Molecular Vision 2004; 10:787-93 ©2004 Molecular Vision Received 22 December 2003 | Accepted 19 August 2004 | Published 13 October 2004

Muscarinic antagonist control of myopia: A molecular search for the M1 receptor in chick

George C. Yin, Alex Gentle, Neville A. McBrien

Department of Optometry and Vision Sciences, The University of Melbourne, Victoria, Australia

Purpose: Pirenzepine, an M1 selective muscarinic antagonist, is effective in slowing the progression of myopia in both humans and experimental animals, including chick. As an M1 selective antagonist, pirenzepine is considered to mediate its effect through M1 receptors. However, there is currently no report of the M1 receptor in chicken. Therefore, if the mechanism of action of pirenzepine is similar across species, either the drug mediates its effect through a non-M1 mecha- nism, or M1 muscarinic receptors are present in chicken. The aim of the present study was to determine whether a genetic template for the M1 receptor was expressed, or even present, in chick. Methods: Polymerase Chain Reaction (PCR), and Southern and northern blotting analyses were used to search for M1 mRNA in chick ocular and brain tissues. PCR and Southern analyses were then used for searching the chick M1 gene and promoter. Appropriate rat positive controls were included throughout the study. Results: Direct mRNA detection by northern analysis showed no evidence of M1 mRNA expression in the chick ocular and brain tissues studied. Identical results were obtained from PCR amplification and were further confirmed by Southern analysis. Similarly, no M1 gene or promoter sequences were detected by PCR or Southern analyses. Our methods were validated in every case by a positive finding in equivalent rat tissue and by detection of M2 and M4 mRNA expression in chick retina. Conclusions: Findings in this study suggest that the chick does not possess an M1 receptor. This finding is of primary interest to vision researchers in that it suggests pirenzepine is unlikely to mediate its inhibitory effect on the progression of myopia through an M1 receptor in chick. Alternative mechanisms of action are discussed.

Topical application of the nonselective muscarinic ace- Despite the efficacy of atropine, pirenzepine, himbacine tylcholine receptor (mAChR) antagonist, atropine, has been and, more recently, the nonselective muscarinic antagonist shown to be effective in slowing the progression of myopia in oxyphenonium [15] in the inhibition of myopic eye growth, humans [1]. The drug inhibits the axial elongation of the eye- their mechanism of action is still unknown. Indeed, the rela- ball, the structural change that causes myopia. The effective- tively high doses required of these agents have led to the sug- ness of atropine has also been demonstrated in animal models gestion that these drugs may mediate their effect through non- and such studies have implicated the retina, choroid, and/or specific or non-receptoral mechanisms [15]. For example, it sclera as potential sites of action for the drug. It has been shown has been shown that ablation of the majority of the retinal that the drug does not work via an accommodative mecha- cholinergic amacrine cells, the major pre-junctional acetyl- nism [2,3]. To date, there have been five distinct mAChR sub- choline source of the retinal cholinergic system, has no ob- types characterized (M1-M5) and atropine binds each of these servable effect on myopia progression in chicks, but atropine subtypes with equal affinity [4,5]. Studies using selective treatment is still effective in preventing myopia progression mAChR antagonists showed that pirenzepine, an M1 selec- in these animals [16]. Furthermore, in vitro application of at- tive antagonist previously used in treatment for gastric ulcer ropine to isolated chick retinal tissue induces a non-specific [6-8], is effective in preventing the progression of myopia in a spreading depression of neuronal activity and leads to an in- dose-dependent manner in both mammalian and avian mod- crease in the general release of retinal neurotransmitters [17]. els [2,9,10]. Recently, pirenzepine has been shown in clinical It has been suggested that this extracellular increase of retinal trials to slow the progression of myopia by approximately 50% neurotransmitters could potentially disrupt the signal for ocu- in children [11,12]. Another selective mAChR antagonist, lar growth. In contrast, studies of the dose-response profile of himbacine (M4/M2 selective), has also been found to inhibit pirenzepine and himbacine demonstrate that myopia is inhib- the progression of myopia in chicks [13]. However, antago- ited in a dose-dependent fashion, suggesting these drugs me- nists selective for M2 (methoctramine and gallamine) and