|Molecular Vision 2007;
Received 14 May 2007 | Accepted 19 July 2007 | Published 20 July 2007
Changes in muscarinic acetylcholine receptor expression in form deprivation myopia in guinea pigs
Liu Qiong, Wu Jie,
Wang Xinmei, Zeng
State Key Laboratory of Ophthalmology Zhongshan Ophthalmic Center, SunYat-sen University, Guangzhou, China
Correspondence to: Dr. Zeng Junwen, State Key Laboratory of Ophthalmology Zhongshan Ophthalmic Center, SunYat-sen University, Guangzhou, 510060, China; Phone: 86-020-87333721; FAX: 86-20-87333271; email: email@example.com
Purpose: Muscarinic receptor signaling is involved in ocular development and implicated in myopia. The aims of this study were to identify the presence of muscarinic acetylcholine receptors (mAChRs) in normal ocular tissues of guinea pigs and to determine if ocular expression of mRNA and protein changes in guinea pigs with or without form-deprived myopia (FDM).
Methods: One- to two-week-old guinea pigs were monocularly treated with a translucent lens. Twenty-one days after the induction of FDM, we collected the retina, choroid, sclera, and iris-ciliary body. We used a semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) to detect changes in mRNA expression for mAChRs. Western blotting analysis was used to investigate changes in protein expression for mAChRs. Levels of mAChRs as well as mRNA and protein expression were statistically compared among FDM, internal, and normal eyes.
Results: We observed expression of mRNA for muscarinic subtypes M1 to M5 in the retina, choroid, sclera, and iris-ciliary body. Proteins for the M1 to M5 subtypes were present in normal ocular tissues. Their molecular weights ranged from 80 kDa for M5 to 52 kDa for M1 as noted on Western blotting. Twenty-one days after the induction of myopia, we observed statistically significant increases in mRNA expression for subtypes M1 (+18.67%) and M4 (+26.48%) as well as in protein expression for M1 (+24.65%) and M4 (+49.11%) in the posterior sclera of FDM-affected eyes (p<0.05 vs. internal control and normal eyes).
Conclusions: The ocular tissues of guinea pigs express muscarinic subtypes M1 to M5. In the posterior sclera, expression of the M1 and M4 subtypes significantly increased in FDM eyes. This finding indicates that muscarinic antagonists may have the potential to act directly on the sclera as a strategy to prevent myopia.
Clinically significant refractive errors are the most common visual disorders with myopia affecting approximately half of the world's young adult population [1-3]. In several Asian countries, the prevalence of myopia may be approaching epidemic proportions .
Deprivation of pattern vision in human infants such as those due to ptosis or hemangioma of the lid causes axial elongation of the deprived eye and high degrees of myopia [5-7]. Studies of many vertebrates including chicks [8,9], guinea pigs , mice , tree shrews , marmosets , and monkeys  have demonstrated a mechanism involving environmental factors, visual mediators, and active emmetropization. Degradation or defocusing of an image falling on the retina may alter retinal neurochemistry, resulting in a signal or signal cascade that traverses the choroid and influencing the growth and remodeling of the sclera. This process may facilitate changes in eye size. Furthermore, this local system can detect signs of defocusing and accurately regulate subsequent changes in eye size with respect to the degree of defocus .
To date, five distinct subtypes of muscarinic acetylcholine receptors (mAChRs), designated M1 to M5, have been characterized . Expression of the mAChRs differs among ocular tissues. The M2, M3, and M4 subtypes were found in the retina, retinal pigment epithelium, choroid, and ciliary body of chicks . Using a reverse-transcription polymerase chain reaction (RT-PCR) in a tree-shrew model of myopia, Truong et al  found the M1 and M4 subtypes in the retina, choroid, ciliary body, and sclera. The M2 subtype was present in only the ciliary body whereas the M3 and M5 subtypes were present in all ocular tissues including the retina, choroid, ciliary body, and sclera. The presence of mAChRs in the sclera has been confirmed in humans .
Muscarinic receptor antagonists such as atropine (a nonselective mAChR antagonist) , oxyphenonium (a nonselective mAChR antagonist) , pirenzepine (an M1-selective antagonist) , and himbacine (an M4-selective antagonist)  can inhibit axial myopia in animals and the structural changes that cause myopia. In clinical practice, topical application of atropine and pirenzepine effectively slows the progression of myopia in humans [24,25]. These observations implicate the retina, choroid, and/or sclera as potential sites of action for muscarinic-active drugs.
