaline and by noradrenaline from cardiac sympathetic fibres. The reduced K+ current leads to delayed repolarization, a prolonged plateau and a high 43 the cardiac myocyte: excitation and contraction frequency of arrhythmias. The prolonged plateau leads to Ca2+ overload and afterdepolarization (see Section 3. How does the action potential induce an order of magnitude increase in sarcoplasmic Ca2+ concentration To prove that sarcoplasmic Ca 2+ causes contraction, Fabiato and Fabiato performed a classic experiment in 1975, in which the sarcolemma was stripped from the myocyte, so that intracellular Ca 2+ could be equilibrated with a known Ca 2+ concentration in the bathing fluid. In junctional regions, there is typically 1 L-type Ca2+ channel for every 10 Ca2+ release channels. The Ca2+ release channels are activated by a rise in free Ca2+ concentration in their local subsarcolemmal environment, brought about by the opening of the adjacent L-type Ca2+ channel. A T-tubule, L-type Ca2+ channel and adjacent cluster of ~10 Ca2+ release channels (only two are shown here) form a functional unit. The trigger Ca2+ from a single L-type channel probably activates a cluster of 6­20 release channels. This raises the sarcoplasmic Ca2+ concentration roughly 10-fold over ~50 ms, from ~0. This does not happen in practice; studies show unequivocally that a small iCa causes a small Ca 2+ transient and weak contraction, while a large iCa causes a large Ca 2+ transient and strong contraction. This is probably because the activation of release channels requires a highly localized, very large rise in subsarcolemmal Ca 2+ concentration, mediated by an immediately adjacent L-type Ca 2+ channel; a lesser, diffuse rise in cytosolic Ca 2+ is not sufficient to activate the release channels. Activation within a given cluster may possibly be all-or-none due to positive feedback (the cluster bomb model), but the number of clusters activated is proportional to the size of i Ca. The release channels activate rapidly, then inactivate slowly, over ~30­100 ms (mechanism unknown). This is part of the process of restitution, the return of the system to its original resting state. Since sarcolemmal expulsion normally equals Ca 2+ entry during the action potential (as iCa), the cell Ca2+ content is unchanged in diastole. As the sarcoplasm Ca 2+ concentration falls, Ca 2+ dissociates from the troponin­tropomyosin complex and the muscle relaxes. The process of restitution, that is, the restocking of the store and restoration of release channel excitability, is normally completed well before the next excitation. The braking effect of phospholamban on the pump is reduced by adrenaline and noradrenaline. Consequently, adrenaline and noradrenaline increase the rate of myocardial relaxation (lusitropic action). This, as well as an increase in iCa, results in an increase of force (inotropic action), as described in the next section. This increases Ca 2+ efflux on the Na+/Ca 2+ exchanger and decreases Ca 2+ entry on the L-type Ca 2+ current. Beta-1 adrenergic stimulation increases the calcium transient and contractility As well as providing the trigger Ca2+, the current iCa continues through the early plateau phase and accounts for ~10%­25% of the rise in sarcoplasmic Ca 2+ concentration. Consequently, 45 the cardiac myocyte: excitation and contraction the force of contraction correlates with the amplitude and duration of iCa. This increases the size of the free Ca 2+ transient, and hence contractile force, through two mechanisms. First, the increase in trigger iCa in the subsarcolemmal space recruits more clusters of release channels, releasing a greater fraction of the Ca 2+ store. Digoxin increases the systolic calcium transient Digoxin, a cardiac glycoside extracted mainly from foxglove, has been used for over two centuries to treat chronic cardiac failure. Its pharmacological target is the sarcolemmal Na+/K+ pump, which it inhibits by ~25%. Since the transmembrane Na+ gradient drives the Na+/ Ca 2+ exchanger, Ca 2+ expulsion is slowed, the Ca 2+ store builds up and cardiac contractility improves. However, the toxic dose of digoxin is only a little higher than the therapeutic dose; at toxic doses, excessive accumulation of intracellular Ca2+ causes store overload. This is a potent trigger for afterdepolarization and arrhythmias; so, a digoxin overdose can trigger arrhythmias (Section 3. Crossbridge formation is induced by a rise in sarcoplasmic free Ca2+ concentration. Store discharge is triggered by the arrival of extracellular Ca2+ as current iCa at the start of an action potential (Ca2+-induced Ca2+ release). During a quiet heartbeat, the free Ca2+ transient is only big enough to activate a fraction of the potential crossbridges. Sympathetic stimulation increases iCa and stored Ca2+, leading to bigger free Ca2+ transients, increased crossbridge formation and increased contractile force. Stretching the cell increases its sensitivity to Ca2+, and therefore increases crossbridge formation and contractile force. Caffeine at very high concentrations in vitro activates Ca2+ release channels, leading to a sustained contraction called a contracture. However, at therapeutic lower levels, caffeine improves contractility by inhibiting phosphodiesterase, as does milrinone, a drug used to treat acute cardiac failure. The bicentennial experiment shown here confirms that foxglove extract increases contractile force and intracellular Ca2+ concentration in ferret papillary muscle. Light emission from the aequorin-injected muscle (top trace) was used to measure systolic free Ca2+ concentration; aequorin, a jellyfish protein, emits blue light in the presence of free Ca2+. When we are at rest, only about 40% of the potential actin­myosin crossbridges are activated during systole, so the heartbeat is gentle. During exercise or stress, the number of crossbridges is increased, so the heart beats harder, that is, with increased force. There are two fundamentally different ways of increasing crossbridge formation: 1. The systolic Ca 2+ transient can be increased, as hearts thus promotes store overload and arrhythmias. Conversely, it is difficult to induce ventricular tachycardia or fibrillation by regional ischaemia in a chronically denervated heart. The overloaded Ca 2+ store is prone to spontaneous, partial discharge during early diastole, possibly exacerbated by increased leakiness of the Ca 2+ release channels (RyR2) due to -adrenoceptor-driven phosphorylation. The diastolic rise in sarcoplasmic Ca 2+ stimulates Ca 2+ expulsion by the sarcolemmal 3Na+/1Ca2+ exchanger. If the afterdepolarization reaches a threshold, a premature action potential follows and may trigger an arrhythmia. Afterdepolarization may occur after the cell has fully repolarized (delayed afterdepolarization) or during its repolarization phase (early afterdepolarization). Delayed afterdepolarizations probably trigger most of the arrhythmias associated with acute cardiac ischaemia, chronic cardiac failure, digoxin toxicity and phosphodiesterase inhibition. Since an increased Ca 2+ current iCa, contributes to this chain of events, the risk of sudden death from arrhythmia after myocardial infarction can be reduced by adrenergic receptor blockers. This is normally brought about by noradrenaline released locally from cardiac sympathetic fibres, and circulating adrenaline from the adrenal gland. Stretching the myocyte in diastole provides a second, fundamentally different way of increasing crossbridge formation. In normal life, the myocardium becomes more stretched whenever diastolic filling increases, for example, every time we lie down. Stretch increases the sensitivity to Ca 2+, rather than the size of the Ca 2+ transient. This is the basis of the fundamental Starling law of the heart or length­tension relation described in Chapter 6. Ischaemia raises intracellular Na+ concentration, partly because the energy supply for the Na+/K+ pump is reduced, and partly because intracellular acidosis stimulates the sarcolemmal Na+/H+ exchanger. Cardiac ischaemia reflexly increases cardiac sympathetic nerve activity, raising noradrenaline and adrenaline levels. American Journal of Physiology 1996; 271: H1151­61, with permission from the American Physiological Society. Voltage dependent Na+ channels conduct a rapid inward current of Na+, iNa, which depolarizes the myocyte (spike of action potential, phase 0). The Na+ channels rapidly inactivate, and a transient outward K+ current, ito, causes an early partial repolarization (phase 1). A second inward current, mainly of extracellular Ca2+ ions, iCa, maintains a prolonged depolarization, the plateau, which lasts 200­400 ms (phase 2). An inward Na+ current through the 3Na+/1Ca2+ exchanger maintains the late plateau.

