Graefe’s Arch Clin Exp Ophthalmol (2001) 239:310–315
L A B O R AT O R Y I N V E S T I G AT I O N
DOI 10.1007/s004170100288
Hagen Thieme Jan Hildebrandt Lars Choritz Olaf Strauss Michael Wiederholt
Received: 1 February 2001 Revised: 6 March 2001 Accepted: 8 March 2001 Published online: 28 April 2001 © Springer-Verlag 2001
H. Thieme (✉) · J. Hildebrandt L. Choritz · O. Strauss · M. Wiederholt Institut für Klinische Physiologie, Universitäts-Klinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany e-mail:
[email protected] Tel.: +49-30-84452791 Fax: +49-30-84454239 H. Thieme Universitätsaugenklinik, Universitäts-Klinikum Benjamin Franklin, Freie Universität Berlin, Berlin, Germany
Muscarinic receptors of the M2 subtype in human and bovine trabecular meshwork
Abstract Background: The trabecular meshwork is a tissue actively involved in the regulation of intraocular pressure via contractile mechanisms. The present study was performed to investigate the effects of muscarinic m2-receptor antagonists on trabecular meshwork contractility and to identify the m2 muscarinic receptor in human and bovine trabecular meshwork cells. Methods: Isometric tension measurements of bovine trabecular meshwork strips were performed using a custom-made force length transducer. Western blot and immunoprecipitation analysis was used to detect the m2-receptor proteins in membrane preparations of human and bovine trabecular meshwork cells. Results: Immunoblotting results showed the expression of an m2-receptor protein band at 56 kDa in both human and bovine trabecular meshwork cells. Two different
Introduction Elevated intraocular pressure (IOP) is considered to be one of the major factors leading to glaucoma. Despite the search for agents that might improve the microcirculation of the eye, treatment of glaucoma mainly focusses on alleviation of increased IOP either by attenuating the production of aqueous humor by the ciliary epithelium (carbonic anhydrase inhibitors, alpha2 agonists and beta blockers) or by enhancing aqueous humor outflow (muscarinic-receptor agonists and epinephrine) [24]. The way in which the latter compound increases outflow facility
m2-receptor antagonists were tested on trabecular meshwork contractility. After carbachol-induced contraction (10–6M set to 100% contractile force), specific m2-receptor antagonists were applied. 3α-Chloroimperaline (10–6M) had no effect on the maximal carbachol-induced contraction in trabecular meshwork strips. Methoctramine induced a significant relaxation at concentrations of 10–7, 10–6 and 5×10–6 M even in the presence of m1- and m3-receptor antagonists. Conclusion: These data indicate that in addition to the m3-receptor subtype present in the trabecular meshwork this tissue also features the m2 receptor. This receptor is partly involved in the regulation of trabecular meshwork contractility, suggesting that outflow facility might be influenced through this receptor.
remains poorly understood. It is generally accepted that contraction of the ciliary muscle (CM) with its extending muscle fibers changes the sizes and shapes of the intratrabecular spaces, leading to increased outflow [13]. However, different methods have demonstrated that trabecular meshwork (TM) has, amongst other features, smooth muscle-like properties and is actively involved in aqueous humor dynamics [26, 31], making this tissue an appropriate target for antiglaucomatous substances. It has been shown that isolated relaxation of the TM leads to increased outflow rates in the model of the perfused anterior chamber with CM detachment [29].
311
Based on information obtained from cloned gene products, at least five different subtypes of the muscarinic acetylcholine receptor (mACHr) exist, designated m1 to m5. This classification has superseded the definition of muscarinic receptors based on pharmacological evidence (M1 to M3) [4,9]. It is generally accepted that the m3 receptor plays a major regulatory function in the outflow pathway [6, 7, 27, 33]. We provide evidence for the expression of the m2-receptor subtype on the protein level in both human and bovine TM. Furthermore, we report the involvement of mACHr of the m2-subtype in the regulation of bovine TM contractility, indicating that outflow facility may be regulated in part by this receptor in addition to the m3 receptor. Inhibition of m2-ACHr might supply a tool for TM relaxation and, possibly, IOP reduction.
