Modd Muscarinic Receptor Function and Expression in Bluegill Retinal Pigment Epithelium

Abstract

Light adaptation in most vertebrates occurs through adjustments in pupillary diameter. Teleosts, however, have a fixed pupil size so they rely on retinomotor movements to regulate the amount of light that reaches the photoreceptors. Retinomotor movements include photoreceptor contraction and elongation as well as the aggregation and dispersion of membrane bound pigment granules located in the retinal pigment epithelium (RPE). Pigment granule dispersion can be induced by using the acetylcholine analog carbachol, and carbachol-induced dispersion can be inhibited by Mooc:l muscarinic receptor specific antagonists. In this study I demonstrate that the native ligand, acetylcholine, also induces pigment granule dispersion but only in the presence of the acetylcholinesterase inhibitor huperzine-A. Previous pharmacological studies have shown that Modd agonists and antagonists are able to mediate carbachol induced dispersion while Meven agents do not. To investigate the involvement of the Ms receptor in light adaptation, isolated bluegill RPE cells were treated with the venom of the Malayan spitting cobra, Naja naja sputatrix. The venom contains an Ms-specific antagonist that inhibited carbachol-induced dispersion. Furthermore, my studies using snake venom revealed possible involvement of phospholipase A2 in carbachol-induced pigment granule dispersion. I also sought to demonstrate Modd receptor expression in RPE by immunolabeling with an anti-human M3 antibody and by using in situ hybridization targeting the Ms receptor transcripts. Labeling with the M3 antibody was seen in the inner and outer nuclear layers and the inner plexiform layer of the bluegill retina. In situ hybridization with the Ms probe indicated a similar distribution of the receptor as that seen with the antibody and also appeared to label structures in the ganglion cell layer. These results suggest that multiple muscarinic receptor subtypes are expressed in the fish retina or that the labels failed to adequately discriminate among Modd receptors.