M3 diate their effects through a receptoral mechanism [10,13]. (4-DAMP and p-F-HHSiD) are not effective at inhibiting However, the identity of specific mAChR subtypes respon- myopia, even at high doses [2,14,15]. sible for the antagonist induced inhibition of myopia is yet to be elucidated. Correspondence to: Neville A. McBrien, Department of Optometry Pirenzepine is generally regarded as selective for the M1 and Vision Sciences, The University of Melbourne, Victoria 3010, receptor subtype and its effectiveness at preventing myopia in Australia; Phone: +61 (03) 8344 7001; FAX: +61 (03) 9349 7474; both chicks and mammals led to the assumption that it was email: [email protected] 787 Molecular Vision 2004; 10:787-93 ©2004 Molecular Vision working via an M1 mediated mechanism [2]. However, the tex, brainstem, and cerebellum dissected out. All tissues were selectivity of pirenzepine for the M4 receptor subtype is only immediately snap frozen in liquid nitrogen. fourfold lower than that for the M1 receptor [4] and a subse- Following enucleation and brain dissection, animals were quent study of the M4/M2 selective mAChR antagonist decapitated and 4 ml of trunk blood collected and stored in himbacine in chicks showed that it was also effective in inhib- EDTA-coated collection tubes (Greiner Vacuettes, Biolab, iting myopia, suggesting the involvement of M2 or M4 recep- Clayton, Australia). The blood samples were kept on ice for tors in the control of myopia. Furthermore, it has been shown 15 min, at -20 °C overnight then stored at -80 °C. that chick M2 receptor has a higher binding affinity for Materials: Agarose, guanidine thiocyanate, RNase-free pirenzepine when compared to mammalian M2 receptor, and DNase I, DNA and RNA loading buffer, M-MLV, RNasin and can act through both M1-3-5-like and M2-4-like transduction oligo deoxythymidine primers were obtained from Promega mechanisms [18]. This suggests that chick M2 receptor may (Annandale, Australia); PCR primers from Proligo (Lismore, have the functional role of an M1 receptor in mammalian spe- Australia); HotStar Taq polymerase, PCR reaction buffer, cies, and may be the possible site of action for pirenzepine- MgCl2, QIAmp DNA Mini Kit, QIAquick Gel Extraction Kit induced myopia inhibition. However, despite the fact that M2- from Qiagen (Clifton Hill, Australia); Megaprime DNA La- selective antagonists (methoctramine, gallamine, and AFDX belling Kit, [32P] dCTPs, Hybond N+ hybridization membrane 116) show high binding affinity towards chick M2 receptor, and RapidHyb buffer from Amersham Biosciences (Baulkham these drugs are ineffective in the control of myopia [13-15]. Hills, Australia); Micro Bio-Spin P-30 purification columns Thus, it is possible that mAChR antagonists prevent myopia from BioRad Laboratories (Regents Park, Australia), all se- progression via M4, rather than M1 or M2, receptors. How- quencing reagents from Beckman Coulter (Gladesville, Aus- ever, such findings are further complicated by the fact that tralia). Phenol:chloroform:isoamyl alcohol and all other gen- although all mAChR subtypes (M1-M5) have been character- eral laboratory reagents were obtained from Sigma (Castle Hill, ized in mammals and a full receptoral complement has been Australia). demonstrated in the mammalian eye [19], only the M2-M5 RNA and genomic DNA isolation and processing: Total subtypes have been characterized in the chicken [18,20-22], RNA was extracted from brain, retina/choroid and iris/ciliary with no reports, to date, of an M1 receptor being identified. body tissue using an established phenol/chloroform purifica- It is important that the mechanism of action of such drugs tion technique described previously [24,25]. Tissue was ho- in myopia-prevention is determined, as widespread use to al- mogenized in appropriate volumes of extraction buffer (4 M leviate the problems associated with the increasing prevalence guanidinium thiocyanate, 25 mM sodium citrate, and 100 mM of myopia is imminent [23]. The aim of this study was to in- β-mercaptoethanol) using a freezer mill, then vestigate a putative M1-mediated mechanism of action of phenol:chloroform:isoamyl alcohol used to purify the RNA. pirenzepine in the inhibition of myopia in chick by searching Isolated RNA was recovered using isopropanol and glycogen. for evidence of an M1 receptor in this species. Initially, spe- The isolated RNA was treated with DNase I to remove any cific genetic probes targeting sequences of the M1 mAChR contaminating genomic DNA and repurified. The quality of mRNA, then probes for the M1 promoter and gene, were used total RNA was checked using spectrophotometry and formal- to attempt to detect the genetic information necessary to pro- dehyde/agarose gel electrophoresis. Complementary DNA duce