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The anatomical organization of muscarinic receptor subtypes in the human eye

University of British Columbia

To help determine the sites of action of cholinergic drugs in man, for the first time, in vitro autoradiographic techniques were employed to characterize the anatomical distributions of muscarinic acetylcholine binding sites and three different muscarinic receptor subtypes in the post-mortem human eye. Additional studies included in situ hybridization localization of one of the muscarinic receptor subtypes, and the examination of the distributions of two second messenger molecules of the inositol phosphate pathway to which several muscarinic receptor subtypes are believed to be coupled. To localize non- subtype specific muscarinic receptor binding sites, in vitro autoradiography was performed on anatomically intact sections of the human eye using the radioligand [³H]quinuclydinyl benzylate (QNB). Muscarinic binding sites were detected in the ocular smooth muscles and the ciliary epithelium in the anterior segment of the eye, but not in the cornea. They were also localized in the retina, retinal pigment epithelium and choroid of the posterior segment. These qualitative results were consistent among all the different eye specimens studied, however there were marked variations in the quantitative densitometric measurements of the relative amounts of binding between different donor eyes. To specifically localize M1. M2. and M3 muscarinic receptor subtype binding sites, [³H]pirenzipine, [³H]oxotremorine, and [³H]4-diphenylacetoxy-N-methyl-piperidine methiodide ([³H]4DAMP) were used respectively. In the anterior segment. [³H]pirenzipine binding sites (M1) were found in the iris, ciliary muscle, and ciliary epithelium while [³H]oxotremorine binding sites (M2) were specifically localized only in the longitudinal portion of the ciliary muscle. The distribution of M3 binding sites determined indirectly by [³H]DAMP competition with pirenzipine was in all structures labelled by [³H]QNB, and in addition, was detected in both the corneal epithelium and the actively dividing cells of the lens epithelium. In the posterior segment, both [³H]pirenzipine and [³H]oxotremorine binding sites were specific for the retina only, in contrast to the M3 binding sites found in the retina, retinal pigment epithelium, and choroid. As the distribution of M3 binding sites was determined indirectly by [³H]DAMP competition with pirenzipine, this subtype was further explored at the mRNA level. Northern Blot Hybridization on total RNA extracted from the human eye anterior segment was performed using a [³²P] m3 oligonucleotide, and detected a single transcript. By in situ hybridization, using an [³⁵S] m3 oligonucleotide, the m3 mRNA was localized in the same structures identified as having M3 binding sites, and in addition, was detected in the trabecular meshwork and corneal endothelium. Two second messenger molecules, IP3 receptor and PKC were localized by in vitro autoradiographic studies with [³H]inositol ( 1,4,5) triphosphate and [³H]phorbol-12, 13-dibutyrate. They were found in all of the structures found to localize M1 and M3 muscarinic receptor subtypes. Muscarinic receptor subtypes were localized by in vitro autoradiography and in situ hybridization. The results may be valuable to understanding the effects and side effects of cholinergic drug therapy, to the rationalization of new therapeutic strategies in diseases of the eye, and to the further research of these and other receptors in normal and pathologically affected human donor eyes.

Text

2011-01-31

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http://hdl.handle.net/2429/31007

Pathology

University of British Columbia

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