|Molecular Vision 2004;
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
Correspondence to: Neville A. McBrien, Department of Optometry and Vision Sciences, The University of Melbourne, Victoria 3010, Australia; Phone: +61 (03) 8344 7001; FAX: +61 (03) 9349 7474; email: email@example.com
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 mechanism, 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 acetylcholine receptor (mAChR) antagonist, atropine, has been shown to be effective in slowing the progression of myopia in humans . The drug inhibits the axial elongation of the eyeball, the structural change that causes myopia. The effectiveness of atropine has also been demonstrated in animal models and such studies have implicated the retina, choroid, and/or sclera as potential sites of action for the drug. It has been shown that the drug does not work via an accommodative mechanism [2,3]. To date, there have been five distinct mAChR subtypes characterized (M1-M5) and atropine binds each of these subtypes with equal affinity [4,5]. Studies using selective mAChR antagonists showed that pirenzepine, an M1 selective antagonist previously used in treatment for gastric ulcer [6-8], is effective in preventing the progression of myopia in a dose-dependent manner in both mammalian and avian models [2,9,10]. Recently, pirenzepine has been shown in clinical trials to slow the progression of myopia by approximately 50% in children [11,12]. Another selective mAChR antagonist, himbacine (M4/M2 selective), has also been found to inhibit the progression of myopia in chicks . However, antagonists selective for M2 (methoctramine and gallamine) and M3 (4-DAMP and p-F-HHSiD) are not effective at inhibiting myopia, even at high doses [2,14,15].
Despite the efficacy of atropine, pirenzepine, himbacine and, more recently, the nonselective muscarinic antagonist oxyphenonium  in the inhibition of myopic eye growth, their mechanism of action is still unknown. Indeed, the relatively high doses required of these agents have led to the suggestion that these drugs may mediate their effect through non-specific or non-receptoral mechanisms . For example, it has been shown that ablation of the majority of the retinal cholinergic amacrine cells, the major pre-junctional acetylcholine source of the retinal cholinergic system, has no observable effect on myopia progression in chicks, but atropine treatment is still effective in preventing myopia progression in these animals . Furthermore, in vitro application of atropine to isolated chick retinal tissue induces a non-specific spreading depression of neuronal activity and leads to an increase in the general release of retinal neurotransmitters . It has been suggested that this extracellular increase of retinal neurotransmitters could potentially disrupt the signal for ocular growth. In contrast, studies of the dose-response profile of pirenzepine and himbacine demonstrate that myopia is inhibited in a dose-dependent fashion, suggesting these drugs mediate their effects through a receptoral mechanism [10,13]. However, the identity of specific mAChR subtypes responsible for the antagonist induced inhibition of myopia is yet to be elucidated.
Pirenzepine is generally regarded as selective for the M1 receptor subtype and its effectiveness at preventing myopia in both chicks and mammals led to the assumption that it was working via an M1 mediated mechanism . However, the selectivity of pirenzepine for the M4 receptor subtype is only fourfold lower than that for the M1 receptor  and a subsequent study of the M4/M2 selective mAChR antagonist himbacine in chicks showed that it was also effective in inhibiting myopia, suggesting the involvement of M2 or M4 receptors in the control of myopia. Furthermore, it has been shown that chick M2 receptor has a higher binding affinity for pirenzepine when compared to mammalian M2 receptor, and can act through both M1-3-5-like and M2-4-like transduction mechanisms . This suggests that chick M2 receptor may have the functional role of an M1 receptor in mammalian species, and may be the possible site of action for pirenzepine-induced myopia inhibition. However, despite the fact that M2-selective antagonists (methoctramine, gallamine, and AFDX 116) show high binding affinity towards chick M2 receptor, these drugs are ineffective in the control of myopia [13-15]. Thus, it is possible that mAChR antagonists prevent myopia progression via M4, rather than M1 or M2, receptors. However, such findings are further complicated by the fact that although all mAChR subtypes (M1-M5) have been characterized in mammals and a full receptoral complement has been demonstrated in the mammalian eye , only the M2-M5 subtypes have been characterized in the chicken [18,20-22], with no reports, to date, of an M1 receptor being identified.
It is important that the mechanism of action of such drugs in myopia-prevention is determined, as widespread use to alleviate the problems associated with the increasing prevalence of myopia is imminent . The aim of this study was to investigate a putative M1-mediated mechanism of action of pirenzepine in the inhibition of myopia in chick by searching for evidence of an M1 receptor in this species. Initially, specific genetic probes targeting sequences of the M1 mAChR mRNA, then probes for the M1 promoter and gene, were used to attempt to detect the genetic information necessary to produce a functional chick M1 mAChR.