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The authors were unable to find any clinical studies confirming these in vitro activities of amantadine allergy testing allergens buy flonase 50 mcg with amex. Picornaviridae Amantadine did not inhibit the entry of poliovirus into cells (Perez and Carrasco allergy treatment ayurvedic buy cheap flonase 50 mcg on-line, 1993) allergy medicine xanax order 50 mcg flonase with visa. Moderate in vitro inhibition of hepatitis A virus replication by amantadine at a concentration of > 100 µM was reported (Widell et al allergy link cheap flonase 50 mcg online. Emerging resistance and cross-resistance There is a very low genetic barrier to adamantane resistance allergy treatment vitamins buy 50 mcg flonase otc, because single mutations in the target viral protein (M2) (see section 3. Mechanism of drug action) result in high-level resistance of influenza A to both amantadine and rimantadine. As a consequence, resistant variants of influenza A may 4526 Amantadine and Rimantadine appear rapidly during treatment or prophylaxis, and there is unequivocal evidence that in vitro resistance is a marker for in vivo resistance. Currently, circulating influenza viruses have a high prevalence of resistance, in animals and humans, including strains that do not regularly cause human infection, such as H5N1. Three publications have reviewed the problem of antiviral drug resistance in influenza, in general and specifically relating to adamantanes (Hayden, 2006; Weinstock and Zuccotti, 2006; Hurt, 2014). There is no cross-resistance between adamantanes and neuraminidase inhibitors because the mechanisms of action are completely different (Govorkova et al. Despite the deficiencies of adamantanes as antivirals, other drugs blocking the M2 pore have recently been discovered (Wu et al. This resistance threshold was partly arbitrary but is relevant to achievable plasma and respiratory secretion concentrations of the drugs (Hayden, 1996). Resistance of influenza A to both amantadine and rimantadine have developed during their administration for either prophylaxis or therapy of patients (Belshe et al. Strains of influenza A resistant to amantadine and rimantadine have been recovered from about 30% of patients receiving treatment and less frequently in those receiving prophylaxis (Hayden and Hay, 1992). The incidence of adamantane-resistant influenza A strains may be equally high in immunocompromised individuals treated with amantadine or rimantadine (Englund et al. These data suggest that children and immunocompromised subjects may be particularly susceptible to the development of adamantaneresistant influenza strains. Adamantane-resistant (as well as neuraminidase inhibitor-resistant) strains of influenza may be shed for prolonged periods by immunocompromised patients, even after adamantane therapy has been discontinued (Klimov et al. In a household setting, where an index case is treated with amantadine and household contacts are given amantadine prophylaxis, prophylaxis has failed because of transmission of amantadine-resistant strains from the treated index case to the household contacts (Hayden and Hay, 1992). Adamantane-resistant viruses that emerge during treatment of patients with amantadine or rimantadine appear to retain full virulence (Sweet et al. Further, adamantaneresistant strains appear to be genetically stable with no evidence of reversion to wild type, even after six passages in an avian model (Bean et al. When reversion does occur in an epidemiologic setting, it appears to be due to reassortment events (Furuse et al. Further, adamantane-resistant influenza strains have the same fitness as wild-type virus in animal passage experiments and thus can be maintained, perhaps indefinitely, without adamantane selection pressure (Bean et al. Amantadine prophylaxis was administered sequentially to all uninfected residents on 9 of 14 wards for 14­31 days per ward to control an influenza outbreak during the 1993­94 influenza season; amantadine treatment was simultaneously provided to 29 sick residents. A total of 16 residents had adamantane susceptibility testing of influenza A isolates, and 12 were amantadine resistant; 4 of the 12 patients had not received any antiviral therapy. Further evidence supports transmission of adamantane-resistant influenza strains, including from infected humans to uninfected family members and other contacts (Belshe et al. This mutation exchanges the M2 wildtype amino acid (serine) that is critical