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Muscarinic GREGOR W. NIETGEN, JOERG SCHMIDT, LUTZ HESSE, CHRISTIAN W. HONEMANN, functioning and MARCEL E. DURIEUX distribution in the : molecular basis and implications for clinical diagnosis and therapy

The role of has generated number of receptors (, considerable interest over the last decade. and others) have been found in all types of Dale's first description in 1914 of the muscarinic ocular tissue: the functional consequence of and nicotinic components of the cholinergic their activation remains elusive but is currently systeml provided an explanation for the effects being investigated with great zeal. of various cholinergic active on the eye. patients are still being treated with G.w. Nietgen J. Schmidt Parasympathetic cholinergic input to the human almost 40 years5 after its introduction to western L. Hesse muscle comes from medicine in 1875.6 The development of specific Zentrum fOr whose make up the ciliary , a muscarinic (acetyl-j3-methylcholine) Augenheilkunde branch of the third cranial nerve. and antagonists () followed. Philipps-Universitat Marburg is released by these neurons onto their target was discovered in 19267 and Marburg, Germany 8 cells, the surrounding the . named in 1932. At the same time was C.W. Honemann Muscarinic acetylcholine receptors on the synthesised,9 a resistant to M.E. Durieux surface of the muscle cells transduce the with a suitable specificity for glaucoma Departments of chemical signal into a which treatment.IO,ll Over the years various , Anaesthesiology and constricts the pupil. It has also been shown that compounds have been investigated regarding Neurosurgery muscarinic cholinergic receptors exist in the their potential to lower University of Virginia Health mammalian , once thought to (lOP), the most prominent pathophysiological Sciences Center receive only noradrenergic input from the feature of glaucoma. The majority of these Charlottesvile sympathetic system? This double reciprocal substances do not affect the muscarinic system Virginia, USA innervation of the iris sphincter follows the of the eye and intervene at different receptor Gregor W Nietgen, MD � general pattern of innervation: stimulation of sites. Zentrum fOr the parasympathetic The , focus of intense Augenheilkunde (cholinergic muscarinic), which functions investigation, contracts through activation of Philipps-Universitat Marburg Robert-Koch-Strasse 4 through the polyphosphoinositide signalling muscarinic receptors. Due to its insertion into 35033 Marburg pathway, leads to contraction. Relaxation is a the it increases aqueous Germany outflow facility, thereby reducing IOpy,13 A result of the activation of the sympathetic Tel: +49 6421 282600 nervous system (beta-adrenergic), which variety of drugs can also reduce lOP, yet by Fax: +49 6421 285678 functions through the cAMP system. very different mechanisms of action. This e-mail: The active secretion of is indicates the pathogenetic complexity of [email protected] carried out by the ciliary and is glaucoma, with its multiple possible causes; Supported in part by therefore a key target for regulation by however, lOP regulation through the National Institutes of Health grant GMS 52387/02 to endogenous regulators and anti-glaucoma muscarinic signalling system appears to be an M.E.D. G.WN. was drugs. Histological evidence indicates that the important component. The ocular muscarinic supported by a grant from receive innervation by both receptor system is not dedicated solely to the the Deutsche sympathetic and parasympathetic . maintenance of pressure though. A Forschungsgemeinschaft Further on, ciliary epithelial cells have been wide distribution of these receptors in the (DFG, Ni 482/1-1). C.WH. is supported by a grant from demonstrated to contain both adrenergic and has been found. Muscarinic the Innovative Medizinische cholinergiC receptors? Interactions between the signalling is involved in Forschung, MOnster, two second messenger systems are important in functions of the ,14 possibly in reparative Germany (lMF Ho-1-6-11/98-8) regulation of smooth muscle tone and are an functions in the corneal and lenticular Received: 14 September important focal point for pharmacological tissue,15,16 and appears to play a major role in 1998 manipulation.4 Besides these well-established the embryonic and postnatal development of Accepted in revised form: functions of the ocular receptor interplay a vast the eye.17 The main distinction between 22 January 1999

Eye (1999) 13, 285-300 © 1999 Royal College of Ophthalmologists 285 muscarinic and nicotinic receptors, though noted very thousand receptor types have now been shown to belong early, hardly explained their distinct roles in cholinergic to the -coupled receptor (GCR) superfamily, of signalling. The past decade, however, has seen the which the muscarinic receptors form a small but molecular cloning of both nicotinic18 and muscarinic19 distinguished cluster. acetylcholine receptors in Numa's laboratory,20 leading GCRs all show the same molecular signature in their to greatly expanded understanding of these systems. In sequence: most are around SOD amino acids in addition, new research techniques such as patch length and include seven stretches of approximately 20 clamping21 and single channel recording22 have hydrophobic amino acids each. These domains are provided additional insights into the functioning of the thought to form a-helices traversing the membrane, cholinergic signalling system. leading to the designation of these proteins as seven­ We now know that, although acetylcholine is the transmembrane, or, more fancifully, serpentine or physiological on both nicotinic and muscarinic heptahelical receptors (Fig. I). receptors, they are completely different entities: the first The G proteins stimulated by receptor activation a multi-subunit, -gated (i.e. an control a number of intercellular systems. Best described ionotropic receptor), the second a single-subunit, G are G proteins stimulating (Gs) and inhibiting (Gi) protein-coupled receptor (i.e. a metabotropic receptor). It adenyl ate cyclase, with corresponding changes in cAMP appears likely that all muscarinic receptor subtypes have levels. C, activated by Gq or Go, generates now been cloned, allowing development of specific trisphosphate (IP , which releases Ca2+ from 3 antibodies,23 detailed mapping of tissue distribution, and intracellular stores) and diacylglycerol (which activates synthesis of subtype-specific agonists and antagonists protein kinase C). In addition, G proteins can activate ion (Table la)?4-26 It has become clear that muscarinic channels, as in the case of Gk (a Gi subtype), which closes signalling plays an important role in multiple locations of a neuronal in response to muscarinic the eye, and that ocular cholinergic drugs interfere stimulation. significantly with this system. This article will focus on the molecular basis of these findings. It will show that the complex distribution of muscarinic receptors in the eye is Five muscarinic receptor subtypes have been cloned only a part of many interacting signalling systems, all resulting in the development and maintenance of vision. Once the DNA sequence of one muscarinic receptor was Following a brief summary of the molecular of known19 other subtypes were isolated in rapid muscarinic receptors, their distribution and function in succession. Thus far, five muscarinic receptor have been the human eye will be described. A description of the cloned,28 designated ml, m2, m3, m4 and mS. The clinical implications of these signalling pathways and existence of this many subtypes was surprising, as their interactions in pathological processes will be pharmacological studies suggested initially only two (MI outlined. and M2). Glandular M2 receptors were designated M3. (Names with a capital 'M' indicate pharmacologically defined subtypes, whereas those with a small 'm' Molecular biology of muscarinic signalling indicate clones.) Four pharmacological subtypes have The first muscarinic receptor was cloned in 1986.19 In the now been defined (MI, M2, M3, M4)?9,30 This apparent 13 years that have passed, a remarkable amount of excess of subtypes is typical for GCRs, and presumably information has been gathered about the molecular allows finer regulation of receptor expression. The five biology of muscarinic signalling. Not only have subtypes fall into two groups - the 'odd' (ml, m3, mS) (presumably) all subtypes of muscarinic receptors been and the 'even' (m2, m4) - based on sequence cloned, but detailed information on their structure­ and second messenger signalling. The odd group signals activity relationship is available, which will prove useful primarily through intracellular Ca2+ ; the even group in the development of new, highly selective agonist and through decreases in cAMP production. In the or antagonist drugs. retina, where signalling systems eventually have to transduce their actions through changes in membrane potential, ml and m3 inhibit a G protein-coupled Muscarinic receptors belong to the G protein-coupled a potassium current (IM) and activate a Ca2 -activated receptor superfamily potassium current (IK(ca», a whereas m2 and m4 When the DNA encoding the muscarinic receptor had receptors inhibit the lea current through voltage­ been isolated, it was compared with previously cloned activated Ca2+ channels (Fig. 2).31 Although the clones sequences, and its closest relative was found to be the were numbered in the order they were identified, the ml visual .19 Although at first this may clone happens to show most of the properties of the appear to be an unusual relationship, the sequence pharmacological Ml type, and the m2 clone those of the similarity relates to the fact that similar intracellular M2 type; similar correspondences exist with the m3/ M3 systems transduce the signals generated by these and m4/ M4 combinations. The presence of the mS molecules. In both cases, a GTP-binding protein (G subtype in several brain regions and in the eye is protein) forms an intermediate between membrane documented, although its function and pharmacological receptor and intracellular second messenger. More than a profile remain to be established.

286 A

-"'--c

B N

Fig. 1. Model of a muscarinic . (A) Linear model. The whole molecule is approximately 500 amino acids long. Seven hydrophobic stretches of approximately 20 amino acids are present, presumably forming a.-helices that pass through the cell membrane, thus forming seven transmembrane domains (tl-t7). Extracellularly the aminoterminus (N) and three outside loops (01---03) are found; intracellularly there are similarly three loops (il-i3), and the carboxy terminus (e). (B) Top-down view. Although in (A) the molecule is pictured as a linear complex, the transmembrane domains are thought to be in close proximity, forming an ellipse with a central ligand-binding cavity (indicated by a dashed circle). Asp and Tyr refer to two amino acids important for ligand interaction. G protein binding takes place at the i3 loop and the carboxy terminus. From Durieux.27

Muscarinic receptor junctions are related to molecular transmembrane regions (t2, t3, t7),32 whereas the i3 loop domains and the carboxy terminus (C) are involved in G protein

The cloned muscarinic receptor subtypes and other binding33 and regulation through phosphorylation.34 In members of the superfamily have been used to determine muscarinic receptors, the G protein binding specificity the intramolecular sites involved in ligand binding and G has been mapped to a remarkably small domain of protein coupling. As the muscarinic subtypes show approximately 20 amino acids in the i3 IOOp?5 As in 89-98% amino acid sequence identity in mammalian adrenergic receptors,36 agonist binding in muscarinic species, specificity of ligand binding and G protein receptors is initiated by contact with a specific aspartate coupling must depend on relatively small changes in residue in t3?7 Exchange of (part of) t6, i3, t7 and i4 structure. In agreement with their functional grouping, between the m2 and m3 subtypes resulted in a change in the odd and even receptors show particularly high G protein coupling and subtype-selective ligand within-group similarity. There is, however, a remarkable binding.38 Mutation studies have shown a series of lack of sequence similarity in the third intracellular loop threonine and residues in t3, t5, t6 and t7 that are (i3, Fig. 1), with the exception of the first and last 15 to 20 of importance in agonist, but not antagonist binding,39 amino acids. Studies of bacteriorhodpsin (a related again demonstrating the role of transmembrane domains molecule for which the three-dimensional structure has for ligand binding. Thus, the functional domains of these been established) and adrenergic receptors have receptors are well established and the importance and demonstrated that ligand binding takes place in a pocket, potential of highly selective ocular drugs for these primarily consisting of the second, third and seventh domains can be foreseen.