Despite these observations, radioligand-binding assay showed no change in the number or affinity of mAChRs in the retina, choroid, or sclera of chick eyes during the development of myopia . Ablation of most retinal cholinergic amacrine cells, the major prejunctional source of acetylcholine in the retinal cholinergic system, had no observable effect on myopic progression in chickens; moreover, treatment with atropine was still effective in preventing the progression . These findings suggest that mAChRs in the retina may not participate in regulating eye growth during the induction of myopia. However, pirenzepine inhibits myopia in a dose-dependent fashion; this finding suggests that these drugs exert their effects by means of receptors .
Given the results described, it is necessary to identify the precise sites and role of mAChR signaling in both normal and myopic eye growth. The aims of this study were to identify the presence of mAChRs in normal ocular tissues of guinea pigs and to determine if the expression of muscarinic receptor subtypes change in the ocular tissues of guinea pigs with form-deprived myopia (FDM).
Forty one- to two-week-old pigmented guinea pigs (Cavia porcellus) were maternally reared and housed in large cages with a cycle of 12 h of darkness and 12 h of white fluorescent lighting. The temperature was maintained at 25 °C. Food and water were available ad libitum. All experiments conformed to the statement of the Association for Research in Vision and Ophthalmology for the use of animals in vision and ophthalmological research.
We randomly assigned the guinea pigs to a formed-deprived myopia (FDM) group (n=24) or a normal group (n=16). The FDM group was raised with a diffuser placed over one randomly selected eye for 21 days and the other eye served as an internal control group. The diffusers were translucent lenses with a diameter of 12 mm and a thickness of 0.8 mm. They were mounted on a matching plastic ring and glued to the periorbital fur of the animals. The diffusers were checked twice a day and if necessary, briefly removed for cleaning. Animals in the normal group were chosen from the same litters as the FDM animals.
Cycloplegia was induced with two drops of tropicamide and refractive errors were measured by means of steak retinoscopy in hand-held, awake animals. Stable refractive errors were generally obtained after 15 min when no pupillary response was observed. All refractive errors referred to the spherical-component refractive error, which was defined as the mean refractive error in the horizontal and vertical meridians. The axial dimensions of the eyes were measured by performing ultrasonography with a 10-MHz transducer while the animals were anesthetized with 10% ether in oxygen. The axial length of the eye was defined as the distance from the front of the cornea to the back of the sclera. Ocular refraction and axial ocular dimensions were collected at the start and end of the experiment.
After specific treatment periods were completed, the animals were given a lethal dose of chloral hydrate and their eyes were enucleated. The conjunctiva, extraocular muscles, and orbital fat were dissected away from the globe. Digital calipers were used to immediately measure the equatorial diameters and axial lengths.
Using a surgical microscope (Topcon, Tokyo, Japan) and razor blade, we cut the eyes of the guinea pigs perpendicular to the anteroposterior axis and approximately 1 mm posterior to the ora serrata on the ice plate. The anterior segment of the eye was discarded except for the iris and the ciliary body. The retina including the adjacent retinal pigment epithelium was separated from the choroid and care was taken to avoid cross-contamination of the tissues. From this point on, we referred to the retina-retinal pigment epithelial complex as the retina. The posterior sclera was excised by using a 7 mm-diameter trephine and the head of the optic nerve was discarded. The iris-ciliary body, retina, choroid, and sclera were snap frozen in liquid nitrogen and stored at -80 °C.
Reverse-transcriptase polymerase chain reaction
Total RNA was isolated from the iris-ciliary body, retina, and choroid tissues with a kit (RNeasy Mini kit; Qiagen, Hilden, Germany) and from the sclera with another kit (RNeasy Fibrous Tissue Mini kit; Qiagen) according to the manufacturer's instructions. The concentration and purity of the RNA were determined by using a spectrophotometer. The absorbance ratio of optical densities at 260 and 280 nm (OD260/OD280) was consistently around 1.90. The integrity of the purified RNA was verified by means of formaldehyde agarose gel electrophoresis followed by ethidium bromide staining.
We then used a reverse-transcriptase polymerase chain reaction (RT-PCR) kit (OneStep; Qiagen, Hilden, Germany) to reverse-transcribe and clone 1.5 μg of the total RNA sample. This step consisted of reverse transcription at 50 °C for 30 min followed by 95 °C for 15 min. PCR cycle parameters were 45 s of denaturation at 94 °C, 45 s of annealing, 1 min of extension at 72 °C, and a final 10-min extension at 72 °C. We performed 31-35 cycles with annealing temperatures of 48-59 °C depending on the abundance of the particular message. Table 1 lists the sequence-specific primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and subtypes M1 to M5 as previously described .