a functional chick M1 mAChR. (cDNA) was synthesized from aliquots of total RNA, via re- verse transcription (RT) as previously reported [25], and stored METHODS at -20 °C for downstream PCR analysis. Animals and tissue collection: Chicks (White Leghorn x Black Genomic DNA was purified from chick and rat blood Australorp) were obtained from a local hatchery and kept in a using the commercially available QIAmp DNA Mini Kit and temperature-controlled brooder, under fluorescent lighting, on the suppliers protocol. Blood samples were thawed and the a 12/12 h light/dark cycle, until they were 1 week old. Adult nucleated cells lysed using the kit components. Genomic DNA Sprague-Dawley rats were obtained from a local supplier and was immobilized on the spin columns, washed, recovered and reared under a similar lighting regimen until they were 7 weeks quantified by spectrophotometry. DNA was stored at -20 °C. old. All experimental protocols conformed to the National PCR primer design: M1 mAChR mRNA sequences were Health and Medical Research Council of Australia’s “Guide- obtained from the genomic databases for human (GenBank lines for the Care and Use of Animals in Research”, which are accession number NM_000738), rat (M16406), mouse comparable to the guidelines of the Institute for Laboratory (NM_007698), pig (X04413) and rhesus monkey (AF026262). Animal Research. These sequences were aligned (Clustal-W version 7.0) and Animals were anesthetized with ketamine (75 mg/kg) and regions that showed highest homology across species were xylazine (5 mg/kg) and sacrificed using sodium pentobarbi- chosen for the positioning of PCR primers. Two sets of prim- tone (150 mg/kg). Enucleated chick eyes were hemisected into ers (M1a and M1b; Table 1) were designed, using the Primer anterior and posterior segments and the retina-choroid com- 3 program (release 1.0; source code available at Primer3; Fig- plex isolated under a dissecting microscope. The iris-ciliary ure 1A). Sequence alignment was also performed on the mRNA body complex was then isolated from the anterior segment. sequences of the four previously characterized chick mAChR Chick and rat skulls were opened mid-sagittally and the cor- subtypes (M2, M3, M4, and M5; M73217, L10617, J05218, 788 Molecular Vision 2004; 10:787-93 ©2004 Molecular Vision and AF201960, respectively) to ensure that the primers did act as a control for probe hybridization. Radiolabeled M1a not fall within areas of corresponding cross-subtype homol- probe or 18S probes (2.5 ng/ml), in RapidHyb buffer, were ogy (Figure 1B). Finally, NCBI BLAST searches were car- hybridized to membranes at 37 °C over a 24 h period, then ried out to ensure there was minimal likelihood of any primer membranes were washed at 50 °C (2X SSC, 0.1% SDS). Hy- pair generating PCR fragments from unrelated sequences. It bridization conditions were relatively low stringency to maxi- was expected that the primers would produce similar size PCR mize chances of M1 detection in chick should significant se- fragments from mRNA and genomic DNA, given that the cod- quence differences exist between the putative chick and rat ing region of the M1 mAChR gene is contained in a single M1 sequence. Phosphoimaging was used to visualize the re- exon [4]. sults. Primers to the M1 promoter (Table 1) were designed, us- Analysis of M1 mRNA expression by PCR and Southern ing a similar protocol to that described above, from M1 pro- blotting: Complementary DNA from chick and rat tissues was moter sequence information for human (AF091492) and rat subjected to PCR analysis, using standard techniques and re- (AF091491). The same primer pairs were subsequently used agents along with the primer pairs M1a and M1b. To maxi- in experiments involving both chick and rat tissues. Two sepa- mize chances of detecting chick M1 cDNA, four primer an- rate primers targeting chick M2 (M73217) and M4 (J05218) nealing temperatures (between 55 and 64 °C) and four mag- receptor sequence were also designed using a similar protocol nesium chloride titrations (1.5, 2.5, 4, and 5 mM) were used. (Table 1). A 40 cycle reaction protocol (95 °C for 45 s, specified anneal- Preparation of radiolabeled probes: Complementary ing temperature for 45 s, and 72 °C for 1 min) was run on a DNA fragments were amplified from rat mRNA and gDNA gradient PCR block, with HotStar Taq Master Mix, allowing templates, using the two sets of M1 mAChR primers and the the four different primer annealing temperatures to be applied M1 promoter primers, separated by electrophoresis, then ex- at once. PCR reaction products were electrophoresed on aga- cised and purified using the commercially available QIAquick Gel Extraction Kit. Sequence identity was confirmed following dye termina- tor cycle sequencing