Ординатура / Офтальмология / Английские материалы / Ophthalmic Drugs Diagnostic and Therapeutic Uses 5th edition_Hopkins, Pearson_2007
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GENERAL PHARMACOLOGICAL PRINCIPLES 17
Figure 1.5 Schematic representation of the neurohumoral transmission of autonomic and motor nerves. Open circles, acetylcholine storage vesicles; solid circles, acetylcholine receptor sites; open triangles, noradrenaline (norepinephrine) storage vesicles; solid triangles, noradrenaline receptor sites. Only the postganglionic sympathetic fibre has vesicles containing noradrenaline. Transmission at sympathetic neuroeffector junctions is by noradrenaline, in contrast to acetylcholine transmission at all ganglia, at the parasympathetic neuroeffectors and
at neuromuscular junctions. It should be noted that this
greatly simplified representation of ganglionic transmission has now been superseded by the concept that such transmission is a far more complex process, involving cholinoceptive and adrenoceptive sites (after Drill 1965 Pharmacology in medicine, 3rd edn. McGraw-Hill, New York).
•A general increase for all ions (chiefly Na+ and K+), in which case a localized depolarization follows: the excitatory postsynaptic potential (EPSP).
•A selective permeability increase for smaller ions only (chiefly K+ or Cl−) followed by stabilization or actual hyperpolarization of the membrane: the inhibitory postsynaptic potential (IPSP).
Initiation of postjunctional activity
A propagated AP in a neuron, or muscle action potential (AP) in most types of skeletal muscle, or secretion in gland cells, is initiated if an EPSP exceeds a certain threshold value. In smooth muscle and certain types of tonic skeletal muscle, propagated impulses do not occur; instead an EPSP activates a localized contractile response. EPSPs initiated by other neuronal sources at the same time and site as an IPSP will be opposed by the latter, the algebraic sum of these effects deciding whether or not a propagated impulse or other response ensues.
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The extraocular muscles are stimulated in the manner described above for most types of skeletal muscle (Koelle 1975), but some differences in the structure and reaction of these particular muscles should be noted. It is usual in most skeletal muscle for one motor neuron to innervate between 100 and 200 muscle fibres, whereas in the extraocular muscles one neuron may supply one between 5 and 20 fibres. Such small motor units permit a precision of control over extraocular muscles not to be found in other skeletal musculature.
This smoothness of ocular movement is further assisted by the exceptionally large amount of elastic connective tissue around the loosely arranged fine extrinsic ocular fibres, not found to the same extent in the dense connective tissue surrounding the bundles of other skeletal muscles.
Vesicles believed to contain acetylcholine are concentrated together with a large number of mitochondria within the axonal terminals of motor nerves. Similar vesicles also occur in the various presynaptic terminals or boutons, but unlike the small, discrete motor endplates in skeletal muscle fibres, the presynaptic and postsynaptic neuronaI processes form complicated patterns of intertwining ramifications amidst the tightly packed cells of the ganglion (for example, approximately 100 000 ganglion cells occur in a cubic millimetre of the superior cervical ganglion). As with the skeletal muscle, the transmitter in the autonomic ganglia has proved to be acetylcholine, which has been obtained from the perfusate of isolated ganglia after stimulation of the preganglionic fibres. The superior cervical ganglion of the cat is often used in such experiments.
Eccles (1964, 1973), Katz (1966), McLennan (1970) and Krnjevic (1974) are only a few of the many research scientists who have contributed much to our understanding of synaptic transmission. Koelle (1975) referring to the work of Katz & Miledi (1972), stated that this demonstrated that immediate postjunctional response (of skeletal muscle) to acetylcholine liberated by stimulation of the motor nerve is the development of a localized depolarization at the motor endplate, the endplate potential (EPP), equivalent to EPSP at postsynaptic sites, which on reaching a critical level generates propagated muscle AP leading to a contraction.
In contrast to cholinergically innervated skeletal muscle, smooth and cardiac muscle exhibit an inherent activity that is independent of a nerve supply, although this property probably does not apply to all smooth muscles equally. This activity can be modified but is not initiated by nerve impulses, and although it is less sensitive to electrical stimuli than striped, smooth muscle it is more sensitive to chemical stimulation. It has been suggested that the inherent activity of smooth muscle might be regulated by acetylcholine that is synthesized and released by the muscle fibres. Spikes or waves of reversed membrane polarization travel from cell to cell at rates considerably slower than the action potentials of axons or of skeletal muscle. Rhythmic fluctuations in the membrane resting potential in smooth muscle seem to initiate spikes that, as in skeletal muscle, in turn initiate contractions that in some tissues pass as a wave along the muscle sheet: for example, peristalsis in the small intestine.