287 +ACh K(Ca) A

PK .. C ACh� B AT)

cAMP

Fig. 2. Intracellular signalling by muscarinic receptors. A composite illustration of the intracellular signalling pathways employed by muscarinic receptors. (A) Signalling through a receptor of the 'odd' group. The receptor (indicated by a stylised 7-transmembrane model) is activated by acetylcholine (ACh) and stimulates two main classes of G protein (G). One class, consisting of members of the Go and Gq, families, activates (PL-C). This results in the breakdown of phosphatidylinositol bisphosphate (PIP2) to inositol trisphosphate (fP3) and diacylglycerol (DAG). IP3 acting through its own receptor (fR) releases Ca2+ from internal stores, which can activate Ca-activated K channels (K(Ca))' However, in neurons, IK(Ca) is often inhibited by muscarinic stimulation via unclear pathways. DAG activiates protein kinase C (PK-C). The other G protein, presumably Gql11, closes K channels (Km) in neuronal membranes through an as yet unidentified intermediary. (B) Signalling through a receptor of the 'even' group. Again several G proteins are involved. One, presumably a member of the G; class, inhibits adenylate cyclase (AC) resulting in a decrease in the conversion of ATP to cAMP, and thus decreased cAMP levels. Another G protein, probably Go, inhibits an N-type Ca channel (Ca) through an unidentified intermediary. In cardiac tissue (and possibly in neurons), activation of Gk directly opens a K;, channel. Specific types of G proteins have not been indicated in the figure, as most have not been formally iden tified in studies. Not all cells expressing muscarinic receptors will show all signalling pathways indicated. From Durieux.27

Pharmacology of muscarinic signalling antagonists have been developed, drugs with selective agonist activity are not as widely available. Acetylcholine Until the first cloning of a muscarinic receptor was and most of the classical parasympathomimetic drugs achieved in 1986/9 investigators depended on (carbachol, , muscarinic and pilocarpine) are pharmacological tools, primarily selective antagonists, to non-selective. In functional studies several experimental define the several subtypes of this receptor family. Unfortunately, none of the known antagonists is compounds have displayed some selectivity for M143 completely selective, so that subtypes had to be defined and M244 receptors. However, these substances exhibit a by measuring the binding properties of several functional selectivity for the receptor subtypes only and compounds. Thus, equilibrium binding studies with show no or only limited selectivity in terms of affinity.45 pirenzipine initially indicated the existence of two classes So far even modestly selective agonists for the M3 and of cerebral muscarinic receptors, named M1 and M2?6,40 M4 subtypes are not available. Overall, since the cloning Kinetic studies allowed differentiation of three of the first muscarinic receptor a remarkable amount of subtypes41 and with the development of novel information has been gathered about the molecular antagonists this number was expanded to four biology of muscarinic signalling. Not only have the main (M1-M4)Y Tables 1a and 1b indicate the relative classes of muscarinic receptor subtypes been cloned, but selectivity of the commonly used muscarinic antagonists, detailed information on their structure-activity and relate the pharmacologically defined types to the relationship is available, which has already proved cloned receptor genes. An excellent recent review of this useful in the development of new, highly selective subject is available?4 Whereas many useful and antagonist drugs.

288 Table la. An overview oj differences in selectivity oj various their stimulation leads to increases in intracellular Ca2+ muscarinic antagonists' and therefore activation rather than inhibition of IK(Ca) eDNA: m1 m2 m3 m4 mS would be expected. Such is indeed seen in transfected Subtype: M1 M2 M3 M4 cells49 but it has not been observed in neurons. The ++ + + ++ + mediator involved has not been defined. + ++ + + + AFDX 116 Inhibitory effects of muscarinic Signalling are found in + ++ + ++ + many neurons, and the best-defined pathway is the Methroctamine ++ ++ + ++ + 4-DAMP + + ++ ++ ++ muscarinic effect on voltage-activated Ca2+ currents pFHHSiD + + ++ ++ ++ (I(Ca». This appears to be mediated by m2 or m4 receptors activating G proteins. 50 The N-type Ca aBased on potencies from functional or radioligand binding o experiments on muscarinic receptors. channel involved is sensitive to the Ca- +, relatively low affinity; + +, relatively high affinity. AFDX 116, O- GVIA but not to dihydropyridines. Another [11-2[[2-(9-diethylamino )methyl]-l-piperidnl]acetyl]-S, 11-dihy­ inhibitory effect of muscarinic signalling, even though dro-6H-pyrido [2,3-b ] [1,4]benzodiazepin ]-6-one, 4-DAMP, 4-di­ phenylacetoxylmethyl piperidine methiodide; pFHHSiD, para­ only documented in cardiac atrial cells, is the activation fluorohexahydrosiladifenidol. of inwardly rectifying K channels (Ki) through M2 stimulation. This is responsible for the cardiac side effects of topically applied ophthalmic Intracellular pathways drugs, and has been shown to result from direct activity of stimulated G; proteins on the channel. Dimming of As stated earlier, Ca2+ and cAMP are the best-described vision, especially reported after use of carbachol,51 might intracellular second messengers of the 'odd' and 'even' be a direct effect on muscarinic signalling. A receptor groups, respectively. In the eye, with its primary recent review on the subject is available. 52 Much interest function of electrical signalling, muscarinic systems also has been generated by findings that the G protein transduce their actions through changes in membrane l3-subunit, traditionally considered inactive, appears to potential. Several ion conductances, mainly in neuronal play an important role in this effect.53,54 Although most cells, have been shown to be affected by muscarinic data have been obtained in atrial cells, there is evidence stimulation, and the effects are most easily classified as that similar pathways exist in retinal14,55 and cerebral depolarising (stimulatory) or hyperpolarising signal transduction. 56 Fig. 2 summarises the intracellular (inhibitory)?4,31 The best-known depolarising effect is by pathways involved in muscarinic signalling. This area is inhibition of a non-inactivating voltage-gated K+ the subject of active investigation, and several recent, channel.(IK(rn» that clamps the membrane at its resting more extensive reviews exist.24,57,58 potential.46 Stimulation of (primarily) Ml receptors inhibits this channel, resulting in a more likely to fire when depolarised by other agonists. This effect has Conclusions been studied in some detail, and has been shown to be The investigations that followed cloning of the mediated through the Gq/ G protein.47 A second ll muscarinic receptors have provided first insights into the depolarising influence of muscarinic signalling is complex action they play in normal physiology and in through inhibition of a Ca2+-dated K+ current (IK(Ca», the diseased eye. Ocular pharmacologically active which normally hyperpolarises the cell when an action substances can not be seen as having one receptor potential leads to influx of Ca2+ through voltage­ subtype at one tissue site within the eye - in the case of activated Ca2+ channels.48 Ml receptors seem to be the muscarinic agents several short-term and long-term primary subtype involved, which is surprising, because effects must be considered. Besides the widely present muscarinic receptors, many classes of other GCRs are present in the eye. Structural studies, as well as Table lb. The selectivity ratios oj different muscarinic antagonists" determination of interactions with their secondary signalling mechanisms, will serve in the understanding and evaluation of phenomena such as elevated lOP or visual disturbances and alterations induced by various drugs. These findings can not be explained alone by the structural features of these substances and their affinity towards one receptor type. The interaction of muscarinic signalling with other signalling systems and the influence on slow pace growth promotion, smooth muscle activation and fast neuronal signalling are woven From Doods.29 together in a complex pattern. Side effects and sometimes QNX, RS-( ± )-quinuclidinyl xanthene-9-carboxylate hemioxalate surprisingly beneficial observations can therefore be hydrate. explained when muscarinic agonists and antagonists are "Significantly different from 1 (p < 0.05). aln in vivo radioligand binding studies of muscarinic binding used in . In the last 15 years highly sites in hippocampus, atrium and submandibular gland. selective drugs for certain muscarinic receptor subtypes bMx My = Ki(y)/Ki(x)' have been discovered. Their clinical usefulness is in

289 many cases still elusive. The understanding of the ocular their specificity on the different muscarinic receptor muscarinic system is therefore of great interest for the subtypes and their binding ratios between the Ml to M3 clinical ophthalmologist, since the selectivity of today's subtypes (Table lb). Muscarinic receptor research has drugs requires a far more detailed understanding for been hindered by the lack of antagonists with high their optimal application. affinity for one receptor subtype coupled with very low affinity for the other four receptor types. This results in