reactions (DTCS Quick Start Master Mix, forward and reverse primers), analysis on the Beckman Coulter CEQ 8000 XL DNA Fragment Analyser and comparison with the rat M1 mRNA sequence and rat M1 promoter sequence in the genomic databases. Radiolabeled probes were synthesized using the Megaprime DNA Labelling Kit and the supplied protocol. Purified PCR fragments were used as templates to produce probes labeled with [32P] dCTP. Labeled probes were purified through Micro Bio-Spin P-30 columns, ensuring a significant probe length, and the specific activity checked by scintillation counting. Probes were stored at -20 °C until use. Pre-exisitng probes for 18S rRNA (generated in a similar fashion to that described above) were also labeled for use in northern analy- sis. Analysis of mRNA expression by northern blotting: Us- ing a protocol previously reported [26], chick and rat total RNA (30 µg) were mixed with RNA sample loading buffer and electrophoresed on a formaldehyde/agarose (18%/1%) gel. Samples were transferred to, and immobilized on, nylon mem- branes, using a standard alkaline capillary transfer procedure [27] and a small ‘dot blot’ of the appropriate probe template (M1 or 18S) placed on the lower corners of the membrane to Figure 1. PCR primer design strategy. PCR Primers were designed

TABLE 1. PCR PRIMERS to target regions of high cross-species homology for the M1 receptor but low cross-subtype homology for other chick muscarinic recep- Product Primers Sense primer Antisense primer size (bp) tors. A: Schematic diagram of the sequence alignment between all ------identified M1 mRNA sequences. Regions with a high degree of se- M1a 5'-CAGCAGCAGCTCAGAGAGG-3' 5'-CCATTGGCATCTTGATCACC-3' 209 M1b 5'-CATGGAGTCCCTCACATCC-3' 5'-ATGGTGCTGTTGACGTAGC-3' 387 quence homology between these M1 sequences were identified (in M1 Promoter 5'-GACAAGCTGGGAGGAGAGC-3' 5'-TGGGTAAACTCAGGTGTGACC-3' 212 Chick M2 5'-GCACAGAAATGAATAACTCAACG-3' 5'-TTGCTGACCACATAATCAAGC-3' 340 boxes), and the two primer sets (M1a and M1b) were designed to Chick M4 5'-TCTTCATTCTCCTTGCAGACC-3' 5'-ACTTTGATGGACAGCATCACC-3' 229 target sites within these areas. B: The regions with a high degree of Designated primer name (based on gene or gene product targeted), sequence homology between the chick mAChR subtype sequences sense and antisense primer sequence and expected product size of (M2 to M5) are highlighted (in boxes). Primers were designed out- PCR primers used in the present study. side these regions to avoid mis-priming of these receptor sequences. 789 Molecular Vision 2004; 10:787-93 ©2004 Molecular Vision rose gels (2%), transferred to nylon membranes, and immobi- RESULTS lized using the method described above. Membranes were mRNA analysis by northern blotting: Northern hybrid- exposed to the M1A or M1B radiolabeled probes for 2.5 h at izations using the [32P] labeled 18S probe confirmed the pa- 45 °C, using RapidHyb buffer, and washed at 65°C using rela- tency of RNA transfer to membranes and the reliability of the tively high stringency conditions (2X SSC, 0.5% SDS). Re- probe hybridization process, showing a strong hybridization sults were visualized using the phosphoimager. In cases where to chick retina/choroid, iris/ciliary body, and brain, as well as a positive PCR and/or hybridization was obtained, the experi- to the rat brain controls (Figure 2). Hybridizations using the ment was repeated, then the appropriate fragments were puri- M1A probe, however, showed a localized hybridization sig- fied and sequenced, as described. nal in the rat brain RNA samples only, with no evidence of Analysis of genomic DNA for M1 gene and promoter by PCR hybridization to any of the chick tissue samples (Figure 2). and Southern blotting: Genomic DNA from chick and rat mRNA analysis by RT-PCR and Southern blotting: Gel was used as a template for PCR reactions, employing either analysis of chick and rat cDNA, following PCR reactions with the M1a, M1b, or M1 promoter primers. PCR reactions were primer sets M1a and M1b, repeatedly showed strong amplifi- run across a range of annealing temperatures and magnesium cation of the rat positive control samples with single bands of chloride concentrations as described above. Products were the expected size (Figure 3). No amplification product of the transferred to nylon membranes and hybridized to the appro- appropriate size was observed in any of the chick tissues, with priate probe. Any positive gel or hybridization result was in- either primer pair, and this observation was confirmed fol- vestigated by sequencing. lowing Southern blotting and hybridization, whereas specific hybridization was only encountered in rat positive controls. Sequencing of specific fragments from positive control reac- tions confirmed their identity as the M1 mAChR. PCR and Southern analysis of genomic DNA for M1 gene: Gel analysis of the various PCR reactions using genomic DNA (chick and rat) and primer pairs (M1a and M1b), always showed strong amplification of a single product of the ex- pected size in rat positive control reactions (Figure 4). The identity of these fragments was confirmed by sequencing. Multiple reaction products of a variety of sizes were obtained after PCR of chick genomic DNA and primer set M1b pro- vided a PCR product of approximately the expected size. How-