GENERAL PHARMACOLOGICAL PRINCIPLES 19
Destruction or dissipation of the transmitter
Experiments have demonstrated that depolarization of the smooth muscle fibres of rabbit colon, occurring after a delay of 400 ms after stimulation of the cholinergic postganglionic fibres to this tissue, produces a spike that persists for approximately 600 ms. As the response occurred simultaneously in all the muscle fibres and the rate of depolarization is proportional to the stimulus, Gillespie (1962) concluded that each muscle fibre is probably innervated by more than one nerve fibre.
Membrane potential changes of single smooth muscle cells of the guinea-pig vas deferens, in which the excitatory fibres from the hypogastric nerve are adrenergic, show a somewhat similar state of affairs, although, on stimulation of the nerve, the delay in depolarization is much briefer. The actions of acetylcholine and adrenaline at membrane level were described by Bulbring (1958). Also in the vas deferens of the guinea-pig, Burnstock & Holman (1961) found miniature potentials similar to those at motor endplates of skeletal muscles, in the absence of nerve stimulation. These were probably due to spontaneous release of small amounts of noradrenaline. Burnstock & Holman (1961) include among their conclusions the remark:
Our results have shown that the mechanism of transmission of excitation from sympathetic nerve to smooth muscle is essentially similar to that of transmission at other neuroeffector junctions, stimulation of the effector nerve producing depolarization of the postjunctional membrane.
As in skeletal muscle, the spike initiates a contraction. Although to the author’s knowledge similar potential changes involving this slow buildup of graded potentials to an action potential have not been recorded from ocular smooth muscle, it would be probable that they are applicable to smooth muscle in general, including ocular.
As Koelle (1975) remarks, when impulses can be transmitted across junctions at frequencies ranging from a few to several hundred a second, an efficient means of disposing of the transmitter subsequent to each impulse is essential. Acetylcholinesterase (AChE) is the highly specialized enzyme available at most cholinergic junctions for disposing of acetylcholine released there as the transmitter. It is abundantly present in skeletal muscle tissue and is concentrated in the region of the motor endplates. Diffusion might account for the termination of action of acetylcholine at some synapses and it seems likely that this contributes to the method (which is mainly uptake by the axon terminals themselves and by other cells) of ending the activity of the adrenergic transmitter, noradrenaline. Two enzymes are capable of metabolizing noradrenaline and adrenaline, catechol-O-methyltransferase (COMT), which is found in practically all tissues, and monoamine oxidase (MAO), which is present in nervous tissue and the liver. These enzymes have not the importance or speed of action of acetylcholinesterase in cholinergic transmission.
Neuroeffector junctions are those junctions where two cells are in more or less close physical relationship, the term being confined to nerve
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and effector cells (which respond characteristically to a stimulus) of cardiac muscle, smooth muscle and gland.
A synapse is the area of proximity between two neurons where impulses are transmitted from one nerve cell to another across an ultramicroscopic gap (approximately 20 nm wide) called the synaptic cleft. Ganglia contain many such synapses. Ganglionic transmission is a highly complex process, incorporating many of the elements of transmission at the myoneural junctions of both skeletal and smooth muscles. It is now thought that interneurons and additional transmitters are also involved. In addition to the primary pathway involving ACh depolarization of postsynaptic sites (described above), secondary pathways for the transmission of excitatory and inhibitory impulses have also been described. Specific non-depolarizing ganglion blocking drugs affect the primary pathway but the secondary pathways are insensitive to these agents (Volle & Koelle 1975). There is some evidence indicating the participation of a catecholamine (dopamine or noradrenaline, from a catecholamine-containing cell or interneuron within the ganglion) acting on the ganglion to cause hyperpolarization (IPSP) (Eccles & Libet 1961). It has been suggested that multiple cholinoceptive and adrenoceptive sites exist in the mammalian superior cervical ganglion (Greengard & Kebabian 1974).
Neuromuscular junctions are the spaces that occur between motor nerve fibre endings and skeletal muscle motor endplates, and are comparable to synaptic clefts at synapses. ‘Myoneural junction’ is a term that embraces neuroeffector junctions with smooth muscle and neuromuscular junctions, that is, it includes all types of motor nerve endings.
EVIDENCE FOR NEUROHUMORAL TRANSMISSION
Evidence for neurohumoral transmission can be deduced from the following experimental data:
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The demonstration, at appropriate sites, of the presence of a physiologically active compound and of the enzymes involved in its metabolism.