Localisation and function of muscarinic receptors in the the necessity to define a particular subtype with eye dissociation constants for a range of selective antagonists.24 Understanding the functional importance of muscarcinic Complementary nucleic acid sequences that are able receptor signalling in the eye requires knowledge of the to hybridise with parts of muscarinic receptor mRNA, exact localisation of the receptors and subtype either in Northern blots from tissues or directly in tissue composition. The indication that a muscarinic receptor sections, as in situ hybridisation, have proved another subtype is expressed in a certain part of the eye is taken powerful tool in receptor mapping in the human eye. The as putative evidence that a functional role for this certain presence of mRNA, when found in in situ tissue subtype exists. Not only anatomical curiosity, but also hybridisations, is usually a very strong indicator for the the search for reliable drugs for glaucoma and other expression of the receptor molecule itself. Since many disorders (e.g. ) drive efforts in mapping the studies have been performed on isolated cell cultures quantity and types of ocular muscarinic receptors. From from eye tissue, especially ciliary cells,60 divergent the observation that cholinergic drugs have effects on results were reported regarding the expression of , refraction and it was deduced that muscarinic signalling must play a role in these processes. receptor subtypes in these tissues. This is not surprising, In addition, the development of new and more specific since cultured cells are isolated and are devoid of the agonists and antagonists is of special interest for the interactive intercellular communication processes treatment and management of ocular . Side regulating receptor expression. Other reasons for, in effects developing from the long-term administration of particular, muscular diversity of muscarinic receptor these substances make it desirable to develop, if possible, expression are that the cell line derived from ocular drugs that do not have many of the undesired effects of tissue can have differential activation capacities for many currently in use. expression and that different rates of transcription exist. Separate from the functions of the mechanical This has been described within the family of muscarinic apparatus are those of retinal signal transduction. receptors previously?4 Therefore, results from cultured Neurochemical processes important to signal tissues are not always comparable with the status in vivo, transduction of visual information are believed to be even if more elegant examination methods in cultured modulated by a wide variety of expressed receptors in tissues exist. various defined structures of the retina and . Monoclonal antibodies against various muscarinic These may require more prolonged efforts in mapping subtypes have been introduced and their use in and characterisation than the rest of the optic system. For localisation studies will eventually give the most reliable the detection of receptors various methods exist, each insight into receptor expression. Most antibodies have with a variety of benefits and disadvantages, that have to been raised against peptide sequences of the third be considered in their interpretation. Three different intracellular loop (i3) of each receptor, since this area has methods of investigation are highlighted here: binding the least sequence homology among subtypes,23 or studies, the use of monoclonal antibodies and the against peptides of the carboxy end (e.g. of the m3 detection of mRNA encoding the specific receptor sequence )?5 Subtypes have been described in various subtype. Using these techniques, a reasonably complete organs and parts of the but no picture of ocular muscarinic receptor distribution can be subtype-specific investigations with monoclonal drawn (Table 2). antibodies in the human eye have yet been performed. Binding studies have historically been the most This leaves a wide gap in the knowledge we have so far prominent type of investigations of muscarinic receptors. achieved from binding and expression studies. The Several specific muscarinic agonists and antagonists necessity actually to identify the expressed receptor exist, with which the distribution of muscarinic receptors in the eye has been defined. The major disadvantage of subtypes is crucial, since their assignment to functional the method is that the specificity of these substances is effects has already been studied with selective only modest in most cases. Substances used to identify muscarinic drugs. Resolution problems, combined with muscarinic receptor subtypes (Ml, M2) do not low sensitivity, are presumably the main obstacles that necessarily bind with similar affinity to the cloned have hindered antibody investigations in ocular tissues. receptor subtypes (ml, m2)?3 However, careful Additionally, the absence of a definitive classification of comparison of binding studies using several antagonists subtypes might not be surprising, since many studies can reveal consistent patterns of receptor distribution. An experience difficulties in conclusively designating a overview of the most commonly used antagonists used specific subtype of receptor with in situ hybridisation, in binding studies is given in Tables la, together with antibodies and functional pharmacological studies.

290 Table 2. The distribution of muscarinic receptor subtypes in distinct anatomical spaces of the human eye and in that of other species

Direct protein ml-mSmRNA ml-mSmRNA MI-M4 radioligand Tissue detection in situ hybridisation northern blot binding QNB binding: ffi a Epithelium m3 m3 + subtypes not specifiedn,o a m4 ffi m3 + mSffi ml or m2 ffi Endothelium m3 ffi i m4ff mSffi a Anterior epithelium m3 + a Trabecular meshwork m3 + a k Ciliary non-pigmented m3 + Ml +++ c epithelium/process Ml ++ c k M2 ++ , M3 ++i M3 +++1 a a c Cililary muscle m3 +++p m3 ++ m3 + M2 + a,d,e Whole muscle ml +p M3 ++ m2 +p m4 +p mS +P b Longitudinal ml + b m2 + b m3 + b m4 + b mS + b Circular ml + b m2 + b m3 + b m4 + b mS + a a c Iris m3 ++ m3 + M2 ++ c M3 ++ a / Sphincter M3 +++ ,d c Ml ++

a Epithelium M3 + c Ml ++ h c Retina ml to mS Ml ++ c M2 ++ ,g,i a M3 +

Sclera Ml ++P The comparison of different kinds of investigative tools (radioligand binding, mRNA detection and direct protein detection with monoclonal antibodies) shows a distribution pattern at some anatomical sites. When comparisons of levels of expression were made in a study these are represented as + for present to ++ + for predominant. References: a;*, 59; b*, 60; c*, 61; d*, 62; e*, 63; f, 64; g, 65; h, 66; i, 67; j, 68; k, 69; l, 70; m, 15; n, 16; 0, 71; p, 72. An asterisk indicates investigations made in human tissue. QNB, quinuclidinyl benzylate.

Muscarinic signalling in the retina impressively demonstrate the crucial role of muscarinic signalling in and in the adult Autoradiographic binding sites for muscarinic agents in eye, since multiple patterns of expression appear to theretina have been difficult to allocate, since the spatial guide the layout of retinal structures and later participate resolution of this technique was not satisfactory?6 in visual function throughout ocular growth. A number Higher-resolution studies and more advanced emulsion of possible mechanisms for the development generation techniques, however, revealed the existence of Ml and of neuronal networks have been postulated on the basis M2 receptors in rat, human and monkey,77-79 as well as of changing receptor densities and appearances in the in calf,80 avian14,81 fish82 and rabbit83 retina, where they embryogenesis of the eye.84 The development of retinal are mainly found in the from early structures appears to be greatly influenced by the stages onwards in the developing eye. These findings expression of muscarinic receptors.85 In different stages

291 of embryological and postnatal development, the Ciliary muscle function is regulated by muscarinic subtypes, number and distribution of the muscarinic receptors receptor proteins change during retinal synaptogenesis.66 A second important and extremely well investigated site In embyronic maturation, muscarinic signalling seems to of muscarinic signalling in the human eye is the ciliary influence formation of retinal structures primarily muscle.98 The and the trabecular meshwork through intracellular Caz+ release,14,86,87 and muscarinic have been in the initial focus of interest regarding signalling is predominantly responsible for the muscarinic signalling, since they are crucial for incurvation of the early embyronic neural retina.17,88 and aqueous outflow. It has become Since precursors of and amacrine cells possess evident that in ciliary muscle a diversification of receptor muscarinic receptors,81,89 the concomitant emergence of distribution exists.99,lOO The general presence of different functional cholinergic receptor subtypes with muscarinic receptors in the ciliary muscle complex was differentiation in vivo suggests that acetylcholine plays soon established7o,101,lOZ but further identification of the diverse and temporarily regulating roles in the receptor subtypes seemed desirable for the explanation developing retina.87,9o of accommodative and aqueous flow mechanisms. In the The subtype composition of muscarinic receptors in hope of finding an agonist of muscarinic signalling for the retina can not be interpreted at present. 4-DAMP the control of lOP, precise mappings of muscarinic labelling revealed binding to muscarinic receptors in the subtypes through binding studies and molecular genetics retina and blocking of Ml receptors with pirenzepine have been undertaken.10o The most prominent effect of presumably indicated the concomitant presence of M3 muscarinic drugs in the eye due to constriction of the receptors in human retina as well.59 Stimulation ciliary muscle is miosis, associated with accommodation experiments for GTPase activity revealed that the major and an increased outflow of aqueous humour.13,103,104 site for muscarinic stimulation in bovine retinal Since the ciliary muscle can be mechanically divided into membranes is pharmacologically similar to M2 receptor a circular portion of muscle fibres responsible for sites67 and in the rat retina phosphoinositide hydrolysis changes in accommodation and a longitudinal portion and adenylate cyclase inhibition were mainly found to be mainly responsible for changes in outflow facility, the induced by Ml subtypes.91 However, the accompanying question was soon asked whether differences in receptor presence of molecularly defined m4 and mS receptors distribution were responsible for this distinctive can not be excluded from these findings, since affinities behaviour. , a selective M2 agonist, binds of the employed antagonist for these receptor subtypes specifically to sites on the longitudinal ciliary muscle.61 exist. The m4 and mS clones in the retina can not be The circular muscles, responsible for accommodation, defined at present with specific antagonists, so that care have lower affinity, and it seems possible to influence is needed in interpreting binding studies and their true outflow only by activating especially the longitudinal correlation with the molecular subtypes. Investigations ciliary muscle fibre subtype. Similar findings were with monoclonal antibodies or mRNA hybridisations of reported with the muscarinic agonist when . . these subtypes, except in the developing ferret retina,66 compared WIt h t hi e non-se echve' pI'1 ocarpme.105 -107 are not available and will ultimately determine exact Dose-effect relationships for intracamerally applied locations of these receptor subtypes. However, the role of doses of aceclidine to determine total outflow and cholinergic neurotransmission by muscarinic as well as accommodative amplitude were carried out in nicotinic receptors is evident, though investigations of cynomolgus monkey in vivo. The results showed a these complex patterns of signal transduction are yet to significantly stronger effect of aceclidine on outflow than be performed. Physiological evidence suggests that on accommodation, giving further evidence for a muscarinic binding sites in the inner plexiform layer are dissociation between the accommodative and outflow associated with amacrine and/ or ganglion cells,92,93 facility functions of the ciliary muscle based on although from other studies it appears that association muscarinic activity.108 Additionally, in the monkey eye with bipolar and horizontal cells is also possible?9 longitudinal ciliary muscle fibres differ ultrastructurally Markers for cholinergic synapases are concentrated in and histochemically from fibres in other regions of the the inner plexiform layer were acetylcholine is possibly ciliary muscle,109 providing further evidence for a being released by discrete populations of amacrine, specialised task in regulating humour dynamics. As displaced amacrine and inner bipolar cells.93,94 previously mentioned, aceclidine, a cholinomimetic, has Functional correlations regarding the transmission of been used therapeutically for lOP reduction in chromatic information, patterns or even whole visual glaucomallO and is known to have far less effect on images have not been defined but the presence of accommodation than pilocarpine.ll1-113 A specific multiple receptor populations and their interactions are receptor subtype for aceclidine action was postulated documented?9 The release of acetylcholine from with a site predominantly on the longitudinal portion of displaced amacrine cells under the influence of in ciliary muscles. 13,105,108 This led in the ensuing period to rabbits has been well documented95 and effects of an intense investigation of muscarinic receptor acetylcholine from these cells on the inner plexiform populations in ciliary and in the trabecular layer appear to play a role in subsequent signal meshwork. Additionally a number of functional studies transduction.96,97 were added to determine precise mechanisms of