Interexperimental variations were avoided by analyzing samples from various groups in the same run. All of the PCR products yielded single bands corresponding to the expected sizes in base pairs (Table 1). PCR reaction products were separated on 2% agarose gels by using ethidium bromide for visualization. The relative abundance of each PCR product was determined by quantitatively analyzing digital photographs of gels by using software (Labworks; UVP Products, Upland, CA). RNase-free water was used to replace the template RNA as a negative control.
Before we semiquantitatively applied the experimental samples, we equalized the amount of RNA in all of them. In addition, optimal conditions (e.g., annealing temperature) for each set of primers were determined. Cycle-dependent reactions were subsequently performed for each mRNA species to determine the linear range of detection with ethidium bromide. After this range was established, PCR was performed with the lowest cycle number that reliably produced a detectable product. To minimize variability, we performed duplicate runs for each mRNA amplified and averaged the data.
To assess levels of various mRNAs in the ocular tissues, all values were normalized to that of the housekeeping gene, GAPDH. Thus, GAPDH acted as an internal standard to correct for any variations in RNA isolation.
Tissues were homogenized in ice-cold extraction buffer (0.01 M Tris-HCl at pH 7.4, 0.15 M NaCl, 1% w/v Triton X-100, 0.1% SDS, 1% deoxycholic acid, 1 mM EDTA) as well as protease inhibitors (1 μM pepstatin, 1 μg/ml leupeptin, and 0.2 mM PMSF). After homogenization, samples were placed on ice for 20 min and centrifuged at 11,000x g for 30 min. The supernatant was decanted and the pellet was discarded. Protein concentrations were determined according to the Bradford method by using a Bradford protein quantization reagent (Shen Neng Bocai, Shanghai, China).
For each experimental condition, 40 μg of total protein per line was mixed with 5X sample buffer for SDS polyacrylamide gel electrophoresis. The mixture was boiled for three min, electrophoresed on an 8% SDS polyacrylamide gel, and transferred to nitrocellulose membranes (Pall Corporation, East Hills, NY). Protein loading and transfer efficiency were monitored by staining the membranes with 1% Ponceau S. The membranes were washed three times with TBST (pH 7.6) and soaked in a blocking solution (5% w/v skim milk powder in 2.5 mM Tris-HCl and 14 mM NaCl plus 0.05% Tween-20) for one h at room temperature. The membranes were incubated overnight with primary antibodies to the M1, M2, M3, M4, and M5 subtypes at a 1:400 dilution (0.5 μg/mL, Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C in blocking solution. The membranes were then washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody at a 1:5000 dilution (0.4 μg/mL; Boster, Wu Han, China) for one h at room temperature. The membranes were again washed three times with TBST. They were then incubated with enhanced chemiluminescent detection reagents (Pierce, Rockford, IL) and exposed to film (Kodak, Rochester, NY). GAPDH (Kang Chen, China) was used as a housekeeping protein to normalize the protein load.
Data and statistical analysis
Ocular refraction and biometric measures data were expressed as absolute values and the mean interocular differences between FDM and control eyes or between the right and left eyes. In the absence of evidence for a skewed distribution, values were analyzed by using t tests. Groups were compared by using a one-way analysis of variance (ANOVA) with a Tukey post hoc test where p<0.05 indicated a statistically significant difference. All analyses were performed with software (SPSS version 11.5; SPSS, Chicago, IL).
Ocular biometry and refraction
At 21 days, monocularly deprived eyes had myopia of -5.52±2.54 D and an axial length of 7.90±0.24 mm (p<0.001 versus normal and internal control eyes, ANOVA and Tukey post hoc test; Figure 1). Axial lengths of the FDM eyes were significantly greater than those of control eyes (8.58±0.16 versus 8.42±0.17 mm, p=0.011, ANOVA and Tukey post hoc test) but equatorial diameters did not significantly differ in FDM versus control eyes (9.38±0.2 versus 9.32±0.15 mm, respectively, p=0.64, ANOVA and Tukey post hoc test; Figure 2).
Reverse-transcriptase polymerase chain reaction and western blotting for muscarinic acetylcholine receptors in normal eyes
Table 1 shows the approximate sizes of the amplified products for each mAChR subtype. M1 to M5 were found in the retina, choroid, sclera, and iris-ciliary body (Figure 3A).