Methods Contractility measurements Enucleated bovine eyes were obtained from a local slaughterhouse. Following established procedures [14], small TM strips were carefully dissected. Briefly, eyes were bisected cutting away vitreous, lens and iris. TM strips approximately 2–4 mm long and 0.5 mm wide were prepared in a circular direction. Isometric tension measurements were performed as previously described [14]. The effects of agents on TM were obtained after the tissues had been allowed to rest under control conditions for at least 1 h. The chamber solution with the isolated strips was maintained at constant temperature and pH. Only strips showing a stable baseline contractility were used for experiments. Isometric contractions are expressed relative to the response obtained with a maximally effective carbachol concentration, 10–6 M, which was tested in each tissue strip as a control. To determine the activity of a compound, it was added to the precontracted tissues. Cell cultures Bovine TM (bTM) cell cultures were established as previously described using freshly enucleated bovine eyes obtained from a local slaughterhouse [3, 15]. Human TM cells (hTM) were isolated by methods based on those of Grierson et al. [10] and Siegner et al. [21]. Experiments on human and bovine tissue were performed in accordance with the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals. History of glaucoma was ruled out by screening patients’ history sheets. Human TM was obtained from human donor eyes or eyes enucleated for posterior malignant melanoma (for details see [25]). Briefly, for aiding visualization of the TM the canal of Schlemm was cannulated using a fine wire probe (0.5 mm diameter). The TM was then carefully dissected and the strips placed under coverslips in 35 mm wells. TM cells were cultured in Dulbecco’s modification of Eagle’s minimal essential medium (DMEM) supplemented with 20% fetal calf serum (FCS), 100 U/ml penicillin and 100 µg/ml streptomycin (all cell culture material from Biochrom, Berlin, Germany). Cells were maintained in a 95% air–5% CO2 atmosphere at 37°C and were passaged using the trypsin-EGTA method after reaching confluency. Only well-characterized normal hTM and bTM cells of early passages (3–6) were used for experiments. Histological characterization was performed by Prof. Elke Lütjen-Drecoll, Universität Erlangen, Department of Anatomy, and showed typical morphology as well as immunostaining as previously described [21].
Western blotting and immunoprecipitation Experimental procedures were followed as described previously [23, 25]. Confluent monolayers of bTM or hTM cells were placed on ice and washed three times with ice-cold phosphate-buffered saline (PBS). Cells were then scraped and lysed in lysis buffer A [20 mM tris(hydroxymethyl) aminomethane (Tris) HCL, pH 7.5, 2 mM EDTA, 0.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N' -tetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml aprotinin] containing additional protease inhibitors (Complete, protease inhibitor cocktail, Boehringer Mannheim, Germany). Cells were lysed for 1 h at 4°C on a rocking platform. After a brief preclearing centrifugation step (5 min at 5000 rpm) the supernatant was centrifuged for 30 min at 50,000 g. The pellet (membranous fraction) was resuspended in 100 µl lysis buffer A. Protein content was determined in the pellet, and the probes were subsequently boiled for 5 min in Lämmli buffer and subjected to SDS-PAGE. The lysates and prestained molecular markers were then separated electrophoretically by polyacrylamide gel electrophoresis using 8.5% polyacrylamide. Equal amounts of protein (25–30 µg) were loaded onto each lane and electrophoresed for 1 h at 150 V in Mini Protean electrophoresis cells (Bio Rad, Life Science Group, USA). After blotting of the proteins to nitrocellulose filters (Polyscreen, NEM, Life Science Products, USA) for 1 h at 100 V, the membranes were blocked in PBS containing 5% bovine serum albumin (BSA) for 1 h at room temperature. Polyclonal antibodies were diluted in the same solution (1:2000; anti m2-receptor antibody, Alomone Labs, Jerusalem, Israel) and the membranes incubated at 4°C overnight on a rocking platform. A three- to fourfold excess of blocking peptide was added to the antibody solution to detect nonspecificity of additional protein bands. After three subsequent washing steps with PBS/Tween (3×10 min), the membranes were incubated with a 1:20,000 dilution of peroxidase-conjugate secondary antibody (Dianova, Jackson Immuno Research Laboratories, USA) for 1 h at room temperature. After three final washing steps (10 min, PBS/Tween), the signals were visualized using a chemiluminescence kit (ECL, Amersham, UK) according to the manufacturer’s instructions. For immunoprecipitation, membrane proteins were suspended under gentle rotation overnight at 4°C with protein-A sepharose beads (Sigma, Deisenhofen, Germany) carrying antibodies against muscarinic m2 receptors (Alomone Labs). Beads were sampled by several gentle washing steps, centrifugation and consequent suspension in 1× Lämmli buffer. To separate precipitates from sepharose beads the suspension was incubated at 95°C for 5 min and centrifuged at 5000 rpm for 2 min prior to electrophoresis. The supernatant was subjected to SDS-PAGE and Western blot analysis with blocking peptide used accordingly. Reagents The following reagents were used for contractility measurements: N,N´-bis[6-(2-methoxybenzyl) amino] hexyl-1,8-octane-diamine tetrahydrochloride (methoctramine) and 3α-chloroimperaline was purchased from RBI, Sigma (Deisenhofen, Germany). Pirenzepine and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) were supplied by Biotrend Chemikalien (Cologne, Germany). All other chemicals were of analytical grade and were also purchased from Sigma. Statistical analysis The results of contractility measurements were expressed as mean values ± standard error of the mean (SEM). Statistical analysis was performed using analysis of variance and Student’s t-test for paired observations (% changes versus carbachol-contracted tis-
312
Fig. 1 A Western blot analysis of trabecular meshwork (TM) membrane preparations in bovine TM cell cultures. B A clear signal is detectable at the expected size of 56 kDa. Membrane preparations of human trabecular meshwork cells also displayed a signal at 56 kDa. C This signal was verified by immunoprecipitation leading to an enrichment of the detected signal
Fig. 2 Contractility curve indicating that 3α-chloroimperaline (10–6 M) had no relaxing effect on a single bovine TM strip
sues). Significance was assumed when P