292 muscarinic receptor interplay.13,106,114--117 Radioligand been shown to occur by a muscarinic-receptor-mediated binding studies revealed that oxotremorine as a weak M2 processY4,115,124 Therefore, it is probable that muscarinic agonist binds selectively to the longitudinal fibres of the receptors also play a role in mediating the inhibitory ciliary process whereas no binding was seen in the iris or effects of parasympathetic nerve stimulation or ciliary epithelium. These results suggest that cholinomimetic drugs on ocular sympathetic oxotremorine, by binding selectively to receptors on the neurotransmission, indicating their crucial role in ciliary longitudinal ciliary muscle and inducing its contraction, muscle cholinergic sensitivity. The trabecular meshwork, may modulate outflow facility independently from even though not a part of the ciliary muscle, is accommodation and miosis via the M2 subtype.61 When additionally involved in outflow regulation, as bovine iris and whole ciliary body were investigated biomechanical studies in the monkey have shown. Here regarding expression of muscarinic subtypes, the ratio of it was noted that pathophysiological changes in the m3 to m4 subtype mRNA expression was found to be outflow apparatus induced by echothiopate are in part 13:1. Absence of ml mRNA in the ciliary process and the mediated by anterior segment muscarinic receptors as iris sphincter was noted, but small quantities of m4 well as mechanical factorsY5 The M3 subtype appears to mRNA were expressed in the ciliary process.64 It is be predominant in culturedl26 and native59 human evident that the predominant muscarinic receptor in trabecular meshwork cells. However, it has to be ciliary structures is the m3/ M3 subtype, both in human mentioned that here again muscarinic signalling is only ciliary epithelium3 and ciliary muscle59,6o,118 as well as in partially responsible for drug effects. Functional other species. 119, 120 muscarinic, a-adrenergic and l3-adrenergic receptors in The complex innervation features of the ciliary bovine trabecular meshwork and ciliary muscle are muscle, however, make a solitary responsibility of differentially modulated by various drugs: cholinergic muscarinic receptors for the distinction between outflow and a-adrenergic agonists induce contraction, whereas 13- and accommodation improbable.121,122 Since the ciliary agonists induce relaxation.120 muscle, like the iris smooth muscle, is innervated by nerves of the sympathetic, parasympathetic and sensory Muscarinic receptors of the ciliary epithelium nervous system, activation or blockade of prejunctional receptors may have an additional influence on ciliary In addition to fluid transport through walls, muscle tone since not only postjunctional muscarinic about 95% of aqueous humour is formed by a secretory effects are responsible for ciliary muscle tone. Therefore, process of the cells of the ciliary epithelium. This it can be difficult to predict what overall effect an agonist secretory mechanism and its regulation are only faintly has, because it may differentially affect various parts of understood and it has become one of the key targets for the nervous system simultaneously. Sympathetic nerve regulation by endogenous mediators and anti-glaucoma terminals in the anterior , for example, contain drugs. Multiple methodologies and conflicting results, prejunctional muscarinic receptors that, upon activation depending on the species used, have created a by agonists, inhibit the neural release of noradrenaline. complex picture of the role of muscarinic receptors in this When the prejunctional effects of muscarinic agents on process.127 When the effects of cholinergic agents on evoked secretion of noradrenaline in iris-ciliary body vasoactive intestinal peptide(VIP)-stimulated cAMP segments were investigated, the M2 type was found to be accumulation were investigated in the rabbit ciliary the primary subtype present, the MI and M3 subtypes epithelium, an inhibition of stimulation was found, playing a minor role.123 indicating that the cholinergic system - via muscarinic When mRNA expression studies in native and receptor stimulation and subsequent inhibition of the cultured tissue from the human eye were performed a ciliary epithelial adenylate cyclase - interferes with clearer picture evolved: human ciliary muscle definitely humour formation.128 It remains open as to the biological expresses the mRNA of subtypes m2, m3 and mS and significance of muscarinic receptors in regulating ciliary may also express the mRNA of ml and m4. Differences epithelial transport, and what their contributions are to in expression level of the m2, m3 and mS subtypes were intraocular responses to cholinergic drugs. Functional observed between the circular and longitudinal portions studies with subtype-specific agonists and mapping of of the ciliary muscle, but quite pronounced expression of the various subtypes are scarce. In human non­ all three subtypes of muscarinic receptors by both pigmented ciliary epithelium the carbachol-specific portions shows that a differential distribution probably is stimulation of inositol phosphates was significantly not solely responsible for the dissociation between inhibited by 4-DAMP (M3 antagonist), showing a outflow facility and accommodation that is seen under distinguishable predominance of M3 receptor subtypes, certain conditions.6o The employment of subtype-specific with a large variety of other receptors triggered by antibodies will be the ultimate confirmation of these neurotransmitters and neuropeptides.70 In the rabbit findings, since recent experiments with indicate non-pigmented epithelium a predominance of that muscarinic receptor subtype distribution plays a muscarinic receptors is seen, in to the pigmented minor role in facilitating outflow and lowering IOp.105 part, which contains mainly aI-adrenergic receptors.128 -induced modulation of functional These muscarinic receptors appear to signal via the cholinergic sensitivity in the parasympathetically turnover of membrane phosphoinoitides, generating the innervated, in contrast to denervated, ciliary muscle has second messengers diacylglycerol and IP ,129 and via the 3

293 inhibition of adenylate cyclase,128 suggesting that at least the pos�unctional M3 subtype is responsible for two of the five molecularly defined subtypes are present contraction of the sphincter muscle in humansl23,148 and in the ciliary epithelium: one of the 'odd' group (m3/ M3 in rats.150 When the potencies of several muscarinic as shown59) and one of the 'even' group, the identity of receptor antagonists in blocking either the autoinhibition which remains unknown. The crosstalk between cAMP­ of acetylcholine release or the muscarcinic contraction of and IP -dependent Ca2+ generation has been the sphincter muscle upon acetylcholine release were 3 summarised in a recent review and provides clearer investigated in the guinea-pig iris, no involvement of the insight into the complicated patterns of signal Ml receptor was noted. The results were consistent with transduction in the human eye.4 The physiological the idea of M2 receptors mediating autoinhibition of significance of muscarinic receptors in the ciliary acetylcholine release and M3-like receptors inducing the epithelium, however, remains to be established. Complex contraction of the sphincter muscle in guinea pigs,148,152 interference with or modulation of ciliary epithelial in contrast with M2-mediated IP accumulation and 3 muscarinic receptors by other hormones, subsequent contraction of the sphincter smooth muscle neurotransmitters and ocular hypotensive drugs makes of the rabbit iris.153,154 the muscarinic system a promising target for the Binding studies of the specific M2 agonist development of lOP-lowering substances,130,131 and oxotremorine revealed that no M2 subtypes are present current data support the strong involvement of in the human iris muscle, in contrast to specific biriding muscarinic cholinergic receptors in lOP regulation?,70,132 of Ml and M3 antagonists.59 These findings are supported by the presence of m3 muscarinic receptor subtype mRNA in native61 and cultured155 human iris. Muscarinic receptors influence iris sphincter function Investigations in and show that the signalling The characteristics of muscarinic receptors mediating pathways by M3 receptors in the iris smooth muscle relaxation and/ or contraction have been intensely involves both intracellular and extracellular Ca2+ investigated since the iris is an ideal model for mobilisation and subsequent stimulation of cAMP innervation from the sympathetic, parasympathetic and production, and that M3 receptors are coupled to the sensory nervous systems.121 It is in addition one of the activation of both phospholipase C and adenylate few smooth muscle organs which can be cyclase.156,157 Contraction of the iris sphincter smooth parasympathetically denervated,l33 making it generally muscle in rabbits in contrast seems to be primarily an interesting model for interaction studies of pre- and mediated by IP only.158 3 pos�unctional receptors, not only of the muscarinic type, In conclusion, the understanding of the overall whose general, non-subtype-specific presence in the iris· complex nature of muscarinic receptor distribution on has been documented in a variety of speciesY4-139 various prejunctional and pos�unctional sites in the iris The first report of putative muscarinic receptors by is complicated by investigations in different species and non-subtype-specific binding studies in the human iris inhomogeneous results. Not all species appear to have a soon followed, showing high densities of muscarinic similar receptor interplay, making the search for a valid receptors in the and lower ones in animal model for drug screening problematic. The direct the dilator muscle, matching with the well-known non­ determination of subtypes in humans has been specific pharmacology of atropine2,140 or carbachol.l41,142 undertaken with radioligand studies; however, their In the rabbit iris sphincter, however, pilocarpine is known limitations should be borne in mind. known as a very weak and also behaves as Investigations of mRNA content or direct protein an antagonist,134 which was explained by the assays with subtype-specific monoclonal antibodies have combination of a small number of spare receptors and a revealed that in the human iris sphincter the threshold phenomenon.l43 Indeed, it was shown that predominant subtype, as stated before, is the m3 type pilocarpine causes miosis in vivo by indirectly decreasing accounting for 60-75% of the muscarinic receptors. the iris dilator tone via prejunctional inhibition of Lower levels of between 5% and 10% were recently noradrenaline release in the dilator.l44 An atypical found for the ml, m2, m4 and even the m5 subtype.lOO muscarinic receptor subtype exists in the rabbit iris, 145 different from the pharmacologically and molecularly Presence of muscarinic receptors in the cornea defined subtypes, and has accounted for these differences, which were at first attributed to the presence Among the various mammalian tissues that have been of M3 receptors.146,147 The pos�unctional M3 receptor studied for acetylcholine content, the subtype appears to be mostly prevalent in ,148 contains the highest concentrations.159,160 Even though bovine64 and human123 iris sphincter and rat dilator corneal epithelium is rich in nerve endings, the muscle.149--151 Functionally this makes , since enormous concentration of acetylcholine in these cells is electrically evoked release of noradrenaline in human iris not in accordance with the level usually found in preparations revealed that prejunctional muscarinic junctional tissues where it serves as a . receptors in the human iris-ciliary body correspond to Suggested functions of acetylcholine might involve the M2 subtype mediating the inhibitory effects of regulation of water and ion transport into corneal parasympathetic nerve stimulation or cholinomimetic epithelium.16o Denervation of corneal epithelium has led drugs on ocular sympathetic neurotransmission, whereas to a 87-100% reduction in corneal acetylcholine and is