Animals and tissue collection
Chicks (White Leghorn x Black Australorp) were obtained from a local hatchery and kept in a temperature-controlled brooder, under fluorescent lighting, on a 12/12 h light/dark cycle, until they were 1 week old. Adult Sprague-Dawley rats were obtained from a local supplier and reared under a similar lighting regimen until they were 7 weeks old. All experimental protocols conformed to the National Health and Medical Research Council of Australia's "Guidelines for the Care and Use of Animals in Research", which are comparable to the guidelines of the Institute for Laboratory Animal Research.
Animals were anesthetized with ketamine (75 mg/kg) and xylazine (5 mg/kg) and sacrificed using sodium pentobarbitone (150 mg/kg). Enucleated chick eyes were hemisected into anterior and posterior segments and the retina-choroid complex isolated under a dissecting microscope. The iris-ciliary body complex was then isolated from the anterior segment. Chick and rat skulls were opened mid-sagittally and the cortex, brainstem, and cerebellum dissected out. All tissues were immediately snap frozen in liquid nitrogen.
Following enucleation and brain dissection, animals were decapitated and 4 ml of trunk blood collected and stored in EDTA-coated collection tubes (Greiner Vacuettes, Biolab, Clayton, Australia). The blood samples were kept on ice for 15 min, at -20 °C overnight then stored at -80 °C.
Agarose, guanidine thiocyanate, RNase-free DNase I, DNA and RNA loading buffer, M-MLV, RNasin and oligo deoxythymidine primers were obtained from Promega (Annandale, Australia); PCR primers from Proligo (Lismore, Australia); HotStar Taq polymerase, PCR reaction buffer, MgCl2, QIAmp DNA Mini Kit, QIAquick Gel Extraction Kit from Qiagen (Clifton Hill, Australia); Megaprime DNA Labelling Kit, [32P] dCTPs, Hybond N+ hybridization membrane and RapidHyb buffer from Amersham Biosciences (Baulkham Hills, Australia); Micro Bio-Spin P-30 purification columns from BioRad Laboratories (Regents Park, Australia), all sequencing reagents from Beckman Coulter (Gladesville, Australia). Phenol:chloroform:isoamyl alcohol and all other general laboratory reagents were obtained from Sigma (Castle Hill, Australia).
RNA and genomic DNA isolation and processing
Total RNA was extracted from brain, retina/choroid and iris/ciliary body tissue using an established phenol/chloroform purification technique described previously [24,25]. Tissue was homogenized in appropriate volumes of extraction buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate, and 100 mM β-mercaptoethanol) using a freezer mill, then phenol:chloroform:isoamyl alcohol used to purify the RNA. Isolated RNA was recovered using isopropanol and glycogen. The isolated RNA was treated with DNase I to remove any contaminating genomic DNA and repurified. The quality of total RNA was checked using spectrophotometry and formaldehyde/agarose gel electrophoresis. Complementary DNA (cDNA) was synthesized from aliquots of total RNA, via reverse transcription (RT) as previously reported , and stored at -20 °C for downstream PCR analysis.
Genomic DNA was purified from chick and rat blood using the commercially available QIAmp DNA Mini Kit and the suppliers protocol. Blood samples were thawed and the nucleated cells lysed using the kit components. Genomic DNA was immobilized on the spin columns, washed, recovered and quantified by spectrophotometry. DNA was stored at -20 °C.
PCR primer design
M1 mAChR mRNA sequences were obtained from the genomic databases for human (GenBank accession number NM_000738), rat (M16406), mouse (NM_007698), pig (X04413) and rhesus monkey (AF026262). These sequences were aligned (Clustal-W version 7.0) and regions that showed highest homology across species were chosen for the positioning of PCR primers. Two sets of primers (M1a and M1b; Table 1) were designed, using the Primer 3 program (release 1.0; source code available at Primer3; Figure 1A). Sequence alignment was also performed on the mRNA sequences of the four previously characterized chick mAChR subtypes (M2, M3, M4, and M5; M73217, L10617, J05218, and AF201960, respectively) to ensure that the primers did not fall within areas of corresponding cross-subtype homology (Figure 1B). Finally, NCBI BLAST searches were carried out to ensure there was minimal likelihood of any primer pair generating PCR fragments from unrelated sequences. It was expected that the primers would produce similar size PCR fragments from mRNA and genomic DNA, given that the coding region of the M1 mAChR gene is contained in a single exon .