Figure 3. M1 receptor mRNA expressed in rat but not chick tissue: PCR and Southern analyses. PCR and Southern analyses of chick and rat mRNA suggested M1 receptor expression was confined to rat tissue alone. PCR amplification with primers M1a (A) and M1b (B) Figure 2. M1 receptor mRNA expressed in rat but not chick tissue: at an annealing temperature of 58 °C. Four different

Northern analysis. Northern analysis of chick and rat mRNA sug- MgCl2concentrations, 1.5 mM, 2.5 mM, 4 mM, and 5 mM, were gested M1 receptor expression was confined to rat tissue alone. The used. The tissue samples used in each MgCl2 titration were rat brain northern membrane labeled with the 18S probe (top picture) showed cDNA (R), negative control (N), chick retina-choroid complex (RC), a strong positive signal for all samples (rat brain: R, chick brain: B, iris-ciliary body complex (IC) and brain (B). DNA fragments of the retina-choroid complex: RC, and iris-ciliary body complex: IC; 30 expected size (see Table 1 for details) were observed in rat positive µg of total RNA). The membrane labeled with the M1 probe (bottom controls (207 and 385 bp for primer pairs M1a and M1b, respec- picture) only showed hybridization to the rat sample (R). The arrow tively). There were no fragments of the expected size in any of the indicates the specific binding region. No chick sample (B, RC, or chick samples. Southern blot and probing with 32P radiolabeled M1 IC) showed any specific hybridization. sequence (insets) confirmed the PCR findings. 790 Molecular Vision 2004; 10:787-93 ©2004 Molecular Vision ever, sequencing of these PCR fragments and larger but quences, it was found that the technique was capable of de- strongly amplified fragments revealed that no product was tecting all five mAChR mRNAs in several tree shrew ocular related to the M1 mAChR sequence. In addition, hybridiza- tissues including retina [19]. For these reasons, the techniques tion of the appropriate probe to Southern blots revealed strong used in this study should be capable of detecting the presence hybridization to rat positive control bands but not to any of of M1 receptor mRNA in chicks, provided that the region of the bands in chick tissues, confirming the sequencing results. the M1 sequence studied is present and homologous in birds PCR and Southern analysis of genomic DNA for M1 pro- and mammals. moter: Gel analysis of PCR reactions using chick and rat ge- nomic DNA, and M1 promoter primers, showed strong am- DISCUSSION plification of a single product of the expected size in rat posi- In this study all results suggest that there is no M1 mAChR tive control reactions (Figure 5). The identity of these frag- mRNA expressed in the chick tissues investigated, which it- ments was confirmed through both sequencing and by hybrid- self indicates that the mRNA template necessary for the pro- ization with the radiolabeled probe. No reaction products of duction of the M1 receptor protein is unlikely to be present in the expected size were obtained following PCR of chick ge- chick. Furthermore, the evidence gathered in this study sug- nomic DNA. Products of other sizes were obtained from these gests that the lack of an mRNA template for the M1 receptor reactions, however, subsequent sequencing revealed that none in chick tissues is due to the fact that the chick does not pos- was related to the M1 promoter sequence. Hybridization re- sess a gene or promoter sequence for the M1 receptor. These sults confirmed those of sequencing reactions in that no prod- findings imply that mAChR antagonists, which are effective ucts in the chick reactions were related to the M1 promoter in the prevention of myopia in the chick, are unlikely to be sequence. working through an M1 mediated mechanism. Furthermore, Primers targeting chick M2 and M4 sequences were used if a common mechanism of myopia control exists across spe- in the PCR amplification. By using the same protocol for chick cies, then it may be concluded that such agents do not control M1 detection, fragments of expected size were found in all mammalian or primate myopia progression through their ac- retinal samples (Figure 6). Furthermore, by using a similar tion at the M1 mAChR. PCR protocol with primers targeting human muscarinic se- Although it is difficult to prove that a particular gene or protein is not expressed in a given organism, this study uti- lized several specific approaches to identify the genetic mate- rial necessary for production of an M1 mAChR in chick. Each technique showed no evidence of the presence of essential genetic material for an M1 receptor in the chick and the result was validated in every case by a positive finding in equivalent rat tissue. It might be argued that differences in the sequence of chick M1 genetic information, relative to other species, could have interfered with these observations. However, it is important to note that in this study all primers and probes used