294 associated with mitotic (growth) inhibition, suggesting a Lenticular sites of muscarinic signalling reparative function of muscarinic receptors in the corneal The lens, despite its lack of innervation and the fact it is epithelium.161,162 Initial investigations regarding an organ of entirely epithelial origin, has high levels of muscarinic receptors showed a lack of binding in rabbit activity.l70 Initially this activity was broken cornea preparations,138 in contrast with other thought to be a defensive mechanism based on the high studies which showed the presence of muscarinic concentrations of acetylcholine generated by such nearby receptors in cultured rabbit corneal cells by ONB sources as the iris and the ciliary apparatus.l71 But soon binding?1 Today the wide presence of muscarinic the functional role of acetylcholine emerged and, receptor subtypes in corneal tissues has been interestingly, interference with acetylcholine homeostasis documented and a picture of their multiple functions is in the lens has been shown to have a remarkable effect on evolving. Considering that every contact of the cornea its clarity. A well-known side effect of anticholinergic with an extraocular object causes a destruction of drugs is their ability to induce in humans,l72 in sensitive corneal epithelium two physiological roles may in vitro experimentsl70,173 and in monkeys.174 In isolated explain the extensive amount of acetylcholine: first the epithelial cell preparations of humans, acetylcholine sensory transmission of the damaging cause and induced intracellular release of calcium from secondly the induction of repair mechanisms, via in these cells. This signalling muscarinic signalling, of an easily damaged thin pattern goes together with an activation of ml, m3 or mS epithelial layer. receptors on the cell surface. Indeed, further Following these thoughts, a cGMP-mediated electrophysiological investigations in human175 and stimulatory role of cholinergic receptors in corneal rabbit whole lens preparations revealed that muscarinic, epithelial growth regulation established that activated but not nicotinic, receptor activation induces the release muscarinic receptors in the cornea are a main signalling of intracellular calcium. So far the only direct step in this procedure.16,163,164 After the presence of determination of muscarinic receptor subtypes in the �-adrenergic, E and muscarinic receptors human lens revealed the presence of m3 mRNA and l in cultured corneal epithelial cells of the rabbit was positive radioligand binding with the M3 antagonist established,71 the interplay between these receptors was 4-DAMP, results matching the physiological evidence. 59 investigated. Hereby it was found that the intercellular Still, the elusive role of muscarinic receptors in lens cAMP/ cGMP ratio was essential for epithelial function has to be clarified. Since there is no innervation proliferation and regrowth. The activation of of the lens, a regulatory effect of muscarinic receptors on prostaglandin and f3-adrenergic receptors increased cell homeostasis, rather than on neuronal signal cAMP, inhibited regrowth and increased basement transduction, seems probable. Here again an membrane production after initial injury to the corneal understanding of the interplay with other receptors will epithelium. Activation of muscarinic receptors led to finally reveal the precise functions of the muscarinic increased cGMP-mediated regrowth of the corneal lenticular system. epithelium. The receptor site remained ambiguous and it was assumed that parts of the receptor protein are localised inside the cell, possibly inside the nucleus Muscarinic receptors influence scleral growth itself.165 Recent investigations in highly purified nuclei of One of the most interesting approaches of the long-term rabbit epithelial and endothelial cell lines have indeed application of antimuscarinic drugs is their influence on demonstrated the pre sense of muscarinic receptors scleral growth. Especially in the prevention of myopia in inside the nucleus.15 Additional whole protein humans, the long-term use of antimuscarinic drugs, such investigations of the molecular subtypes revealed that as , appears promising.176 Experimental studies m3, m4 and mS receptors are present in epithelial and in chicks have shown a marked reduction of myopic endothelial cells and that ml and m2 receptors are progression, accompanied by ocular elongation, when present in epithelial cells only. The majority of these these eyes were treated with atropine.177,178 This receptors are attached to membranes; the mS subtype, elongation has been shown to be present even however, seems likely to regulate nuclear functions, in the absence of a connection between the retina and the along with an uncharacterised 47 kDa receptor-like brain.179 It is now believed that a direct stimulus from protein. This is particularly interesting since the retinal cells on scleral chondrocytes via the muscarinic functional role of mS is the least understood of all the system is present. In recent investigations Ml muscarinic subtypes. 15 The intranuclear presence of muscarinic receptors have been found to be primarily present in receptors in corneal epithelial and endothelial cells (and chick scleral chondrocytes when the growth of these cells other cell types as well) suggests the existence of a was monitored in culture.72 Several other cell types in functional, possibly G-protein-dependent, nuclear humans are growth stimulated by muscarinic signalling system, induced by muscarinic acetylcholine acetylcholine receptor agonists and the results of receptor (or related receptor-like) proteins in response to pending clinical trials for the reduction and prevention of intracellular acetylcholine, employing pathways specific myopia through early onset of muscarinic agonists in for intranuclear signalling.166-169 prone patients are promising.

295 Conclusions accompanied by highly selective muscarinic subtype agonists and antagonists in their clinical applications. The omnipresence of muscarinic receptors with their The knowledge of the composition of the ocular various expression patterns in the human eye is a muscarinic system and its interplay with other receptor startling finding. The methods employed to determine signalling systems will therefore help the subtypes have become more sophisticated over recent ophthalmologist in choosing the optimal treatment in the years and ultimately the quantitative determination of future, since the limits for empirical drug use and receptor protein expression will yield definitive insights development are beginning to be reached. into subtype interactions. In addition to the long-known role of muscarinic receptors on outflow and accommodation regulation in the ciliary apparatus, a References wide range of diverse functions has been found. These include the multijunctional sites of neurotransmission in 1. Dale HH. The action of certain and ethers of and their relation to . J Pharmacol Exp Ther the retina and iris, whose subtype composition is still 1914;6:147-96. under investigation. More exploration will be required to 2. Hutchins JB, Hollyfield JG. Autoradiographic identification clarify the presence of muscarinic signalling in the lens of muscarinic receptors in human iris smooth muscle. Exp and cornea, where these receptors most likely play a role Eye Res 1984;38:515-21. 3. Crook RB, Polansky JR. Neurotransmitters and in the nutritious efforts of these brady trophic tissues. neuropeptides stimulate inositol phosphates and Here muscarinic signalling seems to be important for intracellular calcium in cultured human nonpigmented growth and control between cells and less ciliary epithelium. Invest Ophthalmol Vis Sci important for fast pace neuronal transmission of signals. 1992;33:1706-16. The detection of muscarinic signalling in the cellular 4. Abdel-Latiff AA. Cross talk between cyclic AMP and the polyphosphoinositide signalling cascade in iris sphincter nucleus is particularly exciting, since gene expression and other nonvascular smooth muscle. Proc Soc Exp Bioi mechanisms might additionally be influenced by Med 1996;211:163-77. intracellular G protein coupled receptor mechanisms. 5. Weber A. Die Ursache des Glaukoms. Graefes Arch The overall picture of the muscarinic system of the eye Ophthalmol 1877;23:91. 6. Ringer S, Gould AP. On Jabornadi. Lancet 1875;II:157-9. is continuing to evolve. From the extensive investigations 7. Loewi 0, Navratil E. Uber humorale Ubertrachbarkeit der that have been performed it is probable that the ratio and Herznervenwirkung. XI. Uber den Mechanismus der relationship of receptor subtype expression in the eye Vaguswirkung von Physostigmin und Ergotamin. Pfliigers and other body sites will help to determine their Arch 1926;214:689-96. functional roles in a given system. 8. Stedman E, Easson LH. Cholinesterase: an present in -sera from . Biochemical J 1932;26:2056-66. A new range of muscarinic receptor agonists and 9. Kraitmair H. Die Papavarinwirkung eine Benzylreaktion. antagonists is being tested at present in animal and Arch Pathol Pharmakol 1932;164:509. human models. Their aims are various. Prevention of 10. Militor H. A comparative study of the effects of five choline myopia by muscarinic antagonists is an exciting new compounds used in therapeutics: acetylcholine chloride, acetyl-beta-methylcholine chloride, carbaminoyl choline, aspect. However, fears from toxic side effects of long­ ethyl ether beta-methylcholine chloride, carbaminoyl beta­ term use of substances such as atropine are pushing methylcholine chloride. J Pharmacol Exp Ther 1936;58:337. investigations further into the class of more selective 11. Velhagen K. Die Grundlagen der okularen Pharmakologie muscarinic antagonists. The complex distribution and und Toxikologie des Carbaminocholins. Arch Augenheilk 1933;107:319. expression of all known muscarinic receptor subtypes in 12. Barany EH. The mode of action of miotics on outflow the eye can not make us believe in the solely resistance. A study of pilocarpine in the vervet monkey, accommodative influence of muscarinic drugs. Cercopithecus ethiops. Trans Ophthalmol Soc UK Reparative functions in the corneal epithelium are 1966;86:539-78. enhanced by muscarinic agonists and retinal 13. Poyer JF, Gabelt BT, Kaufman PL. The effect of muscarinic agonists and selective receptor subtype antagonists on the neurotransmission is influenced by substances contractile response of the isolated rhesus monkey ciliary interacting with muscarinic receptors. Glaucoma therapy muscle. Exp Eye Res 1994;59:729-36. has one of its major strongholds in using indirect 14. Sakaki Y, Fukuda Y, Yamashita M. Muscarinic and muscarinic drugs and here again a more selective purinergic Ca2+ mobilisations in the neural retina of early embryonic chick. Int J Dev Neurosci 1996;14:691-9. influence on uveoscleral outflow is hoped to be gained 15. Lind GJ, Cavanagh HD. Identification and subcellular by increased subtype selectivity of new drugs - and the distribution of muscarinic acetylcholine receptor-related selectivity towards one receptor subtype will be needed proteins in rabbit cornea and Chinese hamster ovaries. as the picture evolves of an eye densely populated with Invest Ophthalmol Vis Sci 1995;36:1492-507. muscarinic receptors. 16. Walkenbach RJ, Ye GS. Muscarinic receptors and their regulation of cyclic GMP in corneal endothelial cells. Invest The five molecular subtypes of muscarinic receptors Ophthalmol Vis Sci 1990;31:702-7. are all present in the eye. In distinct structures a different 17. Yamashita M, Fukuda Y. Incurvation of early embryonic pattern of receptor subtype composition can be found. neural retina by acetylcholine through muscarinic receptors. The role of most of them are not yet understood, which is Neurosci Lett 1993;163:215-8. 18. Noda M, Takahashi H, Tanabe T, Toyosato M, Furutani Y, not surprising since they were first distinguished only Hirose T, et al. Primary structure of a-subunit precursor of just over 10 years ago. Atropine (including its various Torpedo californica acetylcholine receptor deduced from enduring derivatives) and pilocarpine might soon be eDNA sequence. Nature 1982;299:793-7.