On western blotting with polyclonal mAChRs antibodies that recognized subtypes, M1 immunoreactivity was detected in tissue extracts of the retina, choroid, sclera, and iris-ciliary body at an apparent molecular mass of about 52 kDa. M2 immunoreactivity was weakly detected in these tissues at an apparent molecular mass of about 62 kDa. An intense M3-immunoreactive band was observed for the sclera, choroid, and iris-ciliary body (molecular mass of about 65 kDa) but the band was weak in the retina. Immunoreactivity for M4 with an apparent molecular mass of about 75 kDa was detected in the retina, choroid, sclera, and iris-ciliary body. A weak M5-immunoreactive band was detected in the retina, choroid, sclera, and iris-ciliary body at molecular mass of about 80 kDa (Figure 3B).
Changes in mRNA expression for muscarinic acetylcholine receptors subtypes
Results from ANOVA of mAChR gene expression normalized to the expression of GAPDH showed no significant differences in the retina, choroid, or iris-ciliary body of FDM eyes relative to the internal control and normal eyes (all p>0.05 for M1 to M5; Figure 4). In the posterior sclera, mRNA expression of FDM eyes was significantly greater than that of internal control or normal eyes for subtypes M1 (p=0.021 or p<0.05, respectively, ANOVA) and M4 (p=0.010 or p<0.05, respectively, ANOVA). However, mRNA expression for the M2, M3, and M5 subtypes was not significantly altered in FDM eyes (p>0.05, ANOVA; Figure 5). Figure 6A shows the effect of myopia induction on the mRNA expression for subtypes M1 and M4. After 21 days of visual deprivation, we found significant increases in M1 (+18.67%, p<0.01) and M4 (+26.48%, p<0.01) compared with the internal control eyes in the same animals.
Changes in protein expression for muscarinic acetylcholine receptors subtypes
Figure 6B shows the effect of myopia induction on the protein expression for the M1 and M4 subtype. After 21 days of visual deprivation, significant increases were found for M1 (+24.25%, p<0. 01) and M4 (+49.11%, p<0.01) compared with the internal control eyes of the same animals. On ANOVA, protein expression significantly increased in the posterior sclera of FDM eyes compared with internal control and normal eyes for M1 (p=0.014 and p<0.05, respectively) and M4 (p=0.007 and p<0.01, respectively). However, protein expression for M2, M3, and M5 was not significantly altered 21 days after the induction of myopia (p>0.05; Figure 7).
When we compared FDM eyes with internal control and normal eyes using ANOVA, we observed no significant alterations in mAChR protein expression in the retina, choroid, or iris-ciliary body (all p>0.05 for M1 to M5; Figure 8).
In guinea pigs, FDM is characterized by an increased axial dimension of the eye . Studies in other animal models and humans have implicated muscarinic signaling in the development of myopia [23,30]. In the current study, RT-PCR and western blotting showed expression of the M1 to M5 subtypes in all ocular tissues of normal guinea pigs. Furthermore, expression of M1 and M4 in the posterior sclera significantly increased during the induction of myopia. To our knowledge, our report is the first to document changes in mAChRs in the ocular tissues of guinea pig during myopic induction. Our finding suggests that mAChR signaling may participate in scleral remodeling during the induction of myopia and that the sclera may be potential sites of action for the mAChRs antagonists currently used to prevent myopia.
Ligand-binding studies have historically been conducted to investigate muscarinic receptors. Several specific muscarinic agonists and antagonists exist and have been used to define the distribution of muscarinic receptors in the eye. The major disadvantage of this method is that the specificity of these substances is modest in most cases. However, in our study, antibodies were raised against peptides sequenced in the third intracellular loop (i3) of each receptor. This area had the least sequence homology among subtypes and the manufacturer confirmed the specificity of the primary antibodies to the mAChRs (M1 to M5) by using preabsorption control. Furthermore, mammalian muscarinic subtypes have 89-98% identity in their amino acid sequences . Antihuman mAChR polyclonal antibodies were used to detect protein expression of the mAChR subtypes and to specifically distinguish the mAChRs of guinea pigs. Moreover, our specific primers were designed to detect mRNA expression of mAChRs and yielded products of about 500 bp based on an alignment of human, mouse, and rat mAChRs sequences. Therefore, we combined RT-PCR with western blotting to investigate specific changes in mAChR expression.