Primers to the M1 promoter (Table 1) were designed, using a similar protocol to that described above, from M1 promoter sequence information for human (AF091492) and rat (AF091491). The same primer pairs were subsequently used in experiments involving both chick and rat tissues. Two separate primers targeting chick M2 (M73217) and M4 (J05218) receptor sequence were also designed using a similar protocol (Table 1).
Preparation of radiolabeled probes
Complementary DNA fragments were amplified from rat mRNA and gDNA templates, using the two sets of M1 mAChR primers and the M1 promoter primers, separated by electrophoresis, then excised and purified using the commercially available QIAquick Gel Extraction Kit.
Sequence identity was confirmed following dye terminator 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 analysis.
Analysis of mRNA expression by northern blotting
Using a protocol previously reported , 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 membranes, using a standard alkaline capillary transfer procedure  and a small 'dot blot' of the appropriate probe template (M1 or 18S) placed on the lower corners of the membrane to act as a control for probe hybridization. Radiolabeled M1a probe or 18S probes (2.5 ng/ml), in RapidHyb buffer, were hybridized to membranes at 37 °C over a 24 h period, then membranes were washed at 50 °C (2X SSC, 0.1% SDS). Hybridization conditions were relatively low stringency to maximize chances of M1 detection in chick should significant sequence differences exist between the putative chick and rat M1 sequence. Phosphoimaging was used to visualize the results.
Analysis of M1 mRNA expression by PCR and Southern blotting
Complementary DNA from chick and rat tissues was subjected to PCR analysis, using standard techniques and reagents along with the primer pairs M1a and M1b. To maximize chances of detecting chick M1 cDNA, four primer annealing temperatures (between 55 and 64 °C) and four magnesium chloride titrations (1.5, 2.5, 4, and 5 mM) were used. A 40 cycle reaction protocol (95 °C for 45 s, specified annealing temperature for 45 s, and 72 °C for 1 min) was run on a gradient PCR block, with HotStar Taq Master Mix, allowing the four different primer annealing temperatures to be applied at once. PCR reaction products were electrophoresed on agarose gels (2%), transferred to nylon membranes, and immobilized using the method described above. Membranes were exposed to the M1A or M1B radiolabeled probes for 2.5 h at 45 °C, using RapidHyb buffer, and washed at 65°C using relatively high stringency conditions (2X SSC, 0.5% SDS). Results were visualized using the phosphoimager. In cases where a positive PCR and/or hybridization was obtained, the experiment was repeated, then the appropriate fragments were purified and sequenced, as described.
Analysis of genomic DNA for M1 gene and promoter by PCR and Southern blotting
Genomic DNA from chick and rat was used as a template for PCR reactions, employing either the M1a, M1b, or M1 promoter primers. PCR reactions were run across a range of annealing temperatures and magnesium chloride concentrations as described above. Products were transferred to nylon membranes and hybridized to the appropriate probe. Any positive gel or hybridization result was investigated by sequencing.
mRNA analysis by northern blotting
Northern hybridizations using the [32P] labeled 18S probe confirmed the patency of RNA transfer to membranes and the reliability of the probe hybridization process, showing a strong hybridization to chick retina/choroid, iris/ciliary body, and brain, as well as to the rat brain controls (Figure 2). Hybridizations using the M1A probe, however, showed a localized hybridization signal in the rat brain RNA samples only, with no evidence of hybridization to any of the chick tissue samples (Figure 2).
mRNA analysis by RT-PCR and Southern blotting
Gel analysis of chick and rat cDNA, following PCR reactions with primer sets M1a and M1b, repeatedly showed strong amplification of the rat positive control samples with single bands of the expected size (Figure 3). No amplification product of the appropriate size was observed in any of the chick tissues, with either primer pair, and this observation was confirmed following Southern blotting and hybridization, whereas specific hybridization was only encountered in rat positive controls. Sequencing of specific fragments from positive control reactions 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 expected 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 provided a PCR product of approximately the expected size. However, sequencing of these PCR fragments and larger but strongly amplified fragments revealed that no product was related to the M1 mAChR sequence. In addition, hybridization of the appropriate probe to Southern blots revealed strong hybridization to rat positive control bands but not to any of the bands in chick tissues, confirming the sequencing results.
PCR and Southern analysis of genomic DNA for M1 promoter
Gel analysis of PCR reactions using chick and rat genomic DNA, and M1 promoter primers, showed strong amplification of a single product of the expected size in rat positive control reactions (Figure 5). The identity of these fragments was confirmed through both sequencing and by hybridization with the radiolabeled probe. No reaction products of the expected size were obtained following PCR of chick genomic DNA. Products of other sizes were obtained from these reactions, however, subsequent sequencing revealed that none was related to the M1 promoter sequence. Hybridization results confirmed those of sequencing reactions in that no products in the chick reactions were related to the M1 promoter sequence.