for adamantane binding, to asparagine (see section 3, Mechanism of drug action). Adamantanes stably plug the M2 pore in wild-type virus, with its contact with serine and valine side chains in the M2 pore. Adamantanes binds poorly to the asparagine, resulting in movement and changes in orientation in the drug, with the adamantane spontaneously moving lower in the central cavity of the M2 channel. Put simply, adamantanes bind without blocking proton transport in the S31N M2 channel, explaining the inability of adamantanes to inhibit replication of influenza strains with that mutation (Gleed et al. Amantadine-resistant mutations appear to be similar or identical in both avian and human strains of group A influenza viruses (Bean et al. Further, amantadine and rimantadine appear to have a similar propensity to induce resistance (Hayden et al. Adamantane antiviral drugs also cannot bind to the M2 ion channel of influenza strains with the A30T and S31N mutations because the mutant amino acids are larger than the wild-type ones at these positions, and their larger size blocks binding (Astrahan et al. If the adamantane drugs are unable to bind to the pore channel, they cannot block hydrogen ion transport (see section 3, Mechanism of drug action). Rare influenza strains have been described in which the binding of amantadine to the M2 transmembrane region remains unchanged but the M2 ion channel continues to function. The mutations mediating this effect were all at codon 27 (L/V27A/G/S/T), and the mutant amino acids were smaller than the wild-type ones at this position. It was hypothesized that the presence of smaller amino acid residues made the M2 pore larger, allowing ion flow even when the adamantane drugs were binding to it, and this hypothesis was confirmed by structural studies (Astrahan et al. It is not surprising that, adamantanes induce no changes in the influenza M1 protein, which is neither an integral membrane protein nor an ion channel (Galabov et al. Influenza viruses are found all over the globe, spread by humans or by waterfowl, swine or other animals. Consequently it is not remarkable that adamantane resistance of influenza strains, whether human or animal, has become global. The persistence of adamantane resistance without ongoing selective pressure from use of these drugs has contributed to its continuing global spread (Bean et al. It is interesting that these genotypes were seen almost exclusively in H1 (41% resistant), H3 (44%), and H5 (28%) influenza A strains, with fewer in H7 (13%) and H9 (23%) and with the others 2% (there were only 3 specimens of H17, all resistant). The H1- and H3-resistant variants were seen in both humans and pigs and rarely in avian species; although, as is well known, H5, H7 and H9 were largely restricted to avians. About 95% of the mutations were S31N, with only 1% with V27A and 14% having S31N with V27A (Dong et al. As was noted previously, the prevalence of M2 resistant variants was generally quite low from 1991 to 1995 (Ziegler et al. The S31N mutation, which mediates complete resistance to amantadine and rimantadine, was not prevalent in influenza-infected humans before 2000, but from that date it has gone from nearly zero to virtually 100%. This mutation became common (~ 50% prevalence) in swine influenza before humans, and it suggests that swine might have been the reservoir infecting the human population. Change in prevalence of M2 adamantane-resistant variants from before 2000 to 2013. The frequency of mutations appear relative to the total number of sequences in the subgroup (rows 1 and 2) or the total number of S31N mutants in the subgroup (row 3). Columns in the graph are labeled according to the host organism associated with each sequence subset. Because adamantane therapy is not used widely in many of the countries where there is a high prevalence of resistant strains, it has been hypothesized that the increasing frequency of adamantane resistance globally may be attributable to its interaction with fitness-enhancing mutations at other genomic sites rather than to direct drug selection pressure. This hypothesis is supported by data showing that adamantane resistance is accompanied by mutations in other influenza proteins, defining the so-called N-lineage of influenza, which may improve the fitness of these strains, thereby allowing their spread beyond regions where there is selective pressure by ongoing use of adamantanes (Simonsen et al. Although amantadine resistance is caused by single amino acid