296 19. Kubo T, Fukuda K, Mikami A, Maeda A, Takahashi H, 39. Wess J, Gdula D, Brann MR. Site-directed mutagenesis of Mishina M, et al. Cloning, sequencing and expression of the m3 muscarinic receptor: identification of a series of complementary DNA encoding the muscarinic threonine and tyrosine residues involved in agonist but not acetylcholine receptor. Nature 1986;323:411--6. antagonist binding. EMBO J 1991;10:3729-34. 20. Kubo T, Makada A, Sugimoto K, Akiba I, Mikani A, 40. Hammer R, Giachetti A. Muscarinic receptor subtypes: M1 Takahashi H, et al. Primary structure of porcine cardiac and M2 biochemical and functional characterisation. Life Sci muscarinic receptor deduced from the eDNA sequence. 1982;31:2991-8. FEBS Lett 1987;209:367-72. 41. Waelbroeck M, Gillard M, Robberecht P, Christophe J. 21. Hammill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Muscarinic receptor heterogeneity in rat central nervous Improved patch-clamp techniques for high-resolution system. I. Binding of four selective antagonists to three current recording from cells and cell-free membrane muscarinic receptor subclasses: a comparison with M2 patches. Pfliigers Arch 1981;391:85-100. cardiac muscarinic receptors of the C type. Mol Pharmacol 22. Neher E, Sakmann B. Single channel currents recorded from 1987;32:91-9. membrane of denervated muscle fibres. Nature 42. Waelbroeck M, Tastenoy M, Camus J, Christophe J. Binding 1976;260:799-802. of selective antagonists to four muscarinic receptors (Ml to M4) in rat . Mol PharmacoI 1990;38:267-73. 23. Levey AI. Immunological localization of m1-m5 muscarinic 43. Freedman SB, Dawson GR, Iversen LL, Baker R, Hargreaves acetylcholine receptors in peripheral tissues and brain. Life RJ. The design of novel muscarinic partial agonists that Sci 1993;52:441-8. have functional selectivity in pharmacological preparations 24. Caulfield MP. Muscarinic receptors: characterisation, in vitro and reduced side-effect profile in vivo. Life Sci coupling and function. Pharmacol Ther 1993;58:319-79. 1993;52:489-95. 25. Curtis CAM, Wheatley M, Bansal S, Birdsall NJM, Eveleigh 44. Ensinger HA, Doods HN, Immel-Sehr AR, Kuhn FJ, P, Pedder EK, et al. Propylbenzilylcholine mustard labels an Lambrecht G, Mendla KD, et al. WAL 2014: a muscarinic acidic acid residue in transmembrane helix 3 of the agonist with preferential neuron-stimulating properties. muscarinic receptor. J Bioi Chem 1989;264:489-95. Life Sci 1993;52:473-80. 26. Hammer R, Berrie CP, Birdsall NJM, Burgen ASV, Hulme 45. van Zwieten PA, Doods HN. Muscarinic receptors and EC Pirenzepine distinguishes between different subclasses drugs in cardiovascular medicine. Cardiovasc Drugs Ther of muscarinic receptors. Nature 1980;283:90-2. 1995;9:159--67. 27. Durieux ME. Muscarinic signalling in the central nervous 46. Brown DA, Adams PR. Muscarinic suppression of a novel system: recent developments and implications. voltage-sensitive K+-current in a neuron. Nature Anesthesiology 1996;84:173-89. 1980;283:673--6. 28. Bonner TI. The molecular basis of muscarinic receptor 47. Caulfield MP, Jones S, Vallis Y, Buckley NJ, Kim G-D, diversity. Trends Neurosci 1989;12:148-51. Milligan G, et al. Muscarinic M-current inhibition via 29. Doods HN, Mathy MJ, Davidesko D, van Charldorp KJ, de G7a

297 58. Jones SV. Muscarinic receptor subtypes: modulation of ion 80. Vanderheyden P, Ebinger G, Vauquelin G. Characterisation channels. Life Sci 1993;52:457-64. of Ml- and M2-muscarinic receptors in calf retina 59. Gupta N, Drance SM, McAllister R, Prasad S, Rootman J, membranes. Vision Res 1988;28:247-50. Cynader MS. Localisation of M3 muscarinic receptor 81. McKinnon LA, Gunther EC, Nathanson NM. subtype and mRNA in the human eye. Ophthalmic Res Developmental regulation of the cm2 muscarinic 1994;26:207-13. acetylcholine receptor gene: selective induction by a 60. Zhang X, Hernandez MR, Yang H, Erickson K. Expression secreted factor produced by embryonic chick retinal cells. J of muscarinic receptor subtype mRNA in the human ciliary Neurosci 1998;18:59-69. muscle. Invest Ophthalmol Vis Sci 1995;36:1645-57. 82. Moreno-Yanes JA, Mahler HR. Muscarinic cholinergic 61. Gupta N, McAllister R, Drance SM, Rootman J, Cynader receptors in goldfish retina. Life Sci 1979;24:1787-9l. MS. Muscarinic receptor Ml and M2 subtypes in the human 83. Neal MJ, Dawson e. Muscarinic cholinergic receptors in eye: QNB, pirenzipine, oxotremorine, and AFDX-116 in vitro rabbit retina. J Pharm Pharmacol 1985;37:60-1. autoradiography. Br J Ophthalmol 1994;78:555-9. 84. Sugiyama H, Daniels MP, Nirenberg M. Muscarinic 62. Chen J, Woldemoussie E. Similarity of muscarinic receptor acetylcholine receptors of the developing retina. Proc Nat! subtype in smooth muscles of iris sphincter, ciliary and Acad Sci USA 1977;74:5524-8. guinea pig ileum. FASEB J 1988;Suppl: 788. 85. Salceda R. Muscarinic receptors binding in retinal pigment 63. Woldemoussie E, Feldman B. Characterisation of epithelium during rat development. Neurochem Res muscarinic receptors in human ciliary and iris smooth 1994;19:1207-10. muscle cells by ligand binding and biochemical response 86. Klein WL. Biochemistry and regulation of signal studies. FASEB J 1988;Suppl: 364. transduction by neuronal acetylcholine receptors. Curr Top 64. Honkanen RE, Howard EF, Abdel-Latif AA. M3-muscarinic Cell Regul 1984;24:129-44. 2 receptor subtype predominates in the bovine iris sphincter 87. Wong RO. Cholinergic regulation of [Ca +]i during cell smooth muscle and ciliary processes. Invest Ophthalmol Vis division and differentiation in the mammalian retina. J Sci 1990;31:590-3. Neurosci 1995;15:2696-706. 65. Skorupa AF, Klein WL. Developmentally regulated secreted 88. Friedman Z, Hackett SF, Campochiaro PA. Human retinal factors control expression of muscarinic receptor subtypes pigment epithelial cells possess muscarinic receptors in embryonic chick retina. J Neurochem 1993;60:2087-97. coupled to calcium mobilisation. Brain Res 1988;446:11-6. 66. Hutchins JB. Development of muscarinic acetylcholine 89. Yamashita M, Yoshimoto Y, Fukuda Y. Muscarinic receptors in the ferret retina. Brain Res Dev Brain Res acetylcholine responses in the early embryonic chick retina. 1994;82:45-61. J Neurobiol 1994;25:1144-53. 90. Siman RG, Klein WL. Differential regulation of muscarinic 67. McIntosh H, Blazynski e. Muscarinic receptor stimulated and nicotinic receptors by cholinergic stimulation in GTPase activity in synaptic membranes from bovine retina. cultured avian retina cells. Brain Res 1983;262:99-108. J Neurochem 1992;59:210-5. 91. Qu ZX, Fertel R, Neff NH, Hadjiconstantinou M. 68. Wax MB, Coca-Prados M. Receptor-mediated Pharmacological characterisation of muscarinic receptors phosphoinositide hydrolysis in human ocular ciliary mediating inhibition of adenylate cyclase activity in the rat epithelial cells. Invest Ophthalmol Vis Sci 1989;30:1675-9. retina. J Pharmacol Exp Ther 1988;246:839-42. 69. Mallorga P, Babilon RW, Buisson S, Sugrue MF. Muscarinic 92. Jardon B, Bonaventure N, Scherrer E. Possible involvement receptors of the albino rabbit ciliary process. Exp Eye Res of cholinergic and amacrine cells in the 1989;48:509-22. inhibition exerted by the ON retinal channel on the OFF 70. Polansky JR, Zlock 0, Brasier A, Bloom E. Adrenergic and retinal channel. Eur J Pharmacol 1992;210:201-7. cholinergic receptors in isolated non-pigmented Ciliary 93. Negishi K, Kato S, Teranishi T, Laufer M. An epithelial cells. Curr Eye Res 1985;4:517-22. electrophysiological study on the cholinergiC system in the 3 71. Colley AM, Cavanah HD. Binding of [ H]dihydroalprenolol carp retina. Brain Res 1978;148:85-93. and (3H1quinuclidinyl benzilate to intact cells of cultured 94. Baughman RW, Bader CR. Biochemical characterisation and corneal epithelium. Metab Pediatr Syst Ophthalmol cellular localisation of the cholinergic system in the chicken 1982;6:75-86. retina. Brain Res 1977;138:469-85. 72. Lind GI, Chew SJ, Marzani 0, Wallman J. Muscarinic 95. Masland RH, Mills JW, Cassidy e. The functions of acetylcholine receptor antagonists inhibit chick scleral acetylcholine in the rabbit retina. Proc R Soc Lond Ser B chondrocytes. Invest Ophthalmol Vis Sci 1998;39:2217-31. 1984;223:121-39. 73. Bonner TI, Buckley NJ, Young AC, Brann MR. Identification 96. Linn OM, Massey Se. Homocysteate-evoked release of of a family of muscarinic acetylcholine receptors. Science acetylcholine from the rabbit retina. J Neurochem 1987;237:527-32. 1996;66: 153-60. 74. Bursztajn S, Berman SA, Gilbert W. Differential expression 97. Neal MJ, Cunningham JR, Hutson PH, Semark JE. Calcium of acetylcholine receptor mRNA in nuclei of cultured dependent release of acetylcholine and gamma­ muscle cells. Proc Nat! Acad Sci USA 1989;86:2928-32. aminobuytric acid from the rabbit retina. Neurochem Int 75. Yasuda RP, Ciesla W, Flores LR, Wall SJ, Li M, Satkus SA, 1992;20:43-53. et al. Development of antisera selective for m4 and m5 98. Nilsson SF. The uveoscleral outflow routes. Eye muscarinic cholinergic receptors: distribution of m4 and m5 1997;11:149-54. receptors in rat brain. Mol PharmacoI 1993;43:149-57. 99. Farahbaksh NA, Cilluffo Me. Synergistic increase in Ca2+ 76. Vilaro MT, Mengod G, Palacios JM. Advances and produced by Al and muscarinic receptor limitations of the molecular of cholinergic activation via a pertussis-toxin-sensitive pathway in receptors: the example of multiple muscarinic receptors. epithelial cells of the rabbit ciliary body. Exp Eye Res Prog Brain Res 1993;98:95-10l. 1997;64:173-9. 77. Hruska RE, White R, Azari J, Yamamura HI. Muscarinic 100. Gil OW, Krauss HA, Bogardus AM, Woldemussie E. cholinergic receptors in mammalian retina. Brain Res Muscarinic receptor subtypes in human iris-ciliary body 1978;148:493-8. measured by immunoprecipitation. Invest Ophthalmol Vis 78. Hutchins JB, Hollyfield JG. Acetylcholine receptors in the Sci 1997;38:1434-42. human retina. Invest Ophthalmol Vis Sci 1985;26:1550-7. 101. Barany EH. Muscarinic sub sensitivity without receptor 79. Zarbin MA, Wamsley JK, Palacios JM, Kuhar MJ. change in monkey ciliary muscle. Br J Pharmacol Autoradiographic localisation of high affinity GABA, 1985;84:193-8. , , adrenergic and muscarinic 102. Gilmartin B, Hogan RE. The relationship between tonic cholinergic receptors in the rat, monkey and human retina. accommodation and ciliary muscle innervation. Invest Brain Res 1986;374:75-92. Ophthalmol Vis Sci 1985;26:1024-8.