Recovery of the compound from the perfusate during stimulation of an innervated organ, the substance not being present (or only in vastly reduced amounts) in the absence of such stimulation.
Appropriate administration of the compound produces the same responses as nerve stimulation.
The demonstration that these responses to nerve stimulation and administration of the compound are modified in the same way by various drugs.
This evidence is further supported by that important feature of junctional transmission, the irreducible latent period, that is, the time lag between the arrival of an impulse at the axonal terminal and the manifestation of the post-junctional potential. The evidence for the existence of neurohumoral transmitters is well substantiated by Otto Loewi’s
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classic experiment in 1921, which demonstrated the release of a vagus substance (‘vagustoff’) on stimulation of the cardiac nerve to the frog’s heart. Loewi stimulated the vagus fibres while the heart (the donor) was perfused with a balanced salt solution. The perfusion fluid was then perfused through a second, isolated, denervated frog’s heart (the recipient). Recordings of the rates of contraction of both hearts were made. A substance was liberated from the donor heart that dissolved in the perfusion fluid and slowed the rate of contraction of the recipient heart. This vagustoff was later identified as acetylcholine. Loewi also discovered that an accelerator substance similar to adrenaline (at first called sympathin and later identified as noradrenaline) was liberated into the perfusion fluid (accelerating the rate of contraction of the recipient heart) when the action of the sympathetic fibres in the cardiac nerve of the frog’s vagus predominated over that of the inhibitory parasympathetic vagus fibres.
The cardiac branch of the frog’s vagus contains a sympathetic accelerator component, the accelerens, as well as parasympathetic fibres; thus it is a mixed nerve and its stimulation at times causes inhibition and at other times acceleration. The particular result varies with the frog and with the time of the year; in winter inhibition dominates and in summer acceleration.
Further substantiation has been produced by identification by various pharmacological, chemical and physiological tests of the substance that is present in perfusate from an innervated structure during the period of nerve stimulation but is not present in the absence of stimulation. In addition, it has been demonstrated that the substance so obtained is capable of producing responses identical to those of nerve stimulation and that both responses are modified in the same manner by various drugs.
Most of the general principles concerning the physiology and pharmacology of the autonomic nervous system and its effector organs are applicable, with some reservations, to the neuromuscular junctions (e.g. those of extraocular voluntary muscles) and in some respects to the central nervous system, although here knowledge of the transmitters involved is far from complete.
The autonomic nervous system (involuntary, visceral or vegetative nervous system) consists of nerves, ganglia and plexuses that innervate the heart, blood vessels, glands, viscera and smooth muscles throughout the body. The motor nerves of this system supply all structures of the body except skeletal muscle. Somatic nerves, with their synapses occurring entirely in the central nervous system, supply the latter, whereas the most distal synaptic junctions in the autonomic system are in ganglia occurring outside the spinal cord (Fig. 1.6), for example, the superior cervical ganglion (SCG) and ciliary ganglion (CG), which are the final relay stations for the sympathetic and parasympathetic autonomic innervation, respectively, of the eye. The motor nerves to skeletal muscle, including the extraocular muscles, are medullated (myelinated), whereas the postganglionic autonomic nerves are non-myelinated, with the exception of the short ciliary nerves.
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Figure 1.6 Autonomic nervous system.
CNS |
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Effector |
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Parasympathetic system |
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Sympathetic |
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Adrenal medulla |
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A = Acetylcholine |
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is the transmitter |
Somatic |
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N = Noradrenaline |
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Skeletal muscle |
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is the transmitter |
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Anomalous sympathetic |
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Sweat |
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gland |
ACETYLCHOLINE
Acetylcholine is the neurohumoral transmitter of all preganglionic autonomic nerve fibres, all parasympathetic postganglionic fibres, and a few postganglionic sympathetic fibres (as previously mentioned, those which innervate the sweat glands, vasodilator fibres to skeletal muscle arteries, and postganglionic fibres to the adrenal medulla).
The complex sequence of enzymatic reactions in the formation of ACh can be outlined as follows (Moses, 1975): choline present in the extracellular fluid is taken up, by active transport, into the axoplasm. Choline acetylase (choline-acetyltransferase), which occurs in all cholinergic nerves, is synthesized within the perikaryon and then transported, by unknown means, along the axon to its terminal. The axonal terminals, in addition to their vesicles, contain the large number of mitochondria in which the acetyl coenzyme A is synthesized. The final step in the synthesis of ACh probably takes place within the cytoplasm, and subsequently most of this transmitter is stored within synaptic vesicles. These are mostly concentrated at the synaptic and neuroeffector junctions and are spherical structures 400–500 nm in diameter. It has been estimated that each synaptic vesicle contains from 1000 to over 50 000 molecules of ACh, and a single motor-nerve terminal contains 300 000 or more vesicles (Koelle 1975).