Figure 4. M1 receptor gene exists in rat but not chick genome. PCR, Southern, and subsequent sequencing analyses of chick and rat ge- Figure 5. M1 receptor gene promoter exists in rat but not chick ge- nomic DNA suggested the M1 receptor gene was confined to the rat nome. PCR, Southern and subsequent sequencing analyses of chick alone. PCR amplification of genomic DNA with primer M1a (A) and rat genomic DNA suggested the M1 receptor promoter region and M1b (B). An annealing temperature of 58°C and four different was confined to the rat alone. PCR amplification with M1 promoter

MgCl2 concentrations, 1.5 mM, 2.5 mM, 4 mM, and 5 mM MgCl2, primers at an annealing temperature of 60 °C. Four different MgCl2 were used. In all rat positive controls (R), DNA fragments of the concentrations, 1.5 mM, 2.5 mM, 4 mM, and 5 mM MgCl2, were expected size (207 and 385 bp with primers M1a and M1b, respec- used. DNA fragments of the expected size (212 bp) were observed in tively), were observed. Fragments amplified from chick (C) genomic rat positive controls (R). Fragments amplified from chick (C) ge- DNA (arrows) were sequenced and found to be related to non-cod- nomic DNA were sequenced and found to be related to non-coding ing regions of chromosomes. PCR findings were confirmed by South- chromosomal regions. Results of Southern analysis (shown on the ern analysis (shown on the right). right) further confirmed the PCR results. 791 Molecular Vision 2004; 10:787-93 ©2004 Molecular Vision for detection of M1 sequence targeted regions of the M1 re- It is reasonable to expect the mechanism of action of mAChR ceptor that are critical for the affinity of pirenzepine binding antagonists in inhibiting myopia progression to be consistent [28]. For this reason, if the sequence within this region were across species. Therefore neither atropine nor pirenzepine acts degenerate, one would not expect the receptor to have the same through the M1 receptor in inhibiting myopia in mammals functional properties as the mammalian M1 receptor. and higher primates. The mechanism of action of atropine and The findings of the current study are consistent with a pirenzepine in the prevention of myopia remains unclear. Iden- previous report which demonstrates pirenzepine has a 10 fold tification of this mechanism warrants urgent further investi- higher affinity for the chick M2 receptor subtype than for it’s gation, as these agents are currently under clinical trial for use mammalian counterpart [18], suggesting that M2 acts as a sur- in the prevention of human myopia even though the mecha- rogate for the M1 receptor in birds. It may be argued that such nism and site of action are unknown. data imply that pirenzepine inhibits myopia progression via the M2 receptor in birds and via the M1 receptor in mammals. ACKNOWLEDGEMENTS Thus pirenzepine may inhibit myopia in birds and mammals Research supported by a grant from the National Health and through differing mAChR subtypes. Furthermore himbacine Medical Research Council of Australia (No. 145738-NMcB). (an M4/M2 selective antagonist), while still unproven in mam- This work has previously been presented at the 2003 meeting mals, could feasibly act through the chick M2 receptor in pre- of the Association for Research in Vision and Ophthalmology venting myopia. If this were the case, one might expect the (Yin GC, Gentle A, McBrien NA. Is regulation of ocular growth M1-like and M2-like receptors to act via the same transduc- in chick mediated via the M1 receptor? ARVO Annual Meet- tion mechanism. Although, the literature shows that, the mam- ing; 2003 May 4-9; Fort Lauderdale (FL); the abstract is avail- malian M1 and M2 receptor subtypes share a separate path- able on the ARVO web site). way [29], a study on the CHO cells expressing chick M2 re- ceptor shows that the chick M2 receptor has the ability to REFERENCES couple with both M2-M4-like and M1-M3-M5-like transduc- 1. Bedrossian RH. The effect of atropine on myopia. Ophthalmol- tion pathways [18]. 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