Muscarinic Receptor MA1 and M2 Subtypes in the AFDX- 1 16 in Vitro
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British JournalofOphthalmology 1994; 78: 555-559 555 ORIGINAL ARTICLES - Laboratory science Br J Ophthalmol: first published as 10.1136/bjo.78.7.555 on 1 July 1994. Downloaded from Muscarinic receptor MA1 and M2 subtypes in the human eye: QNB, pirenzipine, oxotremorine, and AFDX-1 16 in vitro autoradiography Neeru Gupta, Rob McAllister, Stephen M Drance, Jack Rootman, Max S Cynader Abstract 4-DAMP (4-diphenylacetoxy-N-methyl- Muscarinic cholinergic agents are used to piperidine methiodide) respectively.6 lower intraocular pressure in the medical AFDX-116, a muscarinic antagonist and management of glaucoma and subtypes of oxotremorine, a muscarinic agonist, have been muscarinic receptors have now been shown to have high affinity for M2 receptors78 recognised in many tissues including the eye. which appear to be a heterogeneous group.9'0 To localise muscarinic receptors and their Ml The genes for five muscarinic receptor species and M2 subtypes in the human eye, in vitro have been cloned and sequenced (ml to miS)" and ligand binding and autoradiographic evidence indicates that the protein products of techniques with densitometric quantitation on the ml, m2, m3 clones must closely resemble the postmortem eye sections were used. As lig- Ml, M2, and M3 receptors respectively.'2 ands, [3H] quinuclydinyl benzylate (QNB) The subtypes ofmuscarinic receptors involved (non-subtype specific muscarinic antagonist), in lowering intraocular pressure and their intra- [3H]pirenzipine (Ml antagonist), [3H]oxotre- ocular location is not well understood. In this morine (M2 muscarinic agonist), study, we report the localisation and quantitation [3H]AFDX-116(11[{2[diethylaminomethyl]1- of binding sites in human eye sections for QNB, piperidinyl} acetyl]5,1 ldihydro-6H-pyrido pirenzipine (Ml subtype), oxotremorine, and [2,3b][1,4]benzodiazepine-6-one) (M2 antag- AFDX-1 16 (M2 subtypes) using in vitro ligand onist) were studied. Specific binding sites for binding, autoradiography, and densitometric QNB, pirenzipine, and AFDX-116 were techniques as previously described. 3 14 15 localised in the entire ciliary muscle, the iris, http://bjo.bmj.com/ and ciliary epithelium. [3HJoxotremorine localised only in the longitudinal portion ofthe Materials and methods ciliary muscle, and additionally, was not Seventeen human cadaveric eyes were obtained localised in the iris or ciliary epithelium. These within 12 hours after death from the Eye Bank of results suggest that oxotremorine, by binding British Columbia (Table 1). At least seven differ- selectively to receptors on the longitudinal ent specimens were used for each ligand binding ciliary muscle and inducing its contraction, study, and additionally, eight different speci- on September 26, 2021 by guest. Protected copyright. may modulate outflow facility independently mens were studied with all four ligands. The eyes from accommodation and miosis. were frozen in liquid isopentane cooled to - 80°C (BrJ Ophthalmol 1994; 78: 555-559) on dry ice, and stored at -20°C until use. Sections of 20 Fm thickness were cut with a cryostat (Cambridge Instruments, Nussloch, Direct and indirect acting muscarinic cholinergic Germany) and placed on glass slides coated with agoiiists have been mainstays in the medical 1% gelatin. management of primary open angle glaucoma. The sections were thawed at room temperature The stimulation of muscarinic receptors in the and incubation with 1 nM[3H]QNB ([3H]QNB: Department of ciliary muscle causes its contraction, and by Ophthalmology, University of British virtue of its insertion into the trabecular mesh- Table 1 Human cadaveric eyes Columbia, 2550 Willow work, aqueous outflow facility is increased and Street, Vancouver, intraocular pressure reduced.15 Subtypes ofmus- Sexlage (years) Hours postmortem Cause ofdeath British Columbia, carinic receptors now Canada have been recognised in M/75 3 Glioblastoma many tissues and their localisation in the eye is an M/40 3 5 Myocardial infarction N Gupta M/71 4-5 Lung carcinoma R McAllister important step towards an understanding of the F/42 5 Cervical carcinoma S M Drance effects of subtype specific muscarinic drugs and M/41 5 Multiple sclerosis J Rootman M/39 5 Myocardial infarction M S Cynader the rationalisation of new ocular therapies based F/41 5 Multiple sclerosis on their potentially selective actions. M/42 6 Lung carcinoma Correspondence to: F/46 7 Breast carcinoma Neeru Gupta, MD, PhD, Three muscarinic receptor subtypes - namely, M/34 7 Pneumonia Department of Ml, M2, and M3, have been pharmacologically M/30 7 Cardiovascular accident Ophthalmology, University of M/88 7 Myocardial infarction Toronto, East Wing 6-508, defined based primarily on their affinities for M/61 8 Myocardial infarction 399 Bathurst Street, Toronto, pirenzipine, AFDX- 1 16(11 [{2[diethylamino) F/51 9-5 Pulmonary embolus Ontario, Canada, M5T 2S8. M/61 9-5 Drowning Accepted for publication methyl] 1-piperidinyl} acetyl]5,1 1-dihydro-6H- M/63 11 5 Breast carcinoma 15 March 1994 pyrido[2 ,3b][ 1 ,4]benzodiazepine-6-one), and M/75 12 Diabetes 556 Gupta, McAllister, Drance, Rootman, Cynader New England Nuclear, Mississauga, Ontario, Canada) in 20 nM HEPES Tris pH 7 5 contain- ing 10 mM Mg2+ for 60 minutes and washed as described for [3H]oxotremorine. 