298 103. Kaufman PL, Barany EH. Residual pilocarpine effects on 124. Kaufman PL. Parasympathetic denervation of the ciliary outflow facility after ciliary muscle disinsertion in the muscle following retinal photocoagulation. Trans Am cynomolgus monkey. Invest OphthalmoI 1976;15:558-6l. Ophthalmol Soc 1990;88:513-53. 104. Kaufman PL, Barany EH. Subsensitivity to pilocarpine of 125. Lutjen-Drecoll E, Kaufman PL. Biomechanics of the aqueous outflow system in monkey eyes after topical echothiophate-induced anatomic changes in monkey anticholinestersae treatment. Am J Ophthalmol aqueous outflow system. Graefes Arch Clin Exp 1976;82:883--9l. Ophthalmol 1986;224:564-75. 105. Gabelt BT, Kaufman PL. Inhibition of aceclidine-stimulated 126. Shade DL, Clark AF, Pang IH. Effects of muscarinic agents outflow facility, accommodation and miosis in rhesus on cultured human trabecular meshwork cells. Exp Eye Res monkeys by muscarinic receptor subtype antagonists. Exp 1996;62:201-10. Eye Res 1994;58:623--30. 127. Kaufman PL. Mechanisms of actions of the cholinergic 106. Hubbard WC, Kee C, Kaufman PL. Aceclidine effects on drugs in the eye. In: Drance SM, Neufeld AN, editors. outflow facility after ciliary muscle disinsertion. Glaucoma. 1st ed. Orlando: Grune and Stratton, Ophthalmologica 1996;210:303--7. 1984;295-327. 107. Poyer JF, Kaufman PL, Flugel C. Age does not affect 128. Jumblatt JE, North GT, Hackmiller RC. Muscarinic contractile responses of the isolated rhesus monkey ciliary cholinergic inhibition of adenylate cyclase in the rabbit iris­ muscle to muscarinic agonists. Curr Eye Res 1993;12:413-22. ciliary body and ciliary epithelium. Invest Ophthalmol Vis 108. Erickson-Lamy K, Schroeder A. Dissociation between the Sci 1990;31:1103-8. effect of aceclidine on outflow facility and accommodation. 129. Yousufzai SY, Honkanen RE, Abdel-Latif AA. Muscarinic Exp Eye Res 1990;50:143--7. cholinergic induced subsensitivity in rabbit iris-ciliary 109. Flugel C, Barany EH, Lutjen-Drecoll E. Histochemical body: effects on myo-inositol triphosphate accumulation, differences within the ciliary muscle and its function in arachidonate release, prostaglandin synthesis, and accommodation. Exp Eye Res 1990;50:219-26. contraction. Invest Ophthalmol Vis Sci 1987;28;1631-8. 110. Hoskins HD Jr, Kass MA. Cholinergic drugs. In: Becker­ 130. Neufeld AH. The mechanisms of action of adrenergic drugs Schaffer's diagnosis and therapy of glaucoma. 6th ed. St in the eye. In: Glaucoma. Orlando: Grune and Stratton, Louis: CV Mosby, 1989;420-34. 1984;277-3 0l. 111. Fechner PU, Teichmann KD, Weyrauch W. Accommodative 131. Sears M, Mead A. A major pathway for the regulation of effects of aceclidine in the treatment of glaucoma. Am J intraocular pressure. Int Ophthalmol 1983;6:201-12. Ophthalmol 1975;79:104-6. 132. Suzuki Y, Nakano T, Sears M. Calcium signals from intact 112. Keren G, Treister G. Effect of aceclidine(+) isomer and rabbit ciliary epithelium observed with confocal pilocarpine on the intraocular pressure decrease and the microscopy. Curr Eye Res 1997;16:166-75. miosis in glaucomatous eyes: effect on accommodation in 133. Hasegawa N, Imaizumi Y, Watanabe M. Parasympathetic normal eyes of young subjects. Ophthalmologica denervation supersensitivity in the rat iris sphincter muscle: 1980;180:181-7. an in vitro study. Jpn J PharmacoI 1987;43:143--51. 113. Lieberman TW, Leopold IH. The use of aceclydine in the 134. Akesson C, Swanson C, Patil PN. Muscarinic receptors of treatment of glaucoma: its effect on intraocular pressure and rabbit irides. Naunyn Schmiedebergs Arch Pharmacol facility of aqueous humor outflow as compared to that of 1983;322:104-10. pilocarpine. Am J Ophthalmol 1967;64:405-15. 135. Karlsen RL. Muscarinic receptor binding and the effect of 114. Croft MA, Kaufman PL, Erickson-Lamy K, Polansky JR. atropine on the guinea-pig iris. Exp Eye Res 1978;27:577-83. Accommodation and ciliary muscle muscarinic receptors 136. Kloog Y, Heron OS, Korczyn AD, Sachs 01, Sokolovsky M. after echothiophate. Invest Ophthalmol Vis Sci Muscarinic acetylcholine receptors in albino rabbit iris­ 1991;32:3288-97. ciliary body. Mol PharmacoI 1979;15:581-7. 115. Erickson-Lamy KA, Polansky JR, Kaufman PL, Zlock OM. Cholinergic drugs alter ciliary muscle response and 137. Kloog Y, Sachs 01, Korczyn AD, Heron OS, Sokolovsky M. receptor content. Invest Ophthalmol Vis Sci 1987;28:375-83. Muscarinic acetylcholine receptors in cat iris. Biochem 116. Gabelt BT, Kaufman PL. Inhibition of outflow facility and Pharmacol 1979;28:1505-1l. accommodative and miotic responses to pilocarpine in 138. Olsen JS, Neufeld AH. The rabbit cornea lacks cholinergic rhesus monkeys by muscarinic receptor subtype receptors. Invest Ophthalmol Vis Sci 1979;18:1216-25. antagonists. J Pharmacol Exp Ther 1992;263:1133--9. 139. Sachs 01, Kloog Y, Korczyn AD, Heron OS, Sokolovsky M. 117. Gabelt BT, Kaufman PL, Polansky JR. Ciliary muscle Denervation, supersensitivity and muscarinic receptors in muscarinic binding sites, choline acetyltransferase, and the cat iris. Biochem Pharmacol 1979;28:1513-8. acetylcholinesterase in aging rhesus monkeys. Invest 140. Kaumann AJ, Hennekes R. The affinity of atropine for Ophthalmol Vis Sci 1990;31:2431-6. muscarine receptors in human sphincter pupillae. Naunyn 118. Pang IH, Matsumoto S, Tamm E, DeSantis L. Schmiedebergs Arch Pharmacol 1979;306:209-1l. Characterisation of muscarinic receptor involvement in 141. Hagan JJ, van der Heijden B, Broekkamp CL. The relative human ciliary function. J Ocul Pharmacol potencies of cholinomimetics and muscarinic antagonists on 1994;10:125-36. the rat iris in vivo: effects of pH on potency of pirenzepine 119. Daniele E, Villani G, Lograno MD. Effects of phorbol and . Naunyn Schmiedebergs Arch Pharmacol on carbachol-induced contraction in bovine ciliary muscle: 1988;338:476-83. possible involvement of protein kinase C. Eur J Pharmacol 142. Patil PN. Reactivity of human iris-sphincter to muscarinic 1997;330:247-56. drugs in vitro. Naunyn Schmiedebergs Arch Pharmacol 120. Wiederholt M, Schafter R, Wagner U, Lepple-Wienhues A. 1992;346:614-9. Contractile response of the isolated trabecular meshwork 143. Konno F, Takayanagi I. Muscarinic acetylcholine receptors and ciliary muscle to cholinergic and adrenergic agents. Ger in the rabbit ciliary body smooth muscle: spare receptors J Ophthalmol 1996;5:146-53. and threshold phenomenon. Jpn J PharmacoI 1985;38:91-9 121. Fuder H. Functional consequences of prejunctional receptor 144. Bognar IT, Baumann B, Dammann F, Knoll B, Meincke M, activation or blockade in the iris. J Ocular Pharmacol Pallas S, et al. M2 muscarinic receptors on the iris sphincter 1994;10:109-23. muscle differ from those on iris noradrenergic nerves. Eur J 122. Lograno MD, Paoletti R. Human intraocular smooth Pharmacol 1989;163:263--74. muscles: role of receptors in aqueous humour dynamics. 145. Bognar IT, Altes U, Beinhauer C, Kessler I, Fuder H. A Pharmacol Res 1989;21:683--99. muscarinic receptor different from the Ml, M2, M3 and M4 123. Jumblatt JE, Hackmiller RC. M2-type muscarinic receptors subtypes mediates the contraction of the rabbit iris mediate prejunctional inhibition of release sphincter. Naunyn Schmiedebergs Arch Pharmacol in the human iris-ciliary body. Exp Eye Res 1994;58:175-80. 1992;345:611-8.

