Abstract— The subcellular distribution of binding sites for tritiated quinuclidinyl benzylate ([3H]QNB) was studied in cow retina. Primary fractions showing higher specific activity than homogenate were P2 (synaptosomal-mitochondrial) and P3 (microsomal). P2 was subfractionated on a Ficoll gradient with the P2B subfraction exhibiting the greatest enrichment in [3H]QNB binding. Similar subfractionation of P2 on a discontinuous sucrose gradient showed that fractions of particles banding in 0.8m and in the 0.8-1.0 m-sucrose interface also exhibit the greatest enrichment of [3H]QNB binding. When subjected to Scatchard analysis, this reaction shows a density of sites equal to 0.212-0.294 pmol per mg of protein. By plotting the apparent dissociation constant (KD) values vs protein concentration a‘true’KD value of 0.73 nM was obtained. Only one set of binding sites was found using three different concentrations of protein. The reaction was specificially antagonized by atropine (DI50= 7 nM) and scopolamine (DI50= 9 nM) whereas carbamylcholine and d-tubocurarine exhibited DI50's of 0.4 and 0.15 mM, respectively. For P3 the binding of [3H]QNB is characterized by one set of binding sites with ni= 0.250 pmol per mg of protein and an apparent KD of 8.2 nM, and a DI50 for atropine of 15 nM. The [3H]QNB binding sites showed a subcellular distribution similar to that of acetylcholinesterase and choline acetyltransferase.
P1 fractions accounted for 40–60% of the total activity of the three cholinergic markers. Purification of the crude P1 yielded an additional fraction in which the cholinergic markers showed an enrichment with respect to homogenate and P1. Synaptosomes isolated from this fraction exhibited the unusual ultrastructure expected from nerve endings in the outer synaptic layer of retina.
The possible location of the muscarinic cholinergic transmitter system in the vertebrate retina is discussed.