The simultaneous discharge of 100 or more quanta (vesicles), following a latent period of 0.75 ms, occurs when an AP arrives at the motornerve terminal (Katz & Miledi 1965). The AP appears to depolarize the
GENERAL PHARMACOLOGICAL PRINCIPLES 23
terminal, increasing the permeability of the terminal axoplasmic membrane and permitting the inflow of calcium ions. This causes the liberation of ACh into the synaptic cleft by the process of exocytosis, that is, the membranes of the vesicles (ACh is found in clear vesicles and noradrenaline in granulated vesicles) fuse to the nerve cell membrane and the area of fusion breaks down, extruding the contents on the outside of the cell, the membrane of the latter remaining intact (Ganong 1979).
Combination of the transmitter with postjunctional receptors and subsequent effects of this have already been discussed, the effects of this mediator being rapidly terminated by diffusion and/or the antagonistic enzyme AChE. The storage, release and disposal of ACh at synaptic and other cholinergic neuroeffector sites is considered to be essentially the same at neuromuscular junctions.
ACETYLCHOLINESTERASE
This enzyme is present at neuromuscular junctions and in the neurons of cholinergic nerves throughout their entire lengths. It is also found in large amounts in erythrocytes. Also called specific, or true cholinesterase (ChE), AChE is capable of rapidly hydrolysing acetylcholine liberated in the process of cholinergic transmission to choline and acetic acid (the latter has no action, and the choline very little, on cholinergic receptors), in time periods as little as a millisecond. The transmitter acetylcholine is its preferred or only substrate.
Butyrocholinesterase is another enzyme found in the body tissues and fluids (nerves, plasma, liver and other organs) that is also capable of hydrolysing acetylcholine, but at a slower rate than AChE. It is known as non-specific, or pseudocholinesterase, but its physiological function is unknown as its experimental inhibition with certain drugs produces no apparent functional derangement at most sites.
Some drugs, known as anticholinesterases (e.g. physostigmine) neutralize acetylcholinesterase and then the liberated acetylcholine continues to act until it diffuses away. For example, after physostigmine has been instilled in the eye, the constriction of the ciliary and sphincter pupillae muscles continues long after parasympathetic stimulation of these muscles has ceased.
NORADRENALINE
Noradrenaline is the neurohumoral transmitter for the great majority of postganglionic sympathetic fibres, which are termed ‘adrenergic’. Sir Henry Dale (1934) was the original proposer of the terms ‘cholinergic’ and ‘adrenergic’ to describe neurons that liberated acetylcholine and noradrenaline, respectively.
Elliott (1905) suggested that postganglionic sympathetic fibres might transmit their impulses to autonomic effector cells by liberation of an
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Figure 1.7 The enzymatic synthesis of noradrenaline and adrenaline.
adrenaline-like substance, later called sympathin. In the 1940s, Euler conclusively identified sympathin as noradrenaline. This transmitter is released from all stimulated postganglionic sympathetic fibres except those to certain sweat glands and vasodilator fibres in man, which were discovered to be cholinergic (Dale & Feldberg 1934).
The steps in the enzymatic synthesis of noradrenaline and adrenaline (known in the USA as norepinephrine and epinephrine, respectively), proposed by Blaschko (1939) and confirmed by demonstration (using radioactive labelled phenylalanine in rats) by Gurin & Delluva (1947), are shown in Fig. 1.7. Tyrosine is taken up into the neuron from the extracellular fluid; the other steps of the enzymatic synthesis occur within the neuron (the second and third steps taking place in the cytoplasm). Dopamine then enters the granules to be converted into noradrenaline. Most of the noradrenaline in the adrenal medulla leaves the granules and is converted in the cytoplasm to adrenaline, re-entering another group of granules for storage until released. Adrenaline
Phenylalanine
Hydroxylase
Tyrosine
Hydroxylase
Dopa
L-aromatic amino acid decarboxylase
Dopamine
Dopamine p-hydroxylase
Noradrenaline
Phenylethanolamine n-methyltranserase
Adrenaline
GENERAL PHARMACOLOGICAL PRINCIPLES 25
accounts for approximately 80% of the catecholamines in the adult human adrenal medulla, noradrenaline contributing most of the remainder.