mutations in the M2 protein, genomewide adjustment involving multiple genes appears to be necessary to obtain efficient replication and transmission of resistant viruses. Such adjustments are attainable through reassortment of segments among different virus lineages (Zaraket et al. The prevalence is highest among H1N1, H3N2, and H5N1 viruses and least among H2 types, probably simply because H2 types are not circulating at present. Because the situation is fluid, and varies with geographic location, the reader is advised to seek current epidemiologic data that are country specific. However, our firm opinion is that it would be highly inadvisable to rely on adamantane therapy or prophylaxis alone for treatment or prevention of influenza A infection, possibly aside from H2 type infections for which adamantane resistance remains rare (Dong et al. In vitro synergy and antagonism When amantadine has been combined with antiviral agents with a different mechanism of action, effects on influenza A replication have usually been additive or synergistic. The agents studied include interferon alpha, and neuraminidase inhibitors (zanamivir, oseltamivir, and peramivir). Varying combinations of amantadine or rimantadine and human interferon alpha have shown additive to synergistic activity against a number of influenza A subtypes (Hayden et al. Similarly, combination therapy using rimantadine and ribavirin can prolong survival in influenza A/H3N2-infected mice (Hayden, 1986). The nucleoside analog 2-deoxy-2-fluoroguanosine, and zanamivir both show additive in vitro activity with rimantadine against influenza A (Hayden et al. However, these investigators also made the curious (and unexplained) observation that these combinations were antagonistic if assessed by production of cell-associated virus. Most interesting, the combination appeared to have additive effects even against amantadineresistant strains of influenza. Similarly, rimantadine and oseltamivir were synergistic against experimental H3N2 infection of mice (Galabov et al. Recently it has been shown that the combination of amantadine, ribavirin, and oseltamivir is synergistically active against influenza virus in vitro, even against strains resistant to one component of the combination and specifically resistant to amantadine (Nguyen et al. A study of that combination in mice experimentally infected with influenza viruses showed clearly that amantadine added efficacy to the ribavirin­oseltamivir combination, even when the infecting strain was fully resistant to amantadine (Nguyen et al. A phase I study in healthy volunteers showed that the pharmacokinetics of the drugs remained unchanged even when given in combination (Seo et al. Although the adamantane antivirals may inhibit replication of influenza and other viruses by interaction with other targets in the virus life cycle, these are not thought to be relevant at clinically achievable concentrations of adamantanes (Oxford and Schild, 1967; Kato and Eggers, 1969; Hay et al. Hence understanding the mechanism of action of adamantanes requires an understanding of the influenza virus life cycle and the relevance of the M2 protein in it (Pinto and Lamb, 2007). This step triggers receptor-mediated endocytosis, with the cell phagocytosing virus particles forming intracellular phagosomes containing virions (Matlin, 1982; Marsh and Helenius, 1989). These processes are not altered by amantadine or rimantadine (Couch and Howard, 1986; Richman et al. Similarly, amantadine does not appear to act at this step in the replicative cycle of other viruses that are susceptible to the drug in higher concentrations (Superti et al. To initiate virion transcription and translation, the replicative complex of the virus (the polymerase and associated proteins) must move from the interior of the influenza virion in the phagosome to the cell cytosol. It is in this process that the M2 protein, which is a hydrogen ion channel, is critical. Protons enter the channel through a waterfilled cavity that leads into the absolutely conserved tetrad of H37 residues that line the pore. A tetrad of His37 residues are associated with four W41 side chains (also absolutely conserved) in a stabilizing interaction that inhibits reverse flow of protons out of an acidified virus (Chizhmakov et al. The H37 tetrad is primed for conduction by binding two protons with high affinity (Hu et al. The phagosome develops into a lysosome by acidification (increasing hydrogen ion concentration) of its luminal contents. Current