Acetylcholine is a neurotransmitter in the brain, retina, and parasympathetic neurons. It is also involved in regulating the function of basic cells, in cellular differentiation, and in gene expression during development . Cholinergic signaling has been implicated in the regulation of the maturation of organs including ocular structures. For instance, inhibition of cholinesterase activity induced morphological abnormalities of the eye as well as the brain and heart during embryogenesis in chicks . Acetylcholine receptors can be segregated into ionotropic receptors that are selectively activated by nicotine-like ligands and metabotropic receptors that are selectively activated by muscarinic-like ligands (mAChR). The mAChRs belong to a family of receptors that contain seven transmembrane domains and that elicit cellular responses by means of interactions with GTP-binding proteins
Cell culture or tissue studies in mammals have demonstrated the expression of mAChRs in various ocular tissues. Examples of these tissues include the chicken retina (M2-M4) ; the bovine iris sphincter and ciliary processes (M2, M3, and M4) ; cultured rabbit corneal cells including epithelial cells (Ml and M5), endothelial cells (M5), and keratocytes (Ml and M5) ; and cultured human cells including the ciliary smooth muscle and iris sphincter cells (M3) ; and human ciliary smooth muscle tissue (M1 to M5) . Furthermore, researchers have demonstrated the expression of mAChRs in scleral tissues of humans and tree shrews . Immunoreactivity of the M1 subtype was not previously found in the chick eyes  but our study and other mammalian studies have demonstrated M1 expression in the retina. This discrepancy may be due to species-related differences.
Studies revealed the existence of mAChRs in the chicken, rat, and human retina where they were mainly found in the inner plexiform layer [38-40]. Physiological evidence suggests that muscarinic binding sites in the inner plexiform layer are associated with amacrine and/or ganglion cells [41,42]. The release of acetylcholine from displaced amacrine cells under the influence of light in rabbits is well documented  and the effects of acetylcholine from these cells on the inner plexiform layer appear to play a role in subsequent signal transduction [44,45]. In different stages of embryological and postnatal development, the subtype, number, and distribution of the muscarinic proteins change during retinal synaptogenesis . These findings indicate the crucial role of muscarinic signaling in embryonic development. Several patterns of expression appear to guide the layout of retinal structures and later participate in visual function throughout ocular growth. They also suggest that muscarinic receptors may participate in the development of experimental myopia in chicks and mammals, though the location of the mAChRs that participate in growth-regulating pathways in the eye remains unknown. Because regulatory phenomena can occur in eyes separated from central mechanisms by sectioning the optic nerve , the detection of signs of defocusing and the control of eye growth involve local intraocular mechanisms. One or more mAChR subtypes in the retina, retinal pigment epithelium, choroid, or ciliary body may be involved in FDM and in the visual regulation of ocular growth.
We found that expression of the mAChR subtypes was not significantly altered in the retina, choroid, and iris-ciliary body in FDM eyes compared with internal control and normal eyes. This finding indicates that mAChRs of these tissues may not be involved in regulating ocular growth during the induction of myopia. Previous observations support this suggestion. Vessey et al  found that mAChR density and affinity in the retina and choroid were not altered during induction of myopia. Moreover, selective ablation of mAChRs and cholinergic amacrine cells of the retina did not affect myopic development . Also, topical administration of atropine could inhibit axial myopia but did not change the density and affinity of mAChRs in the brain and retina . Retinas of chicks or tree shrews showed no changes in acetylcholine concentrations as a consequence of FDM . Additionally, investigators reported only a weak correlation between the potency of muscarinic antagonists to stimulate ZENK, also known as Zif 269, Egr-1, NGFI-A, and Krox-24, expression in glucagon amacrine cells and their potency to suppress the development of myopia .
The sclera might be the presumed site at which muscarinic antagonists act to prevent myopia since expression of the M1 and M4 subtypes in posterior sclera significantly increased in FDM eyes. In the chicken scleral chondrocytes, pirenzepine (an M1-selective antagonist) inhibited the synthesis of DNA and glycosaminoglycans . The reduction in glycosaminoglycan synthesis were not caused by direct drug toxicity of scleral cells because the changes were reversible and because DNA content was not notably reduced in pirenzepine-treated eyes . These molecular changes could restore the strength of the sclera, inhibit axial elongation of the eye, and therefore prevent axial myopia. In our study, expression of M1 and M4 in the posterior sclera was upregulated in FDM. As reduced choroidal blood flow  and decreased acetylcholine synthesis in chick choroid and ciliary ganglion has been reported in eyes developing myopia, we postulate that the upregulated expression might have resulted from signals in the retina and choroid . Because acetylcholine is a neuromodulator and a ligand of the neurotransmitter mAChRs, it plays an important role in regulating the expression of these receptors.
In conclusion, our study provided a comprehensive profile of the expression of mAChRs in the ocular tissues of guinea pigs. Expression of the M1 and M4 subtypes significantly increased in the posterior sclera of FDM eyes. Therefore, the sclera is a possible site of action for muscarinic antagonists in preventing mammalian myopia.
This study was supported by grant 30572005 from the National Natural Science Foundation, China and by grant SUMS98677 from the CMB Foundation.
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