Under very high magnification (electron micrographs) varicosities that contain noradrenaline stored in granular vesicles can be seen on adrenergic nerve fibres (Ruskell 1967, 1969). The Falck-Hillarp fluorescein technique for the demonstration of catecholamines can be used to show these. Briefly, this involves treating the neurons with formaldehyde vapour and examining the resultant histochemical stain under ultraviolet light. The noradrenaline can be seen within the vesicles as fluorescent material.
Adrenergic fibres can sustain the output of noradrenaline for long periods of stimulation but the maintenance of adequate reserves to allow this is dependent on an unimpaired synthesis and reuptake of the transmitter, by active transport, into the adrenergic neuron terminals. Cocaine inhibits the reuptake of catecholamines by adrenergic nerve endings, which temporarily prolongs the activity of noradrenaline, and instillation of cocaine in the eye results in mydriasis is an additional effect to its local anaesthetic property.
It is thought that some of the noradrenaline within the granules is in a smaller mobile pool in equilibrium with some held in reserve as a salt of ATP (four molecules of the catecholamine to one of ATP) along with a specific protein. A much larger mobile pool of noradrenaline exists in the cytoplasm within the nerve terminal. The cytoplasmic and intragranular mobile pools are kept in equilibrium by active transport mechanisms, passive diffusion, enzymatic synthesis and destruction [by mitochondrial monoamine oxidase (MAO)].
The noradrenaline is discharged rapidly from the neuron terminal by the nerve action potential, the latter requiring the presence of calcium ions. The possible involvement of acetylcholine, contained in the sympathetic neuron, as an essential or facilitatory step in the release of noradrenaline – the Burn & Rand hypothesis (1965) – is still controversial.
According to Koelle (1975), in the adrenal medulla ACh liberated by the preganglionic fibres combines with the receptors on the chromaffin cells to produce a localized depolarization, which is followed by the entrance of calcium.
CHOLINERGIC RECEPTORS
Based on their ability to be stimulated by either nicotine (the well-known alkaloid from tobacco) or muscarine (an alkaloid found in the poisonous toadstool Amanita muscaria) cholinergic receptors are divided into two groups: (1) muscarinic receptors found on the effector cells of autonomic structures and (2) nicotinic receptors, on which acetylcholine acts at autonomic synapses and skeletal muscle. Further subdivisions are necessary for both receptors. There is a difference in the susceptibility of nicotinic receptors at the synapses at the motor endplates of skeletal
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muscle to the effects of blocking agents. The neuromuscular blocking drugs, such as tubocurarine, are less effective on autonomic ganglia whereas the reverse is true for ganglion blocking agents such as hexamethonium. There is also more than one type of muscarinic receptor: M1 receptors are found in autonomic ganglia whereas M2 predominate at autonomic effector sites. Additionally, different organs are effected to different degrees by antimuscarinic agents.
In clinical usage three main types of drug produce their effects by acting on cholinergic receptors:
•stimulant or mimicking drugs
•anticholinesterases
•antagonist or blocking drugs.
Cholinomimetics Although there are chemicals that will produce the same effect as endogenous acetylcholine at nicotinic receptors, these have little clinical significance other than possibly as side-effects. However, drugs acting as muscarinic receptors have been used for many decades to produce effects similar to that of stimulating the parasympathetic nervous system – such drugs are known as the parasympathomimetics. They are divided into two types, depending on their structure and origin.
The older group is the naturally occurring cholinomimetic alkaloids, which have a plant source. They are muscarine, pilocarpine and arecoline. Of these, the most widely, and probably the only one that is regularly used is pilocarpine, whose employment in ophthalmic medicine extends back into the last century.
As the supply of plants from which drugs can be extracted has been erratic and expensive, a search for a synthetic alternative was made. Derivatives of acetylcholine – choline esters – were made and were found to mimic acetylcholine to varying degrees. Methacholine is still broken down by true cholinesterase at a slow rate whereas carbachol and bethanechol are resistant. Methacholine has only muscarinic effects whereas carbachol will affect nicotinic receptors.
Anticholinesterases There is a natural background release of ACh from cholinergic terminals. The ACh is broken down by cholinesterase before it can accumulate in physiologically active quantities. Anticholinesterases are compounds that bind to cholinesterase in a similar manner to ACh. The difference between the two is that acetylcholine can be broken down very quickly whereas anticholinesterases are metabolized slowly. While anticholinesterases are occupying the cholinesterase site they are not available to hydrolyse ACh and the transmitter can build up to pharmacologically active levels.
Anticholinesterases vary greatly in duration of action and can be dividied into two types:
•reversible anticholinesterases
•irreversible anticholinesterases.