evidence suggests that the acidinduced changes in the conformation of the tryptophan residue at M2 protein position 41 results in the "opening" of the pore to water-carrying hydrogen ions (Pinto and Lamb, 2007; Schnell and Chou, 2008). A histidine residue at position 37 in the ion channel blocks passage of molecules larger than hydrogen ions (Pinto and Lamb, 2007). Adamantane molecules, at clinically relevant concentrations, enter the M2 ion channel from the exterior of the virus (through the M2 ectodomain), and a single molecule of these drugs binds to specific hydrophobic amino acid residues in the transmembrane portion of the M2 protein, especially a serine at position 31 (Hu et al. Because these drugs are hydrophobic, they function almost as a "cork" in the M2 pore, blocking passage of water and hydrogen ions (Hay et al. All potent M2 inhibitors, regardless of the amino acid target, all contain a positively charged ammonium moiety, which presumably serves as a mimic of the conducting hydronium ion that forms water-mediated hydrogen bonds with backbone carbonyls of M2 (Wang et al. Alteration of the conformational flexibility of the histidine residue at position 37 by adamantanes may be the mechanism by which they block hydrogen transport (Hu et al. The structure of the amantadine-M2 complex has recently been determined (Cady et al. There are two amantadine binding sites on the M2 protein, one high affinity and the other low affinity; binding to the high-affinity site physically occludes the M2 ion channel. The M2 proteins of influenza A and B have broadly similar structures (homotetrameric membrane proteins that are pH-gated hydrogen ion channels), but their amino acid sequences, including those in the channel of the M2 pore, share almost no homology. The adamantane-binding amino acids in the channel of influenza A M2 proteins are hydrophobic, whereas the channel of the influenza B M2 protein is lined with polar amino acids that do not bind adamantanes (Pinto and Lamb, 2007; Ma et al. Amantadine is also inactive against influenza C; when the influenza C M2 protein (the target for amantadine in influenza A) was expressed in Xenopus laevis oocytes, its ion-channel activity was not inhibited by amantadine (Hongo et al. Mutations that result in resistance to amantadine and rimantadine in influenza A viruses are solely contained in hydrophobic regions of the M2 protein (Hay et al. Concentrations of amantadine or rimantadine many times those achieved clinically prevent acidification of the endosome, thereby blocking virion uncoating (Koff and Knight, 1979; Bukrinskaya et al. Uncoating is the likely target of adamantanes for some viruses, such as vesicular stomatitis virus, which are susceptible to the drug at concentrations higher than can be achieved safely in humans (Superti et al. However, amantadine does appear to have a mechanism of action separate to blocking the M2 pore protein. Newly synthesized viral proteins are then transported to the cell surface via the acidic trans-Golgi. The M2 membrane protein of influenza A facilitates these processes by again functioning as an ion channel (Pinto, 1992). Amantadine has been reported to inhibit the release of influenza A virions from the surface of cells infected with the Rostock (H7N1) strain (Sugrue et al. The M2 protein regulates pH gradients across membranes within the trans-Golgi (Grambas et al. No differences in matrix protein M1 have been observed in rimantadine-resistant and susceptible strains of influenza A (H3N2) (Galabov et al. Mode of drug administration and dosage 4531 either prophylaxis or treatment is not recommended as of this writing (October 2015). There are no parenteral formulations of amantadine or rimantadine available for clinical use. The recommended dosage of amantadine for prophylaxis or treatment of adults < 65 years of age is 100 mg given twice daily, usually at breakfast and lunch (see Table 265. However, in a study of healthy adults aged between 18 and 55 years, prophylactic efficacy of amantadine was maintained at a lower dose of 100 mg/day (Reuman et al. Experience with amantadine and rimantadine suggests that, for greatest efficacy, therapy of patients with influenza A infection must be commenced within 48 hours of onset of symptoms (Mostow, 1987), preferably earlier.

Diseases

• Exstrophy of the bladder-epispadias
• Melanoma, malignant
• Goldberg syndrome
• Malaria
• Cockayne syndrome type 3
• Upton Young syndrome

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