16 Incubation Br J Ophthalmol: first published as 10.1136/bjo.78.7.555 on 1 July 1994. Downloaded from - with 0-5 nM [3H]AFDX-116 ([3H]AFDX-116: 97 0 Ci/mmol, New England Nuclear, 4- 0 Mississauga, Ontario, Canada) in Krebs buffer 0- for 60 minutes at room temperature, was fol- 01 lowed by three 5 minute washes. To evaluate non-specific binding, parallel experiments CD included 10 FtM concentrations of unlabelled 0E atropine sulphate (atropine sulphate, New Eng- land Nuclear Corp, Boston, MA, USA) ,0-0 (muscarinic antagonist). Sections were dried in zI an air stream and apposed to tritium sensitive a film in the dark (LKB Ultrofilm, Amersham, Canada, Oakville, Canada). Exposure time determined empirically was as follows; [3H]QNB - 2 weeks, [3H]oxotremorine - 12 weeks, [3H] pirenzipine - 3 weeks, and [3H]AFDX 116 - 3 weeks. Original eye sections were stained with cresyl violet for Nissl substance for anatomical 6 reference. [3H] QNB (nM) Grey values of were calibrated Figure I Scatchard autoradiograms analysis of[3H]QNB against optical density readings ofthe co-exposed ligand binding studies in density standards (autoradiographic [3H] micro- human iris/ciliary body 32-9 Cilmmol, Amersham, Oakville, 0tntario, scales, Amersham, Canada) and mean (n=3) tissue showing a B.,, of 318fmollmg and KD of Canada) for 60 minutes at room temp4erature densitometric quantitative values were expressed 0 51 nM. in sodium phosphate buffer pH 7-4 was followed in fmol/mg (SD). Image analysis was performed by two 5 minute washes in ice cold buffer. with Aristo light table, Nikon Micro-Nikkor Incubation with 0 5 nM [3H]oxotreimorine 55 mm 1:28, Nikkon F-C adaptor, Panasonic (['H]oxotremorine: 87 Ci/mmol, New England Wv-BD400 CCD camera, 0-002 [tF Capacitor, Nuclear, Mississauga, Ontario, Canada) for 30 Sony Trinitron PVM-8200J monitor, data trans- minutes in 20 nM HEPES Tris pH 7 5 buffer lation quick capture board, Macintosh IIfx containing 10 mM Mg2' at room temp4erature computer, and Image 1.23 program (NIH). was followed by three successive 2 minute 1washes in ice cold buffer and a final rinse in deiionised water. Sections were incubated with 1 nM Results http://bjo.bmj.com/ [3H]pirenzipine ([3H]pirenzipine: 82 Ci//mmol, Characterisation of QNB binding in iris/ciliary body preparations showed a Bm., of 318 fmol/mg and KD of0 5 1 nM by Scatchard analysis (Fig 1). Silver grain densities showed a high concentra- tion ofspecific binding sites for QNB localised in _ the ciliary body and iris, with moderate binding . i ...J X: in the posterior segment of the eye (Fig 2a). The on September 26, 2021 by guest. Protected copyright. binding of 1 nM QNB could be totally displaced "" -.- * by addition of unlabelled atropine sulphate (Fig 2b). Anterior segment binding sites were localised in the ciliary muscle, iris, and ciliary epithelium (Fig 2c). Densitometric quantitation revealed specific binding concentrations of363-7 ; (16-9) fmol/mg in the ciliary muscle, 283 6 (34 1) fmol/mg in the iris, and 159-4 (23 4) fmol/mg in the ciliary epithelium. Comparison of Figure 2c b d with its histological counterpart (Fig 2d) showed binding in the cornea and lens equal to non- ; specific background binding. Specific binding sites for oxotremorine were localised (Fig 3a) in the anterior segment and in the retina. This binding was totally displaced by unlabelled atropine sulphate (Fig 3b) or unlabelled oxotremorine. Comparing the auto- radiogram of the anterior segment in Figure 3c with its histological section in Figure 3d, highly oxotremorine sites were noted in Figure 2 Autoradiographic views of[3H]QNB binding sites in eye sections from a 42-year- specific binding old woman. The binding of I nM [3H]QNB concentrates in the ciliary muscle, iris, c~^iliary the longitudinal ciliary muscle. The concentra- epithelium, retina (detached) and retinal pigment epithelium (a). In the higher power view of tion of specific oxotremorine binding sites was the anterior segment (c), the ciliary muscle, iris, and ciliary epithelium are intensely la bellied 112*1 (9-1) fmol/mg in the longitudinal ciliary and this was completely displaced by 10 pM unlabelled atropine sulphate. (b). A Nis. 'sl stained histological section of(c) is given in (d). Findings were consistent in all 10 eye specimu enls muscle. The amount ofbinding in the remainder studied. of the ciliary muscle, the cornea, lens, iris, and Muscarinic receptorM] andM2 subtypes in the human eye: QNB, pirenzipine, oxotremorine, and AFDX-116 in vitro autoradiography 557 Figure 3 Autoradiographic views of[3H]oxotremorine a.. binding sites in eye sections from a 42-year-old woman.