299 146. Bognar IT, Pallas S, Fuder H, Muscholl E. Muscarinic 162. Sigelman S, Friedenwald JS. Mitotic and wound healing inhibition of [3H]noradrenaline release on rabbit iris in vitro: activities of the corneal epithelium: effects of sensory effects of stimulation conditions on of denervation. Arch Ophthalmol 1954;54:46-57. and pilocarpine. Br J Pharmacol 163. Walkenbach RJ, Ye GS. Muscarinic cholinoceptor regulation 1988;94:890-900. of cyclic guanosine monophosphate in human corneal 147. Honkanen RE, Abdel-Latif AA. Characterisation of epithelium. Invest Ophthalmol Vis Sci 1991;32:610-5. cholinergic muscarinic receptors in the rabbit iris. Biochem 164. Walkenbach RI, Ye GS, Boney F, Dueker DK. Muscarinic Pharmacol 1988;37:2575-83. cholinoceptors in native and cultured human corneal 148. Bognar IT, Wesner MT, Fuder H. Muscarine receptor types endothelium. Curr Eye Res 1993;12:155-62. mediating autoinhibitionof acetylcholine release and 165. Cavanagh HD, Colley AM. Cholinergic, adrenergic, and sphincter contraction in the guinea-pig iris. Naunyn PGE1 effects on cyclic nucleotides and growth in cultured Schmiedebergs Arch Pharmacol 1990;341:22-9. corneal epithelium. Metab Pediatr Syst Ophthalmol 149. Masuda Y, Yamahara NS, Tanaka M, Ryang S, Kawai T, 1982;6:63-74. Imaizumi Y, et al. Characterisation of muscarinic receptors 166. Divecha N, Banfic H, Irvine RF. The polyphosphoinositide mediating relaxation and contraction in the rat iris dilator cycle exists in the nuclei of Swiss 3T3 cells under the control muscle. Br J Pharmacol 1995;114:769-76. of a receptor (for IGF-I) in the plasma membrane, and 150. Shiraishi K, Takayanagi I. Subtype of muscarinic receptors stimulation of the cycle increases nuclear diacylglycerol and mediating relaxation and contraction in the rat iris dilator apparently induces translocation of protein kinase C to the smooth muscle. Gen Pharmacol 1993;24:139--42. nucleus. EMBO J 1991;10:3207-14. 151. Yamahara NS, Tanaka M, Imaizumi Y, Watanabe M. Pertussis toxin-sensitive muscarinic relaxation in the rat iris 167. Divecha N, Rhee SG, Letcher AI, Irvine RF. dilator muscle. Br J Pharmacol 1995;114:777-84. Phosphoinositide signalling in rat nuclei: 152. Fuder H, Schopf I, Unckell J, Wesner MT, Melchiorre C, phosphoinositidase C isoform beta 1 is specifically, but not Tacke R, et al. Different muscarine receptors mediate the predominantly, located in the nucleus. Biochem J prejunctional inhibition of [3H]noradrenaline release in rat 1993;289:617-20. or guinea-pig iris and the contraction of the rabbit iris 168. Lind GJ, Cavanagh HD. Nuclear muscarinic acetylcholine sphincter muscle. Naunyn Schmiedebergs Arch Pharmacol receptors in corneal cells from rabbit. Invest Ophthalmol Vis 1989;340:597-604. Sci 1993;34:2943-52. 153. Abdel-Latif AA, Howe PH, Akhtar RA.Muscarinic-receptor 169. Payrastre B, Nievers M, Boonstra J, Breton M, Verkleij AJ, induced myo-inositol trisphosphate accumulation, Van Bergen en Henegouwen PM. A differential location of light chain phosphorylation and contraction in the rabbit phosphoinositide kinases, diacylglycerol kinase, and iris sphincter smooth muscle. Prog Clin Bioi Res phospholipase C in the nuclear matrix. J Bioi Chern 1987;249:119-32. 1992;267:5078-84. 154. Akhtar RA, Honkanen RE, Howe PH, Abdel-Latif AA. M2 170. Michon J Jr, Kinoshita JH. Experimental miotic cataract. I muscarinic receptor subtype is associated with inositol Effects of miotics on lens structure, cation content, and trisphosphate accumulation, myosin light chain hydration. Arch Ophthalmol 1968;79:79-86. phosphorylation and contraction in sphincter smooth 171. Michon J Jr, Kinoshita JH. Cholinesterase in the lens. Arch muscle of rabbit iris. J Pharmacol Exp Ther 1987;243:624-32. Ophthalmol 1967;77:804-8. 155. Woldemussie E, Feldmann BJ, Chen J. Characterisation of 172. Shaffer RN, Hetherington J Jr. Anticholinesterase drugs and muscarinic receptors in cultured human iris sphincter and . Am J Ophthalmol 1966;62:613-8. ciliary smooth muscle cells. Exp Eye Res 1993;56:385-92. 173. Fraser PJ, Duncan G, Tomlinson J. Effect of a cholinesterase 156. Ding KH, Husain S, Akhtar RA, Isales CM, Abdel-Latif AA. inhibitor on salmonid lens: a possible cause for the Inhibition of muscarinic-stimulated polyphosphoinositide increased incidence of cataract in salmon Salmo salar (L.). 2 hydrolysis and Ca + mobilisation in cat iris sphincter Exp Eye Res 1989;49:293-8. smooth muscle cells by cAMP-elevating agents. Cell Signal 174. Philipson B, Kaufman PL, Fagerholm P, Axelsson U, Barany 1997;9:411-21. EH. Echothiophate cataracts in monkeys: electron 157. Tachado SD, Virdee K, Akhtar RA, Abdel-Latif AA. M3 microscopy and microradiography. Arch Ophthalmol muscarinic receptors mediate an increase in both inositol 1979;97:340-6. trisphosphate production and cyclic AMP formation in 175. Alvarez LJ, Candia OA, Zamudio AC. Acetylcholine iris sphincter smooth muscle. J Ocul Pharmacol modulation of the short-circuit current across the rabbit 1994;10:137-47. lens. Exp Eye Res 1995;61:129-40. 158. Abdel-Latif AA. Polyphosphoinositides, generation of second messengers, myosin light chain phosphorylation 176. Bedrossian RH. The effect of atropine on myopia. and contraction in rabbit iris sphincter smooth muscle. Mol Ophthalmology 1979;86:713-7. Cell Biochem 1988;82:125-30. 177. McBrien NA, Moghaddam HO, Reeder AP. Atropine 159. von Bruecke H, Hellauer HF, Umrath K. Acetylcholin und reduces experimental myopia and eye enlargement via a Inhalt der Hornhaut und seine Beziehungen zur nonaccommodative mechanism. Invest Ophthalmol Vis Sci Nervenversorgung. Ophthalmologica 1949;117:19-35. 1993;34:205-15. 160. Williams JD, Cooper JR. Acetylcholine in bovine corneal 178. Stone RA,Lin T, Laties AM. effects epithelium. Biochem Pharmacol 1965;14:1286-9. on experimental chick myopia. Exp Eye Res 1991;52:755-8. 161. Fitzgerald GG, Cooper JR. Acetylcholine as a possible 179. Troilo D, Gottlieb MD, Wallman J. Visual deprivation sensory mediator in rabbit corneal epithelium. Biochem causes myopia in chicks with optic nerve section. Curr Eye PharmacoI 1971;20:2741-8. Res 1987;6:993-9.

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