V10a93-Yin Pgmkr

Molecular Vision 2004; 10:787-93 <http://www.molvis.org/molvis/v10/a93> ©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 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 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 <http://www.molvis.org/molvis/v10/a93> ©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.

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