Primers targeting chick M2 and M4 sequences were used in the PCR amplification. By using the same protocol for chick M1 detection, fragments of expected size were found in all retinal samples (Figure 6). Furthermore, by using a similar PCR protocol with primers targeting human muscarinic sequences, it was found that the technique was capable of detecting all five mAChR mRNAs in several tree shrew ocular tissues including retina . For these reasons, the techniques used in this study should be capable of detecting the presence of M1 receptor mRNA in chicks, provided that the region of the M1 sequence studied is present and homologous in birds and mammals.
In this study all results suggest that there is no M1 mAChR mRNA expressed in the chick tissues investigated, which itself indicates that the mRNA template necessary for the production of the M1 receptor protein is unlikely to be present in chick. Furthermore, the evidence gathered in this study suggests that the lack of an mRNA template for the M1 receptor in chick tissues is due to the fact that the chick does not possess a gene or promoter sequence for the M1 receptor. These findings imply that mAChR antagonists, which are effective in the prevention of myopia in the chick, are unlikely to be working through an M1 mediated mechanism. Furthermore, if a common mechanism of myopia control exists across species, then it may be concluded that such agents do not control mammalian or primate myopia progression through their action at the M1 mAChR.
Although it is difficult to prove that a particular gene or protein is not expressed in a given organism, this study utilized several specific approaches to identify the genetic material 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 for detection of M1 sequence targeted regions of the M1 receptor that are critical for the affinity of pirenzepine binding . For this reason, if the sequence within this region were degenerate, one would not expect the receptor to have the same functional properties as the mammalian M1 receptor.
The findings of the current study are consistent with a previous report which demonstrates pirenzepine has a 10 fold higher affinity for the chick M2 receptor subtype than for it's mammalian counterpart , suggesting that M2 acts as a surrogate for the M1 receptor in birds. It may be argued that such data imply that pirenzepine inhibits myopia progression via the M2 receptor in birds and via the M1 receptor in mammals. Thus pirenzepine may inhibit myopia in birds and mammals through differing mAChR subtypes. Furthermore himbacine (an M4/M2 selective antagonist), while still unproven in mammals, could feasibly act through the chick M2 receptor in preventing myopia. If this were the case, one might expect the M1-like and M2-like receptors to act via the same transduction mechanism. Although, the literature shows that, the mammalian M1 and M2 receptor subtypes share a separate pathway , a study on the CHO cells expressing chick M2 receptor shows that the chick M2 receptor has the ability to couple with both M2-M4-like and M1-M3-M5-like transduction pathways . However, we present the argument that although the M2 receptor in chick has relatively high affinity for pirenzepine and may have the functional role of M1 receptor in mammalian species, the receptor has a higher affinity for methoctramine, gallamine and AFDX 116, all M2 selective antagonists [18,20]. Therefore, were the M2 receptor acting as a surrogate for M1 in myopia inhibition in the chick, each of these antagonists should inhibit myopia progression in the chick, however, they do not. In fact, several studies have demonstrated that none of these M2 antagonists is effective in inhibiting myopia progression in chick [2,14,15].
In conclusion, the current study presents evidence to suggest that the chick does not possess a functional M1 mAChR receptor. Such evidence implies that muscarinic antagonists which prevent the progression of myopia in the chick either work through another muscarinic receptor subtype, most likely M4, or through non-specific or non-receptoral mechanisms. It is reasonable to expect the mechanism of action of mAChR antagonists in inhibiting myopia progression to be consistent across species. Therefore neither atropine nor pirenzepine acts through the M1 receptor in inhibiting myopia in mammals and higher primates. The mechanism of action of atropine and pirenzepine in the prevention of myopia remains unclear. Identification of this mechanism warrants urgent further investigation, as these agents are currently under clinical trial for use in the prevention of human myopia even though the mechanism and site of action are unknown.
Research supported by a grant from the National Health and Medical Research Council of Australia (No. 145738-NMcB). This work has previously been presented at the 2003 meeting of the Association for Research in Vision and Ophthalmology (Yin GC, Gentle A, McBrien NA. Is regulation of ocular growth in chick mediated via the M1 receptor? ARVO Annual Meeting; 2003 May 4-9; Fort Lauderdale (FL); the abstract is available on the ARVO web site).
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